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Tumor Biology |
The Prostate Centre, Vancouver General Hospital, Vancouver, British Columbia, V6H 3Z6 [H. M., M. E. G.]; Division of Urology, University of British Columbia, Vancouver, British Columbia V5Z 3J5 [H. M., M. E. G.]; and Lady Davis Research Institute of the Jewish General Hospital and Departments of Medicine and Oncology, McGill University, Montreal, Quebec, H3T 1E2 [M. P.], 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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IGF-I and IGF-II are potent mitogens and antiapoptotic factors for many normal and malignant tissues. The biological response of cells to IGFs is regulated by various factors in the microenvironment, including the IGFBPs (3) . To date, at least six IGFBPs have been identified that modulate the biological action of the IGFs through high-affinity binding interactions that influence the ability of IGFs to function as ligands for the type I IGF receptor (4) . However, IGFBP physiology is complex, as evidenced by the fact that both stimulatory and inhibitory effects of IGFBPs on cell proliferation have been reported (5, 6, 7, 8) and also data suggesting that certain regulatory actions of IGFBPs are independent of IGFs (4 , 5 , 9) . Additional complexity is suggested by data documenting that an IGFBP-related protein has tumor suppressor activity for prostate cancer (10) .
Accumulating evidence suggests that the IGF system plays an important role in the pathophysiology of cell turnover and renewal in the prostate. In the normal prostate, IGFs are produced by epithelial and stromal cells, and both IGF-I and IGF-II are mitogenic for epithelial cells (11) . Furthermore, in prostate cancer cells, autocrine stimulation by both IGF-I and IGF-II has been demonstrated (12) . Several IGFBPs are produced by normal prostate epithelial and/or stromal cells, and after castration or treatment with antiandrogens, the expression levels of certain IGFBPs are rapidly and significantly altered (13, 14, 15, 16, 17) . Changes in expression of various IGFBPs in benign and malignant prostatic epithelial cells have also been reported, with an increase in IGFBP-2 and IGFBP-5 and decrease in IGFBP-3 from benign to malignant state (18) . The functional significance of these changes in IGFBP gene expression has not been established.
Controlled study of the complex molecular processes associated with AI progression in prostate cancer has proved difficult because few animal models exist that mimics the clinical course of the disease in men. The Shionogi tumor model is an AD mouse mammary carcinoma xenograft that grows s.c. in male syngeneic hosts. In this model, AD tumors in male mice undergo complete regression after castration but recur as rapidly growing AI tumors after 1 month (19) . The highly reproducible 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 currently 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 surrogate end point of AI progression (20) . Therefore, both the Shionogi and LNCaP tumor models are particularly useful in studying mechanisms controlling castration-induced apoptosis and AI progression.
In this study, to investigate the functional significance of IGFBP-5 up-regulation after castration and during AI progression, we first evaluated the effects of IGFBP-5 overexpression by stable transfection on time to AI progression in the LNCaP tumor model and then tested the effects of antisense IGFBP-5 ODN on Shionogi tumor growth both in vitro and in vivo. Up-regulation of IGFBP-5 expression associated with steroid hormone deprivation had been hypothesized previously to reduce IGF activity and thereby contribute to induction of apoptosis (21) . However, our findings demonstrate that increased IGFBP-5 expression after castration represents an adaptive response to enhance IGF bioactivity, which functions to accelerate time to emergence of an AI phenotype.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 6–8-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 100 and 200 mm3
in volume.
Tumor volume was measured once weekly and calculated by the formula
length x width x depth x 0.5236 (20)
. 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/l (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 (23)
was used in all experiments. Shionogi tumor
cells were maintained in DMEM (Life Technologies, Inc.) supplemented
with 5% heat-inactivated FCS. For in vivo study,
approximately 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. Mice required sacrifice when
tumor mass increased >3 cm3
or 10% of body
weight in accordance with institutional accredited guidelines. Details
of the maintenance of mice, tumor stock, and operative procedures are
described in a previous publication (24)
.
Antisense IGFBP-5 ODN.
