Carcinogenesis, Vol. 24, No. 4, 625-635,
April 2003
© 2003 Oxford University Press
COMMENTARY |
Variability and regulation of O6-alkylguanine–DNA alkyltransferase
1 Cancer Research Uk Carcinogenesis Group, Paterson Institute for Cancer Research, Manchester M20 4BX, UK
2 Centre for Occupational and Environmental Health, University of Manchester, Manchester M13 9PL, UK
3 Institute of Toxicology, Division of Applied Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany
4 Max Delbrück Centre for Molecular Medicine, Robert Rössle Str. 10, Berlin 13 092, Germany
To whom correspondence should be addressed Email: gmargison{at}picr.man.ac.uk
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Abstract |
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 Top Abstract IntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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O6-Alkylguanine–DNA alkyltransferase (ATase) confers resistance to many of the biological effects of certain classes of alkylating agents by repairing the DNA lesions responsible. The role of ATase in the mutagenic and toxic effects of the carcinogenic and antitumour alkylating agents are of interest in relation to the prevention and treatment of cancer in man. In this commentary we specifically focus on the variation in ATase levels and our current understanding of the factors involved in the regulation of ATase expression.
Abbreviations: O6-meG, O6-methylguanine; SAM, S-adenosylmethionine
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Introduction |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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The most important biologically significant DNA lesions produced by both carcinogenic and chemotherapeutic alkylating agents such as nitrosamines, nitrosoureas and related compounds, under most circumstances, are O6-alkylguanine adducts. The ability of these adducts to pair with thymine instead of cytosine during DNA replication is responsible for the increase in the frequency of transition mutations following exposure to alkylating agents, and determines their mutational spectrum. The toxic and recombinogenic effects of O6-alkylguanine lesions, in particular the methyl version, is determined by the action of the post-replication mismatch repair system on O6-methylguanine (O6-meG):T mispairs, although the precise mechanisms of these effects remain to be established.
The DNA repair protein O6-alkylguanine–DNA alkyltransferase [ATase; also known as AGT, AGAT and MGMT (O6-methylguanine–DNA methyltransferase; E.C.2.1.1.63)] reverses O6-alkylation damage in DNA simply by removing the offending alkyl group by covalent transfer to the ATase protein, inactivating it and targeting it for ubiquitination and proteasome-mediated degradation. The mechanisms by which the O6-alkylating agents exert their toxic and mutagenic effects and the role of ATase in protecting pro and eukaryotic cells against these effects have been the subject of several recent reviews (1–5).
This review focuses on the patterns of expression of ATase, the possible mechanisms and factors involved, and their potential relevance for cancer prevention and treatment.
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Alkyltransferase genes |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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Several bacteria, including Escherichia coli possess two proteins with alkyltransferase activity. One of them (encoded by ogt) is constitutively active, whereas the other (encoded by ada) is inducible by exposure to low, non-toxic doses of methylating agents. This results in an increase in resistance to subsequent toxic challenges, a process known as the adaptive response. The mechanism of this is well-defined: in addition to containing an ATase domain the Ada protein also has a phosphotriester alkyltransferase domain. Methyl group transfer to this domain activates the ada protein into a transcription factor that upregulates expression of ada and other genes (6). Homologues of ogt are found in eukaryotes. Recently Caenorhabditis elegans has also been shown to have two ATase genes: one similar to that of other eukaryotes and a second, more distant homologue that acts on O6-meG in DNA, although a substitution disrupts the conserved PCHR motif surrounding the active site cysteine residue (7). Intriguingly the C-terminal half of this protein has some similarity to histone 1C (7).
In silico analysis of genomic sequences suggest the presence of another family of ATase homologues in some prokaryotes and lower eukaryotes. Many of them are annotated as O6-meG–DNA methyltransferases but the acceptor cysteine is substituted by a tryptophan. Thus, in several of these sequences such as the ybaz open reading frame in E.coli, the PCHR motif is replaced by PWHR. Figure 1 summarizes the relationship between the ATases and their ATL homologues. It will be interesting to examine the role, if any, of these proteins in the biological effects of alkylating agents.
