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Tumor Biology |
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 [H. M., C. N., M. E. G.], Canada
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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80% of patients. However, progression to
androgen independence ultimately occurs in nearly all of these cases
(1)
. Several hundred clinical studies using traditional
cytotoxic chemotherapeutic agents document objective response rates of
<10% and no improved survival rates (2)
. Accordingly,
progression to androgen independence remains the main obstacle
to improving the survival and quality of life in patients with advanced
disease, emphasizing the need for novel therapeutic strategies that
target the molecular mechanism of the androgen- and chemoresistant
phenotype of prostate cancer. TRPM-2,3 also known as clusterin, sulfated glycoprotein-2, or apolipoprotein J, was first isolated from ram rete testes fluid (3) and has been proposed to have various biological functions, including tissue remodeling, reproduction, lipid transport, and apoptotic cell death (4) . TRPM-2 was initially regarded as a marker for cell death, because its expression is highly up-regulated in various normal and malignant tissues undergoing apoptosis (5, 6, 7, 8) . Recent studies, however, report conflicting findings on the association between enhanced TRPM-2 expression and apoptotic activity (9, 10, 11) . Similarly, TRPM-2 expression is increased in regressing normal prostate after androgen ablation (5 , 12) , and its up-regulation has been shown to be associated with antiapoptotic activity and disease progression in prostate cancer (13, 14, 15) . We have recently reported that TRPM-2 expression in prostate cancer cells has a protective role against castration-induced apoptosis (16) . However, the functional significance of TRPM-2 expression in apoptosis induced by chemotherapeutic agents has not been investigated.
Controlled study of the complex molecular processes associated with progression to androgen independence in prostate cancer has proved difficult, because few animal models exist that reproducibly mimic the clinical course of the disease in men. The AD Shionogi mouse mammary carcinoma model is particularly useful for testing the efficacy of agents targeting castration-induced apoptosis and their effects on time to progression of androgen independence. AD Shionogi tumors in intact male mice undergo complete regression after castration but recur as rapidly growing AI tumors after 1 month in a highly reproducible manner (17) . Of the available human prostate cancer cell lines, only the LNCaP tumors are AD when xenografted into male immunodeficient mice, 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 surrogate end point of progression to androgen independence (18) .
In the present study, we evaluated the effects of TRPM-2 overexpression on time to progression of androgen independence after castration and paclitaxel treatment in the LNCaP tumor model. We then evaluated the effects of paclitaxel treatment on TRPM-2 gene expression in Shionogi tumor cells and the effects of antisense TRPM-2 ODN on paclitaxel chemosensitivity using the Shionogi tumor model.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antisense TRPM-2 ODN.
Phosphorothioate ODN used in this study was obtained from Nucleic
Acid-Protein Service Unit, University of British Columbia. The
sequences of antisense TRPM-2 ODN corresponding to the mouse
TRPM-2 translation initiation site were
5'-GCACAGCAGGAGAATCTTCAT-3'. A 2-base TRPM-2 mismatch ODN
(5'-GCACAGCAGGAGGATATTCAT-3') was used as control.
LNCaP Sublines.
LNCaP cells were kindly provided by Dr. Leland Chung (University of
Virginia, Charlottesville, VA) and maintained in RPMI 1640 (Life
Technologies, Inc., Gaithersburg, MD) supplemented with 5%
heat-inactivated FCS. Steroid hormone-depleted charcoal-stripped media
were prepared as described previously (19)
. A pRC-CMV
expression vector containing the 1.6-kb cDNA fragment encoding human
TRPM-2 was kindly provided by Dr. Martin Tenniswood (W. Alton Jones
Cell Science Center, Lake Placid, NY). The expression vector was
transfected into LNCaP cells by the liposome-mediated gene transfer
method as described previously (20)
. Briefly, 2 x 105 LNCaP cells were plated in 6-cm
plates. The next day, 5 µg of purified TRPM-2-cloned pRC-CMV or
pRC-CMV alone (as a control) were added to LNCaP cells after a
preincubation for 30 min with 5 µg of LipofectAMINE reagent and 3 ml
of serum-free Opti-MEM (Life Technologies). Drug selection, in 300
µg/ml Geneticin (Sigma), was begun 3 days after the transfection.
