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[Cancer Research 63, 2228-2233, May 1, 2003]
© 2003 American Association for Cancer Research

Molecular Biology and Genetics

Androgen-independent Growth of LNCaP Prostate Cancer Cells Is Mediated by Gain-of-Function Mutant p531

Nancy J. Nesslinger, Xu-Bao Shi and Ralph W. deVere White2

Department of Urology, University of California, Davis, School of Medicine, Sacramento, California 95817


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations of p53 are common in hormone-refractory prostate cancer (CaP), suggesting the possibility that these mutations may be involved in the progression of CaP to androgen-independent (AI) growth. However, at present no direct evidence has been presented linking p53 mutations with AI growth of CaP. We established five stably transfected LNCaP cell lines: four containing gain-of-function (GOF) mutant p53 alleles (G245S, R248W, R273H, and R273C) and one containing a non-GOF p53 mutant allele (P151S). The four GOF p53 sublines were able to grow under androgen-depleted conditions, whereas the LNCaP parental line, vector-only line, and the non-GOF line were unable to grow. To investigate the mechanism of the AI growth displayed by the GOF p53 mutants, Western blotting or ELISA were used to examine the expression of the androgen receptor (AR), the AR-regulated prostate-specific antigen (PSA), as well as Akt and Bcl-2 under androgen-depleted conditions. On androgen ablation, the levels of AR decreased in the four GOF p53 sublines compared with the control lines. This decreased AR expression was accompanied by attenuated receptor activity, because a decrease in prostate-specific antigen levels compared with parental LNCaP cells was also observed. Levels of phosphorylated Akt increased in both the GOF p53 sublines and the control lines. Bcl-2 remains unchanged or showed reduced expression in all of the cell lines in the absence of androgen compared to the presence of androgen. These observations suggest that GOF p53 mutants mediate the AI growth of LNCaP cells in an AR-independent fashion, and that both Akt and Bcl-2 are not involved in this process.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CaP3 is a hormone-dependent tumor that requires androgen to develop, grow, and differentiate (1) . Clinically, CaP is treated differently depending on the stage of the disease. In men with localized disease, radical prostatectomy and definitive radiotherapy are the main curative treatments, achieving clinical cures in ~80% of cases. However, in men with metastatic disease, androgen blockade is the sole treatment available. Androgen ablation usually decreases the volume of the primary and metastatic lesions by inducing apoptosis (2) . However, after this initial response, the tumors recur in an AI form that is unresponsive to additional androgen withdrawal (3) and are resistant to cure by chemotherapy (4) . The mechanism of progression to hormone independence remains unclear.

The p53 tumor suppressor gene is a transcription factor that regulates the expression of genes involved in cell-cycle inhibition, apoptosis, genetic stability, and angiogenesis (5) . Mutation of p53 is one of the most common genetic events occurring in human cancer. In CaP, p53 alterations have been investigated widely. Although the reported frequencies of p53 mutations in primary CaP remain controversial, most agree that mutations of p53 are common in advanced CaP. Navone et al. (6) reported elevated p53 protein expression levels in 94% (16 of 17) of hormone-refractory specimens and 22% (6 of 27) of primary untreated tumors. Heidenberg et al. (7) confirmed p53 gene alterations in 82% (9 of 11) of representative specimens. These authors found a clear progression in the number of cases having p53 alterations from untreated primary to hormone-refractory disease (7) . In our laboratory, we have reported previously the frequency of p53 alterations to be ~39% in surgically resected primary CaPs (8) and 71% in CaP metastases to bone (9) . Thus, investigations of clinical samples are in agreement that there is an increased rate of p53 mutation in advanced CaP. These observations support the hypothesis that p53 mutations may be involved in the progression of CaP. However, to date this hypothesis has not been tested by direct experimentation.

