p21 modulates threshold of apoptosis induced by DNA-damage and ...

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prostate cancer cell lines (MDA PCa 2b and LNCaP) to Dx and growth factor withdrawal. We then studied expression of p53 and p21, cell-cycle kinetics and ...
Carcinogenesis vol.23 no.8 pp.1289–1296, 2002

p21 modulates threshold of apoptosis induced by DNA-damage and growth factor withdrawal in prostate cancer cells

Luis A.Martinez, Jun Yang, Elba S.Vazquez1, Marı´a del Carmen Rodriguez-Vargas, Matilde Olive, Jer-Tsong Hsieh2, Christopher J.Logothetis and Nora M.Navone Department of Genitourinary Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA, 1CIPYPCONICET, Department of Biological Chemistry, Facultad de Ciencias Exactas y Naturales, University of Buenos Aires, Buenos Aires, Argentina and 2The University of Texas Southwestern Medical Center, Dallas, TX 77030, USA To whom correspondence should be addressed Email: [email protected]

Current therapy for advanced prostate cancer is largely based on androgen deprivation and is mostly palliative because all patients eventually relapse with androgenindependent disease. Doxorubicin (Dx), an anthracycline used commonly as a chemotherapeutic agent in relapsed prostate cancer, is a strong inducer of p53 expression and p21CIP1/WAF1 (p21) transactivation. Previous reports suggest that p21 may have a role in the modulation to chemotherapy-induced apoptosis, prostate cancer progression and androgen regulation. In order to investigate if p21 has a pro-survival role in the response of prostate cancer cells to cellular stress, we exposed two androgen-regulated human prostate cancer cell lines (MDA PCa 2b and LNCaP) to Dx and growth factor withdrawal. We then studied expression of p53 and p21, cell-cycle kinetics and apoptosis. We have found that p53 protein accumulated in a doseand time-dependent manner after Dx treatment, while p21 expression increased over time with low but decreased with high Dx doses. Apoptosis occurred in parallel with p21 down-modulation. Dx treatment of p53 knockout cells demonstrated that p21 induction was strictly p53 dependent. Reduction of p21 levels in prostate cancer cells with an antisense p21 adenovirus resulted in sensitization to Dx and accelerated onset of apoptosis in response to growth factor withdrawal. The evidence presented here also suggests that caspase activation mediates the apoptosis in this system and supports that p21 may modulate the threshold of apoptosis in prostate cancer. These observations may thus provide implications onto the integration of chemotherapy and androgen ablation. Introduction The development of therapy for prostate cancer has focused on refining the inhibition of androgen-mediated progression. These efforts have had a striking benefit, however, androgenindependent progression invariably occurs (1,2). The therapeutic options for patients with androgen-independent prostate cancer recently increased as a result of the demonstration that Abbreviations: CDK, cyclin-dependent kinase; Dx, doxorubicin; HPV, human papillomavirus; MOI, multiplicities of infection; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling. © Oxford University Press

chemotherapy may be of benefit even to patients with metastatic cancer (3,4). However, as with androgen ablation, the responses are generally of short duration, and resistance to therapy eventually results in progression (3,4). The development of therapies with enhanced antitumor activity will therefore depend on identifying new therapeutic targets, understanding and overcoming the mechanism of resistance to existing treatment, or optimizing integration with other therapies. Anthracylines have demonstrated antitumor efficacy against prostate cancer (5). Doxorubicin (Dx), a commonly used anthracycline, is a DNA-damaging agent and a strong inducer of p53 expression and p21CIP1/WAF1 (p21) transactivation. Several lines of evidence implicate the p53 pathway in the cellular response to DNA damage (6). In addition, p53 mutations have also been implicated in the progression of prostate cancer and resistance to androgen regulation (7,8). The cyclin-dependent kinase (CDK) inhibitor p21 is involved in p53-mediated growth arrest. Induction of p21 protein expression stops cell-cycle progression by inhibiting the activity of CDKs and by interacting with the proliferation cell nuclear antigen and thereby directly preventing DNA synthesis (9,10). The potential importance of p21 in tumor biology has been highlighted in several reports. Human colon cancer cells with wild-type p53 and altered p21 checkpoints have increased sensitivity to treatment in vivo, and the presence of wild-type p21 protects the cells from apoptosis induced by γ-radiation (11). Constitutive expression of p21 prevents p53-mediated apoptosis in human melanoma cells (12). Also, p21 seems to be required for survival of differentiating neuroblastoma cells (13) and has been shown to protect against prostaglandin A2mediated apoptosis of human colorectal carcinoma cells (14). Mechanisms proposed for the pro-survival role of p21 include the regulation of the G2/M checkpoint (15), inhibition of CDKs (11) and interaction with procaspase 3 (16,17). Recent clinical studies identified p21 expression as an indicator of poor survival in prostate cancer (18–21). Moreover, it has been found that p21 is expressed in a subpopulation of patients with androgen-independent prostate cancer (22). We therefore asked whether p21 has a pro-survival role in prostate cancer cells response to cellular stress. In a first attempt to address this question, we used two human prostate cancer cell lines with wild-type p53 and p21 genes: LNCaP (23–25) and MDA PCa 2b (26,27). Our results support a role for p21 in the threshold to apoptosis induced by Dx and growth factors withdrawal in prostate cancer cells. The data also imply that the caspase cascade participates in the apoptosis induced by Dx in prostate cancer cells. Moreover, as p21 expression may be modulated by androgens, our data suggest that the sequence with which anthracyclines and androgen ablation is used in current protocols may affect the efficacy of therapy. The levels of p21 expression could thus be used as a valuable surrogate marker to optimize treatment schedule. Materials and methods Cell lines and treatment MDA PCa 2b cells (26) were propagated in BRFF-HPC1 (Biological Research Faculty and Facility, Jamesville, MD) with 20% fetal bovine serum (Sigma

