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Dunning Adenocarcinoma in Tissue Culture: Isolation of a Cloned Cell Line, R3327H-G8-A1
0022-5347 /82/1274-0823 ,Vol. :27 Printed in
THE ,JOURNAL OF UROLOGY
Copyright© 1982 by The Williams & Wilkins Co.
1
DUNNING ADENOCARCINOMA IN TISSUE CULTURE: ISOLATION OF A CLONED CELL LINE, R3327H-G8-Al MARY ANN SESTILI, *t JAMES S. NORRIS,:j: LARRY I. LIPSCHULTZ AND ROY G. SMITH§ From the Division of Urology, The University of Texas Medical School, Houston, Texas
ABSTRACT
A cloned cell line, R3327H-G8-Al, was isolated from an explant of the well differentiated androgen dependent Dunning R3327H adenocarcinoma. Preliminary characterization of this cell line indicates that it is epithelial-like, and that it synthesizes and secretes large quantities of acid phosphatase. The cells bind testosterone in a saturable manner with an equilibrium dissociation constant of 0.5 nM and with a capacity of 5000 sites/cell. When these cells were injected subcutaneously into the hind flank of Copenhagen-Fischer rats, or into the dorsal or ventral lobes of the prostate, large tumors were produced 3 months following administration, thus demonstrating the tumorigenicity of the cells. Prostate cancer is the third most frequent cause of cancer death in American men over the age of 55, surpassed only by cancer of the lung and colon-rectum.' There are many questions and controversies surrounding prostate cancer which include etiology 2 and efficacy of types and times of treatment including endocrine manipulation. 3- 5 Variability of the cancer itself is the basis of many of these controversies, since variations are found in both morphologic and biochemical indices. 6 One way to resolve these questions is to develop a model system which permits investigation of the basic biology of the tumor. Although tumor models such as the Noble,7 The Pollard GermFree Wistar Rat 8 and the AXC Rat9 exist, we have selected the Dunning tumor 10 for our investigations, because it is well documented that its morphologic, biochemical and growth characteristics closely mimic those of the human. 11 - 13 In this preliminary report, we describe the isolation and characterization of a cloned cell line, R3327H-G8-Al, derived from the Dunning R3327H adenocarcinoma from a Copenhagen X Fischer F 1 rat. This cell line can serve as a valuable model for prostate cancer since it is derived from 1 cell type and is easily manipulated. Although criticism can be made regarding results obtained from manipulations of cells in culture with regard to the simulation of in vivo events, the only method of investigating the properties of a single cell type is through cloning. A more important criticism is perhaps that a cell's function can be altered during its time in culture. Both drawbacks will be investigated by studying the growth, histology and biochemistry of the tumor resulting from implantation of the cloned cells into Copenhagen-Fischer rats. Comparisons of the effects of various agents on the cells both in vitro and in vivo can then be made. MATERIALS AND METHODS
Tumor explant. A Dunning R3327 tumor (defined R3327H according to reference 12) obtained from Dr. Altman, Miami, Florida, which later resulted in the R3327H-G8-Al cell line, was explanted into culture. Media used to establish the cell line consisted of Ham's F-12 with 10 per cent fetal bovine serum, l Accepted for publication September 30, 1981. * Supported by the National Cancer Institute, Division of Resources Center and Community Activities under an Intergovernmental Personnal Act (IP A) agreement assignment. t Current address: National Cancer Institute, Blair Building, Bethesda, Maryland 20205. :j: Departments of Medicine and Physiology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72201. § Requests for reprints: The University of Texas Medical School, Division of Urology, 6431 Fannin, Suite 6018, Houston, Texas 77030. 823
per cent penicillin-streptomycin (all from GIBCO), 10-s M testosterone (T) and 10-9 M 17 ,8-estradiol (E 2 ) (from Steraloids). Cells were cloned first with microtiter plates and the above media and further cloned in soft agar as described by McPherson. 