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Endocrinology Vol. 141, No. 10 3546-3555
Copyright © 2000 by The Endocrine Society

ARTICLES

Double-Stranded Ribonucleic Acid Decreases C6 Rat Glioma Cell Numbers: Effects on Insulin-Like Growth Factor I Gene Expression and Action1

Mary S. Chacko and Martin L. Adamo

Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900

Address all correspondence and requests for reprints to: Martin L. Adamo, Ph.D., Department of Biochemistry, Mail Code 7760, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900. E-mail: adamo{at}biochem.uthscsa.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Poly(IC), a synthetic double-stranded RNA copolymer of inosinic and cytidilic acids, decreases the growth of normal and tumorigenic cells. We tested the hypothesis that Poly(IC) decreases C6 glioma cell growth by disrupting an autocrine insulin-like growth factor I (IGF-I) growth loop. Addition of Poly(IC) decreased C6 cell number in confluent and sparse cultures in a dose-dependent manner. Addition of exogenous IGF-I partially compensated for the decrease in cell number caused by Poly(IC) in confluent and subconfluent cultures of C6 cells, suggesting that one mechanism of Poly(IC) action is through down-regulation of IGF-I gene expression and/or action. Treatment of confluent C6 cells with 10 and 200 µg/ml Poly(IC) for 24 h decreased IGF-I messenger RNA (mRNA) levels to 50% and 25% of the control value, respectively. Treatment of C6 cells with 200 µg/ml Poly(IC) for 24 h reduced IGF-I receptor mRNA levels to 50% of the control level. IGF-binding protein-1 (IGFBP-1), -2, and -6 mRNAs were not expressed in the C6 cells used in this study. Treatment of C6 cells with 200 µg/ml Poly(IC) for 24 h reduced IGFBP-4 mRNA and IGFBP-5 mRNA levels to 26% and 29% of the control level, respectively. There was no significant change in IGFBP-3, insulin receptor, or actin mRNA levels with Poly(IC) treatment. Treatment of confluent C6 cells with 200 µg/ml Poly(IC) for 24 h decreased levels of immunoreactive IGF-I in conditioned medium (CM) to 55% of the control value, decreased IGF-I receptor ß-subunit levels to 28% of the control value, and decreased levels of IGFBP-3, IGFBP-4, and IGFBP-5 protein in CM to 45%, 50%, and 30% of the control values, respectively. There was no significant change in actin and tubulin protein levels with Poly(IC) treatment. These results suggest that IGF-I gene expression is down-regulated by Poly(IC) treatment and that IGF-I bioavailability and action in C6 cells are also altered due to decreases in IGF-I receptor and binding protein levels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
POLY(IC) IS a synthetic double-stranded (ds) RNA copolymer of inosinic and cytidilic acids. dsRNA has been shown to inhibit the growth of tumors in vivo and transformed cells in culture (1, 2). dsRNA binds to and activates dsRNA-activated protein kinase (PKR). In response to dsRNA, PKR phosphorylates inhibitory factor-{kappa}B (I{kappa}B) and releases an active form of nuclear factor-{kappa}B (NF{kappa}B) (3). This active NF{kappa}B translocates to the nucleus and binds to cognate DNA sequences to regulate the transcription of a variety of genes including type I interferon (IFN). IFN induction is thought to be one of the pathways through which dsRNA exerts its antiproliferative effects. Type I IFNs bind to the type I IFN receptor, leading to the consecutive phosphorylation and activation of Janus kinases (JAKs) and signal transducers and activators of transcription (STATs), which, in turn, lead to the induction of IFN-stimulated genes (4).

IFN-stimulated genes include cellular enzymes such as 2'-5'-oligoadenylate synthetase and PKR. Upon activation, 2'-5'-oligoadenylate synthetase converts ATP into a thermostable compound called 2,5A. This compound, in turn, activates a latent endonuclease, ribonuclease L (RNase L) (5). PKR, once activated, phosphorylates translation initiation factor eIF2-{alpha} and certain histones. Phosphorylated eIF2-{alpha}, in turn, binds to the guanine exchange factor eIF2B and subsequently inhibits initiation of protein synthesis (6). Thus, dsRNA influences gene expression at transcriptional, posttranscriptional, and translational levels, and it has been proposed that a consequence of dsRNA action may be to reduce the level of agents that stimulate cell proliferation and/or maintain survival (5).

Insulin-like growth factor I (IGF-I) is a widely expressed peptide hormone whose functions include stimulation of cell proliferation and cell survival (7). The effects of IGF-I are mediated through the IGF-I receptor (IGF-IR). IGF-I is found bound to a family of binding proteins called the IGF-binding proteins (IGFBPs). The IGFBPs not only prolong the half-life and metabolic clearance of IGF-I, but also modulate IGF-I availability and action at the cellular level (7).

As Poly(IC) has been shown to inhibit the growth of tumors as well as transformed cells, we hypothesized that one mechanism by which this could occur was by down-regulation of IGF-I gene expression and/or action. We found that Poly(IC) decreased cell numbers concomitant with decreased IGF-I levels. Exogenous IGF-I was able to partially prevent Poly(IC) from reducing cell number, thus suggesting that IGF-I expression and action may, in part, mediate the effects of Poly(IC).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture: cell number assay
For Poly(IC) dose-response on C6 cells. C6 cells (American Type Culture Collection, Manassas, VA) were either grown to confluence in 35-mm plates or were plated at 60% confluence and grown for 1 day in 35-mm plates (Corning, Inc., Corning, NY) in Ham’s F-12 medium (Mediatech, Herndon, VA) containing 1 mM glutamine and supplemented with 0.001% (wt/vol) penicillin/streptomycin (PS; Mediatech, Herndon, VA) and 10% (vol/vol) FBS (Life Technologies, Inc., Gaithersburg, MD). The cells were then placed in serum-free Ham’s F-12 medium containing 1 mM glutamine and supplemented with 0.1% BSA (Sigma, St. Louis, MO) and 0.001% (wt/vol) PS for 1 day. The cells were then treated with or without Poly(IC) (Pharmacia, Piscataway, NJ) at final concentrations of 10, 100, and 200 µg/ml for 3 days. Medium was supplemented with fresh Poly(IC) every 24 h. Cell numbers were counted after 2 and 3 days. Monolayers were washed with PBS, and cells were trypsinized and diluted in trypan blue.

