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(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 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 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
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 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
Figure 1:
Growth of untransformed and transformed
fibroblasts in 1% serum and serum-free medium.
Fig. 2shows DNA synthesis in untransformed
and transformed cells cultured in SFM in the presence or absence of
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
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
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
In
contrast, in transformed cells (Fig. 7, panelB) in the absence of competing peptide, both In untransformed fibroblasts (Fig. 7, panelA) in the absence of competing peptide,
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
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
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
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
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
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 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.
Volume 270,
Number 1,
Issue of January 6, 1995 pp. 135-142
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
, 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) .
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) .
Cells
Untransformed and SV 40-transformed MRC-5
human fibroblasts were obtained from Dr. C. Arlett (Medical Research
Council Cell Mutation Unit, University of Sussex, United Kingdom). Full
details of the derivation and characterization of these cells is
described elsewhere(25) . Cells were cultured in Earle's
minimum essential medium (EMEM) supplemented with 10% fetal calf serum
(FCS), 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2 mML-glutamine (Life Technologies, Inc., Paisley, Scotland)
at 37 °C in an atmosphere of 95% air, 5% CO
in a
humidified incubator.Peptides and Radiochemicals
Recombinant IGF-I and
IGF-II were obtained from Bachem Ltd. (Saffron Walden, UK). I-IGF-I (specific activity, 2000 Ci/mmol),
I-IGF-II (specific activity, 2000 Ci/mmol),
[6-
H]thymidine (specific activity, 27 Ci/mmol),
[
-P]dCTP (3000 Ci/mmol),
I-labeled protein A (specific activity, 30 mCi/mg), and
sheep anti-mouse Ig F(ab`)2 fragment (specific activity, 9.2
µCi/µg) were purchased from Amersham International (Aylesbury,
UK). The rabbit anti-IGF-I receptor antiserum (
-IR3) (26) was obtained from Cambridge Bioscience (Cambridge, UK).
The mouse monoclonal antibody IGFR 1-2, which recognizes the C
terminus of the IGF receptor 
subunit(27) , was generously
provided by Professor K. Siddle (Department of Clinical Biochemistry,
University of Cambridge, Addenbrooke's Hospital, Cambridge, UK).
Rabbit antisera directed against IGFBP-1(28) , IGFBP-2 (29) , IGFBP-3(30) , and IGFBP-4 (31) were
obtained from Upstate Biotechnology Inc. Rabbit antisera against
IGFBP-5 and IGFBP-6 and recombinant human IGFBP-5 were obtained from
Austral Biologicals (San Ramon, CA). The recombinant protein was
produced in genetically engineered yeast and purified by affinity
chromatography and high pressure liquid chromatography. Full details of
the binding characteristics and biological activity of the recombinant
IGFBP-5 used in the present study are given elsewhere(32) .Cell Growth Assay
Cells were seeded into 24-well
culture dishes (5 
10
cells/well) in the presence of
10% FCS and cultured for 24 h to allow cell attachment. The medium was
then removed and replaced with medium consisting of EMEM supplemented
with 200 µg/ml bovine serum albumin (BSA) and 10 µg/ml
transferrin or with EMEM containing 1% FCS. Following disaggregation in
200 µl of 0.4% trypsin containing 0.02% EDTA, cells were counted
using a hemocytometer for 5 consecutive days.DNA Synthesis Assay
Cells (5
10/well) were seeded in 96-well plates and were allowed to
adhere in EMEM containing 10% FCS for 24 h. To investigate the effect
of IGF treatment, cells were washed with serum-free medium (SFM) and
incubated in this medium for 24 h. Cells were then cultured in fresh
SFM in the presence or absence of either IGF-I (0.1 nM-1.0
µM) or IGF-II (0.1 nM-1.0 µM), for
24 h followed by incubation in [H]thymidine (0.1
µCi/well) for a further 4 h. The cells were then rinsed twice with
PBS, solubilized in 0.1 M NaOH, 2%
NaCO, 1% SDS, and counted. The effect of IGF-I
receptor blockade on DNA synthesis was investigated by treating cells
in SFM with 10 µg/ml -IR3 for either 24 or 48 h prior to
addition of [H]thymidine. To investigate the
effect of IGFBP-5 treatment on DNA synthesis in transformed
fibroblasts, cells plated in 10% FCS were allowed to adhere, were
washed with SFM, were either incubated in SFM containing IGFBP-5 (100
nM) for 48 h prior to addition of
[H]thymidine or pretreated with IGFBP-5 (100
nM) in SFM for 24 h, and were washed and incubated in SFM for
a further 24 h prior to [H]thymidine addition.
