Optimization of methods for the detection of BCR-ABL activity in Philadelphia-positive cells

Hamilton, A., Alhashimi, F., Myssina, S., Jorgensen, H. G. and Holyoake, T. L. (2009) Optimization of methods for the detection of BCR-ABL activity in Philadelphia-positive cells. Experimental Hematology, 37(3), pp. 395-401. (doi: 10.1016/j.exphem.2008.11.005)

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Publisher's URL: http://dx.doi.org/10.1016/j.exphem.2008.11.005

Abstract

<b>Objective:</b> The recent success in treating chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKI), such as imatinib mesylate (IM), has created a demand for reproducible methods to accurately assess inhibition of BCR-ABL activity within CML cells, including rare stein and progenitor cells, either in vitro or in vivo. The purpose of this study was to develop an enzyme-linked immunosorbent (ELISA) method to measure total tyrosine phosphorylation (P-Tyr) in small samples of cells that express BCR-ABL and to compare to more established methods.<p></p> <b>Materials and Methods:</b> The assay was first validated in BCR-ABL wild-type and mutant vs BCR-ABL-negative cell lines. P-Tyr levels were then measured by ELISA in primary CD34(+) CML cells treated with IM.<p></p> <b>Results:</b> In vitro exposure to TKI resulted in decreases in the level of P-Tyr, in both BCR-ABL-positive cell lines and primary CD34(+) CML samples, which were comparable to the reduction in P-Tyr by flow cytometry and phosphorylation of CrkL by either Western blot or flow cytometry.<p></p> <b>Conclusion:</b> We have developed an accurate ELISA method to measure BCR-ABL, activity within Ph+ cells, which is comparable to other in vitro BCR-ABL, assessment techniques in terms of sensitivity and could be adapted for high throughput.<p></p>

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Hamilton, Ms Ashley and Holyoake, Professor Tessa and Jorgensen, Dr Heather
Authors: Hamilton, A., Alhashimi, F., Myssina, S., Jorgensen, H. G., and Holyoake, T. L.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
Journal Name:Experimental Hematology
Publisher:Elsevier
ISSN:0301-472X
ISSN (Online):1873-2399
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
439231Is Bcr-Able expression relevant for the survival of cancer stem cells in chronic myeloid leukaemiaTessa HolyoakeMedical Research Council (MRC)G0600782Institute of Cancer Sciences