alkylguanine-DNA Alkyltransferase Research Articles

Molecular imaging of alkylguanine-DNA alkyltransferase: further evaluation of radioiodinated derivatives of O6-benzylguanine

An inverse correlation has been established between tumor levels of the DNA repair protein alkylguanine-DNA alkyltransferase (AGT) and a positive outcome after alkylator chemotherapy. Quantitative imaging of AGT could provide important information for patient-specific cancer treatment. Several radiolabeled analogues of O6-benzylguanine (BG), a potent AGT inactivator, have been developed and shown to be capable of labeling pure AGT protein. Herein, two of these analogues--O6-3-[*I]iodobenzylguanine ([*I]IBG) and O6-3-[*I]iodobenzyl-2'-deoxyguanosine ([*I]IBdG)--were further evaluated in two murine xenograft models. (AcO)2-[131I]IBdG, a peracetylated derivative of IBdG, also was investigated as an alternative agent. Several biodistribution studies of radioiodinated IBG and IBdG were performed in TE-671 human rhabdomyosarcoma and DAOY human medulloblastoma murine xenograft models. Mice were treated with BG or its nucleoside analogue dBG to deplete the tumor AGT content. The effect of unlabeled IBG and that of 7,8-benzoflavone (BF), an inhibitor of the cytochrome P-450 isozyme CYP1A2, on the tumor uptake of the tracers was determined. The uptake of (AcO)2-[131I]IBdG along with that of [125I]IBdG in DAOY cells in vitro was determined in the presence and absence of a nucleoside transporter inhibitor, dipyridamole. Pretreatment of mice either with BG or dBG failed to reduce tumor levels of [*I]IBG or [*I]IBdG even though such treatments completely depleted tumor AGT content. Treatment of mice with BF increased tumor uptake of [125I]IBG by 56%; however, differentiation of tumors with and without AGT still was not possible. (AcO)2-[131I]IBdG, a peracetylated derivative of IBdG, had a higher uptake in vitro in DAOY tumor cells. However, its uptake, like that of [125I]IBdG, was blocked by dipyridamole. Taken together, these results suggest that labeled agents that are more specific for cellular AGT and that are more metabolically stable are needed.

Is protracted low-dose temozolomide feasible in glioma patients?

The authors investigated the safety of 75 mg/m2 temozolomide for 21 days every 28 days in glioma patients. This schedule could lead to DNA repair enzyme O6-alkylguanine-DNA alkyltransferase depletion, contributing to overcoming drug resistance. Although Phase III studies are forthcoming, no data are available on the long-term toxicity of temozolomide, which, in this series, incurred prolonged, cumulative lymphopenia, which leads to a high incidence of infections.

Polar, functionalized guanine- O6 derivatives resistant to repair by O6 -alkylguanine–DNA alkyltransferase: implications for the design of DNA-modifying drugs

The protein O6 -alkylguanine–DNA alkyltransferase (Atase) is responsible for the repair of DNA lesions generated by several clinically important anti-cancer drugs; this is manifest as active resistance in those cancer cell lines proficient in Atase expression. Novel O6-substituted guanine analogues have been synthesized, bearing acidic, basic and hydrogen bonding functional groups. In contrast to existing O6-modified purine analogues, such as methyl or benzyl, the new compounds were found to resist repair by Atase even when tested at concentrations much higher than O6-benzylguanine, a well-established Atase substrate active both in vitro and in vivo. The inactivity of the new purines as covalent substrates for Atase indicates that agents to deliver these groups to DNA would represent a new class of DNA-modifying drug that circumvents Atase-mediated resistance.

Inhibition of O6-methylguanine-DNA methyltransferase by an alkyltransferase-like protein from Escherichia coli

