methylguanine-DNA Methyltransferase Gene Research Articles

Proteomic and metabolomic analyses illustrate the mechanisms of expression of the O6 -methylguanine-DNA methyltransferase gene in glioblastoma.

Glioblastoma (GBM) has been reported to be the most common high-grade primary malignant brain tumor in clinical practice and has a poor prognosis. O6 -methylguanine-DNA methyltransferase (MGMT) promoter methylation has been related to prolonged overall survival (OS) in GBM patients after temozolomide treatment. Proteomics and metabolomics were combined to explore the dysregulated metabolites and possible protein expression alterations in white matter (control group), MGMT promoter unmethylated GBM (GBM group) or MGMT promoter methylation positive GBM (MGMT group). In total, 2745 upregulated and 969 downregulated proteins were identified in the GBM group compared to the control group, and 131 upregulated and 299 downregulated proteins were identified in the MGMT group compared to the GBM group. Furthermore, 131 upregulated and 299 downregulated metabolites were identified in the GBM group compared to the control group, and 187 upregulated and 147 downregulated metabolites were identified in the MGMT group compared to the GBM group. The results showed that 94 upregulated and 19 downregulated proteins and 20 upregulated and 16 downregulated metabolites in the MGMT group were associated with DNA repair. KEGG pathway enrichment analysis illustrated that the dysregulated proteins and metabolites were involved in multiple metabolic pathways, including the synthesis and degradation of ketone bodies, amino sugar and nucleotide sugar metabolism. Moreover, integrated metabolomics and proteomics analysis was performed, and six key proteins were identified in the MGMT group and GBM group. Three key pathways were recognized as potential biomarkers for recognizing MGMT promoter unmethylated GBM and MGMT promoter methylation positive GBM from GBM patient samples, with areas under the curve of 0.7895, 0.7326 and 0.7026, respectively. This study provides novel mechanisms to understand methylation in GBM and identifies some biomarkers for the prognosis of two different GBM types, MGMT promoter unmethylated or methylated GBM, by using metabolomics and proteomics analyses.

Effect of β-estradiol on the expression of human MGMT gene in cells in vitro

To determine whether the steroid hormone β-estradiol affects the transcription of the human MGMT gene. The expression status of the DNA repair enzyme O (6)-methylguanine-DNA methyltransferase (MGMT) is important when planning the treatment of patients with oncology, because its presence in tumor cells may reduce the effectiveness of alkylating chemotherapy. Alkylating chemotherapy is combined with hormone therapy in the treatment of tumors, so the study of the effect of β-estradiol on the number of transcripts of the human MGMT gene in cells in vitro has not only theoretical and also practical significance. Methods. We used a variety of methods, including culture, molecular genetic and biochemical, such as RNA isolation, cDNA synthesis, reverse transcriptase PCR, agarose and polyacrylamide gel electrophoresis, and statistical processing of the results. Results. We found a tendency of β-estradiol to downregulate the MGMT gene at mRNA level in both HEp-2 and 293 cells in a range of concentrations, except concentrations 1 and 5 nmol/L of β-estradiol in 293 cells. Conclusions. Our data support the hypothesis that β-estradiol is one of the hormonal regulators of the MGMT gene.

Quantitative Analysis of the MGMT Methylation Status of Glioblastomas in Light of the 2021 WHO Classification.

Simple SummaryThe status of a DNA repair protein called MGMT is a prognostic marker in patients with glioblastomas, the most frequent malignant brain tumor. Epigenetic silencing of this gene predicts increased sensitivity to chemotherapy. Silencing is assessed by analyzing modifications, so-called methylation, of a certain gene region. Whereas most studies report such information only in a qualitative manner, quantitative testing might provide additional information. The aim of our study was to determine a quantitative threshold for better survival among patients with glioblastomas. Among 321 patients suffering from glioblastoma, we found better survival in patients with glioblastomas that have ≥16% methylation of a particular region of the MGMT gene. Above 16% methylation, we found no additional benefit with increasing degree of methylation. We suggest using this threshold for selection in clinical trials and for patient counseling.Background: Glioblastomas with methylation of the promoter region of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene exhibit increased sensitivity to alkylating chemotherapy. Quantitative assessment of the MGMT promoter methylation status might provide additional prognostic information. The aim of our study was to determine a quantitative methylation threshold for better survival among patients with glioblastomas. Methods: We included consecutive patients ≥18 years treated at our department between 11/2010 and 08/2018 for a glioblastoma, IDH wildtype, undergoing quantitative MGMT promoter methylation analysis. The primary endpoint was overall survival. Results: A total of 321 patients were included. Median overall survival was 12.6 months. Kaplan–Meier and adjusted Cox regression analysis showed better survival for the groups with 16–30%, 31–60%, and 61–100% methylation. In contrast, survival in the group with 1–15% methylation was similar to those with unmethylated promoter regions. A secondary analysis confirmed this threshold. Conclusions: Better survival is observed in patients with glioblastomas with ≥16% methylation of the MGMT promoter region than with <16% methylation. Survival with tumors with 1–15% methylation is similar to with unmethylated tumors. Above 16% methylation, we found no additional benefit with increasing methylation.

