• O 6 -Methylguanine methyltransferase activity is increased in rat tissues by ionising radiation

      Margison, Geoffrey P; Butler, John; Hoey, Brigid M; Department of Carcinogenesis, Paterson Laboratories, Christie Hospital Withington, M20 9BX, UK (1985)
    • O(6)-(4-bromothenyl)guanine improves the therapeutic index of temozolomide against A375M melanoma xenografts.

      Middleton, Mark R; Kelly, Jane; Thatcher, Nick; Donnelly, Dorothy J; McElhinney, R S; McMurry, T B; McCormick, J E; Margison, Geoffrey P; Cancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, UK. mmiddleton@picr.man.ac.uk (2000-01-15)
      Tumour resistance to methylating agents is linked to expression of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (ATase). There is considerable interest in improving the efficacy of O(6)-alkylating chemotherapy by prior depletion of ATase. We have tested the ability of a modified guanine base, O(6)-(4-bromothenyl)guanine (4BTG), to inactivate ATase and to enhance the anti-tumour effect of temozolomide in an animal model system. A375M human melanoma xenografts were established in the flanks of nude mice. ATase depletion after a single dose of 4BTG or O(6)-BG (20 mg/kg i.p.) was determined over a 24 hr period. Subsequently, we tested the effect of 4BTG (20 mg/kg i.p. daily) and/or temozolomide (80-175 mg/kg i.p. daily) over a 5-day schedule on tumour growth. 4BTG was an effective inactivator of ATase in tumour, producing complete depletion within 2 hr of dosing. Furthermore, it enhanced the tumour growth delay achieved with temozolomide, increasing the tumour quintupling time by 8.7 days (95% confidence interval 6.1-11.3 days, p < 0.0001). Whilst the delay in tumour growth was indistinguishable from that observed with O(6)-benzylguanine (O(6)-BG) and temozolomide, the 4BTG combination resulted in considerably less toxicity (0/9 vs. 2/9 deaths; 6.84% weight loss vs. 9.48%, p = 0.019). 4BTG is a potent inactivator of ATase and enhances the therapeutic ratio of temozolomide in this model system to a greater extent than O(6)-BG.
    • O(6)-methylguanine-DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib.

      Watson, Amanda J; Middleton, Mark R; McGown, Gail; Thorncroft, Mary R; Ranson, Malcolm R; Hersey, Peter; McArthur, Grant A; Davis, Ian D; Thomson, D; Beith, Jane; et al. (2009-04-21)
      We evaluated the pharmacodynamic effects of the O(6)-methylguanine-DNA methyltransferase (MGMT) inactivator lomeguatrib (LM) on patients with melanoma in two clinical trials. Patients received temozolomide (TMZ) for 5 days either alone or with LM for 5, 10 or 14 days. Peripheral blood mononuclear cells (PBMCs) were isolated before treatment and during cycle 1. Where available, tumour biopsies were obtained after the last drug dose in cycle 1. Samples were assayed for MGMT activity, total MGMT protein, and O(6)-methylguanine (O(6)-meG) and N7-methylguanine levels in DNA. MGMT was completely inactivated in PBMC from patients receiving LM, but detectable in those on TMZ alone. Tumours biopsied on the last day of treatment showed complete inactivation of MGMT but there was recovery of activity in tumours sampled later. Significantly more O(6)-meG was present in the PBMC DNA of LM/TMZ patients than those on TMZ alone. LM/TMZ leads to greater MGMT inactivation, and higher levels of O(6)-meG than TMZ alone. Early recovery of MGMT activity in tumours suggested that more protracted dosing with LM is required. Extended dosing of LM completely inactivated PBMC MGMT, and resulted in persistent levels of O(6)-meG in PBMC DNA during treatment.
    • O6-alkylguanine-DNA alkyltransferase activity in schistosomiasis-associated human bladder cancer.

