Smoking is an important cause of lung cancer but not all smokers or ex-smokers develop lung cancer so there is a need to develop biomarkers that help to identify those individuals at increased risk of developing cancer. Hence, this study investigated novel biomarkers of exposure (DNA adducts) and susceptibility (DNA repair) to tobacco-specific nitrosamines (TSNA), potent carcinogens found in cigarette smoke that would be of use in epidemiological studies of cancer risk. To achieve this, an ELISA method which utilised a newly generated and partially characterised rabbit polyclonal antibody against O6-pyridyloxobutyl deoxyguanine (O6-pobdG) to quantify these lesions in DNA, was developed. In addition, the associations between single nucleotide polymorphisms (SNPs) within or near the N-methylpurine-DNA-glycosylase (MPG) DNA repair gene and MPG activity and lung cancer risk were examined.A competitive binding assay was used to exploit an oligodeoxyribonucleotide (ODN) containing a single O6-pobdG lesion that was annealed with a biotinylated complement to facilitate the binding of this double-stranded ODN (ds ODN) to microtitre plates. Experimental conditions were optimised to detect anti-O6-pobG antibody binding to ds O6-pobG ODN coated microtitre plates. Results showed that the optimum dilution of the primary antibody was 1:3000 and the optimum ds O6-pobG ODN concentration for coating was 6.76μM (676 fmoles per well). An increase in antibody incubation time increased antibody binding with an 8 hr incubation step used routinely. Pre-incubating the primary antibody with single stranded O6-pobG ODN (ss O6-pobG ODN) prior to incubating with the ds O6-pobG ODN coated microtitre plates (or simultaneous incubation of ss O6-pobG ODN with coated microtitre plates) resulted in a negative correlation between the signal and ss O6-pobG ODN. The limit of detection of this assay was of the order of 20 fmoles of O6-pobdG concentration. The associations between MPG genotype and MPG activity and lung cancer risk were studied using DNA samples from 125 participants (80 lung cancer cases and 45 controls). MPG activity had already been determined using a [32P]-labelled ODN cleavage assay. DNA was quantified using a picogreen assay and the samples genotyped using Sequenom® MassARRAY technology and MALDI-TOF MS of individual probes. All 13 SNPs examined conformed to Hardy-Weinberg equilibrium (p > 0.05) and showed no linkage disequilibrium. There was no association between individual genotypes and MPG activity but SNP rs2562164 showed a significant genotypic association (p=0.03) in that the mutant allele is more frequent in cases than in controls. 10 haplotypes were found in this population and there was no association between estimated haplotype frequencies and MPG activity level (p>0.05). Haplotype CCTCGCTGCCGT was significantly associated with lung cancer (p=0.01).In conclusion, the current sensitivity of the ELISA assay for O6-pobG in DNA is insufficient to detect this adduct in the amount of the DNA typically extracted from human tissue samples. Further work would be required to increase assay sensitivity. There was some suggestion that MPG genotypes may be associated with lung cancer risk (but not MPG activity) but further work with larger populations would be required to confirm these observations.
|Date of Award||1 Aug 2015|
- The University of Manchester
|Supervisor||Andrew Povey (Supervisor) & Geoffrey Margison (Supervisor)|