Lung cancer risk and variation in MGMT activity and sequence

O(6)-Alkylguanine-DNA alkyltransferase (MGMT) repairs DNA adducts that result from alkylation at the O(6) position of guanine. These lesions are mutagenic and toxic and can be produced by a variety of agents including the tobacco-specific nitrosamines, carcinogens present in cigarette smoke. Here, we review some of our work in the context of inter-individual differences in MGMT expression and their potential influence on lung cancer risk. In humans there are marked inter-individual differences in not only levels of DNA damage in the lung (N7-methylguanine) that can arise from exposure to methylating agents but also in MGMT activity in lung tissues. In the presence of such exposure, this variability 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.     

Human variants of O6-alkylguanine-DNA alkyltransferase

This article summarizes the current understanding of known variant forms of the MGMT gene that encode an altered protein. Epidemiological studies have been carried out to test whether these alterations are associated with altered cancer risk. Laboratory studies using recombinant proteins and cells expressing the known variants have investigated the possible effects of these sequence alterations on the ability of the encoded O(6)-alkylguanine-DNA alkyltransferase protein to protect cells from alkylation damage and to respond to therapeutic inactivators currently undergoing trials for cancer chemotherapy.     

Alkyltransferase-like proteins

Recent in silico analysis has revealed the presence of a group of proteins in pro and lower eukaryotes, but not in Man, that show extensive amino acid sequence similarity to known O(6)-alkylguanine-DNA alkyltransferases, but where the cysteine at the putative active site is replaced by another residue, usually tryptophan. Here we review recent work on these proteins, which we designate as alkyltransferase-like (ATL) proteins, and consider their mechanism of action and role in protecting the host organisms against the biological effects of O(6)-alkylating agents, and their evolution. ATL proteins from Escherichia coli (eAtl, transcribed from the ybaz open reading frame) and Schizosaccharomyces pombe (Atl1) are able to bind to a range of O(6)-alkylguanine residues in DNA and to reversibly inhibit the action of the human alkyltransferase (MGMT) upon these substrates. Isolated proteins were not able to remove the methyl group in O(6)-methylguanine-containing DNA or oligonucleotides, neither did they display glycosylase or endonuclease activity. S. pombe does not contain a functional alkyltransferase and atl1 inactivation sensitises this organism to a variety of alkylating agents, suggesting that Atl1 acts by binding to O(6)-alkylguanine lesions and signalling them for processing by other DNA repair pathways. Currently we cannot exclude the possibility that ATL proteins arose through independent mutation of the alkyltransferase gene in different organisms. However, analyses of the proteins from E. coli and S. pombe, are consistent with a common function.     

O(6)-methylguanine-DNA methyltransferase (MGMT): impact on cancer risk in response to tobacco smoke

Tobacco, smoked, snuffed and chewed, contains powerful mutagens and carcinogens. At least three of them, N-dimethylnitrosamine, N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, attack DNA at the O(6)-position of guanine. The resulting O(6)-alkylguanine adducts are repaired by the suicide enzyme O(6)-methylguanine-DNA methyltransferase (MGMT), which is known to protect against the mutagenic, genotoxic and carcinogenic effects of monofunctional alkylating agents. While in rat liver MGMT was shown to be subject to regulation by genotoxic stress leading to adaptive changes in its activity, in humans evidence of adaptive modulation of MGMT levels is still lacking. Several polymorphisms are known, which are suspected to impact on the risk of developing cancer. In this review we focus on three questions: (a) Has tobacco consumption by smoking or chewing an impact on MGMT expression and MGMT promoter methylation in normal and tumor tissue? (b) Is there an association between MGMT polymorphisms and cancer risk and is this risk related to smoking? (c) Does MGMT protect against tobacco-associated cancer? There are several lines of evidence for an increase of MGMT activity in the normal tissue of smokers compared to non-smokers. Furthermore, in tumors developed in smokers a tendency towards an increase of MGMT expression was found. The data points to the possibility that agents in tobacco smoke are able to trigger upregulation of MGMT in normal and tumor tissue. For MGMT promoter methylation data is conflicting. There is some evidence for an association between MGMT polymorphisms and smoking-induced cancer risk. The key question whether or not MGMT protects against tobacco smoke-induced cancer is difficult to answer since prospective studies on smokers versus non-smokers are lacking and appropriate animal studies with MGMT transgenic mice exposed to the complex mixture of tobacco smoke have not been performed, which indicates the need for further explorations.     

