(Received for publication, May 11, 1994; and in revised form, October 24, 1994)
From the
The reduced growth factor requirements of murine fibroblasts transformed by simian virus 40 (SV 40) have been attributed to insulin-like growth factor (IGF)-I induction by T antigen and consequent activation of IGF-I receptor signaling. The present study shows that the autonomous growth of SV 40-transformed human fibroblasts also requires type-I IGF-I receptor activation but that this is not due to de novo induction of IGF-I gene expression since untransformed human fibroblasts, which fail to proliferate in the absence of serum, also showed IGF-I gene expression under serum-free conditions. DNA synthesis assays confirmed that untransformed cells were responsive to exogenous IGF and indicated that transformed cells were already maximally stimulated. In untransformed fibroblasts, IGF binding was principally to abundant membrane-associated IGFBP-5, whereas in transformed fibroblasts this protein was minimally expressed, and IGF binding was to IGF receptors. Loss of detectable membrane-associated IGFBP-5 in transformed cells was associated with diminished IGFBP-5 gene expression and with loss of IGF-II gene expression. Exogenous IGFBP-5 associated with the membranes of transformed cells and inhibited the autocrine growth of these cells. These findings suggest that loss of IGFBP-5 in SV 40-transformed fibroblasts facilitates interaction of endogenously produced IGF-I with the IGF-I receptor and increases their sensitivity to autocrine stimulation.
The transformation of cells by simian virus 40 (SV 40) is a
multistep process involving a number of cellular and genetic changes (1, 2, 3, 4) . Although the
mechanisms involved are unclear, evidence suggests both activation of
autocrine growth pathways and the inactivation of negative
growth-regulating proteins. Thus, cells transformed by SV 40 show
secretion of growth-promoting substances (5, 6, 7) and reduced requirement for
exogenous growth
factors(1, 2, 3, 4, 5, 6, 7) .
Large tumor antigen has been shown to interact with and inhibit the
growth-suppressive action of cellular p53 protein and
p110, the product of the retinoblastoma susceptibility
gene (for review, see (8) ). Furthermore, SV 40 small tumor
antigen has been shown to induce cell growth through blockade of
protein phosphatase and deregulation of the mitogen-activated protein
kinase cascade(9) .
A recent study of the effect of SV 40 T
antigen on the growth factor requirements of BALB 3T3 cells provided
evidence that activation of the insulin-like growth factor
(IGF)-I receptor is essential for growth stimulation by T
antigen and that SV 40 T antigen transcriptionally regulates IGF-I gene
expression(10) . Although other functions of T antigen are
probably required for transformation in these cells, these findings are
consistent with other reports, suggesting a major role for the IGFs and
their receptors as mediators of cell transformation. Studies in murine
fibroblasts have shown that overexpression of a functional IGF-I
receptor permits growth in soft agar (11) and that constitutive
expression of IGF-I and its receptor abrogates all requirements for
exogenous growth factors(12) . Transfection of the cellular
proto-oncogene c-myb, which induces IGF-I gene expression, has
been shown to confer growth autonomy on mouse fibroblasts that
constitutively express c-myc(13) . More recently,
studies involving targeted disruption of the IGF-I receptor gene have
demonstrated that IGF-I receptor signaling is an indispensable
component of the SV 40-transformation pathway since SV 40 T antigen is
unable to transform mouse embryonic fibroblasts lacking the type-I
IGF-I receptor(14) .
