Inhibition of Transcription Factor Stat5 Induces Cell Death of Human Prostate Cancer Cells

Abstract

Identifying regulators of prostate cancer cell survival may lead to new therapeutic strategies for prostate cancer. We now report prevalent activation of transcription factor Stat5 in human prostate cancer and provide novel evidence that blocking activation of Stat5 in human prostate cancer cells leads to extensive cell death. Specifically, Stat5 was activated in 65% of human prostate cancer specimens examined based on nuclear location of tyrosine phosphorylated Stat5. Adenoviral gene delivery of a dominant-negative Stat5 mutant (DNStat5), but not wild-type Stat5, induced cell death of both the androgen-independent human prostate cancer cell line CWR22Rv and the androgen-sensitive LnCap cell line. Endogenous Stat5 was active in both CWR22Rv and LnCap cells. In contrast, only low levels of inactive Stat5 proteins were detected in the PC-3 cell line, which correlated with resistance to DNStat5-induced cell death. In CWR22Rv and LnCap cells, inhibition of Stat5 by expression of DNStat5 induced apoptotic cell death as judged from morphological changes, DNA fragmentation, and caspase-3 activation with evidence of a caspase-9-dependent mechanism. We propose that blocking Stat5 function may represent a novel therapeutic approach for prostate cancer. Identifying regulators of prostate cancer cell survival may lead to new therapeutic strategies for prostate cancer. We now report prevalent activation of transcription factor Stat5 in human prostate cancer and provide novel evidence that blocking activation of Stat5 in human prostate cancer cells leads to extensive cell death. Specifically, Stat5 was activated in 65% of human prostate cancer specimens examined based on nuclear location of tyrosine phosphorylated Stat5. Adenoviral gene delivery of a dominant-negative Stat5 mutant (DNStat5), but not wild-type Stat5, induced cell death of both the androgen-independent human prostate cancer cell line CWR22Rv and the androgen-sensitive LnCap cell line. Endogenous Stat5 was active in both CWR22Rv and LnCap cells. In contrast, only low levels of inactive Stat5 proteins were detected in the PC-3 cell line, which correlated with resistance to DNStat5-induced cell death. In CWR22Rv and LnCap cells, inhibition of Stat5 by expression of DNStat5 induced apoptotic cell death as judged from morphological changes, DNA fragmentation, and caspase-3 activation with evidence of a caspase-9-dependent mechanism. We propose that blocking Stat5 function may represent a novel therapeutic approach for prostate cancer. Prostate cancer typically progresses to androgen-independent growth after androgen-ablation therapy. Identification of androgen-independent proteins that control prostate cancer cell survival may lead to more effective therapies. Using long term organ cultures of human and rat prostate tissues, we have documented direct effects of prolactin (Prl) 1The abbreviations used are: Prl, prolactin; Stat5, signal transducer and activator of transcription-5; Jak-2, Janus tyrosine kinase-2; mAb, monoclonal antibody; DN, dominant-negative; WT, wild-type; m.o.i., multiplicity of infection; Ad, adenovirus; ELISA, enzyme-linked immunosorbent assay.1The abbreviations used are: Prl, prolactin; Stat5, signal transducer and activator of transcription-5; Jak-2, Janus tyrosine kinase-2; mAb, monoclonal antibody; DN, dominant-negative; WT, wild-type; m.o.i., multiplicity of infection; Ad, adenovirus; ELISA, enzyme-linked immunosorbent assay. as a mitogen and survival factor for prostate epithelium (1Nevalainen M.T. Valve E.M. Ingleton P.M. Nurmi M. Martikainen P.M. Harkonen P.L. J. Clin. Invest. 1997; 99: 618-627Crossref PubMed Scopus (174) Google Scholar, 2Nevalainen M.T. Valve E.M. Makela S.I. Blauer M. Tuohimaa P.J. Harkonen P.L. Endocrinology. 1991; 129: 612-622Crossref PubMed Scopus (78) Google Scholar, 3Nevalainen M.T. Härkönen P.L. Valve E.M. Ping W. Nurmi M. Martikainen P.M. Cancer Res. 1993; 53: 5199-5207PubMed Google Scholar, 4Ahonen T.J. Harkonen P.L. Laine J. Rui H. Martikainen P.M. Nevalainen M.T. Endocrinology. 