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
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0-306-46861-1 0-793-35878-3
©2002 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow
All rights reserved
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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
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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)
Cell line
Age
FIGO Primary category site
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 COLO-704 46 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
6
Table1 (continued)
Cell line
Age
HAC2/0.1 Nk HAC2/P Nk HeW 59 HEY Nk HMOA 57 HNOA 71 Nk HOC-1 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)
Cell line
Age
FIGO Primary category site 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 Nk MCAS-3 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
8
Table 1 (continued)
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
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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
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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] Epithelial-like monolayer. PE 4% in agar Nk 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 Serous adenocarcinomas in hamsters and Epithelial-like monolayer with hemicyst 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 Grows as clusters and single cells in Nk suspension. Doubling time 30–40 hours Tumorigenic Epithelial-like monolayer 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 Epithelial-like monolayer Nk
COV434SUBCL Granulosa cell tumor COV446B Serous carcinoma COV504 Serous carcinoma COV644 Mucinous carcinoma Well differentiated mucinous DO -S cystadenocarcinoma EFO21 Poorly differentiated serous adenocarcinoma EFO27 Moderately differentiated serous papillary adenocarcinoma
Xenograft pathology
Ovarian Cancer
Table 3 (continued)
Continued on next page
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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
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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
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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
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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 Poorly differentiated papillary OVCAR-3 adenocarcinoma Poorly differentiated papillary OVCAR-3 (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
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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
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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
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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
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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 Epithelial-like monolayer. Doubling time Undifferentiated tumors in nude mice. SC 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
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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
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31
Ovarian Cancer
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
Cell line
34
Table 5 (continued) Genetic changes
Reference 38 35 40 40 40 40 41 42 42 42 42 43 45 45 45 45 45 45 45 45
Continued on next page
Wilson and Garner
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 Mode 72. lp-, 6q+,7p+,11p+,i6p and >30 unidentified markers CI 79-36 CI 80-13A Mode 60. t(1;9)(q11;q12)1q-p+, 6q+,14p+, 15p+, -16,-16,18q+, i21q and 2 indistinct metacentric markers 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
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) Genetic changes
Reference 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 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) OAW 28 (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 OMC-3 Mode 43-44.
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
39
Ovarian Cancer
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
40
Wilson and Gamer
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
43
Ovarian Cancer 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.
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Wilson and Gamer
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
45
Ovarian Cancer 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
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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]
47
Ovarian Cancer 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.
48
Wilson and Gamer
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.
49
Ovarian Cancer 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.
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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.
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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.
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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 ) present, G6PD type B (7) ECACC Small bowel Using isoenzyme analysis, ATCC (9) metastasis G6PD type B Laimins (Northwestern Cervix Expression of involucrin in (10,11) the upper half of the University, Chicago, organotypic culture USA) N/k Cervix (12) (12) N/k Cervix (13) (13) N/k Cervix N/k (14) Cervix Using isoenzyme analyses, (15) (15) G6PD type B Using HLA typing, G6PD (16) (16) Cervix type B Cervix Using CEA levels (17) (17) N/k Cervix N/k (18) N/k Cervix (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 (20) N/k 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
(40)
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
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
Poorly differentiated carcinoma of the cervix C-33 (I, 11) C4 (I, 11) Invasive squamous carcinoma of the cervix Epidermoid carcinoma of the cervix CaSki 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 48 HEC-6 n.a. HEC-50 HEC-59 n.a. HEC-88nu n.a. n.a. HEC-108 n.a. HEC-116 Ishikawa HEC-151 n.a. HEC-155 n.a. n.a. END-1,2 47 SCRC-1 AN3CA n.a. RL95.2 65 KLE 68 68 ECC-1 KCC-1a,b 67 UM-EC-1 66 HHUA 54 52 SNG-P SNG-M 52 61 ETN-1 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 Oncogene expression Chromosome Number/Karyotype
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.
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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
Authentication3
Availability
Primary Reference
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
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)
D
I
ATCC
(Cailleau et al., 1978)
Patient Age
Histopathological Tumor Diagnosis' Stage
Primary Specimen Site Site
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
DU4475 HBL-1004 HMA-1 Hs 578.T MCF-7
62 27 48 74 69
IDC Benign PAC IDC IDC
MCF-10A
36
FCBD
MCF-10F
36
FCBD
MDA-MB-134-V1
47
IDC
MDA-MB-157
44
MDA-MB-175-VII
III II II IV IV IV IV
Breast Breast Breast Breast Breast
Continued on next page
Sutherland et al
Culture Method2
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
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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
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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)
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Table 3 Characteristics of Transplantable Human Mammary Carcinomas
(Peterson et al., 1997) Unpublished data (RL Sutherland) (Price et al., 1990a) Continued on next page
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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
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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
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Table 4 (continued)
low low mod mod
high low low high
Continued on next page
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Table 4 (continued) Cell line
IL-2 R
IL-3 Rα IL-6 R
BT-20 BT-474 BT-483 BT-549 DU4475 HBL-100 Hs 578.T MCF-7 MDA-MB-134-VI
neg
neg
neg neg
MDA-MB-157 MDA-MB-175-VI1 MDA-MB -23 1 MDA-MB-330 MDA-MB-361
CNTF R
low
mod
low
neg
neg
high
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
mod high
neg v. high
mod mod
neg high
neg high
neg neg
neg high
high low
CSF-1 R13
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
Hemopoietin receptor superfamily12 G-CSF R GM-CSF-R a GM-CSF-Rβ
IL-11 R
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
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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
Tumor suppressor genes p5334
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
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
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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.
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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
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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.
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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,
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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
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Drug resistance
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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)
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Table 5
See text - originally described as T-47D variant
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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 1Hamon Center for Therapeutic Oncology Research; 2 Department 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.
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.
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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).
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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).
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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
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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
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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
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(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
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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
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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.
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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.
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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.
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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.
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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.
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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 IT D IT D 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
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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.
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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.
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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
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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
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Table 1. Origins of cell lines derived from human germ cell tumors Cell line
Biopsy site/ patient age
Tumor diagnosis
Tera-1 lung/47 lung/22 Tera-2 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 rpln/25 1777N-RP 1777N-RPdiff rpln25 1685 M 1411HP testis/17 1411HRQ rpl/17 ITO testis/27 NEC-14 testis126 testis120 NEC-15 NEC-8 testis124 lung/39 HAZ-1 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
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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
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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.
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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 cyclin D2 Oncogene/others 12p 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; Reilly, 1993; TNR expansion 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 Dmitrovsky et al. 1990; RA resistant sublines; HOX 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
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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 cyclin D2 Oncogene/others 12p
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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 cyclin D2 12p Oncogene/others
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.
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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
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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.
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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
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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
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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
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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 negligible levels in most choriocarcinoma cells.
4.
COMPARISON OF MORPHOLOGICAL FEATURES BETWEEN CELL LINES AND THE ORIGINAL TUMORS
The morphological characteristics are listed in Table 2. All of the choriocarcinoma cell lines thus far established resemble cytotrophoblasts morphologically. Fused, multinuclear, syncytiotrophoblasts rarely form. There are, however, some notable morphological differences between the cell lines derived from gestational choriocarcinomas and the cell lines derived from nongestational choriocarcinomas. Gestational choriocarcinoma cell lines have more numerous cytoplasmic organelles such as mitochondria and rough endoplasmic reticulum. Exposure of BeWo or JHTK-1 cells to CAMP can induce morphological changes that are consistent with differentiation of cytotrophoblasts into syncytiotrophoblast-like cells. The clonal cell line BeWo b30 differentiates in response to CAMP into a polarized syncytiotrophoblast with biochemically and morphologically distinguishable apical and basolateral membranes (17,28).
5.
KARYOLOGY
The modal number of chromosomes in the choriocarcinoma cell lines is as follows: 86 in BeWo, 78 in JEG-1, 71 in JEG-3, 56 in IMa, and 66 in T3M-3. The chromosome numbers in the JHTK-1 cell line range between 107 and 141.
The tumor of origin was a cerebral BeWo: Grayish-white cystic cells characteristic of cytotrophoblasts; no detectable differentiation to metastasis and the derived xenograft syncytiotrophoblasts in log phase of growth. was morphologically similar. JEG: Several clonal cell lines (JEG 1-8) were derived from the same xenograft which served as the source for BeWo cells. Most JEG clones grow as a monolayer. Transplantation of these clonal cells back into hamster cheek pouch resulted in large cystic structures with necrotic centers without invasion of blood vessels.
Xenograft of the original tumor into hamster cheek pouch resulted in a richly vascularized tissue with a marked tendency to hemorrhagic necrosis; formation of cytotrophoblasts and syncytiotrophoblasts in broad sheets; presence of abundant mitoses; absence of cellular infiltration into the wall of the hamster cheek pouch; the tumor tissue could be peeled away from the surrounding host tissues with practically no hemorrhage at points of separation.
SCH Characteristic features of cytotrophoblasts with very little differentiation into syncytiotrophoblasts. Intermediate cells, morphologically between cytotrophoblasts and syncytiotrophoblasts, were also seen.
The original patient material from a metastatic omental lesion of gastric choriocarcinoma showed histological features characteristic of choriocarcinoma coexisting with a small area of adenocarcinoma producing mucin.
Transplantation of SCH cells into nude mice produced tumors which contained chorionic gonadotropin-positive mono- and multinuclear cells. Intermediate and syncytiotrophoblast cells were present in the xenograft much more frequently than under in vitro cell culture conditions.
IMa: Predominantly small polygonal cells with a prominent nucleus and a small number of multinuclear giant cells.
The tumor of origin was derived from a metastatic lesion of paraaortic lymph nodes (primary tumor was in ovary) which showed histological characteristics of a dysgerminoma coexistent with choriocarcinoma.
Transplantation of IMa cells into hamster cheek pouch produced tumors with histological features of choriocarcinoma. The tumors consisted of cytotrophoblast cells in a sheet-like arrangement and a small number of syncytiotrophoblast cells.
Choriocarinoma
Table 2 Morphological features of choriocarcinoma continuous cell lines in vitro and in vivo Cell line Original tumor Xenograft
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Table 2 (continued) Cell line
Original tumor
Xenograft
JHTK-1: Epithelial cells with poorly developed rough endoplasmic reticulum and very few mitochondria with characteristic features of cytotrophoblasts.
Transplantation into nude mice produced tumors with characteristics of choriocarcinoma. The tumors contained a mixture of cytotrophoblasts and syncytiotrophoblasts.
T3M-3: Epithelial cells with prominent nuclei. Cells grow as a monolayer.
Transplantation of T3M-3 cells into nude mice produced tumors which consisted of closely packed, large, round cells. Mitoses were numerous.
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REFERENCES 1. 2. 3. 4.
Graham CH, Lala PK. Biochem Cell Biol 70: 867, 1992 Lala PK, Hamilton GS. Placenta 17: 545, 1996 Dehner LP. Am J Surg Pathol 4: 43,1980 Shanklin DR. In Tumors of the Placenta and Umbilical Cord, p. 30. B.C. Decker Inc., Philadelphia, 1990 5. Belchis DA, Mowry J, Davis HJ. Cancer 72: 2028, 1993 6. Hertz R. Proc Soc Exp Biol Med 102: 77,1959 7. Patillo RA, Gey GO. Cancer Res 28: 1231, 1968 8. Kohler PO, Bridson WE. J Clin Endocrinol 32: 683, 1971 9. Patillo RA, Ruckert A, Hussa R et al. In Vitro 6: 398, 1971 10. Oboshi S, Yoshida K, Seido T et al. Proc 31st Annual Meeting Japanese Cancer Assoc 115, p. 59. Tokyo: Japanese Cancer Assoc 1972 11. Kameya T, Kuramoto H, Suzuki K et al. Cancer Res 35: 2025,1975 12. Sekiya S, Kaiho T, Shirotake S et al. In Vitro 19: 489, 1983 13. Yamazaki H, Kotera S, Ishikawa H et al. J Urol 137: 548, 1987 14. Okabe T, Sasaki N, Matsuzaki M et al. Cancer Res 43: 4920,1983 15. Cool DR, Leibach FH, Bhalla VK et al. J Biol Chem 266: 15750, 1991 16. Jayanthi LD, Ramamoorthy S, Mahesh VB et al. J Biol Chem 269: 14424, 1994 17. Wice B, Menton D, Geuze H, Schwartz AL. Exp Cell Res 186: 306, 1990 18. Nakamoto 0. Asia-Oceania J Obstet Gynecol 6: 177, 1980 19. Suzumori K, Sugimoto Y, Suzumori K et al. Asia-Oceania J Obstet Gynecol 9: 309, 1983 20. Patillo RA, Gey GO, Delfs E, Mattingly RF. Science 159: 1467, 1968 21. Azizkhan JC, Speeg KV Jr, Stromberg K, Goode D. Cancer Res 39: 1952, 1979 22. Sekiya S, Kaiho T, Shirotake S et al. Am J Obstet Gynecol 146: 57, 1983 23. Patillo RA, Hussa RO, Delfs E et al. In Vitro 6: 205, 1970 24. Kohler PO, Bridson WE, Hammond JM et al. Acta Endocrinol Suppl KBH 153: 137, 1971 25. Ellis SA, McMichael AI. J Immunol 144: 731,1990 26. Kovats S, Main EK, Librach C, et al. Science 248: 220, 1990 27. Kato M, Ohashi K, Saji F, Wakimoto A, Tanizawa O. Placenta 12: 217, 1991 28. Furesz TC, Smith CH, Moe AJ. Am J Physiol 265: C212, 1993
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Chapter 25 Thymomas and Thymic Cancers
H.K. Müller-Hermelink and Alexander Marx Department of Pathology, University of Würzburq, Luitpoldkrankenhaus, Joseph-SchneiderStrab e 2, 97080 Würzburg, Germany. Tel: 0049-931-201-5420; Fax: 0049-931-201-3505
1.
INTRODUCTION
The first description of epithelial tumors of the thymus as thymomas was by Bell in 1917 (58). Thymic epithelial tumors (TETs) have to be distinguished from neuroendocrine tumors, lymphomas, sarcomas or thymic germ cell tumors, as some of these are also epithelial in origin. The first classification of TETs (1, 2) used the ratio of lymphocytes to epithelial cells as the main histological criterion, but when tumor stage was taken into account, this classification was of no independent prognostic value (3). Levine and Rosai (1978) suggested a classification (4) that used invasiveness as the main criterion (5), and distinguished encapsulated “benign thymomas” from invasive “malignant thymomas”. Malignant thymomas were subclassified as follows: 1. Category I malignant thymomas, when the neoplastic epithelial cells are normal or slightly atypical 2. Category II malignant thymomas, when the neoplastic cells are moderately or severely atypical. Category II malignant thymomas are now called thymic carcinomas. Benign thymomas and category I malignant thymomas retain characteristics of the thymus (6). In contrast, thymic carcinomas do not retain the characteristics of normal thymus cells, and are classified like other tumors (Table 1) (7-9). Category I malignant thymomas are the most frequent TETs. We introduced a modified classification for these tumors (10,11), which takes as the main criterion the histological resemblance of the neoplastic epithelial cells to counterparts in the normal thymus, distinguishing a medullary and cortical line
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of differentiation. In contrast to previous histological classifications, cortical differentiation was an independent prognostic factor for invasiveness and outcome (12-14). Certain thymoma subtypes are associated with paraneoplastic myasthenia gravis (15,16). Recently, agreement has been reached by a WHO committee for the classification of thymic epithelial tumors, as shown in Table 1 (Rosai and Sobin, 1999) (59).
2.
PRIMARY CULTURES
Short term primary cultures have been established from most types of human thymic epithelial tumors, including benign thymomas, malignant thymomas and thymic carcinomas. Such cultures have been used for cytogenetic studies (17-23), cell biological investigations (24-30), electrophysiology (31) or the identification of thymoma proteins (32,33). As for non-neoplastic human thymic epithelial cells (24,34-41), explant techniques as well as protease digestion strategies producing single cell suspensions have been applied to thymomas (28,30,32,33,42). Fibroblast overgrowth is a major problem when long term cultures or the significant expansion of cells are required. To circumvent this problem five Table 1 Classification of thymic epithelial tumors according to Levine and Rosai (1978), with modifications for benign and category I malignant thymomas (15) and thymic cancers (9) Classification
WHO type
Histological subtype
Benign thymoma
A B B1 B2 B3 C
Medullary thymoma1 Mixed thymoma1 Predominantly cortical thymoma2 Cortical thymoma Well differentiated thymic carcinoma Squamous cell cancer3 Basaloid cancer4 Mucoepidermoid cancer3 Adenosquamous cancer 3 Adenocarcinoma5 Small cell/neuroendocrine cancer5,6 Lymphoepithelioma-like cancer5 Sarcomatoid cancer5 Clear cell cancer5 Large cell cancer5 Undifferentiated cancer5
Malignant thymoma, category I Malignant thymoma, category II
1Some otherwise typical medullary or mixed thymomas are invasive but non-metastatic and behave in a benign manner. 2Also called "organoid thymoma" (14). 3Low or high grade. 4Low grade. 5High grade. 6Neuroendocrine tumors are considered as a separate category according to the recent WHO classification
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different strategies have been applied in non-neoplastic thymic epithelial cultures: 1. Growth in serum free media (34,43-48). 2. Growth in a serum-containing medium with D-valine replacing L-valine (40,41,49). 3. Elimination of fibroblasts by EDTA washing and subculture on mitomycin C-treated 3T3 mouse fibroblast feeder layers (24). 4. Active depletion of fibroblasts from mixed thymic stromal cultures using complement activating cytotoxic antibodies (50). 5. Sorting thymic epithelial cells with magnetic beads or FACS using monoclonal antibodies against MHC class II molecules after enzymatic digestion of deoxyguanosine-treated thymic tissue (51-53). In thymomas these techniques have not been compared systematically. Positive sorting of thymoma epithelial cells using anti-MHC class II coated magnetic beads is impossible in most cases as thymoma cells usually express low levels or are negative for MHC class II antigens (15,54). Primary thymoma epithelial cultures proliferate less well in vitro than nonneoplastic thymic epithelial cells (24). In our experience this is true for serumfree media as well as for serum-containing media with either conventional amino acids or with D-valine replacing the usual L-valine. While a serum-free medium designed for keratinocytes (Keratinocyte-SFM medium, 17005-034, including EGF and Bovine Pituitary Extract; GIBCOBRL) efficiently promotes growth of non-neoplastic thymic epithelial cells and delays fibroblast proliferation for up to four passages after explant culture, serum-free media in our hands have been inefficient for the establishment of primary thymoma cultures. Cortical thymomas and well differentiated thymic carcinomas benefit from fetal calf serum (5-15%), although fibroblast overgrowth occurs quickly. Our preferred medium for explant cultures of thymoma epithelial primary cultures is as follows: MEM containing D-valine (Gibco), 10 mM HEPES, 10% heat-inactivated FCS, 2 mM glutamine, 10 ng/ml EGF, 5 µg/ml insulin, 100 U/ml penicillin and 100 µg/ml streptomycin. To produce explant cultures, thymoma tissue is minced into pieces of approximately 1 mm3 and agitated in culture medium to remove thymocytes. The explants are placed in plastic cell culture dishes and left without moving the dishes for four days. Thereafter half of the medium is changed twice a week. While this strategy is usually adequate to support the in vitro growth of normal thymus and mixed and cortical thymomas, epithelial cell outgrowth from tissue fragments is often negligible in tumors with a high content of fibrous stroma, including well differentiated thymic carcinoma and category II malignant thymomas. In such cases, outgrowth is often increased by stirring thymoma fragments with a trypsin/EDTA solution (0.1 mg/ml and 10 mg/ml, respectively) in PBS for up to 3h at room temperature. An additional overnight treatment at 37°C with collagenase Type IA (10 U/ml; Sigma) made up
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in DMEM/2% newborn calf serum (34) may further improve the yield of thymic epithelial cells, but this has not yet been investigated in thymomas. While neither serum-free conditions nor D-valine containing media in our hands prevent the overgrowth of fibroblasts in thymoma cultures, complement dependent depletion of fibroblasts from thymoma-derived mixed stromal cultures has resulted in almost pure epithelial cell cultures using the protocol given below: 1. Trypsinize thymic stromal cells (5 × 105 or more). 2. Wash twice in PBS/1mM EDTA, transfer to an Eppendorff, spin down. 3. Add 200 µl of anti-fibroblast mAbAS02 (Dianova, Hamburg) diluted 1:5 with PBS. 4. Rotate for 45 min in a cool room (4°C) to load fibroblasts with antibodies. 5. Wash twice as before. 6. Add 200 µl guinea pig complement (Dianova, Hamburg) diluted 1:3.5 with PBS and leave cells for 45 minutes at 37°C. 7. Wash twice as before. 8. Culture remaining cells as usual and check for purity e.g. by immunocytochemistry or FACS. In principle, the method also works with adherent cells but the quantities of mAbAS02 and complement are much higher than required for cells in suspension. Magnetic beads (five beads per cell) coated with mAbAS02 have also been used for depleting fibroblasts from mixed stromal cultures. Long-term epithelial cell cultures from thymomas may be difficult to establish because the tissue comes from adults. Both mouse (51,52) and human studies with non-neoplastic human thymic epithelial cells have used tissue from neonatal or infantile thymuses (24,29,37,38,41) or fetal thymic tissue (36,41). No attempts have been made to immortalize adult normal or neoplastic thymic epithelial cells.
3.
CONTINUOUS CELL LINES
A few cloned human thymic epithelial cell lines established from nonneoplastic infantile, neonatal or fetal thymic tissue have been reported (37,41). The lines reported by Fernandez et al. (1994) are available from the originator’s laboratory. In contrast, no continuous thymoma epithelial cell lines have been described. The only continuous cell line derived from a thymic epithelial tumor, Ty-82, was established from a pleural effusion in a 22 year old female. The primary was a high grade (G4) thymic carcinoma (category II malignant thymoma, T4N1M1) (55). As there was no indication of a germ cell origin, the tumor from which Ty-82 was derived was assumed to be of thymic epithelial cell origin and classified as undifferentiated thymic carcinoma (18). The tumor
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exhibited a solid growth pattern in a fibrous stroma with no recognizable differentiation. Explants from 150 ml of pleural effusion were grown in RPMI 1640 with 20% FCS and 10% human cord serum at 37ºC in 5% CO2 with media changes every 3 days. Ty-82 cells in vitro grow in suspension in glass Petri dishes or in suspension culture flasks (Greiner, Solingen, Germany), but adhere to Falcon 3013 and 3024 flasks (Becton Dickinson). The cell line is available from the originator. Ty-82 cells exhibit a primitive blastoid morphology with round nuclei and prominent nucleoli, and lack desmosomes, tonofilaments and neurosecretory granules by electron microscopy. The cells are positive for a-naphthyl butyrate esterase and acid phosphatase and are negative for peroxidase, Sudan Black B, chloroacetate esterase and PAS. A small proportion of the cells express epithelial membrane antigen (EMA) and are positive for Ki-67 antigen. Ty-82 cells also react with an antibody specific for subcapsular thymic cortical epithelium (56). The cells are negative for the EBV antigen EBNA and other markers (cytokeratins, CD57, CEA, GFAP, S100, Desmin, MHC molecules, ICAMs, CD30, and various markers of immature and mature T and B cells, monocytes and histiocytes) (55). Ty-82 cells grew in 4 of 5 nude mice inoculated. Tumors reached at least 1 cm in diameter within 43-53 days. The histological features of the xenotransplants were similar to those of the original tumor. Compared to Ty-82 cells in vitro, the xenotransplants exhibited a higher frequency of EMA positive cells (14 versus 8%) and a small fraction of cells expressing cytokeratins (like the primary tumor). The cell line was shown to have the same t(15;19)(q12;p13) translocation as the primary tumor (18). This translocation is probably identical to the translocation described in two other independent cases of high grade thymic carcinoma, from which permanent cell lines could not be established. These latter cases were described as either undifferentiated carcinoma (19) or high grade mucoepidermoid carcinoma with extensive metastases (20). Therefore, the t(15;19)(q12;p13) translocation seems to be a non-random event, not found in other types of cancer, that might be of significance in the development of this type of thymic carcinoma. The 5-82 cell line may not be the ideal model for the study of normal functions of the human thymus. Cloned human thymic epithelial cell lines derived from non-neoplastic fetal or neonatal thymuses might be a better tool in this respect (41). However, even normal thymic epithelial cells after a short time in primary cell culture lose their capacity to fully promote the positive selection of thymocytes in vitro (57). Consequently, organ culture systems might be required to investigate the complex physiology of the human thymus.
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REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
Bematz PE et al. J Thoracic Cardiovasc Surg 542: 424, 1961. Lattes R. Cancer 15: 1224, 1962. Lewis JE et al. Cancer 60: 2727, 1987. Levine GD, Rosai J. Hum Pathol 9: 495, 1978. Yamakawa Y et al. Cancer 68: 1984, 1991. Kirchner T et al. Am J Surg Pathol 16: 1153,1992. Snover DC et al. Am J Surg Pathol 6: 451, 1982. Suster S, J Rosai. Cancer 67: 1025, 1991. Shimosato Y. Epithelial tumors of the thymus, A Marx and HK Muller-Hermelink, Eds. Plenum Press, New York, London, p. 9 1997. 10. Kirchner T, Müller-Hermelink HK. Progress in Surgical Pathology, CM Fenoglio-Preiser, M Wolff and F Rilke, Eds. Field and Wood, Philadelphia, 1989. 11. Müller-Hermelink HK, Marx A, Kirchner T. Recent Advances in Histopathology, P Anthony and MacSween R, Eds. Edinburgh, Churchill Livingstone, p. 49 1994. 12. Kuo TT, Lo SK. Hum Pathol 24: 766,1993. 13. Pescarmona E et al. Am J Clin Pathol 93: 190, 1990. 14. Quintanilla Martinez L et al. Hum Pathol 24: 958, 1993. 15. Muller-Hermelink HK et al. Arch Histol Cytol 60: 9, 1997. 16. Marx A et al. Virchows Archiv 430: 355, 1997. 17. Kristoffersson U et al. Cancer Genet Cytogenet 41: 93, 1989. 18. Kubonishi I et al. Cancer Res 51: 3327, 1991. 19. UR Kees et al. Am J Pediatr Hematol Oncol 13: 459, 1991. 20. Lee AC et al. Cancer 72: 2273, 1993. 21. Dal Cin P et al. Genes Chromosomes Cancer 6: 243, 1993. 22. Dal Cin P et al. Cancer Genet Cytogenet 89: 181, 1996. 23. Deminatti MM et al. Ann Genet 37: 72, 1994. 24. Singer KH et al. Hum Immunol 13: 161, 1985. 25. Papadopoulos T et al. Virchows Arch B Cell Pathol Incl Mol Pathol 56: 363, 1989. 26. Talle MA et al. Thymus 18: 169, 1991. 27. Marx A et al. Thymus 23: 83, 1994. 28. Gilhus NE et al. J Neuroimmunol 56: 65, 1995. 29. Screpanti I et al. J Cell Biol 130: 183, 1995. 30. Schultz A et al. Verh Dtsch Ges Path 80: 250, 1996. 31. Siara J et al. Neurology 41: 128, 1991. 32. Marx A et al. Am J Pathol 134: 865, 1989. 33. Marx A et al. Am J Pathol 148: 1839, 1996. 34. Rimm IJ et al. Clin Immunol Immunopathol 31: 56, 1984. 35. Galy AH et al. Cell Immunol 124: 13, 1989. 36. Mizutani S et al. Acta Paediatr Jpn 29: 539, 1987. 37. Galy AH, Spits H. J mmunol 147: 3823, 1991. 38. Galy et al. Cell Immunol 129: 161, 1990. 39. Le PT, Singer KH. Int J Clin Lab Res 23: 56, 1993. 40. Dalloul AH et al. Blood 77: 69, 1991. 41. Femandez E et al. Blood 83: 3245,1994. 42. Zhang J et al. J Exp Med 179: 973,1994. 43. Schreiber L et al. Immunology 74: 621, 1991. 44. Meilin A et al. Scand J Immunol 42: 185, 1995. 45. Meilin A et al. Int J Immunophannacol 19: 39, 1997.
Thymomas and Thymic Cancers 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.
Ropke C, Elbroend J. Dev Immunol 2: 111,1992. Andersen A et al. Scand J Immunol38: 233,1993. Pedersen H et al. Immunol Lett 41: 43, 1994. Dalloul AH et al. Eur J Immunol 21: 2633,1991. Singer KH et al. J Invest Dermatol 94: 85S, 1990. Jenkinson EJ et al. J Exp Med 176: 845,1992. Anderson G, Jenkinson EJ. Immunology Today 18: 363, 1997. Oostenvegel M et al. Immunity 6: 351, 1997. Willcox N et al. Am J Pathol 127: 447, 1987. Kuzume T et al. Int J Cancer 50: 259, 1992. Takeuchi T et al. Virchows Arch A PatholAnat Histopathol 419: 147, 1991. Anderson G et al. Eur J Immunol 27: 1838,1997. Bell ET. J Nerv Ment Dis 45: 130, 1917. Rosai J, Sobin L. In WHO Int Classification of Tumors. Springer-Verlag, 1999.
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Chapter 26 Kaposi’s Sarcoma
Christopher Boshoff Department of Oncology, University College London, 91 Riding House Street, London WlP 6BT, UK. Tel: 0044-1 71-504-9557; Fax: 0044-1 71-504-9555; E-mail: c.
[email protected]
1.
INTRODUCTION
For over 100 years, Kaposi’s sarcoma (KS) remained a rare curiosity to clinicians and cancer researchers, until it shot to prominence as the sentinel of what we now call AIDS. Classic KS occurs predominantly in elderly male patients of Southern European ancestry (1). A high frequency is also seen in Israel and other Middle Eastern countries. It is not known why this form of the disease is generally not as aggressive as the form originally described by Kaposi, but there may be immunological reasons. In some equatorial countries of Africa, KS has existed for many decades, therefore preceding HIV (known as endemic KS) (2). This form is found in younger patients as well as the elderly. The male :female ratio is greater than 3 : 1 and it is generally a more aggressive disease than classic KS (3). During the past 20 years, the incidence of KS among renal transplant recipients and other patients receiving immunosuppressive therapy has increased (known as post-transplant KS or iatrogenic KS) Patients of Mediterranean, Jewish or Arabian ancestry are over-represented among immunosuppressed patients who develop KS after a transplant (1), indicating that those born in countries where classic KS occurs continue to be at risk of developing KS even if they migrate to “low-risk” countries. These data suggested that there is a genetic predisposition or environmental factor (possibly infectious agent) responsible for KS development.
J.R.W Masters and B. Palsson (eds.). Human Cell Culture Vol. II, 157–166. © 1999 Kluwer Academic Publishers. Printed in Great Britain.
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In 1981, the US Centers for Disease Control and Prevention (CDC) became aware of an increased occurrence of two rare diseases in young gay men from New York City (NY, USA) and California (4): Kaposi’s sarcoma and Pneumocystis carinii pneumonia (PCP). This was the beginning of what is today known as the AIDS epidemic and AIDS-KS is today the most common form of KS.
2.
KAPOSI’S SARCOMA HISTOGENESIS AND CLONALITY
Histologically, KS is a complex lesion. In early KS lesions, which normally appear on the skin, there is a collection of small, irregular endothelium-lined spaces that surround normal dermal blood vessels and these are accompanied by a variable, inflammatory infiltrate of lymphocytes (patch-stage). This stage is followed by the expansion of a spindle-celled vascular process throughout the dermis. These spindle cells form slit-like, vascular channels containing erythrocytes (plaque-stage). The later nodular-stage KS lesions are composed of sheets of spindle cells, some of which are mitotic, and slit-like vascular spaces with areas of hemosiderin pigmentation. The spindle cells form the bulk of established KS lesions and are therefore thought to be the neoplastic component, but there is still some controversy over the histogenesis of spindle cells. Although the majority of the spindle cells stain positive for endothelial cell markers including factor VIII and CD34, some cells express proteins characteristic of smooth muscle cells, macrophages or dendritic cells (5, 6). Some spindle cells simultaneously express antigenic determinants characteristic of several different cell types, suggesting that KS spindle cells might be derived from a pluripotent mesenchymal progenitor cell or a mesenchymal cell experiencing aberrant differentiation. Circulating KS-like spindle cells have been isolated and cultured from patients with AIDS-KS and from those thought for other reasons to be at risk of AIDS-KS (7). These circulating cells have an adherent phenotype and express markers of both macrophage and endothelial cells (8). No cell lines have yet been established from such circulating spindle cells. Whether KS is a neoplastic lesion or a reactive process remains controversial. The exact cell of origin is controversial and especially in early lesions, the “tumor cell” compartment makes up the minority of the tumour bulk where the majority of cells are inflammatory cells. Furthermore, the clinical presentation of multiple skin lesions in a defined distribution and spontaneous remission of lesions also favor a reactive hyperplasia rather than a true malignancy. Rabkin and colleagues showed that individual KS lesions are probably clonal (9) and more recently reported that multiple lesions in the same patient were clonal (10), suggesting that KS is a disseminated monoclonal cancer and
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that the changes that permit the clonal outgrowth of spindle cells occur before the disease spreads. These studies need confirmation. Early KS is probably a non-clonal proliferation of endothelial cells or their precursors (eg angioblasts) (11) with a prominent inflammatory and angiogenic response. Advanced disease probably represents a true clonal malignancy with metastases of clonally derived spindle cells to different sites. This hypothesis is comparable to the scenario in EBV-driven polyclonal lymphoproliferations in immunodeficient individuals which can progress to clonal lymphomas.
3. CYTOKINES KS spindle cells in vitro and in vivo express high levels of certain cytokines including IL-6, bFGF, TNFa, Oncostatin M and g-interferon (12–17). In particular, IL-6, bFGF and g-interferon are angiogenic in vitro and in some in vivo assays. IL-6 is produced by KS spindle cells and exogenous IL-6 can also enhance the proliferation of KS cells in culture (18). Because of the nature of early KS lesions it has been suggested that such lesions are “cytokine-driven”. All these studies were done on short-term cultures established from KS lesions. Most KS cells grown in culture are highly dependent for proliferation on combinations of these cytokines. The more aggressive nature of HIV-associated KS has led to speculation that HIV encoded proteins may enhance KS growth (17). The HIV-1 Tat protein transactivates HIV viral and also some host cell genes (19). Tat can be released by infected cells and act extracellularly (20). Tat can induce a functional program in endothelial cells related to angiogenesis and inflammation including the migration, proliferation and expression of plasminogen activator inhibitor-1 and E-selectin (21) Tat induces growth of KS spindle cells in vitro and is angiogenic in vivo and in transgenic mice (17, 20, 22). AIDS-associated KS is frequently more aggressive than non-HIV related KS and it is possible that the angiogenic properties of Tat contribute to this phemomenon.
4.
AN INFECTIOUS CAUSE?
Studies of AIDS case surveillance data support the existence of a sexually transmissible KS cofactor: KS occurs predominantly in gay and bisexual men with AIDS, less commonly in those acquiring HIV through heterosexual contact and rarely in AIDS patients with hemophilia or in intravenous drug users (23).
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NEW HERPESVIRUS: KSHV OR HHV-8
Yuan Chang and colleagues employed representational difference analysis (RDA) to identify sequences of a new herpesvirus (Kaposi’s sarcoma-associated herpesvirus or human herpesvirus-8) in AIDS-KS biopsies (24). This new virus is a gamma herpesvirus (25) (genus Rhadinovirus) with sequence similarity to the oncogenic viruses herpesvirus saimiri (HVS) and Epstein-Barr virus (EBV). KSHV is present in all epidemiologic types of KS (26) and also in a rare lymphoma called primary effusion lymphoma (PEL) (27). In situ, most of the spindle cells and endothelial cells in KS lesions contain the virus, further supporting a role for KSHV in KS pathogenesis (28, 29). This role may be direct whereby KSHV “transforms” endothelial cells or indirect where viral encoded proteins stimulate the growth of spindle cells through paracrine and autocrine mechanisms. The short term spindle cell cultures established from KS biopsies lose the viral sequences after 4–6 passages in culture. We do not know yet whether this loss is because the cells enter a lytic phase (productive viral infection) in culture or whether the culture conditions are not appropriate to maintain latent viral infection. The only KSHV positive cell lines available are B cell lines established from PEL (30–32). The paucity of continuous KS cell lines provides support for the hypothesis that the majority of KS lesions, especially early tumors, represent multicentric hyperplastic lesions, rather than cancers. KS Y-1 and SLK are the only two continuous lines available for research (see Table 1). KS Y-1 is used most frequently, although it is not confirmed that this cell line is derived from KS (33). The cell line was established from a pleural effusion of a patient with KS of the skin, and it is not known whether the patient actually had lung parenchyma or pleural involvement with KS. The KS Y-1 cell line originated from mononuclear cells isolated from the pleural effusion after removal oflymphocytes, monocytes/macrophages, and fibroblasts. KS Y-1 cells express markers characteristic for smooth muscle cells and endothelium including CD31, CD34 and smooth muscle actin. To confirm that the KS Y-1 cell line was derived from the specified patient with skin KS, HLA typing on a skin biopsy and KS Y-1 cells was performed (33). SLK was derived from a KS biopsy of an HIV negative patient who was on cyclosporin post-renal transplantation. The cells have an endothelial morphology and express the endothelial marker Factor VIII. Both cell lines are available directly from the laboratories that established them. Both lines are negative for KSHV sequences as well as for HIV and the other human herpesviruses including cytomegalovirus and Epstein-Barr virus. Both lines are adherent and grow in RPMI 1640 with 10% FCS. They also express markers characteristic of endothelial cells and are epithelioid or spindle shaped when grown in vitro. Tumors established from these lines after
Kaposi's Sarcoma
Table 1
Characteristics of KS cell lines
Cell line Patient age/sex KS Y-1 SLK
?/M, AIDS-KS 28/M, Post-transplant KS
Primary site
Specimen site
Skin Pleural effusion Skin, oral mucosa Oral mucosa lesion
Culture method
Availability
Primary reference
Explant Explant
Directly from authors Directly from authors
Lunardi-Iskandar et al. 1995a Herndier et al. 1994
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inoculation into immunodeficient mice show prominent angiogenesis, which is reminiscent of KS. However, one of the main characteristics of KS, the presence of KSHV DNA in most spindle cells (28, 29), is absent in both cell lines and therefore also in the tumors induced in mice. This feature distinguishes KS-like lesions from true KS and casts doubt over these lines as being representative of KS lesions. Human chorionic-gonadotropin protein (PHCG) blocks the growth of KS Y-1 cells in vitro and prevents the induction of KS-like lesions by these cells in immunodeficient mice (34). Because KS occurs more frequently in males than females and was noted to regress occasionally during pregnancy, it was hypothesised that bHCG might be responsible for inhibiting the growth of KS cells in vivo (34). This cell line is now frequently used to study the antimitogenic effects of bHCG on cellular proliferation (35). Most KS lesions are diploid. However, KS Y-1 is tetraploid and has numerous chromosomal abnormalities and SLK is diploid with a few chromosomal changes. The karyotypic abnormalities found in KS Y-1 and SLK have not been described in KS lesions. This further indicates that these lines are not typical of the cells in KS tumors, but could represent the occasional outgrowth of clonal cells from a heterogeneous lesion. Apart from these two immortalized cell lines, an array of KS lines that are dependent on growth factors have been described (Table 3). The characteristics of these cells are diverse, some being of fibroblastic origin and others belonging to the endothelial lineage. These cell cultures have been established using a variety of techniques, including the addition of specific or undefined growth factors, the combination of which might be expected to select for or against the various cell types present in the original lesion. It is unclear whether any of these cell lines represent “KS tumor cells”, but they have been widely studied to try to elucidate the pathogenesis of KS. It is difficult to assess the significance of the conflicting data resulting from these studies, especially as the cell lines used are so diverse. The earliest reported KS cell lines had a fibroblastoid morphology and expressed mesenchymal, but not endothelial cell markers (39). Albini et al. reported a KS cell line of fibroblastoid appearance with many characteristics of smooth muscle cells (40,41). Cell lines with a spindle-like morphology were established from pleural effusions from patients with pulmonary KS (13, 42). These cells expressed several endothelial markers, as did cell lines established by an independent group, also from pleural effusions (43). Cell lines with characteristics of endothelial cells have also been established from skin biopsies (44, 45) and peripheral blood cells of KS patients (7). Cell cultures have also been reported which seem to be heterogeneous (44, 48). Nakamura and colleagues showed in 1988 that conditioned medium (CM) from T lymphocytes infected with human retroviruses was mitogenic for
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Table 2 In vitro and in vivo growth of KS cell lines Cell line Tumor pathology KS Y-1
Histopathology of skin lesions show spindle cells and prominent vasculature
SLK
Typical KS lesions described
In vitro characteristics
Xenograft pathology
Polygonal morphology that changes to spindle shaped cells when treated with activated T-cell conditioned medium or when cells become confluent Cells grow as a homogeneous monolayer with an epithelioid morphology. Doubling time is 24–36 hours. No nuclear atypia
Tumors in nude and SCID mice show marked vascularity. Tumor metastases are seen. Tumors are described as KSlike lesions Rapidly growing tumors established in nude mice. Histology shows proliferation of endothelial-like cells surrounding slit-like spaces (“KS-like”). Tumor metastases not seen.
Table 3 KS cell lines dependent on growth factors Morphology
Origin of cell line
Fibroblast
Skin KS AIDS
Fibroblast
Skin KS AIDS Skin KS AIDS
Spind1e
Ribbon Fibroblast
Irregular
Endothelial Fibroblast
Skin KS AIDS Skin KS AIDS
Markers
Tumorigenicity
Reference
Endothelial Smooth muscle Fibroblast Smooth muscle
Non-malignant in nude mice
Delli Bovi et al. (39)
Grows in <2% FCS
Albini et al. (40)
Induction of KS-like Salahuddin et al. (13) lesions of mouse origin in nude mice Smooth muscle Grow in <2% FCS Wittek et al. (41)
Endothelial Smooth muscle
Endothelial Smooth muscle Fibroblast Skin KS Endothelial AIDS Smooth muscle Fibroblast Pleural effusion Endothelial AIDS Skin KS Endothelial AIDS
Table adapted from (38)
Grow in <2% FCS
Roth et al. (44)
Not determined
Way et al. (36)
Not determined
Corbeil et al. (43)
Non-malignant in nude mice
Levy (37)
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spindle-like cells derived from AIDS-KS lesions (42). CM from HTLV-II infected cells had the greatest effect and a 30 Kd protein was identified with a strong mitogenic influence. Reduced mitogenic effects of CM were observed on normal endothelial cells (human umbilical vein endothelial cells, HUVEC). HTLV-II CM allowed the long term culture of the KS cells but not HUVEC cells (42). The 30 Kd protein identified by Nakamura et al (42) as the mitogenic component of HTLV-II CM was later identified as oncostatin M (14), a glycoprotein originally shown to have an inhibitory effect on the growth of a melanoma cell line. Purified oncostatin M was demonstrated to show mitogenic effects on KS cells, but not on HUVEC cells (14). The role played by oncostatin M in the pathogenesis of KS is however unresolved and at least one study found no difference in the levels of serum oncostatin M in KS patients and controls. Roth and colleagues found CM from their KS cell lines induced moderate (two-fold) mitogenic effects both on KS cells (autocrine effect) and fibroblasts and endothelial cells (paracrine effect). They attribute these effects to an unknown fibroblast growth factor-like protein (44). This group failed to show mitogenic effects of their cell lines with HTLV-II CM, as did two others (43). Since the cell lines used in these studies had different characteristics, it is possible that these conflicting results are explained by the different cell types used in each study. Miles and colleagues showed that cultured KS cells produce and respond to IL-6 (15). KS cell cultures were shown to express IL-6 and IL-6 receptors. Recombinant IL-6 was shown to be mitogenic for these KS cells and antibodies to IL-6 significantly reduced the growth of these cell lines. Corbeil and colleagues similarly reported high level expression of IL-6 on KS cells in culture (43). Expression of IL-6 and IL-6 receptor mRNA has been shown by dot blot hybridization (15) and by in-situ hybridization in KS lesions. IL-1 has been shown to have a mitogenic effect on some KS cell lines (42). It has also been shown to be expressed by some KS cell lines (46), possibly indicating an autocrine role in the development of KS lesions. TNF- a was shown to be a potent mitogen for KS cells (42). Some KS cell lines express TNF-a (43), whilst others do not (46). Expression of TNF-a in KS lesions has been studied using in-situ mRNA hybridization, but the results have been inconsistent. Platelet-derived growth factor (PDGF) is essential for the growth of some KS cell lines (47) and these cells express PDGF receptors. Other KS cell lines, however, produce PDGF and thus do not require it (16,43). Studies using insitu hybridization and immunohistochemistry demonstrated PDGF receptors in various cells within KS lesions (6). Basic fibroblast growth factor (bFGF) is produced by KS cell lines with diverse characteristics (42, 43, 45) and promotes the growth of KS cells and endothelial cells. Expression of bFGF has been demonstrated in KS lesions
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using in-situ hybridization and immunohistochemistry (17). The angiogenic effect of cultured KS cells in nude mice could be inhibited by a specific bFGF antisense oligonucleotide (48). HIV-1 Tat protein has a mitogenic effect on some KS cell lines (49). Tat protein induces angiogenesis when injected into nude mice (17, 21) and this effect is potentiated by bFGF (17). Since KS develops in the absence of HIV infection, Tat is clearly not required for disease development. It is possible, however, that by potentiating the effect of cytokines such as bFGF, Tat has a role in the pathogenesis of AIDS-KS, which is more severe than classic KS or iatrogenic KS.
6. CONCLUSIONS KS is a multicentric disease and lesions consist of a complex mixture of cell types. The vast majority of cell cultures established from KS lesions require growth factors for prolonged growth in vitro. Only two cell lines have been described (SLK and KS Y-1) which are continuous and which, when injected into nude mice, give rise to KS-like lesions (33, 34,50,51). However, the most important feature of KS is the presence of the new human herpesvirus KSHV in most spindle cells in situ, and all established long term cell cultures and cell lines lack the presence of KSHV DNA. The usefulness of these lines to study the pathogenesis of KS is therefore questioned.
REFERENCES 1. Franceschi S, Geddes M. Tumori 81: 308-314, 1995. 2. Oettle AG. Geographic and racial differences in the frequency of Kaposi’s sarcoma as evidence of environmental or genetic causes. In: Symposium on Kaposi’s Sarcoma, eds LV Ackerman and JF Murray. Karger, Basel, 1962. 3. Wabinga HR, Parkin DM, Wabwire-Mangen F, Mugerwa JW. Int J Cancer 54: 23-361993. 4. Service PH. MMWR 30: 305-308,1981. 5. Nickoloff BJ, Griffiths CE. Am J Pathol 135: 793-800,1989. 6. Stiirzl M, Brandstetter H, Roth WK. AIDS Research and Human Retroviruses 8: 1753-1764, 1992. 7. Browning PJ et al. Blood 84: 2711-20,1994. 8. Sirianni MC et al. Lancet 349: 255, 1997. 9. Rabkin CS, Bedi G, Musaba E, Biggar RJ. N Engl J Med 1: 257-260,1995. 10. Rabkin CS et al. New England Journal of Medicine 336: 988-993,1997. 11. Risau W. Nature 386: 671674,1997. 12. Samaniego F, Markham PD, Gallo RC, Ensoli B. J Immunol 154: 3582-92,1995. 13. Salahuddin SZ et al. Science 242: 430-3,1988. 14. Nair BC et al. Science 255: 1430-2,1992. 15. Miles SA et al. Proc Natl Acad of Sci USA 87: 4068–4072,1990. 16. Ensoli B, Salahuddin SZ, Gallo RC. Cancer Cells 1: 93-6,1989.
166 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.
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Ensoli B et al. Nature 371: 674-80, 1994. Miles SA et al. Proc Natl Acad Sci USA 87: 4068-4072, 1990. Vaishnaw YN, Wong-Staal F. Annual Rev Biochem 60: 577–630,1991. Ensoli B et al. J Virol 67: 277–87, 1993. Albini A, Barillari G, Benelli R, Gallo RC, Ensoli B. Proc Nat1 Acad Sci USA 92: 4838–42, 1995. Vogel J, Hinrichs SH, Reynolds RK, Luciw PA, Jay G. Nature 335: 606–611, 1988. Beral V, in Cancer, HIV and AIDS. Beral V, Jaffe HW, Weiss RA, Eds. (Cold Spring Harbor Laboratory Press, 1991), vol. 10, pp. 5-22. Chang Y et al. Science 266,1865-9, 1994. Moore PS et al. J Virol 70: 549-58, 1996. Boshoff C, Weiss RA. Current Opinion Infect Diseases 10: 26–31, 1997. Cesarman E, Chang Y, Moore PS, Said JW, Knowles DM. N Engl J Med 332: 1186-91, 1995. Boshoff C et al. Nat Med 1: 1274–8, 1995. Staskus KA et al. Journal of Virology 71: 715–719, 1997. Renne R et al. Nat Med 2: 342–6,1996. Boshoff C et al. Blood 91: 1671-9, 1998. Cesarman E et al. Blood 86: 2708-14,1995. Lunardi-Iskander Y et al. J Natl Cancer Inst 87: 974–981, 1995a. Lunardi-Iskandar Y et al. Nature 375: 64-8, 1995b. Griffiths SJ, Adams, DJ, Talbot SJ. Nature 390: 568, 1997. Way D, Witte M, Bemas M. VI International conference on AIDS. San Francisco. 1990. Levy JA, in AIDS: Pathogenesis and Treatment. Levy JA, Ed. (Marcel Dekker, Inc, New York, 1989). Roth WK, Brandstetter H, Sturzl M. AIDS 6: 895-913, 1992. Delli Bovi P, Donti E, Knowles DM. Cancer Research 46: 6333–6338,1986. Albini A, Mitchell CD, Thompson EW. J Cell Biochem 36: 369-376,1988. Wittek AE, Mitchell D, Armstrong GR. AIDS 5: 1485–1493, 1991. Nakamura S, et al. Science 242: 426-30, 1988. Corbeil J, Evans LA, Vasak E, Cooper DA, Penny R. J Immunol 146: 2972, 1991. Roth WK, Werner S, Risau W, Remberger K, Hofschneider PH. International Journal of Cancer 42: 767–773,1988. Ensoli B et al. Science 243: 223–6,1989. Ensoli B, Barillari G, Gallo RC. Immunol Rev 127: 147–55, 1992. Roth WK. Journal of Cancer Research and Clinical Oncology 117: 186–191, 1991. Ensoli B et al. J Clin Invest 94: 1736-46, 1994. Ensoli B, Barillari G, Salahuddin SZ, Gallo RC, Wong SF Nature 345: 84-6, 1990. Siegal HA et al. American Journal of Public Health 81: 1642-1644,1991. Herndier BG et al. AIDS 8: 575–581, 1994.
Chapter 27 Brain Tumors
Francis Ali-Osman Section of Molecular Therapeutics, Department of Experimental Pediatrics, University of Texas M.D. Anderson Cancer Center; Houston, Texas 77030. Tel: 001-713-745-0669; Fax: 001- 713-794-5514; E-mail:
[email protected]
1.
INTRODUCTION
Human brain tumors are histologically and biologically highly heterogeneous (14,15), and the cellular and molecular processes and mechanisms involved in their initiation, progression and clinical and biological behavior are still only partly understood. Advances in techniques of culture and propagation of cells and tissues of these tumors have, however, significantly facilitated progress in this area. Since the mid 1960s to the 1970s, when a series of human malignant glioma cell lines were developed by Ponten and his colleagues (69,90), a relatively large number of well characterized cell lines of the major categories of brain tumors have been developed (14,24,69,90,91) and many of them are available in cell line repositories, such as the American Type Culture Collection. These cell lines have enabled important biological questions, such as those related to growth kinetics, drug sensitivity, cytogenetics, molecular genetics, and gene expression and regulation to be investigated (1-5,19,21,24, 30,51,57,83). This chapter describes the essentials of techniques for in vitro culture of malignant brain tumor cells, and for establishing brain tumor cell lines. A variety of established brain tumor cell lines described in the literature are listed, with information on some of their important cellular, molecular and other relevant features, where such information is available.
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ESTABLISHMENT AND MAINTENANCE OF BRAIN TUMOR CELL LINES
Two basic techniques have been used to initiate the primary cultures from which the majority of brain tumor cell lines have been derived. The first of these, the tissue explant method, involves placing tumor tissue in a fixed position in culture medium, with or without an additional anchoring support, and allowing the tumor cells to grow out of the tissue as explants. The method is technically relatively simple and was used successfully to generate most of the early brain tumor cell lines (3,5,14,24,57,69,90,91). A large number of brain tumor cell lines have been established from cultures generated by variations of this method. For example, in the late 1960s to the 1970s, Ponten, Westermark and colleagues (69,90,91) established the well-characterized Useries of malignant gliomas from explants on grid-supported lens paper or gelatin foam. The second method of initiating primary brain tumor cultures involves using single cells obtained by dissociating the tumor tissue. This method of brain tumor culture has advanced significantly over the past two decades, due to developments in enzymatic techniques of tissue dissociation that result in little to no trauma to the dissociated tumor cells (3,71). Briefly, the tumor specimen is finely cut up, for example with crossed scalpels, into a “slurry”, and treated with an enzyme cocktail consisting of DNAse Type I, neutral protease, collagenase Type Ia in Ca++/Mg++-free Hank’s Balanced Salt Solution for approximately 60-90 minutes, or until the tumor is completely dissociated. The dissociated cells are pelleted by centrifugation, washed with fresh medium and seeded at an appropriate density, in the range of 1 – 2.5 × 105 cells/ml in Dulbecco’s Minimum Essential Medium containing 15% FCS in a 37°C humidified atmosphere. The cultures are examined once a week until they attain confluency (3,71). The generation and establishment of brain tumor cell lines is generally relatively straightforward once a primary culture has been successfully initiated. The process essentially involves successive passaging and the resulting cell lines are generally stable and can be passaged indefinitely. Table 1 summarizes essential features of cell lines derived from brain tumors and reported in the literature. While this list is quite extensive and includes many of the wellcharacterized cell lines, it does not include every cell line ever developed. Characterization of a cell line is a critical and important part of the process. The major morphological features used to characterize malignant brain tumor cell lines in culture include determining the overall growth pattern of the cell population, degree of cellular pleomorphism, extent of process formation, cytoplasmic and nuclear staining characteristics and the size, shape and number of cell nuclei and nucleoli. Immunocytochemical staining allows an assessment of the level and heterogeneity of expression of various proteins, some of which
Brain tumor cell lines
Cell Line
Age /sex
Histology/Grade/Origin (Site)
Method
Culture Type
Culture Authentication
ASTROCYTOMAS GRADE III Ast812 D54MG 39 yr/F 59 yr/F DBTRG-05MG 45 yr/M GOS 1 55 yr/M GOS 3 H4 37 yr/M 76 yr/M Hs683 6 mo/M IPNT-H 48 yr/M IPSB-18 42 yr/M MGR1 (UWR1) 46 yr/F SA 101 72 yr/M SW 1088 68 yr/M SW 1783 54 yr/F U87MG 50 yr/M U118MG M U399MG
Malignant astrocytoma Mixed anaplastic glioma Anaplastic astrocytoma Astrocytoma Grade II/III Oligoastrocytoma Grade II/III Neuroglia Glioma Pilocytic astrocytoma; Hypothalamus Grade III glioma; temporal lobe Anaplastic astrocytoma Anaplastic astrocytoma Anaplastic astrocytoma Anaplastic astrocytoma Astrocytoma Grade III/Glioblastoma Anaplastic glioma/Glioblastoma Anaplastic astrocytoma
Explant Dissociation Dissociation Dissociation Dissociation Explant Explant Dissociation Dissociation Dissociation Dissociation Explant Explant Explant Explant Explant
Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer
IE; K IE;K Karyotype Karyotype Isozymes Isozymes Isozymes Isozymes Karyotype Karyotype Isozymes Isozymes Isozymes K IE K, IE
GLIOBLASTOMAS GRADE IV A-172 53 yr/M A-382 10 yr/M AM 36 yr/M 68 yr/M CCF-STTG1 D18MG 60 yr /F D37MG 61 yr/F D65MG 65 yr/F
Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme; left occipital Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme
Explant Explant Dissociation Explant Dissociation Dissociation Dissociation
Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer
IE; K Karyotype IE Karyotype K, IE IE; K IE; K
Reference 22 14, 24, 18 53 37 37 25,39 39 62, 68, 63,72 52,62,63,69 2 24,59 39,55 39,55 24, 14,39, 69 24, 14,39, 69 24,90
Brain Tumors
Table 1
24,36,39 36 95 10,39 14,24 14, 24, 18 14,24
169
Continued on next page
Age /sex
Histology/Grade/Origin (Site)
Method
Culture Type
D245MG D247MG D263MG D270MG G22 G62 G44 G59 G60 G61 G62 G63 G112 G118 G120 G121 G123 G130 G140 GATAGM-96 GOS 2 HBT 18 HBT 28 HBT 35 HeRo Hu 70
70 yrlM 58 yr/M 53 yr/M 42 yr/M 56 yr/F – – 66 yrlM – – – – 74 yr/M – – – 68 yrlM – 59 yrlM 80 yr/M 64 yrlM 39 yrlM 45 yr/M 63 yr/M 64 yrlM –
Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme; cerebrum Glioblastoma multiforme; right parietal Glioblastoma multiforme; temporal lobe Glioblastoma multiforme
Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation
Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer
Culture Authentication Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype IE; K Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype
Reference 16, 18, 21 16, 18, 21 16, 18, 21 16, 18, 21 7, 92 7, 92 7, 92 7, 92 7, 92 7, 92 7, 92 7,92 7, 92 7, 92 7, 92 7, 92 7, 92 7, 92 7, 92 10 37 4 4 4 28 34
Continued on next page
Ali-Osman
Cell Line
170
Table 1 (continued)
171
Table1 (continued) Cell Line
–
–
–
–
49 yr/F –
–
–
65 yr/F 57 yr/M 59 yr/M 44 yr 68 yr/F 50 yr/M 8 yr/M 72 yrlF 24 yr/F 67 yr/F 61 yr/M 62 yr/M 48 yr/M 56 yr/M 75 yr/M 60 yr/M
Histology/Grade/Origin (Site)
Method
Culture Type
Culture Authentication
Reference
Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme; frontal lobe Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme; Left frontal Glioblastoma multiforme; Right frontal Glioblastoma multiforme; Left parietal Glioblastoma multiforme; Right parietal Glioblastoma multiforme; Left temporal Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma Glioblastoma multiforme
Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation Explant Explant Explant Explant Explant Explant
Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Suspension Monolayer Monolayer Monolayer Monolayer Monolayer
Karyotype Karyotype Karyotype Karyotype Karyotype Isozymes Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype K; IE K; IE K; IE K, IE K; IE
34 34 34 34 34 52, 62,63 82,24 82 82 2 2 24,59 24,59 24,59 24,59 73 73 73 73 73 39,84 24, 14, 39, 6 9 24, 14,39, 6 9 24, 14,39, 6 9 24, 14,39, 69 24, 14,39, 6 9
Continued on next page
Brain Tumors
Hu 195 Hu 104 Hu 112 Hu 175 Hu 197 IPRM-5 LN 18 LN 229 LN 308 MGR2 (UWR2) MGR3 (UWR3) SA4 SA 44 SA 45 SA 137 SF-126 SF-188 SF-210 SF-268 SF-295 T98G U105MG U139MG U178MG U251MG U343MG
Age /sex
172
Table 1 (continued) Cell Line
Age /sex
Histology/Grade/Origin (Site)
Method
Culture Type
Culture Authentication
Reference
Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer Monolayer
K; IE K, IE K; IE K; IE K; IE K, IE Isozymes Isozymes Isozymes Isozymes Isozymes
21 21 24, 89 24, 89 14, 24 24, 90 24, 90 21, 24 21, 24 21, 24 96 14, 24 18, 21 46 24, 59 24, 59
60 yr/M 60 yr/M – 50 yr/M 42 yr/M M – – – – 49 yr/M
Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme Glioblastoma multiforme; fronto-parietal
GLIOSARCOMAS D32MG D336MG Gl-1 SA 39 SA 134
66 yr/M 68 yr/M 61 yr/M 41 yr/F
Gliosarcoma Gliosarcoma Gliosarcoma Gliosarcoma Gliosarcoma
Dissociation Dissociation Explant Dissociation Dissociation
Monolayer Monolayer Monolayer Monolayer Monolayer
IE; K Karyotype IE; K Karyotype Karyotype
Medulloblastoma; peritoneum Medulloblastoma; cerebellum Medulloblastoma; cerebellum Medulloblastoma; cerebellum Medulloblastoma; cerebellum Medulloblastoma; cerebellum Medulloblastoma; cerebellum
Ascites Dissociation Dissociation Dissociation Dissociation Dissociation Dissociation
Susp/Monol Suspension Suspension Suspension Suspension Suspension Suspension
Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype Karyotype
MEDULLOBLASTOMAS D283Med 6 yr/M D306Med 12 yr/F D341Med 3.5 yr/M D382Med 4 yr/F D386Med 7 yr/M D384Med 17 mo/M 5 yr/M D425 Med
17, 31 17 17, 32 17 17 17, 31 17, 31 Continued on next page
Ali-Osman
U343MGa 31L U343MGGaCI26 U372MG U373MG U410MG U489MG U563MG U1231MG U1240MG U1242MG YH
Explant Explant Explant Explant Explant Explant Explant Explant Explant Explant Dissociation
Cell Line
Age /sex
Histology/Grade/Origin (Site)
Method
Culture Type
Culture Authentication
Reference
Daoy HBT 228 IPNN-8 MCD-1 MHH-MED1 MHH-MED2 MHH-MED3 MHH-MED4 TE 671*(=RD) PINEOCYTOMAS PER 452** PER 453** PER 480
4 yr/M 10 yr/F 13 yr/M 17 yr/M 10 yr/M 6 yr/F 3 yr/F 4 yr/M 6 yr/F
Medulloblastoma Medulloblastoma; Posterior fossa Medulloblastoma; 4th ventricle Medulloblastoma; recurrent; Cerebellum Medulloblastoma; Cerebellum; CSF Medulloblastoma; Cerebellum Medulloblastoma; Cerebellum Medulloblastoma; Cerebellum Medulloblastoma/cerebellum
Explant Dissociation Dissociation Explant Dissociation Dissociation Dissociation Dissociation Explant
Monolayer Monolayer Monolayer Monolayer Suspension Suspension Suspension Susp/Monol Monolayer
Isozymes Karyotype Isozymes Isozymes K; IE K; IE K; IE K; IE Karyotype
30, 47, 39 54 68,72 65 67 67 67 67 39, 60
8 mo/F 8 mo/F 11 mo/M
Pineoblastoma; CSF Pineoblastoma; CSF Pineoblastoma; Bone marrow aspirate
Dissociation Suspension Suspension
Suspension Suspension Suspension
Karyotype Karyotype Karyotype
48 48 50
173
*Contaminated with RD cells **Established from CSF of the same patient obtained 10 months before therapy and after high dose methotrexate therapy. Abbreviations: K, karyotype; IE, immunohistochemical analysis
Brain Tumors
Table 1 (continued)
174
Ali-Osman
are characteristic and serve as markers of the different tumor types. Markers of brain tumor cells that are detected by immunocytochemistry include S-100 protein, galactocerebroside, neuron specific esterase, synaptophysin, glutamine synthase, and cytoskeletal proteins, such as vimentin, neurofilament protein and glial fibrillary acidic protein (GFAP) (3,14,83,94).
3.
EXTENT TO WHICH BRAIN TUMOR CELL LINES REFLECT THE CLINICAL DISEASE
Histologically, brain tumors, particularly those of glial origin, are highly heterogeneous at both the cellular and molecular levels (1,14,5,91). Primary malignant gliomas consist not only of malignant glial cells, but also other cellular components, such as endothelial cells, normal reactive astrocytes and infiltrating lymphocytes and macrophages. When such tumors are established in vitro as cell lines, the non-malignant cellular components are often present during the early phases of the cultures, but do not survive over the relatively high number of passages required to establish them as cell lines. Thus, the cells of most brain tumor cell lines consist, primarily, of malignant glial cells, and often these cultures retain many of the morphological features and differentiation markers of the cells of the tumors from which they were established (Table 2). After prolonged in vitro passage, the cultures of malignant glioma cell lines often assume a single predominant morphology, often astrocytic, bipolar (fibroblastic), epithelial, or a mixture of these. The morphological uniformity is the result of a combination of a cellular selection process during culture, adaptation to 2-dimensional in vitro growth, and absence of in vivo effects, such as cell-cell interactions and growth factors. Although the cellular features of a primary tumor, such as cellular and nuclear pleomorphism and GFAP expression, are often maintained in the tumor cell lines (3,5,14,57), some of the features can be significantly altered. However, this can be due to cross-contamination. For example, the TE671 cell line (60,80,93), which was established from a primary medulloblastoma, was crosscontaminated with the RD rhabdomyosarcoma cell line (80). An important feature of brain tumor cell lines that is of clinical significance is the ability to produce tumors in vivo, often measured by the ability to grow as xenografts when transplanted in nude mice. Several established brain tumor cell lines have been shown to retain their tumorigenic capability (Table 2). The histologies of the tumors they give rise to are often similar to those of the same histological tumor type, and thus provide appropriate models for studying clinicaldisease.
