Analytical and Bioanalytical Chemistry

, Volume 406, Issue 27, pp 7027–7036 | Cite as

Single-cell sphingosine kinase activity measurements in primary leukemia

  • Alexandra J. Dickinson
  • Sally A. Hunsucker
  • Paul M. Armistead
  • Nancy L. Allbritton
Research Paper
Part of the following topical collections:
  1. Single Cell Analysis

Abstract

Sphingosine kinase (SK) is a promising therapeutic target in a number of cancers, including leukemia. Traditionally, SK has been measured in bulk cell lysates, but this technique obscures the cellular heterogeneity present in this pathway. For this reason, SK activity was measured in single cells loaded with a fluorescent sphingosine reporter. An automated capillary electrophoresis (CE) system enabled rapid separation and quantification of the phosphorylated and nonphosphorylated sphingosine reporter in single cells. SK activity was measured in tissue-cultured cells derived from chronic myelogenous leukemia (K562), primary peripheral blood mononuclear cells (PBMCs) from three patients with different forms of leukemia, and enriched leukemic blasts from a patient with acute myeloid leukemia (AML). Significant intercellular heterogeneity existed in terms of the degree of reporter phosphorylation (as much as an order of magnitude difference), the amount of reporter uptake, and the metabolites formed. In K562 cells, the average amount of reporter converted to the phosphorylated form was 39 ± 26 % per cell. Of the primary PBMCs analyzed, the average amount of phosphorylated reporter was 16 ± 25 %, 11 ± 26 %, and 13 ± 23 % in a chronic myelogenous leukemia (CML) patient, an AML patient, and a B-cell acute lymphocytic leukemia (B-ALL) patient, respectively. These experiments demonstrated the challenge of studying samples comprised of multiple cell types, with tumor blasts present at 5 to 87 % of the cell population. When the leukemic blasts from a fourth patient with AML were enriched to 99 % of the cell population, 19 ± 36 % of the loaded sphingosine was phosphorylated. Thus, the diversity in SK activity remained even in a nearly pure tumor sample. These enriched AML blasts loaded significantly less reporter (0.12 ± 0.2 amol) relative to that loaded into the PBMCs in the other samples (≥1 amol). The variability in SK signaling may have important implications for SK inhibitors as therapeutics for leukemia and demonstrates the value of single-cell analysis in characterizing the nature of oncogenic signaling in cancer.

Figure

Phosphorylation of a fluorescent sphingosine kinase reporter was used to measure single-cell SK activity in primary cells from leukemic patients. Peripheral blood mononuclear cells as well as enriched leukemic blasts were analyzed.

Keywords

Single-cell analysis Sphingosine kinase Capillary electrophoresis Leukemia Primary cells 

Notes

Acknowledgments

The authors thank Dr. George Fedoriw and the University of North Carolina Hematolymphoid Malignancies Tissue Procurement Facility for providing peripheral blood samples. This research was supported by the NIH (CA171631 to AJD, EB011763 and CA139599 to NLA and HL113594 to PMA)

Supplementary material

216_2014_7974_MOESM1_ESM.pdf (312 kb)
ESM 1 (PDF 311 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Alexandra J. Dickinson
    • 1
  • Sally A. Hunsucker
    • 2
  • Paul M. Armistead
    • 2
  • Nancy L. Allbritton
    • 1
    • 2
    • 3
    • 4
  1. 1.Department of ChemistryUniversity of North CarolinaChapel HillUSA
  2. 2.Lineberger Comprehensive Cancer CenterUniversity of North CarolinaChapel HillUSA
  3. 3.Department of Biomedical EngineeringUniversity of North CarolinaChapel HillUSA
  4. 4.North Carolina State UniversityRaleighUSA

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