MGMT inhibitors--The Trinity College-Paterson Institute experience, a chemist's perception

The DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase (MGMT) can confer resistance to the cancer chemotherapeutic effects of the class of DNA damaging drugs generally referred to as the O(6)-alkylating agents. Inactivation of MGMT is thus a practical approach to improving the efficacy of such agents. An account is given of the collaboration between groups at Trinity College, Dublin and the Paterson Institute, Manchester which led to the development of the MGMT inactivating drug, Patrin (PaTrin-2, Lomeguatrib). The development of a simpler method of synthesis of O(6)-arylmethylguanines opened up the way to make a series of O(6)-heteroalkylmethyl analogues of the archetypal MGMT pseudosubstrate, O(6)-methylguanine. Of these, the furfuryl and thenyl compounds were the most active against recombinant Human MGMT in an in vitro assay. The 4-bromothenyl derivative was chosen for clinical trial as the most active compound. The MGMT active site tolerates O(6)-substituted guanines where the side chain can be quite large, but does not tolerate those with an aromatic or heteroaromatic ring with an 'ortho' substituent.     

MGMT hypermethylation: a prognostic foe, a predictive friend

Alkylation of DNA at the O(6)-position of guanine is one of the most critical events leading to mutation, cancer, and cell death. O(6)-alkylguanine-DNA alkyltransferase (AGT), also known as O(6)-methylguanine-DNA methyltransferase (MGMT), is the DNA repair protein responsible for removing alkylation adducts from the O(6)-position of guanine in DNA. The promoter CpG island hypermethylation-associated gene silencing of MGMT is associated with a wide spectrum of human tumors. This epigenetic inactivation of MGMT has two main consequences in human cancer. First, it uncovers a new mutator pathway that causes the accumulation of G-to-A transition mutations that can affect genes required for genomic stability. Second, there is a strong and significant positive correlation between MGMT promoter hypermethylation and increased tumor sensitivity to alkylating drugs. These findings underline the importance of MGMT promoter hypermethylation in basic and translational cancer research.     

Variation in DNA repair is a factor in cancer susceptibility: a paradigm for the promises and perils of individual and population risk estimation?

The repair of DNA damage protects the genome of the cell from the insults of cancer causing agents. This was originally demonstrated in individuals with the rare genetic disease, xeroderma pigmentosum, the prototype of cancer genes, and subsequently in the relationship of mismatch repair to colon cancer. Recent studies suggests that individuals with less dramatic reductions in the capacity to repair DNA damage are observed at polymorphic frequency and these individuals have an increased susceptibility to several types of cancer. Screening of individuals for DNA sequence variation in the exons of 9 DNA repair genes has resulted in identification of 15 different polymorphic amino acid substitution variants. Although the studies to relate these variants to reduced DNA repair capacity and cancer status have not been completed, the available information is sufficient to suggest that DNA repair genes should be incorporated into molecular epidemiology and cancer susceptibility studies. The availability of molecular epidemiology data presents exciting opportunities for refinement of risk estimation models and identification of individuals at increased risk of disease, with resultant opportunities for effective surveillance and early intervention and treatment. The opportunities to acquire susceptibility data are associated with possible perils for establishment of regulations for permissible exposures to carcinogenic agents and also stigmatization of ‘at risk’ individuals that may result in decreased access to employment opportunities and health care.     

