0013-7227/90/1276-2963$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society
Vol. 127, No. 6 Printed in U.S.A.
Effect of Transforming Growth Factor-/?i on Proliferation and Death of Rat Prostatic Cells* PAULA MARTIKAINENf, NATASHA KYPRIANOU, AND JOHN T. ISAACS The Oncology Center and the Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231
ABSTRACT. The ability of transforming growth factor Bi (TGF/3!) to inhibit proliferation and activate death of rat ventral prostatic glandular cells was tested both in vivo and in vitro. In vivo administration of 50 ng TGF/Vday directly to the regressed ventral prostate of previously castrated male rats had no effect on the proliferative regrowth of the prostatic glandular cells induced by exogeneous androgen replacement. In addition, androgen-stimulated ventral prostatic cell proliferation in vitro in organ culture was not affected by exposure to 0.1-20 ng/ml TGF/?!. In contrast in vivo administration of 50 ng TGF/Vday directly to the ventral prostate of intact noncastrated male rats resulted in the death of about 25% of the prostatic glandular cells within 7 days of treatment. Such TGFjSi treatment did not lower serum testosterone, nor did it affect the size or DNA
P
ROSTATIC androgen levels decrease rapidly in the male rat after castration (1). This decrease produces profound changes within the ventral prostate, resulting in the rapid regression of the gland (2, 3). This rapid regression is due to a decrease in the rate of prostatic glandular cell proliferation coupled with an increase in the rate of prostatic glandular cell death (2-4). This latter death process is initiated when prostatic androgen levels are decreased to a critical level after castration (5). The death of the prostatic glandular epithelial cells induced by androgen ablation occurs as an active energydependent process which involves a cascade of biochemical changes, collectively referred to as programmed cell death (3, 4, 6). It is the activation of this programmed cell death pathway in glandular epithelial cells that results in the rapid regression of the rat ventral prostate after castration. Like other systems in which programmed cell death occurs (7-10), this type of cell death initially involves fragmentation of genomic DNA. This fragmentation involves enzymatic degradation of the Received August 10, 1990. Address requests for reprints to: Dr. John T. Isaacs, Johns Hopkins Oncology Center, 422 North Bond Street, Baltimore, Maryland 21231. * This work was supported by NIH Research Grant CA-50601. t Supported by a Fogarty Fellowship (1F05 TW04050). Present address: Department of Pathology, University of Turku, SF-20520, Finland.
content of the seminal vesicles, demonstrating the local nature of the response. Likewise, in androgen-maintained ventral prostate organ cultures in vitro, there was a dose-response relationship between glandular cell death and TGF/3! concentration in the medium. These results demonstrate that TGFjSi can induce the death of androgen-dependent prostatic glandular cells even when physiological levels of androgen are present. Previous studies have demonstrated that both the receptor and the mRNA for TGF/?! increase rapidly in the ventral prostate after castration. Taken with the present data, these results suggest that TGF/3! may be a physiological intermediate in the programmed cell death of rat prostatic glandular cells activated after androgen ablation. (Endocrinology 127: 2963-2968,1990)
prostatic DNA into nucleosomal oligomers (i.e. multiples of a 180-nucleotide basepair subunit) lacking intranucleosomal breaks in the DNA (3). This fragmentation of prostatic DNA is a result of activation of a Ca2+-Mg2+-dependent endonuclease present within the cell nucleus induced by elevation of intracellular free Ca2+ occurring after androgen ablation (6). The Ca+2-Mg+2-dependent nuclease selectively hydrolyzes prostatic DNA at sites located between nucleosomal units, thus resulting in the sterotypic ladder of DNA fragments (2). DNA fragmentation is subsequently followed by irreversible morphological changes, histologically defined as apoptosis, which characteristically involve chromatic condensation, nuclear disintergration, cell surface blebbling, and eventually cellular fragmentation into a cluster of membrane-bound apoptotic bodies (11). Comparisons of the temporal induction of DNA fragmentation, appearance of apoptotic bodies, and prostatic regression after castration have demonstrated the fragmentation of genomic DNA is the irreversible commitment step in the death of the androgen-dependent rat ventral prostate glandular cells and does not occur as a result of the cells being dead (4, 6). The intermediates in the pathway involved in increasing intracellular free Ca2+ and the subsequent activation of the Ca2+-Mg2+-dependent endonuclease fragmentation
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TGFfr AND THE PROSTATE
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of genomic DNA in the prostatic glandular cells after androgen ablation have not been resolved. Both the receptor content and mRNA for transforming growth factor-/?! (TGF/?i) increase after castration in the rat prostate (12, 13). TGF/?i decreases the proliferation of rat ventral prostatic cells when maintained in primary cell culture in vitro (14). These results suggest that TGF/?i may function as an intermediate in the inhibition of cell proliferation and/or activation of programmed cell death within the prostate after castration. Therefore, to test these possibilities, the ability of TGF0! to inhibit cell proliferation and/or activate programmed cell death in the rat ventral prostate was determined both in vivo and in vitro.