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L.A.Martinez et al. Chemical, St Louis, MO) and gentamicin (50 µg/ml) (Gibco BRL, Gaithersburg, MD). LNCaP (23), originally obtained from the American Type Culture Collection (Mannassas, VA) was routinely cultured in RPMI 1640 supplemented with 10% fetal bovine serum (Sigma Chemical). LNCaP-Bcl-2 (28) stable transfectants were a kind gift of Dr T.J.McDonnell (M.D. Anderson Cancer Center, Houston). The cells were seed in 100 mm tissue culture dishes (8⫻106 cells/dish) and were left untreated or treated with Dx (0.2, 0.5 and 1.0 µg/ml) and harvested by scraping them into lysis buffer at various times. For growth factor withdrawal experiments, the cells were cultured under standard conditions for 24 h and then were placed in F12k medium (Gibco BRL) supplemented with 10% charcoal striped serum (cFBS) (Hyclone, Logan, UT) for 24, 48 and 6 days. Cell-cycle analysis and apoptosis For analysis of cell-cycle distribution, the cells were collected at various time intervals after treatment with Dx, fixed with 70% ethanol, stained with propidium iodide in an RNAasa solution and analyzed for DNA content by FACS. Cell-cycle distributions were obtained by using Coulter EPICS XLMCL flow cytometer (Coulter, Miami, FL) and MultiCycle analysis software (Phoenix Flow Systems, San Diego, CA). Apoptosis was assessed by flow cytometric analysis of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) using the Apo-Brdu kit (Pharmigen, San Diego, CA) and following the manufacturer instructions. Plasmids and antibodies Expression vector pCMV E6 contains the human papillomavirus (HPV) 16 E6 gene subcloned into pCMV-neo downstream of the cytomegalovirus promoter (29) (kindly provided by Drs M.B.Kastan and K.R.Cho, Johns Hopkins University, Baltimore, MD). Immunoblot analyses were performed with the following antibodies: monoclonal human anti-p53 antibody DO-1 (Dako, Carpenteria, CA), monoclonal anti-p21 antibody Ab-1 (Oncogene Research Products, Cambridge, MA), monoclonal anti-β-actin antibody (Amersham, Arlington Heights, IL) and monoclonal anti-poly(ADPribose) polymerase (PARP) antibody (Pharmigen). Protein extraction and western blotting Expression of p53, p21 and PARP proteins was studied by western blot analysis. Protein extracts from cells growing in monolayers were obtained by using RIPA buffer and following standard procedures (30). Cell lysates were centrifuged at 10 000 g for 10 min at 4°C. The supernatants were normalized for protein content, and 50–100 µg of protein per lane was fractionated on 7.5–12% SDS–PAGE (the percentage depending on the molecular weight of the proteins to be detected). The proteins were blotted to a Hybond-ECL nitrocellulose membrane (Amersham) that was probed and washed according to the instructions for the enhanced chemiluminescence western blotting detection system (Amersham-Pharmacia Biotech). The intensities of each band on western blot were quantified using ImageQuant image analysis software (Amersham-Pharmacia Biotech). The values of p21 bands were normalized for the intensity of a loading control. To obtain p21 relative levels, the normalized p21 values were subsequently normalized for the intensity of p21 value in cells infected with Ad5CMV-312 (Ad-c) at time 0. HPV-16 E6 transfection and clonal isolation Cells were transfected with expression vector pCMV E6 (29). Cells transfected with pCMV-neo alone were used as controls. Liposome-mediated transfection was performed with Lipofectamine (Gibco BRL) essentially as described by the manufacturer. The transfected cells were selected with G418 sulfate (Geneticin, Life Technologies, Gaithersburg, MD) (800 µg/ml), and clones were screened for E6 function by western blot analysis of p53 expression. The viral oncoprotein HPV 16 E6 marks p53 for ubiquitin degradation, and clones expressing high levels of E6 have no basal or inducible p53 expression (29). pCMV E6 transfectant clones were therefore screened by western blot analyses for the absence of basal p53 expression and the absence of p53 induction after exposure to Dx. Controls (pCMV-neo-transfected cells) were screened for basal p53 levels and p53 protein induction after exposure to Dx. Antisense p21 adenovirus and conditions of infection Adenovirus antisense p21 (Ad-ASp21), an expression vector carrying a human cytomegalovirus promoter, the simian virus 40 polyadenylation signal and antisense p21 cDNA inserted into the E1-deleted region of the modified adenovirus 5 (31) was used to reduce p21 expression. Ad-c, an adenovirus vector carrying a minicassette including the human cytomagalovirus promoter and the simian virus 40 polyadenylation signal only, was used as a control virus. Viral titers were assessed by performing the plaque-forming assay on 293 cells. MDA PCa 2b cells were seeded at 50–70% confluence, grown for 48 h, and infected with Ad-ASp21 or Ad-c. p21 expression levels were then measured at 48 h by western blot analysis of cells infected with different multiplicities of infection (MOI). We found lower p21 protein levels in AdASp21 cell lysates at an MOI of 15 plaque-forming units/cell with no apparent