Repair of O(6)-alkylguanine by alkyltransferases

The predominant pathway for the repair of O(6)-methylguanine in DNA is via the activity of an alkyltransferase protein that transfers the methyl group to a cysteine acceptor site on the protein itself. This review article describes recent studies on this alkyltransferase. The protein repairs not only methyl groups but also 2-chloroethyl-, benzyl- and pyridyloxobutyl-adducts. It acts on double-stranded DNA by flipping the O(6)-guanine adduct out of the DNA helix and into a binding pocket. The free base, O(6)-benzylguanine, is able to bind in this pocket and react with the cysteine, rendering it an effective inactivator of mammalian alkyltransferases. The alkylated form of the protein is rapidly degraded by the ubiquitin/proteasomal system. Some tumor cells do not express alkyltransferase despite having an intact gene. Methylation of key sites in CpG-rich islands in the promoter region are involved in this silencing and a change in the nuclear localization of an enhancer binding protein may also contribute. The alkyltransferase promoter contains Sp1, GRE and AP-1 sites and is slightly inducible by glucocorticoids and protein kinase C activators. There is a complex relationship between p53 and alkyltransferase expression with p53 mediating a rise in alkyltransferase in response to ionizing radiation but having no clear effect on basal levels. DNA adducts at the O(6)-position of guanine are a major factor in the carcinogenic, mutagenic, apoptopic and clastogenic actions of methylating agents and chloroethylating agents. Studies with transgenic mice in which alkyltransferase levels are increased or decreased confirm the importance of this repair pathway in protecting against carcinogenesis. Alkyltransferase activity in tumors protects them from therapeutic agents such as temozolomide and BCNU. This resistance is abolished by O(6)-benzylguanine and this drug is currently in clinical trials to enhance cancer chemotherapy by these agents. Studies are in progress to reduce the toxicity of such therapy towards the bone marrow by gene therapy to express alkyltransferases with mutations imparting resistance to O(6)-benzylguanine at high levels in marrow stem cells. Several polymorphisms in the human alkyltransferase gene have been identified but the significance of these in terms of alkyltransferase action is currently unknown.     

Survey of polymorphic sequence variation in the immediate 5' region of human DNA repair genes

Systematic screens have revealed extensive DNA sequence variation existing in the human population. Studies of the role of polymorphic genetic variants in explaining the association of family history with risk of common disease have generally focused on variants predicted to disrupt protein structure and activity. Recent studies have identified genetic variation in the level of expression of many genes, variation that is potentially biologically relevant in explaining individual variation in disease risk. In a survey of data available for 108 DNA repair genes that have been systematically screened for sequence variation, an average of 3.3 SNPs per gene were found to exist at a variant allele frequency of at least 0.02 in the region 2kb upstream from the 5'-untranslated region. One-third of the genes harbored a SNP with an allele frequency of at least 0.02 within a predicted promotor element. These variants are distributed among promoter elements that average 20 elements per gene. The frequency of polymorphic SNPs in CpG islands was 0.8 per gene, while the frequency of SNPs in the 5'-UTR was 0.7 per gene. The recognition of extensive genetic variation with potential to impact levels of gene expression, and thereby exacerbate the impact of amino acid substitution variants on the activity of proteins, increases the complexity of analyses required to explain the molecular genetic basis for the familial contribution to the sporadic incidence of common disease.     

Challenges and complexities in estimating both the functional impact and the disease risk associated with the extensive genetic variation in human DNA repair genes

Individual risk and the population incidence of disease result from the interaction of genetic susceptibility and exposure. DNA repair is an example of a cellular process where genetic variation in families with extreme predisposition is documented to be associated with high disease likelihood, including syndromes of premature aging and cancer. Although the identification and characterization of new genes or variants in cancer families continues to be important, the focus of this paper is the current status of efforts to define the impact of polymorphic amino acid substitutions in DNA repair genes on individual and population cancer risk. There is increasing evidence that mild reductions in DNA repair capacity, assumed to be the consequence of common genetic variation, affect cancer predisposition. The extensive variation being found in the coding regions of DNA repair genes and the large number of genes in each of the major repair pathways results in complex genotypes with potential to impact cancer risk in the general population. The implications of this complexity for molecular epidemiology studies, as well as concepts that may make these challenges more manageable, are discussed. The concepts include both experimental and computational approaches that could be employed to develop predictors of disease susceptibility based on DNA repair genotype, focusing initially on studies to assess functional impact on individual proteins and pathways and then on molecular epidemiology studies to assess exposure-dependent health risk. In closing, we raise some of the non-technical challenges to the utilization of the full richness of the genetic variation to reduce disease occurrence and ultimately improve health care.     

