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(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 onstrated an overall survivability in the order of BPH < NHP < LNCaP < PC3 < PCA < Du 145. Upon growth factor deprivation, NHP/BPH cells rapidly underwent apoptosis, leading to a decreased number of live cells. PCA/PC3/Dul45 cells, in contrast, demonstrated an initial phase of ag gressive growth during which apoptosis rarely occurred, followed by a "plateau" phase in which cell loss by apoptosis was compensated by cell accepted that apoptosis is a dominant feature unless suppressed or overridden by growth/survival factors; therefore, most eukaryotic cells (with the exception of, probably, blastomeres) will undergo apoptosis when deprived of appropriate survival factors (1, 2). In creased cell proliferation, extended cell survival, and diminished apoptosis have all been implicated in tumorigenesis. There has been a close association between apoptosis and prostate cancer initiation, progression, metastasis, and response to treatment. The growth and survival of normal prostate epithelial cells depend on androgens; therefore, castration or chemical blocking of androgen function leads to prostate atrophy. Prostate cancer is composed of androgen-dependent and androgen-independent cells. Androgen abla tion eliminates most androgen-dependent cancer cells by inducing proliferation, followed by a later phase in which apoptosis exceeded the cell proliferation. LNCaP cells demonstrated survival characteristics be tween those of NHP/BPH and PCA/PC3/Dul4S cells. We concluded that the increased survivability in prostate cancer cells results from enhanced cell proliferation as well as decreased apoptosis. The molecular mecha nisms for evasion of apoptosis in prostate cancer cells were subsequently investigated. Quantitative Western blotting was used to examine the pro tein expression of PS3 and P21WAIM, Bcl-2 and Bcl-XL (anti-apoptotic apoptosis but can rarely cure the patients due to the presence of androgen-independent cells and emergence of apoptosis-resistant clones (3, 4). Bcl-2 oncoprotein expression has been correlated with the progression and metastasis of prostate cancer cells and the emer gence of androgen-independent cells, as well as the generation of apoptosis- and therapy-resistant cancer cell clones (5-11). On the other hand, androgen-independent prostate cancer cells still retain proteins), and Bax, Bak, and Bad (proapoptotic proteins). The results revealed that, upon trophic factor withdrawal, NHP and BPH cells upregulated wild-type p53 and proapoptotic proteins Bax/Bad/Bak and down-regulated the expression of P21. Furthermore, NHP and BPH cells endogenously expressed little or no Bcl-2. In sharp contrast, prostate cancer cells expressed nonfunctional P53 and various amounts of Bcl-2 proteins. Upon deprivation, these cancer cells up-regulated P21 and Bcl-2 and/or BciXL, lost response to withdrawal-induced up-regulation of Bax/ appropriate apoptotic machinery and can be induced to undergo apoptosis by a wide spectrum of biological and pharmacological treatments, including wild-type P53 and P212 (12-14), prostate This project was undertaken to study the survival properties of various prostate cells, including normal (NHP), BPH (benign prostate hyperplasia), primary carcinoma (PCA), and metastatic prostate cancer cells (LNCaP, PC3, and Dul45), in the absence of trophic factors. Cell prolif eration and cell death were quantitated by enumerating the number of live cells using MTS/PMS kit and of dead (apoptotic) cells using 4',6-diamidino-2-phenylindole dihydrochloride nuclear staining. These cells dem Bad/Bak or decreased or even completely lost Bax expression and ex pressed some novel proteins such as P25 and P54/55 complex. These data together suggest that prostate cancer cells may use multiple molecular mechanisms to evade apoptosis, which, together with increased prolifer ation, contribute to extended survivability of prostate cancer cells in the absence trophic factors. INTRODUCTION Tissue and organ homeostasis is controlled by balanced cell pro liferation, cell survival, and cell death. Cell proliferation is driven and regulated by various peptide growth factors and/or cytokines. Cell survival represents the cell's intrinsic life expectancy in the absence of survival factors, many of which overlap with the growth factors/ cytokines that drive cell proliferation. Physiological cell death occurs mostly via programmed cell death or apoptosis (1). Apoptosis is a genetically controlled suicidal program that takes place, in many cases, when the cells run out of their survivability in the absence of growth/survival (or trophic) factors, although it can also be triggered by a wide variety of physiological or obnoxious stimuli. It is generally Received 2/5/98; accepted 6/2/98. 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. 1To whom requests for reprints should be addressed, at Department of Radiation Oncology, Wayne State University, 407 Life Science Building, Detroit, MI 48202. Phone: (313)577-2184; Fax: (313)577-2375; E-mail: [email protected]. apoptosis response-^ (15), prevention of adhesion (16), transforming growth factor ß(17), tumor necrosis factor a (18), Fas (19), chemotherapeutic drugs such as cisplatin, camptothecin, teniposide, and vincristine (20, 21), novel chemical entities such as Linomide (quinoline-3-carboxamide; Ref. 22), ß-lapachone (23, 24), phenylbutyrate (25), ribonucleotidase inhibitors (26), diethylstilbestrol (27), ß-L-(-)dioxolane-cytidine (28), and retinoid derivatives (29), and signal transduction modifiers such as phorbol esters (30-32) and thapsigargin (33). We recently reported apoptosis induction by prostate secre tory protein 94 (34) and hydroxamic acid compounds (35, 36) in hormone-refractory human prostate cancer cells. Little information exists in regard to differential properties of normal and cancerous prostate epithelial cells in terms of sensitivity to apoptosis induction. Furthermore, no systematic studies have been performed to investigate the interrelationship among the proliferation, survival, and apoptosis of various types of prostate cells. Recently, a series of normal (NHP2), BPH, and PCA prostate cultures have been established and characterized (37). In this study, we took advantage of these primary cultures and compared their growth and survival prop erties in the absence of trophic factors with those of the established malignant prostate cancer cell lines, i.e., LNCaP, PC3, and Dul45. We further investigated the changes and the potential roles of several apoptosis proteins in these six different cell types upon growth factor withdrawal. The results show that prostate carcinoma cells demon strate a much higher proliferative potential and survive much longer than normal and BPH cells. 2 The abbreviations used are: NHP, normal human prostate; BPH, benign prostate hyperplasia; PCA, primary prostatic carcinoma; DAPI, 4',6-diamidino-2-phenylindole; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; P21, P21WAIM. 3466 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research. APOPTOSIS MATERIALS AND PROSTATE form formazan dyes. Viable cells with active mitochondria cleave the tetrazo- AND METHODS Cell Culture, Antibodies, and Reagents. CANCER CELL SURVIVAL PC3. Du 145, and LNCaP (FGC- 10) cells were obtained from American Type Culture Collection and routinely cultured in RPMI 1640 supplemented with 10% fetal bovine serum. 2 mM L-glutamine. and antibiotics. NHP, BPH. and PCA cells were surgically isolated from clinically diagnosed and pathologically confirmed patients and characterized as detailed previously (37). All of these primary cells were cultured in KBM basic medium (Clonetics) supplemented with 5 fig/ml insulin. 0.5 /^g/ml hydrocortisone. 10 ng/ml epidermal growth factor, 50 /J.g/ml bovine pituitary extract. 