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Endocrinology Vol. 140, No. 2 575-584
Copyright © 1999 by The Endocrine Society

ARTICLES

Overexpression of Insulin-Like Growth Factor-Binding Protein-2 in C6 Glioma Cells Results in Conditional Alteration of Cellular Growth1

Sheri L. Bradshaw2, A. Joseph D’Ercole and Victor K. M. Han

Medical Research Council Group in Fetal and Neonatal Health and Development (S.L.B., V.K.M.H.), Departments of Biochemistry (S.L.B., V.K.M.H.) and Pediatrics (V.K.M.H.), University of Western Ontario, The Lawson Research Institute, London, Ontario, Canada N6A 4V2; and the Department of Pediatrics, University of North Carolina (A.J.D.), Chapel Hill, North Carolina 27514

Address all correspondence and requests for reprints to: Victor K. M. Han, M.D., Room H308, The Lawson Research Institute, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2. E-mail: vhan{at}julian.uwo.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To examine the relationship between the expression of insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) and cell growth in a cell type with a defined IGF/IGFBP system, an ovine IGFBP-2 complementary DNA was overexpressed in C6 glioma cells. C6 cells produce IGFBP-3, IGFBP-4, a negligible amount of IGFBP-2, and IGF-I. An ovine IGFBP-2 complementary DNA was transfected into C6 cells, and nine colonies that stably expressed variable levels of IGFBP-2 messenger RNA were selected. Synthesis of corresponding levels of IGFBP-2 was confirmed by ligand blot and immunoblot analyses of conditioned media. Three clones exhibited significantly reduced growth rates, and the remainder showed growth rates similar to those of the wild-type C6 cells. The clones, which overexpressed high levels of IGFBP-2 and IGF-I, had growth rates similar to the wild-type cells, whereas the three clones that overexpressed IGFBP-2 without a concomitant increase in IGF-I had reduced growth rates. In addition, a cell-associated IGFBP was identified in the slow growing clones, but not in the wild-type or the fast growing clones. This cell-associated IGFBP was deduced to be IGFBP-5 based on its molecular size, detection of IGFBP-5 messenger RNA only in slow growing clones, and competition of its binding by heparin. Growth of the slow growing clone, C6BP2-1, could not be overcome by the addition of exogenous IGF-I, suggesting that the cell-associated IGFBP-5 was the dominant regulator of IGF action. These observations suggested that 1) in C6 glioma cells cellular growth is altered by a disturbance in the equilibrium between IGF-I and IGFBPs and/or the functional properties of the IGFBPs; and 2) C6 cells may have a limited capacity to modulate IGF/IGFBP expression in response to changes in endogenous expression of IGFBPs. Endogenous regulation of the balance between IGFs and IGFBPs may be a model of regulation of cellular growth in tumor cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor (IGF)-binding proteins (IGFBPs) are a family of structurally homologous proteins that bind IGFs with high affinity equal to or greater than that of the IGF receptors (1, 2). A family of six high affinity IGFBPs have been purified, and their complementary DNAs (cDNAs) have been cloned and sequenced (2). Recent studies have indicated that there may be additional members of this family of binding proteins, termed IGFBP-related proteins that have low affinity binding to IGFs (3). IGFBPs have a wide variety of functions, including transporting IGFs in the circulation and extracellular spaces, prolonging the half-life of IGFs, regulating their clearance, and modulating the actions of IGFs on target tissues. In vitro studies have demonstrated that IGFBPs may either inhibit or potentiate the biological actions of IGFs (4, 5, 6, 7, 8). In addition to their modulation of IGF action, direct functions for IGFBPs independent of IGFs have been proposed (9, 10, 11, 12). However, the mechanism of action of IGFBPs at the cellular level in both modulating IGF actions and possible direct functions, remains unclear.

There is now substantial evidence that supports the role of IGFs in the growth and development of the brain. IGF messenger RNAs (mRNAs) have been localized in the rat brain during pre- and postnatal development (13, 14, 15, 16, 17). Both IGF-I and IGF-II receptor mRNAs have been localized to regions of the developing rat brain (18, 19), and specific binding of IGFs has been demonstrated in vivo (20, 21, 22). IGFs stimulate mitogenesis in astroglial and oligodendroglial cells and promote the survival and stimulate the outgrowth of neurons in vitro (23, 24, 25). Transgenic mice overexpressing IGF-I have brains larger than those of wild-type mice (26, 27), whereas mice lacking IGF-I have smaller brains (28). These differences in brain size can be attributed partly to hyper- or hypomyelination, respectively, providing additional evidence for the growth-promoting effects of IGF-I on glial cell types. Additionally, mice that overexpress IGFBP-1 showed brain growth retardation from the second week of postnatal life, suggesting that IGFBP-1 may inhibit the growth-promoting effects of IGF-I during brain development at this time (29).

IGFBP mRNAs have also been localized to developing regions of the rodent brain (13, 16, 30). In particular, IGFBP-2 is expressed in the developing rat brain in vivo, primarily in glial cell types (16, 17, 31). In addition, we and others (32, 33, 34) have demonstrated that cultured astroglial cells synthesize IGFBP-2 in vitro. IGFBP-2 as a modulator of IGF action may, therefore, have a significant role in the growth and differentiation of glial cells.

