©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Diminished Expression of Insulin-like Growth Factor (IGF) Binding Protein-5 and Activation of IGF-I-mediated Autocrine Growth in Simian Virus 40-transformed Human Fibroblasts (*)

(Received for publication, May 11, 1994; and in revised form, October 24, 1994)

Julie G. Reeve (§) Ana Guadaño Jieying Xiong Julie Morgan Norman M. Bleehen

From the Medical Research Council Clinical Oncology and Radiotherapeutics Unit, Medical Research Council Centre, Cambridge, CB2 2QH, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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^1 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.


MATERIALS AND METHODS

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(2) 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-^3H]thymidine (specific activity, 27 Ci/mmol), [alpha-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 (alpha-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 beta 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 times 10^4 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 times 10^3/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 [^3H]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% Na(2)CO(3), 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 alpha-IR3 for either 24 or 48 h prior to addition of [^3H]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 [^3H]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 [^3H]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 times 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 times g for 10 min, and the resulting supernatant was further centrifuged at 50,000 times 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 times 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.

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.

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(3) 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.

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(3), 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`)(2) 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 times 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 times 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 beta 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.


RESULTS

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).


Figure 1: Growth of untransformed and transformed fibroblasts in 1% serum and serum-free medium. box, 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 alpha-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 alpha-IR3 markedly inhibited DNA synthesis in these cells. In contrast, it can be seen that alpha-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 alpha-IR3 prior to [^3H]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 [^3H]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.


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.''



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(r) 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) .


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(r) 135,000 and M(r) >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(r) 135,000, consistent with I-IGF-I binding to the alpha-subunit of the type-I IGF-I receptor on transformed cells(37) . The M(r) >218,000 complex behaved similarly and presumably represents I-IGF-I binding to an incompletely reduced alpha-alpha-dimer as previously described(37) . In contrast to the I-IGF-I-complex with apparent M(r) >218,000, the I-IGF-II-complex with M(r) >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(r) >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 alpha-alpha dimer of the IGF-I receptor. The I-IGF-II-complex with M(r) >135,000 detected in the absence of competing peptide (lane5) behaved in the same way as the I-IGF-I-complex with M(r) >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 alpha-subunit of type I receptor with M(r) 135,000 (lane1) and I-IGF-II binding to the type-II receptor with approximate M(r) >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 alpha-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 beta 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(r) 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).


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.''



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(r) 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(r) 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(r) 38,000 from medium conditioned by untransformed cells and a I-IGF-I-IGFBP-6 complex with M(r) 31,000 from media conditioned by transformed cells.


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.''



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.


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.



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(r) 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.


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 alpha-alpha dimer of the type-I receptor with apparent M(r) >218,000, and the smallsinglearrow indicates the alpha-subunit with M(r) 135,000. Affinity labeling of membranes pre-incubated with IGFBP-5 reveals a prominent I-IGF-I-IGFBP-5 complex with apparent M(r) 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 alpha-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 [^3H]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 ([^3H]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 alpha-IR3 (10 µg/ml) or IGFBP-5 (100 nM) for 48 h prior to addition of [^3H]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 [^3H]thymidine.




DISCUSSION

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 44-223-402353; Fax: 44-223-402180.

(^1)
The abbreviations used are: IGF, insulin-like growth factor; PBS, phosphate-buffered saline; kb, kilobase(s); EMEM, Earle's minimum essential medium; FCS, fetal calf serum; bp, base pair(s); BSA, bovine serum albumin; SFM, serum-free medium; RT-PCR, reverse transcriptase polymerase chain reaction; IGFBP, IGF binding protein.


