Apoptosis-Resistance of Hypoxic Cells
2003; Elsevier BV; Volume: 163; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)63693-0
ISSN1525-2191
AutoresZheng Dong, Jin Zhao Wang, Fu‐Shin X. Yu, Manjeri A. Venkatachalam,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoHypoxia is an important pathogenic factor in ischemic disease and tumorigenesis. Under hypoxia, some cells are irreversibly damaged, whereas others adapt to the stress and may become more resistant to injury. The mechanism underlying such adaptive responses is unclear. Our recent study showed hypoxic induction of inhibitor of apoptosis protein-2 (IAP-2). Here we have investigated the critical steps in the apoptotic cascade that are affected by hypoxia and have identified a role for IAP-2 in apoptosis resistance of hypoxic cells. The results show that cells cultured in hypoxia became resistant to staurosporine-induced apoptosis. Apoptosis resistance of these cells took place at the mitochondria and in the cytosol. At the mitochondrial level, membrane accumulation of the proapoptotic molecule Bax was suppressed. This was accompanied by less cytochrome c (cyt. c) release from the organelles. In the cytosol, hypoxia induced IAP-2; the cytosol with IAP-2 was resistant to cyt. c-stimulated caspase activation. Of significance, immunodepletion of IAP-2 from the hypoxic cytosol restored its competence for caspase activation. Thus, death resistance of hypoxic cells involves multiple factors targeting different stages of apoptosis, with IAP-2 suppressing caspases in the cytosol. Hypoxia is an important pathogenic factor in ischemic disease and tumorigenesis. Under hypoxia, some cells are irreversibly damaged, whereas others adapt to the stress and may become more resistant to injury. The mechanism underlying such adaptive responses is unclear. Our recent study showed hypoxic induction of inhibitor of apoptosis protein-2 (IAP-2). Here we have investigated the critical steps in the apoptotic cascade that are affected by hypoxia and have identified a role for IAP-2 in apoptosis resistance of hypoxic cells. The results show that cells cultured in hypoxia became resistant to staurosporine-induced apoptosis. Apoptosis resistance of these cells took place at the mitochondria and in the cytosol. At the mitochondrial level, membrane accumulation of the proapoptotic molecule Bax was suppressed. This was accompanied by less cytochrome c (cyt. c) release from the organelles. In the cytosol, hypoxia induced IAP-2; the cytosol with IAP-2 was resistant to cyt. c-stimulated caspase activation. Of significance, immunodepletion of IAP-2 from the hypoxic cytosol restored its competence for caspase activation. Thus, death resistance of hypoxic cells involves multiple factors targeting different stages of apoptosis, with IAP-2 suppressing caspases in the cytosol. Hypoxia, a condition of decreased availability of oxygen, poses stresses to mammalian cells and is an important pathogenic factor in several types of devastating diseases.1Semenza GL Agani F Feldser D Iyer N Kotch L Laughner E Yu A Hypoxia, HIF-1, and the pathophysiology of common human diseases.Adv Exp Med Biol. 2000; 475: 123-130Crossref PubMed Google Scholar, 2Saikumar P Dong Z Weinberg JM Venkatachalam MA Mechanisms of cell death in hypoxia/reoxygenation injury.Oncogene. 1998; 17: 3341-3349Crossref PubMed Scopus (238) Google Scholar For example, hypoxia during organ ischemia leads to cell injury and death, which contributes to the development of myocardial infarction, acute renal failure and stroke in the brain.3Cotran RS Kumar V Collins T Cell injury and cellular death.in: Cotran RS Kumar V Collins T Robbins Pathologic Basis of Disease. 