Materials and Methods Animals All animals were maintained in accordance with the NIH guide for the care and use of laboratory animals, and the specific protocols used were approved by the Johns Hopkins Medical Institution Animal Care and Use Committee. Adult male Copenhagen rats (175-200 g BW) were obtained from Harlan Sprague-Dawley (Indianapolis, IN). In vivo experiments Two types of in vivo experiments were performed. In the first type of experiment, the ability of TGF/?i to inhibit the androgen-induced proliferative regrowth of the ventral prostatic glandular cells in previous castrate animals was tested. To do these, animals were castrated via a scrotal incision under Metofane anesthesia (methoxyflurane, Pitman-Moore, Washington Crossing, NJ). The animals were left untreated for 2 weeks to allow regression of the ventral prostate. Exogenous porcine TGF/Sj (R & D Systems, Minneapolis, MN) was then administered in vivo by means of sc Alzet model 2001 miniosmotic pumps (Alza, Palo Alto, CA) connected to a catheter made of Silastic tubing (i.e. catalog no. 602-105, Dow-Corning, Midland, MI) with the open end of the catheter implanted through the capsule of the ventral prostate gland, so that 50 ng/day TGFfr were directly released within the gland. Sterile isotonic saline containing 1% rat albumen (Sigma Chemical Co., St. Louis, MO) was used as the solvent for the TGF/3i solution (i.e. 2 fig TGFft/ml solvent). Implantation of the minipump and catheter was performed while the animals were anesthetized with Nembutal (Abbott Laboratory, North Chicago, IL). After a 1-day period of TGFfr exposure, the animals were replaced with exogeneous testosterone to induce the proliferation regrowth of the prostate. To do this, a 2-cm long Silastic capsule packed with testosterone (Sigma Chemical Co.) was implanted sc in the anesthetized animals. Implants were constructed as described previously (5). This size implant restores the serum testosterone level to a physiological value of about 2 ng/ml (5). After a 7-day period of exogeneous androgen restoration, blood was collected, animals were killed, the ventral prostate was removed, freed of fat and capsule, and weighed,
Endo • 1990 Vol 127-No 6
and the DNA content was determined by the diphenylamine method described by Burton (15), using calf thymus DNA as a standard. Serum testosterone was determined as described previously (5). In the second type of in vivo experiment, the ability of TGF/3] to induce the programmed death of prostate cells in the presence of physiological testosterone was tested. To do this, intact (i.e. noncastrated) male rats were anesthetized with Nembutal and then implanted with a TGF/?i-filled osmotic minipump connected to a catheter placed directly into their ventral prostate, as described above. After 7 days, blood was collected, animals were killed, and serum testosterone and ventral prostate were processed as described above. Organ culture method for determining cell proliferation rates Animals were castrated and after 2 weeks were given 2 daily injections of exogenous testosterone (i.e. 0.3 mg testosterone propionate/day). The animals were killed after 2 days of androgen treatment, and their ventral prostates were removed immediately. Ventral prostate explants were established in organ culture, as described in detail previously (16). To do this, the glands were cut with a razor blade into small pieces of approximately 1 mm3. In each culture, pieces from four prostates were mixed. The excised pieces (15/dish) were transferred onto lens paper strips lying on stainless steel grids in petri dishes, and medium was added up to the bottom level of the pieces. The cultures were maintained for 24 h in medium A [i.e. medium 199 with Earle's salts (Gibco, Grand Island, NY), L-glutamine (120 mg/liter), penicillin (100,000 IU/liter), streptomycin (25 mg/liter), 10~7 M testosterone, and 2 Mg/ml insulin (latter two compounds from Sigma)] containing 0, 1, 10, or 20 ng/ml TGFjS,. After 24 h, [125I]iododeoxyuridine (IDU; Amersham, Arlington Heights, IL) was added to the organ culture medium to a final concentration of 0.1 MCi IDU/ml. The use of [125I] IDU to label glandular and not stromal cells of the rat ventral prostate in vitro in organ culture has been described previously (17). After [125I]IDU incubation for 24 h, the organ cultures were washed with cold saline, and the [125I]IDU incorporated into prostatic DNA was determined as described previously (17). The results are expressed as disintegrations per min of [125I]IDU incorporated into DNA/100 ng DNA. Organ culture method for determining cell death rates Animals were castrated and after 2 weeks were given three daily injections of 0.3 mg testosterone propionate. After 3 days of androgen treatment, the animals were killed, and ventral prostatic organ culture was begun, as described above. The DNA of the explants was labeled for 24 h with [125I]IDU, as described above. After the initial 24-h labeling period, the explants were washed with cold medium A for an additional 24 h to remove the non-DNA-incorporated radioactivity. After this washing period, cultures were randomized to medium containing 1) no additive, 2) 10~7 M testosterone, or 3) 10"7 M testosterone and TGFft (i.e. 0.1, 1.0, 10, or 20 ng/ml). In each separate experiment, three organ culture dishes per grouping (i.e. medium 1, medium 2, and each of the four TGF/3, concentration grouping in medium 3) were individually followed over
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TGFfr AND THE PROSTATE a 2-week organ culture period to determine the daily rate of prostatic glandular cell death for each dish individually using the method described previously (17). To do this, the amount of [125I]IDU radioactivity was measured directly for each dish at the beginning of treatment as well as after every medium change, which occurred every 2 days. For this purpose a Nuclear Spectrometer model 300-C (Specialties Electronic Co., Inc., Mt. Holly, NJ) equipped with a well-type detector capable of accepting organ culture dishes was used. Using the measured [125I]IDU radioactivities retained in the organ culture tissue during the 2-week observation period, the daily rate of ventral prostatic glandular cell death was calculated for each culture dish, as described previously (17). The values for the three dishes for each grouping were averaged from each independent organ culture experiments. The final results are expressed as the mean ± SE of three averages from three independent organ culture experiments. Statistical analyses Numerical values are expressed as the mean ± SE. Statistical analyses of significance were made by a one-way analysis of variance, using the Kruskal-Wallis test.
Results In vivo effect of TGFfix on prostatic cell proliferation
Two weeks after castration, male rats were implanted sc with an osmotic minipump connected to a catheter implanted through the capsule of the ventral prostate, and then exogeneous androgen replacement was begun. The pump released about 24 /ul/day vehicle only or vehicle plus TGF/3j (i.e. 2.1 ng TGF/Vh for a total of 50 ng TGFjSi/day) directly onto the regressed ventral prostate lobes. After 1 week of androgen replacement, the ventral prostate wet weight and DNA content were determined in TGFX-treated vs. untreated animals (Table 1). These results demonstrated that a dose of 50 ng/day TGF/?i did not inhibit the androgen-induced proliferative regrowth of ventral prostatic glandular cells in vivo.
In vitro effect of TGFfii on prostatic cell proliferation Prostatic organ cultures were used to test the effect of TGF/?! on prostatic cell proliferation, since in this in vitro system, the ability to deliver a high concentration of TGF/3] to the tissue is more controllable than in vivo. Two days of exogeneous androgen treatment of 2-week castrated male rats induces the recruitment of prostatic glandular cells into the cell cycle and during the next 24 h (i.e. day 3 of androgen treatment), the majority of such cells will enter the S phase of the cycle (18, 19). TGF& inhibits the proliferation of sensitive epithelial cells in vitro by inhibition the transition of these cells from Gi into the S phase of the cell cycle (20). Therefore, organ cultures were initiated from ventral prostates of animals castrated for 2 weeks, then treated for 2 days in vivo with exogeneous testosterone replacement. These prostatic organ cultures were then maintained for 24 h in medium containing 10"7 M testosterone and 2 jug/ml insulin [i.e. the concentrations of these hormones demonstrated previously to stimulate DNA synthesis in organ culture (16)] in the presence or absence of TGF/3x (i.e. 1, 10, or 20 ng/ ml). After 24 h of TGF& exposure, DNA synthesis was determined and used as an index for the number of prostatic glandular cells entering the S phase during the last 24 h of culture. These results (Table 2) demonstrated that TGFfr at 1, 10, or 20 ng/ml did not inhibit DNA synthesis in vitro in prostate organ culture. In vivo effect of TGFfti on prostatic cell death It has been demonstrated that direct exogeneous treatment of the ventral prostate of intact rats with TGF/3i TABLE 2. In vitro effect of TGF/3i on DNA synthesis during prostatic organ culture
Organ culture medium containing"
TABLE 1. In vivo effect of TGF/Ji on the androgen-induced proliferative regrowth of the ventral prostate of castrated rats
Treatment" Intact, no treatment Castration only Castration, then testosterone replacement, with ventral prostate directly exposed to vehicle only for 7 days Castration, then testosterone replacement, with ventral prostate directly exposed to 50 ng/day TGF/3, for 7 days
Serum tesVentral prostate tosterone Wetwt DNA (fig/ (ng/ml) (mg/gland) gland) 2.3 ± 0.4 0.1 ± 0.1* 2.7 ± 0.3
2.8 ± 0.4
' Six animals per grouping. ' P < 0.05 compared to the intact group.
