O6-甲基鸟嘌岭DNA的修复研究

O⁶-甲基鸟嘌岭DNA是某些烷化剂直接或通过酶转化后与DNA反应生成的一种有致死、致突及致癌作用的产物。细胞内的O⁶-甲基鸟嘌呤DNA甲基转移酶能有效地去除这种损伤,即甲基基团从DNA鸟嘌呤第六位氧原子上特异地转移到受体蛋白的半胱氨酸的残基上,反应产物是S-甲基半胱氨酸。受体蛋白就是酶本身。O⁶-甲基乌嘌呤的修复过程所完成的是一种无错的修复,它可能与减少肿瘤的发生有关。     

O^6—甲基鸟嘌呤—DNA—甲基转移酶的基因表达调控

DNA损伤修复是生命科学研究的重要课题。DNA修复在防止基因突变,保证遗传信息稳定性的过程中起着极其重要的作用。近20年来,人们通过大量研究发现细胞内0~6-甲基鸟嘌呤-DNA-甲基转移酶(O~6-metnylguanine-DNA-methyltransferase,MGMT)能专一性很强地修复DNA烷化损伤,防止细胞癌变和死亡;大量试验还证明细胞内MGMT活性的高低是决定肿瘤细胞对亚硝脲类药物产生耐药性的分子基础。详尽了解MGMT基因表达调控机理对DNA损伤修复的理论研究和克服肿瘤细胞耐药性的实践都具有重要的意义。     

O6-甲基鸟嘌呤的制备

O⁶-甲基鸟嘌呤广泛应用于DNA损伤修复和肿瘤研究中。本文以巯基鸟嘌呤为原料经二步反应而制备。并对其进行了元素分析、紫外吸收光谱、高压液相色谱测定和同位素鉴定。     

MGMT研究进展

DNA修复酶O^6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）是机体修复烷基加合物的关键酶,它可以对抗烷化剂造成的DNA损伤,其在肿瘤发生和化疗中所起的作用对于研究肿瘤的发生及治疗均有十分重要的意义。MGMT在不同个体及组织间的表达具有极大的多样性,其表达调控机制是目前人们研究的热点。     

O6-甲基鸟嘌呤-DNA甲基转移酶研究进展

C⁶-甲基鸟嘌呤-DNA甲基转移酶是细胞中一种重要的DNA损伤修复酶,它在抵御烷化剂所致的细胞突变和死亡中扮演重要的角色。本文综述近年来关于这个酶的研究进展,内容包括酶及其基因,酶与细胞突变,酶与肿瘤化疗的关系。     

人MGMT基因Cdna的克隆表达及表达蛋白修复功能的鉴定

克隆了Hela细胞O6 甲基鸟嘌呤 DNA 甲基转移酶 (MGMT)基因的cDNA序列 ,该序列与国外发表的cDNA完全一致。将此cDNA插入原核表达载体pET 2 1a后转化大肠杆菌BL2 1(DE3)获得表达的重组菌株pET 2 1a MGMT E .coliBL2 1(DE3) ,经IPTG诱导后产生分子量为 2 4kD的蛋白质。烷化类诱变剂致死突变实验表明 ,MGMT蛋白的表达能修复受体菌DNA分子因烷化类诱变剂导致的突变。     

抑癌基因MGMT与肿瘤的发生、诊断和治疗

烷化剂是一种致癌剂,同时也是一种常见的肿瘤化疗药物,而O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）作为一种重要的DNA修复酶,对于保护细胞免受环境中的致癌物质和化疗药物引起基因毒性起到重要修复作用,因此它在细胞对环境致癌物的易感性、肿瘤的基因诊断、肿瘤对烷基化药物的耐药性及其肿瘤患者的个性化化疗等研究领域,受到越来越多研究者的关注.该文介绍抑癌基因MGMT与肿瘤关系的最新研究进展.     

Nafion膜固定的亚甲基蓝为介体的生物传感器

制成了以亚甲基蓝为介体的电流型过氧化氢生物传感器,通过离子交换牢固地固定在Nafion膜中的亚甲基蓝,能有效地在辣根过氧化物酶和玻碳电极之间传递电子.探讨了pH值、温度、工作电位和抗坏血酸等物质对此传感器生物电催化还原H₂O₂的影响.此生物传感器选择性好、灵敏度高,对H₂O₂线性响应范围为5.0×10^-7～2×10^-4 mol/L,响应时间少于30 s.     

