HUMAN CELL CULTURE
Volume II: Cancer Cell Lines Part 2
Human Cell Culture Volume 2
The titles published in this series are listed at the end of this volume.
Human Cell Culture Volume II Cancer Cell Lines Part 2 edited by
John R.W. Masters University College London, 67 Riding House Street, London, UK
and
Bernhard Palsson Dept. of Bioengineering, University of California San Diego, USA
KLUWER ACADEMIC PUBLISHERS NEW YORK / BOSTON / DORDRECHT / LONDON / MOSCOW
eBook ISBN: Print ISBN:
0-306-46861-1 0-793-35878-3
©2002 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow
All rights reserved
No part of this eBook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher
Created in the United States of America
Visit Kluwer Online at: and Kluwer's eBookstore at:
http://www.kluweronline.com http://www.ebooks.kluweronline.com
Contents
Foreword to the Series
vii
Introduction
ix
Chapter 17 Ovarian Cancer ANNE P. WILSON AND CHRIS M. GARNER
1
Chapter 18 Cervical Cancer SWEE Y. SHARP AND LLOYD R. KELLAND
55
Chapter 19 Endometrial Cancer P.G. SATYASWAROOP
71
Chapter 20 Breast Cancer ROBERT L. SUTHERLAND, COLIN K.W. WATTS, CHRISTINE S.L. LEE AND ELIZABETH A. MUSGROVE
79
Chapter 21 Paired Breast Cancer Cell Lines IGNACIO I. WISTUBA, ARVIND K. VIRMANI AND ADI F. GAZDAR
107
Chapter 22 Ovarian Germ Cell Tumors MASUMI SAWADA AND TSUNEHARU MIKI
121
Chapter 23 Testicular Germ Cell Tumors MARTIN F. PERA
127
Chapter 24
Choriocarcinoma VADIVEL GANAPATHY, PUTTUR D. PRASAD AND FREDERICK H. LEIBACH
Chapter 25 Thymomas and Thymic Cancers H.K. MÜLLER-HERMELINK AND ALEXANDER MARX
v
141
149
vi
Contents
Chapter 26 Kaposi’s Sarcoma CHRISTOPHER BOSHOFF
157
Chapter 27 Brain Tumors FRANCIS ALI-OSMAN
167
Chapter 28 Head and Neck Cancers CHRISTOPER D. LANSFORD, REIDAR GRENMAN, HENNING BIER, KENNETH D. SOMERS, SANG YOON KIM, THERESA L. WHITESIDE, GARY L. CLAYMAN, HANS-J WELKOBORSKY AND THOMAS E. CAREY
185
Chapter 29 Gastric Cancer TOSHIMITSU SUZUKI
257 AND
MORIMASA SEKIGUCHI
Chapter 30 Colorectal Cancer MICHAEL G. BRATTAIN, J.K.V. WILLSON, A. KOTERBA, S. PATIL AND S. VENKATESWARLU
293
Chapter 31 Prostate Cancer JAMES M. KOZLOWSKI AND JULIA A. SENSIBAR
305
Chapter 32 Liver Cancer MASAYOSHI NAMBA, MASAHIRO MIYAZAKI AND KENICHI FUKAYA
333
Chapter 33 Wilms’ Tumor and Other Childhood Renal Neoplasms NOEL A. BROWNLEE, GIAN G. RE AND DEBRA J. HAZEN-MARTIN
345
Chapter 34 Retinoblastoma BRENDA L. GALLIE, JUDY TROGADIS
361
Contents of Volume I
AND
LIPING HAN 375
Foreword to the Series This series of volumes is in celebration of Human Cell Culture. Our ability to grow nearly every type of normal and diseased human cell in vitro and reconstruct tissues in 3 dimensions has provided the model systems on which much of our understanding of human cell biology and pathology is based. In future, human cell cultures will provide the tools for tissue engineering, gene therapy and the understanding of protein function. The chapters in these volumes are written by leading experts in each field to provide a resource for everyone who works with human cells in the laboratory. John Masters and Bernhard Palsson
vii
This page intentionally left blank.
Introduction Continuous cell lines derived from human cancers are the most widely used resource in laboratory-based cancer research. The first 3 volumes of this series on Human Cell Culture are devoted to these cancer cell lines. The chapters in these first 3 volumes have a common aim. Their purpose is to address 3 questions of fundamental importance to the relevance of human cancer cell lines as model systems of each type of cancer: 1. Do the cell lines available accurately represent the clinical presentation? 2. Do the cell lines accurately represent the histopathology of the original tumors? 3. Do the cell lines accurately represent the molecular genetics of this type of cancer? The cancer cell lines available are derived, in most cases, from the more aggressive and advanced cancers. There are few cell lines derived from low grade organ-confined cancers. This gap can be filled with conditionally immortalized human cancer cell lines. We do not know why the success rate for establishing cell lines is so low for some types of cancer and so high for others. The histopathology of the tumor of origin and the extent to which the derived cell line retains the differentiated features of that tumor are critical. The concept that a single cell line derived from a tumor at a particular site is representative of tumors at that site is naïve and misleading. It is essential that representative cell lines are selected for study, and it is hoped that the chapters in these volumes will help appropriate selections to be made. The data on the molecular genetics of cancer cell lines has been difficult to gather as it is widely distributed throughout the literature and in a stage of transition. We do not yet know the identity of many of the altered genes for each type of cancer, or what their individual roles are in the progression of the disease. Despite being an essential resource for much of cancer research, established cell lines are associated with problems that are often ignored, but which can invalidate the work. The most important problems are cross-
ix
x
Introduction
contamination between cells of either the same or different species, and contamination with microorganisms (usually Mycoplasma). Both crosscontamination and the presence of Mycoplasma are easily checked by PCRbased methods. Many cell lines are cross-contaminated with other human or animal cell lines. Despite the fact that cell lines called Chang liver, KB and Hep-2 are known to be HeLa, authors often fail to acknowledge the fact. HeLa is just the tip of the iceberg of cell line cross-contamination. For most cell lines there is no proof of origin from a particular individual or tumor by a reliable method (DNA profiling is recommended). Mycoplasma contamination is a widespread and recurring problem. Laboratories that do not test for Mycoplasma contamination often have it, and consequently allow it to spread unchecked. How many putative novel human cancer-associated proteins are derived from Mycoplasma ? Many human cancer cell lines are easy to grow and maintain. With simple precautions and good practice they can provide models that are representative of almost every type of clinically advanced human cancer. Many more cell lines are needed to represent low grade, clinically localized cancer.
Chapter 17
Ovarian Cancer Anne P. Wilson and Chris M. Garner Oncology Research Laboratory, Derby City General Hospital, Uttoxeter Road, Derby DE22 3NE, UK. Tel: 01332-340131, ext 5267
1.
INTRODUCTION
The incidence of ovarian cancer worldwide ranges from 1 per 100,000 in Mali to 17 per 100,000 in France (1). It has the highest fatality amongst gynecological cancers and continues to be a significant problem because of the resistance of relapsed disease. Within the common epithelial cancers of the ovary, there are four main histological subtypes (serous, mucinous, endometrioid, clear cell), four levels of differentiation (well, moderate, poor, anaplastic), four Figo stages (I – confined to ovaries; II – confined to pelvis; III – confined to abdomen; IV – spread beyond abdomen) and a borderline category which carries an excellent prognosis but can be lethal in some cases. The comparatively low incidence of the tumor together with the huge variation in disease characteristics is a stumbling block to clinical progress. The wellcharacterised cell lines which reflect this heterogeneity are therefore a valuable commodity for research. Early attempts to culture ovarian adenocarcinomas have been reviewed (2,3). These studies employed the plasma clot technique and reported some success with outgrowth of epithelial cells using medium supplemented with 50% ascitic and pleural fluid together with a balanced salt solution further supplemented with embryo extract. Attempts to culture ovarian tumor cells increased in the 1960s and paralleled the introduction of anticancer drugs. Success with predictive testing for bacterial sensitivity to antibiotics encouraged the hope that a similar approach could be used for individualising cancer treatment. At this time, the first reports were also appearing of correlations between in vitro drug sensitivity testing results on primary cultures from surgical specimens and clinical response to the drugs
J.R.W. Masters and B. Palsson (eds.), Human Cell Culture Vol. II, 1–53. © 1999 Kluwer Academic Publishers. Printed in Great Britain.
2
Wilson and Garner
(4,5). Further extensive reports on tissue culture and ultrastructural characteristics of cultured ovarian carcinoma cells appeared in the 1970s (6,7) and the first reports of continuous cell lines derived from ovarian adenocarcinomas also appeared about this time, and included cell lines 154 and 163 (8) and also an unnamed cell line from “embryonal carcinoma of the ovarium” (9). In the last twenty years, the number of cell lines has increased dramatically and the literature now contains reports on more than 200. The lines are used extensively as models for human ovarian cancer in ways that reflect the clinical problems presented by the disease. Uses include identification of mechanisms of drug resistance, identification and development of new drugs, analysis of oncogene/tumor suppressor gene expression, development of models for adhesion and metastasis and studies on the control of proliferation via peptide growth factors, cytokines and hormones.
2.
METHODS
The success rate for establishing ovarian tumor cell lines from clinical specimens varies from <1% to ~30%. Solid tumors are usually of soft consistency and may be easily disaggregated by mincing, chopping or enzyme disaggregation using collagenase, dispase or trypsin treatment. Dispase appears to offer an improved chance of success (10) over collagenase or trypsin, but growth does occur from mechanically disaggregated tissue. Media used in the establishment of ovarian tumor cell lines include: RPMI 1640, Dulbecco’s Modification of Eagle’s medium (DMEM), Eagle’s MEM and Hams F12 or a 50/50 mixture of DMEM and Hams F12. These are routinely supplemented with animal serum (usually fetal calf serum, sometimes horse or human umbilical cord serum) at concentrations ranging from 5–20%. Other supplements used include insulin, transferrin, selenium (10), cell-free ascites (11–13), various hormones and non-essential amino acids. Ascitic fluids and pleural effusions often contain large numbers of tumor cells and the success rate for establishing cell lines from these sources is greater than that for solid tumors. Primary cultures set up from both fluids and solid tumors may contain stromal cells as contaminants, including mesothelial cells from ascites and fibroblasts from solid tumors. Mesothelial cells and fibroblasts are similar in that their rate of adherence to plastic and detachment from plastic in response to trypsin treatment is much quicker than that for epithelial cells. Contaminating stromal cells can thus be removed using differential attachment and detachment methods (10). The epithelial origin of ovarian tumor cell lines can be confirmed using epithelial-specific surface markers, such as AUA1 (raised to LoVo, a colon adenocarcinoma cell line) (15) and HMFG2 (raised to human milk fat globulin) (16). The antibody OC125 recognises the tumor marker CA125, which is
Ovarian Cancer 3 frequently elevated in the serum of patients with ovarian cancer. Its surface expression on cultured tumor cells can be used to indicate ovarian origin, although it is not completely specific for ovarian cancer (14). Mesothelial cells are a frequent contaminant of primary cultures developed from ascites, and these cells can show an epithelial phenotype under certain growth conditions (17). The presence of staining with ME1, a monoclonal antibody raised to a mesothelioma cell line (187), and absence of staining with AUA1 confirms the presence of mesothelial cells (188). Analysis of keratins 7, 18 and 20 can distinguish the primary source (18,19) as ovary rather than gut. Growth characteristics routinely determined include a morphological description, doubling time, saturation density, clonogenicity in soft agar and tumorigenicity in nude or immunosuppressed mice. Genetic information includes modal chromosome number and a G-banded karyotype. Detailed informative protocols for establishment and characterization of ovarian tumor cell lines have been published elsewhere (10).
3.
CLINICAL SPECTRUM
Table 1 lists 215 cell lines derived from human ovarian tumors. Documented clinical details include age, Figo stage and specimen site and are recorded for 44%, 36% and 76% respectively of all the cell lines. Clinically, the incidence of ovarian cancer increases in the post-menopausal years and this is reflected in the age distribution of the patients. In the cases where age was recorded, 37% (28/75) of cell lines were derived from 51-60 year old women and 29% (22/75) from 41-50 year old women (see Table 2). Ovarian cancer is asymptomatic in its early stages and women therefore most frequently present with Figo stages III or IV. This again is reflected in the Figo distribution of the patients for whom this has been documented with 57% of the cell lines arising from patients with Figo stage III and 24% from Stage IV (Table 2). The distribution of cell lines from treated and untreated patients is similar, with 54% (57/106) originating from untreated patients and 46% (49/106) from treated patients. The incidence of cell line development is greater from ascites than from solid tumors and this again is reflected in the distribution of sample sites. Ascitic fluids gave rise to 74/132 lines (56%) and solid ovarian tumors to 36/132 lines (27%) (Table 2). The remainder originated from solid metastatic deposits. Overall, the clinical spectrum of advanced ovarian cancer is well represented by the cell lines. However, the cell lines are not representative of either Stage I disease with a good prognosis or the borderline tumors with low malignant potential for which there is one cell line, INT.OV9 in Table 15 (189). There is only one cell line (PM1015) originating from a patient with a family history of ovarian cancer, although cases arising in women with a genetic
4
Table 1 Derivation of cell lines Age
FIGO category Primary site
Specimen site
Culture method
Authentication
Availability
Treated
Primary reference
1 1085 154 163 194 2 (A)2008 204 222 27/87 (A)2774 3 4 436 5 6 67R A1 A10 A121 A1847 A2780 A286 A69 A7 A721
Nk 55 68 45 Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk 70 Nk 44 Nk Nk Nk Nk Nk 68 Nk
IV III III III Nk IV Nk Nk Nk Nk Nk IV IV Nk III III Nk Nk Nk Nk Nk Nk Nk Nk IV Nk
Ovary Omentum Ascites Ascites Nk Pleural effusion Nk Nk Nk Recurrent tumor Ascites Ascites Ascites Nk Ovary Ovary ?Ovary Ascites Ascites Ovary ?Ascites ?Ascites Nk Ascites Ascites Nk
E D D D Nk D Nk Nk Nk Nk D D X Nk E E D D D D D D Nk D D Nk
Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk I Nk Nk I Nk Nk I Nk
Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk
No Nk Radn Radn Nk No No Nk Nk Nk No No No Nk No No Nk Yes Yes No No No Yes Yes No No
20 21 8 8 22 20 23 24 24 25 26 20 20 24 20 20 21 27 28 27 29 29 30 27 28 29
Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary
Continued on next page
Wilson and Garner
Cell line
Age
FIGO Primary category site
A90 AMOC-2 Asano A2224 AZ303 AZ364 AZ382 AZ390 AZ402 AZ403 AZ418 AZ419 AZ504 AZ547 BG-1 BR C30 CAOV-3 CAOV-4 CAVEOC-2 CH1 CI 79-36 CI 80-13A CI80-13S CI 81-1 CKS COLO 110
Nk 60 Nk Nk 68 65 68 68 55 76 79 79 Nk Nk Nk Nk Nk 54 45 54 Nk 62 71 71 64 43 56
Nk III Nk Nk IV III IV IV IV IV IV IV Nk Nk III III Nk Nk II Nk Nk III IV IV Nk Nk III
Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary
Specimen site
Culture method
Authentication
Availability
Treated
Primary reference
Lymph node Ovary Nk Ascites Ascites Ascites Ascites Ascites Omentum Ovary Ovary Ascites Ascites Solid tumor Ovary Mesentery Nk ?Ovary Fallopian tube Cervix Ascites Ascites Ascites Metastasis Ascites Ascites ?Ovary
D X Nk D D D D D D D D D D Nk D D Nk D D D D D D D D D D
Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk I I Nk Nk Nk Nk Nk Nk Nk I
Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk ATCC ATCC Nk Nk Nk Nk Nk Nk Nk Nk
Yes No Nk Nk Yes Yes Yes Yes No No No No Nk Nk No Nk Nk Yes Nk Yes Yes Yes No No Yes Yes No
27 31 32 33 34 34 34 34 34 34 34 34 33 33 35 21 36 37 37 38 39 40 40 40 40 41 42
Continued on next page
5
Cell line
Ovarian Cancer
Table 1 (continued)
6
Table1 (continued) Cell line
Age
Specimen site
Culture method
Authentication
Availability
Treated
Primary reference
IV III III Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk III III I III
Pleural effusion Ascites Ascites Ascites Ascites Ascites Pleural effusion Pleural effusion Sigmoidcolon Bladder Ovary Ovary Ovary Pleural effision Ovary Ascites Ascites Omentum Ovary Ascites ?Ovary Nk Nk Nk Ascites
D D D D D D D D E E E E E D E D D D D D Nk Nk Nk Nk D
I I I DNA Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk I I I I
Nk Nk Nk DSMZ ECACC Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk DSMZ DSMZ Nk Nk ATCC Nk Nk Nk Nk
No No Yes Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk
42 42 42 43 44 45 45 45 45 45 45 45 45 45 45 46 47 47 47 47 48 49 49 49 49
Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary
Continued on next page
Wilson and Garner
COLO316 54 COLO319 69 COLO330 59 46 COLO-704 COLO720E 73 COV318 Nk COV362 Nk COV362.4 Nk COV413A Nk COV413B Nk COV434 Nk COV434SUBCLNk COV446B Nk COV504 Nk COV644 Nk Nk DO-S EFO21 56 EFO27 36 EFO3 43 EFO47 49 47 ES-2 FCO Nk FKO Nk FMA Nk H134 62
FIGO Primary category site
Cell line
Age
HAC2/0.1 Nk HAC2/P Nk HeW 59 HEY Nk HMOA 57 HNOA 71 HOC-1 Nk HOC-21 56 HOC-7 Nk HOC-8 Nk HR Nk HSKTC Nk HTB-77 (seeSKOV-3) HTBOA 51 HTOA 50 HTOG 74 HTOT 74 HUOCA-II 51 HUOT 28 HX/62 Nk Ia288 65 IGROV-1 47 JA1 Nk KF Nk KGN Nk
FIGO Primary category site
Specimen site
Culture method
Authentication
Availability
Treated
Primary reference
Nk Nk Nk Nk Nk Nk III IC III Nk Nk Nk
Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary
Nk Nk ?Ovary Peritoneal deposit Ovary Ovary Ascites Ovary Ascites Ascites Ascites Ovary
Nk Nk E X D X D D D D D Nk
Nk Nk I Nk Nk Nk Nk Nk Nk Nk Nk I
Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Riken
No No No Nk No No No No No Nk Nk Nk
50 50 51 52 53 53 52 54 52 55 56 57
Nk Nk I I Nk I Nk III III III Nk Nk
Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary/?Fallopian tube Ovary Ovary Ovary Ovary
Ovary Ovary Ovary Ovary Ovary Vagina Nk Ascites Ovary Ovary ?Ovary Ovary
D D D D D D X D D D Nk Nk
Nk Nk Nk Nk Nk Nk Nk I Nk I Nk I
Nk Riken Nk Nk Nk Nk Nk Nk Nk Nk Nk Riken
No No No No Yes Yes Yes No No No Nk Nk
59 60 61 61 62 63 39 64 65 66 56 67
7
Continued on next page
Ovarian Cancer
Table 1 (continued)
8
Table 1 (continued) Cell line
Age
Nk Nk Nk Nk IIIC Nk Nk Nk Nk III II-IV Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk IV Nk Nk Nk
Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary ?Peritoneum Ovary Ovary
Specimen site Ascites Nk Nk Nk Ascites ?Ovary Nk Ascites Nk Nk Ovary, ascites, pleural effusion Nk Nk Nk Nk Nk Nk Ascites ?Ovary Ascites Ascites Ovary Ascites Ascites Ascites
Culture method
Authentication
Availability
Treated
Primary reference
D Nk Nk Nk D Nk Nk D Nk Nk D
Nk Nk Nk Nk Nk Nk Nk Nk Nk I Nk
Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk
Nk Nk Nk Nk Yes Nk Nk Yes Nk Nk Mixed
68 69 69 70 71 72 73 68 74 49 75
Nk Nk Nk Nk Nk Nk D Nk D D D D D D
Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk DNA DNA
Nk Nk Nk Nk Nk Nk Nk Nk ECACC ECACC ECACC ECACC ECACC ECACC
Nk Nk Nk Nk Nk Nk Yes No Yes Yes No Yes No No
76 76 76 76 76 76 77 78 79 79 79 79 79 79
Continued on next page
Wilson and Garner
KK Nk KOC-1S 45 KOC-2S 45 Kuramochi Nk MAC-2 53 MCAS Nk MCAS-3 Nk MH Nk MLS/P Nk Nk MRI-H207 N=14(NO Nk (NO NAMES) NOC1 Nk NOM1 Nk NOS1 Nk NOS2 Nk NOS3 Nk NOS4 Nk Nk NZ-OV1 0-129 45 OAW138D 53 OAW180D 53 OAW200D 51 74 OAW253D OAW28 75 OAW41M Nk
FIGO Primary category site
Cell line
Age
FIGO Primary category site
Specimen site
Culture method
Authentication
Availability
Treated
Primary reference
OAW42 OAW59M OAWD13 OAW D16 OAWD206 OC 314 OC 315 OC 316 OC-109 OC-194 OC-222 OC-238 OC-7-NU OCC1 OMC-1 OMC-3 OTN 11 OTN 14 OV1225 OV 166 OV 2774 OV-1063 OV17 OV17R OV25 OV25R
46 65 59 59 60 39 49 60 Nk Nk Nk Nk Nk 47 75 58 28 47 Nk Nk Nk 57 50 50 61 67
1C IA III III III IIIC IIIC IV Nk Nk Nk Nk Nk Nk Nk III Nk IV Nk Nk Nk IV III III III III
Ascites Ascites Ascites Omentum Ascites Ascites Ascites Ascites Ascites Nk Nk Ascites Ascites Ascites ?Ovary Ascites Ascites Ascites Nk Nk Nk Ascites Ascites Ascites Ascites Ascites
D D D D D D D D D Nk Nk D X D Nk D D D Nk Nk Nk D D D D D
Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk
ECACC ECACC Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Riken Nk Nk Nk Nk Nk ATCC Nk Nk Nk Nk
Yes No No No Yes No Yes Yes No Nk Nk No No No Nk No Yes No Nk Nk Nk Yes Yes Yes Yes Yes
79,80 79 81 81 81 82 82 82 83 84 84 83 83 85 86 87 88 89 90 90 90 91 92 92 92 92
Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary
9
Continued on next page
Ovarian Cancer
Table 1 (continued)
10
Table I (continued) Age
FIGO Primary category site
OV56 OV58 OV7 OVAN-4 OVAS-21 OVC-8 OVCA 420 OVCA 429 OVCA 432 OVCA 433 OVCAR-2 OVCAR-3 OVCAR-4 OVCAR-5 OVCAR-8 OVCCR1 OVISE OVISE-3 OVK18 OVKATE OVMANA OVMZ1 OVMZ2 OVMZ3 OVMZ4 OVMZ25
46 52 78 Nk Nk Nk Nk Nk Nk Nk Nk 60 Nk Nk Nk 58 40 Nk Nk Nk Nk 44 55 Nk 64 57
IV III III Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk IIB Nk Nk Nk Nk III IV III IV IV
Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary
Specimen site
Culture method
Authentication
Availability
Treated
Primary reference
Ascites Ascites ?Ovary ?Ovary Nk Nk Nk Nk Nk Nk ?Ascites Ascites ?Ascites ?Ascites Nk Ascites Pelvicmass Nk Ascites Metastatic deposit Ovary Ascites Ascites Ascites Ascites Ascites
D D D Nk Nk Nk Nk Nk Nk Nk D D D D Nk D D Nk D D D D D D D D
Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk I Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk
Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk ATCC Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk
Yes Yes Yes Nk Nk Nk Nk Nk Nk Nk Yes Yes Yes No Nk Nk Yes Nk Nk Yes Yes Yes Yes Yes No No
92 92 92 93 32 36 94 94 94 94 29 95 29 29 77 96 97 73 98 99 99 100 100 100 100 100
Continued on next page
Wilson and Garner
Cell line
Cell line OVMZ6 OVSAHO OVSAYO OVTOKO PA1 PEA1 PEA2 PEO1 PEO14 PEO16 PEO23 PEO4 PEO6 PM1015 PXN/94 RIC-2 RMG-I RMG-II RMUG-L RMUG-S RTSG SCHM-1 SHIN-3 SIB-1 SKOV3 (HTB-77)
Age
FIGO Primary category site
70 Nk Nk 78 12 Nk Nk Nk Nk Nk Nk Nk Nk 47 Nk 59 34 Nk Nk Nk Nk 70 56 39 64
IV Nk Nk IIIB Nk Nk Nk Nk Nk Nk Nk Nk Nk IV Nk IIIC Nk Nk Nk Nk Nk IIC IIC IIC Nk
Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary
Specimen site
Culture method
Authentication
Availability
Treated
Primary reference
Ascites Metastatic deposit Ovary Spleen Ascites Pleural effusion Ascites Ascites Ascites Ascites Ascites Ascites Ascites Pleural fluid Nk Ascites Ascites Ascites Nk Nk Pleural effusion Ascites Ovary Pleural effusion Ascites
D D D D D D D D D D D D D D X D D D Nk Nk D D D D D
Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk I
Nk Nk Nk Nk ATCC,ECACC Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk ATCC,ECACC
No Yes No Yes No No Yes Yes No Yes Yes Yes Yes Yes Yes Yes No Nk Nk Nk Nk Yes No Yes Yes
100 99 99 97,99 101 102 102 102 102 102 102 102 102 103 39 71 104 105 106 106 107 71 108 71 58,109,110
11
Continued on next page
Ovarian Cancer
Table 1 (continued)
12
Table 1 (continued) Age
FIGO Primary category site
SRO-82 SW 626 T222 TO14 TR170 TR175 TYKnu UCI 101 UCI 107 UPN36 UWOV1 UWOV2 UWOV2Sf YAOVBIX1 YAOVBIX3 YAOVDK YAOVFAB YAOVJON YAOVWE1 Yoshikazi
Nk 46 Nk Nk Nk Nk Nk 46 47 Nk 30 52 52 Nk Nk Nk Nk Nk Nk Nk
Nk Nk Nk Nk Nk III Nk III III Nk Nk Nk Nk III III Nk Nk Nk Nk Nk
Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary
Specimen site
Culture method
Authentication
Availability
Treated
Primary reference
Nk Tumor from ovary Nk Omentum Ascites Ascites Ovary Ascites + ovary Ascites + ovary Ascites Ascites Ascites Ascites Ascites Ascites Ascites Ascites Ascites Ascites Nk
Nk D Nk D D D X D D X D D D D D D D D D Nk
Nk I Nk Nk 1 I Nk Nk Nk Nk I I I Nk Nk Nk Nk Nk Nk Nk
ATCC ATCC Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk
Nk Nk Nk No Yes No No Yes No Nk No Yes Yes Nk Nk Nk Nk Nk Nk Nk
22 109,110 112 102 66 66 113 114 115 116 117 117 117 118 118 118 118 118 118 32
Availability: Nk, not known (the cell lines have not been deposited in a culture collection for distribution, but providers of cell lines are cited in the papers and may be possible sources). DSMZ, German Collection of Microorganisms and Cell Cultures; ECACC, European Collection of Animal Cell Cultures; ATCC, American Tissue Culture Collection; Abbreviations: Nk, not known; D, dissociation; E, enzymatic; X, xenograft, I, isozymes; DNA, DNA fingerprints; Radn, radiation treatment.
Wilson and Garner
Cell line
Ovarian Cancer
13
Table 2 Distribution of age, FIGO stage and site of derivation of ovarian cancer cell lines Number Age 20–30 31–40 41–50 51–60 61–70 71–80 Total FIGO stage I II III IV Total Site Ascites Pleural effusion Solid tumor from ovary Solid metastasis Total Treatment Untreated Treated Total
Percentage of total
2 4 22 28 11 8 75
3 6 29 37 15 12
7 5 35 15 62
11 8 57 24
74 9 36 13 132
56 7 27 10
57 49 106
54 46
disposition to the disease represent approximately 5% of all ovarian cancer cases (119). More ovarian cell lines have been described since Table 1 was compiled (see Table15).
4.
PATHOLOGY
The histopathological features of the cell lines are shown in Table 3. Most ovarian tumors are epithelial cancers, which are believed to arise from the surface epithelium of the ovary (120). Other types are the malignant teratomas and sex cord tumors such as granulosa and thecoma (121,190), The pathology of the ovarian epithelial cancers is complex and is believed to reflect the pluripotency of the mesothelium during embryonic development of the female reproductive tract. In the developing embryo, the coelomic epithelium is derived from the mesoderm. This epithelium forms the serous lining (mesothelium) of the body cavities covering both visceral and parietal
14
Table 3 Pathology of cell lines Cell line
Brief description of tumor pathology
In vitro features of cell line
Xenograft pathology
1 1085 154 163 194 2 204 222 2774
Serous carcinoma Poorly differentiated adenocarcinoma Papillarycystadenocarcinoma Cystadenocarcinoma Epithelial Serous carcinoma Epithelial Epithelial Endometrioid carcinoma
Nk Epithelial monolayer Epithelial monolayer Epithelial monolayer Monolayer. Doubling time 45 hours Nk Monolayer. Doubling time 40 hours Monolayer. Doubling time 35 hours Epithelial-like monolayer
3 4 436 5 6 67R A1
Clear cell carcinoma Clear cell carcinoma Epithelial Serous carcinoma Carcinosarcoma Poorly differentiated adenocarcinoma Poorly differentiated adenocarcinoma
A10
Moderately differentiated serous papillary cystadenocarcinoma Mixed miillerian tumor
Nk Nk Monolayer. Doubling time 35 hours Nk Nk Epithelial monolayer Epithelial monolayer. Doubling time 67 hours Epithelial-like monolayer. Doubling time 25 hours Epithelial monolayer. Doubling time 22 hours
Nk Nk Nk Nk Nk Nk Nk Nk Poorly differentiated solid serous carcinoma in nude mice? SC or IP Nk Tbmors in nude mice Nk Nk Nk Nk No tumors in nude mice
Tbmors in nude mice. Morphology similar to original tumor. SC/Solid tumors. Spindle shaped morphology consistent with sarcomatous primary Continued on next page
Wilson and Garner
A121
Nk
Cell line
Brief description of tumor pathology
In vitro features of cell line
Xenograft pathology
A2780
Nk
Adherent monolayer
A286 A69
Nk Endometrioid/serous adenocarcinoma
Monolayer Epithelial monolayer. Doubling time 62 hours
A7
Moderately differentiated adenocarcinoma
A721 A90
Nk Poorly differentiated adenocarcinoma
Epithelial-like monolayer with diffuse growth pattern. Doubling time 36 hours Monolayer Epithelial monolayer. Doubling time 71 hours
Undifferentiated carcinoma in nude mice. Xenograft model [49] SC/Solid Nk Tumors with limited growth in nude mice. Morphology similar to original tumor. SC/Solid. Nk
AMOC-2
Poorly differentiated serous adenocarcinoma
Asano A2224
Serous adenocarcinoma Serous adenocarcinoma
AZ303 A2364
Serous adenocarcinoma Serous adenocarcinoma
A2382,390,402, Serous adenocarcinoma 403,418,419 AZ.504 Granulosa cell tumor
Epithelial-like monolayer. Doubling time 17.4 hours Nk Monolayer. Doubling time 20 hours. PE 10–12% in agar Grows in suspension Epithelial-like monolayer. Doubling time 70 hours. Non-clonogenicin soft agar Nk Monolayer. Doubling time 120 hours. PE 0.4–0.6% in agar
Ovarian Cancer
Table 3 (continued)
Nk Tumors with limited growth in nude mice. Morphology similar to original tumor. SC/Solid Poorly differentiated serous adenocarcinoma in nude mice. SC/Solid tumor Nk Tumorigenic in nude mice Non-tumorigenic in nude mice Non-tumorigenic in nude mice Nk Tumorigenic in nude mice Continued on next page
15
16
Table 3 (continued) Cell line
Brief description of tumor pathology
In vitro features of cell line
Xenograft pathology
AZ547
Undifferentiated
Tumorigenic in nude mice
BG-1
Very poorly differentiated adenocarcinoma
BR C30 CAOV-3
Papillary adenocarcinoma Nk Adenocarcinoma
CAOV-4 CAWOC-2
Adenocarcinoma Endometrioid adenocarcinoma
CH1
Papillary cystadenocarcinoma
Mixed monolayer and suspension culture. Doubling time 38 hours. PE 9% in agar Monolayer of polygonal cells. Multilayering at confluence. Doubling time 20-24hours; clonogenic in soft agar Epithelial monolayer ?Adherent monolayer Epithelial-like monolayer. Doubling time 78 hours. PE 3% in agar Epithelial-like monolayer Adherent monolayer. Doubling time 33 hours. PE 50% in agar. Epithelial-like monolayer. Doubling time 17 hours
CI 79-36 CI 80-13A CI80-13S CI81-1
Serous cystadenocarcinoma Undifferentiated carcinoma Undifferentiated carcinoma Carcinoma
CKS
Serous papillary cystadenocarcinoma
COLO 110
Serous cystadenocarcinoma
Poorly differentiated tumors in nude mice. SC/Solid Nk Nk No tumors in immune deprived mice [52]. SC
Continued on next page
Wilson and Garner
Nk Adenocarcinoma in nude mice. SC/Solid. Staining of primary and xenograft identical. Poorly differentiated papillary cystadenocarcinoma in nude mouse, SC/solid xenograft model PX9/109/TC [191] Nk Epithelial-like monolayer. PE 4% in agar Epithelial-like monolayer. PE 1.4% in agar Nk Epithelial-like monolayer. PE 0.35% in agar Nk Epithelial-like monolayer. PE 0.07-1.25% Nk in agar Epithelial-like monolayer with hemicyst Serous adenocarcinomas in hamsters and nude mice. SC/Solid formation, Doubling time 79 hours (34 hours [70]) Epithelial-like monolayer as tightly packed Serous adenocarcinoma in immunosuppressed colonies which do not reach confluence mice.SC/Solid
Cell line
Brief description of tumor pathology
In vitro features of cell line
COLO 316
Serous cystadenocarcinoma
COLO 319
Serous cystadenocarcinoma
COLO 330
Serous cystadenocarcinoma
COLO-704
Adenocarcinoma
COLO720E COV318 COV362 COV362.4 COV413A COV413B COV434
Papillary adenocarcinoma Serous carcinoma Endometrioid carcinoma Endometrioid carcinoma Serous carcinoma Serous carcinoma Granulosa cell tumor
Epithelial-like monolayer with whorled and Serous adenocarcinoma in immunosuppressed acinar growth mice. SC/Solid Cell aggregates with limited attachment Serous adenocarcinoma in immunosuppressed and 3D growth into medium mice.SC/Solid Cell aggregates in suspension Serous adenocarcinoma in immunosuppressed mice.SC/Solid Nk Grows as clusters and single cells in suspension. Doubling time 30–40 hours Epithelial-like monolayer Tumorigenic Epithelial-like monolayer. PE 2.4% in agar Nk Epithelial-like monolayer. PE 2.2% in agar Nk PE 0.74% in agar Nk Epithelial-like monolayer. PE 0.01% in agar Nk Epithelial-like monolayer. PE 1.6% in agar Nk Epithelial-like monolayer with 3D growth. Nk PE 9.3% in agar Nk Nk Epithelial-like monolayer. PE 3.6% in agar Nk Epithelial-like monolayer. PE 0.2% in agar Nk Epithelial-like monolayer. PE 1.3% in agar Nk Cell aggregates with limited attachment. Undifferentiated mucinous adenocarcinoma in nude mice. SC/Solid. IP/Ascites Doubling time 47 hours, vacuolated cells Nk Epithelial-like monolayer Nk Epithelial-like monolayer
COV434SUBCL Granulosa cell tumor COV446B Serous carcinoma COV504 Serous carcinoma COV644 Mucinous carcinoma DO -S Well differentiated mucinous cystadenocarcinoma EFO21 Poorly differentiated serous adenocarcinoma EFO27 Moderately differentiated serous papillary adenocarcinoma
Xenograft pathology
Ovarian Cancer
Table 3 (continued)
Continued on next page
17
18
Table 3 (continued) Cell line
Brief description of tumor pathology
In vitro features of cell line
Xenograft pathology
EFO3
Epithelial monolayer
Nk
EFO47 ES-2
Well differentiated serous papillary cystadenocarcinoma Poorly differentiated clear cell carcinoma Poorly differentiated clear cell carcinoma
Nk Tumors in nude mice
FCO
Clear cell carcinoma
Epithelial-like monolayer ?Adherent monolayer. Doubling time 24 hours Adherent monolayer
FKO
Poorly differentiated serous adenocarcinoma
Adherent monolayer
FMA
Adherent monolayer
H-134
Moderately differentiated mucinous adenocarcinoma Serous papillary adenocarcinoma
HAC2/0.1
Clear cell adenocarcinoma
HAC2/P
Clear cell adenocarcinoma
HeW HEY
Serous cystadenocarcinoma Moderately differentiated papillary cystadenocarcinoma
HMOA
Well differentiated endometrioid adenocanthoma Moderately differentiated endometrioid adenocarcinoma
Adherent monolayer. Doubling time 46.5 hours. PE 4.3% in agar Adherent monolayer. Doubling time 46.5 hours. PE 4.5% in agar Adherent monolayer of large and small cells Epithelial like monolayer. Whorled growth pattern. Doubling time 30 hours. PE 28.5% in agar. Derived from HX/62 Epithelial-like monolayer with 3D growth. Doubling time 58-72hours Epithelial-like monolayer with 3D growth. Doubling time 24-28hours
Nk Nk Adenocarcinoma produced in immunosuppressed mice. SC/Solid Well-differentiated adenocanthoma in nude mice.SC/Solid Poorly differentiated endometrioid adenocarcinoma in nude mice. SC/Solid Continued on next page
Wilson and Garner
HNOA
Adherent monolayer. PE 50% in agar
Clear cell carcinoma in nude mice. Xenograft model. SC/Solid Moderately differentiated serous adenocarcinoma in nude mice. Xenograft model. SC/Solid Poorly differentiated mucinous carcinoma in nude mice. Xenograft model. SC/Solid Undifferentiated carcinoma in nude mice. Xenograft model. SC/Solid Nk
Table 3 (continued) Brief description of tumor pathology
In vitro features of cell line
Xenograft pathology
HOC-1
Well differentiated serous adenocarcinoma
No tumors in immunosuppressed mice
HOC-21 HOC-7
Moderately differentiated serous papillary cystadenocarcinoma Well differentiated serous adenocarcinoma
HOC-8
Serous adenocarcinoma
HR
Serous cystadenocarcinoma
HSKTC HTBOA
Krukenberg tumor Anaplastic carcinoma
HTOA
Well differentiated serous cystadenocarcinoma Mixed granulosa/theca cell tumor
Monolayer epithelial like, whorled growth pattern, Doubling time 36 hours, no growth in agar Epithelial-like monolayer with whorled growth pattern. Doubling time 28 hours Monolayer epithelial like, whorled growth pattern, Doubling time 16.4 hours. PE 10% in agar Epithelial monolayer. Doubling time 48-72hours. Clonogenicity 0.1% in agar Epithelial-like monolayer. Doubling time 13.2 hours Epithelial-like monolayer Epithelial-like monolayer with 3D growth. Doubling time 24-28hours Epithelial-like monolayer with 3D growth. Doubling time 68-72hours Epithelial-like monolayer. Doubling time 24-28hours Fibroblastic in shape. Doubling time 78 hours Epithelial-like monolayer with 3D growth. Doubling time 24 hours Adherent monolayer with polygonal, columnar oval spindle like cells with 3D growth. Doubling time 72-98hours
HTOG HTOT HUOCA-II HUOT
Mixed granulosa/theca cell tumor Well differentiated papillary clear cell adenocarcinoma Malignant teratoma and anaplastic carcinoma
Papillary adenocarcinoma in hamster cheek pouch. Solid No tumors in immunosuppressed mice
Ovarian Cancer
Cell line
No tumors in nude mice Tumors in nude mice with partial similarity to original tumor. SC/Solid tumor. IP/Ascites Nk Anaplastic carcinoma with granulocytosis in nude mice. SC/Solid Serous cystadenocarcinoma in nude mice and hamster cheek pouch. SC/Solid Sarcomatous diffuse granulosa cell tumor in nude mice. SC/Solid Nk Poorly differentiated clear cell adenocarcinoma in nude mice. SC/Solid Anaplastic carcinomas in nude mice. SC/Solid
Continued on next page
19
20
Table 3 (continued) Cell line
Brief description of tumor pathology
In vitro features of cell line
Xenograft pathology
HX/62
Papillary cystadenocarcinoma
Ia288
Moderate/poorly differentiated papillary adenocarcinoma Mainly endometrioid with serous, clear cell and undifferentiated areas
Epithelial-like monolayer. Doubling time 32 hours Epithelial-like monolayer
Undifferentiated carcinoma in nude mice, SC/solid [191] Papillary tumor in nude mice. SC/Solid
IGROV-1
JA1 KF KGN KK KOC-1S KOC-2S Kuramochi
MCAS
Nude mice fast growth tumors, pathology same as tumor from which derived. SC/Solid tumor. IP/Ascites and solid tumor Tumors in nude mice. SC/Solid with rapid growth Tumors in nude mice Nk Nk Nk Nk Nk Nk Mucinous cystadenocarcinoma in nude mice. ?SC/Solid Continued on next page
Wilson and Garner
MAC-2
Clonogenic in agar, PE 0.35%, Doubling time 19 hours, uniform monolayer of polygonal epithelial cells, floating clusters Bipolar spindle shaped cells. Doubling Poorly differentiated serous papillary cystadenocarcinoma time 32 hours. PE 0.143% in agar Serous cystadenocarcinoma Monolayer with spindle and triangular cells. Doubling time 16 hours ?Monolayer Granulosa cell tumor Clear cell carcinoma , Epithelial-like monolayer. Doubling time 96 hours Well differentiated serous papillary Epithelial-like monolayer. Doubling time adenocarcinoma 43.5 hours Poorly differentiated serous adenocarcinoma Adherent monolayer. Doubling time 29 hours Epithelial-like monolayer. Doubling time Undifferentiated carcinoma 36 hours Poorly differentiated serous Epithelial-like monolayer with 3D growth. cystadenocarcinoma Clonogenic in agar Mucinous cystadenocarcinoma Epithelial-like monolayer. Doubling time 27 hours
Cell line
Brief description of tumor pathology
In vitro features of cell line
Xenograft pathology
MCAS-3
Mucinous cystadenocarcinoma
Epithelial-like monolayer
MH
Serous cystadenocarcinoma
MLS/P MRI-H207
Carcinoma Undifferentiated carcinoma
Adherent monolayer. Doubling time 151 hours Adherent monolayer Adherent monolayer
SC/Cystic and solid tumors with vascular invasion. IP/Multiple disseminated tumors in peritoneum with lymph node metastasis Nk
N=14 (NO (NO NAMES) NOC1 NOM1 NOS1 NOS2 NOS3 NOS4 NZ-OV1
Serous, endometrioid and clear cell carcinoma Clear cell adenocarcinoma Mucinous cystadenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Serous papillary cystadenocarcinoma
0-129
Carcinoma
OAW138D
Serous cystadenocarcinoma
OAW 180D
Papillary adenocarcinoma
Nk ? Adherent monolayer ? Adherent monolayer ? Adherent monolayer ? Adherent monolayer ? Adherent monolayer ? Adherent monolayer Adherent monolayer. Doubling time 65 hours Adherent monolayer. Doubling time 10.8hours Epithelial-like monolayer with large and small cells. Doubling time 60 hours. Nonclonogenic in agar Epithelial-like monolayer. Doubling time 74 hours. PE 1% in agar
Ovarian Cancer
Table 3 (continued)
Nk Undifferentiated carcinoma in nude mice. Xenograft model. SC/Solid Six of 14 formed tumors in nude mice Nk Nk Nk Nk Nk Nk Nk Nk Nk
Nk Continued on next page
21
22
Table 3 (continued) Cell line
Brief description of tumor pathology
In vitro features of cell line
Xenograft pathology
OAW 200D
OAW28
Cystadenocarcinoma
OAW41M OAW 42
Carcinoma Serous papillary cystadenocarcinoma
OAW 59M
Epithelial-like monolayer
Nk
Adherent epithelial-like monolayer
OC 316
Endometrioid carcinoma with clear cell areas Moderately differentiated serous papillary adenocarcinoma Moderately differentiated serous papillary adenocarcinoma Moderately differentiated serous papillary adenocarcinoma Poorly differentiated papillary serous cystadenocarcinoma Well-differentiated papillary serous adenocarcinoma Poorly differentiated adenocarcinoma
Epithelial-like monolayer. Doubling time 52 hours. PE 1% in agar Epithelial-like monolayer. Doubling time 80 hours. Non-clonogenic in agar Cobblestoned epithelial-like monolayer. Doubling time 60 hours. PE 0.2% in agar Cobblestoned epithelial-like monolayer Epithelial-like monolayer, forms hemicysts at confluence. Doubling time 45-50hours, PE 0.8-1.7%in agar Epithelial-like monolayer, elongated cells. Doubling time 48 hours. PE 1% in agar Epithelial-like monolayer. Doubling time 100 hours. PE 0.05% in agar Epithelial-like monolayer.
