International Review of Cytology: A Survey of Cell Biology, Volume 90

INTERNATIONAL REVIEW OF CYTOLOGY VOLUME90 ADVISORY EDITORS H. W. BEAMS DONALD G. MURPHY HOWARD A. BERN ROBERT G . E...

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INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME90

ADVISORY EDITORS H. W. BEAMS DONALD G. MURPHY HOWARD A. BERN ROBERT G . E. MURRAY GARY G. BORISY RICHARD NOVICK PIET BORST ANDREAS OKSCHE BHARAT B. CHATTOO MURIEL J . ORD STANLEY COHEN VLADIMIR R. PANTIC RENE COUTEAUX W. J. PEACOCK MARIE A. DlBERARDlNO DARRYL C. REANNEY CHARLES J . FLICKlNGER LIONEL I . REBHUN JEAN-PAUL REVEL OLUF GAMBORG M. NELLY GOLARZ DE BOURNE JOAN SMITH-SONNEBORN WILFRED STEIN YUKIO HlRAMOTO YUKINORI HlROTA HEWSON SWlFT K. KUROSUMI K. TANAKA GIUSEPPE M ILLON IG DENNIS L. TAYLOR ARNOLD MITTELMAN TADASHI UTAKOJI AUDREY MUGGLETON-HARRIS ROY WIDDUS ALEXANDER YUDlN

INTERNATIONAL

Review of Cytology EDITED BY

G. H. BOURNE

J. F. DANIELLI

S t . George's University School oj Medicine St. George's, Greriadu West Itidies

Danielli Associates Worcester-, Massachusetts

ASSISTANT EDITOR K. W. JEON Department of Zoology University of Tennessee Knosville. Teniwmee

VOLUME90

1984

ACADEMIC PRESS, INC. (Harcourr Brace Jovonovich, Publishers)

Orlando San Diego New York London Toronto Montreal Sydney Tokyo

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United Kingdom Edition published by ACADEMIC PRESS, INC. ( L O N D O N ) LTD. 24/28 Oval Road, London N W I 7DX

LIBRARY OF CONGRESS CATALOG CARD NUMBER: 5 2 - 5 2 03

I S B N 0-12-364490-9 PRINTED IN THE UNITED STATES OF AMERICA 84 85 86 87

9 8 7 6 5 4 3 2 1

Contents

CONTRIBUTORS . . . . . . . . .

ix

Electron Microscopic Study of Retrograde Axonal Transport of Horseradish Peroxidase E R Z S L B ~ FEHER T 1.

I1 . 111.

IV . V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Visualization of Horseradish Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Morphology and Histology of the Reaction Product . . . . . . . . . . . . . . . . . . . . . . Uptake of Horseradish Peroxidase into the Nerve Terminals . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

i 3 5

21 25 25

DNA Sequence Amplification in Mammalian Cells JoyCt

.

L . HAMLIN JEFFREYD . MII.BRANDT, NICHOLASH . HEINTZ. A N D JANL. c . AZIZKHAN

I. I1 .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Occurrence of Amplification Phenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Cytological Manifestations of Gcne Amplification . . . . . . . . . . . . . . . . . . . . . . . IV . Nature of Amplified Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Agents That Increase the Frequency of Amplification . . . . . . . . . . . . . . . . . . . . VI . Proposed Mechanisms of Sequence Amplification . . . . . . . . . . . . . . . . . . . . . . . VII . Concluding Remark:, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

31

33

45 52 64 67 75 77

Computer Applications in Cell and Neurobiology: A Review R. RANNLYM i a 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II . The Microcomputer in the Research Laboretory . . . . . . . . . . . . . . . . . . . . . . Ill . Computer Systems for Microscope Control and Plotting . . . . . . . . . . . . . . . V

83 84 90

vi

CONTENTS

I V. V. VI . V11. V111.

IX . X.

93 98 103 107 Ill 117 117 119

Serial Section Reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Computer-Aided Morphonietric Measurement . . . . .................... Video Image Processing and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Computer Uses in Photometry and Fluorescence Microscopy . . . . . . . . . . . . . . Computer-Automated Autoradiography and Immunocytochemistry . . . . . . . . . . Other Cell Biology Computer Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of Microtubule Inhibitors on Invasion and on Related Activities of Tumor Cells MAHCM . M h H t u

I. I1 . Ill . I V. V.

VI . VII . VIII .

AND

MARC DE ME-IS

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochemistry of Microtubule AssemblylDisassembly . . . . . . . . . . . . . . . . . . . . . Analysis of Microtubules inside Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antiinvasiveness of Microtubule Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antiproliferative and Cytotoxic Effect of Microtubule Inhibitors . . . . . . . . . . . . Directional Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of Microtubule Inhibitors o n Plasma Membrane Functions . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

125 126 134

139 144 148 161 161

162

Membranes in the Mitotic Apparatus: Their Structure and Function

I. I1 . 111. I V. V.

VI . VII . VI11 .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Early Studies o n Mitotic Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ER in the MA of Higher Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membranes in the MA of Lower Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . Golgi and Other Membranes in the MA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane Function: Regulation of [ C a 2 + ] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane Function: A Component in Chromosome Transport . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................

169

170 173 198 205 209 224 230 231

Pollen-Pistil Recognition: New Concepts from Electron Microscopy and Cytochemistry C . DUMAS.R . B . KNOX. A N D T . GAUIX

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. The Mature Viable Pollen Grain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

239 241

CONTENTS

vii

I11. The Receptive Pistil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Male-Female Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Surface Topography of Suspended Tissue Cells Y u . A . ROVENSKY A N D Ju . M . VASILIEV Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Morphology of Surface Microextensions of Suspended Cells . . . . . . . . . . . . . . . Surface Topography of Suspended Tissue Cells of Various Types . . . Mechanisms of Formation of Microextensions . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of Previous Contacts with the Substrate on the Surface Topography of Suspended Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.

I1 . 111. IV . V.

273 274 285 290 299 303 304

Gastrointestinal Stem Cells and Their Role in Carcinogenesis A . I . BYKOREZA N D Yu . D . IVASHCHENKO

I. II . 111. IV .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stem Cells of the Small Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stem Cells of the Colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stem Cells of the Gastric Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Regulation of Proliferation and Differentiation in the Gastrointestinal Epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Stem Cells in Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF PREVIOUS VOLUMES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

309 311 318 323 332 344 363 364 375 379

This P a ge Intentionally Left Blank

Contributors

Numbers in parentheses indicate the pages on which the authors' contributions begin.

JANE C. AZIZKHAN(3 l), Department of Biochemistry, University of Virginia School of Medicine, Charlottesville, Virginia 22908 A. I. BYKOREZ (309), Department of Chemical Carcinogenesis, Kavetsky Institute for Oncology Problems, Academy of Science of the Ukrainian SSR, Kiev 252127, USSR MARCDE METS( 125), Laboratory of Experimental Cancerology , Department of Radiotherapy and Nuclear Medicine, University Hospital, B-9000 Ghent, Belgium C. DUMAS (239), Dkpartement de Biologie Vkgktale et C.M.E.A.B.G., Universite' Claude Bernard-Lyon I , Villeurbanne 69622 Cedex, France ERZSEBET FEHER( 1 ), First Institute of Anatomy, Semmelweis University Medical School, Budapest, Hungary T. GAUDE (239), De'partement de Biologie Vkgktale et C.M.E.A.B.G., Universite' Claude Bernard-Lyon I , Villeurbanne 69622 Cedex, France JOYCE L. HAMLIN(3 1), Department of Biochemistry, University of Virginia School of Medicine, Charlottesville, Virginia 22908 NICHOLAS H . H E I N T Z(3 ~ l ) , Department of Biochemistry, University of Virginia School of Medicine, Charlottesville, Virginia 22908 'Present address: Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218. 2Present address: Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont 05401. ix

CONTRIBUTORS

X

PETERK. HEPLER( 169), Department of Botany, University of Massachusetts, Amherst, Massachusetts 01003 Yu. D. IVASHCHENKO (309), Department of Chemical Carcinogenesis, Kavetsky Institute for Oncology Problems, Academy of Science of the Ukrainian SSR, Kiev 252127, USSR R. B. KNOX (239), School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia MARC M. MAREEL (125), Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, University Hospital, B-9000 Ghent, Belgium JEFFREYD. MILBRANDT3 (3I), Department of Biochemistry, University of Virginia School of Medicine, Charlottesville, Virginia 22908 R. RANNEYMIZE (83), Department of Anatomy and Division of Neuroscience, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163 Yu. A . ROVENSKY(273), Laboratory of Mechanisms of Carcinogenesis, Cancer Research Center of the USSR Academy of Medical Sciences, Moscow, USSR Ju. M . VASILIEV(273), Laboratory of Mechanisms of Carcinogeriesis, Cancer Research Center of the USSR Academy of Medical Sciences, Moscow, USSR STEPHEN M. WOLNIAK (169), Department of Botany, University of Maryland, College Park, Muryland 20742

7Present address: Division of Laboratory Medicine. Washington University School of Medicinc. St. Louis, Missouri 631 10.

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME90

This P a ge Intentionally Left Blank

INTERNATIONAL REVIEW OF CYTOLOGY. VOL 90

Electron Microscopic Study of Retrograde Axonal Transport of Horseradish Peroxidase ERZSEBET FEHER First Institute

of Anatomy, Semmelweis

University Medictil School, Budapest, Hungary

I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Visualization of Horseradish Peroxidase . . . . . . . . . . . . . . . . . . . . . . . Morphology and Histology of the Reaction Product . . . . . . . . . . . . . . A . Localization of Horseradish Peroxidase in the Nerve Cell Bodies.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Localization of Horseradish Peroxidase within Nerve Processes 1V. Uptake of Horseradish Peroxidase into the Nerve Terminals . . . . . . . V . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. 111.

I 3 5 5 17

21 25 25

I. Introduction One of the earliest reports on retrograde transport of materials within axons, i.e., toward the cell body, was the in vitro observation of Matsumoto (1920), who followed the rapid movement of vesicles stained with neutral red within outgrowing sympathetic fibers. Experiments carried out by Kerkut et al. (1967) and by Watson ( 1968) indicated that radioactive labeled materials injected into muscles appeared in the perikarya of nerve cells innervating them. The retrograde axonal transport of horseradish peroxidase (HRP), which was originally demonstrated in peripheral motorneurons by Kristensson and Olsson (1971) and by Kristensson et ul. (197 I ) , has arroused great interest among neuroanatomists. It was shown that HRP is transported intraaxonally from the terminal region of an axon retrogradely to the parent cell body (Kristensson et al., 1971; LaVail and LaVail, 1972; Hanson, 1973; Jones and Leavitt, 1973; Kristensson and Olsson, 1973a,b; Ralston and Sharp, 1973; Warr, 1973; Graybiel and Devor, 1974; Kuypers et al., 1974; Nauta et a/., 1974; Ito ef a / ., 1981; Carlson and Mesulani, 1982a). This technique has now become widely used as an experimental tool for demonstrating neuronal connections both in the central and in the peripheral nervous systems (i.e., Kristensson, 1975; LaVail, 1975; Cullheim and Kellerth, 1976; Kitai et ul., 1976; Adams, 1977; Hedreen and McGrath, 1977; Hunt et d., 1977; Luiten and van der Pers, 1977; Keefer, 1978; Malmgren and Olsson, 1978; Kalia and Davies, 1978; Vanegas et al., 1978; Satomi et a l . , 1979; Contreras et I Copyright 0 1Y84 by Academic Prew. Inc All rights of rcpraluclion In any form rexrvcd ISBN 0-12-364490-9

2

ERZSEBET FEHER

a/., 1980; Panneton and Loewy, 1980; Arvidsson and Gobel, 1981; Kuo et

a/. ,

1981; Nicholson and Severin, 1981; Nomura and Mizuno, 1981, 1982; Ross et a/., 1981; Stuesse, 1982). A major advantage of the use of the enzyme HRP is elucidating the connections of the nervous system, that neuronal cell somata are labeled in a way which enables the determination of the cells inducing a particular fiber pathway (LaVail et al., 1973; Ralston and Sharp, 1973; Sherlock et a l ., 1975; Price and Fisher, 1978). For example, LaVail and LaVail (1974) observed retrograde transport of HRP from the region of retinal ganglion cell bodies after injection of HRP into the chick optic tectum. The enzyme HRP has been shown in electron microscopic studies to flow in an orthograde as well as in a retrograde direction (Hanson, 1973; Lynch et al., 1973, 1974; Reperant, 1975; Winfield e t a l . , 1975; Jones and Hartman, 1978). Some studies have demonstrated that orthograde transport of HRP can be used to reveal the central distribution of afferent fibers of peripheral nerves both at the light microscopic (Reperant, 1975; Scalia and Colman, 1974; Luiten, 1975; Gwyn et a / ., 1979; Mesulam and Brushart, 1979; Kalia and Mesulam, 1980) and the electron microscopic level (Muller and McMahan, 1976; Proshansky and Egger, 1977; Rastad et ul., 1977; Bettie et a/., 1978; Gobel and Falls, 1979; Ohara and Lieberman, I98 I ) . The cells projecting to regions injected with HRP are identifiable with histochemical procedures (Graham and Karnovsky, 1966) due to the accumulation of vesicular packets of reaction product, which presumably represent the pinocytotic incorporation of the protein at axon terminals (LaVail and LaVail, 1972; LaVail, 1975; Hedreen and McGrath, 1977). The distribution of reaction product helps to clarify under the light microscope the type of neuron, on the basis of its shape, size (Wilson and Groves, 1981), and dendritic and axonal arborization pattern. In a subsequent step the synaptic connections of this identified neuron can be examined by electron microscopes (Jankowska et a/., 1976; Muller and McMahan, 1976; Cullheini et a / . , 1977; Ralston et a/., 1978, 1980; Rethelyi et a / . , 1979; Robson and Mason, 1979; Langerback et a / . , 1981). The reaction product of the retrograde transport of HRP is usually filling the soma and proximal dendrites of the neuron; this means, first, that the type of neuron-that is labeled-can be determined, and second, that boutons undergoing anterograde degeneration following lesion of an afferent neuron can be traced to the soma and proximal dendrite. HRP is particularly useful for demonstrating the arbors of axons and should be applicable to the study of the intrinsic organization of any region with well defined afferent connections. The intracellularly applied HRP is an invaluable marker for tracing neuronal projections, to resolve the detailed morphology of individual neurons, and for marking cells in synaptological analysis with the electron microscope

EM STUDY OF HORSERADISH PEROXIDASE

3

(Cowan and Cuenod, 1975; Winer, 1977; Kristensson, 1975; LaVail, 1978; Eckert and Boshek, 1980; Elekes and Szabo, 1982).

11. Visualization of Horseradish Peroxidase

The method for visualizing the activity of HRP at the electron microscopic level was introduced by Graham and Karnovsky (1966) who have used it to study membrane recycling and to follow the path of the retrograde transport from the synaptic region. HRP being a protein of molecular weight of about 40,000 will not pass across cell membranes unless an invagination of the membranes does not occur. Visualization of the reaction product of the enzyme inside the terminal should therefore imply a membrane event of this kind. Membrane infoldings supposedly resulting from the release of transmitters have been described by several groups of workers (Holtzman et al., 197 1; Ceccarelly et ul., 1973). The Sigma Type VI HRP used contains mainly the basic isoenzyme. According to Giorgi and Zahnd (1978) it is only this isoenzyme that is taken up and transported retrogradely at detectable levels by undamaged nerve cells (Bunt et al., 1976; Bunt and Haschke, 1978; Malmgren rt al., 1978). The sections of materials used were processed to demonstrate the presence of HRP using tetramethylbenzidine (TMB) or 3,3’-diaminobenzidine (DAB) according to the method of Mesulam (1978) and to Graham and Karnovsky (1966), respectively. The distribution of reaction product was much greater in the TMB incubated tissue than in the DAB incubated tissue under the light microscope. This is consistent with the previous observations on the greater sensitivity of the TMB method (Mesulam and Rosene, 1979; Dietrichs et al., 1981; Carlson and Mesulam, 1982a). In the dorsal horn according to Carlson and Mesulam (1982b) DAB reaction product was localized within membrane-bound bodies located in synaptic terminals. These labeled bodies were generally larger than synaptic vesicles and some were elongated rather than circular in profile (Figs. 1 and 2). In contrast to the DAB reaction product, the crystalloid TMB reaction product was not confined to membrane-bound organelles and frequently filled significant portions of the entire synaptic terminal. It has been reported by several authors (Beattie et al., 1978; Gobel and Falls, 1979) that application of HRP to the proximal ends of dorsal roots and subsequent ultrastructural examination of DAB reaction product in the spinal cord showed labeling on the cytoplasmic side of the axolemma and on the external surface of synaptic vesicles and mitochondria. They concluded that this suggests such labeling occurs mostly by diffusion within the cytoplasm rather than by membrane-bound transport. According to Somogyi et al. (1979) a highly electron-dense reaction product

EM STUDY OF HORSERADISH PEROXIDASE

5

was formed when 3,3'-diaminobenzidine was used as substrate at pH 7.4. Only slightly electron dense, but of a characteristic appearance reaction product formed when 0.02% o-tolidine is used at the same pH. The reaction from otolidine at pH 7.4 is found in membrane-limited particles, including multivesicular bodies. The cobalt-glucose oxidase method is also used for HRP reaction by Itoh et al. ( 1979) and by Nakamura el ul. ( 198 1). In our observations to obtain information on the ultrastructural localization of HRP, materials were processed by the method of Somogyi et a / . (1979). In each cat 0.3-0.04 (1.1 at 20% solution of HRP (Sigma Type VI) in 0.05 M phosphate buffer was injected into the mesenteric nerves between the layers of the mesothelium under semisterile conditions over a period of 13-20 minutes. After 2 or 3 days survival the animals were perfused through the left ventricle with I % glutaraldehyde and I % paraformaldehyde in 0. I M phosphate buffer (pH 7.4) according to Benedeczky and Somogyi (1975). Small pieces of the intestine (the middle part of the intestine innervated by the injected nerves) were excised and then cut by a Vibratome in 30-km sections. The slices were washed for several hours in phosphate buffer and placed for 30 minutes in a medium containing 0.05% 3,3'-diaminobenzidine and 0.03% hydrogen peroxide in 0.1 M phosphate buffer for 1 hour. The slices were then postfixed in osmium acid, dehydrated, arid embedded in Araldite. Ultrathin sections were mounted on single-hole grids, contrasted with uranylacetate and lead citrate. At the control examination-processed in a similar wayy-of these sections no labeled cells and processes were found either on light or on electron microscopy.

111. Morphology and Histology of the Reaction Product

A. LOCALIZATION OF HORSERADISH PEROXIDASE I N THE NERVECELL BODIES The usefulness of HRP as a neuronal marker at the electron microscopic level has already been demonstrated via the use of intracellularly applied HRP by several authors (Cullheim and Kellerth, 1976; Jankowska et id., 1976; Snow et al., 1976; Rastad, 1978; Rastad et al., 1977; RCthelyi et a l . , 1982). In the labeled neurons large (300-700 nm in diameter), highly electron-dense profiles, identified earlier as residual bodies or secondary lysosomes (Broadwell et al.,

FIG. I . Labeled nerve processes in the myenteric plexus. Bar scale= 1 pm. x42,OOO. FIG.2. Arrows show the labeled membrane-bound bodies in the nerve terminal. Bar scale= I bm. X72.000.

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1980) were found. Multivesicular bodies were also common and had a variable morphology; a portion of their limiting membrane was often coated on its cytoplasmic surface. Electron-lucent vesicles (40-80 nm in diameter), HRPlabeled vesicles with or without an external coat, and vacuoles (100-300 nm in diameter) of various shape were apparent in all the preparations, usually accumulated closely to the Golgi zones but also at other cytoplasmic sites (Fig. 3). The peroxidase reaction product eventually filled many of the lysosomal residual bodies in the perikarya (Colman et al., 1976; Takeuchi e f al., 1982). In vesicles, smooth endoplasmic reticulum and membrane-limited granules, the end product fills the space right up to the limiting membrane; in contrast, dense-core vesicles which are not labeled and occur in all neurons have a granular matrix, usually of higher electron density than the HRP reaction product, and there is a translucent zone between the matrix and the limiting membrane (Figs. 4 and 5 ) . Lipofuscin pigment is normally found in ganglion cells and appears to increase significantly with age. However, an accumulation of pigment that may be misinterpreted as HRP vesicles could be ruled out since these animals were young and the control materials showed the absence of these pigments. The distribution and cytological features of the labeled neurons were carefully examined and compared with those of the unlabeled neurons. The labeled neurons were seen in both the myenteric and the submucosal plexuses. They were medium-sized (30-50 p,m) and spindle-shaped, multipolar, triangular, or oval (Fig. 6). These data are similar to those obtained by light microscopy (FehCr and Vajda, 1982b). The shape and distribution of the labeled neurons resembled the medium-size cells stained by silver impregnation (FehCr and Vajda, 1972). According to Dogie1 (1895) and Type I1 nerve cells in the wall of the intestine once were believed to be sensory in nature. Later, Kadanoff and Spassowa (1959) described the sensory function of the bipolar and unipolar neurons in the gut. Kuntz (1922) traced nerve fibers from the submucosal plexus into the mucous membrane and suggested that some of the fibers were likely to originate from afferent neurons in the submucous ganglia. It has also been proved with degeneration methods (Schofield, 1960, 1968; Fehtr and Vajda, 1974) that some of the enteric neurons project centripetally along mesenteric neurovascular bundles. The combined anatomical and physiological studies by Bulbring et al. (1958) proved the presence of afferent neurons that innervate the mucous membrane. It has also been shown that with regard to ultrastructural features the small intestine contains different types of nerve cells (Fehtr and Csinyi, 1974; Cook and Burnstock, 1976). Physiological studies have also demonstrated that the intrinsic nerve plexus of the small intestine is composed of at least three types of neurons (Milton and Smith, 1956; Wood, 1975; Furness and Costa, 1980). The labeled neurons have oval nuclei, contain the usual cytoplasmic orga-

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FIG. 3 . Labeled nerve cell in the submucous plexus. Arrows point to the HRP-labeled veticles close to thc Golgi zones. Bar scale= I pm. XS4.000.

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FIG. 6. A medium-size oval-shaped neuron. Peroxidase is evident in a variety of sizes of vesicles and tubules of the neuron soma. Bar scale= I pm. X 18,000.

FIG. 4. Cytoplasm of the labeled nerve cell. Arrows show the dense-core vesicles. Bar scale= 1 pm. x42.000. FIG.5 . Cytoplasm of the labeled nerve cell in the myenteric plexus. Note the abundant densecore vesicles (arrows) occurring in the cytoplasm. Bar scale= I pm. X30.000.

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nelles, such as mitochondria, Golgi apparatus, smooth and rough endoplasmic reticulum, lysosomes, polysomes, and multivesicular bodies (Figs. 7 and 8). The retrogradely transported HRP has very high electron density and is distributed in the perikaryon. This observation was in good accordance with that found at light microscopic levels. The granules varied in size from 10 to 100 nm and many of the larger ones showed definite evidence of being, by fusion of smaller individual granules, of varying density. In an earlier study of neurons of the central nervous system, LaVail and LaVail (1 974) attempted to classify the organelles involved in the retrograde axonal transport of HRP. In the nerve cell bodies the labeled organelles were characterized as ( 1) large, approximately 100 nm vesicles, (2) multivesicular bodies, (3) cup-shaped organelles, and (4) tubules of agranular reticulum. Most of the HRP-filled vesicles were ovoid or somewhat elongated in shape and were bound by a single smooth membrane (Figs. 9 and 10) (Brownson et al., 1977), but the spherical 30-50 nm synaptic vesicles were not labeled. HRPcontaining vesicles and lysosomes were found throughout the cytoplasm of the ganglion cells. However, there is a noticeable tendency for HRP vesicles to occupy a perinuclear position (Kristensson and Olsson, 1971; LaVail et al., 1973; Sotelo and Riche, 1974; Ellison and Clark, 1975). The cross-sectional diameter of the individual vesicles ranged from 65 nm to 0.50 km, with many about 0.3 p m (Fig. 11). In many instances the HRP-positive structures were concentrated near the inner aspect of the Golgi complexes. The Golgi sacs and vacuoles themselves were usually free of HRP product, but some of them contained a small amount of reaction product (Brownson et a/., 1977). Broadwell and Brightman (1979) described the uptake of HRP by hypothalamic neurons under osmotic stress and its subsequent orthograde transport to the neurohypophysis without passage through the Golgi complex. The size of the vesicles containing HRP in neuronal somata increased with time. According to LaVail and LaVail (1974) the HRP was found within organelles in ganglion cell bodies of the retina contralateral to the tectal injection and the most of these labeled vesicles were larger, i.e., about 0.5 p m with some almost 1 p m in diameter by 24 hours. Most dense bodies were clearly membrane bound (Hanson, 1973; LaVail and LaVail, 1974; RepCrant, 1975; Weldon, 1975; Schwab, 1977) and within the larger ones there were often relatively clear vacuoles of varying size ( Al-Khafai el a / . , 198 1) (Fig. 12). N o diffuse HRP product was found in any ganglion cells. FIG. 7. Vesicles containing HRP accumulate near the Golgi complex. Cup-shaped organelles are showed by the arrow. Bar scale= I p.m. X42.000. FIG. 8. Large multivesicular bodies (arrows) are in the labeled neurons. Bar acale= I pin. x30,000.

FIG.9. Most of the HRP-filled vesicles are ovoid and irregular shape and are bound by a single smooth membrane (arrows). Bar scale= 1 pm. X54,OOO. FIG. 10. Irregularly shaped HRP positive structures are seen to be membrane bound (arrow). Bar scale= 1 pm. X42.000. 12

FIG. I I . The cross-sectional diameter of the individual vesicles ranged from 0.3 to 0.5 pm. Bar scale= 1 pin. X96.000. FIG. 12. Most of the multivesicular bodies contain relatively clear vacuoles of varying size (arrows), Bar scale= I Km. X54.000. 13

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In several electron micrographs it was noted that typical HRP vesicles had fibrillary bridges between the smooth endoplasmic reticulum and the vesicles (Fig. 13). Processes originating from the labeled neurons also contained HRP granules (Holstege and Dekker, 1979; Robson and Mason, 1979). These granules were larger (300 to 500 nm) and disseminated throughout the cytoplasm (Fig. 14). Occasionally, granular vesicles, 80 to 120 nm in diameter, were present in the HRP-labeled neurons. Moreover, the labeled cells contained abundant rough endoplasmic reticulum, which was oriented parallel to periphery of the perikarya. The morphological features of the labeled neurons were apparently different from those of unlabeled neurons in the small intestine. The HRP-labeled neurons were covered with nerve processes forming synapses with the soma (Figs. 15 and 16). The fact that cell somata containing electron-dense granules of the reaction product were of one single type of cell, and most of the unlabeled cells showed different morphological features, yields further evidence for the identification of the sensory nature of some neurons in the small intestine. Recent studies by means of retrograde axonal transport of HRP have demonstrated that neurons in the wall of the small intestine project toward the celiac ganglion in the cat (Feher and Vajda, 1982b). It appears that the peristaltic reflex is mediated by an intrinsic reflex arc, in which the afferent neurons were labeled by HRP. Synapses on their surfaces suggest that these neurons collect information from other local, possibly interneuronal nerve cell processes and thus influence prevertebral ganglion cells. It is also possible that the intrinsic afferent neurons converge and establish synapses on the HRP-labeled neurons conveying the information to the prevertebral ganglion. Hence, some of the labeled neurons may be considered as interneurons. If our assumption is correct, such labeled neurons with their synapses might, in fact, be units of integration. The ultrastructural features of the labeled neurons are similar to that described as Type I by FehCr and Csinyi (1974) and some of these cells were seen to degenerate after capsaicin treatment (FehCr and Vajda, I982a). The peroxidase reaction product eventually filled many of the lysosomal residual bodies in the perikarya. The small, HRP-containing vesicles enter the soma from the axon and are supposed to coalesce to form large structures, since most of the vesicles in the soma are larger than those in the axons, and coalescing profiles are frequently seen near the Golgi region (Sellinger and Petiet, 1973). Nauta et a / . (1975) observed that the HRP-filled tubular profiles sometimes FIG. 13. At the arrow the HRP-containing multivesicular body is continouos with smoothsurfaced endoplasmic reticulum. Bar scale= 1 pm. X96.000. FIG. 14. Labeled nerve process originating from the cell of the subrnucosal plexus. Bar scale= 1 pm. ~18,000.

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appeared to be branched and not infrequently appeared to be in continuity with HRP-filled lysosomes. Several investigators have described the early incorporation and segregation of extracellular markers within cup-shaped organelles and multivesicular bodies (Brightman, 1965; Birks et d . , 1972; Bunge, 1973; Holtzman et al., 1973). Additional internal vesicles and entire multivesicular bodies may be formed in the cell soma near the Golgi apparatus (Hirsch et al., 1968; Friend, 1969). Holtzman et al. (1967) have stressed the relationship between the agranular reticulum and lysosomal system of neurons. We have also noted that many of the lysosomes containing retrogradely transported HRP are associated with the agranular reticulum, and in some cases these lysosomes appear to have an associated tail containing HRP. Thus, the appearance of HRP in the agranular reticulum and lysosomes is consistent with the comparmentalization, transport, and degradation of any exogenous protein taken up by the neuron. The HRP disappears from the cell in 3-4 days and many of the residual lysosomes in these nerve cells are more electron dense than those in the control nerve cells. The structures with more loosely packed HRP reaction product may represent various stages of degradation of HRP. HRP can also reach the neuron’s soma and dendrites as a diffuse label, however, and both diffuse and agranular labeling may coexist in the same cell cluster or even in the same neuron (Adams and Warr, 1976). In the vicinity of the injection site of HRP into the central nervous system, many of the oligodendrocytes, superficial glial cells contained accumulated HRP diffusely within their cytoplasm, particularly after longer intervals (Krishnan and Singer, 1973; LaVail and LaVail, 1974).

B . LOCALIZATION OF HORSERADISH PEROXIDASE WITHIN NERVEPROCESSES The intraaxonal retrograde transport of exogenous protein in the nervous system is an established phenomenon. Multivesicular bodies are regularly present in presynaptic terminals and axons. It is noted that vesicles contain not only dense granules but also a variety of membranous elements, including multivesicular substructures. When endocytosed tracers such as HRP are present, the multivesicular bodies become labeled among the synaptic vesicles (Villegos, and Fernandez, 1966; Holtzman et a/., 1971 ; Teichberg et a/., 1975). According to Theodosis ( 1982) up to 4 hours after the tracer injections, large vacuoles and cup-shaped figures FIG. 15. Small clear vesicles containing nerve terminal (arrow) synapse with the soma. Bar scale= I pm. X72.000. FIG. 16. The HRP-labeled neurons are covered with the nerve process forming synapse with the soma (arrow). Bar scale= I pm. X30.000.

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(from 100 to 300 nm in diameter) are predominant in intraaxonal organelles labeled with HRP. Both in longitudinally and cross-sectioned axons, the HRPlabeled vesicles were often found interspersed between neurotubules and neurofilaments, centrally located in the axoplasm. Associated with these organelles were smaller vesicles and tubules, with or without reaction product. Profiles of intraaxonal HRP vesicles were identified in several configurations. The fine structure of all profiles of HRP vesicles had in common a single outer limiting membrane of smooth endoplasmic reticulum. The dense core type of HRP vesicles found in the cytoplasm was also observed in axons. However, high resolution micrographs of intraaxonal HRP vesicles revealed a variety of subunits in vesicles. Other investigators (Winfield et al., 1975; Colman et al., 1976; Mizuno et al., 1978) found that the HRP product appeared as small dense membranebound bodies. HRP-labeled organelles were present in both axons and terminals (Figs. 17 and 18). Multivesicular bodies, or vacuoles enclosing smaller vesicles, were also labeled by the enzyme its reaction product usually filling their matrix but not the dense-cored synaptic vesicles. Nauta et al. (1975) observed that HRP was localized in tubular profiles and vesicles of varying size but they were always clearly much larger than 25 nm microtubules and no HRP could be found in association with the 25 nm microtubules. It is also of interest to note that the lysosome-like HRP vesicles observed in axons had frequently varying amounts of subcapsular clear or lucid areas and an occasional contact with neurotubules as previously reported by LaVail et al. ( 1973). Labeled multivesicular bodies were more frequent in preparations fixed 8 hours after the peroxidase injections, as were vacuoles completely filled with reaction product. In addition to those labeled organelles, numerous vacuoles, cup-shaped profiles, and multivesicular bodies, devoid of tracer, were apparent in the axonal cytoplasm at all survival periods studied by Theodosis (1982). The labeling of multivesicular bodies in terminals is enhanced by conditions that promote active transmission by the terminals and thus produce increased uptake of tracers into the synaptic vesicles (Teichberg et al., 1975; Schacher et a/., 1976). The amount of endocytized peroxidase that eventually undergoes retrograde transport is markedly increased in preparations whose synaptic vesicles have become labeled through synaptic activity (Teichberg et a / . , 1975). However, according to Gobel and Falls (1979) the HRP reaction product binds to the neurofilaments, neurotubules, and the cytoplasmic surface of the axolemma in the primary axons of the substantia gelationosa of Rolando. Although the terminal is densly labeled, the size and shape of the synaptic vesicles, and the two synaptic contacts remain clearly visible (Rastad, 1981). FIG. 17. Labeled terminal in the submucosal plexus. Bar scale= I pm. X96,OOO. FIG. 18. Multivesicular bodies, vacuoles are present in the labeled nerve processes in the myenteric plexus (arrows). Bar scale= 1 pm. X 18,000.

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According to Beattie et al. (1978) the reaction product can be observed adjacent to mitochondria1 membranes and appears to surround synaptic vesicles. However, according to Nauta e f al. (1975) and Robson and Mason (1979) no HRP was found within the synaptic vesicles. It is frequently claimed that the peroxidase reaction product is found within synaptic vesicles but a close inspection of the micrographs serving as the basis for such claims suggests that it is on the external surface, and the cores of the vesicles are electron lucid (Ceccarelli et af., 1973; Heuser and Reese, 1973; Ripps et af., 1976). In axons, reaction product can be seen adjacent to the plasma membrane and aggregated in the cytoplasm, occasionally over microtubules (Holstege and Dekker, 1979). Egger et al. (1981) using the intraaxonal injection of HRP were able to stain the functionally identified afferent fibers in the cat spinal cord. The labeled terminals proved to be predominantly axodendritic asymmetric synapses containing round, clear vesicles. Multiple synapses on a single dendrite were also observed, at a 900 nm distance from each other. However, when a bouton was making synaptic contact with an HRP-labeled dendrite, postsynaptic densities were not clearly distinguished (Langerback et al., 1981). The cerebellar-olivary axon terminals were detected by Mizuno et al. (1980) anterogradely with HRP injected into the lateral cerebellar nucleus. In the principal olive contralateral to the HRP injection, electron-dense HRP granules were found in axon terminals contacting dendritic profiles. In these HRP-labeled axon terminals the synaptic vesicles were spherical. Morphometric analysis in the supraoptic nucleus of the rat was made by Theodosis (1982) who found that the estimated mean volume density of the peroxidase-containing profiles was relatively small and tended to increase only slightly up to 8 hours. On the other hand, the proportion of these organelles was much higher in the nuclei of dehydrated animals. At 4 hours after administration of HRP, their volume density was twice as that of controls, and at 8 hours it had increased further to over three times the control value. Kistler and Schwartz (1982) used HRP-conjugated to the lectin, wheat germ agglutinin, which binds with high affinity to cell surfaces, and examined the retrograde transport in a single invertebrate neuron. There was apparently more reaction product in the cells after the transsection than after the ligature of the axon. By electron microscopy, there was a marked difference between cells with transsected and ligated axons. While the cells with ligated axons contained no labeled organelles, some organelles in the cells with cut axons contained reaction product. It is noted that the increased HRP uptake by injured neurons resulted in a heavy homogeneous staining of neuron processes (Kristensson and Olsson, 1974). Labeled synaptic vesicles seem not to participate in large numbers in the retrograde transport (Kristensson et al., 1971; LaVail and LaVail, 1974), but

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multivesicular bodies and other structures that may be precursors or contributors to multivesicular bodies or other lysosomes (elongated sacs, tubules) are prominent among the bodies, that carry endocytozed tracers in a retrograde direction (LaVail and LaVail, 1974; Teichberg et al., 1975). Ligature experiments originally indicated that HRP is transported proximaldistally. According to Gwyn et al. (1982) in the labeled axon the electron-dense reaction product was associated with the microtubules and, in the axoplasm, it was found between the microtubules. The appearance of the HRP reaction product described in vagal terminals by Gwyn et al. (1982) resembled the somewhat dispersed appearance of HRP labeling reported in dorsal root terminals by Beattie er al. (1978) and in mamillothalamic terminals by Holstege and Dekker (1979). In contrast to these findings a report of labeling of cerebello-olivary terminals (Mizuno et al., 1980) showed the HRP reaction product as a small number of electron-dense aggregation. Some HRP-labeled terminals showed degenerating features like shrinkage, glia reactions, etc. (Figs. 19 and 20) (Dekker and Kuypers, 1976). A number of regulatory reactions such as chromatolysis after axon injury (Cragg, 1970), retrograde transsynaptic changes (Cowan, 1970), glial reactions (Sjostrand, 1965), and growth regulation (Prestige, 1970) also exist, especially in the central nervous system. No evidence has been provided for either the extrusion of HRP into the extracellular space, or for its transsynaptic transport of HRP in the small intestine. It has been demonstrated by De Olmos and Heimer, (1977) and Mesulam and Brushart (1979) that neurons labeled retrogradely through one of their long axon collaterals and their other long axon collateral contain also HRP. It is even more likely that short local axon collaterals will become labeled after retrograde transport of HRP along the main axon.

IV. Uptake of Horseradish Peroxidase into the Nerve Terminals Findings considered are pertinent to questions concerning the localization, intracellular movement, and degradation of exogenous protein molecules taken up by neurons from the extracellular space. However, numerous reports suggest that HRP can be taken up from the extracellular space by pinocytosis along the cell surface (Teichberg and Bloom, 1976). The uptake of HRP into isolated nerve terminals (synaptosomes) has been studied by Marchbarks ( 1982), using a spectrophotometric method to determine the enzyme activity. The uptake was not affected by metabolic poisons, while it was reduced at lower temperatures and was not associated with any significant release of cytoplasmic lactate dehydrogenase suggesting an endocytotic mechanism.

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Chan et ul. (198 I ) found that in the in vitro uptake phase (neurit terminal region), as many vacuoles as vesicles and tubules were labeled. In the transport phase (along neurit), labeled vacuoles were predominant. It appeared that these neurons utilized mainly vacuoles as a means to transport endocytozed protein to soma1 lysosomes, reminiscent of cultured neuroblastoma cells (Chan et a/., 1980). It is widely accepted that neuronal perikarya synthesize most of the proteins and many of the other substances required for the maintenance and function of their axons. In the case of local uptake by the cell body, HRP is found in pinocytotic vesicles and ultimately in lysosomes and multivesicular bodies (Becker et al., 1968; Holtzman, 1971; Holtzman et ul., 1967; Holtzman and Peterson, 1969; Nagasawa et al., 197 I ; Sellinger and Petiet, 1973). In the case of local uptake by axon terminals HRP has been found in coated vesicles, cisternae of the agranular reticulum, and synaptic vesicles (Brightman, 1968; Zacks and Saito, 1969; Brightman et ul., 1970; Nagasawa et ul., 197 1 ; Ceccarelli et ul., 1973; Heuser and Reese, 1973). In the nerve terminals the peroxidase-containing sacs and vesicles can be distinguished from most of the large dense-cored vesicles since the latter usually have a translucent zone just inside the membrane (Somogyi et al., 1979). The incorporation of the tracer into the small vesicles is thought by Pysh and Wiley (1974) to be formed at least in part from the loaded plasma membrane. Multivesicular bodies have been implicated in the sequestration of endocytotically derived membrane in numerous systems, including neurons, where they were found to increase with increasing exposure time to the tracer (Theodosis, 1982). It is not unlikely that a first step in the sequestration of the endocytotically derived membrane occurs in multivesicular bodies, either within the terminals, after transformation from the vacuolar and cup-shaped figures, or within the perikarya (Holtzman et a/. , 1977). Since multivesicular bodies can move retrogradely in axon cytoplasm (LaVail e t a / ., 1980; Tsukita and Ishikawa, 1980) they could also have served to transport the endocytotically derived membrane to the perikarya. Ceccarelli et al. (1973) believe the vesicles membrane remain discrete during the process of exocytosis and is withdrawn immediately afterward, so that the vesicle is reformed with a complement of HRP that entered from the extracellular fluid during the release of transmitter. This also appears to be the view of Zimmerman and Denston (1977). Several laboratories have obtained results consistent with the possible involvement of the smooth endoplasmic reticulum in retrograde axonal transport (Sotelo FIG. 19. Arrow shows the degenerated labeled nerve terminal in the myenteric plexus. Bar scale= I pm. X42.000. FIG. 20. Degenerated labeled nerve process in the submucosal plexus. Bar scale= I pm. X 42,000.

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and Riche, 1974; Nauta et al., 1975; Repirant, 1975; Price and Fisher, 1978). Once taken up, HRP seems to be transported retrogradely along the axon inside vesicles or smooth endoplasmic reticulum (Turner and Harris, 1974; Nauta et al., 1975; Teichberg et al., 1975). Results from other in vitro studies using single or serial sections by Birks ef al. (1972), Wessels et al. (1974), Weldon (1973, and Bunge ( 1977) have consistently pointed to the fact that tracer containing tubules in the neurons may be morphologically distinguished from the agranular reticulum. Sotelo and Riche (1974) identified tubules filled with HRP that extended for several microns within the axon of the pars reticulata of the substantia nigra after neostrial injections of HRP. Uptake of HRP by intact neurons (especially at their terminals) takes place normally by endocytosis (Becker et al., 1968; Bunt, 1969; Zacks and Saito, 1969; Nagasawa et a / ., 197 1; Heuser and Reese, 1973; Turner and Harris, 1973; Teichberg et al., 1975) or pinocytosis in regions removed from the synaptic complex as were observed in a number of other neuronal systems (Krishnan and Singer, 1973; AlKhagai et al., 1980). LaVail and LaVail (1974) calculated a 9.2 mm/day rate of movement of the HRP vesicles. The amount of HRP transported in retrograde direction may be even greater than in anterograde direction. This estimate would indicate that the terminal region of only a fraction of HRP retinal cells had access to the HRP (LaVail and LaVail, 1974). Brownson et al. (1977) found that by 48 hours following injection the ciliary processes contained large numbers of HRPpositive vesicles in the nonmyelinated axons. However, by 6 days HRP-labeled axons could not be found in the ciliary processes. The anterograde movement of the HRP label from the postganglionic neuron perikarya in the superior cervical ganglion to terminals in the ciliary body may be as rapid as 4 hours and as prolonged as 48 hours, according to Brownson et al. (1977). In the peripheral nervous system axoplasmic components are transported at different rates (Litchy , 1973; Iversen et al., 1975; Jacobson et al., 1975; McEwan and Grafstein, 1968; Sjostrand, 1970). The observations by Brownson et al. (1977) reported that the retrograde flow rate of HRP in the sympathetic nervous system is 500-700 mm/day. Holtzman et al. (1973) have suggested that multivesicular bodies may be involved in the turnover of cell surface materials as well as in the degradation of secretion granule contents and membranes related to the Golgi apparatus and other structures. In view of the work of Droz et af. (1975), it seemed plausible that HRPlabeled elongated tubular structures are involved in retrograde axonal transport. LaVail et al. (1980) observed further that there might be a portion of the smooth endoplasmic reticulum a part of a continous channel. LaVail and LaVail (1974) suggested that microtubules may be involved in the

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mechanism of retrograde transport. Further support for the involvement of microtubules comes from the findings that low doses of vinblastine block retrograde transport of HRP in retinal ganglion cells in adult rats (Bunt and Lund, 1974). Microtubules have also been associated with the rapid, anterograde axonal transport of proteins (Schmitt, 1968; Samson, 1971; Ochs, 1972) and of synaptic vesicles (Smith, 1971; Droz et af., 1973). In summary, the enzyme appeared to be removed or inactivated from the injection site within 4-5 days of injection, and began to disappear from ganglion cells by the third day after injection. This agrees with the finding of Kristensson and Olsson (1973) and LaVail and LaVail (1974).

V. Concluding Remarks A major advantage of the use of the enzyme HRP in elucidating the connections of the nervous system is that neuronal cell somata are labeled in a way which enables the determination of the cells of origin of a particular fiber pathway. The reaction product can be studied under the electron microscope, to clarify the type of neuron, on the basis of the shape, size, and ultrastructural features of the soma, and its dendritic and axonal morphology. In a further step, the synaptic connections with other unlabeled nerve terminals can be studied. The cytoplasm of the labeled nerve cells was identified as round to irregular shaped vesicles bound by a single membrane. The content of the vesicles is dense and homogeneous. The HRP-labeled vesicles appeared in large number around the nucleus than elsewhere in the cytoplasm. Labeled neurons were also seen in the myenteric and in the submucosal plexuses. The granules varied in size from 10 to 100 nm and many of the larger ones showed definite evidence of being by the fusion of smaller individual granules, of varying density. In several electron micrographs it was noted that typical dense core type HRP vesicles showed fibrillary bridges extending between the smooth endoplasmic reticulum and the vesicle membrane. Processes originating from the labeled neurons also contained HRP granules. The fact that the cell somata containing electron-dense granules of the reaction product were of one type of cell, and most of those which were unlabeled showed different morphological features, yields further evidence for the identification of the neurons in the peripheral and the central nervous systems.

REFERENCES A d a m , J . C. (1977). Neuroscience 2, 141-145. A d a m , J. C . , and Warr, W . B . (1976). J . Comp. Neurol. 170, 107-122.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOI. YO

DNA Sequence Amplification in Mammalian Cells JOYCEL. HAMLIN,JEFFREYD. MILBRANDT,' NICHOLASH. HEINTZ,~ AND JANE C. AZIZKHAN~ Department of Biochemistry, University of Virginia School of Medicine. Charlottesville, Virginia I. Introduction . . . . . .................... 11. Occurrence of Am .................... A. Phylogenetic Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Amplified Loci in Mammalian Cells . . . . . . . ..... C. Amplification of Transfected Genes . . . . . . . . D. Evidence for Amplification during Evolution . . . . . . . . . . E. Known and Probable Sequence Amplifications in Maligna 111. Cytological Manifestations of Gene Amplification . . . . . A. Homogeneously-Staining Regions (HSRs) . . . . . . . . , . . . . . . . . . ....................... B. Double Minutes (DMs) . . . . . . C. Relationship between DMS and H S R s . . . . . . . . . . . . . . . . . . . . . IV. Nature of Amplified Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... A. Amplified Endogenous Genes. . . . . . . . B . Amplified Transfected Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Agents That Increase the Frequency of Amplification . . . . . . . . . . . . A. Agents That Interfere with DNA Metabolism . . . . . . . . . . . . . . . B. Growth-Promoting Substances . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Proposed Mechanisms of Sequence Amplifi A. Unequal Sister Chromatid Exchange . . . . . . . . . . . . . . . . . . . . . . B. Rereplication . . . . . . . . . . . . . . . . . . . . VII. Concluding Remarks . . . ....................... ....................... References . . . . . . . . . . .

31 33 34 38 40 42 43 45 45 50 52 57 58 63 64 64 67 67 68 71 75 77

I. Introduction In the typical eukaryotic somatic cell cycle, each chromosome is precisely duplicated during the DNA synthetic (S) period. The synthesis of a chromosomal DNA fiber occurs through the agency of thousands of tandemly arranged replicons (Huberman and Riggs, 1968), each of which usually functions only once 'Present address: Division of Laboratory Medicine, Washington University School of Medicine, St. Louis, Missouri 631 10. 'Present address: Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont 05401. 'Present address: Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218. 31 Copyright 0 1Y84 by Academic Prew. Inc All rights of rcpraluclion In any form rexrvcd ISBN 0-12-364490-9

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in a given S period to ensure that the fiber is exactly duplicated along its entire length. The result of this process is that the two identical daughter chromatids lie side-by-side, connected by a centromere, until separation of chromatids occurs at mitosis and the ploidy of each daughter cell is restored to the original configuration. There are exceptions to this mode of replication, however, in which parts of chromosomes or the entire chromosomal complement are rereplicated prior to a cell division event, with the consequence that the genetic constitution of the cell can be increased in total, or only at selected loci. During polytenization in the larval stages of certain Dipteran species, the chromosomes replicate over and over again without intervening mitosis, resulting in as many as 8000 copies of the genome in a single cell (Daneholt and Estrom, 1967). The result of this process is that the multiple identical DNA fibers lie together in colinear bundles. A related phenomenon known as endoreduplication occurs infrequently in a variety of cell types, and results in the precise reduplication of the entire chromosomal complement without intervening cytokinesis (Herreros and Gianelli, 1967; Gatti et al., 1973). The frequency of endoreduplication can be increased by several agents, including the mitotic spindle inhibitor colchicine (Weber and Hoegerman, 1980; Sutou, 1981). Both of the above processes seem to affect all chromosomal replicons in the same way, and may reflect the overriding of controls that usually prevent initiation of the S period until after mitosis. During development, there are situations in which a single genetic locus can be preferentially rereplicated during the cell cycle by a process known as amplification. The preferential replication of ribosomal genes during oogenesis is a widespread phenomenon in both animals and plants, and presumably allows the organism to cope with the great demand for protein synthesis during development of the oocyte (See Long and Dawid, 1980, for review). In the follicle cells of certain insects, a developmentally controlled process results in the amplification of selected chorion genes whose products are utilized in egg shell formation (Spradling and Mahowald, 1980). Another type of selective amplification has received a great deal of attention in recent years, and is the major focus of this review. When cultured mammalian cells are selected for resistance to increasingly higher concentrations of certain drugs over the period of many months, cell lines can eventually be isolated that greatly overproduce the target protein (usually an enzyme) for the corresponding drug. In almost every case of extremely high levels of resistance, a DNA sequence containing the gene coding for the target protein has been shown to be selectively amplified. The amplified sequences are located in expanded chromosomal regions or on extrachromosomal double minutes. This latter mode of amplification has generated considerable interest for several reasons. Since drug

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resistance of this type has been observed in patients and cultured cell systems that were treated with anticancer agents such as methotrexate, gene amplification is thought to be a major problem in cancer chemotherapy. In addition, evidence has accumulated for many years that tumor cells taken from patients who have apparently not been subjected to drug treatment often display chromosomal anomalies such as the double minutes and expanded chromosomal regions that are characteristic of gene amplification in experimental systems. With the recent discovery that the overproduction of certain normal gene products from cellular oncogenes can lead to the transformed phenotype (see Bishop, 1983, for review), it is clear that gene amplification could be one underlying mechanism in malignancy. Since amplification has been observed at virtually every genetic locus for which there is a suitable selective agent, the phenomenon is apparently widespread and possibly random. This notion is supported by the vast literature on existing gene duplications in mammalian cells. Through the workings of evolution, the extra copy or copies of a gene can be conserved and can function to produce larger amounts of their gene product than could the diploid complement (as in the case of ribosomal genes); or the extra copies can be mutated, leading eventually to new functions in the cell or to inactivation in the case of pseudogenes. Thus, the amplification process observed during the acute development of drug resistance in cultured cells may be a telescoped version of the duplication and amplification mechanisms that have taken place over millions of years to produce the complex genomes of higher organisms. In this article, we will begin by citing several examples of selective DNA amplification in both prokaryotic and eukaryotic systems. We will then discuss the cytological manifestations of gene amplification, and the stability of the process. We will cite recent studies on the molecular nature of the amplified sequences in selected systems, and the types of agents that might provoke gene amplification. We will then attempt to put the major observations related to this interesting chromosomal phenomenon into perspective by discussing certain models for the mechanisms involved in gene amplification.

11. Occurrence of Amplification Phenomena

We will discuss here specific examples of gene duplication and amplification phenomena that occur throughout the evolutionary spectrum, in order to tabulate the possible mechanisms that may be available to mammalian cells for the type of amplification associated with drug resistance. We will then focus on the various genetic loci that are known to be duplicated or amplified in mammalian cells, in order to indicate the broad spectrum of this phenomenon.

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A. PHYLOCENETIC RANGE Gene duplication and tandem amplification in bacteria are frequent occurrences that are detected in drug selection protocols, and have been observed in several bacterial species and at several genetic loci (Anderson and Roth, 1977). Amplification of a single locus can proceed to the point at which the multiple copies can represent 20% of the bacterial chromosome (Anderson et a/., 1976). There are numerous examples of the amplification of drug resistance genes carried on bacterial plasmids (Clowes, 1972). Exposure to the particular antibiotic selects for cells carrying plasmids with tandemly repeated resistance markers (Rownd, 1982). In the F1 plasmid, PRSDl (Schmitt et al., 1979; Mattes et al., 1979), each gene is flanked by insertion sequences, forming a transposon, which is apparently the unit of amplification. The direct repeats flanking the gene are required for amplification, and the process is apparently dependent on host recombination systems. Phage p is itself a transposable element, and transposition from one genomic site to another probably occurs by a process involving replication of the element (Galas and Chandler, 1981; Harshey and Bukhari, 1981). In the process, the phage can be inserted in multiple tandem copies by a mechanism that may involve breakage of the host chromosome and attachment to a nicked end of the element, followed by continuous replication into the chromosome. The bacteriophages A (Edlund et al., 1980), T4 (Kozinski et al., 1980), and PI (Meyer and lida, 1979) have also been shown to undergo selective and multiple reduplications of particular genetic markers. In the case of T4, the amplified loci lie in the vicinity of the known origins of replication (Kozinski et al., 1980). Gene amplification has also been reported in the chromosomes of yeast. In particular, the gene for metallothionein is amplified in tandem after stepwise selection in cadmium (Fogel and Welch, 1982). In petite mutants of yeast, amplification of mitochondria1 DNA sequences occurs by a process resulting in the formation of multiple, tandem copies of fragments of the original mitochondrial genome arrayed in a circle (Gaillard et ul., 1980). The genetic constitution of the repeated unit varies in different mutants, and each repeated unit contains an origin of DNA synthesis (de Zamaroczy et al., 1981; Hyman et a / . , 1982). In insects, certain forms of resistance to insecticides display several of the properties characteristic of drug resistance in mammalian cells. Resistance appears to result from high levels of the target protein for the insecticide, and can be stable or unstable when the chemical agent is removed from the environment (Goldstein, 1974). The natural developmental process of ribosomal DNA amplification during oogenesis (reviewed in Long and Dawid, 1980) occurs in a large variety of organisms, including hypotrichs, echiuroid worms, clams, insects, fish, amphibia, mammals, and plants. The amplified ribosomal DNA (rDNA) copies are

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usually extrachromosomal, and are arrayed in tandem as linear or circular elements. In the case of Xenopus laevis, the repeated units appear to be homogeneous in length (Wellauer et al., 1976). Since there are multiple integrated rDNA copies of variable length in the Xenopus genome prior to amplification, this finding suggests that amplification occurs from a single integrated unit in a selective way (Wellauer et al., 1976). Furthermore, different oocytes from the same animal can amplify different integrated copies (Wellauer et al., 1976; Bird, 1978). Electron microscopic observations have suggested that rDNA amplification in Xenopus occurs via a rolling circle mechanism analogous to the mode of replication of the single-stranded DNA bacteriophage, XI74 (Hourcade et u l . , 1973; Rochaix er al., 1974; Buongiorno-Nardelli et a!., 1976). A related developmental phenomenon occurs in the ovarian follicle cells of certain insects during eggshell formation. Members of the chorion multigene family are organized into two clusters on different chromosomes in Drosophila (Spradling er a l . , 1980; Griffin-Shea et a/., 1980). During oogenesis, each cluster is amplified in situ (Spradling and Mahowald, 1980). Bidirectional replication proceeds from a fixed origin of DNA synthesis located in a central position in each locus (Spradling, 1981). After multiple initiations, the copy number of sequences flanking the origin decreases with distance from the origin, suggesting that replication forks terminate at random positions. Since the amplified DNA sequences are recovered in high-molecular-weight DNA fractions, it appears that the multiple daughter strands do not separate after replication. This suggestion has recently been confirmed in electron microscopic studies on the follicle cells of Drosophila. Multiple replication bubbles lying side-by-side in an onion skin array were seen to terminate at random positions relative to the center of the replication bubble (Oscheim and Miller, 1983). Another example of selective amplification occurs in the Dipteran, Sciaraidae. Several loci are selectively overreplicated during polytenization in the late larval stage of development, apparently as a result of hormonal stimulation (see Bostock and Sumner, 1978, for review). The extra copies remain associated with the giant polytene chromosomes as DNA puffs, and may result from a process similar to that observed in chorion gene amplification in Drosophila. Considerable information exists about the amplification of several viruses that integrate into the chromosomes of higher eukaryotic cells. In the papova virus group, SV40, Polyoma, and BK viruses can integrate as single copies or as headto-tail tandem repeats in transformed host cells (e.g., Botchan et al., 1980; Birg et a/., 1979; Pater et al., 1982). The sites of insertion into the host genome appear to be nonspecific (Gelb et al., 1971; Sambrook et al., 1975; Kutcherlapati et a / . , 1978), and the inserted copies can give rise to free viral DNA (e.g., Hiscott et a / . , 1981; Bullock and Botchan, 1982; Gattoni et al., 1980; Pater et al., 1982). A single integrated copy can apparently also amplify in situ, provided that it contains an origin of replication (Botchan et al., 1979; Colantuoni et al.,

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1980; Baran et af.,1983). Integration, amplification, and possibly excision seem to require a functional T antigen, a protein required for viral replication (Della Valle et al., 1981; Colantuoni et al., 1982; Botchan et al., 1979). The size of the integrated, tandemly repeated viral unit is conserved during the excision of polyoma; i.e., if the repeated unit is 1.3 equivalents long, then the extrachromosomal element is also 1.3 equivalents long (Gattoni et af., 1980). This suggests that integration and excision events are mediated by some common feature related to homologous recombination. However, models involving concomitant replication and excision of the integrated virus have also been proposed (see Section VI). Studies on BK virus indicate that transformation of semipermissive mouse cells results in the tandem integration of viral DNA into the host chromosome, but in nonpermissive hamster cells, the virus integrates as a single copy (Meneguzzi et af., 1981). However, when the virus is linearized with a restriction enzyme that generates staggered cohesive ends, and hamster cells are subsequently transformed with this DNA, the virus is inserted in tandem arrays (Grossi et al., 1981). This result suggests that the formation of tandem arrays may occur prior to integration in the semipermissive situation, possibly through polymeric DNA replication intermediates. It has also been shown that the DNA damaging agent, mitomycin C, provokes onion skin replication of the integrated polyoma (Baran et al., 1983) and SV40 (Bullock and Botchan, 1982) genomes in inducible cell lines. In the former case, replication was shown to proceed from the viral origin into flanking cellular sequences, and appeared to terminate at fixed positions in the host DNA. The phenomenon of amplification of chromosomal DNA sequences in mammalian cells is a relatively recent discovery, and began with studies on drug resistance in cultured cells. Several years ago, both Fischer (1961) and Hakala et af. (1961) selected mouse cell lines that were resistant to the antifolate drug, methotrexate (MTX), and showed that resistance could be accounted for by an increase in the intracellular level of the target enzyme for this drug, dihydrofolate reductase (DHFR). Littlefield ( 1969) subsequently proposed that overproduction of DHFR in MTX-resistant cells could be due to constitutive expression of a normally repressible gene, if the cell had suffered a mutational loss of the repressor itself. Evidence against this hypothesis was obtained by showing that hybrids between resistant and sensitive cells expressed an intermediate level of drug resistance (Littlefield, 1969), and DHFR synthesis was therefore not turned off by the postulated active repressor supplied by the normal, sensitive cell in the hybrids. As an alternative mechanism for DHFR overproduction, Littlefield suggested that the gene coding for DHFR could be amplified in these cell lines. In 1976, Biedler and Spengler made the critical observation that in cytological preparations of mitotic chromosomes from a series of near-diploid, antifolateresistant Chinese hamster lung cells, a very unusual chromosomal anomaly was

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consistently correlated with very high levels of drug resistance (Biedler and Spengler, 1976a,b). In these cell lines, expanded chromosomal regions were observed that stained uniformly to an intermediate degree in the G-banding protocol (contrasted to the usual alternating light and dark banding pattern seen on most chromosomes), and stained darkly when subjected to C-banding. These homogeneously staining regions (HSRs) were not present in drug-sensitive Chinese hamster cells. They suggested that HSRs could be a manifestation of the in situ amplification of the gene coding for dihydrofolate reductase, thus accounting for increased levels of the enzyme. Schimke and co-workers were then able to show that DHFR messenger RNA (mRNA) was greatly overproduced in MTX-resistant murine S 180 cells (Kellems et al., 1976), and they subsequently cloned cDNAs (DNA complementary to mRNA) representing DHFR mRNA species overproduced by this cell line (Ah et a/. , 1976). Using these cDNAs in solution hybridization studies, they showed that genomic DNA isolated from resistant mouse cells contained several hundred copies of the DHFR gene (Ah et a / . , 1976). Schimke and collaborators also showed that multiple copies of the DHFR gene could be localized to HSRs in MTX-resistant Chinese hamster ovary (CHO) and murine cell lines by demonstrating the selective hybridization of radioactive DHFR cDNA to the HSRs in mitotic chromosomes (Nunberg et al., 1978; Dolnick et al., 1979). However, in some MTX-resistant mouse cell lines, the extra DHFR genes were shown to reside on extrachromosomal double minutes (DMs)-small paired, acentromeric bodies dispersed among the mitotic chromosomes. It was demonstrated that the number of DMs per metaphase spread correlated roughly with the number of amplified DHFR genes, and, further, that the DHFR genes cosedimented with the DM fraction on sucrose gradients (Kaufman et al., 1979; Brown et a / . , 1981). These structures were only seen in unstable variants of murine S180 and L1578Y (i.e., those that quickly lose resistance to MTX after removal of drug from the culture medium). Stable variants of the same origin manifested HSRs, and did not contain DMs (Dolnick et al., 1979). Thus, by early 1980, it was reasonably clear that the predominant mechanism for resistance to MTX in cultured mammalian cells was the amplification of the gene coding for DHFR. The amplification of DHFR genes during development of MTX resistance has now been shown to occur in a variety of mammalian cells. DHFR gene amplification has been observed in murine 3T6 (Brown et a / . , 1981), Sarcoma 180 (Kaufman et al., 1979), the EL4 and L1578Y lymphomas (Bostock and TylerSmith, 1982; Kaufman et al., 1979), the SEWA ascites tumor (Martinsson et al., 1982), and the PG193 melanoma (Bostock and Clark, 1980). MTX-resistant variants of the karyologically stable CHO (aneuploid) and the Chinese hamster lung (near-diploid) cell lines have been extensively characterized (Biedler et a/., 1980; Nunberg et al., 1978; Milbrandt et al., 1981; Flintoff et al., 1982). In

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addition, human leukemias (Srimatkandada et al., 1983; Horns et al., 1984), ovarian adenocarcinomas (Trent et al., 1984), breast cancer cells (Cowan et a l . , 1982), and HeLa cells (Wolman et a l . , 1983), as well as a series of rat hepatomas (Fougere-Deschatrette et a l . , 1982) have all been shown to amplify the DHFR gene in response to treatment with increasing concentrations of MTX. Recently, the gene coding for a bifunctional thymidylate synthetase-dihydrofolate reductase in the parasite, Leishmania tropica, has also been found to be amplified in response to MTX treatment (Coderre et ul., 1983).

B. AMPLIFIED LOCIIN MAMMALIAN CELLS To date, the amplification of DHFR genes in the establishment of MTX resistance is the most extensively studied example of gene amplification in mammalian cells, primarily because it was the first to be described. However, the list of genetic loci that can undergo endoreduplication in mammalian cells is growing, and, indeed, seems limited only by the availability of suitable drugs for isolating resistant variants at any given locus. Stark and co-workers have determined that resistance to the antimetabolite, N-(phosphonacety1)-L-aspartate (PALA), is developed in Syrian hamster cells by the overproduction of the multifunctional CAD protein that catalyzes the first three steps in uridine biosynthesis (Wahl et al., 1979). They have shown that the CAD gene is amplified as much as 100-fold in some cell lines, and the multiple genes can be localized to an HSR in each PALA-resistant cell line (Wahl et al., 1982). The metallothioneins are proteins that sequester toxic metals such as cadmium and zinc, and their synthesis is regulated by both heavy metals and by glucocorticoids (Kagi and Nordberg, 1979; Karin et al., 1980). When cultured murine Hepa 1A (hepatoma), S180 (sarcoma), or Friend erythroleukemia cells are subjected to stepwise increases in cadmium concentration over the period of several months, resistant variants are recovered that overproduce metallothionein and its mRNA, and contain 10-60 copies of the metallothionein gene (Beach and Palmiter, 1981; Beach et al., 1981). The Hepa 1A cells displayed numerous DMs, but neither the S 180 nor erythroleukemia cells displayed karyotypic anomalies that could be related to DMs or HSRs (Beach et al., 1981). Amplification of metallothionein genes also occurs in Chinese hamster ovary cells without apparent karyotypic changes (Gick and McCarty, 1982). The amplification of adenosine deaminase in mouse C 1 fibroblasts is responsible for the resistance developed to stepwise increases in coformycin (Yeung et al., 1983a). Increased levels of adenosine deaminase were observed in this system, resistance was lost upon removal of the drug, and DMs were detected in metaphase spreads (Yeung et a l . , 1983a). Cloned adenosine deaminase cDNA probes were used to show amplification of the cognate gene (Yeung et a l . ,

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1984). The gene for adenosine deaminase is also amplified in human choriocarcinoma cells (Yeung et a / . , 1983b) and in CHO cells (Debatisse et af., 1982) after stepwise increases in selective drug. The X-linked gene for HPRT (hypoxanthine/guanine phosphoribosyltransferase) is amplified in both mouse neuroblastomas (Brennand et al., 1982) and in Chinese hamster cell lines (Fuscoe et af., 1983). In the former case, three copies of the X chromosome are fused in a large, rearranged marker chromosome, but this obvious threefold amplification cannot account for the apparent 50-fold amplification of the gene relative to normal cells (Melton et al., 1981). No HSRs or double minutes were observed in this system. Amplification of the HPRT locus was detected in the Chinese hamster cell system by selecting for revertants of a thermo-sensitive HPRT mutant by growth at 39°C (Fuscoe et a/., 1983). This approach was also used by Chasin and colleagues to isolate amplified, mutant DHFR genes without selection in MTX (Chasin et al., 1982), and seems to work in both of the above cases by the overproduction of a partially active protein. Several other drug treatment regimens have been used to select mammalian cells that overproduce the target protein, and many have been shown to have amplified the corresponding gene. 5-Fluorodeoxyuridine selects for the overproduction of thymidylate synthetase (Baskin et al., 1975; Rossana et ul., 1982), hydroxyurea for the M2 subunit of ribonucleotide reductase (Akerblom et af., 1981), albiizin for asparagine synthetase (Andrulis et al., 1983), compactin for 3-hydroxy-3-methyl glutaryl CoA reductase (Chin et al., 1982), tunicamycin for N-acetylglucosaminyltransferase (Criscuolo and Krag, 1982), and colchicine and vincristine for unidentified (microtubular?) proteins (Kopnin, 198 1 ; Biedler, 1982). In many of these cases, some karyological manifestation of amplification is observed in the form of DMs or HSRs. Multidrug cross-resistance has been described by Baskin and colleagues in uptake mutants of murine neuroblastoma cells that were treated with either maytansine, vincristine, adriamycin, or Baker's Antifol (Baskin et al., 1981). These cell lines contain numerous DMs and have been shown to contain elevated levels of alkaline phosphatase. Biedler and Riehm (1970) have also described multidrug cross-resistance in mouse and hamster cell lines that were selected for resistance to actinomycin D. There are, in addition, numerous examples of the nondevelopmental amplification of rDNA genes in mammalian tumor cell lines, including several human and rat neoplasms (Henderson and Megraw-Ripley, 1982; Murao et al., 1982; Tantravahi et d., 1981, 1982). In these instances, there is no obvious selection pressure that maintains the extra copies in the genome. All of these amplifications appear as HSRs, and many are located at the known positions of nucleolar organizer regions in these cell lines (Henderson and Megraw-Ripley, 1982).

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Interestingly, the sarcoma line, XC, contains an additional unidentified HSR that does not contain rDNA genes, suggesting a possible predisposition for amplification in these cells (Tantravahi et al., 1982).

c. AMPLIFICATION OF TRANSFECTED GENES There is a new class of gene amplification that has arisen as a result of the molecular cloning of selectable genes [e.g., DHFR, CAD, and thymidine kinase (TK)] and the advent of gene transfer techniques for introducing DNA into mammalian cells. Cloned DNA is usually delivered to cells either as a CaPO, coprecipitate which is ingested by pinocytosis (Graham and van der Eb, 1973), or by fusion of cells to bacterial spheroplasts harboring recombinant plasmids (Schaffner, 1980). After long-term culture of the transformed derivatives (obtained by growth in a medium that selects for the function imparted by the transferred gene), the DNA is invariably integrated into the chromosome at one or a few sites, and, depending upon the mode of delivery, can often integrate as multicopy, tandem arrays. In the case of the CAD and DHFR genes, stepwise increases in the corresponding drug results in amplification of the transfected genes. The first such instance reported involved the introduction of the entire CAD gene (contained in a recombinant cosmid) into CAD-deficient or wild-type CHO cells (de Saint Vincent et al., 1981). The ura+ derivatives of the CAD-deficient cells were shown to contain more than 10 copies of the recombinant CAD gene, which were subsequently amplified further upon treatment with increasing concentrations of PALA. Transfectants of wild-type CHO cells were selected in a high concentration of PALA directly, and contained multiple copies of the CAD gene, accounting for PALA resistance. The PALA-resistant clones were shown to contain HSRs on a chromosome distinct from the probable location of the CAD gene in wild-type cells. The entire 25 kb CHO DHFR gene has been cloned in a cosmid, and has been used to rescue a DHFR- CHO cell line to the DHFR phenotype (Milbrandt et al., 1983a). Upon amplification after selection with increasing concentrations of MTX, the extra DHFR gene copies (500-700 per diploid genome) were shown to reside in an HSR on a chromosome distinct from the parental gene location, as shown by the binding of radiolabeled DHFR probes to mitotic chromosomes (Milbrandt et al., 1983b). It was also demonstrated that the amplified sequence included more than 90 kb of DNA in addition to the transfected gene itself. This extra DNA probably represents genomic sequences flanking the site of insertion of the recombinant cosmid into the chromosome. The very large (31 kb) murine DHFR gene has been truncated to smaller versions by a variety of experimental approaches, and these minigenes have been used to study DHFR expression and amplification after introduction into CHO +

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and murine cell lines. Crouse and co-workers have constructed chimaeras of DHFR genomic and cDNA sequences that convert DHFR- CHO cells to the DHFR+ phenotype after transfection (Crouse et a / . , 1983). In all instances, multiple copies of the DHFR plasmids were inserted into the genome of the recipient cell. DHFR transformants with approximately fiva: copies of the recombinant plasmid could be shown to amplify the inserted DNA further when subjected to incremental increases in MTX, and flanking cellular sequences were amplified as well. Interestingly, if carrier mouse embryo DNA was added to the DHFR plasmids during the transfection step, clones were obtained that had integrated very large numbers of the minigene (sometimes more than 400 copies per genome), apparently in tandem arrays. These clones were selected under conditions that presumably required only one or a few copies of the plasmid (i.e., were not subjected to MTX selection). This raises the possibility that certain sequences in the carrier mouse embryo genomic DNA were picked up by the plasmids as a result of intracellular ligation, and facilitated amplification. Gasser et al. (1982) and Kaufman and Sharp (1983) have also used DHFR minigenes to transform DHFR- CHO cells to the wild-type phenotype. They observed that most transformants had integrated multiple copies of the plasmids after selection for wild-type levels of DHFR enzyme. In several transfected clones, pBR322 sequences in the vector portion of the recombinants were lost, possibly through homologous recombination events. Furthermore, in both studies, significant numbers of rescued cell lines showed rearrangements of the input DNA, suggesting the relative instability of inserted DNA in this system. Kaufman and Sharp (1983) showed that a DHFR transformant with multiple inserts at different chromosomal locations amplified only one of the inserts after MTX selection, and that flanking (presumably chromosomal) sequences were coamplified with the DHFR minigene. From one cell line that was transfected with a DHFR minigene that included a large part of the SV40 t antigen gene, subsequent amplification resulted in overproduction of both DHFR and a polypeptide related to t antigen. This important result indicates that selectable and amplifiable cloned genes such as DHFR can be used to amplify any nonselectable colinear gene. Kaufman and Sharp also showed that the amplified copies of this chimaeric plasmid were located in the chromosomes as HSRs after amplification, and were usually at terminal positions or in dicentric chromosomes. These investigators suggest that telomeric regions are favored sites of integration, and that the integration event destabilizes the telomeres, inducing the formation of dicentric chromosomes. This is an interesting suggestion, since many endogenous amplified genes also reside at terminal positions on chromosomes, and dicentric chromosomes have been observed in at least two other cases involving amplification of the DHFR gene (Hamlin, unpublished observations; Fougere-Deschatrette et al., 1982). Murray et al. (1983) have constructed a vector that uses the LTR from Harvey +

+

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sarcoma virus to provide 5’ regulatory sequences required for the expression of a colinear DHFR cDNA. When this DNA is delivered to NIH3T3 cells, colonies are recovered that are resistant to MTX as a result of the insertion of multiple copies of the chimaeric recombinant plasmid. When subjected to further increases in MTX, the plasmid copy number per genomic equivalent goes up, and DMs are detected in those cell lines with high copy numbers. The viral sequences are also expressed in these constructions, as evidenced by the ability of the plasmid to produce transformed foci on 3T3 cells. These foci all exhibit elevated levels of DHFR and are MTX resistant. Axel and Roberts (1982) have carried out a series of studies in which they have transfected APRT-/TK -L cells with a chimaeric plasmid containing a wildtype APRT gene and a truncated, promoter-less TK gene. The initial APRT+/ TK- transformants gave rise to TK+ revertants at a very high frequency ( as a consequence of amplification of the chimaeric plasmid and the resulting overproduction of the partially functional TK gene. A unique finding in a subsequent study was that the tandemly arranged amplicons in these cell lines are integrated into chromosomes, as opposed to the DMS that are usually observed when mouse cells have amplified endogenous or transfected genes (Roberts et al., 1983). It remains to be seen whether or not the amplification processes that occur after DNA transfection exactly mimic those observed during amplification of endogenous genes. We will consider the nature of this process in Section VI. Thus, the range of mammalian cell types that have been shown to selectively amplify specific genes after drug treatment regimens is extremely broad. The number of drugs that can select for variants of this type is also large, and the list of genes that can be amplified will probably be lengthened as cloned probes for individual genes become available, allowing direct quantitation of gene copy number. D. EVIDENCE FOR AMPLIFICATION DURING EVOLUTION Most of the above examples of selective endoreduplication involve the rather extensive amplification of the gene in question, resulting in tens or even thousands of copies of the gene per diploid cell. However, it is appropriate to point out here that there exists in the genomes of virtually all higher organisms compelling evidence for many duplications and amplifications throughout the course of evolution. The reason for maintenance of reduplicated copies of genes is undoubtedly a combination of tolerance, mutation, and selection on the part of the affected organism. The examples of the multicopy rDNA and chorion gene families have been cited, and to these should be added the multimembered families of tRNA (reviewed in Long and Dawid, 1980), histone (Kedes, 1979), and immunoglobulin (Gottlieb, 1980) genes, as well as globins (Maniatis et al.,

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1980), actins (Fyrburg et al., 19801, myosins (Robbins et al., 1982; Epstein et al., 1974), collagens (Solomon, 1980), a2-globulin (Kurtz, 1981), vitellogenin (Wahli et al., 1979), interferons (Allen and Fantes, 1980), histocompatibility antigens (Nathanson er al., 1981), and ovalbumin (Royal et al., 1979), all of which have at least three members per haploid genomic equivalent. A partial list of gene duplications in higher eukaryotes includes hexokinase (McLachlan, 1979), ferrodoxin (Wakabayashi ef al., 1980), ricin (Villafranca and Robertus, 1981), calcitonin (Perez et al., 1982), renin (Piccini et al., 1982), isocitrate dehydrogenase (Sattler and Mecham, 1979), lens y-crystallin (Moorman et al., 1982), alcohol dehydrogenase (Oakeshott et al., 1982), salivary amylase (Pronk ef al., 1982), cytosolic malate dehydrogenase (McMillin and Scandalios, 1980), 6-phosphogluconate dehydrogenase (Rao and Rao, 1980), and phosphoglucose isomerase (Gottlieb and Weeden, 1979). However, in many of these instances, the duplicated genes are juxtaposed to one another without significant amounts of flanking non-gene DNA sequence between them. This contrasts with the DNA arrangement found in the development of drug resistance in experimental systems, in which the unit repeated sequences (amplicons) are usually much larger than the gene itself (see Section IV). This difference may indicate that the duplicated genes themselves are retained because they confer some selective advantage throughout evolution, but any extra DNA amplified along with the gene in the initial event is lost by recombination or deletion mechanisms. Alternatively, the original duplication events that led to tandem arrays of genes without intervening DNA may have occurred via mechanisms different than those observed during the acute development of drug resistance observed in experimental systems.

E. KNOWNAND PROBABLE SEQUENCE AMPLIFICATIONS IN MALIGNANCY A most important recent addition to the list of sequence amplifications relates to cellular oncogenes, the counterparts of viral oncogenes whose overexpression has been implicated in the genesis of cellular transformation (Bishop, 1983). In the human myeloid leukemia cell line, HL60, and in primary leukemia cells of the same patient, an 8- to 16-fold amplification of a cellular oncogene (c-myc) has been demonstrated directly with the use of cloned viral oncogene probes (Dalla Favera er a / . , 1982), and overexpression of the corresponding gene was also shown (Westin et al., 1982). The human neuroendocrine tumor lines, Colo 320 DM and Colo 320 HSR, have also been shown to amplify the cellular oncogene, c-myc (Alitalo et a/., 1983). Both lines exhibit enhanced expression of this gene relative to normal cells, and a radiolabeled c-myc genomic fragment hybridizes to the HSR regions in Colo 320 HSR. The cellular oncogene c-Ki-ras, has also been shown to be amplified 30- to 60-fold in cells of the murine adrenocortical tumor lines, YI-DM and Yl-HSR (Schwab et a/.. 1983). In this

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case, the amplified oncogene has suffered rearrangements, but still expresses inordinately large amounts of the c-Ki-ras mRNA and protein. Finally, the oncogene, c-abl, is amplified 4- to 8-fold in a human myelogenous leukemia cell line (K-562), and may involve a translocation that positions the c-abl sequence next to the gene coding for the immunoglobulin K light chain on the Philadelphia (#22) chromosome (Collins and Groudine, 1983). A causal relationship between oncogene amplification per se and transformation has not been demonstrated in any system as yet, however, and it is important to point out that oncogenes occur naturally in multiple copies in certain species of mouse and hamster (Chattopadhyay et al., 1982). It is possible that amplification of cellular oncogenes is a natural developmental phenomenon characteristic of cells in particular stages of differentiation, and that the cultured tumor cell lines used in the above studies happened to be trapped in these developmental stages. In addition to these examples of known oncogene amplifications in malignancy, there is an extensive list of probable amplifications in various neoplasms in mammals, most of which have not been treated with anticancer drugs. The argument for amplification in these cases derives from the frequent occurrence of either DMs or HSRs in the chromosomal complement. The list of examples is extensive and has recently been reviewed thoroughly by Cowell (1982) and by Barker (1982). We refer to selected reports here in order to indicate the diverse nature of neoplasms in which these chromosomal abnormalities have been observed. In no case has the amplification been shown to be the cause of cellular transformation, and it should be remembered that a hallmark of neoplasms is the vast range of chromosomal abnormalities that they contain. Double minutes were first observed by Spriggs and co-workers in cells from a pleural effusion of a malignant lung tumor (Spriggs et al., 1962). Several cases of double minutes were then reported in tumors of neurogenic origin, particularly those of children. It was soon appreciated, however, that several different kinds of human tumors contained double minutes. Many of the tumors were derived from patients that had never been exposed to chemotherapy or to overt radiation. The list (taken from Barker, 1982) includes carcinomas of the breast, cervix, colon, stomach, bladder, lung, and thyroid; chondro- and osteosarcomas; leukemias and lymphomas; gliomas, medulloblastomas, retinoblastomas, and neuroblastomas; and ovarian and testicular tumors. DMs occur in rodents in a similarly broad range of tumor types, including sarcomas, lymphomas, and neuroblastomas. Several of the murine sarcomas have been induced by Rous Sarcoma Virus or by Polyoma. DMs have been detected in both rats and mice after chemical carcinogenesis or radiation (Barker, 1982). Thus, the presence of DMs is ubiquitous in mammalian tumor cells, and as yet has not been observed in normal cells. However, since DMs are difficult to detect because of their small size and lack of G-banding, it is possible that a renewed effort may detect them in normal tissues.

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HSRs have also been observed in tumor cells of man and rodents. They were first described in G-banded preparations of cultured human neuroblastomas (Biedler and Spengler, 1976b). Since then, they have been observed in human breast and colon carcinoma cell lines, solid human tumors, transformed mouse salivary epithelial cells, mouse adrenocarcinomas, rat hepatomas, mouse lymphomas, sarcomas, and melanomas (see Barker, 1982, for references). The probability that DMs and HSRs are different (but sometimes interchangeable) configurations of the same basic chromosomal phenomenon will be discussed in Section Ill.

111. Cytological Manifestations of Gene Amplification

While the existence of double minutes and abnormally long chromosomes has been recognized for years in the metaphase spreads of mammalian tumor cells, their association with gene amplification was not recognized until 1976, when Biedler and Spengler observed a strong correlation between high levels of MTX resistance in Chinese hamster lung fibroblasts and the presence of HSRs on long marker chromosomes. When cloned DHFR sequences became available, it was possible to show that the HSR in a MTX-resistant Chinese hamster ovary cell line was the site of amplification of the DHFR gene (Nunberg et a / . , 1978). However, most MTX-resistant murine cell lines did not display obvious HSRs when subjected to G-banding protocols. Instead, fluorescent staining with acridine orange clearly showed large numbers of small, paired chromatin bodies known as double minutes, whose number per cell correlated roughly with DHFR gene copy number in the particular MTX-resistant cell line (Brown e t a / ., 1981). In this section, we will consider HSRs and DMs separately with regard to occurrence, staining properties, size, stability, and replication pattern. We will then consider situations in which the two forms appear to interconvert in some cell lines under experimental manipulation or long-term culture. The reader is referred to the excellent recent review by Cowell ( 1982) on the subject of DMs and HSRs. We present here an overview of the critical features of the karyology and behavior of these interesting chromosomal anomalies. A. HOMOGENEOUSLY-STAINING REGIONS(HSRs)

HSRs are detected in G-banded preparations as expanded chromosomal regions that do not exhibit the characteristic irregularly spaced dark vertical bands observed in most chromosomes. Instead, the HSRs stain either uniformly lightly, uniformly darkly, or exhibit very fine bands at regular intervals against a background of lighter uniform staining (see Fig. 1A). HSRs have been observed in tumor cells of hamster, mouse, rat, and human

FIG. 1. Cytological manifestations of sequence amplification. (A) Homogeneously staining regions. Methotrexate-resistant Chinese hamster lung fibroblasts (MQ19, Biedler and Spengler, 1976a) were subjected to colcemid treatment. the mitotic chromosomes were spread on microslides, and were stained by the standard G-banding protocol. The HSR is located on chromosome 2 in this cell line and is indicated by a bracket. The normal 2 homolog is indicated with an m w . Note the relatively uniform, intermediate staining of the HSR when compared to other chromosomal regions. (Picture courtesy of J. L. Biedler.) (B) Double minutes. A G-banded preparation of the human neuroendocrine tumor cell line, Colo 320DM (George and Francke, 1980). Note the relatively uniform size of the lightly staining extrachromosomal bodies (double minutes). Note also that a few of these elements appear to be single. (Picture courtesy of D. George.)

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origin (see previous section), and can range in size from barely detectable to 1520% of the condensed mitotic genome length. There can be one or more than one HSR per cell, often located terminally on chromosomes. In a given stable cell line, virtually every cell in the population displays the same number and position of HSRs. In drug-resistant cells, it can be shown that HSRs often reside on the chromosome in which the original unamplified gene is located. Alternatively, they can be located on other identifiable chromosomes. In some cases, the HSRs are located on unidentified marker chromosomes that could arise by breakage of an elongated HSR from its original site, with subsequent attainment of a centromere. In a MTX-resistant CHO cell line derived by Chasin and associates, the amplified DHFR genes are located on the long arm of chromosome 2 (Nunberg et al., 1978). The DHFR gene in parental drug-sensitive CHO cells has also been shown to reside on chromosome 2 in cell fusionlchromosome mapping studies (Roberts er al., 1980). Flintoff et al. (1982) observed HSRs on chromosomes 2 2 (a rearranged 2 ) , 5, and Z5a in a series of MTX-resistant CHO cells developed in their laboratory. In some of these highly resistant cell lines with as many as 75 copies of the DHFR gene, no obvious HSRs were detected, although one line had suffered a rearrangement of chromosome 2 . Biedler and colleagues have extensively characterized a large number of independently isolated Chinese hamster lung fibroblasts that are highly resistant to MTX. Each of these lines displays a single HSR whose length varies approximately in proportion to the level of drug resistance in each line (Biedler et a l ., 1980). While the HSRs in these cell lines can be located at a variety of positions (e.g., chromosomes 2 , 4, 9, and unidentified marker chromosomes), they are often located terminally on the long arm of chromosome 2 , but not necessarily next to the same G-band on the original 2q in each case. These investigators also examined several cell lines with low DHFR gene copy numbers that exhibited highly rearranged, abnormally banded chromosomes (often chromosome 2 ) (Biedler et al., 1980). In some cell lines, small uniformly stained regions are interspersed with segments that band normally, and in situ hybridization with DHFR probes results in separated clusters of grains on a single chromosome. Biedler and colleagues have interpreted their results to mean that amplification of the DHFR gene may occur extrachromosonially in the initial stages, and that the extra copies then insert randomly into chromosomes (and often back into chromosome 2 ) . However, most of the abnormal chromosomes pictured in this study could also be explained by supposing that amplification occurs in situ at the original location of the DHFR gene in chromosome 2 , but that the resulting tandem sequences are unstable and often break at random positions within the array. The two free ends could then provoke translocations and intrachroniosomal recombinations, leading to dispersion of the amplified sequences and the resulting complex karyotypes observed in these cell lines.

