Abstract
Methylating agents are first-line therapeutics for gliomas and malignant melanomas. They attack DNA at various sites, and both O6-methylguanine and N-methylated base adducts contribute to the killing response. The mechanism of cellular defense against these agents primarily involves O6-methylguanine-DNA methyltransferase (MGMT) and base excision repair (BER). Here, we determined whether a key protein involved in DNA double-strand break (DSB) recognition and signaling, nibrin (NBN alias NBS-1), plays a role in the cellular defense against methylating agents. Comparing NBN mutated fibroblasts and lymphoblastoid cells from patients suffering from Nijmegen breakage syndrome, we show that NBN mutants are clearly more sensitive to N-methyl-N′-nitro-N-nitrosoguanidine and temozolomide than the corresponding wild-type cells. Hypersensitivity was due to the induction of both apoptosis and necrosis. The mismatch repair proteins MSH2, MSH6, MLH1, and PMS2 were expressed at a similar level in the cell lines and BER was not affected by NBN mutation. Because MGMT expression abrogated the hypersensitivity of NBN mutated cells, we conclude that O6-methylguanine-derived lesions are responsible for triggering the response. Down-regulation of NBN in melanoma cells by small interfering RNA rendered them more sensitive to temozolomide, suggesting that NBN is a novel modulator of temozolomide sensitivity. Because NBN is part of the MRN complex, which recognizes DSBs, the data strongly indicate that MRN is critically involved in DSB processing after O6-methylguanine induction. The data provide first evidence that NBN is involved in the cellular defense against O6-methylguanine-inducing agents such as temozolomide and identify NBN as a critical target of methylating anticancer drug resistance.
Footnotes
This work was supported by the Deutsche Forschungsgemeinschaft [Grant KA724/13].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.066076.
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ABBREVIATIONS:
- DSB
- double-strand break
- HR
- homologous recombination
- NHEJ
- nonhomologous end joining
- NBN
- nibrin
- NBS
- Nijmegen Breakage Syndrome
- TMZ
- temozolomide
- MNNG
- N-methyl-N′-nitro-N-nitrosoguanidine
- O6MeG
- O6-methylguanine
- O6BG
- O6-benzylguanine
- MMR
- mismatch repair
- MGMT
- O6-methylguanine-DNA methyltransferase
- BER
- base excision repair
- wt
- wild type
- mt
- mutant
- siRNA
- small interfering RNA
- O6BG
- O6-benzylguanine
- PI
- propidium iodide
- PARP
- poly (ADP-ribose) polymerase
- WST
- water-soluble tetrazolium.
- Received May 4, 2010.
- Accepted August 19, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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