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Endocrinology Vol. 143, No. 6 2144-2154
Copyright © 2002 by The Endocrine Society

GROWTH FACTORS-CYTOKINES-ONCOGENES

Double-Stranded Ribonucleic Acid Decreases C6 Rat Glioma Cell Proliferation in Part by Activating Protein Kinase R and Decreasing Insulin-Like Growth Factor I Levels

Maryanne S. Chacko, Xiuye Ma 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 reported that reduction of autocrine IGF-I by polyinosinic-polycytidylic acid [poly(IC)] was permissive for the poly(IC)-mediated decrease in C6 rat glioma cell number. We now report that poly(IC) caused a block in G1 to S transition in confluent C6 cultures, whereas in subconfluent cultures, poly(IC) decreased the percentage of cells in the G2/M phase. Addition of IGF-I to poly(IC)-treated cells decreased the percentage of cells in G0/G1 phase and increased the percentage of cells in G2/M phase in confluent and subconfluent C6 cultures, indicating the reversal of cell cycle blocks. Inhibition of protein kinase R (PKR) activation partially prevented the poly(IC)-mediated cytostasis of C6 cells. Poly(IC) induced interferon-{alpha} in C6 cells. Both IGF-I and a blocking antibody against type I interferon (IFN) prevented the increase in PKR levels and the decrease in cell proliferation caused by poly(IC). We conclude that poly(IC) induces IFN, which mediates the cytostatic effect of poly(IC) on C6 cells at least in part through PKR. IGF-I prevents IFN from inducing PKR, thus explaining the ability of IGF-I to reverse the cell cycle blocks and the decreased C6 proliferation caused by poly(IC).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
POLYINOSINIC-POLYCYTIDYLIC acid [poly(IC)] is a synthetic double-stranded (ds) RNA copolymer of inosinic and cytidilic acids whose anti-tumor effects have been studied in several tumor cell lines, including prostate cancer (1), myeloma (2), osteosarcoma (2), and fibrosarcoma (3). Poly(IC) treatment of patients with superficial bladder cancer increased survival (4), and 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 as well as increased patient survival (5).

Binding of dsRNA to dsRNA-activated protein kinase R (PKR) promotes dimerization, trans-autophosphorylation, and activation of PKR (6). Activated PKR phosphorylates the {alpha}-subunit of eIF2 on serine 51, eventually inhibiting translation initiation (6). Activated PKR also phosphorylates I{kappa}B, resulting in the release and nuclear translocation of NF{kappa}B, which binds to cognate DNA sequences to stimulate transcription of a variety of genes, including type I interferon (IFN) (7).

PKR is known to have proapoptotic (8, 9) as well as antiproliferative effects (10, 11), but the downstream targets mediating these effects are varied and have not been completely characterized. One candidate is IFN, which also has both antiproliferative (12, 13) and proapoptotic (14, 15) effects. We previously found that poly(IC) decreased IGF-I gene transcription in C6 rat glioma cells in a PKR-dependent, but IFN-independent, mechanism (16). The decrease in IGF-I occurred before a decrease in C6 cell number, and exogenous IGF-I partially blocked the poly(IC)-mediated decrease in C6 cell number (17). Thus, IGF-I could be a direct transcriptional target of PKR. We now report that poly(IC) causes specific blocks in the C6 cell cycle that can be reversed by IGF-I and that the cytostatic action of poly(IC) is dependent on IFN induction. Moreover, exogenous IGF-I prevents both poly(IC) and IFN from stimulating PKR levels, suggesting that the reduction of IGF-I by dsRNA could play a permissive role in the IFN-dependent induction of PKR, which we hypothesize is at least partially responsible for the decreased proliferation of C6 cultures.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
C6 cells (American Type Culture Collection, Manassas, VA) were either plated sparsely and grown for 1 d or were grown to confluence in 60-mm plates (35-mm plates for Fig. 8AGo; Corning, Inc., Corning, NY) in Ham’s F-12 medium (Mediatech, Herndon, VA) containing 1 mM glutamine and supplemented with 0.01% penicillin/streptomycin (PS; Mediatech) and 10% FBS (Life Technologies, Inc., Gaithersburg, MD). Cells were placed in Ham’s F-12 medium containing 1 mM glutamine and supplemented with 1% FBS and 0.01% PS for 24 h. Cells were treated with 200 µg/ml poly(IC) (Pharmacia, Piscataway, NJ), 100 international reference units (IRU)/ml IFN (Sigma, St. Louis, MO; where 1 IRU protects 50% of the indicator cells from viral cytopathology), 33 U/ml neutralizing antibody against IFN or preimmune serum (Lee Biomolecular Research Labs, San Diego, CA), 100 nM IGF-I (Austral, San Ramon, CA), or 10 mM 2-aminopurine (AP; Sigma) as described in the figure legends. Medium was replaced (including treatments) every 24 h, and cells were harvested for trypan blue exclusion assay, cell cycle analysis, RT-PCR, and Western immunoblot analysis as described below. To measure cell death (Fig. 5Go), C6 cells were grown to confluence in 60-mm plates in Ham’s F-12 medium containing 1 mM glutamine and supplemented with 0.01% PS and 10% FBS, placed in serum-free Ham’s F-12 medium containing 1 mM glutamine and supplemented with 0.1% BSA (Sigma), and 0.01% PS for 14 d in the presence or absence of 200 µg/ml poly(IC) with no change in medium. Medium was replaced without or with poly(IC) every 24 h.