O(6)-Methylguanine-DNA methyltransferase (MGMT) in normal tissues and tumors: Enzyme activity, promoter methylation and immunohistochemistry

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the pre-mutagenic, pre-carcinogenic and pre-toxic DNA damage O(6)-methylguanine. It also repairs larger adducts on the O(6)-position of guanine, such as O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine and O(6)-chloroethylguanine. These adducts are formed in response to alkylating environmental pollutants, tobacco-specific carcinogens and methylating (procarbazine, dacarbazine, streptozotocine, and temozolomide) as well as chloroethylating (lomustine, nimustine, carmustine, and fotemustine) anticancer drugs. MGMT is therefore a key node in the defense against commonly found carcinogens, and a marker of resistance of normal and cancer cells exposed to alkylating therapeutics. MGMT also likely protects against therapy-related tumor formation caused by these highly mutagenic drugs. Since the amount of MGMT determines the level of repair of toxic DNA alkylation adducts, the MGMT expression level provides important information as to cancer susceptibility and the success of therapy. In this article, we describe the methods employed for detecting MGMT and review the literature with special focus on MGMT activity in normal and neoplastic tissues. The available data show that the expression of MGMT varies greatly in normal tissues and in some cases this has been related to cancer predisposition. MGMT silencing in tumors is mainly regulated epigenetically and in brain tumors this correlates with a better therapeutic response. Conversely, up-regulation of MGMT during cancer treatment limits the therapeutic response. In malignant melanoma, MGMT is not related to the therapeutic response, which is due to other mechanisms of inherent drug resistance. For most cancers, studies that relate MGMT activity to therapeutic outcome following O(6)-alkylating drugs are still lacking.     

ARLTS1, MDM2 and RAD51 gene variations are associated with familial breast cancer

Genetic factors that contribute to the risk of breast cancer are largely not known and association studies have revealed several genes with low penetrance risk alleles for breast cancer. Analysis of these genes may provide important information on the risk factors affecting carcinogenesis. Variations in the ARLTS1, RAD51 and MDM2 genes have been associated with increased risk of different cancer types but for breast cancer the results are not consistent. In this study we investigated the role of the allelic variants in candidate genes acting in the tumor suppressor, DNA repair and p53 pathways as risk factors for familial breast cancer in 147 patients displaying characteristics of familial disease. Presence of the polymorphic variants were investigated by amplification of the corresponding regions and restriction fragment length polymorphism analysis. Genotype and allele frequencies in the patients were significantly different for all three variants. Our results indicate that the polymorphic variants might affect individual susceptibility towards breast cancer.     

Tissue-specific apoptotic effects of the p53 codon 72 polymorphism in a mouse model

Currently there are several dozen human polymorphisms that have been loosely associated with cancer risk. Correlating such variants with cancer risk has been challenging, primarily due to factors such as genetic heterogeneity, contributions of diet and environmental factors, and the difficulty in obtaining large sample sizes for analysis. Such difficulties can be circumvented with the establishment of mouse models for human variants. Recently, several groups have modeled human cancer susceptibility polymorphisms in the mouse. Remarkably, in each case these mouse models have accurately reflected human phenotypes, and clarified the contribution of these variants to cancer risk. We recently reported on a mouse model for the codon 72 polymorphism in p53, and found that this polymorphism regulates the ability to cooperate with NF-kB and induce apoptosis. Here-in we present evidence that this polymorphism impacts the apoptotic function of p53 in a tissue-specific manner; such tissue-specific effects of polymorphic variants represent an added challenge to human cancer risk association studies. The data presented here support the premise that modeling human polymorphisms in the mouse represents a powerful tool to assess the impact of these variants on cancer risk, progression and therapy.     