6th ed. W. B. Saunders Co., Philadelphia1999: 1-29Google Scholar In solid tumors, malformation and malfunction of blood vessels result in poorly oxygenated regions, where cancerous cells are constantly subjected to hypoxic pressure and the selection for death resistance.4Semenza GL HIF-1 and tumor progression: pathophysiology and therapeutics.Trends Mol Med. 2002; 8: S62-S67Abstract Full Text Full Text PDF PubMed Scopus (946) Google Scholar, 5Brown JM The hypoxic cell: a target for selective cancer therapy: eighteenth Bruce F. 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Such a decrease conserves energy to maintain the essential elements for cell viability. Second, expression of genes that are involved in oxygen delivery and anaerobic ATP production are drastically up-regulated. A major transcription factor governing the expression of these genes is hypoxia-inducible factor-1 (HIF-1).10Semenza GL Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1.Annu Rev Cell Dev Biol. 1999; 15: 551-578Crossref PubMed Scopus (1697) Google Scholar Finally, while hypoxia induces cell injury and death, it also activates pathways for cell survival. Hypoxic activation of the PI3K/Akt survival pathway has been shown in PC-12 cells.11Alvarez-Tejado M Naranjo-Suarez S Jimenez C Carrera AC Landazuri MO del Peso L Hypoxia induces the activation of the phosphatidylinositol 3-kinase/Akt cell survival pathway in PC12 cells: protective role in apoptosis.J Biol Chem. 2001; 276: 22368-22374Crossref PubMed Scopus (214) Google Scholar Moreover, our recent studies have documented a striking induction by hypoxia of IAP-2, an apoptosis inhibitory protein.12Dong Z Venkatachalam MA Wang J Patel Y Saikumar P Semenza GL Force T Nishiyama J Up-regulation of apoptosis inhibitory protein IAP-2 by hypoxia: Hif-1-independent mechanisms.J Biol Chem. 2001; 276: 18702-18709Crossref PubMed Scopus (141) Google Scholar, 13Dong Z Nishiyama J Yi X Venkatachalam MA Denton M Gu S Li S Qiang M Gene promoter of apoptosis inhibitory protein IAP2: identification of enhancer elements and activation by severe hypoxia.Biochem J. 2002; 364: 413-421Crossref PubMed Scopus (36) Google Scholar Despite these observations, it is unclear how the pathways and genes regulate the apoptosis sensitivity of hypoxic cells. IAP-2 is a member of the family of inhibitor of apoptosis proteins (IAPs).14Salvesen GS Duckett CS IAP proteins: blocking the road to death's door.Nat Rev Mol Cell Biol. 2002; 3: 401-410Crossref PubMed Scopus (1586) Google Scholar, 15LaCasse EC Baird S Korneluk RG MacKenzie AE The inhibitors of apoptosis (IAPs) and their emerging role in cancer.Oncogene. 1998; 17: 3247-3259Crossref PubMed Scopus (949) Google Scholar, 16Deveraux QL Reed JC IAP family proteins: suppressors of apoptosis.Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2298) Google Scholar IAPs antagonize apoptosis in a variety of experimental models, probably through their interaction and inhibition of caspases. Regulation of IAPs has been implicated in the development of tissue pathology during neoplasia, neurodegenerative disorders, and brain ischemia.14Salvesen GS Duckett CS IAP proteins: blocking the road to death's door.Nat Rev Mol Cell Biol. 2002; 3: 401-410Crossref PubMed Scopus (1586) Google Scholar, 15LaCasse EC Baird S Korneluk RG MacKenzie AE The inhibitors of apoptosis (IAPs) and their emerging role in cancer.Oncogene. 1998; 17: 3247-3259Crossref PubMed Scopus (949) Google Scholar, 16Deveraux QL Reed JC IAP family proteins: suppressors of apoptosis.Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2298) Google Scholar Our recent studies showed that IAP-2 was induced under hypoxia through gene transcription, whereas by HIF-1-independent mechanisms.12Dong Z Venkatachalam MA Wang J Patel Y Saikumar P Semenza GL Force T Nishiyama J Up-regulation of apoptosis inhibitory protein IAP-2 by hypoxia: Hif-1-independent mechanisms.J Biol Chem. 2001; 276: 18702-18709Crossref PubMed Scopus (141) Google Scholar, 13Dong Z Nishiyama J Yi X Venkatachalam MA Denton M Gu S Li S Qiang M Gene promoter of apoptosis inhibitory protein IAP2: identification of enhancer elements and activation by severe hypoxia.Biochem J. 2002; 364: 413-421Crossref PubMed Scopus (36) Google Scholar Of significance, hypoxic cells expressing IAP-2 became resistant to apoptosis.12Dong Z Venkatachalam MA Wang J Patel Y Saikumar P Semenza GL Force T Nishiyama J Up-regulation of apoptosis inhibitory protein IAP-2 by hypoxia: Hif-1-independent mechanisms.J Biol Chem. 2001; 276: 18702-18709Crossref PubMed Scopus (141) Google Scholar, 13Dong Z Nishiyama J Yi X Venkatachalam MA Denton M Gu S Li S Qiang M Gene promoter of apoptosis inhibitory protein IAP2: identification of enhancer elements and activation by severe hypoxia.Biochem J. 2002; 364: 413-421Crossref PubMed Scopus (36) Google Scholar The current study was designed to identify the critical steps in the apoptotic cascade that are suppressed by hypoxia and to determine the role of IAP-2 in death resistance of hypoxic cells. The results show that cells cultured under hypoxia became resistant to staurosporine-induced apoptosis. Such resistance took place not only at caspase activation but also at upstream levels of the mitochondria. The movement of the pro-apoptotic molecule Bax to mitochondria was significantly suppressed in hypoxic cells, which was accompanied by reduced cyt. c release from the organelles. IAP-2 appeared to be a major inhibitory regulator of caspases in hypoxic cells. The cytosol isolated from hypoxic cells contained high levels of IAP-2 and exhibited a significantly lower capacity for caspase activation. Immunodepletion of IAP-2 from the hypoxic cytosol restored its ability for caspase activation. Thus, while death resistance of hypoxic cells takes place at multiple levels, IAP-2 induction plays an important role in the resistance at the level of caspase activation. The cells used in this study were derived from an immortalized cell line of rat kidney proximal tubular epithelium kindly provided by U. Hopfer (Case Western Reserve University, Cleveland, OH). The cells were cultured and plated for experiments as previously described.12Dong Z Venkatachalam MA Wang J Patel Y Saikumar P Semenza GL Force T Nishiyama J Up-regulation of apoptosis inhibitory protein IAP-2 by hypoxia: Hif-1-independent mechanisms.J Biol Chem. 2001; 276: 18702-18709Crossref PubMed Scopus (141) Google Scholar, 17Saikumar P Dong Z Patel Y Hall K Hopfer U Weinberg JM Venkatachalam MA Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.Oncogene. 1998; 17: 3401-3415Crossref PubMed Scopus (264) Google Scholar Antibodies were from the following sources: monoclonal anti-Bax (clone 1D1) from NeoMarkers (Fremont, CA); monoclonal anti-cyt. c (clone 7H8.2C12) from BD Pharmingen (San Diego, CA); polyclonal anti-Bax, anti-IAP-2, anti-lamin B, and anti-gelsolin from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); monoclonal anti-Cox IV (clone 20E8) from Molecular Probes (Eugene, OR); all secondary antibodies from Jackson ImmunoResearch (West Grove, PA). Protein A/G sepharose was obtained from Santa Cruz Biotechnology Inc. and DEVD.AFC was from Enzyme Systems Products (Dublin, CA). Unless indicated, all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Hypoxic incubation was conducted as previously described.12Dong Z Venkatachalam MA Wang J Patel Y Saikumar P Semenza GL Force T Nishiyama J Up-regulation of apoptosis inhibitory protein IAP-2 by hypoxia: Hif-1-independent mechanisms.J Biol Chem. 2001; 276: 18702-18709Crossref PubMed Scopus (141) Google Scholar, 13Dong Z Nishiyama J Yi X Venkatachalam MA Denton M Gu S Li S Qiang M Gene promoter of apoptosis inhibitory protein IAP2: identification of enhancer elements and activation by severe hypoxia.Biochem J. 2002; 364: 413-421Crossref PubMed Scopus (36) Google Scholar, 17Saikumar P Dong Z Patel Y Hall K Hopfer U Weinberg JM Venkatachalam MA Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.Oncogene. 