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  		Figure 8. The poly(IC)-mediated decrease in C6 cell number requires the induction of IFN. C6 rat glioma cells were grown to confluence in 35-mm plates for trypan blue exclusion assays (A) and in 60-mm plates for Western immunoblots (B), cultured for 24 h in Ham’s F-12 medium supplemented with 1% FBS, and then treated with preimmune serum (IgG), blocking antibody against IFN (Bl), poly(IC), IFN, and/or IGF-I in Ham’s F-12 medium supplemented with 1% FBS. Medium was changed at 24 h. Cells were counted, and lysates were harvested for Western immunoblots after 48 h of treatment. Data were normalized to the cell number of control cultures and represent the mean of two separate experiments. *, The mean is significant (P < 0.05) compared with the control value; **, the mean is significant (P < 0.05) compared with the value after poly(IC) treatment; {dagger}, the mean is significant (P < 0.05) compared with the value after IFN treatment. Bars represent the SEM.

 


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  		Figure 5. Chronic treatment with poly(IC) increases DNA laddering in C6 rat glioma cells. C6 rat glioma cells were grown to confluence in 60-mm plates, cultured for 24 h in Ham’s F-12 medium supplemented with 0.1% BSA,and then treated without (-) or with (+) 200 µg/ml poly(IC). Medium was not changed over the course of the experiment. Cells were harvested after 14 d of treatment. Data are representative of two separate experiments.

 
[3H]Thymidine incorporation
[3H]Thymidine incorporation was performed according to the protocol described previously (18). Briefly, C6 cells were plated at a concentration of 4 x 104 cells in 48-well plates (Corning, Inc.) and were grown for either 24 or 48 h to achieve subconfluent or confluent cultures, respectively. The cells were placed in Ham’s F-12 medium containing 1 mM glutamine supplemented with 1% FBS and 0.01% PS for 24 h and treated with 200 µg/ml poly(IC), 100 nM IGF-I, or both. Cells were pulsed with 1 µCi/ml 3[H]thymidine (SA, 6.7 mCi/mmol; NEN Life Science Products, Boston, MA) for the final 3 h of treatment. The medium was then aspirated, and the cells were washed three times with 1 ml ice-cold PBS. Total DNA was precipitated by the addition of 200 µl 10% trichloroacetic acid (Fisher Scientific, Fairlawn, NJ). After 15 min on ice, DNA was solubilized by the addition of 200 µl 0.1 N sodium hydroxide (EM Science, Gibbstown, NJ) and finally neutralized by the addition of 200 µl 0.1 N hydrochloric acid (EM Science) and counted for radioactivity.

Trypan blue exclusion assay
Conditioned medium (CM), containing unattached cells, was collected. Attached cells were then removed from monolayers by trypsinization and combined with the CM containing unattached cells. An aliquot of the suspension was diluted in trypan blue, and both viable (cells excluding trypan blue) and nonviable cells (cells not excluding trypan blue) were counted using a hemocytometer.

Cell cycle analysis
Fluorescence-activated cell sorting (FACS) analysis was performed at the FACS facility of University of Texas Health Science Center (San Antonio, TX) using the protocol described previously (19). Briefly, cells were trypsinized and centrifuged at 1000 x g for 3 min. The cell pellet was gently washed twice with ice-cold PBS. One milliliter of 70% ethanol (precooled to -20 C) was added, and the cells were incubated at -20 C overnight. Cells were centrifuged at 1000 x g for 3 min, and the pellet was gently washed twice with ice-cold PBS and resuspended in 150 µl PBS. Fifty microliters of 1 mg/ml ribonuclease A (RNase A) were added, and the samples were incubated at 37 C for 30 min. Propidium iodide was added at a final concentration of 33 ng/ml. Immediately before flow cytometry, the cells were filtered through a nylon mesh. FACS analysis was performed using the FACStar Plus (Becton Dickinson and Co., San Jose, CA).

