Lysates prepared from the 3 isogenic pairs of GB cells (LN229/LN229M, U251/U251M, and GL261/GL261M) and the MMR-deficient LN229TR2 and MGMT-positive T98G cells were analyzed by Western-blotting for MGMT expression levels. A rabbit polyclonal antibody that crossreacts with both human MGMT and mouse Mgmt proteins was purchased from Boster Biological Technology (Pleasanton, CA) and used in this Western-blot analysis. This antibody was raised against a synthetic peptide PVFQQESFTRQVLWK, which corresponds to a sequence in the middle region of human MGMT which is different from the related rat and mouse sequences by only one amino acid. Furthermore, to check the functionality of the MGMT protein in LN229M and U251M cells (infected with a human MGMT/ffLuc/EGFP-expressing lentivirus) and in GL261M cells (transfected with a murine Mgmt-expressing plasmid), we completed a colony formation assay (CFA) with these cells seeded in 6-well plates at a density of 50 cells/cm2 and incubated with a range of TMZ or NEO212 concentrations in the presence or absence of the MGMT inhibitor O6-benzylguanine (Millipore Sigma, Burlington, MA) which was added to a final concentration of 40 μM.
O6 benzylguanine
Manufactured by Merck Group
Sourced in United States, France
O6-benzylguanine is a chemical compound used in laboratory research. It functions as an inhibitor of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT).
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 nhs ester, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole dapi nuclear stain, by Thermo Fisher Scientific (1 mentions)
Olaparib, by Selleck Chemicals (1 mentions)
Olaparib, by LC Laboratories (1 mentions)
Potassium phosphate, by Merck Group (1 mentions)
Nadph, by Merck Group (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Zaleplon, by Merck Group (1 mentions)
Hydralazine hydrochloride, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Zoniporide dihydrochloride, by Bio-Techne (1 mentions)
Sgx523, by Bio-Techne (1 mentions)
Methyl methanesulfonate, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
2 3 bis 2 methoxy 4 nitro 5 sulfophenyl 5 phenylamino carbonyl 2h tetrazolium hydroxide xtt, by Merck Group (1 mentions)
Ethyl methanesulfonate, by Merck Group (1 mentions)
1 methoxy 5 methylphenazinium methyl sulfate pms, by Merck Group (1 mentions)
Carmustine bcnu, by Merck Group (1 mentions)
Hat medium, by Thermo Fisher Scientific (1 mentions)
9 protocols using o6 benzylguanine
1
MGMT Expression and Function in Glioblastoma
2
MGMT Expression and TMZ Resistance
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Lysates prepared from wildtype GL261 and TMZ-resistant GL261M cells were analyzed by Western-blotting for MGMT expression levels. A rabbit polyclonal antibody that cross-reacts with the mouse MGMT protein was purchased from Boster Biological Technology (Pleasanton, CA, USA) and used in this Western-blot analysis. This antibody was raised against a synthetic peptide PVFQQESFTRQVLWK, which corresponds to a sequence in the middle region of human MGMT which is different from the related rat and mouse sequences by only one amino acid. Furthermore, to check the functionality of the MGMT enzyme resulting from transfection of the MGMT gene into GL261 cells, we completed a colony formation assay (CFA) with GL261M cells incubated in a range of TMZ concentrations in the presence or absence of the MGMT inhibitor O6-benzylguanine (Millipore Sigma, Burlington, MA, USA).
3
Multi-Modal Assessment of Cytotoxic Effects
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TMZ was purchased from Millipore Sigma (Burlington, MA). NEO212 was synthetized by Norac Pharma (Azusa, CA) and kindly provided by NeOnc Technologies (Los Angeles, CA). Olaparib was purchased from LC Laboratories (Woburn, MA). The monoclonal γH2AX antibody (clone JBW601) was purchased from EMD Millipore (Darmstadt, Germany). A secondary Alexa Fluor 647 (AF647) Fab antibody fragment, the Pacific Blue-labeled Annexin V, an Alexa Fluor 488 NHS ester, and the 4’,6 diamidino-2-phenylindole (DAPI) nuclear stain were purchased from ThermoFisher (Waltham, MA). All other reagents, including the MGMT inhibitor O6-benzylguanine or O6BG, were purchased from Millipore Sigma (Burlington, MA). Olaparib was purchased from Selleck Chemicals (Houston, TX). DMSO solutions of alkylating drugs were prepared fresh from powder for each experiment.
4
Hepatic S9 Fraction Procurement and Characterization
5
Cytotoxicity Assay Reagents Preparation
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Isopropyl methanesulfonate (IPMS), ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT), 1-methoxy-5-methylphenazinium methyl sulfate (PMS), O6-benzylguanine and dimethylsulfoxide (DMSO) were obtained from Sigma. Propyl methanesulfonate (nPMS) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Dulbecco's phosphate buffered saline (PBS) was obtained from Life Technologies (Grand Island, NY, USA). All of the chemicals were dissolved in PBS, except XTT which was dissolved in DMSO.
