O6-methylguanine-DNA methyltransferase (MGMT) plays an important role in repairing alkylated DNA. MGMT activity as well as cellular sensitivity to 1- ( 4- amino- 2-methyl-5-pyrimidinyl) methyl-3- ( 2-chloroethyl)-3-ni...O6-methylguanine-DNA methyltransferase (MGMT) plays an important role in repairing alkylated DNA. MGMT activity as well as cellular sensitivity to 1- ( 4- amino- 2-methyl-5-pyrimidinyl) methyl-3- ( 2-chloroethyl)-3-nitrosourea (ACNU) of 20 Chinese tumor cell strains were assayed. A linear response between MGMT activity and ACNU sensitivity (D10) was observed. The lower the MGMT activity In the cells, the more the sensitivity to ACNU killing. It suggested that assay of MGMT activity in tumor biopsy could be used as a guide to predict the effectiveness of ACNU treatment in chemotherapy of human cancer.展开更多
The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity bein...The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included 1) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, 2) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and 3) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumorselective reduction in MGMT activity exist in human tissue.展开更多
BACKGROUND O_(6)-methylguanine-DNA methyltransferase(MGMT)is a suicide enzyme that repairs the mispairing base O_(6)-methyl-guanine induced by environmental and experimental carcinogens.It can transfer the alkyl group...BACKGROUND O_(6)-methylguanine-DNA methyltransferase(MGMT)is a suicide enzyme that repairs the mispairing base O_(6)-methyl-guanine induced by environmental and experimental carcinogens.It can transfer the alkyl group to a cysteine residue in its active site and became inactive.The chemical carcinogen N-nitroso compounds(NOCs)can directly bind to the DNA and induce the O_(6)-methylguanine adducts,which is an important cause of gene mutation and tumorigenesis.However,the underlying regulatory mechanism of MGMT involved in NOCs-induced tumorigenesis,especially in the initiation phase,remains largely unclear.AIM To investigate the molecular regulatory mechanism of MGMT in NOCs-induced gastric cell malignant transformation and tumorigenesis.METHODS We established a gastric epithelial cell malignant transformation model induced by N-methyl-N’-nitro-N-nitrosoguanidine(MNNG)or N-methyl-N-nitroso-urea(MNU)treatment.Cell proliferation,colony formation,soft agar,cell migration,and xenograft assays were used to verify the malignant phenotype.By using quantitative real-time polymerase chain reaction(qPCR)and Western blot analysis,we detected the MGMT expression in malignant transformed cells.We also confirmed the MGMT expression in early stage gastric tumor tissues by qPCR and immunohistochemistry.MGMT gene promoter DNA methylation level was analyzed by methylation-specific PCR and bisulfite sequencing PCR.The role of MGMT in cell malignant transformation was analyzed by colony formation and soft agar assays.RESULTS We observed a constant increase in MGMT mRNA and protein expression in gastric epithelial cell malignant transformation induced by MNNG or MNU treatment.Moreover,we found a reduction of MGMT gene promoter methylation level by methylation-specific PCR and bisulfite sequencing PCR in MNNG/MNU-treated cells.Inhibition of the MGMT expression by O_(6)-benzylguanine promoted the MNNG/MNU-induced malignant phenotypes.Overexpression of MGMT partially reversed the cell malignant transformation process induced by MNNG/MNU.Clinical gastric tissue analysis showed that MGMT was upregulated in the precancerous lesions and metaplasia tissues,but downregulated in the gastric cancer tissues.CONCLUSION Our finding indicated that MGMT upregulation is induced via its DNA promoter hypomethylation.The highly expressed MGMT prevents the NOCs-induced cell malignant transformation and tumorigenesis,which suggests a potential novel approach for chemical carcinogenesis intervention by regulating aberrant epigenetic mechanisms.展开更多
