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: PLoS ONE

Article Title: Comprehensive Analysis of MGMT Promoter Methylation: Correlation with MGMT Expression and Clinical Response in GBM

doi: 10.1371/journal.pone.0016146

Figure Lengend Snippet: Each row represents an individual patient. For each patient the qMSP P positive result is shown as a black rectangle. The next column shows the results of the qMSP test (Labcorp). PFS is represented by a circle with a blackened circle representing greater than 1 year PFS. Positive MGMT protein expression (>15% tumor cells stained positive in IHC) is shown as black rounded rectangle and negative expression is shown as white rounded rectangle. MGMT mRNA expression is shown using color-coded rectangles. Absence of any of the shapes indicates results not available. All CpG sites belonging to the three regions, R1, R2 and R3 are shown within the blue outlined squares. The four distinct groups are outlined with red squares. The qMSP region is labeled with qMSP primers in R3. The transcription start site (TSS) of the MGMT gene is marked by an arrow.

Article Snippet: Real-time PCR of MGMT was performed on the ABI PRISM 7900 HT detection system using a taqman probe (Hs00172470_m1, Applied Biosystems) and taqman reagents under default conditions: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute with human beta-glucuronidase (hGUS) as endogenous control.

Techniques: Expressing, Staining, Labeling

Journal: PLoS ONE

Article Title: Comprehensive Analysis of MGMT Promoter Methylation: Correlation with MGMT Expression and Clinical Response in GBM

doi: 10.1371/journal.pone.0016146

Figure Lengend Snippet: Significant individual CpG site correlations with MGMT mRNA expression, MGMT protein expression and PFS.

Article Snippet: Real-time PCR of MGMT was performed on the ABI PRISM 7900 HT detection system using a taqman probe (Hs00172470_m1, Applied Biosystems) and taqman reagents under default conditions: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute with human beta-glucuronidase (hGUS) as endogenous control.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Comprehensive Analysis of MGMT Promoter Methylation: Correlation with MGMT Expression and Clinical Response in GBM

doi: 10.1371/journal.pone.0016146

Figure Lengend Snippet: All three regions of the MGMT promoter for each patient are classified as methylated (M) or unmethylated (UM). Based on the status of these three regions, patients are classified into four major groups: Group A) all regions unmethylated, Group B) one region methylated, Group C) two regions methylated, and Group D) all three regions methylated. For correlation analysis, Groups B and C were further subdivided as indicated.

Article Snippet: Real-time PCR of MGMT was performed on the ABI PRISM 7900 HT detection system using a taqman probe (Hs00172470_m1, Applied Biosystems) and taqman reagents under default conditions: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute with human beta-glucuronidase (hGUS) as endogenous control.

Techniques: Methylation

Journal: PLoS ONE

Article Title: Comprehensive Analysis of MGMT Promoter Methylation: Correlation with MGMT Expression and Clinical Response in GBM

doi: 10.1371/journal.pone.0016146

Figure Lengend Snippet: Correlation of MGMT promoter methylation patterns with MGMT mRNA expression, MGMT protein expression and PFS.

Article Snippet: Real-time PCR of MGMT was performed on the ABI PRISM 7900 HT detection system using a taqman probe (Hs00172470_m1, Applied Biosystems) and taqman reagents under default conditions: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute with human beta-glucuronidase (hGUS) as endogenous control.

Techniques: Methylation, Expressing

Journal: PLoS ONE

Article Title: Comprehensive Analysis of MGMT Promoter Methylation: Correlation with MGMT Expression and Clinical Response in GBM

doi: 10.1371/journal.pone.0016146

Figure Lengend Snippet: Kaplan-Meier estimation of PFS according to MGMT promoter methylation status determined using: A) 3R classification method - The median PFS for the methylated group was 540 days vs. 210 days for the unmethylated group (HR = 5.23, 95% CI [2.089–13.097], p<0.0001), B) qMSP P - The median PFS for the methylated group was 373 days vs. 224 days for the unmethylated group (HR = 1.707, 95% CI [0.728–4.003], p = 0.213), and C) MS-MLPA - The median PFS for the methylated group was 540 days vs. 210 days for the unmethylated group (HR = 3.076, 95% CI [1.301–7.27], p = 0.007).

