Mammalian cell cycle progression is regulated by a complex machinery that can respond to a wide variety of extra-cellular signals. Multiple signaling cascades modulate components of the cell cycle machinery to elicit proliferation, differentiation or apoptosis. We have been studying how two major components of the cell cycle machinery, the retinoblastoma tumor suppressor protein (Rb) and its main downstream target, the E2F family of transcription factors, are regulated by signaling cascades. Our studies have shown that the signaling kinases Raf-1 as well as p38 can inactivate Rb in response to proliferative or apoptotic signals. An 8-amino acid peptide derived from Raf-1 disrupts the Rb-Raf-1 interaction in vitro. This peptide could bring about an inhibition of Rb phosphorylation, cell proliferation and angiogenesis when introduced into cells via a carrier molecule. In addition, the volume of a human tumor xenografted into nude mice was reduced by about 79% upon injecting the Raf-1 peptide conjugate. In this proposal we plan to study the mechanisms by which Raf-1 reverses Rb-mediated transcriptional repression. Attempts will also be made to screen a drug library (mainly the Diversity Set from DTP, NCI) for agents that can disrupt the Rb-Raf-1 interaction; such a compound could be expected to have anti-cancer effects. Our experiments will also address the role of cell cycle molecules, especially Rb and E2F, in angiogenesis. These experiments will be done on Human Aortic Endothelial Cells induced to form capillary tubules in matrigel by stimulating with VEGF. We had carried out a micro-array analysis to examine genes induced upon VEGF stimulation. A subset of the genes whose expression was altered appeared to have E2F binding sites in their promoter. The potential contribution of these downstream targets of E2F on the angiogenic process will be examined. Experiments are also proposed to study the regulation of Rb function by apoptotic signals. We had found that Fas stimulation of Jurkat cells leads to an inactivation of Rb by phosphorylation, mainly brought about by the p38/Hog 1 kinase; similarly, TNFalpha stimulation of Jurkat cells leads to the binding of the Apoptosis Signal-regulating Kinase (ASK1) to Rb. We plan to assess the functional role of these interactions and investigate how Rb inactivation contributes to the apoptotic process. We expect that the studies proposed here would help identify the molecular mechanisms underlying different aspects of cell proliferation, angiogenesis and apoptosis. We will also attempt to identify and evaluate the anti-cancer properties of agents that can modulate these processes.
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