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, 1:150) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States) and used for immunohistochemistry.

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, 1:150) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States) and used for immunohistochemistry.

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, 1:150) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States) and used for immunohistochemistry.

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, 1:150) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States) and used for immunohistochemistry.

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

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: International Journal of Molecular Sciences

Article Title: Olaparib Is a Mitochondrial Complex I Inhibitor That Kills Temozolomide-Resistant Human Glioblastoma Cells

doi: 10.3390/ijms222111938

Figure Lengend Snippet: High MGMT protein expression does not account for TMZ resistance in the U373/U373-R isogenic human glioblastoma model. ( a ) O6-methylguanine DNA methyltransferase (MGMT) and β-actin (loading control) protein expression as determined by Western blotting in U373 and U373-R glioblastoma cells in the absence of TMZ, with pictures on the left and quantification on the right ( n = 4). ( b ) Clonogenic assays of the cells that were pretreated for 72 h with increasing concentrations of MGMT inhibitor O6-benzylguanine (O6BG). Displayed are the plating efficiency of the vehicle-treated cells at 72 h (left; n = 6); and the surviving fraction (right; n = 6–12), where the data are normalized to the number of corresponding vehicle-treated cells. ( c ) Number of cells 72 h after treatment with the indicated concentrations of TMZ in combination with 50 µM O6BG (O6BG50; left; n = 4) or 100 µM O6BG (O6BG100; right; n = 4). ( d ) Clonogenic assays of the cells that were pretreated for 72 h with increasing concentrations of TMZ and O6BG. Displayed are the plating efficiency of the cells that were treated ± O6BG (left; n = 6); and the surviving fractions of the cells that received O6BG50 (middle; n = 6) or O6BG100 (right; n = 5–6), where the data are normalized to the number of corresponding vehicle-treated cells. All data are shown as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, ns: p > 0.05 versus the corresponding control; ### p < 0.005 for whole curve comparison; by Student’s t -test ( a , b left), two-way ANOVA with Sidak’s post hoc test ( b right, c , d middle, d right), or one-way ANOVA with Dunnett’s post hoc test ( d left).

Article Snippet: The primary antibodies were mouse monoclonals against human MGMT (Santa Cruz Biotechnology, Heidelberg, Germany; catalogue #sc-56157) and against β-actin (Sigma-Aldrich; catalogue #A5441).

Techniques: Expressing, Western Blot

Journal: Bioengineering

Article Title: Looking for A Place for Dose-Dense TMZ Regimens in GBM Patients: An Experience with MGMT Exploratory Evaluation

doi: 10.3390/bioengineering6010011

Figure Lengend Snippet: Patients’ features.

Article Snippet: After blocking of endogenous peroxidase with 3% H 2 O 2 , the sections were pre-treated in an oven with EnVision Flex TRS buffer and immunostained on a DAKO Cytomation autostainer (DAKO, Glostrup, Denmark), using monoclonal mouse anti-human antibody against MGMT (clone MT3.1, MAB16200 EMD Millipore TM , Billerica, MA, USA).

Techniques: Expressing

Journal: Bioengineering

Article Title: Looking for A Place for Dose-Dense TMZ Regimens in GBM Patients: An Experience with MGMT Exploratory Evaluation

doi: 10.3390/bioengineering6010011

Figure Lengend Snippet: Univariate analysis with Fisher exact test (binomial confidence interval) and multivariate analysis with multiple regression. *: statistically significant.

Article Snippet: After blocking of endogenous peroxidase with 3% H 2 O 2 , the sections were pre-treated in an oven with EnVision Flex TRS buffer and immunostained on a DAKO Cytomation autostainer (DAKO, Glostrup, Denmark), using monoclonal mouse anti-human antibody against MGMT (clone MT3.1, MAB16200 EMD Millipore TM , Billerica, MA, USA).

Techniques: Expressing

Journal: Bioengineering

Article Title: Looking for A Place for Dose-Dense TMZ Regimens in GBM Patients: An Experience with MGMT Exploratory Evaluation

doi: 10.3390/bioengineering6010011

Figure Lengend Snippet: Univariate and multivariate Cox hazard regression analysis for PFS and OS. *: statistically significant.

