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Endocrinology Vol. 138, No. 3 985-993
Copyright © 1997 by The Endocrine Society

Articles

Prostaglandin A2 Specifically Represses Insulin-Like Growth Factor-I Gene Expression in C6 Rat Glioma Cells1

Tim Bui, Chiaoyun Kuo, Peter Rotwein2 and Daniel S. Straus

Biomedical Sciences Division and Biology Department, University of California (T.B., C.K., D.S.S.), Riverside, California 92521-0121; and Departments of Internal Medicine, and Biochemistry and Molecular Biophysics, Washington University School of Medicine (P.R.), St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Daniel S. Straus, Biomedical Sciences Division, University of California, Riverside, California 92521-0121. E-mail: daniel.straus{at}ucr.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cyclopentenone PGs (PGA and PGJ series) inhibit tumor cell proliferation in vitro and tumorigenesis in vivo via mechanisms that are at present poorly understood. The C6 rat glioma cell line synthesizes and secretes insulin-like growth factor-I (IGF-I), which is believed to act as an autocrine factor for these cells. PGA2 inhibits the proliferation of the C6 cells and causes an increase in the fraction of cells in the G1 phase of the cell cycle. The inhibition of cell proliferation by PGA2 is accompanied by a decrease in the abundance of IGF-I messenger RNA (mRNA). This regulation of IGF-I gene expression is specific, as the abundance of hypoxanthine-guanine phosphoribosyl transferase (HPRT) and ubiquitin mRNA is not significantly affected by PGA2. The repression of IGF-I gene expression is observed at PGA2 concentrations as low as 10 µM and is evident within 4 h after treatment of the C6 cells with PGA2. In addition to specifically regulating the expression of the IGF-I gene, PGA2 also decreases the abundance of cyclin D1 mRNA and increases the abundance of Waf1 mRNA. The inhibition of cell proliferation by PGA2 is partially reversed by coaddition of IGF-I, indicating partial dominance of IGF-I action over PGA2 action. To investigate the molecular basis for the regulation of IGF-I gene expression by PGA2, we developed a sensitive RT-PCR assay for IGF-I nuclear transcripts. A similar assay was developed for quantifying HPRT transcripts, which were used as a control. Treatment of the C6 cells with 20 µM PGA2 resulted in approximately a 6-fold decrease in IGF-I mRNA and IGF-I nuclear transcripts. In contrast, HPRT mRNA and nuclear transcript levels were not significantly affected by PGA2. These results indicate that the decrease in IGF-I mRNA abundance that occurs in response to PGA2 is caused largely by a decrease in IGF-I nuclear transcript levels. To identify the cis-acting element that mediates the effect of PGA2 on IGF-I transcription, C6 cells were transiently transfected with IGF-I/luciferase expression constructs in which luciferase transcription is driven by IGF-I P1 promoter fragments extending from -1711 to +328 or from -1114 to +328 relative to the beginning of exon 1. Treatment of cells with PGA2 in these transient transfection assays did not decrease luciferase activity. These results suggest that the cis-acting regulatory element required for the response to PGA2 is located outside the -1711 to +328 promoter interval.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CYCLOPENTENONE PGs (PGA and PGJ series), which are characterized by an {alpha}, ß-unsaturated ketone in the cyclopentane ring, are potent inhibitors of cell proliferation (1, 2, 3, 4). At growth inhibitory (subtoxic) concentrations, PGA1, PGA2, and PGJ2 arrest cells in the G1 phase of the cell cycle, with little if any effect on cell viability over a 24-h treatment interval (5, 6). PGA and PGJ series PGs also inhibit tumor cell proliferation in vivo, raising the possibility that the cyclopentenone PGs might be useful as chemotherapeutic agents (1, 2, 3, 4).

Very little is known at present about molecular mechanism(s) by which the cyclopentenone PGs inhibit cell proliferation. Treatment of cells with PGA2 results in increased expression of a number of stress-related genes, including HSP70 (7, 8), c-fos (9), and gadd153 (10). The increased expression of these genes appears to involve an increase in gene transcription as well as a posttranscriptional mechanism(s) (7, 8, 9, 10). PGA2 has also been shown recently to increase the expression of the cyclin-dependent kinase inhibitor Waf1 (p21) in MCF-7 breast cancer cells, via a p53-independent mechanism (6, 11). The induction of HSP70 gene transcription involves activation of the heat-shock transcription factor (7, 8). Details concerning the molecular mechanism by which PGA2 increases transcription of the other genes are unknown.

