FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2014465321> ?p ?o ?g. }

• W2014465321 endingPage "18271" @default.
• W2014465321 startingPage "18263" @default.
• W2014465321 abstract "Many events in apoptosis have been identified but their temporal relationships remain obscure. Apoptosis in human ML-1 cells induced by etoposide is characterized by intracellular acidification, enhanced Hoechst 33342 fluorescence, DNA digestion, chromatin condensation, and proteolysis of poly(ADP-ribose) polymerase. This proteolysis is a marker for the action of ICE/CED-3 proteases, which are critical activators of apoptosis. We observed that three serine/threonine protein phosphatase inhibitors, okadaic acid, calyculin A, and cantharidin, prevented all of these apoptotic characteristics. To determine which protein phosphatase was involved, we investigated the dephosphorylation of the retinoblastoma susceptibility protein Rb, a substrate for protein phosphatase 1 but not protein phosphatase 2A. Rb was dephosphorylated during apoptosis, and each inhibitor prevented this dephosphorylation at the same concentrations that prevented apoptosis. No increase in protein phosphatase 1 activity was observed in apoptotic cells suggesting that dephosphorylation of Rb may result from loss of Rb kinase activity in the presence of a constant level of protein phosphatase activity. Long term inhibition of protein phosphatase 1 (>8 h) also led to the appearance of dephosphorylated Rb, cleavage of poly(ADP-ribose) polymerase and apoptosis, suggesting these events are not solely dependent upon protein phosphatase 1. Rb dephosphorylation was also observed in several other models of apoptosis. Hence, an imbalance between protein phosphatase 1 and Rb kinase may be a common means to activate ICE/CED-3 proteases resulting in the subsequent events of apoptosis. Many events in apoptosis have been identified but their temporal relationships remain obscure. Apoptosis in human ML-1 cells induced by etoposide is characterized by intracellular acidification, enhanced Hoechst 33342 fluorescence, DNA digestion, chromatin condensation, and proteolysis of poly(ADP-ribose) polymerase. This proteolysis is a marker for the action of ICE/CED-3 proteases, which are critical activators of apoptosis. We observed that three serine/threonine protein phosphatase inhibitors, okadaic acid, calyculin A, and cantharidin, prevented all of these apoptotic characteristics. To determine which protein phosphatase was involved, we investigated the dephosphorylation of the retinoblastoma susceptibility protein Rb, a substrate for protein phosphatase 1 but not protein phosphatase 2A. Rb was dephosphorylated during apoptosis, and each inhibitor prevented this dephosphorylation at the same concentrations that prevented apoptosis. No increase in protein phosphatase 1 activity was observed in apoptotic cells suggesting that dephosphorylation of Rb may result from loss of Rb kinase activity in the presence of a constant level of protein phosphatase activity. Long term inhibition of protein phosphatase 1 (>8 h) also led to the appearance of dephosphorylated Rb, cleavage of poly(ADP-ribose) polymerase and apoptosis, suggesting these events are not solely dependent upon protein phosphatase 1. Rb dephosphorylation was also observed in several other models of apoptosis. Hence, an imbalance between protein phosphatase 1 and Rb kinase may be a common means to activate ICE/CED-3 proteases resulting in the subsequent events of apoptosis. Maintaining tissue and organ homeostasis requires a delicate balance between the rate of cell division and the rate of cell death. Cells that are either metabolically compromised or no longer required are eliminated by a pathway commonly known as apoptosis. This process has been characterized by cell shrinkage, cytoplasmic blebbing, chromosome condensation, DNA digestion, and, finally, non-inflammatory removal of the cell from the tissue (1Wyllie A.H. Kerr J.F.R. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Google Scholar). Apoptosis occurs during development, immune regulation, and normal cell turnover, as well as being induced by many pharmacological insults. Decreased apoptosis can lead to cancer and autoimmune diseases, while increased apoptosis can result in neurodegenerative disorders and AIDS (2Thompson C.B. Science. 