      Badawi, A F; Cooper, Donald P; Mostafa, M H; Aboul-Azm, T; Barnard, R J; Margison, Geoffrey P; O'Connor, Peter J; National Center for Toxicology, Jefferson, Arkansas. (1994)
      O6-Alkylguanine-DNA-alkyltransferase (ATase) activity was measured in extracts of 55 bladder tissue samples (46 tumour and nine uninvolved mucosal tissue) from Egyptian patients with schistosome-associated bladder carcinoma. Activity varied from 2.0 to 16.2 fmole ATase/microgram DNA (mean +/- S.D.; 5.6 +/- 4.0) or from 28 to 351 fmole ATase/mg (117 +/- 71). ATase levels in schistosome-associated bladder cancer tissues (5.6 +/- 4.0 fmole ATase/microgram DNA) tended to be lower than those observed in normal human bladder mucosal tissue (8.5 +/- 4.4 fmole ATase/microgram DNA). In a previous study (Badawi et al., Carcinogenesis, 1992, 13, 877-881) DNA-alkylation damage (O6-methyldeoxyguanosine) was found in 44/46 of these schistosome-associated bladder cancer samples at levels ranging from 0.012 to 0.485 mumole O6-MedG/mole deoxyguanosine. We now report an inverse correlation between the levels of methylation damage and ATase activity (r = -0.67; P < 0.001). These observations encourage further investigations of the possible role of environmental alkylating agents in the aetiology of early bladder cancer associated with schistosomiasis.
    • O6-alkylguanine-DNA alkyltransferase assay.

      Watson, Amanda J; Margison, Geoffrey P; CRC Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester, UK. (2000)
    • O6-alkylguanine-DNA alkyltransferase attenuates triazene-induced cytotoxicity and tumor cell immunogenicity in murine L1210 leukemia.

      Graziani, G; Faraoni, I; Grohmann, U; Bianchi, R; Binaglia, L; Margison, Geoffrey P; Watson, Amanda J; Orlando, L; Bonmassar, E; D'Atri, S; et al. (1995-12-15)
      Methylating and chloroethylating triazene compounds (TZCs) are effective antitumor agents in murine leukemias and can induce the appearance of novel antigens in leukemic cells (chemical xenogenization). Recently, it has been shown that TZCs might have a role in the treatment of patients affected by acute myelogenous leukemias that express low levels of the DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (OGAT). In this report, we have evaluated the role of this DNA repair enzyme in the leukemic cell response to the xenogenizing and cytotoxic properties of TZCs. OGAT-deficient murine leukemic L1210 cells were transfected with a recombinant ecotropic retrovirus containing the coding region for the human OGAT protein. Selected clones expressed the human OGAT transcript and had greatly increased OGAT activity. Compared to OGAT-deficient cells, OGAT-expressing cells were considerably more resistant to the xenogenizing properties of 1-(p-chlorophenyl)-3,3- dimethyl-triazene, measured in terms of leukemia graft rejection, and were less susceptible to the cytotoxic activity of the TZCs 8-carbamoyl-3-methyl-imidazo [5,1-d]-1,2,3,5-tetrazin-4(3H)-one and 8-carbamoyl-3-(2-chloroethyl)imidazo [5,1-d]-1,2,3,5-tetrazin-4(3H)-one. These data suggest that methylation of the O6 position of guanine is involved in the appearance of increased tumor immunogenicity after exposure to methylating TZC and that OGAT is able, at least in part, to counteract the cytotoxic effects of methylating and chloroethylating agents.
    • O6-alkylguanine-DNA alkyltransferase: role in carcinogenesis and chemotherapy.

      Margison, Geoffrey P; Santibanez-Koref, Mauro F; CRC Carcinogenesis Group, Paterson Institute for Cancer Research, Manchester, UK. (2002-03)
      The DNA in human cells is continuously undergoing damage as consequences of both endogenous processes and exposure to exogenous agents. The resulting structural changes can be repaired by a number of systems that function to preserve genome integrity. Most pathways are multicomponent, involving incision in the damaged DNA strand and resynthesis using the undamaged strand as a template. In contrast, O(6)-alkylguanine-DNA alkyltransferase is able to act as a single protein that reverses specific types of alkylation damage simply by removing the offending alkyl group, which becomes covalently attached to the protein and inactivates it. The types of damage that ATase repairs are potentially toxic, mutagenic, recombinogenic and clastogenic. They are generated by certain classes of carcinogenic and chemotherapeutic alkylating agents. There is consequently a great deal of interest in this repair system in relation to both carcinogenesis and cancer chemotherapy.
    • O6-alkyltransferase activity in normal human gastric mucosa.

