IN VITRO Volume 14, No. 3, 1978 All rights reserved9
A TRANSITIONAL
C E L L C A R C I N O M A C E L L L I N E 1~2
GEORGE E. MOORE, 3 ROBERT T. MORGAN, LAURIE A. QUINN, ANDLINDA K. WOODS
Department o/Surgery, Denver General Hospital, IV. 8th Avenue and Cherokee Street, Denver, Colorado 80204 SUMMARY
A transitional cell carcinoma cell line, COLO 232, was derived from a primary urinary bladder tumor in a Caucasian male. In culture, COLO 232 retained distinct uroepithelial phenotypic traits and produced both carcinoembryonic antigen and adrenocorticotropic hormone. COLO 232 had a chromosome mode of 58 and retained the X and Y chromosomes. Ten marker chromosomes were identified. COLO 232 will be of value for biochemical and immunological studies.
Key words: transitional cell carcinoma; human cell line; karyotype; adrenocorticotropic hormone; carcinoembryonic antigen.
INTRODUCTION Well characterized human tumor cell lines have been useful for numerous studies of malignant diseases and tumor-associated antigens. In this report, a new tumor cell line derived from a patient with transitional cell carcinoma of the urinary bladder is described. Case report. A 70-year-old Caucasian male had undergone a transurethral resection of the bladder for a tumor and an ileal loop diversion followed by a course of 4000 rads in late 1975. On January 8, 1976, a radical cystectomy and urethrectomy for stage II B, grade 3 transitional cell carcinoma of the bladder was performed. Tissue from this surgical procedure was submitted for tissue culture. The laboratory findings of the patient's status were unremarkable except for an elevated Ca +§ level (12.2 mg%). During the next 10 months, serum calcium level reached a high of 14 mg% and the patient died on October 23, 1976. The donor's ABO blood group was A-t-.
MATERIAL AND METHODS
Cultures of the transitional cell carcinoma tissue were initiated by finely mincing the tumor 1Presented in part at the 28th Annual Meeting of the
Tissue Culture Association, June 7, 1977. ZThis work was supported by Grant No. CA 15018 awarded by the National Cancer Institute, DHEW, and the Mary B. and L. H. Marshall Fund. 3To whom requests for reprints should be addressed.
specimen in 2-ml volumes of R P M I 1640 medium (1}, G E M 1717 medium (2), and H a m ' s medium F-12 ~3}; each supplemented with 10% heatinactivated fetal bovine serum (FBS}, penicillin (100 U per ml), and streptomycin (50 ~g per mB. The tumor tissue suspensions were seeded to 4-oz. flint-glass culture bottles. The bottles were loosely capped and incubated at 37 ~ C in humidified air with 10% CO2. Weekly, 2 to 5 ml fresh medium was added to each culture bottle until cell growth stabilized. The criteria for establishment of the cultured tumor cells as a permanent cell line were: (a~ monolayer outgrowth from the tissue minces reached semiconfluency; (bj the cells were successfully subcultured several times; and {c~ absence of fibroblasts or other cell types. For subculture, cultured tumor cells were dispersed by scraping the monolayer growth with a rubbertipped rod and seeding the cell suspension into equal volumes of fresh culture medium and spent medium. Successful subcultures were split 1:2 at 2-wcek intervals for over I year. Serial morphologic observations by phase microscopy were made of subcultures growing on cover slips. The electron-microscopy method was summarized previously i4). For chromosome preparations, 7-day-old subcultures of the cultured tumor cells were incubated 5 hr at 37 ~ C with 0.1 /~1 Colcemid (Ciba Pharmaceuticals, Summit, N.J.~ per 10 ml medium. The subcultures were scraped with a
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rubber-tipped rod and the resultant cell suspensions were centrifuged, resuspended in 0.075 M KCI for 12 min, centrifuged, resuspended in fixative (3:1 methanol: acetic acid) for 10 min, centrifuged, and resuspended in fresh fixative for an additional 10 min. Slides were made according to the hot-plate method of Lubs, et al. {5). Giemsabanded chromosomes were obtained using a modification of the method of Seabright (6}. Slides were dipped in a trypsin {1:250; Difco, Detroit, Mich.) buffered-saline solution (pH 6.8) for 30 to 90 sec and stained in a buffered (pH 6.8) 2% Giemsa solution (Harleco, Philadelphia, Pa.). Constitutive heterochromatin banding was obtained using a modification of the method of McKenzie and Lubs (7). Slides that were used to obtain Giemsa-banded preparations were incubated at 60 ~ C in 2X SSC (0.3 M NaCI, 0.03 M Na Citrate) for 10 to 20 min and stained in a buffered (pH 6.8} 5% Giemsa solution. A carcinoembryonic antigen (CEA) kit (Roche Diagnostics, Nutley, N.J. } was used to assay: {a) 16-day-old spent R P M I 1640 medium supplemented with 10% FBS from a culture of 2.1 x 106 total live tumor cells; (b} the supernates from freeze-thaw lysed cells of the same culture; and (c) complete R P M I 1640 medium supplemented with 10% FBS without exposure to tumor