Bortezomib sensitization of recurrent glioblastoma with unmethylated MGMT promoter to temozolomide, a phase II study (NCT03643549).

TPS2081 Background: Patients with glioblastoma with functional O6 methylguanine DNA methyltransferase ( MGMT) DNA repair enzyme gain limited benefit from temozolomide (TMZ). Bortezomib depletes the MGMT enzyme, restoring the tumor ́s susceptibility to TMZ, if the chemotherapy is administered in the precise schedule. Additionally, bortezomib inhibits the tumor growth by blocking autophagy flux. Thus, pre-treatment with bortezomib prior to temozolomide leads to DNA repair enzyme depletion and blockade of autophagy-induced survival signals. Methods: The academic, industry independent, multicenter, open label, single arm, non-randomized phase II trial is designed to investigate the survival benefits for patients treated with bortezomib 48hrs prior to TMZ. Efficacy of this therapy will be compared to historical cohorts receiving standard management. The study will include 53 patients with recurrent or progressive, MGMT unmethylated glioblastoma. The additional 10 patients were treated in the phase IB. All patients will receive a combination of bortezomib 1.3mg/m2 administered IV on days 1, 4, 7, during each 4-week chemotherapy cycle with PO TMZ at 200mg/m2 5 days/week q4w starting on day 3. Dose reduction of TMZ is allowed if reduced bone marrow tolerance is present. Major eligibility criteria include histologically confirmed glioblastoma with unmethylated MGMT promoter, MRI evidence of recurrence within 14 days prior to enrolment, age ≥ 18 years with life expectancy &gt; 8 weeks, KPS ≥ 70, radiologically (MRI) confirmed tumor relapse/progression ≥ 12 weeks since completed radiotherapy, tumor not available for radiosurgery, adequate bone marrow, renal and liver function, no contraindications for bortezomib and/or TMZ. Endpoints: Estimation of the median progression free survival (PFS) and overall survival (OS) of patients with recurrent or progressed glioblastoma as well as progression free rate at 6 months. Secondary objectives: Therapy response assessed by contrast enhanced MRI (RANO criteria) and neurological examination (NANO criteria) as well as identification of novel biomarkers that correlate with treatment response. Twenty-four of the planned 53 patients are enrolled. Five of them receive treatment. The prespecified activity goal for the first stage of the study was met. Discussion: Patients with glioblastoma harboring active MGMT enzyme have median survival of 12.7 months, compared to 21.7 months for patients with the MGMT gene promoter silenced by methylation. This dismal prognosis for the approx. 55% of GBM patients underscores the unmet need for novel combination therapies that sensitize the tumors to chemotherapy. Results from this trial will serve as preliminary evidence of the role of bortezomib administered in a precise manner to abolish the temozolomide resistance of GBM with unmethylated MGMT promotor. The study is currently active and recruiting. Clinical trial information: NCT03643549.

Determination of IDH1, IDH2, MGMT, TERT and ATRX Gene Mutations in Glial Tumors.

