Covalent binding of the anticancer drug ellipticine to DNA in V79 cells transfected with human cytochrome P450 enzymes.
Ellipticine is a potent antineoplastic agent whose mechanism of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms covalent DNA adducts and that the formation of the major adduct is dependent on the activation of ellipticine by cytochrome P450 (CYP). We examined a panel of genetically engineered V79 cell lines including the parental line V79MZ and recombinant cells expressing the human CYP enzymes CYP1A1, CYP1A2 or CYP3A4 for their ability to activate ellipticine. The extent of activation was determined by analysing DNA adducts by 32P-postlabelling. Ellipticine was found to be toxic to all V79 cell lines with IC(50) values ranging from 0.25 to 0.40 microM. The nuclease P1 version of the 32P-postlabelling assay yielded a similar pattern of ellipticine-DNA adducts with two major adducts in all cells, the formation of only one of which was dependent on CYP activity. This pattern is identical to that detected in DNA reacted with ellipticine and the reconstituted CYP enzyme system in vitro as confirmed by HPLC of the isolated adducts. Total adduct levels ranged from 2 to 337 adducts per 10(8) nucleotides, in the parental line and in V79 expressing CYP3A4, respectively. As in vitro, human CYP1A2 and CYP1A1 were less active. The results presented here are the first report showing the formation of CYP-mediated covalent DNA adducts by ellipticine in cells in culture, and confirm the formation of covalent DNA adducts as a new mechanism of ellipticine action.
Division of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. e.frei@dkfz.de
Eva
E
Frei
Christian A
CA
Bieler
Volker M
VM
Arlt
Manfred
M
Wiessler
Marie
M
Stiborová
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001665:Binding Sites; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D018736:DNA Adducts / Q000187:drug effects; D004611:Ellipticines / Q000494:pharmacology*; D006801:Humans; D010761:Phosphorus Radioisotopes; D014162:Transfection
D000970:Antineoplastic Agents; D018736:DNA Adducts; D004611:Ellipticines; D010761:Phosphorus Radioisotopes; C034192:ellipticine; D004247:DNA; D003577:Cytochrome P-450 Enzyme System