Human Bladder Carcinoma: Characterization of Two New Tumor Cell Lines and Search for Tumor Viruses1, 2, 3 Suraiya Rasheed,4 Murray B. Gardner,4 Robert W. Rongey,4 Walter A. Nelson-Rees,s and Paul Arnstein 6 , 7 ABSTRACT-Two newly established human bladder carcinoma cell lines, designated HT-1197 and HT-1376, were characterized. Cells of both cultures exhibited fine structural microvilli and tonofibrils indicative of their epithelial origin. In addition, desmosomes were also present in HT-1197. Marker chromosomes present in HT-1197 and HT-1376 distinguished these from each other and from other known human tumor cell lines. Both cultures grew in soft agar, induced fibrinolytic activity, and were tumorigenic in mice and hamsters. No type C or other virus expression was detected in these cell lines nor in other human urothelial tumors tested.-J Natl Cancer Inst 58: 881-890, 1977.
MATERIALS AND METHODS
Initiation of bladder carcinoma cell lines (HT-1197 and HT-/376).-HT-1l97 was derived from a grade 4 transitional cell carcinoma from a 44-year-old Caucasian male. The tumor was obtained for tissue culture from a resection in December 1972, following seven recurrences since the initial diagnosis in 1957. The patient died in February 1974 with widespread metastatic bladder cancer. HT-1376 was obtained in August 1973 from a transurethral resection of invasive, moderately pleomorphic (grade 3) transitional cell carcinoma of a 58-year-old Caucasian woman. This patient died in November 1973 from complications of locally invasive bladder carcinoma. Neither patient received prior chemotherapy nor radiation therapy; there was no known exposure to environmental or occupational carcinogens. Source of other tissues and procedures.-Fresh surgical specimens for tissue culture were obtained from bladder carcinomas, kidney carcinomas, a Wilms' tumor, and rejected renal allografts (see table 3). The primary tumor biopsies were processed for histopathologic study by standard methods and sections stained with H & E. The renal transplant patients had received Imuran and prednisolone for immunosuppression. Peripheral blood leukocytes were collected from 3 other renal transplant patients on immunosuppressive therapy consisting of antilymphocytic globulin in addition to Imuran and prednisolone. In addition, freshly collected tumors and two in vitro-established cell lines were processed and examined by EM as described (l). Cells.-For virus assays we utilized human sarcoma cell lines RD (2) and 1080 (3) and monkey DBS-FRhL-l cells (4). Dog thymus cell line D17 (5) was kindly supplied by Dr. R. M. McAllister (Children's Hospital of
VOL. 58, NO.4, APRIL 1977
ABBREVIATIONS USED: H & E = hematoxylin and eosin; EM = electron microscopy; MEMS = Eagle's minimum essential medium, supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and 50 u.g gentamicin/ml; G6PD = glucose-6-phosphate dehydrogenase; CF = complement fixation; RDP = RNA-directed DNA polymerase; ATS = antithymocyte serum; UDR = uridine; RIA = radioimmunoassay; MuLV = murine leukemia virus; HaLV = hamster leukemia virus; FeLV = feline leukemia virus; RaLV = rat leukemia virus; GaLV = gibbon ape leukemia virus; SSV-SSAV = simian sarcoma and associated virus; IUDR = 5-iododeoxyuridine. Received June 6, 1976; accepted September 20, 1976. Supported by Public Health Service (PHS) contract NOI CP53500 within the Virus Cancer Program of the Division of Cancer Cause and Prevention of the National Cancer Institute (NCI). 3 The research described in this report involved animals maintained in animal care facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. 4 Department of Pathology, University of Southern California School of Medicine, Los Angeles, Calif. 90033. 5 Cell Culture Laboratory, University of California School of Public Health, Naval Biomedical Research Laboratory, Oakland, Calif. 94625. 6 NCI, National Institutes of Health, PHS, U.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. Currently on assignment to the California Department of Health, Berkeley, Calif. 94704. 7 We thank Dr. W. D. Peterson for G6PD assay and Dr. H. P. Charman for p30 RIA; Mrs: B. Alena for CF; Mrs. E. Toth, Miss E. Chan, Mrs. J. Bruszewski, and Mr. R. Flandermeyer for technical assistance; and Miss A. Dawson for preparation of the manuscript. 1
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Tumor cell lines derived from human tumors are useful reagents to study virologic, immunologic, and biochemical aspects of malignant disease. Long-term cultures from human carcinomas have been especially difficult to establish. Here we characterize two new cell lines derived from bladder transitional cell carcinomas.