We have published previously an extensive functional analysis of 16 mutant p53 alleles derived from CaP (10) . We observed that some of the p53 mutants that can dominate over wt p53 also possessed GOF properties, defined as the induction of genomic instability (11) , tumorigenicity (12 , 13) , or transactivation of the promoters of certain genes, such as MDR-1 and proliferating cell nuclear antigen that are not regulated by wt p53 (14) . To study whether these GOF p53 mutants mediate progression of CaP to androgen independence, we selected four GOF alleles (G245S, R248W, R273H, and R273C), which are frequent in CaP (15) , and a non-GOF mutant allele (P151S). The four GOF alleles possess different GOF properties including the ability to transactivate the promoter of the MDR-1 gene, promotion of growth in soft agar, and/or an increased S phase fraction (10) . We found that the LNCaP cells transfected with each of four GOF mutant alleles were able to grow in the absence of androgens, whereas the non-GOF P151S-transfected LNCaP cells and the LNCaP parental cells could not, indicating that GOF p53 mutations can lead to AI growth of LNCaP cells. In addition, we determined the expression levels of several proteins that are known to be involved in AI growth of CaP tumors, to better understand their role in the AI growth of the GOF p53 sublines. To our knowledge, this is the first report demonstrating the direct involvement of GOF p53 mutations in the progression of CaP to AI growth.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
LNCaP cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were routinely maintained in androgen-containing medium, RPMI 1640 supplemented with 10% FBS (Omega Scientific, Tarzana, CA). The androgenic activity of 10% FBS has been shown to be equivalent to the activity of 0.05 nM R1881 (16) . For the androgen-depletion experiments, cells were grown in androgen-depleted medium, phenol red-free RPMI 1640 supplemented with 5% charcoal/dextran-treated FBS (HyClone, Logan, UT).

Stable Transfections.
The G245S, R248W, R273H, R273C, and P151S alleles were individually cloned into the pCR 3.1 vector (Invitrogen, Carlsbad, CA) downstream of the cytomegalovirus promoter. Each allele was sequenced to ensure the presence of the mutation of interest. The plasmids containing the different p53 mutant alleles or the empty pCR3.1 plasmid (vector-only) were stably transfected into LNCaP cells using 9 µg plasmid DNA and calcium phosphate precipitation with a glycerol shock at 5 h post-transfection. After 48 h, cells were grown under G418 (500 µg/ml) selection for 2–3 weeks until isolated colonies appeared. Colonies were selected and expanded in 24-well plates before being transferred to culture flasks.

Cell Counts.
Cells from the LNCaP parental and mutant p53-transfected sublines were plated in triplicate in androgen-containing medium on 60-mm2 Petri dishes using 1 x 105 cells/dish. Cells were allowed to attach overnight before switching to androgen-depleted medium. At days 0, 1, 3, and 5, the cells were trypsinized, pelleted, and resuspended in 1 ml of medium. A 10-µl aliquot was combined with an equal volume of trypan blue, and the cells were counted using a hemocytometer.

Western Blotting.
Pelleted cells were lysed in lysis buffer [150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.5% sodium deoxycholate, 1% Triton X-100, and 0.1% SDS] supplemented with 10 mg/ml leupeptin, 0.1 M aprotinin, 0.1 M phenylmethylsulfonyl fluoride, 0.1 M NaVO4, and 0.05 units/µl RNase-free DNase. The cell lysates were spun at 12,000 rpm at 4°C, and the supernatants were used for protein analysis. Protein concentrations were determined using the BCA Protein Assay Reagent (Pierce, Rockford, IL). Protein (25–50 µg) was separated on an 8% (for AR), a 12% (for Akt), or a 15% (for Bcl-2) SDS-PAGE mini-gel and transferred to nitrocellulose membranes. The AR antibody (NeoMarkers, Fremont, CA), p-Akt antibody (Cell Signaling Technology, Beverly, MA), and Bcl-2 antibody (BD PharMingen, San Diego, CA) were all used at a 1:1,000 dilution, whereas the ß-actin antibody (Sigma, St. Louis, MO) was used at a 1:10,000 dilution.

