Tyrosine kinase inhibitors reduce bcl-2 expression and induce apoptosis in androgen-dependent cells

Takashi Ohigashi1,2, Munehisa Ueno1, Shoichi Nonaka1, Takashi Nakanoma1, Yusuke Furukawa3, Nobuhiro Deguchi1, and Masaru Murai2

1 Department of Urology, Kidney Center, Saitama Medical School, Moroyamamachi 350-0495; 3 Division of Hemopoiesis, Institute of Hematology and Department of Hematology, Jichi Medical School, Minamikawachimachi 329-0498; and 2 Department of Urology, School of Medicine, Keio University, Tokyo 160-8582, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
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The signal transduction pathway showing how androgen withdrawal induces apoptosis in androgen-dependent cells has not been clearly understood. In these studies, we focused on the behavior of tyrosine kinases in androgen-dependent cells and investigated its correlation with apoptosis and bcl-2 expression. We used SC2G, an androgen-dependent mouse mammary carcinoma cell line, which had been cloned from Shionogi Carcinoma 115 (SC115). When SC2G cells were cultured with herbimycin A (HMA), a potent tyrosine kinase inhibitor, the number of viable cells decreased significantly after 24 h. Terminal deoxyribonucleotidyltransferase-mediated dUTP-biotin nick end labeling and flow cytometric analysis of annexin V staining showed that HMA induced apoptosis of SC2G cells. The level of bcl-2 mRNA in SC2G cells was suppressed by HMA in a dose-dependent manner on RT-PCR. Preincubation with caspase inhibitors protected HMA-induced apoptosis of SC2G cells. When a human bcl-2 gene was transfected in SC2G cells and overexpressed, SC2G cells seemed to acquire tolerance for HMA. These data indicate that HMA-sensitive tyrosine kinase(s) can regulate apoptosis and inhibit bcl-2 expression in SC2G mouse androgen-dependent cells. Tyrosine kinase(s) seemed to be a member of signal transduction between androgen receptor activation and bcl-2 expression.

protooncogene; mouse


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN SOME TYPES OF CELLS, absence of growth factors causes apoptosis (6), which is a physiological form of cell death that exhibits highly characteristic features (16). For androgen-dependent cells, castration results within 1 wk in the apoptotic cell death of the normal prostatic cells (7). Androgen withdrawal also induces apoptosis in rat ventral prostate (18) and in androgen-dependent PC-82 tumor cells grown in nude mice (33).

In mammalian cells, bcl-2, a protooncogene cloned by virtue of its translocation into the immunoglobulin locus in follicular B cell lymphomas (32), has been reported to prevent apoptosis in response to a number of apoptotic stimuli (30). The overexpression of Bcl-2 protein blocks apoptosis in response to withdrawal of interleukin-3 or granulocyte-macrophage colony-stimulating factor in the hematopoietic cell line (26). Some investigators reported that high levels of expression of Bcl-2 protein were observed in androgen-independent prostate cancer (22). In these cases, it may be possible that some gene mutations or other factors enable inappropriate regulation of bcl-2 expression, thereby resulting in aggressive tumor growth without androgen stimulation. However, the incidence of androgen receptor gene mutations in human prostatic tumors is reported to be rather low (9). It is not clear whether the activation of androgen receptor directly enhances bcl-2 gene expression or whether some molecular steps exist between both events. If some molecular steps have important roles in bcl-2 expression, we have the opportunity to control androgen-independent carcinoma cells by blocking these signals.

In the present study, we used androgen-dependent SC2G cells derived from SC115 androgen-responsive mouse mammary tumor, which had been maintained in a male mouse (23). Although LNCaP cells established from human prostate carcinoma are the most widely used model of human androgen-sensitive tumor cell, androgen withdrawal reduces tumor growth but does not, apparently, induce apoptosis in vitro. A mutation in the androgen receptor, which was reported in LNCaP cells (34), may cause this phenomenon. On the contrary, it was reported that androgen withdrawal induced apoptosis of SC115 in a serum-free culture in vitro (11). Thus we consider the SC2G cell line derived from SC115 cells to be an excellent model for investigating the mechanism of apoptosis of androgen-dependent tumor cells after androgen withdrawal. We focused on the behavior of tyrosine kinases in SC2G cells induced by testosterone and its correlation with apoptosis in these studies. We also investigated the relationship of tyrosine kinases and bcl-2 expression in SC2G cells.


