CYP1A1 activation of aminoflavone leads to DNA damage in human tumor cell lines.
OBJECTIVE
Aminoflavone (5-amino-2,3-fluorophenyl)-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one; AF; NSC 686288) is a novel anticancer agent with a unique pattern of growth inhibitory activity in the National Cancer Institute (NCI) 60 tumor cell line screen. Phase I clinical trials with AF will begin soon. We previously demonstrated extensive metabolism of AF by cytochrome P450 (CYP) 1A1 and CYP1A2, metabolic activation, formation of irreversible protein and DNA adducts and p53 stabilization in sensitive, but not resistant, human tumor cell lines treated with AF [9]. The present studies focus on the effects of AF on cellular DNA and cellular responses to DNA damage.


METHODS
Phosphorylation of H2AX in MCF7 cells treated with AF was determined with immunofluorescence. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium) assays were used to determine the effect of cotreatment with caffeine or wortmannin, inhibitors of ataxia-telangiectasia-mutated protein (ATM) and ATR (ATM- and rad3-related protein), on the AF IC(50) values for MCF7 cells. DNA damage in MCF7 cells treated with AF was determined by alkaline elution. DNA-topoisomerase complex stabilization was ascertained by the ICE (in vitro complex of enzyme) assay.


RESULTS
Treatment of sensitive cells with AF resulted in phosphorylation of H2AX, a histone 2A variant that is phosphorylated in response to DNA damage. AF IC(50) values for MCF7 cells were lowered by cotreatment with caffeine or wortmannin, further implicating DNA damage in AF cytotoxicity. There was no evidence of DNA-DNA cross-linking in sensitive cells, but protein-associated single-strand breaks were observed after AF treatment. Although this pattern of DNA damage is commonly associated with topoisomerase poisons, there was no evidence for AF-induced stabilization of either topoisomerase I- or II-DNA complexes.


CONCLUSIONS
These studies further implicate DNA damage in the cytotoxicity of AF and identify biochemical features of that damage including formation of protein-associated single-strand breaks not involving topoisomerase I or II.
Pobst
Lori J
LJ
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Guggenheim 13, 200 First Street S.W., Rochester, MN, 55905, USA.
Ames
Matthew M
MM
D000730:Androstadienes / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D045744:Cell Line, Tumor; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D005260:Female; D005419:Flavonoids / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D006657:Histones / Q000378:metabolism*; D006801:Humans; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005419:Flavonoids; C468783:H2AX protein, human; D006657:Histones; D010726:Phosphodiesterase Inhibitors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D025521:Tumor Suppressor Proteins; C413760:aminoflavone; D002110:Caffeine; D019363:Cytochrome P-450 CYP1A1; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D000077191:Wortmannin