10 ng/ml cholera toxin, and antibiotics (37). Only cultures of passages 2-5 were used throughout this study. The following primary antibodies were used in the immunoblotting experiments, (ai anti-p53: a mouse monoclonal anti-human wild-type p53 (clone DO-1; amino acids 21-25 as the immunogen) from Oncogene Research/Calbiochem and another mouse monoclonal anti-human wild-type p53 (clone DO-1 ; amino acids 37-45 as the immunogen) from Santa Cruz Biotechnology: (b) anti-p21: a rabbit polyclonal anti-human peptide antibody (amino acids 146-164 as the immuno gen) from Santa Cruz: (c) anti-Bcl-2: a mouse monoclonal anti-human peptide Bcl-2 (amino acids 41-54 as the immunogen) from either Dako (clone 124) or Calbiochem (clone 100), and a rabbit polyclonal anti-human Bcl-2 raised against a peptide corresponding to amino acids 4-21 (sc-492) from Santa Cruz; (d) anti-Bcl-Xs/I : a rabbit polyclonal anti-human peptide antibody (amino acids 2-19 as the immunogen) from Santa Cruz; (e) anti-Bax: a rabbit polyclonal anti-human Bax peptide antibody (amino acids 150-165 as the immunogen) from Calbiochem and a rabbit anti-human Bax peptide antibody (amino acids 43-61 as the immunogen) from PharMingen; (/) anti-Bak: a rabbit polyclonal anti-human Bak (amino acids 82-104 as the immunogen) from Santa Cruz; (#) anti-Bad: a rabbit polyclonal anti-mouse Bad (amino acids 185-204 as the immunogen) from Santa Cruz; and (/i) anti-actin: a mouse monoclonal anti-actin from ICN. The secondary antibody was either goat anti-rabbit or anti-mouse IgG (whole molecule) conjugated to horseradish peroxidase (Amersham). ECL reagents were bought from Amcrsham. The Western blotting experiment was performed as described previously (38. 39). For starvation, various cells cultured in regular media were washed (three times) with growth factor-free RPMI medium followed by culturing cells for different periods of time in RPMI. Quantification of Live Cells Using the MIS Assays. Cell survival was determined using the CellTiter MTS/PMS cell proliferation kit (Promega Corp.), as described (39). Briefly, log-phase growing prostate cells were cultured in Hat-bottomed 96-well plates (1 X IO4 cell/well) in either RPMI- lium ring into a visible dark blue formazan reaction product while dead cells are not stained. The viable cells in each well were counted by bright-field light microscopy. Western Blotting. Western blotting protocols were detailed before (38. 39). Whole-cell lysates were prepared by directly scraping control or starved prostate cells into the TNC lysis buffer [10 mM Tris-acetate (pH 8.0), 0.5% NP40. and 5 mM CaCU], and solubilized proteins were analyzed on 4-20% gradient or 12% SDS-PAGE. Isolated proteins were transferred to nitrocellu lose and probed with individual primary antibodies as specified in "Results." The secondary antibody used was goat anti-rabbit or -mouse IgG conjugated to horseradish peroxidase. Then membranes were stripped and reprobed with different antibodies. In some cases, the membrane was incubated with two different primary antibodies. The intensities of individual bands were quantitated by densitometric scanning (41). and the values were normalized against the intensity of actin. Characterization of Bcl-2-related Proteins. The Bcl-2 protein normally runs at M, —¿26.000 on SDS-PAGE under reducing conditions. Two prominent upper bands (M, -27.000 and M, 30.000. respectively) were also detected in this study using a monoclonal anti-Bcl-2 (raised against the 41-54 peptide of human Bcl-2) in both PC3 and/or Dul45 cells (see "Results"). These upper bands may represent phosphorylated matched control monoclonal antibody MOPC-21 (IgGl) were added to the precleared supernatants and incubated at 4°Cfor 2 h under rotating conditions. At the end. 100 ¿dof protein A/G-agarose beads were added, and the mixture was further incubated for l h at 4°C.The beads with bound antigen-antibody complex were washed extensively: TNC lysis buffer (two times). TNC-0.1% SDS (one time). TNC-1 M NaCI (one time), and TNC lysis buffer (one time). Finally, the washed beads were reconstituted in 50 ¡¿I of 2X SDS-PAGE sample buffer [125 mM Tris-HCI (pH 6.8), 10% ß-mercaptoethanol. 4% SDS. 20% glycerol. and 0.001% bromphenol blue]. After boiling. 25 /il of the precipitated sample were loaded onto a 12% SDS-PAGE. and separated proteins were transferred to nitrocellulose membrane and then blotted using the monoclonal or the rabbit polyclonal anti-Bcl-2. In the second set of experi 10% PCS medium (for LNCaP. PC3. and Dul45 cells) or in KBM medium supplemented with various growth factors (for NHP. BPH. and PCA cells). The wells were gently washed with plain media (times three), and then 200 /il of trophic factor-free RPMI were added to each well to start the deprivation ments. 50 /j.g of clarified PC3 or Du 145 cell lysates were incubated with 2 units of alkaline phosphatase at 37°Cfor 4 h. At the end. 25 /ig of the protein were loaded onto a 12% SDS-PAGE. and separated proteins were used for immunoblotting with the monoclonal anti-Bcl2. as described above. experiment. The number of live cells on each day thereafter was determined by the uptake of MTS/PMS dyes and measured on a 3550-UV microplate reader (Bio-Rad) using the/1.,,,,, absorbance. One set of wells were measured just prior to the media change, and the absorbance readings were used as the baseline value (i.e., day 0). The cell number on each day was expressed as the percentage cell number of the day 0. For each day's measurement, five- or six-well repeats were run, and each experiment was performed two to six times (see figure legends). A >100% cell number indicates cell growth during that period. Apoptosis Assays by Light Microscopy and DNA Fragmentation. These methods have been detailed previously (35. 36. 38. 39). Quantitation of Cell Death with DAPI Staining and MTT Assays. Different types of prostate cells cultured on glass coverslips in the absence of growth factors for various days were directly stained with DAPI (5 jag/ml) for 10 min at 37°C.At the end, coverslips were gently rinsed (two times) and mounted onto slides with a small amount of SlowFade anti-fade mounting media (Molecular Probes, Eugene, OR). The slides were observed under a Zeiss fluorescence microscope and pictures were recorded on Tmax-400 black/white films. The cells showing condensed chromatin and/or fragmented nuclei were scored as apoptotic. An average of 500-1000 cells were counted for each slide/day. The results were expressed as the percentage of apoptosis of day 0. Alternatively, cell growth/death were monitored by MTT (40) assays. In this assay, the MTT dyes are metabolized by mitochondria! dehydrogenases to isoforms of Bcl-2, some novel Bcl-2- related proteins, or simply nonspecific products. These possibilities were tested with the following two sets of experiments. In the first. PC3 and Du 145 cells were harvested, and cell lysates were prepared using the TNC lysis buffer, as described above. Five hundred /ig of PC3 or Du 145 cell lysates were used for immunoprecipitation experiments, as described previously (41) with some modifications. Briefly, the cell lysates in 1 ml of TNC lysis were first precleared by incubating with 75 fil protein A/G-agarose (Santa Cruz Biotech nology) at 4°Cfor 1 h. Then 5 /ig of the monoclonal anti-Bcl-2 or isotype- RESULTS Expression of Apoptosis Proteins in Various Prostate Cells. We first examined the expression of various apoptosis-related proteins in different types of prostate cells. The results are plotted in Fig. 1. All cells except PC3 expressed P53, as revealed by a monoclonal anti body. Note that longer exposure of the film also resulted in the p53 band in BPH cells (see also Fig. 7). P53 expressed in the primary cells. i.e., NHP, BPH. and PCA, was shown previously to be wild-type (37). LNCaP cells also express wild-type P53 (12). PC3 cells are P53-null; therefore, no protein could be detected (Fig. 1). Dul45 cells expressed the highest amount of P53 protein because the p53 gene in these cells has two mutations (codons 223 and 