Los Angeles), and chimpanzee lung fibroblasts (6) were from the Naval Biomedical Research Laboratory. Tissue culture techniques. -These techniques have been described (3). In short, the tumors were trimmed free of connective tissue, washed, minced, and suspended in MEMS. The cell suspensions with small fragments were seeded in 75-cm 2 plastic flasks (Falcon Plastics, Oxnard, Calif.) and incubated at 37° C in a humidified atmosphere containing 5% CO 2 , After 2-3 weeks, small areas of epithelial growth were seen together with some fibroblasts. We removed the fibroblasts by rapid trypsinization technique (3) using 0.1% trypsin with 0.2 mg EDT Aim!. The cells were washed twice with trypsin, left at room temperature for 1 minute, and rinsed several times with nutrient medium. This technique selectively detached the fibroblasts but not the epithelial cells. Some of the epithelial cells which dislodged during this procedure formed new colonies. By repeating the rapid trypsinization twice a week, we suppressed the fibroblastic growth and more new colonies of epithelial cells formed and eventually covered the entire surface of the flask. Once a confluent epithelial culture was obtained, the cells were detached by being rinsed with 0.25% trypsin and incubated for 5 minutes at 37° C. All subsequent cultures contained only epithelial cells. Isolation of epithelial cells from the primary cultures by "picking" colonies with a Pasteur pipette did not increase the growth potential of these cells. In fact,
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pared at Flow Laboratories, Rockville, Maryland, for use in these tests. The antisera were made against the p30 antigens of MuLV, HaLV, FeLV, RaLV (15), GaLV (16), SSV-SSAV (17), and the endogenous cat type C virus (RD-114) (18). RDP.-The RDP activity was measured in the presence of exogenous template primer poly r Avoligo dT. To increase the sensitivity of assay, the procedure (19) was slightly modified. Tissue culture fluid (10-20 ml) was centrifuged at 20,000xg for 15 minutes to eliminate cellular debris and concentrated (10 X) by centrifugation at 78,480Xg for 1 hour. The pellet was dissolved in 0.2% Nonidet P40, and portions of the solution were added to the reaction mixture (0.05 ml) that contained 0.03 MTris (pH 8.1), 0.04 M KCI, 2 mM MnCI 2 , 10 mM dithiothreitol, and 2.5 JLCi [3H]TTP (New England Nuclear) together with poly rA· oligo dT 0.02 A 260 units (Collaborative Research, Waltham, Mass.) or without added template primer. After incubation for 1 hour at 37° C, aliquots were adsorbed onto Whatman filter paper discs, precipitated in cold trichloroacetic acid, washed in ethanol, and dried; the level of radioactivity was determined in a liquid scintillation counter. Attempts to activate endogenous type C virus by treatment with IUDR.-HT-1197 cells at passage 20 and HT-1376 cells at passage 10 were treated separately with 25 JLg IUDR/ml (Calbiochem, San Diego, Calif.) in the medium containing 10- 6 M dexamethasone. Three days after IUDR treatment, the culture fluids were tested for RDP activity, and the dishes were divided into two groups. In one group the cells were washed with MEMS and cocultivated with dog sarcoma cells (D 17), and the other group was kept without added cells. All the cultures were refed with medium containing 10- 6 M dexamethasone. Each of the cultures was tested again for RDP activity at 12 and 20 days. The methods used for testing susceptibility of these cell lines to infection with exogenous type C viruses are given in table 2. RESULTS
The primary tumors HT-1197 and HT-1376 (figs. lA, D) were poorly differentiated, transitional cell carcinomas invading the bladder muscularis with scant inflammatory response. In each tumor, nests of large pleomorphic squamous carcinoma cells were interspersed between connective tissue septae. Cell lines HT-1197 and HT-1376 were established from these two in vitrogrown bladder carcinomas. The growth pattern of HT1197 cells rather resembled an organ culture and was comprised of three morphologically distinct cell types: a) large triangular or polyhedral cells with one or more nuclei; b) smaller, squamous epithelial cells growing in separate islands, and c) compact, rounded and spindleshaped cells (fig. IE). Because of the low plating efficiency of these cells, we have so far been unable to establish any of these cell types as clonal cell lines. The second cell line, HT-1376, showed mainly polygonal or rounded epithelial cells (fig. 1B). Morphologically distinct cell colonies such as those exhibited by the HT-1197 culture were absent. VOL. 58, NO.4, APRIL 1977
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during the first few weeks of culture, the growth of some fibroblasts in the same container appeared to stimulate the epithelial cells. Plating efficiency, doubling time, and saturation density assays.-Because of the low plating efficiency of the bladder carcinoma cells in liquid medium, the doubling time assay was slightly modified over that described (3). The cells were trypsinized, counted after trypan blue exclusion, and plated in 24 dishes at 25,000 cells/60-mm 2 dish in 10% MEMS. After 24 hours, we determined the number of cells that settled in these dishes by trypsinizing one dish and counting cells in this dish and five other stained dishes. This number of cells represented zero time. Subsequently, at 24, 48, 72, and 96 hours, the cells from one dish were trypsinized and counted, and at least 50 l-mm" areas were counted from each of the four unstained dishes. The population doubling time was determined by plotting the number of cells against the hours. . Fibroblasts and tumor cells of HT-1197 and HT-1376 cultures at a density of 3 x 105 cells/60-mm dish were separately plated in 12 dishes each. Every 24 hours for 12 days, the cells of one normal and one tumor dish were trypsinized and counted. The number of cells in each tumor culture at the time when corresponding fibroblastic cultures showed no further increase in cell number was taken as saturation density (table 1). Karyology and isoenzyme assay.-Cells were prepared for chromosome analysis by conventional staining methods as well as for Q- and G-banding, as described in (7,8). The mobility pattern for G6PD (9) was determined by Dr. W. D. Peterson. Heterotransplantation.