, Volume 10, Issue 6, pp 1317–1331 | Cite as

Biphasic behavior of changes in elemental composition during staurosporine-induced apoptosis

  • F. Arrebola
  • J. Cañizares
  • M. A. Cubero
  • P. V. Crespo
  • A. Warley
  • E. Fernández-SeguraEmail author


Although the identification of events that occur during apoptosis is a fundamental goal of apoptotic cell death research, little is know about the precise sequence of changes in total elemental composition during apoptosis. We evaluated total elemental composition (Na, Mg, P, Cl, S, and K) in relation to molecular and morphological features in human U937 cells induced to undergo apoptosis with staurosporine, an intrinsic pathway activator. To evaluate total elemental content we used electron probe X-ray microanalysis to measure simultaneously all elements from single, individual cells. We observed two phases in the changes in elemental composition (mainly Na, Cl and K). The early phase was characterized by a decrease in intracellular K (P < 0.001) and Cl (P < 0.001) content concomitant with cell shrinkage, and preceded the increase in proteolytic activity associated with the activation of caspase-3. The later phase started with caspase-3 activation, and was characterized by a decrease in the K/Na ratio (P < 0.001) as a consequence of a significant decrease in K and increase in Na content. The inversion of intracellular K and Na content was related with the inhibition of Na+/K+ ATPase. This later phase was also characterized by a significant increase (P < 0.001) in intracellular Cl with respect to the early phase. In addition, we found a decrease in S content and an increase in the P/S ratio. These distinctive changes coincided with chromatin condensation and DNA fragmentation. Together, these findings support the concept that changes in total elemental composition take place in two phases related with molecular and morphological features during staurosporine-induced apoptosis.


apoptosis apoptotic volume decrease caspases chlorine electron probe X-ray microanalysis Na+/K+ ATPase PARP potassium sodium 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Okada Y, Maeno E, Shimizu T, Dezaki K, Wang J, Morishima S. Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD). J Physiol 2001; 532: 3–16.CrossRefPubMedGoogle Scholar
  2. 2.
    Bortner CD, Cidlowski JA. Caspase independent/dependent regulation of K+, cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis. J Biol Chem 1999; 274: 21953–21962.CrossRefPubMedGoogle Scholar
  3. 3.
    Hughes FM, Bortner CD, Purdy GD, Cidlowski JA. Intracellular K+ suppresses the activation of apoptosis in lymphocytes. J Biol Chem 1997; 272: 30567–30576.PubMedGoogle Scholar
  4. 4.
    McCarthy JV, Cotter TG. Cell shrinkage and apoptosis: a role for potassium and sodium efflux. Cell Death Differ 1997; 4: 756–770.CrossRefGoogle Scholar
  5. 5.
    Dallaporta B, Hirsch T, Susin SA, et al. Potassium leakage during the apoptotic degradation phase. J Immunol 1998; 160: 5605–5615.PubMedGoogle Scholar
  6. 6.
    Yu SP, Yeh CH, Sensi SL, et al. Mediation of neuronal apoptosis by enhancement of outward potassium current. Science 1997; 278: 114–117.CrossRefPubMedGoogle Scholar
  7. 7.
    Yu SP, Yeh CH, Gottron F, Wang X, Grabb MC, Choi DW. Role of the outward delayed rectifier K+ current in ceramide-induced caspase activation and apoptosis in cultured neurons. J Neurochem 1999; 73: 933–941.PubMedGoogle Scholar
  8. 8.
    Wang L, Xu D, Dai W, Lu L. An ultraviolet-activated K+ channel mediates apoptosis of myeloblastic leukemia cells. J Biol Chem 1999; 274: 3678–3685.PubMedGoogle Scholar
  9. 9.
    Nietsch HH, Roe MW, Fiekers JF, Moore AL, Lidofsky SD. Activation of potassium and chloride channels by tumor necrosis factor α. Role in liver cell death. J Biol Chem 2000; 275: 20556–20561.CrossRefPubMedGoogle Scholar
  10. 10.
    Krick S, Platoshyn O, Sweeney M, Kim H, Yuan JXJ. Activation of K+ channels induces apoptosis in vascular smooth muscle cells. Am J Physiol Cell Physiol 2001; 280: C970–C979.PubMedGoogle Scholar
  11. 11.
