The central theme of this project concerns the control of cell proliferation in normal and tumor cells. We intend to elucidate the molecular mechanisms that modify the transformed phenotype of mouse NIH3T3 cells containing the human rasEJ oncogene. Our strategy is to study revertant flat non-transformed cell lines derived from NIH3T3(rasEJ). We have isolated ten flat non-transformed cell lines and shown that six retain the rasEJ oncogene and produce p21ras protein. We propose to determine the molecular basis for the non-transformed phenotypes using the following approaches: (1) DNA. Non-transformed revertants will be compared to their transformed parents using Southern blot hybridization in order to detect possible differences in numbers of copies of rasEJ genes, numbers of copies of a tandemly repeated sequence 3' to ras, or methylation of ras. One non-transformed revertant shown to have an altered ras restriction enzyme map will be analyzed further to determine if it has undergone a deletion or a single base change, perhaps producing an analogue protein which acts as a competitive inhibitor of the p21ras protein. (2) Protein. Non-transformed revertants will be compared to parents using immunoprecipitation to detect possible differences in amount of p21ras protein produced, its rate of processing, or its cellular location. One revertant shown to have altered p23/p21 ratios will be further analyzed by pulse-chase experiments to confirm that it is altered in the rate of protein processing. (3) Cloning. Human DNA sequences which allow rasEJ to be re-expressed when transfected into revertants will be cloned from secondary transfectants using human Alu repetitive DNA probes and EMBL3 phage. Primary transfectants which are retransformed have already been isolated. These approaches whould allow us to elucidate molecular mechanisms that modify the transformed phenotype in cells containing the rasEJ oncogene.
NIH ExPorter
NIH ExPorter