(2004) 23, 3541–3549 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc

Inhibition of function diminishes androgen -mediated signaling in prostate lines

Marcus V Cronauer1, Wolfgang A Schulz1, Tatjana Burchardt1, Rolf Ackermann1 and Martin Burchardt*,1

1Department of Urology, Heinrich-Heine University, Du¨sseldorf D-40225, Germany

Current therapy for advanced is mainly ablation (Feldman and Feldman, 2001). Several me- based on androgen deprivation, although most patients chanisms contributing to androgen-independent pro- relapse to androgen-insensitive disease. Several mechan- gression have been identified including alterations in the isms contributing to androgen-independent growth includ- structure, expression, and regulation of the androgen ing alterations in the structure or expression of the receptor (AR) (Culig et al., 1993; Cronauer et al., 2000; (AR) and its cofactors have been Feldman and Feldman, 2001). The AR is expressed in a identified. Recent evidence suggests that p53 is involved in high proportion of metastatic prostate . In rare androgen signaling. The analysis of the effect of p53 on cases, the receptor is mutated (Culig et al., 1993; androgen signaling was performed in 22Rv1 and LNCaP Feldman and Feldman, 2001). In other cases, the prostate cancer cells that express both p53 and AR. The overexpression of AR and/or changes in the expression overexpression of p53 diminished the androgenic response of different cofactors are observed, which may confer in both cell lines in a reporter assay. Conversely, the increased sensitivity of the AR to low levels of inhibition of p53 by three different p53 inhibitors, circulating androgens (Feldman and Feldman, 2001). Pifithrin-1a (PFT-1a), an inhibitor of p53-dependent Further changes permitting prostate carcinoma cells transactivation; , a regulator of p53 expression; to overcome the effects of androgen ablation occur in and a dominant-negative N-terminally truncated p53 gene pathways regulating apoptotic cell death. One pertinent also reduced transactivation of androgen-dependent re- alteration is of the p53 gene in prostatic tumor porter . The inactivation of p53 by PFT-1a cells. The p53 encodes a nuclear decreased AR- expression in both 22Rv1 and , which is activated and which LNCaP cells. Our findings confirm that the overexpres- accumulates in the cells in response to a variety of sion of wild-type p53 decreases androgen function, stresses inducing growth arrest or . Loss of whereas p53 expression at physiological levels stabilizes p53 function may compromise the ability of carcinoma AR signaling. Thus, our findings suggest that there is a cells to undergo apoptosis in response to genomic balance of AR and p53 expression during the androgen- instability, thereby favoring the accumulation of genetic dependent growth of prostate cancer, which is obliterated changes responsible for androgen independence. In during further progression of the disease. prostate cancer, alterations in the p53 tumor suppressor Oncogene (2004) 23, 3541–3549. doi:10.1038/sj.onc.1207346 gene are clearly associated with progressive disease, like metastases to bone- and androgen-independent Keywords: androgen receptor; p53; prostate cancer; growth (Navone et al., 1993; Aprikian et al., 1994; pifithrin; MDM2; probasin promoter Eastham et al., 1995; Meyers et al., 1998; Burchardt et al., 2001). There is increasing evidence that p53 may also directly regulate androgen signaling. The p53 protein has been Introduction shown to interact with several steroid receptors includ- ing the AR (Yu et al., 1997a, b; Sengupta and Wasylyk, As prostate cancer cells depend on androgens for 2001; Shenk et al., 2001). The overexpression of wild- growth and survival, androgen ablation is a standard type p53 in PC-3 cells decreased transactivation of the treatment for patients suffering from metastatic prostate PSA promoter by the AR in a cotransfection reporter cancer. Androgen withdrawal causes cancer regression assay (Shenk et al., 2001). However, interpretation of by decreasing cell proliferation and increasing apopto- this experiment is complicated by the fact that PC-3 cells sis. However, most metastatic prostatic carcinomas normally express neither p53 nor AR and do not progress to a state of disease insensitive to androgen tolerate the expression of either protein over a pro- longed period of time (Isaacs et al., 1991; Srivastava et al., 1995; Heisler et al., 1997; Eastham et al., 2000; *Correspondence: M Burchardt; E-mail: [email protected] Schumacher et al., 2001). Another study showed that the Received 21 February 2003; revised 14 July 2003; accepted 20 August transfection of one specific mutant p53, R175H, thought 2003 to inhibit wild-type p53 function actually increased p53 and androgen receptor in prostate cancer MV Cronauer et al 3542 androgen-dependent induction of PSA in LNCaP cells, promoters by the AR. Moreover, the expression of a which express both wild-type p53 and AR (Gurova et al., dominant-negative p53 (pCMVDD) as well as the 2002). A different study showed that the downregulation overexpression of MDM2 (pCMVhMdm2), a negative of wild-type p53 function by an antisense approach regulator of p53, repressed androgen-induced transacti- conferred a -resistant to LNCaP vation of the probasin promoter plasmid as well. These cells in nude mice (Burchardt et al., 2001). effects of pCMVDD and PFT-1a on AR-dependent A major problem in the analysis of AR signaling is transactivation were strongly p53 dependent, as trans- that under normal conditions, epithelial cell lines as well fection or treatment of p53-null cells with pCMVDD or as primary epithelial cell cultures rarely express AR PFT-1a had no or only little effect on AR signaling. protein (Peehl, 1994; Cronauer et al., 1997). In addition Most interestingly, subsequent analysis of AR protein to LNCaP, only few cell lines expressing AR protein revealed that the inactivation of p53 function resulted in have been described (Navone et al., 1993; Peehl, 1994; a downregulation of AR-protein expression in andro- Sramkoski et al., 1999; Lee et al., 2001; Sinisi et al., gen-stimulated 22Rv1 and LNCaP cells. 2002). In a systematic analysis of the literature on AR- Our findings suggest that wild-type p53 at basal positive cell strains, we noted that almost all prostate physiological levels is necessary for AR signaling and, in cancer cell lines expressing AR also express wild-type fact, has a protective effect on AR-signaling, whereas p53 (Table 1). This observation prompted us to analyse induced levels block it. Our findings support the concept the effects of the dysregulation of endogenous p53 on of a balance between AR and p53 expression during AR signaling in prostate cancer cell lines. androgen-dependent cell growth and highlight the As the overexpression of p53 downregulates the importance of p53 as a modulator of AR signaling in androgenic response, one would predict that the prostate cells. inhibition of endogenous wild-type p53 should upregu- late the androgenic response in prostatic cells. There- fore, we employed three different inhibitors of p53, Pifithrin-1a (PFT-1a), an inhibitor of p53-dependent Results transactivation (Komarov et al., 1999); MDM2, which interacts directly with p53 promoting its ubiquitylation In our study, we used the androgen-sensitive LNCaP and proteasomal degradation (Momand et al., 1992; and the partially androgen-sensitive 22Rv1 prostate Grossman et al., 1998); and a dominant-negative N- cancer cell lines. LNCaP are fully androgen sensitive, terminally truncated p53 (Shaulian et al., 1992). The that is, exhibit androgen-dependent growth and secrete effect of these p53 inhibitors on androgen response and PSA in response to androgens. 22Rv1 grow in an cell growth were studied in LNCaP and 22Rv1 cells, androgen-independent manner but express a functional which express both AR and p53 (Peehl, 1994; Sramkoski AR. In a first series of experiments, we confirmed that et al., 1999). 22Rv1 express both AR and p53 (Figure 1a and b). In In accord with previous findings, the overexpression contrast to LNCaP cells, 22Rv1 cells expressed not only of wild-type p53 in 22Rv1 and LNCaP cells resulted in a a full-length AR (110 kDa) but also a truncated 78 kDa dramatic decrease in transactivation of androgen- inducible gene promoters by the AR. In addition, we showed physiologically induced levels of p53 to be sufficient for this inhibition. Surprisingly, however, treatment of 22Rv1 or LNCaP cells with p53 inhibitors did not increase transactivation by the AR. Instead, treatment of these cell lines with PFT-1a, an inhibitor of p53, reduced transactivation of the probasin and PSA

