, Volume 23, Issue 7–8, pp 449–455 | Cite as

Staurosporine-induced apoptotic water loss is cell- and attachment-specific

  • Michael A. ModelEmail author
  • Nathan J. Mudrak
  • Priyanka S. Rana
  • Robert J. Clements


Apoptotic volume decrease (AVD) is a characteristic cell shrinkage observed during apoptosis. There are at least two known processes that may result in the AVD: exit of intracellular water and splitting of cells into smaller fragments. Although AVD has traditionally been attributed to water loss, direct evidence for that is often lacking. In this study, we quantified intracellular water in staurosporine-treated cells using a previously described optical microscopic technique that combines volume measurements with quantitative phase analysis. Water loss was observed in detached HeLa and in adherent MDCK but not in adherent HeLa cells. At the same time, adherent HeLa and adherent MDCK cells exhibited visually similar apoptotic morphology, including fragmentation and activation of caspase-3. Morphological changes and caspase activation were prevented by chloride channel blockers DIDS and NPPB in both adherent and suspended HeLa cells, while potassium channel blocker TEA was ineffective. We conclude that staurosporine-induced dehydration is not a universal cell response but depends on the cell type and substrate attachment and can only be judged by direct water measurements. The effects of potassium or chloride channel blockers do not always correlate with the AVD.


Apoptotic volume decrease Staurosporine Intracellular water Transport of intensity equation Transmission-through-dye microscopy Apoptotic bodies 



The work was supported by the Kent State University Research Council.


