Cheng C Y, Ryan R F, Vo T P, Hornsby P J
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912.
Exp Cell Res. 1989 Jan;180(1):49-62. doi: 10.1016/0014-4827(89)90211-5.
In the accompanying work we demonstrated that the decline in expression of steroid 17 alpha-hydroxylase in mass cultures and clones of adrenocortical cells is the result of a stochastic switching process which yields mixtures of expressing and nonexpressing cells. There is an apparent positive correlation between the replicative potential of adrenocortical cell cultures and the number of cells in the culture that can express 17 alpha-hydroxylase. We investigated this by extending the cells' replicative potential by transfecting them with cloned SV40 virus. Cells from a senescent subclone, with very limited remaining replicative potential, were transfected. The cell population showed a progressive increase in growth rate and gave rise to a line of cells that expressed T antigen and which was apparently immortalized. Induction of mRNA for 17 alpha-hydroxylase by cyclic AMP was absent in this line of cells, as it was in the senescent cells prior to transfection. The cells remained responsive to gene induction by cyclic AMP as evidenced by increases in mRNA and activity for cholesterol side-chain cleavage. The absence of 17 alpha-hydroxylase expression in this line was not the result of interference by SV40 T antigen. When early passage cells were transfected with pSV3neo, which contains the early region of SV40 and neo, and were selected with G418, SV40 T antigen-expressing lines were derived which showed high levels of expression of 17 alpha-hydroxylase after induction with cyclic AMP. These cells maintained high levels of expression of 17 alpha-hydroxylase through four successive recloning events, over a period of replication much longer than that achievable by nontransfected cells. Thus, transfection by SV40 can be used to dissociate effects of senescence on growth and differentiated gene expression. T antigen expression selectively affects growth, but preserves the state of expression of a differentiated function gene as it was prior to transfection.
在相关研究中，我们证明了在肾上腺皮质细胞的大规模培养物和克隆中，类固醇17α-羟化酶表达的下降是一个随机转换过程的结果，该过程产生了表达和不表达细胞的混合物。肾上腺皮质细胞培养物的复制潜能与培养物中能够表达17α-羟化酶的细胞数量之间存在明显的正相关。我们通过用克隆的SV40病毒转染细胞来扩展其复制潜能，从而对此进行了研究。转染了来自衰老亚克隆、剩余复制潜能非常有限的细胞。细胞群体的生长速率逐渐增加，并产生了一系列表达T抗原且明显永生化的细胞系。在该细胞系中以及转染前的衰老细胞中，环磷酸腺苷（cAMP）均不能诱导17α-羟化酶的mRNA表达。细胞对cAMP诱导基因的反应仍然存在，胆固醇侧链裂解的mRNA和活性增加证明了这一点。该细胞系中17α-羟化酶表达的缺失不是SV40 T抗原干扰的结果。当用含有SV40早期区域和新霉素抗性基因（neo）的pSV3neo转染早期传代细胞并用G418进行筛选时，获得了表达SV40 T抗原的细胞系，这些细胞系在用cAMP诱导后显示出高水平的17α-羟化酶表达。这些细胞在四个连续的亚克隆过程中都保持了17α-羟化酶的高水平表达，其复制时间远远超过未转染细胞所能达到的时间。因此，SV40转染可用于分离衰老对生长和分化基因表达的影响。T抗原表达选择性地影响生长，但保留了转染前分化功能基因的表达状态。