,
Similarity to Primary Tumor
Tumorigenicity In Vivo
Cell Line
In vitro morphology
ASTROCYTOMAS Ast812 D54MG DBTRG-05MG GOS 1 GOS 3 H4 Hs683 IPSB-18 MGR1 (UWR1) SW 1088 SW 1783 U87MG
Polygonal, astrocytic Astrocytic Fibroblastic, spindle-shaped Spindle-shaped, fibroblastic Spindle-shaped, fibroblastic Epithelial Fibroblastic Bipolar Fibroblastic, astrocytic Pleomorphic Pleomorphic Epithelial
GFAP NF NSE, S-100, Class I HLA, GFAP, S-100, Vim GFAP, S-100, Vim Vim GFAP GFAP GFAP GFAP GFAP FBN; Vim
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
– – – Yes
22 14,24 53 37 37 25,39 39 52, 62, 63, 68 2 39 39 14, 21, 24, 45
GLIOBLASTOMAS A-172 AM CCF-STTG1 D18MG D37MG D54MG D65MG D245MG D247MG D263MG
Fibroblastic, astrocytic Bipolar, stelate; polygonal Polygonal Astrocytic Fibroblastic, spindle-shaped Astrocytic Epithelial Pleomorphic Bipolar, fibroblastic Fascicular
NF GFAP; S-100 GFAP; NSE NF NSE, S-100 NF NF GFAP Vim GFAF'
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
No Yes Yes No No Yes Yes Yes No Yes
24, 36, 37 95 36 14,24 14,24 14,24 14,24 18 18 18
Differentiation Markers
– Yes – Yes Yes No
Reference
Brain Tumors
Table 2 Biological characteristics of brain tumor cell lines
Continued on next page
175
Cell Line
Pleomorphic Fibroblastic, spindle-shaped Bipolar, spindle-shaped Spindle-shaped, fibroblastic Spindle-shaped, fibroblastic Spindle-shaped, fibroblastic Spindle-shaped, astrocytic Spindle-shaped Fibroblastic Spindle-shaped, fibroblastic Epithelial Fibroblastic Bipolar, astrocytic Fibroblastic, astrocytic Pleomorphic Bipolar, fibroblastic Bipolar, stellate Epithelial Pleomorphic Astrocytic Astrocytic Fibroblastic Pleomorphic Fascicular Pleomorphic
Differentiation Markers
Similarity to Primary Tumor
GFAP Yes NSE, S100, Class I HLA, – Yes NF, NSE, Vim Yes GFAP, S-100, Vim Yes GFAP, S-100, Vim Yes GFAP, S-100, Vim Yes GFAP; Vim GFAP; Vim Yes GFAP; Vim Yes Yes GFAP Yes Vim GFAP Yes Yes GFAP Yes GFAP GFAP Yes Yes FBN, lam, Collagen IV Yes FBN, lam, Collagen IV Yes FBN, lam, Collagen IV NSE, S-100, Syn, Vi Yes GFAP, FBN, lam, Collagen IV Yes GFAP Yes FBN FBN; Vim Yes GFAP; Vim; Nestin Yes GFAP, FBN; Vim Yes
Tumorigenicity In Vivo No – – Yes Yes Yes Yes Yes – – – No Yes Yes No No No No No Yes No Yes –
Reference 18 53 10 37 37 37 4 4 4 28 25,39 39 52,62,63 2 2 73 73 73 73 73 84 21,24 14, 21,24,45 14, 21, 24, 45 14, 21, 24, 45
Continued on next page
Ali-Osman
D270MG DBTRG-05MG GATAGM-96 GOS 1 GOS 2 GOS 3 HBT 18 HBT 28 HBT 35 HeRo H4 Hs683 IPRMJ MGR2 (UWR2) MGR3 (UWR3) SF-126 SF-188 SF-210 SF-268 SF-295 T98G U105MG U178MG U251MG U343MG
In vitro morphology
176
Table 2 (continued)
Cell Line
In vitro morphology
Differentiation Markers
Similarity to Primary Tumor
Tumorigenicity In Vivo
Reference
U372MG U373MG U410MG U563MG U1231MG U1240MG U1242MG YH
Pleomorphic Astrocytic Astrocytic Polygonal Fibroblastic/Epithelial Fibroblastic/Fascicular Fibroblastic/Bipolar Bipolar; triangular
NF FBN; Vim GFAP; FBN; Vim GFAP; FBN; Vim GFAP GFAP GFAP GFAP GFAP,S-100
Yes Yes Yes Yes Yes Yes Yes Yes
Yes Yes Yes – – – – Yes
14, 21, 24,45 14,21, 24,45 14,21,24,45 66,69 21,24 21,24 21,24 95
GLIOSARCOMAS D32MG D336MG G1-1
Epithelial Bipolar, fibroblastic Multipolar, spindle-shaped
CK-BB; NF Vim GFAP, NF, NSE, Vim
Yes Yes Yes
No No Yes
14,24 18 46
MEDULLOBLASTOMAS D283Med D306Med D341Med D382Med D384Med D425 Med Daoy HBT 228 IPNN-8 MCD-1 MHH-MED1
Spheroids; small round cells Spheroids; small round cells Spheroids; small round cells Spheroids; small round cells Spheroids; small round cells Spheroids; small round cells Spheroids; small round cells Smallpolygonal Smallpolygonal Bipolar, triangular Aggregates of small cells
NF Vim; Syn Yes – Yes Yes GS, NSE NF; Vim; Syn – Yes NF; Vim; Syn Yes Yes NF; Vim; Syn NF, Vim Yes NSE Yes Yes GFAP,NF,S-100, Syn NSE, Vim, S-100, Syn, N-CAM Yes NSE, Vim Yes
Yes Yes Yes Yes Yes Yes Yes – – No –
16,31, 83 16 16,32, 83 16 16,31, 83 16, 31, 83 47,83 54 62, 68, 63, 72 65 67
177
Continued on next page
Brain Tumors
Table 2 (continued)
178
Table 2 (continued) Cell Line
In vitro morphology
Differentiation Markers
Similarity to Primary Tumor
Tumorigenicity in vivo
Reference
MHH-MED2 MHH-MED3 MHH-MED4 TE 671**
Aggregates of small cells Aggregates of small cells Spheroids/bipolar Polygonal; fuciform
N-CAM, NSE, Syn, Vim N-CAM, NSE, Syn, Vim NSE, Syn, Vim NSE, CK-BB
Yes Yes Yes No
– – – Yes
67 67 67 60
PINEOBLASTOMAS PER 452 PER 453 PER 480
Spheroids, small round cells Spheroids, small round cells Spheroids, small round cells
NSE, GFAP, NF, NSE, Syn, Vim NSE, NF, Syn, Vim
Yes Yes Yes
– – –
48 48 48,49
Abbreviations: CK-BB: creatine kinase BB; DH: dopamine b-hydrolase; GFAP: glial fibrillary acidic protein; GS: glutamine synthase; FBN: fibronectin; lam: laminin; N-CAM: neural cell adhesion molecule; NF neurofilament (any subclass); NSE: neuron specific enolase; TH: tyrosine hydrolase; Syn: synaptophysin; Vim: vimentin. *Unless otherwise stated, in vivo tumorigenicity was determined in nude mice and by inoculation in the cheek pouches of cortisone-treated hamsters. **Based on cytogenetic analysis and DNA fingerprinting, the TE 671 cell line has been reported to be cross-contaminated with the RD rhabdomyosarcoma cell line (80)
Ali-Osman
Brain Tumors
4.
179
MOLECULAR GENETICS OF BRAIN TUMOR CELL LINES
Cell lines will continue to play a critical role in advancing knowledge of the molecular nature of tumors of the nervous system (19). For a number of genes implicated in the genesis and progression of brain tumors, their involvement in these processes was first observed in brain tumor cell lines before subsequent extension of the findings to primary specimens. Generally, cytogenetic analyses have shown a significant number and variety of chromosomal abnormalities in brain tumor cell lines (19,64). Cytogenetics provided the first clue of the existence of the tumor suppressor gene MMAC-1 or PTEN, which encodes a tyrosine phosphatase (23,56,79). Several brain tumor cell lines have been analyzed for abnormalities in a variety of oncogenes, tumor suppressor genes and genes encoding cell cycle regulators, growth factors, growth factor receptors, and the genes and proteins involved in apoptosis (Table 3). Oncogene anomalies observed in brain tumor cell lines have been primarily amplifications and/or activating mutations in genes, such as c-myc, n-ras and erbB1, leading to over-expression of the gene product. For the myc family of oncogenes, it is interesting that only c-myc has been shown to be amplified and/or over-expressed in brain tumor cell lines (16, 34,42,44,63,79,80). Both n-myc and 1-myc are usually not expressed. Several cell lines have also been characterized with respect to erbB1 (EGFR) amplifications, rearrangements and over-expression (18,66). There is evidence that the c-myb oncogene is amplified, rearranged and activated in some malignant glioma cell lines (89). Analysis of tumor suppressor genes in brain tumor cell lines has demonstrated significant heterogeneity in the nature and in the presence or absence of abnormalities in these genes. The most extensively studied tumor suppressor genes include p53, pRB1, p16 and most recently, PTEN/MMAC-1 (9, 10,42,45,53,63,65). A number of cell cycle related genes and genes involved in the apoptotic process have been examined in human brain tumor cell lines. These include cyclins A, B1, D1, D3 and E, and cyclin-dependent kinases, such as cdk2 and cdk4 (27,42). The molecular heterogeneity observed in both oncogenes and tumor suppressor genes in brain tumors indicates both a complexity and a significant diversity in the molecular pathogenesis of these tumors. Tumor cell lines have also been examined for the expression of genes and other molecular/biochemical end-points associated with therapeutic resistance. These include the O6-alkylguanine DNA alkyltransferase and its associated methyl excision repair (mer) phenotype, glutathione and glutathione S-transferases, p-glycoprotein, DNA polymerases and DNA excision repair genes. Table 3 provides a summary of some of the major molecular parameters reported for brain tumor cell lines in the literature.
180
Ali-Osman
Table 3 Molecular characteristics of established brain tumor cell lines reported in the literature Gene/Molecular Parameter
Cell Lines
References
D341, D384, D425 (M) SF188 (G) U343MG, U1242MG, T98G, Hu70, Hu104, Hu112, Hu175, Hu197 (G) A172, U87MG ,U251MG (A) MHH-MED 2, D283Med, D306, D341Med, D382, D425 (M) PER-480 (P)
17, 31 86, 88 17, 34, 45
D341 (M) PER-452, PER-453 (P)
29,48, 49, 50 70, 78, 88, 89
Hero (G)
89
N-ras Over-expression p53 Mutations
Hu 70, Hu104, Hu112, Hu195, Hu197 (G) A172, U373MG, LN18, LN308, G22, G59, G112, G123, G130, G140,T98G (G) MCD1, Daoy, HBT 228 (M) U251MG, IPNT-H (A)
34 6, 8, 9,10, 40,63, 65, 65,82,87
Rb Positive/Overexpression
A172, CCF-STG1, U343MG(G) DBTRG-05MG, H4, HS 683, U251MG (A)
c-Myc Amplification/Rearrangement Over-expression
N-myc Over-expression/amplification c-Myb Amplification/Rearrangement
p16 Deletions/Rearrangements MDM2 Amplification/ Over-expression EGFR (erbB1) Amplification Rearrangement Over-expression
Truncated mRNA PDGFR Over-expression alpha
beta
67
42,45,53
A172, CCF-STG1, T98G (G) Gl-1 (S) MGR2, MGR3 (GS) HS683, U251MG, U87MG (A)
41,42, 38 78
CCF-STTG1 (G) U87MG, U251MG (A)
40
D245MG, D270MG (G) DBTRG-05MG (A) D37MG, D245MG, D247MG, D270MG Hu 70, Hu104, Hu112, Hu197, U410 (G) U87MG (A) U178MG, U343Mga C12:6, U489MG, U1240MG, U1242MG (G) D270, D245 (G) U87MG (A) U178MG, U343MG, U372MG, U1231MG, U1242MG (G) U87MG (A) U178MG, U343MG, U372MG, U1231MG, U1242MG (G)
18 18, 34, 93 66
18,93 21,66
21,66
Continued on next page
Brain Tumors
181
Table 3 (continued) Gene/Molecular Parameter
Cell Lines
Interferon-alpha Deletion
HS683, H4, U87MG (A)
Methyl Excision Repair ( Mer) Negative Positive Cell Cycle Regulators Over-expression: Cyclin A Cyclin B1 Cyclin D1 Cyclin E cdk4 CD44 Positive pp60c-src max Over-expression RSU-1 ( ras suppressor) TGF-alpha TGF-Beta
A172, A382, UlOSMG, HBT28, MGR3 (G), U87MG (A) H4, Hs683 (A) U178MG, U373MG, HBT18 (G)
References
1, 4, 20, 25 4, 20, 25
U343 (G) 27 SF 126, U343, U373 (G) 27 SF 126, U343 (G) 27 SF 126, U343 (G), U87 (A) 27 SF 126, SF 188, U343, U373 (G), U87, U251 (A) 27 IPNT-H, IPSB-18 (A) IPRM-5 (G), IPNN-8 (M) 19,68 GOS -1, GOS-3 (A), GOS -2 (G) 37 U87MG, U251 Sp (A) 45,70 U343MG, U373MG, U1242MG (G) U251 (A) 85 U87MG, U251MG (A) 65,66 U178MG, U343MG, U563MG, U1240MG (G) MCD-1 (M) 65
Abbreviations: A, astrocytoma GIII; G, glioblastoma GIV; GS, gliosarcoma; M, medulloblastoma; P, pineoblastoma
REFERENCES 1. Ali-Osman F, Schofield D. Current Opinion in Oncology 2: 655-665, 1990. 2. Ali-Osman F, Stein DE, Renwick A. Cancer Res 50: 6976-6980,1990. 3. Ali-Osman F. In: Methods in Molecular Medicine, Jones GE (ed.) Human Cell Culture Protocols, Humana Press Inc. pp. 63-80,1996. 4. Ali-Osman F, Young P, Rairkar A. Cancer Biochem Biophys 14: 231-241,1995. 5. Ali-Osman F. In: The Gliomas. Berger MS, Wilson CW (eds). WB Saunders, pp. 134-141, 1999. 6. Antoun G, Ali-Osman F. J Cell Biochem, in press. 7. Anker L, Ohgaki H, Ludeke BI, Herrmann H-D. Kleihues P, Westphal M. Int J Cancer 55: 982-987,1993. 8. Arnstein P, Taylor DOW, Nelson-Rees WA, Huebner RJ, Lennette EH. J Natl Cancer Inst 52: 71-84,1974. 9. Asai A, Miyagi Y, Sugiyama A, Gamanuma M, Hong SI, Takamoto S, Nomura K, Matsutani M, Takakura K, Kuchino Y. J Neuro-Oncology 19: 259-268,1994. 10. Bakir A, Gezen F, Yildiz 0, Ayhan A, Kahraman S, Kruse CA, Varella-Garcia M, Yildiz F, Ayhan Kubar. Cancer Genet Cytogen 103: 46-51, 1997. 11. Barna BP, Bay JW, Chou SM, Hoeltge GA, Jacobs B. In Vitro 19: 275,1983. 12. Biedler JL, Helson L, Sprengler BA. Cancer Res 33: 2643-2652, 1978. 13. Biedler JL, Roffler-Tarlov S, Schachtner M, Freedman LS. Cancer Res 38: 3751-3757, 1978.
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14. Bigner DD, Bigner SH, Ponten J, Westermark B, Mahaley MS, Ruoslathi E, Herschman H, Eng LF, Wikstrand CJ. J Neuropath Exp Neurol 40: 201-229, 1981. 15. Bigner DD, McLendon RE, Brunner JM (editors). Russel and Rubinstein's Pathology of Tumors of the Nervous System. 6th Ed. Arnold and Oxford University Press. London, Sydney, Auckland, 1998. 16. Bigner SH, Friedman HS, Biegel J, Wikstrand CJ, Mark J, Gebhardt R, Eng LF, Bigner DD. Acta Neuropath 72: 86-97, 1986. 17. Bigner SH, Friedman HS, Vogelstein B, Oakes WJ, Bigner DD. Cancer Res 50: 2347-2350, 1990. 18. Bigner SH, Humphrey PA, Wong AJ, Vogelstein B, Friedman HS, Bigner DD. Cancer Res 50: 8017-8022,1990. 19. Bigner SH, Schroeck EV. J Neuropath Exp Neurol 56: 1173-1181, 1997. 20. Bodell WJ, Aida T, Berger MS, Rosenblum ML. Carcinogenesis 7: 879-993, 1986. 21. Bongcam-Rudloff E, Nister M, Betholtz C, Wang J-L, Stenman G, Huebner K, Croce CM, Westermark B. Cancer Res 51: 1553-1560, 1991. 22. Cheng A-J, Liao S-K, Chow S-E, Chen J-K, Wang T-CV Biochem Biophys Res Comm 237: 438-444,1997. 23. Chiariello E, Roz L, Albarosa R, Magnani I, Finocchiaro. Oncogene 16: 541-545, 1998. 24. Collins VP. Int Rev Exp Pathol 24: 136-203, 1983. 25. Day R, Ziolkowski CHJ. Nature 279: 797-799, 1978. 26. de Muralt B, de Tribolet N, Diserens A-C, Stavrou D, Mach J-P, Carrel S. Eur J Cancer Clin. Oncol21: 207-216, 1985. 27. Dirks PB, Hubbarb SL, Murakami M, Rutka JT. J Neuropath Exp Neurol 56: 291-300,1997. 28. Fisher H, Schwechheimer K, Heider M, Bernhadt S, Zang KD. Cancer Gen Cytogen 17: 257-268,1985. 29. Foley J, Cohn SL, Salwen HR, Chagnovich D, Cowan J, Mason KL, Parysek LM. Cancer Res 51: 6338-6345, 1991. 30. Friedman HS, Bigner DD. J Neuropath Exper Neurol 44: 592-605, 1985. 31. Friedman HS, Burger PC, Bigner SH, Trojanowski JQ, Wikstrand CJ, Halperin EC, Bigner DD. J Neuropath Exp Neurol 44: 592-605, 1985. 32. Friedman HS, Burger PC, Bigner SH, Trojanowski JQ, Brodeur GM, Xuanmin H, Kurtzberg J, Berens M, Halperin EC, Bigner DD. Amer J Pathol130: 472-484, 1988. 33. Fults D, Pedone CA, Morse HG, Rose JW, McCay RD. J Neuropath Exp Neurol 51: 272-280, 1992. 34. Gerosa MA, Talarico D, Fognani C, Raimondi E, Colombatti M, Tridente G, de Carli L,Valle D. J Natl Cancer Inst 52: 71-84 1979. 35. Gerson JM, Schlesinger HR, Sereni BS, Moorhead PS, Hummeler K. Cancer 39: 2508-2517, 1977. 36. Giard DJ, Aaronson AA, Todaro GJ, Arnstein P, Kersey JH, Dosik H, Parks WP. J Natl Cancer Inst 51: 1417-1423, 1973. 37. Halfter H, Kremerskothen J, Weber J, Hacker-Klom U, Barnekow A, Ringelstein EB, Stogbauer F. J Neuro-Oncol 39: 1-18, 1998 38. Hama S, Sadamoto T, Yoshioka H, Kurisu K, Tahara E, Naruse I, Heike Y, Saijo N. Anticancer Res 17: 1933-1938, 1997. 39. Hay R, Caputto J, Chen TR, Macy M, McClintock P, Reid Y (editors). American Type Culture Collection. Catalogue of Cell Lines and Hybridomas. 7th Edition, 1992. 40. He J, Reifenberger G, Lu L, Collins VP, James CD. Genes Chromosomes and Cancer 11: 91-96, 1994. 41. He J, Allen JR, Collins VP, Allalunis-Turner MJ, Godbout R, Day III RS, James CD. Cancer Res54: 5804-5807, 1994. 42. He J, Olson JJ, James CD. Cancer Res 55: 4833-4836, 1995.
Brain Tumors
183
43. He X, Skapek S, Wikstrand CJ, Friedman HS, Trojanowski JQ, Kempshead JT, Coakham HB, Bigner SH, Bigner DD. J Neuropath Exp Neurol 48: 48-68,1989. 44. He X, Wikstrand CJ, Friedman HS, Bigner SH, Pleasure S, Trojanowski JQ, Bigner DD. Lab Invest 64: 833-843, 1991. 45. Hirvonnen HE, Salonen R, Sandberg MM, Vuorio E, Vaestrik I, Kotilainen E, Kalimo H. Brit J Cancer 69: 16-25,1994. 46. Iwasaki K, Kikuchi H, Miyatake S-I, Kondo S, Oda Y. Neurosurg 30: 228, 1992. 47. Jacobsen PF, Jenkyn J, Papadimitiou JM. JNeuropath Exp Neurol 44: 572-485, 1985. 48. Kees UR, Biegel JA, Ford J, Ranford PR, Peroni SE, Hallam LA, Parmiter AH, Willoughby ML, Spagnolo D. Genes Chromosomes and Cancer 9: 129-135, 1994. 49. Kees UR, Ford J, Dawson VM, Ranford PR, Armstrong JA. Cancer Genet Cytogen 59: 119-127,1998. 50. Kees UR, Spagnolo D, Hallam LA, Ford J, Ranford PR, Baker DL, Callen DF, Biegel JA. CancerGenet Cytogen 100: 159-164, 1998. 51. Kimmel DW, Shapiro JR, Shapiro WR. J Neurosurg 66: 161-171,1987. 52. Knott JCA, Edwards AJ, Gullan RW, Clarke M, Pilkington G. J Neuropath Appl Neurobiol 16: 489-500,1990. 53. Kruse CA, Mitchell DH, Kleinschmidt-de Masters BK, Franklin WA, Morse HG, Spector EB, Lillehei KO. In Vitro Cell Dev Biol 28A: 609-614, 1992. 54. Kutluk M, Ali-Osman F. In Press. 55. Leibovitz A et al. J Natl Cancer Inst 59: 221-225, 1977 56. Li J, Liaw D, Pogsypanina K, Bose S, Wang SI, Puc A, Miliaresis G, Rodgers L, McCombie R, Bigner SH, Giovanella BC, Ittman M, Tycko B, Hibshosh H, Wigler M, Parsons R. Science 275:1943-1947,1997. 57. Liss L. Prog Exp Tumor Res 17: 93-100,1972. 58. Lo H-W, Ali-Osman F. Proc Amer Assoc Cancer Res 1999. 59. Maunoury R. Acta Neuropathol 39: 33-41, 1977. 60. McAllister RM, Issacs H, Rongey R, Peer M, Au W, Sooukup SW, Gardner MB. Int J Cancer 20:206-212, 1977. 61. Miyakoshi J, Dobler KDJ, Mckean JDS, Petruk K, Allen PBR, Aronyk KN, Weir B, HuyserWierenga D, Fulton D, Urtasun RC, Day III RS. Cancer Res 50: 278-283, 1990. 62. Merzak A, McCrea S, Koochecpoor S, Pilkington GJ. Brit J Cancer 70: 199-203,1994. 63. Merzak A, Raynal S, Rogers JP, Lawrence D, Pilkington GJ. J Neurol Sci 127: 125-133,1994. 64. Mohapatra G, Kim DH, Feuerstein BG. Genes Chromosomes and Cancer 13: 86-93, 1995. 65. Moore KD, Dillon-Carter 0, Conejero C, Poltorak M, Chedid M, Tornatore C, Freed W. J Mol Chem Neuropath 29: 107-126, 1996. 66. Nister M, Liberman TA, Betscholtz C, Petterson M, Claesson-Welsh L, Heldin C-H, Schlessinger J, Westermak B. Cancer Res 48: 3910-3918, 1988. 67. Pietsch T, Scharmann T, Fonatsch C, Schmidt D, Oekler R, Freihoff D, Albreccht S, Wiestler OD, Zeltzer P, Riehm HJ. Cancer Res 54: 3278-3287, 1994. 68. Pilkington GJ, Ankiwunmi J, Ognjenovic N, Rogers JP. NeuroReports 4: 259-262, 1993. 69. Ponten J, Westermark B. Med Biol 56: 184-193, 1978. 70. Raschella G, Romeo A, Negroni A, Pucci S, Dominici C, Castello MA, Bevilacqua P, Felsani A, Calabretta B. Cancer Res 54: 2251-2255, 1994. 71. Rosenblum ML. In: Salmon SE. (editor) Cloning of human Tumor Stem Cells. pp. 259-276. Alan R. Liss, New York, 1980. 72. Rooprai H, Merzak A, Bullock P, Pilkington G. JAnticancer Res 17: 4127-4134, 1997. 73. Rutka JT, Giblin JR, Dougherty DY, Liu HC, McCulloch JR, Bell CW, Wilson CB, Rosenblum ML. Acta Neuropathol 75: 92-103, 1987. 74. Schlesinger HR, Gerson JM, Moorhead PS, Maguire H, Hummeler K. Cancer Res 36: 3094-3100, 1976.
184
Ali-Osman
75. Schlesinger HR, Rorke L, Jamieson R, Hummeler K. Cancer Res 41: 2573-2575,1981. 76. Schwab M, Alittalo K, Klempnauer K-H, Varmus H, Bishop JM, Gilbert F, Brodeur G, Goldstein M, Tyrent J. Nature 305: 245-248, 1983. 77. Seeger RC, Rayner SA, Benerjee A, Chung H, Laug WE, Neustein HB, Benedict WF. Cancer Res37: 1364-1371, 1977. 78. Srivenugopal KS, Ali-Osman F. Oncogene 12: 2029-2034,1996, 79. Steck PA, Perhouse MA, Jasser SA, Yung WKA, Lin H, Lignon AH, Langford LA, Baumgard ML, Hattier T, Davis T, Frye T, Swedlund B, Teng DHF, Tavtigian SV. Nature Genetics 15: 356-362,1997. 80. Stratton MR, Darling J, Pilkington GJ, Lantos PL, Reeves BR, Cooper CS. Carcinogenesis 10: 899-905,1989. 81. Tamura K, Shimizu K, Yamada M, Okamoto Y, Matsui Y, Park KC, Mabuchi E, Moriochi S, Mongami H. Cancer Res 49: 5380-5384,1989. 82. Trepel M, Groscurth P, Malipiero U, Gulbins E, Dichgans J, Weller MJ. Neuro-Oncol 39: 19-32,1998. 83. Trojanowski JQ, Fung K-M, Rorke LB, Tohyama T, Yachnis AT, Lee VM-Y. Mol Chem Neuropathol 21: 219-294. 84. Tsai C-H, Hung LM, Chen J-K. J Cell Physiol 164: 108-116,1995. 85. Tsuda T, Marinetti MR, Masuelli L, Cutler ML. Oncogene 11: 397-403, 1995. 86. Tumilowicz JJ, Nichols WN, Cholon JJ, Greene AE. Cancer Res 30 2110-2118, 1970. 87. Van Meir EG, Kikuchi T, Tada M, Li H, Diserens A-C, Wojcik BE, Huang H-JS, Friedman T, de Tribolet N, Cavenee WK. Cancer Res 54: 649-652, 1994. 88. Wasson JC, Saylors III RL, Zeltzer P, Friedman HS, Bigner SH, Burger PC, Trojanowski JQ, Xuanmin H, Kurtzberg J, Berens M, Halperin EC, Bigner DD, Look T, Douglass EC, Brodeur GM. Cancer Res 50: 2987-2990,1990. 89. Welter C, Henn W, Theisinger B, Fischer H, Zang KD, Blin N. Cancer Letters 52: 57-62,1990. 90. Westermark B, Ponten J, Hugosson R. Acta Pathol Microbiol Scand Section A 81: 791-805, 1973. 91. Westermar B, Larsson E, Brunk U, Lubitz W, Mark J. Int J Cancer 28: 341-351, 1981. 92. Westphal M, Haensel M, Mueller D, Laas R, Kunzmann R, Rohde E, Koenig A, Hoelzel F. Hermann HD. Cancer Res 48: 731-740,1988. 93. Yamazaki H, Fukui Y, Ueyama Y, Tamaoki M, Kawamoto T, Taniguchi S, Shibuya M. Mol CellBiol 8: 1816-1820, 1988. 94. Zeltzer PM, Schneider SL, von Hoff DD. J Neuro-Oncology 2: 35-45, 1984. 95. Zeltzer PM, Schneider SL, Marangos PJ, Zweig MH. J Natl Cancer Inst 77: 625-631,1986. 96. Zumu I, Mineura K, Watanabe K, Kowada M. J Neuro-Oncol 17: 111-121,1993.
Chapter 28 Head and Neck Cancers
Christopher D. Lansford1, Reidar Grenman2, Henning Bier3, Kenneth D. Somers4, Sang Yoon Kim5, Theresa L. Whiteside6, Gary L. Clayman7, Hans-J. Welkoborsky8 and Thomas E. Carey1 Department of Otolaryngology and Head and Neck Surgery, University of Michigan, 1301 East Ann Street, Ann Arbor; MI 48109-0506 1; University of Turku, Finland2; Heinrich Heine University, Düsseldorf Germany3; Eastern Virginia Medical School, Norfolk, Virginia 4; University ofUlsan, Seoul, Korea5; University of Pittsburgh, Pittsburgh, Pennsylvania6; University of Texas, MD Anderson Cancer Center; Houston, Texas’; University of Mainz, Mainz, Germany 8. Tel: 001-734-764-4371; Fax: 001-734-764-0014; E-mail:
[email protected]
1.
INTRODUCTION
1.1
Incidence and etiology
Squamous cell carcinoma of the head and neck (SCCHN) is the 6th most common malignant disease worldwide. In the United States, upper aerodigestive tract (UADT) cancers (oral cavity, pharynx, larynx, esophagus) accounted for 5.6% of all cancer deaths among men and 2.5% of cancer deaths in women in the period from 1973-1990 (Miller et al. 1993). Oral cancer is most prevalent in parts of India and Southeast Asia where betel nut use and various forms of oral tobacco use are widespread (Blot et al. 1994). In western countries the overall incidence is lower but there are surprising differences among populations. Oral cancer is the seventh most common cancer among blacks and the 12th most common among whites in the United States. In Michigan the age adjusted incidence rate of UADT is 77 percent higher in blacks (32.3/100,000) than in whites (18.2/100,000) (Schottenfeld et al. 1992). However, Day et al. (1993) found that the higher incidence of oral cancer among blacks when compared to whites could be accounted for by differences in alcohol consumption among smokers. After adjusting for smoking, heavy alcohol consumption (≥30 drinks/week) resulted in a 17-fold increased risk in blacks and a 9-fold J.R. W Masters and B. Palsson (eds.), Human Cell Culture Vol. II, 185–255. © 1999 KluwerAcademic Publishers. Printed in Great Britain.
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higher risk in whites. Among drinkers, blacks tended to consume more than whites and a significantly higher percentage of blacks (37%) than whites (28%) (p = 0.01) were current smokers. The authors concluded that the higher incidence of oral cancer among blacks could be accounted for by these differences and that in the absence of smoking and heavy alcohol consumption the risks would be nearly equal. The total rate of UADT cancer in Michigan in males is 3.3 times higher than in females (Schottenfeld et al. 1992). The highest incidence of oral and pharyngeal cancer is found in males in France (40/100,000) where heavy smoking is common and wine consumption is high. The highest rates for UADT in females is found in India and other parts of Asia where betel nut and quid chewing are prevalent and oral cancers predominate. One study of Indians living in South Africa found a 1:1.6 male to female ratio among oral cancer patients. The odds ratio for oral cancer in women who only chewed areca nut was 43.9 (van Wyk et al. 1993). Similarly, in the United States there is a 50-fold increased risk of cancers of the gum and buccal mucosa in women who are chronic snuff users (Winn et al. 1981). Generally, the etiology of SCCHN involves heavy tobacco (smoking and oral tobacco) use and heavy alcohol intake. Diets low in fresh fruits and vegetables are also commonly linked to SCCHN. A diet deficient in fruits and vegetables may result in persons with high alcohol consumption because they have a tendency to reduce other caloric intake thereby lowering the proportion of fruits and vegetables consumed.
2.
HEAD AND NECK SQUAMOUS CANCER CELL LINES (HNSCC)
2.1
Early lines and the problem of cross-contamination
In spite of the prevalence of head and neck cancer there were few HNSCC cell lines available prior to the 1980s. This deficit was due to the relative lack of interest in this cancer type and the patience required to develop HNSCC lines, which tend to be slow in becoming established. Paradoxically, head and neck cell lines were among the earliest human tumor cell lines developed. The first putative HNSCC lines, Hep 2 and Hep 3, were established by implanting tumor biopsy specimens in immunosuppressed rats and then later transferring the xenografted tumors to cell culture (Moore et al. 1955). The KB cell line, derived from a 54 year old white male with buccal cancer, was the first oral cavity tumor grown directly in cell culture using the tissue explant method (Eagle, 1955). Unfortunately, there are questions about whether these lines are distinct from HeLa, a cell line established from an adenocarcinoma of the cervix (Gey et al. 1952). The HeLa cell line remains in wide use today and was shared by many of the early cell culture laboratories. Harry Eagle had used the HeLa line to optimize his growth medium prior to the time of the original KB
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culture. Possible concerns of cross contamination are first raised from Eagle’s original description of the establishment of the KB cell line. In it, he reports that the KB culture grew slowly at first, but after trypsinization, 2 weeks after being put in culture, it began to grow rapidly. This is a somewhat suspicious behavior for a newly cultured tumor. More than 10 years later Gartler (1967) examined 18 different human cell lines and found that all expressed a rare glucose-6-phosphate dehydrogenase (G6PD) isozyme. HeLa, KB, and Hep 2 were among the lines he tested. None of the lines he tested expressed the common slowly migrating type B isozyme found in Caucasians. Instead, all had the more rare type A form that is expressed in about 30% of Blacks and was not known to be expressed in Caucasians. Furthermore, all of the cell lines Gartler examined also had the same phosphoglucomutase isozyme PGM1, that is found in 60% of Blacks and Caucasians alike. Based on these findings, he concluded that a cell line that expressed the Type A G6PD contaminated all of the lines. Furthermore, he implicated HeLa as the contaminant since its donor was known to be a black woman, and thus was more likely to have the type A form of G6PD. Nelson-Rees extended these observations by demonstrating that HeLa and multiple other established lines including KB and Hep 2, as well as lines from kidney and breast, have identical chromosome markers (Nelson-Rees et al. 1977). A more complete discussion of the problem of cross-contamination was presented by Carey (1985). The important point to keep in mind is that cross-contamination can occur and all persons using cell cultures should use proper techniques and should select the lines they wish to study with care.
2.2
HNSCC lines in the recent era
Starting in the early 1980s, reports of new well-characterized cell lines derived from head and neck tumors began to appear. There are now many HNSCC cell lines available for study. The extensive studies that have been carried out using these panels of cell lines have greatly extended our knowledge of the biology and genetics of head and neck cancer. Table 1 is a fairly comprehensive list of the head and neck cell lines including some new unpublished contributions from as many investigators as we could identify. Three methods of cell line development have been employed. These are culture from dissociated tumor cells obtained by enzyme digestion of tumor tissue, culture from tumor tissue that has been selected and expanded by first growing the tumor as a xenograft, and by far the most common approach, culture from tumor fragments or explants. Several principles of successful culture have emerged. One is that squamous carcinomas are mostly anchorage dependent, most are dependent on a relatively high cell density for proliferation, and that patience is important in establishing new cell lines. These observations are consistent with the high frequency of reports that use the explant culture method (for additional discussion see Carey, 1994).
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Culture methodology
All culture methods are dependent on obtaining fresh tumor tissue. Our best success has come when the tissue specimen is selected by the surgeon from a non-necrotic and uninfected area of the neoplasm and is taken as early as possible during the surgical procedure. Thus, biopsies that are taken specifically for laboratory use have a fairly high successful culture rate. If tissue is from a resection specimen taken late in the procedure it may have been deprived of a blood supply for protracted periods or allowed to desiccate from exposure to the air during the operative procedure. In such cases, successful culture is less likely. At the University of Michigan the fresh specimen is immediately placed into a sterile specimen container and covered with cold transport medium. The transport medium consists of Puck’s saline A containing penicillin (100units/ml), streptomycin (100 µg/ml), and amphotericin B (0.5 µg/ml). The tissue is then transported to the laboratory, washed three times in fresh transport medium, and minced into tiny cubes 1-2mm in size using two scalpel blades. The explants are then placed in 25 cm2 culture flasks, briefly allowed to stick to the surface, and covered with 2-3 ml of culture medium, such as Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 1% non-essential amino acids, 2mM L-glutamine, penicillin and streptomycin. Epithelial cells usually begin to grow out of the fragments within 2–5 days. We typically change the medium for the first time after 3–5 days and then at weekly or biweekly intervals thereafter. The cultures will usually have a burst of mitotic cells on the first or second day after each feeding and sometimes more frequent feeding will stimulate cell proliferation. Occasionally however, cultures will respond poorly to too frequent feedings and will actually slow their growth when fed too often. Thus it is necessary to observe the cultures frequently and assess the effects of the feeding schedule on cell proliferation. It is important to allow the epithelial cell cultures to grow and expand until the colonies reach a size of 1.5–2 cm in diameter before attempting trypsinization and passage. If the cells are growing slowly or very few mitotic cells are present at the time of an attempted harvest, it is unlikely that the passage to a new flask will be successful. To increase the probability of passage success, it helps to feed the culture one or two days before subculture. Some tumors have a tendency to stratify in culture (Carey, 1994). Such cultures are less likely to be successfully passed on the first attempt because the majority of cells that are released by trypsinization are from the differentiated cells on the top of the stratifying colony. In most cases, these cells are incapable of further growth or reattachment to a new culture surface. Therefore, it is often worthwhile to remove the stratifying layers in stepwise trypsin exposures on subsequent days until the basal layer can be seen. Then it should be verified
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that there are mitotic cells present before attempting passage. As mentioned before, feeding can usually induce mitotic activity. Table 1 lists information about each cell line, including cell line designation, patient age and sex, staging information, specimen site, type of lesion, primary site, prior treatment if any, culture method, authentication, availability and a citation to publications. The lines are given in the table by author or group and are organized from time of first publication from the group. This way all lines from a single group are listed together. Cell line nomenclature is an important consideration. We recommend names that include the initials of the institution, eg UM-SCC-number or UTSCC-number. Here the UM stands for University of Michigan and the UT for University of Turku. It is also important that all users employ the full nomenclature and not an abbreviation. For example if someone uses a line from Turku or Michigan and refers to it only as SCC-25, then it would not be possible to determine if this were the line established by James Rheinwald (Rheinwald and Beckett, 1981) by that name or the UM-SCC-25 or the UTSCC-25lines. Not all information is available for every cell line. Authors for this chapter provided specific information that is not available from the literature. Those who originated new lines as determined from a literature search, but did not provide additional information, are cited in the last column of Table 1. Authentication is an important category for verifying the origin of the tumor cell line as being from the expected donor, but is not always available. However, it should be noted that for every comparison that has been made on the University of Michigan cell lines there is concordance with the normal tissue or cultured normal cells from the same donor. Examples of concordance of polymorphic markers in tumor DNA, normal DNA and tumor cell line DNA as well as concordant loss of heterozygosity in tumor and cell line DNA are illustrated in Frank et al. (1997).
3.
IN VITRO STABILITY AND THE VALUE OF CELL LINES AS REPRESENTATIVES OF THE TUMOR OF ORIGIN
It is important to point out that contrary to oft repeated but unsubstantiated comments in the literature, human tumor cell lines are remarkably stable in culture. In this regard, it is worthwhile to recall that characteristic genetic changes such as the Philadelphia chromosome (t9;22) and the t(8;14) translocation in Burkitt’s lymphoma were consistently observed in cultured cell lines. Similarly, the HeLa and CaSki cell lines over many years have retained the HPV type 18 and HPV16 viruses of the host tumor. There are many other examples, such as the MCF7 breast carcinoma that has retained estrogen
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responsiveness for more than 20 years in vitro and the T24 bladder carcinoma that provided the clues to Ras activation. It may be appropriate to think of cell lines as snapshots of what is going on in the tumor in the patient at the time the biopsy or resection was taken for culture. Our basis for this conclusion is drawn in part from analyses of cytogenetic characteristics and in part from analyses of tumor lines from primary and secondary (either recurrences or metastases) in the same individual. We found identity in karyotypes of primary tumor tissue immediately following excision (direct karyotype preparations), early cell line karyotypes, and karyotypes of long established tumor cells all derived from the same tumor (Carey et al. 1989, Grenman et al. 1990, Worsham et al. 1993). In addition, analysis of primary and metastatic tumor cell lines from the same individual share common cytogenetic markers and are distinct by other markers, but these do not change with long term culture in vitro (Carey et al 1993a, 1993b). Similarly, analysis of tumor tissue and cell lines by either RFLP analysis (Kelker et al. 1996), microsatellite repeat polymorphisms (Jones et al. 1997, Frank et al. 1997), or by fluorescence in situ hybridization (Worsham et al. 1995, 1999) shows identity of the tumor specimen used for culture and the cell line. These observations all demonstrate the genetic stability of tumor cells in vitro. However, it should be noted that in vivo tumors can evolve rapidly and extensively. In the cases we have studied, there is a progression of genetic changes in both the primary tumor and the metastatic or recurrent tumors that develop soon after the populations diverge. Both populations share the common events of the original clone, but soon accumulate changes not present in the original primary tumor clone (Carey et al. 1993a,b). Nevertheless, in these cases the cell lines correspond exactly with the tumor tissue from which they were established (Jones et al. 1997, Frank et al. 1997). In fact, the original clonal markers can be used to trace clonality and establish the genetic origin of so-called second primary tumors as metastatic foci (Worsham et al. 1995).
4.
NASOPHARYNGEAL CARCINOMA CELL LINES
Nasopharyngeal carcinoma (NPC) is a common head and neck tumor in certain parts of Southeast Asia. Typically these tumors arise in the lymphoid rich area of the nasopharynx, known as Waldeyer’s ring, consisting of the lingual, pharyngeal and faucial tonsils. Histologically the tumors range from well differentiated squamous cell carcinomas to non-keratinizing and poorly differentiated epithelial tumors. The majority (60– > 80%, depending on the geographic location) fall into the latter category. The Epstein-Barr virus (EBV) appears to be involved in the development and etiology of NPC, tumors often contain the EBV genome, and patients have high titers of antibody to EBV antigens. A number of NPC cell lines have been established. These are summarized in Part 2 of Table 1.
Head and Neck Cancers
5.
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DO HNSCC CELL LINES RETAIN THE PATHOLOGICAL CHARACTERISTICS OF THE TUMORS OF ORIGIN?
In Table 2, details are given about the tumor from which cell lines were derived and information about the cell line. Comparisons of the histopathology of the original specimen and the ability of the cell line to replicate the histologic features in vitro or in xenograft are given whenever available. Remarkably, those tumors that have been studied in xenografts replicate the original histology so well that the mouse tumor and human tumor are often indistinguishable (Carey, 1985).
6.
DO THE CELL LINES REFLECT THE MOLECULAR GENETICS OF HNSCC?
Table 3 contains partial information about the genetic characteristics of the tumor lines. This is by no means a comprehensive listing and the reader is encouraged to consult the original publications of each cell line developer for more complete data. Information is also given regarding the HPV status of some lines. The presence of human papilloma virus in head and neck tumors is still controversial. In several cases, HPV signals have been detected in the original tumor or in the early passages of the cell lines, but then in later stages HPV cannot be detected. The precise explanation for this is not known, but current wisdom suggests that episomal HPV may not be stable in vitro or may be present in only a minority of cells. That the HPV is lost with time also indicates that it is not essential for the transformed state. There are now a few HNSCC lines that have stable content of HPV DNA. It is emerging that these lines also have wild type p53, but at the time of this writing, this data has not yet been published.
ACKNOWLEDGMENTS Work on this chapter was supported in part by grant 1R01 DE12477 from the National Institute of Dental and Craniofacial Research by Dr. Carey and by a research fellowship from the Deafness Research Foundation to Dr. Christopher Lansford.
Cell line name
Age Gender TNM
Grade Stage Specimen site Type of lesion
Primary location
Prev. tx
larynx
none
NS
larynx
recurrence
Hep 3 (HeLa?) 62 male
NS
lymph node
KB(HeLa?)
64 male
NS
lymph node buccal cavity none metastasis primary buccal cavity none
RPMI2650
?
?
NS
Detroit 562
?
female
FaDu SW579 A-253 T3M-1
56 59 54 33
male male male male
HLaC78
?
male? NS
HSmC78
?
male? NS
HLaC79
?
male?
MC
66 female NS
HN-1 HN-2 HN-3
51 male 49 male 63 male
T2N1M0 T3N0M0 T3N0M0
HN-4 HN-5
57 male 73 male
T2N0M0 T2N0M0
pleural fluid
Authenti- Availability Primary reference cation
Rat xenograft Rat xenograft Explant
Toolan 1953 Moore et al. 1955 Eagle 1955
nasal septum
RT,S
pharynx
none
hypopharynx thyroid salivary gland lower gingiva
none none none none
Cell pellet from fluid Cell pellet from fluid E on RC Explant Explant Nude mouse
larynx
none
Explant
Zenner et al. 1979
submandibular none gland larynx
Explant
Zenner et al. 1979
Explant
Zenner et al.1983
DSMZ
Moore and Sandberg 1964 Peterson et al. 1971 Rangan 1972 Leibovitz 1973 Giard et al. 1973 Okabe et al. 1978
maxillary sinus none
Trypsin digest
III II III
distant metastasis hypopharynx primary thyroid primary salivary gland primary pleural distant effusion metastasis neck metastasis neck metastasis neck metastasis maxillary primary sinus tongue primary larynx recurrence tongue recurrence
tongue larynx tongue
Explant Explant Explant
Nakashima et al. 1980 Easty et al. 1981 Easty et al. 1981 Eastyet al. 1981
II II
larynx tongue
recurrence recurrence
larynx tongue
Explant Explant
Easty et al. 1981 Easty et al. 1981
NS NS NS NS
NS
CX,RT,S CX,S,ND CX,RT, S,ND CX,RT,S CX,RT,S
Continued on next page
Lansford et al.
Hep 2 (HeLa?) 57 male
Previous tx Culture method details
192
Table 1. Head and neck SCC cell lines: Donor information, specimen site, culture details and availability
Cellline name HN-6 HN-7 HN-8 HN-9 HN-10 UM-SCC-1 UM-SCC-2
Age Gender TNM 54 56 56 67 57 73 63
male T2N0M0 male T2N0M0 male T2N0M0 female T2N0M0 male T2N0M0 male T2N0M0 female T2N0M0
Grade Stage Specimen site Type of lesion II tongue recurrence II tongue recurrence II tongue recurrence II tongue recurrence II larynx recurrence MD II floorofmouth recurrence WD II alveolarridge recurrence lymphnode
Primary Prev. Previous tx Culture Authenti- Availability Primary location reference tx details method cation tongue Eastyetal. 1981 CX,RT,S Explant tongue CX,RT,S Explant Eastyetal. 1981 Eastyetal. 1981 Explant RT,S tongue Eastyetal. 1981 Explant RT,S tongue larynx Eastyetal. 1981 RT,S Explant Explant floorofmouth S,RT UM-SCC-AR Krauseet al. 1981 See foot- Nudemouse UM-SCC-AR Krauseetal.1981 alveolarridge S note UM-02 nasal S partial Explant UM-SCC-AR Krauseetal.1981 columella rhinectomy CX See foot- Explant tonsillar UM-SCC-AR Krauseetal.1981 pillar noteUM-04 Explant D S supraglottis UM-SCC-AR Krauseetal.1981 BOT Explant none UM-SCC-AR Krauseetal.1981 alveolus none Explant UM-SCC-AR Krauseetal.1981 alveolus Explant RT UM-SCC-AR Krauseetal.1981 ant.tongue Explant RT UM-SCC-AR Krauseetal.1981
73 female TlNOMO MWD I
UM-SCC-AR Krause et al. 1981 UM-SCC-AR Careyetal. 1983 UM-SCC-AR Careyetal. 1983 UM-SCC-AR Careyetal.1983 UM-SCC-AR Careyetal. 1983 UM-SCC-AR Careyetal.1983 UM-SCC-AR Careyetal.1983 UM-SCC-AR Carey et al. 1983
193
lymph node metastasis UM-SCC-4 47 female T3N2aM0 PD IV BOT persistent primary UM-SCC-5 59 male T2NlM0 PD III supraglottis primary UM-SCC-6 37 male T2N0M0 M-PD II BOT primary UM-SCC-7 64 male T2N1M0 MD III alveolus primary UM-SCC-8 76 female T2N1M0 M-WD III alveolus recurrence UM-SCC-9 71 female T2N0M0 M-WD II persistent ant.tongue primary UM-SCC-10A 57 male T3N0M0 M-WD III truevocalcord primary truevocal cord none Explant D UM-SCC-10B 58 male T3N1M0 M-WD III lymphnode lymph node larynx S total Explant D metastasis laryngectomy epiglottis Explant none IV epiglottis primary UM-SCC-11A 65 male T2N2aM0 UM-SCC-11B 65 male T2N2aM0 Explant IV supraglottic persistant supraglottic CX larynx tumor larynx UM-SCC-12 71 male T2N1M0 MWD III larynx Explant S recurrence larynx UM-SCC-13 60 male T3N0M0 WD III esophagus recurrence larynx RT,S Seefoot- Explant note UM-13 floor of mouth recurrence floor of mouth S UM-SCC-14A 58 female TlNOMO P-MWDI Explant D UM-SCC-14B 59 female TlNOMO PD I floor of mouth recurrence floor ofmouth S,S,RT, Explant D UM-SCC-3
Head and Neck Cancer
Table 1. (continued)
Continued on next page
(continued)
Cell line name
Age Gender TNM
UM-SCC-14C 58 female TlNOMO
Grade Stage Specimen site Type of lesion PD
I
UM-SCC-15 70 male T4N1M0 WD IV UM-SCC-16 61 female T2N0M0 MD II UM-SCC-17A 47 female TlNOMO MWD I UM-SCC-17as 47 female TlNOMO
MWD
UM-SCC-17B 47 female TlNOMO
MWD I
UM-SCC-18 UM-SCC-19 UM-SCC-20
68 male 67 male 67 male
UM-SCC-21A 65 UM-SCC-21B 65
DN1M0 T2N1M0 T2N1M0
PD III M-PD III PD III
male male
T2N1M0 T2N1M0
PD
III III
female female female male male
T2N1M0 T2N1M0 T2N0M0 TlNOMO T3N0M0
MD MD MWD WD PD
III III II I III
MWD III
58 58 36 57 50
UM-SCC-26
50 male
T3N1M0
UM-SCC-27
62 male
TlNOMO
UM-SCC-28
61 female TlNOMO
I WD
I
Prev. tx
Previous tx Culture Authenti- Availability method cation details
floor of mouth S,S,RT, See foot- Explant CX note UM-14C hypopharynx none Explant hypopharynx primary larynx Explant none larynx primary supraglottis RT See foot- Explant supraglottis primary note UM-17A supraglottis RT See foot- Explant supraglottis primary note UM-17A RT See foot- Explant local cartilage supraglottis soft tissuenote UM-17B neck invasion S,RT Explant BOT recurrence BOT Explant BOT primary BOT none RT,S See foot- Explant necknode metastasis larynx note UM-20 Explant S,RT ethmoid sinus local invasion skin of nose Explant S,RT lymph node lymph node skin metastasis Explant hypopharynx none hypopharynx primary Explant neck metastasis metastasis hypopharynx none Explant larynx none larynx primary larynx Explant recurrence true vocal cord RT,S,S RT,S,S Explant neck metastasis larynx floor of mouth recurrence
none lymph node BOT metastasis S,RT neck lymph node ant. tongue metastasis true vocal cord none true vocal cord primary
neck
Explant See foot- Explant note UM-27 Explant
Primary reference
D
UM-SCC-AR Carey et al. 1983
D
not available Carey et al. 1983 UM-SCC-AR Carey et al. 1983 UM-SCC-AR Carey et al. 1983
D
UM-SCC-AR Carey et al. 1983
D
UM-SCC-AR Carey et al. 1983 UM-SCC-AR Carey et al. 1983 UM-SCC-AR Carey et al. 1983 UM-SCC-AR Carey et al. 1983 UM-SCC-AR Carey et al. 1983 UM-SCC-AR Kelker et al. 1996 UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR
Carey et al. 1983 Carey et al. 1983 Carey et al. 1983
Buchhagen et al. 1996 UM-SCC-AR Buchhagen et al. 1996 UM-SCC-AR
UM-SCC-AR Buchhagen et al. 1996 Continued on next page
Lansford et al.
UM-SCC-22A UM-SCC-22B UM-SCC-23 UM-SCC-24 UM-SCC-25
Primary location
194
Table 1.
(continued)
Cell line name
Age Gender TNM
UM-SCC-29
66 male
UM-SCC-30
53
UM-SCC-31
56 male
T3N0M0
UM-SCC-32
58
male
T3N1M0
UM-SCC-33
47
female T4N3aM0 WD
Grade Stage Specimen site Type of lesion
T3N2aM0 WD
female T3N1M0
MD PD WD
IV
alveolus
persistent primary III pyriform sinus persistent primary III tonsil persistent primary III retromolar primary trigone IV neck metastasis
Primary location
Prev. tx
Previous tx Culture Authenti- Availability Primary reference method cation details
alveolus
CX
See foot- Explant note UM-29 See foot- Explant note UM-30 Explant
pyriform sinus CX tonsil
RT
retromolar none trigone maxillary sinus CX
UM-SCC-AR Buchhagen et al. 1996 UM-SCC-AR Buchhagen et al. 1996 UM-SCC-AR
Explant
UM-SCC-AR
See foot- Explant note UM-33 Explant Explant Explant
UM-SCC-AR
UM-SCC-34 49 male T3N1M0 MD tonsillar pillar none III tonsillar pillar primary UM-SCC-35 51 male T4N1M0 MWD IV tonsillar fossa primary tonsillar fossa none false vocal cord none false vocal cord primary UM-SCC-36 46 male T2N0M0 MWD II UM-SCC-37 48 male TZNOMO MWD vallecula persistent vallecula CX,RT See foot- Explant UM-SCC-AR primarynote UM-37 tonsillar pillar none Explant UM-SCC-38 60 male T2N2aM0 MD IV tonsillar pillar primary UM-SCC-39 42 male T3N3aM0 MD IV pyriform sinus primary pyriform sinus none Explant esophagus RT,CX See foot- Explant III esophagus persistent UM-SCC-40 64 male T3N0M0 PD note UM-40 arytenoid Explant none III arytenoid UM-SCC-41 78 male T2N1M0 ? primary UM-SCC-42 56 male T4N3bM0 MD IV neck pyriform sinus S Explant persistent UM-SCC-43 ? male palate Explant primary palate retromolar Explant IV neck UM-SCC-44 50 male T4N2bM0 trigone UM-SCC-45 46 female T3N2bM0 Explant IV neck metastasis floor of mouth ? RT,S larynx See foot- Explant recurrence larynx UM-SCC-46 58 female NS note UM-46 UM-SCC-47 53 male T3N1M0 MWD III lateral tongue lateral tongue Explant
Head and Neck Cancer
Table 1.
UM-SCC-AR UM-SCC-AR Grenman et al. 1991 UM-SCC-AR II UM-SCC-AR Grenman et al. 1991 UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR
195
Continued on next page
Grade Stage Specimen site Typeof lesion
UM-SCC-48
51 male
T4N0M0 WD
UM-SCC-49
63 male
T2N1M0 MWD III
lateraltongue primary
59 female T4N3bM0 WD IV 49 male T3N2bM0 WD IV ketatining UM-SCC-52 48 female T3N3cM0 IV UM-SCC-53 49 male T3N1M0 MWD III III UM-SCC-54 83 male T3N0M0 II UM-SCC-55 65 male
BOT primary floorofmouth
IV neck
UM-SCC-50 UM-SCC-51
UM-SCC-57 ? male UM-SCC-58 ? female UM-SCC-59 71 female T3N2bM0 UM-SCC-60 ? ? UM-SCC-62 47 male T3N1M0 ? UM-SCC-63 82 male NS UM-SCC-65 ? male
male male male male
retromolar trigone lateraltongue BOT
none
supraglottis pyriformsinus truevocalcord recurrence retromolar RT trigone
primary
IV lateraltongue primary hypopharynx III tonsil larynx larynx larynx neck
T4N0M0 MWD IV
lateraltongue hypopharynx tonsillarfossa none Skin
hardpalate
primary lymphnode metastasis primary
larynx larynx larynx hardpalate
larynx
Previous tx Culture Authenti- Availability Primary method cation details reference Explant
UM-SCC-AR
Explant
UM-SCC-ARBuchhagenetal. 1996 UM-SCC-AR UM-SCC-ARBuchhagenetal. 1996
Explant Explant Explant Explant Explant Explant
UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR
Explant Explant Explant Explant Explant Explant Explant
UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR Somers et al. 1992 UM-SCC-AR Buchhagen et al. 1996 UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR
Explant Explant Explant Explant CX
Explant Explant Explant Explant
UM-SCC-AR Grenman et al. 1991 UM-SCC-AR UM-SCC-AR UM-SCC-AR Continued on next page
Lansford et al.
35 ? ? ?
tonsil
Prev. tx
larynx
UM-SCC-66 ? male UM-SCC-67 ? male UM-SCC-68A ? male UM-SCC-68B male UM-SCC-69 UM-SCC-70 UM-SCC-71 UM-SCC-72
supraglottis tonsil
metastasis
Primary location
196
Table 1. (continued) Cell line Age Gender TNM name
UM-SCC-74B 50 UM-SCC-75 UM-SCC-76 UM-SCC-77 UM-SCC-78A
male
Grade Stage Specimen site Type of lesion tongue primary recurrence neck recurrence PD III BOT
T3N0M0
III
? female ? male 58 female ? male
Primary location tongue BOT
intraoral
recurrence
larynx
neck
metastasis
larynx
medial pyriform sinus tonsil
Prev. tx none
Previous tx Culture Authenti- Availability Primary reference details method cation UM-SCC-AR Buchhagen et al. 1996 Explant UM-SCC-AR Explant CX,RT See foot- Explant UM-SCC-AR S note 74A UM-SCC-AR CX,RT See foot- Explant S,S,CX note 74B Explant UM-SCC-AR Explant UM-SCC-AR Explant UM-SCC-AR Explant UM-SCC-AR
UM-SCC-78B ? UM-SCC-79 ?
male male
UM-SCC-80 67 UM-SCC-81A 53
male male
T4N1M0 T2N0M0
WD IV P-MWD II
hypopharynx primary Lfalsecord primary
base of tongue & floor of mouth hypopharynx larynx none
UM-SCC-81B 53 male
T2N0M0
MWD II
tonsillar pillar primary
tonsil
MD
II
lateral tongue primary
lateral tongue none
?
II
BOT
lateral tongue RT
UM-SCC-82A 53
female T2N0M0
UM-SCC-82B 53 female T2N0M0
recurrence
S,S
Explant Explant Explant immuno- Explant D suppression x 20 mo for heart transplant See foot- Explant D note 81B Explant
UM-SCC-AR UM-SCC-AR UM-SCC-AR Kelker et al. 1996 UM-SCC-AR
UM-SCC-AR Frank et al. 1997 UM-SCC-AR Buchhagen et al. 1996 UM-SCC-AR Buchhagen et al. 1996
197
External Explant beam radiation and radiation implants 7 months earlier
Head and Neck Cancer
Table 1. (continued) Cell line Age Gender TNM name UM-SCC-73A ? male UM-SCC-73B UM-SCC-74A 50 male T3N0M0
Continued on next page
Cell line name
Age Gender TNM
UM-SCC-83A UM-SCC-83B UM-SCC-84 UM-SCC-85 UM-SCC-86
79 male 79 male 35 male 58 male -45female
T2N0M0
UM-SCC-87 UM-SCC-88 UM-SCC-89 UM-SCC-90 UM-SCC-91 UM-SCC-92 UM-SCC-93
69 76 53 48 65 ? 64
male female male male male female female
T3N0M0 T3N1M1 T3N0M0 T4N3M0 T4N0M0 T2N0M0 T4N0M0
buccal mucosa buccal mucosa III tongue cribiform plate lateral mid tongue III oropharynx IV hypopharynx III R BOT IV epiglottis IV LBOT II lateral tongue IV larynx or neck
UM-SCC-94 UM-SCC-95 UM-SCC-96 UM-SCC-97 UM-SCC-98 UM-SCC-99 UM-SCC-100
73 62 75 38 75 57 35
male male female female male male female
T4N2aM0 T4N1M0 T3N3M0 TlN0M0 T4N0M0 T3N0M0 T4N3M0
IV IV IV I IV III IV
UM-SCC-101A 65 female T2N3M0 UM-SCC-101B 65 female T2N3M0 55 male T3N0M0 SCC-4
MWD IV MWD IV III
Grade Stage Specimen site Type of lesion
Prev. tx
Previous details
lateral tongue None primary residualtumor nasal vestibule S,S,S
primary primary primary primary primary primary recurrence or met larynx primary pyriform sinus primary epiglottis primary lateral tongue primary larynx primary tonsil primary tonsil persistant primary tonsil neck tongue
Primary location
primary metastasis primary
oropharynx hypopharynx RBOT epiglottis LBOT lateral tongue larynx
None None None None None None S,S,S,Cx
larynx None pyriform sinus None epiglottis None anterior tongue None larynx None tonsil None uvula and Clinical tonsil CR at primary PR in neck tonsil None tonsil None tongue RT,Cx little response to Rx; Cx was Mtx
tx Culture Authenti- Availability Primary reference method cation Explant D Explant D Explant Explant Explant D
not available Frank et al. 1997 UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR Frank et al. 1997
Explant Explant Explant Explant Explant Explant Explant
UM-SCC-AR Frank et al. UM-SCC-AR UM-SCC-AR Frank et al. UM-SCC-AR Frank et al. UM-SCC-AR Frank et al. UM-SCC-AR UM-SCC-AR Frank et al.
D D D D D
1997 1997 1997 1997 1997
Explant D Explant Explant Explant Explant Explant Explant
UM-SCC-AR Frank et al. 1997 UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR UM-SCC-AR
Explant D Explant D T-C-D or E on FFL
UM-SCC-AR UM-SCC-AR Rheinwald and Beckett 1981
Lansford et al.
(continued)
198
Table 1.
Continued on next page
(continued)
Cell line name
Age Gender TNM
SCC-9
25 male
SCC-12B.2
Grade Stage Specimen site Type of lesion
Primary location
Prev. tx
oral tongue
primary
oral tongue
none
60 male
facial skin
primary
facial skin
none
SCC-12F.2a
60 male
facial skin
primary
facial skin
none
SCC-12V
60 male
facial skin
primary
facial skin
none
SCC-13
56 female NS
facial skin
primary
facial skin
RT
SCC-15
55 male
T4N1M0
IV
tongue
primary
tongue
none
SCC-25
70
TlN1M0
III
oral tongue
primary
oral tongue
RT
SCC-35
??
T4N0
IV
pyriform sinus recurrence
SCC-49
??
T2N0
II
recurrence
SCC-61
??
T4N2bx
male
SCC-66
III
oral tongue
floor of mouth primary
T4xN0 60 male ??
T4N0M0 T4N1
primary
WD
IV
IV
tongue soft palate
primary primary
pyriform sinus RT
tonsil
oral tongue
RT
?
T-C-D or E on FFL T-C-D or E on FFL T-C-D or E on FFL T-C-D or E on FFL RT gave a T-C-D or CR with E on FFL recurrence at 5 mo T-C-D or E on FFL rapid tumor T-C-D or growth E on FFL during RT RT gave a E on irr CR with 3T3 FFL recurrence at 24 mo RTgave a E on irr PR with 3T3 FFL recurrence at 14 mo. E on irr 3T3 FFL
floor of mouth tongue soft palate
none none
Explant E on irr 3T3 FFL
Primary reference Rheinwald and Beckett 1981 Rheinwald and Beckett 1981 Rheinwald and Beckett 1981 Rheinwald and Beckett 1981 Rheinwald and Beckett 1981 Rheinwald and Beckett 1981 Rheinwald and Beckett 1981 Weichselbaum et al. 1986 Weichselbaum et al. 1986 Weichselbaum et al. 1986 Weichselbaum et al. 1990 Weichselbaum et al. 1986
Continued on next page
199
SCC-68 SCC-71
T2N1
Previous tx Culture Authenti- Availability method cation details
Head and Neck Cancers
Table 1.
SCC-73
??
200
Table1. (continued) Cell line Age Gender TNM name
Grade Stage Specimen site Type of lesion IV
T4N0
SCC-74 SCC-76
51 female T4N0M0 ?? T4N0
PD
SCC-182
71 male
T3N0M0
MD
SCC-200 SCC-203 SCC-210 SCC-213
74 43 60 71
T2N2M0 WD IV T2N2M0 MWD IV IV T3N0M0 PD T4N0M0 PD IV
SCC-220 JSQ-3
66 male
JSQ-3
male male male male
T4N1M0 T3N0
IV IV III
MWD IV III III
T3N0
RMT
primary
mandible maxillary antrum retromolar trigone BOT alvolar ridge tongue retromolar trigone tongue
primary persistent
T3N1
III
SQ-20B
??
T2N0
II
larynx
SQ-29
??
T3N1
III
RMT
Previous tx Culture Authenti- Availability Primary reference details method cation
RMT
?
E on irr 3T3 FFL Explant tumor grew E on irr duringRT 3T3FFL Explant
pyriform sinus recurrence
recurrence
persistent
none CX,S, RT none none S,RT,CX S,RT S,RT
Explant Explant Explant Explant
none
Explant
buccal cavity pyriform sinus RT
larynx
RMT
RT
CX,S
RT gave a E on irr CRwith 3T3 FFL recurrence at 7 mo. RTgavea Eonirr CRwith 3T3 FFL recurrence at 4 mo. Cx gave a E on irr PR 3T3 FFL
Weichselbaum et al. 1986 Weichselbaum et al. 1986
Weichselbaum et al. 1990 Weichselbaum et al. 1990 Dunphy et al. 1992 Weichselbaum et al. 1986 Weichselbaum et al. 1986 Weichselbaum et al. 1986
Lans ford et al.
??
Prev. tx
mandible maxillary antrum retromolar primary trigone primary BOT recurrence alveolarridge primary tongue recurrence retromolar trigone primary tongue
buccal cavity primary
JSQ-13 SQ-9G
Primary location
Continued on next page
Table 1. (continued) Age Gender TNM
SQ-31
??
T2N0
SQ-38
??
T3N0
SQ-39
??
T3N2a
SQ-43
??
T4N2
SQ-50 HN-SCC-3
??
