Because previous studies indicate that the transition of cells from G2 to M phase of the cell cycle requires Cdc2 cyclin B activity, we also assessed the effect of Rac1 inhibition on the proportion of cells in mitosis. The studies presented in Figures 3B and 5A indicate that IR exposure www.selleckchem.com/products/wortmannin.html of log phase growing MCF 7 cells results in a marked decrease in mitotic cells within 2 hours after IR, and that this effect is significantly inhibited by the incubation of cells with NSC23766 or expression of the N17Rac1 dominant negative mutant. Thus, Rac1 inhibition diminishes IR induced G2 M checkpoint activation and increases the entry of cells from G2 into M phase of the cell cycle in MCF 7 cells exposed to IR. These studies suggest Rac1 as an upstream regula tor of G2 M checkpoint response after exposure of cells to IR.
Cellular response to IR induced DNA damage involves activation of ATM and ATR signaling, which results in activation of the Wee1 kinase that Inhibitors,Modulators,Libraries phosphorylates Cdc2 Tyr15 and inhibition of the Cdc25 phosphatase that dephosphorylates Cdc2 Tyr15. Although it still remains unclear how exactly the ATM and ATR kinases are activated in response to genotoxic stress, evidence suggests that multiple mechanisms might be involved in the regulation of this biologic process. Supporting this speculation, a recent study by Wang et al. reported that the p38MAPK pathway is required for the activa tion of ATR kinase after expression of hepatitis B virus X protein.
Another example is NBS1, Inhibitors,Modulators,Libraries a component of the MRE11 RAD50 NBS1 complex, which not only is involved in certain downstream steps of ATM and ATR dependent DNA damage response but also func tions as an upstream mediator required for the ATM and ATR signaling activation after IR induced DNA damage. The results from the present report sug gest that Rac1 also plays an important role in the activa tion of ATM and ATR signaling after IR exposure of cells. A previous study demonstrated that incubation of MCF 7 cells with Rac1 specific inhibitor NSC23766 at 100 uM for 48 hours results in a G1 cell cycle arrest. However, in the present studies, we observe that incubation of MCF 7 cells with 100 uM NSC23766 for up to 24 hours does not result in a detectable increase in G1 phase cells relative to control untreated cells. Furthermore, incubation of other cells, including MDA MB 231, T47D, and ZR 75 1, with 100 uM NSC23766 for up Inhibitors,Modulators,Libraries to 24 hours, also does not result in an increase in percentage of G1 phase cells.
Inhibitors,Modulators,Libraries Thus, the effect of NSC23766 on G1 phase cells is probably time dependent. Additional stu dies are needed to understand the effect of prolonged Rac1 inhibition Inhibitors,Modulators,Libraries on cell cycle regulation in log phase growing cells. Expression of N17Rac1 dominant negative mutant for 72 hours has been previously shown to result http://www.selleckchem.com/products/17-AAG(Geldanamycin).html in G2 M cell cycle arrest in Rat 2 fibroblast cells.
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