BCR-ABL activity and its response to drugs can be determined in CD34+ CML stem cells by CrkL phosphorylation status using flow cytometry.
In chronic myeloid leukaemia, CD34(+) stem/progenitor cells appear resistant to imatinib mesylate (IM) in vitro and in vivo. To investigate the underlying mechanism(s) of IM resistance, it is essential to quantify Bcr-Abl kinase status at the stem cell level. We developed a flow cytometry method to measure CrkL phosphorylation (P-CrkL) in samples with <10(4) cells. The method was first validated in wild-type (K562) and mutant (BAF3) BCR-ABL(+) as well as BCR-ABL(-) (HL60) cell lines. In response to increasing IM concentration, there was a linear reduction in P-CrkL, which was Bcr-Abl specific and correlated with known resistance. The results were comparable to those from Western blotting. The method also proved to be reproducible with small samples of normal and Ph(+) CD34(+) cells and was able to discriminate between Ph(-), sensitive and resistant Ph(+) cells. This assay should now enable investigators to unravel the mechanism(s) of IM resistance in stem cells.
Section of Experimental Haematology, Division of Cancer Sciences & Molecular Pathology, University of Glasgow, Glasgow, UK.
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Hamilton
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Elrick
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Myssina
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Copland
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Jørgensen
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Melo
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Holyoake
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D018952:Antigens, CD34 / Q000096:biosynthesis*; D001549:Benzamides; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry / Q000379:methods; D016044:Fusion Proteins, bcr-abl / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018922:HL-60 Cells; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D000068877:Imatinib Mesylate; D066298:In Vitro Techniques; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; D018952:Antigens, CD34; D001549:Benzamides; C082869:CRKL protein; D009687:Nuclear Proteins; D010879:Piperazines; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D016044:Fusion Proteins, bcr-abl