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toxicity or effect on cell growth. We therefore selected 15 MOI to perform the studies detailed below.

Results Morphology and survival of prostate cancer cells treated with various concentrations of Dx LNCaP cells treated with 0.2 µg/ml Dx showed no evidence of apoptosis under phase-contrast microscopy (Figure 1A). In contrast, cells treated with 1.0 µg/ml Dx showed morphological evidence of apoptosis (Figure 1B). These results are in agreement with those of TUNEL analysis that showed very low percentage (7.2%) of cells undergoing apoptosis at 0.2 µg/ml Dx and a significant percentage (92%) of cells positive for apoptosis when treated with 1.0 µg/ml Dx for 24 h (Figure 1C, D and E). As caspases participate in many apoptotic processes, and PARP is the best-characterized substrate of caspase during apoptosis (32), we also screened for PARP cleavage in cells treated with Dx. A 24-h exposure to 0.2 µg/ml Dx was not sufficient to induce PARP cleavage, which readily occurred at 1.0 µg/ml Dx (Figure 1F). Similar results were obtained with MDA PCa 2b cells. These findings suggest that two distinct biochemical events occur at 0.2 and 1.0 µg/ml Dx. Expression of p53 and p21 after treatment of cells with Dx As Dx is a strong inducer of p53 protein accumulation and LNCaP and MDA PCa 2b cells have wild-type p53 (24,27), we studied expression of p53 and its downstream target p21 in these prostate cancer cells treated with 0.2, 0.5 and 1.0 µg/ml Dx for 24 h. Western blot analysis revealed that the p53 protein levels increased with increasing concentrations of Dx. The p21 expression levels increased in the presence of 0.2 µg/ml Dx, but remained the same at 0.5 µg/ml and decreased at 1.0 µg/ml Dx (Figure 2A and B). We then studied p53 and p21 protein accumulation over time (Figure 2C and D). Western blot analysis revealed that p53 protein accumulated in a dose and time-dependent manner. Whereas p21 expression increased in a time-dependent manner at a dose of 0.2 µg/ml Dx (Figure 2C), it was undetectable at 1.0 µg/ml Dx (Figure 2D). PARP cleavage is prevented by caspase inhibitor and bcl-2 in LNCaP cells Genetic and biochemical studies indicated that apoptosis is triggered by activation of the members of the CED-3/caspase protease family (33,34). We used z-VaD-Ala-Asp-fluoromethyl ketone (z-VAD-FMK) (35), a general inhibitor of caspases, to assess whether the PARP cleavage we observed was the result of activation of caspase cascades. Briefly, cells were simultaneously treated with Dx (at the indicated doses) and z-VAD-FMK (20 µM). The left panel in Figure 2E shows the presence of the 85 kDa degradation product of PARP in LNCaP cells treated with 1.0 µg/ml Dx alone for 24 h. When z-VAD-FMK was added, the degradation product of PARP was not present. z-VAD-FMK also prevented the morphological changes observed in prostate cancer cells after treatment with 1.0 µg/ml Dx (data not shown). To further determine the involvement of the cell death machinery, we evaluated whether over-expression of bcl-2, a well-known antiapoptotic gene, was also capable of blocking apoptosis. Therefore, we used LNCaP cells stable transfected with bcl-2 (28) and LNCaP cells transfected with empty vector as a control. The right panel in Figure 2E shows that PARP cleavage was prevented

