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Molecular Mechanisms of Androgen-Independent Growth of Human Prostate Cancer LNCaP-AI Cells¹

Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Dr. Ming-Jer Tsai, Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: mtsai{at}bcm.tmc.edu

	Abstract

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The goal of this study is to investigate the molecular mechanisms of androgen-independent growth in prostate cancer. We have established an androgen-independent prostatic carcinoma LNCaP-AI (defined as a LNCaP cell line that is capable of growing in charcoal-stripped serum) from the androgen-dependent LNCaP-FGC cells. In contrast to the androgen-independent PC-3 human prostate cancer cells, LNCaP-AI cells still express a similar level of androgen receptor as their parental cells and are sensitive to androgen stimulation. Compared with the parental LNCaP-FGC cells, LNCaP-AI cells are more resistant to apoptosis induced by 12-O-tetradecanoylphorbol-13-acetate and express a much higher level of antiapoptotic gene bcl-2 and cyclin-dependent kinase inhibitor p21, which may confer an enhanced antiapoptosis phenotype. On the other hand, expression of cyclin-dependent kinase inhibitor p16 is significantly reduced in the LNCaP-AI cells, implying the release of an inhibitory effect of p16 on cell cycle progression. Taken together, our results suggest that multiple factors contribute to the development of androgen-independent growth of prostatic carcinoma cells, including enhancement of cell antiapoptosis function, release of cell cycle inhibition, and stimulation of cell proliferation by alternative signaling pathways.

	Introduction

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PROSTATIC CARCINOMA IS the second leading cause of cancer death in men (1, 2, 3). At the early stage of prostate cancer, the growth of prostatic carcinoma cells is androgen-dependent and can be effectively treated by hormone ablation either using surgical or pharmacological methods (4). However, the hormone ablation therapy only causes a temporary regression of prostate tumors (3, 5), and some tumor cells become androgen-independent in 6–18 months (3, 5). The molecular mechanisms by which prostate cancer cells become androgen independent are unknown and remain the focus of intensive research.

Several factors have been demonstrated to be involved in the development of androgen-independent growth in prostate cancer. For instance, mutation (6, 7), amplification, and overexpression (8, 9) of the androgen receptor (AR) gene have been observed in androgen-independent prostate cancer. Androgen independence may also result from expression of bcl-2, an antiapoptotic gene. The bcl-2 gene is not expressed in the normal secretary epithelial cells of the prostate but is expressed in prostate cancer specimens, suggesting that bcl-2 expression is correlated with the cancer phenotype (10). It was demonstrated that overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen-deprivation therapy in vivo (11). Based on the above findings, we hypothesize that multiple factors contribute to the development of androgen-independent growth of prostatic carcinoma cells.

We undertook the current studies to determine whether certain critical cell cycle regulatory proteins, as well as antiapoptotic proteins, play a role in progression of prostate cancer to the androgen-independent state. We generated an androgen-independent LNCaP-AI prostatic carcinoma cell line from androgen-dependent LNCaP-FGC cells by in vitro cell culture. In comparison with parental LNCaP-FGC cells, LNCaP-AI cells were more refractory to 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced apoptosis. Molecular characterization revealed that changes in expression of multiple factors, such as bcl-2 and cyclin-dependent kinase (CDK) inhibitors p21 and p16, may contribute to the androgen-independent growth of prostatic carcinoma LNCaP-AI cells. These results provide an insight into the molecular mechanisms of development of androgen-independent growth of prostatic cancer.

	Materials and Methods

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Cell culture
The androgen-dependent human metastatic prostate adenocarcinoma cell line LNCaP-FGC (5) (ATCC, Rockville, MD) was maintained in RPMI-1640 (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FBS (HyClone Laboratories, Inc., Logan, UT) at 37 C in 5% CO₂. The hormone-independent PC-3 human prostate adenocarcinoma cells (12) (ATCC) were cultured in DMEM/F12 medium (Life Technologies, Inc.) supplemented with 10% FBS. LNCaP-AI cells are currently maintained in RPMI-1640 supplemented with 10% charcoal/dextran-treated (stripped) FBS.

Reagents
AR agonist R1881 was purchased from Dupont Biotechnology Systems (Boston, MA). TPA and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO). Antibodies against p21, p16, CDK2, CDK4, bcl-2, bax, and AR were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Cell growth
Tumor cell growth was estimated by the MTT assay, as previously described (13). Briefly, LNCaP-FGC and LNCaP-AI cells, cultured in medium supplemented with stripped FBS for 1 week, were harvested by exposure to 0.25% trypsin/0.02% EDTA (wt/vol) and seeded into 96-well microculture plates at a density of 10,000 cells/well in RPMI-10% stripped FBS. After incubation in 5% CO₂ at 37 C overnight, the cells were incubated in the same medium containing AR agonist R1881 (10^-9 M) for 2, 4, and 6 days. At the end of incubation, 20 µl MTT (2.5 mg/ml in PBS) was added to each well, and the cells were further incubated for 2 h at 37 C to allow complete reaction between the dye and the enzyme mitochondrial dehydrogenase in the viable cells. After removal of the residual dye and medium, 100 µl dimethylsulfoxide was added to each well, and the absorbance at 570 nm was measured with an MRX microplate reader (Dynatech Laboratories, Chantilly, VA).

