During attempts to isolate the inhibitor of DNA synthesis produced by senescent cells, we have discovered that fibronectin (fn) production by senescent cells is quantitatively and qualitatively different from young cells. The major goal of this project is to determine the role of changes in fn in cellular senescence. These changes could be directly responsible for the loss of proliferative potential that occurs at senescence. Alternatively, they could be part of a pleiotropic response to changes in gene expression that occur with cellular senescence, and as such provide a prototype gene with which to investigate the mechanisms of altered gene expression during in vitro aging. To achieve this goal, we will determine if the fn produced by senescent cells is structurally different from that produced by young cells, by studying splicing variations in the fn mRNA and postranslational modification of the fn protein. We will determine the response of the endogenous fn gene to a variety of cell types as they age in culture. We will determine the response of the endogenous fn gene to a variety of agents. This will be complementary to experiments with fn promoter-CAT constructs. We will determine whether other genes that are up-regulated in senescence are regulated in a manner similar to fn. To investigate the functional significance of fn changes seen with senescence, we will modulate the expression of fn in young and senescent cells and determine the effect on DNA synthesis. We will add fn extracted from senescent and young cells and human plasma to young and senescent cells to determine the effect on cell proliferation. The significance of the presence of particular epitopes of fn on senescent cells will be investigated by determining whether senescent cells can bind the fn fragment on which the epitope is located.
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