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A highly MTX-resistant CHO line derived by Hamlin and colleagues contains two detectable HSRs, neither of which is on chromosome 2 (Milbrandt et al., 1981). Instead, studies on a cloned series of increasingly resistant cell lines from which this line was derived show that even at the lowest level of resistance, the original HSR was located on the long arm of chromosome 1 (J. L. Hamlin, unpublished observations). As amplification increased, this original HSR lengthened and eventually fragmented to yield a second HSR-bearing marker chromosome, which itself lengthens with increasing resistance. In addition, in situ hybridization studies with a cloned DHFR genomic fragment detects a third site of amplified genes on chromosome 2 4 that is undetectable as an HSR at any drug level. In those MTX-resistant murine lines that exhibit HSRs (as opposed to DMs), the amplified DHFR genes are sometimes on chromosome 2, which may be the site of the parental gene (Dolnick er al., 1979). The amplified DHFR genes in human cells have been observed on chromosomes 4 , 5 , 6 , 10, and 19 (Wolman et al., 1983; Trent et al., 1984; Srimatkandada et al., 1983). The site of the endogenous gene in normal, MTX-sensitive human cells is presently not known. Among the various neoplasms that display HSRs, there is as yet no consistent correlation between the chromosomal positions of HSRs and the type of tumor in which they are observed (e.g., see Gilbert and Balaban, 1982). However, it is possible that the same oncogene is amplified in a given class of tumors, but the resulting HSRs are subjected to translocations and rearrangements that mask the original chromosomal location of the oncogene. Balaban-Malenbaum and Gilbert (1982) have also made the interesting observation that an HSR occurs at chromosomal position Ip34 in both a human retinoblastoma (Y79) and in a neuroblastoma (IMR32), suggesting that the same sequence (oncogene?) may be amplified in both cases. The expanded chromosomal regions originally detected by Biedler and Spengler (1976a) in mitotic preparations of MTX-resistant Chinese hamster lung cells and in certain neuroblastomas were termed HSRs because of their uniform (unstriated), intermediate staining with Giemsa. However, a variety of HSRs have now been described that deviate from this euchromatic appearance. In PALA-resistant Syrian hamster cells, Wahl and co-workers have shown that the amplified sequence includes not only the gene coding for the CAD protein complex, but ribosomal DNA as well (Wahl et al., 1983). Most of the amplified sequences are located at the terminus of chromosome 9, which was also shown to be the location of some of the rDNA copies in parental Syrian hamster cells. The expanded chromosomal region on chromosome 9 is characterized by finely apposed dark bands. In rat erythroleukemia (Murao e? al., 1982), H4 hepatoma (Tantravahi el al., 198 l ) , and XC sarcoma (Tantravahi et al., 1982) cell lines, rDNA genes can be extensively amplified, and are observed as HSRs or finely banding regions at known nucleolar organizer regions in this species. Regularly

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spaced alternating light and dark bands have also been observed in a variety of other cell lines, including human neuroblastomas and melanomas (see Cowell, 1982). As pointed out by Cowell, the appearance of expanded chromosomal regions may be critically dependent on the fine points of the staining technique. Indeed, the HSRs in different stained preparations of the same MTX-resistant CHO cell can appear either finely banded or homogeneously euchromatic (Milbrandt et al., 1981). At the present time, very little is known about the composition of amplified sequences in most cell lines. For example, it is not known how many of the HSRs in different cell lines actually do contain ribosomal DNA, satellite DNA, or other repetitive sequences that could confer the finely banding property often observed. The critical observation is that HSRs appear unusual in Giemsa or Qbanding studies because they exhibit either uniform staining or regularly spaced bands, and can usually be detected even by the untrained eye. Since many of the amplified sequences are now being cloned and characterized with respect to sequence composition, it will soon become possible to relate DNA sequence arrangement to chromatin banding properties. This is an especially promising area of cytogenetics that has been difficult to study on single copy genes in the past. Regardless of cell type, HSRs are relatively stable entities that behave in most respects like typical chromosomal segments. Biedler and colleagues have shown that the amplified DHFR genes that reside in HSRs in some MTX-resistant Chinese hamster lung cell lines can be maintained for years in the absence of MTX selection (Biedler el al., 1983). However, in most cell lines, a gradual decline in DHFR activity was observed over the period of several years in culture in the absence of drug, accompanied by a gradual decline in the length of the HSRs. One cell line that exhibits a prominent HSR on chromosome 2 was observed to lose more than 90% of DHFR activity within about 50 cell doublings (Biedler et nl., 1983). Thus, the stability of HSRs can vary, although the mechanism for such variation is unknown. In general, amplified drug resistance markers contained in HSRs are much more stable than those camed on DMs, which can be completely lost from the population in 20-30 cell doublings (e.g., Biedler et nl., 1983; Brown et al., 1981). HSRs seem to replicate by the same mechanisms that govern replication of the remainder of the karyotype, and since they are carried on chromosomes with legitimate centromeres, the daughter HSRs are distributed equally to the two daughter cells at mitosis. The HSRs that contain amplified DHFR genes have been shown to be early replicating in Chinese hamster cells (Harnlin and Biedler, 1981; Milbrandt et al., 1981) and in one MTX-resistant murine cell line (Kellems er al., 1982). In addition, the HSRs in several human neuroblastomas appear to replicate early in the S period (Biedler and Spengler, 1976b). In MTX-

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resistant CHO cells, it has been shown that each unit repeated sequence (amplicon) contains a functional origin of DNA synthesis (Heintz and Hamlin, 1982), with the result that initiation of DNA synthesis at the beginning of S begins synchronously at multiple loci within the HSR (Milbrandt et al., 1981). Since there is probably only one origin within each unit, an amplicon may be equivalent to a replicon. It follows that amplification may proceed via the agency of replicons. This idea will be discussed in Section V1. It has been suggested that all expressed genes are replicated early during the S period, since early replication has long been correlated with euchromatic (presumably active) elements in mammalian chromosomes (see Bostock and Sumner, 1978, for review). However, Hamlin and Biedler (1981) have shown that the HSRs in two MTX-resistant Chinese hamster lung cell lines are early replicating, even though these HSRs are clearly C-band positive. These HSRs therefore probably contain at least some constitutive heterochromatin, which is normally late replicating (Bostock and Sumner, 1978). Interestingly, in Chinese hamster cells that have been transfected with a cloned DHFR gene (J. L. Hamlin, unpublished observations) or with cloned DHFR cDNA (Kaufman et al., 1983), and subsequently have been subjected to incremental increases in MTX concentration, the resulting HSRs have also been shown to be early replicating. This finding adds strength to the argument that expressed genes are always early replicating, but raises questions as to how the transfected gene becomes subject to this control in a new chromosomal location. It is likely that the cloned DHFR sequences have integrated next to an earlyreplicating origin in a new chromosomal location, possibly because euchromatin is a better substrate for recombination than is late-replicating heterochromatin. Alternatively, sequences that integrate into heterochromatin may not be expressed. It is also possible that early-replicating transformants are preferentially selected, since the gene is normally transcribed in late G,/early S (Mariani et al., 19811, and may require early replication for its expression. In any case, studies on these transformants should shed light on the important questions of amplification itself and time-ordered DNA synthesis in mammalian cells. B. DOUBLEMINUTES (DMs) Typically, double minutes are seen in metaphase spreads as small, paired chromatin bodies that usually stain poorly with G, Q, C, R, and Cd banding protocols, and are therefore believed to be euchromatic in nature (see Fig. 1B). They have been observed in tumor cells and cell lines of mouse, human, rat, and hamster origin (Barker, 1982). They have been shown to contain amplified DHFR genes in several MTX-resistant murine cell lines (e.g., Kaufman et al., 1979; Martinsson et al., 1982), and their presence has been correlated with amplification of many other genes in drug resistance, among them vincristine

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(Kuo et al., 1982), metallothionein (Beach et al., 1981), and adenosine deaminase (Yeung er al., 1983a). In addition, they have frequently been observed in a variety of tumor cells in which the amplified material has not as yet been identified (see Barker, 1982, for review). DMs can be distinguished from very small chromosomes by their lack of Cbanding and unusual behavior during mitosis. DMs can range in size from barely detectable at the light microscopy level to the size of the smallest mammalian chromosomes. Large DMs can, in unusual cases, assume the shapes of rods or rings (e.g., Bostock and Clark, 1980). In a given cell, the DMs are usually approximately the same size. Electron microscopic studies on DMs isolated by sucrose gradient fractionation support the concept that the chromatin structure of DMs is closely related to that of normal chromosomes, with the exception that they lack centromeres (Barker and Stubblefield, 1979). DMs vary in number in different cell lines, or even within a single clonal population. They are sometimes observed only in a small subpopulation of a clonal cell line (e.g., Baskin et al., 1981; Levan et al., 1977). In some cell lines, there may be only one or two DMs per cell, and in others, as many as a thousand per metaphase spread (Cowell, 1982). In the latter case, the DMs are very small. In the case of MTX resistance, it has been possible to relate the number of DMs in murine cell lines with different levels of drug resistance (Brown et al., 1981). In this case, there appears to be a rough correlation between the number of DMs and the gene copy number, and the lines seem to maintain a relatively steady-state level of DMs at a given MTX concentration. DMs lack centromeres, and distribute themselves to daughter cells during mitosis by attaching themselves randomly (and usually in groups) to the ends of other chromosomes at the metaphase plate during chromosome segregation (Levan et al., 1976). They are thus carried along adventitiously to daughter cells in a random fashion, with the result that unequal distribution can occur. In cases where the DMs offer a selective advantage to the cell (e.g., drug resistance), the presence of the selective agent would tend to continuously select for the daughter cell that had received the most DMs. However, balanced against this process is the frequent loss of DMs, due to a failure to attach to chromosomes during mitosis, leading to encapsidation by the reforming nuclear envelope, and eventual extrusion from the cell (Levan and Levan, 1978). Brown et al. (1981) and Biedler et al. (1980) have shown that more than 90% of the DHFR genes carried on DMs can be lost in a matter of weeks in the absence of the selective agent, MTX. It has also been observed that cells with large numbers of DMs grow more slowly than do related cell lines with smaller numbers (Kaufman et al., 1981). Thus, the number of DMs observed in any steady-state condition (e.g., constant selective pressure) must be a product of all these factors. This suggests that the DMs observed in tumor cells that have not been treated with drugs must somehow confer a selective advantage on the host cell; otherwise,

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unequal distribution, loss of DMs by enucleation, and slower growth rate would eventually eliminate cells containing DMs. Since DMs are not integrated in a stable fashion into chromosomes, do they replicate, and, if so, how? It is possible that once they accumulate by whatever means (e.g., HSR fragmentation), they do not replicate, but rather are maintained in the population by unequal segregation. Several observations suggest that DMs do indeed replicate once per cell cycle, and apparently during the early S period, These conclusions derive from three types of experiment: (1) bromodeoxyuridine (BUdR) incorporation followed by Hoescht 33258 staining yields the typical harlequin staining pattern characteristic of a single cycle of semiconservative replication during one S period, and shows that each half of a double minute represents the counterpart of a chromatid (Quinn et al., 1979; Barker and Hsu, 1979); (2) after a brief pulse of [3H]thymidine, autoradiography demonstrates that DMs are labeled only in cells that show early replication patterns on the rest of the chromosomes (Barker et al., 1980); ( 3 ) when premature chromosome condensation is provoked in G , phase cells containing DMs, many single minute structures are also observed; however, in G , phase, double minutes predominate, suggesting that they were replicated during the S phase; once again, the BUdR/Hoescht technique displays the harlequin staining pattern expected after a round of semiconservative replication (Barker et al., 1980). In total, these observations support the concept that DMs behave as minichromosomes with respect to replication, but, as stated before, do not divide at mitosis. Separation of the two chromatids must occur sometime between the end of one S period and the beginning of the next, to account for the observation that DMs do not increase in size in stable cell populations during prolonged culture. However, the occasional failure to separate during one cell cycle could lead to larger DMs, and could explain the observed variability in the size of DMs in some cell types. The question of replication of DMs cannot be completely understood at the present time because the basic structure of DMs is not known. They are thought to be composed of linear or circular tandem arrays of amplicons. By analogy to HSRs, each amplicon in a DM could contain an origin of replication. DNA synthesis could then proceed by a mechanism analogous to bidirectional chromosomal replication from multiple origins, as has been suggested for the amplicons contained in HSRs (Heintz and Hamlin, 1982).

C. RELATIONSHIP BETWEEN DMs

AND

HSRs

If DMs and HSRs are different manifestations of the same phenomenon (amplification), then what determines whether a given amplified sequence will take one form or another? This is a complex question for which there is presently no

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satisfactory answer. HSRs have been observed in cells of murine, hamster, rat, and human origin. DMs have been observed in murine, human, and rat cells, but rarely in hamster cells. However, DMs are a property of tumor cells, and very few cultured hamster tumor lines are available for study. DMs have been detected in vincristine-resistant Chinese hamster embryo fibroblasts (Kuo et al., 1982). However, in this case, there is some question whether the DMs observed actually contain the gene that imparts resistance to vincristine, since the number of DMs does not decrease when the drug is removed, even though vincristine resistance decreases. However, this result does show that hamster cells are capable of generating and maintaining such structures. It is likely, therefore, that taxonomic differences per se do not explain the propensity of a given cell line to maintain amplified DNA sequences in either intra- or extrachromosomal forms. The question then arises whether the particular amplified locus in a cell line is constrained to assume one form or another by unknown mechanisms related to DNA sequence. There is no clear-cut answer to this question either, primarily because few systematic studies have been performed in which a single cloned parental cell line has been treated with a variety of drugs to isolate variants that have amplified different genes. However, Biedler and colleagues have shown that a near-diploid cloned Chinese hamster lung cell line (DC3F) gives rise to HSRs when subjected to increasing concentrations of either MTX or vincristine (Biedler and Spengler, 1976a; Biedler, 1982). Hence, two different loci manifest the same chromosomal form in this case. It would be of interest to subject the parental DC3F cell line to stepwise increments of other drugs to determine whether any locus in this cell line could give rise to DMs. It is clear that certain cell lines display a propensity toward the formation of either DMs or HSRs. In highly MTX-resistant Chinese hamster cells (ovary and lung), amplified DHFR genes invariably reside on chromosomal HSRs (Biedler and Spengler, 1976a; Nunberg et a/., 1978; Flintoff et a/.., 1982; Milbrandt et al., 1981). Syrian hamster cells selected for PALA resistance exhibit HSRs, and DMs have apparently never been observed in this system (Wahl et a / . , 1983). In cultured MTX-resistant human cells, the amplified DHFR genes are usually detected as HSRs (Trent, 1982; Wolman el a / . , 1983; Srimatkandada el a / . , 1983), but Trent and co-workers have recently reported the occurrence of one or two DMs in a small percentage of cells obtained from a MTX-resistant human ovarian adenocarcinoma (Trent et a / ., 1984). In most MTX-resistant murine lines, the amplified DHFR genes are located on DMs (Kaufman et a / . , 1979). However, after long-term culture of cell lines bearing DMs (e.g., murine S180 cells), stably amplified lines can occasionally be derived in which the genes are located on HSRs (Dolnick et a/., 1979). The observation that the same cell line can give rise to sublines that display either DMs or HSRs therefore argues that the two forms are interconvertible in some instances. There are several examples among neoplasms that support this

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suggestion. Cell lines derived from the murine Y 1 adrenocortical tumor can have either DMs or HSRs, but not both in the same cell line (George and Franke, 1980). Utilizing clones from an amplified genomic sequence derived from partially purified DMs, George and Powers (1982) have demonstrated that the same sequence is amplified in the HSR of another Y 1 subline. Thus, in this case, the DMs and HSRs are most likely different manifestations of the same amplification phenomenon in the same genetic background. The human neuroendocrine tumor, Colorado 32 1, can also manifest one or the other chromosome abnormality, and sublines can interconvert in culture (Quinn et al., 1979). The RVP-3 mouse tumor displays DMs early after establishment in culture, but these gave rise to microchromosomes after long-term maintainence, some of which C-band and seem to have centromeres (Sainerova and Svoboda, 1981). A provocative finding by Levan and co-workers is the observation that the amplified sequences in the murine tumor line, SEWAIR, exist in alternate states, depending upon whether the cells are cultured as an ascites tumor in vivo or are maintained in tissue culture (Levan and Levan, 1982). The effect of culture conditions points out that subtle cellular differences may determine the propensity of genetic amplifications to assume one or the other form in a given cell type. Since, in the SEWAIR tumor, the HSRs disappear and are replaced by DMs in vivo, the possibility exists that DMs somehow provide a selective advantage to cells in vivo. This suggestion is supported by their finding that subcutaneous injection of a SEWAIR subline displaying DMs provoked tumors in animals, but sublines without DMs did not form tumors (Martinsson, Dahloff, Sandberg, and Levan, unpublished observations). Martinsson et al. (1 982) made the additional very important observation that a given cell can maintain both DMs and HSRs simultaneously, albeit not at the same genetic locus. When the SEWAIR tumor displaying HSRs (amplified oncogenes'?) was subjected to incremental increases in MTX for several months in vitro, multiple DMs developed which ostensibly arose from the amplification of the DHFR gene, but the original HSRs were maintained. This finding argues against the possibility that individual cells in a population can maintain only one of the two configurations due to ambient intracellular conditions. Another approach to understanding the genesis and/or stability of the alternate chromosomal manifestations of gene amplification involves fusion between cell lines. The MTX-resistant murine tumor line, SEWAIR TC13, when cultured in vitro, maintains a mixed but relatively constant proportion of cells showing either DMs (60% of cells) or HSRs (40% of cells) (Jakobsson et al., 1984). However, upon fusion with Chinese hamster V79 cells, all MTX-resistant hybrids exhibited only HSRs. Thus, in this particular combination of cells, DMs do not appear to be transferred and/or maintained in the hybrids. It could be argued that DMs are easily lost at mitosis during the undoubtedly complex

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adjustments that the hybrids must make to the newly formed karyotype. Hybrids with a large enough number of DMs (therefore DHFR genes) to survive selection in MTX would therefore be very few in number. Alternatively, the milieu contributed by the V79 cell in the hybrid may not be able to support the presence of DMs for unknown reasons, and, thus, only cells with HSRs would be selected in MTX . Contrasted with these results are the observations of Kano-Tanaka et ul. (1982). Mouse neuroblastoma cells showing only DMs were fused with either rat liver, rat glioma, or Chinese hamster brain cells. All hybrids manifested DMs, indicating that under these circumstances, DMs are able to be transferred and maintained in the new hybrid cells, even though in one instance, one partner in the hybrid (the Chinese hamster cell) rarely displays DMs. Another variation involves a human neuroblastoma cell line that displays only HSRs, but when fused to mouse fibroblasts, yields cells displaying only DMs (Balaban-Malenbaum and Gilbert, 1980). The interpretation here would be that the resulting hybrids are not able to support HSRs, or that fragmentation of the HSR occurs during fusion, and for unknown reasons, the hybrid cell is not able to reaggregate and/or integrate the DM sequences into HSRs. The results of all these experiments presently do not allow the formulation of any unifying rules for the maintenance of amplified genes in hybrid cells. Clearly, the DMs or HSRs bearing amplified genes cannot by themselves determine their eventual configuration in hybrid cells. Additional factors related to the new cellular environment created by fusion of two disparate cell types must also be involved. As discussed in an earlier section, cloned amplifiable genes have been transferred to cultured cells, and after stepwise selection with the appropriate drug, the cytological properties of resistant cells have been determined. In these cases, the transfected, amplified genes seem to reside in the same kind of chromosomal structure characteristic of amplified endogenous genes. Both the CAD and DHFR genes are located in stable HSRs after transfection and amplification in hamster cells (Wahl et al., 1983; Milbrandt et al., 1983b). The most telling results were obtained in experiments utilizing a variety of cloned murine DHFR minigenes to transform either murine or Chinese hamster cells. After selection for MTX resistance, murine transformants invariably contained large numbers of DMs (Murray et al., 1983), whereas MTX-resistant Chinese hamster transformants displayed only HSRs (Kaufman et al., 1983; Gasser ef nl., 1982). Either the sequences flanking the site of integration must somehow determine the subsequent chromosomal state of the amplified material, or the cell line itself determines it. By the former argument, it might have been expected that independent transformants of a given recipient cell type would have displayed both alternative chromosomal manifestations, depending on the sequences surrounding the site of integration. However, all independent isolates of a given cell line (e.g., mouse)

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consistently displayed the same abnormality. It is more likely that these studies reflect the general observation that cultured Chinese and Syrian hamster cells most often display HSRs as a corrollary to drug resistance and gene arnplification, whereas the formation of DMs is the more usual manifestation of gene amplification in murine cell lines. Schimke and collaborators have attempted to understand the process of gene amplification in greater detail by examining the early stages of amplification (i.e., single step selections in low levels of drug) and the characteristics of loss and fixation of genes as drug resistance is lost or gained. They have utilized a fluorescent MTX derivative that binds tightly to DHFR as an indirect indicator of the number of DHFR genes in a cell. Because the bound MTX fluoresces, cells incubated with the compound can be individually analyzed and/or separated on a fluorescence-activated cell sorter. In addition, their results, in most cases, have been confirmed by determining gene copy number in sorted cells. They first studied MTX-resistant murine lines derived from 3T6 cells that display numerous DMs. When cultured in the absence of MTX, these cell lines lost DHFR genes (fluorescence) with kinetics identical to the loss of DMs, as determined cytologically (Brown et izl., 1981). These results were confirmed by determining the number of DHFR genes in revertant cell lines by Southern blot analysis, using a radiolabeled cloned DHFR cDNA. These studies thus support the notion that the amplified DHFR genes that impart MTX resistance are located on DMs, and the DMs are unstable entities that are frequently lost or unequally distributed to cells during mitosis. These suggestions were confirmed in studies on DM-containing MTX-resistant murine S180 cells (Kaufman et al., 1981). When grown in the absence of MTX for many doublings, cells with progressively fewer DMs became dominant in the population. A most interesting result was obtained when the development of MTX resistance was studied in CHO cells, which invariably display stable HSRs at high levels of drug resistance after long-term culture. When cells were selected at a low drug level and were subsequently analyzed after relatively short intervals (e.g., 2 weeks), a heterodisperse population with variable DHFR gene copy numbers was observed (Kaufman and Schimke, 1981). Moreover, if sorted cells with a given number of DHFR genes were subsequently cloned and were grown in the absence of MTX for about 20 cell doublings, each clone behaved somewhat differently, indicating clonal variation in the stability of the amplified sequences. The progeny of some clones maintained the original gene copy number in the absence of drug, while the progeny of other clones lost all or a large fraction of the amplified genes. Other clones actually gave rise to cells that had amplified the DHFR gene still further, even in the absence of MTX. DMs were apparently not observed in these cells under any of the above manipulations. These data indicate that shortly after amplification, the initial extra DHFR gene copies need not be stably integrated into the chromosome, even in CHO

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cells. However, it could be argued that only those copies that are stably integrated in the initial event give rise to the stable HSRs observed in highly resistant sublines obtained after incremental MTX selection and long-term culture. Furthermore, since cells can amplify the gene in the absence of drug selection, amplification must be a random event, and administration of drug must select those cells that contain enough DHFR genes to support growth at a particular drug level. In all cases where CHO cells were grown at a given drug concentration for 100 doublings or more, the amplified genes were shown to be maintained stably as HSRs after removal of drug for prolonged periods of time. This suggests that the extrachromosomal copies observed at low drug levels are lost from the population, or are eventually integrated in tandem into the chromosome. Since the initial DHFR gene amplification events observed in both mouse and Chinese hamster cells can be unstable, there must be inherent differences in the ability of these cell lines (S 180 and CHO) to fix and maintain the amplified genes as HSRs after long-term culture in MTX. It may be that at all stages of amplification, CHO cells integrate the duplicated segments covalently into the DNA fiber more efficiently than do the murine cell lines, owing to differences in the structural or functional organization of chromosomes. Alternatively, the murine lines may be more efficient at excising duplicated segments through homologous recombination events, leading to extrachromosomal DMs. The few reported cases of MTX-resistant murine cell lines that bear amplified DHFR genes on HSRs would then presumably represent instances in which the recombination process has somehow been suppressed. This argument could also explain the fact that individual sublines originally derived from a single cell (e.g., the murine Y 1 adrenocortical tumor cells) can display either HSRs or DMs (George and Franke, 1980).

IV. Nature of Amplified Sequences In order to understand the mechanisms involved in the amplification of DNA sequences, it will be necessary to define the nature of the amplified unit. One would like to know the answers to the following questions: 1. Are the multiple copies of an amplified sequence arrayed tandemly in a linear fashion, or do they lie side-by-side in an onion skin configuration'? 2 . How large are the unit repeated sequences? 3. Are the amplified sequences of equal size in a given cell line, i.e., is the unit of amplification precise or imprecise? At a given locus, are the boundaries of an amplicon fixed by some aspect of the nucleotide sequence that itself determines the mechanism? 4. Does the sequence continue to be amplified with precision, or is it trimmed

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JOYCE L. HAMLIN ET AL

or rearranged during the process, i.e., do the characteristics of the amplicons observed in highly drug-resistant lines tell us anything about the initial unit of amplification? A . AMPLIFIED ENDOGENOUS GENES

The most compelling evidence that amplification results in linear tandem arrays of a unit repeated sequence (amplicon) is that the HSRs that contain amplified sequences are elongated rather than thickened, and that an HSR has the same diameter as the rest of the chromosomal complement. Furthermore, Biedler and colleagues have shown in a series of highly resistant Chinese hamster cell lines that the length of the HSR is roughly proportional to the level of drug resistance in each line (Biedler et al., 1980). This phenomenon has also been observed in a cloned series of increasingly MTX-resistant CHO cells by Hamlin and colleagues (unpublished observations). Evidence for a linear tandem arrangement of amplicons is less clear for the DNA in double minutes, since they can appear to be large or small in diameter, and since there is some question as to whether they are circular rather than linear (e.g., Bostock and Clark, 1980). However, since MTX-resistant mouse cells display HSRs and DMs as alternative forms of DHFR gene amplification (Kaufman et a/., 1979), it is reasonable to assume that the amplicons are arranged tandemly in both situations. In addition, in their studies on the nature of the nucleotide sequences in the CAD amplicon, Stark and co-workers have found unique junction fragments that are predicted by end-to-end joining of amplicons (see Ardeshir et a / . , 1983, and discussion below). Original estimates for the size of amplicons in several cell lines were made by determining the length of HSRs, and by determining the number of amplified genes in a given cell line. By assuming a figure for the number of residues in a given length of HSR, Nunberg et a / . , (1978) estimated that the size of the amplicon in a MTX-resistant CHO cell was 500-1000 kb in length. Using the same strategy, Bostock and Clark (1980) estimated that the amplicon could be as large as 3000 kb in a MTX-resistant mouse PG193T lymphoma cell line. The observations of Milbrandt et al. (1981) suggest that in some cell lines, the amplicon can be considerably smaller. The restriction fragments derived from the amplicons in highly MTX-resistant CHO cells can be visualized on ethidium bromide-stained agarose gels, and by summing the lengths of all amplified fragments, they estimated that the unit repeated sequence was approximately I35 kb in length. Since there are 1000 copies of the amplicon in this cell line, a total of 1.35x108 bp, or about 4% of the genome length, represents amplified sequences. However, the length of the HSRs in mitotic spreads totals about 8% of the condensed genome length. This discrepancy might be explained if it is assumed that the restriction fragments that can be visualized on gels represent

DNA SEQUENCE AMPLIFICATION

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only a consensus sequence that is amplified in all amplicons, but that is flanked by more or less DNA in each repeated unit. The 135 kb estimate would then be a lower estimate for the size of the repeating unit. Alternatively, estimates for copy number and length of HSRs may be inaccurate enough to account for the twofold discrepancy. Bostock and Tyler-Smith ( 1982) studied MTX-resistant murine EL4 lymphoma cell lines that were cloned from a resistant population selected in a single, low concentration of MTX. After being subjected to increasing drug levels, the amplicons in the highly resistant derivatives were apparently very similar to one another, and appeared to be about 500 kb in length, as determined by direct visualization of amplified restriction fragments in ethidium bromide-stained agarose gels. This result suggests that in this system, the original amplified unit is maintained during amplifications to higher copy number. Furthermore, Bostock and Tyler-Smith used isolated DMs carrying DHFR genes to transfer MTX resistance to sensitive mouse L cells. After incremental increases in MTX to raise the level of resistance, the amplicons were examined in the derivatives, and were found to contain the same sequences as the amplicon in the donor cell. Since a single DM is apparently transferred in these experiments, Bostock and Tyler-Smith argue that every DM in the EL4 lymphoma must have the same array of amplicons. In order to understand more about the structure of amplified sequences contained in DMs or HSRs, it is obviously necessary to isolate the sequences in question. Molecular cloning of amplified sequences is greatly simplified owing to the much larger number of these sequences per diploid nucleus. The practical result of this is that many fewer bacterial clones from libraries containing recombinant genomic fragment have to be screened in order to isolate overlapping clones spanning several equivalents of the amplicon. Schimke and collaborators have isolated more than 200 kb of the DHFR amplicon from the MTX-resistant cell line, S180, by a procedure that involved isolation of a chromosome fraction enriched in double minutes on sucrose gradients (Schilling et af., 1982). By using cloned DHFR cDNA sequences as radioactive probes, they initially cloned fragments from the DHFR gene itself, and then used the endmost fragments from these clones to “walk” to the right and left of the gene. They have used these overlapping clones as radioactive hybridization probes on restriction digests of other MTX-resistant murine cell lines to ask whether the amplicon is the same or different in independently isolated cell lines. They found that these probes cross-hybridized with different lengths of DNA sequence in each cell line, ranging from 80 kb in a murine 3T6 cell line displaying double minutes, to as large as 200 kb in the S 180 cell line itself. This result could indicate that the actual size of DHFR amplicons in different murine cell lines can be different. However, many of these MTXresistant murine lines derive from different parental lines that have been main-

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JOYCE L. HAMLIN ET AL.

tained in culture for many years, and many of these cells are karyotypically unstable. It is therefore possible that the parental sequences flanking the DHFR gene are not identical, due to deletions and/or rearrangements, and, hence, cross-hydridization is not detected between the divergent sequences. A more convincing case for variable amplicon size arising from the same genetic background could be made if cell lines derived from exactly the same parental cell would be shown to have variable sized amplicons. Hamlin and colleagues have cloned approximately 110 kb of contiguous DNA sequence from the amplicon of a MTX-resistant CHO cell line, by first isolating the DHFR gene itself, followed by chromosomal walking (Milbrandt et al., 1983a). These clones have been used to probe independently isolated MTXresistant cell lines of the same or closely related [Chinese hamster lung (CHL)] parentage (Montoya-Zavala and Hamlin, unpublished observations). Both CHO and CHL cells are extremely stable karyotypically, and the CHL cells have a near diploid chromosome complement (Biedler and Spengler, 1976a). The cloned probes from the MTX-resistant CHO cell line cross-hybridize to virtually identical restriction fragments in the DNA of three other MTX-resistant cell lines (two MTX-resistant CHL lines and another MTX-resistant CHO cell). The major differences so far detected between cell lines can be accounted for by simple restriction site gains or losses, but no major deletions or insertions have been observed. It could be argued that sequences more distant from the DHFR gene that have not been cloned as yet will detect different sized amplicons in each cell line. However, direct visualization of the amplified restriction fragments in ethidium bromide-stained agarose gels indicates that the amplicons in all four of these cell lines are similar in size and composition (Hamlin et al., 1982). Together, these findings suggest that two resistant cell lines with exactly the same parentage (line DC3F in the case of two CHL cells, and CHO K1 in the case of the two CHO cells) are constrained to amplify the same sequence, if the parental cell lines are karyotypically stable. Stark and colleagues have prepared libraries of genomic clones from two different PALA-resistant Syrian hamster cell lines (Acdeshir et al. 1983). Clones derived from the CAD amplicons were identified by using total genomic DNA from PALA-resistant cells as radioactive probes (after removing nonspecific, highly repeated sequence elements). These clones represent 162 and 68 kb of each amplicon, although most of the fragments have not been ordered or shown to overlap with one another as yet, and together probably represent a small part of the CAD amplicon in each cell line (estimated to be approximately 500 kb in length). Nevertheless, they have found several fragments in each library that appear to represent junctions fragments between amplicons. From this result, they argue that amplification proceeds by an imperfect mechanism, and that the amplicons within a given cell line end at different places, but usually straddle the CAD gene (see Fig. 2). This argument is supported by their finding that different

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FIG.2. Different mcdcs uf hrquence amplificaticin. The linear sequence of a chromosomal DNA Lher is represented by thc lcttcrs of the alphdbet. whcrc P might reprcscnt a SclCctdhk gcnc. In the precisc mechanism illustrated ahove (1 in figure), the boundaries of [he amplified unit are p r e d e t e m n e d hy sonic aspcct (if the parcntal DNA eequence ( c . g unit of replication, or Iocatiun of highly rcwated e q u e n c e elements), so that all cells derived from the samc parental ccIl must amplify MNOFQRS in the first duplication step. and in all succeeding amplifications at this locus. Only one new junction fragment. S M . I!. formed in the unpindl duplication. and its copy nurnbcr is equivalent to the n u m k r of repeatcd units in all subscquent steps. In the rnechanicm outlined in verf~on2. (he unit of amplification is not fixcd, and the initial duplicakd sequence can be positioned diffcrcntly m u n d P in dlfferent ceH lincs dcrived from thc same parent. However, in subsequent amplificaticm, the duplicated sequencc i s amplified faithfully. Different junction fragments are therefurc amplified in individual cell lincs dcrived fmm the varne parental cell line. In the impcrfcct mechanism rmtlincd in verhion 3. the initial duplicatcd scqucncr can hc positioned differently around P in different cells from the came parent (as in mechanism Z), and in huhsequent amplificatiwns. diffcrcnt sequences arc ampllficd each time. In this case, w m e fragments are prescnt more oflen than othcrs. and severdl uniquc junction fragmunts rcsull fe.g.. LIB. F A . HI) whose copy number is less than thc copy numbcr of P.

.

62

JOYCE L. HAMLIN ET AL.

fragments are amplified to different extents in a single PALA-resistant cell line. This is an important result that is somewhat disturbing, since it indicates that determining the exact nature of the amplified unit may be difficult, if not impossible. By using these clones as probes on restriction digests of genomic DNA, they also showed that no two independently isolated, highly PALA-resistant clones had exactly the same amplified sequences flanking the gene, although a consensus sequence of about 44 kb (including the CAD gene) was amplified in all cell lines (Ardeshir el al., 1983). This result suggests that major rearrangements have occurred during the amplification process to account for the fact that the CAD gene is flanked by different sequences in each cell line. It is important to point out that all of the clones examined derived from two different parental cell lines (a cloned derivative of BHK 21/13 and an SV40-transformed derivative of this cell line), both of which were heteroploid to begin with, indicating inherent chromosomal instability. Thus, it is possible that some of the DNA sequence rearrangements observed are not involved in the amplification process per se, and could be masking a more simple underlying mechanism. When the parental BHK cell line was subjected to a single-step selection in a low level of PALA, most of the resulting PALA-resistant clones amplified the same large sequence (about 68 kb in length) (Zieg et al., 1983). This result agrees with the observations of Bostock and Tyler-Smith ( 1 982) on MTX-resistant mouse lines that were subjected to single-step selections in drug. However, Zieg el al. also observed that different fragments were amplified to different extents in a given PALAresistant cell line, suggesting that the units of amplification are not all identical in length. George and Powers (1981) have also cloned fragments from the amplicon in the mouse adrenocarcinoma, Y 1 . In this case, random fragments cloned from a fraction enriched in DMs were tested for their presence in the amplicon by assessing their genomic copy number on Southern blots or by in situ hybridization to related Y 1 cell lines displaying HSRs (1982). Several clones behaved in both tests as if they were amplified. These findings confirm their suggestion that the DMs and HSRs observed in different Y 1 sublines are alternate forms of the same amplified sequence. Most recently, Kanda et al. (1983) have isolated a large HSR-bearing chromosome from the 1MR32 human neuroblastoma by fluorescence-activated flow sorting of mitotic chromosomes stained with the fluorescent dye, 33258 Hoescht. This approach is feasible because the HSR resides on the largest chromosome in human cells (chromosome l ) , and the length is greatly increased by the expanded HSR. The HSR in this cell line was apparently translocated from its original position on chromosome 2. Genomic DNA fragments from this sorted fraction were cloned into A phage, and approximately 20% of the recombinant clones were shown by Southern blotting and in situ hybridization to derive from the

DNA SEQUENCE AMPLIFICATION

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HSR in the neuroblastoma. These workers have so far isolated approximately 40 kb of noncontiguous DNA from the amplicon, and one of these clones was found to be amplified in several other neuroblastoma cell lines. Genomic DNAs from all these cell lines, including IMR32, were then screened with a v-myc probe by Southern blotting procedures, and the probe was found to have weak homology to a 2.0 kb EcoRI restriction fragment in all cell lines. This 2.0 kb genomic DNA fragment was then cloned, and was shown to be amplified in these and several other neuroblastomas. Moreover, the EcoRI fragment was shown by somatic cell hybridization/Southern blotting techniques to map to chromosome 2, as did all the cloned probes obtained from the HSR in 1MR32 (Kohl et al., 1983). These studies present compelling evidence that independent neuroblastomas may arise by the amplification of similar cellular sequences that include genes related to v-myc. B. AMPLIFIED TRANSFECTED GENES In all cell lines in which transfected genes such as CAD, DHFR, or TK have been amplified, the unit of amplification is much larger than the gene itself. Milbrandt et al. (1983b) showed that the amplified DHFR gene was flanked by approximately 90 kb of additional (presumably genomic) DNA sequence in transfected MTX-resistant CHO cells. After amplification of the transfected CAD gene in CHO cells, de Saint Vincent et al. (1981) showed that flanking DNA was included in the amplicon, although it was not demonstrated how much additional DNA was amplified in this case. When DHFR minigenes are amplified after transfection into either mouse or Chinese hamster cells, it is also clear in most instances that DNA in addition to the cloned gene is amplified (Crouse et al., 1983; Kaufman and Sharp, 1982; Murray et al., 1983). This result was obtained in the case of minigenes regardless of whether or not carrier DNA was included in the CaPO, precipitation step. Thus, in some cases, the amplified extra DNA could represent sequences flanking the site of insertion into the genome, and in others, the extra sequences could come from carrier DNA that was ligated to the DHFR genes prior to integration into the chromosomes or aggregation into DMs. It therefore appears that a cloned amplifiable gene is probably not capable of amplifying itself in situ in the absence of other genomic sequences. These other sequences could contain origins of replication or repetitive sequence elements that promote high frequency recombination. In none of the above studies is it possible to study the nature of the flanking amplified DNA without actually cloning the entire amplicons in each case. Roberts and Axel (1982) have attempted to obviate this problem by inducing amplification of defined sequences. By utilizing a pool of 20 cloned human genomic DNA sequences as carrier for a chimaeric plasmid containing the APRT

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JOYCE L. HAMLIN ET AL.

gene and a promoter-less thymidine kinase gene, they transfected mouse APRT - / TK- L cells, and selected APRT+/TK- transformants. Each cell line had integrated the chimaeric plasmid along with variable numbers of the carrier clones. From these cell lines, they isolated TK+ revertants that had amplified the chimaeric plasmid and flanking DNA, and therefore overexpressed the partially functional TK gene. By utilizing radiolabeled plasmid (pBR322) DNA to probe digests of genomic DNA from these revertants, they were able to examine the arrangements of the chimaeric plasmid and the carrier clones in the amplicons (Roberts et al., 1983). They observed that the amplified DNA consisted of at least 20 repeating units that ranged in length from 40 to 200 kb, depending on the cell line. Furthermore, by examining cloned DNA fragments from the amplicons, they were able to show that the units were contiguous, and were joined to each other apparently by recombination between homologous repetitive elements in each recombinant clone (often between the pBR322 elements themselves). In a given cell line, the amplicons varied in size, as evidenced by the fact that some fragments are present in larger copy numbers than others. Their data suggest that the chimaeric plasmid integrated into a chromosome along with a concatamer of the carrier plasmids, and that amplification occurred as a result of multiple rounds of replication of this unit. Subsequent homologous recombination events then linked the extra units together and to the chromosome. No genomic DNA flanking the site of the original insertion event seems to be included in the amplicon. Roberts et al. suggest the interesting possibility that one of the carrier plasmids contains an origin of DNA synthesis that is responsible for amplification in this system.

V. Agents That Increase the Frequency of Amplification From the large number of amplified genetic loci that have so far been observed in mammalian cells, it would appear that almost any locus can be amplified, and suggests that it could be an unprovoked, random event. Howeve1 many of the agents used to select for amplification are drugs that interfere with DNA metabolism (e.g., MTX, and hydroxyurea), and could, in fact, directly interfere with DNA replication, recombination, or repair processes whose malfunctiw could be responsible for amplification. ,

A. AGENTSTHATINTERFEREWITH DNA METABOLISM Schimke and colleagues have tested a variety of agents for their ability to increase the initial rate of amplification of the DHFR gene in mouse 3T6 ai. CHO cells (Tlsty et al., 1982; Brown et al., 1983). The experimental protocoi involved pretreatment with hydroxyurea, cytosine arabinoside, or MTX (metabolic inhibitors of DNA synthesis), UV light or carcinogens (e.g., N-acetoxy-N-

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acetoylaminofluorene), and 12-0-tetradecanoyl-phorbol13-acetate (TPA, a tumor promoter). The latter agent was used alone or in combination with the other listed agents. Cells were exposed to the agent at several levels and for various time periods, and were then allowed a recovery time interval in the absence of agent. They were then challenged with various concentrations of the selective drug, MTX, and plating efficiencies were determined. Alternatively, in several experiments, DHFR enzyme levels in individual cells in the resulting populations were determined using the fluorescent MTX derivative and the fluorescenceactivated cell sorter. In some cases, the DNA of the resulting MTX-resistant cloned cell lines was analyzed by quantitive Southern blotting for DHFR gene copy number. The results of these experiments can be summarized as follows: (1) all of the above agents tested in pretreatment regimens caused marked enhancement of the frequency of MTX-resistant colonies subsequently selected (in some cases as much as 1000-fold); in fact, in recent studies, this group has been able to induce amplification in a majority of cells in the population (R. T. Schimke, personal communication); ( 2 ) in general, the effect of the pretreatment was greatest at high selective concentrations of MTX; (3) the length of the recovery time after pretreatment can determine the frequency of MTX-resistant colonies subsequently selected, implying that the inductive effect of the agent can be repaired in some cases; (4) pretreatment with the tumor promoter, TPA, by itself was not able to increase the frequency of occurrence of MTX-resistant colonies; however, in combination with UV light, hydroxyurea, or MTX itslef, TPA markedly increased the frequency of MTX-resistant colonies; (5) after all such treatments, many of the MTX-resistant colonies were shown not to have amplified the DHFR gene, and therefore must have sustained other mutations such as decreased transport of MTX or changes in MTX affinity in the enzyme itself; however, the proportion due to amplification per se increases when higher MTX concentrations were used during selection. As pointed out by Brown et al. (1983), there are several variables in these experiments that are difficult to adequately control (e.g., plating efficiencies due to pretreatment, metabolic coupling, clonal variation, etc.), but there appears to be no doubt that agents which inhibit DNA replication and/or damage DNA markedly enhance the frequency of DHFR gene amplification under these experimental conditions. These studies are supported by the observations of Lavi (198I ) on the carcinogen-mediated amplification of integrated SV40 virus in Chinese hamster embryo cells. After exposure to a variety of carcinogens, including 7,12-dimethylbenz(a)anthracene, a heterogeneous collection of extrachromosomal DNA fragments was observed, each of which contains part of, but usually not all of, the SV40 genome. She found that a functional origin of replication was required for amplification in this system, suggesting that the process is somehow mediated through the normal origin of the virus. Another possible example of induced amplification is the observation that N -

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JOYCE L. HAMLIN ET AL.

methyl-N’-nitro-N’-nitrosoguanidine provokes resistance to mycophenolic acid in Chinese hamster V79 cells by the overproduction of IMP dehydrogenase (Huberman e t a / ., 1981). While it is possible that a regulatory locus has been mutagenized in this instance, causing uncontrolled or constitutive expression of this gene, it is also possible that the IMP dyhydrogenase gene has been amplified. Hanawalt (1982) has suggested a variety of routes by which agents that interfere with DNA metabolism might provoke or enhance amplification. DNA damage in bacteria stimulates daughter strand gap repair and the SOS repair system (Hanawalt et a / ., 1979). Both systems are error prone, and could cause mutations leading to loss of negative control over chromosomal origins of DNA synthesis. This could result in extra rounds of DNA synthesis (amplification) at a single locus, and would guarantee that unscheduled synthesis would occur over and over again at the same locus. SOS repair in bacteria has also been shown to short-circuit the usual DNA synthetic control mechanisms by causing reinitiation at the legitimate chromosomal origin of DNA synthesis, apparently without actually mutating the origin (Kogoma and Lark, 1975). Tatsumi and Straws (1979) have also shown that SOS repair allows initiation of replication at sequences other than the legitimate origin, resulting in extra rounds of DNA synthesis. In addition, thymidine starvation, as would be induced by MTX (acting on DHFR) or 5-fluorodeoxyuridine (acting on thymidylate synthetase), leads to the accumulation of breaks in DNA (Barclay et al., 1981). Thymidine starvation can also lead to the incorporation of dUTP into DNA, whose subsequent repair often leads to errors and mutation (Hanawalt, 1982). Hence, many of the agents used to select for amplification can actually function as mutagens that lead to loss of control through mutation or that induce unscheduled replication at legitimate or illegitimate sites in chromosomes. However, a host of selective agents have no obvious involvement in DNA metabolism, and probably do not by themselves cause or abet amplification. Among these agents are compactin (for HMG CoA reductase), vincristine (microtubular proteins), and cadmium (metallothionein). However, it could be argued that the intricate control mechanisms that coordinate DNA synthesis with mitosis, doubling of cell mass (including membrane structures), and eventual cell division can be interrupted at several points with a consequent imbalance of DNA synthesis itself. Hence, agents interfering with any of these pathways could enhance the frequency of amplification. Viral integration must also be included in the list of insults that could provoke amplification either directly or indirectly, since several tumor cell lines that display HSRs and DMs are transformed with viruses (see Barker, 1982). It is not known whether the presumptive amplification is made possible by a cellular metabolic change induced by the virus (i.e., transformation), or whether the integrated viral DNA is directly responsible for, and becomes a part of, the amplified unit.

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B. GROWTH-PROMOTING SUBSTANCES The synergistic role of TPA in the studies of Brown et al. (1983) is unclear, since its action is thought to be at the cell surface as a mitogenic agent. Varshavsky (1981a) has shown that TPA and related nontoxic phorbol ester tumor promoters are all able to increase the frequency of MTX-resistant 3T6 colonies selected in single-step drug treatments by approximately 100-fold when the phorbol ester is present at optimal concentration at the time of MTX selection. However, TPA analogs that are inactive as tumor promoters (e.g., phorbol or phorbol- 12,13,20-triacetate) do not enhance the frequency of MTX-resistant colonies surviving at any selective concentration of MTX. In most colonies, resistance was shown to be due to DHFR gene amplification. Most surprisingly, the hormones insulin, epidermal growth factor, and arginine vasopressin, all of which are mitogenic for 3T6 cells, act in a manner similar to TPA (Barsoum and Varshavsky, 1983). In addition, the effects of TPA and insulin on increasing the frequency of DHFR gene amplification were approximately additive when used together. Varshavsky points out that the mitogenic potential of TPA and hormones could enhance the colony-forming ability of clones that have amplified the DHFR gene, but which would not survive without the mitogen. Another interpretation is that these mitogenic agents put a larger percentage of the population in a metabolic state that is required for amplification (e.g., the DNA synthetic period), implying that replicon misfiring (Varshavsky, 198 1b) or other aberrant DNA replication is responsible for gene amplification. TPA has also been shown to induce the expression and replication (amplification?) of bovine papilloma virus in mouse cells that normally harbor the virus in a nonproductive, nonreplicating state (Amtmann and Sauer, 1982). Whether its action in this system is related to the synergistic effects of TPA described above is not clear. This result could be interpreted to mean that TPA creates a cellular ambience that favors replication of the virus or unscheduled replication of chromosomal origins of DNA synthesis. Alternatively, it may directly affect controlling elements that interact with the virus to suppress or induce its expression andlor replication.

VI. Proposed Mechanisms of Sequence Amplification Is it possible, at this juncture, to fit the largely phenomenological observations that have been reviewed here into any coherent model for the mechanism(s) involved in gene amplification in mammalian cells? Since there seem to be major differences in the modes andlor manifestations of amplification between cell types, and sometimes between different loci in the same cell, there clearly exist variations of any central mechanism. But we will attempt to outline certain plausible models that may be discounted or supported by future experiments.

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JOYCE L. HAMLIN ET A L

The data to be reconciled can be summarized as follows. Mammalian cells can amplify DNA sequences many hundreds of times, possibly at random positions within the genome. The amplified units (amplicons) that have been examined are almost always very large; in cases of drug resistance, the amplicon is much larger than the gene that confers resistance on the cell. The amplicons are apparently arranged in tandem, linear arrays. Depending upon the cell type and the particular locus in question, the multiple copies can be integrated stably into preexisting chromosomes as HSRs (often at the location of the parental gene), or they can exist as extrachromosomal acentromeric double minutes. In some cell lines, the multiple amplicons appear to be able to shuttle between these alternate configurations. Since the net result of amplification is that a particular locus is now present in a supernumerary amount relative to other loci in the same cell, the mechanism must involve either multiple replications of that locus during the S period, or some other mechanism related to recombination that increases the copy number of a given locus relative to the rest of the genome. Possibly gene amplification is a combination of both processes. A. UNEQUAL SISTERCHROMATID EXCHANGE One suggested model is unequal exchange between sister chromatids, with the result that one chromatid obtains two copies of a sequence for which the other chromatid is now deleted. In order for the initial duplication to occur (according to current concepts of recombination in mammalian cells), homologous DNA sequences have to flank the locus in question (A in Fig. 3) in order to provide a basis for pairing; recombination occurs outside the locus itself. If A includes sequences coding for a selectable gene, then only the cell receiving two copies of A will survive, and the other daughter cell will be killed by drug selection. Once duplication occurs, it is now possible for recombination to occur again, either at the original elements that provided homology (X in Fig. 3), or within the sequence A itself, again by a staggered mispairing mechanism. As the number of amplifications increases, recombination should become more likely, since there will be more possibilities for mispairing. It would also be possible to generate collections of amplicons of different size in a single cell if it is further assumed that staggered recombination can occur in regions both within and flanking the core amplicon, A, at highly repeated elements that are dispersed at different positions throughout the region. Amplification would provide a collection of repeated units that would not have precisely defined boundaries, due to recombination occurring at slightly different positions each time, but a core element (A) should be observed that would usually include the gene itself (which is selected). Many different kinds of junction fragments could be formed by a model of this kind, but it might be expected that at least some vestige of the

DNA SEQUENCE AMPLIFICATION

I

* 3

=&r ,

x x xA x x x x

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69

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FIG. 3 . Unequal sister chromatid exchange. The locus A contains a gene or genes (possibly selectable), and is flanked by repetitive elements (x) that could be ribosomal, satellite, or other tandemly repeated sequences. An unequal, homologous recombination event occurs between two x elements situated on either side of A in the two chromatids (as indicated in 1 above). After mitosis and another round of DNA synthesis, one cell is deleted for A on this chromosome, and the other cell now has two copies of A on this chromosome. If A itself contains dispersed repetitive elements (y), as indicated in 1 , then unequal exchanges can occur within A itself, leading to amplicons of variable length and constitution.

repeated dispersed elements that form the basis for recombination might be maintained in each junction fragment. Another prediction is that some highly repeated elements should flank the original locus in the parental cell which has only one copy of the amplicon per chromosome. A further prediction of this model is that the frequency of sister chromatid exchange in HSRs should be higher than in other chromosomal regions, owing to the greater opportunities for mispairing in a tandem array of repeated amplicons. In addition, agents that provoke recombination and sister chromatid exchange, such as those that damage DNA, might be expected to increase the frequency of amplification events. According to this scheme, double minutes would involve internal recombination between adjacent amplicons on the same Chromatid, releasing circular elements made up of monomers or higher multiples of the amplicon arranged in tandem. The differences between cell lines and between loci in a single cell line in their propensity to form either DMs or HSRs would have to be explained by subtle differences related to intrachromatid recombination, and could depend on the nature of the locus itself with respect to the distribution of the recom-

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binogenic sequence elements. It might be expected that certain loci would have a greater likelihood to be amplified than others which are not flanked on both sides by the proper elements. In order to explain the stable fixation of genes as HSRs after prolonged culture of cell lines that originally contained DMs, tandemly arranged amplicons would have to integrate in such a way that the resulting sequences had little propensity for the intrastrand recombination events that are suggested to lead to extrachromosomal DMs. At the present time, there are experimental observations that support this model and those that conflict with it. The genome of mammalian cells is peppered with hundreds of thousands of simple sequence elements (e.g., Alu and Ah-like sequences, satellite DNAs, etc.) that are dispersed both within and outside of genes (Jelinek and Schmid, 1982). Clearly, elements of this kind could provide the requisite homologies for the recombination events suggested, provided that they are positioned appropriately with respect to the amplicon. Recombination between highly repeated satellite DNAs apparently occurs quite often in mammalian cells, and Bostock and Clark (1980) have shown that satellite DNAs form a major part of the DHFR amplicon in the MTX-resistant murine melanoma, PG19T3. In addition, the karyotype of these cells is very unstable, with the multiple copies shuttling frequently into the chromosomes as HSRs and out as DMs. Bostock and Clark have proposed that recombination between DMs at homologous sequences, with subsequent integration by recombination into chromosomes, could account for the formation of HSRs. A disproportionation by intrastrand recombination would then reverse the process and generate DMs that would presumably be variable in size, depending upon the number of amplicons between the two sites of recombination. It is difficult to see how this process could lead to the multiple, uniformly sized DMs that are observed in a single cell in most other systems, however, unless it is assumed that in these other cases, the original excision event occurred early in the amplification process, and multiple DMs of similar size are generated by the DNA synthetic process in a given S period. Another important observation is that ribosomal DNA, a highly repeated element in mammalian cells, is included in the CAD amplicon in Syrian hamster cells (Wahl et al., 1983), and Wahl and co-workers have suggested that rDNA cistrons flanking the CAD locus could be responsible for the original duplication and subsequent amplification of this gene by the process outlined above. Support for this concept derives from studies in bacteria, in which duplications of DNA sequences flanked by rDNA genes are very frequent occurrences (Anderson and Roth, 1977). In addition, in rat hepatoma (Tantravahi et al., 1981), sarcoma (Tantravahi et al., 1982), and erythroleukemia (Murao et al., 1982) cell lines, the rDNA genes have been shown to be amplified, and the HSRs containing these genes are often located on chromosomes that contain nucleolar organizer regions in parental rat cells. In these systems, it is not known what other sequences are

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contained in the amplicon, but it is clear that the repetitive rDNA elements themselves could be providing the basis for unequal recombination. Furthermore, Henderson and Mcgraw-Ripley (1 982) have pointed out that in many human neoplasms, rDNA genes are frequently involved in translocations and other recombinational rearrangements, and are often amplified, suggesting that the repetitive nature of these sequences aids recombination and amplification. Chasin et al. (1982) have addressed the question whether HSRs display a higher frequency of sister chromatid exchanges, as would be predicted by the recombination model for gene amplification. By utilizing the BUdR/Hoescht 33258 method devised by Latt (1974) to illuminate sister chromatid exchanges in MTX-resistant CHO cells, they found that the frequency within the HSR was not greater than in other chromosomal regions, and, indeed, was somewhat depressed. These data therefore do not support one prediction of the recombination model. B. REREPLICATION The model that has received the most attention in recent years states that a given DNA sequence can undergo multiple rounds of DNA synthesis prior to mitosis (Schimke, 1982; Varshavsky, 1981b; Hamlin et al., 1983). This could occur randomly at a low frequency at any replicon, due to leakiness in the control mechanism that normally prevents reinitiation. Alternatively, a mutation at an origin could allow reinitiation, and would, of course, perpetuate the property in that particular replicon, facilitating further amplifications. Agents that interfere with DNA synthesis or that damage DNA could increase the frequency of this kind of mutation. Alternatively, a variety of agents could provoke reinitiation by somehow transiently affecting the structure of the origin. If mammalian chromosomal replicons are defined by fixed origins and termini, then this model predicts that the unit of amplification may be equivalent to the domain of a chromosomal replicon. The amplification mechanism could be relatively precise and could therefore generate uniformly sized amplicons (both within a given cell and between different cells derived from the same parent). However, it is also possible that the replication forks could terminate at different positions within or outside of the replicon during each amplification event, generating a collection of amplicons whose center is the same, but whose size is different. In the latter case, each amplicon could have more than one origin of DNA synthesis. It is also possible that uncontrolled DNA synthesis could initiate at sites not usually utilized as origins, especially if the cellular DNA is damaged by agents that generate single-stranded breaks, etc. A very heterogeneous collection of amplicons would be expected by this mechanism, since neither the origin nor the termini would be fixed at any sequence.

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Regardless of the mode of rereplication of a given sequence, some mechanism must operate to join the extra DNA copies into tandem arrays (either as HSRs or as DMs), else the onion skin arrays characteristic of chorion gene amplification would result. Thus, some form of recombination must operate in any scheme that invokes multiple rounds of DNA synthesis as the inductive event. In one variation, repeated initiations at one origin would generate side-by-side duplexes with only the parental strands actually covalently integrated into the chromosomal DNA fiber as a whole (Fig. 4A). The nonintegrated supernumerary duplexes would be free to ligate end-to-end, either with themselves to form circles, or with other duplexes to form tandem (and possibly circular) arrays that could be released as DMs. HSRs could be formed by the integration of these single or tandem structures into either of the duplexes containing a parental strand. Alternatively, HSRs could be the result of recombination of the DMs at some later time into sites close to or distant from the original replicon. A mechanism for the joining of supernumerary copies of the amplicon to form tandem repeats (either chromosomal or extrachromosomal) is suggested by the studies of Bullock and Botchan (1982). They have examined the amplification and excision of integrated SV40 viral genomes in transformed rodent cells after treatment with mitomycin C or fusion with permissive monkey cells. In this system, an increase in the viral copy number in high-molecular-weight DNA precedes the appearance of free viral forms. In addition, excision and amplification require a functional T antigen. They therefore conclude that amplification is dependent on replication. The extrachromosomal products recovered are closed circles that are apparently formed by the pairing and recombination of two short homologous sequences, one present within the virus and one present in flanking cellular DNA. From one clonal cell line with a single integrated copy of the virus, many different excised copies can result in which different short homologies are utilized. However, from cell lines with tandemly duplicated viral inserts, a homogeneous collection of unit length autonomous forms results, apparently because in this circumstance, the exact and extensive (4 kb) homology between the two tandem viral copies overrides the short homologies used by single copy inserts. Bullock and Botchan propose that the single-stranded regions generated at replication forks during onion skin rereplication facilitate the pairing of the two homologous sequences that straddle the origin. Depending upon whether recombination occurs between sequences on the same or on different duplexes, the products are released from the chromosome as autonomous elements, or result in the in situ tandem duplication of the region contained between the two regions of homology (see Fig. 4A). They further propose that mitomycin C or fusion with permissive cells induces or activates enzymes that are used for viral replication, which is the triggering event in this model. Whether the mechanism of induced replication of SV40 is related to that responsible for amplification of chromosomal DNA sequences remains to be

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A

-0-

T

T

0

0

FIG.4. Proposed mechanisms for the formation of tandem repeats or extrachromosomal elements after rereplication. (A) If a replicon is reduplicated prior to mitosis, an onion skin structure could result, in which two of the four daughter duplexes are not covalently attached to the original chromosomal DNA fibers. Recombination events could occur between homologous elements that happen to flank the origin of replication, as indicated by X in the diagram. Recombinations between two elements on either side of the origin would result in a closed circle if the elements were on the same duplex, or in a tandem duplication if the elements were on different duplexes. In the latter case. integration into the original chromosomal fiber occurs if one of the duplexes contains a parental strand. (B) If DNA is arranged in a series of loops affixed to a nuclear matrix, each one of which represents a replicon, then rereplication during a single S period could result in the structure pictured above. Three adjacent replicons are shown, only one of which has undergone two cycles of replication. This structure is analogous to the one pictured in A, except that the termini indicated by the arrows in A are juxtaposed in B. Again, recombination is proposed to occur between the homologous elements X, on the same or on different duplexes.

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seen. It is difficult to imagine how the chromosomal amplicons, which are usually hundreds of kilobases long, could arise by this mechanism, since it would be expected that at least some of the time the two short homologous sequences required would flank the origin more closely. However, the experiments with SV40 do illuminate the kinds of operations that cells are capable of performing on DNA, and variations of this mechanism could be involved in the amplification of chromosomal sequences other than viruses. Current ideas about the physical arrangement of DNA in the mammalian nucleus provide another suggestion for the formation of tandem arrays of replicated sequences that could account for the large size of most amplicons. A variety of microscopic and biochemical evidence has demonstrated that chromosomal DNA is arranged in loops attached to a subnuclear, proteinaceous scaffold or matrix (Worcel and Benyajati, 1979; Cook et al., 1976), and it has been proposed that replication occurs by feeding the DNA loops through a replication complex attached to the matrix (Pardoll et al., 1980). Once a replicon has been synthesized, the two daughter loops could end up with their four termini juxtaposed as in Fig. 4 9 , awaiting forks from adjacent replicons to approach in order to complete a complex resolution event (possibly involving topoisomerase) that unwinds any super coils ahead of the replication forks and fuses adjacent replicons. An occasional aberrant recombination event at the termini could then generate a head-to-tail tandem integration of the two DNA loops into one continuous strand, or could release a monomeric circle of the replicon. By this model, amplicons are formally equivalent to replicons, and would be of relatively uniform size in a given cell. It is also possible that recombination could occur at multiple sites located in the general vicinity of the replicon termini, generating amplicons of similar size, but terminating at slightly different positions. Furthermore, it could be imagined that while rereplication would increase the likelihood of this event by providing more than four termini simultaneously, it would not necessarily be required by this model. DNA damaging agents could increase the frequency of incorrect resolution or rereplication, or both. The DMs and HSRs generated by the process of rereplication could then be synthesized by the usual mode, utilizing the legitimate origin in bidirectional replication. Alternatively, the proposed circular form of tandem repeats (DMs) could replicate by the rolling circle mechanism observed in the amplification of extrachromosomal rDNA copies in Xenopus (Hourcade et al., 1973; Rochaix et al., 1974; Buongiorno-Nardelli et al., 1976). The data compatible with rereplication models come from several indirect experiments, none of which absolutely distinguishes between rereplication and recombination. The amplicons that have been analyzed so far all seem to be large, and could all be within the range of mammalian chromosomal replicons. However, since no amplicon has been cloned in its entirety, they may turn out to be much larger than replicons, by arguments discussed previously.

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Other circumstantial evidence that implicates DNA replication in the process of amplification derives from studies on the pattern of synthesis of amplicons. The HSRs in three MTX-resistant Chinese hamster cell lines, as well as in a stable derivative of murine S 180 cells, have been observed to initiate replication at multiple loci along their length in early S, and to complete replication by the mid-S period (Hamlin and Biedler, 198 1; Milbrandt et a / ., 198 1; Kellems et al., 1982). In the murine and CHO cell lines that bear transfected, amplified genes as HSRs, multiple initiations within the HSR in early S have also been observed (Kaufman et al., 1983; Hamlin, unpublished observations). In the latter cases, it might be assumed that the cloned gene was introduced into a chromosomal replicon, and that subsequent amplification occurred via the origin of that replicon. These results imply that there is at least one origin of DNA synthesis per repeated unit, as would be expected if legitimate origins were involved in the amplification process. It has also been shown that there is probably only one origin per DHFR amplicon in a MTX-resistant CHO cell line (Hamlin et al., 1983), which lends weight to the argument that an amplicon is equivalent to a parental replicon. As discussed earlier, Schimke and colleagues have demonstrated that rereplication can be induced in a population by a variety of drugs that interfere with DNA synthesis (Tlsty et al., 1982; Brown et af., 1983). However,the amplifications observed in this experimental situation need not be the primary mechanism that accounts for all amplifications, particularly those observed in the absence of obvious insults and/or selections (e.g., neuroblastomas, carcinomas, etc.). The other point worth making is that there may be no single mechanism by which all amplifications can be explained. Certain chromosomal regions that contain repetitive elements such as satellite or rDNA may undergo a few rounds of unequal sister chromatid exchange that establishes an unstable condition in the nucleus. The extra DNA may not be able to affix itself properly to the matrix, encouraging aberrant resolution events during subsequent DNA synthesis that lead to further amplifications. Alternatively, infrequent rereplication that produces tandem repetitions may stimulate subsequent unequal exchange. Even more complex models can be invoked in which both processes (rereplication/insertion and unequal exchange) occur continuously during the entire amplification process, in order to explain the complex sequence arrangements observed in some systems (e.g., Ardeshir et a l . , 1983).

V11. Concluding Remarks It is clear that much remains to be learned about DNA amplification in mammalian cells. One of the most promising areas of investigation is the analysis of sequences contained in amplicons. When it becomes possible to isolate entire

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amplicons in recombinant clones, as well as the parental domain from which the amplicon was derived, much more will be learned about the mechanisms responsible for this interesting genetic phenomenon. Analysis of junction fragments between repeated units will be particularly illuminating, since they may prove to contain the highly repetitive elements responsible for staggered recombination events. Alternatively, it may be possible to show that the ends of amplicons are equivalent to the termini of replicons. Another exciting area of investigation is the amplification of transfected genes, which locate themselves in new and apparently random chromosomal environments. It may be possible to find homologous sequence elements between these heterogeneous amplicon types that are required for the process itself. A most rewarding consequence of this rapidly expanding area of investigation is that whole new fields of vision have been opened. An understanding of the mechanism of gene amplification will necessarily tell us much about the physical and functional organization of DNA in chromosomes, and the complex processes of DNA synthesis and recombination in mammalian cells. The field of cytogenics will undoubtedly be aided as well by an understanding of the types of sequence that generate particular staining properties to chromatin. The study of gene regulation, which is a difficult endeavor in mammalian cells, may be aided by a kind of pseudogenetic approach in which a nonselectable gene can be cotransfected with an amplifiable gene such as DHFR or CAD, and the two genes can be coamplified by drug selection. The mode of regulation of the passenger gene (or lack of it) should shed light on the nature of the other elements involved in normal gene expression. In addition, the overproduction of virtually any protein for which the gene can be cloned will be allowed by this approach. This will be an important development for the purification of scarce biological peptides whose synthesis involves complex processing steps that could not be engineered in bacteria. Finally, the discovery that cellular counterparts of viral oncogenes can be amplified in some human tumors is a major advance in our understanding of the genesis 01cancer. The important work on the inductive effects of certain agents (including anticancer drugs) on amplification will modify our current drug treatment protocols toward more rational directions.

ACKNOWLEDGMENTS We would like to thank the many colleagues who sent us manuscripts prior to their publication. We would also like to thank Melinda Mills for her expert assistance in the preparation of the manuscript. Work in the authors’ laboratory was supported by grants from the NIH and The March of Dimes. J.D.M., N.H H . , and J.C.A. were supported by NIH postdoctoral fellowships, and J.L.H. was the recipient of an American Cancer Society Faculty Research Award.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL 90

Computer Applications in Cell and Neurobiology : A Review R. RANNEY MIZE Department of Anatomy and Division of Neuroscience, University of Tennessee Center for the Health Sciences, Memphis, Tennessee

I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. The Microcomputer in the Research Laboratory . . . . . . . . . . . . . . . . . 111. Computer Systems for Microscope Control and Plotting . . . . . . . . . .

IV . V. VI. VII. VIII. IX. X.

Serial Section Reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Computer-Aided Morphometric Measurement . . . . . . . . . . . . . . . . . . . Video Image Processing and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . Computer Uses in Photometry and Fluorescence Microscopy . . . . . . Computer-Automated Autoradiography and Immunocytochemistry . . Other Cell Biology Computer Applications . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

83 84 90 93 98 I03 107 111

117 117 119

I. Introduction Computer-aided quantitative analysis has come of age in cell and neurobiology . The development of large scale integrated circuits (LSI) and metal oxide semiconductors (MOS) has literally revolutionized our scientific lives. Inexpensive microprocessors and microcomputers based on these technologies are assisting cell and neurobiologists in almost every scientific activity, from acquiring and analyzing data and controlling instrumentation to writing manuscripts, searching the research literature, and ordering laboratory supplies. The reduction in price of computer hardware, particularly small personal computers like Apple and TRS-80, makes a laboratory computer accessible to almost everyone. In preparing for a recent presentation on microcomputer applications in cell biology for the American Association of Anatomists (Mize, 1983e), it became apparent that there were very few review articles describing the uses of microcomputers in cell and neurobiology research. A number of small noncommercial computer systems have been developed for particular applications in these fields, but I found them difficult to locate because they were published in many different journals. Commercial systems were sometimes also difficult to locate. This review attempts to bring this widely dispersed literature together in an integrated 83 Copyrighl 0 1984 by Academic Pres\, Inc. All nphts of rrproduclion in any h r m reserved ISBN 0-12-364490-Y

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format. The review is divided into application sections describing computer uses in microscope control and plotting, serial section reconstruction, computer-aided morphometric measurement, video image processing and analysis, photometry and fluorescence microscopy, autoradiography and immunocytochemistry, and other cell biology computer applications. These sections are preceded by a section describing microcomputer hardware and software which should prove useful to laboratories contemplating the purchase of a microcomputer. In this review, I have largely restricted my discussion to technical papers which provide detailed descriptions of hardware and software and their application to particular research problems. Although these papers may also include research results, their primary emphasis is on the application rather than the findings of the research. This necessarily excludes a large number of research articles which utilize computer analysis but are principally research reports not concerned with the computer methodology. Although I have included main frame and minicomputer systems in the review, the emphasis is placed upon recently developed systems that use smaller, inexpensive microcomputers or single board microprocessors. Specific mention of computer hardware is included to aid those with a given computer product to locate software developed for those systems.

11. The Microcomputer in the Research Laboratory Two approaches to laboratory computer automation have evolved over the last decade (Doerr, 1978; Enke, 1982; Shipton, 1979). The first approach uses dedicated microprocessors to automate single laboratory functions such as control of analytical instruments or calculation of mathematical functions. The second approach uses more versatile minicomputers or microcomputers which can handle a wide variety of laboratory tasks. Dedicated vs flexible is the key distinction to be made between the microprocessor and microcomputer approach (Enke, 1982). Microprocessor systems are single board central processing units (CPUs) with minimal memory whose logic or programmed steps are usually hardwired into the machine (Doerr, 1978). Microcomputers include large memories, high-level languages, input-output controls, and a wide range of peripherals which make them highly flexible, general purpose instruments. There are advantages and disadvantages to each approach. Microprocessors are ideal for dedicated control of single functions because they are fast and cheap. Many manufacturers incorporate microprocessors into laboratory instruments for control of specific device functions such as sampling a voltage level, controlling a power supply, or readout of data. Microprocessors are also often used to perform repetitive mathematical functions which reduce data to intermediate results. Their principal advantage in data acquisition and

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instrument control is speed. Analytical instruments which sample at high rates are thus well served by microprocessors. Instruments which perform lengthy calculations like Fourier transforms, integrations, peak detections, and linear to log conversions also effectively utilize the high speed of the microprocessor. The dedicated microprocessor nevertheless has several disadvantages in a laboratory environment. It must be programmed in machine language unless special program development devices are available. Programming in machine code is difficult and time consuming for the scientist. Machine codes are microprocessor specific so that the codes cannot easily be transported to other machines. Microprocessors often require the expertise of biomedical instrumentation and electronics shops. These are not always available at smaller institutions and their services can be quite expensive. In short, microprocessors are complicated devices which many scientists find difficult to understand. They often lack flexibility in programming and interfacing to peripherals. They are what the computer industry calls “unfriendly” devices. Microcomputers, by contrast, are friendly and have tremendous flexibility. They can be used as general purpose instruments in a variety of tasks. A microcomputer is a microprocessor-based CPU (central processing unit) to which is added read only memory (ROM) and random access memory (RAM), an input multiplexer, address and data buses, buffers for temporary data storage, a clock for timing functions, and control logic (Fig. 1) (Shapiro et al., 1976). Standard peripherals include a CRT display, keyboard, and some form of mass storage, usually a flexible (floppy) or hard disk. An operating system manages memory allocation and mass storage. One or more high-level languages are resident in ROM or software. Various interface cards are available for communication with external devices. A number of software packages for statistical analysis, graphics, terminal emulation, word processing, data base management, and numerical analysis are usually also available at a modest additional cost. These features offer numerous advantages to the scientist. Input-output control of peripheral devices is handled “automatically” by the microcomputer. A few elementary high level language commands conveniently drive printers, plotters, and graphics devices. Keyboard input, CRT display, memory allocation, and mass storage control are managed automatically by the microcomputer. Housekeeping operations such as file maintenance and program editing are handled using the computer’s operating system. High level languages are another major advantage of microcomputers. Most computer systems offer several interpretive or compiled languages, including BASIC, FORTRAN, PASCAL, and C. An ASSEMBLY language is often available as well. There is thus no need for the scientist to master machine or object code. Program development is much faster using high level languages. Five times fewer lines of code are required compared to ASSEMBLY (Brooks, 1975). Sophisticated editors and program debugging routines further simplify program-

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Pi(, I . Rloch tli;ipr:iiii 0 1 the coiiiponeiit\ of an lritcl XOXO-based iiiicroconiptiter, including tlit' niicroptocehsor, real-tinic clock. input iiiiiltiplcxcr. arid address and data buws with as%ici;ited htilt'er~.RAM iiiitl KOM iiiciiiory. control logic. and an adtlress decoder lor peripheral intcrfacri art' also showii (Modii'icd fro111 Shapiro P I d / ., 1976. by periiiisbiori of Aiiirtrtr/ R e i ~ t w . \ , Palo Alto. c;lll~~~l-r~l~l.~

niing. High level languugc progrnmming is oltcn less cxpcnsivc since students willing to work for $4- 10 a n hour ol'ten know several o f these languages. There nre other vdvuntages to high level language programniing. Programs are easier to unclcrxtand and modify because high level languages require iiiore structure than do machine o r asscmbly cotles. High level l a n g i q e s are rnore easily transported from machine to machine, so programs can bc tnunsterred to other laboratory sltec. The most serious clisadvantage of the niicrocomputer is its slow data ncquisition spccd. Compiled languages such as FORTRAN may limit acquisition rates to 5 kl-lz. Interpretive BASICS may slow acquisition to SO Hz (Saiin. 19821). The slow exccution tiines of these languages can be partially overcome by writing instrument control and data acquisition subroutines in ASSEMBLY. Many microcomputers allow insertion of ASSEMBLY subroutines which high

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level language programs can access. Programmable interrupts can speed data acquisition by allowing the computer to be interrupted by the peripheral when data are ready. Data collection speed can also be increased by using direct memory access (DMA) IiO. Using this mode, binary data can be collected from instruments and stored directly in memory at rates up to several hundred kHz, bypassing CPU control. The data can be converted, reduced, and operated on by the CPU when the acquisition process is complete. High speed data acquisition is thus quite feasible using microcomputers if special programming techniques are employed. There are a vast number of microcomputer systems available on the market today and the choice of a computer is often a difficult and bewildering process for the researcher. Some of the most popular microcomputers available today are listed in Table I . The table lists the computer’s CPU, memory options, the inputoutput ports available, the graphics resolution, operating system, and available languages. Although the list is not complete, it should nevertheless serve as a useful guide for potential microcomputer owners. A typical microcomputer system used in our laboratory is shown in Fig. 2. This Hewlett-Packard 9845T desk-top microcomputer has dual 16-bit NMOS-I1 processors, 187 kbytes of core memory, a medium resolution graphics CRT, built-in tape cartridge drives for mass storage, and a thermal line printer. The computer is interfaced to a digital plotter, an 8-in. flexible disk storage device, and a high-resolution digitizer (Fig. 2 ) . This computer was one of the earliest “micros” produced for use by scientists. We chose the computer for its bit-map graphics capabilities, ease of programming in BASIC, portability, and availability of peripherals, particularly the digitizing tablet. Although comparatively expensive by today’s standards, it has many of the desirable features of less expensive microcomputers available on the market now (Table I). One of the major advantages of the system for our purposes was the enhanced BASIC language. We initially had no expert programmers in our group and the BASIC language was easy to learn, simple to debug, and combined some powerful features of other high level languages. The H-P enhanced BASIC includes multicharacter variable names, I/O and graphics command sets, subprogram capabilities, labeled common, prioritized intempts, character string manipulation, and 6 dimensional numeric and string arrays. PASCAL-like statements provide a structured programming environment. Software can be written as independent modules using callable subprograms which are linked to the main program, similar to FORTRAN. The only major disadvantage of enhanced BASIC is that it is an interpretive language and thus executes quite slowly because the interpreter must translate each statement into machine code during program execution. Compiled languages execute far more rapidly because they are translated to machine code

SELtClEU -

~~

System

TABLE I 8 A N D 16 BIT MICROCOMPUTERS I-OK LABORATORY USE

CPU

Min RAM

Max RAM

1/0port\ dvalhhk

Graphics rewlution

Parallel RS-232

280 X 192 12 in. 640 x 240 12 in.

Applc 11E

6502

64K

I28K

TRS-SO Mudel 12

ZXOA

XOK

128K

Parallel

RS-232

H-P 85B

HP X hit or Z80A

32K

544K

ADVANTAGE

%XOA/HOXX

fJ4K

2S6K

128K

7.4M

8/16

H-P 16

DEC Rainbow

M6XOGil

1CK)

IBM PC XT

UEC PRO 325-350

TRS-80 Mvdel I6

ZXOA/808X

h4K

256K

8088

64K

64OK

F-I 1 (I I/23)

M68000i

64K

5 12K

64K

5 12K

128K

1M

Z80A

IBM INST

cs-9ooo

M6X000

192 X 256 I’arallcl ICBE-~X~ 5 in. RS-232 BCD Parallel 640 X 240 RS-232 12 in.

Parallel RS-232C IEEE-488 BCD RS-423 RS-232 Parallel KS-232

300 x 400 9 in.

Parallel

960

RS-232 IEEE-488 RS-423 Parallel RS-232 Parallel RS-232 1liEE-4XX

800 X 240 12 in. 300 X 400 12 in. X 140 12 in.

Operating systcin

Manufacturcr

BASIC PASCAL. FORTRAN C BASIC BASIC BASIC 1-ORTRAN PASCAI.

Apple Coinpuler, Cupertino, CA Tandy Corp. Radio Shack, Ft. Worth, TX

MS-DOS CPIM-80 GRAPHICS-DOS HP-BASIC CPiM-68K

BASIC- I6 FORTRAN-16 PASCAL- 16

North Star Computer, San Leandro, CA

BASK

Hewletl-Packard, Loveland, CO

CPIM-86/80 MS-DOS IBM-DOS CPlM 86 UCSD-P POS

M BASIC

CPIM-80 DOS TRS-DOS CP/M-BO H-P BASIC CPIM-SO UCSD-p

(RSX-11) UCSD-p

640 x 240 12 in. 768 x 4x0 12 in.

Languages availablc

TRS-DOS

MlJLTITASKlNG RT 0.5

P A X AI HPI.

~

c

BASIC

FORTRAN PASCAL BASIC PASCAL C BASIC FORTRAN BASIC PASCAL FORTRAN

Hcwlett-Packard, Loveland. CO

Digital Equipment, Maynard, MA IBM, Armonk, NY

Digital Quipmenr, Maynard. MA

Tandy Corp, Radio Shack, Ft. Worth, TX IBM, Arinonk, NY

COMPUTER APPLICATIONS IN NEUROBIOLOGY

89

FIG. 2. Microcomputer system. The niicrocomputer (Hewlett-Packard 9845T) includes a 560 X 455 bit-map graphics CRT (A). a thermal line printer (B), and two magnetic tape cartridge mass storage drives (C). The computer is interfaced to a digitizer (D, H-P Model 9874A). a 4-color digital plotter (E, H-P Model 9872A). and a dual-density, double sided floppy disk drive (F, H-P Model 9895A). (From Street and Mize, 1983, by permission of Elsevier Biomedical Press, Amsterdam.)

before the program is executed. The most popular compiled languages for scientific use are FORTRAN, PASCAL, and C. These languages are now usually available on most microcomputers (Table I). Software availability was also an important concern when we chose our microcomputer system. We found Hewlett-Packard software available for many laboratory functions. For example, we use Hewlett-Packard statistical packages for statistical analysis, editing, display, data formatting, and printing of our data. The statistical analysis software includes data editing, summary statistics, analysis of variance, linear and nonlinear regression, distribution analyses, and other parametric and nonparametric tests. We use a statistical graphics package for plotting data on the graphics CRT or digital plotter. Time interval plots, histograms, log-log plots, scattergrams, and 3-D plots are available using this package. Various ‘‘office’’-oriented software makes our microcomputer of great value in laboratory management and manuscript writing. We use a word processing package to prepare manuscripts; a terniinal emulator package for asynchronous communication with the university mainframe computer; data base management

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R. RANNEY MIZE

software to enter, sort, search, and retrieve reprints; and an electronic spreadsheet program to manage laboratory purchases and expenditures. The 9845T is used to run graphics presentation software for preparation of figures, slides, and posters for scientific meetings and journal articles. Despite the availability of these commercial packages, we found it necessary to write our own specialized data acquisition and analysis software for microscope plotting, digitizing, and serial section reconstruction. The following sections describe these computer programs which we use in neuroanatomical research. Many other uses of computers in cell and neurobiology research which have been reported in the literature are also reviewed.

111. Computer Systems €or Microscope Control and Plotting

Computers can be used to control a variety of light microscope functions (Boyle and Whitlock, 1975). The microscope stage can be controlled by a computer if stepping motors are attached to the stage drives. The computer can both advance the stage in small increments and simultaneously keep track of stage position. Focusing can also be controlled by a computer if a drive device is attached to the focus knobs. Algorithms are available to automatically focus light microscopes. Several computer-assisted approaches to automated focusing have been described in the literature (Mason and Green, 1975; Ploem et al., 1979; Shoemaker et al., 1982). By adding a TV camera, photometer, or other imaging device, computers can also be used in analysis of microscope images (see Sections V1, VII, and VIII). Computers are also being used to control functions on electron microscopes (Engel et al., 1981; Herrmann et al., 1978; Hillman et al., 1980; Joy, 1982; Kirkland, 1982; McCarthy et a / . , 1982; Rez and Williams, 1982; Rust and Krahl, 1982; Smith, 1982; Statham, 1982). For example, a number of computer systems have been designed for control of STEMS (scanning transmission electron microscopes). Joy (1982), for instance, has designed software which monitors the operating conditions of a STEM. The system uses an Apple 11 Plus microcomputer with A / D and D/A converter cards. APPLESOFT BASIC programs provide a CRT readout of lens current, gun and specimen chamber vacuums, and stage position. Other applications for image storage and enhancement are discussed as well (Joy, 1982). More elaborate control systems are also available, some of which are sold commercially. These systems are particularly useful for controlling the scanning beam of a STEM in automated X-ray analysis (Herrmann et al., 1982; McCarthy et al., 1982; Rust and Krahl, 1982). Computers have been used for some time for microscope plotting. Computerbased plotters are used to produce maps of tissue being analyzed with the light microscope. The maps are usually produced by coding the stage position of the

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91

microscope. The earliest of these computer microscope plotters was developed by Glaser and van der Loos ( 1965) almost 20 years ago. Computer-aided microscope plotters fall into two categories: dendrite-tracking systems used to study the dendritic branching patterns of cells, and microscope plotters used to map the spatial distribution of organelles within tissue. The dendrite-tracking systems plot the tree structures of neurons and measure such features as branch number, dendrite vector. and dendrite diameter. From these measurements, mathematical models of the functional characteristics of the cells can be generated. There are essentially two types of dendrite-tracking computer system (Capowski and Cruce, 1979). The first type uses an encoding device on the microscope stage to detect the position of dendritic branch points. The second type uses a projected image of the dendrite which is digitized by an external device. Capowski (1977), Capowski and Sedivec (1982). DeVoogd et ul. (1981), Overdijk et a / . (1978), and Wann et a / . (1973) have developed dendrite-tracking systems which record the positions of dendritic branches by encoding stage position. Encoding is accomplished on these systems by attaching stepping motors to the stage drives of the light microscope. Counting the pulses generated to drive the stepping motors and converting these counts to distance provides a measure of stage position. Z axis data to encode the depth of a profile within the tissue is taken from the fine focus knob of the microscope, which can also be fitted with a stepping motor. Branch positions are usually recorded by depressing a function key on the computer when the branch point lies under a cross-hair in the microscope binoculars. Other computer microscope systems use potentiometers or shaft encoders rather than stepping motors to encode position (Glaser and van der Loos, 1965; Mize, 1983a; Reed et al.. 1980). With linear potentiometers, position is read as an analog signal in which voltage is proportional to stage position. A I D converters translate the voltage signal to a digital value for computer input. With shaft encoders, the output pulses of the encoders are counted and translated to a unit of measure to represent stage position. In the second type of microscope tracking system, the microscope image is projected to another surface and measurements are taken from that surface. Such systems include images superimposed by a camera lucida onto a digitizing tablet (Green et a / . . 1979; Haug, 1979) and images transmitted to a television monitor via a video camera (Hillman et a l . , 1977; Lindsay, 1977; Paldino, 1979; Paldino and Harth, 1977; Uylings et a / . , 1981; Yelnik et u l . , 1981). Branch points of dendritic processes are digitized by positioning a CRT screen cursor or digitizer cursor over the branch point and digitizing the point. The completed “stick” reconstructions of the neuronal tree structure are displayed on the CRT. Various measurements can be made from these stick reconstructions, including dendrite diameter, distance between branches, branch length, the number of branches, and branch vectors. These measurements are computed automatically using spe-

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R. RANNEY MIZE

cially developed algorithms. The dendrite-tracking systems have been useful in distinguishing cell classes (Glaser et a l . , 1979; Uylings et a l . , 1981) and in revealing parametric alterations in cell populations after various pathologies or experimental manipulations (Woolsey and Dierker, 1979, 1982). The video-based microscope plotters are faster than those which utilize stage encoders, but image quality and resolution are often sacrificed when the image is projected. Both video and encoder-based microscope plotters are susceptible to errors in the estimation of tissue depth, as Glaser (1982) has pointed out. When using dry objectives, objects will appear foreshortened in the Z axis unless a correction factor is used. This is a common but easily rectified problem in computer microscopy. Automatic video image analysis systems have also been developed which have algorithms for automatic “recognition” of Golgi impregnated or HRP filled neurons (Capowski, 1983; Coleman et al.. 1977; Garvey et a l . , 1973). This allows the computer to locate and trace the neuron so that branch points do not have to be plotted manually. However, the complexity of nerve cells as well as irregularities in staining density mandate close operator monitoring of the procedure to reduce computer misinterpretation of artifact. Other computer microscope systems are specifically designed to map the spatial distributions of organelles within a tissue specimen. These are called computer microscope plotters or pantographs (Curcio and Sloan, 1981; Foote et a l . , 1980; Forbes and Petry, 1979; Mize, 1983a; Reddy et a l . , 1973; Reed et al., 1980; Williams and Elde, 1982). These systems can be used to accurately plot the positions of labeled cells, other organelles, and various reaction products within a tissue sample. Our laboratory, for instance, has developed a microcomputer-assisted plotter for the electron microscope which maps the positions of various profiles within a section of tissue and calculates their distance (in microns) from the surfaces of the section (Mize, 1983a). The densities of the profiles within the tissue can also be calculated with the system. Stage position is measured using optical incremental shaft encoders which are attached to the stage drives of the electron microscope by gears. A display/control unit converts the encoder pulses to binary digits as well as providing an LED display of stage position. The 9845T microcomputer is used to control data input and to store, graph, and analyze the plots (Fig. 3). The software for the system includes four programs: (1) truce, used to draw around the outer edges of the tissue specimen; (2) plot, which maps the positions of profiles (synapses, cells) within the boundaries of the tissue; ( 3 ) analyze which includes algorithms for comparing trace and plot data and calculating distance from the tissue surfaces; (4) density, for sorting and counting profile types, measuring surface areas, and calculating profile densities. Commercial statistical software is used to analyze and graph the data and study profile distributions. The maps can be generated in four colors on a digital plotter (Fig. 4). The maps have demonstrated statistically significant

COMPUTER APPLICATIONS IN NEUROBIOLOGY

93

FIG.3 . Block diagram of an electron microscope plotter, which includes shaft encoders (Sh. Enc.) attached to the stage drives of the microscope, a display control box which converts encoder pulses to 16-bit parallel digital code, two 16-bit parallel interface cards to input the signals to the computer, and the H-P 9845T microcomputer, which includes a printer, CRT, plotter, and floppy disk. (Modified from Mize, 1983a. by permission of Elsevier Biomedical Press, Amsterdam.)

differences in the distributions of synapse populations which overlap qualitatively (Mize, 1983~).A microcomputer plotting system which also uses a Hewlett-Packard 9845 computer has been described by Williams and Elde ( 1982) for mapping the distribution of histochemical label in brain slices. Other mapping systems have been designed to plot the three-dimensional distributions of profiles through a volume of tissue. Foote et al. (1980), for instance, have developed a PDP 11/34 computer system with an Evans and Sutherland Picture System 11 graphics processor for this purpose. The system produces three-dimensional maps of neurons within brain nuclei. Comparisons of the distributions of cell groups in different brains can be made quantitatively using the system. Other computer-based plotters for producing three-dimensional maps have been reported (Curcio and Sloan, 1981; Johnson and Capowski, 1973).

IV. Serial Section Reconstruction The three-dimensional reconstruction of biological tissues is of great value both for analyzing the volume of structures and for modeling their three-dimensional molecular configuration. Three-dimensional information can often be ex-

Fic. 4. Computer-plotted map of synaptic terminals within the cat superior colliculus. The plotter outlines the tissue contours of the spccimen. The positions of retinal ( * ) and cortical (0) synapses are indicated by symbols. (From Mize. 1 9 8 3 ~ .by permission of Springer-Verlag. New York.)

