Lung cancer risk and variation in MGMT activity and sequence

d n a r e p a i r 6 ( 2 0 0 7 ) 1134–1144

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Lung cancer risk and variation in MGMT activity and sequence c ˜ Andrew C. Povey a,∗ , Geoffrey P. Margison b , Mauro F. Santiba´ nez-Koref a b c

Centre for Occupational and Environmental Health, University of Manchester, United Kingdom Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom Institute of Human Genetics, University of Newcastle, Newcastle upon Tyne, United Kingdom

a r t i c l e

i n f o

a b s t r a c t

Article history:

O6 -Alkylguanine–DNA alkyltransferase (MGMT) repairs DNA adducts that result from alky-

Published on line 13 June 2007

lation at the O6 position of guanine. These lesions are mutagenic and toxic and can be produced by a variety of agents including the tobacco-specific nitrosamines, carcinogens

Keywords:

present in cigarette smoke. Here, we review some of our work in the context of inter-

Lung cancer

individual differences in MGMT expression and their potential influence on lung cancer

MGMT

risk. In humans there are marked inter-individual differences in not only levels of DNA

O -Alkylguanine

damage in the lung (N7-methylguanine) that can arise from exposure to methylating agents

Smoking

but also in MGMT activity in lung tissues. In the presence of such exposure, this variabil-

6

ity in MGMT activity may alter cancer susceptibility, particularly as animal models have demonstrated that the complete absence of MGMT activity predisposes to alkylating-agent induced cancer while overexpression is protective. Recent studies have uncovered a series of polymorphisms that affect protein activity or are associated with differences in expression levels. The associations between these (and other) polymorphisms and cancer risk are inconsistent, possibly because of small sample sizes and inter-study differences in lung cancer histology. We have recently analysed a consecutive series of case–control studies and found evidence that lung cancer risk was lower in subjects with the R178 allele. © 2007 Elsevier B.V. All rights reserved.

1.

Introduction

O6 -Alkylguanine–DNA alkyltransferase (O6 -methylguanine– DNA methyltransferase; MGMT, E.C.2.1.1.63) constitutes the first line of cellular defence against the toxic, mutagenic and carcinogenic effects of alkylation of DNA at the O6 -position of guanine. MGMT reverses O6 -alkylation damage by covalent transfer of the alkyl group to the protein itself, leading to its inactivation, ubiquitination and proteasome-mediated degradation. More detailed descriptions of protein function are presented in other articles in this issue and have been recently reviewed elsewhere [1–4].

∗

Corresponding author. Tel.: +44 161 275 5232; fax: +44 161 275 7380. E-mail address: [email protected] (A.C. Povey). 1568-7864/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2007.03.022

In humans there are marked differences in MGMT activity between tissues within an individual as well as between individuals [5]. Given the known role of MGMT, it seems reasonable to expect that variation in activity levels may alter cancer susceptibility but only if, at least under some circumstances, activity levels are not sufficient to repair O6 -alkylguanine adducts present in human tissues. In situations where exposure to alkylating agents is only via endogenous sources, even low MGMT levels may be sufficient to provide protection. Thus the influence of MGMT activity on cancer risk should be best detected in populations known to be exposed to alkylating agents since unexposed populations should show little or no

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Table 1 – Tumour induction in C57BL/J MGMT knockout micea Carcinogen [reference]

Sex

Dose

Tissue

Tumour prevalence n/total (% tumours) −/−

−/+

−/− vs. +/+

+/+

p

Dacarbazine [11]

♀♂

4 mg/kg i.p. × 5

Thymus Lung

2/52 (3.8) 6/52 (11.5)

– –

0/47 (0) 2/47 (4.3)

0.27 0.18

NMU [12]

♀♂

2.5 mg/kg i.p. × 1

Thymus Lung

7/52 (13.5) 11/52 (21.2)

0/43 (0) 2/43 (4.7)

0/52 (0) 1/52 (1.9)

0.006 0.006

DMN [13]

♂

1 mg/kg × 1

♀

5 mg/kg × 1

Liver Lung Liver Lung

5/15 (33.3) 6/15 (40.0) 16/18 (88.9) 14/18 (77.8)

– – – –

4/11 (36.3) 1/11 (9.1) 5/11 (45.5) 3/11 (27.3)

0.60 0.09 0.02 0.01

a

Data is taken from Refs. [11,12].

