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    Leukemia. 2006 Jun;20(6):1035-9.

    BCR-ABL activity and its response to drugs can be determined in CD34+ CML stem cells by CrkL phosphorylation status using flow cytometry.

    Hamilton A, Elrick L, Myssina S, Copland M, Jørgensen H, Melo JV, Holyoake T.

    Source

    Section of Experimental Haematology, Division of Cancer Sciences & Molecular Pathology, University of Glasgow, Glasgow, UK.

    Abstract

    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.

    PMID:
    16572205
    [PubMed - indexed for MEDLINE]

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