Testosterone-repressed Prostate Message-2 Is an Antiapoptotic...

8
[CANCER RESEARCH 60, 170 –176, January 1, 2000] Testosterone-repressed Prostate Message-2 Is an Antiapoptotic Gene Involved in Progression to Androgen Independence in Prostate Cancer 1 Hideaki Miyake, Colleen Nelson, Paul S. Rennie, and Martin E. Gleave 2 The Prostate Centre, Vancouver General Hospital, 2660 Oak Street, Vancouver, British Columbia V6H 3Z6, [H. M., C. N., P. S. R., M. E. G.]; and Division of Urology, University of British Columbia, D-9, 2733 Heather Street, Vancouver, British Columbia V5Z 3J5 [C. N., M. E. G.], Canada ABSTRACT Although initially reported as an androgen-repressed gene in the rat prostate, the functional role of testosterone-repressed prostate message-2 (TRPM-2) in apoptosis remains undefined. Inhibition of castration- induced apoptosis by calcium channel blocker treatment in androgen- dependent Shionogi tumors resulted in the prevention of TRPM-2 gene up-regulation, suggesting that TRPM-2 is not directly androgen-re- pressed, but is regulated by apoptotic stimuli. The overexpression of the TRPM-2 gene in human androgen-dependent LNCaP prostate cancer cells by stable transfection rendered them highly resistant to androgen ablation in vivo. We then tested the efficacy of antisense TRPM-2 oligodeoxynucle- otide (ODN) therapy in the Shionogi tumor model and demonstrated that the systemic administration of antisense TRPM-2 ODNs in mice bearing Shionogi tumors after castration resulted in a more rapid onset of apopto- sis and time to complete regression, as well as a significant delay of emergence of androgen-independent recurrent tumors compared to con- trol ODN treatment. Collectively, these findings illustrate that TRPM-2 is an antiapoptotic rather than an androgen-repressed gene that confers resistance to androgen ablation and thereby helps accelerate the progres- sion to androgen independence. INTRODUCTION No therapy exists that is superior to androgen ablation in patients with advanced prostate cancer. Approximately 80% of patients achieve symptomatic and/or objective response after androgen abla- tion; however, the progression to androgen independence ultimately occurs and remains the main obstacle to improving the survival and quality of life in this disease (1). Traditionally, new nonhormonal therapies have been evaluated in patients with hormone refractory disease. When used in this end-stage setting, no nonhormonal agent has improved survival (2). Because androgen resistance develops, in part, from the up-regulation of antiapoptotic genes after androgen withdrawal, the identification and targeting of genes involved in apoptosis and AI 3 (3) progression may lead to the development of novel therapies capable of delaying hormone refractory recurrences. TRPM-2, also known as clusterin or sulfated glycoprotein-2, was first isolated from ram rete testes fluid (3), and it is associated with a wide variety of physiological and pathological processes, including tissue remodeling, lipid transport, reproduction, complement regula- tion, and apoptotic cell death (4). TRPM-2 has been regarded as a marker for cell death because its expression is up-regulated in various normal and malignant tissues undergoing apoptosis (5–9). Recent studies provide conflicting data on the association between enhanced TRPM-2 expression and apoptotic activity (10 –12). Although it was initially named as an androgen-repressed gene up-regulated in re- gressing prostate tissues after androgen ablation (13), the functional role of TRPM-2 in castration-induced apoptosis remains undefined. In prostate cancer, TRPM-2 expression is associated with apoptotic cell death and AI recurrences. The introduction of TRPM-2 cDNA into LNCaP prostate cancer cells increases the resistance to apoptosis induced by tumor necrosis factor a treatment (14). The increased expression of TRPM-2 in prostate cancer correlates with the increas- ing Gleason score (15). However, the functional significance of TRPM-2 expression during AI progression has not been demon- strated. Controlled study of the complex molecular processes associated with AI progression in prostate cancer has proved difficult because it cannot be replicated in vitro, and few animal models exist that reproducibly mimic the clinical course of the disease in men. The Shionogi tumor model is an AD mouse mammary carcinoma xe- nograft that is particularly useful in studying mechanism of castration- induced apoptosis and AI progression. In this model, AD tumors in male mice undergo complete regression after castration but recur as rapidly growing AI tumors after 1 month (16). The highly reproduc- ible regression and recurrence pattern provides a reliable end point to test the efficacy of agents targeting castration-induced apoptosis and their effects on time to AI progression. Of the available human prostate cancer cell lines, only the LNCaP cell line is AD, PSA- secreting, and immortalized in vitro. As in human prostate cancer, serum PSA levels in the LNCaP tumor model are initially regulated by androgen and directly proportional to tumor volume, with loss of androgen-regulated PSA gene expression after castration as a surro- gate end point of AI progression (17). In the present study, we report for the first time that the TRPM-2 gene is an apoptosis-associated gene rather than androgen-repressed gene. We then evaluated the effects of TRPM-2 overexpression on time to AI progression in the LNCaP tumor model. Finally, we tested whether adjuvant use of antisense TRPM-2 ODNs enhances castra- tion-induced apoptosis and delays AI progression in the AD Shionogi tumor model. MATERIALS AND METHODS Shionogi Tumor Growth. The Toronto subline of the transplantable SC- 115 AD mouse mammary carcinoma (18) was used in this study. For in vitro experiments, Shionogi tumor cells were cultured in DMEM (Life Technolo- gies, Inc., Gaithersburg, MD) supplemented with 5% heat-inactivated FCS. For in vivo study, adult male DD/S strain mice received s.c. injections of ;5 3 10 6 tumor cells. When Shionogi tumors became 1–2 cm in diameter, usually 2–3 weeks after injection, castration was performed through an abdominal incision under methoxyflurane anesthesia. Details of the maintenance of mice, tumor stock, and operative procedures are described in a previous publication (19). Mice were maintained in accordance with institutional accredited guidelines. CCB Treatment. One the day before the castration, male DD/S mice bearing the Shionogi tumor were randomly selected for treatment with CCBs (n 5 5) versus no treatment as the control (n 5 5). Beginning 1 day before castration, 588-mg nifedipine and 100-mg norvasc were adminis- tered p.o. three times daily to each mouse in the CCB group for 14 days. Tumor volume was measured twice weekly and calculated by the formula length 3 width 3 depth 3 0.5236 (20). Received 6/16/99; accepted 10/28/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Grant 009002 from the National Cancer Institute of Canada. 2 To whom requests for reprints should be addressed, at Division of Urology, Univer- sity of British Columbia, D-9, 2733 Heather Street, Vancouver, British Columbia V5Z 3J5, Canada. 3 The abbreviations used are: AI, androgen-independent; TRPM-2; testosterone- repressed prostate message-2; AD, androgen-dependent; PSA, prostate-specific antigen; ODN, oligodeoxynucleotide; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine 59-triphosphate nick end labeling; CCB, calcium channel blocker; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; PARP, poly(ADP-ribose) polymerase. 170 Research. on January 22, 2020. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from

Transcript of Testosterone-repressed Prostate Message-2 Is an Antiapoptotic...