Cells were lysed and counted as described above.Membrane Preparations
Crude membranes were
prepared as previously described with minor modifications(22) .
Briefly, cells were removed from tissue culture flasks using a cell
scraper and were centrifuged at 300 g for 4 min. The
pellet was resuspended in ice-cold lysis buffer consisting of 10 mM Tris-HCl buffer (pH 7.4) containing 4 µg/ml aprotinin, 4
µg/ml leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride
and homogenized by passage through a 26-gauge syringe needle. The
suspension was centrifuged at 450 g for 10 min, and
the resulting supernatant was further centrifuged at 50,000 g for 1 h. The pellet was then resuspended in lysis buffer to
a final protein concentration of approximately 5 mg/ml and stored at
-70 °C until assay.Preparation of Conditioned Media
Semi-confluent
cultures of transformed and untransformed fibroblasts growing in
24-well plates were washed twice with PBS and incubated for 12 h in
SFM. The cells were then washed, and fresh SFM was added for 24 h.
Conditioned medium was harvested, clarified by centrifugation, and
lyophilized.Detection of Membrane IGF Binding Sites by Affinity
Cross-linking
Cell membranes (100 µg) were incubated at 25
°C for 2.5 h with 500,000 cpm of I-IGF-I or
I-IGF-II in 10 mM HEPES (pH 7.4) containing 0.5%
BSA in the presence or absence of unlabeled 100 nM IGF-I, 100
nM IGF-II, and 100 µg/ml insulin. Following centrifugation
at 4500
g, the pellet was resuspended in 0.5 ml of 10
mM HEPES (pH 7.4) without BSA, and the radiolabeled peptide
was cross-linked with disuccinimidyl suberate at a final concentration
of 0.1 mM at 4 °C for 15 min. The reaction was quenched
with 0.5 ml of 50 mM Tris-HCl containing 5 mM EDTA
followed by a second centrifugation. The samples were then solubilized
in SDS sample buffer, boiled 5 min, and subsequently electrophoresed on
a 3-15% acrylamide-separating gel at constant current together
with molecular weight markers (Life Technologies, Inc., Paisley, UK).
The gels were dried at 80 °C for 1 h and exposed to Kodak XAR-2
film.I-IGF-I as described above. Samples were solubilized in
sample buffer and electrophoresed on 3-15% linear gradient gels,
dried, and autoradiographed.
Immunoprecipitation and Immunoblot Analyses
For
immunoprecipitation of membrane-associated IGFBPs, 200 µg of
membrane protein were cross-linked to I-IGF-I/II as
described above. Cross-linked membrane preparations were then
solubilized in 500 µl of 50 mM Tris-base (pH 7.8), 2%
(v/v) Triton X-100, 150 mM NaCl, 0.01% NaN
containing leupeptin, aprotinin, and phenylmethylsulfonyl
fluoride at the concentrations previously given for 1 h at 4 °C
followed by centrifugation at 13500 rpm. Anti-IGFBP antiserum was added
to the supernatant from solubilized membranes at a dilution of 1:500
for anti-IGFBP-1 and anti-IGFBP-4 and at 1:1000 for antisera to
IGFBP-2, -3, -5, and -6, followed by incubation at 4 °C overnight.
40 µl of pansorbin (Calbiochem, Nottingham, UK) was then added to
the solution, followed by incubation at 4 °C for 1 h and
centrifugation at 6500 rpm for 4 min. The pellet was resuspended in
sample buffer and centrifuged, and the supernatant was run under
non-reducing conditions on a 12.5% polyacrylamide gel.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.