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The human ATase gene is located on chromosome band 10q26. There are five exons spanning nearly 300 kb of genomic DNA: the last four exons are coding, and the second intron is particularly large (170 kb). The promoter covers the first exon and part of the first intron, it lacks TATA or CAAT boxes, contains CG-rich regions and is reminiscent of that of housekeeping genes. There are Sp1, AP-1 and AP-2 sites, two glucocorticoid responsive elements and a 59 bp element, located at the first exon–intron boundary, that is required for efficient transcription of reporter constructs (see below). The transcript is
0.95 kb long: no splice variants have been described (8). The half-life of the mRNA, as estimated in different cell lines, is 10 h (9,10). The encoded peptide has 207 amino acids and a molecular weight 24 kDa. |
Physiological role of ATase |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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Inactivation of ATase genes in yeast or E.coli results in viable organisms that have an increase in the rate of spontaneous mutations, suggesting a role in protecting against endogenous methylation damage in DNA. In addition, overexpression of ATase genes in E.coli protects against the toxic and mutagenic effects of exogenous O6-alkylating agents (e.g. ref. 11). In mammalian cells, ATase is again not essential for viability but ATase-deficient cells can be made to be more resistant to the effects of endogenous and exogenous alkylating agents by ATase cDNA (reviewed in refs 4,12). Inactivation or inhibition of the ATase protein in cells expressing the endogenous gene in turn makes them more sensitive to such agents (reviewed in ref. 13).
A recent report suggests that, following its action on methylated DNA, methylated ATase can bind to the oestrogen receptor and inhibit its function as a transcription factor in cell proliferation (14). This acquisition of a new functional activity following methylation bears some resemblance to the effect of repair-mediated methylation of the Ada protein in E.coli (see above). However, the physiological importance of such a mechanism remains to be established.
ATase null mutant (knockout) mice are viable, fertile, outwardly normal and have a normal lifespan, indicating once more that ATase is a non-essential gene (15). Such mice are, however, more susceptible to the biological effects of exogenous alkylating agents. On the other hand, human ATase-overexpressing transgenic mice derived from a strain prone to develop spontaneously hepatocellular carcinoma, show a reduced frequency of liver tumours as compared with controls (16). In addition, a reduction of the number of spontaneous tumours and longer survival times has been reported in ada transgenic mice (17).
ATase overexpression also protects against the effects of O6-alkylation of guanine by exogenous mutagenic and carcinogenic agents. Thus, in carcinogenesis experiments with thymus, liver and colon as targets (18–20) and in the mouse skin two-stage initiation–promotion model (21,22). ATase overexpression significantly reduced tumour formation upon exposure to methylating and chloroethylating agents.
In contrast to the protective effects against O6-alkylating agents, ATase has been shown to increase the toxic and mutagenic effects of dihaloalkanes (DHA) such as dibromoethane (DBE) in E.coli and mammalian cells that are compromised for nucleotide excision repair (23–25). The toxicity is mediated by ATase by its direct interaction with DBE metabolites (26). If DHA are generated endogenously, high levels of ATase activity might be a disadvantage in terms of cell survival.
These observations indicate that the normal physiological role of the ATase protein is the protection of cells against the potential adverse effects of DNA alkylation damage at the O6-position of guanine generated by endogenous alkylating species. As overexpression of ATase can provide protection against the effects of such endogenous damage, the constitutive levels of expression must be insufficient to deal with such damage under all circumstances. It is therefore important to consider what the sources of this alkylation damage might be.