cells. Since the patient died with hypercalcemia, spent R P M I 1640 medium with 10% FBS recovered after 10 days from actively growing tumor cells and a control sample of fresh medium were assayed for parathyroid hormone (PTH) by Dr. S. Krutzik, Nichols Institute, San Pedro, Calif. The P T H radioimmunoassay procedure (8) detects immunoreactive P T H in samples utilizing highly purified [12sI]-labeled antisera raised against intact bovine P T H . A serum pool obtained from patients with hyperparathyroidism serves as reference standard. Glucose-6-phosphate dehydrogenase (G6PD) isoenzyme mobility was determined with a cellulose-acetate electrophoresis kit obtained from Helena Laboratories, Beaumont, Tex. Cultured tumor cells t10 7} were lysed by freeze-thaw in 1 ml 0.9% NaCI solution and the supernate was assayed for G6PD isoenzymes. Adrenocorticotropic hormone tACTH} assays were performed at the University of Colorado Medical Center, Denver, Colo., on 14-day-old spent R P M I 1640 medium with 10% FBS from actively growing cultures and on complete medium without exposure to the cultured cells. The
A C T H was assayed by the method of Eipper and Mains {9}. For heterologous transplantation of the cultured tumor cells, five female C 3 H / H e J mice were irradiated with 600 R, and each mouse was injected subcutaneously with 106 viable tumor cells in 0.1 ml saline. When palpable nodules were observed, they were removed surgically and prepared for histological studies. To determine plating efficiency, 1-ml suspensions of 10s viable tumor cells per ml R P M I 1640 medium with 10% FBS were plated in each of six Petri dishes {35-mmL After 18 hr, all nonadherent cells in the dishes were removed with a phosphate-buffered saline {PBS} rinse. The remaining cells were removed with 0.025% trypsin IGrand Island Biological Co., Grand Island, N.Y.}. The subsequent cell suspensions were centrifuged, resuspended in 0.1 ml PBS, and counted in a hemacytometer. For colony formation in soft agar, equal volumes of a 1% agar ~Grand Island Biological Co. } and a 2X concentration of R P M I 1640 medium with 20% FBS were mixed. An aliquot of 5 ml of the agar-medium mixture was plated into 60- by 15-mm plastic Petri dishes. A 1-ml cell suspension of 104 viable tumor cells were mixed thoroughly with 4.5 ml of a 0.67% solution agarose lGrand Island Biological Co. } and 4.5 ml of a 2X concentration R P M I 1640 medium with 20% FBS. Two ml of the tumor cell agarose-medium suspension then was layered carefully on the hardened agar base. The agarose cultures were incubated at 37 ~ C in humidified air with 10% CO2. After 1 week incubation, colony cell counts were made and efficiency for growth in soft agar calculated. The cuhures were tested for pleuropneumonialike organism ~PPLO} contamination by the agarbroth method 14} and the bisbenzamide staining method (10L The cell line was preserved for the cell bank by slow freezing cell suspensions in R P M I 1640 medium supplemented with 20% FBS, 12% dimethylsulfoxide, penicillin and streptomycin. Each ampul, containing approximately 8 x 106 cells, was stored at - 8 5 ~ C {11 }. RESULTS COLO 232 was established from the primary tumor tissue on February 3, 1976, in F-12 medium supplemented with 10% FBS. COLO 232 is maintained in either R P M I 1640 or G E M 1717 medium supplemented with 10% FBS. An additional cell line was established in G E M 1717 medium with 10% FBS and designated COLO 229.
BLADDER CARCINOMA CELL LINE
FIG. 1. Phase-contrast photomicrograph of COLO 232. x182. COLO 229, which shares similar marker properties with COLO 232, has not been fully characterized.
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COLO 232 grew as muhilayered, compact, epithelial-like colonies with a cell doubling time of 48 hr. Serial phase microscopic studies revealed uniform, small taverage 15/am in diameter), polygonal cells with occasional multinucleated giant cells (Fig. 1). Nuclei were large and round with multiple large nucleoli. Prominent "intercellular bridges" spanned wide intercellular spaces. Numerous small refractile vesicles were contained within the cytoplasm. Electron micrographs showed polygonal neoplastic cells with scattered microvillar protrusions. Demosomal attachment plaques were numerous. Tonofibrils, evident in most cells, predominated in the perinuclear region with some scattered randomly in the cytoplasm (Fig. 2). The cytoplasm of some of the cultured tumor cells contained few organelles, whereas others contained numerous mitochondria, abundant free polyribosomes and dilated endoplasmic retriculum. Inclusion bodies and glycogen granules were observed along with fusiform vesicles and residual bodies of varying size. Golgi apparatus was observed in the cells, although not well developed. No phagosomes were observed. The cultured tumor cells had the distinctive asymmetric cell membrane reported for the surface cells of mammalian uroepithelium i12). No virallike particles were observed.
