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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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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 × 108 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.
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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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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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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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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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DISCUSSION |
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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- (TGF-
)-stimulated growth but
also androgen-stimulated cell proliferation of LNCaP prostate carcinoma
cells. In the other report, androgens stimulated TGF-
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-
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- 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 1) 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.
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ACKNOWLEDGEMENTS |
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We thank Masahiro Iida, Risa Hirata, and Hiroshi Nakazawa for their excellent technical assistance.
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FOOTNOTES |
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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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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Akiyama, T.,
J. Ishida,
S. Nakagawa,
H. Ogawara,
S. Watanabe,
N. Itoh,
M. Shibuya,
and
Y. Fukami.
Genistein, a specific inhibitor of tyrosine-specific protein kinases.
J. Biol. Chem.
262:
5592-5595,
1987
2.
Brown, M. T.,
and
J. A. Cooper.
Regulation, substrates and functions of src.
Biochim. Biophys. Acta
1287:
121-149,
1996[ISI][Medline].
3.
Carmichael, J.,
W. G. Degraff,
A. F. Gazdar,
J. D. Minna,
and
J. B. Mitchell.
Evaluation of a tetrazolium-based semi-automated colorimetric assay: assessment of chemosensitivity testing.
Cancer Res.
47:
936-942,
1984[Abstract].
4.
Chin, L. S.,
S. F. Murray,
K. M. Zitnay,
and
B. Rami.
K252a inhibits proliferation of glioma cells by blocking platelet-derived growth factor signal transduction.
Clin. Cancer Res.
3:
771-776,
1997[Abstract].
5.
Crowder, R. J.,
and
R. S. Freeman.
Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor-dependent sympathetic neurons.
J. Neurosci.
18:
2933-2943,
1998
6.
Duke, R. C.,
and
J. J. Cohen.
IL-2 addiction: withdrawal of growth factor activates a suicide program in dependent T cells.
Lymphokine Res.
5:
289-294,
1986[ISI][Medline].
7.
English, H. F.,
N. Kyprianou,
and
J. T. Isaacs.
Relationship between DNA fragmentation and apoptosis in the programmed cell death of the rat ventral prostate.
Prostate
15:
233-250,
1989[ISI][Medline].
8.
Eray, M.,
G. E. Liwszyc,
A. Paasinen-Sohns,
A. Stahls,
M. Kaartinen,
and
L. C. Andersson.
P72syk protein tyrosine kinase: an early transducer of sIgG-triggered apoptotic signaling in human follicular lymphoma cells.
Int. Immunol.
10:
1573-1581,
1998[Abstract].
9.
Evans, B. A.,
M. E. Harper,
C. E. Daniells,
C. E. Watts,
S. Matenhelia,
J. Green,
and
K. Griffiths.
Low incidence of androgen receptor gene mutations in human prostatic tumors using single strand conformation polymorphism analysis.
Prostate
28:
162-171,
1996[ISI][Medline].
10.
Fotsis, T.,
M. Pepper,
H. Adlercreutz,
G. Fleischmann,
T. Hase,
R. Montesano,
and
L. Schweigerer.
Genistein, a dietary-derived inhibitor of in vitro angiogenesis.
Proc. Natl. Acad. Sci. USA
90:
2690-2694,
1993[Abstract].
11.
Furuya, Y.,
J. T. Isaacs,
and
J. Shimazaki.
Induction of programmed death/apoptosis in androgen-dependent mouse mammary tumor cell line (Shionogi Carcinoma 115) by androgen withdrawal.
Jpn. J. Cancer Res.
86:
1159-1165,
1995[Medline].
12.
Gavrieli, Y.,
Y. Sherman,
and
S. A. Ben-Sasson.
Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.
J. Cell Biol.
119:
493-501,
1992[Abstract].
13.
Hiwasa, T.,
Y. Arase,
Z. Chen,
K. Kita,
K. Umezawa,
H. Ito,
and
N. Suzuki.
Stimulation of ultraviolet-induced apoptosis of human fibroblast UVr-1 cells by tyrosine kinase inhibitors.
FEBS Lett.
444:
173-176,
1999[ISI][Medline].
14.
Hofer, D. R.,
E. R. Sherwood,
W. D. Bromberg,
J. Mendelsohn,
C. Lee,
and
J. Kozlowski.
Autonomous growth of androgen-independent human prostatic carcinoma cells: role of transforming growth factor alpha.
Cancer Res.
51:
2780-2785,
1991[Abstract].
15.
Katagiri, K.,
K. K. Yokoyama,
T. Yamamoto,
S. Omura,
S. Irie,
and
T. Katagiri.
Lyn and Fgr protein-tyrosine kinases prevent apoptosis during retinoic acid-induced granulocytic differentiation of HL-60 cells.
J. Biol. Chem.
271:
11557-11562,
1996
16.
Kerr, J. F.,
A. H. Wyllie,
and
A. R. Currie.
Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.
Br. J. Cancer
26:
239-257,
1972[ISI][Medline].
17.
Kondapaka, B. S.,
and
K. B. Reddy.
Tyrosine kinase inhibitor as a novel signal transduction and antiproliferative agent: prostate cancer.
Mol. Cell. Endocrinol.
117:
53-58,
1996[ISI][Medline].
18.
Kyprianou, N.,
and
J. W. Isaacs.
Activation of programmed cell death in the rat ventral prostate.