Article Snippet: Real-time PCR of MGMT was performed on the ABI PRISM 7900 HT detection system using a taqman probe (Hs00172470_m1, Applied Biosystems) and taqman reagents under default conditions: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute with human beta-glucuronidase (hGUS) as endogenous control.

Techniques: Methylation

Journal: PLoS ONE

Article Title: Comprehensive Analysis of MGMT Promoter Methylation: Correlation with MGMT Expression and Clinical Response in GBM

doi: 10.1371/journal.pone.0016146

Figure Lengend Snippet: Kaplan-Meier estimation of PFS according to MGMT promoter methylation status determined using: A. MS-MLPA (p-value = 0.002) and B. qMSP (p-value = 0.089).

Article Snippet: Real-time PCR of MGMT was performed on the ABI PRISM 7900 HT detection system using a taqman probe (Hs00172470_m1, Applied Biosystems) and taqman reagents under default conditions: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute with human beta-glucuronidase (hGUS) as endogenous control.

Techniques: Methylation

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: Sections were immunostained using an Envision+ Kit (Dako, Tokyo, Japan) with anti-glycogen synthase polyclonal antibody, anti-phospho-glycogen synthase polyclonal antibody specific to serine (S) 641-phosphorylated glycogen synthase (p-GSS641) and anti-MGMT antibody (Cell Signaling Abbreviations: ChIP, chromatin immunoprecipitation; DMSO, dimethyl sulfoxide; DNMT3A, DNA (cytosine-5)-methyltransferase 3A; EGS, ethylene glycol bis(succinimidylsuccinate); GBM, glioblastoma; GS, glycogen synthase; GSK3β, glycogen synthase kinase 3β; MGMT, O6-methylguanine DNA methyltransferase; MSP, methylation-specific PCR; p-GSS641, serine (S) 641-phosphorylated glycogen synthase; qPCR, quantitative real-time PCR; QRT–PCR, quantitative reverse transcriptase–polymerase chain reaction; siRNA, small interfering RNA; TMZ, temozolomide. at U niv of Iow a-L aw L ibrary on M ay 29, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from Fig. 1. (A) The immunohistochemical localization of phosphorylated GS in tissues. a and b, patient 1; c and d, patient 2; a and c, primary tumor; b and d, the GSK3β-inhibiting drug-treated recurrent tumor obtained at autopsy.

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. 3. The combined effect of TMZ and a GSK3β inhibitor (AR-A014418) on GBM cells. (A) The relative cell viability of T98G cells after treatment for 144 h with TMZ (50, 100, 200 and 400 μmol/l) or AR-A014418 (5, 10 and 20 μmol/l), alone or in different combinations shown below the panel, measured by the AlamarBlue assay. The relative viability of the cells treated with DMSO alone was scored as 1.0. *P < 0.05, **P < 0.01, AR, AR-A014418; Mann– Whitney U-test, compared with DMSO-treated cells; bars show standard deviation. (B–G) A comparison of combined effect of TMZ (50, 100, 200 and 400 μmol/l) and AR-A014418 (5, 10 and 20 μmol/l) on T98G, U138 and U87 cells. (B–D) Classic isobologram method. The IC50 values of the combination therapy by TMZ and AR-A014418 determined by the AlamarBlue assay were plotted (TMZ IC50, closed square; AR-A014418 IC50, closed diamond).

Article Snippet: Sections were immunostained using an Envision+ Kit (Dako, Tokyo, Japan) with anti-glycogen synthase polyclonal antibody, anti-phospho-glycogen synthase polyclonal antibody specific to serine (S) 641-phosphorylated glycogen synthase (p-GSS641) and anti-MGMT antibody (Cell Signaling Abbreviations: ChIP, chromatin immunoprecipitation; DMSO, dimethyl sulfoxide; DNMT3A, DNA (cytosine-5)-methyltransferase 3A; EGS, ethylene glycol bis(succinimidylsuccinate); GBM, glioblastoma; GS, glycogen synthase; GSK3β, glycogen synthase kinase 3β; MGMT, O6-methylguanine DNA methyltransferase; MSP, methylation-specific PCR; p-GSS641, serine (S) 641-phosphorylated glycogen synthase; qPCR, quantitative real-time PCR; QRT–PCR, quantitative reverse transcriptase–polymerase chain reaction; siRNA, small interfering RNA; TMZ, temozolomide. at U niv of Iow a-L aw L ibrary on M ay 29, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from Fig. 1. (A) The immunohistochemical localization of phosphorylated GS in tissues. a and b, patient 1; c and d, patient 2; a and c, primary tumor; b and d, the GSK3β-inhibiting drug-treated recurrent tumor obtained at autopsy.

