Defective Mre11-dependent activation of Chk2 by ataxia telangiectasia mutated in colorectal carcinoma cells in response to replication-dependent DNA double strand breaks.
The Mre11.Rad50.Nbs1 (MRN) complex binds DNA double strand breaks to repair DNA and activate checkpoints. We report MRN deficiency in three of seven colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study the involvement of MRN in replication-mediated DNA double strand breaks, we examined checkpoint responses to camptothecin, which induces replication-mediated DNA double strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation was independent of MRN. Chk2 activation was ataxia telangiectasia mutated (ATM)-dependent and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRN-deficient HCT116 cells restored Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 down-regulation by small interference RNA (siRNA) in HT29 cells inhibited Chk2 activation and down-regulated Nbs1 and Rad50. Proteasome inhibition also restored Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in three of four MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, six of seven colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double strand breaks. We propose that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G2 arrest, and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers.
Takemura
Haruyuki
H
Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892-4255, USA.
Rao
V Ashutosh
VA
Sordet
Olivier
O
Furuta
Takahisa
T
Miao
Ze-Hong
ZH
Meng
Linghua
L
Zhang
Hongliang
H
Pommier
Yves
Y
D064007:Ataxia Telangiectasia Mutated Proteins; D002277:Carcinoma / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D064447:Checkpoint Kinase 2; D015179:Colorectal Neoplasms / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology*; D004354:Drug Screening Assays, Antitumor; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D000076228:MRE11 Homologue Protein; D008954:Models, Biological; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D025521:Tumor Suppressor Proteins / Q000378:metabolism / Q000502:physiology*
D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; D025521:Tumor Suppressor Proteins; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; D000076228:MRE11 Homologue Protein