Mutagenesis, Vol. 17, No. 6, 483-487,
November 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Mechanisms of carcinogenicity/chemotherapy by O6-methylguanine
1 Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, Manchester M20 4BX, UK, 2 Max Delbrück Centre for Molecular Medicine, Robert Rössle Strasse 10, Berlin 13 092, Germany and 3 Centre for Occupational and Environmental Health, University of Manchester, Manchester M13 9PL, UK
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Abstract |
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 Top Abstract BackgroundMechanismsO6-meG in human DNAO6-meG in carcinogenesisO6-meG in chemotherapyReferences |
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Alkylating agents are a structurally diverse group of compounds that cause a wide range of biological effects, including cell death, mutation and cancer. DNA damaged by these agents contains widely different amounts of 12 alkylated purines/pyrimidines and two phosphotriester isomers. The biological effects appear to be mediated predominantly by attack at the O6 position of guanine. DNA extracted from various normal human tissues contains detectable levels of O6-alkylguanine, the source of which has not been defined. Given that, following DNA replication, this lesion cannot only generate point mutations but can also initiate mismatch repair-mediated DNA recombination and cell death, it seems worthwhile to consider the possible contribution of these events and cell killing to the aetiology of human cancer. There is increasing evidence that point mutations are not the only mechanism involved in malignant transformation by alkylating agents. Some cancer chemotherapeutic agents exploit the cytotoxic effects of O6-alkylguanine and an understanding of the processing of this lesion has allowed strategies to be developed that should increase the effectiveness of such agents.
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Background |
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TopAbstractBackgroundMechanismsO6-meG in human DNAO6-meG in carcinogenesisO6-meG in chemotherapyReferences |
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Alkylating agents are known or suspected human carcinogens whose biological properties and mechanism of action have been extensively explored in cell and animal model systems over several decades (Druckrey, 1967). As well as being carcinogenic in a wide variety of animal species, these agents are mutagenic, toxic, clastogenic and teratogenic (Magee et al., 1976
). The spontaneous or metabolically driven breakdown of these agents to alkylating species that interact with cellular macromolecules is well documented (Margison and O’Connor, 1979). Reaction with DNA bases produces 12 different nitrogen and oxygen adducts and reaction with the phosphate groups produces phosphotriesters: the amounts of the products are determined by the nature of the alkylating species (O’Connor et al., 1979; Margison and O’Connor, 1990). Many of these lesions have been found to be substrates for DNA repair proteins (Singer and Hang, 1997) and this has allowed significant progress to be made in establishing the relationship between specific DNA lesions and the biological effects of the agents responsible. Attention has focused on O6-alkylguanine and, to a lesser extent, O4-alkylthymine and 3-alkyladenine and the vast majority of work has been undertaken on the methyl version of these lesions. The biological effects of many of the other lesions, and higher alkyl versions, remain to be established.
Some of the earliest revelations that the toxic and clastogenic effects of alkylating agents can be a consequence of the formation of O6-methylguanine (O6-meG) in DNA were obtained following the isolation of a prokaryotic gene encoding the repair protein O6-alkylguanine-DNA alkyltransferase (ATase/MGMT). Transfer and expression of this gene into cultured mammalian cells that did not express endogenous repair activity was used to probe the role of the lesion. The miscoding properties of O6-meG had already been established in in vitro and in vivo polymerase fidelity studies (Saffhill et al., 1985) and it was perhaps not surprising that the overexpression of ATase conferred resistance to mutation induction by methylating agents (see for example Brennand and Margison, 1986; Samson et al., 1986). More surprising, however, was that ATase-expressing mammalian cells became more resistant to the killing and sister chromatid exchange- and chromosome aberration-inducing effects of methylating agents, showing that the lesion was also recombinogenic (see for example White et al., 1986).
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Mechanisms |
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TopAbstractBackgroundMechanismsO6-meG in human DNAO6-meG in carcinogenesisO6-meG in chemotherapyReferences |
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Despite these indications that a single DNA adduct such as O6-meG can be responsible for a wide variety of biological effects, the precise molecular mechanisms of recombination and toxicity remain to be defined, whilst that of point mutations is well established.
G
A transition mutations arise following two rounds of replication of DNA containing O6-meG (Figure 1). In vitro experiments indicate that C and A can be incorporated opposite O6-meG (Loechler et al., 1984; Saffhill et al., 1985) and the frequency of this in intact cells has been examined using shuttle vector systems (Altshuler et al., 1996). The efficiency of miscoding indicates that the normal partner, cytosine, is incorporated preferentially and such measurement should be extended to a wide range of human cell types in order to better assess the risk posed by the presence of the lesion in DNA.