Techniques: Alamar Blue Assay, MANN-WHITNEY, Standard Deviation, Comparison

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: Sections were immunostained using an Envision+ Kit (Dako, Tokyo, Japan) with anti-glycogen synthase polyclonal antibody, anti-phospho-glycogen synthase polyclonal antibody specific to serine (S) 641-phosphorylated glycogen synthase (p-GSS641) and anti-MGMT antibody (Cell Signaling Abbreviations: ChIP, chromatin immunoprecipitation; DMSO, dimethyl sulfoxide; DNMT3A, DNA (cytosine-5)-methyltransferase 3A; EGS, ethylene glycol bis(succinimidylsuccinate); GBM, glioblastoma; GS, glycogen synthase; GSK3β, glycogen synthase kinase 3β; MGMT, O6-methylguanine DNA methyltransferase; MSP, methylation-specific PCR; p-GSS641, serine (S) 641-phosphorylated glycogen synthase; qPCR, quantitative real-time PCR; QRT–PCR, quantitative reverse transcriptase–polymerase chain reaction; siRNA, small interfering RNA; TMZ, temozolomide. at U niv of Iow a-L aw L ibrary on M ay 29, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from Fig. 1. (A) The immunohistochemical localization of phosphorylated GS in tissues. a and b, patient 1; c and d, patient 2; a and c, primary tumor; b and d, the GSK3β-inhibiting drug-treated recurrent tumor obtained at autopsy.

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: Sections were immunostained using an Envision+ Kit (Dako, Tokyo, Japan) with anti-glycogen synthase polyclonal antibody, anti-phospho-glycogen synthase polyclonal antibody specific to serine (S) 641-phosphorylated glycogen synthase (p-GSS641) and anti-MGMT antibody (Cell Signaling Abbreviations: ChIP, chromatin immunoprecipitation; DMSO, dimethyl sulfoxide; DNMT3A, DNA (cytosine-5)-methyltransferase 3A; EGS, ethylene glycol bis(succinimidylsuccinate); GBM, glioblastoma; GS, glycogen synthase; GSK3β, glycogen synthase kinase 3β; MGMT, O6-methylguanine DNA methyltransferase; MSP, methylation-specific PCR; p-GSS641, serine (S) 641-phosphorylated glycogen synthase; qPCR, quantitative real-time PCR; QRT–PCR, quantitative reverse transcriptase–polymerase chain reaction; siRNA, small interfering RNA; TMZ, temozolomide. at U niv of Iow a-L aw L ibrary on M ay 29, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from Fig. 1. (A) The immunohistochemical localization of phosphorylated GS in tissues. a and b, patient 1; c and d, patient 2; a and c, primary tumor; b and d, the GSK3β-inhibiting drug-treated recurrent tumor obtained at autopsy.

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: Sections were immunostained using an Envision+ Kit (Dako, Tokyo, Japan) with anti-glycogen synthase polyclonal antibody, anti-phospho-glycogen synthase polyclonal antibody specific to serine (S) 641-phosphorylated glycogen synthase (p-GSS641) and anti-MGMT antibody (Cell Signaling Abbreviations: ChIP, chromatin immunoprecipitation; DMSO, dimethyl sulfoxide; DNMT3A, DNA (cytosine-5)-methyltransferase 3A; EGS, ethylene glycol bis(succinimidylsuccinate); GBM, glioblastoma; GS, glycogen synthase; GSK3β, glycogen synthase kinase 3β; MGMT, O6-methylguanine DNA methyltransferase; MSP, methylation-specific PCR; p-GSS641, serine (S) 641-phosphorylated glycogen synthase; qPCR, quantitative real-time PCR; QRT–PCR, quantitative reverse transcriptase–polymerase chain reaction; siRNA, small interfering RNA; TMZ, temozolomide. at U niv of Iow a-L aw L ibrary on M ay 29, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from Fig. 1. (A) The immunohistochemical localization of phosphorylated GS in tissues. a and b, patient 1; c and d, patient 2; a and c, primary tumor; b and d, the GSK3β-inhibiting drug-treated recurrent tumor obtained at autopsy.