,
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.Northern Blot Analyses
After heat denaturation in
the presence of glyoxal, 5 µg of poly(A) RNA was
electrophoresed in a 1.4% agarose gel and was transferred by Northern
blotting to nylon filters. Hybridizations were performed at 65 °C
for 16 h with
P-labeled cDNA probes in 1 M NaCl,
0.1 M trisodium citrate (6
SSC), 5% dextran sulfate,
0.02% Ficoll, 0.02% BSA, 0.02% polyvinyl pyrrolidone, 0.1% SDS, and 150
µg/ml sonicated salmon sperm DNA at 65 °C for 18 h. The filter
was washed with 0.1 SSC, 0.1% SDS at 65 °C prior to
autoradiography. The following probes were used for hybridization:
0.6-kb phigf-I cDNA (33) (supplied by Genetech Inc. South San
Francisco, CA), the 1.1-kb phigf-II cDNA probe (33) (supplied
by Dr. G. Bell, Howard Hughes Medical Institute, Chicago, IL), and a
mouse actin probe (PRT3) (kindly donated by Dr. John Rogers,
Laboratory of Molecular Biology, Cambridge, UK). For the detection of
IGFBP-5 gene expression, a 491-bp cDNA was generated by reverse
transcriptase polymerase chain reaction using RNA derived from
untransformed MRC-5 fibroblasts. Briefly, 1 µg of mRNA was reverse
transcribed by addition of 0.5 µg/ml random hexamer primers
(Amersham International), 10 mM dNTP mixture (Pharmacia LKB,
UK), and 0.5 µl of super reverse transcriptase (HT Biotechnology
Ltd, Cambridge, UK) followed by incubation at 37 °C for 90 min. 1
µl of first strand cDNA was added to 5 mM dNTP mix, 2
units of Taq polymerase (Promega), and 20 pmol of
oligonucleotides with the following sequences: 5`-GAGCAAGTCAAGATCGAG-3`
corresponding to nucleotides 378-395 and 5`-AACGTTGCTGCTGTCGAA-3`
complementary to nucleotides 869-852 of the cDNA encoding
IGFBP-5(34) . Amplification was performed as follows: (i)
denaturation at 95 °C for 3 min, (ii) annealing at 55 °C for 1
min, (iii) extension at 72 °C for 2 min, and (iv) denaturation at
95 °C for 30 s. Steps ii-iv were repeated 30 times followed
by 1 min at 55 °C and 10 min at 72 °C. The amplification
mixture was electrophoresed on a 1.4% agarose gel in the presence of
ethidium bromide. The amplification product of 491 bp, detected by
ultraviolet transillumination, was excised and P-labeled
using an oligolabeling kit (Pharmacia LKB, UK).
Detection of IGF-I and IGF-II Gene Expression by Reverse
Transcriptase Polymerase Chain Reaction (RT-PCR)
The sequences
of the oligonucleotide primers used for RT-PCR analysis of IGF-I gene
expression have been published elsewhere(35) . The sequences of
the IGF-II specific primers were 5`-ATGGGAATCCCAATGGGGAA-3`,
corresponding to nucleotides 1-20 of the cDNA sequence of
IGF-II(36) , and 5`-CTTGCCCACGGGGTATCTCC-3`, complementary to
nucleotides 345-316. The protocol for RT-PCR was the same as that
described for the production IGFBP-5 cDNA probe. Amplified products
were detected by Southern blotting and hybridization with either P-labeled phigf-I or phigf-II cDNA probe.
Growth Factor Requirements of Untransformed and SV
40-transformed Cells
Fig. 1shows the growth curves
obtained for untransformed and transformed cells cultured either in the
presence of 1% FCS or in serum-free media. It can be seen that
untransformed cells were unable to grow under either condition. In
contrast, SV 40-transformed cells increased in number with an
approximate population doubling time in 1% FCS of 27.5 h and in SFM of
38.5 h. In 10% FCS, untransformed and transformed cells grew with
approximate population doubling times of 32 and 24.5 h, respectively
(data not shown).
, untransformed
cells in either 1% serum or serum-free medium; , transformed
cells in 1% serum;
, transformed cells in serum-free
medium.
-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.
10 µg/ml
-IR3 prior to
[H]thymidine
incorporation.
or IGF-II
╡ and [
H]thymidine
incorporation.
Characterization of IGF-I and IGF-II Gene
Expression
In preliminary studies, IGF-I and IGF-II gene
expression was very weakly detected by Northern blot analysis in
untransformed cells only (data not shown). Hence, RT-PCR was
subsequently used to study the expression of these genes. Fig. 4shows that when cells were grown in the presence of FCS,
IGF-I gene expression was detected by RT-PCR in untransformed
fibroblasts only, as evidenced by the detection of the expected 302-bp
amplification product in these but not transformed fibroblasts.
However, it can be seen that when cultured in the absence of FCS, IGF-I
gene expression was activated in transformed cells and that both cell
lines showed IGF-I gene expression under serum-free conditions. Fig. 5shows that IGF-I gene expression was not detected by
RT-PCR in transformed cells growing in SFM supplemented with either 10
µg/ml insulin or 10 nM IGF-II.
