Retroviral gene transfer of dominant negative Raf-1 mutants suppresses Ha-ras-induced transformation and delays tumor formation

Activating mutants of ras are among the most frequently found genetic alter ations in human cancers. Therefore, Ras appears to be an attractive target for therapeutic intervention using gene transfer. The protein kinase Raf-1 acts as a direct downstream effector of Ras and is involved in Ras-induced cellular transformation. Using the NIH3T3 fibroblast-derived tumor cell lin e PEJ, which expresses oncogenic Ha-ras(G12V), we analyzed whether dominant negative mutants of Raf-1 can inhibit Ras-mediated transformation. Retrovi ral gene transfer was used to stably transduce PEJ cells with three differe nt dominant negative mutants of Raf-1. This resulted in reversion of the tr ansformed phenotype in vitro as evidenced by an increase in contact inhibit ion and reduced anchorage-independent growth. However, tumor formation in n ude mice was significantly delayed only by one of these mutants. Therefore, dominant negative mutants of the oncoprotein Myc, which is known to synerg ize with Raf-1 in tumor formation, were transduced into PEJ cells expressin g a dominant negative Raf mutant. This leads to killing of the cells. These results indicate that although interference with Ras-induced transformatio n using dominant negative mutants of Raf is feasible and effective in vitro using retroviral vectors, an additional block (e.g., that of Myc) is neces sary to kill PEJ cells. These results also indicate that interference with Ras-dependent signaling is not sufficient for inhibition of tumor formation of PU cells in vivo.