Journal: Chemotherapy Research and Practice

Article Title: Inhibition of NF- κ B by Dehydroxymethylepoxyquinomicin Suppresses Invasion and Synergistically Potentiates Temozolomide and γ -Radiation Cytotoxicity in Glioblastoma Cells

doi: 10.1155/2013/593020

Figure Lengend Snippet: (a) The analysis of the methylation status of the MGMT gene in GBM cell lines revealed two groups: methylated (U87MG, U343MG-a, and LN319) and hemimethylated (U251, T98G and U138MG); however, only T98G and U138MG express this gene as detected by real time quantitative PCR (*a pool of 5 white matter samples was used as calibrator); (b) treatment with DHMEQ efficiently decreases the expression of MGMT after 24 h. Data represents two independent experiments in duplicate and are expressed as mean ± SEM; (c) comet assay showed that TMZ-induced DNA damage significantly increases in T98G and U138MG cells as a probable consequence of reduced MGMT expression after exposure to DHMEQ. Each value represents the mean derived from at least three individual experiments (mean ± SD).

Article Snippet: Real-time RT-PCR reactions were performed in triplicate in 10 μ L reactions using the inventoried TaqMan probes (Applied Biosystems, Foster City, CA, EUA) for BCL2 (Hs00608023_m1), BCL-XL (Hs00236329_m1), XIAP (Hs01597783_m1), MMP-2 (Hs01548727_m1), MMP-14 (Hs00237119_m1), uPA (Hs01547054_m1), TIMP-2 (Hs00234278_m1), and MGMT (Hs01037698_m1), on the ABI Prism 7500 Sequence Detector (Applied Biosystems, Foster City, CA, EUA).

Techniques: Methylation, Real-time Polymerase Chain Reaction, Expressing, Single Cell Gel Electrophoresis, Derivative Assay

Journal: Neuro-Oncology Practice

Article Title: Correlation of commercially available quantitative MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation scores and GBM patient survival

doi: 10.1093/nop/npy028

Figure Lengend Snippet: Cox Regression Analysis of Overall Survival and Progression-Free Survival of 102 Primary Glioblastoma MGMT Unmethylated Patients With Substratification by MGMT Values

Article Snippet: Our patient cohort was derived retrospectively and consisted of newly diagnosed GBM patients seen at UCLA and KPLA between 2011 and 2016 with accessible LabCorp quantitative MGMT methylation values.

Techniques: Biomarker Discovery

Journal: Neuro-Oncology Practice

Article Title: Correlation of commercially available quantitative MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation scores and GBM patient survival

doi: 10.1093/nop/npy028

Figure Lengend Snippet: Kaplan-Meier analysis is used to A, compare the MGMT (1-1.99) vs MGMT (<1) and MGMT (≥2) patients. The MGMT (1-1.99) group’s median OS (25.4 months) falls in between the MGMT (≥2) (38.8 months) and MGMT (<1) (17.3 months) median OS values (Log-rank P = .001). B, PFS showed the same trend, namely the MGMT (1-1.99) group generated a higher median OS of 11.8 months compared to the MGMT (<1) group but lower than the MGMT (≥ 2) group, yielding 11.8 months vs 7.92 months and 18.0 months (Log-rank P < .0001), respectively. MGMT indicates O-6-methylguanine-DNA methyltransferase; mo, months; OS, overall survival; PFS, progression-free survival.

Article Snippet: Our patient cohort was derived retrospectively and consisted of newly diagnosed GBM patients seen at UCLA and KPLA between 2011 and 2016 with accessible LabCorp quantitative MGMT methylation values.

Techniques: Generated

Journal: Neuro-Oncology Practice

Article Title: Correlation of commercially available quantitative MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation scores and GBM patient survival

doi: 10.1093/nop/npy028

Figure Lengend Snippet: Cox Regression Analysis of Overall Survival and Progression-Free Survival of 165 Primary Glioblastoma Patients With Substratification by MGMT Values

Article Snippet: Our patient cohort was derived retrospectively and consisted of newly diagnosed GBM patients seen at UCLA and KPLA between 2011 and 2016 with accessible LabCorp quantitative MGMT methylation values.

