The aim of this proposal is to provide insight into cellular aging mechanisms using a model system of bovine and human adrenocortical cells in culture. A hormonally-inducible, -tissue- specific gene, steroid 17a-hydroxylase, will be used as an indicator for general changes in genome in cellular senescence. The mechanism of loss of induction of 17a-hydroxylase in senescence in adrenocortical cells will be investigated. An in vitro transcript elongation assay will be used to measure synthesis rates for 17a-hydroxylase in nuclei from early and late passage cells. The expression of transfected 17a-hydroxylase cDNA in old cells will be examined to investigate the possibility of increased degradation of 17a-hydroxylase mRNA in old cells. Experiments are proposed to identify and characterize a repressor or transcription factor for the 17a-hydroxylase gene which may change in senescence. If evidence for such changes is obtained, the experiments will be extended to investigate whether other steroidogenic enzyme genes are affected, other inducible gene functions, and whether replication is altered by such a factor. Screening for possible repressors or activators will be done by introduction of nuclear extracts by the erythrocyte loading delivery procedure. Expression of 17a-hydroxylase at the individual cell level will be monitored by an in situ hybridization procedure using fluorescence detection, to provide a means for screening nuclear extracts for the presence of regulatory factors. If such factors are detected, they will be used in in vitro experiments using the genomic clone for 17a-hydroxylase. Assays for the presence of DNA-binding proteins in nuclear extracts from senescent or young cells, in conjunction with an in vitro transcription assay, will be used. Senescent adrenocortical cells will be transfected with plasmids comprising 17a-hydroxylase 5' regulatory sequences fused to chloramphenicol acetyltransferase as a reporter gene. Regulation of CAT expression in senescent cells will be investigated using constructs containing various portions of the 5' flanking region of the gene. The nuclease sensitivity of the 17a-hydroxylase gene in senescent cells in comparison to young cells will be studied. Any changes in nuclease sensitivity of the gene in senescence may be followed up by the use of a genomic footprinting procedure. The effects of an immortalizing oncogene will be examined. Adrenocortical cells will be transfected with pLTRmyc, a plasmid containing c-myc under the regulation of the mouse mammary tumor virus long terminal repeat, which provides hormonal control of c-myc expression. Possible immortalization in these transfectants and the effect or myc expression on 17a-hydroxylase induction will be examined.
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