increased risk associated with MGMT variability. An association between low MGMT activity and a particular type of cancer strongly suggests that alkylating agents play a role in the aetiology of the disease. Assuming that (i) the prime function of MGMT is to remove O6 -alkylguanine lesions and that (ii) O6 -alkylguanine lesions are primarily removed by MGMT, the relative levels of both MGMT and O6 -alkylguanine adducts will influence risk. These levels will depend not only on the degree of exposure to DNA damaging agents, but also on co-exposure to agents that may modulate MGMT activity and on factors, such as polymorphisms, that may alter MGMT function. Therefore they will vary between cell types and also depend on the biological effect being examined. However the situation is more complex than this, as the mismatch repair pathway is an absolute requirement for the toxic effects of O6 -methylguanine adducts [6] and bulky O6 -alkylguanine adducts such as O6 butylguanine can be repaired by other DNA repair pathways [7]. Furthermore, MGMT may play a part in other biochemical pathways. For example methylated MGMT can inhibit transcriptional activation by the estrogen receptor [8] and MGMT has also been shown to be capable of blocking the repair of O6 -alkylguanine by nucleotide excision repair [9,10]. The importance of this latter phenomenon in humans is still unclear, but it may be that higher levels of MGMT expression are associated with a decreased efficiency in the repair of certain lesions. Over the past three decades many groups have investigated the causes and the consequences of MGMT expression variability (see other articles in this issue). Here, we will examine some of our own groups’ results in the context of interindividual differences in MGMT expression and their potential influence on cancer risk. We will focus on cancer of the lung, an organ for which there is clear evidence of environmental exposure to alkylating agents, namely those present in tobacco smoke. We will first discuss animal models of lung cancer induced by alkylating agents and then human exposure to DNA alkylating agents. We will then examine inter individual variation in MGMT expression, review some characteristics of the MGMT locus and recapitulate what is known about the relationship between genetic variation, protein activity and expression levels. Finally we will discuss some results from association studies investigating the relationships between intragenic MGMT polymorphisms and cancer risk.

2. Alkylating agent exposure and rodent lung tumorigenesis A relationship between MGMT activity and cancer risk has been clearly shown in murine models (Table 1). Mice lacking functional MGMT are more susceptible to developing tumours, including lung adenomas, in response to exposure to alkylating agents, though it appears that tobacco-specific nitrosamines have not been specifically tested in these models [11,13]. Perhaps unexpectedly, untreated mice appear phenotypically normal and their lifespan and fecundity are not reduced. Conversely, transgenic mice overexpressing MGMT are protected against the effects of alkylating agents [14,15] including lung adenomas when MGMT is expressly targeted to that tissue [16]. Mice with defects both in MGMT and mismatch repair (MLH1 gene deletion) are still sensitive to alkylating agent induced carcinogenicity but are less sensitive to induced toxicity than MGMT ko mice indicating an important role of mismatch repair in determining which pathways are engaged following O6 -alkylguanine formation [6,17]. Among the alkylating agents present in tobacco smoke, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) has been particularly well studied [18]. NNK is a potent lung carcinogen in rodent species, irrespective of the route of administration leading mainly to adenomas and adenocarcinomas [18]. NNK can be metabolised through two different pathways resulting in active species that can either methylate or pyridyloxobutylate DNA [18,19], but it is the formation of O6 -methylguanine, rather than O6 -[4-Oxo-4-(3pyridyl)butyl]guanine (O6 -pob), that appears to be a key step in the formation of lung tumours in mice [20]. Within Clara cells, NNK generates O6 -methylguanine levels that have been shown to be linearly related to subsequent adenocarcinoma induction [21].

3.

Human exposure to alkylating agents

Humans can be exposed to alkylating agents from a number of different sources including occupation, lifestyle or diet [22–24] (Table 2). In addition, alkylating agents are commonly used as chemotherapeutic agents for a number of different cancers and can potentially be derived from other medications

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Table 2 – Human exposure to alkylating agentsa Exposure

Example [reference]

Exogenous Smoking Occupation Diet

NNK metabolites present in urine of smokers [22] Alkyl DNA adducts present in lymphocytes of workers in rubber industry [23] Volatile nitrosamines present preformed in foodstuffs and drinks [24]

Iatrogenic Chemotherapy Nitrosatable drugs

Temozolomide causes DNA damage in human tissues [25] Piperazine nitrosation products detected in urine [26]

Endogenous Cellular processes

Infections a

DNA damage and other biological effects induced by nitrosated bile acids and peptides and s-adenosyl methionine [27–29] Diets rich in nitrates and amines result in increased formation of volatile nitrosamines [30] Volatile nitrosamines present in urine of patients with schistosomiasis [31]

Data taken from Refs. [22–31].