REFERENCES

  1. Goolsby, C. L., Wiley, J. E., Steiner, M., Bartholdi, M. F., Cram, L. S., and Kraemer, P. M. (1991) Cancer Genet. Cytogenet. 49, 231-248
  2. Defendi, V., Naimski, P., and Steinberg, M. L. (1982) J. Cell. Physiol. 2, (suppl.) 131-140
  3. Sach G. H. (1981) In Vitro 17, 1-19 [Medline] [Order article via Infotrieve]
  4. Risser, R., and Pollack, R. (1974) Virology 59, 477-489 [Medline] [Order article via Infotrieve]
  5. Bowen-Pope, D. F., Vogel, A., and Ross, R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 2396-2400 [Abstract]
  6. Dicker, P., Pohjanpelto, P., and Rozengurt, E. (1981) Exp. Cell Res. 135, 221-227 [Medline] [Order article via Infotrieve]
  7. Kaplan, P. L., Topp, W. C., and Ozanne, B. (1981) Virology 108, 484-490 [Medline] [Order article via Infotrieve]
  8. Fanning, E., and Knippers, R. (1992) Annu. Rev. Biochem. 61, 55-85 [CrossRef][Medline] [Order article via Infotrieve]
  9. Sontag, E., Federov, S., Kamibayashi, C., Robbins, D., Cobb, M., and Mumby, M. (1993) Cell 75, 887-897 [Medline] [Order article via Infotrieve]
  10. Porcu, P., Ferber, A., Pietrzkowski, Z., Roberts, C. T., Adamo, M., LeRoith, D., and Baserga, R. (1992) Mol. Cell. Biol. 12, 5069-5077 [Abstract]
  11. Kaleko, M., Rutter, W. G., and Miller, A. D. (1990) Mol. Cell. Biol. 10, 464-473 [Medline] [Order article via Infotrieve]
  12. Pietrzkowski, Z., Lammers, R., Carpenter, G., Soderquist, M., Limardo, M., Phillips, P. D., Ullrich, A., and Baserga, R. (1992) Cell Growth & Diff. 3, 199-205
  13. Travali, S., Reiss, K., Ferber, A., Petralia, S., Mercer, W. E., Calabretta, B., and Baserga, R. (1991) Mol. Cell. Biol. 11, 731-736 [Medline] [Order article via Infotrieve]
  14. Sell, C., Rubini, M., Rubin, R., Liu, J-P, Efstratiadis, A., and Baserga, R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11217-11221 [Abstract]
  15. Ballard, J., Baxter, R., Binoux, M., Clemmons, D., Drop, S., Hall, K., Hintz, R., Rechler, M., Rutanen, E., and Schwander, J. (1989) Acta Endocrinol. 121, 751-752 [Medline] [Order article via Infotrieve]
  16. Drop, L. S. (1992) Endocrinology 130, 1736-1737 [Medline] [Order article via Infotrieve]
  17. Baxter, R. C., and Martin, J. L. (1989) Prog. Growth Factor Res. 1, 49-68 [Medline] [Order article via Infotrieve]
  18. Lamson, G., Giudice, L. C., and Rosenfeld, R. G. (1991) Growth Factors 5, 19-28 [Medline] [Order article via Infotrieve]
  19. Cohen, P., Fielder, P. J., Hasegawa, Y., Frisch, H., Giudice, L. C., and Rosenfeld, R. (1991) Acta Endocrinol. 124, Suppl. 2, 74-85 [Medline] [Order article via Infotrieve]
  20. Cohen, P., Lamson, G., Okajima, T., and Rosenfeld, R. G. (1993) Mol. Endocrinol. 7, 380-386 [Abstract]
  21. Chen, J-C., Shao, Z-M., Saeed Sheikh, M., Hussain, A., LeRoith, D., Roberts, C. T., and Fontana, J. A. (1994) J. Cell. Physiol. 158, 69-78 [Medline] [Order article via Infotrieve]
  22. Reeve, J. G., Morgan, J., Schwander, J., and Bleehen, N. M. (1993) Cancer Res. 53, 4680-4685 [Abstract]
  23. Blat, C., Villaudy, J., Delbe, J., Golde, A., and Harel, L. (1992) Growth Factors 6, 65-75 [Medline] [Order article via Infotrieve]
  24. Martin, J. L., Willetts, K. E., and Baxter, R. C. (1990) J. Biol. Chem. 265, 4124-4130 [Abstract/Free Full Text]
  25. Huschtscha, L. I., and Holliday, R. (1983) J. Cell Sci. 63, 77-99 [Abstract]
