Androgen signaling and post- transcriptional downregulation ... - Nature

Prostate Cancer and Prostatic Diseases (2004) 7, 158–164 & 2004 Nature Publishing Group All rights reserved 1365-7852/04 $30.00

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Androgen signaling and posttranscriptional downregulation of Bcl-2 in androgen-unresponsive prostate cancer CA Rothermund1, VK Gopalakrishnan1 & JK Vishwanatha1* 1

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA

We previously characterized the LNCaP prostate cancer progression model and showed that despite loss of Bcl-2 protein in the androgen-unresponsive LNCaPunresponsive (UR) cells, these cells maintained an increased resistance to the induction of apoptosis. Since the loss of Bcl-2 protein coincided with the progression to androgen-unresponsiveness, we sought to determine if Bcl-2 expression was regulated through androgen signaling pathways. LNCaPresponsive (R) and -UR cells grown in charcoal-stripped serum conditions for 3 months differentiated to a neuroendocrine (NE)-like morphology. Under these conditions, LNCaP-UR cells regained Bcl-2 protein expression, and LNCaP-R cells overexpressed Bcl-2. Chronic exposure to casodex resulted in differentiation of both LNCaP-R and -UR cells to the NE-type morphology accompanied by a marked downregulation of Bcl-2 protein, while Bax protein levels were unchanged. Downregulation of Bcl-2 was post-transcriptional since Bcl-2 message levels were unchanged in LNCaP cells treated with casodex. These data suggest that Bcl-2 is post-transcriptionally modulated by androgen signaling pathways in LNCaP cells. Prostate Cancer and Prostatic Diseases (2004) 7, 158–164. doi:10.1038/sj.pcan.4500717 Published online 4 May 2004

Keywords: androgen-unresponsive; apoptosis; casodex; Bcl-2 post-transcriptional; neuroendocrine

Introduction Prostate cancer progression is marked by resistance to chemotherapeutic agents, an increase in serum levels of prostate specific antigen, and eventual death due to metastasis. Concurrent with the progression of prostate cancer to the terminal stage is the upregulated expression of Bcl-21 in many, but not all advanced prostate cancers. Prostate cancer is heterogenous, and not all high-grade tumors are Bcl-2 positive. Approximately 31–38% of highgrade prostate tumor biopsy samples were Bcl-2 positive.2,3 Although Bcl-2 expression in vivo shows a positive correlation to androgen-unresponsiveness, prostate can-

*Correspondence: JK Vishwanatha, Department of Biochemistry and Molecular Biology, University Nebraska Medical Center, 984525 Nebraska Medical Center, Omaha, NE 68198-4525, USA. E-mail: [email protected] Received 7 November 2003; revised 20 February 2004; accepted 2 March 2004; published online 4 May 2004

cer cell lines do not show this correlation. The androgenunresponsive cell line, DU-145, does not express Bcl-2, yet is resistant to the induction of apoptosis when compared to other prostate cancer cell lines.4 Our previous studies showed that androgen-unresponsive LNCaP-UR cells displayed a loss of Bcl-2 protein expression but maintained an increased resistance to the induction of apoptosis.5 The androgen-unresponsive LNCaP-UR cells were established by continued subculture of the androgen-responsive LNCaP cells under normal growth conditions. At high passage numbers (beyond 95) these cells become androgen-unresponsive.5 Alterations in Bcl-2 protein stimulated by intracellular signaling cascades has been demonstrated.6 A recent study showed that Bcl-2 expression was inhibited by dihydrotestosterone (DHT) in LNCaP-FGC cells.7 LNCaP-FGC (fast growing cells) are low passage number LNCaP cells that are commercially available from the American Type Culture Collection (Manassas, VA, USA). DHT treatment of LNCaP cells was also shown to increase the resistance to the induction of apoptosis.8

