Normal somatic cells undergo only a limited number of divisions. This property is termed the finite replicative lifespan of cells, and the process that leads to the loss of proliferative potential is termed cellular or replicative senescence. Several lines of evidence suggest that senescent cells accumulate with age in vivo, and that a finite replicative lifespan may be a tumor suppressive mechanism. Replicative senescence is genetically controlled by multiple, dominant-acting loci. It is particularly stringent in human cells, which rarely if ever spontaneously acquire an infinite replicative lifespan. Two important phenotypic changes occur in senescent cells. One occurs in all cells: a stable reversible arrest of growth. The other is cell type- specific: a selective alteration in differentiated functions. In both cases, there are specific modifications in gene expression, but little is known about the prime cause(s) for these changes. This proposal aims to study transcriptional regulators whose expression or activity is altered when human fibroblasts undergo replicative senescence. Three types of molecules will be cloned and/or characterized: 1) basic helix-loop-helix (bHLH) transcription factors that appear to be important in the growth arrest and require a functional retinoblastoma tumor suppressor protein for activity; 2) a novel DNA binding activity expressed by senescent cells that appears to act as a transcriptional repressor; 3) an altered form of the E2F transcription factor that is a dominant negative regulator of E2F activity. The regulation and function of these genes will be studied in cultured human cells. Their biochemical activity (DNA binding, cofactor requirements, transactivation potential) will be determined by in vitro and transfection assays. Biological activity (effects on growth, lifespan, differentiated functions) will be assessed by transfection and microinjection experiments. These studies will provide mechanistic data on how the senescent phenotype is controlled, and insights into the role of cell senescence in tumor suppression and aging.
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