-NIH nude athymic mice (10) at 14-15 days of age were separately inoculated sc with 2.53>< 106 cells of HT-1197 and HT-1376 cultures. After 4051 days, the transplant tumors were removed, grown in vitro, and tested for virus expression by CF and RDP activity in the culture fluid. For histopathologic study, the sections were stained with H & E. Cells from each line were also inoculated in ATStreated adult hamsters (11). Buoyant density assayfor type C virus. -Cells were incubated in medium containing 10 JLCi [5' ,6' -3H]UDR/ml (42 Ci/rnmole) (New England Nuclear, Boston, Mass.); after 24 hours, the culture fluids (10-20 ml) were harvested, clarified at 17,000xg for 10 minutes, and purified by centrifugation through 20% sucrose onto 65% sucrose at 27,000 rpm for 2.5 hours in SW27 rotor (Beckman Instrument, Inc., Fullerton, Calif.). The interface was collected, diluted, and centrifuged to equilibrium on a preformed linear 15-55% (wt/wt) sucrose gradient for 17 hours at 90,000 Xg. Fractions (0.5 ml each) were collected, their sucrose concentrations determined, acid-precipitable material was collected on filters, and radioactivity was counted. Viral antigen detection.-Tissue extracts and sonically disrupted (20-50% wt/wt) of tissue culture cells were; tested by CF (12) and RIA (13) for the presence of group-specific (interspecies-gs 3 and species-specific-gs 1) antigen p30 (14) of several mammalian type C RNA tumor viruses. Highly specific immune sera were pre-
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HUMAN BLADDER CARCINOMA CELL LINES
Karyology and G6PD Isoenzyme
More than 20 metaphases of each cell line were examined at different passage levels. HT-1197 revealed 43101, 36-97, and 44-79 chromosomes at passages 12, 33, and 34, respectively. No V-chromosome fluorescence was detected, though it was expected in these maleTABLE
I.-Properties of established transitional cell carcinoma cell lines Cell lines Properties
EM
Chromosomes (modal No.) Isoenzyme (G6PD) mobility Population doubling time (hr) Saturation density" Growth in agar Plating efficiency Fibrinolytic activity RDP activity in culture fluid Mammalian type C virus interspecies and species-specific p30 antigen Transplantability
HT-1197
HT-1376
Desmosomes, tonofibrils, and microvilli; no virus particles 36-101 (4 markers) Type B
Tonofibrils and microvilli; no virus particles 104-121 (6 markers) Type B
96
50
6x10'/cm 2 Yes 18% Present Absent
1.6 x 107/cm2 Yes 36% Present Absent
No reaction with MuLV, GaLV, SSVSSAV, HaLV, RaLV, RD-114 virus p30 antisera Tumors in NIH nude mice and immunosuppressed hamsters
a Values represent the No. of cells at the time when fibroblasts showed no further increase in population.
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derived cells. Aberrant chromosomes, especially an exceptionally long acrocentric chromosome, were noted in all metaphases by conventional staining. The G-banding technique readily revealed this and three additional rearranged or "marker" chromosomes: M1-M4 (fig. 4A). HT-1376 cells presented a different picture; chromosome numbers varied between 104 and 121 at passages 6, 10, and 13. The large number of chromosomes per metaphase made observation difficult. Not unexpectedly, we detected no Y chromosome and at least six marker chromosomes, M 1-M6 , in host cells (fig. 4B). Of these, M}, possibly the clearest, was apparently derived from a tandem fusion of the long arm of #4. M3, present in duplicate, was most likely an isochromosome of #9. Neither cell line exhibited G- or Q-banded marker chromosomes characteristic of HeLa cells (7). Extracts of HT-1197 cells at passage 35 and of HT1376 cells at passage 14 exhibited type B-band mobility pattern for the enzyme G6PD. Heterotransplantation
Within 40-51 days after inoculation of 2.5 to 3 X 106 cells in NIH nude mice, both HT-1197 and HT-1376 cultures induced tumors at the inoculation site, closely resembling the primary transitional cell carcinomas from which they were derived. The transplant tumors grown in vitro were morphologically and karyologically human epithelial cells. Both cultures also produced carcinomas at the site of inoculation in ATS-treated hamsters (11). The transplant tumors (figs. l C, F) were remarkably similar to the primary tumors in their histologic appearance and also showed little or no host inflammatory reaction. The tumor transplants of HT-1197 and HT-1376 did not become infected in vivo with endogenous type C virus of the NIH nude mouse as happened with other human tumor transplants (10), since the transplant tumors when grown in vitro remained negative for RDP as well as for MuLV p30 antigen. The hamster transplant tumors were not tested for virus activity. Virus Studies
By EM no mycoplasma, type C, or other virus-like particles were seen in HT-1197 cells at passage 22 or in HT-1376 cells at passage 7. There was no incorporation of [3H]UDR at 1.14-1.16/cm 3 density following equilibrium sedimentation in sucrose gradients of the culture fluid from HT-1l97 and HT-1376 cells at passages 20 and 8, respectively; nor did either culture contain the group-specific antigen (p30) of MuLV, HaLV, FeL V, GaLV, SSV-SSA V, and RD-1l4 virus (see "Materials and Methods") or mammalian interspecies gs3 antigen as tested by RIA. The supernatants from both cell cultures at passage levels 1, 10, 14, 15, 20, 25, 30, and 36 remained negative for RDP activity. Induction of type C virus with IUDR (20) in the presence or absence of dexamethasone also proved to be unsuccessful. Thus there was no evidence of RNA tumor virus in these two bladder carcinoma cell lines. Cells of HT-1l97 and HT-1376 showed different susceptibility patterns for various exogenous type C virus J
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By EM the HT-1197 culture showed well-formed desmosomes only in squamous epithelial cells that grew in islands (figs. 2A, B). However, all three cell types in this culture showed cytoplasmic tonofibrils and surface microvilli, indicative of their squamous epithelial origin (fig. 3A). In the HT-1376 cultures (fig. 3B), most of the cells contained cytoplasmic tonofibrils and prominent surface microvilli, but desmosomes were not seen. Both cell cultures, even at early passage levels «passage 20), exhibited properties usually associated with malignant cells. The cells when confluent grew in multiple layers and lost contact inhibition of density-dependent cell proliferation. Although both HT-1197 and HT1376 cells readily formed colonies in soft agar medium [also confirmed by Laug et al. (11)], the plating efficiency of these carcinoma cells in the liquid medium was low compared to that of other tumor cell lines (Rasheed S: Unpublished observations). Only 18% of the HT-1197 cells and 36% of the HT-1376 cells formed colonies 8 days after seeding 500 cellsll00-mm 2 dish in the liquid medium. Both cultures grew to high saturation densities in vitro, exhibiting 6x 105 and 1.6x 107 cells/em", respectively (table 1). The doubling time was 96 hours for HT1197 and 50 hours for HT-1376. Fibroblast cultures from HT-1197 and HT-1376 were also separately obtained from the stroma of these same biopsy tissues.
