Role of codon 160 in the sensitivity of human O6-alkylguanine-DNA alkyltransferase to O6-benzylguanine. 1999

M Xu-Welliver, and J Leitão, and S Kanugula, and W J Meehan, and A E Pegg
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033, USA.

O6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein that provides protection from alkylating agents such as dacarbazine, temozolomide, and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which are used for cancer chemotherapy. O6-Benzylguanine (BG) is an inhibitor of AGT that sensitizes tumors to these agents. BG is currently in clinical trials. It is possible that the presence of resistant forms of AGT may limit the effectiveness of this strategy. Previous studies have shown that the AGT mutant G160R, which may occur naturally as a result of a polymorphism in the AGT gene, is resistant to BG, whereas the mutants G160W and G160A are actually more sensitive to the inhibitor. To examine other mutations at this site, a random sequence was placed at codon 160 in the AGT cDNA, and a plasmid library was constructed to express these sequences in Escherichia coli. After selection with BG and N-methyl-N'-nitro-N-nitrosoguanidine, BG-resistant mutants were obtained and analyzed. Eleven different amino acid substitutions were found to impart BG resistance by this assay. The most resistant mutants contained histidine or arginine, which had EC50 values of 12 and 4.7 microM, respectively, compared with the wild-type EC50 of 0.08 microM, but nine other alterations led to at least a 10-fold rise in the EC50 value. Three additional mutations at codon 160 were constructed by site-directed mutagenesis, and these led to 6- to 11-fold increases in resistance to BG. Comparisons of the properties of mutants G160R and G160E showed that the presence of DNA enhanced the reaction with BG much more strongly when an acidic residue was present at this position. This may account for the lack of selection of the G160E mutation even though it did impart resistance to BG. These results indicate that many alterations of AGT at position 160 can lead to significant resistance to BG.

UI MeSH Term Description Entries
D002478 Cells, Cultured Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others. Cultured Cells,Cell, Cultured,Cultured Cell
D003062 Codon A set of three nucleotides in a protein coding sequence that specifies individual amino acids or a termination signal (CODON, TERMINATOR). Most codons are universal, but some organisms do not produce the transfer RNAs (RNA, TRANSFER) complementary to all codons. These codons are referred to as unassigned codons (CODONS, NONSENSE). Codon, Sense,Sense Codon,Codons,Codons, Sense,Sense Codons
D004351 Drug Resistance Diminished or failed response of an organism, disease or tissue to the intended effectiveness of a chemical or drug. It should be differentiated from DRUG TOLERANCE which is the progressive diminution of the susceptibility of a human or animal to the effects of a drug, as a result of continued administration. Resistance, Drug
D004791 Enzyme Inhibitors Compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction. Enzyme Inhibitor,Inhibitor, Enzyme,Inhibitors, Enzyme
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006147 Guanine
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000970 Antineoplastic Agents Substances that inhibit or prevent the proliferation of NEOPLASMS. Anticancer Agent,Antineoplastic,Antineoplastic Agent,Antineoplastic Drug,Antitumor Agent,Antitumor Drug,Cancer Chemotherapy Agent,Cancer Chemotherapy Drug,Anticancer Agents,Antineoplastic Drugs,Antineoplastics,Antitumor Agents,Antitumor Drugs,Cancer Chemotherapy Agents,Cancer Chemotherapy Drugs,Chemotherapeutic Anticancer Agents,Chemotherapeutic Anticancer Drug,Agent, Anticancer,Agent, Antineoplastic,Agent, Antitumor,Agent, Cancer Chemotherapy,Agents, Anticancer,Agents, Antineoplastic,Agents, Antitumor,Agents, Cancer Chemotherapy,Agents, Chemotherapeutic Anticancer,Chemotherapy Agent, Cancer,Chemotherapy Agents, Cancer,Chemotherapy Drug, Cancer,Chemotherapy Drugs, Cancer,Drug, Antineoplastic,Drug, Antitumor,Drug, Cancer Chemotherapy,Drug, Chemotherapeutic Anticancer,Drugs, Antineoplastic,Drugs, Antitumor,Drugs, Cancer Chemotherapy
D016297 Mutagenesis, Site-Directed Genetically engineered MUTAGENESIS at a specific site in the DNA molecule that introduces a base substitution, or an insertion or deletion. Mutagenesis, Oligonucleotide-Directed,Mutagenesis, Site-Specific,Oligonucleotide-Directed Mutagenesis,Site-Directed Mutagenesis,Site-Specific Mutagenesis,Mutageneses, Oligonucleotide-Directed,Mutageneses, Site-Directed,Mutageneses, Site-Specific,Mutagenesis, Oligonucleotide Directed,Mutagenesis, Site Directed,Mutagenesis, Site Specific,Oligonucleotide Directed Mutagenesis,Oligonucleotide-Directed Mutageneses,Site Directed Mutagenesis,Site Specific Mutagenesis,Site-Directed Mutageneses,Site-Specific Mutageneses
D019853 O(6)-Methylguanine-DNA Methyltransferase An enzyme that transfers methyl groups from O(6)-methylguanine, and other methylated moieties of DNA, to a cysteine residue in itself, thus repairing alkylated DNA in a single-step reaction. EC 2.1.1.63. Methylated-DNA-Protein-Cysteine S-Methyltransferase,O(6)-AGT,O(6)-Methylguanine Methyltransferase,DNA Repair Methyltransferase I,DNA Repair Methyltransferase II,Guanine-O(6)-Alkyltransferase,O(6)-Alkylguanine-DNA Alkyltransferase,O(6)-MeG-DNA Methyltransferase,O(6)-Methylguanine DNA Transmethylase,Methylated DNA Protein Cysteine S Methyltransferase,S-Methyltransferase, Methylated-DNA-Protein-Cysteine

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