DNA laddering
DNA laddering was performed according to the protocol described previously (20). Briefly, attached cells were removed by scraping and were combined with unattached cells in CM. Cells were centrifuged for 5 min at 1500 x g, and cell pellets were resuspended in 500 µl 0.5% Triton X-100, followed by lysis for 15 min at room temperature. Lysates were centrifuged at 10,000 x g for 10 min, and the resulting supernatant was extracted with phenol and chloroform (1:1). Genomic DNA was precipitated with 0.1 M MgCl2 and 2 vol 100% ethanol overnight at 4 C. After centrifugation at 15,000 x g for 20 min, the precipitated DNA was dissolved in Tris-EDTA buffer (pH 8). Samples were electrophoresed through a 1.8% agarose gel, followed by staining with ethidium bromide to view genomic DNA.

RT-PCR
Total RNA was extracted from C6 cells using RNA STAT 60 (Tel-Test, Friendswood, TX) and was quantified by absorbance at a wavelength of 260 nm (21). RT-PCR was performed using a Crocodile II PCR machine (Appligene, Inc., Pleasanton, CA). One microgram of RNA was used in RT-PCR reactions with Superscript (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s specifications. Briefly, the denaturation step was carried out at 94 C for 5 min, the annealing step was carried out at 50 C for 1 min, and the elongation step was carried out for 5 min. Primers of rat IFN{alpha} were: forward primer, 5'-3'-CCT CAG CCT CTT CAC ATC AA; and reverse primer, 5'-3'-TGT GGC TCA GGA CTC ATT TC (PCR product size, 325 bp) (22). Primers of rat IFNß were: forward primer, 5'-3'-GAT ATT TCT CTT CCT TTG; and reverse primer, 5'-3'-TCT GTT TTC CTT TGA CCT TTC (PCR product size, 787 bp) (23). Primers of rat actin were: forward primer, 5'-3'-AGG CAT CCT GAC CCT GAA GTA C; and reverse primer, 5'-3'-GAG GCA TAC AGG GAC AAC ACA G (PCR product size, 249 bp) (24). The amplification products were visualized on a 2% agarose gel stained with ethidium bromide.

Western blot
Western immunoblots were performed according to the protocols described previously (17). Cells were 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 lima bean trypsin inhibitor, and 2 mM ß-glycerophosphate). Lysates were passed through a 21-gauge needle to shear DNA and then were incubated on ice for 30 min. Cell lysates were centrifuged at 12,000 rpm for 20 min at 4 C, and equal amounts of cell lysate supernatant 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 gel and 5% stacking gel) at 200 V. Proteins were transferred to nitrocellulose membrane (Immobilon P, Millipore Corp., Bedford, MA) for 1 h at 100 V. The membrane was incubated for 1 h in 5% dry milk solution in 20 mM Tris HCl (pH 7.4), 500 mM NaCl, and 0.05% Tween 20 and then incubated with monoclonal antimouse PKR antibody with a stock concentration of 200 µg/ml (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), polyclonal antirabbit phospho-eIF2 antibody with a stock concentration of 1 mg/ml (Research Genetics, Inc., Huntsville, AL) at a dilution of 1:1000, or monoclonal antimouse total eIF2 antibody (a gift from Dr. Scott Kimball, Milton S. Hershey Medical Center, Hershey, PA) at a dilution of 1:200 in a solution of 1% dry milk solution in 20 mM Tris HCl (pH 7.4), 500 mM NaCl, and 0.05% Tween 20 overnight. The membrane was washed and incubated with secondary antibody (Pierce Chemical Co., Rockford, IL) at a concentration of 1:1000 for 1 h. 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 was determined using one-way ANOVA in a SIMSTAT3 package (Normand Peladeau, Provalis Research, Montréal, Canada).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Our previous studies indicated that poly(IC) reduced the number of attached cells in both subconfluent and confluent cultures of C6 cells (17). In this series of experiments we first examined whether poly(IC) altered DNA synthesis, as assayed by [3H]thymidine incorporation, in confluent and subconfluent C6 cultures. Poly(IC) treatment of confluent C6 cells decreased [3H]thymidine incorporation to 50% (P < 0.05) of that in control cells after 24 h of treatment (Fig. 1AGo). Treatment with 100 nM IGF-I alone had no significant (P > 0.05) effect on [3H]thymidine incorporation compared with control cells. Addition of 100 nM IGF-I prevented the poly(IC)-mediated decrease in [3H]thymidine incorporation and increased [3H]thymidine incorporation to 82% of the control value after 24 h [P < 0.05 compared with cells treated with poly(IC) alone]. There was no significant difference (P > 0.05) between [3H]thymidine incorporation in control cultures and that in cultures treated with both poly(IC) and IGF-I, suggesting that IGF-I completely reversed the decrease in [3H]thymidine incorporation caused by poly(IC) in C6 cells. In marked contrast, treatment of subconfluent cultures of C6 cells with poly(IC) increased [3H]thymidine incorporation 6-fold (P < 0.05) after 24 h of treatment (Fig. 1BGo). Treatment with 100 nM IGF-I increased [3H]thymidine incorporation 2.5-fold (P < 0.05) compared with that in control cells. Addition of exogenous IGF-I did not alter [3H]thymidine incorporation (P > 0.05) compared with that in cells treated with poly(IC) alone.