[25,26]. Alkylating agents may also be formed endogenously within the body through either normal cellular processes or as a result of transformation (chemical or bacterial mediated) of dietary (or therapeutic) substrates at specific sites within the body [27–31]. It is thus clear that humans are continuously exposed to low levels and may be exposed intermittently to higher levels of alkylating agents. Indeed, alkylation damage can be shown to be present in DNA in a large proportion of individuals in the human population not knowingly exposed to high levels of these agents [32]. This confirms that exposure to agents that generate alkylated adducts, be they exogenous or endogenous, is very widespread, and also suggests that repair capacity is not always sufficient for the complete elimination of the damage from DNA. Whether or not such exposure results in an increased cancer risk is unclear. Substantial human exposure occurs by the inhalation of cigarette smoke, which contains not only tobacco-specific nitrosamines (TSNA) but also other nitrosamines that can be found elsewhere in the environment [33]. The cumulative doses of NNK a smoker can receive are comparable to those shown to induce lung adenocarcinomas in experimental animal models [34]. Indeed exposure to TSNA has been suggested as a causal factor in the formation of human lung adenocarcinomas rather than squamous cell carcinoma [35]. Whilst cigarette smoking is associated with adenocarcinoma formation in humans, the association is stronger with the formation of small cell and squamous cell carcinomas [36]. This may reflect the presence of additional carcinogens in cigarette smoke and/or differences between the species. DNA alkylation products have been detected in numerous human tissues [32] but in contrast to other types of DNA damage (e.g. oxidative DNA damage or “bulky” DNA damage), the number of published studies are low and little systematic work has been carried out. Exposure to alkylating agents is often indirectly assessed by measuring N7-methylguanine levels in the DNA [37]. This lesion has no known toxic or mutagenic effect and is repaired relatively slowly, based on evidence from cell culture and animal models. In Fig. 1, we have summarised a number of different studies that have measured N7-methylguanine adducts in human lung tissue

either obtained by biopsy, on autopsy or by bronchial lavage [38–45]. Absolute levels vary between different studies and this is likely to be due not only to differences in the populations studied (e.g. proportion of smokers) and the cell types studied, but also the analytical techniques used. However whilst there is sufficient evidence to indicate that levels of this particular adduct can vary widely (up to two orders of magnitude), it is unclear whether this variability results solely from differences in exposure or may also stem from inherent variability in carcinogen metabolism and DNA repair. The biologically more relevant adduct, O6 -methylguanine, has been detected also in human lung tissue [46], but the number of studies are even fewer than those which have measured N7-methylguanine. However it is possible to estimate O6 -methylguanine levels from N7-methylguanine levels as the levels of O6 -methylguanine produced in DNA by agents that react via unimolecular nucleophilic substitution (SN 1) are ∼10% of N7-methylguanine levels [47]. Based on this, and the amounts of N7-methylguanine found in human tissue DNA, the predicted amounts of O6 -methylguanine would

Fig. 1 – N7-methylguanine levels in DNA from human lung samples. N7-methylguanine levels measured directly in DNA from lung samples obtained at autopsy [38–41] or surgery [42,43] or by lavage (unpub, [44,45]) and measured by HPLC/32 P-postlabelling [38–41,44,45] or by 32 P-postlabelling [42,43] or immunslotblot (unpublished: unpub* = Harrison et al.). Data presented as mean with the range.

d n a r e p a i r 6 ( 2 0 0 7 ) 1134–1144

Fig. 2 – MGMT activity in normal human lung tissue. MGMT activity measured in lung tissue obtained at autopsy [51] or surgery [51–53] or by lavage [55]. Data presented as mean with the range.

is lacking. In lung tissues, particular attention has been paid to the effects of smoking. One study reported an increase of MGMT activity in smokers [52], while another found no association with smoking [53]. More recently we reported that MGMT activity in bronchial epithelial cells obtained from smokers was lower than that in the cells from non-smokers whereas activity in lymphocytes did not differ [58]. The decrease was limited to subjects that were reported to be currently smoking [58]. This could reflect the presence in cigarette smoke of direct inhibitors of MGMT or simply an increase in the amounts of substrate lesions produced in DNA, leading to a larger amount of protein being inactivated. Irrespective of the mechanism/s involved based on the biological effects of O6 -alkylguanine, decreased MGMT activity would be predicted to result in an increased sensitivity of smokers to the biological effects of further alkylation damage.