Techniques: Biomarker Discovery

Journal: The Journal of Nutrition

Article Title: Fish Oil Contaminated with Persistent Organic Pollutants Induces Colonic Aberrant Crypt Foci Formation and Reduces Antioxidant Enzyme Gene Expression in Rats

doi: 10.3945/jn.117.251082

Figure Lengend Snippet: Colonic gene expression of Ptgs2 (A), Rela (B), Mgmt (C), Ogg1 (D), Sod (E), and Cat (F) in male rats fed the CO, FO, or POP FO diet for 9 wk. Values are means ± SEs (n = 10). Means without a common letter differ, P < 0.05. Cat, catalase; CO, corn oil; FO, fish oil; Mgmt, O6-methylguanine DNA methyltransferase; Ogg1, 8-oxoguanine glycosylase; POP, persistent organic pollutant; Ptgs2, prostaglandin endoperoxide synthase 2; Rela, component of NF-κB; RQ, relative quantification; Sod, superoxide dismutase.

Article Snippet: The mRNA levels of prostaglandin endoperoxide synthase 2 ( Ptgs2 ), component of NF-κB ( Rela ), superoxide dismutase ( Sod ), catalase, O 6 -methylguanine DNA methyltransferase ( Mgmt ), and 8-oxoguanine glycosylase ( Ogg1 ) were analyzed with the use of quantitative real-time PCR (ViiA7; Applied Biosystems).

Techniques: Gene Expression, Quantitative Proteomics

Journal: The Journal of Nutrition

Article Title: Fish Oil Contaminated with Persistent Organic Pollutants Induces Colonic Aberrant Crypt Foci Formation and Reduces Antioxidant Enzyme Gene Expression in Rats

doi: 10.3945/jn.117.251082

Figure Lengend Snippet: Spearman correlations between variables in male rats fed the CO, FO, or POP FO diet for 9 wk 1

Article Snippet: The mRNA levels of prostaglandin endoperoxide synthase 2 ( Ptgs2 ), component of NF-κB ( Rela ), superoxide dismutase ( Sod ), catalase, O 6 -methylguanine DNA methyltransferase ( Mgmt ), and 8-oxoguanine glycosylase ( Ogg1 ) were analyzed with the use of quantitative real-time PCR (ViiA7; Applied Biosystems).

Techniques:

Journal: Molecular Diagnosis & Therapy

Article Title: Prognostic and Predictive Epigenetic Biomarkers in Oncology

doi: 10.1007/s40291-018-0371-7

Figure Lengend Snippet: Methylation: prognostic and predictive biomarkers with diagnostic utility

Article Snippet: There are a number of commercial tests available to evaluate the MGMT methylation level by (1) methylation-specific polymerase chain reaction (PCR): PredictMDx Glioblastoma (MDx Health); (2) real-time PCR: MGMT Methylation Detection Kit (EntroGen); (3) MS-MLPA: SALSA MS-MLPA probe mix ME011 MMR genes (MRC-Holland); and (4) pyrosequencing technology: PyroMark MGMT Kit (Qiagen).