We have shown previously that the exogenous addition of IGFBP-2 purified from BRL-3A cells can inhibit the mitogenic action of IGFs on primary astroglial cultures, and that this inhibition is a result of competition for IGF binding to the receptor (32). However, exogenous addition of IGFBPs may not accurately reflect the true autocrine/paracrine nature of the IGF system, as there is increasing evidence demonstrating the interaction of IGFBPs with either extracellular matrix (8) or cellular components at the membrane level (6, 9). We therefore sought to create a system by which the endogenous expression of IGFBP-2 in glial cells is altered and to address the question of whether the overexpression of IGFBP-2 leads to an altered growth phenotype. The rat C6 glioma cell line was chosen as a model system because it synthesizes IGF-I and expresses IGF receptors (35), and its growth is regulated by IGF-I (36). Primary astroglial cells, which demonstrate regulated growth, synthesize high levels of IGFBP-2, whereas C6 glioma cells, with tumorigenic growth, express very low levels of IGFBP-2 (37). We have therefore transfected an ovine IGFBP-2 cDNA constructed in a constitutive expression vector, into the rat C6 glioma cell line and have demonstrated that expression of components of the IGF system were variably altered and that cell growth was conditionally altered.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human recombinant IGF-I and IGF-II were purchased from Bachem Co. (Torrance, CA). IGFs were iodinated using the chloramine-T method as previously described (38). The bovine IGFBP-2 antiserum was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), human recombinant IGFBP-5 was obtained from Austral Biological (San Ramon, CA), hexadimethrine bromide (1,5-dimethyl-1,5-diazaundecamethylene polymethobromide) was obtained from Sigma Chemical Co. (St. Louis, MO), Geneticin (G418), heparin, and GRGDSP and GRGESP peptides were purchased from Life Technologies (Grand Island, NY), and an EXTRA-3 rabbit ExtrAvidin Peroxidase Staining Kit was obtained from Sigma Chemical Co.

Cell culture
Rat C6 glioma cells, obtained from the American Type Culture Collection (a gift from Dr. C. C. G. Naus, University of Western Ontario), were grown in DMEM supplemented with 10% FBS, penicillin (5 U/ml), streptomycin (5 µg/ml), and gentamicin (25 µg/ml; Life Technologies) in 5% CO2-95% air in humidified tissue culture incubators at 37 C. The medium was changed at cell passage every 3–4 days.

Transfection
A 1.1-kb ovine (o) IGFBP-2 cDNA consisting of the entire coding region (39) was cloned into the HindIII site of the mammalian expression vector pRc/cytomegalovirus (CMV) (40). The oIGFBP-2 cDNA in the antisense orientation and the pRc/CMV vector alone were used as controls. The plasmid was transfected into C6 cells using the polybrene/dimethylsulfoxide (DMSO) technique (41). Cells were plated on 60-mm Falcon tissue culture grade petri dishes (Becton Dickinson and Co., Franklin Lakes, NJ) at a density of 5 x 106 cells/dish in complete medium (CM) and allowed to attach overnight. The medium was aspirated, and 20 µg plasmid DNA were equilibrated with 10 µg/ml polybrene in CM at 37 C and added to the cells with periodic swirling for 16 h. The DNA mixture was aspirated, and an equilibrated mixture of 15% DMSO in CM was added to the cells for 4.5 min at 37 C. The DMSO mixture was removed, and the cells were washed with CM (twice) and allowed to recover for 24 h. Each 60-mm dish was subplated into 3 x 100-mm tissue grade petri dishes (Falcon) and covered with selection medium (1.5 mg/ml G418 in CM). Selection medium was changed every 4–5 days for a total selection period of 14 days. After selection, the cells were grown in CM, and discrete colonies were picked after 4 days.

CM were collected from confluent clones in T-75 flasks (Falcon) after 24 h of incubation in serum-free medium (SFM; medium supplemented with 0.1 mg/ml BSA; Sigma Chemical Co.) and centrifuged at 3000 x g to remove cellular debris, and the supernatant was stored at -20 C until analyzed. Cells from the same cultures were used for RNA extraction.

Ligand and immunoblot analyses
Ligand blot analysis was performed as previously described (42). The same membrane was processed for immunoblotting. The membrane was blocked with 4% BSA-TTBS (0.5% Tween in Tris-buffered saline) for 1 h at room temperature. The membrane was washed in TTBS (three times, 10 min each time) followed by incubation in bovine IGFBP-2 antiserum (1:2000 dilution) in 1% BSA-TTBS overnight at 4 C. The membrane was washed in TTBS (three times, 10 min each time) and incubated with secondary antibody, biotinylated goat antirabbit IgG (1:1000 dilution) in 1% BSA-TTBS, for 1 h at room temperature. After washing in TTBS (three times, 10 min each times), the membrane was incubated in ExtrAvidin-peroxidase (1:1000) in 1% BSA-TTBS for 1 h at room temperature. The membrane was washed in TTBS (twice, 10 min each time) and TBS (once, 10 min) and developed with the chromagen diaminobenzadine (Sigma Chemical Co.) in 0.05 M Tris, pH 7.6. The membrane was washed in 0.05 M Tris, pH 7.6, for 1 h at room temperature, air-dried, and exposed to x-ray film (Biomax, Eastman Kodak Co., Rochester, NY) with intensifying screens at -70 C for 3–7 days. Total protein concentrations of the CM were determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Richmond, CA).

Northern blot analysis
Total RNA was prepared from cell cultures by the guanidine thiocyanate-cesium chloride method and subjected to Northern analysis as previously described (42). The resulting blots on Zeta-Probe nylon membranes (Bio-Rad Laboratories, Inc.) were probed in sequence with 32P-labeled cDNAs encoding ovine IGFBP-2 (39); rat IGFBP-1 (a gift from Dr. M. Rechler, NIH, Bethesda, MD); rat IGFBP-3 (a gift from Dr. A. Herington, Melbourne, Australia); rat IGFBP-4, -5, and -6 (gifts from Dr. S. Shimasaki, San Diego, CA); rat IGF-I (a gift from Dr. L. Murphy, Winnipeg, Canada); and mouse IGF-II (a gift from Dr. G. Bell, Chicago, IL). The cDNA inserts were labeled with [32P]deoxy-CTP (ICN Biomedicals, Inc. Canada, St. Laurent, Canada) to specific activities of 1–2 x 109 cpm/µg by the random priming technique using the Oligo-labeling kit (Pharmacia Canada, Inc., Baie d’Urfe, Canada). The blots were stripped in between hybridizations by washing in 0.01 x SSC (standard saline citrate) and 0.5% SDS at 80 C for 30 min twice. Consistency in the relative amounts of total RNAs being loaded into each lane was checked by probing the blots with a radiolabeled cDNA for 18S ribosomal RNA (a gift from Dr. D. Denhardt, Piscataway, NJ).

