Ecteinascidin (Et-743, Yondelis®), an alkaloid derived from the marine tunicate Ecteinascidia turbinate, binds to the minor groove of DNA and alkylates the N2 position of guanine, with a sequence preference for GC-rich triplets. We have shown previously that Et-743 specifically targets transcription-coupled nucleotide excision repair (TCR) and induces TCR-dependent DNA single-strand breaks. By contrast to others alkylating agents, Et-743 is less active in cells deficient for the components of the TCR machinery (XPA, XPD, XPF, XPG, CSA, CSB) than in the complemented or wild type cells (Takebayashi et al. Nature Medicine 2001; 7:961-966). We now report that histone H2AX knockout cells are less sensitive than their wild-type counterparts. To study whether DNA double-strand breaks (DSBs) are implicated in cellular response to Et-743, we examined histone H2AX phosphorylation (gH2AX), a marker of DSBs, in several cell lines treated with Et-743. Et-743 induced DSBs in all cell lines examined. The gH2AX foci were detected with exposures as short as 30 minutes, and at pharmacological doses (as low as 0.05 nM). gH2AX quantitation and close examination revealed that cell lines could be divided into 2 groups. The first group includes XPD-C (XPD-deficient fibroblasts complemented for XPD) and HCT116-M (HCT116 complemented for Mre11 colon carcinoma cells). XPD-C and HCT116-M showed very strong gH2AX induction both in terms of percentage of positive cells and of staining intensity. The second group includes XPD and HCT116 cells (deficient for TCR and Mre11, respectively) in which gH2AX induction was more moderate. To elucidate whether gH2AX foci formation was related to transcription or replication, we used aphidicolin as a replication inhibitor, and DRB as a transcription inhibitor. In XPD-C and HCT116-M, which exhibit the intense gH2AX response, gH2AX formation was inhibited by DRB, whereas aphidicolin had no effect. By contrast, in XPD (deficient for TCR) and HCT116 (deficient for Mre11), DRB had no effect but aphidicolin completely inhibited gH2AX foci formation. These results demonstrate that gH2AX can be used as a pharmacodynamic biomarker for Et-743 in tumor cells. Moreover, the differential effects of aphidicolin and DRB, and the TCR- and Mre11-dependent responses suggest the following hypothesis. Et-743 can induce DSBs (revealed by gH2AX) by two mechanisms. In cells proficient for TCR and with normal Mre11, the DSBs are primarily due to the trapping of transcription-coupled NER complex by Et-743, and are converted to DSBs by Mre11-dependent mechanisms. By contrast in TCR-deficient cells (XPD) and Mre11-deficient cells (HCT116),

[Proc Amer Assoc Cancer Res, Volume 47, 2006]