1998; 17: 3401-3415Crossref PubMed Scopus (264) Google Scholar, 18Dong Z Saikumar P Patel Y Weinberg JM Venkatachalam MA Serine protease inhibitors suppress cytochrome c-mediated caspase-9 activation and apoptosis during hypoxia-reoxygenation.Biochem J. 2000; 347: 669-677Crossref PubMed Scopus (72) Google Scholar Briefly, cells were washed with phosphate-buffered saline (PBS), transferred to an anaerobic chamber with 95% N2/5% CO2, and incubated in Krebs-Ringer bicarbonate buffer. This buffer was pre-equilibrated with 95% N2/5% CO2. EC Oxyrase, a biocatalytic oxygen reducing agent, was added at 1.2 units/ml to the incubation medium to consume residual O2 and maximize the degree of hypoxia. To minimize cell injury by hypoxic incubation per se and reveal staurosporine-induced apoptosis, 5.5 mmol/L glucose was included in the incubation buffer to facilitate glycolytic ATP production.12Dong Z Venkatachalam MA Wang J Patel Y Saikumar P Semenza GL Force T Nishiyama J Up-regulation of apoptosis inhibitory protein IAP-2 by hypoxia: Hif-1-independent mechanisms.J Biol Chem. 2001; 276: 18702-18709Crossref PubMed Scopus (141) Google Scholar, 13Dong Z Nishiyama J Yi X Venkatachalam MA Denton M Gu S Li S Qiang M Gene promoter of apoptosis inhibitory protein IAP2: identification of enhancer elements and activation by severe hypoxia.Biochem J. 2002; 364: 413-421Crossref PubMed Scopus (36) Google Scholar, 17Saikumar P Dong Z Patel Y Hall K Hopfer U Weinberg JM Venkatachalam MA Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.Oncogene. 1998; 17: 3401-3415Crossref PubMed Scopus (264) Google Scholar For reoxygenation, cells after hypoxic incubation were transferred back to full culture medium in 95% air/5% CO2. Staurosporine was added at 1 μmol/L to hypoxic cells or to normally oxygenated cells in Krebs-Ringer bicarbonate buffer in the presence of 5.5 mmol/L glucose. Morphological examination of apoptotic cells was described in our previous studies.12Dong Z Venkatachalam MA Wang J Patel Y Saikumar P Semenza GL Force T Nishiyama J Up-regulation of apoptosis inhibitory protein IAP-2 by hypoxia: Hif-1-independent mechanisms.J Biol Chem. 2001; 276: 18702-18709Crossref PubMed Scopus (141) Google Scholar Typical apoptotic morphology examined included cellular shrinkage, nuclear condensation and fragmentation, and formation of apoptotic bodies. To reveal nuclear changes, cells were exposed to 5 μg/ml of Hoechst 33342 in PBS for 2 to 5 minutes at room temperature. For each condition, apoptosis was monitored in five fields with ∼200 cells per field. The experiments were repeated at least 4 times with duplicate dishes for each condition in every experiment. The enzymatic activity of caspases was measured using an exogenous fluorogenic peptide substrates DEVD.AFC as described in our previous publications.17Saikumar P Dong Z Patel Y Hall K Hopfer U Weinberg JM Venkatachalam MA Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.Oncogene. 1998; 17: 3401-3415Crossref PubMed Scopus (264) Google Scholar, 18Dong Z Saikumar P Patel Y Weinberg JM Venkatachalam MA Serine protease inhibitors suppress cytochrome c-mediated caspase-9 activation and apoptosis during hypoxia-reoxygenation.Biochem J. 2000; 347: 669-677Crossref PubMed Scopus (72) Google Scholar DEVD.AFC is a substrate with specificity for caspase-3, -6, and -7.17Saikumar P Dong Z Patel Y Hall K Hopfer U Weinberg JM Venkatachalam MA Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.Oncogene. 