IGF-I RIA
Conditioned media (5 ml) were lyophilized and solubilized in 1 M acetic acid at 4 C overnight, and then IGFs were separated from IGFBPs using gel filtration chromatography on a Sephadex G-50 (Pharmacia) column. Serial dilutions of extracted media were assayed for IGF-I as previously reported (43).

Growth curves
Selected clones were plated at a density of 1 x 105 cells in T-25 tissue culture flasks (Falcon) in CM. Cell number was determined in triplicate on the following day (time zero) and every 24 h thereafter for a period of 72 h on a Coulter counter (model Zf, Coulter Electronics, Hialeah, FL). Growth curves were constructed by plotting mean cell number against time. Each experiment was performed three times, and the mean ± SEM doubling time was calculated. Statistical analysis for comparing growth rates among different clones was performed using ANOVA.

Affinity cross-linking
Selected clones were plated at a density of 3 x 105 cells in poly-L-lysine (0.05 mg/ml)-coated six-well tissue culture plates in CM. Upon reaching confluence at 48 h, the cell monolayers were washed in HEPES binding buffer, pH 7.4 (0.1 M HEPES, 7.75 mM sodium phosphate dibasic, 2.25 mM sodium phosphate monobasic, and 0.1% BSA), and subsequently incubated with 5 x 105 cpm [125I]IGF-I or -IGF-II with or without competitors (IGF-I, IGF-II, insulin, heparin, GRGDSP, and GRGESP) in HEPES binding buffer for 6 h at 4 C. The incubating solution was aspirated, the cells were washed thoroughly with cold (4 C) PBS, and then the proteins were cross-linked by incubation in 0.1 mM dissuccinimidyl suberate in cross-linking buffer (0.1 M HEPES, 7.75 mM sodium phosphate dibasic, and 2.25 mM sodium phosphate monobasic) for 30 min at room temperature. The cross-linking solution was aspirated, and the proteins were solubilized by the addition of 1 x Laemmli buffer. The samples were denatured by boiling and were run on a 6–14% gradient SDS-PAGE. The gels were fixed in 25% methanol-10% acetic acid in water, dried, and exposed to Biomax (Eastman Kodak Co.) film for autoradiography.

Immunoprecipitation
Conditioned medium was incubated with 2 x 105 cpm [125I]IGF-II for 1 h at 4 C and subsequently cross-linked with the addition of 10 mM dissuccinimidyl suberate. Cell monolayers were cross-linked with [125I]IGF-II as described above, solubilized in membrane homogenization buffer (20 mM Tris, 0.33 M sucrose, 2 mM EDTA, 0.5 mM EGTA, 1% Nonidet P-40, 2 mM phenylmethylsulfonylfluoride, and 0.3% aprotinin, pH 7.5), and sonicated for three 10-sec bursts with a membrane sonicator. The resulting cell suspensions and CM were incubated with antisera against bovine IGFBP-2 (Upstate Biotechnology, Inc.) at a 1:50 dilution at 4 C overnight. Immune complexes were precipitated with the addition of protein A-Sepharose (Pharmacia) and centrifuged, and the pelleted immune complexes were washed twice with membrane homogenization buffer. Protein complexes were boiled in 1 x Laemmli buffer, boiled, and run on 6–14% gradient SDS-PAGE. The gels were dried and exposed to Biomax film (Eastman Kodak Co.) for autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Secretion of IGFBPs
Wild-type C6 glioma cells secreted IGFBPs of Mr 40–45, 28, and 22 (Fig. 1Go). The 40- to 45-kDa IGFBP has been identified immunologically as IGFBP-3 (33, 34). The 22-kDa IGFBP, although not identified immunologically because of the lack of a specific antiserum, was deduced to be IGFBP-4 based on its molecular size and the detection of IGFBP-4 mRNA in Northern blots. The identity of the 28-kDa IGFBP is presently unknown, but may be a glycosylated variant of IGFBP-4 based on its molecular size and the lack of detectable mRNAs for IGFBP-1, -5, or -6.



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  		Figure 1. A, Ligand blot analysis of CM from wild-type C6 glioma cells (lane 1), the pRc/CMV vector-transfected clone (lane 2), and the pRc/CMV-oIGFBP-2-transfected clones (lanes 3–11). CM were collected from cells after incubation for 24 h in SFM, subjected to SDS-PAGE, transferred to nitrocellulose, incubated with [125I]IGF-I, and visualized by autoradiography. B, Immunoblot analysis of the same membrane as that in A with an antiserum against bovine IGFBP-2, showing an immunoreactive band of 34 kDa in the pRc/CMV-oIGFBP-2-transfected clones.

 
Nine G418-resistant clones with variable levels of a 34-kDa IGFBP (IGFBP-2) in the conditioned media as analyzed by ligand blotting were selected (Fig. 1AGo). Clones C6BP2-8, -10, -11, and -12 secreted relatively high levels of this protein, whereas clones C6BP2-1, -4, -6, -7, and -9 secreted lower levels. The identity of this protein was confirmed by immunoblotting with a specific antiserum against bovine IGFBP-2 (Fig. 1BGo). The levels of immunoreactive IGFBP-2 corresponded to the levels of IGFBP-2 as observed by ligand blotting. The vector-transfected control clone (C6CMV-3) did not express IGFBP-2 and was chosen as an appropriate control over other vector control clones because the IGFBP and IGF-I expression profile was most similar to that of the wild-type C6 cells.

