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Endocrinology Vol. 143, No. 2 525-534
Copyright © 2002 by The Endocrine Society

GROWTH FACTORS-CYTOKINES-ONCOGENES

Double-Stranded RNA Decreases IGF-I Gene Expression in a Protein Kinase R-Dependent, but Type I Interferon-Independent, Mechanism in C6 Rat Glioma Cells

Maryanne 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
 
We previously demonstrated that Poly (IC) decreased the growth of C6 cultures in association with reduced IGF-I synthesis and secretion. In this study we characterized the mechanism(s) by which Poly (IC) decreased IGF-I mRNA in C6 cells. Both Poly (IC) and type I interferon (IFN) decreased IGF-I mRNA. Cycloheximide and a blocking antibody against IFN did not alter the Poly (IC)-mediated inhibition of IGF-I mRNA, but prevented IFN from reducing IGF-I mRNA. Poly (IC) did not alter the stability of IGF-I mRNA. Poly (IC) decreased the abundance of IGF-I pre-mRNA in C6 nuclei, but did not inhibit proximal IGF-I exon 1 promoter/luciferase fusion constructs in transient transfection assays. Poly (IC) activated double-stranded RNA-activated protein kinase (PKR) at 5 min and increased PKR protein levels at 48 and 72 h. Exogenous IGF-I did not prevent Poly (IC) from activating PKR, but inhibited the Poly (IC)-mediated increase in PKR protein levels. The PKR inhibitor 2-aminopurine prevented the Poly (IC) stimulation of eIF2-{alpha} phosphorylation and the Poly (IC)-mediated decrease in IGF-I mRNA. We conclude that Poly (IC) decreases IGF-I gene transcription in a mechanism that requires the activation of preexisting PKR, but not the induction of IFN or PKR proteins in C6 cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
POLY (IC), A synthetic double-stranded (ds) RNA copolymer of inosinic and cytidilic acids, has been used as a tool to mimic the effects of ds RNA intermediates produced during viral infection of cells (1). In the 1970s researchers noticed that cells infected with virus died, possibly as a mechanism to prevent lysis of infected cells and consequent infection of neighboring cells (2), and the idea developed that the growth inhibitory action of Poly (IC) would make it a useful therapeutic agent against cancer (3, 4). More recently, iv injection of vesicular stomatitis virus into nude athymic mice decreased the growth of C6 glial tumors (5). In phase I/II clinical trials, im injection of Poly (IC) into patients suffering from glioblastoma and astrocytomas caused either regression or stabilization of tumors and significantly increased survival (6).

ds RNA binds to two ds RNA-binding domains in the amino-terminus of ds RNA-activated protein kinase (PKR) (7). Binding of ds RNA promotes dimerization and subsequently stimulates trans-autophosphorylation and activation of PKR. Activated PKR phosphorylates the {alpha}-subunit of eIF2 on serine 51, resulting in a 100-fold increase in its affinity for the guanine nucleotide exchange factor, eIF2B (8). Since eIF2 is found in excess over eIF2B in cells, a small percentage of phosphorylated eIF2 can sequester all of the available eIF2B in the cell, thereby inhibiting eIF2B-stimulated GTP/GDP exchange on eIF2 and preventing new rounds of translation (8). Ds RNA also binds to and activates 2',5'-oligoadenylate synthetase. The nucleotide products of this enzyme then activate a latent endonuclease, ribonuclease L (RNase L) (9). Activated RNase L degrades ribosomal RNA, also leading to inhibition of protein synthesis.

In addition to its inhibitory effect on protein biosynthesis, activated PKR phosphorylates inhibitor factor {kappa}B, resulting in the release and nuclear translocation of nuclear factor {kappa}B, which then binds to cognate DNA sequences to stimulate transcription of a variety of genes, including {alpha}/ß (type I) interferon (IFN) (10). The binding of IFN to the type 1 IFN receptor activates Janus kinase 1 (JAK1) and tyrosine kinase 2, which phosphorylate and activate signal transducers and activators of transcription (STAT) 1 and 2 (11). STAT1 and -2 then heterodimerize, translocate to the nucleus, and along with the protein p48 form the IFN-stimulated gene factor 3 transcription complex. The IFN-stimulated gene factor 3 complex stimulates the transcription of IFN-stimulated genes, such as PKR, that contain IFN response elements in their promoters (11). Thus, ds RNA could influence gene expression at translational, posttranscriptional, and transcriptional levels, and it has been proposed that a consequence of ds RNA action may be to reduce the level of signaling molecules that stimulate cell proliferation and/or maintain survival (9).