目的探讨DNA修复酶O-6-甲基鸟嘌呤-DNA甲基转移酶(O-6-methylguanine-DNA methyltransferase,MGMT)在顺铂(cisplatin)激活的胃癌SGC-7901细胞自噬中的作用。方法 Western blot法检测自噬底物蛋白p62水平、自噬标志分子Ⅱ型和Ⅰ型微管相...目的探讨DNA修复酶O-6-甲基鸟嘌呤-DNA甲基转移酶(O-6-methylguanine-DNA methyltransferase,MGMT)在顺铂(cisplatin)激活的胃癌SGC-7901细胞自噬中的作用。方法 Western blot法检测自噬底物蛋白p62水平、自噬标志分子Ⅱ型和Ⅰ型微管相关蛋白轻链3(mircotuble-associated protein light chain 3,LC3)的比值变化、MGMT蛋白水平;GFP-LC3表达质粒转染胃癌SGC-7901细胞后用激光共聚焦显微镜观察顺铂对细胞自噬小体形成的影响。qRT-PCR法检测MGMT基因mRNA表达水平。结果顺铂剂量依赖性激活胃癌SGC-7901细胞自噬水平,显著增加SGC-7901细胞中自噬小体数量;MGMT mRNA及蛋白水平随顺铂浓度的增加而降低(P<0.05);过表达MGMT抑制胃癌SGC-7901细胞的基础自噬水平;过表达MGMT抑制顺铂激活的胃癌SGC-7901细胞自噬。结论 DNA修复酶O-6-甲基鸟嘌呤-DNA甲基转移酶抑制顺铂激活的胃癌SGC-7901细胞自噬。展开更多
AIM:To investigate aberrant DNA methylation of CpG islands and subsequent low-or high-level DNA microsatellite instability(MSI)which is assumed to drive colon carcinogenesis. METHODS:DNA of healthy individuals,adenoma...AIM:To investigate aberrant DNA methylation of CpG islands and subsequent low-or high-level DNA microsatellite instability(MSI)which is assumed to drive colon carcinogenesis. METHODS:DNA of healthy individuals,adenoma(tu-bular or villous/tubulovillous)patients,and colorectal carcinoma patients who underwent colonoscopy was used for assessing the prevalence of aberrant DNA methylation of human DNA mismatch repair gene mutator L homologue 1(hMLH1),Cyclin-dependent kinase inhibitor 2A(CDKN2A/p16),and O-6-methylguanine DNA methyltransferase(MGMT),as well as their rela- tion to MSI. RESULTS:The frequency of promoter methylation for each locus increased in the sequence healthy tissue/adenoma/carcinoma.MGMT showed the highest frequency in each group.MGMT and CDKN2A/p16 presented a statistically significant increase in promoter methylation between the less and more tumorigenic forms of colorectal adenomas(tubular vs tubullovillous and villous adenomas).All patients with tubulovillous/villous adenomas,as well as all colorectal cancer patients,showed promoter methylation in at least one of the examined loci.These findings suggest a potentially crucial role for methylation in the polyp/adenoma to cancer progres- sion in colorectal carcinogenesis.MSI and methylation seem to be interdependent,as simultaneous hMLH1, CDKN2A/p16,and MGMT promoter methylation was present in 8/9 colorectal cancer patients showing the MSI phenotype. CONCLUSION:Methylation analysis of hMLH1,CD- KN2A/p16,and MGMT revealed specific methylation profiles for tubular adenomas,tubulovillous/villous adenomas,and colorectal cancers,supporting the use of these alterations in assessment of colorectal tumorigenesis.展开更多
The emergence of drug resistance is a major obstacle limiting the successful chemotherapy of malignant tumors. Our previous studies have demonstrated that O^6-methylguanine-DNA methyltransferase (MGMT, EC 2.1.1.63) is...The emergence of drug resistance is a major obstacle limiting the successful chemotherapy of malignant tumors. Our previous studies have demonstrated that O^6-methylguanine-DNA methyltransferase (MGMT, EC 2.1.1.63) is an important contributor to tumor cellular resistance toward mono- and bifunctional alkylating agents, such as 1- (4-amino-2-methyl-5-pyrimidinyl) methyl-3- (2-chloroethyl) -3-nitrosourea (ACNU) and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). Mechanistically, MGMT can specifically remove the induced alkyl groups at the O^6-position of guanine which finally would lead to a G→ A transition or a lethal DNA interstrand cross-link unless repaired. Cells展开更多