For IGF-I compensation of Poly(IC) effect on C6 cells. C6 cells were either grown to confluence in 35-mm plates or were plated at 60% confluence and grown for 1 day in 35-mm plates in Ham’s F-12 medium containing 1 mM glutamine and supplemented with 0.001% (wt/vol) PS and 10% (vol/vol) FBS (Life Technologies, Inc.). The cells were then placed in serum-free Ham’s F-12 medium containing 1 mM glutamine and supplemented with 0.1% BSA and 0.001% (wt/vol) PS for 1 day. The cells were then treated with or without Poly(IC) at a final concentration of 10 µg/ml. After 1 day of incubation with Poly(IC) alone, medium was supplemented with fresh Poly(IC) and IGF-I (donated by Genentech, Inc., South San Francisco, CA) at concentrations ranging from 1–100 nM. Cell numbers were counted after an additional 1 or 2 days of incubation. Thus, cells were treated with Poly(IC) for a total of 2 or 3 days, respectively, and with IGF-I for a total of 1 or 2 days. Medium was changed every 24 h. Monolayers were washed with PBS and then trypsinized. Cell suspensions were diluted in trypan blue, and both viable (cells excluding trypan blue) and nonviable cells (cells not excluding trypan blue) were counted using a hemocytometer.

For IGF-I correlation with cell number in proliferating C6 cells. C6 cells were plated sparsely in 35-mm plates in Ham’s F-12 medium containing 1 mM glutamine and supplemented with 0.001% PS and 0.1% BSA. The cells were treated with or without 200 µg/ml Poly(IC) on the day of plating. Cells were counted after 1, 3, 4, 7, and 9 days of Poly(IC) treatment. RNA was extracted from parallel plates. Medium was changed every 2 days. Monolayers were washed with PBS and then trypsinized. Cell suspensions were diluted in trypan blue, and both viable and nonviable cells were counted using a hemocytometer.

RNA extraction
C6 cells were grown to confluence in 100-mm plates in Ham’s F-12 medium supplemented with 0.001% PS and 10% FBS. The cells were then placed in serum-free Ham’s F-12 medium supplemented with 0.1% BSA and 0.001% PS for 24 h. The cells were treated with or without Poly(IC) at a final concentration of 10 or 200 µg/ml. The Poly(IC) treatments were also performed in serum-free Ham’s F-12 medium supplemented with 0.1% BSA. Total RNA was harvested using Ultraspec (Biotecx Laboratories, Houston, TX). Absorbance at 260 nm was used to calculate the RNA concentration. The RNAs were electrophoresed through 1.25% agarose/2.2 M formaldehyde gels to check for degradation. Equal amounts of total RNA were used in solution hybridization/RNase protection assays (RPAs) to study changes in messenger RNA (mRNA) levels of IGF-I, IGF-IR, and the three IGF-binding proteins found in C6 cells, i.e. IGFBP-3, IGFBP-4, and IGFBP-5 (8, 9, 10), and ß-actin.

Solution hybridization/RPA
Synthesis of antisense RNA probes was performed using the MAXIscript kit (Ambion, Inc., Austin, TX). Briefly, the template DNA was linearized using the appropriate restriction enzyme. The transcription reaction was assembled according to kit directions, and run-off transcription was performed using the polymerase specific for the construct (Sp6 or T7) and [{alpha}-32 P]UTP (NEN Life Science Products, Boston, MA). The DNA template was digested with deoxyribonuclease (Promega Corp., Madison, WI) after transcription was complete (11). The transcripts were extracted with phenol and isoamyl alcohol/chloroform, and unincorporated ribonucleotides were removed by two rounds of ethanol precipitation (11).

Probes
IGF-I probe. The antisense RNA probe for IGF-I was generated from a 464-bp IGF-I DNA clone ligated into pGEM-4Z (12). The vector was linearized with EcoRI, and run-off transcription was performed using T7 RNA polymerase.

IGF-IR probe. The antisense RNA probe for IGF-IR was generated from a 265-bp EcoRI- RsaI fragment that had been subcloned into pGEM-3 (13). The vector was linearized with EcoRI, and run-off transcription was performed using SP6 RNA polymerase.

IGFBP-3 probe. A 551-bp IGFBP-3 insert was removed from plasmid pRF1507 using AccI and BamHI, and the insert was ligated into the same sites in plasmid vector pGEM2. The vector was linearized with EcoRI, and run-off transcription was performed using T7 RNA polymerase.

IGFBP-4 probe. A 444-bp IGFBP-4 insert was removed from plasmid pRBP-4 501 using SmaI-HindIII and was cloned into the same sites in plasmid vector pGEM2. The vector was linearized with HindIII, and run-off transcription was performed using T7 RNA polymerase.

IGFBP-5 probe. The antisense RNA probe for IGFBP-5 was generated from a 300-bp HindIII-SacI fragment that had been subcloned into pRBP5–501 The insert was removed from the vector using HindIII and SacI and cloned into the HindIII and SacI sites in pGEM4-Z (14). The vector was linearized with EcoRI, and run-off transcription was performed using T7 RNA polymerase.

IGFBP plasmids pRF1507, pRBP4–501, and pRBP5–501 were gifts from Drs. Shunichi Shimasaki and Nicholas Ling.

Insulin receptor probe. The antisense RNA probe for insulin receptor was generated from a 747-bp genomic fragment that had been subcloned into pGEM-4 (15). The vector was linearized with EcoRI, and run-off transcription was performed using T7 RNA polymerase. The probe included 478 bases complementary to the mature insulin receptor mRNA sequence as reported previously (16). The insulin receptor probe was a gift from Dr. Charles Roberts.

Actin probe. The antisense RNA probe for actin was generated from a 126-bp fragment from the rat cytoplasmic ß-actin complementary DNA (cDNA; Ambion, Inc., Austin, TX). Run-off transcription was performed using T7 RNA polymerase.

RPAs were performed using protocols described previously (11). Twenty micrograms of RNA from either control or Poly(IC)-treated C6 cells were hybridized for about 18 h to the various 32P-labeled probes described above. The unhybridized single stranded regions were digested using RNases A and T1 (Ambion, Inc.). SDS and proteinase K were added to the reaction, and proteins were extracted using phenol and isoamyl alcohol-chloroform. The RNA was precipitated in ethanol and finally resolved on a 6% polyacrylamide gel. The gels were exposed to film overnight, and the bands were quantified using phosphorimager analysis.

IGF-I RIA. The IGF-I RIAs were performed by Dr. Clifford J. Rosen and Julie Burgess at the Maine Center for Osteoporosis Research and Education Laboratory (Bangor, ME) using the protocol described previously (17), which is a modification of the protocol described in (18).