Page 1: Testosterone-repressed Prostate Message-2 Is an Antiapoptotic …cancerres.aacrjournals.org/content/canres/60/1/170.full.pdf · [CANCER RESEARCH 60, 170–176, January 1, 2000] Testosterone-repressed

[CANCER RESEARCH 60, 170–176, January 1, 2000]

Testosterone-repressed Prostate Message-2 Is an Antiapoptotic Gene Involved inProgression to Androgen Independence in Prostate Cancer1

Hideaki Miyake, Colleen Nelson, Paul S. Rennie, and Martin E. Gleave2

The Prostate Centre, Vancouver General Hospital, 2660 Oak Street, Vancouver, British Columbia V6H 3Z6, [H. M., C. N., P. S. R., M. E. G.]; and Division of Urology, Universityof British Columbia, D-9, 2733 Heather Street, Vancouver, British Columbia V5Z 3J5 [C. N., M. E. G.], Canada

ABSTRACT

Although initially reported as an androgen-repressed gene in the ratprostate, the functional role of testosterone-repressed prostate message-2(TRPM-2) in apoptosis remains undefined. Inhibition of castration-induced apoptosis by calcium channel blocker treatment in androgen-dependent Shionogi tumors resulted in the prevention ofTRPM-2 geneup-regulation, suggesting that TRPM-2 is not directly androgen-re-pressed, but is regulated by apoptotic stimuli. The overexpression of theTRPM-2gene in human androgen-dependent LNCaP prostate cancer cellsby stable transfection rendered them highly resistant to androgen ablationin vivo. We then tested the efficacy of antisense TRPM-2 oligodeoxynucle-otide (ODN) therapy in the Shionogi tumor model and demonstrated thatthe systemic administration of antisense TRPM-2 ODNs in mice bearingShionogi tumors after castration resulted in a more rapid onset of apopto-sis and time to complete regression, as well as a significant delay ofemergence of androgen-independent recurrent tumors compared to con-trol ODN treatment. Collectively, these findings illustrate that TRPM-2 isan antiapoptotic rather than an androgen-repressed gene that confersresistance to androgen ablation and thereby helps accelerate the progres-sion to androgen independence.

INTRODUCTION

No therapy exists that is superior to androgen ablation in patientswith advanced prostate cancer. Approximately 80% of patientsachieve symptomatic and/or objective response after androgen abla-tion; however, the progression to androgen independence ultimatelyoccurs and remains the main obstacle to improving the survival andquality of life in this disease (1). Traditionally, new nonhormonaltherapies have been evaluated in patients with hormone refractorydisease. When used in this end-stage setting, no nonhormonal agenthas improved survival (2). Because androgen resistance develops, inpart, from the up-regulation of antiapoptotic genes after androgenwithdrawal, the identification and targeting of genes involved inapoptosis and AI3 (3) progression may lead to the development ofnovel therapies capable of delaying hormone refractory recurrences.

TRPM-2, also known as clusterin or sulfated glycoprotein-2, wasfirst isolated from ram rete testes fluid (3), and it is associated with awide variety of physiological and pathological processes, includingtissue remodeling, lipid transport, reproduction, complement regula-tion, and apoptotic cell death (4). TRPM-2 has been regarded as amarker for cell death because its expression is up-regulated in variousnormal and malignant tissues undergoing apoptosis (5–9). Recentstudies provide conflicting data on the association between enhancedTRPM-2 expression and apoptotic activity (10–12). Although it was

initially named as an androgen-repressed gene up-regulated in re-gressing prostate tissues after androgen ablation (13), the functionalrole of TRPM-2 in castration-induced apoptosis remains undefined. Inprostate cancer, TRPM-2 expression is associated with apoptotic celldeath and AI recurrences. The introduction ofTRPM-2cDNA intoLNCaP prostate cancer cells increases the resistance to apoptosisinduced by tumor necrosis factora treatment (14). The increasedexpression of TRPM-2 in prostate cancer correlates with the increas-ing Gleason score (15). However, the functional significance ofTRPM-2 expression during AI progression has not been demon-strated.

Controlled study of the complex molecular processes associatedwith AI progression in prostate cancer has proved difficult because itcannot be replicatedin vitro, and few animal models exist thatreproducibly mimic the clinical course of the disease in men. TheShionogi tumor model is an AD mouse mammary carcinoma xe-nograft that is particularly useful in studying mechanism of castration-induced apoptosis and AI progression. In this model, AD tumors inmale mice undergo complete regression after castration but recur asrapidly growing AI tumors after 1 month (16). The highly reproduc-ible regression and recurrence pattern provides a reliable end point totest the efficacy of agents targeting castration-induced apoptosis andtheir effects on time to AI progression. Of the available humanprostate cancer cell lines, only the LNCaP cell line is AD, PSA-secreting, and immortalizedin vitro. As in human prostate cancer,serum PSA levels in the LNCaP tumor model are initially regulated byandrogen and directly proportional to tumor volume, with loss ofandrogen-regulated PSA gene expression after castration as a surro-gate end point of AI progression (17).

In the present study, we report for the first time that theTRPM-2gene is an apoptosis-associated gene rather than androgen-repressedgene. We then evaluated the effects of TRPM-2 overexpression ontime to AI progression in the LNCaP tumor model. Finally, we testedwhether adjuvant use of antisense TRPM-2 ODNs enhances castra-tion-induced apoptosis and delays AI progression in the AD Shionogitumor model.

MATERIALS AND METHODS

Shionogi Tumor Growth. The Toronto subline of the transplantable SC-115 AD mouse mammary carcinoma (18) was used in this study. Forin vitroexperiments, Shionogi tumor cells were cultured in DMEM (Life Technolo-gies, Inc., Gaithersburg, MD) supplemented with 5% heat-inactivated FCS. Forin vivostudy, adult male DD/S strain mice received s.c. injections of;5 3 106

tumor cells. When Shionogi tumors became 1–2 cm in diameter, usually 2–3weeks after injection, castration was performed through an abdominal incisionunder methoxyflurane anesthesia. Details of the maintenance of mice, tumorstock, and operative procedures are described in a previous publication (19).Mice were maintained in accordance with institutional accredited guidelines.