NBN Gene Polymorphisms and Cancer Susceptibility: A Systemic Review

The relationship between DNA repair failure and cancer is well established as in the case of rare, high penetrant genes in high cancer risk families. Beside this, in the last two decades, several studies have investigated a possible association between low penetrant polymorphic variants in genes devoted to DNA repair pathways and risk for developing cancer. This relationship would be also supported by the observation that DNA repair processes may be modulated by sequence variants in DNA repair genes, leading to susceptibility to environmental carcinogens. In this framework, the aim of this review is to provide the reader with the state of the art on the association between common genetic variants and cancer risk, limiting the attention to single nucleotide polymorphisms (SNPs) of the NBN gene and providing the various odd ratios (ORs). In this respect, the NBN protein, together with MRE11 and RAD50, is part of the MRN complex which is a central player in the very early steps of sensing and processing of DNA double-strand breaks (DSBs), in telomere maintenance, in cell cycle control, and in genomic integrity in general. So far, many papers were devoted to ascertain possible association between common synonymous and non-synonymous NBN gene polymorphisms and increased cancer risk. However, the results still remain inconsistent and inconclusive also in meta-analysis studies for the most investigated E185Q NBN miscoding variant.     

Repair of O6-alkylguanine by alkyltransferases

The predominant pathway for the repair of O⁶-methylguanine in DNA is via the activity of an alkyltransferase protein that transfers the methyl group to a cysteine acceptor site on the protein itself. This review article describes recent studies on this alkyltransferase. The protein repairs not only methyl groups but also 2-chloroethyl-, benzyl- and pyridyloxobutyl-adducts. It acts on double-stranded DNA by flipping the O⁶-guanine adduct out of the DNA helix and into a binding pocket. The free base, O⁶-benzylguanine, is able to bind in this pocket and react with the cysteine, rendering it an effective inactivator of mammalian alkyltransferases. The alkylated form of the protein is rapidly degraded by the ubiquitin/proteasomal system. Some tumor cells do not express alkyltransferase despite having an intact gene. Methylation of key sites in CpG-rich islands in the promoter region are involved in this silencing and a change in the nuclear localization of an enhancer binding protein may also contribute. The alkyltransferase promoter contains Sp1, GRE and AP-1 sites and is slightly inducible by glucocorticoids and protein kinase C activators. There is a complex relationship between p53 and alkyltransferase expression with p53 mediating a rise in alkyltransferase in response to ionizing radiation but having no clear effect on basal levels. DNA adducts at the O⁶-position of guanine are a major factor in the carcinogenic, mutagenic, apoptopic and clastogenic actions of methylating agents and chloroethylating agents. Studies with transgenic mice in which alkyltransferase levels are increased or decreased confirm the importance of this repair pathway in protecting against carcinogenesis. Alkyltransferase activity in tumors protects them from therapeutic agents such as temozolomide and BCNU. This resistance is abolished by O⁶-benzylguanine and this drug is currently in clinical trials to enhance cancer chemotherapy by these agents. Studies are in progress to reduce the toxicity of such therapy towards the bone marrow by gene therapy to express alkyltransferases with mutations imparting resistance to O⁶-benzylguanine at high levels in marrow stem cells. Several polymorphisms in the human alkyltransferase gene have been identified but the significance of these in terms of alkyltransferase action is currently unknown.     

Transgenic systems in studies on genotoxicity of alkylating agents: critical lesions, thresholds and defense mechanisms

Transgenic systems, both cell lines and mice with gain or loss of function, are being used in order to modulate the expression of DNA repair proteins, thus allowing to assess their contribution to the defense against genotoxic mutagens and carcinogens. In this review, questions have been addressed concerning the use of transgenic systems in elucidating critical primary DNA lesions, their conversion into genotoxic endpoints, low-dose effects, and the relative contribution of individual cellular functions in defense. It has been shown that the repair protein alkyltransferase (MGMT) is decisive for protection against methylating and chloroethylating compounds. Protection pertains also to tumor formation, as revealed by the response of MGMT transgenic and knockout mice. Overexpression of genes involved in base excision repair (N-methylpurine-DNA glycosylase, apurinic endonuclease, DNA polymerase β) is in most cases not beneficial in increasing the protection level, whereas their down-modulation or inactivation increases cellular sensitivity. This indicates that non-repaired base N-alkylation lesions and/or repair intermediates possess genotoxic potential. Modulation of mismatch repair and poly(ADP)ribosyl transferase has also been shown to affect the cellular response to alkylating agents. Furthermore, the role of Fos, Jun and p53 in cellular defense against alkylating mutagens is discussed.     

