TI - Net1/RhoA -dependent activation of p38 MAPK is required for cell survival in intoxicated or irradiated cells . AB - DNA damage was shown to induce activation of p38 MAPK [17] . We therefore tested whether exposure of HeLa cells to CDT or IR stimulates p38 MAPK activity , and whether this is required for protection from cell death . Both CDT intoxication and irradiation induced activation of p38 MAPK within 3h after treatment and this effect was maintained for atleast 24h , as assessed by western blot using a p38 MAPK PHOSphorylated specific antibody ( p-p38 ) (Figure 5A) . The kinetics of p38 MAPK PHOSphorylation is in line with the previously reported kinetics of RhoA activation , which peaks at 4h after exposure to CDT or IR [6] . To determine whether p38 MAPK activation is important for cell survival , the cells were treated with the p38 MAPK specific inhibitors SB203580 or SB202190 ( 20 muM ) prior to irradiation or intoxication . Inhibition of p38 MAPK was associated with a 2 - to 4-fold increase in the number of cells exhibiting chromatin condensation 48h after treatment ( Figure 5B and data not shown ) . The increased rate of cell death was confirmed by monitoring activation and increased expression of Bax ( Figures 5C and 5D ) . Activation of p38 MAPK in response to DNA damage was also observed in the colorectal carcinoma cell line HCT116 upon irradiation ( Figure 6A ) . As in HeLa cells , pre-treatment of HCT116 cells with SB203580 prior to irradiation was associated with increased activation of Bax (Figure 6B) . Quantification of the Bax positive cells in this set of experiments was hampered by the low number of cells that survived irradiation upon pre-treatment with SB203580 (Figure 6C) . These results indicate that activation of p38 MAPK protects the cells from death induced by DNA damage . To assess whether the activation of p38 MAPK was dependent on RhoA and its regulator Net1 , the levels of irradiation-induced PHOSphorylation were assessed in cells where the expression of the two proteins was independently knocked down by RNAi . Transfection with two independent RhoA specific siRNA oligonucleotides consistently induced a 70 to 80 percent reduction of the endogenous levels of RhoA (Figure 7A) , and this effect was associated with a strong inhibition of p38 MAPK activation in response to IR and CDT ( Figures 7B and 7C ) . Similar levels of inhibition were obtained by blocking RhoA activity using the cell permeable inhibitor C3 transferase ( data not shown ) . Knock down of endogenous Net1 expression , by either siRNA or shRNA , resulted in an equally potent inhibition of p38 MAPK activation in irradiated cells ( Figures 8A and 8B ) . These results indicate that Net1 and RhoA are upstream signals in the p38 MAPK activation cascade in response to DNA damage . To determine whether the RhoA activated kinases ROCKI and ROCK II are required for p38 MAPK activation , HeLa cells were treated with the ROCKI/II inhibitors H-1152 or Y27632 prior to irradiation . As expected , pre-treatment of control HeLa cells with both inhibitors altered the organization of the actin cytoskeleton and prevented the formation of actin stress fibers upon irradiation ( Figure 9A ) , confirming that these effectors were efficiently blocked . However , the treatment did not impair the activation of p38 MAPK (Figure 9B) , indicating that ROCKI/II were not required for signalling to this kinase . The MAPK-activated protein kinase 2 ( MK2 ) is a direct SUBstrate of the p38 MAPK alpha-and beta-isoforms [18] . We asked therefore whether this protein is also activated in a Net1 - and RhoA -dependent manner upon induction of DNA damage . As illustrated in Figure 10 , a 2 - to 4-fold increase in the PHOSphorylation of MK2 on its activating site Thr334 ( p-MK2 ) was observed in HeLa cells 4h after irradiation or intoxication (Figure 10A) , and a similar effect was observed in irradiated HCT116 cells (Figure 10B) . As expected , this effect was prevented by pre-treatment with the p38 MAPK specific inhibitor SB203580 (Figure 10A) . Importantly , the PHOSphorylation of MK2 following irradiation was abrogated by knock down of either Net1 or RhoA , indicating that these proteins are required for MK2 activation ( Figures 10C and 10D ) .