Aberrant cellular oncogenes (c-oncs) have been associated with a variety of human tumors. The role of these c-oncs is yet unelucidated partly because of our relative inability to manipulate these resident genes. I propose to use a new technique of antisense mRNA inhibition to attenuate the action of one mutant c-onc, EJ ras. This technique involves the subcloning of a gene that transcribes for mRNA complementary to the native mRNA thus allowing for RNA:RNA interactions. This has been shown to attenuate the function of the herpes simplex TK gene. An antisense EJ ras construct has been made where the EJ ras gene is flipped and is transcriptionally driven in the opposite direction by an MSV LTR; furthermore, this vector has a neo gene for dominant selection, and the dhfr gene for amplification. This antisense construct will be transfected into transformed cells bearing a copy of the sense EJ ras and cells will be selected for resistance to G418. The resultant clones will be scored for the number of revertants as compared to controls. The converse experiment where cells bearing the antisense ras are transfected with the sense EJ ras gene and the number of transformants assessed will also be done. If during the mRNA analysis of these cells, we find the antisense message in suboptimal quantities, amplification of the antisense gene can be accomplished through thedhfr construct. In this well defined murine system, I hope to optimize the conditions for the inhibition of the oncogene function by antisense mRNA. once the system is established, I propose to introduce this antisense EJ ras plasmid into the EJ human bladder carcinoma cell line from which the EJ ras gene was isolated and attempt to inactivate the resident mutant ras. If successful, this work has broad applicability as a technique to test the function of a eukaryotic gene by inactivation.
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| Santos, G F; Scott, G K; Lee, W M et al. (1988) Estrogen-induced post-transcriptional modulation of c-myc proto-oncogene expression in human breast cancer cells. J Biol Chem 263:9565-8 |