  26. Kull, F. C., Jr., Jacobs, S., Su, Y. F., Svoboda, M. E., van Wyk, J. J., and Cuatrecasas, P. (1983) J. Biol. Chem. 258, 6561-6566 [Abstract/Free Full Text]
  27. Soos, M. A., Field, C. E., and Siddle, K. (1993) Biochem. J. 290, 419-426 [Medline] [Order article via Infotrieve]
  28. Busby, W. H., Snyder, D. K., and Clemmons D. R. (1988) J. Clin. Endocrinol. & Metab. 67, 1225-1230 [Abstract]
  29. Clemmons, D. R., Snyder, D. K., and Busby, W. H. (1991) J. Clin. Endocrinol. & Metab. 73, 727-733 [Abstract]
  30. Hill, D. J., and Clemmons, D. R. (1992) Growth Factors 6, 315-326 [Medline] [Order article via Infotrieve]
  31. Camacho-Hubner, C., Busby, W. H., Jr., McCusker, R. H., Wright, G., and Clemmons, D. R. (1992) J. Biol. Chem. 267, 11949-11956 [Abstract/Free Full Text]
  32. Kiefer, M. C., Schmid, C., Waldvogel, M., Schlapfer, I., Futo, E., Masiarz, F. R., Green, K., Barr, P. J., and Zapf, J. (1992) J. Biol. Chem. 267, 12692-12699 [Abstract/Free Full Text]
  33. Bell, G. I., Merryweather, J. P., Sanchez-Pescador, R., Stempien, M. M., Priestley, L., Scott, J., and Rall, L. B. (1984) Nature 310, 775-777 [Medline] [Order article via Infotrieve]
  34. Shimasaki, S., Shimonaka, M., Zhang, H-P., and Ling, N. (1991) J. Biol. Chem. 266, 10646-10653 [Abstract/Free Full Text]
  35. Reeve, J. G., Brinkman, A., Hughes, S., Mitchell, J., Schwander, J., and Bleehen, N. M. (1992) J. Natl. Cancer Inst. 84, 628-634 [Abstract]
  36. Jansen, M., van Schaik, F. M. A., van Tol, H., Van den Brande, J. L., and Sussenbach, J. S. (1985) FEBS Lett. 179, 243-246 [CrossRef][Medline] [Order article via Infotrieve]
  37. Sturm, M. A., Conover, C. A., Pham, H., and Rosenfeld, R. G. (1989) Endocrinology 124, 388-396 [Abstract]
  38. Clemmons, D. R., Elgin, R. G., Han, V. K. M., Casella, S. J., D'Ercole, A. J., and van Wyk, J. J. (1986) J. Clin. Invest. 77, 1548-1556 [Medline] [Order article via Infotrieve]
  39. Conover, C. A., Liu, F., Rosenfeld, R. G., and Hintz, R. L. (1989) J. Clin. Invest. 83, 852-859 [Medline] [Order article via Infotrieve]
  40. Clemmons, D. R., Han, V. K. M., Elgin, R. G., and D'Ercole, A. J. (1987) Mol. Endocrinol. 1, 339-347 [Abstract]
  41. McCusker, R. H., Camacho-Hubner, C., Bayne, M. L., Cascieri, M. A., and Clemmons, D. R. (1990) J. Cell. Physiol. 144, 244-253 [Medline] [Order article via Infotrieve]
  42. Clemmons, D. R., Dehoff, M. L., Busby, W. H., Bayne, M. L., and Cascieri, M. A. (1992) Endocrinology 131, 890-895 [Abstract]
  43. Backeljauw, P. F., Dai, Z., Clemmons, D. R., and D'Ercole, A. J. (1993) Endocrinology 132, 1677-1681 [Abstract]
  44. Irminger, J. C., Schoenle, E. J., Briner, J., and Humbel, R. E. (1989) Eur. J. Pediatr. 148, 620-623 [Medline] [Order article via Infotrieve]
  45. Schofield, P. N., Hill, D. J., Lee, A. J., Cheetham, J. C., James, D., and Stewart, C. (1991) Br. J. Cancer 63, 687-692 [Medline] [Order article via Infotrieve]
  46. Jones, J. I., Gickerman, A., Busby, W. H., Camacho-Hubner, C., and Clemmons, D. R. (1993) J. Cell Biol. 121, 679-687 [Abstract]
  47. Kiefer, M. C., Schmid, C., Waldvogel, M., Schlapfer, I., Futo, E., Masiarz, F. R., Green, K., Barr, P. J., and Zapf, J. (1993) Growth Regul. 3, 56-59 [Medline] [Order article via Infotrieve]
  48. Bach, L. A., Hsieh, S. P., Brown, A. L., and Rechler, M. M. (1994) Growth Regul. 4, Suppl. 1, I169

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.