Casodex and apoptosis regulation in prostate cancer CA Rothermund et al

Whether the inhibition of Bcl-2 by DHT or the increased resistance to the induction of apoptosis after treatment with DHT are transient or the result of a more stable expression pattern of Bcl-2, is not known. We sought to mimic chronic androgen-ablation of prostate cancer in vitro to address the regulation of Bcl-2. We disturbed androgen signaling in two ways: by growth of cells in media containing charcoal-stripped serum and by treatment of cells with casodex. We investigated changes in Bcl-2 and Bax expression under hormone-depleted conditions. We found that growth of cells in media containing charcoal-stripped serum for an extended period resulted in overexpression of Bcl-2 protein, whereas chronic treatment of cells with casodex resulted in a downregulation of Bcl-2 protein. Message levels of Bcl-2 after treatment with casodex were not significantly altered suggesting that regulation of Bcl-2 in prostate cancer as it progresses to the androgenunresponsive state occurs post-transcriptionally.

Materials and methods Materials Casodex (bicalutamide) was provided by AstraZeneca and dissolved in dimethyl sulfoxide (DMSO) to a 50 mM concentration and stored at 201C. Docetaxel (taxotere) was obtained from Rhone-Poulenc Rorer Pharmaceuticals Inc. (Collegeville, PA, USA) and dissolved in ethanol to a 100 mM concentration and stored at 201C. RPMI 1640, phenol red-free RPMI 1640, fetal bovine serum (FBS), penicillin–streptomycin (PS), and Hank’s balanced salt solution (HBSS) were purchased from Life Technologies, Inc. (Grand Island, NY, USA). Keratinocyte SFM was purchased from Clonetics (Palo Alto, CA, USA). Charcoal-stripped FBS was purchased from Hyclone (Logan, UT, USA). Mouse anti-human Bcl-2 antibody (sc509) and rabbit anti-human Bax antibody (sc-498) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phosphoglycerate kinase (PGK) antibody was used as previously described.9

Cell culture LNCaP-R, RF, UR, DU-145, and PC-3 cells were maintained as previously described.5 LNCaP-derived C4-2 cells10 were obtained under a material transfer agreement with UroCor (Oklahoma City, OK, USA). C4-2 cells were maintained in RPMI 1640 with 7% FBS and 1% PS (Life Technologies). HPV-18 C-1 cells were grown in keratinocyte-SFM supplemented with bovine pituitary extract (25 mg/ml) and recombinant epidermal growth factor (0.15 ng/ml). For growth in charcoal-stripped media, LNCaP cells were grown to 80% confluence in RPMI 1640 supplemented with 7% FBS and 1% PS then washed with phenol red-free HBSS and replaced with phenol red-free RPMI 1640 containing 2% charcoalstripped FBS and 1% PS. Media was changed once weekly and cells were passaged as needed. Light micrograph photographs were taken after 3 months. For growth in casodex, LNCaP-R and -UR cells were grown to 70% confluence in phenol red containing RPMI 1640 with 7% FBS, and 1% PS then washed with HBSS and the media replaced with phenol red containing

RPMI 1640 with 7% FBS, 1% PS, and 1.0 mM casodex. Untreated cells were maintained in phenol red containing RPMI 1640 with 7% FBS and 1% PS and passaged as needed. Untreated cells and cells maintained in media with casodex were fed every three days.

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Western blot analysis Protein extracts were prepared from cells and immunoblot analysis was performed as previously described.5 The primary antibodies used were mouse monoclonal anti-human Bcl-2 at 1 : 500 dilution and rabbit polyclonal anti-human Bax at 1 : 400 dilution. Rabbit polyclonal antihuman PGK antibody at a 1 : 2000 dilution was included in the Western blot analysis as an internal control.

RT-PCR and Real-time PCR RT-PCR and PCR was performed as previously described.5 Bcl-2 primers used for PCR and Real-time PCR were sense, 50 -CTTTTGCTGTGGGGTTTTGT-30 and antisense 50 -TCTTCTCCTTTTGGGGCTTT-30 . For Real-time PCR reactions, 1.0 ml of cDNA generated by RT-PCR was added to master mixes containing 1 PCR reaction buffer, 200 mM of each dNTP, 1 GC-rich solution, 2.0 ml of 1 : 1000 diluted SYBR green (Molecular Probes, Inc., Eugene, OR, USA), 2 U of FastStart DNA polymerase (Roche, Maneheim, Germany) and 1.0 mM each of forward and reverse primer. Following an initial incubation of 951C for 10 min, 40 cycles of 951C for 1.5 min, 561C for 45 s, and 721C for 1.5 min, were performed. Reactions, in duplicate, were performed on a BioRad iCycler thermalcycler using the iCycler software version 2.3v.