Table 1 Overview of prostate cancer cell lines expressing AR protein Cell line AR p53 Reference

CWR22 + wt Agus et al. (1999) MDA PCa 2a + wt Navone et al. (1993) MDA PCa 2b + wt Navone et al. (1993) Figure 1 (a) Detection of AR in 22Rv1 and LNCaP cells. Total LNCaP + wt Peehl (1994) protein extract (30 mg) was analysed by Western blotting as 22Rv1 + wt (?) Sramkoski et al. (1999), described in Material and methods. 22Rv1 as well as LNCaP cells Tepper et al. (2002) expressed wild-type AR protein with a molecular weight of EPN + wt (?) Sinisi et al. (2002) 110 kDa. Note that 22Rv1 cells express an AR isoform with an DuCaP + wt (?) Lee et al. (2001) apparent molecular weight of 77 kDa. (b) The induction of p53 in PC-3 À — Peehl (1994), Rubin et al. 22Rv1 and LNCaP after exposure to the DNA-damaging agent (1991) cisplatin. 22Rv1 and LNCaP cells were treated with cisplatin DU-145 À Mutated Peehl (1994), Rubin et al., (10 mg/ml) for 5–10 h. p53 expression was analysed by Western (1991) blotting as described in Material and methods. Cisplatin induced p53 in 22Rv1 as well as LNCaP cells, suggesting an intact p53 + ¼ presence of AR; À¼absence of AR; wt ¼ wild type signaling cascade