However, it is becoming increasingly clear that regulation of cellular processes by the IGFs involves not only their interaction with receptors but also a complex interplay between the IGFs and membrane-associated and secreted IGF binding proteins. Six distinct IGFBPs (designated IGFBP1-6) have been isolated to date, and their cDNAs have been cloned(15, 16) . These proteins have been shown to modulate the proliferative and metabolic effects of the IGFs, with inhibition of IGF action being most frequently observed(17, 18, 19) . The most compelling evidence for IGFBPs as negative regulators of cell proliferation derives from IGFBP-3 gene transfection studies, which demonstrate that the endogenous production of IGFBP-3 in transfected murine fibroblasts has a profound growth inhibitory effect in these cells(20) . In contrast, IGFBP-3 gene transfection into breast carcinoma cells resulted in potentiation of IGF action in these cells, an observation thought to be related to the ability of IGFBP-3 to block IGF-I-induced receptor down-regulation(21) . Although the molecular mechanisms involved in the interaction of the IGFs with IGFBPs are unclear, these molecules appear to regulate the availability of free IGFs for receptor binding(18, 22) . Given the regulatory effects of IGFBPs on IGF action, it is not surprising that in addition to altered IGF expression, changes in IGFBP production have also been observed in transformed animal and human cell lines(23, 24) . Such observations led us to postulate that changes in IGF interaction with regulatory IGFBPs may be of fundamental importance in effecting the autonomous growth potential of transformed cells.
To address this question, the present study investigates the IGF-IGFBP axis in SV 40-transformed human fibroblasts and their untransformed counterparts. We show that IGFBP-5 gene expression and the expression of membrane-associated and secreted IGFBP-5 is markedly diminished in SV 40-transformed fibroblasts and present evidence that this may be causally involved in the increased sensitivity of these cells to IGF-I mediated autocrine stimulation, which confers autonomous growth potential.
The ability of IGFBP-5 to associate with the cell surface of
transformed cells was investigated by incubating either cells or
membranes with 100 nM IGFBP-5 in either SFM (cells) or PBS
(membranes) for 24 h at 37 °C. Cells or membranes were then
extensively washed with ice-cold PBS and were cross-linked to I-IGF-I as described above. Samples were solubilized in
sample buffer and electrophoresed on 3-15% linear gradient gels,
dried, and autoradiographed.
The protocol
for immunoprecipitation of secreted IGFBPs was essentially as described
for membrane-associated IGFBPs except that for cross-linking,
conditioned medium (50 µl) diluted in 0.5 M sodium
phosphate buffer (pH 7.4) was pre-incubated in ice with 200,000 cpm of I-IGF-II for 30 min. Cross-linking was accomplished by
addition of disuccinimidyl suberate to give a final concentration of
0.1 mM; incubation and quenching was as described above.
For Western immunoblot analyses, proteins were electrophoresed on
12.5 (for analysis of IGFBPs) and 7.5% SDS-polyacrylamide gels (for
analysis of IGF-I receptor expression) under reducing conditions and
transferred to cellulose nitrate paper as described
elsewhere(22) . After transfer, additional protein binding
sites on the nitrocellulose paper were blocked by incubation overnight
in 5 mM EDTA, 0.25% gelatin, 0.01 M NaN,
0.15 M NaCl, 0.05 M Tris-base, and Nonidet P-40 (NGA
buffer). The paper was then incubated overnight at 4 °C with either
rabbit anti-IGFBP antisera in NGA buffer (dilutions as for
immunoprecipitation) or mouse monoclonal antibody IGFR 1-2
diluted 1:500 in NGA buffer. After washing, affinity-purified
I-labeled protein A or
I-labeled sheep
anti-mouse Ig F(ab`)
fragment was used to visualize,
respectively, rabbit anti-IGFBP antibody binding and mouse anti-IGF-I
receptor binding.
Figure 1:
Growth of untransformed and transformed
fibroblasts in 1% serum and serum-free medium. , untransformed
cells in either 1% serum or serum-free medium;
, transformed
cells in 1% serum;
, transformed cells in serum-free
medium.
Fig. 2shows DNA synthesis in untransformed
and transformed cells cultured in SFM in the presence or absence of
-IR3, an antibody specific for the IGF-I receptor. It can be seen
that basal DNA synthesis in transformed cells was approximately 3 times
that of untransformed cells and that treatment with
-IR3 markedly
inhibited DNA synthesis in these cells. In contrast, it can be seen
that
-IR3 had no significant effect on the basal level of DNA
synthesis in untransformed cells.
