The enhancement of Raf-1 kinase activity by knockdown of Spry2 is associated with high sensitivity to paclitaxel in v-Ha-ras-transformed NIH 3T3 fibroblasts

We previously demonstrated that the downregulation of Raf-1 kinase may contribute to the development of acquired resistance in paclitaxel-resistant cells. In this study, we determine whether the sensitivities of parental and its v-Ha-ras-transformed NIH 3T3 cells to paclitaxel were dependent on Raf-1 kinase activity. Paclitaxel sensitivity of v-Ha-ras-transformed cells was found to be significantly higher than that of its parental cells. Paclitaxel transiently increased Raf-1 kinase activity in v-Ha-ras-transformed cells while showing no effect on its parental cells, suggesting that the Raf-1-MAP kinase pathway is proapoptotic. Furthermore, using siRNA-mediated Raf-1 knockdown analysis, we showed that Raf-1 knockdown cells were more resistant than control cells to paclitaxel treatment. In particular, the expression of the gene SPRY2, which has been known to act as an inhibitor on Ras/Raf/MAPK signaling, was downregulated after the treatment with paclitaxel. Methylation-specific PCR also revealed that downregulation of Spry2 was associated with altered methylation of the CpG-rich region of the SPRY2 exon 1. In addition, the Spry2 protein knockdown cells were more susceptible to paclitaxel treatment than control cells. Taken together, our results suggest that the enhancement of Raf-1 kinase activity by knockdown of Spry2 is associated with high sensitivity to paclitaxel.