Generation of DNA double strand breaks during trabectedin DNA damage measured through induction of    γ   H2AX Free

1965

Trabectedin (ET-743, Yondelis^®) is a marine derived anticancer agent which forms covalent adducts with guanine residues present in the minor groove of the DNA. In order to investigate whether Trabectedin generates DNA double strand breaks, either directly or during the repair of the DNA damage, we have analyzed the presence of phosphorylated H2AX (γH2AX) nuclear foci in cells exposed to this drug. To this end, MRC-5 fibroblasts and EBV-transformed lymphoblasts were treated with Trabectedin (1, 3 and 10nM), with mitomycine C (MMC; 33 and 100nM) or exposed to 5 Gy of ionizing radiation (IR), and then analyzed for the presence of γH2AX foci. As expected, γH2AX nuclear foci were observed immediately after cell irradiation. In cells treated either with MMC (which generates inter-strands DNA cross-links) or with Trabectedin, a delayed formation of γH2AX foci was, however, clearly observed. Flow cytometry offers the possibility of rapidly analyzing γH2AX in a high number of cells and additionally facilitates the analysis of DSBs along the cell cycle. These analyses confirmed the immediate generation of IR-induced DSBs in both cell types, as well as the delayed induction of DSBs both in MMC- and Trabectedin -treated cells. Additionally, cytometric studies showed a predominant accumulation of DSBs in cells entering into cell division. Our studies suggest that the generation of DSBs constitutes an intermediate process required for the repair of the DNA damage produced by Trabectedin. Further studies are in progress aiming to discern the mechanism involved in the repair of the DNA damage generated by Trabectedin, and also the susceptibility to this new anticancer agent against cancer cells lacking an efficient repair of DBSs.