Article Snippet: After blocking of endogenous peroxidase with 3% H 2 O 2 , the sections were pre-treated in an oven with EnVision Flex TRS buffer and immunostained on a DAKO Cytomation autostainer (DAKO, Glostrup, Denmark), using monoclonal mouse anti-human antibody against MGMT (clone MT3.1, MAB16200 EMD Millipore TM , Billerica, MA, USA).

Techniques: Expressing

Journal: Cancers

Article Title: Rare Stochastic Expression of O6-Methylguanine- DNA Methyltransferase (MGMT) in MGMT-Negative Melanoma Cells Determines Immediate Emergence of Drug-Resistant Populations upon Treatment with Temozolomide In Vitro and In Vivo

doi: 10.3390/cancers10100362

Figure Lengend Snippet: MGMT (O6-methylguanine-DNA methyltransferase) levels and protection from TMZ (temozolomide) toxicity. ( A ) Protein lysates were prepared from four different melanoma cell lines and MGMT protein levels were analyzed by Western blot. Actin was used as the loading control. ( B ) MGMT-negative 2058 cells and MGMT-positive A375 cells were treated with increasing concentrations of TMZ in the absence or presence of 15 µM O6BG. After 12–14 days, emerging colonies were counted. Number of colonies from untreated cells was set at 100% ( n ≥ 5, ±SE).

Article Snippet: The primary antibody was mouse monoclonal anti-human MGMT from Abcam (cat #ab39253).

Techniques: Western Blot, Control

Journal: Cancers

Article Title: Rare Stochastic Expression of O6-Methylguanine- DNA Methyltransferase (MGMT) in MGMT-Negative Melanoma Cells Determines Immediate Emergence of Drug-Resistant Populations upon Treatment with Temozolomide In Vitro and In Vivo

doi: 10.3390/cancers10100362

Figure Lengend Snippet: Selection of A375 clones after high-dose TMZ treatment. ( A ) Treatment schedule was as follows: 10 5 A375 cells (MGMT positive) were seeded into a 10-cm dish and treated with a single dose of 700 µM TMZ for 48 h. Two weeks later, 12 surviving clones (numbered 1 through 12) were isolated for further analysis by WB and CFA. ( B ) All twelve clones were subjected to CFA with increasing concentrations of TMZ. Shown is the average IC50 of each clone, in comparison to parental A375 cells (left bar in black) ( n = 2–5, ±SE). ( C ) Five of these cell clones were lysed, and MGMT protein levels were analyzed by WB with actin as the loading control. Left lane shows lysate from parental A375 cells. Numbers under the blot indicate quantification of MGMT bands, with reference to the actin signal, and relative to A375 cells (left lane, set at 1.0).

Article Snippet: The primary antibody was mouse monoclonal anti-human MGMT from Abcam (cat #ab39253).

Techniques: Selection, Clone Assay, Isolation, Comparison, Control

Journal: Cancers

Article Title: Rare Stochastic Expression of O6-Methylguanine- DNA Methyltransferase (MGMT) in MGMT-Negative Melanoma Cells Determines Immediate Emergence of Drug-Resistant Populations upon Treatment with Temozolomide In Vitro and In Vivo

doi: 10.3390/cancers10100362

Figure Lengend Snippet: Selection of A2058 clones after high-dose TMZ treatment. ( A ) Treatment schedule was as follows: 10 5 A2058 cells (MGMT negative) were seeded into a 10-cm dish and treated with a single dose of 200 µM TMZ for 48 h. Two weeks later, well over a thousand colonies emerged. To enable selection of well-isolated, individual clones, the colonies were pooled and re-seeded at very low density and allowed to form colonies again (in the absence of any further drug treatment). Then, 12 random colonies (numbered 21 through 32) were picked for further analysis by WB and CFA. ( B ) Six of these clones were subjected to CFA with increasing concentrations of TMZ. Shown is the average IC50 of each clone, in comparison to parental A2058 cells (left bar in black) ( n = 2–5, ±SE). ( C ) Eleven of these cell clones were lysed, and MGMT protein levels were analyzed by WB with actin as the loading control. Left lane shows lysate from parental A2058 cells. Numbers under the blot indicate quantification of MGMT bands, with reference to the actin signal, and relative to Clone 21 (right lane, set at 1.0); <0.1 indicates below detection limit.

