Study On Detection Of O~6-methylguanine-DNA Methyltransferase Promoter Methylation In Chemotherapy For Cancers

Background and Objective:To prevent and surmount the resistance of tumor cells treated by chemotherapy is a prompt problem.O⁶-methylguanine-DNA methyltransferase(MGMT) is the DNA repair protein responsible for removing alkylated adducts form the O⁶-position of guanine in DNA.Previous studies showed that MGMT plays an important role in cell to defense against alkylating agents that generate lethal DNA adducts and provides tumors resistance to treatment with alkylating agents.Recently,advances have been made in the study of the epigenetic silencing mechanism of MGMT in cancer.DNA hypermethylation at the promoter CpG island plays a pivotal role in the epigenetic silencing of MGMT.There is a significant positive correlation between MGMT promoter hypermethylation and increased tumor sensitivity to alkylating agents. These findings underline the importance of MGMT promoter hypermethylation in clinical cancer research as well as in cancer treatment.MGMT promoter CpG island methylation may be a good predictive marker for chemotherapy of alkylating agents. This study were to explore the status of MGMT promoter CpG island methylation and its protein expression as well as cells to alkylating agents drug resistance related to each other in human cancers.The aim was to assess the value of detecting the promoter methylation of MGMT gene in chemotherapy for cancers.Methods:Methylation-specific PCR(MSP) was employed to detect MGMT promoter CpG island methylation in 12 cancer cell lines,39 samples taken from surgery and 32 samples of pretreatment serum all from the patients with gliomas,17 tissue samples of Osteosarcoma patients.Western analysis and immunohistochemistry were used to detect protein expression.MTT were employed to detect the sensitivity of cancer cell lines to bifunctional alkylating agent nimustine hydrochloride(ACNU) and monofunctional aklylating agent temozolomide(TMZ).The drug concentration corresponding to 50%cell survival(IC₅₀ value) was set as a measurable indicator.The Kaplan-Meier curve was adopted to estimate the overall survival according to the methylation status of the MGMT promoter.Statistical correlations were analyzed by software SPSS 13.0 for windows,t test,Fisher's Exact Test,Nonparametric statistics test and spearman correlation coefficient were used for the statistics,P≤0.05 was considered statistical significant.Main content and Results:(1) The relationship between the status of MGMT promoter CpG island and protein expression as well as the alkylating agent drug sensitivity of 12 cancer cell lines.In the 12 cancer cell lines,there are 4 cell lines MGMT promoter CpG island were hypermethylated in which 3 cell lines were not the expression of MGMT protein and one cell line was low expression.While,in the 8 cell lines which MGMT promoter CpG island were unmethylated,MGMT protein expression were examined in all of the 8 cell lines.Spearman correlation coefficient was used to analysis the result.The relation was significant,p＜0.05.In the 4 cell lines which MGMT promoter CpG island were methylated,their IC₅₀ value of ACNU and TMZ were remarkably low.The status of MGMT promoter CpG island methylation was significant to sensitivity of cancer cell lines to alkylating agent drug,ACNU and TMZ,p＜0.05 (Spearman test).(2) Treated cell lines SHG-44 and U251 with the DNA demethylating 5-aza-2'deoxycytidine(5-Aza-CdR) and MGMT inhibitor streptozotocin(STZ).To more directly address whether the promoter CpG island methylation was itself inhibiting the expression of MGMT,cell lines SHG-44 and U251 were treated with 5-aza-2'deoxycytidine(5-Aza-CdR) and streptozotocin(STZ).And the changes of the sensitivity of the cell lines to alkylating agent drug were examined after treatment. The results showed that:With promoter CpG island methylation of the MGMT gene, no MGMT protein was expressed in the normal SHG-44 cells,and its IC₅₀ values to ACNU and TMZ were 30.61μg/ml and 11.24μg/ml,respectively,showing sensitive to alkylating agents ACNU and TMZ.After being treated with 5-Aza-CdR,the SHG-44 cells restored expressing MGMT protein followed by MGMT gene demethylation successfully,and its IC₅₀ values to ACNU and TMZ were increased by 2.5 and 3.1 times respectively(both P＜0.05),showing resistive to alkylating agents ACNU and TMZ.Meanwhile,with promoter CpG island unmethylation of the MGMT gene,MGMT protein was expressed in the normal U251 cells and the U251 cells treated with 5-Aza-CdR,and both showing resistive to alkylating agents ACNU and TMZ.On the contrary,SHG-44 and U251 cell were treated with and STZ plus ACNU/TMZ,STZ can improve dramatically the sensitivity of U251 to ACNU and TMZ(both P＜0.05).But no influence of STZ can be detected in SHG-44 with MGMT promoter methylation.