中国人肿瘤细胞中O~6-甲基鸟嘌呤DNA甲基转移酶活性与对嘧啶亚硝脲敏感性的关系

 分析了20株中国人肿瘤细胞的O~6-甲基鸟嘌呤DNA甲基转移酶(O~6-MT)活性及对嘧啶亚硝脲ACNU的敏感性,发现两者间有较好的线性关系,O~6-MT低,对ACNU敏感,提示O~6-MT可作为使用ACNU对肿瘤化疗时的一项予见性指标。     

水稻CPR5基因参与氧化胁迫响应

为探讨水稻(Oryza sativa L.)中CPR5 基因的功能,以其cDNA 文库为基础进行序列比对,并将1 个同源性较高的序列命名为OsCPR5.生物信息学分析表明,OsCPR5 的开放阅读框长度为1551 bp,编码516 个氨基酸.软件预测该编码蛋白可能是1 个具有5 个跨膜区的膜蛋白和核定位蛋白.组织表达和亚细胞定位分析表明,OsCPR5 在根中的表达水平较高,且广泛分布在细胞膜、细胞质和细胞核上.逆境胁迫分析实验表明,植物激素脱落酸(Abscisic acid, ABA)、过氧化氢(Hydrogen peroxide,H₂O₂)、氯化钠(NaCl)、甲基紫精(Methyl viologen, MV)等环境胁迫可诱导OsCPR5 的上调表达,其中氧化胁迫相关的MV 和H₂O₂ 处理效果最为明显.拟南芥(Arabidopsis thaliana)转基因株系atcpr5/OsCPR5 在浓度为0.5 μmol L^-1、 1.0 μmol L^-1 的MV以及6 mmol L^-1、 8 mmol L^-1 的H₂O₂ 处理下,种子萌发率明显高于atcpr5 突变体.这揭示了OsCPR5 基因在植物抗氧化胁迫响应中具有一定的作用.     

Effects of Some Growth Factors and Cytokines on the Expression of the Repair Enzyme MGMT and Protein MARP in Human Cells In Vitro

The inducible repair enzyme O⁶-methylguanine-DNA methyltransferase (MGMT) eliminates O⁶-methylguanine adducts in DNA and protects the cells from damaging effects of alkylating agents. We have found that anti-MGMT antibodies recognize both the MGMT protein with a mol. weight?~?24 kDa and a protein with a mol. weight?~?48 kDa, which was named MARP (anti-methyltransferase antibody recognizable protein). A number of growth factors and cytokines were shown to regulate the expression of MGMT and MARP proteins. The ranges of concentrations of several growth factors and cytokines that caused increasing or decreasing protein amounts in human cell cultures were determined. The results of special biological experiments have allowed us to assume a possible role of MARP in the repair of alkyl adducts in human cells.     

Development of a new application of the comet assay to assess levels of O6-methylguanine in genomic DNA (CoMeth)

O⁶-methylguanine (O⁶meG) is one of the most premutagenic, precarcinogenic, and precytotoxic DNA lesions formed by alkylating agents. Repair of this DNA damage is achieved by the protein MGMT, which transfers the alkyl groups from the O⁶ position of guanine to a cysteine residue in its active center. Because O⁶meG repair by MGMT is a stoichiometric reaction that irreversibly inactivates MGMT, which is subsequently degraded, the repair capacity of O⁶meG lesions is dependent on existing active MGMT molecules. In the absence of active MGMT, O⁶meG is not repaired, and during replication, O⁶meG:T mispairs are formed. The MMR system recognizes these mispairs and introduces a gap into the strand. If O⁶meG remains in one of the template strands the futile MMR repair process will be repeated, generating more strand breaks (SBs). The toxicity of O⁶meG is, therefore, dependent on MMR and DNA SB induction of cell death. MGMT, on the other hand, protects against O⁶meG toxicity by removing the methyl residue from the guanine. Although removal of O⁶meG makes MGMT an important anticarcinogenic mechanism of DNA repair, its activity significantly decreases the efficacy of cancer chemotherapeutic drugs that aim at achieving cell death through the action of the MMR system on unrepaired O⁶meG lesions. Here, we report on a modification of the comet assay (CoMeth) that allows the qualitative assessment of O⁶meG lesions after their conversion to strand breaks in proliferating MMR-proficient cells after MGMT inhibition. This functional assay allows the testing of compounds with effects on O⁶meG levels, as well as on MGMT or MMR activity, in a proliferating cell system. The expression of MGMT and MMR genes is often altered by promoter methylation, and new epigenetically active compounds are being designed to increase chemotherapeutic efficacy. The CoMeth assay allows the testing of compounds with effects on O⁶meG, MGMT, or MMR activity. This proliferating cell system complements other methodologies that look at effects on these parameters individually through analytical chemistry or in vitro assays with recombinant proteins.     