In addition to increasing the expression of certain genes, the cyclopentenone PGs repress the expression of others. For example, PGA2 represses c-myc gene expression in HL60 cells (12), PGA2 and {Delta}12-PGJ2 repress N-myc gene expression in a human neuroblastoma cell line (13), and PGA2 represses cyclin D1 and cyclin-dependent kinase 4 (cdk4) gene expression in MCF-7 breast cancer cells (6). Thus specific negative as well as positive effects on gene expression are observed following treatment of cells with the cyclopentenone PGs. Very little is known at present about the molecular mechanism for repression of gene expression by the cyclopentenone PGs.

The C6 rat glioma cell line synthesizes and secretes insulin-like growth factor-I (IGF-I), which acts as an autocrine growth factor for these cells (14, 15). Transcription of the IGF-I gene in these cells is directed exclusively by the P1 promoter element (14). Interference with IGF-I or IGF-I receptor synthesis by the C6 cells results in suppression of tumorigenicity in vivo (15, 16, 17). The suppression of tumorigenicity that occurs in vivo when IGF-I or IGF-I receptor synthesis is inhibited is characterized by apoptosis of the tumor cells (17, 18). Thus a major role of IGF-I in the autocrine/paracrine stimulation of tumor cell proliferation in vivo may be to protect the cells from undergoing apoptosis. In the present study, we demonstrate that IGF-I messenger RNA (mRNA) abundance is specifically decreased by PGA2 in the C6 cells. Treatment of the C6 cells with PGA2 also leads to a decrease in the abundance of IGF-I nuclear transcripts, suggesting that the effect of PGA2 on IGF-I gene expression is exerted primarily at the transcriptional level. Suppression of autocrine growth factor synthesis represents a previously unknown potential mechanism for the suppression of tumorigenicity by the cyclopentenone PGs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture experiments
Rat C6 glioma cells were obtained from American Type Culture Collection (Rockville, MD) and cultured in MEM with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). PGA2 (dissolved in methyl acetate) was obtained from the Caymen Chemical Co. (Ann Arbor, MI). Before using the PGA2 in experiments, the methyl acetate was evaporated, and the PGA2 was dissolved in ethanol. Human recombinant IGF-I was purchased from R&D Systems (Minneapolis, MN) and dissolved in 10 mM acetic acid at a concentration of 100 µg/ml (14 µM). Growth experiments were carried out using 50 ng/ml (7 nM) IGF-I. For experiments testing the effects of PGA2 on IGF-I gene expression or cell proliferation, cells were plated at a density of 1.5 x 104 cells/cm2 and cultured for 24 h at 37 C. At this time, the cells were in exponential growth phase and were approximately 50–60% confluent. PGA2 or vehicle (ethanol, final concentration 0.067%) was then added to the culture medium. Cells were harvested at various time intervals for counting (using a Coulter ZBI cell counter or hemacytometer), cell cycle analysis, RNA preparation, or luciferase assay. For cell cycle analysis, cells were harvested by trypsinization, resuspended in ice cold PBS, and fixed with methanol (final concentration 67% vol/vol) (19, 20). Cells were then pelleted by centrifugation, stained with propidium iodide (50 µg/ml) in the presence of heat-treated RNase A (1 mg/ml) for 30 min at 23 C, and filtered through 35 µm nylon mesh (19, 20). Cell cycle analysis was performed using a Becton Dickinson FACScan flow cytometer. The percentage of cells in various phases of the cell cycle was analyzed using the CellFIT software program (Becton Dickinson).

DNA clones
DNA clones used as probes for Northern blot analysis were obtained from the following sources: rat IGF-I complementary DNA (cDNA) prigf1-1 (21), G.I. Bell, Howard Hughes Medical Institute, University of Chicago, Chicago, IL; Chinese hamster ubiquitin cDNA clone pH37 (22), A.J. Fornace, NIH, Bethesda, MD; mouse 1.4-kilobase (kb) cyclin D1 cDNA (23), C. Dickson, Imperial Cancer Research Fund Laboratories, London, U.K.; mouse Waf1 (p21) cDNA (24), K. Huppi, NIH, Bethesda, MD; rat 28S ribosomal RNA genomic clone pSPEE6.7 (25), D. Chikaraishi, Tufts University School of Medicine, Boston, MA; mouse hypoxanthine-guanine phosphoribosyl transferase cDNA clone mHPT5, American Type Culture Collection.