Oncogene p53 and androgen receptor in prostate cancer MV Cronauer et al 3543 isoform (Tepper et al., 2002). To confirm that the AR in 22Rv1 cells is able to transactivate androgen-dependent genes, 22Rv1 cells were transfected with an androgen- responsive probasin-luciferase reporter plasmid. In the presence of 10 nM methyltrienolone (R1881), a synthetic androgen, probasin-promoter activity increased three- fold in 22Rv1 cells compared to 25-fold in LNCaP cells (Figure 2). The typical induction of wild-type p53 expression as a result of exposure to the DNA-damaging agent cisplatin was confirmed in both cell lines by Western Blotting (Figure 1b). In both LNCaP and 22Rv1 cell lines, the transfection of wild-type p53 diminished androgen-induced proba- sin-luciferase reporter activity (Figures 2 and 3a). Of note, in these experiments, p53 was expressed by a retrotransposon promoter that yields only about 10% the expression levels that are induced by other viral promoters, thus avoiding extremely supraphysiological levels of p53 (Steinhoff et al., 2002). To analyse the effects of endogenously induced levels of p53 on AR signaling, we treated 22Rv1 cells with cisplatin. The induction of endogenous p53 by 2.5 or 5 mg/ml cisplatin

Figure 3 (a) Effects of p53 on androgenic signaling in 22Rv1 cells. 22Rv1 cells were transiently transfected with the probasin- promoter-driven luciferase-reporter plasmid pGL3Eprob together with pLinep53 or empty vector (control) and pRL-tk-LUC vector at a ratio 1 : 5 : 8. Cells were treated for 30 h with the indicated concentrations of R1881. Reporter gene activity was measured in cell extracts as described in Material and methods; bars ¼ Probasin/ Renilla activity7s.d. (b) Effects of MDM2 on androgenic signaling in 22Rv1 cells. 22Rv1 cells were transiently transfected with pGL3Eprob, pCMVhMdm2 (MDM2 expression plasmid), and pRL-tk-LUC. Controls were transfected with pCMV-neo-Bam instead of pCMVhMdm2. Cells were treated for 30 h with the indicated concentrations of R1881. Reporter gene activity was Figure 2 Reduction of androgen-inducible promoter activity by determined in cell extracts as described in Material and methods; p53 and PFT-1a in human prostate cancer cell lines LNCaP and bars ¼ Probasin/Renilla activity7s.d. (c) Effects of a truncated 22Rv1. LNCaP (a) and 22Rv1cells (b) were cotransfected with the dominant-negative p53 on androgenic signaling in 22Rv1 cells. androgen-responsive reporter plasmid pGL3Eprob and pLinep53 22Rv1 cells were cotransfected with pGL3Eprob reporter plasmid, expression vector or pLine empty vector (control), respectively. pCMVDD, or empty pCMV-neo-Bam vector and pRL-tk-LUC as Subsequently, cells were treated with R1881 (10 nM) in the described in Material and methods. Cells were treated for 30 h with presence/absence of PFT-1a (20 mM) (Calbiochem-Novabiochem, the indicated concentrations of R1881. Reporter gene activity was Marburg, Germany) for 30 h. Reporter gene activity was assessed assessed as described in Material and methods; bars ¼ Probasin/ by the Dual-Luciferase Reporter Assay System as described in Renilla activity7s.d Material and methods; bars ¼ Probasin/Renilla activity7standard deviation (s.d.)