Recently, a positive correlation was found between IGF-I immunoreactivity and the histopathological grade of astrocytomas (12). The levels of IGF-I are up to 4-fold greater in brain tumors than in normal brain tissue (13), and the number of IGF-I receptors are increased in gliomas compared with normal brain tissue (14). Introduction of antisense IGF-I receptor RNA into C6 rat glioma cells inhibited IGF-I-mediated growth of these cells (15). Although injection of wild-type C6 cells caused tumors in rats, C6 cells stably overexpressing antisense IGF-I receptor RNA were nontumorigenic and prevented tumor formation by wild-type C6 cells in these animals (15). Moreover, intracerebral implantation of C6 cells expressing antisense IGF-I receptor RNA elicited an antitumor response in the brain, leading to tumor regression and long-term survival of these animals (16). We therefore hypothesized that Poly (IC) decreased tumor cell growth by decreasing IGF-I gene expression and/or action. In support of this hypothesis, we previously demonstrated that Poly (IC) caused decreased IGF-I mRNA and peptide levels before causing a decrease in C6 cell number (17). Moreover, the addition of exogenous IGF-I partially prevented the growth inhibitory effect of Poly (IC). In this study we characterized the mechanism(s) by which Poly (IC) decreases IGF-I mRNA in C6 cells and specifically asked whether the effect is due to PKR and/or IFN.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
C6 cells (American Type Culture Collection, Manassas, VA) between passages 6 and 10 were grown 3 d postconfluence in 100-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 (Mediatech) and 10% (vol/vol) FBS (Life Technologies, Inc., Gaithersburg, MD). The cells were then placed in Ham’s F-12 medium containing 1 mM glutamine and supplemented with 1% FBS and 0.001% penicillin/streptomycin for 1 d before treatment. Cells were treated with 10 or 200 µg/ml Poly (IC) (Pharmacia Biotech, Piscataway, NJ), 100 international reference units (IRU)/ml IFN (Sigma, St. Louis, MO), 1 µg/ml cycloheximide Sigma, 33 U/ml neutralizing antibody against IFN or preimmune serum (Lee Biomolecular Research Laboratories, San Diego, CA), 100 nM IGF-I (Austral, San Ramon, CA), 75 µM 5,6-dichloro-1-(D-ribofuranosyl benzimidazole) (DRB) Sigma, or 10 mM 2-aminopurine (AP) Sigma as described in the figure legends.

Total RNA extraction
RNA was extracted from whole cells using RNA STAT 60 (Tel-Test, Friendswood, TX) and was quantified by absorbance at a wavelength of 260 nm (18). Equal amounts of RNA were used in solution hybridization/RNase protection assays to study changes in mRNA or pre-mRNA levels.

Nuclear RNA extraction
Nuclei were isolated as described previously (19). Briefly, the medium was removed after treatment of cells, and the plates were placed on ice. Cells were rinsed twice with 5 ml ice-cold PBS and then scraped and collected in a 15-ml centrifuge tube. The cells were centrifuged at 500 x g for 5 min at 4 C. The cell pellet was gently vortexed, and 4 ml lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% (vol/vol) Nonidet P-40] were added. Cells were incubated for 15 min at 4 C, and then lysates were centrifuged at 500 x g for 5 min at 4 C. Cell lysates were examined under the microscope to confirm the presence of free nuclei. The nuclear pellet was resuspended in 4 ml lysis buffer as before and centrifuged at 500 x g for 5 min at 4 C. RNA in the nuclear pellet was extracted immediately and quantitated as described above for total cellular RNA.

Solution hybridization/RNase protection assay (RPA)
Probes: IGF-I mature mRNA probe.
The antisense RNA probe was generated from a 464-bp IGF-I DNA clone ligated into pGEM-4Z (20). The vector was linearized with EcoRI. RPAs of C6 cells resulted in a protected band of 238 nucleotides (nt) reflecting IGF-I mRNA transcribed from the exon 1 promoter.

IGF-I pre-mRNA probe.
The antisense RNA probe was synthesized from a 635-bp IGF-I DNA clone consisting of 35 bp of exon 1 sequence and 600 bp of intron 1 sequence ligated into pGEM-4Z (21). The vector was linearized with EcoRI. RPAs resulted in a protected band of 635 nt reflecting the presence of unspliced pre-mRNA transcribed from the exon 1 promoter.

Insulin receptor (InR) mRNA probe.
The antisense RNA probe for InR was generated from a 747-bp genomic fragment that had been subcloned into pGEM-4 (22). The probe included 478 bases complementary to the mature InR mRNA sequence and 680 bases complementary to the InR pre-mRNA sequence. The vector was linearized with EcoRI.

ß-Actin mRNA probe.
The antisense RNA probe was synthesized from a 126-bp fragment of the rat cytoplasmic ß-actin cDNA (Ambion, Inc., Austin, TX). RPAs resulted in a protected band of 126 nt.

Synthesis of probes and RPA
Synthesis of probes was performed using the Maxiscript kit Ambion, Inc.. The transcription reaction was assembled according to kit directions, and run-off transcription with radioactive labeling was performed using T7 RNA polymerase and [{alpha}-32P]UTP (SA, 800 Ci/mmol; (NEN Life Science Products, Boston, MA). The DNA template was digested with deoxyribonuclease I (Promega Corp., Madison, WI) after a 1-h transcription reaction (18). The antisense transcripts were extracted with phenol and isoamyl alcohol/chloroform, and unincorporated ribonucleotides were removed by two rounds of ethanol precipitation (18). RPAs were performed using protocols described previously (18). Twenty micrograms of total or nuclear RNA (or 2.5 µg total RNA for ß-actin RPA) from C6 cells were hybridized for approximately 18 h to the various 32P-labeled probes described previously. The unhybridized single-stranded regions were digested using RNases A/T1 Ambion, Inc.. SDS and proteinase K were added to stop the RNase reaction, and protected hybrids were extracted using phenol and isoamyl alcohol/chloroform. The protected hybrids were precipitated with ethanol and finally resolved on a 6% polyacrylamide gel. The gels were exposed to film, and the bands were quantified using PhosphorImager (Molecular Dynamics, Inc., Piscataway, NJ) analysis.

Transient transfection assay
C6 cells were grown to 90% confluence in Ham’s F-12 medium as described above. Transient transfection was performed with 2 µg pGL2-Basic DNA or equal molar amounts of pGL2-Control or IGF-I promoter/luciferase fusion constructs, as previously described (23, 24) using the LipofectAMINE Plus system Life Technologies, Inc. in Opti-MEM medium Life Technologies, Inc.. Three hours after transfection, the medium was changed to Ham’s F-12 medium containing 1% FBS. After 1 d, cells were treated with 10 µg/ml Poly (IC) for 24 h, after which cell lysates were prepared and assayed for luciferase activity and protein concentration as described previously (24, 25).