For transient transfections, the -1711/+328 and -1114/+328 IGF-I P1 promoter fragments were subcloned from IGF1711b/LUC (26) into the promoterless luciferase vector pGL3-Basic (Promega, Madison, WI). The -1114/+328 promoter fragment was isolated by restriction digestion with SmaI and BamHI and cloned into the SmaI/BglII sites of pGL3-Basic. The -1711/+328 promoter construct was produced by first cloning the -1711/+328 PstI/BamHI restriction fragment into the PstI/BamHI sites of pBluescript KS(-) (Stratagene, Cincinnati, OH). A KpnI/SmaI fragment containing nucleotides -1711 to -1115 of the P1 promoter was then isolated and cloned directly in front of the -1114/+328 promoter fragment in pGL3-Basic. The cytomegalovirus (CMV)-ßgal expression plasmid, in which ß-galactosidase expression is under control of the human cytomegalovirus immediate-early enhancer plus promoter, was obtained from F.M. Sladek, University of California, Riverside, CA.

Transfections
Cells were transfected by the calcium phosphate precipitate method, essentially as described by Rosenthal (27). Briefly, cells were plated at a density of 1.5 x 105 cells/6 cm dish and incubated for 20 h at 37 C. Precipitated DNA (5 µg luciferase expression construct plus 2 µg CMV-ßgal construct) was added directly to the dishes. After 8 h, the cells were shocked by treating them with 5 ml 10% dimethylsulfoxide (DMSO) in serum-free MEM for 90 sec. The DMSO solution was then aspirated, and 3 ml fresh medium with 10% serum was added to each dish. Cells were allowed to recover for 40 h and were then treated with PGA2. Cells were harvested 24 h later by scraping them into 450 µl lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, and 1% Triton X-100). Luciferase assays were performed using LAR buffer as the assay buffer (20 mM Tricine, pH 7.8, 1.07 mM (MgCO3)4 Mg(OH)2·5H2O, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM dithiothreitol, 270 µM coenzyme A, 530 µM ATP, 470 µM luciferin) (28) in a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). ß-galactosidase activity was determined as described in Rosenthal (27), except that the reaction was stopped by addition of 0.5 ml 1 M glycine (pH 9.8), to yield a final concentration of 0.65 M glycine.

RNA preparation
Whole cellular RNA (cytoplasmic plus nuclear) was extracted from cultured C6 cells using a modification of the guanidine thiocyanate method, as described previously (29). RNA samples to be analyzed by RT-PCR were treated with RNase-free DNase (RQ1 DNase, Promega) at a concentration of 2.5 U/ml in DNase buffer (50 mM Tris-HCl, pH 7.8, 1 mM MgCl2, 1 mM CaCl2, 100 U/ml RNasin) for 20 min at 37 C. Samples were then extracted twice with phenol-chloroform, and reprecipitated with ethanol.

Northern blot analysis
For Northern blot analysis, RNA samples were denatured and electrophoresed in 1% agarose gels containing 2.2 M formaldehyde, and the RNA was transferred onto nylon filters. Even loading of the gels was confirmed by ethidium bromide staining. The DNA probes (gel-purified restriction fragments of cDNA clones) were labeled by random priming with [{alpha}32P]deoxycytidine triphosphate. Filters were prehybridized, hybridized, and washed as described previously (29, 30, 31). Results were quantified by scanning autoradiograms with an LKB UltroScan laser densitometer, exercising caution to stay within the linear range of the film (31).

PCR primers and PCR fragment subcloning
For amplification of IGF-I nuclear transcripts by RT-PCR, oligonucleotide primers were used that correspond to bases 13 through 32 (oligonucleotide IGF1) and bases 314 through 333 (oligonucleotide IGF2) of intron 5 of the rat IGF-I gene sequence (32). [Subsequent to the publication of Shimatsu and Rotwein (32), the exons of the rat IGF-I gene have been renumbered, so that exon 4 in Shimatsu and Rotwein (32) is now exon 5 (26). Also, the correct splice junction between exon 5 and intron 5 is between nucleotides 421 and 422 of the exon 4 sequence presented in Shimatsu and Rotwein (32) Fig. 3Go (33).] The sequences of the oligonucleotides were 5'-CACACCCAGGAGGGGAACAG-3' (IGF1) and 5'-GTGTTGTTGATGCTCCGTCC-3' (IGF2). Primers used for RT-PCR analysis of hypoxanthine-guanine phosphoribosyl transferase (HPRT) nuclear transcripts corresponded to the last 20 bases of exon 7 (oligonucleotide HPRT1) and the last 2 bases of intron 7 plus the first 21 bases of exon 8 (oligonucleotide HPRT2) of the rat HPRT gene (34, 35, 36, 37). The sequences of the oligonucleotides were 5'-GTTGGATACAGGCCAGACTG-3' (HPRT1) and 5'-CTGGAATTTCAAATCCAACAACT-3' (HPRT2).