      Dyke, G W; Craven, J L; Hall, R; Cooper, Donald P; Soballa, G; Garner, R C; Cancer Research Unit, University of York, Heslington, U.K. (1990-11-05)
      The spectrum of activity of the DNA repair enzyme O6-alkyltransferase has been studied in a large series of normal stomachs in order to establish the baseline range of values for this enzyme. Sixty-eight patients with histologically normal stomachs were biopsied during the course of upper gastrointestinal endoscopy and the biopsies assayed for O6-alkyl-transferase activity. A wide spectrum of activity was found with values ranging from 38 fmol O6-guanine extracted/mg protein to over 400 fmol/mg. This suggests that there may be wide inter-individual differences in susceptibility to alkylating actions in the human gastric mucosa.
    • O6-benzylguanine increases the sensitivity of human primary bone marrow cells to the cytotoxic effects of temozolomide.

      Fairbairn, Leslie J; Watson, Amanda J; Rafferty, Joseph A; Elder, Rhoderick H; Margison, Geoffrey P; Cancer Research Campaign Department, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1995-02)
      The sensitivity of human primary bone marrow granulocyte/macrophage precursor cells to the cytotoxic effects of the methylating antitumor agent temozolomide (8-carbamoyl-3- methylimidazo[5,1-d]-1,2,3,5-tetrazin-4-[3H]-1) was investigated using an in vitro colony-forming assay. In the eight samples examined, there was a range of sensitivities with D37 values from 18.2 to > 55 microM. When cells were simultaneously exposed to the O6-alkylguanine-DNA alkyltransferase (ATase) inactivating agent, O6-benzylguanine (O6BeG; 10 microM), the cytotoxicity of temozolomide was substantially increased with D37 values between 5 and 38.5 microM. O6BeG also increased temozolomide sensitivity in the human colon carcinoma cell line, WiDr, and this was shown to correlate with the O6BeG-mediated depletion of ATase activity. Where the extent of sensitization produced by O6BeG could be calculated, there was a correlation between this and the D37 value in the absence of O6BeG (R = 0.996); thus, sensitization was more extensive in the cells that were inherently more resistant to temozolomide. These data have implications for possible increased hematological toxicity in clinical protocols designed to exploit O6BeG or other agents to deplete ATase activity in tumors cells prior to treatment of patients with temozolomide or related agents.
    • O6-Benzylguanine potentiates BCNU but not busulfan toxicity in hematopoietic stem cells.

      Westerhof, G Robbin; Down, Julian D; Blokland, Irene; Wood, Michelle; Boudewijn, Adrie; Watson, Amanda J; McGown, Alan T; Ploemacher, Rob E; Margison, Geoffrey P; Department of Hematology, Erasmus University, Rotterdam, The Netherlands. (2001-05)
      OBJECTIVE: Busulfan (BU) is often used in conditioning regimens prior to bone marrow transplantation, but its mechanism of action remains to be resolved. We have examined the possibility that BU may exert part of its toxic effects via DNA alkylation at the O6 position of guanine as this might provide an approach to improving the conditioning regimen. METHODS: Survival of LAMA-84 and RJKO cells was assessed by colony-forming assay and cell counting, respectively. O6-alkylguanine-DNA alkyltransferase (ATase) activity was assayed by transfer of radioactivity from [3H]-methylated DNA. Colony-forming potential of normal human bone marrow cells (BMC) was measured in the presence of appropriate growth factors as the formation of both granulocyte-macrophage colony-forming units (CFU-GM) or burst-forming unit erythroids (BFU-E) within the same assay. Murine hematopoietic precursors were grown under a bone marrow stromal cell line to allow measurement of the frequency of cobblestone area-forming cells (CAFC) that correspond to CFU-GM, spleen colony-forming units (CFU-S), and the primitive stem cells with long-term repopulating ability. RESULTS: Inactivation of ATase by O6-benzylguanine (O6-BeG) sensitized a human erythromegakaryocytic cell line (LAMA-84) and normal human bone marrow progenitors to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) but not to BU toxicity. BCNU, but not BU, inactivated ATase in LAMA-84 cells. Overexpression of human ATase in cDNA transfected Chinese hamster cells attenuated the toxicity of BCNU but not BU. Finally, the in vivo treatment of mice showed that the depletion of primitive stem cells by BU as measured in the CAFC assay was not affected by addition of O6-BeG. O6-BeG did, however, dramatically potentiate BCNU toxicity in all CAFC subsets, leading to depletion of more than 99% stem cells. CONCLUSION: These data suggest that BU does not elicit toxicity via alkylation at the O6 position of guanine in DNA in a way that can be influenced by ATase modulation.
    • O6-benzylguanine potentiates the in vivo toxicity and clastogenicity of temozolomide and BCNU in mouse bone marrow.

