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Cellular senescence involves stochastic processes causing loss of expression of differentiated function genes:
1Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912.
Abstract:
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.
Insights
Senescence in adrenocortical cells limits steroid 17 alpha-hydroxylase expression. Simian virus 40 (SV40) transfection can immortalize cells, dissociating growth from differentiated gene expression, preserving function.
Area of Science:
- Cell Biology
- Molecular Endocrinology
- Genetics
Background:
- Adrenocortical cell cultures exhibit stochastic loss of steroid 17 alpha-hydroxylase expression.
- A correlation exists between cellular replicative potential and 17 alpha-hydroxylase expression.
- Senescence limits the replicative capacity of adrenocortical cells.
Purpose of the Study:
- To investigate the relationship between cellular senescence and steroid 17 alpha-hydroxylase expression.
- To determine if simian virus 40 (SV40) transfection can overcome senescence-associated gene expression changes.
- To dissociate the effects of SV40 T antigen on cell growth versus differentiated gene function.
Main Methods:
- Transfection of senescent adrenocortical cells with cloned SV40 virus.
- Selection of SV40 T antigen-expressing cell lines using G418.
- Assessment of steroid 17 alpha-hydroxylase mRNA and activity via cyclic AMP induction.
- Measurement of cholesterol side-chain cleavage mRNA and activity.
- Recloning experiments to assess long-term gene expression stability.
Main Results:
- SV40 transfection of senescent cells led to immortalization and increased growth rate.
- Early passage cells transfected with SV40 showed T antigen expression but lost 17 alpha-hydroxylase induction.
- Cells transfected with pSV3neo (SV40 early region and neo) retained 17 alpha-hydroxylase inducibility.
- High levels of 17 alpha-hydroxylase expression were maintained through multiple recloning events in transfected cells.
- SV40 T antigen selectively impacted cell growth, not the pre-existing differentiated gene expression state.
Conclusions:
- SV40 transfection can overcome replicative senescence in adrenocortical cells.
- Cellular immortalization by SV40 does not necessarily abrogate differentiated gene function.
- SV40 T antigen dissociates growth regulation from the maintenance of differentiated gene expression.
- This approach allows for the study of differentiated gene function in long-term cell cultures independent of senescence.