Phosphorothioate ODNs used in this study were obtained from Nucleic
Acid-Protein Service Unit, University of British Columbia (Vancouver,
Canada). The sequences of antisense IGFBP-5 ODN corresponding to the
mouse IGFBP-5 translation initiation site were
5'-GACCACGCTGATCACCAT-3'. Two base IGFBP-5 mismatch ODNs
(5'-GACCACGCTCATGACCAT-3') were used as control.
Treatment of Cells with ODN.
Lipofectin, a cationic lipid (Life Technologies, Inc.), was used to
increase the ODN uptake of cells. Shionogi cells were treated with
various concentrations of ODN after a preincubation for 20 min with 4
µg/ml lipofectin in serum-free OPTI-MEM (Life Technologies, Inc.).
Four h after the beginning of the incubation, the medium containing ODN
and lipofectin was replaced with standard culture medium described
above.
Northern Blot Analysis.
Total RNA was isolated from Shionogi tumor tissues and cultured
Shionogi tumor cells by the acid-guanidinium
thiocyanate-phenol-chloroform method. The electrophoresis,
hybridization, and washing conditions were carried out as reported
previously (19)
. Mouse IGFBP-5 and G3PDH cDNA probes were
generated by reverse transcription-PCR from total RNA of mouse brain
using primers 5'-AGAAAATGGTGATCAGCGTGGT-3' (sense) and
5'-TTCGGATTCCTGTCTCATCTCA-3' (antisense) for IGFBP-5, and
5'-ATGGTGAAGGTCGGTGTGA-ACGGAT-3' (sense) and
5'-AAAGTTGTCATGGATGACCTT-3' (antisense) for G3PDH. Density of bands for
IGFBP-5 was normalized against that of G3PDH by densitometric analysis.
MTT Assay.
The in vitro effects of antisense IGFBP-5 ODN, anti-IGF-I
antibody (Upstate Biotechnology, Lake Placid, NY), and/or recombinant
IGF-I (Sigma Chemical Co.) on growth of Shionogi tumor cells were
assessed by the MTT assay as described previously (22)
.
Briefly, 1 x 104 cells were
seeded in each well of 96-well microtiter plates and allowed to attach
overnight. Cells were then treated once daily with various
concentrations of ODN for 2 days in the media containing 5
nM recombinant IGF-I or 10 µg/ml anti-IGF-I
antibody. Forty-eight h after ODN treatment, 20 µl of 5 mg/ml MTT
(Sigma Chemical Co.) in PBS were added to each well, followed by
incubation for 4 h at 37°C. The formazan crystals were then
dissolved in DMSO. The absorbance was determined with a microculture
plate reader (Becton Dickinson Labware, Lincoln Park, NJ) at 540 nm.
Absorbance values were normalized to the values obtained for the
vehicle-treated cells to determine the percentage of survival. Each
assay was performed in triplicate.
Cell Death Assay.
Shionogi tumor cells were treated as described above. Live and dead
cells from each subculture of cells were counted using trypan blue
48 h after ODN treatment, and the ratio of dead/total cells was
calculated. Each assay was performed in triplicate.
MAPK in Vitro Kinase Assay.
Shionogi tumor cells were treated as described above, and MAPK activity
was measured using a MAP Kinase Assay Kit (New England Biolabs,
Beverly, MA). Briefly, the cells were washed with PBS, lysed in lysis
buffer, sonicated, and microcentrifuged for 20 min at 4°C. The
supernatants was incubated with 1:100-diluted anti-phospho-MAPK
antibody for 4 h. Protein A-agarose beads were then added and
incubated for another 3 h. The pellets were washed twice with
ice-cold lysis buffer and twice with kinase buffer. The pellets were
incubated with 100 mM ATP and 20 mg/ml Elk1 fusion protein,
a substrate of MAPK, for 30 min at 30°C. Samples were boiled,
separated by electrophoresis through a 10% SDS-polyacrylamide gel, and
transferred to polyvinylidene difluoride membranes. The membranes were
incubated for 1 h at room temperature in blocking buffer and then
probed with 1:1000-diluted anti-phospho-Elk1 antibody. After being
washed, the membranes were incubated with a 1:1000-diluted horseradish
peroxidase-conjugated antirabbit immunoglobulin (Amersham Life Science,
Arlington Heights, IL). The immunoreactivity of phosphorylated Elk1 was
determined using an ECL chemiluminescence kit (Amersham Life Science).