FIG. 2. Ultrastructttre of COLO 232 showing tonofibrils, desmosome and asymmetric angular plasma membrane, x31,000.
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FIG. 3. Giemsa-banded marker chromosomes of COLO 232 compared to normal chromosomes from the same cell show probable deviation of the markers, x2400. Chromosome analyses of COLO 232 were made No detectable amount of P T H was found in form 89 metaphase spreads; 41 of these spreads either the spent medium from COLO 232 or the were photographed and 11 karyotyped. The control fresh medium. The detectable limit of the chromosome mode was 58 with a range of 53 to assay was 25/~1 Eq per ml. 59. In all metaphases analyzed, the X and Y Analyses of the freeze-thaw lysate supernate chromosomes were present. No unusual polymor- from COLO 232 for CEA showed a concentration phisms were noted. The marker chromosomes obof 14.4 ng per 106 cells and 32.4 ng per ml in the served are shown in Fig. 3 and were identified as spent medium. No CEA was detected in complete follows: R P M I 1640 medium with 10% FBS. ACTH levels in spent R P M I 1640 medium M, i(1)(qter-~cen-~qter) with 10% FBS from COLO 232 was 4.0 ng per ml M2 t(1 ;7)(1 p t e r - ' l p2? :: 7q3 ?--'7pter) and zero for the control fresh medium. M3 del,t(1;7)(1 pter-~l p2? ::7q3?--'7pl? :) COLO 232 was positive for type-B isoenzyme of M4 del(3)(pter-'pl2:) G6PD. Ms i(5)(pter~cen~pter) Tumors were induced at the inoculation site of M_~ dic(7;9)(7pter-~7p2 ? : :9q2 ?-~9qter) COLO 232 in irradiated C 3 H / H e J mice. HistoM, t(8;19)(8pter-~8ql?:: 19) logic studies revealed that the tumor nodules were Ms t(ll;?)(1 l p t e r - ' l lq2?::?) malignant cells, morphologically similar to a difM9 t( 14; .9)( 14qter~cen ~ .9) ferentiated bladder carcinoma. M~o i(21 )(qter-~cen-~qter) The plating efficiency for COLO 232 was 65%. Five additional abnormal chromosomes of unCOLO 232 grew in soft agar with a 34% effiknown origin were occasionally observed in ciency. COLO 232 cells. COLO 229 has not been extenThe cell line has been maintained in culture sively characterized cytogehetically, hut some since initiation and aliquots have been frozen for markers (M1, Ms, Ms) were common to both cell the cell bank. The cell line has been retrieved suclines. cessfully from the cell bank.
BLADDER CARCINOMA CELL LINE Mycoplasma was detected in COLO 232 cells by the bisbenzamide method. COLO 232 was negative for PPLO contamination by the agarbroth and orcein techniques. The cell line was treated for mycoplasma contamination with tetracycline-HCl I13 }. D ISCUSSION To date, few established transitional cell carcinoma (TCC~ cell lines have been characterized (14-18}. This report concerns a new TCC cell line, COLO 232, established from a primary tumor specimen. After 1 year in culture, COLO 232 retained a well differentiated morphology that was representative of TCC. Epithelial morphology was observed with phase-contrast microscopy. The distinct uroepithelial phenotypic traits of desmosomes, microvillar projections, thick angular plasma membrane and cytoplasmic fusiform vesicles were observed in electron micrographs. Cytogenetic analysis of COLO 232 revealed that 10 marker chromosomes were present in more than 85% of the metaphases. These markers do not appear to be present in banded karyotypes of TCC cell lines reported in the literature (14,18j. Also, the complement of markers does not appear to be the same as any other banded karyotype of a human cell line. Malignant cells have been reported to produce cell products that are not normally produced by the organ from which the tumor originated I19~. Since the patient had hypercalcemia at death and two of our reported squamous cell carcinoma cell lines ~4,20) produced P T H , COLO 232 was assayed for PTH. No P T H was detected. Ectopic ACTH production has been reported in lung and pancreatic tumors ~21 ~. COLO 232 produced a detectable amount of ACTH in vitro. Further studies of hormone production by TCC cell lines may have implications for the future diagnosis and treatment of this disease. CEA has been detected in squamous cell carcinoma cell lines (4,20j and colon carcinoma cell lines ~22,23L COLO 232 elaborated appreciable amounts of CEA in the culture medium. This cell line may be useful in studies of the role of CEA in malignancies, and is available to other investigators for their study. REFERENCES 1. Moore, G. E., R. E. Gerner, and H. A. Franklin. 1967. Culture of normal human leukocytes. J. Am. Med. Assoc. 199: 519-524.
