Characterizing pro-mutagenic O6-alkylguanine adducts in human colorectal tissue

Abstract

Colorectal cancer is the third most commonly diagnosed cancer worldwide. The positive association between red and/or processed meat consumption and human colorectal cancer risk is well established potentially via heme-catalysed formation of carcinogenic N-nitrosocompounds (NNOC). These NNOC can form a range of O6- alkylguanine (O6-alkylG) adducts that may increase mutational and cancer risk. The overall aim of this study was to develop a novel approach to characterize the overall load of O6-alkylG adducts in human colorectal DNA using the known action of the DNA repair protein, O6-alkylguanine O6-alkyltransferase (MGMT) to irreversibly transfer the alkyl group from O6-alkylG to the MGMT active site peptide (MGMT-ASP). Incubation of chemically synthesised single (SS) and double stranded (DS) oligodeoxyribonucleotides (ODNs) containing O6-methylguanine (O6-MeG), O6- carboxyethylguanine (O6-CEG), but only DS O6-carboxymethylguanine (O6-CMG) -23 mer containing ODNs with MGMT resulted in MGMT inactivation. There was a preference for repair of DS ODNs. Recorded IC50 values were 0.17+0.02, 0.07+0.01, 15.7+2.1, 27.00+1.70 and 7.50+0.50 nanomolar (nM) for SS O6-MeG, DS O6-MeG, DS O6-CMG, SS O6-CEG, and DS O6-CEG, respectively. This repair was confirmed using a restriction endonuclease site deprotection assay as O6-MeG and O6-CEG inhibition of PstI digestion of ODNs, whilst, O6-CMG inhibited MboI. This inhibition was removed by pre-incubation with MGMT. Alkyl group transfer from these ODNs to a Maltose binding protein-MGMT (MBP-MGMT) fusion protein was demonstrated as alkylated active site peptides (ASPs) were detected using with Matrix assisted laser desorption/ionizationtime of flight (MALDI-TOF). S-methylcysteine (m/z 1329.7), and S-carboxyethylcysteine (m/z 1387.7) modified ASPs were detected following incubation of MBP-MGMT incubated with SS or DS O6-MeG, and O6-CEG ODNs, respectively. Scarboxymethylcysteine (m/z 1373.7) was detected with DS but not SS O6-CMG 23 mer ODN. Only the unmodified MGMT-ASP (m/z 1315.7) was found after incubating MGMT with unmodified ODN. Quantitative mass spectrometry (MS) findings highlighted that MGMT preferentially repaired specified adducts when presented in DS ODNs over SS ones. Temozolomide (TMZ) methylated calf thymus DNA (CT-DNA) and human colorectal cancer DNA samples were incubated with his tagged MGMT fusion protein (his-MGMT) and his-MGMT recovered using Ni-coated beads, digested in situ with trypsin and analysed by MALDI-TOF. MS analysis of tryptic digests of MGMT incubated with methylated CT-DNA revealed methylated MGMT-ASP, detecting as low as 50 fmole of O6-MeG/mg CT-DNA. This approach was applied to 13 human colorectal DNA samples, 12 of them showed evidence of O6 alkylation damage. Of 13 samples, 12 contained O6-MeG, 8 contained O6-CMG and 1 contained O6-HOEtG. A further 7 peaks were detected with a signal/noise (S/N) ratio of >10, potentially, correspond to alkylated MGMT-ASP ions but identities of the alkyl groups are as yet unknown. These results demonstrate proof of principle and confirm that human colorectal cancer DNA contains a range of O6-alkylG adducts that constitute a fraction of DNA alkyl adductome.

Date of Award	1 Aug 2018
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Povey (Supervisor) & Perdita Barran (Supervisor)