Apoptosis detection by caspase-3 assay Apoptosis was measured using the ApoAlert Caspase-3 activation colormetric assay (Clontech). The caspase-3 colorimetric assay measures proteolytic cleavage of the chromophore p-nitroanilide (pNA). Liberated pNA is monitored colorimetrically by absorbance at 405 nm. For this assay, cells were grown under the different treatment protocols indicated. At the end of the treatment period, cells were lysed and clear supernatant was collected by centrifugation. To the clear supernatant, reaction buffer containing DTT and 1 mM DEVDpNA substrate mix was added and the reaction mixture was incubated at 371C in the dark for 2 h. After the 2-h incubation period, 100 ml of reaction mixtures were placed into a 96-well plate and absorbance was monitored at 405 nm. A standard curve was constructed by diluting pNA stock solution with cell lysis buffer. Sample pNA concentrations were extrapolated from the slope of the standard curve. Appropriate negative controls were included in the assay. Each assay was performed in triplicate and the experiment was repeated.

Results The LNCaP model mimics the progression of prostate cancer We utilize LNCaP cells to study changes in apoptosis as the cells change from androgen-responsive to an Prostate Cancer and Prostatic Diseases


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Table 1 The LNCaP prostate cancer progression model

In Vivo

a

In Vitro

Early Stage Prostate Cancer •Androgen-sensitive •Slow growth

LNCaP-R

Intermediate Stage Prostate Cancer •Androgen-sensitive •Faster growth

LNCaP-RF

Metastatic (Late) Stage Prostate Cancer •Androgen-insensitive •Fast growth

LNCaP-UR

(Androgen-Responsive, Slow growth) passages 20 to 50

(Androgen-Responsive, Fast growth) passages 50 to 95

LNCaP HPV18C-1

R

UR

C4-2

PC-3

DU-145

Bcl-2

A

PGK

B 1

2

3

b

4

5

6

LNCaP HPV18C-1

R

UR

C4-2

PC-3

DU-145

500bp

(Androgen UnResponsive, Fast growth) passages 95 and above

Bcl-2

M

1

2

3

4

5

6

This model represents the progression of prostate cancer as seen in patients. Cells at early passage (LNCaP-R) represent the early stage of disease characterized by androgen-responsiveness and slow growth. Cells at intermediate passage (LNCaP-RF) represent the progression of disease characterized by androgen-responsiveness and faster growth than early stage disease. Cells at metastatic or late stage (LNCaP-UR) represent the progressed disease, are androgen-unresponsive, and fast growing.

Figure 1 Bcl-2 expression in prostate cells. (a) Cells grown under normal conditions were analyzed by Western blot and shows Bcl-2 protein expression. (b) Cells grown under normal conditions were analyzed by RTPCR and shows Bcl-2 message expression. The cell lines shown are HPV 18C-1 (lane 1), LNCaP-R (lane 2), LNCaP-UR (lane 3), LNCaP-C4-2 (lane 4), PC-3 (lane 5), and DU-145 (lane 6). Lane M denotes molecular weight markers.

androgen-unresponsive phenotype. LNCaP prostate cancer cells are androgen-responsive at low passage numbers but become androgen-unresponsive at high passage numbers.11 Table 1 outlines the passage numbers and designations of the LNCaP cells used in this study.