The most frequent and dangerous kind of primary brain tumor is glioblastoma multiforme (GBM). The survival rates associated with GBM are very short and molecular markers for predicting survival are needed. The aim of our study was to evaluate isocitrate dehydrogenase 1 and 2 (IDH1, IDH2), telomerase reverse transcriptase (TERT), O-6- methylguanine-DNA methyltransferase (MGMT) and alpha-thalassemia/mental retardation, X-linked (ATRX) genes with next-generation sequencing (NGS) to find potential pathological mutations and their effect on survival. Thirty patients who had undergone craniotomy and were diagnosed with high-grade glioma were evaluated for this study. Peripheral blood samples were obtained from all participants. IDH1, IDH2, TERT, MGMT and ATRX genes were evaluated with next-generation sequencing from the samples. Survival analysis evaluated the effects of all these mutations on survival. The median age of the patients was 58.5 (range=11- 74) years, and 56.7% (n=17) were under 60 years of age. According to sex, male patients comprised 66.7%. Targeted NGS detected 21 chromosomal aberrations. When more than three chromosomal anomalies were accepted as a reference, anomaly in three or fewer chromosomes negatively affected overall survival (hazard ratio=2.83). Targeted NGS generates therapeutically meaningful information, providing better prognostic information than conventional histology. Our study shows that NGS provides important information on survival by helping to detect chromosomal changes that can be detected in routine blood samples. It is clear that incorporating molecular diagnostics into our standard-of-care routine will help us better understand our patients' outcomes.

Effect of temozolomide combined with radiotherapy on survival and MGMT protein expression in recurrent malignant glioma patients

Purpose: To investigate the effect of temozolomide (TMZ) combined with radiotherapy (RT) on O-6- methylguanine-DNA methyltransferase (MGMT) protein and survival of recurrent malignant glioma patients.&#x0D; Methods: Ninety-two patients with malignant glioma in our hospital from January 2014 to January 2015 were assigned to study and control groups using the random table method. Subjects in the control group received radiotherapy (total dose in the range of 60 – 75 Gy), while those in the study group were given TMZ orally (75 mg/m2) daily in addition to radiotherapy, as well as TMZ at 150 – 200 mg/m2. After treatment, clinical effectiveness was compared for the two groups. Changes in methylation of MGMT gene were determined in the two groups. The patients were followed up for 3 years, and the degrees of survival and recurrence were recorded.&#x0D; Results: Total effectiveness of clinical treatment was markedly higher in the study group (76.09 %) than in the control group (45.65 %; p &lt; 0.05). One month after radiotherapy, significant decrease in MGMT gene methylation was seen in patients in the study group, relative to control patients (p &lt; 0.05). Patients in the study group had lower median recurrence but higher degree of survival in the 2nd and 3rd years, relative to control patients (p &lt; 0.05).&#x0D; Conclusion: The combination of temozolomide and radiotherapy is more effective than radiotherapy in the treatment of recurrent malignant glioma. The combined treatment significantly inhibits tumor recurrence in patients, and improves their prognosis and standard of life.

Clinical validation of a novel quantitative assay for the detection of MGMT methylation in glioblastoma patients

BackgroundThe promoter hypermethylation of the methylguanine-DNA methyltransferase gene is a frequently used biomarker in daily clinical practice as it is associated with a favorable prognosis in glioblastoma patients treated with temozolamide. Due to the absence of adequately standardized techniques, international harmonization of the MGMT methylation biomarker is still an unmet clinical need for the diagnosis and treatment of glioblastoma patients.ResultsIn this study we carried out a clinical validation of a quantitative assay for MGMT methylation detection by comparing a novel quantitative MSP using double-probe (dp_qMSP) with the conventional MSP in 100 FFPE glioblastoma samples. We performed both technologies and established the best cutoff for the identification of positive-methylated samples using the quantitative data obtained from dp_qMSP. Kaplan–Meier curves and ROC time dependent curves were employed for the comparison of both methodologies.ConclusionsWe obtained similar results using both assays in the same cohort of patients, in terms of progression free survival and overall survival according to Kaplan–Meier curves. In addition, the results of ROC(t) curves showed that dp_qMSP increases the area under curve time-dependent in comparison with MSP for predicting progression free survival and overall survival over time. We concluded that dp_qMSP is an alternative methodology compatible with the results obtained with the conventional MSP. Our assay will improve the therapeutic management of glioblastoma patients, being a more sensitive and competitive alternative methodology that ensures the standardization of the MGMT-biomarker making it reliable and suitable for clinical use.