Oncogene p53 and androgen receptor in prostate cancer MV Cronauer et al 3544 led to a subsequent 67–80% decrease in AR-mediated transactivation of the probasin-luciferase reporter (Table 3). In order to prove that the downregulation of AR signaling in prostate cancer cells can be initiated by physiologically induced levels of p53, we performed a series of experiments in PC-3 prostate cancer cells that do not express p53 (p53-null cells). To analyse the effects of different levels of p53 on AR signaling, PC-3 cells were transfected with an AR expression plasmid (pSG5- AR) together with a probasin-reporter plasmid and pMThup53, in which p53 expression is controlled by the zinc-inducible mouse MT-1 metallothionein promoter (Makri et al., 1998). Treatment with ZnCl2 induces the weak MT-1 promoter approximately fivefold (Hasse et al., 1992). Cells transfected with pMT-CAT (Hasse et al., 1992), instead of pMThup53, served as controls. Low levels of p53 expressed from the uninduced MT promoter did not interfere with transactivation of the probasin promoter by AR, and appeared, in fact, slightly stimulatory. Following induction of p53 by ZnCl2, however, pMThup53-transfected PC-3 cells showed a significantly lower AR activity than either cells not treated with ZnCl2 or PC-3 cells transfected with the pMT-CAT control plasmid instead of pMThup53 (Figure 4a). Taken together, these data show that not only supraphysiological levels of p53 used in previous reports but also levels reached during physiological Figure 4 (a) Effect of different p53 levels on AR signaling in PC-3 induction are able to reduce AR-mediated signaling. cells (p53-null cells) using a zinc-inducible p53 expression vector As p53 downregulated the androgenic response upon (pMThup53). PC-3 cells were transiently transfected with AR expression vector pSG5-AR, a probasin-promoter-driven lucifer- induction, one would predict that the inhibition of ase-reporter plasmid pGL3Eprob together with either pMThup53 endogenous wild-type p53 should upregulate the andro- or control vector (pMT-CAT), and pRL-tk-LUC at a ratio genic response in prostate cancer cells. Therefore, we 1 : 3 : 2 : 5. Cells were incubated for 30 h in the presence/absence analysed the effects of different p53 inhibitors on AR of R1881 and in the presence/absence of ZnCl2. Reporter gene signaling. First, we tested PFT-1a, a pharmacological activity was measured in cell extracts as described in Material and methods; bars % Probasin/Renilla activity7s.d.; 0 no ZnCl , compound that has been reported to reduce transactiva- ¼ ¼ 2 Zn ¼ 150 mM ZnCl2.(b) Effects of PFT-1a and pCMVDD in PC-3 tion of p53-dependent genes in various cell types on AR-mediated transactivation. PC-3 cells (p53-null cells) were including prostate cancer cell lines (Komarov et al., transiently transfected with AR expression vector pSG5-AR, a 1999; Javelaud and Besancon, 2002; Lin et al., 2002a; probasin-promoter-driven luciferase-reporter plasmid pGL3Eprob and with pRL-tk-LUC 1 : 3 : 5. In addition, cells were cotransfected M Kelly et al., 2003). Treatment with 20 m PFT-1a with pCMVDD or pCMV-neo-Bam (empty vector). Subsequently inhibited the induction of probasin-luciferase activity cells were grown for 30 h in the presence of R1881 and PFT-1a as by androgen (10 nM R1881) by 95% in LNCaP and by indicated. Reporter gene activity was measured in cell extracts as 96% in 22Rv1 cells (Figure 2). Moreover, PFT-1a was described in Material and Methods; bars ¼ % Probasin/Renilla unable to restore the AR response in cells transfected activity7s.d with pLinep53 (Figure 2). In summary, in cell lines expressing wild-type p53, both inhibition of p53 by most pronounced in LNCaP, which grows in an PFT-a as well as the overexpression of p53 down- androgen-dependent manner, the compound also re- regulated the androgenic response. duced DNA synthesis in 22Rv1 and PC-3, which do not. As the effects of PFT-1a on p53 might not only limit Moreover, it is important to note that PFT-1a exhibited the response to androgens but might also alter the rate slight antiproliferative effects in PC-3 cells that are of cell proliferation and apoptosis, we analysed the known not to express p53 –protein, suggesting that effects of PFT-1a on DNA synthesis and apoptosis in PFT-1a not solely acts through an inhibition of p53. prostate cancer cell lines. DNA synthesis was deter- The rate of apoptosis was determined by the mined by measuring BrdU incorporation. PFT-1a measurement of -3/7 activity. Caspase activity reduced DNA synthesis in PC-3, 22Rv1 and LNCaP in cells grown without androgens but in the presence of cells. In the absence of androgens, PFT-1a (20 mM) PFT-1a (20 mM) was reduced by 25% in LNCaP and was lowered BrdU incorporation in PC-3, 22Rv1, and increased by 13% in 22Rv1 cells. Caspase-3/7 activity LNCaP by 15, 13, and 54%, respectively. In the also increased at higher concentrations of R1881. presence of R1881 (0.1 nM), BrdU incorporation was Treatment with 10 nM R1881 increased caspase activity reduced by 18, 12, and 30%, respectively (Table 2a). in 22Rv1 and LNCaP cells by 120 and 230%, Although the antiproliferative effects of PFT-1a were respectively. Coincubation with PFT-1a (20 mM), in