    MATERIALS AND METHODS
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Reagents. Herbimycin A (HMA; Wako, Tokyo, Japan) was dissolved in DMSO at 10 mM and stored at -20°C. Inhibitors for caspases, acetyl-Tyr-Val-Ala-Asp-H (aldehyde) (Ac-YVAD-CHO; inhibitor for caspase 1) and acetyl-Asp-Glu-Val-Asp-H (aldehyde) (Ac-DEVD-CHO; inhibitor for caspase 3), were purchased from Peptide Institute (Osaka, Japan).

Cell culture and cell viability. The androgen-dependent mouse mammary tumor cell line, SC2G, was a kind gift from Dr. T. Yamaguchi (Shionogi). SC2G cells were maintained in GIT medium (Nippon Seiyaku, Osaka, Japan) supplemented with 5 × 10-8 M testosterone. GIT medium contained insulin (2.0 mg/l), transferrin (2.0 mg/l), ethanolamine (0.122 mg/l), and 3.0 g/l growth factor of serum, which is a 55-70% ammonium sulfate fraction of bovine serum (mostly proteins of molecular weights between 60,000 and 80,000). Cells were cultured in collagen (type I)-coated plastic flasks (Iwaki Glass, Chiba), and the medium was changed every 2 days. To assess the cell viability, a modification of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay (3) was used. Aliquots of 100 µl of medium containing 2 × 104 cells were plated into a 96-well flat-bottom microplate. At the end of the incubation period, 25 µl of MTT solution in PBS (2.5 mg/ml) were added. After 4 h of incubation with MTT, the medium was aspirated, and 100 µl DMSO were added to each well. The absorbance at 490 nm was measured using a multiwell plate reader (model 3550; Bio-Rad, Hercules, CA), with wells containing medium but no cells serving as blank controls. The results are presented as percentage of the baseline value in each experiment.

Apoptosis assays. The method of terminal deoxyribonucleotidyltransferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) originally described by Gavrieli et al. (12) was used for apoptosis staining with some modifications. Initially, 5 × 104 cells were plated on each well of an eight-well slide-glass chamber. At the end of incubation, the cells were fixed with 4% paraformaldehyde in PBS (wt/vol). DNA nick end labeling was performed following the protocol supplied with the Mebstain apoptosis kit (Medical & Biological Laboratories, Nagoya, Japan) with a slight modification. Briefly, nuclei of cells were stripped of proteins by incubation with 1.6 mg/ml proteinase K at 37°C for 7 min. After the slides were washed with distilled water, they were immersed for 10 min in TdT buffer. TdT (final concentration, 0.077 U/ml) and biotynized dUTP in TdT buffer were added, and slides were incubated at 37°C for 90 min. After the slides were rinsed and covered with blocking solution supplied with the kit, they were covered with FITC for 30 min at 37°C, washed with PBS, and mounted. The ratio of FITC-positive nuclei to all nuclei was counted in four different areas from three different plates. Flow cytometric analysis of annexin V staining was also employed for the detection of apoptosis. Annexin V detects phosphatidylserine externalized to the outer surface of the cell membrane in the early stage of apoptosis. FITC-annexin V (PharMingen, San Diego, CA) and propidium iodide (PI) were applied to determine the percentage of cells undergoing apoptosis, according to the manufacturer's protocol. The cells that were stained positive for annexin V and negative for PI were defined to be in the stage of apoptosis.

