O'Donnell, Paul; Barber, Philip V; Margison, Geoffrey P; Povey, Andrew C (1999-07)
The activity of the DNA repair enzyme O6-alkylguanine-DNA-alkyltransferase (ATase) may be a risk factor in the pathogenesis of lung cancer. ATase activity has previously been measured in peripheral blood lymphocytes (PBLs), cell extracts from bronchoalveolar lavage fluid, and cell homogenates from resected lung tissue. However, it is not clear whether ATase activity in these samples correlates well with the activity found in bronchial epithelial cells, the progenitor cells for the main types of lung cancer. In this study, cell extracts were prepared from PBLs, bronchial lavage (BL) fluid, and bronchial brushings from normal lung in 20 patients attending for routine bronchoscopy. Bronchial brushing sampled a significantly greater proportion of bronchial epithelial cells than did BL [88+/-9% (mean+/-SD) versus 39+/-19%; P < 0.0001]. ATase activity was determined in each of the cell extracts and was found to be higher in PBLs than in bronchial brushings (P = 0.005) and higher in bronchial brushings than in BL (P = 0.005). No correlation in ATase levels was observed between any of the three samples. We conclude that bronchial brushing is a more specific and reliable way of sampling bronchial epithelial cells than BL and that it samples enough cells for ATase activity to be determined. In addition, in terms of the activity of this potentially critical DNA repair enzyme, PBLs, and cell extracts obtained from BL may not provide good surrogate tissue for bronchial epithelial cells, the critical targets for carcinogenesis.
Collis, S J; Tighe, A; Scott, S D; Roberts, Stephen A; Hendry, Jolyon H; Margison, Geoffrey P (2001-04-01)
The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20-50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to gamma-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of approximately 2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the alpha parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation.
Chinnasamy, Nachimuthu; Rafferty, Joseph A; Hickson, Ian; Lashford, Linda S; Longhurst, S J; Thatcher, Nick; Margison, Geoffrey P; Dexter, T Michael; Fairbairn, Leslie J (1998-06)
Murine bone marrow cells were transduced ex vivo with a retrovirus encoding an O6-benzylguanine (O6-beG) insensitive, double mutant form of the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATPA/GA). In animals reconstituted with the transduced bone marrow, about 50% of cells in the multipotent spleen colony-forming cells (CFU-S) and lineage restricted granulocyte-macrophage (GM-CFC) haemopoietic progenitor populations were found to be carrying the transgene and this correlated with the frequency of bone marrow cells and spleen colonies which stained positive for hATPA/GA by immunocyto-chemistry. Expression of hATPA/GA was associated with significant in vivo protection of both CFU-S (P = 0.001) and GM-CFC (P < 0.024) against the toxicity of the antitumour methylating agent, temozolomide, given in combination with O6-beG. Expression of hATPA/GA also led to a reduction in the frequency of combined O6-beG/temozolomide-induced micronuclei seen in polychromatic erythrocytes (P < 0.003). This study is the first to demonstrate in vivo protection of multipotent haemopoietic progenitors against the toxic and clastogenic effects of an O6-alkylating agent in the presence of O6-beG. It also represents the first report of reduced clastogenesis as a consequence of expression of an O6-beG-resistant ATase. In the accompanying article we report hATPA/GA-mediated resistance of human CD34+ haemopoietic progenitors to combined O6-beG/O6-alkylating agent toxicity. Together these two reports suggest that a gene therapy strategy whereby protection of normal haemopoietic tissue may be combined with O6-beG-mediated tumour sensitisation may be efficacious in achieving an increase in therapeutic index.
Cardinal, John W; Margison, Geoffrey P; Mynett, Kurt J; Yates, Allen P; Cameron, Donald P; Elder, Rhoderick H (2001-08)
Type 1 diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta-cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta-cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in APNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta-cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(+/+) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.
Catania, J; Keenan, B C; Margison, Geoffrey P; Fairweather, D S (1987-12)
Quantitation of 5-methylcytosine in DNA after acid hydrolysis has been inaccurate because deamination of cytosine and 5-methylcytosine occurs during the hydrolysis procedure. There is little information in the literature regarding the use of hydrofluoric acid (HF) for DNA hydrolysis and we have therefore undertaken a systematic study of this process. The deoxyribonucleotides of cytosine and 5-methylcytosine were shown not to undergo detectable levels of deamination during prolonged periods (up to 24 h) at 80 degrees C in 48% HF. Kinetic studies show that the release of purine and pyrimidine bases was complete by 4 h under these conditions. Analysis of the 5-methylcytosine content of DNA from various tissues gave levels that were very close to the values reported in the literature. This method is ideally suited for the determination of the overall cytosine methylation levels in DNA.
