The overall objective of this proposal is to investigate the molecular basis for the cessation of proliferation in senescent human diploid fibroblasts (HDF). Previous studies have shown that mitogen-stimulated senescent HDF fail to phosphorylate their retinoblastoma protein (pRB) and lack c-fos, cyclin A and cdc2. In addition, preliminary results indicate that mitogen-stimulated senescent HDF have less cdk2 mRNA than do comparably treated quiescent HDF. Since phosphorylation of pRB and synthesis of c-fos, cyclin A, cdk2 and possibly cdc2 are necessary for DNA synthesis in mitogen-stimulated quiescent cells, a deficiency of these events in senescent HDF constitutes a molecular basis for the lack of DNA synthesis in mitogen-stimulated senescent HDF. Consequently, this project seeks to determine the molecular basis for these specific defects in senescent HDF. The first part of this project investigates the molecular basis for the lack of phosphorylation of pRB and the decreased expression of cyclin A, cdc2 and cdk2 in mitogen-stimulated senescent HDF, including the question of whether senescent HDF are deficient in these four late G1 events because they fail to express c-fos early in the mitogenic response pathway. Since it is likely that the decreased phosphorylation of pRB and the decreased expression of c-fos, cyclin A, cdc2 and cdk2 ultimately trace back to earlier deficiencies in mitogenic signal transduction, the second part of this project investigates the ability of mitogen-stimulated senescent HDF to carry out very early signal transduction events that are necessary for both c-fos expression and DNA synthesis in mitogen- stimulated quiescent HDF. Thus, this second set of studies seeks to identify: l) the earliest defects in the mitogen response pathway that lead to the lack of DNA synthesis in mitogen-stimulated senescent HDF, 2) a specific mechanism for the lack of c-fos expression in these cells, and 3) the relationship between the deficiencies in cyclin A, cdk2, cdc2 and phosphorylated pRB late in G1 and deficiencies in early signal transduction events. Understanding the phenomenon of cellular aging will contribute to our understanding of aging in man, and thereby enhance our ability to deal with age-related health problems. Cells aged in culture show declines in both proliferative capacity and differentiated functions. Both of these kinds of deficits can contribute to the overall aging phenotype of man, yet we do not understand the molecular basis for cellular aging. The ultimate goal of this research project is to contribute to our fundamental knowledge of the mechanism for cellular aging in the belief that this basic scientific information is part of the foundation upon which clinical strategies helpful to man can be built.