Flow Cytometric Analysis.
The flow cytometric analysis of propidium iodide-stained nuclei was
performed as described previously (22)
. Briefly, Shionogi
tumor cells were plated at a density of 5 x 106 cells in six-cm dishes and treated as
described above. The cells were trypsinized 48 h after ODN
treatment, washed twice with PBS, and fixed in 70% ethanol for 5 h at 4°C. The fixed cells were washed twice with PBS, incubated with
1 µg/ml RNaseA (Sigma Chemical Co.) for 1 h at 37°C, and
stained with 5 µg/ml propidium iodide (Sigma Chemical Co.) for 1 h at room temperature. The stained cells were analyzed for relative DNA
content on a FACScan (Becton Dickinson Labware).
In Vivo ODN Treatment.
Male DD/S mice bearing Shionogi tumors were castrated and randomly
selected for treatment with antisense IGFBP-5 versus
mismatch control ODN. Each experimental group consisted of eight mice.
Beginning the day of castration, 15 mg/kg antisense IGFBP-5 or mismatch
control ODNs were injected i.p. once daily into each mouse for 50 days.
Tumor volume was measured twice weekly and calculated as described
above. Data points were reported as average tumor volumes ± SD.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, various patterns of changes in IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5
mRNA expression were observed. IGFBP-1 and IGFBP-6 mRNAs are
undetectable in the Shionogi tumor model (data not shown). Of the
remaining IGFBPs, the most dramatic changes in expression were observed
with IGFBP-5. We confirmed our prior observation (25)
that
IGFBP-5 expression is undetectable in AD intact tumors and highly
up-regulated after castration. In addition, Fig. 1
illustrates that
IGFBP-5 expression remains high in AI tumors. The observed pattern
of IGFBP-5 up-regulation in the Shionogi tumor model during AI
progression is similar to that in rat prostate (13)
and
human prostate (18)
cancer.
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A, abundant levels of IGFBP-5 mRNA were detected in four
independent IGFBP-5-transfected clones (LNCaP/IGFBP-5#1 to
LNCaP/IGFBP-5#4), whereas the control vector-transfected cell line
(LNCaP/Co) did not express detectable IGFBP-5 mRNA levels. LNCaP/Co,
LNCaP/IGFBP-5#1, and LNCaP/IGFBP-5#2 were selected for in
vivo study, with 1 x 106 of
each cell line inoculated s.c. in intact male nude mice. No difference
in tumor take rates, tumor volume, or serum PSA levels were apparent
between different LNCaP sublines grown in intact nude mice prior to
castration. After castration, LNCaP/Co tumor growth was inhibited for 4
weeks, after which LNCaP/Co tumor volume increased 2.0-fold by 8 weeks
after castration. In contrast, LNCaP/IGFBP-5#1 and LNCaP/IGFBP-5#2
tumor volume continued to grow after castration, increasing 4.3- and
5.1-fold, respectively, by 8 weeks after castration (Fig. 2B
). Serum PSA in mice bearing LNCaP/Co tumors decreased by
76% by 2 weeks after castration and increased from 4 to 8 weeks after
castration by 1.3-fold. In comparison, serum PSA in mice bearing
LNCaP/IGFBP-5#1 and LNCaP/IGFBP-5#2 tumors decreased by 61 and 59%,
respectively, before increasing beginning 2 weeks after castration to
3.7- and 4.2-fold, respectively, by 8 weeks (Fig. 1C
).
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A and B, daily treatment of Shionogi tumor cells
with antisense IGFBP-5 ODN (50, 100, 500, or 1000
nM) for 2 days reduced IGFBP-5 mRNA levels by 0,
7, 54, or 83%, respectively, whereas IGFBP-5 mRNA expression
was not affected by the two-base mismatch control ODN at any of the
concentrations used.
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Collectively, these data demonstrate that antisense IGFBP-5 ODN used in
this study induces sequence-specific, gene-specific, and dose-dependent
down-regulation of its target gene.
Antiproliferative Effects of Antisense IGFBP-5 ODN on Shionogi
Tumor Cells.