The levels of the other IGFBPs in the various clones were also variably altered. IGFBP-3 was down-regulated, to a variable degree, in all clones. Clones C6BP2-7 and -9 had just detectable levels of IGFBP-3 protein, whereas clones C6BP2-8, -10, and -11 had no detectable levels. IGFBP-4 was also down-regulated in all clones except the vector control C6CMV-3. The 28-kDa IGFBP was detected in only clones C6CMV-3 and C6BP2-10. An additional IGFBP of 29 kDa was detected in clones C6BP2-1, -4, -7, and -9.

Expression of IGFBP genes
Expression of the transfected ovine IGFBP-2 was analyzed by Northern blotting to determine steady-state mRNA levels (Fig. 2Go). IGFBP-2 mRNA was not detected in the wild-type C6 glioma cells or the vector control clone, C6CMV-3. A 1.6-kb band was detected, at variable levels, in the IGFBP-2-secreting clones. The relative levels of IGFBP-2 steady-state mRNAs corresponded to the relative amount of secreted IGFBP-2 protein for each clone. Clones C6BP2-8, -10, -11, and -12 expressed relatively high levels of IGFBP-2 mRNA, whereas clones C6BP2-1, -4, -6, -7, and -9 expressed lower levels of IGFBP-2 mRNA.



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  		Figure 2. Northern blot analysis of total RNAs (20 mg/lane) from C6 glioma (lane 1), a pRc/CMV vector-transfected control (lane 2), and pRc/CMV-oIGFBP-2-transfected clones (lanes 3–11) sequentially probed with 32P-labeled IGFBP-2 (A), IGFBP-3 (B), and IGFBP-4 (C) and 18S ribosomal RNA cDNAs (D). A 1.6-kb IGFBP-2 transcript was readily detectable only in the pRc/CMV-oIGFBP-2-transfected clones. The 2.7-kb IGFBP-3 transcript and the 2.4-kb IGFBP-4 transcript were observed in various clones. Relative consistency in loading and transfer of total RNA is shown in D by hybridization to 18S ribosomal RNA. Rat IGFBP-1, -5, and -6 mRNAs were not detected using 20 µg total RNA (not shown). IGFBP-5 mRNA was detected by Northern analysis using 40 µg total RNA (see Fig. 8Go).

 
A 2.6-kb IGFBP-3 and 2.4-kb IGFBP-4 transcripts were also detected by Northern blotting (Fig. 2Go). The relative amount of steady-state IGFBP-3 mRNA did not necessarily correspond to the amount of secreted IGFBP-3 protein, suggesting that the regulation of protein production occurred at the translational or posttranslational level. IGFBP-3-specific protease activity was not observed in any of the conditioned media, indicating that this posttranslational processing was not responsible. The relative levels of IGFBP-4 steady-state mRNA for each clone corresponded to the levels of secreted IGFBP-4 protein. IGFBP-1 and -6 mRNAs were not detected by Northern analysis using 20 µg total RNA/lane (data not shown). However, IGFBP-5 mRNA was detected only in clones C6BP2-1 and C6BP2-4, but not in others, by Northern analysis using 40 µg total RNA, indicating that in these clones, IGFBP-5 mRNA was expressed in low abundance (Fig. 8Go).



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  		Figure 8. Northern blot analysis of total RNAs (40 mg/lane) from adult rat liver (lane 1), adult rat kidney (lane 2), C6 glioma (lane 3), a pRc/CMV vector-transfected control (lane 4), and pRc/CMV-oIGFBP-2-transfected clones (lanes 5–13) probed with 32P-labeled rat IGFBP-5 cDNA (A) and 18S ribosomal RNA (B). The 6.0-kb IGFBP-5 transcript was detected in kidney and, in low abundance, in C6BP2-1 and -4. IGFBP-1 and IGFBP-6 mRNAs were not detected in C6 cells or in any of the transfected clones. Note that 40 µg total RNA were required to detect IGFBP-5.

 
Expression of the IGF-I gene
IGF-I stable mRNA levels were examined in the wild-type C6 glioma cells and the various clones by Northern analysis (Fig. 3Go). A major 7.5-kb transcript was detected in addition to minor transcripts of 4.0, 2.0, and 1.2 kb. The relative level of IGF-I steady-state mRNA was low in the wild-type C6 cells. Clones C6BP2-1, -4, and -9 and C6CMV-3 had similarly low levels of IGF-I mRNA compared with the wild-type C6 cells. IGF-I steady-state mRNA was up-regulated in clones C6BP2-6, -7, -8, -10, and -12 compared with that in the wild-type C6 cells. The 7.5- and 1.2-kb IGF-I transcripts were preferentially up-regulated in these clones. IGF-II mRNA was not detected (not shown).



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  		Figure 3. Northern blot analysis of total RNAs (20 mg/lane) from C6 glioma (lane 1), a pRc/CMV vector-transfected control (lane 2), and pRc/CMV-oIGFBP-2-transfected clones (lanes 3–11) probed with 32P-labeled rat IGF-I cDNA (A) and 18S ribosomal RNA (B). The clones expressed variable levels of four distinct IGF-I transcripts of 7.5, 4.0, 1.8, and 1.2 kb. IGF-II mRNA was not detected (not shown).