Techniques: Methylation, Diagnostic Assay, Mutagenesis

Journal: Carcinogenesis

Article Title: Glycogen synthase kinase 3β inhibition sensitizes human glioblastoma cells to temozolomide by affecting O6-methylguanine DNA methyltransferase promoter methylation via c-Myc signaling.

doi: 10.1093/carcin/bgt182

Figure Lengend Snippet: Fig. 2. (A) A comparison of the levels of MGMT gene expression in T98G, U138, U251 and U87 GBM cells by QRT–PCR with primers specific to MGMT. The relative value of MGMT messenger RNA expression in T98G was scored as 1.0. *BLQ, below the limit of quantitation; bars show standard deviations in the data. The figure shows the data from three independent experiments. (B) The effect of GSK3β–RNA interference on GBM cell survival. Values of relative cell viability were measured by the AlamarBlue assay and compared between T98G and U251 cells transfected with GSK3β-specific and non-specific siRNA (10 nmol/l each), respectively, for 72 h. The relative viability of the cells treated with non-specific siRNA was scored as 1.0. *P = 0.002; Mann–Whitney U-test, bars show standard deviation. The figure shows the data from experiments carried out in sextuplicates. (C and D) The relative cell viability of T98G and U251 cells was measured by the AlamarBlue assay and compared between cells treated with DMSO (control) or AR-A014418 (5, 10, 20, 40 or 80 μmol/l) for 144 h. The relative viability of the cells treated with DMSO was scored as 1.0. *P < 0.05; Mann–Whitney U-test, bars show standard deviation. The figure shows the data from experiments carried out in sextuplicates. (E) The effect of GSK3β inhibitor (AR-A014418) on the expression of total and tyrosine 216-phosphorylated GSK3β. Equal amounts of whole-cell lysates from T98G, U138, U251 and U87 cells treated with DMSO or 20 μmol/l AR-A014418, respectively, for 72 h were analyzed by western blot of total and phosphorylated GSK3β proteins, with β-actin used as loading control. AR, AR-A014418. The figure shows the representative data from three independent western blots.

Article Snippet: Rabbit monoclonal anti-c-Myc antibody (Epitomics, Burlingame, CA), mouse monoclonal anti-GSK3β antibody and mouse monoclonal antityrosine 216-phosphorylated GSK3β antibody (BD Biosciences, San Jose, CA), anti-DNA (cytosine-5)-methyltransferase 3A antibody (Imgenex, San Diego, CA), anti-β-catenin antibody and anti-MGMT antibody (Cell Signaling Technology) were used at a dilution of 1:5000, 1:10 000, 1:5000, 1:1000, 1:5000 and 1:2500, respectively.

Techniques: Comparison, Gene Expression, Quantitative RT-PCR, RNA Expression, Quantitation Assay, Alamar Blue Assay, Transfection, MANN-WHITNEY, Standard Deviation, Control, Expressing, Western Blot

Journal: Carcinogenesis

Article Title: Glycogen synthase kinase 3β inhibition sensitizes human glioblastoma cells to temozolomide by affecting O6-methylguanine DNA methyltransferase promoter methylation via c-Myc signaling.

doi: 10.1093/carcin/bgt182

Figure Lengend Snippet: Fig. 4. (A) Changes in MGMT gene methylation status in GBM cells by GSK3β inhibition. The effect of GSK3β inhibition with 5, 10 or 20 μmol/l of AR-A014418 for 72 h on the methylation status of the MGMT promoter in T98G, U251 and 138 cells was observed by MSP assay. PCR products in the M lanes and U lanes indicate methylated and unmethylated status of the MGMT promoter, respectively. U87, U87 GBM cell line as a methylated control; U138, U138 GBM cell line as an unmethylated control; AR, AR-A014418. (B) The effect of GSK3β inhibition on methylation status of the MGMT promoter examined with MethyLight assay in T98G, U251 and U138 cells treated for 72 h with DMSO or AR-A014418 (5, 10 or 20 μmol/l). qPCR of bisulfite-converted DNA with the primers and probes specific to the methylated fraction of the MGMT promoter. The level of MGMT promoter methylation in the cells treated with DMSO was scored as 1.0. *P < 0.05, **P < 0.01, AR, AR-A014418; Mann–Whitney U-test, bars show standard deviation. The figure shows the data from three independent experiments. (C) QRT–PCR analysis of the effect of GSK3β inhibition on MGMT gene expression in T98G, U251 and U138 cells treated with DMSO or AR-A014418 (5, 10 or 20 μmol/l), respectively, for 72 h. The level of MGMT messenger RNA expression in the cells treated with DMSO was scored as 1.0. *BLQ, below the limit of quantitation. **P < 0.01; Mann–Whitney U-test, bars show standard deviation. The figure shows the data from three independent experiments.