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  		Figure 3. Time course for effect of PGA2 on IGF-I mRNA (A), cyclin D1 mRNA (B), Waf1 mRNA (C), and 28S ribosomal RNA (D). PGA2 was added at T = 0, and cells were harvested at T = 0, 4, 8, 12, or 24 h. Cultures treated with PGA2: lanes 5, 6, 9, 10, 13, 14, 17, 18; control cultures: lanes 1, 2, 3, 4, 7, 8, 11, 12, 15, 16. Cells were harvested at indicated times, and RNA was extracted for Northern blot analysis.

 
Using the IGF-I primers, a single product with a molecular size of 321 bp was generated by PCR amplification of either rat genomic DNA (spleen DNA prepared from a single Sprague-Dawley rat) or PCR amplification of a 4.2-kb rat IGF-I genomic clone (30) covering exons 4 and 5 and the beginning of intron 5. The 321-bp product obtained by amplification of the 4.2-kb genomic clone was subcloned into the TA cloning vector (Invitrogen, San Diego, CA). A single product with a molecular size of approximately 210 bp was obtained by PCR amplification of rat genomic DNA using the HPRT primers. This fragment was also subcloned into the TA cloning vector.

Reverse transcription-PCR
Before reverse transcription, 0.5 µg total RNA and 3 µg random hexamer primers (GIBCO BRL, Gaithersburg, MD) were preheated to 70 C. After 10 min, the mixture was quick-cooled in a dry ice/ethanol bath. Reverse transcription with 200 U SuperscriptII RNaseH- reverse transcriptase was performed in first-strand buffer (GIBCO BRL), 1 mM deoxynucleotide triphosphates, 10 mM dithiothreitol, 0.4 U/µl RNasin (Promega), and 0.1 µg/µl BSA, in a final volume of 20 µl, at 37 C for 1 h. The reaction product was then diluted to 50 µl with water.

PCR amplification was performed with Taq polymerase (Perkin Elmer, Norwalk, CT) with 2 µl of the diluted reverse transcription product. Amplification with the IGF-I primers was allowed to occur over 20 cycles consisting of 95 C (1.5 min), 60 C (1.5 min), and 72 C (3 min) followed by 15 min of final extension at 72 C. For analysis of HPRT transcript levels, the same reverse transcription product was also amplified in a different tube with the HPRT primers for 27 cycles using the same temperature and time parameters.

The PCR products were electrophoresed in 2% agarose gels and then blotted onto nylon filters. Gel-purified inserts from the DNA subclones corresponding to the amplified IGF-I and HPRT fragments were labeled by random priming and hybridized to the filters. Data were quantified by scanning autoradiograms with an LKB UltroScan laser densitometer or by image analysis with a Molecular Dynamics PhosphorImager. When results were quantified by autoradiography and scanning densitometry, caution was exercised to stay within the linear range of the x-ray film (31). The RT-PCR assay was linear with respect to template RNA (see Results below).

Statistical analysis
The significance of the difference between two means was determined by the unpaired Student’s t test, using P < 0.05 as the cutoff for significance. For comparison of more than two means, data were subjected to ANOVA followed by the Student-Newman-Keuls multiple comparison test, with a probability value {alpha} = 0.05 used to evaluate significance. Linear regression analysis was performed using the GraphPAD graphics program (GraphPAD Software, San Diego, CA). The significance of the difference between the slope of a line and zero was evaluated by the t test, using P < 0.05 as the cutoff for significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A Northern blot illustrating the effect of increasing concentrations of PGA2 on IGF-I mRNA abundance is shown in Fig. 1AGo. Quantitative analysis of the results (Fig. 2AGo) indicated that PGA2 repressed the expression of the IGF-I gene at concentrations >=10 µM. At 20 µM PGA2, the abundance of IGF-I mRNA was decreased by 14-fold. At this concentration, cell growth was inhibited, but there was no obvious sign of cytotoxicity of PGA2 (see below). The effect of PGA2 on IGF-I mRNA was specific, because PGA2 did not have any significant effect on the abundance of HPRT or ubiquitin mRNA (Figs. 1Go, B and C and 2, B and C).



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  		Figure 1. Dose-response experiment showing effect of varying concentrations of PGA2 on IGF-I mRNA (A), HPRT mRNA (B), and ubiquitin mRNA (C). Cells were treated with PGA2 for 24 h. RNA was then extracted, and Northern blots were prepared using 20 µg RNA/gel lane. Lanes 1–2, Control treated with vehicle alone; lanes 3–4, 2.5 µM PGA2; lanes 5–6, 5 µM PGA2; lanes 7–8, 10 µM PGA2; lanes 9–10, 15 µM PGA2; lanes 11–12, 20 µM PGA2. The 1.3 kb ubiquitin mRNA species is product of UbB gene; 3.0 and 3.9 kb mRNA species are product of UbC gene (38).

