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Geriatric Research, Education, and Clinical Center, VA Puget Sound Health Care System (S.E.D., L.M., S.R.P.), Tacoma, Washington 98493; Division of Gerontology and Geriatric Medicine, Department of Medicine, University of Washington (S.E.D., L.M., S.R.P.), Seattle, Washington; and the Department of Pathology (J.L.W.), Medical College of Virginia, Richmond, Virginia 23298
Address all correspondence and requests for reprints to: Stephen Plymate, Geriatric Research, Education, and Clinical Center, VA Puget Sound Health Care System, American Lake Division, Tacoma, Washington 98493. E-mail: splymate{at}u.washington.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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0.01). Colony formation in soft agar was
significantly inhibited up to 14 days after plating in the IGFBP-4
transfected cells when compared with the M12 controls
(P 0.01): however, in the presence of
des(1–3)IGF-I, there was no significant difference between the control
and IGFBP-4 transfectants in colony formation in soft agar. Apoptosis
in an IGFBP-4 transfected cell line was significantly increased in
response to induction by 6-hydroxyurea compared with the control line.
When injected sc into male athymic/nude mice, a marked delay was noted
in tumor formation in animals receiving IGFBP-4 transfected cells
(P 0.01). Interestingly, IGFBP-2 protein levels
were reduced in the conditioned media of all IGFBP-4 transfected cell
cultures. These data indicate that an inhibitory IGFBP may
significantly delay the growth of malignant prostate epithelial cells
and enhance the sensitivity of these cells to apoptosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In several tissue systems, during the transition from the benign to malignant state, qualitative and quantitative changes in various components of the IGF axis frequently occur. Human epithelial breast cancer cells (18, 19) and human pancreatic (20) and parathyroid tumors (21) contained elevated levels of the IGF-IR when compared with the benign state. Work in our laboratory has shown that in prostate epithelium, both in vivo and in vitro there are several changes in the IGF system. IGF-IR levels decrease and IGF-II levels increase in malignant cells (12, 22). Changes in expression of IGFBPs also occur in the progression to prostate cancer: IGFBP-2 and IGFBP-5 levels increase, whereas IGFBP-3 (16, 17) and IGFBP-7 (23) levels decrease from the benign to malignant state.
Immortalization of cells, an early event in the transformation process, requires overexpression of the IGF-IR and an increase in IGF expression in NIH 3T3 and PC-12 cells (24, 25). When either IGF-IR or IGF expression was blocked in these cell lines, immortalization and resulting transformation was inhibited. Rat prostate cancer cells lacking the IGF-IR exhibited suppressed tumor growth and inhibition of invasive growth to surrounding cells in vivo (26), suggesting the IGF system also plays an important role in metastasis of prostate cancer.
We postulated that overexpression of an inhibitory IGFBP could decrease the availability of the already up-regulated IGF-II in prostate cancer cells, and that this would in turn reduce tumor formation and or growth. Of the six well characterized IGFBPs, IGFBP-4 alone appears to act exclusively in an inhibitory fashion with respect to the IGF ligands (3), by sequestering IGF from the IGF-IR. In this study, we attempted to alter the availability of the IGF-II ligand by overexpressing IGFBP-4 in the highly tumorigenic and metastatic M12 human prostate epithelial cell line to determine the resulting effect on tumor cell growth. The M12 cell line, like prostate adenocarcinomas, overexpress the IGF-II ligand and have reduced levels of the IGF-IR (22).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
Derivation of the M12 cell line has been previously described
(27, 28). Briefly, human prostate epithelial cells were immortalized
with SV40T antigen to the produce the benign, immortalized P69SV40T
(P69) cell line. P69 cells were injected sc into athymic nude mice,
producing tumor nodules in 2 of 18 animals after 180 days. These
nodules were reimplanted into athymic nude mice, and after three
passages resulted in the M12 cells, which demonstrated a short latency
period of 7–10 days to tumor formation in all 10 animals receiving sc
injection, and were locally invasive and metastatic when injected
intraprostatically (29). M12 cells were cultured in RPMI-1640 medium
supplemented with 10 ng/ml EGF, 0.1 mM dexamethasone, 5
mg/ml insulin, 5 mg/ml transferrin, and 5 ng/ml selenium, fungizone,
and gentamicin (ITS medium) at 37 C in 5% CO2. All cells
used in these experiments were mycoplasma free as determined by the
Gen-Probe Mycoplasma T.C. Rapid Detection System (Gen-Probe, San Diego,
CA).