14 The largest colony was used in this investigation and was defined as the R3327H-G8-Al. Cell cultures. Cell line R3327H-G8-Al was routinely grown in Falcon Flasks (75 cm. 2 ) with Dulbecco's modified Eagle's medium (MEM), 10 per cent horse serum, and 1 per cent penicillin-streptomycin in a humidified 5 per cent air atmosphere at 37C. Subcultures were made with 0.25 per cent trypsin in Hanks' balanced salt solution (BSS). All reagents were obtained from GIBCO. Animals. Male Copenhagen X Fischer F, rats were obtained from the Papanicolaou Cancer Research Institute in Miami, Florida. At the 7th week of birth, each animal was implanted either subcutaneously in the right hind flank or in the dorsal or ventral lobes of the prostate with 10 6 R3327H-G8-Al cells which had been suspended in Hanks' BSS (0.2 ml.). Animals were housed for 3 months prior to removal of tumors. Acid phosphatase. Acid phosphatase was assayed in cultured media and sonicated cells by the procedure of Roy and associates15 with the Dupont ACA system. Monolayers of R3327HG8-Al cells and rat kidney fibroblasts, used as controls, were scraped in 3 ml. Dulbecco's phosphate buffered saline (PBS, pH 7.4) with a rubber policeman, centrifuged at 1000 X g for 10 minutes in a Sorvall RC-5 refrigerated centrifuge and the supernatant discarded. Cell pellets were resuspended in 5 ml. cold PBS and sonicated with a Cell Disruptor Model W-220F ultrasonic probe (Heat System-Ultrasonics, Inc., Plainview, Long Island, New York) at an output control setting of 4 for 30 seconds at 4C. Supernatants and media were stored at -20C for a maximum of 24 hours if not assayed immediately. Mycoplasm screening. Evidence for mycoplasm contamination was assayed with the Hoeschst compound #33258 fluorescent staining technique. No evidence of mycoplasm contamination was seen when cells were examined by fluorescent microscopy. No fluorescence was seen in the cell cytoplasm or background. Testosterone binding by R3327H-G8-Al cells. Approximately 5 X 105 cells were plated into 4 60 mm. Petri dishes/ assay point in the media described above. The cells were allowed to attain confluency which occurred in 48-72 hours. Twenty-four hours prior to assay, the media was removed and the cells were washed thoroughly with Hank's BSS. Dulbecco's MEM + 1 per cent stripped horse serum were then added to the monolayers which were allowed to incubate for 24 hours.
824
SESTILI AND ASSOCIATES
At the time of assay this media was removed, the cells were washed with Hanks' BSS and then washed with serum free Dulbecco's MEM. Varying concentrations of radiolabeled testosterone (0.25-3 nM) in Dulbecco's MEM were added to the assay dishes. To estimate nonspecific binding a 100-fold excess of radioinert testosterone was added to duplicate dishes of cells. The Petri dishes were then incubated for 1 hour at 37C in humidified 5 per cent C02/95 per cent air incubator. Upon completion of incubation, the dishes were placed in ice, the media removed by aspiration and the cells washed (3 X 3 ml.) with ice-cold Dulbecco's phosphate buffered saline (PBS pH 7.4). The monolayers were then disrupted in Dulbecco's PBS with a rubber policeman, centrifuged at 1000 X g for 5 minutes at 2C and the supernatant discarded. Absolute ethanol (3 ml.) was then added to the pelleted cells, vortexed, and allowed to extract for 18 hours at room temperature. Finally, the cells were pelleted by centrifugation and the supernatant was counted in 10 ml. Scintiverse to measure incorporation of [3H]testosterone. To determine the specificity of [3H]testosterone binding, a similar incubation procedure was used in the presence of a 100fold excess of radioinert estradiol, diethylstilbestrol, dihydrotestosterone and cortisol. DNA analysis was performed on each of the samples of pelleted cells with the method of Burton. 16 RESULTS
At least 6 months elapsed between the time the initial culture was explanted and the time that viable cells were obtained. This type of phenomenon has been seen in other attempts to culture prostate cells. 17 Eventually quantities of cells, as seen in figure 1, were sufficient for cloning and serial passage. The G8-Al cell line normally grows as a continuous monolayer (fig. 2). If left undisturbed, except for media changes, the monolayers become confluent and eventually form tightly packed 3-dimensional colonies. The cells, viewed by phase contrast microscopy, appear epithelial-like and are joined to one another on all sides without any apparent intercellular spaces. All 10 of the rats implanted subcutaneously with 106 G8-Al cells into their hind flank formed subcutaneous tumors which remained localized and encapsulated within the subcutaneous tissue. By 3 months the average tumor size was 2 X 1.5 cm. and the average weight was 4.1 (± 2. 7) gm. Cells were also implanted in the prostate of Copenhagen X Fischer rats, using a lower mid-line incision, and under asceptic conditions the abdomen was entered and the bladder exposed. Inferior and anterior lateral to the bladder neck both the ventral prostate and the
Fm. 2. Phase contrast photomicrographs of R3327H-G8-Al cells in 30th passage grown as a monolayer. Cells appear as broad, flat, polygonal epithelial cells which are joined to one another on all sides without any apparent intercellular spaces. Reduced from xlOO.