cells in phenol red-free media containing charcoalstripped serum. Figure 2A shows LNCaP-R cells grown in normal media display a rounded, epithelial-type morphology. LNCaP-R cells appear to differentiate to the NE-like morphology after 3 months of growth in phenol red-free media containing charcoal-stripped serum (Figure 2B). LNCaP-UR cells show a rounded, epithelial-type morphology when grown in normal media (Figure 2D) and also appear to differentiate to the NE-like morphology after 3 months of growth in phenol red-free media containing charcoal-stripped serum (Figure 2E). We also treated LNCaP cells with 1.0 mM casodex, an androgen-receptor antagonist, to address the role of androgens in LNCaP cells more directly. Figure 2C shows LNCaP-R cells grown 3 weeks in normal media containing 1 mM casodex appear to differentiate to the NE-like morphology similar to LNCaP-UR cells under the same conditions (Figure 2E). We designated the cells grown in charcoal-stripped serum as LNCaP-R NE and LNCaP-UR NE. Besides the morphological change, LNCaP-NE cells displayed greatly reduced growth compared to LNCaP cells and were viable for up to 6 months under these culture conditions. The NE-like LNCaP cells grown under steroid-reduced conditions have been shown to express androgen receptor.16 It was possible to reverse the NElike phenotype after 1 month by changing the media to normal serum. However, beyond 3 months in media containing charcoal-stripped serum, the cell morphology could not be reversed.

Bcl-2 expression in prostate cells We sought to determine the expression of Bcl-2 in immortalized prostate epithelial cells, and androgenresponsive and -unresponsive prostate cancer cells. HPV18 C-1 is an immortalized human prostate epithelial cell line.12 LNCaP-C4-2 is an androgen-unresponsive subline of LNCaP cells,10 and PC-3 and DU-145 are androgenunresponsive cell lines. Figure 1a, (panel A) shows protein expression of Bcl-2 in HPV-18 C-1 (lane 1), LNCaP-R, UR, and C4-2 (lanes 2, 3, and 4), PC-3 (lane 5), and DU-145 (lane 6). The androgen-unresponsive cells LNCaP-UR (lane 3) and DU-145 (lane 6) showed Bcl-2 absent. Phosphoglycerate kinase (PGK) was included as a loading control (Figure 1a, panel B). To determine if the absence of Bcl-2 in DU-145 and LNCaP-UR cells was at the transcriptional level, RT-PCR was performed and showed that all cells tested expressed Bcl-2 message (Figure 1b).

Growth of LNCaP cells in charcoal-stripped media or casodex drives neuroendocrine-like differentiation It was previously shown that LNCaP cells could be induced to undergo transdifferentiation to a neuroendocrine (NE)-like morphology by increases in intracellular cyclic AMP,13 growth in steroid-depleted media,14 or by treatment with interleukin-6.15 We sought to mimic androgen ablation in LNCaP cells to address what changes would occur in Bcl-2 and Bax. We grew LNCaP Prostate Cancer and Prostatic Diseases

LNCaP-UR cells regain expression of Bcl-2 after extended growth in charcoal-stripped serum Given the dramatic morphological changes in the LNCaP cells after 3 months of culture in phenol red-free media containing charcoal-stripped serum, we sought to determine if any changes in Bcl-2 and Bax had occurred. Bcl-2


161

LNCaP-R

A

B

C

E

F

LNCaP-UR

D

Figure 2 LNCaP cells grown in charcoal-stripped serum or casodex appear to differentiate to the NE-like morphology. LNCaP cells were grown in normal media with serum, media containing charcoal-stripped serum, or normal media with serum containing 1 mM casodex. (A) LNCaP-R cells grown in normal media or (B) phenol red-free RPMI 1640 supplemented with 2% charcoal-stripped FBS for 3 months or (C) normal media containing 1 mM casodex for 3 weeks. (D) LNCaP-UR cells grown in normal media or (E) phenol red-free media supplemented with 2% charcoal-stripped FBS or (F) normal media containing 1 mM casodex for 3 weeks. Photomicrographs are 400 magnification.