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  		Figure 1. Poly(IC) alters [3H]thymidine incorporation into DNA of confluent and subconfluent cultures of C6 rat glioma cells. C6 rat glioma cells (4 x 104) were plated in 48-well plates and grown for either 48 h (A) or 24 h (B) in Ham’s F-12 medium containing 10% FBS, and then cultured for 24 h in Ham’s F-12 medium containing 1% FBS. Cells were treated for 24 h with 200 µg/ml poly(IC), 100 nM IGF-I, or both. Data are normalized to trichloroacetic acid-precipitable counts per min from control cultures. Data represent the mean of three separate experiments. *, Data are significant (P < 0.05) compared with the control value; **, the mean is significant (P < 0.05) compared with the value after poly(IC) treatment. Bars represent the SEM.

 
To better understand the reasons for these changes in [3H]thymidine incorporation with poly(IC) treatment, we determined the effect of poly(IC) on cell cycle in confluent and subconfluent cultures of C6 cells. As expected from the [3H]thymidine incorporation experiments, poly(IC) treatment of confluent cultures of C6 cells caused a decrease in the percentage of cells in the S phase of the cell cycle from 5.53% to 3.64% (P < 0.05; Fig. 2AGo). Addition of exogenous IGF-I to poly(IC)-treated cells reversed the decrease in G1 to S phase transition and also increased the percentage of cells in the G2/M phase of the cell cycle from 9.36% to 15.22% (P < 0.05). Addition of IGF-I alone had no significant effect on the percentage of cells in the different stages of the cell cycle compared with that in control cells (P > 0.05; Fig. 2BGo).



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  		Figure 2. Poly(IC) decreases the percentage of cells in the S phase of the cell cycle of confluent cultures of C6 rat glioma cells. C6 rat glioma cells were grown to confluence in 60-mm plates, cultured for 24 h in Ham’s F-12 medium supplemented with 1% FBS, and then treated with 200 µg/ml poly(IC), 100 nM IGF-I, or both in Ham’s F-12 medium supplemented with 1% FBS for 48 h. Cells were harvested for cell cycle analysis as described in Materials and Methods. A, Control (A), poly(IC) (B), and poly(IC) plus IGF-I (C). B, Control (A) and IGF-I (B). Data represent the mean of two separate experiments. The y-axis shows the number of cells, and the x-axis shows the fluorescence intensity.

 
In contrast, poly(IC) treatment of subconfluent cultures of C6 cells caused an increase in the percentage of cells in the S phase of the cell cycle from 5.24% to 13% (P < 0.05; Fig. 3AGo), consistent with the increase in [3H]thymidine incorporation. However, poly(IC) decreased the percentage of cells in the G2/M phase, suggesting that the poly(IC)-treated cells were blocked at the S to G2 transition. Addition of exogenous IGF-I reversed this block, as evidenced by the increased percentage of cells in the G2/M phase of the cell cycle from 6.39% to 12.51% (P < 0.05). Exogenous IGF-I also decreased the percentage of cells in G0/G1, suggesting that it also increased the G1 to S transition in subconfluent cultures. Treatment with IGF-I alone increased the percentage of cells in the S phase of the cell cycle from 13% to 18% (P < 0.05; Fig. 3BGo).



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  		Figure 3. Poly(IC) increases the percentage of cells in the S phase of the cell cycle of subconfluent cultures of C6 rat glioma cells. C6 rat glioma cells were plated sparsely in 60-mm plates, cultured for 24 h in Ham’s F-12 medium supplemented with 1% FBS, and then treated with 200 µg/ml poly(IC), 100 nM IGF-I, or both in Ham’s F-12 medium supplemented with 1% FBS for 48 h. Cells were harvested for cell cycle analysis. A, Control (A), poly(IC) (B), and poly(IC) plus IGF-I (C). B, Control (A) and IGF-I (B). Data represent the mean of two separate experiments.

 
To determine whether poly(IC) also caused increased C6 cell death, both attached and floating cells were collected and assayed for trypan blue exclusion. All cell numbers are normalized to the number of cells in control plates at 48 h, which was set at 100% (Fig. 4AGo). Treatment of cells with 200 µg/ml poly(IC) for 48 h decreased the number of cells to 49% of the control value (P < 0.05). Treatment with IGF-I alone had no significant effect (P > 0.05) on cell number compared with that for control cells at 48 h. Addition of 100 nM IGF-I partially blocked the poly(IC)-mediated decrease in cell number and increased cell number to 70% of that for control cells [P < 0.05 compared with cells treated with poly(IC) alone]. Treatment of cells with 200 µg/ml poly(IC) for 72 h decreased the number of cells to 46% of the control value (P < 0.05). Treatment with IGF-I alone had no significant effect (P > 0.05) on cell number compared with that of control cells at 72 h. Addition of 100 nM IGF-I partially blocked the poly(IC)-mediated decrease in cell number and increased cell number to 80% of that for control cells [P < 0.05 compared with cells treated with poly(IC) alone]. Under all conditions, 97–100% of the total cells were viable, as assayed by trypan blue exclusion (data not shown). There was no significant difference between the number of viable cells in the poly(IC)-treated cultures and that of cells in control plates on d 0 (P < 0.05), and there were no differences in the percentage of nonviable cells (data not shown), indicating that treatment with poly(IC) for 72 h has a cytostatic, but not a cytotoxic, effect on confluent cultures of C6 cells.



