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
现在普遍认为肿瘤的发生与DNA的异常甲基化有关，所以DNA甲基化状态的研究成为了研究肿瘤发生机制的一个热点，MSP(methylation specific PCR)则是检测DNA甲基化状态的一个简便有效的方法。理想的引物是MSP扩增成功与否的关键因素之一...现在普遍认为肿瘤的发生与DNA的异常甲基化有关，所以DNA甲基化状态的研究成为了研究肿瘤发生机制的一个热点，MSP(methylation specific PCR)则是检测DNA甲基化状态的一个简便有效的方法。理想的引物是MSP扩增成功与否的关键因素之一，它决定着MSP扩增的特异性、扩增的效率和扩增的保真度等。展开更多
Ojbective To search for circulating DNA as a biomarker for the cancer burden of patients^[1].Methods Methylation specific PCR(MSP),according to Herman et al,was used to identify this epigenetic modification of DNA fro...Ojbective To search for circulating DNA as a biomarker for the cancer burden of patients^[1].Methods Methylation specific PCR(MSP),according to Herman et al,was used to identify this epigenetic modification of DNA from serum and tissue of 111 patients with different cancers.In parallel the mutant K-ras gene was analyzed in 55 cases for comparison.Results Methylation of the p16 gene was found in serum or cancer tissue in about 50% of patients with a statistically significant difference Results Methylation of the p16 gene was found in serum or cancer tissue in about 50% of patients with a statistically significant difference to healthy controls(P<0.05).There was no difference between the DNA from the tumor and from the neighboring normal tissue or from sereum in early cancer.The positivity rate increased with progression of the tumor.20 days after the operation methylated DNA nearly disappeared.The positivity rate for mutant K-ras was only 30% and thus much lower than that of methlated DNA.The combination of the two markers increased the positivity rate of tumors by 13%.Conclusion The detection of methylation of the p16 gene by MSP combined with mutant K-ras is serum of cancer patients would be a very effective method to estimate the cancer burden.展开更多
目的研究膀胱癌中BLU基因启动子甲基化状态、转录水平及其意义。方法采用甲基化特异性PCR(meth-ylation specific PCR,MSP)技术和逆转录PCR(RT-PCR)技术分别检测54例膀胱癌患者癌组织及45例相应癌周正常组织、3例非肿瘤患者的正常膀胱...目的研究膀胱癌中BLU基因启动子甲基化状态、转录水平及其意义。方法采用甲基化特异性PCR(meth-ylation specific PCR,MSP)技术和逆转录PCR(RT-PCR)技术分别检测54例膀胱癌患者癌组织及45例相应癌周正常组织、3例非肿瘤患者的正常膀胱组织中BLU基因启动子甲基化状态和转录水平。结果①31.5%(17/54)膀胱癌组织中BLU基因高甲基化,相应癌周正常组织和3例正常膀胱组织均未发现该基因高甲基化改变;②甲基化状态与膀胱癌临床病理参数无明显相关性;③在45例膀胱癌组织中,BLU表达缺失率为42.2%(19/45);④BLU mRNA表达缺失与肿瘤临床分期和病理分级相关(P<0.05);⑤在启动子高甲基化的膀胱癌中,92.3%(12/13)BLU mRNA表达异常下调或缺失。结论膀胱癌中频繁发生BLU基因的高甲基化和mRNA表达缺失,其高甲基化可能是基因转录失活的重要原因。展开更多
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.
文摘现在普遍认为肿瘤的发生与DNA的异常甲基化有关，所以DNA甲基化状态的研究成为了研究肿瘤发生机制的一个热点，MSP(methylation specific PCR)则是检测DNA甲基化状态的一个简便有效的方法。理想的引物是MSP扩增成功与否的关键因素之一，它决定着MSP扩增的特异性、扩增的效率和扩增的保真度等。
文摘Ojbective To search for circulating DNA as a biomarker for the cancer burden of patients^[1].Methods Methylation specific PCR(MSP),according to Herman et al,was used to identify this epigenetic modification of DNA from serum and tissue of 111 patients with different cancers.In parallel the mutant K-ras gene was analyzed in 55 cases for comparison.Results Methylation of the p16 gene was found in serum or cancer tissue in about 50% of patients with a statistically significant difference Results Methylation of the p16 gene was found in serum or cancer tissue in about 50% of patients with a statistically significant difference to healthy controls(P<0.05).There was no difference between the DNA from the tumor and from the neighboring normal tissue or from sereum in early cancer.The positivity rate increased with progression of the tumor.20 days after the operation methylated DNA nearly disappeared.The positivity rate for mutant K-ras was only 30% and thus much lower than that of methlated DNA.The combination of the two markers increased the positivity rate of tumors by 13%.Conclusion The detection of methylation of the p16 gene by MSP combined with mutant K-ras is serum of cancer patients would be a very effective method to estimate the cancer burden.
文摘目的研究膀胱癌中BLU基因启动子甲基化状态、转录水平及其意义。方法采用甲基化特异性PCR(meth-ylation specific PCR,MSP)技术和逆转录PCR(RT-PCR)技术分别检测54例膀胱癌患者癌组织及45例相应癌周正常组织、3例非肿瘤患者的正常膀胱组织中BLU基因启动子甲基化状态和转录水平。结果①31.5%(17/54)膀胱癌组织中BLU基因高甲基化,相应癌周正常组织和3例正常膀胱组织均未发现该基因高甲基化改变;②甲基化状态与膀胱癌临床病理参数无明显相关性;③在45例膀胱癌组织中,BLU表达缺失率为42.2%(19/45);④BLU mRNA表达缺失与肿瘤临床分期和病理分级相关(P<0.05);⑤在启动子高甲基化的膀胱癌中,92.3%(12/13)BLU mRNA表达异常下调或缺失。结论膀胱癌中频繁发生BLU基因的高甲基化和mRNA表达缺失,其高甲基化可能是基因转录失活的重要原因。
