The limited proliferative ability of normal animal cells in culture is widely accepted as a manifestation of aging at the cellular level. Most of the investigations of this phenomenon have been devoted to the study of intracellular changes that occur during in vitro aging. However, it is likely that an understanding of the mechanisms leading to the inevitable and essentially irreversable loss of proliferative ability in normal cells will have significance for a large number of biological problems, including the processes of neoplasia and abnormal development. Recent experimental evidence from cell hybridization and cell reconstruction experiments has led to the idea that after a period of active growth in culture normal cells produce a specific substance(s) that prevents DNA synthesis and cell division, thereby resulting in what has been commonly called cellular senescence. We have further found that human diploid fibroblasts held in a nondividing state (quiescent cells) produce an inhibitory substance(s) similar to that produced by senescent cells. Proteins that block the initiation of DNA synthesis can be extracted from the membranes of these cells and, when poly(A)+ mRNA from senescent cells is microinjected into young cells, the initiation of DNA synthesis is blocked. The project proposed in this application will be to identify, isolate and characterize the protein products responsible for the inhibitory activity of senescent and quiescent cells and the mRNAs and genomic DNA coding for them. We will then begin to study the control of expression of the genes and the relationship of in vitro cellular senescence to actual in vivo cellular aging. Monoclonal antibodies to the senescence and quiescence factors will be prepared and used in immunocytochemical studies to determine the cellular localization of the factors in aging human diploid fibroblasts, vascular endothelial cells, and keratinocytes. cDNA probes will be prepared to the mRNA coding for the senescence and quiescence factors. These will be used to perform preliminary structural studies on the senescence factor gene and to study the regulation of senescence and quiescence factors.
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