The alkyltransferase-like (ATL) proteins contain primary sequence motifs resembling those found in DNA repair O6-alkylguanine-DNA alkyltransferase proteins. However, in the putative active site of ATL proteins, a tryptophan (W83) residue replaces the cysteine at the known active site of alkyltransferases. The Escherichia coli atl gene was expressed as a fusion protein and purified. Neither ATL nor C83 or A83 mutants transferred [3H] from [3H]-methylated DNA to themselves, and the levels of O6-methyl guanine (O6-meG) in substrate DNA were not affected by ATL. However, ATL inhibited the transfer of methyl groups to human alkyltransferase (MGMT). Inhibition was reduced by prolonged incubation in the presence of MGMT, again suggesting that O6-meG in the substrate is not changed by ATL. Gel-shift assays show that ATL binds to short single- or double-stranded oligonucleotides containing O6-meG, but not to oligonucleotides containing 8-oxoguanine, ethenoadenine, 5-hydroxycytosine or O4-methylthymine. There was no evidence of demethylation of O6-meG or of glycosylase or endonuclease activity. Overexpression of ATL in E.coli increased, or did not affect, the toxicity of N-methyl-N′-nitro-N-nitrosoguanidine in an alklyltransferase-proficient and -deficient strain, respectively. These results suggest that ATL may act as a damage sensor that flags O6-meG and possibly other O6-alkylation lesions for processing by other repair pathways.

Engineering Substrate Specificity of O6‐Alkylguanine‐DNA Alkyltransferase for Specific Protein Labeling in Living Cells

Fusion proteins of human O(6)-alkylguanine-DNA alkyltransferase (AGT) can be specifically labeled with a wide variety of synthetic probes in mammalian cells; this makes them an attractive tool for studying protein function. However, to avoid undesired labeling of endogenous wild-type AGT (wtAGT), the specific labeling of AGT fusion proteins has been restricted to AGT-deficient mammalian cell lines. We present here the synthesis of an inhibitor of wtAGT and the generation of AGT mutants that are resistant to this inhibitor. This enabled the inactivation of wtAGT and specific labeling of fusion proteins of the AGT mutant in vitro and in living cells. The ability to specifically label AGT fusion proteins in the presence of endogenous AGT, after brief incubation of the cells with a small-molecule inhibitor, should significantly broaden the scope of application of AGT fusion proteins for studying protein function in living cells.

O6-3-[ 125I]iodobenzyl-2′-deoxyguanosine ([ 125I]IBdG): synthesis and evaluation of its usefulness as an agent for quantification of alkylguanine-DNA alkyltransferase (AGT)

The development of O 6-(3-[ 125I]iodobenzyl)-2′-deoxyguanosine ([ 125I]IBdG), the glycosylated analogue of the O 6-3-iodobenzylguanine (IBG), as an agent for the in vivo mapping of the DNA repair protein alkylguanine-DNA alkyltransferase (AGT) is described. Synthesis of its tin precursor, O 6-3-trimethylstannylbenzyl-2′-deoxyguanosine (TBdG) was achieved in four steps from deoxyguanosine. Radioiodination of TBdG in a single step gave [ 125I]IBdG in 70–85% isolated radiochemical yield. [ 125I]IBdG bound specifically to pure AGT with an IC 50 of 7.1 μM. From paired-label assays, [ 125I]IBdG showed a 2- to 3-fold higher cellular uptake than [ 131I]IBG in DAOY medulloblastoma, TE-671 rhabdomyosarcoma, SK-Mel-28 melanoma, and HT-29 colon carcinoma human cell lines. Uptake of both labeled compounds in these cell lines decreased with increasing concentrations of unlabeled O 6-benzylguanine (BG) when BG was present in the medium during incubation with the labeled compounds. Compared to BG, unlabeled IBdG diminished the uptake of [ 125I]IBdG and [ 131I]IBG in DAOY cells more efficiently (IC 50 < 1 μM vs >10 μM for BG). There was no significant change in cell-bound activity of [ 125I]IBdG and [ 131I]IBG when BG was removed from the incubation medium before incubating cells with the tracers, suggesting that only a very small portion of radioactivity taken up by the cells is AGT bound. This was corroborated by gel-electrophoresis performed on extracts from cells treated with varying amounts of BG and then incubated with [ 125I]IBdG in the presence of BG. No radiolabeled AGT band was discernable by phosphor-imaging, signifying low cellular AGT binding of the radiotracer. In contrast, when cell extracts were prepared from BG pre-treated cells and aliquots were incubated with [ 125I]IBdG subsequently, the intensity of radiolabeled AGT band decreased linearly as a function of BG concentration. This suggests that the low level of [ 125I]IBdG that binds to AGT does so in a concentration dependent manner. These data suggest that IBdG is transported across the cell membrane to a higher degree than IBG. However, to be a practical tracer for quantifying cellular AGT, considerable localization of such derivatives need to occur within the cell nucleus where AGT is present predominantly.