Colonies were harvested 2 weeks after drug selection using cloning
cylinders and expanded to cell lines.
Assessment of in Vivo LNCaP Tumor Growth and
Determination of Serum PSA Levels.
One million cells of each LNCaP subline were inoculated s.c. with 0.1
ml of Matrigel (Becton Dickinson Labware, Lincoln Park, NJ) in the
flank region of 6- to 8-week-old male athymic nude mice (BALB/c strain;
Charles River Laboratory, Montreal, Quebec, Canada). Each experimental
group consisted of six mice. Mice were castrated via a scrotal approach
when tumors reached 200–300 mm3 in volume, and
from 10 to 14 days after castration, 0.5 mg of polymeric micellar
paclitaxel was administered once daily by i.v. injection. Tumor volume
was measured once weekly and calculated by the formula length x width x depth x 0.5236
(19)
. Blood samples were obtained with tail vein incisions
of mice once weekly. Serum PSA levels were determined by an enzymatic
immunoassay kit with a lower limit of sensitivity of 0.2 µg/liter
(Abbott IMX, Montreal, Quebec, Canada) according to the manufacturer’s
protocol. Data points were reported as mean values ± SD.
Shionogi Tumor Growth.
The Toronto subline of the transplantable SC-115 AD mouse mammary
carcinoma was used in all experiments (21)
. Shionogi tumor
cells were maintained in DMEM (Life Technologies) supplemented with 5%
heat-inactivated FCS. For in vivo study, 5 x 106 cells of the Shionogi carcinoma were
injected s.c. into adult male DD/S strain mice. 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
(22)
.
Treatment of Cells with ODN.
In vitro-cultured cells were treated with various
concentrations of ODN after a preincubation for 20 min with 4 µg/ml
Lipofectin (Life Technologies) in serum free Opti-MEM. Media containing
ODN and Lipofectin was replaced 4 h later with standard culture
medium described above.
Northern Blot Analysis.
Total RNA was isolated from in vitro-cultured cells and
in vivo tumor tissues by the acid-guanidium
thiocyanate-phenol-chloroform method. Poly(A)+
mRNA was then purified from total RNA using oligodeoxy-thymidylate
cellulose (Pharmacia Biotech Inc., Uppsala, Sweden). Five micrograms of
poly(A)+ mRNA from each sample were subjected to
electrophoresis on 1.2% agarose-formaldehyde gels and transferred to
nylon membranes (Amersham, Arlington Heights, IL) overnight according
to standard procedure (17)
. The RNA blots were hybridized
with a mouse TRPM-2 cDNA probe labeled with
[32P]dCTP by random primer labeling. After
stripping, the membranes were rehybridized with a mouse G3PDH cDNA
probe. These probes were generated by reverse transcription-PCR from
total RNA of mouse brain using primers 5'-AATGAGCTCCAAGAACTG-TCCACT-3'
(sense) and 5'-AAAGAGCGTGTCTATGATGCCAGAT-3' (antisense) for TRPM-2 and
5'-ATGGTGAAGGTCGGTGTGAACGGAT-3' (sense) and 5'-AAAGTTGTCATGGATGACCTT-3'
(antisense) for G3PDH. Density of bands for TRPM-2 was normalized
against that of G3PDH by densitometric analysis.
Western Blot Analysis.
The expression of TRPM-2 and PARP protein in cultured cells and
tumor tissues was determined by Western blot analysis as described
previously (20)
. Briefly, samples containing equal amounts
of protein (15 µg) were electrophoresed on a SDS-polyacrylamide gel
and transferred to a nitrocellulose filter. The filters were blocked in
PBS containing 5% nonfat milk powder at 4°C overnight and then
incubated for 1 h with an anti-human TRPM-2 goat polyclonal
antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-human
PARP mouse monoclonal antibody (PharMingen, Mississauga, Ontario,
Canada), or anti-rat ß-tubulin mouse monoclonal antibody (Chemicon
International Inc., Tumecula, CA). The filters were then incubated for
30 min with horseradish peroxidase-conjugated anti-goat or mouse IgG
antibody (Amersham), and specific proteins were detected using an
enhanced chemiluminescence system (Amersham).