IGFBP ligand blot
The levels of IGFBPs were determined from conditioned medium using the ligand blot protocol described previously (19). Conditioned medium was collected from control and Poly(IC)-treated cells and centrifuged at 3000 rpm for 5 min to remove dead cells and debris. The protease inhibitors aprotinin, leupeptin, and pepstatin at final concentrations of 6.5, 10, and 6.5 µg/ml, respectively, were added to the conditioned medium. Trichloroacetic acid was added to a final concentration of 5%, and the proteins were allowed to precipitate overnight at 4 C (20). Samples were centrifuged for 20 min to collect protein, and the pellets were dissolved in Laemmli buffer [100 mM Tris-HCl (pH 6.8), 2% SDS, 20% glycerol, and 0.04% bromophenol blue]. Samples were subjected to SDS-PAGE (15% resolving gel, with a 5% stacking gel). Proteins were transferred to a nitrocellulose membrane (Immobilon P, Millipore Corp., Bedford, MA) at 150 V for 2 h. The membrane was blocked to prevent nonspecific binding by incubating in 3% Nonidet P-40 in WLB saline [10 mM Tris-HCl (pH 7.4), 8.8 g/liter NaCl, and 0.5 g/liter sodium azide] for 30 min. The membrane was then incubated in 1% Tween-20 solution in WLB saline for 2 h, followed by incubation in 1% BSA solution in WLB saline for 10 min and then incubated overnight in a solution containing 1% Tween-20, 1% BSA, and 2.5 x 105 cpm [125I]IGF-I (NEN Life Science Products) in WLB saline. After this step, the membrane was washed twice in 1% Tween-20 in WLB saline for 15 min, washed once for 15 min in WLB saline, and then washed twice more for 5 min each in WLB saline solution. The membrane was air-dried for 10 min and then exposed to film overnight.

Western blot
IGF-IR, actin, and tubulin Western immunoblots were performed according to the protocols described previously (21). Conditioned medium was removed from control and Poly(IC)-treated cells, and the monolayers were rinsed with cold PBS. Cells were scraped off the plate and lysed in lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.5% Nonidet P-40] containing freshly added protease and phosphatase inhibitors (50 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride, 25 µg/ml aprotinin, 25 µg/ml trypsin inhibitor, and 2 mM ß-glycerophosphate). Cell lysates were passed through a 21-gauge needle several times to shear DNA and then incubated on ice for 30 min. Cell lysates were centrifuged at 12,000 rpm for 20 min at 4 C, and the supernatants were transferred to a new tube. Equal amounts of cell lysate protein [as determined by Bradford assay (22)] from control and Poly(IC)-treated cells were combined with Laemmli buffer [100 mM Tris-HCl (pH 6.8), 2% SDS, 20% glycerol, and 0.04% bromophenol blue, and 2% ß-mercaptoethanol] and subjected to SDS-PAGE (10% resolving, with a 5% stacking gel in all cases) at 200 V. Proteins were transferred to nitrocellulose membrane (Immobilon P, Millipore Corp.) for 1 h and 20 min at 150 V. The membrane was incubated for 3 h in 5% dry milk solution in TBST [20 mM Tris-HCl (pH 7.4), 500 mM NaCl, and 0.05% Tween-20] to block nonspecific binding and then incubated with the respective antibody at a dilution of 1:1000 overnight in a solution of 1% dry milk solution in TBST. The antibodies used were polyclonal anti-IGF-I receptor ß-subunit (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), monoclonal anti-ß-tubulin (Sigma), and monoclonal anti-ß-actin (Sigma). After the incubation with primary antibody, the membrane was washed three times in TBST for 15 min each time. The membrane was then incubated with the respective secondary antibody (antirabbit IgG for IGF-IR ß-subunit and antimouse IgG for both ß-actin and ß-tubulin; Pierce Chemical Co., Rockford, IL) at a concentration of 1:1000 for 1 h and then washed three times in TBST for 15 min each time. The membrane was incubated with enhanced chemiluminescence reagents (Pierce Chemical Co., Rockford, IL) and exposed to film, and the bands were quantified using densitometric analysis.

Statistical analysis
Data are the mean ± SEM for the indicated number of observations. Statistical differences between means was determined using the one-way ANOVA in the SIMSTAT3 package (Normand Peladeau, Provalis Research, Montreal, Canada) or SigmaStat (Jandel Corp., San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Decrease in C6 cell number with Poly(IC) treatment and partial compensation of the Poly(IC) effect by exogenous IGF-I
Confluent cultures of C6 cells were treated with different concentrations of Poly(IC) for 3 days. Cell numbers were counted after 2 (day 2) or 3 (day 3) days of treatment. All cell numbers were normalized and are expressed as the fold change relative to the cell numbers of control plates (i.e. not treated with Poly(IC) on day 2. Treatment of C6 cells with Poly(IC) decreased attached cell numbers in confluent cultures in a dose-dependent manner (Fig. 1AGo). Treatment with 10 µg/ml Poly(IC) decreased cell numbers to 57% of the control value on day 2 and to 80% of the control value on day 3 (P < 0.05). Treatment with 100 µg/ml Poly(IC) decreased cell number to 53% of the control value on day 2 and to 64% of the control value on day 3 (P < 0.05). Treatment with 200 µg/ml Poly(IC) decreased cell number to 33% of the control value on day 2 and to 43% of the control value on day 3 (P < 0.05). Greater than 95% of the cells remaining attached to the plate were viable, as assessed by trypan blue exclusion.



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  		Figure 1. Poly(IC) decreases the cell number of confluent and subconfluent cultures of C6 cells in a dose-dependent manner. C6 cells were either grown to confluence in 35-mm plates (A) or were plated at 60% confluence in 35-mm plates and grown for 1 day (B). The cells were placed in serum-free medium for 1 day. They were then treated with 0 (control), 10, 100, or 200 µg/ml Poly(IC) for 3 days. Medium was supplemented with fresh Poly(IC) every 24 h. Cell numbers were counted after 2 and 3 days. Monolayers were washed with PBS, and cells were trypsinized and diluted in trypan blue. The y-axis represents cell number normalized to the control value on day 2. The data represent three separate experiments. The bars are the SEM.

 
Treatment of C6 cells with Poly(IC) also decreased cell numbers in subconfluent cultures in a dose-dependent manner (Fig. 1BGo). Treatment with 10 µg/ml Poly(IC) decreased cell number to 70% of the control value on day 2 and to 51% of the control value on day 3 (P < 0.05). Treatment with 100 µg/ml Poly(IC) decreased cell number to 57% of the control value on day 2 and to 42% of the control value on day 3 (P < 0.05). Treatment with 200 µg/ml Poly(IC) decreased cell number to 40% of the control value on days 2 and 3 (P < 0.05).

Our hypothesis was that the decrease in cell number observed with Poly(IC) treatment was mediated, at least in part, by down-regulation of IGF-I gene expression and/or action. Therefore, after 1 day of incubation in medium containing 10 µg/ml Poly(IC) alone, the cells were treated with fresh medium supplemented with 10 µg/ml Poly(IC) and IGF-I at concentrations ranging from 1–100 nM. Cell number was counted after either 1 (Day 1) or 2 (Day 2) days of incubation in the new medium. Medium was changed after 1 day.