Endocrinology
122:
552-562,
1988[Abstract].
19.
Lieu, C. H.,
C. C. Liu,
T. H. Yu,
K. D. Chen,
Y. N. Chang,
and
Y. K. Lai.
Role of mitogen-activated protein kinase in taxol-induced apoptosis in human leukemic U937 cells.
Cell Growth Differ.
9:
767-776,
1998[Abstract].
20.
Liu, X. H.,
H. S. Wiley,
and
A. W. Meike.
Androgens regulate proliferation of human prostate cancer cells in culture by increasing transforming growth factor- (EGF)/TGF-
receptor.
J. Clin. Endocrinol. Metab.
77:
1472-1478,
1993[Abstract].
21.
Lowe, C.,
T. Yoneda,
B. F. Boyce,
H. Chen,
G. R. Mundy,
and
P. Soriano.
Osteopetrosis in Src-deficient mice is due to an autonomous defect of osteoclasts.
Proc. Natl. Acad. Sci. USA
90:
4485-4489,
1993[Abstract].
22.
McDonnell, T. J.,
P. Troncoso,
S. M. Brisbay,
C. Logothetis,
L. W. Chung,
J. T. Hsieh,
S. M. Tu,
and
M. L. Campbell.
Expression of the protooncogene bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer.
Cancer Res.
52:
6940-6944,
1992[Abstract].
23.
Mineshita, T.,
and
K. Yamaguchi.
An androgen-dependent tumor derived from a hormone-independent spontaneous tumor of a female mouse.
Steroids
4:
815-830,
1964[ISI].
24.
Moyer, J. D.,
E. G. Barbacci,
K. K. Iwata,
L. Arnold,
B. Boman,
A. Cunningham,
C. DiOrio,
J. Doty,
M. J. Morin,
M. P. Moyer,
M. Neveu,
V. A. Pollack,
L. R. Pustilnik,
M. M. Reynolds,
D. Sloan,
A. Theleman,
and
P. Miller.
Induction of apoptosis and cell cycle arrest by CP-358, 774, an inhibitor of epidermal growth factor receptor tyrosine kinase.
Cancer Res.
57:
4838-4848,
1997[Abstract].
25.
Negrini, M.,
E. Silini,
C. A. Kozak,
Y. Tsujimoto,
and
C. M. Croce.
Molecular analysis of mbcl-2: structures and expression of the murine gene homologous to the human gene involved in follicular lymphoma.
Cell
49:
455-463,
1987[ISI][Medline].
26.
Nunez, G.,
L. London,
D. Hockenberry,
M. Alexander,
J. P. McKearn,
and
S. J. Korsmeyer.
Deregulated bcl-2 gene expression selectivity prolongs survival of growth factor deprived hematopoietic cell lines.
J. Immunol.
144:
3602-3610,
1990
27.
Ohigashi, T.,
M. Ueno,
M. Iida,
R. Hirata,
T. Nakanoma,
and
N. Deguchi.
Suppression of default apoptosis in androgen-dependent cells by testosterone-mediated bcl-2 expression.
Int. J. Urol.
6:
119-124,
1999[ISI][Medline].
28.
Otani, H.,
M. Erdos,
and
W. J. Leonard.
Tyrosine kinase(s) regulate apoptosis and bcl-2 expression in a growth factor-dependent cell line.
J. Biol. Chem.
268:
22733-22736,
1993
29.
Qin, S.,
Y. Minami,
T. Kurosaki,
and
H. Yamamura.
Distinctive functions of Syk and Lyn in mediating osmotic stress- and ultraviolet C irradiation-induced apoptosis in chicken B cells.
J. Biol. Chem.
272:
17994-17999,
1997
30.
Reed, J. C.
Bcl-2 and the regulation of programmed cell death.
J. Cell Biol.
124:
1-6,
1994[ISI][Medline].
31.
Shao, Z. M.,
M. L. Alpaugh,
J. A. Fontana,
and
S. H. Barsky.
Genistein inhibits proliferation similarly in estrogen receptor-positive and negative human breast carcinoma cell lines characterized by P21WAF1/CIP1 induction, G2/M arrest, and apoptosis.
J. Cell. Biochem.
69:
44-54,
1998[ISI][Medline].
32.
Tsujimoto, Y.,
J. Gorham,
J. Cossman,
E. Jaffe,
and
C. M. Croce.
The t(14:18) chromosomal translocations involved in B-cell neoplasms result from mistakes in VDJ joining.
Science
229:
1390-1393,
1985[ISI][Medline].
33.
Van Werden, W. M.,
A. van Kreuningen,
N. M. J. Elissen,
M. Vermeij,
F. H. De Jong,
G. J. van Steenbrugge,
and
F. H. Schroeder.
Castration-induced changes in morphology, androgen levels, and proliferative activity of human prostate cancer tissue grown in athymic nude mice.
Prostate
23:
65-74,
1993.
34.
Veldscolte, J.,
C. Ris-Stalpers,
G. G. J. M. Kuiper,
G. Jenster,
C. Berrevoets,
E. Claassen,
H. C. J. van Rooij,
J. Trapman,
A. O. Brinkmann,
and
E. Mulder.
A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens.
Biochem. Biophys. Res. Commun.
173:
534-540,
1990[ISI][Medline].