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: Cancers

Article Title: Synergistic Antitumor Activity of Talazoparib and Temozolomide in Malignant Rhabdoid Tumors

doi: 10.3390/cancers16112041

Figure Lengend Snippet: MGMT as a potential biomarker of MRT response and the therapy effect on DDR. ( A) Western blot analysis of SMARCB1 (46 kDa), PARP1 (116 kDa)/cleaved PARP1 (cPARP1; 89 kDa), MGMT (21 kDa), and GAPDH (37 kDa) proteins. ( B ) Quantification of band intensities in ( A ) normalized to GAPDH loading control. One-way ANOVA was performed across 6 xenograft tumors. Significant change was reported with p = 0.0038; Tukey’s HSD further identified significant change only between G401 and WT-16 ( p = 0.0022). Data are presented as mean ± SEM of 3 independent tests. Asterisks *** indicate a statistical difference of p ≤ 0.001 ( t -test). ( C ) Inverse correlation between MGMT protein expression and the antitumor activity of PEG~TLZ+TMZ in MRT xenografts. Axis Y shows tumor response categories: 1-MCR (maintained complete response), 2-CR (complete response), 3-PR (partial response), 4-SD (stable disease), and 5-PD (progressive disease). Axis X: MGMT protein expression normalized to GAPDH. This resulted in Pearson correlation coefficient R = 0.88 with a significant p -value of 0.019. ( D ) G401 SMARCB1−/− cell potentiation to TLZ+TMZ by MGMT inhibition. Cells were treated with increasing concentrations of TMZ (0–1 mM), TLZ IC 50 (176 nM), and O 6 BG (25 μM). Alamar Blue was added to the plates at 72 h and incubated for 4 h; fluorescence was measured at 590 nm (excitation 530 nm) on a Clariostar microplate reader. ( E ) Protein array of basal levels of DDR proteins in G401 SMARCB1−/− and G401 SMARCB1+/+ cell lines. Blot was processed 24 h after treatment with DOX (1 μg/mL). The spliced fragments of the same blot are marked with thin white lines. ( F ) Plot quantitating the DDR protein expression (from the DDR protein array assay as in ( E )) in G401 SMARCB1−/− and G401 SMARCB1+/+ cells treated with TMZ (IC 50 = 446 μM for G401 SMARCB1+/+ and 395 μM for G401 SMARCB1−/− ), TLZ (IC 50 = 95 nM G401 SMARCB1+/+ and 176 nM for G401 SMARCB1−/− ), and TLZ+TMZ for 72 h. Original western blots are presented in .

Article Snippet: The membranes were probed with antibodies against SMARCB1 (1:1000, Cell Signaling, 91735S), MGMT (1:1000, Cell Signaling, 58121), PARP1 (1:1000, Cell Signaling, 9542), and GAPDH (1:1000, Cell Signaling, 2118).

Techniques: Biomarker Discovery, Western Blot, Control, Expressing, Activity Assay, Inhibition, Incubation, Fluorescence, Protein Array