274), resulting in accumulation of the mutant protein (12). P21, a P53 target, was expressed in all of the cells including PC3 (Fig. 1). There appear to be some differences in terms of the protein amount of p2lWAF-' in different cells (Fig. 1). When using 25-100 jug of whole-cell lysates, the M, 26,000 Bcl-2 protein was undetectable or. in some cases (e.g.. Fig. 3S), only faintly detectable in NHP cells (Fig. 1). In similar experiments, Bcl-2 was not detected at all in BPH and PCA cells (Fig. 1; see also Figs. 7 and 9) 3467 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research. APOPTOSIS AND PROSTATE CANCER CELL SURVIVAL antibody (data not shown). Western blotting was also performed with three proapoptotic proteins, Bax, Bad, and Bak. All of the prostate cells expressed the M, -22,000 Bad (Figs. 3-7) and Mr -25,000 Bak (Fig. 1). All prostate cells except Dul45 expressed the Mr -21,000 P53 P21 —¿ WAF-l •¿21 kd ,30kd-Bcl-2 3cl-2 (27 kd) x26-kd Bcl-2 Bcl-2 Bel-XL —¿ Bax Bak . products. We performed two sets of experiments to differentiate among these possibilities. In the first, Bcl-2 and/or Bcl-2-related proteins were immunoprecipitated, and the precipitates were further subject to immunoblotting with the polyclonal or monoclonal antiBcl-2. This type of "double-shot" (i.e., immunoprecipitation followed •¿30kd ~ ^. by immunoblotting) protocol has been widely used to confirm the specificity of protein bands identified on Western blot. As shown in Fig. 24, the MT 30,000 band was specifically immunoprecipitated down by the monoclonal anti-Bcl-2 but not by control antibody MOPC-21, suggesting that this protein represents a Bcl-2-related 21 kd •¿25kd Actin Fig. 1. Expression of apoptosis proteins in prostate cells. Twenty five /ig of whole-cell lysates prepared from regularly cultured primary [i.e., NHP (normal), BPH. and PCA (primary) cells] or established prostate cancer cells (i.e., LNCaP, PC3. and Dul45) were resolved on 4-20% SDS-PAGE and used for immunoblotting with a monoclonal anti-P53 (amino acids 21-25 as the immunogen). The membrane was sequentially stripped [62.5 IHMTris-HCI (pH 6.5), 2% SDS, and 100 mM 2-ME, l h at 50°C]and reprobed with: rabbit polyclonal anti-p21v , monoclonal anti-Bcl-2 (amino acids 41-54 as the im munogen), polyclonal anti-Bcl-X^s, polyclonal anti-Bax (amino acids 43-61 as the immunogen), polyclonal anti-Bak, polyclonal anti-Bad (not shown), or monoclonal antiactin (see "Materials and Methods" for details). The same panels of antibodies were used in all of the subsequent experiments unless specified otherwise. As indicated in "Materials and Methods" and other parts of the text, several different antibodies were sometimes used for certain molecules for the purpose of characterization. The secondary antibodies used were either goal anti-rabbit or anti-mouse IgG conjugated to HRP. Left, identities of individual proteins; right, corresponding molecular weights. Actin is detected as a Afr —¿45.000 protein. The monoclonal anti-Bcl-2 consistently detected several different bands in LNCaP (M, 26,000 Bcl-2), PC3 (M, 26,000, M, 27,000, and M, 30,000 Bcl-2) and Du 145 cells (M, 30,000 Bcl-2). The M, 27,000 band is labeled as the phosphorylated Bcl-2 (see Fig. 2). in several repeat experiments, even after extended exposure. The M, 26,000 Bcl-2 protein was detected in LNCaP cells (Fig. 1). In PC3 cells, in addition to the Mr 26,000 Bcl-2, a Mr 27,000 and a Mr 30,000 protein were also detected (Fig. 1). In Dul45 cells, by contrast, only the Mr 30,000 protein was detected (Fig. 1) in addition to the Mr 26,000 Bcl-2, which was detected only when higher amounts of proteins/longer exposure were used (Fig. 2). This pattern of expres sion for Bcl-2 protein was also observed when the membrane was probed with a polyclonal antibody (data not shown). In general, the Bcl-XL protein was detected as a faint Mr —¿30,000protein in all prostate cells (Fig. 1, indicated by an arrow). This polyclonal antibody also recognized two prominent upper bands, the identities of which were unknown and whose intensities varied with different cell types. No Bcl-Xs protein was detected in any cell type with this polyclonal Bax protein, as revealed by an antibody recognizing the amino acids 43-61 of human Bax. The lack of Bax protein expression in Du 145 cells was also confirmed by another polyclonal anti-Bax raised against peptide 150-165 (data not shown). Interestingly, LNCaP and PC3 cells appear to express more Bax proteins than the three primary prostate cells (Fig. 1). The monoclonal anti-Bcl-2 used in our studies did not recognize any other Bcl-2 family proteins (Fig. 1 and data not shown). There fore, the A/r 27,000 and MT30,000 bands detected by this antibody in PC3 and/or Du 145 cells may represent phosphorylated Bcl-2 protein (42-46) or a new Bcl-2-related protein, such as BRAG-1, which migrates at M, —¿30,000 (47), or simply represent nonspecific reaction protein instead of a nonspecific product. Blotting with the polyclonal anti-Bcl-2 antibody produced similar results (data not shown). Note that the status of the Mr 26,000 and Mr 27,000 proteins in this experiment was not very clear due to the proximity of these bands to the immunoglobulin light chain (MT—¿25,000; Fig. 2A). The direct use of whole-cell lysates in immunoblotting again identified the Mr 26,000, Mr 27,000, and M, 30,000 (on the order of Mr 26,000 » Mr 27,000 > Mr 30,000 in terms of intensity) protein bands in PC3 cells, and Air 26,000 and Mr 30,000 (Mr 30,000 » Mr 26,000) protein bands in Du 145 cells (Fig. 24). Only the Mr 26,000 Bcl-2 protein was detected in A431 human epidermoid carcinoma cells (used as a control; Fig. 24, Lane 1). In the second set of experiments, whole-cell lysates from PC3 and Dul45 cells were treated with alkaline phosphatase and then immunoblotted with monoclonal anti-Bcl-2. As shown in Fig. 2B, in PC3 cells, phosphatase treatment dramatically decreased the amount of the Mr 27,000 protein with a simultaneous increase in the quantity of MT 26,000 protein, suggesting that the MT 27,000 protein in PC3 cells is phosphorylated Bcl-2. In contrast, the level of the Mr 30,000 protein in PC3 cells was hardly affected by the phosphatase treatment, which similarly did not affect the mobility of the Mr 30,000 protein in Dul45 cells (Fig. 20). The intensity of the MT 30,000 protein in Du 145 cells appeared to decrease a small amount; however, no increased Mr 26,000 Bcl-2 protein was observed after the phosphatase treatment (Fig. 2B). Taken together, these data suggest that the Mr 27,000 protein represents the phosphorylated isoform of Bcl-2 protein, and the Mr 30,000 protein may represent a novel Bcl-2-related protein, the true molecular identity of which awaits further characterization. Survival of NHP Cells in the Absence of Trophic Factors. As early as 24 h after the withdrawal of growth factors, NHP cells stopped proliferation, as measured by MTS assays (Fig. 3A). There after, interestingly, there appeared to have a slight "bouncing-back" in the cell growth 48-72 h after culturing in the absence of trophic factors (Fig. 3A), as observed consistently in several repeat experi ments. The reason for this observation was not clear. DNA fragmen tation assays (Fig. 3B, inset), DAPI staining (Fig. 3Q, and MTT assays (not shown) revealed that NHP cells died through apoptosis. 