COMPUTER APPLICATIONS IN NEUROBIOLOGY

95

tracted from single sections using stereo pair imaging or optical slice techniques (see Turner, 1981, for an extensive review of this topic, and Ghosh, 1975). Reconstruction from multiple serial thin electron microscope sections is often useful when high resolution is needed, or when the object to be reconstructed is large. Several tasks are required to reconstruct tissue from serial sections. Sections must be stacked, aligned, and displayed. In addition, rotation of the reconstructions is useful for studying their shape and spatial relationships. The history of serial section reconstruction has been reviewed by Gaunt and Gaunt (1978), Mannen (1978), and Ware and LoPresti (1975). Computer-based reconstruction has been reviewed by Macagno et u / . (1979) and Sobel et ul. (1980). A number of computerized electron microscope reconstruction systems were developed during the 1970s, most using specially fabricated instrumentation and minicomputer hardware (Glasser et ul., 1977; Hillman et al., 1977; Llinas and Hillman, 1975; Lubbers, 1977; Macagno et al., 1979; Perkins et al., 1979; Rakic et ul., 1974; Shantz and McCann, 1978; Veen and Peachey, 1977). Paralleling this development of reconstruction hardware were advances in software algorithms for reconstruction and image rotation (Dierker, 1976; Gentile and Harth, 1978; Gordon and Herman, 1977; Kam, 1980; Newman and Sproull, 1979; Veen and Peachey, 1977). Perhaps the best known reconstruction system introduced during this period is CARTOS (Computer Aided Reconstruction and Tracing of Sections), developed by Cyrus Levinthal’s group at Columbia University. The CARTOS system uses a PDP 11/34 computer and an Evans and Sutherland graphics display device. Profiles from serial sections can be entered in the computer system using various digitizing devices (Levinthal and Ware, 1972; Macagno et al., 1979; Sobel et a / . , 1980). A portable loaner system for CARTOS is available for use through a shared instrument resource grant from NIH. The portable system allows investigators to digitize serial sections in their own laboratories. Real-time rotation and analysis of the reconstructions is then performed at the home laboratory site in New York. Stevens et al. (1980) have developed a similar system for neuron reconstruction. Their system employs 35 mm film strips which have been produced from serial electron microscope negatives. The film strips are aligned and analyzed under computer control. The films are viewed with a video camera and displayed on a monitor. Advance of the film strips, fine alignment of the frames, and digitization of profiles are controlled by a specially designed Z80 microprocessor-based unit. The film is surveyed at different magnifications using a video zoom camera. The outlines of reconstructed profiles are digitized directly on a video screen by moving a screen cursor around the profile. Consecutive sections are microaligned by comparing one section with a previous section stored in a video memory. To align, the current and stored images are superimposed on a video monitor. The images are then rapidly alternated on the video

96

R. RANNEY MIZE

screen and one image moved with a joystick until alignment is achieved. Ideal alignment is obtained when apparent movement (“flicker”) is minimized. The logically aligned film strips can be replayed under computer control. Image rotation is accomplished using a DEC graphics package on a PDP 11/34 which runs specially developed rotation software. This approach is quite powerful. The film strips offer a convenient, easily stored record of the reconstructions. The alignment and digitization of the films are relatively rapid. The reconstructions can be reviewed and edited easily. On the other hand, the video hardware is expensive and producing the 35 mm movies is both time consuming and costly. Simpler, less-expensive systems have recently been developed which use microcomputer-based manual digitizers and graphics display devices. The digitizer is used for data entry, the graphics unit is used for aligning and displaying sections, and a floppy or hard disk is used for mass storage (Glasser et al., 1977; Johnson and Capowski, 1983; Macagno et ul., 1979; Perkins et al., 1979; Prothero and Prothero, 1982; Street and Mize, 1983). Our system, for instance, uses the Hewlett-Packard digitizer to trace outlines of cells or synaptic profiles within each serial section. The outlines are traced from 8” x 10” photographs, although projected slides or movies can also be used. Once digitized, two consecutive digitized outlines are displayed on the graphics CRT of the 9845T computer. The sections are aligned by translating and rotating one of the outlines with special arrow keys until it is superimposed over the other outline. The process works well although it is somewhat slow because the graphics screen must redraw each translation. Where there are slight tissue distortions we approximate a “best fit” by eye rather than using a complicated algorithm to match the sections. This works well since the human eye seems well-adapted to making these judgments rapidly. The procedure is continued until all sections are aligned. The X-Y coordinate values of the aligned profiles are then stored on floppy disks. A rotation algorithm allows us to display the reconstructed cells or synapses at different rotations in space with hidden lines removed (Fig. 5) (Street and Mize, 1983, 1985). Our system is programmed in enhanced BASIC. Because the H-P enhanced BASIC is an interpretive language, the rotation algorithms are slow. A solution to this problem is provided by graphics display systems (Glasser et al., 1977; Johnson and Capowski, 1983; Macagno et al., 1979). These devices have hardwired logic for rotation and translation and therefore provide real-time interactive graphics capability. Johnson and Capowski (1983), for instance, use a Neuroscience Display Processor for rapid rotation and translation of reconstructed images. This refreshed vector graphics system is driven by a PDP 11/45. Most of their reconstruction software is written in FORTRAN with callable assembly language subroutines for controlling peripheral devices. Another 3-D reconstruction FORTRAN program for entry and display of images (but with no facility for

COMPUTER APPLICATIONS IN NEUROBIOLOGY

97

FIG. 5. computer reconstruction of a retinal synapse in the cat lateral geniculate nucleus. The synapse has been rotated in three planes. Hidden lines have been removed to give a three-dimensional perspective to the reconstructions. Symbols represent regions of synaptic contact with other cells.

rotation) has been developed for microcomputers running under the CP/ M operating system (Prothero and Prothero, 1982). Computerized reconstruction has been used to study the dendrite structure and spatial relationships between neurons (Glasser et al., 1977; Llinas and Hillman, 1975; Macagno et al., 1979; Stevens et al., 1980; Street and Mize, 1983), the density of synaptic contacts on neurons (Mize et al., 1982; Stevens et al., 1980; Street and Mize, 1983), to develop structural models for viruses and other microbiological organisms (Perkins et a/., I979), and to examine the structure of mitochondria (Tenny et a l . , 1980; Veen and Peachey, 19771, chromosomes (Moens and Moens, 1981), and even single molecules (Perkins et a l . . 1979). Computerized reconstruction offers a major technical advance over manual techniques. The computer can be used to manipulate, measure, and store sectional data automatically. Computations for rotation can be executed rapidly with the computer. The reconstructions can be graphically displayed on computerbased graphics devices. None of these procedures is possible using manual techniques.

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V. Computer-Aided Morphometric Measurement Quantitative analysis of cytological materials is becoming increasingly common in cell and neurobiology . Measurement of various geometric characteristics of cells and organelles is called morphometry. Planimetry and stereology are systematic techniques used in morphometry for measuring parameters such as object density, size, shape, and volume quantitatively. The introduction of microcomputer-based measuring systems greatly facilitates the application of these techniques to microscope materials. Microcomputer systems increase the speed and accuracy of data acquisition, allow for much more efficient mathematical analysis, and provide a convenient source of data storage and display. Computerized planimetry is a direct method for measuring such geometric features as cross-sectional area, perimeter (length), diameter, and shape. Two computerized approaches are used in planimetry: computerized analysis in which image data are entered manually with a digitizing tablet and semiautomatic and automatic video image analysis in which the image is reconstructed electronically. Digitizing tablets are electromechanical devices which have a tablet surface or platen usually embedded with an electrically active wire grid (Fig. 2). The wire grid is electrically referenced to a cursor or stylus (pen). The user collects data by tracing around the outer contours of profiles using the cursor (or an electronic stylus pen). The cursor “senses” the coordinate values representing the profile’s outline and transmits these values to the computer. The computer converts the values to measures of area, length, diameter, and shape. The digitized image can be a micrograph placed on the platen or a back projected slide or movie frame. The coordinate sensing techniques of early digitizers were mechanical (Veen and Peachey, 1977) or sonic (Cowan and Wann, 1973; Dunn e t a / . , 1975, 1977) but most modern digitizer tablets employ electrical wave sensors (Hewlett-Packard, Summagraphics, Talos). Digitizing tablets can be small and inexpensive (circa $300) or large and expensive (circa $10,000). Small data or graphics tablets used to lack resolution and accuracy and were most often used to manipulate cursors or data on a CRT. However, recently developed small tablets have excellent resolution and are quite accurate. Precision measurement digitizers have resolutions of up to 25 p m and often include special features such as function keys and LED displays. Many digitizers have built-in microprocessors which do much of the maintenance work of the digitizer (signal conversion from voltage to digital x, y coordinate values, origin setting, scaling, axis alignment, and skew correction). Digitizers can be interfaced to a microcomputer via any of the standard interface ports (the GP-IB IEEE-488 instrument interface, 8 or 16-bit parallel, and RS232 serial interfaces are most common). The x, y coordinate positions of each data point are usually transmitted to the microcomputer as binary numbers, although ASCIl values are also sometimes used. The microcomputer is used to convert

COMPUTER APPLICATIONS IN NEUROBIOLOCY

99

these data points to decimal values, to perform geometric calculations, and to store the data. The control of data input, storage, and calculation can be managed by assembly language routines or by high level language programs. A number of high level language programs for digitizing morphometric features have been described in the literature, Several FORTRAN programs have been written to drive digitizers interfaced to PDP (DEC) computers (Albright and Sawler, 1981; Cowan and Wann, 1973; Dunn e f al., 1975, 1977). BASIC language programs have also been developed for a variety of microcomputer digitizing systems (Dennino et a / ., 1978; Mize, 1983b; Pullen, 1982). Digitizing systems written in assembly language have also been reported (Green et al., 1979; Peachey, 1982). All of these programs have algorithms for calculating area and perimeter. Many also calculate other parameters such as diameters, Feret dimensions, angles of orientation, center of gravity, and various form or shape factors. Formulas for calculating some of these values are reported by Bradbury (1977). A large variety of inexpensive commercial morphometric digitizing systems are now available on the market (Table 11). Peachey (1982), for instance, describes a simple single board microprocessor-based system for about $2000. The system includes a 280 processor with clock, parallel I/O ports, 1K byte of RAM, and several EPROM (erasable programmable read only memory) boards. This single board computer is interfaced via an 8-bit parallel port to a Summagraphics BITPAD. The programs are written in assembly language and are “stored” on the nonvolatile EPROMS. The programs convert data points, calculate positive and negative areas and length, and print the results on an inexpensive printer. A more elaborate version of this system is available from Laboratory Computer Systems (Cambridge, Mass., Table 11). Other more expensive digitizing tablet systems are available commercially, some of which include flexible general purpose microcomputers (Table 11). Much of the cost of these systems is for the software, as the hardware is generally relatively inexpensive. We have developed a noncommercial, highly flexible digitizing system based around our H-P 9845T microcomputer and H-P 9874A digitizer. The programs are written entirely in H-P enhanced BASIC (Mize, 1983b). The system has been used to measure cell and synapse areas, to measure synaptic vesicle sizes and shapes, to measure the length of immunocytochemically stained collagen fibers, and to measure the size and density of intramembrane particles on freeze-fractured replicas. When measuring cells, the programs allow entry of data values for the number of elements contacting a cell, the number of elements within a cell (such as autoradiographic grains), cell type, and cell depth. These values are entered from the digitizer’s numeric keypad (Fig. 2). The operator then traces around the profile with the digitizer’s cursor. From the trace, the programs calculate cross-sectional area, perimeter, average diameter, and form factor (an index of circularity). The programs also compute contact and element densities

100

R. RANNEY MIZE TABLE I1 SELECTED COMMERCIAL IMAGEANALYSIS INSTRUMENTS

System

Computer

Resolution

Measurementsa

MICROPLAN

Microprocessor

0.025 MM

A,L,P,F,D,S,C

OPTOMAX

Apple IIE

0.10 MM

LADD 40000

Rockwell AIM 65

0.10 MM

A,L,P,F,D,S,C ANGLE, others A,L,P,F,D,S,C others

NUMONICS 1224EM

Microprocessor (8080)

0.25 MM

A,L,P,F,D,S,C ANGLE, others

MOP-30

Microprocessor

MM

A,L,P.F,D,S,C ANGLE, others

Manufacturer/ distributor

Digitizing tablet systems

Laboratory Computer Sys, Cambridge, MA Optomax Inc, Hollis, NH Ladd Research Industries, Burlington,VE Numonics Corporation, Lansdale, PA Carl Zeiss,lnc,New York, NY

Video digitizing systems (include tablets) OPTOMAX

Apple IIE

SMI UNICOMP

Apple IIE

BIOQUANT

Apple IIE

VIDEOPLAN

Microprocessor (Z80A)

A,L,P,F,D,S,C ANGLE, others A,L,P,D,S ANGLE A,L,P,F,D,S,C ANGLE, others A,L,P,F.D,S,C ANGLE, others

Optomax Inc, Hollis NH Southern Micro. Instr., Atlanta, GA R&M Biometrics, Nashville, TN Carl Zeiss Inc, New York, NY

OA, Area; L, length; P, perimeter; F, Feret dimensions; D, diameters; S, shape factors; C, center of gravity; ANGLE, various angles.

based upon the numeric values entered before tracing. The data are stored on floppy disk, printed out on a thermal printer, and can be analyzed and graphed using Hewlett-Packard statistical and graphics packages (Mize, 1983b). A flow diagram illustrating the operation of the system is shown in Fig. 6 . Parametric measures made with our digitizing system have permitted us to quantify properties of projection neurons in the superior colliculus (Caldwell and Mize, 1981; Harrell et al., 1982) and reveal variations in the size and contact densities of retinal axon terminals in different visual system structures (Mize, 1983d; Mize and Homer, 1984). A second major type of morphometric digitizing system employs a video camera attached to a light microscope. Many commercial systems use this approach (Table 11). An image of the microscope specimen is produced by the

COMPUTER APPLICATIONS IN NEUROBIOLOGY

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FIG. 6 . Flow diagram of the program operating steps involved in digitizing and calculating the area, perimeter, average diameter, and form factor of cells and synapses.

camera and displayed on a television or CRT monitor. A screen cursor is superimposed on the TV image. This cursor can be moved remotely by manipulating a “mouse,” a pen on a digitizing tablet, or a joystick. The outlines of profiles within the TV image are digitized by moving the screen cursor around the edge of the profile. A video outline of the trace is usually drawn on the screen as the cursor is moved. Once the profile is outlined, the computer performs measurements similar to the systems described earlier. Area, perimeter, various diameters (maximum, minimum, average, feret), angles, centers of gravity, and shape factors are routinely included with these systems (Table 11). A major advantage of TV camera systems is the automatic reproduction of the microscope image. The costly intermediate steps of photographic reproduction are avoided. The ability to produce a digital image is another advantage. Using appropriate hardware upgrades, the digital image can be stored and manipulated (see Section VI). On the other hand, television images have lower resolution than photographs and remote tracing with the screen cursor is more difficult and usually less accurate than direct tracing on a digitizing tablet. Optical distortions in the image produced by the camera pose a further potential problem with these systems.

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Computers are now also sometimes used in stereology studies. Stereology is a collection of mathematical methods for estimating three-dimensional volumetric information from two-dimensional images. Stereological techniques can be used for estimating the number of objects in a volume of tissue, the fractional surface area and volume of these objects, and the total volume of tissue. The size distributions of the objects in a field can also be determined using stereological methods. Measurements are made using a method called poirzt counting. A lattice of test lines or points is placed over micrographs and the lines or points which bisect selected objects are counted. Estimates of object number, area, length, fractional volume, and distribution density are calculated from these counts. The mathematical procedures underlying the technique have been described in detail (Cruz-Orive, 1976; Weibel, 1979, 1980; Weibel and Bolender, 1973). Microcomputer systems can assist both in the collection of data and in performing the mathematical operations from which the estimates are obtained. Digitizing tablets can be used to count the numbers of test points overlying objects of interest. By using an electronic marking stylus rather than a cursor, points can be marked with ink to avoid double counting. The counts are tabulated automatically by the computer. More commonly, the test points of the grid lattice are counted by hand and the values entered into the microcomputer from a keyboard or numeric keypad (Bolender et al., 1982; Briarty and Fischer, 1981; Poole and Costoff, 1979). Various volumetric and surface area algorithms are then performed by the computer (Bolender et al., 1982; Briarty and Fischer, 1981; Poole and Costoff, 1979; Ratz eta/., 1974). Poole and Costoff (1979), for instance, have developed BASIC language programs with algorithms for calculating numerical densities, object volumes, the surface areas of individual profiles, and volume/surface area ratios. More recently, Bolender et al. (1982) have described a computer system for stereology which utilizes a Tektronix 4052 desktop microcomputer both to assist in counting and for calculation. Special function keys on the computer keyboard are used to enter the counts. The BASIC programs calculate volume density and surface density as well as summary statistics. Error curves are drawn on the graphics CRT to determine the appropriateness of various sampling parameters. The system can also store the data permanently on cartridge tape. Stereological procedures are also useful for measuring the sizes and distributions of intramembrane particles (IMPS) on freeze-fractured replicas. Niedermeyer and Wilke (1982) have developed special algorithms for studying IMP distributions using a digitizer system coupled to an H-P 1000 minicomputer. Particle counts, particle positions, and particle diameters are measured with a MOP DIGIPLAN. The digitizer cursor is placed over each particle and a special key depressed to code the particle count and its position in the field. The diameter of the particle is entered by measuring the distance between two points at opposite edges of the particle, marked with the digitizer cursor. A computer

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program run on the H-P 1000 then calculates a coefficient of dispersion, a measure of the degree of clustering of the particles (Niedermeyer and Wilke, 1982). Particle size distributions and particle densities are also summarized by the computer program. Other stereological methods for cluster analysis, size distribution, and volume estimation have been developed for use with computer image analysis systems (Baudhuin et al., 1979; Mahlhorn and Packer, 1976; Sprumont, 1980; Wree et a/., 1982). Whether computerized planimetry measurements produce more accurate data than stereological point counting has been debated. Mathieu ef al. (1981) have compared manual point counting, planimetric measurements using a digitizer, and video image analysis techniques to measure muscle fibers. When estimating aggregate global parameters alone (Vv and Av), manual point counting was up to 1 I times more efficient and nearly as accurate as direct measurements using a computerized digitizer. A computer-aided Quantimet 720 automatic image analysis system was both faster and more accurate than either manual digitizing or point counting. Object extraction using the image analyzer, however, proved difficult except for highly stereotyped specimens. Although the image analyzer technique was fastest and most accurate for a synthetic specimen, the analyzer was unable to distinguish mitochondria poor muscle fibers in a biological specimen (Mathieu et al., 1981). Planimetry has advantages over stereology for certain applications. Peachey (1982) lists some of these. Measurements of many parameters (area, length, diameter) can be made simultaneously with a digitizer system, but must be calculated separately using point counting methods. Because digitizing allows more random sampling strategies, fewer samples need to be measured. Error corrections are largely unnecessary since no assumptions must be made about object shape or regularity. Finally, planimetry gives more accurate measurements of individual areas and lengths, although the value of this accuracy has been questioned (Mathieu et al., 1981). The choice of method should probably depend upon whether global parameters or local parameters are of greatest interest, the accuracy of measurement needed, and whether the appropriate technology is available.

VI. Video Image Processing and Analysis Video image analysis is another computer technology employed to make morphometric measurements of tissue. Video processing and analysis systems employ scanning TV cameras or microphotometers to produce an array of picture points (pixels). The light intensity of the image at each pixel in the array is also measured. These light intensity values are called grey level discrimination values (or grey scale values). These values are digitized so that they can be manipulated

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by a computer. The values are temporarily stored in a storage and display device, usually a video frame store buffer (Smith, 1982). They can also be permanently stored on a computer mass storage device. The spatial resolution of various systems may vary from 128 X 128 to 2048 X 2048 array points. The grey scale resolution may vary from 8 to 4096 grey scale levels. Effective grey scale resolution is generally far lower than the theoretical limits of the hardware and optics of the system. Spatial and grey scale resolutions can be limited by the scanning device, the storage space of the video frame store buffer, the core memory of the computer, or the inherent limitations of the image itself. Once images have been digitized and stored, they can be enhanced or edited, and specific features can be extracted from them. When objects of interest have been identified and extracted by extraction algorithms or operator interaction, the video image analysis system can count and measure the objects automatically. TV-based image processing and analysis systems are available from a number of commercial sources (Table 111). Although still used primarily in industrial applications such as metalurgy , their potential usefulness in biology is vast, particularly in the fields of pathology and cytology where large numbers of slides must be analyzed rapidly. Image analysis for morphometrics has been reviewed in detail by Bradbury TABLE 111 SEMIAUTOMATIC I M A G E ANALYSIS A N D PHOTOMETRY-FLUORtSCENCt SYSTEMS

System

Computer

Grey level discrimination

Features

Manufacturer/ distributor

Semiautomatic image analysis systems IBAS QUANTIMET 900 OMNICON 3000 LEITZ TAS JOYCE-LOEBL MAGISCAN 2

Z80A Based LSI I 1 123 Z80A Based PDP 11/23 N.A.

256 256 256 256 64

Extraction measurement Extraction measurement Extraction measurement Extraction measurement Extraction measurement

Carl Zeiss Inc, New York, NY Cambridge Instruments, San Diego, CA Bausch and Lomb, Rochester, NY Ernst Leitz, Inc, Rockleigh, NJ Nikon, Inc., Garden City, NY

Photometry-fluorescence systems ZONAX

8080A

MPV-DADS 560

Z80A Micro

256 4096

Photometric area Photometric area

Carl Zeiss, Inc, New York, NY Emst Leitz, Inc, Rockleigh, NJ

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(1977, 1979, 1981). Several discussions of image digitizing hardware have also recently been published (Smith, 1982; Sobel et a / . , 1980). A recent issue of Ulfrarnicroscopy (Rez and Williams, 1982) is devoted to the topic of image processing and analysis in electron microscopy, where there is considerable interest in utilizing computer techniques to enhance and detect images which are difficult to resolve optically. An issue of the Journal c?f’Microscopy(“Computer Techniques in Electron Microscopy and Analysis” 127, 1-126, 1982) is also devoted to computer uses in electron microscopy. This complex area is beyond the scope of the present review. A number of image analysis systems have been developed for use with light microscopes. Noncommercial systems include the LEYTAS (Ploem er al., 1974, 1978, 1979; Van Ingen et al., 1980; Vrolijk et a / . , 1980a, b), FAZYTAN (Erhardt er al., 1980), TULIPS (Bengtsson et a / . , 1977; Holmquist et a l ., 1977, 1981), IMAGIC (Van Heel and Keegstra, 1981), and SEMPIC (Dormer, 1980; Dormer et ul., 1978, 1981) image processing systems, all of which utilize main frame or minicomputers, as do commercial systems like the Leitz T.A.S. and Quantimet 900 (Table 111). Some recently developed commercial image analysis systems incorporate 8-bit microprocessors (Bausch and Lomb Omnicon and Zeiss IBAS) (Table HI), but their cost still remains high, generally exceeding $80,000.00. Several noncommercial microcomputer-based systems have also been reported in the literature. Descriptions of hardware independent software programs for image analysis have also been published (Hegrel and Altbauer, 1982; Nawrath and Serra, 1979; Rink, 1977; Smith, 1978). The software for calculating areas, perimeters, and diameters of extracted objects are well developed on these image analysis systems. Pattern recognition and image extraction algorithms are far more difficult to develop and are generally successful only on highly stereotyped profiles of relatively constant contrast (Bradbury, 1979, 1981; Sobel et u l . , 1980). Most video image analysis systems used for morphometry will therefore operate both in fully automated or semiautomatic, human interactive modes. Automatic pattern recognition procedures have been used to quantify nerve and muscle fiber diameters which tend to be relatively constant in both shape and staining characteristics. Zimmerman et al. (1980), for instance, have developed computer algorithms that are able to recognize, extract, and measure myelinated fibers from peripheral nerve stumps. Light microscope images are viewed by a video camera and digitized by a transient recorder to a grey scale resolution of 256. A DeAnza memory display is used to store and redisplay the digitized images. Recognition algorithms are written in MACRO and run on a PDP 11/34. The algorithms locate center points between light (axon) and dark (myelin) areas of the image. Vectors from the center point are drawn at 4 angles to determine if the light area (axon) is surrounded by a dark ring (myelin). An edge detection algorithm then finely calculates the border of the cylindrical myelin profile. Once the profile has been recognized and traced

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automatically, other algorithms count, size, and evaluate the shapes of the extracted fibers. The software algorithms succeed in identifying about 80% of all fibers with minimal false positives (artifacts). The size distributions of nerve fibers have been studied by other investigators using commercial image analyzers (Bradbury, 1977, 1980). A detailed account of algorithm mathematics for extracting and measuring myelinated fibers has been published by Geckle and Jenkins (1983). Automated image analysis has also been applied to more complex central nervous system tissue. Miller (1981), for instance, has used a Quantimet 720 to count neurons within the human hippocampus. Both cell numbers and cell density were calculated following automatic recognition designed to detect neurons with clearly defined nucleoli (Miller, 1981). A Leitz T.A.S. video image analysis system has been used to automatically recognize individual layers of mouse cerebellum (Gardette et al., 1981). The recognition algorithm is based upon differential densitometry applied to stained sections and can be used to compare the volumes of various layers of cerebellum in normal and pathological material. Pattern recognition has also been applied to the analysis of bone where it is possible to measure quantitative features associated with the load bearing properties of the bone (Green el al., 1981). A variety of other uses in automatic cell recognition have been reported (Preston, 1980; Preston and Dekker, 1980). For less stereotyped profiles, image analyzers must usually be used in a semiautomatic, interactive mode in which the image can be edited and the objects enhanced for extraction under control of the investigator (Schmassmann et al., 1979; Slavin et ul., 1982). Slavin el al. (1982), for instance, have used a Joyce-Loebl MAGISCAN to measure the diameters of muscle fibers. The image of the muscle is displayed on a TV monitor. To measure diameter, the investigator uses a light pen to mark two points at either edge of the muscle fiber. The microcomputer then automatically calculates the length of a line drawn between the two points. This type of interaction is easier than enhancing the image in preparation for automatic recognition and avoids the time-consuming and costly development of pattern recognition algorithms. Specially designed microcomputer-based image analysis systems have also recently been reported. Hainfeld et al. (1982) describe a simple microcomputerbased system for use in studying protein structure. The system utilizes a TV camera coupled to a Lexidata digital video frame store buffer. The frame store buffer can store 5 12 X 5 12 X 8-bit (256) grey level images rapidly and at modest cost. The system is interfaced to a DEC LSI 11/23 computer system with hard disk storage. Images are displayed on a color monitor to highlight grey level data. FORTRAN-based programs for the system perform measurements of mass per unit length and molecular weights of individual proteins. Automated image analysis is also being employed in tissue culture studies (Brown et al., 1979; Goldrosen et al., 1982). Goldrosen et al. (1982), for

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instance, have developed a computerized TV camera system for counting cells in tissue culture. The programs are written in BASIC and run on a Hewlett-Packard 9835A computer. An Omnicon video image analyzer digitizes an image of individual microplate wells. Specially written algorithms determine cell counts. The computer also controls a scanning stage upon which the wells are placed. A complete well plate can thus be scanned automatically. Several commercial systems are also available for counting the numbers of profiles in well plates (Leitz, Inc., Rockleigh. NJ: Artek, Inc.. Farmingdale, NY). Morphometric video image analysis is also being used in other areas of biology. Chromosome analysis is one such application (de France and van den Berg, 1977; DeWald er a / . . 1977; Oosterlinck er ( I / . , 1977; Stengel-Rutkowski er a / . , 1974). I n this field, programs have been developed for karyotyping chromosomes. Automated karyotyping, however. remains inferior to human classification by skilled technicians (Oosterlinck a/., 1977). Lymphocyte classilication has also been automated using computerized image analysis (Durie et crl., 1978: Olson et ( I / , , IY79a,b). Olson cr c d . (1979a.b). for instance, have differentiated subgroups cif inurine T cells by using both ruorphometric parameters (such as relative nuclear area) and microphotometric measures of optical density. Axoplasmic tlow of particuliite mattcr i n living axons has also been studied using computerized image analysis of time delay films (Leestrma and Freeman. 1977). Although therc are ninny examples of successful image extraction and analysis of relatively stereotyped profiles, fully automated image axilysis is not yet possible in many areas of biology. The technology needs to develop more refined imaging devices to detect low contrast images. Extraction algorithms for discriminating irregular profilcs in complex tihsues must also bc improved. Inadequate contrast (grey scale detection) in specimen preparations is a chief problem. Improved staining procedures and photographic enhancenient techniques may alleviate sotile of these difficultics. Various htrategies for improving feature extraction have heen reviewed by Bradbury 1979).

VII. Computer LJsev in Photometry and Fluorescence Microscopy Photonictric measuring devices hav e long heen ~isedto extract information about optical density. transmittancc. absorbance, fluorescence. and othcr spectral characteristics o f tissue (see Ploem. 19x0. for a recent review). Microprocessors and microcomputers greatly assist this process. Coniputers are used both for controlling the instrumentation which collects this information and tor performing the calculations necessary t o derive measures such as optical density and lluorescence. Computer devices have been interfaced to micrciphotometers. spectrophotometers. fluorescence microscopes. and niicrodensitometers to aid in essment of photometric data.

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FIG. 7. Block diagram of a schematic computer-controlled photometry system for light microscopes. The system allows control of the photometer (white boxes), the microscope shutters (light grey boxes), and the scanning stage (dark grey boxes). The schematic is a modified version of a system described by Rundquist (1981).

A typical computerized photometry system can control a number of microscope functions. Three standard functions are shown in Fig. 7. First, the computer can collect and store readings from the photometer. To do this, the amplified analog signal from the photomultiplier tube or photodiode is converted to a digital value with an AID converter. This produces a signal compatible with the digital requirements of the computer. In addition to storing the photomultiplier reading, the computer can also determine the number of readings taken from the photometer by controlling the rate at which the computer samples the A / D converter card. Second, the computer can control the sampling duration, frequency, and measuring sequence of shutters on the microscope. Specimen exposure to fluorescence or other activating light can thus be controlled by the computer. The sequence of exposure can also be placed under program control. The amount of time allowed to take a single photomultiplier reading can also be placed under computer control. A third function that can be controlled is the motorized scanning stage. By interfacing the stage to the computer, the speed, dwell time, stepping distance, and stepping pattern of the stage can be programmed. By controlling these three functions, the computer can reproduce and store a digital image of the specimen. The stored image will contain values for the x and y positions of each picture element (pixel), its grey scale value (i.e., photometric intensity), and the light source and exposure time used to obtain that value. From

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this information, a two-dimensional spatial map of light intensities can be reconstructed and displayed. Color graphics can be used for spectral coding of intensity differences on the CRT screen. The image can also be manipulated (enhanced, modified, reduced) and analyzed quantitatively. Computer-controlled photometers are thus very powerful for controlling instrument functions, for analyzing data, and for displaying that data. A number of automated microscopes have been developed for microphotometry and fluorescence analysis (Brugal et al., 1979; Ploem et al., 1979; Rich and Wampler, 1981; Stewart et al., 1980; Walter and Berns, 1981). Most of these noncommercial laboratory systems utilize minicomputers. Commercial microscope manufacturers are now offering moderate priced photometric systems coupled to 8-bit processor-based microcomputers. Both the Leitz distributed Stahl DADS-560 and the Zeiss Zonax utilize Z80 microprocessors for microscope control and photometric conversions (Table 111). Fully automated microscopes have computer-controlled scanning stages, shutter control for the light source and photomultiplier tubes, and control of photometer sampling rate. The more elaborate automated microscopes may also have computer-controlled focusing devices, monochromators, and projective rotors to change magnification (Ploem et al., 1979). This degree of automation seems excessive for most research applications. Several descriptions of computer control of fluorescence microscopes have been published. Schipper et al. (1978) use an MPV-I1 microphotometer interfaced to a PDP/ 12 minicomputer for fluorescence measurements. A program called APOMOS controls the scanning speed of the stage and the sampling rate of the photometer. Data processing features include histogram plots of intensity values in different fluorescence categories, maps of the fluorescing profiles, and plots of intensity profiles. The quantitative analysis of fluorescence intensity using this computer-based system has revealed reductions in fluorescence after administration of noradrenaline blockers which were not apparent by qualitative observation (Schipper et a l . , 1978). Fluorescence measurements can also be handled by much smaller computers. Rundquist (198 1) has recently described a fluorescence microscope system interfaced to a 280 based ABC-80 personal computer. The computer has 32K bytes of RAM, 16K bytes of ROM, a 12-bit A/D converter, and a TTL 110 interface to link the computer to the fluorescence microscope. Current and voltage measures of fluorescence light and signal amplification are performed by an MPV- 1 photometer. The amplified signal is then fed to an A/D converter inserted into an SlOO bus slot on the microcomputer. A special interface from the computer to the electronic shutter control unit permits computer control of the light source. Software to control the system is written in BASIC with assembly level subroutines for data acquisition and microscope control. The data acquisition sequence includes the following steps: a dark current reading of the photomultiplier

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is converted and stored; the shutter to the activating light source is opened for a program-specified time interval; a value from the photomultiplier is collected, converted, and stored; the data are normalized by comparing it to premeasured standards, background readings, and dark current. Simple statistical summaries and histogram plots are also generated by BASIC level programs run on the microcomputer. Emission spectra can be recorded using a motor-driven interference wedge whose position is coded by the computer. Other computer-based devices for measurement of emission and absorbance spectra have been described (Kleinfeld P f ul., 1979; Kucera et ul., 1979; Kucera and Ribaupierre, 1980; Schmidt, 1982; Sharp et ( I / . , 1981; Thiesen and Thiesen, 1977; Van der Ploeg er ul., 1979). Van der Ploeg e t a / . ( 1979) describe a software system, BIOSCAN, which generates simultaneous measures of absorbance at two wavelengths and integrates the values over the total object area. The programs are run on a PDP 1 I / 10 with data displayed on a Tektronix 4010 storage scope. Nichols et al. ( 1979) have interfaced a Beckman 25 absorption instrument to an Intel 8080-based microcomputer. Digital voltages are sent to the computer via a BCD interface. A machine language subroutine controls the data acquisition sequence, storage, and BCD data conversions. BASIC language programs control the analysis and display of data. Sharp et ul. (1981) describe a specially designed interface which controls data acquisition from a BCD port on the Perkin-Elmer 572 spectrophotometer. Interface control is handled by FORTRAN callable function subroutines run on a North Star Horizon Z80A based microcomputer (Sharp et a/., 1981). Computer-assisted photometric devices have also been developed for many other cell biology applications. Microdensitometers, for instance, have been interfaced to microcomputers (Rasch and Rasch, 1979; Smith et a!., 1980). Rasch and Rasch (1979) describe a specially fabricated interface which collects integrated density and area measurements from a Vickers M86 microdensitometer. The readings are transferred to a SOL-I11 microcomputer. The BASIC programs used with the system display and store the collected values, compute summary statistics and regression and correlation coefficients, and plot histograms of the logarithmically transformed absorbance values (Rasch and Rasch, 1979). A similar BASIC-language software system for use with Vickers instruments has been developed for the Commodore PET series 3032 computer (Smith et al., 1980). Various microdensitometry systems employing computer assisted video analysis have also been reported in the literature (Heintzen, 1978; Heintzen et al., 1982; Kugler, 1981; Mancini et ul., 1978; Robb and Jowsey, 1978). Computer-controlled photometric devices are also useful in electrophoresis studies. Two-dimensional electrophoresis gels and chromatography plates can be analyzed quantitatively using computer interfaced optical density measuring devices. Computers can quantitatively assess variations in both the position and concentration of proteins and polypeptides on gels. Measures of gel density have

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been made using TV based image analyzers (Aycock e t a / . , 1981; Garrels, 1979; Lemkin et a / . , 1979; Lemkin and Lipkin, 1983; Mariash et al., 1982) and scanning microdensitometers (Goldman et al., 1982; Pette and Wimmer, 1980; Widdoss and Ferris, 1978; Wong er al., 1977). These can be linked to minicomputers, microcomputers, or dedicated microprocessors. Lemkin and his colleagues (Lemkin et al., 1978; Lemkin and Lipkin, 1983) describe a group of FORTRAN programs for analysis of two-dimensional gel images. The programs, run on a PDP/8e computer, are designed to convert density measures to estimates of polypeptide concentration. Mariash ef ul. (1 982) have developed a less expensive system to analyze two-dimensional gel radiofluorograms which uses an Apple I1 computer. A TV camera system is linked to a digitizing circuit board installed on the Apple 11. The radiofluorogram is back-lit by an X-ray view box. Its image is digitized by the digitizing hardware interfaced with the Apple 11 computer. Specially written software programs control scanning of the radiofluorogram and calculate mean density from each pixel of the digitized image. Counts per minute per unit area can be calculated from the intensity measurements. The system is inexpensive but has a lower grey scale resolution (64 levels) than more elaborate systems (Garrels, 1979; Goldman et u l . , 1982). Very inexpensive microprocessor-based systems for gel electrophoresis have also been developed. Widdoss and Ferris ( I 978) describe a microprocessorcontrolled scanning densitometer for measuring electrophoresis films. The system employs an LED light source, a silicon photosensor, a Motorola 6800 microprocessor with RAM and ROM memory devices, an 8-bit parallel 1/0port, a clock, and an A/D converter. The CPU is programmed to sample the data, integrate the analog signal, and display the data on an LCD device. The system can calculate the absolute value of each protein fraction on the plate. Although this system is quite inexpensive, it lacks the flexibility of data display and analysis afforded by a general purpose microcomputer.

VIII. Computer-Automated Autoradiography and Immunocytochemistry A number of approaches to quantifying grain densities in autoradiographs have been developed in the last 15 years. Quantitative autoradiography has been discussed by Goldstein and Williams (1971), Dormer and Thiel (1976), and Rogers (1973), among others. Manual counting of individual grains is one solution to quantifying grain density. However, manual counting is both tedious and also inaccurate when grains are clustered. Photometric measurement is another common approach to evaluating grain densities. Reflectance measurements using incident light rnicrophotometry (Dormer and Thiel, 1976) and absorbance measurements using flying-spot microdensitometry (Goldstein and Williams, 197 l ) are two photometric techniques used in autoradiographic grain analysis. Image

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analysis devices have also been used to quantify autoradiographs (Prensky , 197I). Most recently, computer-aided microscopes have been developed to estimate autoradiographic grain counts automatically, Early computer automated microscopes for analyzing autoradiographs were developed by Boyle and Whitlock (1974, 1977) and Wann et al. (1974). The system developed by Wann et al. (1974) uses a PDP-12 computer, a Zeiss Universal microscope, and a closed-circuit television camera. The software is written in assembly language and FOCAL. The counting algorithms described by these authors are based upon detection of individual grains rather than photometric measures of grain density. Boyle and Whitlock (1977) describe several computer approaches for estimating autoradiographic grain density. These include counting image points (pixels) above a threshold intensity, measuring the amount of light reflected by all silver grains in the field, and “blob” counting in which the number of overlapping grains in a blob of silver are calculated from measurements of blob size. The system described by Boyle and Whitlock (1974) also uses a PDP-12 computer, a Zeiss Universal microscope, and a Plumbicon equipped TV camera. The TV image passes through a contrast amplifier to adjust the grey scale range. The image is then digitized into one of 256 grey levels for each of 512 X 512 image points. Software for the system has been developed in several languages. Computer-aided cytophotometric techniques have also been used to quantitatively evaluate autoradiograms. Ruter et al. (1979), for instance, has studied tritium-labeled mouse tumor cells using cytophotometry. Their system includes a PDP 11/50 computer, a Zeiss Axiomat microscope, and a Spatial Data TV camera. With the system, dark-field autoradiographs are digitized to 512 X 480 image points with a resolution of 256 grey levels. The images are analyzed using FORTRAN software. The authors found that autoradiographic grain clusters could be accurately detected if the light intensity threshold was properly set. Total grain area was found to be an accurate measure of incorporated label within the tumor cells. Ruter el al. (1979) argue that identification of individual grains within dense grain clusters is often impossible and conclude that the total grain area measurement is the preferred procedure. More recently, Sklarew (1982a,b) has used a Quantimet 720 computer-based TV image analysis system to evaluate autoradiographs. The software developed for the system includes two separate algorithms. Single grains over individual cells are counted using the ‘‘full feature count” measurement algorithm included with the Quantimet. Where grain density produces clusters of grains in the image, counts are estimated from measurements of integrated grain cluster area. The algorithms for integrated area incorporate predetermined measurements of grain size and shape. Grain phase estimates are generated by the autodetector module of the Quantimet system. The algorithms yield counts which are independent of the clustering patterns and density of grains. Sklarew (1982a,b) has

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used the system to simultaneously measure Feulgen density and ['Hlthymidine labeling in rat kidney cell cultures. The densitometric features of the Quantimet 720 permit measurements of mean optical density of Feulgen stained nuclei at the same time that autoradiographic grains in the cytoplasm are counted. The densitometry measurements also provide a means for evaluating the effects of tritium absorption caused by uneven staining and irregular section thickness. Computer-based image analysis systems have also been developed to evaluate autoradiographically labeled 2-deoxy-d-[ 14C]glucose.The 2-deoxyglucose technique, first developed by Sokoloff and his colleagues (Sokoloff et al., 1977), is a widely used method for measuring metabolic rates in brain tissue. Autoradiograms are obtained by exposing tissue to sensitive X-ray film. The films can be analyzed quantitatively to obtain measures of cerebral metabolism. Computerized image analysis systems provide a means of representing the metabolic rates pictorially. The systems can produce spatially precise, quantitative maps of local rates of glucose utilization from the autoradiograms. The differences in local glucose utilization can be represented by color-coding on a color graphics monitor. The system developed by Sokoloff and his colleagues (Goochee et al., 1980a,b) uses an Optronics rotating-drum scanning densitometer to digitize an image of the X-ray films, a DeAnza Image Display system to store and display the image, and a PDP 11/34 computer to process and analyze the image. A 5 megabyte hard disk is used for permanent storage of the images. The analysis programs are written in PASCAL. The system has an array size of 256 X 256 image points and can digitize an area as small as 6.4 mm2. The system has a resolution of about 200 pm, limited largely by the grain size of the X-ray film. The grey scale resolution of the system is 256. The PASCAL software calculates three measures from the optical density data: the mean optical density for a selected region of tissue, the mean radioactive tracer concentration, and the mean rate of cerebral glucose utilization. The metabolic maps produced by this computer system represent a significant advance in analyzing 2-deoxyglucose autoradiographs. Differences in metabolic rate are readily visualized because color-coded differences can be easily detected by the human visual system. The digital images can also be enhanced and modified under computer control. For example, selected areas of interest can be magnified using a feature called digital zooming. Any 128 X 128 area can be expanded to fill the display screen. It is also possible to enhance the contrast of the digitized image. There are two weaknesses to the system. The size of the image array, limited by the image processor, is smaller than is desirable for large brain sections. In addition, the Optronics P- 1000 rotating drum digitizer is slow, requiring between 0.5 and 2 minutes to completely scan an X-ray film. Commercially available hardware upgrades (Optronics, Inc.) can increase both the speed and array size of

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the system. The newer hardware provides a convenient visual representation of the spatial distribution of metabolic activity and a powerful analytical tool for calculating rates of glucose utilization with fewer limitations in speed and array size. TV-based image analysis systems have also been used for analysis of 2deoxyglucose experiments. Gallistel et al. (1982), for example, have designed a TV camera system to detect and measure local alterations in “metabolically coupled functional activity” from 2-deoxyglucose autoradiograms. This semiquantitative approach compares relative optical densities in the autoradiograms rather than computing actual rates of glucose utilization. The computer system developed by Gallistel et a/. (1982) uses a Spatial Data video digitizing system which consists of a vidicon TV camera, an image refresh memory, and digitizing hardware. The digitizer has an array memory of 320 X 240 image points and a grey scale resolution of 256 density levels. The current measured resolving power of the system is 5 linedmm, but can be increased substantially using microscope optics. The X-ray films are backlit while being digitized. A PDP 11/34 computer with 7.5 megabyte hard disk runs the software. A Conrac color TV monitor is used to display the autoradiographs in color. The system software computes relative optical densities, a normalized rankorder transformation of the optical density measured by the video digitizing system. The transformations are obtained by calculating differences between experimentally relevant brain areas and control areas which do not show signficant glucose metabolism. This approach is simpler because fewer measurements are required, the glucose can be injected intraperitoneally, and arterial blood samples need not be taken. The semiquantitative approach to 2-deoxyglucose experiments has been criticized, but has proven useful in studies where there is interest in demonstrating functional alterations which occur as a result of various behavioral manipulations. The approach has been used to demonstrate changes in brain activity related to self-stimulation reward (Gallistel, 1981), feeding and drinking (Roberts, I980), and other motivationally significant behaviors. The system software has several powerful features. Images can be windowed in order to enhance a limited range of optical densities. A histological image can be superimposed upon the autoradiograph in order to easily locate histological details. The software can also statistically analyze the numerical data and compute normalized indices of the optical densities. The TV camera system developed by Gallistel et al. ( I 982) has less resolution and a smaller array size than the rotating drum densitometer system of Goochee et a!. (1980a,b). However, TV camera-based digitizing systems are less expensive than most microdensitometers and can scan an image far more rapidly than even the newest highspeed scanning densitometers. It is also possible to use photometry-based image processing systems to quan-

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tify imniunohistochemical staining in brain and other tissues. Our lab (in collaboration with Dr. Robert Spencer) has been using a DADS-560 Microscope Photometer Data Acquisition and Display system to estimate the staining intensity of an anterograde lectin tracer-wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). The DADS-560 (Table 111) includes a Leitz MPV compact photometer linked to a Z80A-based data acquisition system. The system is programmed to control the read-rate of the photometer (by controlling the duration and frequency at which the photomultiplier shutter is open), and the speed, dwell, and stepping size of the microscope stage. The maximum array size is 128 x I28 points if the scan is displayed graphically. Without display, the image can be histogram processed into an almost infinite number of bins. Maximum grey scale resolution for each array point is 4096 grey levels. The DADS560 is designed for fluorescence microscopy and is therefore programmed to normalize data to a reference standard. Image array and grey scale data are stored by an Okidata 64K ZXO-based color graphics microcomputer. The digitized microscope image can be manipulated and displayed in several formats with this computer system. intensity differences can be represented as color compressed maps using eight colors to represent different intensity ranges. A black and white image can also be generated by windowing a selected intensity range and displaying it monochromatically. A histogram of the number of pixels (counts) occurring at each intensity lcvel is displayed next to each color-compressed intensity map (Fig. 8). Display of smaller intensity differences can bc obtained by wintiowing an intensity range on the histogram. I t i s also possible t o threshold the data to mask lower or higher intcnsities. 'The DADS-560 programs. written in machine code. will also calculate the percentage area (of the total tissue scanned) rcprescntecl by one o r niore intensities. We have used the system to detect and meastir2 thc relative intcnsitiea 01' wheat germ ~~gglutinin-lioiseradisliperoxiciaae staining contained in retinal afterents within the latel-ul geniculatc nucleus of the cat thalamus. By choosing an appropriate photomultiplier high voltage setting. we have becn able to digitize ii specinien so that most of the label falls in a diffcrcnt grey acale range than the unlabeled tissue. By windowing intensity levels, u c can selectively display the W G A - tlK P I abe I wit ho ti t \i g ti i fi ca t i t con t a m i na t io 11Iro I N 11n 5 t a i ned ce I Is or o t he r artifacts (Fig. 8). Relative differences in the intensity of labeling in diflcrent regions of the nucleus ant1 the perccntagc area of the nucleus containing label of a particular intensity c;tn then bc measured. Thc technique should prove uaeful in comparing innervation densities tollowing such cxperimental manipulations as light deprivation and brain lesions. Making comparative measurements betwecn experimental groups may. of course. require careful normalization procedures and analysis of ;I large number of tissue samples,. The DADS-560 has a high grey scale resolution and superior sensitivity for

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Fic. 8. CRT display from a DADS-560 computerized photometry system, originally photographed in color. Label within retinal terminals in the lateral geniculate nucleus is shown on the right. Greys within the bands of label represent different labeling densities (coded by different colors). Histogram at left shows the distribution of intensities for all picture elements in the scan.

low level light detection. Like other computerized photometric systems, however, it is limited in the specimen size that can be scanned and in the number of array points which can be displayed graphically. The speed with which the unit can scan (and thus digitize) a specimen is also limited in comparison to video digitizing units. Video digitizing systems have also been used 10 analyze immunocytochemically labeled brain tissue. Cassell et al. (1982), for instance, have used a video image analyzer to study the density distribution of various peptides in the amygdala. Dark field photomicrographic negatives of PAP reacted tissue were digitized with an EyeCom I1 camera system (Spatial Data Systems). Negatives were used for image input in order to digitize the optical density present in the film rather than light intensity. Using this approach, the camera can measure transmittance. The EyeCom I1 camera is capable of digitizing 640 x 480 picture elements at a grey scale resolution of 256. By comparing grey level frequency histograms, the authors found differences in the density of different peptides in

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particular nuclei of the amygdaloid complex. They also demonstrated a reduction in peptide density after lesions of selected afferents. It seems likely that both photometric and video image processing systems will be used extensively in the future to analyze autoradiographically and immunocytochemically labeled tissues. Computerized analysis systems provide spatial maps of the distribution of label, a method for measuring relative labeling densities, and the ability to compute the amount of tissue labeled.

IX. Other Cell Biology Computer Applications Computer-assisted measuring devices are used in many other laboratory applications of interest to cell and neurobiologists. Gas chromatograph mass spectrometer computer systems are now quite common (see Shapiro et al., 1976, for a review of some of these instruments). Microcomputer and microprocessor assisted cell sorters and flow cytometry devices are also being used increasingly in biomedical laboratories. Flow cytometry is a technique for separating and analyzing cell types which are suspended in solution. Although the resolution of this technique is relatively low, it is capable of analyzing cells at very high speeds (on the order of 100,000 cells per minute). A detailed discussion of these techniques is beyond the scope of this review, but the reader is referred to the following detailed discussions of the computer-based technique (Amdt-Jovin and Jovin, 1979; Hiebert et al., 1981; Miller et al., 1978; Peters et al., 1982; Salzman et al., 1978; Voet et al., 1982). Computers are also being employed for modeling biological processes. Mathematical modeling of such diverse events as neural plate development in newt embryos, bacterial wall surface growth, and protein structure are presented in a recently edited book by Geisow and Barrett (1983). Computer analysis of radioimmunoassay data is also quite common (see, for instance, English, 1981; Faure et al., 1980). There are a variety of other applications not covered in this review. The reader is referred to articles by Carroll ef al. (1981), Crawley e; al. (1982), and Emmett-Oglesby et al. ( 1982) describing uses of microcomputers in behavioral experiments. Tzikoni et al. (198 1) and Patek and Tomkins (1980) discuss computer programs useful for real-time signal acquisition and analysis in physiological experiments. The use of microcomputers in biochemistry is discussed by Giles (1981). The literature on computer uses in these areas will increase enormously in the next several years.

X. Conclusions This article has reviewed a variety of computer uses in cell and neurobiology and also discussed microcomputer hardware and software. Commercial and non-

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commercial systems for microscope control and plotting, three-dimensional reconstruction, morphometry, image analysis, photometry, fluorescence microscopy, gel electrophoresis, and autoradiographic and immunohistochemical analysis have been described. The review should help direct the reader to the appropriate computer technology for hidher research application. I have tried to include a broad survey of the applications literature rather than a detailed discussion of the merits of individual computer systems. Choice of an appropriate computer system requires careful consideration of many factors. It is difficult to assess the quality of computer systems based solely on written accounts. A detailed critique of available systems is thus beyond the scope of this review. Nevertheless, the information I have included will direct you to literature and system users which should be helpful in evaluating various equipment. Commercial manufacturers are rapidly developing computer-based systems for many cell and neurobiology applications. It is far easier today to find a moderately priced commercial system to fit your research needs than was the case just a few years ago. Many commercial vendors are now also interfacing specialized laboratory instruments to inexpensive general-purpose microcomputers. It is therefore often preferable to purchase a commercial system rather than undertake the time-consuming task of developing your own hardware interfaces and software. However, many computer applications remain highly specialized and are unlikely to be developed commercially. Software exchange among colleagues in these cases is of great value to the scientific community. There is little doubt that almost all laboratory functions involving on-line measurement, control, and analysis will become computer automated over the next decade. The dramatic reduction in the price of large scale integrated circuits, volatile memory, and mass storage makes this revolution inevitable. The major stumbling blocks in the revolution are the training of scientists to use the technology and the development of software for specialized applications. I hope this review contributes to those endeavors.

ACKNOWLEDGMENTS I wish to thank Dr. Jerome Sutin and the American Association of Anatomists for inviting me to prepare a portion of this review for a Workshop on Microprocessors at the AAA Annual Meeting (1983). Mary King Givens and Ellen McDonell, Reference Librarians with the University of Tennessee Mooney Medical Library, contributed by assisting with computer searches of the literature. Linda Homer expertly prepared the figures. Jeffrey Peery and Angela Smith proofed and Mary Gaither typed the manuscript. Some of my laboratory’s research cited in this review was supported by NIH Grant EY02973 from the National Eye Institute, a New Faculty Research Grant from the State of Tennessee, and an Equipment Grant from the College of Medicine. Vashaw Instruments and Leitz, Inc., generously loaned my laboratory the DADS-560 Photometry Data Acquisition and Display System.

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Rez, P., and Williams, D. B. (1982). Ultramicroscopy 8, 247-252. Rich, E. S., and Wampler, J. E. (1981). Clin. Chem. 27, 1558-1568. Rink, M. (1977). Microsc. Acta (Suppl) 1, 189-190. Robb, R. A., and Jowsey, J. (1978). Calcq. Tissue Res. 25, 265-271. Roberts, W. W. (1980). J. Comp. Neurol. 194, 617-638. Rogers, A. W. (1973). In “Techniques of Autoradiography.” Elsevier, Amsterdam. Rundquist, 1. (1981). Hisrochemistry 70, 151-159. Rust, H.-P., and Krahl, D. (1982). Ulrramicroscopy 8, 287-292. Ruter, A,, Aus, H. M., Harms, H., Haucke, M., ter Meulen, V., Maurer- Schultz, B., Korr, H., and Kellerer, A. (1979). J . Hisrochem. Cytochem. 27, 217-224. Salin, E. (1982a). Am. Lab. 14, 80-86. Salin, E. (1982b). Am. Lab. 14, 35-38. Salzman, G. C., Hiebert, R. D., and Crowell, J. M. (1978). Compur. Biomed. Res. 11, 77-88. Schipper, J., Tilders, F. J. H., and Ploem, I . S. (1978). J . Hisrochem. Cytochem. 26, 1057-1066. Schmassmann, A,, Mikuz, G., Bartsch, G., and Rohr, H. (1979). Microsc. Acta 82, 163-178. Schmidt, W. (1982). Anal. Biochem. 125, 162-167. Shantz, M. J., and McCann, G. D. (1978). IEEE Trans. Biomed. Eng. 25, 99-103. Shapiro, M. B., Schultz, A. R., and Jennings (1976). Annu. Rev. Biophys. Bioeng. 5, 177-204. Sharp, T. R., Gopinath, K. R., Brandt, P. W., and Rosenbeny, T. L. (1981). Anal. Biochem. 116, 545-552. Shipton, H. W. (1979). Annu. Rev. Biophys. Bioeng. 8, 269-286. Shoemaker, R. L.. Bartels, P. H., Hillman, D. W., Jonas, J . , Kessler, D., Shack, R. V., and Vukobratovich, D. (1982). IEEE Trans. Biomed. Eng. 29, 82-91. Sklarew, R. J. (1982a). J . Hisrochem. Cyrochern. 30, 35-48. Sklarew, R. J. (1982b). J . Hisrochem. Cytochem. 30, 49-57. Slavin, G., Sowter, C . , Ward, P., and Paton, K. (1982). J . Clin.Pathol. 35, 1268-1271. Smith, K. C. A. (1982). J . Microsc. 127, 3-16. Smith, M. T., Wills, E. D., Drew, K., Maxwell, C., Daly, J. R., Reader, S. C. J., and Robertson, W. R. (1980). Hisrochemistry 68, 321-323. Smith, P. R. (1978). Ultramicroscopy 3, 153-160. Sobel, I., Levinthal, C., and Macagno, E. R. (1980). Annu. Rev. Biophys. Bioeng. 9, 347-362. Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, M. H., Patlak, C. S., Pettigrew, K. D., Sakurada, 0 . .and Shinohara, M. (1977). J . Neurochem. 28, 897-916. Sprumont, P. (1980). Compur. Programs Biomed. 12, 7-13. Statham, P. J. (1982). Ultramicroscopy 8, 309-320. Stengel-Rutkowski, S., Gundlach, H., and Zang, K. D. (1974). Exp. Cell Res. 87, 313-325. Stevens, J. K. (1980). Microsc. Soc. Can. Bull. 8, 4-12. Stevens, J. K., Davis, T. L., Friedman, N., and Sterling, P. (1980). Brain Res. Rev. 2, 265-293. Stewart, S. S., Miller, R. G., and Price, G. B. (1980). Cytornetry 1, 204-211. Street,,C. H., and Mize, R. R. (1983). J. Neurosci. Methods 7, 359-375. Street, C. H., and Mize, R. R. (1984). In “The Microcomputer in Cell and Neurobiology Research” (R. R. Mize, ed.), Elsevier, Amsterdam, in press. Tenny, J. R., Long, J. W., Jr., McFarland, W. D., Vorbeck, M. L., Townsend, J. F., and Martin, A. P. (1980). Comput. Programs Biomed. 12, 1-6. Thiessen, G., and Thiessen, H. (1977). Prog. Hisrochem. Cytochem. 9, 1-156. Turner, J. N. (1981). Methods Cell Biol. 22. Tzikoni, E., Feldman, S., and Kedem, J. (1981). Compur. Programs Biomed. 13, 151-156. Uylings, H. B. M., Parnavelas, J. G., and Walg, H. L. (1981). In “Advances in the Morphology of Cells and Tissues,” pp. 185-192. Liss, New York. Van der Ploeg, M., van den Broek, K., and Mitchell, J. P. (1979). Histochemisrry 62, 29-43.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL YO

Effect of Microtubule Inhibitors on Invasion and on Related Activities of Tumor Cells MARCM . MAREELAND MARCDE METS Laboratory of Esperimental Cancerology, Department qf Radiotherapy and Nuclear Medicine, University Hospitcil, Ghent, Belgium

1.

.....................................

ubule AssemblyiDisassembly . . . . . . . . . . . . . A. General Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Microtubule Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Analysis of Microtubules Inside Cells . . . . . . . . . . . . . . . . . . . . . . . . . IV. Antiinvasiveness of Microtubule Inhibitors A. Observations in Vitro.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Observations in Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Antiproliferative and Cytotoxic Effect of Microtubule Inhibitors. . . . VI. Directional Migration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. An Explanation for the Antiinvasive Activity of Microtubule Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Assays for Cell Migration. . . . . . . . . . . . . . . C. Effect of Microtubule Inhibitors on Migratio .................... D. Mechanisms of Direction Finding E. Directional Migration of Invading Cells. . . . . . . . . . . . . . . . . . . . VII. Effect of Microtubule Inhibitors on Plasma Membrane Functions . . . VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11.

12s 126 126 126 134 139 139 143 144 148 I48 148 153 158 IS9 161 161 162

I. Introduction Invasion marks the onset of malignancy in solid tumors. A consequence of invasion is metastasis leading to the death of the host in many cases. Students of invasion and metastasis hope that the understanding of its mechanisms will lead to the development of a more efficient therapy of cancer. So far, little is known about these mechanisms. Analysis of the tumor activities necessary for invasion is one possible approach to the problem. That migration brings tumor cells into neighboring tissues was already accepted at the end of the nineteenth century, although direct evidence for it was lacking. Later, time lapse films have confirmed this opinion at least for some experimental tumors. The question why tumor cells migrate into neighboring tissues and not for example along tissue boundaries, as it occurs during wound healing, has not been answered. It is postulated that invasive I25 Copyright I: IYX4 by Academic 1’r.e~. Inu All right\ ot reproduction ~n any turni rcwvcd. ISBN n - i ? - 3 w ~ o - ~

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tumor cells respond to factors that determine their direction of migration. Experimental evidence that tumor cells are sensitive to such factors has been obtained both in vitro and in vivo. Some of the evidence comes from experiments with microtubule inhibitors (MTIs). These agents disturb the equilibrium between microtubule (MT) assembly/disassembly . We review here data about the effect of MTIs on invasion, on directional migration, and on other cellular activities that are possibly involved in invasion. We discuss the hypothesis that MTIs inhibit tumor invasion because they interfere with directional migration through disturbance of the cytoplasmic microtubule complex (CMTC). The review includes biochemical experiments (in the test tube), experiments with cultured cells (in virro), and experiments using whole organisms (in vivo).

11. Biochemistry of Microtubule Assembly/Disassembly A. GENERAL CONCEPTS

The building block of MTs is tubulin. Tubulin is a 6 S dimeric protein of 110,000 molecular weight. It consists of 2 nonidentical chains of about 50,000 daltons designated a and p. Polymerization of tubulin dimers produces the protofilaments which organize to form a MT. MTs are labile structures as the equilibrium between the MT and the pool of free tubulin results from continuous assembly/disassembly at both ends of the MT (Margolis and Wilson, 1981). Figure 1 outlines current concepts about MT assembly/disassembly. Tubulin flow from the net assembly end toward the net disassembly end, termed treadmilling, provides the MT with a potential vectorial transport mechanism. GTP hydrolysis normally accompanies MT assembly although it is not an absolute requirement for it (Margolis, 1981). Possible functions of GTP hydrolysis are the maintenance of differences in critical tubulin concentrations, strengthening of dimer-dimer bonds or protecting the assembly capacity of tubulin (Maccioni and Seeds, 1983), and support of subunit treadmilling. Other factors involved in MT assemblyldisassembly are MT-associated proteins called MAPS. These include MAP 1 and 2 and the low-molecular-weight factor tau (Murphy and Borisi, 1975; Sloboda et al., 1976; Weingarten er al., 1976). It is important to notice that the concept is based mainly on observations of MT assembly/disassembly in the test tube, which have not been repeated systematically in living cells in vivo or in vitro. Important factors spatially regulating MT assembly in cells and absent in the test tube are microtubule organizing centers (MTOCs).

B. MICROTUBULE INHIBITORS The term MTIs has been used originally for agents that interfere with MT assembly. In the present review it will be applied to all substances that affect the

MICROTUBULE INHIBITORS

TUBULlN

+

COLCHlClNE

c3+

0

-

127

T UBULIN -C OLC H IC IN E

c3

FIG. I . Schematic representation of microtubule assembly/disassembly under control conditions (top) and in presence of colchicine (bottom). Tubulin flux is from the net assembly end (Ad) toward the net disassembly end (aD). Modified from Margolis and Wilson, (1981) and from Schiff and Horwitz, (1981b).

normal equilibrium between MT assembly and MT disassembly. Table I shows the structure and origin of the MTIs discussed in the present review. From a chemical point of view, knowledge about the interaction of tubulin with various drugs is restricted. The chemical structure of most of these drugs is complex, and substitution of certain groups of atoms does not permit a decision about the importance of such parts of the drug molecule in the interaction with tubulin or with MTs. Therefore, tubulin-drug interactions are of a complex nature, as they are the sum of chemical and of physicochemical processes such as van der Waals interactions, hydrogen bonds, dipole interactions, etc.

MARC M. MAREEL A N D MARC DE METS

128

TABLE I STRUCTURE AND O R I G I N OF SOMEMICROTUBULE INHIBITORS Name and formula

Structure

Colchicine

Origin

MW

Colchicum autumnale

399

37 1

Colcemid Cz I H 25 NO 5 R = CH3

Lumicolchicine C22H25N06

UV irradiation of colchicine

399

Steganacin 424

C24H 24O7

Podophy llotoxin C22H2208

n

OH

0

ocn,

ocn,

Podophyllum peltarum. P . emodi, Juniperus virginiana

414

129

MICROTUBULE INHIBITORS TABLE I Name and formula

(Continued)

Structure

VP- 16-213 C29H3 I

R

=

Origin

MW

Semisynthetic

587

Semisynthetic

655

I3

CH3

on

191

Methylbenzimidazol-2yl-carbaniate (MBC) CYHYN~O~

R:H M B t

30 I

Vinblastine (VLB) C46HSX"LOY Rl = CH1 R 2 = OCOCH, R3 COOCH,

Carharuntus I'OSCUS (periwinkle)

810

cfr VLB

824

(continued)

MARC M. MAREEL AND MARC DE METS

130

TABLE I

Name and formula

RI Rz R3

(Continued)

Structure

Origin

MW

Semisynthetic

153

Maytenus ovatus

692

Taxus brevifoolia

809

CHO OCOCH3 = COOCH3

= =

J

COOCH,

Desacetyl VLB C44H56N408 RI = CH3 R 2 = OH R 3 = COOCH3 Vindesine (VDS) C43HSSNS07 R I = CH3 R2 = OH R 3 = CONH2

Taxol C46H 19N0

14

0 11

CH,-C-0

-0-

0

MICROTUBULE INHIBITORS

131

What we know is that different drugs interact with different areas of the tubulin molecule or of the MT. Such an area is called a binding site. Binding sites may consist of several binding places, the exact localization of both being unknown. 1. The Colchicine Binding Site It has been known for a long time that tubulin can bind the tropolone derivative colchicine. This binding is specific with regard to the binding site and stable; the resulting tubulin-colchicine complex can be used for quantitating tubulin in extracts. The aromatic ring (A) with three methoxy groups is also present in podophyllotoxin (E) that binds to the same site of tubulin as colchicine. This may point to the stereochemical importance of such substituents. Substitution in the amino group or in the position of the oxy and methoxy groups on ring C of colchicine results in derivatives with altered tubulin binding activity. According to different investigations (Shelanski and Taylor, 1967; Weisenberg et al., 1968; Wilson et al., 1974; Margolis et a l ., 1980), tubulin dimers have one high-affinity binding site for colchicine. Values of dissociation constants depend on the method of measurement and on the origin of the tubulin. The kinetics of the binding reaction are temperature dependent. At O"C, colchicine does not react with tubulin, but a preformed colchicine-tubulin complex is stable at 0°C (Wilson, 1975). Garland (1977) has suggested that the binding mechanism entails several conformational changes, the formation rates of which are different. Anions favor the rate of colchicine-tubulin binding, by making the binding site of the C-ring of colchicine more accessible to the tubulin molecule (Bhattacharyya and Wolff, 1976). The amount of colchicine bound per milligram tubulin is decreased by addition of vinblastine, Nocodazole, and bleomycin while the inverse is found for procarbazide and chlorambucil (Brodie et al., 1979). Wilson and Meza (1973) have indicated that the colchicine binding site of tubulin is not expressed on the surface of the intact MT. Polymerization of tubulin can readily be distinguished from its binding ability to colchicine, as demonstrated by Barton (1978). Colchicine is supposed to inhibit MT assembly by sequential binding to soluble tubulin, and this colchicine-tubulin complex blocks the polymerization by binding at the MT end (Fig. 1). It has been demonstrated by electron microscopy that the preformed colchicine-tubulin complex has a greater effect on MT formation than pure colchicine (Margolis and Wilson, 1977). However, the tubulin-colchicine complex partially inhibits tubulin polymerization in concentration ranges about 100- lOOOX in excess of the number of MT ends. An irreversible blocking would be inconsistent with such values. Therefore, MT inhibition by colchicine has been proposed as a reversible binding. Another possibility is that the colchicine-tubulin complex does not block, but impairs further addition of tubulin subunits to the growing MT. This would result in a tubulin/tubulin-colchicine copolymer, the impairment of which de-

132

MARC M . MAREEL AND MARC DE METS

pends on the mole fraction of the incorporated complexes (Sternlicht and Ringel, 1979; Sternlicht et al., 1980). This results in a model of 20 to 30 tubulincolchicine complexes incorporated per MT challenging either reversible or irreversible MT end-blocking. The results obtained by Keates and Mason (1981) indicate that inhibition of MT elongation is consistent with a reversible association of the tubulin-colchicine complex at the MT end. Whether the a or the /3 chain of tubulin contains the colchicine binding site is unknown. It has been reported that colchicine stimulates the GTPase activity of tubulin (David-Pfeuty et al., 1977, 1979) and that the trimethoxybenzene ring (A) of the colchicine molecule is primarily responsible for this stimulation (Lin and Hamel, 198 1). This would indicate that colchicine inhibits MT assembly and, at the same time, creates a condition favorable for MT assembly. Several structural analogs and derivatives of colchicine have been investigated for their abilities to affect MTs. Among these substances, colcemid and lumicolchicine are best known. Lumicolchicine can be obtained by ultraviolet irradiation of colchicine. This substance does not bind to tubulin, illustrating the importance of the tropolone moiety (C-ring) in the colchicine molecule for its interaction with tubulin. Steganacin is another analog of colchicine. Its formula shares the trimethoxybenzene ring with both colchicine and podophyllotoxin. Steganacin has been shown to inhibit MT assembly in the test tube (Wang et al., 1977; Zavala et al., 1980). It is a competitive inhibitor of colchicine binding to tubulin, indicating that its mechanism of action is similar to that reported for colchicine (Schiff and Horwitz, 198 lb). Podophyllotoxin reacts with tubulin at the same site, but not at an identical place as does colchicine. It is supposed that the trimethoxy ring in both drugs is responsible for their reaction with a specific receptor in the tubulin molecule. The binding rate of podophyllotoxin is about 10 times faster than that of colchicine. The reaction proceeds at 0°C and is reversible (Cortese et d . , 1977). VP- 16-2 13 and VM-26 are semisynthetic derivatives of podophyllotoxin with a glucoside moiety at C,. They do not inhibit MT assembly at concentrations 20 times higher than the effective concentrations of podophyllotoxin in the test tube (Loike and Horwitz, 1976; Loike et al., 1978) and do not affect the MT assembly/disassembly equilibrium in vitro (Mareel et al., 1982a). On the other hand, 4'-demethylepipodophyllotoxin,the nonglucoside congener of VP- 16-2 13, inhibits MT assembly. These results suggest that the glucoside moiety could sterically hinder the ability of ring D to bind to tubulin (Loike et al., 1978). The effect of 100 pA4 of podophyllotoxin, colchicine, and vinblastine on the alkylation of tubulin with iodoacetamide has been described by Luduena and Roach (1981a,b). The reaction is inhibited in the range 19-72%, with halfmaximal effectiveness at 3-5 pA4 concentration of the drugs. This indicates that the suppressive effect of the drugs is mediated by their high-affinity binding site. Podophyllotoxin has been shown to inhibit the GTPase activity of tubulin

MICROTUBULE INHIBITORS

133

(David-Pfeuty et ul.. 1977, 1979) in contrast with the effect of colchicine. This inhibitory effect must derive from the tetrahydronaphthol moiety in podophyllotoxin (Lin and Hamel, 1981). Indole derivatives were shown to interact with tubulin. Among these products, methylbenzimidazol-2-yl-carbamate(Davidse and Flach, 1977) binds to tubulin at the colchicine site, but does not inhibit binding of podophyllotoxin. Nocodazole (De Brabander e f ul. , 1975; Hoebeke et al., 1976) blocks MT assembly in the test tube and in vitro, leaving a pool of unpolymerized subunits. The binding to tubulin is a reversible and partly temperature dependent reaction, with a maximum at 25°C (Hoebeke et ul., 1976). It has been reported that Nocodazole binds to both the tubulin dimers and the polymeric form. However, the reconstitution of MTs in the test tube is inhibited by this drug. From a chemical point of view, the sulfiydryl residues of such a nocodazole-tubulin complex become more accessible to chemical modification (Lee et ul. , 1980). Nocodazole stimulates the GTP hydrolysis of tubulin (Lin and Hamel, 1981), as does colchicine. 2. The Vinblustine Binding Site It has been observed that vinblastine binds to tubulin at sites different from the binding sites of colchicine and podophyllotoxin (Bryan, 1972). This binding reaction is very different from that of colchicine: the binding is rapid, reversible, and independent of the temperature (Wilson e t a / ., 1978). Furthermore, calcium, colchicine, or GTP does not affect this binding reaction. The number of binding sites of vinblastine to the tubulin dimer has not been fixed unambiguously, because the use of different methods results in different data. However, it is most probable that tubulin has two binding sites with high affinity for vinblastine, both different from the colchicine site. Apart from this, several low-affinity binding sites may exist, the number of which might depend on the origin of the tubulin. Differential aggregation states of tubulin are induced in vitro by different concentrations of vinblastine: dimers are obtained at concentrations of 2X 10- 5M (Lee ef ul., 1975), while larger structures such as spirals, rings and double helices are obtained at concentrations of 1 X IOP4M (Fugiwara and Tilney, 1975; Erickson, 1975). Analogous effects of vinblastine are observed on membrane protein, actin, DNA, and polyribosomes. David-Pfeuty et ul. (1977,1979) reported that vinblastine inhibits the tubulin-dependent GTP hydrolysis, as does podophyllotoxin, but in contrast with colchicine. Vincristine and desacetylvinblastine are inhibitors of the vinblastine binding (Wilson et ul., 1975). It has been demonstrated that both the catharantine and the vindoline moiety of the inhibitor molecules are involved in the binding reaction with tubulin (Owellen et u l . , 1977). Maytansine competes with vinblastine and vincristine for binding to tubulin (Bhattacharyya and Wolff, 1977). This binding is not inhibited by colchicine, and tubulin seems to have an additional specific binding place for maytansine

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MARC M. MAREEL AND MARC DE METS

(Mandelbaum-Shavit et al., 1976). Maytansine does not initiate aggregation of tubulin (Bhattacharyya and Wolff, 1977) but produces microtubule disassembly in vivo (Schnaitman et al., 1975) and in the test tube (Luduena, 1977). The concept is that maytansine is an alkylating agent and denatures tubulin by reaction with a critical group near its binding site in the MT polymer (Luduena, 1979). It also inhibits GTP hydrolysis of tubulin (Lin and Hamel, 1981), as does vinblastine. 3. Taxol Taxol demonstrates some properties that are opposite to these of MTIs. It is the only drug that promotes microtubule assembly in the test tube, in vitro, and in vivo by decreasing the tubulin concentration critical for polymerization (De Brabander et al., 1981; Heidemann and Gallas, 1980). MTs formed in the presence of taxol do not depolymerize in the cold (4°C) or by addition of 4mM CaCl, (Schiff and Horwitz, 1980). The lag time for MT assembly is decreased and almost completely eliminated by 10 pM taxol (Schiff et al., 1979). There are certain circumstances that influence the action of taxol on the tubulin polymerization. Absence of MAPS in tubulin preparations does not alter assembly in the presence of taxol (Schiff and Horwitz, 1981a). Assembly is also induced by taxol in cells pretreated with Nocodazole, but the pattern depends on the relative concentrations of taxol and Nocodazole (De Brabander et al., 1981). Concentrations of 100 pM taxol and 2-0.2 pA4 Nocodazole result in the production of random MTs as in cells not pretreated with Nocodazole. With 5-2.5 pM taxol and 20 pM Nocodazole, centrosomal MTs are far in excess of free MTs. Parness and Horwitz (1981) suggest that there is a competition between the drugs podophyllotoxin, vinblastine, and taxol for different forms (dimer and polymer) of tubulin, and not for a single binding site. These authors also suggest that taxol stabilizes interactions between tubulin dimers, binds specifically to intact MTs (see also Schiff and Horwitz, 1981b), and stabilizes the formed MTs. Parness et al. (1982) investigated some taxol analogs and semisynthetic derivatives, and concluded from these experiments that the presence of the taxane ring in a molecule is not sufficient to induce MT assembly in the test tube and in virro; they claim that an ester-linkage at C,, is essential for these effects. Taxol does not induce polymerization of purified actin (Parness and Horwitz, 1981).

111. Analysis of Microtubules Inside Cells

The equilibrium of MT assembly/disassembly in the test tube is amenable to quantitative analysis but the status of MT complexes inside cells is difficult to evaluate. Nevertheless, evaluation of the effect of MTIs on MT complexes under the circumstances of each experiment is a prerequisite for drawing conclusions

MICROTUBULE INHIBITORS

135

about the causal relationship between drug-induced alterations of cellular functions and disturbance of MT complexes. Spindle MTs serve a well known function, namely separation of chromosomes during mitosis (De Brabander et af., 1979). Therefore, the effect of MTIs on spindle MTs can be inferred from alterations of mitotic cells such as metaphase arrest and formation of multimicronucleated cells (De Brabander et al., 1976a). The problem is less simple when cytoplasmic MTs are concerned. Immunocytochemical staining with antiserum against tubulin is probably the best method to reveal the CMTC in individual whole cells spread on artificial substrates (Fig. 2 ) . Technical problems associated with such immunostainings have been recently reviewed (De Mey, 1983). One of them is the relative lack of penetration of the primary antiserum and/or of other components of the procedure into cells even when they are permeabilized (Fig. 3). Another problem is that MTs are difficult to be distinguished in round cells (Frankel, 1976; De Mey et al., 1976; Osborn and Weber, 1977) because they are superimposed upon each other or in a direction perpendicular to the plane of focus. In most cells immunocytochemistry allows at best a semiquantitative estimation of the CMTC. For MO, mouse cells cultured on coverslips we have used the following criteria (Fig. 2): the CMTC is normal when bundles of MTs irradiate from the perinuclear area to the cell periphery; it is disturbed when MT bundles are short and/or irregularly scattered over the cytoplasm; MTs are absent when the cytoplasm is diffusely stained (Mareel et al., 1982a). Some MTIs, for example, vinca alkaloids, produce so-called paracrystals (Fig. 2 ) . This phenomenon, however, occurs at drug concentrations that are at least one order of magnitude above these that disturb the CMTC. It should also be noted that MT-mediated cellular activities are affected at concentrations of MTIs below these needed for complete disappearance of the CMTC. Discrete changes of the CMTC such as alterations in the relationship of MTs with the plasma membrane (Eichhorn and Peterkorfsky, 1979) are not visualized in immunocytochemical preparations. Using indirect immunofluorescence Anderson et al. (1982) have counted the number of MTs and measured their length in polymorphonuclear leucocytes. After exposure to a chemotactic gradient definite changes in the average length and range of MT lengths occurred but their number (about 35) remained constant. Colchicine at concentrations between lo-’ and l o p 8 M lowered both the number of MTs per cell and the number of cells with intact MTs. Whether this quantitative method can be applied to other types of cells remain to be examined. Transmission electron micrographs have a tendency to underestimate both the number and the length of MTs even in appropriately fixed cells (Chernoff and Overton, 1979). Such underestimations have been used as an argument against the role of CMTs in the migration of epithelial cells (DiPasquale, 1975; Nakatsuji, 1979). Since the advent of immunocytochemistry most authors agree that epithelial cells have a well elaborated CMTC which is sensitive to MTIs (for reviews see Middleton,

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1982; Vasiliev, 1982). Random ultrathin sections were used by Malech er al. (1977) to count relative numbers of MTs within a radius of 1 .O p m around the centriole. They found that the orientation of polymorphonuclear leukocytes, the position of the centriole, and the number of MTs responded to the same concentration of colchicine as did directional migration. Ultrastructural analysis is

FIG. 2. Light (a,b,c,e,f) and phase contrast (d) micrographs of C3H mouse fibroblastic cells (MO) stained with antiserum against tubulin and showing various aspects of the cytoplasmic microtubule complex (CMTC). a, Normal CMTC; b , disturbed CMTC after incubation for I hour at 4°C; c , c , absence of CMTC and diffuse staining of the cytoplasm after incubation with 1 pg Nocodazolei ml for 2 hours; d , disturbed CMTC and absence of microtubules in peripheral lamella after incubation with I p g taxol/ml for 2 hours; e, paracrystals after incubation with 10 pg epideoxyvincristine for 7 days; f. niultinucleated cell with CMTC after incubation with 0.003 pg vinblastineiml for 2 days. Scale bars = 25 p m .

MICROTUBULE INHIBITORS

I37

Ftc. 3. Transmission electron micrograph of a horizontal section from a culture of MO, cells on plastic. Cells were stained with antiserum against tubulin following the unlabeled antibody enzyme method and embedded for sectioning afterward. The section is more peripheral and more microtubules are visualized in cell A than in cell B. This phenomenon is ascribed to limited penetration of the reaction products into the cells. Scale bar = 5 pm. Inset: stained cells before embedding; scale bar = 20 pm.

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also useful to visualize the effect of MTIs on sections from three-dimensional systems which are relatively unaccessible to immunostaining with antiserum against tubulin. Interference with MT assembly provokes a spectrum of fairly typical ultrastructural alterations: absence or scarceness of MTs, dislocation of the Golgi complex from its perinuclear position with scattering of smooth endoplasmic reticulum over the cytoplasm, accumulation of 10-nm filaments, and in some types of cells appearance of annulate lamellae (George et al., 1965; Journey et al., 1968; Krishan et al., 1968; Krishan and Hsu, 1969; De Brabander and Borgers, 1975; Chemnitz et a l . , 1977). A number of authors have relied on mitotic arrest or on the accumulation of metaphases to control the effect of MTIs on the CMTC. This was criticized by Goldman (in Trinkaus, 1973) who suggested that the CMTC should not necessarily react with MTIs in the same way as does the mitotic spindle. In our experiments (Storme and Mareel, 1980; Mareel et a l . , 1980; Storme et al., 1981; Mareel et al., 1982c) cell proliferation was slightly more sensitive to MTIs than directional migration and the effect of the MTIs on mitosis was less rapidly reversible than that on directional migration. For example, it was not unusual to observe multimicronucleated cells (defectuous mitotic spindle) that were polarized and had a well established CMTC (Fig. 2) after removal of the MTIs. On the other hand experiments were described where a dose of MTI which affected the CMTC had no effect on spindle MTs (Behnke, 1970). Accumulation of cells in metaphase can be used to assess the effect of MTIs only in proliferating cell populations. Here, accumulation of metaphases will depend not only on the effect of MTIs on the mitotic spindle but also on their effect on the progress of cells through other phases of the cycle (Wells et al., 1976; Sweeney et al., 1978; Sluder, 1979). A group of Swedish researchers (Norberg and Uddman, 1973; Bandmann et al., 1974; Bandmann, 1976; Rydgren et al., 1976) have used inhibition of oxalate-induced radial segmentation to assess the effect of MTIs on the CMTC of lymphocytes and monocytes. In this assay threshold concentrations of MTIs for inhibition of radial segmentation are similar to threshold concentrations for metaphase arrest. Methods for the quantitative determination of the ratio between assembled and unassembled tubulin inside cells do exist (Solomon and Magendantz, 1981). They consist of extraction of unassembled tubulin with buffers that preserve most MTs leaving so called cytoskeletons. For determination of assembled tubulin cytoskeletons are treated with depolymerizing solutions. To our knowledge these methods have not been used for the study of the effect of MTIs on the cellular activities discussed in the present review. In our opinion, it is not advisable to rely on data from the literature for the evaluation of the effect of MTIs on MT complexes unless the types of cells and the conditions of the experiments are strictly identical. Striking differences in sensitivity to MTIs have been found between various types of cells (Sweeney et al., 1978; Gupta, 1983; Dexter et al., 1983). We have found that considerably

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139

higher concentrations of MTIs were needed to affect MT complexes in organ cultures on semisolid agar-agar medium as compared to fluid medium (Mareel et al., 1980). This phenomenon has been ascribed to differential binding of MTIs to various proteins in the culture media and to differences in the diffusion of MTIs into the tissues, but other factors could not be excluded. Together, these data stress the need to include a proper evaluation of the MT complexes in experiments about the causal relationship between alterations induced by MTIs and disturbance of MT complexes.

IV. Antiinvasiveness of Microtubule Inhibitors

A. OBSERVATIONS in Vitro Growing interest in tumor invasion has been accompanied by the development of a number of in vitro assays for invasiveness (review by Mareel, 1980). None of these assays has been generally accepted [for discussion see chapters 10 to 16 in “Invasion and Metastasis” (L. Liotta and I. Hart, eds.). Martinus Nijhoff, The Hauge, 19821. For an investigation of the antiinvasive activity of MTIs we have used confrontations of precultured fragments of embryonic chick heart with aggregates of MO, cells in organ culture (Mareel et al., 1979). MO, cells are virally transformed C3H mouse fibroblastic cells. They are malignant by all standards since they produced invasive and metastasizing fibrosarcomas after implantation into syngeneic mice (Meyvisch and Mareel, 1982). Confronting pairs of heart tissue and MO, cell aggregates were incubated individually in fluid medium on a gyrotory shaker and fixed for histologic examination after 2 to 7 days. They were completely serially sectioned and consecutive sections were stained with hematoxylin-eosin and with an antiserum against chick heart (Mareel et al., 1981a). The following criteria of invasiveness were used: occupation of the heart tissue by MO, cells, degenerative alteration of the heart tissue, and progression of both occupation and degeneration. We have recently discussed the value of the method of analysis (Mareel, 1983) and the relevance of the assay for tumor invasion in vivo (Mareel, 1982). When an aggregate of MO, cells with a diameter of 0.2 mm was confronted with a precultured fragment of heart tissue with a diameter of 0.4 mm, the MO, cells replaced the heart tissue to a large extent within 4 days of incubation (Fig. 4). Addition of MTIs to the culture medium has been found to inhibit completely invasion and to cause MO, cells to remain at the periphery of the heart tissue (Fig. 4) (Mareel and De Brabander, 1978a; Mareel et al., 1980, 1982a,b; Storme and Mareel, 1981). Inhibition of invasion by MTIs has also been shown with K12 rat adenocarcinoma cells which in absence of drugs invaded into the heart tissue following a different pattern and more slowly than MO, cells (Schallier et al., 1982). The antiinvasive effect of

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FIG. 4. Light micrographs of 8-pm-thick sections from confrontations of an M 0 4 cell aggregate with a precultured fragment of 9-day-old embryonic chick heart in culture on gyrotory shaker. Fixation after 4 days; consecutive sections were stained with hematoxylin-eosin (a,c,e) and with an antiserum against chick heart (b,d,f). a and b, control culture; c and d, with I pg cisplatiniml; e and f , with 0.3 pg podophyllotoxiniml. At antiproliferative concentrations cisplatin permits invasion and podophyllotoxin inhibits invasion. Scale bars = 100 pm.

MTIs on MO, cells was also obvious when mesonephros was used as a host tissue instead of heart (Mareel and De Brabander, 1978b). It is probable that MTIs have so-called side effects which are not related to their interaction with MT assembly/disassembly. Binding of MTIs to tubulin (see Section 1I.B) was originally demonstrated through precipitation of tubulin

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by MTls in the test tube. This reaction was, however, not specific for tubulin as a number of other cellular proteins were also precipitable with some MTIs (Wilson et al., 1970; Gietzen et al., 1982). Furthermore, MTls have been shown to affect metabolic pathways which probably do not depend on the structural and functional integrity of MT complexes (Creasey and Markiw, 1964; Richards, 1968; Wagner and Roizman, 1968; Creasey, 1981). There are a number of arguments to accept that disturbance of MT complexes are at the basis of the antiinvasive effect of MTIs in our in vitro experiments. First, inhibition of invasion is a common feature to all agents that are known to affect MTs (Table 11). It is unlikely that such a common effect would be due to nonspecific side effects of different MTIs. The antiinvasive effect has been observed with taxol (Mareel, et al., 1983) that disturbs the MT complexes through unordered assembly and with drugs that inhibit MT assembly through binding to tubulin (see Section 11,B). The latter group of MTIs differ in their chemical structures and in their binding places to tubulin. Second, glycosylated podophyllotoxin congeners that have lost their capacity to bind to tubulin and consequently no longer disturb MT complexes (see Section 11, B , I ) are not antiinvasive in contrast to podophyllotoxin (Mareel et al., I982a). Third, immunocytochemical staining with antiserum against tubulin of MO, cells cultured in presence of MTIs reveals disturbances of MT complexes at drug concentrations similar to these that inhibit invasion in organ culture using fluid medium. Fourth, ultrastructural analysis of organ TABLE I1 ANTIPROLIbtRATIVt DRUGS

I N H I B I T O R Y FOR I N V A S I O N Ob

Mod

CELLS IN

ORGAN CULTURt

Bis-vindesine disulfide (LY 1085550; MW 1631; 3.0 pgiml)

Desacetylvinblastine-P-hydroxyethylamide (LY 1039750; MW 797; 1 .O-3.0 pgiml) Epi-deoxyvincristine (LY 119863"; MW 806; 3.0 pg/ml) Flubendazole (R17889h: MW 313; 0.3-1.0 p,g/ml) Methyl[5-(2-(4-fluorophenyl)- 1,3-dioxolan-2-yl)-lH-benzimidazoI-2-yl] carbamate (R34803"; MW 342; 1.O- 10 pgiml) Methyl[5-(2-thienylcarbonyl)-IH-benzimidazol-2-yl] carbamate (Nocodazolek MW 30 I; 0. I- I .O pgiml)

Podophyllotoxin (Aldrich Europe, Beerse, Belgium; MW 414; 0.1-0.3 pgiml) Taxol (NSC-125973-Lr; MW 809; I .O-10 piml) Tubulazole (R46846",d; MW 508; 0.3- I .0 pgiml) Vinblastine sulfate (Velbe"; MW 898; 0. I-I .0 pgiml) Vinblastine-P-chloroethyloxazolidinedione(LY 104208~1;MW 871.5; 0.3-3.0 pgiml) Vincristine sulfate (Oncovin"; MW 923; 0. I- 1 .0 pgiml) Vindesine (EldisinetJ; MW 753; 0.1-1.0 pgiml) "Eli Lilly and Co., Indianapolis, Ind. "anssen Pharmaceutica, Beerse, Belgium. rDrug Synthesis and Chemistry Branch, National Institutes of Health, Bethesda, Md Geuens et al. (1984).