Levels of acid phosphatase in media and sonicated cells measured by the Dupont ACA method Acid Phosphatase (IU/5 ml. from 106 cells)
Sample Media R3327H (GS-Al) Rat kidney fibroblasts Control media Sonicated cells R3327H (GS-Al) Rat kidney fibroblasts
0.70 0.05 0.01 6.0 0.8
dorsal prostate glands were identified. Each was then injected with a cell suspension containing 107 tumor cells per ml. with a no. 24 gauge hypodermic needle and a tuberculin syringe. Approximately 0.1 ml. of cell suspension was injected per gland. The abdomen was then washed with sterile saline and the incision closed with a single layer of 2-0 silk suture. After 12 weeks large masses were observed in the prostate whether G8Al cells were injected into either the dorsal or ventral prostatic lobes. Acid phosphatase was chosen as the marker protein. When 5 ml. of media from 106 R3327H-G8-Al cells were assayed for acid phosphatase and compared to media from rat kidney fibroblasts, there was approximately a 14-fold increase in the media from R3327H-G8-Al cells. Sonicated G8-Al cells show at least a 7-fold increase of acid phosphatase compared to sonicated rat kidney fibroblasts (table). Scatchard analysis on data derived from the whole cell uptake of [3 H]testosterone demonstrated a single class of binding sites of high affinity saturable binding having an equilibrium dissociation constant (Kd) of 0.5 nM. The concentration of the binding sites was 5000/cell. Specificity studies demonstrated that unlabeled 5 a-dihydrotestosterone at a 10-fold excess was as effective as unlabeled testosterone in displacing 50 per cent of [3H]testosterone from the cells. No displacement could be demonstrated using a 10-fold excess of estradiol, diethylstilbestrol, progesterone or cortisol. DISCUSSION
Fm. 1. Phase contrast photomicrographs of cells from R3327H tumor explant before cloning in microtiter plates. Both attached epithelial-like and mitotic cells can be seen. Reduced from xlOO.
We report the isolation and preliminary characterization of a cloned tissue culture cell line, R3327H-G8-Al, which was derived from the Dunning R3327H prostate adenocarcinoma. The G8-Al cell line grows as a continuous monolayer with epithelial like characteristics when viewed by phase contrast microscopy. The fact that the cells form domelike structures when left undisturbed indicates very strongly that they are
1SOL.A~_T:ION OF {JLONED CELL LII~E
epithelial since this condition has been defined others as a characteristic found only in epithelial cells. 18 Neoplastic characteristics of the cell line is verified since tumors are produced following implantation of R3327H-G8-Al cells into host animals. From studies by Kutscher 19 and Gutman20 acid phosphatase is considered to be a marker for prostatic carcinoma; consequently, it was the selected marker protein for this cell line. Indeed, our results show that the GS-Al cells are synthesizing large quantities of acid phosphatase when compared to rat kidney fibroblasts as a control, and that even larger amounts of the enzyme are synthesized and then secreted into the environment, i.e., the media. We have also demonstrated that the cells contain high affinity binding characteristics for testosterone specific binding. This is again similar to that described for the R3327H tumor 12 from which the cell line was cloned. The R3327H-G8-Al clone has maintained through 70 serial passages many of the characteristics of the R3327H tumor from which it was derived. The cells are epithelial-like, tumorigenic, synthesize and secrete acid phosphatase, and bind testosterone in a saturable manner with high affinity (Ki = 0.5 nM) and high specificity. In the future it is planned to conduct in vitro and in vivo growth studies, and more extensive biochemical studies including complete steroid receptor analyses of both the GS-Al cell line and the tumors which result from their implantation into host animals. We also intend to define the epithelial characteristics of the cell line using electron microscopy. It is our belief that the R3327H-G8-Al cell line has potential as a valuable in vitro model to answer basic biologic questions associated with prostate carcinoma. The cell system is derived from a single cell, is easily manipulated for controlled studies, and can be grown in large quantities even after freezing and thawing. It is also an especially useful system since it is derived from the Dunning tumor which has been acclaimed as appropriate to study problems related to prostate cancer in humans. Acknowledgments. We are grateful to Dr. Herb Fritzche, M.D. Anderson Hospital, Houston, for his assistance with the acid phosphatase assays.
