Skip to main content
SpringerLink
Log in

Single-cell sphingosine kinase activity measurements in primary leukemia

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

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.

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Hannun YA, Obeid LM (2008) Nat Rev Mol Cell Biol 9:139–150

    Article  CAS  Google Scholar 

  2. Pyne NJ, Pyne S (2010) Nat Rev Cancer 10:489–503

    Article  CAS  Google Scholar 

  3. Takabe K, Paugh SW, Milstien S, Spiegel S (2008) Pharmacol Rev 60:181–195

    Article  CAS  Google Scholar 

  4. Cuvillier O, Pirianov G, Kleuser B, Vanek P, Coso O, Gutkind J, Spiegel S (1996) Nature 381:800–803

    Article  CAS  Google Scholar 

  5. Ogretmen B, Hannun Y (2004) Nat Rev Cancer 4:604–616

    Article  CAS  Google Scholar 

  6. Siegel R, Ma JM, Zou ZH, Jemal A (2014) CA Cancer J Clin 64:9–29

    Article  Google Scholar 

  7. Sweeney EA, Sakakura C, Shirahama T, Masamune A, Ohta H, Hakomori S, Igarashi Y (1996) Int J Cancer 66:358–366

    Article  CAS  Google Scholar 

  8. Paugh S, Paugh B, Rahmani M, Kapitonov D, Almenara J, Kordula T, Milstien S, Adams J, Zipkin R, Grant S, Spiegel S (2008) Blood 112:1382–1391

    Article  CAS  Google Scholar 

  9. Kim BM, Choi YJ, Lee YH, Joe YA, Hong SH (2010) Apoptosis 15:982–993

    Article  CAS  Google Scholar 

  10. Salas A, Ponnusamy S, Senkal C, Meyers-Needham M, Selvam S, Saddoughi S, Apohan E, Sentelle R, Smith C, Gault C, Obeid L, El-Shewy H, Oaks J, Santhanam R, Marcucci G, Baran Y, Mahajan S, Fernandes D, Stuart R, Perrotti D, Ogretmen B (2011) Blood 117:5941–5952

    Article  CAS  Google Scholar 

  11. Baran Y, Salas A, Senkal CE, Gunduz U, Bielawski J, Obeid LM, Ogretmen B (2007) J Biol Chem 282:10922–10934

    Article  CAS  Google Scholar 

  12. Edmonds Y, Milstien S, Spiegel S (2011) Pharmacol Ther 132:352–360

    Article  CAS  Google Scholar 

  13. Meacham CE, Morrison SJ (2013) Nature 501:328–337

    Article  CAS  Google Scholar 

  14. Burrell RA, McGranahan N, Bartek J, Swanton C (2013) Nature 501:338–345

    Article  CAS  Google Scholar 

  15. Gerlinger M (2012) N Engl J Med 367:976–976

    CAS  Google Scholar 

  16. Ding L, Ley TJ, Larson DE, Miller CA, Koboldt DC, Welch JS, Ritchey JK, Young MA, Lamprecht T, McLellan MD, McMichael JF, Wallis JW, Lu C, Shen D, Harris CC, Dooling DJ, Fulton RS, Fulton LL, Chen K, Schmidt H, Kalicki-Veizer J, Magrini VJ, Cook L, McGrath SD, Vickery TL, Wendl MC, Heath S, Watson MA, Link DC, Tomasson MH, Shannon WD, Payton JE, Kulkarni S, Westervelt P, Walter MJ, Graubert TA, Mardis ER, Wilson RK, DiPersio JF (2012) Nature 481:506–510