Reduction of BCNU toxicity to lung cells by high-level expression of O(6)-methylguanine-DNA methyltransferase

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an important cause of pulmonary toxicity. BCNU alkylates DNA at the O(6) position of guanine. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl groups from the O(6) position of guanine. To determine whether overexpression of MGMT in a lung cell reduces BCNU toxicity, the MGMT gene was transfected into A549 cells, a lung epithelial cell line. Transfected A549 cell populations demonstrated high levels of MGMT RNA, MGMT protein, and DNA repair activity. The overexpression of MGMT in lung epithelial cells provided protection from the cytotoxic effects of BCNU. Control A549 cells incubated with 100 microM BCNU had a cell survival rate of 12.5 +/- 1.2%; however, A549 cells overexpressing MGMT had a survival rate of 71.8 +/- 2.7% (P < 0.001). We also demonstrated successful transfection of MGMT into human pulmonary artery endothelial cells and a primary culture of rat type II alveolar epithelial cells with overexpression of MGMT, resulting in significant protection from BCNU toxicity. These data suggest that overexpression of DNA repair proteins such as MGMT in lung cells may protect the lung cells from cytotoxic effects of cancer chemotherapy drugs such as BCNU.     

Enhancement effect of O6-Fluorobenzylguanines on chloroethylnitrosourea cytotoxicity in tumor cells

O⁶-Alkylguanine derivatives sensitize tumor cells to chloroethylnitrosourea (CENU) chemotherapy by inactivation of O⁶-methylguanine-DNA methyltransferase (MGMT), which repairs CENU-induced O⁶-alkylguanines in DNA by accepting the alkyl group at a cysteine moiety. To test the biological significance of synthesized O⁶-fluorobenzylguanine derivatives, we measured their ability of inactivation of MGMT activity and their effects on the cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in comparison with the effects of O⁶-benzylguanine and O⁶-phenylguanine. The O⁶-(4-and 3-fluorobenzyl)guanines considerably reduced the MGMT activity of HeLa S3 cell-free extract as did O⁶-benzylguanine. In contrast, O⁶-(2-fluorobenzyl)guanine and O⁶-phenylguanine had less of an effect on the activity. Two-hour pretreatment of O⁶-(4-and 3-fluorobenzyl)guanines potentiated ACNU cytotoxicity in HeLa S3 cells to a greater extent than did O⁶-(2-fluorobenzyl)guanine and O⁶-phenylguanine. The enhancement effects were consistent with the depletion of MGMT activity after the pretreatment of O⁶-fluorobenzylguanine derivatives. O⁶-Fluorobenzylguanines with a fluoro-substitution at the 4- or 3-position of the benzyl group were comparable to O⁶-benzylguanine and were powerful MGMT inactivators. The chemical features of the O⁶-benzyl group are a biologically important determinant in the reaction evolution with MGMT.     

O6-methylguanine DNA methyltransferase as a promising target for the treatment of temozolomide-resistant gliomas

Temozolomide (TMZ) is an alkylating agent currently used as first-line therapy for gliomas treatment due to its DNA-damaging effect. However, drug resistance occurs, preventing multi-cycle use of this chemotherapeutic agent. One of the major mechanisms of cancer drug resistance is enhanced activity of a DNA repair enzyme, O⁶-methylguanine-DNA-methyltransferase (MGMT), which counteracts chemotherapy-induced DNA alkylation and is a key component of chemoresistance. MGMT repairs TMZ-induced DNA lesions, O⁶-meG, by transferring the alkyl group from guanine to a cysteine residue. This review provides an overview of recent advances in the field, with particular emphasis on the inhibitors of MGMT and underlying mechanisms. Literature search was performed through PubMed and all relevant articles were reviewed, with particular attention to MGMT, its role in TMZ-resistant gliomas, effects of MGMT inhibitors and the underlying mechanisms. Several strategies are currently being pursued to improve the therapeutic efficacy of TMZ via inhibition of MGMT to reduce chemoresistance and improve overall survival. MGMT may be a promising target for the treatment of TMZ-resistant gliomas.     