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cultures shows the characteristics of MT inhibition (see Section 111) when invasion is arrested. In the experiments with MO, cells MTIs seem to affect both the MO, cells and the heart tissue. Other experiments (Armstrong and Armstrong, 1979) have shown that MTIs inhibit the motility of chick heart cells in aggregate culture. The question could, therefore, be raised whether inhibition of invasion is due to the effect of the MTIs on the MO, cells or on the heart. Preliminary results with murine neuroblastoma cells (M. Mareel, B. Hill, and E. Bruyneel, unpublished results) selected for resistance to vincristine have shown that these cells invade into the heart tissue at concentrations of vincristine that completely inhibit invasion of MO, cells in the same assay. Although further experiments are needed to elucidate the relative contributions of malignant cells and normal tissues to invasion in organ culture, the experiments with neuroblastoma cells indicate that inhibition of invasion is predominantly due to alterations of the MO, cells. So far, we have concluded from our in vitro experiments that MTIs are inhibitors of invasion in vitro and that their effect on invasion has to be ascribed to disturbance of MT complexes. Results of some other experiments (Easty and Easty, 1974; Roos and Van de Pavert, 1982) using different assays and different criteria of analysis do not support this conclusion. Easty and Easty (1974) found that 5 pg colcemid/ml did not alter the capacity of Schmidt-Ruppin virus transformed rat fibroblasts, polyoma virus transformed BHK cells, or Harding-Passey mouse melanoma cells to infiltrate into the chick chorioallantoic membrane in vitro. There are some doubts about the value of the chorioallantoic membrane for the study of tumor invasion (for discussion see Armstrong et al., 1982). One major problem is that malignant cells as well as nonmalignant ones implant on the membtane only at sites of preexisting traumata. Although Easty and Easty (1974) observed colcemid-induced changes in the type of cytoplasmic extension produced by the tumor cells, they did not control whether the drug affected MTs under the conditions of their assay. Roos and Van de Pavert (1982) seeded MB6A murine ascites lymphosarcoma cells and TA3 mammary carcinoma cells on monolayers of adult hepatocytes. They studied the effect of colchicine and Nocodazole on the interaction index (number of interacting tumor cells per hepatocyte nucleus) and on the infiltration index (fraction of interacting cells that was completely encircled by hepatocytes). The MTIs reduced the interaction index with MB6A cells but not with TA3 cells; with both types of tumor cells the infiltration index increased. Large variations in control cultures performed on different occasions were ascribed to disparities between batches of tumor cells from different mice. The in vitro system had morphological characteristics in common with the infiltration of the same tumor cells in the mouse liver in vivo. No evidence was, however, presented that the interaction index or the infiltration index measured invasion. Regardless of whether or not an in vitro assay truely measures invasion it should

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143

be taken into account that the interaction of tumor cells with normal cells or tissues in two-dimensional tissue culture on artificial substrates differs in various aspects from interaction in three-dimensional organ culture (Mareel, 1980; Mareel and Meyvisch, 1981; Van Peteghem and Mareel, 1982). Another laboratory (L. A. Liotta et al., personal communication) confirmed our conclusion about the antiinvasiveness of MTIs in vitro. Using the amnion invasion assay (Russo et al., 1982) they found that MTIs interfered with invasion of malignant cells through the amnion by contrast with antiproliferative agents which did not affect MTs.

B. OBSERVATIONS in Vivo Final proof of the antiinvasive activity of MTIs would be the demonstration that these agents stop invasion of tumor cells in vivo. Extrapolation from the in vitro experiments to the situation in vivo needs caution because of uncertainty about the relevance of the in v i m assay, and because of differences in the pharmacokinetics and in some cases in the basic mechanisms of action of the drugs. In vitro the drug is added to the culture medium at the onset of the experiment and it is assumed that drug concentrations remain roughly the same throughout the experiment although this has not been properly controlled. In vivo, MTIs, for example, the vinca alkaloids, reach a peak concentration in the serum after intravenous injection followed by a steep decay (Nelson et al., 1980). Since in most treatment schedules MTIs are injected at regular (for example weekly) intervals the tumor cells are exposed to the drug repeatedly but for relatively short periods of time, whereas in vitro exposure is probably continuous. About storage of the drugs in tissues in vitro as compared to in vivo virtually nothing is known. Glycosylated podophyllotoxin congeners (Mareel et al., 1982a) are examples of drugs where differences in the basic mechanisms of action might be expected in vivo as compared to in vitro (see Section 11, B, 1). These congeners do not affect MTs in v i m presumably because the glycoside moiety sterically interferes with the ability to bind to tubulin (Loike and Horwitz, 1976; Loike et al., 1978). According to Evans et al. ( 1982) the glycoside moiety might be split off during metabolization so that an aglucone is produced which can be expected to act as a MTI in vivo. Glycosylated podophyllotoxin congeners would thus act as MTIs in vivo via their metabolites by contrast with their activity in vitro where such metabolites are not produced. To our knowledge, in vivo methods to evaluate the antiinvasive effect of MTIs in a way comparable to the in vitro method do not exist. Some of the problems associated with the in vivo observations have been discussed earlier (Mareel, 1980). Methods are available for the qualitative and quantitative analysis of a feature that is frequently associated with invasion namely metastasis. It should, however, be realized that the formation of metastases depends on a number of

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competences of malignant cells: induction of angiogenesis, invasion into blood and lymph vessels, detachment from the primary tumor, survival in the circulation, arrest in the capillary bed, extravasation, and progressive growth at distant sites (Hart and Fidler, 1981). Inhibition of metastasis by MTIs could, therefore, be ascribed to interference with invasion or with one or more of the other steps of metastasis. Atassi et al. (1982) were able to inhibit formation of spontaneous metastases from experimental mouse tumors with the MTI vindesine. We agree with their conclusion that this observation is in accordance with our in vitro observations but does not prove that vindesine acts as an antiinvasive agent in vivo. It is the opinion of Hart and co-workers (1980), who found inhibition of artificial (i.e., after intravenous injection) metastasis by MTIs, that a combination of subtile modifications of host-tumor interactions is a more likely explanation than inhibition of a single activity of the tumor cells. One possible illustration of this concept might be the observation that pretreatment of MO, cell aggregates with MTIs reduced their tumorigenicity after implantation in the pinna (Meyvisch et al., 1983). The interpretation was that a delay in invasiveness caused by pretreatment with the MTIs provided the host with the opportunity to eliminate MO, cells implanted subcutaneously into the pinna.

V. Antiproliferative and Cytotoxic Effect of Microtubule Inhibitors In interphase cells MTs constitute the CMTC; at the beginning of mitosis (early M phase) the CMTC disassembles and the pool of tubulin reassembles to form the mitotic spindle. MTIs that affect specifically one of both complexes and leave the other intact have not been described so far. Recent studies by lzant et a / . (1983) indicate that differences might exist in the MAPs modulating tubulin assembly/disassembly and tubulin function in both complexes. They have found a lymphocyte hybridoma monoclonal antibody raised against MAPs from HeLa cell MT protein that binds to the mitotic apparatus of PtK, cells but not to the CMTC. This observation might open the way to selective inhibition of mitosis by tubulin binding agents. In our experiments MTIs affected both MT complexes at approximately the same concentrations. Inhibition of cell proliferation has long been the major interest of students of MTls (for review see Dustin, 1978), and the antiproliferative activity of MTIs has been at the basis of their use in cancer chemotherapy (Palmer et a/., 1960; Cardinali et ul., 1963; Frei et a / ., 1964; Bichel, 1967; De Brabander et a / ., 1976b; Sweeney et a / ., 1978). In our in vitro experiments antiinvasive concentrations inhibited the proliferation of the MO, cells. Growth pressure has been proposed as one of the mechanisms of tumor invasion (for reviews see Easty, 1975; Mareel, 1980). At first glance, the antiinvasive effect of MTls seems to support this concept. We made, however, a number of

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observations strongly indicating that the antiproliferative effect of MTIs could not be held responsible for inhibition of invasion. First, a series of growth inhibitors permitted invasion (Table 111). None of these drugs that were permissive for invasion was found to affect MTs. This observation suggests that growth and invasion might be basically unrelated activities of malignant cell populations. This is concomitant with the clinical observation that slowly growing tumors may be highly invasive and contradicts the concept of invasive growth pressure. We have recently obtained evidence that in the in vitro assay for invasiveness growth in not necessarily followed by invasion. Lowering the incubation temperature to 29°C permitted growth to about 50% of controls and arrested invasion for at least 10 days (Mareel et al., 1982~).A similar situation was created through addition of the alkyl-lysophospholipid ET- 18-OCH3 to the culture medium (Storme et al., 1984). Second, MTIs were also antiinvasive when chick heart fragments were confronted with larger MO, cell aggregates containing approximately the number of cells present in control cultures without drug at the end of the assay (Mareel et al., 1982a,b). This showed that lowering the number of cells through inhibition of proliferation was not responsible for inhibition of invasion. Third, combined treatment with 5-fluorouracil and MTIs excluded that the antiinvasive effect was due to interference with the spindle TABLE 111 ANTIPROLIFERATIVE DRUGSPERMISSIVE FOR INVASION O t M04 CELLS ORGANCULTURE

IN

Bleomycin sulfate (Bleomycine"; MW 1400; 10-60 pg/mlb) Chlorambucil (Leukeran"; MW 304;30 pg/ml) Cisplatin (Platinol"; MW 300; I pg/ml) Cytosine arabinoside (Cytosard; MW 280; 1 .O-6.0 pgiml) 1,2-Di-(3,5-dioxopiperazin-5-yl)propane(ICRF- 159e; MW 278; 30-60 pg/ml) Doxorubicin (Adriblasting MW 580; 0.1- 1 .O pgirnl) Etoposide (VP-16-213c; MW 587; 10-30 pg/ml) 5-Fluorouracil (Fluorouracila; MW 130; I .O-10 kg/ml) Hydroxyurea (Aldrich Europe, Beerse, Belgium; MW 76; 10-30 pg/ml) L-Phenylalanine mustard (Alkeran"; MW 305;3.0-10 pg/ml) Methotrexate (Ledertrexaat'l; MW 454;0.1- 1 .O &g/ml) Teniposide (VM-26"; MW 655; 1 .O-6.0 pgiml) "N.V. Wellcome, Aalst, Belgium bconcentrations (pgiml) used in the assay for invasiveness; all concentrations inhibited the growth of M 0 4 cell aggregates. r N . V . Bristol Benelux, Brussel, Belgium. dN.V. Upjohn, Puurs, Belgium. =Imperial Chemical Industries Limited, Macclesfield, Cheshire, U.K Wontedison Farmaceutica Benelux, Brussel, Belgium. 8N.V. Roche, Brussel, Belgium. 'Cyanamid Benelux N.V., Louvain-La-Neuve, Belgium.

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t

I

NECROSIS

3

FIG. 5 . Schematic representation of some synchronized cycle events that occur during mitosis. Cytoplasmic microtubules (I) are replaced by spindle microtubules (2-4) and reconstructed after telophase ( 5 ) . Chromosomes condense (2), are separated (3), and decondense ( 5 ) . The nuclear membrane disappears during prophase (2) and is reconstructed during telophase (4). Treatment of the cells with inhibitors of tubulin polymerization does not only block the microtubule cycle but also strongly affects the other cycles, and most cell types become rapidly necrotic during this stage. The derangement in space and time can however better be observed in cells that are able to survive the abortive mitotic phase such as MO cells (10). After having spent several hours in the disorganized mitotic stage (8) (up to 6 hours) the cells begin to form multiple furrows (9) each provided with a

MICROTUBULE INHIBITORS

147

MTs. 5-Fluorouracil blocks cells in early S phase of the division cycle so that spindle MTs do not form. The invasion of such nonproliferative but invasive cells can be inhibited with MTIs (Mareel and De Brabander, 1978a). It has been generally accepted that MTIs are cytotoxic for cells that pass through M phase (Wells et af., 1976). The effect of MTIs on MO, cells, however, was remarkably reversible even after 4 to 6 days treatment at high concentrations (Mareel and De Brabander, 1978a; Storme et al., 1981; De Brabander et af., 1976b; Meyvisch et af., 1983). Inhibition of invasion could, therefore, not be ascribed to cytotoxicity. The question whether cells arrested in metaphase by MTIs could subsequently reenter a normal proliferative cycle has been discussed in a review by Camplejohn (1980) followed by a reply by Klein (1980). Reversibility in cell populations might be due to heterogeneity in drug sensitivity with selective survival of part of the population, to reversible arrest in a particular cycle phase, and to recruitment of nonproliferating cells. In individual cells participating at proliferation reversibility through reentry into the cycle after mitotic arrest appeared to depend on the type of cells (Malawiska et af., 1968; Krishan, 1968; Krishan et af., 1968), the delay since arrest, the concentration of the drug (Journey et af., 1968; George et al., 1965), and the type of MTI. A comparison between data from various experiments is, therefore, very difficult. The phenomena associated with reversible and irreversible mitotic arrest by MTIs as observed in various types of cells are summarized in Fig. 5. In addition, Hamilton and Armstrong-Snyder (1982) have recently described that in PtK, cells upon release from Nocodazole-induced metaphase arrest the remaining portions of the cycle were completed about 35% faster than in normal mitosis, Even in types of cells which are unable to survive metaphase arrest, the cytotoxic effect of MTIs cannot be explained solely on the basis of their interference with the formation of the mitotic spindle. Some observations with vinca alkaloids in vitro (Olah, et af.,1978; Rosner et af., 1975) and in vivo (Stryckmans et af., 1973) suggest that the cytotoxicity of MTIs may be undependent on M phase or even on other phases of the cell cycle. MTI may also be cytotoxic for interphase cells. Hirsimaki et af. (1976) have described necrosis of liver cells after ip injection of vinblastine. The most prominent early effect (after 4 hours) was formation of autophagosomes. The authors presumed that vinblastine interferred with the transport and secretion of the very

circular ring of microfilaments. No separation however occurs. The unseparated chromosomes are separately, or in small groups enveloped into a new nuclear membrane (9) giving rise to multimicronucleated cells (10). Finally, the cells readhere (10). However, they do not assume a polarized shape. This is probably due to the fact the contractile filament system fails to become organized in stable and active parts, which is obvious from the irregular undulating membrane activity all around the cell periphery (6 and 10). From De Brabander et al. (1979) with permission of the authors and the publisher.

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low-density lipoproteins from the parenchymal cells (Hirsimaki and Pilstrom, 1982). The well documented neurotoxicity of the vinca alkaloids (Conrad et a/., 1979; Chan et al., 1980; Gerzon, 1980) is also unrelated to cell proliferation.

VI. Directional Migration In the present discussion we will use the term locomotion for the active movement of individual cells and the term migration for the active movement of groups or sheets of cells. A. AN EXPLANATION FOR THE ANTIINVASIVE ACTIVITY OF MICROTUBULE INHIBITORS We have ascribed the antiinvasive effect of MTls to interference with directional migration: MTIs disturb the CMTC which is necessary for the determination of direction in migrating cells, but not for migration per se. Although the role of cell migration in invasion is not fully understood and although the factors responsible for the direction (i.e., into the normal tissue) of migration are unknown, a good deal of circumstantial evidence supports the opinion that inhibition of invasion by MTIs is related to inhibition of directional migration. In MO, cell populations both invasion and directional migration showed approximately the same dose-responsiveness (Mareel et af., 1982a,b), were equally reversible, were not sensitive to inhibition of cell proliferation (Fig. 6), and could be stopped during their course within the first 24 hours after addition of MTIs (Mareel et al., 1982b). So far, all inhibitors of directional migration were found to be antiinvasive (Mareel et a/., 1982c, and Table 11). This does not imply that permission of directional migration predicts permission of invasion. For example, the alkyl-lysophospholipid ET- 18-OCH3 inhibited invasion at concentrations permitting directional migration (Storme et al., 1984). This observation indicates that directional migration is a necessary activity of invading cells, but that other cellular activities may be needed as well. Our interpretation of the relationship between inhibition of directional migration and the antiinvasiveness of MTIs is indirectly supported by evidence from the literature that MTIs are universal inhibitors of directional migration and that tumor cells are sensitive to factors directing migration. B. ASSAYSFOR CELLMIGRATION Conclusions about the effect of MTIs on cell migration in virro depend to a large extent on the type of migration that is measured in a particular assay. Modifications of the Boyden chamber have been used most frequently for the

I49

MICROTUBULE INHIBITORS

3.5. 3.0-

I 0

. 1

. 2

. 3 4 5 time (days)

6

FIG. 6 . Directional migration of MO, cells from an aggregate explanted on glass. Ordinate: mean diameter of area covered by cells that have migrated from the aggregate; mean and SD from 5 cultures. Abscissa: time of incubation; drugs were added at day 2 (arrow). Control; (a),= with 0. I p g methotrexatelml: to),with 0.3 p g flubendazoleiml.

(A),

study of the migration of leukocytes and macrophages (for discussion see Zigmond and Hirsch, 1973; Wilkinson, 1976). Such chambers consist of an upper and a lower compartment separated by a filter. Russo et 01. (1981) have recently developed an assay using pieces of human amnion to separate both compartments instead of a filter. Cells are put in the upper compartment and their migration is assessed by measuring the distance from the top of the filter to the furthest plane of focus inside the filter which contains at least two cells in focus. Alternatively, numbers of cells are counted at any given depth of the filter, on the bottom of the filter, or on a second filter put in the lower compartment. By using filters with a pore size of 0.45 p m which do not allow migration, Malech et af. (1977) measured orientation of cells (vertical position of cells and position of the centriole) without migration. In Boyden chamber assays random migration (for theoretical considerations see Weiss, 1983), activated random migration, and directional migration were clearly distinguished. When a chemoattractant is involved activated random migration is also called chemokinesis and directional migratiqn is also called chemotaxis. Random migration occurs when saline is present in both compartments of the chamber. When a chemoattractant is present at equal concentrations in both compartments a chemically enhanced nondirectional migration (or chemokinesis) can be observed. In both situations the distribution of cells in the filter after various times was similar to thzt which would

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MARC M . MAREEL AND MARC DE METS

be expected from a uniform population of particles exhibiting random walk: a characteristic linear relationship between the log of the number of cells at given distances vs the square of the distances (Zigmond and Hirsch, 1973). When a gradient of chemoattractant between both chambers exists, the assay measures directional migration (or chemotaxis), a reaction by which the direction of locomotion of cells is determined by substances in the environment (Wilkinson, 1976). Distinction between this three types of migration can also be made with the agarose-plate assay, Here cells and media eventually containing the chemoattractant are put in wells punched out in the agarose and the distance covered by the leading front is measured. According to Daughaday et al. (1981) this method is superior to the Boyden chamber because such factors as cell deformability influencing passage through the filter are involved to a lesser extent. This consideration is not without importance since different cellular functions might well be affected in a different way by alterations of the microtubule complex. By contrast, the agarose-droplet assay is believed to measure random migration (Varani et al., 1979). Here cells are explanted in a droplet of agarose and distances of the leading edge of locomoting cells from the edge of the droplet is determined. Counting numbers of cells migrating beyond the corona may indicate a subpopulation of fast cells. Measurement of the area covered by cells migrating out of a capillary tube is also considered to be an assay for random migration (Cheung et a / . , 1978). It should, however, be mentioned that neither in the agarose-droplet assay nor in the capillary tube assay migration has been analyzed following a random walk model. The locomotory behavior of solitary cells seeded on artificial substrates such as glass or plastic depends on the cell type. Ramsey and Harris (1972) have shown that the locomotion of leukocytes on glass is random: the frequency distribution of net squared displacements was not significantly different from that of randomly diffusing particles. Directional migration (chemotaxis) of leukocytes on glass could be assayed by the dot-like attractant system (Rydgren et a/., 1976). Glass adherent yeast phagocytes serve as the source of chemoattractant and the locomotion of leukocytes is expressed either as the quotient between the straight distance and the actual distance (locomotive index) or as the distance of the cell to the attractant at the beginning of observation minus the distance at the end of observation (net radial dislocation). A mathematical analysis of the turn-angle probability distribution of polymorphonuclear cells sensing chemical gradients from a streak of aggregated y-globulin was described by Nossal and Zigmond (1976). When solitary fibroblasts are seeded on an artificial substrate they first spread radially (Vasiliev and Gelfand, 1976). Later the ruffling activity is constrained to one pole of the cell; this pole becomes the leading edge of a polarized solitary locomoting fibroblast (Fig. 7). Gail and Boone (1970) have demonstrated that such fibroblasts were not pure random walkers but persisted in their direction of

s P H E R 0 ID

@

RADIALLY SPREAD CELL

EPITHELIAL CELL

POLARIZED LOCOMOTION

NOT POLARIZED LIMITED LOCOMOTION

t

AGGREGATE OR CELL SHEET

FIBROBLASTS

POLARIZED MARGINAL CELLS DIRECTIONAL L O C O M O T I O N

FIG. 7. Schematic representation of the locomotory activity of solitary cells (top) and of groups of cells (bottom) in culture on artificial substrate. Horizontal bars indicate inhibition by microtubule inhibitors: explanation in the text. Modified from Vasiliev (1982).

152

MARC M. MAREEL AND MARC DE METS

locomotion. The latter authors charted the position of the cell at specified time lapse intervals and measured both square displacements and angles between successive segments of displacement. By modifying the random walk model to comprehend the persistence effect, Gail and Boone (1970) were able to characterize the locomotion of these persisting cells in terms of an augmented diffusion constant. Most solitary epithelial cells (Fig. 7) do not pass beyond the stage of radial spread; they show no or a very limited locomotory activity (Middleton, 1977). This holds true for solitary epithelial cells on artificial substrates, but not necessarily for epithelial cells on more natural substrates like collagen (Tchao, 1982). The situation is quite different when fibroblasts or epithelial cells are explanted on an artificial substrate as a tissue fragment, an aggregate, or a sheet, because intercellular contacts influence the locomotion of the individual cells (Fig. 7). There is little doubt that under these circumstances radial migration of cells from the explant should be considered as directional migration (Gaillard, 1942; Abercrombie, 1961; Abercrombie and Heaysman, 1966; Badley el al., 1980; Mareel et a / . , I981 b). This does not imply that a chemoattractant is involved. Since other factors may also determine the direction of migration directional migration and chemotaxis should not be considered as synonyms. For chick heart fibroblasts the distribution of directions of migration was such that in 72% out of 4845 readings from films the cell increased its distance from the explant (Abercrombie and Heaysman, 1966). Furthermore, the proportion of readings in which the distance from the explant increased augmented with the number of intercellular contacts, an observation compatible with the theory of contact inhibition of movement. This fact is also supported by the observation that round cell transformants which have lost contact inhibition of movement d o not perform directional migration from an aggregate explanted on glass (Mareel et al., 1981b). In epithelial sheets marginal cells are polarized in that they display a leading lamella from that part of the cell not in contact with other cells: their direction of migration is almost uniquely outward (Middleton, 1977). Here also it is believed that contact inhibition of lamellar activity constrains the activity to the presumptive leading edge of the cell (Vasiliev and Gelfand, 1976). For the same reasons as mentioned with explants, migration of fibroblastic or epitheloid cells at the margins of a wound should be considered as directional migration (Vasiliev et a / . , 1970; Vaughan and Trinkaus, 1966; Wilbur and Chambers, 1942; Tchao and Leighton, 1979) and some authors clearly stated that this type of migration was essentially unidirectional (Selden et al., 1981; Kupfer et al., 1982). Although contact inhibition of lamellar activity and not chemotaxis is most probably governing the direction of locomotion in cells migrating from sheets or explants, no arguments are available to consider this type of migration as random.

MICROTUBULE INHIBITORS

153

C. EFFECTOF MICROTUBULE INHIBITORS O N MIGRATION OF CELLS Data about the effect of MTIs on migration of cells are summarized in Table IV. So far, differences in the sensitivity to MTIs of directional migration by malignant versus nonmalignant cells have not been described. By contrast, differential responses have been described for various cell types. 1. Leukocytes and Mucrophuges In most experiments (Table IV) MTls interfered with the capacity of leukocytes and of macrophages to perform directional migration (chemotaxis) itz vitro (for review see Wilkinson, 1976). The experiments of Daughaday and co-workers (1981) stand as an exception. These authors suggest that inhibition of directional migration in assays using filters is not due to interference with MTs but to effects of the drugs at other sites in the cell, for example the plasma membrane. One shortage of the latter experiments is the lack of evidence that colchicine at a concentration of M interferes with the assembly of the CMTC under the conditions of the agarose plate assay. The opinion that disruption o f t h e CMTC decreases the rigidity of the cells (Crispe, 1976), which should facilitate their passage through a filter and, therefore, counteract inhibition of passage, also invalidates to some extent the criticism raised by Daughaday and co-workers (1981). The effect of MTIs on random migration varied from alteration in the type of movement (Padawar, 1966; Bhisey and Freed, 1971) to increased or decreased rate of migration most probably following variations in drug concentration. Most authors, therefore, have come to the conclusion that the integrity of the CMTC is essential for orientation of locomotion in leukocytes and macrophages but not for locomotion per se.

2 . Fibroblustic Cells Vasiliev and co-workers ( 1970) have first demonstrated that MTIs interfered with the directional migration of fibroblastic cells from the edges of a wound in monolayer culture. Their idea that MTs are necessary for the maintenance of the leading edge by inactivation of the rest of the cell periphery has been followed by most authors (review by Vasiliev, 1982). After treatment with MTIs the pseudopodia1 activity becomes irregularly distributed all around the cell periphery so that polarization of the cell is lost. Such cells no longer migrate in a persistant direction but are still able to exert random walk (Vasiliev et al. , 1970; Gail and Boone, 1971; De Brabander et nl., 1976b; Ivanova et ul., 1976). In presence of comparatively high concentrations of colchicine BHK-21 cells did not seem to move although ruffling occurred at several places around the cell periphery (Goldman, 1971). That MTIs did not interfere either with the formation of pseudopodia or lamellae or with lamellar functions such as clearing of Con A

Effcct on ~

~

Cell iypc

Status of CMTC

Drug

Human granulocyte,+ VLB (10 6-10 - 4 M ) n . t . CI. (10-’-10- 3 M ) DCL (0.01-0. I pgitiil) Oxalare-induccd raSI’l (0.1- 1 .O p,p/rnl) dial scgmcntalivri

hkawrentcnt

r. 111,

Culturc (in plN

Average sprcad

-1

Royden (casein) Culture on

Ccll number

Assay

a.r.m.

dni.

Rcfercncc

R ~ I M C Yand Harris

I

(1972) Bandman er ( 1974)

(I/.

Sped

gIiW

SPl ( 0 . 5

Oxalatc-induccd ra-

+L,g/IIII)

dial scpnicntatioii

Boy den (cawin)

Cell iiumhzr

Ilot-IlkC

Path

Kydprcn el

(11.

( 1976)

dttractaiir Uayden ( E M ) Ccll niiiiiber Distancc Hoyden (cilse1n)

Boydcn (caseSPI ((1.I- I .O kpiinl) CL

10-

M)

Oxaliite-induced radial .scgrncntaticln MT counting

in. BaCb-J Bvydcn (casein) Hoyden

Cell numher

Valcnus (1078)

Distance

Sirdcrslroin

Ccll numhcr

Distancc

(FMLP) VLB.VCR (0.5-2 0

Human mrmcytea

CL

MI

/\Farose drop

Migration ilrca

n.t.

Bvydcn ( E M .

Distaricc

cascin)

Spilhcrg e/ d.

Daughaday

PI

ul.

(1981)

n t.

pp/ 1111)

ul

( 1979)

(GHC, CCF, FMW) Aparose plate

et

(lY79)

HorvLth and B a l k s (1982) Rur~cll(’I a / . (1975)

VLB,CL( 10 8- 10 - 4 M) CL ( I0 I0 M) VLB ( 1 0 - X - I O - 6 M ) CL (10-X-10-2 M )

n , t.

Boyden

Distance

TEM

Speed

n.t.

Culture on glass Capillary tube

CL ( 1 0 - 5 M )

n.t.

Capillary tube

Migration area Path

-

Mouse macrophages

Mouse embryo fibroblasts

VI

BALBl3T3 fibroblasts BHK-2IlC13 cells

-

’-

-

CL (0.1 pmiml) TEM DCL (0.01-0. I pglml) VLB (0 005-0 05 pgiml) Metaphase arrest CL (0 36 pglml) CL (5-40 pglml)

TEM

3T3-fibroblasts

CL (4 x 10-7 M )

MO fibroblastic cells B16 melanoma cells Hela cells

ND ( I pglml)

TEM

VCR (0 01-1 pglml) VLB (0 01-1 pglml) CL ( 1 pg/ml) CL (10-3 M )

Cell shape

Mouse embryo fibroblasts Mouse fibrosarcoma cells M 0 4 fibrosarcoma cells B16BL6 cells

Wound healing

Culture on glass Culture on glass Wound healing Culture on plastic

Migration area

t-1

Crispe ( 1976)

t

t-1 t

1

Path

.1

Path

4

Cell number

1

Path

5

Agarosedroplet

Distance5

Aggregate on glass Agarose droplet

Migration area Distances

1

Bhisey and Freed (1971) Sundharadaa and Cheung (1977); Cheung et a / . (1978) Cheung and Terry ( I 980) VasiIiev t t a / . (1970)

Gail and Boone (1971) Goldman (1971) Yarnell and Schnebli ( 1974) De Brabander et a / . ( 1976)

Varani et ul. (1978)

VLB ( 1 0 - 4 ~ ) ND (0 01-1 0 pglml)h Irnmunocytochemistry n.t. CL (10-5 M )

1

Storme and Mareel ( 1980)

1

Hart ef u / . ( 1980)

(continued)

TABLE 1V

(Corttinirtd)

Ellcct on

Cell type

Drug

Statuh of CMTC

Assay

Measurement

r.m.

a.r.111.

d.111.

Reference

culture o n agar Morphogcncsis New's nicthod 1)istance Wound Distance healing

Cookc ( 1973)

Wound healing Culture on agar

Gastrulation

Ilunlap and Donaldson (1978) Nakatsuji (1979)

n.t.

Boyden (RC'F) Cell nuinher

Spiro and Mundy

n.t.

Boyden (FMLP.

Xtnopu' emhryo

CL (0.05%)

Mitotic arreht

Chick blastodem MtTR kidncy cells, SV40 transtor-

CI. (0 1-1.0 pg/ml) CL (0.1 1.0 p&l"'I)

Mitotic arrest TEM

n.t.

Downie (1975) Vasiliev e/ ul. (1475)

rnant

Newt epidenni5

CI. (10 rngikg)

Mitotic arrest

Xeriopus lurvrs eggs

CL (0.1-1.0 m M ) PPT (0.01 m M ) VLB (0.1 inginil) CL.VLB ( I 10 @I)

Mitotic awht

Walker carcinoma

Distance

(1980)

cells

Walker carcinoma cells

CL

(10-7

M)

Cell numhcr

J.

Wass

CI

ol. (1981)

7AHM. BCF) Bovine aorta endothelial cells K12 rat adenocarcinoma cells

CL. VLB (10-910-5 M ) ND (0.01-1 pgi'ml)

n.t.

Wound healing

Distance

Selden et a/.( 198 1)

Imrnunocytuchemistry

Aggregate on

Migrarioii area

Schallier PI d. (1982)

elah\

UVLH, Vinhlastine; CL, colchicine; DCL, dernecolcin: SPI. podophyllic acid ethylhydrazidc: VCK, vincristine; ND. Nocodazole; PPT, podophyllotoxin. n.t., not tested or rcfercnce to the literamre; CMTC. cytoplasmic microtubule complex; MT. microtubulcs; TEM. transmission clectron microscopy. Boyden. Original Boyden's techniquc or modifications of it; EAS. cndotoxin activated serum; GIIC, glycylhistidyl-glycinc: BaCL. bacterial chcniotactic factor; CCI;, crysralinduced chernotactic factor; FMLP. formyl-inethionyl-leucylphcnylalaninc. ZAHM. Zymosan-activated human seru111:BCI:, bonc derived cheniotactlc factor. r.rn., Kandom migration; a.r.rn..activated random migration; d.n]., dircctional inigrdtion. Effect o f d ~ u g is s expressed as no effect (=), increase ( T ), or dccreascd ( 5 ): horizontal arrows point to effects at higher drug concentrations. "All drugs mentioned in Table I1 had a similar effect as ND.

MICROTUBULE INHIBITORS

157

receptors was also shown by Vasiliev’s group (Domnina et al., 1977; Vasiliev, 1982). Another argument in favor of the role of the CMTC in directional migration is the positioning of the Golgi complex and the MTOC forward of the nucleus in the direction of lamellar extension shortly after wounding of monolayer cultures (Kupfer et al., 1982). Whether MTIs also affect random migration of fibroblastic cells is a difficult question since fibroblasts even when solitary are not pure random walkers (Gail and Boone, 1970). Whether the migration of fibroblasts from agarose-droplets (Varani et af., 1979) corresponds to the criteria of random walk remains to be examined.

3. Epithelial and Endothelial Cells Whether the CMTC plays a role in the directional migration of epithelial cells is not clear (Table IV). Most authors agree that the surface morphology, surface activity associated with locomotion, and the radial spread of epithelial cells are not MT dependent (DiPasquale, 1975; Chernoff and Overton, 1979; Middleton, 1982; Downie, 1975). However, MTIs did affect the cytoplasmic organization (internal polarization) of epithelial cells (De Brabander et al., 1978) and Middleton (1982) noted that oriented spreading associated with intercellular contact (contact induced spreading of pigmented retina epithelial cells) was affected by MTIs. Indirect evidence for the role of MTs in directional migration of endothelial cells at the edges of a wound was provided by the reorientation of the MTOCs (Gotlieb et al., 1981). MTOCs, otherwise randomly oriented, were found in front of the nucleus facing the direction of migration in 80% of the edge cells 4 hours after wounding. As pointed out by the authors, it is not clear whether the reorientation of MTOCs determined the direction of migration or whether the migration of the cells causes a reorientation of MTOCs in a preferred direction. These authors who found no effect of MTIs on the directional migration of epithelial cells have presumed that the orienting function of MTs as it occurs in fibroblasts is apparently performed in epithelial cells by extensive lateral intercellular contacts as put forward by Vasiliev’s group (Vasiliev and Gelfand, 1976; Domnina et al., 1977). So, additional stabilization of certain surface areas through MTs would not be essential to confine lamellar activity to the leading edge of epithelial cells, unlike in fibroblasts. This opinion does not explain why, at least in some epithelial cells, a limited area of contact with another cell is sufficient to induce polarization with restriction of lamellar activity to the leading edge (Van Peteghem and Mareel, 1978). Alternative explanations for the localization of lamellar activity are pH gradients (Weiss and Scott, 1963), contact guidance by the substrate, and local diffusion gradients (Middleton, 1982). Clearly, further experiments are needed to clarify whether the CMTC is necessary for the directional migration of epithelial cells. We would like to emphasize

158

MARC M . MAREEL AND MARC D E METS

here the need for a reliable control of the alteration of the CMTC under the circumstances of each particular experiment. D. MECHANISMS OF DIRECTION FINDING How MTls affect cellular migration is largely unknown. Do they inhibit migration per se? Do they interfere with orientation (direction finding)? Do they disturb direction in migrating cells? Do they act on the factor(s) that determine(s) the direction of migration? Do MTIs act through changes of the cell shape? Is mitotic arrest responsible for alteration of migration? Valerius ( 1978) suggests that colchicine affects the still unidentified membrane transport mechanism involved in the translation of the signal into an appropriate locomotory response. Considering the experiments summarized in Table IV it is not likely that MTIs interfere with migration per se, at least not at concentrations that affect directional migration. Malech and co-workers (1977) have concluded from their experiments that oriented assembly of MTs and consequent orientation of the cell is important in providing the vector of migration during chemotaxis. This is confirmed by Spilberg and co-workers (1979). Incubation of polymorphonuclear leukocytes with chemoattractant before bringing them into a gradient abolished the chemotactic response and this deactivation was inhibited by colchicine. The interpretation was that a relatively diffuse assembly of MTs occurred in the presence of an homogeneous concentration of chemoattractant so that cells could no longer respond to a gradient by anisotropic MT assembly. This is in agreement with the opinion of other authors (Bandman et d . , 1974; Selden et d . , 1981; Gotlieb et al., 1981) that in various types of cells a redistribution of centriole associated MTs is an essential mechanism of directional migration. Little work has been done to examine the effect of MTIs on the chemotactic or other factors themselves. This is not without importance since some authors presented evidence that MTls prevented the release of chemotactic substances from phagocytizing leukocytes in the dot-like attractant assay (Rydgren et uf., 1976; Back et al., 1978). Downie ( I 975) has explained inhibition of the epiboly in chick blastoderms by the effect of the MTIs on the inner cells which do not participate directly in migration. The contractility of these cells lacking the ability to maintain a flattened shape would exert a tension against the centrifugal migration of the edge cells, capable of stopping the migration or even to reverse it. The major argument was that MTIs no longer affected the centrifugal migration when the inner cells were dissected out. The role of MTs in maintenance of cell shape was also accepted by others (Granholm and Baker, 1970; Handel and Roth, 1971). In a review De Brabander and co-workers ( 1977) have refuted the mechanistic concept that MTs produce a rigid skeletal frame supporting directly cell shapes. They considered that MTI induced shape alterations of cells, which were only

MICROTUBULE INHIBITORS

159

seen in the horizontal plane, might be the consequence of an altered pattern of movement brought about by the loss of a regulating system which normally divides the contractile microfilament system into active and stable parts. In their opinion, the cell shape is primarily determined by the migratory capacity of the cell. which is influenced by external factors such as interaction with the substrate or with other cells. Considering the effect of MTIs on the directional migration of cells at the margins of epithelial sheets or at the edges of epithelial wounds should leave us with the conclusion that confinement of lamellar activity to the free edge of the cell is not sufficient to assure directional migration. Presumably, internal polarization of the cell is also involved. Arrest of mitosis was invoked by Nakatsuji (1979) to explain inhibition of gastrulation in Xenopus eggs. This concept cannot be accepted to explain the effect of MTIs on directional migration in a general way. Other authors (Cooke, 1973; Downie, 1975) have shown that morphogenetic movements can continue at least for some time without mitosis. Using aggregates explanted on glass we have shown that inhibitors of proliferation which do not affect MTs hardly affect directional migration for at least 4 days (Storme and Mareel, 1980; Schallier et ul., 1982).

E. DIRECTIONAL MIGRATION OF INVADING CELLS Although the concept that malignant cells invaded other tissues by locomotion was accepted at the end of the previous century, direct evidence for it is scarce. This is due mainly to technical problems associated with the direct observation of cells inside the relatively untransparent three-dimensional array of tissues in vivo or in vitro. It should be noted that the interaction of normal and malignant cells locomoting on glass has been thoroughly investigated by Abercrombie and his group (Abercrombie, 1970; Abercrombie and Heaysman, 1976). These authors, however, have cautioned their readers against extrapolation of interactions of cells on artificial substrates to invasion in vivo. The few microcinematographic observations in vivo are, therefore, of great value for our understanding of the role of migration in invasion. Loconioting tumor cells were shown during extravasation in the mesentery by Sat0 and Suzuki (1972) and in the rabbit ear chamber by Wood ( 1958). Later, Wood el a/. (1967) and others (Thornes er a / ., 1968) were able to demonstrate locomotion of cells invading from the primary tumor into the neighbouring tissues. Most data, however, came from a recently developed combined in vivoiin vitro technique (Haemmerli and Strauli, 1978; Haemmerli et a / . , 1982). These authors injected leukemia cells and V2 carcinoma cells intraperitoneally into syngeneic or into immunoincompetent animals and afterward filmed the behavior of these cells inside the mesentery in vitro. These films have provided convincing evidence that tumor cells locomote inside the mesentery. Most importantly, they suggested that analysis of locomotion of cells on artificial substrates accessible to filming might be to a large extent

160

MARC M. MAREEL AND MARC DE METS

relevant for their locomotion in vivo. The aforementioned in vivo observations confirmed the opinion derived from static pictures. Whether the migration of invading cells meets the criteria of directional migration or represents random walk has not been examined. There is, nevertheless, evidence that tumor cells are sensitive to chemotactic factors both in vivo and in vitro. Using Boyden-type two-compartment assays sensitivity of various tumor cells to the following chemoattractants has been demonstrated: factors from the supernatant of resorbing bone cultures (Orr et a / . , 1979), collagen derived peptides, the synthetic peptide N-formyl-methionyl-leucyl-phenylalanine(FMLP) (Wass et al., 1981a), and a fragment from C,, leukotactic peptide (Orr et al., 1978, 1983). The latter chemoattractant appears to be derived from the same portion of the C, molecule as the leukotactic peptide presumably by cleavage of the amino terminal portion of the leukotactic peptide by a proteolytic enzyme. It is interesting to notice that this proteolytic activity can be generated from normal tissues as well as from tumors (Romualdez and Ward, 1975; Romualdez et al., 1976). Using the collagenous stroma of the human amnion as a barrier instead of a filter in the two-compartment assay, Thorgeirsson and co-workers (1982) have demonstrated that FMLP stimulated the passage of M5076 reticulum sarcoma cells through the stroma. The following arguments were put forward in favor of stimulation of directional migration of FMLP in this experiment: the concentration of FMLP in the lower compartment giving optimal stimulation M) was the same as that found for optimal stimulation of tumor cells in filter assays, and FMLP did not affect either cell proliferation o r collagenase activity. Evidence for a chemotactic response of tumor cells in vivo came from experiments by a group of Japanese workers (Yoshida et a / . , 1970; Hayashi et al., 1970; Ozaki et al., 1971; Koono et al., 1974a,b; Ushijima et al., 1976; and review in Hayashi and Ishimaru, 1981). These experiments suggest that chemotaxis is involved in at least one type of invasion, namely extravasation. The chemotactic factors were purified from a number of animal and human tumors. They were not produced by the tumor cells themselves but appeared to result from the interaction of a tumor protease with precursors in the normal tissues. These factors attracted tumor cells but not rat neutrophils in the Boyden chamber. After intradermal injection they induced extravascular migration of circulating tumor cells which subsequently accumulated and proliferated at the site of injection of the factors. Since permeabilization of the vessels did not occur, the authors concluded that extravasation at this particular site was due to chemotactic migration of the tumor cells. A complementary and/or alternative explanation might be stimulated adherence of tumor cells to the vascular endothelium (for review see Varani, 1982). Few experiments have been done to examine whether MTIs affect chemotactic migration of tumor cells and the results available are controversial (Spiro and

MICROTUBULE INHIBITORS

161

Mundy, 1980; Wass et al., 1981b). It appears to us that testing MTIs in the in vivo assay developed by the Japanese group should be extremely interesting.

VII. Effect of Microtubule Inhibitors on Plasma Membrane Functions So far, most experimental data (see Section V1) indicate that MTIs affect directional migration because they abolish the stabilizing effect of the CMTC on the locomotory activity of certain regions of the plasma membrane. It is, however, not excluded that this destabilizing effect influences other functions of the plasma membrane that are possibly involved in invasion: intercellular contact, transduction of signals, distribution of receptors, expression of so-called malignant glycoproteins, cell differentiation, endocytosis, and secretion of lytic enzymes (De Clerck and De Brabander, 1977) and of collagen (Diegelman and Peterkofsky, 1972; Eichhorn and Peterkofsky, 1979). Description of the effect of MTIs in terms of MT assembly/disassembly might be too simple to understand their action on the plasma membrane. Alterations of the mobility of certain lectin receptors (Yin et al., 1972; Yahara and Edelman, 1973; Berlin et al., 1974; de Petris, 1974, 1975; Oliver, 1976; Creasey, 1981) and of the components of the hormone-sensitive adenylate cyclase system (Rudolph and Malawista, 1980) were ascribed to disruption of MTs rather than to a direct effect of the MTls on the plasma membrane. The synergistic effects of tertiary amine local anesthetics, which act by dissolving into the lipid phase of the membrane, and MTIs on the mobility of cell surface molecules (Poste et al., 1975; Eichhorn and Peterkofsky, 1979; Nicolson and Poste, 1976) or on cell differentiation (Edstrom er ul., 1975) suggest that MTIs may also have a direct effect on the plasma membrane. This opinion is to some extent supported by the finding that in macrophages and polymorphonuclear leukocytes MTIs interfere with the synthesis of phosphatidylcholine, a key component of the plasma membrane (Pike er al., 1980). An alternative explanation for the effect of the tertiary amines is that they disrupt the connection between the CMTs and the plasma membrane. At least in hepatic cells, the negatively charged phospholipids such as phosphatidylserine but not phosphatidylcholine seem to be good candidates for this connection (Reaven and Azhar, 1981). This cursory look into the literature about the effects of MTIs on the plasma membrane is meant only to leave open the possibility that other phenomena, so far unexplored, might be at the basis of the antiinvasiveness of MTIs.

VIII. Conclusions At this time, experiments with microtubule inhibitors allow us to conclude the following:

162

MARC M . MAREEL AND MARC DE METS

1 . Microtubule inhibitors disturb the equilibrium of microtubule assembly/disassembly and, therefore, interfere with the structure and function of the mitotic spindle and of the cytoplasmic microtubule complex. 2. Immunocytochemistry with antibodies against tubulin, the building block of microtubules, is the method of choice to evaluate the StdtUS of microtubule complexes inside cells. 3 . Microtubule inhibitors arrest invasion of malignant cells in organ culture and probably also in vivo. 4. The antiinvasive effect of microtubule inhibitors can be ascribed neither to cytotoxicity nor to inhibition of mitosis. 5. Most observations favor the hypothesis that inhibition of invasion by microtubule inhibitors is due to disturbance of the cytoplasmic microtubule complex and to the consequent inability of tumor cells to perform directional migration.

It is our opinion that microtubule inhibitors are powerful tools for further analysis of the mechanisms of tumor invasion including studies of microtubule assembly/disassembly in normal versus malignant cells and of microtubulemediated cellular activities.

AC.KNOWL.l:DCMENTS The authors thank G . MattIiys.De Sniet for typing the manuscript and J . Roels van Kerckvoorde. G . K . Dc Bruyne, and F. De Bruyn for preparing the illustrations. Research in the authors’ laboratory is supported by grants from the Kankerfonds van de Algeniene Spaar-cn Li.jfrentekas, Brussels, Belgium, from the N.F.W.O. Belgium (20093 and 39.000983), and from Eli Lilly Benelux, Brussels, Belgium.

REFEKENC~S Abercronibic, M . (1961). E.xp. Cell RPS. Sitppl, 8, 188-198. Abercrombie, M. (1970). Eur. J . Cuncrr 6, 7-13. Abcrcronihie, M., and Heaysnian, J . E. M . (1966). Ann. Med. Exp. Fenti. 44, 161-165. Abercrombie, M . , and Heaysman, J . E. M . (1976). J . Null. Cancerfns/. 56, 561-570. Anderscin, D. C., Wible, L. J . , Hughes. B. J., Smith. C. W., and Brinkley, B. R . ( I 982). CP//31, 719-729. Armstrong, M. T . , and Armstrong, P. B. (1979). Exp. Cell Res. 120, 359-364. Armstrong, P. B . , Quigley. J . P . , and Sidcbottom, E. (1982). Cuncer Rrs. 42, 1826-1837. Atassi, G . , Dumont, P . , and Vandendris, M . (1982). Invasion Metasrasis 2, 217-231. Back, O., Bandmann. U., Norberg, B . , and Siiderstroni, U-B. (1978). Scand. J . Haernurol. 20, 108-116.

Badley. R. A , , Couchnian, J . R., and Rees, D. A. (1980). J . Muscle Res. CeIl ,%‘or;/. 1, 5-14.

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Varani, J . (1982). Cancer Mefasfasis Rev. 1, 17-28. Varani, J . , Orr, W . , and Ward. P. A. (1978). Am. J . Pafhol. 90, 159-171. Varani, J . , Orr, W., and Ward, P. A. (1979). Cancer Res. 39, 2376-2380. Vasiliev, .I.M. (1982). I n “Cell Behaviour. A Tribute to Michael Abercrombie” (R. Bellairs, A. Curtis, and C. Dunn, eds.), pp. 135-158. Cambridge Univ. Press, London and New York. Vasiliev, J . M., and Gelfand, I. M. (1976). I n “Cell Motility - Book A: Motility, Muscle and Nonmuscle Cells” (R. Goldman, T . Pollard, and J . Rosenbauni, eds), pp. 279-304. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York. Vasiliev. J . M., Gelfand, I . M . , Domnina. L. V., Ivanova, 0. Y., Komm, S. G., and Olshevskaja, L. V. (1970). J . Emhryol. Exp. Morphol. 24, 625-640. Vasiliev, J . M . , Gelfdnd, 1. M . , Domnina, L. V., Zacharova, 0. S., and Ljubimov, A. V. (1975). Proc. N a f l . Acad. Sci. U . S . A . 72, 719-722. Vaughan, R. B., and Trinkaus, J . P. (1966). J. Cell Sci. 1, 407-413. Wagner, E. K., and Roiznian, B. (1968). Science 162, 569-570. Wang, R. W.-J., Rebhun, L. I., and Kupchan, S. M. (1977). Cancer Res. 37, 3071-3079. Wass, J. A., Varani, J . , Piontek, G . E . , Ward, P. A,, andOrr, F. W. (1981a). C e / I D # e r . 10, 329332. Wass, J . A., Varani, J . , Piontek. G. E.. Goff, D., and Ward, P. A. (1981b). J . N a t l . Cancer Inst. 66, 927-933. Weingarten, M. D., Lockwood, A. H . , Hwo, S . , and Kirshner, M. (1976). Proc. Narl. Acud. Sci. U . S . A . 72, 1858-1862. Weisenberg, R. C., Borisy, G. G . , and Taylor, E. W. (1968). Biochemistry 7, 4466-4479. Weiss, G. H. (1983). Am. Sci. 71, 65-71. Weiss, P., and Scott, B. I . H. (1963). Proc. Narl. Acad. Sci. U . S . A . 50, 330-336. Wells, J . , Berry, R. J., and Laing, A. H. (1976). Eur. J . Cancer 12, 793-796. Wilbur, K. M . , and Chambers, R. (1942). J. Exp. Zoo/. 91, 287-302. Wilkinson. P. C . (1976). In “~mmunobiologyof the Macrophage” (D. S. Nelson, ed.), pp. 349365. Academic Press, New York. Wilson, L. (1975). Awn. N . Y . Acad. Sci. 253, 213-231. Wilson, L., and Meza. I . (1973). J . Cell Biol. 58, 709-719. Wilson, L . , Bryan, J . , Ruby, A . , and Mazia. D. (1970). Proc. N a t l . Acad. Sci. U . S . A . 66,807-814. Wilson, L., Bamburg, J . R . , Mizel, S. B., Grisham, L. M., and Creswell, K. M. (1974). Fed. Proc. Fed. Am. Soc. Ekp. B i d . 33, 158-166. Wilson, L., Creswell, K. M., and Chin. D. 11975). Biochemistrvv 14, 5586-5592. Wilson, L., Morse, A. N. C., and Bryan, J . (1978). J . M o l . B i d . 121, 255-268. Wood, S. (1958). Arch. Pofhol. 66, 550-568. Wood, S., Baker. R. R., and Marzocchi, B. (1967). I n “Endogenous Factors in Influencing HostTumor Balance” (R. W . Wissler. T . L. Dao, and s. Wood, eds.), pp. 223-237. Univ. of Chicago Press, Chicago, Illinois. Yahara, I . , and Edelman, G. M . (1973). Nature (London) 246, 152-155. Yarnell, M. M., and Schnebli, H. P. (1974). J . Cell Sci. 16, 181-188. Yin, H. H.. Ukena, T . E.. and Berlin, R. D. (1972). Science 178, 867-868. Yoshida, K., Ozaki, T., Ushijima, K . , and Hayashi, H. (1970). I n r . J . Cancer 6, 123-132. Zavala. F., Geunard, D., Robin, J., and Brown, E. (1980). J. M e d . Chem. 23, 546-553. Zigmond, S . H., and Hirsch, J . G. (1973). J. Exp. Med. 137, 387-410.

INTERNATIONAL REVIEW OF CYTOLOGY. VOL YO

Membranes in the Mitotic Apparatus: Their Structure and Function PETERK. HEPLERAND STEPHENM. WOLNIAK Deprirtrnent of' Botciny , University of Mnssachusc~rts,Amhrrst, Masscdiusetts. mid Department of Boriinv, University id'Munllnnci, College Park. Mriylnnd I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Early Studies on Mitotic Membranes.. . . . . . . . , , . . . . . . . . . , . . . . . 111. ER in the MA of Higher Organisms.. . . . . . . . . . . . . . . . . . . . . . , . , . A. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Changes in the ER during Mitosis . . . . . . . . . . . . . . . . . . . . . . . . D. Membranes in Isolated M A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Organisms Thought to Lack ER in the M A . . . . . . . . . . . . . . . . . IV. Membranes in the MA of Lower Organisms.. . . . . . . . . . . . . . . , . , . V. Golgi and Other Membranes in the M A . . . . . . . . . . . . . . . . . . . . . , , . A. Golgi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Membrane Function: Regulation of [Ca'+ I . . . . . . . . . . . . . . . . . . . . . 11.

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B. Ca2+ Regulation in Nondividing Systems. . . . . . . . . . . . . . . . . . VII.

VIII.

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C . C a 2 + Regulation in the M A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane Function: A Component in Chromosome Transport . . . . . A. Membranes: An Integral Component of the Mitotic Cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 B. Membranes as a Mitotic Anchor.. . . . . . . . . . . . . . . . . . . . . . . . . 226 227 C . Membranes as Part of the Force Generation S y s t e m . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 ............ . . . . . . _ _ . _ . _ . . 231 References . . . . . . . . . .

I. Introduction Membranes are universal components of the mitotic (or meiotic) apparatus (MA). The nuclear envelope (NE) is present in cells of all eukaryotic organisms from the most primitive to the most advanced and inevitably it becomes closely associated with the spindle fibers during their formation and/or function. In addition to the NE there are other membranes, notably the endoplasmic reticulum (ER) and the Golgi apparatus, that occur in the MA of many different cells. Increasingly it is becoming evident that membranes in one form or another are as ubiquitous in the MA as microtubules. Furthermore, they undergo transforma169 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-3644W-9

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tions in concert with the events of mitosis that may be related to the formation and function of the spindle fibers. Whereas microtubules, ever since their discovery, have been assumed to be involved directly in chromosome transport, a function for membranes has been much less apparent. With the elucidation and characterization of the function of endomembrane system of the muscle cells, the sarcoplasmic reticulum (SR), and with the discovery that microtubule assembly could be controlled by the calcium ion concentration ([Ca2+1) it became attractive to imagine that membranes in the MA, like the SR of muscle, regulate [Ca2+] and thus the formation of the spindle and associated events of mitosis. Membranes, in addition, have been postulated to be structural components of the MA-cytoskeleton and possibly a direct participant in force generation for chromosome motion. Although the last 20 years of studies on mitosis have been dominated by single-minded attention to microtubules, there is a new and steadily growing interest in mitotic membranes. It is, seemingly, only the beginning of a potentially promising area of research and it is appropriate, therefore, to review the subject as it stands today in the hopes of more widely awakening an interest to the problem and in providing direction for future work. The purpose of this article is to show the structural contribution of membranes to the MA, to discuss the variability in this morphology that exists in different cell types, and finally to set forth ideas and experimentation on the function of these membranes. Brief general reviews on membranes in the MA have appeared (Harris, 1978; Hepler, 1977; Hepler er al., 1981; Paweletz, 1981) as well as more detailed accounts of membranes, especially in primitive eukaryotic cells (Fuller, 1976; Heath, 1978, 1980b; Kubai, 1975, 1978), to which the reader is directed for additional information.

11. Early Studies on Mitotic Membranes

The realization that membranes are integral and major components of the MA came from early ultrastructural studies on mitotic cells. Porter and Machado (1960) observed an extensive system of ER in dividing root tip cells of onion that clustered broadly in the polar region and extended around and into the MA. Independently during 1960 and 1961 additional accounts on mitotic membranes appeared that expanded our understanding of this subject. Rebhun (1960) portrayed long chains of rough-surfaced vesicles radiating out from the cell center along astral rays in the surf clam, Spisula solidissirnu. Examining spermatocytes of Drosophila, Ito ( 1 960) described the development of a multilayered system of ER cisternae, called parafusorial lamellae, that virtually encased the MA and totally separated it from the rest of the cell. The parafusorial lamellae, in addition, became continuous with an equally developed system of astral lamellae that radiated out from the spindle poles.

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Subsequent studies further enlarged our appreciation for the general and common occurrence of membranes in the MA by showing examples in a mammalian carcinoma (Buck, 1961) and in sea urchin embryos (Harris, 1961, 1962). Buck (1961) described quadrilayered lamellae of ER radiating out from asters and near chromosomes within the MA. He also depicted examples of vesicles and more conventional bilayered lamellae. Of all the early studies, those of Harris (1961) on sea urchins provide the most dramatic examples of membranes within the MA and the asters. The initiation of mitosis is first seen as a dense accumulation of ER around the aster, and following NE breakdown membranes penetrate the entire MA. She also described fine spindle fibers, and concludes that the MA as a whole consists of two different structural elements, “the coarser material of the astral center and astral rays, which are composed of tubular elements and vesicles of the endoplasmic reticulum; and the chromosomal and continuous fibers, which are straighter, finer, and more regular.” During subsequent years successive ultrastructural studies on mitotic cells depicted membranes, both ER and Golgi in the MA, some notable examples being derived from observations of HeLa (Robbins and Gonatas, 1964; Robbins and Jentzch, 1969; Robbins et al., 1968), spermatocytes of European corn borer (Roth et al., 1966), and pollen mother cells of Trillium (Sakai, 1969b). Thus from the outset it is apparent that membranes were considered key elements within the MA but unfortunately interest in them quickly declined. In brief membranes were displaced by spindle microtubules as the focus of attention. Although microtubeles had been observed in some instances in preparations fixed with osmium tetroxide (Harris, 1961), the introduction of glutaraldehyde as a superior primary fixing agent in the mid-1960s (Sabatini et al., 1963) made the demonstration of spindle microtubules possible and easy (Ledbetter and Porter, 1963) in virtually all cells, plant and animal. Given the long standing interest in the mechanism of chromosome motion and the possible role played by the spindle fibers it is not surprising that investigations became directed toward microtubules, their structure, composition, disposition within the cell, relationship to chromosomes and spindle poles, and changes during mitosis. However, it seems to have gone unnoticed that whereas glutaraldehyde-osmium tetroxide is good for fixing and contrasting microtubules, it is much less successful in its ability to display elements of ER. Ledbetter and Porter (1963) realized this shortcoming and noted that elements of ER are defined in great measure by their rows of attached ribosomes. It follows that smooth tubular profiles of ER might escape attention altogether. In addition to the difficulty in contrasting membranes there has been a lack of interest on membranes which prompted Harris (1975) to state that “even when they are obviously present, they are often ignored by the authors or only mentioned in passing.” Despite the various problems and lack of attention, some conspicuous MAmembrane associations have appeared that could not be ignored. Certainly the sea urchin, by different fix ation procedures, yields striking and compelling im-

FIG. I . The aster center at metaphase in the sea urchin, Arhacia. The centriole is surrounded by microtubules and numerous membrane profiles. The membranes are predominately smooth and vesiculate. Unless specified cell length bars for all figures equal I pm. x37,OOO. (From P. K. Hepler and E. D. Salmon, unpublished.)

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ages of membranes throughout the aster and MA (Harris, 1961, 1962, 1975; Ito 1981) (Figs. 1 and 2). Chains of vesicles radiating along tracks defined by microtubules virtually dominate the view (Harris, 1975). In many higher plants large aggregations of ER occur in the region of the spindle pole (Hepler, 1977) (Figs. 3 and 4); generally in a variety of animal cells the spindle pole or the centriole region is also characterized by enriched accumulations of vesicles and lamellar elements including both Golgi and ER (Paweletz, 1981). In more recent years studies on membranes have benefited from the development of techniques that markedly contrast membrane systems and thus bring them out in strong relief relative to the rest of the MA. Impregnation with osmium tetroxide or a mixture of zinc, iodine, and osmium tetroxide (Hawes rt a / . , 1981) produces intense staining of ER and Golgi but unfortunately, like potassium permanganate, destroys much cytoplasmic fine structure including microtubules. Judicious use of potassium permanganate alone or in combination with glutaraldehyde has proved valuable in delineating the ER and Golgi vesicles in dividing cultured mammalian cells (Moll and Paweletz, 1980). Finally, a combination postfixation that includes osmium tetroxide and potassium ferricyanide (OsFeCN) has been introduced that provides excellent contrast of ER while preserving the cytoplasmic detail similar to that obtained with a conventional glutaraldehyde procedure (Hepler, 1980, 198 1 ) (Fig. 3 ) . All these techniques have been used to portray dividing cells which when coupled with numerous earlier studies provide a rich documentation of spindlemembrane association. Together with the realization that these membranes may regulate the ionic milieu of the MA and/or they may be part of the cytoskeletal transport system for chromosome motion (Harris, 1975; Hepler, 1977), a more complicated but probably more realistic understanding of the MA emerges that may help us to decipher the mechanism of mitosis. etal.,

111. ER in the MA of Higher Organisms

A. GENERAL The ER is a conspicuous but morphologically variable component of the MA. Perhaps its most constant and important general feature in higher eukaryotes is its structural and developmental relationship with the NE. ER is connected to the NE during interphase. When cells enter mitosis the NE breaks and transforms into ER, whereas at the end of mitosis ER becomes appressed to the chromosomes and transforms back into NE (for review, Franke, 1974). Beyond this the ER in different organisms shows some remarkably intimate and specific associations with the MA and in particular with the spindle fibers that may suggest membrane functions underlying the mechanism of mitosis.

FIG. 2. Metaphase in Arbacia. A longitudinal section of the MA shows the chromosomes at the bottom of the figure. Numerous vesicular membrane elements are situated among the spindle microtubules. X32,500. (From P. K. Hepler and E. D. Salmon, unpublished.)

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FIG. 3 . Metaphase in a lettuce (Lacrrrcci) root cell. Elements of the ER aggrcgate at the two spindle poles and appear to form a cap. ER extends along the sides of the MA and a few tubular elements penetrate toward the metaphase plate. Prominently stained plastids occur at each pole as well as less darkly contrasted mitochondria. X 10.000. (From P. K . Hepler, unpublished.)

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FIG.4. A detail of ER at the spindle pole. In dividing barley (Hordeurn)leaf cells the ER at the spindle pole appears both as fenestrated lamellae (FL) and as tubular reticula (TR). These membranes are interconnected and are continuous with a layer of ER at the cell periphery (lower left comer of the micrograph). X30,OOO. (From Hepler, 1980.)

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In this section we discuss the structural relationship of the ER to the MA of cells from several different organisms. We point out the morphological variability of these membranes and show their changes as cells progress through mitosis.

B. MORPHOLOGY The ER consists of elements that may appear lamellar, tubular, or vesiculate. Lamellar ER often occurs at the periphery of the MA (Paweletz, 1981), or especially in plants, aggregated at the spindle pole (Hepler, 1977, 1980) (Fig. 3). It most closely resembles the NE and in contrast to tubular or vesiculate ER may have clusters of ribosomes attached. It seems certain that a portion of the lamellar elements in higher eukaryotes is derived directly from NE, which through breakdown, dispersal, and loss of nuclear pore structure becomes indistinguishable from ER. Even though nuclear pores are absent, interestingly, fenestrae are present (Hepler, 1980; LaFountain and Thomas, 1975). These openings in the ER system resemble nuclear pores to the extent that both are cytoplasmic channels that pass through a double membrane lamella. However, they remain morphologically distinct from nuclear pores in the following ways: they are irregular in size (40-100 nm diameter) and outline, they do not show the 8-fold radial symmetry of the nuclear pore, and they appear not to possess substructure or attached particles (Hepler, 1980). The lamellar fenestrae may be derived initially from nuclear pores and, during reformation of the nuclear envelope at telophase, may be a precursor membrane structure for the reformation of nuclear pores. However, recent work of K. L. McDonald (unpublished observation) reveals that in PtK cells fenestrated lamellae appear around the nucleus before the NE breaks down suggesting that the fenestrae can arise independently of nuclear pores. Although structurally dissimilar to nuclear pores the fenestrae of ER, like pores, may possess properties of vectorial transport. Since the lamellar ER, as discussed below, often forms a sheath around the MA and creates a compartment separate from the cytoplasm, the fenestrae may be avenues of communication and passage for macromolecules between the spindle and its surrounding cytoplasm. Tubular membranes usually occur in the form of an anastomosing network or reticulum. They may be present in the MA interior in large amounts, as for example in oocytes of the strepsipteran parasite, Xenos, in which the spindle region is virtually filled with tubular and vesicular ER (Figs. 5 and 6) (Rieder and Nowogrodzki, 1983). More commonly the MA contains fewer elements of tubuiar ER; in spindles of plants these may extend to the pole regions where they are observed to be continuous with the fenestrated lamellae (Hepler, 1980). As the membranes of different morphologies merge distinctions between them become difficult to make; a close reticulation of the tubular elements renders them similar to the fenestrated lamellae, while an enlargement of the fenestrae causes

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the lamellar membranes to resemble those that are tubular (Fig. 4). Of the various categories of mitotic ER the tubular elements have been the most elusive because they easily escape detection by conventional glutaraldehyde-osmium tetroxide fixation procedures for electron microscopy. The OsFeCN technique, because it stains the ER inner leaflet and especially because it fills the cisternal space with an electron-dense reaction product, has proved particularly valuable in elucidating these membranes (Hepler, 1980, 1981). The dense staining permits identification of small segments of the tubular membranes in thin section that might otherwise go entirely unnoticed. Serial section reconstructions establish that these seemingly isolated segments or vesicles are in reality part of a complex three-dimensional membrane network (Hepler, 1980). In many examples the ER is apparent neither as lamellar nor tubular elements but as vesicles. Sea urchin MAS, for example, contain chains of vesicles (Harris, 1975; Ito et al., 1981) (Figs. 1 and 2 ) and dilated vesicles dominate the view of the pollen mother cell of Iris undergoing meiosis (Ryan, 1980) (Fig. 7). To what extent these differences in membrane morphologies are real and to what extent they are due to fixation artifact is an important but unanswered question. We cannot help but think that to a certain degree, the swollen vesiculate morphology is a preparation artifact. Marine organisms, in particular, are known to be difficult objects for electron microscopy. However, in the example of the pollen mother cell the same investigator using the same technique has produced rather different membrane morphologies in Alliurn as opposed to Iris (Ryan, 1980). In contrast to those of Iris the membranes of AlIium are unswollen and evenly spaced. Differences in membrane morphology probably exist between species but the exact nature and magnitude of these differences await further investigation and the use of alternate preparation procedures. Among membranes that are related to the ER because they too are derived from the NE are paired cisternae. Buck (1961) described quadrilaminar elements in carcinoma cells; they have been found repeatedly in a variety of animal cells (for review, Paweletz, 1981), but not to our knowledge in plants. During breakdown, the NE may fold onto itself, and instances occur in which the smooth inner membranes become tightly appressed, giving rise to the quadrilaminar appearance of paired cisternae. Normally they are found only during mitosis and although they may occur within the MA they show no particular relationship to the spindle structure. The morphology of ER associated with M A is not unique or markedly different from ER in general. It does, however, in certain instances, bear a striking FIGS.5 m i ) 6. Metaphase in the oocyte of the strepsipteran parasite, Xrnos. High voltage electron micrographs of longitudinal (Fig. 5 ) and transverse (Fig. 6) thick (0.25 pni) sections reveal an MA that is packed with smooth tubular and vesicular membranes. The chromosomes are numbered. Small black dots in the vicinity of numbers 6 and 7, and elsewhere in Fig. 6 are microtubules in cross section. X 14,000. (From C. Rieder. unpublished.)

FIG. 7 . Early anaphase in a pollen mother cell of Iris. The spindle pole is especially rich in ER (er) and many elements extend in the MA. ER also serves as a boundary between the MA and nonspindle cytoplasm. Mitochondria cluster along the sides of the MA but they d o not enter the spindle region. Kinetochore microtubules (krnts) and nonkinetochore microtubules (nkmts) can be discerned. Inserts show the same cell by light and low power electron microscopy. Bar for the inserts equals 10 k m . x4000. (From Ryan, 1980.)

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similarity to the SR of muscle. From studies in which the ER has been contrasted with the OsFeCN postfixative it is apparent that both ER and SR stain positively and that both share common structural detail (Hepler, 1980). For example, the fenestrated lamellae of the MA resemble the fenestrated collar of SR where it overlies the “A” band of the muscle sarcomere (Forbes et a / ., 1977; Peachey, 1965). Likewise, the tubular reticulum of mitotic ER resembles the “network” SR, that portion of the muscle membrane system that connects the terminal cisternal to the fenestrated collar. To the extent that muscle SR, a fully interconnected system, is differentiated into regions that are morphologically and functionally distinct, one can ask whether the mitotic membranes are similarly differentiated. The morphological evidence indicates a degree of differentiation between interconnected mitotic membranes which causes us to speculate about a functional specialization regarding Ca2 + release and resequestration that might exist within the mitotic ER (Section VI). IN C . CHANGES

THE

ER

DURING

MITOSIS

1. Prophase During the course of mitosis there are dramatic changes in the distribution and amount of spindle associated ER, and there is the creation of intimate structural associations between membrane elements and spindle microtubules. The intial formation of the spindle in many organisms is closely associated with the NE. In endosperm of Haernanthus polarizing light microscope observations (InouC and Bajer, 1961) and immunogold staining with antitubulin (DeMey et u / . , 1982) show that the spindle first appears in a region of cytoplasm called the “clear zone” that resides adjacent to the NE. Even more intimate structural affinity between the NE and the developing MA occurs in the moss Mniurn in which the regularly spaced microtubules arise in a sheath that encases the NE before its subsequent breakdown (Lambert, 1980). A NE association with, and possible role in MA formation can be inferred from studies on animal cells in which it is shown that centrioles may be closely associated with the NE (Fulton, 1971; Nadezhdina et a/., 1979) and that the separating centriole pairs may move in grooves on the NE (Paweletz, 1974). The NE may be more than just a barrier to microtubule entry; it may participate as a MTOC based on its potential role at maintaining proper ion concentration, especially [Ca2 I (see Section Vl). The early stages of mitosis are also characterized by an accumulation of ER at the spindle pole during prophase. The studies of Harris (1961, 1962), mentioned previously, document the aggregation and growth of a compact mass of membranes and vesicles around the centrioles in dividing sea urchins. But in many different cell types membranes abound in the polar region and one frequently observes these elements radiating along paths defined by microtubules (Dougherty and Lee, 1967; Moll and Paweletz, 1980; Pleshkewych and Levine, 1975; +

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Robbins and Gonatas, 1964; Robbins et al., 1968; Roos, 1973; Roth et al., 1 966). Drosophila spermatocytes have an especially prominent system of membranes; astral lamellae of ER radiate outward from the centriolar region at the spindle poles (Church and Lin, 1982; Fritz-Niggli and Suda, 1972; Ito, 1960). These elements make contact with a multiple layered system of parafusorial ER that accumulates during prophase and ensheaths the nucleus (Ito, 1960). Prior to meiosis, the nucleus with NE is surrounded by mitochondria but no particular membrane system. As prophase progresses elements of ER appear outside of the mitochondria-rich perinuclear zone. Subsequently, as the ER acquires its multilayers, it becomes situated immediately adjacent to the NE. The situation in plants is also quite dramatic because in many respects the spindle pole prior to mitosis is a void, lacking any particular organellar inclusion or specific grouping of existing organelles. lnterphase cells may possess a perinuclear cytoplasm rich in ER but normally one cannot detect any order in this material that indicates the structure and position of the future spindle poles. However, as cells enter prophase lamellar and tubular elements of ER become clustered in regions that become the poles (Allen and Bowen, 1966; Burgess, 1970; Burgess and Northcote, 1968; Cutter and Hung, 1972; Esau and Gill, 1969; Hanzely and Schjeide, 1973; Hepler, 1976, 1977, 1980; Hepler and Wolniak, 1983; Sakai, 1969a,b; Pickett-Heaps, 1967; Ryan, 1980; Wilson, 1970). Between these membrane cisternae segments of microtubules appear radiating outward (Fig. 8); thus to a limited degree the pole morphology of these plants resembles the aster of dividing animal cells. Aggregations of ER characterize the spindle pole in flowering and nonflowering plants alike. The most prominent examples have been observed in dividing pollen mother cells of several different monocots, including Trillium (Sakai, 1969b), Allium (Ryan, 1980), Iris (Ryan, 1980, and Tradescuntia (Wilson, 1970). 2. Prometaphase-Metaphase The breakdown of the NE marks the onset of prometaphase and is the point at which microtubules are first seen in the nuclear region attached to chromosomes. Membranes are also seen in the MA at this time; ER and fragments of NE form a variety of spindle associations from those in which the membrane component seems randomly displaced in the MA to those in which rather specific alignment between microtubules and membranes occurs. A description of some examples below will provide an appreciation for the range of spindle-membrane associations that arise during early prometaphase and develop thereafter. Again, we cite the studies on marine invertebrate embryos as providing incontrovertible evidence that membranes comprise a significant portion of the MA (Harris, 1961, 1962, 1975; lto et a / . , 1981; Rebhun, 1960) (Fig. 2). But similar abundances of intra-MA membranes have been reported for pollen mother cells of Trillium

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FIG. 8. Early prometaphase in lettuce (Lacritca).The nuclear envelope (NE) has broken in a few places. The spindle pole (P) becomes obvious at this time a5 a cluster of ER elements. X 18,000. (From Hepler and Wolniak, 1983.)

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(Sakai, 1969a) and Iris (Ryan, 1980), and for spermatocytes of the European corn borer (Roth et ul., 1966). More commonly, one observes an enrichment of membranes in the spindle pole and a noticeable but smaller amount of ER dispersed within the MA itself. HeLa cells, for example, contain several intraspindle profiles of ER (Moll and Paweletz, 1980; Robbins and Gonatas, 1964; Robbins and Jentsch, 1969; Robbins et ul., 1968) as do a host of other cells of both plant and animal origin. A survey of many published studies showing electron micrographs of dividing cells reveals almost without exception the presence of ER elements within the MA. Of particular interest is the fact that in some species the intraspindle elements of ER possess remarkably specific structural associations with the spindle microtubules. In barley leaf cells, for example, use of the OsFeCN procedure for contrasting ER reveals a tubular ER that extends from the pole specifically along kinetochore microtubules to the point at which the latter attach to the chromosome (Hepler, 1980) (Fig. 9). Serial section analysis indicates the reticulate nature of the tubular ER and shows that membrane elements penetrate throughout the kinetochore fiber and form numerous close appositions with the microtubules (Hepler, 1980) (Fig. 10). A similar situation occurs in spermatocytes of Drosophilu in which Church and Lin (1982), through serial section analysis of a kinetochore, demonstrate that “each microtubule can be tracked to the membrane invagination at the polar regions.” Huemunthus endosperm contains membranes throughout the metaphase MA and in association with the kinetochore fiber bundles (Jackson and Doyle, 1982; Jensen, 1982). The study of Jackson and Doyle (1982) shows that those elements of ER associated with the kinetochore tubules have a more lamellar, oriented appearance than those in the adjacent spindle region among the nonkinetochore microtubules (Fig. 1 I ) . The more rigid-appearing morphology of the kinetochore-ER suggests possibly a degree of association with the microtubule not evident in nonkinetochore regions of the MA. A conspicuous association of metaphase kinetochore microtubules with ER cisternae has been recently described in the moss Funuriu (Schmiedel et a / . . 198 1). Smooth membrane elements intermingle within the bundle of kinetochore microtubules and in some instances appear to ensheath them. While membrane elements generally align parallel to microtubules throughout the MA, Schmiedel et ul. (198 1 ) note that in the region of the kinetochore fiber close to the point of attachment to the chromosome, the ER cisternae tend to lie perpendicular to the FIG. 9. Metaphase in a barley (Hordeurn) leaf mesophyll cell. Densely stained elements of ER aggregate at the poles, and interpenetrate the MA along kinetochore fibers ( * ) , ER also forms a boundary between spindle and nonspindle cytoplasm. From serial sections of this cell we know that those regions appearing as gaps (lower left) in the surrounding ER layer become occupied by membrane elements in successive sections. The kinetochore-membrane association seen in the upper right is shown at higher magnification in Fig. 8 . X 13,000. (From Hepler, 1980.)

FIG. 10. A membrane-kinetochore association in Fig. 7 is shown here in greater detail. A tubular reticular ER entwines in and around the kinetochore microtubules (MT) and extends to the point of fiber attachment to the chromosome (C). Serial sections show that the segments of membranes are part of one interconnected, continuous system. x46.000. (From Hepler, 1980.) Bar equals 0.5 km. FIG. 1 1 . Kinetochore-membrane association in the endosperm of Haemanthus. Because the

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long axis of the microtubules giving the impression that membrane had entered the fiber laterally. Finally, we draw attention to a unique situation in wolf spider spermatocytes in which it is noted that each chromosome and its attached kinetochore fiber is encased in a separate lamella of ER (Figs. 12 and 13). The membrane is not NE since nuclear pores are absent; also NE breakdown occurred earlier in prophase. At the polar region the individual encasements for each chromosome focus toward a centriole and appear fenestrated (Wise, 1982). The few examples of kinetochore fiber-membrane associations demonstrate the range of structural variability that has been found but do not exhaust the number of instances in which the relationship has been observed. Beyond those mentioned there are examples in lettuce root tip (Hepler, unpublished), onion root tip (Hanzely and Schjeide, 1973), onion pollen mother cells (Ryan, 1980), Trillium pollen mother cells (Sakai, 1969b), soybean protoplasts (Fowke et al., 1975), grasshopper spermatocytes (Nicklas ef al., 1979), cricket spermatocytes (A. Harris, unpublished), and rat hepatoma cells (Chang and Gibley, 1968) and therefore we conclude that a kinetochore fiber-ER association may be widespread. Elements of the ER thus construct a variety of associations with the fibrous components of the MA. In most instances the membranes are dispersed within the MA but in others they form specific associations with kinetochore fibers. These structural observations emphasize the existence, in some examples at least, of a high degree of order among spindle membranes and of membrane distributional changes that coordinate closely with those of the spindle fibers during mitosis. From a structural point of view it seems important to ask how these associations arose. Very little information is available on this process. It seems evident that membranes move in from the MA periphery, especially from the poles. Microtubules may also come into the MA from the pole, and in so doing they may carry membrane elements. It is often observed during late prophase that microtubules impinge upon and probably account for the poleward deformation and even the subsequent breakdown of the NE (Bajer and MolC-Bajer, 1969). These observations provide a basis for understanding a process of microtubule penetration that transports membrane into the MA. If however chromosome microtubules arise through assembly and growth from the kinetochore (Borisy and Gould, 1977; McGill and Brinkley, 1975; Snyder and McIntosh, 1975; Telzer et al., 1975) then it becomes difficult to understand how the kinetochore cisternal spaces are not occluded by the dense OsFeCN reaction product as shown in Fig. 8 the membrane elements are less conspicuous. Nevertheless they are present in large number. In Huemanrhus it is noteworthy that the ER within the kinetochore appears to align along the microtubules (*), x25,OOO. (From Jackson and Doyle, 1982.) Bar equals 0.5 pn.

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specific membrane systems orginate, since it would seem the membranes would be required to move inward while microtubules grow outward. However, a long held view concerning chromosomal fiber formation is that the spindle fibers (microtubules), formed at the poles, are captured by the kinetochore (for review, Schrader, 1944; Pickett-Heaps and Tippit, 1978). We are certain that membranes enter the MA from the periphery, most likely the poles, and the simplest explanation is that microtubules do likewise. Besides polar aggregations of ER and intrusions into the MA there is a third general aspect of spindle membrane morphology at metaphase that deserves attention. We refer to the observations showing that the MA is surrounded by layers of ER (Erlandson and De Harven, 1971; Fux, 1974; Hepler, 1980; Ito, 1960; Kubai, 1982; Moll and Paweletz, 1980; Wolf, 1980). Thus despite the fact that the NE has broken and dispersed, the MA remains ensheathed by a membrane system. In some instances such as Drosophila spermatocytes the sheath is multilamellar and highly developed (Ito, 1960). Spermatocytes of Sciaru during meiosis possess a membrane layer around the MA that is much less conspicuous than that observed in Drosophila but serial section reconstruction unambiguously establishes its presence and virtual continuity (Kubai, 1982). Commonly the membrane sheath is one to a few layers thick and in any particular section may show regions of discontinuity (Hepler, 1980; Moll and Paweletz, 1980) (Fig. 14). Often it can be established from serial sections that the elements of ER, in another plane, lie close to the discontinuities. Thus the MA can be viewed as being surrounded by a membrane sheath that is composed of overlapping cisternal elements (Hepler, 1980). Spaces or discontinuities within the sheath may provide pathways, albeit tortuous, for communication and transport between the MA and its surrounding cytoplasm. It can be argued that this membrane sheath results from a simple displacement of cytoplasm during spindle formation. Due to space limitations, for example, membrane elements may become piled up at the spindle periphery and then their presence would be of little consequence to the function of the MA. Evidence against this view is provided by our studies of mitosis in spermatogenous cells of the water fern Marsileu (Hepler, 1977, Hepler, unpublished). In this instance the MA occupies only a small portion of the total cell volume. The cytoplasm is relatively poor in ER, yet the MA possesses a distinct boundary of ER (Figs. 14 and 15). It seems possible that elements from the breaking NE and those recruited from the cytoplasm are specifically accumulated around the MA. We Fras. I 2 AND 13, Kinetochore-membrane association in the Wolf spider (Lvcosci) spermatocyte. A unique situation occurs in which each chromosome becomes enwrapped by a layer of ER. At the point where the kinetochore fiber5 extend from the chromosome to the pole ( K ) so does the ER envelope. creating a separate microspindle for each chromosome. X 11.000. In Fig. 13 the central kinotochore of Fig. I2 is shown at higher magnification. The ER sheath delimits the bundle of kinetochore microtubules. Some nonkinetochore microtubules occur outside of the ER sheath but they are few in number. X22.000. (From D. Wise and D. F. Kubai, unpublished.)

FIG. 14. Metaphase in a spermatogenous cell of the water fern. Marsilea. The MA is small, occupying less than 20% of the total cell volume. In the cytoplasm as a whole there are relatively few 190

FIG. 15. A detail view of the upper spindle pole in Fig. 14. A highly reticulate and mostly smooth ER nlembI'dne system characterizes the region to which the spindle microtubules ( * ) extend and terminate. X32.000. (From P. K . Hepler. unpublished.)

elements of ER. At mitosis much of the ER clusters at the spindle poles (P) and some membranes extend along the sides of the M A . Plastids, mitochondria. and Golgi-dictyosomes are randomly scattered in the cytoplasm outside of the spindle region. The upper spindle pole is shown in Fig. 15. X 11,500. (From P. K . Hepler, unpublished.) 191

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conclude, therefore, that membranes create domains; they separate the MA from the rest of the cytoplasm and within the MA itself they may isolate particular structures or spindle elements such as kinetochore fibers. 3. Anaphase-Telophase Following the relatively static condition of metaphase a period of rapid membrane reorganization occurs once the cell enters anaphase. Because of the speed of the process it has been difficult to obtain a detailed ultrastructural sequence in many different organisms. From the onset of anaphase it may be only a few minutes before the nuclear envelope reappears (Robbins and Gonatas, 1964). Ultrastructural studies on several different plant cells indicate that the membranes remain most heavily accumulated on the poleward side of the advancing chromosomes with relatively few elements in the interzone. In barley the polar membranes become especially aggregated, possibly through compaction as a result of progressive anaphase motion of the chromosomes (Hepler, 1980) (Fig. 16). The specific kinetochore-membrane associations that are evident in metaphase and early anaphase lose their distinctive morphological identity during mid to late anaphase and similarly the ER ensheathing the MA loses its continuity and disperses. Concomitantly some elements of ER become appressed to the poleward faces of the advancing chromosomes and reconstruct the nuclear envelope (Fig. 17). The presence of nuclear pores on those elements closely associated with the chromosomes indicates that the membrane has changed from ER to NE. The conversion of ER to NE appears to involve a host of changes that are only partly understood. Immunological and biochemical studies on the fibrous laminar component of the NE show that there are specific proteins (lamins) which disperse with NE breakdown at prometaphase and reaggregate at NE reformation (Gerace and Blobel, 1980; Grace et al., 1978). Structural studies by Maul (1977) suggest that a portion of the nuclear pore complex remains on the chromosomes themselves and may serve to seed the reformation of new pores during telophase. The structural studies support both a distributional and quantitative change in membranes during mitosis. In HeLa, for example, the electron microscopic studies suggest that membranes increase in amount from prophase to metaphase and that they decline thereafter (Moll and Paweletz, 1980; Robbins and Gonatas, 1964), whereas in barley, a cursory examination suggests that membranes may not reach their peak until anaphase (Hepler, 1980). This issue has been treated more rigorously by Ryan (1980) in his studies on pollen mother cells of Iris. Using a planometric analysis of electron micrographs of pollen mother cells in meiosis I1 he calculated the percentage ER at the different stages. The results indicate that membranes increase in amount by a factor of two from interphase to their highest point at mid prometaphase. Thereafter there is a rather abrupt decline in metaphase without much further change in anaphase or telophase. Perhaps not surprising is the fact that spindle membrane increase is most rapid during and just after nuclear envelope breakdown. However, these quantitative

FIG. 16. Anaphase in a barley leaf cell. An extensive accumulation of densely stained ER occupies the spindle pole while only a few elements occur in the interzone. X 13.000. (From Hepler, 1980.1

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FIG. 17. Telophase in a barley leaf cell. ER at the poles hecomes spread out along the periphery of the cell. Some elements become appressed predominately to the poleward facing portions of the chromosomes and transform into the new nuclear envelope (*). X 16,000. (From P. K . Hepler, unpublished.)

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data by indicating a high point in prometaphase do not support a metaphase or especially anaphase time of maximum membrane accumulation. There, of course, could be marked variation between different organisms and thus these kinds of studies should be performed more widely so that we can develop a sense of quantitative change in membranes as we have for the spindle fibers.

I N ISOLATED MA D. MEMBRANES

MA have been isolated from different organisms but most commonly from marine invertebrates (for review, Zimmerman and Forer, 1981). Since the normal MA as visualized in fixed, embedded preparations contains an abundance of membranes, it is not surprising that the isolates also show numerous membrane profiles. However, without exception the membranes in the isolates look badly damaged and distorted (Forer and Zimmerman, 1974, 1976a,b; Kane, 1962; Rebhun and Sander, 1967); the individual elements are extensively swollen and markedly vesiculate. If the isolation procedure employs a detergent such as Triton X-100 membranes are destroyed altogether either in sea urchins (Salmon and Segall, 1980) or HeLa cells (McIntosh et al., 1979). Thus, while techniques have been developed to preserve the bulk of the birefringent retardation and hence the bulk of the ordered microtubules, the same cannot be said for membranes. One of the best membrane preservations by electron microscopic standards occurs in those MA isolated in hexanediol and subsequentially transferred to H,O buffered at pH 6.0 for fixation (Kane, 1962). In an important study on the content of dry matter in the MA Forer and Goldman ( 1972) found, using interference microscopy, that hexylene glycol isolates lost up to 85% of their dry matter from their in vivo state. There was only a small reduction in birefringent retardation and therefore a dissolution of ordered microtubules could account for only a minor fraction of the total extracted material. Since membranes, as visualized by electron microscopy, comprise a significant proportion of the MA and since these elements suffer considerable distortion and destruction during the isolation procedure, it seems plausible that their loss may account for a major share of the observed reduction in dry matter. Despite the obvious difficulties it is noteworthy that isolated MA contain membranes. Under conditions in which the MA have been mechanically isolated the vesicular material is abundant and apparently capable of pumping Ca2+ (Silver et al., 1980), a point which we will return to in Section VI. Future studies on isolated MA, especially those aimed at developing functional mitotic models, might benefit from a closer attention to the quality of membranes in the preparations. Indeed, one explanation for the relative lack of success in obtaining chromosome motion in isolated MA may be due to the distortion of the mitotic ER and the inhibition of its contribution to the operation of the MA.

