Cell growth and division are controlled by signals acting at the cell surface. These signals initiate biochemical cascades that lead to changes in gene expression. While most known signaling pathways stimulate cell growth, there are some that are inhibitory. Cancer is an aberration of normal growth control resulting from an accumulation of mutations that leads to constitutive activation of stimulatory pathways or inactivation of inhibitory pathways. Understanding cellular regulation of growth control has important implications for cellular responses during growth and repair as well as in transformation. There are also implications for possible pharmacological targets to interfere with the unregulated growth that occurs in carcinogenesis. Rb is a tumor supressor protein which, when functional and hypophosphorylated can bind to and regulate E2F transcription factors. Cyclin dependent kinase phosphorylation of Rb can prevent its binding to E2F, allowing progression through the cell cycle. E2F transcription factors are necessary for the transcription of immediate early genes in the response to growth factors which stimulate cell division. When Rb is bound to E2F the growth signal is downregulated. Id (inhibitor of DNA binding) proteins are a family of helix-loop-helix HLH proteins which are ubiquitously expressed and dimerize with members of class A and B basic HLH proteins producing heterodimers which, due to the absence of the basic region, cannot bind DNA. Id appears to negatively regulate DNA binding of bHLH proteins Specific Aim: To further characterize the Rb protein we will investigate the possibility that Rb protein is modulated by a member of the bHLH protein family. Experiments are designed to show that coexpression of Id protein and Rb protein in an Rb-cell line, Saos-2, can overcome the negative effect of Rb on growth signal stimulation. The Rb and Id transfected Saos-2 cells will be identified by co-transfection with the SV2 neo gene to identify the geneticin (G418) resistant cells and observed for morphologic changes which demonstrate either cell division or signs of differentiation.
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