Article Snippet: The primary antibody was mouse monoclonal anti-human MGMT from Abcam (cat #ab39253).

Techniques: Selection, Clone Assay, Isolation, Comparison, Control

Journal: Cancers

Article Title: Rare Stochastic Expression of O6-Methylguanine- DNA Methyltransferase (MGMT) in MGMT-Negative Melanoma Cells Determines Immediate Emergence of Drug-Resistant Populations upon Treatment with Temozolomide In Vitro and In Vivo

doi: 10.3390/cancers10100362

Figure Lengend Snippet: MGMT protein levels in mass cultures after TMZ treatment. ( A ) Treatment schedule was as follows: 10 5 cells were seeded into the wells of a 6-well dish and treated with a single dose of TMZ at increasing concentrations from 0 to 500 µM for 48 h. Surviving cells were kept in culture until the population had regrown sufficiently to harvest enough cells for WB analysis. ( B ) Cell lysates were prepared from mass cultures of surviving A2058 cells and analyzed by WB for MGMT protein levels. Actin was used as a loading control. A lysate of A375 cells was used as a positive control for MGMT protein. ( C ) The same experiment was also performed with M24 cells. Please note that MGMT protein was undetectable before TMZ treatment in both cell lines. Numbers under the blots indicate quantification of MGMT bands, with reference to the actin signal, and relative to the highest TMZ concentration used (right lane, set at 1.0); <0.1 indicates below detection limit.

Article Snippet: The primary antibody was mouse monoclonal anti-human MGMT from Abcam (cat #ab39253).

Techniques: Control, Positive Control, Concentration Assay

Journal: Cancers

Article Title: Rare Stochastic Expression of O6-Methylguanine- DNA Methyltransferase (MGMT) in MGMT-Negative Melanoma Cells Determines Immediate Emergence of Drug-Resistant Populations upon Treatment with Temozolomide In Vitro and In Vivo

doi: 10.3390/cancers10100362

Figure Lengend Snippet: MGMT protein levels in clones derived from untreated A2058 cells. ( A ) Treatment schedule was as follows: 100 A2058 cells were seeded into a 10-cm dish. After colonies had formed (in the absence of any drug treatment), 12 of them (numbered 41 through 52) were isolated and expanded for further analysis by WB and CFA. ( B ) Cell lysates were prepared from all clones and analyzed by WB for MGMT protein levels. Actin was used as a loading control. A lysate of A375 cells was used as a positive control for MGMT protein, and a lysate of parental A2058 cells was used as the negative control. Except for clone 43, all clones were negative for MGMT protein (clones 49 to 52 were negative as well, but not included here; clone 44 was lost). Numbers under the blot indicate quantification of MGMT bands, with reference to the actin signal, and relative to Clone 43 (set at 1.0); <0.1 indicates below detection limit. ( C ) To confirm drug sensitivity in correlation with MGMT levels, clone 41 and 43 were treated with increasing concentrations of TMZ in the presence or absence of O6BG, and CFA was performed 12 days later ( n = 3, ±SE).

Article Snippet: The primary antibody was mouse monoclonal anti-human MGMT from Abcam (cat #ab39253).

Techniques: Clone Assay, Derivative Assay, Isolation, Control, Positive Control, Negative Control

Journal: Cancers

Article Title: Rare Stochastic Expression of O6-Methylguanine- DNA Methyltransferase (MGMT) in MGMT-Negative Melanoma Cells Determines Immediate Emergence of Drug-Resistant Populations upon Treatment with Temozolomide In Vitro and In Vivo

doi: 10.3390/cancers10100362

Figure Lengend Snippet: Emergence of MGMT protein in MGMT-negative clone. ( A ) Treatment schedule was as follows: 10 5 cells were seeded into the wells of a 6-well dish and treated with a single dose of TMZ at increasing concentrations from 0 to 50 µM for 48 h. Surviving cells were kept in culture until the population had regrown sufficiently to harvest enough cells for WB analysis. ( B ) Cell lysates were prepared from mass cultures of surviving A2058 cells and from clone 41, followed by WB analysis of MGMT protein levels. Actin was used as a loading control. Numbers under the blots indicate quantification of MGMT bands, with reference to the actin signal, and relative to the intensity in the right lane (set at 1.0); <0.1 indicates below detection limit.