Western blot analysis
Aliquots of samples with the same amount of protein, determined by using the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA), were mixed with loading buffer (final concentrations of 62.5 mM Tris-HCl (pH 6.8), 2.3% SDS, 100 mM dithiothreitol, and 0.005% bromophenol blue), boiled, fractionated in a 15% SDS-PAGE, and transferred onto a 0.45-µm nitrocellulose membrane by electroblotting (Bio-Rad Laboratories, Inc.). The membranes were blocked with 2% fat-free milk in PBS and then probed with first antibody (0.05 µg/ml IgG) in PBS containing 0.1% Tween 20 (PBST) and 1% fat-free milk. The membranes were then washed four times in PBST and incubated with horseradish peroxidase-conjugated F(ab')₂ of goat antirabbit secondary antibody (Bio-Rad Laboratories, Inc.) in PBST containing 1% fat-free milk. After washing four times in PBST, the membranes were visualized using the ECL Western blotting detection system (Amersham Pharmacia Biotech, Arlington Heights, IL).

DNA fragmentation assay
The apoptosis was monitored by internucleosomal DNA degradation. Briefly, genomic DNA was isolated using a DNA isolation kit purchased from QIAGEN (Chatsworth, CA). Aliquots of DNA (10 µg/lane) were electrophoresed through a 1.8% agarose gel, which was stained with ethidium bromide in Tris-acetic acid-EDTA buffer. Fluorescent DNA bands were visualized with a UV transilluminator and were photographed. A ladder pattern, representing fragments of DNA in multiples of 180–200 bp, provided evidence for apoptosis.

	Results

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Establishment of an androgen-independent LNCaP-AI cell line
To study the molecular mechanisms of development of androgen-independent growth of prostatic carcinoma, we generated an androgen-independent LNCaP-AI cell line (defined by the ability of this LNCaP subline to proliferate under conditions of charcoal-stripped serum) by long-term culture of androgen-dependent LNCaP-FGC cells in RPMI-1640 medium containing stripped serum. This approach mimics the androgen ablation condition for treating prostate cancer (3, 5). After 3 weeks in culture, some of the LNCaP-FGC cells underwent apoptosis, as demonstrated by the formation of apoptotic bodies, and a majority of the cells showed neuroendocrine-like phenotype (Fig. 1). This phenomenon has been observed by others, in a previous report, stating that after hormone deprivation, LNCaP-FGC cells develop an alternative autocrine mechanism by producing and secreting neurotensin (14). Androgen-stimulated cells do not respond to neurotensin, possibly because of androgen-induced metalloprotease, which degrades neurotensin. More than 99% of cells underwent apoptosis during 6 months of cell culture in medium containing stripped serum. The remaining cells grew in an androgen-independent fashion (Fig. 2A). We named the resulting cell line LNCaP-AI.

View larger version (91K): [in this window] [in a new window]   Figure 1. Neuroendocrine phenotype of LNCaP-FGC cells. LNCaP-FGC cells are cultured in either normal medium (A) or medium containing stripped serum (B) for 3 weeks. Cell morphology was recorded.

View larger version (30K): [in this window] [in a new window]   Figure 2. The effects of androgen on LNCaP-FGC and LNCaP-AI cell growth. A, LNCaP-FGC and LNCaP-AI cells were cultured in medium containing 10% stripped FBS for 1 week. Cells were then seeded into 96-well microculture plates at a density of 10,000 cells/well and cultured for 6 days, with addition of androgen agonist R1881 (10-9 M) on day 0, 2, or 4. The cell density were determined on day 6 by MTT assay. B, AR expression in LNCaP-FGC, LNCaP-AI, and PC-3 cells. LNCaP-FGC and LNCaP-AI cells were cultured in medium containing 10% stripped FBS for 1 week, followed by incubation with AR agonist R1881 (10-8 M) for 48 h. Subsequently, total cellular proteins from LNCaP cells, as well as PC-3 cells, were isolated and subjected to Western blot analysis using 50 µg protein per sample. Ponceau S staining was used as the protein loading control.

