ABSTRACT
Deregulated NOTCH1 has been reported in lymphoid leukaemia, although its role in chronic myeloid leukaemia (CML) is not well established. We previously reported BCR-ABL down-regulation of a novel haematopoietic regulator, CCN3, in CML; CCN3 is a non-canonical NOTCH1 ligand. This study characterizes the NOTCH1CCN3 signalling axis in CML. In K562 cells, BCR-ABL silencing reduced full-length NOTCH1 (NOTCH1-FL) and inhibited the cleavage of NOTCH1 intracellular domain (NOTCH1-ICD), resulting in decreased expression of the NOTCH1 targets c-MYC and HES1. K562 cells stably overexpressing CCN3 (K562/CCN3) or treated with recombinant CCN3(rCCN3) showed a significant reduction in NOTCH1 signalling (> 50% reduction in NOTCH1-ICD, p < 0.05).Gamma secretase inhibitor (GSI), which blocks NOTCH1 signalling, reduced K562/CCN3 colony formation but increased that of K562/control cells. GSI combined with either rCCN3 or imatinib reduced K562 colony formation with enhanced reduction of NOTCH1 signalling observed with combination treatments. We demonstrate an oncogenic role for NOTCH1 in CML and suggest that BCR-ABL disruption of NOTCH1CCN3 signalling contributes to the pathogenesis of CML.
Subject(s)
Fusion Proteins, bcr-abl/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Nephroblastoma Overexpressed Protein/metabolism , Receptor, Notch1/metabolism , Signal Transduction , Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Flow Cytometry , Fusion Proteins, bcr-abl/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Humans , Imatinib Mesylate , K562 Cells/drug effects , K562 Cells/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Piperazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , RNA, Small Interfering , Real-Time Polymerase Chain Reaction , Signal Transduction/drug effects , TransfectionABSTRACT
Capillary electrophoresis (CE) of erythrocytes from different sources under various conditions is reported in this paper. It was found that erythrocyte samples from sheep, duck, and human showed characteristic and reproducible elution peaks, and that the retention times of A-, B-, AB-, and O-type erythrocytes from human blood were distinctively different; even subtle differences, among individuals with the same blood type could be detected by CE. A strictly linear correlation was obtained between the peak area and the amount of human erythrocyte over a range of 4.8 x 10(2)-1.9 x 10(4) cells (r=0.999), indicating that CE could be used for rapid and accurate quantification of erythrocytes. Using this CE protocol, the decrease of the surface electrical charge of erythrocyte during storage was confirmed. Therefore, this work demonstrated that CE could be a useful alternative for characterizing and quantifying erythrocytes or other cells.