  1. 1.
    Bortner CD, Cidlowski JA (2002) Apoptotic volume decrease and the incredible shrinking cell. Cell Death Differ 9:1307–1310CrossRefPubMedGoogle Scholar
  2. 2.
    Bortner CD, Cidlowski JA (2004) The role of apoptotic volume decrease and ionic homeostasis in the activation and repression of apoptosis. Pflugers Arch 448:313–318CrossRefPubMedGoogle Scholar
  3. 3.
    Orlov SN, Platonova AA, Hamet P, Grygorczyk R (2013) Cell volume and monovalent ion transporters: their role in cell death machinery triggering and progression. Am J Physiol Cell Physiol 305:C361–C372CrossRefPubMedGoogle Scholar
  4. 4.
    Model MA (2014) Possible causes of apoptotic volume decrease: an attempt at quantitative review. Am J Physiol Cell Physiol 306:C417–C424CrossRefPubMedGoogle Scholar
  5. 5.
    McCarthy JV, Cotter TG (1997) Cell shrinkage and apoptosis: a role for potassium and sodium ion efflux. Cell Death Differ 4:756–770CrossRefPubMedGoogle Scholar
  6. 6.
    Cohen GM, Sun XM, Snowden RT, Dinsdale D, Skilleter DN (1992) Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation. Biochem J 286:331–334CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Yamada T, Ohyama H (1988) Radiation-induced interphase death of rat thymocytes is internally programmed (apoptosis). Int J Radiat Biol Relat Stud Phys Chem Med 53:65–75CrossRefPubMedGoogle Scholar
  8. 8.
    Wyllie AH, Morris RG (1982) Hormone-induced cell death. Purification ad properties of thymocytes undergoing apoptosis after glucocorticoid treatment. Am J Pathol 109:78–87PubMedPubMedCentralGoogle Scholar
  9. 9.
    Patterson SD, Grossman JS, D’Andrea P, Latter GI (1995) Reduced numatrin/B23/nucleophosmin labeling in apoptotic Jurkat T-lymphoblasts. J Biol Chem 270:9429–9436CrossRefPubMedGoogle Scholar
  10. 10.
    Grover WH, Bryan AK, Diez-Silva M, Suresh S, Higgins JM, Manalis SR (2011) Measuring single-cell density. Proc Natl Acad Sci 108:10992–10996CrossRefPubMedGoogle Scholar
  11. 11.
    Benson RS, Heer S, Dive C, Watson AJ (1996) Characterization of cell volume loss in CEM-C7A cells during dexamethasone-induced apoptosis. Am J Physiol 270:C1190–C1203CrossRefPubMedGoogle Scholar
  12. 12.
    Yurinskaya V, Goryachaya T, Guzhova I, Moshkov A, Rozanov Y, Sakuta G, Shirokova A, Shumilina E, Vassilieva I, Lang F, Vereninov A (2005) Potassium and sodium balance in U937 cells during apoptosis with and without cell shrinkage. Cell Physiol Biochem 16:155–162CrossRefPubMedGoogle Scholar
  13. 13.
    Khmaladze A, Matz RL, Epstein T, Jasensky J, Banaszak Holl MM, Chen Z (2012) Cell volume changes during apoptosis monitored in real time using digital holographic microscopy. J Struct Biol 178:270–278CrossRefPubMedGoogle Scholar
  14. 14.
    Khmaladze A (2017) Examining live cell culture cultures during apoptosis by digital holographic phase imaging and Raman spectroscopy. J Phys Conf Ser 909:012001CrossRefGoogle Scholar
  15. 15.
    Mugnano M, Calabuig A, Grilli S, Miccio L, Ferraro P (2015) Monitoring cell morphology during necrosis and apoptosis by quantitative phase imaging. Proc SPIE 9529:952901–952909CrossRefGoogle Scholar
  16. 16.
    Sharikova A, Saide G, Sfakis L, Park JY, Desta H, Maloney MC, Castracane J, Mahajan SD, Khmaladze A (2017) Monitoring of live cell cultures during apoptosis by phase imaging and Raman spectroscopy. Proc SPIE 10074:100740V–100740V1CrossRefGoogle Scholar
  17. 17.
    Zhang Q, Zhong L, Tang P, Yuan Y, Liu S, Tian J, Lu X (2017) Quantitative refractive index distribution of single cell by combining phase-shifting interferometry and AFM imaging. Sci Rep 7:2532CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Mudrak NJ, Rana PS, Model MA (2017) Calibrated brightfield-based imaging for measuring intracellular protein concentration. Cytometry 93:297–304CrossRefPubMedGoogle Scholar
  19. 19.
    Maeno E, Ishizaki Y, Kanaseki T, Hazama A, Okada Y (2000) Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proc Natl Acad Sci USA 97:9487–9492CrossRefPubMedGoogle Scholar
  20. 20.
    Krumschnabel G, Maehr T, Nawaz M, Schwarzbaum PJ, Manzl C (2007) Staurosporine-induced cell death in salmonid cells: the role of apoptotic volume decrease, ion fluxes and MAP kinase signaling. Apoptosis 12:1755–1768CrossRefPubMedGoogle Scholar
  21. 21.
    l’Hoste S, Chargui A, Belfodil R, Corcelle E, Duranton C, Rubera I, Poujeol C, Mograbi B, Tauc M, Poujeol P (2010) CFTR mediates apoptotic volume decrease and cell death by controlling glutathione efflux and ros production in cultured mice proximal tubules. Am J Physiol Renal Physiol 298:F435–F453CrossRefPubMedGoogle Scholar
  22. 22.
    Arrebola F, Cañizares J, Cubero MA, Crespo PV, Warley A, Fernández-Segura E (2005) Biphasic behavior of changes in elemental composition during staurosporine-induced apoptosis. Apoptosis 10:1317–1331CrossRefPubMedGoogle Scholar
