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-5 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 Ki-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.
Department of Molecular Biology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
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Monia
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Stacey
D002455:Cell Division / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D013936:Thymidine