title: Topoisomerase I-mediated cytotoxicity of N-methyl-N'-nitro-N-nitrosoguanidine: trapping of topoisomerase I by the O6-methylguanine.
abstract: Alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are known to covalently link alkyl groups at the position 6 of guanines (O6MG) in DNA. O6-alkylguanine-DNA alkyltransferase (AGT) specifically removes the methyl group of the O6MG. Using purified human topoisomerase I (Top1), we found an 8-10-fold enhancement of Top1 cleavage complexes when O6MG is incorporated in oligonucleotides at the +1 position relative to a unique Top1 cleavage site. Top1 poisoning by O6MG is attributable to a decrease of the Top1-mediated DNA religation as well as an increase in the enzyme cleavage step. Increased cleavage is probably linked to a change in the hydrogen bonding pattern, such as in the case of the 8-oxoguanine, whereas inhibition of religation could be attributed to altered base pairing, such as abasic sites or base mismatches, because incorporation of a 6-thioguanine did not affect Top1 activity. Top1-DNA covalent complexes are also induced in MNNG-treated CHO cells constitutively lacking the AGT enzyme. Conversely, no increase could be detected in CHO cells transfected with the wild-type human AGT. Moreover, we show that yeasts overexpressing the human Top1 are more sensitive to MNNG, whereas knock-out Top1 strain cells display some resistance to the drug. Altogether, these results suggest a role for Top1 poisoning by alkylated bases in the antiproliferative activity of alkylating agents as well as in the DNA lesions resulting from endogenous and carcinogenic DNA modifications.
authors.affiliation: Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
authors.forename: P
authors.initials: P
authors.lastname: Pourquier
authors.forename: J L
authors.initials: JL
authors.lastname: Waltman
authors.forename: Y
authors.initials: Y
authors.lastname: Urasaki
authors.forename: N A
authors.initials: NA
authors.lastname: Loktionova
authors.forename: A E
authors.initials: AE
authors.lastname: Pegg
authors.forename: J L
authors.initials: JL
authors.lastname: Nitiss
authors.forename: Y
authors.initials: Y
authors.lastname: Pommier
mesh_terms: D000477:Alkylating Agents / Q000633:toxicity; D000818:Animals; D016466:CHO Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000633:toxicity*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology; D014162:Transfection
chemical_list: D000477:Alkylating Agents; D008769:Methylnitronitrosoguanidine; D006147:Guanine; D004247:DNA; C008449:O-(6)-methylguanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D004264:DNA Topoisomerases, Type I