Correlation between ZAP‐70, phospho‐ZAP‐70, and phospho‐Syk expression in leukemic cells from patients with CLL

Kaplan, David R.; Meyerson, Howard; Li, Xiaofeng; Drasny, Carly; Liu, Fangfang; Costaldi, Mark; Barr, Paul M.; Lazarus, Hillard M.

doi:10.1002/cyto.b.20506

Cited by 10 publications

(22 citation statements)

References 31 publications

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“…More recently, a phase I/II clinical trial of fostamatinib disodium, an oral Syk inhibitor showed clinical activity in CLL and non-Hodgkins lymphoma [27]. Another study showed a negative correlation between the expression of ZAP70 with the phosphorylation state of Syk and a positive correlation between p-Syk with p21 cip , a cell cycle inhibitor [55]. Further insights into the relationship of phosphatase activity with BCR signaling molecules and apoptosis could be determined by experiments including specific tyrosine phosphatase inhibitors specifically targeting SHP-1 and/or CD45.…”

Section: Discussionmentioning

confidence: 99%

Association of Reactive Oxygen Species-Mediated Signal Transduction with In Vitro Apoptosis Sensitivity in Chronic Lymphocytic Leukemia B Cells

et al. 2011

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BackgroundChronic lymphocytic leukemia (CLL) is a B cell malignancy with a variable clinical course and unpredictable response to therapeutic agents. Single cell network profiling (SCNP) utilizing flow cytometry measures alterations in signaling biology in the context of molecular changes occurring in malignancies. In this study SCNP was used to identify proteomic profiles associated with in vitro apoptotic responsiveness of CLL B cells to fludarabine, as a basis for ultimately linking these with clinical outcome.Methodology/Principal FindingSCNP was used to quantify modulated-signaling of B cell receptor (BCR) network proteins and in vitro F-ara-A mediated apoptosis in 23 CLL samples. Of the modulators studied the reactive oxygen species, hydrogen peroxide (H2O2), a known intracellular second messenger and a general tyrosine phosphatase inhibitor stratified CLL samples into two sub-groups based on the percentage of B cells in a CLL sample with increased phosphorylation of BCR network proteins. Separately, in the same patient samples, in vitro exposure to F-ara-A also identified two sub-groups with B cells showing competence or refractoriness to apoptotic induction. Statistical analysis showed that in vitro F-ara-A apoptotic proficiency was highly associated with the proficiency of CLL B cells to undergo H2O2-augmented signaling.Conclusions/SignificanceThis linkage in CLL B cells among the mechanisms governing chemotherapy-induced apoptosis increased signaling of BCR network proteins and a likely role of phosphatase activity suggests a means of stratifying patients for their response to F-ara-A based regimens. Future studies will examine the clinical applicability of these findings and also the utility of this approach in relating mechanism to function of therapeutic agents.

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Section: Discussionmentioning

confidence: 99%

Association of Reactive Oxygen Species-Mediated Signal Transduction with In Vitro Apoptosis Sensitivity in Chronic Lymphocytic Leukemia B Cells

et al. 2011

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“…A number of publications have demonstrated expert use of TSA amplification and flow cytometry to quantify surface and intracellular protein levels in single cells (23–29). But to a novice the approach can be cumbersome due to the abundance of interdependent assay variables.…”

Section: Discussionmentioning

confidence: 99%

“…Subsequently, measurement of D cyclins by TSA revealed differential expression in three B cell lymphoproliferative diseases (28). Recently, TSA-based measurement of intracellular signaling activity in leukemic B cells showed that basal levels of ZAP-70 and Syk phosphorylation were negatively correlated to total expression of ZAP-70 protein (29). …”

Section: Introductionmentioning

confidence: 99%

Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry

et al. 2010

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Intracellular flow cytometry permits quantitation of diverse molecular targets at the single-cell level. However, limitations in detection sensitivity inherently restrict the method, sometimes resulting in the inability to measure proteins of very low abundance or to differentiate cells expressing subtly different protein concentrations. To improve these measurements, an enzymatic amplification approach called tyramide signal amplification (TSA) was optimized for assessment of intracellular kinase cascades. First, Pacific Blue, Pacific Orange, and Alexa Fluor 488 tyramide reporters were shown to exhibit low nonspecific binding in permeabilized cells. Next, the effects of antibody concentration, tyramide concentration, and reaction time on assay resolution were characterized. Use of optimized TSA resulted in a 10-fold or greater improvement in measurement resolution of endogenous Erk and Stat cell signaling pathways relative to standard, nonamplified detection. TSA also enhanced assay sensitivity and, in conjunction with fluorescent cell barcoding, improved assay performance according to a metric used to evaluate high-throughput drug screens. TSA was used to profile Stat1 phosphorylation in primary immune system cells, which revealed heterogeneity in various populations, including CD41 FoxP31 regulatory T cells. We anticipate the approach will be broadly applicable to intracellular flow cytometry assays with low signal-to-noise ratios. '

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“…While other findings suggested that lower levels of p21cip1 and/or a defect in p53 activity may account in part for the more aggressive disease course in patients with high levels of ZAP-70 rather than enhanced BCR signaling as has been previously hypothesized. Kaplan et al [22] demonstrated that increased expression of ZAP-70 is correlated with decreased levels of phosphorylated ZAP-70 and phosphorylated Syk measured directly ex vivo using highresolution immunophenotyping. Syk and ZAP-70 phosphorylation appear to be mostly independent of each other.…”

Section: Structure and Function Of Zap-70mentioning

confidence: 99%

Detection methods of ZAP-70 in chronic lymphocytic leukemia

Wang

Fan

et al. 2011

Clin Exp Med

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The clinical course of patients with chronic lymphocytic leukemia (CLL) is highly heterogeneous, with some patients experiencing rapid disease progression and others living for decades without requiring treatment. Immunoglobulin heavy chain variable region (IGHV) mutation status is a powerful prognostic factor in patients with CLL. The presence or absence of IGHV mutation status is currently the gold-standard prognostic factor, but this technique is labor-intensive and costly. The expression of ζ-chain-associated protein kinase 70 kDa (ZAP-70), detected by flow cytometry, has been served as a potential surrogate for the absence of IGHV mutation status since 2003. Given the current problems with the standardization of ZAP-70 assessment by flow cytometry, this chapter reviews the analysis of ZAP-70 expression by flow cytometry and the relative experimental conditions using the associated publications for "ZAP-70" and "detection method" on Medline since 2003. Thus, mean fluorescence intensity (MFI) ratio method yielded a more reproducible and easily adaptable method to routine use in the clinical diagnostic laboratory. In conclusion, though ZAP-70 is a valuable prognostic factor in CLL, detection methods of ZAP-70 remain to be fully standardized.

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Correlation between ZAP‐70, phospho‐ZAP‐70, and phospho‐Syk expression in leukemic cells from patients with CLL

Cited by 10 publications

References 31 publications

Association of Reactive Oxygen Species-Mediated Signal Transduction with In Vitro Apoptosis Sensitivity in Chronic Lymphocytic Leukemia B Cells

Association of Reactive Oxygen Species-Mediated Signal Transduction with In Vitro Apoptosis Sensitivity in Chronic Lymphocytic Leukemia B Cells

Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry

Detection methods of ZAP-70 in chronic lymphocytic leukemia

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