Figure 2:
Effect of an antibody to type-I IGF-I
receptor on basal DNA synthesis in untransformed (UNTF) and
transformed (TF) fibroblasts. Cells were cultured in
serum-free medium for 24 h and then for a further 24 h with ╡ or
without 10 µg/ml
-IR3 prior to
[
H]thymidine
incorporation.
Fig. 3A shows the effect of increasing concentrations of IGF-I and IGF-II on DNA synthesis in untransformed cells cultured in SFM. It can be seen that concentrations of IGF-I between 0.3 and 10.0 nM stimulated DNA synthesis and that outside this concentration range IGF-I had no significant effect. In contrast, untransformed fibroblasts were much less responsive to IGF-II stimulation with little stimulation occurring below a concentration of 300 nM. Fig. 3B shows that neither IGF-I nor IGF-II stimulated DNA synthesis in transformed cells and that concentrations of IGF-I and IGF-II in the range of 100 nM-1.0 µM inhibited DNA synthesis in these cells.
Figure 3:
Effect of IGF-I and IGF-II on DNA
synthesis in untransformed (panel A) and transformed
fibroblasts (panel B). Cells were maintained in serum-free
medium for 24 h prior to stimulation with IGF-I or IGF-II
╡ and [
H]thymidine
incorporation.
Figure 4: RT-PCR analysis of IGF-I gene expression in untransformed (UNTF) and transformed (TF) fibroblasts. Cells were grown in either FCS-containing medium (+FCS) or in the absence of FCS (-FCS) for 48 h prior to RNA extraction and RT-PCR analysis as described under ``Materials and Methods.''
Figure 5: Inhibition of IGF-I gene expression in transformed fibroblasts growing in serum-free medium by insulin and IGF-II. Transformed fibroblasts were either grown in the presence of FCS, in SFM, or in SFM supplemented with either 10 µg/ml insulin (SFM + INS) or 10 nM IGF-II (SFM + IGF-II) for 24 h prior to RNA extraction and RT-PCR analysis as described under ``Materials and Methods.''
It can be seen from Fig. 6that whereas IGF-II gene expression occurred in untransformed cells in the presence or absence of FCS, as evidenced by the detection of the expected 345-bp amplification product, transformed cells showed no detectable IGF-II gene expression under either condition.
Figure 6: RT-PCR analysis of IGF-II gene expression in untransformed (UNTF) and transformed (TF) fibroblasts. Cells were grown in either FCS containing medium (+FCS) or in the absence of FCS (-FCS) for 48 h prior to RNA extraction and RT-PCR analysis as described under ``Materials and Methods.''
Figure 7:
Detection of IGF binding sites on the
membranes of untransformed and transformed fibroblasts. Membrane
proteins from untransformed (panelA) and transformed (panelB) fibroblasts were affinity labeled with I-IGF-I (lanes1-4) and
I-IGF-II (lanes5-8) and
electrophoresed under reducing conditions on 3-15% linear
gradient SDS-polyacrylamide gels. Incubations of membranes with
radiolabeled peptide were performed as follows: lanes1 and 5, without competing peptide; lanes2 and 6, with 100 nM IGF-I; lanes3 and 7, with 100 nM IGF-II; lanes4 and 8, with 10 µg/ml
insulin.
In
contrast, in transformed cells (Fig. 7, panelB) in the absence of competing peptide, both I-IGF-I (lane1) and
I-IGF-II (lane5) were cross-linked
predominantly to high molecular weight proteins with apparent M
135,000 and M
>218,000.
It can be seen in panelB that the presence of
competing IGF-I (lane2), IGF-II (lane3), and to a lesser extent insulin (lane4) inhibited formation of
I-IGF-I-protein
complex with M
135,000, consistent with
I-IGF-I binding to the
-subunit of the type-I IGF-I
receptor on transformed cells(37) . The M
>218,000 complex behaved similarly and presumably represents
I-IGF-I binding to an incompletely reduced
-
-dimer as previously described(37) . In contrast to
the
I-IGF-I-complex with apparent M
>218,000, the
I-IGF-II-complex with M
>218,000 detected in the absence of competing
peptide (lane5) was not abolished by competing IGF-I (lane6) and insulin (lane8),
although both peptides caused some diminution in labeling intensity.