Analysis of the correlation of MGMT and XRCC1 gene expression with the pathogenesis of glioma and its mechanism analysis

Objective To investigate the relationship between the expression of O6- methyl guanine DNA methyltransferase (MGMT), X-ray repair cross complementation gene 1 (XRCC1) and the incidence of glioma. Methods From February 2015 to September 2017, 53 glioma patients (glioma group) in our hospital were enrolled in the study. 50 patients with hypertensive intracerebral hemorrhage were selected as control A group, and 106 healthy volunteers as control B group. Immunohistochemical staining was used to detect the expression of MGMT and XRCC1 in brain tissue of glioma group and control A group, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to detect the polymorphism of MGMT and XRCC1 gene in glioma group and control B group. Results The positive expression rates of MGMT and XRCC1 in the tissues of brain glioma were 47.17% and 39.62%, respectively, which were significantly higher than those in the control A group (P 0.05); There was no correlation between the expression of MGMT and XRCC1 in glioma tissues (rs=0.162, P>0.05); The proportion of XRCC1 genotype AG+ GG in brain glioma group was 58.49%, which was significantly higher than that of control B group (P<0.05); The proportion of MGMT genotype LP+ PP in brain glioma group was 28.30%, which was significantly higher than that of control B group (P<0.05). Conclusions MGMT and XRCC1 are increased significantly in glioma brain tissues, but not correlated with pathological grades; The polymorphism of MGMT and XRCC1 genes may be related to the susceptibility of gliomas. Key words: DNA modification methylases/GE; X-ray repair cross complementing protein 1/GE; Glioma/GE; Polymorphism, genetic

Repair gene O6 -methylguanine-DNA methyltransferase is controlled by SP1 and up-regulated by glucocorticoids, but not by temozolomide and radiation.

Therapy of malignant glioma relies on treatment with the O6 -methylating agent temozolomide (TMZ) concomitant with ionizing radiation followed by adjuvant TMZ. For the treatment of recurrences, DNA chloroethylating drugs are also used. The main killing lesion induced by these drugs is O6 -alkylguanine. Since this damage is repaired by O6 -methylguanine-DNA methyltransferase (MGMT), the repair enzyme represents a most important factor of drug resistance, limiting the therapy of malignant high-grade gliomas. Although MGMT has been shown to be transcriptionally up-regulated in rodents following genotoxic stress, it is still unclear whether human MGMT is subject to up-regulation. Here, we addressed the question whether MGMT in glioma cells is enhanced following alkylating drugs or ionizing radiation, using promoter assays. We also checked the response of glioma cell lines to dexamethasone. In a series of experiments, we found no evidence that the human MGMT promoter is significantly up-regulated following treatment with TMZ, the chloroethylating agent nimustine or radiation. It was activated, however, by dexamethasone. Using deletion constructs, we further show that the basal level of MGMT is mainly determined by the transcription factor SP1. The high amount of SP1 sites in the MGMT promoter likely prevents transcriptional up-regulation following genotoxic stress by neutralizing inducible signals. The regulation of MGMT by miRNAs plays only a minor role, as shown by DICER knockdown experiments. Since high dose dexamethasone concomitant with temozolomide is frequently used in glioblastoma therapy, induction of the MGMT gene through glucocorticoids in MGMT promoter unmethylated cases might cause further elevation of drug resistance, while radiation and alkylating drugs seem not to induce MGMT at transcriptional level.

Abstract 285: Disulfiram, a dual MGMT and aldehyde dehydrogenase inhibitor, sensitizes ER-positive breast cancer cells to temozolomide and cyclophosphamide