T4N2 T3N3bM0
Grade Stage Specimensite Typeof lesion
Primary location
Prev. tx
Previoustx Culture Authenti-Availability details method cation
pyriformsinus CX,RT CXgave a E on irr CR,RT 3T3FFL given for consolidation but Ca recurred at 7 mo. ? E on irr III RMT primary RMT 3T3FFL CX,S Cxgavea Eonirr IV RMT persistent RMT PR 3T3FFL recurrence supraglottis RT RTgave a E on irr IV supraglottis CR, 3T3FFL stomalrecurrence at 7 mo. IV supraglottis recurrence supraglottis RT E on irr 3T3FFL primary softpalate IV softpalate II
pyriformsinus recurrence
HN-SCC-28
larynx
T4N1M0
IV BOT
HN-SCC-68 HN-SCC-80 HN-SCC-104
T3N3aM0
IV pyriformsinus primary
HN-SCC-109A HN-SCC-131
T2NxM0
ant.tongue
primary
primary
BOT
pyriformsinus larynx ant.tongue
Explant
Weichselbaum et al. 1986
Weichselbaum et al. 1986 Weichselbaum et al. 1986 Weichselbaum et al. 1986 Weichselbaum et al. 1986 Weichselbaumetal. 1990 Weichselbaum et al. 1989 Dunphy et al. 1992 Dunphy et al.1992 Weichselbaum et al. 1990 Dunphy et al.1992 Dunphy et al.1992 Weichselbaum et al. 1990 Dunphyet al.1992 Weichselbaum et al. 1990 Continued on next page
201
HN-SCC-29 HN-SCC-42 HN-SCC-58
Primary reference
Head and Neck Cancers
Cell line name
Grade Stage Specimensite Typeof lesion
TlN1M0
HN-SCC-135
III hardpalate
primary
Primary location
T4N3bM0
IV
oropharynx
primary
oropharynx
HN-SCC-151
T3N0M0
III
ant. tongue
primary
ant. tongue
HN-SCC-152 HN-SCC-153
T3N0M0
III
larynx
primary
larynx
HN-SCC-161
T4N1M0
IV
BOT
primary
BOT
HN-SCC-166
T4N0M0
IV
HN-SCC-167 HN-SCC-170A
T3N0M0 TZN0M0
III II
soft and hard primary palate
soft and hard palate
subglottic
subglottic
HN-SCC-170B HN-SCC-294 HN-SCC-296A
TZNOMO T2N0M0
II II
MDA-1386
58 57 38 67 81 54 64
female male male female female male male
NS NS NS NS NS T3N0M0 WD III T3N3aM0 P-MWDIV
72 male T4N3bM0 PD
tongue larynx laIynx buccal mucosa oral mucosa tonsil lymphnode
subglottic RT? oral tongue pyriform sinus
recurrence tongue S,RT recurrence larynx RT,S recurrence larynx RT,S recurrence buccal mucosa RT,S primary oral mucosa ? tonsil primary lymphnode larynx ? metastasis IV lymphnode lymphnode hypopharynx metastasis
Explant Explant Explant Explant Explant Explant Explant
Weichselbaumetal. 1990 Weichselbaum et al. 1990 Weichselbaum et al. 1990 Dunphy et al. 1992 Weichselbaum et al. 1990 Weichselbaum et al. 1990 Weichselbaum et al. 1990 Cowan et al. 1992 Weichselbaum et al. 1990 Dunphy et al. 1992 Cowan et al. 1992 Weichselbaum et al. 1992 Rupniak et al. 1985 Rupniaketal. 1985 Rupniak etal. 1985 Rupniak et al. 1985 Yanagawa et al. 1986 Sacks et al. 1988 Sacks et al. 1989 Beckhardt et al. 1995 Continued on next page
Lansford et al.
TR-126 TR-131 TR-138 TR-146 TYS MDA-183 MDA886LN
subglottic primary oral tongue primary pyriform sinus primary
Previoustx Culture Authenti- Availability Primary method cation reference details
hardpalate
HN-SCC-143
primary
Prev. tx
202
Table 1. (continued) Cellline Age Gender TNM name
Table 1.
(continued)
MDA-1483 MDA-1586 MDA-1986
Age Gender TNM 66 male
T2N1M0
Grade Stage Specimen site Type of lesion WD
III
WD
MDA-686LN HC-2,3,4,7,9 64 female T3N0M0
Primary location
Prev. tx
primary
RMT ? larynx lymph node lymph node tongue metastasis BOT maxillarysinus recurrence maxillary sinus RT,S RMT
Previous tx Culture Authenti- Availability Primary reference method cation details Explant
Sacks et al. 1988
Komiyamaet al. 1989 Heo et al. 1989
PCI-1
65 male
T2N0M0
MWD II
larynx
PCI-2
51 male
T3N0M0
MWD III
floor of mouth primary
floor of mouth none
PCI-3
50 male
T3N0M0
MWD III
51 male
T3N0M0
MWD III
retromolar trigone larynx
none
PC1-4A
retromolar trigone larynx
Nude mouse Explant DNA finger printing Explant DNA finger printing Explant
none
Explant
PCI-4B
51 male
T3N0M0
MWD III
lymphnode
larynx
none
Explant
PCI-5 PCI-6A PCI-6B
63 male 81 male 81 male
hypopharynx none tonsil none tonsil S,RT
Explant Explant Explant
Contact Heo et al. 1989 TLW Contact Heo et al. 1989 TLW Contact Heo et al. 1989 TLW Contact TLW Heo et al. 1989 Contact TLW Heo et al. 1989 Contact TLW Heo et al. 1989
PCI-7 PCI-8
45 male 54 male
BOT none pyriformsinus none
Explant Explant
Contact TLW Heo et al. 1989 Contact TLW Heo et al. 1989
PCI-9A PCI-9B
56 male 56 male
BOT BOT
Explant Explant
Contact TLW Heo et al. 1989 Contact TLW Heo et al. 1989
III
recurrence
primary primary
S
none none
Contact TLW Contact TLW
Heo et al. 1989
Continued on next page
203
lymphnode metastasis T4N1M0 MD III hypopharynx primary T3N3N0 MD II tonsil primary T3N3M0 MD IV lymphnode recurrent lymph node metastasis primary T4N2M0 MD IV BOT lymphnode T3N2M0 MWD IV lymphnode metastasis primary T4N3M0 MD IV BOT T4N3M0 MD IV lymphnode lymphnode metastasis
larynx
Head and Neck Cancers
Cell line name
(continued) Grade Stage Specimen site Type of lesion
PCI-10 PCI-11 PCI-12
61 male 83 male 65 male
T2N1M0 T4N0M0 T4N0M0
MWD III PD IV MD IV
PCI-13 PCI-14
50 male 37 male
T4N1M0 DNOMO
PD WD
III
PCI-15
69 male
T2N1M0
PD
III
PCI-15A PCI-16
69 male 61 male
T2N1M0 T2N1M0
III PD P-MWD III
PCI-17
61
PCI-18 PCI-19 PCI-20
46 male 56 male 66 male
T4N0M0 T3N0M0 TxN3M0
MWD IV MWD III PD
PCI-21 PCI-22A PCI-22B
63 male 59 male 59 male
T3N2M0 T4N1M0 T4N1M0
WD MD MD
PCI-23
65
PCI-24 PCI-25
81 male 65 male
female T2N0M0
female T2N0M0 T2N0M0 T4N1M0
PD
II
IV IV IV
MWD II WD II MWD IV
BOT primary pyriformsinus primary lymphnode recurrent lymph node metastasis RMT primary lymph node recurrent lymph node metastasis neck lymph node metastasis pyriformsinus primary lymph node lymph node metastasis primary Epiglottis (larynx) recurrence larynx primary glottis skin lymph node metastasis pyriformsinus primary buccal mucosa primary neck lymph node metastasis floorofmouth primary tongue neck
Primary location
Prev. tx
Previous tx Culture Authenti- Availability method cation details
Primary reference
BOT none pyriformsinus none pyriformsinus RT,S
Explant Explant Explant
Contact TLW Heo et al. 1989 Contact TLW Heo et al. 1989 Contact TLW Heo et al. 1989
RMT larynx
none S
Explant Explant
Contact TLW Heo et al. 1989 Contact TLW Heo et al. 1989
hypopharynx
none
Explant
Contact TLW Heo et al. 1989
pyriform sinus Epiglottis (larynx) Epiglottis (larynx) larynx glottis skin
none none
Explant Explant
Contact TLW Heo et al. 1989 Contact TLW Heo et al. 1989
S,RT
Explant
Contact TLW Heo et al. 1989
RT none none
Explant Explant Explant
Contact TLW Heo et al. 1989 Contact TLW Contact TLW
pyriformsinus none buccalmucosa none buccal mucosa none
Explant Explant Explant
Contact TLW Contact TLW Sacchi et al. 1991 Contact TLW Sacchi et al. 1991
floorofmouth none
primary tongue lymph node larynx metastasis
none none
Explant Explant Explant
Contact TLW Yasamura et al. 1994 Contact TLW Heo et al. 1990 Contact TLW Sacchi et al. 1991 Continued on next page
Lansford et al
Age Gender TNM
204
Table 1. Cell line name
(continued) Age Gender TNM
Grade Stage Specimen site Type of lesion
PCI-26
64 male
T3N0M0
WD
III
larynx
primary
PCI-27
51
male
T4N0M0
MD
IV
neck
PCI-28
50 male
T3N2M0
MD
IV
PCI-29 PCI-30
70 female T4N0M0 54 male T3N1M0
MWD IV MWD III
epiglottis (larynx) glottis BOT
lymph node floor of mouth metastasis primary epiglottis (larynx) primary glottis primary BOT
PCI-31
48
male
T3N0M0
PD
RMT
primary
PCI-32
61
male
T4N0M0
MWD IV
PCI-33
65
male
TlN0M0
MD
I
PCI-34
67
male
T4N2M0
WD
IV
PCI-35
75
male
T3N1M0
MWD III
PCI-36
65 female TZNOMO
MWD II
PCI-37A
62 male
T3N2M0
MD
IV
PCI-37B
62 male
T3N2M0
MD
IV
PCI-38
56 male
T3N1M0
MWD III
tongue
epiglottis (larynx) recurrent epiglottis lymph node (larynx) metastasis primary intraoral
PCI-39
73 male
TZNOMO
MWD II
epiglottis
primary
III
Previous tx Culture Authenti- Availability method cation details
Primary location
Prev. tx
larynx
none
Explant
none
Explant
none
Explant
RT none
Explant Explant
none
Explant
lymph node pyriform sinus none metastasis none floor of mouth metastasis intraoral
Explant
RMT
neck
retromolar trigone aryepiglottic fold neck epiglottis (larynx) epiglottis (larynx)
Primary reference
Contact TLW Snyderman et al. 1994 Contact TLW Contact TLW Sacchi et al. 1991 Contact TLW Contact TLW Yasamura et al. 1994 Contact TLW Snyderman et al. 1994 Contact TLW Yin et al. 1991
metastasis
intraoral
none
Explant
Contact TLW Snyderman et al. 1994 Contact TLW Yin et al. 1991
primary
aryepiglottic fold tongue
none
Explant
Contact TLW
S
Explant
Contact TLW Snyderman et al. 1994
none
Explant
none
Explant
Contact TLW Snyderman et al, 1994 Contact TLW Snyderman et al. 1994
none
Explant
none
Explant
recurrent lymph node metastasis primary
epiglottis (larynx)
Explant
Contact TLW Yasumura et al. 1994 Contact TLW Snyderman et al. 1994 Continued on next page
205
Cell line name
Head and Neck Cancers
Table 1.
(continued) Age Gender TNM
PCI-40
60
PCI-41
73 female T2N0M0
MD
PCI-42
58
MWD IV
PCI-43 PCI-44 PCI-45
63 male 78 male 47 male
TlN0M0 MWD I T4N0M0 MD IV T4N3M0 WD IV
retromolar trigone glottis glottis neck
PCI-46
46
T2N2M0
neck
PCI-47 PCI-50
57 female T3N0M0 92 male T2N0M0
MWD III WD II
PCI-51 PCI-52
54 43
male male
TlN2M0 TlN2M0
MD IV MWD IV
PCI-100 PCI-101 PCI-102 PCI-103 PCI-104 PCI-105 PCI-106 UT-SCC-1A
72 53 48 68 64 69 70 75
male male male male male male female female
T3N1M0 MD T4N2M0 MD T2N0M0 PD T3N0M0 PD T4N3M0 MD T3N0M0 WD T4N2M0 MD T2N1M0 MD
UT-SCC-1B
75 female T2N1M0
MD
III
UT-SCC-2
60 male
MD
IV
male
male
male
Grade Stage Specimen site Type of lesion
T4N2M0 MD
T4N2M0
T4N1M0
MD
IV
neck
II
BOT
IV
larynx tongue
pyriformsinus aryepiglottic fold III floor of mouth IV tongue II larynx III larynx IV pyriformsinus III hypopharynx IV pyriform sinus III gingiva gingiva
Primary location
Prev. tx
Previous tx Culture Authenti- Availability method cation details
lymph node pyriform sinus none metastasis primary BOT S,RT
Explant
primary
Primary reference
retromolar trigone primary glottis primary glottis lymph node tongue metastasis lymph node BOT metastasis primary larynx primary tongue
RT,S
Explant
Contact TLW Snyderman et al. 1994 Contact TLW Snyderman et al. 1994 Contact TLW
RT,S S none
Explant Explant Explant
Contact TLW Contact TLW Contact TLW
none
Explant
Contact TLW
none none
Explant Explant
primary primary
none none
Explant Explant
primary primary recurrence primary primary primary primary recurrence recurrence
floorofmouth primary
pyriformsinus aryepiglottic fold floor of mouth tongue glottis larynx pyriformsinus hypopharynx pyriform sinus gingiva mandibula
Explant
none none RT,CX none none S,RT RT RT
floorofmouth none
30Gy2 months 30 Gy 4 months
Explant Explant Explant Explant Explant Explant Explant Explant
Contact TLW Contact TLW Yasumura et al. 1993 Contact TLW Sung et al. 1995 Contact TLW Nagashima et al. 1998 Contact TLW Myers et al. 1996 Contact TLW Myers et al. 1996 Contact TLW Contact TLW Contact TLW Myers et al. 1996 Contact TLW Myers et al. 1996 Contact TLW AC Grenman et al. 1991
Explant
AC
Explant
AC
Grenman et al. 1991 Grenman et al. 1991 Continued on next page
Lansford et al.
Cell line name
206
Table 1.
Table 1. (continued) Age Gender TNM
Grade Stage Specimensite Type of lesion
Primary location
Prev. tx
UT-SCC-4
43 female T3N0M0 MD
III neck
UT-SCCJ
58 male
MD
III
tongue
UT-SCC-6A
51 female T2N1M0 WD
III
larynx
UT-SCC-6B
51 female T2N1M0 WD
III
neck
metastasis
supraglottic larynx
RT
UT-SCC-7
67 male
TlNOMO MD
I
neck
metastasis
cutisregio temporalis
RT
UT-SCC-8
42 male
TZN0M0 WD
II
larynx
primary
UT-SCC-9
81 male
T2N1M0 WD
III
neck
metastasis
supraglottic none larynx glotticlarynx RT
UT-SCC-10
62 male
TlNOMO MD
I
tongue
primary
scclinguae
UT-SCC-11
58 male
TlNOMO MD
I
larynx
recurrence glotticlarynx RT
T1N1M0
hypopharynx (RT) supraglottic larynx RT persistent tongue primary recurrence supraglottic RT larynx
metastasis
none
Previoustx Culture Authenti- Availability Primary reference method cation details Seefoot- Explant note UT-04
AC
Pekkola-Heino et al. 1992
50Gy2 months 75 Gy1 year4 months 75Gy 1 year4 months 57Gy 1 year6 months
Explant
AC
Explant
AC
Pekkola-Heino etal. 1992 Servomaaetal. 1996
Explant
AC
Explant
AC
Explant
AC
50Gy6 months
Explant
AC
Explant
AC
70Gy4 months
Explant
AC
Explant
AC
II
skin ofnose
primary
skin ofnose
none
UT-SCC-12B 81 female TZNOMO MD
II
neck
metastasis
cutisnasi
none
Explant
AC
supraglottic larynx tongue
RT
75 Gy5 Explant months 66Gy to Explant primaryand 53 Gy to neck; 2months
AC
UT-SCC-13
53 male
T3N0M0 MD
III
larynx
recurrence
UT-SCC-14
25 male
T3N1M0
III
tongue
primary
MD
RT
AC
Pekkola-Heino etal. 1992 Pekkola-Heino etal. 1992 ibid. Pekkola-Heino etal. 1995 Soukkaetal. 1995 Pekkola-Heino etal. 1995 Servomaaet al. 1996 Johanssonetal. 1997 ibid.
207
UT-SCC-12A 81 female T2N0M0 WD
Servomaaet al. 1996
Head and Neck Cancers
Cell line name
Continued on next page
Cellline name UT-SCC-15
Age Gender TNM 51 male
Grade Stage Specimen site Typeof lesion
TlNOMO WD
I
tongue
recurrence tongue
UT-SCC-16A 77 female T3N0M0 PD
III
tongue
primary
UT-SCC-16B 77 female T3N0M0 PD
III
neck
metastasis
UT-SCC-17
65 male
II
sternum
metastasis
UT-SCC-18
56 male
T3N1M0 MD
III
gingiva
persistent
UT-SCC-19A 44 male UT-SCC-19B 44 male
T4N0M0 MD T4N0M0 MD
IV larynx IV larynx
T2N0M0 PD
UT-SCC-20A 58 female TlNOMO MD
UT-SCC-20B 4
79 male 79 male
MD
T3N0M0 MD III TlNOMO MD I
primary persistent primary
floorofmouth primary
floor ofmouth residual
tongue larynx
primary residual
Prev. tx RT
Previoustx Culture Authenti- Availability Primary method cation reference details
65Gy Explant tongue5 months,51 Gyneck tongue RT 68tongueExplant 3months tongue Explant RT 50Gy neck RT 64 Gy 1 Explant supraglottic larynx year2 months gingiva Explant RT 68Gy 1month glotticlarynx none Explant glotticlarynx RT Explant 62Gy larynx,52 Gyneck, 2months floorofmouth RT Explant 63Gy tongue,55 Gyneck 1 month floorofmouth RT 63 Gy Explant tongue,55 Gyneck 6 months tongue RT 10 Gy Explant glotticlarynx RT Explant 68Gy2 year7 months
AC
Pekkola-Heino etal. 1996
AC
ibid.
AC
ibid.
AC
Kiuru etal. 1997
AC
Johansson etal. 1997 Elomaaetal. 1995 ibid.
AC AC
AC
Pekkola-Heino etal. 1996
AC
ibid.
AC AC
Elomaa et al. 1995
Continued on next page
Lansford et al.
UT-SCC-21 UT-SCC-22
female NS
I
Primary location
208
Table 1. (continued)
Gender TNM Grade Stage Specimen site
UT-SCC-23
66 male
T3N0M0 WD
III larynx
UT-SCC-24A 41 male
T2N0M0 MD
II
UT-SCC-24B 41 male T2N0M0 MD UT-SCC-25
tongue neck
II
tongue
50 male T2N0M0 WD II
Type of lesion
Primary location
persistent primary primary
Prev. Previous tx Culture Authenti- Availability Primary tx details method cation reference
transglottis
RT
tongue
persistent tongue metastasis recurrence tongue
RT RT
UT-SCC-26A 60 male
T1N2M0 MD
IV neck
metastasis
hypopharynx RT
UT-SCC-26B 60 male
T1N2M0 MD
IV neck
metastasis
hypopharynx RT
II
gingivaof recurrence maxilla UT-SCC-28 58 female T2N0M0 WD II floorofmouth persistent primary primary UT-SCC-29 82 male T2N0M0 WD II larynx UT-SCC-30 77 female T3N1M0 WD III oraltongue primary UT-SCC-27 71 male
T2N0M0 PD
UT-SCC-31
58 male
T3N2BM0 M-WD IV floorofmouth primary
UT-SCC-32
66 male
T3N0M0 WD
UT-SCC-33
86 female T2N0M0 MD
III II
tongue
glotticlarynx none tongue none
Explant
AC
Kiuruetal.1997
Explant
AC
Explant 62Gy1 month 65,63Gy Explant tongue,50 Gyneck Explant 60Gy completed 2months previous Explant 60Gy completed 6months previous Explant 64Gy4 months 65Gy1,5 Explant months Explant Explant
AC
Pekkola-Heino et al. 1996 ibid.
AC AC
Explant
AC
Explant
AC
ibid.
Explant
AC
ibid.
floorofmouth none
persistent tongue primary gingivaof primary mandible
RT none
66Gy3 weeks
AC
Johanssonetal. 1997
AC
ibid.
AC
ibid.
AC AC
Kiuruetal.1997 ibid. Elomaaetal.1995 Pekkola-Heino etal.1996 Kiuruetal.1997
Continued on next page
209
gingivaof mandible
RT gingivaof maxilla floorofmouth RT
75Gy8 months none
Head and Neck Cancers
Table1. (continued) Cell line Age name
(continued)
Cell line name
Age Gender TNM
Grade Stage Specimen site Type of lesion
Primary location
Prev. tx
UT-SCC-34
63
male
T4N0M0
WD
IV
none
UT-SCC-35
50 male
T2N0M0
MD
II
supraglottic larynx glottic larynx
UT-SCC-36 UT-SCC-37
46 male T4N1M0 61 female T2N0M0
PD WD
IV II
UT-SCC-38 UT-SCC-39
66 male 81 male
T2N0M0 T2N0M0
MD MD
UT-SCC-40 UT-SCC-41
65 male 76 male
supraglottic larynx larynx
primary
primary primary
II II
floor of mouth gingiva of mandible larynx larynx
primary primary
T3N0M0 WD T3N0M0 WD
III III
tongue tongue
primary recurrence
residual
male
T4N3M0
PD
IV
larynx
primary
UT-SCC-42B
43
male
T4N3M0
PD
IV
neck
primary
UT-SCC-43A
75 female T4N1M0
MD
IV
primary
UT-SCC-43B
75 female T4N1M0
MD
IV
UT-SCC-44
71
gingiva of mandible gingiva of mandible gingiva of maxilla floor of mouth gingiva of maxilla
female T4N2BM0 PD
UT-SCC-45 76 male UT-SCC-46A 62 male UT-SCC-46B
62
male
IV
T3N1M0 TlNOMO
PD PD
III I
TlNOMO
WD
I
gingivaof maxilla
persistent primary persistent primary primary primary primary
supraglottic larynx supraglottic larynx gingiva of mandible gingiva of mandible gingiva of maxilla floor of mouth gingivaof maxilla (retromolar) gingivaof maxilla (retromolar)
Explant
AC
Explant
AC
Explant Dx: lichen Explant ruber planus Explant Explant
AC AC AC AC
Explant Explant
AC AC
Explant
AC
none
Explant
AC
none
Explant
AC
Explant
AC
Explant
AC
Explant Explant
AC AC
Explant
AC
RT none none none none none RT none
RT RT none none none
66 Gy4 months
65 Gy3 weeks
64 Gy 4 weeks 62 Gy 4 weeks
Primary reference Kiuru et al. 1997
Continued on next page
Lansford et al.
UT-SCC-42A 43
floor of mouth gingiva of mandible glottic larynx supraglottic larynx tongue tongue
Previous tx Culture Authenti- Availability method cation details
210
Table 1.
(continued)
Cell line name
Age Gender TNM
Grade Stage Specimen site Type of lesion
Primary location
UT-SCC-47 UT-SCC-48 UT-SCC-49
78 male 81 male 76 male
T2N0M0 T3N0M0 T2N0M0
PD MD MD
II II II
floor of mouth primary parotidgland primary larynx primary
floor of mouth none parotid gland none glottic larynx none
UT-SCC-50
70 male
T2N0
PD
II
larynx
recurrence
glottic larynx
RT
UT-SCC-51
62 male
T2N0M0
M-WD II
larynx
recurrence
glottic larynx
RT
UT-SCC-52
51
T2N1M0
MD
III
tongue
RT
UT-SCC-53 UT-SCC-54A UT-SCC-54B
76 male T4N2cM0 WD 58 female T2N0M0 WD 58 female T2N0M0 WD
IV II II
persistent primary maxillary sinus primary buccal mucosa primary buccal mucosa recurrence
maxillary sinus none none buccal mucosa
UT-SCC-55
76 male
T4N1M0
MD
IV
primary
EV-SCC-1
55 male
T2N0M0
M-WD II
gingiva of mandible alveolus
primary
gingiva of mandible alveolus
EV-SCC-2 EV-SCC-3
52 male 65 male
T2N2aM0 WD IV T2N2bM0 P-MWDIV
lymph node tonsil
metastasis primary
pharynx tonsil
EV-SCC-4
45 male
T3N1M0
floor of mouth primary
floor of mouth
tongue lymph node lymph node
tongue hypopharynx tongue
male
EV-SCC-7 19 male EV-SCC-10M 66 male EV-SCC-14M 59 male
M-WD III
T4N0M0 MD T4N1M0 MD T2N2bM0 MD
IV IV IV
tongue
primary metastasis metastasis
Prev. tx
none
Previous tx Culture Authenti- Availability method cation details Explant Explant Explant
A second primary later diagnosed 62 Gy 12 Explant year rad. induced? 67 Gy 22 Explant months dysplasia 65 Gy3 Explant weeks Explant Explant Dx was Explant lichen ruben Explant Nude mouse Nude mouse Nude mouse
Primary reference
AC AC AC AC
Head and Neck Cancers
Table 1.
AC AC AC AC AC AC AR
Somers et al. 1992
AR AR
Patel et al. 1996 Somers et al. 1992
AR
Somers et al. 1992
AR AR AR
Patel et al. 1996 Patel et al. 1996 Patel et al. 1996
211
Continued on next page
Table I.
(continued) Age Gender TNM
EV-SCC-17P 34 male EV-SCC-17M 34 male EV-SCC-18 67 male 62 62 74 62 71 42 70 77 66 62
AMC-HN-1 AMC-HN-2 AMC-HN-3 AMC-HN-4 AMC-HN-5 AMC-HN-6 AMC-HN-7 AMC-HN-8 AMC-HN-9
54 57 63 65 67 61 59 46 66
T4N0M0 PD IV T4N0M0 ? IV T3N1M0 P-MWDIII
male male male male male male male male female male
T3N1M0 T3N1M0 T2N0M0 T4N0M0 T2N1M0 TlN1M0 T3N0M0 T2N2bM0 NS T2N2M0 NS 41 male TlN2M0 62 male T4N3M0 61 male T2N0M0 87 female NS 72 male T4N2M0 66 male T2N0M0 male male male female male male male male female
T1N0M0 T4N2M0 T3N1M0 T4N0M0 T3N0M0 T4N2M0 T4N2M0 T3N2M0 T4N2M0
tongue lymph node larynx
primary metastasis primary
Primary location
Prev. tx
AR AR AR
tongue tongue larynx
P-MWDIII ? III MD II MD IV MD III PD III WD III MD IV MD PD IV
tongue primary tongue lymph node metastasis tongue glottis none glottis primary retromolar none retromolar primary pyriformsinus none pyriformsinus primary floor of mouth none floor of mouth primary vocalcord none vocalcord primary epiglottis none epiglottis primary floor of mouth none floor of mouth supraglottis none supraglottis primary
PD PD MD PD Un-D MD
IV IV II
MD MD MD WD MD MD WD WD Un-D
I IV III IV III IV IV IV IV
tonsil tonsil vocalcord nasal tip retromolar aryepiglottic fold tongue hypopharynx larynx tongue nasalcavity floorofmouth larynx lymphnode lymphnode
IV II
primary primary primary primary primary primary primary recurrence primary recurrence recurrence primary recurrence metastasis metastasis
Previous tx Culture Authenti- Availability method cation details
tonsil tonsil vocalcord nasal tip retromolar aryepiglottic fold tongue hypopharynx larynx tongue nasalcavity floorofmouth larynx larynx parotid
AR AR
Primary reference Patel et al. 1996 Patel et al. 1996 Patel et al. 1996
none none none none none none
Explant Explant Explant Explant Explant Explant
Patel et al. 1996 Patel et al. 1996 Van Dyke et al. 1994 Van Dyke et al. 1994 Van Dyke et al. 1994 Van Dyke et al. 1994 Van Dyke et al. 1994 Van Dyke et al. 1994 Van Dyke et al. 1994 Van Dyke et al. 1994 Kelkeretal. 1996 Van Dyke et al. 1994 Van Dyke et al. 1994 Van Dyke et al. 1994 Van Dyke et al. 1994 Van Dyke et al. 1994 Van Dyke et al. 1994
none S,RT,CX none S,RT,CX S,RT none S,RT S,RT none
Explant Explant Explant Explant Explant Explant Explant Explant Explant
Kimet al. 1997 Kim etal. 1997 Kim etal. 1997 Kim et al. 1997 Kim et al. 1997 Kimetal.1997 Kimetal.1997 Kim et al.1997 Kim etal.1997
Explant Explant Explant Explant Explant Explant Explant Explant
Continued on next page
Lansford et al.
EV-SCC-19P EV-SCC-19M HFH-SCC-3 HFH-SCC-4 HFH-SCC-6 HFH-SCC-8 HFH-SCC-11 HFH-SCC-12 HFH-SCC-15 HFH-SCC-16 HFH-SCC-17 HFH-SCC-19 HFH-SCC-20 HFH-SCC-28 HFH-SCC-29 HFH-SCC-33 HFH-SCC-42
Grade Stage Specimen site Type of lesion
212
Cell line name
Table 1.
Age Gender TNM 64 male
UD-SCC-2 58 UD-SCC-3 45 47 UD-SCC-4 UD-SCC-5 44 UD-SCC-6 64 HNSCCUM-01T 65 HNSCCUM-02T 44
male male male male male male
Grade Stage Specimen site Type of lesion T3N2BM0 PD IV ant.tongue primary
TlN3M0 T2N2cM0 T3N1M0 T1N1M0 T2N0M0 T2NcM0
PD PD MD PD PD PD
male T3N3bM0 PD
HNSCCUM-03T56 male
T3N2bM0 MD
HNSCCUM-04N52 male TlNlMO
MD
Primary location tonsil
Prev. tx
IV IV III III IV IV
pyriformsinus lymphnode tongue supraglottis tongue vallecula
IV
baseoftongue primary
baseoftongue none
IV
pyriformsinus primary
pyriformsinus none
III
supraglottic
primary
supraglottic
none
primary metastasis primary primary primary primary
pyriformsinus aryepiglottic tongue tongue tongue vallecula
2,ND,RT none none none
HNSCCUM-05N 54
male TlN2bM0 MD
IV oropharynx
primary
oropharynx
none
HNSCCUM-06N65
male T2N2cM0 PD
IV vallecula
primary
vallecula
none
HNSCCUM-07N 53
male T1N2bM0 MD
IV
pyriformsinus primary
pyriformsinus none
III III
labialmucosa primary BOT primary
labialmucosa BOT
TU-138 TU-159
53 male 44 male
female T4N2bM0 WD male T3N0M0 WD female T3N2bM0 MD
IV FOM III larynx IV tonsil
male
T2N2cM0 MD
IV
hypopharynx primary
male male
PD T2N2aM0 MD T2N2cM0 MD
IV IV
tonsil lymphnode
primar primary primary
primary metastasis
FOM larynx tonsil tongue hypopharynx tonsil hypopharynx
Explant Explant Explant Explant Explant
Primary reference Contact H. Bier Ballo et al.1999 Ballo et al. 1999 Ballo et al. 1999 Ballo et al. 1999 Ballo et al. 1999 Ballo et al. 1999
Contact H. Bier Contact H. Bier Contact H. Bier Contact H. Bier Contact H. Bier Contact H-JWelkoborsky Contact H-JWelkoborsky Contact H-JWelkoborsky Contact H-JWelkoborsky Contact H-JWelkoborsky Contact H-JWelkoborsky Contact H-JWelkoborsky Lengyeletal.1995 Beckhardt et al. 1995 Beckhardt et al. 1995 Beckhardtet al. 1995 Beckhardtet al. 1995 Beckhardtetal.1995 Beckhardtetal.1995
Beckhardt et al.1995 Beckhardt etal. 1995 Continued on next page
213
70 TU-I67 54 TU-177 40 TU-182 TU-202 60 TU-212 TU-358B TU-686 (MDA?) 44 TU-158LN 60 TU-212-LN
T3N0M0 WD T3N0M0 MD
Previous tx Culture Authenti- Availability method cation details Explant
Head and Neck Cancers
Cell line name UD-SCC-1
(continued)
(continued)
214
Table 1.
Table 1 Part 2 Nasopharyngeal carcinoma (NF'C) cell Lines Age Gender TNM
Grade Stage Specimen site Type of lesion
NPC/HK1
41
male
WHO
NPC-KT
74
female
Lymphoepithelioma (WHO III)
C15
13
female T4N3M1
WHOIII
nasopharynx
C17
38
male
T3N3
WHOIII
C18
47
male
T4N3
WHO III
I
nasopharynx
recurrent
Primary location nasopharynx
Prev. tx RT
nasopharynx
none
primary
nasopharynx
none
cutaneous met
metastasis
nasopharynx
XRT, cx
lymphnode
metastasis
nasopharynx
CX
nasopharynx
primary
Previous tx Culture Authenti- Availability method cation details
Primary reference
600radsto Explant Nude nasopharynx mouse with total response for 17.5years. Recurrence noted 1 month prior to biopsy Explant of NPC cells fused to adenoid cells using Sendai virus Nude mouse Primary, Nude mouse RT to head and neck followed by bone and skin metastatic relapse treated with bleomycin, cis-platinum, 5-FU 2 mos of Nude bleomycin, mouse cis-platinum, 5-FU
Huang et al. 1980
Takimoto et al. 1984
Busson et al. 1988 Busson et al. 1988
Busson et al. 1988
Continued on next page
Lansford et al.
Cell line name
Table 1. (continued) Cell line name
Age Gender TNM
2117 CG1
47 male
cNE1 CNE2 HNE-1
27 male
NM8
Grade Stage Specimen site
Type of lesion
Primary location
Prev, tx
WHO III
nasopharynx
primary nasopharynx none
WHO II
nasopharynx
primary nasopharynx none
1° squamous nasopharynx primary nasopharynx none CA nasopharynx 1°squamous nasopharynx primary nasopharynx none CA nasopharynx nasopharynx primary nasopharynx none WHO III
nasopharynx
primary
nasopharynx
HNE-3 HONE-I NPC-TW01
64 male
NPC-TW01N1 64 male
Previoustx details
WHOI
nasopharynx primary
WHOI
nasopharynx
none none
NPC-TW02
36 female
WHO I
nasopharynx primary
nasopharynx none
NPC-TW03
36 male
WHO II B
nasopharynx primary
nasopharynx none
none
Culture Authenti- Availmethod cation ability Nude mouse Explant Nude mouse Explant Explant
Primary reference Colbum et al. 1989 Chang et al. 1989 Colbum et al. 1989
Head and Neck Cancers
Table 1 Part 2 Nasopharyngeal carcinoma (NPC) cell lines
Colbum et al. 1989 Nude mouse Nude mouse
Colburn et al. 1989 Yaoetal.1990 Yaoetal.1990 Linetal.1990 Linetal.1990
Linetal. 1990 Linetal. 1993 Continued on next page
215
Nude mouse Explant Nude mouse Fromnude mouse solid tumor mass after injecting NPC-TW01 NPC line Explant Nude mouse Explant Nude mouse
Li et al. 1989
216
Table 1. (continued) Table 1 Part 2 Nasopharyngeal carcinoma (NPC) cell lines Celllinename Age Gender TNM Grade
Specimen Stage Site
TypEof lesion
Primary location
Prev. tx
WHOIIB
nasopharynx primary
nasopharynx none
NPC-TWOS 47 male
WHOI
nasopharynx primary
nasopharynx none
NPC-TW06
58 female
WHOIIB
nasopharynx primary
nasophraynx none
NPC-TW07
75 male
WHOIIB
nasopharynx primary
nasopharynx none
NPC-TW08
50 male
WHOI
nasopharynx primary
nasopharynx none
NPC-TW09
30 male
WHOIIB
nasopharynx primary
nasopharynx none
NPC-TW04
36 female
Previous tx details
Culture Authenti-Availmethod cation ability Explant Nude mouse Explant Nude mouse Explant Nude mouse Explant Nude mouse Explant Nude mouse Explant Nude mouse
primary reference Linetal.1993 Linetal.1993 Linetal. 1993 Linetal.1993 Linetal. 1993 Linetal.1993 Continued on next page
Lansford et al.
Head and Neck Cancers
217
Table1. Footnotes Cell line name: (HeLa?) - See text regarding cross-contamination. For HNSCCUM cell lines the suffix T means primary tumor; N means metastatic lymph node. TNM: Tumor, node, metastasis classification, stage at primary diagnosis only, NS = not staged. Specimen Site column: BOT, back of tongue; RMT, retromolar trigone; FOM, front of mouth Grade column: WD, well differentiated; PD, poorly differentiated; MWD, moderately well differentiated; M-WD, moderately to well differentiated; P-MWD; poorly to moderately well differentiated; Un-D, undifferentiated; WHO = World Health Organization. Culture method column: E on RC, Explant on rat collagen; T-C-D, Trypsin collagenase digest; E on FFL, Explant on fibroblast feeder layer; E on irr 3T3 FFL, Minced tumor fragments (explant) co-cultivated with irradiated 3T3 fibroblasts; SCC-13 requires a feeder layer for survival and proliferation Prior Treatment column: S, surgery; RT, radiotherapy; CX, chemotherapy; ND, neck dissection. Prior Treatment Details column: UM-03, Local excision with partial mandibulectomy and suprahyoid neck dissection; UM-04, CX with BOMM x2 (75% response by primary, apparently complete response of neck node); UM-13, 54 Gy and anterior esophagectomy, partial tracheal resection, total thyroidectomy; UM-14C, 5-FU, Velbam, and methotrexate ctx failed; UM-17A, primary and bilateral lateral necks got rt, total dose = 6600 rads; UM-17B, bilateral lateral necks got rt, total dose = 6600 rads; UM-20, RT was 6500 rads to primary, 4000 rads to ipsilateral neck. S was total laryngectomy; UM-27 primary excision and postop RT of 5000 rad; UM-29, Three courses of pre-op Cx: cis-platinum and MGBG; UM-30, Pre-op Cx: Cis-platinum and MGBG; UM-33, cisplatin and MGBG chemo x 2 courses without tumor response; UM-37, 3 courses of cis-platinum and 5-FU, initially with excellent response, but after 3 months developed a large anterior jugular, left-sided neck node. Then full course RT. UM-40,5FU, Platinol, and 6480 Rads to the neck UM-46, 6000 rads supraglottic, then supraglottic laryngectomy and R radical neck; UM-74A, cisplatin & 5-FU x 3 courses, 6500 rads external + 2500 rads implant, hemiglossectomy & radical neck; UM-74B, cisplatin & 5-FU x 3 courses, 6500 rads external t 2500 rads implant, hemiglossectomy & radical neck, tracheotomy, 5-FU+ CBDCA, UM-81B, 2nd primary of tonsillar pillar in heart transplant recipient (> 34 mos on immunosuppression, supraglottic laryngectomy, laryngectomy; For UT-SCC cell lines XX Gy X months indicates radiation dose given and the time prior to surgery; UT-04,8 years earlier had radiotherapy for the mediastinum for morbus Hodgkin + MOPP chemotherapy x 6. ND = neck dissection. Authentication column: D = DNA comparison or fingerprinting, H = HLA typing, I = isozyme analysis. For the UM-SCC-cell lines, D indicates that the tumor cell line has been compared to microsatellite polymorphisms from normal tissue or cells from the same individual. For examples see Frank et al. 1997 Availability column: AR, Available upon Request. UM-SCC-AR, UM-SCC cell line are Available upon Request. UM-SCC cell lines are intellectual properties of the University of Michigan. Michigan grants permission for other investigators to use these lines provided that they are not distributed and that they are not used for commercial exploitation. The University of Michigan retains ownership of all lines, progeny and products. Contact TLW. To inquire about the use of PCI-SCC cell lines please contact Theresa L. Whiteside. AC, UT-SCC cell lines are available for collaborative studies. Please contact Reidar Grenman. For UD lines, contact Henning Bier; For HNSCCUM lines, Contact Hans-J Welkoborsky. DSMZ, German Tissue Culture Collection. Published resources, including cell lines, should be freely available for non-commercial use and application.
Cell line
Prior RX
RPMI 2650
RT/S
Detroit 562
none
FaDu
none
UM-SCC-1 UM-SCC-2
S
UM-SCC-3
S
UM-SCC-4
cx
UM-SCC-5
S
Treatment description
Original pathology
In vitro morphology
Nude mouse morphology
Continued on next page
Lansford et al.
Monolayer colony forming growth with little tendency to stratify Monolayer colony forming growth with little tendency to stratify Monolayer colony forming growth with little tendency to stratify Regression of tumor in nude mice Local excision with partial Progressive growth in Originally, a 1.5cm SCC in mandibulectomy and nude mice the right lower alveolus extending into the socket of suprahyoid neck dissection a bicuspid tooth Slow growth in the nude mouse; keratin horn formation CX with BOMM x2 Inoculation of 2 nude mice Tumor involves right tongue resulted in progressive (75% response by primary, near tonsillar pillar with a growth in one and stabilized apparently complete response large defect in the BOT tumor in the other of neck node) Monolayer non-stratifying cells forming monolayer cultures. Spreading cells with few or no cellsmigratingindependently from the large islands. Same morphology in passage 4 and passage77
218
Table 2. Head and neck squamous cancer cell line characteristics
Cell line
Prior RX
UM-SCC-6
none
UM-SCC-7
none
UM-SCC-8
RT
UM-SCC-9
RT
Treatment description
Original pathology
In vitro morphology
This specimen was taken from a 4 × 1.5 cm exophytic SCC of the right alveolar process growing down toward the FOM. A 3.5 × 1.5 cm firm mobile metastatic ipsilateral superior jugular digastric node was present at this time Pathologically, a partly necrotic keratinizing well differentiated SCC
The specimen is from a 4 x 1.5 cm exophytic SCC of the left anterior tongue accompanied by erythroplakia extending to the FOM almost to the mandibular alveolus
Stratifying colonies with a limited propensity for mound or dome formation. Some areas were undifferentiated while others had dyskeratosis and involution of the nuclei
Nude mouse morphology Inoculation of 2 nude mice resulted in progressive growth in one and stabilized tumor in the other Progressive tumor growth in 12 of 13 nude mice, with tumor regression in one
Head and Neck Cancers
Table 2. (continued)
Regression of tumor in one nude mouse Regression of tumor in nude mice
Continued on next page
219
UM-SCC-10A
none
UM-SCC-10B UM-SCC-11A
S none
UM-SCC-11B
CX
UM-SCC-12
S
UM-SCC-13
RT/S
Treatment description
Original pathology
In vitro morphology
Infiltrative lesion of Growth primarily in islands epiglottis accompanied by of cells an ipsilateral 8 x 8 cmlymph node metastatic mass Monolayer non-stratifying culture with individual cells migrating independently of colonies
54 Gy and anterior esophagectomy, partial tracheal resection, total thyroidectomy
Nude mouse morphology
220
Table 2. (continued) Cell line Prior RX
Regression of tumor in one and stabilization of tumor in one of two inoculated nude mice Progressive tumor growth Regression of tumor in nude mice
Progressive growth in five and stabilized tumor in four of nine inoculated nude mice Progressive tumor growth in five and stabilized tumor in seven of 12 inoculated nude mice
Continued on next page
Lansford et al.
This recurrence is 9 months post rt and 8 months post anterior esophagectomy, partial tracheal resection, total thyroidectomy and was found at the site of the esophageal anastamosis. Microscopically, a well-differentiated keratinizing SCC with slight variation in nuclear size, some mitoses and intracellular bridges
Prior RX
Treatment description
UM-SCC-14A
S/S/RT/S
UM-SCC-14B
S/S/RT/S/S
UM-SCC-14C
S/S/RT/S/S/CX 5-FU, Velbam, and methotrexate ctx failed
UM-SCC-15
none
Original pathology A recurrent tumor 75 months after initial dx. involving a clinically positive contralateral jugular node and biopsy-proven local recurrence This local recurrence was not clinically evident but obtained from a mandibulectomy for osteoradionecrosis This recurrence is a 3 × 4cm submental tumor with two 3 cm nodes and several smaller nodes in the ipsilateral neck This specimen from an extensive lesion involving the posterior hypopharynx to the level of the prevertebral fascia from the nasopharynx to the introitus of the esophagus. Several positive neck nodes were also present
In vitro morphology
Nude mouse morphology Progressive tumor growth in nude mice
The UM-SCC15 cell culture undergoes spontaneous lysis at passage level 6–8 and thus does not represent a true established cell line
Head and Neck Cancers
Table 2. (continued) Cell line
Regression of tumor in one inoculated nude mouse
Continued on next page
221
Treatment description
none
UM-SCC-17A
RT
Primary tumor and bilateral lateral necks got rt, total dose = 6600 rads
UM-SCC-17B
RT
Primary tumor and bilateral lateral necks got rt, total dose = 6600 rads
A large cancer involving the left true and false vocal cords and extending up on the laryngeal surface of the epiglottis. Microscopic exam showed orderly nonkeratinized stratified squamous epithelium with good vascularization and scant fibrous stroma. Some loss of intercellular bridging, slight size and shape variation in cells and nuclei, and occasional individual cell keratinization were present
A fungating lesion of the left true and false vocal cords with swelling of the left arytenoid
In vitro morphology Primary cultures formed a single, large stratifying mound with very little lateral spread. With time in culture and repeated exposure to limited trypsinization, some cells were separated which themselves reattached in the same flask and formed small islands which also stratified. The domes arise from multipolar cells that form a monolayer in which small stellate clusters or rosettes form at regular intervals. This phenotype unchanged over 50+ passages Stratifying colonies that reach a certain size and then continue to to proliferate without expanding in size. Cells become densely packed within islands without forming confluent monolayers. This phenotype remains stable over 100 + in vitro passages Stratifying colonies that reach a certain size and then continue to proliferate without expanding in size. Cells become densely packed within islands without forming confluent monolayers. This phenotype remains stable over 100+ in vitro passages
Nude mouse morphology Stabilized tumor in one inoculated nude mouse
Regression of tumor in one and stabilization of tumor in one of two inoculated nude mice
Stabilized tumor in one inoculated nude mouse
continued on next page
Lansford et al.
UM-SCC-16
Original pathology
222
Table 2. (continued) Cell line Prior RX
UM-SCC-18
S/RT
UM-SCC-19
none
UM-SCC-20
RT/S
RT was 6500 rads to primary, 4000 rads to ipsilateral neck. S was total laryngectomy
Original pathology Recurrent disease 17 months s/p surgery, RT of a 2 × 2 cm necrotic mass in the right base of tongue extending into the vallecula, right aryepiglottic fold, and pyriform sinus with a 2.5 cm ipsilateral mid internal jugular chain This line comes from a 3 × 4 cm ulcerated mass involving the right BOT, extending just across the midline and anteriorly to the circumvillate papillae. One 3 cm firm jugulodigastric node on the right was also present This second recurrence of tumor was two lcm discrete SCCs of the hypopharynx with a large ipsilateral neck nodal metastasis from which this line was developed
In vitro morphology
Nude mouse morphology Progressive growth in one and stabilized tumor in seven of eight inoculated nude mice
Head and Neck Cancers
Table 2. (continued) Cell line Prior RX Treatment description
Regression of tumor in one and stabilization of tumor in one of two inoculated nude mice
Monolayer non-stratifying culture. Poorly differentiated histology with loose clusters of cells and individual cells migrating independently of colonies. A component of the cells grew in suspension independent of adhesion to a substrate, but these could not be propagated in culture
223
Continued on next page
Original pathology
none
An exophytic, ulcerated 3.5 × 3 an mass extending from the posterior oropharynx circumferentially around the hypopharynx at the level of the cricoid and extending down to the left pyriform sinus. A 1 x 2 cm firm mobile ipsilateral left midjugular vein node was present. This biopsy taken from the posterior arytenoids
UM-SCC-23
none
Tumor was a 1.5 × 1.5cm ulcerated lesion microscopically showing in-situ and invasive SCC
UM-SCC-24
RT/S/S
This specimen taken from the stomal recurrence that followed initial RT, hemilaryngectomy, and then total laryngectomy. Microscopically, a well-differentiated infiltrating neoplasm with finger-like projections
UM-SCC-25
RT/S/S
This recurrent SCC specimen was 5.5 long×3.5cm wide firm, non-fixed tumor of the mid-posterior pharynx extending down from the inferior pole of the tonsil toward the right hypopharynx. Micro had been well differentiated 2 months previously, now infiltrative poorly differentiated with necrosis.
Nude mouse morphology
Cuboidal cells mostly in colonies but with some breaking off to form small new colonies. Cells produce hemidesmosomes in culture
Large cuboidal cells in monolayer colonies
Lansford et al.
UM-SCC-22A
In vitro morphology
224
Table 2. (continued) Cell line Prior RX Treatment description
Continued on next page
Cell line UM-SCC-26
Prior RX Treatment description none
In vitro morphology Original pathology This invasive keratinizing SCC specimen was taken from the left tongue which extended well into the tongue base and up into the mobile tongue on the left side
UM-SCC-27
S/RT
This tumor, found one year after resection and RT, is a 3 cm mid jugular digastric neck node
UM-SCC-28
none
UM-SCC-30
CX
UM-SCC-31
UM-SCC-34
Primary excision and postop RT of 5000 rad
Nude mouse morphology
Head and Neck Cancers
Table 2. (continued)
A 0.6cm finn nodular neoplasm found to be invasive keratinizing well differentiated SCC Pre-op: Cis-platinum and MGBG
Monolayer non-stratifying islandforming culture. Predominantly spreading type of morphology
RT
This specimen is the persistent SCC at the tonsillar pillar. This was clinically accompanied by a 1 cm firm contralateral jugular digastric lymph node metastasis
Stratifying colonies that form domes
none
Tumor of the right anterior tonsillar pillar showing ulceration and moderate differentiation Continued on next page
225
The persistent tumor was a 2cm mass over the right aryepiglottic fold and superior pyriform sinus with a fixed ipsilateral true vocal cord. The neck had a 1 x 1 cm ipsilateral jugular node. Microscopically, keratinization present
Prior RX Treatment description none
UM-SCC-36
none
UM-SCC-37
CX,RT
Nude mouse morphology
Lansford et al.
3 courses of cis-platinum and 5-FU, initially with excellent response, but later developed a large anterior jugular, leftsided neck node
Original pathology In vitro morphology Extensive oropharyngeal carcinoma involving all of the left tonsil, soft palate, and posterior pharyngeal wall with extension into the tongue. Tumor occupies the entire BOT and extends anteriorly into the mobile tongue on the left. A 1 x 1 cm left high jugular node was present An exophytic SCC of the left true vocal cord, ventricle, false vocal cord with extension to the epiglottis. Microscopically, an infiltrating neoplasm composed of nests and cords of malignant cells which have pleomorphic nuclei and clear to eosinophilic cytoplasm, apparently arising in normal squamous mucosa, and containing focal areas of keratinization Extensive tumor involving the posterior pharyngeal wall, both lateral pharyngeal walls, superiorly to the right tonsil and behind the velum and also involving the left tonsillar fossa, and inferiorly to the posterior pharynx into both pyriform sinuses. Micro: infiltrative, moderately well differentiated
226
Table 2. (continued) Cell line UM-SCC-35
Continued on next page
Cell line
Prior RX Treatment description
UM-SCC-38
none
UM-SCC-39
none
UM-SCC-40
RT, CX
UM-SCC-41
none
UM-SCC-42
S
5FU, Platinol, and 6480 Rads to the neck
Original pathology
In vitro morphology
Continued on next page
227
Nude mouse morphology A 3-5cm ulcerative tumor in the Monolayer non-stratifying island right tonsillar fossa extending to forming cultures. Pleomorphic with the right BOT, found infiltrative both cuboidal and spreading cell on micro. A 2cm and a 0.5 cm morphologies and a tendency for nodal metastasis were also some cells to migrate independently present of the colonies A right pyriform sinus mass with Stratifying colonies that reach a a 4 × 4 and a 3 ×3 cm ipsilateral certain size and then continue lymph node neck metastases to proliferate without expanding in size. Cells become densely packed within islands without forming confluent monolayers A large high cervical esophageal carcinoma which eroded into the larynx Tumor involved the right arytenoid. A 1 × 2 cm right jugulodigastric node was also present This specimen represents persistent tumor two months after laryngopharyngectomy with left radical neck dissection for a large, friable left pyriform sinus mass with a 1 cm exophytic mass at the right cricoid. The persistent cancer involved cervical lymph nodes and extended deeply into the prevertebral tissues
Head and Neck Cancers
Table 2. (continued)
228
Table 2. (continued) Cell line Prior RX Treatment description UM-SCC-46
RT/S
UM-SCC-49
UM-SCC-50
none
6000 rads supraglottic, then supraglottic laryngectomy and R radical neck
Original pathology
In vitro morphology
Nude mouse morphology
Continued on next page
Lansford et al.
An unresectable recurrent 4 × 3cm SCC of the larynx with tracheal extension and supraglottic extension to below the contralateral clavical. Microscopically, large fragments of exudate containing parakeratotic keratin pears thought to possibly be neoplastic, although viable SCC epithelium not identified with certainty A large ulcerated mass involving the left half of the tongue from the junction of the middle third of the tongue to include the BOT on the left and extending across the midline. A 2.5 cm firm high jugulodigastric node and a smaller submental node on the left are also present Exophytic tumor of the right base of tongue, 4 × 3 cm, extending to left tonsil and to the midline of the vallecula. A 3 × 3 cm jugulodigastric node and a 4× 4cm submental extension
Prior RX Treatment description
UM-SCC-52
UM-SCC-55
RT
UM-SCC-59
UM-SCC-62
none
Original pathology
In vitro morphology
Nude mouse morphology
A left vallecula carcinoma with extension through the epiglottis to involve the laryngeal surface of the epiglottis and crossing the midline. The cancer extended down the left lateral pharyngeal wall to involve the left aryepiglottic fold, the left arytenoid, the left false vocal cord as well as the right false vocal cord. Pt. had a 5 × 6cm firm, slightly mobile node Monolayer non-stratifying island-forming culture. Has both cuboidal and spreading morphologies This specimen arose from lichen planus and was taken from a 4.5 × 3.5 ulcerative lesion extending from the free lateral margin of the tongue into the floor of the mouth, but not attached to the mandible. A 3.5cm ipsilateral jugulodigastric lymph node was present A 6cm ulcerative lesion in the left tonsillar fossa with extension to the soft palate and base of tongue. A 2 × 2cm left jugulodigastric node was also present
229
Continued on nextpage
Head and Neck Cancers
Table 2. (continued) Cell line
Cell line UM-SCC-80
Prior RX Treatment description
UM-SCC-81A
none
Immunosuppression × 20mo for heart transplant. Hemilaryngectomy initially later total laryngectomy for recurrent cancer
UM-SCC-81B
S,S
Immunosuppression × 34 mo for heart transplant
UM-SCC-82A
none
Nude mouse morphology
Lansford et al.
In vitro morphology Original pathology A 6 × 5 cm mass at the left tonsil, posterior tonsil and hypopharynx, and upper esophagus. Micro: a welldifferentiated, keratinizing SCC with abnormal mitoses. Occasional cells are markedly abnormal and show large hyperchromatic nuclei. High mitotic index. Focal necrotic changes in some fields. A 2 × 3 cm left nodal metastasis present A polypoid mass extending from Thin and relatively large the anterior commissure and cytoplasm involving a portion of the anterior left and right cords as well as a mass extending along the left true and false vocal cords involving the left arytenoid Tonsillar pillar carcinoma. Thought to be second primary cancer On microscopy, an infiltrative, keratinizing SCC in nests, strands and sheets. Varying amounts of cytoplasm with indistinct cell borders. Nuclei are enlarged, pleomorphic and vesicular. Abundant mitoses present and many abnormal. Macronucleoli prominent
230
Table 2. (continued)
Continued on next page
(continued) Prior RX Treatment description
Original pathology
UM-SCC-82B
RT
SCC-12B.2
none
SCC-12F.2a
none
SCC-12V
none
SCC-15
none
This recurrent SCC specimen is from a firm mass at the left BOT, left lateral tongue, tonsillar fossa, retromolar trigone, and soft palate with ulceration. A 4× 6cm left jugular digastric mass was also present. On micro, the SCC deeply invaded tongue muscle and the internal jugular vein and sternocleidomastoid SCC-12 is a heterogeneous tumor harvested from an immunocompromized patient and consisting of three distinct subclones, ‘F.2a,’ ‘B2,’ and ‘V’ SCC-12 is a heterogeneous tumor harvested from an immunocompromized patient and consisting of three distinct subclones, ‘F.2a,’ ‘B2,’ and ‘V’ SCC-12 is a heterogeneous tumor harvested from an immunocompromized patient and consisting of three distinct subclones, ‘F.2a,’ ‘B2,’ and ‘V’ Monolayer colony forming growth with little tendency to stratify
External beam radiation and radiation implants 7 months earlier
In vitro morphology
Nude mouse morphology
231
Continued on next page
Head and Neck Cancers
Table 2. Cell line
S
PCI-2
none
PCI-3
none
PCI-4A
none
PCI-4B
none
Original pathology
In vitro morphology
Nude mouse morphology
Cells grow as sharply demarcated, compact islands with a distinct epithelial morphology and positive keratinization. Doubling time = 66 hr Cells grow as sharply demarcated, compact islands with a distinct epithelial morphology and positive keratinization Cells grow as sharply demarcated, compact islands with a distinct epithelial morphology and positive keratinization
Tumor growth in nude mice pretreated with cyclophosphamide. Moderately well differentiated, as was the original tumor
Tumor growth in nude mice pretreated with cyclophosphamide. Moderately well differentiated, as was the original tumor Tumor growth in nude mice pretreated with cyclophosphamide. Moderately well differentiated histology in contrast to the well differentiated original tumor Cells grow as diffuse, loose Tumor growth in nude mice aggregates that fail to stratify. pretreated with cyclophosThey possess numerous micro- phamide.Welldifferentiated villi and positive keratinization. as was the original tumor Doubling time = 58 hr Cells grow as diffuse, loose Tumor growth in nude mice aggregates that fail to stratify. pretreated with cyclophosThey possess numerous micro- phamide. Well differentiated villi and positive keratinization. as was the original tumor Doubling time = 58 hr Continued on next page
Lansford et al
PCI-1
232
Table 2. (continued) Cell line Prior RX Treatment description
Prior RX Treatment description
Original pathology
PCI-5
none
PCI-6A
none
PCI-6B
S/RT
PCI-7
none
PCI-8
none
Cells grow as sharply demarcated, compact islands with a distinct epithelial morphology and positive keratinization Lymph node extracapsular spread Cells grow as sharply demarcated, compact islands with a distinct epithelial morphology and positive keratinization. Doubling time = 68 hr Cells grow as sharply demarcated, compact islands with a distinct epithelial morphology and positive keratinization. Doubling time = 106 hr Cells grow as sharply demarcated, compact islands with a distinct epithelial morphology and positive keratinization Cells grow as diffuse, loose aggregates that fail to stratify. They possess numerous microvilli and positive keratinization
In vitro morphology
Nude mouse morphology Tumor growth in nude mice pretreated with cyclophosphamide. Moderately well differentiated, as was the original tumor Tumor growth in nude mice pretreated with cyclophosphamide. Moderately well differentiated, as was the original tumor
Head and Neck Cancers
Table 2. (continued) Cell line
Tumor growth in nude mice pretreated with cyclophosphamide. Moderately well differentiated, as was the original tumor Tumor growth in nude mice pretreated with cyclophosphamide. Moderately well differentiated, as was the original tumor Tumor growth in nude mice pretreated with cyclophosphamide. Moderately well differentiated, as was the original tumor
233
Continued on next page
none
PCI-9B
none
PCI-10
none
PCI-11
none
PCI-12
RT/S
Lymph node extracapsular spread
In vitro morphology
Nude mouse morphology
Cells grow as sharply demarcated, compact islands with a distinct epithelial morphology and positive keratinization Cells grow as sharply demarcated, compact islands with a distinct epithelial morphology and positive keratinization Cells grow as sharply demarcated, compact islands with a distinct epithelial morphology and positive keratinization Cells grow as sharply demarcated, compact islands with a distinct epithelial morphology and positive keratinization. Doubling time = 124 hr
Tumor growth in nude mice pretreated with cyclophosphamide. Moderately well differentiated, as was the original tumor Tumor growth in nude mice pretreated with cyclophosphamide
Tumor growth in nude mice pretreated with cyclophosphamide. Moderately well differentiated, as was the original tumor Tumor growth in nude mice pretreated with cyclophosphamide. Poorly differentiated in contrast to the moderately well differentiated original tumor Cells grow as sharply demarcated, Tumor growth in nude mice compact islands with a distinct pretreated with cyclophosepithelial morphology and phamide positive keratinization Continued on next page
Lansford et al
PCI-9A
234
Table 2. (continued) Cell line Prior RX Treatment description Original pathology
PCI-13
none
PCI-14
S
PCI-15
none
PCI-15A
none
PCI-16
none
In vitro morphology
No lymph node extracapsular spread
Cells grow as sharply demarcated, Tumor growth in nude mice compact islands with a distinct epithelial morphology and positive keratinization. Doubling time= 86hr Cells grow as sharply demarcated, Tumor growth in nude mice pretreated with cyclophoscompact islands with a distinct stratified epithelial morphology phamide and positive keratinization. Pleomorphic nuclei with prominent nucleoli. Desmosomes between cells. Bundles of tonofilaments in the cytoplasm Cells grow as sharply demarcated, Tumor growth in nude mice pretreated with cyclophoscompact islands with a distinct epithelial morphology and phamide positive keratinization by IHC. Doubling time = 66 hr Cells grow as sharply demarcated, Tumor growth in nude mice pretreated with cyclophoscompact islands with a distinct epithelial morphology and phamide positive keratinization. Doubling time = 126 hr Cells grow as sharply demarcated, Tumor growth in nude mice compact islands with a distinct pretreated with cyclophosepithelial morphology and phamide positive keratinization
Lymph node extracapsular spread
Nude mouse morphology
Continued on next page
235
Original pathology
Head and Neck Cancers
Table 2. (continued) Cell line Prior RX Treatment description
PCI-17
S/RT
PCI-18
RT
PCI-22A PCI-22B PCI-25 PCI-26 PCI-28 PCI-30 PCI-33 PCI-34 PCI-37A PCI-37B
none none none none none none none none none none
PCI-38 PCI-39
none none
PCI-50
none
Original pathology
In vitro morphology
Cells grow as sharply demarcated, compact islands with a distinct epithelial morphology and positive keratinization Cells grow as sharply demarcated, compact islands with a distinct epithelial morphology and positive keratinization Lymph node extracapsular spread Doubling time = 80 hr Doubling time = 68 hr Lymph node extracapsular spread Doubling time = 200hr Doubling time = 93 hr Lymph node extracapsular spread Doubling time = 240 hr Doubling time = 52 hr Lymph node extracapsular spread Doubling time = 102 hr Doubling time = 73 hr Lymph node extracapsular spread Doubling time = 80 hr Doubling time = 91 hr No lymph node extracapsular spread Doubling time = 82 hr Doubling time = 104 hr
Nude mouse morphology Tumor growth in nude mice pretreated with cyclophosphamide
236
Table 2. (continued) Cell line Prior RX Treatment description
Tumor growth in nude mice pretreated with cyclophosphamide
Lansford et al
Grows as sharply demarcated, compact islands of cells with a distinct epithelial morphology, well differentiated, and with numerous keratinized epithelial pearls. Supernatant factor induces activation, promotes proliferation, and increases antitumor cytotoxicity of natural killer cells and CD4+ T lymphocytes Continued on next page
Table 2. (continued) Prior RX Treatment description
Original pathology
UT-SCC-13 AMC-HN-1
RT none
Laryngeal papilloma prior to cancer
AMC-HN-2
S/RT/CX
AMC-HN-3
none
AMC-HN-4
S/RT/CX
AMC-HN-5
S/RT
75 Gy 5 months
In vitro morphology
Nude mouse morphology
Epithelioid to spindle-shaped cells, relatively little cell-to-cell contact, grows mostly as independent cells until quite confluent, easily detached from flask bottom, tendency to form cell balls when confluent Epithelioid to spindle-shaped cells, relatively little cell-to-cell contact, grows mostly as independent cells until quite confluent, easily detached from flask bottom, tendency to form cell balls when confluent Epithelioid cells that grow in islands with extensive cell-to-cell contact. Grow in monolayers Epithelioid cells that grow in islands with extensive cell-to-cell contact. Grow in monolayers Epithelioid to spindle-shaped cells, relatively little cell-to-cell contact, grows mostly as independent cells until quite confluent, easily detached from flask bottom, tendency to form cell balls when confluent
Recreates histology of original tumor in nude mouse
Head and Neck Cancers
Cell line
Recreates histology of original tumor in nude mouse
Recreates histology of original tumor in nude mouse, includes keratin pearls Recreates histology of original tumor in nude mouse Recreates histology of original tumor in nude mouse
237 Continued on next page
238
Table 2. (continued) Cell line Prior RX Treatment description AMC-HN-6
none
AMC-HN-7
S/RT
AMC-HN-8
SET
AMC-HN-9
none
UD-SCC-1
Original pathology
In vitro morphology
Nude mouse morphology
Continued on next page
Lansford et al
Epithelioid cells that grow in Recreates histology of islands with extensive cell-to-cell original tumor in nude mouse. Includes well formed contact with closely packed infiltrating cords with a cells. Has the capacity to prominent stromal network stratify into thick layers of differentiated cells Epithelioid cells that grow in Recreates histology of islands with extensive cell-to-cell original tumor in nude contact. Grow in monolayers mouse Epithelioid cells that grow in islands with extensive cell-to-cell contact. Grow in monolayers Consists mostly of spindle-shaped Recreates undifferentiated carcinoma histology of cells which do not form islands original tumor in nude mouse even when confluent. Cells are easily detached from the flask Failure to produce tumors Nonstratifying, adherent monolayer cells, sharply demarcated in after xenotransplantation into compact islands. Moderate to nude mice in repeated assays well differentiated SCC histology. Tightly packed cuboidal cell colonies, high N:C ratio, prominent nucleoli
Prior RX Treatment description
Original pathology
Nonstratifying, adherent monolayer cells, sharply demarcated in compact islands. Moderate to well differentiated SCC histology. Small colonies with an elongated spread-out appearance. Loss of cell-to-cell adhesion with individual migrating single cells Nonstratifying, adherent monolayer cells, sharply demarcated in compact islands. Moderate to well differentiated SCC histology. Cuboidal cells in small islands resembling a cobblestone relief Nonstratifying, adherent monolayer cells, sharply demarcated in compact islands. Moderate to well differentiated SCC histology. Large islands of cuboidal cells with occasionally spreading cells at the edge of the islands
UD-SCC-2
UD-SCC-3
S/ND/RT
UD-SCC-4
NPC/HK1
XRT
In vitro morphology
>72 passages. Doubling time 28 hrs. Polygonal cells grow in monolayer in a mosaic pattern with squamous differentiation
Slowly growing tumor in nude mice at 6 wks post sub-renal capsule implant. Solid tumor, frequent mitoses, neovascularization and invasion into normal tissues. Histological appearance generally similar to original biopsy specimen Sub-renal capsule injection into nude mice revealed tumor by subrenal capsule assay only
Slowly growing tumor in nude mice at 6 wks post sub-renal capsule implant. Solid tumor, frequent mitoses, neovascularization and invasion into normal tissues. Histological appearance generally similar to original biopsy specimen Nude mouse xenograft produced a solid 1 cc tumor histologically similar to the biopsy specimen Continued on next page
239
6000 rads to nasopharynx Hong Kong native with total response for 17.5 years. Recurrence noted 1 month prior to bx
Nude mouse morphology
Head and Neck Cancers
Table 2. (continued) Cell line
Cell line
Prior RX Treatment description
Original pathology
NPC-KT
none
Japanese woman with a 2 month >36 passages. Cells grow in history of left neck mass, which monolayer and are larger than the extended from the left nasopharynx Ad-AH cells used to create the into the left nasal septum. EBNA hybrid touch smear (+). Pt. serology: antiVCA/IgA 1:320 (+), and /IgG 1:2560 (+), EA/IgG 1:320, EA/IgA 1:80
c15
none
Moroccan girl with locally advanced No in vitro line tumor and early metastatic spread. Pt. serology: anti-VCA/IgA 1:20, and /IgG 1:2560, IgG EA 1:640, IgA EA 1:10
C17
XRT, CTX
No in vitro line Biopsy taken with bone marrow, liver, and skin mets present 4 years after Dx in this Frenchman. Pt. serology: anti-VCA/IgA 1:40, and /IgG 1:1280, IgG EA 1:160, IgA EA 1:10
Nude mouse morphology Inoculation into nude mice irradiated (5 Gy) prior to implant, drinking water with 0.5 nM estrone. Tumor 1-2 cm3 after 5-7 weeks. NPC cells with no infiltrating mouse cells. No intercellular bridges or keratin. Weak EBNA expression Inoculation into previously irradiated (5 Gy) nude mice, drinking water supplemented with 0.5 nM estrone from inoculation time. 1–2cc tumors recovered 5-7 weeks with loss of non-malignant lymphocytes and homogeneous large syncytial cells with clear nuclei, EBNA+ + Inoculation into previously irradiated (5 Gy) nude mice drinking water supplemented with 0.5 nM estrone from inoculation time. 1-2cc tumors recovered 5-7 weeks with elimination of fibroblasts. No mouse cell infiltration, EBNA+ Continued on next page
Lansford et al
Primary XRT to head and neck followed by bone and skin metastatic relapse treated with bleomycin, cis-platinum 5-FU
In vitro morphology
240
Table 2. (continued)
Cell line
Prior RX Treatment description
C18
CTX
CG1
none
CNE1
none
Chinese patient
CNE2 HNE- 1
none none
Chinese patient
NM 8 HNE-3 HONE-I
none
Original pathology
In vitro morphology
No in vitro line 2mos of bleomycin, cis- Locally advanced cancer with cervical nodal involvement with platinum, 5-FU extra-capsular spread in this Algerian. Pt. serology: anti-VCA/IgA 1:160, and /IgG 1:640, IgG EA 1:320, IgA EA 1:160
>200 generations. Doubling time 20 hrs
Nude mouse morphology Inoculation into previously irradiated (5 Gy) nude mice, drinking water supplemented with 0.5 nM estrone from inoculation time. 1–2cc tumors recovered 5-7 weeks with no infiltrating mouse stromal cells. Weak EBNA Nude mouse xenograft produced a solid tumor mass >1 cc Nude mouse xenograft produced relatively well differentiated squamous pearls and epithelial cell junctions
> 100 passages. Doubling time 45 hrs
Uncloned cell line produced poorly differentiated SCC tumors in 3/3 nude mice
> 90 passages. Doubling time 64 hrs
Uncloned cell line produced poorly differentiated SCC tumors in 6/10 nude mice
241
Continued on next page
Head and Neck Cancers
Table 2. (continued)
none
NPC-TW01N1
none
NPC-TW02
none
NPC-TW03
none
NPC-TW04
none
In vitro morphology
Nude mouse morphology
>100 passages. Doubling time Nude mouse xenograft 10.5 hrs. Spindle morphology with produced a large multilobulated solid tumor mass. bipolar and tripolar processes This mass was moderately undifferentiated SCC with minimal keratinization. Distant metastases to lung, diaphragm, and liver were undifferentiated These cells differ from NPC-TW01 by their multiple nucleoli > 100 passages. Doubling time Nude mouse xenograft 10.8 hrs. Spindle morphology with produced two solid tumor masses with distant metastasis. bipolar and tripolar processes The mass was undifferentiated, in contrast to the primary tumor Nude mouse xenograft 4 month history of palpable mass >100 passages. Bi- or tripolar produced a WHO type IIA spindle cells at low confluence with bony destruction before excision. Large vesicular nuclei with becoming polygonal at confluence solid tumor mass with tumor eosinophilic, prominent nucleoli and emboli indistinct plasma membrane with heavy lymphocytic infiltration. Pt. serology: anti-EBNA (+), antiVCA/IgA 1:20 (+) 3 month history of neck mass and > 100 passages. Bi- or tripolar Nude mouse xenograft spindle cells at low confluence produced a WHO type IIA bloody rhinorrhea. Mild lymphocytic infiltration. Pt serology: anti- becoming polygonal at confluence solid tumor mass with tumor emboli EBNA (+), anti-VCA/IgA 1:10 (+), and /IgG 1:640 (+) Continued on next page
Lansford et al
NPC-TW01
Original pathology
242
Table 2. (continued) Cell line Prior RX Treatment description
NPC-TW05
none
NPC-TW06
none
NPC-TW07
none
NPC-TW08
none
NPC-TW09
none
Original pathology
In vitro morphology
2 year history of AU stiffness, tinnitus, L headache and 1 year history of bilateral neck masses. Pt. serology: anti-EBNA (+), antiVCA/IgA 1:160 (+), and /IgG 1:1280 (+) 3 year history of intermittent epistaxis. Mild lymphocytic infiltration. Pt. serology: anti-EBNA (t) and anti-VCA/IgA (-), and /IgG 1:320 (+) 4 month history of R tinnitus, exophthalmos, ptosis. Some cells showed spindle cell transformation. Less lymphocytic infiltration 1 month history of neck mass and hoarseness. Pt. serology: anti-EBNA (t), anti-VCA/IgA 1:10 (+), and /IgG 1:230 (+)
Nude mouse xenograft > 100 passages. Bi- or tripolar spindle cells at low confluence produced a WHO type I solid becoming polygonal at confluence tumor mass with marked keratin pearl formation
Nude mouse morphology
Nude mouse xenograft > 100 passages. Bi- or tripolar spindle cells at low confluence produced a WHO type IIA becoming polygonal at confluence solid tumor mass with tumor emboli
Head and Neck Cancer
Table 2. (continued) Cell line Prior RX Treatment description
> 100 passages. Bi- or tripolar Nude mouse xenograft produced a WHO type IIA spindle cells at low confluence becoming polygonal at confluence solid tumor mass
Nude mouse xenograft > 100 passages. Bi- or tripolar spindle cells at low confluence produced a WHO type I becoming polygonal at confluence solid tumor mass with marked keratin pearl formation and tumor emboli Nude mouse xenograft 2 month history of neck mass. > 100 passages. Bi- or tripolar Mild lymphocytic infiltration. Pt. spindle cells at low confluence produced a WHO type IIA serology: anti-EBNA (+), antibecoming polygonal at confluence solid tumor mass with tumor emboli early antigen/IgA 1:10 (-), and /IgG 1:10 (t)
243
multiple lines available
UM-SCC-1 UM-SCC-2 UM-SCC-3 UM-SCC-4 UM-SCC-5 UM-SCC-7 UM-SCC-8 UM-SCC-9 UM-SCC-10A
yes
UM-SCC-10B UM-SCC-11A
yes yes
UM-SCC-11B
yes
UM-SCC-14A
yes
UM-SCC-14B
yes
UM-SCC-14C
yes
Genetics
HPV subtypes: 6 (-), 16 (-), 18 (-), 31 (-), Wt p53 present. HPV subtypes: 6 (-), 16 (-), 18. HPV subtypes: 6 (-), 11 (-), 16 (-), 18 (-), 31 (-). HPV subtypes: 6 (-), 16 (-), 18 (-). HPV subtypes: 6 (-), 16 (-), 18 (-). HPV subtypes: 6 (-), 16 (-), 18 (-). HPV subtypes: 6 (-), 16 (-), 18 (-), 31 (-), Hypotetraploid. 3p loss. HPV subtypes: 6 (-), 16 (-), 18 (-). HPV subtypes: 6 (-), 16 (-), 18 (-). No wt p53 present. p53 mutation: GGC->TGC (Gly->Cys) in codon 245. Hypotetraploid. 18q loss: monosomy. No 3p loss. DCC: homozygous. Karyotyped. No wt p53 present. p53 mutation: GGC->TGC (Gly->Qs) in codon 245. HPV subtypes: 6(-), 11 (-), 16 (-), 18 (-). Hypotetraploid. 18q loss: qll.1-qter. No 3p loss. Karyotyped. No Wt p53 present. p53 mutation: TGC->TCC (Cys->Ser) in codon 242. HPV subtypes: 6(-), 16 (-), 18 (-), 31 (-). Hyperdiploid. 18q loss: monosomy. No 3p loss. No Wt p53 present. p53 mutation: TGC–>TCC (Cys–>Ser) in codon 242. Karyotyped. Hypotetraploid. 18q loss: q11.2-q22. DCC homozygous. No 3p loss. No Wt p53 present. p53 mutation: AGA –> AGT (Arg –> Ser) in codon 280 and a 30 bp deletion in exon 8, codons 277-287. Karyotyped. Hypotetraploid. 18q loss: q11.2-q22. DCC: homozygous. 3p loss: p26-pter. No Wt p53 present. p53 mutation: AGA –> AGT (Arg –> Ser) in codon 280. Karyotyped. 18q loss: q11.2-q22.3p loss: p14-pter. No Wt p53 present. p53 mutation: AGA –> AGT (Arg –> Ser) in codon 280. Karyotyped. 18q loss: q11.2-q22. Karyotyped. No Wt p53 present. p53 mutation: CGG–>CTG (Arg->Leu) in codon 248. HPV subtypes: 6 (-), 11 (-), 16 (-), 18 (-), 31 (-), PCR with consensus HPV primer (-). Hypotetraploid. 18q loss: 2-18s/4. 3p loss: p13-pter. Karyotyped. Continued on next page
Lansford et al
UM-SCC-15 UM-SCC-16
244
Table 3 Genetic changes and other known molecular characteristics of head and neck squamous cancer cell lines line name
multiple lines available
Genetics
UM-SCC-17A
yes
UM-SCC-17B UM-SCC-19
yes
HPV subtypes: 6 (–), 11 (–), 16 (–), 18 (–), 31 (–). Hyperdiploid. No 18q loss. DCC: homozygous. Only Wt p53 present. Karyotyped. HPV subtypes: 6 (–), 16 (–), 18 (–), 31 (–). Hyperdiploid. 18q loss, q21.1-q23. No 3p loss. Karyotyped. No wt p53 present. p53 mutation: 10 base pair deletion (frame shift) at codons 148-151. HPV subtypes: 6 (–), 11 (–), 16 (–), 18 (–), 31 (–), PCR with consensus HPV primer (+). HPV subtypes: 6 (–), 11 (–), 16 (–), 18 (–), 31 (–). PCR with consensus HPV primer (–). Near diploid. 18q loss: monosomy. 3p loss: p13-p22. Karyotyped. No 18q loss. No DCC loss. No 3p loss. Karyotyped. Near diploid. No 18q loss. DCC: homozygous. 3p lost. Karyotyped. Near diploid. No 18q loss. 3p lost. No Wt p53 present. p53 mutation: TAT -> TGT (Tyr -> Cys) in codon 220. Karyotyped. HPV subtypes: 6 (–), 16 (–), 18 (–), 31 (+). Wt p53 present. p53 mutation: TGC->TTC (Cys->Phe) in codon 176. 18q loss: q11.2-q23. Karyotyped. HPV subtypes: 6 (–), 11 (–), 16 (–), 18 (–), 31 (–). HPV 16 present, 6 (–), 11 (–), 18 (), 31 (–). HPV subtypes: 6 (–), 16 (–), 18 (–), 31 (–). Hypertriploid/hypodiploid. DNA index 1.64. 18q loss: q11.1-qter. DCC. homozygous. 3p lost. Karyotyped. HPV subtypes: 6 (–), 11 (–), 16 (–), 18 (–), 31 (–). Wt p53 present. p53 mutation: TGC->TTC(Cys->Phe). HPV subtypes: 6 (–), 11 (–), 16 (–), 18 (+), 31 (+), PCR with consensus HPV primer (t). HPV subtypes: 6 (–), 11 (–), 16 (-), 18 (–), 31 (–), PCR with consensus HPV primer (–). 18q loss: q11.2-q22. Karyotyped. 18q loss: q22-qter. Karyotyped. LOH in 18q21.1, no loss in q11.1 (tumor specimen shows corresponding LOH in q21.1). Hypertetraploid. 18p loss: p11.3-pter. No 3p loss. Karyotyped.