Article Snippet: Rabbit monoclonal anti-c-Myc antibody (Epitomics, Burlingame, CA), mouse monoclonal anti-GSK3β antibody and mouse monoclonal antityrosine 216-phosphorylated GSK3β antibody (BD Biosciences, San Jose, CA), anti-DNA (cytosine-5)-methyltransferase 3A antibody (Imgenex, San Diego, CA), anti-β-catenin antibody and anti-MGMT antibody (Cell Signaling Technology) were used at a dilution of 1:5000, 1:10 000, 1:5000, 1:1000, 1:5000 and 1:2500, respectively.

Techniques: Methylation, Inhibition, MSP Assay, Control, MANN-WHITNEY, Standard Deviation, Quantitative RT-PCR, Gene Expression, RNA Expression, Quantitation Assay

Journal: Carcinogenesis

Article Title: Glycogen synthase kinase 3β inhibition sensitizes human glioblastoma cells to temozolomide by affecting O6-methylguanine DNA methyltransferase promoter methylation via c-Myc signaling.

doi: 10.1093/carcin/bgt182

Figure Lengend Snippet: Fig. 5. (A) The effect of GSK3β inhibitor (AR-A014418) on the expression of MGMT and c-Myc. Equal amounts of whole-cell lysates from T98G, U138 and U251 cells treated with DMSO or 20 μmol/l AR-A014418, respectively, for 72 h were analyzed by western blot of c-Myc and MGMT proteins, with β-actin used as loading control, AR, AR-A014418. The figure shows the representative data from three independent western blots. (B) Changes in MGMT gene methylation status in GBM cells by GSK3β and c-Myc inhibition. The effect of combination of DMSO–AR-A014418 (5, 10 or 20 μmol/l) and c-Myc inhibitor 20 μmol/l for 72 h on the methylation status of the MGMT promoter in T98G, U251 and U138 cells was observed by MSP assay. PCR products in the M lanes and U lanes indicate methylated and unmethylated status of the MGMT promoter, respectively. U87, U87 GBM cell line as a methylated control; U138, U138 GBM cell line as an unmethylated control; AR, AR-A014418. (C) The effect of GSK3β and c-Myc inhibition on methylation status of the MGMT promoter examined with MethyLight assay in T98G, U251 and U138 cells treated for 72 h with combination of DMSO–AR-A014418 (5, 10 or 20 μmol/l) and c-Myc inhibitor 20 μmol/l. qPCR of bisulfite-converted DNA with the primers and probes specific to the methylated fraction of the MGMT promoter. The level of MGMT promoter methylation in the cells treated with DMSO was scored as 1.0. **P < 0.01, AR, AR-A014418; Mann–Whitney U-test, bars show standard deviation. The figure shows the data from three independent experiments. 2213

Article Snippet: Rabbit monoclonal anti-c-Myc antibody (Epitomics, Burlingame, CA), mouse monoclonal anti-GSK3β antibody and mouse monoclonal antityrosine 216-phosphorylated GSK3β antibody (BD Biosciences, San Jose, CA), anti-DNA (cytosine-5)-methyltransferase 3A antibody (Imgenex, San Diego, CA), anti-β-catenin antibody and anti-MGMT antibody (Cell Signaling Technology) were used at a dilution of 1:5000, 1:10 000, 1:5000, 1:1000, 1:5000 and 1:2500, respectively.