Quantitative trait locus analysis reveals two intragenic sites that influence O6 -alkylguanine-DNA alkyltransferase activity in peripheral blood mononuclear cells

The repair of specific types of DNA alkylation damage by O6-alkylguanine-DNA alkyltransferase (MGMT) is a major mechanism of resistance to the carcinogenic and chemotherapeutic effects of certain alkylating agents. MGMT expression levels vary widely between individuals but the underlying causes of this variability are not known. To address this, we used an expressed single nucleotide polymorphism (SNP) and demonstrated that the MGMT alleles are frequently expressed at different levels in peripheral blood mononuclear cells (PBMC). This suggests that there is a genetic component of inter-allelic variation of MGMT levels that maps close to or within the MGMT locus. We then used quantitative trait locus (QTL) analysis using intragenic SNPs and found that there are at least two sites influencing inter-individual variation in PBMC MGMT activity. One is characterized by an SNP at the 3' end of the first intron and the second by two SNPs in the last exon. The latter are in perfect disequilibrium and both result in amino acid substitutions-one of them, Ile143Val, affecting an amino acid close to the Cys145 residue at the active site of MGMT. Using in vitro assays, we further showed that while the Val143 variant did not affect the activity of the protein on methylated DNA substrate, it was more resistant to inactivation by the MGMT pseudosubstrate, O6-(4-bromothenyl)guanine. These findings suggest that further investigations of the potential epidemiological and clinical significance of inherited differences in MGMT expression and activity are warranted.

Protein-Functionalized Polymer Brushes

A new strategy for the preparation of protein-functionalized polymer brushes is reported, which is based on a combination of surface-initiated atom transfer radical polymerization (ATRP), p-nitrophenyl chloroformate activation of the surface hydroxyl groups, and subsequent O(6)-benzylguanine (BG) functionalization. The BG-functionalized brushes are used to chemoselectively immobilize O(6)-alkylguanine-DNA-alkyltransferase (AGT) fusion proteins with a defined orientation and surface density. These protein-modified polymer brushes are attractive candidates for the development of protein microarrays.

A bifunctional DNA repair protein from Ferroplasma acidarmanus exhibits O 6 -alkylguanine-DNA alkyltransferase and endonuclease V activities

A recently discovered DNA repair protein of 303 aa from the archaeal organism Ferroplasma acidarmanus was studied. This protein (AGTendoV) consists of a fusion of the C-terminal active site domain of O(6)-alkylguanine-DNA alkyltransferase (AGT) with an endonuclease V domain. The AGTendoV recombinant protein expressed in Escherichia coli and purified to homogeneity repaired O(6)-methylguanine lesions in DNA via alkyl transfer action despite the complete absence of the N-terminal domain and some differences in key active site residues present in known AGTs. The AGTendoV recombinant protein also cleaved DNA substrates that contained the deaminated bases uracil, hypoxanthine, or xanthine in a similar manner to E. coli endonuclease V. Expression of AGTendoV in E. coli GWR109, a strain that lacks endogenous AGT activity, protected against both the killing and mutagenic activity of N-methyl-N'-nitro-N-nitrosoguanidine and was more effective in preventing mutations than human alkyltransferase, suggesting that the endonuclease V activity may also repair a promutagenic lesion produced by this alkylating agent. Expression of AGTendoV in a DNA repair-deficient E. coli nfi(-)alkA(-) strain protected from spontaneous mutations arising in saturated cultures and restored the mutation frequency to that found in the nfi(+) alkA(+) strain. These results demonstrate the physiological occurrence of two completely different but functional DNA repair activities in a single polypeptide chain.

