title: Accurate monitoring of promoter gene methylation with high-resolution melting polymerase chain reaction using the ABCB1 gene as a model.
abstract: Multidrug resistance is the major cause of cancer chemotherapy failure. This phenotype is mainly due to the overexpression of the human ABCB1 gene. Several studies have shown that the transcriptional regulation of this gene is complex. Yet, the impact of this transcriptional regulation has not been well studied in a clinical setting. The acquired expression of ABCB1 is associated with the genomic instability of cancer cells. This includes the occurrence of mutational events that alter chromatin structures through epigenetic modifications such as promoter methylation. Therefore, it is important to introduce new clinical methods to monitor the methylation status of ABCB1 and determine its association with gene expression in order to be able to predict response to therapies. The high-resolution melting (HRM) method has emerged as a highly accurate and sensitive method to quantify methylation status at specific sites of DNA. Here, we established HRM parameters to evaluate the promoter methylation status of the ABCB1 gene. Our study is the first to standardize the HRM dissociation curve to evaluate ABCB1 gene methylation. The association between ABCB1 methylation status and gene expression in established cancer cell lines shows that this method is accurate and reliable.
authors.affiliation: Centro de Estudos de Medula Óssea, Instituto Nacional de Câncer, Rio de Janeiro, RJ, Brasil. amencalha@inca.gov.br
authors.forename: A L
authors.initials: AL
authors.lastname: Mencalha
authors.forename: E F
authors.initials: EF
authors.lastname: Rodrigues
authors.forename: E
authors.initials: E
authors.lastname: Abdelhay
authors.forename: T S
authors.initials: TS
authors.lastname: Fernandez
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020014:K562 Cells; D061986:MCF-7 Cells; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004273:DNA, Neoplasm