The intent of this proposal is to study the effects of clinically relevant epidermal growth factor receptor (EGFR) variants in oncogenic transformation of astrocytes. Immortalized mouse astrocytes and threeglioma associated EGFR cDNA will be used to accomplish the following: (1) the construction and characterization of immortalized astrocyte cell lines expressing glioma associated EGFRs and (2) to define the signal transduction pathways of the wild type and variant EGFRs in immortalized astrocytes. Examination of the growth properties of these cell lines combined with biochemical characterization of the receptors and signaling intermediates may provide insight into the mechanisms of EGFR mediated glial cell transformation. These studies will employ soft-agar colony formation assays, metabolic labeling (DNA and protein), pulse/chase, subcellular fractionation, in vitro kinase assays, chemical cross-linking, immunoprecipitation, SDS-PAGE and immunoblot analyses, and the expression of dominant negative alleles of signaling intermediates. Activation of transactivators will be addressed using Northern and nuclear run-on analysis, gel mobility shift assays and reporter gene activity assays.