In Vitro Cell Growth Assays.
The in vitro growth of LNCaP and Shionogi tumor cells
was assessed by the in vitro mitogenic assay and the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,
respectively, as described previously (19
, 20)
. Briefly,
3 x 103 cells were seeded in each
well of 96-well microtiter plates and allowed to attach overnight.
After treatment with various concentrations of paclitaxel and/or ODN,
LNCaP cells were fixed with 1% glutaraldehyde (Sigma) and stained with
0.5% crystal violet (Sigma), and Shionogi tumor cells were treated
with 20 µl of 5 mg/ml
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma) in
PBS, followed by incubation for 4 h at 37°C. The absorbance was
determined with a microculture plate reader (Becton Dickinson Labware)
at 540 nm. Absorbance values were normalized to the values obtained for
the vehicle-treated cells to determine the percent of survival. Each
assay was performed in triplicate.
DNA Fragmentation Analysis.
The nucleosomal DNA degradation was analyzed as described
previously with a minor modification (20)
. Briefly,
1 x 105 cultured cells were
seeded in 5-cm culture dishes and allowed to adhere overnight. After
the indicated treatment with paclitaxel and/or ODN, cells were
harvested and then lysed in a solution containing 100 mM
NaCl, 10 mM Tris (pH 7.4), 25 mM EDTA, and
0.5% SDS. After the centrifugation, the supernatants were incubated
with 300 µg/ml proteinase K for 5 h at 65°C and extracted with
phenol-chloroform. The aqueous layer was treated with 0.1 volume of 3 M
sodium acetate, and the DNA was precipitated with 2.5 volumes of 95%
ethanol. After treatment with 100 µg/ml RNase A for 1 h at
37°C, the sample was electrophoresed on a 2% agarose gel and stained
with ethidium bromide.
Assessment of in Vivo Shionogi Tumor Growth.
To determine whether combined antisense TRPM-2 ODN and paclitaxel
treatment delays time to AI recurrence after castration compared with
either agent alone, male DD/S mice bearing Shionogi tumors were
castrated and randomly selected for treatment with antisense TRPM-2 ODN
alone (group 1), mismatch control ODN alone (group 2), antisense TRPM-2
ODN plus paclitaxel (group 3), or mismatch control ODN plus paclitaxel
(group 4). Each experimental group consisted of seven mice. Beginning
the day of castration, 12.5 mg/kg antisense TRPM-2 or mismatch control
ODN was injected i.p. once daily into each mouse for 15 days. From 10
to 14 days after castration, 0.5 mg polymeric micellar paclitaxel was
administered once daily by i.v. injection in groups 3 and 4. A second
set of experiments was designed to evaluate the effects of combined
treatment on established AI recurrent tumors. Castrate male DD/S mice
bearing AI Shionogi tumors 0.5 cm in diameter were randomly selected
to receive three treatment regimens as described above. Tumor volume
was measured twice weekly and calculated as described above. Data
points were reported as average tumor volume ± SD.
Statistical Analysis.
The in vitro cytotoxic effects of ODN and/or paclitaxel were
analyzed using a repeated measure ANOVA model. AI recurrence-free
survival curves were calculated by the method of Kaplan-Meier and
evaluated with the Mantel-Cox log rank test. The remaining data were
analyzed by Student’s t test. The level of statistical
significance was set at P < 0.05, and all
statistical calculations were done by use of Statview 4.5 software
(Abacus Concepts, Inc., Berkeley, CA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, abundant levels of both unprocessed (60-kDa) and mature
(40-kDa) forms of TRPM-2 protein were detected in TRPM-2-transfected
clones (LNCaP/T1 to LNCaP/T4) at almost the same levels, whereas the
parental LNCaP (LNCaP/P) and the control vector-transfected cell line
(LNCaP/C) did not express detectable TRPM-2 protein levels. The four
TRPM-2-transfected clones showed almost the same results in the
subsequent experiments; therefore, we hereafter report only the data of
LNCaP/P, LNCaP/C, LNCaP/T1, and LNCaP/T2.
