The proposed studies are designed to identify, molecularly clone and characterize cellular genes which comprise and/or regulate the biochemical pathways essential for the process of malignant transformation. The strategy which has been developed to identify these cellular genes involves the molecular and genetic analysis of non-transformed revertants that arise in populations of tumorigenic cells. These revertants can result from mutations that inactivate transformation effector genes or mutations that activate dominant transformation suppressing genes. The frequency with which such revertants are generated can be increased by random mutagenesis or by DNA mediated gene transfer. The first set of experiments are focused on the continued isolation of new revertants and the characterization of revertants already generated using this approach.
These specific aims i nclude the following. Revertants resulting from mutations in different transformation effector and suppressor genes will be derived from populations of human and rodent cells chemical mutagenesis or retroviral insertional mutagenesis. The transformation effector genes present in normal cells will be identified as open reading frames disrupted by the inserted provirus by their ability to induce retransformation of revertant cells in DNA mediated gene transfer experiments. Dominant transformation suppressing genes will be identified in revertant cells isolated from populations of tumor cells transfected with genomic DNA or cDNA's derived from normal human cells. The transfected transformation effector and suppressor genes will be molecularly cloned from genomic libraries or cDNA libraries prepared from the recipient cells, or by amplification using vector primed polymerase chain reactions. Putative effector and suppressor genes will be subjected to nucleotide sequence analysis. Antisera to each of the cloned effector and suppressor gene products will be developed using synthetic peptides or proteins partially purified from bacteria expressing the cDNA clones. These antisera will be used to study the biochemical properties of their corresponding proteins, their intracellular localization, as well as the relative expression and post-translational processing and modification in normal versus transformed cells. The second set of experiments are focused on defining the mechanism by which the protein ribosomal protein S3a encoded v-fos-transformation effector gene fte-1 contributes to cell transformation.
The specific aims are to include the following. Anti-sense RNA's complementary to the genes encoding fte-l, ribosomal RNA (rRNA) or transcription factors that regulate 45S rRNA will be used to inhibit ribosome biogenesis. These experiments will be used to determine if the fte-1 protein's role as a transformation effector gene is mediated via its function as a ribosomal protein. More importantly, experiments will be performed to test the feasibility of using inhibitors of ribosomal biogenesis as a novel class of chemotherapeutic agents.