Abstract Background: O6 methylguanine DNA methyltransferase (MGMT) is overexpressed in a majority of cancers, including breast cancer. MGMT is a DNA repair protein leading to chemo resistance. MGMT expression directly correlates with ER expression and tamoxifen resistance. Aldehyde Dehydrogenase (ALDH) activity, as a progenitor/stem cell marker, while it has been reported to inversely correlate with MGMT expression in other cancers, has also been linked to chemotherapy and radiation resistance. Methods: We have tested the effect of Antabuse (disulfiram, DSF), as a dual MGMT and ALDH inhibitor, at nontoxic doses, in combination with Temozolomide (TMZ) or/and Cyclophosphamide (CP) on ER+ breast cancer cells. Results: DSF at very low doses (achievable in human serum with standard DSF clinical dosing) decreases ER+ breast cancer cell growth (MCF7, T47D and ZR75) in a dose-dependent manner. DSF further sensitizes breast cancer cells to TMZ or/and CP and significantly inhibits breast cancer growth without causing unwanted side effects on the normal breast epithelial cells. Dose effect and isobologram studies confirm synergistic activity of DSF + CP and moderate synergism for DSF + TMZ. DSF, alone or in combination with TMZ (DSF ± TMZ) and/or CP (DSF ± CP), significantly inhibits expression of MGMT, aldehyde dehydrogenase, ERα, E2F and BIRC5 gene (survivin) - all involved in breast cancer tumorigenesis. DSF, alone or in combination with TMZ (DSF ± TMZ) and/or CP (DSF ± CP) caused significant apoptosis in breast cancer cells. In a dose dependent manner, DSF inhibited colony formation, effect which was further enhanced by addition of TMZ/CP (DSF ± TMZ/CP). Similarly, DSF alone or in combination with TMZ (DSF ± TMZ) and/or CP (DSF ± CP) decreased the metastatic potential of breast cancer cells. Conclusions: Our findings suggest that DSF as dual inhibitor of MGMT and ALDH significantly enhances the antitumor effect of alkylators as CP and TMZ in ER+ breast cancer Citation Format: George C. Bobustuc, Deborah Donohoe, Alisher Holmuhamedov, Srivenugopal Kalkunte, Santhi D. Konduri. Disulfiram, a dual MGMT and aldehyde dehydrogenase inhibitor, sensitizes ER-positive breast cancer cells to temozolomide and cyclophosphamide. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 285.

Abstract 3493: Disulfiram a dual mgmt and aldehyde dehydrogenase inhibitor sensitizes pancreatic cancer to gemcitabine and abraxane

Abstract Background: O6 methylguanine DNA methyltransferase (MGMT) is overexpressed in a majority of cancers, including pancreatic cancer. MGMT has been the focus of significant research for its role in the repair of DNA damage caused by chemotherapeutic agents. Aldehyde Dehydrogenase as a progenitor/stem cell marker has been linked to chemotherapy and radiation resistance. Methods: We tested the administration of Antabuse (disulfiram, DSF) at nontoxic doses in combination with gemcitabine (GEM) and Abraxane (ABX) on pancreatic cancer cells. Results: DSF at very low doses (achievable in human serum with standard DSF clinical dosing) decreases pancreatic cancer cell growth in a dose-dependent manner. DSF further sensitizes pancreatic cancer cells to GEM and ABX and significantly inhibits pancreatic cancer growth without causing unwanted side effects to the normal pancreatic cells. Dose effect and isobologram studies confirm synergistic activity of DSF and ABX combination and moderate synergism for DSF and GEM combination. DSF, either alone or in combination with GEM (DSF ± GEM) and/or ABX (DSF ± ABX), significantly inhibits expression of MGMT, aldehyde dehydrogenase, antigen Ki-67 and BIRC5 gene (survivin) - all involved in pancreatic cancer tumorigenesis. DSF+/-GEM+/-ABX induces the expression of p21Cip1, a cell cycle inhibitor. We show an inverse correlation between aldehyde dehydrogenase activity and MGMT expression, where MGMT expression is high, aldehyde dehydrogenase activity is low and where aldehyde dehydrogenase activity is high, MGMT levels were low. The ALDH3A1 expression is significantly higher in highly metastatic cancer cells (L3.6pl) compared to other metastatic cancer cells (PANC1, Mia PaCa2, ASPC1). We further report that in our model DSF at very low doses (achievable in human serum with standard DSF clinical dosing) effectively inhibits both aldehyde dehydrogenase and MGMT in pancreatic cancer cells. DSF also induces caspase activation indicative of mitochondrial induced apoptosis in these cells. Conclusions: Our findings suggest that DSF as dual inhibitor of MGMT and aldehyde dehydrogenase potentially provides a novel therapeutic approach to inhibiting pancreatic cancer growth/metastasis and synergistically enhances GEM and ABX activity. Citation Format: George C. Bobustuc, Alisher Holmuhamedov, Kalkunte S. Srivenugopal, Jacob C. Frick, JAMES L. WEESE, Santhi D. Konduri. Disulfiram a dual mgmt and aldehyde dehydrogenase inhibitor sensitizes pancreatic cancer to gemcitabine and abraxane. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3493. doi:10.1158/1538-7445.AM2015-3493