Nk
OAW253D
Serous papillary adenocarcinoma with clear cell areas Serous papillary adenocarcinoma
OC-109
Mucinous serous cystadenocarcinoma
Tumors in immunosuppressed mice. SC/Solid. IP/Solid and ascites Tumors in immunosuppressed mice. SC/Solid. IP/Solid and ascites Tumors in immunosuppressed mice. SC/Solid. IP/Solid and ascites Tumors in nude mice. SC
OAW D13 OAW D16 OAW D206 OC 314
Adherent epithelial-like monolayer Adherent epithelial-like monolayer ?Adherent monolayer
Nk Nk Nk
Nk Nk Nk
Continued on next page
Wilson and Garner
OC 315
Nk
Cell line
Brief description of tumor pathology
In vitro features of cell line
Xenograft pathology
OC194 OC-222 OC-238 OC-7-NU OCC1
Nk Nk Serous cystadenocarcinoma Serous cystadenocarcinoma Clear cell carcinoma
Nk Nk Tumors in nude mice. SC Tumors in nude mice. SC Clear cell carcinoma in nude mice. SC/Solid
OMC-1
Well differentiated mucinous cystadenocarcinoma Mucinous cystadenocarcinoma
?Adherent monolayer ?Adherent monolayer ?Adherent monolayer ?Adherent monolayer Epithelial-like monolayer with 3D growth. Doubling time 3638 hours Epithelial-like monolayer. Doubling time 24 hours Epithelial-like monolayer with 3D growth. Doubling time 98-107hours Epithelial-like monolayer with some whorled areas and 3D growth. Doubling time 48 hours Epithelial-like monolayer with 3D growth. Doubling time 28.5 hours
OMC-3 OTN 11
Well differentiated papillary mucinous cystadenocarcinoma
OTN 14
Well differentiated mucinous adenocarcinoma
OV 1225 OV 166 OV 2774 OV-1063
Nk Nk Nk Papillarycystadenocarcinoma
OV17,17R, 25 25R OV56 OV58 OV7
Adenocarcinoma
Epithelial-like monolayer Epithelial-like monolayer Epithelial-like monolayer Cobblestone epithelial-like monolayer. Doubling time 29 hours. PE 1% in agar Adherent monolayer
Serous poorly differentiated carcinoma Serous carcinoma Mixed poorly differentiated carcinoma
Adherent monolayer Adherent monolayer Adherent monolayer
Ovarian Cancer
Table 3 (continued)
Well differentiated mucinous cystadenocarcinoma in nude mice. SC/Solid Moderately differentiated mucinous cystadenocarcinomas in nude mice. SC/Solid Serous adenocarcinomas in nude mice. SC/Solid Poorly differentiated tumors in nude mice. SC/Solid with invasion into surrounding connective tissue and blood vessels Nk Nk Nk Tumor in nude mice Nk Nk Nk Nk
23
Continued on next page
24
Table 3 (continued) Cell line
Brief description of tumor pathology
In vitro features of cell line
Xenograft pathology
OVAN-4
Very poorly differentiated adenocarcinoma
Nk
Undifferentiated tumors in SCID mice. Developed as in vivo model. IP/Solid tumors on peritoneal wall, mesentery and abdominal organs in advanced disease. No vascular invasion. Invasion of muscle seen Nk Nk Nk
Nk ?Adherent monolayer Epithelial monolayer
OVCAR-4
Carcinoma
OVCAR-5
Nk
OVCAR-8
Nk
OVCCR1
Serous cystadenocarcinoma
OVISE
Clear cell adenocarcinoma
Adherent monolayer. Doubling time 25-34hours Adherent monolayer. Doubling time 21 hours Adherent monolayer. Doubling time 24 hours Epithelial-like monolayer. Doubling time 85-96hours Columnar, cuboidal, giant and spindle cells. Doubling time 60 hours
Epithelial-like monolayer. PE 0.01% in agar. Doubling time 48 hours Epithelial-like adherent monolayer. Developed from OVCAR-3 for in vivo growth and proliferation in agar
Non-invasive tumors formed in nude mice. SC/Solid. No invasion of muscle [49] Poorly differentiated serous carcinoma in nude mice. IP/Ascites and diffuse tumor deposits on peritoneal surfaces, viscera and diaphragm. Pulmonary metastases. In vivo passage Nk Nk Nk Nk Clear cell adenocarcinomas in nude mice. SC/Solid. IP/No growth Continued on next page
Wilson and Garner
Clear cell adenocarcinoma OVAS-21 OVC-8 Nk OVCA 420,429 Nk 432,433 OVCAR-3 Poorly differentiated papillary adenocarcinoma OVCAR-3 Poorly differentiated papillary (NU/AG+ ) adenocarcinoma
Cell line
Brief description of tumor pathology
In vitro features of cell line
Xenograft pathology
OVISE-3
Clear cell adenocarcinoma
Epithelial-like monolayer
OVK18 OVKATE
Endometrioid carcinoma Serous papillary adenocarcinoma
Epithelial-like monolayer with 3D growth Adherent epithelial-like monolayer. Doubling time 96 hours
OVMANA
Clear cell adenocarcinoma
OVMZ1 OVMZ2
Moderately differentiated serous adenocarcinoma Well differentiated serous adenocarcinoma
OVMZ3
Well differentiated serous adenocarcinoma
OVMZ4
Moderately differentiated serous adenocarcinoma
Adherent epithelial-like monolayer. Doubling time 67 hours Adherent monolayer. Doubling time 38 hours Adherent monolayer. Doubling time 24 hours Adherent monolayer. Doubling time 52 hours Adherent monolayer. Doubling time 26 hours
Cystic tumors in nude mice with no peritoneal dissemination or lymph node metastasis. SC/ Slow growing cystic tumors with minimal solid components and no vascular invasion. IP/No tumor growth Tumors in nude mice Serous papillary adenocarcinomas in nude mice. SC/Solid. IP/Metastatic tumors and/or dissemination in peritoneal cavity Clear cell adenocarcinomas in nude mice. SC/Solid. IP/No growth Undifferentiated carcinoma in nude mice. SC/Solid and slow growing Undifferentiated carcinoma in nude mice. SC/Solid and slow growing No tumors in nude mice. SC
OVMZS
Well differentiated serous adenocarcinoma
OVMZ6
Poorly differentiated serous adenocarcinoma
Adherent monolayer. Doubling time 40 hours Adherent monolayer. Doubling time 28 hours
Ovarian Cancer
Table 3 (continued)
Moderately differentiated papillary adenocarcinoma in nude mice. SC/Solid and slowgrowing No tumors in nude mice. SC Poorly differentiated adenocarcinoma in nude mice. SC/Solid and slow growing Continued on next page
25
26
Table 3 (continued) Cell line
Brief description of tumor pathology
In vitro features of cell line
Xenograft pathology
OVSAHO
Serous papillary adenocarcinoma
Adherent epithelial-like monolayer. Doubling time 56 hours
OVSAYO
Clear cell adenocarcinoma
OVTOKO
Clear cell adenocarcinoma
Adherent epithelial-like monolayer. Doubling time 88 hours Columnar, cuboidal, giant and spindle cells Doubling time 70 hours
Serous papillary adenocarcinomas in nude mice. SC/Solid. IP/Metastatic tumors and/or dissemination in peritoneal cavity No tumors in nude mice
PA1
Teratocarcinoma
PEA1
Poorly differentiated adenocarcinoma
PEA2
Poorly differentiated adenocarcinoma
PEO 1
Poorly differentiated serous adenocarcinoma Well differentiated serous adenocarcinoma
Nk
Nk
Tumors in immune depressed mice. SC/Solid Nk
Continued on next page
Wilson and Garner
PEO14
Adherent monolayer of small round cells which aggregate. Forms embryoid bodies with differentiation. Doubling time 26-41 hours Epithelial-like monolayer with whorled growth pattern. Doubling time 37 hours. PE 0.05% in agar Epithelial-like monolayer with whorled growth pattern. Doubling time 66 hours. PE 0.01% in agar Cobblestone epithelial-like monolayer. Doubling time 37 hours. PE 3.9% in agar Cobblestone epithelial-like monolayer. Produce hemicysts. Doubling time 108 hours. PE 0.12% in agar
Clear cell adenocarcinomas in nude mice. SC/Solid tumor. IP/Tumors in peritoneal cavity with limited liver and lymph node metastasis Tumors of neuroepithelial cells in nude mice. SC/Solid
Brief description of tumor pathology
In vitro features of cell line
Xenograft pathology
PEO16
Poorly differentiated serous adenocarcinoma
Tumors in immune depressed mice. SC/Solid
PEO23
Well differentiated serous adenocarcinoma
PEO4
Poorly differentiated serous adenocarcinoma
PEo6
Poorly differentiated serous adenocarcinoma
PM1015 PXN/94
Serous papillary carcinoma (familial) Adenocarcinoma
RIC-2
Poorly differentiated adenocarcinoma
RMG-I
Clear cell carcinoma
RMG-II
Clear cell carcinoma
RMUG-L
Mucinous cystadenocarcinoma
Monolayer with stellate cells and hemicysts. Doubling time 40 hours. PE 0.3% in agar Cobblestone epithelial-like monolayer with 3D growth. Produce hemicysts. Doubling time 120 hours. PE 0.1% in agar Cobblestone epithelial-like monolayer. Doubling time 36 hours. PE 18.3% in agar Cobblestone epithelial-like monolayer. Doubling time 77 hours. PE 2.4% in agar Nk Epithelial-like monolayer. Doubling time 23 hours Epithelial-like monolayer. Clonogenic in agar Epithelial-like monolayer with hemicyst formation. Doubling time 60 hours Epithelial-like monolayer. Doubling time 58-86 hours Epithelial-like monolayer
RMUG-S
Mucinous cystadenocarcinoma
Epithelial-like monolayer
RTSG
Mucinous/serous cystadenocarcinoma with poorly differentiated areas
Epithelial-like monolayer with 3D growth. Doubling time 48 hours
Nk
Ovarian Cancer
Table 3 (continued) Cell line
Tumors in immune depressed mice. SC/Solid Tumors in immune depressed mice. SC/Solid Nk Nk Nk Clear cell carcinoma in nude mice. SC No tumors in nude mice Poorly differentiated adenocarcinoma with a mucinous area in nude mice Poorly differentiated adenocarcinoma in nude mice Poorly differentiated adenocarcinoma in nude mice
27
Continued on next page
28
Table 3 (continued) Cell line
Brief description of tumor pathology
In vitro features of cell line
Xenograft pathology
SCHM-1
Serous papillary adenocarcinoma with clear cell differentiation Serous cystadenocarcinoma
Epithelial-like monolayer. Non-clonogenic in agar Epithelial-like monolayer with 3D growth. Doubling time 26.7 hours
Nk
Poorly differentiated endometrioid carcinoma Moderately well-differentiated adenocarcinoma Poorly differentiated mucinous cyst adenocarcinoma
Epithelial-like monolayer. Clonogenic in agar Epithelial-like monolayer. Doubling time 28.8-44 hours. PE 15% in agar [52] Epithelial-like monolayer. Doubling time 29 hours
SRO-82 SW 626
Epithelial Poorly differentiated cystadenocarcinoma
Monolayer. Doubling time 70 hours Epithelial-like monolayer
T222 TO14
Nk Well differentiated serous adenocarcinoma
TR 170
Poorly differentiated cystadenocarcinoma
?Adherent monolayer Cobblestone epithelial-like monolayer. Produce hemicysts. Doubling time 130 hours. PE 0.13% in agar Cobblestone epithelial-like monolayer. Low frequency of tumors in nude mice taking Doubling time 31 hours. PE 2-5%in agar 5 months to establish. SC
SHIN-3
SIB-1 SKOV3 SR8
Serous cystadenocarcinoma in nude mice. SC/Solid with vascular invasion. Distant metastases. IP/Ascites with diffuse peritoneal seeding and distant metastases on uterus and lung Nk Solid adenocarcinomas in nude mice. SC
Continued on next page
Wilson and Garner
Poorly differentiated mucinous adenocarcinoma in nude mice. SC/Solid with muscle invasion. IP/Ascites and multiple tumor deposits in abdomen, liver, diaphragm, mesentery and liver parenchyma Nk Well differentiated papillary adenocarcinoma in nude mice Nk Nk
Cell line
Brief description of tumor pathology
In vitro features of cell line
TR 175
Serous cystadenocarcinoma
TYKnu
Undifferentiated carcinoma
UCI 101
Moderate to poorly differentiated serous papillary cystadenocarcinoma
UCI 107
Papillary adenocarcinoma
UPN36 UWOV1
Moderately well differentiated serous papillary carcinoma Serous papillary cystadenocarcinoma
Epithelial-like monolayer. Whorled growth No tumors in nude mice. SC pattern. Doubling time 34 hours. PE 0.4–0.8% in agar Undifferentiated tumors in nude mice. SC Epithelial-like monolayer. Doubling time 37 hours. PE 13.1% in agar Moderately differentiated papillary Epithelial-like monolayer. Doubling time 24 hours cystadenocarcinoma in athymic Balb C mice. IP/Foci of tumor on viscera and ascites Spindled appearance in monolayer. Tbmors in nude mice. SC/Solid. IP/Ascites and solid tumor nodules Doubling time 24-36hours. PE 4–10% in in agar Epithelial-like monolayer Tumors in nude mice
UWOV2
Serous papillary cystadenocarcinoma
UWOV2Sf
Serous papillary cystadenocarcinoma
YAOVBIX1
Serous papillary cystadenocarcinoma
YAOVBIX3
Serous papillary cystadenocarcinoma
Epithelial-like monolayer. Doubling time 36.5 hours Epithelial-like monolayer. Doubling time 45.6 hours Epithelial-like monolayer. Grows in serumfree medium. Forms domes at confluence. Doubling time 74 hours Adherent monolayer. Clonal origin from YAOVBIX Adherent monolayer. Clonal origin from YAOVBIX
Xenograft pathology
Ovarian Cancer
Table 3 (continued)
Nk Nk Nk
Tbmors in nude mice(IP) with solid omental deposits Tbmors in nude mice(IP) with solid omental deposits Continued on next page
29
30
Table 3 (continued) Cell line
Brief description of tumor pathology
In vitro features of cell line
Xenograft pathology
YAOVDK
Serous papillary cystadenocarcinoma
Adherent monolayer. Clonal origin
YAOVFAB
Carcinoma
?Adherent monolayer
YAOVJON
Carcinoma
?Adherent monolayer
YAOVWE1 Yoshikazi
Serous papillary adenocarcinoma Serous adenocarcinoma
Adherent monolayer. Clonal origin Nk
Tbmors in nude mice(IP) with solid omental deposits Tumors in nude mice(IP) with solid omental deposits Tumors in nude mice(IP) with solid omental deposits No tumors in nude mice Nk
Abbreviations: Nk, not known; SC, subcutaneous; IP, intraperitoneal; PE, plating efficiency
Wilson and Garner
Ovarian Cancer
31
surfaces, as well as the surface of the ovary. Downward migration and differentiation of the coelomic epithelium in embryogenesis gives rise to the fallopian tubes, the lining of the uterus and the endocervix. In the male this development is inhibited by the production of Müllerian inhibiting substance, and Wolffian development occurs instead. The epithelial ovarian cancers include serous, mucinous, and endometrioid cancers, so-called because their histology reflects the different differentiation patterns characterizing the epithelia of the fallopian tubes, endometrium and cervix (122). In addition, there is a clear cell carcinoma group (or mesonephroid carcinoma) the histology of which resembles renal carcinomas. The distribution of the cell lines within the histological subgroups is shown in Table 4. When this is compared with reported clinical incidence (121) there is general similarity within each group, although the Japanese tumor cell lines show a different distribution. Of the 62 cell lines which carried a description of the differentiation of the tumor of origin, 27% (17) were well differentiated, 11% (11) were moderately differentiated, 50% (31) were poorly differentiated and 5% (3) were Table 4 Summary of the cell line histopathology Tumor type Epithelial Serous Mucinous Endome trioid Clear cell Mixed Poorly differentiated Unclassified
Number
Percent
Clinical incidence (%)
62 (7)J 13(6)JJ 8 (3)J 14 (6)J 8 15 18
40 (32)J 8 (27)J 5 (14)J 9 (27)J 5 10 12
50 10 <1 <1 – – –
J
138 Others Teratoma Granulosa Krukenberg Carcinosarcoma
2 4 1 1
1 3 0.5 0.5
2.5 3 – –
8 Differentiation
Number
Well Moderate Poor Undifferentiated
17 11 31 3 ––––––– 62
Percentage of total 27 18 50 5
Cell lines from Japan are shown in parentheses as a separate group because their distribution within the sub-categories is different from the overall distribution J
32
Wilson and Gamer
undifferentiated. Non-epithelial cancers of the ovary are represented by PA1 and HUOT (teratoma), AZ504, COV434, HTOG, KGN (granulosa), HTOT (theca) and HSKTC (Krukenberg, a term used to describe a tumor metastatic to the ovary, usually from the stomach or gastrointestinal tract). The majority of the cell lines grow as adherent epithelial monolayers and express cytokeratins, vimentin, CA125 and epithelial markers. Heterogeneity is demonstrated in size, shape, density, growth pattern, amount of 3D growth, ability of cells to grow in soft agar and doubling time. A number of cell lines demonstrate the properties of transporting epithelium and form hemicysts (CKS, OAW 42, PEO14, PEO16, PEO23, RMG-I, TO14, UWOV2Sf). Doubling times range from 10.8 hours (cell line O–129) to 151 hours (cell line MH), the modal range being < 30 hours for 35 of the cell lines and 31–50 hours for 37 of the cell lines. Not all lines tested were clonogenic in soft agar and those that formed colonies had plating efficiencies which ranged from 0.01% to 50% (cell line CAVEOC-2). AZ303, COLO-704 and COLO 330 were all reported to grow as suspension cultures. Many of the cell lines grow as xenografts in nude mice, immunosuppressed mice or the hamster cheek pouch. Subcutaneous inoculation of tumor cells generally produces subcutaneous solid tumors with no distant metastases, although invasion of surrounding tissue (muscle, lymphatics, blood vessels) is occasionally seen. Intraperitoneal injection produces either peritoneal deposits, ascites or both. Cell lines which produce both ascites and solid tumors by the IP route are IGROV-1, OVCAR-3 (Nu/AG+), SR8, UCI 101 and UCI 107. Cell lines which produce tumor nodules with evidence of invasion by the SC or IP route are MCAS-3, OTN 14, OVAN-4, SHIN-3 and SR8. Cell lines producing metastases are MCAS-3 (lymph nodes- IP route), OVCAR-3 (Nu/AG+) (pulmonary metastases- IP route), OVTOKO (limited liver and lymph node metastases by IP route) and SHIN-3 (distant metastases by SC route). Xenografts of ovarian tumors from biopsies have also been established, including HX/110, PXN/100 and PXN/65 (191). The subcutaneous xenograft models do not provide a fully representative model for human disease in that the metastatic potential of the cell lines is limited. Intraperitoneal injection provides a better metastatic model, although again the xenografts display limited metastasis in comparison with the tumor of origin, and ethical considerations raised by the use of the intraperitoneal route need to be taken into account. The pathology of the xenografts resembles that of the original tumor in the majority of cases.
5.
MOLECULAR GENETICS
The cytogenetic changes for the ovarian cancer cell lines, where this information is available, are shown in Table 5. In addition, the data has been
Cell line
Genetic changes
Reference
154 163 194 204 222 2774 436 A1 A10 A121 A69 AI A90 AMOC-2 Asano AZ303 AZ364 AZ382 AZ390 AZ402 AZ403 AZ418 AZ419 BG-1 CAOV-4
Mode 66 Mode 66. Amplified neu Normal neu Normal neu Mode66-68. M1/t(13q;1p) m2/i(9q) m3/t(17q;21q) Amplified neu Mode 55. T(1:3)(p21->pter).t(1:3),lp+.2q+.5q+.6q+.6q25-,10q+.der(11)(?:11p14->q23:3p12->pter),t(1p:13q).14q+.15q-.der(17) Range 60-135,no mode Mode 60–64. Mode 46. T(1:?)(p21:?).t(1;3)(p21->qter:p12->pter).?iso(3p).3p21-.3p-q+.6q24-.14q+ . DM Range 54-66, mode 62 Mode 60. T(1:3)(p21->qter;p12->pter).6q24-.inv(7)(p15),13p+.der(4).13p+ .13p+ .13p+ .13p+.?13p+.?der(17).der(21) Mode 46. XX, 1q-,5p+,5p-,12q-. Homozygous deletion of p16/cdkn2a and p15/mts2. WT ras Investigated for deletion of chromosome 11p13-11p15.5 Investigated for deletion of chromosome 11p13-11p15.5 Investigated for deletion of chromosome 11p13-11p15.5 Investigated for deletion of chromosome 11p13-11p15.5 Investigated for deletion of chromosome 11p13-11p15.5 Investigated for deletion of chromosome 11p13-11p15.5 Investigated for deletion of chromosome 11p13-11p15.5 Investigated for deletion of chromosome 11p13-11p15.5 Mode 46. XXX,-4,-13, +derdic(4),t(1:4)(p11:q33),t(14:15),+ mar Modal range 60-63. 6 markers iso(3q),dic(6petr--->6q21::?::8q),unknown isochromosome, t(7p?;9q+[hsr]),iso(21q?),del(6)(p21)
8 8 22 24 24 26 24 27 28 27 27 28 27 31 32 34 34 34 34 34 34 34 34 35 37
Ovarian Cancer
Table 5 Cytogenetic changes
Continued on next page
33
Table 5 (continued) Cell line
Genetic changes
Reference
CAVEOC-2 Mode 76-78. <3n> ,X, + del(2q),+ 3,-4,der(s),del(?)(q31), +9,-11, +der(12)t( 21;?)(q24;?), + (13)x3,-16,-17,+ (20)x3,-21,-22, and 5 markers[cp5] CH1 Mean 45 CI 79-36 Mode 72. lp-, 6q+,7p+,11p+,i6p and >30 unidentified markers Mode 60. t(1;9)(q11;q12)1q-p+, 6q+,14p+, 15p+, -16,-16,18q+, i21q and 2 indistinct metacentric markers CI 80-13A Mode 60. As CI 80-13A but also 4p-, 4q+ and extra small acrocentric marker. No i21q CI80-13S Modes of clonal lines 44,44,65,70,85 and 99. Common abnormalities are t(1;3)(p33;p23),6p+, t(9;21)(p11;p10),7p-, CI81-1 ins(11)(q13) and 3 markers CKS Mode 44, monosomy in 1 and trisomy in 5, no markers.(mode37 [70]) COLO 110 Mode 74.t(1;6)(1qter to cen to 6pter),t(1;?)(1pter to 1 q43::?),del(1)(pter to q22),depl(3)(qter to p13:),del(6)(pter to 25:), t(7;?)(7qter to 7p11::?),t(21;?)(21pter to 21q22;;?) and 1-3 markers COLO 316 Mode 68. Numerous abnormalities with 48 markers COLO 319 Bimodal 67 and 69. Numerous abnormalities with up to 55 markers COLO 330 Mode 57. Numerous abnormalities with 27-38 markers COLO-704 Mode 51, DM, XX, +1, +7, +8, +19, +20, i(1q), der(8)t(8;?)(q21:?) COV318 Mode 74. T(X22)(p11;q11),hsr19q+,t(X22),de1(5)(q11q14),der(9)t(5;9)(p11;p22),11p+( llqter cen4::5q12qter) t(13q21q) t(8q14q),i(11p),i(18p).and other abnormalities COV362.4 Mode 69. T(8;11)(p23;q14),mar1q::11q13::hsr,del(X)(q21)xq+,der(5)t(5;17)(p11;q11),t(13q22q),t(2q8q),i(9q),i(11p),i(21q) and other abnormalities COV413B Mode 38 .-X,del(5)(p12p14),dup(5)(q32 to q35),der(11)t(5;11)(q12;p14),der(13)t(7;13)(q12:p11),t(2q8q) and other abnormalities COV434 Mode 47. +5 and other abnormalities COV434SUBMode 48 COV446B Modes 45/96. Del(X)(q24),i(5p),der(19)t(5;19)(q22;q12),t(13q22q)t(13q15q),i(8q),i(5p),i(7q)i(8q),i(11q),i(21q) and other abnormalities COV504 Mode 64. T(X16)(q26;q21)t(13;17)(q11;p11)or(p11q11),t(X16),5p+,t(13q17q) and other abnormalities COV644 Mode 58.2 large mar with hsr,inv(X),der(1)t(1;5)(p21;q14?),i( 13q) and other abnormalities
38 35 40 40 40 40 41 42 42 42 42 43 45 45 45 45 45 45 45 45
Continued on next page
Cell line
Genetic changes
Reference
DO-S EF021 EFO27 EFO3 EFO47 ES-2 HeW HEY HMOA HNOA HOC-1 HOC-21 HOC-7 HOC-8 HR HSKTC HTBOA HTOA HTOG HTOT HUOCA-II HUOT HX/62 Ia288 IGROV-1 JA1
59,XX,+4a,+6c,+e,+f,+g. Mode >100 Mode >100 Mode 49 Mode46 66, XX to 88, XX Mode 87 Range 45-113, del(3)(p12:)t(5:?)(5p15;?) Del (6)(q21-23:) i(8q) del(11)(q21:) and 3-5 unidentified markers Mode 46-47.1 marker 2pMode 46-47 Mode 49, inv(3)(p13q23) Mode 46. Mode 50,nv(3)(p13q23)t(11;13)(q13;p13)t(12;19)(q24;q13) Mode 52. Abr 6q. No DM. Mode 68 Modal range 87-96 Mode 65-68. XX,(5p+)x3,6q-,7q-,12p-.12p+,13p+,14p+,21p+ and 9 markers Modal range 95-105 Mode 80. Mode 46. Mode 46 Modal range 52-56.3 markers. Mean 82 Mode 62-66. 6 markers. m1/7q+;m3/3+;m4/11p+;m613p+ 46,XX,inv(3)(p13p25); t(2;5)(q33;q22) Mode 80-86.dm. See [124] for detailed karyotype
46 47 47 47 47 48 51 52 53 53 52 54 52 55 56 57 59 60 61 61 62 63 39 64 65 66
35
Continued on next page
Ovarian Cancer
Table 5 (continued)
Cell line KK Kuramochi MAC-2 MCAS MH MLS/P NZ-OV1 0-129 OAW 138D
36
Table 5 (continued) Reference
Genetic changes
68 70 71 72 68 74 77 78 79 79
79
79 79 79 79 79 85 87
Continued on next page
Wilson and Garner
Mode 67. Mode 50. Mode 45. Peritetraploid Mode 72, 14-19 markers Mean 68. Multiple numerical aberrations and 18 clonal structural aberrations Mode 80. Mode 40. Range 66-72,-X,der(1)t(1;3)(p36;pl4),de1(3)(p13),add(3)(p13),-4,+6, del(6) (q13q23),+add(7)(p13),der(7)t(3;7)(p25;p22),der(11)t(1:11)(p32;p15),-14,-17,-17,-19 + 7-13 markers and other changes OAW 180D Range 83-96,-X,-X,der(1)t(1;11)(p13;q13)add(l)(q42)x2,+t(l;6)(q10;p10),+der(3)t(3;15)(p11;q11),der(6)t(1;6)(p13;q25) +hsr(7)(p11)add(7)(p11)x2,der(7)(q21),-14,der(14)t(3;14)(q21;q24),-17,add(17)(p11),-19, add(19)(q13), and 5-8 markers and other changes OAW 200D Range 63-69,-X,-X,der(X5)(p10;p10),+add(1)(p13),+2, -3,-5,i(6)(p10),add(6)(p11), add(6)(p21),+7, +der(7)t(1;7)(p13;p13)add(1) (p36),-8,add(8) (p11),-10,-12,add(12)(p11),-13,i(13)(q10),14,-15,-16,-18,-18,-19,+add(20)(q13) +21, +22,+der(?)(?;3)(?;q21) and 1-5 markers OAW253D Range 62-66,X,-X,-X, add (3) (q13),add (3)(q29),der(6)t(1;6) (q21;q21), del(11)(p13),add(16)(q24),inc OAW 28 Range 40-46,X,der(X)t(X,3)(q21;p12),-1, -3, del(3)(q21),der(6)t(3;6)(q1&p11), del(7)(q11),add(11)(q23), +add(11)(p15),add(14) (p11),-17,-17,-19, and 7-13 markers and other changes OAW 41M AS OAW 28 OAW 42 Range 79-84,-X, add (1)(p13),add(1)(q21), +del(1)(p31), +add(1)(p32),-3,add (3)(p13),-9,-9,i(9)(p10),-13,-13 der(14)t(5;14)(q15;p13),add(17)(q25),der(17)add(l7)(p11)add(17)(q25), and 3-8 markers and other changes OAW 59M Range 52-54,XX,+X,+6,+i(6)(p10),+i(7)(q10),-8, +add(11)(p13),-13,-13,der(13;14)(q10;q10),-16,-17,-18,add(19)(q13), and 7-9 markers Mode 70-77. XX,-1,+t (1;14),+ (1;14), +del(1)x2, +2+3+4+5x3 +6+7x2+t(7;?), +8p+,-9, +de1(9)x2,+ 10, + 11, + 12, +del(12),+ 13 + 15 OCC1 + 16p+,del( 17), + 19, +20, +21, and 2 markers Mode 43-44. OMC-3
Genetic changes
Reference
OMC1 OTN 11
Mode 84 Mode 41, XX, -3, -4, -8, -8, -9, -12, -12, -17, -18, -22, + 9 markers. i(3q) OR t(3;12), t(8;?)(q22;?), T(4;18)(p16;q21), t(9;12), t(12;?)(qll;?), Del9q, 17q+, t(4;18) recipr.? Mode 86,3-8 markers Modal range 56-58. DM. Trisomy. Monosomy for X. WT p16/CDKN2a, p15/mts2, and k-ras Modal range 60-70. Markers 1p +q31-,7p +,1 1p+,12q+ ,19q+ ,20q+ ,20p12-q12Mode 57. Abnormalities in chromosome 1. Mode 62. Mode47 Mode 78. 46, XX,+t(15q20q),+t(15p20p),-15-20 inactivation of one X chromosome Aneuploid Aneuploid Mode 41 Aneuploid Aneuploid Aneuploid Mode 41 Mode 41 Mean45 Mode 77 Mode 47. 8 marker chromosomes Hypertriploid Hypodiploid
86 88
OTN 14 OV-1063 OVAS-21 OVCAR-3 OVCCR1 OVISE OK18 OVTOKO PA1 PEA1 PEA2 PEO1 PEO14 PEO16 PEO23 PEO4 PEO6 PXN/94 RIC-2 RMG-I RMG-II RMUG-L RMUG-S
89 91 32 95 96 97 98 97,99 101 102 102 102 102 102 102 102 102 39 71 104 105 106 106
37
Continued on next page
Ovarian Cancer
Table 5 (continued) Cell line
38
Table 5 (continued) Cell line
Genetic changes
RTSG SCHM-1 SHIN-3 SIB-1 SKOV3 SKOV3
Abbreviations: ABR, abnormal banding region; DM, double minutes; HSR, homogeneous staining region; MAR, marker chromosomes
Reference 107 71 109 71 123 124 111 22 125 102 66 66 113 114 115 117 117 117 118 118 118 118 118 118 32
Wilson and Garner
Aneuploid with a tetraploid mode Mode 46 Mode 61.8 trisomies. 6q+11q- and 5 markers Mode 46 Mode 43. Del(1)(q21),der(13)t(1;?;13)(q11;?;q34),der(11)t(11;?)(q12),del(10)(q22) and 3 other markers No mode, Range 69-149. 12 markers reported including del(1) (pter--->q21:),der(l3)t(l:13)(lqter--->lqll::13q34--->13pter) der(11)t(11;?)(11pter---> 11q12::?) Two modes near diploid and near triploid. Range 40-78.der( 1)t(1;17)(p36?;q21.2?)der(6)t(X,6)(q27?;q21?), Der(x)t(x;6) SR8 (q27?;q21?),de1(3)(p13?) Normal neu SRO-82 SW 626 Modal range 83-108.der(2)t(2;5)(q35;q31);de1(8)(q13q22);del(12)(q13);t(9q13q) TO14 Aneuploid TR170 Mode 45-47.dm. See [124] for detailed karyotype TR175 Mode 51.dm. See [124] for detailed karyotype TYKnu Mode 56. Subcentric marker chromosome. UCI 101 Del 6q, abnormalities in chromosome 1 UCI 107 Mode 46. X,der(X)t(X7)(p11;q22),inv dup(1)(q12;q43),t(6;6;11;22)(p21.3;q16;q23.3;q13,3),de~(13)(q14.1) Passage 17 mean 52. 11p+. Passage 57 mean 87 11p+ UWOV1 UWOV2 Mean91. UWOV2Sf Mean 88. YAOVBIX1 Overexpresses neu YAOVBIX3 Does not over-express neu YAOVDK Does not over-express neu YAOVFAB Does not over-express neu YAOVJON Does not over-express neu YAOVWE1 Does not over-express neu Yoshikazi WT p16/CDKN2A, p15/MTS2, and k-ras
Ovarian Cancer
39
summarized to show cell lines with hypodiploid, close to diploid and aneuploid karyotypes (Table 6). Cell lines with specific information relating to oncogene and tumor suppressor gene expression are shown in Table 7. The genetic information is complex because so many structural abnormalities have been identified in ovarian cancer and many of the cell lines are aneuploid with multiple markers, deletions, additions and rearrangements.