1995; 267: 1456-1462Google Scholar). Despite the importance of apoptosis, the signal transduction pathways responsible for the associated morphological and biochemical changes are poorly understood. Much recent emphasis has focused on the identification of proteases of the ICE/CED-3 family that appear essential to this pathway (3Miura M. Zhu H. Rotello R. Hartwieg E.A. Yuan J. Cell. 1993; 75: 653-660Google Scholar, 4Kuida K. Lippke J.A. Ku G. Harding M.W. Livingston D.J. Su M.S.-S. Flavell R.A. Science. 1995; 267: 2000-2003Google Scholar, 5Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Google Scholar), but both the upstream and downstream events remain elusive. While investigating endonucleases that might be involved in apoptosis, we detected and purified deoxyribonuclease II (DNase II), an endonuclease that is active at slightly acidic pH but inactive at pH 7.0 (6Barry M.A. Eastman A. Arch. Biochem. Biophys. 1993; 300: 440-450Google Scholar). To determine whether DNase II might be responsible for the DNA digestion, we measured intracellular pH in various cell models during apoptosis. In each model tested, the apoptotic cells were seen as a distinct population with an acidic shift of 0.5-0.8 pH units (7Barry M.A. Eastman A. Biochem. Biophys. Res. Commun. 1992; 186: 782-789Google Scholar, 8Barry M.A. Reynolds J.E. Eastman A. Cancer Res. 1993; 53: 2349-2357Google Scholar, 9Morana S. Li J. Springer E.W. Eastman A. Int. J. Oncol. 1994; 5: 153-158Google Scholar, 10Li J. Eastman A. J. Biol. Chem. 1995; 270: 3203-3211Google Scholar, 11Reynolds J.E. Li J. Craig R.W. Eastman A. Exp. Cell Res. 1996; 225 (in press)Google Scholar). Recently, other laboratories have confirmed that intracellular acidification occurs during apoptosis (12Perez-Sala D. Collado-Escobar D. Mollinedo F. J. Biol. Chem. 1995; 270: 6235-6242Google Scholar, 13Rebello A. Gomez J. de Aragon A.M. Lastres P. Silva A. Perez-Sala D. Exp. Cell Res. 1995; 218: 581-585Google Scholar). Hence, intracellular acidification appears to represent a common event in the pathway of apoptotic cell death. Although intracellular acidification can activate DNase II (6Barry M.A. Eastman A. Arch. Biochem. Biophys. 1993; 300: 440-450Google Scholar), we have recently established that a low intracellular pH is not required for DNA digestion, suggesting that an additional means of activating DNase II can occur, or that an alternate endonuclease may be involved. Specifically, when CTLL-2 cells were maintained at an extracellular pH of 8.0, DNA digestion and apoptosis still occurred upon removal of interleukin 2, but the intracellular pH only dropped to 7.2, a pH at which DNase II should be inactive (10Li J. Eastman A. J. Biol. Chem. 1995; 270: 3203-3211Google Scholar). Hence, during apoptosis, cells always appear to undergo intracellular acidification, but the low pH that results is not required for the activation of an endonuclease. These observations suggest that both intracellular acidification and DNA digestion are regulated by a common upstream regulator. This paper presents the results of a search for such a regulator. The intracellular acidification that occurs during apoptosis results from selective loss of pH regulation. Furthermore, we have demonstrated that the acidification that occurs in apoptotic CTLL-2 cells results from an alteration in the set point of the Na+/H+-antiport (10Li J. Eastman A. J. Biol. Chem. 1995; 270: 3203-3211Google Scholar). Normally, the Na+/H+-antiport raises the intracellular pH to around neutrality. Although apoptotic CTLL-2 cells still retained functional antiport, it was only able to raise intracellular pH to around 6.3 in apoptotic cells. The set-point of the Na+/H+-antiport has been shown to be regulated by phosphorylation (14Sardet C. Franchi A. Pouyssegur J. Cell. 1989; 56: 271-280Google Scholar, 15Sardet C. Counillon L. Franchi A. Pouyssegur J. Science. 1990; 247: 723-726Google Scholar, 16Schwartz M.A. Lechene C. Ingber D.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7849-7853Google Scholar). A wide variety of external signals, including growth factors and the extracellular matrix, activate a kinase cascade that leads to phosphorylation of the antiport and intracellular alkalization. These same stimuli are also known to enhance cell survival. Many intracellular signaling components, such as protein kinase C and Ha-Ras, also lead to enhanced survival and activation of the antiport (17Swann K. Whitaker M. Nature. 