Prostaglandin A2 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. 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 microM 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 microM 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.
Bui
T
T
Biomedical Sciences Division, University of California, Riverside 92521-0121, USA.
Kuo
C
C
Rotwein
P
P
Straus
D S
DS
D000818:Animals; D015152:Blotting, Northern; D002455:Cell Division / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000187:drug effects*; D005910:Glioma / Q000235:genetics* / Q000473:pathology; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011454:Prostaglandins A / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D011454:Prostaglandins A; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D008156:Luciferases; C100008:prostaglandin A2