| Objective: Previous studies show that the DNA-repair enzyme O~6-methylguanine-DNA-methyltransferase (MGMT) is one of drug resistant factors that affect chemosensibility of glioma. The MGMT gene has been shown to be silenced by promoter methylation in many human tumors. This study explored the relationship between the hypermethylation of CpG islands in the promoter regions of MGMT genes of cell lines and its sensitivity to BCNU; the relationship between the hypermethylation of CpG islands in the promoter regions of MGMT genes of glioma cells and the expression of MGMT and the concordance of the hypermethylation of CpG islands in the promoter regions of MGMT genes between tumor and paired serum of 27 glioma patients.Methods: In this research, 9 human glioma cell lines, 27 primary glioma tissues and paired serum were studied. Genomic DNA was extracted from tissues and paired serum using protocols provided by Gentra Puregene DNA purification kit. The DNA was modified by bisulfite reaction using the CpGenome DNA modification kit. Modified DNA was amplified by methylation-specic PCR (MSP) procedure to analyze the status of methylation. Methylation-specific PCR was used to study the promotermethylation of the MGMT gene in glioma tissues and paired serum; the cell resistance to BCNU in human glioma cell lines was determined by SRB assay; MGMT expression was examined by immunohistochemistry method. The x~2 test, Fisher's Exact Test, Nonparametric statistics test and Spearman correlation coefficient were used for the statistics, P<0.05 was considered significant. Result:1. In the 6 cell lines which were hypermethylated, sensitivity to BCNU was noted in 5 cell lines; in the 3 cell lines which were unmethylated, resistance to BCNU was noted in all of the 3 cell lines. Spearman correlation coefficient was used to analysis the result. The relation was significant, P <0.05.2. The rate of MGMT gene promotor methylation of glioma tissues in maleand female were 55.6%(10/18) and 44.4%(4/9), respectively. However, the difference was not significant, P>0.05; The rate of MGMT gene promotor methylation of paired serum in male and female were 50.0% (9/18) and 44.4% (4/9), respectively. However, the difference was not significant, P >0.05.3. The rate of hypermethylation of the promoter of MGMT gene of glioma tissues was 36.3%(4/l 1) in patients aged not more than 50 years; the rate of hypermethylation of the promoter of MGMT gene was 62.5%(10/16) in patient aged more than 50 years. The differences in methylation rate amongthe both groups were not significant, />>0.05; The rate of hypermethylation of the promoter of MGMT gene of paired serum was 36.3%(4/l 1) in patients aged not more than 50 years; the rate of hypermethy lation of the promoter of MGMT gene was 56.2%(9/16) in patient aged more than 50 years. The differences in methylation rate among the both groups were not significant, P>0.05.4. Of the 27 glioma tissues, the methylation rate of MGMT in Grade I > Grade II > Grade III and Grade IV groups were 0.0% (0/2X 50.0% (5/10), 57.1% (8/14) and 100%, respectively. There were no significant differences among these groups, P>0.05; Of the 27 paired serum, the methylation rate of MGMT in Grade I ^ Grade II ^ Grade III and Grade IV groups were 0.0%(0/2X 40.0% (4/1 OX 57.1% (8/14) and 100%, respectively. There were no significant differences among these groups, P>0.05.5. Among the 27 glioma samples, MGMT expression was noted in 18 samples, in which hypermethylation was detected in 7(38.9%) cases, while MGMT expression was not noted in 9 samples, in which hypermethylation was detected in 7(77.8%) cases. Spearman correlation coefficient was used to analysis the result. The relation was significant, P<0.05.6. MGMT status was studied in DNA extracted from 27 glioma samples and paired serum samples. Among the 27 gliomas samples, MGMT hypermethylation was detected in 14(51.9%) cases, while it was detected in 13(48.1%) paired serum samples; MGMT unmethylation was detected in13(48.1%) gliomas samples, while it was detected in 14(51.9%) pairedserum samples. Spearman correlation coefficient was used to analysis theresult. The relation was significant, P<0.05.Conclusion:The present study demonstrates that the aberrant methylation of MGMTgene promoter may play an important role in the sensibility of glioma toalkylating agents.1. In human glioma cell lines, the relation between the aberrant methylation of CpG islands in the promoter regions of MGMT gene promoter and the cell resistant to BCNU is significant. If the MGMT gene promoter is hypermethylated, the cell lines are sensitive to BCNU, while if the MGMT gene promoter is unmethylated, the cell lines are resistant to BCNU,2. The relationship between the aberrant methylation of MGMT gene promoter and the expression of MGMT in glioma is significant, so is the relationship between the aberrant methylation of MGMT gene promoter and the sensibility of glioma to alkylating agents, While the aberrant methylation of promoter of MGMT gene does not show any significant relationship with clinical condition such as age, gender and pathological grading.3. The relation between the aberrant methylation of promoter of MGMT gene in glioma tissues and paired serum is significant. Methylated MGMT is found in serum DNA of glioma patients, with a good correlation between serum and primary tumor tissue. Methylation-specific PCR assay in serumDNA could be a good predictive tool for selecting glioma patients to be treated with alkylating agents.
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