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2. Woods, L.K., G.E. Moore, C.J. Bainbridge, C.C. Huang, C. Huzeila, and L. A. Quinn. 1973. Lymphoid cell lines established from peripheral blood of persons with Down's syndrome. N.Y. State J. Med. 73: 869-873. 3. Ham, R. G. 1965. Clonal growth of mammalian cells in a chemically defined synthetic medium. Proc. Nat. Acad. Sci. U.S.A. 53: 288-293. 4. Moore, G. E., S. B. Merrick, L. K. Woods, and N. M. Arabasz. 1975. A human squamous cell carcinoma cell line. Cancer Res. 35: 2684-2688. 5. Labs, H. A., W. H. McKenzie, S. R. Patil, and S. Merrick. 1973. New staining methods for chromosomes. In: D. M. Prescott (Ed. j, Methods in Cell Biology. Vol. 6. pp. 346-380. 6. Seabright, M. 1971. A rapid banding technique for human chromosomes. Lancet 2: 971-972. 7. McKenzie, W.H., and H.A. Labs. 1973. An analysis of technical variables in the production of C bands. Chromosoma 41:175-182. 8. Krutzik, S. 1976. Radioimmunoassay Manual. 3rd Edition. Nichols Institute, San Pedro, Calif., pp. 209-220. 9. Eipper, B.A., and R.E. Mains. 1975. High molecular weight forms of adrenocorticotropic hormone in the mouse pituitary and in a mouse tumor cell line. Biochemistry 14: 3836-3844. 10. Chen, T. R. 1975. Microscopic demonstration of mycoplasma contamination in cell cultures and cell culture media. Procedure 75361. TCA Manual 1: 229-232. 11. Mizrahi, A., and G. E. Moore. 1971. Long-term preservation of permanent human hematopoietic cell lines. J. Med. 2: 380-386. 12. Firth, J. A., and R. M. Hicks. 1973. Interspecies variation in the fine structure and enzyme cytochemistry of mammalian transitional epithelium. J. Anat. 116: 31-43. 13. Quinn, L. A., L. K. Woods, S. B. Merrick, N. M. Arabasz, and G. E. Moore. 1977. Cytogenetic analysis of 12 human malignant melanoma cell lines. J. Nat. Cancer Inst. 59: 301-307. 14. O'Toole, C., S. Mayak, Z. Price, W. H. Gilbert, and J. Waisman. 1976. A cell line ISCaBERj derived from squamous cell carcinoma of the human urinary bladder. Int. J. Cancer 17: 707-714. 15. Elliot, A. Y., P. Cleveland, J. Cervenka, A.E. Castro, N. Stein, T . R . Hakala, and E.E. Fraley. 1974. Characterization of a cell line from human transitional cell carcinoma of the urinary tract. J. Nat. Cancer Inst. 53: 1341-1349. 16. Rigby, C. C., and L. M. Franks. 1970. A human tissue culture cell line from a transitional cell tumor of the urinary bladder: Growth, chromosome pattern and ultrastructure. Br. J. Cancer 24: 746-754. 17. Babenik, J., M. Baresova, V. Viklicky, J. Jakoubkova, H. Sainerova, and J. Donner. 1973. Established cell line of urinary bladder carcinoma ~T24) containing tumor-specific antigen. Int. J. Cancer 11: 765-773. 18. Rasheed, S., M.B. Gardner, R.W. Rongey, W.A. Nelson-Rees, and P. Arnstein. 1977. Human bladder carcinoma: Characterization of two new tumor cell lines and search for tumor viruses. J. Nat. Cancer Inst. 58: 881-890.
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19. Liddle, G . W . , W . E . Nicholson, D . P . Island, D. N. Orth, K. Abe, and S. C. Lowder. 1969. Clinical and laboratory studies of ectopic hormonal syndromes. Rec. Prog. Horm. Res. 25: 283-293. 20. Morgan, R. T., L. A. Quinn, L. K. Woods, and G. E. Moore. 1977. Marker properties of tmnor and lymphoid cell lines derived from a patient with squamous cell carcinoma. Cancer Res. 37: 2030-2035.
21. Gerwirtz, G., and R . S . Yalow. 1974. Ectopic ACTH production in carcinoma of the lung. J. Clin. Invest. 53: 1022-1032. 22. Leibovitz, A., J. C. Stinson, W. B. McCombs, III, C. E. McCoy, K. C. Mazui', and N. D. Mabry. 1976. Classification of human colorectal adenocarcinoma cell lines. Cancer Res. 36: 4562-4569. 23. Drewinko, B., M . M . Romsdahl, L.Y. Yang, M. J. Ahearn, and J. M. Trujillo. 1976. Establishment of a human carcinoembryonic antigenproducing colon adenocarcinoma cell line. Cancer Res. 36: 467-475.
