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Vol. 19, Issue 9, 3969-3981, September 2008

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Transcription-coupled DNA Double-Strand Breaks Are Mediated via the Nucleotide Excision Repair and the Mre11-Rad50-Nbs1 Complex

Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255

Submitted February 28, 2008; Revised June 20, 2008; Accepted June 29, 2008
Monitoring Editor: John L. Cleveland

The cellular activity of Yondelis (trabectedin, Ecteinascidin 743, Et743) is known to depend on transcription-coupled nucleotide excision repair (TCR). However, the subsequent cellular effects of Et743 are not fully understood. Here we show that Et743 induces both transcription- and replication-coupled DNA double-strand breaks (DSBs) that are detectible by neutral COMET assay and as -H2AX foci that colocalize with 53BP1, Mre11, Ser¹⁹⁸¹-pATM, and Thr⁶⁸-pChk2. The transcription coupled-DSBs (TC-DSBs) induced by Et743 depended both on TCR and Mre11-Rad50-Nbs1 (MRN) and were associated with DNA-PK–dependent -H2AX foci. In contrast to DNA-PK, ATM phosphorylated H2AX both in NER-proficient and -deficient cells, but its full activation was dependent on H2AX as well as DNA-PK, suggesting a positive feedback loop: DNA-PK--H2AX-ATM. Knocking-out H2AX or inactivating DNA-PK reduced Et743's antiproliferative activity, whereas ATM and MRN tended to act as survival factors. Our results highlight the interplays between ATM and DNA-PK and their impacts on H2AX phosphorylation and cell survival. They also suggest that -H2AX may serve as a biomarker in patients treated with Et743 and that molecular profiling of tumors for TCR, MRN, ATM, and DNA-PK might be useful to anticipate tumor response to Et743 treatment.

Address correspondence to: Yves Pommier (pommier{at}nih.gov)

Abbreviations used: APD, aphidicolin; AT, ataxia telangiectasia; ATM, ataxia telangiectasia–mutated gene; CS, Cockayne syndrome; DNA-PK, DNA-dependent protein kinase; DRB, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole; DSB, DNA double-strand break; Et743, ecteinascidin 743; GGR, global genome repair; -H2AX, histone H2AX phosphorylated on serine 139; MRN, Mre11-Rad50-Nbs1; NER, nucleotide excision repair; RC-DSB, replication-coupled DSB; RPA, replication protein A; TC-DSB, transcription-coupled DSB; TCR, transcription-coupled nucleotide excision repair; XP, xeroderma pigmentosum.