225 ± 13 20 ±3* 134 ± 15*
128 ± 17*
528 ± 12 130 ± 14* 448 ±11*
437 ± 27*
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Testosterone (10 7 M + insulin (2 Mg/ml) Testosterone (10~7) + insulin (2 Mg/ml) +1 ng/ml TGF0, +10 ng/ml TGF& +20 ng/ml TGF/3,
In vitro ventral prostatic DNA synthesis in organ culture (dpm [l25I]IDU incorporated into DNA/100 Mg DNA) 1987 ± 192*
1875 ± 158 2015 ± 89 2154 ± 235
" Cultures were initiated from prostates of rats castrated 2 weeks earlier and then treated for 2 days with exogeneous testosterone in vivo. The organ cultures were then maintained in vitro for 24 h in medium containing various additives, as indicated. After 24-h exposure, [125I]IDU was added, and 24 h later the disintegrations per min of IDU incorporated into DNA were determined. * Values are the mean ± SE from six separate organ culture dishes per treatment.
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TGFfr AND THE PROSTATE
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can induce the death of prostate cells in vivo (13). These studies did not determine, however, whether this is due to a systemic effect on the serum testosterone level or to a direct effect on the ventral prostate itself. To resolve this, intact male rats were implanted with osmotic minipumps to deliver either vehicle only or vehicle containing TGF/?i (i.e. 50 ng/day) directly to the ventral prostate for 1 week. This study demonstrated that 50 ng/day TGF/3i produced a 20-25% decrease in the ventral prostate wet weight and DNA content (Table 3). This effect was local, since the prostatic TGF/?! treatment had no effect on either serum testosterone or the seminal vesicle's wet weight or DNA content in animals whose ventral prostates were decreased. In vitro effect of TGFfii on prostatic cell death
An organ culture system has been developed in which the DNA of prostatic glandular, but not stromal, cells are [125I]IDU labeled, so that the loss of [125I]IDU by the organ culture tissue can be used to determine the daily rate of the death of these prostatic glandular cells in vitro (17). Using this [125I]IDU organ culture method, a dose-response relationship between TGF/?i and prostatic cell death in organ culture was demonstrated (Table 4). At a dose of 20 ng/ml, TGF& was capable of increasing the daily rate of prostatic glandular cell death to nearly the same extent as removal of testosterone from the medium. The dose-dependent ability of TGF& to increase prostatic cell death was also demonstrated, using the loss of total prostatic wet weight and DNA content after 14 days in organ culture as indices (Table 5). Discussion In previously reported in vitro studies, McKeehan and Adams (14) demonstrated that TGFft at 0.1-1.0 ng/ml was inhibitory to the proliferation of ventral prostatic epithelial cells in primary culture. In the present study TGF/?i was not inhibitory to the proliferation of ventral prostatic glandular cells in organ culture in vitro even at
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a concentration of up to 20 ng/ml. These studies also demonstrated that in vivo exposure of ventral prostates to 50 ng/day TGF/?i was not inhibitory to the proliferation of prostatic glandular cells. A major difference between the studies of McKeehan and Adams (14) and the present studies is that in the earlier studies prostatic epithelial cells devoid of prostatic stromal cells were exposed to TGF^i. In contrast, in the in vitro organ culture and in vivo studies reported in this manuscript, both prostatic epithelial and stromal cells are present. The presence of prostatic stromal cells in these latter studies may be significant. Both epidermal growth factorlike material and fibroblast growth factor (FGF) have been demonstrated in the rat ventral prostate (21, 22). Cultured human prostate-derived fibroblasts produce FGF (23). In addition, FGF mRNA levels are increased when regressed prostates of castrated rats are induced to proliferate during exogeneous androgen replacement (24). In the studies of McKeehan and Adams (14), TGF/?! inhibition of proliferation of rat prostatic cancer cells induced by FGF in vitro could be overcome by increasing the concentration of FGF in the medium. Combining all of these data suggests that either TGF/?i is not inhibitory to the proliferation of prostatic glandular cells or that such inhibition can be attenuated by additional stromal factors under certain conditions. For example, the inability of TGFjSi to inhibit prostatic proliferation in the present in vivo and in vitro studies could be due to the prostatic stromal cells producing sufficient FGF to overcome the inhibitory effect of TGF/?i on proliferation of prostatic glandular cells. Concievably, in the castrated rat as the prostatic glandular cell number approaches the value present in an intact animal, FGF levels may decrease to below a critical value, and the inhibitory effect of TGF/?i on proliferation of glandular cells would then be manifest. Such a possibility could explain why in the castrated rat once the prostatic glandular cell number is restored to its normal value by continuous exogeneous androgen replacement, the net proliferation of prostatic glandular cells ceases (5) even though FGF and androgen
TABLE 3. In vivo effect of TGF/?! on the ventral prostate in intact rats
Treatment0 Intact, no treatment 7-Day castrates Intact, with ventral prostate directly exposed to vehicle for 7 days Intact, with ventral prostate directly exposed to 50 ng/day of TGF& for 7 days 0 6
Ventral prostate
Seminal vesicle
Serum testosterone (ng/ml)
Wet wt (mg/gland)
DNA (Mg/ gland)
Wetwt (mg/gland)
DNA (Mg/ gland)
2.1 ± 0.3 0.1 ± O.I6 2.3 ± 0.2
215 ± 19 43 ±T 208 ± 11
510 ± 24 185 ± 356 508 ± 37
428 ± 13 115 ± 12* 442 ± 10
819 ± 31 411 ± 21* 827 ± 30
2.2 ± 0.2
162 ± 19"
401 ± 24*
439 ± 15
805 ± 24
Nine animals per grouping. Statistically lower than intact, no treatment group at P < 0.05.