Rafferty, Joseph A; Wibley, J E; Speers, P; Hickson, Ian; Margison, Geoffrey P; Moody, P C; Douglas, K T (1997-09-26)
O6-Alkylguanine DNA-alkyltransferase (ATase) repairs toxic, mutagenic and carcinogenic O6-alkylguanine (O6-alkG) lesions in DNA by a highly conserved reaction involving the stoichiometric transfer of the alkyl group to the active centre cysteine residue of the ATase protein. In the Escherichia coli Ada ATase, which is effectively refactory to inhibition by O6-benzylguanine (O6-BzG), the residue corresponding to glycine-160 (G160) for the mammalian proteins of this class is replaced by a tryptophan (W). Therefore, to investigate the potential role of the G160 of the human ATase (hAT) protein in determining sensitivity to O6-BzG, site-directed mutagenesis was used to produce a mutant protein (hATG160W) substituted at position 160 with a W residue. The hATG160W mutant was found to be stably expressed and was 3- and 5-fold more sensitive than hAT to inactivation by O6-BzG, in the absence and presence of additional calf-thymus DNA respectively. A similar, DNA dependent increased sensitivity of the hATG160W mutant relative to wild-type was also found for O6-methylguanine mediated inactivation. The potential role of the W160 residue in stabilising the binding of the O6-alkG to the protein is discussed in terms of a homology model of the structure of hAT. The region occupied by G/W-160 forms the site of a putative hinge that could be important in the conformational change that is likely to occur on DNA binding. Three sequence motifs have been identified in this region which may influence O6-BzG access to the active site; YSGG or YSGGG in mammals (YAGG in E. coli Ogt, YAGS in Dat from Bacillus subtilis), YRWG in E. coli Ada and Salmonella typhimurium (but YKWS in Saccharomyces cerevisiae) or YRGGF in AdaB from B. Subtilis. Finally,conformational and stereoelectronic analysis of the putative transition states for the alkyl transfer from a series of inactivators of hAT, including O6-BzG was undertaken to rationalise the unexpected weak inhibition shown by the alpha-pi-unsaturated electrophiles.
The spontaneous hypoxanthine phosphoribosyl transferase deficient (HPRT-) mutants of V79 cells (TG11 and TG15) were transfected with a retrovirus-based plasmid containing a truncated form of the Escherichia coli gene which codes for O6-alkylguanine (O6-AG) DNA alkyltransferase (ATase). The resultant cell lines TG11SB5 and TG15SB7 were G418 resistant and expressed high levels of O6-AG ATase activity. The frequency of revertants induced by equitoxic doses of N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) was 10- to 50-fold higher in TG11 than in TG15. In TG11SB5 and TG15SB7 induced revertant frequencies were reduced relative to TG11 and TG15 by factors of 6-8 and 1.5-3.0, respectively, immediately after treatment. On delayed plating the frequency of MNU-induced revertant colonies decreased at a rate inversely proportional to dose in both TG11 and TG11SB5. In contrast, after exposure of TG11SB5 to ENU (50 or 75 micrograms/ml) initial reversion frequencies were low compared with TG11, but then rose to a plateau frequency by 24 h, which was maintained for up to 72 h. The frequency of reversion observed, the degree of protection afforded by the E.coli O6-AG ATase and the kinetics of expression of revertants were thus cell line specific suggesting that DNA sequence specific alkylation and/or preferential repair may be responsible. The initial protection against mutagenesis is consistent with the hypothesis that MNU- and ENU-induced reversion is the result of miscoding opposite O6-AG or O4-alkylthymine residues. Expression of O6-AG ATase activity was variable when cells were continually cultured over long periods despite the presence of the selective antibiotic G418.
Durrant, Linda G; Margison, Geoffrey P; Boyle, John M (1981)
Exposure of Chinese hamster V79 cells to a non-toxic dose of N-methyl-N-nitrosourea, followed at intervals by exposure to toxic challenging doses of the same agent, resulted in increased survival of colony forming ability when these cells were compared with matched control cells that only received the challenging dose. The extent of the increase was dependent on the time interval between exposures, and rose to a maximum of about two-fold 5 days after the initial dose, declining slowly to control values on subsequent days. Whilst pretreatment enhanced survival, it altered neither the frequency of mutation to 6-thioguanine resistance, nor the formation or loss of 3-methyladenine, 7-methylguanine and O6-methylguanine. Modification of the conditions by which the initial dose was administered led to a reduction or abolition of the survival response. It is suggested that enhanced survival may result from alteration in the ability to recover from cellular damage rather than by improved DNA repair.
Margison, Geoffrey P; Cooper, D P; Smith, R A; Montesano, R; Bresil, H; Planche-Martel, G; O'Connor, Peter J (1984)
N-nitroso compounds and related alkylating agents react with several nucleophilic sites in DNA. One of the products, O6-alkylguanine, may be responsible for the carcinogenic effects of such agents as it can mispair with thymine during DNA replication, thereby affecting the integrity of the genome and consequently the repair of this lesion could be an important determinant in carcinogenesis. The use of both in vivo and in vitro procedures for the assay of the activity of this repair system have shown that it can be enhanced in rat liver by treatment with a variety of agents while in other species, the system is not only less effective but does not appear to respond to such treatments. Differences in the capacity and inducibility of this repair system may be related to the susceptibility of different species to tumour induction by chronic administration of nitrosamines.
In order to investigate the importance of 3-methyladenine in cellular sensitivity to chemical methylating agents we have constructed retroviral vectors for the integration and expression of the Escherichia coli tag gene in mammalian cells. The tag gene encodes 3-methyladenine DNA glycosylase-1 which specifically removes 3-alkyladenines from DNA. The constructs were introduced into Chinese hamster V79 cells by liposome mediated transfection or into murine haemopoietic stem cells by cocultivation with a lipofected, virus-packaging cell line. In both cases, stable transfectants were selected for resistance to the antibiotic, G418, conferred by expression of the neo gene carried by the vector. Measurements of 3-methyladenine DNA glycosylase activity in cell extracts showed an up to 10-fold increase in cell lines with stably integrated tag gene sequences. These cell lines were significantly more resistant to the cytotoxic effects of methylmethanesulfonate and N-methyl-N-nitrosourea than their parent cell lines, indicating that 3-methyladenine repair is a limiting factor in cellular resistance to these methylating agents. Furthermore, the mutation frequency induced by methylmethanesulfonate was reduced to 50% of normal by expression of 3-methyladenine I activity in the Chinese hamster cells, indicating that m3A is not only a cytotoxic but also a premutagenic lesion in mammalian cells. It is concluded that an alkylation repair gene function of a type only thought to be present in bacteria can yield a hyperresistant phenotype when transferred to mammalian cells.
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