p21 and apoptosis in prostate cancer cells

Fig. 1. Phase-contrast microscopy of LNCaP cells cultured in medium with 0.2 µg/ml Dx (A) and 1.0 µg/ml Dx (B) for 24 h. The field shown in (B) is a typical change in morphology suggesting apoptosis: chromatin condensation, formation of membrane-bound apoptotic bodies. Similar findings were obtained with MDA PCa 2b cells. TUNEL analysis of LNCaP cells without treatment (C) and after treatment with 0.2 (D), and 1.0 µg/ml Dx (E) for 24 h. The lines indicate the biotin-16-dUTP signal levels in the control cells. Also shown in each box is the percentage of apoptotic cells. These results are representative of two independent experiments and similar findings were obtained with MDA PCa 2b cells. Western blot analysis of PARP cleavage in control cells and cells treated with 0.2 and 1.0 µg/ml Dx (F). The arrow indicates the cleaved form (85 kDa) of PARP. Similar amounts of proteins were loaded in each lane. These results are representative of two independent experiments and similar findings were obtained with MDA PCa 2b cells.

Fig. 2. Western blot analysis of p53, and p21 in prostate cancer cells treated with Dx (A–D). LNCaP (A) and MDA PCa 2b (B) cells were treated with various Dx concentrations (0.2, 0.5 and 1 µg/ml) for 24 h. MDA PCa 2b cells were treated with 0.2 (C) and 1.0 µg/ml (D) Dx for 6–72 h. Western blotting was performed by standard procedures with monoclonal antibodies to human p53, p21. β-Actin was used as a loading control. These results are representative of two independent experiments. Prevention of PARP cleavage in LNCaP cells treated with Dx (E). Western blot analysis of PARP cleavage in LNCaP cells treated with Dx and z-VAD-FMK (20 µM) (left panel) or LNCaP-Bcl-2 cells treated with Dx (right panel) for 24 h. The generation of the 85 kDa PARP fragment was prevented by z-VAD-FMK and bcl-2. In the right panel LNCaP cells transfected with empty vector was used as control. Similar amounts of proteins were loaded in each lane. Similar findings were observed in an independent experiment.

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Fig. 3. Cell-cycle distribution of prostate cancer cells treated with Dx. MDA PCa 2b, and LNCaP cells were treated with the indicated Dx concentrations and analyzed for DNA content by FACS analysis (A). MDA PCa 2b and LNCap cells were treated with 0.2, 0.5 and 1.0 µg/ml Dx for 24 h and analyzed for cellcycle distribution (B). MDA PCa 2b cells were treated with 0.2, 0.5 and 1.0 µg/ml DX for the indicated times and analyzed for cell-cycle distribution. These results are representative of two independent experiments.

by expression of bcl-2. The data suggest that Dx-induced apoptosis is a specific event mediated by the caspase family and that it can be prevented. Cell-cycle distribution Progression through the cell cycle is driven by the periodic activation of several CDK. CDKs activity is partially regulated by CDK inhibitors. p21 is a CDK inhibitor that mediates arrest at the G1 and G2/M checkpoint (36) and we showed that Dx modifies p21 expression levels in this system. We then studied the cell-cycle distribution of MDA PCa 2a and LNCaP prostate cancer cells treated with Dx and found that these cells do not arrest in G1, but shows increased percentage of cells at G2/M when treated with 0.2 µg/ml Dx (Figure 3A). To assess the effect of Dx on the cell-cycle distribution over time, MDA PCa 2b cells were exposed to various doses of Dx and harvested at different times (Figure 3B). Continuous exposure to 0.2 µg/ml Dx decreased the percentage of cells in G1 and increased S–G2/M cells (Figure 3B). A delayed increase (⬍20%) of cells in sub-G1 is observed only after treatment for 72 h (Figure 3B). After 48 h of treatment with 0.5 and 1.0 µg/ml Dx percentage of cells in G1 and S–G2/M decreased and there was a striking increase of cell DNA in sub G1 phase 1292

(which indicates apoptosis) (Figure 3B). Two distinct biological responses were observed with 0.2 and 1.0 µg/ml Dx; this parallels p21 expression levels in this system. Induction of p21 expression is p53 dependent in cells treated with Dx Expression of the HPV16 E6 gene is an efficient method of inactivating p53 function (29). E6 promotes the degradation of endogenous p53 by a ubiquitin-mediated pathway and has been shown to abrogate both p53 induction and G1 arrest in irradiated cells (29). To determine if p21 induction by Dx is p53 dependent, we exposed HPV E6 transformed cells to different concentrations of Dx and measured p21 expression by western blot analysis. p21 induction by Dx was found to be strictly p53 dependent at all three Dx doses used, as assessed by the absence of p21 protein in the HPV E6 expressing cells (Figure 4A). Similar findings were observed with LNCaP cell line (data not shown). Antisense p21 sensitizes MDA PCa 2b and LNCaP cells to Dx-induced apoptosis p21 expression levels were inversely associated with apoptosis in this system and p21 has been implicated as a survival