RNA extraction and RT-PCR. Total RNA was isolated from SC2G cells using RNeasy total RNA kits (Qiagen, Hilden, Germany) according to the manufacturer's directions. RNA concentrations and purity were determined by spectroscopy on a GeneQuant II spectrophotometer (Pharmacia, Cambridge, UK) and by gel analysis. Two hundred fifty nanograms of total RNA of each sample were applied for RT-PCR. The primer pairs for mouse bcl-2 were synthesized on the basis of the published mouse bcl-2 gene sequence (25) (forward primer, 5'-ATCTTCTCCTTCCAGCCTGA-3'; reverse primer, 5'-TCAGTCATCCACAGGGCGAT-3'). The ideal size of the PCR product was 386 bp. The primer pairs for human bcl-2 were also synthesized (forward primer, 5'-ACTTGTGGCCCAGATAGGCACCCAG-3'; reverse primer, 5'-CGACTTCGCCGAGATGTCCAGCCAG-3'). All of the steps for the reverse transcription and the subsequent amplification were performed in a single reaction tube using a GeneAmp thermostable rTth RT RNA PCR kit (Roche Molecular Systems, Branchburg, NJ). The synthesis of first-strand cDNA was carried out at 70°C for 15 min. The PCR profile was 95°C for 45 s, 65°C for 45 s, and 72°C for 2 min for 35 cycles, followed by 72°C for 7 min. After PCR, aliquots of the reaction were analyzed on 1.2% agarose gel with ethidium bromide (0.5 mg/ml). Results were compared against glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA quantified with the use of the same RT-PCR conditions. The primer pairs for G3PDH were purchased from Clontech (Palo Alto, CA).

Effects of caspase inhibitors on HMA-mediated inhibition of SC2G cell growth. SC2G cells (2 × 104 cells) were preincubated for 2 h with various concentrations of either Ac-YVAD-CHO or Ac-DEVD-CHO. Cells were then incubated for 24 h in the presence or absence of 1 µM HMA. The percentage of viable cells was determined by MTT assay.

Transfection of human bcl-2 cDNA into SC2G cells. The full-length human bcl-2 cDNA was cloned in the Xho I-Xba I site of the plasmid pcDNA3. SC2G cells (5 × 105) were seeded into each well of a six-well culture plate, following transfection for 48 h with the pcDNA3/hbcl-2 plasmid or with a control plasmid. Aliquots containing 1 µg of plasmid and 10 µl of Lipofectamine reagent (Life Technologies, Rockville, MD) in 200 µl of serum-free OPTI-MEM (Life Technologies) were preincubated for 30 min and were then added to a culture well with 800 µl of serum-free OPTI-MEM. The transfection medium was replaced 8 h later with GIT-5% FBS medium; 24 h later, this medium was replaced with GIT containing 500 µg/ml G418 sulfate (Life Technologies). The cells were then cloned with a limiting dilution method after 3 wk. The expression of human bcl-2 gene was evaluated by RT-PCR and Western blot analysis.


    RESULTS
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Effect of tyrosine kinase inhibitors on cell viability and on apoptosis in SC2G cells. When SC2G cells were cultured for 24 h, tyrosine kinase inhibitors caused dose-dependent decreases in their viability. HMA at doses >0.1 µM resulted in significant decreases of absorbance during MTT assay (Fig. 1). Another tyrosine kinase inhibitor, genistein, also decreased absorbance significantly at 20 µM or higher concentrations. TUNEL staining of SC2G cells after 24-h cultures with 1 µM HMA showed 24.3 ± 1.5% positive-staining nuclei, whereas 2.3 ± 0.3% of nuclei were stained in control cells (Fig. 2). In the flow cytometric measurement, the percentage of cells positively stained with annexin V and unstained with PI in SC2G cells cultured for 10 h with 0, 1, and 2.5 µM HMA was 16.9, 29.4 and 64.5%, respectively (Fig. 3).


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Fig. 1.   Effects of herbimycin A (HMA; A) or genistein (B) on the cell growth of SC2G cells. SC2G cells (104 cells) were incubated in 100 µl medium containing 50 nM testosterone in each well of a 96-well microplate with several concentrations of tyrosine kinase inhibitors for 24 h. At the end of incubation, cell viability was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) methods. Absorbance is plotted as percent mean ± SE of medium control. Data were obtained from 4 different determinations. * P < 0.05 from medium control (Tukey's multiple-range test).



