The retinoblastoma protein, pRb, controls entry into the S phase of the cell cycle and acts as a tumor suppressor in many tissues. Re-introduction of pRb into tumor cells results in growth arrest due in part to E2F-dependent transcriptional repression of S phase genes. pRb may also be involved in terminal cell cycle exit as an instigator of E2F-independent senescence or differentiation programs. The irreversibility of pRb-induced cell cycle exit is illustrated by our observation that when shifted to the nonpermissive temperature, SAOS-2 cells previously arrested by temperature-sensitive pRb (tspRb) enter S phase but do not proliferate, and instead die as a result of apoptosis. These results suggest that in addition to repressing E2F-dependent transcription and S phase entry pRb initiates an irreversible growth suppressive program. To better understand these growth inhibitory properties of pRb, we have studied the role of pRb in senescence and differentiation. First, we have found that pRb induces p27kipi post-transcriptionally independent of E2F regulation. This induction is required for pRb-mediated Gi arrest and senescence. While the mechanism of this induction is unclear, our evidence indicates that it is related to cytoskeletal changes that result from alterations in transcriptional patterns soon after pRb expression. We have uncovered a second important function of pRb in cell cycle exit that is the result of pRb's ability to interact with the transcription factor CBFA1. This factor is required for bone development and expression of late markers of osteogenesis. We find that pRb can act as a direct coactivator of CBFA1 by binding to this protein at promoter sites within certain CBFA 1-regulated genes. The physiological relevance of this interaction is illustrated by the observation that cells lacking pRb fail to achieve complete osteogenic differentiation, nor do they undergo CBFA1 dependent growth arrest, even though CBFA1 protein levels are induced normally. These multiple growth suppressive properties of pRb may explain the prevalence of pRb mutations in osteosarcoma. Our current goal is to elucidate the mechanisms of CBFA1 transcriptional activation and senescence induction by pRb and to identify genes regulated by pRb in these processes. To address these issues we will: 1) Explore the mechanism through which pRb activates CBFA 1-dependent transcription. 2) Investigate the pRb-dependent proliferation arrest in senescent cells and in cells expressing CBFA1. 3) Determine the consequences of pRb loss on osteoblast and osteocyte phenotypes in vitro and in vivo. 4) Identify novel changes in transcription patterns in cells expressing temperature-sensitive pRb.
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