Accurate monitoring of promoter gene methylation with high-resolution melting polymerase chain reaction using the ABCB1 gene as a model

Abstract

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 ABCB1 gene methylation analyses 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.

Increasing BMI is associated with reduced expression of P-glycoprotein (ABCB1 gene) in the human brain with a stronger association in African Americans than Caucasians

“…DNA methylation is an active mechanism in the regulation of ABCB1 expression. 30 Epigenetic mechanisms such as DNA methylation could also constitute a possible mechanism by which obesity—as assessed by BMI—could affect P-gp expression. 30 It is well-known that obesity per se is often related to a variety of potentially stressful responses such as insulin resistance, altered nutritional status and chronic low-grade inflammation.…”

“… 30 Epigenetic mechanisms such as DNA methylation could also constitute a possible mechanism by which obesity—as assessed by BMI—could affect P-gp expression. 30 It is well-known that obesity per se is often related to a variety of potentially stressful responses such as insulin resistance, altered nutritional status and chronic low-grade inflammation. 31 , 32 Moreover, obesity is known to be associated with an increased cancer risk, 32 , 33 which might also reflect deleterious epigenetic alterations.…”

Manipulation of primer affinity improves high-resolution melting accuracy for imprinted genes

“…In their assay, the area under the 50% methylation plot was larger than that above, resulting in a large area in which low methylation fractions are dispersed. This results in high accuracy, particularly for low methylation fraction samples, and has proved very pertinent to cancer studies because cancer samples usually have low methylation levels (Wojdacz and Hansen, 2006;Wojdacz and Dobrovic, 2007;Wojdacz et al, 2008Wojdacz et al, , 2009Wojdacz et al, , 2011Mencalha et al, 2013).…”

Association between <em>MDR1</em> polymorphisms and XELIRI and XELOX chemoresistance in Saudi patients with colorectal cancer

“…The expression of the MDR1 gene and the activity of its protein product can differ among individuals due to genetic single nucleotide polymorphisms (SNPs) (7)(8)(9)(10)(11). Since gene polymorphisms have the ability to influence clinical response to chemotherapy, they can also affect the absorption, distribution, metabolism and excretion of drugs (9)(10)(11).…”