To determine the effects of antisense IGFBP-5 ODN on cell
proliferation, we treated Shionogi tumor cells once daily with either 1
µM antisense IGFBP-5 or mismatch control ODN for 2 days
and determined cell number over a 72-h period. Antisense IGFBP-5 ODN
treatment of cells resulted in significant inhibition of Shionogi tumor
cell proliferation over this 72 h, whereas cell growth was not
influenced by treatment with mismatch control ODN (Fig. 4
A).
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). These antiproliferative
effects directly correlated with the degree of IGFBP-5 mRNA reduction
in Shionogi tumor cells by antisense IGFBP-5 ODN. In contrast, no
significant effects were observed on cell proliferation treated with
mismatch control ODN at any of the used concentrations.
To determine whether a decrease in cell proliferation by antisense
IGFBP-5 ODN resulted from apoptosis, the number of live and dead cells
were counted after antisense IGFBP-5 or mismatch control ODN treatment.
The ratio of dead cells to total cell number of antisense IGFBP-5
ODN-treated cells was not significantly different from that of mismatch
control ODN-treated cells (Fig. 4C
). Hence, inhibition of
cell growth after antisense IGFBP-5 ODN treatment is not the result of
enhanced apoptosis.
IGF-I-dependent Effects of Antisense IGFBP-5 ODN on Shionogi
Tumor Cells.
To analyze the relationship between IGFBP-5 and IGF-I in the
regulation of Shionogi tumor cell growth, the effects of antisense
IGFBP-5 ODN treatment on the cell growth in the presence of anti-IGF-I
antibody and/or recombinant IGF-I were evaluated. As shown in Fig. 5
A, recombinant IGF-I increased Shionogi tumor cell growth,
whereas anti-IGF-I antibody inhibited the cell growth. Furthermore,
exogenous recombinant IGF-I treatment could overcome the inhibitory
effects on the cell growth by antisense IGFBP-5 ODN. Treatment of
Shionogi cells with antisense IGFBP-5 ODN and anti-IGF-I antibody did
not produce additive growth-inhibitory effects over that of anti-IGF-I
antibody alone. These findings support an enhancing and IGF-I-dependent
effect of IGFBP-5 on the cell proliferation.
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).
To examine effects of changes in IGFBP-5 expression levels on
cell cycle regulation, flow cytometric analysis was performed in
Shionogi tumor cells. As shown in Fig. 5C
, decreases in
IGFBP-5 expression levels induced by antisense IGFBP-5 ODN treatment
resulted in G1 cell cycle arrest, thereby
reducing the fraction of cells in the S + G2-M phases by >50% compared with mismatch
control ODN treatment.
Antisense IGFBP-5 ODN Delays AI progression of Shionogi Tumors
in Vivo.
Male mice bearing Shionogi tumors were castrated 2–3 weeks after tumor
implantation, at which time tumors were 1–2 cm in diameter and
randomly selected for treatment with antisense IGFBP-5
versus mismatch control ODN. Mean tumor volume was similar
in both groups at the beginning of ODN treatment. Beginning the day of
castration, 15 mg/kg ODN was administered once daily by i.p. injection
for 50 days. As shown in Fig. 6
A, antisense IGFBP-5 ODN treatment delayed recurrence of AI
tumors compared with mismatch control ODN treatment. Although AI tumors
recurred in all mice in both groups during an observation period of 60
days after castration, the median time to emergence of first palpable
AI recurrent tumors was delayed by 7 days in mice treated with
antisense IGFBP-5 ODN (35 days after castration) compared with that in
mice treated with mismatch control ODN (28 days after castration).