Vector preparation
The mammalian expression vector pcDNA3.1 was used to prepare the
pcBP4 construct that expresses the IGFBP-4 cDNA from the constitutive
CMV promoter. A 0.989-kb cDNA fragment containing the full-length
IGFBP-4 coding sequence (30) was ligated into the EcoRI and
XhoI sites, oriented 5' to 3', of pcDNA3.1 using Ready-To-Go
T4 ligation kit (Pharmacia Biotech, Piscataway, NJ). The IGFBP-4 cDNA
contains 189 bp of 5' untranslated region, a 761-bp coding sequence,
and 42 bp of the 3' untranslated region. Subcloning efficient DH5a
Escherichia coli cells (Life Technologies, Gaithersburg, MD)
were transformed with the pcDNA3.1-IGFBP4 ligation and ampicillin
resistant colonies assayed by DNA minipreparations and restriction
digestion.
Cell lines
Cells lines were produced by liposome mediated transfection
using Tfx-50 (Promega) according to the manufacturer’s protocol, using
1.33 mg plasmid DNA per 5.5 x 105 cells in a 60-mm culture
dish. Control cells were produced by transfecting M12 cells with
pcDNA3.1 alone. Both transfected and nontransfected M12 cells were
selected with G418 (800 mg/liter active ingredient) for 7 days until
all the nontransfected M12 control cells died. At this point, the G418
concentration was stepped down to 200 mg/liter, and the cells were
selected for another 2 weeks. After the 2-week period, resistant cells
were pooled to form a polyclonal cell line designated M12BP4pop. Two
clonal cell lines, expressing low and intermediate levels of the
transgenic IGFBP-4, were selected from M12BP4pop culture for analysis
in this study. These clones were picked using the method of
Gibson-D’Ambrosio et al. (31). Briefly, M12BP4pop cells
were plated sparsely on plastic culture dishes and grown until
individual colonies were visible. A sterile nylon membrane (Genescreen,
DuPont, Wilmington, DE) was placed over the colonies, and the medium
was aspirated. After 3 h, the membrane was marked for orientation
on the plate and removed; growth medium was replaced, and cells
remaining on the plate were allowed to grow. The membrane was
immediately probed with IGFBP-4 antibody by Western immunoblotting as
described below. Colonies identified as high expressors of IGFBP-4 were
removed from the master plate using cloning rings and subcultured.
Messenger RNA (mRNA) analysis
Cells were cultured until they reached 90% confluence, at which
time total cytoplasmic RNA was isolated using an acid guanidinium
thiocyanate-phenol-chloroform extraction method. Ten milligrams of each
RNA preparation were separated by electrophoresis through a 1.5%
agarose/2.2 M formaldehyde gel, transferred overnight by
capillary action onto a nylon membrane (Genescreen, DuPont) using
10 x SSC as the transfer solution, and cross-linked to the
membrane by UV irradiation in a Statalinker 1800 (Stratagene, La Jolla,
CA). The Northern blot was probed with the
EcoRI/HindI11 fragment of the IGFBP-4 cDNA
radiolabeled (1 x 109 dpm/ug)with
a-[32P]-dCTP (New England Nuclear-DuPont; specific
activity 3000Ci/mmol) using a random priming kit (Prime-a gene,
Promega) overnight at 42 C in 50% formamide, 5 x SSC, 10 x
Denhardt’s solution, 1% SDS, 100 mg/ml sheared, denatured Herring
sperm DNA. The blot was washed for 30 min in 2 x SSC at RT, 30
min in 2 x SSC, and 1% SDS at RT, and stringently washed at 65 C
in 0.2 x SSC, 1% SDS for 30 min. Blots were exposed to Kodak XAR
film (Eastman Kodak Co., Rochester, NY) for 2 days with one intensifier
screen at -70 C. Bands in the resulting autoradiograph were quantified
using an image analyzer equipped with MCID version 4.2 software
(Imaging Research, St. Catherines, Ontario, Canada).
