Bui T, Kuo C, Rotwein P, Straus D S
Biomedical Sciences Division, University of California, Riverside 92521-0121, USA.
Endocrinology. 1997 Mar;138(3):985-93. doi: 10.1210/endo.138.3.4980.
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.
环戊烯酮类前列腺素（PGA和PGJ系列）通过目前尚不清楚的机制在体外抑制肿瘤细胞增殖，在体内抑制肿瘤发生。C6大鼠胶质瘤细胞系合成并分泌胰岛素样生长因子-I（IGF-I），据信其作为这些细胞的自分泌因子发挥作用。PGA2抑制C6细胞的增殖，并使细胞周期G1期的细胞比例增加。PGA2对细胞增殖的抑制伴随着IGF-I信使核糖核酸（mRNA）丰度的降低。IGF-I基因表达的这种调节是特异性的，因为次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（HPRT）和泛素mRNA的丰度不受PGA2的显著影响。在低至10微摩尔的PGA2浓度下即可观察到IGF-I基因表达的抑制，在用PGA2处理C6细胞后4小时内就很明显。除了特异性调节IGF-I基因的表达外，PGA2还降低细胞周期蛋白D1 mRNA的丰度并增加Waf1 mRNA的丰度。通过共同添加IGF-I可部分逆转PGA2对细胞增殖的抑制，这表明IGF-I作用部分优于PGA2作用。为了研究PGA2调节IGF-I基因表达的分子基础，我们开发了一种用于检测IGF-I核转录本的灵敏逆转录聚合酶链反应（RT-PCR）检测方法。还开发了一种类似的检测方法用于定量HPRT转录本，其用作对照。用20微摩尔PGA2处理C6细胞导致IGF-I mRNA和IGF-I核转录本减少约6倍。相比之下，HPRT mRNA和核转录本水平不受PGA2的显著影响。这些结果表明，响应PGA2而发生的IGF-I mRNA丰度降低主要是由IGF-I核转录本水平降低引起的。为了鉴定介导PGA2对IGF-I转录作用的顺式作用元件，用IGF-I/荧光素酶表达构建体瞬时转染C6细胞，其中荧光素酶转录由相对于外显子1起始位置从-1711延伸至-328或从-1114延伸至+328的IGF-I P1启动子片段驱动。在这些瞬时转染实验中用PGA2处理细胞并没有降低荧光素酶活性。这些结果表明，对PGA2反应所需的顺式作用调节元件位于-1711至+328启动子区间之外。