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  		Figure 4. Poly(IC) has an acute cytostatic effect on C6 rat glioma cells. C6 rat glioma cells were either grown to confluence (A) or plated sparsely (B) in 60-mm plates, cultured for 24 h in Ham’s F-12 medium supplemented with 1% FBS, and then treated with 200 µg/ml poly(IC), 100 nM IGF-I, or both in Ham’s F-12 medium supplemented with 1% FBS for up to 72 h. Medium was changed every 24 h. Both attached cells and those in the conditioned medium were harvested for trypan blue exclusion analysis after 48 and 72 h of treatment. Data were normalized to the cell number of control cultures at 48 h. Data represent the mean of three separate experiments. *, The mean is significant (P < 0.05) compared with control value; **, the mean is significant (P < 0.05) compared with the value after poly(IC) treatment. Bars represent SEM.

 
Trypan blue exclusion assays were also performed on subconfluent cultures of C6 cells. All cell numbers are normalized to the number of cells in control plates at 48 h, which was set at 100% (Fig. 4BGo). Treatment of cells with 200 µg/ml poly(IC) for 48 h decreased the number of cells to 57% of the control value (P < 0.05). Addition of IGF-I alone increased cell number 1.9-fold compared with the control value (P < 0.05). The addition of 100 nM IGF-I partially blocked the poly(IC)-mediated decrease in cell number and increased cell number by 15% compared with the control value [P < 0.05 compared with cells treated with poly(IC) alone]. Treatment of cells with 200 µg/ml poly(IC) for 72 h decreased the number of cells to 75% of the control value (P < 0.05). The addition of IGF-I alone had no significant effect on the number of C6 cells compared with controls (P > 0.05). The addition of 100 nM IGF-I partially blocked the poly(IC)-mediated decrease in cell number and increased cell number by 33% compared with the control value [P < 0.05 compared with cells treated with poly(IC) alone]. All cells were viable, as measured by trypan blue exclusion (data not shown), indicating that, as in confluent cultures, poly(IC) treatment for 72 h had a cytostatic, but not a cytotoxic, effect in subconfluent cultures of C6 cells.

To confirm that 48 h of poly(IC) treatment did not produce a cytotoxic effect on C6 cells, we also measured cell death by DNA laddering and exposure of annexin V to the outer layer of the plasma membrane. Our data demonstrate that there was little difference in cell death as measured by either of these two techniques under the conditions of the experiment up to 72 h of treatment in confluent and subconfluent C6 cultures (data not shown). However, in contrast to our results after short-term treatment with poly(IC), we found that treatment with poly(IC) for 14 d, under conditions of serum starvation and without refeeding nutrients, resulted in increased DNA laddering, indicative of increased C6 cell death (Fig. 5Go). These data suggest that under the stress of nutrient and serum starvation, poly(IC) can cause C6 cell death.

We had previously demonstrated that blocking the activation of PKR prevented poly(IC) from inhibiting IGF-I mRNA (16). We now asked whether blocking the activation of PKR would also block the antiproliferative effects of poly(IC) in C6 cells. All cell numbers were normalized to those of cultures on d 0, the day the treatments began, which was set at 100%. Cell number increased 1.8-fold in control cultures grown in the absence of poly(IC). In cells treated with 200 µg/ml poly(IC) for 48 h, cell number was 105% (P < 0.05) compared with that of control cells (Fig. 6AGo). Treatment with AP, which blocks activation of PKR (26, 27), had no significant effect (P > 0.05) on C6 cell number compared with that of control cells. However, the cell number of cultures treated with both poly(IC) and AP was 142% [P < 0.05 compared with cells treated with poly(IC) alone], suggesting that AP partially blocked the cytostatic effect of poly(IC) in C6 cultures. There was a significant difference (P < 0.05) between the number of cells in control cultures and that of cells that had been treated with both poly(IC) and AP, suggesting that under the conditions of this experiment, blocking PKR activation was not sufficient to block the poly(IC)-mediated cytostatic effect on C6 cells. Western immunoblots confirmed that poly(IC) activated PKR, as measured by increased levels of the approximately 38-kDa phosphorylated eIF2-{alpha}, a substrate of PKR (Fig. 6BGo). The ratio of the change in the levels of p-eIF2 to total eIF2 showed that poly(IC) increased the levels of p-eIF2 by 1.6-fold after 3 h of treatment. Pretreatment with AP decreased the levels of p-eIF2 to 30% of the control value after 3 h of treatment, and pretreatment with AP blocked the induction of p-eIF2 by poly(IC). Treatment with poly(IC) induced the levels of p-eIF2 by 3-fold after 48 h. However, pretreatment with AP only partially blocked the induction of p-eIF2 by poly(IC) after 48 h of treatment. The changes in the levels of eIF2-{alpha} phosphorylaton were not due to increased eIF2-{alpha} protein levels in C6 cells (Fig. 6BGo, bottom panel).