1998; 17: 3401-3415Crossref PubMed Scopus (264) Google Scholar, 18Dong Z Saikumar P Patel Y Weinberg JM Venkatachalam MA Serine protease inhibitors suppress cytochrome c-mediated caspase-9 activation and apoptosis during hypoxia-reoxygenation.Biochem J. 2000; 347: 669-677Crossref PubMed Scopus (72) Google Scholar Cells were extracted with 1% Triton X-100. The lysates of 25 μg of protein were added to the enzymatic reactions containing 50 μmol/L DEVD.AFC. After 60 minutes of reaction at 37°C, fluorescence at Excitation 360 nm/Emission 530 nm was monitored. For each measurement, a standard curve was constructed using free AFC. Based on the standard curve, the fluorescence reading from each enzymatic reaction was translated into the molar amount of liberated AFC to indicate caspase activity. To analyze the redistribution of Bax and cyt. c during apoptosis, cells were fractionated into cytosolic and membrane-bound fractions using low concentrations of digitonin. Selective permeabilization of plasma membranes by digitonin was monitored by microscopy. This method of cellular fractionation for examination of Bax and cyt. c translocation has been used successfully in recent studies.17Saikumar P Dong Z Patel Y Hall K Hopfer U Weinberg JM Venkatachalam MA Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.Oncogene. 1998; 17: 3401-3415Crossref PubMed Scopus (264) Google Scholar, 18Dong Z Saikumar P Patel Y Weinberg JM Venkatachalam MA Serine protease inhibitors suppress cytochrome c-mediated caspase-9 activation and apoptosis during hypoxia-reoxygenation.Biochem J. 2000; 347: 669-677Crossref PubMed Scopus (72) Google Scholar, 19Waterhouse NJ Goldstein JC von Ahsen O Schuler M Newmeyer DD Green DR Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process.J Cell Biol. 2001; 153: 319-328Crossref PubMed Scopus (377) Google Scholar, 20Gottlieb RA Granville DJ Analyzing mitochondrial changes during apoptosis.Methods. 2002; 26: 341-347Crossref PubMed Scopus (69) Google Scholar, 21Mikhailov V Mikhailova M Pulkrabek DJ Dong Z Venkatachalam MA Saikumar P Bcl-2 prevents Bax oligomerization in the mitochondrial outer membrane.J Biol Chem. 2001; 276: 18361-18374Crossref PubMed Scopus (281) Google Scholar Briefly, cells were exposed to 0.05% digitonin in isotonic sucrose buffer (in mM: 250 sucrose, 10 Hepes, 10 KCl, 1.5 MgCl2, 1 ethylenediaminetetraacetate, and 1 EGTA; pH 7.1) for 2 minutes at room temperature to collect the soluble fraction as cytosolic extracts. Digitonin insoluble fraction was dissolved in 2% sodium dodecyl sulfate (SDS) buffer to collect the membrane-bound part. Since Bax/cyt. c redistribution mainly takes place between the cytosol and mitochondria, immunoblot analysis of the membrane-bound part is expected to reveal mainly the mitochondrial content of these molecules. Proteins were analyzed by immunoblotting using NuPAGE Gel Systems as described previously.12Dong Z Venkatachalam MA Wang J Patel Y Saikumar P Semenza GL Force T Nishiyama J Up-regulation of apoptosis inhibitory protein IAP-2 by hypoxia: Hif-1-independent mechanisms.J Biol Chem. 2001; 276: 18702-18709Crossref PubMed Scopus (141) Google Scholar, 17Saikumar P Dong Z Patel Y Hall K Hopfer U Weinberg JM Venkatachalam MA Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.Oncogene. 