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2.-8usceptibility pattern of human bladder carcinoma cells to exogenous type C virus infection» HT-1197 21 days
Virus"
Un infected Control MuLV (WM-1504) MuLV (WM-292) RD-114 FeLV (G-A)
76 days
RPDd
CFe
RDpd
0 48 121 3,437 5,906
0 0 0
20 2,177 7,824 NT NT
+ +
RDc
HT-1376 76 days
21 days
CFe
RDpd
0
10 1,121 29,798 138 183
+ + NT NT
21 days
CFe
RDpd
CFe
RDpd
CFe
0
0 NT NT 70 53
0 NT NT 0 0
98 28,971 17,037 2,511 4,584
+ + + +
+ + 0 0
0
TABLE
3.-Tissues examined for type C RNA tumor virus
Tissue Bladder carcinoma Bladder carcinoma
Bladder carcinoma Kidney carcinoma Kidney carcinoma Rejected renal allograft Buffy coat from renal transplant patients a b
Source
Method
Number positive/ No. tested 0/6
Fresh surgical
Tissue culture
Dimethyl sulfoxide preserved (-96° C) surgical Fresh surgical and tissue culture cells Fresh surgical
Tissue culture
0/6
EM
on a
Tissue culture
0/3 b
Fresh surgical
EM
0/4
Fresh surgical
Tissue culture
0/3
Fresh surgical
Tissue culture
0/3
DISCUSSION
Values include the HT-1197 and HT-1376 cell lines. Values include one Wilms' tumor.
infections. All the four viruses, FeLV (G-A strain), RD114, and two wild mouse MuLV strains (1504 and 292) replicated well in HT-1197, but only wild mouse MuLV propagated in HT-1376, as evidenced by the induction of RD P and CF gs antigen of infecting virus after 21 and 76 days (table 2). Attempts to Isolate Type C Virus From Other Renal Tissues
Type C virus was not isolated from tissue mince or four bladder and kidney carcinoma cell strains from 15 other fresh surgical specimens (table 3). These cell strains consisted of tumor-like epithelial cells, but none established into a cell line. Each of the specimens was
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either cocultivated with, or homogenates assayed directly for virus isolation on, human RD, 1080, dog D17, and monkey DBS cells. No cytopathic effect or RNA virus activity was detected. We also cocultivated three leukocyte samples from the blood of renal transplant patients with human 1080, monkey, and chimpanzee cells. There was no type C virus as judged by the total absence of RDP activity in the fluids of each of these cultures at 20, 30, 40, and 60 days after cocultivation.
We established two new carcinoma cell lines from human bladder transitional cell carcinomas. Based on squamous epithelial cell islands with desmosomes in HT-1197 but not in HT-1376, the HT-1197 cell line appears more differentiated than HT-1376. Not only are these cultures morphologically distinct from each other, but also they differ in other respects (table 1) from the previously reported bladder tumor cell lines RT4 (21, 22), T24 (23), and 253J (24). Our cell lines show many more chromosomes than RT4 does and have a much longer generation time than T24. The 253J cell line was reported to contain virus-like particles (24, 25) which were absent in our cultures. Other cell lines mentioned in the literature (26-29) are not adequately described for comparison. With the exception of a recently described parathyroid hormone-secreting squamous cell carcinoma of skin origin (30), the HT-1197 and HT-1376 exhibit groups of marker chromosomes not previously observed, to our knowledge, in any other human cell lines studied by chromosome-banding techniques (2, 3,7,8,31). Admittedly, the high number of chromosomes in HT-1376 precludes optimal examination, but it is interesting that, while most normal chromosomes are present in three to 10 copies at times, the markers, except for M3 , were apparently only present singly. Although HT-1197 has at least three morphologically distinct cell types, the chromosomes of 20 metaphases show a uniform pattern. This could be due to a VOL. 58, NO.4, APRIL 1977
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a At passage 32 HT-1197 and HT-1376 cells that were preincubated for 18 hr with MEMS containing 2 JLg Polybrene/ml were separately exposed to 0.4 ml undiluted filtered (0.45 JL) MuLV strains, FeLV (G-A), and RD-114 virus. The infected and uninfected control cells were subcultured once a week and tested for RDP activity at 21 days. If the cultures were negative, they were further passaged and tested again at 44 and 76 days. Results on three expts gave similar results. NT=not tested. b Input virus had titers of 10'··, 10'.4, 103 . ' , and 104 TCID50/ml on human 1080 cells for MuLV (1504 and 292), Fe LV (G-A), and RD-114 virus, respectively. c RD=human rhabdomyosarcoma cell, passage 135, exposed to the same viruses as were the positive controls. d RDP [counts per min (cpm/rnl)] in culture fluids at 21 and 76 days after exposure to virus; cpm values were corrected after deducting background and cpm without template. e CF test on cell packs (20% extract) giving positive ( + = 3 + fixation) or negative (0=