1999; 140: 5412-5421Crossref PubMed Google Scholar). These observations have been complemented by demonstration of massive hyperplasia of prostates in transgenic mice overexpressing Prl (5Wennbo H. Kindblom J. Isaksson O.G. Tornell J. Endocrinology. 1997; 138: 4410-4415Crossref PubMed Scopus (157) Google Scholar, 6Kindblom J. Dillner K. Ling C. Tornell J. Wennbo H. Prostate. 2002; 53: 24-33Crossref PubMed Scopus (27) Google Scholar) as well as reduced prostate sizes in Prl-null mice (7Steger R.W. Chandrashekar V. Zhao W. Bartke A. Horseman N.D. Endocrinology. 1998; 139: 3691-3695Crossref PubMed Scopus (92) Google Scholar). Importantly, we have also shown local production of Prl (1Nevalainen M.T. Valve E.M. Ingleton P.M. Nurmi M. Martikainen P.M. Harkonen P.L. J. Clin. Invest. 1997; 99: 618-627Crossref PubMed Scopus (174) Google Scholar, 8Nevalainen M.T. Valve E.M. Ahonen T. Yagi A. Paranko J. Harkonen P.L. FASEB J. 1997; 11: 1297-1307Crossref PubMed Scopus (70) Google Scholar) and expression of Prl receptors in prostate epithelium (1Nevalainen M.T. Valve E.M. Ingleton P.M. Nurmi M. Martikainen P.M. Harkonen P.L. J. Clin. Invest. 1997; 99: 618-627Crossref PubMed Scopus (174) Google Scholar, 9Nevalainen M.T. Valve E.M. Ingleton P.M. Harkonen P.L. Endocrinology. 1996; 137: 3078-3088Crossref PubMed Scopus (83) Google Scholar) and thereby provided evidence for an autocrine loop of Prl action in prostate. Downstream of Prl receptor activation, we have demonstrated that Stat5 is a key signaling protein in normal rat prostate epithelium (10Ahonen T.J. Harkonen P.L. Rui H. Nevalainen M.T. Endocrinology. 2002; 143: 228-238Crossref PubMed Scopus (46) Google Scholar) and that deficiency of Stat5 in Stat5a-null mice is associated with defective prostate tissue architecture (11Nevalainen M.T. Ahonen T.J. Yamashita H. Chandrashekar V. Bartke A. Grimley P.M. Robinson G.W. Hennighausen L. Rui H. Lab. Invest. 2000; 80: 993-1006Crossref PubMed Scopus (36) Google Scholar). Stat5 is one of seven members of the Stat family of transcription factors in mammals (12Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3330) Google Scholar) and consists of two distinct but highly homologous gene products: the 94-kDa Stat5a and 92-kDa Stat5b (13Wakao H. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (709) Google Scholar, 14Liu X. Robinson G.W. Gouilleux F. Groner B. Hennighausen L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8831-8835Crossref PubMed Scopus (452) Google Scholar). In response to Prl, Stat5a and Stat5b become activated by phosphorylation on residue Tyr-694 and Tyr-699, respectively, in the C-terminal domain predominantly by Janus tyrosine kinase-2 (Jak2), which is preassociated with the cytoplasmic domain of the Prl receptor (15Rui H. Kirken R.A. Farrar W. J. Biol. Chem. 1994; 269: 5364-5368Abstract Full Text PDF PubMed Google Scholar). Tyrosine-phosphorylated Stat5 proteins dimerize and translocate to the nucleus where they bind to specific response elements of target gene promoters to regulate transcription (16Gouilleux F. Wakao H. Mundt M. Groner B. EMBO J. 1994; 13: 4361-4369Crossref PubMed Scopus (522) Google Scholar). Based on our previous findings of a survival function of Prl in normal prostate epithelium (4Ahonen T.J. Harkonen P.L. Laine J. Rui H. Martikainen P.M. Nevalainen M.T. Endocrinology. 1999; 140: 5412-5421Crossref PubMed Google Scholar), an autocrine production of Prl by prostate epithelial cells (1Nevalainen M.T. Valve E.M. Ingleton P.M. Nurmi M. Martikainen P.M. Harkonen P.L. J. Clin. Invest. 1997; 99: 618-627Crossref PubMed Scopus (174) Google Scholar, 8Nevalainen M.T. Valve E.M. Ahonen T. Yagi A. Paranko J. Harkonen P.L. FASEB J. 1997; 11: 1297-1307Crossref PubMed Scopus (70) Google Scholar), and the glandular defect of the prostates of Stat5a knock-out mice (11Nevalainen M.T. Ahonen T.J. Yamashita H. Chandrashekar V. Bartke A. Grimley P.M. Robinson G.W. Hennighausen L. Rui H. Lab. Invest. 