The Methylation Status of MGMT and SFRP2 Genes in Iranian Patients With Polyps

Background: Colorectal cancer is the third most common cancer in the world and the forth cause of death by cancer globally. Colorectal cancer starts with a polyp and might develop due to multiple epigenetic and genetic changes that lead to change of normal colon epithelium to adenocarcinoma of colon. Recent studies show that epigenetic changes such as methylation of SFRP2 (secreted frizzled-related2) a gene in negative regulation Wnt signaling pathways and MGMT (methylguanine-DNA methyltransferase) a DNA repair gene is associated with colorectal cancer. Objectives: Due to correlation of polyp and cancer, the current study aimed to evaluate the role of SFRP2 and MGMT methylation in patients with polyp in south of Iran. Materials and Methods: Forty eight tissue samples of polyp and adenoma, and 20 control samples were investigated for methylation status of SFRP2 and MGMT with mutagenically separated Polymerase Chain Reaction (MS-PCR) method. Results: Methylation in at least one gene was reported about 93.8%. The methylation of SFRP2 was reported in 39 out of the 48 subjects (81.2%) whereas methylation of MGMT was observed in 32 subjects (66.6%). Conclusions: The current study results showed that methylation of SFRP2 and MGMT play an important role in polyp formation.

Impact on prognosis of the regional distribution of MGMT methylation with respect to the CpG island methylator phenotype and age in glioma patients.

Clinical and molecular prognostic factors in gliomas include age, IDH mutation, the glioma CpG island methylator phenotype (G-CIMP+) and promoter methylation of the O(6)-methylguanine DNA-methyltransferase (MGMT) gene. Among these markers, a predictive value was reported in glioblastomas (GBM) for MGMT promoter methylation, in particular in elderly GBM patients. In this study, methylation data from 46 glioma samples with the Illumina 450K platform were obtained and extended using external data to include a total of 247 glioma samples. Methylation analysis of the whole MGMT gene with this platform revealed two strongly survival-associated CpG regions within the promoter and the gene body, which were confirmed in a reported dataset of high grade-gliomas. Methylation at the promoter (CpG 25, cg12981137 and the prognostic model MGMT-STP27) and at the gene body CpG 165 (cg07933035), were significantly associated with better overall survival, and strongly correlated with G-CIMP+ status. In this series, the prognostic value of MGMT methylation at the promoter was not observed in G-CIMP- cases, although around 50 % of them were MGMT-methylated. These results were also obtained in an homogeneously-treated series of chemoradiated G-CIMP- GBMs analyzed by MSP and qMSP, and confirmed in a reported pyrosequencing-analyzed series of gliomas. Interestingly, in contrast to the MGMT promoter, gene body methylation was of prognostic value in G-CIMP-patients older than 65 years. Our study highlights the relevance of the prognostic value of the different regions of methylation throughout the MGMT gene that could be affected by specific G-CIMP profiles and age groups.

MGMT gene silencing by promoter hypermethylation in gastric cancer in a high incidence area.

Inactivation of tumor suppressor and DNA repair genes by promoter hypermethylation does commonly occur in human cancers. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that removes methyl groups as well as larger adducts at the O(6) position of guanine. In the absence of MGMT activity, O(6)-methylguanine mispairs with thymine during DNA replication, resulting in G:C to A:T transitions. Promoter hypermethylation of the MGMT gene has been observed in various cancers, including gastric cancer. Here, we aimed at assessing the promoter hypermethylation, mutation and expression status of the MGMT gene in patients from a geographic region with a high incidence of gastric cancer (Kashmir, North India) and to investigate their association with various clinicopathological characteristics. In this study 82 gastric cancer samples and adjacent normal tissues were included. Mutations in the MGMT gene were detected by single stranded conformational polymorphism (SSCP) analysis and direct sequencing. Methylation-specific polymerase chain reaction (MS-PCR) and Western blot analyses were performed to detect promoter hypermethylation and concomitant (loss of) expression of the MGMT gene. Promoter hypermethylation of the MGMT gene was found in 52.44% (43 of 82) of the tumor samples and loss of MGMT protein expression was detected in 45.12% (37 of 82) of the tumor samples. Hypermethylation and loss of expression were significantly associated with higher tumor grades (moderately/poorly differentiated) (P < 0.05) and higher tumor stages (III/IV) (P < 0.05). In addition, MGMT hypermethylation and loss of expression were found to be significantly associated with high salt tea consumption (P < 0.05). Our results indicate that MGMT promoter hypermethylation and concomitant loss of MGMT protein expression may play an important role in the development of gastric cancer in the Kashmiri population. High salt tea consumption may be a risk factor.

MGMT testing--the challenges for biomarker-based glioma treatment.

