Alexander I E, Clarke C L, Shine J, Sutherland R L
Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New South Wales, Australia.
Mol Endocrinol. 1989 Sep;3(9):1377-86. doi: 10.1210/mend-3-9-1377.
The present study was designed to investigate whether inhibition of progesterone receptor (PR) gene transcription and/or regulation of PR mRNA half-life were involved in the progestin-mediated decrease of PR in T-47D human breast cancer cells. Cells were treated with the progestin ORG 2058 and PR mRNA measured by Northern blot analysis of total RNA. A major PR mRNA around 13.5 kilobases and minor species around the 28S ribosomal RNA subunit were decreased upon ORG 2058 treatment. The decrease was not detectable until 2-3 h after treatment and was the same at all ORG 2058 concentrations (1-100 nM) tested. The decrease in PR mRNA was unaffected by actinomycin D in the first 3 h but was inhibited thereafter. There was a partial recovery of PR mRNA levels 24 h after ORG 2058 exposure. Immunoblot analysis showed that immunoreactive PR decreased in parallel with PR mRNA. The rate of protein loss in the first 12 h after progestin treatment was related to the ORG 2058 concentration used. Nuclear run-on experiments showed that ORG 2058 caused a decrease of up to 70% in the transcription rate of the PR gene. The half-life of PR mRNA was shown to be 2-2.5 h by [3H]uridine incorporation, which was much shorter than estimates obtained using actinomycin D, and was unaffected by ORG 2058. In summary, these data have shown that the mechanism by which progestins decrease the concentration of PR includes inhibition of transcription of the PR gene.
本研究旨在调查孕酮受体（PR）基因转录的抑制和/或PR mRNA半衰期的调节是否参与了孕激素介导的T-47D人乳腺癌细胞中PR的减少。用孕激素ORG 2058处理细胞，并通过对总RNA进行Northern印迹分析来测量PR mRNA。ORG 2058处理后，约13.5千碱基的主要PR mRNA和28S核糖体RNA亚基周围的次要种类减少。直到处理后2-3小时才检测到这种减少，并且在所有测试的ORG 2058浓度（1-100 nM）下都是相同的。PR mRNA的减少在最初3小时内不受放线菌素D的影响，但此后受到抑制。ORG 2058暴露24小时后，PR mRNA水平有部分恢复。免疫印迹分析表明，免疫反应性PR与PR mRNA平行下降。孕激素处理后最初12小时内蛋白质损失的速率与所用的ORG 2058浓度有关。核转录实验表明，ORG 2058导致PR基因转录速率下降高达70%。通过[3H]尿苷掺入显示PR mRNA的半衰期为2-2.5小时，这比使用放线菌素D获得的估计值短得多，并且不受ORG 2058的影响。总之，这些数据表明孕激素降低PR浓度的机制包括抑制PR基因的转录。