5. be of a similar magnitude to those that have been reported to cause mutations [48]. However, this may be misleading: SN 2 alkylating agents such as the endogenous agent S-adenosylmethionine produce vastly lower amounts of O6 methylguanine (∼0.01%) relative to N7-methylguanine [47]. So whilst O6 -methylguanine and N7-methylguanine have been detected in human lung tissue, it is not possible to extrapolate with any certainty, the amounts originally generated in DNA nor the chemical nature of the agents responsible. Conversely, considerable heterogeneity in adduct levels has been demonstrated in mouse lung following exposure to NNK [49]: it is thus possible that within certain cell types adduct levels are much higher.

4.

Variation in MGMT activity in the lung

MGMT activity is present at widely different levels in different human tissues and in those tissues that have been examined, there is also marked inter-individual differences in activity [5,50]. For example in human lung tissue, MGMT activity can vary over two orders of magnitude (Fig. 2; [51–54]). The basis of these inter-individual differences remains to be characterised. There have been no studies where samples have been repeatedly taken from the lungs of patients over a period of time but according to a study of MGMT activity in lymphocytes, intra-individual variation accounted for 60% of the variation in activity between samples [55]. Therefore differences in MGMT activity are a consequence of differences between individuals and variation within individuals over time. There have been reports describing circadian variation in MGMT activity in lymphocytes [56] and differences during the menstrual cycle [57], but most attention has focused on differences in expression in response to DNA damage. In rats, and to a lesser extent in other rodents, induction of MGMT expression has been observed in response to a range of treatments. These include exposure to genotoxic agents such as N-nitrosamines, aflatoxin and ionizing radiation or to ␥-interferon, and even partial hepatectomy (see [5] for a review). However, in humans, definite evidence of inducibility

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MGMT activity and lung cancer risk

Associations between MGMT activity and cancer risk can theoretically be studied in a number of different ways. MGMT activity can be assessed directly in lung tissue (or in an appropriate surrogate) of patients with and without lung cancer. Differences in normal tissue activity may then reflect an inherent susceptibility or temporal variation or other environmental exposures. In malignant tissue, MGMT activity may also reflect the disease process, particularly as it is clear that epigenetic silencing occurring within malignant tissue, can directly affect MGMT expression ([59] and elsewhere in this issue). To reduce the confounding effect of the disease process, an alternative approach would be to use a prospective cohort study and analyse samples, collected at recruitment of subjects that, subsequently did or did not develop lung cancer. A more indirect approach is to investigate the associations between polymorphisms at the MGMT locus and cancer risk. This approach still requires an assessment of the influence of polymorphic sites on MGMT activity. However, it has the distinct advantage that genotyping can be carried out on easily accessible material and it is accepted that the genotype represents a stable influence, while direct activity measurements may reflect transient changes in activity. Furthermore, such studies can use a case–control design as the genotype is not likely to be influenced by disease. An initial study reported a significantly lower level of MGMT activity in cultured fibroblasts isolated from lung cancer patients versus controls, suggesting that there may indeed be underlying genetic factors that influence MGMT activity and hence lung cancer susceptibility [60]. However it is more difficult to assess if there is an association between physiological variation of MGMT activity and cancer risk in humans. We have measured MGMT activity in bronchial epithelial cells and peripheral blood mononuclear cells [58] and found no difference between lung cancer cases and controls (Table 3). Table 4 summarises the results of studies investigating the relationship between lung cancer risk and MGMT activity. These and studies of other tissues have reported few associations between MGMT activity and cancer risk [61,62]. It is conceivable that when using material directly, without a cell culture step, the underlying differences become more difficult

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Table 3 – Relative risk of lung cancer and MGMT activitya Tissue

MGMT activity (fmole/␮g DNA)