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TGF& AND THE PROSTATE TABLE 4. In vitro dose-response effect of TGF/3i on the daily rate of prostatic cell death in organ culture
Additive to organ culture medium"
None Testosterone only (10~7 M) Testosterone (10~7 M) + TGFjS, at 0.1 ng/ml 1.0 ng/ml 10 ng/ml 20 ng/ml
Daily rate of prostatic glandular cell death in organ culture (% of glandular cell dying/ day) 14.5 ± 0.7* 5.5 ± 0.4 5.9 ± 0.4 6.5 ± 0.2 8.4 ± 0.3* 11.3 ± 0.4*
° Values are the mean ± SE from six separate organ culture dishes per treatment. * Statistically higher than testosterone only group at P < 0.05.
are still present in the gland. To test this possibility, the regressed ventral prostates of castrated male rats induced to regrow by exogeneous androgen replacement are being directly exposed to TGF0! alone, neutralizing anti-FGF antibody alone, or a combination of the two via osmotic minipumps to test whether any of these treatments can inhibit prostatic glandular cell proliferation. In contrast to the inability of TGF& to inhibit prostatic glandular cell proliferation in vivo and in vitro, as shown in the present studies, TGFjSi is able to induce the programmed death of prostatic glandular cells in vivo and in vitro even in the presence of physiological androgen. Previous studies demonstrated that when rats are castrated, the receptor content and the mRNA for TGFjSi increase in the ventral prostate (12, 13). The temporal induction of these in vivo TGF/?i changes is identical to that involved in the death of prostatic glandular cells after castration (3, 4, 6). These results suggest that increased levels of TGFBx may well be an intermediate step in the cascade of events involved in the programmed
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death of rat prostatic glandular cells activated after androgen ablation. This possibility is further supported by the studies that have demonstrated that androgen-dependent human prostatic cancer cells, like normal rat prostatic glandular cells, retain the ability to activate this programmed cell death pathway after androgen ablation (25). Associated with the activation of this programmed cell death of human prostatic cancer cells after castration is an enhanced expression of TGF/3i, fragmentation of tumor DNA into nucleosomal oligomers, and the histological appearance of apoptotic bodies (25). Acknowledgments The technical expertise of John C. Lamb as well as the secretarial help of Barbara A. Lee are gratefully acknowledged.
References 1. Kyprianou N, Isaacs JT 1987 Biological significance of measurable androgen levels in the rat ventral prostate following castration. Prostate 10:313-324 2. Isaacs JT 1984 Antagonistic effect of androgens on prostatic cell death. Prostate 5:545-557 3. Kyprianou N, Isaacs JT 1988 Activation of programmed cell death in the rat ventral prostate after castration. Endocrinology 122:552562 4. English HF, Kyprianou N, Isaacs JT 1989 Relationship between DNA fragmentation and apoptosis in the programmed cell death in the rat prostate following castration. Prostate 15:233-250 5. Kyprianou N, Isaacs JT 1987 Quantal relationship between prostatic dihydrotestosterone and prostatic cell content: critical threshold concept. Prostate 11:41-50 6. Kyprianou N, English HF, Isaacs JT 1988 Activation of a Ca2+Mg2+-dependent endonuclease as an early event in castrationinduced prostatic cell death. Prostate 13:103-117 7. Wyllie AH 1980 Glucocorticoid induces in thymocytes a nucleaselike activity associated with the chromatin condensation of apoptosis. Nature 284:555-556 8. Umansky SR, Korol BA, Nelipovich PA 1981 In vivo DNA degradation in thymocytes of 7-irradiated or hydrocortisone-treated rats. Biochim Biophys Acta 655:9-17 9. Wyllie AH, Morris RG, Smith AL, Dunlop D 1984 Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol 142:67-77
TABLE 5. In vitro effect of TGF/3i on the weight wet and DNA content of prostatic tissue maintained for 14 days in organ culture Prostatic organ culture Additive to organ culture medium"
Wet wt (mg/organ culture)
DNA (^g/organ culture)
None Testosterone only (10~7 M) Testosterone (10~7 M) + TGF/3, at 0.1 ng/ml 1 ng/ml 10 ng/ml 20 ng/ml
4.8 ± 0.4* 9.3 ± 0.6
11.5 ± 0.8* 19.4 ± 1.1
9.2 ± 0.8 9.3 ± 1.4* 6.9 ± 0.4* 6.6 ± 0.2*
18.7 ± 0.7 19.2 ± 0.5 15.1 ± 0.5* 13.5 ± 0.9*
' Values are the mean ± SE from six separate organ culture dishes per treatment. * P < 0.05 compared to testosterone only group.
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TGFft AND THE PROSTATE
10. Cohen JJ, Duke RC 1984 Glucocorticoid activation of a calciumdependent endonuclease in thymocyte nuclei leads to cell death. J Immunol 132:38-42 11. Kerr JFR, Wyllie AH, Currie AR 1972 Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br J Cancer 226:239-257 12. Kyprianou N, Isaacs JT 1988 Identification of a cellular receptor for transforming growth factor beta in rat ventral prostate and its negative regulation by androgens. Endocrinology 123:2124-2131 13. Kyprianou N, Isaacs JT 1989 Expression of transforming growth factor beta in the rat ventral prostate during castration-induced programmed cell death. Mol Endocrinol 3:1515-1522 14. McKeehan WL, Adams PS 1988 Heparin-binding growth factor/ proatatropin attenuates inhibition of rat prostate tumor epithelial cell growth by transforming growth factor type beta. In Vitro Cell Dev Biol 24:243-246 15. Burton K 1968 Determination of DNA concentration with diphenylamine. In: Grossmann L, Moldave K (eds) Methods in Enzymology. Academic Press, New York, vol 12B:163-166 16. Martikainen P 1987 Maintenance of adult rat ventral prostate in organ culture. Anat Rec 218:166-174 17. Martikainen P, Isaacs JT 1990 An organ culture system for the study of programmed cell death in the rat ventral prostate. Endocrinology 127:1268-1277 18. Carter MF, Chung LWK, Coffey DA 1972 The temporal requirements for androgens during the cell cycle of the prostate gland. In:
19. 20. 21.
22. 23.
24.
25.
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King LR, Murphy GP (eds) Urological Research. Plenum Press, New York, pp 27-38 Bruchovsky N, Lesser B, Van Doom E, Craven S 1975 Hormonal effects on cell proliferation in rat prostate. Vit Horm 33:61-102 Howe P, Leof EB 1990 Transforming growth factor & signalling pathways. J Cell Biochem [Suppl] 14C:259 Matuo Y, Nishi N, Matsui S, Sandberg AA, Isaacs JT, Wada F 1987 Heparin binding affinity of rat prostatic growth factor in normal and cancerous prostates: partial purification and characterization of rat prostatic growth factor in the Dunning tumor. Cancer Res 47:188-192 Jacobs SC, Story MT, Sasse J, Lawson RK 1988 Characterization of growth factors derived from the rat ventral prostate. J Urol 139:1106-1110 Story MT, Livingston B, Baeten L, Swartz SJ, Jacobs SC, Begun FP, Lawson RK 1989 Cultured human prostate-derived fibroblasts produce a factor that stimulates their growth with properties indistinguishable from basic fibroblast growth factor. Prostate 15:355-365 Katz AE, Benson MC, Wise GJ, Olsson CA, Bandyk MG, Sawczuk IS, Tomashefsky P, Buttyan R 1989 Gene activity during the early phase of androgen-stimulated rat prostate regrowth. Cancer Res 49:5889-5894 Kyprianou N, English HF, Isaacs JT 1990 Programmed cell death during regression of the PC-82 human prostatic cancer following androgen ablation. Cancer Res 50:3748-3753
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Vol. 127, No. 6 Printed in U.S.A.
