title: The bacterial alkyltransferase-like (eATL) protein protects mammalian cells against methylating agent-induced toxicity.
abstract: In both pro- and eukaryotes, the mutagenic and toxic DNA adduct O(6)-methylguanine (O(6)MeG) is subject to repair by alkyltransferase proteins via methyl group transfer. In addition, in prokaryotes, there are proteins with sequence homology to alkyltransferases, collectively designated as alkyltransferase-like (ATL) proteins, which bind to O(6)-alkylguanine adducts and mediate resistance to alkylating agents. Whether such proteins might enable similar protection in higher eukaryotes is unknown. Here we expressed the ATL protein of Escherichia coli (eATL) in mammalian cells and addressed the question whether it is able to protect them against the cytotoxic effects of alkylating agents. The Chinese hamster cell line CHO-9, the nucleotide excision repair (NER) deficient derivative 43-3B and the DNA mismatch repair (MMR) impaired derivative Tk22-C1 were transfected with eATL cloned in an expression plasmid and the sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was determined in reproductive survival, DNA double-strand break (DSB) and apoptosis assays. The results indicate that eATL expression is tolerated in mammalian cells and conferes protection against killing by MNNG in both wild-type and 43-3B cells, but not in the MMR-impaired cell line. The protection effect was dependent on the expression level of eATL and was completely ablated in cells co-expressing the human O(6)-methylguanine-DNA methyltransferase (MGMT). eATL did not protect against cytotoxicity induced by the chloroethylating agent lomustine, suggesting that O(6)-chloroethylguanine adducts are not target of eATL. To investigate the mechanism of protection, we determined O(6)MeG levels in DNA after MNNG treatment and found that eATL did not cause removal of the adduct. However, eATL expression resulted in a significantly lower level of DSBs in MNNG-treated cells, and this was concomitant with attenuation of G2 blockage and a lower level of apoptosis. The results suggest that eATL confers protection against methylating agents by masking O(6)MeG/thymine mispaired adducts, preventing them from becoming a substrate for mismatch repair-mediated DSB formation and cell death.
authors.affiliation: Department of Toxicology, University Medical Center, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany.
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Tomaszowski
authors.affiliation: Department of Toxicology, University Medical Center, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany.
authors.forename: Dorthe
authors.initials: D
authors.lastname: Aasland
authors.affiliation: Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX Manchester, UK.
authors.forename: Geoffrey P
authors.initials: GP
authors.lastname: Margison
authors.affiliation: Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX Manchester, UK.
authors.forename: Emma
authors.initials: E
authors.lastname: Williams
authors.affiliation: Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX Manchester, UK.
authors.forename: Sarah I
authors.initials: SI
authors.lastname: Pinder
authors.affiliation: Centre de Recherche en Cancérologie de Marseille, CNRS-UMR7258, Inserm-U1068, Institut Paoli-Calmettes, Université Aix-Marseille, France.
authors.forename: Mauro
authors.initials: M
authors.lastname: Modesti
authors.affiliation: Centre de Recherche en Cancérologie de Marseille, CNRS-UMR7258, Inserm-U1068, Institut Paoli-Calmettes, Université Aix-Marseille, France.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Fuchs
authors.affiliation: Department of Toxicology, University Medical Center, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany. Electronic address: kaina@uni-mainz.de.
authors.forename: Bernd
authors.initials: B
authors.lastname: Kaina
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism*; D000477:Alkylating Agents / Q000633:toxicity*; D000818:Animals; D017209:Apoptosis; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D015254:DNA Modification Methylases / Q000378:metabolism; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000633:toxicity; D019076:Transgenes; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D000477:Alkylating Agents; D018736:DNA Adducts; D029968:Escherichia coli Proteins; D025521:Tumor Suppressor Proteins; D008769:Methylnitronitrosoguanidine; D006147:Guanine; D004247:DNA; C008449:O-(6)-methylguanine; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D019883:Alkyl and Aryl Transferases; C502320:alkyltransferase-like protein, E coli; D045643:DNA Repair Enzymes
keywords: ATL; Alkylating agents; Alkyltransferase; Killing protection; MGMT; O6MeG