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  		Figure 6. The poly(IC)-mediated decrease in C6 cell number requires the activation of PKR. C6 rat glioma cells were grown to confluence in 60-mm plates for trypan blue exclusion assays (A) and Western immunoblots (B) and cultured for 24 h in Ham’s F-12 medium supplemented with 1% FBS. Cells either were not treated with AP (lanes C and P) or were pretreated with 10 mM AP (lanes A and P+A) for 6 h. Medium was then removed, and Ham’s F-12 medium with 1% FBS containing no additions (lanes C and A) or 200 µg/ml poly(IC) (lanes P and P+A) was added. Medium was changed at 24 h. Cells were counted after 48 h of treatment, and lysates were harvested for Western immunoblots after 3 and 48 h of treatment. Data were normalized to the cell number of control cultures on d 0. Data represent the mean of two separate experiments. *, The mean is significant (P < 0.05) compared with the control value; **, the mean is significant (P < 0.05) compared with the value after poly(IC) treatment. Bars represent the SEM.

 
We next tested the hypothesis that the antiproliferative effects of poly(IC) in C6 cells were mediated via the induction of IFN. RT-PCR reactions showed that treatment with 200 µg/ml poly(IC) induced IFN{alpha} mRNA within 24 h of treatment (Fig. 7AGo). Treatment with 100 nM IGF-I alone did not induce IFN{alpha} and also had no effect on the induction of IFN{alpha} by poly(IC) in C6 cells. There were no significant effects on ß-actin mRNA levels over the course of the experiment (Fig. 7BGo). Unlike IFN{alpha}, IFNß mRNA was not detected in either control cultures or cultures treated with poly(IC), IGF-I, or both poly(IC) and IGF-I (data not shown). However, we cannot rule out the possibility that IFNß mRNA was transiently induced in C6 cells, but returned to basal levels by the time the RNA was harvested for RT-PCR.



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  		Figure 7. Poly(IC) induces IFN{alpha} in C6 cells after 24 h of treatment. C6 rat glioma cells were grown to confluence in 60-mm plates, cultured for 24 h in Ham’s F-12 medium supplemented with 1% FBS, and then treated with 200 µg/ml poly(IC), 100 nM IGF-I, or both in Ham’s F-12 medium supplemented with 1% FBS for 24 h. RNA was extracted from control cells (C) and cells treated with poly(IC) (P), IGF-I (I), or poly(IC) and IGF-I (P+I). RT-PCR was performed to detect changes in IFN{alpha}, IFNß, and ß-actin mRNA levels. Data are representative of two separate experiments.

 
Since poly(IC) induced IFN{alpha} in C6 cells, we asked whether the antiproliferative effect of poly(IC) was IFN dependent in confluent cultures. All cell numbers were normalized to the number of cells in control plates, which was set at 100%. Treatment with either control preimmune serum or immune serum against mouse IFN{alpha} and IFNß alone had no significant effect (P > 0.05) on C6 cell numbers compared with those of untreated cells (Fig. 8AGo). In cells treated with 200 µg/ml poly(IC) for 48 h, cell number was 54% of control (P < 0.05), consistent with the cytostatic effect of poly(IC) observed above. The effect of poly(IC) on C6 cell number was not altered by treatment with control preimmune serum. However, treatment with 33 U/ml immune serum against mouse IFN{alpha} and IFNß blocked the poly(IC)-mediated decrease in C6 cell number [P < 0.05 compared with cells treated with poly(IC) alone]. Treatment of cells with 100 nM IGF-I also blocked the poly(IC)-mediated decrease in C6 cell number [P < 0.05 compared with cells treated with poly(IC) alone]. In C6 cells treated with 100 IRU/ml IFN, cell number was 45% of control (P < 0.05), and there was no difference in the number of dead cells between control and IFN-treated cells (data not shown). We therefore concluded that IFN also exerted a cytostatic effect on C6 cells. Addition of preimmune serum did not alter the effect of IFN on C6 cell number (P > 0.05 compared with cells treated with IFN alone). Addition of either immune serum against IFN or 100 nM IGF-I blocked the IFN-mediated decrease in C6 cell number (P < 0.05 compared with cells treated with IFN alone). These results suggested that poly(IC) caused a cytostatic effect by inducing IFN, and that IGF-I might act to block IFN action in C6 cells.

