Clonal variation in response to adrenocorticotropin in cultured bovine adrenocortical cells: Relationship to senescence

Abstract

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.

Loss of expression of a differentiated function gene, steroid 17 alpha-hydroxylase, as adrenocortical cells senescence in culture.

“…In human and bovine adrenocortical cells in culture, expression of 17a-hydroxylase is dependent on the presence of corticotropin or other stimulators of cyclic AMP production; enzyme activity and mRNA levels are undetectably low in the long-term absence of the inducer (3,4,(7)(8)(9)(10)(18)(19)(20). Because stimulation of intracellular cyclic AMP by the physiological stimulator of the adrenocortical cell corticotropin declines as a function of senescence in bovine adrenocortical cells (13,21), we chose to use cholera toxin as a means for producing a sustained increase in intracellular cyclic AMP in these studies. Cholera toxin has been shown (19,(21)(22)(23) to be a very potent stimulus for steroidogenesis and enzyme induction in this culture system.…”

“…Cumulative population doublings were calculated as the log2 of the cumulative increase in the population (13). Cells were cloned using the same medium with the addition of 2% (vol/vol) UltroSer G (Reactifs IBF, Villeneuve-laGarenne, France) (13,14).…”

“…Cells were prepared from bovine adrenal cortex tissue as described (11) and were grown on culture dishes coated with fibronectin (12) in a 1:1 (vol/vol) mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium with 10% (vol/vol) fetal bovine serum and partially purified brain fibroblast growth factor (bFGF) at 100 ng/ml in an atmosphere of 5% 02/85% N2/10% CO2. For long-term growth and estimation ofreplicative potential, cells were grown under these conditions and passed repeatedly using a 1:5 split ratio using Pronase E (Sigma) to remove the cells (13). Cumulative population doublings were calculated as the log2 of the cumulative increase in the population (13).…”

“…For long-term growth and estimation ofreplicative potential, cells were grown under these conditions and passed repeatedly using a 1:5 split ratio using Pronase E (Sigma) to remove the cells (13). Cumulative population doublings were calculated as the log2 of the cumulative increase in the population (13). Cells were cloned using the same medium with the addition of 2% (vol/vol) UltroSer G (Reactifs IBF, Villeneuve-laGarenne, France) (13,14).…”

“…Cumulative population doublings were calculated as the log2 of the cumulative increase in the population (13). Cells were cloned using the same medium with the addition of 2% (vol/vol) UltroSer G (Reactifs IBF, Villeneuve-laGarenne, France) (13,14).Induction and Measurement of 17a-Hydroxylase. For induction of 17a-hydroxylase, cultures were changed to serumfree medium containing DMEM/F-12, 1:1 (vol/vol), bovine serum albumin at 50 ,ug/ml, 20 nM insulin, 100 ,iM ascorbic acid, 20 nM selenite, and 1 ,uM a-tocopherol (12) …”