Advances in Cancer Research Volume 35

ADVANCES IN CANCER RESEARCH VOLUME 35

Contributors to This Volume Gerald L. Bartlett

Monika Graessmann

Morris C. Berenbaum

Berge Hampar

Walter Eckhart

John W. Kreider

Maria E. Ferioli

Lawrence Levine

Adolf Graessmann

Christian Mueller

Giuseppe Scalabrino

ADVANCES IN CANCER RESEARCH Edited by

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

SIDNEY WEINHOUSE Fels Research institute Temple University Medical School Philadelphia, Pennsylvania

Volume 35-7987

ACADEMIC PRESS A Subsidiary of Harcourt Brace Jovanovich, Publishers

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0

COPYRIGHT 1981, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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LIBRARY OF CONGRESS CATALOG CARDNUMBER:52-13360 ISBN 0-1 2-006635-1 PRINTED IN THE UNITED STATES O F AMERICA 81 82 83 84

9 8 7 6 5 4 3 2 1

CONTENTS CONTFUBUTORS TO VOLUME 35

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

ix

Polyoma T Antigens WALTERECKHART

I . Introduction .......................................................... I1. Polyoma T Antigens in Lytically Infected Cells .........................

1 1

Regulatory Signals .................................................... IV. Structural Features of the Pol yoma T Antigens-Comparison with SV40 ............................................................ V. Mutations Affecting the T Antigens ..................................... VI . Cell Transformation and Tumorigenicity ................................ VII . The Role of the Large T Antigen in Lytic Infection ..................... VIII . The Role of the Large T Antigen in Transformation ..................... IX. The Small and Medium T Antigens in Lytic Infection ................... X . The Small and Medium T Antigens in Transformation ................... XI . Functions Associated with the Small and Medium T Antigens ............ References ...........................................................

5

111. The Polyoma DNA Nucleotide S e q u e n c e x o d i n g and

9 11 12 13 15 18 19 21 23

Transformation Induced by Herpes Simplex Virus: A Potentially Novel Type of Virus-Cell Interaction BERCE HAMPAR I . Introduction .......................................................... I1 . Transformation-Definition of Terms ................................... 111. Models for in Vitro Transformation by HSV ............................. IV Properties of Transformed Cells ........................................ V. Persistance of HSV Genetic Material in Transformed Cells ............... VI . Mechanism of Transformation by HSV ................................. References ...........................................................

.

27 28 28 31 37 41 45

Arachidonic Acid Transformation and Tumor Production LAWRENCELEVINE I . Introduction .......................................................... I1 . Arachidonic Acid Transformation ....................................... V

49 49

vi

CONTENTS

I11. Prostaglandin Levels in Tumors

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

52

IV. Arachidonic Acid Transformation and Hypercalcemia .................... V. Arachidonic Acid Transformation and Tumor Promotion .................. VI . Prostaglandins: Their Effects on Cell or Tumor Growth .................. VII Prostaglandins and the Immune Response .............................. VIII . Challenges ........................................................... References ...........................................................

55 58 67 69 71 73

.

The Shope Papilloma-Carcinoma Complex of Rabbits: A Model System of Neoplastic Progression and Spontaneous Regression JOHN

w. KREIDER AND GERALDL . BARTLETT

I . Introduction: Historical Origins ........................................ I1 . Interaction of Shope Papilloma Virus and Host Cells .................... 111. Neoplastic Progression ................................................ IV. Spontaneous Regression ............................................... V. Shope Papilloma-Carcinoma Complex as a Model System ............... References ...........................................................

81 82 93 98 103 107

Regulation of SV40 Gene Expression AWLF GRAESSMANN. MONIKAGRAESSMANN. AND CHEUSTIAN MUELLER

. .

I Introduction .......................................................... I1 . Cell Type Dependence of SV40 and PV Gene Expression ............... 111 Functions of SV40 Tumor Antigens ..................................... IV. Cell Transformation ................................................... V. Microinjection: Applications and Trends ................................ References ...........................................................

111 114 125 139 140 146

Polyamines in Mammalian Tumors. Part I GIUSEPPESCALABRINO AND MARIAE . FERIOLI I . Introduction and Background .......................................... I1. Levels of Polyamines and Their Biosynthetic Enzymes in Fully Developed Experimental Tumors

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

111. Modification in Vivo and in Vitro of Tissue Polyamine

Metabolism by Chemical Carcinogens and Tumor Promoters ............. IV. Biosynthesis and Levels of Polyamines in Cells during the Virus-Induced Transformation Process .................................. V. Changes in Polyamine Biosynthesis and Content of Target Tissues by Physical Carcinogens.. ............................................. References ...........................................................

152 197 205 233 241 244

vii

CONTENTS

Criteria for Analyzing Interactions between Biologically Active Agents MORRISC . BERENBAUM

I . Introduction ................................. I1 . Effect Multiplication ...... .....

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

111. Effect Summation ......... ..... ............ IV. Agent Interaction and Self-Interaction .................................. V. Isoboles and the Interaction Index .................................. VI . Criteria Based on Changes in Dose-Response Curves . . . . . . . . . . . . . . . . . . . VII . Modifications of the Isobole Method .............. ........... VIII Therapeutic Optimization ............................................. IX . Conclusion ..................................... .... References ...........................................................

.

INDEX

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

CONTENTS O F PREVIOUS VOLUMES

269 273 280 285 288 304 313 322 329 332 337 341

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CONTRIBUTORS TO VOLUME 35 Numbers in parentheses indicate the pages on which the authors’ contributions begin.

GERALDL. BARTLETT,Departments of Pathology and Microbiology, The Milton S . Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033 (81) MORRIS C . BERENBAUM,Wellcome Laboratories of Experimental Pathology, Variety Club Research Wing, St. Mary’s Hospital Medical School, London W2 IPG, Great Britain (269) Tumor Virology Laboratory, The Salk Institute, WALTERECKHART, Sun Diego, California 92138 (1) MARIAE . FERIOLI,Znstitute of General Pathology and C.N.R. Centre for Research in Cell Pathology, University of Milan, 20133 Milan, Ztaly (151) ADOLF GRAESSMANN, Znstitut fur Molekularbiologie und Biochemie der Freien, Universitat Berlin, Berlin, Federal Republic of Germany (1 11) MONIKAGRAESSMANN, Znstitut fur Molekularbiologie und Biochemie der Freien, Universitdt Berlin, Berlin, Federal Republic of Germany (111) BERGEHAMPAR, Laboratory of Molecular Virology, National Cancer Institute, Frederick, Maryland 21 701 (27) JOHN W. KEIEIDER, Departments of Pathology and Microbiology, The Milton S . Hershey Medical Center, The Pennsylvania State Uniuersity, Hershey, Pennsylvania 17033 (81) LAWRENCELEVINE,Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254 (49) CHRISTIAN MUELLER,Znstitut fiir Molekularbiologie und Biochemie der Freien, Universitat Berlin, Berlin, Federal Republic of Germany (111) GIUSEPPESCALABFUNO, Institute of General Pathology and C.N.R. Centre for Research in Cell Pathology, University of Milan, 20133 Milan, Ztaly (151)

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POLYOMA T ANTIGENS Walter Eckhart Tumor Virology Laboratory. The Salk Institute, San Diego, California

I. Introduction

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

11. Polyoma T Antigens in Lytically Infected Cells .......................... 111. The Pol yoma DNA Nucleotide Sequence-Coding and

Regulatory Signals .................................................... IV. Structural Features of the Polyoma T Antigens-Comparison with SV40 ............................................................. V. Mutations Affecting the T Antigens ...................................... VI. Cell Transformation and Tumorigenicity ................................. VII. The Role of the Large T Antigen in Lytic Infection ...................... VIII. The Role of the Large T Antigen in Transformation ...................... IX. The Small and Medium T Antigens in Lytic Infection .................... X. The Small and Medium T Antigens in Transformation .................... XI. Functions Associated with the Small and Medium T Antigens.. ........... References ............................................................

1 1 5

9 11 12 13 15 18 19 21 23

I. Introduction

Polyoma ‘r antigens are viral-coded proteins found in infected or transformed cells. They are detected by immunofluorescence or immunoprecipitation, using sera from animals bearing polyoma-induced tumors. Because the T antigens are implicated in malignant cell transformation by polyoma, they have been studied intensively. This review summarizes the current picture of these proteins, including their structure, the regions of the viral genome coding for them, and their possible roles in cell transformation. For background information, the reader should consult the second edition of Molecular Biology of Tumor Viruses, Part 2, DNA Tumor Viruses (Tooze, 1980). In addition, there are several comprehensive reviews concerning the molecular biology of polyoma virus (Ito, 1980) and the T antigens of polyoma (Turler, 1980) and SV40 (Martin, 1981). The 1979 Cold Spring Harbor Symposium on Quantitative Biology (Volume 44, Viral Oncogenes) also contains much up-to-date information. 11. Polyoma T Antigens in Lytically Infected Cells

Polyoma virus produces a lytic infection in mouse cells. The viral replication cycle is divided into early and late phases, corresponding 1 ADVANCES IN CANCER RESEARCH, VOL. 35

Copyright 0 1981 by Academic Press,Inc. All rights of reproduction in any form reserved. ISBN 0-12-006635-1

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WALTER ECKHART

to the times before and after viral DNA replication. The viral genome is likewise divided into early and late regions. The early region encodes the T antigens, which are synthesized before viral DNA replication, The late region encodes the virion proteins, VP1, VP2, and VP3, which are synthesized after viral DNA replication. The viral genome is circular. The early region includes about 2900 base pairs, the late region about 2400 base pairs. The circular viral genome is conventionally represented as a physical map, divided into 100 units, with the EcoRI restriction enzyme cleavage site arbitrarily designated as OD00 map units (Griffin et ul., 1974; Fried and Griffin, 1977). On this physical map, the origin of viral DNA replication is located at about 70 map units. The early region of the viral genome extends clockwise from the replication origin, through the EcoRI site, to about 26 map units. The late region of the viral genome extends counterclockwise from the replication origin to about 26 map units. The overall organization of the genome of SV40 is similar to that of polyoma (Soeda et al., 1980). SV40 produces a lytic infection of monkey cells. The early region of the genome encodes the T antigens, and the late region encodes three virion proteins, VPl, VP2, and VP3, similar 'in size and organization to those of polyoma. The T antigens of SV40 will not be reviewed in detail here (see Martin, 1981), but features of their coding regions, structures, and possible functions in cell transformation will be compared to those of the polyoma T antigens. Although the overall genetic organization of the two viruses appears to be quite similar, the T antigens of polyoma and SV40 show important structural differences and may have functional differences as well. Polyoma T antigens in lytically infected cells have been isolated by immunoprecipitation using antitumor serum and analyzed by S DSpolyacrylamide gel electrophoresis (Ito et al., 1977a,b; Turler and Salomon, 1977; Hutchinson et al., 1978; Schaaausen et al., 1978). This kind of analysis depends on the ability of the antiserum to recognize proteins in the viral-infected cells. Generally, rat or hamster antisera have been used. The sera may have different reactivities against the different T antigens. In addition, other proteins may be precipitated nonspecifically, or specifically because of their association with the viral T antigens. Furthermore, protein modification or proteolysis may result in the appearance of related proteins having different apparent molecular weights. These factors make it necessary to use a variety of biochemical and genetic techniques to characterize the proteins in the immunoprecipitates.

POLYOMA T ANTIGENS

3

The major T antigen is a protein of apparent molecular weight 90,000-100,000 (actual molecular weight about 89,000) (Ito et al., 1977a,b; Turler and Salomon, 1977; Hutchinson et al., 1978; Schaffhausen et al., 1978; Deininger et al., 1980; Soeda et al., 1980). This is referred to as the large T antigen. A second protein, referred to as the small T antigen, has an apparent and actual molecular weight of 22,000-23,000 (It0 et al., 1977c; Hutchinson et al., 1978; Schaghausen et al., 1978; Deiningeret al., 1980; Soeda et al., 1980). A third protein, the “medium” or “middle” T antigen, has an apparent molecular weight of 55,000-60,000(actual molecular weight about 49,000) (It0 et al., 197713; Hutchinson et al., 1978; Schafthausen et al., 1978; Deininger et al., 1980; Soeda et al., 1980). In addition to these three viral-coded T antigens, a number of other proteins are found in the immunoprecipitates. The major virion protein, VP1, is often present, possibly because it occurs in large amounts in cells late during lytic infection. Several additional bands are found in the 50,000-60,000 molecular weight region and in the 30,000-40,000 molecular weight region. Some of these additional proteins have been characterized by peptide analysis (to be discussed later), but so far none of them appears to be virus coded. The intracellular location of the polyoma T antigens has been studied by cell fractionation (Ito et al., 1977b; Silver et al., 1978; Tiirler, 1980).The large T antigen is found mainly in the nucleus (Silveret al., 1978). By contrast, the medium T antigen appears to be localized in the plasma membrane fraction (It0 et al., 1977b). The large T antigen and the medium T antigen are also present in the cytoplasmic fraction, but in decreased amounts compared to their respective presences in the nucleus and the plasma membrane. The majority of the small T antigen is present in the cytoplasmic fraction (Silver et al., 1978). A number of lines of evidence can be used to establish which of the immunoprecipitable proteins are virus coded. One is in vitro translation of virus-specific mRNA from lytically infected cells (Hunter et al., 1978). When polyoma-specific RNA was translated in an mRNAdependent reticulocyte lysate, proteins related to the small, medium, and large T antigen could be immunoprecipitated from the translation product (Hunteret al., 1978). The only other detectable band was VP1, suggesting that the additional proteins observed in the 30,00040,000 and 50,000-60,000molecular weight regions in extracts of infected cells are not primary translation products of viral mRNA. The large T antigen synthesized in vitro has a slightly faster mobility than the large T antigen from infected cells (Hunter et al., 1978).

4

WALTER ECKHART

This difference most likely reflects modification of the large T antigen after translation in the infected cell. In fact, the mobility of the large T antigen, radiolabeled by a short pulse in infected cells, is similar to that of the in vitro product and is converted during a chase to the mobility of the large T antigen labeled continuously (It0 et al., 1977a; Hunter et al., 1980). Translation of polyoma complementary RNA (cRNA) in vitro produces only one immunoprecipitable protein, which has a mobility slightly slower than that of the small T antigen (Hunter et al., 1978). This result suggests that the medium and large T antigens could not be synthesized from unmodified primary transcripts of the viral DNA; it further suggests that the coding region for the small T antigen might contain an intervening sequence which, when present in cRNA, would contribute additional amino acid sequences to the protein synthesized in vitro. These interpretations were supported by subsequent analysis of the polyoma DNA nucleotide sequence (to be discussed later). The sizes of the mRNAs coding for the T antigen were analyzed by separation of the RNAs by gel electrophoresis, followed by translation of individual fractions in vitro (Hunter et al., 1978). The mRNAs for the small and medium T antigens are indistinguishable in size by this method, and both are larger than the mRNA for the large T antigen. The observation that polyoma has three T antigens, with a total apparent molecular weight of about 175,000, initially posed a problem because the coding capacity of the early region is only about 100,000 MW of protein. This problem is resolved by recognizing that the three T antigens are encoded in overlapping, but nonidentical, regions of the viral DNA. The structural relationships among the three polyoma T antigens have been investigated by peptide analysis of the isolated proteins (Hutchinson et al., 1978; Smart and Ito, 1978; Hunter et al., 1979, 1980; Simmons et al., 1979; Ito et al., 1980). Two systems have been employed: (1) separation in two dimensions by thin layer electrophoresis and chromatography (Hutchinson et al., 1978; Hunter et al., 1979, 1980; Ito et al., 1980) and (2) ion exchange chromatography, sometimes followed by paper chromatography (Smart and Ito, 1978; Simmons et al., 1979). There is general agreement on the following observations. The small, medium, and large T antigens share several peptides derived from the amino terminal regions of the three proteins, including a peptide thought to be the N-terminal tryptic peptide. The small and medium T antigens have some peptides not found in the large T antigen. Both the large T antigen and the medium T antigen have unique peptides that are not present in any other T antigens.

POLYOMA T ANTIGENS

5

These observations suggest that the three T antigens are encoded in overlapping but nonidentical regions of the viral genome. A more detailed analysis of the organization of the coding region for the T antigens has been made by comparing the nucleotide sequence of polyoma DNA to the observed peptides of the proteins (Hunter et al., 1979, 1980). The organization of the coding regions is consistent with the structure of early mRNAs and with the properties of mutants affecting the T antigens. I l l . The Polyoma DNA Nucleotide Sequence-Coding

and Regulatory Signals

The nucleotide sequence of polyoma DNA has been of great value in defining the details of the coding regions and regulatory signals for the synthesis of the viral proteins. The complete sequence has been worked out independently by two groups using different plaque isolates of the wild-type virus derived originally from the same strain (Soeda et al., 1980; Deininger et al., 1980). As a result, the sequences show some differences &om one another, especially in the noncoding regions of the genome. The numbering systems for the nucleotides are also different: Soeda et aZ. (1979) use the HpaII restriction enzyme fragment 3/5 junction, at about 70.5 map units, as the beginning for numbering; Friedmann et al. (1978b, 1979) use a nearby run of eight adenine-thymine (AT) base pairs in the region of the origin of replication. This results in a difference of 15 in the numbers of the nucleotides designating the same features in the early coding region, up to about 100 nucleotides beyond the EcoRI site at 0/100 map units, after which the numbers show more variation. For the beginning of the early coding region, then, the sequences are related as follows, Soeda et al. equals Friedmann et al. minus 15; for example, the EcoRI cleavage site is 1560-1565 in Soeda et al. and 1575-1580 in Friedmann et al. The numbering of Friedmann et al. will be used here. The structure of the polyoma early mRNAs is important for defining the intervening sequences removed from the RNAs after transcription. There are at least three early mRNAs, corresponding to the three T antigens. The mRNAs have 5’ ends mapping near nucleotide 165 (Kamen et al., 1980). The ATG codon for the N-terminal methionine residue, shared by the three T antigens, is at nucleotide 188-190. The intervening sequences have been mapped within a few nucleotides by analyzing mRNA-DNA heteroduplexes digested with single-strandspecific nuclease (Kamen e t al., 1980). The positions for splice junctions can be inferred by examining the DNA sequence for “consensus” splice junction sequences (Kamen et al., 1980).

6

WALTER ECKHART

FIG.1. Genomic organization of polyoma.

The genomic organization of polyoma is shown in Fig. 1. The arrangement of the. T-antigen coding regions is shown schematically in Fig. 2. The three T antigens have common N-terminal amino acid sequences encoded between nucleotides 188 and 424. The large T-antigen coding region has an intervening sequence removing nucleotides 425 through 809 from the mRNA. The small and medium T antigens share amino acid sequences encoded between nucleotides 425 and 761, which are not present in the large T antigen because they are within the intervening sequence. The small T antigen has an inter-

DNA

90

80

70 , 0

500

1000

I00 0 I500

10 2000

20 2500

mop units

3000 base pairs 2818

medium T 7 I88

sma"

1

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

761 824

1512

v p \ 8 2 1

188

810

FIG.2. Schematic representation of T-antigen coding region.

POLYOMA T ANTIGENS

7

vening sequence removing nucleotides 762 through 809 from the mRNA. This intervening sequence is within the coding region for the small T antigen. The coding region for the small T antigen continues beyond the intervening sequence for 11nucleotides before encountering a termination codon. The medium T antigen has an intervening sequence removing nucleotides 762 through 823 from the mRNA. This intervening sequence has the interesting feature of having the same proximal splice junction as the small T-antigen intervening sequence, but a different distal splice junction. A striking feature of the organization of the coding region is the use of two different open reading frames over a stretch of about 700 nucleotides for the synthesis of portions of the large and medium T antigens. Support for this feature of the genetic organization comes from the nucleotide sequence itself, which shows that two open reading frames are available in that region (Friedmann et al., 1979; Soeda et al., 1979), from the properties of mutations affecting the T antigens (discussed later), and from a comparison of observed and predicted peptides of the large and medium T antigens (Hunter et al., 1979, 1980). The nucleotide sequence predicts that there should be seven methionine-containing tryptic peptides unique to the medium T antigen encoded in one of the open reading frames between nucleotides 824 (immediately following the distal splice junction) and 1515 (the end of the termination codon for medium T). The number and properties of the observed methionine-containing tryptic peptides, analyzed by two-dimensional thin layer chromatography and electrophoresis, are in agreement with the predicted peptides derived from the presumed reading frame (Hunter et al., 1979,1980). Two of the predicted peptides have been synthesized chemically. The synthetic peptides migrate identically to two of the observed peptides (Hunter et al., 1979, 1980). This evidence, together with the genetic evidence (It0 et al., 1980) (to be discussed later), establishes the reading frame used to encode the medium T antigen. A similar approach has been used to identify the other open reading frame as the one coding for the large T antigen (Hunter et al., 1979, 1980). Several additional features of the polyoma nucleotide sequence are notable. As previously mentioned, there are differences among polyoma strains in the nucleotide sequences of the noncoding region between the origin of viral DNA replication and the initiation codon for the synthesis of the T antigens. In fact, substantial deletions of DNA can occur in this region, and viable deletion mutants, not grossly affected in infectivity or transforming ability, have been isolated (Bendig and Folk, 1979; Magnusson and Berg, 1979; Wells et d., 1979). By

8

WALTER ECKHART

contrast, the regions encoding the small and medium T antigens (about 1300 base pairs) are identical in the two isolates that have been sequenced, except for a single base difference at nucleotide 1232 (Soeda et al., 1980; Deininger et al., 1980). This observation suggests that changes in the noncoding region are tolerated more easily than changes in the coding regions. Ordinarily, this might not be surprising, but the region unique to the small and medium T antigens does not seem to be essential for the viability of the virus, except under certain conditions (see following discussion of mutations). Perhaps alterations in the common N-terminal region of the three proteins are discouraged because they have a deleterious effect on the large T antigen, which is required for viral DNA replication, and alterations in the regions common to the small and medium T antigens are discouraged because these proteins have some essential functions in replication under the tissue culture conditions ordinarily used to grow polyoma. The coding region for the T antigens has two proximal and two distal splice junctions (Kamen et al., 1980). As previously described, the large T-antigen mRNA is apparently spliced between nucleotides 425 and 809, the small T-antigen mRNA is apparently spliced between nucleotides 762 and 809, and the medium T-antigen mRNA is apparently spliced between nucleotides 762 and 823 (Kamen et al., 1980).A fourth splice combination is possible between the proximal junction at nucleotides 424425 and the distal junction at nucleotides 8231824. An mRNA spliced in this manner would produce a protein of 85 amino acids, with a molecular weight of about 9700. So far, such a protein has not been observed, but it might have escaped detection because of its low molecular weight. The signals for mRNA initiation and polyadenylation have not been precisely defined. However, the sequence AATAAA, often found 15-30 nucleotides preceding the sites of polyadenylation in eukaryotic messages, occurs in the polyoma DNA sequence at nucleotides 29372942, near the 3' end of the early mRNAs. This is immediately following the termination codon for the large T antigen in the sequence of Soeda et al. and 15 nucleotides after the termination codon in the sequence of Friedmann et al. (Interestingly, the two sequences predict different C-terminal sequences for the large T antigen owing to a frame shift in one sequence compared to the other because of an extra nucleotide at position 2912 in the sequence of Friedmann et al. This sequence predicts two C-terminal amino acids to be encoded following this position, whereas the sequence of Soeda et al. predicts seven.) Studies of polyoma early mRNAs have detected a minor RNA species with a 3' end near nucleotide 1525, just after the termination

POLYOMA T ANTIGENS

9

codon for the medium T antigen at nucleotides 1513-1515 (Kamen et al., 1980; W. Heiser, personal communication). There is also a potential polyadenylation signal in this region, AATAAA, at nucleotides 14911496. It is not known how this minor RNA species is spliced, that is, whether it codes for the small or medium T antigens or for an unidentified additional protein. IV. Structural Features of the Polyoma T Antigens-Comparison

with SV40

The amino acid sequence of each of the polyoma T antigens can be deduced from the nucleotide sequence of the DNA. The nucleotide sequence also provides a means of making a detailed comparison of the genome of polyoma with that of SV40 (Friedmann et al., 1979; Soeda et al., 1980). Previous comparisons had to rely on nucleic acid hybridization and protein characterization; the nucleotide sequences give a more detailed picture. The overall genetic organization of SV40 is similar to that of polyoma, as previously noted. Both genomes are divided into early and late regions, the early regions encoding the T antigens and the late regions encoding the virion proteins. A major difference occurs in the organization of the T antigens, however. The SV40 genome encodes only two T antigens, a small T antigen (20,500 MW) and a large T antigen (81,600 MW). Examination of the SV40 nucleotide sequence does not reveal any extensive open reading frame which could encode a medium T antigen, such as is found with polyoma (Friedmann et al., 1979; Soeda et al., 1980). The small and large SV40 T antigens have common N-terminal regions, as do the polyoma T antigens, and the C-terminal portion of the SV40 small T antigen is encoded within an intervening sequence removed from the large T-antigen mRNA, as is the case with the polyoma small and medium T antigens. The splicing pattern of the SV40 early mRNAs is similar to that of polyoma in that the small and large T-antigen mRNAs use the same distal splice junction but different proximal splice junctions. However, the splice in the SV40 small T-antigen mRNA is beyond the termination codon for the protein, whereas in pol yoma the splice precedes the termination codons. There is considerable homology between the polyoma and SV40 genomes, in 80%of the early coding region, at both the nucleotide and amino acid sequence levels (Friedmann et al., 1979; Soeda et al., 1980). If the coding sequences in the early regions of the two viruses are aligned so as to maximize homologies, it is apparent that there is a region where the homology is much lower than average: the region

10

WALTER ECKHART

where the large and medium T antigens of polyoma are translated in alternative open reading frames. The homology is also lower than average at the C-terminal ends of the two large T antigens. These similarities and differences in homology between the pol yoma and SV40 T antigens may reflect similarities and differences in the functions of the proteins, but there is not yet sufficient information about the functions of the proteins to allow any firm conclusions to be drawn. An interesting and unusual feature of the polyoma and SV40 small T antigens is the arrangement of cysteine residues (Friedmann et al., 1978a). Eight of the 11 cysteine residues in the polyoma small T antigen can be aligned with eight of the 11 cysteine residues in the SV40 small T antigen. There are two regions in each protein (amino acid residues 120-125 and 148-153) in which the cysteines are clustered in an arrangement Cys x Cys xx Cys. These cysteine clusters are in the portion of the small T antigen not shared with the large T antigen (i.e., the large T-antigen intervening sequence) for both polyoma and SV40. Such arrangements of cysteine residues are uncommon. However, the sequence Cys x Cys xx Cys occurs in the glycoprotein hormones, thyroid-stimulating hormone, luteinizing hormone, and chorionic gonadotropin (Friedmann et al., 1978a). Whether this similarity reflects some functional similarity remains to be determined. Some features of the predicted amino acid sequence for the C-terminal half of the polyoma medium T antigen are notable (Hunter et al., 1979, 1980; Soeda et al., 1980).This half of the molecule has an unusually high proportion of hydrophobic residues (tyrosine, tryptophan, phenylalanine, leucine, isoleucine, and valine): 30% compared to 15%in the large T antigen encoded in the same region. There are some striking hydrophobic clusters, such as 1 of 21 amino acids encoded by nucleotides 1432 to 1494, ending 6 amino acids from the C-terminus of the medium T antigen. There are no charged amino acids in this cluster. Such a sequence might be responsible for anchoring the medium T antigen in a membrane. The distribution of acidic and basic amino acids in the medium T antigen is also unusual (Hunter et al., 1979, 1980; Soeda et al., 1980). The amino acid sequence encoded between nucleotides 958 and 1059 is basic; it contains no acidic residues. The amino acid sequence encoded from nucleotides 1069 to 1236, by contrast, contains 20 acidic residues, including a run of six glutamic acid residues, and only one basic residue. The C-terminal end of the medium T antigen, from nucleotide 1357 to the termination codon at 1513-1515, again is basic; it contains no acidic residues. Although these unusual features are suggestive, it is not yet possible to associate them with functional properties of the T antigens. One

POLYOMA T ANTIGENS

11

promising approach to this problem is site-specific mutagenesis (to be discussed). It may be possible to mutate specific portions of the genome-for example, the region encoding hydrophobic residues that might anchor the medium T antigen in a membrane-and correlate changes in structure with changes in location or functions of the proteins. V. Mutations Affecting the T Antigens

Mutations affecting the T antigens have been useful in defining the coding regions and functions of the proteins. The first mutants isolated were temperature-sensitive mutants, selected on the basis of their ability to form plaques at a permissive temperature but not at a restrictive temperature (Fried, 1965a; Eckhart, 1969; di Mayorca et al., 1969). The temperature-sensitive mutants affecting the large T antigen are called tsA mutants and are defective in viral DNA replication during lytic infection at the restrictive temperature. They are also defective in cell transformation when infection is carried out at the restrictive temperature. Corresponding tsA mutants of SV40 have a similar defect in viral DNA replication at the restrictive temperature and are also defective in cell transformation at the restrictive temperature. The effect of these mutations on maintenance of transformed cell growth properties at the restrictive temperature will be discussed in more detail later. The tsA mutants of polyoma and SV40 have been mapped by heteroduplex marker rescue (Lai and Nathans, 1974, 1975a,b; Feunteun et al., 1976; Miller and Fried, 1976). This technique localizes a mutation to a particular restriction enzyme fragment but does not give a precise location at the nucleotide level. In the case of the polyoma tsA mutants, all of the mutations mapped so far have been localized to the distal portion of the early region of the genome, between about 2 and 26 map units (Feunteun et al., 1976; Miller and Fried, 1976). This localizes them to the coding region of the C-terminal portion of the large T antigen, beyond the termination codon for the medium T antigen. The tsA mutants of SV40 are similarly localized in the C-terminal portion of the SV40 large T antigen (Lai and Nathans, 1974, 1975a,b). It is noteworthy that no tsA mutation among the 15 or so mapped so far occurs in the N-terminal region of the large T antigen shared with the small or medium T antigens. A second class of mutations affecting the T antigens are the hr-t (host range nontransforming) mutations (Benjamin, 1970). These mutants were selected on the basis of their ability to grow on a polyomatransformed mouse cell but not on its normal counterpart. Mutants selected in this way were found to be defective in cell transformation. Subsequently it was found that the hr-t mutants showed variable abil-

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WALTER ECKHART

ity to grow on a variety of normal and transformed cells, and the basis of the host range effect is not yet clear (Benjamin and Goldman, 1975; Goldman and Benjamin, 1975). The hr-t mutations are localized in the portion of the early region of the genome coding for the small and medium T antigens, within the intervening sequence for the large T antigen (Feunteun et al., 1976; Staneloni et al., 1977). Consequently, they affect the small and medium T antigens simultaneously but do not affect the large T antigen (Ito et al., 1977b,c; Schafthausen et al., 1978; Hutchinson et al., 1978; Ito, 1979). Several of the hr-t mutations have been analyzed by DNA sequencing (Benjamin et al., 1980; Hattori et al., 1979; Carmichael and Benjamin, 1980; Soeda and Griffin, 1978). Many of the mutations are deletions which shift the reading frame for the small and medium T antigens, resulting in abnormal termination of the proteins (Benjamin et al., 1980).There is a class of deletion mutations in SV40 located in a corresponding region of the SV40 genome, within the intervening sequence for the large T antigen, affecting the C-terminal portion of the SV40 small T antigen (Cole et al., 1977; Crawford et al., 1978; Shenk et al., 1976). A third class of mutations affecting the T antigens is a deletion mutations, which can be either viable or nonviable depending on their locations and functional effects. Originally the nonviable deletions were propagated together with a nondefective polyoma helper virus. Now the nonviable mutations are generally introduced into viral DNA cloned in bacteria, where the DNA can be propagated as part of a plasmid or phage. The viable deletion mutations isolated so far that affect the polyoma T antigens are located in the region of the genome coding simultaneously for the large and medium T antigens (Bendig et al., 1980; Magnusson and Berg, 1979; Griffin and Maddock, 1979). These mutations have variable effects on cell transformation, which will be discussed later. The tsA and hr-t mutants complement each other for transformation (Eckhart, 1977; Fluck et al., 1977). Mixed infection of cells by a tsA mutant and an hr-t mutant leads to cell transformation at the restrictive temperature, whereas infection by either of the mutants alone does not. This result suggests that the role of the large T antigen in transformation is different from that of the small andlor medium T antigens. VI. Cell Transformation and Tumorigenicity

Injection of polyoma into newborn hamsters leads to multiple tumors, both at the site of injection and at other sites (reviewed by

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Weil, 1978). Infection of tissue culture cells by polyoma leads either to lytic infection, in which the cells are killed, or to an abortive infection, in which the cells survive. A small proportion of the abortively infected cells acquire new growth characteristics, and it is this process which is referred to as cell transformation. In some cases, cells transformed in culture acquire the ability to form tumors in animals. The transformed cells contain viral DNA, usually covalently integrated into cellular DNA, and express virus-specific early RNA and some or all of the T antigens. Transformed cells can differ from their untransformed counterparts in many ways. In general, transformed cells are selected on the basis of their ability to grow to higher saturation densities, resulting in the formation of foci on monolayers of untransformed cells, or on the basis of their ability to grow in an anchorage-independent manner, forming colonies in semisolid agar. Transformed cells often have other properties: They may be agglutinated readily with lectins, often release plasminogen activator activity, tend to be difficult to arrest in depleted medium, and do not usually have prominent actin networks. There is no single phenotype that defines a transformed cell, however, and the spectrum of transformed cell growth properties overlaps that of normal cells. For the purpose of analyzing transformation by polyoma, a cell is considered to be transformed after infection if it is able to multiply to form a focus on a monolayer of parental cells or to form a colony in semisolid agar. Transformation can be either abortive or stable. A large proportion of cells transiently exhibit altered growth properties after infection by polyoma, in particular, the ability to undergo a few cell divisions in agar suspension. This is referred to as abortive transformation. A small fraction of the infected cells go on to be stably transformed, that is, to express altered growth properties permanently. It is likely that stable transformation requires the presence of viral DNA in a state in which it can express the viral information required to alter cell growth properties. Therefore, the difference between abortive and stable transformation may be in whether viral DNA is stably maintained and expressed in the progeny of an infected cell. VII. The Role of the Large T Antigen in Lytic Infection

The T antigens are expressed during the early phase of lytic infection. The role of the large T antigen has been analyzed by studying the effect of mutations in the large T antigen on processes occurring in lytically infected cells. As noted above, the tsA mutants of polyoma

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and SV40 map in the region of the genome coding for the C-terminal portion of the large T antigen and outside the coding region for the small andlor medium T antigens. Therefore, the tsA mutations can be used to analyze the effects of functional or nonfunctional large T antigens in infected cells. From this kind of analysis, the large T antigen appears to be involved in at least two processes important for the life cycle of the virus during lytic infection: viral DNA replication and regulation of early RNA transcription. The large T antigen is required for the initiatioii of viral DNA replication by both SV40 and polyoma (Tegtmeyer, 1972; Francke and Eckhart, 1973). Cells infected by tsA mutants fail to initiate viral DNA replication at the restrictive temperature or after a shift from the permissive to the restrictive temperature. Viral DNA replication, once initiated, appears no longer to require the large T antigen. Permissive cells infected with polyoma or SV40 tsA mutants show increased levels of early mRNA at the restrictive temperature compared to cells infected with the wild-type virus (Reed et aZ., 1976; Alwine et al., 1977; Khoury and May, 1977; Birkenmeier et aZ., 1977; Cogen, 1978). The rate of early mRNA synthesis increases in tsA mutant-infected cells on a shift from the permissive to the restrictive temperature. This implies that a functional large T antigen inhibits the synthesis of early mRNA, thereby regulating its own synthesis (Tegtmeyer et aZ., 1975; Khoury and May, 1977; Edwards et al., 1979). The large T antigen binds to viral DNA preferentially in the region of the origin of replication, presumably reflecting its function in the initiation of viral DNA replication (Prives et al., 1980; Oren e t al., 1980; Tjian, 1978, 1979; Reed et aZ., 1975; Jesse1 et al., 1976). It is not yet clear whether the same interaction of the large T antigen with viral DNA is involved in both replication and regulation of early transcription. This question will probably be clarified by further studies of DNA replication and transcription using mutations affecting the large T antigen and the region of the viral DNA to which it binds. The large T antigens of polyoma and SV40 are rendered thermolabile by tsA mutations; the large T antigens are degraded more rapidly at the restrictive temperature in cells infected by these mutants (Alwine et al., 1975; Kuchino and Yamaguchi, 1975; Tenen et d., 1975; Ito et al., 1977a; Hutchinson et al., 1978; Silver et al., 1978) than in cells infected by the respective wild-type viruses. Because of the increased synthesis of early mRNA, however, the rate of synthesis of the large T antigen also increases in cells infected by tsA mutants at the restrictive temperature. Therefore, the amount of the large T anti-

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gen protein in tsA mutant infected cells may not change greatly following a shift to the restrictive temperature (Edwards et d.,1979). VIII.

The Role of the Large T Antigen in Transformation

The role of the T antigens in transformation has been studied in a variety of ways: by characterizing the T antigens expressed in transformed cells, by studying the effects of mutations in the T antigen on the properties of transformed cells, and by infecting cells with DNA carrying different portions of the early region of the viral genome. The expression of the large T antigen in polyoma-transformed cells is variable, depending on the number and arrangement of the integrated viral DNA sequences. If viral DNA sequences are integrated at several positions, or in several orientations, in the cellular DNA, the pattern of T-antigen expression can become quite complex. Different lines of polyoma-transformed cells show different T antigen patterns with great variation from one cell line to another (It0 et al., 1980; It0 and Spurr, 1980; Lania et aZ., 1980a,b). I n all cases analyzed so far, the small and medium T antigens are present in the transformed cells. The expression of the large T antigen varies from one cell line to another and depends on the species (rat, hamster, or mouse) of the transformed cell. In some lines-particularly rat c e l l s - a full size large T antigen is often expressed. In some lines, truncated forms of the large T antigen are expressed; in others, the protein appears to be larger than the species found during lytic infection. In polyoma-transformed mouse cells, the full-size large T antigen is generally not expressed. The variety of forms of the polyoma large T antigen expressed in transformed cells can be explained by the variety of arrangements of the integrated viral genomes and the interactions of the large T antigen with different species of host cells. In a few cases, the T antigens expressed in polyoma-transformed cells have been correlated with the state of the viral sequences deduced by restriction enzyme digestion and blot hybridization (Lania et al., 1980a,b). Because of the complications introduced by free viral genomes in transformed rat cell lines, the analysis has been confined to cases in which there are no free viral genomes and only a single insert of viral DNA. The insertion of viral sequences occurs at different places in the cellular DNA, and the region of the viral genome joined to cellular DNA is different in different transformed cell lines. In some cases, the viral sequences are organized in head-to-tail tandem duplications (Basilico et al., 1979,

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1980; Birg et al., 1979; Lania et al., 1979). All the cells which did not express the full-size large T antigen are found to have discontinuities in the coding region for the large T antigen, caused either by deletions or by fusion to host sequences. The polyoma mRNAs present in transformed cells also have been characterized and are consistent with the forms of the T antigens expressed (Kamen et al., 1980). Only cells containing free viral DNA contain mRNAs corresponding to the entire early region of the viral DNA. The cell lines containing polyoma inserts with discontinuities in the coding region for the large T antigen do not contain mRNA sequences from the region interrupted by the discontinuity. In some cases, transcription of integrated viral sequences continues into, and terminates in, adjacent cellular sequences. So far, transcription appears to be initiated at viral promotors; no readthrough from cellular promotors has been observed (Kamen et al., 1980). The absence of the full-length large T antigen from transformed mouse cells can be explained by the involvement of the large T antigen in viral DNA replication and the lytic interaction between mouse cells and polyoma (Hutchinson et al., 1978; Fluck and Benjamin, 1979). If a functional large T antigen were present in a permissive mouse cell, the viral DNA would be expected to replicate, resulting in loss of integrated viral DNA and death of the host cell (Vogt, 1970; Cuzin et al., 1970; Folk, 1973; Basilico et al., 1979). Apparently, this situation is avoided by the interruption of the coding region for the large T antigen in permissive transformed cells (Lania et al., 1980a). Complete viral genomes can be maintained in some polyomatransformed mouse cells transformed by tsA mutants and maintained at the restrictive temperature (Fried, 196513; Eckhart, 1969; di Mayorca et al., 1969; Vogt, 1970; Folk, 1973; Bourgaux et al., 1978; Basilico et al., 1979). At the restrictive temperature, the ability of the large T antigen to replicate the integrated viral DNA is presumably blocked, obviating the necessity of selecting for inactive large T antigens by other mechanisms. Another mechanism of maintaining a functional large T antigen in a transformed permissive cell is to mutate or remove the viral origin of replication so that replication cannot occur. This has been done with SV40 (Gluzman et al., 1980)by infecting permissive monkey cells with viral DNA lacking sequences in the region of the origin of replication. The universal presence of the polyoma small and medium T antigens in transformed cells, coupled with the frequent absence of the large T antigen, suggests that the C-terminal portion of the large T

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antigen (encoded from roughly 0 to 26 map units) is not required for the expression of transformation by polyoma. The role of the SV40 large T antigen in transformation, inferred from its presence in transformed cells, appears to be different from that of the polyoma large T antigen. The large T antigen of SV40 is always present in transformed cells, more than 100 transformants having been analyzed so far (Martin, 1981). The role of the polyoma large T antigen in transformation has been studied by testing the properties of tsA mutant-transformed cells grown at the permissive and restrictive temperatures. As previously noted, polyoma tsA mutants fail to transform cells when infection is carried out with virions at the restrictive temperature. Many cells, when transformed at the permissive temperature, retain their transformed characteristics at the restrictive temperature (Fried, 1965b; Eckhart, 1969; di Mayorca et al., 1969; Vogt, 1970; Folk, 1973; Basilico et al., 1979). In retrospect, this is not surprising, as many polyoma transformants do not express the portion of the large T antigen in which the tsA mutations map and therefore would not be expected to show temperature dependence owing to changes in the function of the large T antigen (Kamen et al., 1975; Eckhart, 1977). Some rat cell lines do retain a functional large T antigen, however, and some of these lines display temperature-dependent growth properties when transformation is carried out with tsA mutants (Seif and Cuzin, 1977). The proportion of transformed cells expressing a temperature-dependent phenotype has been reported to depend on the multiplicity of infection and the conditions under which the cells are kept after infection: Seeding at low density favors the appearance of temperature-dependent transformants (Rassoulzadegan et al., 1978a,b, 1980; Rassoulzadegan and Cuzin, 1980). In other cases, there seems to be substantial variability in the temperature dependence of the growth properties of transformants derived from a single cell line using a particular virus, even wild-type (Fluck and Benjamin, 1979). On balance, the evidence available up to now suggests that the polyoma large T antigen is required at an early stage in transformation, at least when infection is carried out using virions, but is not necessary to maintain transformed cell growth properties. The temperature dependence of some polyoma tsA mutant-transformed cells may be explained by some peculiarity of transformed cells that express a fullsize large T antigen, perhaps a regulatory effect on the small and/or medium T antigens. In the case of SV40, the retention of a full-size large T antigen in all transformants tested and the failure to transform

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cells with viral DNA not containing the complete early region suggest that the SV40 large T antigen is necessary for full expression of transformation by SV40. The polyoma large T antigen is involved in the excision of integrated viral DNA molecules from the DNA of transformed cells. This was originally suggested by the induction of virus replication in polyoma tsA mutant-transformed mouse cells after a shift from the restrictive to the permissive temperature (Cuzin et al., 1970). The process has been investigated further using transformed rat cell lines in which free and integrated viral genomes can coexist (Prasad et al., 1976). The free viral DNA molecules occur because of excision and limited replication of the integrated molecules (Zouzias et al., 1977). Only a small fraction of the cells in the population contain free viral DNA at any one time, and the free DNA disappears when tsA mutanttransformed cells are grown at the restrictive temperature, suggesting that the free viral DNA results from spontaneous induction of replication requiring a functional large T antigen (Zouzias et al., 1977; Basilico et al., 1979). The nature of the excision process has been studied by characterizing the free viral DNA molecules appearing in transformed rat cell populations (Gattoni et al., 1980). The transformed cells contain viral DNA molecules whose structure reflects that of the integrated species, which is usually present in a tandem head-to-tail orientation of full length and defective molecules. The results support the notion that homologous recombination is involved in the generation of free viral DNA from the integrated sequences (Gattoni e t al., 1980). Amplification of integrated polyoma DNA sequences can also occur in transformed cells after the initial integration event. This process also requires a functional large T antigen, as it does not occur in tsA mutant-transformed rat cells at the restrictive temperature (Colantuoni et al., 1980). IX. The Small and Medium T Antigens in Lytic Infection

The polyoma small and medium T antigens are affected simultaneously by the hr-t mutations, which map in the region of the genome encoding these two proteins but within the intervening sequence for the large T antigen. The small and medium T antigens are not absolutely required for lytic infection because, in general, the hr-t mutants are viable, although they show differences in ability to grow in different host cells (Benjamin and Goldman, 1975; Goldman and Benjamin, 1975). As discussed previously, the hr-t mutants characterized so far

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are of three kinds: out-of-frame deletions, an in-frame deletion, and a three base pair insertion followed by a single base pair change. The effects of these mutations on the small and medium T antigens are the following: 1. Neither the small T nor the medium T antigen is detected in cells infected with the out-of-frame deletion mutants; presumably, the aberrant proteins formed by premature termination of translation are unstable. 2. A shortened medium T antigen is detected in cells infected with the in-frame deletion mutant. 3. Normal-size medium and small T antigens are detected in cells infected by the insertion mutant, but the amount of the small T antigen seems diminished relative to the amount of wild-type small T antigen (Silver et al., 1978; Benjamin et al., 1980). X. The Small and Medium T Antigens in Transformation

The small and medium T antigens are implicated in transformation by mutations affecting one or both proteins. The hr-t mutants are defective in transformation and complement the tsA mutants for transformation at the restrictive temperature (Eckhart, 1977; Fluck et al., 1977). Therefore, at least two functions involved in transformation are encoded in the early region of the polyoma genome. Because the hr-t mutants affect the small and medium T antigens simultaneously, the properties of the hr-t mutants do not separate the possible functions of the two proteins in transformation. Other experiments, using both polyoma-induced tumors and transformed cells, have shown that fragments of polyoma DNA, able to express the small and medium T antigens but not the large T antigen, are competent for transformation (Israel et al., 1979a,b, 1980a,b; Novak et al., 1980; Hassell et al., 1980).When tumors are induced in hamsters by the inoculation of polyoma DNA, the tumorigenicity of the DNA is increased severalfold if the DNA is linearized by interrupting the early region of the genome in the coding region for the C-terminal half of the large T antigen (Israel et al., 1979b, 1980a). The resulting tumors express the small and medium T antigens but not the large T antigen (Israel et al., 1980b). The enhancement of tumorigenicity when the large T-antigen coding region is interrupted could be due to the elimination of lytic effects of the infection (as suggested in the case of transformed mouse cells discussed earlier) or to the elimination of some immunological response favoring tumor rejection.

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The availability of fragments of the polyoma genome, cloned in bacterial plasmids, has allowed transformation to be tested using rigorously purified portions of the viral genome. The portion of the early region encoding the small and medium T antigens, but lacking the C-terminal portion of the large T antigen between 0 or 2 and 26 map units, is competent for transformation of cells in culture (Novak et al., 1980; Hassell et al., 1980). This supports the conclusion that the C-terminal portion of the large T antigen is not necessary for the expression of transformation and points toward the small and/or medium T antigen as being required. These experiments show that the large T antigen is not necessary for transformation when infection is carried out with viral DNA. The experiments with tsA mutants, described earlier, suggest that the large T antigen is required at least at the initial stages of transformation when infection is carried out with virions. The explanation for the different results is not yet apparent. “Leakiness” of the tsA mutation after DNA infection is unlikely to be the explanation, because the tsA mutants are still blocked in replication during lytic infection at the restrictive temperature when infection is carried out with viral DNA. Perhaps the presence of large amounts of viral DNA, introduced during DNA infection, favors integration of the viral genome, a step for which the large T antigen may be required after infection with virions. In an attempt to separate the effects of the small and medium T antigens on transformation, several mutants have been isolated which carry in-frame deletions in the region of the polyoma genome coding simultaneously for the medium and large T antigens (Griffin and Maddock, 1979; Magnusson and Berg, 1979; Bendig et al., 1980). These mutations could potentially affect the functions of either or both the medium and large T antigens but not the small T antigen. Some of the mutations show no effect on virus replication or transformation (Bendig et al., 1980). Others do not affect virus replication but decrease the frequency of transformation 7- to 25-fold when assayed by colony formation in agar (Magnusson and Berg, 1979). Two mutants, d18 and d123, have deletions which affect the growth properties of transformed cells (Griffin and Maddock, 1979). The d18 mutant induces somewhat larger foci and colonies in agar than the wild-type virus. The d123 mutant forms smaller foci and smaller colonies in agar. The properties of these mutants suggest that they contain alterations in a viral gene product affecting the expression of transformation (It0 et al., 1980). Both the d18 and the d123 mutants produce shortened medium and large T antigens compared to wild type. In the case of the d123 mutant, the reduction in the apparent size of the large T antigen is approx-

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imately what would be expected from the size of the deletion, but the reduction in the apparent size of the medium T antigen is much greater (It0 et al., 1980). Possibly, the amino acids of the medium T antigen removed by the deletion cause abnormally slow migration of the wild-type protein (It0 et al., 1980). So far it is not possible to tell whether the deletion mutations just described are exerting their effects through the large T antigen or the medium T antigen. The fact that viral replication is not abnormal in the mutants showing decreased transformation suggests that the large T antigen is normal, at least in its function for viral DNA replication. Complementation tests with tsA mutants should help to clarify the nature of the functional alterations. Polyoma genomes cloned in bacterial plasmids can be used to make extensive mutational alterations in the viral DNA without regard to potential effects on viral replication. Such mutated genomes, propagated in bacteria, can be used to test the effects of other changes in the medium T antigen on the phenotype of transformed cells. XI. Functions Associated with the Small and Medium T Antigens

Two approaches have been used to identify functions associated with viral transforming proteins: One is to study the properties of mutated transforming proteins; the other is to surmise what function the protein might have and test for it. The effect of the hr-t mutations on functions occurring in cells after infection has been studied extensively (Schlegel and Benjamin, 1978). When rat cells are infected by wild-type polyoma, they undergo morphological changes, lose their well-defined cytoplasmic actin architecture, and show increases in the size of their nuclei and nucleoli (Schlegel and Benjamin, 1978). By contrast, rat cells infected by hr-t mutants fail to undergo these changes. Both wild-type polyoma and hr-t mutants cause the induction of cellular DNA synthesis, followed by cell division, in infected cultures. However, the cell division after hr-t mutant infection appears to be confined to one round, whereas wild-type infection promotes several rounds. Therefore, the small and/or medium T antigens appear to be necessary for changes in cell shape, intracellular architecture, and multiple cycles of cell division after infection. The second approach-trying to guess a function and test for it-has led to attempts to detect a protein kinase activity associated with the polyoma T antigens, as had been shown previously for the Rous sarcoma virus transforming protein, pp6Owc(Collett and Erikson, 1978).

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Using a similar assay, incubation of immunoprecipitates with radiolabeled ATP, it is observed that polyoma T antigen immunoprecipitates contain an activity that preferentially phosphorylates the medium T antigen (Eckhart et al., 1979; S c h a a a u s e n and Benjamin, 1979; Smith et al., 1979).With certain sera, phosphorylation of immunoglobulin is also observed (Smith e t al., 1979).The activity is not observed in extracts of hr-t mutant-infected cells, which lack functional small and medium T antigens (Eckhart et al., 1979; Schaffhausen and Benjamin, 1979; Smith et al., 1979). So far it is not possible to tell whether the activity is a property of the medium T antigen, itself, or is a cellular activity which specifically associates with the medium T antigen and uses it as a substrate. The polyoma medium T antigen-associated kinase activity has the unusual property of specifically phosphorylating residues of tyrosine, rather than of serine or threonine (Eckhart et al., 1979). This unusual activity, for which there was no precedent at the time, led to a reexamination of phosphorylation by the Rous sarcoma virus, pp6OmCprotein kinase, which also was found to phosphorylate tyrosine residues specifically (Hunter and Sefton, 1980). Several protein kinase activities associated with tumor viruses are now recognized to phosphorylate tyrosine residues specifically (reviewed by Hunter and Sefton, 1981), and the phosphorylation of tyrosine residues in target proteins may be directly involved in cell transformation b y the tumor viruses which have this activity (Sefton et al., 1980). It is not yet clear whether phosphorylation by the activity associated with the pol yoma medium T antigen is important in transformation by polyoma. Rous sarcoma virus-transformed cells show increases in the overall levels of phosphotyrosine, compared to normal cells, whereas polyomatransformed cells do not (Sefton et al., 1980). Further characterization of the activity, and of the effects of mutations, should help to resolve the question. The structure and coding regions of the polyoma T antigens are now well characterized b y the combination of genetic experiments, mRNA analysis, peptide analysis, and DNA sequencing. The functions of the T antigens, particularly those of the small and medium T antigens that may be involved in the expression of transformation, remain to be clarified. Experiments in the near future will probably concentrate on purification of the proteins and analysis of a variety of specific mutations designed to alter particular regions of the molecules. The variety of ways in which cell transformation and tumorigenesis can be accomplished should become clearer as more viral transforming proteins become accessible to analysis.

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Goldman, E., and Benjamin, T. L. (1975). Virology 66,372-384. Griffin,B. E., and Maddock, C. (1979).]. Virol. 31,645-656. Griffin, B. E., Fried, M., and Cowie, A. (1974). Proc. Natl. Acad. Sci. U.SA. 71, 20772081. Hassell, J. A,, Topp, W. C., Rifkin, D. B., and Moreau, P. (1980).Proc. Natl. Acad. Sci. U S . A. 77, 3978-3982. Hattori, J., Carmichael, G. G., and Benjamin, T. L. (1979). Cell 16, 505-513. Hunter, T., and Sefion, B. M. (1980). Proc. Natl. Acad. Sci. U.S.A. 77, 1311-1315. Hunter, T., and Sefton, B. M. (1981). In “Molecular Aspects of Cellular Regulation” (P. Cohen and S . van Heyminger, eds.), Vol. 11, ElseviedNorth-Holland Biomedical Press, AmsterdadNew York (in press). Hunter, T., Hutchinson, M. A., and Eckhart, W. (1978).Proc. Natl. Acad. Sci. U.S.A. 75, 5917-5921. Hunter, T., Hutchinson, M. A., Eckhart, W., Friedmann, T., Esty,A., LaPorte, P., and Deininger, P. (1979). Nucleic Acids Res. 7,2275-2288. Hunter, T., Hutchinson, M. A., Eckhart, W., Friedman, T., Esty, A., LaPorte, P., and Deininger, P. (1980). Cold Spring Harbor Syrnp. Quant. Biol. 44, 131-139. Hutchinson, M. A., Hunter, T., and Eckhart, W. (1978). Cell 15, 65-77. Israel, M. A., Chan, H. W., Hourihan, S. L., Rowe, W. P., and Martin, M. A. (1979a).J. Virol. 29,990-996. Israel, M. A., Simmons, D. T., Hourihan, S. L., Rowe, W. P., and Martin, M. A. (1979b). Proc. Natl. Acad. Sci. U S A . 76,3713-3716. Israel, M. A., Chowdhury, K., Ramseur, J., Chandrasekaran, K., Vanderryn, D. F., and Martin, M. A. (1980a). Cold Spring Harbor Symp. Quant. Biol. 44,591-596. Israel, M. A., Vandenyn, D. F., Meltzer, M. L., and Martin, M. A. (1980b).J. B i d . Chem. 255,3798-3805. Ito, Y. (1979). Virology 98,261-266. Ito, Y. (1980).In “Viral Oncology” (G. Klein, ed.), pp. 447-480. Raven Press, New York. Ito, Y., and Spurr, N. (1980). Cold Spring Harbor Symp. Quant. Biol. 44, 149-157. Jto, Y., Spurr, N., and Dulbecco, R. (1977a). Proc. Natl. Acad. Sci. U.S.A. 74, 12591263. Ito, Y., Brocklehurst, J., and Dulbecco, R. (1977b). Proc. Natl. Acad. Sci. U . S A . 74, 4666-4670. Ito,?., Brocklehurst, J., Spurr, N., Griffiths, M., Hurst, J., and Fried, M. (1977~). Colloy. -1nst. Natl. Sante Rech. Med. 69, 145-151. Ito, Y., Spurr, N., and Griffin, B. E. (1980).J.Virol. 35, 219-232. Jessel, D., Landau, T., Hudson, J., Lalor, T., Tenen, D., and Livingston, D. M. (1976). Cell 8, 535-545. Kamen, R., Lindstrom, D., Shure, H., and Old, R. (1975). Cold Spring Harbor Symp. Quant. B i d . 39, 187-198. Kamen, R.,Favaloro, J,, Parker, J., Treisman, R., Lania, L., Fried, M., and Mellor, A. (1980). Cold Spring Harbor Symp. Quant. Biol. 44,63-75. Khoury, G., and May, E. (1977).J. Virol. 23, 167-176. Kuchino, T., and Yamaguchi, N. (1975).J.Virol. 15, 1302-1307. Lai, C.-J., and Nathans, D. (1974). Virology 60,466-475. Lai, C.-J., and Nathans, D. (1975a) Virology 66, 70-81. Lai, C.-J., and Natbans, D. (1975b). Cold Spring Harbor Symp. Quant. Biol. 39,53-60. Lania, L., Griffiths, M., Cooke, B., Ito, Y., and Fried, M. (1979).Cell 18, 793-802. Lania, L., Gandini-Attardi, D., Griffiths, M., Cooke, B., DeCicco, D., and Fried, M. (1980a).Virology 101,217-232.

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Lania, L., Hayday, A., Bjursell, G., Gandini-Attardi, D., and Fried, M. (1980b). Cold Spring Harbor Symp. Quant. Biol. 44,497-603. Magnusson, G., and Berg, P. (1979).J . Virol. 32, 523-529. Martin, R. G. (1981).Ado. Cancer Res. 34, 1-68. Miller, L., and Fried, M. (1976).J. Virol. 18, 824-832. Novak, V., Dilworth, J. M., and Griffith, B. E. (1980). Proc. Natl. Acad. Sci. U S A . 77, 3278-3282. Oren, M., Winocour, E., and Prives, C. (1980).Proc. Natl. Acad. Sci. U.S.A. 77,220-224. Prasad, I., Zouzias, D., and Basilico, C. (1976). J. Virol. 18, 436-444. Prives, C., Beck, Y.,and Shure, H. (1980).J. Virol. 33, 689-696. Rassoulzadegan, M., and Cuzin, F. (1980)J. Virol. 33, 909-911. Rassoulzadegan, M., Perbal, B., and Cuzin, F. (1978a).J. Virol. 28, 1-5. Rassoulzadegan, M., Seif, R., and Cuzin, F. (1978b).J. Virol. 28,421-426. Rassoulzadegan, M., Mougneau, E., Perbal, B., Gaudray, P., Birg, F., and Cuzin, F. (1980). Cold Spring Harbor Symp. Quant. Biol. 44, 333-342. Reed, S. I., Ferguson, J., Davis, R. W., and Stark, G . R. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 1605-1609. Reed, S. I., Stark, G. R., and Alwine, J. C. (1976). Proc. Natl. Acad. Sci. U.S.A. 73, 3083-3087. Schafthausen, B. S., and Benjamin, T. L. (1979). Cell 18, 935-946. Schafthausen, B. S., Silver, J., and Benjamin, T. L. (1978).Proc. Natl. Acad. Sci. U.S.A. 75,79-83. Schlegel, R., and Benjamin, T.L. (1978). Cell 14,587-599. Sefton, B. M., Hunter, T., Beemon, K., and Eckhart, W. (1980). Cell 20,807-816. Seif, R., and Cuzin, F. (1977). J . Virol. 24, 721-728. Shenk, T. E., Carbon, J., and Berg, P. (1976).J. Virol. 18, 664-671. Silver, J.. Schafiausen, B., and Benjamin, T. L. (1978). Cell 15, 485-496. Simmons, D. T., Chang, C., and Martin M. A. (1979).J. Virol. 29, 881-887. Smart, J. E., and Ito, Y. (1978). Cell 15, 1427-1437. Smith, A. E., Smith, R., Griffin, B., and Fried, M. (1979). Cell 18, 915-924. Soeda, E., and Griffin, B. E. (1978). Nature (London) 276, 294-298. Soeda, E., Arrand, J. R., Smolar, N., and Griffin, B. E. (1979). Cell 17, 357-370. Soeda, E., Arrand, J. R., Smolar, N., Walsh, J. E., and Griffin, B. E. (1980). Nature (London) 283,445-453. Staneloni, R., Fluck, M., and Benjamin, T. L. (1977).Virology 77, 598-609. Tegtmeyer, P. (1972).J. Virol. 10, 591-598. Tegtmeyer, P., Schwartz. M., Collins, J. K., and Rundell, K. (1975).J. Virol. 16, 168-178. Tenen, D. G., Baygell, P., and Livingston, D. M. (1975).Proc. Natl. Acad. Sc i. U.S.A.72, 4351-4355. Tjian, R. (1978). Cell 13, 165-179. Tjian, R. (1979). Cold Spring Harho7 Symp. Quant. Biol. 43, 655-662. Tooze, J., ed. (1980). “Molecular Biology of Tumor Viruses,” 2nd ed., Part 2. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Tiirler, H. (1980). Mol. Cell. Biochem. 32, 63-93. Turler, H., and Salomon, C. (1977). Col1oq.-Znst. Natl. Sante Rech. Med. 69, 131-144. Vogt, M. (1970).J. Mol. Biol. 47,307-316. Weil, R. (1978). Biochim. Biophys. Acta 516, 301-388. Wells, R. D., Hutchinson, M. A., and Eckhart, W. (1979).J. Virol. 32, 517-522. Zouzias, D., Prasad, I., and Basilico, C. (1977).J. Virol. 24, 142-150.

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TRANSFORMATION INDUCED BY HERPES SIMPLEX VIRUS: A POTENTIALLY NOVEL TYPE OF VIRUS-CELL INTERACTION Berge Harnpar Laboratory of Molecular Virology, National Cancer Institute, Frederick. Maryland

I. Introduction

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

11. Transformation-Definition of Terms ...................................... 111. Models for in Vitro Transformation by HSV ...............................

27 28 28 29 29 30 30 30 31 31 32 37 37 39 41

A. Use of Nonpermissive Cells .......................................... B. Use of Virus-Host Range Mutants .................................... C. Use of Inactivated Viruses ............................................ D. Use of Temperature-Sensitive (ts) Virus Mutants ....................... E. Use of Virus DNA or Restriction Enzyme-Digested DNA Fragments .... IV. Properties of Transformed Cells .......................................... A. Biochemically Transformed Cells ..................................... B. Morphologically and Tumorigenically Transformed Cells ............... V. Persistence of HSV Genetic Material in Transformed Cells ................. A. Biochemically Transformed Cells ..................................... B. Morphological and Tumorigenic Transformation ........................ VI. Mechanism of Transformation by HSV .................................... References .................................. ........................ 45

I. introduction

Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) have been implicated as etiologic factors in certain human tumors (reviewed by Rawls et al., 1977), and have been shown to be capable of transforming cells in vitro (Duff and Rapp, 1971; Munyon et al., 1971). Attempts to define the role of HSV in transformation have concentrated on finding analogies with transformations induced by other DNA viruses (e.g., papovaviruses, E B virus), although alternative possibilities have been proposed (Hampar et al., 1976; Hampar and Boyd, 1978; Skinner, 1976).The purposes here are (1) to briefly summarize the findings with respect to HSV-induced transformation in vitro, (2) to contrast these findings with transformation induced by other DNA viruses, and (3)to consider mechanisms which may be operative in the HSV system. No attempt is made to review or reference the entire literature relating to HSV-induced transformation. 27 ADVANCES IN CANCER RESEARCH, VOL. 35

Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-006635-1

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II. Transformation-Definition

of Terms

The term transformation refers to the spectrum of phenotypic changes which mammalian cells may undergo either spontaneously or following treatment with one or more “transforming agents” (biological, physical, or chemical). For the purposes of this discussion, the following definitions will apply. Transformation refers to an alteration in one or more properties of a cell and may include the following:

1. Biochemical transformation-the conversion of a thymidine kinase (TK)-negative cell to a TK-positive state following introduction and expression in the cell of the HSV T K gene. The transformation need not be stable; that is, the cell may revert to a TK-negative state (with or without loss of the viral DNA), either in the presence or the absence of TK-selective HAT medium. 2. Morphological transformation-alterations whereby cells express one or more phenotypic properties different from their “normal” counterparts. These properties may include, among others, (a) growth in serum-deficient medium (Dulbecco, 1970), (b) anchorage independence (Macpherson and Montagnier, 1964), and (c) high saturation density and loss of contact inhibition of movement (Abercrombie, 1979). 3. Tumorigenic transformation-the ability of cells to form progressive tumors when inoculated into appropriate hosts. Tumorigenic transformation may occur concomitantly with or in the absence of expression of one or more of the properties usually associated with morphological transformation. Ill. Models for in Vitro Transformation by HSV

Biochemical transformation, originally described by Munyon et al. (1971), involves introduction into TK-negative cells of the HSV T K gene (review by Pellicer et al., 1980b). Cells expressing the viral enzyme can be isolated using TK-selective HAT medium (Littlefield, 1964). Morphological transformation by HSV, originally described by Duff and Rapp (1971), involves morphological alterations which may lead to acquiring tumorigenic potential (reviewed by Rapp, 1974). HSV is similar to other DNA-containing viruses in that productive infection invariably leads to cell death, thus precluding transformation. To circumvent this problem, one can (1)manipulate the system, (2) select for a specific cell or virus strain, or (3) alter the environment in which the infected cultures are maintained to avoid productive infection, as discussed in more detail below.

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A. USE

OF

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NONPERMISSIVECELLS

By definition, cells nonpermissive for a DNA virus do not produce infectious virus, although the cells may undergo a lethal infection depending on the stage during the virus cycle when the infection is aborted. To be useful in transformation assays, nonpermissive cells must abort the DNA virus cycle early enough to avoid cell death. In the case of HSV, the point of irreversible lethal infection has yet to be definitively established, although it must occur relatively early during the productive cycle, and certainly before the time of viral DNA replication. Reliable nonpermissive systems for studying morphological transformation by “small” oncogenic DNA viruses are available (reviewed by Rapp and Westmoreland, 1976). With SV40, for example, nonpermissive mouse cells undergo morphological transformation within a few weeks after infection. Although SV40 does not undergo productive infection in mouse cells, early products of the viral replication cycle are expressed and are apparently responsible for the ensuing morphological transformation. A comparable nonpermissive cell system has not been described for use in morphological transformation by HSV, but one system has been employed for biochemical transformation (McAuslan et al., 1975). Rat cells (XC) transformed by Rous sarcoma virus (RSV) and nonpermissive for HSV (virus functions are expressed only transiently) were made TK-negative by growth in 5-bromodeoxyuridine (BUdR). The XC (TK-negative)cells were infected with wild-type HSV-1 or HSV-2, and cells expressing TK were selected in HAT medium. The enzyme being expressed in the cells was characterized as viral in origin. After approximately 15 subcultures, however, the cells stopped expressing the viral enzyme and expressed a TK that was characterized as cellular in origin, although the cells apparently retained viral DNA. B. USE

OF

VIRUS-HOSTRANGE MUTANTS

The isolation of HSV mutants with a restrictive host range (Koment and Rapp, 1975) is of potential value for studying morphological transformation. The mutant virus must be able to infect the resistant cells, and the productive virus cycle must be aborted at a stage early enough to avoid cell death. A host range mutant of HSV-1 has been isolated which can morphologically transform rat and mouse cells maintained under physiological conditions (B. Ham par, unpublished observations).

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c. USE OF INACTIVATED VIRUSES By far the most widely used system for studying morphological and biochemical transformation by HSV employs virus the infectivity of which has been destroyed by either irradiation (Duff and Rapp, 1971) or photodynamic inactivation (Rapp et al., 1973).The inactivated virus can then be tested for morphological (Duff and Rapp, 1971; Rappet al., 1973) or biochemical (Munyon et al., 1971) transformation in otherwise permissive cells. Variations have been noted in the ability of different strains of HSV to induce morphological transformation following UV inactivation (reviewed in Duff and Doller, 1973). A potential shortcoming of this system is that the inactivation must be sufficient to destroy or substantially reduce viral infectivity without causing a comparable reduction in viral transforming capability (Latarjet et al., 1967). Although the relative target sizes for these viral functions (infectivity and morphological or biochemical transformation) are apparently different enough to allow infectivity to be destroyed while still retaining some transforming activity (Benjamin, 1965), it is likely that portions of transforming regions of the viral genome are inactivated in concert with infectivity. Consequently, the frequency of morphological or biochemical transformation by inactivated virus is probably lower than would be obtained in a system employing intact virus. D. USE

OF

TEMPERATURE-SENSITIVE (ts) VIRUS MUTANTS

An alternative approach to using virus in which infectivity has been destroyed by physical or chemical agents is to use ts virus mutants. Cells infected with these mutants are placed at the nonpermissive temperature (238°C) for varying periods to minimize breakout of infectious virus and cell killing. The cultures are then placed at a physiological temperature (37°C) to assess morphological transformation (Macnab, 1974; Takahoshi and Yamanishi, 1974). Temperature-sensitive mutants have also been employed with permissive cells for obtaining biochemical transfonnants, although UV inactivation of the virus was still required (Hughes and Munyon, 1975).

E. USE OF VIRUSDNA OR RESTRICTION ENZYME-DIGESTED DNA FRAGMENTS Purified HSV DNA is infectious when transfected into cells (Lando and Ryhiner, 1969). The infectivity of the DNA can be destroyed by shearing or otherwise destroying the integrity of the intact molecule.

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Sheared HSV DNA has been employed for inducing both morphological (Wilkie et al., 1974) and biochemical transformation (Wigler et al., 1977; Maitland and McDougall, 1977) using the calcium precipitation technique of Graham and van der E b (1973). In one study (Jariwalla et al., 1979), tumorigenic transformation was induced using unsheared HSV-2 DNA at a concentration (50.01 pg) low enough to avoid breakout of infectious virus. The use of sheared or unsheared HSV DNA to induce transformation offers little advantage over inactivated virus with respect to identifying a specific region(s) of the viral genome with transforming potential. To identify the transforming region(s) of the HSV genome, purified restriction enzyme-digested DNA fragments have been employed in both morphological (Jariwalla et al., 1980; Camacho and Spear, 1978; Reyes et al., 1980) and biochemical transformation (Wigleret al., 1977; Maitland and McDougall, 1977) assays. In some cases, HSV DNA fragments cloned in appropriate vectors have been used (Hsiung et al.,

1980). IV. Properties of Transformed Cells

A. BIOCHEMICALLY TRANSFORMED CELLS Biochemical transformation by HSV has been carried out with established lines of TK-negative cells of human (Davis et al., 1974) and nonhuman (Munyon et al., 1971) origin. Identification of the TK being expressed is necessary to differentiate cells biochemically transformed by HSV and revertant cells expressing the cellular TK. Differentiation of cellular and viral TKs can be achieved by determining the immunological properties, physicochemical properties, or substrate specificities of the expressed enzyme (Klemperer et al., 1967; Kitet al., 1974; Cohen, 1972; Cooper, 1973). One or more of these properties can also be employed for distinguishing HSV-1 T K from HSV-2 TK. Once it has been established that the cells have been transformed by HSV, specific questions can be answered concerning the effect of the retained viral genetic material on cellular properties related to morphological and/or tumorigenic transformation. For example, the HSV TK gene has been employed in co-transfection experiments for introducing non-TK genes into TK-negative cells (Wigler et al., 1979; Mantei et al., 1979). Such experiments could be employed for determining the transforming potential of non-TK genes, provided that nontransformed TK-negative cells are used and that the HSV TK gene, in itself, does not morphologically transform the cells. To answer this

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question, we recently developed a TK-negative mouse cell line (cl. B2) where the cells display a “normal” flat morphology and are nontumorigenic in nude mice (Hamparet al., 1981). The cl. B2 cells, when biochemically transformed with UV-HSV-1, retain a flat morphology, remain nontumorigenic, and express the viral enzyme. In contrast, cl. B2 cells morphologically transformed by UV-HSV-1 in the absence of TK-selective HAT medium are tumorigenic in nude mice and remain T K negative. The findings with cl. B2 cells suggest that morphological and biochemical transformation by HSV-1 are independent and unrelated events. The HSV-1 TK gene would seem a suitable vehicle for introducing non-TK genes into mammalian cells for testing their transforming potential.

B . MORPHOLOGICALLYAND TUMORIGENICALLY TRANSFORMED CELLS 1. Early Passage versus Established Cells

Early passage and established cells have been employed for studying morphological transformation by HSV. Early passage cells of hamster (Duff and Rapp, 1971) or rat (Macnab, 1974) origin have been used for most studies, while a few studies have employed human cells (Darai and Munk, 1973). In other studies, established cell lines (Boyd and Orme, 1975) have been employed. Both cell systems offer certain advantages and disadvantages which must be recognized if they are to be effectively employed. Early passage cells are, essentially, all diploid, which is an obvious advantage, since it is less likely that the cells have begun to traverse the transformation pathway (Foulds, 1954). However, after numerous subcultures, these cells undergo a “crisis” during which the majority of cells succumb. Those cells which survive the “crisis” to form an established cell line acquire altered properties that invariably lead to tumorigenic transformation. Although various procedures have been described to minimize the surviving cells’ rate of progression to transformation [e.g., subculturing at frequent intervals at low cell densities (Aaronson and Todaro, 1968)], none of these procedures can prevent the cells from ultimately reaching a tumorigenically transformed state. Transformation of early passage cells obviously depends on the cells’ ability to survive “crisis.” This raises the question of whether a transforming agent (e.g., HSV) functions directly to induce transformation or indirectly by “immortalizing” the cells (Miller et al., 1974) so that they survive crisis, with morphological and tumorigenic transformation ensuing as normal sequelae of “spontaneous” events. The

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distinction between agents which act directly or indirectly is of paramount importance in understanding the mechanism of morphological and tumorigenic transformation, irrespective of the fact that cells subjected to either type of agent will ultimately acquire the properties of transformed cells. In contrast to early passage cells, established cell lines are, by definition, “transformed,” although they may not express morphological or phenotypic properties characteristic of transformed cells. These cells are usually noneuploid, a distinct disadvantage when compared to early passage cells. However, established cells have an advantage over early passage cells with respect to the availability of adequate controls in transformation assays. Since early passage cell cultures may contain a mixture of cells of diverse origin (e.g., when embryos are employed), their heterogeneity precludes their effective use as controls in transformation assays. Established cell lines, in contrast, can be cloned to give a relatively homogeneous cell population that can be used effectively as controls in transformation assays. For example, if selection is involved in morphological or tumorigenic transformation induced by a specific agent, it would be very difficult to test this with early passage cells, whereas clonal isolates of established cells could be utilized much more effectively. Further, if one wishes to compare the properties of morphologically transformed cells with‘those of their “normal” counterparts, early passage cells are not suitable controls, since the morphologically or tumorigenically transformed cells represent those cells which have survived crisis. Thus, cells initially cultured from animal tissues are diploid and “nontransformed.” After subculture, the cells undergo crisis and either die or survive to form cell lines. The latter are, by definition, “transformed” and will progress to morphological and tumorigenic transformation with continued subculture. Malignancy in vivo or tumorigenic transformation in vitro is a multistep process (Foulds, 1954). Established cell lines employed for in vitro transformation have already progressed some distance along the transformation pathway, whereas early passage cells must survive crisis, which, in itself, is a transforming event.

2 . Frequency of Transformation Morphological and/or tumorigenic transformation by HSV is a relatively inefficient process, with frequencies of 5 compared to SV40, where transformation frequencies of 210-* can be obtained. In most studies with HSV, transformation frequencies are not discernible from the reported data, since distinct foci were either not evident or

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appeared only after one or more passages of the cells. A transformation frequency of 5 10-5 with HSV is based on the number of infected cells initially seeded and is probably an overestimate. A few examples will suffice to demonstrate the point. In their initial study, Duff and Rapp (1971) seeded 5 x 109 hamster embryo cells infected with UV-HSV-2. Foci of morphologically transformed cells appeared in 21-28 days in 2 of the 17 cultures initially seeded. In another study (Rapp et al., 1973), 5 x 1oj hamster embryo cells were infected in suspension with photodynamically inactivated virus and 1-2 x 109 cells were seeded. Foci were evident in 3-4 weeks, but no numbers were given. Finally, rat embryo cells (2 x 10‘) were infected after 24-48 hr with a ts mutant of HSV-2 (Macnab, 1974). The cells were incubated at 38°C for 48 hr and subcultured at 1 : 2 split ratio. After 10 days at 38”C, the cells were incubated at 37°C. Foci appeared after 21 days at an estimated frequency of lo-’. In considering the question of the transformation frequency with HSV, it must be reemphasized that transformation requires either inactivation of viral infectivity or maintenance of the cultures under nonphysiological conditions (see Section 111,A). Since these conditions may minimize the transformation frequency, we have no valid way of determining the true frequencies of morphological and tumorigenic transformation with HSV under optimal conditions. 3. Phenotypic Properties In most cases, HSV transformed cells have been identified morphologically on the basis of their appearance compared to surrounding “nontransformed” cells. The transformed foci are isolated and propagated for testing of phenotypic properties usually associated with morphologically transformed cells. The properties tested include, among others, saturation density, growth in serum-deficient medium, contact inhibition of movement, and anchorage independence. In some, but not all studies, the cells have also been tested for tumorigenic potential. In general, the properties of cells morphologically transformed by HSV are typical of cells transformed either spontaneously or b y other agents. Further, at least some HSV-transformed cells have proved tumorigenic (Duffand Rapp, 1973b) when inoculated into appropriate hosts, although the latency periods may vary widely (Macnab, 1979). In some cases, HSV-transformed cells may not acquire tumorigenic potential until the cells have been passaged in vitro numerous times (Macnab, 1974). Similar findings have been reported with other viral transformation systems (Vogt and Dulbecco, 1960) and are in agree-

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ment with the conclusion that “malignant” transformation is a multistep process (reviewed by Armitage and Doll, 1954; Foulds, 1975). It is well established that phenotypic properties usually associated with a transformed phenotype (e.g., anchorage independence, growth in serum-deficient medium, etc.) are not necessarily correlated with tumorigenicity (Stanbridge and Wilkinson, 1978, 1980; Gallimore et al., 1977). Consequently, studies with HSV in which only the phenotypic properties of the cells are measured cannot be taken as presumptive evidence that HSV also induces tumorigenic transformation of the cells. It should be stressed, however, that with certain cell lines a close correlation may be found between specific phenotypic properties associated with morphological transformation and tumorigenicity (Shin et al., 1975). Morphological transformation by DNA viruses, such as SV40, is usually accompanied by alterations in the cells’ phenotypic properties which can be attributed to expression of viral transforming sequences retained in the cells. A series of experiments were carried out to determine whether transformation by HSV also involved alterations in the cells’ phenotypic properties which could be attributed to retained viral gene sequences (Hampar et al., 1980). A clonal line of BALB/c cells (10E2) was employed. The 10E2 cells, at passage 40, showed foci of spindle-shaped cells which were isolated and shown to be tumorigenic in nude and conventional (immunocompetent) mice. These spontaneously transformed cells, however, did not express phenotypic properties typical of morphologically transformed cells. For example, the tumorigenically transformed 10E2 cells isolated at passage 40 grew poorly in serum-deficient medium, remained contact inhibited, and were anchorage dependent when tested in agarose and agar. When low-passage 10E2 cells (passages 4-30) were transformed with SV40, however, the transformed cells showed high saturation densities, good growth in serum-deficient medium, and anchorage independence in agarose and agar. Interestingly, only 10% of the SV40 morphologically transformed 10E2 cells proved tumorigenic in nude mice. The 10E2 cells, at low passage, were also infected with UV-HSV-2. Foci of spindle-shaped cells appeared in the HSV-infected cultures that were indistinguishable from those observed in high-passage 10E2 cultures undergoing spontaneous transformation. The UV-HSV-2 morphologically transformed 10E2 cells were also similar to spontaneously transformed cells in that neither expressed phenotypic properties characteristic of morphologically transformed cells and both produced tumors in nude and conventional (immunocompetent) mice.