Many patients with malignant gliomas do not respond to alkylating agent chemotherapy. Alkylator resistance of glioma cells is mainly mediated by the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). Epigenetic silencing of the MGMT gene by promoter methylation in glioma cells compromises this DNA repair mechanism and increases chemosensitivity. MGMT promoter methylation is, therefore, a strong prognostic biomarker in paediatric and adult patients with glioblastoma treated with temozolomide. Notably, elderly patients (>65-70 years) with glioblastoma whose tumours lack MGMT promoter methylation derive minimal benefit from such chemotherapy. Thus, MGMT promoter methylation status has become a frequently requested laboratory test in neuro-oncology. This Review presents current data on the prognostic and predictive relevance of MGMT testing, discusses clinical trials that have used MGMT status to select participants, evaluates known issues concerning the molecular testing procedure, and addresses the necessity for molecular-context-dependent interpretation of MGMT test results. Whether MGMT promoter methylation testing should be offered to all individuals with glioblastoma, or only to elderly patients and those in clinical trials, is also discussed. Justifications for withholding alkylating agent chemotherapy in patients with MGMT-unmethylated glioblastomas outside clinical trials, and the potential role for MGMT testing in other gliomas, are also discussed.

Aberrant DNA methylation of MGMT and hMLH1 genes in prediction of gastric cancer.

We aimed to explore the association between aberrant DNA methylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) and human mutL homolog 1 (hMLH1) genes with gastric cancer. A total of 283 gastric cancer patients who were confirmed by pathological diagnosis were included in our study. Aberrant DNA methylation of MGMT and hMLH1 were detected. The proportions of DNA hypermethylation in MGMT and hMLH1 in cancer tissues were significantly higher than those in remote normal-appearing tissues. The DNA hypermethylation of MGMT was correlated with the tumor-necrosis-metastasis stage in gastric cancer tissues. Results showed that individuals with gastric cancer in the N1 and M1 stages had a significantly higher risk of DNA hypermethylation of MGMT in cancer tissues [odds ratio (OR) = 1.97, 95% confidence interval (CI) = 1.15-3.37 for the N1 stage; OR (95%CI) = 5.39 (2.08-14.98) for the M1 stage]. In conclusion, we found that aberrant hypermethylation of MGMT could be a predictive biomarker for detecting gastric cancer.

Glycogen synthase kinase 3β inhibition sensitizes human glioblastoma cells to temozolomide by affecting O 6 -methylguanine DNA methyltransferase promoter methylation via c-Myc signaling

Glycogen synthase kinase 3β (GSK3β) is a serine/threonine protein kinase involved in human cancers including glioblastoma. We have previously demonstrated that GSK3β inhibition enhances temozolomide effect in glioma cells. In this report, we investigated the molecular mechanisms of sensitization of glioblastoma cells to temozolomide by GSK3β inhibition, focusing on O(6)-methylguanine DNA methyltransferase (MGMT) gene silencing. Glioblastoma tissues from patients treated with the GSK3β-inhibiting drugs were subjected to immunohistochemistry and methylation-specific PCR assay. Human glioblastoma cell lines T98G, U138, U251 and U87 were treated with a small-molecule GSK3β inhibitor, AR-A014418 or GSK3β-specific small interfering RNA. The combined effect of temozolomide and AR-A014418 on cell proliferation was determined by AlamarBlue assay and an isobologram method. MGMT promoter methylation was estimated by methylation-specific PCR and MethyLight assay. MGMT gene expression was evaluated by real-time quantitative reverse transcriptase-PCR. c-Myc and DNA (cytosine-5)-methyltransferase 3A binding to the MGMT promoter was estimated by chromatin immunoprecipitation assay. GSK3β inhibition decreased phosphorylation of glycogen synthase and reduced MGMT expression and increased MGMT promoter methylation in clinical tumors. In glioblastoma cell lines, GSK3β inhibition decreased cell viability, enhanced temozolomide effect and downregulated MGMT expression with relevant changes in the methylation levels of the MGMT promoter. Here, we showed for the first time that c-Myc binds to the MGMT promoter with consequent recruitment of DNA (cytosine-5)-methyltransferase 3A, regulating the levels of MGMT promoter methylation. The results of this study suggest that GSK3β inhibition enhances temozolomide effect by silencing MGMT expression via c-Myc-mediated promoter methylation.