A TRANSITIONAL
C E L L C A R C I N O M A C E L L L I N E 1~2
GEORGE E. MOORE, 3 ROBERT T. MORGAN, LAURIE A. QUINN, ANDLINDA K. WOODS
Department o/Surgery, Denver General Hospital, IV. 8th Avenue and Cherokee Street, Denver, Colorado 80204 SUMMARY
A transitional cell carcinoma cell line, COLO 232, was derived from a primary urinary bladder tumor in a Caucasian male. In culture, COLO 232 retained distinct uroepithelial phenotypic traits and produced both carcinoembryonic antigen and adrenocorticotropic hormone. COLO 232 had a chromosome mode of 58 and retained the X and Y chromosomes. Ten marker chromosomes were identified. COLO 232 will be of value for biochemical and immunological studies.
Key words: transitional cell carcinoma; human cell line; karyotype; adrenocorticotropic hormone; carcinoembryonic antigen.
INTRODUCTION Well characterized human tumor cell lines have been useful for numerous studies of malignant diseases and tumor-associated antigens. In this report, a new tumor cell line derived from a patient with transitional cell carcinoma of the urinary bladder is described. Case report. A 70-year-old Caucasian male had undergone a transurethral resection of the bladder for a tumor and an ileal loop diversion followed by a course of 4000 rads in late 1975. On January 8, 1976, a radical cystectomy and urethrectomy for stage II B, grade 3 transitional cell carcinoma of the bladder was performed. Tissue from this surgical procedure was submitted for tissue culture. The laboratory findings of the patient's status were unremarkable except for an elevated Ca +§ level (12.2 mg%). During the next 10 months, serum calcium level reached a high of 14 mg% and the patient died on October 23, 1976. The donor's ABO blood group was A-t-.
MATERIAL AND METHODS
Cultures of the transitional cell carcinoma tissue were initiated by finely mincing the tumor 1Presented in part at the 28th Annual Meeting of the
Tissue Culture Association, June 7, 1977. ZThis work was supported by Grant No. CA 15018 awarded by the National Cancer Institute, DHEW, and the Mary B. and L. H. Marshall Fund. 3To whom requests for reprints should be addressed.
specimen in 2-ml volumes of R P M I 1640 medium (1}, G E M 1717 medium (2), and H a m ' s medium F-12 ~3}; each supplemented with 10% heatinactivated fetal bovine serum (FBS}, penicillin (100 U per ml), and streptomycin (50 ~g per mB. The tumor tissue suspensions were seeded to 4-oz. flint-glass culture bottles. The bottles were loosely capped and incubated at 37 ~ C in humidified air with 10% CO2. Weekly, 2 to 5 ml fresh medium was added to each culture bottle until cell growth stabilized. The criteria for establishment of the cultured tumor cells as a permanent cell line were: (a~ monolayer outgrowth from the tissue minces reached semiconfluency; (bj the cells were successfully subcultured several times; and {c~ absence of fibroblasts or other cell types. For subculture, cultured tumor cells were dispersed by scraping the monolayer growth with a rubbertipped rod and seeding the cell suspension into equal volumes of fresh culture medium and spent medium. Successful subcultures were split 1:2 at 2-wcek intervals for over I year. Serial morphologic observations by phase microscopy were made of subcultures growing on cover slips. The electron-microscopy method was summarized previously i4). For chromosome preparations, 7-day-old subcultures of the cultured tumor cells were incubated 5 hr at 37 ~ C with 0.1 /~1 Colcemid (Ciba Pharmaceuticals, Summit, N.J.~ per 10 ml medium. The subcultures were scraped with a
301
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rubber-tipped rod and the resultant cell suspensions were centrifuged, resuspended in 0.075 M KCI for 12 min, centrifuged, resuspended in fixative (3:1 methanol: acetic acid) for 10 min, centrifuged, and resuspended in fresh fixative for an additional 10 min. Slides were made according to the hot-plate method of Lubs, et al. {5). Giemsabanded chromosomes were obtained using a modification of the method of Seabright (6}. Slides were dipped in a trypsin {1:250; Difco, Detroit, Mich.) buffered-saline solution (pH 6.8) for 30 to 90 sec and stained in a buffered (pH 6.8) 2% Giemsa solution (Harleco, Philadelphia, Pa.). Constitutive heterochromatin banding was obtained using a modification of the method of McKenzie and Lubs (7). Slides that were used to obtain Giemsa-banded preparations were incubated at 60 ~ C in 2X SSC (0.3 M NaCI, 0.03 M Na Citrate) for 10 to 20 min and stained in a buffered (pH 6.8} 5% Giemsa solution. A carcinoembryonic antigen (CEA) kit (Roche Diagnostics, Nutley, N.J. } was used to assay: {a) 16-day-old spent R P M I 1640 medium supplemented with 10% FBS from a culture of 2.1 x 106 total live tumor cells; (b} the supernates from freeze-thaw lysed cells of the same culture; and (c) complete R P M I 1640 medium supplemented with 10% FBS without exposure to tumor