U528824.pdf (25.37 MB)
Biological consequences of DNA adduct formation
thesisposted on 2014-12-15, 10:42 authored by James Anthony. Baker
The role of specific DNA adducts in chromosomal aberration was investigated in a human lymphoblastoid cell line transfected with drug-metabolising enzymes (MCL-5) or vector-only (cHol). The cells were treated with ethylating agents which differ in their DNA ethylation profiles, namely the direct-acting carcinogens N-nitroso-N-ethylurea (ENU) and diethyl sulphate (DES), and the indirect-acting N-nitrosodiethylamine (NDEA). Levels of O6 and N-7 ethylguanine (EtGua) were measured by immunoslot-blot analysis, allowing other adduct levels to be inferred. Equimolar (1 mM) treatment with DES or ENU for 28 hr gave similar levels of total DNA alkylation, but a different distribution of particular DNA adducts. The ability of the cell line to respond to genotoxic insult was assessed by measuring DNA repair, cell cycle arrest and induction of apoptosis. The cells were found to have normal levels of methylpurine-DNA glycosylase but were lacking methylguanine- methyltransferase. The half-lives of N-7 and O6 EtGua were determined to be 37 +/- 4 hr and 29 +/- 14 hr respectively. Cells were unable to arrest in G1 following y-irradiation, indicative of a mutant p53 phenotype. The cell line was also resistant to induction of apoptosis, as determined by internucleosomal cleavage and measurement of DNA fragmentation by flow cytometry. DNA strand breaks (critical lesions in aberration formation) were measured by p53 accumulation and by nick translation with pulsed-field gel electrophoresis. ENU-treated cells gave higher p53 responses than cells treated with equimolar DES, indicating that N- 7 EtGua is not the major adduct responsible for strand breakage. Caffeine co-incubation was shown to abrogate both S-phase and G2/M checkpoints, and also increase strand breakage. The frequency of induction of a specific chromosome aberration [t(14 18)] was investigated, with the effects of ENU (but not DES) and caffeine being synergistic. The important DNA adducts for chromosome aberration formation in this cell line appear to be O6 EtGua, O4 EtThy and phosphotriesters. The interplay between phenotype and cellular consequences of particular DNA adducts is highlighted.
Date of award1997-01-01
Awarding institutionUniversity of Leicester