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The studies with 10E2 cells allowed several conclusions concerning the properties of these cells transformed either spontaneously or following infection with UV-HSV-2 or SV40. First, SV40-transformed 10E2 cells expressed phenotypic properties characteristic of morphologically transformed cells. Expression of these properties could be attributed to retention of viral gene sequences in the cells, as evidenced by the production of T and t antigens. Second, while SV40 was efficient in morphologically transforming 10E2 cells (frequencies of 10-2-10-3),at least 90% of the isolated foci failed to induce tumors in nude mice, and none induced tumors in conventional (immunocompetent) mice. This suggests that transformation of 10E2 cells by SV40 affects primarily those cellular properties responsible for morphological transformation and, to a much lesser extent, those properties associated with tumorigenic transformation. Third, 10E2 cells transformed spontaneously or by UV-HSV-2 were tumorigenic in both nude and conventional (immunocompetent) mice, but the cells did not express phenotypic properties characteristic of morphologically transformed cells. These findings indicate that, at the biological level, 10E2 cells transformed by UV-HSV-2 did not express phenotypic properties that could be ascribed to retention and expression in the cells of viral gene sequences. The results, in agreement with those obtained with SV40transformed cells, also indicate that morphological and tumorigenic transformation in 10E2 cells are separable events that may be only partially related. 4. Properties Related to the Genome of the Transforming Virus Viral transformation, in contrast to transformation induced by chemical or physical agents, is considered unique in that it involves addition to the cells of new genetic information (Rapp and Westmoreland, 1976; Temin, 1980). The addition of new genetic material occurs with both RNA and DNA viruses and is generally accepted as required for viralinduced transformation. It is possible, however, for cells to retain viral transforming sequences without expressing a transformed phenotype (Boettiger, 1974; Porzig et al., 1979). Continuous shedding of virus in the case of cells transformed by DNA viruses would not be compatible with cell survival. In contrast, cells transformed by retrovirus may continue to shed virus without being killed. Consequently, cells transformed by DNA viruses need retain only a portion of the viral genome that, in the case of papovaviruses, is evidenced by expression of viral-coded “tumor” (T) antigens. These viral-coded products are responsible for the transformed cells expressing one or more of the phenotypic properties asso-

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ciated with morphological transformation (see Section III,B,3). Viralcoded proteins comparable to T antigens have not been described in HSV-transformed cells. Cells tumorigenically transformed by DNA viruses, such as SV40, also express surface antigens which can elicit an immune response in immunocompetent animals, resulting in rejection of the tumors. These transplantation rejection antigens are coded for by the virus genome (Chang et al., 1979). Similar viral-coded rejection antigens have not been found in cells tumorigenically transformed in HSV, even in cases where the cells have been reported to express viral-coded proteins (Duff et al., 1973). V. Persistence of HSV Genetic Material in Transformed Cells

A. BIOCHEMICALLY TRANSFORMED CELLS

Biochemical transformation by HSV has been achieved using inactivated virus (Munyon et al., 1971), sheared viral DNA (Bacchetti and Graham, 1977), purified or cloned fragments of viral DNA (Maitland and McDougall, 1977; Wigler et al., 1977),and high-molecular-weight DNA (Minsonet al., 1978) or chromosomes (McBride et al., 1978) from biochemically transformed cells. In each case, persistence of the TKcoding portion of the viral genome has been confirmed by identification of the enzyme being expressed in the cells. Definitive evidence for persistence of a portion of the viral DNA in transformed cells has been obtained by DNA-DNA hybridization (Kraiselburd et al., 1975) or by Southern blot hybridization (Kit et al., 1980) to identify specific viral DNA sequences. From these studies a picture has emerged concerning some of the events associated with biochemical transformation. The HSV-1 TK gene initially localized in the 3.4 kb BamHI fragment (Wigler et al., 1977) has been further localized at map coordinates 0.302-0.311 (Colbere-Garapin et al., 1979). The DNAs of HSV-1 and HSV-2 are essentially colinear, and the HSV-2 TK gene has been localized at map coordinates 0.284-0.314 (Reyes et al., 1980). When intact virus or viral DNA is used for transformation, DNA sequences additional to those coding for the viral TK can be detected in transformed cells. Cells biochemically transformed with UV-HSV-2, for example, all contained viral DNA segments between map coordinates 0.28-0.32, where the viral T K gene is localized (Leiden et al., 1980). In addition, contiguous viral DNA sequences extending from map coordinates 0.14-0.57, 0.14-0.42, 0.2 1-0.32, and 0.28-0.42 were detected in four different transformed cell lines, respectively. Two cell

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lines transformed with HSV-1 DNA were also tested. One line showed a contiguous segment of viral DNA extending from map coordinates 0.27-0.41, and the other showed noncontiguous segments extending from map coordinates 0.11-0.17, 0.29-0.32, 0.34-0.40, 0.52-0.56, and 0.91-0.96. Two regions of the viral genomes, extending from map coordinates 0.00-0.06 and 0.57-0.82, were uniformly absent from most, if not all, of the transformed cells. Interestingly, none of the transformed cells contained viral DNA sequences coding for immediate early (a)proteins (Clements et al., 1977; Jones et al., 1977). While this, in itself, does not preclude similarities between transformation induced b y HSV and papovaviruses, one would anticipate from the findings with papovaviruses that HSV-transforming gene sequences should code for proteins synthesized early during the productive cycle. In some biochemically transformed cells, the viral DNA has been reported to be stably associated with high-molecular-weight nuclear DNA (Pellicer et al., 1978) and to segregate with the chromosomes (Smiley et al., 1978; Kit et al., 1979).The stable association of the viral TK gene with the cell is also evidenced by the finding that tumorderived cell lines of biochemically transformed cells retain the viral enzyme even after passage in animals under nonselective conditions (Pellicer et al., 1980a). In other cases, the viral DNA is apparently not retained in a stable form, as evidenced b y the relatively high rates of reversion of the cells to a TK-negative state with loss of the viral D N A when selected in the presence of BUdR (Kraiselburd et al., 1975). Alternatively, expression of the viral enzyme may be suppressed despite the maintenance in the revertant cells of the viral T K gene (Davidson et al., 1973). Revertant cells have also been described which retain viral DNA sequences outside the TK gene (Bastow et al., 1980). The available evidence by hybridization that integration of the HSV T K gene itself occurs in the cell DNA, as seen with DNA of other viruses (Botchan et al., 1980), must be reevaluated based on recent findings that transfected viral DNA can integrate into the carrier DNA during formation of concatamers in the cotransfected cells (Perucho et al., 1980). Any integration of the TK gene into the cell DNA may occur via the carrier DNA. In addition to expressing the viral enzyme, several biochemically transformed cell lines synthesize other viral proteins, including structural proteins (Chadha et al., 1977). A nuclear antigen (HANA) which is associated with chromosomes and may represent the viral enzyme has also been described in biochemically transformed cells (Kit et al.,

1980). ,J

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In summary, the viral TK gene can be retained in transformed cells in a stable or unstable fashion. In some cases, viral DNA sequences in addition to the viral TK gene are retained in the cells. The transformed cells may express viral-coded proteins in addition to TK. Finally, the presence in cells of the viral TK gene need not result in morphological transformation (see Section IV,A).

B. MORPHOLOGICALAND TUMORIGENIC TRANSFORMATION Efforts to identify regions of the HSV genome with transforming potential have concentrated on testing endonuclease restriction enzyme-digested DNA fragments by transfection. Using XbaIdigested fragments of HSV-1, Camacho and Spear (1978) reported that the 15.5 X 106-dalton XbaI F fragment (map coordinates 0.30-0.45) could morphologically transform hamster embryo cells. The transformed cells expressed a viral antigen(s) detectable by immunofluorescence (FA), and a 113,000-dalton protein, similar in mobility to a precursor of viral protein VP7(Bz), was immunoprecipitable from the transformed cells. The neutralizing activity of anti-VP7 (B,) serum was reduced approximately 90% when adsorbed with the transformed cells. The presence of viral DNA in the transformed cells was not reported. Reyes et aZ. (1980)tested DNA fragments from HSV-1 and HSV-2 for transforming activity using hamster embryo cells and BALB/3T3 cells. Morphological transformation was observed with the 15.8 kb BgZII I fragment of HSV-1 (map coordinates 0.311-0.415) and with the 7.4 kb BgZIl N fragment of HSV-2 (map coordinates 0.582-0.628). These morphological transforming regions, termed mtr-I and mtr-11, respectively, were shown to be nonhomologous. Attempts to detect viral DNA in mtr-I-transformed cells were negative, while cells transformed with mtr-I1 showed DNA sequences which hybridized to the BgZII N fragment of HSV-2. Jariwalla et al. (1980), using hamster embryo cells, reported transforming activity associated with the 16.5 x 106-dalton BgZIIIHpaI fragment CD of HSV-2 DNA at map coordinates 0.43-0.58. The transformed cells were tumorigenic at passages 25, 39, 48 in neonatal hamsters and were positive by complement fixation for viral protein ICP 10, which is immunologically related to antigen AG-4 (Strnad and Aurelian, 1976). The presence of viral DNA in the transformed cells was not reported. Expression of viral proteins in HSV-transformed cells has been detected primarily by immunofluorescence (Duff and Rapp, 1971), with

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staining usually limited to perinuclear and cytoplasmic regions in fixed cells and to the cell surface in unfixed cells. Viral protein expression in transformed cells has also been detected by immunoprecipitation (Gupta and Rapp, 1977) and by demonstrating virus-neutralizing activity in the sera of tumor-bearing animals (Duff and Rapp, 1973a). The persistence of viral genetic material in transformed cells has been deduced from biological studies, where the growth of virus ts mutants has been reported to be enhanced in transformed cells (Benyesh-Melnick et al., 1974), and by the isolation of intratypic variants from transformed cells infected with ts mutants (Park et al., 1980). The first direct demonstration that HSV-transformed cells could retain portions of the viral genome was reported by Collard et al. (1973), who showed the presence of viral RNA in HSV-2-transformed hamster cells b y molecular hybridization. Approximately 11% of the viral genome was transcribed in the transformed cells. Other studies indicated that transformed cells could retain various portions of the viral genome. Frenkel et al. (1976) detected 8-32% of the HSV-2 genome at 1-3 copies per cell in different cell lines and showed that the complexity of the retained sequences decreased with increasing cell passage. Minson et al. (1976) detected 40% of the HSV-2 genome in passage 45 transformed hamster cells, but none at passage 80. Clonal sublines of the transformed cells were negative for viral DNA by molecular hybridization (Minson et al., 1976) but positive for viral RNA by in situ hybridization (Copple and McDougall, 1976). Bibor-Hardy et al. (1979) and Kessous et al. (1979), using HSV-2-transformed hamster cells cloned in agar at passage 28, detected 40% of the genome at 1-6 copies per cell. Tumor derivatives also showed 40% of the viral genome, but at only 2-3 copies per cell. I n situ hybridization (reviewed by McDougall and Galloway, 1978) has also been employed for detecting viral RNA in transformed cells. Using restriction enzyme-digested fragments of HSV-2 DNA, viral RNA was detected in varying amounts in cloned sublines of HSV-2transformed hamster embryo cells (Galloway et al., 1980). The complexity of the retained viral DNA was reduced with cell passage, in agreement with other findings (Frenkel et al., 1976). Two regions of the HSV-2 genome common to most transformed cells were localized at map coordinates 0.21-0.33 and 0.60-0.65 (Galloway et al., 1980). In one study, HSV-2 tumorigenically transformed mouse cells tested between passages 3 and 35, showed no viral proteins by FA or immunoprecipitation and no antibodies in the sera of tumor-bearing animals (Hampar et al., 1980). Similarly, viral DNA was not detected in

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these transformed cells by molecular hybridization, and viral RNA was not detected by in situ hybridization. In summary, the persistence of viral DNA sequences in some, but not all, HSV-transformed cells has been reported. The complexity of the retained viral DNA sequences may diminish with increasing cell passage. Different regions of the viral genome are retained or transcribed in different cell lines. Transformed cells may express viral proteins, but no one protein has been detected consistently in all cells tested. VI. Mechanism of Transformation by HSV

Malignant transformation in uiuo and tumorigenic transformation in vitro are multistep processes. Morphological transformation in uitro is also a multistep process, in the sense that specific phenotypic changes (e.g., saturation density, anchorage independence, etc.) may become more pronounced with increasing cell passage (Hampar et al., 1980). While with some cell lines tumorigenic transformation may be closely correlated with at least one phenotypic property normally associated with morphological transformation (Shin et al., 1975),other cell lines (e.g., 10E2) may show no such correlation (Hampar et al., 1980). When early passage cells are employed for assaying the transforming potential of a specific agent, several alternatives must be considered to account for the ultimate appearance of morphologically or tumorigenically transformed cells. Early passage cells, after a limited number of cell doublings, undergo a “crisis,” during which the cells either die or become “immortalized” to form an established cell line. Once the cells are immortalized, they invariably progress to a state of tumorigenicity. A transforming agent in early passage cells may function in several ways. First, it may function to immortalize the cells, so that they ultimately undergo spontaneous events leading to tumorigenic transformation. Morphological transformation may precede, accompany, or follow tumorigenic transformation. Second, the transforming agent may directly affect the cell genome to cause morphological andlor tumorigenic transformation. It is not clear how these transformation events are related to the crisis stage. It is possible, for example, that cells may undergo transformation and still succumb at the crisis stage. Alternatively, crisis may have to precede transformation or may be circumvented in any cell which has undergone transformation. Finally, the transforming agent may function by both immortalizing and transforming the cells. When HSV is employed as a transforming agent with early passage cells, it is well established that at least some of the morphologically or

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tumorigenically transformed cells retain viral DNA (see Section V,B). It is also well established that these viral gene sequences may diminish in complexity with increasing cell passage or cloning and, in some cases, at least, be eliminated from the cells or fall below the threshold level of detection. How the HSV genome functions in these cells is not known, and there is no convincing evidence that viral DNA sequences must be retained by the cells for maintenance of the transformed state. The viral DNA in transformed cells may function to immortalize the cells, transform the cells, or both. From a holistic viewpoint, an immortaIizing function for the HSV genome, such as occurs with the Epstein-Barr virus in lymphoid cells (Miller et ul., 1974), has certain attractive features. Retention in cells of foreign viral DNA sequences (e.g., SV40) may occur in the absence of applied selective pressures or, as in the case of the HSV TK gene, may require selective pressure. Early passage cells infected with HSV may passively retain portions of the viral genome in the absence of selective pressures or may retain viral gene sequences as a means of surviving crisis, which, in itself, could serve as the selective pressure. In the latter situation, retention in the cells of the viral DNA sequences would not be required once the cells have passed crisis, and, unless the viral DNA has formed a stable association with the nuclear DNA, the viral sequences could subsequently be eliminated or discarded by the cells. This does not exclude the possibility that a small specific portion of the viral genome is retained in the transformed cells and is required for maintenance of the transformed state. Such HSV gene sequences have yet to be identified. When established cells are employed for transformation assays, a different situation exists than with early passage cells. Established cells are, by nature, already “transformed,” since they have survived crisis and are invariably noneuploid. These cells, in a sense, have already progressed an appreciable distance along the pathway leading to tumorigenicity. With most cell lines, morphological transformation is considered a precursor of tumorigenic transformation. This conclusion may be erroneous, however, since it is based almost entirely on the visual identification of transformed cells. Since cells which have undergone morphological transformation are readily distinguished from surrounding “normal-appearing” cells, the presence in cultures of tumorigenic cells that do not appear to be morphologically transformed may go unnoticed. The identification of tumorigenically transformed cells would require testing of the whole cell population for tumor formation in appropriate hosts. Even then, the presence of such

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cells could be missed if they represented only a minor proportion of the cell population. The 10E2 cell system is a case in point (Hampar et al., 1980). At passage 40, the cells proved tumorigenic in nude and conventional (immunocompetent) mice, although foci of morphologically transformed cells were not readily apparent. Tumorigenicity was correlated with foci containing spindle-shaped, contact-inhibited cells. With increasing cell passage, these cells also expressed phenotypic properties characteristic of morphologically transformed cells. When low-passage 10E2 cells were infected with SV40, foci of morphologically transformed cells showing loss of contact inhibition were readily apparent within 3-4 weeks. The majority of the cells in these foci (-90%) proved nontumorigenic in nude mice. The SV40transformed cells expressed both large T and small t antigens, and infectious virus was rescuable by fusion to permissive cells (B. Hampar et al., unpublished). The phenotypic properties of the SV40 morphologically transformed cells could be attributed to retention and expression in the cells of the viral genome. In 10E2 cells, at least, SV40 can best be classified as a virus which is more effective in inducing morphological transformation than tumorigenic transformation. When low-passage 10E2 cells were infected with UV-HSV-2, a pattern distinctly different from SV40 was observed. After one or more passages, the UV-HSV-2-infected cells showed areas of spindleshaped, contact-inhibited cells similar to those identified as tumorigenic in high-passage control cultures. When isolated, these HSV-transformed cells proved tumorigenic in both nude and conventional (immunocompetent) mice. In all respects, the cells transformed by UV-HSV-2 were identical to spontaneously transformed cells. At the biological level, therefore, there was no evidence that 10E2 cells tumorigenically transformed by UV-HSV-2 acquired phenotypic properties which could be ascribed to retained viral gene sequences, as occurred with SV40. Further, the UV-HSV-2-transformedcells showed no evidence of viral protein expression or retained viral DNA. At this point, we can say with some certainty that morphological or tumorigenic transformation by HSV differs from transformation by papovaviruses or EB virus. It is possible that retention of a relatively small, as yet undefined fragment of the HSV genome is required for maintenance of the transformed state in the absence of expression of viral-coded products comparable to papovavirus “tumor” antigen, transplantation rejection antigen, or the nuclear antigen of EB virus [reviewed in Epstein and Achong (1979)l. Alternatively, transforma-

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tion by HSV may occur by a hit-and-run type mechanism (Skinner, 1976; Hampar and Boyd, 1978), where retention in the cells of viral DNA is not required. In considering a hit-and-run mechanism, several properties ascribed to HSV may be relevant. First, HSV can damage cell chromosomes (Hampar and Ellison, 1961). Second, HSV has been reported capable of inducing reparable damage to cell DNA (Lorentz et al., 1977). Finally, HSV can activate endogenous retrovirus in mouse cells (Hampar et al., 1976). Whether any of these properties plays a role in HSV-induced morphological or tumorigenic transformation is not known. The ability of HSV to damage chromosomes and induce reparable DNA damage is consistent with the properties of carcinogenic and/or mutagenic agents. HSV has not been shown to be mutagenic. The ability of HSV to activate endogenous retrovirus indicates that HSV can turn on expression of at least one cellular gene. Retrovirus activation has been localized to specific regions of the HSV genome (Boyd et al., 1980). The retrovirus activated by HSV in BALB/c mouse cells has been classified as xenotropic in host range (Hampar et al., 1977), which indicates that the virus cannot be readily transmitted horizontally to other cells in the population. Further, there is no reason to suspect that this retrovirus has transforming properties. Consequently, we have excluded any possibility that transformation of BALBlc 10E2 cells by UV-HSV-2 results from activation and synthesis of a retrovirus that is transmitted to other cells in the population (Hampar and Boyd, 1978). If retrovirus activation is involved in HSV-induced transformation of 10E2 cells, it must involve those cells in which the retrovirus is activated. Although a hit-and-run mechanism with HSV is obviously speculative, it does require serious consideration, if for no other reason than the fact that evidence for a conventional mechanism of transformation by HSV has not been confirmed, despite several years’ effort. The major problem in elucidating the role of HSV in transformation is the lack of a system in which significant levels of transformation can be obtained reproducibly. It would appear that resolution of this problem should be the first order of business. ACKNOWLEDGMENTS I would like to thank my colleagues, A. Boyd, J. Derge, M. Tainsky, M. Zweig, C. Heilman, and S. Showalter, for their helpful comments during the preparation of this manuscript.

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Pellicer, A., Wagner, E. F., Kareh, A. E., Dewey, M. J., Reuser, A. J., Silverstein, S., Axel, R., and Mintz, B. (1980a).Proc. Natl. Acad. Sci. U S A . 77,2098-2102. Pellicer, A., Robins, D., Wold, B., Sweet, R., Jackson, J., Lowy, I., Roberts, J. M., Sim, G. K., Silverstein, S., and Axel, R. (1980b). Science 209, 1414-1422. Perucho, M., Hanahan, D., and Wigler, M. (1980). Cell 22,309-317. Porzig, K. J., Robbins, K. C., and Aaronson, S. A. (1979). Cell 16, 875-884. Rapp, F. (1974).Adu. Cancer Res. 19,265-302. Rapp, R., and Westmoreland, D. (1976). Biochim. Biophys. Acta 458, 167-211. Rapp, F., Li, J.-L. H., and Jerkofsky, M. (1973).Virology 55,339-346. Rawls, W. E., Bacchetti, S., and Graham, F. L. (1977). Cum. Top. Microbiol. Immunol. 77,71-95. Reyes, G. R., LaFemina, R., Hayward, S. D., and Hayward, G. S. (1980). Cold Spring Harbor Symp. Quant. Biol. 44,629-641. Shin, S . , Freedman, V. H., Risser, R.,and Pollack, R. (1975).Proc.Natl. Acad. Sci. U S A . 72,4435-4439. Skinner, G. R. B. (1976). Br. J . E x p . Pathol. 57,361-376. Smiley, J,. R., Steege, D. A., Juricek, D. K., Summers, W. P., and Ruddle, F. H. (1978). Cell 15, 45-68. Stanbridge, E. J., and Wilkinson, J. (1978). Proc. Natl. Acad. Sci. U . S A . 75, 1466-1469. Stanbridge, E. J., and Wilkinson, J. (1980). Int. J. Cancer 26, 1-8. Strnad, B. C., and Aurelian, L. (1976). Virology 73, 244-258. Takahoshi, M., and Yamanishi, K. (1974). Virology 61,306-311. Temin, H. M. (1980).Cold Spring Harbor Symp. Quant. Biol. 44, 1-8. Vogt, M., and Dulbecco, R. (1960). Proc. Natl. Acad. Sci. U . S A . 46,365-370. Wigler, M., Silverstein, S., Lee, L.-S., Pellicer, A,, Cheng, Y.-C., and Axel, R. (1977).Cell 11,223-232. Wigler, M., Sweet, R., Sim, G. K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, s., and Axel, R. (1979). Cell 16,777-785. Wilkie, N. M., Clements, J. B., Macnab, J. C. M., and Subak-Sharpe, J. H. (1974). Cold Spring Harbor Symp. Quant. Biol. 39,657-666.

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A RA CHID0NIC AC ID TRANS FOR MATI 0N AND TUMOR PRODUCTION Lawrence Levine Department of Biochemistry. Brandeis University. Waltham, Massachusetts

I. Introduction ........................................................... 11. Arachidonic Acid Transformation ........................................ 111. Prostaglandin Levels in Tumors.. ....................................... IV. Arachidonic Acid Transformation and Hypercalcemia ..................... V. Arachidonic Acid Transfonnation and Tumor Promotion ................... A. Effects of TPA on Prostaglandin Production by Cells in Culture .................................................... B. Inhibitors of Prostaglandin Production: Their Effects on the Activities of TPA in Cells in Culture ................................. C. Effects of TPA on Prostaglandin Production in Vim ................... D. Stimulation of Prostaglandin Production by Growth Factors . . . . . . . . . . . VI. Prostaglandins: Their Effects on Cell or Tumor Growth ................... VII. Prostaglandins and the Immune Response ............................... VIII. Challenges ............................................................ References ............................................................

49 49 52 55 58 59 61 65 66 67 69 71 73

I. Introduction

Several observations have encouraged considerable speculation on the relationship between prostaglandins and cancer. Increased prostaglandin levels have been found in blood and/or urine of animals carrying neoplasms, as well as in transformed cells growing in tissue culture. In addition, it has been shown that prostaglandin production is associated with tumor promotion. Whether or not these associations are causally or casually related in such a complex of diseases is not clear. However, the concept that prostaglandins affect tumor growth is a constant theme in the mechanisms proposed to explain the phenomenon. Several reviews on the relationship of prostaglandins to cancer have been published (Bennett, 1979; Easty and Easty, 1976; Goodwin et al., 1980; Jaffe, 1974; Karim and Rao, 1976; Karmali, 1980; Pelus and Strausser, 1977). II. Arachidonic Acid Transformation

Some of the pathways of arachidonic acid metabolism are shown in Fig. 1. Arachidonic acid, which is the unsaturated fatty acid found in 49 ADVANCES IN CANCER RESEARCH, VOL. 35

Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-006635-1

HO LTA

0

Glulolhione S- Transferose

I

c::3

0

o--?-CooH

OOH

ARACHIDONIC ACID TRANSFORMATION AND TUMORS

51

highest concentration in cellular phospholipids, is considered in detail; but other unsaturated fatty acids can also serve as substrates for the cyclooxygenase. The endoperoxides (PGG2 and PGH2) are the immediate products of the cyclooxygenase (Hamberg and Samuelsson, 1973; Nugteren and Hazelhof, 1973) and can undergo enzymatic and nonenzymatic transformations to form thromboxanes (TxA2and TxB2) (Hamberg et al., 1975; Needleman et al., 1976), prostacyclins (PG12) (Moncada and Vane, 1977), and prostaglandins (PGE2, PGFza, and PGD2). These endoperoxide metabolites can exist in classes depending on the degree of unsaturation in their precursor fatty acids; e.g., arachidonic acid (eicosatetraenoic acid) is transformed to class-two (PGEZ, PGFz,, etc.) products, and eicosatrienoic acid is transformed to class-one (PGE1, PGFla, etc.) products. The endoperoxides, and the products formed from the endoperoxides, are potent pharmacologically; and their activities, qualitatively and quantitatively, depend on the nature of the enzymatic (and nonenzymatic) transformations. Arachidonic acid also can be converted by lipoxygenases to hydroperoxyarachidonic acids and the corresponding hydroxyarachidonates (Hamberg and Samuelsson, 1973). Arachidonic acid is substrate for lipoxygenases, which also produce potent pharmacologically active compounds. The product of lipoxygenases acting at the 5 double bond of arachidonic acid can form a 5,6-epoxyarachidonic acid intermediate (leucotriene A). Leucotriene (LT)A can be transformed either to the potent chemotactic lipid 512-dihydroxyarachidonic acid (LTB) or, in the presence of glutathione and glutathione S-transferase, to the glutathione-containing compound (LTC). LTC, in some tissues, can be further metabolized b y y-glutamyl transpeptidase to form the cysteinyl-glycyl-containing compound (LTD). LTC, LTD, and the cysteinyl-containing compound (LTE) comprise a family of pharmacologically active compounds termed slow reactive substances of anaphylaxis (SRS-A) (Borgeat and Samuelsson, 1979;Jakschiket al., 1977; Morris et al., 1980a,b; Murphy et al., 1979; Orning et al., 1980; Parker et al., 1980). Prostaglandins, prostacyclins, and thromboxanes are not stored to any considerable extent in mammalian tissues. Any increase in their levels probably is brought about by a physiological stimulation and their rapid biosynthesis. This synthesis is often limited by the availability of precursor polyunsaturated fatty acids (Lands and Samuelsson, 1968; Vonkeman and van Dorp, 1968). Unsaturated fatty acids do not exist free in cells but are found in the form of phosphoglycerides and triglycerides, which must b e deacylated to provide substrate for the lipoxygenases and cyclooxygenases. The tissue phos-

52

LAWRENCE LEVINE

pholipids are the richest source of these precursor polyunsaturated fatty acids, and it has been postulated that the phospholipases of the cell are part of the sequence of events involved in prostaglandin biosynthesis (Kunze and Vogt, 1971). Deacylation of phospholipids probably occurs by more than one enzymatic pathway. For example, in platelets, three mechanisms of acylhydrolase activity have been proposed: (1) phosphatidylcholine and phosphatidylinositol are substrates for distinct phospholipase A, activities (Bills et al., 1977); (2) phosphatidylinositol is a substrate for sequential activities of phospholipase C (Rittenhouse-Simmons, 1979) and diacylglycerol lipase (Bell et al., 1979); ( 3 ) phosphatidic acid, generated by sequential actions of a phospholipase C on phosphatidylinositol and phosphorylation of the diacylglycerol, stimulates the phospholipase A, attack of phosphatidylcholine (Lapetina et al., 1980). In rat mast cells, a pool of phosphatidylcholine, synthesized from phosphatidylethanolamine by three methylation steps, is attacked by phospholipase A, (Hirata and Axelrod, 1980); in methylcholanthrene-transformed mouse fibroblasts, as a result of stimulation by bradykinin, phosphatidylinositol is deacylated by phospholipase A,; phosphatidylcholine also may be hydrolyzed by phospholipase A, (Hong and Deykin, 1979, 1981). Ill. Prostaglandin Levels in Tumors

Most mammalian cells have the enzyme(s) that synthesize endoperoxides from arachidonic acid. The capacity to generate thromboxanes, prostacyclin, and the prostaglandins from endogenous endoperoxides varies considerably among cells (Levine et al., 1979). For example, in endothelial cells derived from bovine aorta, more than 90% of cyclooxygenase products is PGI,, approximately 80% of the products of endothelial cells derived from bovine adrenal is PGI,, whereas only 26% of the cyclooxygenase metabolites derived from human umbilicus vein endothelial cells is PGI,. Marcus et al. (1978) found that 50% of cyclooxygenase products of endothelial cells derived from umbilical cords is prostacyclin. Cells derived from murine lymphoma cells (WEHI) synthesize relatively large amounts of thromboxane (around 60% of all cyclooxygenase products). PGE, (16%), PGF, plus PGF,, (19%), and 6-keto-PGF1, (about 4%) were also found. This cell line is a macrophage-like cell. Guinea pig macrophages have been reported to produce in culture PGE, (Gordon et al., 1976); and mouse macrophages, PGE, and 6-keto-PGF,, (Humes et al., 1977) and PGE, and

ARACHIDONIC ACID TRANSFORMATION AND TUMORS

53

thromboxane (Brune et al., 1978a). Subsets of monocytes and macrophages appear to be capable of metabolizing arachidonic acid differently. Around 65% of cyclooxygenase products synthesized by WI-38 is PGEZ. Thromboxane (12%), PGF2, (18%), and prostacyclin (3%) are other biosynthetic products. D-550 cells, a fibroblast cell line derived from normal human foreskin, synthesizes mainly PGE, and PGF,,, in a ratio of 4 to 1, respectively. Similar percentages of PGE2 and PGF2, are produced by the adult type I1 alveolar cells isolated from rat lung. Taylor et al. (1979) report that these adult type I1 alveolar cells synthesize mainly PG12: 80% of cyclooxygenase products measured by release of radiolabeled compounds from [SH]arachidonic acid-prelabeled cells was found to be 6-keto-PGF1, . About 70% of the cyclooxygenase products of epithelial-like dog kidney cells is PGF2,. This cell also synthesizes considerable amounts of prostacyclin (22%) and PGE2 (9%).The cyclooxygenase products synthesized by some cells in culture are shown in Table I. Thus, it is not surprising that most extracts of tumors contain cyclooxygenase products. In most of the early studies on prostaglandin content of tumors, only PGE2and PGFzawere measured. The levels of these prostaglandins in tumors or the tumors’ biosynthetic capacities usually were compared to those of the appropriate normal tissue. PGE, and/or PGFza have been found in extracts of medullary, anaplastic and papillary carcinomas of the thyroid (Jaffe and Condon, 1976; Kaplan et al., 1973; Williams et al., 1968), neuroblastoma (Williams et al., 1968), pheochromocytoma (Papanicolaou et al., 1975; Sandler et al., 1968), islet cell tumors (Sandler et al., 1968), colonic carcinoma (Bennett et al., 1977a),breast carcinoma (Bennett et al., 1977b, 1979, 1980b; Rolland et al., 1980; Stamford et al., 1980),bronchial carcinoma (Fiedler et al., 1980), renal cell carcinoma (Zusman et al., 1974), and in several other neoplasms (Goodwin et al., 1980; Husby et al., 1977). Prostaglandins have been found in extracts of experimental tumors. PGE2, as measured by bioassay and identified by gas chromatographic and mass-spectrometric analysis, is present in BP8/ P, ascites cells and in BP8/P1 solid tumors of mice as well as in the mouse sarcoma 180 tumors (Sykes and Maddox, 1972). A large increase in PGE and PGF is found in virus-induced Maloney sarcoma tumors when compared with the levels of those prostaglandins in the normal leg muscles of these mice (Humes and Strausser, 1974; Humes et al., 1974; Strausser and Humes, 1975). We have found that extracts of a mouse fibrosarcoma, HSDM1, contain large amounts of PGE2 (Tashjian et al., 1972), as does the VX, carcinoma of rabbit (Voelkel et al., 1975).

ARACHIWNIC

TABLE I ACID METABOLISM B Y CELLS IN CULTURE" % Cyclooxygenase product in culture fluid

Cell line

Source

Stimulant

Analysis technique

&Keto-PGF,, ______

WEHI-5 Endothelial MDCK WI-38

D-550 RBL- 1

Mouse lymphoma Bovine aorta Dog kidney Normal human embryonic lung Normal human foreskin Mouse leukemia

Melittin (1 &ml) Melittin (1 pg/ml) TPA (1 ng/ml) Melittin (1&ml) Melittin (1pg/ml) A-23187 (1pg/ml)

RIA RIA RIA RIA RIA RIA

4

93 22 3 1

1

TxBI

PGF2,

PGE,

PGD,

58 1 <1 12 1 0

19 3 68 18 19 22

16 2 9 67 79

1 <1 <1 <1 (1 16

~~

60

a The serologic specificities of the appropriate anti-arachidonic acid metabolites have been reported in the following: 6-Keto-PGF1,, Gaudet et al. (1980);TxB,, Alam et al. (1979);PCFZ,, Levine et al. (1971),Pong and Levine (1977);PCEI, Levine et al. (1971), Pong and Levine (1977);PGDz, Gaudet et al. (1980), Levine et al. (1979).

ARACHIDONIC ACID TRANSFORMATION AND TUMORS

55

Possibly of more significance in assessing the relationship of arachidonic acid transformation to cancer are the findings of elevated prostaglandins in the blood and urines of patients with cancers or in animals bearing tumors. Bennett (1979) has extensively reviewed the literature dealing with levels of prostaglandin found in the blood of patients with tumors. Increases have been found in the blood of patients with medullary carcinoma of the thyroid (Jaffe and Condon, 1976; Kaplan et al., 1973; Williams et al., 1968), neuroblastoma (Williams et al., 1968), Kaposi’s sarcoma (Bhana et al., 1971), breast cancer (Bockman and Myers, 1977; Powles et al., 1977; Rolland et al., 1980; Stamford et al., 1980), renal cell carcinoma (Cummings and Robertson, 1977), bronchial carcinoma (Fiedler et al., 1980), and a variety of other cancers (Demers et al., 1977, 1979; Demers and Derck, 1980). Increases in prostaglandin metabolites are present in urines of a variety of patients with cancer (Seyberth e t a l , 1975).

IV. Arachidonic Acid Transformation and Hypercalcemia

In a significant number of patients with cancer, the accompanying hypercalcemia appears to have a humoral etiology; resection of the tumors in such cases leads to remission of the hypercalcemia, and reappearance of the tumor is accompanied by the hypercalcemia (Tashjian, 1978). Production by the tumors of serologically active parathyroid hormone has been implicated, but parathyroid hormone is found only in a minority of such patients (Powell et al., 1973). Some arachidonic acid transformation products are potent bonereabsorbing substances in vitro (Bennett et d., 1980a; Dietrich e t d . , 1975; Klein and Raisz, 1970; Raisz et al., 1977; Santoro et al., 1977; Tashjian et al., 1977~). PGE, is the most potent of the cyclooxygenase metabolites, but other products and their metabolites also possess bone-resorbing activities. The bone-resorbing activities of many of the lipoxygenase products are not known. Constant intravenous infusion of exogenous PGE, in the unanesthetized, intact rat leads to elevated plasma calcium levels (Franklin and Tashjian, 1975), but infusion of PGEz or PGEl into the thoracic aorta does not affect plasma calcium levels in the dog (Belie1 et al., 1973). Intraarterial PGE, infusion in thyroparathyroidectomized, but not intact, rats elevates plasma calcium levels (Robertson and Baylink, 1977). IntraperitoneaI injections of 16,16-dimethyl-PGE,-methylester,a long-acting synthetic analog of PGE,, result in enhanced bone resorption as measured histologically (Santoro e t aZ., 1977). The humoral mediator of hypercalcemia

56

LAWRENCE LEVINE

was suggested to be PGE, (Tashjian et al., 1972) when it was found that a transplantable mouse fibrosarcoma HSDMl produced elevated plasma calcium concentrations two weeks after subcutaneous or intramuscular implantation of the tumor. This tumor does not invade bone. The tumor, as well as clonal strains of HSDMl cells grown in culture, produces large amounts of PGE,, which was identified as the bone-resorbing factor (Levine et al., 1972; Tashjian et al., 1974). Indomethacin, a potent inhibitor of cyclooxygenase activity, 5,8,11,14eicosatetraynoic acid, an inhibitor of both cyclooxygenase and lipoxygenase activities, and hydrocortisone, which blocks both cyclooxygenase and lipoxygenase pathways by preventing formation of substrate, inhibit the formation of PGE, by the cells in culture (Levine et al., 1972; Tashjian et al., 1972, 1974). In uivo, plasma levels of PGE,, and more strikingly its more stable metabolite 13,14-dihydro15-keto-PGE,, are elevated after tumor implantation (Tashjian et al., 1973, 1977a). The elevation of the plasma levels of the metabolite precedes that of the plasma calcium. Treatment of the tumor-bearing mice with indomethacin (Tashjian et al., 1973) or hydrocortisone (Tashjian et al., 1977a) prevents the elevation of both plasma 13,14dihydr0-15-keto-PGE~and plasma calcium, both of which are again elevated upon withdrawal of the indomethacin or hydrocortisone treatment. A second animal model in which the elevated levels of plasma calcium are associated with PGE, production by the tumor is the VX, squamous-cell carcinoma in the rabbit (Voelkel et al., 1975). In this tumor, as with HSDM, fibrosarcoma of the mouse, clonal strains of cells in culture as well as the tumor produce large amounts of PGE,. Both the PGE, and the bone-resorbing activities of the cells and tumors are inhibited by indomethacin. Rabbits have elevated plasma levels of 13,14-dihydro-15-keto-PGE, which precede the elevation of plasma calcium (Alam et al., 1980; Seyberth et al., 1977; Tashjian et al., 1977b) if the tumor is implanted intramuscularly. If, however, the tumor is implanted intra-abdominally, the plasma level of 13,14-dihydro-15-keto-PGEzis again elevated, but not that of plasma calcium (Hubbard et al., 1980).Treatment of the rabbits with indomethacin or hydrocortisone either prevents or reduces the hypercalcemia and elevated levels of 13,14-dihydro-15-keto-PGE,,depending on the time between the treatment and the intramuscular implantation (Tashjian et al., 1977b). The effects of such treatments are reversible. Whereas the elevation in peripheral venous plasma PGE, is difficult to detect, the elevation of its longer lived metabolites is striking. However, the level of PGE, in the venous drainage of the

ARACHIDONIC ACID TRANSFORMATION AND TUMORS

57

V X 2 tumor is higher than that found in systemic venous plasma and that of the opposite femoral vein (Voelkel et al., 1975). The plasma concentrations of two acute-phase reactants, ceruloplasmin and haptoglobin, rise rapidly following implantation of the VX2 tumor (Voelkel et al., 1978). Plasma concentrations of ceruloplasmin rise in parallel with 13,14-dihydro-15-keto-PGEe, and both precede the rise in plasma calcium. Indomethacin prevents the rise in the ceruloplasmin and reduces the elevation once it has occurred. No changes in plasma albumin concentration are noted. It was suggested that arachidonic acid metabolites may play a role in the elevation of these acute-phase proteins in certain patients with malignant tumors, as well as in patients with certain chronic inflammatory diseases (Voelkel et al., 1978). Conditioned media from synovial tissue from patients with rheumatoid arthritis contain bone resorption-stimulating activity. The synovial cultures also produce PGE2.The production of both the bone resorption-stimulating activity and PGE, is inhibited more than 90% by treatment of the synovial cultures with indomethacin. It was suggested that the PGEz produced by rheumatoid synovia may contribute to the destruction of juxta-articular bone in rheumatoid arthritis (Robinson et al., 1975). Bone itself can metabolize arachidonic acid. PGE2 is produced by bone by a complement-mediated reaction (Raisz et al., 1974; Sandberg et al., 1977)as well as after treatment with crude collagenase (Dowsett et al., 1 9 7 6 ~ )Bone . is stimulated to transform arachidonic acid (PGE2 was measured) after stimulation b y 12-0-tetradecanoyl phorbol-13acetate (TPA), a potent tumor promoter, and melittin, a polypeptide isolated from bee venom (Tashjian et al., 1978), as well as epidermal growth factor (Tashjian and Levine, 1978). It was suggested that PGEz could act locally to stimulate bone resorption. The tumor promoters, epidermal growth factor, and melittin stimulate acylhydrolase activity of cells in culture (Hassid and Levine, 1977; Levine and Hassid, 1977a,b), so it is not surprising that arachidonic acid metabolites other than PGE2 are found in bone-culture-conditioned media. Fetal rat bones synthesize PGE2, PGF2,, and PG12 (Raisz et al., 1979). Mouse calvaria also synthesize PGE,, PGF2,, and PG12 and, in addition, oxidize the 15-hydroxy function of these prostaglandins to their respective 15-keto metabolites and reduce the 13,14 double bond to the 13,14-dihydro-15-keto compounds (Voelkel et al., 1980). When incubated with exogenous arachidonic acid, bone synthesizes thromboxane. PG12 stimulates bone resorption of fetal rat bones (Raisz et al.,

58

LAWRENCE LEVINE

1979), but conflicting data have been reported on the activity of PG12 on mouse calvaria (Bennett et al., 1980a; Voelkel e t al., 1980). However, in the VX2 squamous-cell carcinoma of the rabbit, elevations of PGIz in plasma, measured as its more stable product 6-keto-PGF1,, have not been found (Alam et al., 1979). It must be recalled that the bone-resorbing activities of the lipoxygenase products of arachidonic acid transformation have not been reported. The mouse HSDMl fibrosarcoma and the rabbit VX2 squamous-cell carcinoma could be visualized as animal models for tumors that mediate hypercalcemia in human malignancy. This concept is supported by the findings that indomethacin treatment reduces the hypercalcemia of a patient with a renal cell adenocarcinoma (Brereton et al., 1974) and that, in a patient with renal cell carcinoma, the hypercalcemia is accompanied by elevated plasma immunoreactive PGEr but low plasma parathyroid hormone (Robertson et al., 1975). Similar responses to indomethacin treatment, or the findings of elevated plasma levels of prostaglandins, in patients with hypercalcemias associated with a variety of malignancies have been reported (Demers et d., 1977; Seyberth et aZ., 1975). However, many patients with hypercalcemias associated with various malignancies do not respond to indomethacin treatment, nor can an elevated prostaglandin synthesis be demonstrated (Seyberth et aZ., 1978). V. Arachidonic Acid Transformation and Tumor Promotion

Carcinogenesis may be a multistep process (Boutwell, 1974), and it is probable that various environmental factors act at different steps in this process. In one of the best-studied models, the “two-stage carcinogenesis” system in mouse skin (Berenblum, 1969), two distinct stages designated “initiation” and “promotion” have been identified. Additional stages and cofactors may play a role in cancer induction in other tissues and species. The interaction of multiple factors probably is required for the induction of many human cancers. The essential features of the two-stage carcinogenesis model are the following: (1)one application of the initiator alone to mouse skin will produce few or no tumors; (2) many applications of the promoting substance alone will produce inflammation followed by epithelial hyperplasia but will not produce tumors; and (3) one application of the initiator followed by many applications of the promoter results in both benign and malignant tumors. The promoting stage has been separated into at least two stages (Boutwell, 1974). Tumor promoters and

AFIACHIDONIC ACID TRANSFORMATION AND TUMORS

59

mechanisms of tumor promotion have recently been reviewed (Diamond et al., 1980).

A. EFFECTSOF TPA ON PROSTAGLANDIN PRODUCTION BY CELLSIN CULTURE In the simple two-stage model in mouse skin (Berenblum, 1969), croton oil prepared from the seeds of Croton tiglium was the promoting agent. The fractions from the croton oil responsible for the activity have been purified and characterized by Hecker (1968) and van Duuren (1969) and their colleagues. Indeed, the most extensively studied tumor promoters are the phorbol diesters purified from croton oil. Among these, TPA is the most active. TPA stimulates several types of cells to produce prostaglandins (Table 11). The most responsive of these cells is the canine kidney cell line MDCK; as little as to loTLoM TPA is sufficient to stimulate prostaglandin production (Levine and Hassid, 1977b). Phorbol-12,13-didecanoate(PDD) at M and 10-lo M also stimulates prostaglandin production by MDCK cells, but the non-tumor-producing phorbol diester 4a-phorbol-12,13didecanoate (4a-PDD) is inactive even at lo-' M . One of the several activities of TPA on cells is deacylation of cellular

TABLE I1 CELLS IN WHICH AFUCHIDONIC ACID TRANSFORMATION IS STIMULATED BY TPA Cell

Source

Reference

M DCK WEHI-5 HSDMl LC-540 Smooth muscle WI-38 D-550 MC5-5 Macrophages Osteosarcoma

Dog kidney Mouse lymphoma Mouse fibrosarcoma Rat Leydig Bovine aorta Human embryonic lung Human foreskin Transformed mouse fibroblast Mouse peritoneum Human bone

Y-1

Mouse adrenal Mouse epidermis Chick embryo

Levine and Hassid, 1977b Levine et al., 1979 L. Levine, unpublished data L. Levine, unpublished data L. Levine, unpublished data Levine et al., 1979 Levine et al., 1979 Levine et al., 1979 Brune et al., 1978a M. A. Shupnik and A. H. Tashjian, Jr., unpublished data L. Levine, unpublished data Fiirstenberger et al., 1980 Mufson et al., 1979 Crutchley et al , 1980

HEU30 Fibroblasts HeLa

60

LAWRENCE LEVINE

phospholipids (Levine and Hassid, 197713; Ohuchi and Levine, 1978a). TPA and PDD, but not 4a-PDD, stimulate the release of arachidonic acid from the membrane phospholipids of MDCK cells. The hee arachidonic acid can then be metabolized by the cyclooxygenase system to the prostaglandins. TPA and PDD, but not 4aPDD, also alter the morphology of MDCK cells. These effects of TPA, deacylation of phospholipids, prostaglandin production, and alteration of cell morphology, require time for expression. Cycloheximide inhibits all three effects (Ohuchi and Levine, 1978a). Indomethacin inhibits the stimulated prostaglandin synthesis and, at high concentrations, also inhibits TPA’s effect on cell morphology (Levine, 1981). Indomethacin is a potent inhibitor of cyclooxygenase (Lands and Rome, 1976; Smith and Lands, 1971) but is also an inhibitor of phospholipase A2 (Kaplan et al., 1978), phosphodiesterase (Flores and Sharp, 1972), PGE2-9-keto reductase (Hassid and Levine, 1977b; Stone and Hart, 1975), and a CAMP-dependent protein kinase (Kantor and Hampton, 1979); it also inhibits some deacylation of cellular lipids (Ohuchi and Levine, 1978b). Unstimulated macrophages obtained from the peritoneal cavity of mice respond to TPA ( 10-s-lO-s M) and PDD (10”-10-* M) treatment by releasing PGE2 (Brune et al., 1978a). TPA (10-7-10-s M ) rapidly releases arachiodonic acid, PGE2, and PGFZafrom chick embryo fibroblasts, PDD, phorbol- 12,lSdibenzoate and mezerein are also active, whereas phorbol and 4a-PDD are ineffective (Mufson et al., 1979). Both the release of arachidonic acid and the stimulation of prostaglandin production are inhibited by cycloheximide and puromycin. Indomethacin inhibits TPA-induced prostaglandin synthesis but slightly enhances arachidonic acid release. TPA stimulates PGE2 and PGF,, production in HeLa cells (Crutchley et al., 1980), PGE2 biosynthesis in the murine epidermal cell line HEW30 (Fiirstenbergeret al., 1980), and production of PGE2, Fa,, and I2 by bone (Voelkel et al., 1980). TPA stimulates prostaglandin biosynthesis by activating acylhydrolases (Levine, 1979b; Levine and Alam, 1981; Levine and Hassid, 1977b; Levine and Ohuchi, 1978b; Ohuchi and Levine, 1978a). As outlined in Section 11, several biochemical pathways have been recognized within this deacylation reaction. There is selectivity among the acylhydrolases for substrate or enzyme, or both when TPA stimulates MDCK cells; MDCK cells in which the phospholipids are labeled with [‘4C]linoleic acid and [3H]arachidonic acid are stimulated by TPA to release arachidonic acid but not linoleic acid (Ohuchi and Levine, 197813).

ARACHIDONIC ACID TRANSFORMATION AND TUMORS

61

B. INHIBITORS OF PROSTAGLANDIN PRODUCTION: THEIREFFECTS ON THE ACTIVITIES O F TPA IN CELLS IN CULTURE Indomethacin is one of the most potent inhibitors of prostaglandin production (Robinson and Vane, 1974). It prevents formation of endoperoxides by blocking cyclooxygenase activity (Lands and Rome, 1976; Smith and Lands, 1971). However, indomethacin also inhibits phospholipase A2 (Kaplan et al., 1978), acylhydrolase activity in MDCK cells (Ohuchi and Levine, 1978b), phosphodiesterase (Flores and Sharp, 1972), PGE2-9-keto reductase (Hassid and Levine, 1977b; Stone and Hart, 1975), and a CAMP-dependent protein kinase (Kantor and Hampton, 1979). In view of this nonspecificity, the effects of indomethacin may not be attributed solely to inhibition of prostaglandin biosynthesis (Flower, 1974). Indomethacin does inhibit TPAstimulated prostaglandin production by cells in culture. Another series of inhibitors of prostaglandin production are the anti-inflammatory steroids (Gryglewski et al., 1975; Kantrowitz et al., 1975; Lewis and Piper, 1975; Tashjian et al., 1975). In some cells, the corticosteroids have been shown to inhibit prostaglandin production by blocking the expression of acylhydrolase activity (Hong and Levine, 1976; Tam et al., 1977). The glucocorticoids may inhibit acylhydrolase activity by inducing biosynthesis of a phospholipase A2 inhibitor (Blackwell et al., 1980; Flower and Blackwell, 1979; Nijkamp et al., 1976). In rabbit neutrophils, glucocorticoids induce a phospholipase A2 inhibiting protein (Hirata et al., 1980). The mechanism of action of glucocorticoids may vary among cells and tissues. In 3T3 cells, corticosteroids stimulate cyclooxygenase activity, although arachidonic acid release is inhibited under some conditions (Chandrabose et al., 1978). In rheumatoid synovia, release of arachidonic acid is unaffected even under conditions where prostaglandin synthesis is more than 80% inhibited (Robinson et al., 1980). Preincubation of dexamethasone with smooth muscle cells prepared from bovine aorta inhibits the stimulation of prostaglandin production of TPA (L. Levine, unpublished data). The retinoids are another class of inhibitors that block tumor promotion. They are effective both in vitro and in uivo. Retinoids inhibit promoter-induced ornithine decarboxylase (ODC) activity in mouse epidermis (Verma and Boutwell, 1977; Verma et al., 1978). Those retinoids that inhibit TPA-induced ODC activity in mouse epidermis also inhibit TPA-induced ODC activity in phytohemagglutinin-treated bovine lymphocytes (Kensler et al., 1978) and TPA’s comitogenic activity in these lectin-treated cells (Kensler and Mueller, 1978). An-

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other inhibitor of TPA’s comitogenic effect on these cells is 5,8,11,14eicosatetraynoic acid (Wertz and Mueller, 1980), an inhibitor of lipoxygenase as well as the prostaglandin-producing cyclooxygenase. Retinoids, at low concentrations, stimulate plasminogen-activator synthesis in chick embryo fibroblasts, and at suboptimal levels of TPA the effects of retinoic acid and TPA are synergistic (Wilson and Reich, 1978). In MDCK cells the retinoids, cis-retinoic acid, trans-retinoic acid, retinyl acetate, retinol, retinal, retinyl palmitate, and trimethylme thoxyphenyl retinoic acid, do not affect deacylation of phospholipids or prostaglandin production, and at relatively high concentrations they even enhance them (Levine and Ohuchi, 1978a). In chick embryo fibroblasts, trans-retinoic acid ( 10-5-10-6 M ) inhibits TPA-induced arachidonic acid release and prostaglandin production (Mufson et al., 1979). The synthetic retinoid N-(4-hydroxyphenyl)retinamide,at levels ranging from 0.025 to 3.1 p M , inhibits serum- and TPA-stimulated biosynthesis of PGFz., 6-keto-PGF1., and PGEz by MDCK cells (Levine, 1979a). N-(4-hydroxyphenyl)retinamide also is a potent inhibitor of PGEz and PGFzu production by serum-stimulated methylcholanthrene-transformed mouse fibroblasts and normal human fibroblasts. In the presence of 10% fetal bovine serum, N (4-hydroxypheny1)retinamideinhibits prostaglandin production by MDCK cells 4 times less effectively than indomethacin and about 50 times more effectively than aspirin. However, N-(4-hydroxyphenyl)retinamide does not affect the release of radiolabeled materials from TPA-stimulated labeled cells; cyclooxygenase activity appears to be inhibited, but not acylhydrolases (Levine, 1979a). The contributions of the 4-hydroxy- and phenol- functions of N-(4-hydroxyphenyl)retinamide to its prostaglandin-inhibiting effectiveness can be seen in Fig. 2 (L. Levine and M. Sporn, unpublished data). Another inhibitor of TPA-stimulated prostaglandin production in MDCK cells is a-tocopherol (Ohuchi and Levine, 1980). a-Tocopherol inhibits prostaglandin production in TPA-stimulated MDCK cells but not in control cells. This inhibitory effect is observed only if the cells are treated with TPA in the presence of a-tocopherol. a-Tocopherol decreases TPA-stimulated prostaglandin production by inhibiting the binding of [3H]TPA to the cells. The mechanism is not clear since relatively large concentrations ( 102-103mole excess of a-tocopherol to TPA) are required to inhibit binding to the cells. It is possible that TPA is more soluble in a-tocopherol bound in the MDCK cell membrane or in an a-tocopherol micelle. Another possibility is that a receptor site for TPA is being reversibly blocked by a-tocopherol. There appears to be

Retinoid

@

A

A

.

L

O

”

Effect

-

p

on Prostaglondln Production

It

N-(Z-Hydroxyphenyl)retinornide

N- (3-HydroxyphenyNretinomide

N- Phenylret inomide

~ - ( 4 - ~ t h o x y p h e n Iratinornide yl

@

L

b

N

q

-

;

:

~-(4-Hydroxy-3-carboxypheny1)retinomide

N-(3-Methyl-4-hydroxyphenyl lrelinomide

.1 = strong inhibitor; + = no effect; t t = stimulation; = slight inhibition; f = slight stimulation (L.Levine and M. Sporn, unpublished data). FIG.2. Effects of some synthetic retinoids on prostaglandin production.

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some specificity for tumor promoters originating from Croton tiglium L.; a-tocopherol inhibits the effects of the tumor-promoting phorbol esters obtained from this plant but not the effects of diterpenoid esters isolated from the plant genera Daphne or Gnidia. TPA stimulation of prostaglandin synthesis requires protein synthesis. Cycloheximide from 0.05 to 0.5 pg/ml inhibits TPA-stimulated prostaglandin production and deacylation of cellular phospholipids in MDCK cells (Ohuchi and Levine, 1978a). Cycloheximide, 40 pg/ml, and puromycin, 20 pg/ml, inhibit TPA-stimulated prostaglandin production and acylhydrolase activity by more than 90% in chick embryo fibroblasts (Mufson et al., 1979). Inhibition of TPA-stimulated deacylation of cellular lipids and prostaglandin production b y indomethacin, dexamethasone, retinoic acid, N-(4-hydroxyphenyl)retinamide,a-tocopherol, and cycloheximide is summarized in Table 1x1. In MDCK cells, retinoic acid does not alter any of these responses, and at high concentrations it is synergistic with TPA (Levine and Ohuchi, 1978a). Indomethacin inhibits prostaglandin production very effectively, but it also inhibits the deacylation of cellular lipids (Ohuchi and Levine, 1978b). N(4-Hydroxypheny1)retinamideinhibits TPA-stimulated prostaglandin production but does not affect deacylation of lipids (Levine, 1979a). a-Tocopherol inhibits both effects, prostaglandin production and deacylation of cellular lipids; it does this by blocking the binding of TPA to MDCK cells. Dexamethasone, which inhibits prostaglandin

TABLE I11 INHIBITION OF TPA-STIMULATED DEACYLATION OF PHOSPHOLIPIDS AND PROSTAGLANDIN PRODUCTION BY CELLSIN CULTURE Inhibitor Cyc!oheximide" Indomethacin Retinoic acid' N-(4-Hydroxypheny1)retinamide a-TocopherolC Dexamethasoned

0.1 pg/ml. * 5 x 10-5 M (+); 1 x 10-5 M (-). ' MDCK cells. MC5-5 and smooth muscle cells.

Phospholipid deacylation

Prostaglandin production

+

+ + + + +

20

-

+ +

ARACHIDONIC ACID TRANSFORMATION AND TUMORS

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production in several cells by preventing the expression of acylhydrolase activity, inhibits TPA-stimulated prostaglandin production in smooth muscle cells (L. Levine, unpublished data). Cycloheximide (0.1 vg/ml) inhibits both TPA-stimulated reactions, most likely by inhibiting synthesis of a protein that stimulates prostaglandin production and acylhydrolase activity. Whereas the TPA-stimulated prostaglandin biosynthesis is inhibited by cycloheximide of 0.1 pg/ml, that of most other stimulators is not (Levine, 1981). However, cycloheximide at 2.0 pdml inhibits serum-, thrombin-, and bradykinin-stimulated prostaglandin synthesis by MC5-5 cells (Pong et al., 1977). C. EFFECTSOF TPA ON PROSTAGLANDIN PRODUCTION in Vivo There is good correlation between the irritant and promoting activities in mouse skin and PGE2-release in mouse macrophages in a series of diterpine derivatives (Brune et al., 197813). One of the pleiotypic effects of TPA on mouse skin is induction of ornithine decarboxylase (ODC) activity (Diamond et al., 1980). Indomethacin inhibits TPA’s induction of ODC in mouse skin (Verma et al., 1980). Moreover, this inhibition is completely blocked by treatment with PGEl and PGE2,but not by PGF1, and PGF,,. Indomethacin administered one hour before TPA completely inhibits the proliferative response of mouse epidermis to TPA; this inhibition is reversed by applying PGE2,but not PGFz., simultaneously with TPA (Furstenberger and Marks, 1978). TPA-induced skin inflammation is not influenced by indomethacin, suggesting that PGE, (or a closely related compound) mediates the mitogenic effect of TPA in mouse skin. TPA does not compete for hypothetical PGE receptors, at least not on adipose cells (Furstenberger and Marks, 1979a) as suggested by Smythies et al. (1975). Lupulescu (1978) has shown that mice treated with 3-methylcholanthrene and injected concomitantly with 10 pg of PGE, three times weekly for 2 months have increased skin tumors. When PGF, replaces PGE, in the intramuscular injections, no increase is observed. Two peaks of PGE biosynthesis, one at 10 minutes and a second at 60 minutes, are found in mouse skin following a topical application of TPA (Fiirstenberger and Marks, 1980). Pretreatment of the skin with indomethacin abolishes the 10-minute peak of PGE synthesis as well as the proliferative response induced by TPA. However, if the indomethacin is applied 30-60 minutes after application of the TPA, inhibition of the proliferative response is not observed. Thus, the early PGE synthesis was suggested to be an obligatory event for epidermal

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cell proliferation induced b y TPA. Another effective stimulator of prostaglandin production is the calcium ionophore antibiotic A-23187 from Streptomyces chartreusis (Gemsa et al., 1979; Knapp et al., 1977; Marks et al., 1981; Pickett et al., 1977; Rittenhouse-Simmons and Deykin, 1977; Waelbroeck and Boeynaens, 1977; Wantzell and Epand, 1978; Weksler et al., 1978). Topical application of A-23187 on mouse skin induces in vivo prostaglandin-mediated epidermal hyperplasia and inflammation similar to those initiated by TPA (Marks et al., 1981). However, A-23187 does not have tumor-promoting activity. Thus, these two biological effects of TPA, prostaglandin-mediated epidermal hyperplasia and inflammation, are not indicative of tumor-promoting capacity (Marks et al., 1981). Natural and synthetic glucocorticoids inhibit tumor promotion (Belman and Troll, 1972; Ghadially and Green, 1954; Schwarz et al., 1977). Topical application of dexamethasone inhibits TPA-mediated tumor promotion (Scribner and Slaga, 1973). Fluocinolone acitonide is as effective as dexamethasone at lower dose levels and also blocks promoter-induced hyperplasia and DNA synthesis (Schwarz et al., 1977).As summarized above, glucocorticoids also inhibit expression of acylhydrolase activity and consequently prostaglandin production only in some cells (Hong and Levine, 1976; Tam et al., 1977) and tissues (Blackwell et al., 1980; Flower and Blackwell, 1979; Nijkamp et al., 1976). Indomethacin, a more potent inhibitor of prostaglandin production but a weak inhibitor of acylhydrolase activity (Ohuchi and Levine, 1978b), at low doses is only a weak inhibitor of TPA-induced tumor promotion (Viaje et al., 1977) but at high doses is an effective inhibitor of TPA-induced biochemical effects (Fiirstenberger and Marks, 1978; Verma and Boutwell, 1977). Since indomethacin can affect acylhydrolase activity, mediation of the TPA effects by the lipoxygenase products of arachidonic acid transformation (e.g., hydroxy fatty acids and leucotrienes) cannot be excluded. D. STIMULATIONOF PROSTAGLANDIN PRODUCTION BY GROWTH FACTORS Some proteins stimulate acylhydrolase activity and prostaglandin production, e.g., epidermal growth factor and platelet-derived growth factor (Coughlin et al., 1980; Levine and Hassid, 1977a). Several growth factors recently have been described and characterized (Dayer et al., 1977; DeLarco and Todaro, 1978; Meats et al., 1980; Roberts et al., 1980). All these factors stimulate prostaglandin production (Levine, 1981); they probably do so by stimulating acylhydrolase ac-

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tivity. Some of these growth factors have been shown to confer transformed phenotypes on normal cells in vitro (DeLarco and Todaro, 1978; Roberts et al., 1980). A family of such growth factors probably exists in cells; their mitogenic effects and ability to transform phenotypes may be causally related to their capacity to stimulate acylhydrolase activities. VI. Prostaglandins: Their Effects on Cell or Tumor Growth

Addition of exogenous prostaglandins to cells in culture has led to the conclusion that prostaglandins, especially PGE, inhibit cell growth. PGE, added to cultures of mouse 3T3 fibroblasts slows growth of the cells (Johnson and Pastin, 1971, 1972). The growth of murine plasma tumor (MPC-11) is suppressed by 0.1 to 10 pg PGE,; PGE2 is slightly less effective; and PGF2, is ineffective (Naseem and Hollander, 1973). PGE inhibits the growth of mouse EL-4 lymphoma cells; PGEZ, 1-100 pg/ml, also inhibits proliferation as measured by thymidine uptake (Sonis et al., 1977). PGEl, 1 puglml, inhibits cell replication by mouse B-16 melanoma, and in vivo 16,16-dimethylPGEz methylester, 5 pg injected subcutaneously at the site of the tumor cell injection, reduces tumor growth (Santoro et al., 1976). PGF2a, 10-400 ng/ml, added to quiescent Swiss mouse 3T3 cell initiates DNA synthesis and cell proliferation in a small proportion of the cells. PGEl and PGE2also are effective at initiating DNA synthesis but only at high concentrations, and even at high concentrations these prostaglandins are less effective than PGFza (DeAsua et al., 1975). Inhibitors of prostaglandin production also have been used to study the effects of prostaglandins on tumor growth. In general, they stimulate growth, but inhibition also has been observed. It must be recalled that inhibitors of cyclooxygenase by nonsteroidal anti-inflammatory drugs block the production of prostaglandins, prostacyclin, and thromboxanes. Inhibitors such as 5,8,11,14-eicosatetraynoicacid block production of cyclooxygenase and lipoxygenase products. Antiinflammatory steroids also may block the production of cyclooxygenase and lipoxygenase products. Some inhibitors (e.g., indomethacin) have multiple specificities and may affect enzymes other than those mediating prostaglandin synthesis. Indomethacin (0.1-100 nM) increases the replication of HEp-2, L, and HeLa cells; the effect is reversed by adding PGEl (1 pg/ml) to the culture. However, indomethacin at 1p M does not significantly alter growth (Thomas et al., 1974).The growth of mouse B-16 melanomain vitro is enhanced in the presence of indomethacin or hydrocortisone (Santoro et al., 1976).Rep-

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lication of methylcholanthrene-induced fibrosarcoma cells from C3H mice is not significantly affected by 0.1 pgfml indomethacin, but at 20 kg/ml, thymidine uptake is increased 2.5-fold (Lynch et aZ., 1978). On the other hand, indomethacin, aspirin, or sodium salicylate reversibly inhibit growth of rat hepatoma cells; there is no effect with 1-10 p M indomethacin, approximately 20% inhibition with 0.1 mM, and 80% inhibition with 0.5 mM (Hial et al., 1977). Indomethacin does not affect the growth of mouse HSDM, fibroblasts in vitro (Levine et al., 1972), but indomethacin (100-125 pg) administered to mice daily with their food reduces the weights of the HSDMl fibrosarcoma by 27% (Tashjian et al., 1973).Indomethacin (50 pg administered subcutaneously on alternate days) reduces the size of Maloney sarcoma virus-induced tumors in 20-day-old BALB/c mice and delays the onset of tumor growth (Humes et aZ., 1974). In older mice (6-week-old), the same dose of indomethacin is much more effective at reducing the size of the Maloney sarcoma virus-induced tumors (Strausser and Humes, 1975). Bone destruction by the virus-induced tumor is also inhibited by this indomethacin treatment. The growth of a transplanted methylcholanthrene-induced mouse fibrosarcoma is reduced in mice given indomethacin (125 pg/day) interperitoneally for the first 10 to 14 days (Plescia et al., 1975). Treatment of mice with aspirin is also effective at reducing the growth of tumors; aspirin (150 mg/kg administered twice daily by mouth) inhibits the growth of the transplanted mast-cell ascites tumor (P185)and Lewis lung carcinoma. Indomethacin (3-5 mg/kg) is even more effective (Hialet aZ., 1976). In C3H mice bearing a transplantable methylcholanthrene-induced fibrosarcoma, indomethacin or aspirin (administered by way of drinking water from day 7 to day 49 after tumor transplantation) reduces the tumor size measured at day 46 (Lynch et al., 1978). Indomethacin, aspirin, or hydrocortisone also increase survival time of the mice. The nonsteroidal anti-inflammatory drug flurbiprofen, an effective inhibitor of prostaglandin production, in combination with chemotherapy or radiotherapy reduces tumor growth in mice when compared to the treatment by chemotherapy and/or radiotherapy alone (Bennett et al., 1979). In addition, flurbiprofen given with methotrexate in mice following removal of the primary tumor prolongs survival and reduces the incidence of local recurrence (Berstock et al., 1980).Indomethacin inhibits growth of fibrosarcoma in mice and potentiates immunotherapy (Lynch and Salomon, 1979). Administration of indomethacin to rats with Yoshida hepatoma cells reduces tumors (Trevisani et al., 1980). N-(4-Hydroxyphenyl)retinamide, an effective inhibitor of prostaglandin production (Levine, 1979a), inhibits the development

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of breast cancer induced in rats by N-nitroso-N-methylurea when fed orally to rats over a 2-week or 6-month period (Moon et al., 1979). Prostaglandins may mediate metastatic spread of tumors, especially to bone. A correlation between the level of PGE2production by human breast cancer and its metastasis to bone has been reported (Bennett et al., 1975). In rats bearing a Walker carcinosarcoma, treatment with aspirin or indomethacin prevents metastasis to bone but does not slow growth of the tumor (Powles et al., 1978). In rabbits, indomethacin slows the skeletal destruction from tumor metastases but does not affect the development of pulmonary metastases (Galasko and Bennett, 1976). Development of osteolytic bone tumors in rats can be prevented by aspirin and indomethacin (Dowsett et al., 1976a,b; Powles et al., 1973). Benoral, an aspirin-paracetamol conjugate, also prevents bone metastasis in rats (Powles et al., 1980). However, treatment of human patients with breast cancer with aspirin and indomethacin does not significantly reduce metastasis to the bone (Powles et al., 1980). PGD, may play a role in metastases. This conclusion is based on the findings that the highly metastatic malignant melanoma B16Flo forms less PGD2 from PGH, compared to the moderately metastatic parent cell line B 16F1(Fitzpatrick and Stringfellow, 1979). VII. Prostaglandins and the Immune Response

As documented in earlier sections of this review, addition of exogenous prostaglandins or inhibitors of prostaglandin synthesis to cells in culture or in vivo affects cell and tumor growth. The use of inhibitors with multiple specificities, the number of arachidonic acid metabolites (Fig. l), their diverse pharmacological activities, and their lability may explain some of the contradictory findings attributed to a particular metabolic product or inhibitor. Nevertheless, it is clear that neoplastic tissues produce high levels of prostaglandins, probably more than normal tissue. It is also apparent that prostaglandins or inhibitors of prostaglandin synthesis affect cell and tumor growth. In Section VI, several examples of slowing tumor growth in vivo by the administration of inhibitors of prostaglandin synthesis have been cited. One general mechanism that could account for many of these phenomena is that prostaglandins affect the immune response; i.e., increased prostaglandin production suppresses the host’s immune response to the tumor. Inhibition of prostaglandin production by anti-inflammatory drugs (e.g., indomethacin, aspirin, flurbiprofen, flufenamic acid, glucocorticoids, or 5,8,11,14-eicosatetraynoicacid) permits the host’s

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immune system to more effectively reject the tumor. Such a view is supported by the following findings. Mice bearing a virus-induced ascites cell tumor (MCDV-12) or a methylcholanthrene-induced fibrosarcoma (MC-16) do not produce antibodies to sheep erythrocytes. The tumor cells block the immune response to the sheep erythrocytes when added to spleen cells in the presence of the sheep erythrocytes. These tumors synthesize large amounts of PGE2, and PGEz also blocks production of antibodies to the sheep erythrocytes in this in vitro system (Plescia et al., 1975). Administration of indomethacin, flufenamic acid, and aspirin to the syngeneic C57B1/6J mouse blocks the immunosuppressive activity of MC16 cells in vivo (Grinwich and Plescia, 1976). In untreated mice bearing MeC3-1 tumors, no evidence of immunosuppression is found; an enhanced immune response to sheep erythrocytes is reported (Lynch et al., 1978). In tumor-bearing mice, treatment with indomethacin phrtially restores the depressed mitogen responses of spleen cells to phytohemagglutinin and to bacterial lipopolysaccharides (Pelus and Strausser, 1976). Inhibitors of prostaglandin synthesis enhance the natural and antibody-dependent cytotoxicity of lymphocytes for PGEz-producing tumor cells (Droller et al., 1978). PGEz induces suppressor T lymphocytes (Goodwin and Webb, 1980) and also the production of suppressor factor by lymphocytes (Rogers et d., 1980). The source of the PGEz may not be the tumor cells. Macrophages secrete PGEz (Kurland and Bockman, 1978) which may regulate the proliferative response of normal peripheral blood mononuclear cells to T-cell mitogens (Goodwin et al., 1977, 1978). In the presence of indomethacin, the mitogen-induced lymphocyte proliferation is increased (Goodwin and Messner, 1979). PGEz overcomes this enhancement and returns the proliferative response to normal. In mice injected with sheep erythrocytes, administration of indomethacin increases the appearance of anti-sheep erythrocyteproducing spleen cells (Webb and Osheroff, 1976); i.e., the inhibitors of prostaglandin synthesis act on the suppressor cells. Lymphocytes from patients with Hodgkin’s disease incorporate less thymidine and produce increased levels of PGEz in response to phytohemagglutinin (Goodwin et al., 1977). Inhibitors of prostaglandin synthesis block both of these effects. In ten anergic patients with a variety of metastatic solid tumors, indomethacin does not reverse the depressed phytohemagglutinin response in vitro (Kauffman et al., 1978). However, an increase in prostaglandin-mediated immunosuppression may be an important factor in the anergy of some patients with malignancies other than Hodgkin’s disease (Vosixa and Thies, 1979).

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VIII. Challenges

Most cells have the potential to deacylate cellular lipids, to convert the free arachidonic acid to endoperoxides, and to form lipoxygenase-mediated hydroperoxy fatty acids. The endoperoxides are enzymatically and nonenzymatically converted to prostacyclin, thromboxane, and PGE,, PGF,, and PGD2, whereas the hydroperoxyarachidonic acid can be converted to several hydroxy fatty acids, dihydroxy fatty acids, and/or to a family of fatty acids that contain glutathione, cysteinylglycine, or cysteinyl moieties [ slow reactive substances (SRS)]. Many of these end products are unstable chemically and/or metabolically. Most of them are pharmacologically potent and possess diverse activities. This biochemical cascade is controlled at the deacylation reaction since free arachidonic acid is not present in cells; it is esterified. The deacylation reactions that make free arachidonic acid available for the cyclooxygenases and lipoxygenases are triggered by a variety of compounds of diverse biological activities (Levine, 1981). Protein synthesis is required for many of these stimulations; the rapidly turning over protein could be the phospholipase or a protein regulator(s) of phospholipase activity (Pong et al., 1977). We also suggest as one mechanism of action of anti-inflammatory steroids that “the steroids could affect the synthesis of a regulator for the phospholipase activity” (Tam et al., 1977). Such a regulator has been partially purified (Blackwell et d,1980; Hirata and Axelrod, 1980). Most likely, since there is more than one reaction leading to deacylation, more than one regulator of deacylation will be found. Most cells also have the enzymatic capacity to transform the free arachidonic acid. The final products of this transformation are unique to the cell; i.e., the levels and proportions of each product synthesized by the cell are intrinsic properties of that cell (see Table I). A cell that produces high concentrations and proportions of PGIz will produce predominantly PGI, when stimulated.. A cell that produces predominantly PGEz or TxAz will produce PGEz or TxAz in the same proportion when stimulated. It is not improbable that cells of similar embryonic origin but from different species will synthesize similar proportions of arachidonic acid transformation products. Cells also differ in their sensitivity to stimulation; for example, among the cells listed in Table 11, the TPA concentrations needed to stimulate vary by 2 to 3 orders of magnitude (L. Levine, unpublished data). Therefore, it is not surprising that some of the published effects of prostaglandins and prostaglandin synthesis inhibitors on cell and tumor growth conflict. Most of the studies cited suggest that

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arachidonic acid transformation is associated with cancer. The evidence for this association is (1)that neoplastic tissue produces high levels of prostaglandins, (2) that prostaglandins affect cell growth in vitro and tumor growth in vivo, and ( 3 ) that arachidonic acid transformation is associated with tumor promotion. However, whereas nonsteroidal anti-inflammatory inhibitors of prostaglandin synthesis may reduce tumor growth i n vivo or tumor growth in the two-stage mouse skin model of carcinogenesis, their effects are weak at best. Steroids are much more effective in the latter model. The effects of the nonsteroidal anti-inflammatory inhibitors of prostaglandin synthesis on tumor growth, i n vivo, are best observed when combined with chemotherapy or radiotherapy. Nevertheless, a causal relationship between arachidonic acid transformation and cancer has yet to be demonstrated. At least three steps in the arachidonic acid transformation scheme can be considered as being responsible for the previously summarized associations:

1. Acylhydrolase activity can lead to production of lysophospholipids and alteration of membrane fluidity (Hirata and Axelrod, 1980) [TPA treatment, which stimulates deacylation of cellular lipids (Levine, 1981), also alters membrane fluidity (Castagna et al., 1979; Weinstein et al., 1979)l. 2. Cyclooxygenase activity leads to production of the prostaglandins, prostacyclins, and thromboxanes. 3. Lipoxygenase activity leads to formation of the hydroxyarachidonic acids and SRS compounds. As noted, inhibitors of cyclooxygenase activity are only partially effective, at best, at reducing tumors, whereas inhibitors of acylhydrolase activities such as glucocorticoids, which also block reactions (2) and (3), are more effective. Thus, acylhydrolase activity, or possibly lipoxygenase activity, is more likely to be causally related to cancer than cyclooxygenase activity. It is possible that all three activities combine to give the tumor cell a favorable advantage for growth; the altered fluidity increases growth potential and differentiation properties of the cell, and, at the same time, the cell is stimulated to synthesize products that inhibit the immune response. It should be recalled that neoplastic tissue does produce high levels of prostaglandins; thus, acylhydrolase activity of neoplastic tissue and alteration of membrane fluidity is correspondingly high. These considerations suggest that inhibitors of acylhydrolase activity would be effective at reducing tumor growth i n vivo. Although the glucocorticoids inhibit the expression of acylhydrolase activity, they

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do so in some but not all cells; and, in addition, they may affect biochemical reactions other than expression of acylhydrolase activities. The regulators of acylhydrolase activity may be more specific. If arachidonic acid transformation and cancer are indeed causally related, an understanding of how these regulating molecules exert their effects may lead to their use in the control of tumor growth in uiuo. At any rate, the elucidation of their mechanism of action is required and offers a challenging area for future research.

ACKNOWLEDGMENTS The author’s research is supported by grants GM-27256 and CA-17309 from the National Institutes of Health. He is an American Cancer Society Research Professor of Biochemistry (Award PRP-21). Thanks are due to Professor Helen Van Vunakis for critically reading the manuscript.

REFERENCES Alam, I., Ohuchi, K., and Levine, L. (1979).Anal. Biochem. 93,339-345. Alam, I., Voelkel, E. F., Tashjian, A. H., and Levine, L. (1980).Prostaglandins Med. 4, 227-237. Beliel, 0. M., Singer, F. R., and Coburn, J. W. (1973). Prostaglandins 3,237-241. Bell, R. L., Kennedy, D. A., Stanford, N., and Majerus, P. W. (1979). Proc. Natl. Acad. Sci. USA. 76,32383241. Belman, S . , and Troll, W. (1972).Cancer Res. 32, 450-454. Bennett, A. (1979). In “Practical Applications of Prostaglandins and their Synthesis Inhibitors” (S. M. M. Karim, ed.), pp. 149-188. MTP Press Ltd., Lancaster. Bennett, A., McDonald, A. M., Simpson, J. S., and Stamford, I. F. (1975). Lancet 1, 1218-1220. Bennett, A., del Tacca, M., Stamford, I. F., and Zebro, T. (1977a). Br. J . Cancer 35, 881-884. Bennett, A., Charlier, E. M., McDonald, A. M., Simpson, J. S., Stamford, I. F., and Zebro, T. (197713).Lancet 2,624-626. Bennett, A., Houghton, J., Leaper, D. J., and Stamford, I. F. (1979). Prostaglandins 17, 179- 191. Bennett, A., Edwards, D., Ali, N. N., Auger, D., and Harris, M. (1980a).Adu.Prostaglandin Thromboxane Res. 6,547-548. Bennett, A., Berstock, D. A., Harris, M., Raja, B., Rowe, D. J. F., Stamford, I. F., and Wright, J. E . (1980b).A d a Prostaglandin Thrornboxane Res. 6,595-600. Berenblum, I. (1969). Prog. Exp. Tumor Res. 11,21-30. Berstock, D. A., Houghton, J., and Bennett, A. (1980).Adu. Prostaglandin Thromboxane Res. 6, 567-569. Bhana, D., Hillier, K., and Karim, S. M. M. (1971). Cancer 27, 233-237. Bills, T. K., Smith, J. B., and Silver, M. J. (1977).In “Prostaglandins in Hematology” (M. J. Silver, J. B. Smith, and J. J. Kocsis, eds.), pp. 27-55. Spectrum Publ., New York. Blackwell, G. J., Carnuccio, R., DiRosa, M., Flower, R. J., Parente, L., and Persico, P. (1980).Nature (London) 287, 147-149.