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Both recombination and cell death are dependent upon a functional mismatch repair (MMR) system. The hMSH2–hMSH6 heterodimer of the MMR system (mutS
) initially recognizes the O6-meG:T mispair formed following one round of replication of DNA containing O6-meG. Other components of the MMR system are recruited and a long patch (>~1 kb) of the newly replicated strand is removed. The mispaired thymidine is thus excised, only to be replaced during repair synthesis (involving polymerase 
or 
and DNA ligase III or IV) by another thymidine residue. This results in a region of DNA where one strand is constantly being excised and replaced, and this is referred to as a ‘futile’ repair cycle (Figure 1
; Branch et al., 1993).
How MMR is causally connected to apoptotic cell death and recombination has not been established, although mouse models indicate that the apoptotic response is mediated through p53 (Toft et al., 1999). One possibility is that double-strand breaks are produced when O6-meG residues are present on opposite DNA strands and the distance between the residues is short enough to allow the single-stranded regions of DNA, resulting from excision of the newly replicated strand by MMR, to overlap. An alternative possibility is that since another round of DNA replication seems to be required for both death and recombination, some of the MMR single-strand intermediates are replicated (in the absence of cell cycle arrest) and that this results in a double-strand break in one of the daughter strands (Figure 1). It is well established from the effects of ionizing radiation that double-strand breaks are lethal, only 40 being required to kill a cell (Ward, 1981), although, again, the mechanism of this has yet to be defined.
Double-strand breaks are processed by the recombination repair system, of which there are two pathways (Jackson, 2002). One of these, homologous recombination (HR), requires the Rad51, Rad52 and Rad54 gene products and may occur mainly in the late S–G2 stage of the cell cycle. The other, non-homologous end joining (NHEJ), is dependant either on the Ku70/Ku80/DNA PK complex, which appears to be the main pathway in invertebrates, acting in the G1–early S phase, or on the P95 (Nibrin, Nbs1)/Mre11/Rad50 complex. However, many other factors appear to be involved in recombination pathways, including Flap endonuclease 1, ligase IV, XRCC4, p53 and the products of the Fanconi’s anaemia, Cockayne’s syndrome (CSA and CSB), Werner’s syndrome, Bloom’s syndrome and other genes (Bernstein et al., 2002). The molecular interactions occurring during these repair processes are highly complex and much has yet to be resolved. NHEJ and HR are likely to result in different phenotypic changes and in the case of HR this will also depend on the choice of recombination partners, i.e. whether intra- or inter-allelic recombination events occur.
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O6-meG in human DNA |
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TopAbstractBackgroundMechanismsO6-meG in human DNAO6-meG in carcinogenesisO6-meG in chemotherapyReferences |
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Very widely varying levels of O6-meG have been detected in human DNA in several studies of various populations (Kyrtopoulos, 1998
; Povey, 2000). Although this may be a consequence of exposure either to known methylating agents that are present in cigarette smoke or the diet or to unknown exogenous agents, endogenous processes also contribute to DNA alkylation (Marnett & Burcham, 1993; Hecht, 1999). Endogenous processes might include the ‘aberrant’ methylation of guanine by S-adenosylmethionine and the endogenous nitrosation of compounds containing primary amino groups and their subsequent breakdown to methylating species (Sedgwick, 1997). An example of this is the nitrosated bile acid n-nitrosoglycocholic acid that has been shown to be able to methylate DNA in vitro and in vivo (Shuker and Margison, 1997). However, the contribution of this or indeed any other nitroso compound to endogenous damage requires further investigation.
The levels of O6-meG in cancer patients DNA following treatment with methylating antitumour agents are orders of magnitude greater than the levels seen in untreated individuals (Povey, 2000). Many chemotherapy treatment regimes, not least those that include methylating agents, are carcinogeneic in patients surviving long term. In the case of patients with lymphoma, the incidence of acute myeloid leukemia is as high as 20% in patients treated with a regimen that includes alkylating agents (Karp and Smith, 1997), although the contribution of other components of the regime cannot be ignored.