CCB Treatment. One the day before the castration, male DD/S micebearing the Shionogi tumor were randomly selected for treatment withCCBs (n5 5) versusno treatment as the control (n5 5). Beginning 1 daybefore castration, 588-mg nifedipine and 100-mg norvasc were adminis-tered p.o. three times daily to each mouse in the CCB group for 14 days.Tumor volume was measured twice weekly and calculated by the formulalength 3 width 3 depth3 0.5236 (20).

Received 6/16/99; accepted 10/28/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grant 009002 from the National Cancer Institute of Canada.2 To whom requests for reprints should be addressed, at Division of Urology, Univer-

sity of British Columbia, D-9, 2733 Heather Street, Vancouver, British Columbia V5Z3J5, Canada.

3 The abbreviations used are: AI, androgen-independent; TRPM-2; testosterone-repressed prostate message-2; AD, androgen-dependent; PSA, prostate-specific antigen;ODN, oligodeoxynucleotide; TUNEL, terminal deoxynucleotidyl transferase-mediateddeoxyuridine 59-triphosphate nick end labeling; CCB, calcium channel blocker; G3PDH,glyceraldehyde-3-phosphate dehydrogenase; PARP, poly(ADP-ribose) polymerase.

170

Research. on January 22, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 2: Testosterone-repressed Prostate Message-2 Is an Antiapoptotic …cancerres.aacrjournals.org/content/canres/60/1/170.full.pdf · [CANCER RESEARCH 60, 170–176, January 1, 2000] Testosterone-repressed

TUNEL Staining. A modified TUNEL technique (21) was used to detectapoptotic cells in Shionogi tumors using the ApopTagin Situ ApoptosisDetection System (Oncor Inc., Gaithersburg, MD) according to the manufac-ture’s protocol. The number of positively stained cells per high power field infive random fields was counted and averaged.

Expression Plasmid and Transfection of LNCaP Cells.LNCaP cellswere kindly provided by Dr. Leland W. K. Chung (University of Virginia,Charlottesville, VA) and maintained in RPMI 1640 (Life Technologies, Inc.)supplemented with 5% heat-inactivated FCS. pRC-CMV expression vectorcontaining the 1.6-kb cDNA fragment encoding human TRPM-2 was kindlyprovided by Dr. Martin Tenniswood (W. Alton Jones Cell Science Center,Lake Placid, NY). The expression vector was transfected into LNCaP cells bythe liposome-mediated gene transfer method (22). Briefly, 23 105 LNCaPcells were plated in 6-cm plates. The next day, 5mg of purified TRPM-2cloned pRC-CMV or pRC-CMV alone (as a control) was added to LNCaPcells after a preincubation for 30 min with 5mg of lipofectamine reagent and3 ml of serum-free OPTI-MEM (Life Technologies, Inc.). Drug selection in300 mg/ml of Geneticin (Sigma Chemical Co., St. Louis, MO) was begun 3days after the transfection. Colonies were harvested 2 weeks after drugselection using cloning cylinders, and cell lines were expanded forin vivoinjection.

Assessment ofin Vivo LNCaP Tumor Growth and Determination ofSerum PSA Levels.One million cells of each of the LNCaP sublines wereinoculated s.c. with 0.1 ml of Matrigel (Becton Dickinson Labware, LincolnPark, NJ) in the flank region of 6- to 8-week-old male athymic nude mice(BALB/c strain; Charles River Laboratory, Montreal, Canada). Each experi-mental group consisted of six mice. Mice were castrated via a scrotal approachwhen tumors reached 100 and 200 mm3 in volume. Tumor volume wasmeasured once weekly and calculated as described above. Blood samples wereobtained with tail vein incisions of mice once weekly. Serum PSA levels weredetermined by an enzymatic immunoassay kit with a lower limit of sensitivityof 0.2 mg/liter (Abbott IMX, Montreal, Canada) according to the manufac-ture’s protocol. Data points were reported as mean values6 SD.

Antisense TRPM-2 ODNs.Phosphorothioate ODNs used in this studywere obtained from Nucleic Acid-Protein Service Unit, University of BritishColumbia (Vancouver, Canada). The sequences of antisense TRPM-2 ODNscorresponding to the mouseTRPM-2translation initiation site were 59-GCA-CAGCAGGAGAATCTTCAT-39. Two base TRPM-2 mismatch ODNs (59-GCACAGCAGGAGGATATTCAT-39) were used as the control.

Treatment of Cells with ODNs. Lipofectin, a cationic lipid (Life Tech-nologies, Inc.) was used to increase the ODN uptake of cells. Shionogi cellswere treated with various concentrations of ODNs after a preincubation for 20min with 4 mg/ml lipofectin in serum-free OPTI-MEM (Life Technologies,Inc.). After 4 h, the medium containing ODNs and lipofectin was replaced withthe standard culture medium described above.

Northern Blot Analysis. Total RNA was isolated from Shionogi tumortissues and cultured Shionogi tumor cells by the acid-guanidium thiocyanate-phenol-chloroform method. Poly(A)1 mRNA was then purified from totalRNA using oligodeoxythymidylate cellulose. The electrophoresis, hybridiza-tion, and washing conditions were carried out as previously reported (17).Mouse TRPM-2 and G3PDH cDNA probes were generated by reverse tran-scription-PCR from total RNA of mouse brain using primers 59-AAT-GAGCTCCAAGAACTGTCCACT- 39(sense) and 59-AAA-GAGCGTGTC-TATGATGCCAGAT-39 (antisense) for TRPM-2, and 59-ATGGTGAAG-GTCGGTGTGAACGGAT-39 (sense) and 59-AAAGTTGTCATGGAT-GACCTT-39 (antisense) for G3PDH. The density of bands for TRPM-2 wasnormalized against that of G3PDH by densitometric analysis.

Western Blot Analysis. Samples containing equal amounts of protein (15mg) from lysates of the cultured Shionogi cells and Shionogi tumors wereelectrophoresed on an SDS-polyacrylamide gel and transferred to a nitrocel-lulose filter. The filters were blocked in PBS containing 5% nonfat milkpowder at 4°C overnight and then incubated for 1 h with a 1:400-diluted C-18,an antimouse TRPM-2 goat polyclonal antibody (Santa Cruz BiotechnologyInc., Santa Cruz, CA), 1:10000-diluted MAB065, an antiratb-tubulin mousemonoclonal antibody (CHEMICON INTERNATIONAL INC., Tumecula, Cal-ifornia), or 1:600-diluted C2–10, an antihuman PARP mouse monoclonalantibody that reacts with mouse PARP (PHARMINGEN, Mississauga, Cana-da). The filters were then incubated for 30 min with horseradish peroxidase-conjugated antigoat or antimouse IgG antibody (Amersham Life Science,

Arlington Heights, IL), and specific proteins were detected using an enhancedchemiluminescence Western blotting analysis system (Amersham Life Sci-ence).