Table 6 Modal chromosome numbers* < 46 Hypodiploid
46–50 Close to diploid
CKS COV413B COV446B MAC-2 OAW 28 OTN 11 PEO1 PEO4 PEO6 RMUG-S SKOV3
A69 AMOC-2 BG-1 COV434 EFO3 EFO47 HMOA HNOA HOC 1 HOC 21 HOC 7 HTOT HUOCA-II IGROV-1 Kuramochi OVK18 PA1 RMG-1 SCHM-1 SIB-1 SR8 TR170
*Refer to Table 3 for specific values **Bimodal range reported
>50 Aneup1oid 154 2774 A10 A7 CAOV-4 COLO 110 COLO 319** COV318 COV446B** COV644 EFO21 ES-2 HeW HOC 8 HSKTC HTOA HUOT JA1 KK MH OAW 180D OAW 253D OAW 59M OTN 14 OVCAR-3 OVISE PEA1 PEO14 RIC-2 RTSG SKOV3 SW 626 TR175 UWOV2
163 A1 A121 A90 CAVEOC-2 COLO 316 COLO 330 COV362.4 COV504 DO-S EFO27 GG HEY HR HTBOA HTOG Ia288 JC MCAS OAW 138D OAW 200D OAW 42 OCC1 OV-1063 OVCCR1 OVTOKO PEA2 PEO16 RMUG-L SHIN-3 Sr8** TO14 UWOV1 UWOV2Sf
Wilson and Gamer
40 Table 7 Specific genetic changes Gene p53
k-ras
Cell lines Wild type
A2774, A2780, IGROV-1, PA1 OVCA 420, OVCA 429, OVCA 433 OVMZ6 A2780.AD, EC SKOV3 OVCAR-3
[146, 192, 193] [94] [147] [148] [149] [150]
Mutation
OVCAR-3, OVCAR-5, OVCAR-8, SKOV3, SW 626, OVCA 432 OVMZ11, OVMZ18, OVMZ28, OVMZ32 222, 2774, A2780CP, EC, PM1015, CAOV-3, CAOV-4, Kuramochi, PAI,
[146,192,193] [94] [147] [148] [149]
Wild type
Asano, HAC-2,OVAS-21, RMG-I, Yoshikazi
[32]
Mutation
SHIN-3
[32]
Amplification
HOC-8
[55]
HTOA, KF, Kuramochi, HAC-2, MH, PA1 A1847, A2780,OVCAR-2,OVCAR-3,OVCAR-4, OVCAR -7,OVCAR-8 HAC-2, OVAS-21, Yoshikazi 2774, A2780, SKOV6
[144] [128] [32] [152]
Mutation
MCAS, HOV-7 OVCAR -10
[144] [152] [128]
No transcription
KK
[144]
Homozygous deletion
RMG -I, SKOV, TYKnu OVCAR-5, PEO1, SKOV3 Asano, RMG-I, SHIN-3 27/87, CI 80-13S, PEO1, PEO4, SKOV3
[144] [128] [32] [151]
Wild type
HAC-2, OVAS-21, Yoshikazi
[32]
Homozygous deletion
Asano, SHIN-3
[32]
p16 Wild type (CDKN2A) INK4A
P15 (MTS2)
AKT2
Ref
No amplification A2780,OVCAR-2,OVCAR-4, OVCAR 5,OVCAR -7, OVCAR -10
[153]
(19q13)
Amplification
OVCAR-3,OVCAR- 8
[153]
c-fms
Expression
HEY, SKOV3, YAOVBIX, YAOVDK N1 (subclone of HOC-7) HMG, HRA, KF,KK
[140] [154] [151]
No expression
YAOVWE1
[140] Continued on next page
41
Ovarian Cancer Table 7 (continued) Gene pp60c-src activity
PTPN6
Cell lines
Ref
< 1.0 U/106 cells
OVCAR-4, OVCAR-5, OVCAR-8
[156]
>1.0 U/10 6 cells
IGROV-1 (6.7U), OVCAR-3 (3.3U), SKOV3 (2.2U)
[156]
Normal expression
SKOV3
[157]
Over-expression
CAOV-3, DOV13, OVCA 420, OVCA 429, OVCA 432, OVCA 433,OVCAR-3
[157]
hMSH2
Defective
2774 2008A (cisplatin-resistant subline of 2008)
[23] [194] [196]
hMLH1
Defective
SKOV3 9/10 cisplatin-resistant sublines of A2780, but not parent A2780
[194] [195]
hPMS2
Defective
9/10 cisplatin-resistant sublines of A2780, but not parent A2780
[195]
erbB-2
Normal expression
222,204, CAOV-3, OVCAR-3, PAl, SRO-82 T222, UCI 101, UCI 107, HEY, YAOVBIX3, YAOVDK, YAOVFAB, YAOVJON, YAOVWE1
[22] [112] [118] [118]
Over-expression
194, 436, SKOV3 YAOVBIX1 OVCA 429, OVCA 433, OVCAR-3
[22] [118] [158]
222, A2008, CAOV-3, CAOV-4, OVCAR-3
[23]
2774, PA1, SKOV3, UCI 101
[23]
Genetic Stable instability Unstable
Cell lines shown in italics are not included in Table 1 because there appears to be no published information to support their derivation
The karyotypic abnormalities which have been most frequently reported in surgical specimens from ovarian cancer patients involve chromosomes 1, 3, 6, 9, 10, 11, 12 and 14 (126). In a series of primary ovarian tumors the breakpoints of clonal structural abnormalities clustered to 19p13, 11p13–15, 1q21–23, 1p36, 19q13, 3p12–13 and 6q21–23 (127). Loss of heterozygosity has been reported on chromosomes 9 (128), 17p (129,130), 3p (131), 6 (131,132), 11p (131,133), 11q (134), Xp and 13q(135). Oncogenes which have been implicated in ovarian cancer include ras (136, 137), erbB-2 (138,139) and c-fms (140,141,142) whilst tumor suppressor genes include p53 (129,143) and CDKN2A (32,128,144).
42
Wilson and Gamer
Microsatellite instability and defects in mismatch repair have been implicated in ovarian cancer, both in tumor progression and in the development of resistance to cisplatin (194). The subunits hMLH1 and hPMS2 of the MutLea mismatch repair protein complex and hMSH2 of the MutS mismatch repair protein complex have been identified as mutation sites in cell lines with resistance to cisplatin and in biopsies of ovarian cancer (194–196).
6.
CROSS-CONTAMINATION
No HeLa contamination has been reported for any of the cell lines. However, there is indication of cross contamination for several cell lines. These include 41M and OAW 28 which were found to share an identical DNA fingerprint. DNA fingerprinting of uncultured material from OAW 28 has confirmed that both lines originate from this patient and cross contamination was an early event in the life history of 41M (79). Phenotypic differences between the cell lines have been reported (39). Results from a recent report describing the use of microsatellite sequencing for cell line identification showed identical allelic profiles at ten different loci for SKOV3, YAOVBIX1 and OC436 (undocumented cell line originating from the ascites of a patient with Meig’s Syndrome, a term used for an association of ascites, hydrothorax and an ovarian tumor, usually a fibroma) (145).
7.
SPECIAL FEATURES
Other relevant features of cell lines which relate to ovarian cancer include hormone receptor status, drug sensitivity and response to peptide growth factors, particularly EGF and TGFß. Some of the published data is summarised in Table 8. The normal ovary differs from other hormonally sensitive tissues in that estrogen receptor (ER) and progesterone receptor (PR) do not appear to be equally distributed and PR expression is more frequent than ER expression (159). Although the reported incidence of ER and PR in ovarian cancer shows some variability, the general concensus is that there is reduced expression of PR and increased expression of ER compared with normal ovary. Several studies have investigated the relationship between ER/PR status, survival, prognostic features and treatment response, but the conclusions are conflicting (159). The role of hormonal therapy is not clear, although a variety of therapeutic approaches have been tested, including medroxyprogesterone acetate and tamoxifen. Some tumors express HCG receptors and inhibitors of gonadotropin-releasing hormone have produced responses in nude mice bearing xenografted ovarian tumor cells (160). Androgen receptors have also
Ovarian Cancer
43
Table 8 Response to hormones Hormone system
Cell lines
Findings
Ref
Estrogen receptor
OAW 42 OV 1225, OV 166, OV 2774 PEO16, PEO14, PEO23, TO14 OVCAR-3 PEA1, PEA2
Does not express ER Do not express ER Do not express ER ER expression Express low levels of ER (12-23 fmol/mg protein) Express ER (92-132 fmol/mg protein) Secretes estradiol
[161] [90] [162] [94] [162]
PEO1, PEO4, PEO6 Secretion of estradiol OTN 11
[162] [88]
Androgen receptor
OV 2774, OV 1225 OVCAR-3 OV 166
Does not express AR Expresses AR Expresses AR
[90] [94] [90]
Progesterone receptor
OVCAR-3, OVCAR-4, OVCAR-5, A2780, A1847
All express progesterone receptor and are inhibited by mifepristone
[163]
Secretion of HCG
163
Secretes HCG
[164]
Response to HCG and FSH
EFO3, EFO27, EFO21, EFO47 Only EFO3 and EFO27 respond [47] to HCG. None respond to FSH.
LH-RH mRNA and receptor
EFO21, EFO27
Activin receptor. CAOV-4, SKOV3, SW 626, PA1, ES-2 Secretion of inhibin/activin Secretion of follistatin
Both express LH-RH receptor and secrete LH-RH
[165]
All cell lines express the activin [166] receptor. Only PA1 and ES-2 secrete follistatin. All lines except PA1 secrete aBa or Bb subunits of actin
Abbreviations: ER, estrogen receptor; AR, androgen receptor; HCG, human chorionic gonadotropin; FSH, follicle stimulating hormone, LH-RH, luteinising hormone releasing hormone
been detected. The approaches which have been used to study features relating to hormone responsiveness are heterogeneous, but there is published data on a number of cell lines. The response to hormones is summarised in Table 8. Chemotherapy is the treatment of choice for ovarian cancer following debulking surgery and the drugs which have been most frequently used include cisplatin, carboplatin, adriamycin and cyclophosphamide. The response rate to first line chemotherapy is high, but relapse rates are also high and there is no effective second-line chemotherapy for resistant disease.
Wilson and Gamer
44
Paclitaxel was first used as a second line agent in the treatment of ovarian cancer, and the high response rate encouraged its use as a first line drug in combination with cisplatin. Research efforts are strongly focused towards identification of mechanisms of resistance, development of new drugs with novel modes of action and development of new forms of therapy including immunotherapy and the use of biological modifiers. Cell lines are an important resource for such work and this is evident from the number of studies which report drug sensitivities and development of drug resistant lines. The previous treatment of patients from whom cell lines have been developed is summarized in Table 9. The in vitro sensitivities of some cell lines to cisplatin, adriamycin and taxol is shown in Table 10. It should be noted that the relative levels of resistance reported in Table 10 were derived using a number of different assay methods which are not directly comparable. The cell lines from which resistant sublines have been developed are shown in Table 11. The cell lines which originate from the same patient at different sites or different times are shown in Table 12. One of the novel treatment strategies currently under development involves tyrosine kinase inhibitors with specificity for the EGF receptor. TGFß is also of interest as a potential inhibitor of ovarian cancer growth. A number of cell lines have been assessed for their responses to EGF and TGFß and these data are summarized in Table 13. Finally, several lines have been successfully cloned to produce sublines and some lines are reported to grow under reduced serum or serum-free conditions. These data are shown in Table 14.
Table 9 Cell lines derived from treated patients Drug treatment
Cell lines
Radiation
CKS, COLO 330, OTN11, PEO16, SCHM-1, 154, 163
CIS or CARB only
A286, OAW 138D, OAW 180D, OAW 42, OV56
CIS/CARB + others
CAVEOC-2, HUOCA-II, MAC-2, OV-1063, OV17R, OV25, OV25R, OV58, OVCAR-2, OVCAR-3, OVCAR-4, OVISE, OVMZl, OVMZ2, OVMZ3, OVTOKO, PEA2, PEO1, PEO23, PEO4, PEO6, PM1015, RIC-2, SIB-1, TR170, UCI 101, UWOV2
Single alkylating agent
OAW 253D, OV7, SKOV3
Other
A10, AZ303, AZ364, AZ382, AZ390, CAOV-3, HUOT
Untreated
See Table 1
Abbreviations: CIS, cisplatin; CARB, carboplatin
Ovarian Cancer
45
Table 10 Cell line sensitivities to cisplatin, adriamycin and paclitaxel (figures refer to IC50) Cell line 67R 1085 BR CAVEOC-2 ES-2 HAC2/P HAC2/0.1 HEY* HOC-7* HTOA HX/62 Ia288 IGROV-1 JA1 KK MH NZ-OV1 OAW 42[81] OAW 138D[81] OAW 180D[81] OAW 200D[81] OAW 253D[81] OAW 28[81] OAW 59M[81] OAW D13[81] OAW D16[81] OAW D206[81] OMC-3 OVCAR-3 OVCAR-4 OVCAR-5 OVCAR-8 OVC-8 OVISE OVTOKO PM1015 PXN/94 SKOV3 TR175 TR170 UCI 101 UCI 107
CIS (µM)
9.4 low to medium res 0.66 1.584 3.3 0.165 sens 2.5–8.8 5 sens 0.726 0.95 3.28 1.56 19.88 4.36 10.04 10.01 2.47 5.86 32.27 14.15 13.26 20.33
8.58-13.33 8.58-13.33 sens 1.1-1.6
ADM (µm)
1.82 low to medium res
TAX (µM) 9.83 8.78 10.18 3.33
0.24 0.034
3.7[77] 0.1 30,000 0.04 15,000 0.07 < 0.01 0.03 0.04 20,000 0.01 1.62 0.02 0.05
2.5[77] 5.1[77] 4[77]
sens 44.8 44.8 sens 4.4[77]
3.66 3.33 3.3 0.33
0.083 0.06 0.02 0.17
590 29
Unreferenced data is taken from the references referred to in Table 1. Abbreviations: sens, sensitive; res; resistance; IC50, concentration of drug required to reduce control values by 50%; *IC90, concentration of drug required to reduce control values by 90%; CIS, cisplatin; ADM, adriamycin (doxorubicin); Tax, taxolTM, paclitaxel
46
Wilson and Gamer
Table 11 Drug-resistant cell lines Cell line
Drug
A1847
Cisplatin Adriamycin Melphalan Cisplatin Adriamycin Melphalan CPT-IIa Cisplatin
A2780
HAC2 HAC2 (=HAC 2/P) KF TYK KF-1 (cloned from KF) NOS2
[167,168]
[169] [170] [171]
Cisplatin
NOS2CR1 NOS2CR2 NOS2CBR NOS2DR 0-129/DDP4 0-129/DDP8 0-129DDP16 OAW42-A
7x 16 x 8x 6x 2.1 x 4.1 x 6.3 x 68.7 x
[173]
OAW42A OAW42-SR OAW42-A1 OAW-dox OAW-dox OAW-tax OAW-tax OV1/DDP OV1/VCR OV1/DXR SKVLB (0.01) SKVLB (0.03) SKVLB (0.06) SKVLB (1.0) SKVCR (0.015) SKVCR (0.1) SKVCR (0.25) SKVCR (2.0) 41McisR 41MdoxR CH1cisR CH1doxR COLO/DDP
26x 8x 14x 93x 616x 83x 93x 15 x 800 x 20 x 4x 64 x 490 x 2000 x 4x 260 x 1000x 4100 x 4.7x 7x 6.5x 9Ox 2.5x
[197]
2.7x
[201]
Doxorubicin
Doxorubicin Taxol Doxorubicin Taxo1 Cisplatin Vincristine Adriamycin Vinblastine
Vincristine
CH1
[167]
3x 3x 20 x
OAW 42
41M
3x 5x 4x 10 x 100 X–150 x 10 x 9.7 x 2.4 x
Reference
KFr TYK/R KFr
0-129
SKOV3
A1847CP A1847AD A1847ME A2780CP A2780AD A2780ME HAC2/CPT HAC2/0.1
Magnitude of resistanceb
Cisplatin Cisplatin Cisplatin
CBDCA DWA214R Cisplatin
OV1/P (= IGROV-1)
Derived lines
Cisplatin Doxorubicin Cisplatin Doxorubicin Cisplatin
COLO3 16 2008 Cisplatin 2008/DDP a : Camptothecin analog b: Not comparable because different assay methods
[172]
[78]
[174]
[198]
[175]
[176]
[176]
[199] [200] [199] [200] [201]
Ovarian Cancer
47
Table 12 Multiple cell lines derived from the same patient Cell line
Relationship
Reference
YAOVBIX1 YAOVBIX3
Two morphologically distinct clones from same ascites sample
[145]
CI 80-13A CI 80-13S
Two cell lines derived from solid (S) and ascites (A) of same patient at same time
[40]
COV413A COV413B
Metastasis from sigmoid (A) and from bladder dome (B) of same patient at same time
[45]
OAW D13, OAW D16, OAWD206
Peritoneal fluid (D13) and omental deposit (D16) concurrently from same patient pre-treatment and post-treatment sample (D206) from same patient
[81]
PEO1 PE04 PEO6
Established from ascites of same patient during treatment and relapse
[102]
PEA1 PEA2
Established from same patient before and after treatment
[102]
PEO14 T014
Established from ascites (PEO14) and solid metastasis (TO14) from same patient prior to treatment
[102]
PEO14, TO14 PEO23
Established from same patient pre (PEO14, TO14) and post treatment (PEO23)
[102]
RMUG-L RMUG-S
Two morphologically distinct cell lines derived from same tumor sample
[106]
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Boyle P et al. In Ovarian Cancer 4, Pub. Chapman and Hall, Chapter 9: 91, 1996. Southam CM, Cancer; 7, 394, 1954. Rose GG et al. Journal of the National Cancer Institute 11: 1223, 1951. Wright JC et al. Cancer 15: 284, 1962. Limburg HG, Proc Roy Soc Med 62: 361, 1969. Ioachim HL et al. Laboratory Investigation 31: 381, 1974. Ioachim HL et al. Natl Cancer Inst Monogr 42: 45, 1975. DiSaia PJ et al. Gynecologic Oncology 3: 215, 1975. Kimoto T et al .Acta Path Jap 25: 89, 1975. Wilson AP, In Cell and Tissue Culture Laboratory Procedures, John Wiley and Sons, Preparation of Ovarian Cell Cultures, 1996. Uitendaal MP et al. British Journal of Cancer 48: 55, 1983. Broxterman HJ et al. International Journal of Cell Cloning 5: 158, 1987. Mills GB et al. Cancer Research 48: 1066, 1988. Bast RC et al. Journal of Clinical Investigation 68: 1331, 1981. Arklie J, DPhil Thesis Oxford University, 1981.
Wilson and Gamer
48 Table 13 Response to TGFb and EGF Cell line
Concn
% Inhibition
Reference
TGFb DOV13 OVCA 420 OVCA 429 OVCA 432 OVCA 433
10ng/ml 10ng/ml 10ng/ml 10ng/ml 10ng/ml
0 90 0 20 20
[177]
1 ng/ml 1ng/ml 1ng/ml 1ng/ml 1ng/ml 1ng/ml
42 0 0 82 40 29
[178]
1ng/ml 1ng/ml 1ng/ml –– 1 pM
20 50 0
[179]
No response 50
[180]
Concn
% Stimulation
Reference
0.1nM 0.1 nM 0.1nM
~65 ~20 ~60
[181]
A7 OAW 138D OAW 180D OAW 200D OAW 253D OAW 28 OAW 59M
10ng/ml 10ng/ml 10ng/ml 10ng/ml 10ng/ml 10ng/ml 10ng/ml
–32 6 8 0 12 91 5
[182]
OAW 42
10ng/ml
–47
[183]
OAW 180D OAW200D OAW253D OAW28 OAW42 OAW59M PEO1 PEO14 PEO4 IGROV-1 OVCCR1 Cell line EGF PEO 1 PEO14 PEO4
Abbreviations: TGF-b, transforming growth factor beta; EGF, epidermal growth factor 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
Ward BG et al. Cancer 60: 787, 1987. Connel ND et al. Cell 34: 245, 1983. Moll Ret al. Cell 31: 11, 1982. Moll R et al. American Journal of Pathology 140: 427, 1992. Smith A et al. Cancer Genet Cytogenet 24: 231, 1987. Cameron MR et al. Oncology Research 7: 145, 1995. Lichtenstein A et al. Cancer Research 50: 7364, 1990. Orth K et al. Proc Natl Acad Sci(USA) 91: 9495, 1994. Nio Y et al. Cancer Immunol Immunother 27: 246, 1988. Chenevix-Trench G et al. Am Journal of Human Genet 55: 43, 1994. Freedman RS et al. Cancer 42: 2352, 1978. Crickard K et al. Gynecologic Oncology 32: 163,1989. Abu Sinna G et al. Gynecologic Oncology 7: 267, 1979. Hamilton TC et al. Seminars in Oncology 11 : 285, 1990.
Ovarian Cancer
49
Table 14 Cell lines that grow in the absence of or in reduced concentrations of serum Cell line
Clones
Reference
HOC-7
N1,N2,N3 D1,D2,D3
[184]
HR
HR-A HR-I
[185]
OVISE
OVISE-1, -2, -3 (72 Clones obtained)
[186]
COV434
COV434SUBCL.
[45]
COV413B
COV353
[45]
COV362
COV362.4
UWOV2
UWOV2Sf (0%)
[45] 1
OVCCR1 1
[71]
OVCCR1/rs (0.5%)1 OVCCR1/sf (0%)1
[96]
Concentration of serum used
30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.
[117]
MAC-2 (0%) RIC-2 (3%) 1 SCHM-1 (3%) 1 SIB-1 (0%)1 1
Fanning J et al. Gynecologic Oncology 39: 119, 1990. Yabushita H et al. Obst Gynecol Jpn 41: 888, 1989. Ichikawa Y et al. International Journal of Cancer 69: 466, 1996. Neyns B et al. Oncogene 12: 1247, 1996. Vandamme B et al. Cancer Research 52: 6646, 1992. Geisinger KM et al. Cancer 63: 280, 1989. Safrit JT, Gynecologic Oncology 48: 214, 1992. ATCC – Unpublished data deposited by J. Fogh. Griffon G et al.Anticancer Research 16: 177, 1996. Hills CA et al. Cancer 59: 527, 1989. Bertoncello I et al. Australian JEXp Biol & Med Sci 63: 241, 1985. Sekiguchi M et al. Japan J Exp Med 50: 283, 1980. Woods LK et al. Cancer Research 39: 4449, 1979. DSMZ – Unpublished data deposited by G. Moore. ECACC – Unpublished data deposited by G. Moore. Van den Berg-Bakker CA et al., Int Journal of Cancer 53: 613, 1993. Briers TW et al. Cancer Research 49: 5153, 1989. Simon WE et al. Journal of the National Cancer Institute 70: 839, 1983. Duran GE et al. Cancer Chemotherapy & Pharmacology 38: 210, 1996. Molthoff CFM et al. International Journal of Cancer 47: 72, 1991. Niimi S et al. Cancer Research 52: 328, 1992. Patillo RA et al. Cancer Research 39: 1185, 1979. Buick RN et al. Cancer Research 45: 3668, 1985. Ishiwata I et al. Gynecologic Oncology 25: 95, 1986.
50
Wilson and Gamer
Table 15 Cell lines described after Table was compiled Cell line
Histology
Comments
Reference
KEN-3 EC MN-1 3AO and AO
Fibrosarcoma Mixed germ cell tumor Mucinous cystadenocarcinoma NK
[202,203] [202,204] [202,205] [206]
SNU-8 SNU-119 SNU-251 SNU-563 SNU-840 CABA-1 LN1 INT.OV1 INTOV2 INT.OV3 INT.OV4 INT.OV5 INT.OV6 INT.OV7 INT.OV8 INT.OV9 UL-3A,B,C
Serous papillary cystadenoca Serous papillary cystadenoca Endometrioid carcinoma Endometrioid carcinoma Malignant Brenner tumor Papillary adenocarcinoma Mixed Mullerian tumor Serous Serous Mucinous Mucinous Serous Serous Serous Mucinous Borderline Well-differentiated serous adenocarcinoma
De novo resistance to cisplatin Sensitive to cisplatin Sensitive to cisplatin Estrogen and progesteronedependent. Deposited with Chinese cell bank Wild type p53, BRCA1, hMLH1 p53 mutation p53, BRCA1, hMLH1 mutations p53 mutation Wild type p53,RCA1, hMLH1 p53 mutation
UT-OC-1 UT-OC-2 UT-OC-3 UT-OC-4 UT-OC-5 CP70 OVT2 YKT
Mucinous Endometrioid Serous Endometrioid Serous
54. 55. 56. 57. 58. 59. 60. 61. 62. 63.
Serous
The INT series have been characterized for antigens potentially recognisable by HLA-restricted cytotoxic T cells
[207] [207] [207] [207] [207] [208] [209] [189]
A obtained at staging [210] laparotomy, B during treatment with cisplatin and taxol and C after clinical progression [211,212]
Analyzed for TGF- b receptors Analyzed for TGF- b receptors Grows in nude mouse ovary and metastasizes to liver
Yamada T et al. Keio J Med 23: 53, 1974. Filmus J et al. Cancer Research 46: 5179, 1986. Kikuchi Y et al. Biochem Biophys Res Communications 123: 385, 1984. Riken. Source reference not found. Deposited by I. Ishiwata. Uhl-Steidl M et al. Oncology 52: 400, 1995. Ishiwata I et al. Gynecologic Oncology 30: 35, 1988. Ishiwata I et al. Gynecologic Oncology 26: 340, 1987. Ishiwata I et al. Journal of the National Cancer Institute 72: 789, 1984. Ishiwata I et al. Journal of the National Cancer Institute 78: 667, 1987. Ishiwata I et al. Journal of the National Cancer Institute 75: 411, 1985.
[213] [213] [214]
Ovarian Cancer 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114.
Clamon G et al. Gynecologic Oncology 20: 92, 1985. Benard J et al. Cancer Research 45: 4970, 1985. Hill BT et al. International Journal of Cancer 39: 219, 1987. Riken _ Unpublished data. Deposited by Nishi and Yoshiro. Sasa H et al. Human Cell 6: 106, 1993. Morimitsu Y et al. Gynecologic Oncology 48: 155, 1993. Motayama T,Acta Obstet Gynecol Jpn 33: 1197, 1981. Hirte HW et al. Cancer 74: 900, 1994. Kidera Y et al. Acta Obst Gynaec Jpn 37: 1820, 1985. Miyagi E et al. Clinical & Experimental Metastasis 13: 89, 1995. Xu J et al. Cancer Genetics & Cytogenetics 74: 1, 1994. Van Haaften-Day C et al. Cancer Research 43: 3725, 1983. Wakahara Y et al. Oncology 54: 48, 1997. Baguley BC et al. European Journal of Cancer 31A: 230, 1995. Chen G et al. Cell Biology & Toxicology 11: 273, 1995. Wilson AP et al. British Journal of Cancer 74: 722, 1996. Wilson, AP, Journal of the National Cancer Institute 72: 513, 1984. Wilson AP et al. Abstract in 9th NCI- EORTC meeting, Amsterdam, 1996. Alama A et al. Gynecologic Oncology 62: 82, 1996. Maymon R et al. Gynecologic Oncology 55: 265, 1994. Holschneider CH et al. Cancer Chem Pharmacol 34: 66, 1994. Wong WSF et al. Gynecologic Oncology 38: 37, 1990. Hayakawa 0 et al. Sapporo Medical Journal 55: 153, 1986. Yamada T. et al. Gynecologic Oncology 40: 118 1991. Poels PHK et al. Gynecologic Oncology 32: 203, 1989. Van Niekerk CC et al. International Journal of Cancer 42: 104, 1988. Kieback DG et al. Gynecologic Oncology 51: 167, 1993. Horowitz et al. Oncology 42: 332, 1985. Boocock CA et al. Journal National Cancer Institute 87: 506, 1995. Walker W et al. Clinical & Experimental Immunology 101: 494, 1995. Elbendary A et al. Cell Growth and Differentiation 5:1301, 1994. Hamilton TC et al. Cancer Research 43: 5379, 1983. Jozan S et al. In Vitro Cell Dev Biol 28a: 687, 1992. Gorai I et al. Gynecologic Oncology 57: 33, 1995. Uehara S et al.Acta Obst Gynaec Jpn 35: 19, 1983. Yanagibashi T. et al. British Journal of Cancer 76: 829, 1997. Mobus V et al. International Journal of Cancer 52: 76, 1992. Zeuthen J et al. International Journal of Cancer 25: 19, 1980. Langdon SP et al. Cancer Research 48: 6166, 1988. Buller RE et al. Gynecologic Oncology 56: 39, 1995. Nozawa S et al. Human Cell 1: 426, 1988. Yajima M, Keio Med J 66: 817, 1989. Sakayori M et al. Human Cell 3: 52, 1990. Udagawa Y et al. Human Cell 3: 70, 1990. Imai S et al. Oncology 47: 177, 1990. Fogh J et al., J Natl Cancer Institute 58: 209, 1977. Fogh J et al. J Natl Cancer Institute 59: 221, 1977. Han X et al. European Journal of Cancer 32A: 160, 1996. Santin AD et al. Gynecologic Oncology 60: 468, 1996. Yoshiya N, Acta Obst Gynaec Japan 38: 1747, 1986. Fuchtner C et al. Gynecologic Oncology 48: 203, 1993.
51
52 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 161. 162. 163. 164. 165.
Wilson and Gamer Gamboa G et al. Gynecologic Oncology 58: 336, 1995. Cannistra SA et al. Cancer Research 53: 3830, 1993. Golombick T et al. In Vitro Cell Dev Biol 26: 447, 1990. King BL et al. American Journal of Pathology 140: 23, 1992. Williams, C, British Medical Journal 304: 1501, 1992. Parmley TH et al. Am J Obstet Gyn 120: 234, 1974. Fox H & Langley FA, In Tumors of the Ovary William Heinemann, 1976. Scully RE Human Pathology 1: 73, 1970. ATCC data sheet. Sheer D et al. Cancer Genet Cytogenet 26: 339, 1987. ATCC data sheet. Steel CM et al. In Ovarian Cancer 4 Chapman and Hall Medical, Chapter 6: 61, 1996. Pejovic T et al. Genes Chromosomes & Cancer 4: 58, 1992. Schultz DC et al. Cancer Research 55: 2150, 1995. Eccles DM et al. British Journal of Cancer 65: 40, 1992. Schultz DC et al. Cancer Research 56: 1997, 1996. Ehlen T et al. Oncogene 5: 219, 1990. Foulkes WD et al. British Journal of Cancer 67: 551, 1993. Viel A et al. British Journal of Cancer 66: 1030, 1992. Foulkes WD et al. British Journal of Cancer 67: 268, 1993. Yang-Feng TL et al. Int J Cancer 52: 575, 1992. Boltz EM et al. Int J Cancer 43: 428, 1989. Viel A et al. Tumori. 77: 16, 1991. Berchuk A et al. Cancer Research 50: 4087, 1990. Haldane JS et al.J. Pathol. 162: 231, 1990. Kacinski BM et al. American Journal of Pathology 137: 135, 1990. Yokayama Y et al. British Journal of Cancer 76: 977, 1977. Price FV et al. Am J Obstet Gynecol 168: 520, 1993. Wells M et al. In Ovarian Cancer 4, Chapman and Hall Medical, Chapter 3, 27, 1996. Kanuma T et al. Molecular Carcinogenesis 18: 134, 1997. King BL et al. Am J Pathol 144: 486, 1994. Debemardis D et al. J. Pharmacol Exp Therap 279: 84, 1997. Runnenbaum IB et al. Gynecologic Oncology 61: 266, 1996. Skilling JS et al. Gynecologic Oncology 60: 72, 1996. Yaginuma Y et al. Cancer Research 52: 4196, 1992. Havrilesky LJ et al. Obstetrics and Gynaecology 85: 1007, 1995. Shih YC et al. Int J Cancer 70: 508, 1997. Schuyer M et al. British Journal of Cancer 74: 1069, 1996. Cheng JQ et al. Proc Nat Acad Sci. (USA) 89: 9267, 1992. Krupitza G et al. British Journal of Cancer 72: 35, 1995. Suzuki M et al. Oncology 53: 99, 1996. Budde RJ et al. Cancer Biochem Biophys 14: 171, 1994. Mok SC et al. Gynecologic Oncology 57: 299, 1995. Gordon AW et al. Cancer Letters 89: 63, 1995. Leake RE et al. In Ovarian Cancer 1, Chapman and Hall Medical, Chapter 9: 69, 1990. Manetta A et al. Fertility and Sterility 63: 282, 1995. Hamand K – personal communication 1997. Langdon SP et al. British Journal of Cancer, 62,213, 1990. Rose FV et al., Oncogene 12: 999, 1996. Kanabus J et al. Cancer Research 38: 765, 1978. Irmer G et al. Cancer Research 55: 817, 1995.
Ovarian Cancer 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183. 184. 185. 186. 187. 188. 189. 190. 191. 192. 193. 194. 195. 196. 197. 198. 199. 200. 201. 202. 203. 204. 205. 206. 207. 208. 209. 210. 211. 2 12. 213. 214.
53
DiSimone N et al. Endocrinology 137: 486, 1996. Hamilton TC et al Seminars in Oncology 11: 285, 1984. Louie KG et al. CancerResearch 45: 2110, 1985. Kijima T et al. Anticancer Research 14: 799, 1994. Niimi S et al. Cancer Research 52: 328, 1992. Kikuchi Y e t al. Japanese Journal of CancerResearch 88: 213, 1997. Kikuchi Y et al. J Natl CancerInst 77: 1181, 1986. Misawa T et al. Japanese Journal of Cancer Research 86: 88, 1995. Redmond A et al. European Journal of Cancer 29A: 1078, 1993. Voeltzel T et al. Hybridoma 13: 367, 1994. Bradley G et al. Cancer Research 49: 2790, 1989. Berchuk A et al. Cancer Research 50: 4087, 1990. Wilson AP et al. British Journal ofCancer 63: 61 (P145), 1991. Bartlett JMS et al. British Journal of Cancer 65: 655, 1992. Jozan S et al. Proc. Am. Assoc. CancerResearch 32: 83, 1991. Crew AJ et al. European Journal of Cancer 28: 337, 1991. Wilson AP et al. unpublished data. Wilson AP et al. British Journal of Cancer 63: 61 (P144), 1991. Grunt TW et al. Differentiation 53: 45, 1993. Kikuchi Y et al. Cancer Research 47: 592, 1987. Nakazawa T et al.Acta Obstet Gynecol Jpn 43: 1041, 1991. Stahel RA et al. Int J Cancer 41: 218, 1988. Radford H and Wilson AP, Analytical Cell Path 11: 173, 1996. Ramakrishna V et al. Int J Cancer 73: 143, 1997. Fox H and Buckley CH. In Pathology for Gynaecologists, 2nd ed. Edward Arnold, 1991. Harrap KR et al. Annals Oncology 1: 65, 1990. O’Connor PM et al. CancerResearch 57: 4285, 1997. De Feudis et al. Br J Cancer 76: 474, 1997. Boyer JC et al. Cancer Research 55: 6063, 1995. Brown R et al. Oncogene 15: 45, 1997. Aebi S et al. Cancer Research 56: 3087, 1996. Moran E et al. Europ J Cancer 33: 652, 1997. Masanek U et al. Anticancer Drugs 8: 189, 1997. Kelland R et al. Cancer Research 52: 3857, 1992. Sharp SY et al. Br J Cancer 70: 409, 1994. Andrews PA et al. Cancer Research 45: 6250, 1985. Kiyozuka Y et al. Oncology Reports 2: 517, 1995. Kiyozuka Y et al. Acta Obst Gynaecol Jpn 44: 461, 1992. Imamura K et al. Oncology Reports 2: 17, 1995. Yoshida M et al. J Jpn Soc Clin Cytol 32: 1, 1993. Shi-Zhong B et al. Cancer 79: 1944, 1997. Ying Y et al. Gynecologic p66: 378, 1997. Dolo V et al, Oncology Research 9: 129, 1977. Becker JL et al. In Vitro Cell Dev Biol 33: 325, 1997. Yashar C et al.Am JReprod Immunol38: 431, 1997. Engblom P et al. AnticancerResearch 16: 1743, 1996. Engblom P et al. Anticancer Research 17: 1849, 1997. Jakowlew SB et al. Anticancer Research 17: 1849, 1997. Yoshida Y et al. Anticancer Research 18: 327, 1998.
This page intentionally left blank.
Chapter 18 Cervical Cancer Swee Y. Sharp and Lloyd R. Kelland Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK. Tel: 004418-722-4261; Fax: 0044-181-722-4101; E-mail: lloyd@icl:ac.uk
1.
INTRODUCTION
Cancer of the cervix is the second most common cause of cancer death amongwomen worldwide. There are approximately 13,500 new cases and 6000 deaths from the disease per annum in the United States. In the United Kingdom, the overall 5 year survival rate is 58%. Although the prognosis for early stage patients is good through combined surgery and radiotherapy, late stage disease is not particularly chemosensitive. Clinically, 95% of cervical neoplasms are derived from squamous cells and 4-5% are adenocarcinomas. Cervical cancer may hold claim to the origins of tissue culture of human tumors, since the earliest and probably most widely used cell line, HeLa, was established by George Gey and co-workers at Johns Hopkins in 1952 from a carcinoma of the cervix of a 31 year old lady, Henrietta Lacks (1). Now believed to be derived from an adenocarcinoma of the cervix, a detailed historical record of this famous cell line has been published (2). Cell lines derived from cervical cancer have been useful in understanding the tumor biology of the human papillomaviruses (HPV) (3) and the corresponding role of these viruses in the inactivation of the tumor suppressor gene p53. Today, there are approximately 30 commonly used continuous cell lines representative of the disease, including several clonal sublines of HeLa.
