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

被引:69
作者
Hamilton, A.
Elrick, L.
Myssina, S.
Copland, M.
Jorgensen, H.
Melo, J. V.
Holyoake, T.
机构
[1] Univ Glasgow, Div Canc Sci & Mol Pathol, Sect Expt Haematol, Royal Infirm, Glasgow G31 2ER, Lanark, Scotland
[2] Univ London Imperial Coll Sci Technol & Med, Hammersmith Hosp, Dept Haematol, London, England
基金
英国医学研究理事会;
关键词
chronic myeloid leukaemia; stem cells; flow cytometry; CrkL;
D O I
10.1038/sj.leu.2404189
中图分类号
R73 [肿瘤学];
学科分类号
100214 ;
摘要
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.
引用
收藏
页码:1035 / 1039
页数:5
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