Western blot
Western immunoblots were performed according to the protocols described previously (17). Medium was removed from cells, and the monolayers were rinsed with ice-cold PBS. Cells were scraped off the plate and lysed in 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 incubated on ice for 30 min. Cell lysates were centrifuged at 12,000 x g 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 (25)] were combined with Laemmli buffer [100 mM Tris-HCl (pH 6.8), 2% SDS, 20% glycerol, 0.04% bromophenol blue, and 2% ß-mercaptoethanol] and subjected to SDS-PAGE (10% resolving and 5% stacking) at 200 V. Proteins were transferred to nitrocellulose membrane (Immobilon P, (Millipore Corp., Bedford MA) for 1 h and 30 min at 200 V. The membrane was incubated for 1 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 primary antibody [monoclonal antimouse PKR (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), polyclonal antirabbit phosphorylated eIF2 (Research Genetics, Inc., Huntsville, AL), and monoclonal antimouse total eIF2 (a gift from Dr. Scott Kimball at the Milton S. Hershey Medical Center, Hershey, PA)] at a dilution of 1:1000 (1:750 for total eIF2) overnight in a solution of 1% dry milk solution in TBST. After the incubation with primary antibody, the membrane was washed three times in TBST for 15 min each time. The membrane was incubated with the appropriate secondary antibody (Pierce Chemical Co., Rockford, IL) at a concentration of 1:1000 for 1 h and washed three times in TBST for 15 min each time. Finally, the membrane was incubated with enhanced chemiluminescence reagents Pierce Chemical Co. and exposed to film.

Statistical analysis
Data are shown as the mean ± SEM for the indicated number of observations. Statistical differences between means were determined using one-way ANOVA in the SIMSTAT3 package (Normand Peladeau, Provalis Research, Montréal, Canada).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We initially studied the time course of the Poly (IC)-mediated decrease in IGF-I mRNA levels. IGF-I mRNA formed a protected band of 238 nt in RPAs (Fig. 1AGo). Poly (IC) decreased IGF-I mRNA to 52% (P < 0.05), 42% (P < 0.05), and 29% (P < 0.05) of the control value after 6, 12, and 24 h of treatment, respectively (Fig. 1Go). ß-Actin formed a protected band of 126 nt (Fig. 1AGo). There was no significant effect of Poly (IC) on ß-actin mRNA (P > 0.05) over the time course of the experiment (Fig. 1Go).



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  		Figure 1. Poly (IC) decreases steady state IGF-I mRNA in C6 rat glioma cells in a time-dependent manner. C6 cells, 3 d postconfluence, were cultured for 24 h in Ham’s F-12 medium supplemented with 1% FBS and then treated without (-) or with (+) 200 µg/ml Poly (IC) in Ham’s F-12 medium supplemented with 1% FBS up to 24 h. Total RNA was harvested at the indicated times of treatment with Poly (IC), and equal concentrations of RNA from control and treated cells were subjected to RPAs for IGF-I and ß-actin as described in Materials and Methods. The data represent two separate experiments. A, A representative autoradiograph shows the protected band of IGF-I mRNA at 238 nt and the protected band of ß-actin mRNA at 126 nt. B, The quantified data. mRNA values were normalized to the control value at 30 min. *, Mean is significantly different (P < 0.05) from control. Bars represent the SEM.

 
The induction of IFN synthesis and secretion is thought to be one of the pathways through which dsRNA exerts its antiproliferative effects (11). We therefore tested the hypothesis that Poly (IC) decreased IGF-I mRNA levels through the induction of IFN. Treatment of C6 cells with either 200 µg/ml Poly (IC) or 100 IRU/ml of IFN for 24 h decreased the level of steady state IGF-I mRNA to 29% of that in untreated cells (compare lanes 1 vs. lanes 2 and 3 in Fig. 2Go; P < 0.05). Treatment of cells with 1 µg/ml of the protein synthesis inhibitor cycloheximide did not significantly alter IGF-I mRNA in control cultures (compare lanes 1 and 4; P > 0.05). Furthermore, cycloheximide did not prevent Poly (IC) from decreasing IGF-I mRNA to 29% of that in cells treated with cycloheximide alone (compare lanes 4 and 5; P < 0.05). In contrast, cycloheximide completely prevented IFN from reducing IGF-I mRNA (compare lanes 3 and 6). These data demonstrate that the Poly (IC)-mediated decrease in IGF-I mRNA does not require new protein synthesis, whereas the IFN-mediated decrease in IGF-I mRNA does, suggesting that the Poly (IC)-mediated decrease in IGF-I mRNA does not require IFN induction.



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  		Figure 2. The Poly (IC)-mediated decrease in IGF-I mRNA in C6 rat glioma cells does not require new protein synthesis and is independent of the induction of type 1 IFN. C6 cells were grown as described in Fig. 1Go and then treated with either Poly (IC) (P[IC]) or type 1 IFN in the presence or absence of cycloheximide (CH), preimmune serum (IgG), or neutralizing antibody (BL) against IFN. All treatments were in Ham’s F-12 medium supplemented with 1% FBS for 24 h. Total RNA was harvested, and equal concentrations of RNA from control and treated cells were subjected to RPAs for IGF-I and ß-actin as described in Materials and Methods. A, Representative autoradiographs. B, The quantified data. mRNA values were normalized to that of control. *, Mean is significantly different (P < 0.05) from control; **, mean is significantly different (P < 0.05) from control plus CH; #, mean is significantly different (P < 0.05) from control plus IG; ##, mean is significantly different (P < 0.05) from control plus BL. Data represent three independent experiments. Bars represent the SEM.