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E. ORGANISMS THOUGHT TO LACKER

I N THE

MA

Despite the large number of examples showing ER within the MA there are cell types, especially spermatocytes in some species of insects, in which these membranes have not been commonly observed. Reports from different laboratories have failed to reveal ER in the MA of spermatocytes of the crane fly, Nephrotomu (Forer and Brinkley, 1977; Fuge, 1974a,b, 1977a,b, 1980, 1981; LaFountain, 1976) for example. These and other observations have prompted Fuge (1977b) to conclude that, “Endoplasmic reticulum and large cytoplasmic organelles such as mitochondria and Golgi bodies, are generally not present in the spindle region.” We agree that Golgi bodies and mitochondria are not generally present in the spindle, but, as the foregoing section has amply demonstrated ER does normally occur in the MA. Is the observed absence of ER in spermatocytes of the crane fly and possibly other insects a species or cell type anomaly, or are there alternate explanations for the apparent lack of membranes? In part it seems certain that some cell types have few membranes in their M A . However, although the ER may be reduced in amount it must be emphasized that it is not entirely absent. When Nephrotoma spermatocytes were frozen and fractured ostensibly to examine the structure of spindle microtubules the micrographs in addition depicted elements of lamellar, fenestrated ER among the microtubules (LaFountain and Thomas, 1975). We suspect that membranes are more abundant in these organisms than the micrographs from fixed material would lead us to believe. Again we emphasize the inability of conventional glutaraldehyde-osmium fixation to adequately contrast membranes thus accounting, in part, for our failure to visualize these elements. Particularly when membranes are smooth, lacking attached ribosomes, and reticulate-tubular in morphology they may easily escape detection. In support of these assertions we note that the development of newer fixation procedures, the “microfixation” technique (Nicklas et d.,1979), has revealed a remarkable abundance of tubular ER within the M A of grasshopper and cricket (A. Harris, unpublished) spermatocytes, cell types which by other procedures may show only few membrane profiles. In cells fixed in this manner the cisternal space of the tubular ER is moderately electron dense so that the membrane is easily visible as an abundance of curved tubular profiles that extend through the MA and even penetrate kinetochore bundles. Some mammalian cells such as the commonly used PtK line appear to have few ER profiles in their M A (Roos, 1973). A thorough reanalysis of these cells FIG. 18. Prometaphase in a cultured rat kangaroo (PtK) (Potorus) cell. A view of the spindle looking obliquely downward from the pole shows the microtubules radiating from the centriole pair. Among the microtubules is an extensive system of reticulate, tubular ER, revealed through staining with OsFeCN. Portions of three chromosomes (C) are evident. X 10,000. (From K. L. McDonald, unpublished.)

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using the OsFeCN procedure however reveals a considerable amount of smooth lamellar and tubular ER (McDonald, 1982) (Fig. 18). The bulk is situated around the MA periphery but some elements extend into the interior of the spindle. From these few examples we think that it is premature to conclude for any cell type that membranes are absent. The development of newer, improved techniques for enhancing membrane contrast is revealing ER profiles in cell types that heretofore have been thought to lack these structures. The newer procedures, themselves, may be far from perfect or ideal in depicting membrane structure and thus it seems likely that continued progress will reveal more rather than less of these virtually ubiquitous spindle components.

IV. Membranes in the MA of Lower Organisms Among lower eukaryotic organisms, perhaps even more so than in advanced species, there is considerable variation in the structural relationship of membranes to the MA. In most of these instances the membrane under consideration is the NE. Beyond the simple fact that the NE creates a separate compartment for the genetic component of the cell, there are some striking and specific associations between chromosomes and NE on one hand and microtubules and NE on the other hand that suggest NE participation in the formation and function of the MA. In this section we will not attempt to provide a detailed review of all lower organisms that have been studied, rather it is our intention to single out a few examples that display the diversity, range, and specificity of membrane (NE) association and participation in mitosis. Spindle and NE structure in fungi, algae, and protozoans have received considerable attention in the literature; for additional information the reader is directed toward several recent reviews (Fuller, 1976; Heath, 1978, 1980b; Kubai, 1975, 1978; Pickett-Heaps, 1974). A major generalization concerning the NE of many lower organisms is that it remains intact throughout mitosis (Fig. 19). These are referred to as “closed” MA in contrast to the “open” MA of higher organism in which the NE breaks down. It is apparent, however, from the studies summarized in the previous section, that even in higher organisms with “open” spindles the MA remains surrounded by a system of ER. Likewise many “closed” MA undergo a limited amount of NE breakdown, often in the polar region. Thus the structural distinction between open and closed spindles may be simply a matter of degree. Similarly spindle membranes may have comparable functional roles in “closed” and “open” MA. Studies of lower organisms emphasize the important structural role that the NE plays during mitosis; it may be the attachment site for the chromosomes and/or microtubules. Especially good examples occur in dinoflagellates (Cachon and Cachon, 1974; Kubai and Ris, 1969; Oakley and Dodge, 1976), which may represent examples of the most primitive eukaryotes that are known. In most

FIG. 19. Meiotic metaphase in the hollyhock rust fungus, Pucciniu. The MA is entirely closed. Spindle microtubules insert upon a specialized region of the nuclear envelope. A few vesicles (V) occur within the M A . On the lower left the nucleus forms an outpocketing due possibly to growth of the NE. X22,500. (From O’Donnell and McLaughlin, 1981b.)

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examples the NE remains entirely closed throughout mitosis. Chromosomes within the nucleus remain condensed and occur in “ V ” shapes with the point of the V being attached to the inner membrane of the NE. Microtubular spindle fibers occur outside of the NE in cylindrical channels that penetrate through the nucleus. Although Kubai and Ris (1969) assert from their studies of Crypthecodinium cohnii that microtubules do not attach to the NE, the more recent work of Oakley and Dodge (1976) on Amphidinium reveals clear terminations of microtubules onto the outer membrane of the NE at the point at which chromosomes are attached. A primitive kinetochore thus occurs in dinoflagellates and consists of a region or patch of NE to which chromosomes are connected to the inside and microtubules to the outside. Even more sculptured examples of a membrane interface in primitive kinetochores have been revealed in the hypermastigote, Trichonympha (Kubai, 1973) and the parasitic protozoan, Syndinium (Ris and Kubai, 1974). In Trichonympha a three-pronged accumulation of dense material encrusts an opening on the NE and becomes the region to which chromosomes and spindle fibers attach (Fig. 20). These observations together provide compelling support for the view that at least a portion of the kinetochore was originally associated with the NE. Perhaps the most persuasive evidence for microtubule-NE association occurs in those organisms in which the intranuclear spindle fibers insert into a spindle pole body or nucleus associated organelle that resides either close to or directly upon the NE (Fig. 19). An especially well studied example occurs in the fission yeast, Saccharomyces, in which the spindle pole body, consisting of a dense layered plaque, is embedded within a discontinuity in the NE (Moens and Rapport, 197 1 ; Peterson et al., 1972). T o the inside is a layer of amorphous granular material into which the shaft of spindle microtubules terminates, while on the outside rests a layer of fibrillar-granular material which is capped over by a flattened membrane vesicle. The importance of the yeast spindle pole body in microtubule organization is demonstrated by those studies showing that isolates can initiate microtubule polymerization in vitro (Byers et a/., 1978; Hyams and Borisy, 1978). A membrane association with the spindle pole body is made evident not only by the fact that the body itself is directly attached to the NE but also by the fact that a flattened membrane vesicle immediately outside the nucleus overlies the structure (Peterson et a / . , 1972). There are, in addition, organisms lacking spindle pole bodies in which intranuclear microtubules are associated laterally along the NE. In the micronucleus of the protist Tetrahymena (Jaeckel-Williams, 1978; LaFountain and Davidson, 1979, 1980) a sheath of microtubules oriented parallel to the mitotic axis lines the inner surface of the NE. These microtubules appear partially embedded in a fibrillo-granular matrix that is attached to the NE. Furthermore they are crossbridged to one another and also laterally to spindle microtubules that are centrally displaced within the MA. A continuum of structural interactions via microtubules thus can be perceived from the NE inward to the chromosomes.

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FIG.20. Kinetochores in the hypermastigote. Trichotiytnpha. A specialized plaque within the nuclear envelope is the point at which microtubules on the outside and chromatin (C) on the inside attach. X75.000. (From Kubai, 1973.) Bar equals 0.5 pm.

A second example of intranuclear microtubule association with the NE occurs in the green alga Oedogonium (Coss and Pickett-Heaps, 1974; Pickett-Heaps and Fowke, 1970). During mitosis horn-like projections of the NE protrude outward in the axis of the spindle. Electron microscopic examination of these projections reveals that they are filled with microtubules. Again the NE appears to act as a locus to which microtubules associate or upon which they seed and polymerize. Besides serving as attachment sites for chromosomes and microtubules the NE shows a variety of specializations that are temporally and spatially related to the events of mitosis. A great many organisms with “closed” MA develop enlarged openings or fenestrae on the NE in the region of the spindle pole (for review, Fuller, 1976; Heath, 1978, 1980b, 1981; Kubai, 19’75, 1978). Sometimes these are simply enlarged pores (Fig. 21) but often marked discontinuities in the NE

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FIG. 21. Mitosis in the trophozoite of the malaria parasite, Plasmodiurnfufiux. The nucleus ( N ) is closed except for an enlarged pore at the spindle pole (arrow). Spindle microtubules (sf) focus toward the pore and a dense staining material appears to extend between the nuclear and cytoplasmic compartments. x45,OOO. (From Hepler et a / . . 1966.) Bar equals 0.5 p.m.

arise with the concomitant appearance of membrane vesicles. Microtubules both from the intranuclear spindle and those outside of the nucleus focus or aggregate around the fenestrated region, giving the impression that rapid and massive interchange, perhaps even of whole microtubules, can occur at these points. Extensive arrays of membrane (ER) have been observed in some organisms to occur outside of the NE and these too appear to be related to or associated with the MA. In different fungi (Braselton e t a / ., 1975; Dykstra, 1976; Setliff et a/., 1974) and algae (Hudson and Waaland, 1974; Marchant, 1972, 1974; McDonald, 1972; Pickett-Heaps, 1972a) a perinuclear sheath of ER surrounds the nucleus and in some instances appears to be intimately associated with the formation and organization of an extra nuclear array of microtubules. In the red alga Mernbranopteru (McDonald, 1972) microtubules are sandwiched between a perinuclear layer of ER and the NE. Subsequently, the microtubules disappear as the intranuclear spindle forms. In this example the NE is fenestrated at the poles and membrane discontinuities in the ER sheath also occur in the polar region. Another example of a specific accumulation of extranuclear ER occurs in the fungus, Basidiobolus (Gull and Trinci, 1974; Sun and Bowen, 1972; Tanaka, 1970). Here the spindle pole is not focused and covers a broad area. The NE fragments, but pieces of it remain and delineate the spindle boundary. Immedi-

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ately outside the nuclear region an “extensive labyrinth of membranous cisternae” (Tanaka, 1970) develops and persists throughout mitosis. These cisternae are referred to as a spongework and probably consist of ER; it is from the region occupied by the spongework that microtubules penetrate through the NE discontinuities into the MA interior. Finally among the lower organisms we draw attention to the existence of extensive arrays of membranes within the MA. A striking example occurs in the cruciform mitosis of fungi in the plasmodiophorales (Braselton et a / . , 1975; Dylewski ef a / . , 1978; Garber and Aist, 1979) (Fig. 22). The MA is “closed” with polar fenestrae in the NE. Concomitant with the organization of microtubules in the MA is the appearance of several conspicuous elements of membranes. Garber and Aist (1979) suggest that these may be derived from the NE that fragments at the pole, since some profiles contain nuclear pores. Furthermore, they may be carried in by microtubules that penetrate the MA from the pole during spindle formation. Electron micrographs of cells in metaphase reveal an extensive ramification of membranes throughout the MA; in some instances the elements align along kinetochore microtubules thus resembling closely the spindle-membrane configurations in barley leaf cells (Fig. 2 2 ) . While these intraspindle membrane elements may participate in reformation of the NE their appearance in such large amounts and in the early stages of mitosis well before anaphase suggests that their function may extend beyond the construction of a new envelope. Among other examples of intraspindle membranes in lower organisms we draw attention to the recent report of O’Donnell and McLaughlin (1981a,b) demonstrating the presence of intranuclear vesicles in the Hollyhock rust fungus, Puccinia (Fig. 19). The noteworthy feature of these observations is that the MA is entirely closed; a spindle pole body resides on the NE and there are no fenestrae or evident openings to the cytoplasm. Thus it is an unanswered question where these vesicles originate. In structure they resemble those derived from the Golgi but it seems unlikely that they could have entered the MA through the NE. Possibly they are derived from infoldings of the inner membrane of the NE. These observations should cause us to be alert for additional complexities and heretofore unrecognized membrane specializations in spindle structure. In brief it is evident that among lower eukaryotic organisms there are many examples in which membranes show a specific and intimate association with the MA. It seems important to an understanding of membrane function to observe that the NE may be the attachment site for both chromosomes and microtubules. For microtubules the NE may be associated with their kinetochoric or the polar terminations. In addition. microtubules may laterally associate with the NE. Together with the ramification of membranes both inside and outside of the nucleus it is apparent that spindle-membrane associations are diverse and complex; they may underlie key processes essential for the separation of chro-

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Fic;. 22. Metaphase in the club root fungus, Plasmodiophoru. The nuclear envelope has been replaccd by a double layer of perinuclear ER (PER) that surrounds the MA. Within the MA there are prominent nicmbrane intrusions ( * ) some of which extend along kinctochore microtubules. Centrioles at the poles possess a loose surrounding of membranes. some of which are derived from the fragmented nuclear envelope. Chromosomes (C); nucleolus (Nu). X28.000. (From Garber and Aist, 1979.)

mosomes. Aside from the function of these membranes in mitosis of lower organisms it is reasonable to ask which, if aily, of these functions have been retained in cells more advanced. The structural studies provide examples of common membrane-MA affinities between lower and higher organisms and thus comparative investigations on membrane function may prove valuable in deciphering operational similarities as well.

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V. Golgi and Other Membranes in the MA A. GOLGI There may be some who question the presence of ER in the spindle of higher organisms, but all agree that vesicles of one kind or another are present. It is difficult in some instances to define vesicle origin, but for many the Golgi apparatus is the source. The profusion of vesicles around and within the MA, especially in animal cells, has led Paweletz (1981) to conclude that the Golgi apparatus provides a large fraction of the mitotic membranes. Commonly in animal cells the Golgi apparatus, composed of several interconnected dictyosomes, is aggregated around the centriole during interphase. During the course of mitosis studies of several different cells show marked changes in the morphology of the Golgi system that are coupled to the stages of mitosis. Moskalewski et al. (1977), in a thorough study of the Golgi complex during mitosis in cartilaginous cells in vitro, reveal that by early prophase the dictyosomes that had been closely clustered around the centriole begin to disperse. Initially, the dictyosomal morphology remains normal, but before the onset of prometaphase the individual cisternae become reduced in number and smaller size. By metaphase the dictyosomes are small and discrete and in anaphase only a few remain at the spindle poles. The situation reverses itself quickly during telophase as the dictyosomes reemerge and regroup in their juxtanuclear region around the centrioles. Similar changes have been observed in other cell types (Kimura and Onoe, 1970; Melmed et al., 1973); in human melonoma cells the decrease in Golgi structure is so marked during anaphase that Maul and Brinkley (1970) report difficulty in finding any dictyosome cisternae. They are led to suggest that de novo reformation from nearby smooth ER may occur and may account in part for the reemergence of dictyosomes in telophase. Moskalewski et al. (1975) have correlated the change in Golgi morphology with the changes in microtubule number, both during normal mitosis and in the presence of antimicrotubule agents. Colchicine and vinblastine, which lead to the depolymerization of microtubules, cause a dispersal and degeneration of the dictyosomal cisternae similar to that observed during normal mitosis. Concomitant with the decrease in size and structure of the dictyosome is an increase in the number of vesicles. The changes appear not to be one of degenerative fragmentation but a vesiculation process normal for the Golgi system. Vesicles are found within and throughout the MA during mitosis; in several cell types they appear more numerous in the MA than ER. The aforementioned carefully documented dispersal and disorganization of the Golgi apparatus has not been observed in all animal cells. Investigating rat hepatoma cells, Chang and Gibley (1968) note that at all stages of mitosis dictyosomes of normal size and structure are evident, often clustered at the poles.

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They assert that the dictyosomes do not break up, a view that receives support from studies of Dougherty (1964) on hepatic cells of rat and Roth et al. (1966) on spermatocytes of the European corn borer. Changes of the Golgi apparatus similar to those reported above have also not been observed in dividing plant cells. In higher plants the Golgi, even during interphase, is already dispersed throughout the cell as many separate unconnected dictyosomes. Throughout mitosis unaltered dictyosomes are evident, and furthermore, they do not appear to be organized in any particular region although during cytokinesis they may cluster at the edges of the phragmoplast where they apparently provide vesicles for the growing cell plate (Whaley and Mollenhauer, 1963; Hepler, 1982). The vesicles themselves in cells of higher plants and animals seem to be randomly dispersed throughout the MA. No particular structural association or spatial aggregation similar to that noted for ER has been reported, although Paweletz (1981), based on studies of HeLa cells, allows that the vesicles are more abundant in the MA interior while the ER is more heavily clustered at the periphery. In our investigations on membrane distribution in barley leaf and lettuce root tip cells, we have observed small clear vesicles, identified as Golgi in origin because they do not stain with the OsFeCN reagent (Hepler, 1980). Sometimes these vesicles are situated among the kinetochore microtubules but generally they seem to be randomly scattered throughout the MA interior. Investigations on lower organisms have provided excellent examples of close structural affinity between the Golgi apparatus and the MA. There are several instances in fungi (Dykstra, 1976; Garber and Aist, 1979; Kazama, 1974; Porter, 1972) and algae (Heywood, 1978; Lgvlie and Briten, 1970; Marchant, 1972; McDonald and Pickett-Heaps, 1976; Pickett-Heaps, 1973b; Pickett-Heaps and McDonald, 1975) in which the Golgi apparatus resides immediately adjacent to a centriole, the latter occupying the spindle pole close to a fenestrated portion of the NE. Some of the most conspicuous examples of Golgi-MA associations occur in diatoms (Pickett-Heaps et al., 1975, 1978, 1980; Tippit and PickettHeaps, 1977; Tippit et al., 1978) (Fig. 23). Were it not for the remarkable nearly crystalline central microtubular spindle possibly the surrounding Golgi complex might attract more attention. In different species of diatoms Golgi dictyosomes and associated vesicles are conspicuously evident at the spindle pole and extending outward along the sides of the MA. Although fragments of NE may remain at the edge of the spindle in some species [e.g., Pinularia (Pickett-Heaps et al.. 1978) and Diutoma (Pickett-Heaps et al., 1975)] it is the aggregate accumulation of Golgi cisternae and vesicles that appears to form the boundary between the MA and the cytoplasm (Fig. 23). The association between Golgi and the MA occurs early in mitosis; as the polar plate arises and initiates the formation of spindle microtubules Golgi cisternae appear to be positioned nearby and may be the source of the associated vesicles. As mitosis progresses the Golgi cisternae

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FIG. 23. Anaphase in the diatom. Nitzschia. An enormous aggregation of dictyosomes and vesicles occurs at the edge of thc MA, along the right side of the figure. These Golgi derived membranes appear to form a boundary between the spindle and nonspindle cytoplasm. A few vesicles can be seen within the MA itself. X8500. (From Pickett-Heaps et a / . , 1980.)

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appear to extend outward from the pole plate along the sides of the MA. By late anaphase, studies on Fragilaria, for example (Tippit et al., 1978), show four prominent Golgi cisternae positioned two along each side of the elongating MA. Structurally, the Golgi and vesicles in diatoms rather than the ER-NE appear to create an isolated spindle compartment (Fig. 23). In addition to the Golgi-MA proximity in diatoms there is, invariably, a prominent vesicle or group of vesicles closely associated with a specialized mitotic center called a persistent pole complex (Pickett-Heaps et al., 1980; Tippit and Pickett-Heaps, 1977) (Fig. 24). The vesicle is positioned on the distal side of

FIG. 24. Early stage of spindle formation in the diatom, Nirzschiu. The spindle arises between two plates that become the poles of the MA. Of particular interest is the abundance of vesicles, possibly derived from the Golgi apparatus, that intermingle among the microtubules. A second noteworthy feature is that fact that a prominent vesicle (V) is always situated upon an amorphous granular matrix on the distal side of the polar plate. It too may be derived from the Golgi apparatus. X21.000. (From Pickett-Heaps ef a / . . 1980.)

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the persistent pole complex and seemingly attached to it by a granular-fibrillar matrix in a structural relationship that is maintained throughout division. B. MITOCHONDRIA Although mitochondria have been commonly observed in the MA of some cells, e.g., cultured PtK cells (K. L. McDonald, unpublished observation) they are generally excluded and may be clustered at the edges of the spindle (Wilson, 1928). Endosperm cells of the African blood lily, Haemanrhus, possess prominent aggregations of mitochondria and other organelles such as plastids in the region of the spindle pole (Hepler and Jackson, unpublished). In embryonic heart muscle cells mitochondria surround the MA and align along microtubules radiating from the asters (Goode, 1975b). Inspection of the published micrographs suggests in this instance that the mitochondria1 density at the edge of the spindle is greater than in the nearby nonspindle cytoplasm. During the later stages of mitosis mitochondria enter the interzone. Among the best established instances of mitochondria-MA association are several examples in spermatocytes of different insects. Examination by light microscopy reveals prominent phase-dense bands along the sides of the MA (for an example see Figs. 2a, 6 , 9a, b, in Kubai and Wise, 1981). Electron microscopy of these cells indicate that the bulk of the phase dense material is constituted by mitochondria. Long, branched organelles demark the sides of the MA but do not extend into the region of the pole. They create a distinct boundary between the spindle and the rest of the cytoplasm.

VI. Membrane Function: Regulation of [Ca2 ] +

A. GENERAL Of the possible roles that membranes play in the MA the regulation of [Ca2+] has seemed to be the most likely. The obvious structural similarity between spindle-associated ER and muscle SR invites comparison about functional similarities as well. Dougherty and Lee (1967) first hypothesized that smooth membranes in the MA might behave like the “relaxing grana,” the SR, of the myocyte and control contractile events of karyokinesis and cytokinesis through modulation of [Ca2 1. A specific role for Ca2+ in mitosis emerged from the discovery by Weisenberg ( 1972) that microtubules are depolymerized by elevated levels of this ion. These results further stimulated thinking about how the Ca2 itself is regulated in the MA and led to more specific models on spindle membrane function. Briefly it seemed likely that microtubules, a major component of the spindle +

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FIG. 35. Diagrammatic representation of a cell in late metaphase. An extenaive, Ca2+ -containing membrane system occupies the spindle poles and extends into the spindle interior along kinetochore fibers. As the cell progresses from metaphase to anaphase we suggest that therc is a release of Caz+ from the membrane compartment. The resultant increase in free [Ca’+] in the MA may activate one or more motile events associated with chromosome motion. (From Hepler p / a / . , 1981 .)

fibers, would be controlled in their formation and possibly function by fluctuations in the [ C a 2 + ]as brought about by the MA membrane system (Harris, 1975, 1981; Hepler, 1977; Hepler et ul., 1981). During prophase to metaphase, for example, one might expect the [Ca2 1 to be low (- 0.1 @f) to permit formation of microtubules, but with the onset of anaphase the intracellular [Ca2+ ] might increase and thus cause the depolymerization of microtubules, a process that is known to occur as the chromosomes move to the poles (InouC and Sato, 1967) (Fig. 25). In addition to microtubule depolymerization several other processes may participate in mitosis that are also calcium stimulated. For example microtubules may interact with adjacent microtubules or membrane elements and undergo sliding, being driven by a dynein-like ATPase (McIntosh et al., 1969). Evidence for dynein participation in mitosis has recently emerged (Cande, 1982) and based on comparison with its ciliary and flagellar counterpart the enzyme would be expected to be Ca2 regulated (Doughty, 1979). Yet another spindle-associated force-generating complex might be one composed of acto-myosin. Again through similarity with both muscle and nonmuscle motile systems one expects it to be Ca2+ regulated. Finally we draw attention to the possible occurrence of gel-sol conversions within the MA that might accompany a contractile event (McIntosh, 1981). Once more Ca2+ emerges as the likely physiological regulator. The mitotic cell might utilize a combination of the above processes, or others that have not been mentioned, to cause chromosome motion. Of particular note is the fact that of those processes mentioned an increase in [Ca2 ] would be expected to activate or stimulate them. Taken together these several potential mitosis-related events greatly strengthen the Ca2 hypothesis and make apparent the importance of elucidating the role of the spindle-associated membrane system (for review, Hepler and Wolniak, 1983). We hasten to add, though, that changes +

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in free [Ca2+]within the MA have not been observed. Thus, as attractive and compelling as the Ca2+ hypothesis may appear, the key question whether changes in free [Ca2+]occur in the MA during mitosis must be answered in order to establish a factual basis for the theory. Despite our lack of data of this central issue there have been, in recent years, several studies that indicate a role for membrane regulation of Ca2+ in the MA. In order to develop these ideas more completely we first briefly discuss Ca2 regulation in nondividing systems. +

B. Ca2+ REGULATION I N NONDIVIDING SYSTEMS The rapidly expanding body of information on Ca2+ regulation in a host of muscle and nonmuscle cells reveals aspects of the process that probably apply to most systems including the MA and thus it is instructive from a comparative point of view to discuss their common, general features. The resting free [Ca2+], for example, in virtually all cells that have been critically examined is around 0.1-0.2 pM. Changes in this concentration that stimulate a motile process generally involve an elevation to 1.0 or slightly higher. For muscle contraction these ranges of [Ca2'3 have been confirmed by different procedures; increasingly in nonmuscle systems similar concentration increases are observed for the activation of a variety of processes (for review, see Borle, 1981; Kretsinger, 1981). Changes in [Ca2+],may be transitory, as in muscle where resequestration quickly brings the ion concentration to the resting level within seconds (Endo, 1977), or they may occur over longer periods of time, as in several developmentally stimulated processes which appear to require elevated levels of Ca2+ for hours (Borle, 1981; Kretsinger, 1981). Of pertinence to our discussion are the systems and processes within the cell that regulate [Ca2 1. Just the routine maintenance of [Ca2 1, at submicromolar levels while the outside ion concentration is millimolar requires a considerable expenditure of energy to offset the 1000- to 10,000-fold electrochemical gradient across the plasma membrane (Borle, 1981; Godfraind-deBecker and Godfraind, 1980). Calcium buffering by cellular constituents may occur through several mechanisms. In a limited sense, cytoplasmic Ca2+ may be bound directly by macromolecules and thereby maintained at a low free concentraton. For example, calmodulin, with its Ca2+-binding sites could serve, in addition to its enzyme activating capacities, as an effective Ca-chelator in the physiological range of 0.1-4.0 pM free Ca2+ (Means et al., 1982). In cases of severe Ca2+ loading, however, saturation of these sites would probably occur quickly, and, therefore, it is likely that direct binding of the cation by macromolecules may be most important only for precise, short-term buffering of [Ca2+I,. Effective longterm maintenance of [Ca2+], on the other hand, results from the activities of Ca2 -transport ATPases. These transport pumps are located on the endo-

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membrane system (ER or SR), on the outer mitochondrial membrane or on the plasma membrane, and their common function is to reduce [Ca2+],by transporting it into the ER cisternal compartment, into the mitochondrion, or into the the extracellular space, respectively. The characteristics of these pumps have been the topic of investigation in many laboratories. In striated muscle, a very effective Ca2+-ATPase is situated in the endomembrane (i.e., SR) lipid bilayer. Following a massive efflux of Ca2 from the SR during muscle activation, the Ca2 -ATPase rapidly transports Ca2 back into the SR compartment and thereby buffers the myoplasmic [Ca2+] to submicromolar levels, required during muscle relaxation (Endo, 1977). In nonmuscle systems, too, Ca2 uptake into the endomembrane compartment facilitates [Ca2+], buffering. A Ca2+-ATPase situated on ER cisternae, for example in presynaptic nerve terminals, plays a major role in maintaining axonal Ca2+homeostatis (Blaustein et a l . , 1978a,b, 1980a,b; Brinley, 1980). The axonal pump is highly efficient at submicromolar [Ca2+], (Blaustein et a l . , 1980a), having rapid uptake rates at physiological concentrations of Ca2 and requiring ATP hydrolysis for transport (Blaustein et a l . , 1980b). In these respects it is similar to the SR Ca2 -ATPase (Blaustein et a l . , 1980a). In the presence of high levels of Ca2 , however, the axonal pump loses effectiveness, when compared to the SR pump (Blaustein et a l . , 1980a,b). Because of its high activity in low [Ca2‘1 and its low activity in high [Ca2+], the axonal Ca2 -ATPase situated on the ER has been termed the “high-affinity/low-capacity” Ca2 pump (Blaustein et a l . , 1978a). The axonal and SR Ca2+-ATPases exhibit similar sensitivities to transport inhibition by the Ca2+ ionophore A23187 (Blaustein et a l . , 1978a, 1980b), to tetracaine (10 mM) and to mersalyl (50 N )(Blaustein et a l . , 1978a). An important consideration in any cellular scheme for the regulation of [Ca2+] is the role played by mitochondria. Extensive studies show that they are capable of sequestering Ca2+ (Lehninger et a/., 1967; Bygrave, 1978; Carafoli and Crompton, 1978); however in contrast to axonal ER, mitochondria appear to possess a low-affinity/high-capacity pump mechanism that is inhibited by treatments with ruthenium red, FCCP, NaN,, and DNP, agents that have little or no inhibitory effect on ER-based uptake (Blaustein et a l . , 1978a). The low-affinity/high-capacity pump situated on the mitochondrial membrane functions at (Blaustein e t a l . , 1978a, 1980a), a optimal levels when [Ca2+],exceeds 10 condition of severe Ca2 loading. In contrast, the high-affinity/low-capacity pump of the ER appears best suited for Ca2 buffering under conditions of only low to moderate Ca2+ loading (see Blaustein et al., 1980a; Brinley, 1980). That both systems do function in concert to buffer [Ca2+],has been demonstrated in experimental morphological studies conducted at the electron microscope level (Henkart, 1980; Henkart et a l . , 1976, 1978; McGraw et a / . , 1980a,b). Variations in the preloading of axons with Ca2+, followed by oxalate +

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treatment, freeze substitution, and analysis by transmission electron microscopy or by energy dispersive X-ray techniques, reveal that under low to moderate Ca2 loading, high-affinity/low-capacity ER transport predominates and that under severe Ca2+ loading mitochondrial uptake occurs as well (Henkart er a / ., 1978; Henkart, 1980; McCraw e r a / . , 1980a,b). Mitochondria1 uptake does not occur in the presence of FCCP, DNP, or NaN,, even under conditions of severe Ca2 loading (McGraw et a / ., 1980a). Treatment with A23 187 or EGTA prevents oxalate accumulation in the ER (McGraw et al., 1980a,b). [Ca2 Ii buffering by mitochondrial- and ER-situated transport ATPases has been studied in a variety of other systems as well. [Ca2 + I i regulation in response to light adaptation in invertebrate photoreceptors appears to be mediated by the ER (Brown et a / . , 1977); light adaptation involves an increase in [Ca2+Ii (Brown and Blinks, 1974). Ca2 uptake, as determined morphologically by osmium-potassium antimonate precipitation occurs in both mitochondria and in ER cisternae (Perrelet and Bader, 1978; Walz, 1979, 1982). Ca2 uptake in ER microsomes isolated from adipocytes (Bruns et a/.. 1976), rat liver (Moore et al., 1975), and fibroblasts (Moore and Pastan, 1978) varies considerably in its sensitivities to [Ca’ ] and in its rate of C a 2 + uptake in the presence of inhibitors of mitochondrial and nonmitochondrial transport. Microsomal K,, values for Ca2 in these studies were intermediate between ER high-affinityilow-capacity (Kcil = 0 . 4 pM; Blaustein et al., 1978b, 1980b) and mitochondrial low-af10 p M ; Blaustein et a / . . 1978b, 1980b). In that finity/high-capacity (Kca some of the mitochondrial uptake inhibitors will reduce the rate and extent of Ca’+ uptake by liver microsomes (Moore and Pastan, 1978), these results describe either true differences among the transporting enzymes, or, alternatively, heterogeneity in the isolates. Yet another system for regulating [Ca’+ 1, is the plasma membrane Ca’ transport ATPase that extrudes the cation against a steep electrochemical and concentration gradient. Ca’ + efflux is often accompanied by antiport of K , Mg2 , Na , or H ’ ~ .Along with a rather strict specificity for ATP (Lew and Stossel, 1980; Lichtman et d . , 1981), calniodulin has been found to play an integral role in active Ca’+ extrusion across the plasma membrane in several types of cells. In the presence of ATP and calmoddin, outward transport of Ca2 is enhanced 2- to 4-fold in erythrocytes (Jarrett and Penniston, 1977; Raess and Vincenzi, 1980; reviewed by Roufogalis, 1979). macrophages (Lew and Stossel, 1980), and lymphocytes (Lichtman et a / . . 1981), and is reduced substantially by phenothiazine inhibition of calmodulin (Jarrett and Penniston, 1977; Lew and Stossel. 1980; Raess and Vincenzi, 1980; Levin and Weiss, 1980; Lichtman et d.,1981. 1982). Although the sensitivities of these pumps to [Ca2’- l i vary as a function of the experimental conditions (for discussion see Roufogalis. 1979), the ICa’ J i for half-maximai inactivation of “high-affinity” binding, ATPase, and C a 2 + transport falls typically in the 0.1-1 FM range +

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(Ferreira and Lew, 1976; Lew and Stossel, 1980; Lichtman et al., 1981). Plasma membrane Ca2+ pumps appear to be insensitive to ouabain (Lew and Stossel, 1980; Lichtman et al., 1981) and to inhibitors of mitochondria1 Ca2+ uptake, e.g., NaN,, NaCN (Lew and Stossel, 1980; Lichtman et al., 1981); however ruthenium red inhibits ATPase in inverted erythrocyte ghosts (Hinds et al., 1981). Perturbation of the transmembrane gradient by agents that disrupt ER-Ca2+ uptake, e.g., A23187, evokes a similar inhibition of Ca2+ transport by the pumps situated in the plasma membrane (reviewed by Roufogalis, 1979). The overall similarities of the high-affinity pumps located on nonmuscle ER and plasma membranes extend to observed rates of Ca2+ transport as well. These pumps function at a transport rate ranging from 1 to 15 nmol Ca2+/mg proteidminute, which translates to less than 10% of the maximum observed for skeletal muscle SR (reviewed by deMeis, 1981; deMeis and Vianna, 1979; Kaupp and Schnetkamp, 1982). Buffering of [Ca2+ I i by active transport into the ER, mitochondria, or to the extracellular space may be limited to specific regions in the cell (Rose and Loewenstein, 1975). Restriction of a pulsed [Ca2+Ii increase to a localized region in the cytoplasm by highly efficient sequestering mechanisms appears to create discrete “functional domains” that exhibit strikingly different physiological activities as a consequence of this concentration difference. The occurrence of a localized flux of Ca2 has been most graphically demonstrated in the fertilization reaction of Mecfaku (Ridgway et al., 1977; Gilkey et al., 1978). Measured by aequorin bioluminescence, the sudden increase in [Ca2 Ii is initiated at the micropyle when the spermatozoan contacts and fuses with the egg. Aequorin bioluminescence expands toward the vegetal pole as a ring in the cortical cytoplasm; as cortical vesicle breakdown progresses, so too does bioluminescence. Most remarkably, the ring of luminescence is never wider than 10 pm, providing evidence for rapid resequestration of Ca2+ following the event (Ridgway et al., 1977; Gilkey et al., 1978; Jaffe, 1980). +

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Effective buffering of [Ca2+1, to submicromolar levels is a prerequisite for stability of the MA, since spindle rnicrotubules, both in virro and in vivo,display high sensitivity to the cation. In vitro, spindle microtubules will depolymerize within 1-2 minutes if the free [Ca2 ] exceeds 1 pM (Salmon and Segall, 1980). In vivo microinjection of Ca2+ into the MA of echinoderm eggs (Kiehart, 1981) and cultured mammalian cells (Izant, 1983) promotes rapid disintegration of spindle fibers as assayed by polarized light microscopy. Interestingly, however, just minutes after the microinjection, reformation of the spindle and normal anaphase ensue (Kiehart, 198 1; Izant, 1983). Spindle reassembly in echinoderm eggs is inhibited by caffeine treatment, suggesting that buffering of [Ca2+],is +

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facilitated by a membrane transport and sequestration mechanism (Kiehart, 198 1). These results obtained in vivo, when coupled with ultrastructural demonstrations of the close proximity of spindle membranes (i.e., ER cisternae: Harris, 1975, 1978; Hepler, 1980; Hepler et al., 1981) to kinetochore fiber microtubules, suggest that the membranes may function in the regulation of [Ca2+Ii within the domain of the spindle. 1. Localization ofCa'+ in the M A

Using pyroantimonate precipitation as a histological stain at the electron microscope level Wick and Hepler (1980) have shown that the compartments delimited by spindle membranes are sites of dense precipitation. Studies that examine the sensitivity of known standards of K , Mg2 , and Ca2 antimonate to extraction by the chelators, EDTA and EGTA, establish that the deposits in tissue sections are most probably Ca2+ antimonate. Besides in the ER and NE, precipitates are observed over mitochondria and Golgi cisternae as well as in the wall near the plasma membrane and in the vacuoles. However, of all these compartments only the ER shows a close and specific association with the MA and thus from proximity alone appears to be the most likely candidate for regulating spindle [Ca2+J. While it is apparent that the deposits are confined to the cisternal space of the ER or NE it has not been possible to detect any changes in amount or localization during the different stages of mitosis. The deposits probably represent that component of the total Ca2+ which is exchangeably bound (Wick and Hepler, 1982), and in amounts it is of much greater magnitude than that which is free. Although we expect the free Ca2 is derived, at least in part, from this pool, the amount needed to cause a large change in free [Ca2+] constitutes only a small fraction of the bound [Ca2+ ] and thus would probably not be detected even if the technique were much more sensitive. Localization of Ca2+ has in addition been examined in living mitotic cells using the fluorescent chelate probe chlorotetracycline (CTC) (Wolniak et al., 1980, 1981). CTC is an antibiotic that freely passes across membranes, and has the capacity to bind multivalent metal cations. When complexed with a metal ion and illuminated with blue light, CTC emits a yellow-green fluorescence (Caswell, 1979). The physiologically relevant metal cations bound by the probe are Mg2 and Ca2 . Both the absorption and emission maxima are specific for the cation bound; for Mg2 , absorbance is maximal at 375 nm, and fluorescence at 520 nm, while for Ca2 , absorbance is maximal at 390 nm, and fluorescence at 530 nm. Ca2 -based fluorescence can be increased by judicious spectral control (Fabiato and Fabiato, 1979). The Ca2+ signal is augmented several-fold when the Ca2+-CTC complex is in association with a membrane surface (Hallett et al., 1972; Caswell and Warren, 1972; Caswell and Hutchison, 1971; Caswell, 1979) while the Mg2+ signal is affected to a far lesser extent by the polarity of the environment. Since the Ca2+-CTC signal is maximal at the polar/apolar +

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interface, the dye effectively maps the distribution of membrane-associated Ca2+. CTC binds Ca2+ fairly effectively ( K , = 9 pkf in 70% methanol; Caswell, 1979), and it appears that the [Ca2+Ii would have to be somewhat higher than “resting levels” for substantial binding by CTC to the cytoplasmic face of endomembranes (Caswell, 1979). Under conditions in which Ca2+ is sequestered in a membrane-bound compartment and present in high concentrations (> 1 pM), the CTC signal originates from the inner face of the membrane (Caswell and Brandt, 1981; Caswell, 1979; Caswell and Warren, 1972). Using CTC in vivo, on mitotic endosperm cells of Haemanthus, we observe a pattern of fluorescence from the metaphase spindle (Figs. 26, 27, and 28) that coincides with the distribution of the birefringent kinetochore fiber bundles (Fig. 27 vs 28; Wolniak et al., 1981). A massive tubular and reticulate network of ER cisternae is present in the metaphase spindle in close association with the kinetochore fiber microtubules (Jackson and Doyle, 1982) (Fig. l l ) . It appears that the CTC signal originates from these membranes. Although the kinetochore fiber birefringence remains as the cells progress into early anaphase, the discrete coneshaped zones of CTC fluorescence are no longer apparent, rather, spindle fluorescence becomes uniformly bright (Wolniak et al., 1980). CTC-spindle fluorescence in Haemanthus is sensitive to reductions in [Ca2+Ii by treatment with procaine, EGTA, and La3+ (Wolniak et al., 1980) and to increases in [Ca2+Ii by treatment with CCCP and DNP (Wolniak et al., 1980). While each of these perturbing agents has its own set of nonspecific effects, their common influence on [Ca2 Ii,and their predictable qualitative effects on CTC fluorescence suggest that we are monitoring shifts in the quantity of membrane-associated (i.e., bound) Ca2 . If taken at face value, the spindle membranes we detect with CTC fluorescence respond like a high-affinity/low-capacity Ca’ -buffering system. CTC has also been used to follow membrane-associated Ca2+ in dividing cultured mammalian cells (Schatten et al., 1982; Sisken et al., 1981) and isolated MA of sea urchins (Schatten ef al., 1982). Sisken et al. (1 98 1) find that CTC fluorescence during metaphase in HeLa cells is relatively low in the spindle and is more intense in cortical areas located around the MA. In this instance the probe is probably denoting the peripheral localization of Ca2 -rich organelles such as mitochondria. MA of sea urchins isolated in the absence of detergent contain significant CTC fluorescence (Schatten et a l . , 1982) as one would expect from the quantity of membrane vesicles that are known to be present. The fluorescence, as in endosperm of Haemanthus, is both punctate and diffuse and +

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FIGS.26, 27, A N D 28. Phase contrast, fluorescence, and polarized light micrographs of a metaphase endosperm cell of Huemunfhus. The cell has been treated with chlorotetracycline to label membrane-associated Ca2+. The pair of arrows denote a region of the MA that is devoid of chromosome arms (26). This same region contains cones of chlorotetracycline fluorescence (27), and birefringent kinetochore fibers (28). It is noteworthy that the cones of chlorotetracycline fluorescence exactly overlie the birefringent kinetochore fibers. X600. (From Wolniak er a / . . 1981. ) Bar equals 10 ILm.

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PETER K . HEPLER A N D STEPHEN M. WOLNIAK

again probably reflects the distribution of Ca2 -containing organelles and endomembranes. +

2. Membrane-Ca2 Transport in the MA The evidence above establishes that the endomembrane system contains Ca2 but do these membranes have the capacity to transport and thereby sequester Ca2 ? Recently, Silver et a / . (1980) demonstrated, with a mechanical isolation technique, that membrane vesicles are intimately associated with sea urchin spindle microtubules, and that at least some of these vesicles exhibit ATPdependent Ca2 transport. Ca2+ uptake is augmented 180% over controls in the presence of ATP. Uptake is not significantly different from controls in the presence of either AMPPCP or AMPPNP (nonhydrolyzable ATP analogs) or when treated with the Ca2+ ionophore A23187 (Silver et al., 1980). When treated with CTC, these MA emit characteristic yellow-green fluorescence that is sensitive to detergent or Ca2+ perturbation by A23187 (Schatten and Schatten, 1980; R. B. Silver, personal communication). Using a rather different method of isolation, Petzelt and co-workers (Mazia et al., 1972; Petzelt, 1972, 1979; Auel and Petzelt, 1978) have isolated a “mitotic Ca2+-ATPase” from MA in a variety of cell types. This ATPase is most prevalent during periods of high mitotic activity in synchronized cells (e.g., sea urchin embryos, HeLa cells, etc.; see Petzelt, 1979) and appears to be associated with membranes (Auel and Petzelt, 1978). Unlike the Ca2+-ATPase of sarcoplasmic reticulum, or that of many other cells, whose Ca2+-ATPase molecular weight range is approximately 100,000-1 10,000, the mitotic Ca*+-ATPase has a molecular weight of approximately 260,000 (Petzelt, 1979). Attempts at inhibition of the mitotic Ca2+-ATPase by treatment with methylene blue, ruthenium red, oligomycin, ouabain, and vanadate are ineffective (see Petzelt, 1979, for a discussion). Whether or not this protein functions in the type of Ca2+ transport observed in isolated MA by Silver et al. (1980) remains to be demonstrated. Neither Ca2+-ATPase activity, from the spindle as described by Petzelt and coworkers, nor Ca2+ uptake by spindle vesicles as described by Silver and coworkers appears to be activated or regulated by calmodulin (for a review, see Nagle and Egrie, 1981). +

+

+

+

3. [Ca2’] Changes in the MA Increases in [Ca2+Ii accompany a host of developmental processes that in some instances involve mitosis or meiosis. An excellent example occurs in starfish oocytes in which an increase in [Ca2+Iiis brought about by the binding of the hormone 1-methyl adenine to the egg surface that stimulates the breakdown of the nuclear envelope and the progression of the cell through meiosis (Moreau et al., 1978). [Ca2+] increases associated with cell division are also inferred from studies of other systems in which mitosis is induced following application of the ionophore A23 187 and exogenous Ca2 (Luckasen et a l . , +

219

MEMBRANES OF THE MITOTIC APPARATUS

1974; Saunders and Hepler, 1982). A connection between a [ C a 2 + ]increase and mitosis seems likely; yet, in all of these examples the results indicate ion changes that precede, sometimes by several hours, the actual mitotic process. Thus the occurrence of Ca2+ fluxes specifically at the time of mitosis remains undemonstrated. Under the assumption that changes in [Ca2 1 do occur during mitosis and are essential to the normal progression of events attempts have been made to culture actively dividing cells at extremely low (< l o p 8 M ) or extremely high (> l o p 3 M ) concentrations of the ion (Wick and Hepler, in preparation). Using microspores of the water fern Marsileu Wick and Hepler (1984) find that low concentrations, produced by incubation in chelators (EDTA, EGTA), inhibit mitosis whereas high concentrations, generated in culture by the ionophore A23 187 plus 10 mM Ca' , have no effect. The cycling-dividng cell is admittedly complex and it is possible that the restrictive-low [Ca2+ ] inhibits processes other than those involved in spindle function, such as DNA synthesis. We are puzzled though why the high [Ca2 ] failed to produce an effect since we would predict that spindle fibers would be depolymerized and mitosis inhibited. Independent studies showing that A23 187 plus Ca2 stimulated acidification of the medium presumably by an exchange of Ca2+ for H + supports the contention that intracellular [Ca2+] has increased (Wick, 1978). Given the complexity of the system, especially the existence of separate compartments within the cell as created by an extensively ramified membrane system, we cannot be certain that the [Ca2+] specifically within the domain of the spindle has been modified. These experiments require an independent means for detecting lCa2 ] in order to establish that the concentration has been altered. An indication that there is a Ca2 -sensitive step during mitosis itself derives from the recent studies of Izant (1983) and from work in our laboratory (Hepler, 1983). Using cultured PtK- 1 cells Izant (1983) microinjected Ca2 [ 1- 10 p M ] solutions during metaphase and thereby hastened the onset of anaphase. Injection of Ca2+-EGTA buffers in which the [Ca2+] is 0.1 pM or less inhibits the transition from metaphase to anaphase. Similarly we (Hepler, 1983) have shown that culture of dividing Tradescantia stamen hairs in restrictive [Ca2+] (< 0.1 pA4) lengthens, by a factor of two, the time required for a cell to progress from nuclear envelope breakdown to the onset of anaphase. Curiously in both PtK cells and Tradescantia hairs the modified [Ca2+] appears to have no effect on the rate of anaphase chromosome motion. These foregoing data suggest that [Ca2+] changes do occur but their detection has so far proven to be difficult. Rink et al. (1980) have attempted to measure Ca2+ transition in embryos of Xenopus using an ion selective electrode. Although their method is capable of measuring very low [Ca2+] (< l pA4) they failed to detect any changes during mitosis. Moreover, under conditions in which the [Ca2+] was clamped at 0.1 pM they failed to stop cleavage leading them to conclude that Ca2+ fluxes did not accompany cell division. We think that the +

+

+

+

+

+

+

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PETER K. HEPLER AND STEPHEN M . WOLNIAK

conclusions fail to take into account the existence of subcellular compartments as defined by endomembranes. It seems likely that changes in [Ca2+] might occur specifically within the domain of the MA and unless the microelectrode was precisely positioned within this compartment the ion transients would go undetected. With the view that the presumptive ionic changes will be spatially localized within the MA we have sought the existence of these hypothetical fluxes microfluorometrically using permeant dyes in the spindle of living endosperm cells of Haemanthus (Wolniak et al., 1983). CTC has been employed to monitor changes in membrane-associated Ca2 while 8-anilino-1-naphthaline sulfonate (ANS - ) and 3,3’-dipentyl 2,2’-dioxacarbocyanine [diOC,(3) 1 have been used to detect “voltage” changes. To date our studies have been directed at possible changes during the metaphaselanaphase transition since at this time one might expect an increase in [Ca2+] that participates in the breakdown of microtubules. In addition the splitting of the chromatids, as indicated by the recent work mentioned above, appears to be, in part at least, under Ca2+ control. Our results show a decline in CTC fluorescence from a 12 pm diameter spot within the chromosome-to-pole region of the spindle that precedes the onset of anaphase by 810 minutes (Wolniak et al., 1983). A decline in CTC fluorescence represents a decrease in either the amount of membrane-associated Ca2+, or the amount of membrane material present in the zone of measurement. That total membrane quantity remains constant is demonstrated by unchanging fluorescence during metaphase with the fluorescent membrane marker N-phenyl-1-naphthylamine (NPN) (Wolniak et af., 1983). The change in CTC emission intensity then signifies a reduction in membrane-associated Ca2 , and suggests the occurrence of Ca2+ translocation prior to the onset of anaphase. Studies on muscle SR, both in vivo and in vitro,have correlated the decline in CTC fluorescence with an efflux of Ca2 from the membrane compartment. By comparison we suggest that the CTC fluorescence reductions indicate a similar release of membrane bound Ca2+ in the MA; indirectly these data further suggest that the free [Ca2+] within the spindle has risen. The ultrastructure of the dividing Huemanthus endosperm cell shows a profusion of membrane elements. Tubular cisternae of ER entwine around kinetochore microtubules while more loosely arranged, vesiculate lamellae occur between kinetochore bundles (Jackson and Doyle, 1982) (Fig. 11). The possibility emerges that these two somewhat differently disposed membrane systems have different amounts of associated Ca2+ depending on the phase of mitosis. The observations on flattened metaphase endosperm cells reveal that the CTC fluorescence is localized in cone-shaped arrays that specifically overlie the kinetochore bundles (Wolniak et d . , 1981). The interpretation at the ultrastructural level would therefore place the greater quantities of membrane-associated Ca2 in the kinetochore-associated tubular cisternae. During late metaphase and early anaphase, when the fluorescence signal diminishes, the cones also disappear and +

+

+

+

+

22 1

MEMBRANES OF THE MITOTIC APPARATUS

the CTC fluorescence becomes uniformly spread across the spindle. An explanation for these observations may derive from our understanding of Ca2+ movements in striated muscle. Here it is shown that, at rest, Ca2+ is localized in the terminal cisternae of the SR. Upon stimulation Ca2+ is released, and following contraction is resequestered throughout the entire SR (Winegrad, 1970). In the dividing Haemanthus cell the morphological and physiological observations are consistent with a similar explanation. It is possible for example that Ca2 , stored within tubular ER cisternae among the kinetochore microtubules at metaphase is released prior to the onset of anaphase and subsequently becomes associated with or resequestered by all membranes throughout the MA. Fluctuations in [ C a 2 + ] may not be the only ionic events that accompany the transition from metaphase to anaphase. We have observed dramatic changes in fluorescence from the chromosome-to-pole region of the spindle at the onset of anaphase, using two charge-sensitive dyes, the cationic probe 3, 3'-dipentyldioxarcarbocyanine [diOC,( 3)] and the anionic probe 8-anilino- 1 -naphthyline sulfonate (ANS) (Wolniak et a/.. 1983). Although we do not know the mechanisni(s) by which increases in fluorescence intensity occur, we emphasize the fact that they occur specifically within the spindle and are not observed in nonspindle cytoplasm or the organelle-rich spindle pole. We infer that considerable shifts in ion distribution accompany the onset of anaphase. Fluctuations in [H ] or [ K ] may play a central role in mitotic regulation, and may be responsible for the fluorescence shifts we observe with diOCJ3) and ANS. Clearly, further clarification of these changes is required. In a scheme for the regailation of mitosis, we believe i t is important to regard the domain of the spindle as a discrete, or nearly discrete entity in the cytoplasm. It is separated from the nonspindle cytoplasm by the ER-NE complex, and ionic events that can be observed within the spindle using fluorescence probes (Wolniak et (11.. 1983) appear to be restricted to that domain. Based on our fluorescence intensity measurements (Wolniak et u l . , 1983), we also believe that a mitotic regulation scheme must also inciude fluctuations in ion concentrations. Although we do not know the extent to which H , K', or Caz.'. may be involved, it is reasonable to suspect that a shift in ion status in the spindle resulting from ion translocations either triggers directly or provides permissive conditions for chromosome motion. The regulatory role of calmodulin in many other systems and its abundance in the spindle (Means et a / . , 1982) requires serious consideration in any regulatory scheme for mitosis, whether it be involved in triggering motile activity, or simply in buffering [Ca'+Ii. Finally, we believe that a complex combination of factors, rather than a single event may initiate chromosome motion. +

+

+

+

4. Cu' Regulcition during Ei1oli.rtioti of' the M A Throughout the discussion o n Ca' + regulation in the MA repeated emphasis has been placed on the proximity of membranes to the spindle fibers. This +

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PETER K. HEPLER A N D STEPHEN M . WOLNIAK

structural coupling may permit an equally close degree of physiological communication and regulation, and indeed, the studies of Kiehart (1981) and Izant (1983) show that artificially introduced Ca2+ is rapidly sequestered. It thus seems plausible that the fine control of [Ca2+] may have been an important factor in the evolution of spindle-membrane associations; specifically it may have provided the selective pressure that favored the development of an intimate structural apposition of membranes and microtubules. In primitive eukaryotes with “closed” MA the NE resides close to the spindle elements and seems well disposed for the regulation of [Ca2+]. The great number of organisms with closed spindles attests to the evolutionary success of this configuration, yet the more advanced eukaryotes increasingly possess “open” spindles. Because the extensive transformations of the NE and other membranes in open spindles (e.g., dispersal of the NE, formation of a spindle membrane boundary, interpolation of elements within the M A interior, and reformation of the NE) require expenditure of energy and resources, it is reasonable to ask how these transformations confer a selective advantage upon the advanced eukaryotic cell. It has been suggested that the breakdown of the NE may be important for entry into the nucleus of essential spindle components, such as tubulin, that are synthesized in the cytoplasm (Goode, 1975a; Pickett-Heaps, 1974). This idea seems attractive; however, we point out that microtubules do assemble in spindles that are entirely closed (reviewed by Fuller, 1976; Heath, 1978, 1980b, 1981a; Kubai, 1975). Thus, while breakdown of the NE may facilitate the entry of tubulin subunits into the MA it is not essential. The extensive interpenetration of membranes into the spindle region suggests that processes beyond simply a mixing of cyto- and nucleoplasms are involved. The distances over which the fine control of Ca2+ may be exerted may have provided the selective pressure during evolution that favored the close juxtaposition of the Ca2 +-sequestering component (the membrane) and the Ca’ -regulated components (the spindle fiber) (T. Cavalier-Smith, personal conimunication). In lower eukaryotes with closed spindles the nuclei are generally small, often less than 5 p m in diameter (Table I). Distances may be sufficiently close S O that Ca‘+ released by the NE can diffuse quickly and efficiently to the target macromolecules. With the acquisition of greater genetic complexity the size of the genome and nucleus increased. A metaphase M A might now exceed 20 pm or more in diameter and the distance between the MA periphery and the interior spindle fibers might be too great for diffusional processes to deliver or remove Ca2+ quickly and uniformly from all regions of the MA. As a result, chromosome splitting and the onset of anaphase, for example, might lose synchrony. Some chromosomes might move more quickly to the poles while others failed to move at all. In brief the motile events within the MA might lose their coordination that is essential for an exact separation of duplicate chromosomes to +

MEMBRANES OF THE MITOTIC APPARATUS

SILL Ob

223

TABLE I M ~ T A P H ASPINDLL SL 01-P R I M I T IEUKARYOTtS V~ WITH CLOsto MA''

Fungi Algae Protists

1-5 pm

6-10 pm

>I0 p m

23 16 7

0 3 3

0 2 0

"Measurements on nietaphase MA have been made from published micrographs of 54 different species of fungi. algae, and protozoans possessing closed. intranuclear spindles. The table summarizes the number of species in different size categories. All 23 species of fungi with closed MAS, from which measurements were made, have spindles between I and 5 pm across, whereas 16 of 21 algae and 7 of 10 protozoans fall into this category. Only 2 species examined had closed spindles larger than I 0 pni and these are the green algae Ordogonium (17 pni) and Mougeotia ( I 2 pm). In Oedogonium. even though the closed spindle is large. the unique projections of microtubules into horn-like outfoldings of the NE brings the membranes and tubules into very close proximity. Thus the 17 pm width of the spindle may be misleadingly large relative to distances over which ion regulation is presumed to occur. The list of organisins from which measurements were made is as follows. Fungi-Arcyriu cinerea (Mims, 1972), Boletus rubinellus (McLaughlin, 197 I),Coprinus rudiatus (Thielke, 1974), Dic/yostelium discoideum (Moens, 1976; Roos and Camenzind, 198 I).Echinosteliurn minutum (Hinchee and Haskins, 1980), Enrophlyctis sp. (Powell, 1975). Fusariurn mysporum (Aist and Williams. 1972), Lubyrinrhula sp. (Perkins, 1970; Porter, 1972), Phvcomyces hlakesleeanus (Franke and Reau, 1973). Physurum flavicomum (plasmodium) (Aldrich, 1969), Phjwrurn polycephulurn (plasmodium) (Ryser, 1970; Sakai and Shigenaga, 1972), Pilobolus crystallinus (Bland and Lunney, 1975), Plusmodiophora brassicae (Garber and Aist, 1979), Polysphondvlium iiolaceum (Roos. 1975), Pucciniu malvacearum (O'Donnell and McLaughlin, 1981b). Sacchurornyces cerevisiae (Moens and Rapport, 1971; Peterson and Ris, 1976; Peterson et a / . . 1972). Saprolegnia Jerax (Heath, 1980a), Sorodiplophtys stercoreu (Dykstra, 1976). Sorosphaera veronicae (Braselton et a / ., 1975), Thrartstochytrium sp. (Kazania, 1974), Thraitstorheca claiuta (Heath, 1974a), Uromvces phaseoli (Heath and Heath, 1976), and Ustilugo sp. (Poon and Day, 1976). Algae-Acrosiphonia sp. (Hudson and Waaland, 1974). Bulbochaete sp. (Pickett-Heaps, I973a), Cuulerpa hrachvpus (Hori, 198 1). Ch1aurnydomonas reitzhurdtii (Coss, 1974; Johnson and Porter, 1968), Cladophora Jexitosu (Scott and Bullock, 1976), Clurfophora glomerutu (McDonald and Pickett-Heaps, 1976), Coelastrum sp. (Marchant, 1972), Cvlindrocupsu sp. (Pickett-Heaps and McDonald, 1975). Dusya baillouviana (Phillips and Scott, I981 ), Euglena gracilis (Gillott and Triemer, 1978), Microspora sp. (Pickett-Heaps, 1973b), Mougeotia sp. (Beck-Hansen and Fowke, 1972). Oedogoniurn cardiacum (Pickett-Heaps and Fowke, 1970), Pediastrum horqanum (Marchant. 1974). Pedinomonu.s (Pickett-Heaps and Ott, 1974), Polysiphonia harvevi (Scott et a/.. 1980), Porphyridiurn purpureum (Schornstein and Scott, 1982). Tetruedron sp. (Pickett-Heaps, 1972b). Tetraspora sp. (Pickett-Heaps, 1973~).Uhw murabikis (Lwlie and Brlten, 1970). and Vacuokuria virescens (Heywood, 1978). Protozoans-Allogromiu laticolluris (Schwab. 1972). Blepharisma sp. (Jenkins. 1967), Naegleriu gruheri (Schuster, 1975). Paramecium aurellu (micronucleus) (Stevenson and Lloyd, 1971). Plasmodiumfallux (Hepler ef al.. 1966). Tetrahymena (micronucleus) (LaFountain and Davidson, 19791, Tetruhymenu (macronucleus) (Williams and Williams, 1976), Tokophyra infirsionurn (micro- and macronucleus) (Millecchia and Rudzinska, 197 1 ), Trvpanosoma cruzi (Solari, 1980). and Trypanosoma rhodesiense (Vickernian and Preston, 1970). No attempt has been made to develop taxonomic groupings. Also no claim is made with regard to the completeness of this list: very likely several more species could be added. We believe, however, that new examples would fall into the size categories defined and that relatively few, large. closed, intranuclear spindles will be found.

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PETER K. HEPLER AND STEPHEN M. WOLNIAK

daughter cells. The system of juxtaposed elements of membranes and fibers found in open spindles may permit refined temporal and spatial regulation of [Ca2 ] within cells containing large genomes that assures a correct, failsafe separation of chromosomes. +

VII. Membrane Function: A Component in Chromosome Transport In addition to their postulated role in the regulation of [Ca2 j it seems possible that membranes, especially ER, participate in chromosome transport as structural-mechanical components of the MA. The abundance of ER at the spindle pole and its ramifications throughout interior portions of the MA reveal a disposition suited to structural interaction with the spindle fibers as well as to their ionic regulation. The difficulty, however, in developing these arguments stems from the well known structural complexity of the MA. The presence of multiple elements, i.e., both membrane and microtubules (and microfilaments) together at the same time and place within the MA makes it difficult to sort out those elements which contribute to the movement of the chromosomes. The almost universal assumption is that spindle microtubules cause force with a small dissenting group supporting a role for actin microfilaments. Only a few have included membranes as a component of the mechanochemical force generating system (Heath, 1974b, 1978, 1981b; Kubai, 1975, 1978; Nicklas, 1971). This seems to be a great oversight to us and in this section we attempt to strengthen the case for membranes as structural components in chromosome motion. +

A. MEMBRANES: AN INTEGRAL COMPONENT OF THE MITOTIC CYTOSKELETON The cell membrane is a key element of the cytoskeleton; it is the structure to which the fibrous proteins are ultimately attached and upon which those forces controlling cell shape and cell motility are expressed (Weatherbee, 1981). The structure and composition of the membrane-cytoskeleton complex have received considerable attention in studies of mammalian erythrocytes (Branton et al., 1982). Different proteins, including notably spectrin and ankyrin, have been identified and have been shown to form the link between the integral membrane components on the one hand and actin filaments on the other. An especially exciting recent development has been the discovery that macromolecules similar to erythrocyte spectrin and ankyrin occur in membranes of a wide variety of cell types. Proteins that cross-react immunologically with erythrocyte spectrin and possess a similar ultrastructure morphology and ability to bind actin have been derived from several nonerythrocytic cells of chicken (Repasky et al., 1982) and from mammalian brain tissues (Bennett et al., 1982; Burridge et al., 1982;

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225

Glenney et al., 1982). It is especially noteworthy that the (Y subunit of spectrin is immunologically related to the high-molecular-weight, microtubule-associated protein, MAP-2 (Davis and Bennett, 1982). Although spectrin does not copolymerize with microtubules and MAP-2 does not bind to the erythrocyte plasma membrane, both do bind to filamentous actin. Ankyrin has also been shown to occur in nonerythrocytic cells (Bennett and Davis, 1981). Of particular interest is the fact that it cross-reacts with the other major high-molecular-weight microtubule-associated protein, MAP- 1, and copolymerizes with microtubules. Even more pertinent to the present discussion is the ability of antibodies directed against ankyrin to stain the MA of the HeLa cells. The staining has a similarity in distribution to that of microtubules but the observations showing marked fluorescence at the spindle poles with very little in the interzone reveals a pattern that is more closely related to the distribution of membranes (Bennett and Davis, 1981). A second development that underscores the possible close relationship of membranes to the fibrous cytoskeleton has been the discovery that a portion of the cellular tubulin exhibits properties of an integral component of membranes (for review, Weatherbee, 1981). Since tubulin occurs both in microtubules and membranes it is not surprising that these two cytoplasmic elements will bind to one another (Caron and Berlin, 1979; Sherline et al., 1977; Suprenant and Dentler, 1982). The association of pancreatic secretory vesicles with in vitro polymerized microtubules requires MAP-2 and is inhibited by ATP (Suprenant and Dentler, 1982). Electron microscopic observations furthermore reveal structural cross-bridges between the microtubule and the vesicle membrane (Suprenant and Dentler, 1982). The significance of these findings to the function of the MA may be profound. It is already established that membranes are present in the MA and furthermore ultrastructural studies reveal the presence of membrane-microtubule crossbridges (Fux, 1974; Hepler et al., 1970). The biochemical studies now suggest several ways in which the cross-connections might arise. The high-molecularweight microtubule-associated proteins (MAPs) lie at the crux of these associations. MAPs occur in the MA (Connally et al., 1977; Bulinsky and Borisy, 1980); a monoclonal antibody to MAPs that binds the spindle and not interphase tubules suggests the presence of MA-specific proteins that may be important for mitotic regulation (Izant el al., 1982). In addition to their widely accepted role in linking adjacent tubules it seems that within the MA MAPs could also link a microtubule to a neighboring membrane element. The simplest mechanism might be one that involves a MAP bridge between a tubulin subunit on the microtubule lattice and one in the membrane. Alternatively it is possible that MAP binds a microtubule to an actin filament (Griffiths and Pollard, 1978, 1982; Sattilaro et al., 198 1) which is attached by a spectrin-ankyrin complex to the membrane. At the moment too little is known to permit a detailed accounting of specific attach-

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PETER K. HEPLER AND STEPHEN M. WOLNIAK

ment mechanisms; the important point, however, is to realize that membranes might be as central as microtubules to the structure and function of the MA.

B. MEMBRANES AS

A

MITOTICANCHOR

Membranes might act as a scaffolding to which spindle fibers bind and become anchored. A firm attachment or anchoring of the spindle fibers in the region of the pole is essential for the separation of chromosomes at anaphase. As long as forces are generated between the chromosome and the pole to which it faces the spindle fiber must be held firmly enough to resist the load imposed by the dragging of the chromosome through the MA matrix. If the spindle fibers were not sufficiently anchored then the chromosome would remain stationary and the poleward end of the fiber would move toward the metaphase plate. In some models, especially the “treadmilling” scheme (Margolis and Wilson, 198 1; Margolis et al., 1978), the spindle pole becomes the locus for two simultaneous processes, namely the anchoring of the microtubule and the removal of subunits. Margolis and Wilson ( 1981 ) diagram a hypothetical doughnut-shaped structure through which a microtubule is inserted and with which it interacts laterally. We suggest here that the spindle-associated membranes provide the necessary scaffolding for chromosome movement and that stabilization may be achieved by membrane-microtubule crossbridges along the length of the microtubule. A potential problem with having ER as an anchor for spindle fibers is the possible high degree of deformability of the membrane system. One might expect that under tensile stress the membrane would experience strain deformation. Components within the membrane might move or change configuration and thus be unable to serve as an anchor. However, the presence of spectrin-like fibrous proteins on the ER could stabilize and secure microtubule binding sites, and endow the membrane with sufficient molecular stability to hold against shearing forces. A second factor that might help stabilize the ER system and contribute to its possible function as the spindle anchor is its size and the extent of its ramifications. The electron microscopic studies discussed in Section III,A and B reveal in some cells (e.g., sea urchin, barley) a membrane system that not only extends into the MA but permeates the non-MA regions of the cell as well. It is noteworthy in barley and other species of plants that the polar membranes are multilamellar and are continuous with a layer of ER that resides in the cell cortex (Fig. 9). The latter elements in turn possess numerous connections with plasmodesmata and are quite firmly associated with the plasmalemma and cell wall (Hepler, 1982). The ER thus possesses numerous points of close association with itself and with the plasma membrane and these may impart a structural stability that can easily withstand the load imposed by a chromosome moving through the mitotic matrix.

MEMBRANES OF THE MITOTIC APPARATUS

227

C. MEMBRANES AS PARTOF THE FORCE GENERATION SYSTEM

In the foregoing section membranes have been visualized as structural elements to which microtubules bind and achieve a degree of positional stability. Here we introduce the thought that membranes might participate actively in chromosome transport either directly or through shear force interaction with microtubules (or possibly actin microfilaments). The more primitive organisms, in particular, provide numerous examples of direct membrane involvement in chromosome motion. Studies on prokaryotes suggest that the chromosomes attach to the plasmalemma and through the insertion of new membrane material between these attachment points the chromosomes are separated (Ryter, 1968). Membrane growth or flow may also contribute to chromosome separation in a variety of eukaryotes. For example, in Trichonymphu, Kubai (1983) has demonstrated that the chromosomes, which are attached to the NE, undergo considerable movement before chromosomes and the extranuclear microtubules become associated. Microtubules thus may participate in later stages of mitosis but cannot be the force producers in early stages when chromosomes are moving. Kubai (1973) suggests that membrane growth, in a manner similar to that in prokaryotes, contributes to chromosome motion. Dinoflagellates are also cited as example of NE-generated chromosome motion, however, as discussed earlier, a reassessment of the published literature does not, in our view, establish that microtubules are not attached to the NE. In Amphidinium (Oakley and Dodge, 1976) the microtubules clearly are attached while in Crypthecodiniurn (Kubai and Ris, 1969) they may be. Regardless of the situation, the NE to the extent that it is the interface provides, at least, the binding sites for both chromosomes and microtubules and may, in addition, actively contribute to chromosome motion. In fungi there are several examples of NE evagination during mitosis, apparently not caused by growth or pushing of the central spindle shaft, that might indicate the presence of novel processes involved in anaphase separation of chromosomes. McCully and Robinow (197 1) observed in studies of Schizosuccharomyces that the NE between the separating spindle pole bodies was bowed outward and was not stretched or taut as they imagined it should be if the pole bodies were being pushed apart by the intervening central spindle. It seemed that NE growth exceeded spindle elongation and they argued it might be a part of the driving force. A more dramatic example occurs in the yeast Saccharomyces in which it is shown that the nucleus blebs into the daughter bud cell prior to and independently from the central spindle (King and Hyams, 1982). Yet another example occurs in the fungus Thraustotheca (Heath, 1974a) which during mitosis develops long “horn-shaped” projections on its nucleus that are parallel to the axis of the spindle. The horn projections extend well beyond the poles leaving the extranuclear centrioles in deep cytoplasmic pockets.

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PETER K . HEPLER AND STEPHEN M. WOLNIAK

There is no structure within the nucleus that can account for the formation of the horns but there are microtubules aligned along the cytoplasmic surfaces and Heath (1974a,b) proposes that a heretofore unspecified type of shear interaction may cause the formation of the nuclear membrane projections. It also seems likely that the nucelar stretching contributes to anaphase elongation and the movement of chromosomes. Experimental evidence for the presence of an extranuclear force generating system that might act upon the NE comes from the laser microbeam studies of Aist and Berns (1981). Whereas severing the central spindle allows anaphase to proceed up to three times the control rate, irradiation of selected regions outside of the nucleus, but in the vicinity of the spindle pole body, retards the rate of motion. Taken together these various observations cause us to rethink the mechanism of force generation during anaphase. For the examples mentioned the central spindle does not appear to drive the process. It seems reasonable therefore to direct attention toward the NE and to consider at least two possibly interrelated events. First, the insertion of new membrane material is needed to support the extensive elongations that are observed and may contribute to motion itself. Second, the action of cytoskeletal fibers such as microtubules or actin-containing microfilaments, which are closely associated with the NE, may provide the bulk of the driving force and direction for anaphase elongation. Membrane participation in chromosome motion has also been demonstrated or implicated in higher organisms. During meiotic prophase in spermatocytes of Acheta (Rickards, 1975, 1981) and Nephrotomu (LaFountain, 1982) chromosomes undergo extensive saltations despite the fact that they are separated from spindle microtubules by the double membrane of the NE. Of special note is the observation that the portion of the chromosomes being moved is always intimately associated with the inner surface of the NE. The saltations in Acheta (Rickards, 1975, 1981) are rapid, sometimes exceeding rates of anaphase motion by a factor of 10, and are often directed to or from the spindle ‘poles. The sensitivity to low temperature and respiratory poison indicate that these movements in Nephrotoma spermatocytes are driven by metabolic processes (LaFountain, 1982). Curiously in Acheta but not Nephrotoma the motion is inhibited by colcemid. Even so Rickards (1981) argues that while microtubules may guide the direction of saltation they do not provide the force; he favors a membraneassociated actin-microfilament complex as the force generator. On the assumption that the cell uses the same basic system for moving chromosomes in anaphase as it does in prophase Rickards (1981) emphasizes the need and desirability for deciphering these prophase saltations. An explanation of their cause may contain clues about chromosome transport that could apply generally. Because the prophase movements are always dependent upon a close association or attachment between the chromosome and the NE, we are led to

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speculate that a similar membrane association may also be a component of prometaphase congression and anaphase separation. Yet another example of a possible membrane involvement in chromosome movement occurs during karyokinesis in the monopolar spermatocyte of Sciura (Kubai, 1982). In these cells the maternal and paternal sets of chromosomes are grouped separately from one another. Both are focused toward one pole with the paternal set being more distal than the maternal set. Only the paternal chromosomes have attached kinetochore spindle fibers but curiously during anaphase these chromosomes do not move. Rather the maternal chromosomes which lack kinetochore microtubules migrate to the pole at anaphase, and Kubai (1982) suggests that the membrane system, found nearby may participate in transport. Ideas on the mechanism of membrane participation in mitosis may be derived from a comparative analysis of different nonmitotic motile systems in which it is becoming established that cellular membranes are a component of the force generating apparatus. In cilia and flagella the plasma membrane is increasingly recognized as an active component in motility (Dentler, 1981). A Chlamydomonus gamete can attach to another or to a substrate by its flagella and traverse itself relative to the attachment site (Bloodgood, 1981). Bloodgood (1977) has elegantly demonstrated that particles bound to the plasma membrane overlying the flagellum can be transported quickly along the surface. It seems likely that these and possibly other motile phenomena of cilia and flagella involve an interaction between the axonemal doublet microtubules and the plasma membrane. Crossbridges between axonemal doublets and the plasmalemma have been observed and cytochemical evidence shows that they have ATPase activity (Dentler, 1977). In addition biochemical studies on Tetrahymenu and Aequipecten ciliary membranes reveal the presence of tubulin and of high-molecular-weight proteins that have dynein-like ATPase activity. Finally, Dentler et al. (1980) have inhibited motility by a photoactive chemical cross-linking agent that causes the membrane to adhere tightly to the axoneme. Briefly summarized these studies show that there is a structural and even mechanochemical coupling between axonemal microtubules and the plasmalemma that participates in ciliary motility. Whether a similar situation exists in the mitotic cell is unknown. Nevertheless, the close apposition between microtubules and ER, and the existence of cross links between these two elements provide some positive supportive evidence. Moreover, dynein is a component of the MA (Pratt et al., 1980) and recent physiological investigations provide positive support for a role for dynein in anaphase motion of chromosomes (Cande, 1982; Cande and Wolniak, 1978). Although the systems cited above show the movement of a particle or membrane vesicle relative to the microtubule surface, we now introduce the converse possibility, namely that a microtubule moves relative to a membrane surface. If membrane binding sites for spindle fibers possess sufficient structural stability,

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FIG. 29. Diagrammatic representation of a cell in late metaphase as shown in Fig. 25. Here we draw attention to the possible structural interactions between chromosomal microtubules and the interpenetrating membrane system. The microtubules may anchor to and become stabilized by membranes. In addition we suggest that the cross bridge may have mechanochemical properties like dynein that allow the microtubule to crawl along the membrane and pull the chromosome to the pole. A Ca2f release, as depicted in Fig. 25, could stimulate the motile events.

as might be brought about by an ER-associated cytoskeleton, then through the action of mechanochemical crossbridges the microtubule could crawl along the membrane system and in so doing pull its attached chromosome to the pole (Fig. 29).

VII. Summary Membranes constitute an important component of the MA. Elements of the ER and/or vesicles of the Golgi apparatus occur in virtually all spindles. During the process of mitosis these membranes undergo transformations and changes in disposition that correlate with the formation and function of the microtubular spindle fibers. During prophase the nucleus, although surrounded by an envelope, is devoid of membranes. However, upon breakage and dispersal of the NE, in cells of higher eukaryotes, membranes enter the nuclear region, and in addition some elements accumulate at the spindle perimeter and create a boundary between the MA and the rest of the cytoplasm. Within the MA itself accumulation of membranes may occur and in some instances the individual elements may be specifically aligned with the chromosomal spindle fibers. Structurally membranes create domains within which physiological processes might be spatially confined and regulated. It seems plausible that these spindle membranes, like the sarcoplasmic reticulum of muscle, regulate the concentration of Ca2 and control the formation and function of spindle fibers and thus the separation of chromosomes. Considerable evidence supports the idea that Ca2 regulates the motile machinery of the MA, and recent studies emphasize the +

+

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23 1

relationship of Ca2 to the membrane compartments and of ion changes within the MA during mitosis. However, no experiment has yet demonstrated that the free [Ca2 ] actually changes during mitosis; the validity of the membrane-Ca2 theory hinges upon the outcome of studies on this question. In addition to their possible role in the regulation of [Ca2+ ] it seems possible that membranes participate directly as structural elements in chromosome transport. From a phylogenetic point of view the membrane of the cell is recognized as the prime agent responsible for chromosome separation. During the evolution of eukaryotes from prokaryotes it is reasonable to speculate that a portion of this membrane transport system has been retained. We think that it is quite likely that membranes interact with microtubules and microfilaments and contribute to the structural stability of the MA. But beyond this, the possibility emerges that membranes may be a part of the force generating mechanism and directly contribute to the movement of chromosomes. The membrane may contain fibrous components that stabilize its structure and act as binding factors for cytoplasmic fibers. Increasingly it seems evident that membranes are essential components in motility; elucidation of their role in chromosome motion may provide a key to our understanding of the unsolved mechanism of mitosis. +

+

+

ACKNOWLEDGMENTS Many of our colleagues have been extremely generous in providing helpful comments, sharing recent unpublished data, and making available copies of micrographs. For critical reading of the manuscript we thank Z. Cande, B. Jacobson, D. Kubai, K. McDonald and D. Myles. Original figures have been kindly provided by R. Garber, W . Jackson, D. Kubai, K. McDonald, D. McLaughlin, K. O’Donnell, J . Pickett-Heaps, C. Rieder, K. Ryan, and D. Wise. For assistance in the preparation of the manuscript we thank D. Callaham, M. Hepler, and K. Nelson. Finally for helpful discussions in the initial stages of preparation one of us (PKH) thanks T. Cavalier-Smith, B. Gunning, and S. Wick. This study has been supported by Grant GM-25120 from the NIH.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. YO

Pollen-Pistil Recognition: New Concepts from Electron Microscopy and Cytochemistry C. DUMAS,*R. B. K N O X , ~AND ' T. GAUDE* *Dt!partement de Biologie VegPtale et C . M . E . A . B . G . ,Universite' Claude BernardLyon I , Villeurbanne, FrrInre, and 7"Schoolof Botany, University of Melbourne, Parkville, Victoria, Australia Introduction , , . _ .. _ . ,. _ .. ............ The Mature Viable Pollen G ............ 111. The Receptive Pistil. . . . . . . ............ ............ IV. Male-Female Interactions, . A. Attachment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Hydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The Progamic Phase of Fertilization.. . . . . . . . . . . . . . . . . . . . . . D. Pistil Interactions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. The Callose Rejection Response. . . . . . . . .............. F. Molecular Basis for Pollen Infomlation and Pistil Read-Out Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

11.

239 241 251 255 255 259 259 261 263 263 268 269

I. Introduction Following the simultaneous discovery of the mechanism of fertilization in flowering plants by the Russian S. Nawaschin and the Frenchman L. Guignard at the end of the nineteenth century, there has been considerable extension and clarification of the structures involved in reproduction. In terms of male-female recognition, i.e., interactions between pollen grain and pistil, perhaps two of the most exciting discoveries have been the demonstration that the pollen wall may be interpreted as a living structure containing enzymes and other proteins (Tsinger and Petrovskaya-Baranova, 1961), and, on the female side, the demonstration of the outer protein-containing layer of the stigma surface (Mattsson et al., 1974). Heslop-Harrison (1975) provided the first cellular model to explain male-female recognition in flowering plants. He used a new combination of data from electron microscopy and transposed concepts from animal cell-cell communication. This model, together with other more recent hypotheses, attempts to explain how the female partner is able to discriminate the right male partner to comply with the two principles of reproductive biology: increase in hetero239 Copyright 0 1984 by Academic Press. Inc. All right\ of reprcductiun in any form reserved. ISBN 0-12-364490-9

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zygosity and maintenance of stability of the species (see de Nettancourt, 1977; Frankel and Galun, 1977). When we examine these various models critically, we find there is very little evidence to support these usually complex hypotheses of pollen-pistil interactions (Dumas and Gaude, 1981; Knox, 1983a). In this review, we have attempted to clarify this important area of plant cell biology, and propose to interpret the interactions in terms of the key concepts. The first is that the proteins and other molecules carried by the pollen grain mainly in its wall provide prima facie information. The second is the read-out system possessed by the female partner to interpret this pollen information. Both systems operate under control of a complex genetic system, the S locus, which may be interpreted as a sexual recognition gene system (Fig. 1). As a consequence, if information and read-out are compatible, fertilization results. If not, depending on the extent, integrity, and efficiency of the read-out system, the interactions may be partly or totally blocked and incompatibility ensues. The pollen grain is thus unable to transfer its male germ unit, although it may be otherwise quite fertile. The incompatibility reaction that causes the blockage may be expressed as an active rejection response (Fig. 2).

INFORMATION

I

coA T

I

INTERFACE

READ-OUT SYSTEM

FIG. I . Pollen-stigma interface. During male-female recognition, signal molecules from the pollen coat transfer information to the stigma. These molecules interact with receptors on the stigma surface or within the style. An incompatibility between information and read-out system results in rejection characterized by inhibition of pollen germination or tube growth. Their compatibility provides for hydration which involves a flux regulated by osmotic differences between the both partners. This flow induces germination and tube growth. Analysis and precise localization of molecules constituting the information and the read-out system are necessary to modify the recognition system to obtain new plant types. ( I ) Flux of water from stigma to pollen grain. (2) Release of hydrophylic substances from pollen to stigma surface. Closed symbols, informational molecules of the pollen grain. Y-shaped symbols, molecules of the stigma read-out system.

24 1

POLLEN-PISTIL RECOGNITION CONTACT Pollen-Stigma

Incongruity Barrier

-'

Pollen hydration

Pollen germination lnterspecific Barriers

Entry of pollen tube into pistil

I lntraspeci fic Barriers

Growth of pollen tube to ovule

FIG.

2.

Barriers to fertilization: a simplified scheme.

The nature and control of pollen rejection suggest the operation of a sophisticated system to control self and nonself recognition in flowering plants. We have previously drawn parallels between the immune system and the major histocompatibility complex of vertebrates with the recognition systems of the most highly evolved plants, the angiosperms (Clarke and Knox, 1980; Dumas and Gaude, 1982). In this article, we will elucidate the nature and characteristics of pollen information and pistil read-out systems. The interactions between these two systems open a new and exciting area in plant cell biology, which will not only lead to a new understanding of reproductive biology but to the regulation and manipulation of fertilization and seed-setting for the benefit of mankind.

11. The Mature Viable Pollen Grain

The male partner is the mature viable pollen grain. It is virtually a dehydrated organism. The water content at time of dispersal varies widely among different pollen types, generally between 15 and 35% (Stanley and Linskens, 1974). The grains also have virtually no respiratory metabolism. Like spores or seeds, they

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FIG.3 . Bicellular pollen grain of Corylus uvellunu. Transmission electron microscopical (TEM) observation after PATAG treatment of the ultrathin sections showing the polysaccharidic nature of storage compounds. GC. Generative cell; VN, vegetative nucleus. X5700. (Photograph from M. H. Simard, Lyon I . )

utilize this means for dispersion and species survival (Frankel and Galun, 1977). Each grain is surrounded by a complex wall, which not only has mechanical protective properties, but has coat materials (pigments) which screen the hereditary material from damaging UV light (see Zandonella et al., 1981). About twothirds of pollen types are bicellular (Brewbaker, 1957), carrying the progenitor cell of the male gametes (Fig. 3). The remainder are tricellular, and the pair of sperm cells are borne directly within the vegetative cell at maturity e.g., in oil seed rape, Brussica (Fig. 4).

POLLEN-PISTIL RECOGNITION

243

The significance of this difference in the structure of the hereditary materials of the pollen grain becomes apparent when the pollen germinates. In tricellular types, the metabolism of the grain is geared to take advantage of its lead in development, and pollen tube growth rate may be very rapid, with a single phase, so that fertilization may occur within minutes of pollen deposition on the stigmas (20 minutes in grass pollen, 45 minutes in sunflower) (Heslop-Harrison, 1979a,b; Vithanage and Knox, 1977). In contrast, in bicellular types, metabolic changes are needed during germination (Mascarenhas, 1975) as well as cell division. Tube growth may be biphasic (Mulcahy and Mulcahy, 1983). Initially growth is slow, becoming more rapid after about 7 hours in Petunia. CsnSequently, fertilization requires at least 24 hours after pollination. For successful fertilization, pollen must be viable. Even if we collect pollen from a freshly opened flower, how can we be sure it is viable? Viability has traditionally been assessed by success in fertilization and seed set, but this is a time-consuming and laborious process. Today there are more rapid methods. I n vitro culture methods involve germination of pollen on an agar medium (semisolid) or in liquid culture (see Knox, 1983a,b). The medium comprises a carbon source, e.g., sucrose and essential mineral oligoelements including traces of boron and higher amounts of calcium. Within a time period of about 24 hours, it is possible to assess such parameters as percentage germination, pollen tube length, and growth rate. Unfortunately this test is applicable mainly to bicellular pollen types (e.g., Williams et d., 1982), as most tricellular pollen types show a very low percentage germination in vitro (Bar-Shalom and Mattsson, 1977; Roberts er u / . , 1983). Staining tests are also applicable to pollen, but these detect the presence or absence o f cytoplasm, so are indicators of pollen sterility rather than viability. The most rapid test available is the fluorochromatic reaction (FCR) first employed for pollen by HeslopHarrison and Heslop-Harrison ( 1970). The method requires fluorescence niicroscopy which limits its applicability in the field. The selective permeability properties of the plasma membrane of the pollen grain is tested with respect to fluorescein diacetate, and the presence of intracellular esterase activity. A correlation has been demonstrated between FCR data and pollen germination in vitro (Shivanna and Heslop-Harrison, 1981). These tests of pollen viability all involve loss of the sample tested, which is no longer available for subsequent breeding tests or other experiments. A new method developed by Dumas et LII. (1983) overcomes this problem. It depends on the use of nuclear magnetic resonance spectroscopy (NMR). It is based on the changes in water content of the pollen as it becomes nonviable. Traditional methods for estimating water content are unreliable. In nonphotosynthetic tissue, such as pollen grains, expression of water content on a dry weight basis is usually satisfactory, but is unsatisfactory for the same reason if expressed on a fresh weight basis. The most accurate method to determine cellular water content

245

POLLEN-PISTIL RECOGNITION

today is provided by thermigravimetric analysis with isotherms at 85 to 90°C until there is no further mass change. These estimates are accompanied by pollen viability tests by FCR. With this method, values for pollen water content as low as 5.6% have been obtained for pollen of Poputus nigru, and 16% for Brussicu oteraceu. Dead pollen apparently acts like a sponge in absorbing atmospheric water (Dumas er ul., 1984a). The NMR is employed to test the evolution in water content that occurs during loss of viability, e.g., in Brussica pollen (Dumas er al., 1983). The method provides data that are rich in structural as well as dynamic information. It utilizes the principle of resonance of the hydrogen atomic nucleus, [ H , when placed in an intense magnetic field. In the liquid state, the NMR signal has a narrow linewidth (approx. 1 Hz) while in the solid state (as with a dry pollen sample) a broad line-width (approx. 600 Hz) may be obtained. A negative correlation has been obtained between the values of the spin-spin relaxation time ( T I ) and the percentage viability in Brassicu pollen (Dumas ef u l . , 1984a). Relaxation times are computed from the formula 1 / T , = P ,[ I / T 2 ( h ) ]+ ( 1 - P I)[ 1 /T2cf)]where b is bound water molecules, f' is free water molecules, P I is proton spin lattice relaxation time, and T2 is proton spin-spin relaxation time. A model has been employed which includes two types of cellular water, bound water (low T2 values) and free water (high T , ) values. The expectation i s that living pollen contains more bound water than dead grains. Loss of pollen water content has been found to occur in threc steps (Figs. 5 and 6), following increasing temperature treatment and aging at room temperature. NMR results are closely paralleled by loss o f viability data from FCR tests of the same pollen. Thus, loss of viability is correlated with water content. Also. there is no water content expenditure in viable pollen under isothermic conditions, suggesting that water content is regulated within certain limiting temperatures commonly experienced by the pollen. This characteristic is perhaps mediated by the plasma membrane or the wall of the pollen grain with its viscous pollen coat. This may be an inlportant adaptation tor pollen survival during dispersal. A fraction of liquid water (vital water) may be eliminated through the porous plasma membrane of dead grains (Burke c't N I . . I976), but in a living grain the plasma membrane is an effective barrier. What i h the state of the plasma membrane o f mature pollen'? Two types are known: ( I ) those with a poro~isand ineffective plasma membrane, associated generally with a low water content and short viable life. e.g.. Secrrlr pollen (Heslop-Harrison. I979a,b): and ( 7 ) those with a continuous and effective plasma membrane, associated generally with a high water content and longer viable life. e . g . . Brussicw pollen (Dumas and Gaude, 1983). Here, a typical particulate structure characteristic of biological ~~~~

Fic;. 4. Tricrllular pollen grain of.Brmsictr olrrciwtr. TEM cihwwtion after hcavy metal u l t contrast. SCI and SC2. sperm cell\. VN. vrgriativc nucleu\. X S 9 0 0 . IFroni Dumaa CI o l . . 1984h.)