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THE SHOPE PAPILLOMA-CARCINOMA COMPLEX OF RABBITS: A MODEL SYSTEM OF NEOPLASTIC PROGRESSION AND SPONTANEOUS REGRESSION John W. Kreider and Gerald L. Bartlett

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Departments of Pathology and Microbiology The Milton S Hershey Medical Center. The Pennsylvania State University Hershey. Pennsylvania

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I . Introduction: Historical Origins ......................................... I1. Interaction of Shope Papilloma Virus and Host Cells ..................... A . Natural Occurrence and Transmission of SPV ......................... B . Structure of Virion and Genome ..................................... C . The Rabbit Kidney Vacuolating Virus ................................ D . Host Cell Specificity ................................................ E . Factors Influencing SPV Replication .................................. F. Papilloma Arginase ................................................. G . Cell and Tissue Culture Studies ..................................... I11. Neoplastic Progression .................................................. A . Definition and Characteristics ....................................... B . Malignant Transformation ........................................... C . Factors Influencing Neoplastic Progression ........................... D . Role of SPV Genome in Carcinomatous Transformation ................ IV. Spontaneous Regression . . . . . . . . . . . . . . . . . ............................. A . Alterations in Growth Pattern ........ ............................. B . Genetic Factors ................................................ C . Mechanism of Spontaneous Regression ............................... D . Immune Stimulation ................................................ E . Immune Suppression ................................................ V. Shope Papilloma-Carcinoma Complex as a Model System . . . . . . . . . . . . . . . . . A . Theoretical and Practical Considerations ........................ B . Relevance to Human Neoplasia ...................................... References .............................................................

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I. Introduction: Historical Origins

Horned cottontails and jackrabbits were known to the pioneers of the western plains and were first described in popular hunting and fishing magazines in the early 1900s . These animals sported black or gray. hornlike projections from the head and face. This spectacular visage may have given rise to the popular legend of the “jackalope.” These apocryphal creatures allegedly resulted from the miscegenation ofjackrabbits and pronghorn antelopes Folklore attributed to them the unfortunate habit of driving their horns into the posteriors of unsus-

.

81 ADVANCES IN CANCER RESEARCH. VOL . 35

Copyright 0 1981 by Acndemic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-006635-1

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JOHN W. KREIDER AND GERALD L. BARTLETT

pecting farmers, ranchers, and hunters. Horned cottontails were initially documented in scientific literature (Fig. 1) by the famous naturalist Ernest Thompson Seton (1909): The horned cottontail is a well-known freak. The horns are a morbid product of the skin and seem to be the result of irritating attacks by a skin parasite. These growths are ofno particular service to the owner and will hardly be interesting until someone more fully explains their origin. (Seton, 1937)

Cancer research was introduced to the horned cottontail by Richard E. Shope, physician-scientist of the Rockefeller Institute (Shope and Hurst, 1933).One day Shope was in his laboratory examining a rabbit with a cutaneous fibroma induced by the virus he had recently isolated. A laboratory visitor from Cherokee, Iowa, looked disdainfully at the fibroma and said that he had “shot rabbits with horns out of the side of their heads like Texas steers, or out of the top of their noses like a rhinoceros” (Gross, 1970). Shope was invited to hunt these wondrous rabbits and eventually obtained tissue for study. Shope’s initial study demonstrated that the horns of Iowa cottontails were epidermal papillomas, readily transmissible to both cottontails and domestic rabbits by means of cell-free filtrates. Rapid progress resulted from the early studies of Rockefeller Institute investigators, including Shope, ROUS,Beard, and Kidd. It was soon established that the Shope papilloma virus (SPV) was one of the first identified mammalian tumor viruses. The fate of the papilloma was quite variable, with some animals retaining persistent papillomas for the rest of their lives, whereas in others the tumors completely disappeared. The majority of domestic rabbits developed invasive, metastatic, and ultimately lethal epidermoid carcinomas. Factors determining the course of papillomas have been the subject of almost 50 years of inves tigation. This interesting animal model system has contributed to our understanding of fundamental mechanisms in neoplasia, but much remains to be learned from it. II. Interaction of Shope Papilloma Virus and Host Cells

A. NATURALOCCURRENCE AND TRANSMISSION OF SPV

The S hope papilloma has a restricted geographic range, mostly confined to the high plains of the western United States (Fig. 2). Shope described this location as extending “from north-eastern Oklahoma up through eastern Kansas, western Missouri, eastern Nebraska, and probably South Dakota, western Iowa, and up into Minnesota” (Gross,

SHOPE PAPILLOMA-CARCINOMA COMPLEX OF RABBITS

Plate CVl

Horned Cottontails

83

E.ZSe6n

FIG. 1. Appearance of horned cottontails as sketched by Ernest Thompson Seton (1937), reproduced by permission of the copyright owner.

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JOHN W. KREIDER AND GERALD L. BARTLETT

FIG.2. Distribution of naturally occurring Shope papillomas of cottontail rabbits as described by Richard E. Shope in correspondence with Gross (1970).

1970). The reasons for this peculiar geographic range are unknown, but some of the factors might include the coincidental distribution of vectors, cottontail subspecies, trace minerals (Gross, 1970), or other, unsuspected factors. Little is known of the vectors responsible for natural transmission, but in the laboratory SPV has been transmitted with a number of parasites including rabbit ticks (Larson et al., 1936), nematodes (Rendtorff and Wilcox, 1957), mosquitoes, and assassin bugs (Dalmat, 1958). Responsible vectors may be different at various points in the natural range. We have found that naturally occurring papillomas on Kansas cottontails are predominant on the perineum, with a minority on the head and shoulders (J. W. Kreider, unpublished observations). In Minnesota, the warts are predominantly found on the head and neck, a location consistent with passage by biting or sucking insects (Larson et aZ., 1936). The dominant host species for the SPV in its natural range is the cottontail, S ylvilagus jloridanus. There are several S . floridanus subspecies within the papilloma range, but their distribution is not coincidental with that of the papilloma (correspondence with E. R. Hall, mammalogist at the University of Kansas, Museum of Natural History,

SHOPE PAPILLOMA-CARCINOMA COMPLEX OF RABBITS

85

Lawrence, Kansas). A very small number of jackrabbits are also naturally infected; we have studied a single, papillomatous Kansas jackrabbit that yielded no recoverable SPV. Cottontail populations in the papilloma range are subject to cyclic variations in population density ( E. R. Hall, personal communication). The percentage of cottontails with papillomas varies greatly from year to year (E. R. Hall, personal communication). Thus, the amount of SPV available for scientific studies has been highly variable and unpredictable. Trace mineral distribution is a difficult problem to study, especially with the advent of modern fertilizing practices, but there appears to be no relationship of mineral deficiencies or excesses to the papilloma range. Shope papillomas for scientific studies have been almost exclusively provided to investigators for more than 40 years by Earl Johnson, Rago, Kansas. Mr. Johnson died in 1977, and his business interests are maintained by his son. The Shope papillomas were an intriguing sideline to Johnson’s major interest, the trapping of jackrabbits used to train racing greyhounds and the trapping of cottontails for restocking by rabbit hunters, mostly in the eastern United States. Rabbits in Kansas will enter boxtraps only in the winter, when deep snow cover prevents access to food sources other than bait in the traps. Thus, most of the winter-trapped and then restocked cottontails quickly come before the gun in the eastern winter hunting seasons. Although Kansas cottontails have been stocked in large numbers in both New Jersey (Gross, 1970) and Pennsylvania (J. W. Kreider, unpublished observations), the imported rabbits have survived poorly. We are aware of no Shope papilloma recoveries in New Jersey and only a single cottontail with Shope papilloma was identified in Lickdale, Pennsylvania, out of thousands personally examined by one of us (J. W. K.). Kansas cottontails have been successfully stocked on Whidbey Island in Puget Sound, Washington. The Shope papilloma is an indigenous infection in these animals and useful amounts of SPV have been recovered (Evans and Ito, 1966). In summary, naturally occurring SPV is restricted largely to the midwestern high plains. Factors responsible for this localization are completely unknown.

B.

STRUCTURE O F VIRION AND

GENOME

Most early studies with SPV relied on the infectivity of “crude” SPV suspensions consisting of saline extracts (10%w/v) of cottontail papillomas. Differential high- and low-speed ultracentrifugation gave “partially purified” virion suspensions (Beard et d.,1939). “Purified” SPV

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JOHN W. KREIDER AND GERALD L. BARTLETT

became available with the introduction of banding techniques using cesium chloride density gradients (Breedis et al., 1962). The SPV virion produces bands at 1.34 g/cm3 and is an icosahedron of approximately 50-65 nm in preparations negatively stained with phosphotungstic acid (Breedis et al., 1962; Chambers et al., 1966). The capsid contains capsomeres approximately 100 8, long (Breedis et al., 1962).The number of capsomeres in the capsid in unclear; it has variously been reported as 42, 72, 96 (Williams et al., 1960; Klug and Finch, 1965; Mattern, 1962). Cesium chloride density gradients also produce bands of lesser density (1.29 g/cm3)that contain noninfectious, hollow particles with partly disrupted capsids (Breedis et al., 1962; Kass and Knight, 1965). Tubular assemblages of capsomeres, probably representing faulty capsid assemblies, are sometimes also visible in low density bands. Sera from rabbits bearing the Shope papillomaderived Vx7 carcinoma react with a structural polypeptide of human plantar wart papillomavirus HPV-1 (Orth et al., 1978). Conversely, antigenic determinants of SPV capsid are bound to antisera raised against disrupted HPV-1 virions or polypeptides from capsids. These results indicate that both papillomaviruses contain rigidly conserved, common antigenic determinants. The significance of the antigens in resistance to infection are unknown. SPV particles contain a supercoiled, double-stranded DNA genome of approximately 4.2 X lo6daltons (Crawford, 1964). The percentage of guanine and cytosine of SPV DNA range from 47 to 49.5 (Crawford and Crawford, 1963; Watson and Littlefield, 1960). No nucleotide sequence homology between SPV and human papillomavirus DNAs was detected under stringent hybridization conditions (Crawford, 1969). When less stringent (30% formamide) conditions were employed, common sequences were found in DNAs of SPV, HPV-1, and bovine papillomavirus (Howley et al., 1980). It seems reasonable that these conserved sequences may encode at least for the cross-reacting structural polypeptide described in SPV and HPV-1 capsids (Orth et al., 1978). Such conservation across wide differences in host species argues for an important role for these components in the biology of papillomaviruses. Infectious SPV DNA can be phenol extracted from homogenized papillomas and carcinomas (Evans and Ito, 1966). Infectivity of DNA extracts was related to viral content; for example, cottontail papillomas contained more infectious DNA than those of domestic rabbits. The fate of DNA-induced papillomas was similar to the fate of papillomas induced by conventional, SPV particle suspensions. In summary, the SPV virion is a particle of about 55 nm with a

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genome of close to 5.0 X 10‘ daltons. The specific gene products include a structural capsid polypeptide encoded by a sequence conserved in several papillomaviruses. C. THE RABBIT K~DNEYVACUOLATINGVIRUS A second DNA-containing virus was found in some naturally occurring Shope papillomas (Hartley and Rowe, 1964).This “rabbit kidney vacuolating virus” (RKV) was apparently a contaminant or “passenger” virus. The RKV is readily identified b y characteristic cytopathic effects on cultures of domestic rabbit kidney and by hemagglutination of guinea pig erythrocytes. RKV is antigenically distinct from a number of other viruses, including polyoma, SV40, bovine papilloma, K, Kilham, and H-1. The RKV is endemic to cottontail populations from both Kansas (4 positive of 16 tested) and Maryland (13 positive of 46 tested). It does not produce papillomas on skin or buccal mucosa of rabbits and is nonpathogenic for laboratory animals inoculated by a variety of routes. We found no correlations between the amount of RKV in papilloma extracts and either SPV content or papilloma morphology (Goldman et al., 1972).The RKV does not appear to be a SPV substrain and probably plays no role in the pathogenesis of the Shope papilloma-carcinoma complex. However, its unpredictable presence is a potential pitfall, especially in studies of papilloma-associated antigens and of DNA cloning and hybridization.

D. HOSTCELL SPECIFICITY SPV produced papillomas with equal facility on the skins of laboratory-infected jackrabbits (Lepus calijornicus), snowshoe hares (Lepus americanus), domestic rabbits (Oryctolagus cuniculus), and cottontails (Sylvilagus Joridanus) (Beard and ROUS,1935).The papillomas in Lepus species appeared with longer latency and grew more slowly than those in domestic rabbits. The host range can be extended to rats, although with some difficulty. Papillomas cannot be induced in fetal, neonatal, suckling, or adult rat skin by direct inoculation of SPV suspensions (J. W. Kreider, unpublished observations) or infectious DNA (Ito, 1962). However, if fetal rat skin is infected in short-term organ cultures and grafted orthotopically to syngeneic adult rats, papillomas do develop in some of the infected fetal rat skin grafts (Greene, 1953; Kreider and Breedis, 1969).The papillomas appear at very low incidence (
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JOHN W. KREIDER AND GERALD L . BARTLETT

(1-2 mm) and invariably regress. Histologically, the tumors are typical cutaneous papillomas, quite similar to Shope papillomas of rabbits. Host immunosuppression prevents rat papilloma regression but does not increase the yield or growth of induced papillomas. Thus regression may be due to immune mechanisms, but initial resistance to SPV infection is not (Kreider et al., 1971b). Susceptibility to SPV infection is also determined by factors related to cell phenotype. In a classic experiment, Kidd and Parsons (1936) directly inoculated SPV into a variety of rabbit epithelia including tongue, gums, nares, conjunctivae, genitals, and mammary glands. No tumors were produced. SPV was inoculated into a linear pattern of sites which extended from the buccal mucosa across the vermillion border and onto the hair-bearing skin of the lip. Papillomas were induced only on the hair-bearing skin. There have been claims of papillomatous proliferations in SPVinjected rabbit tooth germ (Fleming, 1958) and parotid gland (Fleming, 1960). From the data presented, it is not possible to conclude that these proliferations were true neoplasms. Rabbit epidermal cell transformation by SPV requires interaction with mesenchyme. A variety of mesenchymal types besides dermis will support papillomatous transformation, so the mesenchymal requirement is not specific (Breedis and Kreider, 1970). Vitamin A deficiency or excess can produce striking alterations in the differentiation of epithelia of various types. Vitamin A deficiency results in a keratinizing metaplasia of normally nonsquamous epithelium. Inoculation of these metaplastic epithelia with SPV failed to produce tumors (Kidd and Parsons, 1936). Thus, the induction of keratinization was not a sufficient condition for susceptibility to SPV tumor formation. In contrast, vitamin A excess strongly inhibited the growth of Shope papillomas (McMichael, 1965). The papillary fronds became atrophic, reduced in height, and occasionally sloughed off. When vitamin A was discontinued, the papillomas regrew. These observations demonstrated that papilloma growth could not be sustained in the presence of high vitamin A levels. SPV infectious potential may be restricted completely to a single cell type, viz., migrating wound epithelium derived from the hair follicle sheath cell. This inference is supported by several observations. First, Shope papillomas develop only in hair-follicle-bearing skin (Kidd and Parsons, 1936). Second, epidermal trauma of follicle-bearing skin is absolutely required for SPV infection. Wound healing is accomplished in part by epidermal migration derived from nearby hair follicles, and these cells are quite susceptible to SPV infection

SHOPE PAPILLOMA-CARCINOMA

COMPLEX OF RABBITS

89

(Breedis, 1962). Third, the rate of epidermal migration in organ cultures of rabbit skin is significantly accelerated by SPV infection (Kreider, 1963). Fourth, the rate of growth of Shope papillomas is accelerated by SPV infection of skin during the active phase of the hair growth cycle and is retarded if infection occurs in the inactive phase (Whiteley, 1956). The “wound” epithelial cells represent a phenotypic alteration of hair follicle sheath cells and are capable of not only resurfacing cutaneous wounds but of regenerating hair follicles and sebaceous glands (Breedis, 1954). They are therefore “equipotential” and represent a cell phenotype, which, although transitional, may be distinctly different from other epidermal cells. There is no evident reason for the unique susceptibility of this cell type to SPV, but the mechanism could be dependent on ephemeral cell surface modulation resulting in the temporary expression of specific receptors for SPV.

E. FACTORS INFLUENCING SPV REPLICATION A major limitation to the estimation of infectious SPV titer in various preparations is the insensitivity of the usual infectivity assay consisting of cutaneous scarification followed by inoculation with serial dilutions of virus. One infectious SPV dose may contain 1 x lo* viral particles (Beard, 1956). A slight increase in infectivity was described when hyperplastic skin was used as substrate (Friedewald, 1944). The insensitivity of the conventional SPV assay led to the fallacious view that endogenous host factors might “mask” viral infectivity (Beard, 1956). Variations in papilloma SPV content are properly attributed to differences in viral replication rather than masking. SPV replicates in papillomas of jackrabbits and snowshoe hares but only rarely produces natural infections in jackrabbits and probably never does so in snowshoe hares (Beard and ROUS,1935). Cottontails from Kansas (Shope and Hurst, 1933), New Jersey (Beard and Rous, 1935), New York (Syverton et al., 1950a), Minnesota (Larson et al., 1936), Pennsylvania (J. W. Kreider, unpublished observations), and Washington (Evans and Ito, 1966) replicate SPV to varying degrees. Few direct comparative studies have been conducted, but it appears that naturally infected Kansas cottontails produce the highest SPV titers; even these tumors, however, may occasionally produce little or no SPV (Syverton et al., 1950b,c). Naturally infected Kansas cottontail papillomas produce more SPV than experimentally induced tumors of the same hosts (Kidd, 1939). Comparative studies were conducted on SPV titers in laboratoryinfected cottontail rabbits (Evans, 1964). Viral content was approxi-

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JOHN W. KREIDER AND GERALD L. BARTLETT

mately similar in the multiple papillomas of a single cottontail. Great differences occurred in the viral content of papillomas of different rabbits. The papillomas of longest duration contained the most virus, an observation consistent with relative increases in the amounts of keratin horn in the aging papillomas. Investigators studying Shope papilloma have been handicapped by reliance on naturally infected Kansas cottontails as an SPV source. The supply is variable since it is limited by season, snow depths, cottontail population density, prevalence of papillomas in the population, and SPV titers in individual papillomas. To eliminate some of these uncertainties, some alternative SPV sources have been studied including laboratory passage of SPV in cottontails or domestic rabbits. The most successful laboratory propagation has been the Washington B strain, passaged at least three times in Whidbey Island cottontails (Evans and Ito, 1966). Cottontails do not survive well in the laboratory since they are fragile, excitable, and susceptible to a number of diseases. An alternative to cottontail maintenance was suggested by our studies, in which Shope papillomas grew orthotopically in rabbit skin transplanted to athymic nude mice (Kreider et al., 1979), to immunosuppressed mice (Pass et aZ., 1973), or to exteriorized hamster cheekpouches (Kreider et al., 1971a). Typical Shope papillomas developed in hosts of all three types. Cottontail skin was similarly transformed in athymic mice, but SPV titers have not yet been measured (J. W. Kreider et al., unpublished observations). In contrast to cottontails, domestic rabbits produce papillomas that rarely contain infectious SPV. It is possible to select and serially pass SPV strains that replicate to modest titers in domestic rabbit papillomas. These strains have been independently isolated by scientists in the United States (Shope, 1935), England (Selbie and Robinson, 1947), and Russia (Nartsissov, 1959), but the titers are low so the potential for experimentation is limited. SPV replication in cottontail papillomas is also modulated by phenotypic factors, namely, epidermal maturation and keratinization (Fig. 3). The basal or germinal layers of the papilloma contain a large proportion of cells in DNA synthesis (Rashad and Evans, 1967a), but these cells contain no virion capsid antigen or infectious virus (Noyes, 1959). Maturing papilloma cells in the granular layer take up tritiated thymidine, but similar cells of carcinogen-induced papilloma or normal skin do not (Rashad and Evans, 1967a). Using complementary tritiated RNA to form hybrids in situ, SPV DNA is also detected in granular cell nuclei (Orth et al., 1971; Croissant et al., 1972). Viral

SHOPE PAPILLOMA-CARCINOMA COMPLEX OF RABBITS

-

Epidermal Layer Keratin Keratohyalin SPlnY Germinal

Cell DNA Synthesis

+t

SH).

Viral DNA Synthesis

Viral Capsid Antigen

+

.HH

+m

-

91

+ I

-

FIG. 3. Influence of epidermal cell maturation and keratinization on the replication of SPV. The relative amounts are indicated by an arbitrary scale where (4 = none and (+ + +) = maximum. See text for references.

+

capsid antigen and infectious SPV are detected in the granular and keratin layers (Noyes and Mellors, 1957). These observations are consistent with the interpretation that SPV exists in cottontail papilloma basal cells in a latent, possibly integrated state, but these basal cells are nonpermissive for SPV replication. With papilloma cell maturation and keratinization, the viral genome replicates and is encapsidated. These inferences are strengthened by observations that high SPV titers are linked to characteristic keratinization patterns consisting of a fragmented keratin layer and a welldeveloped granular layer containing many large keratohyalin bodies (Rashad and Evans, 1967b).The apparent linkage between viral replication and epidermal cell maturation may explain the poor replication of SPV in carcinomas and cell cultures, in which epidermal keratinization is often diminished and usually abnormal. Thus, SPV infectivity assays are notoriously insensitive and limit detectaLility of SPV replication. Both genetic and phenotypic factors determine viral replication. Naturally infected Kansas cottontails are the best practical source of infectious SPV. Viral replication is highly dependent on epidermal keratinization.

F. PAPILLOMA ARGINASE Shope papillomas contained high levels of L-arginase (L-arginine amidinohydrolase, EC 3.5.3.1),but normal rabbit skin did not (Rogers, 1959). Rabbits carrying Shope papillomas reportedly developed precipitating antibodies against the papilloma arginase; the antibodies did not neutralize rabbit liver arginase (Rogers and Moore, 1963).The physiochemical properties of papilloma arginase were different from

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JOHN W. KREIDER AND GERALD L. BARTLETT

those of liver arginase. On the basis of these observations, it was concluded that the information for the synthesis of the enzyme was derived from the SPV genome rather than the cell genome. The significance of this finding was thought to be that the arginase, uncontrolled by host homeostasis, depleted cellular arginine, which reduced the synthesis of arginine-rich nuclear histones and released the papilloma cell genome for excessive proliferation (Evans and Rogers, 1967). Rogers measured serum arginine levels in investigators who had previously worked with the SPV and found lower values than in controls; these same sera contained precipitins reactive with the papilloma arginase (Rogers, 1966). Thus, it can be concluded that the SPV persists in man as a harmless passenger. This has led to an attempt at “gene therapy,” in which patients with congenital arginase deficiency and abnormally high serum arginine levels were given injections of SPV. No significant improvement in serum arginine levels followed treatment (Terheggen et al., 1975). Other investigators have attempted to confirm the assertion by Rogers that the papilloma arginase is specified by the SPV genome. Normal rabbit epidermis and 9,10-dimethyl-1,2benzanthracene-induced papillomas contained appreciable arginase activity with physicochemical characteristics identical to Shope papilloma arginase; Shope papilloma bearers lacked arginase-precipitating antibodies (Orth et al., 1967). Subsequent data from an independent laboratory (Satoh et al., 1967) confirmed the negative findings of Orth and associates. In summary, independent studies have confirmed the presence of arginase in Shope papilloma but have not supported the assertion that the enzyme is encoded by the SPV genome.

G. CELL AND TISSUECULTURE

STUDIES

Despite the fact that SPV was one of the first oncogenic viruses that transformed normal cells in vitro (Coman, 1946), further progress in this area has been negligible. Most studies have been unpublished, but those reported have described no effects (Moulton and Lau, 1964; White et al., 1963) or cytotoxic effects (Ptokhov, 1960; Christensen, 1964). On one occasion, cytopathic effects were due to a previously undescribed passenger virus, the RKV (Hartley and Rowe, 1964). Enhanced arginase activity was detected in SPV-infected cottontail skin cultures (Passen and Schultz, 1965). Adsorption of SPV to cell membranes of primary cultures of fetal rabbit skin cells in monolayer culture was demonstrated by immunofluorescence (Kreider et al., 1967). SPV caused an enhanced proliferative activity of epidermal cells in

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organ culture (DeMaeyer, 1962; Kreider, 1963), and that has remained the only independently confirmed effect of SPV on skin cells in uitro. An important property of putatively transformed cells in vitro could be tumorigenicity. Only three studies have demonstrated papilloma formation by SPV-infected cell and organ cultures after transplantation in vivo (Coman, 1946; Kreider, 1963; Kreider et al., 1967). A number of studies have demonstrated nuclear (Shiratori et al., 1967; Yoshida and Ito, 1968; Osato and Ito, 1967; Osato and Ito, 1968), cytoplasmic (Shiratori et aZ., 1969), and membrane-associated (Ishimoto and Ito, 1969, 1971) antigens in cultured cells from SPVinduced papilloma and carcinoma and in SPV-infected normal cells. It is difficult to interpret the significance of these observations since controls for RKV contamination were often not included, and the original findings have not been confirmed in other laboratories. Comparable antigens have not been found in other papillomavirus systems. In summary, it is clear that rabbit skin cells infected in vitro with SPV respond with modest increases in proliferative activity and can form papillomas on transplantation to appropriate hosts. However, the SPV-infected cells show no morphological transformation and there is no compelling evidence for the presence of tumor-associated antigens in these cells. Ill. Neoplastic Progression

A. DEFINITIONAND CHARACTERISTICS

Neoplastic progression, in the sense defined by Foulds (1969), is that process whereby malignant cells arise from abnormal or premalignant cells through a series of cumulative, heritable alterations. These alterations, probably somatic mutations, yield heterogeneous, clonally derived cell populations. The host environment negatively selects for those neoplastic cell clones which are best suited to aggressively exploit host resources. Thus, the development of neoplastic cell populations can be viewed as a form of cellular evolution. The Shope papilloma-carcinoma complex is an excellent model of neoplastic progression. The epidermal cell evolves through recognizable, stepwise stages (Table I) in which host environmental influences probably play a decisive role in determining neoplastic transformation, benign proliferation, spontaneous regression or invasion, and metastasis. Some of these host influences are well known, others are poorly understood, and some are speculative. In the initial stage

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JOHN W. KREIDER AND GERALD L. BARTLETT

TABLE I STAGESIN THE PROGRESSION AND REGRESSION OF THE SHOPE PAPILLOMA-CARCINOMA COMPLEX

Stage

0

I I1

I11 IV

Event

Days postinfection

Latency Papillomas appear Exponential growth Regression Persistence Carcinoma

0- 14 14-21 30- 60 60-90 Indefinite 350-450

Percentage affected hosts

100 100 100 10-40 20-30 40-60

(Stage 0),epidermal cells are infected by cutaneous scarification and virus inoculation with SPV. Approximately 2-3 weeks later, benign papillomas appear at the inoculation sites (Stage I). Exponential growth of these papillomas ensues over the next 30-60 days (Stage 11). Between 60 and 90 days, 10-40% of the papillomas may regress spontaneously. The remainder of the papillomas enter Stage 111, in which no further regressions occur. A variable proportion (20-30%) of the papillomas persist as benign tumors for the lifetime of the animal. The remainder (40-60%) become invasive epidermoid carcinomas (Stage IV). B. MALIGNANTTRANSFORMATION Papillomas which become malignant undergo a characteristic series of gross morphological changes (Rous and Beard, 193513).An ulcerated region covered with brownish, dried exudate and with rolled margins appears at the periphery of the papilloma. The ulceration gradually spreads to adjacent skin and papilloma and may eventually replace the entire papilloma. Malignant transformation is often associated with an increase in mitotic figures. Papillomas characteristically have a diploid chromosome number (2n = 44), but derived carcinomas are hyperdiploid, with no specific chromosome changes (Palmer, 1959; Bayreuther, 1960; McMichael et al., 1963). Mean cellular DNA content of carcinomas is similar to that of advanced papillomas, but there is a wider range of DNA content in the carcinomas (Moulton and Lau, 1967). It is not known whether papilloma-derived carcinomas are monoclonal or polyclonal in origin. Karyotypic analysis has shown single stemlines in some carcinomas, but the stemlines are karyotypically diverse

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(McMichael et al., 1963). The presence of X-chromosome-linked isoenzymes in female rabbits might permit an analysis of clonal heterogeneity in both papillomas and papilloma-derived carcinomas (Murray et al., 1971). Microscopically, cords and nests of epidermoid carcinoma cells with keratin “pearls” penetrate the subjacent dermis and may eventually fix the ulcer to the underlying axial musculature. Lymphatic vessels are frequently filled with extensions of tumor cells in cords. Metastases in regional lymph nodes, especially the axillary nodes, are common. Pulmonary metastases are found in about 25% of rabbits which die with epidermoid carcinomas. We have recently noted severe amyloidosis in most rabbits which have died with epidermoid carcinoma of greater than 3-months duration. Amyloid is present in glomerular mesangium, hepatic sinusoids, and splenic red pulp. It is not clear why amyloidosis develops. Both carcinomatosis and chronic infections (common in rabbits with terminal cancer) can predispose to amyloidosis. C. FACTORS INFLUENCING NEOPLASTICPROGRESSION The study of neoplastic progression in the Shope papilloma has been impeded by several circumstances. It takes about one year of continued growth for Shope papillomas to undergo malignant transformation, and only 50-60% of the animals will develop carcinomas. Mortality due to unrelated extraneous diseases further decreases the yield of of carcinomatous hosts. Therefore, the study of spontaneous malignant transformation is likely to be inefficient, expensive, and to require considerable patience. Serial transplantation of derived carcinomas is infeasible except in syngeneic rabbits. These considerations are responsible for the fragmentary data available on mechanisms of neoplastic progression of Shope papilloma. Host factors are important determinants of carcinomatous transformation. Cottontails either naturally or experimentally infected with SPV develop a lower incidence of carcinomas than domestic rabbits (Syverton et al., 1950a). Rats have never developed carcinomas in induced Shope papillomas (Kreider and Breedis, 1969). Undefined, genetically determined factors are probably responsible for the different risks of carcinoma in the three species. Host age is an additional determinant. Of 6 rabbits infected in utero, 5 developed epidermoid carcinoma within 7 months; 8 of 14 adult control rabbits developed carcinomas within 6-12 months (Fischer and Syverton, 1951). Newborn rabbits inoculated with SPV within the

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first 24 hours after birth developed malignant conversions only after the lapse of 1 year; the percentage of carcinomas was not reported (Seto et al., 1977). Accelerated carcinomatous transformation in papillomas of rabbits infected in utero with SPV (Fischer and Syverton, 1951) might be interpreted as support for the hypothesis that active host immunity normally restricts Shope papilloma to carcinoma development. However, immune tolerance to SPV virion antigen is not present in these rabbits. Further, chronic immunosuppression with methylprednisolone in rabbits (McMichael, 1967) or with methylprednisolone and antithymocyte serum in rats (Kreider et al., 1971b), sufficient to inhibit papilloma regression in both species, resulted in no alteration of the frequency of malignant transformation in rabbits and no appearance of carcinomas in the rats. Since it is difficult to maintain rabbits under chronic immunosuppression for over 1 year, it may be impossible to directly test the role of host immunity in malignant conversion. An alternative approach to this problem was suggested by our studies on the behavior of SPV-infected rabbit skin grafts on athymic mice (Kreider et al., 1979). We have found three malignant carcinomas developing within 4 months in rabbit papillomas grafted to nude mice. This is considerably shorter than comparable periods (1- 2 years) for autochthonous, papilloma-derived carcinomas in our laboratory. It was not possible to estimate the incidence of malignant change in rabbit papillomas in our nude mice because of a high incidence of early (1-4 months) deaths due to murine hepatitis infections. Adequate isolation facilities for athymic mice could permit the conducting of experiments to directly compare malignant transformation in autochthonous and nude-mouse-engrafted papilloma.

D. ROLE OF SPV GENOMEIN

CARCINOMATOUS

TRANSFORMATION

One of the major unsolved problems in the Shope papillomacarcinoma complex is the role of the SPV genome in malignant change. There is no question that the viral genome is conserved in the epidermoid carcinoma cells: 1. Most primary carcinomas contain no detectable infectious SPV (Kidd and ROUS,1940; Syverton et al., 1950c), but infectious virus can sometimes be recovered with improved bioassay methods (Rogers et al., 1960). 2. SPV capsid antigen can be demonstrated by immunofluorescence in sections of carcinomas (Mellors, 1960). A structural, virion-associ-

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ated polypeptide can be detected in the Vx7 carcinoma (Orth e t al., 1978). The Vx7 carcinoma is a Shope-papilloma-derived tumor of domestic rabbit origin, serially transplantable in allogeneic hosts, 3. Shope papilloma tumor rejection antigens persist in Vx7 carcinoma cells (Evans et d., 1962~). 4. Infectious SPV nucleic acid can be extracted from the Vx7 carcinoma (Ito, 1970). 5. Recent studies employing nick-translated, radiolabeled SPV DNA as probe for molecular reassociation experiments have elegantly demonstrated that non-virus-producing, domestic rabbit primary and metastatic carcinomas contain 10-100 copies of the viral genome per diploid cell equivalent DNA (Stevens and Wettstein, 1979). Viral DNA sequences are in a high-molecular-weight form, consisting of linear, head-to-tail tandem repeats and are not in the supercoiled configuration characteristic of virions (Wettstein and Stevens, 1980). It is clear that the SPV genome persists in carcinoma cells, and the conservation of the SPV genome, especially in the long-transplanted Vx7 carcinoma, argues for an important role of the virus in the maintenance of the malignant state. More direct evidence supporting the participation of the SPV genome in malignant transformation is derived from several older observations. The probability of eventual carcinomatous transformation is directly proportional to the dose of SPV used to initiate the papillomas (Rous et al., 1936). Cocarcinogenesis studies in which SPV is used in concert with hydrocarbon carcinogens infer a key role of SPV in malignant transformation. Tarring of rabbit ears produces generalized epidermal hyperplasia and papillomatosis. The papillomas never become malignant and often regress after cessation of tar application (Rous and Beard, 1935a). Following an intravenous SPV injection, extraordinary changes in tarred ears became evident within the astonishingly brief period of 2-3 weeks (Rous and Kidd, 1936). Numerous, discrete, highly anaplastic carcinomas developed, some in preexisting tar papillomas but others where no lesions previously had been visible. Fragments of tar papillomas, infected with SPV during brief incubation in vitro, were grafted intramuscularly and subcutaneously (Kidd and Rous, 1937). Progressively growing papillomas and some carcinomas developed in SPV-infected tar warts, but no tumors were formed in the grafts obtained from uninfected warts. When tar or methylcholanthrene was applied to established Shope papillomas, both the frequency and the latency of malignant transformation were accelerated (Rous and Friedewald, 1944). Simultaneous administration of SPV and carcine

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gen to normal skin both increased the yield and shortened the latency of malignant transformants of Shope papillomas (Rogers and ROUS, 1951). Carcinomas appeared in the remarkably short period of 60-80 days. Thus, carcinomas derived from Shope papillomas often contain multiple copies of SPV genome. SPV infection can increase the transplantability of tar papillomas and is responsible for extremely rapid malignant transformation which never occurs in controls. Polycyclic hydrocarbons shorten the latency and increase the yield of malignant transformation of Shope papillomas. Taken together, these observations strongly support a key role for the SPV genome in the induction and maintenance of the malignant phase of the SPV-transformed cell. Cocarcinogenesis studies with Shope papilloma suggest that a minimum of two events, possibly both consisting of somatic mutations, may be required for the establishment of the malignant state. One such event is provided by the SPV genome and the second by the experimentally applied polycyclic hydrocarbons. In the absence of exogenously applied chemical carcinogens, other undefined environmental influences, accumulated over 1-2 years of papilloma growth, may provide the second event in the “spontaneous” malignant transformation. Such a “two-hit” concept has been proposed for the development of neuroblastoma in man (Knudson and Strong, 1972).

IV. Spontaneous Regression

A. ALTERATIONS IN GROWTHPATTERN Spontaneous regression of established tumors is relatively common for neoplasms induced by papillomaviruses. In our experience, and that of others (Evans et al., 1962a), Shope rabbit papilloma regression follows a highly characteristic kinetic pattern. Tumors which will ultimately regress appear with latency and initial growth rates comparable to those of progressing tumors. The frequency of regression is independent of the SPV concentration but is inversely proportional to the area of inoculated skin and, therefore, papilloma size. Most regressions occur within 30-60 days postinoculation, but some begin as late as 90 days. Once initiated, regression proceeds at a steady pace and is complete within 1-2 weeks. The regression is morphologically the result of the gradual shortening of papillary fronds. The tumor almost never abruptly falls off but, instead, slowly dwindles to insignificance. In

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repeated experiments, the regression frequency is variable but usually within the range of 10-30%.

B. GENETICFACTORS Genetic factors significantly influence the regression frequency. Laboratory infected cottontails had a higher regression incidence (36%) than domestic rabbits (9%); cottontail regressions continue for 18 months postinoculation, whereas domestic rabbit regressions occurred in the first few months (Syverton, 1952). The regression frequency in feral domestic rabbits (San Juan strain) was slightly less than in laboratory strains of domestic rabbits (Evans and Thomsen, 1969). Surprisingly, the chronological pattern of papilloma regression in feral, San Juan domestic rabbits was similar to that described for cottontails. Most regressions in San Juan rabbits occurred as late as 7 months (Evans and Ito, 1966). The reasons for the relatively early regressions in laboratory domestic rabbits and late regressions in both cottontails and feral domestic rabbits are unknown. The difference in these regression patterns could reflect fundamental differences in mechanism. Shope papillomas induced in transplants of SPV-infected fetal rat skin invariably regress within a few weeks after development; the regression is associated with an intense mononuclear leukocytic inNtration quite similar to that seen in regressing Shope papillomas of rabbits (Kreider et al., 1971b).

c. MECHANISMOF SPONTANEOUS REGRESSION Spontaneous regression cannot be attributed to abortive interactions of SPV and papilloma cells. Regression is not due to the disappearance of SPV from the papilloma because infectious SPV can be recovered from regressing papillomas (Kidd, 1938). We have found that regression is unrelated to the SPV concentration in the inocula, since, in an individual rabbit, papillomas induced by high or low SPV concentrations regress simultaneously. Epidermis from regressor rabbits is not resistant to subsequent SPV infection, since in vitro incubation of regressor skin with virus, followed by grafting to the hamster cheekpouch, invariably produces papillomas which do not regress during the period of observation (Kreider, 1963). There are several lines of evidence which support the assertion that

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the spontaneous regression of the Shope papilloma is the result of an immune mechanism. Classical characteristics of immune responses include systemicity, specificity, recall, passive transfer with sera or leukocytes, and lymphoreticular infiltration of target tissues (Kreider, 1963). Systemicity of Shope papilloma regression is supported by the concurrent regression of all papillomas on an individual rabbit. Occasional rare exceptions are noted, but these “regressions” may be the result of mechanical dislodgment through handling, chewing by the host rabbit, or formation of a vascular thrombus with resultant tumor infarction and necrosis. Under these circumstances, papillomas often recur. The specificity of Shope papilloma regressions has never been established because the outbred hosts preclude challenge with transplantable tumors. Demonstration of the recall of immunity to autologous Shope papilloma tissue requires the employment of strategies to circumvent SPVneutralizing antibody present in previously inoculated hosts. These antibodies effectively prevent SPV rechallenge of both progressors and regressors. Two methods have been used to successfully rechallenge progressors and regressors with autologous Shope papilloma. In the fist of these, small fragments of autologous skin were washed free of antiviral antibody and incubated with partially purified virus in short-term organ culture and then grafted to orthotopic sites of the original donor (Kreider, 1963). The second method involved direct cutaneous challenge of progressor and regressor rabbits with infectious viral DNA (Evans and Ito, 1966). Both methods produced complementary results, i.e., progressor rabbits developed secondary papillomas from the challenge inocula, but regressors did not. Rabbits originally immunized with virus alone were not resistant to the secondary challenges (Kreider, 1963). These results infer the presence of a nonvirion-associated, tumor-rejection antigen in the Shope papilloma system, but they are not unequivocal. It is not known whether the immunity is responsible for the regression or simply follows tumor removal. A test for tumor immunity in hosts with surgically excised papillomas would settle this issue. Passive transfer of immunity from regressors to secondary hosts has not been achieved with regressor sera (Evans et al., 1962a; J. W. Kreider, personal observations), excluding an important role for antibody in spontaneous regression. Further, anti-SPV titers in regressors were not higher than in progressors (Kreider, 1963). Attempts to transfer immunity with spleen and lymph node cells have been frustrated by the unavailability, at that time, of syngeneic rabbits (Evans et al., 1962a; J. W. Kreider, personal observations). Under these conditions,

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survival of the allogeneic donor lymphocytes cannot be expected. Since syngeneic rabbits are now available, it should be possible to test this hypothesis. Mononuclear leukocytic infiltration of regressing Shope papillomas has been observed (Kidd, 1938). However, the superficial location of the papillomas provides many opportunities for leukocytic infiltration secondary to mechanical trauma, microbial infection, and vascular thrombi. Progressing papillomas frequently have substantial leukocytic infiltration. Further, the infiltrates are often variably distributed and difficult to evaluate or compare on a subjective basis. We have recently completed a quantitative study of the leukocytic infiltration of regressing Shope papillomas and the relationship of leukocytes to papilloma cell proliferation (Kreider, 1980). Regressing papillomas contained four times as many leukocytes as progressing tumors. The leukocytes were most concentrated at the epithelial basement membrane. The number of papilloma cells labeled with tritiated thymidine was reduced by at least 50% in regressing papilloma. This decline in the growth fraction alone could account for the regression but does not exclude associated, direct tumor cell killing. The inhibition of tumor cell proliferation was independent of the local leukocyte concentration or direct tumor-cell-leukocyte contact. A locally produced lymphokine such as proliferation-inhibitory factor, inhibitor of DNA synthesis, lymphotoxin, or interferon could fulfill this function. The cellular requirements for papilloma regression have not been established; for example, it is not known if papilloma regression will occur in T-cell-depleted hosts. The identities of leukocyte effectors of immunity are unknown. Papilloma and carcinoma cells placed in vitro were killed by both regressor and progressor lymphocytes (Hellstrom et al., 1969). An explanation for spontaneous regression may lie in Hellstrom’s observation that sera from progressors of either Stage I11 or IV blocked lymphocyte cytotoxicity, but regressor sera did not.

D. IMMUNESTIMULATION Strong support for the hypothesis that immune mechanisms are responsible for spontaneous regression was provided by the studies of Evans and associates, who demonstrated that administration of a papilloma vaccine significantly increased the frequency of regressions (Evans et al., 196213). The vaccine was composed of allogeneic, minced papilloma and usually was mixed with Bordetella pertussis as a possible adjuvant. Rapid processing of papilloma tissue to maximize viability was an important determinant of vaccine success, but B . per-

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tussis offered no additional benefit (Evans et al., 196213). Intradermal vaccine injections were made at six or more sites on the same day as a challenge of SPV at a remote site. The vaccine raised the regression frequency to 73% versus 17% in controls. The identity ofthe antigen(s) in minced papilloma vaccines which augment regression is unknown. It does not seem likely that virion-associated antigens are essential, since domestic rabbit papillomas contain little or no SPV. Further, antiviral immunity does not protect against papilloma cell challenge (Kreider, 1963). The possible contribution of other mechanisms to papilloma regression was inferred from the following study. Despite the success of papilloma vaccine in increasing regression frequency in domestic, laboratory-reared rabbits (Evans et al., 1962b), the vaccine was ineffective in feral, San Juan rabbits (Evans and Thomsen, 1969). The potential role of indigenous microbial or viral flora of rabbits in promoting regression was assessed by transferring pooled blood, nasal washings, and fecal suspensions from regressors into San Juan rabbits, which were also inoculated with RKV. Recipients were challenged with SPV and regression frequencies were determined. Pretreatment with the microbial and viral flora inhibited growth of papillomas and significantly increased the frequency of regression. Although alternative interpretations cannot be excluded, these results are consistent with a role for indigenous flora in augmenting papilloma regression through nonspecific immune stimulation. Seto et al., (1980) suggested that cutaneous, anaerobic coryneform bacteria enhance Shope papilloma regression. We have treated Shope papillomas by intratumoral injections of 1.0-mg killed Corynebacterium pamum vaccine given weekly. This schedule produced a significant increase in regression. Less intense treatment schedules were ineffective. E. IMMUNESUPPRESSION The hypothesis that immune mechanisms are responsible for spontaneous regression was also tested in studies of regression frequencies in immunosuppressed rabbits. Regional lymphadenectomy did not alter regression frequency, nor did administration of modest doses of cortisone (Evans et al., 1962a). Methylprednisolone at high dosage strongly suppressed papilloma regression and tumor lymphoreticular infiltration ( McMichael, 1967). Immunosuppression of rats with methylprednisolone or antithymocyte sera prevented spontaneous regression of Shope papillomas induced in fetal rat skin grafts but did not improve the yield or the growth of papillomas (Kreider et al., 1971b).

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Fetal and newborn rabbits are immunologically incompetent (Porter, 1960); SPV injection of rabbits in utero (Syverton, 1952) or in neonatal life (Seto et al., 1980) is associated with a reduction in regression fiequency. Antiviral antibodies did develop in the neonatal hosts, so immune tolerance to SPV was not a likely explanation for reduced regressions. In summary, Shope papilloma regressions are systemic, anamnestic, and associated with tumor leukocytic infiltration-all of which are characteristics suggesting an immune mechanism. Further support for this inference is drawn from observations that the frequency of regression is increased by specific and nonspecific immune stimulation and decreased in immunodepressed and neonatal hosts. Knowledge of the immune mechanism is still fragmentary, but inhibition of tumor cell proliferation by a humoral factor could be an important mediator of Shope papilloma regression. V. Shope Papilloma-Carcinoma Complex as a Model System

A. THEORETICAL AND PRACTICAL CONSIDERATIONS

Animal tumors may have value as models on at least two different levels. First, they may serve as substitutes for certain human neoplasms that they resemble in critical biological or pathological characteristics. In such cases, one assumes that the similarities outweigh the differences and that conclusions drawn from studies of the experimental model are applicable to the human counterpart. In that restricted context, the Shope papilloma system bears resemblance to several human disease complexes, most importantly, those lesions attributable to human papillomavirus infections. Animal tumor-host systems may also be considered models in the broad sense that many of their constituent components are well defined and the system is subject to a reasonable level of control. Those features mean that analysis of basic tumor biology is more feasible in such a model than in poorly defined, highly variable, uncontrollable human neoplastic diseases. The Shope papilloma-carcinoma model has value both as a basic research tool and as a patient surrogate. The Shope papilloma model is fertile ground for a variety of basic investigations. It is important to define the molecular and cellular mechanisms of epidermal cell transformation, progression, and regression. As yet, there is only limited understanding of the role of the SPV in each of those stages of the disease, as well as its contribution to

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maintenance of persistent papillomas and whether the virus functions as an initiator, a promotor, or in other ways. Much more should be learned about how various endogenous factors, such as genetics, hormones, tissue differentiation, specific immunity, and nonspecific defense mechanisms affect tumor pathogenesis. Only a few of the many possible interactions of the Shope system with other exogenous factors have been identified, and those few have not been studied in any detail. Thus, much of importance to basic science remains to be learned from the Shope papilloma-carcinoma complex. Despite the many important and interesting past research contributions of the Shope papilloma, there has been a paucity of recent studies with this model. This is due, at least in part, to several practical difficulties involved in working with the system. The overriding limitation is the lack of suitable techniques for SPV propagation in the laboratory or for the in vitro transformation and recognition of the neoplastic cells. Recent success in cloning SPV genome in the laboratory by Howley and associates (1980) offers hope for bypassing some of the previous technical limitations of the system. There are no convenient morphological, enzymatic, or antigenic markers in SPV-infected cells in vitro. I n vivo studies are hampered, not only by limited supplies of virus, but also by the size and expense of rabbits, the difficulty of doing detailed genetic studies in rabbits, and the limited availability and added expense of inbred rabbits for studies requiring transplantation in syngeneic hosts. Yet, the research potentials of the Shope model as it exists now have not been exhausted, and hopefully, further studies in this area will resolve some of the practical limitations of the system, B. RELEVANCETO HUMANNEOPLASIA One of the human diseases that is similar to the Shope system is a rare condition called epidermodysplasia verrucciformis (EV). Typically, disseminated verruccae of characteristic appearance appear at birth or in early childhood (Lutzner, 1978). Spontaneous regressions do not occur. There is a high incidence of progression to carcinoma in situ (Bowen’s Disease), most commonly in sun-exposed areas; development of invasive or metastatic squamous cell carcinoma is rare (Jablonska et al., 1972; Orth et al., 1979). The latent period for progression from papilloma to carcinoma ranges from 2 to 62 years, with an average of about 24 years (Lutzner, 1978). Patients with a clinical diagnosis of EV can be divided into two classes, those with multiple, nonprogressing verruca plana that contain HPV-3 and those with reddish plaques, pityriasis versicolor-like, or achromic plaques that contain HPV-5 and

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that progress to carcinoma with high frequency (Orth et al., 1979; HPV-5 was called HPV-4 by these authors prior to adoption of standardized nomenclature). Mature virus can be easily detected in the benign warts, but the virus disappears (or is drastically reduced) in the carcinomas (Jablonska et al., 1972). EV tends to occur in family clusters, occasionally with a history of consanguinity. It may be associated with mental retardation and seems to follow an autosomal recessive inheritance pattern (Lutzner, 1978). EV patients commonly have a suppression of cell-mediated immunity (Jablonska et al., 1978).Thus, the similarities between EV and the Shope complex (Table 11) include the viral etiology, the relative amounts of virus in benign and maligTABLE I1 COMPARISON OF SHOPE PAPILLOMA-CARCINOMA COMPLEX AND HUMANPAPILLOMATOSES Feature

Shope complex

Epidermody splasia vermcciformis HPV-5 Childhood' Skin (disseminated) Uncommon

TWO

RELATED

Condyloma acuminatum HPV-Gb Maturityd Genitalia (trauma)d Occasional (postpartum)d Sexuald Raref*' 1 year" Usually in situ Polyclonal' Benign onlyh Unknown

Etiology Onset Distribution Regressions

SPV Maturity Skin (trauma) Common

Transmission Progression Progression latency Type of carcinoma Clonality Virus production Genetics

Unknown Trauma? Commonfl.r*e Common 2-60 years' >1 year Usually in situfl,C*P Invasive Unknown Unknown Benign only' Benign only Familial' (autosomal Cottontail vs. recessive?) domestic Unknown Affects regression Depressed' Sunlight ( ~ l t r a v i o l e t ? )Unknown ~~ Hydrocarbons Enlarge in Unknown Unknown pregnancyd

Host immunity Cocarcinogen Hormonal response

Orth et al., 1979. Gissmann and zur Hausen, 1980. ' Lutzner, 1978. Oriel, 1971. Jablonska et al., 1972. Orth et al., 1977. 'See zur Hausen, 1976, for list of original reports. " Kovi et al., 1974. ' Friedman and Fialkow, 1976. Jablonska et al., 1978. 'I

'

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JOHN W. KREIDER AND GERALD L . BARTLETT

nant lesions, the tempo and incidence of carcinomatous progression, and the importance of host genotype to susceptibility. On the other hand, EV and the Shope complex differ in terms of time of onset, invasiveness of the carcinomas, frequency of spontaneous regression, association with immune depression, evidence for tumor immunity, and nature of cocarcinogens. A second human disease that resembles the Shope system in many ways is condyloma acuminatum (veneral wart, anogenital wart). These verruccae begin to appear during young adulthood. They arise at sites of genital trauma and are limited to the anogenital area (Oriel, 1971). The condylomata appear to be caused by a human papillomavirus (HPV-6) (Gissmann and zur Hausen, 1980), which is evidently transmitted through sexual contact. The lesions may enlarge markedly during pregnancy and may undergo partial or complete regression, either spontaneously or during the puerperium (Oriel, 1971). Although they respond well to treatment with podophyllin, recurrence is common, but progression to carcinoma (usually in situ) is rare (Oriel, 1971; Orth et al., 1977). When it does occur, carcinomatous change develops in a previously benign wart with a fairly short latency (6-8 months) (Kovi et al., 1974). The benign papillomas appear to be polyclonal in origin (Friedman and Fialkow, 1976) and produce mature virus, but virus is usually not detectable in malignant lesions (Kovi et al., 1974). Factors that predispose to carcinomatous progression have not been identified. There is no evidence for or against a significant role of host genotype (other than species restriction) or for host immunity in the pathogenesis of this disease. There have been reports that human papillomavirus is present in some dysplastic lesions of the human cervix (so-called flat condyloma) (Morin and Meisels, 1980) leading to the suggestion that HPV may be etiologically related to cervical carcinoma (zur Hausen, 1976). Similarities between condyloma acuminata and Shope tumors (Table 11) include viral etiology, occurrence of regression or progression, virus content of lesions, and time of onset. Condyloma acuminata and Shope papilloma-carcinomas differ in terms of frequency and tempo of progression, evidence for tumor immunity, evidence for hormonal role in regression, and evidence for cocarcinogenesis. In contrast to most human neoplastic diseases, these syndromes do not pose problems of early diagnosis or occult micrometastases. The clinical problems presented by these papillomatoses are to prevent the initial lesions, to cure the benign papillomas, to prevent malignant progression, or, ultimately, to cure the malignant, locally aggressive

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tumors. Clinical studies to those ends are necessary but difficult. The pathological and biological similarities between the human papillomatoses and the Shope complex suggest that conclusions drawn from investigations of the Shope papilloma may be predictive for the human diseases or may be valid guidelines for the design and pursuit of the relevant clinical research. We believe that the major unsolved problems of the Shope papilloma-carcinoma complex are shared by some of the human papillomaviruses. It is essential to determine the relative contributions to SPV susceptibility of genetic, immunologic, hormonal and other phenotypic factors. The exact role of the SPV genome as well as host and environmental factors in neoplastic progression are unknown. The mechanism of spontaneous regression is imperfectly understood. We do not know the leukocytic requirements or the leukocyte effectors of regression. The role of host immunity in progression of papilloma to carcinoma or in the development of metastases is unknown. Finally, methods for inducing specific and nonspecific resistance to established papillomas and carcinomas are crude and require substantial improvement. Thus, the Shope papilloma-carcinoma system can continue to contribute to our understanding of the mechanisms of neoplasia and the pathogenesis of papillomavirus infections.

A c KNO WLEDGMENTS We thank Michael Farlling for assistance in the preparation of this manuscript and Peggy Siegfried and Jeanne Vozzella for typing. The authors received financial support from USPHS grants CA-22869, CB74158, Cancer Center Grant P30-CA-18450, and the Jake Gittlen Memorial Golf Tournament during the preparation of this manuscript.

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Kreider, J. W., Breedis, C., and Curran, J. S. (1967).JNCZ,J. Natl. Cancer Inst. 38,921. Kreider, J. W., Haft, H. M., and Roode, P. B. (1971a).J . Inuest. Dermatol. 57, 66. Kreider, J. W., Benjamin, S. A., Pruchnic, W. F., and Strimlan, C. V. (1971b).J . Inuest. Dermatol. 56, 102. Kreider, J. W., Bartlett, G. L., and Sharkey, F. E. (1979). Cancer Res. 39, 273. Larson, C. L., Shillinger, J. E., and Green, R. G. (1936). Proc. SOC. E x p . Biol. Med. 33, 536. Lutzner, M. (1978). Bull. Cancer 65, 169. McMichael, H. (1965). Cancer Res. 25, 947. McMichael, H. (1967).JNCZ,J. Natl. Cancer Znst. 39, 55. McMichael, H., Wagner, J. E., Nowell, P. C., and Hungerford, D. A. (1963).JNCI, J . Natl. Cancer Inst. 31, 1197. Mattern, C. F. T. (1962).Science 137,612. Mellors, R. C. (1960). Cancer Res. 20, 744. Morin, C., and Meisels, A. (1980).Acta Cytol. J . 24, 82. Moulton, J. E., and Lau, D. (1964). Cornell Vet. 54, 602. Moulton, J. E., and Lau, D. (1967).Am. J . Vet. Res. 28,219. Murray, R. F., Hobbs, J., and Payne, B. (1971). Nature (London) 232,51. Nartsissov, N. V. (1959). “Pathology and Immunology of Tumors.” Pergamon, Oxford. Noyes, W. F. (1959)J. E x p . Med. 109,423. Noyes, W. F., and Mellors, R. C. (1957).J . E x p . Med. 106, 555. Oriel, J. D. (1971). Br. J . Vener. Dis. 47, 1. Orth, G., Vielle, F., and Changeux, J. P. (1967). Virology 31, 729. Orth, G., Jeanteur, P., and Croissant, 0. (1971). Proc. Natl. Acad. Sci. U.S.A. 68, 1876. Orth, G., Breitburd, F., Favre, M., and Croissant, 0. (1977). Cold Spring Harbor Conf. Cell Proliferation 4, 1043. Orth, G., Breitburd, F., and Favre, M. (1978).Virology 91, 243. Orth, G., Jablonska, S., Jarzabek-Chorzelska, M., Obalek, S., Rzesa, G., Favre, M., and Croissant, 0. (1979). Cancer Res. 39, 1074. Osato, T., and Ito, Y. (1967)J. E x p . Med. 126, 881. Osata, T., and Ito, Y. (1968). Proc. SOC. E x p . Biol. Med. 128, 1025. Palmer, C. G. (1959).JNCZ, J . Nut.?.Cancer Inst. 23, 241. Pass, F., Niimura, M., and Kreider, J. W. (1973).J. Inuest. Dermatol. 61,371. Passen, S . , and Schultz, R. B. (1965). Virology 26, 122. Porter, K. A. (1960).Nature (London) 185, 789. Ptokhov, M. P. (1960). V o p r . Onkol. 6, 14. Rashad, A. L., and Evans, C. A. (1967a). Cancer Res. 27, 1639. Rashad, A. L., and Evans, C. A. (1967b). Cancer Res. 27, 1855. Rendtorff, R. C., and Wilcox, A. (1957).J. Infect. Dis. 100, 119. Rogers, S. (1959).Nature (London) 183, 1815. Rogers, S. (1966).Nature (London) 212, 1220. Rogers, S., and Moore, M. (1963).J . E x p . Med. 117,521. Rogers, S . , and Rous, P. (1951).J . E x p . Med. 93,459. Rogers, S., Kidd, J. G., and Rous, P. (1960).Acta Unio Int. Cancrum 16, 129. Rous, P., and Beard, J. W. (1935a).Proc. SOC. E x p . Biol. Med. 33, 358. Rous, P., and Beard, J. W. (1935b).J . E x p . Med. 62, 523. Rous, P., and Friedewald, W. F. (1944).J. E x p . Med. 79, 511. Rous, P., and Kidd, J. G. (1936).Science 83, 468. Rous, P., and Kidd, J. G. (1937).J . E x p . Med. 67, 399. Rous, P., Kidd, J. G., and Beard, J. W. (1936).J . E x p . Med. 64, 385.

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Satoh, P. S., Yoshida, T. O., and Ito, Y. (1967).Virology 33, 354. Selbie, F. R.,and Robinson, R. H. M. (1947).Br. J . Cancer 1, 371. Seto, A,, Notake, K., Kawanishi, M., and Ito, Y.(1977).Proc. SOC. E x p . Biol. Med. 156,64. Seto, A., Salehi, S. A., Miyanomae, K., Ushijima, T., and Ito, Y. (1980).Oncology 37, 12. Seton, E. T. (1909).“Life Histories of Northern Animals. An Account of the Mammals of Manitoba,” p. 672.Scribner’s, New York. Seton, E. T. (1937).“Lives of Game Animals,” Vol. IV, p. 787.Literary Guild of America, New York. Shiratori, O., Osato, T., and Ito, Y. (1967).Proc. SOC. E x p . Biol. Med. 125,435. Shiratori, O., Osato, T., and Ito, Y. (1969).Proc. SOC. E x p . Biol. Med. 130, 115. Shope, R. E.(1935).Proc. SOC. E x p . Biol. Med. 32,830. Shope, R. E.,and Hurst, E. W. (1933).J. E x p . Med. 58,607. Stevens, J. G., and Wettstein, F, 0. (1979). J . Virol. 30,891. Syverton, J. T. (1952).Ann. N.Y. Acad.‘Sci. 54, 1126. Syverton, J. T., Dascomb, H. E., Koomen, J., Wells, E. B., and Berry, G. P. (1950a). Cancer Res. 10,379. Syverton, J. T., Dascomb, H. E., Wells, E. B., Koomen, J., and Berry, G. P. (1950b). Cancer Res. 10,440. Syverton, J. T., Wells, E. B., Koomen, J., Dascomb, H. E., and Berry, G. P. (1950~). Cancer Res. 10,474. Terheggen, H. C . , Lowenthal, A., Lavinha, F.,Colombo, J. P., and Rogers, S. (1975).2. Kinderheilkd. 119, 1. Watson, J. D., and Littlefield, J. W. (196O).J. Mol. B i d . 2, 161. Wettstein, F.O., and Stevens, J. G. (1980).Cold Spring Harbor Conf. Cell Proliferation 7,301. White, D. O . , Huebner, R. J., Rowe, W. P., and Traub, R. (1963).Aust. J . Exp. Biol. Med. Sci. 41, 41. Whiteley, H. J. (1956). J . Pathol. Bacteriol. 72, 1. Williams, R. C., Kass, S . J., and Knight, C. A. (1960).Virology 12,48. Yoshida, T. O.,and Ito, Y. (1968).Proc. SOC. E x p . Blol. Med. 128,587. zur Hausen, H.(1976).Cancer Res. 36,794.

REGULATION OF SV40 GENE EXPRESSION Adolf Graessrnann, Monika Graessmann, and Christian Mueller lnstitut fur Molekularbiologie und Biochemie der Freien, Unlversitat Berlin, Berlin. Federal Republic of Germany

I. Introduction

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

11. Cell Type Dependence of SV40 and PV Gene Expressio

A. Permissive Cells .................................................... B. Nonpermissive Cells ................................................ C. Semipermissive Cells ............................................... D. Virus-Resistant Cells ................................................ E. Conclusions ........................................................ 111. Functions of SV40 Tumor Antigens ...................................... A. Induction of Cellular and Viral DNA Synthesis ....................... B. Regulation of Late and Early SV40 Gene Expression .................. C. Stimulation of rRNA Synthesis ....................................... D. Helper Function for Adenovirus and U Antigenicity ................... E. Reduction of the Actin Cable Structure ............................... IV. Cell Transformation .................................................... V. Microinjection: Applications and Trends .................................

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

111 114 114 114 120 121 123 125 127 132 136 137 137 139 140 146

I. Introduction

For some 20 years, the two papovaviruses simian virus 40 (SV40) and polyoma virus (PV) have been the subjects of intensive study, and the interest in these viruses is still growing. Whereas the underlying stimulus for these efforts clearly stems from the oncogenic potential of both viruses, the process of viral cell transformation is too complex to be understood without complete knowledge of the organization of the viral genomes and the regulation of their gene expression. Since these relatively simple viruses depend on cellular enzymes for theirfeplication and gene expression, their thorough study has added enormously to and still improves our understanding of the molecular biology of eukaryotic cells. This vast amount of work has been reviewed extensively in recent years (Fried and Griffin, 1977; Fareed and Davoli, 1977; Kelly and Nathans, 1977; Weil, 1978; Lebowitz and Weissmann, 1979; Tooze, 1980). This article deals more specifically with the microinjection approach as a tool for studying SV40 and PV gene expression and extends a previous review on the same subject (Graessmann et al., 1979a). With our system, biological material of virtually any 111 ADVANCES IN CANCER RESEARCH, VOL. 35

Copyright 01981 by Academic Press,Inc. All rights of reproduction in any form reserved. ISBN 0-12-006635-1

112

ADOLF GRAESSMANN ET AL.

source can be transferred with glass micropipettes directly into living, single-tissue culture cells (Fig. 1).The preparation of the pipettes and the instrumentation required, as well as the process of microinjection and the evaluation of experiments, have been recently described in detail (Graessmann et al., 1980a). SV40 and PV are the smallest DNA tumor viruses known. The constituents of the viruses are proteins, cell and virus coded, and one

FIG.1. Assembled instruments for microinjection. Insert shows four steps of the procedure. (From Mueller e t al., 1981, by permission of Springer, Berlin, Heidelberg, and New York.)

REGULATION OF

~ ~ GENE 4 0EXPRESSION

113

double-stranded DNA molecule. Within the virus particles and in infected cells, the DNA is complexed with histones in a nucleosomal structure. Following removal of the histones in vitro, the superhelical DNA form I is obtained. Introduction of a single nick untwists the superhelix, generating the relaxed circular DNA form 11, whereas a double-strand break leads to the linear form 111. Alkaline treatment of DNA form I yields an interlocked, denatured cyclic coil that, on neutralization, may partially or fully renature. The primary structure of the SV40 genome was determined by Fiers and co-workers (1978) and by the group of Weissman (Reddy et al., 1978), and Griffin’s group sequenced the PV DNA (Soeda et al., 1980). Functionally, the viral genomes can be subdivided into two parts, the early and the late genome regions (Fig. 2). The early and late genes are oriented in opposite directions on their respective DNA strands. SV40 and PV have maximized their coding capacity using different reading frames within certain DNA segments. In the case of PV, three proteins, the large, middle, and small tumor (T) antigens, are coded by the early genome region, whereas two early SV40 proteins, the large and small T antigens, have been identified so far. Three capsid proteins are encoded by the late genome part, respectively, and their genes are also, in part, overlapping. Early and late genes are under different control mechanisms: Transcription of the early viral genome regions occurs directly after infection, whereas late gene expression is T antigen delarge T

FIG. 2. Gene maps of SV40 and PV. Both genomes are oriented with the origin of replication (ori) at the top. Map coordinates refer to the respective single EcoRI sites. Arcs with arrowheads cover DNA sequences coding for viral proteins, with the arrowheads pointing to the carboxy terminals. Dotted arcs cover spliced-out sequences. (Based on Tooze, 1980.)

114

ADOLF GRAESSMANN ET AL.

pendent. The transition point between these two phases is the onset of viral DNA replication. The modality of SV40 or PV gene expression is cell type dependent (Tooze, 1980). It. Cell Type Dependence of SV40 and PV Gene Expression

A. PERMISSIVE CELLS Only cells of the native host, i.e., monkey cells for SV40 and mouse cells for PV, support viral multiplication with high efficiency, producing 103-104 plaque-forming units (PFU) of progeny virus per cell. Using antibodies against T antigen and the viral capsid (V antigen), the infection cycle can be followed by immunofluorescence at the singlecell level. Little is known about the very early events of the infection cycle, namely, virus adsorption, penetration, and intracellular release. The nature of the viral receptors on the cell membrane is not known. Treatment of cells with receptor-destroying enzyme (neuraminidase) impairs PV but not SV40 infection. Shortly after infection, membrane vesicles containing typically one virion can be found in the cytoplasm, and morphologically intact virus particles appear in the nucleus within 1-2 hr (Tooze, 1980). We studied the temporal sequence of SV40-specific events in infected monkey c e l l s 4 l ) virus adsorption and penetration, intracellular virus release; (2) intranuclear accumulation of the template DNA; (3) transcription of early mRNAs in appropriate quantities; (4) T-antigen synthesis and its intranuclear accumulation; and ( 5 ) late viral gene expression-by microinjection of the following substances into TC7 monkey cells: SV40 virions, SV40 DNA I, early SV40 RNA, or purified SV40 T antigen. Following injection, recigient cells were fixed at appropriate time intervals and simultaneously stained for T and V antigen. Figure 3 correlates the times required for detectable T and V antigen expression in microinjected cells and in cells infected with SV40 at a multiplicity of 100 PFU per cell.

B. NONPERMISSIVE CELLS Mouse cells infected by the conventional virus adsorption method or by DNA transfection are nonpermissive for SV40. At high virus input multiplicities, 95% of 3T3 mouse cells or primary mouse kidney cells stain positive for T antigen within 72 hr after infection, but capsid protein synthesis is never demonstrable. With prolonged incubation, the number of T-antigen-positive cells decreases again. A small pro-

REGULATION OF

~ ~ GENE 4 0EXPRESSION

115

virus on

hours) time scale

0

1

i

12

16

I

SVlrO infection

12h--ci 16h

SV40 injection(nudeus1

11 h-! 15h

j

DNA I injectionhucleus)

8 h+ 12h

1

early R N A injection (cytoplasm)

5h-

T-antigen injection (cytoplasm)

0.5 h

c:

i

+’

FIG. 3. Temporal sequence of SV40-specific events in TC7 monkey cells. Times of first appearance of intranuclear SV40-specific antigens after inoculation of virus, viral DNA, viral RNA, or T antigen are given below the time scale. The temporal sequence depicted above the time scale is deduced from these experimental observations. (From Graessmann et al., 1979a, by permission of Springer, Berlin, Heidelberg, and New York.)

portion of these cells (0.1-1.0%) may grow out to permanent T-antigen-positive cell lines that may gain the ability to grow anchorage independent and to form tumors upon inoculation into animals. This abortive type of response of mouse cells to SV40 can be converted into a productive infection cycle by fusion of infected mouse cells with noninfected permissive monkey cells. To further investigate the mechanism involved in the abortiveness in mouse cells, we microinjected different concentrations of SV40 DNA and early SV40 RNA into mouse cells and analyzed T- and V-antigen synthesis (Graessmann et al., 1976). Late SV40 gene expression and viral multiplication can be induced in these cells by intranuclear injection of a large number of SV40 DNA I molecules. The percentage of cells supporting late SV40 gene expression is correlated with the number of viral DNA molecules (or intact virions) transferred per cell. At a multiplicity of 2000 to 4000 DNA molecules, 30 to 40% of the recipient mouse cells stain positive for V antigen, whereas after transfer of 125 to

TABLE I

SV40 V ANTIGEN FORMATION IN 3T3 CELLS MICROJNJECTEDWITH SV40 DNA OR WITH A MIXTURE OF SV40 DNA AND PV DNAa Injection of a mixture of SV40 and PV DNA I

Injection of SV40 DNA I Concentration of SV40 DNA ( cLgW

Average number injected DNA molecules/cell

V antigen (%)

1000 500 250 120 60

2000-4000 1000-2000 500-1000 250-500 125-250

38 25 12 4 0

Concentration of SV40 DNA (cLg/ml)

Concentration of PV DNA (CLdmU

Average number injected DNA molecules/cell

V antigen (%)

1000

0 500 750 870 940

2000-4000

38

2000-4000 2000-4000 2000-4000

27 NDb

2000-4000

0

500 250 120

60

Cells were fixed and stained 48 hr after microinjection. Data reproduced with permission from Graessmann et al. (1976). Not determined.

4

REGULATION OF

~ ~ GENE 4 0EXPRESSION

117

250 DNA molecules only T-antigen synthesis is demonstrable. This gene dose correlation is not due to an unspecific degradation of the SV40 DNA transferred, since addition of calf thymus or PV DNA to the injection solution did not affect late SV40 gene expression (Table I). With high multiplicities of injection, mouse cells produce progeny SV40 virus with an efficiency close to that of permissive monkey cells (Table 11). Generally, mouse cells injected with large numbers of SV40 DNA molecules contain larger amounts of T antigen than virus-infected cells, as tested by direct immunofluorescence. In this regard, it is of interest that late SV40 gene expression is also demonstrable after injection of two to four SV40 DNA I molecules mixed with early SV40 RNA (cRNA) containing the information for viral T antigens (see Section II1,A) (Table 111). These observations raise the question of whether sufficient synthesis of T antigen is one of the factors determining permissiveness. If it is, then permissive monkey cells should also exhibit a quantitative correlation between intranuclear T-antigen accumulation and V-antigen synthesis. With differently labeled antibodies against T and V antigen, both proteins can be followed quantitatively by microscope fluorescence photometry in a single cell (Graessmann et d., 1978). Figure 4 shows the time course of intranuclear T-antigen accumulation in SV40-infected TC7 monkey cells. At appropriate time intervals after infection, cells were fixed and stained with fluoresceinlabeled anti-T and rhodamine-B-conjugated anti-V antibodies. In these experiments, the intranuclear T-antigen concentrations of randomly chosen cells were measured in arbitrary units (AU). Identically

PRODUCTION OF

TABLE I1 S V 4 0 IN 3T3 AND TC7 CELLS MICROINJECTED WITH 20004000 DNA I MOLECULES" 3T3 cells Total

Hours postinjection

Number of cells injected

24 48 72

300 300 100

0 0 5 x 104

96

100

1 x 105

plaques

Plaques/ injected cell

TC7 cells, plaques/ injected cell

0 0 5 x 102 1 x 103

2 x 102 2 x 103

0

NDb

Injected cells were harvested at the respective times, sonicated, and plaque-assayed on CV1 monkey cells. * Not determined.

118

ADOLF GRAESSMANN ET AL.

SV40 T- AND V-ANTIGEN

FORhlATION IN 3T3 CELLS MICROINJECTED WITH OF SV40 DNA AND cRNA"

TABLE 111

Concentration of SV40 DNA I (pdml)

Average number of injected DNA moleculeslcell

10

20-40 20-40 2-4

10 1 0 10

0

20-40

Concentration (pdml) of cRNA (origin)

MIXTURES

SV40T antigen (%o)

0

100

500 (SV40) 500 (SV40) 500 (SV40) 500 (PV)

100 100

SV40V antigen (%)

80

0 50 8 0

100

0

*

" Each point is based on a count of 300 injected cells and is reproducible within 10% of the given value. The cells were fixed and stained for T and V antigen 24 hr after microinjection. Data reproduced with permission from Graessmann et al. (1976). treated SV40-transformed human cells (SV80) served as a biological standard. A continuous increase in the amount of the T antigen was observed over a period of 48 hr, regardless of whether the multiplicity of infection was 100 (Fig. 4), 10, or 1 PFU per cell. A threshold correla-

1

1101

8

16 24 32 LO time after infection (hours)

L0

FIG. 4. Time course of SV40 T-antigen concentration in CVl monkey cells infected with 100 PFU per cell. Columns represent the T-antigen fluorescence of 70% of the cells; minimum and maximum values are given by the bars. The T-antigen content of SV80 reference cells is inserted. The dotted line represents the threshold value of T antigen, below which V antigen is not detectable. (From Graessmann et al., 1978, by permission of The Rockefeller University Press, New York.)

REGULATION OF

sv40 GENE EXPRESSION

119

tion between T- and V-antigen synthesis became obvious upon measuring the T-antigen concentration in V-antigen-positive cells. Independent of the virus input or the time after infection, the minimal amount of T antigen in capsid protein-positive cells was at least 28 AU. To learn how many T-antigen molecules are equivalent to this vaIue, purified T antigen was microinjected at different concentrations into the nuclei of TC7 monkey cells. Recipient cells were fixed and stained as in the experiment described above. Figure 5 shows that the intranuclear fluorescence is linearly correlated with the amount of T-antigen molecules transferred per cell and that the threshold value of 28 AU approximately corresponds to the presence of 1-2 X lo6 T-antigen molecules (Graessmann et al., 1980b). The 3T3 mouse cells infected conventionally with SV40 contain significantly less T antigen than do monkey cells. Furthermore, only the number of T-antig6n-positive cells, but not the T-antigen concentration in individual cells, increases with prolonged incubation time. In contrast, mouse cells microinjected with 2000-4000 SV40 DNA I AU

30

25 20

15

10

5

1.0

2.5

4.0

5.0

T-ANTIGEN CONCENTRATION I N MG/ML 2-4

x 105

1-2

x 106

T-ANTIGEN MOLECULES INJECTED/CELL

FIG. 5. Correlation between the intranuclear fluorescence measured (AU) and the amount of T-antigen molecules microinjected per cell nucleus. Identically stained SV80 cells served as a biological standard.

120

ADOLF GRAESSMANN ET AL.

I

T

,I, i 1 4

LLL1

8

16

A

LO 24 32 time after infection (hours)

1

56

FIG, 6. Time course of SV40 T-antigen concentration in 3T3 mouse cells infected with 500 PFU per cell. Intranuclear T-antigen fluorescence of 3T3 cells microinjected with 2000-4000 SV40 DNA I molecules is also given (-.-.-.-). Details as described in Fig. 4. (From Graessmann et al., 1978, by permission of The Rockefeller University Press, New York.)

molecules accumulate T antigen equivalent to 50-90 AU, and in V-antigen-positive cells this value is always above 58 AU (Fig. 6). The experiments described so far show that the nonpermissiveness of mouse cells is not an absolute feature, since late viral gene products are synthesized and progeny virions formed after intranuclear inoculation of large numbers of SV40 DNA molecules. It seems likely that this high amount of viral DNA, which is not accessible in mouse cells conventionally infected with SV40, is required for efficient synthesis of an early SV40 gene product, presumably the T antigen. It remains to be tested whether the early SV40 genome region is a less efficient template in mouse cells than in monkey cells or whether posttranscriptional control steps are involved. However, SV40 T antigen injection experiments indicate that this is not a matter of a higher T antigen turnover rate, as shown in Fig. 7.

C. SEMIPERMISSIVE CELLS Semipermissive cells support SV40 or PV gene expression with a low efficiency exhibiting mostly an abortive type of response. Table IV shows the percentage of T- and V-antigen-synthesizing hamster, rat, and human cells after SV40 or PV infection. The nonsusceptibility of the majority of these cells is caused by one or more steps of the very early events of the infection cycle before decapsidation, since microinjection of 20 to 40 virus particles or DNA molecules induced viral gene

REGULATION OF

Sv40 GENE

EXPRESSION

121

fi T I

I

1

FIG.7. Turnover o f T antigen in CV1 (----) or 3T3 (-) T antigen.

cells microinjected with SV40

expression in a very high percentage of the recipient cells (Tables V and VI).

D. VIRUS-RESISTANT CELLS Resistance to virus infection can be (1) a species-specific quality, (2) acquired during the process of cell differentiation, or (3) a feature gained upon cell transformation. 1. Human cells are resistant to PV infection. Primary or secondary human embryonic cells infected with a high multiplicity (up to 1000 PFU per cell) do not synthesize any virus-specific product in detectable amounts. A permanent human cell line, Wi38, was tested, and synthesized T antigen with a low frequency following PV infection (Table IV). In contrast, secondary human or Wi38 cells microinjected with either PV particles or viral DNA support early and late gene expression with high efficiency (Table VI) (Gruen et al., 1974). Total

122

ADOLF GRAESSMANN ET AL.

TABLE IV SYNTHESIS OF sv40- OR PV-SPECIFIC ANTIGENS IN SEMIPERMISSIVE CELLS AT DIFFERENTTIMESAFTER INFECTION" Percentage of SV40 antigen-positive cells at (hours after infection) 24 Cell source Hamsterb Ratr Human' a

T 0 0 0.5

48 V

T

24

96 T

V

0 0.01 0 5 0 8

Percentage of PV antigen-positive cells at (hours after infection)

V

T

0.01 0 0 0 <0.001 NDd ND 1 2 0 0.5 10

48

V

T

96 V

T

2 0 0.5 0 0.01 5 0 2 <0.001 0 0 0

V

0 0.1 0

Data reproduced with permission from Graessmann et al. (1979a). Baby hamster kidney cells, passage 10. Embryonic rat cells (Wistar strain), passages 3-6. Not determined. Human Wi38 cells.

resistance of cells to SV40 infection is rare. We analyzed SV40 infectivity in flat revertants (1-4) isolated from SV40-transformed rat cells (14B). A feature of this cell line is its absolute resistance to SV40 infection or SV40 DNA transfection (Steinberg et al., 1978).This resistance can be overcome through microinjection of virus particles or of viral DNA. More than 90% of the flat revertant 1-4 cells synthesize T antigen after transfer of 2-4 PFU per cell (Graessmann et al., 1979~). Susceptibility to SV40 infection can also be restored by inserting membrane fragments from permissive monkey cells into the plasma TABLE V SYNTHESIS O F SV40-SPECIFIC ANTIGENS IN SEMIPERMISSIVE CELLS FIXEDAND STAINED 24 HR AFTER MICROINJECTION WITH SV40 DNA I" Percentage of SV40 antigen-positive cells after transfer of (number of DNA I molecules injected per cell) 2000-4000

200-400

20-40

Cell source

T

V

T

V

T

V

Hamsterb Rat' Humand

99 82 99

12 12 55

78 76 99

3

20

2

25 99

0 0

25

" Data reproduced with permission from Graessmann et al. (1979a).

'Baby hamster kidney cells, passage 10.

' Embryonic rat cells (Wistar strain), passages 3-6. Human Wi38 cells.

1

REGULATION OF

~ ~ GENE 4 0EXPRESSION

123

TABLE VI OF PV-SPECIFICANTIGENS IN SEMIPERMISSIVECELLS FIXEDAND SYNTHESIS STAINED24 HR AFTER MICROINJECTION WITH PV DNA I" Percentage of PV antigen-positive cells after transfer of (number of DNA I molecules injected per cell) 2000-4000

200-400

20-40

Cell source

T

V

T

V

T

V

Hamsteld Rat' Humand

80 99 95

32 50

80 99 80

2 40

30 45

0

NDe

0 10 ND

a

10

Data reproduced with permission from Graessmann et al. (1979a). 10. Embryonic rat cells (Wistar strain), passages 3-6. Human Wi38 cells. Not determined.

