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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Hamilton
Ashley
A
Section of Experimental Haematology, Cancer Division, Faculty of Medicine, University of Glasgow, Paul O'Gorman Leukaemia Research Centre, Gartnavel General Hospital, Glasgow, UK.
Alhashimi
Fatma
F
Myssina
Svetlana
S
Jorgensen
Heather G
HG
Holyoake
Tessa L
TL
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D016044:Fusion Proteins, bcr-abl / Q000032:analysis*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000473:pathology*; D008722:Methods; D010766:Phosphorylation; D012107:Research Design; D014443:Tyrosine / Q000032:analysis
D014443:Tyrosine; D016044:Fusion Proteins, bcr-abl