The long term objectives of this proposal are to understand the fundamental mechanisms by which Simian Virus 40 affects cellular gene expression and alters cellular growth properties. Studies of mutant SV40 large T antigens demonstrate that 3 regions of T antigen play critical roles in immortalization and transformation by SV40. Two are the regions through which T antigen forms complexes with p105Rb and p53, tumor suppressor proteins that act by as yet unknown mechanisms to control normal cell growth. A large fraction of human cancers contain mutations affecting Rb, p53, or both. Sequences near the N-terminus of T antigen constitute the third important region and may also interact with a cellular tumor suppressor protein, though none has yet been identified. The experiments proposed in this project are directed at increasing our understanding of how SV40 transforms cells and alters normal cellular growth control. Since SV40 affects growth control by interacting with tumor suppressor proteins, mutants of SV40 T antigen defective for these interactions are potentially powerful tools to investigate how these proteins control cellular growth. Four specific areas will be addressed: (1) Large T antigen induces DNA replication and mitosis in quiescent cells. Recombinant adenoviruses expressing mutant or wildtype large T antigens will be constructed and used to determine which regions and functions of T antigen are required for these cellular responses. (2) Fusion proteins between T antigen and the hormone binding domain of steroid hormone receptors will be constructed and cell lines expressing these fusion proteins will be prepared. Addition of hormone to these cells should activate T antigen, leading to changes in cellular gene expression. Subtracted cDNA libraries will be prepared to identify genes activated or repressed following T antigen expression in quiescent cells. Characterization of these genes will focus on those activated or repressed by wildtype T antigen but not by mutant T antigens defective for binding to one or more tumor suppressor proteins. In this way, genes should be identified whose expression is altered when p53, Rb, and other cellular proteins are sequestered. (3) The vast majority of adult human cancers are of epithelial cell origin. Most studies of transformation and immortalization by SV40 have been conducted in rodent cells or in human fibroblasts. The ability of mutant and wildtype T antigens to extend the lifespan and alter the growth properties of human mammary epithelial cells will be determined. (4) Attempts will be made to identify cellular proteins that interact with T antigen through sequences near the N-terminus. Fusion proteins containing glutathione S-transferase and portions of wildtype or mutant T antigens will be prepared in bacteria and used to screen lambda expression libraries to identify those clones which produce proteins able to interact with the N-terminal portion of T antigen. In an alternative approach, interactions between this portion of T antigen and mammalian proteins will be detected by using a sensitive assay for protein/protein interactions in S. cerevisiae.
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