For a number of years, the principal investigator's laboratory has been actively engaged in isolating potentially novel primary response genes induced during the differentiation of established myeloid cell lines (32D and M1). Several novel cDNAs, referred to as the myeloid differentiation primary response (MyD) genes, have been isolated. Two of these novel genes have been determined to be related to the Gadd family of genes involved in DNA excision repair, which are induced by genotoxic stress including alkylating agents and irradiation. MyD116 is the murine homologue of Gadd34, while MyD118 is related but distinct to Gadd45. A third member of the MyD118/Gadd45 family, termed CR6, has recently been cloned by other investigators. MyD116 and the MyD118 family of genes appear to play a role in the regulation of growth arrest and apoptosis. Physical interactions between MyD/Gadd proteins and proteins associated with DNA replication and cell cycle regulation have been demonstrated; MyD118 and Gadd45 also stimulate DNA repair in an in vitro assay. The focus of this revised renewal proposal is to understand at the molecular, genetic, and biochemical level how MyD116, MyD118, Gadd45, and CR6 influence growth arrest and apoptosis. The investigator now hypothesizes that MyD118 and Gadd45 positively regulate growth arrest and apoptosis, CR6 negatively regulates these pathways, and MyD116 may modulate these pathways in either a positive or negative fashion depending upon the biologic context.
The specific aims of the proposal are to: 1) further characterize MyD116 and the MyD118 family of proteins by studying protein expression and post-translational modification in vivo as well as ascertaining if protein modification and cellular localization are cell cycle regulated; 2) perform genetic and functional analyses of these proteins during growth arrest and apoptosis. Functional studies will be performed by blocking or de-regulating expression using anti-sense technologies and then determining the phenotype in the 32D and M1 cell lines. It will be determined how these genes interact with other proteins, such as c-myc, c-myb, bcl2, and bax, in regulating growth arrest and apoptosis. The role of MyD/Gadd genes will also be addressed using a cell free apoptotic DNA degradation system; and 3) further study the mechanism of action and physical interaction of these genes with cell cycle genes. Additional interacting partners will be cloned and analyzed and the role of MyD/Gadd genes in DNA repair and DNA replication will be further studied.
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