 
To confirm that the inhibitory effect of Poly (IC) on IGF-I mRNA in C6 cells was independent of endogenous IFN induction, cells were treated with 200 µg/ml Poly (IC) in the presence of 33 U/ml of a neutralizing antibody against mouse {alpha} and ß IFN, which, based on the manufacturer’s guidelines, are required to block the activity of 100 IRU/ml IFN. Neither the antibody alone nor preimmune serum altered the level of IGF-I mRNA in nontreated cells (Fig. 2AGo, lanes 7 and 10), nor did they prevent the Poly (IC)-mediated inhibition of IGF-I mRNA in C6 cells (lanes 8 and 11). In contrast, the blocking antibody (but not preimmune serum) prevented IFN from inhibiting IGF-I mRNA (lanes 9 and 12). There was no significant effect (P > 0.05) of any of the treatments on the level of ß-actin mRNA. Based on these observations, we concluded that the Poly (IC)-mediated decrease in IGF-I mRNA did not require new protein synthesis in general or the induction of IFN in particular, whereas the inhibitory effect of IFN on IGF-I mRNA did require ongoing protein synthesis.

The Poly (IC)-mediated decrease in steady state IGF-I mRNA levels could be due to decreased gene transcription and/or decreased mRNA stability. We first asked whether Poly (IC) decreased IGF-I mRNA stability in C6 cells by comparing the abundance of IGF-I mRNA in transcriptionally arrested control and Poly (IC)-treated cultures. Transcriptional arrest was obtained by treating cultures with 75 µM DRB, a specific inhibitor of RNA polymerase II, and mRNA decay was determined after subsequent treatment without or with 200 µg/ml Poly (IC). As shown in Fig. 3Go, there was no significant difference (P > 0.05) in the level of either IGF-I or ß-actin mRNAs between control and Poly (IC)-treated C6 cells at any of the time points after transcriptional arrest. The half-lives of both IGF-I and ß-actin mRNAs were calculated by fitting the data with the standard exponential decay equation. The half-life of IGF-I mRNA was 12.78 ± 1.35 h in the absence of Poly (IC) and 14.43 ± 0.93 h in the presence of Poly (IC). The half-life of ß-actin mRNA was 6.6 ± 1.42 h in the absence of Poly (IC) and 6.27 ± 1.61 h in the presence of Poly (IC). We therefore conclude that under conditions where transcription is blocked, Poly (IC) has no significant effect on IGF-I mRNA stability.



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  		Figure 3. Poly (IC) does not alter the stability of IGF-I mRNA in C6 rat glioma cells. C6 cells were grown as described in Fig. 1Go. The medium of each plate was supplemented with 75 µM DRB, and the cells were treated without (-) or with (+) 200 µg/ml Poly (IC). Three milliliters medium were resupplemented with 3 µl 75 mM DRB after 12 h. Total RNA was harvested after 0, 6, 10, and 24 h of treatment, and equal concentrations of RNA from control and treated cells were subjected to RPAs for IGF-I and ß-actin as described in Materials and Methods. The data represent three separate experiments. A, Representative autoradiographs. B, The quantified data, in which the remaining IGF-I or ß-actin mRNA was expressed as a percentage of that at time zero. Bars represent the SEM.

 
We next asked whether the Poly (IC)-mediated decrease in the steady state level of IGF-I mRNA was mediated by a decrease in IGF-I gene transcription. Other studies have demonstrated that the abundance of nuclear IGF-I pre-mRNA transcripts can be correlated with the rate of transcription of the IGF-I gene (26, 27, 28). Nuclei were isolated from control and Poly (IC)-treated C6 cells, and nuclear RNA was extracted as described in Materials and Methods. RPAs demonstrated that IGF-I and InR pre-mRNA formed protected bands of 635 and 680 nt, respectively (Fig. 4AGo). The abundance of IGF-I pre-mRNA was decreased to 21% of the control value (P < 0.05) in Poly (IC)-treated cultures (Fig. 4BGo). There was no significant difference (P > 0.05) in the abundance of InR pre-mRNA levels with Poly (IC) treatment. This result further demonstrated that the Poly (IC)-mediated decrease in IGF-I pre-mRNA is not the result of a generalized down-regulation in gene transcription in C6 cells. Based on the results from Figs. 3Go and 4Go, we conclude that the Poly (IC)-mediated decrease in steady state IGF-I mRNA was caused primarily by decreased IGF-I gene transcription.



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  		Figure 4. Poly (IC) decreases the abundance of IGF-I pre-mRNA in the nuclei of C6 rat glioma cells. C6 cells were grown as described in Fig. 1Go and were then treated without (0) or with 10 or 200 µg/ml Poly (IC) in Ham’s F-12 medium supplemented with 1% FBS for 24 h. Nuclei were isolated, and nuclear RNA was harvested and subjected to RPAs for IGF-I or InR as described in Materials and Methods. The data represent four separate experiments for IGF-I and three for InR pre-mRNA. A, A representative autoradiograph shows the protected band of IGF-I pre-mRNA at 635 nt and the protected band of InR pre-mRNA at 680 nt. B, The quantified data. Nuclear pre-mRNA values are normalized to the control value (0). *, Mean is significantly different (P < 0.05) from control. Bars represent the SEM.