* Baby hamster kidney cells, passage

membrane of 1-4 cells via reconstituted Sendai virus (Graessmann et al., 1981). 2. Multinucleated muscle cells acquire resistance to virus infection during the course of differentiation from mononucleated myoblasts to myotubes; myoblasts from a variety of species are susceptible to PV or SV40 infection, but the terminally differentiated multinucleated myotubes are not (Fogel and Defendi, 1967). This resistance can be bypassed through microinjection of either intact virions or viral DNA (Table VII) (Graessmann et al., 1973). A point of interest is the observation that postmitotic myotubes as recipient cells support only T-antigen formation upon injection of DNA I, but incorporation of [ 3H]thymidine and V-antigen synthesis were not observed. In contrast, these functions were demonstrable in myotubes when the precursor myoblasts were used as recipients, indicating that stimulation of cell DNA synthesis as a PV and SV40 T-antigen-specific function depended on the differentiation state of the recipient cells when virus inoculation occurred. 3. SV40- and PV-transformed cells are mostly resistant to superinfection by the transforming virus. During recent years we have tested different transformed cell lines and found that they support late viral gene expression upon microinjection, as shown for three SV40transformed cell lines in Table VIII.

E. CONCLUSIONS The preceding sections demonstrate the correlation between the very early events of the infection cycle and the modality of

124

ADOLF GRAESSMANN ET AL.

TABLE VII AFTER SYNTHESIS OF PV-SPECIFIC ANTIGENS IN RAT MYOTUBES" O F FULL PV PARTICLES OR PV DNA I'

MICRO INJECTION^

PV DNA I Full PV particles Number of Virus particles titer injected (PFU/ml) per cell 10"

100-200 10-20 1-2

10'O

109

Percentage of PV antigenpositive cells T

V

63 24 0.1

0 NDd ND

DNA I concentration (mg/ml) 0.1 0.01 0.001

Percentage of PV antigenpositive cells

Number of DNA molecules injected per cell

T

V

100-200 10-20 1-2

80 71 2

0 ND ND

" Rat myotubes were obtained from 16- to 18-day-old rat embryos as described elsewhere (Graessmann et al., 1973). * Cells were fixed and stained 48 hr after microinjection. ' Data reproduced with permission from Graessmann et al. (1979a). ,INot determined. papovavirus gene expression. The nonsusceptibility of "resistant cells" can be bypassed by direct inoculation of virions or viral DNA into these cells. The fact that intact virus particles are also expressed in microinjected cells shows that the restriction is imposed at one or more steps before intranuclear virus decapsidation: virus adsorption, pene-

LATE

TABLE VIII SV40 GENEEWRESSIONI N SV40-TRANSFORMEDCELL OF SV40 DNA I' MICROINJECTION

LINES AFTER

Percentage of SV40 V antigen-positive cells afterb

Cell line (origin)

Infection with SV40 (500 PFU/cell)

Microinjection of SV40 DNA I (number of DNA I molecules injected per cell) 2000-4000

200-400

20-40

12 18

7 2 ND

0 N D' ND

-

SVT2 (mouse) SV80 (human) 14-B (rat)

0 0 0

6

" Data reproduced with permission from Graessmann et al. (1979a). Cells were fixed and stained 48 hr after infection or microinjection.

'Not determined.

REGULATION OF ~

~ GENE 4 0EXPRESSION

125

tration, intracellular release. Susceptibility can also be rendered by inserting membrane fragments from permissive cells. Such host range or cell type restriction is not limited to papovaviruses. Epstein-Barr virus (EBV), for example, is a human lymphotropic herpesvirus with a strong restriction to B-lymphocytes. However, a broad variety of cell types from different species can be infected by microinjection of EBV DNA (Graessmann et al., 1980d). Recently, Klein’s group demonstrated that susceptibility to EBV can be transmitted by inserting membrane fragments from Burkitt lymphoma cells via reconstituted Sendai virus into membranes of EBV-resistant cells (Volsky et al., 1980). Ill. Functions of SV40 Tumor Antigens

The first direct experimental evidence that T antigen is a viruscoded protein came from microinjection experiments with early SV40-specific RNA. To exclude contaminations with cellular RNA in these experiments, we synthesized RNA complementary to SV40 DNA (cRNA)in uitro with E . Coli DNA-dependent RNA polymerase. With superhelical SV40 DNA I, this enzyme preferentially transcribes the early viral DNA strand, and at least 50% of the RNA molecules are of genomic length. Template DNA was removed by exhaustive DNase treatment and Cs2S04 equilibrium density gradient centrifugation. Upon microinjection, T-antigen synthesis was demonstrable in recipient monkey or mouse cells independent of whether cellular RNA synthesis was blocked by actinomycin D (Table IX) (Graessmann et al., 1974; Graessmann and Graessmann, 1976). Meanwhile the exact coding regions for large T and small t antigens are determined. As shown in Fig. 2,large T antigen is encoded by two discontinuous DNA segments. Only splicing of its mRNA allows synthesis of large T antigen, since the intron at map positions 0.59-0.54 contains termination signals in all three reading frames. This explains why, in i n uitro translation systems, the unspliced cRNA directed only the synthesis of T antigen-related proteins with molecular weights up to 70,000 (70K family) but not of full-size large T antigen. Fingerprint analyses suggest that the coding sequences for the 70K family are located at map positions 0.54-0.17. Authentic small t antigen is synthesized under these conditions (Paucha et al., 1978). In contrast, SV40 cRNA directs synthesis of full-size large T antigen upon microinjection into monkey cells. When T antigen extracted and immunoprecipitated from TC7 monkey cells is injected either into the cytoplasm or into the nuclei, it migrates to the same position on SDS-polyacrylamide gels as

126

ADOLF GRAESSMANN ET AL. TABLE IX SV40 T-ANTIGENFORMATIONIN PRIMARY MOUSEKIDNEY CELLS"

Injection SV40 cRNA (0.5 mg/ml) SV40 cRNA (0.5 mg/ml) in actinomycin-D-treated cells SV40 cRNA (0.5 mg/ml) in cycloheximide-treated cells SV40 DNA I (1 mg/ml) in actinomycin-D-treated cells SV40 DNA I (0.1 mg/ml)' + TC7 cell RNA (lmg/ml) in actinomycin-D-treated cells SV40 cRNA, RNase treated ( 8 ) TC7 cell RNA

Cells with T-antigen formation (%)* 41 41 0 0

0 0 0

" Data reproduced with permission from Graessmann and Graessmann (1976). Each measurement is based on a count of 300 injected cells. Cells were fixed and stained 15 hr after injection. Without actinomycin D, this DNA concentration induces T-antigen synthesis in 100% of the injected cells. the wild-type T antigen from infected cells (Fig. 8). De no00 cell protein synthesis is not required for the production of full-size large T antigen, as shown in actinomycin-D-treated microinjected cells, but it is clearly nucleus dependent since cytochalasin-B-enucleated TC7 cells support only the synthesis of the 70K family after microinjection of cRNA (Mueller et al., 1981; Graessmann, Graessmann, and Mueller, unpublished observations). We conclude that the injected cRNA is spliced upon microinjection, that this process is nucleus dependent, and that cRNA injected into the cytoplasm presumably has to find its way into the nucleus. In spite of the fact that only two proteins are identified as early SV40 coded, numerous functions can be attributed to the early viral genome part (Tooze, 1980). Of these, the following were further analyzed by microinjection of SV40 DNA, DNA fragments, cRNA, or purified T antigen: induction of cell DNA synthesis, stimulation of viral DNA replication, regulation of late and early viral gene expression, stimulation of rRNA synthesis, helper function for adenovirus in monkey cells,

REGULATION OF

SV40 GENE

EXPRESSION

127

FIG.8. SDS-polyacrylamide gel electrophoresis of SV40 T antigens: fluorograph of immunoprecipitated proteins from (a) mock-injected TC7 cells, (b) TC7 cells microinjected with SV40 cRNA into the cytoplasm, (c) TC7 cells 40 hr after infection with SV40, and (d) TC7 cells microinjected with SV40 cRNA into the nucleus. The position of the large T antigen is indicated.

reduction of actin cable structure, induction and maintenance of cell transformation, and antigenic sites.

A. INDUCTION

OF CELLULAR AND VIRAL

D N A SYNTHESIS

Upon infection of arrested cell cultures, SV40 stimulates cell D N A synthesis. This property is large T antigen specific, as is directly shown by microinjection of early viral cRNA (Graessmann and Graessmann,

128

ADOLF GRAESSMANN ET AL.

1976) or purified T antigen. The T-antigen-related D2-protein isolated from HeLa cells infected with the adeno-SV4O hybrid virus Ad2+D2 and the T antigen isolated from the human SV40-transformed cell line SV80 induced cell DNA synthesis in about 80% of the recipient cells. Table X demonstrates the temporal correlation between intranuclear T-antigen appearance and the percentage of quiescent primary mouse kidney cells incorporating [ 3H]thymidine after D2-protein or SV80 T-antigen injection. Boiling of the proteins destroyed this activity completely (Tjian et al., 1978). Efficient synthesis of T antigen in terms of quality and quantity is also essential for viral DNA replication. Using temperature-sensitive early SV40 mutants (tsA), Tegtmeyer and his co-workers first showed the correlation between T-antigen synthesis and viral DNA replication. Permissive monkey cells infected at the nonpermissive temperature synthesize T antigen, but viral DNA replication and capsid protein synthesis are not demonstrable. Cells infected at the permissive temperature complete viral DNA synthesis when shifted to the nonpermissive temperature, but new rounds of SV40 DNA replication are not initiated (Cowan et al., 1973). Stimulation of cell DNA synthesis was observed at the nonpermissive temperature (Martin and Chou, 1975). Since T antigen is a multifunctional protein, domains with different functions can be expected. In order to map these.functions more preTABLE X STIMULATION OF DNA SYNTHESIS IN PRIMARY MOUSEU D N E Y CELLS MICROINJECTED WITH SV40 T ANTIGEN OR VIRAL DNA" Injection of SV40 T antigen Labeling period (hours postinfection)

T-antigenpositive cells (%)

0-5 5-10 10-15 15-20 20-25

95 95 85 75 42

Injection of SV40 DNA I

T-antigenpositive cells T-antigenstimulated for positive DNA synthesis (%) cells (%)

T-antigenpositive cells stimulated for DNA synthesis (%) ~~

0 15 80 75 25

0 60 99 99 99

0 0 35 92 50

~

Primary mouse kidney cells grown to confluence on glass slides were transferred into serum-free medium 24 hr before microinjection. L3H]Thymidine was added at a final concentration of 0.5 pCi/ml for the time intervals indicated. Thereafter, cells were washed with phosphate-buffered saline, fixed and stained for T antigen, and finally processed for autoradiography.

REGULATION OF

Sv40 GENE EXPRESSION

129

cisely, the early SV40 genome part was fragmented by digesting DNA I with different restriction enzymes. The first set of enzymes conserved the N-terminal coding region but generated variations toward the C-terminal early coding region (Fig. 9). Through microinjection experiments with these fragments, it was shown that about 20% of the molecular structure of the large T antigen away from the C terminus is totally dispensable for induction of cell DNA synthesis. The HpaIII Pst I-A fragment induced cell DNA synthesis in T-antigen-positive cells as efficiently as intact viral DNA. The HpaIIIHpaI-B fragment failed to stimulate DNA synthesis in confluent cultures of primary mouse kidney cells but exhibited some residual effect in TC7 monkey cells (Table XI). Since this fragment contains the entire coding region for the small t antigen, this protein may not be involved in the process of initiation of cell DNA synthesis. However, Baserga’s group observed stimulation of cell DNA synthesis in ts cell mutants derived from the hamster BHK line (ts13 and tsAF8) at the nonpermissive temperature upon microinjection of the Hpa I-B fragment, although on a relatively high background of DNA-synthesizing uninjected cells

FIG. 9. Assignment of DNA fragments used for microinjection to the physical map of the SV40 genome. (From Mueller et al., 1978, by permission of MIT Press, Cambridge.)

TABLE XI EARLYVIRUS-SPECIFIC FUNCTIONS I N TC7 CELLS INJECTED WITH VARIOUS SV40 NUCLEIC ACIDS"

Type of SV40 nucleic acid injected

Concentration (mg/ml)

Percentage of early genome

DNA I cRNA HPuIIIHPuI-B BumI-A HpaIYPstI-A HpaIIIBamHI-A BglYBamHI-A cRNA (BglYBamHI-A) HpaII/HpaI-C

0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

100 58 69 80 100 100

-

42

Antigenpositive injected cells (%) T

U

100 91 72 64 61 0 63 0 65 0 64 55 0 0 36 17 0 0

Data reproduced with permission from Mueller et al. (1978).

V 100 0

0 0 0 0 0 0 0

T-antigenpositive cells incorporating ["]thymidine ( % ) b 94 93 3 34 84

91

Injected cells positivefor complementation of tsA7 or tsA58 at 41.5"C (%), ND' 45 0 0 0 39

0

0

89 0

ND 0

Injected cells positivefor helper function for Ad2 virus (%)"

85 ND 0 0 0 52 0 ND ND

* Identical results were obtained in primary mouse kidney cells. Values are corrected against a background incorporation of 3%. ' Percentage of cells positive for SV40 V antigen. Percentage of cells positive for Ad2 fiber protein. 'Not determined.

REGULATION OF

~ ~ GENE 4 0EXPRESSION

131

(Floros et al., 1981).To analyze the influence of T-antigen structure on its ability to induce viral DNA replication and late gene expression, we microinjected early SV40 DNA fragments into TC7 monkey cells preinfected with tsA7 or tsA58 mutant virus. Complementation of tsA viruses at the nonpermissive temperature, i.e., capsid protein synthesis, was not obtained with the HpaIIIPst I-A fragment (Mueller et al., 1978). This observation confirms that viral DNA synthesis depends highly on the quality of T antigen, as already suggested by the tsAvirus infection experiments. The failure of this fragment to complement tsA virus is not due to a reduced rate of T-antigen synthesis since injected cells contained T-antigen-reactive material in amounts significantly above the threshold concentration equivalent to 28 AU. In this respect, it is of interest that purified T antigen (D2 protein, SV80 T antigen) also failed to complement tsA virus upon microinjection, although the amount of protein injectable is close to 28 AU, i.e., 1-2 X lo6 molecules (Fig. 5). Since the same T antigen preparations stimulated cell DNA synthesis even at cellular concentrations of 2-4 x 1oj molecules, we assume that, although biologically active, both the D2 protein and the SV80 T antigen are deficient for viral DNA replication. The D2 protein is a defective protein since the coding hybrid virus lacks early SV40 DNA sequences at map positions 0.630.54 (Tjian et al., 1978). The SV80 T antigen also differs in some respect from wild-type T antigen since cell hybrids between TC7 and SV80 cells do not complement tsA mutants at the nonpermissive temperature of 41.5”C (Graessmann, Graessmann, and Mueller, unpublished observation). It is not known so far how T antigen stimulates cellular and viral DNA replication. In this regard, some features of T antigen are of interest. 1. T antigen is a DNA-binding protein with affinity to doublestranded DNAs from many species. For SV40 DNA, Tjian (1978) demonstrated specific binding to three adjacent DNA segments within sequences containing the origin of DNA replication and the putative early and late promoters (Fig. 10). As shown recently, repetitive sequences of mammalian cell DNA, the Alu family, carry a stretch of 14 nucleotides in common with one of the SV40 T-antigen binding sites (Jelinek et al., 1980).These cellular DNA sequences may be a competitor to SV40 DNA for the binding of T antigen. This could explain the differences in T-antigen concentrations required for cellular or viral DNA replication. 2. Tjian et al. (1980) demonstrated the association of at least two enzymatic activities (ATPase, kinase) with T antigen. In uitro, the D2

132

ADOLF GRAESSMANN ET AL.

FIG. 10.Schematic representation of the three T-antigen binding sites I, 11, and 111 within regulatory sequences of the SV40 genome. Differential binding of T antigen is indicated by the density of hatching. (Based on Rio et al., 1980.)

protein caused the transfer of y-phosphate from ATP to the D2 protein itself and to exogenous acceptor molecules (e.g., phosvitin). 3. Last, but not least, T antigen forms complexes with cellular proteins, e.g., a cellular 53K protein, kinase (Tjian et d.,1980; Crawford

et al., 1980). It is tempting to speculate that these properties are crucial for the induction of cellular and viral DNA synthesis.

B. REGULATIONOF

LATE AND

EARLYSV40 GENE EXPRESSION

While T antigen regulates early SV40 gene expression down (autoregulation of T-antigen synthesis) (Alwine et al., 1977; Rio et al., 1980), efficient synthesis of T antigen in terms of quality and quantity is required for capsid protein synthesis, which, in turn, is linked to the onset of viral DNA replication. We analyzed two prominent consequences of DNA replication as a possible regulatory element for capsid protein synthesis: (1) increase in the number of SV40 DNA molecules,

REGULATION O F

~

~ GENE 4 0EXPRESSION

133

and (2) change of conformation of the DNA template during replication. To distinguish between these alternatives we microinjected either high numbers of SV40 DNA I molecules or low copy numbers of replicative intermediate DNA (RI-DNA),nicked SV40 DNA 11, or partially denatured SV40 DNA I (DNA Itlen)at conditions excluding viral DNA replication. Three different methods were used to prevent SV40 DNA replication: chemical inhibitors (araC, FUdR); multinucleated myotubes as recipient cells; tsA DNA at the nonpermissive temperature. Independent of how DNA synthesis was blocked, capsid protein synthesis was not demonstrable after microinjection of 2000-4000 SV40 DNA I molecules per cell. In contrast, transfer of 200-400 RI-DNA molecules induced V-antigen formation in about 30% of the recipient cells (Table XII). However, this percentage indicated that the RI-DNA molecules per se cannot induce capsid protein synthesis since at permissive conditions injection of 1-2 RI-DNA molecules led to T- and V-antigen formation. More likely, only a small fraction of the molecules present in the RI-DNA preparation is used for late gene expression. Since partially denatured SV40 DNA induced V-antigen synthesis in about 16% of the recipient cells and single-nicked DNA I1 induced it in 1% of the recipient cells, we may conclude that accessibility of a control region, for convenience termed “late promoter,” is enhanced by a conformational change of the SV40 DNA template, e.g., via generation of single-stranded regions or nicks at specific sites (Graessmann et aZ., 1977). In this regard, it is of importance that late SV40 gene expression is totally independent of T-antigen synthesis and viral DNA replication when the late genome region is brought artificially under “early promoter” control (see the following). In order to confine the right-hand boundary of the early-promoter region, SV40 DNA I was cleaved with the restriction enzymes BgZI and BamHI (Fig. 9). The BgZI enzyme cuts SV40 DNA about 80 nucleotides upstream from the initiator AUG for large T and small t antigens (Lebowitz and Weissmann, 1979). In spite of the fact that the BgZIIBamHI-A fragment contains the entire early coding region, T-antigen synthesis was not demonstrable after injection into TC7 cells. In contrast, the in vitro transcript of this fragment induced T-antigen synthesis after injection, indicating that the BgZI cut interferes with transcription but not with translation. The 80 nucleotides between the BgZI site and the initiator AUG are not essential for the promoter activity. This was demonstrated by microinjection of fragments containing either the early promoter region or coding sequences (Fig. 11). Upon injection, the Tay IIBam HI fragment (map positions

TABLE XI1 SV40 GENE EXPRESSIONA T CONDITIONS I$ZSTRlCTIVE FOR VIRAL DNA REPLICATION VARIOUS FORMS OF SV40 DNA"

IN

CELLS MICROINJECTED WITH ~

Antigen-positive cells (%) TC7 cells and inhibitors

Type of SV40 DNA injected

Number of SV40 DNA molecules transferred per cell

T

V

T

DNA I RI-DNA DNA I1 DNA Iden tsA7 DNA I tsA7 RI-DNA

2000-4000 200-400 200-400 200-400 2000-4000 200-400

99 99 90 95 99 ND

0 32 2 16 0 ND

99 99 ND 95 ND ND

araC

FUdR

TC7 cells at 41.5"C

Rat myotubes V

T

V

T

V

0

80 80 ND ND ND ND

0 30 ND ND ND ND

99 ND* ND ND 99 99

80 ND ND ND

30 ND 16 ND ND

0 16

" SV40 DNA was extracted from plaque-purified virus and further purified by CsCllethidium bromide equilibrium centrifugation, yielding SV40 DNA I and DNA 11. Form I1 contained, on the average, one random nick per molecule. Replicative intermediate DNA (RI-DNA) was isolated 32 hr after infection of TC7 cells with either wt777 or tsA7 virus. Purification of this DNA included digestions with RNase, RNase H, and Pronase, CsCllethidium bromide equilibrium centrifugation and either velocity centrifugation through sucrose gradients or chromatography on benzoylated-naphthoylated DEAE-cellulose. Neutral denatured SV40 DNA I d c n was obtained by a 3-min exposure of DNA I to pH 13.2, followed by neutralization. Cells were fixed and stained 24 hr after microinjection. Data reproduced with permission from Graessmann et al. (1977). Not determined.

REGULATION OF

U + L HpaII BglI

Sv40 GENE

135

EXPRESSION

early

BamH I

TaqI

late

1

HpaII BglI KpnI Bcl I RG. 11. Assignment of SV40 DNA fragments used for promoter studies. Arrows indicate the direction of transcription, the dotted arrow shows the orientation of the early promoter. (From Mueller et al., 1981, by permission of Springer, Heidelberg, Berlin, and New York.)

0.566-0.144) is totally inactive but becomes active in combination with the H p a IIIBgZI “promoter” fragment. When TC7 monkey, 52.2 rat, or 3T3 mouse cells are injected with the two DNA fragments they are ligated in vitro or as a mere mixture stained positive for SV40 T antigen (Mueller et al., 1981). Furthermore, stable T-antigen-positive cell lines were isolated from 52.2 rat cells injected with the fragment mixture. Protein extracts from these cells contain the 70K family but not the large T or small t antigens, as shown in Fig. 12 (Graessmann, Graessmann, and Mueller, unpublished observation). Different conclusions can be drawn from these experiments: (1) the H p a IIIBgZI fragment contains promoter activity; (2) this promoter activity can be transferred to promoter-negative DNAs; and (3) a former internal AUG is presumably used for translation initiation.

136

ADOLF GRAESSMANN ET AL.

FIG. 12. SDS-polyacrylamide gel electrophoresis of SV40 T antigens: fluorograph of immunoprecipitated proteins from (a) 52.2 rat cells transformed with the fragment mixture H p u IIIBgl I and Tuq IIEco RI-A (cell line TB2), (b) 52.2 rat cells transformed with wild-type SV40 DNA, (c) 52.2 rat cells infected with SV40, and (d) mock-injected 52.2 rat cells. The position of the large T antigen is indicated.

T-antigen-independent V-antigen synthesis was obtained after ligation of the H p a IIIBgZI DNA fragment in a converted orientation to the late KpnIIBclI DNA fragment (Fig. 11).Independent of the cell type used (rat, mouse, or monkey), this assembled DNA induced V-antigen synthesis with a high efficiency. Inhibitors of DNA synthesis have no influence on capsid protein formation under these conditions.

C. STIMULATIONOF rRNA SYNTHESIS

SV40 stimulates not only cell DNA but also rRNA synthesis. Baserga and his co-workers analyzed reactivation of the silent rRNA genes in a human-mouse hybrid cell line (55-54).These cells were infected with

REGULATION O F

Sv40 GENE EXPRESSION

137

nondefective adeno-SV40 hybrid viruses and then microinjected with suitable early SV40 DNA fragments. These experiments allowed the mapping of this function between map coordinates 0.39 and 0.27 (Soprano et ul., 1981).

D. HELPERFUNCTION FOR ADENOVIRUSAND

u ANTIGENICITY

Monkey cells are nonpermissive for adenovirus. TC7 cells infected with adenovirus type 2 synthesize hexon but not fiber protein, both of which are adenocapsid proteins (Tooze, 1980). TC7 cells coinfected with SV40 support fiber protein synthesis and adenovirus maturation. This SV40-mediated helper effect is a large T-antigen function since purified T antigen allows fiber protein synthesis and viral multiplication after microinjection into adenovirus-infected TC7 cells. A 23K hybrid protein, partly coded by an adeno gene and by the SV40 early C-terminal coding region exerts helper activity with the same efficiency as intact large T antigen (Tjian et al., 1978; Fey et al., 1979). Similar results were obtained by microinjection of early SV40 DNA fragments (Mueller et al., 1978). Therefore, this helper function is localized within the last 50-100 amino acids at the C terminus of the large T antigen. Intranuclear U antigen synthesis is a further event associated with early SV40 gene expression. This antigenicity is part of the large T antigen since anti-U antiserum precipitates purified T antigen (Robb, 1977). Anti-23K antiserum also precipitates T antigen and competes with anti-U antiserum, implicating a close correlation between U and 23K antigenicity (Graessmann, Graessmann, and Mueller, unpublished observation).

E. REDUCTIONOF

THE

ACTIN CABLE STRUCTURE

The SV40-induced change of the cytoplasmic actin cable structure is small t-antigen dependent. When 52.2 rat cells are infected with the deletion mutant virus d1884 or injected with 884 DNA I, they retain their well-developed cytoskeleton. This deletion mutant is small t-antigen negative but codes for authentic large T antigen (Crawford et al., 1978; Sleighet al., 1978). Furthermore, cells injected with purified large T antigen (D2 protein, SV80 T antigen) behave like mockinjected cells in this regard. The actin cable reducing activity is localized at map positions 0.65-0.375, as tested by injection of the H p a IIIHpa I-B fragment. This observation suggests that small t antigen is responsible for this early SV40 function. Since the H p a II/Hpa I-B fragment also contains sequences uniquely coding for large T antigen,

138

ADOLF GRAESSMANN ET AL.

a large T-antigen-related protein (maximal size 30K) may act coordinately with small t antigen (Graessmann et ul., 1980~). The hypothesis that the reduction of actin cables is small t-antigen dependent is in conflict with the tsA sensitivity reported elsewhere (Pollack et nl., 1975). However, it was not tested in these experiments whether small t-antigen synthesis was also affected at the elevated temperature. The biological significance of the cytoskeletal change for cell transformation remains to be determined, but it is a common feature of many cell lines transformed by oncogenic DNA or RNA tumor viruses or by chemical carcinogens (Freedman and Shin, 1975). Furthermore, the mechanism by which t antigen may cause this alteration is unclear. In SV40-infected cells, reduction of actin cables occurs with a time lag of several hours after the appearance of T antigen, and cells transformed with RNA tumor viruses thermosensitive in the src gene alter the cytoskeleton considerably faster after the thermoshift (Beug et u l . , 1978; see also McClain et d.,1978).This may indicate that the src gene product (pp60), as an actin-binding protein, causes this change directly, whereas small t antigen acts in an indirect manner. In this regard, it is of interest that growth factors like epidermal growth factor (EGF) mediate the change of the actin network through interaction with the cell membrane: Injection of up to 5 x lo6molecules of E G F

CHANCE OF

TABLE XI11 CYTOSKELETAL STRUCTURE I N MICROINJECTEDREF CELLS"

Material injected into cells T antigen (D2 hybrid protein) Wild-type DNA H p a I + H p a I1 fragment B EGFb into cytoplasm EGFb into nuclei EGF + T-antigen EGF + wild-type DNA H p a I + H p a I1 fragment B EGF added to the medium (25 nglml)

Nuclear T-antigen staining

Reduction of cytoplasm actin cables

+ +

-

-

+ + N D'

' Data reproduced with permission from Graessmann

+

-

+ +

et al. (1980~). the injection conditions used, 5 x loREGF molecules (Collaborative Research Inc.) were microinjected per recipient cell. Cells were fixed and stained 6, 12, 24, 36, and 48 hr after microinjection. Similar results were obtained when rat 1 cells were used for microinjection. Not determined.

REGULATION OF

~ ~ GENE 4 0EXPRESSION

139

into the nucleus or cytoplasm of rat cells had no effect on the actin cable structure, in contrast to EGF added to the culture medium (Table XIII) (Graessmann et d.,1980~). IV. Cell Transformation

SV40 transforms cells of a variety of species (e.g., mouse, rat, hamster, muntjak, and human). Also, permissive monkey cells can be transformed when transfected with UV-irradiated SV40 DNA, with SV40 DNA fragments, or with DNA defective in the origin of replication (Gluzman et al., 1977; Graessmann et al., 1980; Y. Gluzman, personal communication).In vivo, the oncogenicity of SV40 is mainly restricted to hamsters. Analyzing a large number of SV40 transformed cell lines, Pollack and co-workers (1974) found that established criteria of transformation are not fully correlated and that different phenotypes of transformants exist. At least two categories, the maximal and the minimal transformants, could be characterized (Table XIV). Since SV40 mutants, affected either in the large T or small t antigen (tsA mutants; deletion mutants d154/59), exist, the respective roles of these proteins in the process of transformation could be analyzed. Experiments with tsA mutants clearly indicate that wild-type large T antigen is required for the induction and maintenance of the transformed state (Martin and Chou, 1975; Osborn and Weber, 1975; Tegtmeyer, 1975). Furthermore, small t antigen also seems to be involved in the process of cell transformation. The question of whether small t-antigen-negative mutant viruses preferentially induce minimal transformants is somewhat controversial. Different groups have reported isolating only minimally transformed cells upon infection with d1884 virus (or upon transfection with the mutant DNA), whereas Martin and co-workers (1980) obtained maximal transformants with this TABLE XIV PROPERTIES OF MINIMAL AND MAXIMALTRANSFORM ANTS^ Properties

Minimal

Maximal

Clone on plastic Grow in low serum Clone in agar Activate plasminogen Lose adin cables Tumorigenicity in nude mice

