Journal: Cancer gene therapy

Article Title: Human-yeast chimeric repair protein protects mammalian cells against alkylating agents: enhancement of MGMT protection.

doi: 10.1038/sj.cgt.7700605

Figure Lengend Snippet: Figure 1 Construction of the chimeric MGMT-Apn1 construct. The MGMT and Apn1 cDNA sequences were amplified from bacterial clones and the chimeric molecule was created using the overlapping PCR technique.3 The fusion product was subcloned into the pcDNA 3.0 vector, sequenced and used to transfect K562 cells.

Article Snippet: DNA isolation, RNA isolation, Northern and Western blot analyses, and sodium dodecyl sulfate-polyacrylamide eletrophoresis (SDS-PAGE) were performed as has been previously described.3 Western blot analysis was performed with the mouse anti-MGMT monoclonal antibody (MT 23.2; Novus Biologicals, Littleton, CO) and the goat anti-mouse IgG antibody (Chemicon International, Inc., Temecula, CA).

Techniques: Construct, Amplification, Clone Assay, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: Human-yeast chimeric repair protein protects mammalian cells against alkylating agents: enhancement of MGMT protection.

doi: 10.1038/sj.cgt.7700605

Figure Lengend Snippet: Figure 2 Analysis of K562 cells containing chimeric transgene. (a) The MGMT-Apn1 K562 clone and pcDNA vector-transfected control cells were analyzed by Northern blot analysis. Both the radiolabeled MGMT and Apn1 cDNAs hybridized with a 1.75 kb message from total RNA of MGMT-Apn1-transfected cells, but not of vector-control cells. The radiolabeled GAPDH cDNA hybridized to a 1 kb transcript from total RNA of both control cells and MGMT-Apn1-transfected cells. (b) Western blot analysis: The MGMT monoclonal antibody detected the 64 kDa MGMT- Apn1 fusion protein of transfected K562 cells, but not of control cells (lane 3). The antibody also detected the 21 kDa MGMT protein from MGMT- transfected K562 cells (lane 2). (c) MGMT activity analysis: The 18-mer O6-methyl guanine oligonucleotide assay was performed with 50 mg of protein from sonicated K562 cell extract. MGMT activity was detected in both MGMT- and MGMT-Apn1-transfected K562 cells. Presence of the 8-mer fragment indicates MGMT activity. (d) AP endonuclease activity assay: The 26-mer oligonucleotide assay was performed with 5 mg of total protein from sonicated cell extract in the presence of 20 mM EDTA. Apurinic endonuclease activity was detected in MGMT-Apn1-transfected cells, but not control or MGMT-transfected cells. Presence of the 14-mer indicates cleavage of the artificial AP site. Apn1 is Mg2+ independent, EDTA resistant whereas the endogenous mammalian Ape1 protein requires Mg2+ and is inactive in the presence of EDTA.

Article Snippet: DNA isolation, RNA isolation, Northern and Western blot analyses, and sodium dodecyl sulfate-polyacrylamide eletrophoresis (SDS-PAGE) were performed as has been previously described.3 Western blot analysis was performed with the mouse anti-MGMT monoclonal antibody (MT 23.2; Novus Biologicals, Littleton, CO) and the goat anti-mouse IgG antibody (Chemicon International, Inc., Temecula, CA).

Techniques: Plasmid Preparation, Transfection, Control, Northern Blot, Western Blot, Activity Assay, Oligonucleotide Assay, Sonication

Journal: bioRxiv

Article Title: Cannabidiol as a Prophylactic Agent Against Glioblastoma Growth: A Preclinical Investigation

doi: 10.1101/2025.10.29.685440

Figure Lengend Snippet: A) Immunofluorescence staining of MGMT and Ki-67 in GBM tumors from mice pretreated with inhaled CBD for 14 days, 3 days, or placebo. Representative images show that MGMT and Ki-67 levels were significantly reduced in the 14-day CBD pretreatment group compared to both the 3-day CBD and placebo groups (Scale bar: 40 μm). B) Quantification of staining intensity revealed significantly lower levels of MGMT and Ki-67 in the 14-day CBD pretreatment group compared to the 3-day CBD and placebo groups (p < 0.001). These results suggest that CBD pretreatment reduces key biomarkers associated with chemotherapy resistance and tumor proliferation, potentially enhancing therapeutic sensitivity. C) To further validate the effect of CBD on tumor progression, we assessed MGMT expression levels using Western blotting. The blot shows increased MGMT expression in the GBM tumors of mice pretreated with placebo (lane 2) compared to the normal brain tissue used as a negative control (lane 4). Long-term CBD pretreatment (14 days, lane 1) significantly reduced MGMT expression in the tumor compared to acute CBD pretreatment (3 days, lane 3), corroborating the reduction observed in the immunofluorescence analysis.