Peripheral blood mononuclear cells

Bronchial epithelial cells

a

Cases/controls

OR (95% CI)

>11.23 7.96–11.22 5.91–7.95 <5.9

17/17 13/24 14/21 14/20

1.0 0.5 (0.2–1.6) 0.7 (0.2–1.9) 0.7 (0.2–2.0)

>2.11 1.1–2.1 0.61–1.09 <0.6

13/12 10/17 12/15 10/21

1.0 0.5 (0.2–1.9) 0.7 (0.2–2.5) 0.4 (0.1–1.5)

MGMT activity measured in peripheral blood mononuclear cells and bronchial epithelial cells obtained from lung cancer cases and controls at bronchoscopy. Study population is described in greater detail elsewhere [58].

to detect since the activity may still reflect, for example, environmental influences or the average activity of different cell types with potentially a range in MGMT activity. In humans, low MGMT activity within colon adenomas has indeed been associated with the formation of GC-AT transition mutations [63]. Furthermore, in several tumour types, methylation of cytosine residues in CpG islands in the promoter region of MGMT, is associated with the increased formation of GC-AT transition mutations in both oncogenes and tumour suppressor genes [64,65] and thus provides compelling support for the role of alkylating agents in these events.

6. Sequence variation at the human MGMT locus The human MGMT gene is located on chromosome band 10q26. The message is encoded by five exons distributed over 300 kb of genomic DNA: the last four exons are coding, and the second intron is particularly large (170 kb) [66]. The promoter covers the first exon and part of the first intron, it lacks TATA or CAAT boxes and contains CpG rich regions [67]. There are Sp1, AP-1, and AP-2 sites, two possible glucocorticoid responsive elements and a 59 bp element, located at the first exon/intron boundary, which is required for efficient transcription of reporter constructs. The transcript is approximately 0.95 kb long and encodes a peptide with 207 amino acids ([66], see other papers in this issue). A large number of polymorphisms in the MGMT locus have described. At the time of writing of this manuscript (June 2006), there were over a thousand entries in dbSNP that mapped between the first and the last exon. Of these, 438 showed minor allele frequencies above 0.05. The extent of linkage dise-

quilibrium has been characterized by the HAPMAP consortium (2003) and by our group [68]. Regions with high levels of disequilibrium are found in the 5 end of the gene, around the third, and surrounding the fifth exon. In the following paragraphs when referring to a polymorphism we will either use their dbSNP identifier or a designation describing their location and the names of the residues found in the different alleles at the corresponding position.

6.1.

Polymorphisms affecting protein function

The observed inter-individual differences in MGMT activity levels led initially to the search for polymorphisms that might affect activity, and a number of candidate single nucleotide polymorphisms (SNPs) have been reported within the coding region. So far, interest has focussed on those resulting in amino acid substitutions. Initially the R160 allele attracted considerable interest because of a possible association with cancer susceptibility [69] and its resistance to the MGMT inhibitor O6 -benzylguanine [70]. Recently Mijal et al. [71] showed that compared with O6 -methylguanine, O6 -benzylguanine and O6 -pobG are less efficiently processed by the R160 allele than by the common allele (G160). As mentioned above, O6 -pobG is produced by NNK, one of the carcinogens present in cigarette smoke. However, this allele is very rare [72] and subsequent studies failed to confirm the association with cancer [73]. Gene transfer experiments in bacteria and mammalian cells suggest that the protein coded by another allele, W65, is unstable [74]. However this allele is also infrequent and whether carriers are at increased cancer risk remains to be determined. The allele, V143 has recently begun to attract some attention because it changes an isoleucine residue close to the

Table 4 – Case–control studies between activity and cancer riska Material

Phenotype [reference]

Year

Origin of population

Cases

Controls

Association

Lung epithelia (brushings) Peripheral lymphocytes Peripheral lymphocytes Peripheral lymphocytes Cultured primary fibroblasts Cultured primary fibroblasts

Lung cancer [58] Lung cancer [58] Lung cancer [61] Thyroid cancer [62] Lung cancer [60] Cutaneous melanoma [60]

2006 2005 2002 2002 1989 1989

UK UK Seven European countries Turkey Germany Germany

58 45 153 24 45 39

82 65 106 25 29 29

None None None Decreased levels Decreased levels None

a

Data taken from Refs. [57,50–61].