UM-SCC-21A UM-SCC-21B UM-SCC-22A UM-SCC-22B UM-SCC-23
yes yes
UM-SCC-33 UM-SCC-47 UM-SCC-38 UM-SCC-49 UM-SCC-63 UM-SCC-69 UM-SCC-80 UM-SCC-81B UM-SCC-82A
yes
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Continued on next page
Head and Neck Cancer
Table 3 (continued) line name
Genetics
UM-SCC-83A UM-SCC-83B UM-SCC-86 UM-SCC-87 UM-SCC-89 UM-SCC-90 UM-SCC-91 UM-SCC-93 UM-SCC-94 UM-SCC-101A UM-SCC-101B SCC-12B.2 SCC-25 SCC-35 SCC-61 JSQ-3 SQ-9G SQ-20B SQ-31 SQ-38 HN-SCC-135 HN-SCC-15 1 HN-SCC-167 HN-SCC-294 MDA-183
yes yes
No gross 18q loss, smaller regions of loss noted on extensive evaluation. No loss in 18q11.1. LOH 18q12.1-q23. No 18q loss in cell line or tumor. LOH in 18q11.1, q12.2-12.3, q23 (tumor showed AI/LOH for these markers). Tumor and line show no loss in 18q11.1, q12.2-12.3, q23. Line has LOH in 18q21.1, q23. Tumor is LOH/AI for these markers. Tumor and line show no loss at 18q23, MSI at 18q11.1. Line shows LOH at 18q11.1, q21.1, q23. Tumor shows AI/LOH at these markers. Tumor and line show no loss at 18q11.1, 12.2-12.3, q23. LOH 18q11.1-18q23. No loss 18q. DNA index 1.88. Diploid. DNA index 1.93. 72 chromosomes. Triploid. 65 chromosomes. Diploid. DNA index 1.27. 46 chromosomes. Diploid. DNA index 2.88. 57 chromosomes. DNA index 1.53. Triploid. DNA index 1.73. 68 chromosomes. Tetraploid. 85 chromosomes. DNA index 2.64. Triploid. Tetraploid. 82 chromosomes. Triploid. 70 chromosomes. Tetraploid. 80 chromosomes. HPV negative. p53 mutations: (1) C–>T (Pro–>Ser) in exon 5, codon 151. (2) mutation in exon 7 by SSCP. Rb negative by Western. Continued on next page
yes yes
Lansford et al
multiple lines available
246
Table 3 (continued) line name
multiple lines available
MDA-1386 MDA-1483
Yes yes yes yes
yes
yes yes
yes yes
HPV negative. p53 mutation: exon 8 mutated by SSCP Rb negative by Western. HPV 18(+). p53 mutations: (1) A–>A+G (Tyr–>Tyr+Cys) in exon 5, codon 126. (2) G–>G+A in intron 5, position 1, maybe causing alternative splicing. Rb negative by Western. HPV 16 (+), 18 (t). p53 wt by SSCP. del(3) (p11), der(9)t(9;11) (p13;q14) del(3) (p11), +6, der(12)t(9;12) (q12;p11) del(3) (p11), +6 +6, der(12) del(3) (p11), +6, der(12), small marker. PCI-4A has 45 chromosomes and PCI-4B has 80. del(3) (p11), +6, small marker del(3) (p11), +6, der(12), small marker del(3) (p11), +6, small marker del(3) (p11), +6, der(9)t(9;11) (p13;q14), small marker del(3) (p11), +6, der(9)t(9;11) (p13;q14) +6, der(9)t(9;11) (p13;q14) del(3) (p11), +6, small marker del(3) (p11), der (9)t(9;11) (p13;q14), small marker del(3) (p11), small marker +6, der(12)t(9;12) (q12;p11), small marker No wt p53 present. p53 mutation: CGA–>TGA (Arg->Stop) in exon 6, codon 196. (t) Bcl–2 expression. No wt p53 present. p53 mutation: CGA–>TGA (Arg->Stop) in exon 6, codon 196. No wt p53 present. p53 mutation: TGT–>TTT (Cys–>Phe) in exon 8, codon 275. No wt p53 present. p53 mutation: 9 base pair deletion in exon 7, codon 248-250. No wt p53 present. p53 mutation: CCC->CAT (Pro->His) in exon 5, codon 151. Wt p53 present. p53 mutation: one allele lost (LOH) (+) Bcl–2 expression. Wt p53 present. p53 mutation: one allele lost (LOH). Continued on next page
247
MDA-1986 PCI-1 PCI-2 PCI-3 PCI-4A PCI-4B PCI-5 PCI-6A PCI-6B PCI-7 PCI-9A PCI-9B PCI-10 PCI-11 PCI-12 PCI-13 UT-SCC-1A UT-SCC-1B UT-SCC-2 UT-SCC-4 UT-SCC-5 UT-SCC-6A UT-SCC-6B
Genetics
Head and Neck Cancer
Table 3 (continued) line name
248
Table 3
(continued)
line name
multiple lines available
UT-SCC-7 UT-SCC-8 UT-SCC-9 UT-SCC-10 UT-SCC-11 UT-SCC-12A UT-SCC-12B UT-SCC-13
yes yes
UT-SCC-14 UT-SCC-15 UT-SCC-16A
yes
UT-SCC-16B
yes
UT-SCC-17 yes yes yes yes
Wt p53 present. p53 mutation: GGA->GAA (Gly–>Glu) in exon 8, codon 266. No wt p53 present. p53 mutation: ATC–>TTC (Ile–>Phe) in exon 7, codon 255. (+) Bcl–2 expression. No p53 (wt or mutant) present. No Bcl–2 expression. No wt p53 present. p53 mutations: CAG->TAG (Gln–>stop) in exon 5, codon 144, and CGA–>TGA (Arg->stop) in exon 8, codon 306. (+) Bcl–2 expression. No wt p53 present. p53 mutations: 30 base pair deletion exon 6, codons 187-197 (in-frame mutation). No wt p53 present. p53 mutation: CGA–>TGA (Arg–>stop) in exon 10, codon 342. No wt p53 present. p53 mutation: CGA–>TGA (Arg–>stop) in exon 10, codon 342. No wt p53 present. p53 mutation: 10 base pair deletion + 1 base pair insertion exon 6, codons 205-208 (deletion & frame shift mutation). Normal wt p53 present. Normal wt p53 present. No wt p53 present. p53 mutations: CGT–>TGT (Arg–>Cys) in exon 4, codon 110, and ATC–>AAC (Ile–>Asn) in exon 7, codon 232. One Bcl–2 allele deletion. No wt p53 present. p53 mutations: CGT–>TGT (Arg–>Cys) in exon 4, codon 110, and ATC–>AAC (Ile–>An) in exon 7, codon 232. No wt p53 present. p53 mutations: CGT–>CTT (Arg->Leu) in exon 4, codon 110, and CTG–>CAG (Leu–>Gin) in exon 7, codon 257. Wt p53 present. p53 mutation: 3 bp insertion (Ile) at exon 7, codons 254-256. (-) Bcl–2 expression. No wt p53 present. p53 mutation: GAG–>AAG (Glu–>Lys) in exon 8, codon 285. (+) Bcl–2 expression. No wt p53 present. p53 mutation: GAG–>AAG (Glu–>Lys) in exon 8, codon 285. No wt p53 present, p53 mutation: CGG–>TCG (Arg–>Trp) in exon 7, codon 248. No wt p53 present, p53 mutation: CGG–>TCG (Arg–>Trp) in exon 7, codon 248. No wt p53 present. p53 mutation: GAG–>AGG (Glu–>Lys) in exon 8, codon 285. Wt p53 present. p53 mutation: TGT–>TTT (Cys–>Phe) in exon 7, codon 238. Continued on next page
Lanford et al
UT-SCC-18 UT-SCC-19A UT-SCC-19B UT-SCC-20A UT-SCC-20B UT-SCC-21 UT-SCC-22
Genetics
line name
UT-SCC-23 UT-SCC-24A UT-SCC-24B UT-SCC-25 UT-SCC-26A UT-SCC-26B UT-SCC-27 UT-SCC-28 UT-SCC-29 UT-SCC-30 UT-SCC-31 UT-SCC-32 UT-SCC-33 UT-SCC-34 UT-SCC-35 UT-SCC-36 UT-SCC-39 UT-SCC-40 EV-SCC-1 EV-SCC-2 EV-SCC-3 EV-SCC-4 EV-SCC-7 EV-SCC-10M EV-SCC-14M
multiple lines available yes yes yes yes
Genetics
Normal wt p53 present. Normal wt p53 present. Normal wt p53 present. (+) Bcl–2 expression. No wt p53 present. p53 mutation: CGG–>TGG (Arg–>Trp) in exon 7, codon 248. No wt p53 present. p53 mutation: TAC–>TAA (Tyr–>stop) in exon 7, codon 236. No wt p53 present. p53 mutation: TAC–>TAA (Tyr–>stop) in exon 7, codon 236. Wt p53 present. p53 mutation: TGC->TTC (Cys–>Phe) in exon 7, codon 242. No wt p53 present. p53 mutation: CGT–>TGT (Arg–>Cys) in exon 8, codon 273. No wt p53 present. p53 mutation: CAG->TAG (Gln->stop) in exon 4, codon 104. One Bcl–2 allele deletion. No wt p53 present. p53 mutation: CGG–>CCG (Arg->Pro) in exon 8, codon 282. No wt p53 present. p53 mutation: CAA–>TAA (Gln->stop) in exon 4, codon 52. No wt p53 present. p53 mutation: GGA->GAA (Gly–>Glu) in exon 8, codon 266. No wt p53 present. p53 mutation: CGG–>TGG (Arg–>Trp) in exon 8, codon 282. Normal wt p53 present. No wt p53 present. p53 mutation: CGT–>TGT (Arg–>Cys) in exon 7, codon 245. No wt p53 present. p53 mutation: GGC–>AGC (Gly–>Ser) in exon 7, codon 244. No p53 mutation detected. No wt p53 present. p53 mutation: 30 base pair deletion in exon 8, codons 277-287. No wt p53 present. p53 mutation; CGA–>TGA (Arg->stop) in exon 6, codon 213. p53 wt present. p53 mutation: CGG–>CTG (Arg->Leu) in exon 7, codon 248. p53 mutation: 2 bp (TT) addition in exon 8, codon 289-290. p53 mutation in exon 6 (by SSCP). p53 mutation in exon 6 (by SSCP). p53 mutation in exon 5 (by SSCP).
249
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Head and Neck Cancer
Table 3 (continued)
250
Table 3 (continued) line name
Genetics
p53 mutation in exon 8 (by SSCP). p53 mutation in exon 8 (by SSCP). p53 mutation in exon 5,6 (by SSCP). p53 mutation in exon 5,6 (by SSCP). p53 mutation in exon 8 (by SSCP). 18p loss: p11.1-pter. Karyotyped. 18q loss: q11.2-qter. Karyotyped. 18p loss: p11.1-q11.2, q22-qter. Karyotyped. No 18q loss. Karyotyped. No 18q loss. 18q loss: q11.2-pter. Karyotyped. 18q loss: q11.2-pter. DCC not tested. Karyotyped. No 18q loss. Karyotyped. No DCC loss. No 18q loss. Karyotyped. No 18q loss. Karyotyped. No 18q loss. Karyotyped. 18q loss: 2-18s/4. Karyotyped. 18q loss: q11.2-qter. Karyotyped. No 18q loss. Karyotyped. Hyperdiploid. DNA index 2.41. Diploid. DNA index 1.37. Diploid. DNA index 1.45. Hyperdiploid. DNA index 1.6. Hyperdiploid. DNA index 2.13. Continued on next page
Lansford et al
EV-SCC-17P EV-SCC-18 EV-SCC-19P EV-SCC-19M EV-SCC-17M HFH-SCC-3 HFH-SCC-4 HFH-SCC-6 HFH-SCC-8 HFH-SCC-11 HFH-SCC-12 HFH-SCC-15 HFH-SCC-16 HFH-SCC-17 HFH-SCC-19 HFH-SCC-20 HFH-SCC-28 HFH-SCC-29 HFH-SCC-33 HFH-SCC-42 AMC-HN-1 AMC-HN-2 AMC-HN-3 AMC-HN-4 AMC-HN-5
multiple lines available
line name
AMC-HN-6 AMC-HN-7 AMC-HN-8 UD-SCC-1 UD-SCC-2 UD-SCC-3 UD-SCC-4 UD-SCC-5 UD-SCC-6 TU-138 TU-159 TU-167 TU-177 TU-182 Tu-212 TU-686 (MDA?) Tu-158 (LN) TU-212 (LN) NPC/HK1 NPC-KT C15 C17 C18 2117 CG1
multiple lines available
Genetics
Diploid. DNA index 1.38. Diploid. DNA index 1.52. Hyperdiploid. DNA index 1.98. p53 mutation: Exon 3 deleted (codons 25-32). Stop amino acid 43/44; Wt p53 present. Karyotyped. Hypotriploid aberrant. Only Wt p53 present. p53 LOH. Karyotyped. No Wt p53 present. p53 mutation: Glu (GAG) –> STOP (TAG) in codon 224. Karyotyped. No Wt p53 present. p53 mutation: Deletion of 13 nucleotides in codons 150-154. Stop at 169/170. No Wt p53 present. p53 mutation: Codon 179 His (CAT) –> Tyr (TAT). No Wt p53 present. p53 mutation: Codon 220 Tyr (CAT) –> Cys (TGT). p53 mutation: C–>T (Pro–>Ser) in exon 5, codon 151. Rb negative by Western. Rb negative by Western. p53 mutation: G–>A in intron 1, position 1, causing alternative splicing. Rb positive by Western. p53 mutation: C–>T (Pro–>Ser) in exon 5, codon 151. Rb negative by Western. p53 mutation: C–>T (Pro–>Ser) in exon 5, codon 151. Rb negative by Western. p53 mutation: C–>T (Pro–>Ser) in exon 5, codon 151. Rb negative by Western. p53 mutation: C–>T (Pro–>Ser) in exon 5, codon 151. Rb negative by Western. p53 mutation: C–>T (Pro–>Ser) in exon 5, codon 151. Rb negative by Western. EBV (-). Numerical and structural abnormalities on chromosome analysis. EBNA positivity stable at 94-987% from passage 4 to 36. EBV (+), 30 copies. No EA, VCA, or MA complexes or EBV particles detected (non-lytic). No mouse chromosomes. EBV (+), 12 copies. No EA, VCA, or MA complexes or EBV particles detected (non-lytic). No mouse chromosomes. EBV (+), 3 copies. No EA, VCA, or MA complexes or EBV particles detected (non-lytic). No mouse chromosomes. EBV (+). EBV (+). Karyotyped.
251
Continued on next page
Head and Neck Cancers
Table 3 (continued)
line name
CNE1 CNE2 HNE-1 NM 8 HONE-I NPC-TW01 NPC-TW02 NPC-TW03 NPC-TW04 NPC-TW05 NPC-TW06 NPC-TW07
NPC-TW09
Genetics
EBV (-). EBV (-). EBV (+). EBV lost after passage 42. Karyotyped. EBV (+). EBV (+). EBV lost after passage 42. EBV (+) by PCR in earlier passages and (-) in later passages, HPV-16 E2 and E6 (+) by PCR in earlier passages and (-) in later passages, HCMV (+) in both earlier and later passages, adenovirus (-). No specific c-fgr, c-fos, or v-sis expression. Karyotyped. EBV and HPV-16 E6 by PCR in both earlier passages and later passages, HPV-16 E2 (+) in earlier but (-) in later passages, HCMV (+) by PCR in both earlier and later passages, adenovirus (-). No specific c-fgr, c-fos, or v-sis expression. EBV and HPV-16 E2 & E6, and HCMV (+) by PCR in earlier passages, but EBV and HPV-16 E2 and E6 became (-) while HCMV remained (+) by PCR in later passages. Karyotyped. Multiple chromosome abnormalities. EBV, HPV-16 E2 & E6, and HCMV (-) by PCR in both earlier and later passages. Karyotyped. Multiple chromosome abnormalities. EBV (+) by PCR and in-situ hybridization, HPV-16 E2 (+), HPV-16 (-), and HCMV (+) by PCR in early passages. Later passages were EBV (-), HPV-16 E2 & E6 (-), and HCMV (+) by PCR. Karyotyped. Multiple chromosome abnormalities. EBV (+) by PCR and in-situ hybridization, HPV-16 E2 (+), HPV-16 (-), and HCMV (+) by PCR in early passages. Later passages were EBV (-), HPV-16 E2 (+),HPV-16 E6 (-), and HCMV (+) by PCR. Karyotyped. Multiple chromosome abnormalities. EBV (+) by PCR and in-situ hybridization, HPV-16 E2 (+), HPV-16 E6 (-), and HCMV (+) by PCR in early passages. Later passages were EBV (-), HPV-16 E2 (+), HPV-16 E6 (-), and HCMV (+) by PCR. Karyotyped. Multiple chromosome abnormalities. EBV (+), HPV-16 E2 (+), HPV-16 E6 (-), and HCMV (+) by PCR in early passages. Later passages were EBV (-), HPV-16 E2 (t), HPV-16 E6 (-), and HCMV (-) by PCR. Karyotyped. Multiple chromosome abnormalities. EBV (-) by PCR, HPV-16 E2 & E6 (+), and HCMV (-) by PCR in early and later passages. Karyotyped. Multiple chromosome abnormalities.
Lansford et al
NPC-TW08
multiple lines available
252
Table 3 (continued)
Head and Neck Cancers
253
REFERENCES Ballo H, Koldowsky P, Hoffmann T, Balz V, Hildebrandt B, Gerharz CD, Bier H. Anticancer Res 19: 2356-2366,1999. Beckhardt RN et al. Arch Otolalyngol Head Neck Surg 121: 1265-1270, 1995. Blot WJ, McLaughlin JK, Devesa SS, Fraumeni JF. In: Cancer Epidemiology and Prevention, 2nd Edition. Ed. Schottenfeld, D. 1994. Buchhagen DL, Worsham MJ, Van Dyke DL, Carey TE. Head & Neck 18: 529-537, 1996. Busson P et al. Int J Cancer 42: 599–406, 1988. Carey TE. In: Head and Neck Cancer, Ed. Wittes RE. Vol 2. Wiley & Sons, New York. pp 287–314,1985. Carey TE. In: Atlas of Human Tumor Cell Lines. Hay R, Gazdar A and Park J-G (eds.). Academic Press, Inc., Harcourt Brace Jovanovich, Orlando, FL, pp. 79–120, 1994. Carey TE, Van Dyke DL, Worsham MJ, Bradford CR, Babu VR, Schwartz DR, Hsu S and Baker SR. Cancer Res 49: 6098–6107,1989. Carey TE, Kimmel KA, Schwartz DR, Richter DE, Baker SR, Krause CJ. Otolaryngol Head Neck Surg 91: 482–491, 1983. Carey TE, Van Dyke DL, Worsham MJ. Anticancer Res 13: 2561–2568,1993a. Carey TE, Worsham MJ, Van Dyke DL. J Cell Biochem Suppl 17F: 213–222, 1993b. Chang YS et al. Cancer Res 49: 6752–6757,1989. Colburn NH et al. Int J Cancer 44: 1012–1016, 1989 Cowan JM, Beckett MA, Ahmed-Swan S, Weichselbaum RR. J Natl Cancer Inst 84: 793–797, 1992. Day GL, Blot WJ, Austin DF, Bernstein L, Greenberg RS, Preston-Maritn S, Schoenberg JB, Winn DM, McLaughlin JK, Fraumeni JF. J Natl Cancer Inst 85: 465–73, 1993. Dunphy EJ, Beckett MA, Thompson LH, Weichselbaum RR. Radiation Research 130: 166–170, 1992. Eagle H. Science 122: 501-504,1955. Easty DM, Easty GC, Carter RL, Monaghan P, Butler LJ. Br. J. Cancer 43: 772-785, 1981. Elornaa L, Joensuu H, Kulmala J, Klemi P, Grknman R. Acta Otolayngol 115: 340-344,1995. Frank CJ, McClatchey KD, Devaney KO, Carey TE. Cancer Res 57: 824–827, 1997. Gartler SM. Natl Cancer Inst Monog 26: 167–195, 1973. Gey GO, Coffman WD, Kubicek MT. Cancer Res 12: 264–265, 1952 Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH, Dosik H, Parks WP. J Natl Cancer Inst 51: 1417–1423,1973, Grénman R, Carey TE, McClatchey KD, Wagner JG, Pekkola-Heino K, Schwartz DR, Wolf GT, Lacivita LP, Ho L, Baker SR, Krause CJ, Lichter AS. Cancer 67: 2741–2747, 1991. Grknrnan SE, Van Dyke DL, Worsharn MJ, England B, McClatchey KD, Hopkins M, Babu VR, Grenman R, Carey TE. Int J Cancer 45: 920-927, 1990. Grénman R, Pekkola-Heino K, Joensuu H, Aitasolo K, Klemi P, Lakkala T Arch Otolalyngol Head Neck Surg 118: 542–547, 1992. Heo DS, Snyderman C, Gollin SM, Pan S, Walker E, Deka R, Barnes EL, Johnson JT, Herberman RB, Whiteside TL. Cancer Res 49: 5167-5175, 1989. Heo DS, Park J-G, Hata K, Day R, Herberman RB, Whiteside TL. Cancer Res 50: 3681-3690, 1990. Huang DP et al. Int J Cancer 26: 127-132,1980. Johansson N, Airola K, Grknman R, Kariniemi A-L, Saarialho-Kere U, Kahari V-M. Am J Pathol 151: 499–508, 1997. Jones JW, Raval JR, Beals TF, Worsham MJ, Van Dyke DL, Esclamado RM, Wolf GT, Bradford CR, Miller T, Carey TE. Arch Otolaryngol Head Neck Surg 123: 610–614, 1997.
254
Lansford et al.
Kelker W, Van Dyke DL, Worsham MJ, Christopherson PL, James CD, Conlon MR, Carey TE. Anticancer Research 16: 2365-2372,1996. Kim SY, Chu KC, Lee HR, Lee KS, Carey TE. Acta Otolaryngol 117: 775-784, 1997. Kiuru A, Servomaa K, Grénman R, Pulkkinen J, Rytömaa T. Acta Otolaryngol (Stockh) suppl 529: 237–240,1997, Komiyama S, Matsui K, Kudoh S, Nogae I, Kuratomi Y, Saburi Y, Asoh KI, Kohno K, Kuwano M. Cancer 63: 675–681,1989. Krause CJ, Carey TE, Ott RW, Hurbis C, McClatchey KD, Regezi JA. Arch. Otolaryngol 107: 703–710,1981. Lavappa KS. In Vitro 14: 469–475,1978. Leibovitz A. (1973). Reported in: ATCC Catalogue of Cell Lines and Hybridomas, 7th Ed. (1992), pp. 253. American Type Culture Collection, Rockville, Maryland. Lengyel E et al. Cancer Res 55: 963–967, 1995. Leong SS. Advances in thyroid neoplasia. Field Educational Italia, pp. 95-105, 1981. Li GY, Yao KT, Glaser R. Int J Cancer 43,613–618,1989. Lin CT, Wong CI, Chan WY, Tzung KW, Ho JKC, Hsu MM, Chuang SM. Lab Invest 62: 713-724, 1990. Lin CT et al. Lab Invest 68: 716–727, 1993. Myers JN, Yasumura S, Suminami Y, Hirabayashi H, Lin W-Ch, Johnson JT, Lotze MT, Whiteside TL. Clinical Cancer Res 2: 127-135,1996. Miller BA, Ries LAG, Hankey BF et al. SEER Cancer Statistics review: 1973-1990, National Cancer Institute NIH Pub. No. 93-2789,1993. Moore AE, Sabachewsky L, Toolan HW. Cancer Res 13: 598405, 1955. Moore GE, Sandberg AA. Cancer 17: 170–175,1964. Nagashima S, Mailliard R, Kashii Y, Reichert TS, Herberman RB, Robbins P, Whiteside TL. Blood 91: 3850–61,1998. Nakashima T, Makishima K, Hiroto I. Ann Otol 89: 2628,1980. Nelson-Rees WA, Flandermeyer RR. Science 195: 1343, 1977 Okabe T, Sato N, Kondo Y, Asano S, Ohsawa N, Kosaka K, Ueyama Y. Cancer Res 38: 3910-3917,1978. Patel AM, Incognito LS, Schechter GL, Wasilenko WJ, Somers KD. Oncogene 12: 31-35,1996. Pekkola-Heino K, Kulmala J, Grénman R. Head and Neck 14: 196–199, 1992. Pekkola-Heino K, Jaakkola M, Kulmala M, Grénman R. Cancer Res CIin Oncol 121: 452–456, 1995. Pekkola-Heino K, Servomaa K, Kiuru A, Grénman R. Acta Otolaryngol (Stockh) 116: 341-344, 1996. Peterson WD Jr, Stulberg CS, Simpson WF. Proc Soc Exp BiolMed 136: 1187–1191,1971. Rangan SR. Cancer 29: 117–121,1972. Rheinwald JG, Beckett MA. Cancer Res 41: 1657-1663,1981. Rupniak HT, Rowlatt C, Lane EB, Steele JG, Trejdosiewicz LK, Laskiewicz B, Povey S, Hill BT. J Natl Cancer Inst 75: 621–635, 1985. Sacchi M, Klapan I, Johnson JT, Whiteside TL. Arch Otolaryngol Head Neck Surg 117: 321–326, 1991. Sachs PG, Parnes SM, Gallick GE, Mansouri Z, Lichter R, Satya-Prakash KL, Pathak S, Parsons DF. Cancer Res 48: 2858-2866,1988. Sacks PG, Oke V, Amos B, Vasey T, Lotan R. Int J Cancer 44: 926-933,1989. Schottenfeld D, Bradford C, DeGuire P, Spivak G. Upper Aerodigestive Tract Cancers: A Review. In: Cancer Incidence and Mortality Michigan 1992. Michigan Department of Public Health, Lansing, MI Servomaa K, Kiuru A, Grénman R, Pekkola-Heino K, Pulkkinen JO, Rytömaa T. Cell Prolif 29: 219–230,1996.
Head and Neck Cancers
255
Snyderman CH, Kaplan I, Milanovich M, Heo DS, Wagner R, Schwartz D, Johnson JT, Whiteside TL. Otolaiyngol. Head Neck Surg 111: 189–196,1994. Somers KD., Merrick MA, Lopez ME, Incognito LS, Schechter GL, Casey G. Cancer Res 52: 5997–6000,1992. Soukka T, Salmi M, Joensuu H, Häkkinen L, Sointu P, Koulu L, Kalimo K, Klemi P, Grénman R, Jalkanen S. Cancer Res 57: 2281-2289,1997. Sung M-W, Yasumura S, Johnson JT, Van Dongen GAMS, Whiteside TL. Int J Cancer 61: 864–872,1995. Takimoto T, Kamide M, Umeda R. Arch Otorhinolaryngol 239: 87-92,1984. Takimoto T et al. Otolaryngol Head Neck Surg 93: 500-504,1990. Toolan HW. Cancer Res 13: 389, 1953. Van Dyke DL, Worsham MJ, Benninger MS, Krause CJ, Baker SR, Wolf GT, Drumheller T, Tilley BC, Carey TE. Genes Chromosomes Cancer 9: 192-206,1994. Van Wyk CW et al. S African Med J 83: 425–429, 1993. Weichselbaum RR, Beckett MA, Hallahan DE, Kufe DW, Vokes EE. Seminars in Oncology 19: 1420,1992. Weichselbaum RR, Beckett MA, Vijayakumar S, Simon MA, Awan AM, Nachman J, Panje WR, Goldman ME, Tybor AG, Moran WJ, Vokes EE, Ahmed-Swan S, Farhangi E. Radiation Oncology Biol Phys 19: 313-319, 1990. Weichselbaum RR, Dahlberg W, Beckett M, Karrison T, Miller D, Carl J, Ervin TJ. Proc Natl Acad Sci USA 83: 2684–2688,1986, Weichselbaum RR, Dunphy EJ, Beckett MA, Tybor AG, Moran WJ, Goldman ME, Vokes EE, Panje WR. Head and Neck 11: 437–642,1989. Winn DM et al. New Engl J Med 304: 745–749, 1981. Worsham MJ, Carey TE, Benninger MS, Gasser KM, Kelker W, Zarbo RJ, Van Dyke DL. Genes Chromosomes Cancer 7: 158–164,1993a. Worsham MJ, Benninger MJ, Zarbo RJ, Carey TE, Van Dyke DL. Genes Chromosomes Cancer 6: 58–60, 1993b. Worsham MJ, Wolman SR, Carey TE, Zarbo RJ, Benninger MS, Van Dyke DL. Human Path 26: 251–261,1995. Worsham MJ, Wolman SR, Carey TE, Zarbo RJ, Benninger MS, Van Dyke DL. J Clin Path: Molec Path 52: 4246,1999. Yanagawa T, Hayashi Y, Yoshida H, Yura Y, Nagamine S, Bando T, Sato M. Am J Path 124: 496–509, 1986. Yao K et al. Int J Cancer 45: 83-89, 1990. Yasumura S, Weidmann E, Hirabayashi H, Johnson JT, Herberman RB, Whiteside TL. Int J Cancer 57: 297-305,1994. Yasumura S, Hirabayashi H, Schwartz DR, Toso JF, Johnson JT, Herberman RB, Whiteside TL. Cancer Res 53: 1461-1468, 1993. Yin X-Y, Donovan-Peluso M, Whiteside TL, Day R, Herberman RB, Locker J. Genes, Chromosomes Cancer 3: 443-454, 1991. Zenner HP, Lehner W, Herrmann IF. Arch Otorhinolaryngol 225: 269–277, 1979. Zenner HP, Herrmann IF, Bremer W, Stahl-Mauge C.Acta Otolalyngol 96: 371–381, 1983.
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Chapter 29 Gastric Cancer
Toshimitsu Suzuki and Morimasa Sekiguchi Department of Pathology, School of Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima City, 960-1295 Japan and Department of Surgery, Saitama Medical School, 38 Morohongo, Moroyama-machi, Iruma-gun, Saitama, 350-0451 Japan. Fax: 0081-24-5487151; E-mail:
[email protected]
1.
INTRODUCTION
The first gastric cancer cell line, CaVe, was described in 1963 by Dobrynin. Several cell lines (Sugano et al. 1968) were described in the 1960s, but most of them are no longer available. Since then, more than 70 cell lines derived from gastric cancer have been described (Table 1), with about three-quarters derived by Japanese researchers. In Japan, gastric cancer is one of the most frequent cancers in both sexes and accounts for 20 to 30% of all cancer incidence (Tominaga 1992), although its incidence in Japan has been dropping steadily since 1960. Despite the high incidence of gastric carcinoma in China, South America and Eastern Europe, few of the established cell lines are from these areas (see Table 1). The most widely used cell lines are the MKN series (MKN-1, MKN-7, MKN-28, MKN-45 and MKN-74), KATO-III and TMK-1. These cell lines were derived from various histological types of gastric cancer, including intestinal, diffuse or signet-ring cell carcinoma. In the field of molecular genetics, MKN7 and KATO-III have a special position, because the erbB-2 and K-sam oncogenes were cloned from them (Fukushige et al. 1986, Hattori et al. 1990).
2.
CULTURE METHODS
Primary cultures of human stomach cancer are easy to establish in conventional serum-containing commercial culture media. Initial growth can be
J.R.W Masters and B. Palsson (eds.), Human Cell Culture Vol. II, 257-291. © 1999 Kluwer Academic Publishers. Printed in Great Britain.
Name
Patient Patient age sex/race T
AGS
nd
nd
AZ-521 nd
N
M
B
Primary site
Specimen Culture medium site
Primary Culture Authentimethod cation Availability reference ATCC
nd
JCRB
ECC-10 73
m/J
1
+
1
RPMI1640+20% FCS D
K,M
RCB
ECC-12 63
m/J
3
-
1
RPMI1640 + 20% FCS D
K,M
RCB
GCIY
39
f/J
nd nd 1
EMEM +15% FCS
P
K,M
RCB
GK
nd
nd
nd nd nd nd stomach
liver met
DaigoT+5%FCS
nd
nd
GS
nd
nd
nd nd nd nd stomach
liver met
DaigoT+5%FCS
nd
nd
GT3TKB 53
m/J
nd nd nd nd stomach
ascites
DMEM + 10% FCS
nd
K,M
RCB
HC-154 nd
nd
nd nd nd nd stomach
primary
K,M
author
HGC-27 nd
nd
nd
+
RPMI-1640 + 10% FCS D on McCoy + 10% FCS agar nodalmet Ham’s F12+20% FCS D
K,M
author
HGT-1 60
m/M
3
nd 0
HLN- 60 GAC-5 HNGA 36
m/J
nd +
f/J
nd nd nd nd cardia
nd
1
nd body
liver met (autopsy) B-2 cardia skin met (biopsy) B-4 stomach ascites
nd stomach
B-3 postwall, primary body
K,M
B-3 fundus
M
DMEM+10% FCS, E in DMEM + 20% FCS + agar 10% tryptose phosphate broth nodalmet RPMI1640 + 20% FCS E
ascites
nd
S
K,M
author
Cancer Res43: 1983 1703-1709,1983 Br J Cancer 59: 1989 761-765,1989 Niigata Igakkai Zasshi 1988 106: 21-36,1992 Niigata Igakkai Zasshi 1989 106: 21-36,1992 Human Cell 2 (Suppl) 1989 89-90,1989 Proc Jpn Cancer Assoc 1991 50: 173, 1991 Proc Jpn Cancer Assoc 1991 50: 173, 1991 RCB General Catalog 1994 3,1997 Proc Jpn Cancer Assoc 1986 45: 241, 1986
SC Barranco K Imanishi N Ishihara N Ishihara M Nozue Y Imai Y Imai T Todoroki M Usugane
1974
T Akagi
1982
CL Laboisse
Proc Jpn Cancer Assoc 1988 47: 294,1988 Human Cell 2: 335, 1989 1989
S Morikawa
Acta Med Okayama 30: 215-217,1976 Cancer Res 42: 1541-1548,1982
I Ishiwata
Continued on next page
Suzuki and Sekiguchi
K,M
nd
primary Ham’s F10 + 20% FCS E (biopsy) nd nd nd nd stomach nd nd MEM + 10% FCS
nd nd nd nd stomach
Establish- First ment author
258
Table 1 List of gastric cancer cell lines and availability
Name
Patient Patient age sex/race T
N
M
B
Primary site
Specimen Culture medium site
Primary Culture Authentimethod cation Availability reference
Establish- First ment author
HOGT 5mos
m/J
nd nd nd nd stomach
primary
Ham's F12 + 20% FCS
D
K,M
HPEGAC-T HPEGAC-2 HPEGAC-3 HPEGAC-4 HS-746
53
f/J
nd nd nd nd stomach
ascites
RPMI1640 + 20% FCS
P
M
S Morikawa
74
m/J
nd nd nd nd stomach
ascites
RPMI1640 + 20% FCS
P
M
S Morikawa
39
m/J
nd nd nd B-3 body
ascites
RPMI1640 + 20% FCS
P
M
S Morikawa
69
f/J
nd nd nd nd stomach
ascites
RPMI1640 +20% FCS P
M
S Morikawa
74
m/C
nd nd 1
nd
K,M
ATCC
HSC-39 54
m/J
nd nd nd B-4 stomach
K,M
author
HSC-41 45
m/J
nd nd nd nd stomach
nd
author
Cancer Res 53: 5815-5821,1993
1993
K Yanagihara
HSC-42 nd
nd
nd nd nd nd stomach
nd
author
Cancer Res53: 5815-5821,1993
1993
K Yanagihara
HSC-43 56
m/J
nd nd nd B-4 stomach
K,M
author
Int J Cancer 54: 200–207,1993
1992
K Yanagihara
HSC-45 28
f/J
nd nd nd nd stomach
nd
author
Cancer Res 53: 5815-5821,1993
1993
K Yanagihara
HuG-1
nd
nd nd nd nd stomach
DMEM/aMEM (1:1)+ P 10% FCS + 5% horse serum, aMEM + 10% FCS DMEM/aMEM(1:1) E primary + 10% FCS, DMEM + 10% FCS E xenograft DMEM/aMEM (1:1) + 10% FCS DMEM + 10%FCS primary DMEM +3% FCS, E (biopsy) DMEM/Ham's F12 (1:1) + 0.05% BSA fraction DMEM/aMEM (1:1)+ P ascites 10% FCS, DMEM + 10% FCS nd nd nd
nd
JCRB/RCB Proc Jpn Cancer Assoc 1988 47: 396,1988
nd
nd stomach
musclemet DMEM+10% FCS ascites
Exp Pathol 27: 1983 143-151,1985 1976 Cancer 50: 1775-1782,1982 Cancer 50: 1976 1775-1782,1982 Cancer 50: 1976 1775-1782,1982 Proc Jpn Cancer Assoc 1979 45: 198,1986 JNatl Cancer Inst 62: 1979 225-230,1979 1990 Cancer Res 51: 381-386,1991
I Ishiwata S Kanazawa
Gastric Cancer
(Continued)
Table 1
S Kanazawa SKanazawa M Taniguchi HS Smith K Yanagihara
H Imanishi
259
Continued on next page
(Continued) Primary Culture Authentimethod cation Availability reference
Patient Patient age sex/race T
ISLS
69
m/J
nd + nd B-3 stomach
K,M
Ist-1
63
m/J
nd 1
K,M
IT-25
31
m/J
2
JR-St
35
f/J
nd nd nd B-4 stomach
KATO-III 55
m/J
KE-39
77
m/J
KE-97
52
m/J
KKLS
58
m/J
nd nd nd nd stomach pleural recurrent effusion nd nd nd B-3 lesser curv.primary body 4 2 1 B-3 lesser curv.peritoneal body deposit nd + 1 B-1 stomach nodalmet
KMK-2
58
f/J
nd nd + B-4 stomach
KS-1
55
f/nd
nd nd 1
KWS
42
m/J
MAIV
36
m/J
4
nd 1
MKK-1
56
m/J
3
2
MKN-1
72
m/J
nd + nd B-2 body
N M B
2
Primary site
Specimen Culture medium site
nodalmet AIM-V + 20% FCS, D RPMI-1640 + 15% FCS 1 nd stomach liver met S-clone SF-B+ 10% E bovine albumin 0 nd stomach xenograft MEM + 10% FCS E MEM + 10% FCS
P
K,M
P RPMI-1640/EMEM (1:1) + 20% FCS RPMI-1640 + 10% FCS D
K,M K,M
RPMI-1640 + 10% FCS E
K,M
RPMI-1640 + 15% FCS D
K,M
ascites
Ham’s F12 + 20% FCS, S EMEM + 10% FCS nd stomach Krukenberg Ham’s F12 + 10% FCS E tumor
nd nd nd nd stomach
1
CSF
M
B-4 stomach
ascites
pleural effusion B-3 antrum primary
nd Ham’s F10 +20% FCS E
M K,M SK K,M
RPMI-1640+20% FCS E
K,M
nodalmet RPMI-1640 + 20% FCS E
K,M
Establish- First author ment
Human Cell 4 (Suppl) 1991 51,1991 author Jpn J Cancer Res 82 1991 883485,1991 T Iwamura Proc Jpn Cancer Assoc 1986 45: 241,1986 IBL/author Gastroent Jpn 26: 1989 7-13,1991 ATCC/JCRB/Jpn J Exp Med 48: 1974 IBL/HSRRB 61–48,1978 RCB Human Cell 7: 1994 227-232,1994 RCB Nisshoshi 92 19-25, 1991 1995 M Mai Human Cell 2 (Suppl) 1989 87–88,1989 JNCI 64: 1015-1024, 1979 1980 Eur J Cancer Clin 1988 Oncol 24: 1397-1408, 1988 Human Cell 2:331, 1984 1989 Nichiidaishi 52: 1985 125126,1985 Nisshoshi 89: 1991 2645-2654,1992 RCB/IBL Niigata Igakkai Zasshi 1977 91: 737-752,1977
C-D Huang M Terashima K Ite A Terano M Sekiguchi H Uesugi H Uesugi K Sawaguchi H Nomura R Whelan M Sekiguchi K Kuki AIhara H Hojo
Continued on next page
Suzuki and Sekiguchi
Name
260
Table 1
(Continued)
Name
Patient Patient age sex/race T
MKN-7
39
N M B
Primary site
Specimen Culture medium site
Culture AuthentiPrimary method cation Availability reference
m/J
nd + nd B-2 antrum
nodal met RPMI-1640 + 20% FCS E
K,M
MKN-28 70
f/J
nd + nd nd stomach
nodal met RPMI-1640 +20% FCS E
K,M
MKN-45 62
f/J
nd nd 1
nd stomach
liver met
RPMI-1640 +20% FCS E
K,M
MKN-74 37
m/J
nd nd 1
nd stomach
liver met
RPMI-1640 +20% FCS E
K,M
MKO
nd
nd nd nd nd stomach
xenograft
RPMI-1640 + 10% FCS E
K,M
MUSG-1 61
m/J
4
nd nd B-3 antrum
ascites
RPMI-1640 + 20% FCS P
K,M
Mz-Sto-1 54
m/C
ascites
mind
P ascites fluid + CMRL (1:1) + 15% FCS RPMI-1640 + 10% FCS E
K,M
NCI-N87 nd
nd nd nd nd residual stomach nd nd 1 nd stomach
K,M
NKPS
44
f/J
nd nd nd nd stomach
RPMI-1640 + 20% FCS S
K,M
NTAS
78
f/J
pleural effusion nd nd nd B-4 stomach ascites
K,M
NU-GC-2 56
f/J
nd + nd nd stomach
AIM-+20% FCS, P RPMI-1640 + 15% FCS nodal met RPMI-1640+20% FCS E
K,M
NU-GC-3 72
m/J
nd nd 1
nd stomach
muscle met RPMI-1640+20% FCS E
K,M
NU-GC-4 35
f/J
nd + nd nd stomach
nodal met RPMI-1640 +20% FCS E
K,M
OCUM-1 38
f/J
nd nd nd B-4 stomach
pleural effusion
nd
liver
DMEM+10%FCS
P
K,M
Establish- First author ment
RCB/IBL/ Niigata Igakkai Zasshi 1977 HSRRB 91: 137–152,1977 RCB/JCRB/ Niigata Igakkai Zasshi 1977 IBL 91: 737–752,1917 RCB/IBL/HS Niigata Igakkai Zasshi 1977 RRB, DSMZ 91: 737-752,1977 TSuzuki/ Acta Pathol Jpn 36: 1977 IBL/HSRRB 65-, 1986 Proc Jpn Cancer Assoc 1986 45: 240, 1986 St Marianna Ikadaigaku 1984 Zasshi 14: 459–470, 1986 1987 Eur J Cancer Clin Oncol 23: 697-706,1987 ATCC/KCLB Cancer Res 50: 1977 2773-2780,1990 Proc Jpn Cancer Assoc nd 49: 220,1990 Proc Jpn Cancer Assoc 1991 50,113,1991 1983 JCRB Jpn J Surg 18: 438–446,1988 1984 JCRB Jpn J Surg 18: 438–446,1988 1984 author Jpn J Surg 18: 438–446,1988 K Hirakawa Nihon Gekagakkaishi 1988 92 1451–1460,1991
H Hojo H Hojo
Gastric Cancer
Table 1
H Hojo T Motoyama M Usugane T Shimomura WG Dippold J-G Park Y Deguchi K Sawaguchi SAkiyama SAkiyama SAkiyama TKubo
261
Continued on next page
Name
(Continued) Patient Patient age sex/race T
Primary site
Specimen Culture site medium
Culture AuthentiPrimary method cation Availability reference
primary
E
K,M
pleural RPMI-1640+ 20% FCS P effusion nd nd nd nd stomach peritoneal RPMI-1640+20% FCS E deposit
K,M
N M B
f/J
nd nd nd B-4 nd
m/J
nd nd nd nd stomach
SCH
46
m/J
SGC-7901 56
f/Ch
nd t nd
SH101
nd
nd nd nd nd stomach
SK-GT-1 63
m/C
4
1
0
SK-GT-2 72
m/H
4
1
SK-GT-3 69
m/C
4
SK-GT-4 89
m/C
2
SK-GT-5 67
m/C
SNU-1
44
SNU-5
nd
DMEM + 10% FCS
lesser curv. nodal met RPMI-1640 + 40% FCS E ascites
RPMI-1640 + 10% FCS nd
K,M K,M nd
EMEM + 20% FCS
E
K,M
0
nd proximal primary stomach nd fundus primary
EMEM + 20% FCS
E
K,M
2
1
nd fundus
primary
EMEM+20%FCS
E
K,M
1
0
EMEM+20%FCS
E
K,M
EMEM + 20% FCS
E
K,M
m/K
nd Barrett primary epithelium 2 2 0 nd EC primary junction nd nd nd nd stomach primary
RPMI-1640+ 10% FCS E
K,M
33
f/K
nd nd nd nd stomach
ascites
RPMI-1640+ 10% FCS P
K,M
SNU-16 33
f/K
nd nd nd nd stomach
ascites
RPMI-1640+ 10% FCS P
K,M
K Hirakawa Nisshoshi 92: 1995 199-205,1995 1986 JCRB/IBL Acta Pathol Jpn 36: 65-83,1986 JCRB Culture of Human 1972 Cancer Cells, Asakura, Tokyo, 192-202,1975 Chin Med J 97: 1983 831-834,1984 author Int J Cancer 53: 1993 1013-1016,1993 Cancer 72: 649-657, 1988 1993 Cancer 72 649–657, 1989 1993 Cancer 72 649-657, 1989 1993 Cancer 72 649457, 1989 1993 1989 Cancer 72 649–657 1993 ATCCKCLB Cancer Res 50: 1985 2773-2780,1990 ATCCKCLB Cancer Res 50: 1987 2773-2780,1990 ATCCKCLB Cancer Res 50: 1987 2773-2780,1990
M Yashiro T Motoyama S Oboshi C-H Lin M Nishiyama M Altorki N Altorki N Altorki N Altorki N Altorki J-G Park J-G Park J-G Park
Continued on next page
Suzuki and Sekiguchi
OCUM- 49 2M Okajima 38
Establish- First author ment
262
Table 1
(Continued)
Name
Patient Patient age sex/race T
N M B
St 2474
60
2
St 2957
51
m/G
2
2
0
St 3051
66
m/G
4
2
0
m/G
1
0
Primary site
nd stomach
Specimen Culture site medium
Culture AuthentiPrimary method cation Availability reference
nodal met RPMI-1640 + 10% FCS D
K,M
DSMZ/ author
nd stomach
nodal met RPMI-1640+10%FCS D
K,M
DSMZ/ author
nd stomach
primary
RPMI-1640 + 10% FCS D
K,M
DSMZ/ author
RPMI-41640+10%FCS D
K,M
DSMZ/ author
K
JCRB/ author/ S Yanoma JCRB/IBL
St 23132 72
m/G
2
nd stomach
primary
STKM-1 41
f/J
nd + nd B-4 stomach
pleural effusion
TAKIGA 63 WA 72 TE-7
m/J
nd nd nd B-2 stomach
nodal met DM-170 + 20% FCS
E
K,M
m/J
nd nd nd nd cardia
primary
D
K,M
TGBC11T 72 KB TMK-1 21
f/J
nd nd nd nd stomach
nodalmet DMEM + 5% FCS
nd
K,M
m/J
nd nd nd nd body
xenograft
TSG-6
f/J
4
nodal met RPMI-1640+ 20% FCS D
m/J
nd nd nd B-2 stomach
57
no name- 65 1
0
0
nd 1
B-4 stomach
primary
RPMI-1640+ 10% FCS P
nd
RPMI-1640 +10% FCS Ein M soft agar
6052 t 1% dialyzed bovine serum t EGF t ITS
D
RCB E Tahara
K,M
T Suzuki
K,M
author
Establish- First ment author
Virchows Archiv B 1991 Cell Pathol 63: 335-343, 1993 1991 VirchowsArchivB Cell Pathol 63: 335-343, 1993 1991 VirchowsArchivB Cell Pathol 63: 335-343, 1993 1988 VirchowsArchivB Cell Pathol 63: 335-343, 1993 Human Cell 4: 67-70, 1991 1991 J Gastroenterol 30: 1984 589-598,1995 Human Cell 2 (Suppl) 1983 330,1989 RGB General Catalog 1997 3, 1997 1985 Jpn J Cancer Res (Gann) 76: 1064-1071, 1985 Jpn J Surg 24: 420–428,1990 1994 Proc Jpn Cancer Assoc 1989 50: 173,1991
HPVollmers HPVollmers
Gastirc Cancer
Table 1
HPVollmers HP Vollmers A Arimura M Sekiguchi T Akaishi B Kim A Ochiai K Aizawa T Kawaguchi
263
Continued on next page
(Continued)
Name
Patient Patient Primary Specimen Culture medium age sex/race T N M B site site
noname- nd 2
nd
nd nd nd B-4 stomach xenograft DMEM+5% FCS
Primary CultureAuthentimethod cation Availability reference nd
M
Establish- First ment author
Proc Jpn Cancer Assoc 1990 49:220,1990
264
Table 1
Y Imai
Suzuki and Sekiguchi
Abbreviations: nd: not described; B: macroscopic classification of gastric carcinoma, modified, based on Borrmann’s subtyping; Type 1: polyploid, 2: ulcerative, non-infiltrating; 3: ulcerative, infiltrative; 4: diffuse, infiltrative. Culture method: D: dispersed; E: explant; P: cell pellet; S: suspension. Authentication: K: karyotypic analysis; M: morphological analysis. Race: C: Caucasian: Ch: Chinese; G: German; H Hispanic; J: Japanese; K Korean; M: Moroccan. CSF: cerebrospinal fluid List of correspondence for availability ATCC. Fax: 1-703-365-2701. Toll free in the USA & Canada: 1-800-638-6597. Outside the USA: 1-703-365-2700. DSMZ (German Collection for Microorganisms and Cell Lines). Mascheroder Weg lb, 38124 Braunschweig, Germany. Fax: +49-531-2616-150, HSRRB, commercial. (Health Science Research Resources Bank). 1-1-43 Hoenzaka, Chuo-ku, Osaka, 540-0006, Japan. Fax: +81-6-945-2872. IBL (Immunobiological Laboratories), commercial. 1091-1 Naka, Fujioka City, Gunma, 375-0005, Japan. Tel: +81-274-22-2888. Fax: +81-274-23-5746. JCRB (Japanese Cancer Research Resources Bank-Cell). 10-35 Kamiosaki 2-chome, Shinagawa-ku, Tokyo, 141-0021, Japan. KCLB (Korean Cell Line Bank). Cancer Research Institute, Seoul National University College of Medicine. 28 Yongon-dong, Chongno-gu, Seoul, 11-0744, Korea. Tel: +82-2-742-0020. Fax: +82-2-742-0021. RCB (RIKEN Cell Bank). 3-1-1 Koyadai, Tsukuba Science City, Ibaraki, 305-0074, Japan. Tel: +81-298-363611. Fax: +81-298-369130. T Akagi, Professor. Second Department of Pathology, Okayama University, Medical School. 1-5-2 Shikada-chou, Okayama City, Okayama, 700-0914, Japan. Tel: +81-861-23-7151. Fax: +81-862-21-4743. S. Akiyama. Second Department of Surgery, Nagoya University, School of Medicine. 65 Tsurumai-chou, Showa-ku, Nagoya City, Aichi, 466-0065, Japan. Tel: +81-52-741-2111. A Arimura. First Department of Internal Medicine. Yokohama Minami Kyo-sai Hospital. 500 Mutsuura-cho, Kanagawa-ku, Yokohama-City, Kanagawa, 2360032, Japan. Tel: +81-45-782-2101. Fax: +81-45-701-9159. W Dippold, Professor. Johaness Gutenberg-Universitat Mainz, Verfugungsgebaude fur Forschung und Entwicklung, Obere Zahlbacher Str. 63-6500 Mainz, Postfach 3980, Germany. Tel: t49-6131-173300. Fax: t 49-6131-173364. K Hirakawa. First Department of Surgery, Osaka City University Medical School, 1-4-54 Asahi-machi, Abeno-ku, Osaka City, Osaka, 545-0051, Japan. Tel: +816-645-2121. Fax: 81-6-646-6450. T Iwamura. First Department of Surgery, Miyazaki Medical College, 5-200 Ooaza-kihara, Kiyotake-chou, Miyazaki-gun, Miyazaki, 889-1601, Japan. Tel: +81985-85-1510. Continued on next page
(Continued)
M Mai, Professor. Department of Surgery, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa City, Ishikawa, 920-0934. Tel: +8176-265-2799. S Morikawa, Professor. Department of Pathology, Shimane Medical University, 1-89 Ennya-chou, Izumo City, Shimane, 693-0021, Japan. Tel: +81-853-23-2111. e-mail:
[email protected]. M Nishiyama, Professor. Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima City, Hiroshima, 734-0037, Japan. Tel: +81-82-257-5555. Fax: +81-82-256-7105. e-mail:
[email protected]. T Suzuki, Professor. Department of Pathology, Fukushima Medical University, School of Medicine, 1 Hikariga-oka, Fukushima City, Fukushima, 960-1295, Japan. Tel: +81-24-548-2111. Fax: +81-24-548-7151. E-mail:
[email protected]. E Tahara, Professor. First Department of Pathology, Hiroshima University, School of Medicine, 1-2-3, Kasumi, Minami-ku, Hiroshima City, Hiroshima, 7340037, Japan. Tel: +81-82-257-5555. Fax: +81-82-257-5149. e-mail:
[email protected]. A Terano, Professor. Second Department of Medicine, Dokkyo University, School of Medicine, 880 Ooaza-kitakobayashi, Mibu-chou, Shimotsuga-gun, Tochigi, 321-0207, Japan. Tel: +81-282-86-1111, ext 2728. Fax: +81-282-86-6481. M Terashima. First Department of Surgery, Iwate Medical University, School of Medicine, 19-1 Uchimaru, Morioka City, Iwate, 020-0023, Japan. Tel: +81-19651-5111. Fax: +81-19-651-7166. e-mail:
[email protected]. M Usugane. Molecular Genetics, Department of Medical Genetics, Biomedical Education and Research Center, Osaka University Faculty of Medicine, 2-2 Yamada-oka, Suita City, Osaka, 565-0871, Japan. Tel: +81-6-879-5111. HP Vollmers, Professor. Institut fiir Pathologie Universitat Wurzburg, Josef-Schneider-Str. 2,97080 Wurzburg, Germany. Tel: +49-931-201-3898. Fax: +49-931201-3440. e-mail:
[email protected]. K Yanagihara. National Cancer Institute, 5-1-1 Tsukiji, Chuou-ku, Tokyo, 104-0045, Japan. Tel: +81-3-3542-2511. Fax: +81-3-3542-2548. e-mail:
[email protected] K Yanoma. Kanagawa Cancer Center Clinical Research Institute, 54-2 Nakao-chou, Asahi-ku, Yokohama City, Kanagawa, 241-0815, Japan. Tel: +81-45-3915761 - ext 318. Fax: +81-45-366-3157.
