Dysregulation of the factors downstream of the BCR-ABL fusion protein contributes to evasion of programmed cell death and uncontrolled proliferation. Such dysregulation and has been regarded as the driving force for the development of chronic myelogenous leukemia (CML) (1). Though the tyrosine kinase inhibitor imatinib mesylate (Gleevec) has achieved clinical success in the targeting of the BCR-ABL fusion product (2-4), a significant subset of patients has developed resistance to it as a result of mutations affecting the ABL kinase domain (5). Thus, several more specific tyrosine kinase inhibitors are being

From: Methods in Molecular Biology vol. 378: Monoclonal Antibodies: Methods and Protocols Edited by: M. Albitar © Humana Press Inc., Totowa, NJ

developed. A simple and reliable test adaptable for clinical laboratories is needed to monitor BCR-ABL activity and its subsequent inhibition. Measuring the large and unstable BCR-ABL fusion protein itself is difficult (see Note 1). Thus, the activity of BCR-ABL has been monitored by Western blot analysis of downstream effectors such as CRKL, an adaptor protein that is directly phos-phorylated by the BCR-ABL fusion protein in CML and plays a central role in propagating the kinase signal to downstream effectors (6-8). Here, we describe a flow cytometry approach that can quantitate intracellular proteins and their phosphorylated forms.

Our approach is based on the permeabilization of viable cells, antibody staining of specific intracellular proteins, and quantification of the stained protein molecules in each cell. The costaining of each cell with surface and intracellu-lar protein markers allows measurement of the number of molecules in specific cell populations. The monitoring of changes in response to therapy is possible owing to protein-specific modifications (for example, phosphorylation). In the following sections, we describe how quantitative flow cytometry can be applied to monitoring levels of total and phosphorylated CRKL in clinical samples and cell lines. We also describe ex vivo drug manipulation of a cell line to ensure antibody specificity and provide a positive control for the assay. For example, exposing K562 (a BCR-ABL-positive blast crisis CML cell line) to imatinib mesylate down-regulates levels of CRKL phosphorylation.

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