Effect of Transforming Growth Factor-/?i on Proliferation and Death of Rat Prostatic Cells* PAULA MARTIKAINENf, NATASHA KYPRIANOU, AND JOHN T. ISAACS The Oncology Center and the Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231
ABSTRACT. The ability of transforming growth factor Bi (TGF/3!) to inhibit proliferation and activate death of rat ventral prostatic glandular cells was tested both in vivo and in vitro. In vivo administration of 50 ng TGF/Vday directly to the regressed ventral prostate of previously castrated male rats had no effect on the proliferative regrowth of the prostatic glandular cells induced by exogeneous androgen replacement. In addition, androgen-stimulated ventral prostatic cell proliferation in vitro in organ culture was not affected by exposure to 0.1-20 ng/ml TGF/?!. In contrast in vivo administration of 50 ng TGF/Vday directly to the ventral prostate of intact noncastrated male rats resulted in the death of about 25% of the prostatic glandular cells within 7 days of treatment. Such TGFjSi treatment did not lower serum testosterone, nor did it affect the size or DNA
P
ROSTATIC androgen levels decrease rapidly in the male rat after castration (1). This decrease produces profound changes within the ventral prostate, resulting in the rapid regression of the gland (2, 3). This rapid regression is due to a decrease in the rate of prostatic glandular cell proliferation coupled with an increase in the rate of prostatic glandular cell death (2-4). This latter death process is initiated when prostatic androgen levels are decreased to a critical level after castration (5). The death of the prostatic glandular epithelial cells induced by androgen ablation occurs as an active energydependent process which involves a cascade of biochemical changes, collectively referred to as programmed cell death (3, 4, 6). It is the activation of this programmed cell death pathway in glandular epithelial cells that results in the rapid regression of the rat ventral prostate after castration. Like other systems in which programmed cell death occurs (7-10), this type of cell death initially involves fragmentation of genomic DNA. This fragmentation involves enzymatic degradation of the Received August 10, 1990. Address requests for reprints to: Dr. John T. Isaacs, Johns Hopkins Oncology Center, 422 North Bond Street, Baltimore, Maryland 21231. * This work was supported by NIH Research Grant CA-50601. t Supported by a Fogarty Fellowship (1F05 TW04050). Present address: Department of Pathology, University of Turku, SF-20520, Finland.
content of the seminal vesicles, demonstrating the local nature of the response. Likewise, in androgen-maintained ventral prostate organ cultures in vitro, there was a dose-response relationship between glandular cell death and TGF/3! concentration in the medium. These results demonstrate that TGFjSi can induce the death of androgen-dependent prostatic glandular cells even when physiological levels of androgen are present. Previous studies have demonstrated that both the receptor and the mRNA for TGF/?! increase rapidly in the ventral prostate after castration. Taken with the present data, these results suggest that TGF/3! may be a physiological intermediate in the programmed cell death of rat prostatic glandular cells activated after androgen ablation. (Endocrinology 127: 2963-2968,1990)
prostatic DNA into nucleosomal oligomers (i.e. multiples of a 180-nucleotide basepair subunit) lacking intranucleosomal breaks in the DNA (3). This fragmentation of prostatic DNA is a result of activation of a Ca2+-Mg2+-dependent endonuclease present within the cell nucleus induced by elevation of intracellular free Ca2+ occurring after androgen ablation (6). The Ca+2-Mg+2-dependent nuclease selectively hydrolyzes prostatic DNA at sites located between nucleosomal units, thus resulting in the sterotypic ladder of DNA fragments (2). DNA fragmentation is subsequently followed by irreversible morphological changes, histologically defined as apoptosis, which characteristically involve chromatic condensation, nuclear disintergration, cell surface blebbling, and eventually cellular fragmentation into a cluster of membrane-bound apoptotic bodies (11). Comparisons of the temporal induction of DNA fragmentation, appearance of apoptotic bodies, and prostatic regression after castration have demonstrated the fragmentation of genomic DNA is the irreversible commitment step in the death of the androgen-dependent rat ventral prostate glandular cells and does not occur as a result of the cells being dead (4, 6). The intermediates in the pathway involved in increasing intracellular free Ca2+ and the subsequent activation of the Ca2+-Mg2+-dependent endonuclease fragmentation
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TGFfr AND THE PROSTATE
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of genomic DNA in the prostatic glandular cells after androgen ablation have not been resolved. Both the receptor content and mRNA for transforming growth factor-/?! (TGF/?i) increase after castration in the rat prostate (12, 13). TGF/?i decreases the proliferation of rat ventral prostatic cells when maintained in primary cell culture in vitro (14). These results suggest that TGF/?i may function as an intermediate in the inhibition of cell proliferation and/or activation of programmed cell death within the prostate after castration. Therefore, to test these possibilities, the ability of TGF0! to inhibit cell proliferation and/or activate programmed cell death in the rat ventral prostate was determined both in vivo and in vitro.