Oncogene p53 and androgen receptor in prostate cancer MV Cronauer et al 3545 Table 2 Effects of PFT-1a and R1881 on (a) BrdU incorporation (% of untreated controls that were set at 100%7s.d.) and (b) caspase-3/7 activity (% of untreated controls that were set at 100%7s.d.) in 22Rv1 and LNCaP cells LNCaP 22Rv1 PC-3

(a) 0nM R1881 100 100 100 0.1 nM R1881 124719 1007599711 10 nM R1881 72769674 10474 0nM R1881/20 mM PFT-1a 467587758572 0.1 nM R1881/20 mM PFT-1a 70711 88738276 10 nM R1881/20 mM PFT-1a 4473797279711

LNCaP 22Rv1 (b) 0nM R1881 100 100 0.1 nM R1881 17173 17078 10 nM R1881 220712 330712 0nM R1881/20 mM PFT-1a 7575 11375 0.1 nM R1881/20 mM PFT-1a 96711 132711 10 nM R1881/20 mM PFT-1a 224716 182712 general, diminished the induction of caspase activity by R1881 in 22Rv1 and LNCaP cells (Table 2b), except at the highest concentration of R1881 (10 nM) in LNCaP cells. In summary, these findings on the one hand confirm previous observations that high androgen Figure 5 Inhibition of AR-mediated transactivation of different concentrations are associated with higher rates of reporter genes by PFT-1a and a pCMVDD. 22Rv1 cells were spontaneous apoptosis in LNCaP cells (Coffey et al., transiently cotransfected with either pGL3Eprob (a) or PSA-luc 2002). On the other, they demonstrate that the dramatic reporter plasmid (b), and pCMVDD or empty pCMV-neo-Bam decrease in the AR response after PFT-1a treatment is vector, and pRL-tk-LUC as described in Material and methods. One set of cells was treated with 20 mM of PFT-1a. Cells were not due to increased apoptosis. treated for 30 h with the indicated concentrations of R1881. To ensure that the PFT-1a effect on androgenic Reporter gene activity was assessed as described in Material and signaling was predominantly due to its inhibitory action methods; bars ¼ Probasin/Renilla activity7s.d. or PSA/Renilla on p53, we analysed the consequences of p53 down- activity7s.d regulation on AR signaling by the transfection of further p53 inhibitors. For these experiments, we used 22Rv1 cells, which can be transfected at a greater efficiency prostate cancer cells that are p53-null cells. The than LNCaP cells. Like treatment with PFT-1a transfection of a pCMVDD had no effect on AR (Figure 2), the transfection of MDM2 (pCMVhMdm2) signaling in PC-3 cells that do not express endogenous downregulated both basal- and androgen-induced activ- p53. PFT-1a reduced AR transactivation in PC-3 cells ity of the probasin promoter (Figure 3b). Likewise, by 30%. This 30% reduction is minimal compared to transfecting a dominant-negative truncated p53 the downmodulation of AR signaling by 495% in p53- (pCMVDD) to disrupt endogenous p53 signaling positive cell lines (Figure 2), and suggests that the major resulted in strongly diminished transactivation of the part of the PFT-1a effect on AR signaling is due to its probasin promoter in 22Rv1 (Figure 3c). The effects of inhibitory function on p53 (Figure 4b). pCMVDD on probasin promoter activity were compar- To investigate the effects of p53 inhibition on AR able in magnitude to those seen with PFT-1a treatment. expression, LNCaP and 22Rv1 cells were treated with/ In order to ensure that the effects of PFT-1a and without R1881 (10 nM) in the presence/absence of PFT- pCMVDD were not specific to the probasin promoter, 1a for 48 h. Subsequently, AR expression was deter- additional experiments were performed with a PSA- mined by Western blotting (Figure 6). When grown in luciferase reporter plasmid (pPSA61-luc) (Figure 5). As the presence of R1881, PFT-1a-treated LNCaP and in the experiments with the probasin-reporter plasmid, 22Rv1 cells clearly exhibited a downregulation of their PFT-1a and pCMVDD caused a downmodulation of AR protein in comparison to untreated controls, AR-mediated transactivation of the PSA promoter. This suggesting a direct effect of p53 on AR-protein indicates that the disruption of p53 function has a expression (Figure 6). generalized effect on AR transactivation in 22Rv1 cells (Figure 5a and b). In order to prove that the downregulation of AR Discussion signaling in prostate cancer cells by PFT-1a and pCMVDD was indeed mediated through their effects There is little doubt that receptors can on p53, we performed a series of experiments in PC-3 be added to the continually growing list of with