Western ligand and immunoblot analysis of IGFBP-4 expression
IGFBP-4 protein production was assayed by radioligand and
immunoblot analysis. Cells were plated in 60-mm plates in medium
containing serum and allowed to grow to 70% confluence. The cells were
then switched to serum-free medium for 24 h, after which time the
medium was collected, normalized to cell numbers (determined by cell
counts), and concentrated by filtration through nitrocellulose (32).
After concentration, proteins were redissolved in 50 ml denaturing SDS
sample buffer (0.5 M Tris, pH 6.8, with 1% SDS, 10%
glycerol, and 8 M urea) and separated on a 12% SDS-PAGE.
The proteins were then transferred to nitrocellulose by
electroblotting. [125I]-IGF-II (2000 Ci/mmol; Amersham)
was used for radioligand blotting at 1.25 x 105
cpm/ml. Radioactivity was visualized using Kodak XAR-2 film with
intensifying screens at -70 C for 24 h. Band intensities in the
resulting autoradiograms were quantified by densitometry as described
above. Western immunoblotting was prepared as described above through
the transfer step, but the nitrocellulose membrane was probed with
antiserum to human IGFBP-4 at a dilution of 1:4000, and detection was
accomplished with horseradish peroxidase-linked donkey antirabbit
secondary antibody and enhanced chemiluminescence reagents (ECL system,
Amersham) using the manufacturer’s protocol.
Proliferation assays
Cell proliferation was assessed using a colorimetric, MTS assay
for quantification of viable cells using the Cell Titer 96
AQueous kit (Promega). In this assay, 2,500 cells were
added to each well of a 96-well plate; and IGF-II (0–100 ng/ml) was
added to the ITS medium at the time of plating. After 72 h in
culture, the tetrazolium salt and dye solution were added, color
development was allowed to proceed for 2–3 h at 37 C, and absorbance
at 570 nm was measured for each well. Each cell line was tested in
three separate experiments. MTS results were confirmed by cell counts.
The correlation between cell number and the MTS tetrazolium assay in
our laboratory is r = 0.97.
Anchorage-independent growth
For studies of anchorage-independent growth of transfected cell
lines, each well of a 24-well plate was first layered with 0.6%
agarose, 1 x RPMI 1640. A top layer containing an equal volume of
cells (1 x 106) and 2 x RPMI 1640 supplemented
0.3% agar, 200 ng/ml G418 and containing 0 or 50 ng/ml des(1, 2, 3)IGF-I
(DSL, Webster, TX) was added. Plates were maintained at 37 C in 5%
CO2 for 21 days. Colonies greater than 50 µm in diameter
were counted at 14 and 21 days after plating.
FragEL assay for apoptosis
This assay was performed on cell preparations grown directly on
glass slides. 1 x 105 cells were plated on glass
slides and grown in ITS medium/5% FBS at 37 C in 5% CO2
for 24 h to allow attachment. Medium was removed, and specimens
were rehydrated in 1 x TBS buffer and permeabilized with
proteinase K. Endogenous peroxidases were inactivated with hydrogen
peroxide. Some slides were pretreated for 2 h with 50
mM 6-hydroxyurea. The specimens were then end-labeled with
Klenow Labeling Reaction Mix (Oncogene Research Products, Cambridge,
MA) according to the manufacturer’s protocol. The slides were blocked
and stained using the FragEL-Klenow DNA Fragmentation Detection Kit
(Oncogene Research Products) according to the manufacturer’s protocol.