 


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  		Figure 2. Effect of increasing concentrations of PGA2 on abundance of IGF-I mRNA (A), HPRT mRNA (B), and ubiquitin mRNA (C). Data from autoradiograms shown in Fig. 1Go were quantified by scanning densitometry. A, Results for major 8 kb IGF-I mRNA species. C, Results for 1.3 kb UbB mRNA species. Each point represents mean of results obtained with RNA preparations from two different cultures with error bars indicating range for two determinations. Error bars for some points are not shown because they are smaller than the radius of the symbol for those points. All data were analyzed by linear regression analysis. Results presented in A show a significant downward trend with increasing PGA2 concentrations (r = -0.9715, P = 0.0012), whereas results presented in B and C do not show a significant upward or downward trend (r = -0.4550, P = 0.3646 for B and r = 0.1670, P = 0.7519 for C).

 
We next performed experiments to determine the time course for the effect of PGA2 on IGF-I mRNA (Fig. 3AGo). As we reported previously, the abundance of IGF-I mRNA in control, untreated cells increased over time with increasing cell density (38). PGA2 caused a rapid decrease in IGF-I mRNA abundance in treated as compared with control cells, which was evident at times as early as 4 h after addition of PGA2. Furthermore, PGA2 not only prevented the steady time-dependent increase in IGF-I mRNA that occurred in control cells, but it also decreased the abundance of IGF-I mRNA below the T = 0 baseline (Figs. 3AGo and 4AGo). PGA2 has been shown previously to increase the expression of the cdk inhibitor Waf1 in some cell lines and to decrease the expression of cyclin D1 (6, 11). To determine whether PGA2 regulated the expression of these two genes in the C6 cells, the abundance of cyclin D1 and Waf1 mRNA was determined in the time course experiment illustrated in Fig. 3Go. The results indicated that Waf1 mRNA abundance was increased within 4 h after PGA2 addition, whereas cyclin D1 mRNA was repressed by 8 h after PGA2 addition (Figs. 3Go, B and C and 4, B and C).



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  		Figure 4. Results of densitometric scans of blots illustrated in Fig. 3Go. Results were quantified by densitometric scanning of major 8 kb IGF-I mRNA (A), cyclin D1 mRNA (B), and Waf1 mRNA (C). Control cultures {circ}; PGA2-treated cultures •. Each point is mean of results obtained with two different cultures, with error bar indicating range for two determinations. Error bars for some points are not shown because they are smaller than the radius of the symbol for these points.

 
The effect of PGA2 on growth of the C6 cells is shown in Fig. 5Go. PGA2 caused a significant inhibition of growth, which was evident within 12 h after PGA2 addition (Fig. 5AGo). Analysis of cellular DNA content by flow cytometry (FACScan) indicated that PGA2 increased the fraction of cells in the G1 phase of the cell cycle (Fig. 5BGo). PGA2 at a concentration of 20 µM did not have any obvious cytotoxic effect on the cells, nor did it increase the fraction of dead cells in the cultures as indicated by trypan blue staining: the fraction of cells stained with trypan blue was <1% in both control and treated cultures. To determine whether IGF-I might be capable of reversing the growth-inhibitory effect of PGA2, we performed experiments in which cells were treated with PGA2 or PGA2 plus IGF-I. The results (Fig. 5CGo) indicated that IGF-I was able to partially reverse the growth-inhibitory effect of PGA2.



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  		Figure 5. Effect of PGA2 on growth and cell cycle distribution of C6 cells. A, Effect of PGA2 on cell number. PGA2 was added at T = 0, and cells were harvested at T = 12 or 24 h. Each bar represents mean cell count for three different dishes ± SE. *, Significantly lower than control at 12 h (P < 0.05) and 24 h (P < 0.005). B, FACScan analysis of control and PGA2-treated C6 cultures. Right, PGA2 was added to log phase culture at T = 0, and cells were harvested, fixed, and subjected to FACScan analysis 24 h later. Left, Control culture not treated with PGA2. Numbers in panels represent percentage of cells in different phases of cell cycle. C, Effect of exogenous IGF-I on growth of PGA2-treated cells. IGF-I (50 ng/ml, 7 nM) or vehicle (5 nM acetic acid) were added as indicated at T = 0 in presence or absence of PGA2 (20 µM). Cells were harvested and counted at T = 24 h. Data represent pooled results of two experiments, each performed in triplicate; therefore, each bar represents mean cell count from six different cultures ± SE. Data were subjected to ANOVA followed by Student-Newman-Keuls multiple comparison test using {alpha} = 0.05 as cutoff for significance. Means with different letters are significantly different.