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Fig. 2.   Apoptosis staining of SC2G cells. SC2G cells were stained with the TUNEL method after 24 h incubation without (A) or with (B) 1 µM HMA. Nuclei with fragmented DNA were stained with avidin-fluorescein isothiocyanate and developed fluorescence under fluorescence microscopy. All nuclei were also stained with propidium iodide (PI). Therefore, the nuclei of the apoptotic cells appear to be white in the photograph. The photograph shows a representative experiment. Bar, 50 µm.



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Fig. 3.   Histogram of 2-color fluorescence-activated cell sorting analyses of SC2G cells double labeled with FITC-labeled annexin V (horizontal axis) and PI. Panels show results of cells treated with 0, 1, and 2.5 µM HMA for 10 h. Early apoptotic cells are stained positive with annexin V and negative with PI and therefore appear in the lower right quadrant, whereas both upper quadrants accommodate dead cells. Result of a representative experiment.

Effects of HMA on bcl-2 mRNA expression. The change of bcl-2 mRNA expression after the administration of HMA was analyzed by RT-PCR. On RT-PCR, the band of PCR products generated from mouse bcl-2 mRNA weakened at 6 h after the administration of HMA. The suppressive effect on bcl-2 mRNA by HMA occurred in a dose-dependent manner. Figure 4 shows a representative gel for analysis of PCR products. The band almost disappeared with 10 µM HMA, whereas control RT-PCR for G3PDH showed no change. The ratio of bcl-2 to G3PDH in control, 0.625 µM HMA, 2.5 µM HMA, and 10 µM HMA was 44.2%, 12.8%, 1.5%, and 0.7%, respectively.


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Fig. 4.   Effects of HMA on bcl-2 mRNA levels. SC2G cells were incubated with different concentrations of HMA for 6 h. Electrophoresis of RT-PCR products for mouse bcl-2 is shown for a representative experiment. Lane 1, HMA free; lane 2, 0.625 µM HMA; lane 3, 2.5 µM HMA; lane 4, 10 µM HMA; lane 5, molecular marker; lane 6, positive control (mouse M1 cell). Lower gel shows RT-PCR products for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene.

Effects of caspase inhibitors on cell viability cultured with HMA. The preincubation with both caspase inhibitors, Ac-YVAD-CHO and Ac-DEVD-CHO, suppressed the HMA-induced decrease of viable cells (Fig. 5). However, the caspase-3 inhibitor, Ac-DEVD-CHO, seemed to be more potent at suppressing HMA-induced effects. Preincubation of Ac-YVAD-CHO at doses of 200 and 400 µM and Ac-DEVD-CHO at doses >50 µM caused significant suppression of the HMA-induced decrease of cell viability. HMA alone decreased absorbance by 55 ± 2% of untreated controls, whereas HMA decreased only by 75 ± 3% with the preincubation of 400 µM Ac-DEVD-CHO.


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Fig. 5.   Effects of caspase inhibitors on cell viability cultured with HMA. SC2G cells (2 × 104 cells) were preincubated for 2 h with various concentrations of either Ac-YVAD-CHO (A) or Ac-DEVD-CHO (B). Cells were then incubated for 24 h in the presence or absence of 1 µM HMA. Cell viability was determined by an MTT assay. Absorbance is plotted as percent mean ± SE of medium control. Data were obtained from 3 different determinations. * P < 0.05 from control without preincubation with caspase inhibitors (Tukey's multiple-range test).

Effects of HMA on cell viability of human bcl-2 transfected SC2G cells. To examine whether the exogenously transfected human bcl-2 gene has protective effects for HMA-induced apoptosis in SC2G cells, we used SC2G cells overexpressed with the human bcl-2 gene (SC2G-hbcl2). As a control, the same plasmid, which did not contain human bcl-2 cDNA, was also transfected to SC2G cells (SC2G-neo). On RT-PCR, SC2G-hbcl2 showed positive transcription of human bcl-2 mRNA, whereas native SC2G and SC2G-neo were negative. SC2G-hbcl2 lost androgen dependency and featured good cell growth, regardless of the testosterone concentration (Fig. 6). SC2G-hbcl2 cells also seemed to acquire tolerance for HMA as shown in Fig. 7. HMA had less effect on SC2G-hbcl2 cells than on SC2G-neo cells after a 24-h incubation at 2.5 µM.