LNCaP-AI cells are resistant to apoptosis induced by TPA
Hormone ablation causes both normal prostatic epithelial cells and androgen-dependent prostate cancer cells to undergo apoptosis (3, 15, 16). Because LNCaP-AI cells were generated under the hormone-deprivation condition, we investigated whether these cells have developed enhanced antiapoptosis properties during the selection process. Protein kinase activator (TPA) was used as an apoptosis inducer for LNCaP-FGC cells (17). After incubating LNCaP cells with TPA overnight, morphologically, LNCaP cells showed typical apoptotic characteristics, such as chromosome condensation, cell blebbing, and apoptotic body formation (data not shown). Analysis of genomic DNA fragmentation, which is a hallmark of apoptosis, revealed that LNCaP-FGC cells showed an increase of fragmented DNA in a dose-dependent manner, whereas DNA from LNCaP-AI cells was relatively intact (Fig. 3A). This data suggested that LNCaP-AI cells, as compared with parental LNCaP-FGC cells, are much more resistant to apoptosis. Furthermore, the viability of LNCaP-AI and LNCaP-FGC cells was examined upon TPA treatment by trypan blue exclusion counting. LNCaP-AI cells showed a significant higher rate of cell viability and a much lower rate of cell death in response to increased concentrations of TPA stimulation (Fig. 3B). In combination, these results strongly suggest that LNCaP-AI cells have gained an enhanced antiapoptotic phenotype upon androgen ablation selection.

View larger version (29K): [in this window] [in a new window]   Figure 3. Resistance to apoptosis by LNCaP-AI cells. A, Apoptosis of LNCaP-FGC and LNCaP-AI cells induced by TPA. LNCaP-FGC cells were treated with 3, 4, and 5 nM TPA for 24 h. Subsequently, the genomic DNAs from these cells were isolated, and aliquots of 20 µg DNA were subjected to DNA ladder analysis, as described in Materials and Methods. B, Comparison of TPA-induced cell death in LNCaP-FGC and LNCaP-AI cells. LNCaP-FGC and LNCaP-AI cells were treated with 1.25, 2.5, 5, and 10 nM TPA for 24 h. The cell viability was determined by trypan blue exclusion counting.

View larger version (35K): [in this window] [in a new window]   Figure 4. Expressions of CDK2, CDK4, p16, and p21 genes in LNCaP-FGC, LNCaP-AI, and PC-3 cells. LNCaP-FGC, LNCaP-AI, and PC-3 cells were cultured in medium containing 10% stripped FBS for 1 week. Then, the cells were treated with or without AR agonist R1881 (10-9 M) for 48 h, as indicated. The cell extracts were prepared and subjected to Western blot analysis.

Expression of antiapoptotic gene is altered in LNCaP-AI cells
Recently, it has been demonstrated that an increased expression of bcl-2 gene upon hormone deprivation is involved in the development of androgen-independent growth in prostate carcinoma cells (11). The bcl-2 family genes include bcl-2 and bax genes, whose gene products form a heterodimer with one another (17). The relative amounts of bcl-2 to bax determine cell viability during growth factor deprivation. An increased ratio of bcl-2 to bax promotes cell survival, whereas an increased ratio of bax to bcl-2 protein promotes cell death. We, therefore, compared the expressions of bcl-2 and bax genes in LNCaP-FGC, LNCaP-AI, and PC-3 cells. Western blot analysis showed that the basal expression of bcl-2 gene in LNCaP-AI cells was drastically increased to a level that was similar to that in androgen-independent PC-3 cells (Fig. 5). An increased basal expression of bcl-2 gene in LNCaP-AI cells may contribute to an enhanced survival ability of LNCaP-AI cells, under a condition of androgen deprivation. LNCaP-FGC cells expressed similar low basal levels of bcl-2 in the presence or absence of AR agonist R1881 (Fig. 5), which suggested that androgen does not directly regulate bcl-2 expression. The molecular mechanisms of enhanced expression of bcl-2 gene in LNCaP-AI cells are not clear. In contrast to bcl-2, similar levels of bax gene were found in LNCaP-FGC, LNCaP-AI, and PC-3 cells (Fig. 5).

View larger version (50K): [in this window] [in a new window]   Figure 5. Expressions of bcl-2 and bax genes in LNCaP-AI, LNCaP-FGC, and PC-3 cells. LNCaP-FGC, LNCaP-AI, and PC-3 cells were cultured in medium containing 10% stripped FBS for 1 week. Then, the cells were treated with or without AR agonist R1881 (10-9 M) for 48 h, as indicated. The cell extracts were prepared from these cells, followed by Western blot analysis.

