Interestingly, however, in the same cells growth inhibi tion induced by exogenous kinase inhibitor Carfilzomib TGF b1 was clearly enhanced relative to unstimulated controls. As shown by immunoblotting, the Rac1 siRNA, but not the irrelevant control, specifically diminished the level of both total Rac1 protein and prevented the formation of active Rac1 in response to TGF b1 stimulation. Similar data with respect Inhibitors,Modulators,Libraries to TGF b1 induced growth inhibition were obtained for COLO 357 cells. These data show that depletion of Rac1 mimicks the effect of depletion of Smad2 on TGF b1 mediated growth inhibition and led us to conclude that Rac1 antagonizes this cellular function Inhibitors,Modulators,Libraries of TGF b1 in responsive PDAC cells.

Specific inhibition of Rac1 activity potentiates growth inhibition induced by exogenous TGF b1 To scrutinize the role of Rac1 for pancreatic tumour cell proliferation and to evaluate whether Inhibitors,Modulators,Libraries the GTPase function of Rac1 was required for antagonizing TGF b1 induced growth inhibition, we employed previously characterized PANC 1 clones stably expressing dn Rac1 from a retroviral vector. Several individual clones were found to have reduced basal growth and to respond to TGF b1 with more pronounced growth inhibition when compared to empty vector controls or wild type cells supporting our findings on siRNA mediated suppression of RAC1. To exclude the possibility that enhanced apoptosis rather than growth inhibition accounted for lower cell numbers or reduced thymidine incorporation, we measured cell viability in cultures of PANC 1 dnRac1 stable clones and DNA fragmentation on PANC 1 cells transiently transfected with dn Rac1, or GADD45b as control.

Cell viability as assessed by trypanblue exclusion was low and was not significantly Inhibitors,Modulators,Libraries different between control and dn Rac1 expressing cells or between untreated and TGF b treated cells. The observa tion that dn Rac1 lacked a proapoptotic effect was con firmed by a quantitative DNA fragmentation assay. In contrast, ectopic expres sion of GADD45b, a Smad3 dependent TGF b target gene that can mediate TGF b induced apoptosis through p38 activation sensitized PANC 1 Inhibitors,Modulators,Libraries cells to TGF b1 induced DNA fragmentation. then Together these experiments indicated that dn Rac1 sup pressed proliferation rather than increasing apoptosis in both control and TGF b1 treated cells. Next we investi gated how Rac1 interacts with the cell cycle machinery to inhibit the TGF b1 effect. A central mediator of TGF b1 induced growth inhibition in PDAC is the cyclin depen dent kinase inhibitor p21WAF1. Notably, in 3/3 PANC 1 dnRac1 clones analysed, basal and TGF b1 induced levels of p21WAF1 protein were clearly higher than in the wild type and vector controls as demonstrated by immunoblotting, matching results from the Smad2 depletion experiments.