The DNA repair protein that removes this lesion from DNA, ATase, has also been found in varying amounts in many human normal and tumour tissues. In rodents, ATase can be up-regulated by a variety of genotoxic insults (Saffhill et al., 1985). Whilst there is some indication that this may also be the case in man, further studies are required to establish whether this might be an approach to attenuating the biological effects of alkylating agents in normal tissues or enhance them in tumour tissues (see also below). It seems reasonable to suggest that ATase has evolved to protect organisms against the biological effects of alkylating agents and is conserved for the same reason. Whether or not the conservation is to protect against toxicity or mutagenicity is open to debate.
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O6-meG in carcinogenesis |
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TopAbstractBackgroundMechanismsO6-meG in human DNAO6-meG in carcinogenesisO6-meG in chemotherapyReferences |
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There are several mechanisms that could link the presence of O6-meG residues in human DNA to carcinogenesis. Most attention has been focused on the point mutational effects, and the possible contribution of recombination seems to have been largely ignored. There is also experimental evidence to suggest that the toxic effects of this lesion could play a major role in carcinogenesis.
Point mutations
The relationship between carcinogenesis and alkylation damage-mediated GA transition mutations is supported by several lines of evidence. One is the spectrum of tumour-associated mutations that are found in genes that are crucial for malignant transformation. These include the H-ras oncogene, in which transition mutations have been reported in codons 12, 13 and 61 (Barch et al., 1991; Rumsby et al., 1991) and the TP53 tumour suppressor gene, where transitions are found in a number of locations (Ohgaki et al., 1992). These mutations are dependent on exposure to alkylating agents (Sukumar et al., 1983) and the absence or lack of ATase activity (Mitra et al., 1989; Esteller et al., 2001). In one study, colorectal adenomas that contain ras GCAT mutations had significantly lower ATase levels than the surrounding tissue and ATase activity in adenomas without this mutation were similar to surrounding normal tissue (Povey et al., 2002). However, there is also evidence that in mice, cells with pre-existing ras mutations may be present in target tissues (see also below) and alkylating agents can induce tumours without, for example, K-ras GCAT mutations.
Another line of evidence comes from transgenic murine models. ATase over-expressing mice are more resistant and ATase null mice are more susceptible to carcinogenesis by methylating agents (Sakumi et al., 1997; Kawate et al., 1998). However, since the phenotypes of these animals would also affect recombination, it is difficult to eliminate this as a contributing factor (see below). On the other hand, mice lacking both ATase and functional MMR are comparatively resistant to alkylation toxicity but are more prone to develop cancer in response to alkylation damage than ATase-deficient but MMR-proficient animals (Kawate et al., 1998, 2000). It should be noted, however, that the increase in the frequency of tumours is expected since lack of MMR activity on its own predisposes to cancer.
Recombination
The contribution of the recombinational pathway to carcinogenesis by O6-meG has not been extensively investigated. Since recombination depends on the action of the MMR system, as does the toxicity of O6-meG, it is difficult to separate its role from that of the selective pressures generated by toxicity. NHEJ will lead to deletions and translocations (Jackson, 2002). These aberrations can result in the activation of oncogenes and in the deletion of tumour suppressor genes. Recombination between homologous chromosomes can result in loss of information from one of the homologues and render the cell homozygous for an inactive tumour suppressor. Such a mechanism is consistent with the observation that malignant transformation of a human fibroblast cell line with nitrosomethylurea (NMU) was accompanied by inactivation of both TP53 alleles by the same mutation, suggesting a role for homologous recombination (Boley et al., 2000).
Toxicity
The toxic effects of alkylating agents will result in the elimination of cells with, presumably, the highest levels of DNA damage. However, the consequence of this is that sub-lethally damaged cells that, in spite of the presence of O6-meG in their DNA, survive and undertake restorative hyperplasia will replace the dead cells in the tissue and become the predominant population. The impact of this on genome stability is likely to be substantial. Thus, in a repair-deficient Chinese hamster cell line it was estimated that per cell ~7000 O6-meG residues were required for a lethal event, whilst only ~8 were required for a mutation and ~40 for a recombination event (Rasouli-Nia et al., 1994). A sub-lethally damaged cell may thus contain up to 1000 mutations. Whilst this type of study still needs to be carried out in a number of cell types, it suggests that the DNA in surviving cells will have undergone a substantial number of mutations and also recombinational events.