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Endogenous DNA alkylation |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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In man, low but measurable amounts of O6-alkylG are found in the DNA of both normal and tumour tissues (see Figure 2). O6-meG levels in DNA have been reported to be inversely related to ATase levels in some but not all studies (27,28). The papers quoted have used either treated patients or subjects with known increased exposure. The presence of O6-alkylG in DNA probably reflects a steady state between its formation and repair, although the possible accumulation of damage in individuals has not been assessed. Other possibilities are that damage or repair is inter- or intra-cellularly compartmentalized, leading to accumulation in certain compartments, or cyclical.
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Endogenous sources of DNA alkylation damage have not been unequivocally identified, but are probably nitrosated amino or bile acids that decompose spontaneously, or after the action of metabolizing enzymes, to alkylating species (29,30). The nitrosation of primary amines via arginine-dependant formation of nitric oxide is promoted by some cell type, for example pleural mesothelial cells, neutrophils and macrophages. Also, isolated rat hepatocytes release nitrite via the action of induced nitric oxide synthetase.
The methyl group donor, S-adenosylmethionine (SAM) may also be a source of endogenous O6-meG in DNA: SAM has been shown to generate 7-methylguanine in DNA (31,32) by ‘aberrant’ DNA methylation. Given the reaction mechanism, the amounts of O6-meG produced by SAM would be expected to be very small in relation to 7-methylguanine, however, its contribution to endogenous damage cannot, at present, be discounted.
As well as the endogenous sources described above, exogenous sources of alkylation damage are well established and these include cigarette smoking, occupation and diet (33). The relative amounts of exogenously and endogenously induced alkylation damage remain to be determined and it is difficult to see how this would be assessed. It seems reasonable to speculate that factors that can stimulate the formation of endogenous damage, even though they might not be alkylating agents, or indeed genotoxins, are likely to increase cancer risk, unless they result in a concomitant increase in the expression of ATase (see below) and/or other factors that can attenuate the biological effects of such damage.
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Variation in ATase expression levels |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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Cultured cells
Cultured mammalian cells express very widely varying levels of ATase, probably reflecting the ATase activity in their tissue of origin. The activity can closely correlate with their degree of sensitivity to alkylating agents (35). Cell lines in which ATase levels are not detectable have been referred to as methyl excision repair (mer) minus, based on an original assay for host cell reactivation of methylated adenovirus (35) or methyl excision (mex) minus based on survival following exposure to methylating agents (36). However, these definitions do not reflect the variability in expression and sensitivity.
Whilst the majority of cells in newly established lymphoblastoid cell lines show activity (37,38), a substantial proportion of older lymphoblastoid cell lines lack ATase expression (39). Indeed, many immortalized cell lines do not express detectable levels of ATase, immortalization using SV40 (40,41) often being accompanied by loss of activity. However, the appearance of ATase-expressing cells in an ATase negative lymphoblastoid cell line suggests that loss of expression can be reversible (42,43): this might be attributed to promoter methylation (see below), but many other factors might also be involved.
Earlier reports using cultured mammalian cells suggested that there was a significant reduction of MGMT levels and activity prior to, or early in, the S-phase (44–46). However, more recently, it has been shown that there is no variation of ATase message levels during the cell cycle in a variety of mammalian cell lines (47). On the other hand, the state of confluence of cell cultures can modulate ATase expression (48) (see below).
Mammalian tissues
In intact organisms, the wide variation in ATase activity is well documented: there are differences in activity between tissues within an individual, between individuals within one tissue and between cells within a tissue. Expression is usually the highest in liver, followed by colon and lung and is relatively low in brain and myeloid tissues (49). A similar pattern of expression has been described in rat and mouse (50). In man, the tissue-related variation is also overlaid by a large range of interindividual variability (see Figure 3).
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The possible biological bases of these variations have not been widely studied. However, two parameters that have been examined for their potential impact are age and circadian rhythms. ATase activity in paediatric brain tumours was highest in those aged 3–12 years (51) but an inverse correlation between age and ATase expression has been reported in the normal brain tissues, and in the tumours, of patients with cancer of the brain (52–54), whereas for ovarian tumours age-dependence was not found (55). Other studies with normal tissue have also failed to identify age-related differences in ATase expression levels, e.g. in lung (56) or lymphocytes (53). Most studies have reported no relationship between ATase and gender, age and alcohol consumption, whilst for smoking, diet and medication, associations are more frequent (Table I).
