Human diploid fibroblasts in culture exhibit proliferation activity over a limited time span, which has been regarded as a manifestation of cellular aging. This proposal seeks to understand the mechanisms that control the in vitro aging process of human fibroblasts, and to determine how this process influences the cytoskeletal elements, in particular the intermediate filaments (IF), to form an unusually organized network of large entangled bundles. We hypothesize that, in nonproliferating senescent fibroblasts, the lack of total rearrangement of cytoplasmic architecture during mitosis allows the IF system to develop a stable architecture via, in part, persistent interactions among themselves. In this proposal, we plan to ascertain whether, accompanying senescence, there is age-associated quantitative variation in the biosynthesis of the IF core protein, vimentin, and an IF associated protein, p50. We will also investigate age-associated protein modification for vimentin and p50 that may affect the interaction between the two polypeptides. Finally, we will examine the precise conditions controlling p50 binding to vimentin, which should illuminate the possible interfilament linkage function of p50. Understanding of this mechanism can be very useful in elucidating the pathogenesis of Alzheimer's disease, where the permanent involvement of another type of IFs, neurofilaments, is suggested to be associated in the formation of neurofibrillary tangles. We propose that the cessation of proliferation, as the demarcation of the onset of fibroblast senescence, is related to the activation of a series of unique genes. Having already identified a protein, S-30, uniquely present in nonproliferating aging fibroblasts, we plan to explore the function of this senescent-specific protein (SSP) and the mechanism that regulates its expression. Furthermore, we plan to use our well-equipped experience in monoclonal antibody production to search for other putative SSPs, in the hope of establishing an antibody bank of probes demarcating senescent cells. Availability of these probes can provide the means to advance biogerontology from studies of phenomena to the analysis of mechanisms for aging. Furthermore, this antibody bank can be used as powerful tools for in vivo as well as in vitro studies of senescence, differentiation, development and even oncogenesis.
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