FIGURE 8.

Protective effect of PIM1 against docetaxel cytotoxicity depends in part on NFκB transcriptional activity. A, expression of a dominant negative PIM1 (NT81) decreases docetaxel-induced activation of NFκB. RWPE-2 cells stably expressing an NFκB-luciferase reporter gene were infected with retroviruses carrying pLNCX vector or pLNCX/NT81 constructs. Pools were selected with G418 and then treated for 6 h with docetaxel. Luciferase activity was determined. Each bar represents the mean ± S.D. of triplicate determinations of one of three similar experiments. p values were calculated by t tests. **, p < 0.01, showing that the chance of no difference in luciferase activity between vector and NT81-transduced cells is less than 1%. B, p50/NFKB1 and p65/RELA siRNAs inhibit NFκB transcriptional activity in RWPE-2 cells with high or low PIM1 expression. RWPE-2/NFκB-luciferase/PIM1 cells were transfected with control siRNA, siRNAs targeting p50/NFKB1 (si p50), or p65/RELA (si p65). After 48 h, the luciferase activity was measured and compared with that of RWPE-2/NFκB-luciferase/pLNCX cells transfected similarly. Each bar represents the relative luciferase activity of the various cells compared with that of vector-transduced cells treated with control siRNA. The values are the mean ± S.D. of six measurements pooled from two independent experiments. **, p ≤ 0.01 for no difference; *, indicates p < 0.05 for no difference. C, inhibition of NFκB activation by siRNA increases docetaxel-induced cell death. RWPE-2/pLNCX and RWPE-2/PIM1 cells were transfected with the indicated siRNAs and allowed to rest for 24 h. Docetaxel (100 nm) was then added for 48 h. Cell survival was then estimated by MTT assay. Each bar represents the mean ± S.D. of six measurements pooled from two independent experiments. p values were calculated by t test and represent comparisons between PIM1-expressing cells and vector control cells as well as among cells treated with different types of siRNAs. D, MTT survival data from C for docetaxel-treated cells are represented following normalization of the data to the values for control siRNA-treated cells. In this analysis, survival of cells transfected with NFκB-targeting siRNAs is shown as a percentage of the values for the same cells treated with control siRNA. Each bar presents the mean ± S.D. of six measurements pooled from two independent experiments. p values were calculated by t test and represent the likelihood that there is no difference in the sensitizing effect of the siRNA between vector- and PIM1-transduced cells.