 
To determine whether the regulation by PGA2 of IGF-I mRNA abundance in the C6 cells results from a change in IGF-I gene transcription, a sensitive assay for IGF-I nuclear transcripts was developed using RT-PCR. The primers for this assay are two 20-mers corresponding to sequences located in intron 5 of the rat IGF-I gene (Fig. 6Go). This assay measures the abundance of the IGF-I primary nuclear transcript and splicing intermediates still containing intron 5. A similar assay has been developed for quantifying HPRT nuclear transcripts, which are used as a control. This assay involves amplification of intron 7 of the HPRT gene and therefore detects the HPRT primary transcript and splicing intermediates still containing intron 7. All RNA samples used in the RT-PCR assays were treated with RNase-free DNase I before reverse transcription. Control assays in which the reverse transcription step was omitted consistently failed to yield amplified product, indicating that the DNase I treatment removed any genomic DNA that might have been present in the RNA preparations (results not shown).



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  		Figure 6. Location of primers used for RT-PCR amplification of IGF-I nuclear transcripts (A) and HPRT nuclear transcripts (B).

 
Because of the well-known plateau effect in PCR amplification, it is important in quantitative applications of RT-PCR to work below the plateau range with respect to cycle number, so that the assay is linear with respect to RNA template. At 20 cycles of PCR amplification, the assay for IGF-I nuclear transcripts was linear with respect to RNA template in amounts ranging from 0.05–1.5 µg (Fig. 7Go, A and C). At 27 cycles of PCR amplification, the assay for HPRT nuclear transcripts also was linear with respect to RNA template in amounts ranging from 0.05–1.5 µg (Fig. 7Go, B and D).



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  		Figure 7. Linearity of RT-PCR assay of IGF-I (A and C) and HPRT (B and D) nuclear transcripts. Whole cellular RNA was prepared from exponentially growing cells as described in Materials and Methods. Various amounts of total RNA were reverse-tran-scribed and subjected to 20 cycles of PCR using IGF-I primers or 27 cycles of PCR using HPRT primers. Amplified fragments were detected by Southern blot analysis (see Materials and Methods). In A and B, amounts of RNA used were 0.05 µg (lanes 1–2), 0.1 µg (lanes 3–4), 0.3 µg (lanes 5–6), 0.5 µg (lanes 7–8), 1.0 µg (lanes 9–10), and 1.5 µg (lanes 11–12). C and D, Results of phosphorimage analysis of blots shown in A and B. Each point is average of duplicate assays. Data in C and D were subjected to linear re-gression analysis, imposing the requirement that the line pass through the origin (C, r = 0.9954, P < 0.0001; D, r = 0.9980, P < 0.0001).

 
To determine whether PGA2 decreased the level of IGF-I nuclear transcripts, RNA was prepared from control and PGA2-treated cells and analyzed for IGF-I and HPRT nuclear transcripts. The results (Fig. 8Go, A and B) indicated that treatment with PGA2 resulted in a 6.2-fold decrease in IGF-I mRNA and a 6.4-fold decrease in IGF-I nuclear transcripts. These results indicate that the repression by PGA2 of IGF-I gene expression is caused largely by a decrease in IGF-I nuclear transcript levels. HPRT mRNA and nuclear transcripts were not significantly affected by PGA2, indicating that the effect of PGA2 on IGF-I nuclear transcript and mRNA abundance was gene-specific (Fig. 8Go, C and D).



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  		Figure 8. Effect of PGA2 on IGF-I and HPRT mRNA and nuclear transcripts. Six dishes were treated with PGA2 for 24 h, and six dishes were treated with vehicle (control). Whole cellular RNA was prepared from each culture and subjected to Northern blot analysis for detection of IGF-I mRNA (major 8 kb species, A) and HPRT mRNA (C), and RT-PCR for detection of nuclear transcripts (B and D). Results were analyzed by densitometric scanning of autoradiograms. Each bar represents mean of results obtained with RNA preparations from six different cultures ± SE. *, Significantly different from control, P < 0.05.