 
The levels of radioimmunoassayable IGF-I measured in the CM from the wild-type C6 cells ranged from 0.496–4.18 ng/ml, amounts similar to those previously reported by others (34). Clones C6BP2-1, -4, -6, -7, and -9 secreted lower levels of IGF-I than the wild-type C6 cells, whereas clones C6BP2-8, -10, -11, and -12 all secreted higher amounts of IGF-I (Table 1Go). The total IGF-I mRNA levels in various clones correlated with IGF-I peptide concentrations in the conditioned media (r2 = 0.86). In most clones, except clones C6BP2-1 and -4, the concentration of secreted IGF-I corresponded to the relative level of secreted IGFBP-2. Clones C6BP2-8 and -12, which secreted the highest levels of IGFBP-2, also secreted the highest levels of IGF-I.


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  		Table 1. IGF-I concentration in 24-h conditioned medium of C6 (wild-type), CMV-3 (vector control), and C6 IGFBP-2 transfected clones

 
Growth of IGFBP-2-transfected clones
To determine the effect of IGFBP-2 overexpression on glial cell growth, the growth rates of selected clones were determined. Clones C6BP2-10 and -12 were chosen for their high level IGFBP-2 expression, whereas clones C6BP2-1, -4, and -9 were chosen for their moderate expression of IGFBP-2. Growth curves were constructed by counting cell number over a period of 72 h. Representative growth curves for C6 cells and clones C6BP2-1 and -12 are shown in Fig. 4AGo. Clones C6BP2-12 and -10, which highly overexpressed IGFBP-2, exhibited a similar increase in cell number over 72 h, compared with the wild-type C6 cells. In contrast, the moderate expresser of IGFBP-2, clones C6BP2-1, -4, and -9, had significant reductions in cell number at 48 and 72 h, compared with that in the wild-type C6 cells.



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  		Figure 4. A, Growth curves were constructed by plotting the mean cell number against time over a period of 72 h for the wild-type C6 cells (O), C6BP2-1 (D), and C6BP2-12 (µ). The wild-type C6 cells demonstrated an exponential growth rate. C6BP2-12 exhibited a similar growth rate compared with C6 cells. C6BP2-1 exhibited a reduced growth rate compared with C6 cells. The cell number for C6BP2-1 was significantly less than that for C6 cells at 48 and 72 h. *, Statistically significant change in cell number compared with C6 cells (P < 0.05). B, The doubling times were calculated from the growth curves for each clone. The doubling times (mean ± SEM) are shown from three separate experiments. *, Statistically significant difference compared with C6 cells (P < 0.05).

 
The doubling time for each clone was calculated from three separate experiments, and the mean ± SEM were calculated (Fig. 4BGo). Clones C6CMV-3, C6BP2-10, and C6BP-12 had similar doubling times as the wild-type C6 cells. In contrast, the moderate expressers of IGFBP-2, clones C6BP2-1, -4, and -9, had significantly greater doubling times than the C6 cells. Of significance, clones C6BP2-1, -4, and -9 also expressed very low levels of IGF-I mRNA and peptide, whereas clones C6BP2-10 and -12 expressed high levels of IGF-I (Fig. 3Go and Table 1Go).

Affinity cross-linking of IGFs
Cross-linking of [125I]IGF-I or [125I]IGF-II to monolayers of selected clones in the absence or presence of variable concentrations of unlabeled IGF-I, IGF-II, and insulin was used to examine relative IGF receptor binding affinity. When IGF-I was used as the radioligand, all clones examined displayed a 135-kDa band consistent in size with the IGF-I receptor {alpha}-subunit in addition to a 270-kDa band consistent in size with {alpha}-subunit dimers (Fig. 5Go, A–C). [125I]IGF-I binding was competed with unlabeled IGF-I (lanes 2–4), to a lesser degree with IGF-II (lanes 5–7), and with very high concentrations of insulin (lanes 8 and 9). This characteristic binding pattern of the IGF-I receptor was observed with wild-type C6 cells (Fig. 5AGo) and all clones examined, C6BP2-1 (Fig. 5BGo) and C6BP2-12 (Fig. 5CGo), C6CMV-3, and C6BP2-4, -9, and -10 (data not shown). An additional band of 41 kDa was observed in C6BP2-1 (Fig. 5BGo) and C6BP2-4 and C6BP2-9 (not shown). This band was competed with excess IGF-I and IGF-II, but not with insulin, suggesting that it was a membrane-associated IGFBP. IGFs competed more effectively for binding to the 41-kDa membrane-associated IGFBP than for the IGF-I receptor, suggesting that the membrane-associated IGFBP had a greater affinity for IGF-I than the IGF-I receptor.



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  		Figure 5. Affinity cross-linking of [125I]IGF-I to monolayers of wild-type C6 (A), C6BP2-1 (B), and C6BP2-12 (C). All clones displayed a 135-kDa type I receptor {alpha}-subunit (I) monomer and a 270-kDa {alpha}-subunit dimer. Binding was competed by the addition of exogenous IGF-I (lanes 2–4), IGF-II (lanes 5–7) to a lesser degree, and very high concentrations of insulin (lanes 8 and 9). The competition of [125I]IGF-I was similar in all clones examined (C6CMV-3 and C6BP2-4, -9, and -10; data not shown). C6BP2-1 displayed an additional band of 41 kDa (*) that was competed by the addition of IGF-I and IGF-II, but not insulin (B). The 41-kDa band was also observed with C6BP2-4 and -9 (data not shown). [125I]IGF-II was also used to affinity cross-link monolayers of wild-type C6 (D), C6BP2-1 (E), and C6BP2-12 (F). All clones displayed a band of 240 kDa consistent with the type II receptor (II). Binding was competed most effectively by the addition of IGF-II (lanes 11–13), to a lesser degree by IGF-I (lanes 14–16), and not at all by insulin (lanes 17 and 18). The competition of [125I]IGF-II was similar in all clones examined (C6CMV-3 and C6BP2-4, -9, and -10; data not shown). The 41-kDa band (*) was also observed in C6BP2-1 (E) and C6BP2-4 and -9 (data not shown) when IGF-II was used as radioligand. Competition was observed with the addition of IGF-II and IGF-I, but not insulin.

