Progress in high-throughput assays of MGMT and APE1 activities in cell extracts

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Abstract

DNA repair activity is of interest as a potential biomarker of individual susceptibility to genotoxic agents. In view of the current trend for exploitation of large cohorts in molecular epidemiology projects, there is a pressing need for the development of phenotypic DNA repair assays that are high-throughput, very sensitive, inexpensive and reliable.

Towards this goal we have developed and validated two phenotypic assays for the measurement of two DNA repair enzymes in cell extracts: (1) O6-methylguanine-DNA-methyltransferase (MGMT), which repairs the O6-alkylguanine-type of adducts induced in DNA by alkylating genotoxins; and (2) apurinic/apyrimidinic endonuclease 1 (APE 1), which participates in base excision repair (BER) by causing a rate-limiting DNA strand cleavage 5′ to the abasic sites.

The MGMT assay makes use of the fact that: (a) the enzyme works by irreversibly transferring the alkyl group from the O6 position of guanine to a cystein residue in its active site and thereby becomes inactivated and (b) that the free base O6-benzylguanine (BG) is a very good substrate for MGMT. In the new assay, cell extracts are incubated with BG tagged with biotin and the resulting MGMT-BG-biotin complex is immobilized on anti-MGMT-coated microtiter plates, followed by quantitation using streptavidin-conjugated alkaline phosphatase and a chemiluminescence-producing substrate.

A one-step/one-tube phenotypic assay for APE1 activity has been developed based on the use of a fluorescent molecular beacon (partially self-complementary oligonucleotide with a hairpin-loop structure carrying a fluorophore and a quencher at each end). It also contains a single tetrahydrofuran residue (THF) which is recognized and cleaved by APE1, and the subsequently formed single-stranded oligomer becomes a fluorescence signal emitter. Both assays are highly sensitive, require very small amounts of protein extracts, are relatively inexpensive and can be easily automated. They have been extensively validated and are being used in the context of large-scale molecular epidemiology studies.

Introduction

DNA repair is an important parameter contributing to individual susceptibility to the effects of genotoxic carcinogens. The impact of variations in DNA repair ability on individual cancer risk has been demonstrated in population studies of genetic variations at DNA repair-related loci as well as studies based on measurement of repair activity using phenotypic assays (for recent reviews see Refs. [1], [2], [3], [4]). Phenotypic DNA repair assays have an advantage compared to studies based on genetic variation, in that they directly reflect the activity of the repair proteins or multi-protein complexes in question, thus accounting for variations in the level of expression as well as in the catalytic efficacy. On the other hand, they tend to be technically complex and time-consuming, in contrast to genotyping assays, a disadvantage which assumes particular significance in view of the trend towards the use of large population sizes (in the hundreds or even thousands) in molecular epidemiology studies. Thus there is an increasing need for phenotypic repair assays which are relatively simple to conduct, efficient and of low cost. Here we present the development and validation of two phenotypic DNA repair assays which respond to this demand, concerning the repair proteins O6-methylguanine-DNA methyltransferase (MGMT) and apurinic endonuclease 1 (APE1).

MGMT is a protein which repairs O6-methylguanine (O6-meG), a cytotoxic, mutagenic and carcinogenic lesion generated by DNA by methylating agents [5]. If not repaired, these lesions induce cell death by activating apoptotic pathways [6]. Methylating agents include chemicals of environmental relevance (dietary or endogenously formed) such as N-nitroso compounds (e.g. N-nitrosodimethylamine), as well as certain drugs (e.g. temozolomide, procarbazine and dacarbazine) widely used in cancer chemotherapy. Quantification of MGMT is an important biomarker of susceptibility, since human exposure to carcinogenic N-nitroso compounds is ubiquitous [7] and accumulated evidence suggests an influence of MGMT polymorphisms on cancer risk [8]. Interest in the measurement of MGMT activity in human samples also arises from the strong impact of MGMT activity on tumor cell resistance to methylating cytotoxic drugs and therefore, on the clinical response of the patients [9], [10].