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Fig. 6.   A: human bcl-2 mRNA expression in native SC2G, SC2G-neo, and SC2G-hbcl2 cells. Electrophoresis of RT-PCR products for human bcl-2 is shown. Lane 1, native SC2G; lane 2, SC2G-neo; lane 3, SC2G-hbcl2 (clone 1); lane 4, SC2G-hbcl2 (clone 2); lane 5, SC2G-hbcl2 (clone 3); lane 6, a 100-bp size ladder; lane 7, positive control. Clone 3 was used for further experiments. B: effects of testosterone on cell growth of SC2G cells. Aliquots of 5 × 104 SC2G-hbcl2 (104 cells) were incubated in each well of a 96-well microplate with several concentrations of testosterone for 96 h. At the end of incubation, cell viability was tested by MTT methods. Each point represents mean ± SE absorbance obtained from 3 different determinations.



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Fig. 7.   Effects of HMA on cell growth of SC2G-neo cells (open bar) or SC2G-hbcl2 cells (solid bar). Both cells (1 × 104 cells) were incubated in each well of a 96-well microplate with different concentrations of HMA for 24 h. At end of incubation, cell viability was tested by the MTT method. Each point represents the mean ± SE percentage of absorbance cultured in HMA-free medium. Data were obtained from 3 different determinations. * P < 0.05 from cell viability of SC2G-neo cells (Student's unpaired t-test).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recently, many investigators reported that protein tyrosine kinase inhibitors induce apoptosis in a wide variety of cell types. Tyrosine kinase inhibitors not only enhance apoptosis triggered by stimuli such as ultraviolet light (13) or chemicals (19), but they also induce apoptosis when used alone in the growth-factor-dependent cells (4, 24). In nerve growth-factor-dependent neuron cells, the action of tyrosine kinases seems to be necessary for cell survival (5). On the other hand, tyrosine kinases were also reported to mediate apoptotic signals in some cells (8, 29).

There are several reports indicating the relationship between androgen dependency and receptor type tyrosine kinases. Kondapaka and Reddy (17) described that tyrphostin, another tyrosine kinase inhibitor, blocked not only transforming growth factor-alpha (TGF-alpha )-stimulated growth but also androgen-stimulated cell proliferation of LNCaP prostate carcinoma cells. In the other report, androgens stimulated TGF-alpha and epidermal growth factor (EGF) receptor mRNA production and cellular proliferation in the androgen-dependent ALVA101 human metastatic prostate cancer cell line (20). Additionally, an anti-EGF receptor antibody was able to block dihydrotestosterone-induced cell growth (14). Thus the proliferating effects of androgens seemed to be modulated through TGF-alpha or EGF receptors, at least in part.

In this study, we have demonstrated that HMA and genistein, both potent tyrosine kinase inhibitors, induced the inhibition of cell proliferation of SC2G mouse androgen-dependent cells. Tyrosine kinases have two major groups. One major group is the receptor type tyrosine kinases, such as EGF receptor and TGF-alpha receptor. The other major group is the non-receptor-type tyrosine kinases, represented by the Src family. Most Src family tyrosine kinases have been determined to contain a homology sequence called the Src homology (SH) region. Because HMA inhibits tyrosine kinase activity by affecting the SH domain, it is effective for the Src family non-receptor-type tyrosine kinases. On the other hand, genistein inhibits the activities of several protein kinases, including those of growth factor receptor tyrosine kinases and Src family proteins (1). In addition, genistein was reported to have anti-angiogenesis activity, or estrogenic/anti-estrogenic activities (10, 31). Therefore, we selected HMA to focus on the role of non-receptor-type tyrosine kinases in the signal transduction of androgen-dependent cell proliferation.