As expected, Poly(IC) decreased cell number to 78% (P < 0.05) of the control value on day 1 (P < 0.05) and to 30% of the control value (P < 0.05) on day 2 in confluent C6 cells (Fig. 2AGo). Addition of 1 nM IGF-I increased cell number by 46% compared with that in cultures treated with Poly(IC) alone on day 2 (P < 0.05). Addition of 10 nM IGF-I increased cell number by 46% compared with that in cultures treated with Poly(IC) alone on day 2 (P < 0.05). Addition of 100 nM IGF-I increased cell number by 17% compared with that in cultures treated with Poly(IC) alone on day 1(P < 0.05) and increased cell number by 48% compared with that in cultures treated with Poly(IC) alone on day 2 (P < 0.05; Fig. 2AGo).



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  		Figure 2. The decrease in cell number of C6 cells treated with Poly(IC) is partially compensated by addition of exogenous IGF-I. C6 cells were grown to confluence in 35-mm plates (A) or were plated at 60% confluence in 35-mm plates and grown for 1 day (B). The cells were then placed in serum-free medium for 1 day. They were treated without (control) with 10 µg/ml Poly(IC) (P[IC]) for 1 day. After 1 day of incubation with Poly(IC) alone, medium was supplemented with fresh Poly(IC) and IGF-I at concentrations ranging from 1–100 nM. Cell numbers were counted after an additional 1 and 2 days of incubation. The medium was changed after 1 day. Monolayers were washed with PBS, and cells were trypsinized and diluted in trypan blue. The y-axis represents cell number normalized to the control value on day 1. *, Data are significant compared with control on that day. **, Data are significant compared with Poly(IC)-treated cells on that day. The data represent three separate experiments. The bars are the SEM.

 
Also, as expected, Poly(IC) decreased cell number to 57% (P < 0.05) of the control value on day 1 and to 58% of the control value on day 2 in subconfluent C6 cells (Fig. 2BGo). Addition of 10 nM IGF-I increased cell number by 26% compared with that in cultures treated with Poly(IC) alone on day 2 (P < 0.05). Addition of 100 nM IGF-I increased cell number by 49% compared with that in cultures treated with Poly(IC) alone on day 1 (P < 0.05) and increased cell number by 75% compared with that in cultures treated with Poly(IC) alone on day 2 (P < 0.05; Fig. 2BGo).

In all of the cell count experiments, more than 99% of the cells counted excluded trypan blue.

Effect of Poly(IC) on IGF system mRNA levels in confluent and subconfluent C6 cells
In an attempt to correlate the decreased cell number of C6 cells with changes in IGF-I gene expression, we next studied the effect of Poly(IC) on endogenous IGF-I mRNA levels in C6 cells. As IGF-I action is mediated via binding to the IGF-I R, and because the cellular availability of IGF-I to its receptor is modulated by members of the IGFBP family, we also studied the effect of Poly(IC) on gene expression of the members of the IGF system that were expressed in C6 cells, i.e. IGF-IR, IGFBP-3, IGFBP-4, and IGFBP-5 (8, 9, 10, 23).

IGF-I, IGF-IR, IGFBP-3, IGFBP-4, IGFBP-5, insulin receptor, and actin mRNAs formed protected bands of 238, 265, 551, 444, 300, 478, and 126 nucleotides, respectively (Fig. 3Go). The quantified data are presented in Fig. 4Go. The IGF-I mRNA levels were decreased to 50% of the control value at 10 µg/ml Poly(IC) (P < 0.05) and to 25% of the control value (P < 0.05) at 200 µg/ml Poly(IC). The IGF-I R mRNA levels were decreased to 82% of the control value at 10 µg/ml Poly(IC) (P > 0.05) and to 50% of the control value at 200 µg/ml Poly(IC) (P < 0.05). The IGFBP-4 mRNA levels were decreased to 75% of the control value at 10 µg/ml Poly(IC) (P > 0.05) and to 26% of the control value at 200 µg/ml Poly(IC) (P < 0.05). The IGFBP-5 mRNA levels were decreased to 56% of the control value at 10 µg/ml Poly(IC) (P > 0.05) and to 29% of the control value at 200 µg/ml Poly(IC) (P < 0.05). There were no statistically significant changes in mRNA levels of IGFBP-3, insulin receptor, or actin in Poly(IC)-treated cells with respect to control.



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  		Figure 3. Effect of Poly(IC) on mRNA levels of IGF system components in confluent C6 cells. Confluent cultures of C6 rat glioma cells grown in 100-mm plates were cultured 1 day in serum-free Ham’s F-12 medium and were treated without (control) or with Poly(IC) at a concentration of either 10 or 200 µg/ml serum-free Ham’s F-12 medium for 1 day. Total RNA was harvested, and equal concentrations of RNA from control and treated cells were hybridized to the different IGF system and actin probes described in Materials and Methods. Lane 1, RNA from control cells; lane 2, RNA from cells treated with 10 µg/ml Poly(IC); lane 3, RNA from cells treated with 200 µg/ml Poly(IC).

 


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  		Figure 4. Quantified data of effect of Poly(IC) on mRNA levels of IGF system components in confluent C6 cells. Confluent cultures of C6 rat glioma cells grown in 100-mm plates were cultured 1 day in serum-free Ham’s F-12 medium and were treated without (control) or with Poly(IC) at a concentration of either 10 or 200 µg/ml serum-free Ham’s F-12 medium for 1 day. Total RNA was harvested, and equal concentrations of RNA from control and treated cells were hybridized to the different IGF system and actin probes described in Materials and Methods. The data represent three separate experiments. Bars are the SEM.

 
Levels of IGF-I, IGF-IR, and IGFBPs are dependent on cell density (24, 25, 26, 27, 28, 29). Trypan blue exclusion and cell counts showed that there was little difference in the number of viable cells with Poly(IC) treatment after 24 h (Fig. 5Go), although, as expected, cell numbers were decreased at 48 h. Thus, the decrease in mRNA levels in members of the IGF system demonstrated at 24 h cannot be attributed to a change in cell number, and moreover, it occurs before the decrease in cell number.



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  		Figure 5. Changes in mRNA levels in the IGF system at 24 h cannot be explained by changes in cell number in confluent C6 cells. Confluent cultures of C6 rat glioma cells grown in 100-mm plates were cultured for 1 day in serum-free Ham’s F-12 medium and were treated without (control) or with Poly(IC) at a concentration of 200 µg/ml serum-free Ham’s F-12 medium for 24 or 48 h. Monolayers were washed with PBS, and cells were trypsinized and diluted in trypan blue. Viable and nonviable cells were counted using a hemocytometer. The y-axis represents cell number normalized to the number of control cells after 24 h of treatment. The data are from three separate experiments. Bars are the SEM.

