DNA-Protein Cross-links and Replication-Dependent Histone H2AX Phosphorylation Induced by Aminoflavone (NSC 686288), a Novel Anticancer Agent Active against Human Breast Cancer Cells

Ling-hua Meng, Glenda Kohlhagen, Zhi-yong Liao, Smitha Antony, Edward Sausville, Yves Pommier
2005 Cancer Research  
Aminoflavone (5-amino-2,3-fluorophenyl-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one) (NSC 686288) is a candidate for possible advancement to phase I clinical trial. Aminoflavone has a unique activity profile in the NCI 60 cell lines (COMPARE analysis; http://www.dtp.nci.nih.gov/docs/ dtp_search.html), and exhibits potent cellular and animal antitumor activity. To elucidate the mechanism of action of aminoflavone, we studied DNA damage in MCF-7 cells. Aminoflavone induced DNA-protein cross-links
more » ... rotein cross-links (DPC) and DNA single-strand breaks (SSB). Aminoflavone induced high levels of DPC and much lower level of SSB than camptothecin, which induces equal levels of DPC and SSB due to the trapping topoisomerase I-DNA complexes. Accordingly, neither topoisomerase I nor topoisomerase II were detectable in the aminoflavone-induced DPC. Aminoflavone also induced dose-and time-dependent histone H2AX phosphorylation (;-H2AX). ;-H2AX foci occurred with DPC formation, and like DPC, persisted after aminoflavone removal. Aphidicolin prevented ;-H2AX formation, suggesting that ;-H2AX foci correspond to replication-associated DNA double-strand breaks. Accordingly, no ;-H2AX foci were found in proliferating cell nuclear antigen-negative or in mitotic cells. Bromodeoxyuridine incorporation and fluorescence-activated cell sorting analyses showed DNA synthesis inhibition uniformly throughout the S phase after exposure to aminoflavone. Aminoflavone also induced RPA2 and p53 phosphorylation, and induced p21 Waf1/Cip1 and MDM2, demonstrating S-phase checkpoint activation. These studies suggest that aminoflavone produces replication-dependent DNA lesions and S-phase checkpoint activation following DPC formation. ;-H2AX may be a useful clinical marker for monitoring the efficacy of aminoflavone in tumor therapies. (Cancer Res 2005; 65(12): 5337-43) Materials and Methods Cell culture. Human breast cancer MCF-7 cells were obtained from American Type Culture Collection (Manassas, VA) and were grown at 37jC in the presence of 5% CO 2 in DMEM supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY), 100 units/mL penicillin, and 100 Ag/mL streptomycin. Drugs and chemicals. Aminoflavone was obtained from Kyowa Hakko Kogyo. Aphidicolin, nocodazole, bromodeoxyuridine (BrdUrd), camptothecin, and etoposide (VP-16) were purchased from Sigma Chemical Co. (St. Louis, MO). Aminoflavone, aphidicolin, and camptothecin were dissolved at a concentration of 10 mmol/L and nocodazole was dissolved at 2 mg/mL in Requests for reprints: Yves
doi:10.1158/0008-5472.can-05-0003 pmid:15958581 fatcat:7pu3lp63ujc3jbjck4nhqkfu2i