 
In an attempt to identify the cis-acting element that mediates the effect of PGA2 on IGF-I gene transcription, C6 cells were transiently transfected with IGF-I/luciferase expression constructs in which luciferase expression was driven by P1 promoter fragments extending from -1711 to +328 or from -1114 to +328 relative to the beginning of exon 1. Transient transfections were also performed with the pGL3 promoterless luciferase vector, which served as a control. A CMV-ßgal expression construct was cotransfected along with each construct to control for variability in transfection efficiency. The results (Fig. 9Go) indicated that treatment with PGA2 did not repress luciferase expression in cells transfected with the IGF/luciferase constructs, and in fact, increased luciferase expression by about 2-fold. The increase in luciferase expression was nonspecific, because it was also observed with the pGL3 promoterless vector. In the experiment illustrated in Fig. 9Go, luciferase data were normalized to ß-galactosidase activity to correct for differences in transfection efficiency. The activity of ß-galactosidase was slightly increased in the cultures treated with PGA2, so that the increase in normalized luciferase activity was not caused by a decrease in ß-galactosidase activity. An alternative method for normalizing luciferase data is to normalize to total cellular protein (39, 40). The results illustrated in Fig. 9Go were very similar if the data were normalized to cellular protein: again, PGA2 treatment increased rather than decreased normalized luciferase activity (data not shown). Based on the results of the transient transfection experiments, we conclude that a negative PGA2 regulatory element is not located in the -1711 to +328 interval of the IGF-I promoter.



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  		Figure 9. Effect of PGA2 on luciferase expression in cells transiently transfected with IGF-I/luciferase expression constructs. C6 cells were transiently transfected as described in Materials and Methods. Cells were treated for 24 h with vehicle (open bars) or 20 µM PGA2 (cross-hatched bars), harvested, and assayed for luciferase and ß-galactosidase activity. Luciferase/ß-galactosidase ratio is shown for pGL3-Basic (no promoter), pGL3 containing -1114/+328 region of IGF-I P1 promoter, or pGL3 containing -1711/+328 region of IGF-I P1 promoter. *, Significantly different from control, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We present here the novel finding that PGA2 specifically represses IGF-I gene expression in C6 glioma cells. Thus, the IGF-I gene belongs to the family of genes that are repressed by cyclopentenone PGs: this family has been shown previously to include the genes encoding c-myc (12), N-myc (13), cyclin D1 (6), and cdk4 (6). At a concentration of 20 µM, PGA2 slowed the growth of the C6 cells and caused an increase in the fraction of cells in the G1 phase of the cell cycle, without any overt cytotoxic effect on the cells. This pattern of arrest in G1 without overt cytotoxicity is similar to that observed previously with PGA2-treated MCF-7 breast cancer cells (6) and human T-cell leukemia virus type I-immortalized MT-2 cells treated with PGA1 or PGJ2 (5).

IGF-I, acting via the IGF-I receptor, has been shown to protect a number of cell types from apoptosis (17, 18, 41, 42, 43). In addition, it is known that IGF-I serves as an autocrine factor for the C6 glioma cells in vivo, and that disruption of this autocrine loop results in suppression of tumorigenicity characterized by apoptosis of the tumor cell population (15, 16, 17, 18). The cyclopentenone PGs inhibit tumor cell growth in vivo and have been proposed as possible chemotherapeutic agents (1, 2, 3, 4). Inhibition of growth factor production by tumor cells could be a biologically significant action of the cyclopentenone PGs in suppression of tumorigenesis. PGA2 inhibits the growth of a variety of tumor cell types, only some of which express IGF-I. Therefore, inhibition of IGF-I production by tumor cells could be causally associated with growth arrest of only a subset of tumor cell types. The possibility that PGA2 might suppress the expression of autocrine factors other than IGF-I, or the production of growth factors by normal stromal cells, which can act in a paracrine fashion on tumor cells, remains to be explored.

In the present study, we used a simple quantitative RT-PCR assay for IGF-I nuclear transcripts. This method involves amplifying nuclear pre-mRNA sequences by RT-PCR, with primers located within or flanking an intron. The only RNA sequences that can act as templates for this amplification are the primary nuclear transcript and splicing intermediates that still contain the intron. As in previous studies in which we quantified nuclear transcripts by Northern blot analysis or RNase protection assay (29, 44, 45, 46), in the present study nuclear transcripts were used to estimate transcriptional activity. The RT-PCR assay offers the advantage of extreme sensitivity for detection of nuclear transcripts and can therefore be used in situations in which transcript levels are too low to be detected by other methods. RT-PCR has been used previously by others for quantitative measurement of nuclear transcript levels, and in experiments in which both transcription run-on assays and RT-PCR assays were performed, the results of the two assays agreed quite well (47, 48, 49, 50, 51). Thus in most experimental situations, measurements of nuclear transcript levels appear to provide a valid estimate of transcriptional activity (29, 44, 45, 46, 47, 48, 49, 50, 51).