Gastric Cancer
Table 1
265
In vitro features of cell line in comparison to original tumor
Description of tumor pathology
AGS
Adherent, piling up in large colonies. Focally moderately differentiated with recognizable gland formation to poorly differentiated with sheets, clusters, and cords of malignant cells, transmural extension into the pancreas. Mucin production; slight. Well differentiated adenocarcinoma in the Partly floating, partly adherent, with small cell mucosa and small cell endocrine cell aggregates. Neurosecretory granules; positive. carcinoma in the submucosa including foci of squamous cell carcinoma in the latter. Neurosecretory granules; positive. Adherent, not monolayered, cell aggregates with Small cell carcinoma (endocrine cell piling up. Neurosecretory granules: positive. carcinoma) with pseudorosettes. Neurosecretory granules: positive. Poorly differentiated adenocarcinoma, Adherent, monolayer of polygonal cells with piling up. scirrhous, with mucin production. Gastric carcinoma with liver metastasis nd and high serum AFP. Gastric carcinoma with liver metastasis nd and high serum AFP. Adenocarcinoma mucocellulare scirrhosum. Epithelial-like Poorly differentiated adenocarcinoma. Adherent, cell clusters with dispersed growth. Well-differentiated tubular adenocarcinoma Adherent, monolayer, polygonal epithelioid cells. in the mucosa and undifferentiated carcinoma in other areas: nodal metastasis; undifferentiated carcinoma. Ulcerating, infiltrating tumor with serosal Adherent, epithelial-like morphology. involvement, poorly differentiated adenocarcinoma without mucus production.
EC-10(1)
ECC-12 GCIY GK GS GT3TKB HC-154 HGC-27
HGT-1
Xenograft pathology Solid nests of oval cells with areas of necrosis. Some cells had PAS-stained dense cytoplasmic mucin droplets. No gland formation and no Alcian blue-stained mucin. Endocrine cell carcinoma alone. Neurosecretory granules: positive
Endocrine cell carcinoma similar to that of primary tumor. Neurosecretory granules: positive. Poorly differentiated adenocarcinoma with weak positivity of PAS or Alcian blue staining. Take in nm. Take in nm. Take in nm. Take in nm. nd
Solid tumor with same morphology as that of the initial tumor but occasional tubular structures. Continued on next page
Suzuki and Sekiguchi
Name
266
Table 2 Comparison of cell lines with in vivo histology, in vitro growth pattern, and related xenograft morphology
Name
Description of tumor pathology
In vitro features of cell line in comparison to original tumor
Xenograft pathology
HLN-GAC-5
Continued on next page
267
Adenocarcinoma, poorly differentiated with Adherent, spindle-shaped cells. Take in nm. liver and nodal metastasis. Benign tridennal teratoma. HOGT Benign gastric tridermal teratoma. Fibroblast-like cells. Floating, PAS-positive signet-ring cells in early Not taken in nm. HPE-GAC-T(2) Poorly differentiated adenocarcinoma, peritonitis carcinomatosa. stage. HPE-GAC-2(3) Adenocarcinoma, partly poorly Not taken in nm. Floating in small cell clusters. differentiated. HPE-GAC-3 Adenocarcinoma, peritonitis carcinomatosa. Adherent, partly floating. Not taken in nm. HPE-GAC-4 Adenocarcinoma, peritonitis carcinomatosa. Adherent, epithelial but not monolayered, and Not taken in nm. floating with growth. Carcinoma. Very abnormal. Carcinoma. HS-746 HSC-39 Signet-ring cell carcinoma and peritonitis Floating, round cells with loose cell aggregates; Poorly differentiated adenocarcinoma including signet-ring cells with medullary carcinomatosa. PAS-positive. growth; PAS-positive. HSC-41 Moderately differentiated tubular Undifferentiated carcinoma composed of solid Multilayered sheets with clusters upon adenocarcinoma. masses of tumor cells. confluence. Undifferentiated carcinoma composed of solid Well-differentiated tubular adenocarcinoma, Multilayered sheets with clusters upon HSC-42 serially transplanted in athymic mice. mass of tumor cells. confluence. HSC-43 Scirrhous gastric carcinoma. Adherent, piling up and multilayered, cuboidal Poorly differentiated carcinoma, expansive and round cells; PAS and Alcian blue-positive. growth. Multilayered sheets with clusters upon Signet-ring cell carcinoma. Signet-ring cell carcinoma. HSC-45 confluence. 1st-1 Adherent, epithelial-like morphology. nd Moderately differentiated tubular adenocarcinoma and mucin granules in cytoplasm with liver metastasis and paragastric lymph node metastasis. High serum AFP. Signet-ring cell carcinoma; PAS-positive. Adherent, monolayered, mainly polygonal and nd JR-St some round floating cells; PAS-positive. Floating, free round cells and adherent few cells. Not taken in nm. KATO-III Signet-ring cell carcinoma.
Gastric Cancer
Table 2 (Continued)
Name
Description of tumor pathology
KE-39
Poorly differentiated adenocarcinoma.
KE-97
Mucinous carcinoma with component of poorly differentiated adenocarcinoma and signet-ring cell carcinoma. Poorly differentiated adenocarcinoma with multinucleated or bizarre giant cells, medullary growth. PAS-negative. Poorly differentiated adenocarcinoma.
KKLS KMK-2 KS-1 KWS MAIV MKK- 1 MKN-1
MKN-28 MKN-45
In vitro features of cell line in comparison to original tumor
Xenograft pathology
Adherent, epithelial growth as small cell nests that coalesce in time. Adherent, round cells with multi-nucleated giant cells and mucin-containing cells.
Poorly differentiated adenocarcinoma with medullary growth. Poorly differentiated adenocarcinoma with medullary growth.
Adherent monolayer of polygonal cells with piling up or giant cells.
Poorly differentiated adenocarcinoma with medullary growth.
nd. Almost adherent and monolayered with cell aggregates on the monolayer. Poorly differentiated adenocarcinoma with Floating, round cells, numerous loosely packed nd. signet-ring cell component aggregates of variable size. Partly adherent, various sued polygonal cells Poorly differentiated adenocarcinoma. Take in nm. and partly floating, round cells. Signet-ring cell carcinoma, superficial nd. Floating, round cells with clusters, partly adherent. spreading type, scirrhous. Poorly differentiated adenocarcinoma with Adherent, round cells, not-pavement-like Poorly differentiated adenocarcinoma with duodenal invasion. arrangement. PAS and Alcian blue positive. central necrosis. Squamous cell carcinoma with component of Adenosquamous carcinoma; adenocarcinoma Adherent, epithelial-like and partly floating, round cells. poorly differentiated carcinoma. element alone in nodal metastasis for culture. Well-differentiated adenocarcinoma with Adherent, epithelial monolayer, with occasional Not taken in nm. multinucleated giant cells. weak positivity for Alcian blue staining. Well differentiated adenocarcinoma in Adherent, epithelial-like monolayer with piling Well differentiated adenocarcinoma with focal poorly differentiated carcinoma; PAS and/or biopsy specimen and PAS and Alcian blue- up; PAS and/or Alcian blue-positive. Alcian blue-positive. positive in nodal metastasis. Poorly differentiated adenocarcinoma. PASPoorly differentiated adenocarcinoma with Adherent but not sheet-like, piling up and irregular growth pattern; PAS-positive, Alcian positive, Alcian blue-negative. medullary growth and liver metastasis. blue-negative. PAS-positive; Alcian blue-negative. Continued on next page
Suzuki and Sekiguchi
MKN-7
268
Table 2 (Continued)
In vitro features of cell line in comparison to original tumor
Name
Description of tumor pathology
MKO
Poorly differentiated carcinoma.
MUSG-1
Poorly differentiated adenocarcinoma in biopsy specimen and peritoneal deposit. Moderately well-differentiated adenocarcinoma with islets of signet-ring cells; PAS-positive. Well differentiated adenocarcinoma. Adherent, monolayer, epithelial-like, Poorly differentiated adenocarcinoma, Adherent, monolayer, pavement-like. scirrhous. Poorly differentiated adenocarcinoma, Floating, single or small cell clusters; PAS scirrhous. positive. Poorly differentiated adenocarcinoma with Epithelial, polygonal cells with monolayer sheet; PAS-positive, Alcian blue-negative. paragastric nodal metastasis. Poorly differentiated adenocarcinoma, Epithelial, polygonal cells with monolayer sheet; PAS-positive, Alcian blue-negative. metastasis in brachial muscle. Poorly differentiated adenocarcinoma with Adherent, round cells, partly as free floating cells; PAS, Alcian blue-negative. areas of signet-ring cell carcinoma, and paragastric nodal metastasis. Poorly differentiated adenocarcinoma with Floating, round and signet-ring like, partly loosely attached; PAS and Alcian blue-positive. signet-ring cell carcinoma, biopsy. Floating, round or paired cells. Scirrhous gastric cancer.
MZ-Sto-1 NCI-N87 NKPS NTPS NU-GC-2 NU-GC-3 NU-GC-4 OCUM-1 OCUM-2M Okajima SCH(4)
Adherent cell clusters with dispersed growth from the clusters. Mainly adherent, round cells, partly floating from piling-up. Adherent in small islands, epithelial cell-type.
Poorly differentiated adenocarcinoma. Floating with singles or cell aggregates. Primary choriocarcinoma of male stomach, Adherent, monolayer, numerous piling-ups, polygonal and multinucleated giant cells, HCGfocal adenocarcinoma element, autopsy, high urine HCG. producing.
Xenograft pathology Poorly differentiated carcinoma. Poorly differentiated adenocarcinoma weakly positive for PAS, negative for Alcian blue. nd.
Gastric Cancer
Table 2 (Continued)
Well differentiated adenocarcinoma. Carcinoma, CA19-9 and CEA-positive. nd. Not taken in athymic nude mouse even by treatment with anti-asialo GM1 antibody. Poorly differentiated adenocarcinoma. Poorly differentiated adenocarcinoma. Poorly differentiated adenocarcinoma, medullary growth. Poorly differentiated adenocarcinoma with poor fibrosis. Poorly differentiated carcinoma. Choriocarcinoma with HCG-production.
269
Continued on next page
Name
Description of tumor pathology
SGC-7901
Invasive carcinomatous growth along the stomach lesser curvature with nodal and peritoneal involvement; nodal metastasis, adenocarcinoma, PAS-positive, Alcian bluenegative. Well-differentiated tubular adenocarcinoma. Moderately differentiated adenocarcinoma.
SH-101(5) SK-GT-1 SK-GT-2 SK-GT-3 SK-GT-4 SK-GT-5 SNU-1 SNU-5 SNU-16
In vitro features of cell line in comparison to original tumor
Xenograft pathology
Epithelial-like, pavement-like monolayer; PASpositive, Alcian blue negative.
Poorly differentiated carcinoma with PASpositivity and Alcian blue-negativity.
Monolayer or multilayer. Adherent, epithelial as sheets of cells, mainly polygonal but pleomorphic including spindleshaped cells. Adherent, epithelial as small cell nests.
Undifferentiated carcinoma. Histologically similar to the inoculated tumor.
Adherent, epithelial, large fusiform cells with large spindle-shaped nuclei. Adherent, small epithelial cell nests that enlarge and coalesce in time. Adherent, epithelial as sheets of polygonal cells and/or spindle-shaped cells. Poorly differentiated adenocarcinoma. Floating, partly adherent, more round. Poorly differentiated adenocarcinoma. Floating, partly adherent, more round. Poorly differentiated adenocarcinoma. Floating, partly adherent, more round and a small number of goblet cells. Adherent, epithelial-like. Adenocarcinoma, intestinal type. Adherent, epithelial-like. Adenocarcinoma, intestinal type. Adherent, epithelial-like, rather spindle-shaped. Adenocarcinoma, intestinal type. Adherent, epithelial-like. Adenocarcinoma, intestinal type. Poorly differentiated adenocarcinoma partly Adherent, monolayered polygonal cells. signet-ring cell-like.
Histologically similar to the inoculated tumor. Histologically similar to the inoculated tumor. Histologically similar to the inoculated tumor. Histologically similar to the inoculated tumor. Poorly differentiated adenocarcinoma. Not tested. Not tested. Solid tumor. Solid tumor. Solid tumor. Solid tumor. Poorly differentiated adenocarcinoma, PASpositive. Continued on next page
Suzuki and Sekiguchi
St 2474 St 2957 St 3051 St 23132 STKh4-1
Poorly differentiated adenocarcinoma with ulceration. Moderately to poorly differentiated mucin producing adenocarcinoma. Well-differentiated adenocarcinoma arising in Barrett epithelium of the distal esophagus. Poorly differentiated adenocarcinoma.
270
Table 2 (Continued)
Gastric Cancer
Table 2 (Continued) In vitro features of cell line in comparison to original tumor
Name
Description of tumor pathology
TAKIGAWA
Moderately differentiated adenocarcinoma, high serum AFP.
Adherent, cell-islands, without piling-up.
TMK-1
Poorly differentiated adenocarcinoma.
TSG
Loosely adherent, oval or cuboidal-shaped, and loose clusters with piling up, PAS-positive, keratin and vimentin-positive. Floating, round, single cells, PAS-positive.
Signet-ring cell carcinoma, diffusely infiltrative growth pattern. Signet-ring cell carcinoma with diffusely Floating, round, including signet-ring cells; infiltrative growth and peritoneal PAS-positive. dissemination. Poorly differentiated adenocarcinoma, high Loosely, adherent, epithelial-like serum AFP. , Floating Signet-ring cell carcinoma
TSG-6 No name-1 No name-2
Xenograft pathology Papillary adenocarcinoma and squamous cell carcinoma; transplantable in nm by co-innoculation of the cells with polystyrene plate. nd nd nd nd Similar histology to the primary tumor.
The text presented in this table is mostly from the original literature as cited in Table 1, a different origin of data is indicated in the text. Superscript numbers: (1) = Fukuda et al. 1988; (2) = Kubota et al. 1988; (3) = Kotou et al. 1991; (4) Kameya et al, 1975; (5) = Yanagihara et al. 1993a. Abbreviations: AFP: alphafetoprotein; CEA carcinoembryonic antigen; HCG; human chorionic gonadotropin; nm: nude mouse; nd: not described; PAS: periodic acid Schiff reaction.
271
MCNo Marker Chr ras c-myc erbB-2 K-sam c-met p53
GF
GFR
AGS ECC-10(1)
dip1oid hypotetraploid
47 83
nd nd
nd nd nd amp
nd nd
nd nd
nd nd
nd nd
nd GR(2)
ECC-12(1) GCIY
hyperdiploid hypotriploid
54 57
nd none
nd nd
nd nd
nd nd
nd nd
GK GS GT3TKB HC-154 HGC-27 HGT- 1 HLN-GAC-5 HOGT HPE-GAC-T HPE-GAC-2 HPE-GAC-3 HPE-GAC-4 HSC-746 HSC-39(4)
nd nd hyperdiploid hypertetraploid hypertetraploid hypotriploid nd diploid nd nd nd nd hyperdiploid hypotriploid
nd nd 50 97 109-110 57 nd 46 nd nd nd nd nd 66
nd nd nd nd nd +marl-17 nd none nd nd nd nd nd +marl-4
nd nd nd nd nd nd nd nd nd nd nd nd nd nd
nd nd nd nd nd nd nd nd nd nd nd nd nd nd
HSC-43(4)
pseudotetraploid 88
nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nl amp na (Ha, N,K) nd na na
HSC-41(7) HSC-42(7) HSC-45(7) HUG-1 Ist-1
nd nd nd nd hypertriploid
nd nd nd nd 71-77
+marl-3 nd nd nd nd some
nd nd nd nd nd
amp nd
nd nd nd nd nd
nd nd nd nd nd
nd nd nd nd nd nd
nd nd nd nd nd nd
nd nd
Tumor marker
Others
nd ten clones of AGS serotonin (initial CK-BB stage), NSE nd nd nd serotonin,NSE AAAD,CK-BB,NPY,PYY nd nd nd CA19-9,CA-125 +12, +18,-5,-7,-9,-17, –15×2 nd nd EGFR AFP growth enhancement by EGF growth inhibition by EGF nd nd EGFR nd nd nd nd target of CTL(3) CEA LDH nd nd nd nd nd nd nd AT, LAP, LDH nd nd nd nd histamine H2R nd nd nd CEA CK & VM positive nd nd nd AFP/CEA:nl nd nd EGFR CEA nd nd EGFR CEA nd nd EGFR CEA nd nd EGFR CEA nd nd nd nd mut(5) gastrinnd CA19-9, CA125, numerous DMs. nl in L-myc, CEA, SLX, TPA erbA, abl, mos, myb, src. apoptosis by TGF b1, cripto(6) nd TGFa EGFR CA125, CEA, TPA nl in myb, src, abl, mos, erbA. apoptosis by TGF b1 nd nd nd TPA nd nd nd TPA nd nd nd CA19-9,CEA,TPA nd nd nd CA19-9,CEA,TPA PLAP nd nd nd AFP,CA19-9, CEA, TPA Continued on next page
Suzuki and Sekiguchi
Ploidy
272
Table 3 Ploidy, chromosome numbers, important genetic findings, tumor markers, growth factors, growth factor receptors and other characteristics of cell lines. Name
Ploidy
MC No Marker Chr ras c-myc erbB-2 K-sam c-met p53
JR-St hyperdiploid KATO-III hypotetraploid
54 88
none none
KE-39 KE-97
hypotriploid near diploid
62 47
KKLS KMK-2 KS-1 KWS MAIV MKK-1
hypotetraploid nd hypotriploid hypodiploid near diploid triploid
84 nd 60–65 43 48 69
MKN-1
hypodiploid
39
MKN-7 near tetraploid
88-91
GF
nd nd nd nd amp(8) amp(9) del(10) PDGFA (complete)mRNA(11) TGFamRNA(12)
+marl-20 nd nd nd nd nd
nd nd
nd nd
nd nd
nd nd
present nd nd nd nd several nd nd nd nd +marl-5 nd or +marl-4
nd nd nd nd nd nd
nd nd nd nd nd nd
nd nd nd nd nd nd
nd nd nd nd nd nd
none
two
nd nd nd nd nd nd
nd nd
nd nd
protein(24)nd
amp(27) nd protein(24)
na(9)
na(9)
mut(5,10)
mut(5)
Tumor marker
Others
nd CA19-9, CEA, TPA procollagen III 17PLOH; 18, XY, +7A, +2B, EGFR CEA(14) +16C, +4D, +8E, +2F, +2G; mRNA(12) TNFaR(13) APC:del(15) ERK: amp(16) CycliE amp(17), PP60C-SRC(18) TGFb1 activator(19), TGF b1/TGFbR(20), 10q26amp in HSR(21), CCA-1, HLA-DR(22) CA19-9 62,00, +1P, +7q, +11q,–11P nd nd 47, XO, +8, +12. ICAM-1, nd nd CA19-9, CEA AFP:nl nd nd CA19-9, CEA, TPAt(1:17)(p11?:q11?) AFP:nl nd nd a-AMY, LTAP(23) nd nd nd nd 10-30DMs nd nd CEA;nl nd nd CEA nd nd +1, +2, +5, +16, +11×2, CA19-9, CEA +20×2, Robertsonian type translocation in 13 PDGFA EGFR 17pLOH(10), PP6c-src(18), mRNA(11) mRNA(12) TGFbmRNA(20), TGFbIR/ bIIRmRNA(20) Regan isozyme TGFaPDGF-R of HSAP(26), TF(22) rnRNA(12) mRNA(11) HGFR(25) PDGFA EGFR Apc:mut(15), c-jun:amp(28), CyclinE rnRNA(11) mRNA(12) mRNA:amp(17) TGFbmRNA(20), TGFaHGFR(25) Regan isozyme of HSAP(26) mRNA(12)
nd nd
nd nd nd nd
GFR
273
Continued on next page
Gastric Cancer
Table 3 (Continued) Name
Ploidy
MC No Marker Chr ras c-mycerbB-2
K-sam c-met p53
MKN-28 near triploid
59-80
none
nd na
protein nd
MKN-45 near diploid
59-80
none
nd na
nd na(27) protein(24)
MKN-74 hypodiploid
36
nd
nd nd
protein(24)nd
nd none none
NCI-N87 near diploid
43
na
(9)
GF
mut
(5,10)
amp(9) wt(5,10)
na(9)
wt(5,34)
nd nd nd
GFR
PDGFA EGFR mRNA(11) mRNA(12) TGFaHGFR(25) mRNA(12)
bFGF EGFR CEA(30) mRNA(32) mRNA(12) PDGFA IFGI-R(33) mRNA(11) TGFamRNA(24) PDGFA EGFR mRNA(11) mRNA(12) TGFa(12) mRNA nd CEA nd nd nd CEA nd CEA nd
nd nd na(30)
nd nd nd
nd nd nd
nd
nd nd nd nd nl nd (Ha, N, K) nd na
nd
nd
mut(34) nd
nd
VIPR
NKPS
hyperdiploid 52-53
nd
nd nd
nd
nd
amp
nd
nd
nd
NTAS
hypertriploid 71-72
nd
nd nd
nd
nd
amp
nd
nd
nd
none
nd nd
nd
nd
nd
nd
nd
nd
NUGC-2 hypotriploid
62
Tumor marker Others 17pLOH(10), APC:mut(15), CyclinE mRNA arnp(17), PP6c-src(18), CCA- 1, TF, HLA-DR(22), TGFbmRNA(29), TGFb1, bII,b III-R(31), HLA (W6/32)(30), A blood group(30), Lewisa(30) APC:nl(15), CyclinE mRNA amp(17) ERKmRNA:amp(16),PP60c-src(18), TGFbIR, TGFbIIR(20), TF(22), HLA (W6/32)(30), A blood group(30)
APC:n1(15),c-jun:amp(28), PP60c-src(18),TGFbmRNA(29), TGFbIR, TGFbIIR(20), CCA-1, TF, HLA-DR(22) LDH 54, X, -Y, +5A, +5C, -2D, +E nl in erbA,, A2, B, src, raf, myb, sis, rel, fgr, yes, fes, fms, fos, mos, abl; Lewisa, Lewisb CA-19-9, CEA, DMs TAG72 numerous DMs, ERK mRNA: CA19-9 amp(16) CA19-9, CEA, many DMs, almost trisomy TPA CEA +A, +C, +E, +F, -D, -G, -4P, DMs Continued on next page
Suzuki and Sekiguchi
MKO hypertetraploid99 MUSG-1 hyperdiploid 54 Mz-Sto-1 hypertriploid 73
(24)
274
Table 3 (Continued) Name
(Continued)
Name
Ploidy
MC No Marker Chr ras c-myc erbB-2 K-sam c-met p53
GF
GFR
Tumor marker Others
NUGC-3 hypotriploid 58 NU-GC-4 hyperdiploid 52-54
none none
nd nd nd nd
nd nd
nd nd
nd nd
mut(10) wt(10)
nd nd
nd nd
OCUM-1 hyperdiploid 50
+mar
nd nd
nd
nd
nd
nd
nd
EGFR
OCUM- near triploid 70 2M Okajima hypotriploid 62 SCH hypotriploid 60
+ marl~3
nd amp(35) na(35)
nd
amp
nd
nd
EGFR(15)
nd nd
nd nd nd nd
nd nd
nd nd
amp(37) nd nd nd
nd nd
nd nd
SGC7901 hypotriploid 67 SK-GT-1 hyperdiploid 56
nd nd
nd nd nd nd
nd nd
nd nd
nd nd
nd del(34)
CEA, ferritin +A, +C, +E, +F, -5P, 17pLOH CEA, ferritin XX, 53, +2, +4, +8, +11×2, +19, +20 CA19-9,CEA, +1, +7, +8 Span-1 CA19-9, CEA, nl in met-D, met-H, v-erbB, Span-1 TGFbR(36) SLX nd HCG Nagao isozyme of HSAP(26), TGFb(10), TGFbI,II,R(19) + 1, t 14, + 18, t(21q,5p), t(22q,8p) nd nd -11p13-15, actin, TGFb1, b2, b3
SK-GT-2 hyperdiploid 58
nd
nd nd
nd
nd
nd
mut(34)
SK-GT-4 hyperdiploid 59
nd
nd nd
nd
nd
nd
mut(34)
nd nd
nd
nd
nd
mut(34)
SNU-1
near diploid
47
nd
nd na
amp
nd
nd
wt(38)
nd nd PDGFA, nd TGFa PDGFA, nd TGFa aFGF nd bFGF FGF-5 PDGFA PDGFB TGFa PDGFA nd PDGFB TGFa nd VIPR
SNU-5
hypotetraploid 89
nd
nd na
amp
nd
nd
del(38)
nd
SK-GT-5 hypotriploid
63
nd
VIPR
nd
-11p13-15, actin, TGFb1, b2, b3
nd
-11p13-15, actin, TGFb1, b2, b3
nd
Gastric Cancer
Table 3
-11p13-15 actin, TGFb1, b2, b3
CA19-9, CEA, %DMs:28. nl in N-myc, L-myc, c-sis, TAG72 c-myb CA19-9, CEA, %DMs:16. nl in N-myc, L-myc, c-sis, TAG72 c-myb,muscarine/cholinergicR, DDC, 10q26amp in DMs(21)
275
Continued on next page
Name
Ploidy
MC No Marker Chr ras c-myc erbB-2 K-sam c-met p53
SNU-16
tetraploid
92
nd
nd amp amp
St-2474 hyperdiploid St-2957 hyperdiploid St-3051 near diploid St-23132 near diploid STKM-1 hyperdiploid Takigawa hypertriploid
50 51 48 47 52 77
present present present present nd nd
nl nl nl nl nd nd
nd
nd
nd
nd nd
TSG-6 hyperdiploid 50 no name-1 hypertetraploid 96
nd nd
nd nd nd nd amp amp
nd nd
nd nd
nd nd
nd
nd
nd nd
nd
nd
nd
TMK-1
no name-2nd
amp amp amp na nd nd
nd nd nd nd nd nd
nd
nd
mut(4)
nd nd nd nd nd nd
nd nd nd nd nd nd
nd nd nd nd mut(39) nd
protein(23)nd
nd
nd
mut(5,10)
GF nd
GFR
276
Table 3 (Continued) Tumor marker Others
CA19-9, CEA, %DMs:12. %HSR:24 nl in N-myc, TAG72 L-myc, c-sis, c-myb, DDC, 10q2bamp in DMs(21) CA19-9, CEA nl in c-myb, erbB-1, c-sis nd nd CA19-9, CEA nl in c-myb, erbB-1, c-sis nd nd nd nd CA19-9, CEA nl in c-myb, erbB-1, c-sis nd nd CA19-9, CEA nl in c-myb, erbB-1, c-sis nd nd CA19-9 nd nd AFP, CEA, HCG(-), IL-6: low TPA,CA19-9: low PDGFA(11)EGFR(12) nd 17pLOH(10) APC:wt(15), Cyclin PDGFR(11) EmRNA amp(17) ERKmRNA amp(16), TGFbmRNA(31), TGFbIR(31) EGF EGFR(40) nd nd nd AFP (initial nl in hst-1 culture) nd nd CEA, Dupan VIPR
Continued on next page
Suzuki and Sekiguchi
Abbreviations: AAAD: aromatic L-amino-acid decarboxylase; AMY: amylase; AFP: a fetoprotein; amp: amplified; AT adenosine triphosphatase; bFGF basic fibroblast growth factor; CCA-1: coagulant cancer antigen-1; CEA carcinoembryonic antigen; chr: chromosome; CK cytokeratin; CK-BB: creatine kinase of brain isozyme; CT: calcitonin; CTL: cytotoxic T lymphocyte; DDC: L-DOPA decarboxylase; DM: double minute; EGF epidermal growth factor; G: gastrin; GF growth factor; GFR: growth factor receptor; HCG: human chorionic gonadotropin; HSAP: heat stable alkaline phosphatase; HSR: homogeneously staining region; ICAM-1: intercellular adhesion molecule-1; KGF keratinocyte growth factor; LAP: leucine aminopeptidase; LDH: lactic dehydrogenase; LTAP: liver type alkaline phosphatase; McNo: modal chromosome number; mut: mutated; na: not amplified; nl: within normal; NPY: neuropeptide Y; NSE: neuronspecific enolase; PG: prostaglandin; PLAP: placental alkaline phosphatase; PYY: peptide YY, SC secretory component; SLX: sialyl Lewisx; TAG72: tumor associated glycoprotein 72; TF: tissue factor; TGF transforming growth factor; TFA: tissue polypeptide antigen; VIP: vasoactive intestinal peptide; VM: vimentin; wt: wild type
Gastric Cancer
Table 3 (Continued) Subscript numbers are used to indicate source of description of the genetic change or ploidy etc. other than stated by the original report(s). Superscript numbers: 1 = Fujiwara T et al. 1993,2 = Matsushima Y et al. 1994, 3 = Liu SQ et al. 1995,4 = Yanagihara K, Tsumuraya M. 1992.5 = Matter et al. 1992,6 = Kuniyasu H et al. 1991,7 = Yanagihara K et al. 1993a, 8 = Hattori Y et al. 1990,9 = Kuniyasu H et al. 1992; 10 = Motozaki T et al. 1992a, 11 = Tsuda T et al. 1989, 12 = Yoshida K et al. 1990a, 13 = Sugiyama Y et al. 1996, 14 = Sekiguchi M et al. 1983, 15 = Nishimura K et al. 1995, 16 = Kiyokawa E et al. 1979, 17 = Akama Y et al. 1995,18 = Takekura N et al. 1990,19 = Horimoto M et al. 1995,20 = Yamamoto M et al. 1996,21 =Mor et al. 1991,22 = Adachi T et al. 1997,23 = Tokumitsu S et al. 1979,24 = Kameda T et al. 1990,25 = Takeuchi K et al. 1996,26 = Aizawa K, 1988,27 = Fukushiga S et al. 1986,28 = Nagamine K et al. 1996,29 = Yoshida K et al. 1989,30 = Dippold WG et al. 1987,31 = Ito M et al. 1992b, 32 = Tanimoto H et al. 1991,33 = Durrant LG et al. 1991, 34 = Nabeya Y et al. 1995,35 = Yashiro M et al. 1995b, 36 = Inoue T et al. 1997,37 = Faletto L et al. 1992,38 =Kim J-H et al. 1991,39 = Takahashi M et al. 1997,40 = Aizawa K et al. 1994
277
278
Suzuki and Sekiguchi
obtained from about 30% of samples, but only a small fraction of these cultures become continuous cell lines. The main causes for the failure of long term culture are fibroblast overgrowth or failure to passage. However, the cell lines that have been established are generally easy to maintain as monolayers or suspensions in culture. The mean population doubling time of the 44 cell lines for which it is reported is 33.56 ± 11.37 (range 17–66) hrs. MKN-7 has a doubling time of 66 hrs (Hojo 1977) and TAKIGAWA has an exceedingly long population doubling time, about 158hrs (Sekiguchi et al. 1995). The stage of the cancer appears to influence the success of cell line establishment, since about 85% of stomach cancer cell lines are derived from secondary sites, mainly from nodal or liver metastases and cancerous effusions (see Table 1). Rarer sites include cerebrospinal fluid (JR-St, Terano et al. 1991), bone (MATSUURA, Yasumoto et al. 1992) and Krukenberg tumor (KS-1, Whelan et al. 1988). A few lines grew in serum-free culture from the initiation of cultivation; including ISt-1 (Terashima et al. 1991), HSC-43 (Yanagihara et al. 1993b) and no name-1 (Kawaguchi et al. 1991), and it is possible to adapt MKN-28, MKN45 and MKN-74 to serum-free conditions (Suzuki unpublished). A serum-free environment is particularly suitable for investigations on cancer cell products, such as HCG or alkaline phosphatase by SCH (Kameya et al. 1975); serotonin, neutropeptide Y, and peptide YY by ECC-12 (Fujiwara et al. 1993); a-fetoprotein by GK (Imai et al. 1991), ISt-1 (Terashima et al. 1991), and TAKIGAWA (Sekiguchi et al. 1995); a-amylase by KMK-2 (Nomura et al. 1980); and gastrin by HSC-39 (Yanagihara et al. 1991) and MKN45G (Watson et al. 1990). In addition, serum-free culture conditions are useful for analysis of tumor markers, growth factors or cytokines, which are produced in vitro by most of the gastric cancer cell lines (Table 3).
3.
DO THE CELL LINES AVAILABLE REPRESENT THE CLINICAL DISEASE?
In many industrialized nations the incidence of stomach cancer has shown a marked decrease, but this cancer remains the second most common cause of cancer-related deaths worldwide. Among stomach cancers, however, there has been a steady rise in the incidence of adenocarcinoma of the proximal stomach (cardia) and the gastroesophageal junction. In contrast, the incidence of distal (antral) cancers has remained largely unchanged or has decreased slightly (Blot et al. 1991). More men than women are affected. The stage of stomach cancer remains the most important determinant of prognosis (Alexander et al. 1993) and a comprehensive tumor-node-metastasis (TNM) staging system (UICC, 1987) has been used in the USA and Europe. The TNM staging of stomach cancer, however, has not been prevalent in Japan and instead the proposals of the Japanese Research Society for Gastric Cancer (1993) have been adopted.
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279
Superficial and surgically curable gastric carcinomas produce few symptoms. Consequently, at the time of presentation, the disease is often locally advanced or metastatic (Alexander et al. 1993), although endoscopic intervention for mucosal or superficial carcinoma is widely and successfully performed. Tumor markers are not useful for the diagnosis of stomach carcinoma at an early stage, but carcinoembryonic antigen (CEA) levels are high in 40 to 50% of patients with metastases, while similar elevations are seen in only 10 to 20% of patients with surgically resectable disease (Posner and Mayer, 1994). In addition, a-fetoprotein (AFP) and CA19-9 levels are elevated in 30% of patients with gastric carcinoma (Posner and Mayer, 1994), often in patients with incurable disease. The characteristics of the stomach cancer cell lines shown in Tables 1–3 indicate that cell lines have been established from both sexes, primary cancers, various metastatic lesions, malignant effusions and more rarely xenografts. Cell lines which produce tumor markers in vitro are common. Among 16 cell lines originating from the primaIy site, there are four cell lines from cardiac lesions, including one cardiac carcinoma (SKGT-1, Altorki et al. 1993), two carcinomas of esophagocardiac junction (SKGT-5, Altorki et al. 1993; TE-7, Akaishi 1989) and one Barrett carcinoma (SKGT-4, Altorki et al. 1993). There is one cell line from antral carcinoma (MKK-1, Ihara, 1992). However, most reports have not described the precise site of the primary from which the cell line was obtained. The cell lines originate from various histological subtypes (Table 2). Histological concordance between cell line xenografts and the primary is rare, and only seen in xenografts of MKN-28 (Hojo, 1977), NCI-N87 (Park et al. 1990) and SKGT-1, -3 and -4. Cell lines derived from T0 or T1 stage carcinoma are lacking. The earliest stage is T2 carcinoma, from which SKGT-4 and -5 and ST23132 (Vollmers et al. 1993) were established. Cell lines from earlier stages are needed, and such cultures might be established, since cell lines have been derived from biopsy specimens of stomach cancer (AGS, Barranco et al. 1983; HSC-43, Yanagihara et al. 1993b). Biopsy specimens of early stage carcinoma would be valuable, although contamination with microorganisms must be overcome for cultivation. Authentication of the cell lines listed in Table 1 is limited to karyotyping and morphological features.
4.
COMPARISON OF CELL LINES WITH THE TISSUE OF ORIGIN
The gastric mucosa in the human is subdivided into pyloric antral, fundic and cardiac mucosa. The surface of the mucosa is lined by superficial mucus cells, together with various specialized cells. The cardiac mucosa harbors cardiac glands, the fundic mucosa contains parietal cells, neck mucus cells and chief cells and the antral mucosa is characterized by pyloric glands. In addition, endocrine cells are scattered throughout the mucosa, most frequently in the
280
Suzuki and Sekiguchi
pyloric mucosa. Amongst the endocrine cells, gastrin-producing cells and somatostatin-producing cells are most abundant. Gastric mucosa is often affected by chronic inflammation and intestinal metaplasia, which is defined as the replacement of antral or fundic gastric mucosa by glands composed of epithelium resembling that of intestine. The metaplasia has been classified into complete (small intestinal) and incomplete (colonic) types (Taglbjaerg and Nielsen 1978). Also, precancerous lesions, chronic atrophic gastritis with or without metaplasia (Morson 1955), intestinal metaplasia, adenoma (Hirota et al. 1984) and hyperplastic polyp (Diabo et al. 1987) are described. Correa et al. (1970) and Correa (1988) proposed the hypothesis of multistage carcinogenesis in the stomach, suggesting that the progression from normal epithelial cells to carcinoma cells involves at least six stages: superficial gastritis, chronic atrophic gastritis, intestinal metaplasia of the complete type followed by incomplete type, gastric adenoma, dysplasia, and carcinoma. More than 90% of stomach cancers are adenocarcinomas, and most of the remainder are non-Hodgkin’s lymphoma or leiomyosarcomas (Rotterdam 1989). Cell lines established from these other gastric tumors are lacking (Sekiguchi and Suzuki 1994). Adenocarcinoma can be subdivided into two categories: an intestinal type characterized by cohesive neoplastic cells forming gland-like tubular structures, and a diffuse type in which cell adhesion is absent, so that individual cells infiltrate and thicken the stomach wall without forming a discrete mass (Laurén 1965). In Japan, adenocarcinoma of the stomach is graded as papillary adenocarcinoma (pap), well-differentiated tubular adenocarcinoma (tubl), moderately differentiated tubular adenocarcinoma (tub2), poorly differentiated adenocarcinoma (por) with solid (porl) or non-solid growth (por2), signet-ring cell carcinoma (sig) and mucinous adenocarcinoma (muc) (Jpn Res Soc Gastric Cancer, 1993). In addition, a special type composed of adenosquamous carcinoma, squamous cell carcinoma and carcinoid is included. The pap, tubl, tub2 and largely muc are identical to intestinal type, and por and sig to the diffuse type described by Laurén. Carcinoma cell lines have been established from both intestinal and diffuse type (see Table 2). There is also an adenosquamous carcinoma cell line (MKN1), endocrine cell carcinoma lines (ECC10 and ECC12, Ishihara 1992) and a choriocarcinoma cell line (SCH, Oboshi 1975) (see Tables 1 and 2). AFP-producing gastric carcinoma, initially termed as hepatoid carcinoma and later named as AFP-producing adenocarcinoma (Ishikura et al. 1986), is characterized by a poorer prognosis than that of other gastric adenocarcinomas because of its frequent lymphatic and vascular invasion. The cell lines GK, GS, ISt-1, and TAKIGAWA were established from gastric cancer patients with high serum AFP and in vitro AFP-production is confirmed except for GS. In general, morphological differentiation becomes poorer with subcultivation (see Table 2). For example, xenografts of MKN-74, which was tub2 type in origin, were adenocarcinomas with a tubular structure during early culture stages, but after prolonged culture became poorly differentiated carcinomas
Gastric Cancer
281
(Motoyama et al. 1986). Xenografts of MKN-1, which originated from an adenosquamous cell carcinoma, showed only squamous cell carcinoma (Suzuki, unpublished data). TAKIGAWA, derived from a nodal metastasis from a tub2 tumor, contains squamous cell carcinoma when growing as a xenograft (Sekiguchi et al. 1995). The close association of Epstein-Barr virus (EBV) with a subset of gastric carcinomas has been noted, especially in carcinoma with lymphoid stroma or medullary carcinoma with lymphocytic infiltration (Minamoto et al. 1990) or undifferentiated gastric carcinoma with intense lymphoid infiltration. The EBV genome has also been detected in tubular (intestinal) type carcinoma of the stomach (Shibata and Weiss 1992, Fukayama et al. 1993, Tokunaga et al. 1993a,b). The overall frequency of EBV association with gastric cancer is 6.7% in Japan (Tokunaga et al., 1993a) and 16% in the USA (Shibata and Weiss 1992). The frequency rises to 20.1 % in poorly differentiated gastric adenocarcinomas in Japanese (Ojima et al. 1997). The EBV-associated cancers occur equally in cardia and body (Fukayama et al. 1993) or predominantly in the corpus or antrum (Shibata and Weiss 1992). More men than women are affected (ratio approximately 3 : 1). Overexpression of p53 protein is infrequent in EBV-associated gastric tumors (Ojima et al. 1997). The biologic importance of the EBV genome in gastric cancer is debatable. EBV infection seems to occur irrespective of malignant transformation, since dysplastic and even metaplastic gastric glands also harbor EBV genomes (Hayashi et al. 1996, Arikawa et al. 1997). Systematic surveillance for EBV genomes in gastric cancer cell lines has not been done, but KATO-III, HS756T and AGS are EBV-negative by PCR (Shibata and Weiss 1992). The RF-48 cell line is EBV-positive but the cell line, derived from malignant ascites (Shaver et al. 1989), is keratin negative and leukocyte common antigen (CD45) positive by immunohistochemical analysis (Shibata and Weiss 1992), suggesting a lymphoid origin for this tumor. Recently two EBV-positive cell lines, GT38 and GT39, have been established (Tajima et al. 1998). GT38 is a cancer cell line and GT39 is an immortalized gastric epithelial cell line. These cell lines might be useful for elucidating the relationship between gastric carcinoma and EBV. The MKN-28 cell line is susceptibie to infection and proliferation of herpes simplex virus (Kodama et al. 1996a) or adenovirus type 11 (Kodama et al., 1996b). An association of H. pylori infection and gastric carcinogenesis has been observed. The International Agency for Research into Cancer has designated H. pylori as a group 1 carcinogen (IARC Monogr 1994). Research on H. pylori with gastric cancer cell lines has not been reported.
5.
GENETIC PROPERTIES OF GASTRIC CANCER
In addition to frequent LOH (loss of heterozygosity) on chromosome 17p (p53 locus) (Sano et al. 1991), several other chromosomal loci are deleted in
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gastric cancers. LOH at lq and 18q (the locus for DCC, deleted in colon cancer) is frequently detected in well-differentiated carcinomas (Sano et al. 1991, Uchino et al. 1992). Deletion on 5q, separate from the APC locus, 9p without p16 gene mutation, or 7q is also noted in differentiated carcinomas (Sano et al. 1991, Sakata et al. 1995, Tamura et al. 1996, Nishizuka et al. 1997). Loss of lp is relatively common in advanced poorly differentiated gastric carcinomas (Sano et al. 1991) and LOH at 7q within the BRCA1 locus is common in scirrhous gastric cancer, especially in young patients (Kuniyasu et al. 1994). Detailed chromosomal analysis of the gastric cancer cell lines is generally lacking, but LOH at 11p13-15 is detected in the SK-GT series. The molecular genetics of gastric cancers has been reviewed (Wright and Williams 1993, Fuchs and Mayer 1995, Tahara 1995, Yokozaki et al. 1997). An accumulation of genetic alterations is found in both intestinal- and diffusetype gastric cancers. Unlike colon and pancreatic cancers, gastric cancers rarely have mutations in the ras oncogene (Nanus et al. 1990). Mutations in the c-erbB-2, APC (adenomatous polyposis coli) and p53 genes are found mainly in intestinal-type gastric cancers. The frequency of allelic deletion of MCC (mutated in colon cancer), APC and p53 tumor suppressor genes has been reported to be 33, 34 and 64% of gastric cancers, respectively (Rhyu et al. 1994). p53 gene mutations have been detected in early stage cancers (Yokozaki et al. 1992, Uchino et al. 1993). K-sam and c-met genes are detected in diffuse type gastric cancers (Yokozaki et al. 1997). These disparities between the mutations associated with intestinal and diffuse types of gastric cancer indicate a different sequence of genetic events. Overexpression of the ERK gene, an EPH family receptor protein tyrosine kinase, has been reported in gastric cancer cell lines irrespective of intestinal type or diffuse type origin (Kiyokawa et al. 1994, Table 3). Gastric cancers express growth factors, growth factor receptors, gut hormones and cytokines, including EGF, TGF-α, amphiregulin, cripto, TGFβ1, PDGF, IGF-I or II, bFGF, gastrin, serotonin, IL-1a, IL-6 and IL-8 (Tahara et al. 1994, Tanimoto et al. 1991, Ito et al. 1993, Yokozaki et al. 1997, see Table 3). Of these, the EGF family of growth factors (EGF, TGF- a, cripto, and amphiregulin) are the most frequent growth enhancers seen in gastric cancer. EGF stimulation of EGF-R positive cancer cells occasionally causes growth inhibition, for example in the GS cell line (see Table 3). In addition to the EGF family, poorly differentiated gastric cancers, including scirrhous type, frequently show overexpression of PDGF, IGF-II and bFGF (Tahara et al. 1994). The growth rates of MKN45 and TMK-1 are stimulated by gastrin or combined stimulation with gastrin, TGFa and IGF (Ochiai et al. 1985, Watson et al. 1988, Durrant et al. 1991). Insulin, glucagon and hydrocortisone also stimulate the growth of OCUM-1 (Kubo et al. 1991b). IL-l a or IL-6 acts as an autocrine and/or paracrine growth factor for some gastric cancer cell lines (Ito et al. 1993, 1997). Growth stimulation of gastric cancer cell lines by IL-3 or granulocyte-macrophage colony stimulating factor has also been described (Dippold et al. 1991). TGF-β, an inhibitor of growth, is commonly overexpressed in gastric cancer, especially in scirrhous type, and
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in TMK-1, MKN-1, MKN-7, MKN-28 and MKN-74 (Yoshida et al. 1989). TGF- bl-activator protein, a type of serine protease, has been identified from KATO-III. This cell line produces inactive TGFb1 (Horimoto et al. 1995), but its culture medium contains active TGF-b1. Expression or reduction of TGFb1 receptor has been reported in gastric cancers and cell lines (Ito et al. 1992a,b, see Table 3), and genetic alterations including deletions and amplifications in the type II TGF-b receptor have been detected in four (SNU5, SNU-668, SNU-601, and SNU-719) of seven gastric cancer cell lines that are resistant to TGF-b1-mediated growth inhibition (Park et al. 1994). Mutation of the TGF-b II receptor gene has also been described in primary gastric cancers (Myeroff et al. 1995). The precise biological role of TGF-b1 and its receptor system in gastric cancer remains to be elucidated.
6.
CELL LINE CROSS-CONTAMINATION
Cross-contamination in gastric cancer cell lines has not been systematically studied. The MKN-7 cell line has been mislabeled. The mislabeled MKN-7 grew in suspension culture and has a lymphoid cell morphology. In addition, surface marker for leukocyte common antigen (CD45) and surface pan-B cell marker Leu12 were positive (Suzuki personal observation, 1987). From this evidence, the cell line is regarded as lymphoblastic and the “MKN-7” cell line has been deleted from the list of cell lines in the JCRB cell bank. The epithelial nature of gastric cancer cell lines should be confirmed using pan-cytokeratin staining. Mycoplasma contamination was once observed in the MKN series (Suzuki, personal observation) but they are now free from infection. Generally, however, screening for Mycoplasma contamination has not been done. Under these circumstances, it should be assumed that the cell lines are Mycoplasma positive until proven otherwise. There appear to be some interlaboratory differences between gastric cancer cell lines. MKN-74 exhibits a non-sense mutation in the p53 oncogene in one laboratory (Motozaki et al. 1992), but no mutation in the same gene in another (Matter et al. 1992). The MKN-74 from the latter laboratory was confirmed to have wild-type p53 in an independent laboratory (Nabeya et al. 1995). Another example is MKN-45. Watson et al. (1989a) reported that MKN-45 possesses 3×103 gastrin receptors per cell and that the growth of the cells is augmented by gastrin stimulation (Watson et al. 1989b). In contrast, MKN-45 lacks expression of the gastrin receptor gene in another report (Matsushima et al. 1994). Watson et al (1990) selected a subline MKN-45G from MKN-45. The subline produces gastrin in vitro at twice the amount of the parent cell line, but its gastrin receptor number remains almost the same. According to Hojo’s original description of MKN-45, the material for cultivation contained a few tumor cells with numerous neurosecretory granules by electron microscopy. The shape and size of the dense-cored granules shown in the figure in the paper resemble those of gastrin granules. Therefore, Watson et al. (1990)
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succeeded in cultivating predominantly cancer cells with a neuroendocrine nature, while MKN-45 in other laboratories may have lost its endocrine character.
7.
CELL LINES WITH SPECIAL FEATURES
HSC-39 was established from malignant ascites and HSC-40A from a xenotransplanted tumor in a nude mouse formed by inoculation of cancer cells from the same ascites (Yanagihara et al. 1991). Subclones of HSC-39, HSC40A, HSC-41, HSC-42, HSC-43, HSC-45 and SH101 are also described as HSC-39K6, HSC-40A1, HSC-41E6, HSC-42H, HSC-43C1, HSC-45M2 and SH101-P4, respectively (Yanagihara et al. 1993a). Ten subclones of AGS show similar biological characteristics (Barranco et al. 1983). Cell lines derived from germ cell tumors of the stomach are reported (see Table 1). SCH was derived from a primary choriocarcinoma in a man’s stomach and has biological features similar to those of gestational choriocarcinoma. The primary tumor of the stomach, however, contained a portion of tubular adenocarcinoma. Therefore the choriocarcinoma probably originated from the adenocarcinoma through dedifferentiation of the cancer cells (Oboshi 1975). Another germ cell tumor cell line is HOGT, cultured from a tridermal mature teratoma in an infant. HOGT cells have a fibroblast-like morphology in vitro, but xenografts in nude mice have the histological appearance of tridermal teratoma (Ishiwata et al. 1985). Despite the rare occurrence of endocrine cell carcinoma (ECC), two cell lines from gastric ECC exist, ECC10 and 12. Human gastric cancers usually form more prostaglandins than their associated normal mucosa through active cyclo-oxygenase 2 (Soydan et al. 1997). In addition, stimulation by hepatocyte growth factor augments prostaglandin production in TMK-1 (Hori et al. 1993). Telomerase RNA is expressed in gastric cancer and intestinal metaplasia, indicating that telomerase expression is an early event in gastric carcinogenesis. The gastric cancer cell lines, TMK-1, KATO-III HSC-39 and five cell lines of the MKN series exhibit increased expression of telomerase RNA (Kuniyasu et al. 1997). With poorer differentiation, epithelial cancer cells lose cohesiveness and show fibroblastic morphology or become round cells. In accordance with this phenomenon, cancer cells rarely express both epithelial and mesenchymal intermediate filaments. HLN-GAC-5 is an example (Morikawa et al. 1988), since the cell line expresses keratin (epithelial cell marker) and vimentin (mesenchymal cell marker) simultaneously. More frequent occurrences are abnormalities in adhesion molecules or their related proteins such as Ecadherin, and a- and b-catenins. Scirrhous stomach cancers and/or poorly differentiated stomach cancers frequently have abnormalities (low expression or absence) of E-cadherin and/or a-catenin (Shimoyama and Hirohashi 1991, Ochiai et al. 1994, Yasui et al. 1995). Mutations in the E-cadherin gene are
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observed in 50% of poorly differentiated gastric cancers (Becker et al. 1994). Amino acid deletion in the NH2-terminal region of the b-catenin molecule has been detected in HSC-39 (Oyama et al. 1994). Due to this abnormality, acatenin cannot bind to E-cadherin, resulting in poor cohesiveness of the cancer cells. Chemotherapy for stomach cancer has limited value, although expression of the multidrug resistance protein in gastric adenocarcinoma is low in frequency, being about 34% (Takebayashi et al. 1998). A few reports on the drug sensitivities of the cell lines have been documented (Motoyama et al. 1979, Akiyama et al. 1988, Whelan et al. 1988). A cisplatin-resistant cell line without mdr-1 gene involvement has been established (Nitta et al. 1997). A number of regimens for modulation of cancer cell growth or viability in vitro have been reported, as shown in Table 4. FAS antigen, an apoptosis receptor protein, is expressed in some differentiated adenocarcinoma cell lines (MKN-7, -28 and –4), the poorly differentiated or scirrhous carcinoma cell lines (TMK-1, KATO-III, HSC-39, and MKN-45) and the adenosquamous carcinoma cell line (MKN-1) (Ito et al. 1997). Clear differences of FAS antigen expression exist between intestinal and diffuse type stomach carcinomas, being high in diffuse type carcinoma (50% or more) and very low or absent in intestinal type carcinoma (Vollmers et al. 1997a). Expression of the p53 protein in these two types of carcinoma reveals the reverse relationship to that of FAS antigen, indicating further biological differences between these two types. Models for metastasis or peritonitis carcinomatosa have been established using stomach cancer cell lines. Liver metastasis by inoculation of KKLS, KATO-III or MKN-45 in the gastric wall of nude mice (Watanabe et al. 1993, Watanabe 1994, Watanabe et al. 1997) and blood-borne liver metastasis in chick embryo by inoculation of KKLS in chorioallantoic membrane vein (Tsuchiya et al. 1994) have been described. In the former model, intact cancer tissue (xenograft of the cell line) implanted at an orthotopic site is more likely to result in liver metastasis than inoculation of a cell suspension in the nude mouse stomach (Furukawa et al. 1993). In the chick embryo model, metastasis is associated with collagenase activity (Tsuchiya et al. 1994) or reduced expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in the cancer cells (Tsuchiya et al. 1993). Metastasis is inhibited by transfection of the TIMP1 gene into KKLS cells in the chick embryo model (Tsuchiya et al. 1993) or in nude mice (Watanabe et al. 1996). The model of peritonitis carcinomatosa has been produced with the cell line OCUM-2MD3, a subline of OCUM-2M. The cell line was isolated from a peritoneal deposit in a nude mouse, formed by the implantation in the mouse stomach of a subcutaneous OCUM-2M xenograft from another mouse (Yashiro et al. 1994a). In this model, various factors promote invasive growth or migration of the cancer cells, including CD44 (Nishimura et al. 1995a), TGFβ1 (Yashiro et al. 1995b), a2b1 integrin or a3b1 integrin (Nishimura et al. 1996), interaction of OCUM-2MD-3 with peritoneal or gastric fibroblasts (Yashiro et al. 1994b, Yashiro et al. 1996a), HGF and TGFb produced by gastric fibroblasts (Inoue et al. 1997), exogenous HGF and TGFb (Shibamoto et al.
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Table 4 Factors modulating the growth of gastric cancer cell lines
Cell line
Regimen
Enhancement (E) Growth inhibition (I) or Apoptosis (A)
EGF 5-FU-sensitivity, E flavopiridol MMC-sensitivity, E (PKC inhibitor) paclitaxel-sensitivity, E 8-chloro-cyclic AMP I TMK-1 KATO-III (analog of CAMP) MKN-7 MKN-28 MKN-45 MKN-74 TMK-1 erbstatin EGF-induced or serum(inhibitor of tyrosine kinase) stimulated growth MKN-1 MKN-7 MKN-28 MKN-45 MKN-74 KATO-III PGE2,PGE1,PGE2a, I modified PGE1, dbcAMP, forskolin, calcitonin MKN-1 transfection of wild type p53 I MKN-28 TMK-1 TGFb I I, also in vivo MKN-45 RC-3095 (GRP antagonist) RC-160 (somatostatin analog) AGS transfer of EGFR antisense I KKLS MKN-28 I MKN-28 TNF-a MKN-45 I KATO-III A HSC-39 TGFb1 A TSG-6 MKN-74
HSC-43 HSC-39 MKN-74 KATO-III NuGC-3 SC-M1 HPEGAC-2 MKN-74 MKN-45 KATO-III TMK-1 ST23132
Reference Aizawa et al. 1994 Schwartz et al. 1997 Takanashi et al. 1991
Takekura et al. 1991
Nakamura et al. 1991 Motozaki et al. 1992 Ito et al. 1992a Pinski et al. 1994
Sawada et al. 1997 Sugiyama et al. 1996 Yanagihara and Tsumuraya 1992
ADR A 5-FU A TGFb1 A paclitaxel (inhibitor of micro- A tubule assembly) IFN-b and IFN-g A
Ikeguchi et al. 1995 Yamamoto et al. 1996 Yamamoto et al. 1996 Chang et al. 1996
IFN-g and anti FAS
Ito et al. 1997
A
A SC-1 (monoclonal antibody) A in the coculture OCUM-2M tranilast (fibroblast growth inhibitor) with fibroblast CDDP-induced A, E STKM-1 caffeine
Nagao et al. 1997
Vollmers et al. 1997b Yashiro et al. 1997 Takahashi et al. 1997
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1992) or both peritoneal fibroblasts and b1-integrin (Yashiro et al. 1996b). Serum-free conditioned medium from OCUM-2MD3 contains factor(s) which cause rounding-up of mesothelial cells and growth of submesothelial fibroblasts resulting in peritoneal fibrosis (Yashiro et al. 1996c, Yashiro et al. 1996d). Gastric fibroblasts secrete a growth factor (about 10,000 in MW), which is distinct from those produced by the poorly differentiated cancer cell lines OCUM-1, OCUM-2M and KATO-III (Yashiro et al. 1996a). Production of collagen-degrading metalloproteinases in association with tissue inhibitors of metalloproteinases (Sato et al. 1992) or several kinds of matrix serine proteinases by human gastric cancer cell lines (Koshikawa et al. 1992) is also associated with cancer cell invasion. Invasion and metastasis consist of a series of linked, sequential processes (Fidler 1990). Membrane-type matrix metalloproteinases, which assist the activation of pro-matrix metalloproteinase-2 and are found in gastric carcinomas (Nomura et al. 1995), have a role in gastric tumor metastasis (Sato et al. 1994, Sato and Seiki 1996). Gastric cancer cells produce procollagen. Sakakibara et al. (1982) first reported that cloned MKN-28 and MKN-74 produce procollagen in vitro and in their xenografts. Procollagen mRNA is detectable in TMK-1, KATO-III, MKN-1, MKN-28 and MKN-45 (Yoshida et al. 1990a). In addition, KATO-III, MKN-28 and MKN-45 can stimulate collagen synthesis by human skin fibroblasts (Naito et al. 1984). Growth factors also modulate such functions. EGF or TGFa cause up-regulation of the procollagen gene as well as interstitial collagenase and stromelysin (Yoshida et al. 1990a). Interaction between cancer cells and interstitial cells seems to be crucial in controlling the behavior of the cancer cells in vivo.
REFERENCES Adachi T et al. Nisshoshi 94: 513, 1997. Aizawa K. Niigata Igakkai Zasshi 102: 8, 1988. Aizawa K et al. Jpn J Surg 24: 420, 1994. Akagi T, Kimoto T. Acta Med Okayama 30: 215, 1976. Akaishi T Human Cell 2 (Suppl) 24: 330, 1989. Akama Y et al. Jpn J Cancer Res 86: 617, 1995. Akiyama S et al. Jpn J Surg 18: 438, 1988. Alexander H et al. in De Vita V, Hellman S, Rosenberg S (eds) JB Lippincott, Philadelphia, 1993, pp. 818–848. Altorki N et al. Cancer 72: 649, 1993. Arikawa J et al. Pathol Int 47: 730, 1997. Arimura A et al. Human Cell 4: 67, 1991. Barranco SC et al. Cancer Res 43: 1703, 1983. Becker KF et al. Cancer Res 54: 3845, 1994. Blot WJ et al. JAMA 265: 1287, 1991. Chang Y-F et al. Cancer 77: 14, 1996. Correa P et al. JNCI 44: 297, 1970.
288
Suzuki and Sekiguchi
Correa P. Cancer Res 48: 3554, 1988. Deguchi Y et al. Proc Jpn CancerAssoc 49: 220,1990. Diabo M et al. Am J Gastroenterol 82: 1016, 1987. Dippold WG et al. EurJ Cancer Clin Onclo 23: 697, 1987. Dippold WG et al. Gastroenterol 100: 1338, 1991. Dobrynin Ya V. JNCI 31: 1173,1963. Durrant LJ et al. Br J Cancer 63: 67,1991. Faletto L et al. Oncogene 7 1149, 1992. Fidler IG. Cancer Res 50: 6130, 1990. Fuchs CS, Mayer RJ. New Engl J Med 333: 32, 1995. Fujiwara T et al. Int J Cancer 54: 965, 1993. Fukayama M et al. Lab Invest 71: 73, 1994. Fukuda T et al. Am J Gastroenterol 83: 1176, 1988. Fukushige S et al. Mol Cell Biol 6: 955, 1986. Furukawa T et al. Cancer Res 53: 1204,1993. Hattori Y et al. Proc Natl Acad Sci USA 87: 5983, 1990. Hayashi K et al. Am J Gastroenterol 91: 1481, 1996. Hirota E et al. In Ming SC (ed), Praeger, New York, 1984, pp. 235-252. Hojo H. Niigata Igakkai Zasshi 91: 737, 1977. Hori T et al. FEBS Letters 334: 331, 1993. Horimoto M et al. Br J Cancer 72: 676, 1995. Huang C-D et al. Human Cell 4 (Suppl): 51,1991. IARC Monogr Carcinog Risks Hum 61: 218, 1994. Ihara A. Nisshoshi 89: 2645,1992. Ikeguchi M et al. in Nishi M, Sugano H, Takahashi T (eds), Monduzzi Editore, Bologna, 1995, pp. 1611-1616. Imai Y et al. Proc Jpn Cancer Assoc 49: 220, 1990. Imai Y et al. Proc Jpn CancerAssoc 50: 173, 1991. Imanishi H et al. Proc Jpn Cancer Assoc 47: 396, 1988. Imanishi K et al. Br J Cancer 59: 761, 1989. Inoue T et al. Jpn J Cancer Res 88: 152, 1997. Ishihara N. Niigata Igakkai Zasshi 106: 21, 1992. Ishikura H et al. Cancer 58: 119,1986. Ishiwata I et al. Exp Pathol 27: 143, 1985. Ishiwata I et al. Human Cell 2 335, 1989. Ite K et al. Proc Jpn Cancer Assoc 45: 241, 1986. Ito H et al. In Tahara E (ed), Springer, Tokyo, 1997, pp.81–92. Ito M et al. Cancer Res 52: 295, 1992a. Ito M et al. Jpn J Cancer Res 83: 86, 1992b. Ito R et al. Cancer Res 53: 4102, 1993. Ito R et al. Jpn J Cancer Res 88: 953, 1997. Jpn Res SOC Gastric Cancer: The General Rules for the Gastric Cancer Study, 12th ed, 1993. Kameda T et al. Cancer Res 50: 8002,1990. Kameya T et al. Cancer Res 35: 2025, 1975. Kanazawa S et al. Cancer 50: 1775,1982. Kawaguchi T et al. Proc Jpn CancerAssoc 50: 173,1991. Kim B, Ohno T. RGB Genaral Catalog 3: 140,1997. Kim J-H et al. JNCI 83: 938, 1991. Kiyokawa E et al. Cancer Res 54: 3645,1994. Kodama E et al. Microbiol Immunol 40: 359, 1996a. Kodama E et al. Antiviral Res 31: 159, 1996b.
Gastric Cancer
289
Koshikawa N et al. Cancer Res 52: 5046, 1992. Kotou T et al. Proc Jpn Res Soc Gastroenterol Carcinogenesis 3: 273, 1991. Kubo T. Nihon Gekagakkai-shi 92: 1451,1991a. Kubo T et al. J Jpn Soc Cancer Ther 26: 2387, 1991b. Kubota H et al. Gastroenterol Jpn 23: 371, 1988. Kuki K et al. Nichi-idaishi 52 123, 1985. Kuniyasu H et al. Jpn J Cancer Res 82: 969, 1991. Kuniyasu H et al. BBRC 189: 227, 1992. Kuniyasu H et al. Int J Cancer 59: 597, 1994. Kuniyasu H et al. Jpn J Cancer Res 88: 103, 1997. Laboisse CL et al. Cancer Res 42: 1541, 1982. Laurén P. Acta Pathol Microbiol Scand 64: 31, 1965. Lin C-H et al. Chin Med J 97: 831, 1984. Liu SQ et al. Nature Med 1: 267, 1995. Matsushima Y et al. Jpn J Cancer Res 85: 819, 1994. Matter R et al. Oncol (Life Sci Adv) 11: 7, 1992. Minamoto T et al. Cancer 55: 945,1990. Mor 0 et al. Nucleic Acids Res 19: 117, 1991. Morikawa S et al. Proc Jpn Cancer Assoc 47: 294, 1988. Morson BC. Br J Cancer 9: 377, 1955. Motoyama T et al. Acta Med Biol 27: 49, 1979. Motoyama T et al. Acta Pathol Jpn 36: 65, 1986. Motozaki T et al. BBRC 182 215,1992a. Motozaki T et al. Cancer Res 52: 4335, 1992b. Myeroff LL et al. Cancer Res 55: 5545, 1995. Nabeya Y et al. Int J Cancer 64: 37, 1995. Nagamine K et al. Eur J Biochem 236: 476,1996. Nagao S et al. Human Cell 10: 95, 1997. Naito Y et al. Virchows Archiv [Cell Pathol] 46: 145, 1984. Nakamura A et al. Gastroenterol 199: 910, 1991. Nanus DM et al. Gastroenterol 98: 955, 1990. Nishimura S et al. In Nishi M, Sugano H, Takahashi T (eds), Monduzzi Editore, Bologna, 1995a, pp. 975-979 Nishimura K et al. Int J Oncol 7: 587, 1995b. Nishimura S et al. Br J Cancer 74: 1406, 1996. Nishizuka S et al. Br J Cancer 76: 1567, 1997. Nitta A et al. Cancer Chemother Pharmacol 40: 94, 1997. Nomura H et al. JNCI 64: 1015,1980. Nomura H et al. Cancer Res 55: 3263, 1995. Nozue M et al. Human Cell 2 (Suppl): 89,1989. Oboshi S, in Oboshi S and Sugano H (eds), Asakura, Tokyo, 1975, pp. 192-202. Ochiai A et al. Jpn J Cancer Res (Gann) 76: 1064, 1985. Ochiai A et al. Jpn J Cancer Res 85: 266, 1994. Oda K et al.Am J Pathol 143: 1063, 1993. Ojima H et al. Jpn J Cancer Res 88: 262, 1997. Oyama T et al. Cancer Res 54: 6282,1994. Park J-G et al. Cancer Res 50: 2773, 1990. Park K et al. Proc NatlAcad Sci USA 91: 8772, 1994. Pinski J et al. Int J Cancer 57: 574, 1994. Posner MR, Mayer RJ. Hematol Oncol Clin North Am 8: 533,1994. Rhyu M-G et al. Gastroenterol 106: 1584, 1994.