246

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I

0

\*

1

0

,

I

C

'

'

"

I

I

I

"

10

5

'

"

"

+

15

Days

FIG. 5 . Loss of water content during the pollen aging in Brussicu. Three steps (A to C) are visible. au. Arbitrary units. (From Dumas c( u / . , 1983.)

membranes has been detected in the plasma membrane by freeze-fracture (see Fig. 7 and Dumas et al., 1984a). The first type belongs to plants whose pollen is dispersed in air currents; the grain wall is generally thin with pollencoat reduced or absent (e.g., poplar, Populus, Fig. 8a). In the second type, the pollen is generally dispersed by specific animal vectors, and the wall is thick and reinforced, with a copious covering of sticky pollen coat (e.g., Brassicu, Fig. 8b).

t

100

50

Days

F K . 6. The viability of Bnrs.tic.rr pollen grain decreases from 90 to 0% after 2 week storage in a small closed elas\ container at room temperature (20-23°C). Here also three steps ( A to C ) are visible. (From Duma5 c/ d . . 1983.)

POLLEN-PISTIL RECOGNITION

247

Differences in the chemistry of the pollen coat are suggested by the ultrastructural studies of Hesse (1979. 19801, who has detected “active” and “inactive” types distinguished by their electron density and heterogeneity, and site at the exine surface. While the pollen envelopes play a critical role in regulating water loss and hence grain viability, they are also the site of initial contact with the stigma surface in male-female recognition. What potential has the wall in recognition’? It contains extracellular enzymes-as shown by Tsinger and Petrovskaya-Baranova (1961) and confirmed and greatly extended by Knox and Heslop-Harrison (1969, 1970). Later, further work showed that the wall proteins occurred in two quite different domains (Heslop-Harrison cr a/. 1973, Heslop-Harrison, 1975; Knox et a / ., 1975): ( I ) diploid parentally specified proteins held in a labyrinth of arcades in the outer wall, the exine (sporophytic domain); and (2) haploidspecified proteins laid down in the inner wall layer, the intine (gametophytic domain). A considerable number of proteins and glycoproteins (proteins with sugar chains attached covalently) are present in the pollen wall (see Table I). The proteins housed in the exine are first to make contact with the pistil, while those in the intine may be released more gradually during germination (see Table 11).

FIG. 7. Freeze-fracture of Brassic-u pollen wall showing the plasma membrane of the vegetative cell with a typical particulate structure characteristic of biological membranes. 1. Intine: pl, plasmalemma; arrowhead. direction of the shadowing. X83.700. (From Gaude. 1982.)

248

C. DUMAS ET AL.

FIG. 8. TEM observations of the pollen wall from the two types of grain according to their water content. (a) Pollen wall of Pcpirlus alba possessing a low water content. The exine is thin with a reduced pollen coat. The cytochcniicsl treatment used, e.g., cationized ferritin, allows visualization of the presence of numerous polyanionic sites on the exine surface. X25.000. (Photograph from M. Gaget, Lyon 1 .) (b) Pollen wall of Brassica oleracea grain with high water content. The thick exine carries a copious and sticky pollen coat which may induce adhesion between grains. E, Exine; I , intine; PC, pollen coat. X 12,300.

TABLE I NATUREOF SOME PROTEINS LOCAL.17.ED I N [ N T l N t POLLENGRAINS"

AND

EXINt: SITES

Ol-

Present in Protein Dehydrogenases NADH and succinic dehydrogenase Oxidases Cytochrome oxidase Transferases Phosphorylase Ribonuclease Hvdrolases Acid phosphatase Amylase Cellulase (p-I .4-glucanase) Esterase Invertase (p-fructofuranosidase) Polygalacturonase (Pectinase) Protease Allergens Ragweed Antigen E

Intine

Exine

-

+

-

+

(Rapidly diffusible)

+

-

+ +

-

+

(Rapidly diffusible)

+

+

(External to plasmalemma) (Rapidly diffusible)

+

+

+

+

<'From Knox el al. (1975). TABLE II AVERAGE TIMEOF EARLIEST DETtCTABLE PROTEiN Dlr;kLlSlON

Species and pollen type Alopecurus prarensis. Dacfvlis glo,nerma. Gramineae (tectate; single operculate aperture; smooth exine) Silene tdgaris. Caryophyllaceae (tectate; many operculate apertures; moderately smooth exine) Cosmos bipinnaius. Compositae (tectate; three colpi: spiny exine) Ambrosia irifida, Compositae (tectate; three colpi; exine with low spines) lbcris sempervireris, Cruciferae (murate; three colpi) Hibiscus rosa-sineiisis. Malvaceae (tectate; many nonoperculate apertures; moderately smooth exine)

UFrom Heslop-Harrison el al. (1975b).

FROM

POLLEN WALL."

Emission of sporophytic fraction from sexine sites (seconds)

Emission of gametophytic fraction from intine sites (minutes)

<25

2-3

<30

1-5

2-5

10-20

2-5

-0.5

<20

6- 10

<30

4-5

250

C. DUMAS ET AL.

There is thus evidence for a considerable number of potential informational molecules at the surface of the male partner. There is now biochemical and immunological evidence that many are specifically synthesized in the pollen grain, while others are common with those of somatic tissues of the same plant, e.g., Gladiolus (Clarke et al., 1977), Prunus (Raff et al., 1979), and Zea (Porter, 1981). Is there any evidence for a recognition system which might transmit signals from the pollen surface to the plasma membrane or sperm cells, or for structures at the surface of the pollen grain which may be involved in signaling with the pistil? Recently Gaude ( I 982) detected a surface exinic layer which may fulfil at least some of these properties. It has some of the characteristics of a membrane, including trilamellar structure, and characteristic appearance by freeze fracture microscopy, and coats the surface of the exine in mature pollen. This layer could have the potential to provide direct membrane-membrane contact with the stigma surface (Gaude and Dumas, 1984). Ultimately receipt of signals or their emission must reside with the nucleus of the vegetative cell or the sperm cells themselves. Two laboratories have now demonstrated the presence of physical connections between these elements of heredity. In Plumbago zeylanica, one sperm cell is linked by a long connection to the embayments of the nucleus of the vegetative cell (Russell and Cass, 1981). The pair of sperm cells are held within a common but discontinuous wall. We have recently demonstrated a similar nuclear association in Brassica oleracea pollen, but here the connections are multiple (Dumas et al., 1984b). Long processes, pseudopodia, link the sperm cells together in a tail-tail configuration, rather than in a head-tail or head-head association. The pseudopodia of the sperm cell adjacent to the vegetative nucleus are apparently contiguous with the nuclear envelope (Fig. 9). We have termed this complex the male germ unit, as it appears to be preprogrammed for effective fertilization.

FIG. 9. Scheme of the male germ unit as described in Brassica pollen. ps, Pseudopodia; SCI and SC2, sperm cells; VN, vegetative nucleus. (Built from Dumas et al., 1984b.)

POLLEN-PISTIL RECOGNITION

25 1

A further refinement with important consequences for mode of transmission of heritable organelles, e.g., maternal inheritance, is that the two sperm cells within one grain may exhibit preferential transmission of plastids and/or mitochondria. In Plumbago, mitochondria are largely restricted to the sperm cell adjacent to the vegetative nucleus, while the second sperm cell contains most of the plastids (Russell and Cass, 1983). The significance of this arrangement depends on which of the pair of sperm cells fuses with the egg, and whether any sperm cytoplasm is transmitted. In Brassicu olerucea pollen, there are no plastids in the sperm cells, and mitochondria are largely confined to the sperm adjacent to the vegetative nucleus (Dumas et a / . , 1984b). Thus the maternal inheritance of plastids observed in several genera of Cruciferae may be explained by their absence in the sperm cells. Hagemann (1979) has classified the generative cells of some angiosperms into three types: ( I ) those that completely lack plastids, e.g., Antirrhinum, Gossypium, Lycopersicon, Mirabilis, and Zea; ( 2 ) those that contain no plastids at maturity since they degenerated during differentiation, e.g., Beta, Hostu, Mimulus, Oryza, and Solanum; and (3) those that contain numerous plastids, e.g., Oenothera, Pelargonium. The patterns of maternal transmission of plastids by sperm cells have been reviewed by Sears (1980), who found four types: ( 1 ) exclusion of plastids during spermatogenesis, (2) loss from motile sperm, (3) exclusion during fertilization by sperm cleansing, and (4) degradation within the embryo. All these conclusions have been reached from the study of relatively few genera of angiosperms. They show a wide variety of different patterns of behavior, suggesting that cytoplasmic transmission by the sperm cells, as with nuclear, is far from a chance event. In Brusssica sperm cells, modifications have been observed prior to maturation within the anther that may be analogous to capacitation in mammalian sperm cells. Dramatic changes occur at the membrane interface between sperm and vegetative cell, which we have interpreted as the possible acquisition of recognition capacity, in a manner similar to the glycocalyx of animal cells. Indeed, the sperm cells of Brassica are remarkably similar to animal sperm cells, since they have no cell wall and no plastids (Dumas et a/., 1984b). The male germ unit thus shows specializaticn at both the nuclear and cytoplasmic levels, suggesting that future work may reveal membrane specificity and the means for cellular communication.

111. The Receptive Pistil

The female partner is the receptive pistil. This structure not only houses the female gametes, but also receives the pollen, and allows for germination and pollen tube growth and selective discrimination of right from wrong pollen types for successful fertilization and seed set. There are three important parts: (1) the

252

C. DUMAS ET AL.

stigma, where pollen is received, and whose surface may possess receptors capable of identifying the pollen type; (2) the ovary, a special female germ unit, is contained within the parental tissue of the ovary where the syngamic stage occurs in the ovule(s). After fertilization, the germ unit provides the seed, while the ovary provides the fruit; and (3) the style, which provides a mechanical facilitation pathway for the pollen tube during its traverse from the stigma to the ovary and during which the main part of the progamic phase occurs. The cytology of these structures has recently been extensively reviewed (Tilton and Horner, 1980; Knox, 1983b; Dumas and Gaude, 1983); here, we will consider only the aspects important for intercellular communication. The stigma is a gland, covered by specialized receptive cells, usually elongate papillae (see reviews by Heslop-Harrison and Shivanna, 1977; Heslop-Harrison, 1981). There are two broad, but overlapping types: wet stigmas which bear a copious secretion of exudate and dry types covered by a thin film of adhesive material termed the pellicle (Mattsson et a f . , 1974). In wet stigmas, both cytological and biochemical analyses have been carried out, and two subtypes identified. In the first, e.g., Lilium, the exudate is hydrophilic. Nearly 99% of the exudate is polysaccharides, proteins, and water (Aspinall and Rosell, 1978; Gleeson and Clarke, 1980a,b). Among the polysaccharides, more than 90% of the residues are galactose, arabinose, rhamnose, and glucuronic acid. One of the components is a stickly and much branched polymer, containing 0-rhamnopyranosyl-( 1 + 4)-glucopyranosyl uronic acid-( 1 + 6)galactopyranose (Aspinall and Rosell, 1978). This arabinogalactan has a galactose:arabinose ratio of 2: 1 (Gleeson and Clarke, 1980a,b). The cytological route of secretion of the exudate involves a granulocrine pathway of secretion, from ER and Golgi apparatus through the plasma membrane (see Dashek et al., 1971). This is a classical route for glycoprotein secretion (Chrispeels, 1976). A similar mechanism has been found in Aptenia stigmas (Kristen, 1977). In the second type of wet stigma, the lipophilic type, the most detailed analyses have been carried out in Forsythia. The exudate includes neutral lipids, especially triglycerides and fatty acids C, to C,,, terpenes, and phospholipids (Dumas, 1977). This exudate is sticky, acting as a liquid cuticle (as in Petunia, Konar and Linskens, 1966a,b). This prevents stigma dehydration in the absence of a cuticle. The route of secretion is mainly holocrine in type, involving smooth ER in the biosynthesis step, vacuoles in the transitory vacuolation period, and periplasmic accumulation before transport through the cell walls. Among dry-type stigmas, only a small number of types have been investigated by transmission electron microscopy (see Heslop-Harrison, 1981). A classic example is the stigma of Brassica, whose pellicle layer was first to be discovered by Mattsson et ul. (1974) and its properties further characterized by HeslopHarrison et al. (1975a,b). The cytoplasm of the stigma papilla is surrounded by a plasma membrane, a thick polysaccharide wall, a discontinuous cuticle, and the

253

POLLEN-PISTIL RECOGNITION

outermost layer, the pellicle. The pellicle is heterogeneous in ultrastructural appearance. In Brassica stigmas, it is not visible by transmission electron microscopy with Reynold's stain. Specific cytochemical methods need to be employed to visualize the pellicle (see Table 111). In other genera, this is not the case: in Populus and Saponaria the pellicle is readily detected by Reynold's stain. An interesting feature of the techniques employed to reveal the pellicle in Brassica is that not all give uniform continuous binding. Both Con A and mannosyl-ferritin bind in distinct patches, perhaps indicative of a capping-like phenomenon that occurs on animal cell surfaces. This may indicate that the pellicle is capable of changing its state.

TABLE 111 ELECTRONMICROSCOPE CYTWHEMICAI.TREATMENTS USED TO V I S U A L ~THE Z ~ Pti.i.ici.t. DRYSTIGMA Srecits Treatment Localization of enzymatic activities Nonspecific esterasel'

ATPase Ltxalization of a pemieahility barrier Colloidal lanthsnum nitrate

Species

IN

Reference

Mattsaon ct ul. ( 1974)

Heslop-Harrison ( I97Sa) Caude (1982)

cf

01.

Heslop-Harrison

CI

id.

(197Sa) Gaude (1982)

Localization of polyanionic SltCS

Cetylpyridiniuni chloride Cationized ferritin Localization of glyciwmjupatca (u-iiiannosyl and aplucosyl rcsiducs) Con A-peroxidase DAB

Con A-iron dextran Con A-ferritin Con A-niannosyl ferritin

Clarke 1'1 trl. (19x0) Caudc (1982)

Hcrlop-Harrison (1976) Pettitt (1980)

Herd and Beadle (1980) Clarke and Knox ( 1978) Clarke cv ul ( 1 980) Caude (19x2)

"The presence of a nonspecific estcrase activity on the surface of dry stigmas has been extended for all species (Heslop-Harriscin and Shiviinna. 1977; Heslop-Harrison. 198 I ).

254

C. DUMAS ET AL.

An interesting feature of Con A binding to the pellicle is that it has been demonstrated to be specific only once (Knox et ul., 1976), i.e., capable of inhibition by specific sugars. This experiment was carried out by biochemical analysis; all subsequent cytochemical analyses by transmission electron microscopy have given a similar density of reaction product in test and controls. Exceptions are fluorescent-labeled lectin in Gladiolus (Knox et al., 1976) and peroxidase-labeled lectin in Phalaris (Heslop-Harrison, 1976). In the other cases (Table III), the methods may not be sensitive enough to detect any specific binding above background adhesiveness of the stigma surface. Ontogenesis of the stigma pellicle remains to be convincingly demonstrated at transmission electron microscope level. Some properties of the pellicle during its transition to receptivity were demonstrated by Heslop-Harrison et al. ( 1975a). An important feature of stigma function is the effective period of pollination, i.e., when the stigma is receptive to the right kind of pollen for seed setting. The duration of this period varies widely: from a few hours in an Australian mimosa, Acaciu retinodes (Bernhardt et al., 1983), to nearly 10 days in the grape vine, Vitis vinifera (Carraro et al., 1979). The age of the flower, the time of day, and the presence or absence of stigmatic exudate all may influence receptivity. How is stigma receptivity determined experimentally? There are three basic ways: ( 1 ) the morphological appearance of the stigma, including papillar movements, e.g., Hibiscus (Buttrose et al., 1977); (2) correlation with seed-setting ability; and (3) differential staining in dry-type sitgmas (see Table 111). In some cases, the end of receptivity may be indicated by the appearance of the cell wall polysaccharide callose which spreads progressively across the stigma, e.g., avocado (Sedgley, 1979). This may be interpreted as indicating the aging or senescence of the stigma cells. The ovules, of which there may be one or many within the ovary, also have a distinct period of viability, when they are prepared to receive pollen tubes. leading to successful fertilization. In pome and stone fruits, including avocado, the loss of viability of ovules for pollen tubes is indicated by the cytological appearance of callose in the cell walls, and its progressive spread across the cells of unpollinated aging ovules (see Dumas and Knox, 1983). The callose may again be an indicator of tissue senescence, perhaps through control of cellular autolysis by hydrolytic enzymes. An important feature of ovule viability is that it is part of the pistil viability calendar. It is possible for the growth period of pollen tubes in apple pistils to be so delayed that the ovules have lost viability by the time the pollen tubes reach them (Anvari and Stosser, 1978, 1981; MartinezTellez and Crossa-Reynaud, 1982). For pollination to lead to successful fertilization, the stigma must be receptive and the ovules viable. In most reproductive systems, the ovules contain fully differentiated female gametophytes-the embryo sac. These are generally present when the stigma is receptive and pollination anticipated. However, in a few groups of angiosperms,

POLLEN-PISTIL RECOGNITION

255

and in most gymnosperms, the ovary does not commence differentiation until after pollination, when pollen tube growth in the style has commenced. The pollen tube enters a period of dormancy at the base of the style until ovule differentiation is complete. This system occurs in hazelnut, oak, and certain types of orchid (see Knox, 1983a,b). In understanding the structure of the female germ unit, perhaps the major advance has come from recent studies of the embryo sac by transmission electron microscopy. Jensen (1974), a pioneer in this field, developed the concept of the female gametic complex, incorporating the egg cell with its pair of synergids. These cells are the first to be contacted by the male germ unit, after release from the pollen tube. The central cell needs to be added as part of the female germ unit, because of its role in endosperm formation following double fertilization. The central cell is surrounded by bounding membranes, while the synergids and egg cell have apical regions where the cell wall is absent, permitting direct membrane contact with the sperm cells.

IV. Male-Female Interactions What are the cellular events that occur in the pollinated pistil during the progamic phase leading to fertilization'? First of all, pollen is deposited on the stigma, where its species- and genotype-specific information is read-out by the stigma or other pistil tissues. This information and read-out system is encoded in a complex genetic system, of which the most common and simplest type is the monofactorial S locus, with many alleles (see de Nettancourt, 1977; Lewis, 1979). It is likely that the S supergene controls many processes of reproduction, including certain interspecific incompatibility responses, as well as preventing self-fertilization. In any event, pollen acceptance or rejection is based on the consequences of a dialogue between the gene products controlled by the S supergene. The process as a whole is a system to achieve fertilization and options for continuing or aborting the interaction occur at each subsystem encountered (see Knox and Clarke, 1980; Williams, et al., 1982). A. ATTACHMENT Adhesiveness is a fundamental cellular property which plays an important role in cell-cell interactions and recognition (Frazier and Glaser, 1979). Pollen adhesion to the stigma depends on the deposition and sedimentation of the pollen with the substratum (biological or physical) and on the subsequent formation of attachement bonds. It is accepted today that pollen adhesion is achieved by a series of steps, namely pollen deposition, contact, attachment, and hydration. It is

256

C. DUMAS ET AL TABLE IV PHYSICOCHEMICAL APPROACH TO POLLEN-STIGMA ADHESION SUGGESTED BY COMPOSITION Ol- A N IDEAL ADHESIVE ACCORDING TO R ~ Y N O L D ( 1 S971 )"

T H ~

Candidate

Component

Characteristic

Adhesive base Branched polymer of high molecular weight Plasticizer Thickener

Tackifier

Detergent

Pollen surface

Stigma surface

'?

Arabinogalactans

Prevent adhesive becoming brittle Increase viscosity of adhcsive

Diffusible mono- Free nionosacsaccharides charides Glycoproteins Glycoproteins

Resin-like cornpounds enhancing adhesive properties Wetting agent

Pigments of the pollen coat

Glycolipids

?

Glycolipids

Reference Clarke et a / . (1979); Gleeson and Clarke (1980a,b) Clarke et a / . (1979)

Knox et a / . ( 1976); Gaude (1982); Nishio and Hinata (1978) Heslop-Harrison (1968); Clarke et al. (1979) Clarke et a / . (1979); Gaude (1 982)

<'Based on Clarke et a / . (1979) and Dumas and Gaude (1982)

likely that each of these steps gives rise to an increasing degree of adhesion or binding (Dumas and Gaude, 1981). The contact step which initiates the interaction is probably mainly physical in nature, depending on surface charge phenomena. Five negative charges per 100 A2 are considered to be necessary for contact to be achieved (see Maroudas, 1977). Electrostatic forces have yet to be demonstrated on pollen surfaces, although histochemical data may be interpreted as indicating the presence of negatively charged groups within the exine (see Knox, 1983a,b). Some of the parameters have also been reviewed by Wottiez and Willemse (1979). Attachment takes two forms: nonspecific (irreversible) and specific (reversible). Nonspecific attachment is considered to be mainly chemical in nature. Clarke et al. (1979) pointed out that all the components of an ideal adhesive are present when both pollen and stigma surfaces are taken together (see Table IV). Specific attachment has often been interpreted from work with animal cells as a reversible attachment, e.g., with lectins or glycosyltransferases. Evidence for specific adhesion has come from binding of lectins to Gladiolus stigmas (Knox et al., 1976) where binding was assessed using radiolabeled lectin, and from in-

257

POLLEN-PISTIL RECOGNITION

terpretation of pollen adhesion assays in Brassica (Roberts et al., 1979; Stead et al., 1980). The pollen-stigma interface is very different in terms of adhesion between compatible and self-incompatible situations. The size of the interface is much reduced following self-matings, but greatly increased following cross (compatible) matings. One interpretation of these data is that the physical forces governing adhesion are strongest in compatible matings where there is specific adhesion. What kind of factors may be involved in specific adhesion? Several classical molecular models have been proposed to explain the nature of the bonds between cell surface components in specific or nonspecific adhesion. Three models are especially important. The first is the glycosyltransferase model of Roth (1973). He suggested that the enzymes present on one cell may interact with appropriate substrates on a second cell to form a stable enzyme-substrate complex, which would constitute the bond of attachment. While no glycosyltransferase activity has yet been found in pollen or stigma surfaces, an enzyme with associated glycosyltransferase activity has recenty been located in the pollen wall of Brassica, namely P-galactosidase (Singh et al., 1983). Further, pollen grains defective in the enzyme are extremely inefficient in adhesion to the stigma surface. The second model was suggested by Bowles (l979), and deals with the participation of lectins in intercellular communication. Lectins are proteins which exhibit specific, reversible carbohydrate-binding activity and can be multivalent (Goldstein el al., 1980). In this model, the lectins are membrane-bound, but their receptors may be similar or soluble components. Two main states can occur: either the complementary pairs are closed and self-neutralized, or alternatively the pairs are open and not complexed. This model is not incompatible with the first, since regulation may be mediated by glycosyltransferases, glycosidases, or proteases. The question arises whether lectins are present at either pollen or stigma interface'? Both lectins and lectin-like compounds are present (Table V) while

LECTINSOR LECTIN-LIKt

FOUND

Localization

TABLE V (AGGLUTININS A N D MITOGENS) IN POLLEN A N D PISTIL

COMPOUNOS

Activity

Reference

Pollen Cyiiodon dactylon

Platanus acrrifolia Brassica oleraceu Populus curamericana Pistil Primula obconica

Leukocyte agglutinins Lymphocyte mitogens Hemagglutinins Hemagglutinins

Lindberg el a/. (1982) Anfosso er a / . ( I 983) Gaude et a/. (1983) Gaude et a / . (1983)

Hemagglutinins

Golynskaya et ul. ( I 976)

25 8

C. DUMAS ET AL

CON A BINDING

ON

POLLEN

TABLE VI STIGMA SHOWING THE PRESENCE OF GLYCOSYL RESIDUES BOTHINTERACTING SURFACES

AND

Localization Pollen Grasses Brassica oleracea; B . napus Saponaria officinalis

Stigmao Gladiolus gandavensis

ON

Treatment

Reference

Double diffusion tests; immunocytology FITC-Con A; Con A-mannosyl ferritin FITC-Con A

Watson ef a / . (1974)

Helianthus annuus

FITC-Con A FITC-Tridacnin FITC-Con A

Brassica oleracea; B . napus Saponaria afficinalis

FITC-Con A FITC-Con A

Gaude ( I 982) Gaude and Dumas (unpublished data)

Knox et a/. (1976) Clarke ei al. (1979) Vithanage and Knox ( 1977) Gaude (1982) Gaude and Dumas (unpublished data)

“Electron microscope cytochemical treatments using labeled Con A are listed in Table 111.

polysaccharides and glycoconjugates with sugar residues that may bind to labelled lectins (Table VI) are also present. Unfortunately, little is known of their chemical nature and their interactions at pollination. In vitru, lectins such as Con A may have widely different effects: stimulating pollen tube growth (Southworth, 1975), blocking pollen tube penetration of the stigma surface (Knox et al., 1976), and affecting the read-out of pollen information at the stigma surface (Kerhoas et al., 1983). The presence of agglutinins in Brassica pollen has been demonstrated by a pollen-rosetting technique with red blood cells (Fig. 10, and Gaude et al., 1983). This agglutination could not be inhibited by a range of mono- and disaccharides suggesting that the agglutinin may be membrane-bound, possibly in the plasma membrane or in the exinic outer layer. In contrast to this finding, Gaude et al. (1983) also found a soluble agglutinin in the diffusate of Pupulus pollen. Agglutination could be prevented by heating extracts to 80°C or by trypsin treatment. Barondes (1980) devised the term cell adhesion molecules (CAMS) for those molecules capable of binding cells to other cells, or for extracellular binding components. CAMS may be nonspecific, binding all cells together regardless of origin, or highly specific to one kind of cell. Cell adhesion is considered to result from interactions between a surface protein and complementary receptors on other cells.

POLLEN-PISTIL RECOGNITION

259

FIG. 10. Scanning electron microscopic observation of the binding of native rabbit erythrocytes to a Brussicu pollen grain. Po, Pollen grain. X 1100. (From Gaude P I a/.. 1983.)

B . HYDRATION Hydration occurs in response to specific adhesion; water passes from the stigma to the pollen grain, and the subsequent enlargement of the grain constitutes the first morphological and physiological event of pollination. The water potential of a mature pollen grain is considerably lower than that of the surrounding moist substrate of the stigma surface. Consequently, water flows in the direction of decreasing water potential, from the stigma to the pollen. The grains swell, may undergo a change in shape, and become properly hydrated. The kinetics of the process in grass pollen have been elegantly demonstrated by Heslop-Harrison (1 979a,b).

C. THEPROCAMIC PHASEOF FERTILIZATION Several important biochemical modifications occur during the early events of pollen-pistil interactions. First, cutinases and probably carbohydrases are acti-

260

C. DUMAS ET AL

vated and lyse the stigma cell wall around the tip of the pollen tube. Active cutinases have been shown to be present in the pollen of nasturtium, Tropaeolum mujus (see Kolattukudy, 1981). The enzymes differ substantially from fungal cutinases, which also exhibit tip growth. In other systems, active cutinases were present only during pollen-stigma interactions, and could not be detected in the mature pollen (Heslop-Harrison, 1977). Perhaps the active enzyme is formed by subunits contributed from both pollen (pollen tube) and stigma. Christ ( 1959) provided a simple model to explain pollen-stigma interactions in Cruciferue in terms of cutinase activation. He considered that the cuticle acts as an important barrier in the self-incompatibility response of Cruciferae. He showed that two possible combinations were active: ( 1) in self-incompatible matings, active cutinase of pollen is inhibited by stigma, preventing cuticle erosion, and (2) in cross-compatible matings, an inactive precursor form in pollen is activated by stigma factors, leading to cuticle erosion. Later, Linskens and Heinen (1962) supported this hypothesis, noting that some erosion of the cuticle occurs in self-pollinations. The cutinase model alone is inadequate to explain the phenomena of pollen tube arrest following selfmatings (Dickinson and Lewis, 1973). After penetration, the pollen tube is guided through the intercellular system of the cell walls of the style, mostly comprising a fluid or mucilage. This extracellular system, whether it is in the form of a canal (e.g., lily), furrow (e.g., date palm), or solid tissue (e.g., tomato), serves as a mechanical facilitation pathway for the pollen tubes, often direct to the ovules themselves. Most pollen grains range in size from 20 to 200 km (an exception is certain seagrasses whose filamentous grains may be up to 5000 pm long (see Ducker er al., 1978). Yet they are capable of producing pollen tubes that may be several centimeters in length. How is this growth achieved'? The living cytoplasm, containing the male germ unit, is borne in the tip of the tube. As the tube grows, turgor pressure within it decreases, inducing physical stress which may result in the formation of a callose plug which seals the living tip from the remains of the pollen grain, and restores turgor pressure to the system. In this way, with continued growth, a series of callose plugs is formed, so that the elongating tube finally resembles a ladder. The question then arises whether the pollen grain has sufficient resources to sustain such growth? An elegant demonstration has been provided by Loewus and Labarca (1973). They used two different types of methodology: (1) in vifro pollen culture with incorporation of ['H]myoinositol (a pectin precursor forming the dominant wall polysaccharide in the tubes), and (2) in vivo tube growth in excised styles, where the tubes emerged into a medium containing the radiolabeled compound or it is injected into whole styles. The radioactive component accumulated in the style mucilage and became incorporated into the tube wall

POLLEN-PISTIL RECOGNITION

26 1

during its biosynthesis. (see also Miki-Hirosige and Nakamura, 198 1; Kroh and Knuiman, 1981). Because the pollen tube, as vector for the sperm cells, obtains part of its nutrition from the female tissues, it may be useful to compare it with a plant parasite. Why does the female tissue not reject this parasite? There is an obvious parallel in the acceptance of the fetus during pregnancy in mammals, controlled by the HLA system. The pollen tube may have a symbiotic association tolerated by the female tissue to ensure success of fertilization. In flowering plants, the pollen tube as vector of the male germ unit carries it to the female gametic complex, and employs the same strategy: an apparently passive transmission mediated by the pollen tube during its tip growth through the style. In contrast, less-evolved types of plants employ a more hazardous system of sperm cell transfer, which involves direct sperm-egg contact by swimming sperm cells in an aqueous medium. D. PISTILINTERACTIONS Information exchange and read-out for the stigma take place at the pellicle in dry-type stigmas. We have recently demonstrated (Kerhoas et al., 1983) the importance of the pellicle and its receptors in read-out of information from Brussica pollen. In this system, callose synthesis is triggered in the stigma cells within minutes of self (incompatible) pollination, but not by cross (compatible) pollination. The callose can be monitored by staining tissues with aniline blue fluorochrome, and fluorescence microscopy. It is thus possible to investigate the nature of the pollen information that induces the stigma response (see Dumas and Knox, 1983). Pollen information is provided by extracts of known protein content, obtained as whole pollen diffusates; after diffusing for a few minutes, the pollen is removed by filtration, and the extract applied as a drop to the stigmas. These diffusates contain wall-held proteins, glycoconjugates, and other molecules, i.e., pollen information. This is able to mimic the response of viable pollen in callose responses of stigmas. Self-pollen information induces a stigma callose response, while compatible information does not. When stigmas are first treated with the lectin, concanavalin A , known to bind to the pellicle in this and other systems, the subsequent response to self-pollen information is blocked. The lectin may mask the pellicle, so that the stigma is unable to read-out the pollen information. Also, when stigmas are first treated with a detergent, Triton X- 100, the subsequent response to self-pollen information is blocked. It is likely that the detergent modifies or partly solubilizes the pellicle, so that once again it is unable to read-out the pollen information. Does the pollen grain read-out stigma information'? Evidence from several systems where the pollen grain produces callose following self (incompatible)

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but not after cross (compatible) pollinations (see Dumas and Knox, 1983) suggests that the pollen grain does receive information from the stigma, and responds to it. We must also assume, although there is as yet no conclusive evidence, that information exchange occurs at each diagnostic landmark in the pollination system (see Fig. 2 ) . There is some evidence that specific signals are exchanged between pollen or pollen tube, and a structure that is widely separated at the time of exchange: the ovule. Signals are received in the ovules of Petunia at about 9 hours and between 18 to 30 hours after pollination, in both cases, prior to pollen tube contact with the ovary (Deurenberg, 1976a,b). The signals are detected by increases in protein synthesis in the ovules of pollinated plants which do not occur in unpollinated control plants. The signals have the following characteristic features: ( 1) primary signal indicates arrival of pollen tube in style; and ( 2 ) secondary signal indicates whether self- or crosspollen is present in style. When concomitantly the ovule is viable, and the stigma is pollinated, one of the synergids within the gametic complex of the embryo sac is transformed. It changes dramatically in structure and composition, appearing to have degenerated. It is this synergid which the pollen tube finally enters. Jensen et al. (1983) have induced this change in vitro in one of the two synergids by treating cultured ovules of cotton with the plant hormone, gibberellic acid. They detected high levels of calcium in the transformed synergid, and draw a parallel with in vitro pollen tube growth experiments where calcium has been shown to have a chemotropic effect. In cotton, syngamy is made possible since the synergid and egg cell walls are incomplete around the apical parts of these cells. Sperm cell cleansing apparently occurs during syngamy in Petunia. Van Went (1970) has shown that the two sperm cells fuse with the plasma membranes of the egg or central cell, and only the sperm nuclei appear within the cells prior to nuclear fusion. During this germ unit confrontation, the question arises as to whether the pair of sperm cells fuse at random with egg or central cell, or whether there is some predetermined specificity. Russell and Cass (1983) claim this exists for fertilization in Plumbago. Certainly the structure and composition of the germ units in both Brassica and Plumbago suggest that the process is not random. Also, the mobility of the sperm cells within the pollen tube confers on them the advantage in selection, since the female partners are static as far as is known. However, how much reliance should we place on the possession of a particular heritable organelle as an indicator of sperm cell specificity? Does it in fact reflect any different binding specificity at the plasma membranes of the gametic complex? The demonstration of sperm cell specificity in structures that are only a few micrometers in size will require considerably more sophistication than has been employed to date.

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E. THECALLOSE REJECTIONRESPONSE Callose is implicated in the active rejection response observed in stigmas of self-pollinated Brassica, as noted earlier in this review. There have been considerable speculations on the possible role that the callose might play: ( I ) prevent tissue hydration through control of cell water equilibrium through antagonistic roles of calcium and potassium ions (Eschrich, 1975); (2) pool of nutrients available after hydrolysis in accordance with the transitory nature of callose deposits (Currier, 1957; Sedgley, 1977); or (3) active defense reactions in isolating or sealing pollen from the stigma (Aist, 1976; Lewis, 1980). It is possible that the compatible pollen grain might actively destroy the rejection mechanism through the action of a pool of enzymes following stigma surface recognition reactions (see Fig. 11). This means that callose will be actively and continuously degraded in a compatible pollination (Linskens, 1975, 1976). In the incompatible interaction, boron may be sequestered by callose, producing a boron deficiency leading to alteration of polyphenol metabolism. Phytoalexin-like components may then be synthesized (Lewis, 1980). The phytoalexin, rishitin, is known to inhibit pollen tube growth in vitro (Hodgkin and Lyon, 1979). The compatible situation has been compared with the host plant resistant to fungal disease (see Dumas and Knox, 1983). F. MOLECULAR BASISFOR POLLENINFORMATION A N D PISTIL READ-OUTSYSTEMS Most of the data available today concern the pistil read-out systems, and little is available for pollen information. We will review this latter material first. 1. Pollen Information Systems The existence of antigens with specificity directed toward the alleles of the S supergene of Oenothera orgunensis pollen was demonstrated by Lewis (1952). The S-specific antigen comprised about 20% of the pollen proteins and diffused from moistened pollen in isotonic media within 30 minutes (Makinen and Lewis, 1962). Individual pollen grains produced precipitates when sprinkled on gel containing S-specific antisera (Lewis et al., 1967). Unfortunately no further progress has been made with this system in identifying and characterizing the active protein. Linskens (1960) also identified antigens with S allele specificity in pollen of Petunia. A comparison of pollen antigens with those from other plant tissues has been made in Gladiolus (Clarke et al., 1977) and Prunus (Raff et al., 1979) which has a self-incompatibility system. Allergens, proteins with the ability to bind to a specific immunoglobulin E in man and provoke the allergic response (seasonal asthma and hay fever), have also been isolated from many types of pollen. Their function in the plant is unknown (see Howlett and Knox, 1983).

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HOST/ PATHOGEN

0 CALLOSE

J.

ElHxH Callose degradation

Enzyme Pool

Callose accumulation

No inhibitor

Complexing

synthesis

of borate

Inhibitor synthesis (e.g., phytoalexin)

El COMPATIBLE

I

El INCOMPATIBLE

FIG. I 1 . Summary scheme showing current hypotheses concerning callose synthesis, degradation by hydrolytic enzymes, borate complexing, and inhibitor production (e.g., phytodlexin-like compound) in self-incompatible pollination. (See details in text; from Dumas and Knox, 1983.)

Enzymes present in the pollen wall may also be important components of the information system. In Brassica pollen defective for P-galactosidase, the efficiency of adhesion to the stigma is drastically reduced, suggesting that the enzyme plays a role in stigma communication (Singh et al., 1983).

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2. Stigma Read-Our Systems

In some cases, stigma read-out is located on the surface, at the pellicle. The read-out system appears to be S gene specified. Using isoelectric focusing methods, Roberts et al. (1979) demonstrated that the appearance of glycoproteins binding to the lectin Con A is correlated in time with the acquisition of selfincompatibility in Brassica. Previously, Nishio and Hinata ( 1977, 1979) had shown that a glycoprotein with S allele specificity (S, allele) showed a characteristic affinity for Con A. Later, Gaude (1982) found that Con A binds to the pellicle of Brassica, suggesting that the S-specific glycoprotein is pellicle located. Recently, Ferrari et al. (1981) isolated and partly characterized a glycoprotein specific for the S, allele. It has been found to regulate pollen germination in v i m , and to modify the behavior of compatible pollen at the stigma surface. Similar pollen growth inhibition may be obtained after treating the Brassica stigma surface with Con A (Kerhoas et af., 1983). Previously, S-specific antigens had been analyzed by immunodiffusion and immunoelectrophoresis (see Nasrallah, 1979) while the molecular size and nature of the S-specific macromolecules have been determined by a variety of physicochemical methods (Table Vll). It is likely that these potential recognition molecules may act in the stigma pellicle in Brassica. Their presence may be correlated with stigma receptivity, i.e., they provide an efficient, functioning read-out system. The Brassica system, controlled by a sporophytically inherited S gene system, with read-out and inhibition at the stigma surface, provides an ideal experimental system. In contrast, gametophytic systems, where only the site of rejection of pollen tubes is known, usually deep within the style, are much more difficult to approach experimentally. These systems appear to provide for retardation in expression of the read-out system. Is pollen information completely preprogrammed and available in the mature bicellular pollen grain? Most gametophytic systems are associated with this pollen type (Brewbaker, 1957). Furthermore, they generally have wet-type stigmas, in which the pellicle may be destroyed or partly solubilized, so that a new read-out system is needed. Where is it located? Is it adjacent to the site of tube inhibition'? Inhibition occurs within the mechanical facilitation pathway of the style-in furrow, canal, or transmitting tissue-in an intercellular mucilaginous medium. If the read-out system does not remain at the stigma, then this poses another problem. The pollen information system will have to be located at the growing tip of the pollen tube. It is also significant that tube arrest in gametophytic systems may occur at the point of transition into the parasitic state for the pollen tubes (Mulcahy and Mulcahy, 1983; Raff et a/., 1981).

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TABLE VII NATUREOF MATERIAL WHICHCORRELATES W I T H S GENOTYPE DETECTED I N POLLEN OR STIGMA EXTRACTS<’ Nature of putative S gene product

System Sporophytic Brassica olrracea

Brassica campesrris

Gametophytic Oenothera organensis Petunia sp Prunus uviutn

Nicotiana aluru

Stigma: S2 antigen: glycoprotein; p/ high MW 54,000 Stigma: S antigen: Con A-binding glycoprotein; p/ 10.3; MW 57,000 Stigma: S22 antigen: Con A-binding glycoprotein; pl 1 I . I ; MW 60,000-65.000 Stigma: S7 antigen: Con A-binding glycoprotein: pl 10.6; MW 57,000 Stigma: S7 antigen: Con A-binding glycoprotein; pl 5.7; MW 57,000

Pollen: S allele corresponding antigens Pollen and style: S allele corresponding antigcns Style: S antigen: Lectin-binding glycoprotein; pl high; MW 37.000-39.000 Style: S allele corresponding products

Reference

Ferrari et u / . (1981) Nishio and Hinata ( 1982) Nishio and Hinata (1982) Nishio and Hinata (1982) Nishio and Hinata ( 1979)

Makinen and Lewis ( 1962) Linskens (1960)

Mau et a / . (1982)

Bredemeijer and Blass (1981)

“Completed from Mau et a / . (1982)

In the pistil of Prunus uvium,which has a wet-type stigma, five antigens have been identified, including two in the style, antigens P and S (Raff et ul., 1981). Antigen P is common to all genotypes, while antigen S is specific to S,S, genotypes. Their properties are given in Table VII. Both are glycoproteins, and glycoprotein S strongly inhibits self-pollen tube growth in vitro (Williams et al., 1982). One question that arises is whether this component is an S gene product, or is it a receptor for S gene product from the pollen? The answer is not yet known, but several types of intercellular communication between the male information and the female read-out system may exist. Models have been proposed by Gleeson and Clarke (1980a,b). An elegant molecular model to explain S gene action was proposed by Lewis

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(1965): the oppositional or inhibitory model. The S gene is expressed in both pollen and pistil-ither producing identical dimers which fuse together to give an active tetrameric inhibitor; or complementary stereospecific molecules interact, the complex produced by fusion being inhibitory. Molecular interactions adapted for the control of hydration step of the pollen grain have been recently proposed (Gaude, 1982; Dumas and Gaude, 1983). Following the pollen-stigma contact, a reorganization of the pollen information and read-out system occurs (Gaude, 1982). This event may play a preponderant role in pollen adhesion and recognition processes in assuring control of the water flow from stigma to pollen. The pollen hydration level after pollination seems directly correlated to the acceptance or rejection of the grain, as shown by the work of Roberts et al. (1980). Once more, overcoming incompatibility by increasing the relative humidity confirms the importance of the hydration step in recognition mechanism (Carter and Mac Neilly, 1976). These facts led us to propose a molecular model to explain male-female recognition in sporophytic systems (Gaude, 1982; Dumas and Gaude 1983). This model is based on the control of the water flow from stigma to pollen by the reorganization of cell surface components of both interacting partners (Fig. 12). The pollen hydration

a

FIG. I ? .

Model of molecular interaction between pollen and stigma surface components in interface during compatible combination. The specific interaction between S-products from the pollen information and rcad-out system would lead to a reorganization o f cellular surface components. Lipids and proteins of the interface settle according to their respective affinities (electrostatic. hydrophobe. hydrogen liaiwna. van der Waals forces) The rnacrornolecular edifice thus formed would present facilitated ways for water flow and ensure a full pollcn hydration. The hydrated grain germinates and the pollen tube can fertilize the embryo sac. (b) Pollen-stigma interface during incompatible combination. The S-product interaction would induce the formation of a hydrophobic macromolecular structure which would constitute a barrier to efficient hydration of the pollcn grain. Such a weak hydrated pollen is rejected on the stigma surface. The pollen coat (PC) and the stigma pellicle (Pe) are composed principally by lipids (L). proteins. and glycoproteins ( P ) . Arrows indicate water flows from stigma to pollen.

Brc4ssic.u. (From Gaude. 1982.) ( a ) Pollcn-\tigma

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must no longer be considered as a simple physical phenomenon depending on an osmotic pressure difference between male and female organisms, but constitutes the first recognition event involving membrane-like structures (Gaude and Dumas, 1984). The interactions between these compounds and the modifications which are flowing would regulate the water flux and, thus, the acceptance or rejection of the pollen grain.

V. Conclusions In this new area of cell biology of fertilization in flowering plants, the concepts of pollen information interacting with pistil read-out systems are strongly supported by the available data. This is especially true for systems like Brassica where the major events of recognition occur on the stigma surface. Today, the data suggest that these systems operate through glycoconjugates and related molecules. This view is in agreement with recent work on animal systems (Koszinski and Kramer, 1981). These recognition molecules appear to act in a way analogous to antigen-antibody, lectin-sugar residue, or enzyme-substrate interactions. This analogy has been extended to include the possibility of direct membranemembrane contact between pollen and stigma surfaces in Brassica. Pollen information is generally regarded as being housed within the two domains of the pollen wall, i.e., in the pollen coat or within the intine polysaccharide matrix. In Brusuicu pollen, the membrane-like exinic outer layer described earlier in this review may contain many of the recognition determinants. On the female side, there is increasing evidence that the pellicle of dry-type stigmas, the remarkably plastic surface layer, appears to have many of the properties of a membrane, if not its structural characteristics. These two surface layers provide the possibility for direct contact and interactions, although these have yet to be demonstrated. These new concepts in plant cell biology are especially valuable in interpreting several techniques used empirically in plant breeding to overcome incompatibility responses. These methods involve modifications to the surfaces of pollen or pistil partners, either by physical or chemical treatments (see Clarke and Knox, 1978). Today, wc interpret these techniques as modifying the pollen information or pistil read-out systems (see Dumas and Knox, 1983; Dumas et d.,1984a). This approach is especially useful to begin to understand the physiology ofS gene action. Genetic manipulation ofthe natural route for fertilization and seed-setting becomes a practical possibility, through manipulation of the information or read-out systems. The possibilities of the utilization of the male germ unit for the transfer of genes through the vehicle of the pollen tube may be realized. We may anticipate rapid progress in the future in understanding and nianipula-

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tion of fertilization through the characterization of mutants, such as that for pollen defective in the enzyme P-galactosidase (Singh er al., 1983). The use of mutants provides experimental systems in which the reproductive process is blocked at specific points, because of the expression of the mutant gene in the haploid pollen grains. The discovery that many isozymes of pollen are held in common with other parental tissues indicates the potential for gametophytic selection through the medium of the pollen tube at the haploid level (see Mulcahy, 1979). Thus the process of fertilization in flowering plants is open to manipulation for the benefit of mankind.

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Jensen, W. A. (1974). I n “Dynamic Aspects of Plant Ultrastructure” (A. W. Roberts, ed.), pp. 481-503. Academic Press, New York. Jensen, W. A,, Ashton, M. E., and Beasley, C. A. (1983).I n “Pollen: Biology and Implications for Plant Breeding” (D. L. Mulcahy and E. Ottaviano, eds.), pp. 67-72. Elsevier, Amsterdam. Kerhoas, C., Knox, R. B., and Dumas, C. (1983). Ann. Boi. 52, 597-602. Knox, R. B. (1983a). In “Encyclopedia Plant Physiol.” (J. Heslop-Harrison and H. F. Linskens, eds.), Vol. 14. Springer-Verlag, Berlin and New York (in press). Knox, R. B. (1983b). I n “Embryology of Angiosperms” (B. M. Johri, ed.). Springer-Verlag, Berlin and New York (in press). Knox, R . B., and Clarke, A. E. (1980). I n “Contemporary Topics in Immunobiology. Self-non-self Discrimination” (J. J . Marchalonis and N . Cohen, eds.), Vol. 9, pp. 1-36. Plenum, New York. Knox, R. B., and Heslop-Hamson, J. (1969). Naiure (London) 223, 92-94. Knox. R. B., and Heslop-Harrison, J. (1970). J . Cell Sci. 6, 1-27. Knox, R. B., Heslop-Harrison, J . , and Heslop-Harrison, Y. (1975). I n “The biology of the Male Gamete” (J. L. Duckett and P. A. Racey, eds.), pp. 177-188. Academic Press, New York. Knox, R. B . , Clarke, A. E., Harrison, S . , Smith, P., and Marchalonis, J . J . (1976). Proc. Narl. Acad. Sci. U . S . A . 73, 2788-2792. Kolattukudy, P. E. (1981). Annu. Rev. Plant Physiol. 32, 539-567. Konar, R. N., and Linskens, H. F. (1966a). Plania 71, 356-371. Konar, R. N., and Linskens, H. F. (1966b). Planra 71, 372-387. Koszinowski, U. H., and Krdmer, M. (1981). Naiure (London) 289, 181-184. Kristen. V. (1977). Protoplasma 92, 243-252. Kroh, M., and Knuiman, B. (1981). Acia Soc. Bot. Pol. SO, 75. Lewis, D. (1952). Proc. R . Soc. London Ser. B 140, 127-135. Lewis, D. (1965). Genet. Todaji 3, 657-663. Lewis, D. (1979). “Sexual Incompatibility in Plants.” Arnold, London. Lewis, D., Burrage, S . . and Walls, D. (1967). J . Exp. Bot. 18, 371-378. Lewis, D. H. (1980). New Phviol. 84, 261-270. Lindberg, R. E., Pinnas, J. L., and Jones, J. F. (1982). J . Allergy Clin. Immunol. 69, 388-396. Linskens, H. F. (1960). Z. Bof. 48, 126-135. Linskens, H. F. (1975). Proc. R . Soc. London Ser. B 188, 299-311. Linskens, H. F. (1976). I n “Specificity in Plant Diseases” (R. K. S . Wood and A. Graniti, ed.), pp. 31 1-325. Plenum, New York. Linskens, H. F., and Heinen, W. (1962). Z . Bor. 50, 338-347. Loewus, F., and Labarca, C. (1973). In “Biogenesis of Plant Cell Wall Polysaccharides” (F. Loewus, ed.), pp. 175-194. Academic Press, New York. Makinen, Y. L. A., and Lewis, D. (1962). Genei. Res. 3, 352-363. Maroudas, N. G. (1977). J . Cell. Phvsiol. 90, 5 1 1-520. Martinez-Tellez, J., and Crossa-Reynaud, P. (1982). Agronomic 2, 333-340. Mascarenhas, J . P. (1975). Boi. Rev. 41, 259-314. Mattsson. O., Knox, R . B., Heslop-Harrison. J., and Heslop-Harrison, Y. (1974). Nafure (London) 247, 298-300. Mau, S . L., Raff, J., and Clarke, A. E. (1982). Planra 156, 505-516. Miki-Hirosige, H., and Nakamura, S . (1981). Acta Soc. Bor. Pol. SO, 77-82. Mulcahy, D. L. (1979). Science 206, 20-23. Mulcahy, G. B., and Mulcahy. D. L. (1983). In “Pollen: Biology and Implications for Plant Breeding” (D. L. Mulcahy and E. Ottaviano, eds.), pp. 29-34. Elsevier, Amsterdam. Nasrallah, M. E. (1979). Herrdity 43, 259-264. Nettancourt, D., de ( 1977). “Incompatibility in Angiosperms.” Springer-Verlag, Berlin and New York.

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Nishio, T., and Hinata, K. (1977). Heredify 38, 391-396. Nishio, T., and Hinata, K. (1978). Jpn. J. Genet. 53, 27-35. Nishio, T., and Hinata, K. (1979). Jpn. J. Genet. 54, 307-31 I . Nishio, T., and Hinata, K. (1982). Genetics 100, 641-647. Pettitt, J. M. (1980). Ann. Eof. 45, 257-272. Porter, E. K. (1981). Environ. Health Perspect. 37, 53-60. Raff, J . W., Hutchinson, J. F . , Knox, R. B., and Clarke, A. E. (1979). Diferentiarion 12, 179-186. Raff, J . W., Knox, R. B., and Clarke, A. E. (1981). Planta 153, 124-129. Reynolds, G. E. T. (1971). In “Aspects of Adhesion” (D. J . Alner, ed.), pp. 96-1 1 I . Univ. of London Press, London. Roberts, 1. N., Stead, A. D., Ockendon, D. J . , and Dickinson, H. G. (1979). Planta 146, 179-183. Roberts, I. N . , Stead, A. D., Ockendon. D. J . , and Dickinson, H. G . (1980). Theor. Appl. Genet. 58, 241-246. Roberts, 1. N., Gaude, T . , Harrod, G . , and Dickinson, H. G . (1983). Theor. Appl. Genet. 65,231238. Roth, S . (1973). Q . Rev. Eiol. 48, 541-563. Russel, S . D., and Cass, D. D. (1981). Protoplasma 107, 85-107. Russel, S. D., and Cass, D. D. (1983). In “Pollen: Biology and Implications for Plant Breeding” (D. L. Mulcahy and E. Ottaviano, eds.), pp. 135-140. Elsevier, Amsterdam. Sears, B . B. (1980). Plasmid 4, 233-255. Sedgley, M. (1977). Sci. Hortic. Amsterdam 7, 27-36. Sedgley, M. (1979). J. Cell Sci. 38, 49-60. Shivanna, K. R., and Heslop-Harrison, J . (1981). Ann. Eot. 47, 759-770. Singh, M. B., Morgenson, R., and Knox, R. B. (1983). Nature (London) (in press). Southworth, D. (1975). Nature (London) 258, 600-602. Stanley, R. G., and Linskens, H. F. (1974). “Pollen.” Springer-Verlag, Berlin and New York. Stead, A. D., Roberts, I . N., and Dickinson, H. G. (1980). J . Cell Sci. 42, 417-423. Tilton, V. R., and Homer, H. T. (1980). Am. J. Eot. 67, I 113-1 131. Tsinger, N. V., and Petrovskaya-Baranova, T. P. (1961). Dokl. Akad. Nauk S.S.S.R. 138,466-469. Vithanage, H. I . M. V., and Knox, R. B. (1977). Phytomorphology 27, 168-179. Watson, L., Knox, R. B., and Creaser, E. H. (1974). Nature (London) 249, 574-576. Went, J . L., van (1970). Acfa Eot. Neerl. 19, 313-322. Williams, E. G., Ramm-Anderson, S . , Dumas, C., Mau, S. L., and Clarke, A. E. (1982). Plania 156, 517-519. Wottiez, R. D., and Willemse, M. T. M. (1979). Phytomorphology 29, 57-63. Zandonella, P., Dumas, C., and Gaude, T. (1981). Apidologie 12, 383-396.

INTERNATIONAI REVIEW OF CYTOI.OGY. VOI. 90

Surface Topography of Suspended Tissue Cells Yu. A. ROVENSKYA N D Ju. M . VASILIEV Laboratory of Mechanisms of Carcinogenesis, Cancer Research Center of’ the USSR Acudemy of Medicul Sciences, Moscow, USSR I. 11. 111. IV. V.

VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Morphology of Surface Microextensions of Suspended Cells. . . . . . . Surface Topography of Suspended Tissue Cells of Various Types . . Mechanisms of Formation of Microextensions . . . . . . . . . . . . . . . . . . Effects of Previous Contacts with the Substrate on the Surface Topography of Suspended C e l l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

273 274 285 290 299 303 304

I. Introduction Most cells of multicellular organisms can exist in two alternative states: they can be either attached to extracellular matrices and to other cells or suspended in fluids. Suspended and attached cells usually have substantially different morphology. Transition from the attached to suspended state can occur spontaneously, e.g., during hematopoiesis, or can be induced artificially by dissociating agents. Reversible transitions between the attached and suspended states can be easily induced and observed in cultures of various tissue cells. Spreading of cultured cells on artificial substrates is the best studied variant of these transitions (see reviews: Revel et a / . , 1974; Vasiliev and Gelfand, 1977, 1981; Grinnell, 1978, 1980; Heaysman et a / . , 1982; Vasiliev, 1982a,b). Spreading can be regarded as a simplified prototype of cell interaction with its territory, that is, with the noncellular matrices to which these cells are attached in tissues in vivo. Deficient spreading is a characteristic feature of neoplastically transformed cells (see reviews in Vasiliev and Gelfand, 1977, 1981). Due to numerous investigations performed during the last years we already know much about cell alterations accompanying spreading and about the properties of cells in the substratespread state. In contrast, little is known about the properties of cells in an alternative morphological state, that is, about suspended cells. Obviously, one cannot hope to gain complete understanding of the mechanism of spreading without detailed knowledge of the initial point of this process, of the suspended state. The importance of investigating this state is increased by facts showing that 273 Copyright G 1984 hy Academic Prcv. Inc. All righis u i reproduction in any fnmi reserved ISBN 0-12-364400-0

274

YU. A. ROVENSKY AND JU. M. VASILIEV

certain characteristics of normal and neoplastic cells are different not only in the substrate-spread state but also in suspension. In particular, suspended nontransformed and transformed cells may have a different ability to multiply in suspension (Macpherson and Montagnier, 1964; Stoker et al., 1968; Shin et a/. , 1975; Tucker et al., 1977; Colburn et al., 1978). Suspended cells of primary cultures, of minimally transformed lines and of fully transformed neoplastic lines have different ability to synthesize proteins, mRNA, ribosomal RNA precursors, and DNA (Benecke et a/., 1978, 1980; Wittelsberger et al., 1981; Raz and BenZe’ev, 1982) and also different agglutinability by concanavalin A and other lectins (Aub et al., 1965; Burger, 1969, 1973; Nicolson, 1976). We do not know, however, any structural features of suspended cells of various types which correlate with these differences of behavior. One of the important morphological features of suspended cells had been revealed by scanning electron microscopy: it was found that the surface of these spherical cells usually was not smooth but covered by numerous microextensions such as microvilli, blebs, or folds. These microextensions probably play important roles in the physiology of suspended cells, especially, in their reactions to microenvironment. The purpose of this article is to review the main results of the investigations of surface topography of suspended cells and to discuss certain unsolved problems which have arisen in the course of these investigations. We will limit our discussion to the surface topography of suspended cultured tissue cells, especially, of normal and transformed fibroblasts. For comparison we will quote some data about the cells permanently or almost permanently living in a suspended state, e.g., about blood cells or the cells of ascites tumors.

11. Morphology of Surface Microextensions of Suspended Cells

Three main morphological types of microextensions can be distinguished: microvilli, blebs, and folds (see reviews: Vesely and Boyde, 1973; Rovensky, 1979). Microvilli are cylindrical elongated processes about 0.1-0.2 pm in diameter; their length can vary from 0.2-0.5 to 5-6 pm (Figs. 1 and 7). The shape of microvilli can be straight or bended; sometimes they have bulbous ends; the branched microvilli are occasionally observed at the surface of suspended cells (Fig. 1). Detailed morphometric studies characterizing distribution of the sizes of microvilli in various cells and on the surface of one cell are still lacking. From

FIG. I . Transformed mouse fibroblast of the L line in suspension. Microvilli and blebs; some microvilli are branched and have bulbous ends. Field width, 11.8 p m . FIG. 2. Mouse embryo cell in suspension. Blebs of varying diameter. Occasional microvilli. Field width, 1 I .8 b m .

FIG. 1

FIG.2

276

YU. A. ROVENSKY AND JU. M. VASILIEV

visual observations one gets the impression that the diameter of various microvilli on the same cell usually remains relatively constant. Their length, although having considerable variations, seems not to exceed a certain maximum. Quantitative studies are needed to confirm or to refute these impressions. Blebs can be described as rounded surface protrusions with the smooth surface and with the diameter varying usually from less than I to 2-3 IJ-m(Figs. 2, 5, and 6). Very large blebs are sometimes seen on the surface of cytochalasintreated cells: the diameter of a single bleb in these cells may become almost equal to that of the main cell body (Fig. 3). Usually the shape of each bleb is nearly spherical or hemispherical; elongated ellipsoid blebs are not seen on the surface of suspended cells. We will designate all the protrusions of flattened shape as folds. Their thickness is usually 0.1-0.4 IJ-m but their width and length can vary considerably. Large folds of complex shape are often called ruffles (Figs. 4 and 8). Usually classification of a microextension as a microvillus, fold, or bleb presents no difficulties. However, there are, of course, some extensions which

FIG. 3 . Cytochalasin B-treated (2 hours; 2 pglml) LSF cell in suspension. Very large single bleb. Field width. 23.5 pni.

SURFACE TOPOGRAPHY OF SUSPENDED CELLS

FIG. 4. wm.

271

Mouse ascites hepatoma AH-2223 cell in suspension. Focus of ruffles. Field width, 11.8

have a shape intermediate between these three types. Very short microvilli may become almost indistinguishable from very small blebs. Swollen folds or microvilli may become similar to blebs. The whole surface of a suspended cell can be covered by protrusions of one type or of several types; these reliefs can be designated respectively as homogeneous and mixed (Figs. 5- 10). The combination of blebs and microvilli seems to be the most common among mixed reliefs (Figs. 1 and 10). Usually extensions of different types and sizes are distributed more or less evenly on various parts of the surface of one suspended tissue cell; one cannot distinguish any “zones” of this surface having various surface reliefs. There are, however, numerous exceptions to this rule. For instance, at the surface of mouse ascitic hepatoma AH-22a cells (Rovensky et (11.. 1980). one could see discrete zones covered with microvilli, which were much longer than those at the other areas of cell surface; foci of ruffles were also present on these surfaces (Figs. 4 and 11). Similar “foci” of altered microrelief were seen on the surface of human tumor cells growing in peritoneal or pleural exudates (Domagala and Koss, 1977; Koss and Domagala, 1980). Functionally different zones with various microreliefs are characteristic of

FIG. 5

FIG.6.

SURFACE TOPOGRAPHY OF SUSPENDED CELLS

279

FIG. 7 . GR/mt (mouse mammary tumor cell line) in suspension. Homogeneous microvillous surface relief. Field width, 23.5 pm.

certain types and states of blood cells and macrophages (Berlin and Oliver, 1978). Usually microextensions cover the whole surface of suspended cell or, at least, its large part. Are there any types of suspended tissue cells characterized by the smooth surface without extensions? Several papers contain descriptions of populations of suspended cultured cell lines (Temmink and Collard, 1977; Collard, 1979) and of blood leukocytes (Polliack et al., 1973, 1974) in which the cells with smooth surfaces were predominant. In our experiments with normal and neoplastic fibroblastic cells and ascites tumor cells we did not observe regularly any population in which most of the suspended cells had smooth surfaces. The smooth cells were seen occasionally in suspensions of various lines (Samilchuk and Rovensky, 1980) (Fig. 12), but their relative frequency was usually small. It is not clear whether the smooth surface is a normal variant of surface topography FIGS. 5 AND 6 . Mouse embryo cells in suspension. Homogeneous blebbed surface reliefs. Blebs have high (Fig. 5 ) or low (Fig. 6 ) density. Field width, 23.5 pm.

FIG. 8.

FIG. 9.

SURFACE TOPOGRAPHY OF SUSPENDED CELLS

28 1

FIG. 10. Mouse L cell in suspension. Mixed (blebbed and microvillous) surface relief. Field width, 23.5 p,m.

of suspended cell or a result of some secondary changes occurring in the course of preparation of suspension, e.g., of cell injury leading to swelling. Several technical remarks can be made with regard to the examination of surface topography of suspended cells. Of course, preparation of suspended cells for SEM should meet all the general standards of adequate SEM examination. The major problems arising with regard to the various stages of the preparation of suspended and attached cells (fixation, drying etc.) for SEM had been adequately reviewed (Boyde, 1976; Boyde and Vesely, 1972; Lin et al., 1973; Barber and Burkholder, 1975; Brunk et al., 1975; Boyde et al., 1977, 1981; Roath et al., 1978; Schneider et al., 1978; Boyde and Maconnachie, 1979; Schiff and Gennaro, 1979), and will not be discussed here. Adequate collection of fixed suspended cells on some supporting surface is essential for their SEM examination. FIG. 8. Mouse ascites sarcoma 37 cell in suspension. Homogcneous folded surface relief. Field width. 23.5 pm. FIG.9. Mouse ascites sarcoma 37 cell in suspension. Mixed folded and microvillous surface relief. Field width, 23.5 bm.

FIG. 11.

FIG. 12.

SURFACE TOPOGRAPHY OF SUSPENDED CELLS

283

FIG. 13. Mouse embryo fibroblast fixed in suspension and then air-dryed. Substrate-attached pseudoextensions form circular lamella and filopodia. Field width, 23.5 pm.

Various collecting surfaces were proposed and used with satisfactory results (Flechon et al., 1975; Sanders et al., 1975; Vial and Porter, 1975; Vergara et al., 1977; Rovensky, 1978). When one starts to use a new collection method it is advisable to make a quantitative estimation of the efficiency of collection. This is easily done by comparing the number of cells in suspension covering the supporting surface before collection and the number of cells counted on this surface in final SEM specimen. The methods collecting not less than 80-90% suspended cells can be usually regarded as satisfactory. Even the short-term contact of living suspended cells with the substrate can alter their surface topography (Wetzel et al., 1974; Alexander and Wetzel, 1975; de Harven et al., 1975). Therefore the cells should be fixed in suspension and collected on the supporting substrate only after fixation. By the same reason the ~

11. Mouse ascites hepatoma AH-22a cell in suspension. Focus of long microvilli and ruffles. Field width, 58.8 p m . FIG. 12. Mouse embryo fibroblast in suspension. Smooth cell surface. Field width, 11.8 pm. FIG.

284

YU. A . ROVENSKY AND JU. M . VASILIEV

cells that were not completely detached from the substrate should not a priori be regarded as being identical in cell surface topography to suspended cells. Formation of “pseudoextensions” in the basal parts of suspended cells after fixation deserves special attention. These artifacts can be formed when the cells fixed in suspension are attached to the collecting surfaces and then prepared for SEM. Pseudoextensions have shapes similar to those of long substrate-attached filopodia or small circular lamella (Figs. 13 and 14). We (Rovensky, 1979) often observed such pseudoextensions in the preparation of various cells types prefixed in suspensions, attached to various supporting surfaces (aluminium foil, glass, polymers), and then air-dried; use of hypotonic fixatives increased the frequency of those structures. Adequate fixation, followed by critical point drying, considerably decreased the number of pseudoextensions seen in the specimens of suspended cells. However, occasionally long “pseudofilopodia” were found even in the critical point dried specimens (Fig. 14). The mechanism of formation of pseudoextensions is not clear. Probably, they are squeezed from prefixed substrate-attached cells by the pressure exerted upon these cells during drying (Boyde et al., 1977, 1981; Boyde and Maconnachie, 1979).

FIG. 14. IAR-2 cell fixed in suspension and then critical point dryed. Substrate-attached filopodial pseudoextensions. Field width, 5 8 . 8 pm.

285

SURFACE TOPOGRAPHY OF SUSPENDED CELLS

111. Surface Topography of Suspended Tissue Cells of Various Types

Comparative investigations of surface topography of the suspended cells of primary fibroblastic cultures, of minimally transformed lines, and of their more transformed descendants were performed by several groups of authors and gave rather variable results (see Table I). Thus, in the experiments of Ukena and TABLE I VARIOUSTYPESOF SURFACE M I C R O R E L IIN ~ FNORMAL. S AND

Cells Embryo mouse fibroblasts (primary culture) Swissi3T3 (“nornial” cell line) SVT-2 (BALBI3T3 cells transformed with SV40 virus; clone of SV 3T3 line) L 929 (transformed mouse fibroblasts) Nil-1CI (hamster embryo cells spontaneously transformed) IC-21 (mouse macrophages transformed with SV40 virus) SwissI3T3

NEOPI-ASTIC

CELL. SUSPENSIONS

Detaching agent

Predominant cell surface relief

EDTA

Microvillous

Ukena and Karnovsky (1977)

EDTA

Microvillous

EDTA

Microvillous

Ukena and Karnovsky (1977) Ukena and Karnovsky (1977)

EDTA

Microvillous

EDTA

Microvillous

EDTA

Microvillous

Ukena and Karnovsky (1977)

EDTA

Microvillous

Collard and Teinmink (1976): Temmink and Collard (1977): Collard (1979) Collard and Temmink (1976): Temmink and Collard (1977); Collard (1979) Collard and Ternmink (1976): Temmink and Collard ( 1977); Collard ( 1979) Collard and Temmink (1976): Temmink and Collard ( 1977); Collard (1979)

3T3-4 (“normal” cell line)

EDTA

Microvillous

SwissiSV 3T3

EDTA

Smooth

L-929

EDTA

Microvillous

+ folded

+ folded + folded

References

Ukena and Karnovsky (1977) Ukena and Karnovsky (1977)

286

YU. A . ROVENSKY AND J U . M. VASILIEV TABLE I

Cells

Detaching agent

(Co~tinuerl)

Predominant cell surface relief

EDTA

H7W (clone of Chinese hamster ovary line, CHO) T14 (human bladder carcinoma cell line) BALBI3T3 (“normal” cell line) SVT-2

EDTA Trypsin

Microvillous Microvillous

EDTA EDTA t trypsin ECTA

Microvillous + blebbed Microvillous + blebbed Microvillous

EDTA

Microvillous

+ folded

3T3 f (3T3 cells spontaneously transformed

Blebbed

+ folded

SwissI3T3 3T3-4

EDTA EDTA Trypsin

Blebbed Smooth Microvillous

L929

EDTA

Microvillous

Trypsin(’

Blcbbed

Trypsin” Trypsin”

Blebhed Blchhed

BHK-21 (“normal” hamster cell line) BHK-2 I BALBI3T3

References Collard and Temmink (1976): Temmink and Collard (1977); Collard (1979) Noonan and Ukena (1978) Vcrgara rf al. (1977) Gershman and Rosen (1978) Cershman and Rosen (1978) Rosen and Culp ( 1977) Willingham and Pastan (1975): PaStan and Willingham (1978) Willingham and Pastan (1975); Pastan and Willinghain (1978) Erickson and Trinkaus ( 1976) Revel er ul. ( 1974) Furcht and Wendelschafer-Crabh ( 1978)

“The cells were examined 10 minutes after their attachment to substratc. ”The cells were examincd just before their detachment from substrate.

Karnovsky (1977) all the examined cells had similar microvillous relief and n o consistent changes accompanying transformation were observed. Several authors (Collard and Temmink, 1976; Collard, 1979; Gershman and Rosen, 1978) reported the disappearance of microvillous relief after transformation of 3T3 cells by SV40 virus. Willingham and Pastan (1975) suggested that suspended transformed cells can have microvillous relief more often than nontransformed ones and that these differences in surface topography are correlated with different agglutinability of these cells by lectins. In our laboratory we examined quantitatively relative frequencies of the sitspendcd cells with various reliefs in the populations of many different types of cultures. The results of these examinations (Bershadsky et a / ., 1976; Rovensky. 1978, 1979; Samilchuk and Rovensky, 1980: Rovensky. unpublished results) are summarized in Table 11. As seen from the table there was n o examined population in which all the cells

PERCENTAGESOF VARIOUS TYPES

Ol-

TABLE I1 SCIRI-Act MICRORELIEFS IN NORMAL AND N~OPLASTIC CtLL SUSPtNSIONS" Surface microrelief types

Number

Cells Primary cultures Mouse embryo fibroblasts Rat embryo fibroblasts Chicken embryo fibroblasts Human embryo fibroblasts Hamster embryo fibroblasts

6

Mouse cell lines L (fibroblasts transformed in virro with 20-methylcholantrene) S40 (the line obtained from plastic film-induced sarcoma) 84/16 (the line obtained from plastic film-induced sarcoma) 130/32 (the line obtained from plastic film-induced sarcoma) M-22 (embryo fibroblasts transformed with SV40 virus) BALBI3T3 (CI 4)

Detaching agent

Blebbed

EDTA + trypsin EDTA + trypsin EDTA trypsin EDTA + trypsin EDTA + trypsin EDTA Trypsin

49.1 f 2.6 28.8 rt 3.9 60.5 f 6.2 47.0 ? 5.0 48.5 f 15.5 79.1 f 3.1 39.0 2 3.8

+

Microvillous

11.6 22.2 24.5 I I .O

f 1.1

3.6 2 9.3 rt 3.0 1 1 . 1 f 6.3 5.7 ? 1.9 49.7 f 4.1 rt

(blebbed

Mixed + microvillous)

39.2 48.3 12.2 42.0 38.2

?

2.3

2 1.0 rt

2 rt

1.6 8.0 7.7

-

45.7

%

3.8

f 4.7

Trypsin

2.0 f 1 . 1

82.0

?

3.4

Trypsin

-

90.0

f

2.1

6.0

%

2.8

66.0

f

4.3

30.0

f

3.7

Trypsin

27.9

Trypsin

2.1

+ 3.8

1.0 2 0.5

59.7 f 2.5

43.4

55.6

7.7

-

2.2

-

10.1 f 2.2 3.8 2 1.6

16.0 -t 3.2

12.4 lr- 5.8 ?

?

2.5

50.0

2

-

rt

1.1

4.0

-

0.6 2 1.9 2

-

2.1

Smooth

-

7.5

4.3

Trypsin

0.6 2.7

S.1 2 1 . 5

Trypsin

2

Folded

2

4.0

f

1.2

-

7.8 (continued)

TABLE I1

(Conrinued) Surface microrelief types ~~

Number 12 13 14 15 N io 00

16 17

18

19 20 21

Cells BALB/SV 3T3 (cl9) 152/ 19 (spontaneously transformed fibroblasts) 82CTi 15T (spontaneously transformed fibroblasts) AH-22aK (the line obtained from ascites hepatoma AH-22a) MFTR (kidney cells transformed in virro with SV40 virus) LSF in serum-free medium (subline of L cells which in serum-free medium reverses to “normal” phenotype) LSF (in scrum-containing medium) Rat cell lines XC (the line obtained from sarcoma induced by Rous virus) NRK (kidney cells) Ki-MSV (NRK cells transformed with mouse Kirsten sarcoma virus)

Detaching agent

Blebbed

Microvillous

Trypsin Trypsin

21.9 i 2.6 5.9 i 1.2

16.8 i 4.2 37.2 i 5.1

61.3 i 6.4 54.9 ? 7.8

39.4 i 1.4

60.5 t 1.4

Trypsin Trypsin

2.0

0.3

98.0 2 2.1

Trypsin

7.7 i 1.2

59.0 -+ 4.3

EDTA

-40

EDTA

EDTA

+ trypsin

EDTA EDTA

+ trypsin + trypsin

-t

-4-6

9.0 + 5.0 2.8 2 1.1

-20-25

(blebbed

Mixed + microvillous)

30.8 2 5.6 -30

>90

98.0 2 2.0

2.0 t 2.0

42.0 i 4.0 74.9 5 6.9

49.0 2 1.0 18.4 t 5.4

Folded -

2.0

5

~

1.2

-

2.6

5 -

Smooth

-

1.1

-

-

22 23 24

25

26 27 t.l m \D

28

29 30

IAR 2 (liver-derived epithelial nontumorigenic rat line) IAR-2-3 I RT6 (tumorigenic line obtained from IAR-2) IAR 6-1 RT7A (tumorigenic liver-derived rat line] Bovine cell line FBT (epithelial line derived from tracheal Hamster cell lines BHK-21 (line from hamster kidney) HEK-40 (spontaneously transformed embryo fibroblasts) SV-KX (embryo fibroblasts transformed with SV40 virus) Mink cell lines MvlLu (iiiink lung cell line) Ki-Mv ILu (Mvl Lu cells transformed with Kirsten murine sarcoma virus)

EDTA

A

trypsin

34.8

EDTA

itrypsin

EDTA

EDTA

8.9

14.0 f 4.9

38.6 t 4.7

2.5

?

0.5

6.7

* 2.9

81.8 t 6.1

10.6 f 2.9

0.9

2

0.9

+ trypsin

25.8

* 4.4

34.1 2 5 . 5

40.1

r

trypsin

7.7

74.4

3.9

15.4 2 4.2

2.6 t 1.2

EDTA

A

trypsiil

1.0 2 1.0

68.1 t 2.1

23.9 f 5.5

7.1

EDTA

t

trypsin

6.5

14.5 % 6.7

67.5

2.0 2 0.8

EDTA Tr;ypsin

4.3 k 1.2 2.1 2 1.9

63.1 t 3.9 69.7 2 4.2

26.3 f 2.8 23.9 t 3.1

5.8 ? 1.4 4.2 r. 2.1

EDTA Trypsin EDTA Trypsin

21.8 L 3.0 11.5 2 2.5 26.1 f 2.7 8.2 2 1.8

35.6 2 2.7 53.3 2 3.2 26.1 2 2.8 46.8 -t 4.1

37.9 t 3.1 29.2 ? 3.0 41.6 5 3.2 40.5 2 2.9

4.6 f 1.5 6.1 ? 2.1 6.2 2 1.3 4.4 t 1.2

16.0

f

?

1.2

f

“Data of Bershadsky cf ul. (1976) (N17. 18). Rovensky (1978. 1979, and unpublished) ( N 1-5.6-16. 30).

f

2

-

9.9

5.7

i_

0.8

19-27). and Samdchuk and Rovensky (1980) ( N 5 , 28-

290

YU. A. ROVENSKY AND J U . M. VASILIEV

had the same single type of relief. All the three main patterns of relief (blebbed, microvillous, and mixed) were present in almost all the populations. There were, however, considerable differences in the relative frequencies of reliefs between various populations. In particular, high proportions of cells with purely blebbed relief were characteristic of suspended primary cultures of embryo fibroblasts of several species; the cells with purely microvillous relief were in the minority in these cultures. Considerable decreases of the relative frequency of cells with blebbed relief and increases of the frequency of microvillous cells were typical of most populations of permanent cell lines as compared with primary cultures. This type of distribution was characteristic not only of fully transformed lines but also of minimally transformed ones such as BALB/3T3 (N 1 1 in Table 11) or NRK (N 20). Additional transformation of NRK cells by Kirsten mouse sarcoma virus led to a further increase of the frequency of microvillous cells (compare N 21 with N 20). Similarly, the tumorigenic rat liver-derived line IAR 2-3 1 RT6 (N 23) had a much higher frequency of microvillous cells than its nontumorigenic ancestor, IAR-2 line (N 22). The case of LSF cells (NN 17,18 in Table 11) deserves special mention. These cells transferred from usual serum-containing medium into the medium without serum reversibly acquired a less transformed phenotype: they spread better on the substrate and their agglutinability with lectins and efficiency of colony formation in semifluid media decreased (Bershadsky et d., 1976). This phenotypic reversion was also accompanied by a considerable decrease of the fraction of microvillous cells in suspension. Two lines of cells transformed with SV40 virus (BALB/SV3T3 and M-22) had unusual distributions of reliefs different from those of the lines transformed by other agents: they contained relatively high proportion of blebbed cells and decreased proportion of microvillous cells. As already mentioned, other authors (see Table 1) also observed the disappearance of microvilli after transformation with SV40 virus. Taken together these data show that microvilli and blebs are the two most common types of microextensions in suspended fibroblastic cultures. Relative frequencies of the cells with both types of microreliefs seem to be characteristic of each type of culture. Often, although not always, a change in the frequency of microvillous cells is increased in the course of multistep neoplastic evolution in cultures (see Vasiliev and Gelfand, 198 1 , for discussion of these stages). In the following sections we will discuss some of the factors which may determine relative frequencies of microvilli and blebs on the cell surface.

IV. Mechanisms of Formation of Microextensions Transmission electron microscopy of the sections of microextensions always reveals two main components: the plasma membrane and submembranous cortical layer. Probably, alterations of these two structures play leading roles in the

SURFACE TOPOGRAPHY OF SUSPENDED CELLS

29 1

formation of microextensions. The cortical layer is the submembranous zone consisting mainly of actin microfilaments. Microfilaments of the cortical layer of the substrate-spread cells form a complex network consisting of individual microfilaments and bundles of microfilaments (review in Vasiliev and Gelfand, 1977, 1981). At least some of these microfilaments are, probably, anchored to the cell membrane components and through these components to the extracellular matrix; the mechanisms of this anchorage are at present being intensively investigated. In particular, the ends of microfilament boundles are attached from the inside to the membrane in the specialized sites of cell-substrate adhesion called focal contacts. Specific proteins, such as vinculin (Geiger, I98 1 ; Rohrschneider e t a l . , 1981), are found in these contacts. These proteins, probably, are essential for anchorage of microfilaments to membrane components. The cortical layer of substrate-spread cell anchored to the membrane and to the substrate exerts traction on the substrate (Harris, 1982). Probably, the same centripetal force exerted by the cortex leads to the contraction of cell detached from the substrate and to the acquisition of spherical shape by the suspended cell. During detachment some parts of the cytoplasm (so-called “footpads.” “isolated contacts”) may be detached from the cell and left on the substrate (Revel et a/., 1974; Culp, 1976; Rosen and Culp, 1977; Badley et al., 1978; Culp et al., 1979). The cells in suspended state have no large microfilament bundles. Electron microscopic examination of sections of the cortical layer of these cells shows that blebs and microvilli have a somewhat different structure. The matrix cortical layer is usually seen at the periphery of blebs while their central parts are usually filled with ribosomes; occasionally other cytoplasmic organelles, e.g., mitochondria, are seen within the blebs (see, e.g., Fig. 8 in Vasiliev and Gelfand, 1977). In contrast, microvilli do not have the central part with cytoplasmic organelles; they usually contain only the thin cortical layer in which groups of parallel microfilaments can be occasionally seen. Detailed studies of the organization of the cortical layer in various types of protrusions remain to be done. The cortical layer may determine the shape of protrusions in several possible ways. 1. Microfilaments or other cytoskeletal fibrils may remain anchored to certain sites of the membrane of suspended cells; then these “anchoring sites” will be pulled inside by the contracting cortical layer more strongly than other parts of the membrane. As a result, infoldings and protrusions will be formed at the surface (see Fay, 1976). 2. Extension of protrusions may be a result of directional local centrifugal flow of intracellular fluid. In particular, the contracting cortical layer may exert hydrostatic pressure on the internal cytoplasm (see Bereiter-Hahn et al., 198 1, for discussion). Due to this pressure the cytoplasm may flow to the periphery through the discontinuities (“openings”) in the cortical layer. Obviously, the shape of protrusions will depend on the size and form of these openings.

292

YU. A. ROVENSKY AND JU. M. VASILIEV

3. Extension of protrusions may be the result of local polymerization of new actin microfilaments pushing the membrane outwards. Distribution of sites of this polymerization may be determined by the structure of preexisting cortical layer and the membrane. Various possible mechanisms may be combined in different ways, e.g., the flow of cytoplasm containing unpolymerized actin may be followed by polymerization of microfilaments within the protrusion. The crucial role of actin microfilaments in the determination of the shape of microextensions is confirmed by the results of experiments with cytochalasins. Cytochalasins specifically affect cortical actin-containing structures: they inhibit polymerization of microfilaments from actin and have been reported to induce in certain conditions depolymerization of these microfilaments (Flanagan and Lin, 1980; Lin et al., 1980; MacLean-Fletchner and Pollard, 1980; Maruyama ef al., 1980). Vlodavsky and Sachs ( I 977) using darkfield light microscopy have found that cytochalasin B in large doses inhibits formation of microvilli on the surface of suspended transformed hamster fibroblasts but not on the surface of normal progenitors of these cells. Shortening of microvilli and formation of blebs were observed in the cytochalasin-treated cultures of HeLa line grown in suspension (Godman et af.,1975). Incubation of the ascites cells of mouse sarcoma 180 with cytochalasin B led to a decrease of the number of microvilli and to the appearance of folds (Oppenheimer et a / ., 1977). In these experiments large doses of cytochalasins causing profound disorganization of cortical layer were used. In our recent experiments (Allikmets et af., 1983) we studied the effects of short-term incubation of cells with the relatively low doses of cytochalasins B and D which caused only partial alterations of the organization of cortical structures. Suspended cells of several types (suspended substrate-grown transformed fibroblasts of the L line; LS variant of L cells adapted to growth in suspension and mouse Ehrlich ascites tumor cells) were used. Suspensions of all these cell types were incubated for 30- I80 minutes with low concentrations of cytochalasins (2 pg/ml of cytochalasin B or 0.2 kg/ml of cytochalasin D). This treatment had decreased the incidence of cells with microvillous relief and increased the incidence of cells with blebbed relief (see Table 111; Figs. 15-18). Microvilli remaining at the surface of cytochalasin-treated cells were shorter than in control suspensions and their density was decreased; very short microvilli could become indistinguishable from small blebs (Fig. 16). Morphology of blebbed relief was not changed except that single very large blebs were occasionally seen (Fig. 3). Sometimes the blebs were concentrated on one pole of the cell surface (Fig. 18). These experiments suggest that mechanisms of formation of microvilli and blebs are different. Formation of microvilli requires the normal state of actin cytoskeleton and is inhibited by cytochalasins. Formation of blebs can occur in the cells with partially desorganized actin cytoskeleton; small doses

293

SURFACE TOPOGRAPHY OF SUSPENDED CELLS

TABLE 111 ACTIONOF CYTOCHALASIN B (2 )Lg/ml) ON T H t SUKI-ACE TOPOGKAPHY OF SUSPtNDtD cL;LLS"

Period of incubation (minutes)

Percentage of cells with different microreliefs Cell treatment

Microvillous

Control Cytochalasin B Control Cytochalasin B Control Cytochalasin B

43.3 t 9.0 66.7 2 8.6 63.2 t 11.0 -

Mixed

Blebbed

L cells

30 60 I80

46.7 t 9.1 12.5 t 8.1 26.7 .t 8.1 31.8 ? 9.9 36.8 .t 11.0 25.0 t 9.7

10.0 2 87.5 t 6.7 2 68.2 2

14.0 C _ 6.3 65.5 2 8.6 13.0 2 7.0 -

3.0

5.5 8.1 4.6 9.9

-

75.0 t 9.7

Ehrlich ascites tumor cells

30

Control Cytochalasin B Control Cytochalasin B

180

83.0 r. 6.8 34.5 2 8.6 82.6 t 7.8 -

3.1

4.3 ? 4.0 100.0

<"Dataof Allikmets ct al. (1983)

FIG. 15.