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, LNCaP/T1 and LNCaP/T2 exhibited significantly higher
resistance to paclitaxel compared with LNCaP/P and LNCaP/C both in
normal and charcoal-stripped media (P < 0.01
for both). Overexpression of TRPM-2 in LNCaP cells increased the
IC50 of paclitaxel 5-fold (from 1.5 to 8
nM) in normal media and 20-fold (from 0.01 to 0.4
nM) in charcoal-stripped media (Fig. 1B)
.
The induction of apoptosis in LNCaP sublines in normal media
treated with 1 nM paclitaxel for 72 h was assessed
by DNA degradation assay and Western blot analysis of PARP protein,
a substrate of the caspases activated during the process of
apoptotic execution (23)
. The characteristic apoptotic
DNA ladders were detected in LNCaP/P and LNCaP/C but not in LNCaP/T1
and LNCaP/T2 (Fig. 1D)
. Similarly, the
Mr 116,000 intact form of PARP was
observed in all of LNCaP sublines, whereas the
Mr 85,000 PARP cleavage fragment was
detected after paclitaxel treatment only in LNCaP/P and LNCaP/C (Fig. 1E)
.
Acquisition of Resistant Phenotype to Paclitaxel by Overexpression
of TRPM-2 in the LNCaP Tumor Model in Vivo.
To determine whether TRPM-2 overexpression confers resistance to
paclitaxel treatment in vivo, 1 x 106 cells of each cell line (LNCaP/P, LNCaP/C,
LNCaP/T1, or LNCaP/T2) were inoculated s.c. in male nude mice. When
tumors reached 200–300 mm3, mice were castrated.
Beginning 10 days after castration, 0.5 mg of polymeric micellar
paclitaxel was administered i.v. once daily for 5 days. LNCaP/P and
LNCaP/C tumor growth decreased by 61 and 57%, respectively, by 4 weeks
after castration and remained below precastrate volumes by 10 weeks
after castration. In contrast, LNCaP/T1 and LNCaP/T2 tumor volume
decreased by 11 and 28%, respectively, by 4 weeks after castration and
thereafter increased 2.4- and 1.9-fold, respectively, by 10 weeks after
castration (Fig. 2A)
. Serum PSA in mice bearing LNCaP/P and LNCaP/C tumors
decreased by 77 and 75%, respectively, by 1 week after castration and
remained below precastrate levels by 10 weeks after castration. In
comparison, serum PSA in mice bearing LNCaP/T1 and LNCaP/T2 tumors
decreased by 51 and 55%, respectively, before increasing 1.6- and
1.4-fold above precastrate levels, respectively, by 10 weeks after
castration (Fig. 2B)
.
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, TRPM-2 mRNA induction increased in a dose-dependent manner
by paclitaxel treatment at concentrations up to 10
nM. Time course experiments demonstrated that
paclitaxel-induced TRPM-2 mRNA up-regulation peaked by 48 h after
treatment and began decreasing by 72 h after treatment (Fig. 3B)
.
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, 500 nM antisense
TRPM-2 ODN combined with 10 or 50 nM paclitaxel
decreased TRPM-2 mRNA levels by 85 or 70%, respectively, compared with
500 nM mismatch control ODN treatment.
Synergistic Effects of Antisense TRPM-2 ODN and Paclitaxel
Treatment on Induction of Apoptosis in Shionogi Tumor Cells.
To examine whether treatment with antisense TRPM-2 ODN enhances the
paclitaxel-induced cytotoxicity, Shionogi tumor cells were treated with
various concentrations of antisense TRPM-2 ODN once daily for 2 days
and then incubated with various concentrations of paclitaxel for 2
days. As shown in Fig. 4A
, antisense TRPM-2 ODN treatment significantly enhanced
paclitaxel chemosensitivity in a dose-dependent manner
(P < 0.01), reducing the
IC50 of paclitaxel from 100 to 25
nM, whereas mismatch control ODN had no effect.
Dose-dependent synergy between antisense TRPM-2 ODN and paclitaxel was
also observed by increasing the antisense ODN concentration when
paclitaxel concentration was fixed at 10 nM
(P < 0.01; Fig. 4B
).
|
. Similarly, cleavage of PARP protein was detected only
after combined antisense TRPM-2 ODN and paclitaxel treatment (Fig. 4D)
.