MATERIALS AND METHODS
Initiation of bladder carcinoma cell lines (HT-1197 and HT-/376).-HT-1l97 was derived from a grade 4 transitional cell carcinoma from a 44-year-old Caucasian male. The tumor was obtained for tissue culture from a resection in December 1972, following seven recurrences since the initial diagnosis in 1957. The patient died in February 1974 with widespread metastatic bladder cancer. HT-1376 was obtained in August 1973 from a transurethral resection of invasive, moderately pleomorphic (grade 3) transitional cell carcinoma of a 58-year-old Caucasian woman. This patient died in November 1973 from complications of locally invasive bladder carcinoma. Neither patient received prior chemotherapy nor radiation therapy; there was no known exposure to environmental or occupational carcinogens. Source of other tissues and procedures.-Fresh surgical specimens for tissue culture were obtained from bladder carcinomas, kidney carcinomas, a Wilms' tumor, and rejected renal allografts (see table 3). The primary tumor biopsies were processed for histopathologic study by standard methods and sections stained with H & E. The renal transplant patients had received Imuran and prednisolone for immunosuppression. Peripheral blood leukocytes were collected from 3 other renal transplant patients on immunosuppressive therapy consisting of antilymphocytic globulin in addition to Imuran and prednisolone. In addition, freshly collected tumors and two in vitro-established cell lines were processed and examined by EM as described (l). Cells.-For virus assays we utilized human sarcoma cell lines RD (2) and 1080 (3) and monkey DBS-FRhL-l cells (4). Dog thymus cell line D17 (5) was kindly supplied by Dr. R. M. McAllister (Children's Hospital of
VOL. 58, NO.4, APRIL 1977
ABBREVIATIONS USED: H & E = hematoxylin and eosin; EM = electron microscopy; MEMS = Eagle's minimum essential medium, supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and 50 u.g gentamicin/ml; G6PD = glucose-6-phosphate dehydrogenase; CF = complement fixation; RDP = RNA-directed DNA polymerase; ATS = antithymocyte serum; UDR = uridine; RIA = radioimmunoassay; MuLV = murine leukemia virus; HaLV = hamster leukemia virus; FeLV = feline leukemia virus; RaLV = rat leukemia virus; GaLV = gibbon ape leukemia virus; SSV-SSAV = simian sarcoma and associated virus; IUDR = 5-iododeoxyuridine. Received June 6, 1976; accepted September 20, 1976. Supported by Public Health Service (PHS) contract NOI CP53500 within the Virus Cancer Program of the Division of Cancer Cause and Prevention of the National Cancer Institute (NCI). 3 The research described in this report involved animals maintained in animal care facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. 4 Department of Pathology, University of Southern California School of Medicine, Los Angeles, Calif. 90033. 5 Cell Culture Laboratory, University of California School of Public Health, Naval Biomedical Research Laboratory, Oakland, Calif. 94625. 6 NCI, National Institutes of Health, PHS, U.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. Currently on assignment to the California Department of Health, Berkeley, Calif. 94704. 7 We thank Dr. W. D. Peterson for G6PD assay and Dr. H. P. Charman for p30 RIA; Mrs: B. Alena for CF; Mrs. E. Toth, Miss E. Chan, Mrs. J. Bruszewski, and Mr. R. Flandermeyer for technical assistance; and Miss A. Dawson for preparation of the manuscript. 1
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Tumor cell lines derived from human tumors are useful reagents to study virologic, immunologic, and biochemical aspects of malignant disease. Long-term cultures from human carcinomas have been especially difficult to establish. Here we characterize two new cell lines derived from bladder transitional cell carcinomas.
