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Versatile electroanalytical bioplatforms for simultaneous determination of cancer-related DNA 5-methyl- and 5-hydroxymethyl-cytosines at global and gene-specific levels in human serum and tissues Eloy Povedano, Víctor Ruiz-Valdepeñas Montiel, Alejandro Valverde, Fernando NavarroVilloslada, Paloma Yanez-Sedeno, María Pedrero, Ana Montero-Calle, Rodrigo Barderas, Alberto Peláez-García, Marta Mendiola, David Hardisson, Jaime Feliú, Jordi Camps, Elisabet Rodríguez-Tomàs, Jorge Joven, Meritxell Arenas, Susana Campuzano, and José M. Pingarrón ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01339 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018
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ACS Sensors
Versatile electroanalytical bioplatforms for simultaneous determination of cancer-related DNA 5-methyl- and 5-hydroxymethylcytosines at global and gene-specific levels in human serum and tissues. Eloy Povedano,a Víctor Ruiz-Valdepeñas Montiel, a Alejandro Valverde,a Fernando Navarro-Villoslada,a Paloma Yáñez-Sedeño,a María Pedrero,a Ana Montero-Calle,a Rodrigo Barderas,b Alberto PeláezGarcía,c Marta Mendiola, c,d David Hardisson,c Jaime Feliú, d,e Jordi Camps, f Elisabet RodríguezTomàs, f,g Jorge Joven,f Meritxell Arenas,f Susana Campuzano,a,* José M. Pingarróna,* a
Departamento de Química Analítica, Facultad de CC. Químicas, Universidad Complutense de Madrid, E-28040 Madrid (Spain). b CROSADIS, UFIEC, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid (Spain). c Molecular Pathology and Therapeutic Targets Group Hospital Universitario La Paz IdiPAZ, Madrid (Spain). d Center for Biomedical Research in the Cancer Network (Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Instituto de Salud Carlos III, Madrid (Spain). e Translational Oncology Group Hospital Universitario La Paz IdiPAZ, Madrid (Spain). f Unitat de Recerca Biomèdica, Hospital Universitari Sant Joan, Institut d´Investigació Sanitària Pere Virgili, Universitat Rovira i Virgili, Reus (Spain). g Department of Radiation Oncology, Hospital Universitari Sant Joan, Institut d´Investigació Sanitària Pere Virgili, Universitat Rovira i Virgili, Reus (Spain). KEYWORDS: global and gene-specific DNA methylation; 5-mC; 5-hmC; electrochemical platforms; cancer; human serum; paraffin-embedded colorectal tissues.
ABSTRACT: This paper reports the preparation of versatile electrochemical biosensing platforms for the simple, rapid and PCRindependent detection of the most frequent DNA methylation marks (5-methylcytosine, 5-mC, and/or 5-hydroxymethylcytosine, 5hmC) both at global and gene-specific levels. The implemented strategies, relying on the smart coupling of immuno-magnetic beads (MBs), specific DNA probes and amperometric detection at screen-printed carbon electrodes (SPCEs), provided sensitive and selective determination of the target methylated DNAs in less than 90 min with a great reproducibility and demonstrated feasibility for the simultaneous detection of the same or different cytosine epimarks both at global level and in different loci of the same gene or in different genes. The bioplatforms were applied to determine global methylation events in paraffin-embedded colorectal tissues and specific methylation at promoters of tumor suppressor genes in genomic DNA extracted from cancer cells and paraffinembedded colorectal tissues, and in serum without previous DNA extraction from cancer patients.
Methylation is an important epigenetic DNA modification transcriptional function to genes and are currently considered governing gene expression. The genomic level of methylated as important indicators of the initiation and progression of DNA and its derivatives may serve as important indicator for cancers.2-4 1,2 the initiation and progression of cancer and other diseases. Locus-specific DNA hypermethylation of relevant tumor Modification of cytosine (C) to 5-methylcytosine (5-mC) is suppressor genes such as Ras association domain family 1 the most studied epigenetic alteration and has attracted much isoform A (RASSF1A)5 and O-6-methylguanine-DNA methylattention because of its important role in several cellular protransferase (MGMT)6,7 at the promoter region leads to their cesses including embryonic development, genomic imprinting, silencing and have been proposed as prognostic and diagnostic cellular differentiation and carcinogenesis. Apart from 5-mC, biomarkers in clinical settings. Unlike locus-specific changes, the Ten-Eleven Translocation (TET) enzymes mediate sequenalterations of epigenetic marks (epimarks) at the global level is tial oxidation of 5-mC to 5-hydroxymethylcytosine (5-hmC; rapidly gaining prominence as they generate additional celluCH2OH group at cytosine C-5), then to 5-formylcytosine (5lar epigenetic heterogeneity, which can be used to define prefC; -CHO at cytosine C-5) and finally to 5-carboxylcytosine viously unrecognized subsets of cancer patients with distinct (5-caC; -COOH at cytosine C-5). These intermediates in the clinical outcomes. Alteration of 5-mC global levels has been demethylation pathway have been shown to confer unique ACS Paragon Plus Environment
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suggested as a prognostic and diagnostic biomarker depicting early neoplasia and solid tumors and being strongly associated with tumor aggressiveness, regardless of cancer tissue origin.4 In addition, recently it has been reported that the loss of the relatively stable intermediate of active DNA demethylation, 5hmC, is broadly and tightly associated with solid tumor development: glioblastoma, astrocytomas gastric, prostate, breast, liver, brain, pancreatic, lung and colon cancers compared to the matched surrounding normal tissues.3,4,8-10 Therefore 5hmC is regarded as a novel epigenetic hallmark of cancer and its detection is gradually becoming a hotspot on epigenetics.9,11 Quantitative analysis on genome-wide and locus-specific distribution of these epimarks is useful in identifying cancer in its early stages for prognosis or monitor the success of administered therapies in cancer patients.4 However, the low abundance of these cytosine variants in humans: 5-mC makes up about 1 % of the total DNA bases, the 5-hmC abundance is ∼ 10 to 100-fold lower than that of 5-mC (0.5 –