	Discussion

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It has been reported previously that cells from androgen-independent prostate cancers express a high level of bcl-2 and overexpression of bcl-2 protects prostate cancer cells from undergoing apoptosis upon androgen depletion in vivo (10, 11). On the contrary, the normal secretary prostatic epithelial cells do not express bcl-2. Consistent with the above finding, we found that androgen-independent LNCaP-AI cells express a much higher level of bcl-2 and are more refractory to TPA-induced apoptosis than the parental LNCaP-FGC cells. The expression level of bax in LNCaP-AI cells is similar to those in LNCaP-FGC and PC-3 cells. In addition, bcl-2 expression is not affected by AR agonist R1881. These results suggest that bcl-2 may not be a regulator in preventing the normal prostatic epithelial cells from undergoing apoptosis under conditions such as castration but plays a key role in an antiapoptotic effect in androgen-independent prostate cancer.

Previously, we have shown that androgen down-regulates the expression of CDK inhibitor p16 gene at both the messenger RNA and protein levels, suggesting that androgen releases the inhibitory effect of p16 on cell cycle by down-regulating the gene expression (19). The results from the present study demonstrated that the basal expression of the p16 gene is decreased, in comparison with that in the parental LNCaP-FGC cells. These results extend our previous finding in that a low basal expression of the p16 gene in LNCaP-AI cells shows a negative impact on the cell cycle blocking in androgen-independent prostate cancer cells. However, the molecular mechanisms, by which the selection pressure of hormone deprivation results in a sustained decreased expression of the p16 gene and hence releases the inhibitory effect of p16 on cell cycle progression in androgen-independent LNCaP-AI cells, remain unclear. It has been demonstrated that the promoter of the p16 gene is not methylated in normal prostate tissue and in androgen-dependent LNCaP-FGC cells but in androgen-independent PC-3 cells (20). Deletion and methylation combine to inactivate p16 gene in a subset of prostate tumor, and alteration of this gene may represent a late event in prostate cancer progression. It is speculated that the decreased expression of cell cycle inhibitor can enhance the survival ability of LNCaP-AI cells under an aberrant condition such as androgen ablation.

The CDK inhibitor p21 is a cell-cycle repressor. It is particularly interesting to note that the basal expression of the p21 gene in androgen-independent LNCaP-AI cells increases. Moreover, consistent with our previous findings in LNCaP-FGC cells (16), expressions of the p21 gene in LNCaP-AI, as well as in LNCaP-FGC cells, were further stimulated by AR agonist R1881. Because androgen stimulates the proliferation of both LNCaP-FGC and LNCaP-AI cells, the functional significance of increased expression of the p21 gene in response to androgen treatment is not likely to cause cell growth arrest. In addition to inhibiting cell cycle progression, p21 has been demonstrated to be involved in DNA repair and antiapoptosis (21, 22, 23). Activation of CDK2 is required for apoptosis events to occur (24). Compelling evidence indicates that p21 binds to the cyclin-CDK complex and inhibits the CDK2 activity. Hence, inactivation or degradation of p21 enables CDK2 activation and permits cells to undergo apoptosis (25). The C-termini of CDK inhibitors p21 and p27 are truncated by caspases in apoptotic cells, which leads to activation of CDK2 (25). To support our observations, it was reported that in contrast to normal prostatic epithelial cells and benign prostate hyperplasia, prostate cancer cells may use multiple mechanisms to evade apoptosis in response to trophic factor deprivation, such as up-regulation of p21 and bcl-2 genes and down-regulation or lost expression of bax gene (26). Taken together, our results suggested that overexpression of p21 gene in androgen-independent LNCaP-AI cells is one of the factors required for the cell survival under the hormone ablation condition.

It has been well-documented that castration causes the prostatic epithelial cells to undergo apoptosis, suggesting that androgen is essential for maintenance of the integrity of the prostatic epithelium (15, 16). However, the target genes that play a role of antiapoptosis function, stimulated by androgen, have not been identified. Because immunohistochemistry analysis showed that the prostatic epithelial cells, but only a few stroma cells, express p21 protein, it is tempting to speculate that p21 may be one of the candidates responsible for maintaining the integrity of the prostatic epithelium in an androgen-dependent manner (18). Further determination of this p21 function in prostatic epithelial cells should shed some light on this important process.

In summary, we presented in this study an in vitro model of androgen-independent prostatic cancer. Because LNCaP-AI cells were generated under androgen deprivation conditions that mimic the androgen ablation therapy for human prostate cancer, this cell line should be a useful tool for studying the molecular mechanisms of androgen-independent growth and for testing drugs against androgen-independent prostate cancer, which is critical for developing strategies to treat the advanced prostate cancer. Our data suggest that multiple factors contribute to the development of androgen-independent growth in prostate cancer cells, including enhancement of cell antiapoptosis function, release of cell cycle inhibition, and stimulation of cell proliferation by alternative signaling pathways.

	Acknowledgments

 
We thank Miss Naomi Lee for assistance in manuscript preparation.

	Footnotes

 
¹ This work was supported by Baylor Special Program of Research Excellence in prostate cancer (CA58204).

Received January 8, 1999.

	References

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