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IKK? regulates the repair of DNA double-strand breaks induced by ionizing radiation in MCF-7 breast cancer cells.

ABSTRACT: Activation of the IKK-NF?B pathway increases the resistance of cancer cells to ionizing radiation (IR). This effect has been largely attributed to the induction of anti-apoptotic proteins by NF?B. Since efficient repair of DNA double strand breaks (DSBs) is required for the clonogenic survival of irradiated cells, we investigated if activation of the IKK-NF?B pathway also regulates DSB repair to promote cell survival after IR. We found that inhibition of the IKK-NF?B pathway with a specific IKK? inhibitor significantly reduced the repair of IR-induced DSBs in MCF-7 cells. The repair of DSBs was also significantly inhibited by silencing IKK? expression with IKK? shRNA. However, down-regulation of IKK? expression with IKK? shRNA had no significant effect on the repair of IR-induced DSBs. Similar findings were also observed in IKK? and/or IKK? knockout mouse embryonic fibroblasts (MEFs). More importantly, inhibition of IKK? with an inhibitor or down-regulation of IKK? with IKK? shRNA sensitized MCF-7 cells to IR-induced clonogenic cell death. DSB repair function and resistance to IR were completely restored by IKK? reconstitution in IKK?-knockdown MCF-7 cells. These findings demonstrate that IKK? can regulate the repair of DSBs, a previously undescribed and important IKK? kinase function; and inhibition of DSB repair may contribute to cance cell radiosensitization induced by IKK? inhibition. As such, specific inhibition of IKK? may represents a more effective approach to sensitize cancer cells to radiotherapy.

SUBMITTER: Wu L

PROVIDER: S-EPMC3072401 | biostudies-other | 2011 Apr

REPOSITORIES: biostudies-other

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IKKβ regulates the repair of DNA double-strand breaks induced by ionizing radiation in MCF-7 breast cancer cells.

Wu Lixian L Shao Lijian L An Ningfei N Wang Junru J Pazhanisamy Senthil S Feng Wei W Hauer-Jensen Martin M Miyamoto Shigeki S Zhou Daohong D

PloS one 20110407 4

Activation of the IKK-NFκB pathway increases the resistance of cancer cells to ionizing radiation (IR). This effect has been largely attributed to the induction of anti-apoptotic proteins by NFκB. Since efficient repair of DNA double strand breaks (DSBs) is required for the clonogenic survival of irradiated cells, we investigated if activation of the IKK-NFκB pathway also regulates DSB repair to promote cell survival after IR. We found that inhibition of the IKK-NFκB pathway with a specific IKKβ ...[more]

PMID: 21490922

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Project description:Reptin/RUVBL2 is overexpressed in most hepatocellular carcinomas and is required for the growth and viability of HCC cells. Reptin is involved in several chromatin remodeling complexes, some of which are involved in the detection and repair of DNA damage, but data on Reptin involvement in the repair of DNA damage are scarce and contradictory. Our objective was to study the effects of Reptin silencing on the repair of DNA double-strand breaks (DSB) in HCC cells. Treatment of HuH7 cells with etoposide (25 ?M, 30 min) or ? irradiation (4 Gy) increased the phosphorylation of H2AX by 1.94 ± 0.13 and 2.0 ± 0.02 fold, respectively. These values were significantly reduced by 35 and 65 % after Reptin silencing with inducible shRNA. Irradiation increased the number of BRCA1 (3-fold) and 53BP1 foci (7.5 fold). Depletion of Reptin reduced these values by 62 and 48%, respectively. These defects in activation and/or recruitment of repair proteins were not due to a decreased number of DSBs as measured by the COMET assay. All these results were confirmed in the Hep3B cell line. Protein expression of ATM and DNA-PKcs, the major H2AX kinases, was significantly reduced by 52 and 61 % after Reptin depletion whereas their mRNA level remained unchanged. Phosphorylation of Chk2, another ATM target, was not significantly altered. Using co-immunoprecipitation, we showed an interaction between Reptin and DNA-PKcs. The half-life of newly-synthesized DNA-PKcs was reduced when Reptin was silenced. Finally, depletion of Reptin was synergistic with etoposide or ? irradiation to reduce cell growth and colony formation. In conclusion, Reptin is an important cofactor for the repair of DSBs. Our data, combined with those of the literature suggests that it operates at least in part by regulating the expression of DNA-PKcs by a stabilization mechanism. Overexpression of Reptin in HCC could be a factor of resistance to treatment, consistent with the observed overexpression of Reptin in subgroups of chemo-resistant breast and ovarian cancers.

Dataset Information

IKK? regulates the repair of DNA double-strand breaks induced by ionizing radiation in MCF-7 breast cancer cells.

Publications

IKKβ regulates the repair of DNA double-strand breaks induced by ionizing radiation in MCF-7 breast cancer cells.

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