3468 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research. APOPTOSIS Anti-Bcl-2 Whole cell lysates s 97.4 —¿ 66 —¿ 46 —¿ 30 —¿ 21.5 —¿ AntiBcl-2 Blotting MOPC --pn kî à Srià D AND PROSTATE CANCER CELL SURVIVAL B Anu-Bcl-2 I.P PC3 pi u fi + U —¿Ij; heavy chain ^-* •¿Â»â€¢*-i light 97.466- Cells - Alk. phos. + •¿â€” —¿Â» -BSA 4630- chain 14.3—¿ - Blotting Dul45 21.5—¿ ,-30kd-Bcl-2 =^©-Bcl-2 (27 kd) Bcl-2 (26 kd) Fig. 2. Characterization of Bcl-2-related proteins. In A. the M, 30,000 protein represents a specific Bcl-2-related protein. Five hundred /ig whole-cell lysates from either PC3 (Lanes 4 and 6) or Dul45 (¡Mnes5 and 7) cells were immunoprecipitated with either monoclonal anti-Bcl-2 (Lanes 4 and 5) or isotype-matched control antibody MOPC-21 (Lanes 6 and 7). The immunoprecipitates were resolved on a 12% SDS-PAOE. and the membrane was probed with the monoclonal anli-Bcl-2. As controls, 5 fig of total cell lysates from A431 (human epidermoid carcinoma; Lane /), 50 ng of whole-cell lysates from Du 145 (Lane 3). or 70 fig of lysates from PC3 cells (Lane 2) were directly used for Western blotting. Left, molecular weight markers. Right. Ihe immunoglobulin heavy (M, ~54,000) and light (M, —¿25.000) chains under reducing conditions. The M, —¿66,000 band in the whole-cell lysates (Lanes 1-3) represents the BSA (the monoclonal anti-Bcl-2 was raised against a peptide conjugated to BSA). The Afr 30,000 Bcl-2 protein was precipitated by anti-Bcl-2 but not by MOPC-21 (arrowhead on the right). B, the M, 27,000 band is the phosphorylated Bcl-2 protein. For details, see "Materials and Methods" and other parts of the text. In untreated PC3 cells (indicated by the "-" sign), the M, 26,000, M, 27,000, and M, 30,000 proteins were identified by the monoclonal anli-Bcl-2, which detected the M, 30.000 and the faint M, 26,000 protein in untreated Du 145 cells. Phosphatase (Alk. phos. ) treatment (indicated by the " + " sign) resulted in the disappearance of the M, 27.000 with simultaneous increase in the amount of M, 26,000 Bcl-2 in PC3 cells, without affecting the mobility of the M, 30.000 band in either PC3 or Dul45 cells. Increased apoptosis (i.e., positive DAPI labeling) was observed 24 h after growth factor withdrawal. On day 2, —¿20% cells were apoptotic (Fig. 3, B and C, middle panel). By day 7, 60-70% cells were dead (Fig. 3, B and C. right panel). Characterization of cell growth and cell death by several different methods (i.e., MTS assays, DAPI staining, DNA fragmentation assays, and MTT) overall produced consistent results; NHP cells do not proliferate and undergo steady apoptotic death upon removal of trophic factors (Fig. 3 and data not shown). Note that the DNA ladder formation in all three primary cells (Fig. 3; see also Figs. 6 and 8) was not as clear-cut as in established cell lines (see Figs. 10, 12, and 14). This is not surprising because apoptosis in primary cultures often is not accompanied by distinct DNA ladder formation (reviewed in Ref. 4). Western blotting was used to examine the changes of apoptosis proteins. As shown in Fig. 4, all proteins except P21 increased immediately after deprivation, as determined by densitometric scanning after normalizing the protein levels to that of actin (Fig. 5: see legend for details). The most significant increase was observed with Bcl-XL (> 15 fold by day 6; Figs. 4 and SA). The NHP cells expressed barely detectable levels of Bcl-2, which appeared to increase marginally by days 3 and 4 (Fig. 4). In contrast, all three proapoptotic proteins, i.e., Bax, Bad, and Bak, significantly increased with time (Figs. 4 and 5A). P53 also demonstrated a 2-3-fold increase, whereas its target. P21, did not show any corresponding changes and whose levels actually declined sharply by day 5 (Figs. 4 and 5/4). Therefore, the P21 expression in NHP cells appears to be p53 inde pendent. Survival of BPH Cells in the Absence of Trophic Factors. Like NHP cells, BPH cells did not grow, upon growth factor withdrawal, as revealed by MTS assays (Fig. 6). In fact, BPH cells appeared to be more sensitive than NHP cells to deprivation because they demon strated an even faster loss in the number of live cells (compare Figs. 6A and 3/4). For example, —¿40% of BPH cells were apoptotic (Fig. 6, B and C, middle) by day 2 compared with —¿20% apoptosis in NHP 3, after which its levels gradually decreased (Figs. 7 and 5ß).The P21 level did not undergo appreciable changes and was not affected by P53 up-regulation. No Bcl-2 was detected in these cells (Fig. 7). The Bcl-XL level was also very low, which showed only a marginal increase (Figs. 7 and 5B). As in NHP cells, Bax and Bad proteins persistently increased with time; however, Bak did not show any changes (Figs. 7 and 5B). Survival of PCA Cells in the Absence of Trophic Factors. PCA cells behaved completely differently with respect to their survival in the absence of trophic factors. Immediately (i.e., 24 h) after growth factor withdrawal, PCA cells proliferated (>2.5 fold increase in cell number) as determined by MTS assays (Fig. 8). PCA cells did not demonstrate significant (15-20%) apoptosis until days 5-6, as sup ported by faintly increased DNA ladder formation (Fig. 8ß,inset). MTT assays (data not shown) also confirmed increased cell prolifer ation and rare cell death. The total number of live cells did not decrease, even by day 5 when increased apoptosis was observed (Fig. 8, B and C, middle panel), suggesting that continued cell proliferation compensated for the cell loss. By day 11, nearly the same number of cells (i.e., the same number of cells as initially input or 10,000 cells/well; see "Materials and Methods") were still alive (Fig. 8, A and C, right panel). The most significant changes in apoptosis proteins in PCA cells after trophic factor deprivation were the up-regulation of p2]WAF-i and BCI_XL around day 4 (Figs. 9 and 5Q, prior to the initiation of apparent DNA fragmentation (Fig. 8S, inset). Like NHP and BPH cells, PCA cells expressed extremely low levels of Bcl-2, which was undetectable (Fig. 9). In sharp contrast to NHP and BPH cells, all other proteins, including P53, Bax, Bad, and Bak, were not up-regulated (Fig. 9). However, interestingly, three prominent protein bands, i.e., a MT -25,000 protein and a M, 54,000/55,000 doublet, were simultaneously detected on day 4 after growth factor withdrawal, when using the monoclonal anti-P53 for the Western blotting (Fig. 9, top panel). These two novel entities of proteins were designated P25 cells at the same time (Fig. 3). By day 7, >80% cells were dead (Fig. and P54/55, respectively. Survival of LNCaP Cells in the Absence of Trophic Factors. 6, B and C, right panel). Western blotting revealed quite different patterns of changes in apoptosis proteins than found in NHP cells. The LNCaP cells demonstrated marginal proliferation upon serum starva P53 up-regulation was most dramatic, with —¿30-fold increase by day tion, as revealed by MTS assays (Fig. 10/4). However, the cells did not 3469 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research. APOPTOSIS AND PROSTATE proteins except P21 increased (2-10-fold) in expression levels upon serum starvation (Figs. 11 and 5D). Also like in NHP cells, the P21 levels decreased in a time-dependent manner, which was barely de tectable by day 7 (Fig. 11). The only significant difference is that the up-regulation of Bcl-XL in LNCaP cells was not as dramatic as in NHP cells. Interestingly, the anti-P53 detected a lower band in addi tion to the expected M, 53,000 protein. The molecular nature of this lower band is unclear, although it might be a proteolytic product (48). Survival of PC3 Cells in the Absence of Trophic Factors. PC3 cells behaved like PCA cells when deprived of serum growth factors. These cells demonstrated a steady growth, which peaked on day 3 (Fig. \2A). The cell proliferation plateaued on day 4 (Fig. 1X4), when increased apoptosis (DAPI labeling and DNA fragmentation) was observed (Fig. 12, B and C, middle panel). This increased cell death clearly negated the cell proliferation; therefore, the net number of live cells did not further increase on day 4 (Fig. \2A). Starting from day 5, the number of live cells began to decrease (Fig. \2A), suggesting that, by this time, cell death (Fig. 125) began to surpass the cell prolifer ation. Direct quantitation of apoptosis demonstrated that by day 8, —¿60% of the cells were apoptotic (Fig. 12, B and C, righi panel). This loo- 20 •¿ 0 1 2 3 4 5 CANCER CELL SURVIVAL C Time (days post growth factor withdrawal) B observation was consistent with the MTS measurement showing