FUNCTIONAL ASPECTS OF THE RETINOBLASTOMA GENE

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A long term objective of this proposal is to understand the function of the human retinoblastoma (Rb) gene and its role in the initiation and progression of various human tumors including those which occur as second malignancies in individuals with hereditary retinoblastoma. Its role in such common cancers such as breast and lung will also be investigated as well as its possible function ingrowth regulation, immortalization, and differentiation. Specific aims include determining the effect of placing either a normal 13 chromosome or the Rb cDNA itself into human tumor cell types in which the loss of both Rb alleles has been implicated. These include retinoblastoma, osteosarcoma, fibrosarcoma, carcinoma of the breast, small cell carcinoma of the lung, and bladder cancer. A major emphasis will be placed on the Rb gene product level using a high affinity Rb antibody which has been produced in our laboratory. Studies in this area will include examining the presence of absence of the Rb protein at the single cell level, correlating the phosphorylation pattern of the Rb gene product with immortalization, growth regulation, differentiation, or tumorigenesis and characterizing two Rb associated proteins whose presence we have recently documented. In addition the mechanism responsible for the marked diminution in unphosphorylated Rb protein and concomitant increase in phosphorylated Rb protein forms found in spontaneously transformed fibroblasts will be studied and other spontaneously transformed cells of human epithelial origin also will be examined for similar Rb protein changes. Various methodologies will be used for these studies. These include gene transfer by transfection, chromosome transfer by microcell fusion, protein analysis by immunoprecipitation. Western blotting, immunohistochemistry and 2-D gel electrophoresis and the analysis of Rb and Rb-associated protein by microsequencing. These studies should provide critical information on the role of the Rb gene in the initiation and progression of numerous human cancers as well as the role of specific changes in phosphorylation of the Rb gene products in immortalization, growth regulation and differentiation of human cells.