TY - JOUR
T1 - RAC1 GTPase plays an important role in γ-irradiation induced G 2/M checkpoint activation
AU - Yan, Ying
AU - Greer, Patrick M.
AU - Cao, Phu T.
AU - Kolb, Ryan H.
AU - Cowan, Kenneth H.
N1 - Funding Information:
We thank Dr. Helen Piwnica-Worms for the GST-Cdc25C construct, Dr. Toren Finkel for Ad.N17Rac1 and Ad.dl312 adenoviral vectors, Dr. Vimla Band for MCF-10A and 76N cells, Dr. Charles Kuzynski, Megan Michalak, and Victoria Smith for assistance on the flow-cytometry analysis; and Dr. Janina Baranowska-Kortylewicz for assistance on the operation of Mark I 68A Cesium-137 Irradiator. This work was supported by Nebraska DHHS-LB506 grant 2010-40 to YY, NCI Training Grant (NCI T32 CA009476) to RK, and NCI Cancer Center Support Grant (P30CA036727) to KC.
PY - 2012/4/11
Y1 - 2012/4/11
N2 - Introduction: In response to gamma-irradiation (IR)-induced double-strand DNA breaks, cells undergo cell-cycle arrest, allowing time for DNA repair before reentering the cell cycle. G 2/M checkpoint activation involves activation of ataxia telangiectasia mutated (ATM)/ATM- and rad3-related (ATR) kinases and inhibition of Cdc25 phosphatases, resulting in inhibition of Cdc2 kinase and subsequent G 2/M cell-cycle arrest. Previous studies from our laboratory showed that the G 2/M checkpoint activation after IR exposure of MCF-7 breast cancer cells is dependent on the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signaling. In the present studies, we investigated the role of Ras-related C3 botulinum toxin substrate 1 (Rac1) guanosine triphosphatase (GTPase) in IR-induced G 2/M checkpoint response and ERK1/2 activation, as well as in cell survival after IR.Methods: With Rac1-specific inhibitor, dominant negative mutant Rac1 (N17Rac1) and specific small interfering RNA, the effect of Rac1 on IR-induced G 2/M checkpoint response and ERK1/2 activation was examined in human breast cancer cells. In addition, the effect of Rac1 on cell survival after irradiation was assessed by using Rac1-specific inhibitor.Results: IR exposure of MCF-7 breast cancer cells was associated with a marked activation of Rac1 GTPase. Furthermore, inhibition of Rac1 by using specific inhibitor, dominant-negative Rac1 mutant, or specific siRNA resulted in attenuation of IR-induced G 2/M arrest and concomitant diminution of IR-induced activation of ATM, ATR, Chk1, and Chk2 kinases, as well as phosphorylation of Cdc2-Tyr15. Moreover, Rac1 inhibition or decreased Rac1 expression also abrogated IR-induced phosphorylation of mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) and ERK1/2. Ultimately, inhibition of Rac1 markedly increased cellular sensitivity to IR exposure, which involves induction of apoptosis.Conclusion: Studies in this report suggest that Rac1 GTPase plays an essential role in the activation of IR-induced ERK1/2 signaling and subsequent G 2/M checkpoint response. Furthermore, results also support a role for Rac1 in promoting cell survival after irradiation treatment.
AB - Introduction: In response to gamma-irradiation (IR)-induced double-strand DNA breaks, cells undergo cell-cycle arrest, allowing time for DNA repair before reentering the cell cycle. G 2/M checkpoint activation involves activation of ataxia telangiectasia mutated (ATM)/ATM- and rad3-related (ATR) kinases and inhibition of Cdc25 phosphatases, resulting in inhibition of Cdc2 kinase and subsequent G 2/M cell-cycle arrest. Previous studies from our laboratory showed that the G 2/M checkpoint activation after IR exposure of MCF-7 breast cancer cells is dependent on the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signaling. In the present studies, we investigated the role of Ras-related C3 botulinum toxin substrate 1 (Rac1) guanosine triphosphatase (GTPase) in IR-induced G 2/M checkpoint response and ERK1/2 activation, as well as in cell survival after IR.Methods: With Rac1-specific inhibitor, dominant negative mutant Rac1 (N17Rac1) and specific small interfering RNA, the effect of Rac1 on IR-induced G 2/M checkpoint response and ERK1/2 activation was examined in human breast cancer cells. In addition, the effect of Rac1 on cell survival after irradiation was assessed by using Rac1-specific inhibitor.Results: IR exposure of MCF-7 breast cancer cells was associated with a marked activation of Rac1 GTPase. Furthermore, inhibition of Rac1 by using specific inhibitor, dominant-negative Rac1 mutant, or specific siRNA resulted in attenuation of IR-induced G 2/M arrest and concomitant diminution of IR-induced activation of ATM, ATR, Chk1, and Chk2 kinases, as well as phosphorylation of Cdc2-Tyr15. Moreover, Rac1 inhibition or decreased Rac1 expression also abrogated IR-induced phosphorylation of mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) and ERK1/2. Ultimately, inhibition of Rac1 markedly increased cellular sensitivity to IR exposure, which involves induction of apoptosis.Conclusion: Studies in this report suggest that Rac1 GTPase plays an essential role in the activation of IR-induced ERK1/2 signaling and subsequent G 2/M checkpoint response. Furthermore, results also support a role for Rac1 in promoting cell survival after irradiation treatment.
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U2 - 10.1186/bcr3164
DO - 10.1186/bcr3164
M3 - Article
C2 - 22494620
AN - SCOPUS:84859513105
SN - 1465-5411
VL - 14
JO - Breast Cancer Research
JF - Breast Cancer Research
IS - 2
M1 - R60
ER -