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Diurnal variation in ATase activity in mononuclear blood cells has recently been reported (57): the extent of variation differed between individuals but was on average
30%, with a peak around midnight. Variation in ATase levels have also been reported in rat mammary epithelial cells during the estrous cycle (58). |
Pathological conditions |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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Because of its relevance to chemotherapy, there have been numerous investigations of ATase expression levels in normal and tumour tissues of cancer patients. Relative to adjacent normal tissue, increased activity has been observed in tumours of the breast (59–62), brain (54) and colon (63,64) (see Figure 3). Activity levels in tumours are more variable than in normal tissues and individual tumours can sometimes be devoid of ATase activity. The proportion of gliomas lacking ATase expression is comparatively high and it is likely that this is the basis of their treatment with O6-alkylating agents (65).
Immunohistochemical analysis reveals that within tumours, regions with and without ATase can be found and different metastases can also display different levels of expression (66). Such variation may explain the lack of correlation between ATase activity in tumour biopsies from melanoma patients and their response to Temozolomide therapy (67). However, it is clear that ATase is only one of many factors that determine the response of tumours to such therapy.
Another pathological condition in which ATase expression levels have been examined is hepatitis. In chronic hepatitis there is a 2.5-fold increase in mRNA levels irrespective of the aetiology, whilst in cirrhotic liver mRNA levels are attenuated (68,69). Acute hepatitis B infection has been reported to result in substantial changes in the intracellular distribution of ATase with the protein present in the cytoplasm, rather than the nucleus, in regions of affected livers (70).
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Induction of ATase expression in response to exogenous agents |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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In rodents, particularly rat, a number of factors have been found to be able to upregulate expression of the ATase gene (71,72). Inducers include not only alkylating agents, but also ionizing radiation and other genotoxins and partial hepatectomy. The increase in ATase activity can be explained by transcriptional stimulation of the ATase gene, as demonstrated by RNA synthesis inhibition experiments (10) and by transfection of reporter constructs containing the ATase promoter (73,74). It should be noted that the extent of induction is rather modest: generally 5-fold at most in rats and 2–3-fold in mice, but usually less than this. In a study with adult rats treated with 2-acetylaminofluorene, northern analysis indicated a
10-fold induction of ATase mRNA whereas activity was induced only 4-fold (75). The peak of induction is, depending on the model, between 24 and 48 h after exposure to the inducing agent, suggesting that a cascade of events is required to affect the response. Growth phase may also affect response since, at least for rat cells subjected to ionizing radiation, induction is less pronounced in confluent than exponentially growing cells (76). In rat liver cells in vitro, induction was related to the state of differentiation as revealed by TAT expression suggesting again cell type-specific factors to be involved (77). ATase modulation has been extensively studied in rodent cells and tissues and it is worth noting that agents that are effective inducing agents in rat issues are not always effective in mouse and other rodents. Neither is the magnitude of induction the same in mouse and rat tissue using agents that are effective in both. This suggests that the mechanisms may be different in these species and that it might be unsafe to extrapolate from rodents to man.
The mechanism of ATase upregulation has yet to be defined and the diverse spectrum of inducing agents suggests that different factors may be operating. However, the possibility of a common feature for all agents, with the possible exception of hormones that are likely to operate through hormone responsive elements in the promoter (see below), cannot be excluded. It has been speculated that this common factor might be DNA double-strand breaks (dsb) particularly as the introduction of restriction endonucleases into cells can also upregulate expression (78,79). Experiments using H4 rat hepatoma cells treated with 3-aminobenzamide suggest that inhibition of poly (ADP-ribose) polymerase can enhance the induction of ATase activity by MNNG (80) and one possible explanation for this might be an increase in the frequency of DNA dsb.