that approximately the same number of cells (i.e., 100% cell number) as initially input (i.e., 10,000 cells/well) were still alive (Fig. 12A) by day 8, considering that PC3 cells initially had a >2-fold proliferation (Fig. 12A). Western blotting examination of apoptosis proteins also re vealed similar alterations as observed in PCA cells in that PC3 cells i Time (days post growth factor withdrawal) Days after growth factor withdrawal 0 1 Actin -/ P21 WAF-l Bcl-2 Fig. 3. Survival of NHP cells in the absence of trophic factors. A. quantitation of live cells using the MTS assays. The percentage cell number (compared to DO, which is 100%) represents means derived from three independent experiments; bars, SE. B. quantitation of apoptosis by DAPI staining. Inset, DNA fragmentation. C, micrographs showing DAPI-stained NHP cells starved for 0 (¡eft).2 (middle), or 7 (right) days. Apoptotic cells were brightly stained due to chromatin condensation. Note that in the middle and righi panels, some apoptotic cells turned necrotic in culture and thus degraded. Therefore, an overall decreased cell number was noticed. The arrowheads in the left and middle panels illustrate apoptotic NHP cells, whereas the arrowheads in the right panel indicate very few live cells. X100. die immediately (as did NHP and BPH cells) until days 3-4 (Fig. 10ß).Prominent apoptosis (DAPI labeling and DNA fragmentation) was observed around day 4 (Fig. 10, B and C, middle panel), yet the total cell number (i.e., the same number as initially input) was main tained up to day 6, after which the cell number went down (Fig. 10A). These observations suggest that the cell loss due to apoptosis between days 4-6 was partially compensated by cell proliferation. By day 8, —¿70% cells were apoptotic (Fig. 10, A-C, right panel). Immunoblotting examination of various apoptosis proteins revealed quite similar patterns of alterations as observed in NHP cells. Specifically, all Bax Bak —¿Â«.' (22 kd) Fig. 4. Alterations in apoptosis proteins in NHP cells after growth factor withdrawal. Thirty u.g of whole-cell lysates prepared from unstarved NHP cells or from cells cultured in the absence of growth factors for various days were resolved on a 12% SDS-PAGE. The same membrane was sequentially probed with individual or mixed primary antibodies (see Fig. 1). Left, individual molecules. The arrowhead on the right points to the M, —¿30.000 Bcl-XL. 3470 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research. APOPTOSIS AND PROSTATE CANCER CELL SURVIVAL 10.0 Vi 4) a a. xv w '•O £ "3 06 5.0 v äs 2.5 0.0 DU Dl D2 D3 D4 Ãœ5 D6 D7 DO Dl D2 Time (days) DS D6 e ^o 'Z vt o o. X 4) D4 Time (days) 4- W D3 3- s. X 4) 4) •¿^ 2- —¿ —¿ "w i» 1- —¿l— DO Dl —¿l— D2 D3 —¿l— D4 —¿l— DS D6 DO Dl D2 D3 Time (days) D4 DS D6 D7 D8 Time (days) o VI P21 Bcl-2 Bcl-XL 4- Bak Bad vt 2o. 4l hl =X 9) X v 3- W 2: 2H —¿ "«3 W ai 1- DO Dl D2 D3 D4 DS D6 D7 DO Time (days) Dl D2 D3 Time D4 DS D6 D7 (days) Fig. 5. Quantitative presentation of scanning data as determined by densitometry. The intensity of each individual protein band was first determined by scanning (41) and then normalized to the intensity of actin protein band. After normalization, the individual protein levels are plotted as "relative expression" {i.e., fold changes) compared with unstarved cells (i.e.. DO samples), the protein expression levels of which are considered as \.A, NHP cells. B. BPH cells. The values for P53 are: 1, 1.8, 9.7, 30.9, 15.4, 17.5, and 15 from DO to D6, respectively. C, PCA cells. Note that the data for P53 were not plotted because the P53 protein on immunoblot overlapped with the P54/55 doublet. D, LNCaP cells. E. PC3 cells. The values for Bcl-XL are: 1, 1.1, 0.9, 1.0, 1.0, 1.2, and 1.1 from DO to D7. respectively. F. Dul45 cells. Bcl-2 represents the M, 30,000 Bcl-2 protein band. 3471 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research. APOPTOSIS AND PROSTATE CANCER CELL SURVIVAL with the M, 26,000 Bcl-2 protein (Fig. 13). In contrast, the Bcl-XL protein did not change significantly (Figs. 13 and 5£). Survival of Dul45 Cells in the Absence of Trophic Factors. Dul45 cells demonstrated the highest survival potential (Fig. 144). Upon serum removal, these cells gradually proliferated, nearly dou bling the cell number by day 3 when DNA fragmentation was first observed (Fig. 14ß),suggesting that the cell proliferation exceeded cell death by apoptosis. The death rate in starved Du 145 cells dem onstrated a very slow kinetic (Fig. 14ß),from ~10% on day 3, to -15% on day 6 (Fig. 14C, middle), and to -25% by day 12, at which time still about the same number (i.e., 10,000 cells/well initially plated) of cells were alive (Fig. 14C, right panel). A portion (—5%) 1234567 Time (days post growth factor withdrawal) of the Du 145 cells in the culture survived up to 30 days after serum withdrawal (data not shown). Western blotting of apoptosis proteins revealed that the proapoptotic proteins Bak and Bad did not undergo significant changes in their expression levels (Figs. 15 and 5F), as in Days after serum withdrawal n 3 l 4 6 P53 234567 Time (days post growth factor withdrawal) WAF-l P21 Bcl-2 Bax Fig. 6. Survival of BPH cells in the absence of trophic factors. A, quantitation of live cells using the MTS assays. The percentage cell number (compared to DO. which is 100%) represents means derived from three independent experiments; bars, SE. B, quantitation of apoptosis by DAPI staining. Inset. DNA fragmentation. C micrographs showing DAPI-stained NHP cells starved for 0 (left), 2 (middle), or 7 (right) days. Apoptotic cells were brightly stained because of chromatin condensation. Note that in the middle and right panels, some apoptotic cells turned necrotic in culture and thus degraded. Therefore, an overall decreased cell number was noticed. The arrowheads in the left and middle panels illustrate apoptotic BPH cells, whereas the arrowheads in the right panel indicate very few live cells. X100. lost their response in up-regulating all three proapoptotic proteins upon serum deprivation (Figs. 13 and 5£).In fact, the Bax protein actually went down dramatically by day 6 (Figs. 13 and 5£).Also similar to PCA cells, the P21 levels significantly increased by day 3 prior to the initiation of DNA fragmentation (Figs. 13 and 5£). Another aspect of the similarity between PC3 cells and PCA cells was revealed by the detection of the P54/55 doublet in PC3 cells on day 5 after serum starvation (Fig. 13). Interestingly, the P25 band detected in PCA cells was not observed in PC3 cells (Fig. 13). Different from PCA cells, PC3 cells expressed the Mr 26,000 Bcl-2 protein, the levels of which significantly increased upon serum starvation (Figs. 13 and 5E). The M, 27,000 Bcl-2 protein levels also elevated simultaneously Bak - „¿ Bad Actin Fig. 7. Alterations in apoptosis proteins in BPH cells following growth factor with drawal. Thirty ¿tgof whole cell lysates prepared from unstarved BPH cells or from cells cultured in the absence of growth factors for various days were resolved on a 12% SDS-PAGE. The same membrane was sequentially probed with individual primary antibodies (see Fig. 1). Left, individual molecules. The arrowheads on the right point to the M, -30,000 Bcl-X, and M, -22,000 Bad, respectively. Note the upper band in the Bad blot was incompletely deprobed Bak. 3472 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research. APOPTOSIS AND PROSTATE CANCER CELL SURVIVAL A 3fl0 (37). All three types of cells were diploid in karyotype, positive for both keratinocytes and PSA, and negative for ras and p53 mutations (37). When cultured in defined medium containing various growth factors, these cells demonstrated progressively accelerating growth properties; the population doubling times of NHP, BPH, and PCA cells were 51, 37, and 29 h, respectively (37). Here we compared the growth and survival properties of these primary prostate cells with those of three established prostate cancer cell