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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Rowley JD . A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature 1973; 243: 290–293.
Deininger MW, Goldman JM, Melo JV . The molecular biology of chronic myeloid leukemia. Blood 2000; 96: 3343–3356.
Oda T, Heaney C, Hagopian JR, Okuda K, Griffin JD, Druker BJ . Crkl is the major tyrosine-phosphorylated protein in neutrophils from patients with chronic myelogenous leukemia. J Biol Chem 1994; 269: 22925–22928.
Nichols GL, Raines MA, Vera JC, Lacomis L, Tempst P, Golde DW . Identification of CRKL as the constitutively phosphorylated 39-kD tyrosine phosphoprotein in chronic myelogenous leukemia cells. Blood 1994; 84: 2912–2918.
ten Hoeve J, Arlinghaus RB, Guo JQ, Heisterkamp N, Groffen J . Tyrosine phosphorylation of CRKL in Philadelphia+ leukemia. Blood 1994; 84: 1731–1736.
Hochhaus A, Kreil S, Corbin A, La Rosee P, Lahaye T, Berger U et al. Roots of clinical resistance to STI-571 cancer therapy. Science 2001; 293: 2163.
White D, Saunders V, Lyons AB, Branford S, Grigg A, To LB et al. In vitro sensitivity to imatinib-induced inhibition of ABL kinase activity is predictive of molecular response in patients with de novo CML. Blood 2005; 106: 2520–2526.
Holyoake T, Jiang X, Eaves C, Eaves A . Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia. Blood 1999; 94: 2056–2064.
Holyoake TL, Jiang X, Jørgensen HG, Graham S, Alcorn MJ, Laird C et al. Primitive quiescent leukemic cells from patients with chronic myeloid leukemia spontaneously initiate factor-independent growth in vitro in association with up-regulation of expression of interleukin-3. Blood 2001; 97: 720–728.
Graham SM, Jørgensen HG, Allan E, Pearson C, Alcorn MJ, Richmond L et al. Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro. Blood 2002; 99: 319–325.
Elrick LJ, Jørgensen HG, Mountford JC, Holyoake TL . Punish the parent not the progeny. Blood 2005; 105: 1862–1866.
Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001; 344: 1031–1037.
Corbin AS, La Rosee P, Stoffregen EP, Druker BJ, Deininger MW . Several Bcr-Abl kinase domain mutants associated with imatinib mesylate resistance remain sensitive to imatinib. Blood 2001; 101: 4611–4614.
Schultheis B, Szydlo R, Mahon FX, Apperly JF, Melo JV . Analysis of total phosphotyrosine levels in CD34+ cells from CML patients to predict the response to imatinib mesylate treatment. Blood 2005; 105: 4893–4894.
Roche-Lestienne C, Soenen-Cornu V, Grardel-Duflos N, Laï J-L, Philippe N, Facon T et al. Several types of mutations of the Abl gene can be found in chronic myeloid leukemia patients resistant to STI571, and they can pre-exist to the onset of treatment. Blood 2002; 100: 1014–1018.
Kreuzer K-A, Le Coutre P, Landt O, Na I-K, Schwarz M, Schultheis K et al. Pre-existence and evolution of imatinib mesylate-resistant clones in chronic myelogenous leukaemia detected by a PNA-based PCR clamping technique. Ann Hematol 2003; 82: 284–289.
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.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.leu.2404189
Keywords
This article is cited by
-
Efficiency of nilotinib to target chronic phase-chronic myeloid leukaemia primary mature CD34− and immature CD34+ cells
Scientific Reports (2021)
-
Mtss1 is a critical epigenetically regulated tumor suppressor in CML
Leukemia (2016)
-
Pharmacokinetics and pharmacodynamics of dasatinib in the chronic phase of newly diagnosed chronic myeloid leukemia
European Journal of Clinical Pharmacology (2016)
-
Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines
Nature Protocols (2015)
-
Second generation tyrosine kinase inhibitors prevent disease progression in high-risk (high CIP2A) chronic myeloid leukaemia patients
Leukemia (2015)