6
Combination Therapy for Cancer Treatment
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O6-benzylguanine (Sigma) and carmustine/BCNU (Sigma) were made fresh for each injection. First, O6BG (120 mg/m2) was infused i.v. over 15 to 20 min (flow rate ∼600 mL/h). BCNU was given ∼30–45 min after the end of O6BG infusion. The BCNU doses were 10, 20, and 30 mg/m2 as indicated. O6BG infusion (120 mg/m2) was repeated 7–8 h after the end of the first infusion. Neutrophil counts decreased after each round of O6BG/BCNU treatment. The subsequent dose of O6BG/BCNU was therefore given only after neutrophil counts recovered (2–4 weeks).
7
Measuring Microsatellite Instability and HPRT Mutation
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MMR status was determined by fluorescent multiplex PCR-based analysis of five microsatellite markers [30 (link)]. For HPRT mutation assay, HCT116-GFP and HCT116-RIP140 cells were incubated in HAT medium (Life Technologies, Paisley, UK) to eliminate cells harboring preexisting hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutants. After 3 days, cells were seeded in Petri dishes, incubated with 20 µM O6-benzylguanine (Sigma-Aldrich, Saint-Quentin, France) for 2 h to deplete O-6-methylguanine-DNA methyltransferase (MGMT) and then supplemented or not with 1 µM methyl-nitro-nitroso-guanidine (MNNG) (TCI Europe) for 1 h. Cells were seeded in 6-well plates (200 cells/well), and cloning efficiency was analyzed 10 days later. In parallel, 105, 2 × 105 or 5 × 105 cells per well were plated with 5 µg/mL 6-thioguanine (6-TG, Sigma-Aldrich, Saint-Quentin, France) and cultured for 30 days. After staining with crystal violet, the mutation frequency was calculated (number of colonies/cloning efficiency × 105).
8
DNA Damage Response Assays
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Where indicated, cells were treated with 0.2 ug/ml neocarzinostatin (NCS) (Sigma-Aldrich, N9162–100UG) for 30 minutes, 20 μg/ml bleomycin (Selleck, S1214) for 3 hours, 40 J/m2 UVC (254 nm) for 15 minutes, 50–250 μM H2O2 (Sigma-Aldrich, H1009–100ML) for 20 minutes, 500 μM MMS (Sigma-Aldrich, 129925–5G) for 1 hour, 10 μM methylnitronitrosoguanidine (MNNG) (TCI, M0527) and 10 μM O6-benzylguanine (Sigma-Aldrich, B2292–50MG) for 2 hours, 1 μM CPT (EMD Millipore, 208925–50MG) for 1 hour, 100 μg/ml cycloheximide (Sigma-Aldrich, C7698–1G), 1 μg/ml doxycycline (Sigma-Aldrich, D9891–1G), 10 μM EdU (Thermo Fisher Scientific, 1149–100), 10 μM MG132 (Peptides International, IZL-3175v) and 2.5 μM MLN4924 (Active Biochem, A-1139), 1 μM Palbociclib (Selleck Chemicals).
9
Lentiviral Transduction and Temozolomide Treatment in Glioma Cells
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The human glioma cell lines U251 was kindly provided by Eric Holland and U87 (HTB-14) was purchased from ATCC. The Gp2-293 packaging cell line was purchased from Clontech (Cat.
631458). Cells were cultured in DMEM (Sigma-Aldrich, Cat. D5796) + 10% FBS (Sigma-Aldrich, Cat. F7524). All the cell lines were routinely checked for Mycoplasma contamination by PCR analysis. DNA fingerprinting has been performed for authentication of the glioma cell lines (data available upon request).
Lentiviruses were generated by co-transfection of lentiviral plasmids (pKLV-U6gRNA-PGKpuro2ABFP and lentiCas9-Blast) and 2nd generation packaging vectors (pMD2G and psPAX2) in Gp2-293 cells using calcium-phosphate precipitate. High-titer virus was collected at 36 and 60 hours following transfection and used to infect cells in presence of 7 μg/ml polybrene (Sigma-Aldrich, Cat. H9268-5G) for 12 hours. Transduced cells were selected with Blasticidin (3 μg/ml) (Gibco, Cat. A11139-03) and Puromycin (1.5 μg/ml) (Sigma-Aldrich, Cat. P8833-25MG).
Temozolomide was purchased from Selleckchem (Cat. S1237). O 6 -Benzylguanine was from Sigma-Aldrich (Cat. B2292-50MG).
631458). Cells were cultured in DMEM (Sigma-Aldrich, Cat. D5796) + 10% FBS (Sigma-Aldrich, Cat. F7524). All the cell lines were routinely checked for Mycoplasma contamination by PCR analysis. DNA fingerprinting has been performed for authentication of the glioma cell lines (data available upon request).
Lentiviruses were generated by co-transfection of lentiviral plasmids (pKLV-U6gRNA-PGKpuro2ABFP and lentiCas9-Blast) and 2nd generation packaging vectors (pMD2G and psPAX2) in Gp2-293 cells using calcium-phosphate precipitate. High-titer virus was collected at 36 and 60 hours following transfection and used to infect cells in presence of 7 μg/ml polybrene (Sigma-Aldrich, Cat. H9268-5G) for 12 hours. Transduced cells were selected with Blasticidin (3 μg/ml) (Gibco, Cat. A11139-03) and Puromycin (1.5 μg/ml) (Sigma-Aldrich, Cat. P8833-25MG).
Temozolomide was purchased from Selleckchem (Cat. S1237). O 6 -Benzylguanine was from Sigma-Aldrich (Cat. B2292-50MG).
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