2000; 80: 993-1006Crossref PubMed Scopus (36) Google Scholar), we hypothesized that Stat5 may act as a survival protein in human prostate cancer and that blocking Stat5 function could induce death of prostate cancer cells. We investigated activation of Stat5 in 40 human prostate cancer samples by immunohistochemical analysis using activation state-specific anti-phospho-TyrStat5 antibody. To specifically block Stat5 activity in human prostate cancer cells, we created a dominant-negative mutant of Stat5 (DNStat5) in an adenoviral transfer vector. In this work, we demonstrate that transcription factor Stat5 is activated in a significant number of human prostate cancer specimens and that blocking Stat5 activity induces extensive apoptosis of prostate cancer cells. Cell Culture—CWR22Rv, LnCap, and PC-3 cells (ATCC, Manassas, VA) were cultured in RPMI 1640 medium (Biofluids, Gaithersburg, MD), and T47D cells (ATCC) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mm l-glutamine, and penicillin-streptomycin (50 IU/ml and 50 μg/ml, respectively) at 37 °C with 5% CO2. LnCap cells were cultured in the presence of 1 nm dihydrotestosterone (5α-androstan-17β-ol-3-one, Sigma). Immunohistochemical Detection of Phosphorylated Stat5 in Human Prostate Cancer—Tissue sections of formalin-fixed prostate cancer samples from 40 patients were immunostained for activated Stat5 as described previously (17Nevalainen M.T. Xie J. Bubendorf L. Wagner K.U. Rui H. Mol. Endocrinol. 2002; 16: 1108-1124Crossref PubMed Scopus (65) Google Scholar). Tissue sections were deparaffinized in xylene followed by rehydration in graded alcohol. To unmask the epitopes, the slides were microwave-treated with antigen-retrieval solution AxAR1 (Advantex BioReagents, Conroe, TX) or AxAR2 for use with anti-pTyrStat5 mAb or anti-pan-Stat5 mAb, respectively. Endogenous peroxidase activity was blocked by incubating the slides in 0.3% hydrogen peroxide, and the tissue sections were incubated in normal goat serum (BioGenex Laboratories Inc., San Ramon, CA) for 2 h to block unspecific binding of immunoglobulins (IgGs). The anti-pTyrStat5 mAb was diluted in 1% bovine serum albumin in phosphate-buffered saline at a final concentration of 0.6 μg/ml (Advantex BioReagents). The anti-pan-Stat5 mAb was used at a concentration of 2 μg/ml (Advantex Bioreagents). Antigen-antibody complexes were detected using anti-mouse-biotinylated goat secondary antibody followed by streptavidin-horseradish-peroxidase complex (BioGenex). 3,3′-Diaminobenzidine was used as chromogen, and hematoxylin was used as counterstain. For negative controls, parallel slides were immunostained with subtype-specific mouse IgG, and lactating human mammary gland was used as positive control tissue (17Nevalainen M.T. Xie J. Bubendorf L. Wagner K.U. Rui H. Mol. Endocrinol. 2002; 16: 1108-1124Crossref PubMed Scopus (65) Google Scholar). Generation of Adenovirus for Gene Delivery of Dominant-negative and Wild-type Stat5—Expression vector for murine Stat5a (pXM-Stat5a) was kindly provided by Xiuwen Liu and Lothar Hennighausen (National Institutes of Health, Bethesda, MD) (14Liu X. Robinson G.W. Gouilleux F. Groner B. Hennighausen L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8831-8835Crossref PubMed Scopus (452) Google Scholar). A dominant-negative variant of Stat5 (Stat5aΔ713) was derived by truncation after amino acid residue Ala-713 of pXM-Stat5a using a PCR fragment generated using 5′-TAATACGACTCACTATAGGG-3′ (sense) and 5′-GCTCTAGACTAGGCATCTGTGGATGCATTG-3′ (antisense) primers followed by EcoRI and XbaI digestion and subcloning into the EcoRI-XbaI-digested pXM-Stat5a. The DNA sequence of the resulting construct pXM-Stat5aΔ713 was verified before use. The ability of our DNStat5 (Stat5aΔ713) expression construct to completely suppress both Stat5a- and Stat5b-mediated transcriptional activation has been reported previously (18Yamashita H. Iwase H. Toyama T. Fujii Y. Oncogene. 