Oncogene p53 and androgen receptor in prostate cancer MV Cronauer et al 3546 Table 3 Effect of p53 inducing stress on AR-mediated transactivation of the probasin promoter Cisplatin 0 mg/ml 2.5 mg/ml 5 mg/ml

Untreated control 35407849 ND ND 20 nM R1881 7221773661 3010173492 150297701

22Rv1 cells were cotransfected with the androgen-responsive reporter plasmid pGL3Eprob and pRL-tk-LUC Renilla vector as described in Material and methods. Subsequently, cells were treated with R1881 (20 nM) in the presence/absence of different concentrations of cisplatin for 30 h. Reporter gene activity was assessed by the Dual-Luciferase Reporter Assay System as described in Material and methods. Results are expressed in Probasin/Renilla activity7s.d.; ND ¼ not determined

interaction, to induction of IL-6, or to other mechan- Figure 6 Effect of PFT-1a on the expression of AR-protein. LNCaP and 22Rv1cells were grown for 48 h in the presence/ isms. Accordingly, the overexpression of several p53 absence of R1881 (10 nM) and of PFT-1a (20 mM) Subsequently, mutants frequent in advanced prostate cancer (G245S, cells were lysed and AR-protein expression was analysed by R248W, R273H, and R273C) was recently shown not to Western blotting as described in Material and methods; upper increase PSA in stably transfected LNCaP cells (Nes- panel: AR protein; lower panel: a-tubulin (loading control) slinger et al., 2003). Here, we confirmed that that the 22Rv1 prostate carcinoma cell line like the LNCaP cell line expresses which p53 directly interacts (Yap et al., 1996; Yu et al., AR and p53 (Figure 1). In agreement with a recent 1997a, b; Shenk et al., 2001). Interactions of p53 with report (Tepper et al., 2002), we found that 22Rv1 cells estrogen, glucocorticoid, and thyroid receptors are well express, in contrast to LNCaP cells, not only a full- established (Yap et al., 1996; Yu et al., 1997a, b). length AR but also a truncated form of the AR. Overexpressed p53 represses AR induction of the PSA Androgen sensitivity of 22Rv1 cells was demonstrated gene, although direct binding of p53 to the AR has not by transfection assays with two different androgen- been demonstrated (Shenk et al., 2001). The ability of responsive reporter plasmids (pPSA61-luc, pGL3Eprob) p53 to modulate AR-mediated transcription was initi- (Figure 5). ally studied in cotransfection assays using the human Using different cotransfection assays, we confirmed prostate cancer cell line, PC-3, which expresses neither that the overexpression of p53 blocks androgen signal- p53 nor AR (Carroll et al., 1993; Peehl, 1994; Rubin ing in 22Rv1, LNCaP, and PC-3 cells (Figures 2, 3a, and et al., 1991). Interpretation of these results is compli- 4). In order to mimic physiological levels of p53, we used cated by the fact that PC-3 cells do not tolerate long- p53 expression vectors under the control of different term expression of either protein in vitro (Heisler et al., promoters (i.e. Line, MT), which yield only about 1– 1997; Jacobberger et al., 1999; Eastham et al., 2000; 10% the expression levels of the usual viral promoters Schumacher et al., 2001). Thus, the repression of AR- (Hasse et al., 1992; Steinhoff et al., 2002), thus avoiding mediated transcription by p53 is achieved in a transient extremely supraphysiological levels of p53 achieved in state in cells that are likely to be destined for cell death. other studies (Shenk et al., 2001). Moreover, physiolo- A second study on this issue reported that the gical induction of p53 with cisplatin in 22Rv1 cells was transfection of one specific mutant p53 (R175H) sufficient to block androgen signaling in these cells increased the induction of PSA in LNCaP cells, which (Figure 1b, Table 3). Our results indicate for the first contain functional AR and wild-type p53 (Gurova et al., time that typically induced levels of p53 can interfere 2002). Only this mutant, but none of the others, with AR function. exhibited this effect, which the authors ascribed to a However, our data show that basal levels of p53 have dominant-negative function of this particular mutant a very different effect. If basal levels of wild-type p53 (Gurova et al., 2002). However, the function of R175H were limiting for androgen responses, one would expect in different cell types is controversial. It is certainly inhibitors of p53 to increase androgen signaling. debatable to regard R175H simply as a dominant- Surprisingly, three different treatments aimed at inhibit- negative inhibitor of p53 (Pocard et al., 1996; Coursen ing endogenous p53 decreased rather than increased et al., 1997; Kremenetskaya et al., 1997; Gurova et al., androgenic responses in these cell lines. 2002; Shi et al., 2002). Specifically, R175H is known to PFT-1a, a synthetic reversible inhibitor of p53 in induce the interleukin 6 (IL-6) in different cell various cell types including prostate cancer cells (Lin systems (Runnebaum et al., 1994; Asschert et al., 1999). et al., 2002a; Kelly et al., 2003), dramatically inhibited Recent studies have demonstrated that IL-6 is a potent the induction of probasin-luciferase activity and PSA- inducer of AR signaling in LNCaP cells (Hobisch et al., luciferase activity in LNCaP and 22Rv1 cells. One might 1998; Chen et al., 2000; Culig et al., 2002). Therefore, it attribute this finding to toxic effects of PFT-1a unrelated remains to be elucidated whether the increased induc- to p53. The analysis of the effect of PFT-1a on the tion of PSA in LNCaP cells after transfection with proliferation of different prostate cancer cell lines indeed mutant p53, R175H, is due to the inhibition of p53–AR showed that PFT-1a to some extent negatively affected