Article Snippet: The primary antibody was mouse monoclonal anti-human MGMT from Abcam (cat #ab39253).

Techniques: Control

Journal: Cancers

Article Title: Rare Stochastic Expression of O6-Methylguanine- DNA Methyltransferase (MGMT) in MGMT-Negative Melanoma Cells Determines Immediate Emergence of Drug-Resistant Populations upon Treatment with Temozolomide In Vitro and In Vivo

doi: 10.3390/cancers10100362

Figure Lengend Snippet: MGMT promoter methylation status. ( A ) DNA was isolated from A2058 cells and several derived clones, and analyzed for methylation status of the MGMT promoter. Reference DNA from RKO and HCT8 colorectal carcinoma cell lines was included as negative (neg. ctr.) and positive controls (pos. ctr.), respectively. PMR: percentage of methylated reference. ( B ) A2058 cells were treated (Tx) with 1, 2, 3, or 4 cycles of 5-AzaC. Thereafter, cell lysates were prepared and analyzed for MGMT protein levels by WB. Actin was used as the loading control. Cell lysate labeled 4* was from cells that were cultured for an additional 4 weeks after 4 cycles of 5-AzaC treatment.

Article Snippet: The primary antibody was mouse monoclonal anti-human MGMT from Abcam (cat #ab39253).

Techniques: Methylation, Isolation, Derivative Assay, Clone Assay, Control, Labeling, Cell Culture

Journal: Cancers

Article Title: Rare Stochastic Expression of O6-Methylguanine- DNA Methyltransferase (MGMT) in MGMT-Negative Melanoma Cells Determines Immediate Emergence of Drug-Resistant Populations upon Treatment with Temozolomide In Vitro and In Vivo

doi: 10.3390/cancers10100362

Figure Lengend Snippet: Drug resistance after high-dose TMZ plus O6BG treatment. ( A ) Treatment schedule was as follows: 3 × 10 6 A2058 cells were seeded into a 25-cm plate and treated with 100 µM TMZ in the presence of O6BG. After about 6 weeks, one colony of surviving cells (called clone 61) emerged and was cultured for further analysis by WB and CFA. ( B ) Cell lysate was prepared from clone 61 and analyzed by WB for MGMT and MSH6 protein levels. Lysates from A375 and A2058 cells were included as MGMT-positive and -negative controls, respectively; lysates from HCT116 and DLD1 cells were included as MSH6-positive and -negative controls, respectively. ( C ) To confirm drug sensitivity in correlation with MMR deficiency, clone 61 was treated with increasing concentrations of TMZ in the presence or absence of O6BG, and CFA was performed 12 days later ( n = 3, ±SE). ( D ) Clone 61 was treated with increasing concentrations of TMZ (single dose, as per schedule listed in ). After complete recovery, mass cultures were analyzed for MGMT protein levels by WB analysis with actin as the loading control.

Article Snippet: The primary antibody was mouse monoclonal anti-human MGMT from Abcam (cat #ab39253).

Techniques: Cell Culture, Control

Journal: Cancers

Article Title: Rare Stochastic Expression of O6-Methylguanine- DNA Methyltransferase (MGMT) in MGMT-Negative Melanoma Cells Determines Immediate Emergence of Drug-Resistant Populations upon Treatment with Temozolomide In Vitro and In Vivo