PSA ELISA.
The ELISA reactions were carried out using 1 x 105 cells grown in 24-well plates in 500 µl of androgen-containing or androgen-depleted medium. The cells were grown for 3 days before plating in 24-well plates. Cells were grown for additional 2 days, and medium was collected. A PSA ELISA kit (Medicorp, Montreal, Quebec, Canada) was used to measure PSA protein in 20 µl of medium in duplicate as per the manufacturer’s directions. Dilutions were made as necessary to ensure that the sample readings fell within the standard curves. The experiment was performed three times and the results averaged.

PSA RT-PCR.
RNA was extracted from cell pellets using TRIzol Reagent (Life Technologies, Inc., Grand Island, NY). cDNA synthesis was carried out using Moloney murine leukemia virus Reverse Transcriptase (Promega, Madison, WI). The following primers were used to amplify the PSA transcripts: PSA-forward: 5'-TGGGAGTGCGAGAAGCATTC and PSA-reverse: 5'-GCACACAGCATGAACTTGGTCAC. The following amplification conditions were used in a MJ PTC-100 thermal cycler (MJ Research, Incline Village, NV): an initial denaturation for 5 min at 94°C, 30 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 1 min, followed by a final extension for 10 min at 72°C.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Establishment of Stably Transfected LNCaP Sublines.
Four GOF p53 mutant alleles (G245S, R248W, R273H, and R273C) and the non-GOF P151S mutant allele were selected for the present study. All are dominant negative, with the R248W, R273H, and R273C mutants dominant over two wt copies of p53, and the G245S and P151S alleles dominant over one wt copy of p53. The four mutant alleles were able to activate the MDR-1 promoter, to grow in soft agar, and/or to increase S phase, exhibiting GOF activity (10) . Each of the five mutant alleles was stably transfected into LNCaP cells to yield four GOF and a non-GOF p53 subline. As a control, the empty pCR3.1 plasmid (vector-only) was also stably transfected into LNCaP cells. The expression of p53 in these stable sublines was confirmed by Western blotting. In each case, the transfected subline demonstrated increased p53 protein compared with the LNCaP parental cells (Fig. 1)Citation , indicating that the exogenous p53 was expressed. To examine whether these exogenous p53 mutants dominate over the LNCaP endogenous wt p53, the expression level of p53-regulated p21waf1 protein after 10-Gy radiation was tested using Western blotting analysis. As shown in Fig. 1Citation , reduced p21waf1 levels were observed in these GOF sublines as compared with the parental LNCaP cells, suggesting a dominant-negative effect.



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  		Fig. 1. Expression of p53 and p21waf1 protein in the mutant p53-transfected LNCaP cells. Top, Western blotting demonstrates the overexpression of p53 protein in each subline compared with the parental LNCaP, confirming the presence of exogenous p53 in these sublines. Middle, Western blotting analysis of p21waf1 expression in mutant p53-transfected LNCaP sublines 4 h after 10-Gy radiation. The parental LNCaP cells, after induction by radiation, express high levels of p21waf1, whereas the four GOF p53 sublines have comparatively lower p21waf1 levels, suggesting a dominant-negative effect. Bottom, the ß-actin, used as a standard to confirm equivalent loading, was derived from the same protein preparations as was used for the p53 and p21 Western blotting. Each lane contains 50 µg protein.

 
AI Growth of LNCaP Cells Expressing GOF Mutant p53.
The LNCaP parental cells, the vector-only subline, the P151S subline, and the four GOF p53 sublines were grown for 5 days in androgen-depleted medium. The parental LNCaP cells and the P151S subline underwent a growth arrest (Fig. 2A)Citation . Similarly, the vector-only subline demonstrated growth arrest (data not shown). In contrast, the GOF p53 sublines continued to grow in androgen-depleted medium (Fig. 2A)Citation , thus exhibiting an AI phenotype. The growth rates of these lines in androgen-depleted medium were examined by counting cells on days 0, 1, 3, and 5. As shown in Fig. 2BCitation , the three control lines (the parental LNCaP, vector-only subline, and the P151S subline) failed to grow in androgen-depleted medium, whereas the four GOF p53 sublines were able to proliferate in the medium with a doubling-time of ~2.5 days for three of the sublines. We additionally compared the four GOF p53 sublines to determine the effect of different GOF p53 mutants on the growth of LNCaP cells. Cells were grown for 5 days in the presence or absence of androgens, and the fold increases in cell number were calculated by dividing the cell number in androgen-containing medium by cell number in androgen-depleted medium. We observed that the growth of three GOF sublines (G245S, R248W, and R273C) was slightly faster in androgen-containing medium than in androgen-depleted medium, whereas the R273H subline had similar growth rates in the two media (Fig. 2C)Citation . Overall, these experiments demonstrate that only GOF mutant p53 alleles resulted in the AI growth of LNCaP cells.



