Experimental hematology · 2009
Optimization of methods for the detection of BCR-ABL activity in Philadelphia-positive cells.
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Hamilton Ashley, Alhashimi Fatma, Myssina Svetlana, Jorgensen Heather G, Holyoake Tessa L
Аннотация
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.
MATERIALS AND 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.
CONCLUSION: 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.
MATERIALS AND 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.
CONCLUSION: 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.