      Chinnasamy, Nachimuthu; Rafferty, Joseph A; Hickson, Ian; Ashby, John; Tinwell, Helen; Margison, Geoffrey P; Dexter, T Michael; Fairbairn, Leslie J; Cancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1997-03-01)
      The effects of treatment of mice with O6-benzylguanine (O6-BeG) on the levels of O6-alkylguanine-DNA alkyltransferase (ATase) in the hematopoietic compartment and on the in vivo sensitivity of hematopoietic progenitor cells to the toxic and clastogenic effects of the antitumor agents 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) and temozolomide were studied. When the overall effects of BCNU alone or with O6-BeG pretreatment were compared, dose potentiating factors of 4.17 for marrow cellularity, 4.57 for granulocyte macrophage-colony forming cells (GM-CFC) and 8.25 for colony forming unit-spleen (CFU-S) in O6-BeG pretreated versus nonpretreated animals were observed. A similar trend of dose potentiation was observed for temozolomide, although it was of lower magnitude: 1.20 for marrow cellularity, 1.63 for GM-CFC, and 1.68 for CFU-S. When the clastogenic effects of BCNU and temozolomide were examined in the mouse bone marrow micronucleus assay, a significantly (P < .05 to .001) higher frequency of micronuclei formation was observed in mice that received O6-BeG pretreatment compared with mice that received no pretreatment. These data suggest that the use of O6-BeG as a tumor-sensitizing agent before treatment of patients with O6-alkylating agents may lead to more severe hematological toxicity and possibly to an increased incidence of secondary leukemias as a result of elevated mutation frequencies in these patients.
    • O6-methylguanine formation, repair protein depletion and clinical outcome with a 4 hr schedule of temozolomide in the treatment of advanced melanoma: results of a phase II study.

      Middleton, Mark R; Lee, Siow Ming; Arance, Ana; Wood, Michelle; Thatcher, Nick; Margison, Geoffrey P; Cancer Research Campaign Department of Medical Oncology, Christie Hospital NHS Trust, Manchester, UK. mmiddleton@picr.man.ac.uk (2000-11-01)
      O6-Methylguanine-DNA methyltransferase (MGMT) is a major determinant of resistance to temozolomide. Its levels are depleted in lymphocytes after drug administration, but there is partial recovery by 24 hr, the usual time of subsequent dosing. Administering subsequent doses of temozolomide at the MGMT nadir could enhance its effectiveness, by increasing the amount of O6-methylguanine (O6-meG) in DNA. We evaluated the efficacy of such a schedule of temozolomide and determined the kinetics of MGMT depletion and O6-meG formation in DNA following treatment. Thirty patients with advanced malignant melanoma were treated with temozolomide 1,000 mg/m2 equally split into 5 doses over a 16 hr period every 28 days. O6-meG formation was determined in peripheral blood mononuclear cell (PBMC) DNA and, in a subset of patients, in tumor tissue during the first treatment cycle. MGMT levels fell rapidly with dosing, reaching a nadir in PBMCs of 18.0 +/- 2.26% of initial levels. O6-meG levels increased during the treatment period, peaking at 11.1 +/- 1.25 micromol/mol dG in PBMCs and at 4.25 +/- 0.79 micromol/mol dG in tumor biopsies. The main toxicities were grade IV thrombocytopenia in 12 patients (42.8%) and grade IV neutropenia in 11 patients (39.2%), associated with fever in 8 cases. There were 7 responses (1 complete), for an overall response rate of 23.3%; median overall survival was 6.1 months. The compressed schedule has activity against melanoma, with greater MGMT depletion and O6-meG formation than previously reported for O6-alkylating agent regimens. Myelosuppression precludes its wider application, but MGMT in PBMCs predicted the dose intensity of temozolomide that patients could sustain, suggesting a means by which individuals suitable for this approach might be identified.
    • O6-methylguanine-DNA methyltransferase in pretreatment tumour biopsies as a predictor of response to temozolomide in melanoma.