Delayed Hormone-refractory Recurrence of Shionogi Tumors
in Vivo by Combined Antisense TRPM-2 ODN and Paclitaxel
Treatment.
Male mice bearing Shionogi tumors between 1 and 2 cm in diameter
were randomly selected for treatment with either antisense TRPM-2 ODN
alone, mismatch control ODN alone, antisense TRPM-2 ODN plus micellar
paclitaxel, or mismatch control ODN plus micellar paclitaxel. Mean
tumor volume was similar at the beginning of treatment in all four
treatment groups. Beginning the day of castration, 12.5 mg/kg antisense
TRPM-2 or mismatch control ODN was administered i.p. once daily for 15
days. Beginning 10 days after castration, 0.5 mg of polymeric micellar
paclitaxel was administered i.v. once daily for 5 days. During an
observation period of 60 days after castration, AI tumors recurred in
four of seven mice after a median of 53 days in antisense TRPM-2 ODN
plus micellar paclitaxel treatment group, whereas AI tumors recurred in
all mice after a median of 28, 39, and 42 days in the mismatch control
ODN treatment group, antisense TRPM-2 ODN treatment group, and mismatch
control ODN plus micellar paclitaxel treatment group, respectively
(P < 0.01). Mean tumor volume at day 60
after castration was 2789, 3481, 712, and 1276
mm3 in the antisense TRPM-2 ODN, mismatch control
ODN, antisense TRPM-2 ODN and paclitaxel, and mismatch control ODN and
paclitaxel treatment groups, respectively (Fig. 5, A and B)
.
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, all mice treated with antisense TRPM-2 ODN alone were
sacrificed by day 21 after initiation of treatment because of tumor
mass >10% of body weight. Time to sacrifice was delayed in the other
two treatment groups. Combined antisense TRPM-2 ODN plus paclitaxel
treatment resulted in the most significant delay in tumor progression,
producing a mean tumor volume 30–40% lower than in the mismatch
control ODN plus paclitaxel treatment group (P < 0.05).
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. Furthermore, the
Mr 85,000 PARP cleavage fragment was
detectable in AI Shionogi tumors only after combined treatment with
antisense TRPM-2 ODN and micellar paclitaxel (Fig. 6C)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(13)
. Furthermore, Steinberg et
al. (14)
reported a close correlation between
staining intensity of TRPM-2 by immunohistochemical analysis and
Gleason pattern in human prostate cancer specimens. We also
demonstrated that TRPM-2 expression renders prostate cancer cells more
resistant to androgen ablation and helps mediate progression of
androgen independence after castration (16)
. Collectively,
these findings suggest a protective role of TRPM-2 against apoptosis
induced by various types of stimuli; however, the significance of
TRPM-2 expression in chemotherapy-induced apoptosis has not been
evaluated. The efficacy of chemotherapy for patients with prostate cancer remains limited for various reasons, including inherent chemoresistance, pharmaceutical mechanism of chemotherapeutic action, and inability of elderly patients to tolerate its toxicity (2 , 24) . To date, no chemotherapeutic agent has demonstrated improved survival in patients with advanced prostate cancer, emphasizing the need for novel therapeutic strategies that target the molecular basis of androgen resistance and chemoresistance of prostate cancer. We have recently shown that antisense Bcl-2 ODN delayed progression to androgen independence (17) and enhanced paclitaxel chemosensitivity in the Shionogi tumor model (24) . These findings illustrate that targeting an antiapoptotic gene with sequence-specific antisense ODN can result in enhanced apoptosis after androgen withdrawal and conventional cytotoxic chemotherapy. The objectives of this study were to examine whether TRPM-2 overexpression confers resistance to paclitaxel and to determine whether antisense TRPM-2 ODN could enhance paclitaxel chemosensitivity and delay emergence of AI tumors beyond that achieved with either agent alone.
We initially evaluated the effects of TRPM-2 overexpression on paclitaxel chemosensitivity using TRPM-2-transfected LNCaP cells and observed that TRPM-2 transfectants were more highly resistant to paclitaxel both in vitro and in vivo through the inhibition of apoptotic cell death. These findings provide the first evidence that TRPM-2 overexpression protects prostate cancer cells from paclitaxel-induced apoptosis, and its up-regulation may contribute to the chemoresistant phenotype in prostate cancer.
