Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1996-09-16
1999-03-09
Eisenschenk, Frank C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435193, 435975, 436518, 436530, 53038826, G01N 33577, C12N 910, C07K 1640
Patent
active
058798994
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to methods of assaying human O.sup.6 -methylguanine-DNA methyltransferase (MGMT) and/or active site alkylated derivatives (R-MGMT) thereof.
Human MGMT is an important DNA repair enzyme which can transfer alkyl groups from O.sup.6 -alkylguanine and O.sup.4 -alkylthymine residues in DNA to a cysteine residue (Cys 167) at the active site of the enzyme. Such alkylated nucleotide residues, which can give rise to transition mutations in DNA sequences by virtue of their miscoding behaviour, can be produced by carcinogenic alkylating agents such as N-nitroso compounds, e.g. N-methylnitrosourea (NMU) and N-ethylnitrosourea (NEU) and N,N-dialkylnitrosamines.
Various human genes associated with tumour formation have been found to carry point mutations, for example ras, p53 and ERCC-3. These mutations are widely believed to be crucial in the activation (oncogenes) or suppression (tumour suppressor genes) of these genes and much evidence supports point mutations arising through the action of alkylating agents on DNA being involved in the etiology of certain human cancers. Thus, it has been shown that a single dose of NMU produces mammary tumours in rats as a result of a G.C to A.T transition mutation in codon 12 of the rat H-ras oncogene. This point mutation can also be generated in vivo by substituting O.sup.6 -methylguanine for guanine in DNA. As O.sup.6 -methylguanine is one of the alkylated nucleotides formed by action of alkylating carcinogens on DNA and it can produce the G.C to A.T transition mutation, it is thought to be involved in the formation of mammary tumours in rats exposed to NMU (Zarbl et al. Nature (1985) 315, 382-385; Mitra et al. Proc. Natl. Acad. Sci. USA (1989) 86, 8650-8654).
N,N-dialkylnitrosamines are environmental alkylating agents and have also been shown to be potent tumour inducers in experimental animals (Magee and Barnes Br. J. Cancer, (1956) 10, 114-122). Hence, these chemicals and other alkylating agents which react by the SNI mechanism are considered likely causative agents of human cancers through their ability to produce mutagenic DNA base adducts in vivo (Bartsch et al eds., Relevance of N-Nitroso Compounds to Human Cancer: Exposures and Mechanisms, IARC Scientific Publication no. 84 (1987): International Agency for Research on Cancer, Lyon, France; O'Neill et al eds., Relevance to Human Cancer of N-Nitroso Compounds, Tobacco and Mycotoxins. ibid. no.105 (1991); P. D. Lawley, in Chemical Carcinogens Chapter 7, vol I, Ed. C. E. Searle, ACS Monograph 182, American Chemical Society, Washington, (1984); Vogelstein and Kinzler Nature (1992) 335, 209-210).
Since MGMT can specifically repair these mutagenic lesions in DNA produced by alkylating agents, the level of this enzyme in cells is believed to be a crucial factor in determining the sensitivity of cells towards the tumour-inducing mutagenic action of alkylating carcinogens. (Lindahl et al, Annu. Rev. Biochem. (1988) 57, 133-157; A. E. Pegg, Cancer Res. (1990) 50, 6119-6129). In support of this, it has recently been observed that transgenic mice carrying the human gene for MGMT, targeted for expression in T-cells, exhibit a lower incidence of thymic lymphomas when treated with NMu compared with control non-transgenic mice similarly treated (Dumenco et al. Science (1993) 259, 210-222). Furthermore, MGMT-deficient cell lines have been shown to be highly susceptible to the cytotoxic and mutagenic action of SN1 type alkylating agents (D. B. Yarosh Mutation Research (1985) 145, 1-16; A. E. Pegg, ibid.).
Cellular MGMT also has implications for tumour treatment with chemotherapeutic alkylating agents such as the bifunctional chloroethylating agent 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and other chloroethylnitrosoureas, e.g. 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). The chemotherapeutic effect of such drugs depends upon formation of lethal DNA cross-linking base adducts which can be effectively removed by the suicidal repair action of MGMT. Thus, tumour cell lines which are devoid of this enzyme
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Eisenschenk Frank C.
National University of Singapore
Rabin Evelyn
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