p21 and apoptosis in prostate cancer cells

Fig. 4. Western blot analysis of p53 and p21 expression after Dx treatment of HPV 16 E6-transfected MDA PCa 2b cells (A). MDA PCa 2b cells were transfected with HPV 16 E6, and then treated with the indicated doses of Dx for 24 h. MDA PCa 2b cells transfected with empty vector were used as controls. β-Actin was used as a loading control. Similar findings were observed with LNCaP cell line. Western blot analysis of PARP in MDA PCa 2b cells infected with Ad-ASp21 at various MOI during 48 h (B). β-Actin was used as a loading control. Western blot analysis of p21 expression in MDA PCa 2b cells infected with Ad-ASp21 and Ad-c and treated with 0.2 µg/ml Dx for 24 h (C). β-Actin was used as a loading control. Western blot analysis of PARP in MDA PCa 2b cells infected with Ad-ASp21 and Ad-c and treated with 0.2 Dx for 24 h (D). Similar amounts of proteins were loaded in each lane. Western blot analysis of PARP in MDA PCa 2b cells infected with Ad-ASp21 and Ad-c and treated with 0.5 µg/ml Dx for the indicated times (E). Similar amounts of proteins were loaded in each lane. Similar findings were observed with LNCaP cell line. TUNEL analysis of MDA PCa 2b cells infected with AD-ASp21 and Ad-C and treated with 0.2, 0.5, and 1.0 µg/ml Dx for 24 h (F). The lines indicate the biotin-16-dUTP signal levels in the control cells. Also shown in each box is the percentage of apoptotic cells. Similar findings were observed with LNCaP cell line.

factor in several reports (11,12,17,37). This suggests that p21 modulates the threshold of Dx-induced apoptosis in this system. We tested this hypothesis by decreasing p21 protein levels.

Western blot analysis of MDA PCa 2b cells infected with Ad-ASp21 at 5–20 MOI showed no cleavage of PARP (Figure 4B). MDA PCa 2b cells were then infected with Ad-ASp21 1293

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Fig. 5. Western blot analysis of p21, p53 and PARP in MDA PCa 2b cells grown in cFBS for 24 h, 48 h, and 6 days (A). β-Actin was used as a loading control. Western blot analysis of p21 expression in cells infected with Ad-ASp21 or Ad-c and challenged with growth factor withdrawal for 0–48 h (B). Similar amounts of proteins were loaded in each lane. MDA PCa 2b cells infected with AD-ASp21 and AD-c and placed in cFBS for 0, 24, 48 and 72 h were harvested for TUNEL analysis (C). The lines indicate the biotin-16-dUTP signal levels in the control cells. Also shown in each box is the percentage of apoptotic cells. Similar findings were observed with LNCaP cell line.

or Ad-c at 15 MOI. p21 protein levels in cells infected with Ad-ASp21 were decreased by 40% when compared with cells infected with Ad-c (Figure 4C). The difference in the level of p21 expression was more pronounced after the infected cells were treated with 0.2 µg/ml Dx; this resulted in a significant reduction (100%) of p21 induction as compared with cells infected with Ad-c and treated with Dx (Figure 4C). Accordingly, PARP cleavage was seen in cells infected with Ad1294

ASp21 but not in controls (Figure 4D). Furthermore, when the infected cells were treated with 0.5 µg/ml Dx, PARP cleavage was seen earlier than in controls (Figure 4E). We subsequently assessed apoptosis by TUNEL assay. Figure 4F shows that after treatment with Dx, apoptosis in cells infected with Ad-AS-p21 was significantly higher than in controls. LNCaP cells infected with Ad-AS-p21 were also sensitized to Dx (data not shown).