290
Suzuki and Sekiguchi
Rotterdam H. In Rotterdam H, Enterline HT (eds), Springer-Verlag, New York, 1989, pp. 142–204. Sakakibara K et al. Cancer Res 42: 2019, 1982. Sakata K et al. Jpn J Cancer Res 86: 333, 1995. Sano T et al. Cancer Res 51: 2926, 1991. Sato H et al. Oncogene 7: 77, 1992. Sato H et al. Nature 370: 61, 1994. Sato H, Seiki MJ. Biochem 119: 209,1996. Sawada H et al. In Siewest JR, Rader JD (eds), Mondezzi Editore, Bologna; 1997, pp. 677–681. Sawaguchi K et al. Human Cell 2 (Suppl): 87, 1989. Sawaguchi K et al. Proc Jpn Cancer Assoc 50: 173, 1991. Schwartz GK et al. In Siewest JR and Rader JD (eds), Mondezzi Editore, Bologna, 1997, pp. 627–629. Sekiguchi M et al. Jpn J Exp Med 48: 61,1978. Sekiguchi M et al. Soshiki Baiyo 9: 201, 1983. Sekiguchi M et al. Human Cell 2: 331, 1989. Sekiguchi M, Suzuki T. In Hay RG, Park J-G, Gazdar A (eds), Academic Press, San Diego New York Boston London Sydney Tokyo Toronto, 1994, pp. 287–316. Sekiguchi M et al. J Gastroenterol 30: 589, 1995. Shaver W et al. Gastroenterol 96: 465a, 1989. Shibamoto S et al. Cell Struct Funct 17: 185, 1992. Shibata D and Weiss LM. Am J Pathol 14: 769, 1992. Shimomura T, St Marianna Ikadaigaku Zasshi 14: 459,1986. Shimoyama Y, Hirohashi S. Cancer Res 51: 2181, 1991. Smith HS. JNCI 62: 225, 1979. Soydan AS et al. Eur J Cancer 33: 1508,1997. Sugano H et al. Proc Jpn Cancer Assoc 27: 169,1968. Sugiyama Y et al. Surgery Frontier 3: 238, 1996. Taglbjaerg P, Nielsen HO. Acta Pathol Microbiol Scand [A] 86: 351, 1978. Tahara E et al. In Nishi M, Ichikawa H, Nakajima T, Maruyama K, Tahara E (eds), Springer, Tokyo, 1994, pp. 163-173. Tahara E. Cancer 75: 1410, 1995. Tajima M et al. Jpn J Cancer Res 89: 262, 1998. Takahashi A et al. Jpn J Cancer Res 82: 325, 1991. Takahashi M et al. In Siewest JR and Rader JD (eds), Mondezzi Editore, Bologna, 1997, pp. 623–626. Takebayashi Y et al. Cancer 82 661, 1998. Takekura N et al. Int J Cancer 45: 847, 1990. Takekura N et al. Int J Cancer 47: 938,1991. Takeuchi K et al. Exp Cell Res 223: 420, 1996. Tamura G et al. Cancer Res 56: 612, 1996. Taniguchi M et al. Proc Jpn Cancer Assoc 45: 198, 1986. Tanimoto H et al. Virchows Archiv B Cell Pathol 61: 263, 1991. Terano A et al. Gastroenterol Jpn 26: 7, 1991. Terashima M et al. Jpn J Cancer Res 82: 883, 1991. Todoroki T RGB General Catalog 3: 157, 1997. Tokumitsu S et al. Cancer Res 39:4732, 1979. Tokunaga M et al. Acta Pathol Jpn 43: 574, 1993a. Tokunaga M et al. Am J Pathol 143: 1250, 1993b. Tominaga S. Jpn J Cancer Chemother 19: 1113, 1992. Tsuchiya Y et al. Cancer Res 53: 1397, 1993.
Gastric Cancer
291
Tsuchiya Y et al. Int J Cancer 56: 46,1994. Tsuda T et al. Jpn J Cancer Res 80: 813, 1989. Uchino S et al. Cancer Res 52:3099, 1992. Uchino S et al. Int J Cancer 54: 759, 1993. Uesugi H et al. Human Cell 7: 227, 1994. Uesugi H, Naka H. Nisshoshi 92: 19, 1995. UICC International Union Against Cancer, TMN classification of malignant tumors. Cancer, Springer, Berlin Heidelberg New York London Paris Tokyo, 1987. Usugane M et al. Proc Jpn CancerAssoc 45: 240, 1986. Vollmers HP et al. Virchows Archiv B Cell Pathol 63: 335, 1993. Vollmers HP et al. Cancer 79: 433, 1997a. Vollmers HP et al., In Siewest JR and Rader JD (eds), Mondezzi Editore, Bologna, 1997, pp. 617–622b. Watanabe M et al. Nichi-Ge-Kaishi 94: 1164, 1993. Watanabe M. J Juzen Med SOC 103: 357, 1994. Watanabe M et al. Cancer 77: 1676, 1996. Watanabe M et al. In Siewest JR and Rader JD (eds), Mondezzi Editore, Bologna, 1997, pp. 797–800. Watson SA et al. Br J Surg 75: 342, 1988. Watson SA et al. Br J Surg 76: 639, 1989a. Watson SA et al. Br J Cancer 59: 554, 1989b. Watson SA et al. Int J Cancer 45: 90, 1990. Whelan R et al. Eur J Cancer Clin Oncol 24: 1397, 1988. Whelan SC, Parkin DM, Masuyer E (eds) IARC Sci Pub, Lyon, 1993. Wright F', Williams G. Gut 34: 145, 1993. Yamamoto M et al. Cancer 77: 1628,1996. Yanagihara K et al. Cancer Res 51: 381, 1991. Yanagihara K, Tsumuraya M. Cancer Res 52: 4042, 1992. Yanagihara K et al. Cancer Res 53: 5815, 1993a. Yanagihara K et al. Int J Cancer 54: 200, 1993 b. Yashiro M et al. Nichi-Ge-Kaishi 95: 288, 1994a. Yashiro M et al. Jpn J Cancer Res 85: 883,1994b. Yashiro M et al. Nisshoshi 92: 199, 1995 a. Yashiro M et al. Br J Cancer 72: 1200, 1995b. Yashiro M et al. Br J Cancer 74: 1096, 1996a. Yashiro M et al. Clin Exp Metastasis 14: 43, 1996b. Yashiro M et al. Cancer 77: 1668, 1996c. Yashiro M et al. Int J Cancer 67: 289, 1996d. Yashiro M et al. Anticancer Res 17: 895, 1997. Yasui W et al. Oncol Rep 2: 111, 1995. Yasumoto K et al. JBiol Chem 31: 22506, 1992. Yokozaki H et al. J Cancer Res Clin Oncol119: 67, 1992. Yokozaki H et al. In Tahara H (ed), Springer, Tokyo, 1997, pp. 55-70. Yoshida K et al. Int J Cancer 44: 394, 1989. Yoshida K et al. Jpn J Cancer Res 81: 43, 1990a. Yoshida K et al. Jpn J Cancer Res 81: 793, 1990b.
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Chapter 30 Colorectal Cancer
Michael G. Brattain1, J.K.V. Willson2, A. Koterba1, S. Patil1 and S.Venkateswarlu1 1 Department of Surgery, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7840 and 2Ireland Cancer Center of Case Western University and University Hospitals, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 441 06. Tel: 001-210-567-4482; Fax: 001-210-5674664; E-mail:
[email protected]
1.
INTRODUCTION
The generation of colorectal carcinoma cell lines from human specimens has become relatively routine since the initial attempts dating back to the 1970s. The early years of colon carcinoma cell line development were characterized by a very low success rate. For example, in a pioneering study by Leibovitz et al, continuous cell lines were developed from about 10% of over 163 specimens attempted (1). Thus, there was a concern that the low percentage of successful cell line development was not reflective of the “typical” colon cancer. During the 1980s inter- and intratumoral heterogeneity of the malignant cell compartments in various types of tumors including colon cancer became widely appreciated. Consequently, an important goal became the development of tissue culture methods that would permit the development of cell lines from a high percentage of the specimens obtained from surgery in order to provide better representation of the malignant cell types found in colon carcinoma. Several laboratories were successful in developing different broadly applicable methods generating a variety of phenotypes based on intestinal cell markers, morphology, tumorigenicity in athymic mice and histology (2-4). In addition to concerns about how representative cultured cell lines were with respect to the malignant cells in the original tumors, there were concerns that maintenance of cultures in serum-containing medium would
J.R. W. Masters and B. Palsson (eds.), Human Cell Culture Vol. II, 293–303. © 1999 KluwerAcademic Publishers. Printed in Great Britain.
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generate artifacts, particularly with respect to growth studies. Thus, the 1980s also saw the development of the use of chemically defined media for colon carcinoma cell lines (4–6,11,27). The late 1980s and 1990s have witnessed an explosion in our knowledge of the molecular genetics of cancer. Colon cancer is one of the best characterized tumor types with respect to molecular genetics. The widespread availability of human colon carcinoma cell lines has been an important contribution to the generation of molecular genetic models for the development of colon cancer (7).
2.
ESTABLISHMENT AND MAINTENANCE OF CULTURES
A wide variety of techniques have been applied to the initiation of colon carcinoma cell lines from primary specimens (Table 1). When cell suspensions have been used better success has been obtained using multi-cellular aggregates rather than single cells. High initial inocula are generally helpful for primary culture establishment. Growth of early stage malignant cells and benign adenomatous cells appears to be greatly aided by the use of a collagen substitute or a fibroblast feeder layer, both of which promote cell adhesion. Subculture of primary cultures is generally not attempted until vigorous malignant cell growth has been observed. Cell lines can be serially propagated after suspending cells by non-enzymatic as well as enzymatic methods (3,9). More differentiated cells show stronger adherence to culture surfaces than poorly differentiated cells and may, therefore, require different passaging techniques. Early passages require lower split ratios when passaging. Thus, even though success rates for the culture of individual specimens have been greatly improved and there are a wide variety of cellular phenotypes available, cell line development is an extremely selective process, both during initiation and serial passage. Moreover, earlier stage neoplasms appear to be more difficult to establish (9) and are more fastidious in their culture requirements. Normal colon epithelial cell lines have been established by immortalization. Medium requirements vary widely as a function of the individual laboratory in which cultures were initiated. Most cell lines are dependent upon serum supplements and many laboratories include additives such as growth factors, selenite, triiodothyronine and hydrocortisone. Establishment of cultures in serum-free medium is rare, but has been accomplished (4) and some cell lines have been converted to growth in serum-free medium after initial cultures have been passaged and established (4–6,11). Gazdar and colleagues (4) utilized a chemically defined medium (similar to other media) to initiate cultures and compared the success rate with the same specimens initiated in serum-containing medium. The success rate in defined medium was 38% while that obtained using serum supplemented medium was 25%. However, given
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the small numbers of specimens, it does not seem likely that the different success rates were operationally significant. The series of cell lines initiated by Gazdar and colleagues is also noteworthy in that they avoided the use of exposed surface tumor, instead choosing to utilize invasive areas and secondary sites when possible (4). Not surprisingly, one of the major difficulties in culturing primary human colon carcinoma specimens is microbial contamination of cultures (particularly yeast). This strategy was employed in an attempt to minimize the chances of microbial contamination, but may select for cell types in the later stages of the disease, since the invasive areas of tumors were favored by this technique.
3.
DEGREE TO WHICH CELL LINES REFLECT THE CLINICAL DISEASE
Large numbers of human colon carcinoma cell lines have been established as seen in Table 1. This abundance is in part due to the availability of the tissue and the relatively high success rate of cell line development from surgical specimens. Given the large number of cell lines it is not surprising that the whole spectrum of the disease is seen at the morphological level and, in so far as can be determined, at the behavioural and molecular levels as well. There is, however, only one example of a cell line established from a Duke’s A tumor (SW802, Table 1). This deficit is in part because this stage of colorectal cancer occurs in conjunction with polyps containing non-malignant but transformed epithelial cells. On the other hand, most other subgroups of colorectal carcinomas are well represented by the cell lines listed in Table 1. For example, adult colon does not express small intestine specific enzymes, but fetal colon does. About 5% of carcinomas express these fetal colon enzymes, as reflected by cell lines such as CaCo-2 and HT29 (13,14). Highly mucinous carcinomas, which are relatively rare, can also be found among the cell lines in Table 1 (eg NCI-H498). As discussed below, examples of the various histological types of colorectal carcinoma are abundant, both with respect to morphological differentiation (or the lack of it) and specialized function in tissue culture, and in subcutaneous xenografts of cell lines in athymic mice. Colorectal cancer often invades and metastasizes. Consequently, it would be expected that cell lines would reflect these properties. Much less work has been done in these aspects of colon cancer progression using cell lines than in differentiation patterns and molecular genetics (discussed below). In vitro invasion studies have identified both invasive and non-invasive cell lines (15,16). Subcutaneous xenografts of colorectal tumors are rarely invasive or metastatic (NCI-H716 is one exception). Consequently, orthotopic cecal implantation has been performed for some cell lines (16,17). Sublines of LS174T and KM12 which metastasize to the lymph nodes and liver after cecal
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transplantation have been selected for increased metastatic performance through repeated in vivo passages via the cecal route (16) or via splenic injection, which also generates liver nodules (17,18). It is not known whether any of the cell lines listed in Table 1 would not be metastatic by the cecal injection route.
4.
DEGREE TO WHICH CELL LINES REFLECT THE PATHOLOGY OF COLORECTAL CANCER
Colorectal cancer is characterized by multiple cell types reflecting the kinds of cells seen in normal crypts. Consequently, it is not surprising that colon cancer cell lines show a wide spectrum of cell types and in some cases can show a wide spectrum of morphologies within a single cell line (2,11,12). The colonic functions of lubrication by mucous production and absorption of water through vectorial transport are carried out by goblet and columnar cells, respectively. Stem cells are responsible for renewing differentiated crypt cells lost at the cell surface. One additional, but rare, cell type in normal colon is the endocrine cell. Each cell type except the endocrine cell is well represented by colon carcinoma cell lines (Table 2). Colonic carcinomas are evaluated for differentiation on the basis of glandlike formation and classified as being well, moderately or poorly differentiated. Interestingly, all 3 types of primary specimens as well as specimens from secondary sites can give rise to cell lines which show differentiated features (4,11,12). For example, the LIM1863 cell line derived from a poorly differentiated ulcerated carcinoma which extended through the colonic wall grows as organoids with both columnar and goblet cells (11). Thus, while this particular cell line does not appear to be representative of the tumor from which it is derived, it is representative of other types of colorectal tumors. Most cell lines derived from poorly differentiated carcinomas are characterized by undifferentiated growth in culture (both monolayer and suspension). The formation of gland-like structures in tissue culture is generally associated with cell lines which grow in a non-adherent or semi-adherent fashion in a 3 dimensional manner (3,4,11). Differentiation in adherent cells is generally associated with a columnar phenotype showing baso-lateral polarity with microvilli which can form transport domes when confluent (4,13). Endocrine features have not been examined in detail in most cell lines, although Park et al. (4) noted endocrine markers in a number of their colorectal cell lines.
5.
MOLECULAR GENETICS
The genetics of colorectal cancer are well studied, particularly in respect to changes associated with disease progression (7). There are numerous cell lines
Age/sex
Caco-2 Colo320DM *Colo205 DLD1 HCT8 HCT15 HT29 *HCT116 LS123 *LS 174T LoVo NCI-H498 NCI-H508 NCI-H716 NCI-H742 NCI-H747 NCI-H768 NCI-H548 NCI-H630 NCI-H684 NCI-H958 SNU-C1 SNU-C5 SNU-C4 SNU-C2A SNU-C2B
72/M 55/F 70/M – 67/M – 44/F –/M 65/F 58/F 56/M 56/M 55/M 33/M 58M 69/M 33/F 52/M 60/M 65/M 54/M 71/M 77/F 35/M 43/F 43/F
Duke’s stage – – – – – – B B D – – – – – – – – – – – – – – –
Grade
Pathology
Primary site
Specimen site
2
AC AC AC AC AC AC AC C AC AC AC AC AC AC AC AC AC AC AC AC AC AC AC AC C C
colon colon colon colon cecum colon colon colon colon colon colon cecum cecum cecum rectosigmoid cecum cecum colon rectum colon colon colon cecum colon cecum cecum
I•
1
ascitic fluid I• I• 1• I• I• I• I• shoulder mets peritoneum peritoneum ascites Il liver duct I• 1• Liver mets Liver mets Liver mets peritoneum I• I• I• I•
Culture
Authentication
Reference
E D D D D D E D D D D D D D D D D D D D D D X X X X
isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme
19 20 21 21 23 21 19 12 24 25 26 4 4 4
isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme
4 4 4 4 4 4 4 4 4 4 4
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Cell Line
Colorectal Caner
Table 1
Age/sex
SW48 SW403 SK-CO-1 SW480 KM12 SW802 SW620 SW837 SW948 SW1116 SW1417 SW1463 T84 WiDr VACO1 VACO3 VACO5 VACO4 VACO6 VACO8 VACO9 VACO9M VACO10 VACO10M VACO206 VACO241
82/F 51/F 65/M 51/M – 51/M 53/M 81/F 73/M 53/F 66/F 72/M 78/F 42/F 66/M 78/F 59/M 63/M 56/M 67/M 67/M 72/F 72/F – –
Duke’s stage
Grade 4 3
D 4 B A 4 4 3 2 3 2/3 – – D C C D C D D D D D B D
Pathology AC AC C AC AC AC AC AC AC AC AC AC C AC AC AC AC AC AC AC AC AC AC AC AC AC
Primary site
Specimen site
colon colon colon colon colon colon colon rectum colon colon colon rectum colon colon colon colon cecum rectum cecum cecum rectum rectum cecum cecum colon colon
I• lung mets ascites ab wall I• I• lung mets I• I• I• I• I• lung mets I• liver mets mesenteric node I• I• I• I• I• I• liver mets omental mets colon liver mets
Culture E E D D X E E E E E E E X D D D D D D D D D D D E E
Authentication isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme isoenzyme karyotype karyotype karyotype karyotype karyotype karyotype karyotype karyotype karyotype karyotype karyotype karyotype
Reference 1 1 ATCC 1 17 1 1 1 1 1 1 1 27 28 3 3 3 3 3 3 3 3 3 3 9 9
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Cell Line
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Table 1 (continued)
Colorectal Cancer
Table 1 (continued) Cell Line
Age/sex
Duke’s stage
LIM1215 LIM1863 HCA-2 HCA-7 HCA-16 HCA-19 HCA-24 HCA-46
34/M 74/F 83/F 58/F 56/M 66/M 68/M 53/F
D D C B B B B C
Grade
Pathology
Primary site
Specimen site
AC AC AC AC AC AC AC AC
colon cecum colon colon rectum colon colon colon
omental mets I• I• I• I• I• I• I•
Culture E E E E E E E E
Authentication
Reference
isoenzyme – – – – – – –
29 11 10 10 10 10 10 10
Duke’s stage (A-D), Broder’s Grade 1-4,I• = primary tumor, D = dissociated cells (either mechanical or enzymatic), X = xenograft, E = explant culture
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Table 2 Cell Line
Differentiation of Specimen
In Vitro
Xenograft Histology
NCI-H548 NCI-H6300 NCI-H684 NCI-H958 NCI-H508 NCI-H742 NCI-H768 SNU-C1 SNU-C2A SNU-C4 SNU-C5 NCI-H716 NCI-H498 LIM1863 LIM1215 VACO9 CA2 HCA7 HCA16 HCA19 HCA24 HCA46 CaCO2 T84 VACO206 VACO241 HCT116
W M M M W M M M M M M P P P P P W M M W W P W W W P P
D D D D D D D U U U U U D D D D D D D D D U D D D U U
Consistent Consistent Consistent No xenografts formed Consistent No xenografts formed Consistent Consistent Consistent Consistent Consistent Consistent Consistent Rare xenografts – – Consistent Consistent Consistent Consistent Consistent Consistent Consistent Consistent Consistent Consistent Consistent
W = Well-differentiated; M = Moderately differentiated; P = Poorly differentiated; D = Differentiated cells in culture (goblet and/or columnar); U = Undifferentiated. The last column indicates whether the xenograft histology is consistent with the histology of the original specimen; “–” indicates xenograft histology was not described
representative of the various types of mutations commonly seen in primary and metastatic specimens (Table 3). Many of the mutations are frequent in pre-cancerous lesions (eg APC, Ki-ras). However, it is clear that development and progression of colorectal cancer occurs as an accumulation of mutations rather than a linear progression of mutations starting with APC (7). Thus, it would not be expected that each cell line would have all of the common mutations. The APC gene is important because it is targeted in familial adenomatous polyposis. Analysis of replication error by mismatch repair genes (RER+) has indicated that it is associated with another familial syndrome termed hereditary non-polyposis colorectal cancer (HNPCC). Over 90% of HNPCC patients show RER+, which reflects the loss of mismatch repair function due
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Colorectal Cancer Table 3 Genetic Changes
Cell Lines
Reference
APC Mutation
Colo 201 Colo 320 DLD 1 SW1222 SW832 HT29 SW403 Caco 2 SW480 SW948 HRA19 Lovo SW1417 SWCO1 SW837 HCT116 SW403 SW480 DLD-1 HCT15 SW1116 SW480 Colo 320 HCI H747 SW1116 HCT15 SW837 HCT116 SW48 SW1417 SW1116 SW403 SW1463 VACO 6 NCH H630 sw1222 SW480 HCT116 DLD-1 LS174T SW48
33 33 33 33 33 33 33 33 33 33 33 33 33 33 34 35 36 36 37 37 37 30 32 31 31 32 38 39 39 39 39 39 39 39 39 40 40 41 43 42 42
Ki-ras
p53
DCC
RER+
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to mutations on chromosome 3 in colorectal cancer (41,42). A significant proportion of sporadic colorectal cancers are also RER+. Both types of RER+ patients show mutational inactivation of the TGFb type II receptor (41,42) in more than 90% of cases. Replacement of the type II receptor in RER+ cell lines leads to reversion of malignant properties (44). There are indications that mutations associated with the transformation and progression of colorectal cancer in RER+ may be different from those in non RER+ sporadic cancer. For example, most RER+ colorectal cancer cell lines show wild type APC and p53 while non-RER+ cell lines rarely show inactivation of the type II TGFb receptor. The predominant site of mutational inactivation in the TGFb type II receptor resides in a long repeat of 10 adenines in the amino terminal half of the gene (44), a type of structure targeted by repair defects. However, stretches of long repeats are relatively rare in coding regions, and no other comparable genes have been identified. The chromosomes associated with the mutations summarized in Table 3 are 5 (APC), 17 (p53) and 18 (DCC) for sporadic cancer. Non-RER+ cell lines frequently show aberrations in these chromosomes, but also display many other genetic changes. RER+ tumors are associated with loss of the 3p region which contains the genes for a mismatch repair subunit and the type II receptor. Alterations in chromosome lp also appear to be frequent in colorectal cancer, but as yet have not been associated with a tumor suppressor function.
REFERENCES 1. Leibovitz A, Stinson JC, McCombs MB, McCoy CE, Mazur KC, Mabry ND. Cancer Res 36: 4562,1976. 2. Brattain MG, Fine WD, Khaled FM, Thompson J, Brattain DE. Cancer Res 41: 1751, 1981. 3. McBain JA, Weese JF, Meisner LF, Wolberg WH, Willson JKV. Cancer Res 44: 5813, 1984. 4. Park JG, Oie HK, Sugarbaker RH, Henslee JG, Chen TR, Johnson BE, Gazdar A. Cancer Res47: 6710, 1987. 5. Boyd D, Levine AE, Brattain DE, McKnight MK, Brattain MG. Cancer Res 48: 2469, 1988. 6. Mulder KM, Brattain MG. Mol Endo 3: 1215, 1989. 7. Fearon ER. Adv Internal Med 39: 123,1994. 8. Brattain MG, Levine KE, Chakrabarty S, Yeonin LD, Willson JKV, Long B. Cancer Metastasis 3: 177, 1984. 9. Willson JKV, Bittner GN, Oberley TD, Meisner LF, Weese JL. Cancer Res 47: 2704, 1987. 10. Kirkland SC, Bailey IC. Br J Cancer 53: 779,1986. 11. Whitehead RH, Jones JK, Gabriel A, Lukies RE. Cancer Res 47: 2683,1987. 12. Brattain MG, Marks ME, McCombs J, Finely,W, Brattain DE. Br J Cancer 47: 373, 1983. 13. Chantret I, Barbat A, Dussaulx E, Brattain MG, Zwiebaum A. Cancer Res 48: 1936, 1988. 14. Zwiebaum A, Hauri HF', Sterchi E, Chantret I, Haffen K, Barat J, Sordat B. Int J Cancer 34: 591,1984. 15. Schlecte W, Brattain M, Boyd D. Cancer Communications 2: 173, 1990. 16. Bresalier RS, Raper SE, Hujanen ES, Kim YS. Int J Cancer 39: 625, 1987. 17. Morikawa K, Walker S, Nakajima M, Pathak S, Jessup JM, Fidler IJ. Cancer Res 48: 6863, 1988.
Colorectal Cancer 18. 19. 20. 21. 22. 23.
303
Morikawa K, Walker S, Jessup JM, Fidler IJ. Cancer Res 48: 1943, 1988. Fogh J, Wright,WC, Loveless JD. J Natl Cancer Inst 58: 209, 1977. Quinn LA, Moore GE, Morgan RT, Woods LK. Cancer Res 39: 4914,1979. Semple TU, Quinn LA, Woods LK, Moore GE. Cancer Res 38: 1345, 1978. Dexter DL, Barbosa JA, Calabresi P. Cancer Res 39: 1020, 1979. Tompkins WA, Watrach AM, Schmale JD, Schultz RM, Harris J. JNatl Cancer Inst 52: 11001, 1974. 24. Rutzy LP, Giovanella BC, Tom BH, Kaye U, Naguchi PD, Kahan BD. In Vitro 19: 99, 1983. 25. Tom BH, Rutzley LP, Jakstys M, Oyasu R, Kaye CI, Kahan BD. In Vitro 12: 180, 1976. 26. Drewinko B, Romsdahl MM, Yang LY, Ahearn MJ, Trujillo JM. Cancer Res 36: 467,1976. 27. Murakami H, Masui H. Proc Natl Acad Sci USA 77: 3464,1980. 28. Noguchi P, Wallace R, Johnson J, Earley EM, O’Brien S, Ferrone S, Pellegrino MA, Milstein J, Needy C, Browne W, Petricciani J. In Vitro 15: 401, 1979. 29. Whitehead RH, MacRae FA, St. John DJ, Ma J. J Natl Cancer Inst 74: 759, 1985. 30. Rodrigues NR, Rowan A, Smith ME, Kerr IB, Bodmer WF, Gannan V, Lane OP. Proc Natl Acad Sci USA 87: 7555,1990. 31. Peinado MA, Fernandez-Renert M, Capella G, Wilson L, Perucho M. Int J Oncol 2: 123,1993. 32. Cottu PH, Muzeau F, Estreicher A, Flejou JF, Iggo R, Thomas G, Hamelin R. Oncogene 13: 2727, 1996. 33. Ilyas M, Tomlinson IPM, Rowan A, Pignatelli M, Bodmer WF. Proc Natl Acad Sci USA 94: 10330, 1997. 34. Wada M, Miller CW, Yokota J, Lee E, Mizoguchi H, Koeffler HP. J Mol Med 75: 139, 1997. 35. Bujard A, Zipfel PA, Frey RS, Mulder KM. Int J Cancer 67: 539, 1996. 36. Fujita J, Yoshida 0, Ebi Y, Nakayama H, Onoue H, Rhim JS, Kitamura Y. Urol Res 16: 415, 1988. 37. Wojciechowicz DC, Park PY, Paty PB. Biochem Biophys Res Commun 212: 758, 1995. 38. Baker SJ, Preisinger AC, Jessup JM, Paraskeva C, Markowitz S, Willson JKV, Hamilton S, Vogelstein B. Cancer Res 50: 7717, 1990. 39. Fearon ER, Chok R, Nigro JM et al. Science 247: 49, 1990. 40. Mackinnon WB, Huschtscha L, Dent K, Hancock R, Paraskeva C, Mountford CE. Int J Cancer 59: 248,1994. 41. Markowitz S, Wang J, Meyeroff L et al. Science 268: 1336,1995. 42. Vincent F, Nagashima M, Takenoshita S, Khan MA, Gemma A, Hagiwara K, Bennett WP. Oncogene 15: 117, 1997. 43. Mancuso T, Aguilar F, Pescarolo MP, Clerico L, Russo P, Parodi S. Nucleic Acids Res 25: 3643, 1997. 44. Wang J, Sun L-Z, Myeroff L, Wang X, Gentry LE, Yang J, Liang J, Zborowska E, Markowitz S, Willson JKV, Brattain MG. JBiol Chem 270: 22044, 1995.
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Chapter 31 Prostate Cancer
James M. Kozlowski and Julia A. Sensibar Northwestern University Medical School, Department of Urology, 303 East Chicago Avenue, Tarry 11-715, Chicago, IL 60611-3008. Tel: 001-312-908-8145; Far: 001-312-908-7275; E-mail: j-kozlowski @nwu.edu
1.
INTRODUCTION
Adenocarcinoma of the prostate (CaP) is the most prevalent cancer in men. Occult CaP has been identified in over 30% of autopsy specimens in males older than 50 years of age (Holund 1980, Kozlowski and Grayhack, 1996). The rate of detection of occult CaP increases progressively with age, tripling by the ninth decade. Most of these histologic or occult carcinomas exhibit a slow growth rate and will not adversely affect the quality or duration of life. In contrast, prostate cancers which become clinically manifest often acquire phenotypes which facilitate tumor invasion and metastasis. CaP is the most common clinically detected cancer in American men. Projected 1999 estimates from the American Cancer Society suggest that CaP will account for 29% (179,300) of newly diagnosed cancers in this population. Approximately 25% of men with clinically apparent CaP die of the disease. This neoplasm is the second most common cause of cancer death among American men and is projected to account for 13% (37,000 events) of male cancer deaths in 1999 (Landis et al, 1999). About 70% of CaP originate within the peripheral zone. Transition zone origin is observed in 25% of cases (Kozlowski and Grayhack, 1996). Those tumors contained within the confines of the anatomic capsule are potentially curable with radical prostatectomy or radiation therapy. Unfortunately, only palliative treatment is available for those patients with locally advanced tumors or those with metastasis to the lymphatic and/or skeletal systems.
J.R. W Masters and B. Palsson (eds.), Human Cell Culture Vol. II, 305–331. © 1999 Kluwer Academic Publishers. Printed in Great Britain.
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Approximately 75% of patients with metastatic CaP demonstrate evidence of both objective and subjective improvement following the initiation of androgen-ablative therapy. The average duration of benefit is 18-24 months (Kozlowski and Grayhack, 1996). The uncontrolled proliferation of androgenindependent tumor subpopulations accounts for the majority of relapses in this cohort. About one half of these patients will die within the first year following relapse; the majority of the remainder will succumb to the disease process or related causes within two years (Kozlowski and Grayhack, 1996). These statistics have not changed appreciably despite the development of a diverse array of new therapies, both conventional and experimental. Despite recent advances, our basic perceptions concerning the biology of prostate cancer remain surprisingly primitive. In large part, this inadequacy can be attributed to the existing deficiency of appropriate animal models and in vitro systems. Indeed, an urgent clinical and scientific need exists for the development of truly relevant experimental systems that would accelerate our understanding of the biology of human prostate cancer and lead to the development of novel, effective, and well-tolerated therapies. Useful information has been derived from investigations performed using rodent models for CaP which include the Noble (Noble, 1977; Drago et al. 1979, 1981), ACI (Ward et al. 1980; Shain et al. 1975), Pollard (Pollard, 1973, 1980), and Dunning R-3327 (Dunning 1963; Isaacs and Coffey, 1979; Lubaroff et al. 1980) tumors. Despite their acknowledged utility, investigations performed using these systems cannot substitute for a multifaceted analysis of human CaP as assessed following successful serial propagation in vitro or xenografting in immune deficient animals. The scarcity of well-characterized, immortalized cell lines and xenograft tumor systems can be attributed to the complex and stringent growth requirements of these tumors. The following sections will briefly highlight the major features associated with those prostate tumor systems for which reliable information is available.
2.
CULTURE OF HUMAN PROSTATE CELLS
The majority of the well characterized human CaP cell lines were derived from metastatic foci from patients with hormone-refractory disease. With the exception of LNCaP and possibly MDA PCa 2, these tumor systems were preselected for their absolute or relative androgen insensitivity. In each instance, a fresh tissue harvest was followed by rapid tissue processing. The latter involved very simple approaches, often utilizing mechanical disruption followed by enzymatic dissociation and/or explanting. Collagen-coated dishes were used in most instances. In each case, successful attachment, propagation, passaging, and subsequent immortalization took place in a growth environment consisting of basic tissue culture media plus the addition of fetal calf serum.
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Testosterone concentrations in fetal calf serum (approximately 0.2 ng/ml) are lower than those found in the circulation in normal males (5–12 ng/ml). As fetal calf serum is used at a concentration of 5-20%, the level of testosterone available to cells in culture is less than 1% of the physiologic level. The human prostate is a complex organ which exhibits well-defined zonal, compartmental (epithelial versus stromal), and cellular heterogeneity (Kozlowski and Grayhack, 1996). The ‘dynamic reciprocity’ which typifies epithelial–stromal interaction is well-recognized, but poorly understood. Normal and aberrant function of this prostatic ‘tissue matrix system’ appears to be closely linked to the mechanisms underlying hyperplastic and malignant prostatic growth (Cunha et al. 1987). The difficulties associated with the long term culture of malignant prostatic epithelial cells is attributable to our relatively primitive understanding of the complex nurturing influences present within this microenvironment. In addition, the process of immortalization is probably dependent on the mutation, deletion, hypermethylation, or amplification of specific genes (Goldstein, 1990; Peehl, 1994). Prostate cancers are biologically heterogeneous and consist of a diverse array of cellular subpopulations differing from one another in a wide variety of phenotypes (including androgen sensitivity; the production of growth factors and their receptors; invasiveness and metastatic capacity (Kozlowski et al. 1988). This phenotypic diversity, which permits selected variants to develop from the primary tumor, accounts for the differences frequently noted between the parental tumor and its metastases (Kozlowski et al. 1984a,b). It is therefore not surprising that the majority of immortal CaP cell lines have been derived from metastatic foci which have flourished in patients exhibiting the hormone-refractory phenotype. Over the past 15 years, many of the longstanding obstacles to the successful in vitro propagation of benign and malignant prostate epithelial cells have been overcome. These improvements, which are discussed in detail in several reviews (Kozlowski et al. 1988; Peehl, 1992, 1994), include: (1) the acquisition of fresh prostatic tissue (primary and metastatic) from biopsy, surgical, and autopsy specimens; (2) the immediate histologic or cytologic confirmation that the tissues removed for experimentation are composed predominantly of malignant cells; (3) prompt mechanical dissociation, followed by collagenase digestion, the duration of which is predicated on tissue volume; (4) immediate separation of epithelial and stromal components using a 5-step discontinuous Percoll gradient technique or the immediate transfer of the cell suspensions in growth media to collagen-coated dishes; (5) the use of serum-free medium, such as PFMR-4a (Peehl and Stamey, 1986) WAJC-404 (Chaproniere and McKeehan, 1986; McKeehan et al. 1984) and PrEGM (BioWhittaker), each of which contains relatively low ionic calcium concentrations and has been defined specifically for cultivation of prostatic epithelial cells; (6) the addition of essential growth factors and additives, such as zinc-stabilized insulin, transferrin, selenous acid, bovine serum albumin, linoleic acid, epidermal growth
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factor, prolactin, polyvinyl pyrrolidone, cholera toxin, and bovine pituitary extract; (7) refeeding the primary cultures with growth media every 3–4 days until the cells are semiconfluent; (8) subculture of actively dividing subconfluent cells (using trypsin/EDTA) and distribution of approximately 5 × 104 cells per 100-mm collagen-coated dish for serial culture; and (9) cryopreservation of aliquots at the time of each passage, since strains of benign and malignant prostatic epithelial cells generally survive only 3–5 passages (15–20 populations doubling) before the onset of senescence (Kozlowski et al. 1988; Peehl, 1992). The rapid elimination of stromal contaminants is highly desirable. This is accomplished in a number of ways. First, the collagenase digest is relatively toxic to stromal cells. Second, the use of a discontinuous Percoll gradient technique permits the rapid separation of epithelial cells (which are more dense) from stromal passengers (Cooke and Littleton, 1985; Kozlowski et al. 1988). Alternatively, the use of multiple, brief centrifugations of the cellular digest may produce similar results (Peehl, 1992). Finally, serum-free growth is not conducive to the proliferation of stromal cells. These techniques permit the isolation and expansion of virtually pure epithelial populations. The latter perception has been validated utilizing discriminatory immunohistochemistry, as well as 2-dimensional gel electrophoresis (Kozlowski et al. 1988; Sherwood et al. 1989, 1990; Peehl 1992). It should be emphasized that fresh prostate cancers (particularly high Gleason grade tumors which lack a distinct acinar configuration) are relatively fragile and their exposure to dissociating enzymes should be limited (Peehl, 1992, 1994). Unfortunately, normal, hyperplastic, and malignant prostate epithelial cell cultures cannot be reliably distinguished on the basis of morphological features. Similarly, there are no cancer-specific antibodies suitable for this purpose. Establishing the cytokeratin (CK) profile of these cultures is mandatory in order to validate their epithelial origin. Sherwood and associates (1990) demonstrated that epithelial cells harvested from fresh BPH tissue by Percoll gradient centrifugation and propagated in vitro using selective culture techniques showed alterations in CK expression compared to intact human prostates. Specifically, CKs 6, 14, 16 and 17 were noted in cultured BPH epithelial cells but not fresh normal prostate or BPH tissue. Immunoblot analysis of the established CaP cell lines PC-3, DU-145, and LNCaP showed expression of CKs 8 and 18 but not CKs 5,7, and 15 which were observed in benign prostate. These findings are in concert with loss of the basal cell CK phenotype (5/15) and maintenance of the adluminal or secretory cell CK phenotype (8/18) in prostate cancer. Prostatic acid phosphatase (PAP) and prostate specific antigen (PSA) are the prototypic markers of the adluminal or secretory phenotype. Growth in monolayer culture is associated with a number of aberrations including: (1) loss of androgen responsiveness; (2) a shift from a differentiated to a proliferative phenotype (Fong et al. 1991; Lee et al. 1995); and (3) loss of the cells’ ability to synthesize/secrete these tissue-specific markers. Overcoming this
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environmental distortion may require plating cells at high density and utilizing growth on natural substrates (collagen or Matrigel) (Peehl 1992, 1994; Fong et al. 1991, 1992). These approaches facilitate maintenance of cell polarity and critically important cell-cell interactions which are required for differentiated function. Of the reasonably well characterized CaP cell lines listed in Table 1, only LNCaP (and possibly MDA PCa 2) maintain androgen responsiveness and the ability to express these marker proteins. Before concluding this section, it is important to reemphasize several points. First, most of the well-characterized CaP cell lines were established utilizing relatively simple tissue culture techniques. Second, our enhanced understanding of prostate biology and the development of sophisticated tissue culture techniques have not translated into the establishment of a diverse array of new, well-characterized, continuous cell lines.
3.
CONTINUOUS CELL LINES
Cancers originating from the epithelial cells of the prostate constitute over 95% of all prostatic malignancies (Kozlowski and Grayhack, 1996). The Gleason classification is currently the most popular system used to grade prostatic carcinoma. It employs low-power magnification (×40-100) to assess the acinar pattern of the tumor and its relationship to the stromal compartment. Five tumor grades progressing from the most (grade 1) to the least (grade 5) differentiated are recognized. About 50% of prostate cancers manifest more than one Gleason histologic pattern. That the presence of a histologically heterogeneous tumor has biologic significance led to the use of the sum of the primary and secondary patterns as the Gleason (pattern) score (Gleason 1977; Kozlowski and Grayhack, 1996). Possible pattern scores range from 2–10. This permits the separation of tumors into well-differentiated (2–4), moderately-differentiated (5–7), and poorly-differentiated (8–10). Histologic tumor grade is the most important predictor of tumor progression for clinically localized disease and the Gleason tumor score correlates with a variety of adverse phenotypes including: invasive and metastatic capacity, the subsequent development of lymphatic and skeletal metastasis; ureteral obstruction; and cancer death rate (Catalona, 1984; Gleason, 1977; Kramer et al. 1980). With the advent of PSA screening, there has been a distinct ‘stage shift’ in that most patients present with clinical organ-confined disease. The majority of these prostate cancers possess an intermediate Gleason pattern score. In about 30% of cases, the tumor is under-staged by clinical criteria and evidence of extracapsular extension is documented following step-section analysis of radical prostatectomy specimens (Kozlowski and Grayhack, 1996). It is currently uncommon for patients to present with bulky, locally advanced tumors or exhibit overt evidence of metastatic disease. Nonetheless, about 25% of
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men with CaP die as a direct result of the disease (Catalona, 1994). Their demise is directly attributable to the uncontrolled proliferation and dissemination of tumor subpopulations which are endowed with invasive and metastatic capacity. From a histologic standpoint, these tumors are generally poorly differentiated. The ability of these tumor systems to express PSA is often blunted by their undifferentiated state, as well as the selection pressure induced by androgen-ablative therapy (Kozlowski and Grayhack, 1996). Nonetheless, the majority of these cancers maintain some ability to express PSA, in some cases at very high levels. The idealized library of human CaP cell lines should span the spectrum of clinical and biological diversity summarized in Table 4 and in the preceding paragraph. It would be highly desirable to establish cell lines derived from primary, organ-confined prostate cancers possessing low, intermediate, and high Gleason pattern scores. In addition, this idealized library should consist of cell lines derived from locally advanced tumors and representative metastatic foci (lymph nodes, bone). This latter group should be composed of treatmentnaive (ie, androgen sensitive) and post-treatment (ie, hormone refractory) tumor systems. Other desirable features would include maintenance of invasive and metastatic capacity, as well as the ability to express PSA. A careful analysis of the 8 immortalized CaP cell lines depicted in Table 1 reveals that they fall far short of the idealized library described in the preceding paragraph. Only 2 of these cell lines (JCA-1, ND-1) were derived from primary prostate cancers (Muraki et al. 1990; Narayan and Dahiya, 1992). The 6 remaining lines were derived from metastatic foci, including: brain (DU-145) (Stone et al. 1978), lymph node (LNCaP, TSU-PR1, DuPro-1) (Horoszewicz et al. 1980, 1983; Iizumi et al. 1987; Gingrich et al. 1991), and bone (PC-3, MDA PCa 2a, b) (Kaighn et al. 1978, 1979; Navone et al. 1997). The primary-derived cell lines have not been as rigorously characterized or evaluated as those derived from metastases. Nonetheless, a few features are worth additional commentary. ND-1 was obtained from a radical prostatectomy specimen in a treatment-naive patient whose tumor exhibited a Gleason pattern score of 3+3. Over 80% of such tumors have the ability to express PSA and exhibit androgen responsiveness (Kozlowski and Grayhack, 1996). In contrast, ND-1 is relatively or absolutely hormone-refractory (Narayan and Dahiya, 1992). Similarly, JCA-1 was derived from a treatment-naive patient who underwent radical prostatectomy. The tumor of origin exhibited a Gleason pattern score of 4+4. Again, one would anticipate some degree of androgen responsiveness and the ability to express PSA. However, JCA-1 exhibits an androgen-independent phenotype (Muraki et al. 1990). Of the cell lines derived from metastatic foci, four are absolutely hormonerefractory (DU-145, PC-3, TSU-PR1, DuPro-1). In each instance, the cell lines were derived from patients who had been heavily pre-treated, which exerts a selection pressure which favors the emergence of pleomorphic, undifferentiated, androgen-insensitive prostate tumor cells.
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Only cell lines LNCaP and MDA PCa 2a, b maintain their androgen sensitivity and the ability to express PSA in vitro and in vivo. It must be emphasized that the patients from whom these tumor cell lines were derived were also heavily pre-treated. LNCaP has been much more rigorously evaluated than MDA PCa 2a, b. The androgen receptor in LNCaP cells has a point mutation in the ligand-binding domain (Trapman et al. 1990). As a result, testosterone, dihydrotestosterone, estradiol, progesterone, flutamide, and hydroxyflutamide can transactivate target genes (Umekita et al. 1996). Presumably, these continous cell lines represent the unique clonal expansion of androgen-sensitive tumor subpopulations which were also endowed with phenotypes permitting in vitro propagation. There are additional CaP cell lines that have been reported in the literature, but whose characterization is insufficient for inclusion in Table 1. The cell line LRVA-4 was established from a primary prostate tumor (Gleason score of 8) in a 70 year old man (Fan 1988). The tumor tissue was obtained at autopsy 4 hours post-mortem. A simple explant technique was used to establish the line. The culture medium used was a 50:50 mixture of Ham’s F-12 and Eagle’s MEM. Initially, 25% fetal calf serum was added to the culture media. This was later decreased to 10%. In vitro, the cell line apparently displayed 2 distinct subpopulation growth patterns (monolayer and multicellular spheroids). Cell line BM1604 was established from a 67 year old man with localized prostate cancer (van Helden et al. 1994). The tumor tissue was removed from a radical prostatectomy specimen and exhibited a Gleason score of 5. Tissue fragments were subjected first to enzymatic dissociation followed by explanting. The explants were cultured utilizing two different approaches. The first employed growth on standard uncoated tissue culture flasks utilizing RPMI1640 media containing 5% fetal calf serum. Alternatively, explants were cultured on ECM-coated tissue culture flasks in the absence of serum using an enriched Ham’s F-12 medium. The cells apparently grew as islands which fused when the cultures became confluent, and did not maintain their ability to express PSA/PAP. DNA fingerprinting demonstrated that the DNA from the tumor was essentially the same as that in the patient’s blood, thus confirming the origin of the cell line. A total of 6 locus-specific differences were identified between the blood and tissue sample DNA. Karyotype analysis demonstrated the absence of the Y chromosome. The ALVA-31 cell line was established by Loop and associates (1993) from a radical prostatectomy specimen. The tumor of origin was a well-differentiated adenocarcinoma (Stage B2). The primary specimen was positive for PSA by immunohistochemistry. Needle biopsies were obtained from the main tumor mass and subjected to further mincing. Tissue explants were seeded into culture flasks containing RPM1-1640 + 10% fetal calf serum. The initial tumor cultures (up to passage 3) were supplemented with 10%-conditioned medium obtained from confluent lung adenocarcinoma cell line cultures. The latter
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Table I General background information regarding human CaP cell lines Cell line
Patient Patient race, age blood type
DU-145
69
PC-3
62
LNCaP
50
TSU-PR1 73 JCA-1
67
DuPro-1
57
ND-1
na
Caucasian/O+ T4 Nx Mlc HR AP nl Caucasian/na T3b(?),Nx,Mx HR AP (-) Caucasian/B+ T3-4(?),N1, Mla-b HR AP (-) Asian/na T3-4(?) Nx Mla-b HRAP(-) Caucasian/A+ T2a(?) NO (?) MO (?) AP(nl) na/na Tx Nx Mla HR AP (nl) na/na
T2b N0 M0 ?AP ?PSA
Black/na
Tx Nx M1b HR-PSA
Pathology stage*
Pathology Primary Specimen grade site site
pT4 N1 Mlc MD-PD
prostate brain
prostate pT4(?) N PD M1 b-c pT3-4(?) N1 MD prostate M1 b(?) pT3-4(?) MD prostate Mx(?) Mla-b pT2a(?) N1 MD-PD prostate M0(?) pTx Nx M1a PD prostate pT3a NO MO MD Gleason 3+3 pTxNxM1b PD
bone
Culture Authenmethod tication Availability E
CH, K, I ATCC, DSMZ Stone et al. 1978
D
CH,I,K ATCC
lymph node E (supraclavicular) lymph node D (cervical) prostate E lymph node X (supraclavicular) DÞ E
CH, K CH, K CH, K CH, K
Kaighn et al. 1979
ATCC, DSMZ Horoszewicz et al. 1980 Tatsuo Iizumi Iizumi et al. 1987 John Isaacs Muraki et al. 1990 J. Muraki
E
CH, K
J. Gingrich P. Walther K.Webb P. Narayan
prostate paraspinal mass D
CH, K
N. Navone
prostate prostate
Primary reference
Gingrich et al. 1991
Navone et al. 1997
Narayan and Dahiya, 1992
Abbreviations: HR, hormone refractory; E, explant; CH, comparative histology; K, Karyotype, cell line; I, isoenzyme analysis, cell line; AP, acid phosphatase; D, dissociation; MD, moderately differentiated; PD, poorly differentiated; PSA, prostate specific antigen; X, xenograft. *See Table 4 for staging information
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MDA PCa 63 2a, b
TNM category (clinical)*
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technique was designed to stimulate tumor growth until adequate cell density was achieved. Individual epithelial cell colonies were isolated from contaminating stromal cells by removing them from the monolayer with a Pasteur micropipet. By passage 3, the cells were adapted to growth in semisolid media. With continued passage, PSA immunoreactivity was lost. The modal chromosome number was 59 (range: 24–112) and all metaphases were aneuploid. Other characteristics of the karyotype included: absence of the Y chromosome; a large marker chromosome mapped between chromosomes 3 and 4; loss of chromosome 10; a 5/9 translocation and additional small marker chromosomes. ALVA-31 is tumorigenic in intact male, castrate male, and female nude mice. Although testosterone depletion did not influence the overall tumor induction rate (>90%), tumor growth rate was significantly reduced. Metastases were not observed following subcutaneous injection. Despite low level expression of the cytosolic androgen receptor, ALVA-31 cells have the capacity to reduce testosterone to dihydrotestosterone. In conclusion, the growth of the cell line is modulated by androgens, but ALVA-31 is not androgen-dependent (Loop et al. 1993). The 1013L cell line was established from an undifferentiated primary prostate cancer which exhibited both acinar and ductal features (Williams 1980). It represents the only human CaP cell line that grows in stationary suspension culture (not as a monolayer). 1013L cells proliferate as small spheroids (in suspension) in RPM1-1640 medium supplemented with 10% fetal calf serum (Billstrom et al. 1995). Cytogenetic studies have demonstrated that 1013L has a modal chromosome number of 75; retained the Y-chromosome until passage 44; and exhibits an unusual pattern of interstitial C-bands (Hartley-Asp et al. 1989). The tumor expresses CKs 7, 8, 18, and 19. 1013L is hormone-independent and does not express PSA. This tumor appears to lack invasive potential and has virtually undetectable production of uPA (Billstrom et al. 1995). 1013L is tumorigenic in both male and female SCID mice, but must be implanted in a sterile gelatin sponge for local tumor formation to occur following subcutaneous implantation. The frustration associated with attempts to establish new human CaP cell lines has understandably stimulated interest in the use of DNA transfection. The majority of these attempts have involved the use of SV40T antigen introduced into neonatal (Kaighn et al. 1989), benign adult prostatic epithelial cells (Cussenot et al. 1991), and non-neoplastic prostate cancer epithelial cells (Jackson-Cook et al. 1996; Bright et al. 1997), immortalization by human papillomaviruses (Weijerman et al., 1994) or transfection with oncogenes (Rhim et al. 1994). These aspects have been reviewed (Webber et al. 1977). More recently, introduction of the telomerase gene to facilitate the immortalization of cell lines has been reported (Bodnar et al. 1998).
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4.
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COMPARISON OF TUMOR PATHOLOGY WITH IN VITRO FEATURES AND XENOGRAFT PATHOLOGY
Ideally, there should be a close phenotypic correlation between the prostate cancer of origin and the continuous cell line. The latter should possess in vitro and in vivo (xenograft) characteristics that closely link it with the original tumor. Shared phenotypes should include: histopathology, expression of tumor markers, invasive potential, tumorigenicity, and metastatic potential. The subsequent brief discussion will highlight the fact that the cell lines listed in Table 2 do not perfectly parallel the human tumor of origin. The phenotype most closely maintained is that of histopathology. In virtually all instances, the original tumor exhibited characteristics of either moderately-differentiated or poorly-differentiated prostate adenocarcinomas. In no instance has there been a significant discordance between the xenograft pathology and that exhibited by the original tumor of origin. The issue of tumor marker expression is more difficult to evaluate. In most instances, only sketchy information is available with respect to important patient-related characteristics. The latter include information regarding serum PAP/PSA levels and whether the patient-derived tumor was evaluated immunohistochemically for the expression of these markers. As stated previously, the only cell lines that maintain androgen responsiveness, possess functional androgen receptors, and are capable of PSA production are LNCaP and MDA PCa 2a, b. In the case of LNCaP, the available patient information does not permit an accurate comparison. With respect to MDA PCa 2, PSA expression was a distinct feature of the patient’s tumor. Of interest, cell line DuPro-1 was derived from a xenograft system (DU 5683) whose features included: moderately-differentiated to poorly-differentiated histology; androgen sensitivity and PSA expression. Once that xenograft was adapted to in vitro growth, the resulting cell line assumed an androgen-insensitive phenotype. For each of the cell lines, the pathologic stage assigned to the tumors of origin clearly indicates acquisition of the invasive phenotype. The documentation of extracapsular disease (ND-1) and frank metastasis (all other lines) is indicative of tumor systems capable of degrading extracellular matrix (ECM). The development of a modified Boyden chamber chemoinvasion assay (Albini et al. 1987) facilitates a qualitative and quantitative analysis of a given cell line’s invasive capacity. Of the cell lines listed in Table 2, this type of analysis has been performed for DU-145, PC-3, LNCaP and TSU-PR1. Under standard assay parameters and utilizing non-selected parental cell lines, only PC3 and TSU-PR1 demonstrate a significant propensity for invasion through lamininrich basement membrane biomatrix designated Matrigel (Kozlowski et al. 1988; Lee et al. 1993; Gaylis et al. 1989; Keer et al. 1991). This feature correlates with increased expression of urokinase-like plasminogen activator (uPA)
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and the elaboration of other matrix-degrading proteases by these respective tumor cell lines (Gaylis et al. 1989; Keer et al. 1991). Despite the fact that DU145 and LNCaP were derived from metastatic foci, their constitutive ability to penetrate this basement membrane barrier is weak. Consistent with their transformed phenotype, all of the cell lines listed in Table 2 (with the exception of LNCaP) manifest tumorigenic potential following direct subcutaneous inoculation into athymic male nude mice. With respect to LNCaP, its tumorigenic potential must be facilitated by co-injection with prostate or bone fibroblasts (Gleave et al. 1991, 1992) or Matrigel (Lim et al. 1993). Matrigel co-injection was also used to facilitate the xenografting of MDC PCa 2a, b (Navone et al. 1997). Of importance, LNCaP xenografts maintain their androgen responsiveness (Lim et al. 1993; Gleave et al. 1992). Indeed, studies utilizing this murine model have demonstrated a significant correlation of serum PSA to tumor volume and weight. Castration leads to involution of these tumors and stabilization of the serum PSA levels. Other investigators also utilized this model system to demonstrate that decreases in circulating PSA following anti-androgen therapy may not always reflect a corresponding reduction in tumor volume (Gleave et al. 1992). Of the cell lines listed in Table 2, 6 are derived from metastatic foci. Ideally, these tumor systems should maintain the metastatic potential demonstrated in the natural host. It is now acknowledged that the subcutaneous deposition of human tumor cell suspensions into the flank regions of young athymic nude or Scid mice may underestimate the metastatic potential (Kozlowski et al. 1984; Kozlowski et al. 1988). Early studies have demonstrated that metastatic capacity might be better demonstrated by utilizing alternative routes of injection, including intravenous (tail vein), intrasplenic or intracardiac. More recently, the search for the most “fertile microenvironment” has logically led to the concept of orthotopic implantation (ie, direct intraprostatic injection) (Stephenson et al. 1992). The only parental (unselected) cell lines which exhibit metastatic potential are the PC3 and TSU-PR1 systems. Of note, the castration-induced regression of established LNCaP xenografts ultimately leads to the emergence of an androgen-insensitive variant (Wu et al. 1994). Induced by this selection pressure, the androgen-insensitive LNCaP variant is capable of metastasis to lymph nodes and bone following orthotopic implantation (Thalmann et al. 1994).
5.
MOLECULAR GENETICS
Brothman and associates (1990) described the major karyotypic changes associated with prostate cancer. Common abnormalities included: loss of chromosomes 1, 2, 5 and Y; gains of chromosome 7, 14, 20 and 22; and rearrangements involving 2p, 7q and 10q. Table 3 reveals the frequency with
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Table 2 Comparison of original tumor pathology with respective CaP cell liness Cell Line
Tumor pathology
In vitro features
Xenograft pathology
DU-145
MD-PD (+/–) AP PD, (?) AP
MG, PD, MEM/RPMI-1640 + 10% FCS AI (+) SAG, (+/–), AP, (-) PSA, (+/–) Invasive MG, PD, F12K/RPM1-1640 + 5-10%FCS, AI, (+), SAG, (-) AP, (-) PSA (+)Invasive Disorderly MG, loosely attached, MD, AS, (+) AR, RPMI-1640 + 10% FCS, (+) SAG (+) AP, (+) PSA, (-) Invasive
grade 2, (+) T in male and female NM (+/–) MP, (-) AP, (-) PSA PD, (+) T in male and female NM (+) MP, (–) AP, (–) PSA MD-PD (+) T with stroma/Matrigel male >>>female NM AS (+) AP (+) PSA (+) AR (-)MP- parental line S.C. site PD (+) T male=female NM AI (+) MP (–) AP, (–) PSA
PC-3
MD (?) AP
TSU-PR1
MD (?) AP
JCA-1
MD-PD Gleason 4+4 (+/–) AP (+ /–) PSA
DuPro–1
Patient = PD ?AP Donor Xenograft = MD-PD, (+) PSA, AS
MG, tightly packed PD AI (-) AR DM160/RPMI-1640 + 10-15% FCS (+) SAG (-) AP, (-) PSA, (+) Invasive MG MD-PD AI RPMI-1640 + 10% FCS (+/–) AP, (-) PSA, ? Invasive MG Isolated islands, PD RPMI-1640 + 10% FCS (+) SAG, (-) AP, (-) PSA, AI, ? Invasive
MD-PD (+)T male> >female NM AI (+) AP, (–) PSA, (–) MP PD (+) T, male=female NM AI (–) AP, (–) PSA, ?MP Continued on next page
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LNCaP
Prostate Cancer
Table 2 (continued) Cell Line
Tumor pathology
In vitro features
Xenograft pathology
ND-1
MD-PD Gleason 3 +3 ?AP ? PSA PD + PSA
MG, tightly packed MD-PD (+) SAG, (+/–) PSA DMEM + 10% FCS, ? Invasive Grow in clumps, form layers, PD BRFF-HPC1 or F12K (+) and 20% FBS (+)SAG,(+)AS,(+)AR (+) PSA, ? Invasive
MD-PD (+)T male NM (+/–) AP, (+/–) PSA ?AI, ?MP ?PD (+) T male NM AS (+) PSA, (–) MP
MDA PCa 2a, b
Abbreviations: MG, monolayer growth; AI, androgen independent; SAG, soft agar growth; AP, acid phosphatase; PSA, prostate specific antigen; NM, nude mice; MP, metastatic potential; T, tumorigenic; MD, moderately differentiated; PD, poorly differentiated AS, androgen-sensitive; AR, androgen receptor.
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which the well-characterized CaP cell lines demonstrate these features. A number of more recent and equally important observations have been forthcoming with respect to the genomic changes associated with prostate cancer. Step-section analysis performed on whole gland specimens containing prostate cancer usually reveals the presence of multiple tumor foci (Kozlowski and Grayhack, 1996). It has been debated whether these observations reflect a ‘field change’ phenomenon (Grayhack 1974) or merely represent a manifestation of intraglandular spread following unifocal tumor development. The studies conducted by Cheng and associates (1998) involved a molecular analysis of microsatellite alterations in the DNA from separate tumors in the same prostate. Their study focused on factors for the putative tumor suppressor gene on chromosome 8p (Macoska et al. 1995) and for the BRCA1 gene on chromosome 17q. The pattern of allelic loss documented in that study was compatible with independent tumor origin in 15/18 informative cases. Prostatic intraepithelial neoplasia (PIN) represents the precursor to most invasive prostate cancers originating within the peripheral zone (Helpap et al. 1995; Bostwick 1995; Grignon and Sakr, 1996). A number of recent studies have demonstrated that frequent allelic loss at chromosome 7q31-q35, 8p1221, 8p22, 8q22, 8q22.2, and 8q12.2, occur in both PIN and invasive prostate cancer (Emmert-Buck et al. 1995; Zenkhausen et al. 1994; Vocke et al. 1996; Bostwick et al. 1996). The implications associated with another potential precursor lesion designated atypical adenomatous hyperplasia (AAH) have been more controversial. It is thought by some that AAH may be related to low-grade adenocarcinomas originating within the transition zone (Helpap et al. 1995; Grignon and Sakr, 1996), but others have refuted this association Table 3 Karyotypic abnormalities in CaP and selected cell lines Main genetic changes associated with prostate cancer
Cell lines which exhibit these genetic changes
Double Minutes Marker Chromosomes Hyperdiploidy (–) 1 (–) 2 (–) 5 (–) Y (+) 7 (+) 14 (+) 20 (+) 22 RA 2P RA 7q RA 10q
PC-3; DU-145 PC-3; TSU-PRI; DU-145; LNCaP; JCA-1; ND-1; Du-Pro-1 PC-3; TSU-PRI; DU-145; LNCaP; JCA-1; ND-1; Du-Pro-1 PC-3 PC-3 PC-3; JCA-1 PC-3; DuPro-1 DU-145; PC-3; LNCaP; TSU-PR1; DuPro-1; ND-1 DU-145; PC-3; LNCaP; TSU-PR1; JCA-1; ND-1; DuPro-1 DU-145; PC-3; LNCaP; TSU-PR1; JCA-1; DuPro-1 DU-145; LNCaP; TSU-PR1; JCA-1; DuPro-1; ND-1 MDA PCa2 JCA-1; MDA PCa2 TSU-PR1; JCA-1
Abbreviations: (–), loss of chromosome; (+), gain of chromosome; RA, rearrangement of ch r o m o s o m e
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Prostate Cancer Table 4 Staging designations for carcinoma of prostate
Description
Clinical Stage UICC* Hopkins Memorial AJCC, 1992 (Modified Jewett) (Modified Whitmore, 1990)
Disease localized to prostate A A Clinically unsuspected T1 Incidental histologic finding Focal, low-grade A 1† A1‡ T1a† Intragland lump diffuse or high grade T1b§ A2 A2 Tumor identified, needle biopsy T1c (e.g., PSA elevated) Risk recognized clinically (confined T2 B B to prostate) Tumor confined to 1 lobe surrounded by normal tissue; B1 <2 cm (Whitmore) B1N B1 >2 cm B2 Half a lobe or less T2a More than half a lobe, but not both T2b Tumor in both lobes T2c B2 B3 Disseminated disease Periprostatic, extends through capsule T3 C C Lateral sulcus C1 Unilateral T3a Bilateral T3b Base of seminal vesicle and/or T3c C2 lateral sulcus > Base of seminal vesicle and/or T3c C3 other structure Tumor fixed: invades adjacent structure T4 other than seminal vesicle Bladder neck, extend sphincter and T4a rectum Levator or pelvic wall T4b Distant D D Pelvic lymph nodes N1-3** D1 D1 Bones, lung, etc. M1a–1c*** D2 D2 Elevated acid phosphatase only D0 D0 *UICC (International Unon Against Cancer); AJCC (American Joint Committee on Cancer) 1992. Note that T0 category is now listed as no evidence of primary tumor (Schroeder, 1992); † Tumor present in 5% or less of tissue; ‡ Tumor present in more than three microscopic foci. § Tumor present in more than 5% of tissue. **N1– one regional code < 2 cm; N2 - one regional node >2 cm <5 cm or multiple regional nodes, none 5 cm; N3 - regional node >5 cm. *** M1a – nonregional nodes; Mlb –bone; M1c – other site.
(Srigley, 1988). Recent studies by Cheng and associates (1998) demonstrated that 47% of AAH cases were associated with allelic imbalance at the same 5 microsatellite polymorphic markers typical of PIN. This data provides a genetic link between prostate cancer and AAH.