From the results of TUNEL staining and flow cytometric analysis of annexin V staining, it is indicated that growth inhibition with HMA occurred by apoptosis. We demonstrated from RT-PCR that HMA also suppresses the expression of bcl-2 mRNA. We have previously reported that bcl-2 mRNA levels and protein levels decreased after androgen withdrawal in SC2G cells (27). We reported that the blockade of Bcl-2 protein synthesis with bcl-2 antisense oligonucleotides had cytotoxic effects on SC2G cells. Therefore, we hypothesize that androgen depletion decreases the amount of Bcl-2, a protein that prevents default apoptosis of SC2G cells, and results in the activation of an intrinsic suicide program.

The inhibitor of tyrosine kinase, HMA, seemed to have a reaction similar to the depletion of androgens in SC2G cells. These inhibitory effects were observed even in the presence of a sufficient level of testosterone. Therefore, it is suggested that functioning tyrosine kinase pathways are required for bcl-2 expression in androgen-dependent cell growth. However, the kind of Src family members actually playing roles in the signal transduction between androgen receptor activation and bcl-2 expression is not clear. Srk family tyrosine kinases are reported to be the intermediates for many kinds of messages in cells (2). Recently, much attention has been paid to the role of Srk in bone absorption (21). In B lymphoma cells, Lyn also seemed to play a role as a member in the signal transduction pathway preventing apoptosis (15). However, there has been no report indicating the relationship between the Srk family and androgen receptors.

In our hypothesis, SC2G cells activate the intrinsic pathway inducing apoptosis, unless a sufficient level of bcl-2 is continuously expressed. If bcl-2 expression is suppressed by HMA, the downstream pathway is likely to activate. To block the final step of the apoptotic pathway, we applied the specific inhibitors of caspases. Inhibitors for both caspase-1 and caspase-3 prevented HMA-induced growth inhibition in SC2G cells, although the caspase-3 inhibitor was more potent. All the caspases induce apoptosis when overexpressed in cultured cells. Among them, caspase-3, formerly named CPP32 or YAMA, has a direct proteolytic action on poly(ADP-ribose) polymerase. On the other hand, caspase-1 (interleukin-converting enzyme 1beta ) induces apoptosis by activating caspase-3. Therefore, the inhibition of caspase-3 is conceivably more selective for blocking HMA-induced apoptosis. In our experiment, both YVAD and DEVD did not recover cell viability to the baseline of untreated cells. Because the nonesterified peptides used in our experiment have poor permeability, the doses used to block the caspases were rather high. At concentrations >400 µM, the caspase inhibitors themselves seemed to inhibit cell proliferation.

As expected, human bcl-2 was not present in SC2G cells. However, because of their homology to mouse bcl-2, murine cells acquire resistance to apoptotic stimuli when human bcl-2 is overexpressed (28). In this study, SC2G cells acquired a resistance to the HMA-induced cell death when human bcl-2 was overexpressed by transfection. This result seemed to confirm the hypothesis that HMA inhibited tyrosine kinase in the upstream of bcl-2 expression in androgen-dependent cells.

In conclusion, our data indicate that HMA-sensitive tyrosine kinase(s) can regulate apoptosis and inhibit bcl-2 expression in SC2G mouse androgen-dependent cells. Tyrosine kinase(s) seemed to be a member of signal transduction between androgen receptor activation and bcl-2 expression. It remains unclear, however, which type of tyrosine kinase actually plays a role and by what mechanism the androgen receptor regulates tyrosine kinases. Further study may provide new insights into the molecular mechanism of this pathway.


    ACKNOWLEDGEMENTS

We thank Masahiro Iida, Risa Hirata, and Hiroshi Nakazawa for their excellent technical assistance.


    FOOTNOTES

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

Address for reprint requests and other correspondence: T. Ohigashi, Dept. of Urology, School of Medicine, Keio Univ., 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan (E-mail: ohigashi{at}med.keio.ac.jp).

Received 4 December 1998; accepted in final form 26 August 1999.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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