Apoptotic cells were then identified and quantified (per 100 cells) by
light microscopy.
Fragmentation gel assay for apoptosis
Media collected from cells grown for the above FragEL assay were
spun at 2000 rpm to collect and concentrate cells that had detached
from the plates. Harvested cells were resuspended in 15 ml of a 1:1
mixture of sample buffer (10% glycerol, 10 mM Tris, pH
8.0, 0.1% (wt/vol) bromophenol blue) and RNase A (10 mg/ml) and loaded
onto an agarose gel divided at the wells into a lower 2% portion and
an upper portion containing 1% agarose, 2% SDS, and 64 mg/ml
proteinase K. DNA was electrophoresed for 16 h at 60 V. Gels were
stained in water containing 2 mg/ml EtBr in water for 1 h, then
washed 3 times in 3 liters of water for several hours (33). DNA was
visualized by UV transillumination to reveal characteristic ladders
indicative of apoptosis.
Isolation of cells from tumors
Tumors that developed in nude, athymic male mice after sc
injection of 1 x 106 cells of either M12-pcCNA3 or
-BP4C were removed at 10 weeks after injection and digested with 0.1%
collagenase (Type 1) and 50 µg/ml DNAse (Worthington Biochemical
Corp., Freehold, NJ) according to the protocol of Peehl and Stamey
(34). Dispersed cells were plated in ITS medium/5% FBS at 5%
CO2, 37 C for 24 h to allow attachment. After 24
h, cultures were switched to serum-free ITS medium for 24 h, and
used for Western ligandblot analysis as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Two
clonal cell lines M12-BP4A and -BP4C also expressed the 2.0-kb
transgenic IGFBP-4 transcript at approximately 4- and 8-fold higher
levels respectively than the endogenous IGFBP-4 mRNA (data not shown).
The control cells (M12-pcDNA3) transfected with the empty pcDNA3.1
vector, did not express the 1.3-kb transcript.
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). The results were quantified by
densitometry. We observed a greater than 50-fold increase in the signal
of IGFBP-4 (
24 kDa) protein band from medium conditioned by
M12-BP4pop, whereas the band intensities of the IGFBP-4 band in the
clonal cell lines M12-BP4C and -BP4A were 40- and 15-fold higher,
respectively, than medium conditioned by control M12-pcDNA3 cells. The
band seen at 26 kDa is the glycosylated form of IGFBP-4. There is a
decrease in IGFBP-2 (32 kDa) expression in medium conditioned by all
the IGFBP-4 overexpressing cell lines (Fig. 2, A and B). This decrease
was more pronounced in the cell lines expressing higher levels of
IGFBP-4.
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). The IGFBP-4
antiserum reacts with low affinity to IGFBP-2 (32 kDa). From this
immunoblot it is also possible to detect the same pattern of IGFBP-2
expression as seen in the radioligand blot (Fig. 2
). The higher levels
of IGFBP-4 expression are associated with lower levels of IGFBP-2.
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). Each experiment was repeated three
times. In the absence or presence of 50 mM IGF-II, the two
M12-BP4 cell lines proliferated significantly more slowly than the
controls. However, the increase in rate of proliferation of M12BP4-C
cells from 1 to 10 and 10 to 100 ng/ml IGF-II was statistically
significant (P < 0.01). The M12BP4-pop cell line
showed a significant increase (P < 0.01) in
proliferation from 10–100 ng/ml IGF-II.
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). When the cell lines were grown
in soft agar in the presence of 50 ng/ml des(1, 2, 3)IGF-I, there was no
significant difference (P < 0.01) in number of
colonies formed between control cells and those overexpressing IGFBP-4,
indicating that the effect of IGFBP-4 overexpression was due to
inhibition of IGF, and not to an IGF-independent effect (Fig. 5B).