d n a r e p a i r 6 ( 2 0 0 7 ) 1134–1144

alkyl acceptor cysteine residue in the active site at position 145. The I143V allele is in near perfect disequilibrium with K178R. The frequency of I143 varies across populations. The data from the HAPMAP consortium report a frequency of 18% in the probands of European origin (CEPH), but fail to detect any carriers in samples from Nigeria (YRI) or Japan (JPT) (HapMap release 16c.1). Our own results suggest that this substitution does not affect the ability of the protein to react with O6 -methylguanine in DNA, or its thermal stability. It does however reduce its activity towards low molecular weight pseudo-substrates including O6 -benzylguanine and O6 -(4bromothenyl)guanine, both of which are in clinical trials as MGMT inactivating agents [68]. However, a striking difference between the V143-R178 and the I143-K178 alleles, as reported by Mijal et al., is that the ability of only the latter to repair O6 pobG is sequence context sensitive [75]. This would suggest that lesions that V143-R178 carriers maybe able to repair are processed more slowly by the I143-K178 allele, suggesting that V143-R178 may afford a superior degree of protection against the detrimental effects of certain O6 -alkylating agents.

6.2. Polymorphisms associated with differences in expression levels Another strand of inquiry has been the investigation of the relationship between polymorphisms and variation of expression at the mRNA level. Initial work led to the identification of polymorphisms in the promoter region [76–78]. Some of these polymorphisms affect potential transcription factor binding sites [76] and experiments using reporter constructs revealed a slight but detectable influence of rs16906252 on transcription in vitro [78]. In human PBMC and in lung tissue, the two MGMT alleles are frequently expressed at different levels. This suggests that polymorphisms can affect transcription by acting in cis [79]. This is consistent with a recent study that found an association between the two intragenic polymorphisms analysed (L84F and I143V) and the genotoxicity of NNK in peripheral blood lymphocytes assessed as the frequency of chromosome aberrations [80]. However, too few markers were investigated to map the sites involved and it is not clear if the results reflect differences in protein function or in expression levels. More recently, quantitative trait locus analysis revealed that at least two intragenic sites influence MGMT activity in PBMC [68]. One is located in the 5 half of the gene, the most significant association being with a marker in the first exon (rs12268840). However, the authors were not able to exclude the possibility that this association was due to polymorphism(s) affecting transcription that are located in the promoter region. A second region showing significant association with protein activity is characterized by two markers in the fifth exon, rs2308321 and rs2308327 (I143V and K178R). Since one of these changes modifies the activity of the protein (see above), it is possible that this association may not reflect an influence upon transcription, but differences in the ability of one of the variants to react with some so far unidentified substrate, resulting in their preferential inactivation. Together these two regions account for 20% of the observed variation in expression between samples. As mentioned above, previous data [55] indicated that inter-individual variation accounts for some 40% of variation in activity in PBMC, suggesting that

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intragenic polymorphisms account for a substantial proportion of inter-individual variation. Most of these studies have been carried out using peripheral blood cells, and the questions remains whether polymorphisms affecting expression levels in blood also affect expression in other tissues such as the lung.

7.