      Middleton, Mark R; Lunn, J M; Morris, Charles; Rustin, G; Wedge, S R; Brampton, M H; Lind, Michael J; Lee, Siow Ming; Newell, D R; Bleehen, N M; et al. (1998-11)
      Resistance of tumour cells to methylating and monochloroethylating agents in vitro and in vivo has been linked to levels of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). In a clinical trial of temozolomide in advanced malignant melanoma, the relationship between pretreatment MGMT levels in biopsies of cutaneous tumours and involved lymph nodes and clinical response to the drug has been studied. Among 50 evaluable patients, there were three complete responses (CR), four partial responses (PR), six with stable disease (SD) and 37 with progressive disease (PD), with an overall response rate of 14%. In 33 patients in whom MGMT level and clinical response could be evaluated, the tumour MGMT levels (fmol mg(-1) protein) were: CR, 158 +/- 119; PR, 607 +/- 481; NC, 171 +/- 101; PD, 185 +/- 42.3. Thus, measurements of pretreatment levels of MGMT in melanoma did not predict for response to temozolomide.
    • O6-Methylguanine-DNA methyltransferase inactivation and chemotherapy.

      Verbeek, Barbara; Southgate, Thomas D; Gilham, David E; Margison, Geoffrey P; Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. (2008)
      INTRODUCTION: Alkylating agents are frequently used in the chemotherapy of many types of cancer. This group of drugs mediates cell death by damaging DNA and therefore, understandably, cellular DNA repair mechanisms can influence both their antitumour efficacy and their dose-limiting toxicities. SOURCES OF DATA: This review focuses on the mechanism of action of the DNA repair protein, O(6)-methylguanine-DNA methyltransferase (MGMT) and its exploitation in cancer therapy and reviews the current literature. AREAS OF AGREEMENT: MGMT can provide resistance to alkylating agents by DNA damage reversal. Inhibition of tumour MGMT by pseudosubstrates to overcome tumour resistance is under clinical evaluation. In addition, MGMT overexpression in haematopoietic stem cells has been shown in animal models to protect normal cells against the myelosuppressive effects of chemotherapy: this strategy has also entered clinical trials. AREAS OF CONTROVERSY: MGMT inhibitors enhance the myelotoxic effect of O(6)-alkylating drugs and therefore reduce the maximum-tolerated dose of these agents. Retroviral vectors used for chemoprotective gene therapy are associated with insertional mutagenesis and leukaemia development. GROWING POINTS: The results of ongoing preclinical and clinical research involving various aspects of MGMT modulation should provide new prospects for the treatment of glioma, melanoma and other cancer types. AREAS TIMELY FOR DEVELOPING RESEARCH: Tissue- and tumour-specific approaches to the modulation of MGMT together with other DNA repair functions and in combination with immuno- or radiotherapy are promising strategies to improve alkylating agent therapy.
    • Obesity and cancer: pathophysiological and biological mechanisms.