+ +

+ + + + + +

~

~~~

Based on Pollack et al. (1974) and Topp et al. (1979).

140

ADOLF GRAESSMANN ET AL.

virus in in vivo and in vitro infections. Using 52.2 rat cells as recipients for the d1884 DNA, we isolated transformants with the same frequency, 10-20% of the injected cells, as with wild-type DNA, but none of the mutant-transformed cell lines formed colonies of more than 16 cells in soft agar (Graessmann, Graessmann, and Mueller, unpublished observation). The ultimate goal of the SV40 DNA fragment injection experiments is to provide some clue to the early SV40-specific functions involved in the process of cell transformation. So far, stable T-antigen-positive maximal transformants were only obtained from cells injected with fragments carrying the entire early coding region. Those fragments lacking sequences coding for the C terminus of T antigen (Fig. 9) expressed their functions only for up to 120 hr postinjection (Graessmann et al., 1980). It is unclear at the moment why these fragment-injected cells become T-antigen negative after this period of time. It is of interest in this regard that stable transformants appear with a frequency of 10-20% in rat cells injected with the fragment combination HpaIIIBgZI and Taq IIEcoRI-A (used instead of the TaqI/ BamHI fragment, see Fig. 11).These transformed cells stain to 99% positive for T antigen (70K family; see Fig. 12) but do not grow in soft agar and exhibit a cell morphology close to the parental 52.2 cell line. V. Microinjection: Applications and Trends

A major purpose of this article is to emphasize the applicability of the microinjection technique as a tool to study the relationship between eukaryotic genes and their functions, to unravel phenotypic changes exerted by purified gene products, to investigate the regulation of DNA replication and transcription, and to get some deeper understanding of the process of DNA integration and malignant cell transformation. To sum up this approach, Fig. 13gives a synopsis of the early SV40 genome region, its products, and their functions, based on the data discussed in Section 111. Interestingly, microinjection turned out to be especially suited for experiments aiming at the stable integration of foreign DNA into the cellular genome. As described in Section IV, up to 20% of rat cells microinjected with various SV40 DNAs grew out to stably transformed clones. Comparable results were obtained by Capecchi (1980) with mouse LMTK- cells: 20% of these cells gave rise to stable TK+ colonies when microinjected with a pBR322-derived plasmid containing SV40 DNA sequences and the herpes simplex virus thymidine kinase gene. Similar findings were reported by Anderson and co-workers (1980) and by P. Oudet and D. Arndt-Jovin (personal communications).

REGULATION OF

Sv40 GENE

141

EXPRESSION

EcoRI

HpaII Bgll V

W

I 1.0

.9

IS

.I7

-

HpaI

TaqI

-

PstI

v

SV40 early genome region

.I5

.5

13

T-antigen

Bad1 V

.2

11

I

proteins

r

Istimulation o f viral DNA synthesis stimulation o f cell

functions

I

DNA synthesis

U-antig .

lW.13. Functional map of the early SV40 coding region. (From Mueller et al., 1981, by permission of Springer, Berlin, Heidelberg, and New York.)

The microinjection approach has been used with advantage by several other groups. With an experimental set-up similar to ours but developed independently by Diacumakos et al. (1970), Diacumakos and Gershey were able to show that, on the average, one intact SV40 virion transferred to the nucleus of a monkey cell is sufficient to induce the synthesis of viral T antigen and the production of progeny virus (Diacumakos and Gershey, 1977; Gershey and Diacumakos, 1978). Stacey et al. (1977) investigated the mechanism by which the avian leukosis virus RAV-2 complements a Rous sarcoma virus deficient in the formation of the viral envelope glycoprotein [RSV(-)I. This helper effect is brought about more efficiently in RSV( -)-transformed chicken cells by microinjection of a 21s mRNA than by microinjection of a 35s

0.0

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ADOLF GRAESSMANN E T AL.

mRNA extracted from RAV-2-infected cells. With intact RAV-2 virion RNA, it was shown later that complementation of RSV(-), i.e., release of infectious RSV virions, occurred only when the helper RNA was injected into the nucleus but not when it was injected into the cytoplasm of RSV( -) transformants, arguing for a nuclear step required to modify the genomic 35s RAV-2 RNA to a functional envelope mRNA (Stacey, 1980).Studies on Epstein-Barr virus (EBV), a DNA virus regularly associated with two human neoplasms-Burkitt's lymphoma and nasopharyngeal carcinoma-has been, so far, severely hampered by the host range restriction of the virus to human or primate B lymphocytes. By microinjection, EBV DNA can be introduced into primary human amnion epithelial cells (Klein et ul., 1980) or into fibroblasts of human, monkey, or rat origin (Graessmann et ul., 1980d), where EBV-specific antigens are detected. Mapping experiments with purified EBV DNA fragments are currently under way. Westphal and co-workers analyzed intracellular translation of microinjected adenovirus 2 (Ad2) early and late mRNA and were able to detect authentic early and late viral polypeptides on polyacrylamide gels loaded with immunoprecipitates from about 500 injected cells labeled with [35S]methionineand [35S]cysteine.The helper function of Ad2 for the growth of the adenovirus-associated parvovirus AAV2 was shown to be promoted by early Ad2 gene products (Richardson et ul., 1980). Various nucleic acids of nonviral origin have been tested for their biological activity using the microinjection approach. The first report in this regard shows that RNA extracted from Harding-Passey mouse melanoma tumor cells induces the incorporation of melanin precursors in terminally differentiated rat muscle cells (Graessmann and Graessmann, 1971). More specifically, Stacey and Allfrey (1976a) observed correct translation of heterologous mRNA in human HeLa cells that synthesized avian globin after microinjection of duck globin mRNA. Recently, Huez and co-workers (1981)analyzed the synthesis and degradation of rabbit a- and 0-globins in human HeLa cells after microinjection of purified rabbit globin mRNA. As detected on fluorograms of two-dimensional polyacrylamide gels run with extracts from 150 injected cells, authentic a- and 0-globins were made. However, the translational stability of the a-globin mRNA decreased significantly faster than that of the 0-globin mRNA, and deadenylated globin mRNA seems to have too short a half-life in somatic cells to yield any detectable product. Liu and co-workers (1979)found human fibroblast interferon in mouse L cells injected with partially fractionated mRNA from human fibroblasts superinduced to synthesize interferon; thymidine kinase, hypoxanthine phosphoribosyltransferase,

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adenine phosphoribosyltransferase, and propionyl-CoA-carboxylase activities were found in the respective enzyme-deficient cells upon transfer of mRNA from human HeLa cells. Thus, microinjection seems suited as a screening procedure for the isolation of specific mRNAs. Similarly, Celis (1978) published preliminary work aiming at the screening of nonsense mutations in mammalian cells by microinjection of suitable suppressor tRNAs. A. Graessmann, R. Gesteland, and T. Grodzicker (unpublished) were able to suppress amber and ocher mutations in adeno-SV40 hybrid viruses isolated from Ad2+ND1 by microinjection of yeast suppressor tRNAs into mutant-infected monkey cells. The functionality and fate of proteins or peptides within living cells have been less broadly studied by microinjection. Stacey and Allfrey (1976b) investigated the turnover of fluorescently labeled heterologous and homologous proteins in HeLa cells, showing that intracellular degradation varies greatly depending on the size and the origin of the protein under view. The in vivo structure and turnover of the cytoskeleton and its constituents have been studied by microinjection of fluorescently labeled actin (Kreis et al., 1979) and a-actinin (Feramisco, 1979). The cyclic peptide phalloidin, which promotes actin polymerization, was shown to interfere in a concentrationdependent manner with locomotion and growth of microinjected cells, indicating that these functions require a delicately balanced reversible equilibrium between free actin and its different polymerization states (Wheland et al., 1977). McClain et al. (1978) have evidence through microinjection of extracts from cells transformed with either wild-type RSV or mutant RSV thermosensitive in the src gene that the src gene product is involved in the dissolution of the cellular microfilament bundles, thereby afflicting the regulation of cell growth. Clearly, the scope of possible applications of the microinjection technique has not been fully exploited; it will, we think, develop continuously with the elaboration of techniques fit to handle and analyze samples from a single up to some thousand cells. The reisolation of injected, intracellularly modified materials (e.g., processed cRNA) or of newly made products is greatly facilitated when giant cells generated by fusion (e.g., with polyethylene 1000, paramyxoviruses) of mononucIeated cells are used as recipients for microinjection (Graessmann et al., 1979b; Huez et al., 1980). Whereas with standard culture cells the injection volume per cell may vary within 10-lo-lO-'l ml, these fused cells tolerate up to 10-5 ml in a single injection. For a comparison with related techniques developed for the transfer of biomaterials into eukaryotic cells (for reviews, see Celis et al., 1980;

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TABLE XV GENERAL FEATURES OF THE MICROINJECTION TECHNIQUE Single adherent tissue culture cells Recipient cells Material transferable

Suspension culture cells, fixed to dish

Primary or secondary Lymphocytes cells; all permanent cell lines Lymphocytes Cell organelles, viruses, DNA, RNA, proteins, etc. Nucleus, cytoplasm Nucleus, cytoplasm

Site of application Injection volume per cell (ml) Ways of analysis Biological, biochemical 90-100% Efficiency

Biological 90-100%

Fused cells; PEG 1000 as fusion agent Permanent cell lines; lymphocytes Permanent cell lines; lymphocytes Cytoplasm

Biochemical, biological 70-90%

Baserga et al., 1980), the following list of general features of the microinjection system may be helpful (see also Table XV): 1. Every tissue cell line tested so far has proved suitable for microinjection; suspension culture cells, e.g., lymphocytes, are prepared for microinjection by binding them to a substrate via suitable linkers (Fig. 14) (Graessmann et al., 1980d). 2. As demonstrated in this article, virtually no limitations exist regarding the material to be transferred. Intact virions, DNAs, RNAs, and proteins, as well as small metabolites or substances unrelated to cellular metabolism, can be introduced in purified form without the involvement of helper macromolecules or chemical treatment. The number of molecules transferred is directly correlated with the concentration of the injection solution and with the volume transferred per cell. Eveil intact cell organelles, e.g., cell nuclei (Graessmann, 1968), can be implanted into culture cells by microinjection. 3. The site of inoculation within the recipient cell-nucleus or cytoplasm-an be chosen by the investigator.' This allows studies on intracellular transport and compartmentalization, compartmentdependent modification steps, etc. (Tjian et al., 1978; Graessmann et al., 1980c; Stacey, 1980; Gershey and Diacumakos, 1978). Interestingly, Capecchi (1980) claims that the TK+ phenotype could be re! With more emphasis on the technical aspects of microinjection, even intramitochondrial injections seem possible (Diacumakos, 1980).

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sv40 GENE EXPRESSION

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FIG.14. Friend leukemia cells unattached (top) and attached to the plastic surface of a Petri dish pretreated with concanavalin A (bottom).

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stored only upon microinjection of TWplasmid DNA into the nucleus of LMTK- mouse cells. Injections into the cytoplasm proved to be ineffective. Similarly, we found that expression of cytoplasmically administered DNA strongly depends on the structure of the DNA itself. Closed circular superhelical SV40 DNA I is expressed with the same efficiency in cells receiving nuclear or cytoplasmic injections, whereas expression of nicked circular DNA I1 or linearized DNA I11 is greatly hampered when transferred into the cytoplasm (Graessmann, Graessmann, and Mueller, unpublished observation). We therefore assume that it is the direct access to the nucleus that makes the microinjection technique so efficient in transformation experiments. 4. A sample volume of about 2 pl is sufficient for microinjection; most of this material is retained for other experiments. 5. The number and localization of recipient cells is known. 6. Injected cells respond efficiently and remain as viable as their uninjected neighbors on condition that the material transferred has no cytopathic effects. However, the microinjection technique is restricted to the use of cultured cells as recipients; it is hardly applicable to intact organisms. Yet it seems conceivable to isolate cells from individuals (e.g., bone marrow cells), to passage them in vitro and to clone out lines from injected cells. The donor individual could then be the recipient for these cells.

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Jelinek, W. R., Toomey, T. P., Leinwand, L., Duncan, C. H., Biro, P. A., Choudary, P. V., Weissman, S. M., Rubin, C. M., Houck, C. M., Deininger, P. L., and Schmid, C. W. (1980).Proc. Natl. Acad. Sci. U.S.A. 77, 1398-1402. Kelly, T. J., Jr., and Nathans, D. (1977).Ado. Virus Res. 21,85-173. Klein, G., Luka, J., and Zeuthen, J. (1980).Cold Spring Harbor Symp. Quant. Biol. 44, 253-261. Kreis, T. E., Winterhalter, K. H., and Birchmeier, W. (1979).Proc. Natl. Acad. Sci. U.S.A. 76,3814-3818. Lebowitz, P., and Weissmann, S. M. (1979).Curr. Top. Microbiol. Immunol. 87,43-172. Liu, C.-P., Slate, D. L., Gravel, R., and Ruddle, F. H. (1979).Proc. Natl. Acad. Sci. U.S.A. 76,4503-4506. McClain, D. A., Manes, P. F., and Edelman, G. M. (1978).Proc. Natl. Acad. Sci. U.S.A. 75,2750-2754. Martin, R. G., and Chou, J. Y. (1975).J. Virol. 15,599-612. Martin, R. G., Setlow, V. P., Chepelinsky, A. B., Seif, R., Lewis, A. M., Jr., Scher, C. D., Stiles, C. D.. and Avila, J. (1980).Cold Spring Harbor Symp. Quant. Biol. 44, 311424. Mueller, C., Graessmann, A,, and Graessmann, M. (1978).Cell 15,579-585. Mueller, C.. Graessmann, M., and Graessmann, A. (1981).In “International Cell Biology 1980-1981” (H. G. Schweiger, ed.), pp. 119-127.Springer-Verlag, Berlin and New York. Virol. 15,636-644. Osborn, M., and Weber, K. (1975).J. Paucha, E.,Harvey, R., and Smith, A. E. (1978)J.Virol. 28, 154-170. Pollack, R.,Riser, R., Conlon, S. R., and Rifkin, D. (1974).Proc. Natl. Acad. Sci. U.S.A. 71,4792-4796. Pollack, R., Osborn, M., and Weber, K. (1975).Proc. Natl. Acad. Sci. U.S.A. 72,994-998. Reddy, V. B., Thimmappaya, B., Dhar, R., Subramanian, K. N., Zain, B. S., Pan, J., Celma, M. L., Gosh, P. K., and Weissman, S. M. (1978).Science 200,494-502. Richardson, W. R., Carter, B. J., and Westphal, H. (1980). Proc. Natl. Acad. Sci. U.S.A. 77, 931-935. Rio, D., Robbins, A., Myers, R.. and Tjian, R. (1980).Proc. Natl. Acad. Sci. U.S.A. 77, 5706-5710. Robb, J. A. (1977).Proc. Natl. Acad. Sci. U.S.A.74,447-451. Sleigh, M.J., Topp, W. C., Hanich, R., and Sambrook, J. (1978).Cell 14,79-88. Soeda, E., Arrand, J. R., Smolar, N., Walsh, J. E.. and Griffin, B. E. (1980).Nature (London)283,445-453. Soprano, K. J., Jonak, G. J., Galanti, N., Floros, J., and Baserga, R. (1981).Virology 109, 127-136. Stacey, D. W. (1980).In “Transfer of Cell Constituents into Eukaryotic Cells” (J. E. Celis, A. Graessmann, and A. Loyter, eds.), pp. 29-54. Plenum Press, New York. Stacey, D. W., and Allfrey, V. G. (1976a).Cell 9,725-732. Stacey, D. W., and Allfrey, V. G. (197613). J . Cell Biol. 75, 807-817. Stacey, D. W., Allfrey, V. G., and Hanafusa, H. (1977).Proc. Natl. Acad. Sci. U.S.A. 74, 1614-1618. Steinberg, B., Pollack, R., Topp, W. C., and Botchan, M. (1978).Cell 13, 19-32. Tegtmeyer, P. (1975). J. Virol. 15,613-618. Tjian, R. (1978).Cell 13, 165-179. Tjian, R., Fey, G., and Graessmann, A. (1978).Proc. Natl. Acad. Sci. U.S.A. 75, 12791283. Tjian, R., Robbins, A., and Clark, R. (1980).Cold Spring Harbor Symp. Quant. Biol. 44, 103-11 1.

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Tooze, J., ed. (1980).“DNA Tumor Viruses.” Cold Spring Harbor Lab., Cold Spring Harbor, New York. Topp, W. C., Rifkin, D., Graessmann, A., Chang, C. M., and Sleigh, M. J. (1979).In “Hormones and Cell Culture” (G. Sato and R. Ross, eds.), pp. 361-370.Cold Spring Harbor Lab., Cold Spring Harbor, New York. Volsky, D. J., Shapiro, I. M., and Klein, G. (1980).Proc. Natl. Acad. Sci. U.S.A. 77, 5453-5457. Weil, R. (1978).Biochim. Biophys. Acta 516, 301-388. Wheland, J., Osborn, M., and Weber, K. (1977).Proc. Natl. Acad. Sci. U.S.A. 74,56135617.

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POLYAMINES IN MAMMALIAN TUMORS Part I Giuseppe Scalabrino and Maria E. Ferioli Institute of General Pathology and C.N.R. Centre for Research in Cell Pathology. University of Milan. Milan, Italy

Nil minus est hominis occupati quam vivere: nullius rei difficilior scientia est. Professores aliarum artium vulgo multique sunt, quasdam vero ex his pueri admodum ita percepisse visi sunt, ut etiam praecipere possent: vivere tota vita discendum est et, quod magis fortasse miraberis, tota vita discendum est mori. SENECA, “De Brevitate Vitae,” 7, 3 L’ignorance qui estoit naturellement en nous, nous l’avons, par longue estude, confirmbe e t averbe. MONTAIGNE, “Essais,” L. 11, C. 12

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I. Introduction and Background , , , . , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Structure-Function Relationship of Polyamines , . . . . . . . . . . . . . . .. .. B. Properties of the Biosynthetic Enzymes of Polyamines . . . . . . . . . . . . . C. S-Adenosyl-L-Methionine . . . . . . . . . . . . . . . , , . .. . . . . .. .. .. . D. 5’-Methylthioadenosine , . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Catabolism of the Major Polyamines in Mammalian Organisms . . . F. Conjugation Products and Excretion Products . . . . . . . . . . . . . . . . . . . . . . . G. Natural Antipolyamine Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . 11. Levels of Polyamines and Their Biosynthetic Enzymes in Fully Developed Experimental Tumors . . . .. . . . . . . . , . . . . . . . . . . . , . . . . . . . . . . . . . . . 111. Modification in Vivo and in Vitro of Tissue Polyamine Metabolism by Chemical Carcinogens and Tumor Promoters . . . . . . . . . . . . . . . . . . . . . . . A. Effects of a Single Administration of a Carcinogen or Tumor Promoter on Polyamine Biosynthesis and Content in the Target Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Effects of Repeated or Prolonged Administration of a Carcinogen or Tumor Promoter on Polyamine Biosynthesis and . .. . . . Content in the Target Tissues.. . . . . . . . . . . . . . . . . . . . . . C. Multistage Carcinogenesis , . . . . . . , . . . . . . . . . . . . . , , . . . . . . . . . . . ..... D. Mutagenic Action and Antimutagenic Properties of Polyamines . . . . . . . . E. Polyamine Levels in Urine, Sera, and Erythrocytes of Rats during Chemical Carcinogenesis or Bearing Several Experimental Tumors . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Biosynthesis and Levels of Polyamines in Cells during the Virus-Induced Transformation Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Effects of Oncogenic RNA Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Effects of Onbgenic DNA Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Changes in Polyamine Biosynthesis and Content of Target Tissues by Physical Carcinogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

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I. Introduction and Background

Afker the discovery of polyamines, it became clear in time that they are ubiquitous in mammalian body tissues. Moreover, an everincreasing number of important regulatory functions have been attributed to the polyamines in the different fields of cellular biochemistry in eukaryotes. Excellent reviews of the physiological functions fulfilled by polyamines, or of some particular aspects of these, are available in the enormous literature on these substances (Tabor et al., 1961; Tabor and Tabor, 1964, 1972; Bachrach, 1970b; Raina and Janne, 1970a, 1975; Stevens, 1970; Smith, 1971, 1972; Cohen, 1972; Williams-Ashman, 1972; Williams-Ashman et al., 1972a, 1973; Morris and Fillingame, 1974; Barbiroli et al., 1976; Caldareraet al., 1976; Cli, et al., 1976; Russell et al., 1976a; Sakai and Cohen, 1976c; C. W. Tabor and Tabor, 1976; Karpetsky et al., 1977; Pardee et al., 1978; Canellakis et al., 1979; Cohen and McCormick, 1979; Maudsley, 1979; Quash and Roch, 1979; Stevens and Winther, 1979; Williams-Ashman and Canellakis, 1979; Heby and Andersson, 1980; McCann, 1980; Russell, 1980; Theoharides, 1980). Furthermore, the roles of polyamines in the biochemistry of particular organs or metabolic pathways are included in some monographs or reviews (Raina, 1963; Janne, 1967; Siimes, 1967; Williams-Ashman et al., 1969, 1976, 1980; Janne et al., 1976; Algranati and Goldenberg, 1977; Kramer et al., 1979; Shaw, 1979; Slotkin, 1979; Lesiewicz and Goldsmith, 1980; Lowe, 1980). Three comprehensive books have also been published, by Cohen ( 1971), Bachrach (1973), and Gaugas (1980a). A series of volumes, of which the two that have appeared so far were edited by Campbell et al. ( 1978a,b), present recent advances in the polyamine field in a variety of disciplines, and thus indicate the growing importance of polyamines in biochemistry and medicine. The proceedings of two symposia have been edited by Kremzner (1970) and Herbst and Bachrach (1970), in which most of the information about the metabolism and function of polyamines obtained before 1970 is elucidated. Finally, there is one short, but critical, article (Cohen, 1978) about particular biochemical functions of polyamines. Although many of the reviews cited include one (generally short) paragraph on the polyamines in mammalian tumors, only a symposium volume edited by Russell (1973a) and one book more recently published by Russell and Durie (1978), and five reviews by Russell (1973b,c), Bachrach (1976b), Janne et al. (1978), and Milano et al. (1980) are devoted to the data accumulated during the past ten years on this topic and to highlighting the behavior and significance of

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polyamines and their biosynthetic enzymes in oncology, in both animals and humans. The present review is intended not as a comprehensive summary nor, even less, as an analytical discussion of the different aspects of the biochemistry and physiology of polyamines in the different types of living organisms. For an in-depth discussion of those subjects we refer the reader to the above-mentioned references. However, in this introductory section some material concerning the structure, distribution, and function of polyamines, almost exclusively limited to mammalian cells, will b e included. General information about the biosynthetic and degradative pathways of the main polyamines in mammalian cells will also be up-dated. This we consider to be a necessary and useful preamble to the sections that follow, which will be strictly devoted to mammalian neoplasms and in which some concepts about the physiology of polyamines and the regulation of their metabolism in normal cells will be discussed. Our aim is to review selectively and in detail the extensive literature on the connections between the polyamines and cancer in mammals and to judge whether these connections are coincidental or characteristic of the neoplastic disease process. Because of the numerous recent developments in this area, another purpose of the present review is to appraise its present status. A customary and widely accepted definition of a tumor is that it is a mass of abnormal cells with altered phenotypes; its many differences from normal cells, in both morphological and biochemical features, lead to excessive growth of the tumorous cells. Thus, the essence of the concept of neoplasm is the seemingly autonomous and uncontrolled cell multiplication. Consequently, the modifications in polyamine biosynthesis and content observed in several biological models of controlled cell growth in mammals-such as liver regeneration, regenerating nerves, different target organs after treatment with growthpromoting hormones, myocardial hypertrophy, kidney hypertrophy, prostatic hyperplasia, wound healing, EGF-stimulated cultured epidermal cells or fibroblasts, mitogen-stimulated cultured lymphocytes, and NGF-stimulated neurons-will be deliberately left out of this review; the reader will find the appropriate references in the reviews and books cited.

A. STRUCTURE-FUNCTION RELATIONSHIP OF

POLYAMINES

The aliphatic polyamines are nonprotein, polycationic substances that are widely distributed in living organisms (animals, bacteria, vi-

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ruses, yeasts, and plants) and in biological fluids. The main physiological polyamines present in different types of tissues are putrescine and its propylamine derivatives, spermidine, and spermine. Cadaverine and 1,Sdiaminopropane are found only rarely in animal tissues and in biological fluids. The common name of putrescine is derived from its first isolation from cultures of Vibrio cholerae and from cadavers undergoing bacterial decomposition, and that of spermine to the discovery of spermatozoa together with crystals of the phosphate salt of spermine in human seminal fluids (Cohen, 1971).During synthesis of spermine, another base, which was identified as spermidine phosphate, was obtained. From a chemical point of view, the term “polyamines” is, in one respect, a misnomer, since it suggests a compound with multiple amine residues. To circumvent this problem, it has been suggested recently that the term polyamines can be replaced with the term “oligoamines,” which is chemically more precise (Stevens and Winther, 1979). Putrescine is, strictly speaking, a simple diamine, whereas spermidine is a triamine and spermine a tetraamine. However, the metabolic and functional similarities of these compounds are so close that it is convenient and conventional to classify and handle them together. The formulae of the aforementioned polyamines are as follows: HzNCHZCH2CHJVHZ 1,3-Diaminopropane

H2NCH2CHzCHZCHZNHz 1,4-Diaminobutane or 1,CTetramethylenediamine (putrescine) HzNCHpCHzCHzCH&HZNH2 l,5-Diaminopentane (cadaverine)

HZNCHZCHZCH~NHCH&H&H&H~NH~ N-(Spropylamine)-1,4-diaminobutane or 4-Azaoctane-1,8-diamine or N-(3-aminopropy1)-tetramethylene-1,Cdiamine (spermidine)

HJVCHzCHzCHzNHCHzCHzCHzCHzNHCH&HzCHzNHz N,N‘-bis(3-propylamine)-1,4-diaminobutaneor 4,9-Diazadodecane-l,12-diamine or N,N’-bis(3-aminopropyl)-tetramethylene-l,4-diamine (spermine)

The polyamine pattern may vary markedly from one species to another, and in a given species it differs greatly among various tissues and also depends on growth conditions, growth rate, age, etc., of the tissue. In general, spermidine and spermine are the main polyamines synthesized in eukaryotes, usually present in millimolar concentrations, whereas smaller amounts of putrescine are synthesized, usually in nanomolar concentrations. Prokaryotes have higher concefitrations of putrescine than spermidine and lack spermine, except for a few

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bacterial species. Thus, spermine synthesis appears to be either very rare or utterly lacking in bacteria, suggesting that spermine has some nuclear function. Using mouse cells, it was found that, although spermidine and spermine occur in both nuclear and cytoplasmic fractions, the concentration of these t w o polyamines in nuclei surrounded by membranes (the so-called karyoplasts) is at least three times higher than that in enucleated cells (the so-called cytoplasts) or in whole cells (Clark and Greenspan, 1977; McCormick, 1977, 1978a). The ability of nucleated eukaryotic cells to synthesize spermine may be considered to be an evolutionary acquisition of function in which spermidine is converted to a novel functional compound by the addition of an aminopropyl moiety. However, an intriguing, but still unsolved, question is whether spermine has its own qualitatively specific properties in the cell biochemistry of eukaryotes, such that it cannot be replaced at all by spermidine, or whether it is a duplicate of spermidine and consequently could be considered to be a bonus biomolecule. Unfortunately, although several mutants lacking in one of the polyamines or various polyamine biosynthetic enzymes have been isolated and characterized from yeasts and bacteria (Hirshfield et al., 1970; Maas et al., 1970; Morris and Jorstad, 1970, 1973; Young and Srinivasan, 1972, 1974; Morris and Hansen, 1973; Algranati et al., 1975; CunninghamRundles and Maas, 1975; Echandi and Algranati, 1975a,b; Goldemberg and Algranati, 1977; Hafner et al., 1977,1978,1979; Cohn et al., 1978a,b,c, 1980; Geiger and Morris, 1978a,b; Taboret al., 1978; Whitney and Morris, 1978; Whitney et al., 1978; Igarashi et al., 1979), no analogous mutants of any mammalian cell lines are available. Only mutant mammalian cell lines showing decreased polyamine transport have been described (Mandel and Flintoff, 1978). In eukaryotes, however, few instances have been found thus far in which spermidine and spermine are not interchangeable to some degree. Throughout the growth of fish and rat brain and in the livers of mice starved or treated with phenobarbital, spermine levels correlate with those of DNA, whereas spermidine levels show excellent correlation with the RNA content in the tissue (Seiler et al., 1969; Seiler, 1973; Seiler and Lamberty, 1975; Seiler and Schmidt-Glenewinkel, 1975). Analogous localizations of spermidine and spermine have been found in viruses. In fact, in herpes simplex virus type 1 (HSV-l), which mature their virions in the nucleus of the infected cell, spermine is associated with the nucleocapsid, whereas spermidine is isolated with the viral envelope (Gibson and Roizman, 1971; Cohen and McCormick, 1979); in vaccinia virus, which, like pox viruses, multiplies exclusively in the cytoplasm of the infected cell, spermine is associated with the DNA-

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containing cores and spermidine with the membrane material (Lanzer and Holowczak, 1975). Furthermore, in explants of mouse mammary gland, spermidine but not spermine can replace glucocorticoids in stimulating the synthesis of a-lactalbumin and casein (Oka, 1974; Oka and Perry, 1974a,b; Kano and Oka, 1976; Rillema et al., 1977; Houdebine et al., 1978; Oka et al., 1978a; Bolander and Topper, 1979). However, it has been recently demonstrated that this action of spermidine in mammary gland seems to differ from species to species (Bolander and Topper, 1979). Finally, it has been reported that the rat ventral prostate contains an acid protein that is localized in the cytosol, is induced by androgens, and binds spermine much more tightly than spermidine or other natural diamines (Liang et al., 1978; Liao et al., 1979; Mezzetti et al., 1979). Just recently, in order to differentiate between those effects of spermine due to its polycationic nature and its effects as a specific component needed for maximum efficiency in protein synthesis, an immunochemical approach has been developed (Quash et al., 1976; Bartos and Bartos, 1978; F. Bartos et al., 1979, 1980; Niveleau and Quash, 1979). Using specific antispermine antibodies, an inhibition of protein synthesis in the wheat germ cell-free system programmed with exogenous mRNA was shown and was found to be specific, since it was not overcome by putrescine or spermidine but only by spermine (Niveleau and Quash, 1979). In addition to the aforementioned polyamines, another group of polyamines has been recently identified in plants, algae, bacteria (especially thermophilic bacteria), some multicellular marine organisms (e.g., arthropods), and mammals (Johnson and Markhan, 1962; Kullnig et al., 1970; Kuttan et al., 1971; Imaoka and Matsuoka, 1974; Oshima, 1975, 1979; De Rosa et al., 1976, 1978, 1980; Nickerson and Lane, 1977; Stillway and Walle, 1977; Kneifel et al., 1978; Rosano et al., 1978; Zappia et al., 197813; Aleksijevic et al., 1979; Kremzner and Sturman, 1979; Yamamoto et al., 1979). Among these so-called new polyamines it seems important to mention the following compounds: caldine, thermine, and thermospermine. The formulae of these new polyamines and of other polyamines of minor importance are as follows: HzNCHzCHOHCHpCHSNHi 2-Hydroxyputrescine

HpNCHzCHOHCHzCHzNHCHzCHzCHzNHz 2-H ydroxy spermidine HSNCH~CH&H&JHCH&~HZCH~NH, Sym-norspermidine or Bis(3-aminopropy1)amine or 1,7-Diamino-4-azaheptane (caldine)

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HzNCH&HzCHzCHzNHCHZCHzCH2CHzNHz Bis(4-aminobuty1)amine(homospermidine)

HOOCCHzCHzNHCH2CHzCH&HzNHCHzCHzCOOH N , N '-bis(2-carboxyethyl)-1,4-diaminobutane(spermic acid)

H~NCH&HZCH~NHCH~CHZCH~NHCH&HZCH~NH~ 1,l l-Diamino-4,8-diazaundecaneor N,N'-bis(3-aminopropyl)-1,3-diamino-propane or Norspermine or Sym-norspermine or N,N'-bis(3-aminopropyl)-tri-methylene-1,3-diamine (thermine)

H~NCH~CH~CH~NHCH~CH~CH~NHCH~CH~CH~CH~NH~ 1,12-Diamino-4,8-diazododecane(thermospermine)

Although some of these new polyamines have also been found in eukaryotes, their biological significance and specificity in these organisms remains to be clarified. In mammals, although remarkable amounts of polyamines are present in the main biological fluids (especially in seminal fluid), these substances fulfill their paramount biochemical roles inside the cell. Most of the natural functions of polyamines in different kinds of biological systems are closely connected with their physicochemical properties. The three most widely distributed polyamines (putrescine, spermidine, and spermine) are very soluble in water, have weak chelating capacity, and are stable polycations with pK values between 8.0 and 11.0 for the primary amino group and between 9.0 and 10.9 for the secondary amino group. At physiological pH, i.e., around neutrality, all of the primary and secondary amino groups of putrescine, spermidine, and spermine are protonated, so that they have two, three, or four basic centers, respectively. Most of the various biological effects of these polyamines are undoubtedly related to this polybasic structure. In fact, polyamines are known to have high affinity for negatively charged compounds and molecules, e.g., the phospholipids of cell membranes and of myelin-rich structures and nucleic acids (Ames and Dubin, 1960; Kropinski et al., 1973; Levy et al., 1974; Gabbay et al., 1976; Gosule and Schellman, 1976, 1978; Osland and Kleppe, 1977; Bolton and Kearns, 1978; Chattoraj et al., 1978; Damaschun et al., 1978; Giorgi, 1978; Minyat et al., 1978; Becker et al., 1979). In uitro, polyamines directly stimulate various DNA and RNA polymerases, methylases, nucleotidyltransferases, hydrolases, and ribonucleases and affect reactions involving tRNA, ribosomal RNA, and mRNA molecules, thus contributing to polyribosomal protein synthesis (Janne et al., 1976; Canellakis et al., 1979; Cohen and McCormick, 1979; Williams-Ashman and Canellakis, 1979). Therefore, polyamines appear to be implicated in virtually every step of the synthesis and ultimate metabolic fate of RNA. In the sequence DNA+ RNA+ protein the polyamines influence, at the transcriptional level, strand

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selection and chain initiation, extension, or termination and, at the translational level, preparation of tRNA, aminoacylation, messenger binding to the ribosomal subunits, the translation of natural and synthetic mRNAs, and the assembly of the ribosomal subunits (Raina and Janne, 1970a; Barbiroli et al., 1976; Caldarera et al., 1976; Janne et al., 1976; Maudsley, 1979). Additionally, polyamines stabilize the structures of tRNA, ribosomes, and polysomes and promote the attachment of free ribosomes to endoplasmic reticular membranes (Cohen, 1971; Bachrach, 1973; Janne et al., 1976; Cohen and McCormick, 1979; Maudsley, 1979).Always because of the polybasic characteristic of the polyamines, these nitrogenous bases are known to specifically bind to the polyanionic nucleate, thus stabilizing nucleic acids against denaturation and digestion by nucleases (Cohen, 1971; Bachrach, 1973; Janne et al., 1976; Williams-Ashman and Canellakis, 1979). The multitudinous effects that spermidine and spermine have in cell-free systems that synthesize or degrade nucleic acids or proteins may be both stimulatory and inhibitory, depending not only on the concentrations of the polyamines but also on the experimental conditions, such as ionic strength (particularly of Mg ions), pH, and temperature (Raina and Janne, 1975; C. W. Tabor and Tabor, 1976). Although no direct evidence proves that polyamines have these same functions in vivo in intact cells, a large body of indirect evidence suggests that the close association between nucleic acids and polyamines also exists in vivo in prokaryotic and eukaryotic organisms. Another important feature of the molecular structure of polyamines is the possibility of rotation around the carbon-carbon and carbonnitrogen bonds, which confers considerable conformational flexibility to the polyamines (Sakai and Cohen, 1976c; Cohen, 1978; Cohen and McCormick, 1979). Therefore, spermidine and spermine can assume an extended conformation (as in the case of condensation with DNA) or a more locked conformation (as in the case of binding to tRNA) (Sakai and Cohen, 1976c; Cohen, 1978; Cohen and McCormick, 1979).X-Ray analysis has suggested several possible configurations for the complex formed between the polyamines and double-stranded DNA. Spermidine and spermine can bind to DNA through the interactions of phosphate groups with each positively charged amino group, the tetramethylene portion of the polyamine bridges the narrow (minor) groove of the helix between the two strands, and the trimethylene portion bridges adjacent phosphate groups (Sakai and Cohen, 1976c; Cohen, 1978; Cohen and McCormick, 1979). Alternatively, polyamines can bind in the minor groove of' DNA by hydrogen bonding of the amines to phosphate oxygens (Sakai and Cohen, 1976c;

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Cohen, 1978; Cohen and McCormick, 1979). It is likely that all these configurations can be adopted in viuo in either a random or nonrandom manner, so that the polyamines can be attached like the histones along sections of DNA molecule. According to what has been demonstrated so far, the role of spermine and spermidine in gene expression seems to lie in the induction of supramolecular structures of the DNA, in competition with the histones and nonhistone proteins, or of the RNAs in RNA-protein complexes. The secondary and tertiary structures of all sorts of polynucleotides can be profoundly affected by polyamines. Molecules of tRNA can be converted from their inactive configuration to a more compact active form by the addition of monovalent cations or of much lower concentrations of Mg2+or polyamine. Moreover, specific tight binding sites for spermidine exist in the tRNA molecules of yeast and Escherichia coli (Ladner et al., 1972; Pochon and Cohen, 1972; Santi and Webster, 1975; Takeda and Ohnishi, 1975; Evans and Deutscher, 1976; Prinz et al., 1976; Bolton and Kearns, 1977a,b; de Varebeke and Augustyniak, 1977). It seems opportune to mention here that recent crystallographic studies have defined the position of spermine within the molecule of yeast phenylalanine tRNA (Cohen, 1978; Quigley et al., 1978). This is probably the first example of elucidation of the function of a polyamine in terms of molecular structure. However, differences in experimental results, probably due to the different experimental conditions, have been obtained, and their interpretations are still the object of discussion. In many aspects, the polyamines may be regarded functionally as a group of compounds acting like organic cations (Veloso et al., 1973; Igarashi et al., 1975; Imai et al., 1975; Fukuyama and Yamashita, 1976; Nakai and Glinsmann, 1977b; Tanigawa et al., 1977; Lovgren et al., 1978; Kitada et al., 1979). In many in vitro systems studied so far, the effectiveness of the polyamines follows a cationic progression, i.e., spermine, the strongest base, is the most effective, followed by spermidine and then by putrescine, whereas in a few other studies a precise structural demand for a specific polyamine has been shown. Interchangeability between polyamines and Mg2+ and other cations has been described in several steps of protein biosynthesis, albeit Mg2+may not be able to substitute for all the polyamine molecules (Khawaja and Raina, 1970; Khawaja, 1971, 1972). It has been shown that M$+ ions can substitute, to some extent, for spermidine and spermine in the process of DNA synthesis in cultured cells (Melvin and Keir, 1979). In the case of yeast tRNAPhe,previously mentioned, the role of polyamine, which is to modify the structure of the mole-

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cule by pulling strands together and producing bends in particular regions, is quite different from that of Mg2+ (Cohen, 1978; Cohen and McCormick, 1979). Similarly, the binding of M$+ to a nucleotide is strictly one to one, whereas interactions between polyamines and nucleotides are affected not only by charge interactions but also by structural features of polyamine molecules, resulting in variable and multiple binding. The polyamines can also compete with M g + for complex formation with AMP, ADP, ATP, and other nucleotides. There are other biochemical reactions in which Mg2+ and polyamines are not interchangeable. Mg2+ and spermine within certain concentration ranges seem to be synergistic in elevating the respiratory control ratio associated with mitochondria1 oxidation of P-hydroxybutyrate, but the spermine concentrations involved are much lower than those of Mgz+ (Chaffee et al., 1977, 1978, 1979). Moreover, spermine cannot completely substitute for MgZfin enhancing the phosphorylation of nonhistone chromatin proteins (Imai et al., 1975) or in stimulating the choline kinase reaction (Fukuyama and Yamashita, 1976), and the inhibiting effect of spermine on lipid peroxidation is much stronger than that of Mgz+(Kitada et al., 1979). Finally, spermine promotes the ADP-ribosylation of nonhistone proteins, whereas Mg2+promotes that of histones (Tanigawa et al., 1977). Functionally, the polyamines offer a selective advantage over inorganic cations in that intracellular synthesis is possible. This permits fine adjustments of the intracellular polyamine concentrations according to different physiological conditions. However, a set of very complex systems for maintaining the cellular homeostasis of magnesium and other metal cations does exist, although many aspects of it are yet undefined (Rasmussen and Bordier, 1974). In the case of magnesium, this is demonstrated by the facts that mammalian cells are able to retain nearly normal MgZ+content despite extracellular Mg2+deficiency and that, conversely, Mg2+uptake declines when hypermagnesemia develops (Rasmussen and Bordier, 1974). Unlike polyamines, the MgZ+content of the mitochondria is generally greater than that in the cytosol (Rasmussen and Bordier, 1974). Within cells, MgZfand other metal cations are obtained from the bloodstream, whereas polyamines are produced within the cells and then released into the blood stream. However, there is an important point of contact between the cellular regulation of polyamine biosynthesis and Mg2+concentration in that both are under the influence of several hormones. Naturally occurring aliphatic polyamines have been shown to influence a number of biochemical reactions involving membrane functions. Several authors, using membranes purified from different tissues,

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have reported that spermine and spermidine inhibit Na+,K+-ATPase activity, which is generally considered to be the enzymic mechanism of cation transport across the cell membrane (Ahmed and WilliamsAshman, 1969; Peter et al., 1973; Tashima and Hasegawa, 1975; Heinrich-Hirsch et al., 1977; Quarfoth and Ahmed, 1977; Tashima et aZ., 1977, 1978; Quarfoth et aZ., 1978). A similar inhibitory effect of spermine and spermidine on the activity of CaZ+,Mg2+-ATPasehas been shown in the muscular tissue (De Meis and De Paula, 1967; Nagai et al., 1969), in which polyamines also promote the polymerization of actin (Oriol-Audit, 1978). Therefore, it is quite possible that the polyamines are involved, among others, in physiological contraction processes, e.g., as regulators of ATP concentrations. Interestingly enough, a regulatory effect on membrane-bound acetylcholinesterase has also been demonstrated (Kossorotow et al., 1974; Anand et al., 1976; Max and Oh, 1977), hinting that the polyamines may also have a role in synaptic transmission. Polyamines have been found to have important effects on different membranes with which they are possibly associated in uiuo. A marked stabilization of membrane structure against lysis or swelling has been reported for several microorganisms and mammalian subcellular fractions (Mager, 1959; Anderson and Norris, 1960; Herbst and Witherspoon, 1960; Tabor, 1960; Quigley and Cohen, 1969). The mechanism of this stabilization remains to be elucidated, but it is conceivable that the polyamines, as polycations, strongly bind to membranes, which usually have a net negative charge. A further molecular explication for such stabilization may reside in the recently reported demonstration that polyamines markedly protect mitochondria1 membranes against the destabilizing action of exogenous phospholipases (Sechi et al., 1978). It is well known, in fact, that the endogenous phospholipases bound to many membranes, such as those of lysosomes and mitochondria, operate in vivo to seriously alter membrane structure. During recent years, new biochemical reactions in the regulation of which the polyamines are physiologically involved have been identified. Thus, we attach paramount importance to the fact that these substances can act as modulators of the metabolism of cyclic nucleotides, of the activities of protein kinases, and of glycerolipid biosynthesis. The role of polyamines in the biosynthesis and the inactivation of the cyclic nucleotides will be discussed in detail in Part 11, Section I of this article in Volume 36. The effect of polyamines on the activity of protein kinases in normal cells depends on the type of protein kinase tested (Imai et aZ., 1975; Lee and Iverson, 1976; Murray et al., 1976; Takai et aZ., 1976; Maen-

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paa, 1977; Nakai and Glinsmann, 1977a; Ahmed et al., 1978; FarronFurstenthal and Lightholder, 1978; Hochman et aZ., 1978; Job et d., 1979). Polyamines have been shown to inhibit the activity of CAMPdependent protein kinases and, on the contrary, to stimulate the activities of both cGMP-dependent and cyclic nucleotide-independent protein kinases (Lee and Iverson, 1976; Murray et al., 1976; Takai et al., 1976; Maenpaa, 1977; Hochman et al., 1978; Job et al., 1979). When such regulation involves the phosphoprotein kinases localized in the nucleus, this would mean that the polyamines could also assume a regulatory role in genetic expression, since it is well established that nuclear kinases have a key role in the regulation of chromosomal protein phosphorylation and gene expression. Germane to the stimulation by spermine and spermidine of the chromatin-associated noncyclic AMP-dependent protein kinases are the reports that polyamines can directly enhance certain nucleoside diphosphate kinase reactions (Nakai and Glinsmann, 1977a) and the aforementioned choline kinase reaction (Fukuyama and Yamashita, 1976). Finally, studies have recently reported that spermidine and spermine stimulate the synthesis of triglyceride (Jamdar, 1977, 1978).

B. PROPERTIES OF THE BIOSYNTHETIC ENZYMES OF POLYAMINES Four enzymes are known to be involved in the biosynthesis of polyamines in eukaryotic organisms: two decarboxylases and two synthases. L-Ornithine decarboxylase (L-ornithine carboxyl-lyase, EC 4.1.1.17) catalyzes the formation of putrescine from L-ornithine. S-Adenosyl-L-methionine decarboxylase (SAMD) (EC 4.1.1.50) produces S-methyladenosylhomocysteamine (“decarboxylated Sadenosylmethionine,” also defined as S-adenosyl-(5’)-3-methylthiopropylamine) from S-adenosyl-L-methionine (SAM). This enzyme is of paramount importance for the syntheses of spermidine and spermine. Spermidine synthase catalyzes the transfer of the propylamine group to putrescine to yield spermidine. Spermine synthase transfers the propylamine group from S -methyladenosylhomocysteamine to spermidine to form spermine. In both the latter reactions, methylthioadenosine and one proton are the other reaction products. For these four enzymes, a large body of experimental evidence points out the key role of enzyme ornithine decarboxylase (ODC) as the rate-limiting step in the biosynthetic pathway of the polyamines. The scheme for the biosynthesis of putrescine, spermidine, and spermine in mammalian tissues is presented in Fig. 1. The closely related connections between the biosynthetic pathway of polyamines

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OR

S - METHYLADENOSYL-HOMOCYSTEAMlflE OR

S -ADENOSYL - (5') - 3 PHZ N&C,C/N\ I I HC+N/c\NGH

SPERMIOINE CH (CHoH),C

L - 0 1 5'- MET HY LT H 10ADENOSINE + H+

SPERM INE

@ L-ORNITHINE CARBOXY-LYASE (ORNITHINE DECARBOXYLASE) (EC 4 1 1 17) @ ATP L-METHIDNINE -S-ADENOSYLTRANSFERASE(S-ADENOSYLMETHIONINE SYNTHETASE)(EC2 5 16)

0

@

@

S-AOENOSYL-L-METHIONINE CARBOXY-LYASE (S-ADENOSYL-L-METHIONINE DECARBOXYLASE)(EC41150) S-METHYLADENOSYLHOMOYSTEAMINE PUTRESCINE AMINOPROPYLTRANSFERASE (SPERMIDINE SYNTHASE) (EC 2 5 1 16) S-METHYLAOENOSYLHOMOCYSTEAMINE SPERMlOlNE AMINOPROPYLTRANSFERASE (SPERMINE SYNTHASE )

FIG.1. Biosynthetic pathway of the chief polyamines in mammalian tissues.

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and some other major pathways of intermediary metabolism in mammalian cells are shown in Fig. 2. A second distinct pathway of putrescine biosynthesis is present in bacteria; in this pathway, agmatine is formed from arginine by arginine decarboxylase and, in turn, is hydrolyzed to putrescine and PROLINE

\

A’-PVRROLINE - 5CARBOXVL AT E

ASPARTATE

-succp

CITRUL(INE

CARBAMVL - PHOSPHATE GLUTAMIC GLUTAMIC A C I D +~-SEMIALDEHVDE

/

7L-oRNITHINE

CYSTATHIONINE +CVSTEINE

t

PUTRESCINE

HOMOCVSTEINE

L-METHIONINE

.

+

S-ADENOSVLL -METHIONINE

DECAREOXVLATED ADENOSVLMETHIONINE

7

ARGININO-

FUMARAT E

POLYAMINES I N MAMMALIAN TUMORS

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urea by agmatine ureohydrolase (Bachrach, 1973; C. W. Tabor and Tabor, 1976). 1. Ornithine Decarboxylase Mammalian ODC is a pyridoxal phosphate-requiring enzyme, like most of the decarboxylases, and shows a specific requirement for thiols, with certain dithiols, such as dithiothreitol, being especially effective. In the absence of thiol compounds, the enzyme appears to polymerize and loses enzymic activity. The enzyme is specific for L-omithine, with an apparent K, value for its substrate between 0.1 and 0.2 mM. D-Omithine is not attacked. Mammalian ODC from several rat tissues has been extensively purified, and its principal molecular properties have been characterized. The basal ODC activity of most resting adult mammalian tissues is extremely low, with the notable exception of the prostate. Most ODC activity is localized in the soluble cytosol, only a little being detectable in some cellular organelles and in the nucleus (Pegg and Williams-Ashman, 1968a; Ono et al., 1972; Richman et al., 1975; Eloranta et al., 1976a; Murphy and Brosnan, 1976; McCormick, 1977, 1978a; Ferioli et al., 1980). ODC displays a well-documented circadian rhythm in many organs of the rat so far investigated, thus providing a good model for studies of enzymic chronobiology (Hayashi et al., 1972; Stone et al., 1974; Walker and Potter, 1974; Nicholson et al., 1976; Fujimoto et al., 1978; Noguchi et al., 1979; Scalabrino et al., 1979a; Yarygin et al., 1979). In the rat, the fine regulation of the circadian rhythm seems to reside, at least in part, in the pineal gland (Scalabrino et al., 1979a; Yarygin et al., 1979). Moreover, the circadian rhythm and the activity of ODC can be influenced by external cyclical habits and environmental conditions, such as the alternation of food-starvation and/or light-dark periods, and by diet composition (Hopkins et al., 1973; McAnulty and Williams, 1975, 1977; Yanagi et al., 1975; Maudsley et al., 1976; Farwell et al., 1977; Morrison and Goldsmith, 1978; Rozovski et al., 1978). Two remarkable attributes of mammalian ODC must be mentioned here. 1. In most adult organs so far investigated, the ODC activity can be modulated simultaneously in vivo by several hormones, which are organ specific. When a hormone, whatever its chemical nature, has an anabolic and/or differentiating effect on its target organ, the increase in ODC activity is an early event in the cellular response to the hormone. Very frequently, similar enhancement of the ODC activity also occurs in several mammalian tissues in response to other nonhormonal agents

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that can stimulate cell growth and/or induce cell differentiation. All these experimental facts imply a striking capacity of ODC to be induced. Such an increase in the levels of ODC activity could also be part of the coordinated growth response of a cell, i.e., of the so-called pleiotypic program (Hershko et al., 1971). The inducibility of ODC activity by hormonal and nonhormonal effectors has also been confirmed in isolated organs, as well as in cultured or isolated cells (Cohen et al., 1970; Aisbitt and Barry, 1973; Mallette and Exton, 1973; Hogan et al., 1974a; Antony et al., 1976; Haselbacher and Humbel, 1976; Lumeng, 1976, 1979; Nissley et al., 1976; Oka and Perry, 1976; Friedman et al., 1977b; Jefferson and Pegg, 1977; Jefferson et al., 1977; Osterman and Hammond, 1977, 1978, 1979; Scheinman and Burrow, 1977; Scheinman et al., 1977; Spaulding, 1977; D’Amore et al., 1978; Levine et al., 1978; Ostermanet at., 1978; Rupniak and Paul, 1978; Sakaiet al., 1978; Smith and Stange, 1978; Yanget al., 1978; Piik et al., 1979; Takigawa et al., 1979, 1980; Veldhuis and Hammond, 1979; Veldhuis et al., 1979, 1980; Kapyaho, 1980; Klingensmith et al., 1980; Lin et al., 1980; Parker and Vernadakis, 1980). Interestingly enough, it has been demonstrated using enucleated cells that ODC activity can be induced in the cytoplasts but not in karyoplasts and, more important, that the kinetics of ODC induction in the cytoplasts are similar in time course to those in the whole cell (Clark and Greenspan, 1979). On the contrary, when a hormone is antianabolic, a rapid decrease in ODC activity in the target tissue has been reported, as noticeably occurs in the lymphatic part of rat thymus when glucocorticoids are given (Richards, 1978; Scalabrino et al., 197913). In the majority of biological systems so far tested, the induction of ODC activity appears to be the result of the synthesis of new enzymic proteins and not of the activation of preexisting enzyme molecules, since it is prevented or at least greatly reduced by concomitant use of some inhibitors of protein and/or RNA syntheses and since it is simultaneous with an increase in the amount of the immunoreactive enzyme protein (Holtta, 1975; Canellakis and Theoharides, 1976; Scheinman et ul., 1977). However, several studies carried out in experimental systems under growing and nongrowing conditions, both in vivo and in vitro, have suggested the existence of another path of ODC induction, in which no new mRNA synthesis is required. The induction of ODC activity in these instances is completely or partially actinomycin D resistant; the ODC activity can even be stimulated by the drug (Russell and Snyder, 1969; Fausto, 1971; Kay and Cooke, 1971; Beck et al., 1972; Kay et al., 1972; Kay and Lindsay, 1973b; Clark, 1974; Hogan et al., 1974a,b; Byus and Russell, 1975; Oka and Perry, 1976;

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Yamasaki and Ichihara, 1976; Chen and Canellakis, 1977; Costa, 1978b; Grillo et al., 1978b; Lau and Slotkin, 1979; Inderlied et al., 1980; Lin et al., 1980; Perry and Oka, 1980). Therefore, ODC biosynthesis seems to be able to happen either contingently or not contingently on prior production of new ODC mRNA molecules and to be separately regulated at both transcriptional and translational levels (Heby and Emanuelsson, 1979). The underlying mechanisms by which a wide variety of factors stimulate mammalian ODC activity are still unclear. Some experiments with biological systems under growing conditions both in vivo and in vitro have been claimed to show that increased intracellular levels of CAMP are necessarily involved in this process. In turn, cAMP activates intracellular protein kinase. In more detail, ODC induction can occur if protein kinase type I alone is stimulated; a subsequent activation of protein kinase type I1 does not seem to be necessary. After the activation of protein kinase, the synthesis of new mRNAs follows, and, on translation of the newly synthesized ODC mRNAs, there is an increase in ODC activity (Byus and Russell, 1974, 1975, 1976a,b; Hogan et al., 1974a; Mizoguchi et al., 1975; Reddy and Villee, 1975; Russell and Stambrook, 1975; Short et al., 1975; Zusman and Burrow, 1975; Canellakis and Theoharides, 1976; Costa et al., 1976; Oka and Perry, 1976; Russell et al., 1976a; Yamasaki and Ichihara, 1976; Byus et al., 1977, 1978a,b; Jungmann and Russell, 1977; Scheinman and Burrow, 1977; Costa, 1978a; Costa and Nye, 1978; Hochman et al., 1978; Insel and Fenno, 1978; Levine et al., 1978; Manen et al., 1978; Osterman and Hammond, 1978; Osterman et al., 1978; Rupniak and Paul, 1978; Klimpel et al., 1979; Rosenfeld and Barrieux, 1979; Russell and Haddox, 1979; Combest and Russell, 1980; Costa et al., 1980; Hibasami et al., 1980c; Madhubala and Reddy, 1980; Nichols and Prosser, 1980). However, there is no compelling evidence to indicate that involvement of cAMP is mandatory in the process of ODC induction, since in a large body of experimental results there was no relationship between the enhancement in ODC levels and the changes in cyclic AMP levels within the cell (Mangan et al., 1973; Thrower and Ord, 1974; Eloranta and Raina, 1975; Richman et al., 1975; Oka and Perry, 1976; Scalabrino and Ferioli, 1976; Collawn and Baggett, 1977; Jefferson and Pegg, 1977; Johnson and Sashida, 1977; Mufson et al., 1977; Chadwick et al., 1978; Insel and Fenno, 1978; Lau and Slotkin, 1979; Marks et al., 1979; Piik et al., 1979; Veldhuis and Hammond, 1979; Veldhuis et al., 1979; Combest and Russell, 1980; Gpyaho, 1980; Klingensmith et al., 1980). The rise in cAMP level and the increased ODC activity within the cell could be merely parallel manifes-

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tations of the interaction of the hormone with the cell. Therefore, although CAMP may increase ODC activity in some experimental systems, generalizations regarding the mandatory intracellular regulation of ODC activity by this cyclic nucleotide appear to be premature and not sufficiently grounded, until several additional normal and pathological tissues in many different growth states have been studied. The control of ODC activity by different hormones in different mammalian tissues has been extensively reviewed (Morris and Fillingame, 1974; Raina and J h n e , 1975; C. W. Tabor and Tabor, 1976; Janneet al., 1978). 2. Generally speaking, ODC has a fast rate of turnover in mammalian cells, as demonstrated by measuring its tile after inducing it and then measuring the rate of loss of its activity after inhibiting protein synthesis. In many mammalian tissues, the apparent half-life of ODC, determined after using different procedures for inducing the enzyme, is 10-30 min which is one of the shortest molecular turnover times among mammalian enzymes (Bachrach, 1973; Schimke, 1973; Janne et al., 1976; C. W. Tabor and Tabor, 1976). Interestingly, using rat liver ODC antiserum and an immunotitration method, Obenrader and Prouty (197%) have demonstrated that in rat liver the tllz for antigen is longer than that for enzyme activity loss by approximately 9 min. This result is in keeping with the hypothesis that the ODC molecule is inactivated prior to being degraded. However, a large number of studies both in uitro and in uiuo have demonstrated that the half-life of ODC may not be an absolute and constant value, valid for every tissue and every growth rate condition. In fact, in different organs of the rat or using a variety of mammalian cell cultures (normal or neoplastic), isolated cells, perfused organs (e.g., rat liver), lower eukaryotes, and even enucleated cells, a rather wide range of half-lives for ODC has been reported, making it evident that the half-life of ODC is influenced by a number of biological parameters, such as the growth conditions, the composition of the growth media, the growth rate of the cells, and even the type of inhibitor of protein synthesis used (Russell and Snyder, 1969; Russsll et al., 1970; Hannonen et at., 1972; Kay et al., 1972; Melvin et al., 1972; Aisbitt and Barry, 1973; Kay and Lindsay, 1973a; Mitchell and Rusch, 1973; Clark, 1974; Hogan and Murden, 1974; Hogan et al., 197413; Janne and Holtta, 1974; Lembach, 1974; O’Brien et al., 1975a; Bachrach, 1976a; Heller et al., 1976b; Prouty, 1976; Yamasaki and Ichihara, 1976; Chen and Canellakis, 1977; Clark and Greenspan, 1977,1979; Conroy et al., 1977; Jefferson and Pegg, 1977; Kallio et al., 1977a; Obenrader and Prouty, 1977b; Canellakis et al., 1978; McCormick, 1978a; Minaga et al., 1978; Poso et al., 1978; Pegg and McGill, 1979; Veldhuis et al., 1979; Lau and Slotkin, 1980). Gen-

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erally speaking, one can conclude that there is good evidence that in cell cultures the half-life of ODC is a variable function, whereas in in vivo systems the tllzfor the enzyme is relatively more constant. There is evidence that ODC exists in different molecular forms, since multiple forms of ODC have been separated from regenerating or hepatocarcinogen-treated rat livers, from different kinds of growing cell cultures, from lower eukaryotes, and from bacteria. In exponentially growing mouse fibroblasts, two forms of ODC have been found that differ with respect to pyridoxal5’-phosphate affinity but that are without appreciable difference in their K, for ornithine and that have similar activity half-lives (Clark and Fuller, 1976a; Fuller et al., 1978). Two separable forms of ODC with different K, values for ornithine but identical antigenic properties and molecular sizes have been isolated from livers of rats injected with thioacetamide and from regenerating rat liver (Prouty and Obenrader, 1976; Obenrader and Prouty, 1977a). The increases observed in heart ODC activity of mature rats after T3or isoproterenol administration or the innately higher ODC of 2-day-old neonates always involve a form of the enzyme with an increased affinity for substrate (Lau and Slotkin, 1979). In addition, the developmental decline of heart ODC activity in the rat can be explained by the disappearance of the high-affinity kinetic form of the enzyme (Lau and Slotkin, 1980).In a primitive eukaryote (Physarurn polycephalurn) the ODC activity consists of two interconvertible forms, which differ in their K, for pyridoxal phosphate and their molecular size, though their K , for the substrate, ornithine, is unchanged. In this case these forms appear to be two distinct states, i.e., active and less active, of a common enzyme protein (Mitchell, 1974, 1975; Mitchell and Sedory, 1974; Mitchell et al., 1976, 1978b; Mitchell and Carter, 1977; Sedory and Mitchell, 1977; Mitchell and Kottas, 1979). Escherichia coli also contains two ODCs: a “biosynthetic” enzyme found during normal exponential growth and a “biodegradative” one induced by growth at low pH in culture media enriched with amino acids. They are distinct but very similar proteins, with similar molecular weight and similar kinetic properties, but the antibody to the purified biodegradative ODC does not cross-react with the biosynthetic enzyme (Morris and Pardee, 1965,1966; Applebaum et al., 1975,1977). Moreover, the K, values for ornithine have been demonstrated to be significantly different in uninfected HeLa cells than in vaccinia virus-infected cells, being higher in the latter than in the former (Hodgson and Williamson, 1975). Besides the above-reported existence of multiple ODC forms in a given tissue or cell, there is also the possibility that ODC from different tissues in the same animal species can display different enzymic prop-

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erties. Comparative kinetic studies of ODC activity in brain and liver of the rat indicate that the brain ODC has a higher affinity for the substrate than the liver ODC (Butler and Schanberg, 1976). The exact relationship between the various forms of ODC in different tissues has not yet been completely elucidated. It is not yet known whether multiple enzyme forms represent different transcriptional products (i.e., isozymes) or posttranslational modifications (e.g., proteolytic cleavage). Nevertheless, however these questions are answered in the future, it seems to us important to point out the following: (1) it is not by chance that these multiple forms of ODC have been clearly identified and characterized in tissues and cells which were stimulated to growth by various stimuli or, at least, in which the ODC activity was increased or decreased by suitable variations of the media’s osmolarity; (2) thus, in 3T3 fibroblasts stimulated to growth, in hepatic hyperplasia either surgically or chemically induced, in Physarum polycephalum after osmotic shock, and in vaccinia virusinfected HeLa cells, the change in the growth state and the subsequent increase in ODC activity are generally accompanied by a predominant presence of those forms of ODC which show higher affinity for the substrate and/or for the coenzyme; (3) consequently, the possibility that the K, of the ODCs for the coenzyme and/or for the substrate might change represents, along with a concomitant change in the enzyme’s half-life, one of the key mechanisms available to the cell to fit ODC activity to the cell growth state and to environmental conditions. In fact, several lines of experimental evidence obtained in cells in culture suggest that frequently the increase in the half-life of a rapidly turning-over enzyme such as ODC represents an important, albeit not the only available, mechanism for the increase in cellular enzyme activity. However, conflicting and quite opposite results have been reported in this regard (Clark, 1974). Furthermore, it is important that in normal rat liver the ODC activity declined exponentially after treatment with cycloheximide, with a half-life rather similar to that observed in regenerating liver, in which there is intense stimulation of the enzyme activity in response to partial hepatectomy (Russell and Snyder, 1969). In conclusion, for the biological significance of the multiple forms of ODC, as for the half-life of ODC and for mediation by CAMP of ODC induction, no general rule can yet be drawn. Specific ODC antibodies have been prepared in different laboratories with purified enzyme preparations (Friedman et al., 1972; Holtta, 1975; Canellakis and Theoharides, 1976; Theoharides and Canellakis, 1976; Kallio et al., 1977c; Piik et al., 1977; Scheinman et al., 1977;

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Kallio, 1978; Poso et al., 1978). By immunological techniques it was possible to ascertain that the rise in ODC activity in some organs, such as liver or thyroid, of the rat after in vivo or in vitro induction of the enzyme by hormones (GH or TSH) is accompanied by increased amounts of immunoprecipitable protein, which indicates de novo synthesis of the enzyme protein (Holtta, 1975; Scheinman et al., 1977). This result has been confirmed in cultured neoplastic cells (Canellakis and Theoharides, 1976). Conversely, the decrease in ODC activity in the regenerating liver or the ventral prostate of the rat after in vivo injections of inhibitors (such as putrescine, 1,3-diaminopropane, and cycloheximide) were usually, if not always, associated with a similar and parallel decay in the amount of immunoreactive protein, as revealed by immunotitration of the enzyme (Kallio et al., 1977c; Piik et al., 1977; Kallio, 1978; Poso et aZ., 1978). The different preparations of antiserum to rat liver ODC showed no cross-reactivity with ODC derived from E. coli (Theoharides and Canellakis, 1976). Finally, the induced ODC was identical with the noninduced one, as demonstrated by titration with an antibody monospecific for this enzyme and by heat stability (Canellakis and Theoharides, 1976). ODC activity itself has been suggested to be a regulatory factor for RNA polymerase I activity and is even considered to be a trigger for the stimulation of RNA polymerases (Manen and Russell, 1975a,b, 1976, 1977a,b; Russell et al., 1976a; Manen et al., 1977; Haddox and Russell, 1980). However, several results with different biological systems have indicated that this is not always so (Spaulding, 1977; Ferioli et al., 1980). Particularly, it should be mentioned that in postischemic rat liver the activation of ODC can be abolished without impairing the activation of RNA synthesis that occurs in this experimental system (Ferioli et aZ., 1980). Therefore, one can conclude that, if there is a link between ODC and RNA metabolism, it is by no means a necessary one. Without any doubt, the regulation of ODC activity in eukaryotic cells represents at the present one of the most intriguing questions in the control of polyamine biosynthesis. This problem is also of paramount importance because ODC catalyzes the rate-limiting step in the polyamine biosynthetic pathway. Unlike ODC from E. coli, in which this enzyme appears to be largely under the regulation of certain guanine nucleotides (Holtta et al., 1972, 1974; Sakai and Cohen, 1976a,b), mammalian ODC is controlled not by low-molecular-weight effectors (Pegg and Williams-Ashman, 1968a; Janne and WilliamsAshman, 1971b; Friedman et al., 1972; Clark, 1974) but by several distinctly different mechanisms. Mammalian ODC does not exhibit

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allosteric behavior, since it is not at all directly modulated by small biomolecules, with the exception of thiols. Experiments carried out in the assay systems in vitro and aimed at demonstrating direct effects of the product of the ODC reaction (i.e., putrescine) and of the subsequent polyamines (spermidine and spermine) on the decarboxylase have shown that these molecules are only very weak direct competitive inhibitors of catalytic activity of ODC (Pegg and WilliamsAshman, 1968a; Raina and Janne, 1968; Morris et al., 1970; Janne and Williams-Ashman, 1971b; Ono et al., 1972; Clark, 1974; Morley and Ho, 1976; Pegg and McGill, 1979). By contrast, using in vivo and in vitro biological living systems (i.e., intact animals, cultured cells, and even cytoplasts) exogenous polyamines, particularly putrescine, are very effective in depressing ODC activity and in preventing the rise in ODC activity caused by several means. It has been observed, as well, that administration of certain other diamines not normally present in living organisms decreases ODC activity in various rat tissues in vivo and in cultured cells (Schrock et al., 1970; Kay and Lindsay, 1973b; Janne and Holtta, 1974; Clark and Fuller, 1975, 1976b; Canellakis and Theoharides, 1976; Fong et al., 1976; Heller et al., 1976b, 1977b, 1978; Poso and Janne, 1976a,b; Friedman et al., 1977a; Guha and Janne, 1977; Jefferson and Pegg, 1977; Kallio et al., 1977a-d; McCann et al., 1977; Piik et al., 1977; Poso, 1977; Poso et al., 1977; Kallio, 1978; Mitchell et al., 1978a; Pegg et al., 1978; Bethel1 and Pegg, 1979a; Clark and Greenspan, 1979; Grillo et al., 1980; Klingensmith et al., 1980; Stoscheck et al., 1980). The mechanisms by which exogenous polyamines and synthetic diamines suppress the expression of ODC activity (i.e., the so-called amine repression of ODC) are not well understood. In summary, for such a regulation of ODC activity, two main different, though not conflicting, explanations have been proposed. Evidence has been presented that this inhibitory effect on ODC activity of di- and polyamines may be mediated through a decline in the synthesis of the enzyme at some posttranscriptional steps. The rapidity of the amine action, which is comparable with that of cycloheximide and other inhibitors of eukaryotic protein synthesis, is consistent with this hypothesis (Janne and Holtta, 1974; Clark and Fuller, 1975; Kallio et al., 1977a,c).Furthermore, some evidence supports the idea that the action of the diamines might involve transcriptional control elements of gene expression (Janne and Holtta, 1974; Kallio et al., 1977a). Thus, the inhibitory action of putrescine on ODC activity is not a simple direct feedback inhibition. In any case, this decrease in ODC activity by

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putrescine appears to be due to the diamine per se rather than to a subsequent polyamine, i.e., spermidine or spermine (Pegg et al., 1978). In fact, in vivo treatment of rats with MGBG, which prevents spermidine synthesis by an inhibition of SAMD activity, did not prevent the putrescine-induced decrease in rat liver ODC activity (Pegg et al., 1978). Further, some nonphysiological diamines, structural analogs of putrescine that are poor substrates for the propylamine transferase reaction in which spermidine is formed and that are thus converted into polyamine analogs to a very limited extent, were also able to decrease ODC activity in vivo (Pegget al., 1978). However, the picture is further complicated by the finding that the reduction in ODC activity of the primitive eukaryote Physarum polycephalum brought about by putrescine, spermidine, or spermine is due not to a decrease in enzyme molecules but rather to the rapid conversion of the active enzyme to a stable, catalytically less active form (Mitchell et al., 1978a). An alternative, fundamentally different explanation of the inhibitory effects of di- and polyamines on ODC activity in vivo has been recently put forward by Canellakis and his co-workers. These authors found that the addition of putrescine to cell cultures or the injection of partially hepatectomized rats with putrescine swiftly induced in the cell or in the liver the formation of a nondiffusible and noncompetitive inhibitor of ODC activity, which they called “ODC antizyme” (Fong et al., 1976; Heller et al., 1976a,b, 1977a,b, 1978; Heller and Canellakis, 1980).The production of this ODC antizyme has been confirmed in other biological systems and tissues by other authors (Friedman et al., 1977a; Jefferson and Pegg, 1977; Kallio et al., 1977c, 1979; McCann et al., 1977; Minaga et al., 1978; Pegg et al., 1978; Grillo et al., 1980; Klingensmith et al., 1980; Stoscheck et al., 1980; Weekes et al., 1980), with very few negative reports (Clark and Fuller, 1976b; Mitchell and Carter, 1977; Piik et al., 1977; Mitchell et al., 1978a; Pegg et al., 1978; Weekes et al., 1980). Certainly, the discovery of an ODC antizyme is a very important addition to the understanding of molecular regulation of the biosynthesis of polyamines. The antizyme appears to be a protein, since it is sensitive to proteinase but not to nucleases, and its induction by putrescine is inhibited by cycloheximide but not by actinomycin D, indicating that synthesis of protein is occurring on stable mRNA templates. The mechanism of action of this macromolecular inhibitor seems to involve reversible binding of the inhibitor to ODC, resulting in a loss of catalytic activity of the enzyme and disappearance of the free inhibitor. Interestingly, eukaryote ODC activity is also inhibited by a negative effector of E.

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coli ODC, which, although it has been isolated from this bacterial strain, shows characteristics similar to those of the ODC antizyme present in eukaryotic cells (Kyriakidis et al., 1978). A positive effector of ODC activity has also been isolated in E . coli (Kyriakidis et al., 1978). Finally, it is worthwhile to mention that in mammalian cells the ODC antizyme is a normal component, present at least in the livers of rats untreated with putrescine or other diamines (Heller et al., 1977b). In uninduced rat liver cells the largest part of the ODC antizyme activity is localized in the nuclei (in the nucleolus as well as in the nucleoplasm), the remaining activity being found in some subcellular particles, i.e., in the smooth endoplasmic reticulum and in the rough endoplasmic reticulum (Heller et al., 197713). Regardless of the mechanisms that may be involved in regulating the ODC activity in eukaryotic cells by physiological polyamines, putrescine certainly has a peculiar and prominent role in comparison with spermidine and spermine in such a regulation. In fact, putrescine may regulate the activities of both ODC and SAMD in eukaryotes, since this natural diamine sharply depresses ODC activity, on the one hand, and, on the other hand, markedly stimulates SAMD (see Section I,B,2). Surprisingly, it has been observed that, in general, the ODC of most cells is sensitive to external polyamine concentrations several orders of magnitude lower than that found internally. This greater sensitivity of mammalian ODC to exogenous polyamines, despite the presence of much higher endogenous levels of polyamines, hints at the possibility that the mechanism controlling ODC activity may be regulated by a polyamine-sensitive site on the external cell membrane (Canellakis et al., 1978). Although there is, as yet, no conclusive evidence that extracellular di- and polyamines can regulate ODC activity at such a site, particularly because these amines have been demonstrated to be taken up into the cell by an active-transport system (Pohjanpelto, 1973, 1976; Lajtha and Sershen, 1974; Pateman and Shaw, 1975; Kano and Oka, 1976), some experimental evidence exists for the presence of membrane-associated sites that affect ODC activity (Quash e t al., 1971, 1972, 1976, 1978; Richman et al., 1975; Chen et al., 1976a; Canellakis et al., 1978; Gibbs et al., 1980). In fact, agents known to affect the membrane via the cytoskeleton (such as colchicine, cytochalasin, and vinblastine) inhibit the induction of ODC in vivo and in cultured cells (Richman et al., 1975; Chen et al., 1976a), and, furthermore, putrescine has been proved to be associated with sites on the surface of different types of cells (Quash et al., 1971, 1972, 1976). The regulation of cellular ODC activity is not limited to the different

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intracellular control mechanisms, such as those discussed previously (i.e., RNA synthesis, antizyme induction, changes in the enzyme's half-life, changes in the molecular form of the enzyme, translational control exerted by polyamines). Extensive studies indicate that many environmental parameters, including the ion content of the media, can profoundly influence the levels of ODC activity in different kinds of tissues. More exactly, decreasing the osmolarity of the culture medium results in marked stimulation of ODC activity, whereas hyperosmotic culture medium prevents an increase in ODC activity inside the cells (Munro et al., 1975; Friedman et al., 197713; Mitchell and Kottas, 1979; Perry and Oka, 1980). Furthermore, high external levels of certain cations (Ca", Na+, K+, and M$+) inhibit ODC activity (Chen et al., 1976b; Otani et al., 1980), whereas high extracellular Ca2+levels are strictly required for both of the separate pathways of ODC induction, i.e., the cAMP dependent and the cAMP independent (D'Amore and Shepro, 1978; Gibbs et al., 1980). It seems to be of great interest to note (a) that there is a very close relationship between some inorganic cations and the organic polyamine polycations in regulating ODC activity and (b) that inorganic cations, with the notable exception of calcium, seem to act in controlling ODC activity like organic polyamine polycations, since changes in the pools of the cations cause inverse responses in the ODC activity. The reader can obtain further and more detailed information about the different mechanisms involved in the control of ODC activity in mammalian cells from the excellent reviews provided by Canellakis et al. (1979), Bachrach (1980), and McCann (1980). 2. S-Adenosyl-LMethionine Decarboxylase In most organisms, S-adenosyl-L-methionine decarboxylase (SAMD) (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) plays a pivotal role in polyamine biosynthesis by contributing S-methyladenosylhomocysteamine (i.e., decarboxylated S-adenosyl-L-methionine), which in turn donates its propylamine moiety to putrescine to give spermidine and then to spermidine to give spermine. Contrary to earlier reports (Pegg and Williams-Ashman, 1968b, 1969; Feldman et al., 1971, 1972; Manen and Russell, 1974), it is now well established that the decarboxylation of S-adenosyl-L-methionine and the propylamine transfer reaction are catalyzed by different enzymes (Coppoc et d., 1971; Janne and Williams-Ashman, 1971a; Janne et al., 1971a,b; Pegg, 1974; Sturman, 1976). Three types of SAMD activities with different in vitro biochemical features have

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been described: (1)prokaryotic enzymes, which are Mgz+dependent but putrescine insensitive (Wickner et al., 1970; Coppoc et al., 1971; Poso et al., 1976); (2) enzymes of lower eukaryotes, which are not influenced either by divalent cations or by putrescine (Mitchell and Rusch, 1973; Poso et al., 1975a,b, 1976); and (3) the enzymes from higher organisms and from yeasts, which are stimulated markedly by putrescine (also at physiological concentrations of this diamine) and, to a lesser extent, by spermidine and cadaverine in the assay system (Coppocet al., 1971; Janne and Williams-Ashman, 1971a; Janne et al., 1971a; Zappia et al., 1972; Poso et al., 1975a, 1976; Sakai et al., 1979; Wilson et al., 1979). The important effect of putrescine on the activity of mammalian SAMDs must be connected with the remarkable decrease caused by the diamine in the apparent K,,, value of the enzyme for S-adenosyl-L-methionine (Hannonen, 1975; Poso et al., 1975a; Oka et aZ., 1978b; Sakai et aZ., 1979). In the presence of a saturating concentration of the activator putrescine, SAMD has apparent K , values for adenosylmethionine ranging from 0.09 mM to 0.02 mM, depending on the tissue source of the enzyme (Coppoc et al., 1971; Hannonen, 1976; Porta et al., 1977; Oka et al., 197813). It is therefore likely that the activity of SAMD in vivo can be significantly modulated by changes in the amount of putrescine present inside of the cell, as consequences of corresponding changes in ODC activity. Amazingly enough, however, Sakai et al. (1980) recently demonstrated that the increases in the intracellular levels of putrescine, induced in cultured mouse mammary tissue by different means, resulted in a decrease of both the activity and the amount of SAMD in tissue. At present, this dissociation between the effects of putrescine on SAMD in the assay system and in cells remains to be explained. The absence of putrescine activation of prokaryotic and some lower eukaryotic SAMDs can conceivably be attributed to the near absence of spermine and its synthase in these species (Poso et al., 1976). Analogously, the stimulation by putrescine of eukaryotic SAMD has evolutionary significance, since this property presumably enables the cell to have adequate amounts of decarboxylated S-adenosyl-L-methionine for polyamine biosynthesis. Another unique characteristic of SAMD isolated from eukaryotic sources is the strong in vitro inhibition of the enzyme by S -methylhomocystearnine, i.e., by the product of the reaction (Yamanoha and Samejima, 1980).The presence of such an inhibition is another difference between SAMD from prokaryotic and SAMD from eukaryotic organisms (Raina and Janne, 1975).

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Like ODC, mammalian SAMD is an enzyme located in both the cytosol fraction (Schmidt and Cantoni, 1973; Sturman, 1976; Symonds and Brosnan, 1977; Wilson et al., 1979) and the nucleus of the cell (McCormick, 1977, 1978a). It has been purified from different organs of the rat and from baker’s yeast and its molecular weight determined. Like ODC, mammalian SAMD has a short biological half-life, which ranges between 20 and 120 min, depending on the tissue studied (Russell and Taylor, 1971; Hannonen et al., 1972; Russell and Potyraj, 1972; Fillingame and Morris, 1973; Kay and Lindsay, 1973a; Mitchell and Rusch, 1973; Pegg et al., 1973; Janne and Holtta, 1974; Pegg and Jefferson, 1974; O’Brien et al., 1975a; Eloranta et al., 1976a; Janne et al., 1976; Jefferson and Pegg, 1977; Grillo et al., 1978a; Poso et al., 1978; Pegg, 1979). We have no convincing evidence thus far for the existence of multiple forms of SAMD in mammalian cells. Although earlier works suggested that pyridoxal 5-phosphate is a cofactor for eukaryotic SAMD (Feldman et al., 1972; Sturman and Kremzner, 1974; Hannonen, 1975), later studies have demonstrated that, despite other differences, the putrescine-activated SAMD (e.g., that of yeasts and of rat liver) resembles the prokaryote SAMD (e.g., that of E. coli) in having a covalently bound pyruvate cofactor (Wickner et al., 1970; Cohn et al., 1977; Pegg, 1977a; Demetriou et al., 1978). Pertinent to these findings are several reports indicating that hepatic SAMD activity is not at all affected in rats fed on a diet deficient in vitamin Be (Eloranta et al., 197613; Hannonen, 1976; Pegg, 197%). The substrate specificity of SAMD from eukaryotic sources is quite rigorous, although some analogs of S-adenosyl-L-methionine containing selenium have been shown to be attacked by the enzyme (Pegg, 1969; Zappia et al., 1972). Like ODC, mammalian SAMD activity is influenced by the nutritional state (Eloranta and Raina, 1977) and shows circadian rhythm in several organs of the rat (Scalabrino et al., 1979a). The SAMD activity can increase in mammalian tissues (both in vivo and in cultured cells) in response to various stimuli for cell growth or differentiation, such as partial hepatectomy or some anabolic hormones, so that SAMD is also an inducible enzyme (Kaye et al., 1971; Russell and Lombardini, 1971; Russell and Taylor, 1971; Hannonen et al., 1972; Oka and Perry, 1974a; Eloranta and Raina, 1977; Feil et al., 1977; Sakai et al., 1977, 1978; Igarashi et al., 1978). However, the observed enhancement of SAMD levels after these stimuli is usually smaller than that observed in ODC activity under the same experi-

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mental conditions. Moreover, although ODC and SAMD activities are frequently enhanced in a coordinated way in the tissues in response to stimuli, in many tissues large increases in ODC activity are often not followed by proportionate rises in SAMD activity (Williams-Ashman et al., 1977; Williams-Ashman and Canellakis, 1979; Ferioli et al., 1980). Highly purified specific antibodies to SAMD from the livers of rat and mouse have been prepared (Pegg, 1979; Sakai et al., 1979),and by immunotitration it was shown that the increase in SAMD activity in the tissues during regenerative growth or after appropriate hormonal stimulation is due to an increase in the amount of enzyme protein (Pegg, 1979). It was also found that, after treatment of the rats with cycloheximide, the antigen disappeared, with a tllzslightly longer than that for the enzyme activity (Pegg, 1979). These data for SAMD are quite similar to those for ODC and may mean that, as in the case of ODC, the enzyme is first inactivated and then degraded. Convincing evidence in vivo or in cultured cells for the regulation of SAMD activity by di- and polyamines, comparable to the so-called amine repression of ODC activity, is for the moment lacking. In fact, either quite opposite or ambiguous findings have been reported about the effects of in vivo administration of putrescine or spermidine on SAMD levels in different organs of the rat. An in viuo inhibitory action of spermidine on the hepatic and muscular enzymes was found by some authors (Janne and Holtta, 1974; Hopkins and Manchester, 1980), and this inhibition was also confirmed in cultured lymphocytes (Kay and Lindsay, 1973b). However, in vivo stimulation by spermidine of hepatic SAMD in the chicken has been reported (Grillo et al., 1978a). Furthermore, putrescine was found to not affect or to scarcely stimulate SAMD levels in liver of rats or chickens when given intraperitoneally (Janne and Holtta, 1974; Grillo et al., 1978a) or when added to cultured lymphocytes (Kay and Lindsay, 1973b). The injection of the same diamine or of 173-diaminopropanebrought about a decrease in the activity of this decarboxylase in the seminal vesicle but not in the prostate of the rat (Piik et al., 1977). As recently demonstrated by Zappia and his co-workers, decarboxylated SAM is also the precursor of some of the newly identified polyamines, i.e., sym-norspermidine and sym-norspermine. Two molecules of this decarboxylated SAM are required for the biosynthesis of s ym-norspermidine and three molecules for the biosynthesis of symnorspermine (De Rosa et al., 1978; Zappia et al., 197913). The peculiarity of the biosynthetic pathway of these “new polyamines” lies in the fact that S-5’-deoxyadenosyl-(5’)-3-methylthiopropylamine, i.e., decarboxylated SAM , contains the entire carbon skeleton of the mole-

POLYAMINES I N MAMMALIAN TUMORS

179

cules of sym-norspermidine and sym-norspermine, which are also called caldine and thermine.

3. Spermidine Synthase This enzyme (S-methyladenosylhomocysteamine:putrescine aminopropyltransferase, EC 2.5.1.16) catalyzes the transfer of the propylamine moiety of decarboxylated SAM to putrescine to form spermidine, thiomethyladenosine, and one proton. When measured under optimal assay conditions in soluble extracts of most eukaryotic tissues examined so far, the activity of this synthase is noticeably higher than the activities of the two polyamine biosynthetic decarboxylases (Raina and Janne, 1975). Spermidine synthase from eukaryotic sources has not been fully purified and characterized. No cofactor requirement has been reported for this aminopropyltransferase. The K , value of spermidine synthase for synthetic decarboxylated SAM has been found to be below 4 p M (Samejima and Nakazawa, 1980), and a similar value was determined with naturally occurring decarboxylated SAM (Raina and Janne, 1975). Unlike the aminopropyltransferase of prokaryotes, for which not only putrescine but also cadaverine and spermidine can act as acceptors in the aminopropyl transfer reaction (Bowman et al., 1973), mammalian spermidine synthase can use putrescine and, to a slight degree, l74-diarnino-2-butene, but not spermidine or 1,3-diaminopropane7as acceptors of the aminopropyl group from decarboxylated SAM (Samejima and Nakazawa, 1980). Conflicting reports are available as to whether 1,Sdiaminopentane (cadaverine) can be a substrate for spermidine synthase from eukaryotic organisms (Kallio et aZ., 1977d; Hibasami and Pegg, 1978a; Samejima and Nakazawa, 1980). Therefore, we can conclude that the mammalian enzyme is quite specific for the formation of spermidine. Moreover, the mammalian spermidine synthase transfers only the aminopropyl group, not the aminoethyl or aminobutyl groups (Samejima and Nakazawa, 1980). Unlike the two polyamine bios ynthetic decarboxylases, spermidine synthase has a rather long apparent biological half-life (Haina and Janne, 1975; Oka et al., 1977). This enzyme is markedly inhibited in vitro by SAM, by its synthetic or natural analogs and derivatives, and by cadaverine (1,5-diaminopentane) (Hibasami and Pegg, 197813; Hibasami et al., 1980b). Spermidine synthase activity can be increased by hormones in an inducible manner (Oka et al., 1977; Kapyaho et al., 1980). Interestingly, in some prokaryotes that have no detectable SAMD

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GIUSEPPE SCALABRINO AND MARIA E. FERIOLI

activity, a novel pathway of spermidine synthesis has been described, in which spermine synthase is absent (Tait, 1976). According to the proposed scheme, L-aspartic-P-semialdehyde condenses with putrescine to yield a Schiff base, which is then reduced to carboxyspermidine by an NADPH-dependent step. Carboxyspermidine, in turn, undergoes a pyridoxal 5-phosphate-dependent enzymatic decarboxylation to give rise to spermidine. Of further relevance to this is the recent demonstration of the coexistence of the two pathways, i.e., the classical (which includes SAMD and spermidine synthase) and the new one (in which P-aspartylsemialdehyde is precursor), for spermidine biosynthesis in Lathyrus sativus seedlings (Srivenugopal and Adiga, 1980). 4. Spemnine Synthase

This enzyme (S-methyladenosylhomocysteamine:spermidine aminopropyltransferase) catalyzes the synthesis of spermine, using decarboxylated SAM as the donor of the propylamine group and spermidine as the acceptor. Other products of the reaction are 5-methylthioadenosine and one proton. This enzyme has been highly purified and characterized and shows a high affinity for S -methyladenosylhomocysteamine,with a K, value of about 0.6 p M (Pajula et al., 1978, 1979; Pajula and Raina, 1979). Purified spermine synthase is inhibited by putrescine and even more by one of the reaction products, methylthioadenosine (Pajula and Raina, 1979; Hibasami et aZ., 1980b). Putrescine was found to be a competitive inhibitor of spermine synthesis (Pegg and WilliamsAshman, 1970; Hannonen et al., 1972). SAM is also a strong in vitro inhibitor of the activity of the enzyme, which is much more depressed by this compound than is spermidine synthase (Hibasami et al., 1980a,b). Another physiological in vitro inhibitor of spermine synthase is 175-diaminopentane (Hibasami and Pegg, 1978b). No cofactor or coenzyme appears to be needed for the synthesis of spermine by spermine synthase. This enzyme is also inducible by hormones (Gpyaho et al., 1980). However, we do not know at present whether the in vitro inhibition of the aminopropyltransferases by SAM, decarboxylated SAM, cadaverine, or 5’-methylthioadenosine also occurs in vivo, because the concentrations of SAM in the tissues are low (Eloranta et al., 1976b; Eloranta, 1977, 1979; Eloranta and Raina, 1977; Hoffman et aZ., 1979) and those of decarboxylated SAM (Hibasami et al., 1980d) and cadaverine are very low. Very few measurements of cellular 5’-methylthioadenosine content have been made (Rhodes and WilliamsAshman, 1964; Seidenfeld et aZ., 1980).

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181

C. S -ADENOSYL-L-METHIONINE S-Adenosyl-L-methionine (SAM) has become increasingly important in cell biochemistry. In fact, SAM links two biochemically important processes: biological methylations and polyamine biosynthesis. SAM is employed as a methyl donor in some, but by no means all, enzymic methyltransferase reactions. Additionally, t w o recently found roles for SAM appear to be of particular importance: (1) SAM is required for both bacterial chemotaxis and the chemotaxis of human monocytes (Aswad and Koshland, 1974,1975; Springer and Koshland, 1977; Pike et al., 1978); these findings support a relationship between SAMmediated transmethylation reactions and immune and inflammatory functions; (2) SAM is required, with ATP and magnesium ions, by the class I type of bacterial restriction enzymes (Linn et al., 1977; Cat0 and Burdon, 1979; Smith, 1979). SAM is also of paramount importance in the normal and pathological biochemistry of the mammalian central nervous system [e.g., it is involved in the methylation of some neurotransmitters (Wurtman, 1979), and its abnormal metabolism was proposed to be connected with some psychotic disorders (Baldessarini et al., 1979)] and in preventing, or at least reducing, the severe cell hepatic toxic injury induced by several drugs and molecules (Stramentinoli et al., 1978b, 1979). Just as for the three major polyamines, an active transport system with high affinity for SAM has been described in mammalian cells (Mizoguchi et al., 1972; Pezzoli et al., 1978; Stramentinoli et al., 1978a; Zappia et al., 1978a). Finally, at least one rate-limiting factor of SAM synthesis in mammalian tissues seems to be the tissue concentration of methionine available to the cell (Eloranta, 1977, 1979). The pathways of SAM metabolism and catabolism and the interconnections between transmethylation and transsulfuration reactions, on the one hand, and polyamine synthesis, on the other hand, are presented in Fig. 3. For further details about the different roles played by SAM in the biochemistry of eukaryotic and prokaryotic cells, we refer the reader to two books on these topics (Salvatore et al., 1977; Zappia et al., 1979a).

D.

5’-METHYLTHIOADENOSINE

It is noteworthy that concomitant with the reactions of both spermidine and spermine synthases there is stoichiometric production of 5‘-methylthioadenosine (5’-MTA). In microorganisms this nucleoside is synthesized through several different pathways from the common

182

GIUSEPPE SCALABFUNO AND MARIA E. FEFUOLI

L - METHIONINE METHYL1 HIOADENOSINE

P; + PP;

PUTRESCINE S-METHYLADENOSVL HOMOCYSTEAMINE

A-ACCEPTORS

0 S-ADENOSYL-1- HOMOCYSTEINE ~

@

A T P c AMP+

II

IMP+

ADENOSINE

L - HOMOCYSTE INE

INOSINE

L - CYSTATHI0 NINE

HVPOXANTHINE

@

\I/

L-HOMOSEAINE + L-CYS ElNE

$~o+zo~

WJ2

CoA

URIC ACID

TAURINE

-TL-cvsT ZH

GLUTAT HI ONE

0 METHIONINE ADENOSYLTRANSFERASE (EC 2 5 1 6 ) @ S-ADENOSYL-L-METHIONINE DECARBOXYLASE (EC 4 1 1 5 0 ) 0 METHYLTRANSFERASES @

S-ADENOSYL-L-HOMOCYSTEINE HYDROLASE (EC 3 3 1 1 )

@

ADENOSINE DEAMINASE ( E C 3 5 4 17)

@

INOSINE -GUANOSINE PHOSPHORYLASE ( E C 2 4 2 1 )

@ @ @ @ @ @ @

XANTHINE

OXIDASE (EC 1 2 3 2 )

ADENOSINE KINASE

(EC 2 7 1 2 0 )

HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE (EC 2 4 2 8 ) N5-METHYL FH4 HOMOCYSTEINE METHYLTRANSFERASE BETAINE

L-HOMOCYSTEINE-S-METHYLTRANSFERASE (EC 2 1 1 5)

CYSTATHIONINE

0-SYNTHASE

(EC 4 2 1 2 2 )

CYSTATHIONINE T-LYASE (CYSTATHIONASE) (EC 4 4 1 1)

FIG. 3. Pathways of S-adenosyl-L-methionine synthesis and catabolism in mammalian tissues. Interconnections between the transmethylation and transsulfuration reactions are also shown.

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POLYAMINES IN MAMMALIAN TUMORS

precursor, SAM. Only two of these biosynthetic routes are operative in mammalian tissues. One reaction that leads to the formation of this thioether is the direct cleavage of SAM by a specific SAM lyase (or SAM-splitting enzyme) to form 5’-MTA and a-amino-y-butyrolactone. The second route is the transfer of a propylamine moiety from decarboxylated SAM to putrescine or spermidine to yield spermidine or spermine, respectively; this one is quantitatively more important. By this reaction, one molecule of 5’-MTA is produced per molecule of spermidine formed, and 2 molecules of 5’-MTAare produced per molecule of spermine formed (Ferro et d., 1976). Larger amounts of 5’-MTA are formed in thermophilic bacteria during the biosynthesis of some newly identified polyamines (e.g., sym-norspermidine and sym-norspermine), since two molecules of 5’-MTA are produced per molecule of sym-norspermidine and three molecules of 5’-MTA per molecule of sym-norspermine (De Rosa et al., 1978; Carteni-Farina e t al., 1979; Zappia et al., 1980). The pathways for 5’-MTA biosynthesis in mammalian cells are presented in Fig. 4. It has long been customary to regard 5’-MTA as a waste product of the last steps of the polyamine biosynthetic pathway. However, several more recent lines of experimental evidence have attributed important biological roles to 5’-MTA. In fact, 5’-MTA has been found to inhibit the proliferation of human lymphocytes stimulated to multiply by mitogens (Vandenbark et al., 1980) and to inhibit the incorporation SPERMIDINE

+

S’-METHYLTHIOADENOSINE]

PUTRESCINE

SPERMlDlNE

coq

DECARB~XYLATED

S - ADENOSY L-L-MET HlONlNE

,A SpERMlNE +

I5’-METHYLTHIDADENOSlNE

]

S -ADENOSYL - L‘- METHIDNINE

HOMOSEAINE LACTONE

I 5’-METHYLTHIOADENOSINE 0 S-ADENOSYL-L-METHIONINE-SPLITTING FIG.

ENZYME

4. Routes of biosynthesis of 5’-methylthioadenosine in mammalian cells.

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GIUSEPPE SCALABRINO AND MARIA E. FERIOLI

of labeled uridine into RNA in explanted salivary glands from Drosophtla melanogaster (Law et al., 1976). On the other hand, 5’-MTA or some metabolite of it has been demonstrated to be required for the growth of certain cell lines in culture (Toohey, 1977, 1978). Moreover, the ubiquitous distribution and the high activity of the enzyme 5’-MTA phosphorylase (EC 2.4.2.1), which degrades this compound to 5’-methylthioribose-l-phosphate and adenine in mammalian tissues (Cacciapuoti et al., 1978; Garbers, 1978; Zappia et al., 1978c, 1980; Ferro et al., 1979), accounts for the low intracellular levels of 5’-MTA in mammalian cells. Any accumulation of 5’-MTA would probably be deleterious to cells not only because of the adverse effect of this nucleoside on transmethylation but also because it would deplete the adenine nucleotide pool. However, although the metabolic fate of 5’-methylthioribose-l-phosphate, produced by enzymic cleavage of 5’-MTA, remains largely unknown, the adenine produced by the same metabolic reaction is available to reenter the pool of adenine nucleotides. Although 5‘-MTA is a strong in uitro inhibitor of spermine synthase (Pajula and Raina, 1979), the real inhibition of polyamine production displayed in uiuo by 5’-MTA may be only of little importance because of the rapid degradation of this nucleoside. Williams-Ashman et al. (1973, 1977), Williams-Ashman and Canellakis (1979), and Hibasami et al. (1980b) have suggested that the polyamine biosynthetic pathway may have as its major function the production of 5’-MTA rather than that of polyamines. However, partially clarifying this question, quite stimulating observations have been provided by Nicolette et al. (1980) and Seidenfeld et al. (1980). These authors demonstrated that, in both the ventral prostate and the uterus of the rat, 5’-MTA phosphorylase activity was markedly reduced by gonadectomy and, conversely, markedly and selectively enhanced by the administration of the appropriate sex hormones to the gonadectomized rats. Therefore, it is very interesting to notice that, at least in the prostate or uterus of the rat, 5’-MTA phosphorylase and the two biosynthetic decarboxylases show the same coordinated behavior in response to castration and stimulation by sex hormones, since the activities of all three enzymes are decreased by castration and increased by hormone administration (Seidenfeld et al., 1980). It is conceivable that the similar behaviors of these three enzymes are a synergistic and integrated response of the mammalian target tissues to hormonal stimulation, leading simultaneously to a stimulation of polyamine biosynthesis and to maintenance of low levels of 5‘-MTA inside the cell. In this case, the well-known stimulating effects

POLYAMINES IN MAMMALIAN TUMORS

185

of polyamines on cell growth and differentiation would be strengthened by a concomitant decrease in the inhibitory effect on cell growth of 5’-MTA. Therefore, although it is still premature to draw any final conclusion about the major function of the polyamine biosynthetic pathway, it is tempting to speculate that all of the final products of polyamine biosynthesis (i.e., polyamines and 5’-MTA)are of physiological impor: tance and that 5’-MTA phosphorylase may also act in a coordinate way with some enzymes involved in the biosynthesis of polyamines. Thus, the same biosynthetic route could give rise at the same time to compounds acting in quite opposite ways on cell growth: both stimulating compounds (i.e., polyamines) and inhibiting compounds (i.e., 5’MTA). This would allow the cell to delicately regulate its growth and differentiation under different biological situations by suitably adjusting the intracellular concentrations of these compounds.

