New strategy to address DNA-methyl transferase activity in ovarian cancer cell cultures by monitoring the formation of 5-methylcytosine using HPLC-UV.
Methylation of mammalian genomic DNA is catalyzed by DNA methyltransferases (DNMTs). Aberrant expression and activity of these enzymes has been reported to play an important role in the initiation and progression of tumors and its response to chemotherapy. Therefore, there is a great interest in developing strategies to detect human DNMTs activity. We propose a simple, antibody-free, label-free and non-radioactive analytical strategy in which methyltransferase activity is measured trough the determination of the 5-methylcytosine (5mC) content in DNA by a chromatographic method (HPLC-UV) previously developed. For this aim, a correlation between the enzyme activity and the concentration of 5mC obtained by HPLC-UV is previously obtained under optimized conditions using both, un-methylated and hemi-methylated DNA substrates and the prokaryotic methyltransferase M.SssI as model enzyme. The evaluation of the methylation yield in un-methylated known sequences (a 623bp PCR-amplicon) turned to be quantitative (110%) in experiments conducted in-vitro. Methylation of hemi-methylated and low-methylated sequences could be also detected with the proposed approach. The application of the methodology to the determination of the DNMTs activity in nuclear extracts from human ovarian cancer cells has revealed the presence of matrix effects (also confirmed by standard additions) that hampered quantitative enzyme recovery. The obtained results showed the high importance of adequate sample clean-up steps.
Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, C/Julian Clavería 8, 33006 Oviedo, Spain.
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Iglesias González
Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, C/Julian Clavería 8, 33006 Oviedo, Spain. Electronic address: eblancog@uniovi.es.
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Blanco-González
Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, C/Julian Clavería 8, 33006 Oviedo, Spain. Electronic address: montesmaria@univovi.es.
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Montes-Bayón
D044503:5-Methylcytosine / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D018899:CpG Islands; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000032:analysis / Q000378:metabolism*; D019175:DNA Methylation; D057075:Enzyme Assays / Q000379:methods; D005260:Female; D006801:Humans; D010051:Ovarian Neoplasms / Q000201:enzymology*; D010053:Ovary / Q000201:enzymology
D044503:5-Methylcytosine; D004247:DNA; C427203:calf thymus DNA; D004248:DNA (Cytosine-5-)-Methyltransferases
Activity; DNA methylation; HPLC-UV; Methyl transferases