Abstract
Apoptosis is often associated with acidification of the cytosol and since loss of lysosomal proton gradient and release of lysosomal content are early events during apoptosis, we investigated if the lysosomal compartment could contribute to cytosolic acidification. After exposure of U937 cells to tumor necrosis factor-α, three populations; healthy, pre-apoptotic, and apoptotic cells, were identified by flow cytometry. These populations were investigated regarding intra-cellular pH and apoptosis-associated events. There was a drop in cytosolic pH from 7.2 ± 0.1 in healthy cells to 6.8 ± 0.1 in pre-apoptotic, caspase-negative cells. In apoptotic, caspase-positive cells, the pH was further decreased to 5.7 ± 0.04. The cytosolic acidification was not affected by addition of specific inhibitors towards caspases or the mitochondrial F0F1-ATPase. In parallel to the cytosolic acidification, a rise in lysosomal pH from 4.3 ± 0.3, in the healthy population, to 4.8 ± 0.3 and 5.5 ± 0.3 in the pre-apoptotic- and apoptotic populations, respectively, was detected. In addition, lysosomal membrane permeability increased as detected as release of cathepsin D from lysosomes to the cytosol in pre-apoptotic and apoptotic cells. We, thus, suggest that lysosomal proton release is the cause of the cytosolic acidification of U937 cells exposed to TNF-α.
Similar content being viewed by others
References
Foghsgaard L, Wissing D, Mauch D, et al (2001) Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J Cell Biol 153:999–1010
Werneburg NW, Guicciardi ME, Bronk SF, Gores GJ (2002) Tumor necrosis factor-alpha-associated lysosomal permeabilization is cathepsin B dependent. Am J Phys—Gastrointest Liver Phys 283:G947–956
Johansson AC, Steen H, Öllinger K, Roberg K (2003) Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine. Cell Death Differ 10:1253–1259
Roberg K, Öllinger K (1998) Oxidative stress causes relocation of the lysosomal enzyme cathepsin D with ensuing apoptosis in neonatal rat cardiomyocytes. Am J Pathol 152:1151–1156
Roberts LR, Kurosawa H, Bronk SF, et al (1997) Cathepsin B contributes to bile salt-induced apoptosis of rat hepatocytes. Gastroenterology 113:1714–1726
Gottlieb RA, Nordberg J, Skowronski E, Babior BM (1996) Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification. Proc Natl Acad Sci USA 93:654–658
Li J, Eastman A (1995) Apoptosis in an interleukin-2-dependent cytotoxic T lymphocyte cell line is associated with intracellular acidification. Role of the Na+/H+-antiport. J Biol Chem 270:3203–3211
Matsuyama S, Llopis J, Deveraux QL, Tsien RY, Reed JC (2000) Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis. Nat Cellbiol 2:318–325
Matsuyama S, Reed JC (2000) Mitochondria-dependent apoptosis and cellular pH regulation. Cell Death Differ 7:1155–1165
Thangaraju M, Sharma K, Leber B, Andrews DW, Shen SH, Srikant CB (1999) Regulation of acidification and apoptosis by SHP-1 and Bcl-2. J Biol Chem 274:29549–29557
Beem E, Holliday LS, Segal MS (2004) The 1.4-MDa apoptosome is a critical intermediate in apoptosome maturation. Am J Physiol - Cell Physiol 287:C664–672
Segal MS, Beem E (2001) Effect of pH, ionic charge, and osmolality on cytochrome c-mediated caspase-3 activity. Am J Physiol - Cell Physiol 281:C1196–1204
Roy S, Bayly CI, Gareau Y, et al (2001) Maintenance of caspase-3 proenzyme dormancy by an intrinsic “safety catch” regulatory tripeptide. Proc Natl Acad Sci USA 98:6132–6137
Matsuyama S, Schendel SL, Xie Z, Reed JC (1998) Cytoprotection by Bcl-2 requires the pore-forming alpha5 and alpha6 helices. J Biol Chem 273:30995–31001
Schendel SL, Azimov R, Pawlowski K, Godzik A, Kagan BL, Reed JC (1999) Ion channel activity of the BH3 only Bcl-2 family member, BID. J Biol Chem 274:21932–21936
Schendel SL, Xie Z, Montal MO, Matsuyama S, Montal M, Reed JC (1997) Channel formation by antiapoptotic protein Bcl-2. Proc Natl Acad Sci USA 94:5113–5118
Schlesinger PH, Gross A, Yin XM, et al (1997) Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2. Proc Natl Acad Sci USA 94:11357–11362
Xie Z, Schendel S, Matsuyama S, Reed JC (1998) Acidic pH promotes dimerization of Bcl-2 family proteins. Biochemistry 37:6410–6418