2.
ESTABLISHMENT OF CELL LINES
Continuous cell lines of cervical carcinoma grow as monolayers with an epithelioid morphology. Cell lines can be established using protocols for the
J.R.W Masters and B. Palsson (eds.),Human Cell Culture Vol. II, 55- 70. © 1999 Kluwer Academic Publishers. Printed in Great Britain.
56
Sharp and Kelland
cultivation of epidermal keratinocytes and other human squamous carcinomas (4). This involves fine chopping and enzymatic disaggregation of tumor biopsies using a cocktail of enzymes (collagenase 0.2 mglml, pronase 0.5 mg/ml, DNaseI 0.2 mg/ml; 37°C 10 mins) followed by culture in a 5% CO2 atmosphere in Dulbecco’s Modified Eagle’s medium or Hams F12 supplemented with 20% fetal bovine serum plus the following growth factors: hydrocortisone at 0.4 µg/ml, insulin 5 µg/ml and transferrin 5 µg/ml. In addition, many primary cultures retain dependence on fibroblast feeder layer support and are unable to grow in semisolid (agar) medium. The feeders usually are a lethally irradiated (200 Gy) layer of the Swiss mouse embryonic fibroblast line 3T3 added at 2 × 105cells/25 cm2 flask. Control of fibroblast overgrowth is critical in the early stages of primary culture. Such control may be achieved by a combination of careful removal of underlying connective tissue from initial biopsies and selective removal by detachment using a 30 second incubation of the primary culture with 0.02% EDTA or physical detachment using a rubber policeman. Typically, after 15 to 20 passages, cells exhibit less stringent growth requirements and may be maintained in medium containing 10% fetal bovine serum plus hydrocortisone and insulin but without feeder layer support. Colony forming efficiencies generally remain low in soft agar (<1%), relative to monolayer cultures on plastic in the presence or absence of feeder layer support (5-50%) (5). In addition to the cell lines described in Table 1, four cell lines have been described with no detailed information: CC801 (42), CAC-1 (43), JSK-1 (43) and MS-757 (44). There are also various HeLa sublines (e.g. HeLa S3 (1,23)). No contamination of cervical cancer cell lines with HeLa cells has been reported. Clinically, 95% of cervical cancer is derived from squamous cell cancers with only 4-5% adenocarcinomas. The continuous cell lines reflect this, with the majority (HeLa is an exception) being of classical epithelial morphology. Since cervical cancer does not often metastasize, the majority of the lines were established from primary cervical biopsies. The lines are derived from patients of a wide age-range (25 for HX171 to 66 for C-33 and Me-180). Some of the more recently described lines (e.g. HX151, HX156 and HX171) were derived from women of less than 35 years of age.
3.
HISTOPATHOLOGY
The majority of the cell lines reported in Table 2 are able to form xenografts in nude or SCID mice. The morphological appearance of the xenografted tumors appeared to be indistinguishable in most cases from that of the original patient tumor biopsy (e.g. (5) for the HX lines), with the exception of the W12 cells (40) and SFCC cells (31). In addition to xenografts being derived from
Origins and establishment of cervical cancer cell lines
Cell line C-33 (I, 11) C 4 ( I ,
Patient age (years) 66 1 1 )
4 1
Path stage and grade
Primary site
Specimen site
N/k S t a g e 1 1 , grade IV N/k
Cervix C e r v i x
Cervix C e r v i x
CaSki
40
CC-1 and CC-2
N/k
N/k
Cervix
CC2/CUHK2 CC3/CUHK3 CC7T/GH CUMC-3
N/k N/k N/k 32
N/k N/k Stage IV Stage IIB
Cervix Cervix Cervix Cervix
CUMC-6
31
Stage 1B
Cervix
CX Cx106 EC-50 HeLa
48 N/k 57 31
Stage IIA N/k Stage IIIA Cervix
Cervix Cervix Cervix Cervix
HKMUS HKUS HOG-1
50 70 34
Stage IB N/k N/k
Cervix Cervix Cervix
HOKUG
53
Stage IIA
Cervix
Cervix
Authentication
Availability
Primary reference
57
ATCC (6) N/k M a r k e r c h r o m o s o m e s A T C C a n d ( 8 ) ECACC present, G6PD type B (7) Small bowel Using isoenzyme analysis, ATCC (9) metastasis G6PD type B Cervix Expression of involucrin in Laimins (Northwestern (10,11) the upper half of the University, Chicago, organotypic culture USA) Cervix N/k (12) (12) Cervix N/k (13) (13) Cervix N/k N/k (14) Cervix Using isoenzyme analyses, (15) (15) G6PD type B Cervix Using HLA typing, G6PD (16) (16) type B Cervix Using CEA levels (17) (17) Cervix N/k N/k (18) Cervix N/k (19) (19) G6PD type A (7) Cervix ATCC, ECACC and (1,2) DSMZ Cervix Using CEA levels N/k (20) Cervix By CEA production N/k (21) Cervix By immunostaining for keratin (22) (22) and vimentin intermediate filaments Using CEA levels N/k (20) Cervix Continued on next page
Cervical Cancer
Table1
58
Table1 (continued) Patient age (years)
Path stage and grade
Primary site
Specimen site
Authentication
Availability
Primary reference
HT-3
58
N/k
Cervix
(23)
30
Stage IB
Cervix
(5)
(5)
HX155
44
Stage IB
Cervix
Cervix
(5)
(5)
HX156
31
Stage IIB
Cervix
Cervix
(5)
(5)
HX160
63
Stage IIB
Cervix
Cervix
(5)
(5)
HX171
25
Stage IIB
Cervix
Cervix
(5)
(24)
Me-180 MS751
66 47
N/k N/k
Cervix Cervix
Omentum Lymph node metastasis
Using isoenzyme analysis, G6PD type B By immunostaining for keratin and vimentin intermediate filaments By immunostaining for keratin and vimentin intermediate filaments By immunostaining for keratin and vimentin intermediate filaments By immunostaining for keratin and vimentin intermediate filaments By immunostaining for keratin and vimentin intermediate filaments N/k Using isoenzyme analysis, G6PD type B
ATCC
HX151
Lymph node metastasis Cervix
ATCC ATCC
N/k
Cervix
Cervix
N/k
N/k
(25) N/k, derived by J. Sykes 1974 (ATCC) (26,27)
Stage II
Cervix
Metastatic lesion Nk
(28)
N/k NHIK3016, 3017,3025,3043 OMC-1 52
(28) Continued on next page
Sharp and Kelland
Cell line
Patient age (years)
Path stage and grade
Primary site
Specimen site
Authentication
Availability
Primary reference
OMC-4
47
Stage IB
Cervix
Cervix
(29)
(29)
QG-U SFCC SiHa SKG-I SKG-II (a, b) SKG-III (a, b)
N/k 39 55 N/k 50 38
N/k Stage IB Grade II N/k N/k Stage II
Cervix Cervix Cervix Cervix Cervix Cervix
Cervix Cervix Cervix N/k Cervix Cervix
(30) (31) ATCC N/k (34) (35)
(30) (31) (32) (33) (34) (35)
SW756 TC-140 TC-146 (a, b) TC-S
N/k 39 36 78
N/k N/k N/k Stage IIB
Cervix Cervix Cervix Cervix
N/k (37) (37) (38)
(36) (37) (37) (38)
TMCC-1
N/k
N/k
Cervix
N/k Cervix Cervix Metastatic inguinal lymph node Cervix
Using tumor marker production (CA125, CA19-9) N/k N/k N/k N/k Marker chromosomes present Using isoenzyme analysis, G6PD type B N/k N/k N/k Using immunohistochemistry to determine CEA production N/k
(39)
W12
22
Cervix
Cervix
N/k
XH1 (a, b)
32
Low grade cervical lesion (CIN 1) N/k
Dr M Sakamoto (Tokyo Medical College, Japan) (40)
Cervix
Cervix
Using two minisatellite hypervariable DNA probes, MS1 and MS31
(41)
(41)
Cerival Cancer
Table 1 (continued) Cell line
(40)
N/k = not known; FCS = fetal calf serum; FBS = fetal bovine serum; ATCC = American Type Culture Collection; ECACC = European Collection of Animal Cell Cultures; CEA = carcinoembryonic antigen; G6PD = glucose 6-phosphate dehydrogenase. Sublines are indicated in parentheses.
59
Cell line
Tumor pathology
C-33 (I, 11) Poorly differentiated carcinoma of the cervix C4 (I, 11) Invasive squamous carcinoma of the cervix CaSki Epidermoid carcinoma of the cervix CC-1 and CC-2 Invasive squamous cell carcinoma, undifferentiated ' CC2/CUHK2 Moderately differentiated squamous cell carcinoma CC3/CUHK3
Invasive squamous cell carcinoma
CC7T/VGH CUMC-3
N/k Invasive large cell nonkeratinizing squamous cell carcinoma. Poorly differentiated Invasive large cell nonkeratinizing squamous cell carcinoma. Moderately well differentiated
CUMC-6
In vitro features of cell line Undifferentiated epithelial cells Pleomorphic, highly stratified, epithelial sheets Epithelial N/k Doubling time of 72hr, 2.2% plating efficiency, epithelial Doubling time of 42hr, 8% plating efficiency, epithelial Doubling time of 34hr, epithelial Doubling time of 48hr, 28% plating efficiency, epithelial Doubling time of 36hr, 32% plating efficiency, epithelial
Squamous cell carcinoma N/k Moderately well differentiated, large cell type, epidermoid carcinoma
Doubling time of 20hr, epithelial N/k Doubling time of 16hr, epithelial
HeLa
Adenocarcinoma of the cervix, resistant to radiation
HKMUS
Large cell nonkeratinizing squamous cell carcinoma
Doubling time of 24hr, 45% plating efficiency, epithelial Doubling time of 35hr, 63% plating efficiency, epithelial
Undifferentiated carcinoma N/k N/k N/k N/k Forms tumors. Show prominent desmosomes and tonofilaments Tumorigenic in nude mice Transplanted tumors closely resembled the original tumor The transplanted tumor gave rise to fast growing tumors in nude mice and was more poorly differentiated than the original tumor Not tumorigenic Forms tumors in nude mice Tumors consisted of squamous cell carcinoma and histologically resembled the original tumor biopsy material Produced tumors (45) Produced tumors similar to the original carcinoma in the subcutis of mice, but not in their abdominal cavities Continued on next page
Sharp and Kelland
CX Cx106 EC-50
Xenograft pathology
60
Table2 In vivo and in vitro properties of cervical cancer cell lines
Cell line
Tumor pathology
In vitro features of cell line
HKUS
Small cell non-keratinizing squamous cell carcinoma
Doubling time of 24hr, 72% plating efficiency, epithelial
HOG-1
Moderately differentiated squamous cell carcinoma
Epithelial
HOKUG HT-3
Glassy cell carcinoma (mixed adenosquamous carcinoma) N/k
Doubling time of 44hr, 55% plating efficiency, epithelial Poorly differentiated, epithelial
HX151
Poorly differentiated squamous cell carcinoma
Epithelial, 4-5% plating efficiency
HX155
Epithelial, 24% plating efficiency
HX171 Me-180
Moderately differentiated infiltrating keratinizing squamous cell carcinoma Poorly differentiated squamous cell carcinoma Moderately well-differentiated squamous cell carcinoma Poorly differentiated squamous cell carcinoma Invasive squamous carcinoma of the cervix
MS751
N/k
HX156 HX160
Epithelial, 32% plating efficiency Epithelial, 8% plating efficiency Epithelial, 30% plating efficiency Epithelial Epithelial
Xenograft pathology Heterotransplantation of cells into nude mice reveals a poorly differentiated adenocarcinoma. Squamous cell carcinoma was not detected histologically in any tumor A polygonal to spindle cell carcinoma developed in nude mice within 2-3 weeks of inoculation Easily transplanted into nude mice and metastasized Forms tumors in nude mice, poorly differentiated epidermoid carcinoma, grade III Poorly differentiated invasive carcinoma (46) Well differentiated keratinizing tumor (46) Poorly differentiated tumor (46) Moderately well differentiated (46) N/k Well-differentiated epidermoid carcinoma, grade I Poorly differentiated epidermoid carcinoma, grade III
61
Continued on next page
Cervical Cancer
Table 2 (continued)
62
Table 2 (continued) Cell line
Tumor pathology
NHIK3016, N/k 3017, 3025,3043 Epithelial OMC-1 Large cell non-keratinizing squamous cell carcinoma
Xenograft pathology
Doubling time for NHIK3025 is 17hr
N/k
Doubling time of 43hr, 25% plating efficiency, epithelial
After heterotransplantation into nude mice, cells grew into solid large cell non-keratinizing squamous cell carcinoma, which resembled the original tumor Tumor grew well and the histology resembled the original tumor N/k Palpable tumors developed in nude mice within 1 month, producing typical clear cell adenocarcinomas, similar to the primary tumor Poorly differentiated epidermoid carcinoma, grade III N/k 1.5-2 cm tumor developed 3 months after subcutaneous inoculation in nude mice. These tumors were epidermoid cancers and closely resembled the original tumor
OMC-4
Well differentiated endocervical adenocarcinoma
Doubling time of 63hr, 18% plating efficiency
QG-U SFCC
Cervical squamous cell carcinoma Clear cell adenocarcinoma of the uterine cervix
N/k Doubling time of 67.6hr, epithelial
SiHa
Squamous cell carcinoma of the cervix
SKG-I SKG-II (a, b)
N/k Cervical epidermoid carcinoma from an untreated patient
Epithelial, 80% plating efficiency Doubling time of 28hr, epithelial, 58% plating efficiency
SKG-III (a,b) SW756
N/k N/k
N/k N/k
Homogenous, undifferentiated, epithelial
N/k N/k
Continued on next page
Sharp and Kelland
In vitro features of cell line
Cell line TC-140
Tumor pathology Keratinizing, moderately well-differentiated squamous cell carcinoma
In vitro features of cell line Epithelial
TC-146 (a, b)
Large cell nonkeratinizing, moderately well differentiated epidermoid carcinoma of the cervix
Epithelial
TC-S
Large cell keratinizing squamous cell carcinoma
Doubling time of 29.8hr, 85.5% plating efficiency, epithelial
TMCC-1
Poorly differentiated adenocarcinoma of the endocervix Low grade squamous intra-epithelial neoplasia
N/k
Invasive focally keratinizing adenosquamous carcinoma
Epithelial
W12
XH1 (a, b)
Keratinocyte morphology with minimal stratification, doubling time of 24-30hr, 7-10% plating efficiency
Xenograft pathology Formed benign-appearing cysts in nude mice, similar in morphology to welldifferentiated areas of the primary tumor Underwent keratinization after inoculation into nude mice. Produced invasive, but not progressively growing tumors Tumors formed in nude mice showed keratinizing squamous cell carcinoma, which resembled those of the original cervical cancer N/k
63
Not tumorigenic. When grafted into the flank of nude mice they reform an epithelium with basal cell crowding and koilocytic appearance (characteristic of HPV infection) Tumors derived from XH1 and XH1a cells showed morphology similar to that of original cervical caricnoma. XH1b cells produced invasive tumors and consist of poorly differentiated squamous carcinoma
Cervical Cancer
Table 2 (continued)
64
Sharp and Kelland
cell lines, a series of xenografts derived directly from subcutaneous implantation of patient tumor biopsy has been described (46). Nine xenograft lines from 23 original biopsies were established including xenograft counterparts of the HX151, HX155, HX156 and HX160 cell lines. These serially transplantable xenografts, which broadly maintain the morphological appearance of the original patient biopsies, have proven useful in determining in vivo radiobiological and chemosensitivity. In addition, another xenograft (YIK-1) (47) has been described, which was established from a 31 year old woman with the rare cervical tumor, argyrophil small cell carcinoma of the uterine cervix (ASCC). YIK-1 maintained the characteristics of the original ASCC and contains multiple copies of HPV16 DNA.
4.
MOLECULAR GENETICS
In the past decade there has been interest in the role of human papilloma DNA viruses (HPV) in the etiology of cervical cancer. The majority of cervical carcinoma cell lines contain integrated HPV (especially types 16 and 18) DNA sequences (49,53,54). W12 cells are unique as they represent an early stage in HPV16 related cervical disease (40). Two of the HPV gene products, E6 and E7, possess transforming properties through complexes with p53 (55) and RB (56) respectively, leading to their inactivation. Interestingly, in the HPV negative cervical carcinoma cell lines (e.g. C-33A and HT-3), mutations of p53 and/or RB have been reported (48). However, one HPV negative, p53 mutation negative line (CX) has been described (17). In human cervical cancer, alterations of the c-myc gene have also been linked with HPV DNA, as observed with the CUMC-6 cell line (16). Due to widespread cross-contamination of cell lines with HeLa, especially in the 1960s, the reported chromosomal markers of HeLa are of particular importance in tumor cell biology. Four typical HeLa marker chromosomes are recognised: M1 is a rearranged long arm and centromere of chromosome 1 and the long arm of chromosome 3, M2 is a combination of short arm of chromosome 3 and long arm of chromosome 5, M3 (4-5 copies) is an isochromosome of the short arm of chromosome 5 and M4 (2 copies) consists of the long arm of chromosome 11 and an arm of chromosome 19. The majority of the cervical carcinoma cell lines have been shown to possess chromosomes distinct from those of HeLa. Although Mycoplasma contamination was reported in some cervical carcinoma cell lines (e.g HT-3, Me-180, SiHa), this contamination was subsequently eradicated. In addition to chromosome analysis, the isoenzyme analysis of the X-linked G6PD (glucose 6-phosphate dehydrogenase) has helped to distinguish HeLa cells which, being derived from a Negro patient, are of G6PD type A. The
Cell line
Karyotype
HPV status
p53 status
Ras status
pRb status
Other oncogenes/tsp/ mutator phenotype
C-33 (I, 11)
Modal chromosome number = 44-46. Hypodiploid C4-1; Modal chromosome number = 44-45. Hypodiploid C4-11; Modal chromosome number = 44. Hypodiploid Modal chromosome number = 77. Aneuploid N/k
No detectable HPV
Mutation (48)
N/k
Mutation (48)
N/k
C4-1; HPV18 C4-II; HPV18 (3)
N/k
N/k
N/k
N/k
HPV16 with 300 to 500 copies (49) CC-1; HPV18 CC-2 HPV16 HPV16 No detectable HPV
N/k
N/k
N/k
N/k
N/k
N/k
N/k
N/k
N/k N/k
N/k
N/k N/k
N/k N/k
N/k
N/k N/k
N/k
N/k
C4 (I, II)
CaSki CC-1 and cc-2 CC2/CUHK2 CC3/CUHK3 CC7T/VGH CUMC-3 CUMC-6
CX Cx106 EC-50
Modal chromosome number = 75 Modal chromosome number = 83. Hypotetraploid N/k Modal chromosome number = 78. Hypotetraploid Diploid. Structural abnormality observed was the elongation of short arm of chromosome 1 Modal chromosome number = 46 Modal chromosome number = 72. Hypotetraploid Aneuploid. Translocations, fragmentations and rearrangements among chromosomes occurred with high frequency
No detectable HPV HPV16
No mutation N/k
N/k N/k
N/k N/k
N/k No amplification of c-myc Amplification and rearrangement of c-myc N/k N/k
N/k
N/k
N/k
N/k
N/k
HPV16 (50) HPV16 HPV16 and HPV18
N/k Mutation (exons 4 to 9) Mutation (exons 5 to 9)
Cervical Cancer
Table3 Main genetic changes of cervical cancer cell lines
Continued on next page
65
Cell line
HeLa
HKMUS HKUS HOG-1 HOKUG HT-3 HX151 HX155 HX156 HX160
Modal chromosome number = 82. Aneuploid. Four marker chromosomes (see text below) Modal chromosome number = 42. Aneuploid Modal chromosome number = 45. Diploid Aneuploid Modal chromosome number = 42. Aneuploid Modal chromosome number = 65 Modal chromosome number = 71. Aneuploid Modal chromosome number = 74. Aneuploid Modal chromosome number = 75. Aneuploid Modal chromosome number = 73. Aneuploid Aneuploid Modal chromosome number = 63. Aneuploid N/k Aneuploid
MS751 NHIK3016, 3017,3025,3043 OMC-1 Aneuploid
HPV status
p53 status
Ras status
pRb status
Other oncogenes/tsp/ mutator phenotype
HPV18 (3)
N/k
N/k
N/k
N/k
HPV18 (51)
N/k
N/k
N/k
N/k
N/k N/k HPV16 (51)
N/k N/k N/k
N/k N/k
N/k N/k N/k
N/k N/k N/k
No detectable HPV HPV16
Mutation (48) N/k
N/k N/k
Mutation (48) N/k
N/k N/k
HPV16
N/k
N/k
N/k
N/k
HPV16
N/k
N/k
N/k
N/k
HPV16
N/k
N/k
N/k
N/k
N/k HPV18 (49)
N/k N/k
N/k
N/k N/k
N/k N/k
HPV18 (49) N/k
N/k N/k
N/k N/k
N/k N/k
N/k N/k
N/k
N/k
N/k
N/k
N/k Continued on next page
Sharp and Kelland
HX171 Me-180
Karyotype
66
Table 3 (continued)
Cell line
Karyotype
HPV status
OMC-4
Modal chromosome number = 51. Aneuploid N/k N/k N/k
No detectable HPV
QG-U SFCC
Modal chromosome number = 74 Modal chromosome number = 42-45. Hypodiploid SKG-III (a, b) Modal chromosome number = 42-43. Hypodiploid SW756 N/k TC-140 Modal chromosome number = variable. Aneuploid TC-146 (a, b) Modal chromosome number = variable TC-S Modal chromosome number = 100-115. Hyperploid TMCC-1 N/k SKG-1 SKG-II (a, b)
w12 (a, b)
Chromosome numbers range from 53 to 100. Aneuploid Modal chromosome number = 78. Aneuploid. Has many rearranged chromosomes
N/k with 1 to 500 copies (49) HPV18 (52) HPV18 (52) (52) HPV18 (3) (100-200 copies) N/k HPV18 in high copy number
status
Missense mutation (43) N/k N/k
status
Ras status
Other oncogenes/tsp/ mutator phenotype
N/k
N/k
N/k
N/k
N/k N/k N/k
N/k N/k N/k
N/k N/k
N/k
N/k N/k
N/k N/k
N/k
N/k
N/k
N/k
N/k N/k
N/k
N/k N/k
N/k N/k
N/k N/k
N/k
N/k N/k
N/k N/k
N/k
N/k
Normal p53 gene (43) N/k
N/k
N/k
N/k
N/k
N/k
N/k
N/k
Cervical Cancer
Table 3 (continued)
67
68
Sharp andKelland
absence of HeLa cell contamination in 169 cell lines, including the Me-180, SW756, SiHa, HT-3, C-33 and C4I has been confirmed (7). In addition, all of the HX series of cervical carcinoma cell lines possessed a G6PD type B phenotype (5).
5.
CELL LINES WITH SPECIAL FEATURES
Compared to other gynecological malignancies, cervical cancer is not particularly responsive to drugs. Cisplatin is the most active single agent, conferring objective response rates in the region of 25% (57). In accordance with the clinical observations, the HX series of 5 cervical cancer cell lines has been shown to be relatively resistant to cisplatin (and carboplatin) (58). Our recent studies with these lines suggest that their relatively high levels of metallothionein may contribute to their intrinsic resistance to cisplatin (58). In addition, two cell line models of acquired cisplatin resistance have been described: HX/155cisR (around 9-fold resistant) (5) and NHIK3025/DDP (around 11 fold-resistant) (59). The HX/155cisR line exhibited multiple mechanisms of resistance to cisplatin, including reduced cellular accumulation (2.4 fold), and elevated glutathione and metallothionein combining to reduce the amount of binding to DNA (5). In addition it is possible, as for other cisplatinresistant cells, that an enhanced removal of platinum-DNA adducts may also contribute to the resistance phenotype. Similarly, with the acquired cisplatinresistant NHIK3025/DDP line, reduced platinum accumulation accounted for at least part of the resistance. In agreement with many other acquired cisplatin resistant cell lines, the multidrug resistance (mdr1) gene (commonly overexpressed following in vitro exposure of cell lines to doxorubicin) was not expressed in the NHIK3025/DDP cells. In other studies, the Me-180 cell line was made resistant to tumor necrosis factor (TNF) and was shown to be 3.5 fold more sensitive to cisplatin (60). Cisplatin-induced cytotoxicity in the TNF resistant Me-180 cell line appears to be mediated at the level of DNA repair. There are a few reports of independent cell lines being derived from the same patient although from a single biopsy rather than obtained sequentially (i.e pre and post-treatment). These include the SKG series of lines derived in Japan where SKGII a and b and SKGIII a and b are described (34,35), as well as the C4-I and II lines (8), and the XH1 a and b lines (41).
REFERENCES 1. Gey GO et al. Cancer Res 12: 264, 1952. 2. Jones H.W et al. Obstet Gynecol/38: 945, 1971. 3. Schwarz E et al. Nature 314 : 111, 1985. 4. Rheinwald JG, Beckett MA. Cancer Res 41: 1657,1981.
Cervical Cancer 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54.
69
Kelland LR et al. Cancer Res 47: 4947, 1987. Auersperg N. J Natl Cancer Inst 32: 135, 1964. Fogh J et al. J Natl Cancer Inst 58: 209, 1977. Auersperg N, Hawryluk AP. J Natl Cancer Inst 28: 605, 1962. Pattillo RA et al. Science 196: 1456, 1977. Radar JA et al. Oncogene 5: 576,1990. Benbrook DM et al. Tissue Cell 27: 269, 1995. Ho TH et al. Anticancer Res 13: 967, 1993. Chew EC et al. Anticancer Res 10: 213, 1990. Ko J et al. Chin J Microbiol Immunol 13: 273, 1980. Kim JW et al. Gynecol Oncol 57: 47,1995. Kim JW et al. Gynecol Oncol 62 230,1996. Chou CY et al. Cancer Letters 102: 173, 1996. Kitchener HC. Br J Obstet Gynaecol 95: 182, 1988. Porter JC et al. Am J Obstet Gynecol130 487,1978. Ishiwata I et al. Int Acad Cytology Anal Quantitative Cytology Histol 12: 290, 1990. Ishiwata I et al. Acta Obstet Gynaec Jpn 40: 616, 1988. White JO et al. Int J Cancer 52: 247, 1992. Fogh J, Trempe G. In: Human tumor cells in vitro. p115: 1975. Kelland LR, Steel GC. Radiotherapy Oncol 13: 225, 1988. Sykes JA et al. J Natl Cancer Inst 45: 107, 1970. Nordbye K, Oftebro R. Exp Cell Res 58: 458, 1969. Oftebro R, Nordbye K. Exp Cell Res 58: 459, 1969. Ueda M et al. Human Cell 2: 401, 1989. Yamada T et al. Acta Obstet Gynaec Jpn 39: 859, 1987. Shirasawa H et al. J Gen Virol 70 1913, 1989. Ochiai A et al. Cancer 64: 854-859, 1989. FriedI F et al. Proc Soc Exp Biol Med 135: 543, 1970. Nozawa S et al. In: Carcinoma of the Cervix: Biology and Diagnosis. p142: 1982. Ishiwata I et al. Acta Obst Gynaec Jpn 30: 731, 1978. Nozawa S et al. Cancer Res 43: 1748, 1983. Davis JW et al. Nat Immun Cell Growth Regul 3: 203, 1983. Braun L et al. Am J Pathol 143: 832, 1993. Kawabata M. Acta Obst Gynaec Jpn 36: 2619, 1984. Zheng PS et al. Gynecol Oncol 60 245, 1996. Stanley MA et al. Int J Cancer 43: 672, 1989. Han X et al. Br J Cancer 64: 645,1991. Jinwen H, Jin G. Proc CAMS PUMC 4: 162,1989. Iwasaka T et al. Gynecol Oncol 48: 104,1993. Norskov-Lauritsen N, Ebbesen P. In Vivo 7: 481, 1993. Fogh J et al. J Natl Cancer Inst 59: 221, 1977. Kelland LR, Tonkin KS. In: Establishment and response to chemotherapy of human cervical carcinoma xenografts. eds. Winograd B et al., Springer-Verlag p57: 1988. Ichimura H et al. Jpn J Cancer Res 79: 1255, 1988. Scheffner M et al. Proc Natl Acad Sci USA 88: 5523,1991. Yee CY et al. Am J Pathol 119: 361, 1985. Choo KB et al. J Virol 63: 782, 1989. Ishiwata I et al. IntAcad Cytology Anal Quantitative Cytology Histol 13: 363, 1991. Takebe N et al. Biochem Biophys Res Comm 143: 837, 1987. Spence RP et al. Cancer Res 48: 324,1988. Tsunokawa Y et al. Int J Cancer 37: 499, 1986.
70 55. 56. 57. 58. 59. 60.
Sharp and Kelland Werness BA et al. Science 248: 76, 1990. Dyson N et al. Science 243: 934,1989. Alberts DS et al. Semin Oncol 18: 11, 1991. Mellish KJ et al. Br J Cancer 68: 240, 1993. Melvik JE et al. Cellular Pharmacology 1: 9, 1993. Nishikawa K et al. Cancer Res 52: 4758, 1992.
Chapter 19 Endometrial Cancer P.G. Satyaswaroop† Department of Obstetrics and Gynecology, The Milton S Hershey Medical Center, Pennsylvania State University, Hershey, PA 17033.
1.
INTRODUCTION
Endometrial cancer is the most common gynecologic malignancy in the Western World. In the United States it is estimated that 36,100 new cases were identified and 6300 died from this disease in 1998 (1). Endometrial cancer is predominantly a postmenopausal disease and its incidence appears to have increased in the past 50 years, mostly due to increased life expectancy and improved detection methods. Surgery and radiation therapy result in 80-85 percent cure rates in early stages. Five year survival in patients in whom tumor had spread regionally at the time of diagnosis (2) was 66%. Recurrences in grade I, stage I patients were only 4%, while those in stage I, grade III patients were 41%. Endometrial adenocarcinomas are the most frequent uterine tumors. Among these, the clear cell carcinomas, adenosquamous carcinomas and papillary serous carcinomas comprise about 15%. The five year survival in these histologic subtypes is relatively poor and ranges from 44 to 68% in stage I disease (3,4). Thus, different endometrial tumor types have distinct patterns of malignant behavior and detailed studies on the biology of these subtypes may enhance our understanding and lead to better treatment strategies. Hormonal therapy and chemotherapy are the treatments of choice for recurrent or metastatic endometrial carcinomas. Normal endometrial epithelium is a steroid-sensitive tissue: estradiol-17b stimulates and progesterone inhibits endometrial epithelial cell proliferation. Therefore, progestins have been commonly used in the treatment of metastatic endometrial cancers with about 25 percent response rates (5,6). Cytotoxic chemotherapy generally
Deceased
†
J.R.W Masters and B. Palsson (eds.), Human Cell Culture Vol. II, 71–78. © 1999 KluwerAcademic Publishers. Printed in Great Britain.
72
Satyaswaroop
resulted in about 30 percent response rates (7). Development of tests which predict the response of tumors to hormone therapy or chemotherapy will aid in patient selection for either of these treatments. Hence, many of the experimental studies have focussed on the detection of steroid receptors and the sensitivity of endometrial cancers to steroids, antisteroids and chemotherapeutic agents in vitro as a way of predicting response (8).
2.
METHODS
Development of cell lines from tumors of human endometrium has proven difficult and few cell lines have been established. This poor success rate was reviewed by Kuramoto et al. (9), who established the first endometrial carcinoma cell line (10,11). During a 22 year study with 210 cases, using mainly enzymatic digestion of tumor tissue followed by culture of isolated tumor cells in nutrient medium, they reported a success rate of 4.8%. The well differentiated endometrial adenocarcinomas were especially difficult to establish by this procedure. There are a few successful efforts at establishing cell lines from ascites and other metastatic sites and simultaneous cultures of primary and metastatic tumor lesions (Table 1). Permanent cell lines from clear cell and papillary serous endometrial carcinomas were also established and are listed in Table 1. Although the number of cell lines of different tumor types currently available is limited, they do represent a broad spectrum of human endometrial carcinomas. In general, the surgically removed tumors from the primary or metastatic sites were cut and minced into 2 to 3 mm3 pieces and cultured in plastic dishes in a nutrient medium containing 15-20 percent fetal bovine serum (FBS) in humidified, 5 percent CO2 atmosphere until confluent monolayers were observed. The monolayers, which may be comprised of epithelial as well as stromal cells, were repeatedly trypsinized. Endometrial tumor explants appear to yield more active cultures than enzymatic procedures, which yield isolated endometrial glands or single epithelial cells (12). Several of the cell lines were also established in culture following their growth in athymic mice. In these studies, the minced tumor pieces were transplanted subcutaneously in athymic mice and cells were later established in culture from solid tumors that grew during serial transplantation (13,14). Selection procedures which repeatedly monitored for progesterone receptor expression by immunocytochemistry were critical for establishing an estrogen receptor positive, estrogen responsive cell line, ECC-1, from a nude mouse grown estrogen-responsive endometrial carcinoma (14,15). The method used to develop this cell line is outlined below.
Cell Line
Origin of endometrial cancer cell lines Patient Age (years)
71 HEC-1 HEC-6 48 HEC-50 n.a. HEC-59 n.a. HEC-88nu n.a. HEC-108 n.a. HEC-116 n.a. Ishikawa HEC-151 n.a. n.a. HEC-155 n.a. END-1,2 SCRC-1 47 AN3CA n.a. RL95.2 65 KLE 68 ECC-1 68 KCC-1a,b 67 UM-EC-1 66 HHUA 54 SNG-P 52 52 SNG-M ETN-1 61 SK-UT2 49 SPEC-1;-2 57 n.a. = not available
Histopathology: grade and type
Site of Cancer
Culture method
Availability
Primary reference
G2, adenoca adenoacanthoma G3, adenoca G2, adenoca adenoacanthoma G3, adenoca G2, adenoca G1, adenoca G2, adenoca serous adenoca adenoca G1, adenoca adenoca G2, adenosquamous G2/4, adenoca G2, adenoca clear cell ca G3, adenoca G1, adenoca G2, adenoca lymph node met. G2, adenoca, adenosquamous G3, adenoca G3, papillary serous adenoca
primary primary ascites primary primary primary primary primary primary primary endomet endomet lymph node metastasis endomet endomet endomet endomet endomet endomet endomet lymph node skin met endomet endomet
plasma clot culture cells cells cells xenotransplant cells cells cells cells cells xenotransplant explant explant explant explant xenotransplant cells cells/explant cells cells cells explant explant cells
ATCC originator originator originator originator originator originator originator originator originator originator Originator ATCC, DSMZ ATCC ATCC originator originator originator originator originator originator originator originator originator
(10) (9) (9) (9) (9) (9) (9) (25) (9) (9) (13) (26) (27) (28) (18) (14) (29) (30) (31) (32) (32) (33) (34) (35)
Endomental Cancer
Table1
73
74
Satyaswaroop
EnCa-101 (Transplantable, human endometrial carcinoma, estrogen (ER) and progesterone receptor (PR) positive) ↓ collagenase digestion Glandular units ↓ cultured in 10% FBS medium + insulin, cholera toxin, transferrin, estrogen (E2) Epithelial monolayer (101 AE7) ↓ Cytokeratin+ve, ER +ve, PR -ve, E2 nonresponsive for 25 passages for 1 year 101 AE7 (passage 26) ↓ 5% cells intensely positive for PR by immunocytochemistry, ↓ cloned twice and selected for PR positivity ECC-1 cell line in continuous culture (ER positive,E2 responsive) It is worth observing that the in vivo xenotransplantation system consistently resulted in the maintenance of differentiated characteristics and the hormonal sensitivity of the original endometrial tumor. The differentiated EnCa-101 tumor which was maintained by serial transplantation predictably responded to administered estrogen with increased growth and PR expression for several years (16).
3.
BIOCHEMICAL CHARACTERISTICS
Early studies of established continuous tumor cell lines focussed on morphologic characteristics, such as polygonal or cobblestone growth pattern of cells by light microscopy, the presence of desmosomes and microvilli by electron microscopy, and epithelial markers such as the expression of cytokeratin. It soon became evident that the endometrial cells in culture were either devoid of sex steroid receptors or expressed aberrant receptors, and that they were not responsive to steroids added in vitro (17,18). It is worth remembering that endometrium is remarkably sensitive to estrogen and exhibits dramatic increase in growth following exposure to estradiol in vivo. However, there are very few endometrial cells in culture which contain steroid hormone receptors and which respond to added steroids in vitro, even when the cells were derived from tumors that were originally positive for steroid receptors. This deficit is presumably due to the poor stability of steroid receptors or the selective outgrowth of receptor-negative cells under culture conditions. We previously reported on the dramatic decrease in progesterone receptors in human endometrial carcinomas compared to normal endometrial tissue in vitro and suggested that this lability could account for the absence of steroid responsiveness of cultured endometrial tumor tissues (19).
Endometrial Cancer
75
Tumor heterogeneity and differential expression of steroid receptors in endometrial carcinomas are well recognized (20,21) and selective growth of receptor-negative cells among this heterogeneous cell population remains a distinct possibility. It is of note that among the recently established endometrial tumor cell lines with steroid receptors and response to steroid hormones only one cell line, Ishikawa, was established directly in monolayer cultures following enzymic digestion of tumor tissue (25). The other two cell lines, ECC-1 and HEC-88nu, were derived from athymic mouse grown tumors whose receptors and hormonal sensitivity were maintained during xenotransplantation (9,22). While the presence of steroid hormone receptors or responsiveness to steroids could rarely be demonstrated in established cells or upon growth of these cells as solid tumors in athymic mice, many of these cells recapitulated the histologic characteristics of the original endometrial tumor from which the cell lines were established (Table 2). The patterns of histologic differentiation in two human endometrial adenocarcinoma cell lines _ ECC-1 (differentiated) and AN3CA (poorly differentiated) – were compared in monolayer culture, three dimensional culture in matrigel as well as growth as xenografts in athymic mice (23). The poorly differentiated carcinoma cell line AN3CA failed to form glands in matrigel or in athymic mice while the differentiated adenocarcinoma cell line ECC-1 showed the re-emergence of the original architectural differentiation and functional characteristics.
4.