Furthermore, growth of recurrent AI tumors was substantially inhibited
in the antisense IGFBP-5 ODN treatment group compared with the mismatch
control ODN group, and the time to sacrifice of mice was significantly
prolonged in the antisense IGFBP-5 ODN treatment group,
i.e., all mice required sacrifice in mismatch ODN group
after a median of 53 days after castration compared with only one of
eight mice in the antisense IGFBP-5 ODN treatment group 60 days after
castration.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, we reported a dramatic increase in IGFBP-5 expression after castration in the Shionogi tumor model, and that IGFBP-5 expression is directly regulated by apoptosis-inducing stimuli rather than androgen (25) . Our results agree with previous findings that IGFBP-5 expression changes most substantially among several IGFBPs in prostate tissues after androgen withdrawal (13, 14, 15, 16, 17) . Although various functional roles of IGFBP-5 expression have been suggested in different model systems, these data are varying and conflicting. For example, IGFBP-5 has been reported to either stimulate or inhibit cell proliferation under different experimental conditions (4, 5, 6 , 28, 29, 30, 31) , and these effects are exerted dependent and/or independent of its well-characterized actions associated with modulation of IGF bioavailability (4 , 5) . Furthermore, to date, there has been no data demonstrating the functional significance of IGFBP-5 up-regulation after androgen ablation in prostate cancer progression.
In this study, we generated several IGFBP-5-overexpressing LNCaP cell lines to characterize the functional role of IGFBP-5 up-regulation in AI progression of prostate cancer. Although tumor incidence, tumor growth rates, and serum PSA levels were similar among the LNCaP sublines growing in intact mice, tumor growth and serum PSA levels increased severalfold faster in mice bearing IGFBP-5-transfected LNCaP tumors after castration than those bearing control LNCaP tumors. These results provide the first clear evidence that IGFBP-5 up-regulation in prostate cancer cells after castration accelerates time to AI progression.
To further define the functional significance of IGFBP-5 up-regulation after castration, we used the AD mouse Shionogi tumor model that shares similar characteristics with human prostate cancer. Like human prostate cancer, Shionogi tumors have a functional androgen receptor, undergo extensive castration-induced apoptosis after androgen withdrawal, and later recur as AI tumors in a highly reproducible manner. At the molecular level, the Shionogi tumor model also shares a number of characteristics with human prostate cancers, such as up-regulation of Bcl-2, TRPM-2, and IGFBPs after castration (19 , 23 , 25) . In the present study, we confirmed that the up-regulation of IGFBP-5 after castration is maintained in rapidly growing recurrent AI Shionogi tumors, which along with accelerated growth of IGFBP-5-overexpressing LNCaP tumors, identifies IGFBP-5 overexpression as a potential mediator of progression in this model.
Antisense ODNs, chemically modified stretches of single-stranded DNA that are complementary to mRNA regions of a target gene and thereby effectively inhibit gene expression by forming RNA/DNA duplexes (32) , offer one strategy to specifically target IGFBP-5 gene expression. Phosphorothioate ODNs are water soluble, stable agents manufactured to resist nuclease digestion. After parenteral administration, phosphorothioate ODNs become associated with high-capacity, low-affinity serum binding proteins (33) . In this study, phosphorothioate antisense IGFBP-5 ODN corresponding to the mouse IGFBP-5 translation initiation site was used that inhibited expression of IGFBP-5 mRNA in Shionogi tumor cells in a dose-dependent and sequence-specific manner. Furthermore, antisense IGFBP-5 ODN inhibited cell proliferation and induced cell cycle arrest in Shionogi tumor cells in a time- and dose-dependent manner. Antisense IGFBP-5 ODN treatment did not appear to induce apoptosis either in vitro or in vivo, which suggests that antisense IGFBP-5 ODN activity occurs via inhibition of cell proliferation rather than induction of apoptosis. We subsequently showed that the growth-inhibitory effects of antisense IGFBP-5 ODN could be overcome by exogenous IGF-I and that antisense IGFBP-5 ODN treatment caused no additional inhibition of cell proliferation when IGF-I activity was neutralized by anti-IGF-I antibody. We also identified a direct association between cell growth rate and MAPK activity in Shionogi cells after antisense IGFBP-5 ODN treatment. Collectively, these findings demonstrate that antisense IGFBP-5 ODN inhibited the cell proliferation, at least in part, through an IGF-I-dependent mechanism involving inactivation of MAPK.
