Wei L L, Krett N L, Francis M D, Gordon D F, Wood W M, O'Malley B W, Horwitz K B
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
Mol Endocrinol. 1988 Jan;2(1):62-72. doi: 10.1210/mend-2-1-62.
We have used AB-52, a monoclonal antibody which recognizes both the A (94,000 daltons) and B (120,000 daltons) proteins of human progesterone receptors (hPR), and hPR-50, a PR complementary DNA probe isolated from a T47D-pcD library, to study the structure and hormonal regulation of the hPR mRNAs and proteins in human breast cancer cells. RNA blot hybridization analysis of poly(A+) RNA shows that T47DCO, an estrogen resistant human breast tumor cell line in which PR are constitutively expressed, contain at least six PR mRNAs ranging in size from 2.5 to 11.4 kilobases. All six are mature cytoplasmic messages that are also present in normal human endometrium and in PR-positive MCF-7 breast cancer cells, but not in PR-negative cells. Using hPR-50 RNA synthesized in vitro as a 1.3 kilobase standard, we calculate that MCF-7 cells contain approximately 16 message molecules per cell which are increased to approximately 45 by estradiol treatment; T47DCO cells contain approximately 90 message molecules per cell constitutively expressed. Treatment of T47DCO cells with progesterone leads to down-regulation of immunoreactive A- and B-receptors in the first 8-12 h, followed by their replenishment during the next 48 h. In parallel, hPR message levels initially decrease and then return to pretreatment levels. The synthetic progestin R5020 chronically down-regulates A- and B-receptors; the proteins are profoundly suppressed for at least 48 h, while PR mRNAs fall to less than 15% of control. However, with both hormones, parallel changes in protein and message levels are observed, suggesting that progestational agonists autoregulate the levels of their own receptors by inhibiting transcription of the PR gene. Antagonists appear to have different effects. With the antiprogestin RU 486 there is discordance between hPR protein and message levels which may be due to an ineffective inhibitory interaction between the antagonist-occupied receptors and PR genes, thereby disrupting the negative feedback loop.
我们使用了AB - 52（一种可识别人类孕酮受体（hPR）的A蛋白（94,000道尔顿）和B蛋白（120,000道尔顿）的单克隆抗体）以及hPR - 50（从T47D - pcD文库中分离出的PR互补DNA探针）来研究人乳腺癌细胞中hPR mRNA和蛋白的结构及激素调节。对聚腺苷酸加尾（poly(A+)）RNA的RNA印迹杂交分析表明，T47DCO（一种雌激素抗性的人乳腺肿瘤细胞系，其中PR持续表达）含有至少六种大小在2.5至11.4千碱基之间的PR mRNA。所有这六种都是成熟的细胞质信使RNA，它们也存在于正常的人子宫内膜和PR阳性的MCF - 7乳腺癌细胞中，但不存在于PR阴性细胞中。以体外合成的1.3千碱基的hPR - 50 RNA作为标准，我们计算出MCF - 7细胞每个细胞含有约16个信使RNA分子，经雌二醇处理后增加到约45个；T47DCO细胞每个细胞持续表达约90个信使RNA分子。用孕酮处理T47DCO细胞会导致免疫反应性A受体和B受体在前8 - 12小时下调，随后在接下来的48小时内得到补充。同时，hPR信使RNA水平最初下降，然后恢复到预处理水平。合成孕激素R5020会长期下调A受体和B受体；这些蛋白至少48小时受到深度抑制，而PR mRNA降至对照的不到15%。然而，对于这两种激素，都观察到蛋白和信使RNA水平的平行变化，这表明孕激素激动剂通过抑制PR基因的转录来自动调节其自身受体的水平。拮抗剂似乎有不同的作用。使用抗孕激素RU 486时，hPR蛋白和信使RNA水平之间存在不一致，这可能是由于拮抗剂占据的受体与PR基因之间的抑制性相互作用无效，从而破坏了负反馈回路。