MOLECULAR GENETICS
While cytogenetic features are routinely examined in established human endometrial carcinoma cell lines, there are few studies on oncogenes and tumor suppressor genes. While ras, p53 and PTEN expression and mutations in these genes were examined in primary and metastatic tumors of different histologic grade and stage, few studies have examined the alterations in oncogenes in established endometrial cancer cell lines (24,36). These are listed in Table 3. One of these studies concluded that amplification or overexpression of several oncogenes frequently seen in other human gynecologic and breast malignancies are not prevalent in endometrial carcinoma. The ras oncogene mutations appear to be relatively common.
5.
CELL LINES WITH SPECIAL FEATURES
Among the established human endometrial carcinoma cell lines, the steroid hormone receptor positive, hormonally responsive cell lines – Ishikawa and ECC-1, have been widely used. The HEC-1 cell line with defective estrogen
76 Table 2
Satyaswaroop Histopathologic characteristics
Cell Line
Doubling time (hr)
Tumor pathology
Growth in nude mice
Xenograft pathology
HEC-1
31
G2, adenoca
+
HEC-6 HEC-50 HEC-59 HEC-88nu HEC-108 HEC- 116 Ishikawa HEC-151 HEC-155 END-1,2 SCRC-1 AN3CA
56 30 25 48 26 27 28 38 n.a. n.a. 42
mod differentiated adenoca adenoca adenoca adenoca adenoca adenoca adenoca well differentiated adenoca – adenoca well differentiated poorly differentiated
RL95.2 KLE ECC-1 KCC-1a,b UM-EC-1 HHUA SNG-P SNG-M ETN-1
22-34 114 75; +E2 40h 87 24 34 33 30 72
SK-UT2 SPEC-1,2
48 48
adenoacanthoma G3, adenoca G2, adenoca adenoacanthoma G3, adenoca G2, adenoca G1, adenoca G2, adenoca serous adenoca ad en o c a G1, adenoca primary endo. ca cells from met G2, adenosquamous G3, adenoca G2, adenoca clear cell ca G3, adenoca G1, adenoca G2, adenoca G2, adenoca endometrial adenoca cells from skin met G3, adenoca G3, papillary serous
+ + + + + + + + n.d. + + + n.d. + + + + + n.d. n.d. + + +
– poorly differentiated mod differentiated clear cell adenoca poorly differentiated adenoca n.d. n.d. well differentiated adenoca poorly differentiated poorly differentiated
n.a. = not available; n.d. = not done
receptor has also been extensively used in the study of growth factors, their receptors and their mediation in endometrial growth. The recently established cell lines of different human endometrial carcinoma subtypes essentially cover the entire clinical spectrum of the disease.
ACKNOWLEDGMENT Some of the studies listed here were supported by the National Cancer Institute grant CA-62211.
HEC-1 HEC-6 HEC-50 HEC-59 HEC-88nu
47,1 marker 46, pseudodiploid, no markers 56 47 3n
HEC-108 HEC-116 Ishikawa HEC-151 HEC-155 END-1,2 SCRC-1 AN3CA RL.95.2 KLE ECC-1
48 41 46, pseudodiploid 3n hyperdiploid 46, normal female 46, N1, female, G&Q banding
KCC-1a,b UM-EC-1
la-33-79: 1b-64-82 43; XX; monosomy 4, 19, other deletions, rearrangements wide variation, aneuploid 46, marker inconsistent 46, marker inconsistent 61 (39-107); 7 markers 104-124, several del & rearrange 80-120, aneuploid
HHUA SNG-P SNG-M ETN-1 SK-UT2 SPEC-1
47, XX+8 51-56,6-8 markers by G band
Ki-ras mutation; Ki/12 no ras mutations no ras mutations Ha-ras mutation; Ha/61 no ras mutations
K-ras, N-myc, c-myc amplified Ki-ras mutation; Ki/12 no ras mutations
Unique features ER-nonfunctional ER, PR +ve, E-responsive ER, PR +ve, steroid responsive serous adeno ca ER +ve ER defective cloned, selected for PR expression E, responsive E,, E, producing, clear cell Ca -
Endometrial Cancer
Table3 Cytogenic characterization, oncogene expression and unique features Cell Line Chromosome Number/Karyotype Oncogene expression
ER, PR +ve primary and met from same pt. i.p. produces ascites and distant metastasis i.p. inj - forms ascites papillary serous adenoca
77
78
Satyaswaroop
REFERENCES 1. Cancer Facts and Figures, 1997. American Cancer Society World Wide Web, 1998. 2. DiSaia PJ, Creaseman WT. In Clinical Gynecologic Oncology. Third Edition. CV. Mosby Co. Washington DC. pp 161-197,1989. 3. Christopherson WN, Alberhansy RC, Connelly PJ. Am J Obstet Gynecol 77: 534, 1982. 4. Christopherson WN, Alberhansy RC, Connelly PJ. Cancer 49: 1511, 1982. 5. Kelley RM, Baker WH. N Engl J Med 264: 216, 1960. 6. Satyaswaroop PG, Podczaski ES, Mortel R. In Gynecological Malignancies, Principles and Practice of Gynecologic Oncology. Eds, Hoskins WJ et al. J.B. Lippincott, Philadelphia. pp 151-166,1992. 7. Deppe E. In Chemotherapy Of Gynecologic Cancer, Alan Liss, New York, 1984. 8. Hung YC, Tabibzadeh S, Satyaswaroop PG. In Atlas of Human Tumor Cell lines, Eds. RJ Hay et al., Academic Press, New York, pp 359-386, 1994. 9. Kuramoto H, Nishida M, Morisawa T, Hamano M, Hata H, Kao Y, Ohno E, Iida T.Ann NY Acad Sci 622: 402,1991 10. Kuramoto H, Tamura S, Notake Y. Am J Obstet Gynecol 114: 1012, 1972. 11. Kuramoto H.Acta Obstet Gynaec Jap 19: 47, 1972. 12. Satyaswaroop PG, Fleming H, Bressler RS, Gurpide E. Cancer Res 38: 4367, 1978. 13. Merenda C, Sordat B, Mach JP, Carrel S. Int J Cancer 16: 559,1975. 14. Satyaswaroop PG, Sivarajah A, Zaino RJ, Mortel R. Prog. Cancer Res Therapy 35: 430, 1988. 15. Tabibzadeh S, Kaffka KL, Kilian PL, Satyaswaroop PG. In Vitro 26: 1173,1991. 16. Satyaswaroop PG, Mortel R. J Steroid Biochem Mol Biol 37: 997,1990. 17. Fleming H, Blumenthal R, Gurpide E. J Steroid Biochem 20: 5, 1984 18. Richardson GS, Dickersin GR, Atkins L, MacLaughlin DT, Raam S, Merk LP, Bradley FM. Gynecol Oncol 17: 213, 1984 19. Satyaswaroop PG, Mortel R. Cancer Res 42: 1322,1982. 20. Mortel R, Zaino RJ, Satyaswaroop PG. Cancer 53: 113,1984. 21. Zaino RJ, Clarke CL, Mortel R, Satyaswaroop PG. Cancer Res 48: 1889, 1988. 22. Clarke CL, Satyaswaroop PG. Cancer Res 45: 5417,1985. 23. Satyaswaroop PG, Tabibzadeh SS. Cancer Res 51: 5661,1991. 24. Boyd J, Risinger JI. Mol Carcinogenesis 4: 189, 1991. 25. Nishida M, Kasahara K, Kaneko M, Iwasaki H. Acta Obstet Gynaec Jap 37: 1103,1985. 26. Gorodecki J, Mortel R, Ward SP et al. Am J Obstet Gynecol 135: 671, 1979. 27. Dawe CJ, Banfield WG, Morgan WD et al. J Natl Cancer Inst 33: 441,1964. 28. Way DL, Grosso DS, Davis JR et al. In Vitro 19: 147, 1983. 29. Katoaka A, Kojiro M, Yakashiji M, Abe H. Cancer 67: 1588, 1991. 30. Grenman SE, van Dyke DL, Worsham MJ, Rosario F, Roberts JA, McClatchey KD, Schwartz DR, Babu VR, Carey TE. Cancer Res 48: 1864, 1988. 31. Ishiwata I, Ishiwata C, Soma M, Arai J, Ishikawa H. Gynecol. Oncol 17: 281, 1984. 32. Ishiwata I, Nozawa S, Inoue T, Okumura H. Cancer Res 37: 1777, 1977. 33. Kruitwagen RFPM, Poels LG, Willernsen WNP, Croes HJE, Smeets DFCM, Beck JLM, Hanselaar AGJM, Ramaekers FCS, Roilland R. Int J Cancer 43: 1098, 1989. 34. Rubin SC, Federich MG, Lloyd KO, Lewis JL, Hoskins WJ. Gynecol Oncol 45: 273,1992. 35. Boyd JA, Siegal GP, Kaufman DG. Gynecol Oncol 33: 301,1989.
Chapter 20 Breast Cancer Robert L. Sutherland, Colin K.W. Watts, Christine S.L. Lee and Elizabeth A. Musgrove Cancer Research Program, Garvan Institute of Medical Research, St Vincent’s Hospital, Darlinghurst, Sydney, NSW 2010, Australia. Tel: 0061-2-9295-8322; Fax: 0061 -2-9295-8321; E-mail:
[email protected]
1.
INTRODUCTION
Breast cancer is second only to lung cancer as a cause of cancer deaths in women in industrialised societies. Major advances have been made in the last decade, a significant proportion of which have emanated from studies employing human breast cancer cell lines as experimental models. Propagation of human breast carcinoma cells in long term culture proved initially to be particularly difficult, even at a time when significant advances were forthcoming from cancers at other sites. Although some earlier attempts are documented, the first stable, continuous culture of a human breast cancer cell line, BT-20, was not achieved until 1958 (Lasfargues and Ozzello, 1958). This achievement was followed in 1963 by the establishment of the CaMa line by Dobrynin (Dobrynin, 1963); the only success from 33 mammary carcinomas placed in primary culture. During the early 1970s several new lines were isolated; the first of these, HBT-3 and HBT-39, were subsequently shown to be HeLa cells (Nelson-Rees et al., 1977; Engel et al., 1978a) but whether or not this contamination arose during their isolation, or following contamination of stable mammary carcinoma cell lines is unclear. The last of these early lines to be developed was the SK-Br-3 cell line (Trempe and Fogh, 1973; Fogh and Trempe, 1975), which became a popular model in the 1990s due to amplification and overexpression of the erbB2 receptor tyrosine kinase, an important oncogene in human breast cancer. A significant development subsequently occurred when Cailleau and colleagues discovered that breast carcinoma cells from pleural effusions could
J.R. W. Masters and B. Palsson (eds.), Human Cell Culture Vol. II, 79–106. © 1999 Kluwer Academic Publishers. Printed in Great Britain.
80
Sutherland et al.
more readily adapt to in vitro culture than cells derived from explants of solid tumors. This discovery led first to the propagation of the MDA-MB-134, -175 and -231 cell lines (Cailleau et al., 1974) and subsequently to several others. Interestingly, these workers had previously had no success in establishing cell lines from over 200 breast carcinomas. The success with pleural effusions highlighted the limitations of establishing continuous cultures from solid tumor explants, i.e. the low yield of viable epithelial cells and overgrowth by fibroblasts. This methodological advance subsequently led to the establishment of several more cell lines. However, despite the subsequent major contributions of these cell lines to breast cancer biology, none of them demonstrated responsiveness to estrogen, a critical etiological factor in the development of breast cancer (Henderson et al., 1988; 1996). Thus, it was the establishment of the ER positive cell line, MCF-7, and the subsequent demonstration of its sensitivity to a range of different hormones and growth factors that heralded the arrival of the breast cancer cell line as the predominant tool in the study of human breast cancer biology (reviewed in Levenson and Jordan, 1997). Equally importantly, the subsequent demonstration that breast carcinoma cells express a broad spectrum of receptors for steroid and peptide hormones, growth factors and cytokines, and often retain responsiveness to their cognate ligands, has extended the use of these cell lines far beyond breast cancer. The cell lines are used to study the receptors themselves, their signalling pathways, and their control of gene expression and cellular function. Thus breast cancer cell lines have become major experimental models, not only for breast cancer research but for dissecting basic molecular mechanisms controlling diverse aspects of epithelial cell biology.
2.
CLINICAL SPECTRUM OF CONTINUOUS CELL LINES
A rapid expansion in the number of breast cancer cell lines available has resulted from a more detailed appreciation of the conditions required to establish continuous cultures of breast carcinoma cells and the priority for research on this disease. In preparing this chapter, we searched both literature and cell line databases in an attempt to generate an extensive list of the current availability of breast cancer cell lines. From this list we selected cell lines for further documentation based on the criteria that: they were continuous breast carcinoma cell lines, were of human origin, were the subject of at least two peer reviewed publications listed in the Medline database and appeared to be readily available through an established cell culture bank or from the laboratory of origin, These criteria excluded a number of important cell lines that are likely to be of considerable interest to workers in the field, e.g. primary
Breast Cancer
81
cultures of normal breast epithelial cells, normal cells transformed by chemical carcinogens or viral and cellular oncogenes, and potentially interesting cell lines in the early stages of their characterization. The most important of these exclusions are the Human Mammary Epithelial Cultures (HMEC) derived by Dr Martha Stampfer and colleagues which include transformed and tumorigenic cell lines derived from the original primary cultures (Stampfer et al., 1980; 1992; Hammond et al., 1984), see also www.lbl.gov/~mrgs/; a series of human luminal epithelial cell lines transformed by SV40 virus (Bartek et al., 1991); lines of the 21NT series (21MT-1,-2,21NT, 21PT) (Band et al., 1989a, 1990); cultures of the NBL series consisting of primary epithelial cells and mixed cultures of stromal and epithelial cells that are available from ATCC; and the SUM series of 11 cell lines available through the University of Michigan Human Breast Cell Bank that were isolated under, and have been continually maintained in, growth factor defined conditions (Ethier et al., 1993, 1996); see also www.cancer.med.umich.edu/breast_cell/umbnkdb.htm. More recently an important series of paired tumor and non-tumor cell lines have been established from breast cancer patients (Gazdar et al., 1998, Wistuba et al., 1998), and these are described in the following chapter. Table 1 provides a summary of selected features of the most commonly studied breast cancer cell lines currently available. An earlier review (Engel et al., 1978a) also covers additional properties of many of the cell lines summarised here. Table 2 lists the primary references for cell lines that have been reported in the literature but were excluded from further documentation according to the criteria outlined above. Although the exact sequence of events in the development of breast cancer has yet to be established, a histological model of progression from normal breast epithelium through proliferative disease without atypia, atypical ductal hyperplasia, ductal carcinoma in situ to invasive cancer has been proposed as a model of the phenotypic and genetic evolution of the disease (Dupont et al., 1985; O’Connell et al., 1994). Continuous cell lines representative of each of these histological stages would be invaluable models for studying these genetic and molecular changes. While some of the normal breast epithelial cell lines transformed with chemical carcinogens, viral oncogenes or individual cellular oncogenes may provide reasonable models of early cellular transformation, there are very few cell lines that have been established from premalignant lesions. The HBL-100 line was initially derived from normal mammary epithelial cells isolated from milk (Gaffney, 1982) but was subsequently shown to contain SV40 sequences (Caron de Fromentel et al., 1985) which presumably resulted in its transformation and immortalization. The more recently isolated MCF10A and MCF-10F cell lines (Soule et al., 1990) were derived from a patient with fibrocystic breast disease. These cells express several breast epithelial cell antigens, display characteristics of luminal ductal cells, have a normal or near
Patient Age
Histopathological Tumor Diagnosis' Stage
Primary Specimen Site Site
Culture Method2
Authentication3
Availability
Primary Reference
BT-20 BT-474 BT-483 BT-549 CaMa EFM-19
74 60 25 72 28 50
IDC IDC IDC IDC MC IDC
Breast Breast Breast Breast Breast Breast
E E E E E D
H, I I I I I I
ATCC ATCC, DSMZ ATCC ATCC Original source DSMZ
(Lasfargues et al., 1958) (Lasfargues et al., 1978) (Lasfargues et al., 1978) (Dobrynin, 1963) (Simon et al., 1984)
E D D E D
I I I I H, I
ATCC ATCC Original source ATCC ATCC, DSMZ
(Langlois et al., 1979) (Gaffney, 1982) (Matano et al., 1991) (Hackett et al., 1977) (Soule et al., 1973)
Breast
Breast Breast Breast Breast Breast Pleural effusion Skin Breastmilk Ascites Breast Pleural effusion Breast
DU4475 HBL-1004 HMA-1 Hs 578.T MCF-7
62 27 48 74 69
IDC Benign PAC IDC IDC
MCF-10A
36
FCBD
E
I
ATCC
Breast
Breast
E
I
ATCC
IV
Breast
D
I
ATCC
MC
IV
Breast
D
I
ATCC
(Young et al., 1974)
56
IDC
IV
Breast
D
I
ATCC
(Cailleau et al., 1974)
MDA-MB-231
51
IDC
IV
Breast
D
I
ATCC
(Cailleau et al., 1974)
MDA-MB-330
43
ILC
IV
Breast
Pleural effusion Pleural effusion Pleural effusion Pleural effusion Pleural effusion
(Soule et al., 1990; Tait et al., 1990) (Soule et al., 1990; Tait et al., 1990) (Cailleau et al., 1974)
MCF-10F
36
FCBD
MDA-MB-134-V1
47
IDC
MDA-MB-157
44
MDA-MB-175-VII
D
I
ATCC
(Cailleau et al., 1978)
III II II IV IV IV IV
Breast Breast Breast Breast Breast
Continued on next page
Sutherland et al
Cell line
82
Table 1 Origin, Pathology and Source of Commonly Used Human Breast Cancer Cell Lines
Cell line
Patient Age
Histopathological Diagnosis'
Tumor Stage
Primary Specimen Site Site
MDA-MB-361 MDA-MB-415
40 38
AC IAC
IV IV
Breast Breast
MDA-MB-435S
31
IDC
IV
Breast
MDA-MB-436
43
IDC
IV
Breast
MDA-MB-453
48
AC
IV
Breast
MDA-MB -468
51
AC
IV
Breast
MFM-223
> 50
IDC
IV
Breast
PMC-42
68
PAC
IV
Breast
SK-Br3
43
AC
IV
Breast
T-47D
54
IDC
IV
Breast
UACC-812 UACC-893 ZR-75-1 ZR-75-27
42 57 63 47
IDC IDC IDC IDC
IV II IV IV
Breast Breast Breast Breast
ZR-75-30
47
IDC
IV
Breast
Brain Pleural effusion Pleural effusion Pleural effusion Pleural effusion Pleural effusion Pleural effusion Pleural effusion Pleural effusion Pleural effusion Breast Breast Ascites Pleural effusion Ascites
Culture Method2
Authentication3
Availability
Primary Reference
D D
I I
ATCC ATCC
(Cailleau et al., 1978) (Cailleau et al., 1978)
D
I
ATCC
D
I
ATCC
D
I
D
I
ATCC, DSMZ ATCC
D
I
(Cailleau et al., 1978; Siciliano et al., 1979) (Cailleau et al., 1978; Siciliano et al., 1979) (Cailleau et al., 1978; Siciliano et al., 1979) (Cailleau et al., 1978; Siciliano et al., 1979) (Hackenberg et al., 1991)
D D
I
D
I
E E D D
I I
D
I
Breast Cancer
Table 1 (continued)
Original source, DSMZ Original (Whitehead et al., 1983) source ATCC (Trempe and Fogh, 1973; Fogh and Trempe, 1975) ATCC (Keydar et al., 1979) ATCC ATCC ATCC Original source ATCC
(Meltzer et al., 1991) (Meltzer et al., 1991) (Engel et al., 1978b) (Engel et al., 1978b) (Engel et al., 1978b)
IDC, infiltrating ductal carcinoma; MC, medullary carcinoma; PAC, papillary adenocarcinoma; FCBD, fibrocystic breast disease; ILC, infiltrating lobular carcinoma; AC, adenocarcinoma; IAC, invasive adenocarcinoma. 2E, explant; D, dissociated tissue. 3H, HLA typing; I, isozyme analysis. 4Epithelial cells from breast milk (Gaffney, 1982) subsequently shown to contain SV40-derived sequences (Caron de Fromentel et al.,1985). 1
83
Sutherland et al.
84 Table 2 Less Commonly used Human Breast Cancer Cell Lines Cell Line
Reference
8701-BC BSMZ Ca2-83 CALc18 CAL51 CAL85 -1 CAL148 EFF-3 HMT-3909 Ia-270 IBEP-1, -2, -3 KPL-1 WL3C 21NT 21PT 21MT-1,-2 MA-11 MAST M25-SF OCUB-1 SUM-44PE SUM-52PE SUM-102PT SUM-149PT SUM-1315MO2 SUM-159PT SUM- 185PE SUM-190PT SUM-206CWN SUM-225CWN SUM-229PE SW613-S UISO-BC-1, -2 YMB-1
(Minafra et al., 1989) (Watanabe et al., 1992) (Rudland et al., 1985) (Gioanni et al., 1985) (Gioanni et al., 1990) (Gioanni et al., 1993) (Gioanni et al, 1993) (Healicon et al., 1993) (Petersen et al., 1990) (Loh et al., 1985) (Siwek et al., 1998) (Kurebayashi et al., 1995) (Kurebayashi et al., 1996) (Band et al., 1990) (Band et al., 1990) (Band et al., 1989b, 1990) (Rye et al., 1996) (Zoli et al., 1996) (Katano et al., 1994) (Sawada et al., 1994) (Ethier et al., 1993) (Ethier et al., 1996) (Sartor et al., 1997) www.cancer.med.umich.edu/breast_cell/umbnkdb.htm www.cancer.med.umich.edu/breast_cell/umbnkdb.htm www.cancer.med.umich.edu/breast_cell/umbnkdb.htm www.cancer.med.umich.edu/breast_cellh/umbnkdb.htm www.cancer.med.umich.edu/breast_cellhmbnkdb. htm www.cancer.med.umich.edu/breast_cellhmbnkdb. htm www.cancer.med.umich.edu/breast_cellhmbnkdb.htm www.cancer.med.umich.edu/breast_cellhmbnkdb. htm (Modjtahedi et al., 1985) (Sasi et al., 1991) (Yamane et al., 1984)
normal karyotype and consequently are probably representative of ductal hyperplasia in vivo. The majority of the other cell lines listed in Table 1 were derived from invasive cancers and, since many were derived from pleural effusions, are representative of late stage metastatic disease. In agreement with the frequency of diagnosis the majority of available cell lines are derived from infiltrating ductal carcinoma. Other pathologies are generally poorly represented although the CaMa and MDA-MB-157 lines are derived from medullary carcinoma and HMA-1 from a papillary carcinoma. Both these pathologies
Breast Cancer
85
represent rare forms of infiltrating ductal carcinoma. Importantly, there is only one cell line, MDA-MB-330, derived from a lobular carcinoma, although this pathology constitutes about 20% of newly diagnosed breast cancers. Despite these potential limitations, ductal carcinoma presents as a wide spectrum of biological phenotypes reflecting different degrees of differentiation and malignancy which are correlated with the subsequent course of the disease. Thus several of the cell lines retain a number of important features of differentiated mammary epithelium including: morphological characteristics, expression of estrogen receptor (ER) and progesterone receptor (PR), and markers of differentiated function (e.g. expression of milkfat globule antigen and milk proteins). Of these, MCF-7 cells are by far the most studied and some of the more important findings have been reviewed recently (Levenson et al., 1997). This cell line shares with a number of others (e.g. T-47D, ZR-75-1, MDA-MB-361 and BT-474) a complete dependence on estrogen for tumor growth in athymic nude mice, thus reflecting the properties of the hormonedependent phenotype in vivo. The corollary is also apparent. A number of the well studied cell lines are autonomous, requiring only low concentrations of calf serum and no added hormones for their continuous passage in culture or for tumor growth in nude mice. These cell lines, of which MDA-MB-231, BT20, SK-Br-3 and MDA-MB-435 are probably the most studied, produce relatively high levels of autocrine growth factors, tend to overexpress growth factor receptors of the erbB family, lack responsiveness to steroid hormones in vitro and in vivo and under some circumstances form metastases in nude mice (reviewed in Diekson and Lippman, 1987, 1995). These properties are shared by the hormone unresponsive phenotype of primary breast cancer, where the absence of steroid hormone receptors in the primary tumor is associated with resistance to all forms of endocrine therapy. Thus, the currently available cell lines provide a range of biological properties that cover the majority of phenotypes typical of the spectrum of invasive ductal carcinomas currently diagnosed. As noted above there is inadequate representation from cell types associated with the earliest stages of disease development, with carcinoma in situ and some of the less commonly diagnosed pathological types, particularly invasive lobular carcinoma, which may well be associated with quite distinct molecular lesions.
3.
HISTOPATHOLOGY OF BREAST CANCER XENOGRAFTS
In marked contrast to several other tumor types, where continuously passaged xenograft lines derived directly from primary cancers have provided the bulk of new knowledge, the most widely studied xenografts in breast cancer have been those derived from established human breast cancer cell lines. This
86
Sutherland et al.
difference is probably due to the fact that surgical breast carcinoma has a particularly low efficiency of viable grafts in nude mice. However, breast cancer cell line xenografts have provided valuable models for studying tumor/host interactions, including the influence of endocrinological and immunological factors, and provided important in vitro models for testing therapeutic responsiveness (reviewed in Clarke et al., 1993; Clarke, 1996). Cell lines, and indeed primary tumors, grow better when inoculated into the mammary fat pad than at other sites, presumably because the mammary stroma offers an environment conducive to growth of these epithelial cells. An additional requirement for the growth of the hormone-dependent phenotype is exogenous estrogen, normally administered as a subcutaneous pellet. Generally speaking such xenografts have little propensity to metastasize, but the MDA-MB-435 cell line will metastasize to the lymph nodes, lungs and viscera. Models of distant metastasis have been produced following intravenous or intra-arterial inoculation of cell lines (Price and Zhang, 1990b). Table 3 summarizes some properties of the more commonly studied breast cancer xenografts. Few serially transplantable human breast cancer xenografts have been well characterized and widely employed as experimental models. This deficit presumably was due to an initial lack of availability and the widespread use of the more readily manipulable, better characterized, human breast cancer cell lines. There are, however, a small number of xenograft models that have been used predominantly for studies of hormone and therapeutic responsiveness. These include: • Br-10, which was derived from a pleural effusion of a 43 year old premenopausal woman with infiltrating ductal carcinoma of the breast (Hirohashi et al., 1976,1977; Shimosato et al., 1976). This tumor contains ER but not PR and is estrogen dependent in vivo (Hirohashi et al., 1977). A less-studied ER negative and hormone unresponsive xenograft line, Br-13, that can be transplanted in both ascitic and solid forms, was derived by the same workers (Hirohashi et al., 1976). • MX-1, which was established from cancer tissue removed from a 29 year old female with breast cancer (Ovejera et al., 1978) has been employed widely in preclinical testing of potential new therapeutic agents. • T61, derived from a mastectomy specimen from a 54 year old postmenopausal woman and T60 obtained from a 31 year old premenopausal woman (Brünner et al., 1983, 1985a,b). The former is ER-positive and PR-positive but remarkably responds to estrogen administration with complete tumor regression. T60 lacks hormone receptors and is unresponsive to estrogens (Brünner et al., 1983, 1985a). Xenografts derived from breast cancer cell lines have been used to address questions of hormone responsiveness, disease progression, invasiveness and metastasis. Despite the wide spectrum of cell lines available, the majority of studies have focussed on MCF-7, representative of the hormone-responsive
Cell Line
Origin
ER Status
Estrogen responsiveness
Metastasis’
Independent Independent/responsive
11-BR-G Br-10
Human xenograft Human xenograft
Br-13 Hs 578T KPL-1 KPL-3C MA-11 MCF-7
Human Human Human Human Human Human
MCF-7ADR MCF-7/MIII
MCF-7 variant selected for adriamycin resistance MCF-7 variant
+
Independent/stimulated
±
MCF-7LCC1
MCF-7 variant
+
Independent/stimulated
±
MCF-7/LCC2 R27 MCF-7/MKS1 MDA-MB -231 ML- a
+ + + – +
Independent/stimulated Independent/responsive Independent/inhibited Independent/unresponsive Independent/inhibited
MDA-MB -33 1 MDA-MB-361
MCF-7 variant MCF-7 variant MCF-7 transfected with FGF-4 Human breast cancer cell line MDA-MB-23 1 transfected with ER Human breast cancer cell line Human breast cancer cell line
+ +
Dependent Dependent
MDA-MB -435
Human breast cancer cell line
–
Independen t/unresponsive
xenograft breast cancer breast cancer breast cancer breast cancer breast cancer
– +
cell cell cell cell cell
line line line line line
+ – + + – + –
–
Independent/unresponsive Independent/unresponsive
± +2 + –
Dependent Independent/unresponsive
–
– + +
– +
References (Bover et al., 1991) (Hirohashi et al., 1976, 1977; Fukutomi et al., 1989) (Hirohashi et al., 1976, 1977) (Thompson et al., 1992) (Kurebayashi et al., 1995) (Kurebayashi et al., 1996) (Rye et al., 1996) (Soule et al., 1973; Thompson et al., 1993) (Vickers et al., 1988; Thompson et al., 1992) (Clarke et al., 1989a; Thompson et al., 1993) (Brunner et al., 1993a; Thompson et al., 1993) (Brunner et al., 1993b) Nawata et al., 1981a, 1981b) (Kurebayashi et al., 1993) (Price et al., 1990a) (Jiang et al., 1992)
Breast Cancer
Table 3 Characteristics of Transplantable Human Mammary Carcinomas
(Peterson et al., 1997) Unpublished data (RL Sutherland) (Price et al., 1990a) Continued on next page
87
88
Table 3 (continued) Cell Line
Origin
ER Status
Estrogen responsiveness
MDA-MB-435/LCC6 MDA-MB-468 MX-1
Human breast cancer cell line Human breast cancer cell line Human xenograft
–
Independent Independent
MX-2A SW 613-S T60 T61 T-47D
Human xenograft Human breast cancer cell line Human xenograft Human xenograft Human breast cancer cell line
–
Independent
– – +
Independent/unresponsive Independent/inhibited Dependent
–
ZR-75-1
Human breast cancer cell line
+
Dependent
–
-, rare metastases; ± , occasional metastases;
1'
Metastases + +2
–
References (Leonessa et al., 1996) (Price et al., 1990b) (Ovejera et al., 1978; Fukutorni et al., 1989) (Peterson et al., 1997) Modjtahedi et al., 1985) (Briinner et al., 1983, 1985a,b) (Brünner et al., l983,1985a,b) (Engel et al., 1978a; Thompson et al., 1992) (Engel et al., 1978a; Thompson et al., 1992)
, Microscopic lesions
2
Sutherland et al
Breast Cancer
89
phenotype, and MDA-MB-231, representative of the hormone-unresponsive phenotype. Several other cell lines share these properties, as outlined in Table 3, but have been studied much less extensively. Cell lines that have been selected in vitro for various hormone-responsive phenotypes including increased sensitivity to estrogen or resistance to antiestrogens and lines resistant to cytotoxic chemotherapy have also been serially passaged in nude mice with conservation of the phenotype through several generations. These have provided increasing insight into several aspects of the disease and in particular the role of estrogen in tumor growth, invasion and metastasis. More recently, cell lines overexpressing an exogenous gene with a potential role in disease causation or progression have become available from studies in vitro. The successful establishment of xenografts of these lines, as has already been achieved for the MCF-7/MKS-1 that overexpresses fibroblast growth factor (Kurebayashi et al., 1993; McLeskey et al., 1993), will provide greater insight into the pathophysiology of breast cancer.
4.
PHENOTYPIC CHARACTERISTICS OF BREAST CANCER CELL LINES
Table 4 lists some of the features of the most commonly used cell lines, concentrating on expression of nuclear and cell surface receptors, signal transduction molecules and cell cycle regulatory molecules, since these are components of known oncogenic pathways in breast cancer. Expression levels have been categorized as very low to very high, with the expression in normal breast epithelial cells in general defined as moderate. In some cases, mutations, deletions or amplification of the corresponding genes account for the changes in gene expression, but more often the underlying mechanisms are unknown. Thus, the presence of amplification, mutation or deletion is only noted where there is evidence that this genetic change is involved in the change in gene expression. Table 4 has been restricted to alterations in known genes but it is likely that a significant number of genes frequently altered in breast cancer remain to be identified. Some candidate loci harboring oncogenes or tumor suppressor genes identified by cytogenetic analysis are summarised in Tripathy and Benz (1993), but new techniques for identifying such regions (e.g. comparative genomic hybridization (CGH) Kallioniemi et al., 1994, and whole genome scans with microsatellite markers) will accelerate progress towards cataloging the molecular changes important for breast cancer initiation and progression. Summarized below are some specific issues related to the classes of genes listed.