    Article  CAS  Google Scholar 

  17. Hope KJ, Jin LQ, Dick JE (2004) Nat Immunol 5:738–743

    Article  CAS  Google Scholar 

  18. Ley TJ, Miller C, Ding L, Raphael BJ, Mungall AJ, Robertson AG, Hoadley K, Triche TJ, Laird PW, Baty JD, Fulton LL, Fulton R, Heath SE, Kalicki-Veizer J, Kandoth C, Klco JM, Koboldt DC, Kanchi KL, Kulkarni S, Lamprecht TL, Larson DE, Lin L, Lu C, McLellan MD, McMichael JF, Payton J, Schmidt H, Spencer DH, Tomasson MH, Wallis JW, Wartman LD, Watson MA, Welch J, Wendl MC, Ally A, Balasundaram M, Birol I, Butterfield Y, Chiu R, Chu A, Chuah E, Chun HJ, Corbett R, Dhalla N, Guin R, He A, Hirst C, Hirst M, Holt RA, Jones S, Karsan A, Lee D, Li HI, Marra MA, Mayo M, Moore RA, Mungall K, Parker J, Pleasance E, Plettner P, Schein J, Stoll D, Swanson L, Tam A, Thiessen N, Varhol R, Wye N, Zhao YJ, Gabriel S, Getz G, Sougnez C, Zou LH, Leiserson MDM, Vandin F, Wu HT, Applebaum F, Baylin SB, Akbani R, Broom BM, Chen K, Motter TC, Nguyen K, Weinstein JN, Zhang NZ, Ferguson ML, Adams C, Black A, Bowen J, Gastier-Foster J, Grossman T, Lichten-Berg T, Wise L, Davidsen T, Demchok JA, Shaw KRM, Sheth M, Sofia HJ, Yang LM, Downing JR, Eley G, Alonso S, Ayala B, Baboud J, Backus M, Barletta SP, Berton DL, Chu AL, Girshik S, Jensen MA, Kahn A, Kothiyal P, Nicholls MC, Pihl TD, Pot DA, Raman R, Sanbhadti RN, Snyder EE, Srinivasan D, Walton JS, Wan YH, Wang ZN, Issa JPJ, Le Beau M, Carroll M, Kantarjian H, Kornblau S, Bootwalla MS, Lai PH, Shen H, Van den Berg DJ, Weisenberger DJ, Link DC, Walter MJ, Ozenberger BA, Mardis ER, Westervelt P, Graubert TA, DiPersio JF, Wilson RK (2013) N Engl J Med 368:2059–2074

    Article  Google Scholar 

  19. Perez OD, Nolan GP (2002) Nat Biotechnol 20:155–162

    Article  CAS  Google Scholar 

  20. Bendall SC, Simonds EF, Qiu P, Amir EAD, Krutzik PO, Finck R, Bruggner RV, Melamed R, Trejo A, Ornatsky OI, Balderas RS, Plevritis SK, Sachs K, Pe'er D, Tanner SD, Nolan GP (2011) Science 332:687–696

    Article  CAS  Google Scholar 

  21. Perez OD, Krutzik PO, Nolan GP (2004) In: “Flow Cytometric Analysis of Kinase Signaling Cascades,” Hawley TS, Hawley TS (eds) Flow Cytometry Protocols, 2nd edn. Humana, Totowa

  22. Zhang J, Campbell RE, Ting AY, Tsien RY (2002) Nat Rev Mol Cell Biol 3:906–918

    Article  CAS  Google Scholar 

  23. Camera E, Picardo M, Presutti C, Catarcini P, Fanali S (2004) J Sep Sci 27:971–976

    Article  CAS  Google Scholar 

  24. Moroz LL, Dahlgren RL, Boudko D, Sweedler JV, Lovell P (2005) J Inorg Biochem 99:929–939

    Article  CAS  Google Scholar 

  25. Whitmore CD, Olsson U, Larsson EA, Hindsgaul O, Palcic MM, Dovichi NJ (2007) Electrophoresis 28:3100–3104

    Article  CAS  Google Scholar 

  26. Whitmore CD, Hindsgaul O, Palcic MM, Schnaar RL, Dovichi NJ (2007) Anal Chem 79:5139–5142

    Article  CAS  Google Scholar 

  27. Proctor A, Wang QZ, Lawrence DS, Allbritton NL (2012) Anal Chem 84:7195–7202

    Article  CAS  Google Scholar 

  28. Meredith GD, Sims CE, Soughayer JS, Allbritton NL (2000) Nat Biotechnol 18:309–312

    Article  CAS  Google Scholar 

  29. Lee J, Kotliarova S, Kotliarov Y, Li AG, Su Q, Donin NM, Pastorino S, Purow BW, Christopher N, Zhang W, Park JK, Fine HA (2006) Cancer Cell 9:391–403