Hirpara JL, Clement MV, Pervaiz S (2001) Intracellular acidification triggered by mitochondrial-derived hydrogen peroxide is an effector mechanism for drug-induced apoptosis in tumor cells. J Biol Chem 276:514–521
Meisenholder GW, Martin SJ, Green DR, Nordberg J, Babior BM, Gottlieb RA (1996) Events in apoptosis. Acidification is downstream of protease activation and BCL-2 protection. J Biol Chem 271:16260–16262
Liu D, Martino G, Thangaraju M, et al (2000) Caspase-8-mediated intracellular acidification precedes mitochondrial dysfunction in somatostatin-induced apoptosis. J Biol Chem 275:9244–9250
Brunk UT, Svensson I (1999) Oxidative stress, growth factor starvation and Fas activation may all cause apoptosis through lysosomal leak. Redox Report 4:3–11
Zhao M, Eaton JW, Brunk UT (2000) Protection against oxidant-mediated lysosomal rupture: a new anti-apoptotic activity of Bcl-2? FEBS Lett 485:104–108
Guicciardi ME, Deussing J, Miyoshi H, et al (2000) Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest 106:1127–1137
Nilsson C, Kågedal K, Johansson U, Öllinger K (2003) Analysis of cytosolic and lysosomal pH in apoptotic cells by flow cytometry. Methods Cell Sci 25:185–194
Brunk UT, Dalen H, Roberg K, Hellquist HB (1997) Photo-oxidative disruption of lysosomal membranes causes apoptosis of cultured human fibroblasts. Free Radic Biol Med 23:616–626
Reers M, Smith TW, Chen LB (1991) J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30:4480–4486
Smiley ST, Reers M, Mottola-Hartshorn C, et al (1991) Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci USA 88:3671–3675
Hishita T, Tada-Oikawa S, Tohyama K, et al (2001) Caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line P39. Cancer Res 61:2878–2884
Yuan XM, Li W, Brunk UT, Dalen H, Chang YH, Sevanian A (2000) Lysosomal destabilization during macrophage damage induced by cholesterol oxidation products. Free Radic Biol Med 28:208–218
Li W, Yuan XM, Ivanova S, Tracey KJ, Eaton JW, Brunk UT (2003) 3-Aminopropanal, formed during cerebral ischaemia, is a potent lysosomotropic neurotoxin. Biochem J 371:429–436
Li W, Yuan X, Nordgren G, et al (2000) Induction of cell death by the lysosomotropic detergent MSDH. FEBS Lett 470:35–39
Chen JW, Murphy TL, Willingham MC, Pastan I, August JT (1985) Identification of two lysosomal membrane glycoproteins. J Cell Biol 101:85–95
Dubowchik GM, Gawlak SL, Firestone RA (1995) The in vitro effect of three lysosomotropic detergents against three human tumor cell lines. Bioorg Med Chem Lett 5:893–898
Kågedal K, Zhao M, Svensson I, Brunk UT (2001) Sphingosine-induced apoptosis is dependent on lysosomal proteases. Biochem J 359:335–343
Stennicke HR, Salvesen GS (1997) Biochemical characteristics of caspases-3,-6,-7, and -8. J Biol Chem 272:25719–25723
Roberg K, Johansson U, Öllinger K (1999)Lysosomal release of cathepsin D precedes relocation of cytochrome c and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress. Free Radic Biol Med 27:1228–1237
Turk B, Dolenc I, Turk V, Bieth JG (1993) Kinetics of the pH-induced inactivation of human cathepsin L. Biochemistry 32:375–380
Song J, Xu P, Xiang H, Su Z, Storer AC, Ni F (2000) The active-site residue Cys-29 is responsible for the neutral-pH inactivation and the refolding barrier of human cathepsin B FEBS Lett 475:157–162
Reiners JJ, Jr., Caruso JA, Mathieu P, Chelladurai B, Yin XM, Kessel D (2002) Release of cytochrome c and activation of pro-caspase-9 following lysosomal photodamage involves Bid cleavage. Cell Death Differ 9:934–944
Stoka V, Turk B, Schendel SL, et al (2001) Lysosomal protease pathways to apoptosis. Cleavage of bid, not pro-caspases, is the most likely route. J Biol Chem 276:3149–3157
Heinrich M, Neumeyer J, Jakob M, et al (2004) Cathepsin D links TNF-induced acid sphingomyelinase to Bid-mediated caspase-9 and -3 activation. Cell Death Differ 11:550–563
Cirman T, Oresic K, Mazovec GD, et al (2004) Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins. J Biol Chem 279:3578–3587
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Nilsson, C., Johansson, U., Johansson, AC. et al. Cytosolic acidification and lysosomal alkalinization during TNF-α induced apoptosis in U937 cells. Apoptosis 11, 1149–1159 (2006). https://doi.org/10.1007/s10495-006-7108-5
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10495-006-7108-5