Los Angeles), and chimpanzee lung fibroblasts (6) were from the Naval Biomedical Research Laboratory. Tissue culture techniques. -These techniques have been described (3). In short, the tumors were trimmed free of connective tissue, washed, minced, and suspended in MEMS. The cell suspensions with small fragments were seeded in 75-cm 2 plastic flasks (Falcon Plastics, Oxnard, Calif.) and incubated at 37° C in a humidified atmosphere containing 5% CO 2 , After 2-3 weeks, small areas of epithelial growth were seen together with some fibroblasts. We removed the fibroblasts by rapid trypsinization technique (3) using 0.1% trypsin with 0.2 mg EDT Aim!. The cells were washed twice with trypsin, left at room temperature for 1 minute, and rinsed several times with nutrient medium. This technique selectively detached the fibroblasts but not the epithelial cells. Some of the epithelial cells which dislodged during this procedure formed new colonies. By repeating the rapid trypsinization twice a week, we suppressed the fibroblastic growth and more new colonies of epithelial cells formed and eventually covered the entire surface of the flask. Once a confluent epithelial culture was obtained, the cells were detached by being rinsed with 0.25% trypsin and incubated for 5 minutes at 37° C. All subsequent cultures contained only epithelial cells. Isolation of epithelial cells from the primary cultures by "picking" colonies with a Pasteur pipette did not increase the growth potential of these cells. In fact,
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pared at Flow Laboratories, Rockville, Maryland, for use in these tests. The antisera were made against the p30 antigens of MuLV, HaLV, FeLV, RaLV (15), GaLV (16), SSV-SSAV (17), and the endogenous cat type C virus (RD-114) (18). RDP.-The RDP activity was measured in the presence of exogenous template primer poly r Avoligo dT. To increase the sensitivity of assay, the procedure (19) was slightly modified. Tissue culture fluid (10-20 ml) was centrifuged at 20,000xg for 15 minutes to eliminate cellular debris and concentrated (10 X) by centrifugation at 78,480Xg for 1 hour. The pellet was dissolved in 0.2% Nonidet P40, and portions of the solution were added to the reaction mixture (0.05 ml) that contained 0.03 MTris (pH 8.1), 0.04 M KCI, 2 mM MnCI 2 , 10 mM dithiothreitol, and 2.5 JLCi [3H]TTP (New England Nuclear) together with poly rA· oligo dT 0.02 A 260 units (Collaborative Research, Waltham, Mass.) or without added template primer. After incubation for 1 hour at 37° C, aliquots were adsorbed onto Whatman filter paper discs, precipitated in cold trichloroacetic acid, washed in ethanol, and dried; the level of radioactivity was determined in a liquid scintillation counter. Attempts to activate endogenous type C virus by treatment with IUDR.-HT-1197 cells at passage 20 and HT-1376 cells at passage 10 were treated separately with 25 JLg IUDR/ml (Calbiochem, San Diego, Calif.) in the medium containing 10- 6 M dexamethasone. Three days after IUDR treatment, the culture fluids were tested for RDP activity, and the dishes were divided into two groups. In one group the cells were washed with MEMS and cocultivated with dog sarcoma cells (D 17), and the other group was kept without added cells. All the cultures were refed with medium containing 10- 6 M dexamethasone. Each of the cultures was tested again for RDP activity at 12 and 20 days. The methods used for testing susceptibility of these cell lines to infection with exogenous type C viruses are given in table 2. RESULTS
The primary tumors HT-1197 and HT-1376 (figs. lA, D) were poorly differentiated, transitional cell carcinomas invading the bladder muscularis with scant inflammatory response. In each tumor, nests of large pleomorphic squamous carcinoma cells were interspersed between connective tissue septae. Cell lines HT-1197 and HT-1376 were established from these two in vitrogrown bladder carcinomas. The growth pattern of HT1197 cells rather resembled an organ culture and was comprised of three morphologically distinct cell types: a) large triangular or polyhedral cells with one or more nuclei; b) smaller, squamous epithelial cells growing in separate islands, and c) compact, rounded and spindleshaped cells (fig. IE). Because of the low plating efficiency of these cells, we have so far been unable to establish any of these cell types as clonal cell lines. The second cell line, HT-1376, showed mainly polygonal or rounded epithelial cells (fig. 1B). Morphologically distinct cell colonies such as those exhibited by the HT-1197 culture were absent. VOL. 58, NO.4, APRIL 1977
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during the first few weeks of culture, the growth of some fibroblasts in the same container appeared to stimulate the epithelial cells. Plating efficiency, doubling time, and saturation density assays.-Because of the low plating efficiency of the bladder carcinoma cells in liquid medium, the doubling time assay was slightly modified over that described (3). The cells were trypsinized, counted after trypan blue exclusion, and plated in 24 dishes at 25,000 cells/60-mm 2 dish in 10% MEMS. After 24 hours, we determined the number of cells that settled in these dishes by trypsinizing one dish and counting cells in this dish and five other stained dishes. This number of cells represented zero time. Subsequently, at 24, 48, 72, and 96 hours, the cells from one dish were trypsinized and counted, and at least 50 l-mm" areas were counted from each of the four unstained dishes. The population doubling time was determined by plotting the number of cells against the hours. . Fibroblasts and tumor cells of HT-1197 and HT-1376 cultures at a density of 3 x 105 cells/60-mm dish were separately plated in 12 dishes each. Every 24 hours for 12 days, the cells of one normal and one tumor dish were trypsinized and counted. The number of cells in each tumor culture at the time when corresponding fibroblastic cultures showed no further increase in cell number was taken as saturation density (table 1). Karyology and isoenzyme assay.-Cells were prepared for chromosome analysis by conventional staining methods as well as for Q- and G-banding, as described in (7,8). The mobility pattern for G6PD (9) was determined by Dr. W. D. Peterson. Heterotransplantation.