However, when colony numbers were counted at 21 days in soft agar in
serum-free medium, there were no significant differences in colony
number between any of the four cell lines (data not shown). Each
experiment was repeated three times.
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). Microphotographs of contol M12-pcDNA3
or -BP4C cells treated with and without hydroxyurea for 2 h before
DNA fragment end-labeling are shown in Fig. 6, A–D. The dark staining
bodies indicated with arrows are cells undergoing apoptosis.
Photomicrographs A and B are M12-pcDNA3 cells grown in the presence or
absence of 50 mM 6-hydroxyurea for 2 h before DNA
end-labeling. The percentage of M12-pcDNA3 cells undergoing apoptosis
were 0.5% ± 0.3 and 2.1% ± 0.3 in the absence or presence of
hydroxyurea, respectively. Photomicrographs C and D represent M12-BP4C
cells treated without and with hydroxyurea respectively. M12-BP4C cells
underwent apoptosis at 11% ± 2.0 and 19% ± 4.0 in the absence or
presence of hydroxyurea, respectively. M12-BP4C cells demonstrated a
higher level of DNA end-labeling in both treatments when compared with
the control cells, indicating that apoptosis was increased in IGFBP-4
overexpressing cells.
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), a marked increase in apoptosis
was seen in the M12BP-4C clone (lane 3) compared with the control clone
(lane 2) as indicated by the increased DNA fragmentation. Lane 1 is DNA
from attached M12-pcDNA3 cells that did not receive 6-hydroxyurea
treatment and is included as a negative control for apoptosis.
Effect of overexpression of IGFBP-4 on tumorigenesis in vivo
To determine the effect of IGFBP-4 overexpression in
vivo, 1 x 106 cells from each of three M12
derived cell lines (M12-pcDNA3, -BP4A, and -BP4C) were injected sc into
sets of 10 nude, athymic, male mice and tumor growth, determined as
number of mice developing new tumors over a 9-week-period, was
determined (Fig. 7). The total number of
tumors formed in mice receiving injections of M12-BP4A and -BP4C clonal
cells was lower than in mice receiving control cells over the 9-week
postinjection period. At the end of 9 weeks, all 10 control mice had
developed tumors, whereas only 5 and 6 mice had developed tumors in the
M12-BP4C and -BP4A treated mice, respectively. When comparing the rate
of new tumor formation, at weeks 5 and 6 there was a significantly
(P < 0.01) lower rate of new tumor formation in both
M12-BP4A and-BP4C treated mice, whereas at week 7 only the M12-BP4C
treated mice showed a significantly (P < 0.01) lower
rate of new tumor formation compared with the M12-pcDNA3 control cells.
By week 8 there was no significant difference in rate of new tumor
formation between any of the treatments.
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). Therefore, the loss of inhibition of
tumorigenesis was not due to a loss of IGFBP-4 expression. Preliminary
data (unpublished) comparing levels of IGFBP-4 in conditioned medium of
the M12-BP4C and -pcDNA3 tumor derived cells, as assayed by Western
ligand blot analysis, and grown in the presence or absence of 100 ng/ml
IGF-II indicate no decrease in IGFBP-4 levels in the presence of
IGF-II. These preliminary data suggest that proteolysis of IGFBP-4,
which is enhanced by the presence of IGFs (for review see Ref. 3) is
not a factor contributing to the loss of inhibition of tumor growth in
the M12-BP4C tumor derived cells in vitro. The expression of
IGFBP-2 also remained low in the M12-BP4C derived tumor cells, which is
consistent with expression patterns seen in the M12-BP4 cell lines
in vitro.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Overexpression of IGFBP-4 did not, however, result in long-term inhibition of anchorage-independent growth of M12 cells in vitro, nor inhibition of tumor development in vivo. Both colony numbers in vitro and tumor growth in vivo approximated control values by the end of the experiments, despite a significant lag phase in early growth rates. Western radioligand blot analysis of cells derived from tumors demonstrated that the loss of inhibition was not due to loss of expression of transgenic IGFBP-4 over extended time, indicating the interaction of an additional compensatory mechanism.
