Different individuals possess slightly different genetic information and show genetically-determined differences in several enzyme activities due to genetic variability. Following an integrated approach, we studied the polymorphisms and methylation of sites contained in the 5' flanking region of the metabolizing enzyme CYP2E1 in correlation to its expression in both tumor and non-neoplastic liver cell lines, since to date little is known about the influence of these (epi)genetic elements in basal conditions and under induction by the specific inductor and a demethylating agent. In treated cells, reduced DNA methylation, assessed both at genomic and gene level, was not consistently associated with the increase of enzyme expression. Interestingly, the Rsa/Pst haplotype differentially influenced CYP2E1 enzyme expression. In addition, regarding the Variable Number of Tandem Repeats polymorphism, cells with A4/A4 genotype showed a greater expression inhibition (ranging from 20% to 30%) compared with others carrying the A2/A2 one, while those cells bringing A2/A3 genotype showed an increase of expression (of 25%, about). Finally, we demonstrated for the first time that the A2 and A3 CYP2E1 alleles play a more important role in the expression of the enzyme, compared with other (epi)genetic factors, since they are binding sites for trans-acting proteins.
Keywords: 5-Azacytidine; 5-azacytidine; BSA; CYP; CYP2E1; CYP2E1 gene polymorphisms; DMSO; DNA; DNA methylation; DTT; EDTA; EMSA; Electrophoretic Mobility Shift Assay; Ethanol; HCC; HRP; Hepatocellular carcinoma cell lines; MSRE-PCR; MTT; MeSAP-PCR; Methylation Sensitive Restriction Endonuclease-Polymerase Chain Reaction; PBS; PCR; Polymerase Chain Reaction; RFLP; ROS; Reactive Oxygen Species; Restriction Fragment Length Polymorphism; VNTR; Variable Number of Tandem Repeats; ZCyd; [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]; bovine serum albumin; cytochrome p450; cytochrome p450 2E1; deoxyribonucleic acid; dimethyl sulfoxyde; dithiothreitol; ethylene diamine tetraacetic acid; hepato-cellular carcinoma; horse radish peroxidase; methylation sensitive arbitrarily primed-polymerase chain reaction; phosphate buffered saline.
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