-NIH nude athymic mice (10) at 14-15 days of age were separately inoculated sc with 2.53>< 106 cells of HT-1197 and HT-1376 cultures. After 4051 days, the transplant tumors were removed, grown in vitro, and tested for virus expression by CF and RDP activity in the culture fluid. For histopathologic study, the sections were stained with H & E. Cells from each line were also inoculated in ATStreated adult hamsters (11). Buoyant density assayfor type C virus. -Cells were incubated in medium containing 10 JLCi [5' ,6' -3H]UDR/ml (42 Ci/rnmole) (New England Nuclear, Boston, Mass.); after 24 hours, the culture fluids (10-20 ml) were harvested, clarified at 17,000xg for 10 minutes, and purified by centrifugation through 20% sucrose onto 65% sucrose at 27,000 rpm for 2.5 hours in SW27 rotor (Beckman Instrument, Inc., Fullerton, Calif.). The interface was collected, diluted, and centrifuged to equilibrium on a preformed linear 15-55% (wt/wt) sucrose gradient for 17 hours at 90,000 Xg. Fractions (0.5 ml each) were collected, their sucrose concentrations determined, acid-precipitable material was collected on filters, and radioactivity was counted. Viral antigen detection.-Tissue extracts and sonically disrupted (20-50% wt/wt) of tissue culture cells were; tested by CF (12) and RIA (13) for the presence of group-specific (interspecies-gs 3 and species-specific-gs 1) antigen p30 (14) of several mammalian type C RNA tumor viruses. Highly specific immune sera were pre-
883
HUMAN BLADDER CARCINOMA CELL LINES
Karyology and G6PD Isoenzyme
More than 20 metaphases of each cell line were examined at different passage levels. HT-1197 revealed 43101, 36-97, and 44-79 chromosomes at passages 12, 33, and 34, respectively. No V-chromosome fluorescence was detected, though it was expected in these maleTABLE
I.-Properties of established transitional cell carcinoma cell lines Cell lines Properties
EM
Chromosomes (modal No.) Isoenzyme (G6PD) mobility Population doubling time (hr) Saturation density" Growth in agar Plating efficiency Fibrinolytic activity RDP activity in culture fluid Mammalian type C virus interspecies and species-specific p30 antigen Transplantability
HT-1197
HT-1376
Desmosomes, tonofibrils, and microvilli; no virus particles 36-101 (4 markers) Type B
Tonofibrils and microvilli; no virus particles 104-121 (6 markers) Type B
96
50
6x10'/cm 2 Yes 18% Present Absent
1.6 x 107/cm2 Yes 36% Present Absent
No reaction with MuLV, GaLV, SSVSSAV, HaLV, RaLV, RD-114 virus p30 antisera Tumors in NIH nude mice and immunosuppressed hamsters
a Values represent the No. of cells at the time when fibroblasts showed no further increase in population.
VOL. 58, NO.4, APRIL 1977
derived cells. Aberrant chromosomes, especially an exceptionally long acrocentric chromosome, were noted in all metaphases by conventional staining. The G-banding technique readily revealed this and three additional rearranged or "marker" chromosomes: M1-M4 (fig. 4A). HT-1376 cells presented a different picture; chromosome numbers varied between 104 and 121 at passages 6, 10, and 13. The large number of chromosomes per metaphase made observation difficult. Not unexpectedly, we detected no Y chromosome and at least six marker chromosomes, M 1-M6 , in host cells (fig. 4B). Of these, M}, possibly the clearest, was apparently derived from a tandem fusion of the long arm of #4. M3, present in duplicate, was most likely an isochromosome of #9. Neither cell line exhibited G- or Q-banded marker chromosomes characteristic of HeLa cells (7). Extracts of HT-1197 cells at passage 35 and of HT1376 cells at passage 14 exhibited type B-band mobility pattern for the enzyme G6PD. Heterotransplantation
Within 40-51 days after inoculation of 2.5 to 3 X 106 cells in NIH nude mice, both HT-1197 and HT-1376 cultures induced tumors at the inoculation site, closely resembling the primary transitional cell carcinomas from which they were derived. The transplant tumors grown in vitro were morphologically and karyologically human epithelial cells. Both cultures also produced carcinomas at the site of inoculation in ATS-treated hamsters (11). The transplant tumors (figs. l C, F) were remarkably similar to the primary tumors in their histologic appearance and also showed little or no host inflammatory reaction. The tumor transplants of HT-1197 and HT-1376 did not become infected in vivo with endogenous type C virus of the NIH nude mouse as happened with other human tumor transplants (10), since the transplant tumors when grown in vitro remained negative for RDP as well as for MuLV p30 antigen. The hamster transplant tumors were not tested for virus activity. Virus Studies
By EM no mycoplasma, type C, or other virus-like particles were seen in HT-1197 cells at passage 22 or in HT-1376 cells at passage 7. There was no incorporation of [3H]UDR at 1.14-1.16/cm 3 density following equilibrium sedimentation in sucrose gradients of the culture fluid from HT-1l97 and HT-1376 cells at passages 20 and 8, respectively; nor did either culture contain the group-specific antigen (p30) of MuLV, HaLV, FeL V, GaLV, SSV-SSA V, and RD-1l4 virus (see "Materials and Methods") or mammalian interspecies gs3 antigen as tested by RIA. The supernatants from both cell cultures at passage levels 1, 10, 14, 15, 20, 25, 30, and 36 remained negative for RDP activity. Induction of type C virus with IUDR (20) in the presence or absence of dexamethasone also proved to be unsuccessful. Thus there was no evidence of RNA tumor virus in these two bladder carcinoma cell lines. Cells of HT-1l97 and HT-1376 showed different susceptibility patterns for various exogenous type C virus J
NATL CANCER INST
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By EM the HT-1197 culture showed well-formed desmosomes only in squamous epithelial cells that grew in islands (figs. 2A, B). However, all three cell types in this culture showed cytoplasmic tonofibrils and surface microvilli, indicative of their squamous epithelial origin (fig. 3A). In the HT-1376 cultures (fig. 3B), most of the cells contained cytoplasmic tonofibrils and prominent surface microvilli, but desmosomes were not seen. Both cell cultures, even at early passage levels «passage 20), exhibited properties usually associated with malignant cells. The cells when confluent grew in multiple layers and lost contact inhibition of density-dependent cell proliferation. Although both HT-1197 and HT1376 cells readily formed colonies in soft agar medium [also confirmed by Laug et al. (11)], the plating efficiency of these carcinoma cells in the liquid medium was low compared to that of other tumor cell lines (Rasheed S: Unpublished observations). Only 18% of the HT-1197 cells and 36% of the HT-1376 cells formed colonies 8 days after seeding 500 cellsll00-mm 2 dish in the liquid medium. Both cultures grew to high saturation densities in vitro, exhibiting 6x 105 and 1.6x 107 cells/em", respectively (table 1). The doubling time was 96 hours for HT1197 and 50 hours for HT-1376. Fibroblast cultures from HT-1197 and HT-1376 were also separately obtained from the stroma of these same biopsy tissues.
