TY - JOUR T1 - Induction of Biphasic DNA Double Strand Breaks and Activation of Multiple Repair Protein Complexes by DNA Topoisomerase I Drug 7-Ethyl-10-hydroxy-camptothecin JF - Molecular Pharmacology JO - Mol Pharmacol SP - 742 LP - 748 DO - 10.1124/mol.61.4.742 VL - 61 IS - 4 AU - Jiaxi Wu AU - Ming-biao Yin AU - Gunnar Hapke AU - Károly Tóth AU - Youcef M. Rustum Y1 - 2002/04/01 UR - http://molpharm.aspetjournals.org/content/61/4/742.abstract N2 - Camptothecins demonstrate a broad spectrum of antitumor activity. Although they are known to trap DNA topoisomerase I on DNA, form cleavable complexes, and generate DNA breaks upon collision with DNA or RNA polymerases, the precise mechanisms predictive for antitumor activity remain to be identified. Recent studies using panels of colorectal and breast cancer cell lines indicate that events downstream of cleavable complexes are more relevant. In this study, we chose SN-38, an active metabolite of irinotecan, to characterize DNA double strand breaks and repair mechanisms induced by this type of drugs using a human head and neck squamous cell carcinoma cell line A253. The results showed that 2-h exposure of cells to an IC50concentration of SN-38 induces biphasic DNA double-strand break (DSBs): an immediate phase, which was greatly reduced within 8 h, and a lagging phase, culminating 24 h after drug removal. Three DNA double-strand break repair protein complexes were activated: DNA-dependent protein kinase (DNA-PK), NBS1-MRE11-RAD50, and BRCA1. Aphidicolin, a DNA polymerase inhibitor, abolished both phase I DSBs and the activation of repair protein complexes, suggesting that they resulted from the collision between the cleavable complex and DNA polymerase of S-phase cells. This is in contrast to ionizing radiation-induced activation of DNA-PK and NBS1-MRE11-RAD50 complexes that occur predominantly among non–S-phase cells. The trigger for phase II DSBs cannot be abolished by aphidicolin. The data also indicate that DNA fragments in the size of 50 to 200 kilobases were detected in the lagging phase. This suggests that the late DNA DSBs were associated with apoptotic cell death. ER -