This project focuses on the mechanisms of cell transformation by oncogenes which act through the MAP kinase signaling pathway. We had isolated a phenotypic revertant of rat cells transformed by the mos oncogene which is resistant to some, but not all oncogenes, acting through MAP kinase. Using the revertant cells and their transformed parent, we identified a novel gene, DRM, whose expression is down- regulated in transformed cells and whose overexpression in normal cells blocks proliferation. We are currently characterizing drm and its biological function. Cell fractionation studies indicate that DRM is predominantly localized in the nucleus and immunofluorescent microscopy of cells transfected with a construct expressing DRM fused with green fluorescent protein (GFP) confirms this localization and indicates tht DRM is concentrated within discrete regions of the nucleus whose number and location vary from cell to cell. Double fluorescent labeling and confocal microscopy indicated DRM did not co-localize to nucleoli or to spliceosomes. Analysis of DRM expression in diploid human fibroblasts indicates that the level of DRM RNA and protein is cell cycle regulated and is increased when cell proliferation is inhibited by serum starvation, contact inhibition or treatment with TGF-beta. When induced to re-enter the cell cycle, arrested cells exhibit a reduction in the level of DRM protein. Conversely, cells induced to overexpress DRM by transient transfection of DRM expression constructs results in accumulation of cells in the G0/G1 phase with a concomitant decrease in the number of cells in the S phase. Our data suggests DRM may act to inhibit cell growth by arresting cells in the G0/G1 phase of the cell cycle. We have also isolated cDNA clones of the murine homolog of DRM and confirmed the high degree of conservation of DRM. Rat and mouse drm show only 1/184 amino acid differences. We have isolated the genomic locus of drm from mice and confirm that, like the rat, the mouse drm is encoded in a single exon containing a 184 amino acid open reading frame. In conjunction with our previous results, our continued analysis of DRM indicates that it represents a novel gene with the ability to alter progression through the cell cycle and may play a critical role in cell growth and differentiation.