 
When [125I]IGF-II was used as the radioligand, all clones displayed a 240-kDa band consistent in size with the IGF-II receptor in C6 cells (Fig. 5Go, D–F). This band was competed most effectively by unlabeled IGF-II, to a lesser degree by IGF-I, and not at all by insulin, consistent with the competition profile of the IGF-II receptor. The binding competition profile of [125I]IGF-II was similar in the wild-type C6 cells and in all clones examined (C6CMV-3 and C6BP2-4, -9, and -10; not shown). The 41-kDa band was also observed in C6BP2-1 (Fig. 5EGo) and C6BP2-4 and -9 (data not shown) when [125I]IGF-II was used as radioligand. Its binding was competed by unlabeled IGF-I and IGF-II, but not by insulin, again suggesting its identity as a membrane-associated IGFBP. IGFs competed more effectively for binding to the 41-kDa membrane-associated IGFBP than for the IGF-II receptor, again suggesting that it had a greater affinity for IGF-II than the IGF-II receptor.

Characterization of the cell-associated IGFBP
An antiserum against bovine IGFBP-2 was used to immunoprecipitate [125I]IGF-II cross-linked proteins from conditioned media and cell monolayers of selected clones (Fig. 6Go). In the conditioned media of C6BP2-12, as expected, a large amount of [125I]IGF-II-IGFBP-2 cross-linked complexes were immunoprecipitated, whereas a lesser amount from C6BP2-1 and -4 and a very small amount from C6 were immunoprecipitated. The size of the [125I]IGF-II-IGFBP-2 immunoprecipitated complex from the conditioned media was slightly larger than that of the [125I]IGF-II-IGFBP cross-linked complex associated with the C6BP2-1 cells.



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  		Figure 6. [125I]IGF-II was affinity cross-linked to both monolayer cultures and conditioned medium from C6, C6BP2-1, -4, and -12; immunoprecipitated with antiserum against bovine IGFBP-2; and subjected to SDS-PAGE, and immune complexes were visualized by autoradiography. Immunoreactive [125I]IGF-II-IGFBP complexes were observed in the CM of C6 and C6BP2-1, -4, and -12. An additional [125I]IGF-II-IGFBP complex of smaller size was immunoprecipitated from CM and cell monolayers of C6BP2-1 and -4, which migrated at the same relative mol wt as the [125I]IGF-II cross-linked species from C6BP2-1. Lane 1, C6BP2-1 [125I]IGF-II cross-linked only; lanes 2–5, immunoprecipitated [125I]IGF-II-IGFBP complexes from cell monolayers of C6 and C6BP2-1, -4, and -12; lanes 6–9, immunoprecipitated [125I]IGF-II-IGFBP complexes from CM of C6 and C6BP2-1, -4, and -12.

 
The IGFBP-2 antiserum immunoprecipitated an additional [125I]IGF-II-IGFBP complex of smaller size. This was detected in clones C6BP2-1 and -4 in both the conditioned media and the cells. This [125I]IGF-II-IGFBP complex migrated at the same position as the membrane-associated IGFBP found on C6BP2-1 detected by cross-linking alone, suggesting that the membrane-associated IGFBP was not molecularly identical to IGFBP-2 secreted in the conditioned media, but was an IGFBP that cross-reacted with the IGFBP-2 antiserum. Cross-linking of the [125I]IGF-I-IGFBP complex with the cells of clone C6BP2-1 could be inhibited by the addition of heparin (data not shown). In contrast, peptides containing the Arg-Gly-Asp integrin recognition motif did not inhibit the binding of [125I]IGF-I to the cell-associated IGFBP (data not shown). These results suggested that this IGFBP may associate with the extracellular matrix or with the cell surface, and that the latter did not involve interaction with integrins.

Ligand blot analysis demonstrated that the 29-kDa IGFBP found in the conditioned media of clones C6BP2-1, -4, and -9, but not -12, migrated at the same relative molecular size as IGFBP-5 produced by FRTL-5 cells (Fig. 7Go). Total RNA from the different clones was reexamined by Northern blotting for the expression of IGFBP-1, -5, and -6 mRNAs. A 6.0-kb IGFBP-5 transcript was just detectable in C6BP2-1 and -4 (Fig. 8Go). These data suggest that the 29-kDa IGFBP found in the conditioned media and associated with the cell of the slow growing clones C6BP2-1, -4, and -9 may be IGFBP-5. Immunological characterization of the cell-associated IGFBP was not possible due to the lack of reactivity of the commercially or collaboratively available IGFBP-5 antiserum with various sources of rat IGFBP-5 in Western blotting and immunoprecipitation.



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  		Figure 7. Ligand blot analysis of CM from C6BP2-1, -4, and -12 (lanes 1–3) and FRTL-5 (lane 4), a rat thyroid cell line that secretes predominantly IGFBP-5. The 29-kDa IGFBP secreted by C6BP2-1 and -4, but not by C6BP2-12, comigrates with IGFBP-5 secreted by FRTL-5 cells.

 
To determine whether exogenous purified IGFBP-5 could associate with the cells of the wild-type C6 cells, C6 cells and clone C6BP2-1 were preincubated with human recombinant IGFBP-5 for 24 h and subsequently cross-linked with [125I]IGF-II (Fig. 9Go). The expected 260-kDa type 2 receptor was observed in both C6 cells and clone C6BP2-1. Without preincubation with IGFBP-5, the 41-kDa cell-associated IGFBP was observed in clone C6BP2-1, but not C6 cells. When IGFBP-5 was preincubated for 24 h before cross-linking, a 41-kDa band was observed in C6 cells. Also, a less intense, smaller band of 38 kDa was observed on both the C6 cells and clone C6BP2-1, with preincubation of IGFBP-5, which may be a proteolytic fragment of IGFBP-5. These results suggest that C6 glioma cells have the capacity to associate with IGFBP-5.



