Transcription-coupled DNA double-strand breaks are mediated via the nucleotide excision repair and the Mre11-Rad50-Nbs1 complex.
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 gamma-H2AX foci that colocalize with 53BP1, Mre11, Ser(1981)-pATM, and Thr(68)-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 gamma-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-gamma-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 gamma-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.
Guirouilh-Barbat
Josée
J
Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
Redon
Christophe
C
Pommier
Yves
Y
D017766:Acid Anhydride Hydrolases; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002470:Cell Survival; D020552:Comet Assay; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004149:Dioxoles / Q000494:pharmacology; D006657:Histones / Q000737:chemistry; D006801:Humans; D000076228:MRE11 Homologue Protein; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D044005:Tetrahydroisoquinolines / Q000494:pharmacology; D000077606:Trabectedin; D014158:Transcription, Genetic
D018906:Antineoplastic Agents, Alkylating; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D004149:Dioxoles; C468783:H2AX protein, human; D006657:Histones; C121766:MRE11 protein, human; C112156:NBN protein, human; D009687:Nuclear Proteins; D044005:Tetrahydroisoquinolines; D051747:DNA-Activated Protein Kinase; C497264:Prkdc protein, mouse; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes; D000077606:Trabectedin