    Krick S, Platoshyn O, McDaniel SS, Rubin LJ, Yuan JXJ. Augmented K currents and mitochondrial membrane depolarization in pulmonary artery myocyte apoptosis. Am J Physiol Lung Cell Mol Physiol 2001; 281: L887–L894.PubMedGoogle Scholar
  12. 12.
    Trimarchi JR, Liu L, Smith PJS, Keefe DL. Apoptosis recruits two-pore domain potassium channels used for homeostatic volume regulation. Am J Physiol Cell Physiol 2002; 282: C588–C594.PubMedGoogle Scholar
  13. 13.
    Maeno E, Ishizaki Y, Kanaseki T, Hazama A, Okada Y. Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proc Natl Acad Sci USA 2000; 97: 9487–9492.CrossRefPubMedGoogle Scholar
  14. 14.
    Thompson GJ, Langlais C, Cain K, Conley EC, Cohen GM. Elevated extracellular [K+] inhibits death-receptor- and chemical-mediated apoptosis prior to caspase activation and cytochrome c release. Biochem J 2001; 357: 37–145.CrossRefGoogle Scholar
  15. 15.
    Brevnova EE, Platoshyn O, Zhang S, Yuan JXJ. Overexpression of human KCNA5 increases and enhances apoptosis. Am J Physiol Cell Physiol 2004; 287: C715–C722.CrossRefPubMedGoogle Scholar
  16. 16.
    Szabò I, Lepple-Wienhues A, Kaba KN, Zoratti M, Gulbins E, Lang F. Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis in T lymphocytes. Proc. Natl. Acad. Sci. USA 1998; 95: 6169–6174.PubMedGoogle Scholar
  17. 17.
    Bortner CD, Hughes FM, Cidlowski JA. A primary role for K+ and Na+ efflux in the activation of apoptosis. J Biol Chem 1997; 272: 32436–32442.CrossRefPubMedGoogle Scholar
  18. 18.
    Bortner CD, Cidlowski JA. Uncoupling cell shrinkage from apoptosis reveals that Na+ influx is required for volume loss during programmed cell death. J Biol Chem 2003; 278: 39176–39184.CrossRefPubMedGoogle Scholar
  19. 19.
    Thornberry NA, Lazebnik Y. Caspases: Enemies within. Science 1998; 281: 1312–1316.CrossRefPubMedGoogle Scholar
  20. 20.
    Zimmerman KC, Bonzon C, Green DR. The machinery of programmed cell death. Pharmacol Ther 2001; 92: 57–70.Google Scholar
  21. 21.
    Cain K, Langlais C, Sun XM, Brown DG, Cohen GM. Physiological concentration of K+ inhibit cytochrome c-dependent formation of the apoptosome. J Biol Chem 2001; 276: 41985–41990.CrossRefPubMedGoogle Scholar
  22. 22.
    King KL, Jewell CM, Bortner CD, Cidlowski JA. 28S ribosome degradation in lymphoid cell apoptosis: Evidence for caspase and Bcl-2-dependent and -independent pathways. Cell Death Differ 2000; 7: 994–101.CrossRefPubMedGoogle Scholar
  23. 23.
    Rassola A, Far DF, Hofman P, Rossi B. Lack of internucleosomal DNA fragmentation is related to Cl efflux impairment in hematopoietic cell apoptosis. FASEB J 1999; 13: 1711–1723.Google Scholar
  24. 24.
    Courageot MP, Lépine S, Hours M, Giraud F, Sulpice JC. Involvements of sodium in early phosphatidylserine exposure and phospholipid scrambling by P2X7 purinoreceptor in thymocytes. J Biol Chem 2004; 279: 21815–21823.CrossRefPubMedGoogle Scholar
  25. 25.
    Andersson C, Roomans GM. Determination of chloride efflux by X-ray microanalysis versus MQAE-fluorescence. Microsc Res Tech 2002; 59: 531–535.CrossRefPubMedGoogle Scholar
  26. 26.
    Haddad P, Beck JS, Boyer JL, Graf J. Role of chloride ions in liver cell regulation. Am J Physiol Gastrointest Liver Physiol 1991; 261: G340–G348.Google Scholar
  27. 27.
    Fernández-Segura E, Cañizares FJ, Cubero MA, Warley A, Campos A. A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: Application to scanning and transmission electron microscopy. J Microsc 1999; 196: 19–25.PubMedGoogle Scholar
  28. 28.
    Warley A, Skepper JN. Long freeze-drying times are not necessary during the preparation of thin sections for X-ray microanalysis. J Microsc 2000; 198: 116–23.CrossRefPubMedGoogle Scholar
  29. 29.