(3) The status of MGMT promoter CpG island methylation in glioma and Osteosarcoma tissue samples.Methylation of MGMT promoter CpG island were detectable in 46.2%(18/39) of glioma tissues and 23.5%(4/17) in Osteosarcoma tissues,but not in any normal tissues.The status of MGMT promoter CpG island methylation was association with the protein level of MGMT(both P＜0.05).In 39 glioma patients,the prevalence of methylation in MGMT showed no significant difference between early stage and late stage(P＞0.05);or between less malignant(Ⅰ～Ⅱ) and high malignant(Ⅲ～Ⅳ) cancer (P＞0.05).The results also indicated that the methylation of MGMT was not associated with age or gender of the patients.Overall survival were estimated by the Kaplan-Meier method and compared with the use of the log-rank test.The result showed that the presence of MGMT CpG island methylation was associated with a statistically significant increase in overall survival. For the entire population of 39 glioma patients who were enrolled in operating treatment combined with radiotherapy and chemotherapy with alkylating agents including ACNU,there was a significant difference in overall survival between patients with methylated MGMT promoter and those with an unmethylated MGMT promoter(P＜0.05 by the log-rank test).(4) Prevalence of promoter CpG island hypermethylation in serum samples of glioma. Methylated gene could also be detected in serum of glioma patients.In all 32 serum samples in the study,methylation of MGMT promoter CpG island was detected in about 40.6%of serum.Of the 32 serum samples obtained,21 of them had paired tumor tissue available.We found a similar pattern of methylation changes for MGMT in the serum samples to their paired tumor tissues.MGMT promoter CpG island methylation were detected in 42.9%(9/21) of tissue samples,and in 33.3%(7/21) of these paired serum samples.Of the patients with MGMT promoter CpG island methylation in the tissue samples,MGMT promoter CpG island methylation detected in serum was 77.8%(7/9).And no methylated sequence was detected in serum from those negative for methylation in paired tissues.In contrast,methylated DNA was not detectable in any of the 30 normal serum samples.Patients without MGMT promoter CpG island methylation in glioma tissues were found to have no MGMT promoter CpG island methylation in their serum either.The occurrence of methylation for MGMT in serum was associated with that in their paired tumor tissues(P=0.018, Fisher's Exact Test)Conclusions:The status of MGMT promoter CpG island methylation is closely correlated to MGMT protein expression in cancers.Hypermethylation of the CpG island in the promoter region is an important mechanism responsible for the loss function of DNA repair protein and impacts on the therapeutic response to alkylating agents.The status of MGMT promoter CpG island methylation can report the capacity of cell to induce MGMT protein expression,and it can be used as a molecular marker of projecting the sensitivity of cancer tissues to alkylating agent drugs.MGMT promoter CpG island methylation could also be detected in serum of glioma patients.Furthermore,the occurrence of MGMT promoter CpG island methylation in serum was associated with that in their paired tumor tissues.The presence of tumor-specific methylated gene sequences in peripheral blood may be developed as a noninvasive approach for cancer detection.Further analysis in a much larger size of samples is warranted.Determination of the status of MGMT promoter CpG island methylation by methylation-specific PCR may allow the selection of patients who are most likely to benefit from alkylating agents treatment.For patients whose tumors are not methylated at the MGMT,alternative treatments with a different mechanism of action or methods of inhibiting MGMT should be developed.Methylation analysis appears to be a promising predictive factor in primary glioma and a noninvasive tumor marker in serum DNA.