p21 and apoptosis in prostate cancer cells

p21 modulates the threshold for apoptosis in MDA PCa 2b cells after growth factor withdrawal When MDA PCa 2b cells were grown in medium supplemented with cFBS, p21 expression increased at 48 h and decreased at 6 days (Figure 5A), and minimal apoptosis (as indicated by PARP cleavage) was detected only after 6 days of culture (Figure 5A). We then assessed whether p21 expression modulated the threshold of apoptosis in prostate cancer cells growing in cFBS by infecting cells with Ad-ASp21 or Ad-c. p21 expression and TUNEL analysis were subsequently assessed at 0–48 h and at 0–72 h after growth factor withdrawal respectively. p21 expression levels were lower in cells infected with Ad-ASp21 than those infected with Ad-c (Figure 5B). TUNEL analysis of cells infected with Ad-c showed 1.2–4.7% apoptotic cells while cells infected with Ad-ASp21 showed ⬎70% apoptotic cells after 48 h of growth factor withdrawal (Figure 5C). These results identify p21 as an important player in the survival of prostate cancer cells after growth factor withdrawal and suggests that p21 provides pro-survival signals to prostate cancer cells. Discussion In this study we examined the induction of apoptosis in human prostate cancer cells that express wild-type p53. Our findings suggest that in these cells, the threshold for apoptosis can be modulated by the expression of p21, a protein controlled by the p53 gene. Although, there is ample evidence that p21 has a role in the response of cells to stress in various systems, there is a paucity of data on the role of p21 in prostate cancer and the normal prostate. The results presented in this report demonstrate that reducing p21 protein levels sensitizes the prostate cancer cell lines, MDA PCa 2b and LNCaP, to apoptosis induced by growth factor deprivation and the DNAdamaging agent Dx. These results suggest that p21 may protect prostate cancer cells from apoptosis induced by different cellular stresses. Several reports have documented that p21 protects against the apoptosis induced by a variety of different cellular stresses (17,23) and that p21 plays a central role in cellular resistance to a variety of anticancer agents [γ-radiation, paclitaxel (Taxol), and doxorubicin] in different systems (11,12,16,17). Mechanisms proposed for the pro-survival role of p21 include the regulation of the G2/M checkpoint (36), inhibition of Cdks (29) and interaction with procaspase 3 (17,26,37). The results shown in Figures 1 and 2 suggest that the apoptosis induced by Dx is mediated by activation of caspases during continuous genotoxic stress. p21 expression can be induced by p53dependent and p53-independent mechanisms. Induction of p21 gene expression by DNA damage is widely believed to be p53 dependent and accounts for cell-cycle arrest in G1 in several systems (11,36,38). We showed that p21 induction by Dx in this system is strictly p53 dependent (Figure 4A). However, exposure of the cells to Dx, did not result in G1 arrest, instead a G2 arrest was observed at 0.2 µg/ml, a Dx dose that did not induce apoptosis (Figure 3). That treatment of the cells with 0.5 and 1.0 µg/ml Dx resulted in apoptosis (sub-G1 accumulation of cells DNA) (Figure 3B), suggest that the lack of p21 induction with 1.0 µg/ml Dx may be an underlying mechanism for the inability to prevent apoptosis under these experimental conditions. We subsequently tested whether p21 levels had an impact in the threshold for apoptosis in MDA PCa 2b and LNCaP cells treated with 0.2 µg/ml and found

that decreasing p21 expression levels rendered cells more susceptible to the apoptotic stimuli. We also found that p21 expression levels modulate the apoptosis threshold for growth factor withdrawal. This supports a pro-survival role for p21 in prostate cancer. Studies of patient material showed that nearly all cancers continue to express the androgen receptor regardless of clinical stage or hormone status (22). Moreover, ligand-independent activation of the androgen receptor pathway was recently suggested as a mechanism of androgen-independent growth (39). Intriguingly, recent evidence indicated that androgens could up regulate the expression of the Cdk inhibitor p21 through a functional androgen response element in the p21 promoter (40,41). It is therefore reasonable to believe that activation of the androgen receptor signaling pathway during the androgen independent growth of prostate cancer may up regulate p21 expression. Accordingly, recent clinical studies identified p21 expression as an indicator of poor survival in prostate cancer (18–21), and it was suggested to be associated with androgen independent progression (22). The observations described here thus suggest that androgen ablation, which is widely used early in the treatment of prostate cancer, may influence acquired resistance to chemotherapy. These observations may have implications in the integration of chemotherapy and androgen ablation in that the sequence with which anthracyclines and androgen ablation are used may impact efficacy of therapy. Acknowledgements This work was supported by grants from the National Institutes of Health (CA75499), and by The Association for the Cure of Cancer of the Prostate.