In Vivo ODN Treatment. Male DD/S mice bearing the Shionogi tumorwere castrated and randomly selected for treatment with antisense TRPM-2versusmismatch control ODNs. Each experimental group consisted of sevenmice. Beginning the day of castration, each mouse received injections i.p. of12.5 mg/kg of antisense TRPM-2 or mismatch control once daily for 15 days.The tumor volume was measured as described above. Data points werereported as mean tumor volumes6 SD.

RESULTS

TRPM-2 mRNA Expression Increases after Castration in theShionogi Tumor Model. Northern blot analyses were used to char-acterize changes in TRPM-2 mRNA expression in AD intact tumorsbefore castration, regressing tumors 4 and 7 days after castration, andAI recurrent tumors 28 days after castration. As shown in Fig. 1,compared to AD intact tumors before castration, TRPM-2 mRNAexpression is up-regulated;14-fold and 10-fold 4 and 7 days aftercastration, respectively and is maintained at 8-fold higher levels in AItumors. The pattern of TRPM-2 up-regulation in the Shionogi tumormodel after castration is similar to that in the rat ventral prostate (5)and human prostate cancer xenografts (9).

Inhibition of Castration-induced Apoptosis and TRPM-2 Up-regulation in Shionogi Tumors by CCBs. To determine whetherchanges of TRPM-2 expression after castration result from up-regu-lation of an androgen-repressed gene or are directly associated withapoptosis, mice bearing Shionogi tumors were treated beginning 1 daybefore castration with oral CCBs (nifedipine and norvasc) three timesper day for 14 days. After castration, Shionogi tumors in mice treatedwith CCBs continue to grow with a doubling time of 8 days, comparedto complete regression of Shionogi tumors in control mice (data notshown). As reported in our recent study (23), Shionogi tumor regres-sion after castration was associated with a 10-fold increase in thenumber of apoptotic bodies detected by TUNEL staining, between 3and 5 days postcastration. By 4 days postcastration, numerous apop-totic bodies (;20/high power field) in untreated control tumors wereobserved, but not in tumors treated with CCBs (Fig. 2,A and B).Furthermore, TRPM-2 up-regulation in Shionogi tumors after castra-tion was significantly inhibited by CCB treatment. TRPM-2 mRNAlevels in tumors treated with CCBs were 90%, 86%, and 77% lowerthan controls at 4, 7, and 10 days after castration, respectively (Fig. 2,C andD).

Overexpression of TRPM-2 Accelerates Time to AI Progres-sion in the LNCaP Tumor Model. To determine the effects ofTRPM-2 overexpression on time to AI progression after androgenablation, LNCaP cells were transfected withTRPM-2 cDNA ex-

Fig. 1. Changes in TRPM-2 mRNA expression in the Shionogi tumor model. Poly(A)1

RNA was extracted from Shionogi tumors harvested before and at several time points aftercastration, and it was analyzed for TRPM-2 and G3PDH levels by Northern blotting.Lane1, AD tumor before castration;Lane 2, regressing tumor 4 days after castration;Lane 3,regressing tumor 7 days after castration;Lane 4, AI recurrent tumor 28 days aftercastration.

171

TRPM-2 IS INVOLVED IN PROGRESSION TO ANDROGEN INDEPENDENCE

Research. on January 22, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 3: Testosterone-repressed Prostate Message-2 Is an Antiapoptotic …cancerres.aacrjournals.org/content/canres/60/1/170.full.pdf · [CANCER RESEARCH 60, 170–176, January 1, 2000] Testosterone-repressed

pression vector pRC-CMV/TRPM-2 or the pRC-CMV vector aloneas a control. As shown in Fig. 3A, abundant levels of TRPM-2mRNA were detected in four independent TRPM-2-transfectedclones (LNCaP/T1 to LNCaP/T4), whereas the parental LNCaP(LNCaP/P) and the control vector-transfected cell line (LNCaP/C)did not express detectable TRPM-2 mRNA levels. LNCaP/P, LN-CaP/C, LNCaP/T1, and LNCaP/T2 were selected forin vivo study,with 1 3 106 of each cell line inoculated s.c. in intact male nudemice. After castration, LNCaP/P and LNCaP/C tumor growth wasinhibited for 4 weeks, after which LNCaP/P and LNCaP/C tumorvolumes increased 2.5- and 2.3-fold, respectively by 9 weeks aftercastration. In contrast, LNCaP/T1 and LNCaP/T2 tumor volumescontinued to grow after castration, increasing 4.5- and 4.7-fold,respectively by 9 weeks after castration (Fig. 3B). Serum PSA inmice bearing LNCaP/P and LNCaP/C tumors decreased by 64%and 71%, respectively by 1 week after castration and increasedfrom 4 to 9 weeks after castration by 2.1- and 2.3-fold, respec-tively. In comparison, serum PSA in mice bearing LNCaP/T1 andLNCaP/T2 tumors decreased by 40% and 54%, respectively beforeincreasing to 3.9- and 4.1-fold, respectively by 9 weeks (Fig. 3C)beginning 2 weeks after castration.

Antisense ODN-mediated Inhibition of TRPM-2 Expression inShionogi Tumor Cells. Antisense TRPM-2 ODNs corresponding tothe mouseTRPM-2 translation initiation site and mismatch controlODNs containing two base changes were used in this study. The effectof treatment with antisense TRPM-2 ODNs on TRPM-2 mRNAexpression in Shionogi tumor cells was evaluated by Northern blotanalysis. As shown in Fig. 4,A and B, daily treatment of Shionogitumor cells with antisense TRPM-2 ODNs (50, 100, 500 or 1000 nM)for 2 days reduced TRPM-2 mRNA levels by 2, 15, 81, or 98%,respectively. In contrast, TRPM-2 mRNA levels were unchanged bythe two-base mismatch control ODNs at the used concentrations.

To determine whether decreases in TRPM-2 mRNA levels inducedby antisense ODNs were accompanied by corresponding decreases inprotein levels, Western blot analysis was used to measure changes inTRPM-2 protein levels in Shionogi tumor cells after daily treatmentwith antisense TRPM-2 ODNs for 4 consecutive days. Substantialinhibition of both unprocessed (60-kDa) and mature (40-kDa) formsof TRPM-2 protein were observed with 1000-nM antisense TRPM-2ODNs, but not with mismatch control ODN treatment (Fig. 4C).