LNCaP RF

R

LNCaP NE R UR

UR

a

LNCaP-R

µM Casodex

A

B

C PGK

1

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8

Figure 3 Bcl-2 protein expression is regained in LNCaP-UR cells. Cells grown 3 months in normal media containing serum or phenol red-free media containing charcoal-stripped serum were subjected to Western blot analysis to probe for Bcl-2 and Bax. Panel A: Bcl-2 protein expression is present in LNCaP-R and RF cells grown in normal media (lanes 1–4) but absent in LNCaP-UR cells (lanes 5 and 6). Bcl-2 is overexpressed in LNCaP-R NE cells (lane 7) and in LNCaP-UR NE cells (lane 8) after growth in media containing charcoal-stripped serum. Panel B: The membrane from panel A was stripped and probed for Bax and shows Bax is present in all LNCaP samples. Panel C. PGK was included as a loading control.

protein was detected in LNCaP-R and RF cells (Figure 3, panel A, lanes 1–4) and, as previously shown, was not present in LNCaP-UR cells (Figure 3, panel A, lanes 5 and 6). Interestingly, Bcl-2 protein expression was regained in LNCaP-UR NE cells (Figure 3, panel A, lane 8) and LNCaP-R NE cells appeared to overexpress Bcl-2 (Figure 3, panel A, lane 7). Bax protein expression appeared unchanged in all samples (Figure 3, panel B). PGK (Figure 3, panel C) was included as a control.

LNCaP-R and RF cells downregulate Bcl-2 when treated with casodex, while Bax is unchanged We treated LNCaP cells with casodex, a nonsteroidal antiandrogen, in media containing phenol red and 7%

1

LNCaP-RF

10

100

0

1

10

LNCaP-UR 100 0

1

10 100

Bcl-2

BCL-2

Bax

0

PGK

A

1

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1

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4

5 4

6

7

6

7

5

8

9 8

10 9

11 12 B

10 11

12

b µM Casodex 0

LNCaP-R 1 10 100

0

LNCaP-RF 1 10 100

0

LNCaP-UR 1 10 100 A

Bax

B

PGK

1

2

3

4

5

6

7

8

9

10

11 12

Figure 4 Bcl-2 and Bax expression in casodex-treated LNCaP cells. Cells were treated with increasing concentrations of casodex for 5 weeks then subjected to Western blot analysis to probe for Bcl-2 and Bax protein expression. (a) There was downregulation of Bcl-2 protein in LNCaP-R and RF cells (panel A, lanes 2–4 and lanes 6–8, respectively) with no Bcl-2 detected in LNCaP-UR cells (panel A, lanes 9–12). (b) Bax protein levels remained unchanged in the LNCaP model (panel A). PGK was included as a loading control (a, panel B and b, panel B).

FBS to address more directly the role of androgen in Bcl-2 protein expression in LNCaP cells. As shown in Figure 4a, Bcl-2 protein was downregulated in LNCaP-R and -RF cells (panel A, lanes 2–4, and 6–8, respectively) after 5 weeks of treatment with 1, 10, and 100 mM casodex. As expected, LNCaP-UR cells (Figure 4a, panel A, lanes 9–12) showed no Bcl-2 expression. Figure 4b shows that Bax protein expression was unchanged in LNCaP cells treated with 1, 10, and 100 mM casodex for 5 weeks. PGK was included as a control (Figure 4a panel B and 4b panel B). Prostate Cancer and Prostatic Diseases


Bcl-2 message expression in LNCaP cells treated with casodex

Pretreatment of LNCaP cells with casodex for 5 weeks results in increased resistance to induction of apoptosis by treatment with docetaxel We have previously shown that treatment of the LNCaP model with docetaxel induced apoptosis in both LNCaPR and -UR cells.5 To determine if pretreatment of cells with casodex affected the ability of docetaxel to induce apoptosis in the LNCaP model, we pretreated LNCaP cells for 5 weeks in casodex, then treated with 5 nM docetaxel for 48 h. Figure 7 shows apoptosis as evidenced by caspase-3 activation in the LNCaP model in cells grown in media with or without casodex, and then either left untreated or treated with 5 nM docetaxel for 48 h (Po0.05). Cells grown in media with casodex for 5 weeks show increased basal apoptosis, and this basal apoptosis is similar in both the LNCaP-R and LNCaP-UR cells. Treatment of cells grown in media without casodex with 5 nM docetaxel induces significant apoptosis. The extent of apoptosis induction is greater in LNCaP-R cells compared to LNCaP-UR cells. However, in LNCaP-R cells pretreated with 1 mM casodex for 5 weeks, treatment