Characterization of Membrane IGF Binding Sites
Fig. 7shows affinity labeling of membrane proteins from
untransformed and transformed fibroblasts in the absence or presence of
competing peptides. In untransformed cells (panelA)
in the absence of competing peptide, both I-IGF-I (lane1) and
I-IGF-II (lane5) were predominantly cross-linked to a low molecular
weight protein, which migrated as a complex with apparent M
38,000. It can be seen in panelA that the presence of competing IGF-I in the affinity labeling
reaction completely abolished cross-linking of I-IGF-I (lane2) and partially inhibited cross-linking of
I-IGF-II (lane6) to the low molecular
weight protein. Cross-linking of both
I-IGF-I and
I-IGF-II to the low molecular weight protein was
completely abolished in the presence of competing IGF-II (lanes3 and 7) but not by insulin (lanes4 and 8). These findings in untransformed
fibroblasts indicate that in the absence of competing peptide, IGF-I
and IGF-II predominantly bind to a low molecular weight IGF binding
protein, which has a higher affinity for IGF-II(37) .
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.
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) .
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.
Immunoblot Analysis of IGF-I Receptor
Expression
The monoclonal antibody IGFR 1-2 reacted with
the denatured IGF-I receptor subunit and also with the
incompletely denatured receptor on immunoblots as previously
reported(27) . No difference in the levels of IGF-I receptor
expression in untransformed and transformed fibroblasts was detected
(data not shown).Immunoprecipitation and Immunoblot Analysis of Membrane
IGFBPs
IGFBP-1, IGFBP-4, and IGFBP-6 were not detected in the
membranes of either untransformed or transformed cells by
immunoprecipitation or by immunoblotting. Antisera directed against
IGFBP-2 and IGFBP-3 were reactive with membrane proteins of
untransformed and transformed cells, but any differences between the
expression of these proteins between untransformed and transformed
fibroblasts were minimal, and neither of these antisera
immunoprecipitated the major low molecular weight IGFBP detected in the
membranes of untransformed fibroblasts. Incubation of I-IGF-II cross-linked membrane proteins from
untransformed and transformed cells with a rabbit antiserum to IGFBP-5
is shown in Fig. 8A. It can be seen that the
I-IGF-II-complex with approximate M
38,000 is immunoprecipitated from the membranes of untransformed
cells (lane2) but not from transformed cells (lane4). Immunoblotting studies confirmed the
differential expression of IGFBP-5 in the two cell types (Fig. 8B).
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.''
Characterization of Secreted IGFBPs
IGFBP-1 was
not detected in the conditioned media from either cell line, and no
major differences in the secretion of IGFBP-2 and IGFBP-3 between
untransformed and transformed fibroblasts were observed by
immunoblotting (data not shown). It can be seen in Fig. 9that
an anti-IGFBP-4 antiserum immunoprecipitated a I-IGF-I-IGFBP-4 complex with M
31,000 from medium conditioned by untransformed cells. Since this
antibody shows 50% cross-reactivity with IGFBP-2, a I-IGF-I-IGFBP-2 complex with M
38,000 was immunoprecipitated from medium conditioned by
transformed cells. An antiserum specific for IGFBP-5 immunoprecipitated
a I-IGF-I-IGFBP-5 complex from medium conditioned by
untransformed cells only (Fig. 9). The anti-IGFBP-6 antiserum,
which shows cross-reactivity with IGFBP-5, immunoprecipitated a
I-IGF-I-IGFBP-5 complex with M
38,000 from medium conditioned by untransformed cells and a I-IGF-I-IGFBP-6 complex with M
31,000 from media conditioned by transformed cells.
I-IGF-II and immunoprecipitated with
antisera directed against IGFBP-4, IGFBP-5, and IGFBP-6. Antisera
cross-reactivities are given in
``Results.''
IGFBP-5 Gene Expression in Untransformed and Transformed
Fibroblasts
Northern blot analysis of IGFBP-5 gene expression in
untransformed cells revealed a prominent 6.0-kb and a weaker 1.7-kb
IGFBP-5 transcript in these cells (Fig. 10). In contrast,
IGFBP-5 gene expression was only weakly detected in transformed cells.
It can be seen that in both untransformed and transformed cells, the
level of IGFBP-5 gene expression is no different in cells grown in the
presence or absence of serum.
IGFBP-5 Binding to Transformed Cells and Growth
Inhibition
It can be seen from Fig. 11that when
membranes from transformed fibroblasts were incubated with IGFBP-5 and
subsequently cross-linked to I-IGF-I, a M
38,000 I-IGF-I-protein complex was detected,
indicating that IGFBP-5 is able to associate in a stable manner with
the membranes of these cells. This result was also obtained when whole
cells were incubated with IGFBP-5 (data not shown). It can be seen from Fig. 11that when this protein is bound to the cell membrane,
I-IGF-receptor complexes (arrows) are more
weakly detected than when this protein is absent from the membrane.
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.
-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).
-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.
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.
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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