doi: 10.3390/cancers10100362

Figure Lengend Snippet: Drug resistance after repeated low-dose TMZ plus O6BG treatment. ( A ) Treatment schedule was as follows: 3 × 10 6 A2058 cells were seeded into a 25-cm plate and treated with 10 µM TMZ in the presence of O6BG once daily over 5 days. After about 15 days, there were approximately 250 colonies, which were pooled and expanded. After sufficient proliferation, 3 × 10 6 of these cells were seeded again into a 25-cm plate and exposed to a single treatment of 100 µM TMZ in the presence of O6BG. Another 15 days later, approximately 2000 colonies emerged, which were sub-cultured to allow for the isolation of 12 individual clones (numbered 71 through 82), which were analyzed by WB and CFA. ( B ) Cell lysates were prepared from all clones and analyzed by WB for MGMT and MSH6 protein levels. Lysates from A375 and A2058 cells were included for comparison. (Clones 81 and 82 are not included in this figure; clone 76 was lost.) Numbers under the blot indicate quantification of MGMT bands, with reference to the actin signal, and relative to Clone 79 (set at 1.0); <0.1 indicates below detection limit. ( C ) To confirm drug sensitivity in correlation with MGMT or MMR deficiency, clones 71 and 79 were treated with increasing concentrations of TMZ in the presence or absence of O6BG, and CFA was performed 12 days later ( n = 3, ±SE).

Article Snippet: The primary antibody was mouse monoclonal anti-human MGMT from Abcam (cat #ab39253).

Techniques: Cell Culture, Isolation, Clone Assay, Comparison

Journal: Cancers

Article Title: Rare Stochastic Expression of O6-Methylguanine- DNA Methyltransferase (MGMT) in MGMT-Negative Melanoma Cells Determines Immediate Emergence of Drug-Resistant Populations upon Treatment with Temozolomide In Vitro and In Vivo

doi: 10.3390/cancers10100362

Figure Lengend Snippet: MGMT expression in tumor tissue. ( A ) Nude mice were injected subcutaneously with 4 × 10 6 MGMT-positive A375 cells or with MGMT-negative A2058 cells. Once pea-sized tumors had developed, tumor tissue was harvested and subjected to immunostaining for human MGMT. While A375 tissue showed positive staining in most cells, A2058 tissue revealed positivity only in a small minority of cells (2 examples are indicated by arrows in the enlarged section). ( B ) Western blot was performed with crude lysates from cells cultured in vitro (A375, A2058, M24, and MEF) and from xenograft tumor tissues (A1–A4: tumors derived from implanted A2058 cells; M1–M3: tumors derived from implanted M24 cells), using a human-specific MGMT antibody. A1, A2, M1 and M2 are tumors from mice that only received vehicle treatment (control); A3, A4 and M3 are tumors from mice that received TMZ therapy (M4 animal was lost). To confirm the presence of human tumor cells in tissues harvested from mice—especially in those lanes where the MGMT signal was negative—we used an antibody against TRA-1-85/CD147, an epitope that is present only on human cells, but not mouse cells. Please note that specificity of this antibody was confirmed by lack of reactivity with the lane containing lysate from mouse embryo fibroblasts (MEF). Actin was used as the loading control. Numbers under the blot indicate quantification of MGMT bands, with reference to the actin signal, and relative to A4 sample (set at 1.0); <0.1 indicates below detection limit; only lanes with tumor tissue samples were measured.

Article Snippet: The primary antibody was mouse monoclonal anti-human MGMT from Abcam (cat #ab39253).

Techniques: Expressing, Injection, Immunostaining, Staining, Western Blot, Cell Culture, In Vitro, Derivative Assay, Control

Journal: Cancers

Article Title: Rare Stochastic Expression of O6-Methylguanine- DNA Methyltransferase (MGMT) in MGMT-Negative Melanoma Cells Determines Immediate Emergence of Drug-Resistant Populations upon Treatment with Temozolomide In Vitro and In Vivo

doi: 10.3390/cancers10100362

Figure Lengend Snippet: Model of selection of drug-resistant melanoma cell populations during treatment with TMZ, with or without added O6BG. Please note that rare MGMT-positive cells (blue) and even rarer MMR-deficient cells (orange) already are present in the parental cell population before the onset of drug treatment. Based on our results, there can also be mixed MGMT-positive/MMR-deficient cells (not displayed here); in such cells, MGMT would ensure survival in case of TMZ treatment, and MMR-deficiency would ensure survival in case of TMZ + O6BG combination treatment. See further details in Discussion and Conclusions sections.

Article Snippet: The primary antibody was mouse monoclonal anti-human MGMT from Abcam (cat #ab39253).

Techniques: Selection