Phosphatidylinositol 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog 1 (AKT) signaling in cancer is implicated in various survival pathways including regulation of recombinase (RAD51). suppression of RAD51 in FLO-1 cells, significantly increased the anticancer activity of wortmannin in these cells, both in vitro and in vivo. We show that PI3K signaling and hsRAD51, through distinct roles in DNA damage response and repair pathways, provide survival advantage to BAC cells. In cells with inherent low expression of AKT, RAD51 is unaffected by PI3K suppression and provides an additional survival pathway. BMS-777607 Simultaneous suppression of PI3K and RAD51, especially in cells with lower AKT expression, can significantly reduce their proliferative potential. and (Figure 3A). A prominent (3-fold) reduction in telomere maintenance gene regulator of telomere elongation helicase BMS-777607 1 (HUS1 checkpoint homolog (was not affected by wortmannin (data not shown). These data indicate that wortmannin down-regulates the ATRCCHEK1 pathway without affecting the key Rabbit polyclonal to LIMK2.There are approximately 40 known eukaryotic LIM proteins, so named for the LIM domains they contain.LIM domains are highly conserved cysteine-rich structures containing 2 zinc fingers. genes implicated in the CHEK2 pathway. Although several DNA damage-inducible genes were up-regulated (Figure 3B), many key DNA repair genes including DNA ligase I, antitumor activity, inspite of a relatively weak antiproliferative activity (Figure 5A, panel II). RAD51 suppression was also associated with elevated expression of DNA damage-inducible genes (Figure 5A, panel II). These data indicate that RAD51 suppression is associated with suppression of and other genes from both checkpoint pathways. Figure 5 Impact of recombinase (RAD51) suppression in FLO-1 cells. FLO-1 cells were transduced with control (CS) or RAD51-specific (RS4) shRNAs and selected in puromycin. A: (I) RS4 lentiviruses mediate a strong suppression of RAD51. The expression profile of … Impact of RAD51 suppression on growth of FLO-1 cells as tumors in mice To evaluate the impact of RAD51 suppression on the growth of FLO-1 cells as tumors, cells transduced with control or RAD51-specific shRNAs (cultured for ~37 days) were injected subcutaneously into the mice as described above and tumor sizes were measured at different time points. The tumors in mice injected with RAD51-suppressed cells were significantly smaller than those injected BMS-777607 with control cells (Figure 5C), indicating an antitumor effect. The growth rate of RAD51-suppressed cells was reduced by 20C30% (data not shown). Impact of RAD51 suppression on efficacy of wortmannin in FLO-1 cells Since wortmannin suppressed the expression of several important recombination/repair genes in FLO-1 cells, except RAD51, we evaluated the impact of RAD51 suppression on the proliferative potential of these cells. FLO-1 cells, transduced with control or RAD51-specific shRNAs, were cultured in the presence or BMS-777607 absence of wortmannin and were evaluated for cell viability. Following treatment, the viability of cells transduced with control and RAD51-specific shRNAs was reduced by 18% and 71%, respectively (Figure 6A). Consistent with previous observations, the treatment of untransduced FLO-1 cells with wortmannin reduced cell viability by only 27% (Figure 6A). These data indicate that suppression of RAD51 significantly enhances the antiproliferative activity of wortmannin in BAC cells. Telomere length in wortmannin-treated cells, RAD51-suppressed cells, and wortmannin-treated RAD51-suppressed cells was reduced by 12%, 34%, and 58%, respectively (Figure 6B). This indicates that simultaneous suppression of PI3K and RAD51 has a great impact on telomere maintenance. Transduced cells were cultured in the presence or absence BMS-777607 of wortmannin for 72 h and analyzed by western blotting for levels of phosphorylated H2AX, a marker for DNA damage, and poly(ADP-ribose) polymerase (PARP), a protein which is involved in DNA damage response and undergoes caspase-3-mediated cleavage during apoptosis. H2AX and PARP were also investigated following exposure of these cells to UV light.