The relationships between differentiation, proliferation and senescence are fundamental to cell biology, recapitulated in multiple tissue systems. Molecules identified either as regulators of differentiation or of cellular proliferation have turned out to possess pleiotropic and often unexpected roles in other systems. For example, the retinoblastoma protein (pRb) was initially identified as a tumor suppressor protein and cell cycle regulator critical to the genesis of the childhood tumor, retinoblastoma, but has since been shown to play key roles both in cellular senescence and development in a wide range of cell types and associated tumors. Despite this clear association of pRb with tumor-suppressive mechanisms in most human tumors, a detailed understanding of pRb's role in these processes is lacking. We have found that aspects of the senescent state engendered by the pRb tumor suppressor depend on activation of cdk5 and modification and increased expression of ERM proteins. The experiments described below are designed to elucidate pRb's function in activation of cdk5 and to explore the consequences of this on senescent cell biology in the context of tumor growth. Intriguingly, our work on the role of pRb in differentiation of normal mouse osteoblasts both in vivo and in culture suggests that pRb may also work through cdk5 to influence the osteoblast's ability to sense cell-cell contact and undergo proliferative arrest. Further, we find that loss of pRb has multiple effects on gene expression patterns during osteoblast differentiation that result in accumulation of cells with apparent osteoprogenitor potential, thus likely predisposing osteoblasts to tumorigenic conversion. In order to understand the mechanisms involved in pRb-mediated senescence and differentiation, and in particular to ascertain pRb's role in a terminal cell cycle exit program associated with these functions, four specific aims are proposed: (1) Determine the mechanism of activation of cdk5 in senescent cells. (2) Ascertain the effects of constitutive cdk5 or ERM """"""""knock-down"""""""" or loss on proliferation and tumorigenesis. (3) Investigate the mechanistic role of pRb in osteoblast differentiation in detail through use of targeted knockout mouse strains and genomic and proteomic analyses of osteoblasts derived from them. (4) Investigate the role of pRb and cdk5 in controlling cell surface expression of N-cadherin and ?-catenin in primary and immortal osteoblasts. Cancer results in part from the loss of mechanisms that restrict cell division in the end stages of tissue development and after cellular stress. Recent work from our lab and others has identified the retinoblastoma protein, pRb, as a key effector of this growth suppression, and indeed pRb function is lost in most cancer cells. We propose to investigate the molecular mechanism(s) used by pRb to prevent abnormal cell division in order to identify proteins and processes appropriate for eventual therapeutic intervention.
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