Drug resistance is still one of the major problems in cancer therapy. However, during the past several years, many efforts have been focused on reversing or depleting the molecular basis of drug resistance along with ...Drug resistance is still one of the major problems in cancer therapy. However, during the past several years, many efforts have been focused on reversing or depleting the molecular basis of drug resistance along with the elucidating of the mechanisms involved.Nitrosourea compounds such as 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) and 1, 3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) are an important class of useful antitumor drugs. They exert their antitumor activity by alkylating the O^6 position of guanine and leading to a DNA interstrand cross-link. Introcellular O^6-methylguanine-DNA methyltransferase (O^6-MT) can repair the mentioned DNA lesions and thus plays an important role in determining the sensitivity of展开更多
Background O^6-methylguanine-DNA-methyltransferase （MGMT） is a specific DNA revising enzyme transferring alkylated groups from DNA to its cysteine residue to avoid the abnormal twisting of DNA. Therefore, it is one ...Background O^6-methylguanine-DNA-methyltransferase （MGMT） is a specific DNA revising enzyme transferring alkylated groups from DNA to its cysteine residue to avoid the abnormal twisting of DNA. Therefore, it is one of the drug resistant genes targeted in the treatment of cancer. This study explored the protective effect of MGMT gene transferred into mammalian cells. Methods Mammalian expression vector containing the MGMT gene cloned from human hepatocytes by RT-PCR was constructed and transferred into K562 cells and human peripheral blood mononuclear cells （PBMCs） via liposome, then assayed for gene expression at RNA and protein levels. MIF assay was used to check the drug resistance of cells transfected with MGMT gene. Results MGMT gene was successfully cloned. Real-time PCR showed that the mRNA expression in gene transfected groups in K562 cell line and PBMC were 13.4 and 4.0 times that of the empty vector transfected groups respectively. Results of Western blotting showed distinct higher expression of MGMT in gene transfected group than in other two groups. The IC50 values increased to 7 and 2 times that of the original values respectively in stable transfected K562 cells and transient transfected PBMC. Conclusion The alkylating resistance of eukaryotic cells is enhanced after being transfected with MGMT gene which protein product performs the protective function, and may provide the reference for the protective model of peripheral blood cells in cancer chemotherapy.展开更多
Two kinds of human tumor cell strains having different activity of O^6-methylguanine-DNA methyltransferase (O^6-MT) were transplanted into nude mice. Then the mice were inject-ed intraperitoneally with bifunctional al...Two kinds of human tumor cell strains having different activity of O^6-methylguanine-DNA methyltransferase (O^6-MT) were transplanted into nude mice. Then the mice were inject-ed intraperitoneally with bifunctional alkylating agent 1--(4--amino--2-methyl--5--pyrimidinyl)methy1-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU). The tumors with low O^6-MTactivity were quickly suppressed or cured. The result suggests that some tumors, if previ-sionally determined with low O^6-MT activity, might be efficiently cured by treatment withACNU. This probably opens a new way for human cancer chemotherapy.展开更多
文摘O6-methylguanine-DNA methyltransferase (MGMT) plays an important role in repairing alkylated DNA. MGMT activity as well as cellular sensitivity to 1- ( 4- amino- 2-methyl-5-pyrimidinyl) methyl-3- ( 2-chloroethyl)-3-nitrosourea (ACNU) of 20 Chinese tumor cell strains were assayed. A linear response between MGMT activity and ACNU sensitivity (D10) was observed. The lower the MGMT activity In the cells, the more the sensitivity to ACNU killing. It suggested that assay of MGMT activity in tumor biopsy could be used as a guide to predict the effectiveness of ACNU treatment in chemotherapy of human cancer.
文摘The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included 1) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, 2) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and 3) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumorselective reduction in MGMT activity exist in human tissue.