Possible explanations for the loss of inhibition of tumorigenesis by IGFBP-4 in the M12 background are as follows:
1) Proteolysis of IGFBP-4. There are well documented examples of proteolysis of IGFBP-4 in several systems (6). The IGFBP-4 proteolyzing enzyme Cathepsin, which is stimulated by IGF, is expressed in cultured prostatic carcinoma cells (40). However, our in vivo data indicate that the effect of proteolysis of IGFBP-4 was probably minimal in this system. Western radioligand blot analysis indicated high levels of expression of IGFBP-4 in cells derived from a 10-week-old M12-BP4C tumor, a time when inhibition of tumorigenesis was lost. Unpublished data from preliminary experiments using control and M12-BP4C tumor-derived cells indicate there is no change in IGFBP-4 protein levels in the medium of these cell cultures in the presence or absence of 100 ng/ml IGF-II. Although preliminary, these results support the suggestion that proteolysis is not a significant factor determining the loss of inhibition of tumorigenesis in M12-BP4C derived tumors.
2) Production of autocrine IGF goes up sufficiently to titrate out the elevated levels of IGFBP-4, thus increasing the amount of free IGF available to bind to the IGF-IR. Data are not currently available to support or refute this possibility; however, our results warrant further investigation to determine whether an increase in IGF expression does occur over time in M12-BP4 cell lines and tumors.
3) That the IGF axis is bypassed. Although it is apparent that the IGF axis is required for normal development of the prostate and for initial cell transformation in cancer development (41, 42), it may be less important in later stages of carcinogenesis. In the progression from the benign, immortalized P69 cells (the parental prostate cell line from which M12 was derived) to the M12 cell line, there is a concomitant decrease in the number of IGF-IRs and proliferative response to IGF (22, 43). M12 cells have been shown to be less responsive to IGF-stimulated proliferation than the parental P69 cell line, where receptor number is high and IGF-II levels are lower. In M12 cells, the IGF-IRs appear to be maximally stimulated by the high levels of autocrine IGF-II, and addition of exogenous IGF has little proliferative effect (22). This progressive decrease in the number of IGF-IRs and increase in IGF-II levels, as cells become more malignant, is mirrored in vivo (12). Although it is unclear what effect this down-regulation of the receptor may play in cancer formation, it may indicate that as cancer progresses, there is either a lower dependence on the IGF axis for growth, or an increase in postreceptor signaling. However, we saw an initial inhibition of tumor growth with overexpression of IGFBP-4 and no indications of an IGF-independent IGFBP-4 effect, indicating the IGF ligand still exerts a growth potentiating function in the highly malignant M12 cells.
Western ligand blot analysis of media collected from cells overexpressing IGFBP-4 had lower levels of IGFBP-2. This down-regulation of IGFBP-2 associated with IGFBP-4 overexpression was seen in cultures of the transfected cells as well as cultures of tumor-derived cells. Cell lines M12-BP4pop and -BP4C, which express IGFBP-4 at high levels, had the lowest levels of IGFBP-2 expression, whereas M12-BP4A cells expressed low to intermediate levels of IGFBP-4 and demonstrated a higher level of IGFBP-2 expression. The control M12-pcDNA3 cells, which have the lowest IGFBP-4 expression, also had the highest level of IGFBP-2 protein expression. The decrease in IGFBP-2 expression in M12-BP4 cell lines is probably due to a decrease in IGF availability. In a recent study by Wang et al. (44), it was shown that IGFBP-2 expression was down-regulated in glioma cells expressing an IGF-I antisense transcript. This study also found that IGFBP-2 acted synergistically with IGF-I to enhance glioma cell growth. IGFBP-2 expression is increased in many cell lines derived from solid tumors (45), and expression is up-regulated by IGF-I in breast cancer cells (46). We have found IGFBP-2 expression increased in both M12 cells and in prostate adenocarcinomas (16). The role IGFBP-2 plays in prostate cancer is not known, but its expression appears to be positively correlated with increasing degrees of prostate cell malignancy and its role in the development or progression of prostate cancer warrants further investigation.
