REVERSION OF RAS ONCOGENE MEDIATED CELL TRANSFORMATION

Project Details

Description

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.
StatusFinished
Effective start/end date1/1/903/31/90

Funding

  • National Cancer Institute
  • National Cancer Institute
  • National Cancer Institute

ASJC

  • Genetics

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