[CANCER RESEARCH 55, 4438-4445, October 1, 1995] Overexpression of bcl-2 Protects Prostate Cancer Cells from Apoptosis in Vitro and Confers Resistance to Androgen Depletion in Vivo1 Anthony J. Raffo, Harris Perlman, Min-Wei Chen, Mark L. Day, Jack S. Streitman, and Ralph Buttyan2 Department of Urology, College of Physicians and Surgeons of Columbia University, Neu- York, Neu- York 10032 ¡A.J. R.. H. P.. M-W. C., J. S. S., R. B.¡,and Michigan Prostate Institute, University of Michigan. Ann Arbor. Michigan 48109 ¡M.L D.¡ ABSTRACT Normal (nonneoplastic) human prostatic secretory epithelial cells do not express the hcl-2 protein. However, a recent immunohistochemical .survey of neoplastic human prostate tissues showed that a fraction of primary untreated prostate adenocarcinoma cells expressed this apopto- sis-suppressing oncoprotein at significant levels (Colombe! el al.. Am. J. Pillimi., 143: 390-400, 1993). Additionally, a number of hormone-refrac tory prostatic adenocarcinomas obtained from hormonally-treated pa tients (subsequent to surgical or drug castration therapy) were examined and were found to be uniform in their elevated expression of bcl-2 oncoprotein. The results of this preliminary survey imply that bcl-2 expression distinguishes a subgroup of primary human prostate cancers and that the expression of this protein might be a factor enabling prostate cancer cells to survive in an androgen-deprived environment. The current study was undertaken to determine the degree to which overexpression of bcl-2 can protect human prostate cancer cells from apoptotic stimuli in vitro and in vivo. Human prostate cancer cells (LNCaP) were transfected with a neomycin-selectable eucaryotic expression vector containing cDNA encoding human bcl-2. Transfected clonal variants that express bcl-2 protein (LNCaP/bcl-2) were unaltered with regard to their basal growth rate in 10% serum-containing medium, or with regard to their expression of the differentiated human prostate cell gene products prostate-specific antigen or androgen receptor protein. The bcl-2-transfected clones were altered, however, with regard to their growth rate in charcoal-stripped serum lacking dihydrotestosterone. Additionally, in contrast to the paren tal or control-transfected cell lines, LNCaP/bcl-2 cells were highly resist ant to a variety of apoptotic stimuli in vitro including serum starvation and 10 MMphorbol ester (phorbol 12-myristate 13-acetate) supplementation of the medium. Lastly, the overexpression of bcl-2 by these prostate cancer cells altered their tumorigenic potential in a nude mouse assay, s.c. injections of Id1'LNCaP/bcl-2 cells into male nude mice resulted in earlier and larger tumor formation compared to an equivalent injection of pa rental or control-transfected LNCaP cells. When these variant cell lines were injected into castrated male nude mice, only the LNCaP/bcl-2- transformed cells gave rise to tumors. Moreover, LNCaP/bcl-2 tumors grown in intact male nude mice were refractory to the growth-inhibiting effects of castration demonstrated by parental LNCaP cells. Data obtained in this study demonstrate that the bcl-2 oncoprotein can protect prostate cancer cells from apoptotic stimuli hi vitro and suggest that such protec tion correlates with the ability to form hormone-refractory prostate tu mors in vivo. INTRODUCTION Therapy for the advanced form of prostate cancer generally in volves either surgical gonadectomy to remove the major source of androgens or drug treatments that suppress androgen production and action. These treatments are at least palliatively effective because they Received 4/4/95; accepted 8/2/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by United States Public Health Service Grants N1H- CA58089 and NIH-CA47848, as well as grants from the CaPCure Foundation and the Koch Foundation. M-W. C. is a research fellow of the American Foundation for Urological Disease. • To whom requests for reprints should be addressed, at Department of Urology, Columbia University, College of Physicians and Surgeons, 630 West loKth Street, New York, NY 10032. can initiate apoptosis of prostate cancer cells. Unfortunately, some fraction of the prostate cancer cells inevitably survives these therapies and continues to grow. It is clear that these cells have a means of avoiding or inhibiting the cellular mechanism that activates the cell death pathway so intrinsic to normal prostate epithelial cells after androgen withdrawal. Therefore, our ability to comprehend the mech anism of this apoptotic inhibition and overcome it has significant ramifications for the development of new and more effective therapies to successfully treat this malignancy. On the basis of the rapidly increasing knowledge of the genetic components involved in the apoptotic pathway, we recently screened a large number of human prostatic tissues to characterize whether any of these tissues expressed the bcl-2 oncoprotein, a potent and broad- spectrum suppressor of apoptosis (1, 2). The results of this immuno histochemical survey showed that no normal cells of the adult human prostatic secretory epithelium expressed this protein. In contrast, a subset of primary prostatic adenocarcinomas obtained from untreated prostate cancer patients positively immunostained for bcl-2, as did all of the hormone-refractory prostatic adenocarcinomas obtained from hormone-treated patients (3). This intriguing pattern establishes the potential involvement of bcl-2 in the developmental pathway of prostate cancer leading to hormone resistance. The results that we obtained by experimental manipulation of bcl-2 expression in prostate cancer cells, presented here, further support this hypothesis. When exogenously expressed in cultured cells and in transgenic mouse systems, bcl-2 has been shown to protect against apoptotic stimuli as diverse as chemotherapeutic agents and growth factor deprivation (1, 2, 4—6).This protection extends to certain forms of hormone-activated apoptosis, most prominently, glucocorticoid-in- duced apoptosis of thymocytes (7). To date, the question of whether bcl-2 expression can protect endocrine-dependent cells from hormone withdrawal has not yet been established. To test this for prostate cells, we genetically manipulated a commonly used prostate cancer cell line, LNCaP, so that these cells would overexpress the human bcl-2 pro tein. This cell line is a popular model for the study of prostate cancer (8, 9) because it retains some of the most prominent differentiated features of the human prostate cell, including the production of the prostate secretory protein, PSA1 (10), the prostate-specific membrane antigen (11), and the AR (12). Moreover, LNCaP cells have proven to be growth responsive to androgenic steroids in vitro (13). Addition ally, these cells show an androgen-dependent phenotype in vivo with regard to their ability to form tumors in male but not castrated male nude mice (8, 9). In this series of experiments, we examined whether exogenously expressed bcl-2 protein can alter the expression of the differentiated prostate gene products or the basal growth rate of LNCaP cells. Furthermore, we tested whether enhanced bcl-2 expression could alter the LNCaP cell growth response to DHT or the apoptotic response of these cells to serum withdrawal or phorbol ester supplementation in vitro. Finally, the results of a study of LNCaP tumor formation in intact and castrated male nude mice strongly support the hypothesis 1 The abbreviations used are: PSA, prostate-specific antigen; FBS, fetal bovine serum; TPA, phorbol 12-myristate 13-acetate; AR. androgen receptor; DHT. dihyrotestosterone. 4438 Research. on September 10, 2021. © 1995 American Association for Cancer cancerres.aacrjournals.org Downloaded from