Techniques: Expressing, Western Blot, Control, Methylation, Inhibition, MSP Assay, MANN-WHITNEY, Standard Deviation

Journal: Carcinogenesis

Article Title: Glycogen synthase kinase 3β inhibition sensitizes human glioblastoma cells to temozolomide by affecting O6-methylguanine DNA methyltransferase promoter methylation via c-Myc signaling.

doi: 10.1093/carcin/bgt182

Figure Lengend Snippet: Fig. 6. Epigenetic silencing of MGMT expression by GSK3β inhibition. (A and B) Comparison by ChIP assay of the binding of histone H3, c-Myc, DNMT3A and FLAG to the E-box site in the MGMT promoter between the GBM cells treated with DMSO or with a GSK3β inhibitor (AR-A014418). T98G and U138 cells were treated for 72 h with DMSO or 5 μmol/l of AR-A014418. The amount of DNA coprecipitated with the antibody to each molecule was measured by

Article Snippet: Rabbit monoclonal anti-c-Myc antibody (Epitomics, Burlingame, CA), mouse monoclonal anti-GSK3β antibody and mouse monoclonal antityrosine 216-phosphorylated GSK3β antibody (BD Biosciences, San Jose, CA), anti-DNA (cytosine-5)-methyltransferase 3A antibody (Imgenex, San Diego, CA), anti-β-catenin antibody and anti-MGMT antibody (Cell Signaling Technology) were used at a dilution of 1:5000, 1:10 000, 1:5000, 1:1000, 1:5000 and 1:2500, respectively.

Techniques: Expressing, Inhibition, Comparison, Binding Assay

Journal: Scientific Reports

Article Title: MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma

doi: 10.1038/s41598-024-61240-x

Figure Lengend Snippet: MGMT expression levels in GBM, GSCs and melanoma cell lines. ( A and D ) The methylation-specific PCR (MSP) analyses of the MGMT promoter from GBMs, GSCs, and melanoma cell lines. Note the bands in the unmethylated (U, 93 bp) and methylated (M, 81 bp) lanes for GBM, GSCs, and melanoma cell lines, reflecting the unmethylated/methylated MGMT promoter. Percentages represent the proportion of methylation (M) and unmethylation (U) on the MGMT promoter in each cell line. ( B and E) Transcript levels of MGMT mRNA in GBM, GSCs, and melanoma cell lines were determined by qRT-PCR. ( C and F) MGMT levels in GBM, GSCs, and melanoma cell lines were determined by immunoblot analysis. Data are the mean ± SEM for three independent experiments.

Article Snippet: MGMT mRNA levels were determined by quantitative real-time PCR (qRT-PCR) using the TaqManTM Universal Master Mix II, with UNG (Thermo Fisher Scientific) and MGMT Taqman probes (Hs00172470_m1; Thermo Fisher Scientific).

Techniques: Expressing, Methylation, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma

doi: 10.1038/s41598-024-61240-x

Figure Lengend Snippet: The effect of siMGMT on the radioresponse of MGMT-producing cells. ( A and B ) ACPK1, GBMJ1, A375, and MM415 cells were transfected with or without 25 nM siMGMT for 48 h. MGMT levels were determined by immunoblot analysis. ( C and D ) ACPK1, GBMJ1, A375, and MM415 cells were transfected with 25 nM of si (negative control) and siMGMT before radiation. Surviving fraction (Log) curves were generated after normalizing for the cytotoxicity generated by siMGMT alone. Data are the mean ± SEM for three independent experiments. * P < 0.05 and ** P < 0.005 by Student’s t-test.

Article Snippet: MGMT mRNA levels were determined by quantitative real-time PCR (qRT-PCR) using the TaqManTM Universal Master Mix II, with UNG (Thermo Fisher Scientific) and MGMT Taqman probes (Hs00172470_m1; Thermo Fisher Scientific).