1985; 314: 274-277Google Scholar, 18Maly K. Uberall F. Loferer H. Doppler W. Oberhuber H. Groner B. Grunicke H.H. J. Biol. Chem. 1989; 264: 11839-11842Google Scholar). Furthermore, the protein phosphatase inhibitor okadaic acid has been shown to activate the antiport (19Bianchini L. Woodside M. Sardet C. Pouyssegur J. Takai A. Grinstein S. J. Biol. Chem. 1991; 266: 15406-15413Google Scholar). These observations all support the notion that protein kinase cascades protect cells through pathways that also regulate intracellular pH. Accordingly, we have investigated the role of protein phosphatases as potential mediators of intracellular acidification and apoptosis. Previous results have suggested that the protein phosphatase inhibitors okadaic acid and calyculin A can prevent the onset of apoptosis (20Song Q. Baxter G.D. Kovacs E.M. Findik D. Lavin M. J. Cell. Physiol. 1992; 153: 550-556Google Scholar, 21Song Q. Lavin M.F. Biochem. Biophys. Res. Commun. 1993; 190: 47-55Google Scholar). Those results are confirmed and extended here. We demonstrate that apoptosis, as assessed by DNA digestion, intracellular acidification, changes in membrane permeability, and morphology, can be prevented by a number of protein phosphatase inhibitors. Furthermore, proteolytic cleavage of poly(ADP-ribose) polymerase (PARP), 1The abbreviations used are: PARPpoly(ADP-ribose) polymeraseCHOChinese hamster ovaryICEinterleukin 1β-converting enzymepHiintracellular pHPP1protein phosphatase 1PP2Aprotein phosphatase 2A. a marker for the action of ICE/CED-3 proteases, was inhibited at the same concentration of each protein phosphatase inhibitor. Dephosphorylation of the retinoblastoma susceptibility protein Rb was also associated with the onset of apoptosis, and this was inhibited by the same concentrations of each inhibitor that were effective at blocking apoptosis. The dephosphorylation of Rb is normally mediated by protein phosphatase PP1 at the onset of mitosis (22Ludlow J.W. Glendening C.L. Livingston D.M. DeCaprio J.A. Mol. Cell. Biol. 1993; 13: 367-372Google Scholar, 23Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W.-H. Elledge S.J. Genes Dev. 1993; 7: 555-569Google Scholar), thereby suggesting that aberrant regulation of PP1, or the counteracting protein kinases, leads to the activation of ICE/CED-3 proteases and the subsequent morphological and biochemical events associated with apoptosis. poly(ADP-ribose) polymerase Chinese hamster ovary interleukin 1β-converting enzyme intracellular pH protein phosphatase 1 protein phosphatase 2A. Etoposide, okadaic acid, calyculin A, cantharidin, acridine orange, ethidium bromide, Percoll, other chemicals, and enzymes were purchased from Sigma. The acetoxymethyl ester of carboxy-SNARF-1 was obtained from Molecular Probes (Eugene, OR). Cell culture supplies and the protein phosphatase assay kit were purchased from Life Technologies, Inc. The G3-245 anti-Rb mouse monoclonal antibody used in initial studies was purchased from Pharmingen (San Diego, CA); the C15 anti-Rb rabbit polyclonal antibody used in subsequent studies was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-PP1 polyclonal antibody was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). The C-2-10 monoclonal antibody to poly(ADP-ribose)polymerase was a gift of Dr. Guy Poirier (Laval University Hospital, Quebec, Canada). ML-1 myeloid leukemia cells were maintained in 5% CO2 at 37°C in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin. All cultures were maintained in logarithmic growth at a density <1 × 106 cells/ml. Cells were incubated with etoposide for 30 min after which they were centrifuged, washed twice, and resuspended in completed medium without drug. The phosphatase inhibitors were added only for the incubation period following removal of etoposide. In most experiments, cells were harvested and analyzed 4 h after removal of etoposide. DNA digestion was measured by agarose gel electrophoresis as described previously (6Barry M.A. Eastman A. Arch. Biochem. Biophys. 