Tissue-specific apoptotic effects of the p53 codon 72 polymorphism in a mouse model

Currently there are several dozen human polymorphisms that have been loosely associated with cancer risk. Correlating such variants with cancer risk has been challenging, primarily due to factors such as genetic heterogeneity, contributions of diet and environmental factors, and the difficulty in obtaining large sample sizes for analysis. Such difficulties can be circumvented with the establishment of mouse models for human variants. Recently, several groups have modeled human cancer susceptibility polymorphisms in the mouse. Remarkably, in each case these mouse models have accurately reflected human phenotypes, and clarified the contribution of these variants to cancer risk. We recently reported on a mouse model for the codon 72 polymorphism in p53, and found that this polymorphism regulates the ability to cooperate with NFκB and induce apoptosis. Here-in we present evidence that this polymorphism impacts the apoptotic function of p53 in a tissue-specific manner; such tissue-specific effects of polymorphic variants represent an added challenge to human cancer risk association studies. The data presented here support the premise that modeling human polymorphisms in the mouse represents a powerful tool to assess the impact of these variants on cancer risk, progression and therapy.Key words: p53, polymorphism, apoptosis, codon 72, NFκB     

O-Methylguanine-DNA methyltransferase (MGMT) in normal tissues and tumors: Enzyme activity,promoter methylation and immunohistochemistry

O⁶-Methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the pre-mutagenic, pre-carcinogenic and pre-toxic DNA damage O⁶-methylguanine. It also repairs larger adducts on the O⁶-position of guanine, such as O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine and O⁶-chloroethylguanine. These adducts are formed in response to alkylating environmental pollutants, tobacco-specific carcinogens and methylating (procarbazine, dacarbazine, streptozotocine, and temozolomide) as well as chloroethylating (lomustine, nimustine, carmustine, and fotemustine) anticancer drugs. MGMT is therefore a key node in the defense against commonly found carcinogens, and a marker of resistance of normal and cancer cells exposed to alkylating therapeutics. MGMT also likely protects against therapy-related tumor formation caused by these highly mutagenic drugs. Since the amount of MGMT determines the level of repair of toxic DNA alkylation adducts, the MGMT expression level provides important information as to cancer susceptibility and the success of therapy. In this article, we describe the methods employed for detecting MGMT and review the literature with special focus on MGMT activity in normal and neoplastic tissues. The available data show that the expression of MGMT varies greatly in normal tissues and in some cases this has been related to cancer predisposition. MGMT silencing in tumors is mainly regulated epigenetically and in brain tumors this correlates with a better therapeutic response. Conversely, up-regulation of MGMT during cancer treatment limits the therapeutic response. In malignant melanoma, MGMT is not related to the therapeutic response, which is due to other mechanisms of inherent drug resistance. For most cancers, studies that relate MGMT activity to therapeutic outcome following O⁶-alkylating drugs are still lacking.     

Telomerase and DNA repair in glioma

Both DNA repair (MGMT) and immortalisation (telomerase) have been linked to the root of cancer. In glioma, MGMT expression is negatively regulated through promoter methylation and its absence is associated with enhanced chemosensitivity. However, recent studies indicate that telomerase is positively regulated through methylation and its elimination enhances chemotherapy. These observations suggest that suppression of telomerase in combination with MGMT may have additional anti-proliferative and anti-apoptotic effects, which may lead to increased patient survival rates. However, different approaches may be required to compliment the epigenetic events that regulate these genes. Nevertheless, given that median survival of glioma patients is less than a year, this review focuses on the recent approaches used to target MGMT and telomerase, with a view to increase life expectancy of patients while limiting side effects.