-+ -

L cell in suspension. Microvillous surface relief. Field width, 23.5 prn

294

Y U . A. ROVENSKY AND J U . M. VASILIEV

FIG. 16. Cytochalasin B-treated ( 1 hour; 2 kg/ml) L cell in suspension. Microvilli become much shorter; their density is decreased. Field width, 23.5 pin.

of cytochalasins do not inhibit formation of blebs and may even favor it. Possibly, protrusion of microvillus involves the local polymerization of microfilaments, which form the core of the microextension. In contrast, formation of blebs may be a result of the flow of cytoplasm through the “openings” in the cortical network. Cytochalasin may then favor the extension of blebs by increasing the size and number of the openings in the cortical layer: at the same time the drug may prevent formation of microvilli by inhibiting actin polymerization. This suggestion is in agreement with electron microscopic observations (see above) showing the presence of cytoplasmatic organelles within the blebs but not within the microvilli. Rate of diffusion of receptors within the membrane of blebs was found to be much higher than that within the membrane of the other parts of cell surfaces (Barak and Webb, 1982). This result indicates that blebbing leads to release of membrane components from the constraints imposed by submembranous structures. In other words, during formation of blebs anchorage of membrane components to cortical cytoskeleton may be destroyed. The suggestion that formation of blebs may require partial disorganization of actin cytoskeleton is also in good agreement with the finding that blebbing is an

SURFACE TOPOGRAPHY O F SUSPENDED CELLS

295

early sign of the injury of isolated suspended hepatocytes produced by bromobenzene and other toxic chemicals in these cells (Jewel1 et a / ., 1982). As already mentioned, extension of microvilli, in contrast to that of blebs, can involve polymerization of actin microfilaments and formation of new “anchorages” between these microfilaments and membrane proteins. Interesting facts indirectly supporting this suggestion were obtained in the experiments of Carraway, Carraway, and collaborators (Carraway et al., 1979, 1983; Huggins et d., 1980). These authors compared the properties of two sublines of rat ascites mammary carcinoma. The cells of one of these sublines had branched microvilli at the surface and low mobility of surface receptors. Isolated membranes of the microvilli of these cells contained actin, cell surface glycoprotein with a MW about 70,000-80,000 and a protein with MW 58,000. This last protein was absent in the preparations of microvilli from other subline, whose cells had shorter unbrached microvilli at the surface and more mobile surface receptors. The authors suggest that the 58,000 MW protein stabilizes association of the cell surface glycoprotein with microfilaments, thereby stabilizing the microvilli and

FIG. 17. Cytochalasin B-treated ( 1 hour: 2 Kgiml) L cell in suspension. Microvillous surface relief was changed for a blebbed one. Field width, 23.5 p.m.

296

Y U . A . ROVENSKY AND JU. M. VASILIEV

FIG. 18. Cytochalasin B-treated ( 3 hours; 2 pgiml) Ehrlich ascites tumor cell in suspension. Blebs concentrated on one pole of the cell surface. Field width, 23.5 pm.

restricting cell surface receptor mobility. Further studies in this direction seem to be very promising. Besides actin microfilaments the cells contain at least two other systems of cytoskeletal fibrils: microtubules and intermediate filaments. We do not know anything about their roles, if any, in the determination of surface topography of suspended tissue cells. One possible way to approach this problem is to examine in detail the effects of colchicine and other microtubule-depolymerizing drugs on suspended cells. This question deserves detailed study, especially, because colchicine has been found to change the shapes of substrate-attached tissue cells (Vasiliev and Gelfand, 1977) and also to induce development of large protuberances in polymorphonuclear leukocytes, lymphocytes, and macrophages (Oliver and Berlin, 1982a,b). What factors induce the protrusion of microextensions? Blood platelets are the suspended cells in which mechanism of the induction of pseudopods was studied in more detail than in other cells. Formation of pseudopods in platelets is induced by their incubation with certain activating agents such as ADP and serotonin (Barnhart, 1978; White and Gerrard, 1980). These agents act as ligands interact-

SURFACE TOPOGRAPHY OF SUSPENDED CELLS

297

ing with corresponding membrane receptors. Suspended tissue cells, unlike platelets, protrude microextensions spontaneously, without application of any special activating factors. Possibly, some standard components of the humoral medium of culture act as activating factors continually inducing formation of microextensions. It would be important to study systematically the effects of various modifications of the composition of medium on the formation of protrusions. Vlodavsky and Sachs (1977) observed inhibition of the formation of microvilli after incubation of suspended transformed hamster fibroblasts with an ionophore which increased permeability of cell membrane for calcium. Osborn and Weber (1980) described the disappearance of microvilli at the surface of substrateattached HeLa cells after incubation with trifluoperazine-a drug, that inhibits interaction of calcium ion with an intracellular calcium-binding protein, calmodd i n . Calmodulin mediates calcium regulation of diverse intracellular processes. Alterations of calcium ion homeostasis may also be essential for the formation of blebs. Jewel1 et al. (1982) had found that the degree of blebbing of suspended hepatocytes was proportional to extramitochondrial concentration of this ion. The possible role of calcium and of other membrane-generated messengers in the induction of microextensions in suspended cells deserves further study. In summary, there is reason to suggest that formation of microextensions is induced by some membrane changes leading to alterations of underlying cortical microfilaments. The exact mechanism of these changes remains unknown. Formation of blebs and of microvilli seems to require various types of reorganizations of the cortical cytoskeleton. The factors involved in formation of folds are completely obscure. Extensions formed by the substrate-attached cells (pseudopods and lamellae) have a number of characteristic properties: they are contractile, they have a distinctive ability to form adhesions and to clear their surface from the receptors cross-linked by corresponding ligands (see review in Vasiliev, 1982a). It would be important to find out whether microextensions of suspended tissue cells have similar characteristics. Are there any mechanisms controlling global distribution of extensions at the surface of suspended cells? The surface of certain unspread blood cells, such as polynuclears, macrophages, and lymphocytes, can be differentiated into several morphologically and functionally distinct regions; one particular manifestation of this differentiation is the ability of these suspended cells to move cross-linked receptors into certain areas of the surface, that is, their ability to perform the capping of receptors (de Petris, 1977, 1978; see review in Oliver and Berlin, 1982a). Substrate-spread fibroblasts and epithelial cells also have highly polarized surfaces. In particular, formation of pseudopods in the substrate-attached cells is usually localized only in certain active zones of the surface; other parts of the surface remain nonactive. Distribution of pseudopodial activity was found to

298

Y U . A . ROVENSKY AND JU. M. VASILIEV

depend on the orientation of microtubules and, probably, of microfilaments (see review in Vasiliev, l982b). The whole surface of suspended tissue cells in contrast to that of substrate-attached cells is usually covered with microextensions. Suspended fibroblasts, in contrast to the substrate-attached ones, do not collect cross-linked receptors into caps (Domnina and Pletjushkina, 198I ) . Thus, the surface of these cells seems to be uniform in its properties. However, in certain cases nonrandom localization of microextensions had been observed on the surface of suspended cells: several examples of these observations had been given in previous sections. These observations suggest that at least some suspended tissue cells, like substrate-attached cells and blood cells, may have mechanisms regulating formation of microextensions in various parts of the surface. Dependence of surface topography on the amount of available surface material is another question that needs further investigation. Measurements performed by Erickson and Trinkaus (1976) in the scanning electron micrographs of unspread and substrate-spread BHK 21 cells had shown that the total area of the cell surface is not changed in the course of spreading; they suggested that surface material stored within microextensions of suspended cells is used up during spreading. Dynamic observations of the formation of blebs at the surface of small cytoplasmic fragments detached from the main bodies of 3T3 cells were performed by Albrecht-Buehler (1981). He had shown that extension of a new bleb from the fragment surface is usually accompanied by the contraction and disappearance of some other bleb present at the same surface. These results are in good agreement with the suggestion of Trinkaus (1980). He supposed that the limitation of the amount of surface material may control the extension of pseudopods so that the increased pseudopodial activity in one region should be accompanied by a corresponding decrease of activity in other areas. It would be very important to perform detailed morphometric studies of various cells in the course of spreading, detachment, and other reorganizations in order to find out whether the surface area and volume remain constant during all these changes. Examination of suspended cells with smooth surfaces, if any such cells were found (see previous section), would be of special interest: obviously, these cells cannot spread without altering the total area of their surface or without changing the volume. Detailed quantitative studies of the alterations of cell surface area in the course of mitotic cycle are also needed in order to understand better how the cell regulates the amount of its surface material. The topography of the cell surface was found to be different at various stages of the mitotic cycle in suspension cultures of several lines of neoplastic cells (Enlander et al., 1975; Knutton et al., 1975; Toth, 1981) and in cultures of substrate-attached cells (Porter et al., 1973; Hale et al., 1975; Collard and Temmink, 1976; Lundgren and Roos, 1976; Wetzel et al., 1977, 1978). These cycle-dependent alterations observed in differ-

SURFACE TOPOGRAPHY OF SUSPENDED CELLS

299

ent lines had no obvious common features. At present we do not yet know exactly how the cell accumulates and stores the new surface material needed for mitosis. Alterations of surface topography associated with differentiation also remain almost unstudied. Investigation of these associations can give interesting results as indicated by the findings of Brown and Klotz (1980) with the Friend murine erythroleukemia cells induced to differentiate with dimethyl sulfoxide. Whereas immature undifferentiated cells had uniform density of small microvilli fully differentiated cells were covered with large blebs.

V. Effects of Previous Contacts with the Substrate on the Surface Topography of Suspended Cells Dependence of the surface relief of suspended cells on the previous morphology of the same cells in the substrate-spread state was first demonstrated by Harrison and Allen (1979). These authors have shown that bipolar fibroblast-like cells of the CHO line and discoid isometrically spread epithelioid cells of the same line acquire various surface topography after their detachment from the substrate by trypsin: fibroblast-like cells become covered with blebs and epithelioid cells with microvilli. Our experiments (Rovensky, 1983) have also shown that surface topography of suspended cells depends on their previous history, more specifically, on the degree of their spreading before detachment. In these experiments we used the method developed by Folkman and Moscona (1978). Plastic culture dishes were covered by adhesion-decreasing polymer poly(2-hydroxyethyl methacrylate) [poly (HEMA)]. By varying the concentration of poly (HEMA) i n ethanol solution one could obtain the substrates with various degrees of adhesiveness. In f x t , normal mouse fibroblasts seeded on the Falcon dishes precovered with 11250- 1 / 1000 ethanol solutions of poly (HEMA) 24 hours later were much less spread than control cells seeded on the untreated Falcon dishes (Figs. 19-21). Suspensions prepared from the cultures poorly spread on the poly (HEMA) had a lower percentage of bleb-covered cells and higher percentage of cells covered with microvilli than control suspensions prepared from well-spread cells (Table 1V). Similar results were obtained in the experiments with normal mouse fibroblasts seeded on the grooved plastic (polyvinylchloride) substrates. As shown carlier (Kovensky er d., 197 I : Vesely ct t i / . , 198 1 ), fibroblasts avoid the grooves and are concentrated on the cylindrical substrate surfaces located between the grooves. The fibroblasts on the grooved substrate surfaces were oriented and less spread than those seeded at the same density on control flat substrates (Fig. 2 2 ) . Suspensions prepared from the cultures grown on grooved

300

YU. A. ROVENSKY A N D JU. M. VASILIEV

FIG: 19. Mouse embryo fibroblasts cultivated on Falcon plastic substrate. The cells are well spread and have a smooth surface. Field width, 117.7 pm.

substrates had a higher percentage of microvillous cells than control suspensions (Table IV). Thus, the suspended cells brought in suspension from a poorly spread state develop microvillous surface relief more often than those suspended from the well-attached state. Normal fibroblasts poorly spread on the poly (HEMA) and those well-spread on usual plastic have considerable differences in the organization of cytoskeleton. In particular, according to our observations, only the wellspread cells have bundles of actin microfilaments revealed by immunofluorescence. As discussed above, formation of blebs is, possibly, the result of some degree of disorganization of cortical layer. Detachment and contraction of cell during preparation of suspension are accompanied by profound reorganization of cytoskeleton. Detachment of the well-spread cell, probably, leads to more profound changes of the cell and of its cytoskeleton; therefore the cells in suspension FIGS. 20 A N D 21. Mouse embryo fibroblasts cultivated on Falcon plastic precovered with poly (HEMA). The cells are poorly spread and their surfaces have niicrovilli, folds. or ruffles. Field width, 117.7 pm (Fig. 20) and 58.8 p m (Fig. 21).

FIG.

20

FIG. 21

302

Y U . A. ROVENSKY AND JU. M. VASILIEV

FIG. 22. Mouse embryo fibroblasts cultivated on the grooved plastic substrate. The cells on cylindrical substrate surface are oriented and less spread than those on flat substrate surface. Field width, 117.7 km.

TABLE IV PERCENTAGES OF CELLS WITH VARIOUS TYPES OF SURFACt MICRORELIEFS IN SUSPENSIONS OBTAINED PROM CULTURES OF MOUSEEMBRYO FIBROBLASTS GROWNON VARIOUS SUBSTRATES

Falcon dishes with various adhesiveness Type of microrelief Blebbed Microvillous Mixed (blebbedmicrovillous) Folded

Polyvinilchloride films with various geometry of the surface

Covered with poly (HEMA) Uncovered

111000

I1250

40.3 +. 3.8 12.8 2 2.4 46.3 t 2.5

35.0 k 2.4 15.4 2 2.4 49.4 k 2.4

22.9 k 2.8 32.4 2 5.7 44.5 t 3.4

0.6 +- 0.6

0.2 k 0.2

0.3 t 0.3

Cylindrical Flat

(R = 55 pin)

62.8 t 3.7 7.9 2 1.4 28.4 2 3.7

40.1 -C 4.8 20.8 2 3.8 38.1 k 4.4

0.8

* 0.5

0.9

k

0.9

SURFACE TOPOGRAPHY OF SUSPENDED CELLS

303

more often have a partially disorganized cytoskeleton associated with blebbing. In any case, these experiments show that suspended cells have a certain “memory” about the degree of their previous spreading. Mechanisms of this “memory” require further study. In certain cases suspended cells, possibly, retain the local signs of the sites of previous contacts with the substrates. These sites were observed at the surface of suspended cells of culture of human bladder carcinoma (Vergara et af., 1977), and of mesothelium (Koss and Domagala, 1980). They were distinguished from other parts of the surface by their smoother microrelief. The structure of these “sites of former contacts” remains unknown.

VI. Conclusion Microextensions can be regarded as reversible specializations of cell surface formed with the participation of the cortical cytoskeleton and of the membrane. The data available at present give reason to suggest that different types of alterations of cytoskeleton may be involved in the formation of blebs and microvilli. Protrusion of blebs, possibly, involves partial destruction of cortical network of microfilaments, formation of “holes” in this network, and flow of cytoplasmic content into these holes. Polymerization of actin microfilaments may be the main process leading to the extension of microvilli. Study of the cytoskeleton of various microextensions with modern methods, especially, the preparation of replicas (Heuser and Kirschner, 1980) can be essential for further progress in the elucidation of structural differences between blebs and microvilli. Probably, the formation of blebs and of microvilli has different biological significance. Blebs may be manifestations of the disorganization of cortex in pathological conditions, e.g., after the action of toxic agents. In physiological conditions blebs may be formed at the intermediate stages of reorganization of cortex, in the course of transition of cell cortex from one stable state into another, e.g, during mitosis or during detachment from the well-spread state. Microvillous relief may be a sign of a more stable state of the cell, better adapted for the life in suspension. It may be significant in this respect that many types of ascites tumor cells permanently living in suspension usually have microvillous relief (Oppenheimer et al., 1977; Rovensky, 1979; Rovensky et al., 1980). Years ago Tomkins and collaborators (Evans et ul., 1974) suggested that formation of microvilli may be a form of cell adaptation for better intake of sugars. Possibly, microvilli of suspended tissue cells belong to the same group of surface specializations, involved in transport phenomena, as the brush border microvilli of intestinal cells. These brush border microvilli are known to have a well-developed and specialized actin cytoskeleton including the core bundle of microfilaments connected with the underlying terminal web (Matsudaira and Burgess, 1981; Moos-

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eker et al., 1981; Weber and Glenney, 1982). The exact mechanisms of participation of various types of microvilli in transport phenomena remain to be studied. Of course, microvilli and other microextensions may also have many other still obscure functions. For instance, they may be used by the cell to probe its immediate environment (Albrecht-Buehler, 1976) or for the storage of surface material. With regard to this last possibility it would be important to study in detail alterations of surface areas and of cell volume accompanying transitions between the suspended and spread states of various tissue cells. In the course of multistep neoplastic evolution the cells become progressively better adapted to the life and multiplication in suspension. As already mentioned in the introduction, cells with increasingly transformed phenotype progressively lose the response of metabolism and proliferation to detachment from the sustrate. Increased frequency of microvillous cells often observed in suspensions of neoplastic cultures may be somehow related to these alterations of the shape dependence of metabolism; both groups of changes may be manifestations of increased adaptation to the life in suspended state. As we have seen, the relief in suspension may depend on the degree of previous spreading on the substrate. Transformed cells are less spread on the substrate than their normal progenitors. This may be one of the reasons why detached transformed cells more easily reorganize their cytoskeleton and acquire microvillous relief. Possibly, the degree of other alterations of cells induced by detachment, e.g., of metabolic changes, can also depend on the previous spreading. Analysis of surface topography of suspended cells can be regarded as a special aspect of the studies of responses of normal and transformed cells to alterations of shape and of cell-substrate contacts. Investigations of mechanisms of these responses are still beginning. The importance of these investigations is obvious.

REFERENCES Albrecht-Buehler, G . (1976). In “Cell Motility” (R. Goldman, T. Pollard, and J . Rosenbaurn, eds.), pp. 247-264. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Albrecht-Buehler, G. (1981). Cold Spring Harbor Symp. Quanr. B i d . 46, 45-49. Alexander, E. L . , and Wetzel, B . (1975). Science 188, 732-734. Allikmets, E. J u . , Vasiliev, Ju. M . , and Rovensky, Yu. A. (1983). Eyull. Eksp. B i d . Med. 5, 8487. Aub, J . C . , Sanford, D. H . , and Cote, M. N. (1965). Proc. Narl. Acad. Sci. U.S.A. 54, 396-399. Badley, R. A , , Lloyd, C. W . , Woods, A . , Carruthers, L., Allcock, C., and Rees, D. A . (1978). Exp. Cell Res. 117, 231-244. Barak, L. S . , and Webb, W. W. (1982). J . Cell Biol. 95, 846-852. Barber, T. A . , and Burkholder, P. M. (1975). In “Scanning Electron Microscopyil975” (0.Johari, ed.), pp. 369-378. 11T Res. Inst., Chicago, Illinois. Barnhart, M. I. ( 1978). Mol. Cell. Eiochem. 22, 1 13- 136.

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Gastrointestinal Stem Cells and Their Role in Carcinogenesis A. I. BYKOREZAND Yu. D. IVASHCHENKO Department of Chemical Carcinogenesis, Kavetsky Institute for Oncology Problems, Academy of Science of the Ukrainian SSR,Kiev, USSR 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Stem Cells of the Small Intestine , . . . . . . . , . . . , . . . . . , . , . . . , . . , , A. General Characteristic . . . . . . . . . . . . . . . . . . , . . . . . . . , . . . , . . , B . Response of Stem Cells to Various Types of Damaging Agents 111. Stem Cells of the Colon . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . , , A. The Origin of Various Differentiated Cells , , . . , . , . . , . . . , . . , B. Peculiarities of Proliferation of Low Differentiated Enterocytes. C. Quantitative Characterization of Stem Cells. . . , . . , . . . . . . . . , , IV. Stem Cells of the Gastric Glands , . . . . . . . , , . . , , . . , . , . , , , . . , . . . A. Cardiac Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Fundic Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Pyloric Glands . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . D. Differentiation and Renewal of Endocrine Cells . . . . . . . . . . . , , V. Regulation of Proliferation and Differentiation in the Gastrointestinal Epithelium. . . , . . . . . . . . . . . , . . . . . . . . . . , , . A. Systemic Modulators of Cellular Proliferation . . , . . . . . . . . . . , . B. Paracrine Regulation . , , . . . , . . . . . . . . , . . . . . . , . . . . . , . . . , . . C. Regulatory Mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Stem Cells in Carcinogenesis , . . . . . . . . . . . . . . . . , . . . . . . . . . . . . , . A. Peculiarities of Cytotypical Differentiation in Tumors . . . . . . . , B. Disturbances in Proliferation during Carcinogenesis . . . . . . . . . . C. The Markers of Stem Cell Differentiation Disturbances.. . . . . , D. Problems of Gastrointestinal Neoplastic Growth Morphogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Factors Affecting Differentiation of Tumor Cells . . . . . . . . . . . . VI1. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . , .

309 31 I 31 I 313 318 318 320 322 323 323 323 330 330 332 332 333 331 344 344 341 352 351 361 363 364

I. Introduction Numerous successful studies performed during the last several decades on the epithelial proliferation and differentiation peculiarities in the gastrointestinal mucosa, both in animals and humans, still leave the histogenesis of different cell populations forming the morphofunctional structures insufficiently clear. It is surprising that in spite of an increasing number of investigations aimed at stem 309 Copyripht ii, 1984 hy Acadcmic Prcs. Inc. All right, ot reproduction in any Iorni reserved ISBN n - 1 2 - 3 w w ~

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cell identification in the intestinal and gastric epithelium there is no distinct “image” of these cells. The “stem cell” is a collective term, for this cellular type includes the cells known as “clonogenic,” “progenitor,” “cambial,” i.e., the cells capable of unlimited self-replication as well as of giving rise to various types of the differentiated cells. The most reasonable definition of stem cells has been suggested by Potten: “the cells ultimately responsible for all cell replacement, i.e. of both specialised cells and themselves, throughout adult life: the long-lived, ‘fixed’ or ‘anchored’ cells that are the ancestors of any recognisable or hypothetical cell lineages within tissues” (Potten, 1980). In many cases, the absence of any signs of cytotypical differentiation serves as a marker of the cell “sternness.” However, a low degree of differentiation (morphological, biochemical, functional) may also be characteristic of the cornmitted cells. The absence of reliable morphological markers and methods of cloning isolated normal epithelial cells of the gastrointestinal mucosa in vitro accounts for the fact that classifying cells as stem cells depends equally upon both the specific biological characteristics of the cells under study and the peculiarities of the investigative technology. Therefore, the behavior of stem cells (the ideal would be to obtain quantitative and qualitative parameters of the morphological, dynamic, metabolic, and structural features of these cells) both within normal and pathologically changed structures of the intestinal tube remains obscure in many respects. This is especially true regarding the factors responsible for regulation of the stern cell renewal, the differentiation pathways of their progeny, and cooperation between stem and other cells. Several promising hypotheses concerning the role of stem cells of the gastrointestinal mucosa in carcinogenesis have been developed and corroborated experimentally in recent years, although some of them may prove to be rather false than true. Presumably, the extension of our knowledge in a field of increasing cornplexity-structure and functioning of the hierarchical renewing cell populations under normal conditions, promotes the understanding of the regularities of carcinogenesis and vice versa. The general problems concerning the identification of stem cells by different methods, and their interrelationships with descendant populations, have been discussed in a number of monographs and excellent reviews (see Cairnie, 1976; Appleton et ul., 1980; Steel, 1977; Potmesil et al., 1977; Lajhta, 1979; Potten et al., 1979; Mattioli, 1982; Till, 1982). Therefore, the present review chiefly dwells upon the role played by stem cells of the gastrointestinal epithelium in the renewal of various differentiated cells both under normal conditions and during carcinogenesis. Taking into account that considerably less reliable information is available

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concerning stem cells of the colonic and especially gastric mucosa, we chose primarily to present data on small intestinal stem cells.

11. Stem Cells of the Small Intestine A. GENERAL CHARACTERIST~C 1 . Cellular Composition of Small Inrestine Epithelium

The epithelial lining of the mammalian small intestine is composed of heterogeneous cell populations, in which one may distinguish the following cell types: low differentiated crypt cells, Paneth cells, columnar (absorptive) cells, goblet cells, enteroendocrine cells, and tuft cells (fibrillovesicular cells) (Trier and Madara, 1981). In the small intestine of guinea pigs, rabbits, and monkeys there has recently been recognized a new type of epithelial cell, termed cup cells, located on the villi amid absorptive enterocytes (Madara, 1982). As to their structure, these cells resemble some of the enteroendocrine cells (motilin-producing as well as argyrophobic) but their functional specialization and life cycle so far remain obscure, The enterocytes of a particular type, the so-called M cells (“microfold” or “membranous”), which are present within follicle-associated epithelium, overlying the Peyer’s patches in ileum from a variety of different mammals, are derived from the epithelium of crypts, adjacent to follicles. However, the M cells seem to originate not from low differentiated cells of crypts, as is the case with all other cellular types (goblet cells, columnar cells, Paneth cells) but from young fully differentiated columnar enterocytes beyond the proliferative zone, i .e., resulting from structural reconstruction of the cells which are already incapable of proliferation (peculiar type of modulation). The origin of M cells is directly associated with the establishment of close contacts among some columnar enterocytes and lymphocytes of the Peyer’s patches, where a single M cell usually interacts simultaneously with several lymphoid cells (Smith et d., 1980; Smith and Peacock, 1980). M cells have been found to be responsible for direct transport of antigens from the intestinal cavity to the gut associated lymphoid tissue (see von Rosen et a l . , 1981). 2. Localization and Ultrastructural Peculiarities of Stem Enterocytes In the course of the ultrastructural study (Cheng and Leblond, 1974a-c; Leblond and Cheng, 1976), there were observed marked structural changes in the morphology of columnar enterocytes, occupying different cellular positions in the crypts and on the villi. The investigators have demonstrated that in the basal

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portions of crypts are located various subpopulations of enterocytes, including the subpopulation of stem cells, represented by undifferentiated, embryonic-like proliferating columnar enterocytes, situated in the bottom and in some higher positions. The cytoplasm of these cells contains a minimal amount of organelles (separate cysternae of rough endoplasmic reticulum, undeveloped Golgi apparatus, scarce mitochondria) but it exhibits the presence of a large number of free ribosomes. The nucleus is characterized by a diffuse structure of chromatin and large nucleolus, displaying an irregular shape and composed of a network of fibrillar, granulofibrillar and granular material, divided by interstitial spaces (Altmann and Leblond, 1982). This type of nucleolar structure, known as an “open” one, can also be observed in the stem cells of other renewing cell populations and may, probably, be regarded as one of their distinguishing characteristics (Leblond, 198I ) . In the course of differentiation of enterocytes, while migrating from the crypt bottom up to the extremity of the villi, the nucleolus decreases in size, its shape becomes more regular, and after passing the midportion of the villi height, the enterocyte nucleolus acquires a spherical shape, whereas the interstitial spaces disappear, and components of pars fibrosa are converted into pars granulosa components with subsequent reduction in the area of the latter portion. This type of nucleolus structure is defined as “dense” or atrophic (Leblond, 1981; Altmann and Leblond, 1982). Bjerknes and Cheng (1981a-e) have shown that ( 1 ) differentiated cells, and primarily the Paneth cells, distributed in the stem cells’ localization zone (i.e., within the limits of 1-4 cellular position) are being differentiated beyond the borders of this zone in positions 5-7 with subsequent migration at various rates toward the crypt base. Thus, the Paneth cells, enteroendocrine cells, and mucous cells are incapable of differentiating in situ; (2) the death of differentiated cells in the zone of stem cells results not from the cell position, but from the cellular age, as the downward cell migration exhibits a turbulent nature; (3) despite the fact that neonatal animals manifest an absence of concentration gradient in the Paneth cells by positions (in the adult animals this gradient appears to be well pronounced), the first Paneth cells occur in positions 5 and higher, that is, above the zone of stem cells; (4) the columnar villous and enteroendocrine enterocytes are also differentiated above the fourth position from the descendants of the stem cells, whereas highly differentiated cells of these types seen within the limits of positions 1-4 reach that area while migrating in a downward direction; and (5) the committed proliferating cells (columnar, mucous, enteroendocrine) traveling in a downward direction on entering the zone of the stem cells’ location are noted to discontinue their proliferation. 3. Mitotic Cycle of Stem Cells The majority of authors support the view that in mice and rats the cell cycle time (T,) of the enterocytes lining the bottom and 8-10 basal positions of small

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intestinal crypts is longer than in cells of the proliferative zone, which are located higher. In rats it was demonstrated by the fraction of labeled mitoses method (FLM) (Caimie et a / ., 1965; Al-Dewachi et al. , 1979), the stathmokinetic technique (Wright et a/., 1972a), and by repeated administration of [3H]thymidine (Pozharisski et a(., 1977a; Wright, 1978). By the stathmokinetic technique the above is confirmed for mice as well (AI-Dewachi et a / ., 1975a), although, as the authors of this publication suggested, visible changes in enterocytes’ T, in various crypt positions may to a greater extent reflect variability of growth fraction value for each position, rather than true variability of the T,. According to Cairnie et al. (1965), Al-Dewachi e t a l . (1975a), and Potten et al. (1974), the T, in basal enterocytes equals approximately 15-35 hours, mean value 24 hours, whereas Pozharisski et ul. (1977a) reported that T, in basal enterocytes was in the range 12-20 hours while in the uppermost cells of the proliferation zone the T, amounts to 9- 13 hours in mice (Al-Dewachi et a[., 1975b) and 1 1 - 12 hours in rats (Pozharisski et a / . , 1977a). The T, in stem cells (clonogenic) of small intestinal crypts is found to range from 40 to 60 hours (Hanson et al. , 1979). The rhythm of proliferation of these cells shows no circadian peculiarities, characteristic of the above lying enterocytes (Al-Dewachi et al., 1976).

B. RESPONSE OF STEMCELLSTO VARIOUSTYPESOF DAMAGING AGENTS 1. Fluctuations in Cell Cycle Time

The slowest rate of proliferation is observed in the enterocytes of the eight prcbottom positions in small intestinal crypts of mice and rats. These are the cells which according to Lamerton (1972) are able to ensure repopulation of crypts following exposure to irradiation or cytotoxic agents. These cells are less likely to be in a susceptible phase of the mitotic cycle in a moment of exposure compared with the more rapidly proliferating cells in the amplification compartment. However, the behavior of this enterocytes’ subpopulation following exposure to various damaging agents appears to be far from stable and resistance to radiation injury and S phase-specific agents’ effects are also rather variable. The facts available support the hypothesis that slowly proliferating cells are the first to start repopulation of the crypts. So, Al-Dewachi et al. (1977) have revealed that if a large proportion of proliferating crypt cells of rat small intestine is killed following administration of hydroxyurea (HU) then the index of DNA-synthetizing cells is abruptly increased amid basal enterocytes and they manifest considerable reduction in the cell cycle time. This created an impression that the above cells are really both the “functional” stem cells (i.e., they ensure the restoration of all cellular types in epithelium of crypts under normal conditions, as was shown by Cheng and Leblond, 1974a-c), and they appear to be “potential”

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stem cells, displaying clonogenic capacity and being capable of sufficient divisions to restore crypt cell population after the death of rapidly proliferating cells. However, it was found that a single injection of cytosine arabinoside (ara-C), which exerts a selective effect on the cells in the DNA synthesis phase, results in the death of 80% of clonogenic cells. It is not easy to explain the above observation except to assume that these cells are the slow proliferating ones (Boarder and Blackett, 1976). These data have been confirmed by Al-Dewachi et a1 (1980), who demonstrated that following a single ara-C injection no DNA-synthetizing cells can be observed at all for 6 hours, but later low labeling index occurs in all cellular positions within the zone of proliferation. The authors of this work managed to show that diminution of mean cell cycle time occurring after the end of exposure to ara-C is most pronounced in the enterocytes of 10 basal positions in the crypts, but this reduction was not substantial enough to serve as proof of a decisive role played by these cells in the processes of epithelium repopulation. The results suggested that the majority of clonogenic cells in small intestinal crypts have a relatively short cycle and, apparently, they are located within the active proliferation zone. However, Al-Dewachi et al. emphasize that proportional I, increase is more marked in the basal positions compared to the cells farther up the crypts. In basal enterocytes in addition to T, diminution a higher rate of mitotic activity is also observed. However, while assessing data concerning mitotic cycle time, one should bear in mind that by employing a labeled mitoses curve it is possible to determine only the mean T, value for the most rapidly proliferating subpopulations of cells, and, generally speaking, the problem of statistical comparison of kinetic parameters, obtained in FLM analysis, is still far from being solved (Steel, 1977). While plotting FLM for cells located in various positions, it seems a laborious task to correct the error resulting from cellular migration in the period following [3H]TdR administration. In our laboratory data were obtained indicating that if rats were treated with 5 doses of hydroxyurea, the duodenal crypts show simultaneous occurrence of labeled cells throughout the whole proliferative zone. The regeneration processes in the survived crypts start with proliferation at all levels, whereas the proliferative zone is extended up to the crypto-villous junction (Ivashchenko and Bykorez, unpublished). The assumption that some rapidly proliferating enterocytes, situated in the maximal proliferation zone, may probably have clonogenic properties was first confirmed by the findings of the experiments performed by Hageman and Lesher (197 l ) , who demonstrated that administration of HU prior to irradiation exerts considerable influence on the shape of the clonogenic cell survival curve, shifting it to the left, which seems to be in reasonably good correlation with the initial premises. In the work of Boarder and Blackett (1976) other S phase-specific cytotoxic agents were used and crypts' survival in mice was found to diminish by 60-80%, if they were irradiated either after administration of [ "]thymidine

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with high specific activity (('HJTdR-HSA) or of ara-C. The authors considered that the sharp decrease in survival rate of the crypts is in agreement with the data that a part of the clonogenic cells is the subpopulation of rapidly proliferating cells. Hageman (1980) pointed out that stem cells of intestinal crypts are the rapidly cycling population, as following synchronizing with HU phase-dependent radiosensitivity is revealed, which reaches its maximum in late G , and early S phases, and falls to its minimum in the middle of S phase. The alternative viewpoint is expressed by Hanson et a/. ( 1980) who believe that stem cells have a longer G , phase, and, in response to the HU effect, they enter the mitotic cycle being partially synchronized and after this pass through at least one more cycle. If HU was administered 15 minutes prior to irradiation, the survival curve remains unchanged, but if it was done 2 hours before irradiation, the survival curve is displaced to the left (the stem cells become more radiosensitive). When this period reaches 6 hours, the survival curve is displaced to the right. The modifications in radiosensitivity of the stem cells following exposure to HU suggest that clonogenic cells may have a short cell cycle, but in that case they would be killed by colchicine and ['HITdR-HSA. Therefore, it is more likely that HU causes rapid recruitment of stem cells from G, or extended G , phase into S phase. However, Potten (1980) failed to reveal any effect of pretreatment with HU or ["H]TdR-HSA on survival of clonogenic crypt cells after irradiation; this has been interpreted as an indication that the majority of clonogenic cells are not in the S phase. Phelps (1980) disclosed that the greater part of cryptogenic cells manifests high sensitivity to phase-specific cytotoxic agents, such as HU, vincristin, colcemid, or ara-C, and it suggests that they are in the rapid cycle.

2 . Quantitative Characteristic of the Stem Cells Withers and Elkind (1969, 1970) supposed that all proliferating cells of the intestinal crypts (about 130-150) can be regarded as stem cells. Hageman et al. (1971) reported that the number of stem cells in the crypt amounts to approximately 10. Hendry and Potten (1 974) tried to determine the number of stem cells by calculation of microcolonies following split-dose irradiation and achieved a value of 44 however, subsequently they obtained more accurate data, and arrived at the conclusion that crypts comprise 86 k 48 stem cells (Potten and Hendry, 1975). Other researchers (Masuda and Withers, 1977) in the course of study aimed at investigation of regeneration of crypts following fractional irradiation obtained data revealing the existence in crypts of about 140 stem cells, i.e., the supposition of Withers and Elkind that all proliferating cells of crypts can be considered stem cells was practically confirmed. The results obtained by Cairnie and Millen (1975). Potten and Hendry (1975), and Potten et al. (1979) enabled the authors to develop a hypothesis on the subdivision of crypt clonogenic cells into two classes: rapidly proliferating and

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slowly proliferating cells. Proceeding from this hypothesis the authors explained the high experimental quantitative values of stem cells by the fact that an experimentally ascertained number of clonogenic stem cells per one crypt represents the maximal value, as some of regeneratory microcolonies can probably originate also from non-stem cells. Under these conditions, when following irradiation, a considerable part of the stem cells is noted to die, some of the surviving rapidly proliferating cells, instead of continuing their differentiation and migration in an upward direction, can be transformed into functional stem cells. Additional data in favor of the above hypothesis are adduced by Yau and Cairnie (1979). The authors came to the conclusion that radiobiological experiments provide the possibility to establish only the number of potential stem cells, that is cells capable of clone formation, while the real number of functioning stem cells in nonirradiated crypts seems to be considerably less, and, probably, does not exceed 10%of the total number of all proliferating cells in crypts, which is found to be in good agreement with autoradiographic and electron microscopic data, regarding the amount of stem cells, as reported by Leblond and Cheng (1976). Potten et al. (1979) indicate that the best model to suit the data available is that of the small intestinal crypts, which comprises about 20 constantly functioning and slowly renewing stem cells and approximately 60 rapidly proliferating cells (situated above the localization zone of stem cells) with the capacity to form colonies and therefore constituting a pool of potentially stem enterocytes. According to calculations by Moore and Broadbent (1980) the quantity of clonogenic cells in the mice jejunal crypts equals approximately 60 cells (calculated from the cell survival curve following y-irradiation). Similar data on the amount of clonogenic cells are reported by Moore et al. (19821, that is 54 &? 29 However, the investigations performed by these authors with the view to estimate the number of clonogenic cells after adriamycin administration revealed that per one crypt there occurred only 1-2 cells. For this reason the supposition was made that populations of clonogenic cells are heterogeneous as regards their sensitivity to various damaging agents, and, for instance, a subpopulation of the most radioresistant clonogenic cells appears to be extremely sensitive to the cytotoxic action of some chemical substances (Dethlefsen and Riley, 1979). According to Cheng and Leblond (1974~)the enterocytes of the bottom and two prebottom positions are considered “functioning” stem cells. Taking into account the findings of radiobiologic experiments carried out by Potten and Hendry (1975), who found that about 80 cells in the small intestine crypts are clonogenic by nature, Al-Dewachi rt uf. (1979) also arrived at the conclusion that slowly proliferating cells in the basal 8-9 positions of the crypts include both “functional” stem cells (in two of the most basal positions) and “potential” stem cells. Bjerknes and Cheng (1981a) on the basis of a geometrical model and after detailed quantitative analysis of proliferation and differentiation of cells of various types have established that jejunal crypts comprise about 14 stem cells in the basal 4-5 positions.

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317

3. Impact of Age on Proliferative Activity of the Stem Cells To elucidate the discrepancies in data available on survival rate of crypts following exposure to irradiation, suppositions were made on the possible role of age differences in animals used for the experiments, and species of animals, and the influence of circadian rhythms on the population of stem cells (Potten et ul., 1979). Hanson et al. (1979) ascertained that animal age exerts considerable effect on the proliferative characteristics of clonogenic cells. Under conditions when rapidly proliferating committed cells die, the stem clonogenic cells manifest a gradual shortening in the cell cycle. This conclusion was made by Hanson et al. (1979), who observed that the survival rate of the clonogenic enterocytes following single irradiation is seen to decline with increase in time of animals' exposure to colcemide prior to irradiation. The insignificant decrease in survival rate of clonogenic cells after first and second colcemide injections is followed with a abrupt reduction in the number of surviving cells in animals, subjected to 3-7 injections of colcemide. Administration of four doses of colcemide results in a decrease of the total number of enterocytes in the crypts of the small intestine and the clonogenic cell survival curve was shifted to the left (i.e., reduction in number of clonogenic cells), while Do is almost diminished twice, when experiments were performed on 50-day-old mice. However, in the adult ( 1 10- 130 days old) similarly treated animals, in spite of the decrease in overall number of enterocytes, the survival curve position was not affected. Nevertheless, following 24-hour administration of colcemide the survival rate of clonogenic cells in these animals is noted to decline sharply. The similar effect is observed in the adult animals after administration of S-toxic agent that is of ['HITdR-HSA. If the clonogenic cells appeared to be the rapidly proliferating ones, then in this case the survival curve following [3H]TdR-HSA should have been shifted to the left. But the negative result is indicative of the fact that only a paucity of clonogenic cells is in the S phase during ['HITdR-HSA administration and the above can be regarded as indirect proof of the longer T, in the stem cells. The reduction in the survival rate of clonogenic cells 24 hours afrer colcemide injection can be associated either with the death of a part of the clonogenic cells in mitosis or with a decrease of cellular involvement in the radioresistant phases of the cell cycle compared with the amount of radioresistant cells following colcemide 12-hour administration. If prior to administration of colcemide to B6CF,/ An1 adult mice, they were exposed to irradiation with y rays or fast neutrons in doses under 50 rad then following application of a resolution dose of 1100 rad the survival rate of clonogenic cells also declined sharply (Hanson et al., 1980). Evidently low irradiation doses stimulate entrance of some clonogenic stem cells into DNA synthesis. In the young mice administration of three colcemide doses prior to irradiation proved to be sufficient to induce an abrupt decrease in the survival rate of the clonogenic cells. Thus, the cell cycle time of clonogenic cells in young animals

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is likely to be shorter than in the adult ones, and colcemide administration results in the death of the major part of the stem cells (Hanson et ul., 1979). The same authors demonstrated that in B6CF, adult mice administration of HU 15 minutes prior to y-irradiation had no effect on the survival rate of clonogenic cells, whereas the young mice of the same line following HU administration produced 3-fold reduction in the survival rate of clonogenic cells. Both these data and the results obtained after colcemide administration indicate that in young mice the clonogenic cells have a higher proliferation rate (T, is shorter and growth fraction is greater). In the light of these findings it is easy to see why in the experiments performed by Boarder and Blackett (1976), the [3H]TdR-HSA and ara-C manifested such high cytotoxicity in relation to the clonogenic enterocytes. The point is that the above authors carried out their experiments on 56- to 84-day-old mice.

111. Stem Cells of the Colon

A. THE ORIGIN OF VARIOUS DIFFERENTIATED CELLS Rats show substantial differences as to the cellular composition of crypts in various parts of the large intestine. In the ascending colon, the lower one-third of the crypts contains predominantly mucous cells (goblet cells and cells with small amounts of mucoid, i.e., oligomucous cells), whereas the upper one-third mainly has high columnar cells. In contrast, crypts of the descending colon contain only a small amount of mucous cells in basal positions (Shamsuddin and Trump, 1981a). It should be noted that the data concerning the structure of the epithelial lining of the lower portion of the colonic crypts in rodents are conflicting. It has been found that the basal portion of the colon crypts of the mouse contains vacuolated, mucous, columnar, enteroendocrine, and “caveolated” cells, the latter corresponding to fibrillovesicular, or tuft cells (Chang and Leblond, 1971a,b; Nabeyama, 1974). According to Nabeyama (1975), all these cells are not fully differentiated and pass through intermediate stages of differentiation from the vacuolated cells, which have the lowest level of differentiation, to various types of differentiated enterocytes. The vacuolated cells are more numerous in the basal two-thirds of the descending colonic crypts in the mouse whereas columnar cells are predominant in the upper one-third. In the course of their migration and proliferation, the vacuolated cells transform into columnar enterocytes. The differentiation of mucous cells is associated with the proliferating oligomucous cells that arise from the vacuolated cells with low level of differentiation (Chang and Leblond, 197la). The data concerning the renewal and origin of the enteroendocrine and caveolated cells from the vacuolated cells (Tsubouchi and Leblond, 1979; Tsubouchi, 1981) cannot be regarded as absolutely reliable.

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In the basal parts of the colonic crypts there have also been found cells with signs of cytodifferentiation of two types: some vacuolated cells contain mucous granules, and some vacuolated cells have endocrine-type secretory granules (Nabeyama, 19751, and the mucous-argentaffin cells (Corriveau et ul., 1974). However, these cells are very seldom seen and either are in the process of development into mono-specialized enterocytes or reflect possible anomalous ways of differentiation of stem enterocytes. According to Nabeyama (1975), it is impossible to find cells without signs of cytotypic differentiation in colon crypts, and the cells with the lowest level of differentiation (presumptive stem cells) are the vacuolated cells near or at the cryptal base. The studies of Shamsuddin and Trump (1981a) have shown, however, that the enterocytes of the rat colonic crypts can be divided into the following four main groups: columnar, mucous, enteroendocrine, and undifferentiated. Many authors have described the vacuolated enterocytes and considered them as stem cells, but Shamsuddin and Trump regard them as a subtype of mucous cells because their vacuoles, which remain unstained after periodic acid-Schiff (PAS) reaction, are stained with alcian blue and thus contain sialomucins. As revealed by electron microscopy, undifferentiated enterocytes showed no signs of secretory activity, had a high nucleocytoplasmic ratio, and contained moderate amounts of free ribosomes and fewer number of mitochondria, thus resembling the stem cells of small intestinal crypts (Shamsuddin and Trump, 1981a). The authors also observed the cells at intermediate stages of differentiation: from undifferentiated (stem) cells to all the abovedescribed types of mature enterocytes. The presence of undifferentiated enterocytes within the rat colonic epithelium was also reported by Pierce et al. (1977). They were cuboidal cells invariably lying on the basement membrane and having insufficiently developed rough endoplasmic reticulum (RER), a nonactive Golgi apparatus, and numerous free ribosomes. The apical end of these cells did not reach the lumina of the crypts, and they looked “walled-up” in the depths of the epithelial lining which made them resemble intraepithelial lymphocytes. Their lymphocytic origin was rejected, however, by the presence of desmosomes that connected them with the neighboring cells. These “undifferentiated colon cells” were unlabeled 2 hours following injection of [3H]TdR, thus representing a slowly proliferating population (Pierce et al., 1977). Taking their histochemical findings as a basis, Shamsuddin and Trump (1981a) classified the cells with the PAS-positive secretory granules, which are mucous according to Chang and Leblond (1971a), together with the sialomucinproducing vacuolated cells as mucous cells. However, Chang (1981) considers this concept to be ill-founded because, ultrastructurally, the sulfomucin-containing cells differ from the sialomucin-containing vacuolated cells in that the former have well-developed Golgi apparatus and RER, granular and fibrillar content of the mucous granules compared to the fine fibrillar content of the vacuoles. Chang

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(1981) has stated that the vacuolated cells are cytotypically less differentiated, and resemble the “undifferentiated cells” described by Shamsuddin and Trump (1981a). The vacuolated cells have been found in rectal crypts of mice, rats, rabbits, and humans. These cells have been described as sialomucin-containing nongoblet mucous cells in the rectosigmoidal colon of rodents (Wetzel et d., 1966). However, while having highly developed vacuoles in mice and rabbits, these cells are in structure very different in rats and humans. Chang (1981) believes that as the mucous cells migrate and differentiate, they transform into goblet cells, whereas the vacuolated cells, while migrating upward, transform into columnar cells and, while migrating downward, give rise to the mucous line of enterocytes (Chang and Leblond, 1971a; Chang and Nadler, 1975). According to Chang (1981), the main question which has to be answered is how the cells containing secretory vacuoles with sialomucins lose them as they migrate, and transform into absorptive columnar cells. Shamsuddin and Trump (198 Ic) raised a number of objections to classifying vacuolated cells as stem cells after Chang (198 1). The authors believe that the ability of the vacuolated cells to undergo division does not rule out a high degree of differentiation. Thus Shamsuddin and Trump (1981a,c) have no doubt that the colonic crypts of all mammals contain actual undifferentiated stem cells, although they admit that future studies will be needed for convincing morphological identification of stem cells. B. PECULIARITIES OF PROLIFERATION OF Low DIFFERENTIATED ENTEROCYTES The renewal of colonic epithelium has a number of peculiarities. In the small intestine, the proliferative zone occupies nearly two-thirds of the crypt, whereas in the large intestine, particularly in the descending segment, the proliferative zone is much shorter taking up not more than one-half of the length of the crypt (Pozharisski et al., 1977a). In crypts of the descending colon, the index of DNAsynthesizing cells at the basal positions differs only slightly from that in the maximal proliferation zone (Chang and Nadler, 1975; Pozharisski et al., 1977a). However, the issue of cell cycle duration heterogeneity of colonic enterocytes is controversial (Pozharisski et al., 1977a; Sawicki and Rowinski, 1980). As shown by Pozharisski and co-workers (1977a), the proliferating enterocytes of the colonic crypts, especially those of the bottom and basal positions, in all segments of the rat colon are significantly heterogeneous in respect to their T,. It is possible to detect 3 or 4 subpopulations having T , ranging from 11-12 to 30 hours and even more. As reported by Sawicki and Rowinski (1980), the crypts of the guinea pig ascending colon contain subpopulations in the upper and middle thirds of the crypts having short length of cell cycle (13-16 hours), subpopulations in the

GASTROINTESTINAL STEM CELLS

32 1

middle and basal thirds of the crypts having T , ranging between 22 and 26 hours, and a subpopulation of the crypt base having T, > 26 hours, with a part of the cells probably in the Go phase. However, as revealed by studies of the duration of phases and entire cell cycle with labeled mitoses method (FLM) for the enterocytes occupying different positions (Chang and Nadler, 1974; Appleton et a / ., 1980), these values do not differ significantly for enterocytes with different degrees of differentiation. For the low differentiated vacuolated enterocytes situated near or at the crypt base of the descending colon, only tcz is significantly longer than r,, of the upper enterocytes, whereas T, is equal for all the studied subpopulation; of the proliferating enterocytes-23.2 hours (Chang and Nadler, 1975), 15.5 hours in the descending colon, 21.2 hours in the transverse colon, and 18.9 hours in the ascending colon (Appleton et a / . , 1980). For the cells of the basal positions in the ascending colon t,, amounts to 3 hours, whereas for the cells near the upper border of the proliferation zone t,, equals about only 1 hour (Sawicki and Rowinski, 1980). According to the data obtained by Rijke and Cart (1979) and Sunter (1980), the T , of enterocytes of the rat descending colon is 50-60 hours, while the growth fraction within proliferative compartment approaches I .O. Having calculated the value of growth fraction (I,) for each position, Appleton et a/. (1980) showed that in all parts of the large bowel I, was considerably less than I .0 (the average value equals 0.5). It was found that after several doses of [3H]TdR unlabeled colonic cells were detectable only at the crypt base, whereas 10-15 days later a number of labeled cells in this zone was much greater compared with the middle and upper segments of the crypts (Pozharisski et ul., 1977b). All these results indicate that the stem cells both in the large and small intestinal crypts occupy the bottom and basal positions. It has been shown that the division of stem enterocytes occurs in two basal segments if, conventionally, the crypts are divided into 10 segments (Chang and Nadler, 1975). The descendants of the stem cells undergo another mitosis within 3-7 segments after which they stop proliferating and migrate upward to the zone of differentiated cells. The mucous cells preserve the capability to proliferate in segments 1-6. Like columnar cells, they go through two mitotic cycles (within proliferative zone). The number of mitotic cycles for each type of cells depends upon where the undifferentiated uncommitted cell transforms into a specialized enterocyte. If, prior to this moment, the low differentiated cell has passed through 1 mitotic cycle and is located within segment 1 , the mucous enterocyte, while migrating within segments 2-6, will divide twice more. If the transformation occurs within segments 2-4, the daughter cells will divide only once before leaving the proliferative zone, but the undifferentiated precursor cells will have by then undergone two divisions. Therefore in any case the cells of each type, when differentiating and maturing from stem cells to nonproliferating specialized cells, in most cases go through three cycles. The only exception are the enteroen-

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docrine cells: low differentiated cells of this type pass through 1-2 mitotic cycles (Chang and Nadler, 1975). When migrating and differentiating in the guinea pig colonic crypts, the cells undergo approximately 7 divisions (Sawicki and Rowinski, 1980) whereas in the jejunum of mice and rats these numbers are 4 and 14, respectively (Leblond and Cheng, 1976; Pozharisski et al., 1975).

c. QUANTITATIVE CHARACTERIZATION OF STEM CELLS Quantitative studies of cryptogenic (clonogenic, colony-forming) cells of the descending colon mucosa resulted in important findings. According to the data published by Hamilton (1979), the crypts of the descending colon contain, on average, 2.09-2.60 cryptogenic cells per crypt, the maximal number of cryptogenic cells contained in one crypt being 1 I . The author found that many crypts began to regenerate not from the basal segments where, according to some data, the stem cells are located. From this study Hamilton (1979) concluded that the precise localization of the cryptogenic cells is, at the moment, unlikely to be possible. Hamilton ( I 979) believes that the most probable site of localization of the cryptogenic cells is the space between the ninth and the eighteenth cell positions (taking the cells located at the crypt bottom as position 1). In this case, however, it is very unlikely that cryptogenic cells are capable of performing the functions of stem cells since the crypt epthelium above the base is transitional. It has been shown that radiosensitivity of crypts varies in the course of time (Hendry, 1975). The survival rate of the crypts following irradiation is seen to be minimal when the proliferation indices are the highest (Potten e t a / ., 1977) which suggests synchronous movement of clonogenic cells through the mitotic cycle as there is good evidence for minimal radiosensitivity in S phase and maximal in G, and M phases. The existence of partially synchronized populations in colonic crypts has been revealed (within the limits of the ninth and twenty-eighth positions) but the data available are not sufficient for the alternative explanations to be either finally accepted or refuted (Hamilton, 1980): ( I ) cryptogenic cells are located between positions 9 and 12 and proliferate slowly; (2) these cells are distributed among the amplifying cell populations situated in cell positions 1 to 9 and proliferate with the same speed as nonclonogenic cells; and (3) the sensitivity of existing techniques is insufficient for registration and examination of only 2 or 3 cryptogenic cells dispersed among 300-400 other crypt cells. Having assumed that the stem cells are localized in basal segment of the crypts, Sawicki and Rowinski (1980) estimated that there may be I to 3 stem cells per one crypt provided the parameters of their proliferation (/,, T,, t,) equal 2.5%, 26 hours, and 7.5 hours, respectively, and the total amount of stem and non-stem cells in basal parts of guinea pig crypts of the ascending colon is 15 to 30. Therefore it can be concluded that the data obtained thus far are not sufficient for a reliable identification of cryptogenic cells with any subpopulation of pro-

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liferating cells. Using various techniques of analysis of clonogenic cell survival curves after irradiation, Yau and Cairnie (1979) found that the number of stem cells per one crypt ranges from 3 1 to 170 depending on the mathematical model and the method of statistical analysis of experimental data used. Therefore, these authors made a very important conclusion: radiobiological experiments of this kind cannot provide a final answer to the question of the number of stem cells per one crypt.

IV. Stem Cells of the Gastric Glands A. CARDIAC GLANDS The cardiac glands consist mostly of two types of cells: cardiac mucous cells (producing neutral mucosubstances) and cells of surface-foveolar epithelium, which form the foveolar portions of cardiac glands. The cardiac mucous cells as to their characteristic features, including the ultrastructural peculiarities of organelles and secretory granules, resemble the mucous neck cells of the fundic glands. In the cardiac glands opening into the groove lumen, there are cells of a particular type, the so-called cardiac serous cells (Wattel and Geuze, 1978), the existence of which is questioned by other researchers (Lee el a / ., 1982). The undifferentiated cells in cardiac glands are localized on the border between zones of mucous cells and foveolar epithelium. In the same zone of cardiac glands there are also present immature mucous, foveolar, and serous cells. However, 1 hour after the rats were treated with [‘Hlthymidine, only undifferentiated cells or cells starting to differentiate (immature cells) appeared to be labeled, whereas no labeling was observed either in mature mucous or in serous cells (Wattel and Geuze, 1978). The fibrillovesicular cells, which have been described in the gastrointestinal mucosa by various authors under different names such as “tuft” cells (Isomaki, 1973), “nondifferentiated” (Johnson and Young, 1968), “S” cells (Ferguson, 1969), and fibrillovesicular cells (Hammond and Ladeur, 1968), are especially numerous in the cardiac glands and in the rat gastric groove. These cells were never seen to incorporate [’Hlthymidine. These facts permited Wattel and Geuze (1978) to conclude that all types of differentiated cells in cardiac glands arise from a common undifferentiated precursor. B . FUNDICGLANDS 1. Characteristic of Low Differentiated Cells-Presumable Stem Cells The cellular composition of the proliferative zone in the fundic glands appears to be rather heterogeneous. One hour after [3H]TdR administration the labeled cells were observed in the foveolar epithelium, as well as in separate mucous

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neck cells and in undifferentiated isthmic cells, the paucity of which is revealed by electron microscopy (Wattel et a l . , 1977). However, it should be noted that data concerning the low differentiated cells, their localization, and renewal appear to be conflicting. The fibrillovesicular cells, which Johnson and Young (1968) described as low differentiated cellsprecursors of all other types of cells in the gastric glands, turned out to be highly differentiated specialized cells. The true low differentiated cells in the gastric glands were found by a number of researchers (Rubin et a / . , 1968; Kataoka, 1970; Tahara, 1971; Nabeyama, 1975; Uchida et a / . , 1977; Wattel et a / . , 1977; Leblond and Lee, 1979). These cells are described under various names: “immature mucous neck cells” (Corpron, 1966), “young, actively proliferating cells” (Shibasaki, 1961), and “primitive stem cells” (Helander, 1969). As Helander (1969) points out these problems are purely terminologic, whether to define these cells as “primitive stem cells” or low differentiated mucous cells. According to Wattel et a/. (1977) in the fundic glands of rat stomach mucosa the undifferentiated cells are concentrated in the isthmic area. Applying electron microscopic autoradiography the authors were able to show that only undifferentiated isthmic cells, low differentiated foveolar cells, which are also situated in the isthmic zone, and neck mucous (with different levels of differentiation) cells can incorporate [3H]thymidine. In the course of electron microscopic autoradiography only 8 cells out of 84 labeled isthmic and neck cells appeared to be the cells without signs of cytotypical differentiation, and which, probably, could be regarded as stem cells of the fundic glands (Wattel et d., 1977). The main characteristic features of undifferentiated cells are the absence of secretory granules, insufficiently developed rough endoplasmic reticulum as well as the Golgi apparatus, scarcity of mitochondria, and abundance of free polyribosomes. It is also pointed out that in undifferentiated isthmic cells of the fundic glands, the nucleocytoplasmic ratio seems to be considerably higher than in all other epithelial cells of the glands (Tahara, 1971; Uchida et al., 1977). The investigation of undifferentiated cells is hindered by the fact that they are located amid substantially larger sized parietal cells, and, because of this, the cytoplasm and nucleus of undifferentiated cells are found to be strongly deformed. 2 . Heterogeneity of Proliferating Cells of Gastric Glands in Regard to Cell Cycle Time In Table I kinetic parameters of cell populations of neck-foveolar epithelium in fundic and pyloric glands of rat stomach are given. The data presented indicate that most distinct differences in time parameters of populations analyzed are seen in the length of S phase and entire mitotic cycle. In the course of comparison of cell cycle parameters, determined with the FLM method and CLI (cumulative labeling index curve), facts were obtained indicative of heterogeneity in populations of proliferating cells in fundic and pyloric glands as regards the cell cycle

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TABLE I CELLPOPULATION KINETIC PARAMETERS OF PYLORIC A N D FUNDIC GLANDSI N INTACT RATS" Neck-foveolar epithelium Parameters of cell population kinetics

Pyloric glands

Mitotic index (%) Index of DNA-synthetizing cells for entire population (I,, %) Index of DNA-synthetizing cells in zone of maximal proliferation (5%) Duration of mitosis, t,,, (hours) Duration of S phase, I,(hours) Duration of G 2 phase. I ~ (hours) , Cell cycle time, measured from FLM curve (hours) Cell cycle time, measured from cumulative labeling index curve Cell cycle time, estimated by stathinokinetic technique (hours) Growth fraction calculated from a labeling index distribution curve ([p, 9 ) Growth fraction calculated from proportion cycle paranieters and labeling index:

7.2 ? 1.5 6.8 2 0.8 22.4

?

3.5

0.8 7.6

Fundic glands 4.2 4.0

2 ?

0.66 0.6

12.2 2 3.7 1.1

19.0 31.6

10.0 0.95 30.0 55.0

19.2

27.8

20.0

15.0

16.3

12.0

0.8

"Based on data from Bykorez and Ivashchenko (1982a)

time. It was found that T, value for fundic glands determined by CLI amounts to = 55 hours and for pyloric glands it equals 30 hours. Taking as a basis the results of CLI and FLM analysis one can come to the conclusion that in the zone of maximal proliferation of pyloric glands all cells are actively proliferating (growth fraction I , = 1 .O) and it is possible to distinguish conditionally two subpopulations in relation to cell cycle time: with T, = 16-19 and = 30 hours, respectively. The analysis of the CLI for the zone of maximal proliferation of fundic glands permits the conclusion to be drawn that heterogeneity of proliferating cells in respect to their T, is higher in these glands (T, ranges 28-55 hours) than that in the pyloric ones. It is, therefore, likely that a part of the cells located in the zone of proliferation may have a considerably longer cell cycle time or they are in a state of relative rest ( I , 1 .O). The above data enable us to conclude that in the basal layers of the proliferative zone in gastric glands there are concentrated undifferentiated stem cells, exhibiting considerably longer cell cycle time than their descendants in the zone of maximal proliferation. The subpopulation of stem proliferating cells in the pyloric glands

*

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displays an important special feature as compared with the fundic ones, that is, their mitotic cycle is shorter. And if in the pyloric glands the major part of stem cells is evidently proliferating, then in the fundic glands a substantial number of stem cells are probably in a state of relative rest. Masuda el a/. ( 1978), employing the technique of plotting and analysis of survival curves for the clonogenic cells following fractional irradiation and taking into account data reported by Chen and Withers (1972) on the relationship dose (after single irradiation)-survival rate of stem cells in the gastric mucosa have calculated that in the mice stomach one gland comprises approximately 21 stem cells. The gastric stem cells were found to be most radioresistant as compared to the stem cells of small and large intestinal crypts (Masuda and Withers. 1977). Following a single HU treatment the first poorly labeled cells in pyloric glands occurred primarily within 4 positions, adjacent to the body. that is precisely in the zone of localization of the least differentiated cells. The fundic glands 4.5 hours after HU administration exhibit occasional poorly labeled DNA-synthetizing cells, which are localized in the deep portions of the foveola. No labeled cells were observed amid highly differentiated cells (chief, parietal, and cells of the pyloric body). The periglandular fibroblasts also remained unlabeled (Ivashchenko et a / ., unpublished). The first labeled cells to appear in the pyloric glands 4 hours after administration of 5 HU doses (with 3 hour intervals) were also the cells of the most basal isthmic positions, adjacent to the body. In the fundic glands the DNA-synthetizing cells, observed 4 hours later, were poorly labeled, and though the I , value was close to the control one, that is 3.2 ? 0.8%, the spatial distribution of the labeled cells differed significantly from the normal one. Thus, if in normal fundic glands 1 hour after [3H]TdR administration 85% of the labeled cells are noted to be the foveolar (isthmic) cells, and only 15% are represented by low differentiated neck cells, then after 5 doses of HU the low differentiated neck cells appear to be the first to start synthesis of DNA, and their proportion among labeled cells amounts to 84.6 +- 4.6%, whereas the quantity of foveolar labeled cells reaches only 15.6 f 1.8% (Ivashchenko et a / ., unpublished).

3 . Differentiation of Mucous Cells In the region of isthmus a special type of cell has been described which can be regarded as transitional forms, that is, the cells being in the process of differentiation either to foveolar or to mucous neck cells (Spicer et al., 1978). The differentiation and maturation of foveolar cells start in the isthmic zone and are linked with proliferation and migration of some of the undifferentiated cells’ descendants in the direction to the foveolar mouth. Thus, some isthmic cells incorporating L3H]thymidine demonstrate a small number of secretory granules along with an increase in RER cisternae number, compared to undifferentiated cells. Among cells incorporating [3H]TdR and situated in the isthmus of fundic glands, the majority of cells exhibit these special features and thus can be

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considered as immature cells differentiating in the direction of foveolar epithelium (Wattel et a l . , 1977; Spicer et al., 1978). The differentiating immature mucous neck cells, containing characteristic secretory mucous granules, are found in the immediate vicinity of the isthmic lower border (or neck upper border), though the number of such cells is significantly less than that of immature foveolar cells (Tahara, 1971; Wattel et al., 1977) which is probably associated with a considerably longer duration of life cycle in the mucous neck cells. Besides, not only immature cells, but also mature highly differentiated mucous neck cells are capable of proliferation, and therefore it seems that there is not much need for an influx of young low differentiated cells to the neck zone. It has been suggested that with maturation the mucous neck cells migrate downward, i.e., in the opposite direction in relation to foveolar cells (Kataoka, 1970; Hattori and Fujita, 1976). We have shown that in the neonatal rats epithelial conglomerates in the gastric mucosa are composed of low differentiated cells and some of these cells contain mucous secretory granules, the ultrastructure of which is similar to that of secretory granules of the surface epithelium. But in contrast to the highly differentiated surface cells, the secretion of which is distinctly PAS-positive, the secretion of low differentiated cells appears to be stained following paradoxical Con A-staining according to Katsuyama and Spicer (1978), which is indicative of identity between these cells and mucous neck cells in adult animals. Therefore, in the process of ontogenic development of the stomach glandular apparatus the differentiation of mucous cells of foveolar and neck types also appears to be spatially dissociated. The cells with secretory granules of both types, neck and foveolar, were observable in electron microscopic study of fundic glands in rats and mice (Tahara, 1971; Wattel et al., 1977). It is rather doubtful whether such cells can be considered as an intermediate stage of cell differentiation from foveolar to mucous cells, or vice versa, as their number is considerably less than the amount of low differentiated cells with scarce secretory granules of either type, namely of foveolar or neck cells. For this reason, it is most likely that all types of mucous cells originate from undifferentiated stem cells, located mainly in the isthmic zone of fundic glands. And cells exhibiting signs of cytotypic differentiation characteristic of both neck and foveolar epithelium can probably be regarded as a particular cellular type. Spicer et al. (1978) believe that similar cells have their own particular specialization, as they are noted not to harbor their secretion in the cytoplasm for a long time, but release it at once in the glandular lumen. Wattel et al. (1977) indicated that such cells, if the necessity arises, can serve as an additional source for replenishment of populations of both foveolar and neck cells, as they preserve their proliferative capacity. 4. Renewal of Parietal and Chief Cells

The problem of renewal of highly differentiated cells in the stomach glands even nowadays attracts much interest on the part of researchers, but so far it

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remains a controversial issue. In numerous publications it has been pointed out that parietal and chief cells are incapable of mitotic division and their renewal is associated with differentiation of proliferating cells in the regeneration zone of fundic glands (Hunt and Hunt, 1962; Lipkin et al., 1963; McDonald et a / . , 1964). Some authors, however, believe that parietal cells are able to synthetize DNA and undergo mitosis (Timashevich, 1978; Chen and Withers, 1975). It is worth mentioning here that only Harm (1910) reported that he had observed the mitosis of parietal cells, though none of the succeeding researchers could reveal the same phenomenon (Ragins et al., 1968; Willems et a / ., 1972; Willems and Lehy, 197.5). It has been demonstrated that only 0.02% of parietal cells appear to be labeled following the single administration of ['Hlthymidine (Chen and Withers, 1975). The other investigators (Willems et al., 1972; Willems and Lehy, 1975; Wattel et ul., 1977; Bykorez and Ivashchenko, 1982b) disclosed no DNA-synthetizing parietal cells at all, whereas following administration of 6 ['Hlthymidine doses with intervals of 8-14 hours, 1.8-2.2% of parietal cells were found to be labeled (Ragins et a / . , 1968; Willems and Lehy, 1975). By day 7-15 after the last ['Hlthymidine injection, their number increased up to 4-6% (Willems and Lehy, 1975; Bykorez and Ivashchenko, 1982b). Such rapid increment in the number of labeled parietal cells with such a low index of DNAsynthetizing cells cannot be explained only by the intrinsic mitotic activity of these cells. Both these data and the findings of electron microscopic study, confirming the existence of immature forms of parietal cells in the proliferation zone among low differentiated glandular neck cells (Hattori, 1974; Kaku, 1966; Lawn, 1960; Tahara, 1971; Wattel et al., 1977), can serve as proof that renewal of the parietal cell population takes place due to their differentiation from low differentiated descendants of the stem cells. The majority of researchers hold the view that the chief cells exhibit the capacity for proliferation and these authors characterize this population as capable of self-maintenance (Willems et a/., 1972; Willems and Lehy, 1975; Pansu et a / ., 1977). However, researchers investigating the mitotic activity of the chief cells((Zimmerman, 1925; Creamer et a / ., 196 I ; Hunt and Hunt, 1962) suggested that chief cells are renewing by means of differentiation from the neck mucous epithelium, having failed to reveal the chief cells undergoing mitotic division. At the same time electron microscopic study showed no transitional-type cells between immature neck cells and chief cells (Rubin et d., 1968; Chen and Withers, 1975). One hour after administration of ['Hlthymidine the labeled chief cells could be seen both in the vicinity of the neck and in deep portions of the glandular body (Myhre, 1960; Willems et al., 1972; Chen and Withers, 1975; Willems and Lehy, 1975). However no labeled chief cells were found in electron microscopic autoradiography (Wattel et al., 1977). In the process of ontogenetic development the chief cells occur later than all other types of differentiated epithelial cells (Tatematsu et al., 1975). The find-

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ings of our studies indicate that during the first 3-5 days of postnatal development in the forming fundic glands of rat stomach the basal parts are composed of nondifferentiated cells, cells with signs of mucous neck type (characteristic granules revealed by ultrastructural study and by specific staining pattern after paradoxical Con A reaction), and low-differentiated cells, which, we believe, can be regarded as committed precursors of the chief cells. There are some facts to support the above statement: the amount of rough endoplasmic reticulum in the latter cells appears to be considerably higher than in the neck and foveolar cells; the secretory granules differ from the mucous ones, though they do not correspond to pepsinogen granules seen in mature chief cells; and some of the cells display PAS-positive secretion in the form of 1-2 granules (on semithin sections). It is unlikely that processes of differentiation of the chief cells are linked with the disappearance of mucous secretion from the mature highly differentiated mucous neck cells. If it were the case, then it would be logical to assume that during the first days of the neonatal period the developing body of fundic glands would be composed only of the neck mucous cells. For this reason we maintain that chief cells arise in the following sequence: ( 1 ) proliferation of undifferentiated descendants of the stem cells, committed toward differentiation into chief cells; (2) increase in amount of rough reticulum cisternae in these cells and synthesis of occasional granules of propepsinogen and mucoid under the conditions of continuing proliferation; and (3) activation of pepsinogen synthesis and abrupt retardation of proliferation (lvashchenko et a / ., unpublished). The presence of a small amount of mucous secretory granules in low differentiated cells of the stomach glands, as well as in some of the differentiated cells (chief, parietal, endocrine) indicates that commitment of stem cells either under the influence of inductors or caused by expression of some unknown endogenous determinants most probably occurs in the direction of mucous-producing cells, especially as the latter are seen to be the predominant cellular type for all types of gastric glands. There is a series of convincing arguments that differentiation of the chief cells is not associated with modulation in the differentiation of neck mucous cells. The cells, similar to the neck mucous ones, are produced in the intermediate (mucoparietal) and pyloric glands, but the chief cells are not seen to arise. In pseudopyloric metaplasia of the fundic glands the chief cells are replaced with the neck mucous ones, and the occurring changes appear to be of a stable nature. In this case no pepsinogen 1 (characteristic for chief cells only) is found in metaplastic glands, but there is preserved production of pepsinogen 11, characteristic of both neck mucous cells (Lechado et a / . , 1976; Samlof, 1982) and glandular cells of pyloric glands and epithelium of the Brunner’s glands. Thus, it is likely that for differentiation of stem cells to chief cells, extremely specific microenvironmental conditions are needed, created only in case of balanced concentrations in the glands of neck mucous, parietal, and endocrine cells. For

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all these reasons, after various damage to the fundic glands’ epithelium, the chief cells are substituted for the mucous ones, as there exists, probably, fixed ontogenetic priority in the commitment of the stem cells in the direction of mucoidproducing cells.

C. PYLORICGLANDS In the pyloric glands of the rat gastric mucosa one can distinguish the following types of mucous-producing cells: surface-foveolar mucous cells, low differentiated mucous cells of the isthmic zone, and glandular pyloric cells of the body (Bykorez and Ivashchenko, 1982a; Lee et al., 1982). The surface-foveolar epithelium of pyloric glands is not seen to differ qualitatively from corresponding cells of fundic glands, but the quantity of foveolar cells per one pyloric glands is higher than in the fundic glands. As regards the stem cells in pyloric glands, they are thus far scantily explored. So, for instance, Nabeyama (1975) has found that even the least differentiated cells of pyloric glands, localized in the isthmic zone, display a small amount of mucous-type granules. In the isthmus of pyloric glands the number of cells without signs of cytotypic differentiation is significantly less than that of low differentiated cells with occasional secretory small-sized granules. On the basis of electron microscopic examination Leblond and Lee (1979) distinguish the following cell types in the isthmic zone: ( 1) nondifferentiated, (2) immature foveolar, (3) immature mucous glandular, and (4) intermediate (with secretion granules of foveolar and glandular type), which are more numerous than nondifferentiated cells, but less numerous than immature cells of foveolar and glandular types. All cells of the above types are capable of mitotic division, including also immature endocrine cells, originating either from immediate descendants of the stem cells or from immature mixed mucous-endocrine cells (Tahara, 1971 ; Nabeyama, 1975; Ratzenhofer and Aubock, 1980; Bykorez and Ivashchenko, 1982a). D. DIFFERENTIATION AND RENEWAL OF ENDOCRINE CELLS

The identification difficulties and slow rate of renewal of the majority of enteroendocrine cells lead to a discrepancy in facts available on the ways and cellular sources of the physiologic regeneration of cells of the diffuse neuroendocrine system. The works of Leblond and co-authors dealing primarily with the renewal of enterochromaffin cells and performed with employment of electron microscopy, enabled the authors to draw the conclusion that endocrine cells have no capacity for self-replication, and that endocrine cells, along with all other types of differentiated exocrine cells, are derived from the common stem cells (Ferreira and

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Leblond, 197 1; Cheng and Leblond, 1974b; Tsubouchi and Leblond, 1979). This view is also supported by other investigators (Sidhu, 1979; Bykorez and Ivashchenko, 1982a; Hattori et al., 1982a; Prdks and Felt, 1982; Tahara et al., 1982). However, the DNA-synthesizing cells were described among the argentaffin cells in human rectal mucosa (Deschner and Lipkin, 1966) and mice duodenal mucosa (Odartchenko et al., 1970). gastrin-producing cells in stomach pyloric glands of mice (Lehy and Willems, 1976) and hamsters (Fujimoto et a l ., 1980). But despite the presence of scarce labeled [3H]TdR DNA-synthesizing G cells, Fujimoto et al. (1980) suggested, that the major part of G cells originates from immature precursors cells. At the same time Lehy and Willems (1976), admitting the existence of a mechanism which ensures the origination of G cells through differentiation, believe that the majority of cells in this population are capable of self-renewal. The autoradiographic study (following single and multiple administration of ["H]TdR) of sections of gastric walls in rats, mice, and guinea pigs, with immunocytochemical detection of D cells, revealed (Lehy, 1982) that somatostatinproducing cells of pyloric glands also exhibit a capacity for DNA replicative synthesis and mitotic division. The number of DNA-synthetizing D cells approximates the amount of labeled G cells. The special study of the endocrine cells in the process of regeneration in the pyloric and fundic parts of the rat gastric mucosa showed that endocrine cells originate from the proliferating low-differentiated cells-common sources of epithelium regeneration (Hattori et al., 1982b). The existence of common stem cells for exocrine and endocrine-paracrine epithelium is supported by the fact that during regeneration amphicrine cells (cells with endocrine and zymogenic or mucous granules (Tahara, 1971; Hattori et al., 1982a); in the cases of chronic gastritis and intestinal metaplasia mucoargentaffin and mucoargyrophil cells were found, while in appendix carcinoids and in regenerates located in the vicinity of ulcer mucoargyrophobic cells were seen (Ratzenhoffer and Aubock, 1980). The endocrine-exocrine cells are observed mainly while exploring the gastrointestinal mucosa, which, for some reason, manifests an increased proliferation rate. However, in the normal epithelium, the amphicrine cells are rarely encountered, namely in the mouse large intestine (Nabeyama, 1975) and small intestine (Cheng and Leblond, 1974c) and in the gastric glands of rats (Capella et al., 1971), mice (Kataoka, 1969; Nabeyama, 1975), and man (Ratzenhofer and Aubock, 1980). The existence of a small population of mixed cells reflects the variability in morphogenetic potentialities (plasticity) of stem cells and indicates that processes of endogenous determination (commitment) are preceded with the peculiar stage of "dichotomous selection. Evidently, only separate descendants of the stem cells, being exposed to the equal effect of various differentiation stimuli, are unable to choose some particular way of development. "

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V. Regulation of Proliferation and Differentiation in the Gastrointestinal Epithelium The regulatory mechanisms of stem cells’ proliferation in the gastrointestinal mucosa under physiologic conditions and, particularly, when exposed to the action of toxic or carcinogenic agents are far from being finally clarified. Nevertheless, to date there is a range of disclosed and in part elucidated factors, interfering with the processes of epithelium renewal in crypts and glands. Naturally, one has to admit that in the majority of cases the target cells are poorly characterized, especially as regards their receptors for different gastrointestinal and other hormones. Only some of the factors discussed below exert a direct effect on the stem cells, but the involvement of stem cells in both physiological and pathological processes is indubitable. Below we shall present data both on exogenous regulatory factors and on certain mechanisms of intrasystemic local control, despite the fact that the role of information transmitters, acting within the latter system, is only speculative. A. SYSTEMIC MODULATORS OF CELLULAR PROLIFERATION 1 . Significance of Nutrition

During complete or partial fasting the number of proliferating enterocytes is rapidly decreasing, the migration is retarded, the cell cycle is lengthened, and hypermature columnar enterocytes are seen to appear (Hopper et al., 1972; Levine et al., 1974; Al-Dewachi et al., 1975a; Hagemann and Stragand, 1977; Koga and Kimura, 1979). The total parenteral feeding prevents the development of postresectional hyperplasia in the mucosa of the intestinal remnant in rats and dogs (Feldman et al., 1976; Levine et al., 1976; Morin et al., 1978). Evidently, such effects of nutrition are realized through trophic factors, contained in the food, and especially in saliva (e.g., EGF, NGF, gastrin, glucagon, Barka, 1980). 2. Neurovuscular Factors Norepinephrine is known to enhance the cell proliferation in the stomach and a-adrenoblocking agents eliminate this effect, whereas the impact of epinephrine, inhibiting the proliferation, is suppressed by P-adrenoblockers (Tutton and Helme, 1974). The cholinergic drugs and electrostimulation of the mesenteric nerves enhance the mitotic activity, whereas sympathectomy leads to its inhibition (Tutton, 1977).

3. Hormonal Factors Estrogens, stimulating proliferation in the target organs, exert an inhibiting effect on intestinal epithelium proliferation, inducing partial block of G , 4, which appears to be longer after single dose administration than following re-

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peated treatments, thus resembling the tachyphylactic effect (Chang and Hoff, 1980). Furthermore, the duration of S phase of the proliferating cells in duodenum, jejunum, and large and small intestine of ovariectomized animals was significantly reduced (Galand et al., 1967); that, however, cannot be regarded as proof of proliferation acceleration, though probably it may serve as one of the estrogen action alternative ways, modulating the cell cycle in the organs, which are not their direct targets (Hoff et al., 1981). The existence and quantitative assessment of estrogenic receptors in the epithelial cells of gastrointestinal tract thus far remain a controversial issue, though there are data available supporting the presence of such receptors (Alford et al., 1979) in the normal enterocytes and even in the cells of large bowel carcinomas. However, because of scarce evidence, no definitive conclusion can be drawn on the role of estrogens in regulation of proliferation of normal and neoplastically transformed enterocytes (Agrez and Spencer, 1982). Androgens. The stimulating effect of androgenic hormones on proliferation in the small intestinal mucosa has been described in many publications (Tuochimaa and Niemi, 1968; Wright er al., 1972b). The large intestinal cells display a low level of androgen receptors, while it is seen to be considerably higher in the cells of colonic tumors, induced by 1,2-dimethylhydrazine (Mehta et a l . , 1980). Testosterone may enhance proliferation in the jejunal epithelium while it fails to affect the large intestinal epithelium proliferation-a difference which is so far hardly explicable (Tutton and Barkla, 1982). Hydrocortisone induces partial G , -S block in the mice forestomach epithelium and stimulates differentiation of low differentiated isthmic cells to chief cells (Frankfurt, 1968; Tatematsu et al., 1975). Prednisolone causes an increase in intracellular content of prostaglandins, exhibiting a cytoprotective effect (Derelanko and Long, 1982). B.

PARACRINE

REGULATION

The gastrointestinal peptide hormones are believed to play a particular role in the regulation of proliferation of gastrointestinal epithelium in mammals. These hormones are secreted by the paracrine-endocrine cells of the diffuse neuroendocrine system. 1 . Gastrin

Gastrin stimulates epithelium proliferation in all portions of the gastrointestinal mucosa (Johnson, 1976, 1977a, 1979). The gastrin-producing cells (G cells) in the pyloric glands of the stomach and G cells of the duodenal crypts serve as a major source of gastrin secretion. The exogenously administered gastrin or pentagastrin are seen to enhance the proliferation of low differentiated cells in the gastric fundic glands, and, probably, cause an increase in the number of progenitor cells differentiating into parietal cells (Willems and Lehy, 1975),

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as well as in the number of proliferating cells in pyloric glands (Lehy rt a / . , 19791, duodenum, large intestine, and pancreas (Mainz et a / . , 1974; Johnson rt ul., 1975; Mak and Chang, 1976; Johnson, 1977a). However, there are data available indicating that gastrin not only fails to stimulate the synthesis of DNA and proteins in the pyloric glands (Johnson, 1976), but it may even inhibit proliferation there (Casteleyn et a / ., 1977). The insufficient endogenous production of gastrin following antrectomy results in development of mucosal hypoplasia in all portions of the gastrointestinal tract (Dembinski and Johnson, 1979). It is essential to note that gastrin infusion or its repeated injections enhance DNA synthesis in the gastric mucosa of adult animals, but fail to produce a similar effect in neonatal animals or sucklings (Takeuchi et d.,1981). The gastrin trophic effect on the rat glandular epithelium is not revealed until the twentieth day of postnatal growth which reflects the appearance of receptors for gastrin on the target cells in the gastric mucosa around the twentieth day (Majumdar and Johnson, 1982). In fact, only by day 15-20 of life does the abrupt activation of mucosal growth occur in the stomach and intestine (Al-Nafussi and Wright, 1982a). However, the proliferation rate in the epithelium of stomach and intestine is rather high in the period preceding the twentieth day, which is indicative of the fact that apart from gastrin other stimulators of proliferation also exist during the early developmental period.

2. Glucagon Glucagon has been shown to stimulate proliferation in the fundic glands and large intestine, though its effectiveness reached only 40% of pentagastrin activity (Johnson, 1977b). 3. Secretin and Vasoactive Intestinal Peptide (VIP) Secretin and VIP manifest inhibitory action in relation to gastrin trophic effect (Johnson and Guthrie, 1974; Johnson, 1977b). VIP is considered to be the principal stimulator of the Brunner’s glands secretory activity controlled by VIPcontaining nerve terminals (Kirkegaard et a l . , 1981).

4. Somatostatin Konturek et al. (1981) pointed out that somatostatin might enhance the DNA synthesis in the fundic glands of rat stomach. However, as Lehy et al. (1979) reported, the somatostatin prolonged infusion has caused considerable inhibition of low differentiated cell’s proliferation rate in the fundic and pyloric glands, while in the duodenal and jejunal epithelium, somatostatin exerts less pronounced antiproliferative effect. Its inhibitory impact appears to be rather shortterm, about 4 hours, which together with peculiarities of the mechanisms’ action (inhibition of cell proliferation by blocking cell movement through the cell cycle on the boundary of S-G, and G,-M phases) is rather similar to the effect of

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chalones. The somatostatin inhibitory influence is observed to be longer in proliferating cells, stimulated by gastrin (Lehy et al., 1979). The studies are now underway to elucidate the distribution of somatostatin receptors on the gastric epithelial cells as well as the mechanism of somatostatin action (Reyl et al., 1979; Reyl and Lewin, 1982).

5 . Epidermal Growth Factor (EGF) EGF is noted to enhance DNA and RNA synthesis in the epithelium of fundic glands in rat stomach (Schewing et al., 1979; Johnson and Guthrie, 1980), to stimulate ornithine decarboxylase activity in the gastric and duodenal epithelium of 8-day-old mice, and decarboxylase in its turn activates RNA polymerase 1 (Feldnian et al., 1978). Administration of EGF in a dose of 20 pg/kg exerts the same stimulating effect on proliferation as pentagastrin, though it is limited to the stomach niucosa, and appears to be less marked in the duodenum and large intestine (Johnson and Guthrie, 1980). Somewhat controversial data were reported by Al-Nafussi and Wright (1928b) who found no acceleration in cell production in gastric glands of mice and rats following administration of six doses of EGF (i.e., with the same regimen of administration as described by Johnson and Guthrie. 1978). The authors also observed a significant increase in the cell production in the duodenal and ileal crypts, as well as in large intestinal crypts of mice, whereas with rats the acceleration of proliferation was seen to occur only in jejunal and ileal crypts (Al-Nafussi and Wright, 1982b). The reasons for such discrepancies in the data presented thus far remain obscure. Under physiological conditions EGF secreted primarily by submaxillary salivary glands has been shown to induce a variety of biological effects, when released to the gastrointestinal tract with saliva (Barka, 1980). The specific EGF receptors are found in the isolated enterocytes (Forgue-Lafitte et a / . , 1980) and epithelial cells of stomach mucosa (Gregory et al., 1978). So far it is unclear what the role of EGF, produced by the Brunner’s glands, is in the regulation of intestinal epithelium renewal (Heitz et a / . , 1978; Elder et al., 1978) and whether EGF is secreted intraluminally, or it acts in a paracrine manner on the cells of Brunner’s glands. In addition, EGF is indicated to accelerate processes of intestinal mucosa morphogenetic maturation in mice embryos (Calvert et a / ., 1982). 6 . Histamine ECL cells and A-like cells in some rodents (rats, mice, Praomys natalensis) are found to produce histamine (Hakanson et a / . , 1976). The involvement of histamine as a trophic factor, stimulating proliferation in the gastrointestinal mucosa, is mediated probably via H, receptors on epithelial cells (Tutton, 1976). Indirect proof of the apparent role of histamine in the stimulation of epithelium proliferation in the stomach and small and large intestines is the suppression of

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DNA synthesis in the above organs following administration of cimetidine, known to be a highly specific antagonist to H, receptors (Andre et al., 1978). However, there are some findings available which are inconsistent with the above observation. The lack of any substantial cimetidine impact on the proliferation in the normal gastric mucosa was demonstrated as well as enhancing cell proliferation on the edges of ulcers (Kasajima et al., 1982). These differences can be evidently attributed to the existence in the target cells of stomach glandular epithelium of two classes of binding sites for histamine: ( I ) H2 receptors, the stimulating effect of which is mediated through the adenylate cyclase-CAMP system and (2) receptors which are not linked with this system (Batzri et a / . , 1982). 7. Chalones The substa;ces exhibiting chalone-like activity have been isolated from the animal small intestinal mucosa (Brugal, 1976; Bergeron and Sassier, 1980). Inasmuch as these substances are known to be low-molecular-weight peptides and display tissue-specific properties, they induce reversible inhibition in proliferation of enterocytes on the boundary of phases G ,-S. The level of reduction in the number of DNA-synthetizing cells caused by chalones is seen to be identical to that produced by secretin (Johnson and Guthrie, 1974) or pancreozymin, which are extracted from the small intestine, though the action of chalones is manifested more promptly. 8. Interaction of Various Factors The development of steady intestinal crypts hyperplasia following resection of 30-70% of small intestinal length may be putatively associated with the action of a particular humoral factor of nongastrin character (most likely candidates: enteroglucagon, anterior pituitary hormone, mineralcorticoids) which, probably, stimulates the increase in the stem cell numbers, though the localization zone of the latter remains in the same limits. The percentage of stem cells becomes higher in the total number of cells situated within the range of 1-4 positions in the jejunal crypts (Williamson and Malt, 1980; Bjerkness and Cheng, 1981d). However, some authors have doubts if any of the previously described hormones can play this role, and suggest that the local growth factors, produced by the paracrine cells of diffuse neuroendocrine system can be expected to provide this effect (Sharp et a/., 1980). These authors believe that stable paracrine stimulation of the stem cells can be considered the basis of postresectional hyperplasia, as their data have demonstrated that the influence of various systemic hormones (thyroid, sex, steroid, neurotransmitters, gastrin) is mediated predominantly on the level of committed cells-precursors and differentiating cells. Following proximal resection of the small intestine in rats, the compensatory hypertrophy of the distal portion is accompanied with sharp acceleration in the

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rate of cellular migration. The occurring disbalance between the enlargement of sizes of morphofunctional structures and migration rate results in shortening of enterocytes’ life span (Menge et u l . , 1982). Inasmuch as the decrease in cell cycle time takes place simultaneously with deceleration of transport rate of amino acids and glucose, it has been suggested that functional status is determined primarily by the age of the cells, rather than by their position on the villus (i.e., not by positional information) (Robinson er al., 1982). However, despite the extension of the proliferation zone to the vicinity of the crypto-villous junction, the maturity of villous base enterocytes remains the same as in controls (Van Dongen et al. 1977; Gutschmidt et a l . , 1982), though the enterocytes of hypertrophic villi display no incremental activity in some enzymes in the process of migration. Thus this example indicates that each particular factor involved in regulation of proliferation and differentiation appears to have a relative significance. Robinson et al. ( 1 982) suggested that development of immature cell populations may include two pathways: either underdifferentiation at the level of crypts or at the expense of accelerated migration along the villus, depending on the stimulus, which induces hyperproliferation. C. REGULATORY MECH~NISMS 1 . Role of Endogenous Determinant5

The investigations of morphological features in regeneration of large intestinal mucosal epithelium in vitro under the conditions of prolonged organ culture have demonstrated that initially observed phase of degenerative alterations in the course of time is succeeded with activation of regeneration and restoration of the epithelial structural integrity (Autrup, 1980; Senior et a/., 1982). The active regeneration is seen to occur only after complete destruction of crypts, when occasional islands of viable cells have survived, which resembles intestinal epithelial regeneratory microcolonies following radiation injury. The complete reconstruction of large intestinal crypts along with all types of differentiated cells lining crypts is indicative of the fact that the stem colony-forming cells prove to be the sources of regeneration. As Senior et al. (1982) during their experiments supplemented the culture medium with no hormonal regulatory factors, then the peculiarities in cell proliferation, migration, and differentiation in the process of regeneration are influenced mainly by the morphogenetic potencies of both stem cells and their descendants, by the intercellular interactions, and, probably, affected by pericryptal stromal elements. The impact of various hormones (Chang and Hoff, 1980; Sharp et al., 1980), cyclic nucleotides (Tutton and Barkla, 1980), diet factors (Levine et al., 1974; Lehnert, 1979), bile acids Deschner and Raicht, 1979), and autonomous nervous system (Lachat and Goncalves, 1978) on the renewal processes of gastrointestinal epithelium is limited, apparently, by the action, modifying the endogenous mechanisms of control.

