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 known to play a key role in normal cell proliferation and to be frequently mutated in naturally occurring tumors, it remains unclear which of these proteins functions to regulate growth in normal cells, Gene-specific oligonucleotides (oligos) against c-Ki-RAS (ISIS 6957), c-Ha-RAS (ISIS 2503), and oncogenic Ha-RAS (ISIS 2570) were used to analyze the requirement for individual RAS proteins in the proliferation of diploid human lung fibroblasts (MRC-5), and human bladder carcinoma cell lines with (T24) or without (J-82) a RAS mutation. The oncogenic Ha RAS oligo substantially inhibited T24 cell proliferation, whereas the c-Ki-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 inhibiting 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 the 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 demonstrated 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 most effectively reduced with the oncogenic Ha-RAS oligo, These results demonstrate that oncogenic Ha-RAS plays an important role in the proliferation of T24 cells, whereas c-Ki-RAS contributes predominantly to the proliferation of normal MRC 5 cells.