    Roomans GM. Quantitative electron probe X-ray microanalysis of biological specimens. J Electron Microsc Tech 1988; 9: 19–44.PubMedGoogle Scholar
  30. 30.
    Warley A. X-ray microanalysis for biologists. London: Portland Press 1997.Google Scholar
  31. 31.
    Bortner CD, Gómez-Angelats M, Cidlowski JA. Plasma membrane depolarization without repolarization is an early molecular event in anti-Fas-induced apoptosis. J Biol Chem 2001; 276: 4304–4314.CrossRefPubMedGoogle Scholar
  32. 32.
    Bertrand R, Solary E, O'Connor P, Kohn KW, Pommier Y. Induction of common pathway of apoptosis by staurosporine. Exp Cell Res 1994, 211: 314–321.CrossRefPubMedGoogle Scholar
  33. 33.
    Mills JC, Stone NL, Erhardt J, Pittman RN. Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation. J Cell Biol 1998, 140: 627–636.CrossRefPubMedGoogle Scholar
  34. 34.
    Abraham EH, Breslow JL, Epstein J, Chang-Sing P, Lechene C. Preparation of individual human diploid fibroblasts and study of ion transport. Am J Physiol 1985; 248: C154–C164.PubMedGoogle Scholar
  35. 35.
    Zierold K. Effects of cadmium on electrolyte ions in cultured rat hepatocytes studied by X-ray microanalysis of cryosections. Toxicol Appl Pharmacol 1997; 144: 70–76.CrossRefPubMedGoogle Scholar
  36. 36.
    Fernández-Segura E, Cañizares FJ, Cubero MA, Warley A, Campos A. Changes in elemental content during apoptotic cell death studied by electron probe X-ray microanalysis. Exp Cell Res 1999; 253: 454–462.PubMedGoogle Scholar
  37. 37.
    Skepper JN, Karydis I, Garnett MR, et al. Changes in elemental concentrations are associated with early stages of apoptosis in human monocyte-macrophages exposed to oxidized low-density lipoprotein: An X-ray microanalytical study. J Pathol 1999; 188: 100–106.CrossRefPubMedGoogle Scholar
  38. 38.
    Salido M, Vilches J, Lopez A, Roomans GM. Neuropeptides bombesin and calcitonin inhibit apoptosis-related elemental changes in prostate carcinoma cell lines. Cancer 2002; 94: 368–377.CrossRefPubMedGoogle Scholar
  39. 39.
    Salido M, Vilches J, Roomans GM. Changes in elemental concentration in LNCaP cells are associated with a protective effect of neuropeptides on etoposide-induced apoptosis. Cell Biol Int 2004; 28: 397–402.CrossRefPubMedGoogle Scholar
  40. 40.
    Okada Y, Maeno E, Shimizu T, Manabe K, Mori S, Nabekura T. Dual roles of plasmalemmal chloride channels in induction of cell death. Pflügers Arch 2004; 448: 287–295.CrossRefPubMedGoogle Scholar
  41. 41.
    Dallaporta B, Marchetti P, de Pablo MA, et al. Plasma membrana potential in thymocyte apoptosis. J Immunol 1999; 162: 6534–6542.PubMedGoogle Scholar
  42. 42.
    Vu CCQ, Bortner CD, Cidlowski JA. Differential involvement of initiator caspases in apoptotic volume decrease and potassium efflux during Fas- and UV-induced cell death. J Biol Chem 2001; 276: 37602–37611.CrossRefPubMedGoogle Scholar
  43. 43.
    Porcelli AM, Ghelli A, Zanna C, Valente P, Ferroni S, Rugolo M. Apoptosis induced by staurosporine in ECV304 cells require cell shrinkage and upregulation of Cl conductance. Cell Death Differ 2004; 11: 655–662.PubMedGoogle Scholar
  44. 44.
    Platoshyn O, Zhang S, McDaniel SS, Yuan JXJ. Cytochrome c activates K+ channels before inducing apoptosis. Am J Physiol Cell Physiol 2002; 283: C1298–C1305.Google Scholar
  45. 45.
    Remillard CV, Yuan XJJ. Activation of K+ channels: An essential pathway in programmed cell death. Am J Physiol Lung Cel Mol Physiol 2004; 286: L49–L67.Google Scholar
  46. 46.