 
Transient transfection experiments were performed with rat IGF-I exon 1 promoter/luciferase fusion constructs to map potential Poly (IC) response element(s) in C6 cells. Treatment with 200 µg/ml Poly (IC) reduced the activities of pGL2-Basic and pGL2-Control/luciferase constructs at 3 h (data not shown). This led to the concern that the Poly (IC) effect on the IGF-I promoter could be masked by the nonspecific effect of Poly (IC) on luciferase activity. We therefore decided to test the effect of 10 µg/ml Poly (IC) on IGF-I exon 1 promoter constructs, as we had previously demonstrated that 10 µg/ml Poly (IC) decreased C6 cell IGF-I mRNA to 50% of the control value after 24 h of treatment (17). However, the promoter activities of the constructs containing 1500, 1000, and 500 bp of the 5'-flanking region and 319 bp of exon 1 sequence, and the activity of the construct containing exon 1 promoter sequence from +75 to +319 (where +1 is the designation given to transcription start site 1) all remained unaltered (P > 0.05) in C6 cells treated with 10 µg/ml Poly (IC) for 24 h (Fig. 5Go). These data suggest that under the conditions of these experiments, the IGF-I exon 1 promoter region between -1500 and +319 bp does not contain any Poly (IC) response elements in C6 cells.



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  		Figure 5. Poly (IC) does not inhibit proximal IGF-I exon 1 promoter activity. C6 rat glioma cells were transfected with the indicated IGF-I exon 1/luciferase fusion constructs (- refers to nucleotides upstream of IGF-I exon 1 transcription start site 1 and + refers to nucleotides downstream of transcription start site 1) and then treated without (control) or with 10 µg/ml Poly (IC). The luciferase activities determined after 24 h were corrected for protein concentrations of the cellular lysates and normalized to the activity of the promoterless pGL2-Basic. The promoter activity of pGL2-Control is shown as 0.01x to fit into the graph. Data represent two independent experiments. Bars represent the SEM.

 
Having determined that IFN did not mediate the inhibitory effects of ds RNA on IGF-I gene expression, we next sought to determine whether ds RNA could inhibit IGF-I by activating PKR. Western immunoblots demonstrated that treatment of C6 cells with 200 µg/ml Poly (IC) increased the amount of phosphorylated eIF2-{alpha}, indicative of PKR activation, within 5 min of treatment (Fig. 6AGo). The effect was maintained at least up to 6 h after the addition of Poly (IC). The addition of 100 nM IGF-I along with 200 µg/ml Poly (IC) did not inhibit the Poly (IC) stimulation of eIF2-{alpha} phosphorylation. Moreover, the increase in eIF2-{alpha} phosphorylation caused by Poly (IC) was not due to increased eIF2-{alpha} protein levels (Fig. 6BGo). Since Poly (IC) stimulated phosphorylation of eIF2-{alpha} as early as 5 min, we hypothesized that activation of preexisting PKR was required for the Poly (IC)-mediated decrease in IGF-I mRNA. To test this hypothesis we used AP, a known inhibitor of PKR activation (29, 30). As expected from our previous results, treatment with 200 µg/ml Poly (IC) decreased IGF-I mRNA to 27% of the control value (P < 0.05; Fig. 7Go, A and B). Treatment with either AP or IGF-I alone had no significant effect (P > 0.05 compared with control cells) on IGF-I mRNA. Treatment with AP blocked the Poly (IC)-mediated decrease in IGF-I mRNA (P > 0.05 compared with control cells). Treatment with Poly (IC) in the presence of 100 nM IGF-I did not alter the inhibition of IGF-I mRNA caused by Poly (IC) alone [P > 0.05 compared with cells treated with Poly (IC) alone]. None of these treatments had any significant effect on ß-actin mRNA (P > 0.05; Fig. 7Go, A and B). Western blots performed on cell lysates from parallel plates indicated that 24 h of Poly (IC) treatment increased eIF2-{alpha} phosphorylation (Fig. 7CGo), whereas AP or IGF-I had little effect on the level of phosphorylated eIF2-{alpha}. However, AP effectively blocked the Poly (IC)-mediated increase in eIF2-{alpha} phosphorylation. IGF-I did not alter eIF2-{alpha} phosphorylation in cells also treated with Poly (IC). Moreover, the changes in the level of phosphorylated eIF2-{alpha} could not be explained by changes in the level of total eIF2-{alpha} (Fig. 7DGo).



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  		Figure 6. Poly (IC) causes phosphorylation of eIF2-{alpha} in C6 rat glioma cells. C6 cells were grown as described in Fig. 1Go and treated without (-) or with (+) 200 µg/ml Poly (IC) in the absence (-) or presence (+) of 100 nM IGF-I in Ham’s F-12 medium supplemented with 1% FBS. Western immunoblotting was performed on 15 µg cell lysate protein using antibodies against phosphorylated eIF2-{alpha} (A) or total eIF2-{alpha} (B). The band representing eIF2-{alpha} migrated at about 36 kDa. The data represent one of three separate experiments.

 


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  		Figure 7. The Poly (IC)-mediated decrease in IGF-I mRNA requires PKR activation in C6 rat glioma cells. C6 cells were grown as described in Fig. 1Go. Cells were either not treated with AP (lanes C, P, I and P+I) or were pretreated with 10 mM AP (lanes A and P+A) for 1 h. Medium was then removed, and Ham’s F-12 medium with 1% FBS containing no additions (lanes C and A), 200 µg/ml Poly (IC) (lanes P and P+A), 100 nM IGF-I (lane I), or Poly (IC) and IGF-I (lane P+I) was added. Total RNA was harvested after 24 h of treatment, and equal concentrations of RNA from control and treated cells were subjected to RPAs for IGF-I and ß-actin as described in Materials and Methods. The data represent two separate experiments. A, Representative autoradiographs show the protected bands of IGF-I and ß-actin mRNAs. B, The quantified data. mRNA values were normalized to that of control (ctrl, lane C in A). Bars represent the SEM. Western immunoblotting for phosphorylated eIF2-{alpha} (C) and total eIF2-{alpha} (D) was performed on 15 µg cell lysate protein from parallel plates.