Inhibition of AI Progression in the Shionogi Tumor Model byAntisense TRPM-2 ODN Treatment. Male mice bearing Shionogitumors 1–2 cm in diameter were castrated and randomly selected for

Fig. 2. Effect of CCB treatment on apoptosis and TRPM-2 expression in the Shionogi tumor model.a andb, 4 days after castration, Shionogi tumors were harvested for TUNELstaining from CCB-treated (a) and untreated control (b) mice to identify apoptotic cells.c andd, 4, 7, and 10 days after castration, Shionogi tumors were harvested for Northern blotanalysis from CCB-treated and untreated control mice, poly(A)1 RNA was extracted from tumor tissues, and TRPM-2 and G3PDH levels were analyzed.Lane 1, AD tumor beforecastration;Lanes 2, 3,and4, untreated tumors 4, 7, and 10 days after castration, respectively;Lanes 5, 6,and7, CCB-treated tumors 4, 7, and 10 days after castration, respectively;Lane 8,AI tumor 28 days after castration (c). Quantitative analysis of TRPM-2 mRNA levels after normalization to G3PDH mRNA levels in Shionogi tumors after CCB treatmentwas performed by using the laser densitometer. Each column represents the mean value with SD (d).

172

TRPM-2 IS INVOLVED IN PROGRESSION TO ANDROGEN INDEPENDENCE

Research. on January 22, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 4: Testosterone-repressed Prostate Message-2 Is an Antiapoptotic …cancerres.aacrjournals.org/content/canres/60/1/170.full.pdf · [CANCER RESEARCH 60, 170–176, January 1, 2000] Testosterone-repressed

treatment with antisense TRPM-2versusmismatch control ODNs.Mean pretreatment tumor volume was similar in both groups. Begin-ning the day of castration, 12.5 mg/kg of ODNs were administeredonce daily by i.p. injection for 15 days. As shown in Fig. 5, Shionogi

tumors regressed faster, and complete regression occurred earlier inmice treated with the antisense TRPM-2 ODN compared to thosetreated with mismatch control ODNs. Furthermore, antisenseTRPM-2 ODN treatment significantly delayed the recurrence of AItumors compared to mismatch control ODN treatment. After an ob-servation period of 50 days postcastration, the mean tumor volume inthe mismatch-treated control group was six times that of the antisense-treated group. AI tumors recurred in five of seven mice after a medianof 46 days in the antisense TRPM-2 ODN treatment group, whereasAI tumors recurred in all mice after a median of 35 days in themismatch ODN treatment group. No side effects, including weightloss, gait disturbances, or anorexia were observed with antisenseTRPM-2 or mismatch ODN treatment over the 50-day observationperiod.

Fig. 3. Effect of TRPM-2 overexpression on LNCaP tumor growth and serum PSA innude mice after castration.a, total RNA was extracted from PC3 (positive control for thescreening of TRPM-2 mRNA expression), LNCaP/P, LNCaP/C, and four clones ofTRPM-2 transfectants (LNCaP/T1 to LNCaP/T4) and analyzed for TRPM-2 and G3PDHlevels by Northern blotting.b, male nude mice received injections s.c. of each LNCaPsubline, and after castration, tumor volume was measured once weekly. Each pointrepresents the mean tumor volume in each experimental group containing six mice withSD.c, blood samples for the measurement of serum PSA levels were obtained with the tailvein of the mice after castration once weekly. Serum PSA levels were determined by anenzymatic immunoassay kit according to the manufacturer’s control (Abbott IMX, Mon-treal, Canada). Each point represents the mean PSA level in each experimental groupcontaining six mice with SD.

Fig. 4. Sequence-specific inhibition of TRPM-2 expression by the antisense TRPM-2ODN in Shionogi tumor cells.a, Shionogi tumor cells were treated daily with variousconcentrations of the antisense TRPM-2 ODN or a two-base TRPM-2 mismatch ODN asa control for 2 days. poly(A)1 RNA was extracted from culture cells, and TRPM-2 andG3PDH levels were analyzed by Northern blotting.No Tx,untreated cells.b, quantitativeanalysis of TRPM-2 mRNA levels after normalization to G3PDH mRNA levels inShionogi tumor cells after treatment with various concentrations of antisense TRPM-2 ormismatch control ODNs was performed by using laser densitometer. Each point representsthe mean of triplicate analyses with SD.c, Shionogi tumor cells were treated with variousconcentrations of antisense TRPM-2 or mismatch control ODN once daily for 4 days,protein was extracted from culture cells, and TRPM-2 andb-tubulin protein levels wereanalyzed by Western blotting. Unprocessed form of TRPM-2, size5 60 kDa; mature formof TRPM-2, size5 40 kDa.No Tx, untreated cells.

173

TRPM-2 IS INVOLVED IN PROGRESSION TO ANDROGEN INDEPENDENCE

Research. on January 22, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 5: Testosterone-repressed Prostate Message-2 Is an Antiapoptotic …cancerres.aacrjournals.org/content/canres/60/1/170.full.pdf · [CANCER RESEARCH 60, 170–176, January 1, 2000] Testosterone-repressed

We then examined the effects ofin vivo ODN treatment onTRPM-2 mRNA expression in Shionogi tumors by Northern blotting.In this experiment, beginning the day of castration, each of threetumor-bearing mice were administered 12.5 mg/kg of antisenseTRPM-2 or mismatch ODNs i.p. once daily, and tumor tissues wereharvested for RNA extraction 3 days after castration. AntisenseTRPM-2 ODN treatment resulted in a 70% reduction in TRPM-2mRNA levels in Shionogi tumors compared to mismatch controlODN-treated tumors (Fig. 6).

To determine whether more rapid regression of antisense ODN-

treated tumors resulted from an earlier onset of castration-inducedapoptosis, Western blotting of tumor tissues was used to evaluate thecleavage of the PARP protein, a substrate of the caspase activatedduring the final process of apoptotic execution (24). Proteins wereextracted 3 days postcastration from each of three Shionogi tumors inmice administered antisense TRPM-2 or mismatch control ODNsunder the same treatment schedule described above. The 116-kDaintact form of PARP was observed in both antisense TRPM-2 ODN-treated and mismatch control ODN-treated Shionogi tumors, whereasthe 85-kDa PARP cleavage fragment was clearly detectable only inantisense TRPM-2 ODN-treated Shionogi tumors (Fig. 7).

