Defective Mre11-dependent Activation of Chk2 by Ataxia Telangiectasia Mutated in Colorectal Carcinoma Cells in Response to Replication-dependent DNA Double Strand Breaks

Abstract

The Mre11⅐Rad50⅐Nbs1 (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 MRNdeficient 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 G 2 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.

PKB/Akt promotes DSB repair in cancer cells through upregulating Mre11 expression following ionizing radiation

“…Ding et al, 2006;Williams et al, 2007Williams et al, , 2008. It also can stabilize Rad50 and Nbs1 polypeptides and act as a chaperone for the MRN complex (Takemura et al, 2006). In this study, we found that knockdown of Mre11 obviously impaired the repair of radiation-induced DNA-DSB damage in CNE2 and HeLa cells (Figure 2).…”

Cellular processing determinants for the activation of damage signals in response to topoisomerase I-linked DNA breakage

“…Consistent with activation of multiple DDR, CPT treatment caused the time-and dose-dependent phosphorylation of p53 at Ser15 (p53 15p and its elevation in protein level), Chk2 at Thr68 (Chk2 68p ), RPA2 (RPA p ) (Supplementary information, Figure S1B-S1D). In addition, we have also found that both phosphorylation of H2AX (γ-H2AX) and Chk1 at Ser345 (Chk1 345p ) were stimulated in response to CPT treatment (Supplementary information, Figure S1E) [11][12][13]16]. Using the elevated p53 protein level as an example, we showed that the CPT-induced DDR was similarly activated in two types of treatment protocols, the short-term treatment with higher CPT doses (1-20 µM, Supplementary information, Figure S1B) and the long-term exposure with the clinically relevant doses (1-64 nM, Supplementary information, Figure S1C).…”

“…The structural information [8][9][10] and recent studies on the roles of cellular activities and pathways for TOPcc-induced DDR [11][12][13][14][15][16] have suggested that the interactions between TOPcc and cellular activities, such as proteasomal degradation, might process DNA break embedded in the enzyme center into more detectable DNA lesions. These DNA lesions are differentially detected and then followed by the activation of various DDR.…”

Knockdown of Chk1 sensitizes human colon carcinoma HCT116 cells in a p53-dependent manner to lidamycin through abrogation of a G2/M checkpoint and induction of apoptosis

“…The Mre11, Rad50 and Nbs1 proteins (MRN) complex recruits ATM at sites of DNA damage and directly activates the ATM-Chk2 pathway. 43,44 However, MRN complex is deficient in HCT116 cells due to the deficiency of its central subunit Mre11. 43 It seems that Chk2 can not been activated by ATM in Mre11 deficient HCT116 cells.…”

“…43,44 However, MRN complex is deficient in HCT116 cells due to the deficiency of its central subunit Mre11. 43 It seems that Chk2 can not been activated by ATM in Mre11 deficient HCT116 cells. By contraries, our data showed that Chk2 was activated by LDM in HCT116 cells.…”