Formation of this complex was, however, completely inhibited by IGF-II (lane7). These findings suggest that the
I-IGF-II band with M
>218,000
predominantly represents
I-IGF-II binding to the type-II
IGF receptor and also to a lesser extent
I-IGF-II binding
to the incompletely reduced
-
dimer of the IGF-I receptor.
The
I-IGF-II-complex with M
>135,000 detected in the absence of competing peptide (lane5) behaved in the same way as the
I-IGF-I-complex with M
>135,000 (lane1), being inhibited by competing IGF-I (lane6) and IGF-II (lane7) and by
insulin (lane8). These findings indicate that in
transformed fibroblasts in the absence of competing peptide,
I-IGF-II binds to both the type-I and the type-II IGF
receptor(37) .
In untransformed fibroblasts (Fig. 7, panelA) in the absence of competing peptide, I-IGF-I binding to the
-subunit of type I receptor
with M
135,000 (lane1) and
I-IGF-II binding to the type-II receptor with approximate M
>218,000 (lane5) was only
weakly detected. Both IGF-I (lane2) and IGF-II (lane3) and to a lesser extent insulin (lane4) inhibited
I-IGF-I binding to the
-subunit of the type-I IGF-I receptor. However, it can be seen
that whereas IGF-II completely abolished the binding of
I-IGF-II to the type-II receptor (lane7), the presence of competing cold IGF-I markedly
potentiated the cross-linking of
I-IGF-II to IGF-II
receptor (lane6). This potentiation of type-II
receptor binding was associated with partial inhibition of
I-IGF-II cross-linking to the low molecular weight IGFBP
by cold IGF-I. Taken together, these findings demonstrate that IGF-II
interacts predominantly with membrane-associated IGFBP in the absence
of IGF-I and with the type-II receptor in the presence of competing
IGF-I, this probably reflecting differences in the relative affinities
of the IGFBP and the type-II receptor for the radiolabeled IGF-II and
the cold IGF-I.
Figure 8:
A,
immunoprecipitation of IGFBP-5 protein in the membranes of
untransformed (UNTF) and transformed (TF)
fibroblasts. Lanes1 and 3, membrane
proteins affinity labeled with I-IGF-II; lanes2 and 4, membrane proteins affinity labeled with
I-IGF-II and immunoprecipitated by anti-IGFBP-5
antiserum. B, immunoblotting of IGFBP-5 protein in the
membranes of untransformed and transformed fibroblasts. Membrane
proteins from untransformed and transformed fibroblasts were
electrophoresed on a 12.5% SDS-polyacrylamide gel, transferred to
cellulose nitrate paper, and immunoblotted with rabbit anti-IGFBP-5
antisera. Antibody binding was visualized using
I-protein
A and autoradiography as described under ``Materials and
Methods.''
Figure 9:
Characterization of IGFBP-4, IGFBP-5, and
IGFBP-6 secretion by untransformed (UNTF) and transformed (TF) fibroblasts. Proteins in conditioned medium were affinity
labeled with I-IGF-II and immunoprecipitated with
antisera directed against IGFBP-4, IGFBP-5, and IGFBP-6. Antisera
cross-reactivities are given in
``Results.''
Figure 10: Northern blot analysis of IGFBP-5 gene expression in untransformed (UNTF) and transformed (TF) fibroblasts. Cells were grown in either FCS containing medium (+FCS) or in the absence of FCS (-FCS) for 48 h prior to RNA extraction and Northern blot analysis as described under ``Materials and Methods.'' Filters were reprobed with an actin cDNA probe to confirm approximately equal loading of RNA in all tracks.