    Dupéré-Minier G, Hamelin C, Desharnais P, Bernier J. Apoptotic volume decrease, pH acidification and chloride channel activation during apoptosis requires CD45 expression in HPB-ALL T cells. Apoptosis 2004; 9: 543–551.PubMedGoogle Scholar
  47. 47.
    Shimizu T, Numata T, Okada Y. A role of reactive oxygen in apoptotic activation of volume-sensitive Cl(-) channel. Proc Natl Acad Sci USA 2004; 101: 6770–6773.PubMedGoogle Scholar
  48. 48.
    Widlak P, Garrard WT. Discovery, regulation, and action of the major apoptotic nucleases DFF40/CAD and endonuclease G. J Cell Biochem 2005; 94: 1078–1087.CrossRefPubMedGoogle Scholar
  49. 49.
    Krep H, Lefurgey A, Graves SW, Hockett D, Ingram P, Hollenberg NK. Elemental composition of Na pump inhibited rabbit aorta VSM cells by electron probe X-ray microanalysis. Am J Physiol 1996; 271: H514–H520.PubMedGoogle Scholar
  50. 50.
    Yu SP, Regulation and critical role of potassium homeostasis in apoptosis. Prog Neurobiol 2003; 70: 363–386.CrossRefPubMedGoogle Scholar
  51. 51.
    Barbiero G, Duranti F, Bonelli G, Amenta J, Baccino FM. Intracellular ionic variations in apoptotic death of L cells by inhibitors of cell cycle progression. Exp Cell Res 1995; 217: 410–418.CrossRefPubMedGoogle Scholar
  52. 52.
    Nobel CSI, Aronson J, van den Dobbelsteen DJ, Slater AFG. Inhibition of Na+/K+-ATPase may be one mechanism contributing to potassium efflux and cell shrinkage in CD95-induced apoptosis. Apoptosis 2000; 5: 153–163.CrossRefPubMedGoogle Scholar
  53. 53.
    Mann CL, Bortner CD, Jewell CM, Cidlowski JA. Glucocorticoid-induced plasma membrane depolarization during tymocytes apoptosis: Association with cell shrinkage and degradation of the Na+/K+-adenosine triphosphatase. Endocrinology 2001; 142: 5059–5068.PubMedGoogle Scholar
  54. 54.
    Xiao AY, Wang XQ, Yang A, Yu SP. Slight impairment of Na+,K+-ATPase synergistically aggravates ceramide- and β-amyloid-induced apoptosis in cortical neurons. Brain Res 2002; 955: 253–259.CrossRefPubMedGoogle Scholar
  55. 55.
    Xiao AY, Wei L, Xia S, Rothman S, Yu SP. Ionic mechanism of ouabain-induced concurrent apoptosis and necrosis in individual cultured cortical neurons. J Neurochem 2002; 22: 1350–1362.Google Scholar
  56. 56.
    Wang XQ, Xiao AY, Sheline C, et al. Apoptotic insults impair Na+,K+-ATPase activity as a mechanism of neuronal death mediated by concurrent ATP deficiency and oxidant stress. J Cell Sci 2003; 116: 2099–2110.PubMedGoogle Scholar
  57. 57.
    Düssmann H, Rehm M, Kögel D, Prehn JHM. Outer mitochondrial membrane permeabilization during apoptosis triggers caspase-independent mitochondrial and caspase-dependent membrane potential depolarization: a single-cell analysis. J Cell Sci 2003; 116: 525–536.PubMedGoogle Scholar
  58. 58.
    Arrebola F, Zabiti S, Cañizares FJ, Cubero MA, Crespo PV, Fernández-Segura E. Changes in intracellular sodium, chlorine, and potassium concentrations in staurosporine-induced apoptosis. J Cell Physiol 2004; 204: 500–507.Google Scholar

Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • F. Arrebola
    • 1
  • J. Cañizares
    • 1
    • 2
  • M. A. Cubero
    • 1
  • P. V. Crespo
    • 1
    • 2
  • A. Warley
    • 3
  • E. Fernández-Segura
    • 1
    • 2
    • 4
    Email author
  1. 1.Department of Histology, Faculty of MedicineUniversity of GranadaGranadaSpain
  2. 2.Institute of NeurosciencesUniversity of GranadaGranadaSpain
  3. 3.CUI King's College LondonLondonUK
  4. 4.Department of Histology, Faculty of MedicineUniversity of GranadaGranadaSpain

Personalised recommendations