1993; 300: 440-450Google Scholar, 24Eastman A. Methods Cell Biol. 1995; 46: 41-55Google Scholar). Briefly, 106 cells were added directly to the wells of a 2% agarose gel, where they were lysed and digested with ribonuclease A and proteinase K. The gel was electrophoresed for 16 h, and the DNA was stained with ethidium bromide and visualized under ultraviolet light. In this method, high molecular weight DNA (>20 kilobase pairs) remains trapped in or near the well, while smaller fragments (down to 180 base pairs) are resolved in the gel. Chromatin condensation in apoptotic cells was assessed by staining with acridine orange, while membrane integrity was measured simultaneously by exclusion of ethidium bromide (25Duke R.C. Cohen J.J. Coligan J.E. Kruisbeek A.M. Margulies D.H. Shevach E.M. Strober W. Current Protocols in Immunology. John Wiley & Sons, Inc., New York1992: 3.17.1Google Scholar). A 25-µl aliquot of cells in medium was stained by addition of 1 µl of a stock solution of 100 µg/ml acridine orange plus 100 µg/ml ethidium bromide in phosphate-buffered saline. A minimum of 200 cells were scored under a fluorescence microscope for apoptotic morphology. For analysis of Rb and PP1, 105 cells were lysed in 100 µl of 50 mM Tris-HCl (pH 6.8), 2% SDS, 0.1% bromphenol blue, 10% glycerol, 0.1%β-mercaptoethanol. For analysis of Rb, 20 µl of this total cell lysate was electrophoresed in a 6% SDS-polyacrylamide gel (MiniPROTEAN II; Bio-Rad). The proteins were electroblotted to a polyvinylidene fluoride membrane (Immobilon-P, Millipore, Marlborough, MA), blocked with 1% bovine serum albumin, and probed with anti-Rb monoclonal antibody at a dilution of 1/1,000. The secondary antibody was goat anti-mouse at a dilution of 1:2,000 (Bio-Rad). Alternately, an anti-Rb polyclonal antibody was used at 1:100 dilution with a goat anti-rabbit secondary antibody diluted 1:3,000. Immunoreactivity was detected with the enhanced chemiluminescence detection kit (ECL, Amersham Corp.). For analysis of PP1, a 10-µl aliquot of the cell lysate was electrophoresed in a 15% SDS-polyacrylamide gel, probed with an anti-PP1 rabbit polyclonal antibody diluted 1/3,000, and then with a goat anti-rabbit secondary antibody (Bio-Rad) at a dilution of 1:2,000. For analysis of PARP, 106 cells were lysed by sonication on ice in 100 µl of lysis buffer (50 mM Tris-HCl, pH 6.8, 4 M urea, 5%β-mercaptoethanol, 2% SDS). 40 µl of this lysate was electrophoresed on a 6% SDS-polyacrylamide gel. Proteins were electroblotted to membranes and blocked in TBSTM (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% (v/v) Tween-20, 5% (w/v) nonfat dried milk) for 2 h at room temperature. The membranes were probed with an anti-PARP mouse monoclonal antibody (C-2-10) diluted 1:10,000 in TBSTM. The secondary antibody was goat anti-mouse at a dilution of 1:3,000, followed by ECL. For the measurement of intracellular pH, cells were loaded for 1 h with 1 µM carboxy-SNARF-1 acetoxymethyl ester, and analyzed on a Becton-Dickinson FACScan Plus flow cytometer with excitation at 488 nm and emission measured at 585 and 640 nm (8Barry M.A. Reynolds J.E. Eastman A. Cancer Res. 1993; 53: 2349-2357Google Scholar, 24Eastman A. Methods Cell Biol. 1995; 46: 41-55Google Scholar). Cells were maintained at 37°C in complete medium including bicarbonate during analysis. The pH measurements were obtained by ratioing the fluorescence emissions at the two appropriate wavelengths, and comparing to a pH calibration curve. Apoptotic cells were also detected by staining with Hoechst 33342. This dye is thought to preferentially stain apoptotic cells due to very early changes in membrane permeability (26Ormerod M.G. Sun X.-M. Snowden R.T. Davies R. Fearnhead H. Cohen G.M. Cytometry. 1993; 14: 595-602Google Scholar). Cells were incubated with 1 µg/ml Hoechst 33342 for 5 min and analyzed on the flow cytometer with excitation at 355 nm and emission measured at 440 nm. In the experiments shown here, cells were stained with carboxy-SNARF-1 for 55 min, followed by the addition of Hoechst 33342 for 5 min prior to simultaneous analysis of the fluorescence of both dyes. Cell cycle analysis was performed on cells that had been fixed in ethanol and stained with propidium iodide as described previously (27Sorenson C.M. Eastman A. Cancer Res. 1988; 48: 4484-4488Google Scholar). Apoptotic cells were separated from normal cells by their increase in buoyant density. Cells (5 × 106) were suspended in 4 ml of completed culture medium plus 4 ml of a Percoll solution (9 volumes of Percoll plus 1 volume of 10 × minimal essential medium) and 0.1% Pluronic F68 surfactant to limit cell adhesion to the centrifuge tubes. After centrifugation at 