Oncogene p53 and androgen receptor in prostate cancer MV Cronauer et al 3547 the proliferation of prostate cancer cells, independent of the inactivation of p53 as well diminishes AR-mediated their p53 status (Table 2a). This finding is supported by signaling. Our findings suggest that physiological levels a recent paper reporting PFT-1a not to act solely of wild-type p53 are necessary and have a protective through p53-dependent mechanisms (Komarova et al., effect on AR signaling in androgen-responsive prostate 2003). However, its moderate inhibitory effects on cell cancer cell lines. In contrast, elevated levels of p53 block growth and apoptosis cannot account for the 90% androgen signaling. Our observations support the decrease in androgen signaling caused by the compound. concept that androgen-dependent growth of prostate Nevertheless, the specific inhibitory effect on p53 cancer cells occurs at a balanced level of p53 and AR signaling may appear exaggerated due to toxic effects expression. Disruption of this balance may play a crucial of the compound. Therefore, additional experiments role in the progression to androgen-insensitive prostate with other p53 inhibitors were crucial. Thus, we used cancer. a pCMVDD that represents the C-terminal residues 302–390 of wild-type p53 preceded by 14 N-terminal residues of wild-type p53 (Shaulian et al., 1992). In Materials and methods contrast to mutated p53 R175H, pCMVDD is a bona fide dominant-negative inhibitor of p53 that inhibits p53 Plasmids DNA-binding activity, thereby disrupting endogenous The probasin-promoter luciferase-reporter plasmid (pGL3E- p53 signaling (Shaulian et al., 1992). Like PFT-1a, this prob) and the pSG5-AR were a gift from Dr Z Culig, pCMVDD strongly inhibited transactivation of the Innsbruck, Austria. The PSA-luciferase plasmid (pPSA61-luc) probasin and PSA promoters (Figures 3c and 5). The was supplied by Dr Jan Trapman, Erasmus University, effects were comparable in magnitude to those obtained Rotterdam, The Netherlands, via Dr A Baniahmad, University by PFT-1a, thereby confirming that the effects of PFT- of Giessen, Germany. The pCMVDD and pCMVhMdm2 1a on androgen signaling cannot be attributed to toxic expression plasmids were kindly provided by Dr M Oren, effects unlinked to p53. This observation is furthermore Weizman Institute of Science, Rehovot, Israel and Dr B supported by the fact that the same pCMVDD had no Vogelstein, Johns Hopkins University Oncology Center, Baltimore, MD, USA. The pLinep53 plasmid and its effect on AR signaling in PC-3 cells, which are p53-null corresponding cDNA-less plasmid pLine as well as pMThup53 (Figure 4b). Similarly, PFT-1a reduced AR transactiva- and pMT-CAT have been described by our laboratory (Hasse tion in PC-3 cells by only 30% (Figure 4b). et al., 1992; Makri et al., 1998; Steinhoff et al., 2002). A recent clinical study suggested that MDM2 over- expression is a frequent mechanism of p53 inactivation Tissue culture in prostate cancer (Osman et al., 1999). The transfection of MDM2 into 22Rv1 cells downregulated basal as well 22Rv1, LNCaP, and PC-3 prostatic carcinoma cells were routinely maintained in RPMI-1640, supplemented with 10% as androgen-induced activity of the probasin promoter, fetal bovine serum and antibiotics. During experiments, cells albeit not as pronounced as pCMVDD or PFT-1a were cultured in RPMI-1640 with 2.5% charcoal-treated (Figure 3b). Recently, it has been demonstrated that steroid-free fetal bovine serum, cFBS (PAA Laboratories, MDM2 is essential for Akt-mediated ubiquitylation and Linz, Austria), and antibiotics supplemented with the in- degradation of the AR in vitro (Lin et al., 2002b). As the dicated amounts of the synthetic androgen, milbolerone, MDM2 gene is itself under transcriptional control of R1181 (New England Nuclear, Dreieichenhain, Germany). p53, creating an autoregulatory loop, it is conceivable that p53 may control AR-protein levels and Transfection assays its activation indirectly via MDM2. Cells were grown on 24-well plates. The probasin-promoter Our data indeed show that one level at which p53 luciferase-reporter plasmid, pGL3Eprob, was mixed with affects androgen signaling is through the regulation of either pLinep53, pCMVhMdm2 or pCMVDD expression AR expression or stability. The inactivation of p53 by vector, or the respective cDNA-less vectors at a ratio of 1 : 5. PFT-1a decreased AR-protein expression in 22Rv1 and pRL-tk-LUC Renilla vector (Promega, Mann-heim, Ger- LNCaP cells (Figure 6). This relationship may explain many) was used to correct for transfection efficiency. the strikingly tight relationship between AR expression Transfection was performed using FuGene6 (Roche Diagnos- and expression of wild-type p53 in vitro revealed by a tics Corporation, Basel, Switzerland), according to the survey of the literature (Table 1). Most recently, in new manufacturer’s instructions. After an incubation period of 12 h in RPMI-1640 with 5% cFBS, the medium was changed androgen-independent prostate cancer cell lines derived to RPMI-1640 containing 2.5% cFBS, with or without from LNCaP via in vitro deprivation (Patel et al., 2000), androgens. Reporter activity was assessed after a 30 h low levels of p53 were reported along with almost incubation period using the Dual-Luciferase Reporter Assay undetectable levels of PSA mRNA and reduced levels of System (Promega, Mannheim, Germany). AR mRNA (Freedland et al., 2003). Similarly, in a consistent relationship appears to exist between Immunoblotting of p53 and AR the expression of (ER) and wild-type Prostate cancer cell lines, 22Rv1 and LNCaP, were cultured in p53 expression (Caleffi et al., 1994). T25 culture flasks for 48 h. Cell protein extracts were prepared There is little doubt that a overexpression of p53 as described (Marth et al., 1990). Protein extracts (30 mg) were reduces transactivation of androgen-dependent genes electrophoresed through a 7% SDS–PAGE gel and electro- (Shenk et al., 2001; Nesslinger et al., 2003). However, blotted onto PVD membranes. Nonspecific binding was for the first time, we present experimental evidence that blocked by incubating the membrane with 5% nonfat dry

