Biological markers [35]. The list of the mechanisms controlling TET expression in leukemic cells is growing and involves microenvironmental factors [12], hypoxia, reactive oxygen species (ROS), and miRNAs such as miR-15a and miR-29 [36]. Additional experiments are required to better understand the mechanisms controlling active DNA demethylation in CLL and their consequences. Relevant limitations of our study include the following: (i) global 5-mCyt and 5-hmCyt analysis is likely to reflect variations of repetitive elements (half of the genome) than gene regulatory sequence, which may render comparison with EWAS studies difficult; (ii) the absence of a well-established control B cell for CLL B cells, making difficult the comparison of the transcriptional analysis between normal and tumor cells; and (iii) a transcriptional analysis for DNMTs and TETs, which does not test enzyme activity. In conclusion, our study established that loss in the 5Cyt derivatives, 5-mCyt and, to a lesser extent, 5-hmCyt, during disease progression, better predict the CLL patients’ outcome when associated with the classical cytogenetic analysis. Future work is required to develop routine assays for monitoring DNA methylation during disease course and to test their utility as therapeutic response predictors.France), and B cells were further enriched using the Pan B cell Isolation Kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Cell purity was assessed by fluorescence-activated cell sorting (FACS) analysis and was over 95 for B cells (CD19+). Fluorescenceactivated cell sorting (FACS) antibodies (Abs) used were purchased from Beckman Coulter.DNA sample preparation and global DNA 5-mCyt and 5hmCyt level assessment by ELISA.Materials and methodsCells and sample preparationClinical and biological data were retrospectively NIK333MedChemExpress Peretinoin obtained for 127 untreated patients diagnosed with CLL according to the World Health Organization (WHO) classification [37], and samples were collected for 60 CLL patients and 15 healthy volunteers at the Brest University Hospital. Consent was obtained from all individuals and the protocol approved by the Ethical Board at the Brest University Hospital (OFICE, November 26, 2015; ClinicalTrials.gov: NCT03294980; CRB Brest, collection 2008-214), in accordance with the Declaration of Helsinki. Lymphocytes were isolated from peripheral blood mononuclear cells (PBMC) by Ficoll-Hypaque density gradient centrifugation (Eurobio, Courtaboeuf,DNA was extracted from purified B cells using Biosprint 15 DNA Blood Kit (Qiagen, Hilden, Germany). Next, DNA was quantified and its purity assessed using the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). An ELISA previously developed in the laboratory was used and adapted to measure global 5-mCyt and 5-hmCyt [38]. Briefly, high affinity microplates (Thermo 269620, Thermo Fisher Scientific) were pre-coated 90 min at room temperature (RT) with 100 l poly-L-lysine 0.01 (Sigma-Aldrich, St. Louis, MI) to attach DNA. Next, DNA samples adjusted at 2 ng/l in carbonate/bicarbonate buffer 0.1 M pH 9.6 were denatured at 95 for 6 min, kept on ice 5?0 min, and then 100 l dispensed in each well, in duplicates. The plates were next incubated overnight at 4 , three washes with phosphate-buffered saline (PBS)-Tween 0.01 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28242652 were performed, and 200 l of PBS with bovine serum albumin (BSA) 1 was added in each well as blocking solution. After 1-h incubation at RT and extensive washing, 100 l of mouse IgG.
