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Extended Survivability of Prostate Cancer Cells in the Absence Of (CANCER RESEARCH 58. 3466-3479. August 1. 1998] Extended Survivability of Prostate Cancer Cells in the Absence of Trophic Factors: Increased Proliferation, Evasion of Apoptosis, and the Role of Apoptosis Proteins Dean G. Tang,1 Li Li, Dharam P. Chopra, and Arthur T. Porter Department of Radiation Oncology, Wayne State University, Detroit, Michigan 48202 ID. G. T., A. T. P.¡; Karmanos Cancer Institute, Detroit, Michigan 48201 [D. G. T., A. T. P.]; Biomide Corporation, Grosse Pointe Farms. Michigan 48236 [D. G. T., L. L.]; and institute of Chemical Toxicologv, Wavne State University, Detroit, Michigan 48226 ID. P. C.I ABSTRACT accepted that apoptosis is a dominant feature unless suppressed or overridden by growth/survival factors; therefore, most eukaryotic This project was undertaken to study the survival properties of various cells (with the exception of, probably, blastomeres) will undergo prostate cells, including normal (NHP), BPH (benign prostate hyperpla- sia), primary carcinoma (PCA), and metastatic prostate cancer cells apoptosis when deprived of appropriate survival factors (1, 2). In (LNCaP, PC3, and Dul45), in the absence of trophic factors. Cell prolif creased cell proliferation, extended cell survival, and diminished eration and cell death were quantitated by enumerating the number of live apoptosis have all been implicated in tumorigenesis. cells using MTS/PMS kit and of dead (apoptotic) cells using 4',6-dia- There has been a close association between apoptosis and prostate midino-2-phenylindole dihydrochloride nuclear staining. These cells dem cancer initiation, progression, metastasis, and response to treatment. onstrated an overall survivability in the order of BPH < NHP < LNCaP The growth and survival of normal prostate epithelial cells depend on < PC3 < PCA < Du 145. Upon growth factor deprivation, NHP/BPH cells androgens; therefore, castration or chemical blocking of androgen rapidly underwent apoptosis, leading to a decreased number of live cells. function leads to prostate atrophy. Prostate cancer is composed of PCA/PC3/Dul45 cells, in contrast, demonstrated an initial phase of ag androgen-dependent and androgen-independent cells. Androgen abla gressive growth during which apoptosis rarely occurred, followed by a "plateau" phase in which cell loss by apoptosis was compensated by cell tion eliminates most androgen-dependent cancer cells by inducing proliferation, followed by a later phase in which apoptosis exceeded the apoptosis but can rarely cure the patients due to the presence of cell proliferation. LNCaP cells demonstrated survival characteristics be androgen-independent cells and emergence of apoptosis-resistant tween those of NHP/BPH and PCA/PC3/Dul4S cells. We concluded that clones (3, 4). Bcl-2 oncoprotein expression has been correlated with the increased survivability in prostate cancer cells results from enhanced the progression and metastasis of prostate cancer cells and the emer cell proliferation as well as decreased apoptosis. The molecular mecha gence of androgen-independent cells, as well as the generation of nisms for evasion of apoptosis in prostate cancer cells were subsequently apoptosis- and therapy-resistant cancer cell clones (5-11). On the investigated. Quantitative Western blotting was used to examine the pro tein expression of PS3 and P21WAIM, Bcl-2 and Bcl-XL (anti-apoptotic other hand, androgen-independent prostate cancer cells still retain proteins), and Bax, Bak, and Bad (proapoptotic proteins). The results appropriate apoptotic machinery and can be induced to undergo revealed that, upon trophic factor withdrawal, NHP and BPH cells up- apoptosis by a wide spectrum of biological and pharmacological regulated wild-type p53 and proapoptotic proteins Bax/Bad/Bak and treatments, including wild-type P53 and P212 (12-14), prostate down-regulated the expression of P21. Furthermore, NHP and BPH cells apoptosis response-^ (15), prevention of adhesion (16), transforming endogenously expressed little or no Bcl-2. In sharp contrast, prostate growth factor ß(17), tumor necrosis factor a (18), Fas (19), chemo- cancer cells expressed nonfunctional P53 and various amounts of Bcl-2 proteins. Upon deprivation, these cancer cells up-regulated P21 and Bcl-2 therapeutic drugs such as cisplatin, camptothecin, teniposide, and and/or BciXL, lost response to withdrawal-induced up-regulation of Bax/ vincristine (20, 21), novel chemical entities such as Linomide (quin- Bad/Bak or decreased or even completely lost Bax expression and ex oline-3-carboxamide; Ref. 22), ß-lapachone(23, 24), phenylbutyrate pressed some novel proteins such as P25 and P54/55 complex. These