The data for cultured human cells is much less extensive, suggesting that many human cell types might not be capable of ATase upregulation. Thus, human diploid fibroblasts and HeLa cells do not upregulate ATase following genotoxin treatment although they do express the protein (10). Also, irradiation of human lymphocytes with gamma-rays does not induce ATase expression (Figure 3B) in contrast to their effect in rat tissues. There is however, a small but significantly higher level of ATase activity in the lungs (81), placentae (82) and in lung tumours (83) of smokers as compared with non-smokers. Interestingly, the level in lymphocytes of smokers is not enhanced (see Figure 3B) suggesting a cell type specificity of this phenomenon. Whether or not this is due to DNA damage-mediated induction of ATase expression, or selection of cells expressing higher levels of ATase has not been addressed. There have also been reports that ATase expression is attenuated in the lymphocytes of patients undergoing surgery (84), embalmers (85) and of clinical workers handling cancer chemotherapeutic agents (86).
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Mechanisms underlying inherent variability |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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There are a number of factors that are known to influence the activity and the levels of ATase protein expressed in mammalian tissues and in cultured cells. These are described below and summarized in Figure 4.
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Polymorphisms and other changes at the genomic level |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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The extent of interindividual differences in ATase expression levels has led to the search of polymorphisms that might affect activity and a number of base changes have been reported in the human gene: Table II shows the most informative single nucleotide polymorphisms within the transcribed region. So far, interest has focused on those resulting in amino acid substitutions, their effect on the activity of the coded protein and the possible association with cancer. Initially the G160 allele attracted considerable interest because of a possible association with cancer susceptibility (87) and its resistance to the ATase inhibitor O6-benzylguanine (88). However, other studies suggested that this allele is very rare (89) and failed to confirm the association with cancer (90). Gene transfer studies in bacteria and mammalian cells suggest that the protein coded by another allele, W65 is unstable (91) and its impact remains to be examined. Recently an association between allele V143 and lung cancer has been reported (90). This change affects the isoleucine residue close to the alkyl acceptor cysteine residue at position 145. There have been no studies investigating if there is an association between genotype and ATase activity levels in different individuals, although substitutions affecting elements within the ATase promoter have been reported (92).
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Deletions and other chromosomal aberrations do not seem to be a frequent cause of the lack of ATase activity in tumours and immortalized cell lines. However, glial tumours do often show loss of heterozygosity in 10q26 (93) suggesting that the lack of one allele may contribute to changes in activity levels. There has been only one report describing ATase mutations and deletions and this was in a substantial proportion of tumours of the oesophagus (94).
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Transcription factors |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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Experiments using a 1.2 kb 5'-fragment of the ATase gene with promoter activity showed a 1000-fold variation in potency between different cell lines. The basis of the variation was not defined; however, the levels of the ubiquitously expressed Sp1 transcription factor did not correlate with transcriptional activity (8). Transfection of c-Fos or c-Jun expression plasmids into F9 cells expressing very low levels of AP-1 increased the expression of ATase reporter constructs and the effect was dependent on the integrity of one of the AP-1 sites in the promoter (95). This is consistent with the observed binding of AP-1 to the ATase promoter fragment and an increase of ATase mRNA levels by protein kinase C activators such as diacylglycerol and 12-O-tetradecanoylphorbol-13-acetate (TPA) or by inhibitors of protein phosphatases such as okadaic acid. This indicates that the transcription factor, AP-1 is involved in ATase regulation (95). TPA stimulation of HeLa cells resulted in a 4-fold increase in ATase mRNA levels which, though modest, was sufficient to elicit an increased resistance to the toxic effects of the O6-alkylating agent, BCNU (95). Similarly, a role for the glucocorticoid response elements (GRE) was shown by dexamethasone treatment of rat H4IIE and HeLa S3 cells which increased the expression both of reporter constructs and ATase mRNA levels (73). Footprinting assays demonstrated the binding of the glucocorticoid receptor to the GREs within the ATase promoter. Dexamethasone stimulation, although only 2-fold, was sufficient to increase resistance to BCNU (96).