Techniques: Transfection, Western Blot, Negative Control, Generated

Journal: Scientific Reports

Article Title: MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma

doi: 10.1038/s41598-024-61240-x

Figure Lengend Snippet: The effect of lomeguatrib on radioresponse of MGMT-producing cells. ( A and B ) ACPK1, GBMJ1, A375, and MM415 cells were treated with a gradient (25 µM, 50 µM, 100 µM, and 150 µM) of lomeguatrib for the indicated times (24 h and 48 h). MGMT levels were determined by immunoblot analysis. ( C and D ) ACPK1, GBMJ1, A375, and MM415 cells were treated with the designated concentrations of lomeguatrib (ACPK1, A375, and MM415—100 µM and GBMJ1—50 µM) for 16 h before radiation. Surviving fraction (Log) curves were generated after normalizing for the cytotoxicity generated by lomeguatrib alone. * P < 0.05 and ** P < 0.005 by Student’s t-test.

Article Snippet: MGMT mRNA levels were determined by quantitative real-time PCR (qRT-PCR) using the TaqManTM Universal Master Mix II, with UNG (Thermo Fisher Scientific) and MGMT Taqman probes (Hs00172470_m1; Thermo Fisher Scientific).

Techniques: Western Blot, Generated

Journal: Scientific Reports

Article Title: MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma

doi: 10.1038/s41598-024-61240-x

Figure Lengend Snippet: The effect of lomeguatrib on radiation-induced γH2AX foci and mitotic catastrophe in MGMT-producing cells. ( A ) The quantitative assessment of γH2AX foci per cell at 1 h and 24 h after radiation is shown. Foci were counted in at least 50 cells per experiment and representative histograph images obtained from control, lomeguatrib-only, radiation (4 Gy)-only, and lomeguatrib/radiation combination treatment. Data are the mean ± SEM for three independent experiments, and statistical significance was determined by Student’s t-test. *P < 0.05 and **P < 0.005 (radiation versus lomeguatrib/radiation). ( B ) The quantitative assessment of nuclear fragmentation per cell at 24 h and 72 h after radiation is shown. Nuclear fragmentation (defined as the presence of two or more distinct lobes within a single cell) was evaluated in at least 100 cells per treatment per experiment and representative histograph images obtained from control cells and cells pretreated with lomeguatrib alone, radiation (4 Gy) alone, and combination treatment of lomeguatrib with radiation. Data are the mean ± SEM for three to four independent experiments. Statistical significance was determined by Student’s t-test. *P < 0.05 and ***P < 0.0005 (radiation versus Lomeguatrib + radiation).

Article Snippet: MGMT mRNA levels were determined by quantitative real-time PCR (qRT-PCR) using the TaqManTM Universal Master Mix II, with UNG (Thermo Fisher Scientific) and MGMT Taqman probes (Hs00172470_m1; Thermo Fisher Scientific).

Techniques: Control

Journal: Scientific Reports

Article Title: MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma

doi: 10.1038/s41598-024-61240-x

Figure Lengend Snippet: The effect of MGMT overexpression on the radioresponse in non-MGMT-producing cells. ( A ) MGMT protein expression was assessed via western blot analysis following transfection of 1 µg GFP-MGMT vector in non-MGMT-producing cells (OSU61, NSC11, WM852 and WM266-4) for 48 h. ( B and C ) OSU61, NSC11, WM852 and WM266-4 cells were transfected with 1 µg of GFP-control vector and GFP-MGMT vector before radiation. Surviving fraction (Log) curves were generated after normalizing for the cytotoxicity generated by GFP- MGMT vector alone. DMF values were calculated at a surviving fraction (Log) of 0.1. Data are the mean ± SEM for three independent experiments. * P < 0.05 by Student’s t-test.

Article Snippet: MGMT mRNA levels were determined by quantitative real-time PCR (qRT-PCR) using the TaqManTM Universal Master Mix II, with UNG (Thermo Fisher Scientific) and MGMT Taqman probes (Hs00172470_m1; Thermo Fisher Scientific).

Techniques: Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation, Control, Generated