On the basis of the present in vitro data, we hypothesized that targeting IGFBP-5 up-regulation precipitated by androgen withdrawal using antisense strategy may inhibit progression to androgen independence by inhibiting IGFBP-5-enhanced IGF-I activity. In our in vivo experiments, administration of antisense IGFBP-5 ODN after castration delayed time to AI progression and inhibited AI recurrent tumor growth. Consistent with our in vitro treatments, treatment of mice bearing Shionogi tumors with antisense IGFBP-5 ODN also inhibited the IGFBP-5 mRNA expression in vivo. These findings illustrate that in vivo systemic administration of ODN can result in sequence-specific down-regulation of a target gene in tumor tissues.
As described above, the biological activity of IGFBP-5 varies depending upon various cell types, which may reflect differential regulation of extracellular matrix interactions (3 , 4 , 29 , 30) or posttranslational modification (31) . Indeed, even cell growth conditions will significantly affect IGFBP-5 expression, as shown in Shionogi cells grown in vitro (high expressing) versus AD tumors in vivo (no expression). Therefore, although our present data show that IGFBP-5 enhances IGF-I bioactivity and increases cell proliferation, studies using additional prostate tumor systems (34 , 35) are needed to clarify tissue-specific interactions between IGF-I and IGFBP-5 and to better define the relative importance of IGFBP-5 after androgen ablation in prostate cancer.
A rational strategy to delay AI progression should be based on molecular mechanisms and would target the adaptive changes in gene expression precipitated by androgen withdrawal, rather than the conventional approach of treating patients with established hormone refractory disease. Integration and appropriate timing of combination therapies, based on biological mechanism of progression and castration-induced changes in gene expression, may provide means to inhibit AI progression in a major way. The present study provides the first direct evidence to support a functional role for IGFBP-5 in AI progression. Dramatic up-regulation of IGFBP-5 after castration helps potentiate the mitogenic activity of IGF-I. Furthermore, reduction of IGFBP-5 gene expression using antisense IGFBP-5 ODN delays recurrence and growth of AI tumors. These preclinical data identify a novel target and therapy using antisense IGFBP-5 ODN after androgen ablation in patients with advanced prostate cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Grant 009002 from the
National Cancer Institute of Canada. 
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: AI, androgen
independent; AD, androgen dependent; IGF, insulin-like growth factor;
ODN, oligodeoxynucleotide; IGFBP, insulin-like growth factor binding
protein; PSA, prostate-specific antigen; CMV, cytomegalovirus; MAPK,
mitogen-activated protein kinase; G3PDH, glyceraldehyde-3-phosphate
dehydrogenase; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Received 7/30/99. Accepted 3/31/00.
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REFERENCES |
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 E. Tonner, M. C. Barber, G. J. Allan, J. Beattie, J. Webster, C. B. A. Whitelaw, and D. J. Flint Insulin-like growth factor binding protein-5 (IGFBP-5) induces premature cell death in the mammary glands of transgenic mice Development, January 10, 2002; 129(19): 4547 - 4557. [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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 M. W. Elmlinger, I. Mayer, D. Schnabel, B. S. Schuett, D. Diesing, G. Romalo, H. A. Wollmann, W. Weidemann, K.-D. Spindler, M. B. Ranke, et al. Decreased Expression of IGF-II and Its Binding Protein, IGF-Binding Protein-2, in Genital Skin Fibroblasts of Patients with Complete Androgen Insensitivity Syndrome Compared with Normally Virilized Males J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4741 - 4746. [Abstract] [Full Text] [PDF]
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 O. Kucuk, F. H. Sarkar, W. Sakr, Z. Djuric, M. N. Pollak, F. Khachik, Y.-W. Li, M. Banerjee, D. Grignon, J. S. Bertram, et al. Phase II Randomized Clinical Trial of Lycopene Supplementation before Radical Prostatectomy Cancer Epidemiol. Biomarkers Prev., August 1, 2001; 10(8): 861 - 868. [Abstract] [Full Text] [PDF]
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T. Nickerson, F. Chang, D. Lorimer, S. P. Smeekens, C. L. Sawyers, and M. Pollak In Vivo Progression of LAPC-9 and LNCaP Prostate Cancer Models to Androgen Independence Is Associated with Increased Expression of Insulin-like Growth Factor I (IGF-I) and IGF-I Receptor (IGF-IR) Cancer Res., August 1, 2001; 61(16): 6276 - 6280. [Abstract] [Full Text] [PDF]
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