lines in the absence of trophic factors. We first characterized the expression of several apoptosis-related proteins in different types of prostate cells, from normal to malignant. All cells express P21, Bcl-XL, Bad, and Bak, and all cells except Du 145 express Bax protein. However, primary cells generally express little or no Bcl-2, whereas LNCaP cells express abundant Bcl-2 protein. In PC3 and Du 145 cells, a more complicated pattern of Bcl-2 expression was observed. In addition to the regular Mr 26,000 Bcl-2, PC3 cells also express two minor upper bands that migrate at M, 27,000 and M, 30,000, respectively. In contrast, Du 145 cells 25*' 200 lso ,„ 50 2 4 6 g 10 12 Time (days post growth factor withdrawal) Days post growth factor withdrawal 23456789 1 0123451 i ii 6 Time (days post growth factor withdrawal) P53 Actin -/ P21 P54/55 i WAF-l Bcl-2 Fig. 8. Survival of PCA cells in the absence of trophic factors. A, quantitation of live cells using the MTS assays. The percentage cell number (compared to DO. which is 100%) represents means derived from three independent experiments; bars, SE. B, quantitation of apoptosis by DAP] staining. Inset, DNA fragmentation. C. DAPI-stained NHP cells starved for 0 (left), 5 (middle), or 11 (right) days. Apoptotic cells were brightly stained due to chromatin condensation. The arrowheads illustrate apoptotic PCA cells. X100. Bcl-XL PCA and PC3 cells. Also different from the PCA/PC3 cells, Du 145 did not express Bax protein, as tested by several antibodies raised against different regions of the molecule (Fig. 15; data not shown). The most salient feature is the expression of the Mr 30,000 Bcl-2 protein in Du 145 cells, the levels of which increased steadily after serum starvation (Figs. 15 and 5F). Other aspects in the changes of apoptosis proteins after serum starvation in Du 145 cells were similar to those observed in PC3 cells. Specifically, P21 protein levels went up (nearly 2-fold), whereas the Bcl-XL underwent a slight increase (Figs. 15 and 5F). Also, like in PC3 cells, the P54/55 doublet was detected in Du 145 cells on day 3 after serum starvation (Fig. 15). Interestingly, the P25 protein band was also detected (Fig. 15). DISCUSSION Recently, NHP, BPH, and PCA cells were established, and their growth properties and nutritional requirements were characterized —¿â€”^ Bax Bak Bad ___. Fig. 9. Alterations in apoptosis proteins in PCA cells after growth factor withdrawal. Thirty fig of whole-cell lysates were prepared from unslarved PCA cells or from cells cultured in the absence of growth factors for various days were resolved on a 12% SDS-PAGE. The same membrane was sequentially probed with individual primary antibodies (see Fig. 1). Left, individual molecules. The arrowhead on the right points to the M, -30,000 Bcl-X,. Right, novel P25 and P54/55 proteins. 3473 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research. APOPTOSIS AND PROSTATE CANCER CELL SURVIVAL treatment established the Mr 27,000 band but not the Mr 30,000 band as the phosphorylated Bcl-2 protein. Therefore, the Mr 30,000 Bcl-2 protein identified in PC3 and Du 145 cells is different from the Mr 30,000 Bcl-2 in HT29 cells, which is a hyperphosphorylated form of the Mr 26,000 Bcl-2 (46). It is not yet known whether the Mr 30,000 Bcl-2 protein in PC3/Dul45 cells is or is related to the newly reported Mr 30,000 BRAG-1, a Bcl-2-related protein primarily expressed in the brain (47). Both Mr 27,000 and Mr 30,000 Bcl-2 proteins are detected in PC3 and/or Du 145 cells without any prior stimulation, suggesting that these proteins are endogenously expressed in both cells. 2468 Time (days post serum withdrawal) B 10° Days after serum withdrawal I 01234567 MO 1 2 3 4 5 (days) I 8 CO 4» 20 2468 P21 10 WAF-l Time (days post serum withdrawal) Bcl-2 Bel-XL Bax Fig. 10. Survival of LNCaP cells in the absence of trophic factors. A, quantitation of live cells using the MTS assays. The percentage cell number (compared to DO, which is 100%) represents means derived from three independent experiments; bars, SE. B, quantitation of apoptosis by DAPI staining. Inset, DNA fragmentation. C, DAPI-stained NHP cells starved for 0 (left), 4 (middle), or 8 (right) days. Apoptotic cells were brightly stained because of chromatin condensation. The arrowheads illustrate apoptotic LNCaP cells. XI00. ^„.»-»-^^ Bak Bad appear to primarily express the Mr 30,000 band with very low levels of the Mr 26,000 Bcl-2 protein. It is well known that Bcl-2 can undergo phosphorylation on serine residues, resulting in either Mr 27,000 Bcl-2 in multiple cell types such as leukemia, MCF-7, and PC3 cells (42-46) or Mr 30,000 Bcl-2 in HT-29 colon cancer cells (46). The Mr 30,000 Bcl-2 protein has not been reported in either PC3 or Du145 cells, even after treatment with taxol or okadaic acid (42,43, 45). We therefore further characterized the Mr 27,000 and A/r 30,000 bands detected in PC3 and/or Du 145 cells by the monoclonal antiBcl-2 antibody. Immunoprecipitation followed by Western blotting first established the Mr 30,000 band in both cell types as a specific Bcl-2-related protein instead of nonspecific antibody binding. Subse quently, a dephosphorylation experiment with alkaline phosphatase Fig. 11. Alterations in apoptosis proteins in LNCaP cells after growth factor with drawal. Thirty (ig of whole-cell lysates prepared from unstarved LNCaP cells or from cells cultured in the absence of serum for various days were resolved on a 12% SDS-PAGE. The same membrane was sequentially probed with individual primary antibodies (see Fig. 1). Left, individual molecules. The arrowhead on the right points to the Mr -30,000 Bcl-XL. Note that the anti-P53 detected the expected M, 53,000 protein and a lower band, whose identity is presently unknown. 3474 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research. APOPTOSIS AND PROSTATE CANCER CELL SURVIVAL in the absence of trophic factors, than in PC3/PCA/Dul45 cells (summarized in Fig. 16), suggesting that cancerous prostate cells naturally possess a longer life span (or survivability) than NHP/BPH cells. LNCaP cells, initially isolated from the lymph node metastasis of a prostate cancer patient, demonstrate a survivability between that of NHP/BPH and that of PC3/PCA/Dul45 cells. These cells behave differently from other cancer cells in that they do not proliferate significantly in the absence of trophic factors. However, LNCaP cells also behave differently from NHP/BPH cells in that they do not die immediately after serum withdrawal. Instead, these cells keep a rela tively constant cell number for 5-6 days, although the apoptosis is initiated at approximately day 4. The mechanism(s) for the unique survival characteristics of LNCaP cells are presently unknown. Taken together, the present data suggest that prostate cancer cells generally demonstrate longer survivability than their normal counterparts due to increased proliferative capability and decreased cell death rate. The increased cell proliferation results from the production by cancer cells of various growth factors (49), whereas the decreased death rate 300 250 200 150 100 * 2 4 6 H Time (days after serum withdrawal) B 100 80 L co ä. •¿< 20" .2 Days post serum withdrawal 6543210 Days after serum withdrawal 2468 10 6 234 7 Time (days after serum withdrawal) P21 WAF-l _._ _ «M * =: P54/55 ©-Bcl-2 (27 kd) Bcl-2 Bcl-Xt Bax Fig. 12. Survival of PC3 cells in the absence of trophic factors. A. quantitation of live cells using the MTS assays. The percentage cell number (compared to DO, which is 100%) represents means derived from six independent experiments; bars, SE. B. quantitation of apoptosis by DAP1 staining. Inset. DNA fragmentation. C, DAPl-stained NHP cells starved for 0 (left). 