1 µM 10 µM 100 µM

0.035

Bcl-2 message levels

In view of the downregulation of the bcl-2 protein levels in the casodex-treated LNCaP-R and LNCaP-UR cells, we sought to determine if casodex affected Bcl-2 message expression. RT-PCR analysis (Figure 5) shows that Bcl-2 message expression is present in LNCaP-R cells (lanes 1–4) and LNCaP-UR cells (lanes 5–8) cells treated with 1, 10, and 100 mM casodex, respectively, for 5 weeks. While RT-PCR is not a quantitative analysis, our data show that Bcl-2 message is present while the Bcl-2 protein is lost in these cells. To further determine if casodex treatment caused any changes in Bcl-2 message levels, real-time PCR was performed. Expression of Bcl-2 message in LNCaP-UR cells appeared to increase at 1 and 10 mM treatment with casodex (Figure 6). No significant differences in Bcl-2 message expression was detected by real-time PCR analysis in LNCaP-R cells (Figure 6) when comparing the control samples with samples treated with 1, 10, and 100 mM casodex for 5 weeks.

C 0.040

0.030

0.025

0.020

0.015

0.010

0.005

0.000

LNCaP-R

LNCaP-UR

Figure 6 Real-time PCR analysis of Bcl-2 expression in the LNCaP model treated with casodex. RNA extracted from LNCaP-R and UR cells either untreated (control) or treated for 5 weeks in 1, 10, or 100 mM casodex were reverse transcribed, then analyzed by real-time PCR. Samples were run in duplicate and CT (threshold cycle where amplification is in logarithmic phase) scores were averaged and the reciprocal, averaged CT score of each sample reported. The threshold baseline cycle number was determined by the iCycler software version 2.3. Data shown are 7s.e.m. (Po0.05).

4 Control

Relative Apoptosis Induction

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1 µM Cas 5 nM Doc 1 µM Cas + 5 nM Doc

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LNCaP-R µM

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500bp

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500bp

β -Actin M

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Figure 5 Bcl-2 message expression in LNCaP cells treated with casodex. LNCaP cells were untreated or treated for 5 weeks in 1, 10, and 100 mM casodex, then RNA was extracted and subjected to RT-PCR analysis to probe for Bcl-2 and b-actin message expression. Lane 1, LNCaP-Runtreated control; Lanes 2–4, LNCaP-R treated 5 weeks with 1, 10 and 100 mM casodex; Lane 5, LNCaP-UR-untreated control. Lanes 6–8, LNCaP-UR treated 5 weeks with 1, 10 and 100 mM casodex; Lane M, DNA molecular weight marker. Upper panel shows Bcl-2 message expression and the lower panel shows b-actin expression used as an internal control. Prostate Cancer and Prostatic Diseases

LNCaP-UR

Figure 7 Casodex treatment selects for apoptosis-resistant cells. LNCaPR and LNCaP-UR cells were either grown in normal media (Control) or in the presence of 1 mM casodex (1 mM Cas) for 5 weeks. Apoptosis was induced by incubation of cells with 5 nM docetaxel for 48 h. Apoptosis was monitored by the ApoAlert Caspase 3 activation colormetric assay (Clontech). The data show apoptosis induction relative to the untreated control (cells grown in normal media). Each experiment was run in triplicate and the experiment was repeated. Vertical lines above each bar represents the s.e.m. (Po0.05).

with docetaxel for 48 h appears to result in a significantly decreased ability to induce apoptosis. LNCaP-UR cells, under the same conditions, are resistant to apoptosis induction by docetaxel. These results suggest that casodex may select for apoptosis-resistant LNCaP-R cells or drive differential gene expression that results in resistance to apoptosis.