PKR is thought to be one of the mediators of the antiproliferative effects of IFN (28), and our current results implicated PKR in poly(IC) and IFN cytostasis in C6 cells. Thus, we also asked whether IGF-I could interfere with poly(IC) or IFN induction of PKR protein in C6 cells. Addition of either preimmune serum or blocking antibody against IFN did not alter the level of the approximately 68-kDa PKR protein in C6 cells (Fig. 8BGo). Treatment with 200 µg/ml poly(IC) for 48 h increased the levels of PKR protein 9-fold over that in control cells. Addition of poly(IC) along with preimmune serum did not prevent the increase in PKR protein. However, the addition of blocking antibody against IFN prevented the poly(IC)-mediated increase in PKR protein. Moreover, the addition of 100 nM IGF-I prevented the poly(IC)-mediated increase in PKR protein. Treatment of C6 cells with 100 IRU/ml IFN for 48 h increased PKR protein 9-fold compared with the control value. Addition of IFN along with preimmune serum had no effect on the increase in PKR protein. However, the addition of either blocking antibody against IFN or 100 nM IGF-I prevented IFN from increasing PKR protein levels.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Prior studies have shown that dsRNA has an antitumorigenic action against gliomas (5, 29), but the mechanism of dsRNA action has not been completely elucidated. dsRNA is known to activate PKR, which has proapoptotic as well as antiproliferative effects (8, 9, 10, 11). However, the growth inhibitory targets of PKR have not yet been completely characterized. Our prior studies show that dsRNA reduces IGF-I synthesis and secretion in a PKR-dependent mechanism and that exogenous IGF-I blocks the poly(IC)-mediated decrease in C6 cell number (16, 17). We also found that dsRNA reduced IGF-I transcription, but not mRNA half-life, indicating that IGF-I may be a directly repressed, transcriptional target of dsRNA action (16).

Our present data suggest that the lower cell number in poly(IC)-treated cultures compared with control cultures in 1% FBS and at 48–72 h is primarily caused by decreased cell proliferation. Blocking the activation of PKR partially blocked the cytostatic effect of poly(IC) on C6 cells. Poly(IC) induced IFN{alpha} in C6 cells, suggesting that IFN could mediate the cytostatic effect of poly(IC) on C6 cells. Our data also show that an immune serum against IFN prevented poly(IC) action, further suggesting that the induction of IFN mediated the cytostatic effect of poly(IC) on C6 cultures. Type 1 IFN decreases the proliferation of various cells, including gliomas (12, 13, 30, 31), and PKR is one of the targets through which IFN exerts its antiproliferative effects (28). Indeed, both poly(IC)- and IFN-mediated cytostatic effects were associated with increased PKR protein levels in C6 cultures. Moreover, treatment with the blocking antibody against IFN blocked not only the cytostatic effect on C6 cells, but also the increase in PKR protein. We hypothesized that IGF-I blocked the poly(IC)-mediated cytostatic effect on C6 cell number by preventing PKR induction. In support of this hypothesis, we found that the decreased cytostatic effect in C6 cells treated with both poly(IC) and IGF-I or IFN and IGF-I was associated with decreased induction of PKR protein.

In confluent cultures, poly(IC) decreased the G1 to S transition and DNA synthesis, whereas in subconfluent cultures, poly(IC) decreased the S to G2 transition, with the result that DNA synthesis was increased, but mitosis was decreased. As the cytostatic effects of poly(IC) were prevented by an IFN-neutralizing antibody, it is reasonable to speculate that autocrine IFN was responsible for these cell cycle blocks. Type I IFN has been known to cause a block in either the G1 phase or the G2/M phase of the cell cycle, resulting in a decrease or an increase in the percentage of cells in the S phase of the cell cycle, respectively (32, 33, 34, 35). The blocks in G1 as well as G2/M phases of the cell cycle eventually result in decreased cell proliferation (13, 36). The IFN-mediated inhibition of glioblastoma cell cycle progression was associated with increased levels of the cyclin-dependent kinase inhibitor p21WAF1 (37). IFN{alpha} caused cell cycle arrest of lymphoid cultures coupled with induction of p21WAF1, decreased activity of cyclin-dependent kinases, and increased levels of hypophosphorylated retinoblastoma protein (Rb) (38). Treatment with IFN{alpha} also caused decreased levels of cyclin D3 mRNA and cyclin-dependent kinase activity associated with cyclins A and E (39).

Our data show that poly(IC) treatment increased PKR levels by inducing IFN in C6 cells. Activation of PKR led to decreased levels of cyclins D, E, and A in NIH-3T3 cells (40). PKR also caused decreased myogenic cell proliferation by increasing the level of p21WAF1 and the levels of hypophosphorylated Rb (41). A similar phenomenon could be occurring in C6 cells. Activation of PKR could result in decreased synthesis of cyclins required for passage through the cell cycle. Increased levels of cyclin-dependent kinase inhibitors and hypophosphorylated Rb could also prevent progress through the cell cycle. Thus, our results are consistent with the concept that the cytostatic effect of poly(IC) and possibly IFN is due at least in part to activation of PKR.

