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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

¹ Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland and ² Greenebaum Cancer Center, University of Maryland, Baltimore, Maryland

Requests for reprints: Yves Pommier, Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Building 37, Room 5068, 37 Convent Drive, Bethesda, MD 20892-4255. Phone: 301-496-5944; Fax: 301-401-0752; pommier{at}nih.gov.

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 (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.

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