Figure 11:
Association of IGFBP-5 with the membrane
of transformed cells. Untreated membranes from transformed
cells(-) or membranes treated with 100 nM IGFBP-5 for 24
h (+) were affinity labeled with I-IGF-I and
electrophoresed under reducing conditions on a 3-15%
SDS-polyacrylamide gel. The smalldoublearrows indicate incompletely reduced
-
dimer of the type-I
receptor with apparent M
>218,000, and the smallsinglearrow indicates the
-subunit with M
135,000. Affinity labeling of
membranes pre-incubated with IGFBP-5 reveals a prominent
I-IGF-I-IGFBP-5 complex with apparent M
38,000.
Fig. 12A shows that addition of IGFBP-5 to cultures
of transformed cells growing under serum-free conditions inhibited DNA
synthesis in these cells to a level similar to that seen with
-IR3. Fig. 12B shows that DNA synthesis in cells
pretreated with IGFBP-5 for 24 h and subsequently incubated for a
further 24 h in the absence of IGFBP-5 prior to
[
H]thymidine addition is markedly less than that
in untreated cells. In contrast, IGFBP-5 had no effect on the basal DNA
synthesis of untransformed cells ([
H]thymidine
incorporation, 881 ± 110 dpm (mean ± S.D.) in absence of
100 nM IGFBP-5 versus 855 ± 82 dpm (mean
± S.D.) in presence of IGFBP-5).
Figure 12:
IGFBP-5 inhibition of DNA synthesis in
transformed cells. PanelA, cells were cultured in
serum-free medium in the absence (control) or presence of
either -IR3 (10 µg/ml) or IGFBP-5 (100 nM) for 48 h
prior to addition of [
H]thymidine. PanelB, pretreated cells were incubated in IGFBP-5 (100
nM) in serum-free medium for 24 h, extensively washed, and
incubated for a further 24 h in serum-free medium prior to addition of
[
H]thymidine.
Previous studies have shown that untransformed human
fibroblasts secrete IGFBP-3, IGFBP-4, and IGFBP-5 (31, 38, 39) and that IGFBP-3 and IGFBP-5 are
able to associate with the cell surface of
fibroblasts(31, 38) . Indeed, cell surface-associated
IGFBPs have been shown to represent the majority of I-IGF-I binding sites on human fibroblasts (38) with IGFBP-3 being the main form of IGFBP binding to
fibroblast surfaces studied to
date(38, 40, 41) . In contrast, in the
present study IGFBP-5 was identified as the major membrane-associated
IGFBP present on human untransformed MRC-5 fibroblasts, suggesting that
the surface expression of IGFBPs can differ between fibroblast strains.
Importantly, the association of IGFBPs with cell membranes has been
shown to change IGF-I cellular binding in a manner suggestive of direct
alteration of binding to the type-I receptor(31, 38) .
This raises the possibility that membrane-associated IGFBPs are
important regulators of IGF action and that a quantitative change in
the expression of these proteins has the potential for altering
cellular responsiveness to IGF stimulation. These observations are
particularly pertinent given the findings of the present study, which
demonstrate a marked reduction in surface expression of IGFBP-5 in
cells transformed by SV 40 T antigen. The different levels of
membrane-associated IGFBP-5 detected in untransformed and transformed
fibroblasts clearly alters the nature of IGF-cell interaction as
evidenced by membrane-cross-linking studies, which show that in
untransformed cells, IGF binding is principally to a 31-kDa protein
identified as IGFBP-5 by immunoprecipitation, whereas in transformed
fibroblasts IGF binding is to the type-I and type-II receptors. These
observations are consistent with the contention that surface IGFBP-5
acts as a reservoir of IGF binding sites, which effectively competes
with IGF receptors for ligand binding.