Techniques: Expressing, Inhibition, Comparison, Binding Assay

Journal: World Journal of Gastrointestinal Oncology

Article Title: O 6 -methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

doi: 10.4251/wjgo.v14.i3.664

Figure Lengend Snippet: O 6 -methylguanine-DNA methyltransferase expression is enhanced in early stage gastric cancer. A: Representative images of immunohistochemistry staining for O 6 -methylguanine-DNA methyltransferase (MGMT) in early stage gastric tumor and normal tissues ( n = 19); B: The mRNA level of MGMT in early stage gastric tumor and adjacent normal tissues ( n = 19). The mRNA expression was normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH); C: MGMT mRNA and protein expression in normal gastric epithelial cells and cancer cells by quantitative real-time polymerase chain reaction and immunoblot assays. GAPDH was used to normalize MGMT expression. The analyses were repeated three times, and the results are expressed as the mean ± SD. a P < 0.05; b P < 0.01. MG-C: MNNG-induced malignant transformed cell; MU-C: MNU-induced malignant transformed cell; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Mouse monoclonal anti-human MGMT antibody (dilution, 1:1000) and mouse monoclonal anti-human GADPH antibody (dilution, 1:2000) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction, Western Blot, Transformation Assay

Journal: World Journal of Gastrointestinal Oncology

Article Title: O 6 -methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

doi: 10.4251/wjgo.v14.i3.664

Figure Lengend Snippet: O 6 -methylguanine-DNA methyltransferase is downregulated in N-nitroso compound-induced gastric epithelial cell malignant transformation. A and B: O 6 -methylguanine-DNA methyltransferase (MGMT) mRNA and protein expression in transformed gastric epithelial cells induced by N-methyl-N’-nitro-N-nitrosoguanidine (MNNG)/N-methyl-N-nitroso-urea (MNU) for 1, 4, and 8 wk; C: Cell anchorage-independent growth on soft agar for subcolones of MNNG/MNU-induced cells. C1-28: Different subcolones of MNNG/MNU-induced cells; MGMT(+): MGMT expression is upregulated in these subcolones; MGMT(-): MGMT expression is downregulated or no-changed in these subcolones; D: Apoptosis assay of MNNG/MNU-transformed subcolones after doxycycline treatment. The analyses were repeated three times, and the results are expressed as the mean ± SD. a P < 0.05; b P < 0.01. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Mouse monoclonal anti-human MGMT antibody (dilution, 1:1000) and mouse monoclonal anti-human GADPH antibody (dilution, 1:2000) were purchased from Santa Cruz Biotechnology.

Techniques: Transformation Assay, Expressing, Apoptosis Assay

Journal: World Journal of Gastrointestinal Oncology

Article Title: O 6 -methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

doi: 10.4251/wjgo.v14.i3.664

Figure Lengend Snippet: DNA hypomethylation contributes to O 6 -methylguanine-DNA methyltransferase upregulation in cell malignant transformation. A: Luciferase reporter assay in control and N-nitroso compound-transformed cells using PGL3-O 6 -methylguanine-DNA methyltransferase ( MGMT ) promoter; B and C: Methylation specific polymerase chain reaction and bisulfite genomic sequence analysis of the DNA methylation level of N-methyl-N’-nitro-N-nitrosoguanidine/N-methyl-N-nitroso-urea-induced transformed cells compared with control cells; D: Correlation of MGMT expression and DNA methylation level of MGMT promoter based on the CCLE database; E: ChIP assay with anti-DNMT1 and anti-H3K9Me3 and H3K4Me2 antibodies for analyzing the DNMT1 binding to the MGMT promoter and the H3K9Me3 and H3K4Me2 levels in the MGMT promoter. The analyses were repeated three times, and the results are expressed as the mean ± SD. a P < 0.05; b P < 0.01. M: Methylated; U: Unmethylated; IgG: Immunoglobulin G.

Article Snippet: Mouse monoclonal anti-human MGMT antibody (dilution, 1:1000) and mouse monoclonal anti-human GADPH antibody (dilution, 1:2000) were purchased from Santa Cruz Biotechnology.

