Journal
LEUKEMIA
Volume 20, Issue 6, Pages 1035-1039Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/sj.leu.2404189
Keywords
chronic myeloid leukaemia; stem cells; flow cytometry; CrkL
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Funding
- Medical Research Council [G84/6317] Funding Source: Medline
- Chief Scientist Office [SCD/04] Funding Source: Medline
- Medical Research Council [G84/6317] Funding Source: researchfish
- MRC [G84/6317] Funding Source: UKRI
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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-AbI 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-AbI 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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