5,000 × g for 30 min at 36°C, a continuous gradient of Percoll with banded cells was obtained. The gradients were fractionated from the top with a pipette, and the cells were collected by diluting with medium and recentrifuging. Protein phosphatase PP1 and PP2A activity were analyzed with the Protein Phosphatase Assay System (Life Technologies, Inc.), which utilizes 32P-labeled phosphorylase A as a specific substrate for PP1 and PP2A in vitro (28Cohen P. Klumpp S. Schelling D.L. FEBS Lett. 1989; 250: 596-600Google Scholar). Phosphorylase B is converted to phosphorylase A by coincubation of kinase/substrate solution with phosphorylation reaction buffer and 0.5 mCi of [γ-32P]ATP (30°C for 1 h), followed by protein concentration via ammonium sulfate precipitation and centrifugation. ML-1 cell lysates were prepared and assayed according to an adaptation of the manufacturer's protocol. Cells were washed with phosphate-buffered saline, pelleted, and 105 cells were lysed in 100 µl of 50 mM Tris-HCl, pH 7.0, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% (v/v) β-mercaptoethanol, 0.5% (v/v) Triton X-100, 25 µg/ml leupeptin, and 25 µg/ml aprotinin. PP1 and PP2A activity in the cell extracts were immediately assayed by combining 20 µl of cell lysate, 20 µl of protein phosphatase assay buffer, and 20 µl of [32P]phosphorylase A solution, followed by incubation at 30°C for 10 min. The solubilized 32P was separated from residual phosphorylase A as well as cellular proteins by precipitation with trichloroacetic acid, followed by centrifugation. Soluble 32P was then quantified by scintillation counting. PP1 activity was defined as the phosphatase activity inhibited by addition of 1.25 µg/ml inhibitor-2 peptide, and PP2A activity was defined as the activity inhibited by 2 nM okadaic acid. The sum of these two activities was close to 100% of the total phosphatase activity. Many lymphoid cell lines undergo DNA digestion and apoptosis rapidly following a variety of insults. We have previously shown that HL-60 and ML-1 cells show chromatin condensation, DNA fragmentation, and intracellular acidification within 4 h of incubation with the topoisomerase II inhibitor etoposide (8Barry M.A. Reynolds J.E. Eastman A. Cancer Res. 1993; 53: 2349-2357Google Scholar, 9Morana S. Li J. Springer E.W. Eastman A. Int. J. Oncol. 1994; 5: 153-158Google Scholar). In the current experiments, we have further defined the etoposide concentration and kinetics required to induce apoptosis in ML-1 cells. In each experiment, cells were incubated with etoposide for 30 min; the drug was then removed so that the induction of apoptosis would occur subsequent to the primary insult rather than under conditions of increasing etoposide-mediated damage. ML-1 cells were incubated with 0-80 µg/ml etoposide for 30 min, followed by removal of drug and incubation for an additional 4 h. DNA fragmentation was assessed by agarose gel electrophoresis (Fig. 1A). Control cells showed only high molecular weight DNA in or near the well of the gel, whereas damaged cells showed a dose-dependent formation of internucleosomal DNA fragmentation. A concentration of 20 µg/ml showed extensive internucleosomal DNA fragmentation. The kinetics of DNA fragmentation were determined following incubation of cells with 20 µg/ml etoposide (Fig. 1B). At the time of removal of etoposide (30 min), there was an increase in high molecular weight DNA fragments migrating about 1 cm into the gel. These fragments result from the cleavable complexes formed by etoposide and topoisomerase II, but disappear rapidly upon removal of the drug. The majority of internucleosomal DNA fragmentation appeared 3-4 h after removal of etoposide. For HL-60 cells, we previously showed that the cells most susceptible to apoptosis were those in S phase at the time of incubation with etoposide; the remaining cells eventually progressed to, and arrested in, the G2 phase of the cell cycle before undergoing apoptosis (8Barry M.A. Reynolds J.E. Eastman A. Cancer Res. 1993; 53: 2349-2357Google Scholar). Similar experiments in ML-1 cells also showed that the S phase cells were most susceptible (shown below with regard to Fig. 8), but the remaining surviving cells stayed arrested in the G1 phase (data not shown). This is consistent with the ML-1 cells expressing the wild-type p53 tumor suppressor protein, in contrast to HL-60 cells. Hence, the apoptosis observed in this paper is independent of p53 status, although expression of p53 does contribute to cell cycle perturbations in the surviving cells. To determine the number of cells undergoing apoptosis at each time or drug concentration, cells were analyzed by flow cytometry for increased fluorescent staining with Hoechst 33342. A number of reports have shown that apoptotic cells accumulate Hoechst 33342 at an increased rate, and this can be assessed by a short incubation (<5 min) with the dye (10Li J. Eastman A. J. Biol. Chem. 1995; 270: 3203-3211Google Scholar, 26Ormerod M.G. Sun X.