Immunoreactivity of Stat5 phosphorylated on tyrosine as a cell‐based measure of Bcr/Abl kinase activity

Abstract

Background: Stat5 1 (Signal Transducer and Activator of Transcription 5) is normally phosphorylated and activated by Janus kinases. In cells transformed with BCR/ABL, Stat5 is constitutively activated by promiscuous phosphorylation. Cytometry of intracellular antigens can be used to evaluate cell treatments affecting gene expression, because it precisely provides the fraction of affected cells and the quantitative change in expression. Here, we asked whether we could measure a phosphorylated epitope on Stat5 by cytometry, and whether that measurement would respond to Bcr/Abl inhibition. Methods: Chronic myelogenous leukemia (CML) cell lines or control Bcr/Abl-negative cells were treated or not with imatinib mesylate, fixed and permeabilized with formaldehyde followed by methanol; reacted with rabbit polyclonal and mouse monoclonal antibodies against an epitope including tyrosine 694 of Stat5a (pSTAT5); reacted with antibodies that mark mitotic cells; counterstained with secondary fluorescent antibodies and 4Ј,6-diamidino-2-phenylindole (DAPI); and then subjected to flow cytometry. Western blotting was performed with pSTAT5 and Stat5 antibodies.

Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations

“…However, although we do not have evidence that our original technique provides biologically relevant measurements, we do know that the signal is equivalent to western blot analysis. In general, our experience indicates that fluorescence-based cytometric measurements are more precise but less sensitive than western blots (5,16). Further, by comparing western blotting with cytometry for samples that have been extracted with various solutions (Frisa and Jacobberger, unpublished data), we have come to the expected conclusion that we detect only a fraction of the intracellular epitope by cytometry, i.e., a significant portion of the epitope is not available for staining after alcohol fixation/permeabilization and staining at antibody saturation.…”

“…Thus, different anticoagulants, overnight storage and shipment, whole blood staining, and several erythrocyte lysis techniques result in reproducible, convenient, and flexible assays. Recently, interest has been stimulated in potentially less robust assays of cell signaling components based on the relatively recent availability of phosphorylation state-specific antibodies (1)(2)(3)(4)(5)(6). Because of the quantitative and correlative natures of cytometry, this represents a novel and potentially powerful approach to classification of hematologic malignancies including the selection of patients for molecular targeted therapies and monitoring drug effects in individual patients (1,(6)(7)(8).…”

“…Analysis of signal transduction pathways by flow cytometry presents technical problems that are not currently encountered in routine clinical applications (1)(2)(3)(4)(5). The phosphorylation states of individual signaling elements change rapidly in response to stimuli and therefore may be subject to changes due to sample collection, storage, preparation, and staining that may obscure the signaling state of the condition under study.…”

Validation of a multiparameter flow cytometry method for the determination of phosphorylated extracellular‐signal‐regulated kinase and DNA in circulating tumor cells

“…In this study, we present the development and validation of a multiparameter fluorescence-activated cell sorting (FACS) method for the quantification of pERK, and the detection of nucleated CTCs isolated from peripheral human blood. Phosphorylation state-specific antibodies (19)(20)(21)(22)(23)(24) have recently become available and were used for the quantification of pERK. The applicability of the method was demonstrated in peripheral blood from patients with advanced metastatic breast, colon, lung, ovarian, and urothelial cancer.…”

A look at where we came from and where we are going

“…Many of these early and successful targeted therapies, such as the bcr:abl kinase inhibitor therapy, to mention only one, are kinase inhibitors. Cytometric assays to monitor kinase activity are rapidly being developed (1)(2)(3) and are a part of most clinical trials of these new generations of targeted therapies as key analyses to help understand drug action, emergence of drug resistance, and interactions with other therapies used in combination with these targeted therapies. It is clear that these cell-based cytometric assays are going to move to the clinical laboratory in the near future, thus ensuring a growing role of clinical cytometry in patient therapeutic decisions and monitoring.…”