E. CATABOLISM O F THE MAJORPOLYAMINES MAMMALIAN ORGANISMS

IN

Historically, one of the earliest demonstrated physiological roles of polyamines is the activity of these substances as growth factors for certain types of microorganisms (Herbst and Snell, 1948). However, only one year after the first report, it was found (Rozanski et al., 1949) that human semen inhibited the growth of certain bacteria. Although the exact mechanisms of this inhibition were not completely understood at once, it was established that the active principle for the inhibition was associated with the liquid part of the semen and not with the spermatozoa. Later, it became clear that one of the unique properties of human semen is its high content of the triamine and tetraamine (mostly spermine and, to a lesser extent, spermidine), in comparison with other body fluids and tissues, and its high diamine oxidase activity, which yields the cytotoxic oxidized derivatives of these polyamines. Therefore, in an interval of only one year, the theoretical and biochemical bases were laid that later would be fully developed to the conception of the polyamines and their oxidized derivatives as parts of an integrated biochemical system devoted to the regulation of cell proliferation and growth, by means of stimulating substances (i.e., polyamines) and inhibiting substances (i.e., oxidized derivatives of polyamines). The existence of a strict, functional connection between the polyamines and their catabolic products has been recently confkmed at the enzymological level by an interesting report in which

186

GIUSEPPE SCALABFUNO A N D MAIUA E. FEFUOLI

diamine oxidase activity was shown to parallel ODC activity in rat small intestine mucosa (Baylin et al., 1978). Although there are a variety of amino oxidase activities in various animal tissues, the exact role played b y these enzymes in controlling tissue levels of polyamines remains unclear. At present, the classification of amine oxidases is quite confused. 1. Oxidation of Spemnidine and Spermine Amine oxidases are enzymes that are widely distributed in animal tissues and are classically divided into two distinct families: (1) monoamine oxidases [ MAO; amine : oxygen oxidoreductase (deaminating) (flavin-containing); E C 1.4.3.41, which act on primary, secondary, and tertiary amines and (2) diamine oxidases [DAO; diamine :oxygen oxidoreductase (deaminating) (pyridoxal-containing); E C 1.4.3.61, which act on aliphatic diamines, including histamine, and polyamines, and on primary monoamines. The identity of histaminase and diamine oxidase is generally accepted (Zeller, 1965). However, there are considerable differences in the substrate affinities of the DAO enzyme(s) between species and between tissues. The most quantitatively important catabolic pathway of polyamines is by oxidation. Putrescine, spermidine, and spermine are oxidized or cleaved by various enzymes from different sources: mammalian tissues, bacteria, plants, and such physiological fluids as plasma of many ruminants, but not of nonruminants, pregnancy plasma, seminal plasma, and amniotic fluid (Tabor, 1951; Hirsch, 1953b; Tabor et al., 1954; Blaschko and Hawes, 1959; Blaschko et al., 1959; Kobayashi, 1964; Caldarera et al., 1965, 1969; Yamada et al., 1967; Tryding and Willert, 1968; Argento-Cerh et al., 1973a,b; Beaven and De Jong, 1973; Janne et al., 1973; Bardsley et al., 1974; Nakano et al., 1974; Baylin and Margolis, 1975; Holtfa et al., 1975; Yasunobu et al., 1976; Holtta, 1977; A. C. Andersson et al., 1978a, 1980b; Illei and Morgan, 1979a,b, 1980; Bieganski et al., 1980; Gahl et al., 1980; Morgan, 1980; Morgan et al., 1980). Essentially, those enzymes capable of oxidizing or cleaving spermidine and spermine are collectively called polyamine oxidases, irrespective of whether they also act on mono- or diamines (Morgan, 1980). Only seldom is a monoamine oxidase able to attack polyamines. Additionally, a fraction purified from an L-aminoacid oxidase of viper venom has been recently demonstrated to oxidize polyamines as well (Braganca et al., 1979). However, it is not yet well established whether there is one enzyme capable of oxidizing all the polyamines or a separate enzyme oxidizing each. Some findings have supported the existence of separate specific

POLYAMINES IN MAMMALIAN TUMORS

187

oxidases, from different sources, for each polyamine, i.e., a putrescine oxidase composed of two different subunits (Gahl and Pitot, 1979; M. Okada et al., 1979a,c; Gahl et al., 1980), a spermidine oxidase (M. Okadaet al., 1979b; Gahl et al., 1980), and a spermidine and spermine oxidase, collectively named polyamineoxidase (PAO) (Holtta, 1977). These reports are in keeping with earlier ones (Hirsch, 195313; Blaschko and Hawes, 1959; Blaschko et al., 1959; Bachrach, 1962). The topic of amine oxidases and the polyamine oxidases has been reviewed (Blaschko, 1962, 1963; Zeller, 1963; Buffoni, 1966, 1980; A. C. Andersson et al., 1980b; Morgan, 1980). The products of oxidative cleavage of the polyamines differ noticeably in relation to the source from which the enzyme employed was isolated (e.g., from mammalian or bacterial systems). 1. Those mammalian amine oxidases capable of oxidizing polyamines, (noticeably active in the intestines, placenta, ovary, semen, serum, and kidneys of various animals) catalyze the oxidative deamination of one or both terminal -CH2NH2 groups of the polyamines to the corresponding mono- or dialdehydes (Unemoto, 1963; Taboret al., 1964a,b). The products of the enzymic reaction are the following: an iminomonoaldehyde from the oxidation of spermidine or an iminodialdehyde from spermine, plus two molecules of ammonia and hydrogen peroxide in the case of spermine, and one molecule of ammonia and hydrogen peroxide in the case of spermidine (Unemoto, 1963; Tabor et al., 1964a). The dioxidation of spermine and the monooxidation of spermidine are represented by the following equations : H2N(CH2)sNH(CH2)iNH(CH2)3NH2+ 202 + 2H2O + Spermine 0 0

\\

H

//

+ 2NH3 + 2H20

C(CH2)2NH(CHp)dNH(CHz)&

/

'H Dioxidized Spermine

0HIN(CH~)$JH(CH&NH~ + 02 + HpO +

\\ /

C(CH~)~NH(CH~)INHI+ NH3 + HzO2

H' Spermidine

Monooxidized Spermidine

The products of oxidized polyamines were found to be quite unstable and to undergo successive and spontaneous metabolic cleavage because dioxidized spermine yields monooxidized spermidine and acrolein

188

GIUSEPPE SCALABFUNO AND MARIA E. FERIOLI

0 CH,,=CH--C

// \

H

whereas monooxidized spermidine decomposes to putrescine and acrolein (Tabor et al., 1964a; Kimes and Morris, 1971a). The monoaldehyde derived from oxidation of spermidine can be metabolized to putreanine, a condensation product of putrescine and P-alanine, [H2N(CH2)4NH(CH2)2COOH, i.e., N-(4-aminobutyl)-3-aminopropionic acid], by an aldehyde dehydrogenase (Nakajima, 1973). Putreanine has been found in the brain and liver of mammals (Kakimoto et al., 1969; Shiba and Kaneko, 1969; Nakajima, 1973; Kremzner and Sturman, 1979). 2. Those bacterial amine oxidases capable of cleaving polyamines catabolize polyamines by splitting the molecules into smaller fragments. The different kinds of oxidative degradation of spermidine and spermine by bacterial suspensions entail the formation either of 1,3-diaminopropane and y -aminobutyraldehyde [H2N(CH2),CHO] (which spontaneously cyclizes to yield A'-pyrroline)

A'-Pyrroline

or of 1,4-diaminobutane and P-aminopropionaldehyde [or 3-aminopropionaldehyde, H2N(CH2)&H0, which is further oxidized to palanine, H2N(CH2)2COOH or converted to a dialdehyde, i.e., malondialdehyde, OHCCH2CHO]. This difference in the products of the catabolic reaction depends on the type of bacteria employed (Razin et al., 1958, 1959; Weaver and Herbst, 1958a,b; Bachrach et al., 1960; Bachrach, 1962; Padmanabhan and Kim, 1965; Okada et al., 1979b). An alternative origin of p-aminopropionaldehyde from 1,3diaminopropane has also been clearly demonstrated (Quash and Taylor, 1970). It is also worthwhile to note that some bacterial oxidases, mainly spermidine oxidase, do not utilize molecular oxygen but require the addition of electron acceptors; they are therefore called spermidine dehydrogenases rather than oxidases (Tabor and Kellogg, 1970; M, Okada et al., 1979b). 3. Polyamine oxidase (PAO) is a single flavin enzyme, newly identified from rat liver (Holtfa, 1977), which catalyzes the oxidation of both spermidine and spermine. These molecules are cleaved at the secondary amino groups to yield 3-aminopropionaldehyde and putrescine from spermidine or spermidine from spermine. Recently, it has

POLYAMINES I N MAMMALIAN TUMORS

189

also been shown that some acetyl derivatives of spermine and spermidine (namely,N ' - andN8-acetylspermidine,N1-acetylspermine, and N1,N1*-diacetylspermine)are natural substrates of this enzyme (Holtta, 1977; Seiler et al., 1980b). Undoubtedly, the mono- and dialdehydes, which arise from the extracellular enzymic oxidation of spermine and spermidine, are, from the biological point of view, more important than other catabolic derivatives of polyamines. In fact, dioxidized spermine has been shown to inhibit growth of various bacteria (Hirsch and Dubos, 1952; Taylor and Morgan, 1952; Hirsch, 1953a; Tabor et al., 1954; Tabor and Rosenthal, 1956; Razin and Rozansky, 1957; Bachrach and Persky, 1964; Kimes and Morris, 1971b),to have a phagocidal action (Bachrachet aZ., 1963, 1971; Bachrach and Leibovici, 1965a,b, 1966; Fukami et al., 1967; Oki et aZ., 1968, 1969), and to inactivate animal and plant viruses (Bachrachet al., 1965,1971; Bachrach and Don, 1970,1971; Kremzner and Harter, 1970; Bachrach and Rosenkovitch, 1972). It is important to recall here that it has been realized since the earliest investigations (Hirsch, 1953a) of this topic that the presence of an appropriate plasma or seminal liquid in the incubation medium was essential for rendering spermine inhibitory for the bacterial growth, since spermine did not inhibit the multiplication of tubercle bacilli in a synthetic medium without plasma. In this regard, it is likely that the oxidized polyamines can be part of the mechanisms of native aspecific immunity, as factors present in blood and in tissues that are active against bacterial and viral growth. Additionally, the enzymatically oxidized polyamines cause a loss of motility of sperm cells and powerfully inhibit some important metabolic processes in the spermatozoa (Tabor and Rosenthal, 1956; Janne et al., 1973; Pulkkinen et al., 1978). Finally, the oxidation products of spermine and spermidine are powerful negative effectors of cellular proliferation, as has been demonstrated in cultures of fibroblasts (Gahl et al., 1976; Gahl and Pitot, 1978; Gaugas and Dewey, 1979; Webber and Chaproniere-Rickenberg, 1980), mitogen-stimulated lymphocytes (Byrd and Jacobs, 1977; Byrd et al., 1977, 1978; Gaugas and Curzen, 1978; Allen et al., 1979; Gaugas and Dewey, 1979; Gaugas, 1980b; Swanson and Gibbs, 1980), and unstimulated thymocytes (Gaugas and Dewey, 1979; Morgan et al., 1980). The interaction of polyamine oxidase with one of the new bacterial polyamines, i.e., thermine, has also been shown to potently inhibit lymphocyte proliferation (Gaugas and Dewey, 1979; Gaugas, 1980b). The activity of these drugs against bacteria and viruses can be explained by their binding to cellular nucleic acids and by their capacity

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to inhibit the syntheses of nucleic acids and mRNA in microorganisms and in mammalian cells (Bachrach and Persky, 1966, 1969; Bachrach and Eilon, 1967, 1969a,b; Persky et al., 1967; Eilon and Bachrach, 1969).As was cleverly ,suggested by Bachrach and his co-workers, who excellently and thoroughly investigated the products of oxidative catabolism of polyamines and their biological properties, these monoand dialdehydes resemble bifunctional alkylating agents, such as nitrogen mustards, and also behave like those antibiotics that inhibit DNA-directed RNA polymerase by binding to the DNA template. The biological actions of short-chain aliphatic aldehydes on different eukaryotic and prokaryotic systems have been extensively studied in our institute by Ciaranfi and his co-workers, who have obtained some important results (such as the inhibition of protein synthesis and of cellular proliferation, especially in neoplastic cells, and the essential nature of the presence of an aldehydic group for the inhibitory properties of the molecules), and these actions are analogous to those of the long-chain mono- and dialdehydes arising from oxidation of spermidine and spermine (Guidotti et al., 1964,1965; Ciaranfi et al., 1965, 1971; Perin et al., 1972, 1978; Sessa et al., 1977). Thiazolidin-4carboxylic acid, which is the condensation product of some aliphatic aldehydes with L-cysteine both in vitro and in vivo (Guidotti e t al., 1965; Loreti et al., 1971), is one of the pharmacological agents which has recently been found to be able to restore “contact inhibition” in tumor cell cultures (Gosalvez et al., 1979). Some very exciting perspectives about the biological actions and significance of the oxidized derivatives from polyamines have been put forward by some investigators. On the grounds of the experimental data showing high and increasing activity of DAO in the placenta and plasma of mammals during pregnancy (Kobayashi, 1964; Tryding and Willert, 1968; Bardsley et al., 1974; Baylin and Margolis, 1975; A. C . Anderson et al., 1978a; Illei and Morgan, 1979a,b), a high polyamine content of mammalian placenta (Porta et al., 1978), and, finally, the powerful suppression of the mitogen-stimulated lymphocyte proliferation elicited in vitro by the products of the ruminant sera-polyamine interaction, it has been argued that such an immunodepressant effect might also operate in vivo in the intervillous circulation of the placenta. Such a mechanism might represent a natural and localized immunological barrier, which can protect, at least in part, the conceptus from immunological rejection by the mother caused by the immunological incompatibility between the fetoplacental unit and the mother (Byrd et al., 1978; Gaugas and Curzen, 1978; Morgan and Illei, 1980). Further credence is given to such a hypothesis by the following

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findings: (1)high levels of ODC activity were found in the developing rat placenta; (2) most of the ODC activity was found in the fetal part of the rat placenta; (3)in the rat placenta during pregnancy, the time course of ODC activity was similar to that of DAO activity; (4) in contrast to the pattern of distribution for ODC activity, most DAO activity was found to be localized in the maternal part of the rat placenta (Maudsley and Kobayashi, 1977). Although P-aminopropionaldehyde has been identified as a normal constituent of human serum (Quash and Taylor, 1970) and acrolein has been demonstrated to be formed in significant amounts from enzymatically oxidized spermine and spermidine in vitro (Alarcon, 1966,1970, 1972),the physiological significance of the former aldehyde remains to be established, whereas the cytotoxic property (Alarcon, 1966, 1970, 1972) and the antimicrobial activity (Kimes and Morris, 1971b) of the latter aldehyde have been clearly demonstrated, although its antiviral activity is still highly doubtful (Bachrach et al., 1971; Bachrach and Rosenkovitch, 1972). Furthermore, the real role of acrolein as possible inhibitor of cell proliferation has also been questioned (Gaugas and Dewey, 1979; Gaugas, 1980b). Finally, a further possible elucidation of the molecular mechanism involved in the cytostatic effect of the PAO-spermine interaction has been proposed by Gaugas and Dewey (1980).Accordingly, 02-.produced during oxidative catabolism of spermine by PA0 could react with dioxidized spermine to generate free radicals that could be additional antimitotic agents. The production of 02-dependentfree radicals or H202, also generated by polyamine catabolism, is apparently not directly involved in in vitro cytostatic effect, since this effect was found to be independent of catalase activity and the addition of superoxide dismutase paradoxically potentiated that effect. The topic of oxidized spermidine and spermine and their biological roles has been thoroughly reviewed several times by Bachrach (1970a,b, 1973). 2. Oxidative and Nonoxidative Catabolism of Putrescine and Cadaverine In mammals, putrescine catabolism occurs through several multistep pathways. 1. First, the oxidative deamination of putrescine involving mammalian DAOs leads to y-aminobutyraldehyde (4-aminobutyraldehyde) formation, which nonenzymically cyclizes into an internal aldimine ring, A1-pyrroline. One molecule each of ammonia and hydrogen peroxide are also formed during this reaction. This direct pathway

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catalyzed by DAO is quantitatively the most important. Putrescine oxidase from some bacteria also cleaves putrescine to give the same final products (Okadaet al., 1979~). The catabolic oxidative pathway of putrescine is presented in the following scheme: HzN(CH2)dNHZ + HzO + Putrescine

+ HzOp + NHD y-Aminobutyraldehyde

0 2 + HzN(CH&CHO

A'-Pyrroline

However, from a theoretical point of view, y-aminobutyraldehyde will only be quantitatively transformed into A'-pyrroline when the tissue which contains the DAO activity is utterly devoid of any aldehyde-metabolizing enzymes. Most mammalian tissues are able to metabolize y-aminobutyraldehyde as substrate. This aldehyde can be oxidized by an aldehyde dehydrogenase to yield y-aminobutyric acid (GABA) or reduced by an aldehyde reductase or an alcohol dehydrogenase to yield 4-amino-l-butanol (Fogel et al., 1978). In recent years, an ever-increasing number of reports on the conversion of putrescine to GABA in vivo and i n vitro in different organs of mammals have appeared (Seiler and Knodgen, '1971; Seiler et al., 1971, 1973a, 1979a; Seiler, 1973; DeMello et al., 1976; Henningsson and Rosengren, 1976; Konishi et al., 1977; Tsuji and Nakajima, 1978; Sobue and Nakajima, 1978; Anderson and Henningsson, 1980a; Andersson et al., 1980a). It has also been shown in these reports that the transformation of putrescine into GABA implies the presence of a direct pathway which does not include glutamic acid as an intermediate, since it is well established that in eucaryotes GABA can also be synthesized from glutamic acid, catalyzed by L-glutamate l-carboxy-lyase (or glutamic acid decarboxylase, EC 4.1.1.15). The enzyme mainly involved in the in vivo conversion of putrescine to GABA is again DAO (Seiler and Eichentopf, 1975; Sourkes and Missala, 1978; Tsuj and Nakajima, 1978). Evidence has been presented that ornithine is also a precursor for GABA in adult mammalian tissues, probably via putrescine (Seiler and Knodgen, 1971; Murrin, 1980). Recently, the regulatory interrelations between polyamines and GABA have been carefully reconsidered by Seiler et al. (1979b, 1980a). 2. Experimental evidence has been accumulated in favor of the existence of another catabolic pathway for putrescine, in which this diamine is first acetylated to monoacetylputrescine, which is, in turn, degraded to GABA, through the different steps shown in Fig. 5 (Seiler

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N -acetylS-AB-CHO

1

ALD O H

ALD-DH

0 R

0

SI I

N - a t e l yI S-AB-COOH

enzyme

COOH

a r-AB-COOH I I

b Succinic acid

C t t r a t e cycle

COt

1 , 4 - 0 A B = 1.4-DIAMINOBUTANE (PUTRESCINE ) r - A B - C O O H = r-AMINOBUTYRIC ACID r - A B - C H O = r-AMINOBUTYRALDEHYDE M A 0 AMlNE OXIDASE ( F L A V I N - CONTAINING) ( E C 1 4 3 4 ) OAO = AMlNE O X I D A S E (COPPER-CONTAINING) ( E C 1 4 3 6 ) A L O - O H = AMINOBUTYRALDEHYDE DEHYDROGENASE (EC 1 2 1 1 9 ) ACETYLATING ENZYME = PUTRESCINE ACETYLTRANSFERASE (EC 2 3 1 57) DEACETYLATING ENZYME = N-ACETYL 4-AMINOBUTYRIC AClO DEACETYLASE

@ @

EXTRAMITOCHONDRIAL PATHWAY MITOCHONDRIAL PATHWAY

FIG.5. Different pathways of putrescine catabolism in mammalian tissues.

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and Al-Therib, 1974a,b; Seiler and Eichentopf, 1975; Seiler et al., 1979a). The enzyme acetylating putrescine, i.e., the acetyl-CoAdependent N-acetyltransferase, was found to be located mostly in the nucleus (Seiler and Al-Therib, 1974a; Seiler et al., 1975). However, this catabolic pathway of putrescine is probably a minor one. In summary, one can state that two of the three main catabolic routes for 1,Cdiaminobutane in mammalian cells lead to the formation of GABA, which in the extramitochondrial compartment, is practically a metabolic end product but which in the mitochondrial compartment is an intermediate product, further metabolized in the citrate cycle. Surprisingly enough, it should be noted that, at least in the brain, the mitochondrial catabolic pathway for putrescine includes a step in which a typical diamine is deaminated by MA0 (Seiler and Al-Therib, 1974b). This peculiar behavior of M A 0 has recently been confirmed for the acetyl derivative of another diamine, i.e., monoacetylcadaverine, deaminated by rat liver M A 0 (Suzuky et al., 1980). 3. A third catabolic pathway of putrescine has been detected and demonstrated to be independently active, though only in some mammalian organs. In this case, putrescine is converted via A'-pyrroline to 2-pyrrolidone (a lactam not previously identified in any biological system) in liver, spleen, and lung but not in kidneys, brain, heart, and muscle (Lundgren and Hankins, 1978; Lundgren et al., 1980). This lactam is then metabolized into 5-hydroxy-2-pyrrolidone7another compound new to the biological systems (Lundgren and Fales, 1980). Nevertheless, this newly identified catabolic route for putrescine needs deeper and more careful studies to clarify the enzymatic basis of the formation of these derivatives. The three main catabolic pathways of putrescine in mammals are presented in Fig. 5. 4. Putrescine may also be converted into y-aminobutyraldehyde by a nonoxidative mechanism, catalyzed by a diamino-a-ketoglutarate transaminase, present in a variety of bacteria (Kim and Tchen, 1962; Kim, 1964; Michaels and Kim, 1966). In this enzymic reaction, free ammonia is not released nor is oxygen consumed. 5 . Finally, 1,s-diaminopentane is oxidized by animal DAO to A'piperidine with production of ammonia and hydrogen peroxide and with oxygen consumption (Bachrach, 1973), according to the following t

0 2+

HeN(CH2)rCHO + HzOZ + NHS

6 N

Hzo

A'-Piperidine

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195

F. CONJUGATION PRODUCTS AND EXCRETION PRODUCTS As far as the conjugation products of polyamines are concerned, quite similar conjugated forms have been identified in prokaryotes and in eukaryotes. In fact, in bacteria, polyamines can be conjugated with glutathione or be acetylated (mono- or diacetylated) or linked to peptides (Dubin, 1959; Dubin and Rosenthal, 1960; C. W. Tabor and Tabor, 1970; H. Tabor and Tabor, 1975, 1976). This is also essentially true for the eukaryotes. Monoacetylputrescine [ HzN(CHz),NHC CHa](N’-acetyl-l,4-diaminobutane)

1I

0

has been found in vertebrate tissues (Seiler et al., 1973b), in human brain (Perry et al., 1967), in normal human lymphocytes (Menashe et al., 1980), and in the urines of normal humans and rats (Noto et al., 1978; Seiler and Knodgen, 1979b). The presence of such a molecule must be connected with that catabolic pathway of putrescine which begins with the acetylation of this molecule. A y-glutamyl derivative of putrescine has been detected in rat brain (Nakajima et al., 1976). Both spermidine and spermine, but not diamines, have been demonstrated to serve as substrates for acetyl-CoA-dependent N acetyltransferase activity present in the nuclei of liver of calf and rat and in isolated chromatin from rat liver and kidney, and undergo N-acetylation in this system (Blankenship and Walle, 1977; Libby, 1978a, 1980). In the case of spermidine, the product of the enzymatic reaction is acetylspermidine B (or N1-acetylspermidine). This compound can be further metabolized by PA0 (Holtta, 1977), whereas acetylspermidine A (or iV-acetylspermidine) is deacetylated by a specific cytosol enzyme present in rat tissues to yield acetic acid and spermidine (Blankenship, 1978; Blankenship and Walle, 1978). N 1 Acetylspermidine, alternatively, can be enzymatically converted to putrescine (Blankenship, 1979) or enzymatically deacetylated (Libby, 1978b).

!

H,N(CHz)aH(CHz)$IH CHS N ‘-Monoacetylspermidine [ N ‘-(3-acetamidopropyl)-1,4diaminobutane]

E

HSC NH(CHz)4NH(CHz)sNHp N8-Monoacety lspermidine [N-(4-acetamidobutyl)-1,3diaminopropaneJ

Monoacetylspermidines (A and B forms) are normal constituents of human urine (Nakajima et al., 1969; Noto et al., 1978; Seiler and

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Knodgen, 1979b). l-N-Acetylspermidine occurs normally in human serum (Smith et al., 1978) and in human lymphocytes (Faber et al., 1980; Menashe et al., 1980). Putreanine and N-(3-aminopropyl)-4aminobutyric acid are other normal catabolites of spermidine in human urine (Noto et al., 1978; Asatoor, 1979). The occurrence of the diamines (putrescine and cadaverine) and polyamines (spermidine and spermine) in human urine as Schiff bases, in which the polyamines conjugate with pyridoxal or pyridoxal phosphate has recently been recognized (Aigner-Held et al., 1979). In the blood of normal subjects, cadaverine is present as monoacyl derivatives, i.e., monoacetylcadaverine and monopropionylcadaverine (Dolezalova et al., 1978). The products of conjugation of polyamines with peptides in some biological fluids, such as human plasma and human amniotic fluid, have been very carefully investigated by Rennert and his group. These investigators found that the polyamines are conjugated to peptide carriers (Chan et al., 1978, 1979; Seale et al,, 1979a,b). In plasma, this peptide conjugate contains, in largest amount, putrescine and a small amount of spermidine, but no spermine (Seale et al., 1979a). In the amniotic fluid, putrescine, spermine, and spermidine are associated with peptides, but spermine can also be present as the acetylated derivative (Chan et al., 1978,1979; Seale et al., 1979b). Putrescine can also be transported in human plasma bound to fibronectin (Roch et al., 1980). Conjugation is a process not involving any structural alteration of polyamines and is a biochemical reaction well known to occur typically in tbe liver in uiuo. On the one hand, no conjugation of polyamines of any kind takes place in vitro between labeled polyamines and plasma or whole blood (Rosenblum et al., 1976), and, on the other hand, near-total hepatectomy of the rat prevents the formation of detectable polyamine conjugates (Rosenblum et d., 1976; Rosenblum and Russell, 1977). Although acetylated derivatives of polyamines are probably the most significant fraction of the conjugated polyamines, the newly identified polyamine-peptide conjugates may provide important new insights into the various metabolic transformations of polyamines in man and other mammals, both healthy and diseased. Therefore, one can conclude that-as far as is known-the free polyamines are present in the physiological body fluids of mammals in small or even in negligible amounts, with the conjugated forms clearly quantitatively predominant (Janne et al., 1978). However, whether acetylated polyamines play active biochemical roles, are only physio-

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logically inactivated polyamines, or are only excretion forms of these compounds [it is noteworthy that polyamine acetyltransferase has also been found in kidney (Blankenship and Walle, 1977)] remains to be worked out. Polyamines are also included in the molecular structures of some antibiotics and some antineoplastic agents. Edeine A contains spermidine in covalent linkage to a pentapeptide (Hettinger and Craig, 1968; Hettinger et al., 1970). Edeine B contains N-guanyl-N’-(S aminopropyl)-1,4-diaminobutane (guanylspermidine) (Hettinger et al., 1968, 1970; Kid0 et al., 1980). Among the active bleomycins, which are a group of natural and synthetic antitumor drugs, bleomycin A5 contains the triamine spermidine and bleomycin & the tetraamine spermine (Cohen and I, 1976; Lapi and Cohen, 1977; Cohen, 1979; Kid0 et al., 1980). G. NATURALANTIPOLYAMINEANTIBODIES Normal human and rabbit sera have been shown to contain naturally occurring antipolyamine antibodies. These natural antibodies react specifically with polyamines and have been classified as IgG immunoglobulins, regardless of the species in which they have been identified (Roch et al., 1978,1979; D. Bartos et aZ., 1980; Ripoll et al., 1980). The biological significance of these antibodies and their susceptibility to quantitative change in different kinds of diseases, including neoplasms, will be of theoretical and practical concern. II. Levels of Polyamines and Their Biosynthetic Enzymes in Fully Developed Experimental Tumors

The aim of this section is to summarize present knowledge about the relationships between polyamines and some fully developed experimental tumors. For this purpose, we will consider the changes in polyamine content observed in different types of tumor, the alterations in polyamine biosynthetic decarboxylase activities, and the applicability of these parameters as biological markers of tumor growth or regression. Starting from the earliest reports (Russell and Snyder, 1968; Snyder and Russell, 1970; Snyder et al., 1970), which showed high levels of ODC activity in some hepatomas and sarcomas, a great deal of research has been done in this specific area. It is necessary to state as a preliminary that, in analyzing the polyamine contents of neoplasms, one needs to establish their physiological levels and the normal changes in those levels in the corresponding normal tissues before one can correlate increases of these

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GIUSEPPE SCALABRINO AND MARIA E. FERIOLI

substances with neoplastic transformation or with different degrees of malignancy. Ornithine is an interesting amino acid since it appears not to be a true building block in the biosynthesis of any major protein, but it is a key metabolite for which several enzyme systems and metabolic pathways compete. In the livers of mammals, ornithine is produced by the action of arginase on L-arginine and is thereafter utilized in several metabolic pathways: it may be (1)channeled by ODC into polyamine synthesis, (2) siphoned off into the urea cycle by ornithine transcarbamylase (OCT), and (3) metabolized by the transaminase reaction catalyzed by L-ornithine : 2-oxoacid aminotransferase (ornithine transaminase) (OTA) into glutamic-y-semialdehyde synthesis. Of the enzymes involved in the metabolic pathways of ornithine, only ODC is at present known to be constantly increased in rapidly growing rat liver tumors, and its increase has been positively correlated with the growth rate of hepatomas tested. In fact, a systematic study carried out with several rat hepatomas with different growth rates resulted in the discovery of a shift in ornithine metabolism that could confer a selective biological advantage on the cancer cell and that is linked with tumor growth rate (Weber, 1972, 1973a,b; Weber et al., 1972a,b; Williams-Ashmann et al., 1972a,b, 1973; Tomino e t al., 1974). The term “imbalance” was applied to this shift by Weber to indicate alterations from the normal state of metabolic balance, which denotes dynamic equilibrium in steady-state conditions. For the metabolic pathways of ornithine in rat hepatomas, the imbalance is represented by progressively elevated ODC activity, in parallel with the increase in the hepatoma growth rate, whereas OTA and OCT activities progressively declined in parallel with the increase in the hepatoma growth rate. This imbalance in ornithine metabolism in tumor cells was accompanied by other imbalances in the metabolic pathways of carbohydrates, pyrimidine, and cyclic nucleotides (Weber, 1972, 1973a,b; Weber et al., 1972b). Consequently, as a result of these quantitative modifications of the activities of the three key enzymes (ODC, OTA, OCT) which compete for ornithine, in the rapidly growing neoplastic hepatomas only the one (ODC) that channels ornithine into the polyamine biosynthetic pathway is selectively increased, and the other two (OTA, OCT) are selectively and markedly decreased. Therefore, the biochemical advantages that arise from the imbalance in ornithine metabolism and are able to support the high growth rate of the hepatoma cells are essentially two, occurring concomitantly: (a) increased polyamine biosynthesis, starting with the increased ODC activity; (b) increased purine and pyrimidine biosynthesis, due to the

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decreased OCT activity, which decrease allows lesser utilization of carbamyl phosphate and aspartate in the urea cycle and increased utilization for the synthesis of DNA and RNA. It is well known, in fact, that the urea cycle competes with purine and pyrimidine biosynthesis for two important precursors, carbamyl phosphate and aspartate. However, it is still an open question whether such a derangement of the activities of the three enzymes that compete for ornithine utilization is typical of all neoplasms or is characteristic only of the hepatomas, since no other such systematic studies are available for neoplasms of other organs. However, the simultaneous presence of all these imbalances in carbohydrate, pyrimidine, nucleic acid, ornithine, and cyclic nucleotide metabolism appears to be specific for neoplastic liver, since no similar patterns have been found in fetal, normal, differentiating, or regenerating rat liver (Weber, 1972, 1973a,b; Weber et al., 197213). Two other pertinent considerations must be added: (1)the SAMD activities of almost all the hepatomas tested were either within the normal range or even decreased, only very rarely being higher than in normal liver; (2) putrescine concentrations generally tended to be highest in the most rapidly growing hepatomas, but the steady-state concentration of this diamine did not always exactly mirror the ODC activity of the same sample of tumor tissue (Williams-Ashman et al., 1972a,b, 1973). Furthermore, in some Morris hepatornas, ODC levels were also found to be dissociated from concentrations of spermidine and spermine, which were even lower than those in resting liver (Cavia and Webb, 197213). These findings are particularly important because these reports are among the earliest to stress the possibility that ODC, rather than the polyamine content or pattern, might be a better indicator of the growth rate of a neoplastic tissue. However, Pariza et al. (1976a) questioned whether the increase in ODC activity is a good indicator of the hepatoma growth rate, because they found in circadian rhythm studies that ODC activity in Morris hepatoma 7800 was lower than in normal liver throughout most of the day. For the same hepatoma, Cavia and Webb (1972a,b) reported just the opposite, with the ODC activity level higher than in control liver. The ODC level of the same hepatoma was decreased after bilateral adrenalectomy of the host, demonstrating the ODC, at least in this case, was still hormone responsive (Cavia and Webb, 1972a). The picture is further complicated by the fact that another indicator for the growth rate of the neoplasms has been proposed, the spermidine/spermine ratio (Russell, 1973b; Russell and Durie, 1978). Elevation of this biochemical ratio, whether or not accompanied by high putrescine content, has been demonstrated to be greater in fast-

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GIUSEPPE SCALABRINO AND MARIA E. FERTOLI

growing hepatomas than in slow-growing ones (Russell, 1973b; Russell and Durie, 1978) and to occur also in both solid and ascites Rd13 sarcoma (Neish and Key, 1967, 1968), in Ehrlich ascites tumor cells, in rat mammary tumor, and in L1210 leukemia (see discussion later in this section). It is noteworthy that spermine stimulated in uitro the activities of all three RNA polymerases from a rapidly growing hepatoma when native DNA was used as template and, even more, that the stimulation of all these tumor enzymic activities was greater than those of the corresponding activities from normal rat liver (Rose et al., 1976). The stimulation by spermine of DNA-dependent RNA-polymerase activity has been confirmed in in uitro studies with enzyme purified from nuclei of Ehrlich ascites tumor cells (Blair and Mukherjee, 1973). Some other effects induced by polyamines at the molecular level have been investigated in some Morris hepatomas. Spermine stimulated the total ADP ribosylation of nuclear proteins in two Morris hepatomas and in host liver, while the effect of spermidine was less marked (Perrella and Lea, 1979). Moreover, the effect of spermine on the relative proportion of labeled ADP-ribose incorporated into the different nuclear fractions was to further strengthen the trend, already evident in its absence, in a spectrum of homogeneous tissues with different proliferation rates (such as resting liver, regenerating liver, and some Morris hepatomas), to increase the incorporation of ADPribose into nonhistone proteins, and to decrease concomitantly the incorporation into histones (Perrella and Lea, 1979). Further studies focused on the effects of spermine on the distribution of ADP-ribose molecules bound to histones in hepatomas and host liver. Interestingly, spermine once again caused a shift in the incorporation of labeled ADP-ribose in the histones of the nuclei from hepatomas and host liver (Perrella and Lea, 1979). In detail, spermine caused a shift in the distribution of labeled ADP-ribose from the core histones (i.e., the histones which form the core of the nucleosomal structure) to the H 1 internucleosomal histones, without significantly changing total incorporation into histones (Perrella and Lea, 1979). This is another indication of the role of spermine in regulating DNA-to-protein interactions. Ehrlich ascites carcinoma, growing in the peritoneal cavities of mice, is a tumor particularly well studied for polyamine biosynthesis and content. The putrescine concentration in the tumor cells markedly increased in the first week after tumor inoculation, i.e., during the phase of extremely rapid cell multiplication, but thereafter it dropped by nearly one-half (Anderson and Heby, 1972, 1977). This time course

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was not confirmed by Kallio et al. (1977d), who observed only small changes in cellular putrescine concentrations during tumor growth. The patterns of change in the intracellular concentrations of spermidine, spermine, DNA, and RNA were quite well correlated reciprocally with each other (Andersson and Heby, 1972). The spermidine concentration was found by some authors (Bachrach et al., 1967; Andersson and Heby, 1972; Kallio et al., 1977d; Uehara et al., 1980), but not by others (Siimes and Janne, 1967), to be always much higher than that of spermine. These data lend further support to possible connections of putrescine concentration with rapid growth phase and of spermidine and spermine concentrations with nucleic acids. Indeed, a high positive correlation between the cellular content of spermidine and spermine and that of nucleic acids has been reported (Andersson and Heby, 1977). In this respect, spermidine stimulated in vitro the RNA synthesis of Ehrlich ascites tumor cells when it was present in the incubation medium at low concentrations, but it had exactly the opposite effect at higher concentrations (Raina et al., 1968). The ability of Ehrlich ascites cells to form spermidine from putrescine in uivo and to interconvert spermidine and spermine in ui tro was also demonstrated in early studies of this type of tumor (Bachrach et al., 1967; Siimes and Janne, 1967). As for the activities of the polyamine biosynthetic enzymes in Ehrlich ascites cells during tumor growth, ODC and SAMD were found to change very little during the first week after the i.p. inoculation, whereas spermidine and spermine synthases increased significantly, in the last 3 days of the same period (Kallio et al., 1977d). In contrast with this, ODC activity is markedly increased in Ehrlich ascites tumor cells, reaching a peak 4 days after tumor inoculation, i.e., at nearly the time when cell proliferation is fastest (Noguchi et al., 1976a,b). More careful sequential studies demonstrated that ODC activity increased dramatically within a few hours after the i.p. inoculation of the ascites tumor cells (Harris et al., 1975; Andersson and Heby, 1977; G. Andersson et al., 1978b). It was also possible to establish that there are high positive correlations between the activities of ODC and SAMD and the specific growth rate of the tumorous cells (Andersson and Heby, 1977, 1980; G. Andersson et al., 1978b) and between the peak in ODC activity and the peak of the [3H]thymidine index, i.e., the ODC increase paralleled the surge of cells from the Gl-Go into the S phase (Harris et al., 1975). Very surprisingly, when the activities of the two polyamine biosynthetic decarboxylases began to decline, the decrease was not accompanied by a concomitant decrease in the cellu-

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GIUSEPPE SCALABRINO AND MARIA E. FERIOLI

lar spermidine and spermine contents, which instead continued to rise during plateau phase growth, when the tumor growth rate had decreased (G. Andersson et al., 1978b; Andersson and Heby, 1980). From E hrlich ascites tumor cell-free fluid, a factor has been purified that is a heat-labile, alkali-stable, acidic protein and is able to stimulate in uiuo ODC activity in liver and spleen but not in kidneys of normal mice (Noguchi et al., 1976a,b; Kashiwagi et al., 1979). ODC induction by this factor had a very slow time course after in uiuo injection and seemed to occur independently of a release of hypophyseal or adrenal hormones, since induction was also observed in hypophysectomized or adrenalectomized mice (Noguchi et al., 1976a; Kashiwagi et al., 1979). The ODC-stimulating factor appears to be specific for Ehrlich ascites tumor fluid and cells, since in vivo injection of homogenates of various normal tissues into mice caused no stimulation of ODC activity in the livers of the recipient normal animals (Noguchi et al., 1976a). This factor, released from tumor cells, is also thought to be responsible for the observed increases in ODC activity in the liver and spleen of the host animal a few days after the i.p. inoculation of Ehrlich ascites tumor cells, since these changes were not accompanied by infiltration or metastasis of tumor cells in these organs (Noguchi et al., 1976a,b). In contrast, the renal ODC activity in mice with Ehrlich ascites carcinoma greatly decreased during the tumor growth in the days after the inoculation (Noguchi et al., 1976a,b). According to an attractive hypothesis (Andersson and Heby, 1977, 1980; Heby et al., 1979; Linden et al., 1980), the extracellular polyamines released into the ascites fluid as a result of tumor cell death could be responsible for the inhibition of intracellular polyamine biosynthesis, which in turn brings about a marked reduction in the rate of DNA synthesis and cell proliferation (see also Section 111,E). Putrescine, when administered in uiuo in repeated injections, strongly inhibited the tumor cell proliferation and ODC activity by eliciting the formation of the ODC antizyme, and, when added to the in vitro assay mixture, it markedly inhibited ODC activity in the cytosol fraction from tumor cells stimulated to grow (Andersson and Heby, 1977,1980; Hebyet al., 1979; Lindenet al., 1980). On the other hand, multiple injections of putrescine into mice with Ehrlich ascites carcinoma cells neither affected the SAMD activity of neoplastic cells nor decreased their incorporation of tritiated thymidine into DNA (Linden et al., 1980). A factor not yet chemically identified and not tumor specific, but able to stimulate ODC activity in uitro has been demonstrated in ascites fluids taken from murine hosts at progressive stages of growth

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of different types of tumor, both chemically induced and spontaneously generated, e.g., fibrosarcoma, mammary carcinoma, ovarian carcinoma, leukosis L1210 (Vaage and Agarwal, 1979). This ODC stimulation was observed in the same neoplastic cells first isolated from the foregoing neoplasms grown in uivo and cultured in uitro. This stimulation of ODC by ascites fluid was greater during the initial period of malignant growth and then declined as growth continued, the only exception being leukemia L1210 (Vaage and Aganval, 1979). In serum and in ascites fluid from mice with malignant tumors, namely, fibrosarcoma, mammary carcinoma, and ovarian carcinoma, a “negative” factor with a markedly depressive effect on ODC activity of both normal unstimulated lymphocytes and PHA-activated lymphocytes is present (Vaage et aZ., 1978). An analogous inhibitory effect on ODC activity of PHA-activated lymphocytes taken from tumor hosts was also displayed by ascites fluids taken from mice given neoplastic implants (Vaage et al., 1978). This ODC inhibition was more pronounced, the more advanced the neoplastic disease (Vaage et d., 1978). Whether such factor(s) in serum and/or ascites fluid taken from tumor hosts could explain the general anergy frequently associated with advanced cancer remains a matter of speculation. In addition to rat hepatomas and Ehrlich ascites tumor cells, rat mammary carcinoma and mouse leukosis are ideal for studies of polyamine biosynthesis and accumulation. The ratio of spermidine to spermine in rat mammary carcinoma was higher when the tumor was growing (Russell et al., 1974c; Russell and Durie, 1978). Polyamine concentrations in rat mammary carcinoma were profoundly modified by castration plus removal of the pituitary gland, which induced the process of tumor regression. Putrescine and spermidine levels dropped significantly shortly after surgery, whereas the spermine level, surprisingly, increased (Russell et al., 1974c; Russell and Durie, 1978). Consequently, the spermidine/spermine ratio fell. In the tumor’s interstitial fluid, an increase in spermidine level was detected during tumor regression, whereas putrescine concentration was essentially unchanged (Russell et al., 1974c; Russell and Durie, 1978). In L1210 leukemia, both solid and ascites forms, ODC activity increased rapidly within a few days after inoculation and then dropped to very low levels just prior to the death of the mice (Russell, 1970; Russell and Levy, 1971; Russell and Durie 1978). SAMD activity was also elevated in the earliest phases of tumor growth, but, unlike ODC, it remained at high levels (Russell, 1970; Russell and Levy, 1971; Russell and Durie, 1978). As for polyamine concentrations, a high spermidine/spermine ratio in L1210 leukemia was due to an increase

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in the concentration of the former, concurrent with a slight decrease in the latter (Russell and Levy, 1971; Newton et al., 1976; Russell and Durie, 1978). The activities of nuclear DNA-dependent RNA polymerases I, 11, and I11 isolated from L1210 leukemia cells were stimulated by spermine more than by spermidine (Blair et al., 1980). In AKR leukemic cells, the spermidine and putrescine contents were similar in the Goand G1phases of the cell cycle, whereas the spermine level decreased from Go to GI (Heby et al., 1973). Furthermore, the cellular contents of three main polyamines increased progressively as the cell traversed the cell cycle phases from Gl to M (Heby et al., 1973). Ornithine metabolism has been measured in control animals and in rats with either Walker 256 carcinoma or methylcholanthrene-induced tumors by measuring the labeled COz liberated from [ l-14C]ornithine injected i.p. (Buftkin et al., 1978).This follow-up study revealed that ornithine metabolism was significantly increased over that in normal animals and that this increase can reasonably be connected with the growth rate of the neoplasm since the regression of the tumor occurred concurrently with a reduction in ornithine metabolism (BufIkin et al., 1978). Last, but not least, it is very interesting that some isozymes of S-adenosyl-L-methionine synthetase have been identified in some hepatomas as well as during chemical hepatocarcinogenesis and that no isozymes of the enzymes involved in the polyamine biosynthetic pathway have been identified so far in neoplasms. Briefly, there are two widely accepted classifications of the isozymes of Sadenosyl-L-methionine synthetase: (1)they are distinguished by their molecular weights and sensitivities to dimethylsulfoxide into a,p, and y forms; (2) they are distinguished by their different K, values for methionine and are called low K, intermediate K,, and high K, forms. The activities of the a-and /3-isozymes progressively decreased, whereas the kidney-type y-isozyme (which is present in kidney, in most other tissues, and in fetal liver) progressively increased during the rat liver carcinogenesis by N-2-fluorenylacetamide (Tsukada and Okada, 1980), in some Morris rat hepatomas, and in Yoshida ascites hepatoma AH130 (G. Okada et al., 1979). A neoplastic abnormal isozyme of S-adenosylmethionine synthetase, with an intermediate K,, has been found in neoplastic nodules induced by a hepatocarcinogen and in various transplantable hepatocellular carcinomas (Liau et al., 1979a,b). This type of isozyme has been detected in a large spectrum of human malignant tumors xenografted into athymic nude mice, but it has never been detected in any normal human tissue examined (Liau et al., 1980).

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Ill. Modification in Vivo and in Vitro of Tissue Polyamine Metabolism by Chemical Carcinogens and Tumor Promoters

A. EFFECTSOF A SINGLE ADMINISTRATIONOF A CARCINOGEN OR TUMORPROMOTER ON POLYAMINE BIOSYNTHESIS AND CONTENT IN THE TARGETTISSUES Generally speaking, a single administration of a carcinogenic drug (whether or not at carcinogenic dosages) or of a tumor promoter makes it easier to determine the molecular events occurring in the cells after penetration ,of the drug. Among hepatocarcinogenic molecules, a single injection of thioacetamide, a weak carcinogenic agent that stimulates RNA synthesis, is able to induce in vivo a large increase in hepatic ODC activity in the rat (Fausto, 1969,1970; Raina and Janne, 1970b; Cavia and Webb, 1972a; Ono et al., 1972,1973; Pegg et al., 1978). This kind of induction presents some particular features: (1)its peak occurs much later than those of ODC induced by hormones; (2) it is not mediated by the hormones, since adrenalectomy or hypophysectomy did not significantly modify the induction of ODC in rat liver by the drug (Cavia and Webb, 1972a; Ono et al., 1973; Suzuki et al., 1973); ( 3 ) it is quantitatively greater by far than the ODC induced by several hormones active on the liver, so that ODC has been commonly purified extensively from thioacetamide-treated rats (Ono et al., 1972). In fact, the marked elevation of liver ODC produced by the drug is roughly comparable to that observed during the earliest phases of rat liver regeneration (Fausto, 1969). Obviously, enzyme enhancement of this degree is accompanied by increased hepatic concentrations of putrescine and spermidine, but spermine concentration decreases (Fausto, 1970; Raina and Janne, 1970b). Other thioamide derivatives, such as acylthioureas, are able to induce hepatic ODC in vivo in the rat (On0 et al., 1973). In addition, the livers of rats with ODC induced by injection of thioacetamide contain multiple forms of the enzyme (Obenrader and Prouty, 1977a). Lastly, treatment with thioacetamide greatly prolongs the apparent half-life of ODC activity (Obenrader and Prouty, 197713; Poso et al., 1978) and also apparently stabilizes the enzyme, as revealed by markedly reduced sensitivity of the enzyme to inhibition by cycloheximide or 1,3-diaminopropane. Both these phenomena have also been observed for SAMD activity, i.e., lengthening of the SAMD half-life (Poso et al., 1978) and decreased sensitivity of SAMD activity from thioacetamidetreated rats to inhibition by some drugs (Poso et al., 1978). Similarly, a single treatment with another hepatocarcinogen, i.e.,

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carbon tetrachloride, induces ODC and SAMD activities in rat liver and stabilizes both the enzymes, since the inhibition of these enzymes by cycloheximide was less in animals treated with CCl, than in controls (Holtta et al., 1973; Poso et al., 1978). The time course of ODC induction by CC1, is roughly similar to that observed in thioacetamide-treated animals (Holtta et al., 1973). An accumulation of putrescine also occurs in the liver after treatment with carbon tetrachloride (Holtta et al., 1973). It should be noted that both these drugs, i.e., thioacetamide and CC14, are powerful hepatotoxic agents, so that it is quite difficult to assess how much of the ODC induction is connected with a specific effect of the drug on the enzyme and how much is a secondary effect due to the regeneration after liver cell destruction. Both the aforementioned drugs have another common effect on polyamine metabolism in the livers of treated animals. They very greatly stimulate the conversion of spermidine to putrescine, which is usually quantitatively negligible in untreated livers (Holtta et al., 1973). This effect is due most probably to a substantial increase in spermidine acetylation, which has been observed in liver extracts of rats pretreated with either carbon tetrachloride (Matsui and Pegg, 1980a) or thioacetamide (Matsui and Pegg, 1980b; Seiler et al., 1980~). These results further support the concept that monoacetylation of spermidine is a prerequisite for its conversion to putrescine, since, as previously mentioned in the introductory section, N'-acetylspermidine can be attacked by polyamine oxidase to yield putrescine. Accordingly, a single treatment with thioacetamide produced delayed increases (compared with that in ODC) in both polyamine oxidase activity and acetylpolyamine deacetylase activity (Seiler et al., 1980~). There is a wide variety of carcinogenic molecules and tumor promoters which, when administered in uiuo, specifically induce rat liver microsomal enzymes responsible for their metabolism and activation. These drugs have a common inductive pathway, which consists of the following events: (1)an increase in CAMPcontent and activation of the type I CAMP-dependent protein kinase, (2) ODC induction, and (3) induction of the specific microsomal drug-metabolizing enzymes. This sequence has been well established for rat liver after a single administration of 3-methylcholanthrene (Russell, 1971; Byus et al., 1976; Russell et al., 1976a; Manen et al., 1978; Russell and Durie, 1978; Russell and Haddox, 1979). The specificity of the aforementioned biochemical sequence has been elegantly demonstrated in an inbred strain of mice, called aryl hydrocarbon nonresponsive because hepatic aryl hydrocarbon hydroxylase (i.e., the niicrosomal monooxygenase enzyme

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metabolizing polycylic hydrocarbons) cannot be induced in this strain by the administration of polycyclic hydrocarbons. 3-Methylcholanthrene injected into these animals was unable to induce any significant increase in the activities of CAMP-dependent protein kinase, ornithine decarboxylase, or RNA polymerase I (Manen et al., 1978; Russell and Durie, 1978; Russell and Haddox, 1979). Recently, we have gained further insight into the genetic control of the induction of numerous enzyme activities (including ODC) in the livers of rodents by polycyclic aromatic compounds. Nebert et al. (1980) have elegantly demonstrated that there is a strong association between the Ahb regulatory allele and ODC induction by 3-methylcholanthrene and other noncarcinogenic xenobiotics. The presence of a cytosol receptor for polycyclic aromatic inducers in genetically responsive (Ahbfihb)and heterozygotic (Ahb/Ahd)mice is essential for the process of enzymic induction. On the contrary, the genetically nonresponsive (Ahd/Ahd) mice have-as a result of a mutation-an altered cytosol receptor with a very low affinity for the inducing compounds. This cytosol receptor is therefore the major regulatory gene product. Probably, the interreaction between polycyclic aromatic compounds and a single cytosol receptor initiates the sequential induction of a series of enzymes, which includes two forms of cytochrome P-450, microsomal UDP glucuronosyltransferase, NAD(P) : menadione oxidoreductase, and ODC (Nebert et al., 1980). The Ah locus thus seems to include at least two regulatory genes, five structural genes, and perhaps one temporal gene (Nebert et al., 1980). AhblAhb-and Ahb/Ahd-responsivemice, which have suitable amounts of the cytosol receptors, respond to the polycyclic aromatic inducers with high ODC induction. Moreover, the peak in ODC activity seems to precede the peak of stimulation of total cellular RNA synthesis, which in turn precedes the peak of aryl hydrocarbon hydroxylase induction (Nebert et al., 1980). Hepatic ODC activity was induced by GH to similar extents in both Ah-responsive and Ah-nonresponsive inbred strains (Nebert et al., 1980). However, in disagreement with the results cited earlier (Byus et al., 1976; Russell et al., 1976a; Manen et al., 1978; Russell and Durie, 1978; Russell and Haddox, 1979), Nebert et al. (1980) found 3-methylcholanthrene to be without effect on hepatic CAMP-dependent protein kinase activity. A single injection of either 3,4-benzopyrene7a well-known carcinogen, or phenobarbital, a tumor promoter (Diamond et al., 1980), has been shown to induce hepatic ODC activity in rats and mice (Russell, 1971; Byus et al., 1976; Russell et al., 1976a; Manen et al., 1978; Russell and Durie, 1978; Russell and Haddox, 1979). Phenobarbital is

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known to behave quite peculiarly as a promoter of hepatocarcinogenesis; it enhances the neoplastic development if given after short-term treatment with suitable hepatocarcinogens, but, if given prior to or together with the hepatocarcinogens, it inhibits tumor development in the liver (Wattenberg, 1978; Diamond et al., 1980). Phenobarbital, like 3,4-benzopyrene, is also known to induce an increase in the hepatic microsomal mixed-function oxidase system, which metabolizes it (Wattenberg, 1978). Therefore, phenobarbital has been shown to induce the same sequence of biochemical events previously described for 3-methylcholanthrene, including the induction of ODC activity, even in mice defective in aryl hydrocarbon hydroxylase (Byus et al., 1976; Russell et al., 1976a; Manen et al., 1978; Russell and Durie, 1978; Russell and Haddox, 1979). Phenobarbital, 3-methylcholanthrene, and 3,4-benzopyrene were also shown to cause an enhancement of hepatic SAMD activity in the rat, but this induction occurs later than that of ODC activity (Russell, 1971). Again, as with other carcinogens, the effects of a single carcinogenic dose of diethylnitrosamine (DENA) were a rapid increase in the hepatic CAMP-dependent protein kinase, followed by ODC induction (Olson and Russell, 197913). However, a peculiarity after such a treatment was that hepatic ODC activity remained elevated for many days, a response which has never been seen after injection of any of the physiological growth stimuli (Olson and Russell, 1979b). The modifications in hepatic polyamine levels caused by a single injection of a hepatocarcinogen have been very little investigated. Single treatments with 2-acetylaminofluorene (2-AFF) or DL-ethionine increased spermidine concentration in rat liver (regardless of the sex of the animals), whereas a treatment with 3’-methyl-4-dimethylaminoazobenzene (3’-Me-DAB) had the opposite effect, decreasing spermidine and spermine contents (Raina et al., 1964; Neish, 1967). Great increases in hepatic concentrations of putrescine, monoacetylputrescine, spermidine, and N’-acetylspermidine were observed at different times after injection in rats treated only one time with thioacetamide (Raina and Janne, 1970b; Seiler et al., 1980~). Like the liver, the urinary bladder of rodents is susceptible to a variety of chemical carcinogens. ODC and SAMD activities of urinary bladders of mice or rats were induced b y topical or p.0. administration of N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide (FANFT) or 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT), which are potent bladder carcinogens for rodents (Matsushima et al., 1979; Matsushima and Bryan, 1980). It was also observed that (1)nearly 80% of the stimulated ODC activity was located in the bladder epithelim, i.e., the cellular

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elements susceptible to carcinogenesis; and (2) the induction of ODC activity was greater than that of SAMD activity and occurred sooner after the administration of the drug than did ODC induction by hepatocarcinogens in liver (Matsushima and Bryan, 1980). As for the relationships between the chemical structure of a molecule and its ability to induce ODC, a study of a variety of 5-nitrofuran analogs demonstrated that the nitro group is essential for inducing the enzymic activity (Ertiirk et al., 1980). In studies on the connections between the synthesis of di- and polyamines in various kinds of chemically induced neoplastic growth, mammary carcinomas of female rats are one of the experimental models of choice. This neoplasm was induced by a single dose of 7,12dimethylbenz(a)anthracene (DMBA), and polyamine biosynthesis and content in the neoplastic tissue were investigated (Andersson et al., 1976). The mammary tumors showed greater ODC activity than normal mammary glands. Surprisingly, this enzyme enhancement was not accompanied by a concomitant increase in the product of this enzymatic reaction, i.e., putrescine. Of the main polyamines, the levels of spermidine and spermine, but not of putrescine, were by far higher in neoplastic tissue than in the normal control (Andersson et al., 1976). Colon carcinogenesis can be obtained by administering Nmethyl-N’-nitro-N-nitrosoguanidine(MNNG). Intrarectal instillation of this drug resulted in a dramatic and rapid induction of colonic ODC and SAMD activities (Takano et al., 1980). Very interestingly, the instillation of some physioIogica1 compounds, such as the bile salts, which are strongly suspected of having a promoting effect in colon carcinogenesis (Diamond et al., 1980), also resulted in marked ODC stimulation (Takano et al., 1980). Finally, as for polyamine catabolism in chemically induced tumors, the route of acetylation of putrescine was investigated in rat gliomas induced by a single injection of nitrosoethylurea (Seiler et al., 1975).A dramatic increase in putrescine acetylase activity and an increase in putrescine concentration were found in neoplastic tissue (Seiler et al., 1975).

B. EFFECTS O F REPEATED OR PROLONGED ADMINISTRATIONOF A CARCINOGEN OR TUMORPROMOTER ON POLYAMINE BIOSYNTHESIS AND CONTENT I N THE TARGETTISSUES Determining the polyamine content and the activities of the bios ynthetic enzymes involved after chronic treatment with a carcinogenic agent or, even better, monitoring all of them during tumor

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development helps to establish which are early derangements during neoplastic transformation, which are later ones that occur only in the developed neoplasm, and whether all or some of these derangements are characteristic of the neoplastic growth. In the rat, the changes in polyamine metabolism and content in the liver have been investigated after repeated administration of the widely used ethionine or diethylnitrosamine and during continuous feeding of 4-dimethylaminoazobenzene (DAB). In the livers of ethionine-treated rats, spermidine decreased transiently during the first days of treatment, gradually increased in the following weeks, and was still high after 6 months (Raina et al., 1964; Wainfan et al., 1978). Large doses of ethionine caused a marked fall in hepatic spermine content (Raina et al., 1964), although this was not confirmed in a later study (Wainfanet al., 1978).It is difficult to explain why these changes in hepatic polyamine patterns should be caused by ethionine, which is a well-known antimetabolite to methionine and is transformed into S-adenosylethionine. Daily treatment for several days with noncarcinogenic doses of diethylnitrosamine caused an activation of CAMP-dependent protein kinase and subsequent induction of ODC in rat liver (Olson and Russell, 197913). The extent of the ODC induction was found to be proportional to the length of treatment (Olson and Russell, 1979b). The modifications of polyamine biosynthesis and content during the hepatocarcinogenesis in rat liver due to DAB feeding have been very carefully studied and monitored by two teams almost simultaneously (Perin and Sessa, 1978; Scalabrino et al., 1978). The derangements of the polyamine biosynthetic enzymes were shown to display peculiar time courses. The activities of both polyamine biosynthetic decarboxylases, i.e., ODC and SAMD, showed marked increases as early as 1month after commencement of the dye-containing diet (Scalabrino et al., 1978). However, the increase in ODC activity was of a greater order of magnitude than that of SAMD. These increases in both hepatic enzymic activities were transient, and both enzyme activities markedly decreased during the next 2 months on the oncogenic diet (Scalabrino et al., 1978). Thereafter, a second increase in the enzyme activities occurred in the third and fourth months of azo-dye feeding, and both ODC and SAMD remained at elevated levels during the last period of the experiment. These biphasic time courses of ODC and SAMD activities, with two peaks, appear to be specific both for the liver and for the enzymes, since (a) the time course of the activities of the same enzymes in the kidneys of the same animals on the azo-dye diet from which the livers were taken were completely different from

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those in the livers and (b) hepatic tyrosine aminotransferase, an easily inducible enzyme with a very short half-life (i.e., an enzyme with some important biochemical properties in common with the two polyamine biosynthetic decarboxylases), did not display any biphasic response like that observed for ODC and SAMD in liver during azodye hepatocarcinogenesis (Scalabrino et al., 1978). The activities of spermidine and spermine synthases were constantly significantly lower in the livers of rats fed the oncogenic diet than in the control animals, the only exception being an increase in the activity of spermidine synthase at the beginning of the treatment (Scalabrino et al., 1978). As for the polyamine concentrations in rat liver during DAB carcinogenesis, the changes in putrescine concentration roughly followed the fluctuations in ODC activity, and the levels were always higher than in the controls through the entire period of experimentation (Perin and Sessa, 1978; Scalabrino et al., 1978). However, quite contradictory results have been reported for hepatic spermidine and spermine concentrations, since the significant increases in both of these polyamines observed by Perin and Sessa (1978), particularly in the last part of the experimentation, were not found by Scalabrino et al. (1978),who described decreased spermidine and spermine concentrations in livers of animals given the oncogenic diet. The rate of protein synthesis correlated well with the concentration of total polyamines in normal livers and in the livers of rats eating the DAB diet, killed every month throughout the period of treatment (Perin and Sessa, 1978). Using 3'-methyl-DABYwhich is a derivative of DAB and has greater oncogenic power than DAB, the chronobiology of the circadian rhythm of ODC in rat liver was investigated at regular time intervals from zero time until complete tumor development (Scalabrino et al., 1981). After a transient disappearance of the ODC's circadian rhythm during the first month on the oncogenic diet, this rhythm was reestablished in the livers of the rats at 60 and 90 days and then disappeared for the next 2 months. When present, the ODC rhythm in 3'-methy-DAB-treated rats had the same daily temporal pattern as that of the controls. Conversely, in the livers of rats treated with 1-naphthylisothiocyanate (a-NIT), which causes bile duct hyperplasia but never hepatic neoplasia, circadian rhythm of ODC was never detectable after only 1 month of feeding (Scalabrino et al., 1981). Therefore, although at the end of the experimental period disappearance of the hepatic ODC circadian rhythm was common to both kinds of pathological processes (i.e., hyperplasia and neoplasia), during its development each pathological process had a different, specific, well-defined change in the chronobiological pattern of the ODC rhythm. Moreover, disappear-

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ance of the ODC circadian rhythm is not a general and constant biochemical feature of all kinds of fully developed hepatomas, inasmuch as other studies have demonstrated that this rhythm disappeared in rat Morris hepatoma 7800 but not in rat Morris hepatoma 5123-C (Pariza et al., 1976a,b) (see Section 11). The alternation of presence and absence of ODC circadian rhythm might reflect changes in the cell population during neoplastic transformation. The chronobiological differences in ODC rhythm between the group fed 3’-methyl-DAB and the group fed a-NIT could be related to the different types of proliferating cells involved in the hepatic responses to the two drugs. Moreover, it has been demonstrated that, on the whole, a complete liver carcinogen such as 3’-methyl-DAB is more active in elevating ODC activity than is a simple liver hyperplastic agent such as a-NIT (Scalabrino et al., 1981). This is in good agreement with what has been demonstrated in mouse skin, where some hyperplastic agents that promote cancer weakly or not at all generally have little stimulatory effect on ODC activity, as compared with the effects of powerful tumor promoters such as the esters of the phorbol series isolated from croton oil (see Section 111,C). When we take into consideration the effects of prolonged administration of some tumor promoters, such as phenobarbital and butylated hydroxytoluene (BHT), we note that continuous feeding of the rats with either of these two drugs, surprisingly, did not enhance at all the ODC activity in liver or in lungs (Saccone and Pariza, 1978). This is in striking contrast with what has been described in Section II1,A in regard to the stimulation of hepatic ODC activity by a single injection of phenobarbital. However, the hepatic polyamine content was modified by prolonged treatment with phenobarbital, since another study demonstrated that the RNA content of mouse liver increases proportionally with the increase in spermidine and that the enhancement of DNA content was directly proportional to the spermine increase (Seiler et al., 1969). During renal carcinogenesis in the hamster due to repeated administration of 17-P-estradio1,an increase in ODC, but not in SAMD, activity was found (Nawata et al., 1980). Chronic estradiol treatment produced increases in putrescine and spennidine content in the kidneys of the treated animals, without any significant difference in spermine level between treated and control animals (Nawata et al., 1980). In a brain tumor originally induced by weekly injections of N-nitrosomethylurea and subsequently grown in tissue culture and then inoculated into the flanks of rats, the polyamine concentrations and the activities of the polyamine biosynthetic decarboxylases were

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significantly higher at the periphery of the tumor than in the center (Marton and Heby, 1974). This correlated well with the faster growth rate of the periphery than of the center of the solid tumor, as demonstrated by a markedly lower central mitotic index (Marton and Heby, 1974). C. MULTISTAGE CARCINOGENESIS Animal experiments on chemical carcinogenesis have clearly indicated that cancer development is usually a multifactorial and multistep process. Mouse skin tumors induced by aromatic polycyclic hydrocarbons are one of the most useful experimental models for studying cancer formation in uiuo. Mouse skin is certainly one of the best defined systems for studying multistage carcinogenesis. One can clearly see at least two distinct phases during chemical carcinogenesis: initiation and promotion. These two steps can be elicited by different groups of compounds (e.g., aromatic polycyclic hydrocarbons act as initiators and phorbol esters as promoters). The reader can consult the excellent reviews for details on the concept of two-stage carcinogenesis and tumor promotion, particularly in mouse skin (Boutwell, 1964, 1974; Berenblum, 1974, 1975; Scribner and Suss, 1978; Diamond et al., 1980). Here, it seems to us to be enough to recall the concepts fundamental to this topic, which are propaedeutic for understanding the derangements in polyamine biosynthesis engendered in the target tissues by this kind of carcinogenic process. A tumor promoter is a specific type of cocarcinogen that is effective only when administered after initiating action with a carcinogen given at noncarcinogenic dosages. When the sequence of the administration of the two drugs is reversed, neoplasms do not arise. In the classical model of two-stage carcinogenesis, a low, subcarcinogenic dose of a carcinogen (the initiator) is applied to mouse skin. Frequent and repeated applications of the promoter follow. Eventually, papillomas arise, but if the treatment with the promoter is further prolonged, carcinomas develop. The initiating action of the carcinogen is irreversible, whereas the effects of the promoter, which is not itself carcinogenic, are reversible. If treatment with the promoter is too meager or infrequent or discontinued too soon, tumors will not develop. Initiation could be viewed as the process of induction of some neoplastic “dormant” cells capable of being “reawakened,” whereas promotion is the process producing the appearance of the tumor and its subsequent growth (Berenblum, 1974, 1975). The conversion by the initiator of a normal cell into a “dormant” tumor cell is essentially an irreversible

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process, and a delay in the interval between completion of initiating action and the start of promoting action could not seriously affect the incidence of tumors but merely cause a corresponding delay in the time of their appearance (Berenblum, 1975). Scribner and Suss (1978) suggest that the minimal effect of an initiator is to produce isolated cells which are phenotypically identical to their unaffected neighbors with regard to functional proteins and membrane structure; these cells differ only in their ability to express altered information upon extended exposure to a promoting influence. Since only cells already transformed by initiating action can be promoted, reversal of the procedure, i.e., applying the promoting agent before the initiating agent, is ineffective in producing tumors (Berenblum, 1975). There are some conceptual points involved in two-stage carcinogenesis that are controversial and need, therefore, to be discussed briefly. First, since mutagenesis is often but by no means mandatorily connected with the carcinogenic process, the idea that initiation equals mutagenicity is quite shaky. Many strong mutagens have quite poor initiating potency (Scribner and Suss, 1978). Second, according to a former misconception, there was a prevailing tendency to consider “ cocarcinogenic action” and “promoting action” as synonymous terms, but this has been corrected (Berenblum, 1974,1975).Promoting action is only one special form of cocarcinogenic action, which has a far broader connotation. In fact, a cocarcinogen is any exogenous or even sometimes endogenous factor capable of augmenting tumor induction when it is administered with or is present with a suboptimal dose of carcinogen. Further support of the clear distinction between the two concepts of cocarcinogen and tumor promoter lies in the fact that most of the cocarcinogenic molecules do not act at all as tumor promoters in the sense defined above (Berenblum, 1974). Third, inflammation does not seem to b e a critical and obligatory step in the promotion process, since, although almost all promoters are inflammatory, not all inflammatory agents are promoters (Scribner and Suss, 1978). Therefore, an important role of inflammation in the sequential events in tumor promotion appears quite unlikely. Fourth, the real importance of the hyperplasia of the target tissues induced very frequently by promoters as an essential part of tumor promotion is uncertain, since agents inducing extensive hyperplastic stimulation have been shown to be very weak or even inactive as promoters (Scribner and Suss, 1978; Diamond et al., 1980). Experimental two-stage carcinogenesis is not limited to the mouse skin model but has been described for other organs of rodents, such as the liver, urinary bladder, colon, thyroid, kidney, lungs, mammary

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glands, stomach mucosa, and haematopoietic tissue (Berenblum, 1974; Diamond et al., 1978, 1980). Croton oil, from the seeds of the plant Crotona tiglium, has been classically employed as a tumor promoter in the mouse skin model. The promoting action of croton oil and TPA is effective for the skin of the mouse and rat, but not that of the guinea pig or rabbit. The biologically active components of croton oil are some diesters of the diterpene alcohol, phorbol. Phorbol itself is inactive in mouse skin, but, when administered intraperitoneally, is a promoter of leukemia and neoplasias of the lungs, liver, and mammary glands in rodents that have received an initiating dose of a carcinogen (Diamond et al., 1980). The relative promoting potencies of the various phorbol diesters in mouse skin are connected with their molecular structures, such as the following: (1)the steric arrangement of the phorbol molecule; (2) the presence of a free primary allylic hydroxyl group at C-20; (3)the presence of an a-@unsaturated keto group at (2-3; (4) the types of fatty acids esterified at the 12 and 13 positions; ( 5 ) among the phorbol12,13-diesters, two major groups have been identified, i.e., the A series, with a long-chain fatty acid on C-12 and a short-chain fatty acid at C-13, and the B series, with a long-chain fatty acid at C-13 and a short-chain fatty acid at (2-12, (6) esterification at C-20 or oxidation to either the aldehyde or acid at the same 20 position and the loss of the hydroxy group greatly reduce the promoting potency of the molecule; (7) the promoting activity ofthe diesters increases with increasing chain length up to 8 carbons, but, beginning with the 12-carbon length, activity markedly decreases and progressively disappears with further increasing chain length; (8)the proper steric configuration at the ring junction of C-4 and C-10 is essential for a high promoting activity of the phorbol diesters; the C-20 hydrogen and C-4 hydroxyl must be trans to maintain biological activity; when they are cis, there is great or total loss of biological activity (Fujiki et al., 1980);(9) in general, a powerful tumor promoter must have both highly lipophilic and hydrophilic portions of the molecule (Diamond et al., 1978, 1980). Other different types of diterpene esters with tumor promoting properties in mouse skin have been isolated from plants other than Crotona tiglium. Moreover, in addition to the diterpene esters, many other compounds with tumor-promoting activity in mouse skin and with a great variety of chemical structures have been identified (reviewed by Diamond et al., 1980). Among all the phorbol diesters, 12-0-tetradecanoyl-phorbol-13acetate (TPA) has been shown to have the highest tumor promoting activity in the mouse skin model. In fact, its biological and biochemi-

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cal effects were first noted and then extensively investigated in this experimental model. The structures of phorbol and TPA are shown in Fig. 6. Even a single topical application of one of the potent phorbol esters induces myriad cellular responses, both biological and biochemical. Therefore, one of the problems in the study of the mechanism of promotion has been how to distinguish between those biochemical effects elicited by the promoter that are essential for the promotion process and those effects that are collateral. Here, we will review the studies that suggest an important role of polyamines in the tumor promotion process, carried out with mouse skin, rat liver, and cell

COMPOUND

Phorbol Phorbol- 12,13- d i e s t e r s 1PA

SUBSTITUE NTS A1

12

H Fatty acid Tetradecanoate

F a t t y acid Acetate

H

FIG. 6. Structures of phorbol and its biologically active derivatives, phorbol-12,13diesters, in which the five- and seven-membered rings of the tigliane moiety are trans interlinked.

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cultures. For further details on the cellular morphological changes and the biochemical effects other than those induced by tumor promoters on polyamine biosynthesis, some excellent reviews can be consulted (Boutwell, 1964,1974,1976; Diamond et al., 1978,1980; Scribner and Suss, 1978). 1. I n Vivo Systems a. Mouse Skin Model f o r Two-Stage Carcinogenesis. With a systematic approach, O’Brien and Boutwell and their co-workers tested the effects of a large number of tumor promoters on polyamine metabolism in mouse epidermis and showed that the induction of ODC and SAMD activities is an essential component in the sequential biochemical events of promotion (O’Brien et al., 1975a,b; O’Brien, 1976, 1980). Inductions of these enzymes are among the earliest changes in biochemical systems known to occur in mouse epidermis after promoter treatment and before the general increase in total RNA and protein synthesis. In particular, the induction of ODC activity by tumorpromoting agents had been considered to be a phenotypic change essential for mouse skin carcinogenesis (0’Brien, 1976, 1980). A single topical application of croton oil or TPA, but not of phorbol, induces an astounding increase in the level of ODC activity in mouse epidermis (O’Brien et al., 1975a; O’Brien, 1976, 1980). ODC induction in mouse epidermis by TPA seems to be influenced by the age of the animals, since unresponsiveness of ODC to TPA was observed in newborn mice and in mice a few days after birth (Lichti and Yuspa, 1976). As had been observed when ODC activity was induced by other physiological or pharmacological agents, the increased epidermal ODC activity induced by TPA is predominantly located in the soluble fraction (Vermaet al., 1979a). Moreover, the induction of this enzyme activity by these tumor promoters is rapid and transient and follows a kinetics roughly similar to that observed during ODC induction by hormones in other organs of rodents, with a very sharp peak of activity 5-6 hr after the treatment followed by a return to control level after another 6 hr (O’Brien et al., 1975a; O’Brien, 1976, 1980). The stimulation of SAMD activity by croton oil and TPA was less pronounced than that of ODC, rising more slowly and showing a broader peak and a slower fall to the control level (O’Brien et al., 1975a; O’Brien, 1976, 1980). Obviously, the large increases in epidermal polyamine biosynthetic decarboxylases led to subsequent tissue accumulations of polyamines, especially of putrescine and spermidine (O’Brien, 1976). Stimulation of both the polyamine biosynthetic decarboxylases by tumor-promoting stimuli

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was dose dependent (O-Brien et al., 1975a; O’Brien, 1976). The effects of single topical applications of the different phorbol esters on ODC and SAMD activities of mouse epidermis correlated well with their promoting potencies (O’Brien et al., 197513). A possible correlation between the degree of the induction of ODC activity and the incidence of skin papillomas per mouse with the doses of TPA emerged when the amounts of TPA administered were increased (Verma and Boutwell, 1980a). The same correlation for skin carcinomas could be seen with the lowest, but not the highest, doses of TPA (Verma and Boutwell, 1980a). Pretreatment with cycloheximide at a fixed time interval before giving the tumor promoter, which was done at variable time intervals before killing the animals, resulted in an inhibition of ODC enhancement, whereas SAMD induction was prevented or decreased in some instances but not in others (O’Brien et al., 1975b; O’Brien, 1976; Boutwell et al., 1979). This would indicate that induction of the two polyamine biosynthetic decarboxylases in mouse epidermis is regulated independently (O’Brien et al., 1975a; O’Brien, 1976). In contrast, pretreatment with actinomycin D always failed to block the enzyme responses to the promoters (O’Brien et al., 1975a; O’Brien, 1976). The apparently universal ability of promoters to cause induction of the polyamine biosynthetic decarboxylases was further indicated by the observation that other promoting compounds (such as iodoacetic acid, anthralin, and Tween 60, which are chemically quite different from the phorbol esters), when applied to mouse skin in an appropriate dose schedule, also induced ODC and SAMD activities in mouse epidermis (O’Brien et aZ., 1975b; O’Brien, 1976; Boutwell et al., 1979). In addition, the kinetics of the enzyme responses after single applications were different for each of these three compounds, and each differed from the responses to the promoting phorbol esters (O’Brien et al., 197513). Most surprisingly, the kinetics of ODC induction after multiple applications of one of these three compounds closely resembled the kinetics of the enzyme response after single or multiple TPA applications (O’Brien et al., 1975b; Boutwell et al., 1979). But, the kinetics of SAMD stimulation after multiple applications of iodoacetic acid, anthralin, or Tween 60 did not greatly differ from that observed after a single application of the same drugs (O’Brien et al., 1975b). The response of ODC appears to be specific, within certain limitations, for promoter agents, whereas SAMD can be stimulated in epidermal hyperplasia produced by either promoters or hyperplastic nonpromoter agents (O’Brien, 1976,1980; Boutwell e t al., 1979). Some

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hyperplastic agents, e.g., acetic acid and cantharidin, which promote weakly or not at all, caused very little stimulation of epidermal ODC activity (and delayed that) but at the same time intensely stimulated SAMD activity (O’Brien et al., 1975b; O’Brien, 1976; Boutwell et al., 1979). A very minimal stimulation of mouse epidermal ODC was elicited by a compound such as 4-0-methyl-TPA, which is a pure hyperplasiogenic agent with very weak tumor-promoting activity (Marks et al., 1979). Therefore, the enhancement of ODC activity brought about by the promoters does not seem to be related to the stimulation of cell proliferation by these drugs, since these two biochemical and biological events, i.e., ODC induction and cell proliferation, can be suitably dissociated in mouse epidermis, as occurs with hyperplastic agents that stimulate cellular multiplication but not ODC activity. A single topical application of DMBA or other carcinogenic hydrocarbons, such as benzo [a ] pyrene, 3-methylcholanthrene, and 1,2,5,6-dibenzanthracene7at initiating doses (i.e., at low doses) to mouse skin did not at all affect epidermal polyamine biosynthetic decarboxylase activities (O’Brien et al., 1975b; O’Brien, 1976; Boutwell et al., 1979). However, when the same molecules were tested at high dosages, i.e., when they acted as complete carcinogens, epidermal ODC and SAMD activities were intensely stimulated (O’Brien et al., 1975b; O’Brien, 1976; Boutwell et al., 1979). Noncarcinogenic hydrocarbons were ineffectivein inducing ODC (O’Brien, 1976; Boutwell et al., 1979). This differential response of ODC activity to initiating and carcinogenic doses of a drug seems to further support the idea that ODC enhancement is strictly related to the promotion process (O’Brien et al., 197513; Boutwell et al., 1979). Most interestingly, ODC levels, though not SAMD levels, have been shown to correlate well with the degree of malignancy of mouse skin two-stage neoplasms, because papiIlomas had much lower ODC levels than carcinomas, and the values for both kinds of tumors were higher than those of normal epidermis (O’Brien et al., 1975b; O’Brien, 1976; Boutwell et al., 1979). It is also worth noting here that the ODC in cutaneous papillomas, in turn, had a half-life time much longer than the half-life of ODC in normal skin (O’Brien, 1976). Conversely, the half-lives of SAMD were not significantly different in normal skin and in cutaneous papillomas (O’Brien, 1976). On the grounds of all the above, O’Brien (1976) came to the conclusion that the induction of ODC activity is at least one of the essential, possibly obligatory, biochemical events in carcinogenesis of mouse

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skin. In other words, the promotion process may cause a constant increase in the ODC activity in the initiated cells and thus provide them with a biochemical tool useful for uncontrolled and prolonged growth. A large number of later studies have further clarified some other molecular aspects of epidermal ODC induction after topical application of tumor promoters. First, the induction of this enzyme by TPA is much lower in mice maintained for a suitable time on a vitamin Bedeficient diet (Murray and Froscio, 1977). It is well known that ODC requires pyridoxal-5-phosphate as a cofactor. Complementing this, the administration of pyridoxal-5-phosphate to vitamin Be-deficient mice again allowed normal induction of epidermal ODC activity by TPA, as in the control animals (Murray and Froscio, 1977). Unlike what has been described for ODC, application of TPA induced marked and similar enhancements of epidermal cell proliferation in both mice maintained on a complete diet and mice on a vitamin B6-deficientdiet (Murray, 1978). Consequently, these results suggest that there is no causal or mandatory relationship between ODC induction and induction of hyperplasia by TPA. Second, ODC induction by TPA does not appear to be mediated by earlier increases in the epidermal levels of CAMP and/or cGMP, because a single topical application of this phorbol diester at doses inducing ODC caused no increases in the levels of either of these cyclic nucleotides (Mufson et al., 1977; Mufson, 1978; Boutwell et al., 1979; Marks et al., 1979). This confirmed once more that the regulation of ODC induction in some mammalian tissues by cyclic nucleotides is by no means a general phenomenon (see Section 1,B). However, a conflicting report has appeared showing that a single topical preapplication of 3-isobutyl-l-methylxanthine, a well-known potent inhibitor of cyclic nucleotide phosphodiesterases, before the TPA application acted synergistically with TPA in increasing ODC activity, with a smaller TPA dose needed to obtain the same or even higher values of ODC activity (Perchellet and Boutwell, 1980a). Therefore, whether cyclic nucleotides are really the second messengers for the growth-related increases in RNA and protein syntheses that occur in the epidermis after TPA application remains a moot question. Third, it has been claimed that prostaglandins El and Ez are involved selectively in the induction of epidermal ODC activity by TPA, since application of PGEl and PGE2, but not of PGFl and PGFk, with the application of TPA resulted in removal of the inhibition of ODC induction caused by pretreatment with indomethacin or acetylsalicylic acid, well-known inhibitors of prostaglandin synthesis (Verma et al., 1978). But indomethacin pretreatment did not prevent the induction of SAMD by TPA (Verma et al., 1978). Furthermore, simulta-

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neous application of PGE2, but not of PGF2, and TPA reversed the blockage of the cell proliferative response of mouse epidermis to TPA induced by pretreatment with indomethacin (Furstenberg and Marks, 1978). Therefore, unlike vitamin B6deficiency, the PGEs seem to be factors involved in controlling both ODC induction and cell proliferation caused by TPA. Fourth, since one of the routes by which ornithine is synthesized is via hydrolysis of arginine by arginase to yield ornithine and urea, the effect of TPA application on arginase activity in mouse epidermis has been tested (Vermaet al., 1979a). Arginase activity was unresponsive to such treatment, indicating that this enzyme is not a limiting step in the stimulation of polyamine biosynthesis by TPA (Verma et al., 1979a). The foregoing leading idea that epidermal ODC activity is nearly exclusively stimulated by tumor-promoting agents, whereas nonpromoting stimulators do not have this effect, has been critically revised in some recent studies. It has been demonstrated that epidermal ODC activity is strongly increased not only by TPA but also by Ti 8*, which is a TPA structural analog that has essentially no tumorpromoting power but still has mitogenic and irritant properties similar to those of TPA (Marks et al., 1979). Mezerein, too, which is a diterpene ester of plant origin, like TPA, and has many close structural similarities to TPA, has been tested for its biological and biochemical effects (Mufson et al., 1979). Mezerein, although it is equipotent with TPA on an equimolar basis in inducing hyperplasia, in inflammatory activity, and especially in inducing ODC activity in mouse epidermis, is a very weak mouse skin tumor promoter (Mufson et al., 1979). Ethylphenylpropiolate (EPP), another good inflammatory and hyperplasiogenic compound with weak tumor-promoting power, caused a marked increase in both ODC and SAMD activities in mouse epidermis after a single topical application (Takigawa et al., 1977; Boutwell et al., 1979). In this case, too, the induction of ODC activity changed in parallel with concomitant changes in skin putrescine concentrations (Takigawa et al., 1977). These authors doubt .the idea that the tumor-promoting potency of a compound can be exactly mirrored by its effect in inducing ODC activity. In order to further clarify the links, if any, between ODC induction and the tumor-promotion processes, another experimental approach has been employed in recent years that consists of the replacement of chemical tumor-promoting stimuli with mechanical ones. Cutaneous papilloma development has been “initiated” by a carcinogen mole* Ti 8; 12-0-tetradeca-2-cis,4- trans-6,8-tetraenoylphorbol-l8acetate.

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cule, i.e., DMBA, administered in a subthreshold dose; thereafter, the mechanical treatments were either skin massages (which cause cell proliferation and are devoid of any tumor-promoting power) or skin wounding (which also induces cell proliferation and is, additionally, a promoting stimulus). A marked enhancement of epidermal ODC activity was observed after skin wounding but not after skin massages (Clark-Lewis and Murray, 1978). On the other hand, TPA has been found to stimulate ODC activity in rat skin, a tissue in which it was not previously known to have been a tumor promoter (Lesiewicz et al., 1979, 1980).The ODC activity of rat skin was stimulated additively by TPA application plus hair plucking (Lesiewicz et al., 1979, 1980). The ODC stimulation by either TPA or hair plucking has been found to be much greater in the rat epidermis than in the rat dermis (Lesiewicz et al., 1980). The time course of the ODC response to TPA differs in several aspects from that to hair plucking (Lesiewicz et al., 1980). Aerosolized TPA was also shown to induce pulmonary ODC activity in mice in a dose-dependent manner (Dinowitz et al., 1980). In conclusion, one can reasonably think that all these results combined argue for the idea that mouse skin tumor-promoter stimuli, both chemical and mechanical, generally cause hyperplasia, irritation, and ODC induction, but the reverse is not true, i.e., no stimulus that elicits all of these three biological responses has necessarily to be considered a tumor promoter. Consequently, this triad of biological and biochemical reactions to the tumor promoters is nearly a constant in the tumorpromotion process but is not sufficient per se to accomplish this process. Therefore, the relevance of these and other enhancements of some cellular activities induced by the promoting phorbol esters to the mechanism of skin tumor promotion is still unsolved. Similarly, it is still a matter of speculation whether there is a causal relationship between ODC induction and the tumor-promotion process, on the one hand, or between ODC induction and cell proliferation, on the other. The particular aspects of the stimulation of polyamine metabolism in the two-stage protocol of mouse skin carcinogenesis have been reviewed by Slaga et al. (1978), Boutwell et al. (1979), Lesiewicz and Goldsmith (1980), and Lowe (1980). b. Models of Two- or Multistage Carcinogenesis in Livers of Rodents. There are very few studies available in the literature on this subject. No enhancement was seen in ODC activity in the livers of rats that received diethylnitrosamine (DENA) in drinking water and then, after discontinuation of this nitrosamine, were given phenobarbital in the diet, although phenobarbital greatly increased the incidence of liver tumors induced by DENA, i.e., was very effective as a tumor promoter for rat liver (Farwell et al., 1978). Neither was hepatic ODC

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activity induced by DENA alone or by phenobarbital alone (Farwell et al., 1978). Again, most surprisingly, no increase in ODC activity was found in fully developed hepatocarcinomas induced by DENA only or resulting from phenobarbital promotion of DENA carcinogenesis (Farwell et al., 1978). A three-stage model of carcinogenesis for rat liver has been proposed by Solt and Farber (1976). The typical sequence of such a protocol consists of a DENA injection as initiator, followed by a low level of 2-acetylaminofluorene (AFF)in the diet for a short time period, acting as a selective growth inhibitor, and finally followed by partial hepatectomy, acting as a generalized potent growth stimulus. This sequence of treatments cannot be reversed without greatly reducing the incidence of hepatic neoplasms. In this multistage carcinogenesis model, CAMP-dependent protein kinase and ODC activities were investigated (Olson and Russell, 1979a). The activities of these two enzymes were enhanced, first one and then the other, shortly after DENA administration and remained elevated for several days. After waning to the control levels, the activities of both these enzymes were stimulated once again by partial hepatectomy and remained high for two weeks thereafter (Olson and Russell, 1979a). Therefore, in this three-stage model of hepatic carcinogenesis, enhancement of ODC activity in the target organ appears to be tightly linked with the carcinogenetic process, which is quite different from what has been previously described for the two-stage model for the same organ. As in the skin protocol, one of the two stimuli in the two-stage scheme for hepatic carcinogenesis can be surgical. It is well established that rapidly proliferating rat hepatocytes, namely, regenerating liver, are more sensitive to the carcinogenic effects of urethan than resting cells. Injection of this drug has been shown to suppress to similar extents both the typical biphasic ODC enhancement and the increase in intracellular cAMP levels, both induced in the liver remnant by partial hepatectomy (Matsui et al., 1978, 1980). However, after this early suppression phase, ODC and SAMD activities again rose in regenerating livers of rats that were given urethan, i.e., enzyme inductions were only delayed in comparison with regenerating controls (Matsui et al., 1980).Unlike liver regeneration, the enhancement of hepatic ODC activity by several hormones was not suppressed and the intracellular cAMP level not modified b y administration of urethan (Matsui et al., 1978). Inasmuch as urethan is known to inhibit in vivo hepatic DNA and RNA syntheses, it must be clarified whether the parallelism between the suppression of ODC induction and the impairment of DNA synthesis expresses a cause-effect relationship and whether the inhibition of ODC activity is an obligatory step in the

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molecular mechanism by which urethan induces neoplasms. This last would represent an interesting and perhaps unique exception in ODC responses to carcinogens, since, as previously demonstrated by a large body of evidence, carcinogens generally have the ability to induce ODC activity in target organs.

2. In Vitro Systems TPA and other promoting phorbol esters have been tested for ODC induction and other effects on polyamine metabolism in different in vitro cellular systems, which can be classified into those consisting of mouse epidermal cells and those consisting of cells other than epidermal ones. Such a division is justified in order to tentatively compare the effects in in vitro systems with those in in vivo systems, although this extrapolation must be made with great caution. In fact, at present it is still questionable whether the in vitro tumor promotion process is an exact mirror of the same process in viuo. Nevertheless, all in vitro systems can generally be used as tools for investigating and dissecting the molecular mechanisms involved in tumor promotion. Certainly the mouse epidermal cell culture system appears to be by far the most appropriate for studying the promotion process in vitro, since the tumor promotion concept was first defined in the mouse skin model of carcinogenesis in vivo. a. Cultures of Mouse Epidermal Cells. The effects of a series of phorbol esters, including both those with a wide range of tumorpromoting potency and those without any, on DNA synthesis and ODC activity have been investigated in mouse epidermal cell cultures (Yuspa et al., 1976). A good correlation between the effectiveness of the phorbol esters as tumor-promoting agents in mouse skin in vivo and their ability to stimulate DNA synthesis and ODC activity in vitro was found (Yuspa et al., 1976). The kinetics of the ODC induction were roughly similar to those observed in the same cells in vivo, and this enzymic stimulation chronologically occurs much earlier than stimulation of DNA synthesis (Yuspa et al., 1976). By analogy, similar results were obtained in cultured epidermal cells from newborn mice (Lichti and Yuspa, 1976; Lichti et al., 1978a). Moreover, the induction of ODC activity by TPA in cultured mouse epidermal cells was completely prevented by the previous or contemporaneous presence of cycloheximide in the medium, indicating that the enzyme induction requires de novo protein synthesis (Lichti et al., 1978b). In contrast, actinomycin D at doses that completely inhibit total RNA synthesis paradoxically enhanced ODC induction by TPA (Lichti et al., 1978b). ODC induction by TPA in isolated epidermal cells appeared to be CAMP and cGMP dependent, because TPA treatment enhanced the