Materials and Methods Animals All animals were maintained in accordance with the NIH guide for the care and use of laboratory animals, and the specific protocols used were approved by the Johns Hopkins Medical Institution Animal Care and Use Committee. Adult male Copenhagen rats (175-200 g BW) were obtained from Harlan Sprague-Dawley (Indianapolis, IN). In vivo experiments Two types of in vivo experiments were performed. In the first type of experiment, the ability of TGF/?i to inhibit the androgen-induced proliferative regrowth of the ventral prostatic glandular cells in previous castrate animals was tested. To do these, animals were castrated via a scrotal incision under Metofane anesthesia (methoxyflurane, Pitman-Moore, Washington Crossing, NJ). The animals were left untreated for 2 weeks to allow regression of the ventral prostate. Exogenous porcine TGF/Sj (R & D Systems, Minneapolis, MN) was then administered in vivo by means of sc Alzet model 2001 miniosmotic pumps (Alza, Palo Alto, CA) connected to a catheter made of Silastic tubing (i.e. catalog no. 602-105, Dow-Corning, Midland, MI) with the open end of the catheter implanted through the capsule of the ventral prostate gland, so that 50 ng/day TGFfr were directly released within the gland. Sterile isotonic saline containing 1% rat albumen (Sigma Chemical Co., St. Louis, MO) was used as the solvent for the TGF/3i solution (i.e. 2 fig TGFft/ml solvent). Implantation of the minipump and catheter was performed while the animals were anesthetized with Nembutal (Abbott Laboratory, North Chicago, IL). After a 1-day period of TGFfr exposure, the animals were replaced with exogeneous testosterone to induce the proliferation regrowth of the prostate. To do this, a 2-cm long Silastic capsule packed with testosterone (Sigma Chemical Co.) was implanted sc in the anesthetized animals. Implants were constructed as described previously (5). This size implant restores the serum testosterone level to a physiological value of about 2 ng/ml (5). After a 7-day period of exogeneous androgen restoration, blood was collected, animals were killed, the ventral prostate was removed, freed of fat and capsule, and weighed,
Endo • 1990 Vol 127-No 6
and the DNA content was determined by the diphenylamine method described by Burton (15), using calf thymus DNA as a standard. Serum testosterone was determined as described previously (5). In the second type of in vivo experiment, the ability of TGF/3] to induce the programmed death of prostate cells in the presence of physiological testosterone was tested. To do this, intact (i.e. noncastrated) male rats were anesthetized with Nembutal and then implanted with a TGF/?i-filled osmotic minipump connected to a catheter placed directly into their ventral prostate, as described above. After 7 days, blood was collected, animals were killed, and serum testosterone and ventral prostate were processed as described above. Organ culture method for determining cell proliferation rates Animals were castrated and after 2 weeks were given 2 daily injections of exogenous testosterone (i.e. 0.3 mg testosterone propionate/day). The animals were killed after 2 days of androgen treatment, and their ventral prostates were removed immediately. Ventral prostate explants were established in organ culture, as described in detail previously (16). To do this, the glands were cut with a razor blade into small pieces of approximately 1 mm3. In each culture, pieces from four prostates were mixed. The excised pieces (15/dish) were transferred onto lens paper strips lying on stainless steel grids in petri dishes, and medium was added up to the bottom level of the pieces. The cultures were maintained for 24 h in medium A [i.e. medium 199 with Earle's salts (Gibco, Grand Island, NY), L-glutamine (120 mg/liter), penicillin (100,000 IU/liter), streptomycin (25 mg/liter), 10~7 M testosterone, and 2 Mg/ml insulin (latter two compounds from Sigma)] containing 0, 1, 10, or 20 ng/ml TGFjS,. After 24 h, [125I]iododeoxyuridine (IDU; Amersham, Arlington Heights, IL) was added to the organ culture medium to a final concentration of 0.1 MCi IDU/ml. The use of [125I] IDU to label glandular and not stromal cells of the rat ventral prostate in vitro in organ culture has been described previously (17). After [125I]IDU incubation for 24 h, the organ cultures were washed with cold saline, and the [125I]IDU incorporated into prostatic DNA was determined as described previously (17). The results are expressed as disintegrations per min of [125I]IDU incorporated into DNA/100 ng DNA. Organ culture method for determining cell death rates Animals were castrated and after 2 weeks were given three daily injections of 0.3 mg testosterone propionate. After 3 days of androgen treatment, the animals were killed, and ventral prostatic organ culture was begun, as described above. The DNA of the explants was labeled for 24 h with [125I]IDU, as described above. After the initial 24-h labeling period, the explants were washed with cold medium A for an additional 24 h to remove the non-DNA-incorporated radioactivity. After this washing period, cultures were randomized to medium containing 1) no additive, 2) 10~7 M testosterone, or 3) 10"7 M testosterone and TGFft (i.e. 0.1, 1.0, 10, or 20 ng/ml). In each separate experiment, three organ culture dishes per grouping (i.e. medium 1, medium 2, and each of the four TGF/3, concentration grouping in medium 3) were individually followed over
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TGFfr AND THE PROSTATE a 2-week organ culture period to determine the daily rate of prostatic glandular cell death for each dish individually using the method described previously (17). To do this, the amount of [125I]IDU radioactivity was measured directly for each dish at the beginning of treatment as well as after every medium change, which occurred every 2 days. For this purpose a Nuclear Spectrometer model 300-C (Specialties Electronic Co., Inc., Mt. Holly, NJ) equipped with a well-type detector capable of accepting organ culture dishes was used. Using the measured [125I]IDU radioactivities retained in the organ culture tissue during the 2-week observation period, the daily rate of ventral prostatic glandular cell death was calculated for each culture dish, as described previously (17). The values for the three dishes for each grouping were averaged from each independent organ culture experiments. The final results are expressed as the mean ± SE of three averages from three independent organ culture experiments. Statistical analyses Numerical values are expressed as the mean ± SE. Statistical analyses of significance were made by a one-way analysis of variance, using the Kruskal-Wallis test.
Results In vivo effect of TGFfix on prostatic cell proliferation
Two weeks after castration, male rats were implanted sc with an osmotic minipump connected to a catheter implanted through the capsule of the ventral prostate, and then exogeneous androgen replacement was begun. The pump released about 24 /ul/day vehicle only or vehicle plus TGF/3j (i.e. 2.1 ng TGF/Vh for a total of 50 ng TGFjSi/day) directly onto the regressed ventral prostate lobes. After 1 week of androgen replacement, the ventral prostate wet weight and DNA content were determined in TGFX-treated vs. untreated animals (Table 1). These results demonstrated that a dose of 50 ng/day TGF/?i did not inhibit the androgen-induced proliferative regrowth of ventral prostatic glandular cells in vivo.
In vitro effect of TGFfii on prostatic cell proliferation Prostatic organ cultures were used to test the effect of TGF/?! on prostatic cell proliferation, since in this in vitro system, the ability to deliver a high concentration of TGF/3] to the tissue is more controllable than in vivo. Two days of exogeneous androgen treatment of 2-week castrated male rats induces the recruitment of prostatic glandular cells into the cell cycle and during the next 24 h (i.e. day 3 of androgen treatment), the majority of such cells will enter the S phase of the cycle (18, 19). TGF& inhibits the proliferation of sensitive epithelial cells in vitro by inhibition the transition of these cells from Gi into the S phase of the cell cycle (20). Therefore, organ cultures were initiated from ventral prostates of animals castrated for 2 weeks, then treated for 2 days in vivo with exogeneous testosterone replacement. These prostatic organ cultures were then maintained for 24 h in medium containing 10"7 M testosterone and 2 jug/ml insulin [i.e. the concentrations of these hormones demonstrated previously to stimulate DNA synthesis in organ culture (16)] in the presence or absence of TGF/3x (i.e. 1, 10, or 20 ng/ ml). After 24 h of TGF& exposure, DNA synthesis was determined and used as an index for the number of prostatic glandular cells entering the S phase during the last 24 h of culture. These results (Table 2) demonstrated that TGFfr at 1, 10, or 20 ng/ml did not inhibit DNA synthesis in vitro in prostate organ culture. In vivo effect of TGFfti on prostatic cell death It has been demonstrated that direct exogeneous treatment of the ventral prostate of intact rats with TGF/3i TABLE 2. In vitro effect of TGF/3i on DNA synthesis during prostatic organ culture
Organ culture medium containing"
TABLE 1. In vivo effect of TGF/Ji on the androgen-induced proliferative regrowth of the ventral prostate of castrated rats
Treatment" Intact, no treatment Castration only Castration, then testosterone replacement, with ventral prostate directly exposed to vehicle only for 7 days Castration, then testosterone replacement, with ventral prostate directly exposed to 50 ng/day TGF/3, for 7 days
Serum tesVentral prostate tosterone Wetwt DNA (fig/ (ng/ml) (mg/gland) gland) 2.3 ± 0.4 0.1 ± 0.1* 2.7 ± 0.3
2.8 ± 0.4
' Six animals per grouping. ' P < 0.05 compared to the intact group.