90
Table 4 Patterns of gene expression/mutation in breast cancer cell lines' ER2
PR3
BT-20 BT-474 BT-483 BT-549 DU4475 HBL-100 Hs 578.T MCF-7 MDA-MB-134-VI MDA-MB-157 MDA-MB-175-VII MDA-MB -23 1 MDA-MB -330 MDA-MB -36 1 MDA-MB-415 MDA-MB-435S MDA-MB-436 MDA-MB-453 MDA-MB-468 SK-Br3 T-47D ZR-75-1
mRNA but no binding mod high neg neg neg neg high v. high neg mod neg mRNA but no binding high
neg high
neg neg mod neg high mod
Steroid hormone and retinoid receptors AR4 GR5 RARα6
neg neg mod neg
neg mod neg neg neg neg neg mod mod
neg neg high
neg neg mod
neg high
v.high mod
neg mod high
mod low low high neg mod mod low low mod low mod mod low
mod mod mod low low low
RARβ6
RARγ6
high high
low low
mod mod
mod mod high high
mod high neg neg
mod mod mod mod
mod mod high
mod mod low
mod mod mod
mod
neg
mod
high
neg
mod Continued on next page
Sutherland et al
Cell line
Cell line BT-20 BT-474 BT-483 BT-549 DU4475 HBL-100 Hs 578.T MCF-7 MDA-MB-134-VI MDA-MB-157 MDA-MB-175-VI1 MDA-MB-231 MDA-MB-330 MDA-MB-361 MDA-MB-415 MDA-MB-435S MDA-MB-436 MDA-MB-453 MDA-MB-468 SK-Br3 T-47D ZR-75-1
EGF-R7
erbB28
Receptor tyrosine kinases erbB39
v. high/amp v.low/wt v. lowhvt modhvt neghvt low/wt lowhvt v. lowhvt neghvt lowhvt v. lowhvt mod/wt mod/wt low/wt
high/wt v. high/amp high/wt modhvt mod/wt modhvt modhvt modhvt mod/wt mod/wt high/wt modhvt modhvt v. high/amp
v. high v. high v. high high high high high v. high v. high high v. high mod mod v. high
modhvt neghvt v. high/amp modhvt v.low/wt lowhvt
modhvt v. high/amp mod/wt v. high/amp mod/wt high/wt
mod v. high v. high v. high v. high
erbB410
FGFR111
FGFR211
mod
neg
low
mod
low
neg
low v. high
neg low
high
mod
Breast Cancer
Table 4 (continued)
low low mod mod
high low low high
Continued on next page
91
92
Table 4 (continued) Hemopoietin receptor superfamily12 G-CSF R GM-CSF-R a GM-CSF-Rβ
Cell line
IL-2 R
IL-3 Rα IL-6 R
IL-11 R
CNTF R
BT-20 BT-474 BT-483 BT-549 DU4475 HBL-100 Hs 578.T MCF-7 MDA-MB-134-VI
neg
neg
low
mod
low
neg
neg
high
neg neg
neg neg
low neg
mod high
high low
high neg
neg v.low
low neg
neg
neg
neg
high
neg
neg
neg
v. low
neg neg
neg neg
low low
mod high
low high
neg neg
neg neg
neg neg
neg
low
low
high
neg
neg
neg
v. low
neg
neg
low
high
high
neg
high
low
MDA-MB-157 MDA-MB-175-VI1 MDA-MB -23 1 MDA-MB-330 MDA-MB-361
mod high
neg v. high
mod mod
neg high
neg high
neg neg
neg high
high low
Prl R14
GH R
low
neg high high high/neg neg low/neg neg high high mRNA/ no binding neg low low neg high mRNA/ low binding
neg
neg
low
neg low neg high/low high mod
mod neg mod high high
neg low
low high
Continued on next page
Sutherland et al
MDA-MB-415 MDA-MB-435S MDA-MB-436 MDA-MB-453 MDA-MB-468 SK-Br3 T-47D ZR-75-1
CSF-1 R13
Cell line BT-20 BT-474 BT-483 BT-549 DU4475 HBL-100 Hs 578.T MCF-7 MDA-MB-134-VI MDA-MB-15 7 MDA-MB- 175-VI1 MDA-MB-231 MDA-MB-330 MDA-MB-361 MDA-MB-415 MDA-MB-435S MDA-MB-436 MDA-MB-453 MDA-MB-468 SK-Br3 T-47D ZR-75-1
Signalling molecules Grb1418 PLC g 19 Shc19
Ras15
Grb216
Grb717 neg v. high neg neg neg neg high neg
neg neg low neg neg
high v. low mod high high
high mod low high
activated Ha-ras amplified N-ras
low low low low low low low high/amp
neg low
mod high
mod low
mod low low mod mod high/amp
neg neg neg neg neg v. high
low neg neg neg low low
mod high high high high mod
v. low v. high/amp mod v. low low low
neg high neg high neg neg
neg neg low neg high mod
mod high mod v. low mod mod
activated Ki-ras
Nck19
Ras-GAP19
EMS120
mod high low mod
mod mod mod mod
neg mod mod high mod neg
low low mod low low low mod mod
mod mod mod mod mod mod high mod
wt/mod wt/mod wtimod wtimod wt/low wt/mod wtlmod ampimod-high amp/v. high amp/mod amp/high wt/mod wt/mod wtlmod
mod low low neg low
v. high low mod low low
mod mod high mod mod mod
wtimod amp/high wt/mod wt/mod wtimod amp/high
Breast Cancer
Table 4 (continued)
Continued on next page
93
94
Table 4 (continued) Cyclin D121
Cyclin E22
BT-20 BT-474 BT-483 BT-549 DU4475 HBL-100 Hs 578.T MCF-7 MDA-MB-134-VI MDA-MB-157 MDA-MB-175-VI1 MDA-MB-231 MDA-MB-330 MDA-MB-361 MDA-MB-4 15 MDA-MB-435s MDA-MB-436 MDA-MB-453
lowhvt modht modht v. lowhvt lowhvt lowhvt modht mod-highhvt v. high/amp modht high/amp modhvt higNamp mod/amp
mod (truncated) mod low
lowhvt mRNA high (truncated) protein mod/amp lowhvt lowiwt modhvt mod/amp
high (truncated)
MDA-MB-468 SK-Br3 T-47D ZR-75-1
Cdk6u23
mod
mod
mod-high mod mod high v. high (amp)
low
mod
mod mod v. low
low-high
high mod mod
low v. low mod
low
Cell cycle regulatory molecules Cdc25A24 p1525 p1626 low mod mod mod mod
del wt
low mod mod low low mod low mod
wt wt
p’morph
wt del del wt p’morph wt
low neg
wt
low low low low
p’morph wt wt/deP1 wt
pW27
p1928
del LOH wt wt/ovxp wt/meth
mut wt
wt wt
wt
wt wt
del del wt/meth ovxp wt del del LOWmut
wt wt wt
wt wt wt wt
wt
wt
wt wt wt
wt wt wt
wt
wt
wt wt mut wt
wt wt
Ovxp wt
wt/ovxp wt/meth wt
wt
p2lZ9 low/p’moxph low mod vlow mod low mod mod low mod low low low/mRNAmcd
p2730 high low
mod-high high high low mod high
low low
low v. low mod mod
mod
Continued on nextpage
Sutherland et al
Cell line
Cell line
c-Myc32
BT-20 BT-474 BT-483 BT-549 CaMa-1 DU4475 HBL100 Hs 578.T MCF-7 MDA-MB-134-VI MDA-MB-157 MDA-MB-175-VII MDA-MB-23 1 MDA-MB-330 MDA-MB-361 MDA-MB-415 MDA-MB-435s MDA-MB-436 MDA-MB-453 MDA-MB-468 SK-Br3 T-47D ZR-75-1
low/wt mod
Transcription factors ERM33 PEA333
ER8133
RB34
mod low low mod
mod low low mod
high neg neg v. high
wt wt wt mut
mod mod low neg low low mod mod mod
mod low v. low neg v. low mod mod mod low
low mod neg neg neg neg v. high v. high low
del SV40-bound wt wt wt wt wt wt wt wt
mod low
mod neg
v. high neg
neg low neg
low low neg
neg neg neg
Tumor suppressor genes p5334 mut mut
Breast Cancer
Table 4 (continued)
mut
wt
low mod/wt low
modhvt mod low
amp low/wt
mut wt mut wt wt wt
SV40-bound mut wt mut mut wt
mut mut mut mut mut Continued on next page
95
96
Table 4 Footnotes 1Gene expression is classified as very low (v. low), low, moderate (mod), high, or very high (v. high), or undetectable (neg). Amp, amplification; del, deletion; mut, mutation; LOH, loss of heterozygosity; p’morph, polymorphisms; wt, wild-type; ovxp, overexpression. 2Estrogen receptor: (Horwitz et al., 1978; Hall et al., 1990); Unpublished data from this laboratory (R. Sini, Y-E Chiew, A. deFazio). 3Progesterone receptor: (Howitz et al., 1978; Hall et al., 1990). 4Androgen receptor: (Hall et al., 1992). SGlucocorticoid receptor: (Hall et al., 1990); Unpublished data from this laboratory (Y-E Chiew, A. deFazio). 6Retinoic acid receptors: (Roman et al., 1992). 7Epidermal growth factor receptor (erbB1): (Hall et al., 1990; Lee et al., 1990); Unpublished data from this laboratory (A. deFazio; R. Sini, Y-E Chiew). 8erbB2MER2/neu: Unpublished data from this laboratory; (R. Sini, Y-E Chiew, A. deFazio). 9Unpublished data from this laboratory (Y-E Chiew, A. deFazio). 10ErbB4 (Plowman et al., 1993). “Fibroblast growth factor receptors: (Luqmani et al., 1992). 12Interleukin receptors (IL-2 R, -3 Ra, -6 R, -11 R), ciliary neurotrophic factor receptor (CNTF R), granulocyte colony-stimulating factor receptor (G-CSF R), granulocyte/macrophage colony-stimulating factor (GM-CSF R), growth hormone receptor (GH R): (Douglas et al., 1997). 13Colony-stimulating factor-1 receptor/fms (CSF-1 R): (Kacinski et al., 1991). 14Prolactin receptor: (Douglas et al., 1997; Ormandy et al., 1997). 15Reviewed in Salomon et al., 1991. 16Grb2 (Daly et al., 1994). 17Grb7 (Stein et al., 1994). Unpublished data from this laboratory (G. Sanderson, R. Fiddes, RJ Daly). I8Grbl4 (Daly et al., 1996). 19Unpublished data from this laboratory (M. Binder, RJ Daly). 20EMS1 (Campbell et al., 1996). 21Amplification: (Buckley et al., 1993); Expression: (Buckley et al., 1993; Tam et al., 1994; Sweeney et al., 1998). 22Cyclin E protein: (Keyomarsi et al., 1993; Sweeney et al., 1998) NB truncated forms are frequently expressed in breast cancer cell lines but not normal breast epithelial cells; Cell lines other than MDA-MB-157 generally express similar levels of mRNA (Buckley et al., 1993; Keyomarsi et al., 1993). 23Cdk6 (Tam et al., 1994; Sweeney et al., 1998). 24Unpublished mRNA data from this laboratory (J. Ball). 25p15/INK4B/MTS2: (Musgrove et al., 1995; Zariwala et al., 1996a). 26p16/INK4A/MTS1: Inactivated by gene methylation (meth) (Herman et al., 1995; Musgrove et al., 1995), unpublished data from this laboratory (R. Hui). 27p18/INK4C: (Lapointe et al., 1996; Zariwala et al., 1996a). 28p19DNK4D: (Zariwala et al., 1996b). 29p21/WAF1/CIP1/SDI1: (Musgrove et al., 1995; Sweeney et al., 1998). 30p27/KIP1 (Musgrove et al., 1995). 31As noted in the text several T-47D variants exist. Those from this laboratory have wild-type p15 (Musgrove et al., 1995) while the gene is deleted in those from the ATCC (Zariwala et al., 1996a). 32Expression: unpublished data from this laboratory (EA Musgrove); copy number (Kozbor et al., 1984). 33ETS family members: (Baert et al., 1997). 34References summarized in Musgrove et al., 1995.
Sutherland et al
Breast Cancer 4.1
97
Steroid hormone and retinoid receptors
Members of the nuclear receptor gene superfamily, including the steroid hormone and retinoid receptors, are ligand-activated transcription factors intimately involved in the control of cell proliferation and differentiation. Given the importance of sex steroids in the etiology of breast cancer (Henderson et al., 1996) and their importance in defining breast cancer phenotype (Dickson et al., 1987), most breast cancer cell lines have been characterized for the presence or absence and level of expression of these receptors. In general the receptors for the sex steroids, ER, PR and androgen receptor (AR), are co-expressed (Hall et al., 1990). The classic estrogenresponsive breast cancer cell line is MCF-7, but other lines including T-47D and ZR-75-1 have frequently been used for studies of estrogen action. Two cell lines, BT-20 and MDA-MB-330, express low levels of ER mRNA but are functionally ER-negative. The T-47D cell line expresses PR at very high levels and has become the cell line of choice for studies of progestin action. While some of the other cell lines also express PR, they are much less sensitive. MDA-MB-453 cells express AR in the absence of ER and PR, providing a model in which androgen effects can be examined in the absence of crossreactivity with the other steroid hormone receptors. Retinoids have major growth-inhibitory and differentiation-inducing effects in mammary epithelium and RARb has been proposed as a potential tumor suppressor gene in these cells (Swisshelm et al., 1994). RARa is more highly expressed in ER-positive cell lines and is under estrogen control, RARb is seldom expressed and only in the ER-negative phenotype while RARg expression is ubiquitous (Roman et al., 1992, 1993). This spectrum of cell lines provides useful models for further assessing the role of retinoids in breast cancer, including the identification of potential new therapeutically active molecules. Other transcription factors with a suspected or potential role in breast cancer are c-myc (Tripathy et al., 1993; Watson et al., 1996) and several members of the ETS family (Baert et al., 1997; Benz et al., 1997; Chang et al., 1997). There is limited information on their expression and regulation in these cell lines. 4.2
ErbB receptor family
The EGF receptor (erbB1) is expressed inversely to the ER (Lee et al., 1990; deFazio et al., 1997), so that all the cell lines expressing high levels of the EGF-R are hormone unresponsive (Dickson et al., 1987,1995). Other members of this family are also overexpressed in various breast cancer cell lines, sometimes as a consequence of gene amplification. Given the ability of receptors within this family to heterodimerize, the choice of cell line for a particular
98
Sutherland et al.
application will be dependent on the suite of receptors expressed. The recent development of novel therapies targetting members of this receptor family makes many of these cell lines and their xenografted counterparts excellent models for preclinical testing. 4.3
Hemopoietin receptor superfamily
Unexpectedly, some breast cancer cell lines express high levels of mRNA for receptors of the hemopoietin receptor superfamily. All lines tested to date express the common receptor subunit gp130 and the leukemia inhibitory factor (LIF) receptor (Douglas et al., 1997). Since some of the cognate ligands, particularly oncostatin M, have major differentiation-inducing activity in vitro (Douglas et al., 1998) it is likely that selected cell lines will play an increasing role in further defining these effects at the molecular level. The prolactin and growth hormone receptors are structurally related to the other cytokine receptors and are expressed in the majority of breast cancer cell lines, consistent with the roles of prolactin and growth hormone in mammary development. Interestingly, the prolactin receptor appears to be coordinately expressed with, and coregulated by, the sex steroid receptors (Ormandy et al., 1997). 4.4
Signalling molecules
Data documenting amplification of Grb2 (Daly et al., 1994), coamplification of Grb7 and erbB2 (Stein et al., 1994) and amplification of the gene for the cSrc substrate EMS1 in breast cancer (Fantl et al., 1993) provide strong evidence for amplification downstream of signalling pathways in the pathology of breast cancer. Selected cell lines with specific aberrations provide useful models for ongoing studies. 4.5
Cell cycle regulatory molecules
The cyclins, CDKs and CDK inhibitors associate combinatorially and thus aberrant expression of any one of these molecules can influence the association of others. Variations in expression are significant for cyclins D1 and E (Buckley et al., 1993; Keyomarsi et al., 1993) but less pronounced for the remaining cyclins. Similarly, Cdc2, Cdk2 and Cdk4 are universally expressed at similar levels but Cdk6 expression is variable and apparently absent in some cell lines (Tam et al., 1994; Sweeney et al., 1998). Since aberrant expression and regulation of cyclin D1, cyclin E and p27 (Buckley et al., 1993; Keyomarsi et al., 1993, 1994; Bartkova et al., 1994; Gillett et al., 1994; Catzavelos et al., 1997; Porter et al., 1997) appear to be common features of clinical breast cancer, several of the cell lines are proving valuable models in assessing the effects of aberrant expression on phenotype and therapeutic responsiveness.
Breast Cancer
99
5. CROSS-CONTAMINATION AND STABILITY Cross-contamination and misclassification was a major problem in the early establishment of breast cancer cell lines (Engel et al., 1978a), with evidence of contamination from a number of different sources, but predominantly by HeLa cells (Nelson-Rees et al., 1977). With the advent of DNA fingerprinting for definitive classification of genotypes this theoretically is no longer a problem. Few laboratories incorporate this procedure into their routine quality control, and prefer to ignore the still prevalent problems of crosscontamination between cell lines and contamination with Mycoplasma. Although the phenotypic characteristics of the majority of the cell lines under routine study are reasonably stable, marked differences in some features, particularly hormone responsiveness, have been reported. Whilst some of these, especially estrogen responsiveness, may be attributable to differing culture conditions, there is clear evidence of altered sensitivity to antiestrogens under standard conditions (Reddel et al., 1985). Similarly, T-47D cells maintained in different laboratories show differing levels of ER and PR expression and radically different levels of responsiveness to progestins. The phenotype of T-47D from ATCC is quite resistant to the growth-inhibitory effects of progestins (Kalkhoven et al., 1995) while the cells maintained in this laboratory (Sutherland et al., 1988; Kalkhoven et al., 1995) and the T-47Dco (Horwitz et al., 1985) are exquisitely sensitive. Studies from two laboratories have reported both phenotypic and genotypic drift in T-47D, which appears to be one of the least stable cell lines. These studies have led to the evolution of sublines and clones of differing ploidy and sensitivity to estrogen, antiestrogens and progestins (Reddel et al., 1988; Graham et al., 1990). One of these variant sublines, T-47D-5 (Reddel et al., 1988), was later shown to be a contamination by MCF-7 cells.
6.
VARIANTS WITH ALTERED DRUG AND HORMONE SENSITIVITY
The central role of estrogen in the etiology of breast cancer and the consequent importance of antiestrogens in breast cancer therapy has focussed much attention on factors regulating the loss of hormone sensitivity in breast cancer. Several distinct phenotypes exist: i.e. estrogen-dependent, estrogenindependent but estrogen-responsive, and estrogen-unresponsive. Several laboratories have isolated derivatives, mainly of the MCF-7 cell line, that fit these criteria (Table 5) and these have provided important models for dissecting out potential mechanisms for estrogen sensitivity. Equally valuable are similar series of cell lines selected for resistance to tamoxifen and other nonsteroidal antiestrogens, and to the newer classes of steroidal antiestrogens,
100
Sutherland et al.
although fewer of the latter are available. These antiestrogen-resistant cell lines have provided some insight into potential mechanisms of tamoxifen resistance and may identify strategies for overcoming this clinically important problem. Altered sensitivity to progestins has received much less attention, but as noted previously T-47D variants which have lost sensitivity are available. Progestin resistance in PR-positive cells is represented by a sole cell line resistant to the clinically relevant progestin, MPA (Gibelli et al., 1994). The importance of autocrine and paracrine growth factor production in regulating steroid responsiveness has been studied in considerable detail (reviewed in Dickson and Lippman, 1987,1995). As a consequence, some cell lines have been engineered to produce growth factors under the control of constitutive or inducible promoters (Daly et al., 1991; Kurebayashi et al., 1993). These and similar lines expressing other relevant growth factors are likely to prove invaluable models for ascertaining the roles of growth factor production on in vitro steroid sensitivity and disease progression and on responsiveness in vivo when xenografted to appropriate animal hosts. As has been the case with most other cancer types, cell lines resistant to various chemotherapeutic regimens have been derived and provide important insights into the causes and consequences of drug resistance. Some of the more widely studied models are listed in Table 5.
6.
CONCLUSIONS
The large battery of human breast cancer cell lines currently available has made invaluable contributions to the contemporary knowledge of breast cancer biology. Further development of cell lines representing preneoplastic lesions and carcinoma in situ would be extremely valuable additions to the current series. Equally important are likely to be engineered cell lines expressing candidate genes of potential importance in the evolution and progression of breast cancer, particularly as these can be studied both in vitro and in vivo in xenograft models.
ACKNOWLEDGMENTS Research in this laboratory is supported by research grants from the National Health and Medical Research Council of Australia and the New South Wales State Cancer Council. We are indebted to our past and present colleagues in the Cancer Research Program who have provided unpublished data for inclusion in Table 4.
Parent Cell Line
A. Estrogens
EFM-19 MCF-7 MCF-7 MCF-7 MCF-7 MCF-7 MCF-7 MCF-7 MCF-7
EFM-19T AL-1 or TAMR-1 CG4 CG5 K3 LCC1 MIII McG MKS
MCF-7 T-47D T-47D T-47D ZR-75-1
RL-3 T-47D:C4 T-47D:C4:2W (clone of T-47D:C4) T-47Dco ZR-PR-LT
MCF-7 MCF-71 MCF-7 MCF-7 MCF-7 MCF-7
182R-6 5-23 AL-1 or TAMR-1 LCC2 LCC9 LY-2
MCF-7 MCF-7 MCF-7 MCF-7 ZR-75
R-27 RL-3 Rtx6 TOT ZR-75-9a1
B. Antiestrogen therapy
Derivative Cell Line
Resistance stim. by TAM inhibited by IC1 164,384 and IC1 182,780 inc. sensitivity/response to estrogen inc. sensitivity/response to estrogen estrogen independent, estrogen supersensitive? estrogen independent estrogen independent estrogen dependent estrogen independent, inhibited by estrogen, antiestrogen (TAM) sensitive estrogen responsive ER-,PR-; estrogen unresponsive estrogen unresponsive
Reference (Simon et al., 1992) (Lykkesfeldt et al., 1986) (Natoli et al., 1983) (Natoli et al., 1983) (Katzenellenbogen et al., 1987) (Clarke et al., 1989a; Brunner et al., 1993a) (Clarke et al., 1989a; Brunner et al., 1993a) (Osborne et al., 1987; Chen et al., 1998) (Kurebayashi et al., 1993; McLeskey et al., 1993) (Wiseman et al., 1993) (Murphy et al., 1990) (Pink et al., 1996)
estrogen independent, antiestrogen resistant estrogen independent, TAM sensitive
(Horwitz et al., 1982) (van den Berg et al., 1990)
IC1 182,780 tamoxifen tamoxifen 4-OH-TAM, TAM IC1 182,780 tamoxifen, LY 117018, nafoxidine, 4-hydroxy-TAM, IC1 164,384 tamoxifen tamoxifen tamoxifen, LY 117018 4-hy droxy -TAM tamoxifen
(Lykkesfeldt et al., 1995) (Reddel et al., 1988) (Lykkesfeldt et al., 1986) (Briinner et al., 1993b) (Briinner et al., 1997) (Bronzert et al., 1985; Clarke et al., 1989b) (Nawata et al., 1981a) (Wiseman et al., 1993) (Faye et al., 1983; Borras et al., 1994) (Herman et al., 1996) (van den Berg et al., 1989) Continued on next page
101
Drug resistance
Breast Cancer
Table 5 Cell lines with Altered Sensitivity to Cytotoxic Chemotherapy, Antiestrogen Therapy and Steroid Hormones
(continued)
Drug resistance
Parent Cell Line Derivative Cell Line
C. Progestins
MCF-7 T-47D
D. Growth MCF-7 factor expression MCF-7McG E. Cytotoxic chemotherapy
MCF-7 MCF-7 MCF-7 MCF-7 MCF-7 MCF-7 MCF-7 CG5 MDA-MB-231 ZR-75
Resistance
Reference
MCF-7/MPA T-47D-Y and subsequently T-47D-YA and T-47D-YB
progestin (MPA) unresponsive progestin unresponsive
(Gibelli et al., 1994) (Sartorius et al., 1994)
MKS
constitutive FGF-4 expression
M15, M17?
IGF-2
(Kurebayashi et al., 1993; McLeskey et al., 1993) (Daly et al., 1991)
MCF-7ADR AdRVp HC MelR -R VP CG5/Dx MDA-VP MTXR ZR-75
adriamycin adriamycin, melphalan cyclophosphamide methotrexate, melphalan multi-drug resistant e toposide adriamycin etoposide methotrexate
(Vickers et al., 1988) (Chen et al., 1990) (Frei III et al., 1988) (Moscow et al., 1993,1995) (Ojima et al., 1996) (Schneider et al., 1994) (Gibelli et al., 1996) (Asano et al., 1996) (Cowan et al., 1984)
102
Table 5
See text - originally described as T-47D variant
1
Sutherland et al
Breast Cancer
103
REFERENCES Asano T, An T, Zwelling LA et al. Oncology Res 8: 101, 1996. Baert JL, Monte D, Musgrove EA et al. Int J Cancer 70: 590, 1997. Band V, Sager R. Proc Nutl Acad Sci USA 86: 1249,1989a. Band V, Zajchowski D, Stenman G et al. Genes, Chromosomes & Cancer 1: 48, 1989b. Band V, Zajchowski D, Swisshelm K et al. Cancer Res 50: 7351, 1990. Bartek J, Bartkova J, Kyprianou N et al. Proc NatlAcad Sci USA 88: 3520, 1991. Bartkova J, Lukas J, Muller H et al. Int J Cancer 57: 353, 1994. Benz CC, O’Hagan RC, Richter B et al. Oncogene 15: 1513, 1997. Borras M, Jin L, Bouhoute A et al. Biochem Pharmacol 48: 2015, 1994. Bover L, Barrio M, Slawtsky I et al. Breast Cancer Res Treat 19: 47, 1991. Bronzert DA, Greene GL, Lippman ME. Endocrinology 117: 1409,1985. Briinner N, Spang-Thomsen M, Vindelov L et al. Br J Cancer 47: 641, 1983. Briinner N, Bastert GB, Poulsen HS et al. Eur J Cancer Clin Oncol 21: 833, 1985a. Briinner N, Spang TM, Vindeløv L et al. Eur J Cancer Clin Oncol 21: 1349, 1985b. Briinner N, Boulay V, Fojo A et al. Cancer Res 53: 283, 1993a. Briinner N, Frandsen TL, Holst HC et al. Cancer Res 53: 3229, 1993b. Briinner N, Boysen B, Jirus S et al. Cancer Res 57: 3486, 1997. Buckley MF, Sweeney KJE, Hamilton JA et al. Oncogene 8: 2127,1993. Cailleau R, Young R, Olive M et al. J Nat Cancer Inst 53: 661, 1974. Cailleau R, Olive M, Cruciger QV. In vitro 14: 911, 1978. Campbell DH, deFazio A, Sutherland RL et al. Int J Cancer 68: 485, 1996. Caron de Fromentel C, Nardeux PC, Soussi T et al. Exp Cell Res 160 83, 1985. Catzavelos C, Bhattacharya N, Ung YC et al. Nature Medicine 3: 227, 1997. Chang CH, Scott GK, Kuo WL et al. Oncogene 14: 1617, 1997. Chen Y, Martinez LA, Lacava M et al. Oncogene 16: 1913,1998. Chen YN, Mickley LA, Schwartz AM et al. J Biol Chem 265: 10073, 1990. Clarke R, Briinner N, Katzenellenbogen BS et al. Proc Natl Acad Sci USA 86: 3649,1989a. Clarke R, Briinner N, Thompson EW et al. J Endocrinol 122: 331, 1989b. Clarke R, Thompson E, Leonessa F et al. Breast Cancer Res Treat 24: 227, 1993. Clarke R. In: Diseases of the Breast. Harris JR, Lippman ME, Morrow M et al. (eds). p. 245. Lippincott-Raven Publishers, Philadelphia, 1996. Cowan KH, Jolivet J. J Biol Chem 259: 10793,1984. Daly RJ, Harris WH, Wang DY et aI. Cell Growth Differ 2: 457, 1991. Daly RJ, Binder MD, Sutherland RL. Oncogene 9: 2723,1994. Daly RJ, Sanderson GM, Janes PW et al. J Biol Chem 271: 12502,1996. deFazio A, Chiew Y-E, McEvoy M et al. Cell Growth & Differ 8: 903, 1997. Dickson RB, Lippman ME. Endocr Rev 8: 29, 1987. Dickson RB, Lippman ME. Endocr Rev 16: 559, 1995. Dobrynin YV J Natl Cancer Inst 31: 1173, 1963. Douglas AM, Goss GA, Sutherland RL et al. Oncogene 14: 661, 1997. Douglas AM, Grant SL, Goss GA et al. Int J Cancer 75: 64, 1998. Dupont WD, Page DL. N Engl J Med 312: 146, 1985. Engel LW, Young NA. Cancer Res 38: 4327,1978a. Engel LW, Young NA, Tralka TS et al. Cancer Res 38: 3352,1978b. Ethier SP, Mahacek ML, Gullick WJ et al. Cancer Res 53: 627, 1993. Ethier SP, Kokeny KE, Ridings JW et al. Cancer Res 56: 899, 1996. Fantl V, Smith R, Brookes S et al. Cancer Surveys 18: 77, 1993. Faye JC, Jozan S, Redeuilh G et al. Proc Natl Acad Sci USA 80: 3158,1983.
104
Sutherland et al.
Fogh J, Trempe G. In: Human tumor cells in vitro Fogh J (ed). p. 115,1975. Plenum Press, New York. Frei III E, Teicher B, Holden S et al. Cancer Res 48: 6417, 1988. Fukutomi T, Yamaguchi K, Kubota T et al. Jpn J Surg 19: 718, 1989. Gaffney EV Cell Tissue Res 227: 563,1982. Gazdar AF, Kurvari, V, Virmani A et al. Int J Cancer 78: 766, 1998. Gibelli N, Zibers C, Sica G et al. Anticancer Res 14: 37,1994. Gibelli N, Zibera C, Asti A et al. Anticancer Res 16: 1675, 1996. Gillett C, Fantl V, Smith R et al. Cancer Res 54: 1812, 1994. Gioanni J, Courdi A, Lalanne CM et al. Cancer Res 45: 1246, 1985. Gioanni J, Le Francois D, Zanghellini E et al. Br J Cancer 62: 8, 1990. Gioanni J, Mazeau C, Zanghellini E et al. Bull Cancer 80: 472, 1993. Graham ML, Krett NL, Miller LA et al. Cancer Res 50: 6208, 1990. Hackenberg R, Luttchens S, Hofmann J et al. Cancer Res 51: 5722,1991. Hackett AJ, Smith HS, Springer EL et al. J Natl Cancer Inst 58: 1795, 1977. Hall RE, Lee CSL, Alexander IE et al. Int J Cancer 46: 1081,1990. Hall RE, Tilley WD, McPhaul MJ et al. Int J Cancer 52: 778, 1992. Hammond SL, Ham RG, Stampfer MR. Proc Natl Acad Sci USA 81: 5435, 1984. Healicon RM, Westley BR, May FE. Int J Cancer 53: 388,1993. Henderson B, Ross R, Bernstein L. Cancer Res 48: 246,1988. Henderson BE, Bernstein L. In: Diseases of the Breast. Harris JR, Lippman ME, Morrow M et al. (eds). p. 185. Lippincott-Raven Publishers, Philadelphia, 1996. Herman ME, Katzenellenbogen BS. J Steroid Biochem Mol Biol 59: 121, 1996. Herman JG, Merlo A, Mao L et al. Cancer Res 55: 4525,1995. Hirohashi S, Shimosato Y, Nagai K et al. Gann 67: 431, 1976. Hirohashi S, Shimosata Y, Kameya T et al. Cancer Res 37: 3184, 1977. Horwitz KB, Zava DT, Thilagar AK et al. Cancer Res 38: 2434, 1978. Horwitz KB, Mockus MB, Lessey BA. Cell 28: 633, 1982. Horwitz KB, Freidenberg GR. Cancer Res 45: 167, 1985. Jiang SY, Jordan VC. J Natl Cancer Inst 84: 580,1992. Kacinski BM, Scata KA, Carter D et al. Oncogene 6: 941,1991. Kalkhoven E, Kwakkenbos-Isbriicker L, Mummery CL et al. Int J Cancer 61: 80,1995. Kallioniemi A, Kallioniemi OP, Piper J et al. Proc Natl Acad Sci USA 91: 2156, 1994. Katano M, Matsuo T, Morisaki T et al. Cancer Immunol Immun 39: 161, 1994. Katzenellenbogen BS, Kendra KL, Norman MJ et al. Cancer Res 47: 4355,1987. Keydar I, Chen L, Karby S et al. Eur J Cancer 15: 659,1979. Keyomarsi K, Pardee AB. Proc NatlAcad Sci USA 90: 1112,1993. Keyomarsi K, O’Leary N, Molnar G et al. Cancer Res 54: 380, 1994. Kozbor D, Croce CM. Cancer Res 44: 438, 1984. Kurebayashi J, McLeskey SW, Johnson MD et al. Cancer Res 53: 2178, 1993. Kurebayashi J, Kurosumi M, Sonoo H. Br J Cancer 71: 845,1995. Kurebayashi J, Kurosumi M, Sonoo H. Br J Cancer 74: 200, 1996. Langlois AJ, Holder W Jr, Iglehart JD et al. Cancer Res 39: 2604, 1979. Lapointe J, Lachance Y, Labrie Y et al. Cancer Res 56: 4586, 1996. Lasfargues EY, Ozzello L. J Natl Cancer Inst 21: 1131, 1958. Lasfargues EY, Coutinho WG, Redfield ES. J Natl Cancer Inst 61: 967, 1978. Lee CSL, Hall RE, Alexander IE et al. Growth Factors 3: 97, 1990. Leonessa F, Green D, Licht T et al. Br J Cancer 73: 154, 1996. Levenson AS, Jordan VC. Cancer Res 57: 3071, 1997. Loh PM, Clamon G, MacIndoe J et al. Breast Cancer Res Treat 5: 23, 1985. Luqmani YA, Graham M, Coombes RC. Br J Cancer 66: 273,1992.
Breast Cancer
105
Lykkesfeldt AE, Briand P. Br J Cancer 53: 29, 1986. Lykkesfeldt AE, Larsen SS, Briand P. Int J Cancer 61: 529, 1995. Matano S, Ohuchi N, Hirakawa H et al. Tohoku J Exp Med 164: 169,1991. McLeskey SW, Kurebayashi J, Honig SF et al. Cancer Research 53: 2168, 1993. Meltzer P, Leibovitz A, Dalton W et al. British Journal of Cancer 63: 727, 1991. Minafra S, Morello V, Glorioso F et al. Br J Cancer 60: 185, 1989. Modjtahedi N, Lavialle C, Poupon MF et al. Cancer Res 45: 4372, 1985. Moscow JA, Johnston PG, Cole D et al. Biochem Phamacol 49: 1069, 1995. Moscow JA, Swanson CA, Cowan KH. Br J Cancer 68: 732,1993. Murphy CS, Pink JJ, Jordan VC. Cancer Res 50: 7285,1990. Musgrove EA, Lilischkis R, Cornish AL et al. Int J Cancer 63: 584, 1995. Natoli C, Sica G, Natoli V et al. Breast Cancer Res Treat 3: 23,1983. Nawata H, Bronzert D, Lippman ME. J Biol Chem 256: 5016,1981a. Nawata H, Chong MT, Bronzert D et al. J Biol Chem 256: 6895,1981b. Nelson-Rees WA, Flandermeyer RR. Science 195: 1343,1977. O’Connell P, Pekkel V, Fuqua S et al. Breast Cancer Res Treat 32: 5, 1994. Ojima I, Slater J, Michaud E et al. J Med Chem 39: 3889,1996. Ormandy CJ, Hall RE, Manning DL et al. J Clin Endocr Metab 82: 3692,1997. Osborne CK, Hobbs K, Trent JM. Breast Cancer Res Treat 9: 111, 1987. Ovejera AA, Houchens DP, Barker AD. Ann Clin Lab Sci 8: 50,1978. Petersen OW, van Deurs B, Nielsen KV et al. Cancer Res 50: 1257, 1990. Peterson JA, Blank EW, Ceriani RL. Cancer Res 57: 1103, 1997. Pink J, Bilimoria MM, Assikis J et al. Br J Cancer 74: 1227, 1996. Plowman GD, Green JM, Culouscou JM et al. Nature 366: 473, 1993. Porter PL, Malone KE, Heagerty PJ et al. Nature Medicine 3: 222, 1997. Price JE, Polyzos A, Zhang RD et al. Cancer Res 50: 717, 1990a. Price JE, Zhang RD. Cancer Metast Rev 8: 285, 1990b. Reddel RR, Murphy LC, Hall RE et al. Cancer Res 45: 1525, 1985. Reddel RR, Alexander IE, Koga M et al. Cancer Res 48: 4340,1988. Roman SD, Clarke CL, Hall RE et al. Cancer Res 52 2236, 1992. Roman SD, Ormandy CJ, Manning DL et al. Cancer Res 53: 5940,1993. Rostagno P, Moll JL, Birtwisle-Peyrottes I et al. Anticancer Res 14: 2025, 1994. Rudland PS, Hallowes RC, Cox SA et al. Cancer Res 45: 3864, 1985. Rye PD, Norum L, Olsen DR et al. Int J Cancer 68: 682, 1996. Salomon DS, Ciardiello F, Valverius EM et al. Cancer Treat Res 53: 107, 1991. Sartor CI, Dziubinski ML, Yu CL et al. Cancer Res 57: 978, 1997. Sartorius CA, Groshong SD, Miller LA et al. Cancer Res 54: 1994. Sasi R, Hoo JJ, Samuel IP et al. Cancer Genet Cytogenet 51: 239, 1991. Sawada T, Chung YS, Nakata B et al. Human Cell 7: 138, 1994. Schneider E, Horton JK, Yang CH et al. Cancer Res 54: 152, 1994. Shimosato Y, Kameya T, Nagai K et al. J Natl Cancer Inst 56: 1251, 1976. Siciliano MJ, Barker PE, Cailleau R. Cancer Research 39: 919, 1979. Simon WE, Trams G, Holzel F .Arch Gynecol Obstet 253: 131, 1992. Simon WE, Hansel M, Dietel M et al. In Vitro 20: 157, 1984. Siwek B, Larsimont D, Lacroix M et al. Int J Cancer 76: 677, 1998. Soule HD, Vazguez J, Long A et al. J Natl Cancer Inst 51: 1409, 1973. Soule HD, Maloney TM, Wolman SR et al. Cancer Res 50: 6075, 1990. Starnpfer M, Hallowes RC, Hackett AJ. In Vitro 16: 415, 1980. Stampfer MR, Yaswen P. In: Transformation of human epithelial cells: Molecular and oncogenetic mechanisms. George E, Milo BCC, Charles F. Shuler (eds). p. 117. CRC Press, Inc., Boca Raton, Florida, 1992.
106
Sutherland et al.
Stein D, Wu J, Fuqua SA et al. EMBO J 13: 1331, 1994. Sutherland RL, Hall RE, Pang GYN et al. Cancer Res 48: 5084,1988. Sweeney KJ, Swarbrick A, Sutherland RL et al. Oncogene 16: 2865, 1998. Swisshelm K, Ryan K, Lee X et al. Cell Growth & Differentiation 5: 133, 1994. Tait L, Soule HD, Russo J. Cancer Res 50: 6087,1990. Tam SW, Theodoras AM, Shay JW et al. Oncogene 9: 2663,1994. Thompson EW, Paik S, Brunner N et al. J Cell Physiol 150: 534, 1992. Thompson EW, Brunner N, Torn J et al. Clin Exp Metastasis 11: 15, 1993. Trempe G, Fogh J. In Vitro 8: 433,1973. Tripathy D, Benz CC. In: Oncogenes and Tumor Suppressor Genes in Human Malignancies Benz CC and Liu ET (eds). p. 15. Kluwer Academic Publishers, Boston, 1993. van den Berg Hw, Lynch M, Martin J et al. Br J Cancer 59: 522,1989. van den Berg HW, Martin J, Lynch M. Br J Cancer 61: 504,1990. Vickers PJ, Dickson RB, Shoemaker R et al. Mol Endocnnol 2: 886,1988. Watanabe M, Tanaka H, Kamada M et al. Cancer Res 52: 5178,1992. Watson PH, Singh R, Hole AK. Curr Topics Microbiol Zmmunol 213: 267, 1996. Whitehead RH, Bertoncello I, Webber LM et al. J Natl Cancer Inst 40: 649,1983. Wiseman LR, Johnson MD, Wakeling A et al. Eur J Cancer 29A: 2256,1993. Wistuba 11, Behrens C, Milchgrub S et al. Clin Cancer Res 4: 2931, 1998. Yamane M, Nishiki M, Kataoka T et al. Hiroshima J Med Sci 33: 715, 1984. Young RK, Cailleau RM, Mackay B et al. In Vitro 9: 239, 1974. Zariwala M, Liu E, Xiong,Y. Oncogene 12: 451, 1996a. Zariwala M, Xiong Y Oncogene 13: 2033,1996b. Zoli W, Roncuzzi L, Flamigni A et al. J Cancer Res Clin Oncol 122: 237, 1996.
Chapter 21 Paired Breast Cancer Cell Lines Ignacio I. Wistuba1,3, Arvind K. Virman1,2 and Adi E Gazdar1,2 Hamon Center for Therapeutic Oncology Research; 2Department of Pathology, University of Texas Southwestern Medical Center; 5323 Harry Hines Boulevard, Dallas, TX 75235-8593 and 3 Department of Pathology, Pontificia Universidad Catolica de Chile, PO Box 114-D, Santiago, Chile. Tel: 001 -214-648-4921; Far: 001-214-648-4924; E-mail:
[email protected] 1
1.
INTRODUCTION
The usefulness of the available breast cancer cell lines has been restricted by the absence of a readily available source of DNA of normal cells from the patients of origin. Further, most of these breast cancer cell lines have been established from metastatic tumors (Leibovitz, 1994 and Chapter 20). As discussed below, we have reported the establishment and characterization of a relatively large panel of paired tumor and non-malignant human cell lines and strains derived from patients with primary and metastatic tumors, including cell lines derived from patients with germ-line mutations in tumor suppressor genes and from patients with possible genetic predispositions (Gazdar et al. 1998). This panel compensates for many of the shortcomings associated with available breast tumor cell lines and provides important new reagents for breast cancer research. Since the description of the HeLa tumor cell line in 1952 (Scherer et al. 1953), which was established from a patient with cervical carcinoma, permanent cultures derived from human tumors have been widely used to investigate almost every aspect of cancer biology. Despite the pivotal role played by human tumor cell lines in biomedical research, there is a widespread belief in the scientific community that they are not representative of the tumors from which they were derived. Tumor cell lines, including breast carcinoma cell lines, have extensive chromosomal rearrangements, oncogene mutations and multiple sites of allelic loss and gene amplification (Ahmadian et al. 1997; Buckley et al. 1993; Muleris et al. 1994; Virmani et al. 1998). Thus, many investigators presume that alterations of phenotypic properties and
J.R.W Masters and B. Palsson (eds.), Human Cell Culture Vol. II, 107–119. © I999 Kluwer Academic Publishers. Printed in Great Britain.
108
Wistuba et al.
additional molecular changes develop during the prolonged time required for cell culture establishment and subsequent passage. However, until our recent report (Wistuba et al. 1998) no detailed comparison of the properties of human cell lines with those tumors from which they were derived has been published for any cancer type. Our studies indicate that the breast cancer cell lines are remarkably similar in most respects to the tumors from which they were derived, even after some years in continuous culture.
2.