Increased expression of TRPM-2 after paclitaxel treatment and androgen withdrawal is likely an adaptive response, which helps the cell survival against a cell death signal. It follows that inhibition of TRPM-2 up-regulation precipitated by castration and paclitaxel treatment may delay progression of androgen independence through enhanced castration- and paclitaxel-induced apoptosis. Antisense ODNs are chemically modified single-stranded DNA fragments complementary to mRNA regions of a target gene, which form RNA-DNA duplexes and thereby reduce gene expression (26) . The potential problems of rapid intracellular degradation can be overcome by phosphorothioate modification of ODNs, which are more resistant to nuclease digestion. After parenteral administration, phosphorothioate ODN becomes associated with high-capacity, low-affinity serum-binding proteins (27) . Antisense ODNs therefore offer one strategy to specifically target TRPM-2 gene expression.
Phosphorothioate antisense TRPM-2 ODN corresponding to the mouse TRPM-2 translation initiation site used in this study inhibited TRPM-2 mRNA expression in a dose- and sequence-dependent manner, even after paclitaxel treatment, which increases TRPM-2 expression. Furthermore, treatment of Shionogi cells with antisense TRPM-2 ODN reduced the IC50 of paclitaxel by 75% and enhanced paclitaxel-induced apoptosis. Systemic administration of antisense TRPM-2 ODN and micellar paclitaxel in vivo significantly delayed time to emergence of AI tumors compared with either agent alone and also enhanced regression of established AI tumors. Although early adjuvant antisense TRPM-2 ODN therapy after castration delayed progression of androgen independence, treatment with antisense TRPM-2 ODN alone had no effect on growth rates of established AI tumors. However, combined treatment with antisense TRPM-2 ODN plus paclitaxel decreased TRPM-2 mRNA expression and accelerated apoptosis induction in AI Shionogi tumors in vivo. These findings illustrate the efficacy of combined antisense TRPM-2 ODN and paclitaxel treatment for cooperatively delaying progression to androgen independence.
Integration and appropriate timing of combination therapies, based on changes in expression of functionally relevant genes after androgen ablation, may help delay progression to androgen independence. The results in the present study provide proof of principle for two potential strategies to delay emergence of the AI phenotype. The first strategy would initiate treatment earlier to enhance castration-induced apoptosis by targeting the antiapoptotic TRPM-2 gene up-regulation by androgen ablation with antisense TRPM-2 ODN. The second strategy would attempt to enhance sensitivity to conventional chemotherapy by reduction of TRPM-2-mediated chemoresistance with antisense TRPM-2 ODN. The preclinical data presented here provide support for clinical studies with combined antisense TRPM-2 ODN and paclitaxel therapy for advanced prostate cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by National Cancer
Institute of Canada Grant 009002 and the American College of Surgeons
George A. H. Clowes Career Development Award. 
2 To whom requests for reprints should be
addressed, at Division of Urology, University of British Columbia, D-9,
2733 Heather Street, Vancouver, British Columbia V5Z 3J5, Canada.
3 The abbreviations used are: TRPM-2;
testosterone-repressed prostate message-2; AI, androgen-independent;
AD, androgen-dependent; PSA, prostate-specific antigen; ODN,
oligodeoxynucleotide; G3PDH, glyceraldehyde-3-phosphate dehydrogenase;
PARP, poly(ADP-ribose) polymerase; CMV, cytomegalovirus;
poly(A)+ mRNA, polyadenylated mRNA.
Received 10/27/99. Accepted 3/ 6/00.