4.
5. 6. 7.
8. 9. 10. 11.
12.
13. 14.
15.
16. 17.
18. REFERENCES
1. Garfinkel, L., Poindexter, C. E. and Silverberg, E.: Cancer Statistics
1980. C. A., 31: 13, 1981. 2. Franks, L. M.: Etiology, epidemiology and pathology of prostatic cancer. Cancer, 32: 1092, 1973. 3. Nesbit, R. M. and Baum, W. C.: Endocrine control of prostate
19. 20.
carcinoma: clinical and statistical survey of 1,818 cases. J. A. M A., 143: 1317, 1950. Byar, D. P. and the Veterans Administration Cooperative Urological Research Group: Survival of patients with incidentally found microscopic cancer of the prostate: results of a clinical trial of conservative treatment. J. Urol., 108: 908, 1972. Scott, W. W.: Rationale and results of primary endocrine therapy in patients with prostatic cancer. Cancer, 32: 1119, 1973. Sandberg, A. and Gaunt, R.: Model systems for studies of prostate cancer. Seminars in Oncology, 3: 177, 1976. Noble, R. L.: The development of prostatic adenocarcinoma in the Nb rat following prolonged sex hormone administration. Cancer Res., 37: 1929, 1977. Pollard, M.: Spontaneous prostate adenocarcinomas in aged germfree Wistar rats. J. Natl. Cancer Inst., 51: 1235, 1973. Shain, S., McCullough, B. and Segaloff, A.: Spontaneous adenocarcinomas of the ventral prostate of aged AxC rats. J. Natl. Cancer Inst., 55: 177, 1975. Dunning, W. F.: Prostate cancer in the rat. Natl. Cancer Inst. Mongr., 12: 351, 1963. Smolev, J. K., Heston, W. D. W., Scott, W.W. and Coffey, D.S.: Characterization of the Dunning R3327H adenocarcinoma: an appropriate model for prostatic cancer. Cancer Treatment Reports, 61: 273, 1977. Isaacs, J. T., Heston, W. D. W., Weissman, R. M. and Coffey, D.S.: Animal models of the hormone-sensitive and insensitive prostatic adenocarcinomas, Dunning R-3327-H, R3327-HI, and R3327-AT. Cancer Res., 38: 4353, 1978. Lubaroff, D. M., Canfield, L. and Reynolds, C. W.: The Dunning tumors. In: Models for Prostate Cancer. Edited by G. P. Murphy. New York: Alan R. Liss, Inc., p. 133, 1980. MacPherson, I. N.: In: Tissue Culture Methods and Applications. Edited by P. Kruse, Jr. and M. Patterson, Jr. New York: Academic Press, p. 267, 1973. Roy, A. V., Brower, M. E. and Hayden, J. E.: Sodium thymolphthalein monophosphate: a new acid phosphatase substrate with greater specificity for the prostatic enzyme in serum. Clin. Chem., 17: 1093, 1971. Burton, K. H.: A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of DNA. Biochem. J., 62: 315, 1956. Norris, J. S., Bowden, C. and Kohler, P. 0.: Description of a new hamster ventral prostate cell line containing androgen receptors. In vitro, 13: 108, 1977. Lever, J. E.: Inducers of mammalian cell differentiation stimulate dome formation in a differential kidney epithelial cell line (MDCK). Proc. Nat. Acad. Sci. U.S.A., 76: 1323, 1979. Kutscher, W. and Wolbergs, H.: Prostataphosphatase. Z. Physiol. Chem., 236: 237, 1935. Gutman, A. B. and Gutman, E. B.: An "acid" phosphatase occurring in the serum of patients with metastasizing carcinoma of the prostate gland. J. Clin. Invest., 17: 473, 1938.
THE ,JOURNAL OF UROLOGY
Copyright© 1982 by The Williams & Wilkins Co.