Atomic Insight into the Altered O6-Methylguanine-DNA Methyltransferase Protein Architecture in Gastric Cancer

O⁶-methylguanine-DNA methyltransferase (MGMT) is one of the major DNA repair protein that counteracts the alkalyting agent-induced DNA damage by replacing O⁶-methylguanine (mutagenic lesion) back to guanine, eventually suppressing the mismatch errors and double strand crosslinks. Exonic alterations in the form of nucleotide polymorphism may result in altered protein structure that in turn can lead to the loss of function. In the present study, we focused on the population feared for high exposure to alkylating agents owing to their typical and specialized dietary habits. To this end, gastric cancer patients pooled out from the population were selected for the mutational screening of a specific error prone region of MGMT gene. We found that nearly 40% of the studied neoplastic samples harbored missense mutation at codon¹⁵¹ resulting into Serine to Isoleucine variation. This variation resulted in bringing about the structural disorder, subsequently ensuing into a major stoichiometric variance in recognition domain, substrate binding and selectivity loop of the active site of the MGMT protein, as observed under virtual microscope of molecular dynamics simulation (MDS). The atomic insight into MGMT protein by computational approach showed a significant change in the intra molecular hydrogen bond pattern, thus leading to the observed structural anomalies. To further examine the mutational implications on regulatory plugs of MGMT that holds the protein in a DNA-Binding position, a MDS based analysis was carried out on, all known physically interacting amino acids essentially clustered into groups based on their position and function. The results generated by physical-functional clustering of protein indicated that the identified mutation in the vicinity of the active site of MGMT protein causes the local and global destabilization of a protein by either eliminating the stabilizing salt bridges in cluster C3, C4, and C5 or by locally destabilizing the “protein stabilizing hing” mapped on C3-C4 cluster, preceding the active site.     

O-Methylguanine-DNA methyltransferase (MGMT) in normal tissues and tumors: Enzyme activity,promoter methylation and immunohistochemistry

O⁶-Methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the pre-mutagenic, pre-carcinogenic and pre-toxic DNA damage O⁶-methylguanine. It also repairs larger adducts on the O⁶-position of guanine, such as O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine and O⁶-chloroethylguanine. These adducts are formed in response to alkylating environmental pollutants, tobacco-specific carcinogens and methylating (procarbazine, dacarbazine, streptozotocine, and temozolomide) as well as chloroethylating (lomustine, nimustine, carmustine, and fotemustine) anticancer drugs. MGMT is therefore a key node in the defense against commonly found carcinogens, and a marker of resistance of normal and cancer cells exposed to alkylating therapeutics. MGMT also likely protects against therapy-related tumor formation caused by these highly mutagenic drugs. Since the amount of MGMT determines the level of repair of toxic DNA alkylation adducts, the MGMT expression level provides important information as to cancer susceptibility and the success of therapy. In this article, we describe the methods employed for detecting MGMT and review the literature with special focus on MGMT activity in normal and neoplastic tissues. The available data show that the expression of MGMT varies greatly in normal tissues and in some cases this has been related to cancer predisposition. MGMT silencing in tumors is mainly regulated epigenetically and in brain tumors this correlates with a better therapeutic response. Conversely, up-regulation of MGMT during cancer treatment limits the therapeutic response. In malignant melanoma, MGMT is not related to the therapeutic response, which is due to other mechanisms of inherent drug resistance. For most cancers, studies that relate MGMT activity to therapeutic outcome following O⁶-alkylating drugs are still lacking.     

