Abstract
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.
Highlights
The Ras family consists of three highly homologous proteins, each of which can function to transduce physiological signals
Growth Inhibitory Effects of Anti-RAS Oligonucleotides in T24 Cells—Antisense oligonucleotides were first tested in T24 cells, which are known to contain a mutation at codon 12 in the c-Ha-RAS gene [36]
Proliferation was substantially inhibited by the oncogenic Ha-RAS oligo (ISIS 2570; 17 nucleotides in length, complementary to the mRNA region containing the oncogenic mutation)
Summary
The Ras family consists of three highly homologous proteins, each of which can function to transduce physiological signals. Ras proteins play central roles in the control of normal and transformed cell growth, as well as other important biological processes (4 – 6) The evidence supporting this conclusion comes from: 1) the fact that oncogenic ras mutations have been found in a variety of animal tumors as well as 10 –25% of all human malignancies [7,8,9]; 2) the alteration of growth properties of a variety of cell lines following expression of oncogenic ras mutants or the high level expression of normal Ras proteins (for example, microinjection of Ha-Ras protein into serum-starved NIH/3T3 fibroblasts can both stimulate DNA synthesis and induce morphological transformation without requirement for mitogens [10]); and 3) the inhibition of cellular proliferation following neutralization of cellular Ras activity. These molecules might be involved in other functions of Ras, such as its involvement in the control of cellular migration [21], differentiation, and cytoskeletal rear-
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