This proposal aims to understand the mechanisms that control the termination of cell replication at the final stage of fibroblasts' life span; we hypothesize that activation of a series of genes, whose products function directly or indirectly as inhibitors, incapacitates the nonproliferating senescent fibroblasts from traversing the cell cycle. Towards this end we have identified statin, a protein expressed only in nonproliferating cells, and characterized as a marker for G0-quiescent cells. In this proposal, we plan to: (1) characterize biochemically how statin is synthesized, modified and degraded; (2) purify statin in native and denatured forms needed for biochemcial analysis, protein sequencing and antibody production; (3) analyze how statin interacts with components of the nuclear envelope; (4) clone and characterize the statin gene; (5) analyze the cellular functions of statin; (6) analyze the relationship between in vitro and in vivo aging, using statin as a probe; and (7) identify other satin-like proteins. Structural aspects of protein localization will be performed by immunolabelling techniques at both light and electron microscopic levels. Biochemcial studies will be pursued by cell fractionation followed by 1- and 2-D gel analysis, coupled with immunoprecipitation and immunoblotting assays. Proteins will be purified by electrophoretic separation, various column chromatographies, and HPLC. Molecular cloning will be performed by construction and screening of a statin-containing c- DNA library. Functional studies will be performed by microinjection of single cells with monospecific antibodies, purified proteins and c-DNA clones. Histochemcial examination of statin in fibrocytes in tissues derived from animals of differing age groups will be the starting attempt to relate in vitro fibroblast aging to in vivo organismic aging. Other statin-like proteins will be identified by production of unique monoclonal antibodies capable of recognizing rare, nonproliferation- dependent proteins. Results of our studies should advance biogerontology from phenomenological observation towards the mechanisitic investigation of cellular aging. Tools such as monoclonal antibodies, purified proteins and c-DNA clones, developed in this work, will create a bank of powerful experimental probes useful not only in studies of aging fibroblasts, but also in analysis of cell growth in general. Answers to questions raised here are not only central to cellular aging, but also crucial for the understanding of regulation in development, differentiation and oncogenesis.
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