Cells in which the MMR system is attenuated or non-functional are relatively resistant to the toxic effects of alkylation damage and, in fact, in vitro exposure to methylating agents has been used to derive MMR-deficient cell lines. Loss of MMR is associated with a mutator phenotype and the resulting genome-wide increase in mutation rate is believed to predispose to cancer (e.g. by inactivation of a caretaker gene) (Kinzler and Vogelstein, 1997). In man, inheritance of one inactive allelle of certain of the components of the MMR system is associated with a tumour-prone condition that is characterized by an increase in frequency of tumours in organs such as colon and uterus. In the tumours of individuals with such cancer-prone conditions the second allele is inactive and MMR activity is lost. This suggests that tumours that have arisen through alkylation damage may tend to lose MMR.
There is, however, no experimental evidence to suggest that exposure to exogenous alkylating agents is associated with MMR-deficient tumours. It may be that tumours promoted directly by the mutagenic effects of alkylating agents predominate over those arising via an indirect effect through the abolition of the MMR system. It may be that in most cases where exposure to alkylating agents contributes to tumour formation the genetic lesions arising directly from the mutagenic effects of these agents, be they point mutations or recombinations, are sufficient to promote malignant transformation, so that there is little selective pressure favouring loss of MMR activity. An alternative explanation is that lack of MMR is carcinogenic because it abolishes a pathway leading to apoptosis (Tomlinson et al., 1996; Toft et al., 1999; Tomlinson and Bodmer, 1999). Thus, cells lacking MMR are more likely to become malignant not because they are mutation prone, but because they do not die so readily. However, MMR is only the initial factor in the pathway that mediates cell death as a response to the presence of O6-meG. This lesion results in an increase in the selective pressure to lose any of the downstream components of this pathway. At the same time this would increase the frequency of point mutations (see above) and, hence, the probability of generating the appropriate mutants. Such a mechanism is consistent with the observation that mammary tumors in mice exposed to NMU seem to arise by selecting cells with pre-existing GGAGAA mutations in codon 12 of the H-ras gene (Cha et al., 1994). Studies of this type need to be extended to other carcinogenesis models so that a more complete understanding of the events involved in cancer induction can be obtained.
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O6-meG in chemotherapy |
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TopAbstractBackgroundMechanismsO6-meG in human DNAO6-meG in carcinogenesisO6-meG in chemotherapyReferences |
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As it becomes increasingly evident that O6-meG plays a major role in the antitumour effects of clinically used DNA methylating agents, such as dacarbazine (DTIC), streptozotocin, procarbazine and temozolomide, strategies are being developed to exploit this in order to improve the effectiveness of such treatment. The main approach is based on the observation that many tumours express high levels of ATase, making them resistant to this type of chemotherapy. Low molecular weight pseudosubstrate inactivators of ATase (Pegg et al., 1995
; Middleton et al., 2000) increase the persistence of O6-meG in tumour DNA so that the MMR system operates more efficiently to kill greater numbers of tumour cells (Baer et al., 1993; Middleton et al., 2000). Phase I clinical trials of such agents are underway and the results of phase II trials are awaited with interest. In the case of the chloroethylating agent carmustine, in phase I clinical trials the effect of the introduction of an ATase inactivating agent was to considerably reduce the maximum tolerated dose of carmustine, while recent phase II trials of this combination showed no clinical benefit (Quinn et al., 2002). It remains to be seen if the preclinical promise of this strategy is borne out in patients treated with methylating agents and ATase inactivators.
Another line of attack is to enhance the removal of the lesion from the DNA of bone marrow cells, in which the dose-limiting toxic effects of methylating agents are manifest. This involves the in vitro transduction of harvested bone marrow cells using a retrovirus that contains an ATase cDNA. In order to combine the pseudosubstrate and gene therapy approaches, versions of the human ATase cDNA that encode inactivator-resistant mutant ATase protein have been used. These approaches, which have been shown to be successful in murine models (Chinnasamy et al., 1998), are approved for imminent clinical trials (Gerson, 2002).
Whilst these strategies should increase tumour response and reduce collateral toxicity, it remains to be seen if the patients given such therapies survive long term and, if so, whether there is an increased risk of iatrogenic cancers (Andersen et al., 1998) that might be attributed to more persistent O6-meG in other tissues.
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Acknowledgments |
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M.S.K. would like to thank Prof J.G.Reich for the opportunity to visit his department during the completion of this review. G.P.M. gratefully acknowledges the support of Cancer Research UK. A.C.P. thanks the British Lung Foundation and the Association for International Cancer Research for their support.
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Notes |
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4 To whom correspondence should be addressed. Email: gmargison{at}picr.man.ac.uk
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Received on July 1, 2002; accepted on August 5, 2002.
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