Zaidi, N H; Potten, Christopher S; Margison, Geoffrey P; Cooper, Donald P; O'Connor, Peter J (1993-10)
Several potential cancer risk factors have been monitored concurrently in the upper gastrointestinal tract of young male Wistar rats given N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) via the drinking water, a regimen that induces a high yield of tumours in the pylorus and to a lesser extent in the duodenum. Radioimmunoassay was used to determine the amounts of O6-methyl-2'-deoxyguanosine (O6-MedG) formed in the tissue DNA of rats given MNNG at doses of 40 or 80 micrograms/ml for periods of 3, 6 and 12 weeks. The highest adduct concentration was found in the pylorus with progressively lower concentrations in the corpus and duodenum, jejunum, forestomach and oesophagus. Between 3 and 12 weeks these adduct levels decreased in all tissues and there was no evidence of a dose dependent accumulation of O6-MedG. When analysed by immunohistochemistry the distribution of cells with nuclei containing O6-MedG was seen to be heterogeneous in the various tissues. O6-Alkylguanine-DNA alkyltransferase activity increased during the 12 weeks of MNNG treatment in oesophagus and forestomach, but decreased to approximately 50% of the initial value in the corpus, pylorus, duodenum and jejunum. The major changes in DNA synthesis and cell proliferation were the marked upward expansion (i.e. towards the lumen) of the zone of replicating cells in the glands of the pylorus and the greatly increased numbers of replicating damaged cells (i.e. cells that contained O6-MedG whilst undergoing DNA synthesis) as determined by sequential immunohistochemical analysis and autoradiography. Such cells are the probable target cells in this chronic dose carcinogenesis regime. Although similar changes also occurred in the glands of the corpus these were of lesser extent and the changes of labelling index in the oesophagus and forestomach were relatively minor. In the duodenum, MNNG treatment led to erosion of the upper part of the glands so that the zone of cells containing O6-MedG overlapped with the zone of proliferating cells resulting in the formation of many replicating damaged cells. Thus, as in the single dose study (see preceding paper) the distribution of replicating damaged cells coincides with the tumour yield in the tissues of the upper gastrointestinal tract. As in the case of single doses of MNNG the risk factors for carcinogenesis are, a significant level of DNA damage, a lower capacity for DNA repair and an increased DNA synthetic activity, again suggesting that carcinogenic risk cannot readily be determined by studying risk factors individually.
Jackson, Peta E; O'Connor, Peter J; Cooper, Donald P; Margison, Geoffrey P; Povey, Andrew C (2003-03)
Putative risk factors (DNA damage) and risk modifying factors (DNA repair and cell proliferation) were examined in an experimental mouse model in which treatment with dimethylhydrazine (6.8 mg/kg DMH i.p. once weekly) for up to 20 weeks induces colon tumours in a site specific manner with 0, 43 and 87% of animals having proximal, mid and distal colon tumours respectively at the highest cumulative dose. Levels of the pro-carcinogenic DNA adduct, O(6)-methylguanine (O(6)-MeG), in colonic DNA were found to vary with time after final treatment and with location within the colon but not with total DMH dose. O(6)-MeG levels were generally lowest in proximal colon DNA and highest in distal colon DNA. Steady state O(6)-MeG levels were obtained at the highest cumulative DMH dose with O(6)-MeG levels in mid and distal colon DNA being 5 and 10 times higher those in proximal colon DNA. O(6)-alkylguanine-DNA alkyltransferase (MGMT) activity, and cell proliferation indices in the colon were also found to vary with time after final treatment but not with either location within the colon or total DMH dose. O(6)-MeG levels, MGMT activity and cell proliferation indices at specific time points as well as basal MGMT activity were not associated with differences in tumour yield within the colon. However tumour yield was associated with the cumulative amount of O(6)-MeG present in DNA over the treatment period and with the treatment induced cumulative increase in cell proliferation, particularly within regions of the colon crypt where stem cells reside but not with cumulative changes in MGMT activity. Results are consistent with an increased cancer risk arising from an increased mutation load in the target stem cell population due to increased adduct formation/persistence and cell proliferation but also suggest that other cell specific factors may help to determine tumourigenic response.
Potter, P M; Rafferty, Joseph A; Cawkwell, L; Wilkinson, M C; Cooper, Donald P; O'Connor, Peter J; Margison, Geoffrey P (1991-04)
A rat O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA has been isolated from a rat liver cDNA library by hybridization with the human homologue. The candidate 806 bp cDNA was sequenced and shown to contain a 630 bp open reading frame that could encode a protein of 22.2 kd. Fluorography of labelled ATase indicates a 24 kd protein which is consistent with several previous reports. The derived amino acid sequence demonstrated 81% similarity with the human ATase and 94% identity over a 67 residue region encompassing the putative alkyl acceptor site. Peptide sequences derived from cleaved homogeneous rat ATase have been located in the predicted protein providing additional confirmation of the identity of the cDNA. A 1.05 kb mRNA has been detected in rat liver by Northern analysis; treatment of adult rats with 2-acetylaminofluorene causes an approximately 10-fold induction of this message in liver. Following site directed mutagenesis of the 806 bp cDNA, the 630 bp protein coding sequence has been ligated into an Escherichia coli expression vector to achieve ATase levels of greater than 3% of total protein in bacterial extracts.
Jackson, Peta E; Hall, C N; O'Connor, Peter J; Cooper, Donald P; Margison, Geoffrey P; Povey, Andrew C (1997-07)
O6-alkylguanine DNA-alkyltransferase (ATase) provides protection against the toxic, mutagenic and carcinogenic effects of alkylating agents, principally by removing the promutagenic lesion O6-alkylguanine from DNA. Differences in ATase activity in human tissue may thus determine mutational susceptibility. As GC-->AT transitions, which can be induced by O6-alkylguanine in DNA, are commonly observed in the K-ras oncogene of alkylating agent induced animal tumours and in human colorectal tumours, we have examined whether differences in ATase activity may affect the risk of K-ras mutations in humans with colorectal tumours. NTase activity in normal tissue from individuals with a K-ras mutation in colorectal tissue and more specifically a GC-->AT transition (but not a transversion mutation) was significantly lower than that in individuals without a mutation (P < 0.01). Thus, individuals with low ATase activity in normal tissue (i.e. below the median) were at increased risk of having a transition (OR 10.1; 95% CI 1.9-99.0), but not a transversion mutation (OR 1.7; 95% CI 0.3-12.2). There were no significant differences in tumour ATase activity in individuals with or without a mutation. These results suggest that ATase can protect colorectal tissue against the mutagenic effects of alkylating agents and furthermore, that alkylating agent exposure plays a role in the aetiology of colorectal tumours containing a GC-->AT transition in the K-ras oncogene.
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