 
To determine whether Poly (IC)-stimulated PKR activity was secondary to increased levels of PKR protein, we performed Western immunoblots for total PKR protein. Treatment with 200 µg/ml Poly (IC) had little effect on the levels of PKR protein at 24 h after addition (Fig. 8AGo), but strongly stimulated PKR protein level after 48 and 72 h of treatment. The Poly (IC)-mediated induction of PKR protein levels at 48 and 72 h was completely repressed by 100 nM exogenous IGF-I. However, 100 nM IGF-I alone had no effect on PKR levels in C6 cells even after 72 h of treatment (Fig. 8BGo). Thus, our data suggest that the Poly (IC)-mediated decrease in IGF-I mRNA requires activation of preexisting PKR, but does not require the induction of PKR protein levels.



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  		Figure 8. The effect of Poly (IC) on PKR protein levels. C6 rat glioma cells were grown as described in Fig. 1Go and then treated without (-) or with (+) 200 µg/ml Poly (IC) in the absence (-) or presence (+) of 100 nM IGF-I in Ham’s F-12 medium supplemented with 1% FBS for 24, 48, and 72 h (A). Medium was changed every 24 h. Western immunoblotting was performed on 12.5 µg cell lysate protein. The band representing PKR migrated at about 67 kDa. B, Cells were treated for 24, 48, and 72 h with 100 nM IGF-I, followed by Western immunoblotting. Medium was changed every 24 h. The data represent one of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ds RNA was recently shown to be effective in the treatment of patients with glioma and anaplastic astrocytomas (6). We previously demonstrated that Poly (IC) decreased C6 rat glioma cell numbers in association with decreased IGF-I mRNA and peptide secretion (17). Moreover, the Poly (IC)-mediated decrease in cell number was partially prevented by the addition of exogenous IGF-I. In this series of experiments we studied the mechanism(s) of the Poly (IC)-mediated decrease in IGF-I mRNA levels.

Our data demonstrate that the Poly (IC)-mediated decrease in the steady state level of IGF-I mRNA occurs independently of ongoing protein synthesis in general and IFN induction in particular. Earlier studies have identified ds RNA-induced genes that do not require IFN induction. Studies of fibroblasts treated with cycloheximide to block protein synthesis and with IFN antibodies to block IFN action have demonstrated that Poly (IC) induces the transcription of certain genes even under these conditions (31, 32). The induction of these transcripts was blocked by AP, and the researchers suggested that the induction of transcripts by ds RNA could occur via activation of PKR. Our results clearly show that inhibition of activation of PKR prevented Poly (IC) from reducing IGF-I mRNA. Coupled with our observation that Poly (IC) reduced IGF-I transcription, we propose that ds RNA activates preexisting PKR, which initiates a signaling cascade leading to reduced IGF-I transcription. ds RNA-activated transcription factors include nuclear factor {kappa}B (10), IFN response factor 1 (33), and activating transcription factor 2 (34). Although these factors are not generally known to directly suppress transcription, they could stimulate the transcription of factors that eventually inhibit IGF-I gene transcription. However, we were unable to identify a Poly (IC) response element in 1.8 kb of the IGF-I exon 1-proximal promoter region. One explanation could be that 10 µg/ml Poly (IC) was not sufficient to have a measurable effect on the IGF-I promoter. It is also possible that in C6 cells, the Poly (IC) response element(s) is not located in this region or the decrease in IGF-I promoter activity by Poly (IC) cannot be detected in transient transfection assays.

The lack of effect of Poly (IC) on the stability of IGF-I mRNA suggests that activation of RNase L does not lead to direct degradation of IGF-I mRNA. However, RNase L could play an indirect role by decreasing the stability of mRNAs involved in IGF-I gene transcription. If so, this effect would still appear to depend on PKR activation, as blocking PKR activation prevents Poly (IC) from inhibiting IGF-I mRNA.

Recent studies have also described the stimulation of the p38 MAPK and the c-Jun NH2 terminal kinase (JNK) pathways in cells treated with ds RNA (35). Stimulation of the p38 MAPK pathway did not require either RNase L or PKR, while stimulation of JNK required both. Since the Poly (IC) effect to inhibit IGF-I mRNA required PKR activation, it is unlikely that the effect is mediated through the p38 MAPK pathway. The JNK pathway, however, could be a candidate pathway for the Poly (IC) effect on IGF-I gene expression. Thus, the inhibition of PKR activation by AP could theoretically prevent the activation of JNK. Future studies are required to assess this possibility.

Although the inhibitory effect of Poly (IC) on IGF-I gene expression did not require IFN, IFN itself decreased IGF-I mRNA in C6 cells. This effect, unlike the Poly (IC) effect on IGF-I mRNA, required new protein synthesis. Pretreatment with IFN-{alpha} suppressed STAT5 phosphorylation in megakaryocyte cells treated with thrombopoietin (36). The suppression of STAT5 phosphorylation was accompanied by the induction of suppressor of cytokine signaling-1 gene expression by IFN-{alpha}. Studies of STAT5b knockout mice showed decreased liver IGF-I mRNA (37). C6 cells are known to express STAT5 (38). Thus, IFN may inhibit IGF-I mRNA in C6 cells by inducing the synthesis of suppressor of cytokine signaling-1, resulting in decreased STAT5 phosphorylation. Alternatively, IFN could also decrease IGF-I gene expression by inducing PKR synthesis, which would then become activated and inhibit IGF-I mRNA. A goal of future studies will be to elucidate the mechanism by which IFN itself decreases IGF-I mRNA.

