Staels B, Hum D W, Miller W L
Department of Pediatrics, University of California, San Francisco 94143-0978.
Mol Endocrinol. 1993 Mar;7(3):423-33. doi: 10.1210/mend.7.3.8387159.
NCI-H295 is a recently described human adrenocortical carcinoma cell line that makes a variety of steroid hormones. We sought to determine if steroidogenesis in these cells employs the same enzymes as those used in normal adrenal steroidogenesis, and if the genes encoding those enzymes exhibit characteristic responsiveness to activators of the protein kinase-A and -C pathways of intracellular second messengers. Northern blots show that NCI-H295 cells contain abundant mRNAs for three key steroidogenic enzymes, cytochrome P450scc, cytochrome P450c17, and cytochrome P450c21. These mRNAs accumulated in a time- and dose-dependent fashion in response to 8-bromo-cAMP (8Br-cAMP), forskolin, cholera toxin, and 3-isobutyl-1-methylxanthine, all activators of the protein kinase-A pathway. Nuclear run-on assays and actinomycin-D transcriptional inhibition experiments show that cAMP regulates the expression of all three genes primarily at the transcriptional level. Inhibition of protein synthesis with cycloheximide did not prevent the cAMP-induced accumulation of P450scc or P450c17 mRNAs, but did inhibit accumulation of P450c21 mRNA, suggesting that cAMP is acting through a mechanism dependent on protein synthesis to promote accumulation of P450c21 mRNA. Stimulation of the protein kinase-C pathway with phorbol ester decreased P450scc and P450c17 mRNAs, but stimulated the accumulation of P450c21 mRNA. RNase protection experiments, Northern blot hybridizations, and reverse transcription-polymerase chain reaction show that NCI-H295 cells express both the 11 beta-hydroxylase (P450c11 beta) encoded by the P450c11B1 gene and the aldosterone synthetase (P450c11AS) encoded by the P450c11B2 gene. 8Br-cAMP increased the abundance of both of these mRNAs with similar kinetics, with maximal accumulation of both after about 24 h. NCI-H295 cells also contain the mRNAs for aromatase and insulin-like growth factor-II. 8Br-cAMP increased the abundance of aromatase mRNA and decreased the abundance of IGF-II mRNA. These studies show that NCI-H295 cells express most of the enzymes needed for human adrenal steroidogenesis, and that the genes encoding these enzymes respond to stimulation of second messenger pathways in a manner similar to that of human adrenals. NCI-H295 cells appear to be a good model for studying the molecular regulation of human adrenal steroidogenesis.
NCI-H295是一种最近被描述的能产生多种甾体激素的人肾上腺皮质癌细胞系。我们试图确定这些细胞中的甾体激素生成是否使用与正常肾上腺甾体激素生成相同的酶，以及编码这些酶的基因是否对细胞内第二信使的蛋白激酶-A和-C途径的激活剂表现出特征性反应。Northern印迹显示，NCI-H295细胞含有三种关键甾体激素生成酶——细胞色素P450scc、细胞色素P450c17和细胞色素P450c21的丰富mRNA。这些mRNA在响应8-溴-cAMP（8Br-cAMP）、福斯可林、霍乱毒素和3-异丁基-1-甲基黄嘌呤（均为蛋白激酶-A途径的激活剂）时呈时间和剂量依赖性积累。核转录分析和放线菌素-D转录抑制实验表明，cAMP主要在转录水平调节这三种基因的表达。用环己酰亚胺抑制蛋白质合成并不能阻止cAMP诱导的P450scc或P450c17 mRNA的积累，但确实抑制了P450c21 mRNA的积累，这表明cAMP通过一种依赖蛋白质合成的机制促进P450c21 mRNA的积累。用佛波酯刺激蛋白激酶-C途径可降低P450scc和P450c17 mRNA，但刺激P450c21 mRNA的积累。核糖核酸酶保护实验、Northern印迹杂交和逆转录-聚合酶链反应表明，NCI-H295细胞表达由P450c11B1基因编码的11β-羟化酶（P450c11β）和由P450c11B2基因编码的醛固酮合成酶（P450c11AS）。8Br-cAMP以相似的动力学增加了这两种mRNA的丰度，两者在约24小时后均达到最大积累。NCI-H295细胞还含有芳香化酶和胰岛素样生长因子-II的mRNA。8Br-cAMP增加了芳香化酶mRNA的丰度并降低了IGF-II mRNA的丰度。这些研究表明，NCI-H295细胞表达人类肾上腺甾体激素生成所需的大多数酶，并且编码这些酶的基因对第二信使途径刺激的反应方式与人类肾上腺相似。NCI-H295细胞似乎是研究人类肾上腺甾体激素生成分子调节的良好模型。