884
RASHEED, GARDNER, RONGEY, NELSON-REES, AND ARNSTEIN TABLE
2.-8usceptibility pattern of human bladder carcinoma cells to exogenous type C virus infection» HT-1197 21 days
Virus"
Un infected Control MuLV (WM-1504) MuLV (WM-292) RD-114 FeLV (G-A)
76 days
RPDd
CFe
RDpd
0 48 121 3,437 5,906
0 0 0
20 2,177 7,824 NT NT
+ +
RDc
HT-1376 76 days
21 days
CFe
RDpd
0
10 1,121 29,798 138 183
+ + NT NT
21 days
CFe
RDpd
CFe
RDpd
CFe
0
0 NT NT 70 53
0 NT NT 0 0
98 28,971 17,037 2,511 4,584
+ + + +
+ + 0 0
0
TABLE
3.-Tissues examined for type C RNA tumor virus
Tissue Bladder carcinoma Bladder carcinoma
Bladder carcinoma Kidney carcinoma Kidney carcinoma Rejected renal allograft Buffy coat from renal transplant patients a b
Source
Method
Number positive/ No. tested 0/6
Fresh surgical
Tissue culture
Dimethyl sulfoxide preserved (-96° C) surgical Fresh surgical and tissue culture cells Fresh surgical
Tissue culture
0/6
EM
on a
Tissue culture
0/3 b
Fresh surgical
EM
0/4
Fresh surgical
Tissue culture
0/3
Fresh surgical
Tissue culture
0/3
DISCUSSION
Values include the HT-1197 and HT-1376 cell lines. Values include one Wilms' tumor.
infections. All the four viruses, FeLV (G-A strain), RD114, and two wild mouse MuLV strains (1504 and 292) replicated well in HT-1197, but only wild mouse MuLV propagated in HT-1376, as evidenced by the induction of RD P and CF gs antigen of infecting virus after 21 and 76 days (table 2). Attempts to Isolate Type C Virus From Other Renal Tissues
Type C virus was not isolated from tissue mince or four bladder and kidney carcinoma cell strains from 15 other fresh surgical specimens (table 3). These cell strains consisted of tumor-like epithelial cells, but none established into a cell line. Each of the specimens was
J NATL CANCER INST
either cocultivated with, or homogenates assayed directly for virus isolation on, human RD, 1080, dog D17, and monkey DBS cells. No cytopathic effect or RNA virus activity was detected. We also cocultivated three leukocyte samples from the blood of renal transplant patients with human 1080, monkey, and chimpanzee cells. There was no type C virus as judged by the total absence of RDP activity in the fluids of each of these cultures at 20, 30, 40, and 60 days after cocultivation.
We established two new carcinoma cell lines from human bladder transitional cell carcinomas. Based on squamous epithelial cell islands with desmosomes in HT-1197 but not in HT-1376, the HT-1197 cell line appears more differentiated than HT-1376. Not only are these cultures morphologically distinct from each other, but also they differ in other respects (table 1) from the previously reported bladder tumor cell lines RT4 (21, 22), T24 (23), and 253J (24). Our cell lines show many more chromosomes than RT4 does and have a much longer generation time than T24. The 253J cell line was reported to contain virus-like particles (24, 25) which were absent in our cultures. Other cell lines mentioned in the literature (26-29) are not adequately described for comparison. With the exception of a recently described parathyroid hormone-secreting squamous cell carcinoma of skin origin (30), the HT-1197 and HT-1376 exhibit groups of marker chromosomes not previously observed, to our knowledge, in any other human cell lines studied by chromosome-banding techniques (2, 3,7,8,31). Admittedly, the high number of chromosomes in HT-1376 precludes optimal examination, but it is interesting that, while most normal chromosomes are present in three to 10 copies at times, the markers, except for M3 , were apparently only present singly. Although HT-1197 has at least three morphologically distinct cell types, the chromosomes of 20 metaphases show a uniform pattern. This could be due to a VOL. 58, NO.4, APRIL 1977
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a At passage 32 HT-1197 and HT-1376 cells that were preincubated for 18 hr with MEMS containing 2 JLg Polybrene/ml were separately exposed to 0.4 ml undiluted filtered (0.45 JL) MuLV strains, FeLV (G-A), and RD-114 virus. The infected and uninfected control cells were subcultured once a week and tested for RDP activity at 21 days. If the cultures were negative, they were further passaged and tested again at 44 and 76 days. Results on three expts gave similar results. NT=not tested. b Input virus had titers of 10'··, 10'.4, 103 . ' , and 104 TCID50/ml on human 1080 cells for MuLV (1504 and 292), Fe LV (G-A), and RD-114 virus, respectively. c RD=human rhabdomyosarcoma cell, passage 135, exposed to the same viruses as were the positive controls. d RDP [counts per min (cpm/rnl)] in culture fluids at 21 and 76 days after exposure to virus; cpm values were corrected after deducting background and cpm without template. e CF test on cell packs (20% extract) giving positive ( + = 3 + fixation) or negative (0=