基金Supported by National Natural Science Foundation of China,No.81472543 and No.81772919Zhejiang Provincial Natural Science Foundation of China,No.LY18H160024 and No.LY20H160040
文摘BACKGROUND O_(6)-methylguanine-DNA methyltransferase(MGMT)is a suicide enzyme that repairs the mispairing base O_(6)-methyl-guanine induced by environmental and experimental carcinogens.It can transfer the alkyl group to a cysteine residue in its active site and became inactive.The chemical carcinogen N-nitroso compounds(NOCs)can directly bind to the DNA and induce the O_(6)-methylguanine adducts,which is an important cause of gene mutation and tumorigenesis.However,the underlying regulatory mechanism of MGMT involved in NOCs-induced tumorigenesis,especially in the initiation phase,remains largely unclear.AIM To investigate the molecular regulatory mechanism of MGMT in NOCs-induced gastric cell malignant transformation and tumorigenesis.METHODS We established a gastric epithelial cell malignant transformation model induced by N-methyl-N’-nitro-N-nitrosoguanidine(MNNG)or N-methyl-N-nitroso-urea(MNU)treatment.Cell proliferation,colony formation,soft agar,cell migration,and xenograft assays were used to verify the malignant phenotype.By using quantitative real-time polymerase chain reaction(qPCR)and Western blot analysis,we detected the MGMT expression in malignant transformed cells.We also confirmed the MGMT expression in early stage gastric tumor tissues by qPCR and immunohistochemistry.MGMT gene promoter DNA methylation level was analyzed by methylation-specific PCR and bisulfite sequencing PCR.The role of MGMT in cell malignant transformation was analyzed by colony formation and soft agar assays.RESULTS We observed a constant increase in MGMT mRNA and protein expression in gastric epithelial cell malignant transformation induced by MNNG or MNU treatment.Moreover,we found a reduction of MGMT gene promoter methylation level by methylation-specific PCR and bisulfite sequencing PCR in MNNG/MNU-treated cells.Inhibition of the MGMT expression by O_(6)-benzylguanine promoted the MNNG/MNU-induced malignant phenotypes.Overexpression of MGMT partially reversed the cell malignant transformation process induced by MNNG/MNU.Clinical gastric tissue analysis showed that MGMT was upregulated in the precancerous lesions and metaplasia tissues,but downregulated in the gastric cancer tissues.CONCLUSION Our finding indicated that MGMT upregulation is induced via its DNA promoter hypomethylation.The highly expressed MGMT prevents the NOCs-induced cell malignant transformation and tumorigenesis,which suggests a potential novel approach for chemical carcinogenesis intervention by regulating aberrant epigenetic mechanisms.
文摘目的探讨DNA修复酶O-6-甲基鸟嘌呤-DNA甲基转移酶(O-6-methylguanine-DNA methyltransferase,MGMT)在顺铂(cisplatin)激活的胃癌SGC-7901细胞自噬中的作用。方法 Western blot法检测自噬底物蛋白p62水平、自噬标志分子Ⅱ型和Ⅰ型微管相关蛋白轻链3(mircotuble-associated protein light chain 3,LC3)的比值变化、MGMT蛋白水平;GFP-LC3表达质粒转染胃癌SGC-7901细胞后用激光共聚焦显微镜观察顺铂对细胞自噬小体形成的影响。qRT-PCR法检测MGMT基因mRNA表达水平。结果顺铂剂量依赖性激活胃癌SGC-7901细胞自噬水平,显著增加SGC-7901细胞中自噬小体数量;MGMT mRNA及蛋白水平随顺铂浓度的增加而降低(P<0.05);过表达MGMT抑制胃癌SGC-7901细胞的基础自噬水平;过表达MGMT抑制顺铂激活的胃癌SGC-7901细胞自噬。结论 DNA修复酶O-6-甲基鸟嘌呤-DNA甲基转移酶抑制顺铂激活的胃癌SGC-7901细胞自噬。
基金Supported by A 2-year grant of the Greek Ministry of Health and Welfare,No.111K/56
文摘AIM:To investigate aberrant DNA methylation of CpG islands and subsequent low-or high-level DNA microsatellite instability(MSI)which is assumed to drive colon carcinogenesis. METHODS:DNA of healthy individuals,adenoma(tu-bular or villous/tubulovillous)patients,and colorectal carcinoma patients who underwent colonoscopy was used for assessing the prevalence of aberrant DNA methylation of human DNA mismatch repair gene mutator L homologue 1(hMLH1),Cyclin-dependent kinase inhibitor 2A(CDKN2A/p16),and O-6-methylguanine DNA methyltransferase(MGMT),as well as their rela- tion to MSI. RESULTS:The frequency of promoter methylation for each locus increased in the sequence healthy tissue/adenoma/carcinoma.MGMT showed the highest frequency in each group.MGMT and CDKN2A/p16 presented a statistically significant increase in promoter methylation between the less and more tumorigenic forms of colorectal adenomas(tubular vs tubullovillous and villous adenomas).All patients with tubulovillous/villous adenomas,as well as all colorectal cancer patients,showed promoter methylation in at least one of the examined loci.These findings suggest a potentially crucial role for methylation in the polyp/adenoma to cancer progres- sion in colorectal carcinogenesis.MSI and methylation seem to be interdependent,as simultaneous hMLH1, CDKN2A/p16,and MGMT promoter methylation was present in 8/9 colorectal cancer patients showing the MSI phenotype. CONCLUSION:Methylation analysis of hMLH1,CD- KN2A/p16,and MGMT revealed specific methylation profiles for tubular adenomas,tubulovillous/villous adenomas,and colorectal cancers,supporting the use of these alterations in assessment of colorectal tumorigenesis.