4 (middle), or 8 (right) days. Apoptotic cells were brightly stained due to chromatin condensation. X 100. Growth and Survival of Various Prostate Cells upon Trophic Factor Withdrawal. NHP and BPH cells do not grow and demon strate the lowest survival capacity upon trophic factor withdrawal. Apoptosis is observed in these cells as early as 24 h after growth factor withdrawal. In contrast, primary (PCA) and metastatic (PC3 and Dul45) carcinoma cells survive much longer, and these cells keep dividing and proliferating in the absence of trophic factors. These three cancer cell types follow very similar growth/survival patterns upon removal of trophic factors: an initial aggressive growth phase (the first 2-3 days) in which apoptosis rarely occurs; a second "coun terbalancing" phase in which cell loss by apoptosis (taking place on days 3-4) is compensated by sustained cell proliferation; and a third "death" phase in which apoptosis surpasses the cell growth. Direct measurement of cell death by DAPI staining and MTT assays (not shown) has demonstrated a much higher death rate in NHP/BHP cells. Bak —¿ Bad Actin _ Ä _. . ^ Fig. 13. Alterations in apoptosis proteins in PC3 cells after scrum withdrawal. Thirty Hg of whole-cell lysates prepared from unstarved PC3 cells or from cells cultured in the absence of serum for various days were resolved on a 12% SDS-PAGE. The same membrane was sequentially probed with individual primary antibodies (see Fig. 1). Left, individual molecules. The arrowhead on the right points to the M, -30.000 Bcl-X,. The Bcl-2 proteins and Ihe P54/55 doublet are also labeled on the riditi. Note that Lanes 2 and 3 (i.e., days I and 2) loaded less protein, as evidenced by the actin band. Thus, the intensities of these two lanes and all of the rest of the lanes were normali/ed against that of the actin band. 3475 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research. APOPTOSiS AND PROSTATE trum of human tumor cells (4). In this study, there appears to have a correlation between the P53 response and apoptotic sensitivity of various prostate cells. NHP, BPH, and LNCaP cells, which are the cell types sensitive to apoptosis induction by trophic factor withdrawal, demonstrated a time-dependent increase in P53 protein expression. BPH cells demonstrated the most robust (~30-fold increase by day 3) and longest up-regulation of P53, and these cells are most sensitive to apoptosis induction. In contrast, PCA cells, although expressing wildtype P53, do not demonstrate up-regulation of P53, and these cells survive much longer than NHP, BPH, and LNCaP cells (Fig. 16). Similarly, PC3 and Dul45 cells, which do not express functional P53, also possess longer survivability in the absence of trophic factors. Taken together, these observations correlate an up-regulation of func tional P53 with increased sensitivity of prostate cells to apoptosis induction by deprivation (Fig. 16). Quite contrasting alterations are also seen with P21 in sensitive 3 2468 10 Time (days after serum withdrawal) B 100 CANCER CELL SURVIVAL Days post serum withdrawal 0123-156 Ml Days after serum withdrawal 40 012345 2468 P21 10 67 - WAF-l Time (days after serum withdrawal) P54/55 Bcl-2 30kd-Bcl-2 Bel-XL Bax Fig. 14. Survival of Du 145 cells in the absence of trophic factors. A. quantitation of live cells using the MTS assays. The percentage cell number (compared to DO. which is 100%) represents means derived from four independent experiments: bars, SE. B, quantitation of apoptosis by DAPI staining. Insel, DNA fragmentation. C, DAPI-stained NHP cells starved for 0 (left}, 6 (middle), or 12 (righi) days. Apoptotic cells were brightly stained because of chromatin condensation. X100. results from evasion of apoptosis. It is of note that PCA, one of the primary cell types used, demonstrates even higher survivability than LNCaP and PC3 cells, which are established cell lines kept in culture for many generations. Therefore, it is unlikely that any of the differ ences observed among these cells may have resulted from tissue culture artifacts. Role of p53 and P21 in Prostate Cell Growth/Apoptosis upon Trophic Factor Withdrawal. p53 plays two essential roles in main taining the cellular homeostasis: introducing "checkpoints" to arrest cell cycle; and triggering apoptosis to self-destroy those cells beyond repair (50). p53 mediates its biological functions by transcriptionally regulating (either activating or inhibiting) many of its targets, among which is p21, a CIP/KIP family cyclin-dependent kinase inhibitor. In response to genotoxic shocks, cells generally halt cell cycle progres sion by up-regulating P21 protein in a p53-dependent manner. Overexpression of wild-type p53 induces apoptotic death of a wide spec- Bad Actin —¿ •¿â€¢Â» —¿ —¿ —¿â€¢ « Fig. 15. Alterations in apoptosis proteins in Du 145 cells after serum withdrawal. Thirty fig of whole-cell lysates prepared from unstarved Du 145 cells or from cells cultured in the absence of serum for various days were resolved on a 12% SDS-PAGE. The same membrane was sequentially probed with individual primary antibodies (see Fig. 1). Left, individual molecules. The arrowhead on the right points to the A/r —¿30,000 Bcl-X,. The M, 30,000 Bcl-2, P25, and P54/55 doublet are also labeled on the right. Note that the regular Mr 26.000 Bcl-2 protein was barely detectable in this experiment. Note that Lane 2 (i.e., day 1) loaded less protein, as evidenced by actin band. Thus, the intensities of this lane and all of the rest of the lanes were normalized against that of the actin band. 3476 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research. APOPTOSIS AND PROSTATE CANCER CELL SURVIVAL mutantP21:A —¿ ABcl-XL: Bcl-2: Bcl-2: ABcl-XL: p53:iP21:-Bcl-2: p53:AP21:fBcl-2: RiiBcl-2: ABad/Bak: little/noBcl-XLlBax/Bad/Bak: —¿Bad/Bak: fBcl-2: 1: —¿Bax: little/noBcl-XL: A,Bcl-XL:ABax/Bad/Bak: little/noBcl-XL:ABax/Bad/Bak:AGrowth —¿Bax:JP25/P54/55: —¿P25/P54/55: deletedP25/P54/55: A.Bax/Bad: yesGrowth yesGrowth yesGrowth AGrowth AGrowth +++Apop -Apop -Apop +++Apop +++Apop +Apop +•Dul45 ++£PC3~^0wtp53: +• +++BPH +++NHP ++i nullP21:A Awt Fig. 16. A, the overall survivability of various prostate cells in the absence of trophic factors is presented: BPH < NHP < LNCaP < PC3 < PCA < Dul45. Shown above are changes in apoptosis proteins. Refer to text for details. —¿, no change; upward arrowhead, increase; downward arrow head, decrease; little/no, little or no expression. Shown in the middle are the cell growth and apop tosis properties of various prostate cells in the ab sence of trophic factors, as deduced from preceding figures. —¿, no growth. + to +4-+, different levels of apoptosis. BPH and NHP cells do not proliferate at all and demonstrated fast apoptotic kinetics. LNCaP cells demonstrated a low level of proliferation and an intermediate level of apoptosis. PC3 cells demonstrated very aggressive growth and interme diate levels of apoptosis. PCA and Du 145 cells, in contrast, demonstrated a rapid cell proliferation as well as a much delayed and decreased levels of apoptosis. B, summary of molecular events leading to differential survivabilities of normal versus can cerous prostate epithelial cells in the absence of trophic factors. Note thai there exist exceptions to this generalized scheme. For example, PCA cells possess wild-type P53 and express little/no Bcl-2. -^wt B ~^0wtp53:AP2 LNCaP-^p53: PCA -^p53: Normal Prostate Epithelium androgen-dependence growth factor dependence long timewild-type population doubling upregulation P53 P21: no change or decrease litue/no Bcl-2DeprivationDeprivationP53 Bax/Bad/Bakupregulalion Bcl-2: little/no expression Prostate No cell proliferation •¿Apoptosis >V Decreased .—^ cell number Cancer Cells androgen-independence £e^No autocrine growth factor production short population doubling timenon-functional P53 upregulalion —¿ ^ survivabili ty P53 of -^ P21 upregulation Bcl-2low/no high BaxDeprivationDeprivation Bax/Bad/Bak:decrease .