MGMT repairs O6-meG in DNA by a suicide mechanism involving the covalent transfer of the methyl group from DNA to a reactive cystein residue at its active site [5] with concomitant inactivation of the protein. The most common method used for assaying this activity requires use of a [3H]-methylated DNA substrate (prepared using N-[3H]-methyl-N-nitrosourea), incubation of cell extracts with this substrate, selective precipitation and extensive washing of the protein fraction, finally followed by measurement of the radioactivity transferred to it [11]. The main disadvantages of this method relate to the need for poorly available N-[3H]-methyl-N-nitrosourea and the multiple steps of the assay which largely preclude automation and make the assay labour intensive. Although several attempts were made for the development of a simple and high throughput assay [12], [13], [14], the above mentioned one was proven to be the most reliable and is currently used, almost exclusively, for MGMT quantification [15], [16], [17]. We report here the development and validation of a convenient, high-throughput ELISA-type method for measuring MGMT activity in extracts of human tissues. The method makes use of the fact that MGMT readily reacts with O6-benzylguanine free base (BG), covalently transferring the benzyl group to its active-site cystein residue.

Human APE1 is the main apurinic/apyrimidinic endonuclease in human tissues and plays a critical role in the base excision repair of multiple DNA base lesions (including alkylated and oxidized bases, abasic sites, strand breaks) by incising the abasic sites generated after removal of the damaged bases by glycosylases, generating a 3′-OH nick which is eventually sealed by other proteins. hAPE1 activity therefore contributes to the overall efficiency of base excision repair which is known to affect individual susceptibility to carcinogenesis [18]. An influence of hAPE1 activity on the overall efficiency of base excision repair may lie behind reports of small to moderate effects of different genetic polymorphisms of hAPE1 on cancer risk [19], [20]. In addition to its impact on individual cancer susceptibility via its involvement in base excision repair, hAPE1 has attracted interest as a possible target for chemotherapy [21], and in view of its additional functions in relation to the regulation of gene expression and the control of oxidative stress [22].

Previously reported assays of hAPE1 activity involved the use of a 32P-labelled DNA substrate [23]. While a more convenient phenotypic assay has also been reported [24], it appears to have been employed only in the context of mechanistic studies [25], [26]. This assay is based on the use of a molecular beacon-type oligonucleotide. Molecular beacons are defined as partially self-complementary oligomers which carry a fluorophore (6-FAM) and a quencher (Dabcyl) at the complementary 5′ and 3′ ends respectively. When in solution, molecular beacons adopt a double stranded stem–loop structure; the FAM and Dabcyl residues end up in close proximity to each other, causing the emitted florescence to be quenched by fluorescence resonance energy transfer. When a molecular beacon containing a single tetrahydrofuran (THF) residue is incised by hAPE1 at the 3′ site of this residue (hAPE1 structurally resembles an apurinic site and therefore is a substrate for the enzyme), the 5′ fluorescein-bearing oligonucleotide fragment is removed from the 3′ fluorescence quenching, Dabcyl-bearing fragment, and thus, becomes strongly fluorescent. We have re-established this assay and evaluated its performance in relation to its potential for use in population studies.

Section snippets

Materials and methods

The 96-well high-binding microtiter plates, white for the luminescence emission-based BG-Biotin MGMT ELISA (BBME assay) and black for the fluorescence emission-based molecular beacon-APE1 assays were from Greiner Labortechnik (Germany). Antisera were mainly obtained from CHEMICON Inc. (Millipore, Temecula, CA), while specific reagents for ELISA such as I-block (casein) and CDP-Star with Emerald II were obtained from Tropix (Bedford, MA). Purified and titrated human APE1 and the BG-biotin

Sandwich-ELISA for the detection of MGMT

BG-Biotin (Covalys, New England Biolabs Inc.) is a cell-permeable substrate (Fig. 1A) based on biotin with an amidocaproyl linker. It was designed for applications such as biotinylation of proteins fused with MGMT in living cells and their subsequent detection with streptavidin fluorophore conjugates or for in vitro labelling for further analysis. The principle of our method is based on the intrinsic property of mammalian MGMT to recognize as substrates and react irreversibly not only with O6

Discussion

We have developed and validated new phenotypic assays for 2 DNA repair proteins, which are suitable for use in large-scale population studies. Both assays are based on the incubation of crude extracts of blood lymphocytes with the corresponding substrates, followed by measurement of the generated signals using methods which can be readily automated, thus allowing a potential for high-throughput.

The levels of MGMT in human tissues and their influence on cancer risk have been studied in the past,

Conflicts of interest statement

No potential conflicts of interest were disclosed by any of the authors.

Acknowledgement

This work was partly supported by the ECNIS (Environmental Cancer, Nutrition and Individual Susceptibility) Network of Excellence of the European Union (contract no. 513943).

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