Our results reinforce the importance of the IGF system in the development and progression of prostate cancer. Although it is generally agreed that the IGF-IR is responsible for the mitogenic, transforming, differentiation, and antiapoptotic effects of the IGF ligands, enhancement of apoptosis may also be a function of the IGF-IR. When the IGF-IR was reexpressed in M12 cells to a level comparable to that expressed in the benign parental P69 cell line, there was inhibition of tumorigenesis and an increase in apoptosis (43, 47) by an as yet unknown mechanism. Recent studies by Liu et al. and O’Conner et al. suggest there is a proapoptotic domain on the carboxy-terminus of the b-subunit of the IGF-IR (48, 49). It has also been suggested that receptor number per cell may be one factor controlling activation of various functions of the IGF-IR (50). The IGF system may have multiple functions in tumor development that may include a gain in tumorigenic function by stimulation through increased IGF production, and/or a loss of antitumorigenic function as may occur with a loss of a putative pro-apoptotic activity.
The present study was undertaken to determine whether inhibition of IGF action via overexpression of an inhibitory IGFBP would result in decreased growth of prostate cancer cells. There was an initial period of significant inhibition of tumorigenesis in both clonal and polyclonal M12-BP4 cell lines in vitro and in vivo as assayed by anchorage independent growth and tumor development respectively. Although inhibition of tumorigenesis was transient in the M12 transfected cell lines, the increased sensitivity to apoptosis seen in vitro suggests that in combination with other treatments that predispose cells to apoptosis, overexpression of IGFBP-4 may provide improved therapy in the treatment of prostate cancer.
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Acknowledgments |
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Footnotes |
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Received December 23, 1997.
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References |
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S. Mazerbourg, J. Zapf, R. S. Bar, D. R. Brigstock, C. Lalou, M. Binoux, and P. Monget Insulin-Like Growth Factor Binding Protein-4 Proteolytic Degradation in Ovine Preovulatory Follicles: Studies of Underlying Mechanisms Endocrinology, September 1, 1999; 140(9): 4175 - 4184. [Abstract] [Full Text]
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Z.-D. Xu, L. Oey, S. Mohan, M. H. Kawachi, N.-S. Lee, J. J. Rossi, and Y. Fujita-Yamaguchi Hammerhead Ribozyme-Mediated Cleavage of the Human Insulin-Like Growth Factor-II Ribonucleic Acid in Vitro and in Prostate Cancer Cells Endocrinology, May 1, 1999; 140(5): 2134 - 2144. [Abstract] [Full Text]
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C. C. Sprenger, S. E. Damon, V. Hwa, R. G. Rosenfeld, and S. R. Plymate Insulin-like Growth Factor Binding Protein-related Protein 1 (IGFBP-rP1) Is a Potential Tumor Suppressor Protein for Prostate Cancer Cancer Res., May 1, 1999; 59(10): 2370 - 2375. [Abstract] [Full Text] [PDF]
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], M12-BP4pop [
], M12-BP4C cells 
in the presence of
0–100 ng/ml IGF-II using the 3-[4,5
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay described
in Materials and Methods. Note the M12-pcDNA3 controls
demonstrate significantly greater proliferation at all concentrations
of IGF-II. The M12-BP4pop and M12-BP4C cells demonstrate significant
proliferation responses to higher doses of IGF-II. Each value
represents the mean ± SEM for three replicate
experiments. *, (P 
and M12-BP4C
in male, athymic/nude mice. Measurements were performed at 1-week
intervals. Note significantly slower rates of new tumor development in
mice receiving -BP4C and -BP4A cells at weeks 5 and 6 (*,
P 