β-Glucuronidase-Cleavable Prodrugs of O6-Benzylguanine and O6-Benzyl-2‘-deoxyguanosine

Glucuronic acid linked prodrugs of O(6)-benzylguanine and O(6)-benzyl-2'-deoxyguanosine were synthesized. The prodrugs were found to be quite stable at physiological pH and were more than 200-fold less active as inactivators of O(6)-alkylguanine-DNA alkyltransferase (alkyltransferase) than either O(6)-benzylguanine or O(6)-benzyl-2'-deoxyguanosine. Beta-glucuronidase from both Escherichia coli and bovine liver cleaved the prodrugs efficiently to release O(6)-benzylguanine and O(6)-benzyl-2'-deoxyguanosine, respectively. In combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), the prodrugs were not effective adjuvants for HT29 cell killing. However, as expected, incubation of these prodrugs with beta-glucuronidase in the culture medium led to much more efficient cell killing by BCNU as a result of the liberation of the more potent inactivators, O(6)-benzylguanine and O(6)-benzyl-2'-deoxyguanosine. These prodrugs may be useful for prodrug monotherapy of necrotic tumors that liberate beta-glucuronidase or for antibody-directed enzyme prodrug therapy with antibodies that can deliver beta-glucuronidase to target tumor cells.

Drug Selection Enrichment of Type II Pneumocytes Following Transplant of P140K MGMT Lentiviral Transduced Hematopoietic Stem Cells.

Recent reports have shown that bone marrow derived cells can differentiate into epithelial cells of the lung. Data indicate that this event occurs in the absence of cell fusion, however the numbers of donor-derived cells are present at a low frequency. In this study, we investigated whether hematopoietic donor-derived Type II pneumocytes could be enriched by lentiviral or retroviral expression of P140K MGMT. MGMT encodes O6 alkylguanine DNA alkyltransferase (AGT), a major drug resistance protein for DNA damage induced by BCNU. This gene transfer approach provides a selective advantage to wild type donor cells by transduction with a mutant human AGT (P140K MFG-MGMT) which confers resistance to BG and BCNU. P140K transduced whole bone marrow or Sca+kit+lin- cells were transplanted into lethally irradiated wild type recipients followed by two or three doses of 30 mg/kg BG and 7.5–10 mg/kg BCNU. After drug selection, human AGT expressing murine cells comprised up to 99% of bone marrow in mice harvested 5–7 months after transplant (n=8). These mice also showed enrichment for the total number of donor derived alveolar cells after drug treatment from 25% (n= 2 untreated mice) to 46% ± 9.7 (n=4 mice; p=.0001). The high percentage of donor cells was attributed to the hematopoietic cells present in the lung, however there were also donor-derived cells with epithelial morphology. Similar bone marrow plasticity studies have elicited controversy, largely due to the technical limitations of in situ detection of organ specific donor cells. To overcome the inability to accurately detect single cells in lung tissue, we used dispase mediated cell dissociation followed by analysis of single cell suspensions by fluorescent microscopy. Type II pneumocytes were detected using a Pro-surfactant protein C antibody that is specific for alveolar Type II cells. Donor P140K transduced cells were co-detected using an antibody to AGT. Using this method, 10–12% of total cells isolated from the lung were Type II pneumocytes and 1.5–8% of these cells co-expressed AGT in mice treated with BG and BCNU (n=3). This indicated drug selection mediated expansion of transplanted marrow-derived cells with characteristics of lung epithelial Type II pneumocytes. These numbers represent an increase compared to untreated mice in which we were unable to detect donor-derived Type II cells (n=2) and published results indicating 0.2–0.6% of total lung cells are bone marrow-derived epithelial cells. This study indicates that MGMT mediated drug selection of hematopoietic cells, using lentiviral MGMT gene transfer, enhances their capacity for organ specific selection and repopulation of lung alveolar epithelial cells. Drug selection gene transfer may augment transdifferentiation and have therapeutic implications for pulmonary disorders.

Imaging drug resistance with radiolabeled molecules.