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  		Fig. 2. Growth of LNCaP parental and mutant p53-transfected sublines in androgen-depleted medium. A, the LNCaP parental cells and mutant p53-transfected cells grow in androgen-containing medium and/or in androgen-depleted medium for 5 days. Growth arrest was seen for the LNCaP cells and the non-GOF P151S subline (LNCaP+P151S) in androgen-depleted medium. In contrast, the GOF p53 sublines (LNCaP+G245S, LNCaP+R248W, LNCaP+R273H, and LNCaP+R273C) continued to grow in the androgen-depleted medium. B, the growth curves show that the four GOF p53 sublines continued to proliferate in androgen-depleted medium, whereas the LNCaP parental, vector-only, and P151S sublines do not proliferate in this medium. C, comparison of the effect of different GOF p53 mutants on the growth of LNCaP in the presence or absence of androgens. The Y axis is the fold-change expressed as the cell number in androgen-containing medium divided by the cell number in androgen-depleted medium on day 5. The LNCaP parental and P151S subline are used as controls, and have a large fold increase, reflecting the growth arrest of these cells in androgen-depleted medium.

 
Expression and Activity of the AR.
LNCaP cells express a mutant AR that has been identified frequently in CaP patients who have AI disease (17) . Despite this mutation, the LNCaP AR has been shown to bind androgens and maintain its ligand-dependent transactivational activity (18) . To investigate how this mutant receptor is involved in the GOF mutant p53-mediated AI growth, cells were grown in androgen-containing or androgen-depleted medium for 5 days, and AR expression was evaluated by Western blotting. As shown in Fig. 3Citation , the expression levels of AR protein were similar in the LNCaP parental cells, vector-only, and five mutant p53-transfected sublines when grown in androgen-containing medium, indicating that transfection of these mutant p53 alleles into LNCaP cells did not affect the basal levels of receptor expression. When these lines were grown in androgen-depleted medium, a slight decrease in the AR levels was observed in the parental, vector-only, and P151S lines. However, in the case of the four GOF p53 sublines, the expression of AR was markedly down-regulated after 5-day growth in androgen-depleted medium. These results suggest that the AR is not required for GOF p53 mutant-induced AI growth of LNCaP cells. The reduction of AR expression after androgen depletion in the GOF p53 sublines prompted us to examine the transactivational activity of the AR. We analyzed the expression levels of PSA protein, a downstream gene product regulated by the AR in the presence of androgen. Consistent with AR expression levels, both the LNCaP parental cells and the P151S cells expressed high levels of PSA protein in androgen-containing medium, whereas the PSA levels were markedly decreased in androgen-depleted medium (Fig. 4A)Citation . In contrast, introduction of the GOF p53 alleles into LNCaP cells resulted in significant reduction of the levels of PSA protein (P < 0.05) when these cells were grown in androgen-containing medium, although their AR expression levels remained similar to those of the control cell lines. Furthermore, when these four GOF sublines were cultured for 5 days in androgen-depleted medium, their PSA was undetectable using the ELISA approach (Fig. 4A)Citation . In one experiment, ELISA was also performed on cells containing empty plasmids (vector-only control). The PSA levels were comparable with those of the parental LNCaP cells (data not shown). We additionally analyzed PSA mRNA expression using RT-PCR amplification of cDNA derived from these lines. As shown in Fig. 4BCitation , there is decreased PSA mRNA expression in three GOF p53 sublines (R248W, R273H, and R273C) when grown in androgen-containing medium, whereas PSA gene expression was undetectable in the four GOF p53 sublines when grown in androgen-depleted medium. Therefore, the decreased PSA expression at both protein and mRNA levels may be the result of reduced AR expression and/or reduced AR activity.