  23. 23.
    Krick S, Platoshyn O, McDaniel SS, Rubin LJ, Yuan JX (2001) Augmented K+ currents and mitochondrial membrane depolarization in pulmonary artery myocyte apoptosis. Am J Physiol Lung Cell Mol Physiol 281:L887–L894CrossRefPubMedGoogle Scholar
  24. 24.
    Wible BA, Wang L, Kuryshev YA, Basu A, Haldar S, Brown AM (2002) Increased K+ efflux and apoptosis induced by the potassium channel modulatory protein KChAP/PIAS3β in prostate cancer cells. Biol Chem 277:17852–17862CrossRefGoogle Scholar
  25. 25.
    Porcelli AM, Ghelli A, Zanna C, Valente P, Ferroni S, Rugolo M (2004) Apoptosis induced by staurosporine in ECV304 cells requires cell shrinkage and upregulation of Cl- conductance. Cell Death Differ 11:655–662PubMedGoogle Scholar
  26. 26.
    Yurinskaya VE, Rubashkin AA, Vereninov AA (2011) Balance of unidirectional monovalent ion fluxes in cells undergoing apoptosis: why does Na+/K+ pump suppression not cause cell swelling? J Physiol 589:2197–2211CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    O’Reilly N, Xia Z, Fiander H, Tauskela J, Small DL (2002) Disparity between ionic mediators of volume regulation and apoptosis in N1E 115 mouse neuroblastoma cells. Brain Res 943:245–256CrossRefPubMedGoogle Scholar
  28. 28.
    Dezaki K, Maeno E, Sato K, Akita T, Okada Y (2012) Early-phase occurrence of K+ and Cl efflux in addition to Ca2+ mobilization is a prerequisite to apoptosis in HeLa cells. Apoptosis 17:821–831CrossRefPubMedGoogle Scholar
  29. 29.
    Model MA (2015) Cell volume measurements by optical transmission microscopy. Curr Protoc Cytom 72:12.39.1–12.39.9Google Scholar
  30. 30.
    Gregg JL, McGuire KM, Focht DC, Model MA (2010) Measurement of the thickness and volume of adherent cells using transmission-through-dye microscopy. Pflugers Arch 460:1097–1104CrossRefPubMedGoogle Scholar
  31. 31.
    Kasim NR, Kuželová K, Holoubek A, Model MA (2013) Live fluorescence and transmission-through-dye microscopic study of actinomycin D-induced apoptosis and apoptotic volume decrease. Apoptosis 18:521–532CrossRefPubMedGoogle Scholar
  32. 32.
    Hazama A, Okada Y (1988) Ca2+ sensitivity of volume-regulatory K+ and Cl channels in cultured human epithelial cells. J Physiol 402:687–702CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Model MA, Khitrin AK, Blank JL (2008) Measurement of the absorption of concentrated dyes and their use for quantitative imaging of surface topography. J Microsc 231:156–167CrossRefPubMedGoogle Scholar
  34. 34.
    Model MA (2012) Imaging the cell’s third dimension. Microsc Today 20:32–37CrossRefGoogle Scholar
  35. 35.
    Gibbons BA, Robinson LC, Kharel P, Synowicki RA, Model MA (2016) Volume measurements and fluorescent staining indicate an increase in permeability for organic cation transporter substrates during apoptosis. Exper Cell Res 344:112–119CrossRefGoogle Scholar
  36. 36.
    Model MA, Petruccelli JC (in press) Intracellular macromolecules in cell volume control and methods of their quantification. Curr Top Membr 81Google Scholar
  37. 37.
    Theillet FX, Binolfi A, Frembgen-Kesner T, Hingorani K, Sarkar M, Kyne C, Li C, Crowley PB, Gierasch L, Pielak GJ, Elcock AH, Gershenson A, Selenko P (2014) Physicochemical properties of cells and their effects on intrinsically disordered proteins (IDPs). Chem Rev 114:6661–6714CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Ellis RJ (2001) Macromolecular crowding: obvious but underappreciated. Trends Biochem Sci 26:597–604CrossRefPubMedGoogle Scholar
  39. 39.
    Benítez-Rangel E, López-Méndez MC, García L, Guerrero-Hernández A (2015) DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonate) directly inhibits caspase activity in HeLa cell lysates. Cell Death Discov 1:15037CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Liu AH, Cao YN, Liu HT, Zhang WW, Liu Y, Shi TW, Jia GL, Wang XM (2008) DIDS attenuates staurosporine-induced cardiomyocyte apoptosis by PI3K/Akt signaling pathway: activation of eNOS/NO and inhibition of Bax translocation. Cell Physiol Biochem 22:177–186CrossRefPubMedGoogle Scholar
  41. 41.
    Doughty JM, Miller AL, Langton PD (1998) Non-specificity of chloride channel blockers in rat cerebral arteries: block of the L-type calcium channel. J Physiol 507:433–439CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Liu K, Samuel M, Ho M, Harrison RK, Paslay JW (2010) NPPB structure-specifically activates TRPA1 channels. Biochem Pharmacol 80:113–121CrossRefPubMedGoogle Scholar
  43. 43.
    Macias WL, McAteer JA, Tanner GA, Fritz AL, Armstrong WM (1992) NaCl transport by Madin Darby canine kidney cyst epithelial cells. Kidney Int 42:308–319CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biological SciencesKent State UniversityKentUSA

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