CHARACTERISTICS OF CURRENTLY AVAILABLE CANCER CELL LINES
Since the establishment of the first human breast carcinoma cell line in 1958 (Lasfargues & Ozzello, 1958), many attempts have been made to establish additional permanent breast tumor cell lines; however, they are difficult to establish in culture (Smith et al., 1984, 1987). Cailleau et al. (1978) reported culture success rate of about 10% for metastatic tumors, while culture attempts from approximately 300 primary breast carcinomas were completely unsuccessful. In another study, only one of 136 (0.7%) primary breast tumors were successfully cultured (Amadori et al. 1993). McCallum and Lowther (1996) established long-term cultures from 10 (7.4%) of 135 primary tumors. However, the axillary node status of the patients whose tumors were successfully cultured was not stated. Moreover, breast tumor cell lines are frequently initially contaminated with normal epithelial, stromal or mesothelial cells making it difficult to determine the source of the proliferating cultured cells (McCallum & Lowther, 1996). Although about 70 human breast cancer lines have been described in the literature, the number of breast tumor cell lines that have been adequately characterized and which are widely used is only about 20 (Leibovitz, 1994 and Chapter 20). The majority of breast carcinoma cell lines have been initiated from tumor metastases, in particular malignant pleural effusions (Band & Sager, 1989; Mahacek et al. 1993), and thus were from patients with advanced stage disease (Leibovitz, 1994). By contrast, relatively few breast cancer cell lines have been established from primary tumors (Ethier et al. 1993; Petersen et al. 1990), and almost all of those primary tumor cell lines were derived from patients who also had nodal metastases (Leibovitz, 1994). Additional problems with the use of currently available breast cancer cell lines include slow growth rates in vitro (McCallum & Lowther, 1996), and lack of hormonal response. MCF-7 is the most widely studied breast carcinoma cell line because of its steroid receptor status and estrogen sensitivity (reviewed in Levenson & Jordan, 1997), whereas other cell lines that have low steroid receptor expression (such as PMC42) are not widely used (Leibovitz, 1994).
Paired Breast Cancer Cell Lines
3.
109
ESTABLISHMENT OF NEW BREAST CANCER CELL LINES
Recently, we reported the establishment and characterization of 21 new breast cancer cell lines, most of which (18 of 21) were derived from primary breast cancers, including 5 from node-negative tumors (Table 1) (Gazdar et al. 1998). The success rate for primary breast cancers was 18 (10%) of 177 attempts, whereas the success rate for metastatic tumors was 3 (25%) of 12 attempts. The success rate for all tumors was 21 of 189 or 11%. A notable feature was the establishment of paired normal and tumor cell lines from 19 individual patients. Although non-malignant cell lines and strains are needed for comparative studies on genetic predisposition and allelotyping, currently only a few paired breast cell lines are available (Band et al. 1990). We have established the largest collection of paired cell lines, and characterized these using DNA fingerprinting and other molecular analysis (Gazdar et al. 1998). For 19 of these breast cancer cell lines we also established one or more corresponding non-malignant cell strains or B lymphoblastoid (BL) lines, which included 16 BL lines, 2 breast epithelial and 5 stromal cell strains (Table 1). Human breast tumor-derived cells require exogenous growth factors for culture (Band & Sager, 1989; Ethier et al. 1993). Because normal breast cells differentiate and cease to grow in serum-containing media (Band & Sager, 1989; Ethier et al. 1993), special media formulations have been used to establish short- or long-term cell lines from primary and metastatic tumors (Band et al. 1990). However, the success rates of these studies have been low. We used such a formulation for the growth of epithelial and stromal strains from the non-malignant areas of the mastectomy specimens (Band & Sager, 1989; Ethier et al. 1993). For establishment of tumor lines, the methods of tumor handling, disaggregation and culture conditions used in our studies were not unusual (Gazdar et al. 1998). Thus, one major factor in the relatively high success rate may have been extreme patience and prevention of overgrowth by fibroblasts. Breast cancer cell lines have been utilized for a number of biological and biochemical studies, including expression of receptors for growth factors and steroid receptors (see also Chapter 20). Compared with non-malignant breast epithelial cell strains, all of our breast carcinoma cell lines express increased amounts of HER2/neu, an important prognostic marker and a member of the tyrosine kinase growth factor receptor superfamily whose expression is upregulated several-fold in many breast carcinomas (Gazdar et al. 1998). The expression of this protein ranged from 2- to 30-fold greater than the values measured in non-malignant epithelial cell strains. In 11 cell lines, this increase was modest (2- to 4-fold), whereas in 10 cell lines it was considerable (7- to 30fold).
110
Wistuba et al.
Table1 Summary of the clinical and pathologic data concerning the tumors used to establish the new breast cancer cell lines Features Initiation Periods (months) Paired Line/Strain
Clinico-Pathologic Features Age Race
Tumor Source
Histologic
TNM Stage
Tumor Size Histology Grade Lymph Node Metastasis Survival
Median (range)/Number of Cases B Lymphoblastoid Line Breast Stroma Breast Epithelium
White African-American Hispanic Asian East Median Primary Pleural effusions Lymph node Type Ductal Metaplastic Squamous cell Lobular I IIA IIB IIIA IV II III Negative Positive
Median 12.5 months (range 4-44)a 16 5 2 Median 49 years (range 24-82) 10 6 2 1 1 18 2 1 18 1 1 1 2 5 5 6 3 Median 5 cm (range 1.5-16) 6 15 5 11 Median 12 months (range 5-29)a
Of thirteen patients with primary breast tumors receiving curative intent mastectomy, 7 were dead after a mean period of about 10 months (range 5-22 months) a
Of the patients from whom the 21 new breast cancer cell lines were established, ten were investigated for a genetic predisposition (Gazdar et al. 1998). Evidence suggestive or conclusive for a genetic predisposition was discovered in 5 of these 10 subjects (50%), a relatively high incidence. They included patients with germ-line mutations of the BRCA1 gene at 17q21 (cell line HCC1937) (Tomlinson et al. 1998) and the FHIT gene at 3p14.2 (cell line HCC1569) (Ahmadian et al. 1997). The breast tumor cell line HCC1937 was established from a primary breast carcinoma from a 24-year-old patient with a germ-line BRCA1 mutation (insertion C at nucleotide 5382) (Tomlinson et al. 1998). BRCA1 analysis revealed that the tumor cell line is homozygous for BRCA1 mutation, whereas the patient's lymphocyte DNA was heterozygous for the same mutation. This tumor cell line is marked by multiple additional
Paired Breast Cancer Cell Lines
111
genetic changes including a high degree of aneuploidy, an acquired mutation of TP53 with wild-type allele loss, an acquired homozygous deletion of the PTEN gene, and loss of heterozygosity at multiple loci known to be involved in the pathogenesis of breast cancer. This breast tumor-derived cell line may provide a useful model system for the study of familial breast cancer pathogenesis and for elucidating BRCA1 function and localization. The cell line HCC1569 carries a germ-line FHIT gene mutation (G → T substitution at nucleotide 651) in a primary breast tumor arising in a 70 year-old woman. This represents the first germ-line mutation reported in this recently cloned gene (Ohta et al. 1996), and it was also present in one of her two thus far unaffected daughters. Cell lines were also initiated from three women with a family history of and/or early onset breast cancer. Cell lines containing homozygous deletions are useful for the identification of the putative tumor suppressor genes in the deleted regions. Four homozygous deletions on chromosome 3p were identified in the 21 new breast tumor cell lines (Gazdar et al. 1998). These regions are 3p12 (Sundaresan et al. 1998), FHIT gene at 3p14.2 (Ahmadian et al. 1997), and 3p21.3 (Sekido et al. 1998). Because the putative recessive oncogenes at two of these regions (3p12 and 3p21.3) have not been identified, these cell lines provide new resources for gene localization, cloning and characterization. Further information regarding these breast tumor-derived cell lines is available on the World Wide Web (Breast Tissue Repository at the Hamon Center for Therapeutic Oncology Research, The University of Texas Southwestern Medical Center at Dallas; http://www.swmed.edu/bcrep). The breast tumor cell lines and their corresponding normal strains (B lymphoblastoid cell lines) have been deposited with the American Type Culture Collection (ATTC; Rockville, MD) for distribution to the scientific community.
4.
COMPARISON OF BREAST CANCER CELL LINES AND CORRESPONDING TUMORS
Breast cancer pathogenesis is characterized by multiple molecular changes, including activation of oncogenes and loss of known and putative tumor suppressor genes (TSG) (Callahan et al. 1993). Several studies have indicated that breast cancer cell lines have advanced genetic abnormalities and many chromosomal aberrations by cytogenetic analysis (Smith et al., 1984; Smith & Hackett, 1987), high frequencies of deletion by allelotyping studies (Ahmadian et al. 1997), and many sites of chromosomal amplification by comparative genomic hybridization (CGH) analysis (Lushinikova et al. submitted). To investigate these phenomena we compared the morphologic, phenotypic and genetic changes in the cell lines and in their corresponding tumor tissues (Wistuba et al. 1998). The properties of a series of 18 human breast cancer cell
112
Wistuba et al.
lines (cultured for a median period of 25 months, range 9-60) and their corresponding archival tumor tissues (Wistuba et al. 1998) were compared, including morphological characteristics, ploidy and immunohistochemical expression of estrogen (ER) and progesterone (PR) receptors, HER2/neu and p53 proteins. For 17 of these cases, we also tested for allelic losses at 18 chromosomal regions frequently deleted in breast tumors using 51 polymorphic microsatellite markers, and determined the TP53 gene mutation status in exons 5 to 10. Briefly, there was an excellent correlation between the breast tumor cell lines and their corresponding tumor tissues for morphological features (100%), presence of aneuploidy (87%), immunohistochemical expression of ER (87%), PR (73%), HER2/neu (93%) and p53 proteins (100%) (Table 2) (Wistuba et al. 1998). There was an almost perfect correlation between the morphological features of the primary tumors and their corresponding cell lines. Most cell lines were established from poorly differentiated tumors and they grew as epithelioid monolayers lacking obvious morphologic evidence of differentiation and exhibiting high grade nuclear atypia. In comparison, the few cell lines derived from moderately differentiated tumors grew as floating cells organized into duct-like and hollow morula-like structures (Fig. 1, panel A). Aneuploidy (defined as an abnormal nuclear content of DNA) has been considered as evidence of widespread genetic damage and DNA instability (Loeb, 1991). The majority of reports indicate that approximately two-thirds of breast cancers are aneuploid (Smith et al., 1984); however, almost all of the longestablished human breast cancer cell lines are aneuploid (Smith et al., 1984). In our series, most of the tumors (12 of 15,80%) and cell lines (14 of 15,93%) demonstrated one or more aneuploid populations, but the correlation between the specific degree of aneuploidy of the tumors and their corresponding cell lines was variable. Approximately 45% to 65% of primary breast carcinomas are estrogen (ER) and/or progesterone (PR) receptor positive (Tavassoli, 1992). Expression of steroid receptors by breast tumors correlates well with low histologic grade and responsiveness to hormonal manipulations (McGuire & Clark, 1992). As determined by an immunohistochemical method, only 20% of our cell lines (mostly derived from poorly differentiated tumors) demonstrated ER or PR expression. The correlation between the paired tumors and cell lines was 87% for ER expression and 73% for PR expression. HER2/neu oncogene abnormalities have been extensively studied in breast carcinomas by measuring both gene amplification and HER2/neu oncoprotein over-expression (Press et al. 1993; Slamon & Clark, 1988). HER2/neu amplification/over-expression has been reported to occur in 10-30% of breast carcinomas, especially in the more aggressive, poorly differentiated carcinomas that are ER negative, aneuploid and have lymph node metastases (Press et al. 1993). HER2/neu immunohistochemical expression was detected in 6 of 14
Paired Breast Cancer Cell Lines
113
(40%) of the breast cancer cell lines and in 5 of 15 (33%) of their corresponding tumors, showing a very high correlation (93%) between tumor samples and their corresponding cell lines (Fig. 1, panel A). Point mutations in the TP53 gene may result in variant p53 proteins that have an increased half-life and thus can be detected by immunohistochemical techniques which fail to immunostain the low amounts of wild type p53 protein in cells without TP53 mutations (Baas et al. 1994). TP53 gene mutations have been detected in about 40% of breast carcinomas and p53 protein overexpression has been reported in about 30% of tumors and is associated with shorter survival in breast carcinoma patients (Silvestrini et al. 1993). p53 protein expression, as demonstrated by an immunohistochemical method, was detected in 80% of the breast tumor cell lines and their corresponding tumor tissues, indicating complete correlation (100%). However, TP53 gene mutations in exons 5 to 10 were detected in only 47% of the tumor cell lines. Of interest, six of the corresponding tumor tissues exhibited the identical TP53 gene mutation (75% correlation) (Fig. 1, panel A). We determined allelic loss at 18 chromosomal regions frequently deleted in breast cancers using 51 polymorphic microsatellite markers (Table 2) (Wistuba et al. 1998). Nearly identical high LOH frequencies at all chromosomal regions analyzed were detected in tumors and their corresponding cell lines (Fig. 1, panel B). The fractional allelic loss (FAL) indices (an indicator of total allelic loss) were similar or identical in all but two tumor/cell line comparisons. For all of the individual markers there was an excellent correlation between tumors and cell lines (mean concordance of 96%). In all but two of the 275 (99%) comparisons, when allelic loss of a particular microsatellite was present in both the tumor and corresponding cell line, the identical parental allele was lost in both, confirming that allelic loss present in the cell lines originated in the parental tumor tissues (Fig. 1, panel B). Our studies were performed on cell lines cultured for a median period of 26 months. The concordance of allelic loss between tumors and cell lines for all of the comparisons was 100% for two of the three pairs cultured for 56 months or longer, indicating that the properties of cell lines usually closely resemble those of their parental tumors for periods of up to five years. Recently Sekido et al (1998), using one of these breast cancer cell lines, reported a homozygous deletion at the 3p21.3 region that was also present in the corresponding invasive carcinoma and its ductal carcinoma in situ component. Our recent data (Wistuba et al. 1998) and previously reported data in breast cancers (Aldaz et al. 1995; Borg et al. 1992; Li et al. 1997) indicate that LOH frequencies detected at chromosomal regions 6p, 13q14 (RB gene) and 10q23-24 (PTEN/MMCA1 gene) are similar to the LOH frequencies detected in those whose culture status is indeterminate (“sporadic” tumors). However, our tumors (and their corresponding cell lines) have demonstrated a higher incidence of allelic loss compared to the “sporadic” tumors for the BRCA1
114
Wistuba et al.
Paired Breast Cancer Cell Lines
115
gene (17q21), 6q, 8p, TP53 gene (17p13), 5q (APC-MCC genes region), 3p, 11q13, and 9p21 (CDKN2a gene) (An et al. 1996; Andersen et al. 1992; Devilee et al. 1989; Eiriksdottir et al. 1995; Fujii & Gabrielson, 1996; Gudmundsson et al. 1995; Kirchweger et al. 1994; Nagai et al. 1994; Orphanos et al. 1995; Singh et al. 1993; Thompson et al. 1993) (Table 2). Of these, significant differences in the LOH frequencies were detected at 5q (APCMCC region), 6q, 8p, 9p (CDKN2a gene) and 17q (BRCA1 gene) regions (Table 2). In addition, our unpublished allelotyping data performed on the short arm of chromosome 3 (3p) (Wistuba et al. in preparation) and chromosome 4 (Shivapurkar et al. in press) indicate that the sizes of the deletions are bigger in successfully cultured breast cancers than in sporadic tumors. Thus, our recent findings suggest that breast tumors that can be successfully cultured tend to have more advanced genetic changes. In addition to allelic losses, alterations in microsatellite size (microsatellite alterations, MAs) are another genetic change associated with several human cancers (Fishel, 1996). The mechanisms underlying MAs are currently unknown, but they probably represent a form of genomic instability (Fishel, 1996). Although the frequency of MAs in both breast cancer cell lines and their corresponding tumors were low, cell lines demonstrated a higher incidence of MA (Table 2), and most of the MAs detected in the cell lines and their corresponding tumors were different. Although successfully cultured breast carcinomas demonstrated higher mean MA index (an indicator of overall frequency of MAs), this difference was not significant (Table 2). Genomic instability persists after tumor development, resulting in the frequent appearance of multiple subclonal populations (Nowell, 1976), and, presumably, the instability continues during culture life. Because cell cultures frequently have population doubling times considerably shorter than those of in vivo tumors, the frequency of mutational change in cultures may be more rapid than in their corresponding tumors. Although this may explain the higher incidence of MAs in breast cancer cell lines than their corresponding tumors, our data indicate breast carcinoma cultures retain many of the phenotypic and genotypic properties of their corresponding tumors for long periods of time.
5.
PORTRAIT OF SUCCESSFULLY CULTURED BREAST CANCER
Analysis of our established breast cancer cell lines and the primary tumors from which they were derived indicates a profile of the subset of primary tumors that are most likely to develop into permanent cell lines (Table 3). These features include: a) large tumor size with or without axillary lymph node metastases; b) hyperploidy or aneuploidy; c) relatively poor degree of
116
Wistuba et al.
Table 2 Comparison of properties between successfully cultured and uncultured breast carcinomas Feature
Frequency Cell Lines
Cultured Tumors
“Sporadic” Tumors(a)
Aneuploidy
93%
80%
40%(b)
Immunohistochemistry Estrogen Receptor Progesterone Receptor HER2/neu protein p53 protein
20% 20% 40% 73%
7% 20% 33% 73%
45 % (b) 65%(b) 10-30%(b) 30%
TP53 gene mutation (exons 5-10)
47%
35%
40%(b)
Loss of heterozygosity 3p 5q ( APC/MCC genes) 6p 6q 8p 9p ( CDKN2a gene) 10q (PTEN/MMCA1 gene) 11q 13q (RB gene) 17p (TP53 gene) 17q (BRAC1 gene)
71% 63% 29% 82% 81% 46% 50% 50% 46% 79% 88%
71% 63 % 29% 77% 75 % 46% 50% 50% 46% 79% 88%
50% 21%(c) 30%(b) 12%(c) 25 %(c) 0(c) 50%(b) 35% 26% 47% 28%9(c)
Fractional Allelic Loss (FAL) Index(d) Mean
0.52
0.56
0.2(c)
Microsatellite Alteration (MA) Index(e) Mean
0.04
0.006
0.002
‘‘Sporadic’’ tumors = those whose culture status is indeterminate. Data reported in the literature. (c) Cultured vs. “sporadic” tumors, P < 0.05. (d) FAL index, an indicator of overall LOH frequency. (e) MA index, an indicator of overall MA frequency.
(a)
(b)
differentiation; d) steroid receptor negative; e) HER2/neu overexpression; f) positive immunostaining detection of p53 protein expression and TP53 gene mutation; and g) multiple sites of allelic loss involving 3p, 5q, 8p, llq, 17p13 (TP53 gene) and 17q21 (BRCA1 gene) chromosomal regions. Some of these properties have been associated with a poor prognosis. Of interest, more than half of the patients who received curative intent mastectomies and whose tumors were successfully cultured died within 10 months of their surgery (Gazdar et al. 1998). Thus successful culture of a breast tumor appears to be a powerful negative prognostic factor.
117
Paired Breast Cancer Cell Lines Table 3 Portrait of successfully cultured breast carcinomas Feature
Frequency
Genetic Predispositions Poorly Differentiated Tumors (Grades II/III) Large Size (> 2 cm.) Lymph Node Metastasis (+) Poor Survival (< 29 months survival)(a)
5 / 10 15 / 21 16 / 20 11 / 16 6 / 13
Aneuploidy
12 / 15 (80%)
Estrogen/Progesterone Receptors (-) HER2/neu Overexpression p53 Immunostaining (+)
3 / 15 (20%) 5 / 15 (33%) 11 / 15 (73%)
3p LOH 5q LOH 6q LOH 8p LOH l lq LOH 9p21 ( CDKN2a gene) LOH 17p (TP53 gene) LOH 17q (BRCA1 gene) LOH
12 / 17 (71%) 7 / 11 (63%) 13 / 17 (77%) 12 / 16 (75%) 7 / 14 (50%) 6 / 13 (46%) 11 / 14 (79%) 14 / 16 (88%)
(50%) (71%) (80%) (69%) (46%)
After receiving curative intent mastectomy.
(a)
6.
SUMMARY
In summary, we have established a panel of 21 new breast cancer cell lines that included 18 cell lines derived from primary tumors and three derived from metastatic lesions, with an 11% culture success rate. For 19 of these breast cancer cell lines, we also established one or more corresponding nonmalignant cell strains or B lymphoblastoid lines. Cell lines included those from patients with germ-line BRCA1 and FHIT gene mutations. While our studies indicate that only a subset of primary breast carcinomas having several features indicative of advanced tumors with poor prognosis can be successfully cultured, the cell lines retain the properties of their parental tumors for lengthy culture periods and thus provide suitable model systems for biomedical studies of at least one major form of breast cancer.
ACKNOWLEDGMENTS Supported in part by the U.S. Army Grant DAMD17-94-J-4077, and the Komen Foundation.
118
Wistuba et al.
REFERENCES Ahmadian M, Wistuba II, Fong KM, Behrens C, Kodagoda DR, Saboorian MH, Shay J, Tomlinson GE, Blum J, Minna JD, Gazdar AF. Cancer Res 57: 3664-8, 1997. Aldaz CM, Chen T, Sahin A, Cunningham J, Bondy M. Cancer Res 55: 3976-81,1995, Amadori D, Bertoni L, Flamigni A, Savini S, De Giovanni C, Casanova S, De Paola F, Amadori A, Giulotto E, Zoli W. Breast Cancer Res Treat 28: 251-60, 1993. An HX, Niederacher D, Picard F, van Roeyen C, Bender HG, Beckmann MW. Genes Chromosomes Cancer 17: 14-20, 1996. Andersen TI, Gaustad A, Ottestad L, Farrants GW, Nesland JM, Tveit KM, Borresen AL. Genes Chromosomes Cancer; 4: 113-21, 1992. Baas IO, Mulder JW, Offerhaus GJ, Vogelstein B, Hamilton SR. J Pathol 172: 5-12, 1994. Band V, Sager R. Proc Natl Acad Sci USA 86: 1249-53, 1989. Band V, Zajchowski D, Swisshelm K, Trask D, Kulesa V, Cohen C, Connolly J, Sager R. Cancer Res 50: 7351-7, 1990. Borg A, Zhang QX, Alm P, Olsson H, Sellberg G. Cancer Res 52: 2991-4, 1992. Buckley MF, Sweeney KJ, Hamilton JA, Sini RL, Manning DL, Nicholson RI, deFazio A, Watts CK, Musgrove EA, Sutherland RL. Oncogene 8: 2127-33,1993. Cailleau R, Olive M, Cruciger QV. In Vitro 14: 911-5, 1978. Callahan R, Gallahan D, Smith G, Cropp C, Merlo G, Diella F, Liscia D, Lidereau R. Ann NY Acad Sci 698: 21-30, 1993. Devilee P, van den Broek M, Kuipers-Dijkshoorn N, Kolluri R, Khan PM, Pearson PL, Cornelisse CY. Genomics 5: 554-60, 1989. Eiriksdottir G, Sigurdsson A, Jonasson JG, Agnarsson BA, Sigurdsson H, Gudmundsson J, Bergthorsson JT, Barkardottir RB, Egilsson V, Ingvarsson S. Int J Cancer 64: 378-82, 1995. Ethier SP, Mahacek ML, Gullick WJ, Frank TS, Weber BL. Cancer Res 53: 627-35, 1993. Fishel R. J Natl Cancer Inst 88: 1608-1609, 1996. Fujii H, Gabrielson E. Genes Chromosomes Cancer 16: 35-39, 1996. Gazdar AF, Kurvari V, Virmani A, Gollahon L, Sakaguchi M, Westerfield M, Kodagoda D, Stasny V, Cunningham T, Wistuba 11, Tomlinson G, Tonk V, Ashfaq R, Leitch M, Minna JD, Shay JW. Int J Cancer 78: 766-774, 1998. Gudmundsson J, Barkardottir RB, Eiriksdottir G, Baldursson T, Arason A, Egilsson V, Ingvarsson S. Br J Cancer 72: 696-701, 1995. Kirchweger R, Zeillinger R, Schneeberger C, Speiser P, Louason G, Theillet C. Int J Cancer 56: 193-9,1994. Lasfargues EY, Ozzello L. J Natl Cancer Inst 21: 1131-47, 1958. Leibovitz A. An Atlas of Human Tumor Cell Lines. Hay R, Park J-G, Gazdar AF (eds). Academic Press: Philadelphia, pp 161-184, 1994. Levenson AS, Jordan CV. Cancer Res 57: 3071-9,1997. Li J, Yen C, Liaw D, Podyspanina K, Bose S, Wang SI, Puc J, Miliaresis C, Rodgers L, McCombie R, Bigner S, Giovanella BC, Ittmann M, Tycko B, Hibshoosh H, Wigler MH, Parsons R. Science 275: 1943-7, 1997. Loeb LA. Cancer Res 51: 3075-9, 1991. Mahacek ML, Beer DG, Frank TS, Ethier SP. Breast Cancer Res Treat 28: 267-76, 1993. McCallum HM, Lowther GW. Breast Cancer Res Treat 39: 247-59, 1996. McGuire WL, Clark GM. N Engl J Med 326: 1756-61,1992. Muleris M, Almeida A, Gerbault-Seureau M, Malfoy B, Dutrillaux B. Genes Chromosomes Cancer 10:160-70,1994. Nagai MA, Pacheco MM, Brentani MM, Marques LA, Brentani RR, Ponder BA, Mulligan LM. Br J Cancer 69: 754-8, 1994.
Paired Breast Cancer Cell Lines
119
Nowell PC. Science 194: 23-8, 1976. Ohta M, Inoue H, Cotticelli MG, Kastury K, Baffa R, Palazzo J, Siprashvili Z, Mori M, McCue P, Druck T, Croce CM, Huebner K. Cell 84: 587-97,1996. Orphanos V, McGown G, Hey Y, Boyle JM, Santibanez-Koref M. Br J Cancer 71: 290-3,1995. Petersen OW, van Deurs B, Nielsen KV, Madsen MW, Laursen I, Balslev I, Briad P. Cancer Res 50: 1257-70, 1990. Press MF, Pike MC, Chazin VR, Hung G, Udove JA, Markowicz M, Danyluk J, Godolphin W, Sliwkowski M, Akita R, Paterson MC, Slamon DJ. Cancer Res 53: 4960-70, 1993. Scherer WF, Syverton JT, Gey GO. J Exp Med 97: 695-709,1953. Sekido Y, Ahmadian M, Wistuba 11, Latif F, Bader S, Wei MH, Duh FM, Gazdar AF, Lerman MI, Minna JD. Oncogene 16: 3151-7,1998. Shivapurkar N, Sood S, Wistuba 11, Virmani AK, Maitra A, Milchgrub S, Minna JD, Gazdar AF. Cancer Res, in press. Silvestrini R, Benini E, Daidone MG, Veneroni S, Boracchi P, Cappellend V, DiFronzo G, Veronesi U. J Natl Cancer Inst 85: 965-70, 1993. Singh S, Simon M, Meybohm I, Jankte I, Jonat W, Maass H, Goedde HW. Hum Genet 90: 635-40,1993. Slamon DJ, Clark GM. Science 240: 1795-8,1988. Smith HS, Wolman SR, Hackett AJ. Biochem Biophys Acta 738: 103-23, 1984. Smith HS, Hackett AJ. J Lab Clin Med 109: 236-43, 1987. Smith HS, Wolman SR, Dairkee SH, Hancock MC, Lippman M, Leff A, Hackett AJ. J Natl Cancer Inst 78: 611-5, 1987. Sundaresan V, Chung G, Heppell-Parton A, Xiong J, Grundy C, Roberts I, James L, Cahn A, Bench A, Duglas J, Minna J, Sekido Y, Lerman M, Latif F, Bergh J, Li H, Lowe N, Ogilvie D, Rabbits P. Oncogene 1723-9, 1998. Tavassoli FA. Pathology of the breast. Appleton & Lange: East Norwalk, pp 384-90, 1992. Thompson AM, Morris RG, Wallace M, Wyllie AH, Steel CM, Carter DC. Br J Cancer 68: 64-8,1993. Tomlinson GE, Chen TT, Stastny VA, Virmani AK, Spillman MA, Tonk V, Blum JL, Schneider NR, Wistuba 11, Shay JW, Minna JD, Gazdar AF. Cancer Res 58: 3237-42,1998. Virmani AK, Fong KM, Kodagoda D, McIntire D, Hung J, Tonk V, Minna JD, Gazdar AF. Genes Chromosomes Cancer 21: 308-19, 1998. Wistuba 11, Montellano FD, Milchgrub S, Virmani AK, Behrens C, Chen H, Ahmadian M, Nowak JA, Muller C, Minna JD, Gazdar AF. Cancer Res 57: 3154-8, 1997. Wistuba II, Behrens C, Milchgrub S, Syed S, Ahmadian M, Virmani AK, Cunningham TH, Ashfaq R, Minna JD, Gazdar AF. Clinical CancerRes 4: 2931-8, 1998.
This page intentionally left blank.
Chapter 22 Ovarian Germ Cell Tumors
Masumi Sawada1 and Tsuneharu Miki2 Department of Obstetics and Gynecology and 2Department of Urology, Osaka University Medical School, 2-2 Yamadaoka Suita Osaka, Japan. Tel: 0081-6-6879-3351; Fax: 0081-66879-3359 1
1.
INTRODUCTION
Depending on their degree of differentiation, germ cell tumors may contain many different types of cell, including seminoma, embryonal carcinoma, teratoma, choriocarcinoma and yolk sac tumor. Dysgerminoma (seminoma), the most common type of human germ cell tumor, does not occur spontaneously in mice and cannot be induced experimentally in any animal (Damjanov, 1986), and choriocarcinoma is frequently found in human but not murine germ cell tumors. Consequently, human cell lines and xenografts derived from germ cell tumors are particularly valuable models (Andrews and Damjanov, 1994).
2.
PATHOLOGY
Germ cell tumors are derived from primitive germ cells. According to the concept proposed by Teilum (1965), dysgerminoma is a primitive germ cell neoplasm which has not acquired the potential for further differentiation. In contrast, embryonal carcinoma (EC) is composed of multipotential cells capable of differentiating in extragonadal directions (yolk sac tumor and choriocarcinoma) and in somatic directions (teratoma). Dysgerminoma is the most frequent malignant ovarian germ cell tumor (Scully, 1979; Talerman, 1987). The cells have large prominent round nuclei with one or two prominent nucleoli, and abundant cytoplasm which contains
J.R.W Masters and B. Palsson (eds.), Human Cell Culture Vol. II, 121 –125. © 1999 KluwerAcademic Publishers. Printed in Great Britain.
Sawada and Miki
122
glycogen and lipid. Dysgerminoma frequently occurs in a pure form, although it may be associated with other germ cell tumor elements. In occasional cases human chorionic gonadotropin (HCG) can be demonstrated within the cytoplasm of some dysgerminoma cells (Zaloudek, 1981). Lactic dehydrogenase (LDH) and its isoenzyme LDH-1 is found in the tumor cells (Awais, 1983; Fujii et al., 1985). Yolk sac tumor (YST) is a highly malignant neoplasm and the second most common ovarian malignant germ cell neoplasm occurring in pure form (Talerman, 1987). Yolk sac tumor is encountered most frequently in the second and third decades, and is very rare after the menopause, with only a few well documented cases (Talerman, 1987). Microscopically, YST usually shows a variety of histologic patterns, including microcystic, endodermal sinus, papillary, glandular-alveolar, solid, myxomatous, macrocystic, polyvesicular vitelline, hepatoid and primitive intestinal patterns (Talerman, 1987; Jacobsen and Talerman, 1989). The demonstration of alpha-fetoprotein (AFP) within the tumor tissue by immunohistochemistry and elevated levels in the serum are diagnostic. Immature teratoma is an uncommon tumor, accounting for less than 1% of ovarian teratomas (Talerman, 1992). The tumors are usually large and contain cystic areas. Microscopically they are composed of immature tissues derived from the three primitive germ layers and show varying degrees of maturity. The most common constituents are neuroectodermal tissue, different types of mesenchymal tissue and various types of epithelium. A number of marker proteins can be demonstrated in teratomas. AFP and alpha-1-antitrypsin may be found in the epithelium of glandular structures and in foci of hepatic differentiation. CEA may be found in glandular epithelium showing intestinal differentiation (Jacobsen and Talerman, 1989). Nongestational choriocarcinoma is very rare (Jacobs et al. 1982; Vance and Geisinger, 1985). Choriocarcinoma is now seen more frequently as a component of mixed germ cell tumors due to better and more extensive sampling (Talerman, 1992). The tumor produces large amounts of HCG and elevated serum levels are present. HCG is a valuable marker for patients with ovarian germ cell tumors containing choriocarcinoma. Comprehensive overviews of the pathology of human germ cell tumors have been produced by Damjanov (1983,1986), Talerman (1987,1992) and Jacobsen and Talerman (1989).
3.
CELL LINES
The origins of the published human ovarian germ cell tumor-derived cell lines are shown in Table 1.
Cell lines and xenografts derived from female germ cell tumors KURATOU
Patient age TNM category Primary site Specimen site Culture method Culture medium Antigens Number of passages Tumor pathology Xenograft pathology Ploidy Primary reference
25 T3 Ovary Primary
PA-1
12 Recurrence Ovary Ascitic fluid metastasis DIS DIS DM 170 Eagle’s MEM HLA HLA 41 249 D IT D IT Triploid Diploid Tanaka et al. Zeuthen et al. (1989) (1980)
HUOT
IMa
YST-1
YST-2
YST-3
28 Recurrence Ovary Vaginal metastasis X Ham’s F12 AFP 50 IT AC Hyperdiploid Ishiwata et al. (1985)
25 Recurrence Ovary Lymph node metastasis E RPMI 1640 HCG 22 D, C C Hypotriploid Sekiya et al. (1983)
38 Recurrence Ovary Peritoneal metastasis X Eagle’s MEM LDH isozyme 54 Y Y Normal female Sawada et al. (1982)
24 Recurrence Ovary Omental metastasis X Eagle’s MEM AFP 27 Y Y Normal female Sawada et al. (1982)
14 Recurrence Ovary Peritoneal metastasis X Eagle’s MEM AFP, HCG 28 Y, IT Y Normal female Sawada et al. (1982)
Ovarian Germ Cell Tumors
Table 1
Abbreviations: IT, immature teratoma; D, dysgerminoma; C, choriocarcinoma; Y, yolk sac tumor; EC, embryonal carcinoma; AC, anaplastic carcinoma; DIS, dissociated tissue; E, explant; X, xenograft; AFP, alphafetoprotein; HCG-b, human chorionic gonadotropin-b; LDH, lactic dehydrogenase
123
124
Sawada and Miki
The KURATOU cell line (Tanaka et al. 1989) is the only cell line with characteristics of pure dysgerminoma. In culture, small polygonal cells with a prominent nucleus predominate, admixed with a small number of polynuclear giant cells. The original tumor and the xenograft in nude mice were composed only of dysgerminoma. However, an immunohistochemical study demonstrated HCG in almost all of the cultured cells. Kimoto et al. (1975) described the establishment of a cell line from the ascites of a 63-year-old female patient with embryonal carcinoma containing yolk sac elements. However, the cell of origin of this cell line remains unclear as the cultured cells assumed the morphology of mesothelial cells or fibroblasts and there is no description of AFP production. The PA-1 cell line was established in nude mice by Giovanella et al. (1974) from the ascitic fluid of a patient with a recurrent immature teratoma of the ovary, and was characterized by Zeuthen et al. (1980). The cells have a fairly homogeneous appearance, similar to embryonal carcinoma. However, an alternative suggestion is that PA-1 may be immature neuroectodermal teratoma, because these cells are unlike other human embryonal carcinoma cells in culture, both morphologically and in their expression of characteristic surface antigen markers (Andrews and Damjanov, 1994). The PA-1 cell line has been extensively used in studies of carcinogenesis (Tainsky et al. 1984, 1988), extracellular matrix components (Fukuda et al. 1986; Kawata et al. 1991) and cell surface antigens (Andrews et al. 1996). The HUOT cell line was established by Ishiwata et al. (1985) from a recurrent human ovarian immature teratoma. The cultured cells were polygonal, columnar or spindle-like and formed round colonies. In contrast to PA-1, HUOT cells produced large amount of AFP during the stationary growth phase, characterized by the formation of cysts. The culture material and primary ovarian tumor were composed of tridermal organoids. However, the cultured cells produced anaplastic carcinomas following xenotransplantation to nude mice. Kikuchi et al. (1984) established and characterized the YK cell line derived from an immature teratoma. The YK cells grew as xenografts with the histological appearance of embryonal carcinoma. YK cells produce AFP and high levels of LDH. However, the YK cell line is no longer available (personal communication). The IMa cell line was established by Sekiya et al. (1983) from a xenograft, the histology of which was pure choriocarcinoma. The original tumor was a dysgerminoma containing a small area of choriocarcinoma and IMa cells produce HCG. Their response to interferons was studied (Sekiya et al. 1987). The IMa cell line provides a useful tool for clarifying the biological differences between nongestational and gestational choriocarcinoma cells.
Ovarian Gem Cell Tumors
4.
125
XENOGRAFTS
In addition to the small number of cell lines derived from female germ cell tumors, there are also a small number of xenografts (Sawada et al. 1982). YST1, 2 and 3 xenografts retain a histology similar to that of the original tumor, and their main characteristics are included in Table 1.
REFERENCES Andrews PW and Damjanov I. In: Atlas of human tumor cell lines (Hay RJ, Park JG and Gazdar A eds.) Academic Press, San Diego, p.443, 1994. Andrews PW et al. Int J Cancer 66: 806, 1996. Awais GM. Obstet Gynecol 61: 99, 1983. Damjanov I. In: The human teratomas (Damjanov I, Knowles B and Solter D eds.) Human Press, Clifton, New Jersey, p.23, 1983. Damjanov I. In: Pathology of the testis and its adnexa (Talerman A and Roth LM eds.) Churchill Livingstone, New York, p.193, 1986. Fujii S et al. Gynecol Oncol 22: 65,1985. Fukuda MN et al. J Biol.Chem 261: 5145,1986. Giovanella BC et al. J. Natl Cancer Inst 52: 921, 1974. Ishiwata I et al. J Nat1 Cancer Inst 75: 411, 1985. Jacobs AJ et al. Obstet Gynec Surv, 37: 603, 1982. Jacobsen GK and Talerman A. Atlas of germ cell tumours (Jacobson GK and Talerman A eds.) Munksgaard, Copenhagen, 1989. Kawata M et al. Cancer Res 51: 2655, 1991. Kikuchi Y et al. Cancer Res 44: 2952, 1984. Kimoto T et al. Acta Pathol Jap 25: 89, 1975. Sawada M et al. Gynecol Oncol 13: 220, 1982. Scully RE. In: Atlas of tumor pathology, second series, fascicle 16, Armed Forces Institute of Pathology, Washington D.C., 1979. Sekiya S et al. In Vitro 19: 489, 1983. Sekiya S et al. Differentiation 33: 266, 1987. Tainsky MA et al. Science 225: 643, 1984. Tainsky MA et al. Anticancer Res 8: 899, 1988. Talerman A. In: Blaustein's pathology of the female genital tract (Kurman RJ ed.) Springer Verlag, New York, 1987. Talerman A. In: Gynecological tumors. (Sasano N ed.) Springer Verlag, Berlin Heidelberg, p.165, 1992. Tanaka K et al. Acta Obst Gynaec Jpn 41: 1360, 1989 Teilum G. Acta Pathol Microbiol Scand 64: 407, 1965. Vance RP and Geisinger KP. Cancer 56: 2321, 1985. Zaloudek JC et al. Am J Surg Path 5: 361, 1981. Zeuthen J et al. Int J Cancer 25: 19, 1980.