A major obstacle to successful cancer chemotherapy is drug resistance. Multidrug resistance (MDR) is often seen with chemotherapeutic agents such as anthracycline derivatives, vinca alkaloids and taxanes. Multiple aspects of cellular biochemistry have been implicated in the MDR process. Cellular mechanisms of resistance are due to the presence of efflux pumps, P-glycoprotein (P-gp) and multiple resistance-associated protein (MRP), which belong to the ATP-binding cassette (ABC) family of transporters. Another form of drug resistance is involved in the chemotherapy of cancers with alkylating agents such as nitrosourea derivatives and nitrogen mustards. The cytotoxicity of these agents is primarily due to alkylation of the DNA guanine residues at their O6-position, which leads, via a cascade of events, to DNA strand breaks. The DNA repair protein, alkylguanine-DNA alkyl transferase (AGT) removes the alkyl groups from the lesions stoichiometrically to a cysteine in its active site. This process is irreversible and results in the degradation of the protein and its recovery is entirely from de novo synthesis. Noninvasive methodologies for monitoring the transport activity of these efflux pumps and determining tumor content of AGT could serve as critical tools for optimizing chemotherapeutic protocols on a patient-specific basis and gaining an understanding of the dynamics of resistance in living patients. In this review, we will describe the efforts made to date to synthesize radioactive probes of chemotherapy resistance and their use to quantitate these transporters and DNA repair protein by radionuclide imaging.

Phase II trial of O6-benzylguanine (O6BG) and BCNU in patients with advanced melanoma

7524 Background: While alkylating agents such as BCNU are active in melanoma, they give response rates of only 10–15%. BCNU induces DNA damage by formation of a chloroethyl adduct at the O6 position of guanine. This modification can be repaired by alkylguanine DNA alkyltransferase (AGT), and melanoma tumor cells can express high levels of AGT. O6BG is a potent inhibitor of AGT. We hypothesized that addition of O6BG would improve the clinical response to BCNU in patients with advanced melanoma. Methods: Patients had measurable disease, adequate organ function, PS≤ 2, and a corrected DLCO of ≥ 70% predicted. Patients were accrued into 2 cohorts based on whether or not they received prior chemotherapy. O6BG (120 mg/m2) was administered i.v. over 1 hour, followed by BCNU (40 mg/m2) i.v. over 1 hour, as an outpatient. PBMC were collected pre- O6BG and 18 hours later to assess for AGT depletion. Treatment cycles were repeated every 6 weeks and response using RECIST criteria was assessed every 2 cycles. Results: 42 patients were enrolled, 22 chemo-naive and 20 with prior chemo. In the chemo-naive cohort there were 1 CR, 4 SD, 13 PD, and 4 non-evaluable (NE) patients; the median time to progression (TTP) was 80d and the median survival (MS) was 211d. In the prior chemo cohort there were no objective responses, 3 SD, 15 PD, and 2 NE patients; the median TTP was 40d and the MS was 120d. The NE patients had clinical disease progression. AGT was effectively depleted from PBMC in the 15 patients tested (p<0.001). Grade 3/4 toxicities included thrombocytopenia (n=24), neutropenia (19), anemia (10), lymphopenia (7), fever (2), fatigue (2), hyponatremia (2), neuropathy (1), vomiting (1), decreased DLCO (1), and death (1). Toxicities were similar in the two cohorts. Conclusions: O6BG/BCNU was successfully administered on an outpatient basis and depleted AGT from PBMC. However, significant myelosuppression was seen and the clinical response rate was not greater than what is expected with BCNU alone. Although this regimen should not be explored further, administration of O6BG with other alkylating agents with greater single agent activity should be considered. No significant financial relationships to disclose.

Phase II trial of O6-benzylguanine (O6BG) and BCNU in patients with advanced melanoma

7524 Background: While alkylating agents such as BCNU are active in melanoma, they give response rates of only 10–15%. BCNU induces DNA damage by formation of a chloroethyl adduct at the O6 position of guanine. This modification can be repaired by alkylguanine DNA alkyltransferase (AGT), and melanoma tumor cells can express high levels of AGT. O6BG is a potent inhibitor of AGT. We hypothesized that addition of O6BG would improve the clinical response to BCNU in patients with advanced melanoma. Methods: Patients had measurable disease, adequate organ function, PS≤ 2, and a corrected DLCO of ≥ 70% predicted. Patients were accrued into 2 cohorts based on whether or not they received prior chemotherapy. O6BG (120 mg/m2) was administered i.v. over 1 hour, followed by BCNU (40 mg/m2) i.v. over 1 hour, as an outpatient. PBMC were collected pre- O6BG and 18 hours later to assess for AGT depletion. Treatment cycles were repeated every 6 weeks and response using RECIST criteria was assessed every 2 cycles. Results:...

Labeling of fusion proteins with synthetic fluorophores in live cells.