225 ± 13 20 ±3* 134 ± 15*
128 ± 17*
528 ± 12 130 ± 14* 448 ±11*
437 ± 27*
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Testosterone (10 7 M + insulin (2 Mg/ml) Testosterone (10~7) + insulin (2 Mg/ml) +1 ng/ml TGF0, +10 ng/ml TGF& +20 ng/ml TGF/3,
In vitro ventral prostatic DNA synthesis in organ culture (dpm [l25I]IDU incorporated into DNA/100 Mg DNA) 1987 ± 192*
1875 ± 158 2015 ± 89 2154 ± 235
" Cultures were initiated from prostates of rats castrated 2 weeks earlier and then treated for 2 days with exogeneous testosterone in vivo. The organ cultures were then maintained in vitro for 24 h in medium containing various additives, as indicated. After 24-h exposure, [125I]IDU was added, and 24 h later the disintegrations per min of IDU incorporated into DNA were determined. * Values are the mean ± SE from six separate organ culture dishes per treatment.
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TGFfr AND THE PROSTATE
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can induce the death of prostate cells in vivo (13). These studies did not determine, however, whether this is due to a systemic effect on the serum testosterone level or to a direct effect on the ventral prostate itself. To resolve this, intact male rats were implanted with osmotic minipumps to deliver either vehicle only or vehicle containing TGF/?i (i.e. 50 ng/day) directly to the ventral prostate for 1 week. This study demonstrated that 50 ng/day TGF/3i produced a 20-25% decrease in the ventral prostate wet weight and DNA content (Table 3). This effect was local, since the prostatic TGF/?! treatment had no effect on either serum testosterone or the seminal vesicle's wet weight or DNA content in animals whose ventral prostates were decreased. In vitro effect of TGFfii on prostatic cell death
An organ culture system has been developed in which the DNA of prostatic glandular, but not stromal, cells are [125I]IDU labeled, so that the loss of [125I]IDU by the organ culture tissue can be used to determine the daily rate of the death of these prostatic glandular cells in vitro (17). Using this [125I]IDU organ culture method, a dose-response relationship between TGF/?i and prostatic cell death in organ culture was demonstrated (Table 4). At a dose of 20 ng/ml, TGF& was capable of increasing the daily rate of prostatic glandular cell death to nearly the same extent as removal of testosterone from the medium. The dose-dependent ability of TGF& to increase prostatic cell death was also demonstrated, using the loss of total prostatic wet weight and DNA content after 14 days in organ culture as indices (Table 5). Discussion In previously reported in vitro studies, McKeehan and Adams (14) demonstrated that TGFft at 0.1-1.0 ng/ml was inhibitory to the proliferation of ventral prostatic epithelial cells in primary culture. In the present study TGF/?i was not inhibitory to the proliferation of ventral prostatic glandular cells in organ culture in vitro even at
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a concentration of up to 20 ng/ml. These studies also demonstrated that in vivo exposure of ventral prostates to 50 ng/day TGF/?i was not inhibitory to the proliferation of prostatic glandular cells. A major difference between the studies of McKeehan and Adams (14) and the present studies is that in the earlier studies prostatic epithelial cells devoid of prostatic stromal cells were exposed to TGF^i. In contrast, in the in vitro organ culture and in vivo studies reported in this manuscript, both prostatic epithelial and stromal cells are present. The presence of prostatic stromal cells in these latter studies may be significant. Both epidermal growth factorlike material and fibroblast growth factor (FGF) have been demonstrated in the rat ventral prostate (21, 22). Cultured human prostate-derived fibroblasts produce FGF (23). In addition, FGF mRNA levels are increased when regressed prostates of castrated rats are induced to proliferate during exogeneous androgen replacement (24). In the studies of McKeehan and Adams (14), TGF/?! inhibition of proliferation of rat prostatic cancer cells induced by FGF in vitro could be overcome by increasing the concentration of FGF in the medium. Combining all of these data suggests that either TGF/?i is not inhibitory to the proliferation of prostatic glandular cells or that such inhibition can be attenuated by additional stromal factors under certain conditions. For example, the inability of TGFjSi to inhibit prostatic proliferation in the present in vivo and in vitro studies could be due to the prostatic stromal cells producing sufficient FGF to overcome the inhibitory effect of TGF/?i on proliferation of prostatic glandular cells. Concievably, in the castrated rat as the prostatic glandular cell number approaches the value present in an intact animal, FGF levels may decrease to below a critical value, and the inhibitory effect of TGF/?i on proliferation of glandular cells would then be manifest. Such a possibility could explain why in the castrated rat once the prostatic glandular cell number is restored to its normal value by continuous exogeneous androgen replacement, the net proliferation of prostatic glandular cells ceases (5) even though FGF and androgen
TABLE 3. In vivo effect of TGF/?! on the ventral prostate in intact rats
Treatment0 Intact, no treatment 7-Day castrates Intact, with ventral prostate directly exposed to vehicle for 7 days Intact, with ventral prostate directly exposed to 50 ng/day of TGF& for 7 days 0 6
Ventral prostate
Seminal vesicle
Serum testosterone (ng/ml)
Wet wt (mg/gland)
DNA (Mg/ gland)
Wetwt (mg/gland)
DNA (Mg/ gland)
2.1 ± 0.3 0.1 ± O.I6 2.3 ± 0.2
215 ± 19 43 ±T 208 ± 11
510 ± 24 185 ± 356 508 ± 37
428 ± 13 115 ± 12* 442 ± 10
819 ± 31 411 ± 21* 827 ± 30
2.2 ± 0.2
162 ± 19"
401 ± 24*
439 ± 15
805 ± 24
Nine animals per grouping. Statistically lower than intact, no treatment group at P < 0.05.
