High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion.

DNA methylation is a major regulatory process of gene transcription, and aberrant DNA methylation is associated with various diseases including cancer. Many compounds have been reported to modify DNA methylation states. Despite increasing interest in the clinical application of drugs with epigenetic effects, and the use of diagnostic markers for genome-wide hypomethylation in cancer, large-scale screening systems to measure the effects of drugs on DNA methylation are limited. In this study, we improved the previously established fluorescence polarization-based global DNA methylation assay so that it is more suitable for application to human genomic DNA. Our methyl-sensitive fluorescence polarization (MSFP) assay was highly repeatable (inter-assay coefficient of variation = 1.5%) and accurate (r2 = 0.99). According to signal linearity, only 50-80 ng human genomic DNA per reaction was necessary for the 384-well format. MSFP is a simple, rapid approach as all biochemical reactions and final detection can be performed in one well in a 384-well plate without purification steps in less than 3.5 hours. Furthermore, we demonstrated a significant correlation between MSFP and the LINE-1 pyrosequencing assay, a widely used global DNA methylation assay. MSFP can be applied for the pre-screening of compounds that influence global DNA methylation states and also for the diagnosis of certain types of cancer.

Pharmacoepigenetics of the role of DNA methylation in µ-opioid receptor expression in different human brain regions.

AIM: Exposure to opioids has been associated with epigenetic effects. Studies in rodents suggested a role of varying degrees of DNA methylation in the differential regulation of µ-opioid receptor expression across the brain. METHODS: In a translational investigation, using tissue acquired postmortem from 21 brain regions of former opiate addicts, representing a human cohort with chronic opioid exposure, µ-opioid receptor expression was analyzed at the level of DNA methylation, mRNA and protein. RESULTS & CONCLUSION: While high or low µ-opioid receptor expression significantly correlated with local OPRM1 mRNA levels, there was no corresponding association with OPRM1 methylation status. Additional experiments in human cell lines showed that changes in DNA methylation associated with changes in µ-opioid expression were an order of magnitude greater than differences in brain. Hence, different degrees of DNA methylation associated with chronic opioid exposure are unlikely to exert a major role in the region-specificity of µ-opioid receptor expression in the human brain.

Disagreement between two common biomarkers of global DNA methylation.

BACKGROUND: The quantification of global DNA methylation has been established in epigenetic screening. As more practicable alternatives to the HPLC-based gold standard, the methylation analysis of CpG islands in repeatable elements (LINE-1) and the luminometric methylation assay (LUMA) of overall 5-methylcytosine content in "CCGG" recognition sites are most widely used. Both methods are applied as virtually equivalent, despite the hints that their results only partly agree. This triggered the present agreement assessments. RESULTS: Three different human cell types (cultured MCF7 and SHSY5Y cell lines treated with different chemical modulators of DNA methylation and whole blood drawn from pain patients and healthy volunteers) were submitted to the global DNA methylation assays employing LINE-1 or LUMA-based pyrosequencing measurements. The agreement between the two bioassays was assessed using generally accepted approaches to the statistics for laboratory method comparison studies. Although global DNA methylation levels measured by the two methods correlated, five different lines of statistical evidence consistently rejected the assumption of complete agreement. Specifically, a bias was observed between the two methods. In addition, both the magnitude and direction of bias were tissue-dependent. Interassay differences could be grouped based on Bayesian statistics, and these groups allowed in turn to re-identify the originating tissue. CONCLUSIONS: Although providing partly correlated measurements of DNA methylation, interchangeability of the quantitative results obtained with LINE-1 and LUMA was jeopardized by a consistent bias between the results. Moreover, the present analyses strongly indicate a tissue specificity of the differences between the two methods.

Methadone induces hypermethylation of human DNA.

BACKGROUND: Increased global DNA methylation in the blood of patients chronically exposed to opioids had been interpreted as an indication of an epigenetic action of this drug class. MATERIALS & METHODS: To strengthen the causality, human MCF7 cells were cultured in media with the addition of several known or potential modulators of DNA methylation including methadone. RESULTS: Following 3 days of incubation with several different known or potential epigenetic modulators, global DNA methylation, quantified at LINE-1 CpG islands, showed a large variability across all treatments ranging from 27.8 to 63%. Based on distribution analysis of the global methylation of human DNA exposed to various potential modulators, present in vitro experiments showed that treatment with the opioid methadone was associated with an increased probability of hypermethylation. CONCLUSION: This strengthens the evidence that opioids interfere with mechanisms of classical epigenetics.

AMP-activated protein kinase is activated by non-steroidal anti-inflammatory drugs.

AMP-activated kinase (AMPK) is a cellular energy sensor, which is activated in stages of increased adenosine triphosphate (ATP) consumption. Its activation has been associated with a number of beneficial effects such as decrease of inflammatory processes and inhibition of disease progression of diabetes and obesity. A recent study suggested that salicylate, the active metabolite of the non-steroidal anti-inflammatory drug (NSAID) acetyl-salicylic acid (aspirin), is able to activate AMPK pharmacologically. This observation raised the question whether or not other NSAIDs might also act as AMPK activators and whether this action might contribute to their cyclooxygenase (COX)-independent anti-inflammatory properties. In this study, we investigated mouse and human neuronal cells and liver tissue of mice after treatment with various NSAIDs. Our results showed that the non-selective acidic NSAIDs ibuprofen and diclofenac induced AMPK activation similar to aspirin while the COX-2 selective drug etoricoxib and the non-opioid analgesic paracetamol, both drugs have no acidic structure, failed to activate AMPK. In conclusion, our results revealed that AMPK can be activated by specific non-steroidal anti-inflammatory drugs such as salicylic acid, ibuprofen or diclofenac possibly depending on the acidic structure of the drugs. AMPK might therefore contribute to their antinociceptive and anti-inflammatory properties.