References 1. Gittes,R.F. (1991) Carcinoma of the prostate. N. Engl. J. Med., 324, 236–245. 2. Catalona,W.J. (1994) Management of cancer of the prostate. N. Engl. J. Med., 331, 996–1004. 3. Ellerhorst,J.A., Tu,S.M., Amato,R.J., Finn,L., Millikan,R.E., Pagliaro,L.C., Jackson,A. and Logothetis,C.J. (1997) Phase II trial of alternating weekly chemotherapy for patients with hormone-refractory adenocarcinoma of the prostate. Clin. Cancer Res., 3, 2371–2376. 4. Tu,S.M., Pagliaro,L.C., Banks,M.E., Amato,R.J., Millikan,R.E., Bugazia,N.A., Madden,T., Newman,R.A. and Logothetis,C.J. (1998) Phase I study of suramine combined with doxorubicin in the treatment of androgen-independent prostate cancer. Clin. Cancer Res., 4, 1193–1201. 5. Culine,S., Kattan,J., Zanetta,S., Theodore,C., Fizazi,K. and Droz,J.P. (1998) Evaluation of estramustine phosphate combined with weekly doxorubicin in patients with androgen-independent prostate cancer. Am. J. Clin. Oncol., 21, 470–474. 6. Ko,L. and Prives,C. (1996) p53: puzzle and paradigm. Genes Dev., 10, 1054–1072. 7. Navone,N.M., Troncoso,P., Pisters,L.L., Goodrow,T.L., Palmer,L.P., Nichols,W.W., von Eschenbach,A.C. and Conti,C.J. (1993) p53 protein accumulation and gene mutation in the progression of human prostate carcinoma. J. Natl Cancer Inst., 85, 1657–1669. 8. Navone,N.M., LaBate,M.E., Troncoso,P., Pisters,L.L., Conti,C.J., von Eschenbach,A.C. and Logothetis,C.J. (1999) p53 mutations in prostate cancer bone metastases suggest that selected p53 mutants in the primary site define foci with metastatic potential. J. Urol., 161, 304–308. 9. Li,R., Waga,S., Hannon,G.J., Beach,D. and Stillman,B. (1994) Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature, 371, 534–537. 10. Li,R., Hannon,G.J., Beach,D. and Stillman,B. (1996) Subcellular distribution of p21 and PCNA in normal and repair-deficient cells following DNA damage. Curr. Biol., 6, 189–199. 11. Waldman,T., Zhang,Y., Dillehay,L., Yu,J., Kinzler,K., Vogelstein,B. and Williams,J. (1997) Cell-cycle arrest versus cell death in cancer therapy. Nat. Med., 3, 1034–1036.

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L.A.Martinez et al. 12. Gorospe,M., Cirielli,C., Wang,X., Seth,P., Capogrossi,M.C. and Holbrook,N.J. (1997) p21 (Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells. Oncogene, 14, 929–935. 13. Poluha,W., Poluha,D.K., Chang,B., Crosbie,N.E., Schonhoff,C.M., Kilpatrick,D.L. and Ross,A.H. (1996) The cyclin-dependent kinase inhibitor p21 (WAF1) is required for survival of differentiating neuroblastoma cells. Mol. Cell. Biol., 16, 1335–1341. 14. Gorospe,M., Wang,X., Guyton,K.Z. and Holbrook,N.J. (1996) Protective role of p21 (Waf1/Cip1) against prostaglandin A2-mediated apoptosis of human colorectal carcinoma cells. Mol. Cell. Biol., 16, 6654–6660. 15. Yu,D., Jing,T., Liu,B., Yao,J., Tan,M., McDonnell,T.J. and Hung,M.C. (1998) Overexpression of ErbB2 blocks Taxol-induced apoptosis by upregulation of p21Cip1, which inhibits p34Cdc2 kinase. Mol. Cell., 2, 581–591. 16. Gervais,J., Seth,P. and Zhang,H. (1998) Cleavage of CDK-inhibitor p21CIP1/WAF1 by caspases is an early event during DNA damage-induced apoptosis. J. Biol. Chem., 273, 19207–19212. 17. Levkau,B., Koyama,H., Raines,E.W., Clurman,B.E., Herren,B., Orth,K., Roberts,J.M. and Ross,R. (1998) Cleavage of p21Cip1/Waf1 and p27Kip1 mediates apoptosis in endothelial cells through activation of Cdk2: role of a caspase cascade. Mol. Cell., 1, 553–563. 18. Aaltomaa,S., Lipponen,P., Eskelinen,M., Ala-Opas,M. and Kosma,V.M. (1999) Prognostic value and expression of p21 (waf1/cip1) protein in prostate cancer. Prostate, 39, 8–15. 19. Sarkar,F.H., Li,Y., Sakr,W.A., Grignon,D.J., Madan,S.S., Wood,D.P. and Adsay,V. (1999) Relationship of p21 (WAF1) expression with disease-free survival and biochemical recurrence in prostate adenocarcinomas (PCa). Prostate, 40, 256–260. 20. Baretton,G.B., Klenk,U., Diebold,J., Schmeller,N. and Lohrs,U. (1999) Proliferation- and apoptosis-associated factors in advanced prostatic carcinomas before and after androgen deprivation therapy: prognostic significance of p21/WAF1/CIP1 expression. Br. J. Cancer, 80, 546–555. 21. Lancombe,L., Maillete,A., Meyer,F., Veilleux,C., Moore,L. and Fradet,Y. (2001) Expression of p21 predicts PSA failure in locally advanced prostate cancer treated by prostatectomy. Int. J. Cancer, 95, 135–139. 22. Fizazi,K., Martinez,L.A., Sikes,C.R. et al. (2002) The association of p21 (WAF–1/CIP1) with progression to androgen-independent prostate cancer. Clin. Cancer Res., 8, 775–781. 23. Horoszewicz,J.S., Leong,S.S., Kawinski,E., Karr,J.P., Rosenthal,H., Chu,T.M., Mirand,E.A. and Murphy,G.P. (1983) LNCaP model of human prostatic carcinoma. Cancer Res., 43, 1809–1818. 24. Isaacs,W.B., Carter,B.S. and Ewing,C.M (1991) Wild-type p53 suppresses growth of human prostate cancer cells containing mutant p53 alleles. Cancer Res., 51, 4716–4720. 25. Papandreou,C.N., Bogenrieder,T., Scher,H.I., Albino,A.P. and Nanus,D.M. (1996) Expression and sequence analysis of the SDI1/WAF1/CIP1/p21 tumor suppressor gene in prostate cancer cell lines. Int. J. Oncol., 8, 1237–1241. 26. Navone,N.M., Olive,M., Ozen,M. et al. (1997) Establishment of two human prostate cancer cell lines derived from a single bone metastasis. Clin. Cancer Res., 3, 2493–2500.