-M. Snowden R.T. Davies R. Fearnhead H. Cohen G.M. Cytometry. 1993; 14: 595-602Google Scholar, 29Dive C. Gregory C.D. Phipps D.J. Evans D.L. Milner A.E. Wyllie A.H. Biochim. Biophys. Acta. 1992; 1133: 275-285Google Scholar). Simultaneously, cells were analyzed for intracellular pH with the pH-sensitive fluorescent ratio dye carboxy-SNARF-1. The results are visualized as a scatter plot in which Hoechst fluorescence is shown on the ordinate and intracellular pH (expressed as the emission ratio of 545/640 nm) is shown on the abscissa (Fig. 2). Interestingly, we have found that these two fluorescent dyes detect exactly the same population of cells and, as a result, the combination of dyes helps to discriminate otherwise overlapping populations (10Li J. Eastman A. J. Biol. Chem. 1995; 270: 3203-3211Google Scholar). The normal cells appear in the lower left quadrant and the majority of apoptotic cells in the upper right quadrant; the number of apoptotic cells is recorded in each panel. The number of apoptotic cells increases from about 2% at 5 µg/ml etoposide to almost 80% at 80 µg/ml. With respect to kinetics following incubation with 20 µg/ml etoposide, the number of apoptotic cells increases rapidly between 3 and 4 h after treatment in concert with the time of appearance of DNA digestion. The flow cytometry analysis was performed while the extracellular pH was maintained at 7.1, leading to an intracellular pH of 7.15 in the normal cells and 6.5 in the apoptotic cells. Cells were also stained with acridine orange and scored under a microscope for chromatin condensation and alterations in nuclear morphology that occur during apoptosis (25Duke R.C. Cohen J.J. Coligan J.E. Kruisbeek A.M. Margulies D.H. Shevach E.M. Strober W. Current Protocols in Immunology. John Wiley & Sons, Inc., New York1992: 3.17.1Google Scholar). Control samples consistently showed less than 5% apoptotic cells, whereas 4 h after incubation with etoposide, 30-41% of the cells exhibited apoptotic morphology. These values are of a similar order as the results obtained by flow cytometry. In two other models, we have previously sorted cells by flow cytometry on the basis of intracellular pH and established that only the acidic cells exhibit these morphological changes (8Barry M.A. Reynolds J.E. Eastman A. Cancer Res. 1993; 53: 2349-2357Google Scholar, 10Li J. Eastman A. J. Biol. Chem. 1995; 270: 3203-3211Google Scholar). The current experiments further confirm that enhanced Hoechst fluorescence and intracellular acidification are valid markers of apoptosis. In all subsequent experiments, apoptosis was induced with 20 µg/ml etoposide, with DNA fragmentation and other parameters of apoptosis measured after an additional 4-h incubation. The serine/threonine phosphatase inhibitor okadaic acid has been shown to activate the Na+/H+-antiport involved in intracellular pH regulation (19Bianchini L. Woodside M. Sardet C. Pouyssegur J. Takai A. Grinstein S. J. Biol. Chem. 1991; 266: 15406-15413Google Scholar). Okadaic acid, as well as another serine/threonine phosphatase inhibitor calyculin A, have also been reported to inhibit apoptosis (20Song Q. Baxter G.D. Kovacs E.M. Findik D. Lavin M. J. Cell. Physiol. 1992; 153: 550-556Google Scholar, 21Song Q. Lavin M.F. Biochem. Biophys. Res. Commun. 