cells. Since the patient died with hypercalcemia, spent R P M I 1640 medium with 10% FBS recovered after 10 days from actively growing tumor cells and a control sample of fresh medium were assayed for parathyroid hormone (PTH) by Dr. S. Krutzik, Nichols Institute, San Pedro, Calif. The P T H radioimmunoassay procedure (8) detects immunoreactive P T H in samples utilizing highly purified [12sI]-labeled antisera raised against intact bovine P T H . A serum pool obtained from patients with hyperparathyroidism serves as reference standard. Glucose-6-phosphate dehydrogenase (G6PD) isoenzyme mobility was determined with a cellulose-acetate electrophoresis kit obtained from Helena Laboratories, Beaumont, Tex. Cultured tumor cells t10 7} were lysed by freeze-thaw in 1 ml 0.9% NaCI solution and the supernate was assayed for G6PD isoenzymes. Adrenocorticotropic hormone tACTH} assays were performed at the University of Colorado Medical Center, Denver, Colo., on 14-day-old spent R P M I 1640 medium with 10% FBS from actively growing cultures and on complete medium without exposure to the cultured cells. The
A C T H was assayed by the method of Eipper and Mains {9}. For heterologous transplantation of the cultured tumor cells, five female C 3 H / H e J mice were irradiated with 600 R, and each mouse was injected subcutaneously with 106 viable tumor cells in 0.1 ml saline. When palpable nodules were observed, they were removed surgically and prepared for histological studies. To determine plating efficiency, 1-ml suspensions of 10s viable tumor cells per ml R P M I 1640 medium with 10% FBS were plated in each of six Petri dishes {35-mmL After 18 hr, all nonadherent cells in the dishes were removed with a phosphate-buffered saline {PBS} rinse. The remaining cells were removed with 0.025% trypsin IGrand Island Biological Co., Grand Island, N.Y.}. The subsequent cell suspensions were centrifuged, resuspended in 0.1 ml PBS, and counted in a hemacytometer. For colony formation in soft agar, equal volumes of a 1% agar ~Grand Island Biological Co. } and a 2X concentration of R P M I 1640 medium with 20% FBS were mixed. An aliquot of 5 ml of the agar-medium mixture was plated into 60- by 15-mm plastic Petri dishes. A 1-ml cell suspension of 104 viable tumor cells were mixed thoroughly with 4.5 ml of a 0.67% solution agarose lGrand Island Biological Co. } and 4.5 ml of a 2X concentration R P M I 1640 medium with 20% FBS. Two ml of the tumor cell agarose-medium suspension then was layered carefully on the hardened agar base. The agarose cultures were incubated at 37 ~ C in humidified air with 10% CO2. After 1 week incubation, colony cell counts were made and efficiency for growth in soft agar calculated. The cuhures were tested for pleuropneumonialike organism ~PPLO} contamination by the agarbroth method 14} and the bisbenzamide staining method (10L The cell line was preserved for the cell bank by slow freezing cell suspensions in R P M I 1640 medium supplemented with 20% FBS, 12% dimethylsulfoxide, penicillin and streptomycin. Each ampul, containing approximately 8 x 106 cells, was stored at - 8 5 ~ C {11 }. RESULTS COLO 232 was established from the primary tumor tissue on February 3, 1976, in F-12 medium supplemented with 10% FBS. COLO 232 is maintained in either R P M I 1640 or G E M 1717 medium supplemented with 10% FBS. An additional cell line was established in G E M 1717 medium with 10% FBS and designated COLO 229.
BLADDER CARCINOMA CELL LINE
FIG. 1. Phase-contrast photomicrograph of COLO 232. x182. COLO 229, which shares similar marker properties with COLO 232, has not been fully characterized.
303
COLO 232 grew as muhilayered, compact, epithelial-like colonies with a cell doubling time of 48 hr. Serial phase microscopic studies revealed uniform, small taverage 15/am in diameter), polygonal cells with occasional multinucleated giant cells (Fig. 1). Nuclei were large and round with multiple large nucleoli. Prominent "intercellular bridges" spanned wide intercellular spaces. Numerous small refractile vesicles were contained within the cytoplasm. Electron micrographs showed polygonal neoplastic cells with scattered microvillar protrusions. Demosomal attachment plaques were numerous. Tonofibrils, evident in most cells, predominated in the perinuclear region with some scattered randomly in the cytoplasm (Fig. 2). The cytoplasm of some of the cultured tumor cells contained few organelles, whereas others contained numerous mitochondria, abundant free polyribosomes and dilated endoplasmic retriculum. Inclusion bodies and glycogen granules were observed along with fusiform vesicles and residual bodies of varying size. Golgi apparatus was observed in the cells, although not well developed. No phagosomes were observed. The cultured tumor cells had the distinctive asymmetric cell membrane reported for the surface cells of mammalian uroepithelium i12). No virallike particles were observed.
