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Research Service, Department of Veterans Affairs Puget Sound Health Care System (R.H.D., C.S., K.T., S.R.P.), Tacoma, Washington 98493; and Department of Medicine, University of Washington (R.H.D., S.R.P.), Seattle, Washington 98195
Address all correspondence and requests for reprints to: Dr. Rolf H. Drivdahl, Research Service (151), Veterans Affairs Medical Center, American Lake, Tacoma, Washington 98493. E-mail: drivdahl.rolf_h{at}seattle.va.gov
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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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Endocrine activity of the IGF system is accomplished via a series of complex interactions among the ligands, the IGF-I and IGF-II surface receptors (IGF-IR and IGF-IIR), and a group of high affinity IGF-binding proteins (IGFBPs) (1, 8). In addition, numerous proteases have been described that regulate IGFBP levels (9). Most of the activities of IGFs, including protection against apoptosis, are mediated by the IGF-IR (10, 11). Some or all components of the IGF system are dysregulated in cancer cells (2, 12): in normal prostate, stromal cells are the principal source of both IGFs and IGFBPs, but increased IGF-II messenger RNA (mRNA) and protein were reported in malignant prostate epithelial cells, concomitant with reduced levels of IGF-IR and altered patterns of IGFBP production (7, 13, 14). These data are consistent with numerous studies demonstrating overproduction of IGF-II in human tumor cells (15, 16). Clinical studies have indicated that elevated serum levels of IGF-I represent a significant risk factor for the development of prostate cancer (17).
Six IGFBPs have been cloned and sequenced (18, 19), and recently several proteins with limited homology to the IGFBPs and reduced IGF-binding capacities have been identified and designated IGFBP-related proteins (20). Greater than 90% of circulating IGFs are bound to IGFBPs, and it was originally thought that their primary function was to stabilize the ligands while acting as both a transport vehicle and a means to sequester IGFs from their receptors, thereby inhibiting IGF activity. It has become increasingly clear that IGFBPs can also enhance or potentiate IGF effects and in some instances may act independently of the ligands (21). Control of IGFBP synthesis is therefore an additional and intricate mechanism for modulation of IGF activity. In malignant prostate cells, the pattern of IGFBP expression differs markedly from that seen in normal epithelium; in situ hybridization studies have demonstrated increased IGFBP-2 and decreased IGFBP-3 levels in adenocarcinoma tissue (13). IGFBP-2 was also reported to be elevated, and IGFBP-3 reduced, in serum from patients with prostate cancer (22, 23). The increased IGFBP-2 was positively correlated with serum PSA.
Work in our laboratory indicates that IGFBP-4 and -6 are poorly expressed in normal epithelial cells, but are abundant in several tumor cell lines. We found high constitutive expression of IGFBP-4 mRNA and protein in the ALVA-31, -41, and-101 prostate tumor lines as well as in PC-3 (24). IGFBP-4 production was not regulated by treatment of cells with factors that alter cell growth and expression of other IGFBPs, such as 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], IGF-II, or dihydrotestosterone. Overexpression of IGFBP-4 in the M12 prostate tumor cell line delayed, but did not prevent, tumor formation in nude mice, and the researchers concluded that the observed effects were most likely due to prevention of ligand access to the IGF-IR (25). However, this study and others have also suggested coordinate regulation of IGFBPs, such that altered production of one binding protein may profoundly influence the levels of others. In the M12 line, for example, high IGFBP-4 levels were associated with reduced IGFBP-2. As our work has indicated that IGFBP-4 expression is a constitutive feature of tumor cell lines, we have investigated the consequences of inhibiting IGFBP-4 production in two prostate tumor cell lines, in terms of both tumorigenic potential and synthesis of other IGFBPs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell lines and culture
The ALVA-31 prostate cell line was developed from a primary
tumor obtained by radical prostatectomy, as described by Loop et
al. (26). The M12 line was derived from tumors
developed in nude mice injected with p69SV40T cells; these are human
prostate epithelial cells immortalized with simian virus 40 T antigen
(27). Cells were maintained at 37 C in RPMI 1640
supplemented with 10 ng/ml epidermal growth factor, 0.1
µM dexamethasone, 5 µg/ml insulin, 5 µg/ml
transferrin, 5 ng/ml selenium, and 50 µg/ml gentamicin (RPMI/ITS) in
a 95% air/5% CO2 atmosphere. FCS (5%) was
included at plating, and the medium was replaced with the defined
medium after 24 h. Culture conditions for individual assays are
described below; all in vitro experiments were performed a
minimum of three times.
