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Chronic Myeloproliferative Disorders

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

Leukemia volume 20, pages 1035–1039 (2006)Cite this article

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 <104 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.

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Acknowledgements

We acknowledge the support of Dr Richard Rockefeller (Rockefeller Philanthropy Advisors), and Drs Michael Deininger and Brian Druker (Portland, Oregon) for laboratory training for LJE and for providing the BAF3/BCR-ABL cell lines. AH, LE and SM were funded by the Leukaemia Research Fund, UK; MC by Medical Research Council, UK and Leukaemia Research Trust for Scotland; and HJ is a William Thyne Fellow.

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Authors and Affiliations

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

    A Hamilton, L Elrick, S Myssina, M Copland, H Jørgensen & T Holyoake

  2. Department of Haematology, Imperial College, Hammersmith Hospital, London, UK

    J V Melo

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  1. A Hamilton
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  2. L Elrick
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  3. S Myssina
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  4. M Copland
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  5. H Jørgensen
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  6. J V Melo
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  7. T Holyoake
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Correspondence to T Holyoake.

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Hamilton, A., Elrick, L., Myssina, S. et al. BCR-ABL activity and its response to drugs can be determined in CD34+ CML stem cells by CrkL phosphorylation status using flow cytometry. Leukemia 20, 1035–1039 (2006). https://doi.org/10.1038/sj.leu.2404189

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