Changes in TRPM-2 mRNA levels in various normal mouse organsafter antisense ODN treatment were also evaluated. The Shionogitumors, spleen, kidney, brain, and prostate were harvested 3 dayspostcastration for RNA extraction from mice administered antisenseTRPM-2 or mismatch control ODNs under the same treatment sched-ule described above. Antisense TRPM-2 had no effect on TRPM-2expression levels in the spleen, kidney, and brain, whereas TRPM-2mRNA levels in the AD Shionogi tumor and mouse prostate tissueswere reduced after antisense TRPM-2 ODN treatment compared tomismatch control ODN treatment (Fig. 8).

DISCUSSION

TRPM-2was initially considered an androgen-repressed gene as-sociated with the regressing rat prostate gland after castration andregarded as a marker and putative mediator of apoptosis (4, 5). Inmore recent studies, however, TRPM-2 up-regulation was dissociatedfrom apoptosis (10–12, 14, 15, 25). For example, TRPM-2 expressionwas not associated with increased apoptotic activity in sex-hormone-

Fig. 5. Effect of antisense TRPM-2 ODN administration on Shionogi tumor growth.Beginning on the day of castration, 12.5 mg/kg of antisense TRPM-2 or mismatch controlODNs were injected i.p. once daily for 15 days, and tumor volume was measured twiceweekly. Each point represents the mean tumor volume in each experimental groupcontaining seven mice with SD.

Fig. 6. Effect of antisense TRPM-2 ODN administration on TRPM-2 mRNA levels inShionogi tumors by day 3 postcastration.a, beginning on the day of castration, each ofthree Shionogi tumor-bearing mice were treated daily with antisense TRPM-2 or mis-match control ODNs at a dose of 12.5 mg/kg, poly(A)1 RNA was extracted from Shionogitumors 3 days postcastration, and TRPM-2 and G3PDH mRNA levels were analyzed byNorthern blotting.Lanes 1, 2,and 3, Shionogi tumors in mice administered antisenseTRPM-2 ODNs;Lanes 4, 5,and 6, Shionogi tumors in mice administered mismatchcontrol ODNs.b, quantitative analysis of TRPM-2 mRNA levels after normalization toG3PDH mRNA levels in Shionogi tumors after treatment with antisense TRPM-2 ormismatch control ODNs performed by using the laser densitometer. Each column repre-sents the mean value with SD.

Fig. 7. Effect of antisense TRPM-2 ODN administration on the cleavage of PARP inShionogi tumors by day 3 postcastration. Beginning the day of castration, each of threeShionogi tumor-bearing mice were treated daily with antisense TRPM-2 or mismatchcontrol ODNs at a dose of 12.5 mg/kg; proteins extracted from Shionogi tumors 3 dayspostcastration were analyzed by Western blotting with a PARP-specific antibody. Un-cleaved intact PARP, size5 116 kDa; cleaved PARP, size5 85 kDa.Lanes 1, 2,and3,Shionogi tumors in mice administered antisense TRPM-2 ODN;Lanes 4, 5,and 6,Shionogi tumors in mice administered mismatch control ODN.

Fig. 8. Effect of antisense TRPM-2 ODN administration on TRPM-2 mRNA levels innormal organs. Beginning the day of castration, Shionogi tumor-bearing mice were treateddaily with antisense TRPM-2 or mismatch control ODN at a dose of 12.5 mg/kg.Poly(A)1 RNA was extracted from the Shionogi tumor, spleen, kidney, brain, and prostate3 days postcastration, and TRPM-2 and G3PDH mRNA levels were analyzed by Northernblotting.

174

TRPM-2 IS INVOLVED IN PROGRESSION TO ANDROGEN INDEPENDENCE

Research. on January 22, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 6: Testosterone-repressed Prostate Message-2 Is an Antiapoptotic …cancerres.aacrjournals.org/content/canres/60/1/170.full.pdf · [CANCER RESEARCH 60, 170–176, January 1, 2000] Testosterone-repressed

induced prostatic dysplasia in the Noble rat (10). Similarly, TRPM-2expression was enhanced in the absence of increased apoptotic activ-ity and reduced androgen stimulation in rat prostate cancer and ventralprostate tissues (25). The overexpression of TRPM-2 in LNCaPprostate cancer cells enhances the resistance to apoptosis induced bytumor necrosis factora (14). Collectively, these conflicting data donot clearly define the functional role of TRPM-2 associated withapoptosis induced by various types of stimuli.

TRPM-2 mRNA is highly up-regulated in Shionogi tumors aftercastration and in AI recurrent tumors. These changes in TRPM-2 aresimilar to previously reported findings in the rat prostate (5) and thehuman prostate cancer xenograft (9). To differentiate between andro-gen-repressedversusapoptosis-associated up-regulation, CCBs wereadministered before castration to prevent castration-induced apoptosisbecause an early event in apoptotic cascade involves increases in theintracellular-free calcium concentration, with subsequent endonucle-ase activation (26, 27). Castration-induced apoptosis and up-regula-tion of TRPM-2 was prevented by CCB treatment, thereby supportingTRPM-2 as an apoptosis-related rather than androgen-repressed gene.

To determine whether TRPM-2 up-regulation after androgen abla-tion helps mediate the progression to androgen independence, wegenerated TRPM-2 overexpressing LNCaP prostate cancer cell lines.Although tumor take rates and growth in intact male mice weresimilar between parental and overexpressing TRPM-2 tumors, aftercastration, tumor growth and serum PSA increased severalfold fasterin TRPM-2-tranfected tumors. These findings provide the first clearevidence that increased TRPM-2 results in the acquisition of androgenresistance and accelerates time to AI progression.

The up-regulation of TRPM-2 in Shionogi tumors after castrationand the acquisition of androgen resistance with TRPM-2 overexpres-sion suggests that preventing TRPM-2 up-regulation precipitated byandrogen ablation may enhance castration-induced apoptosis and de-lay the AI progression of prostate cancer. Antisense ODN, a chemi-cally modified stretch of single-stranded DNA that is complementaryto mRNA regions of a target gene and thereby effectively inhibitsgene expression by forming RNA/DNA duplexes (28), offers onestrategy to specifically targetTRPM-2gene expression. Phosphoro-thioate ODNs are water soluble, stable agents manufactured to resistnuclease digestion. After parenteral administration, phosphorothioateODNs become associated with high capacity, low affinity serumbinding proteins (29). In this study, the phosphorothioate antisenseTRPM-2 ODN corresponding to the mouseTRPM-2 translation ini-tiation site reduced TRPM-2 expression levels in a sequence-specificand dose-dependent manner.In vivo administration of antisenseTRPM-2 ODN accelerated castration-induced tumor regression anddelayed the time to AI progression compared to that of mismatchcontrol ODNs. Consistent with thein vitro data,in vivo treatment withthe antisense TRPM-2 ODN also reduced TRPM-2 mRNA levels. Theearlier detection of PARP cleavage fragments in Shionogi tumorsafter antisense TRPM-2 ODN treatment suggests that the inhibition ofTRPM-2 up-regulation after castration results in an earlier inductionof castration-induced apoptosis.