Vector preparation and transfection
A 505-bp EcoRI/HindIII fragment of the
human IGFBP-4 cDNA was inserted using T4 DNA ligase into
pcDNA3.1Zeo+ cut with EcoRI and
HindIII, resulting in the antisense orientation relative to
the cytomegalovirus promoter. The ligation reaction was used to
transform competent Escherichia coli DH5
cells; cells
were plated on Luria-Bertoni agar containing 50 µg/ml ampicillin, and
miniprep DNA from individual colonies was analyzed by restriction
enzyme digestion for the presence of IGFBP-4 cDNA. Plasmid DNA from a
positive colony was linearized with PvuI and introduced into
ALVA31 and M12 cells by liposome-mediated transfection with pFx-5
(Invitrogen) according to the manufacturer’s
instructions. Control cells were prepared by transfection with
pcDNA3.1Zeo+ alone; these are designated ALVA31pC
and M12pC. After 48 h, cells were passaged into selective medium
containing 100 µg/ml zeocin and cultured for 10 days. Individual
colonies were isolated from the plate by trypsinization in cloning
rings and were maintained in growth medium containing 50 µg/ml
zeocin. IGFBP-4 mRNA and protein expression were determined by
ribonuclease (RNase) protection assays and Western blotting as
described below.
Western blotting
Proteins in culture medium (serum-free) were bound to
nitrocellulose in a dot blot apparatus, washed with water, and dried.
Circles containing the individual samples were cut out with a cork
borer, transferred to microfuge tubes, and boiled in 100 µl SDS
sample buffer [0.05 M Tris (pH 6.8), 2% SDS, and 0.025%
bromophenol blue] containing 8 M urea. Samples were then
subjected to electrophoresis in 12% SDS-polyacrylamide gels and
transferred electrophoretically to nitrocellulose as described by
Hossenlopp et al. (28). The transfer buffer
contained 15 mM Tris base, 120
mM glycine, and 5% methanol. Membranes were
washed successively in Tris-buffered saline [TBS: 20
mM Tris-HCl (pH 7.5) and 0.15
M NaCl] and then with TBS containing 0.3% Tween
20 (TBST). They were blocked in TBST with 5% nonfat milk, incubated
overnight at 4 C with appropriate antibodies in TBST/5% nonfat milk,
and washed extensively with TBST. Bands were detected using horseradish
peroxidase-linked antirabbit secondary antibody and enhanced
chemiluminescence reagents (ECL system, Amersham Pharmacia Biotech, Arlington Heights, IL), according to the
manufacturer’s protocol.
RNA extraction and analysis
RNA was extracted from cells by a minor modification of the
procedure described by Chomczynski and Sacchi (29).
Briefly, the medium was removed, and the cell layer was covered with a
small volume of lysis solution containing 4 M guanidine
isothiocyanate, 0.5% sarcosyl, and 25 mM sodium citrate,
pH 7.0. The lysate was acidified with 0.1 vol sodium acetate, pH 4.0,
and vigorously extracted with a 3:1 mixture of phenol/chloroform. The
aqueous layer was separated by centrifugation at 10,000 x
g, and the RNA was precipitated with isopropanol. RNA was
redissolved in lysis solution, precipitated with ethanol, and
redissolved in 100% formamide. The RNA concentrations were calculated
from the optical densities at 280 and 260 nm.
RNA was fractionated by electrophoresis in 1.25% agarose gels containing 0.66 M formaldehyde and 20 mM MOPS buffer, pH 7.2 (30, 31). Before loading, samples containing 10 µg RNA were denatured at 70 C in 50% formamide, 2.2 M formaldehyde, and 20 mM MOPS (pH 7.2); 0.5 µg ethidium bromide was added to each sample to stain the RNA. After electrophoresis, gels were washed extensively in water and then in 10 x SSC (1 x SSC is 0.015 M sodium citrate and 0.15 M NaCl, pH 7.0), and the RNA was visualized by UV illumination on a Fotodyne, Inc. (New Berlin, WI) transilluminator. Visual analysis of ethidium bromide staining of the 28S and 18S RNA bands was used as a preliminary indication of the integrity and uniform loading of RNA. RNA was then transferred to Gene Screen by capillary blotting in 10 x SSC and cross-linked to the membrane with the Stratalinker apparatus from Stratagene (La Jolla, CA).
cDNA probes and hybridization
Partial cDNA sequences for IGFBPs were obtained in the plasmid
vector pBluescript SK+ (Stratagene).
