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Research Articles: Therapeutics, Targets, and Development

The Mre11/Rad50/Nbs1 complex interacts with the mismatch repair system and contributes to temozolomide-induced G₂ arrest and cytotoxicity

Department of Neurological Surgery and The Brain Tumor Research Center, UCSF Comprehensive Cancer Center, The University of California-San Francisco, San Francisco, California

Requests for reprints: Russell O. Pieper, UCSF Cancer Center, The University of California-San Francisco, Room N219, 2340 Sutter Street, San Francisco, CA 94115-0875. Phone: 415-502-7132; Fax: 415-502-6779. E-mail: rpieper{at}cc.ucsf.edu

Abstract

The chemotherapeutic agent temozolomide produces O⁶-methylguanine (O6MG) in DNA, which triggers futile DNA mismatch repair, DNA double-strand breaks (DSB), G₂ arrest, and ultimately cell death. Because the protein complex consisting of Mre11/Rad50/Nbs1 (MRN complex) plays a key role in DNA damage detection and signaling, we asked if this complex also played a role in the cellular response to temozolomide. Temozolomide exposure triggered the assembly of MRN complex into chromatin-associated nuclear foci. MRN foci formed significantly earlier than -H2AX and 53BP1 foci that assembled in response to temozolomide-induced DNA DSBs. MRN foci formation was suppressed in cells that incurred lower levels of temozolomide-induced O6MG lesions and/or had decreased mismatch repair capabilities, suggesting that the MRN foci formed not in response to temozolomide-induced DSB but rather in response to mismatch repair processing of mispaired temozolomide-induced O6MG lesions. Consistent with this idea, the MRN foci colocalized with those of proliferating cell nuclear antigen (a component of the mismatch repair complex), and the MRN complex component Nbs1 coimmunoprecipitated with the mismatch repair protein Mlh1 specifically in response to temozolomide treatment. Furthermore, small inhibitory RNA–mediated suppression of Mre11 levels decreased temozolomide-induced G₂ arrest and cytotoxicity in a manner comparable to that achieved by suppression of mismatch repair. These data show that temozolomide-induced O6MG lesions, acted upon by the mismatch repair system, drive formation of the MRN complex foci and the interaction of this complex with the mismatch repair machinery. The MRN complex in turn contributes to the control of temozolomide-induced G₂ arrest and cytotoxicity, and as such is an additional determining factor in glioma sensitivity to DNA methylating chemotherapeutic drugs such as temozolomide. [Mol Cancer Ther 2006;5(11):2757–66]

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Received 4/ 3/06; revised 8/24/06; accepted 9/11/06.

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