Techniques: Transformation Assay, Luciferase, Reporter Assay, Control, Methylation, Polymerase Chain Reaction, Sequencing, DNA Methylation Assay, Expressing, Binding Assay

Journal: World Journal of Gastrointestinal Oncology

Article Title: O 6 -methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

doi: 10.4251/wjgo.v14.i3.664

Figure Lengend Snippet: Inhibition of O 6 -methylguanine-DNA methyltransferase contributes to the N-nitroso compound-induced cell malignant phenotype. A and B: Cell anchorage-independent growth on soft agar and cell colony formation of N-methyl-N’-nitro-N-nitrosoguanidine/N-methyl-N-nitroso-urea-induced cells after O 6 -BG treatment; C: Cell anchorage-independent growth on soft agar of cells with O 6 -methylguanine-DNA methyltransferase (MGMT) knock-down; D: Knock-down efficiency of MGMT detected by Western blot; E and F: Cell anchorage-independent growth on soft agar and cell colony formation of MGMT overexpressing cells; G: The mRNA expression of MGMT in gastric endoscopic biopsy samples. The analyses were repeated three times, and the results are expressed as the mean ± SD. a,c P < 0.05. c P < 0.05, precancerous lesion and early cancer vs advanced cancer. EV: Empty vector; MGMT: MGMT overexpression; MGMT: O 6 -methylguanine-DNA methyltransferase.

Article Snippet: Mouse monoclonal anti-human MGMT antibody (dilution, 1:1000) and mouse monoclonal anti-human GADPH antibody (dilution, 1:2000) were purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Knockdown, Western Blot, Expressing, Plasmid Preparation, Over Expression

Journal: Cancer Cell International

Article Title: Hedgehog/Gli1 signaling pathway regulates MGMT expression and chemoresistance to temozolomide in human glioblastoma

doi: 10.1186/s12935-017-0491-x

Figure Lengend Snippet: Relationship between MGMT expression, Gli1 nuclear staining and MGMT promoter methylation status in 48 cases of surgically-resected primary GBM tissues ( a , b ). Representative photomicrographs showed two GBM cases with distinct Gli1 activity ( c ). The top two photos represent a Gli1-positive GBM with visible nuclear staining of Gli1 (left) and the same sample stained with MGMT antibody showed that the percentage of MGMT staining was far more than 5% (right). The bottom two photos represent a Gli1-negative case with invisible nuclear staining of Gli1 (left). The same sample stained with MGMT antibody showed the ratio of MGMT staining was less than 5% (200×). The scale bar on the top left panel applies to the rest of the photographs and indicates 20 μm

Article Snippet: Rabbit polyclonal anti-Gli1 antibody (1:100, ab49314, Abcam) and mouse monoclonal anti-MGMT antibody (1:100, ab39253, Abcam) were used to stain GSP cells for immunofluorescence assay.

Techniques: Expressing, Staining, Methylation, Activity Assay

Journal: Cancer Cell International

Article Title: Hedgehog/Gli1 signaling pathway regulates MGMT expression and chemoresistance to temozolomide in human glioblastoma

doi: 10.1186/s12935-017-0491-x

Figure Lengend Snippet: Measurement of Gli1 and MGMT expression by real-time quantitative PCR ( a ) and western blot analysis ( b ) in the three GBM lines, including A172, U251, and U87. The expression of the ubiquitously expressed β-actin gene was measured as the control. The MGMT promoter methylation status was also as showed ( c ). The lane labelled as ‘M’ means methylated; ‘U’ stands for unmethylated. Gli1 expression was increased by pcDNA3.1-Gli1 transfection in A172 cells measured at mRNA and protein levels since 48 h and thereafter ( d – f ), as well as MGMT expression and promoter status. When exposed to TMZ treatment, cell viability in the A172 cells transfected with pcDNA3.1-Gli1 was significantly higher than that in the control since 96 h and thereafter ( g ). The percentage of apoptotic cells at 120 h after pcDNA3.1-Gli1 transfection was significantly lower than in the control cells ( h ). *p < 0.05; **p < 0.01