Plasmids were used to transform competent cells of E. coli
DH5 by heat shock, and recombinant bacteria were grown in
Luria-Bertoni broth containing 100 µg/ml ampicillin
(32). Plasmid DNAs were isolated by a standard alkaline
lysis technique, purified by chromatography on Sephacryl S-400, and
digested with appropriate restriction enzymes to release inserts.
Inserts were separated on 1.5% low melt agarose gels and recovered by
phenol extraction and ethanol precipitation.
Northern blots were prehybridized at 43 C in a Hybaid roller bottle oven (Intermountain Scientific, Salt Lake City, UT); the prehybridization solution was 50% formamide, 6 x SSC, 5 x Denhardt’s solution, 0.1 M NaPO4 (pH 7.2), 10 mM sodium pyrophosphate, and 50 µg/ml sonicated herring sperm DNA (33). 32P-Labeled probes were prepared from cDNA inserts by the random primers technique (34), denatured in 0.3 M NaOH, neutralized, and added directly to the prehybridization solution. Hybridization proceeded overnight at 43 C. The blots were washed at moderate stringency (1 x SSC and 0.1% SDS, 65 C) and exposed to Kodak X-OMAT AR5 film with an intensifying screen at -70 C. Bands were quantitated with an image analyzer equipped with the MCID version 4.2 software (Imaging Research, Inc., St. Catherine, Canada).
Hybridization to a labeled 18S oligonucleotide probe was used as an
additional control for uniform loading. The oligonucleotide was end
labeled with [
-32P]ATP and T4 polynucleotide
kinase and purified on a Sephadex G-25 spin column. Hybridization was
performed at 43 C in the same solution as for cDNA probes, except that
the formamide concentration was 20%. Blots were washed twice at 35 C
in 5 x SSC/0.1% SDS (10 min each), once at 50 C in 5 x
SSC/0.1% SDS (30 min), and finally in 5 x SSC at 35 C for 10
min.
RNase protection assays
Cells from transfected clones were grown to near confluence in
standard RPMI/ITS growth medium, and RNA was extracted as described
above. RNase protection assays (35) were used for specific
detection of the antisense IGFBP-4 transcript. The IGFBP-4 cDNA in
pBluescript SK+ was linearized with
HindIII, and the complementary RNA sense strand probe was
synthesized with T3 RNA polymerase in transcription buffer containing
40 mM Tris-HCl (pH 7.9); 6
mM dithiothreitol; 10 mM
NaCl; 2 mM spermidine; 0.4
mM each of ATP, UTP, and GTP; 1 µg plasmid DNA
template; and 50 µCi [32P]CTP. The reaction
was stopped by adding EDTA to 20 mM final
concentration, and the probe was purified by passage through a Sephadex
G-25 spin column. Approximately 5 x 105 cpm
probe were hybridized overnight with RNA from cultured cells at 45 C in
80% formamide. Single stranded (nonhybridized) RNA was then digested
sequentially with an RNaseA/RNase T1 mixture and with proteinase K, and
the remaining hybrids were extracted with phenol/chloroform and
precipitated in ethanol. The samples were recovered by centrifugation,
redissolved in water, and fractionated in a 6% polyacrylamide gel in
Tris/borate/EDTA buffer, pH 8.3. The gel was dried and exposed to x-ray
film at -70 C with an intensifying screen.
Cell proliferation assays
Relative cell number was assessed by measurement of total DNA
content and by direct cell counting. Cells were seeded in 60-mm dishes
(250,000 cells/dish), and assays were performed after 6 days in
culture. For DNA determinations, medium was removed from cells, and the
monolayer was washed with PBS, followed by extraction with 200 µl 0.5
N NaOH, neutralization with 100 µl O.5 M Tris
HCl (pH 7.4) containing 1 M HCl, and determination of DNA
content by a fluorometric assay with Hoechst 33258, using excitation at
365 nm and measuring emission at 458 nm (36). For direct
measurement of cell number, cells were removed from plates by
trypsinization, resuspended in 1 x PBS, and counted in a
hemocytometer. In control experiments, there was a linear relationship
between total DNA and the cell number determined by hemocytometer
counting.
