Cancerous transformation removes the normal control mechanisms that limit cell growth, resulting in invasive tumors composed of morphologically abnormal cells. Many oncogenes have been identified whose misexpression results in such transformation, including Ras, Dbl, Vav, and Bcr. Similarly, anti-oncogenes such as KRev-1 have been identified which act to reverse Ras-dependent transformation. In most cases the signalling pathways by which these oncogenes regulate cell growth, and which would provide logical targets for the development of therapeutic strategies, are not well understood. Characterization of these signalling pathways in human cancers requires the isolation of relevant proteins in those pathways, but doing so presents formidable technical difficulties because of the relatively limited nature of experimental manipulations in humans. To circumvent these difficulties, the investigators have exploited the fact that Dbl, Vav, Bcr, and KRev-1 possess strongly conserved homologs in the yeast S. cerevisiae that can functionally interact with the human genes. All the yeast homologs are members of the yeast budding pathway. The investigators have used a library that expresses human proteins in yeast, and have performed a morphological screen to identify novel human proteins whose overexpression causes yeast to bud pseudohyphally. Preliminary studies indicate that these human genes, designated HEF genes (for Human Enhancers of Filamentous growth) are indeed relevant to specification of cell morphology and to control of cell division. The current proposal seeks to characterize the HEF genes in detail, and in particular to analyze the role of these genes in human cancers. HEF genes will be overexpressed in NIH3T3 cells and PC12 cells both individually and in combination with a series of relevant genes, including the oncogenes Ras and Dbl, and the anti-oncogene KRev-1. Cells containing overexpressed genes will be monitored for cell viability, cell morphology, cell division time, response to growth factors, and response to environmental stress, with particular focus on HEF gene modification of oncogene and anti-oncogene function. A series of genetic experiments will also be performed in yeast which should help identify where HEF genes function in the oncogenic signalling pathway. In complementary studies, KRev-1, Dbl, and a HEF gene will be used as probe proteins in a novel yeast strategy (The Interaction Trap, co-developed by the PI) designed to isolate proteins that physically associate with the probes. Interacting proteins will initially be characterized in respect to their expression patterns in normal and malignant cells.
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