A sensitive dual-fluorescence reporter system enables positive selection of ras suppressors by suppression of ras-induced apoptosis.
We have developed a novel dual-fluorescence reporter system incorporating green (GFP) and red (RFP) fluorescent proteins to monitor expression of the N-ras(m) gene and an N-ras(m) suppressor, respectively. Retroviral vectors were produced in which human N-ras(m) (codon 13 mutation) was coexpressed with GFP, and a ribozyme specifically targeting N-ras(m) was coexpressed with RFP. N-Ras(m) suppression was monitored by measurement of GFP fluorescence in dual-fluorescent (GFP and RFP) cells. We demonstrated that the degree of N-ras(m) suppression was dependent on the ribozyme dose, proportional to red fluorescence, in dual-fluorescent cells. We further showed that ribozyme-mediated N-ras(m)suppression inhibited growth of NIH3T3 and CD34-positive TF-1 cells. In these cultures, ras suppressor activity resulted in the depletion of suppressor-positive cells due to inhibition of cell growth. In contrast, N-ras(m) suppression produced a growth advantage to human leukemic K562 cells, presumably by inhibiting N-ras(m)-induced apoptosis. In K562 cells, ras suppression resulted in the outgrowth of suppressor-positive cells. This provides a platform to identify suppressors of ras that is based on function.
D000818:Animals; D017209:Apoptosis; D002070:Butadienes / Q000494:pharmacology; D045744:Cell Line, Tumor; D002867:Chromones / Q000494:pharmacology; D005434:Flow Cytometry; D005453:Fluorescence; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D020935:MAP Kinase Signaling System; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D041681:NIH 3T3 Cells; D009570:Nitriles / Q000494:pharmacology; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism
D002070:Butadienes; D002867:Chromones; D009025:Morpholines; D009570:Nitriles; D016337:RNA, Catalytic; C113580:U 0126; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D018631:ras Proteins