 
As the Poly(IC)-mediated decrease in cell number was partially prevented by exogenous IGF-I in subconfluent as well as confluent cultures of C6 cells, the effect of Poly(IC) on IGF-I mRNA was also studied in rapidly proliferating C6 cells. The levels of IGF-I mRNA increased 16-fold with increasing cell number in the cells that were not treated with Poly(IC) (Fig. 6Go, A and B). The increase in IGF-I mRNA as a function of cell growth in the Poly(IC)-treated cells was only 3-fold (P < 0.05 compared with the 16-fold increase seen in control cultures). The difference in the fold induction of IGF-I mRNA suggests that Poly(IC) inhibits the cell number- mediated induction of IGF-I mRNA in C6 cells (29). The cells proliferated in the presence of Poly(IC), but cell numbers in the Poly(IC)-treated cells were lower than those in the control cells by an average of 55% over the course of the experiment. The lower levels of IGF-I mRNA seen in Poly(IC)-treated cells cannot, however, be explained solely by a lower cell number. Comparing day 7 for control cells and day 9 for Poly(IC)-treated cells, the cell numbers were equivalent, but in the Poly(IC)-treated cells the IGF-I mRNA was 4-fold lower than at a similar cell number in control cells. The decrease in cell proliferation rate may be due to the decreased induction of IGF-I mRNA in the Poly(IC) cultures. Actin mRNA did not show significant changes over the course of the experiment (Fig. 7Go, A and B; P > 0.05).



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  		Figure 6. The decrease in cell number in proliferating C6 cells treated with Poly(IC) can be correlated with a decrease in IGF-I mRNA. C6 cells were plated in 35-mm plates and grown for 1 day. The cells were then cultured for 1 day in serum-free Ham’s F-12 medium and treated without (control) or with 200 µg/ml Poly(IC). The medium was changed every 2 days. Cells were harvested for cell number after 1, 3, 4, 7, and 9 days of Poly(IC) treatment. Monolayers were washed with PBS, and cells were trypsinized and diluted in trypan blue. Total RNA was harvested from parallel plates, and equal concentrations of RNA from control and treated cells were hybridized to the rat IGF-I mRNA probe described in Materials and Methods. Lane 1, RNA from control plates after 9 days; lane 2, RNA from Poly(IC)-treated plates after 9 days; lane 3, RNA from control plates after 1 day; lane 4, RNA from Poly(IC)-treated plates after 1 day; lane 5, RNA from control plates after 3 days; lane 6, RNA from Poly(IC)-treated plates after 3 days; lane 7, RNA from control plates after 4 days; lane 8, RNA from Poly(IC)-treated plates after 4 days; lane 9, RNA from control plates after 7 days; lane 10, RNA from Poly(IC)-treated plates after 7 days. The bands in A were quantified using PhosphorImager analysis, and the quantitative data are shown in B. The line graphs represent cell number, and the bar graphs represent IGF-I mRNA. The data are from two separate experiments. Bars are the SEM.

 


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  		Figure 7. Poly(IC) has little effect on actin mRNA in proliferating C6 cells. C6 cells were plated in 35-mm plates and grown for 1 day. The cells were then cultured for 1 day in serum-free Ham’s F-12 medium and treated without (control) or with 200 µg/ml Poly(IC). The medium was changed every 2 days. Cells were harvested for cell number after 1, 3, 4, 7, and 9 days of Poly(IC) treatment. Monolayers were washed with PBS, and cells were trypsinized and diluted in trypan blue. Total RNA was harvested from parallel plates, and equal concentrations of RNA from control and treated cells were hybridized to the rat actin mRNA probe described in Materials and Methods. Lane 1, RNA from control plates after 9 days; lane 2, RNA from Poly(IC)-treated plates after 9 days; lane 3, RNA from control plates after 1 day; lane 4, RNA from Poly(IC)-treated plates after 1 day; lane 5, RNA from control plates after 3 days; lane 6, RNA from Poly(IC)-treated plates after 3 days; lane 7, RNA from control plates after 4 days; lane 8, RNA from Poly(IC)-treated plates after 4 days; lane 9, RNA from control plates after 7 days; lane 10, RNA from Poly(IC)-treated plates after 7 days. The bands in A were quantified using PhosphorImager analysis, and the quantitative data are shown in B. The data are from two separate experiments. Bars are the SEM.

 
Effect of Poly(IC) on IGF system protein levels in confluent C6 cells
The decrease in mRNA levels of some of the IGF system components led us to assay the changes in peptide levels of IGF-I, IGF-IR, and the IGFBPs-3, -4, and -5 in Poly(IC)-treated cells. IGF-I RIA was used to quantify IGF-I peptide in the conditioned medium of confluent C6 cells. The immunoreactive IGF-I level in unconditioned medium was 0.63 ± 0.11 ng/ml. Treatment of confluent C6 cells with 200 µg/ml Poly(IC) for 24 h decreased immunoreactive IGF-I levels to 58% of the control value from 2.25 ± 0.05 to 1.3 ± 0.05 ng/ml (P < 0.05; Fig. 8Go).



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  		Figure 8. Poly(IC) decreases IGF-I peptide levels in conditioned medium from confluent C6 cells. Confluent cultures of C6 rat glioma cells grown in 100-mm plates were cultured for 1 day in Ham’s F-12 medium supplemented with 1% FBS and treated without (control) or with 200 µg/ml Poly(IC) in Ham’s F-12 medium supplemented with 1% FBS for 1 day. Conditioned medium from control and Poly(IC)-treated cells and unconditioned medium were subjected to RIA as described in Materials and Methods. The data represent the mean of three separate experiments, except for unconditioned medium where n = 2. Bars are the SEM.

 
The IGFBPs were visualized using Western ligand blots (Fig. 9AGo). Three bands that bound [125I]IGF-I were observed, the largest of which migrated between the 46- and 30-kDa molecular mass markers. The other two bands migrated near the 30-kDa and 21.5-kDa markers, respectively. Based on the mRNA data described earlier, the knowledge that C6 cells express IGFBP-3, -4, and -5 (8, 9, 10), and the apparent molecular masses of these three proteins, they were identified as IGFBP-3 (whose molecular mass is known to be between 39–44 kDa) (30), IGFBP-5 (whose molecular mass is known to be 31 kDa) (31), and IGFBP-4 (whose molecular mass is known to be 24 kDa) (30), respectively. The quantified data are presented in Fig. 9BGo. Treatment of confluent C6 cells with 200 µg/ml Poly(IC) decreased IGFBP-3 peptide levels to 45% of the control value (P < 0.05), decreased IGFBP-4 levels to 50% of the control value, and decreased IGFBP-5 peptide levels to 30% of the control value (P < 0.05), respectively.