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Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric repeats into chromosomal ends using a segment of its RNA component as a template. Telomerase activity is intimately linked with the maintenance of the telomere, and its activation may play a role in cell immortality (Rhyu 1995). The telomeric repeat amplification protocol (TRAP) is a highly sensitive PCRbased assay which permits the evaluation of telomerase activity (Kim et al. 1994) This approach has documented telomerase activity in most human malignancies and cancer cell lines, suggesting that its activation may be a critical step in cell immortalization and oncogenesis (Royle 1996). Studies by Zhang and associates (1998) identified telomerase activity in 92% of prostate cancers. Of great interest and potential importance were the observations of telomerase activity in a wide spectrum of tissues located adjacent to cancer foci, including: PIN (73%), BPH (50%), atrophy (16%), and normal-appearing tissues (36%). Possible explanations include the presence of occult cancer cells in these non-tumor foci or the presence of early molecular alterations of cancer that were histologically inapparent. Utilizing FISH analysis, Takahashi and associates (1994) analyzed needle biopsy cores from randomly selected radical prostatectomy specimens. Common numerical chromosome alterations were gains of chromosome 7 and 8, which were noted in 76% and 59% of aneuploid tumors. Gains of chromosome 7 and 8 were correlated with a higher Gleason score. Gain of chromosome 7 was significantly correlated with advanced tumor pathological stage. In a follow-up study, a similar analysis was performed on paraffin-embedded specimen blocks (following radical prostatectomy). Takahashi and associates (1995) noted that gains of chromosome 8, aneusomy of chromosome 8, and aneusomy of chromosome Y correlated highly with systemic cancer progression. Multivariate analysis subsequently demonstrated that gains of chromosome 8 and aneusomy of chromosome Y were significant independent predictors of systemic cancer progression. Carter and associates (1990) and Kunimi and associates (1991) performed prostate cancer allelotyping studies which revealed frequent loss of heterozygosity (LOH) on chromosomes 8p (50%), 10p (55%), 10q (30%), 16q (31– 60%), and 18q (17–43%). These observations were expanded by Visakorpi and associates (1995) who performed CGH to screen for DNA sequence copy number changes along all chromosomes in 31 primary and 9 recurrent uncultured prostate carcinomas. They detected that 74% of primary prostate cancers showed DNA sequence copy number changes. In that study, losses were five times more common than gains and most often involved 8p (32%), 13q (32%), 6q (22%), 16q (19%), 18q (19%), and 9p (16%). Of note, frequent gains of 7,8q, and X were associated with prostate cancer progression and the development of hormone-independent growth. Cher and associates (1996) also used CGH to study prostate cancer metastases in patients who had received no prior treatment compared to primary or
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recurrent tumors in patients who had received long-term androgen-deprivation therapy. In treatment-naive metastatic foci, several altered chromosomal regions were documented including: 8q gain (85%), 8p loss (80%), 13q loss (75%), 16q loss (55%), 17p loss (50%), and 10q loss (50%). Of interest, the observations generated in the “treated” group were very similar to those of the “treatment-naive” group. The study also demonstrated a number of previously undetected regions of frequent loss including 2q (42%), 5q (39%), 6q (39%), and 15q (39%). Gains of chromosomes llp (52%), lq (52%), 3q (52%), and 2p (45%) were also documented. It is suspected that the regions of loss contained known or candidate tumor suppressor genes. Chromosome 5q31 contains the a-catenin gene which is an obligatory component of the E-cadherin-mediated cell adhesion complex (Furukawa et al. 1994). PC-3, DU-145, and TSU-PR1 have reduced or absent levels of a-catenin or E-cadherin (Morton et al. 1993). LOH at 7q31.1 (c-met oncogene locus) is correlated significantly with a higher Gleason score and lymph node metastasis (Takahashi et al. 1995; Pisters et al. 1995). Chromosome 10q22.1-qter harbors the candidate tumor suppressor gene Mxil (Eagle et al. 1995). The Mxil protein is thought to repress c-Myc activity and loss of this suppression may lead to c-Myc activation (Zervos et al. 1993). LOH at 10q23.3, a region commonly deleted in prostate cancer, may involve the loss of the candidate tumor suppressor gene designated PTEN/MMAC1 (Suzuki et al. 1998). Inactivation of this gene may contribute to the acquisition of metastatic potential in prostate cancer. Another putative metastasis suppressor gene has been mapped to human chromosome 11p11.2 and has been designated KAI1 (Dong et al. 1995). KAI1 expression is significantly reduced in human CaP cell lines derived from metastatic foci (PC-3, LNCaP, TSU-PR1, and DU145). The retinoblastoma susceptibility gene (RB1) is located on 13q14. About one-third of prostate cancers exhibit LOH at this locus (Cher et al. 1996; Cooney et al. 1996). Chromosome 16q contains the E-cadherin locus. E-cadherin is required for normal calcium-mediated cell-cell adhesion. Its expression is frequently lost in high grade, androgen-independent prostate cancers associated with invasive and metastatic potential (Umbas et al. 1994, 1992). Recent studies suggest that there is a separate region of 40% loss at 16q24 that may contain another important tumor suppressor gene (Cher et al. 1996). Over 50% of prostate cancers analyzed demonstrate allelic loss of at least one locus on chromosome 17q which contains the BRCA1 gene (Gao et al. 1995). Finally, the p53 tumor suppressor gene is located on 17p and is known to be mutated in 20–25% of metastatic prostate cancers (Cher et al. 1996). Of note, PC3 is a p53 negative prostate cancer cell line (Rajah et al. 1997). Regions of gain contain dominant oncogenes whose expression is amplified with increased copy number. The epidermal growth factor receptor (erbB-1) is located on chromosome 7p (Cher et al. 1996). Chromosome 7 trisomy is associated with higher grade and advanced-stage prostate cancers (Bandyk et
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al. 1994; Alcaraz et al. 1994). The c-met oncogene maps to chromosome 7q31 and is amplified in 40% of metastatic and androgen-independent prostate cancers (Pisters et al. 1995). Chromosome 8q24 harbors the cMyc oncogene. Of note, CGH analysis suggest the presence of a potentially important oncogene at 8q21.3 (Cher et al. 1996). The H-ras oncogene is located at 11p15.5. CGH analysis indicates a gain in the region of chromosome 17q that includes BRCA1 (Cher et al. 1996). In contrast, the PCR-based analysis conducted by Gao and associates (1995) noted a frequent loss of LOH at that locus. The erbB-2 oncogene is located at 17q12 (Kuhn et al. 1993). Chromosome Xq12 contains the androgen receptor gene which demonstrates a significant degree of amplification in patients with tumor recurrence following protracted androgen ablative therapy (Cher et al. 1996). Increased frequency of gains in the region 4q25-28 is a prominent feature of prostate cancer developing in African-Americans (Cher et al. 1996). It is theorized that a gene at that locus may be increased in activity and responsible for the more rapid rate of disease progression demonstrated in this cohort (Pienta et al. 1995; Brawn et al. 1993). The existing human CaP cell lines have been incompletely characterized with respect to many of the recently described genomic alterations. Understandably, most of the recent molecular studies have targeted fresh or archival prostate cancer specimens. Of interest, Konig and associates (1989) performed a cytogenetic characterization of several androgen-responsive and unresponsive sublines of LNCaP. The hormone-responsive sublines did not show any aberrations in chromosome 8. In contrast, the unresponsive sublines showed rearrangement of the short arm of chromosome 8, resulting in deletion of the 8p23→pter region. Thus, partial deletion of the 8p region may be linked with the development of androgen-independence.
6.
CROSS CONTAMINATION
In 1974, Okada and Schroder reported the establishment of cell line EB33. The line was derived from a patient who underwent a radical perineal prostatectomy for the treatment of a moderately-differentiated prostate adenocarcinoma. Subsequent reports questioned the origin of this cell line, and indicated contamination with HeLa cells (Kaighn et al. 1979; Peehl, 1994). In 1970, Fraley and associates reported the establishment of cell line MA160. This line was supposedly derived from a spontaneous in vitro neoplastic transformation of human benign prostatic epithelium. This line is also suspect, as it expresses several markers unique to HeLa cells (Nelson-Rees et al. 1974; Webber et al. 1977; Pontes et al. 1979). In 1989 Brothman and associates reported the establishment of cell line PPC-1. That cell line was derived from the prostate of a 67 year old black male with metastatic (bone), poorly-differentiated, prostate cancer. Tissues were
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obtained following transurethral resection. In 1993, Chen reported that the cell line PPC-1 is a clonal derivative of cell line PC-3. The two cell lines share numerous common chromosome characters.
7.
PROSTATE CANCER CELL LINES: UNIQUE FEATURES
The cell lines highlighted in the Tables were derived from patients whose tumors exhibited invasive and/or metastatic capacity. Ideally, these CaP cell lines should manifest some, if not most, of the phenotypes commonly associated with poorly-differentiated prostate cancers. The characteristics include: (1) high Gleason pattern score (Kozlowski and Grayhack, 1996); (2) tetraploid or aneuploid DNA content (Shankey et al. 1993); (3) abnormal cell shape, motility, and negative surface charge (Mohler et al. 1987; Carter and Coffey, 1988); (4) decreased nuclear androgen receptor expression (Trachtenberg and Walsh, 1982); (5) androgen receptor gene mutations (Gaddipati et al. 1994); (6) elaboration of angiogenic peptides (Brawer et al. 1994; Ferrer et al. 1998; Joseph et al. 1997); (7) enhanced expression of urokinase-type plasminogen activator and its receptors (Gaylis et al. 1989; Keer et al. 1991; Kozlowski et al. 1995); (8) aberrant expression of various growth factors, their receptors, and binding proteins (Rajah et al. 1997; Hofer et al. 1991; Fong et al. 1992; Pieterzkowski et al. 1993); (9) p53 mutations (Bookstein et al. 1993; Navone et al. 1993; Henke et al. 1994); (10) inhibition of programmed cell death by upregulation of the bc1-2 gene (McDonnell et al. 1992); (11) decreased tumor cell cohesiveness and increased invasiveness associated with reduced expression of the E-cadherin gene and/or deletion of alpha-catenin (Umbas et al. 1992, 1994; Morton et al. 1993; Frixen and Nagamine, 1993); (12) enhanced tumor motility due to up-regulation of thymosin beta 15 (Bao et al. 1996); (13) increased endothelin expression (Nelson et al. 1996); (14) elevated levels of TGF-b1 (Thompson et al. 1993; Eastham et al. 1995); (15) loss of expression of TGF-b1 type I and/or type II receptors (Kim et al. 1996a,b,c; Guo and Kyprianou, 1998); (16) increased interleukin-6 expression (Twillie et al. 1995; Okamoto et al. 1997); (17) decreased expression of neutral endopeptidase 24.11 (Papandreou et al. 1998); (18) preferential adhesion to human bone marrow endothelial cells (Lehr and Pienta, 1998); (19) loss of cyclin-dependent kinase inhibitor p27Kip1 (Tsihlias et al. 1998); and (20) the genomic alterations (losses and gains) characteristic of advanced-stage prostate cancers (see previous section). Dahiya and associates (1996) investigated the mRNA expression of a variety of growth factors (TGF- a, TGF-b1, TGF-b2, TGF-b3, KGF, EGF) and selected growth factor receptors (EGF-R and KGF-R) using a panel of CaP cell lines using RT-PCR. They demonstrated that the human primary CaP line
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(ND-1) showed mRNA transcripts for all growth factors except EGF and KGF. In contrast, LNCaP, DU-145, and PC-3 express mRNA transcripts for all growth factors except KGE KGF-R mRNA was absent in the PC-3 cell line, but present in the others. PC-3 has also been shown to constitutively produce large quantities of TGF- a, and the exaggerated production of that growth factor correlates with aberrant autocrine/ paracrine growth (Hofer et al. 1991). Under normal circumstances, TGF-b is a negative growth regulator that has been postulated to play a role in the control of prostate growth as a result of its ability to inhibit cell proliferation and induce programmed cell death (Thompson 1990; Martikainen et al. 1990; Kyprianou and Isaacs, 1989). These biological responses are mediated via interaction with two major cell surface receptors (type I and type 11) which belong to the serine-threonine kinase family (Lin et al. 1992; Wrana et al. 1994; Franzen et al. 1994). Once bound to TGF-β1 receptor type II, TGF-b1 can recruit receptor type I to form a ternary complex. The cyclin-dependent kinase inhibitors p151NK4, p21WAF-1/CiP and p27Kip1 are postulated as downstream effectors for the intracellular transduction of TGF-b negative growth signals (Guo and Kyprianou, 1998). Prostate cancer is associated with dysfunctional TGF-b signaling, primarily due to loss of TGF- b receptors (Kim et al. 1996 a, b, c; Lamm et al. 1998). PC-3, TSUPR1, and DU-145 express high mRNA levels of both TGF-b type I and type II receptors. In contrast, LNCaP (which is refractory to the growth inhibitory effects of TGF-b1) appears to be receptor deficient. With respect to the latter, controversy exists as to whether receptor type I (Kim et al. 1996a) or type II (Guo and Kyprianou, 1998) is missing in LNCaP cells. TSU-PR1 cells, although they express both TGF-b receptors, are growth stimulated rather than inhibited by TGF-b1 (Lamm et al. 1998). IL-6 is a pleiotropic cytokine whose expression is up-regulated in many prostate cancers (Twillie et al. 1995). Cell lines DU-145 and PC-3 synthesize and release interleukin-6 into the conditioned medium in far greater quantities than LNCaP (Okamoto et al. 1997). These studies also suggest that interleukin-6 functions as a paracrine growth factor for LNCaP and as an autocrine growth factor for DU-145 and PC-3. IL6 expression appears to correlate with an aggressive phenotype. The CaP cell lines have been used to study the mechanisms underlying the coordinated loss of androgen regulation of vascular endothelial cell growth factor (VEGF) expression, which is associated with the progression of prostate cancer to an androgen-independent state. VEGF is constitutively expressed and up-regulated in TSU-PR1, PC-3 and DU-145 by cellular hypoxia, but not by androgens. In contrast, VEGF levels are regulated by androgen in LNCaP and androgen ablation induces growth inhibition in LNCaP xenografts and produces a greater than 60% reduction in tumor VEGF levels (Joseph et al. 1997).
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These cell lines have also assisted in the evaluation of novel therapies. For example, TSU-PR1, LNCaP, PC-3 and DU-145 all demonstrate preferential adhesion to human bone marrow endothelial cells compared with human umbilical vein endothelial cells (Lehr and Pienta, 1998). Adhesion is inhibited in a dose-dependent manner by the galactose-containing carbohydrate modified citrus pectin. Modified citrus pectin has also been shown to have an antiproliferative effect on JCA-1 cells in vitro, which may be correlated with downregulation of cyclin B (Hsieh and Wu, 1995). The existing CaP cell lines have facilitated the evaluation of other novel therapies. For example, LNCaP cells are sensitive to tumor necrosis factor (TNF), while JCA-1 and PC-3 are TNF-resistant (Nakamima et al. 1996). The sensitivity of LNCaP to TNF can be blocked by the overexpression of sulfated glycoprotein-2 (clusterin) by stable transfection. The latter observation reaffirms the hypothesis that clusterin depletion, rather than its expression, is intimately linked with apoptosis (Sensibar et al. 1995). The synthetic retinoid 4-HPR induces a significant reduction of JCA-1 proliferation in vitro (Hsieh et al. 1995). In contrast, all-trans retinoic acid has a biphasic effect on the proliferation of LNCaP cells (low concentrations stimulate; high concentrations inhibit). Of importance, PSA secretory activity is increased at those concentrations of all-trans retinoic acid that inhibit proliferation (Fong et al. 1993). This dichotomy has obvious clinical implications and mandates caution with respect to the interpretation of serum PSA levels in patients receiving differentiation-inducing therapy. JCA-1 fails to express detectable numbers of nuclear vitamin D receptors (VDR) and does not demonstrate growth inhibition in the presence of lα, 25-dihydroxy vitamin-D3 (Hedlund et al. 1996). In contrast, the ALVA-31 cell line possesses abundant VDR and exhibits growth inhibition in the presence of vitamin-D3. Finally, epidermal receptor monoclonal antibody inhibits constitutive receptor phosphorylation, reduces autonomous growth, and sensitizes PC-3 and DU-145 cells to TNF-a (Fong et al.1992).
8.
XENOGRAFTS
For many years, xenografting of human prostate cancers into immune deficient animals was a rare event and serial transplantation was rarely possible (Shankey and Fogh, 1979). More recent observations have revealed that the growth and metastasis of human tumors implanted into nude mice is not a rare event (Kozlowski et al. 1984a,b; Kozlowski et al. 1988). In fact, this animal model can be used with great success for the propagation, expansion and in vivo selection of aggressive subpopulations (Kozlowski et al. 1988). Successful xenografting is contingent on a number of factors, including (1) the harvesting of high-grade primary tumors or metastatic foci; (2) prompt tissue acquisition
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and processing, (3) the use of young 4-6 week old athymic nude or Scid mice; (4) housing the animals in Specific Pathogen Free conditions to minimize immunogenic stimulation; (5) the use of viable finely minced tumor fragments embedded in Matrigel and deposited subcutaneously overlying the anterior aspect of the lateral thoracic wall; (6) the use of tumor cell suspensions coinjected with Matrigel into orthotopic sites; (7) the use of a large tumor inoculum; (8) prolonged experimental duration; (9) hormonal supplementation; and (10) the detailed characterization of the resulting xenograft to validate that it has maintained the features characteristic of the human tumor of origin (Kozlowski et al. 1988; Lim et al. 1992) . In 1980 Hoehn and associates established the transplantable human CaP line designated PC-82. The original tumor was a moderately differentiated adenocarcinoma of the prostate and was obtained following radical perineal prostatectomy. Tumor fragments were implanted subcutaneously into nude mice of the Balb/c background. This cell line exhibits a slow growth rate, hormone dependence, castration-induced regression, and the ability to secrete prostatic acid phosphatase (van Steenbrugge et al. 1984). Ito and Nakazato (1984) established the serially transplantable human CaP line designated HONDA. The patient was a 46 year old Japanese man with obstructive voiding symptoms and a hard nodule in the right lobe of the prostate. His serum AP levels were markedly elevated, strongly suggesting the presence of an invasive or metastatic tumor. He was initially placed on diethylstilbestrol but showed only transient improvement. Ultimately he developed bilateral testicular enlargement and orchidectomy was performed. Histologic analysis of the testes showed a moderately-differentiated adenocarcinoma. PSA immunohistochemistry confirmed prostatic origin. Tumor fragments were implanted in the right flank region of athymic nude mice. The tumor expressed high levels of PAP and PSA. Like PC-82, Honda exhibits androgen-dependence and castration-induced regression. Of note, electron microscopy demonstrated particles resembling type A retroviruses in the endoplasmic reticulum and particles resembling type C retroviruses in the intercellular space of the tumor cells. Pretlow and associates (1993) established the CWR22 transplantable xenograft. The primary prostate tumor that gave rise to CWR22 had a Gleason pattern score of 9. That patient also had evidence of skeletal metastasis. The tumor was minced finely to fragments that would pass through a 16 gauge needle and was mixed with Matrigel. Subcutaneous injections were administered to 4–8 week old male nude mice. Sustained release testosterone pellets were also utilized. After androgen withdrawal, CWR22 regresses and PSA levels drop accordingly. Some animals demonstrate tumor relapse, in which case the PSA starts to rise after approximately 2–7 months and the tumor begins to grow 3–10 months following castration (Nagabhushan et al. 1996). Studies of mRNA from the androgen-dependent CWR22 variant demonstrated the
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expression of PSA, erbB1, erbB2/neu, erbB3, and the neu differentiation factor (Wainstein et al. 1994). Ellis and associates (1996) established the androgen-sensitive, PSAproducing CaP xenograft designated LuCaP23. The patient was a 63-year-old Caucasian male with a stage D1 prostate cancer that exhibited a Gleason pattern score of 3+5. The patient underwent external beam radiation, androgen ablation, and cytotoxic chemotherapy. At death, the patient’s serum PSA level was about 8000 ng/ml. A sterile autopsy was performed within 2 hours of death. The xenograft designated LuCaP23.1 was derived from a lymph node metastasis. Tissue fragments were implanted subcutaneously in 6-week old male athymic mice. Following androgen deprivation, there is a temporary decrease in PSA secretion and tumor size. Ultimately, the tumors become androgen-independent and grow in the castrate host. Analysis of the hormoneindependent tumors demonstrates that they express neuron-specific enolase, which is a characteristic of neuroendocrine differentiation (Liu et al. 1996). In addition the hormone-independent tumors express bc1-2, which inhibits programmed cell death (Liu et al. 1996). With respect to the unselected cell line, castration is associated with an increase in the number of apoptotic cells and a decrease in proliferative activity. The androgen-independent variant which develops following castration demonstrates a low apoptotic index with no increase in proliferative activity. This latter observation may be related to amplified bc1-2 expression (Bladou et al. 1996). Comparative genomic hybridization and molecular cytogenetic characterization of LuCaP23.1 reveals a gain of all or part of chromosomes 3, 5, 6, 7, 8, 11, 12, and the X chromosome. It also exhibits loss of all or part of chromosomes 2,3,6,8,9,10, 17 and 18 (Williams et al. 1997). Finally, several less well-characterized xenograft models have been reported in the literature. Hoehn and associates (1984) report the establishment of the androgen-dependent PC-EW xenograft model. In 1996 van Weerdern and associates reported the development of 7 new CaP tumor xenograft models. The tumors designated PC-295, PC-310, PC-329, and PC-346 are androgendependent. The PC-324, PC-339, and PC-374 are androgen-independent.
ACKNOWLEDGMENT The authors wish to express their gratitude to Lisa-Marie Johnson for her invaluable assistance in the preparation of this manuscript.
REFERENCES Achbarou A et al. Cancer Res 54: 2372, 1994. Albini A, Iwamoto Y, Kleinman HK et al. Cancer Res 47: 3239, 1987.
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Kozlowski and Sensibar
Alcaraz A et al. Cancer Res 54: 3998, 1994. Bandyk MG et al. Genes Chromosomes & Cancer 9: 19, 1994. Bao L, Loda M, Janmey PA et al. Nature Medicine 2: 1322-1306, 1996. Billstrom A, Lecander I, Dagnacs-Hansen F, Dahllof B, Stenram U, Hartley-Asp B. The Prostate 26: 94, 1995. Bladou F, et al. Int. J. Cancer 67: 785, 1996. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB et al. Science 279: 349,1998. Bookstein R, MacGrogan D, Hilsindeck SG et al. Cancer Res 53: 3369,1993. Bostwick DG et al. Prostate 29: 117, 1996. Bostwick DG. Cancer (Phila.) 75: 1823, 1995. Brawer MK, Deering RE, Brown M et al. Cancer 73: 678, 1994. Brawn PN et al. Cancer (Phila.) 71: 2569, 1993. Bright RK et al. Cancer Res 57: 995, 1997. Brothman AR, Lesho LJ et al. Int J Cancer 44: 898,1989. Brothman AR, Peehl DM, Patel AM, McNeal JE. Cancer Res 50: 3795, 1990. Carter BS et al. Proc NatlAcad Sci USA 87: 8751, 1990. Carter HB, Coffey DS. J Urol 140: 173,1988. Catalona WJ. Prostate Cancer. New York, Grune and Stratton, Inc. 1984. Catalona WJ. New Engl JMed 331: 996, 1994. Chaproniere DM, McKeehan WL. Cancer Res 46: 819,1986. Chen TR. Cytogenet Cell Genet 62:183, 1993. Cheng L et al. Cancer Res 58:389, 1998a. Cheng L et al. J Natl Cancer Inst 90: 233, 1998b. Cher ML et al. Cancer Res 56: 3091, 1996. Cooke DB, Littleton GK. The Prostate 7: 209, 1985. Cooney KA et al. Cancer Res 56: 1142,1996. Cunha GR, Donjacour AA, Cooke PS, Mee S, Bigsby RM, Higgins SJ, Sugimura Y. Endocrine Reviews 8. 3: 338, 1987. Cunningham JM et al. Cancer Res 56: 4475, 1996. Cussenot 0 et al. J Urol 143: 881, 1991. Dahiya R et al. Urology 48: 963, 1996. Dong JT et al. Science 268: 884, 1995. Drago JR, Goldman LB, Worgul T, Gershwin E. J Surg Oncol 16: 265, 1981. Drago JR, Maurer RE, Goldman LB, Gershwin ME. Cancer Chemother Pharmacol 3: 167,1979. Dunning WF. In: ‘Biology of the Prostate and Related Tissues.’ National Cancer Institute Monograph 12: 351, 1963. Eagle LR et al. Nat Genet 9: 249, 1995. Eastham JA, Hall SJ, Sehgal I et al. Cancer Res 55: 5151-5155, 1995. Ellis WJ et al. Clin Cancer Res 2: 1039, 1996. Emmert-Buck MR et al. Cancer Res 55: 2959,1995. Fan K. J Urol 139: 318,1988. Ferrer FA et al. Urology 51: 161, 1998. Fong CJ, Sherwood ER, Sutkowski DM, Abu-Jawdeh GM, Yokoo H, Bauer KD, Kozlowski JM, Lee C. Prostate 19: 221, 1991. Fong CJ, Sherwood ER, Mendelsohn J et al. Cancer Res 52: 5887,1992. Fong CJ, Sherwood ER, Braun EJ, Berg LA, Lee C, Kozlowski JM. The Prostate 21: 121, 1992. Fong CJ, Sutkowski DM, Braun EJ, Bauer KD, Sherwood ER, Lee C, Kozlowski JM. J Urol 149: 1190,1993. Fraley EE, Ecker S, Vincent MM. Science 170: 540, 1970. Franzen P et al. Cell 67: 785, 1994.
Prostate Cancer
329
Frixen UH, Nagamine Y. Cancer Res 53: 3618, 1993. Furukawa Y et al. Cytogenet Cell Genet 65: 74. 1994. Gaddipati JP, McLeod DG, Heidenberg HB et al. Cancer Res 54: 2861, 1994. Gao X et al. Cancer Res 55: 1002, 1995. Gaylis FG, Keer HN, Wilson MJ et al. J Urol 142: 193,1989. Gingrich JR, Tucker JA, Walther PJ, Day JW, Poulton SHM, Webb KS. J Urol 146: 915, 1991. Gleason DF. In Tannenbaum M. (Ed.) Urology Pathology: The Prostate. Philadelphia, Lea and Febiger, p. 17, 1977. Gleave ME et al. Cancer Res 51: 3753, 1991. Gleave ME et al. J Urol 147: 1151, 1992a. Gleave ME, et al. Cancer Res 52 1598, 1992b. Goldstein S. Science 249: 1129, 1990. Grayhack JT. Urol Digest 13: 19, 1974. Grignon DJ, Sakr WA. Eur Urol 30: 206, 1996. Guo Y, Kyprianou N. Cell Growth & Differentiation 9: 185, 1998. Hartley-Asp B, Billstrom B, Kruse E. Int J Cancer 44: 161, 1989. Hedlund TE, Moffatt KA, Miller GJ. J Steroid Biochem Mol Biol 58: 277, 1996. Helpap BG et al. Virchows Arch 426: 426, 1995. Henke RP, Kruger E, Ayagn N et al. J Urol 152: 1297, 1994. Hoehn W et al. Prostate 5: 445, 1984. Hofer B, Sherwood E, Bromberg W et al. Cancer Res 51: 2780, 1991. Holund B. Scand J Urol Nephrol 14: 29, 1980. Horoszewicz JS et al. Models for Prostate Cancer, p. 115, 1980. Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H, Chu TM, Mirand EA, Murphy GP. Cancer Res 43: 1809, 1983. Hsieh TC, Ng C, Wu JM. Biochemistry & Molecular Biology International 37: 499, 1995. Hsieh TC, Wu JM. Biochemistry & Molecular Biology International 37: 833, 1995. Iizumi T, Yazaki T, Kanoh S, Kondo I, Koiso K. J Urol 137: 1304,1987. Isaacs JJ, Coffey DS. In: ‘Prostate Cancer,’ UICC Technical Report Series 48: 195, 1979. Ito YZ, Nakazato Y. J Urol 132: 384, 1984. Jackson-Cook C et al. Cancer Genet Cytogenet 87: 14, 1996. Joseph IBJK, Nelson JB, Denmeade SR, Isaacs JT. Clin Cancer Res 3: 2507,1997. Kaighn ME et al. Cancer Res 49: 3050, 1989. Kaighn ME, Lechner JF, Narayan KS, Jones LW. National Cancer Institute Monograph 49: 17, 1978. Kaighn ME, Narayan KS, Ohnuki Y, Lechner JF, Jones LW. Invest Urol 17: 16, 1979. Keer HN, Gaylis FD, Kozlowski JM et al. Prostate 18: 201, 1991. Kim IY et al. Cancer Res 56: 44, 1996a. Kim IY et al. Experimental Cell Research 222: 103,1996b. Kim IY et al. Endocrinology 137: 991, 1996c. Kim IY, Ahn HJ, Zelner DJ et al. Clinical Cancer Research 2: 1255-1261, 1996d. Kim JH, Sherwood ER, Krengel SS et al. J Urol 143: 242A, 1990. Kim NW, Piatszek MA et al. Science Washington DC, 266: 2011, 1994. Konig JJ, Kamst E, Hagemeijer A, Romijn JC, Horoszewicz J, Schroder FH. Urol Res 17: 79, 1989. Kozlowski JM et al. JNCI 72: 913, 1984a. Kozlowski JM et al. Cancer Res 44: 3522, 1984b. Kozlowski JM et al. Tumor Progression and Metastasis. Nicolson GL, Fidler IJ (Eds). Alan R. Liss, Inc. Pp. 189-231, 1988. Kozlowski JM, Grayhack JP. In Gillenwater JY, Grayhack JP, Howards SS, Duckett JW, eds. Adult Pediatric Urology Yearbook Medical Publishers, Chicago, 2nd edition, 2: 34: 1277, 1991.
330
Kozlowski and Sensibar
Kozlowski JM, Keer HN, Gaylis FD et al. In: Fibrinolysis in Disease: The Malignant Process, Interventions in Thrombogenic Mechanisms and Novel Treatment Modalities, Glas-Greenwalt P. (Ed), CRC Press, Inc. 7: 41, 1995. Kozlowski JM, Grayhack JT. In: Adult and Pediatric Urology, 3rd ed., Gillenwater JY et al. (Eds), Volume 2, p. 1575, 1996. Kramer SA, Spahr J, Brendler CB. J Urol 124: 223, 1980. Kuhn EJ et al. J Urol 150: 1427, 1993. Kunimi K et al. Genomics 11: 530, 1991. Kyprianou N, Isaacs JT Mol Endocrinol 3: 1515,1989. Lamm et al. Endocrinology 139: 787,1998. Landis SH, Murray T, Bolden S, Wingo PA. CA Cancer J Clin 49: 8, 1999. Lee C, Shevrin DH, Kozlowski JM. Cancer and Metastasis Reviews 12: 21, 1993. Lee C, Sutkowski DM, Sensibar JA, Zelner D, Kim I, Amsel I, Shaw N, Prins GS, Kozlowski JM. Endocrinology136: 796, 1995. Lehr JE, Pienta KJ. J Natl Cancer Inst 90: 118,1998. Lim DJ et al. The Prostate 22: 109, 1993. Lin HY et al. Cell 68: 775, 1992. Liu AY et al. Int J Cancer 65: 85, 1996. Loop SM, Rozanski TA, Ostenson RC. Prostate 22: 93,1993. Lubaroff DM, Caufield L, Reynolds CW. In Murphy G., (ed): ‘Models for Prostate Cancer.’ New York Alan R. Liss, Inc., pp. 243, 1980. Macoska JA et al. Cancer Res 55: 5390, 1995. Martikainen P et al. Endocrinology 127: 2936, 1990. McDonnell TJ, Troncoso P, Brisbay SM et al. Cancer Res 52: 6940, 1992. McKeehan WL et al. Cancer Res 44: 1998,1984. Mohler JL, Partin AW, Coffey DS. J Urol 138: 168, 1987. Morton RA et al. Cancer Res 53: 3585, 1993. Muraki J et al. Invest Urol 36: 79, 1990. Nagabhushan M et al. Cancer Res 56: 3042,1996. Nakajima Y, et al. Prostate 29(5): 296, 1996. Nakhla AM, Rosner W. Steroids 59: 586, 1994. Nakhla AM, Rosner W. Endocrinology 137: 4126,1996. Narayan P, Dahiya R. J Urol 148: 1600,1992. Navone MM, ’Roncoso P, Pisters LL et al. J Natl Cancer Inst 85: 1657, 1993. Navone NM et al. Clinical Cancer Res 3: 2493, 1997. Nelson, JB, Chan-Tack, K, Hedican, SP, Magnuson, SR, Opgenorth, TJ, Bova, GS, Simons, JW. Cancer Res 663-668,1996. Nelson-Rees WA, Flandermeyer RR, Hawthorne PK. Science 184: 1093, 1974. Noble RL. Cancer Res 37: 1929,1977. Okada H, Schroder FH. Urol Res 2: 111,1974. Okamoto M, Lee C, Oyasu R. Cancer Res 57: 141, 1997. Papandreou CN et al. Nature Med 4: 50, 1998. Peehl DM. In ‘Culture of Epithelial Cells’, Freshney RI. (ed). Wiley-Liss, Inc., pp. 159, 1992. Peehl DM. In ‘Atlas of Human Tumor Cell Lines’., RJ Hay et al. (Eds) Academic Press, pp. 387, 1994. Peehl DM, Stamey TA. In Vitro 22: 82, 1986. Pienta KJ et al. Urology 45: 93, 1995. Pieterzkowski Z, Mulholand G, Gomella L et al. Cancer Res 53: 1102, 1993. Pisters LL et al. J Urol 154: 293, 1995. Pollard M. The Prostate 1: 207, 1980. Pollard M. J Natl Cancer Inst 51: 1235, 1973.
Prostate Cancer
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Pontes JE et al. In Vitro 15: 469, 1979. Pretlow TG et al. J Natl Cancer Inst 85: 394, 1993. Rajah R, Valentinis B, Cohen P. Journal of Biological Chemistry 272: 12181, 1997. Rhim JS et al. Proc Natl Acad Sci USA 91: 11874,1994. Rhyu MS. J Natl Cancer Inst 87: 884, 1995. Royle NJ. J Pathol 180: 233, 1996. Sensibar JA, Sutkowski DM, Raffo A, Buttyan R, Griswold MD, Sylvester SR, Kozlowski JM, Lee C. Cancer Res 55: 2431,1995. Shain SA, McCullough B, Segaloff A. J Natl Cancer Inst 55: 177,1975. Shankey TV, Kallioniemi O-P, Kozlowski JM et al. Cytometry 14: 497, 1993. Sharkey FE, Fogh J. Int J Cancer 24: 733,1979. Shenvood ER, Berg LA, McEwan RN, Pasciak RM, Kozlowski JM, Lee C. J Cell Biochem 40: 201, 1989. Shenvood ER, Berg LA, Mitchell NJ, McNeal JE, Kozlowski JM, Lee C. J Urol143: 167, 1990. Srigley JR. Semin Diagn Pathol 5: 254, 1988. Stephenson RA et al. J Natl Cancer Inst 84: 951, 1992. Stone KR, Mickey DD, Wunderli H, Mickey GH, Paulson DF. Int J Cancer 21: 274, 1978. Suzuki H, Freije D et al. Cancer Res 58: 204, 1998. Takahashi S et al. Clinical Cancer Res 2: 137, 1996. Takahashi S et al. Cancer Res 55: 4114, 1995. Takahashi S et al. Cancer Res 54: 3574, 1994. Thalmann GN et al. Cancer Res 54: 2577, 1994. Thompson TC. Cancer Cells 2: 345, 1990. Trachtenberg J, Walsh PC. J Urol 127: 466, 1982. Trapman J et al. J Steroid Biochem Molec Biol 37: 837, 1990. Tsihlias J et al. Cancer Res 58: 542, 1998. Twillie DA et al. Urology 45: 542, 1995. Umbas R et al. Cancer Res 54: 3929, 1994. Umbas R, Schalken JA, Aalders TW et al. Cancer Res 52: 5104, 1992. Umekita Y et al. Proc NatlAcad Sci USA 93: 11802, 1996. Van Helden PD et al. Br J Cancer 70: 195, 1994. Van Steenbrugge GJ et al. J Urol 131: 812,1984. Van Weerden WM et al. Am J Path 149: 1055, 1996. Visakorpi T, Kallioniemi AH et al. Cancer Res 55: 342, 1995. Vocke CD et al. Cancer Res 56: 2411, 1996. Wainstein MA et al. Cancer Res 54: 6049, 1994. Ward JM, Reznik G, Stinson SF, Lattuada CP, Longellow GD, Cameron TP. Lab Invest 43: 517, 1980. Webber MM, Horan PK, Bouldin TR. Invest Urol 14: 335, 1977. Webber MM, Bello D, Quader S. Prostate 30: 58, 1997. Weijerman PC et al. Cancer Res 54: 5579, 1994. Williams BJ, Jones E, Kozlowski JM, Vessella R, Brothman AB. Genes, Chromosomes & Cancer 18:299, 1997. Williams RD. Invest Urol 17: 359, 1980. Wrana J et al. Nature, Lond. 370: 341, 1994. Wu HC et al. Int J Cancer 57: 406, 1994. Zervos AS et al. Cell 72: 223, 1993. Zhang W, Kapusta LR et al. Cancer Res 58: 619, 1998.
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Chapter 32 Liver Cancer
Masayoshi Namba, Masahiro Miyazaki and Kenichi Fukaya Institute of Cellular and Molecular Biology, Okayama University Medical School, 2-5-1 Shikata, Okayama 700-8558, Japan. Tel: 0081-86-235-7393; Fax: 0081-86-235- 7400; E-mail:
[email protected]
1.
INTRODUCTION
Primary liver cancers are of two types: hepatocellular carcinoma (HCC) which originates from liver parenchymal cells (hepatocytes) and cholangioma, derived from intrahepatic biliary epithelial cells. HCC is one of the major malignant diseases of the world, being responsible for about one million deaths per year (Okuda et al. 1993). The causative factors associated with liver cancer development remain elusive. Epidemiological and experimental data suggest that infection with hepatitis B or C virus (HBV, HCV), ingestion of aflatoxin B1 (AFB1)-contaminated foods, alcoholic cirrhosis, and other factors associated with chronic inflammatory and hepatic regenerative changes are important risk factors for hepatocarcinogenesis (Hsu et al. 1993). However, which oncogenes or tumor suppressor genes are critical for hepatocarcinogenesis is unresolved (Lea 1993).
2.
CELL CULTURE
Since the first human hepatoma cell line (HLE) was described (Doi et al. 1975), about 40 other HCC lines have been established (Table 1). Amongst these, HLE, PLC/PRF/5, HepG2, Hep3B, HuH-6, and HuH-7 (Miyazaki and Namba 1994), are widely used. Human cholangioma cell lines are listed in Table 2.
J.R.W Masters and B. Palsson (eds.), Human Cell Culture Vol. II, 333–343. ©1999 KIuwer Academic Publishers. Printed in Great Britain.
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Table 1 Human hepatocellular carcinoma cell lines Cell line
Patient age/sex
Pathology & TNM category
HLE* HLF* HuH-6*
68 yr/M 68 yr/M 1 yr/M
C-Hc-4
45yr/M
HCC (undifferentiated) HCC (undifferentiated) Hepatoblastoma (well-differentiated) M1 (lung & brain) HCC
C-Hc-20
30yr/M 24 yr/M 15 yr/M 8 yr/M
PLC/PRF5*,** HepG2** Hep3B**
53yr/M 51 yr/M 57 yr/M 52yr/M 34 yr/M 76 yr/M 53yr/M 50yr/M 63 yr/M 50yr/M 57yr/M 51 yr/M 50yr/M 57yr/F
Culture method
liver liver liver
DMEM+ 10% FBS DMEM+10% FBS DMEM+ 10% FBS
liver
RPMI-1640+20% FBS, HB101, HB102 RPMI-1640+10% FBS DMEM+ 10% FBS DMEM+ 10% FBS DMEM+ 10% FBS
liver liver & lung liver liver liver liver liver liver liver liver liver liver liver liver ascites liver liver liver
References Doi et al. 1975 Doi et al. 1975 Doi 1976 Sasaki et al. 1988 Human Cell 1: 106,1988 Alexander et al. 1976 Aden et al. 1979 Aden et al. 1979
Huh et al. 1981 DM-160+15% FBS DM-160+15% FBS Huh et al. 1981 Nakabayashi et al. 1982 DMEM+ 10% FBS DMEM+10% FBS +2mM glutamine Chang et al. 1983 DMEM+10% FBS +2mM glutamine Hu et al. 1986 RPMI-1640+10% FBS Watanabe et al. 1983 HB101 Murakami 1984 Ham’s F-12+10% FBS Matsuura 1983 DMEM+ 10% FBS Nohara 1987 DMEM+ 10% FBS He et al. 1984 Homma 1985 Williams’ E+ 10% FBS Homma 1985 Williams’ E+ 10% FBS Homma 1985 Williams’ E+ 10% FBS Williams’ E+10% FBS Human Cell: 117, 1988 Williams’ E+ 10% FBS Human Cell: 117, 1988 Continued on next page
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huH-1 huH-4 HuH-7* HA22T/VGH HA47T/VGH HCC-M KIM-1 KG55T HH2 FOCUS JHH- 1 * JHH-2* JHH-4* JHH-5* JHH-6*
HCC HCC, M1 (lung) Hepatoblastoma HCC (well-differentiated trabecular) M1 (lung) HCC HCC HCC (well-differentiated) HCC HCC HCC (well-differentiated, trabecular) HCC (well-differentiated, trabecular) HCC HCC HCC (poorly differentiated) HCC HCC HCC HCC HCC
Specimen site
(continued)
Cell line
Patient age/sex
Pathology & TNM category
Specimen site
KYN-1
58 yr/M
HCC (well-differentiated trabecular)
liver
Hep-Tabata HPT-NT/D3
60 yr/M 85yr/F
HCC HCC
liver peripheral blood
KYN-2 Tong/HCC
52 yr/M 59 yr/M
HCC (pleomorphic) HCC
liver liver
TH-1 OHR HCC-T JHH-7* PLC/AN/2
2 yr/M 4 mo/M 69 yr/M 53 yr/M 55 yr/M
liver ascites liver liver liver
HAK-1A HAK-1B RBHF-1
55yr/M 55 yr/M 67 yr/M
Hepatoblastoma, T3N0M0 Hepatoblastoma (poorly differentiated) HCC (well-differentiated, trabecular) HCC HCC (well-differentiated, trabecular & tubular) HCC HCC HCC (pleomorphic)
liver liver liver
Culture method
References
HB101 + 1mM pyruvate, +400 µg/ml Yano et al. 1986 glutamine, DMEM+ 10% FBS Human Cell: 119,1988 DM170+ 10% FBS DMEM+10% FBS +5 µg/ml insulin, Human Cell: 112, 1988 +20 ngiml dexamethasone +5 µg/ml transferrin +20 ng/ml EGF Serum free medium Human Cell: 115, 1988 DMEM/Hams F12, + 1% FBS Stevenson et al. 1987 +25 ngiml EGF +5 µg/ml transferrin, +5 µg/ml insulin +20 ngiml hydrocortisone +6.6 ngiml selenic acid + 10 ngiml liver cell growth factor +500 ngiml glucagon, +674 ng/ml ornithine DM-160+15% FBS Yaoita et al. 1989 Kanno et al. 1989 DMEM+ 10% FBS Saito et al. 1989 RPMI-1640+10% FBS Williams’ E+ 10% FBS Homma et al. 1990 Bagnarelli et al. 1990 RPMI-1640+20% FBS DMEM+5% FBS DMEM + 5% FBS RPMI-1640+10% FBS
Liver Cancer
Table 1
Yano et al. 1993 Yano et al. 1993 Sing et al. 1994
*These cell lines are available from HSRRB in Osaka Branch of NIHS, 1-1-43 Hoen-Zaka, Chuo-ku, Osaka, 540, Japan, Fax:+81-6-945-2872. **These cell lines are available from ATCC, 12301 Parklawn Drive, Rockville, MD 20852, USA.
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Cell line
Patient age/sex
Pathology Adenoca
HChol-Y 1
Specimen site Autopsy specimen of primary liver adenoca Ascites
Culture method Ham’s F12±0.1% FBS Williams’ E + 10% FBS
oz
71/M
CHGS
72/M
Well-differentiated papillary adenoca and poorly differentiated adenoca Tubular adenoca of common bile duct
HUH-28
37/F
Undifferentiated cholangioma
Primary cholangiocellular ca
RPMI-1640/Ham’s F12 (1:1) + 10% FBS RPMI-1640 t 10% FBS
HuCC-T1
56/M
Ascites
RPMI-1640 + 10% FBS
KMBC
73/M
Mod differentiated adenoca in the intrahepatic biliary tree Extrahepatic bile duct ca
Surgically resected tumor
DMEM + 5% FBS
KMC-1
62/M
Cholangiocellular ca
DMEM + 5% FBS
CC-SW-1
55/F
Mod differentiated adenoca
Surgically resected cholangiocellular ca tissue from periphery of tumor Surgically resected liver tumor
DMEM + 5-15% FBS
CC-LP-1
48/F
Mod-poorly differentiated adenoca
Surgically resected liver tumor
DMEM + 5-15% FBS
Primary cholangiocellular ca
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Table 2 Human cholangioma cell lines References Yamaguchi N et al. JNCI 75: 29, 1985 Nagamori et al. Jikeikai Med J 32: 289,1985 Katoh H et al. Human Cell 1: 101, 1988 Kusaka Y et al. Res Exp Med 188: 367,1988 Miyagiwa et al. In Vitro 25: 503,1989 Yano H et al. Cancer 69: 1664, 1992 Iemura A et al. J Hepatol 15: 288,1992 Shimizu Cancer Shimizu Cancer
Y et al. Int J 52: 252,1992 Y et al. Int J 52: 252,1992
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HepG2 and HUH-7 cells show liver-specific differentiated phenotypes in culture (Ding et al. 1994; Lea 1993; Nakabayashi et al. 1982). HUH-7, HUH-6 and PLC/PRF/5 cell lines can grow in serum-free media with some supplements (Nakabayashi et al. 1982), which has made it easier to detect which proteins these cultured liver cells are producing. Establishing long-term cultures of human liver cancer cells is not easy by conventional culture methods because the culture media and methods available may not be suitable, and the cancer cells may have a finite replicative lifespan. A high proportion of human liver cancers are infected with hepatitis viruses such as HBV and HCV. Therefore, special caution should be taken when handling primary cultures.
3.
CHARACTERISTICS OF LIVER CANCER CELLS IN CULTURE
Cultured liver cancer cells retain several differentiated features of hepatocytes (Table 3), including the expression of a-fetoprotein, albumin, transferrin, hemopexin, a1-antitrypsin, a2-macroglobulin, glucose-6-phosphatase, cytokeratin 18, urea cycle-related enzymes, tyrosine aminotransferase, alanine aminotransferase, g-glutamyl transferase, phosphoenolpyruvate carboxykinase, aldolase-B and apolipoprotein (Lea 1993). Some of these differentiated features can be induced by sodium butyrate, dimethylsulfoxide, hexamethylene bisacetamide and retinoic acid (Lea 1993). These liver cell-specific differentiated features are often used to confirm a derivation from liver cells. Production of albumin and a-fetoprotein are the markers most frequently used. However, markers such as these cannot exclude cross-contamination. To solve this problem, DNA fingerprints and isozymes of each HCC and cells from the patient should be analyzed. This has rarely been done in the past, but it should be done in the future. HepG2 cells have measurable activity of the 1A2, 2A6, 2B7, 2E1, 2F1 and 3A5 forms of cytochrome P450 (Lea 1993). Because many drugs are metabolized by these enzymes, this cell line is useful for toxicological studies. Other HCC lines tested so far have no activity of any P450 enzymes.
4.
HEPATITIS VIRUS
As shown in Table 4, hepatitis B surface antigen (HBsAg) and X antigen have been detected in PLC/PRF/5, Hep3B, HB611, huH 1, huH 2 and huH 4 by PCR (Hsu et al. 1993; Knowles et al. 1980). Integration of the HBV genome has also been reported in PLC/PRF/S, Hep3B and JHH-7 cells (Homma et al.
Cell line
Take in nude mouse
Doubling time (h)
Chromosome distribution (mode)
HLE* HLF HuH-6* cHc-4 PLC/PRF/5 * HepG2* Hep3B huH-1 huH-4 HUH-7*
+, mod diffd ND +, poorly diffd + , poorly diffd +, poorly diffd – + ND ND +
34 53 48 20 35–48 49 ND ND ND 36
40–100 hypertetraploid 40–90 hypotriploid hypotriploid (56) heteroploid (55) heteroploid (60) heteroploid (69) heteroploid (70)
HA22T/VGH HCC-M KIM-1 KG55T FOCUS JHH- 1 * JHH-2 JHH-4 JHH-5 JHH-6
+ +, Edmondson type III +, trabecular – t + , poorly diffd ND ND ND ND
ND 24 60 50–60 42-48 44-50 150 55 78 ND
hypertriploid (73) hypotriploid (63) hypertriploid (79) triploid (69) 61-70 hypertetraploid (95) hypertriploid (70) hypertriploid (75)
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Table 3 Characteristics of human hepatocellular carcinoma cell lines Liver specific AFP(+) ® (–) AFP(–) AFP, AL AFP, CEA AFP(–) AAG, AAT, AFP, AL, AMG, BLP, C3, C4, CP, FN, HP, PL, TF AAG, AAT, AFP, AL, AMG, BLP, C3, C4, CP, FN, HP, PL, TF TAT AAT, AFP, AMG, BLP, BMG, C3, C4, CEA, CP, FIB, FN, G6Pase, HP, HX, PRE, TF AAT, AL, ALT, AMG, BLP, C3, C4, CP, GGT, HP, PL, TAT, TF AL, G6PDH: type B, AFP(–), HBsAg(-) AFP, BMG, TF AL, AAT, AMG, TAT AFP, AST, ATT, CEA, FIB, G6Pase ALP, CEA, GGT, LDH, AL(-) AFP(–) AL, ALP, FER, GGT, LDH, AFP(–) AFP, AL, GGT LDH AFP, AL FER
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Liver Cancer
Table 3 (continued) Cell line
Take in nude mouse
Doubling time (h)
Chromosome distribution (mode)
Liver specific
JHH-7 KYN-1 Tong/HCC HCC-T PLC/AN/2 HAK- 1A HAK-1B RBHF-1
+ +, adenoca – +, Edmondson type II ND – + –
26 31 50 24 26 27 20 24
hypertriploid (74) 61-74 hypotriploid (64) hypotriploid (64) hyperdiploid (57) hypodiploid (41) hypertriploid (78) hypodiploid (45)
AFP, AL, CEA, FER AAT, AFP, AL, BMG, CEA, FER, HBcAg(–), HBsAg(–) AAT, AFP, AL, AMG, CP', PL, Integration of HBV DNA AFP, AL(-), HBsAg(-), HBeAg(–) AAT, AL, ALT, AST, FN, G6Pase, Hexokinase AFP(-), CEA(–) AAG, AAT, AL, C3, C4, FN, AFP(-) AAT, AL, C3, C4, FN, PRE, AFP(-), Prothrombin AL, AFP, ALT, AMG, AST, FN, LDH
*Pu et al. 1997. AAG: a1-acid glycoprotein, AAT a1-antitrypsin, AFP: a -fetoprotein, AL: albumin, ALP: alkaline phosphatase, ALT: alanine aminotransferase, AMG: a 2-macroglobulin, AST aspartate aminotransferase, BLP: b-lipoprotein, BMG: b2,-microglobulin, C3 and C4: Complement component, 3 and 4, CEA carcinoembryonic antigen, CP: ceruloplasmin, FER ferritin, FIB: fibrinogen, FN: fibronectin, G6Pase: glucose 6-phosphatase, G6PDH: glucose-6-phosphate dehydrogenase, GGT g -glutamyltransferase, HP: haptoglobin, HX: hemopexin, LDH: lactic acid dehydrogenase, ND: not described, PL plasminogen, PRE: prealbumin, TAT tyrosine aminotransferase, TF. transferrin.
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Table 4 Genetic features of human hepatoma cell lines Cell line
Genetic features
HLE*
p53 codon 249 position 3 G→C, HBV(–), no ras mutation, no expression of Rb, p16 & p15: normal p53 codon 244 position 2 G→ C, HBV(–), no expression of Rb, p16 & p15: normal wild type p53, HBV(–), no ras mutation p53 codon 220 position 2 A →G, HBsAg(–), no ras mutation, mutant Rb, p16 & p15: normal wild type p53, no ras mutation, HBsAg(–) mutant p53, HBsAg(–) mutant pS3, HBsAg(–) mutant p53 mutant p53 wild type p53, integration of HBV DNA p53 codon 249 position 3 G →T, integration of HBsAg, no ras mutation, normal Rb, p16 & p15: normal, G6PDH: type A, rearrangements of chromosome 1 wild type p53, HBsAg( +), rearrangements of chromosome 1 wild type p53, N-ras codon 61 position 2 A →T, HBV(–), mutant Rb, p16 & p15: normal, rearrangements of chromosome 1 wild type p53, HBsAg( +), rearrangements of chromosome 1 wild type p53, HBsAg( +) p53 deletion of 18 bases (codon 264 to codon 270), HBsAg( +) wild type p53, HBV( +) HBsAg(–), rearrangements of chromosome 1 mutant p53: codon 242 position 2 G→A hemochromatosis, HBV(–), HVC(–), deletion of the long arm of chromosome 1
HLF HuH-6* HUH-7 JHH-1* JHH-2* JHH-4* JHH-5* JHH-6* JHH-7* PLC/PRF/S* Hep3B HepG2* huH 1 huH 2 huH 4 HB611 HA22T/VGH HAK-1A & -lB RBHF-1
*The status of p53 and other genetic characteristics have been described (Tsuji et al. 1998; Pu et al. 1997)
1990; Marion et al. 1980; Twist et al. 1981). However, there is no description of HBV or HCV virus production in the continuous HCC cell lines.
5.
HEPATOCYTE GROWTH FACTOR
Hepatocyte growth factor (HGF) is an important regulator of liver regeneration in response to injury (Nakamura et al. 1984). In addition, HGF is a potent mitogen for mature hepatocytes in vitro (Nakamura et al. 1987). Thus, HGF appears to be a likely candidate for autocrine stimulation of HCC. However, treatment of HCC (HepG2, Hep3B and HuH-7 cell lines) with HGF produced a marked inhibition of cell growth (Shiota et al. 1992). In contrast, the growth of HuH-6 cells was enhanced by treatment with HGF (Miyazaki et al. 1992), although expression of met, the receptor of HGF, was detectable in HuH-6 cells at the same level as HepG2 cells (unpublished data).
Liver Cancer
6.
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TUMOR SUPPRESSOR GENES AND ONCOGENES
Mutations in the p53 tumor suppressor gene are associated with increasing histological grade and early recurrence, suggesting that defective p53 function is significant and confers increased clinical aggression (Hayashi et al. 1995). The finding of p53 mutations in a number of HCCs suggests that hepatocellular transformation is linked to the loss of both the structural and functional integrity of p53 (Hsu et al. 1993). Amongst the HCC cell lines, no significant associations were observed between the pattern of p53 and Rb changes and the extent of differentiation of the cell lines (Kaino 1997). About 70% of HCC do not show any p53 abnormalities (Oda et al. 1992). However, dysfunctional p53 is probably more common in HCC than is appreciated from standard genetic screens of the hotspot regions. As shown in Table 4, seven (HLE, HUH-7, PLC/PRF/5, JHH-2, -4, -5, and -6) out of 12 human hepatic cell cancer cell lines examined using the FASAY method showed p53 mutations (Tsuji et al. 1998). The hepatitis B virus-encoded X antigen (HBxAg) heterodimerizes with and inactivates wild-type p53 in vitro and in vivo (Wang et al. 1994). Thus, p53 mutation may not be necessary for hepatocarcinogenesis, although a low frequency of p53 mutations in HBxAg-positive HCCs has been reported (Greenblatt et al. 1997). Exposure to aflatoxin B1 (AFB1) is associated with a characteristic point mutation in p53 (Liang 1995), and almost all of the mutations in AFB1-associated tumors are G:C to T:A transversions at codon 249 (AGG to AGT), changing arginine to serine (Hsu et al. 1991; Greenblatt et al. 1994). These findings indicate that p53 dysfunction may play an important role in hepatocarcinogenesis, but it is not known at which stage the p53 dysfunction is important. To address this question, a mouse model of targeted disruption of the p53 gene will be useful (Bellamy et al. 1997). The status of other oncogenes, such as c-erbB-2, fos, jun, myc, raf, ras and Rb has been described (Lea 1993), but critical gene changes in HCC remain to be identified.
7.
CROSS CONTAMINATION
In 1954, a human liver cell line was established from normal human liver tissue and called the Chang liver cell line (Chang 1954). Later it was found to be cross-contaminated with the HeLa cell line (Gey et al. 1952; Lavappa 1978; Nelson-Rees et al. 1981).
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REFERENCES Aden DP, Fogel A, Plotkin S et al. Nature 282: 615, 1979. Alexander JJ, Bey EM, Geddes EW et al. S Afr Med J 50: 2124, 1976. Bagnarelli P, Devescovi G, Manzin A et al. Hepatology 11: 1024, 1990. Bellamy COC, Clarke AR, Wyllie AH et al. FASEB J 11: 591, 1997. Chang RS. Proc Soc Exp Biol 87: 440,1954. Chang, C, Lin Y, 0-Lee T et al. Mol Cell Biol 3: 1133,1983. Ding S-F, Michail NE, Habib NA. J Hepatol 20: 672, 1994. Doi I. Gann 67: 1, 1976. Doi I, Namba M, Sato J. Gann 66: 385,1975. Gey GO, Cofman WO, Kubicek MT Cancer Res 12: 264,1952. Greenblatt MS, Feitelson MA, Zhu M et al. Cancer Res 57: 426, 1997. Greenblatt MS, Bennett WP, Hollstein M. Cancer Res 55: 4855, 1994. Harris CC. Carcinogenesis 17: 1187,1996. Hayashi H, Sugio K, Matsumata T et al. Hepatology 22: 1702,1995. He L, Isselbacher KJ, Wands JR et al. In Vitro 20: 493, 1984. Homma S. Jikeikai Med J 32: 289,1985. Homma S, Nagamori S, Fujise K et al. Human Cell 3: 152, 1990. Hsu IC, Metcalf RA, Sun T et al. Nature 350: 427, 1991. Hsu IC, Tokiwa T, Bennett W. Carcinogenesis 14: 987,1993. Hu CP, Han SH, Lui WY et al. Hepatology 6: 1396,1986. Huh N and Utakoji T Gann 72: 178, 1981. Kaino M. J Gastroenterol 32: 40, 1997. Kanno S, Tsunoda Y, Shibusawa M et al. Human Cell 2: 211,1989. Knowles BB, Howe CC, Aden DP. Science 209: 497,1980. Lavappa KS. In Vitro 14: 469,1978. Lea MA. Int J Biochem 25: 457,1993. Liang TJ. Hepatology 22 1330, 1995. Lin YM, Hu CP, Chou CK et al. Chung Hua Min Kuo Wei Sheng Wu Chi Mien I Hsueh Tsa Chih 15: 193, 1982. Marion PL, Salazar FH, Alexander JJ et al. J Virol 33: 795, 1980. Matsuura H. Acta Med Okayama 37: 341,1983. Miyazaki M, Gohda E, Tsuboi S et al. Cell Biol Int Rep 16: 145,1992. Miyazaki M and Namba M, In Atlas of Human Tumor Cell Lines, eds. RJ Hay et al. Academic Press, 1994, pp. 185-212. Murakami T. Acta Hepatol Jap 25: 532, 1984. Nakabayashi H, Taketa K, Miyano K et al. Cancer Res 42: 3858, 1982. Nakamura T, Nawa K, Ichihara A. Biochem Biophys Res Commun 122: 1450,1984. Nakamura T, Nawa K, Ichihara A et al. FEBS Lett 224: 311,1987. Nelson-Rees WS, Daniels DW, Flandermeyer RR. Science 212: 446,1981. Nohara T. Gastroenterol Jpn 22: 722, 1987. Oda T, Tsuda H, Scarpa A et al. Cancer Res 52: 6358,1992. Okuda K, Kojiro M, Okuda H. in Diseases of the Liver, Lippincott, 1993, p. 1244. Pu H, Tsuji T, Kondo A et al. Acta Med Okayama 51: 313, 1997. Saito H, Morizane T, Watanabe T et al. Cancer 64: 1054, 1989. Sasaki F, Kameda H, Hata Y et al. Human Cell 1: 89,1988. Shiota G, Rhoads DB, Wang TC et al. Proc Natl Acad Sci USA 89: 373, 1992. Sing GK, Pace R, Prior S et al. Hepatology 20: 74,1994. Stevenson D, Lin JH, Tong MJ et al. Hepatology 7: 1291,1987.
Liver Cancer Tsuji T, Miyazaki M, Fushimi K et al. Biochem Biophys Res Commun 242:317, 1998. Twist EM, Clark HF, Aden DP et al. J Virol 37: 239, 1981. Wang XW, Forrester K, Yeh H et al. Proc NatlAcad Sci USA 91: 2230, 1994. Watanabe T, Morizane T, Tsuchimoto K et al. Int J Cancer 32: 141,1983. Yano H, Kojiro M, Nakashima T. In Vitro Cell Dev Biol 22 631,1986. Yano H, Iemura A, Fukuda K et al. Hepatology 18: 320, 1993. Yaoita S, Ohoi R, Hayashi Y et al. Human Cell 2: 201, 1989.
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Chapter 33 Wilms’ Tumor and Other Childhood Renal Neoplasms
Noel A. Brownlee, Gian G. Re and Debra J. Hazen-Martin Department of Pathology and Laboratoiy Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425. Tel: 001-843-792-4557; Fax: 001-843792-4157; E-mail:
[email protected]
1.
INTRODUCTION
Renal tumors are one of the most common intra-abdominal neoplasms of early childhood and cover a wide clinical and histological spectrum (1). Because these tumors are diagnosed from infancy through the first several years of life, they are likely to represent pathologies of normal kidney morphogenesis (2). However, the relationships between these childhood renal tumors remains unresolved. The development of a well-characterized cell culture-based model system has been and will continue to be beneficial in delineating both pathological mechanisms in tumors and the biological features of normal nephrogenesis.
1.1
Wilms’ Tumor
Wilms’ tumor, or nephroblastoma, is one of the most common solid tumors of childhood, affecting 1 in 10,000 children, usually within the first five years of life. The term Wilms’ tumor has been employed in the past to describe a wide clinical spectrum of childhood renal tumors. The National Wilms’ Tumor Study recognizes several histologically and clinically distinct renal tumors of childhood (3). By far the most common of these tumors is the classical or triphasic Wilms’ tumor which comprises 80% of all pediatric renal malignancies. This tumor is composed of heterogeneous elements which may
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include, in different combinations, immature renal blastema, stroma, and epithelial elements. These three cell types recapitulate structures visible in the developing fetal kidney, a feature that underscores pediatric renal tumors as a developmental aberrancy. While the triphasic Wilms’ tumor is generally curable with combination therapy, tumors with features of anaplasia (particularly those demonstrating diffuse anaplasia) are characterized by increased resistance to standard therapy and, therefore, an increased incidence of recurrence and metastasis (4). Approximately 5% of Wilms’ tumors contain anaplastic cells, a feature hypothesized to represent tumor progression (5).
1.2
Congenital Mesoblastic Nephroma
Congenital mesoblastic nephroma (CMN) is a benign tumor of neonates first delineated from Wilms’ tumor in the late 1960s (6,7). Two subtypes of this tumor have been described based on histological appearance and comprise 5% of all childhood kidney tumors. The cellular or atypical variant is composed of closely apposed cells with a high mitotic index and may recur if resection is incomplete. The classical variant of the tumor histologically resembles a uterine leiomyoma. Both variants are hypothesized to be derived from renal stroma.
1.3
Clear Cell Sarcoma and Malignant Rhabdoid Tumor
Clear cell sarcoma of the kidney (CCSK) and the malignant rhabdoid tumor of the kidney (RTK) were demarcated from Wilms’ tumor in the late 1970s and early 1980s (8). Together these tumors comprise only 7% of all cases, but contribute to the majority of treatment failures and deaths. CCSK specimens demonstrate varying histological features, but primarily include cells with a high nuclear-cytoplasmic ratio, nuclei with scant heterochromatin, the feature responsible for the ‘clear cell’ designation, and an array of anastomosing blood vessels. This tumor also tends to metastasize to the bone (9). RTK cells are characterized cytologically by prominent nucleoli and intracytoplasmic vimentin whorls. Rhabdoid tumors may also occur in extrarenal sites such as the liver, neck, heart and thymus (10). Children with this tumor fare poorly and usually die with widespread metastases (11). The histogenesis of these tumors is unresolved. However, it has been speculated that the CCSK and MRTK are derived from pluripotent renal mesenchymal cells and neuroectodermal cells, respectively (12,13).
1.4
Derivation and Molecular Genetics
The etiology of Wilms’ tumor is unresolved. However, classical Wilms’ tumors have been associated with nephrogenic rests, areas of immature renal tissue that are hypothesized to represent genetic precursors to Wilms’ tumor
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(14). This tumor type has also been associated with several genetic syndromes including the WAGR complex (Wilms’ tumor, aniridia, genitourinary abnormalities and mental retardation), Beckwith-Wiedemann syndrome, and the Denys-Drash syndrome (15). Neither nephrogenic rests nor these genetic syndromes have been associated with the other renal tumors of childhood. The molecular genetic etiology of Wilms’ tumor is complex and is undoubtedly dependent upon numerous genetic abnormalities (16). Deletion or mutation of the WT-1 gene locus at chromosome 11p13 has been hypothesized to represent the key genetic lesion in Wilms’ tumors, but abnormalities of this gene are limited to only 10% of all cases (17). Mutations, deletions, and expression abnormalities of a variety of other genes have been described and include the fetal mitogen insulin-like growth factor-II and the cyclindependent kinase inhibitor p57KIP2, both localized to chromosome 11p15 (18), mutations of the p53 tumor suppressor gene at chromosome 17p13 in anaplastic tumors (19,20), and loss of heterozygosity of chromosomes 1p36 and 16q (21). Linkage studies have also identified familial Wilms’ tumor loci on chromosomes 17 and 19 (22,23). The genetic factors leading to the development of anaplastic Wilms’ tumors, CMN, CCSK, and the RTK are even more enigmatic due to the rarity of these tumor types. Therefore, the genetic relationships between these tumors and Wilms’ tumor remain unresolved.
2.
CELL CULTURE
Past attempts to develop an in vitro model for the study of Wilms’ tumor achieved mixed success. Cell cultures were established from several animal sources as reviewed by Hard (24). The first successful establishment of a nonhuman Wilms’ tumor culture using Wistar rats was reported in 1967 (25). Others have reported the development of two porcine Wilms’ tumor cell cultures that were successfully propagated to passage 45 (26). In addition, the carcinogen dimethylnitrosamine was used to induce rat renal mesenchymal tumors which histologically resemble CMN and CCSK and established fibroblast-like cultures (27). The first report of a Wilms’ tumor cell culture derived from a human source occurred in 1957. This culture, designated CCRF-6, was characterized by cells growing in a pavement-like monolayer with an ‘epithelioid’ appearance (28). Dobrynin later established another human culture, TuWi, described as having an epithelial morphology (29). TuWi cells also grew as mouse heterotransplants, even though the histology of the resulting xenografted tumor failed to demonstrate the features of a Wilms’ tumor. TuWi cells were maintained by the American Type Culture Collection (ATCC) until evidence arose which indicated that these cells had been cross-contaminated with HeLa cells. More recently, Fogh described the development of two additional Wilms’ tumor cell
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lines: SK-NEP-1 and Wiltu-1 (30). Wiltu-1 was characterized as a fibroblastoid culture whereas SK-NEP-1 cells, derived from a pleural effusion of a Wilms’ tumor patient, were epithelial-like and grew in suspension. SK-NEP-1 cells (also known as HTB-48) are also available from the ATCC. Unlike TuWi cells, SK-NEP-1 cells maintained the cytomorphological features of Wilms’ tumor upon nude mouse heterotransplantation (31). The ATCC also maintains the G401 cell line (CRL-1441) that has been used extensively in the study of Wilms’ tumorigenesis (32,33). More recent cell and molecular biological examination of this cell line has demonstrated that these tumor cells may be derived from a RTK, not a classical Wilms’ tumor (34). In addition to G401 and SK-NEP-1 cells, the ATCC also has available four additional Wilms’ tumor cell lines: TE79.T (CRL-7733), TE138.T (CRL-7946), Hs794.T (CRL-7887), and Hs775.T (CRL-7504). The latter four cell lines were originally developed at the Naval Biosciences Laboratory (Oakland, California) and later transferred to the ATCC. No cell lines are available from commercial sources for CMN and CCSK even though successful cultures of both tumors have been established and reported previously (35,36). Several additional renal rhabdoid tumor cell lines have been established and include: RTK, also designated RTlK(37,40), STM9101 derived from a metastatic lung lesion (38), and SWT1 and SWT-2 (39). More recently, additional CMN, CCSK, and RTK cell lines have been developed and well-characterized (40,41). Further molecular characterization of these cell cultures will be necessary in order to evaluate their relationship with the corresponding primary tumors. We have cultured the blastema, epithelium, and anaplastic epithelium of Wilms’ tumor (42–44). In addition, the development of a Wilms’ tumor culture with evidence of skeletal muscle differentiation was described (45). The RM1 (or W4) culture, derived from an anaplastic Wilms’ tumor that later metastasized and killed the patient, has been used in the evaluation of WT-1 splice variants (46) and is a p53 null cell line which overexpresses the MDR-1 gene (20,47). The propagation of human Wilms’ tumors in nude mice has also provided additional tissue with which to initiate cell culture (48). Nude mouse heterotransplantation of tumor often provides a continuing supply of fresh tumor tissue for extraction of nucleic acid and protein, in vivo experimentation, and cell culture use. Establishment and successful propagation of eight Wilms’ tumor xenografts has been previously reported (48). In this study, primary Wilms’ tumor xenografts maintained the histological composition of the primary tumor. With serial passage of these tumors, the epithelial and stromal components of triphasic Wilms’ tumors were eventually lost. One xenografted anaplastic tumor, used to initiate the W4 anaplastic culture, retained the histologic features of the primary tumor. Neither anaplastic progression of the xenografted classical Wilms’ tumors nor metastasis have been reported. Cells in culture may also be used to establish xenografts. Both anaplastic Wilms’ tumor cultures, W4 and W16, CMN-1, and rhabdoid
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tumor cultures may be used to establish xenografts. This method has been reported previously (34).