data (25), ribonucleotidase inhibitors (26), diethylstilbestrol (27), ß-L-(-)- together suggest that prostate cancer cells may use multiple molecular dioxolane-cytidine (28), and retinoid derivatives (29), and signal mechanisms to evade apoptosis, which, together with increased prolifer transduction modifiers such as phorbol esters (30-32) and thapsigar- ation, contribute to extended survivability of prostate cancer cells in the gin (33). We recently reported apoptosis induction by prostate secre absence trophic factors. tory protein 94 (34) and hydroxamic acid compounds (35, 36) in hormone-refractory human prostate cancer cells. INTRODUCTION Little information exists in regard to differential properties of normal and cancerous prostate epithelial cells in terms of sensitivity to Tissue and organ homeostasis is controlled by balanced cell pro apoptosis induction. Furthermore, no systematic studies have been liferation, cell survival, and cell death. Cell proliferation is driven and performed to investigate the interrelationship among the proliferation, regulated by various peptide growth factors and/or cytokines. Cell survival represents the cell's intrinsic life expectancy in the absence of survival, and apoptosis of various types of prostate cells. Recently, a series of normal (NHP2), BPH, and PCA prostate cultures have been survival factors, many of which overlap with the growth factors/ cytokines that drive cell proliferation. Physiological cell death occurs established and characterized (37). In this study, we took advantage of mostly via programmed cell death or apoptosis (1). Apoptosis is a these primary cultures and compared their growth and survival prop genetically controlled suicidal program that takes place, in many erties in the absence of trophic factors with those of the established cases, when the cells run out of their survivability in the absence of malignant prostate cancer cell lines, i.e., LNCaP, PC3, and Dul45. growth/survival (or trophic) factors, although it can also be triggered We further investigated the changes and the potential roles of several by a wide variety of physiological or obnoxious stimuli. It is generally apoptosis proteins in these six different cell types upon growth factor withdrawal. The results show that prostate carcinoma cells demon Received 2/5/98; accepted 6/2/98. strate a much higher proliferative potential and survive much longer The costs of publication of this article were defrayed in part by the payment of page than normal and BPH cells. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Department of Radiation 2 The abbreviations used are: NHP, normal human prostate; BPH, benign prostate hyperplasia; PCA, primary prostatic carcinoma; DAPI, 4',6-diamidino-2-phenylindole; Oncology, Wayne State University, 407 Life Science Building, Detroit, MI 48202. Phone: (313)577-2184; Fax: (313)577-2375; E-mail: [email protected]. MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; P21, P21WAIM. 3466 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1998 American Association for Cancer Research. APOPTOSIS AND PROSTATE CANCER CELL SURVIVAL MATERIALS AND METHODS form formazan dyes. Viable cells with active mitochondria cleave the tetrazo- lium ring into a visible dark blue formazan reaction product while dead cells Cell Culture, Antibodies, and Reagents. PC3. Du 145, and LNCaP (FGC- are not stained. The viable cells in each well were counted by bright-field light 10) cells were obtained from American Type Culture Collection and routinely microscopy. cultured in RPMI 1640 supplemented with 10% fetal bovine serum. 2 mM Western Blotting. Western blotting protocols were detailed before (38. L-glutamine. and antibiotics. NHP, BPH. and PCA cells were surgically 39). Whole-cell lysates were prepared by directly scraping control or starved isolated from clinically diagnosed and pathologically confirmed patients and prostate cells into the TNC lysis buffer [10 mM Tris-acetate (pH 8.0), 0.5% characterized as detailed previously (37). All of these primary cells were NP40. and 5 mM CaCU], and solubilized proteins were analyzed on 4-20% cultured in KBM basic medium (Clonetics) supplemented with 5 fig/ml gradient or 12% SDS-PAGE. Isolated proteins were transferred to nitrocellu insulin. 0.5 /^g/ml hydrocortisone. 10 ng/ml epidermal growth factor, 50 /J.g/ml lose and probed with individual primary antibodies as specified in "Results." bovine pituitary extract. 10 ng/ml cholera toxin, and antibiotics (37). Only The secondary antibody used was goat anti-rabbit or -mouse IgG conjugated to cultures of passages 2-5 were used throughout this study. The following horseradish peroxidase. Then membranes were stripped and reprobed with
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