1993; 190: 47-55Google Scholar). Hence, we investigated the effect of these inhibitors on DNA fragmentation in our model of apoptosis. ML-1 cells were induced to undergo apoptosis with etoposide, and each inhibitor was then added at various concentrations during the subsequent incubation. We found that 1 µM okadaic acid and 10 nM calyculin A were the minimum concentrations required to markedly inhibit DNA digestion (Fig. 3, Fig. 4). We also investigated the effect of another protein phosphatase inhibitor, cantharidin, on DNA fragmentation; this inhibitor was found to prevent DNA fragmentation at 20-30 µM (Fig. 5).Fig. 4Calyculin A prevents DNA fragmentation, PARP cleavage, and dephosphorylation of Rb. The experimental protocol was identical to Fig. 3, with the exception that the cells were incubated with indicated concentrations of calyculin A rather than okadaic acid.View Large Image Figure ViewerDownload (PPT)Fig. 5Cantharidin prevents DNA fragmentation, PARP cleavage, and dephosphorylation of Rb. The experimental protocol was identical to Fig. 3, with the exception that the cells were incubated with indicated concentrations of cantharidin rather than okadaic acid.View Large Image Figure ViewerDownload (PPT) Next, we investigated the effect of each inhibitor on the increased Hoechst fluorescence and intracellular acidification that occurs during apoptosis (Fig. 6). Incubation of cells with 1 µM okadaic acid for 4 h neither enhanced Hoechst fluorescence nor caused intracellular acidification. However, when added to cells following incubation with etoposide, 1 µM okadaic acid completely prevented both enhanced Hoechst fluorescence and intracellular acidification. Incubation with 0.1 µM okadaic acid was unable to prevent acidification or increased Hoechst fluorescence consistent with its inability to inhibit DNA fragmentation (Fig. 3). Results obtained with calyculin A and cantharidin were slightly different from okadaic acid in that each inhibitor alone induced a slight acidic shift (Fig. 6). The intracellular acidification induced by calyculin A (mean pHi = 6.8) was more marked than for cantharidin (pHi = 6.9), but in neither case was it as dramatic as in apoptotic cells (pHi = 6.5), and neither inhibitor enhanced Hoechst fluorescence. However, the concentration of each inhibitor that prevented etoposide-induced DNA digestion (Fig. 4, Fig. 5) was still able to prevent the etoposide-mediated increase in Hoechst fluorescence and intracellular acidification. A lower concentration of either inhibitor was ineffective at preventing the etoposide-induced changes. The morphological changes detectable by staining with acridine orange were also investigated for each of these conditions. Etoposide treatment alone induced chromatin condensation in 30-41% of the cells, whereas the addition of either okadaic acid, calyculin A, or cantharidin to etoposide-treated cells reduced this value to 12.6, 7.0, or 5.0%, respectively (average of three separate experiments). Hence, all three inhibitors were able to prevent apoptosis as assessed by chromatin condensation, increased Hoechst fluorescence, and intracellular acidification, and this protection occurred at the same concentrations that also prevented DNA fragmentation. Considering the recent reports that demonstrate a role for ICE/CED-3 proteases in apoptosis, we investigated whether the inhibitors used here functioned at a step upstream or downstream of this protease. PARP has been shown to be proteolytically cleaved from a 116-kDa protein to a 85-kDa fragment by ICE/CED-3 proteases (30Lazebnik Y.A. Kaufmann S.H. Desnoyers S. Poirier G.G. Earnshaw W.C. Nature. 1994; 371: 346-347Google Scholar). Most recently, this cleavage has been associated with the CPP32/Yama/apopain homolog of this protease family (5Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Google Scholar, 31Nicholson D.W. Ali A. Thornberry N.A. Vaillancour" @default.
• W2014465321 created "2016-06-24" @default.
• W2014465321 creator A5016470161 @default.
• W2014465321 creator A5022241128 @default.
• W2014465321 creator A5031261744 @default.
• W2014465321 creator A5044712943 @default.
• W2014465321 creator A5047181455 @default.
• W2014465321 creator A5053729580 @default.
• W2014465321 date "1996-07-01" @default.
• W2014465321 modified "2023-09-29" @default.
• W2014465321 title "The Involvement of Protein Phosphatases in the Activation of ICE/CED-3 Protease, Intracellular Acidification, DNA Digestion, and Apoptosis" @default.
• W2014465321 cites W1489532715 @default.
• W2014465321 cites W1541528064 @default.
• W2014465321 cites W1563078713 @default.
• W2014465321 cites W1589415602 @default.
• W2014465321 cites W1775327385 @default.
• W2014465321 cites W1964977785 @default.
• W2014465321 cites W1976217600 @default.
• W2014465321 cites W1977707442 @default.
• W2014465321 cites W1984872319 @default.