Poly (IC) clearly increased the levels of PKR protein at 48 and 72 h. Moreover, the addition of IGF-I blocked the induction of PKR protein by Poly (IC) in C6 cells. Induction of IFN by ds RNA leads to transcription of PKR via the IFN-dependent phosphorylation of JAK1 and tyrosine kinase 2 and the activation of STAT1 and -2 (11). IGF-I stimulated the phosphorylation of STAT1 and -3 in neonatal rat cardiomyocytes (39), and there was increased phosphorylation of JAK1 and -2 in NIH-3T3 cells overexpressing the IGF-I receptor (40). Recruitment of JAK1 by signaling through the IGF-I receptor could favor phosphorylation of STAT1 and -3 compared with STAT1 and -2 and consequently inhibit PKR gene expression. Alternatively, if the amount of STAT3 protein is higher than the amount of STAT2 protein in C6 cells, or if STAT1 has a higher affinity for heterodimerization with STAT3 than with STAT2, then IGF-I signaling could also lead to decreased PKR gene expression due to decreased STAT1 and -2 heterodimerization.

In summary, we have shown that PKR activation leads to decreased IGF-I transcription in C6 rat glioma cells. It is also apparent that stimulation of increased IFN or PKR levels is not required for Poly (IC) to reduce IGF-I mRNA. However, IGF-I prevents Poly (IC) from increasing the level of PKR protein. We hypothesize that only after IGF-I levels are decreased can Poly (IC) signaling lead to increased PKR protein. These results thus provide a model to potentially explain the opposing actions of IGF-I and ds RNA/IFN on cellular proliferation.


   Acknowledgments
 
We are grateful to Dr. Charles T. Roberts at Oregon Health Sciences University (Portland, OR) for providing us with the clone used to generate the insulin receptor probe, to Dr. Bret Hassel at the University of Maryland Medical Center (Baltimore, MD) for donating the blocking antibody against IFN and the preimmune control serum, and to Dr. Scott Kimball at the Milton S. Hershey Medical Center (Hershey, PA) for donating the antibody against total eIF2.


   Footnotes
 
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 07 from Children’s Cancer Research Center, University of Texas Health Science Center (to M.L.A.).

Abbreviations: AP, 2-Aminopurine; DRB, 5,6-dichloro-1-(D-ribofuranosyl benzimidazole); ds, double-stranded; IFN, type I interferon; InR, insulin receptor; IRU, international reference units; JAK, Janus kinase; nt, nucleotides; JNK, c-Jun NH2 terminal kinase; PKR, ds RNA-activated protein kinase; RNase, ribonuclease; RPA, ribonuclease protection assay; STAT, signal transducer and activator of transcription; TBST, 20 mM Tris-HCl (pH 7.4), 500 mM NaCl, and 0.05% Tween 20.

Received June 1, 2001.