Discussion The goal of this study was to determine if Bcl-2 was regulated by androgen signaling in prostate cancer. As shown in Figure 1a, the expression of Bcl-2 protein in prostate cancer cell lines is variable. LNCaP-UR and DU145, androgen-unresponsive prostate cancer cell lines, show Bcl-2 absent at the protein level (Figure 1a, lanes 3 and 6); however, Bcl-2 message is expressed (Figure 1b, lanes 3 and 6). We focused on the LNCaP model since androgen-responsive LNCaP-R cells expressed Bcl-2, whereas androgen-unresponsive LNCaP-UR cells did not express Bcl-2 protein.5 We observed that growth of LNCaP-R and -UR cells in media containing charcoal-stripped serum resulted in differentiation to the NE-like morphology (Figure 2B and E, respectively). Concurrent with this differentiation was the gain of expression of Bcl-2 protein by LNCaP-UR cells (Figure 3, panel A, lane 8), which do not express Bcl2 under normal growth conditions (lanes 5 and 6). These results showed that the Bcl-2 transcriptional machinery in LNCaP-UR cells was functional and prompted us to question whether the regain of Bcl-2 protein expression in the LNCaP-UR cells was related to some perturbation in androgen-unresponsive signaling in media containing charcoal-stripped serum. The presence of NE-like cells in prostate cancer is thought to be indicative of androgenunresponsiveness and a marker for poor patient prognosis.17 Our data show that differentiation of LNCaP-UR cells by growth in media containing charcoal-stripped serum results in the regain of expression of Bcl-2 protein supporting the view that NE-like differentiation in prostate cancer and the expression of Bcl-2 may be concurrent events in the progression to androgenunresponsiveness. To demonstrate whether this NE-like differentiation was a result of androgen signaling perturbation, we treated LNCaP cells with casodex to inhibit androgen signaling specifically. We observed NE-like differentiation within 3 weeks of treatment with casodex in both LNCaP-R and -UR cells (Figure 2C and F, respectively). At this stage, there was no change in Bcl-2 protein expression in LNCaP-R or RF cells (data not shown). Continued treatment with casodex for 5 weeks resulted in a downregulation of Bcl-2 protein expression in LNCaP-R and RF (Figure 4a, panel A, lanes 2–4 and 6– 8, respectively) cells, but Bcl-2 protein expression was not detected in LNCaP-UR cells (Figure 4a, panel A, lanes 9–12). When probed for Bcl-2 message expression, we found that casodex treatment did not appear to affect message levels in the LNCaP-R and -UR cells (Figure 5). Real-time PCR analysis showed no significant changes in Bcl-2 expression in LNCaP-R cells after treatment with casodex, although an increase in Bcl-2 message was measured in LNCaP-UR cells after treatment (Figure 6). These data suggest that regulation of Bcl-2 occurs at the post-transcriptional level. Post-transcriptional regulation of Bcl-2 has been shown previously by other groups.18–21 In colorectal cancer, Berney et al19 showed that the expression of Bcl-2 protein was gradually and significantly lost during the progression from moderately dysplastic adenoma to primary colorectal cancer, whereas the cellular expression of Bcl-2 mRNA was gradually increased during this transition. Differences

between bcl-2 mRNA levels and protein levels were also observed in oral carcinomas.20 Previously, we showed that Bax protein was present and its expression levels were similar in LNCaP cells.5 We did not observe any changes in Bax protein expression in LNCaP cells treated with casodex for 5 weeks (Figure 4b, panel A). These findings suggest that Bax may not play a critical role in apoptosis regulation in LNCaP cells, or there may be other proapoptotic mechanisms independent of Bax governing the antiapoptotic function of Bcl-2.

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Conclusions The results of this study show that Bcl-2 protein expression is regulated by androgen signaling, consistent with a recently published report;7 however, our study suggests that chronic androgen-ablation results in a posttranscriptional regulation of Bcl-2. Our results showed that androgen-ablation negatively regulated Bcl-2 expression post-transcriptionaly in androgen-responsive LNCaP-R and RF cells. The major point of this manuscript is to demonstrate that despite apparent loss of Bcl2 protein expression, cells are still androgen-independent and apoptosis-resistant (DU-145 and specimens from patients with androgen-independent prostate cancer). Bcl-2 protein expression was regained in LNCaP-UR NE cells after extended growth in charcoal-stripped serum suggesting that NE-like cells, which are commonly found localized with high-grade tumors22 may contribute to the progression to androgen-unresponsiveness in prostate cancer.

Acknowledgements This work was supported by grants from the Pfeiffer (Gustavus and Louise) Foundation, UNMC/Eppley Cancer Center (LB595), and AstraZeneca LP.

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