      Renehan, Andrew G; Roberts, Darren L; Dive, Caroline; Department of Surgery, School of Cancer and Imaging Sciences, University of Manchester, Christie Hospital NHS Foundation Trust, Wilmslow Road, Manchester, UK. arenehan@picr.man.ac.uk (2008-02)
      Excess body weight (overweight and obesity) is characterized by chronic hyperinsulinaemia and insulin resistance, and is implicated both in cancer risk and cancer mortality. The list of cancers at increased risk of development in an "obesogenic" environment include common adult cancers such as endometrium, post-menopausal breast, colon and kidney, but also less common malignancies such as leukaemia, multiple myeloma, and non-Hodgkin's lymphoma. The pathophysiological and biological mechanisms underpinning these associations are only starting to be understood. Insulin resistance is at the heart of many, but there are several other candidate systems including insulin-like growth factors, sex steroids, adipokines, obesity-related inflammatory markers, the nuclear factor kappa beta (NF-kappa B) system and oxidative stresses. With such as diversity of obesity-related cancers, it is unlikely that there is a "one system fits all" mechanism. While public health strategies to curb the spread of the obesity epidemic appear ineffective, there is a need to better understand the processes linking obesity and cancer as a pre-requisite to the development of new approaches to the prevention and treatment of obesity-related cancers.
    • Obesity and hepatosteatosis in mice with enhanced oxidative DNA damage processing in mitochondria.

      Zhang, H; Xie, C; Spencer, H; Zuo, C; Higuchi, M; Ranganathan, G; Kern, P; Chou, M; Huang, Q; Szczesny, B; et al. (2011-04)
      Mitochondria play critical roles in oxidative phosphorylation and energy metabolism. Increasing evidence supports that mitochondrial DNA (mtDNA) damage and dysfunction play vital roles in the development of many mitochondria-related diseases, such as obesity, diabetes mellitus, infertility, neurodegenerative disorders, and malignant tumors in humans. Human 8-oxoguanine-DNA glycosylase 1 (hOGG1) transgenic (TG) mice were produced by nuclear microinjection. Transgene integration was analyzed by PCR. Transgene expression was measured by RT-PCR and Western blot analysis. Mitochondrial DNA damage was analyzed by mutational analyses and measurement of mtDNA copy number. Total fat content was measured by a whole-body scan using dual-energy X-ray absorptiometry. The hOGG1 overexpression in mitochondria increased the abundance of intracellular free radicals and major deletions in mtDNA. Obesity in hOGG1 TG mice resulted from increased fat content in tissues, produced by hyperphagia. The molecular mechanisms of obesity involved overexpression of genes in the central orexigenic (appetite-stimulating) pathway, peripheral lipogenesis, down-regulation of genes in the central anorexigenic (appetite-suppressing) pathway, peripheral adaptive thermogenesis, and fatty acid oxidation. Diffuse hepatosteatosis, female infertility, and increased frequency of malignant lymphoma were also seen in these hOGG1 TG mice. High levels of hOGG1 expression in mitochondria, resulting in enhanced oxidative DNA damage processing, may be an important factor in human metabolic syndrome, infertility, and malignancy.
    • Observations on the settling and recoverability of transplanted hemopoietic colony-forming units in the mouse spleen.

      Lord, Brian I; Hendry, Jolyon H; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, England (1973-03)
    • Occupational exposure in medicine--a review of radiation doses to hospital staff in north-west England.

      Hughes, J S; Roberts, G C; Stephenson, S K; National Radiological Protection Board, Chilton, Didcot, Oxon (1983-10)
      The personal monitoring service operated by the Regional Physics Department at the Christie Hospital and Holt Radium Institute, Manchester, monitors staff involved with the uses of ionising radiations at all hospitals and clinics administered by the North Western Region Health Authority in England. Monitoring results relating principally to exposure during 1981 have been collated and examined. The analysis indicates that the doses received by staff are for the most part very low and provide little reason for concern. The only area of work in which worthwhile and cost-effective dose reductions could probably be achieved is that involving the use of pre-loaded applicators in gynaecological intra-cavitary therapy. Some relatively high staff exposures result from the use of this technique, and very significant reductions in these doses are confidently expected from a programme which has now commenced for the increasing use of remotely-controlled after-loading equipment housed in shielded treatment rooms.
    • Oestrogens, Beatson and endocrine therapy

      Howell, Anthony; Clarke, Robert B; Anderson, Elizabeth (1997)