Accepted for publication October 15, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Castelli J, Wood KN, Youle RJ 1998 The 2–5A system in viral infection and apoptosis. Biomed Pharmacother 52:386–390[CrossRef][Medline]
  2. Singh B and Postic B 1970 Enhanced resistance of mice to virulent Japanese B encephalitis virus following inactivated vaccine and poly I:C. J Infect Dis 122:339–342[Medline]
  3. Fisher JC, Cooperband SR, Mannick JA 1970 Inhibition of tumor growth with a new synthetic ribonucleic acid. Surg Forum 21:122–124[Medline]
  4. Kreibich G, Suss R, Kinzel V, Hecker E 1970 Biochemical mechanism of tumorigenesis in mouse skin. III. Decrease in tumor yields by Poly I-C administered during initiation of skin by an intragastric dose of 7,12-dimethyl-benz(a)anthracene. Z Krebsforsch 74:383–389[CrossRef][Medline]
  5. Balachandran S, Porosnicu M, Barber GN 2001 Oncolytic activity of vesicular stomatitis virus is effective against tumors exhibiting aberrant p53, ras, or myc function and involves the induction of apoptosis. J Virol 75:3474–3479[Abstract/Free Full Text]
  6. Salazar AM, Levy HB, Ondra S, Kende M, Scherokman B, Brown D, Mena H, Martin N, Schwab K, Domovan D, Dougherty D, Pulliam M, Ippolito M, Graves M, Brown H, Ommaya A 1996 Long term treatment of malignant gliomas with intramuscularly administered polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose: an open pilot study. Neurosurgery 38:1096–1104[CrossRef][Medline]
  7. Patel RC, Sen GC 1992 Identification of the double-stranded RNA-binding domain of the human interferon-inducible protein kinase. J Biol Chem 267:7671–7676[Abstract/Free Full Text]
  8. Kaufman RJ 1999 Double-stranded RNA-activated protein kinase mediates virus-induced apoptosis: a new role for an old actor. Proc Natl Acad Sci USA 96:11693–11695[Free Full Text]
  9. Haines DS, Strauss KI, Gillespie DH 1991 Cellular response to double-stranded RNA. J Cell Biochem 46:9–20[CrossRef][Medline]
  10. 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]
  11. Barber GN 2001 Host defence, viruses and apoptosis. Cell Death Differ 8:113–126[CrossRef][Medline]
  12. Hirano H, Lopes MBS, Laws ER, Asakura T, Goto M, Carpenter JE, Karns LR, Vandenberg SR 1999 Insulin-like growth factor-1 content and pattern of expression correlates with histopathologic grade in diffusely infiltrating astrocytomas. Neuro-Oncology 1:109–119[Abstract]
  13. Sandberg AC, Engberg C, Lake M, von Holst H, Sara VR 1988 The expression of insulin-like growth factor-1 and insulin-like growth factor-2 genes in human fetal and adult brain and in glioma. Neurosci Lett 93:114–119[CrossRef][Medline]
  14. Merrill MJ, Edwards NA 1990 Insulin-like growth factor-1 receptors in human and glial tumors. J Clin Endocrinol Metab 71:199–209[Abstract]
  15. 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-I) receptor are nontumorigenic and induce regression of wild-type tumors. Cancer Res 54:2218–2222[Abstract/Free Full Text]
  16. 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]
  17. Chacko MS and Adamo ML 2000 Double stranded ribonucleic acid decreases C6 rat glioma cell numbers: effects on IGF-I gene-expression and action. Endocrinology 141:3546–3555[Abstract/Free Full Text]
  18. 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. San Diego: Academic Press; 421–425
  19. Greenberg ME, Bender TP 1997 Identification of newly transcribed RNA. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, eds. Current protocols in molecular biology. New York: Wiley & Sons; 4.10.1–4.10.11
  20. Adamo ML, Ben-Hur H, LeRoith D, Roberts Jr CT 1991 Transcription initiation in the two leader exons of the rat IGF-I gene occurs from disperse versus localized sites. Biochem Biophys Res Commun 176:887–893[CrossRef][Medline]
  21. Yeh LC, Adamo ML, Duan C, Lee JC 1998 Osteogenic protein-1 regulates insulin-like growth factor 1 (IGF-I), IGF-II, and IGF binding protein-5 (IGFBP-5) gene expression in fetal rat calvaria cells by different mechanisms. J Cell Physiol 175:78–88[CrossRef][Medline]
  22. 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-I or growth hormone. Endocrinology 140:4850–4860[Abstract/Free Full Text]
  23. Wang L, Wang X, Adamo ML 2000 Two putative GATA motifs in the proximal exon 1 promoter of the rat insulin-like growth factor 1 gene regulate basal promoter activity. Endocrinology 141:1118–1126[Abstract/Free Full Text]
  24. 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]
  25. 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]
  26. Bichell DP, Kikuchi K, Rotwein P 1992 Growth Hormone rapidly activates insulin-like growth factor 1 gene transcription in vivo. Mol Endocrinol 6:1899–1908[Abstract]
  27. Kikuchi K, Bichell DP, Rotwein P 1992 Chromatin changes accompany the developmental activation of insulin-like growth factor 1 gene transcription. J Biol Chem 267:21505–21511[Abstract/Free Full Text]
  28. Bichell DP, Rotwein P, McCarthy TL 1993 Prostaglandin E2 rapidly stimulates insulin-like growth factor 1 gene-expression in primary rat osteoblast cultures: evidence for transcriptional control. Endocrinology 133:1020–1028[Abstract]
  29. Tiwari RK, Kusari J, Kumar R, Sen GC 1988 Gene induction by interferons and double-stranded RNA: selective inhibition by 2-aminopurine. Mol Cell Biol 8:4289–4294[Abstract/Free Full Text]
  30. Wathelet MG, Clauss IM, Paillard FC, Huez GA 1989 2-aminopurine selectively blocks the transcriptional activation of cellular genes by virus, double-stranded RNA and interferons in human cells. Int J Biochem 184:503–509
  31. Memet S, Besancon F, Bourgeade M, Thang MN 1991 Direct induction of interferon-{gamma} and interferon-{alpha}/ß inducible genes by double stranded RNA. J Interferon Res 11:131–141[Medline]
  32. Nakaya T, Sato M, Hata N, Asagiri M, Sumori H, Noguchi S, Tanak N, Taniguchi T 2001 Gene induction pathways mediated by distinct IRFs during viral infection. Biochem Biophys Res Commun 283:1150–1156[CrossRef][Medline]
  33. Kormilas AE, Cantin C, Craig AWB, Jagus R, Hiscott J, Sonenberg N 1995 The interferon-inducible protein kinase PKR modulates the transcriptional activation of immunoglobulin {kappa} gene. J Biol Chem 270:25426–25434[Abstract/Free Full Text]
  34. Bandhopadhyay SK, de La Motte CA, Williams BR 2000 Induction of E-selectin expression by double-stranded RNA and TNF-{alpha} is attenuated in murine aortic endothelial cells derived from double-stranded RNA-activated kinase (PKR)-null mice. J Immunol 164:2077–2083[Abstract/Free Full Text]
  35. Iordanov MS, Paranjape JM, Zhou A Wong J, Williams BR, Meurs EF, Silverman RH, Magun BE 2000 Activation of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase by double-stranded RNA and encephalomyocarditis virus: involvement of RNase L, protein kinase R, and alternative pathways. Mol Cell Biol 20:617–627[Abstract/Free Full Text]
  36. Wang Q, Miyakawa Y, Fox N, Kaushansky K 2000 Interferon-{alpha} directly represses megakaryopoiesis by inhibiting thrombopoietin-induced signaling through induction of SOCS-1. Blood 96:2093–2099[Abstract/Free Full Text]
  37. Davey HW, Xie T, McLachlan MJ, Wilkins RJ, Waxman DJ, Grattan DR 2001 STAT5b is required for GH-induced liver IGF-I gene expression. Endocrinology 142:3836–3841[Abstract/Free Full Text]
  38. 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]
  39. Takahashi T, Fukuda K, Pan J, Kodama H, Sano M, Makino S. Kato T, Manabe T, Ogawa S 1999 Characterization of insulin-like growth factor-1-induced activation of the JAK/STAT pathway in rat cardiomyocytes. Circ Res 85:884–891[Abstract/Free Full Text]
  40. Gual P, Baron V, Lequoy V, Van Obberghen E 1998 Interaction of Janus kinases JAK-1 and JAK-2 with the insulin receptor and the insulin-like growth factor-1 receptor. Endocrinology 139:884–893[Abstract/Free Full Text]



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