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  		Figure 9. Poly(IC) decreases IGFBP-3, IGFBP-4, and IGFBP-5 protein levels in confluent C6 cells. Confluent cultures of C6 rat glioma cells grown in 100-mm plates were cultured for 1 day in Ham’s F-12 medium supplemented with 1% FBS and treated without (control) or with 200 µg/ml Poly(IC) in Ham’s F-12 medium supplemented with 1% FBS for 1 day. Conditioned medium from control and Poly(IC)-treated cells and unconditioned medium were subjected to ligand blot as described in Materials and Methods. The bands in A were quantified using PhosphorImager analysis, and the quantitative data are shown in B. Lane 1, Conditioned medium from control cells; lane 2, conditioned medium from cells treated with 200 µg/ml Poly(IC). The data are the mean of three separate experiments. Bars are the SEM.

 
IGF-IR ß-subunit was visualized using Western blotting (Fig. 10AGo). Bands representing the IGF-IR ß-subunit migrated between the 83 and 114 kDa molecular mass markers. Bands representing ß-actin and ß-tubulin protein migrated near the 37- and 62-kDa markers, respectively. The quantified data are presented in Fig. 10BGo. Treatment of confluent C6 cells with 200 µg/ml Poly(IC) decreased IGF-IR ß-subunit levels to 28% of the control value. There was no significant effect on ß-actin and ß-tubulin protein levels.



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  		Figure 10. Poly(IC) decreases IGF-R ß subunit levels in confluent C6 cells. Confluent cultures of C6 rat glioma cells grown in 100-mm plates, were cultured 1 day in Ham’s F-12 medium supplemented with 1% FBS and were treated without (control) or with 200 µg/ml Poly(IC) in Ham’s F-12 medium supplemented with 1% FBS for 1 day. Lane 1, Cell lysate from control cells; lane 2, cell lysate from Poly(IC)-treated cells. Fifty micrograms of cell lysate from control and Poly(IC)-treated C6 cells were used to detect IGF-IR ß-subunit. One hundred micrograms of cell lysate were used to detect ß-tubulin. Fifteen micrograms of cell lysate from control and Poly(IC)-treated C6 cells were used to detect ß-actin. The bands in A were quantified using densitometric analysis, and the quantitative data are shown in B. The data are the mean of two separate experiments. Bars are the SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Organisms are continuously adapting to changes in the environment and have evolved mechanisms to protect themselves against damage by harmful changes, i.e. stresses to their environment. Cellular stress is induced by insults such as nutrient starvation, viral or bacterial infection, proinflammatory mediators, radiation, and oxidative damage. The stress response involves biochemical changes that affect protein biosynthesis and gene transcription. Stressors cause inhibition of cellular proliferation and induction of apoptosis, possibly by changes in the production of locally acting growth factors. The stress inducer used in this series of experiments, Poly(IC), mimics the stress of viral infection (1). Moreover, Poly(IC) has been shown to inhibit the growth of tumors in vivo and transformed cells in culture (1, 2). One mechanism by which Poly(IC) could decrease the proliferation of cells is by decreasing the levels of agents that stimulate cell proliferation and/or maintain survival at transcriptional, posttranscriptional, or translational levels (5).

Poly(IC) decreased the cell number of confluent and proliferating cultures of C6 cells in a dose-dependent manner. The decrease in cell number could be due to decreased cell proliferation, increased cell death, or both. The decrease in cell number in C6 cells led us to hypothesize that one mechanism by which Poly(IC) acted was via down-regulation of a growth factor, such as IGF-I, that is involved in cell proliferation and/or survival. Addition of exogenous IGF-I to cells treated with Poly(IC) partially compensated for the decrease in cell number seen when cells were treated with Poly(IC) alone. Addition of exogenous IGF-I did not bring the cell number to that of control cells that were not treated with Poly(IC), suggesting either that IGF-I action was also affected by Poly(IC) treatment or that other growth and/or survival signals could also be inhibited by Poly(IC).

Consistent with the hypothesis that decreased cell number was due to inhibition of IGF-I expression and signaling, we found that IGF-I and IGF-IR mRNA were down-regulated in C6 cells treated with Poly(IC). Moreover, these changes in gene expression were accompanied by decreased IGF-I peptide levels in conditioned medium and decreased levels of IGF-IR ß-subunit.

The role of IGF-IR in cell proliferation, apoptosis, and tumorigenicity has been reviewed (32). IGF-I R has been shown to be essential for the growth of T98G human glioblastoma cells (33). The effect of IGF-IR on the survival of C6 cells has been the subject of different studies. When C6 cells were transfected with mutant IGF-IRs, it was found that some mutants could act as dominant negatives in growth assays, but none could induce apoptosis (34). Decreased proliferation of C6 cells by ethanol treatment was accompanied by decreased IGF-I signaling (35). C6 cells expressing antisense IGF-IR RNA were no longer tumorigenic in rats. These cells protected the rats from subsequent tumor challenge and also caused the regression of established tumors (36). Implantation of these C6 cells elicited an antitumor response, leading to regression of the tumor and long-term survival of the rats (37). Injection of these cells resulted in the proliferation of cytotoxic CD8+ lymphocytes, suggesting that stimulation of the cellular immune response could be one mechanism for the antitumor effect seen in rats (38).

IGF-I bioavailability and action are also modulated by IGFBPs. Previous studies have shown that C6 cells express IGFBP-3, -4, and -5 (8, 9, 10). RPAs showed that Poly(IC) treatment had little effect on IGFBP-3 mRNA levels, but decreased IGFBP-3 peptide levels. IGFBP-3 has been shown to cause apoptosis in prostate cancer cells (39) and to either enhance (40) or inhibit (41) the effects of IGF-I depending on the cell type. Thus, the decrease in IGFBP-3 seen with Poly(IC) treatment could be either a compensating attempt by the cells to protect against cell death or a mechanism to further promote the growth inhibitory effect of Poly(IC) by decreasing the availability of the already low levels of IGF-I to cells.

RPAs showed that Poly(IC) treatment decreased the mRNA levels of IGFBP-4 and IGFBP-5, and Western ligand blot showed that Poly(IC) decreased IGFBP-4 and IGFBP-5 peptide levels. IGFBP-4 inhibits IGF-I action in all systems studied to date (42). It is thus possible that the C6 cells are compensating for the decreased levels of IGF-I by decreasing IGFBP-4 levels, so that the maximum amount of existing IGF-I would be available to the cells for cell proliferation and survival. IGFBP-5 can either inhibit or enhance the effects of IGF-I depending on the cell type. The decrease in IGFBP-5 seen with Poly(IC) treatment could be an attempt by the cells either to most efficiently use the available IGF-I or to decrease the availability of the already low levels of IGF-I to cells. Future experiments will determine whether the changes in IGFBP-3, -4, and -5 are caused directly by Poly(IC) or are a result of a change in IGF-I gene expression.