Article Snippet: Membranes were probed simultaneously with anti-mouse MGMT monoclonal antibody (O6-methylguanine-DNA methyltransferase, Novus Biologicals USA, Cat# NB100-168SS) and β-actin antibody (Thermo Fisher) for 24 hours.

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Negative Control

Journal: Cancer Epidemiology, Biomarkers & Prevention

Article Title: O 6 -Methylguanine-DNA Methyltransferase Gene: Epigenetic Silencing and Prognostic Value in Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1055-9965.967.13.6

Figure Lengend Snippet: Figure 1. MSP results from five representative HNSCC cases. Both unmethylated- (U) and methylated-specific (M) MSP primer sets for MGMT were used. Samples 1, 3, and 4 were negative for MGMT promoter methylation; therefore, only the unmethylated (U) amplicons were detected. By contrast, samples 2 and 5 were positive for promoter methylation, as indicated by the presence of both unmethylated (U) and methylated (M) amplicons. Blank (water, sample 6), negative (N; human placental DNA), and positive (P; universal methylated human DNA; Intergen) controls were also included in the PCRs. M ( far left on the panel), molecular marker.

Article Snippet: IHC stainings were done on formalin-fixed, paraffin-embedded tissue sections with a monoclonal antibody against MGMT protein (Novus Biologicals, Littleton, CO; 1:1000 dilution).

Techniques: Methylation, Marker

Journal: Cancer Epidemiology, Biomarkers & Prevention

Article Title: O 6 -Methylguanine-DNA Methyltransferase Gene: Epigenetic Silencing and Prognostic Value in Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1055-9965.967.13.6

Figure Lengend Snippet: Figure 2. IHC staining for the MGMT protein in HNSCC. A and C are from a case strongly positive for the MGMT protein, whereas B and D are from a case that is negative for the protein. In normal squamous epithelia overlying the invasive carcinoma, intense nuclear staining is seen, particularly in the basal and parabasal layers (A, B). In image B, the surface squamous epithelium shows an abrupt transition from mild dysplasia (on the right) to carcinoma in situ (on the left). B. MGMT protein is present in the dysplastic epithelium but absent in the carcinoma in situ. C. Invasive tumor cells in the positive case are mostly positive for the MGMT protein. D. By comparison, the carcinoma cells in the negative case were mostly negative.

Article Snippet: IHC stainings were done on formalin-fixed, paraffin-embedded tissue sections with a monoclonal antibody against MGMT protein (Novus Biologicals, Littleton, CO; 1:1000 dilution).

Techniques: Immunohistochemistry, Staining, In Situ, Comparison

Journal: Cancer Epidemiology, Biomarkers & Prevention

Article Title: O 6 -Methylguanine-DNA Methyltransferase Gene: Epigenetic Silencing and Prognostic Value in Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1055-9965.967.13.6

Figure Lengend Snippet: Figure 3. Five-year cause-specific survival among 94 HNSCC patients. Cause-specific survival was calculated with Kaplan- Meier method according to MGMT MSP results (A: positive- dotted line; negative-solid line) and IHC staining results (B: positive-solid line; negative-dotted line) in HNSCC patients. MGMT promoter hypermethylation or apparent loss of protein expression is significantly correlated with decreased 5-year cause-specific survival, independent of other prognostic factors. The 5-year cause-specific survival rate for HNSCC patients with methylated MGMT promoter or apparent loss of protein expression is only 16% and 36%, respectively. By contrast, the cause-specific survival rate for HNSCC patients with unmethylated MGMT promoter or high levels of MGMT protein reaches 64% and 58%.

Article Snippet: IHC stainings were done on formalin-fixed, paraffin-embedded tissue sections with a monoclonal antibody against MGMT protein (Novus Biologicals, Littleton, CO; 1:1000 dilution).

Techniques: Immunohistochemistry, Expressing, Methylation