• W2014465321 cites W1984972991 @default.
• W2014465321 cites W1988886218 @default.
• W2014465321 cites W1989524887 @default.
• W2014465321 cites W1989911861 @default.
• W2014465321 cites W1991865867 @default.
• W2014465321 cites W1993206112 @default.
• W2014465321 cites W1996180957 @default.
• W2014465321 cites W1999330214 @default.
• W2014465321 cites W2004991062 @default.
• W2014465321 cites W2010298858 @default.
• W2014465321 cites W2025100148 @default.
• W2014465321 cites W2026327508 @default.
• W2014465321 cites W2043771696 @default.
• W2014465321 cites W2051602790 @default.
• W2014465321 cites W2057886275 @default.
• W2014465321 cites W2070257066 @default.
• W2014465321 cites W2081145901 @default.
• W2014465321 cites W2083560313 @default.
• W2014465321 cites W2084570691 @default.
• W2014465321 cites W2091319839 @default.
• W2014465321 cites W2101228347 @default.
• W2014465321 cites W2106104578 @default.
• W2014465321 cites W2139360111 @default.
• W2014465321 cites W2142946131 @default.
• W2014465321 cites W2157786523 @default.
• W2014465321 cites W2168946040 @default.
• W2014465321 cites W2435978650 @default.
• W2014465321 doi "https://doi.org/10.1074/jbc.271.30.18263" @default.
• W2014465321 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/8663484" @default.
• W2014465321 hasPublicationYear "1996" @default.
• W2014465321 type Work @default.
• W2014465321 sameAs 2014465321 @default.
• W2014465321 citedByCount "147" @default.
• W2014465321 countsByYear W20144653212012 @default.
• W2014465321 countsByYear W20144653212013 @default.
• W2014465321 countsByYear W20144653212014 @default.
• W2014465321 countsByYear W20144653212015 @default.
• W2014465321 countsByYear W20144653212018 @default.
• W2014465321 countsByYear W20144653212019 @default.
• W2014465321 countsByYear W20144653212020 @default.
• W2014465321 countsByYear W20144653212021 @default.
• W2014465321 countsByYear W20144653212022 @default.
• W2014465321 crossrefType "journal-article" @default.
• W2014465321 hasAuthorship W2014465321A5016470161 @default.
• W2014465321 hasAuthorship W2014465321A5022241128 @default.
• W2014465321 hasAuthorship W2014465321A5031261744 @default.
• W2014465321 hasAuthorship W2014465321A5044712943 @default.
• W2014465321 hasAuthorship W2014465321A5047181455 @default.
• W2014465321 hasAuthorship W2014465321A5053729580 @default.
• W2014465321 hasBestOaLocation W20144653211 @default.
• W2014465321 hasConcept C178666793 @default.
• W2014465321 hasConcept C181199279 @default.
• W2014465321 hasConcept C185592680 @default.
• W2014465321 hasConcept C190283241 @default.
• W2014465321 hasConcept C2776714187 @default.
• W2014465321 hasConcept C2781357212 @default.
• W2014465321 hasConcept C43617362 @default.
• W2014465321 hasConcept C552990157 @default.
• W2014465321 hasConcept C55493867 @default.
• W2014465321 hasConcept C79879829 @default.
• W2014465321 hasConcept C86803240 @default.
• W2014465321 hasConcept C95444343 @default.
• W2014465321 hasConceptScore W2014465321C178666793 @default.
• W2014465321 hasConceptScore W2014465321C181199279 @default.
• W2014465321 hasConceptScore W2014465321C185592680 @default.
• W2014465321 hasConceptScore W2014465321C190283241 @default.
• W2014465321 hasConceptScore W2014465321C2776714187 @default.
• W2014465321 hasConceptScore W2014465321C2781357212 @default.
• W2014465321 hasConceptScore W2014465321C43617362 @default.
• W2014465321 hasConceptScore W2014465321C552990157 @default.
• W2014465321 hasConceptScore W2014465321C55493867 @default.
• W2014465321 hasConceptScore W2014465321C79879829 @default.
• W2014465321 hasConceptScore W2014465321C86803240 @default.
• W2014465321 hasConceptScore W2014465321C95444343 @default.
• W2014465321 hasIssue "30" @default.
• W2014465321 hasLocation W20144653211 @default.
• W2014465321 hasOpenAccess W2014465321 @default.
• W2014465321 hasPrimaryLocation W20144653211 @default.

• next