The signaling cascade induced by Poly(IC) has not yet been completely elucidated. However, it is known that Poly(IC), acting directly or via induction of type I IFN, induces 2',5'-oligoadenylate synthetase, which eventually results in the activation of RNase L (5). The decrease in the steady state levels of IGF-I, IGF-IR, IGFBP-4, and IGFBP-5 mRNAs could thus be due to activation of RNase L, leading to selective destabilization of these mRNAs.

In addition to its effect on mRNA stability, dsRNA has been shown to regulate gene transcription via activation of PKR and NF{kappa}B (3). Further, IFN acting through the JAK/STAT pathway results in the transcription of IFN-stimulated genes. NF{kappa}B can have proapoptotic (43) as well as antiapoptotic (44, 45) effects. NF{kappa}B could directly inhibit IGF-I gene transcription or, alternatively, stimulate the expression of a factor that reduces IGF-I transcription or one that decreases mRNA stability. Alternatively, activation of PKR could act to directly decrease IGF-I gene transcription or mRNA stability independently of type I IFN. GH induces IGF-I promoter activity in C6 cells via activation of the JAK/STAT pathway (46). It is possible that IFN activates a combination of JAKs and STATs, which eventually leads to the down-regulation of IGF-I, IGF-IR, and IGFBP gene expression. Future experiments will attempt to determine which of these mechanisms underlies the Poly(IC)-mediated decrease in IGF system components in C6 cells.


   Acknowledgments
 
We are grateful to Drs. Shunichi Shimasaki and Nicholas Ling for generously providing us with the cDNAs used to generate the IGFBP probes; to Drs. Haim Werner, Derek LeRoith, and Charles Roberts for providing us with the cDNA used to generate the IGF-I R probe; and to Dr. Charles Roberts for providing us with the cDNA used to generate the insulin receptor probe. We are grateful to Dr. Clifford J. Rosen and Julie Burgess at the Maine Center for Osteoporosis Research and Education Laboratory (Bangor, ME) for performing the IGF-I RIAs. We are also grateful to Melissa Lloyd, Ricardo Garza-Gongora, and Ma Xiuye for technical support.


   Footnotes
 
1 This work was supported by NIDDK NIH Grant DK-47357 (to M.L.A.), Grant AQ-1385 from the Robert A. Welch Foundation (to M.L.A.), and Grant No. 07 from the Children’s Cancer Research Center of UTHSCSA (to M.L.A.). Back

