Hornsby P J, Aldern K A, Harris S E
J Cell Physiol. 1986 Dec;129(3):395-402. doi: 10.1002/jcp.1041290319.
During cellular senescence, non-clonal cultures of bovine adrenocortical cells show a continuous decline in the rate of production of cyclic AMP (cAMP) stimulated by adrenocorticotropin (ACTH), without changes in the rate of forskolin- or prostaglandin E1-stimulated cAMP production. We investigated the possible mechanisms for loss of response to ACTH by examining the properties of clones of bovine adrenocortical cells. ACTH-stimulated cAMP production rates were measured in clones immediately after isolation, during long-term growth following isolation, and after subcloning. ACTH-stimulated rates were compared with cAMP production in response to forskolin, which acts directly on the catalytic subunit of adenylate cyclase. The results show that cloning is not necessarily associated with a loss of response to ACTH, but that clones with high ACTH response can give rise to subclones with low response. Clones of adrenocortical cells, at the same approximate population doubling level (PDL), showed ACTH response levels that ranged from 12 to 135 pmol cAMP/10(6) cells/min, whereas mass cultures at this PDL showed approximately 50 pmol/10(6) cells/min. Forskolin-stimulated cAMP production rates in clones varied only over the range of 59-119 pmol/10(6) cells/min and showed no correlation with the ACTH-stimulated rates. All clones were adrenocortical cells, as shown by mitogenic response to angiotensin II and cAMP-inducible 17 alpha-hydroxylase activity. The replicative potential of clones varied widely, and there was no apparent correlation between ACTH response levels and growth potential. The level of ACTH response in each clone was stable during proliferation through at least 25 PD beyond the stage at which the clone was isolated. When clones were subcloned, a clone with a high ACTH response level produced sister subclones that had ACTH response levels ranging from 3% of that of the parent clone to a level slightly greater than that of the parent clone. The growth potential of sister subclones varied widely, as for the parent clones, and there was no obvious correlation between growth potential and ACTH response. Two subclones were cloned; in sub-subclones, levels of ACTH response were again different from each other and also from the parent subclone; in one case, the level of ACTH response was approximately eight-fold higher than that of the parent subclone. These experiments show that clonal variation in the extent of expression of a differentiated property may occur in a normal differentiated cell in culture. The loss of ACTH response and the loss of proliferative potential appear to be independent stochastic events.
在细胞衰老过程中，牛肾上腺皮质细胞的非克隆培养物显示，促肾上腺皮质激素（ACTH）刺激的环磷酸腺苷（cAMP）生成速率持续下降，而福斯高林或前列腺素E1刺激的cAMP生成速率没有变化。我们通过研究牛肾上腺皮质细胞克隆的特性，来探究对ACTH反应丧失的可能机制。在克隆分离后立即、分离后的长期生长过程中以及亚克隆后，测量ACTH刺激的cAMP生成速率。将ACTH刺激的速率与对直接作用于腺苷酸环化酶催化亚基的福斯高林的cAMP生成反应进行比较。结果表明，克隆不一定与对ACTH的反应丧失相关，但具有高ACTH反应的克隆可能产生低反应的亚克隆。肾上腺皮质细胞克隆在相同的大致群体倍增水平（PDL）下，ACTH反应水平范围为12至135 pmol cAMP/10⁶细胞/分钟，而处于该PDL的大规模培养物显示约为50 pmol/10⁶细胞/分钟。克隆中福斯高林刺激的cAMP生成速率仅在59 - 119 pmol/10⁶细胞/分钟范围内变化，且与ACTH刺激的速率无相关性。所有克隆均为肾上腺皮质细胞，这通过对血管紧张素II的促有丝分裂反应和cAMP诱导的17α - 羟化酶活性得以证明。克隆的增殖潜能差异很大，ACTH反应水平与生长潜能之间没有明显相关性。每个克隆在从分离阶段开始的至少25次群体倍增的增殖过程中，ACTH反应水平保持稳定。当克隆进行亚克隆时，一个具有高ACTH反应水平的克隆产生的姐妹亚克隆，其ACTH反应水平范围从亲本克隆的3%到略高于亲本克隆的水平。姐妹亚克隆的生长潜能差异很大，与亲本克隆一样，生长潜能与ACTH反应之间没有明显相关性。对两个亚克隆进行了克隆；在亚亚克隆中，ACTH反应水平再次彼此不同，也与亲本亚克隆不同；在一个案例中，ACTH反应水平比亲本亚克隆高约八倍。这些实验表明，在培养的正常分化细胞中，分化特性表达程度的克隆变异可能会发生。ACTH反应的丧失和增殖潜能的丧失似乎是独立的随机事件。