The O6-methylguanine-DNA methyltransferase from the hyperthermophilic archaeon Pyrococcus sp. KOD1: a thermostable repair enzyme

The enzyme O⁶-methylguanine-DNA methyltransferase (MGMT) is the most common form of cellular defense against the biological effects of O⁶-methylguanine (O⁶-MeG) in DNA. Based on PCR amplification using primers derived from conserved amino acid sequences of MGMTs from 11 species, we isolated the DNA region coding for MGMT from the hyperthermophilic archaeon Pyrococcus sp. KOD1. The MGMT gene from KOD1 (mgtk) comprises 522 nucleotides, encoding 174 amino acid residues; its product shows considerable similarity to the corresponding mammalian, yeast and bacterial enzymes, especially around putative methyl acceptor sites. Phylogenetic analysis of MGMTs showed that archaeal MGMTs were grouped with their bacterial counterparts. The location of the MGMT gene on the KOD1 chromosome was also determined. The cloned KOD1 MGMT gene was overexpressed using the T7 RNA polymerase expression system, and the recombinant protein was purified by ammonium sulfate fractionation, heat treatment, ion-exchange chromatography and gel filtration chromatography. The purified recombinant protein was assayed for its enzyme activity by monitoring transfer of [³H]methyl groups from the substrate DNA to the MGMT protein; the activity was found to be stable at 90°?C for at least 30?min. When the mgtk gene was placed under the control of the lac promoter and expressed in the methyltransferase-deficient Escherichia coli strain KT233 (Δada, Δogt) cells, a MGMT was produced. The enzyme was functional in vivo and complemented the mutant phenotype, making the cells resistant to the cytotoxic properties of the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine.     

The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase

The consumption of red meat is a risk factor in human colorectal cancer (CRC). One hypothesis is that red meat facilitates the nitrosation of bile acid conjugates and amino acids, which rapidly convert to DNA-damaging carcinogens. Indeed, the toxic and mutagenic DNA adduct O⁶-carboxymethylguanine (O⁶-CMG) is frequently present in human DNA, increases in abundance in people with high levels of dietary red meat and may therefore be a causative factor in CRC. Previous reports suggested that O⁶-CMG is not a substrate for the human version of the DNA damage reversal protein O⁶-methylguanine-DNA methyltransferase (MGMT), which protects against the genotoxic effects of other O⁶-alkylguanine lesions by removing alkyl groups from the O⁶-position. We now show that synthetic oligodeoxyribonucleotides containing the known MGMT substrate O⁶-methylguanine (O⁶-MeG) or O⁶-CMG effectively inactivate MGMT in vitro (IC₅₀ 0.93 and 1.8 nM, respectively). Inactivation involves the removal of the O⁶-alkyl group and its transfer to the active-site cysteine residue of MGMT. O⁶-CMG is therefore an MGMT substrate, and hence MGMT is likely to be a protective factor in CRC under conditions where O⁶-CMG is a potential causative agent.     

O-Methylguanine-DNA methyltransferase in glioma therapy: Promise and problems

Gliomas are the most frequent adult primary brain tumor, and are invariably fatal. The most common diagnosis glioblastoma multiforme (GBM) afflicts 12,500 new patients in the U.S. annually, and has a median survival of approximately one year when treated with the current standard of care. Alkylating agents have long been central in the chemotherapy of GBM and other gliomas. The DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT), the principal human activity that removes cytotoxic O⁶-alkylguanine adducts from DNA, promotes resistance to anti-glioma alkylators, including temozolomide and BCNU, in GBM cell lines and xenografts. Moreover, MGMT expression assessed by immunohistochemistry, biochemical activity or promoter CpG methylation status is associated with the response of GBM to alkylator-based therapies, providing evidence that MGMT promotes clinical resistance to alkylating agents. These observations suggest a role for MGMT in directing adjuvant therapy of GBM and other gliomas. Promoter methylation status is the most clinically tractable measure of MGMT, and there is considerable enthusiasm for exploring its utility as a marker to assign therapy to individual patients. Here, we provide an overview of the biochemical, genetic and biological characteristics of MGMT as they relate to glioma therapy. We consider current methods to assess MGMT expression and discuss their utility as predictors of treatment response. Particular emphasis is given to promoter methylation status and the methodological and conceptual impediments that limit its use to direct treatment. We conclude by considering approaches that may improve the utility of MGMT methylation status in planning optimal therapies tailored to individual patients.