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. , Jorgensen, H.G., Melo, J.V. and Holyoake, T.L. (2006) 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(6), pp. 1035-1039. (doi:10.1038/sj.leu.2404189)

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Abstract

In chronic myeloid leukaemia, CD34<sup>+</sup> stem/progenitor cells appear resistant to imatinib mesylate (IM) <i>in vitro</i> and <i>in vivo</i>. 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<sup>4</sup> cells. The method was first validated in wild-type (K562) and mutant (BAF3) BCR-ABL<sup>+</sup> as well as BCR-ABL<sup>-</sup> (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<sup>+</sup> CD34<sup>+</sup> cells and was able to discriminate between Ph<sup>-</sup>, sensitive and resistant Ph<sup>+</sup> cells. This assay should now enable investigators to unravel the mechanism(s) of IM resistance in stem cells.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Holyoake, Professor Tessa and Jorgensen, Dr Heather and Copland, Professor Mhairi
Authors: Hamilton, A., Elrick, L., Myssina, S., Copland, M., Jorgensen, H.G., Melo, J.V., and Holyoake, T.L.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
Journal Name:Leukemia
ISSN:0887-6924

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
354131Novel drug combinations for eradication of Ph+/Bcr-Abl+ haemopoietic stem cells in chronic myeloid leukaemia (CML)Mhairi CoplandMedical Research Council (MRC)G84/6317Institute of Cancer Sciences