文摘The emergence of drug resistance is a major obstacle limiting the successful chemotherapy of malignant tumors. Our previous studies have demonstrated that O^6-methylguanine-DNA methyltransferase (MGMT, EC 2.1.1.63) is an important contributor to tumor cellular resistance toward mono- and bifunctional alkylating agents, such as 1- (4-amino-2-methyl-5-pyrimidinyl) methyl-3- (2-chloroethyl) -3-nitrosourea (ACNU) and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). Mechanistically, MGMT can specifically remove the induced alkyl groups at the O^6-position of guanine which finally would lead to a G→ A transition or a lethal DNA interstrand cross-link unless repaired. Cells
基金Project supported by the National Natural Science Foundation of China.
文摘Drug resistance is still one of the major problems in cancer therapy. However, during the past several years, many efforts have been focused on reversing or depleting the molecular basis of drug resistance along with the elucidating of the mechanisms involved.Nitrosourea compounds such as 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) and 1, 3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) are an important class of useful antitumor drugs. They exert their antitumor activity by alkylating the O^6 position of guanine and leading to a DNA interstrand cross-link. Introcellular O^6-methylguanine-DNA methyltransferase (O^6-MT) can repair the mentioned DNA lesions and thus plays an important role in determining the sensitivity of
基金the grant from the National Natural Science Foundation of China(No.30460127)
文摘Background O^6-methylguanine-DNA-methyltransferase （MGMT） is a specific DNA revising enzyme transferring alkylated groups from DNA to its cysteine residue to avoid the abnormal twisting of DNA. Therefore, it is one of the drug resistant genes targeted in the treatment of cancer. This study explored the protective effect of MGMT gene transferred into mammalian cells. Methods Mammalian expression vector containing the MGMT gene cloned from human hepatocytes by RT-PCR was constructed and transferred into K562 cells and human peripheral blood mononuclear cells （PBMCs） via liposome, then assayed for gene expression at RNA and protein levels. MIF assay was used to check the drug resistance of cells transfected with MGMT gene. Results MGMT gene was successfully cloned. Real-time PCR showed that the mRNA expression in gene transfected groups in K562 cell line and PBMC were 13.4 and 4.0 times that of the empty vector transfected groups respectively. Results of Western blotting showed distinct higher expression of MGMT in gene transfected group than in other two groups. The IC50 values increased to 7 and 2 times that of the original values respectively in stable transfected K562 cells and transient transfected PBMC. Conclusion The alkylating resistance of eukaryotic cells is enhanced after being transfected with MGMT gene which protein product performs the protective function, and may provide the reference for the protective model of peripheral blood cells in cancer chemotherapy.
基金Project supported by the National Natural Science Foundation of China.
文摘Two kinds of human tumor cell strains having different activity of O^6-methylguanine-DNA methyltransferase (O^6-MT) were transplanted into nude mice. Then the mice were inject-ed intraperitoneally with bifunctional alkylating agent 1--(4--amino--2-methyl--5--pyrimidinyl)methy1-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU). The tumors with low O^6-MTactivity were quickly suppressed or cured. The result suggests that some tumors, if previ-sionally determined with low O^6-MT activity, might be efficiently cured by treatment withACNU. This probably opens a new way for human cancer chemotherapy.