Received October 18, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hubbel HR, Liu R, Maxwell BL 1984 Independent sensitivity of human tumor cell lines to interferon and double-stranded RNA. Cancer Res 44:3252–3257[Abstract/Free Full Text]
  2. Hubbel HR 1986 Synergistic antiproliferative effect of human interferons in combination with mismatched double- stranded RNA on human tumor cells. Int J Cancer 37:359–365[Medline]
  3. Kumar A, Haque J, Lacoste J, Hiscott J, Williams BRG 1994 Double-stranded RNA-dependent protein kinase activates transcription factor NF{kappa}B by phosphorylating I{kappa}B. Proc Natl Acad Sci USA 91:6288–6292[Abstract/Free Full Text]
  4. George RS, Kerr IM, Williams BRG, Silverman RH, Schreiber RD 1998 How cells respond to interferons. Annu Rev Biochem 67:227–264[CrossRef][Medline]
  5. Haines DS, Strauss KI, Gillespie DH 1991 Cellular response to double-stranded RNA. J Cell Biochem 46:9–20[CrossRef][Medline]
  6. Clemens MJ 1997 PKR: a protein kinase regulated by double-stranded RNA. Int J Biochem Cell Biol 29:945–949[CrossRef][Medline]
  7. Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–25[CrossRef][Medline]
  8. Bradshaw SL, Naus CC, Zhu D, Kidder GM, D’Ercole AJ, Han VK 1993 Alterations in the synthesis of insulin-like growth factor binding proteins and insulin-like growth factors in rat C6 glioma cells transfected with gap junction connexin43 cDNA. Regul Pept 48:99–112[CrossRef][Medline]
  9. Bradshaw SL, Naus CC, Zhu D, Kidder GM, Han VK 1993 Insulin-like growth factor binding protein 4 gene-expression is induced by transfection of gap junction connexin43 gene in C6 glioma cell line. Growth Regul 3:26–29[Medline]
  10. Ocrant I, Fay CT, Parmelee JT 1990 Characterization of insulin-like growth factor binding proteins produced in the rat central nervous system. Endocrinology 127:1260–1267[Abstract]
  11. Adamo ML, Stannard B, LeRoith D, Roberts Jr CT 1993 Approaches for the purification, quantitation, and analysis of hormone and receptor mRNAs. In: De Pableo F, Scanes CG, Weintraub BD (eds) Handbook of Endocrine Research Techniques. Academic Press, San Diego, pp 421–425
  12. Adamo ML, Ben-Hur H, Roberts Jr CT, LeRoith D 1991 Regulation of start site usage in the leader exons of the rat IGF-1 gene by development, fasting and diabetes. Mol Endocrinol 5:1677–1686[Abstract]
  13. Werner H, Woloschak M, Adamo ML, Shen-Orr Z, Roberts Jr CT, LeRoith D 1989 Developmental regulation of the rat insulin-like growth factor 1 receptor gene. Proc Natl Acad Sci USA 86:7451–7455[Abstract/Free Full Text]
  14. Yang H, Ney DM, Peterson CA, Lo H, Carrey HV, Adamo ML 1997 Stimulation of intestinal growth is associated with increased insulin-like growth factor binding protein 5 mRNA in the jejunal mucosa of insulin-like growth factor-1-treated parenterally fed rats. Proc Soc Exp Biol Med 216:438–445[Abstract]
  15. Ney DM, Huss DJ, Gillingham MB, Kritsch KR, Dahly EM, Talamantez JL, Adamo ML 1999 Investigation of insulin-like growth factor (IGF)-1 and insulin receptor binding and expression in jejunum of parenterally fed rats treated with IGF-1 or growth hormone. Endocrinology 140:4850–4860[Abstract/Free Full Text]
  16. Goldstein BJ, Dudley AL 1990 The rat insulin receptor: primary structure and correlation of tissue specific alternative messenger RNA splicing. Mol Endocrinol 4:235–244[Abstract]
  17. Grogean T, Verault D, Millard PS, Kiel D, McClain D, Orwoll E, Greenspan S, Rosen CJ 1997 The comparative analysis of methods to measure IGF-I in human seurm. Endocr Metab 4:109–114
  18. Breier B, Gallaher B, Gluckman P 1991 Radioimmunoassay for insulin-like growth factor-I: solutions to some potential problems and pitfalls. Endocrinology 128:347–357
  19. Hossenlopp P, Seurin D, Seovia-Quinson B, Hardouin S, Binoux M 1986 Analysis of serum insulin-like growth factor binding proteins using Western blotting; use of the method for titration of the binding proteins and competitive binding studies. Anal Biochem 154:138–143[CrossRef][Medline]
  20. Knecht EM 1998 The Analysis of Insulin-Like Growth Factor Binding Proteins in Fetal Rat Calvaria Cells and the Effect of Osteogenic Protein 1 on Protein Expression. MS Thesis, University of Texas Health Science Center, San Antonio, TX
  21. Yang H, Jiang D, Li W, Liang J, Genhy LE, Brattain MG 2000 Defective cleavage of membrane bound TGF{alpha} leads to enhanced activation of the EGF receptor in malignant cells. Oncogene 19:1901–1914[CrossRef][Medline]
  22. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  23. Kiess W, Lee L, Graham DE, Greenstein L, Tseng LY, Rechler MM, Nissley SP 1989 Rat C6 glial cells synthesize insulin-like growth factor (IGF-I) and express IGF-I receptor and IGF-II/mannose-6-phosphate receptors. Endocrinology 124:1727–1736[Abstract]
  24. Blat C, Villaudy J, Hasel L 1994 Density-dependent inhibition of mouse embryo fibroblast growth: involvement of IGFBP-3. Exp Cell Res 215:114–118[CrossRef][Medline]
  25. Scott CD, Martin JL, Baxter RC 1984 Rat hepatocyte insulin-like growth factor I and binding protein: effect of growth hormone in vitro and in vivo. Endocrinology 116:1102–1107[Abstract]
  26. Duan C, Clemmons DR 1998 Differential expression and biological effects of insulin-like growth factor binding proteins-4 and -5 in vascular smooth muscle cells. J Biol Chem 273:16836–16842[Abstract/Free Full Text]
  27. Clemmons DR 1985 Variables controlling the secretion of a somatomedin-like peptide by cultured porcine smooth muscle cells. Circ Res 56:418–426[Abstract/Free Full Text]
  28. Rosenfeld RG, Dollar LA, Conover CA 1984 Density associated loss of functional receptors for somatomedin-c/insulin-like growth factor 1 (SM-C/IGF-1) on cultured human fibroblast monolayers. J Cell Physiol 121:419–424[CrossRef][Medline]
  29. Wang L, Adamo ML 2000 Cell density influences IGF-I gene-expression in a cell-type specific manner. Endocrinology 141:2481–2489[Abstract/Free Full Text]
  30. Fottner C, Engelhardt D, Weber MM 1999 Characterization of insulin-like growth factor binding proteins (IGFBPs) secreted by bovine adrenocortical cells in primary culture: regulation by insulin-like growth factors (IGFs) and adrenocorticotropin (ACTH). Horm Met Res. 31:203–281
  31. Monget P, Pisselet C, Monniaux D 1998 Expression of insulin-like growth factor binding protein-5 by ovine granulosa cells is regulated by cell density and programmed cell death in vitro. J Cell Physiol 177:13–25[CrossRef][Medline]
  32. Rubin R, Baserga R 1995 Insulin-like growth factor 1 receptor. Its role in cell proliferation, apoptosis and tumorigenicity. Lab Invest 73:311–331[Medline]
  33. Ambrose D, Resnicoff M, Coppola D, Sell CM, Miura M, Jameson S, Baserga R, Rubin R 1994 Growth regulation of human glioblastoma T98G cells by insulin-like growth factor-1 and its receptor. J Cell Physiol 159:92–100[CrossRef][Medline]
  34. Burgaud JL, Resnicoff M, Baserga R 1995 Mutant IGF-I receptors as dominant negatives for growth and transformation. Biochem Biophys Res Commun. 214:475–481
  35. Resnicoff M, Rubini M, Baserga R, Rubin R 1994 Ethanol inhibits insulin-like growth factor-1-mediated signaling and proliferation of C6 rat glioblastoma cells. Lab Invest. 71:657–662
  36. Resnicoff M, Sell C, Rubini M, Coppola D, Ambrose D, Baserga R, Rubin R 1994 Rat glioblastoma cells expressing an antisense RNA to the insulin-like growth factor-1 (IGF-1) receptor are nontumorigenic and induce regression of wild-type tumors. Cancer Res. 54:2218–2222
  37. Resnicoff M, Tjuvajev J, Rotman HL, Abraham D, Curtis M, Aiken R, Baserga R 1996 Regression of C6 rat brain tumors by cells expressing an antisense insulin-like growth factor I receptor RNA. J Exp Ther Oncol 1:385–389[Medline]
  38. Resnicoff M, Li W, Basak S, Herlyn D, Baserga R, Rubin R 1996 Inhibition of rat C6 glioblastoma tumor growth by expression of insulin-like growth factor I receptor antisense mRNA. Cancer Immunol Immun 42:64–68[CrossRef][Medline]
  39. Rajah R, Khare A, Lee PD, Cohen P 1999 Insulin-like growth factor-binding protein-3 is partially responsible for high-serum-induced apoptosis in PC-3 prostate cancer cells. Endocrinology 163:487–494
  40. Tanaka H, Moriwake T, Matsuoka Y, Nakamura T, Seino Y 1998 Potential role of rhIGF-I/IGFBP-3 in maintaining skeletal mass in space. Bone [Suppl 5] 22:145S–147S
  41. Schmid C, Keller C, Schlapfer I, Veldman C, Zapf J 1998 Calcium and insulin-like growth factor I stimulation of sodium-dependent phosphate transport and proliferation of cultured rat osteoblasts. Biochem Biophys Res Commun. 245:220–225
  42. Clemmons DR, Busby WH, Arai T, Nam TJ, Clarke JB, Jones JI, Ankrapp DK 1995 Role of insulin-like growth factor binding proteins in the control of IGF actions. Prog Growth Factor Res 6:357–366[CrossRef][Medline]
  43. Jung M, Zhang Y, Lee S, Dritschilo A 1995 Correction of radiation sensitivity in ataxia telangectasia cells by truncated I{kappa}B-{alpha}. Science 268:1619–1621[Abstract/Free Full Text]
  44. Beg AA, Baltimore D 1996 An essential role of NF{kappa}B in preventing TNF-{alpha} induced cell death. Science 274:782–784[Abstract/Free Full Text]
  45. Wang CY, Mayo MW, Baldwin AS 1996 TNF-{alpha} and cancer therapy-induced apoptosis: potentiation by inhibition of NF{kappa}B. Science 274:784–787[Abstract/Free Full Text]
  46. Benbassat C, Shoba LNN, Newman M, Adamo ML, Frank SJ, Lowe Jr WL 1999 Growth hormone-mediated regulation of insulin-like growth factor 1 promoter activity in C6 glioma cells. Endocrinology 140:3073–3081[Abstract/Free Full Text]



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