ANTISENSE OLIGONUCLEOTIDES DEMONSTRATE A DOMINANT ROLE OF C-KI-RAS PROTEINS IN REGULATING THE PROLIFERATION OF DIPLOID HUMAN FIBROBLASTS

Although members of the RAS protein family (Ha-, Ki-, and N-RAS) are k nown to play a key role in normal cell proliferation and to be frequen tly mutated in naturally occurring tumors, it remains unclear which of these proteins functions to regulate growth in normal cells, Gene-spe cific oligonucleotides (oligos) against c-Ki-RAS (ISIS 6957), c-Ha-RAS (ISIS 2503), and oncogenic Ha-RAS (ISIS 2570) were used to analyze th e requirement for individual RAS proteins in the proliferation of dipl oid human lung fibroblasts (MRC-5), and human bladder carcinoma cell l ines with (T24) or without (J-82) a RAS mutation. The oncogenic Ha RAS oligo substantially inhibited T24 cell proliferation, whereas the c-K i-RAS and control (ISIS 1966) oligos had little effect, Interestingly, in MRC-5 cells the c-Ki-RAS but not c-Ha-RAS oligo was effective in i nhibiting cell proliferation, No inhibition was seen in the J-82 cells with either oligo, In Western analysis, p21 RAS protein was decreased following treatment with the oncogenic Ha-RAS oligo in T24 cells or t he c-Ki-RAS oligo in MRC-B cells, whereas no reductions were observed in J-82 cells with either oligo, The specificity of these oligos was d emonstrated in Northern analyses in which both Ha-RAS and Hi-RAS oligo treatment resulted in reduced levels of their respective mRNAs in all three cell lines, whereas the mutant Ha-RAS mRNA in T24 cells was mos t effectively reduced with the oncogenic Ha-RAS oligo, These results d emonstrate that oncogenic Ha-RAS plays an important role in the prolif eration of T24 cells, whereas c-Ki-RAS contributes predominantly to th e proliferation of normal MRC 5 cells.