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cellular levels of both cyclic nucleotides, and the simultaneous presence of 3-isobutyl-1-methylxanthine (a well-known inhibitor of cyclic nucleotide phosphodiesterase) potentiated the ODC induction by the promoting agent (Perchellet and Boutwell, 1980a,b). In the aforementioned system of cultured cells, the magnitude of ODC induction was also dependent on the duration and manner of TPA treatment, and, what is more, it changed considerably at various times after plating, since the cells first lost and then regained their responsiveness to TPA (Lichti et al., 1978a). Furthermore, the correlation between the intensity of the stimulation of ODC activity in vitro and the potency of the tumor promoters in vivo is not a general and constant feature of tumor-promoting agents tested in uitro, since this correlation was barely observed for several Tween compounds with a wide range of tumor-promoting potencies (Lichti et al., 1978a). Unlike its effects on ODC activity, TPA treatment was found to produce a dramatic and quite unexpected fall in SAMD activity in cultured epidermal cells, which was also unlike the stimulating effect on SAMD activity observed in vivo (Lichti et al., 1978a). Further evidence of the dissociation of induction of ODC activity from stimulation of DNA synthesis, both caused by TPA in cultured mouse epidermal cells, has been provided by Hennings et al. (1978), who investigated the effects of varying cell culture conditions, such as pH and serum content of the medium, on these two biochemical parameters. TPA induced identical increases in DNA synthesis regardless of differences in serum levels during treatment, although an optimal serum level is required for maximal cellular proliferative responses to TPA. But the absence of serum in the medium greatly reduced ODC induction by TPA, thus indicating that the increase in ODC activity, which always chronologically precedes DNA synthesis, is not a necessary prerequisite for the stimulation of DNA synthesis. Again, lowering of the pH of the culture medium affected ODC induction and stimulation of DNA synthesis b y TPA to different extents, since DNA synthesis and cell proliferation were inhibited at pH 6.7 more than ODC response (Hennings et a,?., 1978). The presence of serum in the incubation medium for mouse skin explants allows much more protracted ODC induction than that observed in the same cultures in the absence of serum (Verma and Boutwell, 1980b).As for the cultured epidermal cells, the induction of ODC activity in incubated mouse skin explants is dependent on the concentration of TPA in the medium (Verma and Boutwell, 1980b). b. Cultures of Cells Other than Epidermal Cells. The relevance of the studies with cultures of nonepidermal cells reported here can be questioned, since the types of cells used are not the natural target cells

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for TPA’s promoting activity in wiuo. In cultures of hamster embryo cells (fibroblasts), a variety of tumor-promoting phorbol diesters was found to induce ODC activity (O’Brien and Diamond, 1977; O’Brien et al., 1979). The same result was found in another cell line derived from hamster embryo cells transformed malignantly b y benzo(a)pyrene. TPA caused a much greater induction of ODC activity in the transformed cells than in the normal ones, although the basal ODC levels of the two types of cell lines were quite similar (O’Brien and Diamond, 1977, 1978a, 1979; O’Brien, 1980). ODC activity was also induced by adding fresh medium to both kinds of cell line cultures, but the magnitude of the enzyme induction was greater in transformed cells than in normal ones (O’Brien and Diamond, 1977, 1978a, 1979; O’Brien, 1980). Likewise, when fresh medium and TPA were added simultaneously, the stimulating effects on ODC activity in normal cells were approximately additive, whereas they were synergistic in transformed cells (O’Brien and Diamond, 1977, 1978a; O’Brien et al., 1979, 1980). This and the earlier results suggest that ODC activity in malignant cells is more sensitive to positive stimuli, either promoting or not, than are normal cells. Other examples, besides that of ODC activity, of enhanced responses to TPA by transformed cells are known and involve morphological changes or metabolic activities, e.g., prostaglandin synthesis (the corresponding references are cited in Diamond et al., 1978). The potentiation of ODC induction by combined TPA and fresh medium appears to be specific for this enzyme, since no similar synergistic or additive effect for SAMD activity was seen in the same cultured cells, either normal or transformed, under the same experimental conditions (O’Brien, 1980; O’Brien et al., 1980). On the other hand, TPA alone induced SAMD activity in both cell types, i.e., normal and transformed (O’Brien, 1980; O’Brien et al., 1980). In transformed cells, ODC enhancement by TPA produced a parallel effect in putrescine concentration but not in spermidine and spermine (O’Brien and Diamond, 1977; O’Brien, 1980). In contrast, no significant changes in polyamine concentrations were observed in normal cells after treatment with TPA (O’Brien, 1980). Unlike ODC induction in cultured normal mouse epidermal cells, the TPA-induced increase in enzyme activity in transformed cells was completely inhibited by cycloheximide and by actinomycin D (O’Brien and Diamond, 1977, 1978a, 1979). Another difference in biological responses to TPA of cultured normal epidermal mouse cells and hamster embryo fibroblasts, whether transformed or not, is that in the latter type of cells DNA synthesis and cell division were not affected by exposure to tumor promoters (O’Brien and Diamond, 1977, 1978a, 1979; O’Brien, 1980).

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Other than in hamster embryo cells, TPA stimulated ODC activity in 3T3 mouse cells and PHA-treated bovine lymphocytes, but not in human fibroblasts or rat embryo fibroblasts (Kensler et al., 1978; O’Brien et al., 1979). Furthermore, TPA stimulated DNA synthesis only in 3T3 mouse cells and human fibroblasts (O’Brien et al., 1979). Therefore, no mandatory relationship between ODC induction by TPA and stimulation of DNA synthesis by the same molecule can be drawn. TPA loses its ability to induce ODC in both normal and transformed hamster embryo fibroblasts when it is completely converted by the cells into phorbol-13-acetateYwhich is not further metabolized and has no ODC inducing activity (O’Brien and Diamond, 1978b). Surprisingly, although some human cell lines were found to be unable to metabolize TPA, the same cell cultures were unable to induce ODC activity by TPA (O’Brien, 1977, 1980; O’Brien and Diamond, 1978b; O’Brien et al., 1979). Several recent studies have suggested that an early and possibly primary site of action of tumor-promoting phorbol esters is the cell membrane, where they might modify structure or function. Recently, it has been demonstrated that the phorbol ester tumor promoters act, at least in part, through a specific hormonal pathway, since these molecules and the neurohypophyseal hormone vasopressin stimulate some biochemical parameters of quiescent cultured mouse Swiss 3T3 cells by a common mechanism (Dicker and Rozengurt, 1980). In fact, TPA and vasopressin can substitute for each other in stimulating DNA synthesis and 2-deoxyglucose uptake and in inducing ODC activity (Dicker and Rozengurt, 1980). TPA and vasopressin added simultaneously to the culture medium show neither additive nor synergistic effects in enhancing all these biological activities. In contrast, TPA has been shown to act synergistically with some well-known growthstimulating polypeptides or hormones, such as insulin and epidermal growth factor (EGF), in stimulating some biochemical parameters (cited in Dicker and Rozengurt, 1980). However, the combination of TPA and insulin was synergistic for ODC induction in chemically transformed hamster embryo fibroblasts, but not in hamster fibroblasts (O’Brien et al., 1980). A very important question, as yet not fully understood, is whether removal of regulation from polyamine biosynthesis could be a key difference between neoplastic transformation and normalcy. Besides the differences in polyamine metabolism between normal and transformed cells previously reported, support for this idea has been provided by O’Brien et d . (1980), who demonstrated that there is normal production in untransformed hamster fibroblasts of the ODC antizyme elicited by putrescine, whereas there was little or no production in

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transformed cells similarly treated with putrescine. This difference in ODC antizyme production occurred despite nearly equal sensitivity of the ODC activities of both cell lines to the inhibitory effects of exogenous putrescine on the enzyme activity (O’Brien et al., 1980). The polyamine metabolic abnormalities plus other metabolic derangements induced by tumor-promoting phorbol esters (especially TPA) in different cell lines in vitro have been reviewed by Diamond et al. (1978, 1980) and O’Brien (1980). To summarize, some major points should be recalled and stressed here : 1. From the results so far reported, it is quite clear that there is no one unique and common behavior of polyamine metabolism after the different phorbol ester treatments in uitro. This is not at all surprising and is in perfect agreement with a large body of experimental evidence that illustrates the great diversity of biological effects produced by promoters in different in vitro systems. Therefore, no general rule valid for every in vitro system and for the effects of every tumorpromoting agent can be drawn. Consequently, the exact role played by polyamines in the tumor-promotion process in vitro is still a matter of speculation. Frequently, however, some similarities in the responses to phorbol esters observed in comparable in vitro systems can be seen and tentatively interpreted. 2. TPA and related compounds induce a large number of biological alterations in cell cultures that mimic those often associated with cell transformation by chemical carcinogens or oncogenic viruses (Diamond et al., 1978). 3. The expression of certain cellular “markers” of transformation in phorbol ester-treated cultures depends on the continuous presence of the agent, so that the normal cells exposed to a promoting phorbol ester can revert to their previous phenotype once the agent is removed. Clearly, this is in striking contrast to the situation in malignant cells, in which the expression of these properties is autonomous. 4. When the biological actions of initiating carcinogens (usually complete carcinogens by themselves) and promoting agents are compared, the most striking difference lies in the fact that initiators can yield electrophil and bind covalently to cellular macromolecules, such as proteins and nucleic acids, whereas there is, up to now, no evidence that promoters bind covalently to these macromolecules. 5. The possibility that phorbol esters have completely different effects in different tissues and species, converting and recognizing the

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initiated cells to neoplastic cells in one species or tissue but not in another, cannot be ruled out. 6. The importance of the studies with two- or multistage carcinogenesis models is not at all limited to the experimental field, since there is ever-increasing evidence that tumor-promoting agents, known or unknown, in the environment may be factors that contribute to the induction of tumors in human beings.

D. MUTAGENICACTION A N D ANTIMUTAGENIC PROPERTIES OF POLYAMINES

1. Mutagenic Efects of Nitroso Derivatives of Spermidine and Spermine It is well known that the “mutation” theory and the “aberrant differentiation” theory have long been discussed as separate mechanisms for carcinogenesis; recently, the “mutation” theory has received more support, since most carcinogens have been found to be mutagens. The ability of carcinogens to bind to DNA and chromatin, resulting in altered structure and function of genetic material, can easily explain their mutagenicity. In this respect the finding that nitrosated spermidine has mutagenic properties in a biological assay system utilizing Salmonella typhimurium is of great interest (Kokatnur et al., 1978; Hotchkiss et al., 1979). Both spermidine and spermine contain secondary and primary amine groups which can be nitrosated by nitrite. Nitrosation can occur during the digestion of numerous foods containing large amounts of spermidine, spermine, and nitrite. Nitrite is produced in human saliva, and its involvement in N-nitroso compound formation in the gastrointestinal tract is reasonable to suppose. It is well known that the most recent views on the etiology of human gastric cancer have focused on the hypothesis that some N-nitroso compounds formed in the stomach are directly responsible, as mutagenic carcinogens, for this type of neoplasia. A variety of N-nitrosamines which arise from nitrosation of spermidine, many of which have been demonstrated to be or are strongly suspected to be carcinogenic, have been identified and characterized (Hildrum et al., 1975; Hotchkiss et al., 1977). Some of these products are volatile, some are not. The mutagenic action of nitroso derivatives of polyamines and the involvement of these products in the etiology of cancer has recently been reviewed (Murray and Correa, 1980).

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2. Antimutagenic Properties of Spermidine and Spermine Spermidine and spermine, because of their basic nature, bind noncovalently to the phosphate residues of DNA and neutralize their negative charges. Thus, the ordered structure assumed by DNA in the presence of spermidine prevents the intercalation of 3,4-benzo[a] pyrene into DNA (Liquori et al., 1967). Spermine, on the other hand, again because it binds to the minor groove of DNA and subsequently stabilizes DNA structure, inhibits the methylation of both chromatin DNA and free DNA induced by N-methyl-N-nitrosourea (MNU), a well-known and potent carcinogenic and mutagenic agent, in eukaryotic cells (Rajalakshmi and Sarma, 1978; Rajalakshmi et al., 1978a,b). It is not known at present whether the protective effects of spermidine and spermine against carcinogen-DNA interaction are limited to methylating agents, such as MNU, and polycyclic hydrocarbons, such as 3,4-benzo[a]pyrene or can be extended to other types of carcinogens, The antimutagenic properties of spermine and other polyamines in microbial systems have been reviewed (Clarke and Shankel, 1975).

E. POLYAMINE LEVELSIN URINE, SERA, AND ERYTHROCYTES OF CHEMICAL CARCINOGENESIS OR BEARINGSEVERAL TUMORS EXPERIMENTAL

RATS DURING

As has been done in humans with different types of neoplasms (see Section 11, Part I1 of this chapter in Vol. 36), the urinary polyamine levels in experimental animals undergoing chemical carcinogenesis or acute treatment with carcinogens have been monitored by several authors. A significant increase in urinary putrescine only was observed in mice which had received a single carcinogenic dose of 3,4-benzo[al pyrene given subcutaneously (Fujita et al., 1978).I n more detail, the urinary putrescine level began to be significantly increased 2 months after the 3,4-benzo[ alpyrene administration, then became even more pronounced 1 month later, reaching a maximum at the fourth month (Fujita et al., 1978). Significant increases in the urinary levels of total polyamines, as well as of each of the chief polyamines in DABhepatoma-bearing rats have been recorded, but the increases of putrescine levels were less than those of spermidine and spermine (Perin and Sessa, 1978). In rats with mammary carcinomata induced by DMBA, only the 24-hr urinary excretion of putrescine was found to parallel tumor

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growth and mass (G. Andersson et al., 1980). But, when the neoplasm was in the final stages of its progression, the correlation disappeared, since the urinary concentration of putrescine decreased unexpectedly (G. Andersson et al., 1980). In a previous study, other authors found only slight increases in the urinary excretion of putrescine and spermidine in rats with the same type of neoplasm induced by the same carcinogen (A. C. Andersson et al., 1976). In the urine of rats with experimental gastric tumors induced by chronic administration of N-methyl-N-nitroso-N’-nitrosoguanidine, there was a significant and early elevation in the putrescine level that became even more marked during the final phases of gastric carcinogenesis (Fujita et d., 1976). In the urine of rats that had received a subcutaneous implant of an immunocytoma, a statistically significant circadian rhythm was apparent for the excretion of putrescine, spermine, and cadaverine, but not for spermidine (Halberg et al., 1976). At all six time points of the day, the rate of excretion for all four polyamines was higher in tumorbearing rats than in control rats, with the overall rate being about twice as great as in controls (Halberg et aZ., 1976). As for serum, rats with chemically induced brain tumors transplanted into flanks had serum spermidine concentrations markedly higher than the serum values of control animals (Marton and Heby, 1974). The same was found in rats with mammary tumors (Russell et al., 1974b,c; Russell and Durie, 1978). When this tumor regressed after castration and hypophysectomy, the serum level of spermidine behaved bimodally, first increasing and then decreasing (Russell et d., 1974b,c; Russell and Durie, 1978). The increase in the serum paralleled an analogous trend of the spermidine levels in the tumor’s interstitial fluid (Russell et al., 1974b,c; Russell and Durie, 1978).These data suggest that the elevated level of spermidine in the sera of rats with tumors in regression phase is mainly a result of spermidine release from the tumor tissue, due to cell death. In further support of this interpretation, the same bimodal time course was observed for the spermidine level in the sera of rats with a Morris hepatoma after effective antineoplastic chemotherapy (Russell et al., 1974a,d; Russell and Durie, 1978). Again, shortly after a single injection of the antineoplastic drug used, 5-fluorouracil, the spermidine concentration within the tumor markedly decreased owing to a marked decrease in the number of hepatoma cells, just at that time when the spermidine concentration in the serum doubled (Russell et al., 1974a,d; Russell and Durie, 1978). By studying the polyamine levels in the sera of rats with this same tumor after local radiation, a rapid increase in spermidine was

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once again seen, but an unexpected similar change in putrescine level was found concomitantly (Russell et al., 1976b; Russell and Durie, 1978).As was observed after chemotherapy, the spermidine concentration within the hepatoma rapidly decreased after local radiation (Russell et al., 1976b; Russell and Durie, 1978). Other studies have carefully investigated the importance of the cellloss factor of a neoplasm in determining elevations of or modifications in the levels of the extracellular polyamines in the sera of animals with different types of tumors. Actually, the concentrations of putrescine and spermidine in cell-free ascites fluid and serum of mice with Ehrlich ascites tumor cells were found to increase greatly not only during tumor growth but also when there was a considerable decrease in the rate of cell proliferation (Heby and Anderson, 1978). That tumor cell death is the cause of increased polyamine levels in physiological fluids in cancer has been cleverly confirmed by inducing tumor cell death by heterologous transplantation of the same type of mouse tumor into gerbils and by following the tumor cell loss (G. Anderson et al., 1978a; Heby et al., 1978a).The concentrations of polyamines in cell-free ascites fluid and serum showed patterns similar to those of the activity of tumor lactate dehydrogenase in the same fluids, the peaks of the time courses for polyamines and lactate dehydrogenase being closely coincident with the time of maximal tumor cell death (G. Andersson et al., 1978a; Heby et al., 1978a). As in patients with malignant disease, polyamine levels in peripheral erythrocytes have been measured in animals with different types of neoplasms. A positive correlation between spermidine and spermine levels in red blood cells of mice with Ehrlich ascites carcinoma cells and the tumor growth has been established, since the increases in the levels of these two polyamines occurred very soon after i.p. inoculation of the ascites carcinoma cells (Uehara et al., 1980). By analogy, erythrocyte spermidine concentration increased with increasing tumor mass in rats transplanted subcutaneously with a fast-growing solid hepatoma (Shipe et al., 1980; Wills et al., 1980). In contrast, in this situation erythrocyte spermine concentration did not at all reflect increases in tumor mass (Shipe et al., 1980). The levels of the main polyamines have been compared in plasma and in red blood cells of mice transplanted with melanoma (Takami and Nishioka, 1980). The levels of all the measured polyamines in both erythrocytes and plasma rose as the number of days after tumor inoculation increased (Takami and Nishioka, 1980). However, the levels of putrescine, spermidine, and spermine in erythrocytes showed progressive increases much greater than those in plasma (Takami and Nishioka, 1980).

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Mathematical models have been formulated to relate tumor cell number to intracellular polyamine concentrations and, utilizing the pharmacokinetics, to predict the polyamine compartmentalization between plasma and organs in which neoplasms have been developed (Woo and Simon, 1973; Himmelstein et al., 1976; Woo and Enagonio, 1977; Himmelstein and Rosenblum, 1980). From all the results reported here concerning the polyamine levels in the urine, the fluid part of blood, and/or the erythrocytes of experimental animals with different types of neoplasms, no single, clear, unique indication has emerged about whether or not an increase of one or more polyamines in the physiological fluids or cells is of any real help in evaluating the growth rate of the experimental tumors. Therefore, the value of polyamine determinations in these biological compartments as a tool for diagnosis or prognosis of cancer remains questionable. One must also keep in mind that high polyamine levels in physiological fluids and/or erythrocytes of experimental animals have been observed in nonpathological conditions, such as during pregnancy and lactation or after hormone administration in the rat (A. C . Andersson et al., 1978b; Lundgren and Oka, 1978; Rojansky et al., 1979; Andersson and Henningsson, 1980b) or in mice after intraperitoneal injection of bovine serum albumin (Uehara et al., 1980). These conclusions on the nonspecificity of the increases of polyamine levels for tumors and on their promise for evaluating the effectiveness of antineoplastic therapy are in keeping with what will be stressed and discussed in the section on human oncology (see Section 11, Part I1 of this chapter in Vol. 36). IV. Biosynthesis and Levels of Polyamines in Cells during the Virus-Induced Transformation Process

Oncogenic viruses are not only of great importance in the etiology of some animal tumors, but some human tumors are also associated with and actually strongly suspected to be caused by viruses. Since the original demonstration by Rous of sarcoma induction by a chicken virus, two distinct classes of cancer-causing viruses have been identified in a range of vertebrate species. Some are DNA viruses and others RNA viruses. Among the DNA viruses of vertebrates, some members of the papova-, adeno-, herpes-, and poxvirus groups are known to be oncogenic for different animal species. Among the RNA viruses, all known oncogenic viruses belong to one group, i.e., the retraviruses, also called retrovirus or oncornavirus (Fraenkel-Conrat, 1974).

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A normal cell population is transformed by infection with an oncogenic virus, followed by isolation of colonies of cells with altered properties. Alterations are found in cellular morphology, there is piling up of cells that normally appear to grow as monolayers, and there are a myriad of biochemical changes at both the surface and the nonsurface level of the cell. Therefore, transformation is defined operationally; it is useful for the identification of virus-induced changes in cultured cells, but it is not a universal correlate of oncogenicity. Most DNA tumor viruses and the sarcoma-inducing retroviruses cause cellular transformation, while leukemia-causing oncornaviruses usually cause cell growth without transformation, with some exceptions. For this reason, the leukemia viruses are often called “nontransforming viruses’’. Furthermore, many of the properties used to define transformation can be transiently induced in normal cells by experimental manipulations, e.g., treatment with proteolytic enzymes. Tumor viruses are obviously defined by their capacity to induce neoplasms in animals. By the same token, transformed cells are neoplastic only if they can grow into tumors in an appropriate host. Nonetheless, although cancer is a disease definable only in whole animals, an analog of malignancy called “cellular transformation” provides an in vitro model on which almost all work with oncogenic viruses is based. In fact, the phenotypic changes which serve to identify transformation of cultured cells are indispensable aids in the investigation of tumor viruses. The normal cells used for transformation studies generally are of two types: cells taken from embryos, often from chicks or rodents, and permanent lines of mammalian cells that can be cloned. Experimental models that involve the use of oncogenic viruses have been devised to test the correlation of polyamine synthesis with neoplastic growth. These models have definite advantages, stemming from the rapidity of transformation and from the ability to perform kinetic experiments with normal and malignant cells derived from the same source and grown under well-controlled and identical experimental conditions. In this section, the changes in polyamine biosynthesis and content of different cells undergoing neoplastic transformation by the oncogenic viruses will be discussed. Polyamine metabolism has been studied in several virus-infected cell systems. Among the systems most extensively studied in vitro are cell transformations by the DNA papovaviruses, such as polyoma or SV40, and by the RNA viruses, such as Rous sarcoma virus or murine sarcoma virus.

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A. EFFECTSO F ONCOGENIC RNA VIRUSES

RNA tumor viruses possess attractive features for study of the mech. anisms involved in cell transformation. These viruses cause rapid and highly efficient transformation which is reproducible under welldefined cellular conditions. The changes can be induced synchronously, so that sequential events can be analyzed. Systematic studies of transformation of chick embryo fibroblasts by different strains of Rous sarcoma virus were carried out by Bachrach and his co-workers. These authors found that transformed chick embryo fibroblasts did not differ from normal counterpart cells in their changes in protein and RNA content occurring during growth (Bachrach et al., 1973, 1974). However, transformation is accompanied by significant alterations in polyamine content, since a continuous rise in putrescine content was observed in transformed cells but not in normal ones, in which the intracellular content of this diamine reached a plateau when the cell became confluent (Bachrach et al., 1973, 1974; Bachrach, 1978).The difference between normal and transformed cells was magnified during subculturing or in old cultures (Bachrach et al., 1974). No significant differences between normal and transformed cells were noticed in the intracellular concentrations of spermine and spermidine (Bachrach et al., 1973, 1974). A connection of this increase in intracellular putrescine content with the viral transformation has been demonstrated with a temperature-sensitive mutant of Rous sarcoma virus, which induces transformation only at the permissive temperature (37°C) but multiplies at both the permissive and the nonpermissive (42°C) temperatures. The chick embryo fibroblasts increased their putrescine content only when viral infection by the temperaturesensitive mutant was carried out at permissive temperature (Don et al., 1975; Bachrach, 1978). When the temperature of transformed chick embryo fibroblasts was shifted from 37°C to 42"C, putrescine content markedly decreased, whereas putrescine content rose dramatically when the temperature shift was reversed, from 42°C to 37°C (Don and Bachrach, 1975; Don et al., 1975; Bachrach, 1978). The same was found to be true for in vivo infections of chorioallantoic membranes of chick embryos with the same strains of Rous sarcoma virus. Infection at the permissive temperature caused an increase in spermidine and putrescine content, and no significant change in the content of these two polyamines occurred between uninfected membranes and membranes infected at the nonpermissive temperature (Don et al., 1975). This makes it possible to distinguish between virus multiplication and the transformation process and to see if either is

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connected with some specific alterations in intracellular polyamine content. Further proof of this was provided by showing that chorioallantoic membranes infected with different types of nononcogenic viruses had the same polyamine levels as uninfected membranes (Don et al., 1975). ODC activity has been measured in the chick embryo fibroblasts using a temperature-sensitive mutant of Rous sarcoma virus and shifting of the temperature (Don and Bachrach, 1975; Bachrach, 1978). ODC activity has been shown to behave similarly and in parallel with that described above for intracellular putrescine content, i.e., it rose after viral transformation at the permissive temperature and dropped nearly to control levels when the temperature was shifted to the nonpermissive level (Don and Bachrach, 1975; Bachrach, 1978). It is worth noting that Rous sarcoma virus induced ODC enhancement before the appearance of morphological alterations in the cell (Don and Bachrach, 1975; Bachrach, 1978). Therefore, ODC elevation is also an early metabolic change in the cells related to viral transformation. Moreover, one of the effects of cell transformation with different strains of Rous sarcoma virus is a lengthening of the ODC half-life, which also occurred only at the permissive temperature (Bachrach, 1976a, 1978). Like ODC, the SAMD activity was increased by viral transformation under permissive conditions, whereas uninfected cells and those infected at the nonpermissive temperature had nearly the same low levels of enzyme activity (Bachrach and Wiener, 1980).Also like ODC, SAMD responded to temperature shifting from nonpermissive to permissive, but the increase in the enzyme activity was slower than that of ODC (Bachrach and Wiener, 1980). Unlike ODC, the half-life of SAMD did not significantly change in chick embryo fibroblasts transformed with the same temperature-sensitive mutant of Rous sarcoma virus at the permissive temperature (Bachrach and Wiener, 1980). The increase in ODC activity in virus-transformed cells has also been demonstrated with other cell types and with other RNA viruses. In fact, using mouse BALB/3T3 cells and murine sarcoma virus, which contains both a transforming virus and a nontransforming strain of murine leukemia virus acting as a “helper” virus, it was demonstrated that ODC activity markedly rose in cells acutely infected and transformed by murine sarcoma virus (Gazdar et al., 1976; Bachrach, 1978; Kilton and Gazdar, 1978). In contrast, infection with the nontransforming virus, the helper virus, had no effect on the cellular ODC levels (Gazdar et al., 1976; Bachrach, 1978; Kilton and Gazdar, 1978). Again, infection with Rauscher leukemia virus, another nontransforming virus, caused no enhancement of cellular ODC levels

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(Gazdaret al., 1976).The enhancement of cellular ODC activity seems truly to be connected with the acute transformation process, since ODC levels fell with successive cell passages, while different permanently transformed cell clones showed a wide range of ODC activities (Kilton and Gazdar, 1978). The cultured rat fibroblasts transformed by Rous sarcoma virus had a higher resting ODC level than the normal cells (Haddox and Russell, 1979). Like normal cell lines (see Section 111,C72),cultured fibroblasts transformed by either Rous sarcoma virus or temperature-sensitive mutant responded to the addition of either TPA or serum by enormously increasing ODC activity (Haddox et al., 1979,1980). However, the stimulation of ODC activity by serum, unlike that by TPA, was followed by increased intracellular content of polyamines in both normal and transformed cell lines tested (Haddox et al., 1979). Serum-dependent ODC induction in both normal and transformed cells was inhibited completely or nearly so by cycloheximide or actinomycin D (Haddox et al., 1980). The ODC increase in transformed cells in response to fresh media and serum addition was greater than in the normal cells (Haddox and Russell, 1979). Again, in cells infected with a thermosensitive mutant of Rous sarcoma virus, the induction of ODC activity after the addition of medium containing fresh serum was much greater at the permissive temperature than at the nonpermissive temperature (Haddox et al., 1980). It is of importance to note that ODC activity is induced to a greater extent during the G1 phase in viral-transformed fibroblasts than in the G1 phase of their normal counterparts (Haddox and Russell, 1979; Haddox et al., 1980), at which phase of the cell cycle the cells are well known to be more susceptible to malignant transformation by chemicals or by viruses (Baserga, 1977). This is notably due to the increased accessibility of chromatin in G1 cells to carcinogenic agents (Baserga, 1977). Furthermore, the enzyme increase in normal cells was totally dependent on serum growth factors, whereas in the Rous sarcoma virustransformed cell lines it was not, since addition of fresh medium alone to the transformed cells was enough to induce ODC activity (Haddox et al., 1980). Another difference between normal and transformed cells in ODC regulation is the lesser sensitivity of serum-dependent ODC induction to inhibition by putrescine in transformed cells than in normal ones (Haddox et al., 1980). All these results raise the possibility that altered regulation of ODC activity is one of the key features of the neoplastic transformation by viruses. Finally, polyamines can regulate the genome expression of mouse

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mammary tumor virus in mouse mammary tumor cells grown in semisynthetic medium (Svec and Links, 1977). In the absence of serum, the production of mouse mammary tumor virus was stimulated by spermidine, but not by spermine (Svec and Links, 1977). However, the stimulation by spermidine was by far less than that elicited by dexamethasone under the same experimental conditions (Svec and Links, 1977). In cultures preincubated with serum, no increase in the synthesis of mouse mammary tumor virus after spermidine treatment was observed (Svec and Links, 1977). B. EFFECTSOF ONCOGENICDNA VIRUSES Using mouse 3T3 cells transformed by SV40 virus, it was possible to show some differences between normal and transformed cells in the ODC responses to the same inducing stimuli (Lembach, 1974). First, untransformed cells required a significantly higher serum concentration to maintain comparable levels of ODC activity than did the transformed cells; this is in keeping with analogous results of Haddox et d. (1980) with a different experimental model. Second, marked increases in the level of ODC activity were observed in both untransformed and transformed cells when the extracellular concentration of serum was increased, but the increases in transformed cells were invariably higher than those in the normal ones (Lembach, 1974). This point was confirmed by Bethel1 and Pegg (1979a). Third, low serum levels that failed to induce ODC activity in 3T3 cells elicited increase in the enzyme level in transformed cells. Fourth, a serum free of gammaglobulins had little stimulating effect on ODC activity of normal cells, but in transformed cells significant increases in enzyme levels were elicited by that kind of serum (Lembach, 1974). Fifth, the SV40-3T3 cells required much greater concentrations of putrescine or spermidine to produce a decrease in ODC activity similar to that in their normal counterparts (Bethel1 and Pegg, 1979a,b). This suggests a possible difference between transformed and nontransformed cells in sensitivity to a decrease in ODC activity caused by the diamine or the polyamine. Also consistent with the idea that important qualitative differences is ODC regulation between normal and virus-transformed cells really exist in the significant finding that the specific activity of the ODC purified from an SV40-transformed 3T3 cell line is approximately eight times that of the ODC from normal tissues (Boucek and Lembach, 1977). In keeping with and in further support of the foregoing idea are the results of the investigations of Isom (1978, 1979). Arrested human fibroblast cells infected with human cytomegalovirus

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(which is a member of the herpesvirus group and is strongly suspected to be somehow involved in causing some human neoplasias) produced a stimulation of ODC activity far greater than that observed in mock infected cells (Isom, 1978, 1979). The ODC stimulation was specific for the viral infective process, since human cytomegalovirus inactivated by UV irradiation did not stimulate cellular ODC activity and human serum containing antibodies neutralizing the cytopathic effect of the virus in vitro also prevented the ODC stimulation (Isom, 1979). Moreover, by selectively blocking the activity of viral DNA polymerase without preventing the stimulation of cell DNA synthesis induced by the virus, ODC induction by virus was remarkably inhibited, suggesting that viral DNA synthesis is required for ODC induction (Isom, 1979). The significance of the abnormal ODC regulation in virus-infected cells is as follows: (1) The enzyme induction by virus was not sensitive to the inhibiting effect of putrescine added to growth medium, unlike the enzyme induction triggered by high serum medium in uninfected cells (Isom, 1978, 1979), but ODC prepared from either infected or uninfected cells was sensitive to the inhibitory action of polyamines in vitro (Isom, 1979; Isom and Backstrom, 1979). This insensitivity of ODC to putrescine was found to be peculiar to the late-passage cells (Isom and Backstrom, 1979). (2)In cells transformed by cytomegalovirus, the addition of fresh medium containing a high serum percentage enhanced ODC activity to a much greater extent than in normal cells (Isom and Backstrom, 1979). (3) ODC of the early-passage transformed cells was completely resistant to the inhibitory activity of spermidine, whereas ODC of the late-passage cells was less inhibited than that of the normal ones by this polyamine (Isom and Backstrom, 1979). Infection of mouse kidney cell cultures with polyoma virus caused biphasic increases in the activities of ODC and SAMD as well as in the intracellular levels of the three chief polyamines (Goldstein et al., 1976; Heby et al., 1976, 197813).This biphasic response consisted of a first peak that occurred shortly after the infection but before the onset of virus-induced host cell DNA synthesis and a second peak that occurred much later, corresponding temporally with the peak of virusinduced cell DNA synthesis (Goldstein et al., 1976; Heby et al., 1976, 1978b).With different types of inhibitors, it was possible to investigate better the connections between polyamine biosynthetic decarboxylases and DNA and rRNA syntheses. When 5-fluorodeoxyuridine completely blocked the virus-induced synthesis of cellular DNA, the time courses of the changes in ODC and SAMD activities and the polyamine levels in the infected cells treated with the inhibitor were

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virtually the same as those in the untreated cells (Goldstein et al., 1976; Heby et al., 1978b). Using actinomycin D at some critical values for its concentrations in the medium and the time of the exposure of the cells to the inhibitor increased ODC activity in virus-infected cells, i.e., ODC “superinduction” occurred (Goldstein et al., 1976; Heby et al., 1976, 1978b). SAMD activity was very seldom superinduced. Cycloheximide inhibited the activities of both the polyamine biosynthetic decarboxylases in the polyoma virus-infected cells (Goldstein et al., 1976; Heby et al., 1976, 197813). All these results show that in certain instances polyamine biosynthesis can be uncoupled from DNA and rRNA syntheses and that ODC and SAMD syntheses can be regulated independently. Polyamine uptake and metabolism have also been investigated in different cell lines transformed by different herpesviruses. The rate of putrescine uptake into MRC-5 cells was found to be increased markedly immediately after infection by human cytomegalovirus (Tyms and Williamson, 1980). The incorporated putrescine served as a precursor for spermidine and spermine syntheses, since the syntheses of these two polyamines were remarkably enhanced in the cells after viral infection and paralleled the putrescine changes (Tyms et al., 1979; Tyms and Williamson, 1980). Increase in the synthesis of virus DNA was also observed concomitantly with. the stimulation of polyamine metabolism (Tyms and Williamson, 1980).Conversely, the conversion of labeled ornithine or putrescine to labeled spermidine and spermine in different cell lines (both normal and neoplastic) infected with herpesvirus-1 or herpesvirus-2 was decreased (Gibson and Roizman, 1971, 1973; McCormick and Newton, 1975; McCormick, 1978b; Tyms et al., 1979), although an initial increase in the rate of putrescine uptake has been observed after cell infection with this kind of herpesvirus-1 (McCormick and Newton, 1975). These effects seem to be the result of inhibition of host protein synthesis after infection (McCormick and Newton, 1975). Unfortunately, the inhibition of polyamine metabolism in cultured cells infected by herpesvirus was not confirmed by Francke (1978), who found a large and steady increase in cell putrescine concentration and a small increase in spermidine concentration without changes in the spermine. There was also a difference in polyamine metabolism between normal and neoplastic cells, both infected by herpes simplex virus type 1 (HSV-l), in that mouse fibroblasts (L-cells) became extremely permeable to polyamines early in infection, whereas neoplastic cells (HeLa cells) did not (McCormick, 1978b).

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Finally, spermidine and, more especially, spennine, which is associated with the viral DNA in HSV-1 (Gibson and Roizman, 1971; Roizman and Furlong, 1974), have been shown to be positive effectors of the HSV-1 DNA polymerase (Wallace et al., 1980). In contrast, putrescine, which is not present in the virion, had little effect on the polymerase reaction (Wallace et al., 1980). Furthermore, the triamine and the tetraamine, but not the diamine, inhibited the deoxyribonuclease induced in KB cells by HSV-1 or HSV-2 ( H o h a n n and Cheng,

1978). In conclusion, the qualitative differences in ODC regulation between normal and virus-transformed cells seem to us of particular interest as a promising direction to follow using different cell lines transformed by several oncogenic viruses. It is, in fact, well known that the viral transformation process of the cells causes alterations in the cell membrane and cell functions (Nicolau, 1978). It is therefore possible that changes in the receptor sites for putrescine and spermidine are responsible for the decreased sensitivity of the ODCs of some different virus-transformed cell lines to the inhibiting effects of the diamine and the polyamine. This concept is in keeping with the suggestion of Canellakis et al. (1978)that malignant cells continue to synthesize and excrete putrescine and polyamines even in the presence of extracellular polyamine concentrations that are known to normally depress ODC activity inside the cell. V. Changes in Polyamine Biosynthesis and Content of Target Tissues by Physical Carcinogens

Among the physical carcinogens, only the effects of ultraviolet light on polyamine biosynthesis in target tissue have been tested. The UV spectrum, the portion of the electromagnetic spectrum between visible light and X rays, is conventionally divided into three major regions: short-wave, with a wavelength of 250-290 nm (also called “germicidal” light), sunburn (UV-B), and long-wave, with a wavelength of 320-400 nm. Ultraviolet light is a complete carcinogen, possessing both initiating and promoting properties. The effective carcinogenic range of UV light is 250-320 nm (Freeman, 1975). ODC has been induced in the epidermis of several species of mammals by UV of different wavelengths. In hairless mice, very shortly after exposure to UV light (UV-B, mostly 290-320 nm), the epidermal ODC began to rise, reaching a peak at approximately the end of the

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first day after irradiation, and then declining gradually (Lowe et al., 1978; Boutwell et al., 1979; Verma et aZ., 1979b; Lowe and Breeding, 1980a,b; Peterson et al., 1980). ODC induction was found to increase progressively with increased numbers of exposures of mouse skin to ultraviolet light in the sunburn range (Lowe and Breeding, 1980b). The induction of epidermal ODC activity by UV-B irradiation was found to be dose dependent (Boutwell et al., 1979; Verma et al., 1979b). The magnitude of ODC induction also paralleled the histopathological lesions that could be observed (Verma et al., 1979b; Lowe and Breeding, 1980b). The time course of incorporation of tritiated thymidine into epidermal cells was not at all parallel to the time course of ODC in the same cells, since DNA synthesis decreased during the entire time that ODC activity increased and was enhanced when ODC declined (Lowe et aZ., 1978; Verma et al., 1979b). ODC induction in mouse epidermis by UV-B light appears to require de nooo synthesis of both protein and RNA, since injection of cycloheximide shortly before killing or 5-azocytidine shortly before UV-B irradiation greatly diminished the ODC stimulation (Verma et al., 1979b). SAMD activity was also induced in mouse epidermis after UV-B irradiation, but the increase was slightly delayed with respect to the ODC response (Boutwell et al., 1979; Verma et al., 197913). ODC induction by UV has also been observed in depilated human epidermis, but the maximum of the induction was later than in mice, occurring 48 hr after irradiation (Lowe et al., 1980). Germicidal ultraviolet light was shown to induce ODC activity in cultured mouse epidermal cells (Lichti et al., 1979, 1980) and to modify the polyamine pattern of mouse epidermis in vivo (Seiler and Knodgen, 1979a). The ODC induction was biphasic and was prevented by exposing the cells to actinomycin or cycloheximide, suggesting the involvement of both transcriptional and translational control of ODC induction (Lichti et al., 1979, 1980). ODC induction was dose dependent, within limits (Lichti et aE., 1980). When the cells were irradiated with germicidal ultraviolet light and then treated with TPA, the effects were additive only under certain particular experimental conditions, suggesting that induction of ODC by UV and its induction by TPA can be reasonably assumed to go through at least partially separate pathways (Lichti et al., 1980). The epidermal polyamine pattern was modified in vivo by germicidal ultraviolet light (Seiler and Knodgen, 1979a). Putrescine concentration rapidly increased within a few hours after irradiation and then declined gradu-

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ally. Spermidine concentration increased as well, but at a much slower rate than that of putrescine, and remained at elevated levels for a much longer time than putrescine. Spermine concentration decreased and, amazingly, reached its minimum when DNA synthesis was greatest (Seiler and Knodgen, 1979a). Whether ODC induction in epidermis by ultraviolet light is relevant to the carcinogenesis triggered by it is still only speculative. Part I of this review has covered polyamines and their metabolism in normal tissues and in chemical, physical, and viral carcinogenesis and cell transformation. Part I1 will appear in Volume 36 and will cover various aspects of polyamines in cancer; namely, polyamine biosynthesis and concentrations in different lines of cultured neoplastic cells; polyamines in human oncology; diamine oxidase activity in human or experimental neoplasms; physiological and pharmacological inhibitors of polyamine biosynthesis in neoplastic tissues or cells; and concluding remarks and speculations for both Parts I and 11.

Ac KNOWLEDGMENTS First, we are very grateful to Professor S. Weinhouse (Philadelphia), for his understanding of our delays. Thereafter we wish to thank those authors who kindly sent to us manuscripts of unpublished but accepted papers: Dr. E. S. Canellakis (New Haven), Dr. K. Y. Chen (New Brunswick), Dr. S. S. Cohen (Long Island), Dr. H. Desser (Vienna), Dr. J. M. Gaugas (Northwood), Dr. 0. Heby (Lund), Dr. U. Lichti (Bethesda), Dr. P. S. Mamont (Strasbourg), Dr. P. McCann (Cincinnati), Dr. D. Morris (Washington), Dr. K. Nishioka (Houston), Dr. G. Quash (Lyons), Dr. A. M. Roch (Lyons), Dr. N. Seiler (Strasbourg), and Dr. T. Slotkin (Durham). Although we have continued to reevaluate the topic while writing and have included new papers as they appeared, we know that we have not avoided all errors or lacunae. We apologize to those investigators whose works we have inadvertently not cited. We are deeply indebted to Professor E. Ciaranfi (Milano), who several years ago introduced us to this field and taught us to love polyamines. We also thank Professor A. Bemelli-Zazzera (Milano) for his interest and advice. We gratefully acknowledge the helpful criticism by Professor U. Bachrach (Jerusalem)of our outline for this work. One of us (G. S.) also thanks Professor J. Janne (Helsinki), in whose laboratory he had the opportunity several years ago to deepen his understanding of some modem aspects of polyamine biosynthesis regulation. To our young co-workers, Dr. M. Puerari and Dr. D. Modena, we express our gratitude for their patient help in organizing and revising the manuscript. Last, but not least, to Dr. B. Rubin (Milano) we extend our warmest thanks for her invaluable editorial assistance in revising the English of the manuscript.

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CRITERIA FOR ANALYZING INTERACTIONS BETWEEN BIOLOGICALLY ACTIVE AGENTS Morris C. Berenbaurn Wellcome Laboratories of Experimental Pathology. Variety Club Research Wing, St. Mary’s Hospital Medical School, London, Great Britain

Introduction .......................................................... Effect Multiplication .................................................. Effect Summation.. ................................................... Agent Interaction and Self-Interaction .................................. V. Isoboles and the Interaction Index ..................................... A. Homergic Combinations ........................................... B. Heterergic Combinations .......................................... C. Isobole Construction from Limited Data ............................ D. Inadequacy of 2 x n Experimental Designs ......................... E . Diagonal Arrays ................................................... VI. Criteria Based on Changes in Dose-Response Curves ................... VII. Modifications of the Isobole Method ................................... A. Response Isoboles ................................................. B. Additivity Envelopes .............................................. VIII. Therapeutic Optimization ............................................. IX. Conclusion ........................................................... References ...........................................................

I. 11. 111. IV.

269 273 280 285 288 288 294 295 298 301 304 313 313 317 322 329 332

I. Introduction

The idea that different agents may interact at the pharmacological level to modify each other’s effects is familiar to all engaged in cancer chemotherapy, with its predilection for combinations of agents, and to those interested in environmental carcinogenesis. One agent may affect another’s absorption, metabolism, or excretion; it may alter tissue sensitivity to another agent; it may even react with it physically or chemically. Unequivocal evidence of such interactions may be obtained when one of the agents in a combination has a particular effect of interest (e.g.,an antitumor effect) and the other has not. Combinations of methotrexate and folinic acid, of 6-mercaptopurine and xanthine oxidase inhibitors, and of cyclophosphamide and thiols are of this nature. In such cases, the antitumor activity of the combination is attributable to only one agent. If there were no interaction between the agents, we would expect the effect of the combination to be just the 269 ADVANCES IN CANCER RESEARCH, VOL.35

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effect of the active agent. If, however, the effect of the combination differs from that of the active agent given alone, this necessarily implies that the agent’s effect has been modified and, therefore, that an interaction has taken place. It is also generally held that, in some sense, we may envisage interactions between agents that share the effect of interest. Indeed, there is a large literature that discusses biochemical and other mechanisms responsible for such interactions (Venditti and Goldin, 1964; Sartorelli, 1965; Handschumacher, 1965; Grindey et al., 1975; Gillette, 1976; Steel and Peckham, 1979). However, it is difficult to define what is meant by these sorts of interactions at the level of the observed effect. The problem is that, if two or more agents in a combination have, say, an antitumor effect, then each contributes its share to the effect of the combination. If we knew what effect to expect if the constituents of the combination did not interact, then we would be able to tell whether the observed effect of the combination was equal to this expected effect and, thus, whether its constituents did or did not interact. Unfortunately, in contrast to the case in which the effect is attributable to one constituent only, there appears to be no obvious way to determine this expectation. Do we expect the effect of the combination to be the sum of the effects of its constituents, or their product, or some more complex function of the individual effects? None of these possibilities seems more self-evidently correct than any other. Accordingly, a variety of different criteria have been devised for deciding whether the agents in a combination interact pharmacologically, and the purpose of this review is to examine these critically, for progress in this field is undoubtedly hampered by the incorrect conclusions that are frequently drawn on the basis of faulty or inappropriately applied criteria. It must be said at the outset that this field is characterized by widespread confusion in basic ideas accompanied by near anarchy in terminology. There is a remarkable compartmentalization between different groups of investigators; cancer chemotherapists, radiobiologists, pharmacologists, microbiologists, and immunologists all tend to use more or less exclusive sets of concepts and criteria. Even within these subgroups, there is no consensus as to what is meant by such commonly used terms as synergy and additivism. Different words are used to describe phenomena which appear to be the same, and one and the same term is used in entirely different senses by different authors. Matters are not helped by a fondness for inventing compIex sub-

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classifications that, again, vary from one author to another. In fact, some reviewers have expressed the opinion that, although consistent terminology and criteria would be highly desirable, the discord is so great that this is simply impossible to obtain (Goldin and Mantel, 1957; Tyrrell, 1978). The sophisticated reader must therefore be warned that much of what follows is presented in what may seem an unduly simple way. No apology is made for this. Confusion is so entrenched in this field, and contradictory opinions so firmly held, that no attempt to clarify matters has any hope of success unless every step in the argument is explicitly justified. For the purposes of this review, only three classes of interaction will be recognized. The way in which they are described is intentionally left imprecise for the present, as the required precision will be derived from the discussion that follows. The interactions are (1) zero interaction, in which the effect of a combination is precisely what is “expected.” This may also be termed additivism, but it should be understood that this term in itself implies nothing about how the operation of “addition” is performed in the context of drug interactions; (2) positive interaction, or synergy, in which the response is greater than “expected’’; and (3) negative interaction, or antagonism, in which the response is less than “expected.” As they stand, these definitions are intuitive and vague rather than formal. They imply the existence of a frame of reference against which expectation may be judged, but they do not state what that frame of reference is. Their merit is that they constitute probably the largest area of common ground among workers in this field and, thus, enable us to avoid preconceived ideas in developing the argument. This primary classification into three classes of interaction does not preclude finer subdivision, but that is not the concern of this review, which has as one of its main aims a clearing of the ground. In the relevant literature, synonyms for what is here called zero interaction or additivism are independence and indifference. Synonyms for positive interaction or synergy are potentiation, augmentation, sensitization, supraadditiveness, etc. Synonyms for negative interaction or antagonism are depotentiation, desensitization, infraadditiveness, negative synergy, etc. At the risk of further confusing the reader, it should be noted that some authors (Dewey et al., 1971; Han and Elkind, 1978) use the term additivism to include cases of synergy, and others (Gaddum, 1943, 1959) use the word synergy to include additivism and even some cases of antagonism.

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As indicated above, the idea of an interaction between agents carries with it the implication that the effect of the combination is greater or less than what is expected from the effects of the agents given alone. The crux of the matter, then, is what is to be “expected.” Plainly, expectation here is not to be derived from an understanding of mechanisms of action of the agents because, in that case, given sufficient information, the effects of all combinations would eventually be shown to be precisely what was “expected” and all would then have to be deemed to show zero interaction. By expectation, we mean that, if we know the magnitude of the effects of agents A, B, C, etc., then we would somehow know how to calculate the magnitude of the effect of a combination of these, given that they did not interact pharmacologically. A comparison of the expected with the observed effect of the combination would then tell us whether the agents in the combination did interact and, presumably, allow us to calculate the extent of the interaction. It should be pointed out at this stage that, in this review, a combination of agents always means a combination of particular doses of these agents, and these doses are then termed the constituents of the combination. This particularity is necessary, for different dose combinations of the same set of agents may have very different effects and show different types of interactions. The following plan will be adopted in this review. First, two commonly used criteria for determining interactions will be described. These are, respectively, effect multiplication and effect summation; they attempt to calculate the effect of a zero interaction combination from the effects of its constituents. These criteria may be justified by reference to simple and biologically plausible models, which will be used to define the different types of interaction quantitatively. It will be shown, however, that these two criteria are valid only for agents with particular types of dose-response curves and that the effectmultiplication criterion requires, in addition, homogeneity of the responding population. The need for a more general framework will thus be apparent. As this more general set of criteria must be independent of the forms of dose-response curves or population characteristics, it cannot be “explained” by reference to one particular biological model. Instead, we examine precisely what is meant by pharmacological interaction between agents (as opposed to the pharmacological interaction of an agent with itself) and thus construct a zero-interaction model that is generally valid and that can be described algebraically and unambiguously, without any need for ill-defined concepts such as expectation.

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It. Effect Multiplication

According to this criterion, it is assumed that when agents do not interact the effect of a combination is the product of the effects of its constituents. To see under what circumstances this sort of relationship might hold, we may use an analogy put forward by Wilcox (1966). Suppose that nails are being thrown randomly at a collection of 1000 eggs and that any egg hit by one or more nails breaks. The nails act quite independently of each other since, as they are discrete objects and are thrown at random, the probability of an egg being hit by any one nail has no effect at all on the probability of its being hit by another and, since one hit is enough to break an egg, there can be no cooperation between nails in breaking individual eggs. Now suppose that 1 bushel of nails (1 dose unit) breaks 90% of the eggs, leaving 100 intact. A second dose unit also hits 90%of the eggs, including 90%of those that are still intact, so that now 10 eggs survive. A third dose breaks 90% of these, leaving one. As the nails all act independently of each other, the effect of throwing any fixed number will be the same, irrespective of how the total dose is subdivided. For example, if 2 dose units are thrown, 10 eggs will remain, whether the nails are all thrown simultaneously, as 2 doses of 1 unit each, or even one nail at a time. Now, if we regard a dose of 2 units as being constituted by 2 doses of 1 unit, then, in terms of fractional survival, the effect of 2 units is precisely the product of the effects of its constituents, i.e., 10/1000 = 100/1000 x 100/1000, and similarly, the effect of a dose of 3 units (survival of 1/1000)is the product of the effect of 3 doses of 1 unit (or of a dose of 2 units and a dose of 1 unit). Here, then, is an example of an agent that behaves as if the effect of any dose is the product of the effects of its constituent doses. The doseeffect relation here is obviously given by the simple exponential-type equation SD = lo-”, where S D is fractional survival after dose D (Fig. 1). The biological relevance of this model lies in the fact that it convincingly explains the shapes of cell survival curves that may be given by ionizing radiation. When a single “hit” by a radiation particle on a single critical cellular target is sufficient to kill the cell, then the doseresponse curve for cell survival will be of this simple exponential type, as shown by classical target theory (Crowther, 1924; Condon and Terril, 1927; Lea, 1955). Survival curves given by alkylating agents also may be of this form (Walker and Helleiner, 1963; Crathorn and Roberts, 1965; Skipper et al., 1965; Bruce et al., 1966; Berenbaum, 1969), and we can postulate here that cell death is caused by the

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-m

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reaction of a single molecule of agent with a single critical cell target. If more than one radiation particle or drug molecule is required to destroy a target, if there is more than one critical target per cell, or if the cell has mechanisms that repair the damage, the curve is not of a simple exponential type but has an initial shoulder (Atwood and Norman, 1949; Lea, 1955; Elkind and Whitmore, 1967; Turner, 1975). Such curves may be produced, not only by ionizing radiations, but also b y ultraviolet light (Haynes, 1964; Patrick and Haynes, 1964; Han and Elkind, 1977), hyperthermia (Dewey et al., 1977), and alkylating agents such as nitrogen mustard (Haynes and Inch, 1963), dimethyl myleran (Goldenberg, 1968; Alexander, 1969), methyl methanesulfonate (Fox and Nias, 1968), cyclophosphamide (De Wys and Kight, 1969), and BCNU (Hahn et al., 1974; Leenhouts e t al., 1980). The discussion in this section does not refer to curves of this kind but only

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

275

to simple exponential curves. Exponential curves with shoulders will be examined extensively later. Now let us extend this useful analogy. Suppose we also throw pebbles at the eggs and, as before, any egg hit by one or more pebbles breaks. The effects of the nails and the pebbles are also independent of each other, i.e., these two different agencies do not interact, for the probability of an egg being hit by a nail is unaffected by the probability of its being hit by a pebble and vice versa. Suppose that 1 dose unit of pebbles hits and breaks half the eggs. After doses of 1,2, and 3 units of pebbles, the fractions of eggs surviving are 500/1000, 250/1000, and 125/1000, respectively. I. Note again that the effect of any “dose” of pebbles is precisely the product of the effects of its constituents (Fig. l ) . ] Suppose now that we throw both nails and pebbles at the eggs. As the effects of the nails and the pebbles are independent, it does not matter whether we imagine them to be thrown simultaneously or one after the other. To simplify matters, let us suppose that 1 unit of nails is thrown first, leaving 100 unbroken eggs. Then a unit of pebbles is thrown, and this breaks 50%of these, leaving 50/1000 surviving. Now the effect of this combination (1 unit nails plus 1 unit pebbles) is precisely the product of the effects of its constituents (lOO/lOOO x 500/1000 = 50/1000). The reader can easily confirm that the same result would follow if the order in which the agents are delivered is reversed, that this general relationship holds for any combination of doses, and that it can be generalized to combinations of more than two different agents. Thus it appears that, when agents behave as if the effect of a dose of any one agent equals the product of the effects of its constituent doses, the combinations of these agents behave in the same way provided the agents do not interact. Explicitly, if E,, E b , etc., are the effects of particular doses ofA, B, etc., then Ea+b

= Ea

Eb

(1)

Synergistic combinations are more effective than expected, and antagonistic agents less effective. Therefore, according to this criterion, the relationship for the former is E,+b > E , X E b and for the latter E a + b < E, X E b . This analogy may be placed on a more general footing as follows. The dose-effect relation for the effect of nails is given by the equation S, = lo-” where S, is fractional survival after a “dose” n of nails. Similarly, the relation between fractional survival Sp and “dose” p of pebbles (in units) is S, = 2-P. To bring both expressions to the same basis, we note that

276

MORRIS C. BERENBAUM

S = e-Atn

and

S, = e-A2P

where hl and h2 are constants (in this case hl = In 10 and A t = In 2). In general, therefore, agents to which this sort of argument is applicable have dose-response curves that are of a simple exponential type, i.e., they are straight lines when survival on a logarithmic scale is plotted against dose on a linear scale (Fig. 1).Then, if al,a2 are two doses of agent A, Sa1 = e - h a i , s0 2 = e-Aaz = e-A(a~+az) = e-Aa~ . e-A@2 = S a1 * sa, (2) Sal+aZ That is, the effect of a combination of any two doses of A is the product of the effects of those doses. This may be generalized to any number of doses, For any particular combination of two agents A and B, we have SA = e-AtA SB = e-A& SA+* = e-AtA-ArB = e - h ~ A . e-A& = S A . S B (3) 9

That is, the effect of a combination of A and B is the product of their individual effects, as required by the effect-multiplication criterion. This may be generalized to any number of agents with dose-response relationships of this sort, provided the agents do not interact with each other. A possible source of confusion should be mentioned here. Some authors, in using the effect-multiplication criterion, assert that they are adding effects (Jawetz and Gunnison, 1953; Martignoni and Haselbacher, 1979). However, it is apparent from the context in these cases that it is not the effects as measured that are being added, but the logarithms of these effects, which is equivalent to multiplying the effects themselves, not adding them. Although this simple model sufficiently validates this criterion when the agents are of the specified type, many workers appear to have derived the criterion from probability theory, apparently assuming that if agents do not interact, their effects may be treated as statistically independent events as defined by probability theory. Their reasoning is not explicit, but appears to be as follows. Let A and B be statistically independent events. Then, from probability theory (Mosteller et al., 1970; Whittle, 1970), if the probabilities ofA and B are P (A) and P (B),respectively, then the probability of at least one of A or €3 occurring, P(A U B), is given by P(A U B ) = P(A) + P ( B ) - P(A) * P ( B )

(4)

The next step in the argument is to equate the probabilities of events A, B, andA U B with fractional kills of individuals or cells (Bliss, 1939;

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

277

Finney, 1942, 1952; Valeriote and Lin, 1975) or fractional inhibitions of enzymes (Webb, 1963; Grindey et al., 1975) after administration of agentsA, B , orA + B. Now fractional kill ofcells or fractional inhibition of an enzyme F = 1 - S, where S is fractional survival or fractional residual enzyme activity. In the model examined above, a hit by either A or B (Le., eventA U B) is sufficient to inactivate the target, so it is the probability of this event that is relevant here. Therefore, Eq. (4) is rewritten as

1 - SA+B

=

(1 -

s.4)+ (1 - S B ) - (1 - s.4) (1 - s,) ’

(5)

Therefore, S A + B = SA * Ss, which is Eq. (3) for a zero-interaction combination ofA and B. The effect-multiplication criterion is probably the one most widely used by cancer research workers; it is based on a biologically plausible model and gives rise to no inconsistencies when applied to agents that themselves have dose-response curves that obey the effectmultiplication rule. Unfortunately, it is widely and incorrectly assumed that this criterion can be applied to any sets of agents, irrespective of the nature of their dose-effect curves. The error lies’in equating the effects of agents with statistically defined events and their probabilities. However, the independence of events as defined in probability theory refers to all the events under consideration, so that not only do events attributable to agent A have to be independent of those attributable to agent B ,but the events attributable to each of these agents separately must also all be independent of each other, as in the analogy of nails and pebbles described previously. In other words, it is correct to multiply effects for combinations of agents only when it is correct to multiply them for each agent separately, i.e., when each agent has a simple exponential dose-response curve. Let us apply this criterion to agents that do not have simple exponential curves, e.g., the agents whose curves are shown in Fig. 2. Here, 1 mg of each agent leaves 30% of cells surviving, and according to the effect-multiplication criterion, if A and B do not interact with each other, a combination of 1 mgA and 1 mgB should leave 30% X 30% = 9%cells surviving. If, in fact, it is found experimentally that a combination of 1 mgA and 1 mg B leaves only 1%survivors, then according to the effect-multiplication criterion the combination is highly synergistic. However, the dose-response curves in Fig. 2 show that the effect of 2 mg of this combination is exactly the same as the effect of 2 mg ofA or 2 mg B alone, so that we can hardly sustain the proposition that these agents are more effective in combination than separately. In

278

MORRIS C. BERENBAUM

0

1

A (mg/kg)

2

0

1

2

B (mg/ kg)

FIG.2. Survival curves for A and B , which may be different or identical agents. This model is used to test various criteria for zero interaction (see text). Note that, in contrast to Fig. 1, survival after any particular dose D is not the product of the survivals after any smaller doses that add up to D.

fact, precisely the same results would be obtained ifA and B were one and the same agent, giving the same dose-response curve as Fig. 2A or B. In this case, the assertion that combinations of A with B are more effective than either agent alone is equivalent to saying that combinations of A with A are more effective than A on its own, a proposition that is hardly logical. It seems inescapable that when dose-response curves are not simply exponential it is not permissable to assume that the effect of a noninteractive combination equals the product of the effects of its constituents. Further, even when effect multiplication can correctly be applied to the effects of a noninteracting combination on a homogeneous population, it fails when applied to a heterogeneous population of varying sensitivites. Sapareto et al. (1978) and Dewey (1979) postulated a population consisting half of cells in S-phase and half of GI cells. It is supposed that a particular dose of agent A kills 90% of GI cells and 10% of S-phase cells, while the converse is true for agent B. Both agents have simple exponential dose-response curves, but the slopes of the curves differ for the two cell types. We have then the situation set out in Table I. It is obvious that, when effects on the whole population are considered, the effect of the combination expected according to the effect-multiplication criterion is not at all the observed effect. Thus, in

TABLE I INFLUENCEOF POPULATION HETEROGENEITY ON EFFECTOF COMBINATION OF AGENTS A THAT OBEY THE EFFECT-MULTIPLICATION RULE" S-Phase

GI-Phase

AND

B

Whole population

SA+B Dose

SAb

SB

SA+B'

SA

SB

SA+B

SAd

SB

1 2 3

0.1 0.01 0.001

0.9 0.81 0.729

0.09 0.0081 0.000729

0.9 0.81 0.729

0.1 0.01 0.001

0.09 0.0081 0.000729

0.5 0.41 0.365

0.5 0.41 0.365

Observede

Expected'

0.09 0.0081 0.000729

0.25 0.168 0.133

The population consists half of S-phase and half of Gl-phase cells. One unit ofA kills 90% of S cells and 10%of GI cells. One unit ofB kills 10% of S cells and 90% of GI cells. Both agents have simple exponential dose-response curves (cf.Sapareto et al., 1978;Dewey,

1979). S A or S B (survival after any fixed dose D ofA or B ) is the product of the effects of any smaller doses that add up to D . SA+B for each cell type equals S A . S B for cells of that type (effect-multiplication rule). Survival of the whole population after any dose of A or B is the mean of the survivals of S-phase and GI-phase cells, respectively, for that dose. SA+Bobserved for the whole population is the mean of survivals of S-phase and GI-phase cells, respectively, for that combination of A and B. ' S A + B expected for the whole population, according to the effect-multiplication criterion, is S A . S B for the whole population.

280

MORRIS C . BERENBAUM

spite of the fact that this criterion is quite correctly applied to each component of the population (as shown by the column for A + B under S-phase and G1-phase cells respectively), it cannot be applied to the whole population. Therefore, although simple exponential doseresponse curves are a necessary condition for the use of the multiplication criterion, they are not in themselves sufficient. We require in addition that the population be homogeneous in sensitivity to each agent. 111. Effect Summation

Another approach to the problem of calculating what to expect from a combination of different agents is simply to sum the effects of its constituents. Accordingly, a combination A + B is said to show zero interaction if its effect equals the sum of the effect of its constituents, i.e., if E , + b = E , + E b , synergy if Ea+b > Ea E b and antagonism if E a + b < E , + E b . This approach may appear naive in view of the complexity of biological phenomena. Nevertheless, it is correct in some circumstances. An example of its use is provided by Livingston and Dethlefsen ( 1979), who measured the number of sister-chromatid exchanges (SCE) per cell produced in Chinese hamster ovary cells in vitro by exposure to X-radiation and hyperthermia, separately and together. Their data are illustrated in Fig. 3. They found that for both agents the increase in SCE was linearly related to dose. When a fixed dose of hyperthermia was followed by various doses of X rays, the number of SCE produced by each combination was about equal to the sum of the numbers produced by each constituent of the combination alone, but there was a slight excess at high radiation doses, suggesting a positive interaction between X-radiation and hyperthermia in this system. It is important to note that, over the range of doses studied and within the limits of experimental error, the dose-effect curve for each of the agents was truly linear, i.e., effect was simply proportional to dose. In these circumstances, if any particular dose of one of the agents is regarded as a combination of its “constituents” (e.g., if 200 rads is regarded as a combination of two doses of 100 rads each), then the effect of any such “combination” is clearly the sum of the effects of its “ constituents.” If two or more such agents did not interact pharmacologically, it would be not unreasonable to expect that the effect of a combination of these different agents would also be the sum of the effects of its constituents. To formalize this relationship, if d, and d2 are two doses of a single agent showing a linear dose-effect relation, Edl = pdl and Ed%= p& where p is a constant, and E d l + d 2 = p(dl + &) =

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

0 X Rays(Rad)

Min at 44’C

200

400

281

600

X Rays (Rad)

Use of the effect-summation criterion (data from Livingston and Dethlefsen, 1979). (A) Numbers of sister-chromatid exchanges (SCE) induced per cell by X rays; (B) the same for hyperthermia. Effect is proportional to dose in both cases; consequently the effect of any dose D is the sum of the effects of any smaller doses that add up to D. For example, the effect of 600 rad (17.5 SCE) is the sum of the effects of 200 rad and 400 rad (6 and 11.5 SCE). (C) The effects of combinations of various doses of X rays with 20 minutes of hyperthermia. According to the effect-summation criterion, the expected effect of each combination is the sum of the effects of its constituents. F I G . 3.

Ed, + Ed*. Then, for two agents A and B which do not interact, and which show linear dose-effect relations,

where A and B are the doses of the two agents and p1 and p 2 are constants. Biologically plausible models that account for linear dose-response curves are not quite so easily constructed as the model for simple exponential curves. There are, of course, examples in physics and chemistry of linear dose-effect relationships that may be extended to include combined effects of more than one agent. For example, the absorbance of light by a colored solution is, over a wide range, linearly related to its concentration, and if two or more different absorbing materials were present in a solution and did not interact optically, then

282

MORRIS C. BERENBAUM

the absorbance of the solution would simply be the sum of the absorbances of its constituents. However, it is difficult to conceive of analogous mechanisms in living organisms. One model, of rather restricted application, that does give linear dose-response curves, is the following. Consider the case of a cytotoxic agent for which survival of a cell population is a simple exponential function of dose (i-e., SD = e-ADwhere SD is fractional survival after dose D and A is a constant), and suppose that the cell population grows exponentially at the same rate before and after treatment (i.e., N t = No e k f where ) N o and N t are cell numbers at times 0 and t, respectively, and k is a constant (Skipper et al., 1964, 1965). Then the growth delay, i.e., the time taken for the population to reach its pretreatment level L after dose D is calculated as follows: 9

Population size after dose D = LePm Population size after growth delay t = L+e-AD

... e k t

= eAD

A k

:.t = - * D

-

ekt

=L

(7)

That is, t is proportional to dose, and a curve relating growth delay to dose, both on linear scales, would be a straight line through the origin. A good example of such a situation is given b y Crathorn and Roberts (1965), who measured the growth of lymphoma cells in vitro after exposure to different concentrations of mustard gas. Their results are illustrated in Fig. 4. The survival curve is a simple exponential up to mustard concentrations of 0.2 pg/ml, and the cells grow exponentially with a doubling time of 0.55 days. For exposures to nitrogen mustard up to concentrations of 0.4 pg/ml, the growth delay is directly proportional to the concentration of mustard. Other examples of proportionality of recovery time to dose where this mechanism may reasonably be supposed to act are provided by the recovery of immunologically competent cells from X-radiation (Taliaferro and Taliaferro, 1964; Brent and Medawar, 1966). In practice, the most likely explanation for the occurrence of apparently linear dose-response curves is that measurements are being made in the low-dose range on curves that at higher ranges would be seen to be nonlinear. Typically, biological dose-response curves for cytotoxic agents are exponential, with or without a shoulder, where random hits on discrete targets are involved (Lea, 1955; Elkind and Whitmore, 1967; Turner, 1975), hyperbolic, where the underlying mechanism is competitive inhibition such as that by antimetabolites (Webb, 1963; Berenbaum, 1968, 1969), or they may have plateaus, where part of the cell population is resistant to the agent (Bruce et al.,

INTERACTIONS O F BIOLOGICALLY ACTIVE AGENTS

283

A

0

0.1

0.2

0.3

D (Mustard Gas, pg/ml) 10 -

-+a

r m 8-

u)

-$

T=

-

/ 2

4

6

8

t (Days)

lF5 D

6

Q

a

5 3

4-

D (Mustard Gas, pg/ml)

FIG.4. Generation of a linear dose-response curve by composition of two exponential curves (data from Crathorn and Roberts, 1965). (A) Survival of mouse lymphoma cells after treatment with various concentrations of mustard gas. (B) Growth of untreated lymphoma cells. (C) Observed growth delay after treatment with mustard gas. This fits the equation T = AD/k where T is growth delay, -k is the exponential constant in (A), A is the exponential constant in (B), and D is dose.

1966). As Lea (1955) pointed out for simple exponential curves, if the dose is such that only a small proportion of targets is hit, the number of targets hit is approximately proportional to dose. This approximation to linearity in the low-dose region applies also to many other types of dose-response curves. This may explain the apparent linearity of

284

MORRIS C. BERENBAUM

dose-response curves for mutations, chromatid breaks, sisterchromatid exchanges, and other chromosomal abnormalities for many agents (Lea, 1955; Elkind and Whitmore, 1967; Auerbach, 1976; Bradley and Sharkey, 1977; Carrano et al., 1978; Livingston and Dethlefsen, 1979; Virsik et al., 1980). Finally, consider the dose-effect relation for a particular carcinogen that acts in the presence of other environmental carcinogens. Carcinogen X merely contributes to this preexisting effect, and, at low dose levels, statistical argument shows that the extra effect is always more or less proportional to the dose of the extra agent, irrespective of the form of its dose-response curve (Crump et al., 1976; Guess et al., 1977; Peto, 1978). (It is implicit here that the carcinogens do not interact in producing tumors.) An example is shown in Table 11, in which carcinogen X and the background carcinogens have highly nonlinear dose-effect relations, yet the relation between the additional effect due

APPROXIMATELINEARITY

Dose of X

Total effective dose

OF

TABLE 11 EXTRAEFFECT OF ONE ADDITIONAL CARCINOGEN"

(XD)

(D)"

Total effectd

0 0.025 0.050 0.075 0.100 0.125 0.150

1.000 1.025 1.050 1.075 1.100 1.125 1.150

1.Ooo 1.077 1.158 1.242 1.331 1.424 1.521

Extra effect due toX (E)

Expected effect of X with linear responsee

0 0.077 0.158 0.242 0.331 0.424 0.521

0 0.083 0.167 0.250 0.333 0.417 0.500

Deviation from linearity

0 - 0.006 - 0.009 - 0.008 - 0.002

+0.007 +0.021

a According to Crump et 01. (1976),Guess et al. (1977),Pet0 (1978).See text for explanation. X is a carcinogen added at various doses XI, to a set of existing environmental carcinogens, the levels of which remain constant and which do not interact pharmacologically with X. The effective dose of existing environmental carcinogens is taken as 1, and the total effective dose D is taken as 1 + X D . The effects of the environmental carcinogens and of X are taken to be the cube of the dose of each, so the total effect is D 3 . (This effect might be, for instance, the number of tumors per exposed animal or the percentage of individuals with tumors.) A linear equation, approximately fitting the relation between the extra effect E due to X and the dose of X, is E = 3.33X D . NOTE. Although the dose-response relations of the environmental carcinogens and of X are highly nonlinear, the extra effect due to X is almost linear with doses up to a 50% increase in total effect.

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

285

to X and its dose would experimentally be indistinguishable from one of simple linearity over the dose range shown. To summarize, the effect-summation criterion may be used when the effects of all the agents in a combination are directly proportional to dose. When the effects of the agents in a combination are not proportional to dose, the effect-summation criterion may not be used. For example, consider again the agents with dose-response curves shown in Fig. 2. A combination of 1 mg of each agent is found to kill 99% of the cells, as does 2 mg of either agent alone. Thus, as shown before, 2 mg of the combination has the same effect as 2 mg of either agent, so the combination is not more effective than either agent used alone. The same result would be obtained ifA and B were the same agent, in which case it would not make sense to assert that the combination was more effective than the agents used alone. However, the effect of the combination (99% kill) undoubtedly exceeds the sum of the effects of its constituents (30% 30% kill). Evidently, when dose-response curves are not strictly linear, it is not permissible to assume that the effect of a noninteractive combination equals the sum of the effects of its constituents. Consideration of the effect-summation and effect-multiplication criteria shows that they may be appropriate for combinations of different agents only when they can be applied to each agent in the combination separately. However, it is not apparent how one would apply them to combinations in which one of the agents had a linear dose-response curve and the other had a simple exponential curve, nor is it apparent from these examples how we could derive suitable criteria where the curves have neither of these simple forms. A criterion is required, therefore, that is independent of the forms of the dose-response curve of the agents or the homogeneity or otherwise of the responding population and that can accordingly be applied to combinations of any agents and to any quantifiable response.

+

IV. Agent Interaction and Self-Interaction

The key to any criterion for examining interactions between different agents lies in the definition of zero interaction. Earlier, this question was left open; zero interaction was described simply as that in which the effect of the combination was that which was “expected.” In the course of discussing the effect-summation and effect-multiplication criteria applied in appropriate circumstances, it was shown that expectation was related to the forms of the dose-response curves of the agents in the combination. In simple cases, one could use the dose-

286

MORRIS C. BERENBAUM

response relations to calculate an expectation for the effect of the combination, An observed effect exceeding this expectation was deemed to show positive interaction, and the converse to show negative interaction. No such simple approach is possible when agents have less simple dose-response curves, and we are thus forced to consider whether we can define interaction between agents in a way that does not depend on the forms of their dose-response curves. A conceptual difficulty arises here because the term interaction may be applied to two entirely different phenomena. When examining the effect of a single agent, we may obtain evidence suggesting either that the individual molecules or particles of the agent act independently (Fig. 1) or that they do not, i.e., that the agent “interacts” with itself. For example, an exponential dose-response curve with a shoulder is not consistent with the independent action of different molecules of drug (or particles of radiation) acting on discrete targets. Such a case may be explained, however, by the existence of repair mechanisms that are also damaged by the agent, or by the necessity to damage more than one target in order to kill a cell, or by the need to hit a target more than once (Lea, 1955; Elkind and Whitmore, 1967; Turner, 1975). In all such cases, there is necessarily cooperation between different molecules or particles of agent; the molecules or particles of one and the same agent indubitably interact with each other in a physically real sense in producing the observed effect. Conversely, molecules of an antimetabolite compete with each other for the binding site of an enzyme. Thus, these molecules do not act independently to produce the observed effect; in fact, they positively interfere with each other and, in consequence, curves relating residual enzyme activity to drug concentration in vitro are typically hyperbolic (Webb, 1963), and, perhaps as a reflection of this, the survival curves of proliferating cells exposed to antimetabolites are also generally hyperbolic (Berenbaum, 1968,1969).Again, there is a physical interaction between molecules of agent in producing the effect being measured. Accordingly, it is perfectly justifiable to claim that these agents interact with themselves. However, such self-interactions are not the concern of this review. Here, we are interested in interactions between different agents, and it must be emphasized that these two types of interaction are quite dissociated. Agents that show no self-interaction do, in some cases, interact with each other to produce some effect, and in other cases they show no interaction. Conversely, agents that show self-interaction may or may not show interaction with each other, as the case may be.

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

287

For example, let us postulate a cell population consisting half of cells in cycle and half of resting cells. Suppose we have two agents, both with simple exponential dose-response curves (and which, accordingly, show no self-interaction), one of which kills only cells in cycle and the other only resting cells. Now, no matter how large a dose of either agent alone is given, it would be impossible to kill more than half the population. Yet, it is possible to kill any desired fraction of the whole population by using appropriate combinations of both agents (Table 111). Thus, at the level of the cell population, these two agents interact, since combinations can produce an effect that cannot be produced with either agent alone. Yet, each agent acting on its own target population shows no self-interaction. Conversely, we can construct a model in which two agents show self-interaction but do not interact with each other. Let us suppose that a cell has two distinct targets which are damaged selectively by two different types of radiation. Let us also suppose that destruction of either target is of the multihit type, and that sublethal damage to either target does not affect the susceptibility to damage of the other. Under these circumstances, each agent shows self-interaction, as evidenced TABLE I11 DISSOCIATION BETWEEN SELF-INTERACTION O F AGENTS AND INTERACTION BETWEEN AGENTS: POSITIVE INTERACTION BETWEEN AGENTSSHOWING NO SELF-INTERACTION' Proliferating cells Dose

SA

SB

1 2 3

0.1 0.01 0.001

1.0 1.0 1.0

SA+B~ 0.55 0.505 0.5005

Resting cells SA

SB

1.0 1.0 1.0

0.1 0.01

0.001

Whole population

SA+B

SAC

SB

SA+B~

0.55 0.505 0.5005

0.55 0,505 0.5005

0.55 0.505 0.5005

0.1 0.01 0.001

Agents that show no self-interaction are those in which the effect of each molecule or particle is independent of the effect of any other; their dose-response curves are of the simple exponential type and obey the effect-multiplication rule (see text). In this case the population consists half of proliferating and half of resting cells. One unit of A kills 90% of proliferating cells but no resting cells; one unit ofB kills 90% ofresting cells but no proliferating cells. * S A + B for either cell type is the mean of S A and S B for those cells. SA or SB for the whole population is the mean of survivals of proliferating and resting cells, respectively. As A has no effect on resting cells and B no effect on proliferating cells, S A + B for the whole population is the mean of S A for proliferating cells and S B for resting cells. NOTE. Neither agent alone, at any dosage, can kill more than half the population yet any level of kill can be obtained by appropriate combinations of both agents.

288

MORRIS C. BERENBAUM

by a shouldered exponential survival curve, but the two agents may not interact in causing cell death. Other models with these properties may be derived without necessarily postulating multihit mechanisms. For example, it may be supposed that each target has its own repair mechanism that is damaged by the agent that selectively damages that target, or that the cell has multiple targets of each type. As an extreme example of the dissociation between self-interaction and interaction between agents, a case will be discussed later (Fig. 22, Section VI1,B) in which two agents interact positively with themselves but negatively (Lea,antagonistically) with each other. We must, therefore, clearly distinguish between these two sorts of interactions. The study of interactions between agents is concerned only with the ways in which different agents interact to produce effects, whether the individual agents are self-interactive or not. V. lsoboles and the Interaction index

A. HOMERGICCOMBINATIONS

In the pharmacological literature, a combination of agents all of which produce the effect being observed is termed homergic. When not all the agents in a combination produce the effect (examples were given in the Introduction), the combination is called heterergic. It is convenient to discuss these two classes of combination separately, although, as will be seen, the same criteria for interaction or noninteraction are applied to both. The problem before us is to find a way of establishing a relation between the doses and the effects of different agents, given separately and in combination, that will indicate whether the agents are interacting with each other in producing the effect. We therefore require a model for zero interaction between agents which can be used as a frame of reference for detecting and measuring departures from zero interaction. Now, a combination that must, by definition, always show zero interaction between agents is the spurious “combination” of an agent with itself, in any arrangement of doses. This must hold, irrespective of the nature of the dose-response curve of the agent or the type of effect measured. Whether the agent shows self-interaction or not, it is axiomatic that a combination of particular doses of one and the same agent must have the same effect as the sum of those doses, because the “combination” and the sum are identical. To examine the effect of such combinations, let us divide our supply of one and the same agent into two lots, labeledA and B , respectively,

289

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

and proceed as if we believed A and B to be different agents, measuring the effects of different doses of each, separately and in combination. (For instance, A and B might be two identical lots of the same drug or two identical radiation sources.) Let the dose-response curve for A (and thus, the curve for B) be that shown in Fig. 5A.A graph is constructed (Fig. 5B),the axes of which represent the doses ofA and B , and on the graph are plotted lines joining all doses of A and B and combinations of these that have equal effects. Clearly, as long as the same total dose of the agent is given, the effect will be the same, whether the total is made up entirely ofA, entirely of B, or any mixture ofA and B. Thus, in Fig. 5B,doses ofA and B with the same specified effect and combinations of A and B with this same effect are represented by points that lie on a straight line. This will be true whatever the effect being measured and whatever the shape of the doseresponse curve of the agent. Different effects will be represented by different lines and different dose-response curves will give differently placed lines, but the lines will always be straight. Moreover, while mixture of, say, B with an inert agent will alter its dose-response curve, this simply has the effect of scaling the graph by a constant factor in the B axis. If, for instance, B were mixed with an equal amount of inert agent (let us call the mixture C), then any effect produced by a dose d of B will also be produced by a dose 2d of C

5-

B

4-

0

DOSE OF A

1

DOSE

2

OF A

FIG.5. A model for zero interacti0n.A and B are the same agent; C is this agent mixed with equal parts ofinert material. (A) Dose-response curves forA, B , andC. (B) Isoboles for the effects of combinations of A and B . All combinations giving the same log,, fractional survival are represented by points on the same straight line. (C) Isoboles for the effects of Combinations ofA and C .

290

MORRIS C . BERENBAUM

(Fig. 5A). The result is shown in Fig. 5C, which illustrates the fact that scaling the lines in Fig. 5B by constants in any axis does not alter their linearity. In such graphs, lines joining points representing equi-effective doses or dose combinations are termed isoboles. They were introduced by Fraser (1870-1871, 1872), who rightly claimed that, in using this device, “the results of the experiment will be rendered apparent b y a mere glance at the diagram.” He further showed that the results of a three-variable experiment could be represented by an isobolar surface in three dimensions. Fraser applied this method to the study of drug antagonism only, and it was extended to the analysis of other types of interaction by Loewe and Muischnek (1926) and Loewe (1928). After Loewe published an account of the method in the English language (Loewe, 1953), it was adopted by a number ofworkers in cancer chemotherapy (Elion et al., 1954; Rubin et al., 1964; Smith e t al., 1970; Werkheiser, 1971; Grindey et al., 1972; Grindey and Nichol, 1972; Werkheiser et al., 1973; Muller and Zahn, 1979; Muller et al., 1976, 1979). However, its impact in this field has been relatively limited, for reasons that are not clear. Certainly, Loewe’s (1953) paper is not easy to follow because of complex and idiosyncratic terminology, awkward mathematical symbolism, and a tendency to split hairs excessively, and the other publications cited make little (and generally no) attempt to explain the rationale of the method. Nevertheless, these deficiencies hardly justify the neglect of this powerful method by most cancer workers. To express the relationships shown in Fig. 5 algebraically, let the doses ofA and B that produce some specified effect be A, and Be, and let their doses in a combination that also has this same effect be A, and B,. Then the straight lines in Figs. 5B and C are given by the equation

The different lines correspond to different effects and therefore to different values of A, and B,. For example, if the effect under consideration is 10+ cell survival, Fig. 5C shows that this is given b y 2 units A , 4 units C , or any combination on the straight line joining these points, for example, by 1.25 units A with 1.5 units C ; using Eq. (8)in this case (1.25/2.0) (1.5/4.0) = 1. This then is our model for zero interaction with two agents. In an isobologram plotted as in Fig. 5B or C, we locate the point representing a given combination and also the points representing the doses of the agents that, given alone, have the same effect as that combination

+

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

291

(the equi-effective doses). If there is no interaction between the agents in that combination, these three points will lie on a straight line and Eq. (8) is satisfied. Definitions of synergy and antagonism follow directly from this. Avoiding preconceived ideas, let us adopt the simple view that when agents are synergistic the effects of a combination should exceed expectation. The converse will be true when agents are antagonistic. Inspection of Fig. 6A suggests immediately how such deviations from expectation might be detected. cell survival Suppose that 2 units of A or 4 units of C produce as before but that the combination of 1.25 units A with 1.5 units C produces survival. Evidently, this combination would be more effective than expected from the zero-interaction model (in which it gave lo-* survival), and the isoboles for survival could not be and straight but would have to be concave upwards. The doses ofA and C producing survival must exceed 2 and 4 units, respectively; let us say they are 2.5 and 5 units (Fig. 6A). We now insert these values, and the appropriate values for the combination, into Eq. (8): (1.2512.5)+ (1.5/5.0)= 0.8. The sum of the fractions is now less than 1, which expresses the upward concavity of the isobole for this effect. Therefore a combination of two agents is synergistic if it gives a value < 1 in Eq.

0

1 2 DOSE OF A

0

1

2

D O S E OF A

FIG. 6. (A) Isoboles showing synergy. For all combinations represented by points on these isoboles, the sum of fractions in Eq. (8) is less than 1, hence the upward concavity of the isoboles. (B) Isoboles showing antagonism. For all points on these isoboles, the sum of fractions in Eq. (8) exceeds 1, hence their downward concavity.

292

MORRIS C. BERENBAUM

(8).The point representing it in an isobologram lies below and to the left of the straight line joining the doses of the agents that, given alone, have the same effect as the combination. If all the combinations with the same effect are synergistic, the isobole for that effect is concave upwards throughout. Conversely, suppose that the combination of 1.25 units A and 1.5 units C gives lo-' cell survival. It would then be less effective than expected from the zero-interaction model, and the isoboles for lo-' and lo-* survival would have to be concave downwards, as in Fig. 6B. The doses of A and C giving lo-' cell survival must be less than 2 and 4 units, respectively. If they are, for example, 1.5 and 3 units, then Eq. (8) gives us (1.25/1.5)+ (1.5/3.0) = 1.33. This sum of fractions exceeds 1, expressing the downward concavity of the isobole for this effect. A combination of two agents is therefore antagonistic if it gives a value > 1 in Eq. (8).In an isobologram, the point that represents it lies above and to the right of the straight line joining the doses of its constituents that, when given alone, have the same effect as this combination. If all the combinations having that effect show antagonism, the isobole for that effect is concave down throughout. It is, of course, entirely possible that for any specified effect some combinations may be synergistic, others antagonistic, and still others additive. In such cases, the isobole for that effect crosses the additive line one or more times and may be described as bimodal or multimodal. Good examples of this in microbial chemotherapy are shown by McAllister (1974), Feldman (1978), and Ayisi et al. (1980). This shows that the direction of the interaction between two agents is not necessarily the same for all combinations having the same effect (although, in practice, it usually is). Nor should it be supposed that the interactions for quantitatively or qualitatively different effects produced by combinations of the same set of agents are necessarily all in the same direction. It is possible for a combination of agents to show synergy for one sort of effect and antagonism for another (as will be seen in Figs. 23-26). Expressed algebraically, the relationships discussed above are:

< 1 for synergy 1 for zero interaction > I for antagonism =

(9)

For combinations of three agents, the two-dimensional graph of Figs. 5 and 6 is replaced by one in three dimensions, and the isobolar lines by isobolar surfaces (Fraser, 1872; Berenbaum, 1977, 1978) (Fig. 7). Expression (9) then extends to

INTERACTIONS O F BIOLOGICALLY ACTIVE AGENTS

293

< 1 for synergy

& + & + G [= 1for zero interaction Be

ce

> 1 for antagonism

(10)

When this expression equals 1, it is the equation of the plane passing through the points representing A, B, and C, on the three coordinate dose axes (Fig. 7A). Thus, let a combination ofA, B , and C have a particular effect E , and let doses ofA, B , and C that each have that effect when given separately (the equi-effective doses) be A, Be, and C,, respectively. Then, if the combination shows zero interaction, the point representing it with reference to the three coordinate dose axes lies in the plane through A, Be, and C,.If the combination is synergistic, the point will lie below this plane and, if all combinations with this effect are synergistic, they make up a concave-up isobolar surface (Fig. 7B). Antagonistic combinations with this effect are represented by points above the plane, and when all such combinations are antagonistic, they make a concave-down isobolar surface (Fig. 7C). These criteria are easily generalized to combinations of any number of agents. For combinations of more than three agents, no graphic

FIG. 7. Isobolar surfaces for combinations of three agents, A, B, and C. (A) Zero interaction, or additivism. Effect E is produced by 4.5units A, 6 units B, or 9 units C. These are the equi-effective doses A,, Be, and C, for this effect. Combination X, which has effect E , consists of 2 units A, 1 unit B, and 3.5units C. The interaction index [Eq. (lo)]for combination X is thus (2/4.5) + (1/6)+ (3.5/9) = 1, and the surface through X, A,, Be, and C. is a flat plane. (B) Synergy. Combination 1; which has effect E [as in (A)], consists of 1.5units A, 2 units B, and 1 unit C. Its interaction index is thus (1.5/4.5) + (2/6) (1/9)= 0.78< 1, and the surface through Y, A,, Be, and C, is concave up. (C) Antagonism. Combination Z, which has effect E, consists of 2 units A, 3 units B, and 4 units C. Its interaction index is thus (2/4.5) + (3/6)+ (4/9)= 1.39> 1, and the surface through Z, A,, Be, and C, is concave down.

+

294

MORRIS C. BERENBAUM

construction is possible, but Eq. (10) may be generalized for any number of agents (A,B,C, , N) to

...

B A, + 2 +Ae

Be

*

"

+2 Ne

< 1 for synergy =

1 for zero interaction

> 1 for antagonism

(11)

The sum offractions in Eqs. (8),(9),(lo),and (11)* may be termed the interaction index, and its value measures the degree of synergy or antagonism. These criteria are independent of the nature of the effects under consideration and do not depend on any assumptions as to the mechanisms of action of the agents, homogeneity of the target population, and so on. It is also important to note that they are independent of the shapes of the dose-response curves, so that they cover cases such as those described above in which effect multiplication and effect summation may also be used (Sections I1 and 111).

B. HETERERCICCOMBINATIONS

The interactions that can be analyzed by constructing isoboles or calculating interaction indices are not restricted to cases in which all agents in a combination produce the effect under consideration. Consider a heterergic combination of two agents, A and B, in which A produces the effect under consideration and B does not. Figure 8 shows three possible types of isoboles. If there is no interaction between A and B, the isobole will be a straight line parallel to the B dose axis. Since no dose ofB produces the required effect, its equi-effective dose B e may be regarded as infinite. Thus, B , / B e is zero and the relevant equation is:

If A and B are synergistic, B increases the effect of A so that the dose o f A required to produce a given effect will be reduced, i.e., A, < A, and so AJA, < 1. Therefore, the isobole will diverge toward the B axis; it will not meet it, as no dose of B alone can produce the effect, and thus BJBe = 0 as before. Accordingly, the isobole will be concave up and the relevant expression is:

* Strictly speaking, expressions (9), (lo), and (11) are not equations as they include inequalities, but the distinction is unnecessary in the present context and they will be termed equations for simplicity.

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

0

2

4

6

8

295

10

A

FIG.8. The three types of interaction for a heterergic combination of two agents; A produces the effect being measured and B does not. When A and B do not interact, the isobole for the effect is a straight line parallel to the B axis. When A and €3 are synergistic, the isobole deviates asymptotically toward the B axis and, when A and B are antagonistic, it deviates away from the B axis.

Conversely, if A and B are antagonistic, B increases the dose of A required to produce a given effect. Therefore, A , > A , and so A J A , > 1. The relevant expression is thus

It appears, then, that construction of isoboles or calculation of interaction indices enables one to determine the type of interaction between agents, and to measure it, irrespective of the nature of their dose-response curves, the effect measured, the population on which it is measured, or whether the combinations are homergic or heterergic. It requires no assumptions about mechanisms of action of the agents. For combinations of two or three agents, either method may be used; the former is simply the geometrical representation of the latter. For combinations of more than three agents, geometrical constructions are not possible, but interaction indices may be calculated for any number of agents.

c, ISOBOLE CONSTRUCTION FROM LIMITEDDATA The objection is sometimes made to isobole construction that it is laborious to have to find a number of combinations, all with precisely

296

MORRIS C. BERENBAUM

the same effect, for the purpose of drawing a line on a graph (Mitchell, 1976). However, there is no need for such precision. After all, it is perfectly proper to determine LD50 by investigating doses on either side of this value without necessarily finding precisely the dose that kills 50%of animals. It is, in fact, often possible to draw isoboles and to carry out corresponding algebraic calculations with surprisingly little information. For example, Fig. 9 illustrates the effects on L5178Y lymphoma cells of methyl-6-mercaptopurine ribonucleoside (Me-6-MPR) and 6-mercaptopurine (6-MP), separately and in two combinations (Paterson and Moriwaki, 1969). Both combinations reduced cell growth to 4.6% of control levels, but neither of the two agents did so at the concentrations at which they were tested alone. Evidently, if the isobole for 4.6% growth meets the individual dose axes, it must do so at points beyond the levels tested, and so it must be concave up, indicating synergy. Algebraically, if Me-6-MPR is agentA and 6-MP is agent €3, we have, for the 4.6% growth effect, A, > 1.5 X M, Be > 5 X M . One of the combinations consisted of 0.75 X lo-' M Me-6-MPR and 2.5 X 5- 21.3

-3 4 yo

\5

n

3-

T

(D

2-

1-

33.3

0 100 0

0.5

1.o

L

1.5 Me- 6- MPR (10-7M)

FIG.9. Construction from limited data of an isobole showing synergy (Paterson and Moriwaki, 1969, Table 2, Exp. 11). Growth of L5178Y mouse lymphoma cells was measured in one concentration each of methyl-6-mercaptopurine ribonucleoside ( M e - 6 MPR) and 6-mercaptopurine (6-MP) and in two combinations of these agents. Values show growth as percentages ofthat of untreated cells. The isobole for 4.6% growth must meet the Me-6-MPR axis (if it meets it at all) at some concentration above 1.5 x lo-' M and the 6-MP axis at some concentration above 5 x lo-' M and therefore must be concave up, indicating that the two combinations are synergistic.

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

297

lo-" M 6-MP, and the relevant algebraic expression is thus 0.75 A,

+

2.5 < 0.75 Be 1.5

I

2.5 5.0

Therefore, the interaction index is less than 1 in this case. The other combination consisted of 1 x lo-' M Me-6-MPR and 1.33 x lo-" M 6-MP, and the relevant expression is thus

-1.0 +A,

1.33 < 1.0 Be 1.5

I

1.33 < 5.0

As a further example, Fig. 10 shows the effect of vincristine and cyclophosphamide on life span in mice with L1210 leukemia (Mulder et al., 1979). The combination gave a mean survival time of 14.9 days, which is less than that given by cyclophosphamide alone when given in the same dosage as that used in the combination. Accordingly, the isobole for 14.9-days survival meets the cyclophosphamide dose axis at some dose less than that used in the combination. Therefore, the equi-effective dose for cyclophosphamide is less than its dosage in the combination and so the fraction representing this agent in Eq. (11)

- 11.8

0

14.9

-10.6

0

200

Cyclophosphamide (rng/kg)

FIG.10. Construction from limited data of an isobole showing antagonism (Mulder et al., 1979, Table 2, Exp. C145). Mice with L1210 leukemia given cyclophosphamide, vincristine, or both simultaneously in a 2 x 2 design (see text). Values are mean survival times (MST). The isobole for a MST of 14.9 days must meet the cyclophosphamide dose axis at some dose less than 200 mg/kg and, thus, is either concave down or divergent from the vincristine dose axis, showing that the combination is antagonistic.

298

MORRIS C. BERENBAUM

must exceed 1. This guarantees that the interaction index for the combination exceeds 1 and, therefore, that it shows antagonism. The other end of the isobole could meet the vincristine dose axis at some dosage above that used in the combination, in which case it would be concave down (indicating antagonism), or, conceivably, it might never meet it. In the latter case, the combination would be heterergic for the effect under consideration, and the isobole would be equivalent to that for antagonism shown in Fig. 8. We have thus established unequivocally the nature of the interaction in both cases without being able to draw precise isoboles and without knowing what doses of either agent produce the effect under consideration.

D.

INADEQUACY OF

2 X n EXPERIMENTAL DESIGNS

In much published work, the following experimental design is used. Two agents are tested alone at doses X and Y, respectively, and also in a combination X + Y of these same doses, and conclusions as to the interaction of these agents are drawn by comparing the effect of the combination X Y with the effect of X and Y separately. For example, Segaloff and Maxfield (1971) found that rats given a 5 mg diethylstilbestrol implant had 1.7 tumors per mammary chain, rats in which the mammary region had been exposed to 800 rads X-irradiation had 1.1 tumors, and rats exposed to both agents had 5.6 tumors (Fig. 11). They concluded that the two agents acted synergistically. Unfortunately, no such conclusion may be drawn from these results as shown by the indeterminate shape of the isobole in Fig. 11. Expression (11) shows that, if a combination is to be deemed synergistic, the sum of fractions in this expression must be less than 1. Now it is quite impossible to prove this if each agent is tested only at one fixed dose alone and at the same dose level in the combination. If the combination happened to have a greater effect than any of its constituents, it would follow that the equi-effective dose of each constituent would exceed the dose used in the combination, i.e., A, < A,, B , < Be, and so on, so that each of the fractions in Eq. (11) would be less than 1, but this is obviously not sufficient to guarantee that the sum of these fractions is less than 1. To guarantee this, w e have to have at least a minimum estimate, or lower bound, for the equi-effective dose of each agent, and only if these values are each large enough may we conclude that the sum of fractions is less than 1 and the combination synergistic. It follows that, to show that a combination is synergistic, each agent must be tested on its own at least one dose level higher than that used

+

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

-P

299

a ul

5.6

800

a x

0

1.7

I t 0

I

5 Diethylstilbestrol (mg)

FIG.11. A 2 x 2 design showing the impossibility of determining whether the isobole for the effect of a combination is concave up, straight, or concave down, and, thus, that this design cannot generally show whether a combination is synergistic, additive, or antagonistic. Values show number of tumors per mammary chain in rats given either a 5 mg diethylstilbestrol implant, 800 rads to the mammary chain, or both (Segaloff and Maxfield, 1971).

in the combination. In fact, the levels must be high enough to allow us to conclude that the sum of fractions in Eq. (11) is less than 1. To ensure this requires some prior knowledge of the dose-response curves of the agents in the appropriate ranges or, at least, some good guesswork. An example of the successful use of such a design is shown in Fig. 9. Similar considerations apply to the demonstration of additivity, except that the sum of fractions here must equal 1. Experimental designs with two agents in which only one dose level is used for each agent alone and in the combination may be termed 2 x 2 designs for reasons that are clear from Fig. 11. This inability to demonstrate the existence of synergy (or additivism) occurs with all designs in which any of the agents is tested only at the dose level used in the combination, irrespective of the number of dose levels of the other agent(s). An example of such a 2 X n design is the experiment of Drewinko et al. (1976),who exposed human lymphoma cells to several different concentrations of bleomycin, with or without one fixed concentration (0.25 pg/ml) of adriamycin. Inappropriate use of the effect-multiplication criterion led the authors to conclude that these agents were synergistic but Fig. 12A shows that, with this 2 X 6 rectangular design, it is impossible to determine the overall shape of

300

MORRIS C. BERENBAUM 0.25r

21 11

4.7

0

25

1.6

0.51

0.16

2

75 100 Bleomycin (pg/rnl) 50

B

J

0

25

50

75

100

BCNU bg/ml)

FIG. 12. Two 2 x 6 designs (Drewinko et al., 1976). Human lymphoma TI cells exposed to two different levels of adriamycin (0 and 0.25 pLg/ml) and (A) six levels of bleomycin (0 to 100 pglml) or (B) six levels of BCNU (0 to 100 pdml). Values show cell growth as percentages of control. (A) Parts of the isoboles for various levels of cell growth msly b e located approximately and are drawn here as straight line segments. However, it is impossible to determine the subsequent direction of those that continue above the 0.25 pglml adriamycin level, and the overall shape of all of them is indeterminate. Thus, the type of interaction present cannot b e determined with this design. (B) Growth inhibition with combinations of 10 and 25 p d m l BCNU with 0.25 j&ml adriamycin is less than that with 0.25 p d m l adriamycin alone, and therefore the isoboles for the effects of these combinations must be concave down, as shown. Thus, it is possible to demonstrate antagonism with a 2 x n design if it is so marked that the effect of a combination is actually less than that of one (or both) of its constituents. The shapes of the other isoboles are indeterminate, as in (A).

any of the isoboles and, thus, to determine whether the combinations were synergistic, additive, or antagonistic. It may be possible to detect antagonism with such designs, but only when it is so marked that the effect of the combination is actually less than that of one (or more) of its constituents (Figs. 10 and 12B). If this constituent is, say, A , then A, < A, and so A,/A, > 1,and therefore the sum of fractions in Eq. (11)necessarily exceeds 1. However, as shown in Fig. 6B, antagonism may be present even when the effect of a combination exceeds the effect of each of its constituents, and the 2 x n

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

30 1

design is incapable of detecting this interaction under such circumstances.

E. DIAGONAL ARRAYS Although, in principle, Eq. (11) shows how the nature and degree of interaction may be determined for any combination of any number of agents, it does not immediately suggest how, given a set of agents, one should go about looking for synergistic or antagonistic combinations. Suppose it was proposed to investigate only four dose levels for each agent in a set of two agents. There would be 16 possible combinations to test, and the one showing the most marked synergy or antagonism might conceivably be any one of these 16. For a set of three agents tested in the same way, there would be 64 combinations, for four agents there would be 256, and so on. The problem, then, is one of logistics. It may be impossible in practice to test more than a small selection of the possible combinations. The question, then, is how this selection should be made so as to give the greatest chance of detecting any interaction that may be present. An early approach to this problem, with combinations of two agents, was made by Stock et al. (1953) and Clark (1958). Doses X and Y of each agent are chosen that produce measurable (and preferably marked) effects; these effects are not necessarily equal. Then the following arrangements of doses are tested: X, Y, X + Y, 0.5X, 0.5Y, 0.5X + 0.5Y, 0.25X, 0.25Y, 0.25X + 0.25Y. If these combinations are plotted in an isobologram, it is seen that the procedure amounts to titrating the combination X + Y in a series of doubling dilutions down the diagonal joining the point representing X + Y to the origin of the graph. These authors claimed that, if the combination 0.25X + 0.25Y had about the same effect as X or Y, this would indicate synergy. If the combination with this effect were 0.5X + 0.5Y, this would show additivism. Antagonism “would be evident when the full doses were used in combination” (presumably, if the effect o f X + Y equalled or was less than that ofX or Y). A difficulty may arise with this method when the effects o f X and Y are substantially different. In these circumstances, it may be found that the effect of OSX + 0.5Y exceeds that of X , for example, but is less than that of Y, in which case no conclusion can be reached. However, the method may be refined and applied to combinations of more than two agents in the following way (Berenbaum, 1977, 1978). If the agents do not interact, the isobole or isobolar surface is linear (the term surface here includes hypersurfaces in more than three dimensions); when

302

MORRIS C. BERENBAUM

there is an interaction, the isoboles depart from linearity. The problem is then one of selecting the point on the linear isobole or isobolar surface that is likely to undergo the most marked deflection from linearity if an interaction is present. Now, clearly, all isoboles meet the various dose axes at the equieffective dose levels for each agent, i.e., even if isoboles show interaction, they must meet additive isoboles at these points, so there is no departure from linearity there. Intuitively, therefore, we would expect any deviation to be small near the dose axes and to be greatest at about the midpoint of the isobolar line or surface (Figs. 6 and 7). Examination of the few isoboles published for combinations of two antitumor agents (Elion et al., 1954; Smith et al., 1970; Werkheiser, 1971; Grindey and Nichol, 1972; Werkheiser et d., 1977; Muller and Zahn, 1979; Muller et al., 1979) and of the much larger number published for combinations of two antibiotics (for example, O’Grady, 1975; Kerry et al., 1975; Bourque et aZ., 1976; Feldman, 1978; F u and Neu, 1976) shows that this is usually the case. This property is illustrated in Fig.

13A. The combination at the midpoint of a two-agent additive line consists of half the equi-effective dose of each agent (Fig. 13A), and the combination at the midpoint of a three-agent additive plane consists of one-third of the equi-effective doses of each agent (Fig. 13B). In general, the combination at the mid-point of an n-agent additive plane (in

n dimensions) consists of lln of the equi-effective doses of each of the n agents. Therefore, for a set of n agents, interactions are most efficiently detected using a reference combination made up of l/n of the equieffective dose of each agent for any specified effect. The procedure is most easily visualized in the two-dimensional case shown in Fig. 13A. A specified effect is produced by 3 units ofA or 5 units of B and the reference combination X therefore consists of 1.5 units ofA and 2.5 units ofB. When synergy is present, X has an effect greater than that specified, so a titration is carried out down the line XO to find a combination that has just the specified effect. In Fig. 13A, this is Y, which is the most synergistic combination on the relevant isobole. Conversely, if antagonism is present, X will have less than the specified effect, so titration is carried out in increasing dosage along the extrapolate of OX. This locates Z , which is the most antagonistic combination on the appropriate isobole. In the three-dimensional case, we find the doses of agents A, B , and C that each produce a specified effect. A reference combination X ’ is made consisting of one third of each of these doses, and fractions or multiples of this reference combination are tested, i.e., we titrate along

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

1

2

3

303

A

A

FIG.13. Diagonal arrays. (A) Combinations of two agents. For effect E, the equieffective doses ofA and B are 3 and 5 units, respectively, and thus combination X, at the midpoint of the additive line, consists of 1.5units A and 2.5 units B. If all combinations having effect E are synergistic, the corresponding isobole is concave up throughout. Testing fractions ofX to find a combination with effect E is equivalent to exploring the diagonal XO. This locates Y, composed of 0.65 units A and 1.2units €3, with interaction + (12’5)= 0.46.This index equals length OY/length OX. If the isobole is index (0.65/3) symmetric about OX, the ratio will be smaller for Y than the corresponding ratio for any other point on the isobole, i.e., Y shows greater synergy than any other combination on the isobole, as shown by their larger interaction indices. Conversely, if all combinations with effectE are antagonistic, testing multiples ofX to find a combination with effectE is equivalent to exploring the extrapolate of diagonal OX. This locates combination Z, consisting of 2.3 units A and 3.9 units B and with interaction index 1.55.This index equals length OZ/length OX. If the isobole is symmetric about the diagonal, the ratio is greater for Z than the corresponding ratio for any other point on this diagonal, as shown by their smaller interaction indices. (B) Combinations of three agents, with a concave-up isobolar surface. The equi-effective doses for A , B , and C are 4,5 and 10 units respectively, and combinationx’, at the midpoint ofthe additive plane, consists of one-third of each of these. Exploration down diagonal X‘O locates combination X. If the isobolar surface is symmetric about OX’ , X will have a lower interaction index than any other combination on this surface. The index equals length OX/length OX’. the IineX’O or the extrapolate of OX’ in Fig. 13B until a combination X is found that has the specified effect. The fraction or multiple of the reference combination that combination X represents is precisely the interaction index of expression (11). This method has been used with combinations of two antibiotics and results in a considerable saving of labor (Sanderson and Drabu, 1979). It is most efficient when synergy or antagonism are most marked along the line joining the origin of the graph to the reference combination, but even with markedly skewed isoboles, this procedure would in

304

MORRIS C. BERENBAUM

most cases reveal the nature of the interaction fairly efficiently, although it would not then determine where it is most pronounced. The method may, however, be misleading in the minority of cases in which the isoboles are bi- or multimodal (McAllister, 1974; Feldman, 1978; Ayisi et al., 1980), and if such anomalies are suspected, exploration along lines other than that specified above would be required. VI. Criteria Based on Changes in Dose-Response Curves

Many workers in fields that customarily involve the accurate construction of fractional survival curves have long been aware of the difficulties that the effect-multiplication criterion creates when survival curves are not simply exponential. In such cases, multiplying the effects of two doses of the same agent does not correctly predict the effect of the sum of those doses. Perhaps for this reason attempts have been made to examine interactions between pairs of agents by analyzing the effect of a fixed dose of one agent on the shape of the whole dose-response curve of the other. For example, Haynes (1964) stated that, when two agents acted synergistically, the effect of a fixed dose of one on the survival curve of the other might be to remove an initial shoulder, increase its limiting slope, or both. Analysis of such changes was taken further by Dewey et aZ. (1971) and Han and Elkind (1978). Four main possibilities were described (Fig. 14).

A. If the dose-response curve of agent X is simply displaced downwards when each of its doses is used in combination with a fixed dose of agent Y, then the normalized curve for these combinations coincides with the curve for X used alone. This is said to show that X and Y act independently, i.e., cells surviving treatment with Y are not damaged and respond to X in the same way as untreated cells. Of course, this is simply a restatement of the effect-multiplication criterion for zero interaction because, when survival for agent X is plotted logarithmically, a uniform displacement downward is equivalent to multiplying cell survival at each dose by the fractional cell survival produced by a fixed dose of agent Y. B. If the dose-response curve for X is simply displaced to the left when it is used with a fixed dose of Y, the normalized curve has the same final slope as the curve forX alone, but it has a lower extrapolation number and a smaller shoulder or none. This is said to show thatX and Y interact “additively,” and that cells surviving treatment with Y are damaged and respond toX as would cells surviving a dose ofX that

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

305

reduced their survival to the same level as the fixed dose of Y employed. C. If the dose-response curve for X shows an increased final slope when it is used with agent Y, but the normalized curve has an unchanged extrapolation number, this is said to show synergy, and that

Dose

14. Criteria for interactions between agents X and Y based on changes in the dose-response curve for X induced by a fixed dose of Y that, alone, reduces survival to 0.1 of controls. Dose-response curves for X alone and with fixed dose of Y (-). Normalized dose-response curve forX with fixed dose ofY(---). Extrapolate of normalized curve to indicate position of extrapolation number on survival axis (------). (A) The doseresponse curve for X is uniformly displaced downwards by Y. The normalized curve coincides with the original dose-response curve and so has unchanged slope, shoulder, and extrapolation number. This is said to show zero interaction or independence of effect. (B) The dose-response curve for X is uniformly displaced to the left by Y. The normalized curve has unchanged slope but a reduced or absent shoulder and a reduced extrapolation number. This is said to indicate “additivism.” (C) The dose-response curve for X given with Y has increased slope and a reduced shoulder. The normalized curve has an unchanged extrapolation number. This is said to show synergy. (D) The dose-response curve for X given with Y has reduced slope. This is said to show antagonism. (Haynes, 1964; Dewey et al., 1971; Han and Elkind, 1978). FIG.

306

MORRIS C. BERENBAUM

cells surviving treatment with Y are not damaged but are made more sensitive to X . D. If the final slope for X is decreased by Y, the interaction is one of antagonism or protection. Dewey et al. (1971) also described a case they termed “additive plus synergistic,” but their description is unclear; the normalized curve shows an increased slope but, although these authors state that the extrapolation number is unchanged, in which case “synergistic” and “synergistic plus additive” curves would be indistinguishable, their illustration shows a curve with a reduced extrapolation number. Several problems arise from the use of these complex criteria. It is clear that, if changes in slope, shoulder, and extrapolation number are all taken into account, then many more than these five possibilities exist (in fact, there are 13), and it is by no means obvious how to interpret some of these in a way consistent with the five already described. For instance, Piro et al. (1975) found that treatment of Chinese hamster cells with actinomycin D before irradiation decreased the slope of the survival curve (antagonism, according to Han and Elkind, 1978), and also decreased its shoulder (synergy, according to Haynes,

1964). Tyrrell (1978) has pointed out that the nature of the interaction in Case (B) is indeterminate, and that it might include examples of zero interaction and synergy. He suggested the term “positive interaction” to cover all cases, including synergy, in which survival was less than in Case (A), reserving the term synergy for cases in which the slope was increased. Further, it is not clear how combinations of more than two agents could be evaluated using these criteria, although, in principle, a criterion used in deciding whether two agents interact should be applicable equally to combinations of any number of agents, for these may also interact. However, all these difficulties are relatively unimportant since it is generally not possible, from examining changes in the dose-response curve of one agent when used with a fixed dose of another, to say whether the interaction is one of synergy or antagonism, or even whether there is an interaction at all. As will be shown below, agents that are antagonistic may appear to be synergistic when examined by these criteria, and vice versa. In fact, this experimental design is of the 2 x n type discussed previously, in which one of the agents is used at a single fixed dosage and which, as shown in Section V,D, cannot provide the information needed to demonstrate synergy or additivism (although it could demonstrate antagonism that was sufficiently marked).

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

307

The possibilities for error in using these criteria are illustrated in Figs. 15 and 16. Figure 15A shows the isoboles for combinations of two different agents, A and B . These isoboles are concave up, therefore A and B are synergistic. From these isoboles, it is possible to deduce the effects of various combinations ofA and B in the region covered by the isoboles, and so it is possible to plot, as in Fig. 15B, dose-response curves for A in combination with particular doses of B in the manner of Dewey et al. (1971) and Han and Elkind (1978). For instance, to draw the dose-response curve for A in combination with fixed doses of 4 units B ,a horizontal line is drawn at the level of the 4-unit dose of B in Fig. 15A. This cuts the isoboles, for -1.0, -1.5, -2.0, -2.5, and -3.0 log,, fractional-survival curves at doses ofA of 1.0,2.5,4.2,6.0,and 7.4 units, respectively; therefore, the curve in Fig. 15B forA with 4 units of B (curve F) is drawn through these points. It is evident that all the curves for A in combination with various fixed doses of B have a reduced slope compared with the curve for A alone. Therefore, according to the criteria of Dewey et al. (1971) and Han and Elkind (1978),A and B are antagonistic, yet the isoboles in Fig. 15A show that they are undoubtedly synergistic. A similar exercise has been performed in Fig. 16. The concave-down isoboles in Fig. 16A show that A and B are antagonistic. Again, horizontal lines drawn at various dose levels of B enable dose-response curves for A i n combination with fixed doses of B to be drawn, and these are shown in Fig. 16B. These curves all have increased slope compared with that for A alone, indicating, according to the criteria we are considering, that A and B are synergistic when, in fact, they are antagonistic. It seems that use of these criteria may lead to conclusions diametrically opposed to the correct ones. Now the critical factor determining the slopes for the combinations of agents is the degree of horizontal separation of the isoboles at the level of the fixed doses of B compared with their separation in the dose axis forA used alone. In Fig. 15A, the horizontal distance between the isoboles at each of the fixed-dose levels of B chosen exceeds their separation in the dose axis of A. It follows directly that the slopes of the dose-response curves of A in combination with these fixed doses of B must be less than that of A alone, and this is borne out in Fig. 15B. Conversely, if the horizontal separation of the isoboles at any fixeddose level ofB is less than it is in the dose axis ofA, as in Fig. 16A, the dose-response curves of A with that fixed dose of B must have greater slope than A alone, as is seen in Fig. 16B. Finally, if the horizontal separation of the isoboles is the same at any dose level of B as it is in

308

MORRIS C. BERENBAUM

A

B

-0.5

-I

4

-3.0 0

2

4

8

6

10

12

14

DOSE

FIG 15. (A) Concave-up isoboles for A and B , showing that these agents interact synergistically. The values on the isoboles are log,, fractional survivals. (B) Dose-

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

309

the dose axis ofA, there will be no difference in slope between the dose-response curves of A alone and A with that dose of B. Now the degree of horizontal separation of the isoboles depends partly on their curvature and partly on the shapes of the dose-response curves for A and B used alone. Geometrical considerations suggest the following: concave-up isoboles (synergy) would tend to give reduced horizontal separation (increased slope) and concave-down isoboles (antagonism) would tend to give increased horizontal separation (decreased slope). On the other hand, a larger shoulder on the dose-response curve for A than on that for B would tend to give increased separation of the isoboles at fixed doses of B and a smaller shoulder would tend to give reduced separation. Accordingly, whether or not the use of criteria based on the slopes of dose-response curves yields the correct conclusion is to some extent fortuitous. If the disparity between the shoulders on the dose-response curves for the two agents is not great, the conclusion based on slopes alone will often be correct; if this disparity is marked, conclusions may well be incorrect. If the curves are simply exponential without shoulders, then no such disparity can arise; the horizontal isobolar separation will always be reduced when there is synergy and increased when there is antagonism, and changes in slope of the dose-response curve will always correctly indicate the type of interaction. It will be noted in Figs. 15B and 16B that the shoulders on the dose-response curves for A diminish and eventually disappear when the curves for A are determined in the presence of increasing fixed doses of B, whether the interaction between A and B is synergistic or antagonistic, and this is equally bound to occur in the intermediate case, that of zero interaction between A and B . It follows that diminution or loss of a shoulder cannot be used to indicate what type of interaction is present. Two examples will show how these considerations affect examination of the interaction between agents in practice. It should first be pointed out that much of the literature cannot be analyzed satisfactorily. The widespread conviction that the criteria discussed here enable

response curves forA and B alone and for combinations ofA with various fixed doses ofB [at dose levels C, D,. . . ,L indicated in (A)]. Each curve forA with a fixed dose of B is drawn through the points where the isoboles in (A) are cut by the line representing the appropriatedose ofB (for example, curve C is the dose-response curve forA given with 1 unit ofB). The slopes of the dose-response curves forA are reduced by each of these doses ofB, soA and B would be deemed antagonistic according to standard radiobiological criteria, although the isoboles in (A) show that they are synergistic.

3 10

MORRIS C . BERENBAUM

A

DOSE OF A

\

\ \

\

'\

0

2

4

a

6

10

12

DOSE

FIG. 16. (A) Concave-down isoboles for A and B , showing antagonism. (B) Doseresponse curves forA and B alone and for combinations ofA with various fixed doses ofB ( C , D,. . , L ) . Procedure as in Fig. 15B. The slopes ofthe dose-response curves forA

.

INTERACTIONS O F BIOLOGICALLY ACTIVE AGENTS

311

one to determine the nature of an interaction between two agents has led to numerous investigations in which one of the agents is given only at one dose, for this is sufficient according to these criteria. However, as shown previously, isoboles indicating synergy or zero interaction between two agents cannot be drawn when one (or both) is given at a single dose level only, nor can the information required for Eq. (11)be obtained in this way. Therefore, such experiments can never provide enough information for the detection of synergy or additivism. Accordingly, the only studies that can be analyzed satisfactorily are those where both agents have been used over an adequate range of dose levels. Even then, many studies cannot be analyzed because they give only normalized results or because, even when there is marked variation in effect from one experiment to another, the eEects of the agents on their own and in combinations have nevertheless been determined in separate experiments. These drawbacks considerably restrict the range of studies available for examination, and the two examples analyzed here are selected because their data are unusually adequate and they lack the faults found in so many others. Han and Elkind (1977) studied the effects of X rays and ultraviolet light (UVL) on hamster kidney cells in uitro. Both agents gave exponential dose-response curves with shoulders (curves X and U in Fig. 17A). Their effect in combination exceeded the product of their separate effects, so, using the effect-multiplication criterion, the authors concluded that cell killing was enhanced by combining the agents. When a fixed dose of either agent was given just before graded doses of the other, the final slope of the curve was unchanged, but the shoulder was diminished or removed, so the type of enhancement observed was described as “additive” in the terminology adopted by these authors and by Dewey et al. (1971).[It would be classed as a type of synergy in the terminology of Haynes (1964) and Tyrrell(1978).] Their curves are replotted in Fig. 17A, and isoboles are drawn in Fig. 17B and C for fractional cell survivals at 0.5 log,, intervals, the positions of the required points being read directly from Fig. 17A. The isoboles are concave down, showing that X rays and UVL were antagonistic in these experiments, contradicting Han and Elkind’s (1978) conclusion that cell killing was enhanced when these agents were combined. Another example is provided by Patrick and Haynes’s (1964) study of the effects of ethylmethanesulfonate (EMS) and X rays on yeasts. are increased whenA is given withB (and the shoulder on the dose-response curve forA is abolished), so A and B would be deemed synergistic according to standard radiobiological criteria, although the isoboles in (A) show that they are antagonistic.

3 12

MORRIS C . BERENBAUM 0

A

-0.5 Z

0

+

0

a a LL

0

f

5

-1.0

a

3

cn

0 0

2 -1.5

-2.0 0

200

400

UVL (ERGS/MM*)

0

0.4

X RAYS(KRAD)

UVL f E R G S / M M 2 )

0.8

INTERACTIONS O F BIOLOGICALLY ACTIVE AGENTS

313

These agents were claimed to interact synergistically since, when yeasts were treated with various fixed concentrations of EMS and then irradiated, the X-ray dose-response curve showed increased slope and loss of shoulder. The relevant curves taken from these authors’ study (Patrick and Haynes, 1964, Fig. 9A, “immediate plating”) are shown in Fig. MA, and the isoboles derived from this in Fig. 18B. Again, in spite of the increased slope and loss of shoulder, these agents are clearly antagonistic as shown by their concave-down isoboles. Other studies giving information adequate for the construction of isoboles in the manner of the foregoing two examples are those by Uretz (1955), Barendsen et al. (1960), Durban and Grecz (1969), Martignoni and Smith (1973), Railton et al. (1975), Tyrrell (1976), Lai and Ducoff (1977), Webb et al. (1978), Correia and Tyrrell (1979), Szumiel and Nias (1980), Hume and Marigold (1981),and Petin and Berdnikova (1981). Overall, the authors’ conclusions based on changes in slopes and shoulders of dose-response curves appear, when compared with conclusions based on isoboles, to be correct in about 40% of cases, so it is clear that the former criteria are often misleading.

VII. Modifications of the lsobole Method

A. RESPONSE ISOBOLES The idea that the axes for constructing isoboles might be scaled according to drug effects rather than drug doses appears from time to time in the literature (Loewe, 1953; Gessner, 1974). Tattersall et d. (1973) made this the basis of a modified isobole method for studying the effects of combinations of drugs on the growth of cells in uitro. The concentration ofa drug that reduces cell counts by 50% (IDso)is assigned a value of 1 and the concentrations that reduce cell counts by 0.75, 0.5, and 0.25 of this reference reduction (i.e., by 37.5, 25, and 12.5%)are assigned values of 0.75, 0.5, and 0.25, respectively, and so on. This gives a linear scale for each agent, and the isoboles are constructed using these scales as axes. Now, it is fairly obvious that this Frc. 17. Effect ofX rays (XR) and ultraviolet light (UVL)on hamster kidney cells (data of Han and Elkind, 1977, Figs. 2 and 3). (A)X,U : dose-response curves for XR and UVL separately. A, B : dose-response curves for XR preceded by 100 or 175 ergs/mm2 UVL respectively. C , D :dose-response curves for UVL preceded by 450 or 700 krads XR respectively. The slopes of the curves are unchanged but the shoulders are reduced or abolished, suggesting synergy according to standard radiohiological criteria. (B) Isoboles for combinations of XR and UVL, UVL given after XR. (C) lsoboles for comhinations ofXR and UVL, XR given after UVL. The isoboles in (B) and (C) are constructed from the data in (A); both sets are concave down, showing antagonism.

314

MORRIS C. BERENBAUM

X RAYS

( K RAD)

INTERACTIONS O F BIOLOGICALLY ACTIVE AGENTS

315

method cannot be correct with agents that have nonlinear doseresponse curves. Consider the case of “combinations” of an agent with itself, with the axes of the isobolograms drawn according to Tattersall et al. (1973). A straight line isobole (indicating equal effect) drawn from the IDsoon one axis to the IDsoon the other will pass through the point representing the “combination” of two ID2JIs. But if the doseresponse curve is nonlinear, twice the IDz5will not equal the IDs0,and the conclusion will be reached that the same effect (50% growth inhibition) is produced by two different doses of the same agent. The same argument applies to any specified effect; thus, the method is applicable only when response is strictly proportional to dose, in which case it does not matter whether dose or response is used as the scale. This method was used by Jackson et al. (1976) to examine the effects of combinations of D-galactosamine (Gal-N) and 3-deazauridine (DAU). The authors’ hypothesis was that, as Gal-N is selectively toxic to the liver and DAU to proliferating cells, combinations might act synergistically in damaging malignant hepatic cells that retain some biochemical resemblance to normal liver. When response isoboles were plotted as described above, maximum synergy was shown towards the Morris hepatoma lines 3924A, 8999R, and 89995, which retain some liver biochemical functions, and little or no synergy was shown toward the Novikoff hepatoma, which is extensively dedifferentiated and has little or no remaining hepatic-type function. Only slight synergy was shown toward the nonhepatic cell lines L1210 leukemia and LS fibroblasts. The finding was therefore taken to support the authors’ hypothesis. How do isoboles plotted according to these authors differ from orthodox isoboles with drug doses or concentrations on linear scales? Jackson et al. (1976) give concentration-effect curves for the two drugs acting on the cell lines used, and the concentrations reducing cell counts by any given percentage in the range of interest can be read off these curves and enable one to transform the scales of the isoboles illustrated by these authors to linear scales. The two sets of isoboles are shown in Fig. 19, using 3924A cells and Novikoff cells as examples. It is clear that, in some cases, the two methods of plotting isoboles may give markedly discrepant results. It is particularly relevant that FIG. 18. Effect of ethylmethanesulfonate (EMS) and X rays (XR) on yeasts (data of Patrick and Haynes, 1964, Fig. 9A). (A) E , X : dose-response curves for EMS and XR separately. A , B , C: dose-response curves for XR after exposure to 6.5, 8.0, and 9.0 x M EMS, respectively, showing increased slope and progressive reduction of shoulder and thus suggesting synergy according to standard radiobiological criteria. (B) Isoboles for combinations of XR and EMS. Curves constructed from data in (A). These isoboles are concave down, showing antagonism.

3 16

MORRIS C. BERENBAUM

--.

A

\

\

'.

I

I

I

t

'. '. \

0.4

\

\

\ ID50

0

0.4

02

0.6

0.8

mM

1.8

mM

D-Galactosamine

NM

ID50

1

0

0.45

0.9

1.35

D-Galactosamine

FIG. Comparison between response isoboles and dose isoboles for effec E of combinations of D-gdactosamine and 3-deazauridine on rat hepatoma cells. Data and method of plotting response isoboles are from Jackson et al. (1976). Isoboles for 50% growth inhibition (IDw effect) are shown on two scales. The inner scale shows drug concentrations giving 1,0.75,0.5, and 0.25ofthe IDW effect, and the outer scale shows 1, 0.75,0.5, and 0.25 of the concentration giving the ID60 effect. Because of the nonlinear relation between concentration and effect, these scales differ, and the arrows from the inner to the outer scales show concentrations on the latter corresponding to particular effect levels on the former. Isobole for the IDWeffect on the effect scale (-0-0-0-). Isobole for the IDw effect on the concentration scale.)-.-.( (A) 3924A cells. Transformation from the effect scale to the concentration scale converts an isobole suggesting synergy to one showing antagonism. (B) Novikoff hepatoma cells. Transformation from the effect scale to the concentration scale converts an isobole suggesting additivism or slight synergy to one showing fairly marked synergy.

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

317

the isoboles for lines 3924A (and for 8999R, not shown here), which show marked synergy when the isobole is plotted according to Jackson et al. (1976), show antagonism when the isobole is plotted correctly and that the isobole for the Novikoff hepatoma, which shows additivism or slight synergy when plotted according to Jackson et d . (1976), shows the most marked synergy of any of the lines tested when isoboles are constructed in orthodox fashion. Therefore, the results, when reanalyzed using orthodox isoboles, militate strongly against the authors’ hypothesis-the cell line for which combinations of Gal-N and DAU show the most pronounced synergy is the Novikoff hepatoma, which has little or no residual hepatic features, and they show antagonism when tested against two lines with marked hepatic characteristics (3924A and 8999R).

B. ADDITIVITYENVELOPES Steel and Peckham (1979)recently proposed an isobolar method that entails constructing an envelope of additivity rather than a single additive line. Their argument was as follows. With agents giving linear dose-response curves (or curves that can be linearized by an appropriate transformation), it is reasonable to expect that, if the agents do not interact, the effect of a combination will be the sum of the effects of its constituents, with d u e account taken of any mathematical transformation used to linearize the dose-response curves. (As has been shown previously, this is correct; when effects are proportional to dose or simple exponential functions of dose, we sum the effects in the one case, and in the other we sum their logarithms.) However, when the dose-response curves are nonlinear and cannot be linearized by any biologically sensible transformation, there is no obvious way in which to perform this summation. Therefore, for any pair of agents with known survival curves, an additivity envelope is constructed that represents, in effect, a region bounded by confidence limits, necessitated by this uncertainty in how to perform the summation. Only those combinations that have the specified effect but which lie outside this region may reasonably be considered as showing interaction. Additivity envelopes are constructed as follows. Using the survival curves of two agents A and B, we try to deduce the composition of those combinations that would produce a given survival fraction S ifA and B did not interact. A dose ofA is selected that reduces survival to SA, which is not as low as S. We then determine, from the survival curve for B, the dose of B that would, on its own, give a logarithmic reduction in survival sufficient to make u p the difference between S A and S . As survival curves are generally nonlinear on semilogarithmic

318

MORRIS C. BERENBAUM

plots, the determination of the required complementary dose of B depends on which part of its survival curve is used. Limiting values of B are obtained if the complementary dose is measured both to the right fiom zero (Mode I) and to the left from the point on the curve corresponding to survival S (Mode 11).Finally, on a graph with axes representipg doses of A and B on linear scales, the positions of all combinations of A and B calculated to give survival S are plotted, each combination consisting of a particular dose of A and its calculated complementary dose of B . For any given dose of A there is a range of possible values of B (the extremes of which are given by the Mode I and Mode I1 determinations, respectively). Thus, over a range of values ofA, the extreme values of B define an envelope, and all combinations represented by points within the envelope are said to be additive. Combinations giving survival S and lying below the envelope are said to be supraadditive, as they require less of the agents to give survival S than would be expected from this zero-interaction model. Conversely, combinations giving survival S and lying above the envelope are said to be subadditive. Let us apply the additivity envelope method to the examination of “combinations” ofA and B where these are, in fact, the same agent. Let the survival curve for A (and B) be as shown in Fig. 20A, and let us find combinations of A and B giving a survival of The survival curve shows that 16 units o f A gives a survival of lo-’.’, and the Mode I method for finding the complementary dose of B shows that we need, fall in survival, to in addition, 16 units of B , giving another reduce survival to lo+. But A and B are the same agent, so 16 units A plus 16 units B is the same as 32 units A. Now, the survival curve shows that the dose ofA giving survival is 20 units, so the Mode I method leads to the conclusion that 32 units ofA has the same effect as 20 units ofA, which is contradicted by the survival curve on which the calculations were based. It follows that the Mode I method of construction is incorrect in this case. The complete isobole for survival calculated according to Mode I is shown in Fig. 20B. The Mode I1 calculation gives a straight line isobole, as must be the case with combinations of an agent with itself, so in this instance the Mode I1 construction is correct (Fig. 20B). However, with combinations of agents with different nonlinear dose-response curves, the Mode I1 calculation generally yields curved additive isoboles (Steel and Peckham, 1979; Steel, 1979; Deen and Williams, 1979; Szumiel and Nias, 1980), although additive isoboles drawn by the orthodox method are straight. Further, the position of the Mode I1 isobole depends on whether it is supposed that agent A complements agent B or vice versa (Steel, 1979), even if they are given simultaneously. I n other

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

319

Dose of A or B

Dose of A

FIG. 20. Construction of an additivity envelope (Steel and Peckham, 1979) for combinations ofA and B when these are the same agent. (A) Dose-response curve ofA and B . (B) Mode I and Mode I1 isoboles for combinations ofA and B giving cell survival (see text for method of construction).

320

MORRIS C. BERENBAUM

words, the type of interaction detected depends on the investigator's suppositions as to modes of action and might change if these suppositions changed, although the experimental observations would not. This is hardly a tenable position. Deen and Williams (1979) used this method to study the effects of BCNU and X rays on 9L rat brain cells grown in vitro (Fig. 21). Most of the points representing the combinations used lay within or touched the lower edges of these envelopes and were thus regarded as additive; one point lay below its appropriate envelope and was regarded as showing supraadditivism. This study gives enough information to allow the construction of conventional isoboles. Figure 22A shows the X-ray survival curves of 9L cells after exposure to various fixed concentrations of BCNU, from 0 to 7.5 pg/ml, and the survival curve for BCNU alone (data from Deen and Williams, 1979, Fig. 3). The dose of X rays required to give any specified fractional survival when used in combination with any of the fixed concentrations of BCNU may be BCNU,lS hr, X RAY

BCNU CONC. IC19/mlI FIG.21. Additivity envelopes for combinations of BCNU and X rays giving lo-', lo-*, and survival of 9L rat brain tumor cells (Fig. 4 of Deen and Williams, 1979, reproduced with permission). Most combinations are located within or almost within additivity envelopes, suggesting no departure from zero interaction. One lies below its appropriate envelope, suggesting synergy. FIG.22. Data of Fig. 3 of Deen and Williams (1979) replotted to construct conventional isoboles. (A) Dose-response curves for BCNU, X rays, and X rays with 1,3,5, or 7.5 to pglml BCNU, respectively. (B) lsoboles for survival plotted from (A). For instance, (A) shows that lo-' survival is given by the following: 5 pg/ml BCNU alone, 3 pg/ml BCNU with 5.5 Gy X rays, 1 pg/ml BCNU with 8.6 Gy X rays, and 10.1 Gy X rays alone. Thus, the isobole for lo-' survival is plotted in (B)with these values as coordinates. All isoboles are concave down, showing antagonism.

INTERACTIONS OF BIOLOGICALLY ACTIVE AGENTS

32 1

322

MORRIS C. BERENBAUM

read directly off Fig, 22A, and this allows isoboles for these specified effects to be constructed as in Fig. 22B. Clearly, all the isoboles are concave down, showing that X rays and BCNU acted subadditively (i,e., antagonistically) in these experiments and not additively or supraadditively as had been concluded using the envelope method, It may be noted that, although BCNU and X rays each show positive self-interaction of the type discussed earlier, as shown by the shoulders on their survival curves, nevertheless the concave-down isoboles in Fig. 22B show that these two agents interact negatively with each other in combination. This illustrates the point that there is no necessary connection between the self-interaction (or noninteraction) of an agent and the type of interaction it shows with other agents (Section IV). VIII. Therapeutic Optimization

The whole of the preceding discussion has been concerned only with the interactions between different agents with respect to single specified effects. However, one and the same agent may have many therapeutically relevant effects. In particuIar, cytotoxic agents damage not only neoplasms but also essential normal tissues; consequently, the attainment of the desired therapeutic effect is usually limited by host toxicity. In an early examination of this problem as it appeared in the treatment of leukemia in experimental animals, Goldin et al. (1955) put forward three pertinent questions: 1. Is there any evidence for synergism with respect to effect on the leukemia alone, disregarding any effects on the host? 2. Is there any evidence of synergism with respect to effect on the host, disregarding any effects on the tumor? 3. If the drugs are synergistic in the sense suggested in (2), what are the optimal proportions in which they should be used to attain maximum antileukemic damage for fixed cost in toxicity to the host? In fact, the third question is still valid even when drugs are not synergistic with respect to host toxicity and, with this modification, these questions encapsulate the problem of what has been termed therapeutic synergy.” This was defined by Venditti and Goldin (1964) as the ability of drugs in combination to produce a therapeutic response superior to the maximum response to either drug alone. In many ways, therapeutic synergy is an unfortunate term, for it confuses two essentially different issues. One is the way in which dif‘I

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323

ferent agents interact to produce some specified effect, for example, destruction of tumor cells, destruction of normal cells, prolongation of life, loss of weight, and so on, a positive interaction with respect to a specified effect being termed synergy. The other issue is the way in which the different interactions (synergistic, additive, or antagonistic) for these different effects in one and the same individual may be exploited so as best to achieve the aims of therapy. As will be shown, for any set of agents, there is generally a combination that will best achieve specified therapeutic aims, irrespective of whether the interactions of these agents in regard to particular effects are synergistic, antagonistic, or additive. Accordingly, the term therapeutic synergy is inappropriate, and it is proposed here to replace it with the term “optimal therapy,” which carries no connotations about the sorts of interaction present and expresses accurately what is being sought (Wampler et al., 1978). Therapeutic optimization requires a review of its own, and that task will not be attempted here. What is proposed is a limited analysis in the light of the foregoing discussion of drug interaction, in the hope that this will clarify some of the issues involved. Suppose that two agents A and B show positive interaction with regard to their antitumor effect and negative interaction with regard to their effect on some critical host tissue, such as bone marrow. Then the isoboles for the various levels of antitumor effect and antibone marrow effect might be, for example, those shown in Fig. 23. Now, suppose that we are willing to accept a reduction in bone marrow cells to, say, 30%of normal but no lower. The isobole for this effect runs from a dose of 1 mg/kg on the dose axis for A to 2 mg/kg on the axis for 23. These doses ofA and B alone give, respectively, 4%and 2%survival of tumor cells. Now we investigate various combinations ofA and B along this isobole and, as we pass from one dose axis to the other, the effect on tumor cells rises to a maximum and then falls, without any change in bone marrow toxicity. The maximum kill of tumor cells on this isobole is given by the combination of 0.8 mg/kg A and 1.5 mg/kg B, which leaves only 1% of tumor cells surviving. This, therefore, is the maximum therapeutic effect attainable at a cost of 30% survival of bone marrow cells. It should be noted from Fig. 23 that 1% survival of tumor cells could also be achieved by giving 3 mg/kg ofA alone or 3.5 mg/kg B alone, but at greater costs in marrow toxicity. Strictly speaking, therefore, therapeutic synergy should not be defined as the achievement by a combination of an effect greater than that which could be obtained by any of

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1

2

3

4

A (mg/kg)

FIG.23. Therapeutic optimization for two agents, A and B. Isoboles for tumor cell survival (-) are shown for 5.2, 1,0.1, and 0.01%of controls. Isoboles for survival of normal bone marrow cells (---) are shown for 30, 20, and 10% of controls. For any specified limit of toxicity to bone marrow, there is an optimum combination ofA and B giving a minimum tumor cell survival. For instance, if 30% bone marrow survival is the maximum acceptable level of toxicity, the optimum combination consists of 0.8 mg/kg A and 1.5 mg/kg B, which gives 1%tumor cell survival. UsingA or B alone, this level of tumor cell survival could b e achieved by 3 mg/kg A or 3.5 mglkg B , each of which leave only 15% of marrow cells surviving.

its constituent agents alone. A more precise definition would entail adding to the definition of Venditti and Goldin (1964) the phrase, “subject to a specified maximum level of toxicity.” In Fig. 23, the degree of bone marrow toxicity accepted fixes the maximum level of tumor destruction obtainable. If 20% survival of marrow cells is considered acceptable, 0.1%survival of tumor cells may be obtained (with a combination of 1.5 mg/kgA and 2.5 mg/kg B); if 10% survival of marrow cells is acceptable, 0.01%survival of tumor cells can be achieved, and so on. Everything depends on how much we are willing to pay in order to destroy tumor cells. We are not limited to specifying only one kind of toxicity. For instance, we might, in a clinical setting, demand that the antitumor effect be the maximum subject to blood neutrophils not falling below 103/pl, and platelets not falling below 5 x 104/pl.We would then have more than one host toxicity isobole as a limit, as in Fig. 24, and the maximum attainable antitumor effect would have to be found within these limits (or as many limits as we chose to set). A good example of the simultaneous measurement of antitumor and host toxicity effects is the work of Fodstad and Pihl (1980) on the

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5

4

33

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m 1

0

1

2

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5

A hg/kg) FIG. 24. Therapeutic optimization with two different toxicity limits. The specified limits are that blood neutrophils fall to not less than 103/gland platelets to not less than 5 x 104/gl;isoboles for these two effects are shown. The isoboles.for percentage of tumor show that, if neutrophil levels had been the sole limit, the maximum cell survival (-) antitumor effect attainable would have been 1% tumor cell survival, with a combination of 2.75 mg/kgA and 1.5mg/kgB. If platelet levels had been the only limit, the maximum antitumor effect (again, 1% survival) would have been produced by a combination of 1.3 mg/kgA and 3 mg/kgB. With both toxicity limits in force, the optimum combination is 2 mg/kg A and 2 mg/kg B, which leaves 2% tumor cells surviving.

effects of adriamycin and ricin on L1210 leukemia. These authors established cell survival curves for leukemic cells, resting bone marrow cells, and proliferating marrow cells for each agent separately and for various combinations. Their detailed data enable isoboles to be drawn for effects down to a 2 loglo fall in survival of leukemic cells and down to a 0.5 loglo fall in normal marrow cells. It is evident from the concave-up isoboles in Fig. 25A that the two agents act synergistically in killing leukemic cells and from the concave-down isoboles in Fig. 25B that they act antagonistically in killing normal bone marrow cells. This antagonism is reflected in the concave-down isobole for the LDS0 effect in nonleukemic animals shown in Fig. 25C. One would expect from these curves that combinations of the agents could be found that would produce in vivo a greater antitumor effect than either agent used alone, subject to some limiting level of toxicity, although the authors did not examine this point in any detail. Now consider the situation shown in Fig. 26. Here, the isoboles for tumor-cell kill show negative interaction and those for bone marrowcell kill show positive interaction. Selecting 20% bone marrow survi-

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R i c h (pg/ kg) FIG. 25. Effects of adriamycin and rich on L1210 leukemia cells and normal marrow cells in mice (Fodstad and Pihl, 1980). (A) Isoboles for survival of leukemic cells at of controls (data from the authors’ Figs. 1-3). Marked synergy is levels of lo-’.’ to

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2

3

327

4

A (mg/kg)

Therapeutic optimization for agents showing antagonism for the antitumor and synergy for host-toxicity (---). Values show percentage survival of effect (-) tumor cells and normal bone marrow cells, respectively. If, say, 20% bone marrow survival is the limiting toxicity, the greatest antitumor effect is obtained with 4 mglkg B alone, which gives 0.1% tumor cell survival. No combination ofA and B gives an effect as great as this without exceeding the toxicity limit, so there is no advantage in combining A and B, and optimal therapy is provided by a single agent. FIG. 26.

Val as the maximum acceptable toxicity, we see that, as we pass along the isobole for this effect from one dose axis to the other, the antitumor effect decreases to a minimum and then increases again. The maximum antitumor effect compatible with this level of toxicity (0.1% survival of tumor cells) is, in fact, obtained only along one of the dose axes (in this example, at a dose of 4.0 mg B given alone). Clearly, there is no therapeutic advantage here in combining A and B , for no combination of agents produces an antitumor effect as great as that achievable by one or other of the constituents used alone, subject to a specified maximum level of host toxicity. Fig. 27 shows that an optimal therapy may be achieved by a combination of agents even if the isoboles for the antitumor effect show antagonism, provided that those for host toxicity show even more marked antagonism. Conversely, if the isoboles for the antitumor effect

seen. (B) Isoboles for survival of bone marrow cells (data from the authors’ Figs. 1-3),showing antagonism for proliferating cells and marked antagonism for resting cells. (C) Isobole for the LDW in nonleukemic mice. Values shown are numbers of survivors in groups of four mice (data from authors’ Table I11 and text). Marked antagonism is seen.

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FIG.27. Therapeutic optimization with agents showing antagonism for both antitumor effect (-) and host toxicity (---). Values are percentage survivals of tumor cells and normal bone marrow cells, respectively. With 20%bone marrow survival as the limiting toxicity, a maximum antitumor effect (1%cell survival) is produced by a combination of 2.75 mg/kg A and 1.75 mg/kg B. No dose ofA or B alone produces this effect without excessive toxicity (<20% marrow survival). Thus, a combination o f A and B provides optimal therapy, even when these agents are antagonistic in their antitumor effect.

show synergy but those for host toxicity show even more pronounced synergy, then the optimum effect will be obtained by giving one of the agents alone (Fig. 28). We may now examine the shapes of the isoboles for increase in life span when this is limited both by tumor growth, on the one hand, and drug toxicity on the other. As we increase the dose of any one agent, we increase both the antitumor effect and host toxicity. It is then usually the case that life span increases to a maximum at some dose level and then falls again as increasing host toxicity begins to outweigh the benefit due to tumor destruction (see Venditti and Goldin, 1964, for review). Now suppose two agents have isoboles for antitumor effect and host toxicity as in Figs. 23 or 25. At low doses of both agents, host toxicity is of minor degree and has little effect on life span. Accordingly, in the low-dose region, the isobole for prolongation of life span will reflect the predominating antitumor effect and will thus be concave up. At excessive doses of either or both agents, host toxicity predominates, for the extent of the antitumor effect is immaterial when the host is killed by the agents. Accordingly, in the high-dose region, the isobole for prolongation of life span will now reflect the predominating host toxicity effect and will thus be concave down. These features show that the isoboles for increase in life span in such a case will in general form

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329

4

A h d k g )

FIG. 28. Therapeutic optimization with agents showing synergy for both antitumor effect (-) and host toxicity (---). Values are percentage survivals of tumor cells and bone marrow cells, respectively. With 20% bone marrow survival as the limiting toxicity, the greatest antitumor effect (5% cell survival) is achieved with a combination of 0.5 mg/kgA and 1.5 mg/kg B. No dose ofA or B alone can produce this effect without excessive toxicity (<20%marrow survival). Thus, a combination of A and B provides optimal therapy even when these agents are synergistic in their toxic effect on the host.

the contours of a hill with a summit not on any dose axis. A typical example of this is shown in Fig. 29 for the interaction of cyclophosphamide and carminomycin in the treatment of L1210 leukemia (Avery and Cruze, 1978). The distinction between the interactions of agents with respect to single effects, which is the main subject of this review, and therapeutic optimization is perhaps illustrated most clearly by the possibility that an optimum combination may exist even for agents that show zero interaction for antitumor and host toxicity effects. Figure 30 illustrates such a case. All isoboles are straight, showing zero interaction between the agents. Two types of toxicity limit the amount of drug that may be given and, as a result, there is a region near the intersection of the two limiting toxicity isoboles containing combinations that produce a greater antitumor effect than is possible with either agent alone if these toxic limits are not to be transgressed. IX. Conclusion

The existence of any substantial interaction between different agents that are used clinically or to which man is exposed environmentally is

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88

88

60

72

63

100

120

140

160

180

I

0.2

0

21

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0

Cyclophosphamide (mg/kg)

FIG, 29. Life span isoboles for mice with L1210 leukemia treated with combinations of cyclophosphamide and carminomycin (Avery and Cruze, 1978, Table I). Values are percentage increases in life span, and isoboles are shown for percentage increases of 50 to 300%,in steps of50%. At low dose levels, isoboles are concave up, reflecting synergy for the antitumor effect with negligible host toxicity. At high dose levels, they are concave down, reflecting antagonism for host toxicity, which is the predominating factor limiting survival in this dose region. The isoboles delineate a hill with a peak corresponding to the combination of 140 mg/kg cyclophosphamide and 0.65 pg/kg carm inomycin,

potentially of great importance. In the one case, the consequence may

be a gratifying improvement in efficacy or a catastrophic increase in toxicity compared with what is expected. In the other, the presence in the environment of numerous carcinogens at low levels creates an enormous potential for interaction. Despite the possible role of such interactions in human carcinogenesis (Gibson et al., 1964; Selikoff et al., 1968; Bross and Natarajan, 1972; Rothman and Keller, 1972; Hammond and Selikoff, 1973; Doll, 1972; Band et al., 1980), they remain virtually uninvestigated, a fact that suggests the need for the utmost conservatism in adding to the number of environmental contaminants or in increasing the level of any one of them. Of course, lack of interaction does not necessarily mean lack of biological significance. The rather artificial example illustrated in Fig. 30 shows how biologically important properties may be possessed b y combinations of agents that do not interact with respect to any important effect. Further, the detailed discussion of interactions presented here should not be allowed to obscure what is obvious, that the absolute magnitude of an effect is important, irrespective of how it is pro-

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0

2

4

6

33 1

8

A (mg/kd

FIG. 30. Therapeutic optimization with agents that show zero interaction for both show antitumor and host toxicity effects. Values on the antitumor effect isoboles (-) percentage tumor cell survival. Host toxicity isoboles (---) and (-.-.-) show limiting toxicities of two different kinds. The maximum antitumor effect obtainable with agentA alone is 10%survival, at a dose of 3 mg/kg. The maximum obtainable with B alone is 5 % survival, at a dose of 4 mgkg. However, a combination of 2 mg/kgA and 3 m a g B gives 2% tumor cell survival. Thus, a combination of A and B gives optimum therapy even when these agents show no interaction for any relevant effect.

duced. When different agents share the same effect, their combinations will inevitably have a greater effect than any of their constituents, except in cases of marked antagonism (to the degree shown in Figs. 10 and 12B).Excluding such cases, and provided always that a combination lies within the region in which toxicity or other limits are inoperative (see Section VIII), the exact nature of an interaction, whether it be synergistic, additive, or moderately antagonistic, may in some circumstances be less important than the fact that an increased effect is obtained. Rothman (1978), for example, points out that, even if it were conclusively shown that two environmental carcinogens had zero interaction in producing cancer, no one would suggest that individuals already exposed to one could with impunity also be exposed to the other.

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The therapeutic significance of interactions between agents requires careful consideration. Most work has been concerned with life span measurements (Venditti and Goldin, 1964; Goldin et al., 1974). In a sense, these have made the problem seem rather simpler than it is in reality. When life span is the measure of effectiveness, both tumor damage and host toxicity are comprehended in the same parameter. The problem then appears to be simply one of measuring a single effect (prolongation of life) for various drug combinations, and one may, indeed, ask whether the combinations show synergy for this effect and assume correctly that the answer has direct therapeutic relevance. However, it is only in such circumstances that synergy for a therapeutic effect and so-called therapeutic synergy may be equated. For any other sort of antitumor effect or any measure of host toxicity other than death, synergy for the therapeutic effect and “therapeutic synergy” are two entirely different things, since the latter (i.e., the provision of optimal therapy by combinations rather than by single agents) may be found irrespective of whether the agents show synergy, additivism, or even antagonism (Fig. 27) so far as the antitumor effect itself is concerned and even when all relevant effects, antitumor and antihost, show zero interaction (Fig. 30). Therefore, in all studies that go beyond simple life span measurements as an index of both antitumor effect and host toxicity, we have to specify what antitumor effect is to be measured, what host toxicity (or toxicities) are important, and what limits we wish to set to these. Accordingly, the measurement of different types of effect, therapeutic and toxic, is inescapable in this sort of investigation.

Ac m oWLEDGMENTS I am grateful to the Cancer Research Campaign and Medical Research Council for continued support.

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INDEX

A Actin, reduction of cable structure, SV40 tumor antigens and, 137-139 S-Adenosyl-L-methionine, polyamines and, 181 Adenovirus, helper function, SV40 tumor antigens and, 137 Agents effect multiplication, 273-280 effect summation, 280-285 interaction and self-interaction, 285288 interactions criteria based on changes in doseresponse curves, 304-313 therapeutic optimization, 321-329 Antibodies, to polyamines, 197 Arachidonic acid, transformation of, 4952 hypercalcemia and, 55-58 tumor promotion and, 58-67 Arginase, of SPV, 91-92

C Carcinogen( s) modification of polyamine metabolism by repeated administrations, 209-213 single administration, 205-209 physical, changes in biosynthesis and content of polyamines in target tissues by, 241-243 Carcinogenesis chemical, polyamine levels in urine, sera and erythrocytes and, 230-233 multistage, polyamines and, 213-229

337

Cell(s) growth, prostaglandins and, 67-69 transformation and tumorigenicity, polyoma and, 12-13 lytically infected, polyoma T antigens in, 1-5 nonpermissive, models for transformation by HSV, 29

D Deoxyribonucleic acid HSV, model for transformation by, 3031 induction of cellular and viral synthesis, SV40 tumor antigens and, 127-132 nucleotide sequence of polyoma, 5-9

H Herpes simplex virus biochemically transformed cells persistence of genetic material in, 37-39 properties of, 31-32 mechanism of transformation by, 41-44 models for transformation by, 28 inactivated virus, 30 nonpermissive cells, 29 temperature-sensitive virus mutants, 30 virus DNA or fragments, 30-31 virus-host range mutants, 29 morphologically and tumorigenically transformed cells persistence of genetic material in, 39-41 properties of, 32-37

INDEX

Hypercalcemia, arachidonic acid transformation and, 55-58

I Immune response prostaglandins and, 69-70 to 5PV, 101-103 Isobole(s), interaction index and diagonal arrays, 301-304 heterergic combinations, 294-295 homergic combinations, 288-294 inadequacy of 2 x n experimental design, 298-301 isobole construction from limited data, 295-298 Isobole method, modifications of additivity envelopes, 317-321 response isoboles, 313-317

L Lytic infection role of large T antigen in, 13-15 small and medium T antigens in, 18-19

M 5'-Methylthioadenosine, polyamines and, 181-185 Microinjection, applications and trends, SV40 virus and, 140-146 Multiplication, interactions between biologically active agents and, 273-280 Mutants host range, model of transformation by HSV and, 29 temperature-sensitive, model for transformation by HSV and, 30 Mutations, of polyoma T antigens, 11-12

N Neoplasia, human, relevance of Shope papilloma-carcinoma to, 104-107

P Polyamines S-adenosyl-L-methionine and, 181 biosynthesis and levels during virusinduced transformation, 233-234

DNA viruses, 238-241 RNA viruses, 235-238 biosynthesis enzymes levels in tumors, 197-204 properties of, 162-180 catabolism in mammals, 185-194 changes in biosynthesis and content of target tissues by physical carcinogens, 241-243 conjugation and excretion products, 195-197 levels in experimental tumors, 197-204 5'-methylthioadenosine and, 181- 185 of metabolism modification in oioo and in oitro, effects of repeated administration of a carcinogen, 209-213 effects of single administration of a carcinogen, 205-209 levels in urine, sera and erythrocytes during carcinogenesis, 230-233 multistage carcinogenesis and, 213229 mutagenic and antimutagenic properties, 229-230 natural antibodies to, 197 structure-function relationship of, 150162 Polyoma T antigen(s) large role in lytic infection, 13-15 role in transformation, 15-18 in lytically infected cells, 1-5 mutations affecting, 11-12 small and medium functions associated with, 21-22 small and medium lytic infection and, 18-19 transformation and, 19-21 structural features, comparison with SV40,9-11 Polyoma virus cell transformation and tumorigenicity, 12-13 DNA nucleotide sequence, 5-9 gene expression, cell type dependence Of, 114-125 Prostaglandin( s) effects on cell and tumor growth, 67-69 immune response and, 69-70 levels in tumors, 52-55

339

INDEX

production effects of TPA on, 59-60,65-66 inhibitors of, 61-65 stimulation by growth factors, 66-67

permissive cells, 114 semipermissive cells, 120-121 virus-resistant cells, 121-123 comparison with polyoma T antigens,

R

function of tumor antigens, 125-127 helper function for adenovirus and U antigenicity, 137 induction of cellular and viral DNA synthesis, 127-132 reduction of actin cable structure,

9-11 Rabbit kidney vacuolating virus, SPV and,

87 Ribonucleic acid, ribosomal, stimulation by SV40 virus, 136-137

137-139

S Shope papilloma-carcinoma complex, as model system relevance to human neoplasia, 104-107 theoretical and practical considerations,

103- 104 Shope papilloma virus historical origins, 81-82 interaction with host cells cell and tissue culture studies, 92-93 factors influencing replication, 89-91 host cell specificity, 87-89 natural occurrence and transmission,

82-85 papilloma arginase, 91-92 rabbit kidney vacuolating virus, 87 structure of virion and genome, 85-

87 neoplastic progression definition and characteristics, 93-94 factors influencing, 95-96 malignant transformation, 94-95 role of genome in carcinomatous transformation, 96-98 spontaneous regression alterations in growth pattern, 98-99 genetic factors, 99 immune stimulation, 101-102 immune suppression, 102-103 mechanism of, 99- 10 1 Summation, interactions between biologically active agents and, 280-285

SV40 cell transformation and, 139-140 cell type dependence of conclusions, 123-125 nonpermissive cells, 114-120

regulation of late and early gene expression, 132-136 stimulation of rRNA synthesis, 136-

137 microinjection: applications and trends,

140- 146

T TPA, effects on prostaglandin production by cells in culture, 59-60 in oivo, 65-66 Transformation biosynthesis and levels of polyamines in cells DNA viruses and, 238-241 RNA viruses and, 235-238 definition of terms, 28 by HSV, in oitro models of, 28-31 role of large T antigen in, 15-18 small and medium T antigens in, 19-21 by SPV, 94-95 role of genome in, 96-98 SV40 virus and, 139-140 Tumor(s) growth, prostaglandins and, 67-69 levels of polyamines and their biosynthetic enzymes in, 197-204 promotion by arachidonic acid transformation, 58 effects of TPA on prostaglandin production by cells in culture, 59-60 in uiuo,65-66 inhibitors of prostaglandin production, 61-65 stimulation of prostaglandin production by growth factors, 66-67 prostaglandin levels in, 52-55

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CONTENTS OF PREVIOUS VOLUMES

Volume 1 Electronic Configuration and Carcinogenesis C. A . Coulson Epidermal Carcinogenesis E. V. Cowdry The Milk Agent in the Origin of Mammary Tumors in Mice L. Dmochowvki Hormonal Aspects of Experimental Tu morigenesis T. U.Gardner Properties of the Agent of Rous No. 1 Sarcoma R. J . C. Harris Applications of Radioisotopes to Studies of Carcinoge nesi s and Tumor Metabolism Charles Heidelberger The Carcinogenic Aminoazo Dyes James A. Miller and Elizabeth C . Miller The Chemistry of Cytotoxic Alkylating Agents M . C. J . Ross Nutrition in Relation to Cancer Albert Tannenbaum and Herbert Silverstone Plasma Proteins in Cancer Richard J . Winzler AUTHOR INDEX-SUBJECT INDEX

Volume 2 The Reactions of Carcinogens with Macromolecules Peter Alexander Chemical Constitution and Carcinogenic Activity G. M . Badger

Carcinogenesis and Tumor Pathogenesis 1. Berenblum Ionizing Radiations and Cancer Austin M . Brues Survival and Preservation of Tumors in the Frozen State James Craigie Energy and Nitrogen Metabolism in Cancer Leonard D. Fenninger and C . Burroughs Mider Some Aspects o f the Clinical Use of Nitrogen Mustards Calvin T. Klopp and Jeanne C. Bateman Genetic Studies in Experimental Cancer L. W . Law The Role of Viruses in the Production of Cancer C . Oberling and M . Cuerin Experimental Cancer Chemotherapy C. Chester Stock AUTHOR INDEX-SUBJECT INDEX

Volume 3 Etiology of Lung Cancer Richard Doll The Expenmental Development and Metabolism of Thyroid Gland Tumors Harold P. Morris Electronic Structure and Carcinogenic Activity and Aromatic Molecules: New Developments A. Pullman and B. Pullman Some Aspects of Carcinogenesis P. Rondoni Pulmonary Tumors in Experimental Animals Michael B. Shimkin

341

342

CONTENTS OF PREVIOUS VOLUMES

Oxidative Metabolism of Neoplastic Tissues Sitlrii,y W(Grihousi, AUTHOR INDEX-SUBJECT INDEX

Volume 4 Advances in Chemotherapy of Cancer in Man Siilrwy Farbor. Rictlolf Tach, Etlicwtl Morrrrirrg Svars. cirri1 Dorrtiltl Pirrkol The Use of Myleran and Similar Agents in Chronic Leukemias D.A . G. Galrorr The Employment of Methods of Inhibition Analysis in the Normal and TumorBearing Mammalian Organism Ahraharrr Goldirr

Some Recent Work on Tumor Immunity P . A . Gor1.r Inductive Tissue Interaction in Development ClgJi)rt/ Groh.s/i,iri Lipids in Cancer Frarrc.os L . Hai.c,rr urrtl W . R . Bliior The Relation between Carcinogenic Activity and the Physical and Chemical Properties of Angular Benzacridines A . Lci(.tr.Ssag/ll,, N . P . B ~ i Hoi. t R. Dulli l c ~ l .arid F . Zujlc4u The Hormonal Genesis of Mammary Cancer 0.~Miihlhoi~h AUTHOR INDEX-SUBJECT INDEX

Volume 5 Tumor-Host Relations R. W . Bvgg Primary Carcinoma of the Liver Charli,s Berrriirir Protein Synthesis with Special Reference to Growth Processes both Normal and Abnormal

P. N . Ctr/rrphc.ll

The Newer Concept of Cancer Toxin Waro Nakaliaru arid Furtriko FuLuoXu Chemically Induced Tumors of Fowls P . R. P ( w i ~ o i , k Anemia in Cancer Virrcc~rrtE . Pric.1. und Rohrrt E . Grocvdii~ld Specific Tumor Antigens L . A . Zilhar Chemistry, Carcinogenicity, and Metabolism of 2-Fluorenamine and Related Compounds Elizahoth K . Wi,ishurgc,r arid Johrr H . Woi.shurgc,r AUTHOR INDEX-SUBJECT INDEX

Volume 6 Blood Enzymes in Cancer and Other Diseases Oscar Bodansky

The Plant Tumor Problem Arrniti C . Braurr arid H1.nr.v N . W o o d Cancer Chemotherapy by Perfusion Oscw C r i w h , Jr. arid Edic*ard T . KreIm~lit?

Viral Etiology of Mouse Leukemia Lirilii~icAGross Radiation Chimeras P. C. K o / / ( v - . A . 1. S . Davic,s, arid Sheila M . A . DooL Etiology and Pathogenesis of Mouse Leukemia J . F. A . P . Millcr Antagonists of Purine and Pyrimidine Metabolites and of Folic Acid C. M . Tinirriis Behavior of Liver Enzymes i n Hepatocarci noge nesi s George Wi4x.r AUTHOR INDEX-SUBJECT I N D E X

Volume 7 Avian Virus Growths and Their Etiologic Agents J. W . Beard

CONTENTS O F PREVIOUS VOLUMES

343

Mechanisms of Resistance to Anticancer Agents R . W. Brockman Cross Resistance and Collateral Sensitivity Studies in Cancer Chemotherapy Dorris J . Hutchison Cytogenic Studies in Chronic Myeloid Leukemia W . M. Court Brown and lshbel M . Tough Ethionine Carcinogenesis Emmanuel Farber Atmospheric Factors in Pathogenesis of Lung Cancer Paul Kotin and Hans L. Falk Progress with Some Tumpr Viruses of Chickens and Mammals: The Problem of Passenger Viruses G. Negroni

The Relation of the Immune Reaction to Cancer Louis V . Caso Amino Acid Transport in Tumor Cells R. M . Johnstone and P. G . S c h o l r j X l Studies on the Development, Biochemistry. and Biology of Experimental Hepatomas Harold P. Morris Biochemistry of Normal and Leukemic Leucocytes, Thrombocytes. and Bone Marrow Cells 1. F . Seitz

AUTHOR INDEX-SUBJECT INDEX

Carcinogens, Enzyme Induction, and Gene Action H. V . Gelboin In Vitro Studies on Protein Synthesis by Malignant Cells A. Clark Griffin The Enzymatic Pattern of Neoplastic Tissue W. Eigene Knox Carcinogenic Nitroso Compounds P. N . Magee and J . M. Barnes The Sulfhydryl Group and Carcinogenesis J . S. Harrington The Treatment of Plasma Cell Myeloma Daniel E. Bergsagel. K. M . GrgJth. A . Haut. and W .J . Stuc4lr.v. Jr.

Volume 8 The Structure of Tumor Viruses and Its Bearing on Their Relation to Viruses in General A. F. Howatson Nuclear Proteins of Neoplastic Cells Harris Busch and William J . Steelr Nucleolar Chromosomes: Structures, Interactions, and Perspectives M. J . Kopac and Gladys M . Mazeyko Carcinogenesis Related to Foods Contaminated by Processing and Fungal Metabolites H. F. Kraybill and M. B. Shimkin Experimental Tobacco Carcinogenesis Ernest L. Wynder and Dietrich Hoffman AUTHOR INDEX-SUBJECT INDEX

Volume 9 Urinary Enzymes and Their Diagnostic Value in Human Cancer Richard Stambaugh and Sidney Weinhouse

AUTHOR INDEX-SUBJECT INDEX

Volume 10

AUTHOR INDEX-SUBJECT INDEX

Volume 11 The Carcinogenic Action and Metabolism of Urethran and N-Hydroxyurethan Sidney S. Mirvish Runting Syndromes, Autoimmunity, and Neoplasia D. Keast Viral-Induced Enzymes and the Problem of Viral Oncogenesis Saul Kit

344

CONTENTS O F PREVIOUS VOLUMES

The Growth-Regulating Activity of Polyanions: A Theoretical Discussion of Their Place in the Intercellular Environment and Their Role in Cell Physiology wi//iani Rrgtlsori Molecular Geometry and Carcinogenic Activity of Aromatic Compounds. New Perspectives Joseph C . Arcos arid Mary F . Argus AUTHOR INDEX-SUBJECT INDEX CUMULATIVE INDEX

Volume 12 Antigens induced by the Mouse Leukemia Viruses G. Pastc,rtiuk Immunological Aspects of Carcinogenesis by Deoxyribonucleic Acid Tumor Viruses G . 1. Di~ichnlcrn Replication of Oncogenic Viruses in VirusInduced Tumor Cells-Their Persistence and interaction with Other Vi-

ruses H . Hanajiisa Cellular immunity against Tumor Antigens Karl Erik Hi4striitir and I n g i ~ g dHcllsfroin Perspectives in the Epidemiology of Leukemia Irving L. Krssler and Abraham M . Lilien-

,feu AUTHOR INDEX-SUBJECT

INDEX

Volume 13 The Role of lmmunoblasts in Host Resistance and lmmunotherapy of Primary Sarcomata P . Alexander und J . G . Hall Evidence for the Viral Etiology of Leukemia in the Domestic Mammals Oswald Jarrert

The Function of the Delayed Sensitivity Reaction as Revealed in the Graft Reaction Culture Haiin Girisburg Epigenetic Processes and Their Relevance to the Study of Neoplasia Gajanati V . Shivbet The Characteristics of Animal Cells Transformed in Vifro lari Mac.phorsoti Role of Cell Association in Virus Infection and Virus Rescue J . Stwbocla and 1. Hloirinok Cancer of the Urinary Tract D . B . Clayson and E . H . Cooper Aspects of the EB Virus M. A . Epsteiir AUTHOR INDEX-SUBJECT INDEX

Volume 14 Active Immunotherapy Giwrgi>sMathP The Investigation of Oncogenic Viral Genomes in Transformed Cells by Nucleic Acid Hybridization EriiiJst Winocour Viral Genome and Oncogenic Transformation: Nuclear and Plasma Membrane Events Giwrgc. M q e r Passive lmmunotherapy of Leukemia and Other Cancer Roland Motfa Humoral Regulators in the Development and Progression of Leukemia Donald Mrtca/f Complement and Tumor Immunology Kusuya Nishioka Alpha-Fetoprotein in Ontogenesis and Its Association with Malignant Tumors G . 1. Abclev Low Dose Radiation Cancers in Man A1ic.c. Strn3art AUTHOR INDEX-SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

Volume 15 Oncogenicity and Cell Transformation by Papovavirus SV40: The Role of the Viral Genome J . S . Butt/. S . S . T l , t . ~ t h ia/ld ~ . J . L . MPI/lid

Nasopharyngeal Carcinoma (NPC) J . H . C. Ho Transcriptional Regulation in Eukaryotic Cells A . J . MucGillii*ruy.J . PNIII. rrtirl G. T h r ~ l jirll Atypical Transfer RNA's and Their Origin in Neoplastic Cells Err11~srBoreh coitl Syliicr J . Korr Use of Genetic Markers to Study Cellular Origin and Development of Tumors in Human Females Philip J . Firrlkoii. Electron Spin Resonance Studies of Carcinogenesis Horolil M . Siiwrt: Some Biochemical Aspects of the Relationship between the Tumor and the Host

v. s. Shcrpot Nuclear Proteins and the Cell Cycle Gary S t ~ i t crtiil i RCIIN~ 5crsi~rjy.o O AUTHOR INDEX-SUBJECT INDEX

345

I ,3-Bis(2-Chloroethyl)-l-Nitrosourea (BCNU) and Other Nitrosoureas in Cancer Treatment: A Review S I P ~ ~ KC. I Cartcr. I Franh M . Schabel. Jr., Lawwnce E. Brodrr, atid Thomas P. Johtiston AUTHOR INDEX-SUBJECT INDEX

Volume 17 Polysaccharides in Cancer: Glycoproteins and Glycolipids Vjjui N . Nigain utid Antonio Cantcro Some Aspects of the Epidemiology and Etiology of Esophageal Cancer with Particular Emphasis on the Transkei, South Africa Gc~ralilP. Warii.ick atid John S . HaringlOll

Genetic Control of Murine Viral Leukemogenesis Friitrh Lilly c i ~ r dTheodorc Piticus Marek's Disease: A Neoplastic Disease of Chickens Caused by a Herpesvirus K . No;i,riari Mutation and Human Cancer Aljircd G. Ktir~ilsori.Jr. Mammary Neoplasia in Mice S . Ncirrtli crtitl Chur1r.s M. McGrcrth AUTHOR INDEX-SUBJECT INDEX

Volume 16 Polysaccharides in Cancer Vjicri N . Nigcoir crrril Aiito~rioC c r ~ i t ~ ~ r i ~ Antitumor Effects of Interferon l o r i Gri~ssi~r Transformation by Polyoma Virus and Simian Virus 40 Joi, Sat~ihrooh Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing? Sir Ali~xcrtrik~~r Hotlilo~i~ The Expression of Normal Histocompatibility Antigens in Tumor Cells Ali.riu L~ri.qc,roi~i

Volume 18 Immunological Aspects of Chemical Carcinoge nesi s R . W . Baltlii,in Isozymes and Cancer Frrtrtiy Schapircr Physiological and Biochemical Reviews of Sex Differences and Carcinogenesis with Particular Reference to the Liver Y w Chrr Toh Immunodeficiency and Cancer Johri H . Karscy. Becitrii.c, D . S p r c t o r , arid Rohrrt A . Good

346

CONTENTS OF PREVIOUS VOLUMES

Recent Observations Related to the Chemotherapy and Immunology of Gestational Choriocarcinoma K . D. Bagshave Glycolipids of Tumor Cell Membrane Sen-itiroh Hakomori Chemical Oncogenesis in Culture Charles Heidelbcrger AUTHOR INDEX-SUBJECT INDEX

Volume 19 Comparative Aspects of Mammary Tumors J . M. Hamilton The Cellular and Molecular Biology of RNA Tumor Viruses, Especially Avian Leukosis-Sarcoma Viruses, and Their Relatives Howard M. Temin Cancer, Differentiation, and Embryonic Antigens: Some Central Problems J . H . Coggin, Jr. and N . G. Andc.rson Simian Herpesviruses and Neoplasia Fredrirh W . Deinhardt. L a u w n c e A . Falk, and Lauren G. Wope Cell-Mediated Immunity to Tumor Cells Ronald B. Herberman Herpesviruses and Cancer Fred Rapp Cyclic AMP and the Transformation of Fibroblast s Ira Pastan and George S. Johnson Tumor Angiogenesis Judah Folkman SUBJECT INDEX

Volume 20 Tumor Cell Surfaces: General Alterations Detected by Agglutinins Annette M. C . Rapin and Max M. Burger

Principles of Immunological Tolerance and lmmunocyte Receptor Blockade G. J . V . Nossal The Role of Macrophages in Defense against Neoplastic Disease Michad H . Lc.t*y and E. Fri&ric~k Wlic,rl0C.L Epoxides in Polycyclic Aromatic Hydrocarbon Metabolism and Carcinogenesis f. Sitns and P. L . Grot*cr Virion and Tumor Cell Antigens of C-Type RNA Tumor Viruses Heiriz Bauer Addendum to "Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing?" Sir Alexandiv Hadilow SUBJECT INDEX

Volume 21 Lung Tumors in Mice: Application to Carcinogenesis Bioassay Michad B. Shimkin and Gary D. Stoner Cell Deazh in Normal and Malignant Tissues E. H . Cooper, A . J . Bcjdji)rcl. and T . E . KiJnny

The Histocompatibility-Linked Immune Response Genes Baruj Brnacerraj arid David H . Kat; Horizontally and Vertically Transmitted Oncornaviruses of Cats M. Esscx Epithelial Cells: Growth in Culture of Normal and Neoplastic Forms Kee$A. Rajferty, Jr. Selection of Biochemically Variant, in Some Cases Mutant, Mammalian Cells in Culture G. B . Clemrnts The Role of DNA Repair and Somatic Mutation in Carcinogenesis J a m s E . Trosko and Ernest H . Y . Chu SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

347

Volume 22

Volume 24

Renal Carcinogenesis J . M. Hamilton Toxicity of Antineoplastic Agents in Man: Chromosomal Aberrations, Antifertility Effects, Congenital Malformations, and Carcinogenic Potential Susan M . Sieber and Richard H . Adam-

The Murine Sarcoma Virus-Induced Tumor: Exception or General Model in Tumor Immunology? J . P. Levy and J . C . Leclerc Organization of the Genomes of Polyoma Virus and SV40 Mike Fried and Beverly E. Gr#fin @,-Microglobulin and the Major Histocompatibility Complex Per A . Peterson. Lars Rusk. und Lars Osrberg Chromosomal Abnormalities and Their Specificity in Human Neoplasms: An Assessment of Recent Observations by Banding Techniques Joachim Mark Temperature-Sensitive Mutations in Animal Cells Claudia Basilica Current Concepts of the Biology of Human Cutaneous Malignant Melanoma Wallace H . Clark, Jr., Michael J . Mastrangelo, Ann M . Ainsiiwrrh, David Berd. Robert E. Belleis and Evelina A . Bernardino

son

Interrelationships among RNA Tumor Viruses and Host Cells Raymond V . Gilden Proteolytic Enzymes, Cell Surface Changes, and Viral Transformation Richard Roblin, lih-Nan Chou, and Paul H . Black lmmunodepression and Malignancy Osias Siutman SUBJECT INDEX

Volume 23 The Genetic Aspects of Human Cancer W .E. Heston The Structure and Function of lntercellular Junctions in Cancer Ronald S. Weinsicin. Frederick B. Merk. and Joseph A h o y Genetics of Adenoviruses Harold S. Ginsberg and C . S. H . Young Molecular Biology of the Carcinogen, 4-Nitroquinoline I-Oxide Minako Nagao and Takashi Sugimura Epstein-Barr Virus and Nonhuman Primates: Natural and Experimental lnfection A. Frank, W . A . Andiman, and C . Miller Tumor Progression and Homeostasis Richmond T. Prehn Genetic Transformation of Animal Cells with Viral DNA or RNA Tumor Viruses Miroslav Hill and Jana Hillova SUBJECT lNDEX

SUBJECT lNDEX

Volume 25 Biological Activity of Tumor Virus DNA F. L. Graham Malignancy and Transformation: Expression in Somatic Cell Hybrids and Variants Harvey L. Ozrr and Krishna K . Jha Tumor-Bound Immunoglobulins: I n Siiu Expressions of Humoral Immunity Isaac P. Wirz The A h Locus and the Metabolism of Chemical Carcinogens and Other Foreign Compounds Snorri S. Thorgeirsson and Daniel W . Neberi

348

CONTENTS OF PREVIOUS VOLUMES

Formation and Metabolism of Alkylated Nucleosides: Possible Role in Carcinogenesis by Nitroso Compounds and Alkylating Agents Atithony E . Pegg Immunosuppression and the Role of Suppressive Factors in Cancer Isao Kamo and Hijrnian Fricvlman Passive lmmunotherapy of Cancer in Animals and Man Sti~wriA.

Rosenberg and William D .

Terry SUBJECT INDEX

The Choice of Animal Tumors for Experimental Studies of Cancer Therapy Harold B. H m i t t Mass Spectrometry in Cancer Research John Roboz

Marrow Transplantation in the Treatment of Acute Leukemia E . Dontiall Thomas. C. Dean Buchiicr. Alexander F&. Paul E . Niirnari, ancl Ruiner Storh Susceptibility of Human Population Groups to Colon Cancer Martin Lipkin

Natural Cell-Mediated lmmunity Ronald B . Hi~rbermaii arid Howard T.

Volume 26

Ho1dt.n SUBJECT I N D E X

The Epidemiology of Large-Bowel Cancer Pdayo Corriw ancl William Haiwszrl

lnteraction between Viral and Genetic Factors in Murine Mammary Cancer J . Hilgers arid P. B ~ w r i d z e i i Inhibitors of Chemical Carcinogenesis Let

W . Wattenberg

Volume 28 Cancer: Somatic-Genetic Considerations F . M. Burnet Tumors Arising in Organ Transplant Recipients

Latent Characteristics of Selected Herpesviruses Israel Ppiiii Jack G . Stiw)tis Antitumor Activity of Corync~bac'ti~riurn Structure and Morphogenesis of Type-C Retroviruses partwm Rotiald C . Monti4uro and Dani P. BolLuka Milas and Martin T. Siwtr SUBJECT INDEX

OgIIPSi

BCG in Tumor lmmunotherapy Rohivv W . Bnldicin and Malidm V.

Volume 27 Translational Products of Type-C RNA Tumor Viruses John R. Stephenson. Sushilkuniar G . Devare, and Fred H . Reynolds.Jr. Quantitative Theories of Oncogenesis Alice S. Whittemori>

Gestational Trophoblastic Disease: Origin of Choriocarcinorna, lnvasive Mole and Choriocarcinoma Associated with Hydatidiform Mole, and Some Immunologic Aspects J . 1. Bri,icw. E . E . Torok. B . D. Kahan. C. R. Stanhope, and B . Halpiwi

Pim 171

The Biology of Cancer Invasion and Metastasis Isaiah J . Fidlar. D(~uglasM . Gerstm. uncl Ian R . Hart

Bovine Leukemia Virus Involvement in Enzootic Bovine Leukosis A . Burny. F . BPX. H . Chantriwne, Y . Cleutcv. D. Dekcgel, J . Ghysdael, R . Kettmann. M . L w k r c q . J . Leuni>n, M. Mammcvidx. arid D. Portetelle Molecular Mechanisms of Steroid Hormone Action St1.phi.n J. Higgins uric1 Ulrich GcJhring SUBJECT I N D E X

CONTENTS OF PREVIOUS VOLUMES

Volume 29 Influence of the Major Histocompatibility Complex on T-cell Activation J . F. A. P. Miller Suppressor Cells: Permitters and Promoters of Malignancy? David N a w Retrodifferentiation and the Fetal Patterns of Gene Expression in Cancer Jose Uriel The Role of Glutathione and Glutathione S-Transferases in the Metabolism of Chemical Carcinogens and Other Electrophilic Agents L. F. Chasseaud a-Fetoprotein in Cancer and Fetal Development Erkki Ruoslahti and Markku Seppala Mammary Tumor Viruses Dan H . Moore, Carole A. Long, Akhil B. Vaidya, Joel B. Shefield, Arnold S . Dion, and Etienne Y. Lasfargues Role o f Selenium in the Chemoprevention of Cancer A. Clark G r i f i n SUBJECT INDEX

Volume 30

349

The Molecular Biology of Lymphotropic Herpesviruses Bill Sugden, Christopher R. Kintner, atid Willie Mark Viral Xenogenization o f Intact Tumor Cells Hiroshi Kobuyashi Virus Augmentation of the Antigenicity of Tumor Cell Extracts Faye C . Austin and Charles W. Bootie INDEX

Volume 31 The Epidemiology of Leukemia Michael Alderson The Role of the Majar Histocompatibility Gene Complex in Murine Cytotoxic T Cell Responses Hermann Wagner, Klaus Pfizenmaier, and Martin Rollinghof The Sequential Analysis of Cancer Development Emmanuel Farber and Ross Camerson Genetic Control of Natural Cytotoxicity and Hybrid Resistance Edward A . Clark and Richard C. Harmon Development af Human Breast Cancer Sefton R . Wellings INDEX

Acute Phase Reactant Proteins in Cancer E. H . Cooper and Joan Stone Induction of Leukemia in Mice by Irradiation and Radiation Leukemia Virus Variants Nechama Haran-Ghera and Alpha Peled On the Multiform Relationships between the Tumor and the Host V. S . Shapot Role of Hydrazine in Carcinogenesis Joseph Bald Experimental Intestinal Cancer Research with Special Reference to Human Pathology Kazymir M. Pozharisski, Alexei J . Likhaocheo, Valeri F. Klimasheoski, and Jacob D. Shaposhnikoo

Volume 32 Tumor Promoters and the Mechanism of Tumor Promotion Leila Diamond, Thoinas G. O’Brien, and William Xi. Baird Shedding &omthe Cell Surface of Normal and Cancer Cells Pnul H . Blnck Tumor Antigens on Neoplasms Induced by Chemical Carcinogens and by DNA- and RNA-Containing Viruses: Properties of the Solubilized Antigens Lloyd \t’. Law, Michael J . Rogers, and Ettore Appella Nutrition and Its Relationship to Cancer Bundaru S . Reddy, Leonard A . Cohen,

350

CONTENTS OF PREVIOUS VOLUMES

G . David McCoy, Peter Hill, John H. Weisburger, arid Ernst L. Wynder INDEX

Volume 33 The Cultivation of Animal Cells in the Chemostat: Application to the Study of "hmor Cell Multiplication Michael G . Tovey Ectopic Hormone Production Viewed as an Abnormality in Regulation of Gene Expression Hiroo Imura The Role of Viruses in Human "hmors Harald Zur Hausen The Oncogenic Function of Mammalian Sarcoma Viruses Poul Andersson Recent Progress in Research on Esophageal Cancer in China Li Mingxin ( L i Min-Hsin), Li Ping, and Li Baorong ( L i Pao-Jung) Mass Transport in Tumors: Characterization and Applications to Chemotherapy Rakesh K. Jain, Jonas M . Weissbrod, andJames Wei INDEX

Volume 34 The Transformation of Cell Growth and Transmogrification of DNA Synthesis by Simian Virus 40 Robert G. Martin Immunologic Mechanisms in UV Radiation Carcinogenesis Margaret L. Kripke The Tumor Dormant State E. Frederick Wheeltick, Kent J . Weinhold. and Judith Levich Marker Chromosome 14q+ in Human Cancer and Leukemia Felix Mitelman Structural Diversity among Retroviral Gene Products: A Molecular Approach to the Study of Biological Function through Structural Variability James W. Gautsch, John H . Elder. Fred C . Jensen, and Richard A . Lerner Teratocarcinomas and Other Neoplasms as Developmental Defects in Gene Expression Beatrice Mintz and Roger A. Fleischmnn Immune Deficiency Predisposing to Epstein-Barr Virus-Induced Lymphoproliferative Diseases: The X-Linked Lymphoproliferative Syndrome as a Model David T. Purtilo INDEX