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  		Figure 9. [125I]IGF-II was cross-linked to C6 cells (lanes 1 and 2) and clone C6BP2-1 (lanes 3 and 4) after a 24-h preincubation with human recombinant IGFBP-5 (2.5 µg/ml; lanes 2 and 4) or SFM alone (lanes 1 and 3).

 
Effect of IGF-I addition on growth of clones
To test whether a change in the balance between IGF-I and IGFBPs would alter the growth of clones, the wild-type C6 and clones C6BP2-1 and -12 were analyzed in medium containing 1% FBS with or without the daily addition of exogenous IGF-I (200 ng/ml; Fig. 10Go). C6 and C6BP2-12 grew at similar rates and were faster than C6BP2-1. Cell number at 72 h was significantly greater for both C6 and C6BP2-12 than for C6BP2-1. Addition of IGF-I to both C6 and C6BP2-12 resulted in a significant increase in cell number at 72 h compared with their cell number in 1% FBS alone. In contrast, the addition of IGF-I to clone C6BP2-1 (or clones C6BP2-4 or -9) did not result in any change in cell number at 72 h compared with 1% FBS alone. These observations suggest that in these clones, factors other than the balance between IGF-I and IGFBP-2, such as the cell-associated IGFBP-5, are the dominant regulators of cell growth.



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  		Figure 10. Growth curves of C6 ({circ}), C6BP2-1 ({blacktriangleup}), and C6BP2-12 ({blacksquare}) in medium containing 1% FBS with (filled symbols) or without (open symbols) the daily addition of IGF-I (200 ng/ml). Statistically significant increases in cell number at 72 h with the addition of IGF-I compared with those without IGF-I for the same clones are designated by * and {ddagger} (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have overexpressed the oIGFBP-2 cDNA in C6 glioma cells to address the biological role of endogenous IGFBP-2 in astroglial cell growth. This study was based on the observation that cells with regulated growth (primary astroglia) express abundant IGFBP-2, whereas those with unregulated growth (glioma cells) express very little IGFBP-2 (37). To study cells with varying capacities to synthesize IGFBP-2, several clones were selected based on the level of expression of IGFBP-2. We found that changes in the growth rate of the clones from the wild-type C6 cells did not correspond to the level of overexpression of IGFBP-2. This finding was unexpected, as we have previously demonstrated that exogenous IGFBP-2 inhibits IGF-stimulated [3H]thymidine incorporation of primary astroglial cells (32). To our surprise, we noted that clones overexpressing very high levels of IGFBP-2, also expressed correspondingly high levels of IGF-I mRNA and peptide and had growth rates similar to those of the wild-type C6 cells. In contrast, clones overexpressing moderate levels of IGFBP-2, with no increase in IGF-I mRNA and peptide, and with the cell-associated IGFBP had significantly reduced growth rates compared with the wild-type C6 cells. These findings suggest that the growth of C6 cells is closely linked to their capacity to regulate the synthesis of both IGF-I and IGFBPs and the capacity of specific IGFBPs to associate with the cells.

Not only was the level of IGFBP-2 synthesis by the various clones different, but other endogenous C6 IGFBPs were variably altered. The amounts of IGFBP-3 and IGFBP-4 detected in the conditioned medium were less than those observed with the wild-type C6 cells. Differences between steady-state mRNA levels and the amount of protein detected in the conditioned medium suggest that posttranscriptional and/or posttranslational processing may have occurred, such as proteolytic processing. Specific protease activity has been described for IGFBP-3 (44, 45) and for IGFBP-4 (46). However, in the cells studied, IGFBP proteases did not appear to play a role in the changes in IGFBP-3 and -4. An increase in the levels of serum IGFBP-3 and IGFBP-4 has been observed in mice lacking the IGF-II receptor gene (47). Additionally, mice lacking the IGFBP-2 gene also have increased serum levels of IGFBP-3 and IGFBP-4 (48). The changes in IGFBP expression in response to a perturbation in IGF system components may be a compensatory response designed to maintain an appropriate balance of growth-promoting and growth-inhibiting factors.

Based on the growth rates of selected clones, we divided the clones into two separate groups: those that grew at rates similar to the wild-type cells and those that grew about 30% slower than the wild-type cells. Interestingly, the clones that had very high levels of expression of IGFBP-2 grew at the same rate as the wild-type cells, whereas those that moderately overexpressed IGFBP-2 grew at slower rates. Those clones that highly overexpressed IGFBP-2 had compensatory up-regulation of IGF-I, which may account for the maintenance of a wild-type growth rate. This inference was supported by the fact that in those clones (e.g. C6BP2-12) that did not express the cell-associated IGFBP-5, the addition of exogenous IGF-I further increased the growth rates. The mechanism for this up-regulation of IGF-I expression is presently unknown. Regardless of the reason for up-regulation of IGF-I gene expression, it is important to note that this compensatory affect is associated with the maintenance of a normal (wild-type) growth rate, whereas the failure to do so results in impaired growth.

IGF-I has been shown to be an important regulator of C6 cell growth. Reduction of endogenous IGF-I production in C6 cells by transfection of an antisense cDNA, resulted in the loss of tumorigenicity of C6 cells in vivo (36). A similar effect was observed when expression of the IGF-I receptor was blocked by antisense oligonucleotides or cDNA in C6 cells (49). Monolayer growth was inhibited, and tumorigenicity was lost both in vitro and in vivo. Similar studies in other cell types have demonstrated that loss of expression of IGF-I or the IGF-I receptor leads to reduced growth and tumorigenicity (50, 51, 52). We have previously shown that when C6 cells are transfected with the gap junction cDNA connexin 43, the reduction in growth rate of the clones overexpressing the connexin 43 cDNA is also associated with a reduction in the level of expression of IGF-I (37). Our present results support the hypothesis that the interaction of IGF-I with its receptor is an important regulator of C6 cell growth.