The finding that the transformed phenotype in these cells is associated with loss of membrane-associated and secreted IGFBP-5 is particularly interesting given that the capacity of SV 40-transformed fibroblasts for autonomous growth is mediated, at least in part, by the activation of an IGF-I autocrine loop. Hence in the present study, the autocrine growth of transformed cells in serum-free medium was associated with activation of IGF-I gene expression and was inhibited by an antibody specific for the type-I IGF-I receptor. These findings are consistent with a previous report demonstrating that in murine fibroblasts, the growth-stimulatory effect of SV 40 T antigen requires interaction of IGF-I with its receptor(10) . In the latter study, no evidence for IGF-I gene expression was observed in untransformed cells, and the ability of transformed murine fibroblasts to grow under conditions of serum deprivation was ascribed to transcriptional activation of IGF-I gene expression by T antigen. Since in the present study both untransformed and transformed fibroblasts showed IGF-I gene expression when cultured in the absence of serum, the unique property of transformed human fibroblasts to proliferate in the absence of exogenous growth factors can not be attributed to activation of de novo IGF-I gene expression by T antigen. One possible explanation is that loss of membrane-associated IGFBP-5 in transformed fibroblasts facilitates interaction of endogenously produced IGF-I with the IGF-I receptor, thereby increasing the sensitivity of these cells to IGF-I-mediated autocrine stimulation. Indeed, Kiefer et al.(32) have previously demonstrated that IGFBP-5 is a potent inhibitor of IGF-I-stimulated DNA synthesis in osteosarcoma cells, a finding compatible with the 40-fold higher affinity of IGFBP-5 for IGF-I compared with that of the type-I receptor for IGF-I(32) .
Several lines of evidence support the contention that loss of IGFBP-5 may be causally involved in the activation of IGF-I-mediated autocrine growth in SV 40-transformed fibroblasts. First, binding of exogenously added IGFBP-5 to the membranes of transformed fibroblasts was associated with inhibition of IGF-I receptor binding as evidenced by a diminution in the affinity labeling of IGF receptors in the presence of membrane-bound IGFBP-5. Second, treatment of transformed fibroblasts with IGFBP-5 markedly inhibited DNA synthesis in serum-free medium. Importantly, DNA synthesis was inhibited in transformed fibroblasts pretreated with IGFBP-5 and subsequently washed free of unbound protein, confirming that the presence of membrane-associated IGFBP-5 alone is sufficient to inhibit the IGF-I-stimulated autocrine growth of these cells (although we cannot rule out that IGFBP-5 exerts its inhibitory effect subsequent to release from the cell surface). These findings in transformed cells treated with IGFBP-5 parallel those in untransformed cells, where the expression of membrane-associated and secreted IGFBP-5 presumably similarly diminishes interaction of endogenously produced IGFs with IGF receptors, thereby resulting in the observed inhibition of cell proliferation under serum-free conditions. Third, in the absence of IGFBP-5, the basal level of DNA synthesis in transformed fibroblasts was markedly higher than that of untransformed cells and did not increase further in the presence of exogenous IGF-I or IGF-II over a concentration range of 0.1 nM-1 µM, suggesting that they are already maximally stimulated by endogenous IGF-I. Indeed, concentrations of IGF-I and IGF-II in excess of 100 nM were growth inhibitory in transformed cells. In contrast, untransformed cells were able to proliferate in serum-free medium only after stimulation with concentrations of 1.0-100 nM IGF-I, which presumably overcome the inhibitory effects of endogenous IGFBP-5 production. Interestingly, concentrations of IGF-II in excess of 100 nM were required to stimulate DNA synthesis in untransformed cells, this possibly reflecting the much higher affinity of IGFBP-5 for IGF-II(32, 42) . The affinities of membrane-associated and secreted IGFBP-5 produced by the untransformed human fibroblasts used in the present study have not been determined, and it is possible that these may be different from those previously reported(32) . However, our findings are consistent with the observations of Kiefer et al.