title: RAC1 GTPase plays an important role in γ-irradiation induced G2/M checkpoint activation.
abstract: BACKGROUND
  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. G2/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 G2/M cell-cycle arrest. Previous studies from our laboratory showed that the G2/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 G2/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 G2/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 G2/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.
  
  
  CONCLUSIONS
  Studies in this report suggest that Rac1 GTPase plays an essential role in the activation of IR-induced ERK1/2 signaling and subsequent G2/M checkpoint response. Furthermore, results also support a role for Rac1 in promoting cell survival after irradiation treatment.
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE, USA. yyan@unmc.edu
authors.forename: Ying
authors.initials: Y
authors.lastname: Yan
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Greer
authors.forename: Phu T
authors.initials: PT
authors.lastname: Cao
authors.forename: Ryan H
authors.initials: RH
authors.lastname: Kolb
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Cowan
mesh_terms: D000634:Aminoquinolines / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology / Q000532:radiotherapy*; D016203:CDC2 Protein Kinase; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000528:radiation effects; D000071877:Checkpoint Kinase 1; D019926:Cyclin B / Q000378:metabolism; D018844:Cyclin-Dependent Kinases; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D059565:G2 Phase Cell Cycle Checkpoints / Q000528:radiation effects*; D005720:Gamma Rays; D006801:Humans; D059566:M Phase Cell Cycle Checkpoints / Q000528:radiation effects*; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000634:Aminoquinolines; D018797:Cell Cycle Proteins; D019926:Cyclin B; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C487513:NSC 23766; D011743:Pyrimidines; C107953:RAC1 protein, human; D025521:Tumor Suppressor Proteins; D014443:Tyrosine; D011494:Protein Kinases; C482128:MAP2K2 protein, human; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482125:MAP2K1 protein, human; D020830:rac1 GTP-Binding Protein