Because TRPM-2 plays a critical role in some normal organs,including the brain, kidney, spleen, and prostate (4, 30, 31), the effectsof antisense TRPM-2 ODNs on TRPM-2 expression levels in theseorgans were also examined. Although antisense ODNs significantlyreduced TRPM-2 levels in the AD prostate and tumor tissues under-going castration-induced apoptosis, TRPM-2 expression was not al-tered by antisense ODNs in the other organs examined. We reportedsimilar differential decreases in Bcl-2 levels after treatment withmouse antisense Bcl-2 ODNs in Shionogi tumors (32). We speculatethat tissues undergoing apoptosis may be more sensitive to antisenseTRPM-2 ODN treatment relative to intact organs because of the

preferential uptake of ODNs in these tissues for reasons of biodistri-bution or increased membrane permeability. However, reduced targetmRNA levels in mouse tissues have been reported by Moniaet al.(33) after i.v. administration of antisense C-raf ODNs, although nosignificant toxicity was observed. A phase I dose-escalation trial usingantisense Bcl-2 ODNs in nine patients with lymphoma reported tumorresponses with no significant toxicity (34). These findings suggest thattherapeutic doses of systemic phosphorothioate antisense ODNs tar-geted against important cellular proteins do not cause significanttoxicity in normal tissues.

Integration and appropriate timing of combination therapies, basedon biological mechanism of progression and castration-inducedchanges in gene expression, may provide means to delay AI progres-sion in a major way (32, 35). New nonhormonal therapies for prostatecancer have traditionally been evaluated in patients with hormonerefractory disease, and when used in this end-stage setting, none hasdemonstrated improved survival (2). A more rational strategy to delayemergence of the AI phenotype would initiate treatment earlier toenhance castration-induced apoptosis by targeting the adaptivechanges in gene expression precipitated by androgen withdrawal. Thepresent study provides evidence for a functional role of TRPM-2 inandrogen resistance and AI progression, and it demonstrates thatreduction ofTRPM-2gene expression by antisense TRPM-2 ODNscan delay progression to androgen independence.

ACKNOWLEDGMENTS

We thank Mary Bowden and Virginia Yago for their excellent technicalassistance.

REFERENCES

1. Denis, L., and Murphy, G. P. Overview of phase III trials on combined androgentreatment in patients with metastatic prostate cancer. Cancer (Phila.),72: 3888–3895,1993.

2. Oh, W. K., and Kantoff, P. W. Management of hormone refractory prostate cancer:current standards and future prospects. J. Urol.,160: 1220–1229, 1998.

3. Blaschuk, O., Burdzy, K., and Fritz, I. B. Purification and characterization of acell-aggregating factor (clusterin), the major glycoprotein in ram rete testis fluid.J. Biol. Chem.,258: 7714–7720, 1983.

4. Rosenberg, M. E., and Silkensen, J. Clusterin: physiologic and pathophysiologicconsiderations. Int. J. Biochem. Cell. Biol.,27: 633–645, 1995.

5. Sensibar, J. A., Griswold, M. D., Sylvester, S. R., Buttyan, R., Bardin, C. W., Cheng,C. Y., Dudek, S., and Lee, C. Prostatic ductal system in rats: regional variation inlocalization of an androgen-repressed gene product, sulfated glycoprotein-2. Endo-crinology,128: 2091–2102, 1991.

6. Connor, J., Buttyan, R., Olsson, C. A., D’Agati, V., O’Toole, K., and Sawczuk, I. S.SGP-2 expression as a genetic marker of progressive cellular pathology in experi-mental hydronephrosis. Kidney Int.,39: 1098–1103, 1991.

7. Kyprianou, N., English, H. F., Davidson, N. E., and Isaacs, J. T. Programmed celldeath during regression of the MCF-7 human breast cancer following estrogenablation. Cancer Res.,51: 162–166, 1991.

8. Wright, P. S., Cross-Doersen, D., Th’ng, J. P., Guo, X. W., Crissman, H. A.,Bradbury, E. M., Montgomery, L. R., Thompson, F. Y., Loudy, D. E., Johnston, J. O.,and Bitonti, A. J. A ribonucleotide reductase inhibitor, MDL 101,731, inducesapoptosis and elevates TRPM-2 mRNA levels in human prostate tumor xenografts.Exp. Cell Res.,222: 54–60, 1996.

9. Kyprianou, N., English, H. F., and Isaacs, J. T. Programmed cell death duringregression of PC-82 human prostate cancer following androgen ablation. Cancer Res.,50: 3748–3753, 1990.

10. Ho, S. M., Leav, I., Ghatak, S., Merk, F., Jagannathan, V. S., and Mallery, K. Lackof association between enhanced TRPM-2/clusterin expression and increased apop-totic activity in sex-hormone-induced prostatic dysplasia of the Noble rat. Am. J.Pathol.,153: 131–139, 1998.

11. Schwochau, G. B., Nath, K. A., and Rosenberg, M. E. Clusterin protects againstoxidative stressin vitro through aggregative and nonaggregative properties. KidneyInt., 53: 1647–1653, 1998.

12. French, L. E., Sappino, A. P., Tschopp, J., and Schifferli, J. A. Distinct sites ofproduction and deposition of the putative cell death marker clusterin in the humanthymus. J. Clin. Invest.,90: 1919–1925, 1992.

13. Montpetit, M. L., Lawless, K. R., and Tenniswood, M. Androgen-repressed messagesin the rat ventral prostate. Prostate,8: 25–36, 1986.

14. Sensibar, J. A., Sutkowski, D. M., Raffo, A., Buttyan, R., Griswold, M. D., Sylvester,S. R., Kozlowski, J. M., and Lee, C. Prevention of cell death induced by tumor

175

TRPM-2 IS INVOLVED IN PROGRESSION TO ANDROGEN INDEPENDENCE

Research. on January 22, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 7: Testosterone-repressed Prostate Message-2 Is an Antiapoptotic …cancerres.aacrjournals.org/content/canres/60/1/170.full.pdf · [CANCER RESEARCH 60, 170–176, January 1, 2000] Testosterone-repressed

necrosis factor a in LNCaP cells by overexpression of sulfated glycoprotein-2(clusterin). Cancer Res.,55: 2431–2437, 1995.