(32) that IGF-II is 50-100 times less potent than IGF-I in stimulating DNA and glycogen synthesis in osteosarcoma cells and, importantly, that IGFBP-5 is a more potent inhibitor of IGF-II than of IGF-I. Indeed, in the latter studies a 5 M excess of IGF-II failed to reverse the inhibitory effect of IGFBP-5. These findings, together with those of the present study, indicate that IGFBP-5 is a major determinant of IGF responsiveness and support the likely though unproven hypothesis that the diminished expression of IGFBP-5 in transformed fibroblasts is causally involved in the increased sensitivity of transformed cells to IGF-I autocrine stimulation. We propose that loss of IGFBP-5 binding sites in transformed fibroblasts increases cellular sensitivity to IGF stimulation such that, under serum-free conditions, low concentrations of endogenously produced IGF promote cell proliferation and higher concentrations of exogenously added IGF lead to receptor down-regulation and the observed growth inhibition. It is important to note in this context that while IGF-I gene expression was detected in transformed fibroblasts growing under serum-free conditions, IGF-I gene expression was not detected by RT-PCR when these cells were grown in the presence of serum, suggesting that IGF-I gene expression in transformed fibroblasts is suppressed by one or more factors present in fetal calf serum. The finding that IGF-I gene expression in transformed fibroblasts was inhibited following addition to serum-free medium of either insulin or IGF-II suggests that insulin-like peptides in serum may be responsible for transcriptional repression of IGF-I gene expression in transformed cells. These findings indicate that IGF-I gene activity in these cells is linked to environmental concentrations of IGF-I by IGF-I feedback inhibition, providing a mechanism whereby transformed fibroblasts are able to regulate extracellular concentrations of IGF-I to sustain growth stimulation.
The loss of membrane-associated IGFBP-5 protein seen in transformed fibroblasts is associated with a marked decrease in IGFBP-5 gene expression in these cells, suggesting that SV 40 T antigen, which has been shown to down-regulate the activity of certain genes(8) , may directly or indirectly inhibit IGFBP-5 gene transcription. Although IGFBP-5 gene expression has been shown to be transcriptionally regulated by IGF-I in rat FRTL-5 cells(43) , the activation of IGF-I gene expression seen when transformed fibroblasts were grown in the absence of serum did not result in a concomitant increase in IGFBP-5 gene expression. However, the present study also demonstrates loss of IGF-II gene expression in transformed cells in addition to decreased IGFBP-5 gene expression, and it is possible that these two events are related. Further studies are required to characterize transcriptional regulation of IGF-II and IGFBP-5 in human fibroblasts and the mechanism by which SV 40 antigen(s) alter the expression of these genes in transformed cells. Although reactivation of IGF-II gene expression has been described in several human tumors, a reduction in IGF-II gene dosage has also been described in developmental neoplasms(44) . Importantly, the forced expression of IGF-II from retroviral constructs in tumorigenic fibroblasts suppressed tumor formation in grafts into nude mice(45) . This finding may indicate that loss of IGF-II expression, as seen in the present study, may contribute to deregulated growth cessation typical of transformed cells.
It has been recently shown that IGFBP-5 is able to bind to the extracellular matrix and in particular has been shown to bind to types III and IV collagen, laminin, and fibronectin(46) . Given that IGFBP-5 associates with the cell surface of human fibroblasts, it is tempting to speculate that membrane-associated IGFBP-5 plays an important role in cellular anchoring via its interaction with the extracellular matrix and that its loss from the membranes of transformed fibroblasts contributes to the alterations in cell-cell and cell-matrix adhesion exhibited by these cells.
The findings presented here indicate the importance of loss of IGFBP-5 expression in the activation of the IGF-I autocrine loop in transformed fibroblasts. However, the present study also demonstrates that unlike untransformed cells, transformed fibroblasts failed to secrete detectable IGFBP-4. Since this protein has been shown to be a potent inhibitor of IGF-I action, its loss may also facilitate IGF-I-receptor interaction. The significance of IGFBP-6 secretion by transformed cells is not known. However, this protein has a much higher affinity for IGF-II than for IGF-I(24) , and while it is a potent inhibitor of IGF-II action(47, 48) , it is reported to have little impact on IGF-I effects(47) . On the basis of the findings of the present study, the secretion of IGFBP-6 by transformed fibroblasts clearly does not interfere in a major way with the IGF-I driven autonomous growth of these cells.