Oncogene p53 and androgen receptor in prostate cancer MV Cronauer et al 3548 milk in phosphate-buffered saline. Afterwards, membranes Roche Basel, Switzerland). The test was performed according were exposed to the AR-specific rabbit antiserum PG-21 to the manufacturer’s instructions. (Upstate, Lake Placid, NY, USA), or to mouse anti-p53, DO-7 Caspase-3/7 activity as an indicator of apoptosis was (Sigma, Taufkirchen, Germany) (Cronauer et al., 2000; determined using the Apo-ONEt Homogeneous Caspase-3/7 Burchardt et al., 2001). Immunoreactive bands were detected Assay (Promega, Madison, WI, USA). Caspase activity was with peroxidase-labeled mouse anti-rabbit or anti-mouse measured according to the manufacturer’s instructions using a antibodies (1 : 5000), respectively. AR and p53 bands were Wallac 1420 multilabel counter (Wallac, Turku, Finland). visualized by enhanced chemiluminescence (Amersham Phar- macia Biotech, Freiburg, Germany). Acknowledgements We thank Dr A Baniahmad, Dr Z Culig, Dr M Oren, Dr J Trappman and Dr B Vogelstein for kindly providing plasmid Cell proliferation and apoptosis assay constructs used in this study. This work was supported by the Cellular proliferation was assessed by means of BrdU Forschungskomission der Medizinischen Fakulta¨ t der Hein- incorporation during DNA synthesis using a commercial rich-Heine Universita¨ t, Du¨ sseldorf and Action Lions – Vaincre colorimetric BrdU ELISA (Cell Proliferation ELISA, BrdU, le Cancer, Luxembourg.

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