    Article  CAS  Google Scholar 

  30. Essaka DC, Prendergast J, Keithley RB, Palcic MM, Hindsgaul O, Schnaar RL, Dovichi NJ (2012) Anal Chem 84:2799–2804

    Article  CAS  Google Scholar 

  31. Phillips RM, Dailey LA, Bair E, Samet JM, Allbritton NL (2014) Anal Chem 86:1291–1297

    Article  CAS  Google Scholar 

  32. Proctor A, Herrera-Loeza SG, Wang Q, Lawrence DS, Yeh JJY, Allbritton NL (2014) Anal Chem 84:4573–4580

    Article  Google Scholar 

  33. Dickinson AJ, Armistead PM, Allbritton NL (2013) Anal Chem 85:4797–4804

    Article  CAS  Google Scholar 

  34. Pai JH, Wang Y, Salazar GT, Sims CE, Bachman M, Li GP, Allbritton NL (2007) Anal Chem 79:8774–8780

    Article  CAS  Google Scholar 

  35. Sims CE, Meredith GD, Krasieva T, Berns M, Tromberg B, Allbritton NL (1998) Anal Cem 70:4570–4577

    Article  CAS  Google Scholar 

  36. Kovarik ML, Shah PK, Armistead PM, Allbritton NL (2013) Anal Chem 85:4991–4997

    Article  CAS  Google Scholar 

  37. Wong ECC, Maher VE, Hines K, Lee J, Carter CS, Goletz T, Kopp W, Mackall CL, Berzofsky JA, Read EJ (2001) Cytotherapy 3:19–29

    Article  CAS  Google Scholar 

  38. Schnute ME, McReynolds MD, Kasten T, Yates M, Jerome G, Rains JW, Hall T, Chrencik J, Kraust M, Cronin CN, Saabye M, Highkin MK, Broadus R, Ogawa S, Cukyne K, Zawadzke LE, Peterkin V, Iyanar K, Scholten JA, Wendling J, Fujiwara H, Nemirovskiy O, Wittwer AJ, Nagiec MM (2012) Biochem J 444:79–88

    Article  CAS  Google Scholar 

  39. Wilson E, Wang E, Mullins RE, Uhlinger DJ, Liotta DC, Lambeth JD, Merrill AH (1988) J Biol Chem 263:9304–9309

    CAS  Google Scholar 

  40. Casanovas RO, Slimane FK, Garand R, Faure GC, Campos L, Deneys V, Bernier M, Falkenrodt A, Lecalvez G, Maynadie M, Bene MC (2003) Leukemia 17:515–527

    Article  Google Scholar 

  41. Legrand O, Simonin G, Perrot JY, Zittoun R, Marie JP (1998) Blood 91:4480–4488

    CAS  Google Scholar 

  42. Abbott BL, Colapietro AM, Barnes Y, Marini F, Andreeff M, Sorrentino BP (2002) Blood 100:4594–4601

    Article  CAS  Google Scholar 

  43. Amir ED, Davis KL, Tadmor MD, Simonds EF, Levine JH, Bendall SC, Shenfeld DK, Krishnaswamy S, Nolan GP, Pe'er D (2013) Nat Biotechnol 31:545–554

    Article  CAS  Google Scholar 

  44. Bendall S, Nolan GP (2012) Nat Biotechnol 30:639–647

    Article  CAS  Google Scholar 

Download references

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)

Author information

Authors and Affiliations

  1. Department of Chemistry, University of North Carolina, Chapel Hill, NC, 27599, USA

    Alexandra J. Dickinson & Nancy L. Allbritton

  2. Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599, USA

    Sally A. Hunsucker, Paul M. Armistead & Nancy L. Allbritton

  3. Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC, 27599, USA

    Nancy L. Allbritton

  4. North Carolina State University, Raleigh, NC, 27695, USA

    Nancy L. Allbritton

Authors
  1. Alexandra J. Dickinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sally A. Hunsucker
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paul M. Armistead
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nancy L. Allbritton
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nancy L. Allbritton.

Additional information

Published in the topical collection Single Cell Analysis with guest editors Petra Dittrich and Norbert Jakubowski.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 311 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dickinson, A.J., Hunsucker, S.A., Armistead, P.M. et al. Single-cell sphingosine kinase activity measurements in primary leukemia. Anal Bioanal Chem 406, 7027–7036 (2014). https://doi.org/10.1007/s00216-014-7974-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-014-7974-6

Keywords

Search

Navigation