2003; 22: 1638-1652Crossref PubMed Scopus (76) Google Scholar). Replication-defective human adenovirus (Ad5) carrying wild-type Stat5 (WTStat5) or DNStat5 was generated using the AdEasy vector system (Qbiogene, Carlsbad, CA). The open reading frame sequences of DNStat5 and WTStat5 were released from respective plasmids by 1) digestion with EcoRI, 2) blunt-ending by Klenow DNA polymerase, and 3) digestion with HindIII, and the resulting fragments were subcloned into the Klenow DNA polymerase bluntended BglII site and the unmodified HindIII site of the pShuttle-CMV transfer vector. Homologous recombination of WTStat5 or DNStat5 transfer vectors with the pAdEasy vector was performed in BJ5183 Escherichia coli by electroporation. Recombined clones were screened by kanamycin-resistant growth and confirmed by PacI digestion to yield two bands of 30 and 4.5 kb. The recombinant viruses were packaged in QBI-293A cells, and resulting clones were selected from plaques and amplified. Expression of WTStat5 and DNStat5 from adenoviral stocks was verified by Western blotting using an anti-pan-Stat5 antibody (Transduction Laboratories, Lexington, KY). Selected recombinant viral stocks were expanded in large scale cultures, purified by double cesium chloride gradient centrifugation, and titered side by side by a standard plaque assay method in QBI-293A cells as per the manufacturer's instructions. Protein Solubilization and Immunoblotting—Pellets of prostate cancer cells were solubilized in lysis buffer (10 mm Tris-HCl, pH 7.6, 5 mm EDTA, 50 mm sodium chloride, 30 mm sodium pyrophosphate, 50 mm sodium fluoride, 1 mm sodium orthovanadate, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, and 2 μg/ml leupeptin). Cell lysates were rotated end-over-end at 4 °C for 60 min, and insoluble material was pelleted at 12,000 × g for 30 min at 4 °C. For immunoprecipitations, the protein concentrations of clarified cell lysates were determined by simplified Bradford method (Bio-Rad). The proteins were analyzed by and using were to diluted in blocking buffer at the mAb μg/ml, anti-pan-Stat5 mAb μg/ml, Laboratories, or to Stat5a or Stat5b The were in buffer (50 mm Tris-HCl, pH 7.6, mm and incubated with goat to mouse or μg/ml, was used to the manufacturer's for antibody Cell was determined by a assay in which a is by cells into a in direct to the number of cells in Prostate cancer cells were on and the with of of adenovirus carrying or with control or cells as after the the assay was performed to the manufacturer's and the was at Cell of DNA after cell death induced by inhibition of transcription factor Stat5 was determined by Detection Prostate cancer cells were on and the with adenovirus carrying or or with control were and at × and cytoplasmic containing DNA were to that been incubated with monoclonal antibody. The of DNA of to antibody was by monoclonal antibody using as a at and LnCap cells × were in phosphate-buffered pelleted at × and in 5 of phosphate-buffered a centrifugation, cells were in of phosphate-buffered saline and by of of the cell been for 1 h at 4 cells were with μg/ml and with A for 30 min at 37 °C. The cells were analyzed by using a cell and of caspase-3 and was determined by Prostate cancer cells were on and the with adenovirus carrying or or with control were and at × and cytoplasmic containing activated were to or with the were to the was generated to the of activated in the cell to of the and determined at Stat5 in Human Prostate have described a highly in method for activation of Stat5 in which is based on immunohistochemical of phosphorylated Stat5 that is the cell nucleus (10Ahonen T.J. Harkonen P.L. Rui H. Nevalainen M.T. Endocrinology. 2002; 143: 228-238Crossref PubMed Scopus (46) Google Scholar, M.T. Xie J. Bubendorf L. Wagner K.U. Rui H. Mol. Endocrinol. 2002; 16: 1108-1124Crossref PubMed Scopus (65) Google Scholar). complemented with immunohistochemical of nuclear Stat5 was to a material of 40 human prostate cancer and significant activation of Stat5 was detected in 65% of of human prostate cancer specimens of samples Stat5 activation by or nuclear human prostate a and In contrast, Stat5 was negative and nuclear for Stat5 was not in normal prostate epithelial cells. human epithelium is as a positive control for activated human Stat5 (17Nevalainen M.T. Xie J. Bubendorf L. Wagner K.U. Rui H. Mol. Endocrinol. 