Characterization of cellular senescence-associated proteins; modulation of their function by immortalizing viral oncogenes

The aim of this proposal is to study molecular details of cellular senescence in cultured human fibroblasts and keratinocytes. We will study alterations in the expression and/or function of cell cycle regulators in senescent cells that may explain the observation that these cells arrest in the G1 phase of the cell cycle. For genes the expression of which is deregulated in senescent cells, we will analyze cis-acting elements in the promoter and the corresponding trans-acting factors driving senescence-specific transcription. A major emphasis will be on regulation of the genes coding for cyclin E and cyclin A, and on the role of the cellular transcription factor E2F in controlling their expression in senescent cells. It is also planned to study alterations in the metabolic stability of key cell cycle regulators in senescent cells. In a second step, we will analyze growth-regulatory functions of several gene products known to be overexpressed in senescent cells, in order to define the mechanism(s) by which these genes may contribute to the senescent phenotype. In particular, we plan to analyze the role of several IGF- binding proteins in the alteration of cellular growth control and apoptosis that are observed during the establishment of cellular senescence. Human fibroblasts and keratinocytes can both be immortalized by human papillomaviruses, and immortalization involves two viral oncogenes, designated E6 and E7. In the second part of the project, we intend to use the immortalizing potential of the E7 gene as genetic tool to study factors that contribute to the senescent phenotype. First, we will analyze the influence of E7 on the expression of known senescence-associated genes. Second, we propose to study the influence of E7 on the function of cellular proteins that are known to be overexpressed in senescent cells. These analyses should provide new insight into the process of replicative senescence in human fibroblasts and keratinocytes; furthermore, we anticipate that the project will provide new information on the ability of human papillomaviruses to break the senescent phenotype.