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Volume 271, Number 45, Issue of November 8, 1996 pp. 28259-28265
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Antisense Oligonucleotides Demonstrate a Dominant Role of c-Ki-RAS Proteins in Regulating the Proliferation of Diploid Human Fibroblasts

(Received for publication, May 6, 1996, and in revised form, August 8, 1996)

Guan Chen , Susan Oh , Brett P. Monia and Dennis W. Stacey

From the Department of Molecular Biology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 and  Isis Pharmaceuticals, Carlsbad, California 92008

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.

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