2.1
Primary Culture
Different approaches to developing cultures from triphasic Wilms’ tumor have been attempted. Successful culture of the heterogeneous cellular components of Wilms’ tumor through the use of dissociation and gradient centrifugation have been reported previously (49). Cell cultures with both stromal and epithelial characteristics were derived using Percoll gradient centrifugation. Successful subculture and propagation of the different elements of Wilms’ tumor were not described in this study, however. Alternatively, differential media formulations may be employed to select for the growth of the different components of Wilms’ tumor. The latter method has proven to be successful in both the establishment of primary culture and continued subculture. This method will be described below. Cell cultures of pediatric renal tumors may be established using either dissociated or explanted, primary or heterotransplanted tumor tissue. The appropriate procedure to use depends on the consistency of the tissue. Classical and anaplastic Wilms’ tumor, cellular CMN, and RTK tumors are generally very friable and may be dissociated using collagenase, or simply minced finely and placed directly into a culture flask. Wilms’ tumors, classical CMN, and CCSK specimens that are composed predominantly of stromal-like cells are generally more difficult to dissociate and may require direct explant of tissue fragments into the culture dish. The optimal substrate material for the successful propagation of primary pediatric renal tumor cell cultures appears to be type I collagen (Collagen Corporation, Palo Alto, California) with adsorbed fetal calf serum proteins (42,43). Type IV collagen, laminin, and tissue culture flask plastic alone are generally not suitable for the growth of these tumor cells. However, the more aggressive tumors, which include the RTK and anaplastic tumors, may not require a type I collagen coated substrate for growth and will grow on plastic alone. Before manipulation, tumor tissue is maintained in chilled 15% v/v fetal calf serum in Dulbecco’s modified Eagle’s medium (DMEM). The specimen may then be cut into small pieces (approximately 1 mm3) and explanted directly into tissue culture flasks (Note: turn flask upside down for 15–20 minutes before adding medium in order to enhance attachment). Alternatively, tissue may be dissociated within a sterile trypsinizing flask containing prewarmed (37°C) Hank’s Balanced Salt Solution (HBSS) with collagenase (1.4 mg/ml) and deoxyribonuclease (1.0 mg/ml), hereafter referred to as dissociation medium. Both collagenase and deoxyribonuclease are available from Sigma Chemical Company (St. Louis, Missouri). Enough dissociation medium should be placed in the trypsinizing flask to cover both stir bar and tissue fragments. The flask
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is then placed on a stir plate within a 37°C water bath for 15 minutes. This step may be repeated depending upon the consistency of the tumor specimen. Dissociated cells are then poured through a fabric filter (90µm mesh diameter) into a 50 ml centrifuge tube. The filter should be washed two times with HBSS to bring the filtrate volume in the centrifuge tube to 50ml. The cell suspension should be centrifuged at 1000 RPM for 10 minutes. The resulting cell pellet may be resuspended in growth medium and aliquoted into tissue culture flasks. Cultures are then incubated at 37°C in a 95% air/5% CO2 incubator. If cells have attached to the substrate after 24 hours, the cultures may be agitated in order to remove cellular debris and replenished with fresh growth medium. Medium should be exchanged with fresh medium three times weekly. Cell subculture is achieved by washing the attached cells twice with phosphate buffered saline (PBS) followed by the addition of 1.0ml of 0.05% trypsin/0.02% EDTA (appropriate for a 25cm2 flask). Both PBS and trypsin should be warmed to 37°C before use. Trypsin may be inactivated by adding an equal volume of fetal calf serum to the cell suspension. The cell suspension is then centrifuged at 1000rpm before being dispersed among additional tissue culture flasks. Primary pediatric renal tumor cell cultures have been propagated in both serum-containing and serum-free growth medium. Either DMEM or a 1:1 mixture of DMEM and Hams F12 (F12), with serum or growth factors, respectively, has been used for the successful initiation of pediatric renal tumor cell cultures. The use of the serum-free DMEM-F12 mixture with growth factors to propagate renal tumor cells was based on its previously described success in the long-term culture of normal kidney epithelium (50) and was especially useful in the culture of a predominantly epithelial Wilms’ tumor (43). In the latter study, serum-free DMEM-F12 was supplemented with the following (values represent final concentration): insulin (5 µg/ml), transferrin (5 µg/ml), selenium (5ng/ml), hydrocortisone (36ng/ml), and triiodothyronine (4pg/ml), and epidermal growth factor (10 ng/ml). The latter media formulation has been employed to successfully culture blastemal components of Wilms’ tumor with the addition of putrescine (10 ng/ml), bovine pituitary extract (30 µg/ml), prostaglandin El (10 nglml), and ethanolamine (1.2 µg/ml). Tumors with stromal characteristics (i.e. stromal predominant Wilms tumor, congenital mesoblastic nephroma, and clear cell sarcoma) have been grown successfully in DMEM-F12 with insulin, transferrin, selenium, hydrocortisone, triiodothyronine (concentrations as those previously noted; this medium formulation hereafter referred to as minimal DME-F12), and either 2%, 10%, or 20% (v/v) fetal calf serum. Malignant rhabdoid tumors grow readily in 2% (v/v) fetal calf serum in minimal DMEM-F12. The ATCC has described the use of several different types of media for its commercially available cell lines. These include 10% (v/v) fetal calf serum in either McCoys 5a, DMEM, or minimal essential media (Eagle) in Earle’s
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balanced salt solution with non-essential amino acids. Further information regarding ATCC cell lines may be obtained on-line at www.atcc.org. Cultures derived from pediatric renal tumor tissue propagated in nude mice have been established for many of the cell lines listed in Table 1. Although tumor tissue maintained in nude mice provides a valuable continuing resource of tumor tissue, cultures that are established using this method will need to be evaluated for mouse fibroblast contamination. This can be achieved by performing chromosomal analysis of the cultures. Acrocentric mouse chromosomes are easily discriminated from human chromosomes providing an easy and direct method of assessing contamination.
3.
DO THE CELL LINES AVAILABLE REFLECT THE CLINICAL SPECTRUM OF DISEASE?
Pediatric renal tumors cover a wide clinico-pathologic spectrum. A representative sample of pediatric renal tumor cell cultures that were developed in this laboratory, the ATCC and the Naval Biosciences Laboratory are listed in Table 1. With the exception of the RTK cell line G401, the Wilms’ tumor cell lines maintained by the ATCC have not been well characterized with regard to evaluating the extent of similarity between the derived culture and the respective primary tumor.
3.1
Classical Wilms’ Tumor
The collection of classical Wilms’ tumors included in Table 1 illustrates the variety of clinical behaviors exhibited by this tumor type. Non-anaplastic, blastemal-predominant Wilms’ tumors are potentially aggressive (51) even though success with the culture of blastemal-predominant Wilms’ tumors in this laboratory has been mixed. For example, the W13 cell culture was derived from a blastemal-predominant tumor which had metastasized to the liver upon initial diagnosis. Blastemal cells of the W13 culture grow very rapidly and may be used to initiate nude mouse heterotransplants. The heterotransplant tissue is then used to initiate subsequent cultures. The W13 culture was derived from a patient who died four years after treatment. In contrast, primary and metastatic tissue from another blastemal-predominant Wilms’ tumor, W1, failed to grow either in culture or nude mice even though the patient died with metastatic disease (unpublished observations). The W9 culture was derived from a predominantly epithelial Wilms’ tumor that did not harbor anaplastic cells. Heterotransplants of the W9 classical tumor were developed, but cultures from this tissue were blastematous. Only two of eighteen cases of primary classical Wilms’ tumors, one of which was W9, produced an epithelial culture (43).
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Table 1. Pediatric renal tumor cell lines Tumor Type Wilms’
CMN CCSK RTK
Culture
Patient age
Patient Sex
TNM Categorya
W13 W9 W4 W16 TE79.Te TE138.Te Hs794.Te Hs775.Te SK-NEP-1 CMN-1 CMN-2 CCSK-BG1 CCSK-2 RTK RT2K G401
3 years 2.5 years 3 years 4 years 7 years 2 years 9 months 13 years 25 years 5 months NA 10 months lyear newborn 7 months 3 months
Male Female Male Male Male Male Male Male Female Male NA Male Female Male Female Male
T2,N0,M1 T2,N0,M0 T2,N1,M1 T2,N0,M0 NA NA NA NA NA T2,N0,M0 NA T2,N0,M0 T2,N0,M0 T2,NX,M1 T2,N0,M1 NA
f
Pathologic Stage* IV I IV I NA NA NA NA IV I NA I I IV IV NA
Primary Tumor Site
Culture Methodb
Right Kidney Left Kidney Left Kidney Left Kidney NA NA NA NA NA Right Kidney NA Left Kidney Left Kidney Right Kidney Left Kidney NA
DandX D DandX D and X NA NA NA NA E D andX DandX E D and E D DandX D
Authenticationc Availabilityd Ref. D D D D NA NA NA I D and I D D D D D D D and I
MUSC MUSC MUSC MUSC ATCC ATCC ATCC ATCC ATCC MUSC MUSC MUSC MUSC MUSC MUSC ATCC
Classification published in TNM Classification of Malignant Tumors, 4th Edition, International Union Against Cancer, 1987. D, dissociated; E, explant; X, xenograft. cD, DNA fingerprinting; I, isoenzyme analysis; H, HLA typing; D, Northern analysis of gene expression (i.e. IGF-2,WT-1, etc.) and transmission electron microscopy dMUSC, tumor developed by Department of Pathology and Laboratory Medicine, Medical University of South Carolina; ATCC, Cell line available from American Type Culture Collection. edeveloped by the Naval Biosciences Laboratory (Oakland, California) and deposited at the ATCC. falso designated RT1K in reference 41. * National Wilms’ Tumor Study staging designation. NA, information is not available
55 43 20 20 None None None None 31 40 40 40 40 37 41 34
a
b
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Anaplastic Wilms’Tumor
A small fraction of Wilms’ tumors (5%) demonstrate morphological signs of anaplasia (i.e. cytological features including hyperchromatic nuclei, presence of multipolar mitotic figures, and a threefold increase in nuclear size in comparison to adjacent cells). Anaplastic tumors are uncommon in children younger than two years of age and are associated with a poorer outcome than classical tumors (52). Two anaplastic Wilms’ tumor cell lines have been developed in this laboratory. W4 and W16 cells were derived from a diffusely and focally anaplastic tumor, respectively. The patient from which W4 cells were derived succumbed to widespread metastatic disease whereas patient W16 remains disease free nine years after initial treatment. These observations are consistent with previous reports indicating that those children with diffusely anaplastic tumors fare more poorly than those with focally anaplastic tumors (4). Surprisingly, another Stage IV diffusely anaplastic tumor, derived from a 21 year old female, grows poorly in culture.
3.3
CMN, CCSK and RTK
Before the 1970s, CMN, CCSK, and RTK were classified as Wilms’ tumors. However, closer study has shown that these tumors are clinically, histologically and molecularly distinct neoplasms of childhood (8,53,54). Unlike the cell cultures described above, the congenital mesoblastic nephroma cultures, CMN1 and CMN-2, are composed of interlacing layers of cells with a high mitotic rate and fibroblast-like appearance. The features of these tumors are consistent with that of the cellular variant of this tumor. Although the high mitotic rate of these tumors may be disconcerting, both classical and cellular mesoblastic nephroma are benign neoplasms. CCSK-2 and CCSK-BG1 were derived from Stage I tumors and have been passaged 13 times in culture. In contrast to previous reports describing epithelial differentiation in this tumor type (36), neither nude mouse xenografts nor cell cultures derived from CCSK primary tumors demonstrate evidence of this form of differentiation. Unlike CMN-1, CCSK cells grow relatively slowly in culture. Table 1 also lists two rhabdoid tumor cell lines. G401 cells were long considered Wilms’ tumors until morphological and gene expression analysis demonstrated that this tumor demonstrated rhabdoid features (34). G401, RT2K, and RTK cells contain prominent intracytoplasmic intermediate filaments and large nucleoli (34,37,41). RT2K cells also have the tendency to grow in grape-like clusters that detach from the substrate and float in the media. Both G401 and RT2K cells grow rapidly in culture and readily form nude mouse heterotransplants, features that are consistent with the aggressive clinical behavior of this tumor.
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Other Cell Lines
Most of the cell lines available from the ATCC have not been characterized. The SK-NEP-1 and G401 cells represent an exception. SK-NEP-1 cells were derived from a pleural effusion and are cytologically epithelial. Cells injected into nude mice formed tumors with features of a Wilms’ tumor even though no histological documentation was provided (31). The Stage IV characteristics and hypertriploid karyotype of this tumor suggest that it may be an anaplastic Wilms’ tumor. Further characterization of the ATCC cell lines will be necessary in order to classify them appropriately.
4.
COMPARATIVE PATHOLOGY OF PRIMARY TUMORS, XENOGRAFTS AND CELL CULTURES
4.1
Wilms’ Tumor
Wilms’ tumors and the other renal tumors affecting young children vary with regard to their state of differentiation. However, epithelial elements of the both classical and anaplastic Wilms’ tumors have been shown to retain features of normal renal epithelium (see Table 2). For example, the W9 tumor was found to contain a prominent epithelial component which was subsequently cultured (43). Phase microscopic evaluation of the W9 culture demonstrated the presence of dome formation (a feature indicative of active membrane transport) interspersed among other cells forming a cobblestone-like pattern. In addition, ultrastructural evaluation and freeze-fracture analysis of these cultured cells provided evidence of junctional complexes. W9 cultured cells were also immunoreactive with cytokeratin and epithelial membrane antigen monoclonal antibodies. These features are consistent with the cellular and molecular features of normal tubular epithelial cells. The well-differentiated features of W9 cells contrast with the lack of epithelial polarity and organization of cells derived from the anaplastic tumor W4 (44), even though these cells demonstrate tight cellular opposition and desmosomes which are features of renal epithelial cells. In contrast to the culture of Wilms’ tumor epithelium, the culture of the blastematous component of Wilms’ tumor has proven to be more difficult. The lack of an appreciable number of blastematous cultures has made it difficult to assess the degree of similarity between normal blastema of the developing kidney and the blastematous component of Wilms’ tumor. Previous study has shown that blastema grows in spherical cell clusters with emanating epitheliallike cells (42). Cell cultures derived from another blastemal-predominant Wilms’ tumor, W13, were found to overexpress the fetal mitogen IGF-2, establishing an autocrine growth loop (55). The growth of W13 cells was blocked with the
Tumor type
Culture
Primary tumor pathology
In vitro features
Wilms’
W13
Wilms’ tumor: blastema and stroma
Blastema
W9 W4 W16 SK-NEP1
CMN
TE79.T TE138.T Hs794.T Hs775.T CMN-1
CCSK-2
Clear cell sarcoma of the kidney
RTK
RT1K
Rhabdoid tumor of the kidney
RT2K
Rhabdoid tumor of the kidney
G401
Rhabdoid tumor of the kidney
* descriptions provided by the ATCC
Epithelial,
non-anaplastic
Epithelial, anaplastic Epithelial,
.
anaplastic
Epithelial, growth in suspension* Monolayer* Epithelial* Epithelial * Stromal’ Stromal with high mitotic index Stromal Stromal, relatively slowgrowing Stromal and polygonal cells Rapidly growing spindle and polygonal shaped cells Rapidly growing polygonal cells; cytoplasmic inclusions Epithelioid*
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CCSK-BG1
Wilms’ tumor Wilms’ tumor Wilms’ tumor Wilms’ tumor Congenital mesoblastic nephroma, cellular variant Congenital mesoblastic nephroma, cellular variant Clear cell sarcoma of the kidney
CMN-2 CCSK
Wilms’ tumor: prominent epithelial component, blastema, stroma Anaplastic Wilms’ tumor: blastema and epithelium; diffuse anaplasia Anaplastic Wilms’ tumor: blastema, epithelium, stroma; focal anaplasia Unknown; derived from pleural effusion
Xenograft pathology Pathological features of the primary tumor are retained (currently to passage 70) Pathological features of primary tumor are retained (growth cessation at passage 20) Cystic nodules with prominent papillary-like anaplastic epithelium (passaged > 100 times) Pathological features of primary tumor are retained (currently to passage 85) Tumors contain small cells consistent with Wilms’ tumor NA NA NA NA Pathological features of primary tumor are retained (currently to passage 93) Pathological features of primary tumor are retained (currently to passage 56) Pathological features of primary tumor are retained (currently to passage 2) Pathological features of primary tumor are retained (currently to passage 4) Pathological features of primary tumor are retained (passaged 50 times) Pathological features of primary tumor are retained (currently to passage 60) Rounded, eosinophilic cells consistent with rhabdoid tumor
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Table 2. Comparative pathology of primary tumor, xenografts and cell cultures
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use of an antibody directed against the IGF-2 cognate receptor, the insulinlike growth factor I receptor. High levels of IGF-2 expression are also a common feature of developing renal tissue, particularly renal blastema (56). Therefore, W13 cells recapitulate the IGF-2 gene expression profile of the normal, developing renal blastema.
4.2
CMN, CCSK and RTK
Cell cultures of CMN, CCSK, and RTK maintain features that are consistent with their respective primary tumors. Cultures of CMN-1 contain cells with a fibroblastic appearance (likely primitive renal interstitial cells) that grow in multiple, overlying sheets with a high mitotic index, all features of cellular CMN. CMN-1 cells may also be used to establish nude mouse heterotransplants that retain the histological features of the primary tumor (40). CCSK-2 and CCSK-BG1 cultures contain cells growing in two to three sheets with a fibroblastic appearance. In contrast to CMN-1 cells, these cells are slower growing. Light microscopic evaluation of CCSK-BG1 cells demonstrates cells with a single, small nucleolus, a characteristic finding in CCSK primary tumors. G401, RT2K, and RTK cultures contain cells with prominent inclusion-like nucleoli and intracytoplasmic intermediate filaments. These cultures may also be used to initiate nude mouse heterotransplants. Relating CCSK and RTK cells back to a corresponding normal kidney cell remains difficult as the cell of origin for both tumors is not known. However, G401 cells were previously reported to be immunoreactive with monoclonal cytokeratin antibodies, a feature supportive of epithelial differentiation (34).
5.
MOLECULAR GENETICS
A variety of genetic alterations, including gross chromosomal abnormalities and mutations (see Table 3), have been described in pediatric renal tumor specimens (16). Overexpression of IGF-2 is a common feature of Wilms’ tumors (57).The blastemal cell culture W13 has been shown to overexpress the IGF-2 gene creating an autocrine growth loop mediated through the IGF-1 receptor (55). In addition, WT-1 was found to regulate the transcription of both the IGF-2 and IGF-1 receptor genes in transfection experiments (58,59). Interestingly, the WT-1 tumor suppressor is not mutated or deleted in this tumor type. Therefore, the W13 cell line would provide a model to use in the evaluation of other means of WT-1 inactivation and the control of IGF-2 and IGF-1 receptor gene expression in this tumor type. In addition, the W13 cell line would provide a model for the evaluation of new drugs which target components of the IGF2 autocrine growth loop.
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Table 3. Summary of genetic abnormalities in pediatric renal tumor cell cultures Genetic alterations/gene expression/gene overexpression WT-1 mutation p53 mutation IGF-II overexpression MDR-1 overexpression N-myc expression c-myc expression chromosome 22q LOHd
Cell lines demonstrating genetic alteration/gene expression profile* W4a W4b, W16c W13,CMN- 1 W4 W4 RTK (akaRT1K), RT2K, G401 RT2K
Reference 46 20 40,55 47 44 37,41 41
* Except for G401, the molecular biological features of the ATCC cell lines remain uncharacterized. de12 variant, exon 2 of WT-1 is spliced out. b homozygous mutant cheterozygous point mutation in codon 273 dLOH = loss of heterozygosity a
In several studies, mutation of the p53 tumor suppressor gene was a frequent finding in anaplastic Wilms’ tumors (19,20). Two anaplastic Wilms’ tumor cell lines developed within this laboratory harbor p53 mutations (20). The W4 culture is a p53 null cell line whereas W16 cells are mutant at the p53 gene locus. WT-1 mutant W4 cells have also been used in studies that assess the role of WT-1 splice variants in tumor suppression (46). Cell cultures of CMN, CCSK, and RTK will be useful in understanding of the relationship between these tumors and Wilms’ tumors. Since CMN and CCSK are likely representative of an abnormality in the developing renal stroma, both cell lines may be useful in the study of normal stromagenesis, a facet of normal kidney development that remains uncharacterized. The use of rhabdoid tumor cell lines has already proven to be useful in the narrowing of a rhabdoid tumor suppressor locus on chromosome 22 and in understanding the histogenesis of these rare tumor types (60,61). G401 cells, in particular, will be useful in further characterizing the rhabdoid loci on chromosomes 11 and 22 affected by the translocation t(11;22)(p15.5;q11.23).
6.
CROSS -CONTAMINATION
Neither ATCC Wilms’ tumor nor pediatric renal tumor cell lines developed within this laboratory demonstrate evidence of cross-contamination with other cell lines, mycoplasma, or viruses. TuWi is cross-contaminated with HeLa cells.
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CELL LINES WITH SPECIAL FEATURES
W4 and W16 are both anaplastic Wilms’ tumor cell lines with p53 mutations. W4 cells do not demonstrate nuclear reactivity with p53 monoclonal antibodies, presumably due to the production of a labile truncated p53 protein. In contrast, W16 cells do demonstrate strong nuclear reactivity with p53 monclonal antibodies. In addition to being a p53 null cell line, W4 cells also overexpress MDR-1, a gene which encodes the ATP-dependent cell membrane bound ‘pump’ which actively extrudes chemotherapeutic agents from the cytoplasm (47). Pglycoprotein function was inhibited by the addition of the calcium channelblocker verapamil in in vitro toxicity experiments using W4 cells. Two cell lines depend upon an IGF-2 autocrine growth loop. W13 cell growth is inhibited by the addition of the polysulfonated naphthyl urea compound, suramin, in both in vitro and in vivo experiments (55). CMN-1 cells, another rapidly growing cell line, are also sensitive to treatment with suramin andaIR3, a monoclonal antibody specific for the IGF-1 receptor (unpublished observation). Conditioned media derived from G401 cultures appears to contain nephroblast growth factor, a protein that appears to be necessary for the primary culture of nephroblasts (33).
REFERENCES 1. Parham DM. Pediatic Neoplasia: Morphology and Biology. Lippincott-Raven, New York, 1996. 2. Gonzalez-Crussi F. Wilms’ Tumor (Nephroblastoma) and Related Renal Neoplasms of Childhood. CRC Press. Boca Raton, Florida, 1984. 3. Murphy WM et al. Atlas of Tumor Pathology: Tumors of the Kidney, Bladder, and Related Urinary Structures. Third series, Fascicle 11, Armed Forces Institute of Pathology, Washington, DC. 4. Faria P et al. American Journal of Surgical Pathology 20: 909, 1996. 5. Re GG et al. Seminars in Diagnostic Pathology 11: 126, 1994. 6. Bolande RP et al. Pediatrics 40: 272, 1967. 7. Wigger HJ et al. American Journal of Clinical Pathology 51: 323, 1969. 8. Beckwith JB et al. Cancer 41: 1937, 1978. 9. Sotelo-Avilia C et al. Human Pathology 16: 1219, 1985. 10. Fisher HP et al. Pathology Research & Practice 184: 541, 1989. 11. Weeks DA et al. American Journal of Surgical Pathology 13: 439, 1989. 12. Haas JE et al. Cancer 54: 2978,1984. 13. Haas JE et al. Human Pathology 12: 646,1981. 14. Park S et al. Nature Genetics 5: 363, 1993. 15. Clericuzio CL et al. Medical Pediatric Oncology 21: 182, 1993. 16. Pritchard-Jones K et al. Lancet 349: 663, 1997. 17. Grundy P et al. Hematology Oncology Clinics of North America 9: 1157, 1995. 18. Feinberg AP et al. Medical Pediatric Oncology 27: 484, 1996. 19. Bardeesy N et al. Nature Genetics 7: 91, 1994.
Wilms’ Tumor and Other Childhood Renal Neoplasms 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. 55. 56. 57. 58. 59. 60. 61.
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El-Bahtimi R et al. Modern Pathology 9: 238, 1996. Grundy PE et al. Cancer Research 54: 2331, 1994. Rahman N et al. Nature Genetics 13: 461, 1996. McDonald JM et al. Cancer Research 58: 1387, 1998. Hard GC. Tumor Biology: In Vitro Culture and Transplantation Models of Wilms’ Tumor. In: Wilms’ Tumor Clinical and Biological Manifestations, Pochedly C and Baum E editors, Elsevier, 1984. Babcock VI et al. Proceedings Society Experimental Biology and Medicine 124: 217, 1967. Pirtle EC et al. American Journal of Veterinay Research 31: 1601, 1970. Hard GC et al. Journal of the National Cancer Institute 54: 1085, 1975. Foley GE et al. Annals New York Academy of Science 76: 506, 1958. Dobrynin YV et al. Journal National Cancer Institute 31: 1173, 1963. Fogh J. National Cancer Institute Monograph 49: 5, 1978. Fogh J et al. Journal National Cancer Institute 59: 221, 1977. Weissman BE et al. Science 236: 175, 1987. Burrow CR et al. Proceedings National Academy of Sciences USA 90: 6066, 1993. Garvin AJ et al. American Journal of Pathology 142: 375, 1993. Nuss D et al. Journal Pediatric Surgery 15: 297, 1980. Ishii E et al. Cancer Research 49: 5392, 1989. Gansler T et al. Human Pathology 22(3): 259, 1991. Ota S et al. Cancer 71: 2862, 1993. Hirose M et al. International Journal of Cancer 67: 218, 1996. Brownlee NA et al. in preparation. Rosson GB et al. Modern Pathology, submitted. Garvin AJ et al. American Journal of Pathology 129: 353,1987. Hazen-Martin DJ et al. American Journal of Pathology 142: 893,1993. Hazen-Martin DJ et al. American Journal of Pathology 144: 1023, 1994. Sens MA et al. Archives of Pathology and Laboratory Medicine 108:58, 1984. Haber DA et al. Science 262: 2057, 1993. Re GG et al. Modern Pathology 10: 129, 1997. Garvin AJ et al. Pediatric Pathology 8: 599, 1988. Velasco S et al. International Journal of Cancer 53: 672, 1993. Detrisac CJ et al. Kidney International 25: 383, 1984. Beckwith JB et al. Medical Pediatric Oncology 27: 422, 1996. Bonadio JF et al. Journal Clinical Oncology 3: 513, 1985. Tomlison GE et al. Cancer 70: 2358, 1992. Yun K. American Journal of Pathology 142: 39, 1993. Vincent TS et al. Cancer Letters 103: 49, 1996. Scott J et al. Nature 365: 764, 1985. Reeve AE et al. Nature 317: 258, 1985. Nichols KE et al. Cancer Research 55: 4540, 1995. Werner H et al. Proceedings National Academy of Sciences USA 90: 5828,1993. Biegel JA et al. Genes Chromosomes Cancer 16: 94, 1996. Ota S et al. Cancer 71: 2862, 1993.
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Chapter 34 Retinoblastoma
Brenda L. Gallie1, Judy Trogadis2 and Liping Han1 Ontario Cancer Institute, University Health Network, 610 University Ave., Toronto M5G 2M9, Canada. Tel: 001-416-946-2324; Fax: 001 -416-813-8883; E-mail:
[email protected] and
[email protected] and 2Vision Science Research Program, Toronto Western Hospital Research Institute, 399 Bathurst St., Toronto M5T 2S8, Canada. Tel: 001 -416-6035088; Fax: 001-416-603-5126; E-mail: judy@playfair:utoronto.ca 1
1.
INTRODUCTION
Studies of the rare embryonic cancer of the retina, retinoblastoma, have contributed fundamentally to the understanding of human cancer. The clinical distribution of tumors in predisposed children led to the concept of tumor suppressor genes (Knudson, 1971). Proof of this concept was obtained by molecular genetic studies of retinoblastoma tumor cell lines and xenografts, that revealed loss of heterozygosity (LOH) at the locus mapped to the inherited predisposition to retinoblastoma (Cavenee et al. 1983; Godbout et al. 1983). A cloned DNA fragment at the RB locus was identified that was totally deleted from one retinoblastoma tumor (Dryja et al. 1986), and was used to clone the RB1 gene (Friend et al. 1986). The RB1 gene (Friend et al. 1986) is mutated in all retinoblastoma tumors, but is also mutated or dysregulated in many human cancers that are not initiated by RB1 mutation. Much of this knowledge was obtained from a few retinoblastoma cell lines. The first and most widely studied cell line, Y79, was the 79th attempt of a group at Yale to establish retinoblastoma in culture (Reid et al. 1974). Y79 and all subsequent established retinoblastoma cell lines grow predominantly in suspension. Intraocular retinoblastoma is never biopsied, since the risk of dissemination is too great. The most successful way to establish retinoblastoma in culture is by first growing the surgical specimen obtained from the enucleated eye as a xenograft in the eyes of athymic nude mice (Gallie et al.
J.R. W Masters and B. Palsson (eds.), Human Cell Culture Vol. II, 361 –374. © 1999 kluwer Academic Publishers. Printed in Great Britain.
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1977), for subsequent passage to tissue culture. Feeder layers also work well to support initial growth of retinoblastomas that subsequently become cell lines (Gallie et al. 1982c). The most aggressive retinoblastomas obtained after therapy fails or derived from metastatic extraocular sites, grow best in vitro. However, retinoblastoma tumors remain very difficult to establish in tissue culture, have a notoriously long doubling time, are almost completely refractory to transfection of DNA, require special conditions to maintain growth in vitro once established, and can rarely be cloned from single cells. The retinoblastoma cell lines available are listed in Table 1 and their characteristics summarized in Table 2.
2.
CULTURE CONDITIONS
Surgical specimens of intraocular retinoblastoma are obtained by opening the enucleated eye as soon as possible after removal. Since more than 50% of the cells in fresh tumor samples are not viable, it is important to obtain the largest possible specimen by opening the unfixed eye along the pupillary-optic nerve axis across the main tumor bulk, which can then be removed without disturbing the tumor-optic nerve relationship for subsequent clinically relevant histological examination. Tumor is immediately placed in tissue culture medium supplemented with serum. Tumor clumps are dispersed simply by vortexing and enzymatic digestion is unnecessary. Retinoblastoma tumor cells prefer to grow at high density (1–5×105/ml) in suspension, adhering to surfaces only under specific conditions (Reid et al. 1974; Gallie et al. 1982c). Several different culture media have been used successfully including RPMI-1640 with 10% FBS, but the best results are obtained with RB Medium: Iscove’s modified Dulbecco’s medium with 15% Fetal Clone III (Hyclone Laboratories, Inc), 10µg/ml insulin, and 55µ M 2mercaptoethanol. Since viable RB cells aggregate and single dead cells remain in suspension, the flasks are gently shaken to liberate dead cells attached in the periphery of the viable clumps. The flask is tilted to one side for a few minutes to allow the viable clumps to settle, then most of the medium containing the dead cells is removed, being careful not to disturb the clumped live cells.
2.1
Feeder Layers
The success of primary culture is increased if the cells are initially placed on a feeder layer prepared from rat smooth muscle cells (Bogenmann and Mark, 1983), human embryonic or murine fibroblasts (Albert et al. 1970; Gallie et al. 1982c; Griegel et al. 1990a,b), retinal pigment epithelium (Weiner et al. 1983) or mouse CB17 bone marrow stromal layers as described by Dexter (Dexter et al.1977).
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Table 1 Derivation of retinoblastoma cell lines Name Y79
WERIRBI RB606 RB617 RB1213 RB1238 RB1256 RB1368 RB610 RB430 RB267
Disease type
Availability
Primary reference
Year estab.
First author
unilateral familial
1,2,5
J National Cancer Inst3: 1347–1360: 1974
1974
Reid TW
unilateral
1,5
bilateral unilateral bilateral unilateral unilateral unilateral unilateral unilateral metastatic bilateral
2 2 2 2 2 2 2 2 2
RB302A bilateral
2
RB369
unilateral
2
RB383
unilateral
2
RB381
unilateral
2
RB414
unilateral
2
RB447
unilateral
2
RB247C bilateral
2
RB265
bilateral
2
RB355
unilateral
2
RB412
unilateral
2
RB429R bilateral
2
RB522A bilateral
2
RB405A unilateral
2
RB409
bilateral
2
RB544
unilateral metastatic
2
Cancer Res 37: 1003,1977
Anticancer Res. 9: 469, 1989 Cancer Res. 42: 301,1982 Cancer Res. 42: 301,1982 Cancer Res. 42: 301,1982 Hum Genet 66: 46,1984 Hum Genet 66: 46, 1984 Hum Genet 66: 46,1984 Hum Genet 66: 46,1984 Hum Genet 70: 291,1985 Hum Genet 70: 291,1985 Nature 304: 451, 1983 Nature 304: 451, 1983 Nature 304: 451, 1983 Nature 304: 451, 1983 Nature 304: 451, 1983 Nature 304: 451, 1983 Nature 304: 451, 1983
References
1987 1988 1995 1995 1995 1997 1987 1981
29,14,13,16,43,18, 20,21,22,26,27, 28,30,32,36,40,2, 4,9,11,12,23,6,19 McFall RC 26,25,43,16,12,8, 6,19 Gallie BL Gallie BL Gallie BL 15 Gallie BL Gallie BL Gallie BL Gallie BL Chan SL 6,5,41
1982
Gallie BL
10,33,6,5,41
1982
Gallie BL
10,33,35
1982
Gallie BL
10,33,35
1984
Squire J
1977
1984
Squire J
35,14,13,33,15,6, 34,41 3533
1983
Squire J
35,33
1984
Squire J
35,33,34
1982
Squire J
13,14,15,6,34,5,41
1982
Squire J
33
1982 1982
Godbout R 35,33,13,14,15,34, 6,5,41 Godbout R 12,35,33,6,5,41
1983
Godbout R 12,35,33,6,41
1983
Godbout R 12
1982
Godbout R 35,33,15,34,5,41
1982
Godbout R 42,35,33,6,5,41 Godbout R 12,41 Continued on next page
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Gallie et al.
Table 1 (Continued) Name
Disease type
Availability
RB507
bilateral
0
LA-RB66 bilateral
0
LA-RB1
bilateral
0
LARB59 LARB62 LARB64 LARB65 LA-RB9
unilateral
0
unilateral
0
unilateral
0
unilateral
0
bilateral
0
LA-RB8
unilateral
0
LARB10 LA-RB2
unilateral
0
unilateral
0
LARB13 LA-RB6
unilateral
0
unilateral
0
unilateral
0
unilateral
0
LARB80 LARB81 LARB69 TOTL-1
unilateral
0
unilateral
0
RBL13
unilaeral
0
RBL14
unilateral
0
RBL18
unilateral
0
RBL7
bilateral
0
RBL30
unilateral
0
RBL15
bilateral
0
Primary reference
Year First estab. author
References
Anticancer Res. 9: 469,1989 Cancer Genet Cytogenet 10: 311,1983 Cancer Genet Cytogenet 10: 311,1983 Cancer Genet Cytogenet 10: 311, 1983 Cancer Genet Cytogenet 10: 311,1983 Cancer Genet Cytogenet 10: 311,1983 Cancer Genet Cytogenet 10: 311,1983 Cancer Genet Cytogenet 10: 311,1983 Cancer Genet Cytogenet 10 311,1983 Cancer Genet Cytogenet 10: 311,1983 Cancer Genet Cytogenet 10: 311, 1983 Cancer Genet Cytogenet 10: 311,1983 Cancer Genet Cytogenet 10: 311, 1983 Cancer Genet Cytogenet 10: 311,1983 Cancer Genet Cytogenet 10: 311,1983 Cancer Genet Cytogenet 10: 311,1983 Cell Struct Funct 14: 331,1989 Int J Cancer 46: 125, 1990 Int J Cancer 46: 125, 1990 Int J Cancer 46 125, 1990 Int J Cancer 46: 125, 1990 Int J Cancer 46: 125, 1990 Int J Cancer 46: 125, 1990
1985
6
ChanSL
1983
Benedict WF
1
1983
Benedict WF
1
1983
Benedict WF
1
1983
Benedict WF
1
1983
Benedict WF
1
1983
Benedict WF
1
1983
Benedict WF
1,3
1983
Benedict WF
1
1983
Benedict WF
1
1983
Benedict WF
1
1983
Benedict WF
1
1983
Benedict WF
1
1983
BenedictWF
1
1983
Benedict WF
1
1983
Benedict WF
1,5
1989
Wakabayashi K 36,39
1990
Griegel S
13
1990
Griegel S
13
1990
GriegelS
13
1990
Griegel S
13
1990
Griegel S
13
1990
Griegel S
13
Continued on next page
Retinoblastoma Table 1
365
(Continued) Avail- Primary ability reference
Name
Disease type
RBL20
bilateral
0
WERIRB27
bilateral
0
WERI RB-24
unilateral
0
LA-RB3 bilateral LA-RB14 LA-RB12 RB475 bilateral
0 0 0 0
Int J Cancer 46: 125, 1990 J Pediatr Ophthalmol Strabismus 27: 212, 1990 J Pediatr Ophthalmol Strabismus 27: 212, 1990 JNCI 70: 95, 1983 JNCI 70: 95, 1983 JNCI 70: 95,1983 Nature 322: 555, 1986
Year estab.
First author
1990
Griegel S
13
1990
Sery TW
17,24,38,7
1990
Sery TW
31
Bogenmann E BogenmannE BogenmannE Squire J
44,1 44 44 34
1983 1983 1983 1984
References
Availability: 0 = not available; 1 = American Type Tissue Collection; 2 =Brenda Gallie, Ontario Cancer Institute; 3 =Japanese Collection of Research Bioresources Cellbank; 4 =European Collection of Animal Cell Culture, 5 = Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; 6 = Cellbank, Russian Academy of Medical Sciences, Moskow; 7 = Instituto Zooprofilafitco Sperimentale, Brescia. References: 1, (Benedict et al. 1983); 2, (Bernard and Klein, 1996); 3, (Bogenmann and Mark, 1983); 4, (Campbell and Chader, 1988a); 5, (Cavenee et al. 1983); 6, (Chan et al. 1989); 7, (Chen et al. 1992); 8, (Cowell et al. 1997); 9, (del Cerro et al. 1992); 10, (Gallie et al. 1982c); 11, (Gallie et al. 1978); 12, (Godbout et al. 1983); 13, (Griegel et al. 1990b); 14, (Griegel et al. 1990a); 15, (Gupta et al. 1996); 16, (Herman et al. 1989); 17, (Huang et al. 1988); 18, (Klaidman et al. 1993); 19, (Kondo et al. 1997); 20, (Koole and Schipper, 1990); 21, (Kyritsis et al. 1986b); 22, (Kyritsis et al. 1986~); 23, (Lee et al. 1984); 24, (Madreperla et al. 1991b); 25, (McFall et al. 1978); 26, (McFall et al. 1977); 27, (Olianas et al. 1992); 28, (Rajagopalan et al. 1993a); 29, (Reid et al. 1974); 30, (Schmidt-Erfurth et al. 1997); 31, (Sery et al. 1990); 32, (Skubitz et al. 1994); 33, (Squire et al. 1985); 34, (Squire et al. 1986); 35, (Squire et al. 1984); 36, (Wakabayashi et al. 1989); 38, (Xu et al. 1991); 39, (Yokoyama et al. 1992); 40, (Zhang et al. 1996); 41, (Zhu et al. 1992); 42, (Godbout et al. 1983); 43, (Jiang et al. 1984); 44, (Bogenmann, 1986)
Since retinoblastoma preferentially metastasizes to bone marrow, bone marrow stromal cells may preferentially provide essential factors. Dexter cultures are prepared from a suspension of mouse femur bone marrow cells cultured at 33°C in 5% CO, in Alpha minimum essential medium supplemented with 20% horse serum and 10-6M hydrocortisone. After 4 weeks, the hematopoietic cells have largely shed into the medium, leaving a bone marrow stromal layer onto which primary retinoblastoma cells can be placed. The culture medium is then changed to RB medium, described above. Initially, the retinoblastoma cells will adhere to the stroma, grow as colonies, and after several weeks to months, move off the feeder layer into suspension. The Dexter feeder layer eventually peels off the plastic surface and the retinoblastoma cells are transferred to a new flask, with or without a feeder layer, depending on their vigour.
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Table 2 In vitro characteristics of retinoblastoma cell lines Name Y79
Growth properties in suspension culture Differentiation
Doubling Cloning MDR time (hours) efficiency % phenotype
Chains, loose clumps Adherent and clumps Loose clumps Tight clumps
Undifferentiated, neuronal 33,36 potential WERIUndifferentiated, neuronal 96,44 RB1 potential RB383 44 RB447 Flexner-Wintersteiner Rosettes RB247C Tight clumps 44 RB355 Adherent and clumps 81 RB412 Loose clumps 44 RB429R Tight clumps 66 RB409 Chains, loose clumps 36 RB507 Tight clumps 66 LA-RB3 Tight clumps Flexner-Wintersteiner Rosettes LATight clumps Flexner-Win tersteiner RB14 Rosettes LA-RB12 Tight clumps Undifferentiated TOTL-1 Tight clumps Neuronal 160
6,40
No
0
No
24
No
Yes Yes Yes Yes Yes No
Abbreviation: MDR phenotype = multidrug resistance phenotype
2.2
Defined Media
Y79 can proliferate or be induced to differentiate in serum-free medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with transferrin (10 mg/ml), putrescine (8.8 ng/ml), sodium selenite (5 ng/ml), insulin (5mg/ml), and progesterone (6.3ng/ml) (Rubin et al. 1981).
2.3
Monolayer Culture
Unlike normal cells and many tumor-derived cell lines, retinoblastoma cells do not readily adhere to negatively charged plastic tissue culture flasks to form monolayers, but will adhere to polyornithine coated or poly-D-lysine positively charged, or laminin-coated surfaces, while retaining their original morphology (McFall et al. 1978; Kyritsis et al. 1984; Kyritsis et al. 1986a).
2.4
Cloning
The cloning efficiency of retinoblastoma cell lines in agar, agarose, or methylcellulose is generally very low. Agarose is toxic to both WERI-RB1 and Y79 (Griegel et al. 1990a,b). If aggregates are dispersed mechanically, the single cells either die from the disruption or reform into aggregates before
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resuming proliferation. Cloning of Y79 and WERI-RB1 has been reported (Inomata et al. 1986; Rootman et al, 1986).
2.5
Xenograft
Xenografting into the eyes of immune deficient mice is more successful than tissue culture for propagation of primary surgical retinoblastoma specimens (Gallie et al. 1977, 1978), and increases the chance that the tumor will subsequently grow in tissue culture. The established tissue culture cell lines generally take as subcutaneous tumors in SCID mice, but it is rare that a primary specimen will proliferate in this location (Phillips et al. 1989).
3.
DO THE CELL LINES AVAILABLE REPRESENT THE CLINICAL SPECTRUM OF RETINOBLASTOMA ?
In comparison to other types of tumors, there is a very narrow clinical spectrum of intraocular retinoblastoma, the main variation being the degree of differentiation. The least differentiated tumors are established most easily in culture, while the most differentiated are very difficult to establish. The benign or ‘pre-malignant’ retinoma (Gallie et al. 1982b) is the most differentiated and does not proliferate, and has never been grown in tissue culture. Undifferentiated retinoblastoma tumors consist of small, round cells containing little cytoplasm and large hyperchromatic nuclei. The most common differentiated feature is the formation of Flexner Wintersteiner rosettes (Flexner, 1891; Popoff and Ellsworth, 1969), spheres of columnar tumor cells surrounding central lumens. The cells forming a rosette show features of photoreceptor cells when examined by electron microscopy: cilia similar to those that form the outer segment of photoreceptor cells protrude into the lumen from the apical (inner) side, the nucleus is situated on the basal side, and the lumen contains hyaluronic acid, similar to the subretinal space. Occasionally, cells within rosettes have more distinct characteristics of photoreceptor cells, such as polarized shape and stacks of lamellated membranes resembling photoreceptor outer segments (Bogenmann, 1986; Reid et al. 1974). Many intraocular retinoblastoma tumors show extensive apoptosis and necrosis. Pseudo-rosettes are clumps of viable tumor cells surrounding blood vessels, to a depth of 10 to 20 cells, beyond which the tumor is necrotic. When retinoblastoma grows into the vitreous, without a blood supply, the opposite pattern arises: the vitreous seeds have necrotic cores, surrounded by a 10 to 12 cell thick rim of viable cells, nourished by diffusion through the vitreous (Gallie et al. 1990).
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Retinoblastoma cell lines are generally similar to the original tumor specimens. Undifferentiated tumor cells are small, round, with sparse cytoplasm and large hyperchromatic nuclei, and form loose chains and clumps of cells in suspension (Gallie et al. 1990). This phenotype is best represented by Y79 (Reid et al. 1974), by far the most frequently studied retinoblastoma cell line. If the original tumor demonstrated Flexner Wintersteiner rosettes, cells in culture will also form Flexner Wintersteiner rosettes, which roll around in the suspension culture. The central lumen is easily seen through the transparent columnar epithelial wall of the rosette. As the tumor becomes established as a cell line, the ability to form rosettes is generally lost, and the undifferentiated cells take over. Rosette formation can be increased with certain feeder layers (Bogenmann, 1986). Rosette formation in xenografts of retinoblastoma in the eyes of immune deficient mice also correlates with the original tumor histology (Bogenmann, 1986; Gallie et al. 1977, 1990). More differentiated retinoblastoma tumors grow in tight clumps which sometimes grow into balls of cells with necrotic centers, large enough to be easily observed floating in the tissue culture flask (Gallie et al. 1990). Such clumps are very resistant to dispersion, both mechanically and by protease treatment (Sery et al. 1990). Proliferation may cease when large growing clumps are dissociated (Wakabayashi et al. 1989). Growth is maintained by smaller clumps budding off the larger ones. All retinoblastoma lines have a very long doubling time, compared to other types of cultured cells. In general, undifferentiated cells have a faster doubling time than differentiated cells. For example, the doubling time for the most undifferentiated cell line, Y79, is 33 hours, for WERI-Rbl is 96 hours and for the highly differentiated TOTL-1 cells a slow 160 hours (Wakabayashi et al. 1989). Telomerase activity is detected in the large majority of tumors. Telomerase activity associated with short telomeres was, however, observed in only 50% of retinoblastomas and retinoblastoma-derived cell lines, suggesting that telomerase activity may not be a marker for acquisition of the malignant phenotype, at least in the case of this embryonic tumor which is relatively small at the time of excision (Gupta et al. 1996). Retinoblastoma tumors also do not show p53 mutations (Nork et al. 1997; Gallie et al. 1999), and usually have a high fraction of cells undergoing apoptosis.
4.
DO THE CELL LINES AVAILABLE REPRESENT CHARACTERISTICS OF EMBRYONIC RETINA?
Retinoblastoma cell lines express many genes characteristic of neuronal retinal cells, and occasionally express glial markers (Griegel et al. 1990a, 1990b; Kivela and Tarkkanen, 1986; Kivela et al. 1986; Klaidman et al. 1993; Messmer
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et al. 1985; Tso et al. 1970) (Table 3). Retinoblastomas arise from pluripotent, developing retinal precursor cells, which retain the ability to display retinal cell characteristics, depending on culture conditions (Campbell and Chader, 1988a). Studies of retinoblastoma cell lines have provided some information on the potential cell of origin (Albert et al. 1992; Albert et al. 1988; Cohen et al. 1988; Howard et al. 1991; Jiang et al. 1984; Kyritsis et al. 1984; Messmer et al. 1985; Perentes et al. 1987; Shternfeld et al. 1996; Tarlton and Easty, 1993; Virtanen et al. 1988; Yuge et al. 1995) and some important clues about the events that are required for full malignancy subsequent to the loss of both RB1 alleles (Squire et al. 1985). Retinoblastoma cell lines, particularly Y79, have also been used to define the biochemical pathways likely to be operational in the retina. Examples are retinoic acid induction of melatonin pathway genes (Bernard and Klein, 1996), expression of insulin receptors (Saviolakis et al. 1986; Campbell and Chader, 1988b; Yorek et al. 1985), insulin-like growth factor, serotonin and dopamine (Yorek et al. 1987), retinal S-antigen (Donoso et al, 1985; Song et al, 1995; Mirshahi et al, 1986), glial fibrillary associated protein (Craft et al. 1985; Jiang et al. 1984), myelin basic protein (Tsokos et al. 1986), expression of the photoreceptor-specific protein, IRBP (Campbell and Chader, 1988b; Fong et al. 1988; Rodrigues et al. 1987), and interaction with the extracellular matrix attachment molecule, laminin, causing neuron-like differentiation in Y79 cells (Campbell and Chader, 1988b). Interestingly, retinoblastoma tumors express cone-specific transducin (TC) but not rod-specific transducin (TR) (Bogenmann et al. 1988; Hurwitz et al. 1990; Rajagopalan et al. 1993a). Functional binding sites for corticotropin-releasing hormone, a neuropeptide whose function remains unclear, have been found on Y79 cells (Olianas et al. 1992).
5.
MOLECULAR GENETICS
The original studies that identified the RB1 gene utilized retinoblastoma surgical specimens (Friend et al. 1986). Subsequently, retinoblastoma tumors and cell lines confirmed mutation of the RB1 gene (Dunn et al. 1988). Since identification of the individual mutations in this large gene is difficult (Gallie et al. 1995), it is not yet proven that all retinoblastoma tumors contain mutations in RB1, although this is very likely. The common RB1 mutation is a ‘null’ allele, in which the mutation causes premature termination of translation, leading to unstable RB protein and mRNA (Dunn et al. 1989). RB1 mutations that are ‘in frame’ result in a stable RB protein and are usually associated with fewer retinoblastoma tumors, so called ‘low penetrance’ retinoblastoma (Gallie et al. 1995; Lohmann et al. 1994). These observations mean that practical strategies for clinical RB1 mutation identification depend on studying genomic DNA from blood of bilaterally
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affected persons with a presumed germline mutation, and from tumor of unilaterally affected persons without a family history, who have only a 15% risk of a germline RB1 mutation. Sufficient tumor DNA of high quality must be available for a large series of tests, so old samples in paraffin histology specimens may not be adequate. All retinoblastoma tumors have additional mutations, including either the i(6p) specific karyotypic rearrangement, or extra copies of chromosome lq (Squire et al. 1985; Squire et al. 1984). N-myc is expressed in all retinoblastoma tumors, consistent with their embryonic retinal origin, but is amplified in the genome as double minute chromosomes or as an intrachromosomal homogeneously staining region in a few cell lines, such as Y79 (Squire et al. 1986). A DEAD box protein which may be involved in cell growth and division was found to be co-amplified with N-myc in Y79 and RB522A (Godbout and Squire,1993).
5.1.
Genetic Modification
Genetic modification of retinoblastoma cell lines is difficult. Transfection of DNA occurs with extremely low efficiency by any of the current techniques: calcium phosphate precipitation, electroporation and all of the commercially available techniques. Viral constructs are therefore frequently used to genetically manipulate these cells (Chen et al. 1992; Huang et al. 1988; Muncaster et al. 1992).
5.2.
Transfection of retinoblastoma cells with the wild-type RB1 gene
In order to rigorously test the function of RB1 as a tumor suppressor gene, several groups have reconstituted retinoblastoma tumor cell lines with wild type RB1. The results have been wide ranging (Chen et al. 1992; Huang et al. 1988; Muncaster et al. 1992), depending on the experimental design, the cell lines studied, and the vectors and promoters used, and strongly suggest that the ability of RB1 to control cell proliferation is dependent on many factors including the state of differentiation and array of additional mutations in the tumor. For example, reconstitution of WERI-RB27 did not alter tissue culture morphology, but did affect tumorigenicity following xenotransplantation (Chen et al. 1992). Reconstitution of Y79 and WERI-RB1 did not result in any detectable phenotypic change (Muncaster et al. 1992).
5.3.
Retinoblastoma Cell Lines as Models for Therapy
The important role of multidrug resistance in treatment failure in retinoblastoma came from drug sensitivity studies of cell lines (Chan et al. 1989).
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Strategies to circumvent multidrug resistance mechanisms have led to the bestyet cure rates (Gallie et al. 1996), which will now be clarified by multicenter clinical trials (Ferris and Chew, 1996). Photodynamic agents are suggested to be effective against Y79 in vitro (Schmidt-Erfurth et al. 1997). These embryonic, generally undifferentiated tumor cells respond in vitro to a variety of agents that induce differentiation (Bogenmann and Mark, 1983; del Cerro et al. 1992; Gallie et al. 1990; Herman et al. 1989; Perentes et al. 1987; Rajagopalan et al. 1993b). Induction of terminal differentiation in cancer cells is interesting as a possible therapy. For example, agents such as retinoic acid, sodium butyrate, dbcAMP, and hexamethylene bis-acetamide can cause differentiation of Y79 cells into non-proliferating cells (Howard et al. 1991; Kyritsis et al. 1986b; Nakagawa and Perentes, 1987; Rajagopalan et al. 1993a; Zhang et al. 1996). Vitamin D analogs may have a similar effect, or may act on tumors by inhibiting angiogenesis (Shokravi et al. 1995; Shternfeld et al. 1996). Y79 cells can be induced to differentiate by retinoic acid or sodium butyrate and when injected into host rat retina, remain irreversibly mitotically arrested, form synapses with host retina, resemble photoreceptor cells, express neural markers such as neuron-specific enolase and interphotoreceptor retinal binding protein, and maintain the glial markers S-antigen and GFAP (del Cerro et al. 1992). With the combination of chemically induced differentiation and xenografting under the retina, the malignant tumorigenic phenotype is suppressed, while the cells are not rejected by the host. Growth of cell lines as intraocular xenografts has been used to assess clinical therapies (Cowell et al. 1997; Gallie et al. 1982a; Totsuka and Minoda, 1982; White et al. 1988, 1989a,b,c). However, such experiments might be better carried out in transgenic mice, in which retinoblastoma tumors form without surgical manipulation, because the RB protein is inactivated by expression of the viral protein, SV40 large T antigen (Howes et al. 1994; Murray et al. 1996a,b).
6.
CROSS-CONTAMINATION
The continuous cell line, Y79, has cross-contaminated at least two other retinoblastoma cell lines. By using molecular analyses of genetic polymorphisms, one laboratory showed that in their hands three cell lines that had originated from separate patients in widely separate laboratories, were cross-contaminated with Y79 (Madreperla et al. 1991a). One report suggested that retinoblastoma cell lines express lymphopoietic receptors: much more likely is that these particular lines were contaminated with lymphoblast cultures (Stein et al. 1981).
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CELL LINES WITH SPECIAL FEATURES
The Y79 cell line has characteristics not shared by other retinoblastoma cell lines. Y79 is undifferentiated but can attach to a substrate for detailed morphological and biochemical studies (Kyritsis et al. 1986a). Perhaps Y79 represents one of the earliest stages of retinoblastoma development, with an unusually broad differentiation potential and a high rate of proliferation. Other retinoblastoma cell lines are notoriously difficult to manipulate experimentally, limiting the studies that can be done on them.
REFERENCES Albert DM et al. Invest Ophthalmol Vis Sci 33: 2354, 1992. Albert DM et al. Invest Ophthalmol 9:64-72: 1970. Albert DM et al. Arch Ophthalmol 106: 536,1988. Benedict WF et al. Cancer Genet Cytogenet 10: 311, 1983. Bernard M et al. J Neurochem 67: 1032,1996. Bogenmann E, Int J Cancer 38: 883,1986. Bogenmann E et al. Science 240: 76,1988. Bogenmann E and Mark C. Journal of the National Cancer Institute 70: 95, 1983. Campbell M and Chader GJ. Ophthalmic Paediatr Genet 9: 171,1988a. Campbell MA and Chader GJ. Invest Ophthalmol Vis Sci 29: 1517,1988b. Cavenee WK et al. Nature 305: 779,1983. Chan SL et al. Anticancer Res 9: 469, 1989. Chen PL et al. Cell Growth Differ 3: 119, 1992. Cohen SM et al. Arch Ophthalmol 106: 541,1988. Cowell JK et al. EurJ Cancer 33: 1070, 1997. Craft JL et al. Exp Eye Res 40: 647, 1985. del Cerro M et al. Brain Res 583: 12, 1992. Danoso LA et al. Arch Ophthalmol 103: 855, 1985. Dexter TM et al. J Cell Physiol 91: 335, 1977. Dryja TP et al. Proceedings of the National Academy of Science, USA 83: 7391,1986. Dunn JM et al. Science 241: 1797, 1988. Dunn JM et al. Mol Cell Biol 9: 4594, 1989. Ferris FL et al.Archives of Ophthalmology 114: 1412, 1996. Flexner S. Johns Hopkins Hosp. Bull. 2: 115, 1891. Fong SL et al. Cancer Res 48: 1124, 1988. Friend SH et al. Nature 323: 643, 1986. Gallie BL et al. Invest Ophthalmol Vis Sci 16: 256, 1977. Gallie BL et al. Archives of Ophthalmology 114: 1321, 1996. Gallie BL et al. New York, Gustav Fischer Inc., p 641, 1982a. Gallie BL et al. Br J Cancer 45: 513, 1982b. Gallie BL et al. Retinoblastoma: for the next generation, in J. Cowell, ed., Cancer Genetics, London, Bios Scientific Publishers, p. 1. 1995. Gallie BL et al. Cancer Res 42: 301, 1982c. Gallie BL et al. Lab Invest 62: 394, 1990. Gallie BL et al. Jap J Ophthalmol 22: 345, 1978. Gallie BL et al. Cancer Res, in press, 1999.
Retinoblastoma
373
Godbout R et al. Nature 304: 451, 1983. Godbout R et al. Proc Natl Acad Sci USA 90: 7578, 1993. Griegel S et al. Differentiation 45: 250, 1990a. Griegel S et al. Int J Cancer 46: 125, 1990b. Gupta J et al. J Natl Cancer Inst 88: 1152, 1996. Herman MN et al. Amer J Path 134: 115, 1989. Howard MA et al. Invest Ophthalmol Vis Sci 32: 1711, 1991. Howes KA et al. Investigative Ophthalmology & Visual Science 35: 342, 1994. Huang HJ et al. Science 242: 1563, 1988. Hurwitz RL et al. J Clin Invest 85: 1872, 1990. Inomata M et al. Invest Ophthalmol Vis Sci 27: 1423, 1986. Jiang Q et al. Exp Eye Res 39: 207, 1984. Kivela T et al. Acta Ophthalmol (Copenh) 64: 664, 1986 Kivela T et al. Invest Ophthalmol Vis Sci 27: 1075, 1986. Klaidman LK et al. Cancer Lett 68: 207, 1993. Knudson AG. Proceedings of the National Academy of Science, USA 68: 820,1971. Kondo Y et al. Experimental Cell Research 236: 51, 1997. Koole P et al. Int J Hyperthermia 6: 203, 1990. Kyritsis AP et al. In Mtro Cell Dev Biol22: 418, 1986a. Kyritsis AP et al. Anticancer Res 6: 465, 1986b. Kyritsis AP et al. Nature 307: 471, 1984. Kyritsis AP et al. Invest Ophthalmol Vis Sci 27: 1760, 1986c. Lee WH et al. Nature 309: 458, 1984. Lohmann DR et al. Hum Genet 94: 349,1994. Madreperla SA et al. Ophthalmic Paediatr Genet 12: 49,1991a. Madreperla SA et al. Cancer Res 51: 6381, 1991b. McFall RC et al. Cancer Res 38: 2827, 1978. McFall RC et al. Cancer Res 37: 1003, 1977. Messmer EP et al. Ophthalmology 92: 167, 1985. Mirshahi M et al. Cancer 57: 1497, 1986. Muncaster MM et al. Cancer Res 52: 654, 1992. Murray TG et al. Archives of Ophthalmology 114: 1376, 1996a. Murray TG et al. Archives of Ophthalmology 114: 1385, 1996b. Nakagawa Y and Perentes E. Ophthalmic Res 19: 205, 1987. Nork TM et al. Arch Ophthalmol 115: 213, 1997. Olianas MC et al. Brain Res 593: 304, 1992. Perentes E et al. Am J Ophthalmol 103: 647, 1987. Phillips, R A et al. In The SCID mouse, Characterisation and Potential Uses, ed M Bosma. Springer-Verlag, Heidelberg, p. 259, 1989. Popoff, N et al. J Ultrastructure Res 29: 535, 1969. Rajagopalan S et al. J Histochem Cytochem 41: 1331, 1993a. Rajagopalan S et al. Ophthalmic Paediatr Genet 14: 117, 1993b. Reid TW et al. J National Cancer Inst 53:1347-360: 1974. Rodrigues MM et al. Ophthalmol 94: 378, 1987. Rootman J et al. Arch Ophthalmol 104:1693, 1986. Rubin NA et al. Vision Res 21:105, 1981. Saviolakis GA et al. J Neurochem 47: 70, 1986. Schmidt-Erfurth U et al. Br J Cancer 75: 54, 1997. Sery TW et al. J Pediatr Ophthalmol Strabismus 27: 212, 1990. Shokravi MT et al. Investigative Ophthalmology and Visual Science 36: 83, 1995. Shternfeld IS et al. Arch Ophthalmol 114: 1396, 1996.
374 Skubitz AP et al. Invest Ophthalmol Vis Sci 35: 2820, 1994. Song Y et al. Yen Ko Hsueh Pao 11: 22, 1995. Squire J et al. Hum Genet 70: 291, 1985. Squire J et al. Nature 322: 555, 1986. Squire J et al. Hum Genet 66: 46, 1984. Stein PC et al. Invest Ophthalmol Vis Sci 21: 550, 1981. Tarlton JF et al. Br J Ophthalmol 77: 805, 1993. Totsuka S et al. Ophthalmologica 185: 158, 1982. Tso MOM et al. Am Jm Ophthalmol 69: 339, 1970. Tsokos M et al. Am J Pathol123: 542, 1986. Virtanen I et al. Lab Invest 59: 649, 1988. Wakabayashi K et al. Cell Struct Funct 14: 331, 1989. Weiner MJ et al. Invest Ophthalmol Vis Sci 24: 943, 1983. White L et al. In Mvo 3: 315, 1989a. White L et al. Br J Cancer 57: 455, 1988. White L et al. Ophthalmic Paediatr Genet 10: 99, 1989b. White L et al. Cancer Chemother Pharmacol 23: 63, 1989c. Xu HJ et al. Cancer Res 51: 4481, 1991. Yokoyama T et al. Experimental Eye Research 55: 225,1992. Yorek MA et al. J Biol Chem 262: 10986, 1987. Yorek MA et al. J Neurochem 45: 1590, 1985. Yuge K et al. Virchows Arch 426: 571, 1995. Zhang Z et al. Anticancer Res 16: 269, 1996. Zhu X et al. Cytogenetics & Cell Genetics 59: 248, 1992.
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Contents of Volume I
Foreword to the Series
vii
Introduction
ix
Chapter 1
Sarcomas BEVERLY A. TEICHER
Chapter 2
Neuroblastoma CAROL J. THIELE
21
Chapter 3
Ewing’s Sarcoma Family of Tumors FRANS VAN VALEN
55
Chapter 4
Mesothelioma MARJAN A. VERSNEL
87
Chapter 5
Pancreatic Tumors TAKESHI IWAMURA and MICHAEL A. HOLLINGSWORTH
107
Chapter 6
Adrenal Cortex Tumors WILLIAM E. RAINEY and JAMES J. MROTEK
123
Chapter 7
Thyroid Gland Tumors THOMAS HOELTING
137
Chapter 8
Pituitary Gland Tumors LEO J. HOFLAND and STEVEN W. J. LAMBERTS
149
Chapter 9
Salivary Gland Tumors MITSUNOBU SATO
161
1
Chapter 10 Esophageal Cancers YUTAKA SHIMADA
179 375
376
Contents of Volume I
Chapter 11 Bladder Cancer RUTH KNUECHEL and JOHN R. W. MASTERS
213
Chapter 12 Renal Cell Cancer THOMAS EBERT, ARISTOTELES ANASTASIADIS and NEIL H. BANDER
231
Chapter 13 Skin Cancer (Non-Melanoma) PETRA BOUKAMP
251
Chapter 14 Melanoma: The Wistar Melanoma (WM) Cell Lines MEI-YU Hsu, DAVID A. ELDER and MEENHARD HERLYN
259
Chapter 15 Melanoma: Brussels Melanoma Cell Lines FRANCIS BRASSEUR
275
Chapter 16 Melanoma: Milan Melanoma Cell Lines ANDREA ANICHINI, ROBERTA MORTARINI, CLAUDIA VEGETTI, ALESSANDRA MOLLA, ALESSANDRA BORRI and GIORGIA PARMIANI
283
Human Cell Culture 1.
J.R.W. Masters and B. Palsson (eds.): Human Cell Culture, Vol. I. 1998 ISBN 0-7923-5 143-6
2.
J.R.W. Masters and B. Palsson (eds.): Human Cell Culture, Vol. II. Cancer Cell Lines Part 2. 1999 ISBN 0-7923-5878-3
3.
M.R. Koller, B.O. Palsson and J. R. W. Masters (eds.): Human Cell Culture, Volume IV. Primary Hematopoietic Cells. 1999 ISBN 0-7923-5821-X
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