Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA.
J Steroid Biochem Mol Biol. 1992 Dec;43(8):951-60. doi: 10.1016/0960-0760(92)90323-B.
Bovine adrenocortical cells undergo a process in which expression of steroid hydroxylases is lost progressively as a function of population doubling level (PDL) in culture. Each cytochrome P450 shows a characteristic rate of loss of expression as a function of PDL (in order of rates of loss: CYP11B >CYP21 >CYP17 >CYP11A). CYP11B and CYP21 require insulin-like growth factor I as well as cyclic AMP; these are the only factors required for induction in the primary culture. Middle- and later passage cells do not express CYP11B and CYP21 under the same conditions, but will do so when cells are grown in extracellular matrix Matrigel. In late-passage cells neither CYP17, CYP21, nor CYP11B are expressed, even in the presence of Matrigel; only CYP11A is expressed in late-passage cultures. When the different environmental factors required for induction of CYP11B and CYP21 are taken into account, induction of these genes disappears with the same kinetics as previously shown for CYP17 as a function of PDL. The primary cause of the loss of expression of these genes is likely to be a phenotypic switching event similar to that previously demonstrated for CYP17 by in situ hybridization. The mechanism of phenotypic switching is unknown. However, one HpaII site at -2.3 kb of CYP17 was methylated in the bovine adrenal cortex in vivo but showed rapid and complete demethylation when adrenocortical cells were placed in culture. This indicates a unique, reproducible, environmentally determined change in methylation, with as yet undetermined consequences. However, data from reporter constructs suggest that phenotypic switching does not result from a simple loss of regulatory factors that act within 2.5 kb of the promoter. Previous data suggested that SV40 T antigen may affect phenotypic switching, and thus that SV40 may be useful for the derivation of functional adrenocortical cell lines. Adaptation of methods previously used for bovine cells to human adrenocortical cells to produce SV40 T antigen-transfected clones yielded data indicating preservation of essential aspects of the human adrenocortical cell differentiated phenotype.
牛肾上腺皮质细胞在培养过程中经历一个过程，其中随着倍增水平（PDL）的增加，类固醇羟化酶的表达逐渐丧失。每种细胞色素 P450 都表现出与 PDL 相关的表达丧失特征（按丧失速率顺序：CYP11B>CYP21>CYP17>CYP11A）。CYP11B 和 CYP21 需要胰岛素样生长因子 I 和环 AMP；这些是初级培养物中诱导所必需的唯一因素。在相同条件下，中晚期传代细胞不会表达 CYP11B 和 CYP21，但当细胞在细胞外基质 Matrigel 中生长时，它们会表达。在晚期传代细胞中，即使存在 Matrigel，也不会表达 CYP17、CYP21 或 CYP11B；只有 CYP11A 在晚期传代培养物中表达。当考虑到诱导 CYP11B 和 CYP21 所需的不同环境因素时，这些基因的诱导与之前所示的 CYP17 随 PDL 变化的动力学一致。这些基因表达丧失的主要原因可能是表型转换事件，类似于之前通过原位杂交证明的 CYP17。表型转换的机制尚不清楚。然而，CYP17 基因的 -2.3kb 处有一个 HpaII 位点在体内的牛肾上腺皮质中被甲基化，但当肾上腺皮质细胞被置于培养中时，该位点迅速且完全去甲基化。这表明存在一种独特的、可重复的、环境决定的甲基化变化，其后果尚未确定。然而，来自报告基因构建体的数据表明，表型转换不是由于作用于启动子 2.5kb 内的调节因子的简单丢失。先前的数据表明，SV40 T 抗原可能影响表型转换，因此 SV40 可能对衍生功能性肾上腺皮质细胞系有用。将先前用于牛细胞的方法适应于人肾上腺皮质细胞，以产生 SV40 T 抗原转染的克隆，获得的数据表明，人肾上腺皮质细胞分化表型的重要方面得到了保留。