MGMT genotype and lung cancer risk

The possible association between MGMT polymorphisms and lung cancer risk has been examined in a small number of studies ([73,78,81–83]; Table 5). Because of its proximity to the alkyl acceptor cysteine at position 145, attention has focused mainly on the I143V polymorphism, although some studies use K178R as a surrogate since it is in nearly perfect disequilibrium with I143V. So far, studies of lung cancer in smoking populations have been inconclusive, although one report showed twofold increased risk associated with the V143 allele in both Caucasians and African Americans [73]. While the pooled data achieved a marginal significance with a 95% confidence interval of 1.01–4.7, for the two groups separately, these increases were not significant (see Table 5; [73]) Surprisingly, V143 was the allele associated with higher protein activity in our QTL analysis [68]. Other studies have failed to find any association [78,83]. Interestingly, a second study has reported a significant association between the I143V genotype and lung cancer risk but this was a multi-centre study of lung cancer in non-smokers [82]. Here, the risk associated with the V143 allele was most pronounced in cases of adenocarcinoma (OR: 2.67; 95% CI: 1.21–5.87). However, as mentioned above, V143 is associated with increased activity levels in PBMC. We have carried out three consecutive series of case–control studies which used the same source population, namely patients attending a particular bronchoscopy clinic ([45,58], Crosbie et al., unpublished data). For this analysis, cases were defined as patients newly diagnosed with a tumour of the lung, trachea or bronchus: patients with a history of lung or any other cancer were excluded. The control group consisted of all other patients who were free of benign and malignant tumours both at the time of, and prior to, examination. Subjects below the age of 40 were also excluded. This population (n = 617) was genotyped for two polymorphisms that we had previously shown [68] to be associated with MGMT activity (K178R and rs12268840). In this study, we found evidence that the 178R allele (and by implication the V143 allele) was associated with reduced lung cancer risk: 2.2% of controls but only 0.4% of lung cancer cases were RR homozygotes. The trend for a decreased risk with the number of R alleles was significant (p = 0.02; Table 6). After adjustment for sex, age, smoking and series, the OR (95% CI) for a 178R heterozygote was 0.68 (0.46–1.03) and for a 178R homozygote was 0.18 (0.02–1.52): of the nine subjects who were homozygous for the R allele only one was a case. Two recent studies, one on colorectal and the other on endometrial cancer similarly found that the V143 allele was associated with a decrease in risk [84,85]. The increased risk of endometrial cancer was restricted to smokers [84]. Overall then, the findings offer a rather confusing picture. For I143V, some reports find no association with cancer risk,

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Table 5 – Lung cancer risk associated with MGMT polymorphismsa Genotype

Population [reference] Korea [81] Poland [78]

F84L (FF/FL/LL)

Poland [78]

Korea [81]

Korea [81] I143V (II/IV/VV)

Caucasian [73] African Americans [73] Poland [78] Multi-centre Caucasian [82]

G160R (GG/GR/RR)

Caucasian [73]

K178R (KK/KR/RR)

African Americans [73] Caucasian [83]

a b

Cases (432): mixed histology; control (432): healthy controls Cases (96): men with 1◦ NSCLC; controls (96): unrelated healthy men Cases (432): mixed histology; control (432): healthy controls Cases (96): men with 1◦ NSCLC; controls (96): unrelated healthy men Cases (432): mixed histology; control (432): healthy controls Cases (53): mixed histology; controls (55): hospital-diagnosis unrelated to smoking Cases (81): mixed histology; controls (81): hospital-diagnosis unrelated to smoking Cases (96): men with 1◦ NSCLC; controls (96): unrelated healthy men Cases (136): nonsmokers with known histology; controls (133): hospital (non-tobacco related diseases) and population registries (healthy individuals) Cases (53): mixed histology; controls (53): hospital-diagnosis unrelated to smoking Cases (81): mixed histology; controls (80): hospital-diagnosis unrelated to smoking Cases (92): mixed histology; control (85): urological and orthopedic surgery clinics

Data taken from references [73,78,81–83]. Odds for rare allele carriers vs. odds for the common allele homozygous.

ORb

Genotype distribution (%)

Matching

Case 42/46/13; control 48/44/8

1.25

Age/sex

Case 81/19/0; control 78/21/1

0.82 (0.41–1.67)

None

Case 79/20/1; control 78/19/2

0.95

Age/sex

Case 70/24/6; control 77/18/5

1.46 (0.76–2.78)

None

Case 80/19/1; control 79/19/2

0.96

Age/sex

Case 77/23/0; control 85/15/0

2.0 (0.78–5.7)

Age, sex, smoking

Case 86/14/0; control 76/5/0

2.3 (0.73–8.3)

Age, sex, smoking

Case 78/21/1; control 78/21/1

1.0 (0.5–1.98)

None

Case 75/24/1; control 86/14/0

2.05 (1.03–4.07)

Country

Case 99/1/0; control 96/4/0

0.32 (0.01–4.12)

None

Case 100/0/0; control 98/2/0

–

None

Case 78/22/0; control 81/19/0

1.19

Age, sex, smoking, education

d n a r e p a i r 6 ( 2 0 0 7 ) 1134–1144

485C-A (CC/CA/AA) rs1625649 1099C-T (CC/CT/TT) (rs16906252) L53L (CC/CT/TT)

Population description (n)

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Table 6 – Analysis of lung cancer risk associated with the codon K178R and rs12268840 polymorphism in Caucasiansa Polymorphism

Genotype

Numbers of cases/controls (% cases)