Article Snippet: Rabbit polyclonal anti-Gli1 antibody (1:100, ab49314, Abcam) and mouse monoclonal anti-MGMT antibody (1:100, ab39253, Abcam) were used to stain GSP cells for immunofluorescence assay.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Methylation, Transfection

Journal: Cancer Cell International

Article Title: Hedgehog/Gli1 signaling pathway regulates MGMT expression and chemoresistance to temozolomide in human glioblastoma

doi: 10.1186/s12935-017-0491-x

Figure Lengend Snippet: The mRNA and protein levels of Gli1 and MGMT in U251 cells were significantly inhibited 48 h after treated by 5 μM cyclopamine ( a and b ). The MGMT promoter statement remained unchanged like the A172 treatment ( c ). When exposed to TMZ, cell viability treated with cyclopamine was significantly lower than that in the control 72 h after the treatment ( d ). The percentage of apoptotic cells in U251 cells treated with cyclopamine was significantly higher than in the control at 120 h post-treatment ( e )

Article Snippet: Rabbit polyclonal anti-Gli1 antibody (1:100, ab49314, Abcam) and mouse monoclonal anti-MGMT antibody (1:100, ab39253, Abcam) were used to stain GSP cells for immunofluorescence assay.

Techniques:

Journal: Cancer Cell International

Article Title: Hedgehog/Gli1 signaling pathway regulates MGMT expression and chemoresistance to temozolomide in human glioblastoma

doi: 10.1186/s12935-017-0491-x

Figure Lengend Snippet: Location and sequence homology of the potential Gli1 binding sites (numbers 1–3) in relation to the structure of the MGMT gene. Blank frames represent the five exons and their length ( a ). The CHIP assay showed only the primers used for the Homo MGMT2 produced the DNA band in the anti-Gli1 lane. ChIP-qPCR also confirmed the result. Human RegIV promoter here served as a positive control for Gli1 binding site ( b ). Gli1 and MGMT expression were inhibited 72 h after treated by 5 μM cyclopamine in U87 GSP cells measured by immunofluorescence staining and western blot ( c ). Green (Gli1); red (MGMT); blue (DAPI)

Article Snippet: Rabbit polyclonal anti-Gli1 antibody (1:100, ab49314, Abcam) and mouse monoclonal anti-MGMT antibody (1:100, ab39253, Abcam) were used to stain GSP cells for immunofluorescence assay.

Techniques: Sequencing, Binding Assay, Produced, Positive Control, Expressing, Immunofluorescence, Staining, Western Blot

Journal: Frontiers in Oncology

Article Title: AI and High-Grade Glioma for Diagnosis and Outcome Prediction: Do All Machine Learning Models Perform Equally Well?

doi: 10.3389/fonc.2021.601425

Figure Lengend Snippet: Number of patients and label distributions for label-sequence combination.

Article Snippet: We used EntroGen’s MGMT Methylation Detection Kit (MSPCR, Cat. No. MGMT-RT44), a semiquantitative real-time PCR-based essay for detection of MGMT promoter methylation within the DMR2 locus, distinguishing between methylated and non-methylated cytosines.

Techniques:

Journal: Frontiers in Oncology

Article Title: AI and High-Grade Glioma for Diagnosis and Outcome Prediction: Do All Machine Learning Models Perform Equally Well?

doi: 10.3389/fonc.2021.601425

Figure Lengend Snippet: MGMT best results (reported as mean ± standard deviation).

Article Snippet: We used EntroGen’s MGMT Methylation Detection Kit (MSPCR, Cat. No. MGMT-RT44), a semiquantitative real-time PCR-based essay for detection of MGMT promoter methylation within the DMR2 locus, distinguishing between methylated and non-methylated cytosines.

Techniques:

Journal: Frontiers in Oncology

Article Title: AI and High-Grade Glioma for Diagnosis and Outcome Prediction: Do All Machine Learning Models Perform Equally Well?

doi: 10.3389/fonc.2021.601425

Figure Lengend Snippet: Best ROC curve for MGMT prediction: AB classifier with FLAIR sequence on CET ROI.

Article Snippet: We used EntroGen’s MGMT Methylation Detection Kit (MSPCR, Cat. No. MGMT-RT44), a semiquantitative real-time PCR-based essay for detection of MGMT promoter methylation within the DMR2 locus, distinguishing between methylated and non-methylated cytosines.

Techniques: Sequencing