Poly(ADP-ribose) polymerase (PARP) assays
Cells grown to near confluence were incubated in basal RPMI for
24 h; etoposide was added to a final concentration of 5
µM for the final 4 h. Monolayers were then washed
twice with 1 x PBS and scraped into 1 ml PBS; an aliquot of this
suspension was saved for total protein determination, and the remaining
cells were recovered by centrifugation at 1000 x g for
5 min. The pellet was frozen and thawed twice and resuspended in RIPA
buffer (PBS containing 1% Nonidet P-40, 0.1% SDS, and 0.5% sodium
deoxycholate). The suspension was drawn several times through a
22-gauge needle to shear DNA and then centrifuged to remove debris.
Aliquots were then mixed with an equal volume of 2 x SDS sample
buffer and prepared for PAGE and Western blotting as described above.
Equal amounts of total protein (50 µg) were loaded in each lane.
Samples were fractionated on a 7% gel and transferred to
nitrocellulose, and the resulting blot was incubated with antibody to
PARP and processed in the same manner as described for IGFBP
immunoblots. Antibody to ß-actin was used as a loading control.
Growth in soft agar
Approximately 200,000 cells were seeded in 60-mm petri dishes in
0.25% agar on a 1% agar underlayer (37). Agar contained
RPMI growth medium buffered with 15 mM HEPES, pH 7.4, and
plates were maintained at 37 C in 5% CO2. IGF-II
(or appropriate vehicle control) was added to the top layer at the time
of plating. Colonies greater than 50 µm in diameter were counted 14
and 21 days after plating.
Growth of tumors in nude mice
Nude athymic male mice were injected sc with either M12asBP4 or
M12pC cells (1 x 106 cells/mouse) and
maintained on a laboratory diet ad libitum for 10 weeks.
Tumors were counted and measured weekly. After 10 weeks, tumors were
removed and digested with 0.1% type I collagenase and 50 µg/ml
deoxyribonuclease I by the technique of Peehl and Stamey
(38). Dispersed cells were plated in RPMI growth medium
with 5% FCS for 24 h, and the medium was then replaced with
defined serum-free medium. IGFBP-4 expression was analyzed by Western
blotting as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Expression of this transcript
correlated with reduced quantities of mature protein in the culture
medium; in two of the ALVA31 clones and two of the M12 clones, IGFBP-4
protein was less than 20% of the control value, and these cells also
exhibited the highest expression of antisense RNA. Clones A and B from
ALVA31 and clones A and C from M12 were chosen for further experiments
described below; they are designated ALVA31asBP4A, ALVA31asBP4B,
M12asBP4A, and M12asBP4C.
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, ALVA31asBP4 and M12asBP4 proliferated
at less than 70% of the rate of cells containing vector only. IGF-II
was weakly mitogenic in controls, but contrary to expectations, it was
even less stimulatory in the asBP4 cells.
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). In
the experiment shown, inhibition after 3 weeks was 70% and 56% in
ALVA31asBP4A and ALVA31asBP4B, respectively, and 87% and 82% in
M12asBP4A and M12asBP4C. As with the cellular proliferation studies,
IGF-II had a small stimulatory effect on colony formation in controls,
but virtually no effect in asBP4 cells. Although inhibition of growth
in soft agar was demonstrated after 14 days in M12 cells overexpressing
IGFBP-4 (25), the differences were no longer apparent
after 21 days; in the asBP4 cells, inhibition was maintained for 25
days, at which point the experiments were terminated.
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Cells were maintained in the presence or absence of 100 ng/ml IGF-II
for 24 h before extraction, and apoptosis was induced by addition
of etoposide to a final concentration of 5 µM for the
final 4 h. Figure 4 demonstrates
that although both M12 and ALVA31 are highly resistant to etoposide
induction, the asBP4 lines produce readily detectable quantities of the
85-kDa PARP fragment. Using the intensity of this band as an index of
the extent of apoptosis, the data indicate that apoptosis was initiated
to a lesser extent in the ALVA31asBP4 cells. Preliminary TUNEL assays
(not shown) support this conclusion.
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demonstrates that 8 of the
10 control mice developed tumors, compared with only 2 of the mice
receiving M12asBP4. Additionally, the tumors in the M12asBP4-treated
mice were significantly smaller; tumor volume in mice injected with
M12asBP4 was only 6% of that in control mice (Fig. 5). No delayed increase in tumor
formation in the later stages of the experiment was observed.
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show that inhibition of IGFBP-4
expression, relative to that in the M12 controls, was still evident in
the tumors from M12asBP4-injected mice. The formation of these tumors
was therefore not simply due to a loss of antisense inhibition in
vivo.