1
DUNNING ADENOCARCINOMA IN TISSUE CULTURE: ISOLATION OF A CLONED CELL LINE, R3327H-G8-Al MARY ANN SESTILI, *t JAMES S. NORRIS,:j: LARRY I. LIPSCHULTZ AND ROY G. SMITH§ From the Division of Urology, The University of Texas Medical School, Houston, Texas
ABSTRACT
A cloned cell line, R3327H-G8-Al, was isolated from an explant of the well differentiated androgen dependent Dunning R3327H adenocarcinoma. Preliminary characterization of this cell line indicates that it is epithelial-like, and that it synthesizes and secretes large quantities of acid phosphatase. The cells bind testosterone in a saturable manner with an equilibrium dissociation constant of 0.5 nM and with a capacity of 5000 sites/cell. When these cells were injected subcutaneously into the hind flank of Copenhagen-Fischer rats, or into the dorsal or ventral lobes of the prostate, large tumors were produced 3 months following administration, thus demonstrating the tumorigenicity of the cells. Prostate cancer is the third most frequent cause of cancer death in American men over the age of 55, surpassed only by cancer of the lung and colon-rectum.' There are many questions and controversies surrounding prostate cancer which include etiology 2 and efficacy of types and times of treatment including endocrine manipulation. 3- 5 Variability of the cancer itself is the basis of many of these controversies, since variations are found in both morphologic and biochemical indices. 6 One way to resolve these questions is to develop a model system which permits investigation of the basic biology of the tumor. Although tumor models such as the Noble,7 The Pollard GermFree Wistar Rat 8 and the AXC Rat9 exist, we have selected the Dunning tumor 10 for our investigations, because it is well documented that its morphologic, biochemical and growth characteristics closely mimic those of the human. 11 - 13 In this preliminary report, we describe the isolation and characterization of a cloned cell line, R3327H-G8-Al, derived from the Dunning R3327H adenocarcinoma from a Copenhagen X Fischer F 1 rat. This cell line can serve as a valuable model for prostate cancer since it is derived from 1 cell type and is easily manipulated. Although criticism can be made regarding results obtained from manipulations of cells in culture with regard to the simulation of in vivo events, the only method of investigating the properties of a single cell type is through cloning. A more important criticism is perhaps that a cell's function can be altered during its time in culture. Both drawbacks will be investigated by studying the growth, histology and biochemistry of the tumor resulting from implantation of the cloned cells into Copenhagen-Fischer rats. Comparisons of the effects of various agents on the cells both in vitro and in vivo can then be made. MATERIALS AND METHODS
Tumor explant. A Dunning R3327 tumor (defined R3327H according to reference 12) obtained from Dr. Altman, Miami, Florida, which later resulted in the R3327H-G8-Al cell line, was explanted into culture. Media used to establish the cell line consisted of Ham's F-12 with 10 per cent fetal bovine serum, l Accepted for publication September 30, 1981. * Supported by the National Cancer Institute, Division of Resources Center and Community Activities under an Intergovernmental Personnal Act (IP A) agreement assignment. t Current address: National Cancer Institute, Blair Building, Bethesda, Maryland 20205. :j: Departments of Medicine and Physiology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72201. § Requests for reprints: The University of Texas Medical School, Division of Urology, 6431 Fannin, Suite 6018, Houston, Texas 77030. 823
per cent penicillin-streptomycin (all from GIBCO), 10-s M testosterone (T) and 10-9 M 17 ,8-estradiol (E 2 ) (from Steraloids). Cells were cloned first with microtiter plates and the above media and further cloned in soft agar as described by McPherson. 14 The largest colony was used in this investigation and was defined as the R3327H-G8-Al. Cell cultures. Cell line R3327H-G8-Al was routinely grown in Falcon Flasks (75 cm. 2 ) with Dulbecco's modified Eagle's medium (MEM), 10 per cent horse serum, and 1 per cent penicillin-streptomycin in a humidified 5 per cent air atmosphere at 37C. Subcultures were made with 0.25 per cent trypsin in Hanks' balanced salt solution (BSS). All reagents were obtained from GIBCO. Animals. Male Copenhagen X Fischer F, rats were obtained from the Papanicolaou Cancer Research Institute in Miami, Florida. At the 7th week of birth, each animal was implanted either subcutaneously in the right hind flank or in the dorsal or ventral lobes of the prostate with 10 6 R3327H-G8-Al cells which had been suspended in Hanks' BSS (0.2 ml.). Animals were housed for 3 months prior to removal of tumors. Acid phosphatase. Acid phosphatase was assayed in cultured media and sonicated cells by the procedure of Roy and associates15 with the Dupont ACA system. Monolayers of R3327HG8-Al cells and rat kidney fibroblasts, used as controls, were scraped in 3 ml. Dulbecco's phosphate buffered saline (PBS, pH 7.4) with a rubber policeman, centrifuged at 1000 X g for 10 minutes in a Sorvall RC-5 refrigerated centrifuge and the supernatant discarded. Cell pellets were resuspended in 5 ml. cold PBS and sonicated with a Cell Disruptor Model W-220F ultrasonic probe (Heat System-Ultrasonics, Inc., Plainview, Long Island, New York) at an output control setting of 4 for 30 seconds at 4C. Supernatants and media were stored at -20C for a maximum of 24 hours if not assayed immediately. Mycoplasm screening. Evidence for mycoplasm contamination was assayed with the Hoeschst compound #33258 fluorescent staining technique. No evidence of mycoplasm contamination was seen when cells were examined by fluorescent microscopy. No fluorescence was seen in the cell cytoplasm or background. Testosterone binding by R3327H-G8-Al cells. Approximately 5 X 105 cells were plated into 4 60 mm. Petri dishes/ assay point in the media described above. The cells were allowed to attain confluency which occurred in 48-72 hours. Twenty-four hours prior to assay, the media was removed and the cells were washed thoroughly with Hank's BSS. Dulbecco's MEM + 1 per cent stripped horse serum were then added to the monolayers which were allowed to incubate for 24 hours.