FIG. 2. Ultrastructttre of COLO 232 showing tonofibrils, desmosome and asymmetric angular plasma membrane, x31,000.
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FIG. 3. Giemsa-banded marker chromosomes of COLO 232 compared to normal chromosomes from the same cell show probable deviation of the markers, x2400. Chromosome analyses of COLO 232 were made No detectable amount of P T H was found in form 89 metaphase spreads; 41 of these spreads either the spent medium from COLO 232 or the were photographed and 11 karyotyped. The control fresh medium. The detectable limit of the chromosome mode was 58 with a range of 53 to assay was 25/~1 Eq per ml. 59. In all metaphases analyzed, the X and Y Analyses of the freeze-thaw lysate supernate chromosomes were present. No unusual polymor- from COLO 232 for CEA showed a concentration phisms were noted. The marker chromosomes obof 14.4 ng per 106 cells and 32.4 ng per ml in the served are shown in Fig. 3 and were identified as spent medium. No CEA was detected in complete follows: R P M I 1640 medium with 10% FBS. ACTH levels in spent R P M I 1640 medium M, i(1)(qter-~cen-~qter) with 10% FBS from COLO 232 was 4.0 ng per ml M2 t(1 ;7)(1 p t e r - ' l p2? :: 7q3 ?--'7pter) and zero for the control fresh medium. M3 del,t(1;7)(1 pter-~l p2? ::7q3?--'7pl? :) COLO 232 was positive for type-B isoenzyme of M4 del(3)(pter-'pl2:) G6PD. Ms i(5)(pter~cen~pter) Tumors were induced at the inoculation site of M_~ dic(7;9)(7pter-~7p2 ? : :9q2 ?-~9qter) COLO 232 in irradiated C 3 H / H e J mice. HistoM, t(8;19)(8pter-~8ql?:: 19) logic studies revealed that the tumor nodules were Ms t(ll;?)(1 l p t e r - ' l lq2?::?) malignant cells, morphologically similar to a difM9 t( 14; .9)( 14qter~cen ~ .9) ferentiated bladder carcinoma. M~o i(21 )(qter-~cen-~qter) The plating efficiency for COLO 232 was 65%. Five additional abnormal chromosomes of unCOLO 232 grew in soft agar with a 34% effiknown origin were occasionally observed in ciency. COLO 232 cells. COLO 229 has not been extenThe cell line has been maintained in culture sively characterized cytogehetically, hut some since initiation and aliquots have been frozen for markers (M1, Ms, Ms) were common to both cell the cell bank. The cell line has been retrieved suclines. cessfully from the cell bank.
BLADDER CARCINOMA CELL LINE Mycoplasma was detected in COLO 232 cells by the bisbenzamide method. COLO 232 was negative for PPLO contamination by the agarbroth and orcein techniques. The cell line was treated for mycoplasma contamination with tetracycline-HCl I13 }. D ISCUSSION To date, few established transitional cell carcinoma (TCC~ cell lines have been characterized (14-18}. This report concerns a new TCC cell line, COLO 232, established from a primary tumor specimen. After 1 year in culture, COLO 232 retained a well differentiated morphology that was representative of TCC. Epithelial morphology was observed with phase-contrast microscopy. The distinct uroepithelial phenotypic traits of desmosomes, microvillar projections, thick angular plasma membrane and cytoplasmic fusiform vesicles were observed in electron micrographs. Cytogenetic analysis of COLO 232 revealed that 10 marker chromosomes were present in more than 85% of the metaphases. These markers do not appear to be present in banded karyotypes of TCC cell lines reported in the literature (14,18j. Also, the complement of markers does not appear to be the same as any other banded karyotype of a human cell line. Malignant cells have been reported to produce cell products that are not normally produced by the organ from which the tumor originated I19~. Since the patient had hypercalcemia at death and two of our reported squamous cell carcinoma cell lines ~4,20) produced P T H , COLO 232 was assayed for PTH. No P T H was detected. Ectopic ACTH production has been reported in lung and pancreatic tumors ~21 ~. COLO 232 produced a detectable amount of ACTH in vitro. Further studies of hormone production by TCC cell lines may have implications for the future diagnosis and treatment of this disease. CEA has been detected in squamous cell carcinoma cell lines (4,20j and colon carcinoma cell lines ~22,23L COLO 232 elaborated appreciable amounts of CEA in the culture medium. This cell line may be useful in studies of the role of CEA in malignancies, and is available to other investigators for their study. REFERENCES 1. Moore, G. E., R. E. Gerner, and H. A. Franklin. 1967. Culture of normal human leukocytes. J. Am. Med. Assoc. 199: 519-524.