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  		Fig. 3. Western blotting analysis of AR expression in GOF p53 sublines and control lines in the presence or absence of androgens. There is no change in AR expression in the LNCaP parental, vector-only, and P151S sublines grown in androgen-containing medium or slight AR reduction in androgen-depleted medium. However, in the GOF p53 sublines, there is a marked reduction in AR expression after androgen withdrawal. Each lane contains 30 µg of protein, and ß-actin was used to ensure equivalent loading. The four blots shown at the right of the figure are from a separate assay.

 


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  		Fig. 4. PSA production in the GOF p53 sublines. A, ELISA analysis of PSA expression. The LNCaP parental and P151S subline grown in androgen-containing medium produce high levels of PSA, whereas the four GOF p53 sublines have lower levels of PSA. On androgen depletion, PSA levels were much lower in the LNCaP parental and P151S cells, with levels undetectable in the four GOF mutant p53 sublines. Error bars represent an average of three experiments; bars, ±SE. B, RT-PCR amplification of PSA. Expression of PSA was highest in the LNCaP parental and P151S subline, whereas very little PSA expression was seen in the four GOF mutant p53 sublines. N-ras was used as control.

 
Expression of Akt in GOF Mutant p53 Sublines.
Because Akt has been implicated in promoting CaP cell survival and growth in the absence of androgen (19) , we examined the levels of p-Akt. All of the cell lines were grown for 5 days in either androgen-containing or androgen-depleted medium, and the p-Akt levels were determined using Western blot analysis of the extracted protein. As shown in Fig. 5Citation , the transfection of GOF mutant p53 alleles into LNCaP cells did not significantly alter p-Akt expression when the cells were grown in androgen-containing medium. However, in each of the cell lines, the p-Akt levels were increased markedly when the cells were grown in androgen-depleted medium. These results indicate that up-regulation of p-Akt after androgen depletion is not a specific response of LNCaP cells to expression of the GOF p53 mutants but rather is a general response to androgen withdrawal.



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  		Fig. 5. Western blotting analysis of p-Akt and Bcl-2 expression in GOF p53 sublines and control lines. Increased p-Akt was detected in all of the cell lines in the absence of androgens, indicating a general response to androgen withdrawal. Bcl-2 expression increased in G245S, R248W, R273H, and P151S sublines in the presence of androgen, and their expression decreased after androgen withdrawal. Levels of Bcl-2 were similar before and after androgen withdrawal in the remaining three lines. ß-Actin was used to ensure equivalent loading. Each lane contains 30 µg of protein.