This page intentionally left blank.
Chapter 23 Testicular Germ Cell Tumors
Martin F. Pera Institute of Reproduction and Development, Monash Medical Centre, Monash University, Clayton, Victoria 3168, Australia. Tel: 0061-3-9594- 7318; Fax: 0061-3-9594- 7311; E-mail:
[email protected]. au
1.
INTRODUCTION
Experimental investigation of germ cell tumors began in the mouse, with the work of Barry Pierce and Roy Stevens (reviews, Pierce et al. 1978, Stevens, 1983). These investigators made major contributions to the stem cell concept of cancer differentiation and our understanding of the pathogenesis of germ cell tumors. Following the development of methods for in vitro culture of mouse teratocarcinomas, these cell lines gained wide acceptance as models for the early embryo (Martin, 1980). Perhaps the most significant contribution of teratocarcinoma studies in the mouse was to provide the experimental background and intellectual impetus for the derivation of diploid embryonic stem cells (reviewed by Evans and Kaufman, 1983), one of the most important tools in experimental biology today. Human germ cell tumors were among the many types of cancer from which Fogh and co-workers (Fogh and Trempe, 1975) developed permanent cell lines in the 1970s. Other workers who described the derivation of cell lines from human germ cell tumors include Bronson et al. (1983), Cotte et al. (1981) and Pera et al. (1987). Andrews and co-workers, using cell lines established by Fogh and Bronson, were the first to undertake the systematic characterization of cell lines from germ cell tumors of the testis (Andrews et al. 1980). The first reports of clonally derived human teratoma cell lines capable of differentiation into defined cell lineages were in 1984 (Andrews et al. 1984; Thomson et al. 1984) and ironically these were both based on one of the oldest cell lines, the Tera-2 line derived by Fogh (above). Since then, these lines and others have
J.R. W. Masters and B. Palsson (eds.), Human Cell Culture Vol. II, 127–140. ©1999 KluwerAcademic Publishers. Printed in Great Britain.
128
Pera
been used in a variety of studies, most of which relate to cell differentiation and chemosensitivity. The recent derivation of diploid embryonic stem cell lines from monkey blastocysts (Thomson et al., 1995) has shown that embryonal carcinoma cells are indeed similar to their normal embryonic counterparts, validating their use as a model for embryonic cell differentiation and confirming Pierce’s concept of malignancy as a caricature of normal differentiation. The properties of cell lines derived from human germ cell tumors have been the subject of a previous review (Andrews and Damjanov, 1994).
2.
DERIVATION OF CELL LINES
Primary culture of germ cell tumors of the testis was reviewed by Pera (1991). The two major classes of germ cell tumors of the testis are the seminomas and non-seminomas, and while there are no convincing reports of cell lines derived from the former, cell lines have been derived from most histological subtypes of non-seminomas: teratocarcinoma, embryonal carcinoma, yolk sac tumors, choriocarcinoma and differentiated teratoma. The majority of cell lines have been established directly from biopsy specimens, as disaggregated tissue fragments, under routine cell culture conditions employing standard media supplemented with fetal calf serum in plastic vessels. In some cases, feeder cell layer support has been used during establishment and subsequent cultivation of the cell lines, the rationale for this being that as feeder cell layers were required for establishment of diploid embryonic stem cells, their inclusion would provide an environment which was less selective against cell lines with extensive differentiation capacity. The available data do not permit a definitive assessment of this hypothesis but it is clear that some cell lines which are pluripotent definitely require feeder support, similar to stem cell lines derived from monkey blastocysts. Many investigators have found that cell lines from human germ cell tumors are much easier to passage as aggregates than as single cells. Culture at high density often favours stem cell renewal, whereas culture at lower density can lead to death or differentiation. Thus routine passage may be best achieved by gentle use of trypsin, scraping, or by dispase treatment. For culture at clonal density of embryonal carcinoma, the use of feeder cells such as Swiss 3T3 embryo fibroblasts is strongly recommended for most cell lines, whether or not they require feeder layers for routine passage. Extended serum-free cultivation of germ cell tumor cell lines is difficult. IGF-2 is critical for the survival of these cells (Biddle et al. 1988). Vitronectin is a serum protein necessary for adhesion and subsequent growth of embryonal carcinoma and is therefore a useful addition to serum-free media (Cooper and Pera, 1987). Some types of yolk sac carcinoma can survive and grow in the absence of feeder cell layers, probably due to their production of vitronectin and other factors. In this case, the only protein supplements used are
Testicular Germ Cell Tumors
129
transferrin, insulin, and albumin (Cooper and Pera, 1987). These same supplements may be used to support survival and slow growth of certain embryonal carcinoma cell lines after initial subculture in serum-containing medium; this approach is useful for experiments aimed at harvest and subsequent purification of proteins secreted by the cell lines.
3.
DO THE CELL LINES MODEL THE CLINICAL SPECTRUM OF GERM CELL TUMORS ?
Table 1 lists information relating to the derivation of the available cell lines from germ cell tumors of the testis. It may be noted that although seminoma has been present in many of the specimens from which lines have been derived, no cell line with the properties of seminoma has been described. No cell lines corresponding to carcinoma in situ, the precursor lesion of all types of germ cell tumors, are available either. Most classes of non-seminoma are well represented with the exception of choriocarcinoma, although there are a number of cell lines derived from gestational trophoblastic tumors (see Chapter 24). Most cell lines from non-seminomas are equivalent to embryonal carcinomas, that is, they are composed almost entirely of stem cells which do not differentiate under basal conditions, though some may respond to inducers of differentiation such as retinoic acid. A few lines show spontaneous differentiation in culture. Yolk sac carcinoma is not uncommon and there are several cell lines representative of the various histological subtypes of this category. There are cell lines derived from testicular primary tumors as well as metastatic lesions, and there are cell lines derived from patients prior to and after chemotherapy. It has not proven necessary in most cases to derive xenografts from the tumor prior to the establishment of cell lines. However, in several cases a mixed cell population from a given isolate has been cloned and found to contain cells with strikingly different properties. For example, the line Tera-2 was only found to contain pluripotent cells after it was subjected to clonal analysis, and the cell line GCT27 gave rise to two subclones, one pluripotent and the other nullipotent. Some cell lines would appear to represent differentiated but immortalized cell populations derived from pluripotent cells present in the original tumors (Andrews et al. 1996). In none of the lines reported have DNA analyses or other genetic markers been used to confirm the origin from a particular patient. The cell line NCCIT is of interest because while it is clearly pluripotent, its phenotype is not that of the classical embryonal carcinoma cell. It has long been speculated that seminoma may progress to embryonal carcinoma, and it is possible that this cell line represents an intermediate in that progression. In embryonal carcinoma, study of the cellular immunophenotype in vitro and in vivo using stem cell specific markers has provided a set of criteria for
130
Pera
Table 1. Origins of cell lines derived from human germ cell tumors Cell line
Biopsy site/ patient age
Tumor diagnosis
Tera-1 lung/47 Tera-2 lung/22 SuSa testis130 833K abdomen/l9 1242B testis123 12550 testis122 1156QE testis/22 577MF forehead/24 577ML lung/24 577MR rpln/24 2044L rpln/24 2061H lung/19 2102Ep testis/23 2102ERP rpln/23 U1161 med/26 1218E testis/23 1428A testis/22 1242B testi/23 LICR-LON-HT1 testis/22 LICR-LON-HT3 testis/26 LICR-LON-HT5 testis/24 LICR-LON-HT-7 testis/32 LICR-LON-HT39/7 testis/40 ER testis 1GH testis 1HL testis 1075Hep liver 1075 Lung lung 1777N-Pr testis/25 1777N-RP rpln/25 rpln25 1777N-RPdiff 1685 M 1411HP testis/17 1411HRQ rpl/17 ITO testis/27 NEC-14 testis126 NEC-15 testis120 NEC-8 testis124 HAZ-1 lung/39 HAZ-2 lung/39 thigh/39 HAZ-3 rpln UM-TC-1
EC EC,T,YS EC,YS T, YS, SE EC, YS EC, CH EC T D IT D IT D EC, T, YS, SE
GCT 27 GCT 35 GCT 44
EC, T EC, T EC, YS
testis testis paln
EC, T EC, T EC, T EC,T,C,SE EC EC,T,SE EC,CH,SE EC, T EC, T EC, T EC EC, T EC,T,YS EC,T,YS EC,T, IT, SE EC, SE EC, YS EC EC,T,CH EC, YS EC,T,YS,SE EC, YS EC EC, T EC, T EC, T EC, CH EC, CH EC EC
Culture methodology D D D D,E D,E D,E D,E D,E D,E D,E D,E D,E D,E D,E D,E D,E D,E D,E D D D D X D,E D,E D,E D,E D,E D,E D,E D,E D,E E E
D D D
Reference Fogh and Trempe, 1975 Fogh and Trempe, 1975 Hogan et al. 1977 Bronson et al. 1980 Wang et al. 1980 Wang et al. 1980 Andrews et al. 1980 Wang et al. 1980 Wang et al. 1980 Wang et al. 1980 Wang et al. 1980 Wang et al. 1980 Wang et al. 1980 Wang et al. 1980 Sundstrom et al. 1980 Wang et al. 1980 Wang et al. 1981 Wang et al. 1980 Cotte et al. 1981 Cotte et al. 1981 Cotte et al. 1981 Cotte et al. 1981 Cotte et al. 1981 Harzmann et al. 1982 Harzmann et al. 1982 Harzmann et al. 1982 Bronson et al. 1983 Bronson et al. 1983 Bronson et al. 1983 Bronson et al. 1983 Bronson et al. 1983 Bronson et al. 1984 Vogelzanget al. 1985 Vogelzang et al. 1985 Sekiya et al. 1985 Sekiya et al. 1985 Sekiya et al. 1985 Sekiya et al. 1985 Oosterhuis et al. 1985 Oosterhuis et al. 1985 Oosterhuis et al. 1985 Grossman and Wedemeyer, 1986 Pera et al. 1987 Pera et al. 1987 Pera et al. 1987 Continued on next page
Testicular Germ Cell Tumors
131
Table 1. Continued Cell line GCT 46 GCT 48 GCT 72 H12.1 H12.5 H12.7 JHTK-1 NCC-EC-1 NCC-EC-2 NCC-EC-3 NCC-EC-IT 169A 218A 228A 240A
Biopsy site/ patient age
Tumor diagnosis
Culture methodology
lung testis testis (scrotum) testis testis testis testis testis123 testis/28 testis/44 med/24
EC, YS EC EC EC, T, CH, SE EC. T, CH, SE EC, T, CH, SE EC EC, T EC, SE EC,CH,T EC, YS, T EC, SE EC
D D D
X,D E E E E
Reference Pera et al. 1987 Pera et al. 1987 Pera et al. 1987 Casper et al. 1987 Casper et al. 1987 Casper et al. 1987 Yamazaki et al. 1987 Teshima et al. 1988 Teshima et al. 1988 Teshima et al. 1988 Teshima et al. 1988 Houldsworth et al. 1997 Houldsworth et al. 1997 Houldsworth et al. 1997 Houldsworth et al. 1997
Abbreviations: rpln, retroperitoneal lymph node; paln, para-aortic lymph node; med, mediastinal tumor; SE, seminoma; EC, embryonal carcinoma; IT, immature teratoma; T, teratocarcinoma (tumor containing EC stem cells plus differentiated tissue); YS, yolk sac carcinoma; CH, choriocarcinoma; E, explant of tumor fragments; D, dissociated tissue; X, cell line established from xenograft tumor in immunosuppressed mouse. 1GH and HL have identical DNA fingerprints.
the validation of the cell lines. Established stem cell markers include SSEA-3, SSEA-4, TRA-1-60, GCTM-2 and CD30 (Andrews et al. 1996; Pera et al. 1997). Some cell lines formerly regarded as embryonal carcinoma have been reassigned to other categories on the basis of marker expression. The identification of specific markers for yolk sac carcinoma and choriocarcinoma stem cells is less advanced, but there are many gene products whose expression is characteristic of the later stages of differentiation along these cell lineages (eg alphafetoprotein, human chorionic gonadotropin). Yolk sac or choriocarcinoma cell lines may express such markers, though they do not always do so.
4.
APPLICATIONS
The principal applications of cell lines from germ cell tumors of the testis are as models for human embryogenesis and in the study of tumor pathogenesis and sensitivity to treatment. Table 2 lists cell lines which have been used in recent studies as models of early human development. In terms of differentiation capacity, only a few cell lines have been shown to exhibit pluripotentiality, the ability to differentiate
132
Table 2. Selected applications of cell lines in studies of differentiation Cell line
Phenotype
Differentiation in vitro Inducer Cell types
Xenograft histology/ differentiation
SuSa 8333 2102Ep
EC EC EC
NONE NONE LDC
NONE NONE UDC
ND EC EC
1411H NTera-2
YS EC
NONE RA
YS/EPO, AFP neurons + UDC
EC, YS/AFP, HCG EC, ENDO, NEURO
UDC UDC UDC neurons + UDC
Tera-2 clone 13
EC
NEC-14 GCT 27C-4 GCT 27X-1
EC EC EC
HMBA BMP-7 activin RA activin HMBA NONE LDC
GCT 35 GCT48 GCT 44
YS/PE
GCT 46 GCT 85 GCT 72 JHTK-1
YSPE YSPE YS/PE CH
EC/NEURO
basal basal basal basal dbcAMP
PE PE VYS/AFP + others TB/HCG TB/HCG
YST ND YST (solid form)/AFP CH/HCG
EC/AFP,HCG EC YST/AFP
Andrews et al. 1980 Andrews et al. 1980 Andrews et al. 1982 Damjanov and Andrews 1983 Lanford et al. 1991 Andrews et al. 1984 Andrews et al. 1990 Andrews et al. 1994 Caricasole et al. 1997 Thomson et al. 1984 Caricasole et al. 1997 Sekiya et al. 1990 Pera et al. 1989 Pera et al. 1989 Roach et al. 1994 Pera et al. 1987 Pera et al. 1987 Pera et al. 1987; Cooper and Pera, 1987;Roach et al. 1994 Pera et al. 1987; Roach et al. 1994 Pera, unpublished Pera et al. 1987; Roach et al. 1994 Yamazaki et al. 1987 Continued on next page
Pera
EC ECIAFP TC, ENDO, ECTO, MESO, YS, SYT/AFP, HCG
RA basal NONE basal
UDC NONE basal or endo neurons, UDC endo YS, TB NONE PE, VYS/ECM
Reference
Cell line
Phenotype
Differentiation in vitro Inducer Cell types
Xenograft histology/ differentiation
Reference
NCCIT
SE/EC
basal
EC Multiple UDC
Teshima et al. 1988 Damjanov et al. 1993
AFP,HCG RA
Testicular Germ Cell Tumors
Table 2. Continued
**Abbreviations: EC, embryonal carcinoma; ND, not done; LDC, low density culture; YS, yolk sac; EPO, erythropoietin expression; AFP, alphafetoprotein expression; HCG, human chorionic gonadotropin expression; RA, retinoic acid; UDC, unclassified differentiated cell, a cell which has lost characteristic stem cell markers but whose lineage is uncertain; ENDO, endodermal differentiation; NEURO, neuronal differentiation; HMBA, hexamethylene bisacetamide; MESO, mesodermal differentiation; SYT syncytiotrophoblast present; TB, trophoblast differentiation; PE, primitive endoderm; SE/EC; seminoma/embryonal carcinoma postulated intermediate; ECM, extracellular matrix production; CH, choriocarcinoma present (cytotrophoblast and syncytiotrophoblast); dbcAMP, dibutyryl cyclic AMP.
133
134
Pera
into a wide variety of cell types. These include two derivatives of Tera-2, GCT 27X-1, and NCCIT; the latter has not been cloned and thus rigorously shown to contain pluripotent cells. Several well-characterized systems for the in vitro differentiation of pluripotent cells exist, based upon treatment with retinoic acid, hexamethylene-bisacetamide, or members of the transforming growth factor beta superfamily. The primary endpoints are neural or endodermal differentiation, but in most sytems studied there are a range of cells generated which, while clearly distinct from stem cells, have not been unambiguously identified. It is likely that these represent primitive progenitor cells for multiple tissue lineages; this area will provide a fertile ground for future research as basic embryological studies produce better markers for these cell types. Although it is not yet clear which cell type in the early embryo the embryonal carcinoma stem cell corresponds to, available evidence indicates that the pluripotent human embryonal carcinoma cells are close in marker expression and growth requirements to diploid embryonic stem cells derived directly from monkey blastocysts. Yolk sac carcinoma cells express markers of epithelia, lack characteristic embryonal carcinoma cell markers, and may synthesize transcription factors, extracellular matrix molecules, and secreted serum proteins characteristic of the secretory epithelial cells of the secondary yolk sac in the human embryo. The yolk sac carcinoma cell lines probably represent various stages in endodermal differentiation. Some may provide good models for paracrine interactions between the primitive endoderm and embryonic ectoderm, the tissue destined to give rise to the embryo proper. More recently cell lines have been used to study genes involved in germ cell tumor pathogenesis. The cell lines are somewhat limited as models, since they represent a late stage in the natural history of the tumors. Their increasing application in the molecular analysis of pathogenesis reflects difficulties with other approaches. For example, the mouse is of limited use as a model for human germ cell tumors, studies of familial susceptibility have not yet yielded good clues as to the critical genes involved, and there is a real need for experimental systems to test hypotheses generated from cytogenetic investigations. Table 3 lists some of the features of the available cell lines. The key genetic lesions in the development of germ cell tumors of the testis remain controversial, despite a good deal of molecular cytogenetic analysis, and there are no mechanistic studies. An isochromosome of 12p is found in most testicular germ cell tumors; and even in those which lack it, there is some form of overrepresentation of sequences on the short arm of chromosome 12. Most cell lines examined also have isochromosome 12p or some form of overrepresentation of 12p, usually in its entirety. The consistent retention of these lesions during extended propagation in vitro might suggest that they are of functional relevance to the growth of the cultured cells, but this point has not been systematically addressed. Specific genes mapping to the short arm of
Cell line
Drug sensitivity Parent Sublines
SuSa 833K
CP CP
2102Ep
CP
CP res CP res
Genetics and gene expression Oncogene/others 12p cyclin D2 wt p53+, Bax+, ki-ras+
i12p+
cycd2+
ki-ras+, GDF3+
i12pt
cycd2 +
i12p-
cycd2+
1411H NTera-2
Bax+, ki-ras+ GDF-3 +
Tera-2
LOH at llp, Wilms locus; ki-ras+ ki-ras+; LOH 11p
Tera-1
CP
CP res
i12pt
cycd2 +
Other features
References
Walker et al. 1990 Hsp 27 overexpressing clones; Dmitrovsky et al. 1990; TNR expansion Reilly, 1993; Chresta et al. 1996; Richards et al. 1996; Houldsworth et al. 1997; King et al. 1997 Oosterhuis et al. 1985; Dmitrovsky et al. 1990; Caricasole et al. 1998; Houldsworth et al. 1997 Sicinski et al. 1996; Henegariu et al. 1998 RA resistant sublines; HOX Dmitrovsky et al. 1990; gene cluster activation by RA Simeone et al. 1990; Moasser et al. 1996; in parent Sicinski et al. 1996; Boersma et al. 1997; Caricasole et al. 1998 Dmitrovsky et al. 1990; Smith and Rukstalis, 1995
Continued on next page
135
Oosterhuis et al. 1985; Dmitrovsky et al. 1990; Smith and Rukstalis, 1995; Timmer-Bosscha et al. 1993
Testicular Germ Cell Tumurs
Table 3. Selected applications of cell lines in studies of drug response, gene expression and cancer genetics
Tera-2 clone 13
Other features
Relaxation of imprinting; GDF-3 + N-myc + ; ¯ diff
NEC-14 GCT27C-4
Genetics and gene expression Oncogene/others 12p cyclin D2
136
Table 3. Continued Drug sensitivity Cell line Parent Sublines
CP,EP
GCT 27X-1
CP res
wt p53+, Bax+; GDF-3 +
GDF-3 +
i12p+
cycD2+
CD30, CD30 ligand+
i12p+
cycD2+
CD30, CD30 ligand +
GCT 35
CP
GDF-3+
cycD2+
CD30, CD30 ligand +
GCT 48
CP
GDF-3 +
cycD2+
CD30, CD30 ligand +
GCT 44
CP
GDF-3-
cycD2+
CD30 ligand+
GCT 46
CP
i12p-
cycD2+
References Caricasole et al. 1998; Rachmilewitz et al. 1996 Hasegawa et al. 1991; Hara et al. 1993 Pera et al. 1987 Chresta et al. 1996; Kelland et al. 1992; Henegariu et al. 1998 Pera et al. 1997; Caricasole et al. 1998; Pera, unpublished Henegariu et al. 1998; Pera et al. 1997; Caricasole et al. 1998; Pera, unpublished Pera et al. 1997; Pera unpublished Pera et al. 1997; Pera, unpublished Pera et al. 1997; Caricasole et al. 1998; Henegariu et al. 1998; Pera, unpublished Pera et al. 1987;
Pera
Pera, unpublished Continued on next page
Cell line
Drug sensitivity Parent Sublines
Genetics and gene expression Oncogene/others 12p cyclin D2
GCT 85 GCT 72
GDF-3 +
i12p+
NCCIT
GDF-3 +
i12p-
cycD2+ cycD2-
Other features CD30-, CD30 ligand+
References
Testicular Germ Cell Tumors
Table 3. Continued
Pera, unpublished Henegariu et al. 1998; Pera et al. 1997; Pera, unpublished Damjanov et al. 1993
Abbreviations: CP, cisplatin sensitive relative to other types of cultured human tumor or diploid fibroblast. Many if not all cisplatin-sensitive cell lines show varying degrees of cross sensitivity to other DNA-damaging agents; CP res, cisplatin resistant subline; EP, etoposide sensitive; wt53+, wild type p53 gene expressed; Bax+, Bax-2 gene expressed; ki-ras+, Kirsten ras gene expressed; GDF-3+, GDF-3 expressed; HOX, homeobox; i12p+, isochromosome of 12p present; cycD2+, cyclin D2 expressed; TNR, trinucleotide repeat; RA, retinoic acid; LOH, loss of heterozygosity; N-myc+, n-myc gene expressed and decreased on induction of differentiation; CD30, CD30 ligand+, CD30 and its ligand expressed.
137
138
Pera
chromosome 12 which have been investigated in the pathogenesis of germ cell tumors include cyclin D2, the ki-ras oncogene, and the TGF-b superfamily member GDF-3. Cyclin D2 expression has been reported in a number of cell lines. Ki-ras is present in multiple copies and is expressed in the cell lines investigated, but there is no evidence that it is mutated. Many EC cell lines express GDF-3; this is a marker for immortalized pluripotent cells whose expression is conserved from mouse to man and which maps to 12p. CD30 is a tumor necrosis factor receptor superfamily member which is a specific marker for human embryonal carcinoma cells. It may play some role in stem cell renewal; and it is known to be expressed by some cell lines. Some cell lines retain other genetic lesions characteristic of the cancers in vivo, such as trinucleotide repeat expansion, loss of heterozygosity at certain loci, and relaxation of imprinting. Molecular pharmacologists have studied the cell lines to elucidate the basis of their unusual chemosensitivity. The sensitivity of the cells to chemotherapeutic agents has been well documented (Walker et al. 1987), and drug resistant sublines have been developed from several cell lines (see Table 3). Although the basis of this sensitivity is not yet clear, current thinking centers around the notion that the cells are particularly sensitive to apoptosis induced by DNA damage. This may be due to the low levels of XPA protein expressed by testis tumor cells (Köberle et al. 1999).
5.
PROBLEM AREAS
The main difficulties in working with cell lines from germ cell tumors of the testis relate to pluripotentiality. This is a property which may be stably preserved during many generations in cell culture, but as in the case of germ line competence in mouse embryonic stem cells, sub-optimal conditions may select for cells with limited differentiation capacity. Thus a degree of care in handling the cell lines is required, and it is advisable to check the phenotype from time to time using available stem cell markers such as TRA-1-60, the cell surface proteoglycan recognised by GCTM-2, or cell surface CD30. Many of the available cell lines have not been subjected to rigorous clonal analysis, so that they may consist of mixed populations of cells with different developmental properties. Certain lines appear to represent differentiated cell populations derived from pluripotent cells within the original isolate, as discussed above.
6.
SPECIAL FEATURES AND FUTURE PROSPECTS
Most biological studies relate to the capacity of cell lines to differentiate. The most useful lines in this regard are the Tera-2 derivatives N-Tera 2 and Tera2
Testicular Gem Cell Tumors
139
clone 13, GCT 27X-1, and NCCIT N-Tera2 and its clones have been particularly useful in the study of neuronal differentiation and there are sublines which are considered to be committed to this fate. However, as noted above, even among the few well studied pluripotent cell lines there are a number of differentiation pathways which remain uncharacterized. Cell lines exist which may be useful for the study of the genetic control of differentiation pathways. For example, there are derivatives of N-Tera2 which are resistant to retinoic acid induced differentiation. GCT 27C-4 and GCT 27X-1 are sister clones derived from the same parental strain which show different capacity for undergoing differentiation in vitro and in vivo.
REFERENCES Andrews PW et al. Int J Cancer 26: 269, 1980. Andrews PW Int J Cancer 30: 567, 1982. Andrews P. Dev Biol 103: 285, 1984. Andrews PW et al. Lab Invest 50: 147, 1984. Andrews PW et al. Differentiation 43: 13 1, 1990. Andrews PW and Damjanov. In: Cell lines from human germ-cell tumours. In Atlas of human tumour cell lines. eds. Hay R et al. New York, Academic Press, 1994. Andrews PW et al. Lab Invest 71: 243, 1994. Andrews PW et al. Int J Cancer 66: 806,1996. Biddle C et al. J Cell Sci 90: 475, 1988. Boersma AWM et al. Cytometry 21: 275, 1997. Bronson DL et al. Cancer Res 40: 2500,1980. Bronson DL et al. In vitro differentiation of human embryonal carcinoma stem cells. In: Teratocarcinoma Stem Cells L. Silver, G. Martin and S. Strickland, (eds.) Cold Spring Harbor Press, Cold Spring Harbor, 1983. Bronson DL et al. J Gen Virol 65: 1043, 1984. Caricasole AAD et al. Analysis of the response of human embryonal carcinoma cells to activin A. In: T Aono, H. Siguno, and W.W. Vale, (eds.) Inhibin, activin and follistatin: recent advances and future views. Springer-Verlag, New York , 1997. Caricasole AAD et al. Oncogene 16: 95 1980. Casper J et al. Int J Androl 10: 105, 1987. Chresta CM et al. Cancer Res 56: 1834, 1996. Cooper S and Pera, MF. Development 104: 565,1987. Cotte C et al. Cancer Res 41: 1422, 1981. Cotte C et al. In Vitro 18: 739, 1982. Damjanov I and Andrews PW. Cancer Res 43: 2190,1983. Damjanov I et al. Lab Invest 68: 220,1993. Dmitrovsky E et al. Oncogene 5: 543,1990. Evans MJ and Kaufman, MH. Cancer Surveys 2: 185,1983. Fogh J and Tempe, G. New human tumor cell lines. In Human Tumor Cells in Vitro. J. Fogh, (ed.) 115–159, Plenum, New York, 1975. Grossman Hara E et al. Oncogene 8: 1023, 1993. Harzmann R et al. J Urol 128: 1055,1982.
140
Pera
Hasegawa T et al. Differentiation 47: 107, 1991. Henegariu 0 et al. JMol Med 76: 648, 1998. Hogan B et al. Nature 270: 515, 1977. Houldsworth J et al. Cell Growth &Differentiation 8: 293, 1997. Kelland LR et al. Cancer Res 52: 1710, 1992. King BL et al. Cancer Res 57: 209, 1997. Koberle B et al. Current Biology 9: 273, 1999. Lanford RE et al. In vitro Cell Dev Biol 27A: 205, 1991. Martin GR. Science 209: 768, 1980. Moasser MM et al. Differentiation 60:251, 1996. Oosterhuis JW et al. Int J Cancer 34: 133 1984. Oosterhuis JW et al. Cancer Genet Cytogenet 15: 99, 1985. Pera MF et al. Int J Cancer 40:334, 1987a. Pera MF et al. Cancer Res 47: 6810, 1987b. Pera MF et al. Differentiation 42:10, 1989. Pera MF. Testicular germ cell tumours. In: JRW Masters, (ed.), Human cancer in primary culture. Kluwer, Dordrecht, 1991. Pera MF et al. Lab Invest 76: 497, 1997. Pierce GB et al. Cancer: a problem of developmental biology. Prentice-Hall, Englewood Cliffs, New Jersey. 1978. Rachmlewitz J et al. Oncogene 13:1687,1996. Reilly PA et al. Cancer Genet. Cytogenet. 68: 114, 1993. Richards EH et al. Cancer Res 56: 2446,1996. Roach S et al. Exp Cell Res 215: 189, 1994. Sekiya S et al. Differentiation 29: 259, 1985. Sekiya S et al. Gynecol Oncol 36: 69, 1990. Sicinski P et al. Nature 384: 470, 1996. Simeone A et al. Nature 346: 763, 1990. Smith RC and Rukstalis DB. J Urol 153: 1684, 1995. Stevens LC. The origin and development of testicular, ovarian and embryo-derived teratomas. In Teratocarcinoma Stem Cells L. Silver, G. Martin and S. Strickland, (eds.) Cold Spring Harbor Press, Cold Spring Harbor, 1983. Sundstrom C et al. Acta Pathol Microbiol Scand Sect. A 88: 189, 1980. Teshima S et al. Lab Invest 59: 328, 1988. Thompson S et al. J Cell Sci 72: 37, 1984. Thomson JA et al. Proc Natl Acad Sci USA 927844,1995. Timmer-Bosscha H et al. Cancer Res 53: 5707, 1993. Walker MC et al. J Natl Cancer Inst 79: 213, 1987. Walker, MC et al. Eur J Cancer 26: 742, 1990. Vogelzang NJ et al. Cancer 55: 2584, 1985. Wang N et al. Cancer Res 40: 796, 1980. Wang N et al. Cancer Res 41: 2135, 1981. Yamazaki H et al. J Urol 137: 548, 1987.
Chapter 24 Choriocarcinoma
Vadivel Ganapathy1,2, Puttur D. Prasad1,2 and Frederick H. Leibach1 Departments of 1 Biochemistry and Molecular Biology and 2Obstetrics and Gynecology, Medical College of Georgia, Augusta, GA 30912-21 00. Tel: 001 -706- 721-7652; Fax: 001 -706- 721-6608
1.
INTRODUCTION
Trophoblast cells represent the first epithelium of embryogenesis and form the functional unit of the placenta. There are several types of trophoblast cells (1,2). Most of the chorionic villi are lined by two distinct trophoblast layers: an inner cytotrophoblast layer and an outer syncytiotrophoblast layer. The cytotrophoblasts in the inner layer are highly proliferative stem cells. The syncytiotrophoblast is a multinucleated cell layer formed by the fusion of differentiated cytotrophoblasts and represents the outermost layer of the placenta that is in direct contact with the maternal blood. The syncytiotrophoblast performs a number of functions essential for the maintenance of pregnancy and for the growth and development of the embryo. This includes the production of various peptide hormones that are specific for the placenta and a number of steroid hormones, principally progesterone. Another obligatory function of this cell layer is to transfer essential nutrients (including glucose, amino acids, vitamins and minerals) from the mother to the developing embryo and to remove metabolic waste products. A distinct subset of chorionic villi, identified as anchoring villi, are involved in the attachment of the placenta to the uterine wall. The cytotrophoblast stem cells lining these anchoring villi give rise to extravillous cytotrophoblasts that are highly migratory, proliferative, and invasive. These cells migrate out of the tips of anchoring villi and invade the uterine decidua, thus facilitating the attachment of the placenta to the uterine wall. Choriocarcinoma is an uncommon and highly malignant tumor of trophoblast cells. Choriocarcinoma consists of two distinct forms: gestational and
J.R. W Masters and B. Palsson (eds.), Human Cell Culture Vol. 11, 141 –147. © 1999 Kluwer Academic Publishers. Printed in Great Britain.
142
Ganapathy et al.
nongestational (3-5). Gestational choriocarcinoma arises from or in association with a gestational event. In this form, the placenta is the site of origin of the tumor. The incidence increases after spontaneous abortions or a molar pregnancy or ectopic pregnancy. Neonatal or infantile choriocarcinoma is a rare form of gestational choriocarcinoma that occurs in newborns and infants. In these cases, the placenta is the primary site of the tumor, but it metastasizes to other organs of the developing fetus and presents as choriocarcinoma in the neonatal or infantile stage. Nongestational choriocarcinoma is unrelated to pregnancy and occurs in extraplacental tissues of a nongravid person (male or female) due to gonadal or extragonadal germ cell tumor. Testis in the male and ovary in the female are the principal sites of nongestational choriocarcinoma (see Chapters 22 and 23). Extragonadal sites of nongestational choriocarcinoma include mediastinum, retroperitoneum, esophagus, stomach (see Chapter 29), lung, kidney, and bladder. In all cases of choriocarcinoma, gestational or nongestational, the tumor is characterized by the secretion of chorionic gonadotropin, resulting in increased levels in the serum. Other trophoblast-specific markers such as the pregnancy-specific glycoprotein and the placental isoform of alkaline phosphatase provide additional tools for diagnosis and monitoring of choriocarcinoma.
2.
MAINTENANCE AND CULTURE CONDITIONS
The first report on the successful transfer and maintenance of choriocarcinoma was by Hertz (6) in 1959, who transplanted tissue taken at autopsy from a cerebral metastasis to the cheek pouch of the hamster. Recipient hamsters were treated with cortisone at the time of transplantation and every third day thereafter for two weeks. Serial transfers could then be made for several generations in the hamster without cortisone treatment. Transfers of the tumor tissue subcutaneously in rats in the right flank area failed to grow with cortisone treatment alone. However, significant growth of the tumors was observed in the rat if the host had been irradiated as well as treated with cortisone, although the rate of tumor growth in the rat was much lower than that in the hamster. One of the choriocarcinomas maintained in the hamster cheek pouch by Hertz was then used by Patillo and Gey (7) to establish the first choriocarcinoma cell line BeWo. Based on the known nutritional requirements of the normal placenta, Patillo and Gey (7) used a culture medium containing high glucose levels. The culture medium was Waymouth’s MB752 with 5 g/L glucose, 40% Gey’s balanced salt solution and 10% placental cord serum. The final glucose concentration in the medium was 3.4 g/L. Reconstituted tropocollagen or human fibrinogen clotted with thrombin was used as the
Choriocarcinoma
143
matrix. The tumor specimens taken from the hamster cheek pouch were dissected in the presence of the culture medium and approximately 1 mm2 fragments were used for culture. One tumor colony from a single culture gave rise to the BeWo cell line. Kohler and Bridson (8) used the same choriocarcinoma tissue to develop 8 different clonal choriocarcinoma cell lines (JEG 1-8). The culture medium in this case was Ham’s F10, containing 13.5% horse serum and 3.2% fetal bovine serum. Tumor tissue fragments cultured in this growth medium were dispersed using trypsin and EDTA and used for the establishment of the cell line. The Jar cell line was established from a trophoblastic tumor of the placenta (9). The original culture medium for the Jar cell line was the same as that used for the establishment of the BeWo cell line (7). Other choriocarcinoma cell lines were cultured in either RPMI-1640 medium or Ham’s F10 medium, supplemented with 20% fetal calf serum (10-14). In our laboratory, we routinely culture choriocarcinoma cells using the following growth media (15,16): RPMI-1640 medium supplemented with 10% fetal bovine serum for Jar cells, Dulbecco’s Modified Eagle Medium/Ham’s nutrient mixture F12 (1:1) supplemented with 10% fetal bovine serum for BeWo cells, and minimum essential medium supplemented with 10% fetal bovine serum for JEG-3 cells.
3.
CONTINUOUS CELL LINES
The continuous cell lines derived from gestational and nongestational choriocarcinoma are listed in Table 1. BeWo, Jar, and JEG cell lines were derived from gestational choriocarcinoma (6-9). The other choriocarcinoma cell lines (SCH, IMa, JHTK-1, and T3M-3) were derived from nongestational choriocarcinoma (10-14). Using the original BeWo culture (7), Wice et al. (17) have isolated a clonal cell line, called BeWo b30, which maintains the morphological, biochemical, and endocrine characteristics of the parental cells. There are also reports in the literature concerning the establishment of Table 1 Continuous cell lines derived from choriocarcinomas Cell line
Patient sex/age
Primary site
Metastatic site
Tumor Specimen Culture site method
Reference
BeWo JEG Jar SCH IMa
F F F/24 M/46 F/25
Placenta Placenta Placenta Stomach Ovary
Brain Brain
Brain Brain Placenta Omentum Paraaortic lymph node Testis
X/D X/D D D X
(6,7) (6,8) (9) (10,11) (12)
X/D X
(13) (14)
Omentum Paraaortic lymph node
JHTK-1 M/45 Testis T3M-3 X, xenograft; D, dispersion of the original tumor tissue
144
Ganapathy et al.
additional choriocarcinoma cells (e.g., HCCM-5 (18) and NUC-1 (19)). Of these continuous cell lines, three are commercially available from the American Type Culture Collection (Rockville, MD, USA): BeWo (the original cell line developed by Patillo and Gey), Jar and JEG-3. All of the continuous choriocarcinoma cell lines, listed in Table 1, produce chorionic gonadotropin, but there are quantitative differences (7-14,20-22). In addition to chorionic gonadotropin, BeWo, Jar, and JEG cells produce placental lactogen and progesterone (9,23,24), SCH cells express the placentaspecific alkaline phosphatase (11), and T3M-3 cells produce progesterone (14). The expression of the human class I histocompatibility antigens (HLAs) has been studied (25-27). JEG cells express HLA-G whereas Jar cells do not. BeWo cells express HLA-G at very low levels. Unlike other classes of HLAs, HLA-G is nonpolymorphic and its expression is restricted to the extravillous cytotrophoblasts in the first trimester placenta. Thus, JEG cells resemble extravillous cytotrophoblasts with respect to HLA expression. The other class I histocompatibility antigens HLA-A, HLA-B and HLA-C are expressed at neg