Although the variable expression of IGF-I among the different clones could account for the differences in growth, we have observed that different growth patterns are associated with changes in the levels of IGF-I and IGFBPs and IGFBP cellular localization. Consistent with the observation of concomitant increased expression of IGF-I and IGFBP-2, is the hypothesis that IGFBP-2 is acting as a competitive inhibitor for IGF-I binding to its receptor. When changes in the balance between IGFBP-2 to IGF-I occurred, as in those clones with IGFBP-2 overexpression and IGF-I underexpression, growth rates were reduced significantly. However, the slow growing clones exhibited a cell-associated IGFBP in addition to the reduced levels of IGF-I. The cell-associated IGFBP displayed preferential affinity for IGFs compared with the receptors and therefore may be the primary determinant for their slow growth.

Immunoprecipitation of the cytosolic fraction with the IGFBP-2 antiserum yielded faint bands of a smaller size compared with those obtained using IGFBP-2 immunoprecipitated from the conditioned medium. As the antiserum is known to have some cross-reactivity with other IGFBPs, it is possible that the immunoprecipitated cell-associated IGFBP may be another IGFBP. The slow growing clones were found to express IGFBP-5 mRNA and have a secreted IGFBP that corresponded in size to a known IGFBP-5 standard. Preincubation with IGFBP-5 before cross-linking resulted in increased levels of the cell-associated IGFBP in the slow growing clone and the wild-type C6 cells. In addition, coincubation with heparin, but not RGD peptides, reduced cell association of the IGFBP, which strongly indicates that the cell-associated IGFBP is IGFBP-5 (8). This was confirmed by the demonstration of a cell-associated IGFBP after incubation with C6 cells. The association can occur either with the cell surface or the extracellular matrix. The appearance of a second smaller cross-linked species may be due to the proteolytic processing of IGFBP-5. These collective results provide evidence to suggest that the cell-associated IGFBP is IGFBP-5. Whether the IGFBP-5 exerts an IGF-dependent or -independent biological action (4) remains to be delineated.

The observation that IGF-I further stimulated the growth of C6 and C6BP2-12, but not C6BP2-1, supports our hypothesis that the cell-associated IGFBP is acting to inhibit the growth of these clones by preventing the interaction of IGF-I with its receptor. Similar effects have been observed with IGFs and cell-associated IGFBPs in different cell systems. Fibroblasts transformed by simian virus 40, which have tumorigenic growth, displayed an increase in expression of IGF-I and loss of cell-associated IGFBP-5. In untransformed cells, which have normal growth, IGF-I binding was principally to cell-associated IGFBP-5, which inhibited its interaction with the type 1 receptor (53). Additionally, IGFs were found to bind predominantly to a cell-associated IGFBP-2, which was identified on a small cell lung tumor cell line (54). These cells did not respond to IGFs in a DNA synthesis assay, suggesting that the cell-associated IGFBP-2 inhibited IGF action by competing with IGF receptors for IGF binding.

A decrease in IGF-I receptors or a reduction in IGF binding affinity may also explain the reduction in growth of C6BP2-1, -4, and -9. However, this did not appear to be the case in the cells studied because IGF receptor binding affinity was similar in all of the clones, but the affinity cross-linking studies indicate semiquantitatively that there was not an alteration in IGF-I receptor binding. An accurate quantification of IGF receptor number was not possible due to the presence of the cell-associated IGFBP on some clones. A reduction in IGF receptor number could also account for the lack of growth response of C6BP2-1 to exogenous IGF-I. It is possible that expression of other endogenous growth factors was altered by the transfection process, and this may also account for the observed changes in growth; however, this was not examined.

The exact mechanism(s) controlling the growth of the various C6BP2 clones remains to be determined; however, it is clear that the interactions between IGF-I and IGFBPs remain an important determinant of their growth. This study suggests that glial cells may have a limited capacity to modulate various components of the IGF system to maintain normal growth, as evidenced by the concomitant up-regulation of IGF-I and down-regulation of endogenous IGFBPs among various clones. It also highlights the importance of examining all components of the IGF system before formulating conclusions on their role in cellular growth. The growth of astroglial cells, like that of many other cells, is regulated by a balance between growth-promoting and growth-inhibiting factors. Therefore, a decrease in a growth factor, IGF-I, or an increase in a growth inhibitory factor, IGFBP-2, should result in decreased growth. However, this study suggests that the regulation of growth of glial cells is complex, and a coordinated interaction between IGFs and IGFBPs, and specific properties of the latter (i.e. cell association), are required to determine the growth of these cells. Such a mechanism of regulation of glial cell growth most likely exists in the developing brain.


   Acknowledgments
 
We thank Dr. M. Rechler, NIH (Bethesda, MD), for the rat IGFBP-1 cDNA; Dr. A. Herington (Melbourne, Australia) for the rat IGFBP-3 cDNA; Dr. S. Shimasaki (San Diego, CA) for the rat IGFBP-4, -5, and -6 cDNAs; Dr. L. Murphy (Winnipeg, Canada) for the rat IGF-I cDNA; Dr. G. Bell (Chicago, IL) for the mouse IGF-II cDNA; and Dr. D. Denhardt, Rutgers University (Piscataway, NJ), for the 18S ribosomal cDNA.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada (to V.K.M.H.). Back

2 Recipient of a Medical Research Council Studentship. Back

Received July 23, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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