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.
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 (gamma-H2AX). Gamma-H2AX foci occurred with DPC formation, and like DPC, persisted after aminoflavone removal. Aphidicolin prevented gamma-H2AX formation, suggesting that gamma-H2AX foci correspond to replication-associated DNA double-strand breaks. Accordingly, no gamma-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. Gamma-H2AX may be a useful clinical marker for monitoring the efficacy of aminoflavone in tumor therapies.
Meng
Ling-hua
LH
Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
Kohlhagen
Glenda
G
Liao
Zhi-yong
ZY
Antony
Smitha
S
Sausville
Edward
E
Pommier
Yves
Y
D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000096:biosynthesis; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000494:pharmacology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects; D004273:DNA, Neoplasm / Q000187:drug effects / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins / Q000096:biosynthesis; D010766:Phosphorylation / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D051736:Proto-Oncogene Proteins c-mdm2; D051716:Replication Protein A; D016196:S Phase / Q000187:drug effects / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D003432:Cross-Linking Reagents; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005419:Flavonoids; C468783:H2AX protein, human; D006657:Histones; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D016159:Tumor Suppressor Protein p53; C413760:aminoflavone; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2