Research on DNA methylation of human osteosarcoma cell MGMT and its relationship with cell resistance to alkylating agents

The objective of this study was to explore the O(6)-methylguanine-DNA methyltransferase (MGMT) gene methylation status and its protein expression, as well as the effects of demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-CdR) on MGMT gene expression and its resistance to alkylating agents, and to elucidate MGMT expression mechanism and significance in osteosarcoma. The human osteosarcoma cell lines Saos-2 and MG-63 were collected and treated with 5-Aza-CdR for 6 days. The cells not treated with 5-Aza-CdR were set as a negative control. The genomic DNA was extracted from the Saos-2 and MG-63 cells using methylation-specific PCR to detect the promoter CpG island methylation status of the MGMT gene. Cell sensitivity to alkylating agents before and after drug administration was detected by the MTT method. The variation in MGMT gene mRNA and protein was detected by reverse transcription PCR (RT-PCR) and Western blotting. The MGMT promoter gene of normal Saos-2 cells was methylated, with reduced MGMT mRNA and protein expression; the MGMT mRNA and protein expression of Saos-2 cells treated with 5-Aza-CdR was obviously enhanced, and its sensitivity to alkylating agents was reversed. Meanwhile, with promoter CpG island unmethylation of the MGMT gene, MGMT protein was expressed in the normal MG-63 cells and the MG-63 cells treated with 5-Aza-CdR, and both showed resistance to alkylating agents. The methylation status of the MGMT gene promoter in human osteosarcoma cells reflected the cells' ability to induce MGMT protein expression and can be used as a molecular marker to project the sensitivity of cancer tissues to alkylating agent drugs.

Type-3 metabotropic glutamate receptors regulate chemoresistance in glioma stem cells, and their levels are inversely related to survival in patients with malignant gliomas

Drug treatment of malignant gliomas is limited by the intrinsic resistance of glioma stem cells (GSCs) to chemotherapy. GSCs isolated from human glioblastoma multiforme (GBM) expressed metabotropic glutamate receptors (mGlu3 receptors). The DNA-alkylating agent, temozolomide, killed GSCs only if mGlu3 receptors were knocked down or pharmacologically inhibited. In contrast, mGlu3 receptor blockade did not affect the action of paclitaxel, etoposide, cis-platinum, and irinotecan. mGlu3 receptor blockade enabled temozolomide toxicity by inhibiting a phosphatidylinositol-3-kinase/nuclear factor-κB pathway that supports the expression of O(6)-methylguanine-DNA methyltransferase (MGMT), an enzyme that confers resistance against DNA-alkylating agents. In mice implanted with GSCs into the brain, temozolomide combined with mGlu3 receptor blockade substantially reduced tumor growth. Finally, 87 patients with GBM undergoing surgery followed by adjuvant chemotherapy with temozolomide survived for longer time if tumor cells expressed low levels of mGlu3 receptors. In addition, the methylation state of the MGMT gene promoter in tumor extracts influenced survival only in those patients with low expression of mGlu3 receptors in the tumor. These data encourage the use of mGlu3 receptor antagonists as add-on drugs in the treatment of GBM, and suggest that the transcript of mGlu3 receptors should be measured in tumor specimens for a correct prediction of patients' survival in response to temozolomide treatment.

The Changes in MGMT Promoter Methylation Status in Initial and Recurrent Glioblastomas

To evaluate the mechanism of the development of therapeutic resistance after temozolomide treatment, we focused on changes in O(6)-methylguanine DNA methyltransferase (MGMT) and mismatch repair (MMR) between initial and recurrent glioblastomas. Tissue samples obtained from 24 paired histologically confirmed initial and recurrent adult glioblastoma patients who were initially treated with temozolomide were used for MGMT and MMR gene promoter methylation status and protein expression analysis using methylation-specific multiplex ligation probe amplification (MS-MLPA), methylation-specific polymerase chain reaction (MSP), and immunohistochemical staining. There was a significant decrease in the methylation ratio of the MGMT promoter determined by MS-MLPA, which was not detectable with MSP, and MGMT protein expression changes were not remarkable. However, there was no epigenetic variability in MMR genes, and a relatively homogeneous expression of MMR proteins was observed in initial and recurrent tumors. We conclude that the development of reduced methylation in the MGMT promoter is one of the mechanisms for acquiring therapeutic resistance after temozolomide treatment in glioblastomas.