Development and Application of a Sensitive and Rapid Immunoassay for the Quantitation of N7-Methyldeoxyguanosine in DNA Samples

Kl, Harrison; Wood, Michelle; Np, Lees; Cn, Hall; Margison, GP; Povey, A.

doi:10.1021/tx000071b

Cited by 31 publications

(27 citation statements)

References 28 publications

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“…There was slow and incomplete inactivation following treatment with TMZ and more rapid, total, depletion with LM/TMZ, consistent with our previous trial (Ranson et al, 2006). N7-methylguanine was detectable, albeit at very low levels, in 12% of the pre-treatment PBMCs, consistent with previous measurements in the blood of non-smokers (Harrison et al, 2001). This may reflect exposure of these individuals to environmental or endogenous methylating agents and/or deficiency in AAG.…”

Section: Discussionsupporting

confidence: 90%

O6-methylguanine-DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib

et al. 2009

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We evaluated the pharmacodynamic effects of the O 6 -methylguanine-DNA methyltransferase (MGMT) inactivator lomeguatrib (LM) on patients with melanoma in two clinical trials. Patients received temozolomide (TMZ) for 5 days either alone or with LM for 5, 10 or 14 days. Peripheral blood mononuclear cells (PBMCs) were isolated before treatment and during cycle 1. Where available, tumour biopsies were obtained after the last drug dose in cycle 1. Samples were assayed for MGMT activity, total MGMT protein, and O 6 -methylguanine (O 6 -meG) and N7-methylguanine levels in DNA. MGMT was completely inactivated in PBMC from patients receiving LM, but detectable in those on TMZ alone. Tumours biopsied on the last day of treatment showed complete inactivation of MGMT but there was recovery of activity in tumours sampled later. Significantly more O 6 -meG was present in the PBMC DNA of LM/TMZ patients than those on TMZ alone. LM/TMZ leads to greater MGMT inactivation, and higher levels of O 6 -meG than TMZ alone. Early recovery of MGMT activity in tumours suggested that more protracted dosing with LM is required. Extended dosing of LM completely inactivated PBMC MGMT, and resulted in persistent levels of O 6 -meG in PBMC DNA during treatment.

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Section: Discussionsupporting

confidence: 90%

O6-methylguanine-DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib

et al. 2009

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“…Alkylation of DNA at the O 6 position of guanine is an important step in the formation of G-to-A mutations in cancer. Human colorectal DNA has long been known to contain O 6 -methylguanine and N 7 -methylguanine adducts that arise from exposure to methylating factors and agents (16,63,64). The sources of these exposures are unknown but can potentially be dietary/lifestyle and occupational characteristics, endogenous alkylating agents, and in situ formation typically mediated by the bacterial or chemical nitrosation of amines (65 -67).…”

Section: Discussionmentioning

confidence: 99%

Epigenetic-Genetic Interactions in theAPC/WNT, RAS/RAF, andP53Pathways in Colorectal Carcinoma

Suehiro

Wong

Chirieac³

et al. 2008

Clinical Cancer Research

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Purpose: Early events in colorectal tumorigenesis include mutation of the adenomatous polyposis coli (APC) gene and epigenetic hypermethylation with transcriptional silencing of the O 6 -methylguanine DNA methyltransferase (MGMT), human mut L homologue 1 (hMLH1), and P16/CDKN2A genes. Epigenetic alterations affect genetic events: Loss of MGMT via hypermethylation reportedly predisposes to guanine-to-adenine or cytosine-to-thymine (G:C!A:T) transition mutations in KRAS and P53, and silencing of hMLH1 leads to high levels of microsatellite instability (MSI-H)/mutator phenotype, suggesting that epigenetic-genetic subtypes exist. Experimental Design: We evaluated the relationships of aberrant methylation of APC, MGMT, hMLH1, P16, N33, and five MINTs to mutations in APC, KRAS, BRAF, and P53 in 208 colorectal carcinomas. Results: We found that APC hypermethylation was age related (P = 0.04), in contrast to the other genes, and did not cluster with CpG island methylator phenotype (CIMP) markers. Hypermethylation of APC concurrently with either MGMT or hMLH1 was strongly associated with occurrence of G-to-A transitions in APC [odds ratio (OR), 26.8; P < 0.0002 from multivariable logic regression model], but C-to-T transitions had no associations. There was no relationship of hypermethylation of any gene, including MGMT, with G-to-A or C-to-T transitions in KRAS or P53, although APC hypermethylation was associated with P53 mutation (P < 0.0002). CIMP with MSI-H due to hMLH1 hypermethylation, or CIMP with loss of MGMT expression in nonM SI-H tumors, was associated with BRAF mutation (OR, 4.5; P < 0.0002). CIMP was also associated with BRAF V600E T-to-A transversion (OR, 48.5; P < 0.0002).Conclusions: Our findings suggest that the heterogeneous epigenetic dysregulation of promoter methylation in various genes is interrelated with the occurrence of mutations, as manifested in epigenetic-genetic subgroups of tumors.The development of most colorectal carcinomas (CRC) is thought to be initiated by inactivation of the APC/b-catenin/ Wnt signaling pathway, usually by mutation of one copy of the APC gene followed by a second event that inactivates the other allele (1-4). The second inactivating event is usually described as allelic deletion or mutation but can be epigenetic methylation of cytosines in CpG islands of the promoter region of APC that results in transcriptional silencing (5, 6). Altered APC has wideranging downstream effects on cell-cell adhesion, transcriptional regulation, chromosomal instability, cell migration, proliferation and cell cycle control, differentiation, and apoptosis (1-4).Somatic APC mutation is associated with the development of intraepithelial neoplasia (dysplasia) in aberrant crypt foci and early adenomas (7). The initiating alterations in APC in these early lesions persist whereas additional genetic and epigenetic events accumulate and drive tumor progression. Somatic mutation in the APC gene is present in as many as 80% of sporadic colorectal adenomas and carcinomas (7 -9), and a mutati...