2002; 16: 1108-1124Crossref PubMed Scopus (65) Google Scholar) and parallel sections of lactating human with mouse were negative and Stat5 in CWR22Rv and LnCap Cell but in PC-3 examined Stat5a and Stat5b protein expression and activation in human prostate cancer cell the androgen-independent CWR22Rv and PC-3 cells, and the androgen-sensitive LnCap cell line. For this of Stat5a or Stat5b was followed by with the anti-phospho-TyrStat5 antibody or with to Stat5a or Stat5b was the Stat5 protein in CWR22Rv and LnCap cells both growth and in In both CWR22Rv and LnCap cells at low and at cell Stat5b was phosphorylated on the In contrast, activation of Stat5a or Stat5b was detected in PC-3 cells, with low or levels of Stat5 protein in cells of Stat5 in Human Prostate Cancer Cell on the prevalent activation of Stat5 in human prostate cancer tissues, we hypothesized that Stat5 may act as a survival protein in human prostate cancer. we blocking Stat5 function in human prostate cancer cells induce cell death. To block activated Stat5 proteins in human prostate we generated an adenovirus for effective delivery of DNStat5 and a control carrying The of the adenoviral vector carrying dominant-negative Stat5a and the is described and of Stat5a after amino acid the transcriptional activation domain and a Stat5a mutant that is to the the Stat5a mutant blocked the function of both and Stat5b (18Yamashita H. Iwase H. Toyama T. Fujii Y. Oncogene. 2003; 22: 1638-1652Crossref PubMed Scopus (76) Google Scholar). We have both the and the viral in for gene tyrosine and DNA binding not To blocking Stat5 activity induce death of human prostate cancer cells, we examined the of of on the of the androgen-independent CWR22Rv cell line. For the cell we used an assay the activity of the cells. CWR22Rv cells were cultured to in and for min to or at to m.o.i., after which the was diluted in medium as h of cells were analyzed for cell A and of the expression of DNStat5 on cell was with at of and of cell by at In contrast, cells with or not significant of of CWR22Rv cells with an control was not reduced not The of in CWR22Rv cells was in that were and more and with titered of Prostate Cancer Cell by DNStat5 of the of on CWR22Rv cell confirmed extensive cell death expression of CWR22Rv cells to at for h extensive cell death as by cell and which morphological with apoptotic cell death. In contrast, was evidence of reduced cell in response to but not induced cell death also in the androgen-sensitive human prostate cancer cell line, LnCap and that of were to induce cell death in LnCap cells with CWR22Rv cells not an that correlated with adenoviral gene delivery of Stat5 proteins into LnCap cells. Specifically, of viral protein delivery that the of induced levels of DNStat5 or WTStat5 expression in LnCap cells in CWR22Rv cells shown by of cell lysates also that levels of DNStat5 and WTStat5 by the respective adenoviral vectors the that the cell death in response to is to specific dominant-negative and not by In contrast, were of cell death in PC-3 cells after h to at as judged from cell not To that DNStat5-induced of prostate cancer cells was to apoptotic cell prostate cancer cells were analyzed for DNStat5-induced of CWR22Rv cells were to and a of at for A of DNStat5 on of DNA was as analyzed by an of DNA fragments a in DNA was detected in cells to that detected in WTStat5 cells with DNStat5-induced death of LnCap cells was also associated with DNA as by a and in DNA levels in cells on at with we that of Stat5 induces apoptotic cell death in both CWR22Rv and LnCap cells as by of In contrast, of PC-3 cells, which not active Stat5, to not of DNA with cells WTStat5 or with cells with control or cells cell death of prostate cancer cells DNStat5 was also verified by cell In both CWR22Rv and LnCap cells DNStat5 but not in cells extensive DNA was detected in the of a large of cell fragments In CWR22Rv cells, this was to of DNA at of using m.o.i., which was that in parallel cells In LnCap cells, the of cell fragments with DNA from in cells to in cells. cells and cells with control or were