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, IGFBP-3 mRNA was strongly up-regulated in the M12asBP4 and ALVA-31asBP4
clones relative to their respective parental lines. The increase in
mRNA in different experiments was consistently 5- to 10-fold; both
parental lines (particularly ALVA-31) produce very low amounts of
IGFBP-3. IGFBP-6 was enhanced 3- to 5-fold in asBP4 cells; the increase
was typically less pronounced in ALVA-31asBP4 clones, because
constitutive production of IGFBP-6 was significantly higher in the
ALVA-31 controls. Western immunoblotting confirmed the results from
Northern analysis, demonstrating markedly enhanced secretion of both
IGFBP-3 and -6 into the culture medium (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have demonstrated that severe inhibition of IGFBP-4 expression by antisense RNA in two prostate cancer cell lines results in slower proliferation in monolayer culture, dramatically decreased colony formation in soft agar, and a heightened sensitivity to the induction of apoptosis by etoposide. In addition, tumor growth in mice was effectively blocked in M12 cells carrying the antisense IGFBP-4 construct, and the inhibitions of both anchorage-independent growth and tumor formation in vivo were maintained for the duration of the experiments.
Overexpression of IGFBP-4 in M12 cells was reported to delay tumorigenesis, and the effect was attributed to sequestration of autocrine IGF and consequent interference with its mitogenic and antiapoptotic activities (25). Logically, underexpression might be expected to exert opposite effects by permitting increased IGF availability. An important difference between the two conditions is the change observed in the expression of other IGFBPs. Overexpression of IGFBP-4 was associated with a decline in IGFBP-2 levels; however, reduced IGFBP-4 correlated with enhanced production of both IGFBP-3 and -6. We have previously found that reduced proliferation and anchorage-independent growth in ALVA-31 and PC-3 cells treated with 1,25-(OH)2D3 are also associated with increased expression of IGFBP-3 and -6. If one of the principal functions of the binding proteins were to prevent survival-promoting actions of IGF, then the increases in IGFBP-3 and IGFBP-6 would afford a more than adequate compensatory mechanism for ligand binding despite the reduced IGFBP-4. This conclusion is substantiated by the lack of response to exogenous IGF-II in the growth experiments here. The primary form of IGF in prostate is IGF-II, and given the emphatic preference of IGFBP-6 for IGF-II, the conditions do not favor free ligand.
Increased IGFBP expression has frequently been invoked as a mediator for suppression of cellular proliferation by hormonal agents. Rozen et al. (41) reported a 15- to 20-fold increase in IGFBP-5 in MCF-7 cells in response to administration of 1,25-(OH)2D3 and several analogs with antiproliferative capability. In PC-3 cells, 1,25-(OH)2D3 and the EB1089 analog stimulated IGFBP-3 synthesis (42), and Nickerson and Hyunh (43) demonstrated dramatic increases in several IGFBPs in rat ventral prostate, concomitant with significant ventral prostate regression induced by EB1089. Transforming growth factor-ß and retinoic acid were also reported to up-regulate IGFBP-3 in PC-3 cells (44). All of these reports concluded that growth inhibition was partially mediated by IGFBPs. We found a correlation between specific IGFBP levels and the extent of transformed cell growth. The mechanism of IGFBP action in asBP4 cell lines is unclear, but is unlikely that simple sequestration of IGF is sufficient to explain why tumorigenesis is delayed in the presence of low IGFBP-2 and high IGFBP-4, but largely prevented in the presence of high IGFBP-3 and -6.
The IGFBPs investigated in this work are diversified in both their physical characteristics and their known physiological actions. IGFBP-3 in particular has been extensively investigated in prostate, and it has been proposed as a prognostic indicator for both prostate and breast cancer. A study of patients with prostate cancer indicated that serum levels of IGFBP-3 were reduced as the disease progressed, whereas IGFBP-2 levels increased (22, 23); the increased IGFBP-2 was positively correlated with serum PSA. Guenette and Tenniswood (45) theorized that IGFBP-3 triggers apoptosis in the prostate after androgen ablation. In vitro work has shown that in addition to its effects on cellular proliferation, IGFBP-3 interacts with cell cycle and apoptotic pathways. It either initiated or potentiated apoptosis in breast cancer cells and in the PC-3 prostate tumor line (46, 47), and in our current study up-regulated IGFBP-3 is associated with enhanced susceptibility to apoptosis induced by etoposide. Constitutive overexpression of IGFBP-3 in M12 cells suppresses the ability to form tumors in mice, concomitant with increased apoptosis and G1 arrest (Plymate, S., personal communication). Other reports indicate that IGFBP-3 is up-regulated by p53 (48, 49). The p53 connection may help to explain the variable quantities of the protein in different cell lines and in clinical studies, given the frequency of p53 mutations in tumors and consequent diminished or undetectable activity (50). IGFBP-3 is also notable among the binding proteins in exertion of IGF-independent effects (21, 51), including the inhibition of cellular proliferation and potentiation of apoptosis. Specific IGFBP-3 receptors have been proposed as mediators of these effects, although the actual proteins have not been identified.