OR (95% CI) adjustedb

pc

K178R

KK KR RR

200/257 (43.8) 50/93 (35.0) 1/8 (11.1)

1.0 0.67 (0.45–1.01) 0.10 (0.01–0.93)b

0.0082

rs12268840

CC CT TT

125/163 (43.4) 97/151 (39.1) 25/41 (37.9)

1.0 0.82 (0.57–1.18) 0.78 (0.44–1.58)b

0.268

a

b c

Lung cancer cases and controls were identified through bronchoscopy clinics in three consecutive studies ([45,58], Crosbie et al., unpublished). Data adapted from Crosbie et al., submitted. Adjusted for age, sex, pack years and series. Trend test.

Fig. 3 – Variation of odds ratio for lung adenocarcinoma risk with a varying proportion of lung adenocarcinomas in the case population. Assuming that the tobacco-specific nitrosamines are causative factors for lung adenocarcinomas and not squamous cell carcinomas, the numbers of subjects required to detect an odds ratio of 2 or 5 will vary depending upon the admix of adenocarcinomas and squamous cell carcinomas in the case population. To calculate n, we have assumed a true risk (OR) of either 2 or 5 and an ˛ of 0.05 and a power of 80% and a varying genotype prevalence of between 5 and 25%.

while others find an increased risk associated with either I143 or V143. This may then reflect the lack of a genuine association between cancer risk and sequence variation at the MGMT locus or alternatively publication bias. Interestingly while the earlier reports, mainly based on small sample size, found no association, or an increase risk, with V143, the more recent reports are consistent with a protective role for this allele. An alternative explanation is that the relationship between genotype and outcome is more complex and is modulated by environmental influences. In the case of lung cancer, it is also possible that variation in MGMT activity predominantly affects the risk of one type of histology, for example it has been suggested that the tobacco-specific nitrosamines are associated more with adenocarcinomas than squamous cell carcinomas [35]. If the case population then is a mixture of patients with different histological types of lung cancer, effects specific to a particular subgroup, such as adenocarcinoma, become difficult to detect. Fig. 3 shows that the number of subjects required to detect an odds ratio of 2 (or 5) depend significantly on the histological mix of cases if it is assumed that tobacco-specific nitrosamines cause only adenocarcinomas and not squamous cell carcinomas. The higher the proportion of non-adenocarcinomas in the case population, the greater the number of subjects required. Therefore in order to detect an effect, a large sample size will be required

when a mixture of histological types is included among the cases. This effect may also then be modulated by the degree of environmental exposure as for example damage by DNA alkylating agents maybe not be important in the aetiology of lung cancer in non-smokers, in whom other aspects of MGMT function, such as interference with nucleotide excision repair, may contribute to cancer risk. Another possible activity of MGMT is the modulation of the activity of the oestrogen receptor [8]. This may be of particular interest in view of the recent data suggesting that colorectal cancer risk is only influenced by MGMT polymorphisms in females [85] and that the V143 allele is protective against endometrial cancer in smoking women [84]. In summary, to better determine cancer risk associated with variability in MGMT at the gene and expression levels, a better understanding is required of the role of MGMT in different biochemical pathways, phenotype–genotype correlations and the extent and sources of human exposure to alkylating agents, both endogenous and exogenous.

Acknowledgements GPM thanks Cancer Research UK for three decades of support. ACP thanks the Colt Foundation and the British Lung Foun-

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dation for recent support. The authors greatly acknowledge the scientific contributions and collaborative efforts of R.M. Agius, N.M. Cherry, K.L. Harrison, S.J. Lewis (Centre for Occupational and Environmental Health, University of Manchester), M. Gittens (Biostatistics Group, University of Manchester), P.V. Barber, P.A.J. Crosbie, K. DeFreyne (deceased), R. McLNiven, P.N.S. O’Donnell (North West Lung Centre, Wythenshawe Hospital, Manchester), G. McGown, S.J. Pearson, M.R. Thorncroft, A.J. Watson (Paterson Institute for Cancer Research, Manchester), J. Heighway (University of Liverpool Cancer ¨ Research Centre, Liverpool), K. Rohde (Max-Delbruck-Centrum ¨ Molekulare Medizin, Berlin-Buch), D. Donnelly, S. McElfur hinney (deceased), T.B.H. McMurry (Chemistry Department, Trinity College, Dublin).

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