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“…For the determination of 7-mG, and other modified nucleotides, a variety of analytical techniques have been described in the literature, as follows: immunoassays [19,20], 32 P-postlabeling methods [7,20], capillary electrophoresis coupled with electrospray ionization mass spectrometry (CE-ESI-MS) [9], liquid chromatographytandem mass spectrometry (LC-MS/MS) [12,[21][22][23] and high-performance liquid chromatography coupled with UV-Vis detection [2,3,24,25] and electrochemical detection [11,14,26]. Regarding the electrochemical detection in the case of 7-mG, glassy carbon electrode is normally used, being presented the 7-mG response about 1 V versus standard Ag/AgCl reference electrode.…”

Section: Introductionmentioning

confidence: 99%

Boron-doped diamond electrodes explored for the electroanalytical detection of 7-methylguanine and applied for its sensing within urine samples

Sanjuán

Brotons

Hernández-Ibáñez

et al. 2016

Electrochimica Acta

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Please cite this article as: Ignacio Sanjuán, Ariadna Brotons, Naiara Hernández-Ibáñez, Christopher W.Foster, Craig E.Banks, Jesús Iniesta, Boron-doped diamond electrodes explored for the electroanalytical detection of 7-methylguanine and applied for its sensing within urine samples, Electrochimica Acta http://dx.doi.org/10.1016/j.electacta. 2015.11.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.  SWV provides good resolution of the anodic peak for 7-mG and the main nucleic bases interferences, such as adenine and guanine. It is plausible the simultaneous determination of 7-mG and another of the most common modified nucleobases, 8-oxoguanine (8-oxoG). 7-mG electrochemical determination within a human urine sample is feasible with linear regression between peak current intensity versus 7-mG concentration. 3 AbstractEpigenetic modifications have been associated by many studies with several types of diseases and metabolic dysfunctions. Specifically, N7-methyl modification of guanine (7-mG) is well established to be used as a biomarker for the detection and determination of DNA methylation. The use of an electrochemical sensor has the potential to provide a simpler and more economic sensing methodology for the determination of 7-mG compared to traditionally utilised laboratory based approaches. In this paper we demonstrate the feasibility of an electrochemical sensor which could potentially be easily applied towards the determination of 7-mG within biological samples, such as human urine. A practical electrochemical configuration was employed consisting of a boron-doped diamond electrode (BDD) as the working electrode and a screen-printed graphite electrode (SPE) providing the counter electrode and the reference electrode.With this new protocol, the electrochemical behaviour of 7-mG has been investigated via cyclic voltammetry (CV) and square wave voltammetry (SWV) using a BDD electrode with a simplified electrochemical set-up The electrochemical behaviour of 7-mG within acetate buffer solutions at a BDD electrode has been compared and contrasted to a glassy carbon electrode with the following parameters studied: voltammetric scan rate, solution pH, 7-mG concentration and electrode surface pretreatment. The oxidative mechanism elucidation has been performed at controlled potential and such results have provided the dimer formation as the major product. The simultaneous electroanalytical identification of 7-mG together with the presence of guanine, adenine and 8-oxoguanine has been investigated under the optimum experimental conditions. Furthermore, the feasibility of using a BDD electrode for the ...

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Development and Application of a Sensitive and Rapid Immunoassay for the Quantitation of N7-Methyldeoxyguanosine in DNA Samples

Cited by 31 publications

References 28 publications

O6-methylguanine-DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib

O6-methylguanine-DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib

Epigenetic-Genetic Interactions in theAPC/WNT, RAS/RAF, andP53Pathways in Colorectal Carcinoma

Boron-doped diamond electrodes explored for the electroanalytical detection of 7-methylguanine and applied for its sensing within urine samples

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