Mobility shift experiments using cell extracts and reporter constructs have demonstrated the presence of a factor termed the MGMT-enhancer binding protein, that binds to a 9mer within the 59 bp enhancer located at the first intron–exon boundary. In cells lacking ATase expression, MEPB is absent from the nucleus but present in the cytoplasm: translocation of the protein can occur under conditions that upregulate ATase expression (97).
In mice, the radiation-mediated increase in ATase activity is dependent on functional p53 (98). Experiments using reporter constructs transfected into rat hepatoma cells show that elements within the human ATase promoter are responsibe for mediating this response (99). The p53-dependant upregulation of ATase in response to radiation in mice is different from the effect of p53 itself, overexpression of which directly suppresses transcription of the gene (99,100). Consequently, basal ATase activity is inversely correlated with wild-type p53 expression in some tumour groups (55,101).
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Chromatin structure and promoter methylation |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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The inhibition of histone deacetylation using Trichostatin-A leads to a 2–3-fold increase of ATase mRNA levels in MIA PaCa-2 cells and this requires the integrity of the AP-1 sites in the promoter (102). Immunoprecipitation and overexpression of adenovirus E1A, a protein that binds the transcriptional coactivators CBP and p300, indicate that histone acetylation in the ATase promoter region and the associated remodeling of chromatin structure are involved in the regulation of ATase expression (102). In cells expressing ATase, the positioning of nucleosomes in the promoter/CpG island region is invariant but this is lost in non-expressing cells (103).
DNA cytosine methylation can affect ATase expression and this is determined by the location of these events within the gene: methylation in the promoter correlates with decreased expression (104) whereas methylation of the body of the gene correlates with an increased expression (105). Sublines of IMR90 cells revealed a substantial heterogeneity of methylation within the ATase gene. In one case, ATase transcription was initially silenced in spite of relative low levels of promoter methylation, which increased with increasing passage number (106). Confluent cells also show an increased level of methylation of the ATase promoter and this methylation pattern is reversed when cells are returned to logarithmic growth (48).
ATase promoter methylation has also been extensively studied in DNA extracted from tissue biopsies. Whilst methylation has not been observed in normal tissues (107), it is one of the common epigenetic changes in a variety of tumours. It can be detected in 40% of gliomas and colorectal carcinomas and in approximately a quarter of non-small cell lung carcinoma, lymphomas and head and neck tumours (107). However, in very few studies has this been examined in relation to the levels of ATase expression within the same tissue sample. Given the heterogeneity of ATase expression as seen by immunohistochemical staining (see above) it seems likely that those cells in which no ATase can be detected may be promoter methylated, however, this has yet to be demonstrated experimentally. It is worth noting that the methodology for promoter methylation is not quantititative. Nevertheless, promoter methylation in several different types of human tumours seems to correlate with their prognosis after treatment with O6-alkylating agents (65,108).
It is intriguing that, in several tumour types, promoter methylation has been reported to correlate with poor prognosis even though surgery was the only treatment (109,110). This might indicate that methylation of the ATase promoter reflects the status of the promoters of other genes that determine disease outcome.
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ATase phosphorylation |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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ATase phosphorylation has been shown to decrease the activity of the protein (111) and novel protein kinases appear to be responsible for this (112). Phosphorylation on ser 204 makes the protein more resistant to proteolytic degradation (113). The extent and functional role of ATase phosphorylation in vivo is unclear.