 
Bcl-2 Expression in GOF Mutant p53 Sublines.
The results described above suggest that the GOF mutant p53-mediated AI growth may be through a pathway that bypasses the AR. Because Bcl-2 is a potent inhibitor of apoptosis that has been implicated in the development of hormone resistance in CaP (20) , we examined these sublines for their expression of Bcl-2 protein, an obvious bypass candidate gene. As shown in Fig. 5Citation , transfection of the mutant p53 alleles into LNCaP cells resulted in increased Bcl-2 expression in the G245S, R248W, R273H, and P151S sublines when grown in androgen-containing medium, as compared with the parental and vector-only cells. The up-regulation of Bcl-2 expression may result from a dominant-negative effect of mutant p53 over wt p53. The exception is the R273C mutant, which had lower expression than the control lines. This was a consistent result seen in three independent experiments. When these seven lines were grown in androgen-depleted medium for 5 days, their Bcl-2 levels were down-regulated to a greater or lesser extent compared with the corresponding cells grown in androgen-containing medium. These results suggest that down-regulation of Bcl-2 expression is not a specific response of LNCaP cells to the GOF p53 mutants but is a general response to androgen withdrawal.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Once CaP becomes unresponsive to androgen withdrawal, no additional curative treatment is available for this disease. Understanding the molecular changes that occur during progression to androgen independence is essential for the development of targeted therapy. Because p53 mutations are found at high frequencies in CaP cell lines, and in clinical samples from AI and metastatic tumors (6 , 7 , 9 , 21 , 22) , this suggests that p53 mutations may play a role in the development of hormone-refractory CaP. This could occur through two mechanisms. First, because the primary effect of p53 mutation is loss of tumor suppressor function, inactivating or reducing wt p53 function could result in androgen independence. This theory is supported by several published studies. One showed that CaP tumor in which p53 was inactivated by the large T antigen of SV40 progressed to androgen independence in transgenic mice (23) . Burchardt et al. (24) reported recently that a hormone-resistant phenotype could be induced in LNCaP cells by reduction of wt p53 function as the result of transfection of either p53 antisense transcripts or a truncated, dominant-negative form of p53. These studies demonstrated that loss of wt p53 function could contribute to the androgen resistance of CaP cells. Second, it is well established that, in addition to the loss of wt p53 function, p53 mutations can also result in GOF characteristics. In other types of cancer, some GOF p53 mutants have been shown to enhance tumorigenic potential (12) or to up-regulate genes involved in cell proliferation (10 , 25) . On the basis of these observations, we hypothesized that, beyond their ability to inhibit the activity of endogenous wt p53, GOF p53 mutants may directly contribute to AI growth of CaP cells. To test this hypothesis, five dominant-negative p53 mutant alleles (four having and one lacking GOF properties) were transfected into LNCaP cells, which normally express wt p53. The ability of these p53 mutants to dominate over the endogenous wt p53 was evidenced by showing that these sublines had reduced p21waf1 expression and increased Bcl-2 expression, because a p53-dependent negative response element has been identified in the Bcl-2 gene (26 , 27) . Among these five mutants, only the four GOF p53 mutant sublines were able to grow in androgen-depleted medium, whereas the non-GOF P151S subline was unable to grow under these conditions. Thus, the data reported here offers strong support to our hypothesis.

In an effort to elucidate the molecular mechanism(s) through which GOF p53 mutants induce AI growth, we examined whether the expression or transactivational activity of the AR was altered by the transfected p53 mutants. In the GOF p53 LNCaP sublines, we failed to observe an increase in AR expression or in AR-dependent PSA expression when grown in androgen-containing medium, whereas we observed a marked decrease in AR and PSA expression when grown in androgen-depleted medium. This indicates that AI growth of these GOF p53-transfected cells can occur in an AR-independent fashion. We observed that these GOF p53 sublines expressed a significantly low level of PSA in androgen-containing medium when compared with the parental LNCaP line and the P151S subline, suggesting that these p53 mutants repress AR activity. This is consistent with observations reported by other groups. Eastham et al. (28) reported that wt p53 was able to inhibit the expression of PSA in CaP tumors grown in nude mice. Shenk et al. (29) observed recently that in AR-transfected PC3 cells the R248W p53 mutant weakly repressed AR transactivation of the PSA promoter in the presence of androgen. This negative activity of p53 is independent of specific DNA binding (30) , and requires the p53 DNA-binding domain and COOH terminus (29) . Thus, the four transfected GOF p53 mutants that contain mutations in the DNA-binding domain and possess the COOH terminus may be able to down-regulate the transactivational activity of the AR, resulting in the reduced PSA expression. However, the extent of down-regulation may be dependent on the cellular context. We additionally found that the expression of AR protein was markedly repressed when these GOF mutant p53-transfected LNCaP sublines were grown for 5 days in androgen-depleted medium. This was accompanied by a marked repression of PSA expression at both the protein and the mRNA levels. It is difficult to attribute this down-regulation of PSA expression to specific inhibition of AR expression by GOF p53 mutants in an androgen-depleted environment. More probably, the decreased AR levels that we observed are attributable to degradation of AR protein in the absence of androgen. This is in accord with the observation that the synthetic androgen R1881 increases AR stabilization (31) . The fact that the GOF mutant p53 sublines that express very low levels of AR protein are nonetheless able to grow in androgen-depleted medium additionally supports our hypothesis that AI growth of these sublines is probably via an AR-independent pathway.