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K. Yamanaka, M. E. Gleave, I. Hara, M. Muramaki, and H. Miyake Synergistic antitumor effect of combined use of adenoviral-mediated p53 gene transfer and antisense oligodeoxynucleotide targeting clusterin gene in an androgen-independent human prostate cancer model Mol. Cancer Ther., February 1, 2005; 4(2): 187 - 195. [Abstract] [Full Text] [PDF]
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 A. Caporali, P. Davalli, S. Astancolle, D. D'Arca, M. Brausi, S. Bettuzzi, and A. Corti The chemopreventive action of catechins in the TRAMP mouse model of prostate carcinogenesis is accompanied by clusterin over-expression Carcinogenesis, November 1, 2004; 25(11): 2217 - 2224. [Abstract] [Full Text] [PDF]
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Y. Bayon, M. A. Ortiz, F. J. Lopez-Hernandez, P. H. Howe, and F. J. Piedrafita The Retinoid Antagonist MX781 Induces Clusterin Expression in Prostate Cancer Cells via Heat Shock Factor-1 and Activator Protein-1 Transcription Factors Cancer Res., August 15, 2004; 64(16): 5905 - 5912. [Abstract] [Full Text] [PDF]
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I. P. Trougakos, A. So, B. Jansen, M. E. Gleave, and E. S. Gonos Silencing Expression of the Clusterin/Apolipoprotein J Gene in Human Cancer Cells Using Small Interfering RNA Induces Spontaneous Apoptosis, Reduced Growth Ability, and Cell Sensitization to Genotoxic and Oxidative Stress Cancer Res., March 1, 2004; 64(5): 1834 - 1842. [Abstract] [Full Text] [PDF]
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L. V. July, E. Beraldi, A. So, L. Fazli, K. Evans, J. C. English, and M. E. Gleave Nucleotide-based therapies targeting clusterin chemosensitize human lung adenocarcinoma cells both in vitro and in vivo Mol. Cancer Ther., March 1, 2004; 3(3): 223 - 232. [Abstract] [Full Text]
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K. S. Leskov, D. Y. Klokov, J. Li, T. J. Kinsella, and D. A. Boothman Synthesis and Functional Analyses of Nuclear Clusterin, a Cell Death Protein J. Biol. Chem., March 21, 2003; 278(13): 11590 - 11600. [Abstract] [Full Text] [PDF]
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 T. Zellweger, K. Chi, H. Miyake, H. Adomat, S. Kiyama, K. Skov, and M. E. Gleave Enhanced Radiation Sensitivity in Prostate Cancer by Inhibition of the Cell Survival Protein Clusterin Clin. Cancer Res., October 1, 2002; 8(10): 3276 - 3284. [Abstract] [Full Text] [PDF]
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H. B. Deng, H. K. Parekh, K.-C. Chow, and H. Simpkins Increased Expression of Dihydrodiol Dehydrogenase Induces Resistance to Cisplatin in Human Ovarian Carcinoma Cells J. Biol. Chem., April 19, 2002; 277(17): 15035 - 15043. [Abstract] [Full Text] [PDF]
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 V. D. Shultz, S. Phillips, M. Sar, P. M. D. Foster, and K. W. Gaido Altered Gene Profiles in Fetal Rat Testes after in Utero Exposure to Di(n-butyl) Phthalate Toxicol. Sci., December 1, 2001; 64(2): 233 - 242. [Abstract] [Full Text] [PDF]
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H. Miyake, I. Hara, S. Kamidono, and M. E. Gleave Synergistic Chemsensitization and Inhibition of Tumor Growth and Metastasis by the Antisense Oligodeoxynucleotide Targeting Clusterin Gene in a Human Bladder Cancer Model Clin. Cancer Res., December 1, 2001; 7(12): 4245 - 4252. [Abstract] [Full Text] [PDF]
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 T. Zellweger, H. Miyake, S. Cooper, K. Chi, B. S. Conklin, B. P. Monia, and M. E. Gleave Antitumor Activity of Antisense Clusterin Oligonucleotides Is Improved in Vitro and in Vivo by Incorporation of 2'-O-(2-Methoxy)Ethyl Chemistry J. Pharmacol. Exp. Ther., September 1, 2001; 298(3): 934 - 940. [Abstract] [Full Text]
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X.S. Ouyang, X. Wang, D.T.W. Lee, S.W. Tsao, and Y.C. Wong Up-regulation of TRPM-2, MMP-7 and ID-1 during sex hormone-induced prostate carcinogenesis in the Noble rat Carcinogenesis, June 1, 2001; 22(6): 965 - 973. [Abstract] [Full Text] [PDF]
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