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2. Woods, L.K., G.E. Moore, C.J. Bainbridge, C.C. Huang, C. Huzeila, and L. A. Quinn. 1973. Lymphoid cell lines established from peripheral blood of persons with Down's syndrome. N.Y. State J. Med. 73: 869-873. 3. Ham, R. G. 1965. Clonal growth of mammalian cells in a chemically defined synthetic medium. Proc. Nat. Acad. Sci. U.S.A. 53: 288-293. 4. Moore, G. E., S. B. Merrick, L. K. Woods, and N. M. Arabasz. 1975. A human squamous cell carcinoma cell line. Cancer Res. 35: 2684-2688. 5. Labs, H. A., W. H. McKenzie, S. R. Patil, and S. Merrick. 1973. New staining methods for chromosomes. In: D. M. Prescott (Ed. j, Methods in Cell Biology. Vol. 6. pp. 346-380. 6. Seabright, M. 1971. A rapid banding technique for human chromosomes. Lancet 2: 971-972. 7. McKenzie, W.H., and H.A. Labs. 1973. An analysis of technical variables in the production of C bands. Chromosoma 41:175-182. 8. Krutzik, S. 1976. Radioimmunoassay Manual. 3rd Edition. Nichols Institute, San Pedro, Calif., pp. 209-220. 9. Eipper, B.A., and R.E. Mains. 1975. High molecular weight forms of adrenocorticotropic hormone in the mouse pituitary and in a mouse tumor cell line. Biochemistry 14: 3836-3844. 10. Chen, T. R. 1975. Microscopic demonstration of mycoplasma contamination in cell cultures and cell culture media. Procedure 75361. TCA Manual 1: 229-232. 11. Mizrahi, A., and G. E. Moore. 1971. Long-term preservation of permanent human hematopoietic cell lines. J. Med. 2: 380-386. 12. Firth, J. A., and R. M. Hicks. 1973. Interspecies variation in the fine structure and enzyme cytochemistry of mammalian transitional epithelium. J. Anat. 116: 31-43. 13. Quinn, L. A., L. K. Woods, S. B. Merrick, N. M. Arabasz, and G. E. Moore. 1977. Cytogenetic analysis of 12 human malignant melanoma cell lines. J. Nat. Cancer Inst. 59: 301-307. 14. O'Toole, C., S. Mayak, Z. Price, W. H. Gilbert, and J. Waisman. 1976. A cell line ISCaBERj derived from squamous cell carcinoma of the human urinary bladder. Int. J. Cancer 17: 707-714. 15. Elliot, A. Y., P. Cleveland, J. Cervenka, A.E. Castro, N. Stein, T . R . Hakala, and E.E. Fraley. 1974. Characterization of a cell line from human transitional cell carcinoma of the urinary tract. J. Nat. Cancer Inst. 53: 1341-1349. 16. Rigby, C. C., and L. M. Franks. 1970. A human tissue culture cell line from a transitional cell tumor of the urinary bladder: Growth, chromosome pattern and ultrastructure. Br. J. Cancer 24: 746-754. 17. Babenik, J., M. Baresova, V. Viklicky, J. Jakoubkova, H. Sainerova, and J. Donner. 1973. Established cell line of urinary bladder carcinoma ~T24) containing tumor-specific antigen. Int. J. Cancer 11: 765-773. 18. Rasheed, S., M.B. Gardner, R.W. Rongey, W.A. Nelson-Rees, and P. Arnstein. 1977. Human bladder carcinoma: Characterization of two new tumor cell lines and search for tumor viruses. J. Nat. Cancer Inst. 58: 881-890.
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19. Liddle, G . W . , W . E . Nicholson, D . P . Island, D. N. Orth, K. Abe, and S. C. Lowder. 1969. Clinical and laboratory studies of ectopic hormonal syndromes. Rec. Prog. Horm. Res. 25: 283-293. 20. Morgan, R. T., L. A. Quinn, L. K. Woods, and G. E. Moore. 1977. Marker properties of tmnor and lymphoid cell lines derived from a patient with squamous cell carcinoma. Cancer Res. 37: 2030-2035.
21. Gerwirtz, G., and R . S . Yalow. 1974. Ectopic ACTH production in carcinoma of the lung. J. Clin. Invest. 53: 1022-1032. 22. Leibovitz, A., J. C. Stinson, W. B. McCombs, III, C. E. McCoy, K. C. Mazui', and N. D. Mabry. 1976. Classification of human colorectal adenocarcinoma cell lines. Cancer Res. 36: 4562-4569. 23. Drewinko, B., M . M . Romsdahl, L.Y. Yang, M. J. Ahearn, and J. M. Trujillo. 1976. Establishment of a human carcinoembryonic antigenproducing colon adenocarcinoma cell line. Cancer Res. 36: 467-475.