used as controls, and the of cells not DNStat5-induced of CWR22Rv and LnCap of the family of cell death. A has been to caspase-3 as a key of a of apoptotic cell death H. M. X. X. Cell Biol. 1999; PubMed Scopus Google Scholar). To apoptosis in prostate cancer cells induced by expression of DNStat5 is by activation of we analyzed caspase-3 activation by a specific caspase-3 assay. of CWR22Rv or LnCap cells to for or respectively, activated caspase-3 in both cell Specifically, in CWR22Rv cells, activation of caspase-3 was in cells DNStat5 with cells. In LnCap cells, the was with we that DNStat5-induced apoptosis in both CWR22Rv and LnCap human prostate cancer cells the activation of apoptosis may induced activation of in a or M. M. Mol. Biol. PubMed Scopus Google Scholar). of Stat5 activation in CWR22Rv and LnCap cells induced activation of we to DNStat5-induced activation of caspase-3 the activation of activity was in response to DNStat5 in both CWR22Rv and LnCap cells for to A of was detected at h in CWR22Rv cells DNStat5 In LnCap cells, the was but the was not We that DNStat5-induced cell death in CWR22Rv cells is by caspase-3 a caspase-9-dependent mechanism. activation a apoptotic M. M. Mol. Biol. PubMed Scopus Google Scholar). to the sequence of that in DNStat5-induced prostate cancer cell The prevalent of activated Stat5 in human prostate cancer specimens that Stat5 is a therapeutic target in a of prostate A by and W. F. J. 2002; PubMed Google Scholar) that in human prostate tissue was only a low of Stat5 binding to an to the of the rat in W. F. J. 2002; PubMed Google Scholar). is that prostate or factors with binding of Stat5 to this In this using our highly in which Stat5 activation at a cell we have demonstrated that Stat5 is activated in normal human and mouse mammary epithelial cells of an activity that is to by assay using tissue of mammary (17Nevalainen M.T. Xie J. Bubendorf L. Wagner K.U. Rui H. Mol. Endocrinol. 2002; 16: 1108-1124Crossref PubMed Scopus (65) Google Scholar). that blocking activation of Stat5 in human prostate is a new therapeutic Delivery of into cancer cells is by of effective delivery but gene therapeutic delivery and both viral and strategies C. Clin. 2002; PubMed Scopus Google Scholar, T. L. Gene 2002; PubMed Scopus Google Scholar). prostate cancer gene based on delivery of DNStat5 may the of the Stat5 tyrosine for Stat5 activation in prostate cancer could represent an and more effective approach to Stat5 in human prostate cancer with more in our to the Stat5 tyrosine for Stat5 in prostate cancer. In this the tyrosine is a this is for tyrosine phosphorylation of Stat5 by a of receptors for prolactin Prl receptor associated signaling of Prl is in prostate epithelium an autocrine loop of Prl action in prostate (1Nevalainen M.T. Valve E.M. Ingleton P.M. Nurmi M. Martikainen P.M. Harkonen P.L. J. Clin. Invest. 1997; 99: 618-627Crossref PubMed Scopus (174) Google Scholar, 8Nevalainen M.T. Valve E.M. Ahonen T. Yagi A. Paranko J. Harkonen P.L. FASEB J. 1997; 11: 1297-1307Crossref PubMed Scopus (70) Google Scholar). In to tyrosine as or as well as members of the tyrosine family or receptor tyrosine also Stat5 in prostate cancer P.M. F. Rui H. 1999; PubMed Scopus Google Scholar). We in this using in of phosphorylated and nuclear Stat5 that Stat5 was activated in a of prostate cancer specimens that inhibition of Stat5 as a therapeutic approach could have a in the of prostate cancer. The that both CWR22Rv and androgen-sensitive LnCap cells to of Stat5 with cell death that apoptosis induced by blocking the activation of Stat5 is of to the Stat5 activation in prostate cancer cell and to DNStat5 that immunohistochemical of activated Stat5 in prostate cancer may to this therapeutic We to Xiuwen Liu and Lothar Hennighausen for for mouse Stat5a and to Yamashita for with subcloning and of and DNStat5 expression plasmids (18Yamashita H. Iwase H. Toyama T. Fujii Y. Oncogene. 2003; 22: 1638-1652Crossref PubMed Scopus (76) Google Scholar). We also Bubendorf for normal human Cancer and for in cell and Cancer and for in cell and tissue