We examined several additional factors that have been implicated in the AI growth of CaP. Akt is a serine-threonine protein kinase that inhibits the transactivational activity of the AR (32) . Furthermore, Akt expression has been shown to increase under conditions of androgen withdrawal. This increased expression is sustained throughout the progression to androgen independence (33) . Phosphorylation of Akt results in full activation of this kinase (34) . We found that expression of the active, phosphorylated form of Akt is considerably higher in all of the LNCaP cell lines when grown in androgen-depleted conditions. This appears to be a general response to androgen ablation, because it is not specifically induced by GOF p53 mutants.

Bcl-2 overexpression has been associated with the progression of CaP to a metastatic phenotype and correlates with the development of therapeutic resistance (24 , 35) . Despite these observations, Bcl-2 expression was somewhat decreased in all of our cell lines maintained in androgen-depleted medium. This down-regulation may reflect the effect of short-term androgen-withdrawal treatment.

A recent study shows that p53 mutants up-regulate the promoter of the BAG-1 gene that expresses a multifunctional antiapoptotic protein (36) . The BAG-1L protein that arises from translation initiation at a noncanonical CUG codon, has been shown to increase AR transactivational activity in the presence of low concentration of androgen (37) . Results from our pilot experiments have demonstrated increased BAG-1L protein expression in GOF p53 sublines compared with the controls, whereas the expression of BAG-1 and its other isoform BAG-1M was not affected in these lines. Whether and how BAG-1L is involved in the GOF p53 mutant-mediated androgen independence is currently under investigation in our laboratory.

The contribution of other gene pathways to AI growth in the presence of GOF p53 mutants is currently under investigation in our laboratory. Microarray assays of these sublines have identified 84 genes as being differentially expressed in GOF p53 sublines compared with vector-only subline. Overall, 61 genes are down-regulated, whereas 23 are up-regulated. These genes encode proteins having a broad range of functions including metastasis, tumor suppression, cell cycle control, G-protein signaling, translation, or detoxification. Elucidation of the relative importance of these gene changes will shed light on GOF p53 mutant-mediated AI growth in CaP.

In conclusion, we have developed a cell model and demonstrated that GOF p53 mutants contribute to AI growth of LNCaP cells in vitro. In the presence of these GOF p53 mutants, the expression and transactivational activity of the AR are down-regulated, indicating that an activated AR is not needed for GOF p53 mutant-mediated proliferation in the absence of androgens. Furthermore, Bcl-2 expression is not involved in this AI growth. Whereas increased Akt expression was not induced directly by GOF p53 mutants, it may be associated with androgen ablation and the development of androgen independence. Taken together, these observations suggest that there are additional pathways involved in the GOF p53 mutant-induced androgen independence. Our LNCaP sublines should be valuable models for investigating these pathways.


   ACKNOWLEDGMENTS
 
We thank Dr. Arline Deitch for insightful discussions and editorial assistance. We also thank Dr. Philip C. Mack for assistance with the statistical analysis and helpful discussions, and Dr. Clifford G. Tepper for analysis of the microarray data.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants NCI RO1 CA 77662 and NCI RO1 92069. Back

2 To whom requests for reprints should be addressed, at Department of Urology, University of California, Davis, School of Medicine, 4860 Y Street, Suite 3500, Sacramento, CA, 95817. Phone: (916) 734-2824; Fax: (916) 734-8094; E-mail: rwdeverewhite{at}ucdavis.edu Back

3 The abbreviations used are: CaP, prostate cancer; AI, androgen-independent; wt, wild-type; GOF, gain-of-function; MDR, multidrug resistance; FBS, fetal bovine serum; PSA, prostate-specific antigen; RT-PCR, reverse transcription-PCR; AR, androgen receptor; p-Akt, phosphorylated Akt; BAG-1L, long BAG-1. Back

Received 8/21/02. Accepted 3/ 5/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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