^ apoptosisT^Extended or deletion Bcl-2: upregulation Novel proteins: P25; P54/55 (?)proliferationEvasion calcineurin), or the death-promoting activity of Bad (through Raf-1; Refs. 55 and 56). The precise mechanism(s) for the antideath effects of Bcl-2/Bcl-XL is still unknown and most likely is cell type and inducer dependent. Bcl-2 has been shown to be phosphorylated by Raf-1 and some other protein kinases, and this phosphorylation has been hypothesized to inactivate the cytoprotective effect of Bcl-2 (42-46). In addition, both Bcl-2/Bcl-XL and Bax have been shown to be transcriptionally regulated by p53 (57, 58). Increased Bcl-2/Bcl-X, protein levels are generally associated and P21 in apoptosis of prostate cells is independent of each other, as with cytoprotection and extended cell survival. In this study, reported by others in oncogene-mediated fibroblast apoptosis (52); NHP/BPH/PCA cells express little or no Bcl-2, and growth factor withdrawal fails to up-regulate Bcl-2, although all three cell types and (c) increased P21 expression is protecting PCA/PC3/Dul45 cells express wild-type p53. In contrast, Bcl-2 in LNCaP (wild-type from apoptosis, as observed in other cell systems (53, 54). The fact that P21 levels go up before apoptosis suggests a causal rather than a p53) and PC3 cells (p53-null) steadily and significantly increases coincidental relationship between the two phenomena. The increased upon deprivation. More interestingly. Du 145 cells primarily ex press the Mr 30,000 Bcl-2. a very stable protein, the levels of which P21 could be inducing a cell cycle arrest because the peak level of induction of this protein also coincides with the time when cells stop remain persistently elevated throughout the starvation period. With respect to Bcl-X,, all cells except PC3 demonstrate a certain proliferation. It is known that dividing cells are much more prone to degree of up-regulation of this protein in response to starvation. apoptosis induction (55); therefore, it is quite likely that the increased P21 will put a "brake" on the proliferative machinery in prostate Based on these results, it appears that; (a) the up-regulation of cancer cells to avoid "mitotic catastrophe" (55). Bcl-2 and/or Bcl-X, in various prostate cells represents a general Role of Bcl-2 and Bcl-X, in Prostate Cell Growth/Apoptosis defensive response to stresses such as deprivation. The up-regula upon Trophic Factor Withdrawal. Bcl-2 and Bcl-XL are two tion of Bcl-X, may be the major defensive (survival) mechanism in members in the Bcl-2 family that possess anti-apoptosis functions. NHP cells; the significant up-regulation of this protein in NHP Many biological activities have been assigned to these proteins for cells may also explain why these cells are relatively more resistant their death-sparing effects: forming membrane channels; modulat to apoptosis induction than BPH cells. However, the cytoprotective ing mitochondria permeability transition pore; retarding cytoeffect of up-regulated Bcl-XL will be overridden by the simulta chrome c and/or AIF apoptosis-inducing factor release from mito neous up-regulation of Bax, Bad. and Bak: (/?) the up-regulation of Bcl-2 and/or Bcl-X, may not depend on p53: and (c) the differ chondria; working as antioxidants; preventing calcium fluctuation; ential up-regulation of Bcl-2 and/or Bcl-X, in various prostate sequestering proapoptotic proteins (such as Bax. Bak, and Bik); and indirectly modulating caspase activity (through CED-4). cell cells might partially contribute to their differential survival abili cycle (through p53BP-2), NF-AT transcriptional activity (through ties (Fig. 16). 3477 (i.e.. BPH, NHP, and LNCaP) versus resistant (i.e.. PCA, PC3, and Du 145) cells. In the former, upon trophic factor withdrawal, P21 either does not change significantly (BPH) or gradually decreases (NHP and LNCaP) while in the latter; the P21 levels invariably increase either prior to or simultaneously with the appearance of DNA fragmentation. These quite distinct changes in the two groups of cells suggest that: (a) the P21 expression is unrelated to or independent of the P53 status, which is consistent with data showing that P21 ex pression can be p53-independent (50, 51); (b) the involvement of P53 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research. APOPTOSIS AND PROSTATE CANCER CELL SURVIVAL Role of Bax, Bad, and Bak in Prostate Cell Growth/Apoptosis upon Trophic Factor Withdrawal. In contrast to Bcl-2 and Bcl-XL, proapoptotic proteins Bax, Bad, and Bak demonstrate very distinct alterations in their expression levels that highly correlate with the sensitivity to apoptosis induction in individual cell types (Fig. 16). Specifically, upon growth factor withdrawal, all sensitive cells, i.e., NHP, BPH, and LNCaP, demonstrate a time-dependent up-regulation of these proteins. In contrast, long-living cells (PCA, PC3, and Du 145) invariably demonstrate a loss of this up-regulation response. More dramatically, PC3 cells demonstrate a gradual decrease, and Du 145, the most resistant cells, demonstrate a complete loss in Bax expression. The Bax/Bad/Bak protein expression appears to be cor related with the p53 functionality; their levels are increased in cells with wild-type p53 (NHP, BPH, and LNCaP), and their levels show no increase or show decrease or loss in cells with no or mutant p53 (PC3 and Du 145). PCA cells, although expressing functional p53, somehow do not up-regulate this protein in response to deprivation and therefore resemble PC3 and Du 145 cells in terms of p53 func tionality. Role of Novel Proteins in Prostate Cell Growth/Apoptosis upon Trophic Factor Withdrawal. In addition to the novel M, 30,000 Bcl-2 protein detected in Du 145 cells, several other proteins, specif REFERENCES i. Raff. M. C. Social controls on cell survival and cell death. Nature (Lond.), 356: 397-400, 1992. Raff. M. C.. Barres. B. A., Bume, J. F., Coles, H. S.. Ishizaki, Y., and Jacobson, M. D. Programmed cell death and the control of cell survival: lessons from the nervous system. Science (Washington DC), 262: 695-700. 1993. 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The present study uses a simple model to compare the growth/survival characteristics of normal versus cancerous prostate cells cultured in the absence of trophic factors. Fig. 16 summarizes the sequential molecular events that occur as these cells progress from a sensitive to a resistant stage with respect to apoptosis induction. NHP and BPH cells express wild-type p53 and little/no Bcl-2. In response to growth factor withdrawal, these cells stop proliferation and rapidly up-regulate p53, which may then up-regulate proapoptotic proteins Bax/Bad/Bak. Cells rapidly enter an apoptotic phase. In contrast, prostate cancer cells, upon deprivation, still proliferate aggressively due to autocrine production of various growth factors. These cells (except PCA) endogenously express much higher levels of Bcl-2. They express either mutant p53 (PC3 and Dul45) or wild-type p53, which somehow does not respond to starvation by up-regulating its expression (PCA), leading to the loss of up-regulation or decrease of proapoptotic proteins. Furthermore, cancer cells such as Du 145 even completely delete a proapoptotic protein (i.e., Bax). At the same time, these cells up-regulate P21, which could induce cell cycle arrest at an appropriate time (because proliferating cells are generally more sen sitive to apoptosis induction; Ref. 55), and Bcl-2, which by itself could extend cell survival. Last but not least, the long-living cells express some novel immunoglobulin or immunoglobulin-like proteins that might also help maintain the cell survivability. Cells with inter mediate life span, i.e., LNCaP, demonstrate a variety of characteristics lying between the above two cell types. 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Oncogene. 9: 1799-1805, 1994. 3479 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research. 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, Li Li, Dharam P. Chopra, et al. Cancer Res 1998;58:3466-3479. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/58/15/3466 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research.