Prostaglandin A(2) specifically represses insulin-like growth factor-I gene expression in C6 rat glioma cells

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. PGA(2) inhibits the proliferation of the C6 cells and causes an increase in the fraction of cells in the G(1) phase of the cell cycle. The inhibition of cell proliferation by PGA(2) 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 PGA(2). The repression of IGF-I gene expression is observed at PGA(2) concentrations as low as 10 mu M and is evident within 4 h after treat ment of the C6 cells with PGA(2). In addition to specifically regulating the expression of the IGF-I gene, PGA(2) also decreases the abundance of cyclin D1 mRNA and increases the abundance of Waf1 mRNA. The inhibition of cell proliferation by PGA, is partially reversed by coaddition of IGF-I, indicating partial dominance of IGF-I action over PGA(2) action. To investigate the molecular basis for the regulation of IGF-I gene expression by PGA(2), 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 oi the C6 cells with 20 mu M PGA, 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 PGA(2). These results indicate that the decrease in IGF-I mRNA abundance that occurs in response to PGA, is caused largely by a decrease in IGF-I nuclear transcript levels. To identify the cis-acting element that mediates the effect of PGA(2) 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 PGA(2) in these transient transfection assays did not decrease luciferase activity. These results suggest that the cis-acting regulatory element required for the response to PGA(2) is located outside the -1711 to +328 promoter interval.