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TGF& AND THE PROSTATE TABLE 4. In vitro dose-response effect of TGF/3i on the daily rate of prostatic cell death in organ culture
Additive to organ culture medium"
None Testosterone only (10~7 M) Testosterone (10~7 M) + TGFjS, at 0.1 ng/ml 1.0 ng/ml 10 ng/ml 20 ng/ml
Daily rate of prostatic glandular cell death in organ culture (% of glandular cell dying/ day) 14.5 ± 0.7* 5.5 ± 0.4 5.9 ± 0.4 6.5 ± 0.2 8.4 ± 0.3* 11.3 ± 0.4*
° Values are the mean ± SE from six separate organ culture dishes per treatment. * Statistically higher than testosterone only group at P < 0.05.
are still present in the gland. To test this possibility, the regressed ventral prostates of castrated male rats induced to regrow by exogeneous androgen replacement are being directly exposed to TGF0! alone, neutralizing anti-FGF antibody alone, or a combination of the two via osmotic minipumps to test whether any of these treatments can inhibit prostatic glandular cell proliferation. In contrast to the inability of TGF& to inhibit prostatic glandular cell proliferation in vivo and in vitro, as shown in the present studies, TGFjSi is able to induce the programmed death of prostatic glandular cells in vivo and in vitro even in the presence of physiological androgen. Previous studies demonstrated that when rats are castrated, the receptor content and the mRNA for TGFjSi increase in the ventral prostate (12, 13). The temporal induction of these in vivo TGF/?i changes is identical to that involved in the death of prostatic glandular cells after castration (3, 4, 6). These results suggest that increased levels of TGFBx may well be an intermediate step in the cascade of events involved in the programmed
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death of rat prostatic glandular cells activated after androgen ablation. This possibility is further supported by the studies that have demonstrated that androgen-dependent human prostatic cancer cells, like normal rat prostatic glandular cells, retain the ability to activate this programmed cell death pathway after androgen ablation (25). Associated with the activation of this programmed cell death of human prostatic cancer cells after castration is an enhanced expression of TGF/3i, fragmentation of tumor DNA into nucleosomal oligomers, and the histological appearance of apoptotic bodies (25). Acknowledgments The technical expertise of John C. Lamb as well as the secretarial help of Barbara A. Lee are gratefully acknowledged.
References 1. Kyprianou N, Isaacs JT 1987 Biological significance of measurable androgen levels in the rat ventral prostate following castration. Prostate 10:313-324 2. Isaacs JT 1984 Antagonistic effect of androgens on prostatic cell death. Prostate 5:545-557 3. Kyprianou N, Isaacs JT 1988 Activation of programmed cell death in the rat ventral prostate after castration. Endocrinology 122:552562 4. English HF, Kyprianou N, Isaacs JT 1989 Relationship between DNA fragmentation and apoptosis in the programmed cell death in the rat prostate following castration. Prostate 15:233-250 5. Kyprianou N, Isaacs JT 1987 Quantal relationship between prostatic dihydrotestosterone and prostatic cell content: critical threshold concept. Prostate 11:41-50 6. Kyprianou N, English HF, Isaacs JT 1988 Activation of a Ca2+Mg2+-dependent endonuclease as an early event in castrationinduced prostatic cell death. Prostate 13:103-117 7. Wyllie AH 1980 Glucocorticoid induces in thymocytes a nucleaselike activity associated with the chromatin condensation of apoptosis. Nature 284:555-556 8. Umansky SR, Korol BA, Nelipovich PA 1981 In vivo DNA degradation in thymocytes of 7-irradiated or hydrocortisone-treated rats. Biochim Biophys Acta 655:9-17 9. Wyllie AH, Morris RG, Smith AL, Dunlop D 1984 Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol 142:67-77
TABLE 5. In vitro effect of TGF/3i on the weight wet and DNA content of prostatic tissue maintained for 14 days in organ culture Prostatic organ culture Additive to organ culture medium"
Wet wt (mg/organ culture)
DNA (^g/organ culture)
None Testosterone only (10~7 M) Testosterone (10~7 M) + TGF/3, at 0.1 ng/ml 1 ng/ml 10 ng/ml 20 ng/ml
4.8 ± 0.4* 9.3 ± 0.6
11.5 ± 0.8* 19.4 ± 1.1
9.2 ± 0.8 9.3 ± 1.4* 6.9 ± 0.4* 6.6 ± 0.2*
18.7 ± 0.7 19.2 ± 0.5 15.1 ± 0.5* 13.5 ± 0.9*
' Values are the mean ± SE from six separate organ culture dishes per treatment. * P < 0.05 compared to testosterone only group.
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TGFft AND THE PROSTATE
10. Cohen JJ, Duke RC 1984 Glucocorticoid activation of a calciumdependent endonuclease in thymocyte nuclei leads to cell death. J Immunol 132:38-42 11. Kerr JFR, Wyllie AH, Currie AR 1972 Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br J Cancer 226:239-257 12. Kyprianou N, Isaacs JT 1988 Identification of a cellular receptor for transforming growth factor beta in rat ventral prostate and its negative regulation by androgens. Endocrinology 123:2124-2131 13. Kyprianou N, Isaacs JT 1989 Expression of transforming growth factor beta in the rat ventral prostate during castration-induced programmed cell death. Mol Endocrinol 3:1515-1522 14. McKeehan WL, Adams PS 1988 Heparin-binding growth factor/ proatatropin attenuates inhibition of rat prostate tumor epithelial cell growth by transforming growth factor type beta. In Vitro Cell Dev Biol 24:243-246 15. Burton K 1968 Determination of DNA concentration with diphenylamine. In: Grossmann L, Moldave K (eds) Methods in Enzymology. Academic Press, New York, vol 12B:163-166 16. Martikainen P 1987 Maintenance of adult rat ventral prostate in organ culture. Anat Rec 218:166-174 17. Martikainen P, Isaacs JT 1990 An organ culture system for the study of programmed cell death in the rat ventral prostate. Endocrinology 127:1268-1277 18. Carter MF, Chung LWK, Coffey DA 1972 The temporal requirements for androgens during the cell cycle of the prostate gland. In:
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King LR, Murphy GP (eds) Urological Research. Plenum Press, New York, pp 27-38 Bruchovsky N, Lesser B, Van Doom E, Craven S 1975 Hormonal effects on cell proliferation in rat prostate. Vit Horm 33:61-102 Howe P, Leof EB 1990 Transforming growth factor & signalling pathways. J Cell Biochem [Suppl] 14C:259 Matuo Y, Nishi N, Matsui S, Sandberg AA, Isaacs JT, Wada F 1987 Heparin binding affinity of rat prostatic growth factor in normal and cancerous prostates: partial purification and characterization of rat prostatic growth factor in the Dunning tumor. Cancer Res 47:188-192 Jacobs SC, Story MT, Sasse J, Lawson RK 1988 Characterization of growth factors derived from the rat ventral prostate. J Urol 139:1106-1110 Story MT, Livingston B, Baeten L, Swartz SJ, Jacobs SC, Begun FP, Lawson RK 1989 Cultured human prostate-derived fibroblasts produce a factor that stimulates their growth with properties indistinguishable from basic fibroblast growth factor. Prostate 15:355-365 Katz AE, Benson MC, Wise GJ, Olsson CA, Bandyk MG, Sawczuk IS, Tomashefsky P, Buttyan R 1989 Gene activity during the early phase of androgen-stimulated rat prostate regrowth. Cancer Res 49:5889-5894 Kyprianou N, English HF, Isaacs JT 1990 Programmed cell death during regression of the PC-82 human prostatic cancer following androgen ablation. Cancer Res 50:3748-3753
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