The addition of IGF-I blocked the effect of poly(IC) on C6 cells. In both confluent and subconfluent cultures, IGF-I increased the percentage of cells in the G2/M phase of the cell cycle, demonstrating that it removed cell cycle blocks in each case and increased progression through the cell cycle and consequently the percentage of cells undergoing mitosis. We showed that PKR induction was one specific step in poly(IC) and IFN action that was blocked by IGF-I. IGF-I and PKR have opposing effects on several cell cycle components. For example, IGF-I increased the number of thyroid cells in the G2/M phase of the cell cycle, increased the levels of cyclins D, E, and A, and decreased the level of the cyclin-dependent kinase inhibitor p27Kip1 (42). Our results suggest that IGF-I could prevent the increase in PKR protein caused by poly(IC) or IFN treatment and thus block the consequent pathways leading to decreased C6 cell proliferation.

Treatment with the immune serum against IFN completely blocked the cytostatic effect of poly(IC) on C6 cells. However, our data also demonstrate a significant difference between the number of cells in control cultures and that in cultures treated with both poly(IC) and AP, suggesting that either AP did not completely block activation of PKR under the conditions of the experiment or PKR is not the sole pathway through which poly(IC) exerts its cytostatic effect on C6 glioma cells. Western immunoblots demonstrate that AP is unable to block PKR activation at 48 h, suggesting that the partial rescue of the cytostatic effect of poly(IC) on C6 cells could be due to decreased effectiveness of AP in preventing PKR activation. However, we cannot rule out the existence of PKR-independent pathways in the poly(IC) effect on C6 cells. In addition to PKR, dsRNA binds to and activates 2',5'-oligoadenylate synthetase (OAS). Activation of OAS leads to the formation of 2'5'-oligoadenylates, which activate a latent endonuclease, RNase L (6), which, in turn, degrades ribosomal RNA and mRNA, leading to inhibition of protein synthesis. RNase L has antitumor effects (43) and thus could be a potential candidate for the effect of dsRNA on C6 cells, especially since IFN stimulates the transcription of OAS (6). Recent studies have described the dsRNA-mediated stimulation of the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun NH2-terminal kinase (JNK) pathways (44). Stimulation of the p38 MAPK pathway did not require either RNase L or PKR, whereas stimulation of JNK required both. Our data suggest the possible activation of both of these pathways in C6 cells treated with poly(IC). As signaling through the JNK pathway required PKR, activation of this pathway could result in the PKR-mediated cytostatic effect on C6 cells. However, preventing the activation of PKR resulted in only a partial block of the cytostatic effect of poly(IC) on C6 cells, suggesting the existence of a PKR-independent pathway as well, indicating the possible role of the p38 MAPK pathway in mediating the effect of poly(IC) on C6 cells.

In summary, we suggest the following model of the effect of poly(IC) on C6 cells. Poly(IC), in an IFN-independent pathway, decreases IGF-I synthesis and secretion in C6 cells. Poly(IC) also induces IFN in C6 cells, which, in turn, increases PKR protein levels. Type 1 IFN mediates all of the cytostatic effects of poly(IC) on C6 cells, whereas increased PKR activation mediates part of the cytostatic effect of poly(IC) on C6 cells. We hypothesize that the reduction of IGF-I allows IFN to increase PKR protein. Future studies will focus on determining the pathways through which IGF-I blocks IFN-stimulated PKR expression and growth inhibition in C6 cells as well as the molecular mechanisms of the PKR-independent effect of poly(IC) on C6 cells.


   Acknowledgments
 
We thank Xiuye Ma for technical assistance with the [3H]thymidine incorporation assays. We are grateful to Dr. Yair Gazzit and Charles Thomas (University of Texas Health Science Center, San Antonio, TX) for intellectual discussion and technical support with the FACS analysis, respectively. We are also grateful to Dr. Bret Hassel (University of Maryland Medical Center, Baltimore, MD) for donating the blocking antibody against IFN and the preimmune control serum, and to Dr. Scott Kimball (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 Grants 07 and 02-022 from Children’s Cancer Research Center (University of Texas Health Science Center; to M.L.A.).

Abbreviations: AP, 2-Aminopurine; CM, conditioned medium; ds, double-stranded; FACS, fluorescence-activated cell sorting; IFN, type I interferon; IRU, international reference units; JNK, c-Jun NH2-terminal kinase; OAS, 2',5'-oligoadenylate synthetase; PKR, protein kinase R; poly(IC), polyinosinic-polycytidylic acid; PS, penicillin/streptomycin; Rb, retinoblastoma protein; RNase, ribonuclease.

Received October 20, 2001.

Accepted for publication February 14, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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