A general approach for the sequential labeling of fusion proteins of O(6)-alkylguanine-DNA alkyltransferase (AGT) with different fluorophores in mammalian cells is presented. AGT fusion proteins with different localizations in the cell can be labeled specifically with different fluorophores, and the fluorescence labeling can be used for applications such as multicolor analysis of dynamic processes and fluorescence resonance energy transfer measurements. The facile access to a variety of different AGT substrates as well as the specificity of the labeling reaction should make the approach an important tool to study protein function in live cells.

DNA binding and nucleotide flipping by the human DNA repair protein AGT.

O(6)-alkylguanine-DNA alkyltransferase (AGT), or O(6)-methylguanine-DNA methyltransferase (MGMT), prevents mutations and apoptosis resulting from alkylation damage to guanines. AGT irreversibly transfers the alkyl lesion to an active site cysteine in a stoichiometric, direct damage reversal pathway. AGT expression therefore elicits tumor resistance to alkylating chemotherapies, and AGT inhibitors are in clinical trials. We report here structures of human AGT in complex with double-stranded DNA containing the biological substrate O(6)-methylguanine or crosslinked to the mechanistic inhibitor N(1),O(6)-ethanoxanthosine. The prototypical DNA major groove-binding helix-turn-helix (HTH) motif mediates unprecedented minor groove DNA binding. This binding architecture has advantages for DNA repair and nucleotide flipping, and provides a paradigm for HTH interactions in sequence-independent DNA-binding proteins like RecQ and BRCA2. Structural and biochemical results further support an unpredicted role for Tyr114 in nucleotide flipping through phosphate rotation and an efficient kinetic mechanism for locating alkylated bases.

The repair of the tobacco specific nitrosamine derived adduct O6-[4-Oxo-4-(3-pyridyl)butyl]guanine by O6-alkylguanine-DNA alkyltransferase variants.

The tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent pulmonary carcinogen, both methylates and pyridyloxobutylates DNA. Both reaction pathways generate promutagenic O6-alkylguanine adducts. These adducts, O6-methylguanine (O6-mG) and O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG), are repaired by O6-alkylguanine-DNA alkyltransferase (AGT). In this report, we demonstrate that pyridyloxobutyl DNA adducts are repaired by AGT in a reaction that results in pyridyloxobutyl transfer to the active site cysteine. Because minor changes within the binding pocket of AGT can alter the ability of this protein to repair bulky O6-alkylguanine adducts relative to O6-mG, we explored the ability of AGTs from different species as well as several human AGT variants and mutants to discriminate between O6-mG or O6-pobG adducts. We incubated proteins with equal molar amounts of oligodeoxynucleotides containing site specifically incorporated O6-mG or O6-pobG and measured repair. Bacterial AGTs poorly repaired O6-pobG. Mouse and rat AGT repaired both adducts at comparable rates. Wild-type human AGT, variant I143V/K178R, and mutant N157H repaired O6-mG approximately twice as fast as O6-pobG. Human variant G160R and mutants P140K, Y158H, G156A, and E166G did not repair O6-pobG until all of the O6-mG was removed. To understand the role of adduct structure on relative repair rates, the competition experiments were repeated with two other bulky O6-alkylguanine adducts, O6-butylguanine (O6-buG) and O6-benzylguanine (O6-bzG). The proteins displayed similar repair preference of O6-mG relative to O6-buG as observed with O6-pobG. In contrast, all of the mammalian proteins, except the mutant P140K, preferentially repaired O6-bzG. These studies indicate that the rate of repair of O6-pobG is highly dependent on protein structure. Inefficient repair of O6-pobG by bacterial AGT explains the high mutagenic activity of this adduct in bacterial systems. In addition, differences observed in the repair of this adduct by mammalian proteins may translate into differences in sensitivity to the mutagenic and carcinogenic effects of NNK or other pyridyloxobutylating nitrosamines.

Exon 5 polymorphisms in the O6-alkylguanine DNA alkyltransferase gene and lung cancer risk in non-smokers exposed to second-hand smoke.