1296

27. Navone,N.M., Rodriquez-Vargas,M.C., Benedict,W.F., Troncoso,P., McDonnell,T.J., Zhou,J.H., Luthra,R. and Logothetis,C.J. (2000) TabBO: a model reflecting common molecular features of androgen-independent prostate cancer. Clin. Cancer Res., 6, 1190–1197. 28. Beham,A.W., Sarkiss,M., Brisbay,S., Tu,S.M., von Eschenbach,A.C. and McDonnell,T.J. (1998) Molecular correlates of bcl-2-enhanced growth following androgen-ablation in prostate carcinoma cells in vivo. Int. J. Mol. Med., 1, 953–959. 29. Keiss,T.D., Slebos,R.J., Nelson,W.G., Kastan,M.B., Plunkett,B.S., Han,S.M., Lorincz,A.T., Hedrick,L. and Cho,K.R. (1993) Human papillomavirus 16 E6 expression disrupts the p53 mediated cellular response to DNA damage. Proc. Natl Acad. Sci. USA, 90, 988–992. 30. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. 2nd Edn, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 31. Ruan,S., Okcu,M.F., Pong,R.C. andreeff,M., Levin,V., Hsieh,J.T. and Zhang,W. (1999) Attenuation of WAF1/Cip1 expression by an antisense adenovirus expression vector sensitizes glioblastoma cells to apoptosis induced by chemotherapeutic agents 1,3-bis (2-chloroethyl)-1-nitrosourea and cisplatin. Clin. Cancer Res., 5, 197–202. 32. Song,Q., Lees-Miller,S.P., Kumar,S. et al. (1996) DNA-dependent protein kinase catalytic subunit: a target for an ICE-like protease in apoptosis. EMBO J., 15, 3238–3246. 33. Steller,H. (1995) Mechanisms and genes of cellular suicide. Science, 267, 1445–1449. 34. Alnemri,E.S., Livingston,D.J., Nicholson,D.W., Salvesen,G., Thornberry,N.A., Wong,W.W. and Yuan,J. (1996) Human ICE/CED-3 protease nomenclature. Cell, 87, 171. 35. Wood,D.E. and Newcomb,E.W. (1999) Caspase-dependent activation of calpain during drug-induced apoptosis. J. Biol. Chem., 274, 8309–8315. 36. Bunz,F., Dutriaux,A., Lengauer,C., Waldman,T., Zhou,S. and Brown,J.P. (1998) Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science, 282, 1497–1501. 37. McDonnell,T.J., Meyn,R.E. and Robertson,L.E. (1995) Implications of apoptotic cell death regulation in cancer therapy. Semin. Cancer Biol., 6, 53–60. 38. Bissonnette,N. and Hunting,D.J. (1998) p21-induced cycle arrest in G1 protects cells from apoptosis induced by UV-irradiation or RNA polymerase II blockage. Oncogene, 16, 3461–3469. 39. Craft,N. and Sawyers,C.L. (1999) Mechanistic concepts in androgendependence of prostate cancer. Cancer Metastasis Rev., 17, 421–427. 40. Lu,S., Liu,M., Epner,D.E., Tsai,S.Y. and Tsai,M.-J. (1999) Androgen regulation of the cyclin-dependent kinase inhibitor p21 gene through an androgen response element in the proximal promoter. Mol. Endocrinol., 13, 376–384. 41. Lu,S., Jenster,G. and Epner,D.E. (2001) Androgen induction of cyclindependent kinase inhibitor p21 gene: role of androgen receptor and transcription factor Sp1 complex. Mol. Endocrinol., 14, 753–760. Received February 1, 2002; revised April 19, 2002; accepted April 29, 2002