824
SESTILI AND ASSOCIATES
At the time of assay this media was removed, the cells were washed with Hanks' BSS and then washed with serum free Dulbecco's MEM. Varying concentrations of radiolabeled testosterone (0.25-3 nM) in Dulbecco's MEM were added to the assay dishes. To estimate nonspecific binding a 100-fold excess of radioinert testosterone was added to duplicate dishes of cells. The Petri dishes were then incubated for 1 hour at 37C in humidified 5 per cent C02/95 per cent air incubator. Upon completion of incubation, the dishes were placed in ice, the media removed by aspiration and the cells washed (3 X 3 ml.) with ice-cold Dulbecco's phosphate buffered saline (PBS pH 7.4). The monolayers were then disrupted in Dulbecco's PBS with a rubber policeman, centrifuged at 1000 X g for 5 minutes at 2C and the supernatant discarded. Absolute ethanol (3 ml.) was then added to the pelleted cells, vortexed, and allowed to extract for 18 hours at room temperature. Finally, the cells were pelleted by centrifugation and the supernatant was counted in 10 ml. Scintiverse to measure incorporation of [3H]testosterone. To determine the specificity of [3H]testosterone binding, a similar incubation procedure was used in the presence of a 100fold excess of radioinert estradiol, diethylstilbestrol, dihydrotestosterone and cortisol. DNA analysis was performed on each of the samples of pelleted cells with the method of Burton. 16 RESULTS
At least 6 months elapsed between the time the initial culture was explanted and the time that viable cells were obtained. This type of phenomenon has been seen in other attempts to culture prostate cells. 17 Eventually quantities of cells, as seen in figure 1, were sufficient for cloning and serial passage. The G8-Al cell line normally grows as a continuous monolayer (fig. 2). If left undisturbed, except for media changes, the monolayers become confluent and eventually form tightly packed 3-dimensional colonies. The cells, viewed by phase contrast microscopy, appear epithelial-like and are joined to one another on all sides without any apparent intercellular spaces. All 10 of the rats implanted subcutaneously with 106 G8-Al cells into their hind flank formed subcutaneous tumors which remained localized and encapsulated within the subcutaneous tissue. By 3 months the average tumor size was 2 X 1.5 cm. and the average weight was 4.1 (± 2. 7) gm. Cells were also implanted in the prostate of Copenhagen X Fischer rats, using a lower mid-line incision, and under asceptic conditions the abdomen was entered and the bladder exposed. Inferior and anterior lateral to the bladder neck both the ventral prostate and the
Fm. 2. Phase contrast photomicrographs of R3327H-G8-Al cells in 30th passage grown as a monolayer. Cells appear as broad, flat, polygonal epithelial cells which are joined to one another on all sides without any apparent intercellular spaces. Reduced from xlOO.