15. Steinberg, J., Oyasu, R., Lang, S., Sintich, S., Rademaker, A., Lee, C., Kozlowski,J. M., and Sensibar, J. A. Intracellular levels of SGP-2 (clusterin) correlate with tumorgrade in prostate cancer. Clin. Cancer Res.,3: 1701–1711, 1997.

16. Bruchovsky, N., Rennie, P. S., Coldman, A. J., Goldenberg, S. L., To, M., andLawson, D. Effects of androgen withdrawal on the stem cell composition of theShionogi carcinoma. Cancer Res.,50: 2275–2282, 1990.

17. Gleave, M. E., Hsieh, J. T., Wu, H. C., von Eschenbach, A. C., and Chung, L. W.Serum prostate specific antigen levels in mice bearing human prostate LNCaP tumorare determined by tumor volume and endocrine and growth factors. Cancer Res.,52:598–1605, 1992.

18. Rennie, P. S., Bruchovsky, N., Buttyan, R., Benson, M., and Cheng, H. Geneexpression during the early phases of regression of the androgen-dependent Shionogimouse mammary carcinoma. Cancer Res.,48: 6309–6312, 1988.

19. Bruchovsky, N., and Rennie, P. S. Classification of dependent and autonomousvariants of Shionogi mammary carcinoma based on heterogeneous patterns of andro-gen binding. Cell,13: 272–280, 1978.

20. Gleave, M., Hsieh, J. T., Gao, C., von Eschenbach, A. C., and Chung, L. W.Acceleration of human prostate carcinoma growthin vivo by factors produced byprostate and bone fibroblasts. Cancer Res.,51: 3753–3761, 1991.

21. Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. Identification of programmed celldeath in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol.,119:493–501, 1992.

22. Miyake, H., Hanada, N., Nakamura, H., Kagawa, S., Fujiwara, T., Hara, I., Eto, H.,Gohji, K., Arakawa, S., Kamidono, S., and Saya, H. Overexpression of Bcl-2 inbladder cancer cells inhibits apoptosis induced by cisplatin and adenoviral- mediatedp53 gene transfer. Oncogene,16: 933–943, 1998.

23. Nickerson, T., Miyake, H., Gleave, M. E., and Pollak, M. Castration-induced apo-ptosis of androgen-dependent Shionogi carcinoma is associated with increased ex-pression of genes encoding insulin-like growth factor-binding proteins. Cancer Res.,59: 3392–3395, 1999.

24. Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W. C.Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE.Nature (Lond.),371: 346–347, 1994.

25. Brandstrom, A., Westin, P., Bergh, A., Cajander, S., and Damber, J. E. Castrationinduces apoptosis in the ventral prostate but not in an androgen-sensitive prostaticadenocarcinoma in the rat. Cancer Res.,54: 3594–3601, 1994.

26. He, H., Lam, M., McCormick, T. S., and Distelhorst, C. W. Maintenance of calciumhomeostasis in the endoplasmic reticulum by Bcl-2. J. Cell Biol.,138:1219–1228, 1997.

27. Marin, M. C., Fernandez, A., Bick, R. J., Brisbay, S., Buja, L. M., Snuggs, M.,McConkey, D. J., von Eschenbach, A. C., Keating, M. J., and McDonnell, T. J.Apoptosis suppression by bcl-2 is correlated with the regulation of nuclear andcytosolic Ca21. Oncogene,12: 2259–2266, 1996.

28. Crooke, S. T. Therapeutic applications of oligonucleotides. Annu. Rev. Pharmacol.Toxicol., 32: 329–376, 1992.

29. Saijo, Y., Perlaky, L., Wang, H., and Busch, H. Pharmacokinetics, tissue distribution,and stability of antisense oligodeoxynucleotide phosphorothioate ISIS 3466 in mice.Oncol. Res.,6: 243–249, 1994.

30. Hammad, S. M., Ranganathan, S., Loukinova, E., Twal, W. O., and Argraves,W. S. Interaction of apolipoprotein J-amyloidb-peptide complex with lowdensity lipoprotein receptor-related protein-2/megalin. A mechanism to preventpathological accumulation of amyloidb-peptide. J. Biol. Chem.,272: 18644 –18649, 1997.

31. Michel, D., Moyse, E., Trembleau, A., Jourdan, F., and Brun, G. Clusterin/ApoJexpression is associated with neuronal apoptosis in the olfactory mucosa of the adultmouse. J. Cell Sci.,110: 1635–1645, 1997.

32. Miyake, H., Tolcher, A., and Gleave, M. E. Antisense Bcl-2 oligodeoxynucleotidesinhibit progression to androgen-independence after castration in the Shionogi tumormodel. Cancer Res.,59: 4030–4034, 1999.

33. Monia, B. P., Johnston, J. F., Geiger, T., Muller, M., and Fabbro, D. Antitumoractivity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-rafkinase. Nat. Med.,2: 668–675, 1996.

34. Webb, A., Cunningham, D., Cotter, F., Clarke, P. A., di Stefano, F., Ross, P., Corbo,M., and Dziewanowska, Z. BCL-2 antisense therapy in patients with non-Hodgkinlymphoma. Lancet,349: 1137–1141, 1997.

35. Gleave, M., Tolcher, A., Miyake, H., Nelson, C., Brown, B., Beraldi, E., and Goldi,L. Progression to androgen independence is delayed by adjuvant treatment withantisense Bcl-2 oligodeoxynucleotides after castration in the LNCaP prostate tumormodel. Clin. Cancer Res.,5: 2891–2898, 1999.

176

TRPM-2 IS INVOLVED IN PROGRESSION TO ANDROGEN INDEPENDENCE

Research. on January 22, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 8: Testosterone-repressed Prostate Message-2 Is an Antiapoptotic …cancerres.aacrjournals.org/content/canres/60/1/170.full.pdf · [CANCER RESEARCH 60, 170–176, January 1, 2000] Testosterone-repressed

2000;60:170-176. Cancer Res   Hideaki Miyake, Colleen Nelson, Paul S. Rennie, et al.   Independence in Prostate CancerAntiapoptotic Gene Involved in Progression to Androgen Testosterone-repressed Prostate Message-2 Is an

  Updated version

  http://cancerres.aacrjournals.org/content/60/1/170

Access the most recent version of this article at:

   

   

  Cited articles

  http://cancerres.aacrjournals.org/content/60/1/170.full#ref-list-1

This article cites 34 articles, 15 of which you can access for free at:

  Citing articles

  http://cancerres.aacrjournals.org/content/60/1/170.full#related-urls

This article has been cited by 38 HighWire-hosted articles. Access the articles at:

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  [email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/60/1/170To request permission to re-use all or part of this article, use this link

Research. on January 22, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from