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Of paramount importance for renewal of epithelial cryptal and glandular lining, there appear to be intercellular contacts and epithelial-stromal interrelationships which is emphasized by the facts showing the difficulties in maintaining the proliferation in cultures of cells isolated from intestinal and gastric mucosa, even if these cells exhibit high viability after isolation (Raul et af., 1978; Pothier and Hugon, 1980; Haffen et al., 1979). Naturally, one cannot rule out the probability that failures in such experiments may be caused to a large extent by inadequate methods of isolation of cells and culture media, or by absence of appropriate underlayers, and other similar factors, as some authors described the possibilities to receive proliferating cultures in those cases when for cultivation they made use of cell fractions, enriched with low-differentiated cells (Chopra el al., 198 1 ; Terano e t a / ., 1982) or epithelium either of embryonal or neonatal intestinal mucosa (Negrel et al., 1979; Quaroni el a l . , 1979). 2. Microenvironmental Influence on Stem Cells In analyzing the present state of knowledge on the stem cells in various tissues one of the major issues considered appears to be the question concerning the existence of “niches” (or micromilieu) with their specific microenvironment, which protects the stem cells from the effect of stimuli causing differentiation. According to Lajtha (1979), as such niches or sites for localizations of “anchored” stem cells there can serve such loci in the cellular system structure, where there is the highest probability of occurrence of stem cells in the Go phase, for instance, because of high concentration of hypothetical inhibitors of proliferation. The fluctuations in microenvironment in these niches can lead to various directions of differentiation for descendants of the stem pluripotent cells. Potten and co-workers (1979) indicated that hypothesis on the existence of special niches for the stem cells permits to formulation of a reasonably good explanation of the asymmetry observed in the division of stem cells. Only a single daughter cell can stay in the “niche” and thus be protected by the microenvironment from the influence of stimuli, which seem to induce differentiation. At the same time it has been suggested that some of the closest descendants of stem cells for some period exhibit a strikingly low probability to begin differentiation and because of this they are able, if the necessity arises, to occupy the “vacant niche” and to take the role of stem cells. The concrete morphological meaning of the term “niche” thus far is limited to a description of corresponding compartments in the bone marrow (Lichtman, 1981; Maloney et al., 1982) in multilayer epithelium of skin and tongue, in the intestinal crypts (Potten et af., 1979). However, in the intestine it refers only to peculiarities of the epithelial elements proper (Potten et al., 1979; Bjerkness and Cheng, 198 la-e) and does not cover the characteristics of mesenchymal elements of the stem cell’s zone. The renewal of pericryptal fibroblasts takes place regardless of enterocytes’ proliferation in the cryptal proliferative zone, and so far no correlation was

GASTROINTESTINAL STEM CELLS

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disclosed between the behavior of cells in these populations (Neal and Potten, 198 1 a ,b). In the fundic glands of hamster gastric mucosa (Hattori, 1974) a peculiar stronial sheath of the proliferation zone has been demonstrated, which is thought to ensure optimal conditions for rapidly proliferating cells. However, no special studies were performed with the view to elucidate the role of the periglandular sheath (stromal skeleton of gastric glands) nor to reveal the character of interaction between parenchymal and mesenchymal elements in the processes of gastric epithelium renewal. In the small intestinal crypts in bottom and basal positions there are observed to occur intermittently stem cells and differentiated cells (the Paneth cells, the goblet cells) and that for a long period of time has been interpreted as a possibility for in situ differentiation of a proportion of stem cells. Such an interpretation was inconsistent with the hypothesis on the existence of “niches” with microenvironment eliminating the possibility for differentiation. However, Bjerknes and Cheng (1981a-e) presented convincing data which support the existence of the spatial demarcation of stem and differentiating cells in crypts that, therefore, confirms the reality of particular microenvironment in the stem positions, excluding differentiation in situ. One of the components determining the radiosensitivity of stem cells may be their position in the niche (“helper function” or “anchorage,” Potten, 1980). The clonogenic cells are seen to differ widely as to their radiosensitivity, and approximately 3-4 cells at the base of the crypt die following irradiation in a dose of 4- 10 rad, whereas the other cells manage to survive even after exposure to 1200 rad (Potten, 1977). Potten et al. (1978) have also reported some findings supporting the hypothesis framed by Cairns ( 1975) on the capacity of epithelial stem cells to distribute the old and new DNA strands in a selective manner, thus preventing the build-up of erroneous new replicas in the daughter stem cells. The dynamic model of hierarchical arrangement within the population of intestinal stem cells, put forth by Potten (1980), substantiates to a certain degree the variations observed in the radiosensitivity of stem cells. The model suggests that radiosensitivity is reduced due to limitations in actively functioning gene pool, the further the cell migrates from the “stem positions.” It remains unclear what determines which of the surviving cells are able to return to the “stem status” to replace the dead highly radiosensitive cells. 3. Mesenchymal- Epithelial Interactions Mesenchymal-epithelial interactions with involvement of basement membrane seem to be of major importance for regulation of proliferation and migration activity of the epithelium. It has been suggested that tissue-specific inductors and proliferation inhibitors can affect epithelium through the basement membrane (Sharp e f al., 1980). Hageman (1980) considers that enlargement of the

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A. I. BYKOREZ AND YU. D. IVASHCHENKO

contact area between the stem cells and basement membrane induced by migration shifts of the overlying cells can be regarded as one of the stages in the mechanism of regulation of stem cells’ proliferation. The flattened stem cell is exposed to the effect of a large amount of a hypothetical “growth factor” which influences the length of the G, phase and capacity of cell to make a choice between self-replication and commitment to a particular line of differentiation. This mechanism depends on the density of cells in the epithelial lining of crypts or glands and resembles the regulation of proliferation in the cellular cultures. Following exposure to different damaging agents, such as ionizing radiation, hydroxyurea, there is observed the phenomenon of flattening of survived cells on the basal membrane, while some part of cells is subjected to apophysis. Therefore, this mechanism on the one hand promotes the preservation of epithelial layer continuity, and on the other hand, it can trigger the acceleration of cellular proliferation, aimed at restoring the physiological number of cells per unit of surface of pericryptal sheath and basement membrane. It can be assumed that depending on the epithelial cell position in the crypt or gland, there have to exist distinct variations in the structure er number of intercellular and cellular basement membrane contacts, particularly on the level of localization of stem cells (rigidly “anchored”), though to now no investigation was performed to disclose special features in junctions in relation to the topography of cells. The proliferation and differentiation of epithelial cells of crypts and glands are to a certain extent influenced by lymphocytes of lamina propria and intraepithelial ones (Manson-Smith et al., 1979; Miller and Nawa, 1979). It has been suggested that lymphoid cells as well as mast cells release substances resembling Iymphokines, which can act as paracrine modulators of differentiation of epithelial stem cells (for review see Castro, 1982). 4. Positional Information It may be considered as established fact that positional information plays a major role in the intricate system of regulatory interactions between separate cells, cell populations, epithelium, and mesenchyme, which determine the spatial arrangement of crypts and glands (demarcation of populations of differentiated and proliferating cells, borders of proliferative zone, functional compartment, and height of structures as a whole). The most essential elements of this information are as follows: epithelial cells themselves which, depending on the level of their intrinsically determined information can occupy different positions; lumen of crypts or glands, occurring as a result of polarization inherent in epithelium, and thereby creating conditions for contacts of cells with regulatory stimuli of the environment (the lumen of crypts and glands may appear to be one of the pathways of paracrine regulation as, for instance, it is ascertained for gastrin, secreted intraluminally), pericryptal fibroblast sheath (Hattori, 1974; Bjerknes and Cheng, 1981a; Neal and Potten, 1981a,b). It has been found that

GASTROINTESTINAL STEM CELLS

34 I

the border between proliferative zone and zone of differentiated cells in crypts is always seen to be perpendicular to the longitudinal axis of the crypts, i.e., positional information determining the coordinate parameters of proliferative zone is the derivative of the coordinate system related to the longitudinal axis; whereas the border between the zone of stem cells’ localization and zone of rapidly proliferating commited cells is primarily influenced by positional information concerning muscularis mucosae (Bjerknes and Cheng, 198 le).

5 . Regulation of Proliferation on the Basis of Feedback One of the commonly accepted theories on growth control in the continuously renewing cell populations states that there is a negative feedback between mature functioning cells and proliferating low differentiated cells. As regards the rapidly renewing epithelium of the gastrointestinal tract, many authors have obtained experimental findings supporting the existence of negative feedback mechanism between villus and crypt cell populations (Cairnie, 1976; Rijke, 1980; see Wright and Al-Nafussi, 1982). The critical analysis of works dealing with this issue and the results of their own studies enabled Wright and Al-Nafussi (1982) to confirm the presence of negative feedback in the system of small intestinal crypto-villous interrelationships. It was also shown that cell loss in villous epithelium stimulates proliferation in crypts as well as the influx of cryptal cells to the villus. However, Wright and Al-Nafiissi failed to exclude the effect of a depletion in the crypt cell number as an additional regulatory factor of proliferation rate in crypts and influx rate of cells from crypts to the villus. In the course of study of regeneration in stomach and intestine following repeated HU administration we have also obtained data revealing the presence of negative feedback proliferation regulation, depending on several factors. The duodenal mucosa after 5-6 doses of HU with 3-hour intervals displayed a sharp decrease in the crypt cell numbers (the crypts’ height was reduced from 40 to 10 positions), whereas the villi height remained unchanged as long as 20 hours. In the shortened crypts right up to the twentieth hour the index of DNA-synthetizing cells was seen to be under the normal value, despite the enlargement of the proliferative zone. However, starting from the twentieth hour, when numerous villi exhibited the destructive changes, the nutnber of DNA-synthetizing cells in the survived crypts strikingly increased and amounted to 200% as compared with the controls. At the same time the reduction in number of proliferating cells in the gastric glands was significantly less marked: for the period of DNA synthesis suppression (exposure to 6 doses of HU during 20 hours), the glandular height was diminished in average by 6 positions (due to the death of 13% of differentiated cells resulting from the migration and 7%’ of proliferating cells killed by the first HU injection) though by the eighth to tenth hour after the last HU injection sharp activation of proliferation was observed. The index of DNA synthetizing cells reached 270-300% in relation to the control value and was

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A. I . BYKOREZ AND YU. D. IVASHCHENKO

seen to increase in all positions-in the zone of localization of stem cells and rapidly proliferating committed cells. Consequently, these data provide no opportunity to answer the question of which factor is the most essential for feedback control; either it is the decline in differentiated cell number or loss in the compartment of proliferating cells (stem cells included) (Ivashchenko and Bykorez, unpublished). Both these phenomena seem to be of certain value. 6. Insufficiently Explored Factors of Regulation of Stem Cell Proliferation Following administration of 5-6 doses of HU the I , in pyloric glands in remote terms is seen to remain substantially higher than that in the controls (12.2 0.9%" weeks after last HU administration) and amounts to 180% in relation to the control value (Bykorez and Ivashchenko, unpublished). During the same period the glands' height is being normalized (30.2 & 2.5 cell positions), though the proliferation zone remains extended, i.e., 11.5 0.5 cellular positions, compared to 6.1 k 0.6 cell positions in controls. Thus, the renewing cellular systems in the stomach glands demonstrate the development of evidently new status in dynamic equilibrium with increased rate of proliferation, migration, and death of cells. This phenomenon resembles to some extent the state observed in the intestinal remnant after proximal partial resection. However, after the resection the proliferative zone in crypts is extended proportionally with enlargement of the crypts as a whole, and, therefore, the index of DNA-synthetizing cells, calculated for the whole cryptal epithelium, is not changed (Hanson et a l . , 1977a-c). Bjerknes and Cheng (1981d) indicated that extension of the proliferative zone in the course of postresectional hyperplasia is not associated with the increase in division number of migrating enterocytes within the limits of this zone; rather it is determined by an incremental influx of cells from the zone of stem cell localization. However, so far it remains unclear whether the zone of stem cell localization in the gastric glands is extended following repeated treatment with hydroxyurea. It has been demonstrated that single administration of HU to animals (mice) resulted in a considerable rise of proliferation rate of hemopoietic pluripotent stem cells (PSC). Ten-fifteen hours after H U injection 60-70% of PSC synchronously enter the S phase, whereas under normal conditions only less than 10%of PSC is usually found in the phase of DNA synthesis. Administration of two doses of HU with a 2-hour interval producing no marked cytotoxic effect on bone marrow, increases PSC number in the S phase to 8090%, 13 hours later after second treatment with H U . On the basis of data obtained it was suggested that more effective recruitment of PSC from G, to mitotic cycle depends on lengthening of period, when DNA-synthetizing cells are absent in the bone marrow. Our data on sharp proliferation increase of pyloric low-differentiated isthmic cells and neck cells of fundic glands, following repeated administration of HU, indicate that stem cells synchronously enter the S

*

*

343

GASTROINTESTINAL STEM CELLS

phase, and it leads in turn to an increase in number of rapidly proliferating committed cells. Therefore, it is likely that recruitment of slow proliferating stem cells in various tissues to the rapid proliferation obeys the common regularity which maintains that the principal stimulus proves to be the absence of DNAsynthetizing cells in all compartments (stem, transit) of the renewing system. However, the hypothetical stimulators (and inhibitors as well) of stem cell proliferation remain to be determined. Interestingly, there are data available that the blood of animals with a raised amount of proliferating PSC in the bone marrow (after HU administration) displays the appearance of PSC proliferation inhibitors (Necas, 1982). If the presence of such inhibitors were confirmed this would be essential proof in favor of the existence of an autonomous system of local regulation of stem cell’s proliferation (e.g., system of short-range acting factors). To elucidate these questions it is of major importance to perform further studies of behavior of slow renewing populations similar to the cells of Brunner’s glands. When the stem cells in pyloric and fundic glands as well as in intestinal crypts are exposed to the action of stimuli, inducing acceleration in their proliferation as a consequence of a massive cell death in foveolar and cryptal epithelium (e.g., after X-irradiation or HU administration), there is seen to occur the phenomenon of sharp acceleration in proliferation of some slowly renewing cell populations in the stomach and intestine. Thus, the Brunner’s glands composed of extremely slowly dividing cells ( I , in control amounts to 0.29 0.08%), following 5-6 doses of HU (500 mg/kg body weight) showed by the fourteenth hour over 100-fold rise of the I , . The cells of pyloric glands body whose proliferative capacity is brought in question by numerous authors displayed a striking rise in amount of DNA-synthetizing cells by 14-16 hours following fifth injection of the HU. The I , value was noted to reach 9.2 2.4% (in controls 0.67 0.12%). One week after administration of 6 doses of HU there was observed a sharp increase in number of DNA-synthetizing chief cells of fundic glands: I , = 1.45 0.10%, as compared to I , < 0.1% in control (Osipova and Ivashchenko, 1983). The abrupt triggering into the cycling stage of the nonproliferating (G,,) Brunner gland cells may be due to the need to augment the yield of epidermal growth factor, produced by the Brunner’s glands for stimulation of proliferation in all segments of the gastrointestinal tract. Hypothetically it can be assumed that under the conditions of epithelium severe damage, the production of urogastrone (epidermal growth factor) by cells of the Brunner’s glands is seen to increase to such an extent that it induces stimulation of proliferation of the producer cells themselves by the mechanism of autocrine stimulation. The cells of Brunner’s glands can probably be related to the monopotent stem cells. Their synchronous recruitment to rapid cell cycle after repeated treatment with HU is not associated with the cytotoxic effect, and supports the possible role of already described various growth factors as the regulators of the stem cells proliferation.

*

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*

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A. I. BYKOREZ A N D YU. D. IVASHCHENKO

VI. Stem Cells in Carcinogenesis In principle, there are three possible sources of tumor growth in the gastrointestinal mucosa: ( I ) nonproliferating differentiated cells, some of which can return to the mitotic cycle, and as a result of the malignant transformation, lose the ability to return to the nonproliferating state (see Gelfant, 1977); (2) actively proliferating cells with different degrees of cytodifferentiation; and (3) undifferentiated pluripotent uncommitted or extremely low differentiated committed cells with different rates of proliferation. A. PECULIARITIES OF CYTOTYPICAL DIFFERENTIATION IN TUMORS The hypothesis of Pierce (1974; Pierce et al., 1977) suggests that the major role in carcinogenesis is played by the stem cells which are presumably cells most at risk of malignant transformation, that is, they are acceptors of carcinogenic effects. The evidence to support this suggestion has been presented by various authors and different aspects of stem cell behavior during carcinogenesis has attracted much attention. In colonic and gastric adenocarcinomas induced by chemical carcinogens, there have been found all types of differentiated and low differentiated cells characteristic of normal crypts or glands (Tahara et a / ., 1975; Kobori et al., 1977; Pierce et al., 1977; Uchida et al., 1977; Bykorez and Ivashchenko, 1982a). The simplest explanation of this fact (thus satisfying the principle of “Occam’s razor”) is the assumption of the existence in the tumor of cells with the properties of stem cells arising as a result of malignant transformation of normal stem cells. This obviously does not imply that all other properties of the tumor stem cells are identical to those of the normal ones. Thus, in tumors the differentiation of descendants of the stem cells does not necessarily lead to the appearance of nonproliferating cells; furthermore, some of the most differentiated cells of adenocarcinomas preserve the ability to divide (Pierce et al., 1977). Moreover, while in the normal epithelium of the gastrointestinal tract the clonogeneity is characteristic only of the proliferating un- and low differentiated cells, primarily of the stem cells, in the tumors, the differentiation is likely to be a reversible process, which is not always accompanied by the loss of clonogenic properties. The resting tumor cells cannot be regarded as being in the Go state, because this is characteristic only of a certain part of the steadily renewing cell populations in which the Go cells can be recruited to the mitotic cycle depending on the requirements of the cell population, whereas in tumors the transition of cells to the state of relative rest is determined by nonspecific influences of the microenvironment rather than by the mechanisms of homeostatic control of proliferation (Kallman et al., 1980).

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345

This, however, does not rule out the existence in tumors of subpopulations of cells in different phases of relative rest as well as the appearance of postmitotic differentiated (“end”) cells that are unable to proliferate, although in most cases these cells lose the ability to proliferate only temporarily (Pozharisski et a / ., 1977; Gelfant, 1977; Ivashchenko and Bykorez, 1979b). Some other important peculiarities of tumor stem cells include the following: their properties may change so that the daughter cells entirely lose the ability to reach the level of histotypical maturity in the process of cytodifferentiation, which, likely, takes place during formation and growth of the low-differentiated tumors. In some cases the disturbances of cytotypical differentiation may lead to the production of low differentiated descendants which are capable of forming tissue-like structures (for instance, during morphogenesis of tubular, papillary, or villous adenocarcinomas), but the complete loss of the structure-forming ability may be observed in other cases. This appears to be associated with the disturbed expression of proteins and glycoproteins of the surface membranes as well as with the decreased amount of junctional complexes (or their complete disappearance) on transformed cells. Under these conditions, if the cells preserve the ability to differentiate, then adenocarcinomas, including mucinous and signet ring cell carcinomas can arise. It is known that the signet ring cells have differentiation signs similar both to those of the intestinal goblet cells and of mucoid-producing cells of the stomach glands (Sasano et al., 1969; Nevalainen and Jarvi, 1977; Yamashiro e t a / . , 1977; Pozharisski and Chepik, 1978). The insufficient differentiation of tumor cells is usually accompanied by distortion of differentiation which causes the appearance of the cells characteristic of no normal tissue of the organism. Morphogenetic Potentialities of Transformed Stem Cells

As revealed by light microscopy, malignant epithelial neoplasms of the gastrointestinal tract have a homogeneous structure, whereas electron microscopic investigations indicate heterogeneity of their cell composition with a substantial amount of well-differentiated cells (Pierce and Cox, 1978). The human gastric adenocarcinomas frequently show a considerable number of endocrine and amphicrine (endo-exocrine) cells (Ratzenhofer and Aubock, 1980; Procs and Feit, 1982; Tahara et a / . . 1982). The endocrine cells occurred even in cell cultures of adenocarcinomas that previously displayed no signs of differentiation (Kobori and Oota, 1979). Therefore these cells arise in the process of differentiation of proliferating tumor cells along with the other types of differentiated cells. The above can be interestingly illustrated by the well-known facts of the presence in the pancreatic carcinoids of G cells (gastrinomas) although the pan-

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A. I . BYKOREZ AND YU. D. IVASHCHENKO

creas of adult humans and animals show no G cells. At the same time in the stomach, where most of G cells are concentrated, gastrinomas do not arise, while in the duodenum (where the quantity of gastrin-producing cells is smaller than in the stomach) gastrinomas are quite frequent though not so frequent as in the pancreas (Larsson, 1980). The pancreas of embryos is known to contain numerous gastrin-producing cells that vanish during the postnatal period of development. These facts suggest high morphogenetic potentialities of stem cells of the gastrointestinal tract and make it possible to explain the presence in malignant neoplasms of various types of differentiated cells which are absent in unchanged morphofunctional structures of the organ and may arise as a result of the widening of morphogenetic potentialities of the transformed stem cells. It has been shown that in the adenocarcinoma with areas of signet ring cell transformation 90% of the tumor cells containing PAS-positive secretion had M , and M, antigens (characteristic of fetal stomach cells) or M, antigen and gastrin (amphicrine cells) whereas the remaining 10% of tumor cells that phenotypically corresponded to immature mucoid cells of intestinal type, like the fetal enterocytes contained M, and M, antigens. In the highly differentiated cells, only the M, antigen has been revealed (Bara, 1980; Bara et ul., 1980; Prade et al., 1982). According to Prade et al. (1982), these findings give indisputable evidence in support of the origin of all types of tumor cells from common stem cells. Among tumor cells of malignant gastrointestinal neoplasms, many contain excessive amounts of microfilaments or intermediate filaments and the increased quantity of the latter being observable already at early stages of carcinogenesis (Gabiani, 1979; Bannash et a l . , 1981). The bundles of tonofilaments and tonofibrils have been found in the cells of duodenal carcinoids (Carstens et al., 1976; Carstens and Broghammer, 1978), as well as in the MNNG-induced adenocarcinomas of the rat stomach (Kobori et al., 1977; Uchida et al., 1977) and in the low differentiated round cell carcinoid-like tumors of the large intestine (Petrelli et al., 1981). These findings confirm the origin of various clones of differentiated cells of tumors from pluripotent stem cells as a result of the divergent differentiation occurring at the expense of the epigenetic reexpression of the genetic information (Mintz, 1978). As revealed by investigations of adenosquamous carcinomas of the rat trachea, the presence in such tumors of various types of differentiated cells results from the unstable differentiation of the pluripotent cells rather than from the existence of several types of stem cells with stable differentiation. This lends support to the fact that despite the wide variety of cell clones in mixed tumors, they can arise as a result of malignant transformation of a single pluripotent cell. In some cases the descendants of the transformed cells during a long period of time can be committed only in one direction of differentiation, in another case the instability appears

GASTROINTESTINAL STEM CELLS

347

at once, in still another the instability of differentiation may arise against the background of the preceding relative stability (Steele and Nettesheim, 198 I ) . We still lack knowledge of the factors that determine the variant of differentiation which is predominant in the tumor, whether it be adenocarcinoma, signet ring cell carcinoma, carcinoid, adenocarcinoid, adenoacanthoma, or carcinoidacanthoma. B. DISTURBANCES I N PROLIFERATION DURING CARCINOGENESIS 1. The Kinetics of Cell Proliferation in Tumors According to Pozharisski and co-workers (1975, 1977c, 1979), the proliferating stem cells of intestinal crypts are the target for the transforming effect of such carcinogen as I ,2-dimethylhydrazine. This conclusion was prompted by the facts that the tumors are most frequent in those segments of the rat large intestine that have the largest number of stem enterocytes with the shortest T,. It was found that in the process of DMG-induced carcinogenesis the subpopulations of enterocytes within the zone of proliferation and near the base of crypts showed changes in their compositions and the percentage of rapidly proliferating cells increased. The kinetic parameters of the cell populations in carcinomas in situ as well as in the superficial cancers, small adenocarcinomas, and microscopically unchanged mucosa displayed a number of similar changes during carcinogenesis. Tumors also showed a relatively small percentage of slowly proliferating cells ( R , subpopulation) which probably represent the proper stem subpopulation of tumor cells (Pozharisski et al., 1 9 7 7 ~ ) . As reported by Chang et a l . , (1979) the T, of the cells of DMG-induced large intestine tumors in mice was equal to that of the normal crypts, although there is a disproportion between duration of certain cell cycle phases: in tumor cells t\ is shorter and tc, is longer; the duration of the G, phase in tumor cells was intermediate as compared with the tc2 of the stem enterocytes of the crypt base and of the rapidly proliferating enterocytes. The tumor cells showed a much greater variability of T, and t,, than the normal enterocytes, while the growth fraction in tumors was 1.5-5 times higher compared to the unchanged crypts (Chang, 1980). Thus the data of cell kinetics studies correlate well with the hypothesis of the origin of tumors from transformed stem cells. As revealed by the data obtained in our laboratory during investigations of the cell population kinetics in MNNG-induced adenocarcinomas of the rat stomach as well as in transitional mucosa and in zones remote from the growth area of the tumor despite a slight increase in T, in tumor cells, growth fraction (as determined from cell cycle parameters with the aid of mathematical modeling) becomes 2.5-3 times greater than in the normal pyloric glands (see Table 11). (Ivashchenko and Bykorez, 1979b; Bykorez and Ivashchenko, 1982a).

TABLE I1 THE KINETICPARAMETERS OF CELLPOPULATIONS OF ADENWARCINOMAS AND PYLORIC GLANDSOF THE RAT STOMACH AT LATESTAGES OF N-METHYL-N-NITRO-N-NI~OSOGUANIDINE-INDUCED CARClNoCENESlS" Duration of cell cycle and its phases

Growth fraction

T, (hours) Cells

Labeling index, I, ('%I

Isthmic-foveolar epithelium of pyloric glands of expenmental animals

7.8 2 1.2

Cells of well-differentiated adenocarcinomas Isthmic-foveolar epithelium of pyloric glands of control animals

Mitotic index (8)

9.0

?

2.5

18.0 +- 4.5

20.5 2 7.4

6.8 2 0.8

7.2 2 1.5

FLMb

Colchicinec CLCd

t,

(hours)

(32

tm

1,

(hours)

(hours)

(%)e

19.8-21.68

18.4

26.0

22.98

-

-

9.1-9.38

1.4-1.78

1.O-I.oX

-

19.0

19.2

31.6

7.6

0.8

0.8

20.0

03ased on data from Bykorez and Ivashchenko (1982a). bMeasured from the fraction of labeled mitoses curve. "Stathmokinetic studies. dMeasured from the cumulative labeling index curve. eMeasured from the labeling index distribution curve. /Calculated from phase duration and labeling index. RObtained with the aid of the mathematical model by Takahashi (1974).

7 . 9 ? 8.0s 0.7-0.89

0 . 8 - 0 . 9 ~ 25.0

1, (%V-

19.0

45.3-50.0

16.3

I , (%I8

-

57.08

-

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349

The index of the DNA-synthetizing cells in the colonic adenomas and adenocarcinomas amounts to 25-30% (Hohn, 1979; Pozharisski et a / . , 1977c), which is somewhat less than that within the crypt proliferative zone. A similar situation has been observed during experimental carcinogenesis in the stomach mucosa where in the zones of adenomatous hyperplasia and in the highly differentiated adenocarcinomas I , varies from 18 to 25% which is more than I , values for epithelium of the pyloric glands as a whole but less than those for the zone of maximal proliferation of the latter (Ivashchenko and Bykorez, 1979a). Studies of the kinetics of cell populations of the large intestinal crypts following the administration of 1,2-dimethyIhydrazine have revealed complex changes occurring in the renewal rate and in the composition of enterocyte populations during carcinogenesis. The principal alterations of the epithelium differentiation are supposed to reflect the disturbances that take place at the level of stem enterocytes subpopulations under conditions in which a part of them becomes malignant while the remaining normal cells proliferate with an increased rate (concomitantly, the percentage of the semistem cells that are ready to differentiate, probably decreases) which causes the above changes both among the rapidly proliferating enterocytes and the differentiated ones (Pozharisski et al., 1977c; Barthold, 1981; Richards, 1981; Sunter et a/., 1981). Taking into account that such changes occur not in one (and not in all) crypt, it can be regarded as an established fact that carcinogenesis proceeds multifocally but apparently with a different rate in different crypts. It is important to emphasize that the disturbances in proliferation and differentiation of the cells as estimated by the extent of the proliferative zone, the number of atypical mitoses, and the heterotopic cell proliferation, the structural atypia are differently pronounced even in the adjacent crypts or glands which is indicative of the autonomous character of the regulation of structural stability of the morphofunctional systems as well as of the fact that initiation and progression of carcinogenesis take place not in a group of crypts or glands but in a single structural unit. It seems likely that the hyperplastic changes observed in epithelium of the organ as a whole such as the increase in dimensions of structures and in amount of cells (regeneratory hyperplasia) are mostly caused by the cytotoxic effects of the carcinogen and can be induced not only by carcinogens but also following the damage caused by small doses of radiation as well as the administration of cytostatic preparations (Pozharisski et al., 1979; Kunze et a/., 1979; Chang, 1982). The increased percentage of proliferating stem cells in the mucosa of gastrointestinal or intestine-intestinal anastomosis leads to more frequent development of tumors arising predominantly from epithelium of such zones either after administration of carcinogenic substances or even “spontaneously” (Pozharisski, 1975; Dahm et a l . , 1979; Meister and Schlag, 1979; Schlake and Nomura, 1979; Harte er al., 198 I ) . Of decisive significance for the initiation of carcinogenesis under such circumstances is the increased pool of proliferating

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stem cells rather than the shortening T, of committed rapidly proliferating cells; this is proved by the following: ( 1 ) under normal circumstances the T, minimum of non-stem cells is also very short (Cairnie et a/., 1975; Appleton et ul., 1980; Wright, 1980); ( 2 ) in tumor cells of the stomach and intestine the T, usually increases somewhat rather than decreases (Pozharisski et a/., 1 9 7 7 ~Ivaschenko ; and Bykorez, 1979b); and (3) the short-term experimentally induced ischemia of the large intestine is followed by the increased rate of proliferation of enterocytes which, however, is not accompanied by expansion of the proliferative zone, whereas for the DMH-induced carcinogenesis, the most characteristic features are expansion of the proliferative zone and a slight alteration of the T, (Rijke and Gart, 1979). 2 . The Heterotopic Proliferation of Stem Cells and Expansion of the Proliferative Zone during Carcinogenesis It is supposed that the predominant localization of atypical cells forming the carcinoma in situ outside the proliferative zone in large intestinal crypts (Lipkin, 1974; Pierce er al., 1977) is associated with the disturbance in differentiation of malignantly transformed stem cells as well as their heterotopic proliferation under abnormal conditions beyond “the niches” (Pozharisski et a/., 1977c, 1979). It is likely that the stem cell transformation alone is not sufficient for the initiation of the tumor growth which is impossible without a certain change in epithelial-mesenchymal relationships caused by the shift of stem cells to that zone of the crypts where usually only nonproliferating cells are located. It appears that such a migration of stem cells is always preceded by a change in composition of cell populations. Thus at early stages of carcinogenesis in the intestinal crypts and gastric glands there can be seen an expansion of the proliferative zone and the appearance of single DNA-synthesizing cells and mitoses near the gastrointestinal lumen (Lipkin, 1974; Pozharisski et ul., 1977c; Deschner, 1978), whereas the even more characteristic feature for carcinogenesis in gastric glands, where unlike the crypts, the proliferative zone fails to reach the gland base, is the widening of the proliferative zone toward the bottom of the glands (Deschner et a/.,1979; Ivashchenko and Bykorez, 1979b; Kunze er al., 1979; Quimby and Eastwood, 1981). Hohn (1 979) considers that the macroscopic forms of DMH-induced colonic cancer are determined by the position of the “proliferative center” in the crypts with expanded proliferative zone: if the maximum of proliferation is concentrated near the crypts’ mouth, there arise the exophytically growing tumors; the most active proliferation of the cells in the middle one-third of the crypts causes exophytic-endophytic tumors similar to adenomas with a wide base and adenocarcinomas only slightly rising over the surface; whereas localized in the basal part of the crypts the cellular foci of maximal inclusion of [‘HJthymidine give rise to the carcinomas with a high degree of malignancy that develop without the

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preceding stage of hyperplastic changes and rapidly invade the deeper layers of the intestinal wall. As a rule, histologically the latter are low differentiated adenocarcinomas or solid undifferentiated carcinomas. After administration of MNNG to the rats. the endophytically growing gastric adenocarcinomas of the tubular type and signet-ring cell cancer arise usually from the foci with highly pronounced dysplasia of the isthmic and basal segments of the pyloric glands in areas with the proliferative zone shifted toward the bottom of the glands (Deschner et d.,1979; Kunze et d., 1979; Bykorez and Ivashchenko, 1982b). This was accompanied by replacement of the mature mucoid-producing epithelium of the pyloric glands body with low differentiated cells which suggests that one of the consequences of the malignant transformation (or initiation?) of stem cells is their shift (possibly, hyperplasia with expansion of stem cells’ localization zone) toward the zones where there are usually located only differentiated cells. It is possible that the proliferation of stem cells outside “the niches” causes disturbances in production of committed cells thus making the microenvironment still more atypical as well as promoting the development of “deafness” of the stem cells to regulatory stimuli. It should be noted that the development of gastric carcinoma from the basal parts of glands appears to be a general pattern of the tumor morphogenesis both under conditions of experimentally induced carcinogenesis and in humans. This is clearly illustrated by the reports of multifocal development of adenomatous diverticulas in the human stomach mucosa against the background of atrophichyperplastic gastritis (Schauer and Kunze, 1979). The question that must be answered is why in the separate foci an increase of degrees of structural and cellular atypia is observed, whereas other foci remain “quiet” (“persistent incomplete carcinogenesis,” Schauer and Kunze, 1979), and the signs of further progress are absent. According to Pozharisski et ul. (1977b,c, 1979), the widening of the proliferative zone in crypts during DMG-induced carcinogenesis should not be regarded as an indication of hyperplasia. In the event of ordinary hyperplasia the zones of proliferating and differentiated cells remain spatially separated with the absolute quantity of the cells rising, whereas in the process of carcinogenesis the size of crypts and glands may remain unchanged while the production of differentiated cells becomes disturbed and the low differentiated cells spread unevenly over the entire length of the structures. The widening of the proliferation zone as a rule has been observed during the intestinal metaplasia of the stomach epithelium (Ivashchenko ef a/. , 1981). However, the expansion of the proliferative zone in the metaplastic glands is of precancerous significance only in case of parallel development of disturbances in cyto- and histotypical differentiation (the so-called intestinal metaplasia of the large intestinal type) (Cuello and Correa, 1979; Heilman and Hopker, 1979). Thus, it appears that the role played by the expansion of the proliferative zone

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depends on the nature of the cells that proliferate dystopically. If there is no significant reduction in number nor severe damage of the qualitative properties of the stem cells, the restitution of cellular composition (in case of predominant impairment of differentiated cells) can be achieved without activation of the stem cells’ proliferation, but merely at the expense of the increased number of the divisions that the committed cells have to pass. In case of more severe damage (e.g., that caused by large doses of ionizing radiation), the increase of the proliferation rate of the stem cells is an inevitable component of regeneration, but the localization zone of the stem cells becomes clearly localized already at early stages of colony formation (Bykorez and Ivashchenko, 1982a). The changes of some, thus for unstudied properties of the stem cells caused by carcinogenic agents leads to the dystopic proliferation of cambial elements of the gastrointestinal mucosa with the progressive increase of disturbances in cytotypical differentiation of their descendants as well as in histotypical differentiation of the functional structures as a whole. C. THE MARKERS OF STEMCELLDIFFERENTIATION DISTURBANCES 1 . Disturbances in Dysplastic Lesions It still remains unclear what kind of changes occur in epithelium of the dysplastic lesions in the gastric and intestinal mucosa in animals treated with various carcinogenic factors. It appears that in these zones there occurs an accumulation of descendants of the initiated stem cells resulting from partially disturbed differentiation. This phenomenon can be compared with the appearance of enzymealtered foci of hepatocytes during experimental hepatocarcinogenesis that are immediate progeny (clonal by origin) of the initiated cell (Pitot and Sirica, 1980). Unfortunately, one can judge stable inherited genetic disturbances (of mutational or epigenetic nature) of the stem cells during carcinogenesis only on the basis of altered differentiation of their descendants. Pozharisski and Chepick ( 1978) found no histochemical differences between the microscopically unchanged colonic mucosa and the epithelium in areas adjacent to the tumor during the DMH-induced carcinogenesis. The first histochemical alterations were observed by these authors only in carcinomas in situ which contradicts the data reported by others. The histochemical study of the large intestinal mucosa in areas adjacent to adenocarcinomas (transitional mucosa) revealed foci of pronounced epithelial dysplasia that secreted sialomucins, the production of which can be observed under normal conditions only among low differentiated cells. As the normal mucoid cells migrate upward in the crypts they display enhanced synthesis of sulfomucins with parallel disappearance of sialomucins (see Filipe, 1979). In the

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areas of epithelial dysplasia of the gastric glands the increased production of sulfomucins is observed more frequently though in case of highly pronounced dysplasia the acidic mucosubstances may totally disappear while the synthesis of the neutral ones may sharply decrease (Bykorez and Ivashchenko, unpublished). Utilization of lectins for the identification of different classes of mucosubstances at the levels of light and electron microscopy made it possible to reveal highly specific differences in secretory composition of normal and transformed cells. The differences in lectin bindings are partly accounted for by sharply reduced activity of N-acetylgalactosaminyltransferase,and galactosyltransferase in human colon cancer cell membranes (Boland et al., 1982), because this leads to the appearance of incompletely glycosylated side chains in glycoproteins molecules. Of particular interest is the fact that peanut agglutinin (PNA) did not bind the goblet cell mucin even in low differentiated oligomucoid proliferating cells in the upper or lower normal colonic crypts, while PNA avidly binds to the mucins secreted into the glandular lumens in colonic cancer. This indicates that the low differentiated normal cells differ from the phenotypically similar malignant cells in that the latter undergo aberrant differentiation. In the transitional mucosa the mucoid-producing cells, though not so active in binding Dolichos biyorus, soybean, Ricinus communis and Bauhinia purpurea agglutinins, become able to bind PNA thus acquiring certain features of differentiation characteristic of the tumor cells (Boland et al., 1982). Histochemical studies also revealed marked changes of the composition of glycoproteins in the mucoid cells contained within epithelium of this zone: sulfomucins disappear, and sialomucins show reduced percentage of 0-acylated derivatives (Filipe, 1979). The structural signs of weakly pronounced dysplasia are combined with insufficient differentiation of most of the cells in the middle and upper thirds of the crypts. Goblet cells preserve the ability to incorporate [3H]galactose in surface segments of the crypts which is indicative of their immaturity (Dawson and Filipe, 1982a). The proliferative zone increases and the number of immature cells grows. This is associated with decreased activity of enzymes responsible for the transfer of sugars during synthesis of glycoproteins. Synthesis of incomplete glycoproteins in goblet cells is accompanied by the activation of synthesis of glycoproteins in absorptive cells which results in the appearance of the vesiculated “intermediate” cells. The latter manifest increased incorporation of [3H]threonine which is characteristic of the immature cells actively synthesizing proteins (Dawson and Filipe, 1982b). Underdevleoped intercellular connective complexes in adenocarcinomas are a constant and pronounced sign distinguishing them from adenomas (Loewenstein, 1979). Electron microscopy in the zones of large intestinal dysplasia reveals the increase in number of undifferentiated cells in the middle and upper third of the

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crypts and even amid the surface epithelium (Shamsuddin and Trump, 198 I b). The intermediate type cells show resemblance both to absorptive and secretory cells. The absorptive cells display a reduction in microvilli number and appearance of electron-dense bodies (Mughal et al., 1981). The amount of such cells is found to be in reasonably good correlation with the degree of dysplasia markedne Similar peculiarities have been described for the dysplasia zones in adenopapillary polyps, where the authors reported the sharply pronounced stratification of undifferentiated cells (Balazs, I98 I ) . The epithelial stratification or pseudostratification, being an important diagnostic sign of dysplasia (Kozuka, 1975), reflects the recapitulation of early ontogenetic stages in intestinal epithelium differentiation in the course of carcinogenesis. Different researchers presented controversial views concerning the precancerous significance of large intestinal adenomas as well as their morphogenesis (Oohara et al., 1981; Muto and Bussey, 1975; Pozharisski and Chepik, 1978). On the basis of histological structure, the adenomas are usually classified as tubular, tubulovillous, and villous. It has also been suggested that the best indicator of adenoma malignant potential appears to be the dysplasia degree, rather than the character of growth (Morson and Konishi, 1980). However, the assessment of structural and cellular atypia degree, which is employed to characterize dysplasia, is largely influenced by subjective estimations. Among the most promising histochemical markers of dysplastic changes in gastrointestinal epithelium which have been studied the following can be named: antigens of blood groups, carcinoembryonal antigen (CEA), secretory component, and cellular IgA content. However, the specificity of blood groups antigens has been found insufficient (Feizi, 1982). The data concerning CEA specificity are very contradictory. Burtin et ul. (1972) found increased amount of CEA in well-differentiated adenomas, whereas Tappeiner et al. ( 1973) reported high CEA content in low differentiated ones. According to Isaacson and Le Vaan (19761, the presence of CEA in adenomatous polyps is a reliable sign of epithelial malignancy. The absence of correlation between the degree of dysplasia and the intensity of CEA staining was demonstrated by Skinner and Whitehead (1981) and Rognum et al. (1982). It is suggested that the production of CEA by the cells depends either on the loss of intercellular contacts or on the sharp reduction in their number (Wagener et al., 1981). This, however, cannot be regarded as an explanation of the discrepancy of the above data. The conflicting results of immunocytochemical studies of CEA appear to be accounted for by the fdCt that CEA is an extremely complex molecule with numerous antigenic epi topes. An interesting hypothesis was framed by Pozharisski et al. (1979). The workers suggested that CEA is secreted by stem cells of the colonic crypts and that the

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increase in number of stem cells caused by various inflammatory-regeneratory and hyperplastic processes may result in increased CEA content in blood plasma and intestinal mucosa. However, immunocytochemical investigations have failed to reveal any dependence of the specific differences in CEA content on the degree of differentiation of the cells in tumors and hyperplastic crypts. Furthermore, the highest concentration of CEA has been found in surface epithelium of hyperplastic crypts (Rognum et al., 1982). At the same time, the immunocytochemical detection of the secretory component and IgA makes it possible to determine the degree of dysplasia more clearly, since the content of these antigens decreases as a result of atypia of epithelium (Rognum et al., 1982). In the zones of epithelial dysplasia where sulfomucins disappeared and only sialomucins were observed, the distribution of CEA was the same as in the unchanged large intestinal mucosa (O’Brien er al., 1981). Perhaps the currently emerging utilization of monoclonal antibodies against CEA will very soon more clearly indicate its effectiveness as a marker of malignant transformation (Lindgren et al., 1982). MNNG, administered during several days, is capable of causing a stable disturbance in differentiation of glandular cells in the body of the pyloric glands, which is indicated by the interruption of their pepsinogen I production (Tatematsu et al., 1980). It appears that the changes caused by MNNG in the genetic apparatus of the stem cells result in disturbed expression of pepsinogen 1 in differentiating cells. Similar alterations in synthesis of pepsinogens have been observed following ingestion of some other gastrotropic carcinogens (Tatematsu et al., 1980), but not after X-irradiation of the stomach which leads to intestinal metaplasia and only occasionally causes carcinoma of the stomach (Jin et al., 1982). The fact that this effect is associated with the stem cells rather than with the differentiated ones is confirmed by the disappearance of pepsinogen I only 810 days after the administration of MNNG, i.e., the period of time required for replacement of pyloric glands body differentiated cells (which develop from normal stem cells), deficient as to the synthesis of pepsinogen cells which arose from the stem cells damaged by MNNG. Moreover, the renovation of pepsinogen 1 synthesis was not observed even 40-450 days after the termination of MNNG intake. It appears that these changes are the first stage in the process of multistep transformation of the stem cells during carcinogenesis. The above data once again provide evidence in support of the plausible hypothesis that the morphological and histochemical features of the differentiated cells in the zones of dysplasia depend on the development of these cells from the initiated stem cells. However, future studies will be needed to elucidate this issue, since the available data indicating the reversible character of sulfomucins disappearance in colonic goblet cells after administration of DMH (Naito, 1982) as well as of the possible regression of dysplasia in the stomach mucosa (Oehlert,

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1979) can be regarded as evidence of the existence of various mechanisms which cause disturbances in genetic expression but of which not all appear to be associated with the initiation, although they are capable of imitating it very closely.

2 . Disturbances during Metaplnsia Of special interest are the processes of metaplasia in gastric mucosa, both of intestinal and pyloric type, which quite frequently either accompany the processes of dysplasia or occur concomitantly with them. Analysis of different types of gastric epithelial metaplasia reemphasizes the significance of the intercellular transfer of information and the highly specific character of relationships between the stem cells and the transitory and differentiated cells. The pyloric metaplasia leads to the formation of typical pyloric glands in the fundic mucosa, whereas the parietal and chief cells disappear and do not arise again (Bykorez and Ivashchenko, 1982a; Hattori et a / . , 1982b). In metaplastic structures, the differentiated cells develop from the low differentiated cells, i.e., the general sequence of renewal of epithelium is not disturbed whereas the morphogenetic potentialities of stem cells undergo irreversible changes. Similar processes also occur during intestinal metaplasia. Some facts indicate that the processes of epithelial metaplasia of the gastrointestinal mucosa are chiefly based on the modification of stem cells, rather than o f their progeny, i.e., these processes result from indirect metaplasia (lvashchenko et a / . , 1981). The intestinal mucosa has a number of endocrine cells that are absent in the gastric glands. Therefore, if the endocrine cells do develop from the stem cells in the stomach and intestine, and the intestinal metaplasia results from changes in morphogenetic information of stem cells of the gastric glands, the zones of intestinal metaplasia should contain endocrine cells (rather, a set of endocrine cells) characteristic of the intestinal mucosa. At the same time, some endocrine cells are substantially less frequent in the intestine than in the gastric glands. Therefore, it can be expected that the zones of intestinal metaplasia have a smaller amount of such cells compared with the surrounding unchanged glands. The data obtained by various authors indicate that all these suppositions are true. Thus, within the zones of intestinal metaplasia the number of gastrin-producing cells (which are much more frequent in the unchanged pyloric mucosa of the stomach than in the small intestinal mucosa) sharply decreases, whereas the amount of enterochromaffin EC cells increases (Tahara et ul., 1975). The number of somatostatin-producing D cells in crypt-like structures of the intestinal metaplasia areas is 3-4 times less compared to the surrounding pyloric glands (Bordi and Ravazzola, 1979). The glycentin (enterog1ucagon)-producingas well as the motilin- and cholecystokinin-containing cells, which are absent in the normal stomach inucosa but present in the intestinal crypts, can be observed in metaplastic crypts within gastric mucosa. At the same time, the intestinal endocrine cells that are localized only in vilous epithelium (secretin and neurotensin-

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containing) are not seen in the zones of intestinal metaplasia (Bordi and Ravazzola, 1979) since the formation of villi does not occw during the process of intestinal metaplasia. It is to be noted that, among the cells of the zones of intestinal metaplasia of the stomach mucosa in humans, some other differentiated cells have been described that are unusual for intestinal epithelium, being mainly characteristic of respiratory and genitourinary epithelium. Okuda and Ogata (1976) were the first to find such cells in epithelium of the human pyloric mucosa and to show their similarity to the ciliated cells of the bronchi, uterus, uterine tubes, and paranasal sinuses. These cells were found in epithelium of the zones of intestinal metaplasia and rarely among the cells forming a polyp in the stomach mucosa. Similar data were reported by Yamashiro et al. (1977) who, among typical enterocytes of the zone of intestinal metaplasia of the human stomach mucosa, found cells with numerous cilia which become connected with the neighboring intestinal-type cells by means of desmosomes from which the authors concluded that the ciliated cells arise as a result of abnormal differentiation of metaplastic stem cells. Therefore, the histogenetic regression manifesting itself in the replacement of complex morphofunctional structures with more simple ones during the process of metaplasia is based on the genetic reprogramming of the stem cells (within the limits of morphogenetic information) resulting from sharp activation of proliferation of stem cells against the background of chronically acting regeneratory stimuli. It happens that the disturbance in the normal microenvironment and appearance of unusual inductive stimuli play one of the major roles in metaplastic conversion of the stem cells. However, the molecular basis of this reprogramming remains absolutely unclear.

D. PROBLEMS OF GASTROINTESTINAL NEOPLASTICGROWTHMORPHOGENESIS According to the views expressed by Pierce ( 1 974) and Pierce and Cox (1978), all intestinal tumors, including both the highly differentiated and low differentiated ones, arise as a consequence of transformation of proliferating cells. It is supposed that the malignant transformation of the pluripotent stem cells of the crypt base leads to the growth of solid (signet ring cell or anaplastic) carcinomas with predominantly endophytic spreading. In case it is proliferating enterocytes migrating upward in the crypts that undergo transformation, then, depending on the level (and, consequently, on the degree of differentiation) of transformation of the cell, either adenomas or adenocarcinomas arise. Therefore, according to Pierce, the degree of differentiation (commitment) of target cells for carcinogenic factors determines the degree of differentiation of the tumors that arise. Pierce believes that the earlier appearance (following the administration of carcinogens) of benign tumors is evidence supporting his views, for, in his opinion, the microenvironment required for the transformed cells of benign

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tumors practically does not differ from that existing in normal tissue of the organ. From the set-up suggested by this hypothesis it can therefore be logically concluded that there is an increased risk of cancer resulting from benign polyps. Not all authors, however, support this view. Thus, according to Knudson (1977), in the case of familial polyposis of the large intestine the risk of malignant transformation of each polyp is not greater than that of polyps in general, while the increased frequency of cancer entirely depends on the increase of the absolute quantity of polyps. In compliance with data obtained by Ward (1974), Pozharisski et al., (1977c, 1978, 1979), Maskens (1981), and Chang (1980, 1982), colonic adenocarcinomas induced by DMH arise de nova, without the preceding stage of polyp formation. According to Wittig et al. (1971), the tumors may arise either de nova or as a result of malignant transformation of initiably benign polyps. As revealed by the detailed study of this question carried out by Pozharissky et al. (1977b,c, 1979), initially in the large intestinal mucosa there arises a focus of carcinoma in situ which directly develops into invasive adenocarcinomas or signet ring cell cancer. These findings have been supported by data obtained by Maskens and Dujardin-Loits (1981) who also found that the adenomatous polyps cannot be a precancer condition, for they arise much later compared to carcinomas. In the view of Maskens and Dujardin-Loits (1981), the fact that adenocarcinomas arise in the intestine de nova implies only the absence of the stage of hyperplastic benign changes in carcinogenesis, but does not deny a possible role in carcinogenesis of other changes in mucosa that precede carcinoma, such as focal atypia. Similar views have been expressed by some other investigators who observed development of focal dysplasia of cryptal epithelium starting from the first days of administration of carcinogen (Deschner, 1978). The authors expressed doubts as to the possibility of providing a clear answer to the question of specificity or nonspecificity of the above-mentioned lesions of mucosa in the process of carcinogenesis. At the same time, Pozharisski et LEI.(1977c, 1979). on the basis of detailed histological and autoradiographic investigations, concluded that the foci of atypia observed in some crypts are not precancer lesions but carcinomas in situ. It should be taken into account, however, that the data concerning the nature of the earliest DMH-induced lesions in animal colonic mucosa are very contradictory. This concerns not only morphological lesions as such, but also the time of their detection. The absence of changes in cryptal epithelium up to the moment of the appearance of foci of carcinomas in situ (at 5e6 months of carcinogenesis) was emphasized by Pozharisski et a / . (1977c, 1979). But some investigators found substantial disturbances in structural and cytotypical differentiation even in early weeks after administration of DMH (Deschner, 1978; Naito, 1982). According to Chang (1980, 1982), during the first stage of carcinogenesis

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there occurs the replacement of normal differentiated enterocytes lining the crypts with a homogeneous population of basophilic cells that resemble the undifferentiated cells of the crypt base and represent, evidently, transformed stem cells. However, the author suggests that some of these atypic crypts may not cause the growth of tumors. Morphologically, if we compare microphotos of “atypical crypts” from Chang (1980, 1982) with “carcinoma in situ” of Pozharisski and Chepik (1978), it appears that the authors have described practically identical lesions. However, Chang (1980) defines such lesions as severe dysplasia, regarding the accumulation of transformed stem cells in the upper part of the large intestinal crypts as the beginning of tumor growth. The species of animals is of great significance for experimental carcinogenesis in the large intestine (Evans ef a l . , 1977; Naito, 1981): in most of rat lines adenocarcinomas arise de novo whereas adenomatous polyps either are absent or appear later than carcinomas (Maskens and Dujardin-Loits, 1981); at the same time, in noninbred ICRiHa mice adenomas are predominant, whereas carcinomas in situ are very rare. Judging from the fact that in mice of the same line, but inbred, the frequency of adenocarcinomas is considerably higher compared to that of adenomas (Evans et al., 1974), the genetic predisposition plays a very important role. It was shown that strongly pronounced dilation, distortion, and hypercellularity of some rat colonic crypts after azoxymethane injections are irreversible, and actually such foci are none other than microscopic carcinomas, often invasive. As for the changes observed prior to focal atypia (dysplasia), e.g., changes in secretion of mucosubstances, they appear to reflect the alterations occurring in composition of populations of differentiated colonocytes and probably also disturbances in these cells differentiation (Shamsuddin et al., 198 1). Unlike the mechanism of new crypts formation by way of bifurcation or longitudinal fissure occurring in the course of postnatal development or after the death of a part of the stem cells following irradiation or administration of large doses of carcinogen (Cairnie, 1975; Deschner, 1978; Maskens, 1978), the chronic effect of carcinogenic factors is accompanied by the appearance of a new type of cryptal morphogenesis: epithelial budding somewhere along the proliferative compartment of a crypt with formation of the pouch consisting of atypical cells and subsequent enlarging and branching of the pouch are concomitant with replacement of the epithelial lining in the mother crypt by atypical cells “overflowing” from the pouch (Chang, 1982). Unicryptally, in the large intestine there can arise adenomas and hyperplastic crypts (Oohara et af.,1981) and carcinoma in situ (Pozharisski et al., 1977c, 1979; Shamsuddin et al., 1981). Therefore, morphogenesis of colon carcinoma has two main variants: either growth de novo or in the following sequence: adenoma (hyperplastic polyp)-dysplasia-carcinoma. Inasmuch as the zones of dysplasia within polyps invariably show expansion of the proliferative zone (a

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sign which is characteristic of the early stages of carcinogenesis arising de n o w ) , it is quite clear that, in principle, there is no difference between these two variants. As can be seen from the above data, carcinoma of the large intestine initially appears in one or several crypts (independently of each other), starting from accumulation of atypical cells (neoplastically transformed stem cells) which form carcinoma in situ or microinvasive cancer (more frequently signet ring cell carcinoma). It should be noted that these processes of carcinogenesis may take place both in unchanged crypts and in polyp-forming crypts. In respect to morphogenesis of gastric carcinoma, however, the issue of tumor growth de novo against the background of the unchanged mucosa looks very problematic. This is chiefly due to the difficulties of early diagnosis of carcinoma in situ (Kraus and Cain, 1979). Moreover, carcinoma of the stomach usually is detected in the mucosa with considerable structural changes: atrophic gastritis, intestinal metaplasia, and adenomatous hyperplasia. The most probable explanation, based on data of numerous clinical and experimental investigations, is that the leading role in gastric carcinogenesis is played by epithelial dysplasia which may evolve simultaneously with all above-mentioned pathological processes (Grundman, 1975; Meister et al., 1979; Morson et al., 1980; Potet and Camillieri, 1982). It should be noted that the stage of carcinoma in situ in the gastric mucosa seems to be very short and unamenable to verification. The appearance of well-differentiated adenocarcinomas of the stomach is associated with the spreading of transformed cells of “associated” type (according to Fujita and Hattori, 1977) over the basement membrane with replacement of the normal glandular and foveolar epithelium. Having arisen in the proliferative zone, the undifferentiated cancer cells of “dissociating” type immediately invade lamina propria through the defects of the basement membrane which results in the formation of the “double-layer structure” (Murakami et al., 1978; Watanabe et al., 1979). When considering the morphogenesis of the differentiated adenocarcinomas, some authors (Hattori and Fujita, 1979, 1980; Taki and Kuwabara, 1981) attach great importance to the appearance (in the process of intestinalization of mucosa and during adenomatous hyperplasia) of isolated cystic structures (resembling cryptal “pouches” lined by atypical cells) within which the transformed or atypical cells are protected from being naturally extruded by the flow of migrating normal cells. The available data suggest that the following two mechanisms may determine the morphogenetic evolution of tumors. 1. The main role has the state of the stem cell at the moment of initiation. If we assume that the commitment of the stem cell depends on its remoteness from the “borders of the niche” and that the direction of commitment is determined by the needs of the cellular system as a whole, then the frequency of tumors with definite character of cytotypical differentiation should correspond to the propor-

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tion of stem cells that are committed in the corresponding direction. In general, this assumption can be corroborated. Thus, for instance, adenocarcinoma, representing the most frequent histological type of gastric cancer (in humans and experimental animals), is supposed to arise from the transformed stem cells committed in the direction of mucoid differentiation which is the most probable variant for the normal stem cells (in view of great speed of renewal of mucoid cells). However, if the frequency of one or another histological type of cancer is regarded not as a whole but with the sex-age distribution taken into account, the above considerations will prove to be erroneous. Thus, it appears that in young human patients, particularly among women, it is low differentiated adenocarcinomas and signet ring cell carcinomas that are the predominant forms of cancer. Still more significant in this respect are the data of experimental studies. The MNNG-induced gastric cancer in male rats of different lines has histological structure predominantly of well-differentiated adenocarcinomas. However, administered to female rats, MNNG either causes carcinoids (Tahara et d., 1981) or produces no tumors at all (Furukawa et al., 1982). Injections of gastrin after the end of the period of MNNG administration sharply changes the histological spectrum in the fully developed carcinomas: most of the animals develop low differentiated and signet ring cell carcinomas instead of well-differentiated adenocarcinomas (Tahara and Haizuka, 1975; Tatsuta er al., 1977, 1980). Moreover, with the above hypothesis it is practically impossible to explain the presence in the well-differentiated adenocarcinomas of differentiated cells with signs not only of mucoid but also of the chief, parietal, endocrine, fibrillovesicular, goblet, and Paneth's cells (Sasano ef uf., 1969; Uchida er al., 1976; Kobori et al., 1977; Bykorez and Ivashchenko, 1982b). 2. The condition of the stem cell (in the sense of direction of commitment) does not play a decisive role at the moment of initiation. The differentiation occurring in one or another direction is determined by the combined effect of different factors of which some stimulate the differentiation ( hormones), and some inhibit it (e.g., various promotors inhibiting differentiation and disturbing intercellular metabolic cooperation). These factors continue to have an influence on stem cells after the initiation has finished and even after the completion of malignant transformation. It appears that this explanation is reasonably reliable and in good agreement with the data thus far obtained, although one cannot completely rule out the nonalternative character of the proposed possibilities which can be consequently realized in the course of tumor development.

E. FACTORSAFFECTINGDIFFERENTIATION OF TUMORCELLS Gastrin, which under normal conditions is a major trophic factor for epithelial cell populations of the gastrointestinal tract, stimulates synthesis of DNA, RNA,

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and proteins in the cells of gastric and large intestinal carcinomas (Kobori et al., 1982; McGregor et al., 1982). Some authors believe that the increased frequency of gastric cancer during pernicious anemia with concomitant atrophic gastritis and achlorhydria is associated with the increased production of gastrin acting as promotor of carcinogenesis (Elsborg and Mosbech, 1979). Inasmuch as gastrin causes hyperplasia of ECL cells, the appearance during pernicious anemia of carcinoids consisting of ECL cells is also believed to be associated with the high concentration of gastrin against the background of the increasing content of carcinogenic N-nitrosamines in the achylic stomach (Wilander, 1981). Thus, it appears that on the transformed cells there remain specific receptors for regulatory-trophic factors. Patients with Gardner’s syndrome often show hyperplasia of Brunner’s glands and substantially increased frequency of duodenal polyps and adenomas as well as of periampullar carcinomas (see St. Hilaire and Jones, 1982). Ablation of the submandibular salivary glands (sialoadenectomy) in mice considerably decreases the frequency of carcinoma of the large intestine following administration of DMH (Li et al., 1980). These findings indicate that the epidermal growth factor plays a major role in processes of transformation and proliferation of normal and transformed cells. Receptors for EGF have been found on cells of colon and rectum adenocarcinomas. The stimulation of DNA synthesis by means of EGF has been also confirmed (St. Hilaire e t a / . , 1980). It should be noted, however, that the loss of receptors for EGF by the cells is considered as the important sign of transformation (Todaro et al., 1981; Weinstein, 1981). It is likely that in adenocarcinomas only the cells committed to terminal differentiation may have such receptors. An interesting example of the exogenous factors effect on cellular differentiation in tumors is reported by Pinto et al. (1982). The replacement of glucose with galactose in the medium of HT-29 cells (taken from carcinoma of the large intestine) led to reversible differentiation: the undifferentiated epithelioid cells acquired strongly pronounced signs of cytotypical differentiation of absorptive enterocytes. Especially unexpected was the presence in the brush border of the enzymes previously observed only in the small intestinal enterocytes. The niechanisms of this phenomenon remain unclear. Under normal conditions, the growth factors stimulate proliferation of the stem cells and differentiation of part of them within the limits of their morphogenetic potentialities. The action of promotors (including “the transforming growth factors”) causes unlimited proliferation of the aberrant (initiated) stem cells and reactivation of abnormal differentiation programs in these cells (Weinstein, 1981) which are manifested in the appearance of the increased number of cells with unusual and mixed phenotypic signs of differentiation. As revealed in a series of studies, the promotors have a double effect: in some initiated cells

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they stimulate differentiation (which is frequently aberrant) whereas in others they inhibit differentiation simultaneously stimulating proliferation (Yamasaki, 1980). It is suggested that the mechanisms of stabilization of the transformed phenotype may be similar to that acting under normal conditions and ensuring stability of normal differentiation (Weinstein, 1981). It is not unlikely that both the ontogenetic maturation of stem cell populations of different tissues and the transformation of stem cells in the course of carcinogenesis depend upon gene rearrangements based on the existence of movable genetic elements (Cairns, 1981; Till, 1982).

VII. Conclusion Comparing the modem level in the biology of gastrointestinal stem cells with that achieved in the hemopoietic stem cell studies, we can say, using Till’s expression (1982), that at present the “morphological phase” in the investigations of the former is continuing. Still urgent is the problem of morphological verification of presumable stem cells in colonic crypts and gastric glands. Also important is to clarify the ways of renewal of the well-differentiated but low proliferating activity endocrine cells, the chief cells of the fundic glands, and the cells of Brunner’s glands. Further studies of interaction between epithelial and stromal cells are needed. The knowledge of relationships between stem cells and their differentiated “neighbors” in the niches, as well as of peculiarities of contacts between epithelial cells proper and of the cells with the basement membrane is of major importance. It is also necessary to clarify the mechanisms of the stem cells “anchoring” as well as of the disturbances of this important property during carcinogenesis. Briefly summarized below are the most essential, in our opinion, directions of the study of stem cells; some of these have already been started, whereas others can only be predicted.

I . The isolation from normal tissues of the fractions of intact cells enriched with the stem cells, with their subsequent culture. 2. The clarification of the factors governing the regulation of renewal of stem cells: specific stimulators and inhibitors of proliferation; and characterization of the receptor apparatus of stem cells, particularly for the enteropancreatic hormones. 3. The antigenic characteristic of stem cells. 4. The study of molecular peculiarities of DNA replication of clonogenic cells.

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5 . The molecular aspects of commitment and differentiation of stem cells.

6. The quantitative and qualitative characteristics of stem cells in the process of carcinogenesis. 7. The qualitative evaluation of the dynamics of formation and disappearance of carcinogenic DNA adducts in stem cells. 8. The clarification of interspecies features of stem cells as well as of their rnorphogenetic potentialities in onto- and phylogenesis. 9. The characterization of metaplastic changes in stem cells.

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This P a ge Intentionally Left Blank

Index

A

Autoradiography. computer-autoniated. I I I I17

c Callose rejection response. pollen-pistil interaction and, 263 Carcinogenesis, stem cells in disturbances in proliferation, 347-352 factors affecting differentiatitrn of tunior cells. 361-363 markers of differentiation disturbances. 352357 peculiarities of cytotypical differentiation in tumors, 344-347 problems of gastrointestinal neoplastic growth morphogenesis, 357-361 Cell(s) suspendcd effects of previous contacts with substrate on surface topography of. 199-303 morphology of surface microcxtensions, 274-284 surface topography of, 285-290 Chromatids. sister, unequal exchange of. 6871 Chromosomes. meinbrane function in transport of as componcnt of mitotic cytoskeleton. 224226 as mitotic anchor, 226 as part of force generation system. 227-230

Colon, stem cells origin of differentiated cells, 3 18-320 peculiarities of proliferation of low differentiated enterocytes, 320-322 quantitative characterization of. 322-323 Computer automated autoradiography and imniunocytochemistry and. I I I - I 17 other cell biology applications, I 17 uses in photometry and fluorescence microscopy, 107-1 1 I Computer system for microscope control and plotting, 90-Y3 niorphonietric measurements and, 98- 103 serial section reconstruction and, 93-97 video image processing and analysis, 103 107

D Deoxyrihonucleic acid sequence amplification in mammalian cells agent increasing frequency of growth-promoting suhbtances, 67 interference with DNA metabolism and. 64-66 cytological manifestations of double minutes, 50-52 homogeneously-staining regions, 45-50 relationship between DMs and HSRs, 5257 nature of amplified sequences, 57-58 endogenous genes, 58-63 transfected genes. 63-64

375

376

INDEX

occurrence of, 33 amplified loci in, 38-40 evidence for amplification during evolution, 42-43 known and probable amplifications in malignancy, 43-45 phylogenetic range, 34-38 transfected genes and, 40-42 proposed mechanisms of, 67-68 rereplication, 71-75 unequal sister chromatid exchange, 6871 Double minutes gene amplification and, 50-52 relationship to HSRs, 52-57

E Endoplasmic reticulum, in mitotic apparatus of higher organisms changes during mitosis, 181-195 general, 173-177 in isolated MA, 195 morphology, 177- 181 organisms thought to lack ER in MA, 196I98 Evolution, evidence for gene amplification during, 42-43

F Fertilization, progamic phase of, 259-261 Fluorescence microscopy, computer uses in, 107- I 1 I

G

Gastric glands, stem cells of cardiac, 323 differentiation and renewal of endocrine cells, 330-331 fundic, 323-330 pyloric, 330 Gastrointestinal epithelium, regulation of proliferation and differentiation paracrine, 333-337 regulatory mechanisms, 337-343 systemic modulators of cellular proliferation. 332-333

Genes endogenous, amplification of, 58-63 transfected, amplification of, 40-42, 63-64 Golgi membranes, in mitotic apparatus, 205209

H Homogeneously-staining regions gene amplification and, 45-50 relationship to DMs, 52-57 Horseradish peroxidase localization of reaction product in nerve cell bodies, 5-17 within nerve processes, 17-21 uptake into nerve terminals, 21-25 visualization of. 3-5

I Immunocytochemistry, computer-automated, 111-117

M Malignancy, DNA amplification in, 43-45 Membranes function in chromosome transport as component of /xitotic cytoskeleton, 224-226 as mitotic anchor, 226 as part of forcp generation system, 227230 function in regulation of [CaZ+ j general, 209-21 I in mitotic apparatus, 214-224 in nondividing systems. 21 1-214 Microcomputer, in research laboratories, 8490 Microextensions mechanism of formation of, 290-299 of suspended cells, morphology of, 274-284 Microscope, control and plotting, computer systems and, 90-93 Microtubules analysis inside cells, 134-139 biochemistry of asaembly/disassembly general concepts, 126 microtubule inhibitors, 126- I34

377

INDEX directional migration and assays for cell migration. 148-152 effect of inhibitors, 153 explanation for antiinvasive activity of inhibitors, 148-158 of invading cells, 159-161 mechanisms of direction finding, 158- 159 Microtubule inhibitors antiinvasiveness of in vitro, 139-143 in vivo. 143-144 antiproliferative and cytotoxic effect of, 144-148 effect on plasma membrane functions, 161 Mitochondria, in mitotic apparatus, 209 Mitotic apparatus endoplasmic reticulum of, in higher organisms changes during mitosis. 18 I - 195 general, 173- 177 in isolated MA, 195 morphology, 177-181 organisms thought to lack ER in MA, 196-198 membranes, early studies on, 170- 173 membrane function in regulation of [Ca2+] general, 209-21 1 in mitotic apparatus, 214-224 in nondividing systems, 21 1-214 membranes of, in lower organisms, 198204 other membranes in Golgi, 205-209 mitochondria, 209 Morphometric measurements, computer-aided, 99- 103

N Nerve localization of horseradish reaction product

in cell bodies, 5-17 processes, 17-2 1 uptake of horseradish peroxidase into terminals, 21-25

P Photometry, computer uses in, 107- I I I Pistil, receptive, 251-255

Plasma membrane, functions, effect of microtubule inhibitors on, 161 Pollen grain, mature and viable, 241-251 Pollen information. pistil read-out systems and, molecular basis for, 263-268 Pollen-pistil interactions attachment, 255-259 callose rejection response, 263 hydration, 259 molecular basis for pollen information and pistil readout systems, 263-268 pistil interactions, 261 -262 progamic phase of fertilization, 259-261

R Rereplication, gene amplification and, 7 1-75

S Serial sections, reconstruction, computer systems and, 93-97 Small intestine, stem cells of general characteristics of, 31 1-3 13 response to damaging agents, 3 13-3 I8 Stem cells carcinogenesis and disturbances in proliferation, 347-352 factors affecting differentiation of tumor cells, 361-363 markers of differentiation disturbances, 352-357 peculiarities of cytotypical differentiation in tumors, 344-347 problems of gastrointestinal neoplastic growth morphogenesis, 357-361 of colon ongin of differentiated cells, 318-320 peculiarities of proliferation of low differentiated enterocytes, 320-322 quantitative characterization of, 322-323 of gastric glands cardiac, 323 differentiation and renewal of endocrine cells, 330-33 1 fundic, 323-330 pyloric, 330 of small intestine general characteristics, 31 1-313 response to damaging agents, 313-318

378

INDEX

T Tumor cells, differentiation, factors affecting. 361-363

V Video images, processing and analysis, 103I07

Contents of Recent Volumes Volume 70

Volume 72

Cycling Noncycling Cell Transitions in Tissue Aging, Immunological Surveillance, Transformation, and Tumor GrowthSEYMOUR GELFANT The Differentiated State of Normal and Malignant Cells or How to Define a “Normal” Cell in CUhre-MINA J. BISSELL On the Nature of Oncogenic Transformation of CellS4ERALD L. CHAN Morphological and Biochemical Aspects of Adhesiveness and Dissociation of Cancer C e l l s AND YASUJI ~SHIMARU HIDEOHAYASHI The Cells of the Gastric MUCOSB-HERBERT F. HELANDER Ultrastructure and Biology of Female Gametophyte in Flowering Plants-R. N. KAPILA N D A. K. BHATNACAR

Microtubule-Membrane Interactions in Cilia and Flagek-wILLIAM L. DENTLER The Chloroplast Endoplasmic Reticulum: Structure, Function, and Evolutionary Significance-SARAH P. GIBES DNA Repair-A. R. LEHMANNAND P. KARRAN Insulin Binding and Glucose Transport-RusSELL HILF,LAURIE K. SORGE,AND ROGER J. GAY Cell Interactions and the Control of Development in Myxobactena Populations-DAVID WHITE Ultrastructure, Chemistry, and Function of the Bacterial Wall-T. J. BEVERIDCE INDEX

INDEX

Volume 73 Volume 71 Integration of Oncogenic Viruses in Mammalian Celk
Protoplasts O f Eukaryotic Algae-MARTHA D. BERLINER Polytene Chromosomes of Plants-WALTER NAGL Endosperm-Its Morphology, Ultrastructure, AND and Histochemistry-S. P. BHATNAGAR VEENASAWHNEY The Role of Phosphorylated Dolichols in Membrane Glycoprotein Biosynthesis: Relation to Cholesterol Biosynthesis-JOAN TUGEND H A t T MILLSA N D ANTHONY M. ADAMANY Mechanisms of Intralysosomal Degradation with Special Reference to Autophagocytosis and Heterophagocytosis of Cell OrganellesHANS GLAUMANN, JAN L. E. ERICSSON, A N D LOUISMARZELLA Membrane Ultrastructure in Urinary TubulesLELIOORCI, FAEIENNE HUMBERT,DENNIS BROWN,A N D ALAINPERRELET Tight Junctions in Arthropod Tissues-NANCY J. LANE Genetics and Aging in PrOtOZoa-JOAN SMITHSONNEBORN

INDEX

INDEX

379

380

CONTENTS O F RECENT VOLUMES

Organization and Expression of Viral Genes in Adenovirus-Transformed Cells-S. J. FLINT The Plasma Membrane as a Regulatory Site in Highly Repeated Sequences in Mammalian Genomes-MAXINE F. SINGER Growth and Differentiation of Neuroblastoma CellS-SlEGFRlED w. DE LAAT Moderately Repetitive DNA in EvolutionROBERTA. BOUCHARD AND PAULT. V A N DER SAAG Mechanisms That Regulate the Structural and Structural Attributes of Membranous Organelles in Bacteria-CHARLEs C. REMSEN Functional Architecture of Cell SurfacesD. BERLIN Separated Anterior Pituitary Cells and Their ReJANETM. OLIVERAND RICHARD sponse to Hypophysiotropic HormonesGenome Activity and Gene Expression in Avian AND MARIA Erythroid C d s K A R L E N G . GASARYAN CARLDENEF,Luc SWENNEN, ANDRIES Morphological and Cytological Aspects of Algal Calcification-MICHAEL A. BOROWITZKAWhat Is the Role of Naturally Produced Electric Current in Vertebrate Regeneration and HealNaturally Occurring Neuron Death and Its Reging?-RICHARD B. BORGENS ulation by Developing Neural PathwaysMetabolism of Ethylene by PlantS-JOHN TIMOTHYJ. CUNNINGHAM A. HALL DODDSAND MICHAEL The Brown Fat Cell-JAN NEDERCAARD AND INDEX OLOVLINDBERG

Volume 74

INDEX

Volume 75

Volume 77

Mitochondria1 Nuclei-TsuNEYOsHI KUROIWA Calcium-Binding Proteins and the Molecular Basis of Calcium Action-LINDA I. VAN ELSlime Mold LeCtinS-JAMES R. BARTLES, DIK, JOSEPHG. ZENEDECUI, DANIELR . WILLIAMA. FRAZIER,AND STEVEND. MARSHAK,A N D D. MARTINWATTERSON Ros EN Lectin-Resistant Cell Surface Variants of Eu- Genetic Predisposition to Cancer in Man: I n karyotic Cells-EvE BARAKBRILES Vifro Studies-LEVY KOPELOVICH Cell Division: Key to Cellular Morphogenesis in Membrane Flow via the Golgi Apparatus of Higher Plant Ceh-DAVlD G . ROBINSON the Fission Yeast, SchizosaccharomycesBYRONF. JOHNSON,CODEB. CALLUA, A N D U w KRISTEN BONGY. Yoo, MICHAELZUKER,AND IAN Cell Membranes in Sponges-WERNER E. G. J. MCDONALD MULLER Microinjection of Fluorescently Labeled Pro- Plant Movements in the Space Environmentteins into Living Cells, with Emphasis on DAVIDG. HEATHCOTE Cytoskeletal hOteinS-THOMAS E. KREIS Chloroplasts and Chloroplast DNA of Acefabularia mediferranea: Facts and HypothesesAND WALTERBIRCHMEIER Evolutionary Aspects of Cell DifferentiationANGELALUTTKE AND SILVANOBONOTTO Structure and Cytochemistry of the Chemical R. A. FLICKINGER SynapSeS-sTEPHEN MANALOV A N D WLADStructure and Function of Postovulatory FolliI M I R OVTSCHAROPF cles (Corpora Lutea) in the Ovaries of Nonmammalian Vertebrates-SRiNivAs K. SAI- INDEX DAPUR INDEX

Volume 78

Volume 76 Cytological Hybridization to Mammalian ChroInOSOmeS-ANN s. HENDERSON

Bioenergetics and Kinetics of Microtubule and Actin Filament Assembly-DisassemblyTERRELLL. HILL AND MARCW. KIRSCHNER

CONTENTS OF RECENT VOLUMES Regulation of the Cell Cycle by Somatomedins-HOWARD ROTHSTEIN Epidermal Growth Factor: Mechanisms of ActiOn-MANJUSRI DAS Recent Progress in the Structure, Origin, Composition, and Function of Cortical Granules in Animal Egg-SARDUL S. GURAYA

38 1

lmmunofluorescence Studies on Plant C e l l d . E. JEFFREE,M. M. YEOMAN, AND D. C. KILPATRICK Biological Interactions Taking Place at a LiquidSolid hkTfaCe-ALEXANDRE ROTHEN INDEX

INDEX

Volume 81 Volume 79 Oxidation of Carbon Monoxide by BacteriaYOUNG M. KIM AND GEORGED. HEGEMAN The Formation, Structure, and Composition of the Mammalian Kinetochore and Ki- Sensory Transduction in Bacterial Chemotaxi S 4 E R A L D L. HAZELBAUER AND SHlGEAKl netochore FiberXoNLu L. RIEDER HARAYAMA Motility during Fertilization4tRAm SCHATThe Functional Significance of Leader and TrailTEN er Sequences in Eukaryotic mRNAs-F. E. Functional Organization in the NucleusBARALLE RONALDHANCOCKA N D T E N I BOULIKAS The Relation of Programmed Cell Death to De- The Fragile x ChrOmOSOme~RANT R. SUTHERLAND velopment and Reproduction: Comparative Psoriasis versus Cancer: Adaptive versus Studies and an Attempt at ClassificationIatrogenic Human Proliferative DiseasesJACQUES BEAULATON AND RICHARDA. SEYMOUR GELFANT LOCKSHIN Cryofixation: A Tool in Biological Ultrastruc- Cell Junctions in the Seminiferous Tubule and the Excurrent Duct of the Testes: Freezeturd1 Research-HELMUT PLATTNER A N D Fracture Studies-TosHio NAGANO A N D LUISBACHMANN FUMIE SUZUKI Stress Protein Formation: Gene Expression and Environmental Interaction with Evolutionary Geometrical Models for Cells in Tissues-HisAO HONDA Significance<. ADAMSA N D R. W. RINNE Growth of Cultured Cells Using Collagen as INDtX Substrate-JASON Y A N G A N D s. NANDI INDEX

Volume 80 DNA Replication Fork Movement Rates in Mammalian Cells-LEON N. KAPP AND ROBERTB. PAINTER Interaction of Virsues with Cell Surface Receptors-MARC TARDIEU,ROCHELLEL. EPSTEIN, A N D HOWARD L. WEINER The Molecular Basis of Crown Gall InductionW. P. ROBERTS The Molecular Cytology of Wheat-Rye Hybrids-R. APPELS Bioenergetic and Ultrastructural Changes Associated with Chloroplast Development-A. R. WELLBURN The Biosynthesis of Microbodies (Peroxisomes, G1yoxysomes)-H. KINDL

Volume 82 The Exon:Intron Structure of Some Mitochondrial Genes and Its Relation to Mitochondria1 Evolution-HENRY R. MAHLER Marine Food-Borne Dinoflagellate ToxinsDANIELG. BADEN Ultrastructure of the Dinoflagellate AmphieSmit-LENmA c. MORRILLAND ALFREDR. LOEBLICH111 The Structure and Function of Annulate Lamellae: Porous Cytoplasmic and Intranuclear Membrane+-hCHARD G. ~ S E Morphological Diversity among Members of the

L

382

CONTENTS O F RECENT VOLUMES

Gastrointestinal Microflora-DWAYNE SAVAGE

C.

INDEX

Cell Surface Receptors: Physical Chemistry and Cellular Regulation-DoucLAs LAUFFENBURGER A N D CHARLES DELIS Kinetics of Inhibition of Transport Systems-R. M. KRUPKAAND R. D E V ~ S INDEX

Volume 83 Transposable Elements in Yeast-VALERIE MoROZ WILLIAMSON Techniques to Study Metabolic Changes at the

85

Receptors for Insulin and CCK in (he Acinar and Organ R' DEPancreas: Relationship to Hormone ActionFURIAAND MARYK. DYGERT IRA D. COLDFINE AND JOHNA. WILLIAMS Form and Function The Involvement of the Intracellular Redox State in Vivot Their Potential in Toxicology and and pH in the Metabolic Control of StimPathologyARoBERT AND ulus-Response C o u p l i n g - Z y c ~ u ~ o ROTH. I. ORD NAOMICHAYEN,AND SHABTAYDIKSTEIN Heterogeneity and Territorial Organization of of DNA Synthesis in Cultured Rat the Nuclear Matrix and Related StructuresHepatoma VAN Wl,K M. BOUTEILLE,D. BOUVIER,A N D A. P. Somatic Cell Genetics and Gene Mapping-FASEVE TEN KAO Associated Tubulin Isotypes and the Multigene Tubulin Changes in Membrane COWAN A N D L, with Cellular Aging-A. MACIEIRA-COELHO Families-N, Retinal Pigment Epithelium: Proliferation and The Ultrastructure of Plastids in Differentiation during Development and ReM , WHATLEY A N D VICgeneration4LcA G. STROEVA The Confined Function Model of the Golgi TOR I. MITASHOV Complex: Center for Ordered Processing of

*'

INDEX

Biosynthetic Products of the Rough Endoplasmic Reticuhm-ALAN M. TARTAKOW Problems in Water Relations of Plants and Cells-PAUL J. KRAMER Volume 84 Phagocyte-Pathogenic Microbe lnteractionsControls to Plastid Division-J. V. POSSINGHAM ANTOINETTERYTER AND CHANTAL~t AND M. E. LAWRENCE CASTELLIER Morphology of Transcription at Cellular and INDEX Molecular LeVelS-FRANCINE PUVIONDUTILLEUL An Assessment of the Evidence for the Role of Volume 86 Ribonucleoprotein Particles in the Maturation Toward a Dynamic Helical Model for the Influof Eukaryote mRNA-J. T. KNOWLER ence of Microtubules on Wall Patterns in Degradative Plasmids-J. M. PEMBERTON PlantsXLlvE W. LLOYD Regulation of Microtubule and Actin Filament Assembly-Disassembly by Associated Small Cellular Organization for Steroidogenesisand Large Mo~ecu~es-TERRELLL. HILL PETERF. HALL AND MARCW. KIRSCHNER Cellular Clocks and Oscillators-R. R. KLEVECZ,S . A. KAUFFMAN, AND R. M. Long-term Effects of Perinatal Exposure to Sex SHYMKO Steroids and Diethylstilbestrol on the Reproductive System of Male MammalsMaturation and Fertilization in Starfish YASUMASA A M , TAKAO MOW, YOSHIHIDE OOCYteS-LAURENT MEIJER A N D PIERRE GUERRIER SUZUKI.AND HOWARDA. BERN

CONTENTS OF RECENT VOLUMES

383

Development of the Cotton Fiber-AMARJIT S . BASRAA N D c. P. MALlK The Neuronal Organization of the Outer Plex- Cytochemistry of Fat Absorption-YuKARI A N D TOSHIMI MIZUNUMA iform Layer of the Primate Retina-ANDREW TAKAHASHI Electrical Activation of Arterial MuscleP. M A R I A N I INUbX DAVIDR. HARDERA N D ALANWATERS Aging of Cells in CUltUre-JAMES R . SMITH A N D D. W. LINCOLN, I1 Chemotactic Factors Associated with Leukocyte Volume 87 Emigration in Immune Tissue Injury: Their Separation, Characterization, and Functional The Modeling Approach-BARBARA E. Specificity-HioEo HAYASHI, MITSUOHONWmiwr DA, YASUO SHIMOKAWA, A N D MlTSUOMl Protein Diffusion in Cell Membranes: Some BiHIRASHIMA ological ~ m p ~ i c a t i o n s - M l c ~ ~ ~ lMc. Neural Organization and Cellular Mechanisms Cl.OSKbY A N D MLJ-MING PO0 of Circadian Pacemakers-JON W. JACKLET ATPases in Mitotic Spindles-M. M. PRATT Cytobiology of the Ovulation-Neurohormone Nucleolar StructureAuY GOtsstNs Producing Neuroendocrine Caudo-Dorsal Membrane Heterogeneity in the Mammalian Cells of Lvmnaea stagnalis-E. W. ROUBOS Spermatozoon-W. V . HOI.T Cell Biology of Trypanosomu CtW-WANDERLEY DE

SOUZA

Capping and the Cytoskeleton-LILLY Y . W GERARD J B O U R G ~ I I G N O ANN D BOLJKGUIGNON The Muscle Satellite Cell: A Review-DENNIS R. CAMPION Cytology of the Secretion in Mammalian Sweat KUROSUMI,SUSLJMU Glands-KAruMAsA S H I B A S A K I . A N D TOSHIO IT0

INDEX

Volume 90

Electron Microscopic Study of Retrograde Axonal Transport of Horseradish PeroxidaseERZSEBETFEHER 1NI)t.X DNA Sequence Amplification in Mammalian Cells-Jouc~ L. HAMLIN,JEFFREY D. MILBRANDT, NICHOLAS H. HEINTZ,A N D Volume 88 JANE c. AZIZKHAN Computer Applications in Cell and NeuLysosomal Functions in Cellular Activation: robiology: A Review-R. RANNEYMIZE Propagation of the Actions of Hormones and Effect of Microtubule Inhibitors on Invasion and Other Effectors-CLARA M. SZtGO A N D on Related Activities of Tumor Celk-MARC RICHARDJ . PlETRAS M. MAREELA N D MARCDE METS Neuronal Secretory SyStemS-MONA CASTEL, Membranes in the Mitotic Apparatus: Their H.-DIETER HAROLD GAINER, A N D Structure and FUnCtiOn-PETER K. HEPLER DELLMANN A N D STEPHENM. WOLNIAK INDEX Pollen-Pistil Recognition: New Concepts from Electron Microscopy and Cytochemistry-C, DUMAS,R. B. KNOX,A N D T. GAUDE Surface Topography of Suspended Tissue Volume 89 Cells-Yu. A. ROVENSKYAND Ju. M. VASILIEV Histochemistry of Zinc and Coppe-PETER Gastrointestinal Stem Cells and Their Role in A N D PETERKASA SZERDAHELYI AND Yu. D. Carcinogenesis-A. 1. BYKOREZ Histochemistry of Adenylate Cyclase-G. AND IVASHCHENKO POEGGEL, H. LUPPA,H . 4 . BERNSTEIN, INDEX J . WEISS

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