Levels of acid phosphatase in media and sonicated cells measured by the Dupont ACA method Acid Phosphatase (IU/5 ml. from 106 cells)
Sample Media R3327H (GS-Al) Rat kidney fibroblasts Control media Sonicated cells R3327H (GS-Al) Rat kidney fibroblasts
0.70 0.05 0.01 6.0 0.8
dorsal prostate glands were identified. Each was then injected with a cell suspension containing 107 tumor cells per ml. with a no. 24 gauge hypodermic needle and a tuberculin syringe. Approximately 0.1 ml. of cell suspension was injected per gland. The abdomen was then washed with sterile saline and the incision closed with a single layer of 2-0 silk suture. After 12 weeks large masses were observed in the prostate whether G8Al cells were injected into either the dorsal or ventral prostatic lobes. Acid phosphatase was chosen as the marker protein. When 5 ml. of media from 106 R3327H-G8-Al cells were assayed for acid phosphatase and compared to media from rat kidney fibroblasts, there was approximately a 14-fold increase in the media from R3327H-G8-Al cells. Sonicated G8-Al cells show at least a 7-fold increase of acid phosphatase compared to sonicated rat kidney fibroblasts (table). Scatchard analysis on data derived from the whole cell uptake of [3 H]testosterone demonstrated a single class of binding sites of high affinity saturable binding having an equilibrium dissociation constant (Kd) of 0.5 nM. The concentration of the binding sites was 5000/cell. Specificity studies demonstrated that unlabeled 5 a-dihydrotestosterone at a 10-fold excess was as effective as unlabeled testosterone in displacing 50 per cent of [3H]testosterone from the cells. No displacement could be demonstrated using a 10-fold excess of estradiol, diethylstilbestrol, progesterone or cortisol. DISCUSSION
Fm. 1. Phase contrast photomicrographs of cells from R3327H tumor explant before cloning in microtiter plates. Both attached epithelial-like and mitotic cells can be seen. Reduced from xlOO.
We report the isolation and preliminary characterization of a cloned tissue culture cell line, R3327H-G8-Al, which was derived from the Dunning R3327H prostate adenocarcinoma. The G8-Al cell line grows as a continuous monolayer with epithelial like characteristics when viewed by phase contrast microscopy. The fact that the cells form domelike structures when left undisturbed indicates very strongly that they are
1SOL.A~_T:ION OF {JLONED CELL LII~E
epithelial since this condition has been defined others as a characteristic found only in epithelial cells. 18 Neoplastic characteristics of the cell line is verified since tumors are produced following implantation of R3327H-G8-Al cells into host animals. From studies by Kutscher 19 and Gutman20 acid phosphatase is considered to be a marker for prostatic carcinoma; consequently, it was the selected marker protein for this cell line. Indeed, our results show that the GS-Al cells are synthesizing large quantities of acid phosphatase when compared to rat kidney fibroblasts as a control, and that even larger amounts of the enzyme are synthesized and then secreted into the environment, i.e., the media. We have also demonstrated that the cells contain high affinity binding characteristics for testosterone specific binding. This is again similar to that described for the R3327H tumor 12 from which the cell line was cloned. The R3327H-G8-Al clone has maintained through 70 serial passages many of the characteristics of the R3327H tumor from which it was derived. The cells are epithelial-like, tumorigenic, synthesize and secrete acid phosphatase, and bind testosterone in a saturable manner with high affinity (Ki = 0.5 nM) and high specificity. In the future it is planned to conduct in vitro and in vivo growth studies, and more extensive biochemical studies including complete steroid receptor analyses of both the GS-Al cell line and the tumors which result from their implantation into host animals. We also intend to define the epithelial characteristics of the cell line using electron microscopy. It is our belief that the R3327H-G8-Al cell line has potential as a valuable in vitro model to answer basic biologic questions associated with prostate carcinoma. The cell system is derived from a single cell, is easily manipulated for controlled studies, and can be grown in large quantities even after freezing and thawing. It is also an especially useful system since it is derived from the Dunning tumor which has been acclaimed as appropriate to study problems related to prostate cancer in humans. Acknowledgments. We are grateful to Dr. Herb Fritzche, M.D. Anderson Hospital, Houston, for his assistance with the acid phosphatase assays.
4.
5. 6. 7.
8. 9. 10. 11.
12.
13. 14.
15.
16. 17.
18. REFERENCES
1. Garfinkel, L., Poindexter, C. E. and Silverberg, E.: Cancer Statistics
1980. C. A., 31: 13, 1981. 2. Franks, L. M.: Etiology, epidemiology and pathology of prostatic cancer. Cancer, 32: 1092, 1973. 3. Nesbit, R. M. and Baum, W. C.: Endocrine control of prostate
19. 20.
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