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Preventive/therapeutic manipulation of ATase activity |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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Given its presence in human DNA and its mutagenic, toxic and recombinogenic potential, it seems unavoidable to conclude that O6-alkG can potentially be responsible for human cancer. However, whilst environmental alkylating agents have been extensively studied, the contribution of such exposures to cancer in the general population is not known. An understanding of the mechanisms of endogenous formation of this lesion and indeed of any exogenous agents that might enhance endogenous formation, will help to define the relative contributions of exogenous and endogenous damage. This may lead to the identification of strategies that will reduce the levels of O6-alkG or its biological impact. It is also possible that further exploration of the factors affecting ATase expression might eventually allow modulation of ATase levels by means of exogenous agents, perhaps even in a tissue-specific manner. However, this does not appear to be a practical proposition at the present time.
In terms of cancer therapy, however, agents that decrease ATase activity in tumours can make them more sensitive to the toxic effects of O6-alkylating agents, at least in experimental models. This is a strategy that is currently being pursued in cancer patients (for reviews see ref. 114): low molecular weight pseudosubstrates of ATase are in clinical trial (115–117), and other approaches, including antisense and genetic suppressor elements are being explored in model systems. However, unless these strategies can be directed towards tumour cells, normal tissues are also likely to be sensitized to the toxic side effects of such therapies. Indeed this is the case in the trials thus far reported: it has been necessary to reduce the dose of the alkylating agents because of increased myelosuppression (118). Normal cell specific upregulation may be an approach to circumvent this, but as already stated, it is not yet known how to effect this using exogenous agents. However, in the case of myelosuppression, ex vivo haemopoietic stem cell transduction with retrovirus harbouring ATase is being actively pursued (118,119). This approach could be used in the absence of repair inhibitors as it would be expected to protect against bone marrow toxicity and even allow O6-alkylating agent dose escalation. Nevertheless, current strategies are exploiting mutant versions of ATase that are resistant to inactivation by pseudosubstrates (2,120) so that tumour sensitization and normal tissue protection can be achieved simultaneously by the use of inactivating agents: clinical trials of this technology should soon be underway.
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Summary |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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In the past few years, considerable progress has been achieved in understanding the factors involved in the regulation of ATase expression. Whilst none of the pathways have yet to be fully resolved, it is conceivable that, in the future, this knowledge will influence the management of any cancers that would be treated with O6-alkylating agents. However, for the present, inhibitors are the only practical method to modulate ATase activity and these are currently been assessed in clinical trials (115–117). The agents being used for this are at present not tumour-targeted and therefore increase the toxic side effects of the co-applied O6-alkylating agents (121). It is thus likely that a reduction in the severity of such side effects will be achieved by targeting the inhibitors to tumour tissues (122), or by providing protective therapies to the affected normal tissues (114–117,119–121,123,124).
In contrast, the processes underlying inter-individual differences in the levels of endogenous DNA damage and of ATase activity, and the extent to which such differences are determined through inherited and/or environmental factors are far less well characterized. Further progress in this area could contribute to an understanding of individual variations both in cancer susceptibility and the relationship of this to environmental exposure. This may eventually provide approaches to decreasing the incidence of human cancer.
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Note added in proof |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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The E.coli ATase-like (ATL) protein has very recently been expressed from its open reading frame: it inhibits the action of ATase on O6-meG in DNA and binds to DNA containing O6-alkylguanine, although its physiological function has yet to be defined (A.E.Pegg, personal communication). The S.pombe ATL has now been similarly expressed and has also been shown to inhibit the action of the human ATase on O6-meG in methylated DNA (S.Pearson, M.Santibáñez-Koref and G.P.Margison, unpublished observations).
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Acknowledgments |
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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. B.K. acknowledges the generous support by Deutsche Forschungsgemeinschaft and Stiftung Rheinland-Pfalz. M.S.K. would like to thank Prof. J.G.Reich for the opportunity to visit his department during the completion of this review.
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References |
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TopAbstractIntroductionAlkyltransferase genesPhysiological role of ATaseEndogenous DNA alkylationVariation in ATase expression...Pathological conditionsInduction of ATase expression...Mechanisms underlying inherent...Polymorphisms and other changes...Transcription factorsChromatin structure and promoter...ATase phosphorylationPreventive/therapeutic...SummaryNote added in proofReferences |
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