Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-alkylguanine.

Abstract

O6-Methylguanine-DNA methyltransferase (MGMT; DNA-O6-methylguanine:protein-L-cysteine S-methyltransferase, EC 2.1.1.63), a unique DNA repair protein present in most organisms, removes the carcinogenic and mutagenic adduct O6-alkylguanine from DNA by stoichiometrically accepting the alkyl group on a cysteine residue in a suicide reaction. The mammalian protein is highly regulated in both somatic and germ-line cells. In addition, the toxicity of certain alkylating drugs in tumor and normal cells is inversely related to the levels of this protein. The cDNA of the human gene, henceforth named MGMT, has been cloned in an expression vector on the basis of its rescue of a methyltransferase-deficient (ada-) Escherichia coli host. A 22-kDa active methyltransferase encoded entirely by the cDNA contains an amino acid sequence of 61 residues that bears 60-65% similarity with segments of E. coli methyltransferase (products of the ada and ogt genes), which encompass the alkyl-acceptor residues. The human cDNA has no sequence similarity with the ada and ogt genes, due in part to differences in codon usage, and shows no detectable homology with E. coli genomic DNA. However, it hybridizes with distinct restriction fragments of human, mouse, and rat DNAs. The lack of methyltransferase observed in many human cell lines is due to the absence of the MGMT gene or to lack of synthesis and/or stability of its 0.95-kilobase poly(A)+ RNA transcript.

O6‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

“…MGMT is one of the most important genes for ACNU resistance. 6,7,10,30 According to our results, the tumor with RQV of MGMT м1 in real-time quantitative RT-PCR should not be treated by ACNU.…”

“…[1][2][3] Drug-resistance genes are some of the most important elements of tumors themselves in determining drug-resistance, 4 and O 6 -methylguanine-DNA methyltransferase (MGMT) is a drug-resistance gene for nitrosoureas. 5,6 The cross-linking of doublestranded DNA by alkylating agents is inhibited by the cellular DNA-repair protein MGMT. MGMT rapidly reverses alkylation at the O 6 position of guanine, thereby averting lethal cross-linking.…”

3-aminobenzamide and/or O6-benzylguanine evaluated as an adjuvant to temozolomide or BCNU treatment in cell lines of variable mismatch repair status and O6-alkylguanine-DNA alkyltransferase activity

“…It is therefore possible that the inhibition of one or more DNA-repair processes, by appropriate pharmacological intervention, may circumvent such resistance. This approach has been most widely examined with inhibitors of AGT, a DNA-repair protein responsible for the stoichiometric removal of adducts produced at the 06-position of guanine (Pegg, 1983;Tano et al, 1990). O6-guanine adduct removal irreversibly inactivates AGT, requiring de novo synthesis of the protein to restore activity (Pegg, 1990).…”

Potentiation of temozolomide and BCNU cytotoxicity by O6-benzylguanine: a comparative study in vitro

“…These interruptions in the daughter strands inhibit replication in the subsequent S-phase (Plant and Roberts, 1971;Ceccotti et al, 1993) and account for methylating cytotoxicity being only apparent after at least two rounds of cell division (Catapano et al, 1987). Since the anti-tumour activity of temozolomide is thought to depend upon the formation of 06-methylguanine, its clinical utility may be limited by the cytoprotective DNA repair protein, 06-alkylguanine -DNA alkyltransferase (AGT), which removes 06-alkylguanine adducts in a stoichiometric, autoinactivating reaction (Pegg, 1983;Tano et al, 1990). Resistance to temozolomide or MTIC readily induced in vitro is attributed to this DNA repair process (Hayward and Parsons, 1984;Catapano et al, 1987).…”

“…Although 06-BG may exacerbate the relatively mild myelosuppression produced by temozolomide (Fairburn et al, 1995) this could, if necessary, be managed by autologous bone marrow infusion and the administration of haematopoietic growth factors. It is also possible that the gene for AGT (Tano et al, 1990) could be transfected into bone marrow cells before infusion and thereby confer greater haematological resistance to such therapy.…”