IGFBP-6 is unique in its dramatically higher (50- to 70-fold) affinity for IGF-II, relative to other binding proteins (52). IGF-II expression is elevated in many tumor cell types (16), and its production is increased in malignant prostate (7). One might therefore anticipate a role for IGFBP-6 in regulating tumor growth as well. It associates to only a limited extent with the cell surface, and O-glycosylation renders it resistant to proteolysis, affording the opportunity of posttranslational regulatory mechanisms (53). It has not been studied extensively in prostate, but both mRNA and protein are expressed in normal prostate tissue (14) and at much higher levels in the ALVA-31 and PC-3 cell lines (24, 54). In neuroblastoma cells, transfection with an IGFBP-6 cDNA effected reduced cell proliferation, and the inhibition was observed even in the presence of des(1, 2, 3)IGF-I, suggesting possible IGF-independent effects (55). In general, however, it appears to act by modulation of IGF-II action, usually manifest as inhibition. In L6A1 myoblasts increased IGFBP-6 expression resulted in inhibition of differentiation due to blockade of precursor cell mitosis (56).
We have proposed a tentative model in which IGFBP-6 and IGFBP-3 could act in concert to mediate growth inhibitory effects of 1,25-(OH)2D3. According to this concept, the principal function of IGFBP-6 would be sequestration of IGF-II, whereas IGFBP-3 contributes to as yet undefined mechanisms in cell cycle arrest and/or apoptosis. However, the means by which blockade of IGFBP-4 expression leads to the up-regulation of the other IGFBPs remains problematic. IGFBP-4 has not been shown to have ligand-independent effects, and it has been thought to act primarily through limiting IGF access to the IGF-IR. A temporarily increased availability of IGFs, resulting from the diminished IGFBP-4 expression, may activate a linked production of IGFBP-3 and -6, but this fails to explain the sustained synthesis. Alternatively, IGF might act in concert with IGFBP-4 to suppress the expression of IGFBP-3 and -6, perhaps by chaperoning IGF to the receptor. This hypothesis is consistent with data reported by Menouny et al. (57), who found increased IGFBP-6 and decreased IGFBP-4 in neuroblastoma cells treated with retinoic acid. In an analogous situation in colon cancer cells, IGF-II enhanced glioma cell growth via a synergistic interaction with IGFBP-2 (58), although this effect was not attributed to changes in the production of other IGFBPs. However, we have found that IGFBP-2 mRNA production is very low in ALVA31 and M12 controls, suggesting that IGFBP-4 may be a more significant contributor to malignant growth in these cells.
As noted above, overexpression of IGFBP-4 in M12 cells was also reported to inhibit growth; an important distinction from the present work is that overexpression delayed, but did not prevent, tumorigenesis. Western ligand blot analysis indicated a decrease in IGFBP-2 production, but confirmation of this result by immunoblotting or mRNA expression was not presented, and levels of IGFBP-3 and -6 were not specifically determined. However, abnormally high quantities of IGFBP-4, rather than facilitating IGF activity, may actually interfere with (but not prevent) IGF binding to the receptor and thereby temporarily reduce the rates of growth and tumorigenesis. It would also be interesting to know whether the putative IGFBP-4-specific protease (9) undergoes compensatory up-regulation in overexpressing cells, leading to a delayed increase in free IGF; recent evidence also suggests that the activity of this enzyme is dependent on direct interaction between IGF-II and IGFBP-4 (59).
Control of IGFBP synthesis involves transcriptional, translational, and posttranslational mechanisms as well as an extensive array of proteases that degrade the mature proteins. However this is accomplished, increasing evidence suggests coordinate control of production of the various IGFBPs, resulting in specific IGFBP patterns that contribute to maintenance of the benign or malignant state.
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Footnotes |
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Received September 6, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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