Ryan R F, Hancock J P, McDonald J J, Hornsby P J
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912.
Exp Cell Res. 1989 Jan;180(1):36-48. doi: 10.1016/0014-4827(89)90210-3.
When grown for long periods in culture, bovine adrenocortical cells lose the expression of a differentiated function gene, steroid 17 alpha-hydroxylase. Previously, we documented a decline in 17 alpha-hydroxylase mRNA with increasing culture passage level after induction with cyclic AMP (P. J. Hornsby et al., 1987, Proc. Natl. Acad. Sci. USA 84, 1580). We used in situ hybridization to investigate the loss of expression of this gene during cellular senescence at an individual cell level. In primary cultures, cells were uniformly positive for hybridization with cDNA for 17 alpha-hydroxylase after cyclic AMP induction. After two passages, cultures comprised a mixture of hybridizing and nonhybridizing cells. Cells appeared either to hybridize at a level comparable to that in primary cultures or to be nonhybridizing. When in situ hybridization was combined with immunofluorescence, cells positive for immunofluorescence were also positive for hybridization. Senescing mass cultures showed decreasing numbers of positive cells, and after 30 passages cultures comprised entirely nonhybridizing cells. Thus, the previously observed decline in overall 17 alpha-hydroxylase mRNA levels results from a decline in the fraction of expressing cells in the culture, and the rate of loss of expressing cells is in agreement with the rate of loss of total 17 alpha-hydroxylase mRNA. Primary clones, even when isolated at an early stage of clonal expansion, had mixtures of subclones of hybridizing and nonhybridizing cells. On recloning, hybridizing subclones usually produced uniformly nonhybridizing sub-subclones. Some subclones within primary clones had a morphology associated with replicative senescence (flattened cells with sparse intercellular contacts), yet had high numbers of hybridizing cells. We conclude that, in both mass and clonal populations, cells initially expressing 17 alpha-hydroxylase rapidly give rise to clones of nonexpressing cells. Such cells are continually derived by a stochastic process from cells originally expressing the gene.
在培养物中长时间生长时，牛肾上腺皮质细胞会失去一种分化功能基因——类固醇17α-羟化酶的表达。此前，我们记录了用环磷酸腺苷诱导后，随着培养传代水平的增加，17α-羟化酶mRNA水平下降（P. J. 霍恩斯比等人，1987年，《美国国家科学院院刊》84卷，第1580页）。我们使用原位杂交技术在单个细胞水平上研究细胞衰老过程中该基因表达的丧失情况。在原代培养中，用环磷酸腺苷诱导后，细胞与17α-羟化酶的cDNA杂交均呈阳性。传代两次后，培养物中包含杂交细胞和非杂交细胞的混合物。细胞要么以与原代培养相当的水平杂交，要么不杂交。当原位杂交与免疫荧光结合时，免疫荧光阳性的细胞杂交也呈阳性。衰老的大规模培养物中阳性细胞数量减少，传代30次后，培养物完全由非杂交细胞组成。因此，之前观察到的总体17α-羟化酶mRNA水平下降是由于培养物中表达细胞比例的下降，且表达细胞的丧失速率与总17α-羟化酶mRNA的丧失速率一致。原代克隆，即使在克隆扩增的早期阶段分离，也有杂交和非杂交细胞亚克隆的混合物。再次克隆时，杂交亚克隆通常产生均匀的非杂交亚亚克隆。原代克隆中的一些亚克隆具有与复制性衰老相关的形态（细胞扁平，细胞间接触稀疏），但杂交细胞数量较多。我们得出结论，在大规模和克隆群体中，最初表达17α-羟化酶的细胞会迅速产生不表达细胞的克隆。此类细胞不断地通过随机过程从最初表达该基因的细胞中产生。