The objective of the study was to examine the association of three exon 5 variants in the O(6)-alkylguanine DNA alkyltransferase (AGT) gene involved in the repair of the mutagenic DNA lesion O(6)-alkylguanine formed by nitrosamines, with lung cancer risk in never-smokers. Exon 5 of the AGT gene was sequenced in genomic DNA from 136 cases and 133 hospital- or population-based controls for whom questionnaire information on second-hand smoke and diet was available to determine the frequencies of the Gly(160)Arg, Ile(143)Val, and Lys(178)Arg variant alleles. No codon (160)Arg variant alleles were found in the study population. The codon (143)Val and (178)Arg variant alleles, present at allele frequencies of 0.07, showed 100% linkage. The odds ratio (OR) of lung cancer for these variant carriers was 2.05 [95% confidence interval (CI) 1.03-4.07]. The risk varied between the different lung cancer pathologies with an increased risk for adenocarcinoma (OR 2.67, 95% CI 1.21-5.87) or small cell carcinoma (OR 4.83, 95% CI 0.91-25.7) but not for squamous cell carcinoma (OR 1.07, 95% CI 0.27-4.18). Compared with individuals carrying the mutant alleles unexposed to second-hand smoke, the OR for exposed variant carriers was 1.95 (95% CI 0.53-1.15); a similar interaction, although not significative, was observed for low consumption of cruciferous vegetables and for green vegetables and tomatoes. These results point toward a role of AGT polymorphisms in lung cancer susceptibility among never-smokers, in particular among subjects exposed to environmental carcinogens.

Molecular predictors of response to chemotherapy in lung cancer

Overall, chemotherapy falls short of the high expectations for improved survival in surgically resected non-small cell lung cancer patients and prolonged survival in the metastatic setting. Conventional chemotherapy trials, even those including new cytotoxic drugs or novel targeting approaches, are hampered by a lack of genetic information. Within the global genomic repair pathway, overexpression of excision repair cross-complementing 1 (ERCC1) has been associated with poor response and survival in cisplatin-treated patients. The lack of DNA adducts in cell nuclei indicates an efficient global genomic repair pathway, which leads to cisplatin resistance. Several xeroderma pigmentosum (XP) genes, including XPD, play an important role in determining the efficiency of the transcription-coupled repair pathway. XPD polymorphism has been related to lower DNA repair capacity and enhanced cisplatin sensitivity. Other DNA repair systems are the base excision repair pathway, in which apurinic/apyrimidinic endonuclease 1 (Ape 1) plays a pivotal role, and the one-step repair pathway, where O 6−alkylguanine-DNA alkyltransferase (MGMT) has a key function. MGMT methylation can be assessed in serum DNA. By assessing ERCC1 mRNA, cisplatin adducts, XPD polymorphism, Ape 1, and MGMT, we can obtain a complete genetic profile, which can be used in real translational research.

Reduced MGMT activity in human colorectal adenomas is associated with K-ras GC->AT transition mutations in a population exposed to methylating agents.

There is increasing evidence to suggest that O(6)-alkyl guanine DNA-alkyltransferase (MGMT) activity provides protection against alkylating agent induced formation of GC-->AT transition mutations in the K-ras oncogene of colorectal tumours. As this mutagenic event occurs during the growth of adenomas, both biomarkers of exposure (N7-methylguanine levels in DNA) and susceptibility (MGMT activity) were measured in biopsy samples obtained from normal and adenomatous tissue from 34 patients with large adenomas (>10 mm in size). There was no correlation between MGMT activity in the adenoma and in matched normal tissue. However, MGMT activity was significantly lower in adenoma tissue than in adjacent normal mucosa (5.18 versus 7.05 fmol/microg DNA, P = 0.01), particularly in men and those whose age was greater than the median. Upon stratification by K-ras mutational status, MGMT activity was lower in adenomas bearing a K-ras GC-->AT transition mutation (mean 4.21 fmol/microg DNA) than in adjacent normal tissue (mean 7.7 fmol/microg DNA; P < 0.004). In contrast, there was no significant difference in MGMT activity in adenomas lacking a K-ras GC-->AT transition mutation and adjacent normal mucosa. N7-methylguanine levels however did not vary with age, gender, K-ras mutational status or MGMT activity. These results are consistent with the acquisition of K-ras GC-->AT transition mutations in adenomas with low MGMT activity as a result of unavoidable exposure to methylating agents.