The decrease in abundance of IGF-I mRNA in response to PGA2 was accompanied by a nearly identical decrease in the abundance of IGF-I nuclear transcripts. These changes were specific, as HPRT mRNA and nuclear transcripts were not decreased in PGA2-treated cells. These results suggest that the decreased expression of the IGF-I gene in response to PGA2 results from a specific repression of IGF-I gene transcription. Because the steady-state level of IGF-I nuclear transcripts is determined by both rate of synthesis and rate of splicing, we cannot formally rule out the possibility that IGF-I nuclear transcripts were regulated by a specific change in the rate of splicing of IGF-I nuclear transcripts. This seems unlikely, however, because a decrease in the rate of splicing would be expected to cause a decrease in IGF-I mRNA but an increase in IGF-I nuclear transcripts (52), which is not what we observed. Conversely, an increase in the rate of splicing would not be expected to caused a decrease in IGF-I mRNA.

The molecular basis for IGF-I gene repression by PGA2 remains an interesting question. The related cyclopentenone prostaglandin PGJ2 is an isomer of PGA2. Recently, a metabolite of PGJ2, 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2), has been shown to activate the transcription of genes involved in adipogenesis by binding to the {gamma}-isoform of the peroxisome proliferator-activated receptor (PPAR{gamma}) (53, 54). However, other ligands of the PPAR{gamma} receptor that are active in inducing adipogenesis do not inhibit cell proliferation, leading to speculation that the growth-inhibitory activity of 15d-PGJ2 may be mediated by another PPAR receptor isoform such as PPAR{delta} (53). PGA1 and PGA2 are also strongly growth inhibitory, and the potential interaction of these two PGs with the various PPAR receptor isoforms has not yet been characterized fully. However, it has been demonstrated recently that in transient transfection assays PGA1 and PGA2 are both capable of activating the PPAR{delta} receptor, with PGA1 being the more potent activator (55). Unlike the PPAR{alpha} and {gamma} receptors, which have limited tissue distribution, the PPAR{delta} receptor is ubiquitously expressed (56) and is therefore very likely present in the C6 glioma cells. Although activation of PPAR receptors would normally be associated with the activation of gene transcription, a number of other nuclear receptors are known to either activate or repress gene transcription, depending on the context (57, 58, 59). Alternatively, PGA2 activates a cascade of cellular events culminating in decreased phosphorylation of Rb, which is predicted to have a repressive effect on genes such as c-myc that utilize E2F for activation of transcription (6, 60). Based on the results of the transient transfection assays, the cis-acting element that mediates the repressive effect of PGA2 on IGF-I gene transcription appears to be located outside the -1711 to +328 P1 promoter interval. The location of this element remains to be determined, as does the molecular mechanism for negative regulation of gene expression by PGA2.

Previous studies have demonstrated that PGA2 represses cyclin D1 gene expression in MCF-7 breast cancer cells, and increases the expression of Waf1 in some but not all cell types (6, 11). Both of these two effects of PGA2 were observed in the C6 glioma cells. Cyclin D1 stimulates and Waf1 inhibits the G1 cyclin-dependent protein kinase cdk4 (61). Thus, the concerted induction of Waf1 and repression of cyclin D1 could be causally associated with growth arrest in the G1 phase of the cell cycle. Because IGF-I partially reversed the growth-inhibitory effect of PGA2, IGF-I is at least partially dominant over PGA2 at the doses used in this study. This result is consistent with a cause-effect relationship between the repression by PGA2 of IGF-I gene expression and cell cycle arrest, although it is also consistent with a model in which PGA2 and IGF-I regulate the same endpoint (i.e. progression through the G1 phase of the cell cycle) independently and in opposite directions. It has been shown previously that IGF-I increases cyclin D1 expression (62). The decrease in cyclin D1 mRNA occurred after the decrease in IGF-I mRNA; thus, it is conceivable that decreased IGF-I expression contributed to the decrease in cyclin D1 mRNA in the C6 cells. In contrast, the induction of Waf1 mRNA occurred at least as early as the repression of IGF-I mRNA. Therefore, it is very unlikely that the decreased expression of IGF-I is causally associated with Waf1 induction. Further knowledge of the molecular pathway(s) by which PGA2 regulates gene expression should help clarify which of the effects of PGA2 on gene expression are related to each other in a cause-effect manner and which occur independently.


   Acknowledgments
 
We thank G. Robertson for generous assistance with the FACScan analysis.


   Footnotes
 
1 This research was supported by NIH Grants DK-39739 (to D.S.S.) and DK-37449 (to P.R.). Back

2 Present address: Division of Molecular Medicine, Department of Medicine, Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97201. Back

Received August 1, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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