1998; 17: 3401-3415Crossref PubMed Scopus (264) Google Scholar, 18Dong Z Saikumar P Patel Y Weinberg JM Venkatachalam MA Serine protease inhibitors suppress cytochrome c-mediated caspase-9 activation and apoptosis during hypoxia-reoxygenation.Biochem J. 2000; 347: 669-677Crossref PubMed Scopus (72) Google Scholar, 21Mikhailov V Mikhailova M Pulkrabek DJ Dong Z Venkatachalam MA Saikumar P Bcl-2 prevents Bax oligomerization in the mitochondrial outer membrane.J Biol Chem. 2001; 276: 18361-18374Crossref PubMed Scopus (281) Google Scholar To analyze Bax and cyt. c translocations, cytosolic and membrane fractions extracted from the same amounts (∼1 × 105) of cells were subjected to electrophoresis. For other immunoblots, 50-μg proteins were loaded for each lane. After electrophoresis on 10% or 12% Bis-Tris gels in MES running buffer, the proteins were electroblotted onto polyvinylidene difluoride membranes. The membranes were subsequently blocked with 5% milk or bovine serum albumin and exposed to primary antibodies overnight at 4°C. After extensive wash in PBS containing Tween-20, the membranes were exposed to the horseradish-peroxidase-conjugated secondary antibodies. Antigens on the blots were revealed by exposure to chemiluminescent substrates (Pierce, Rockford, IL). Cells were grown on collagen-coated glass coverslips. After experimental incubation, cells were fixed with 4% paraformaldehyde in PBS for 1 hour at room temperature. Cells were then permeabilized for 5 minutes with 0.1% SDS, followed by 1 hour blocking in 5% normal goat serum. The cells were subsequently exposed to a mixture of primary antibodies (rabbit anti-Bax and mouse anti-Cox IV) for 1 hour and blocked again in normal goat serum. Finally, the cells were exposed to a mixture of secondary antibodies (Cy-3-labeled goat anti-rabbit IgG and FITC-labeled goat anti-mouse IgG) for 1 hour. The same cells were examined for fluorescence of Cy-3 (red: Bax signal) and fluorescein isothiocyanate (FITC) (green: Cox signal) by laser-scanning confocal microscopy. Cytosolic capacity for caspase activation was determined by in vitro reconstitution assays as described in our previous study.18Dong Z Saikumar P Patel Y Weinberg JM Venkatachalam MA Serine protease inhibitors suppress cytochrome c-mediated caspase-9 activation and apoptosis during hypoxia-reoxygenation.Biochem J. 2000; 347: 669-677Crossref PubMed Scopus (72) Google Scholar Cytosols were extracted from normoxic or hypoxic cells with 0.05% digitonin as described above. The cytosols were concentrated to 4 to 5 mg/ml with 3K cutoff microconcentrators. For reconstitution, 1 μl of 0.5 mg/ml rat heart cyt. c and 1 μl of 10 mmol/L dATP were added to 7.5 μl cytosolic extracts with 25 μg protein, and incubated for 1 hour at 30°C. After incubation, 5 μl of the reconstitution mixture was transferred to 200 μl enzymatic reaction buffer with 50 μmol/L DEVD.AFC. The cleavage of DEVD.AFC was monitored as described above to determine reconstituted caspase activity. Cells were subjected to 3 hours of hypoxia in the presence of glucose. Cytosol was extracted with 0.05% digitonin as described in Cellular Fractionation. Protein concentration of the cytosolic extracts was adjusted to 0.5 μg/ml. For immunodepletion, a polyclonal antibody against IAP-2 was added to 200 μg cytosol and incubated at 4°C for 1 hour by mixing on a rocker. Protein A/G sepharose of 30 μl was then added for overnight mixing at 4°C. The mixture was centrifuged to collect the supernatant for immunoblot analysis of IAP-2 to determine the efficacy of immunodepletion. The IAP-2-depleted samples were also analyzed for their capacity of caspase activation by in vitro reconstitutions using exogenous cyt. c. To measure ATP, cells were extracted with trichloroacetic acid. ATP in cell extracts was measured by luminometry of the luciferin firefly luciferase reaction.22Dong Z Venkatachalam MA Weinberg JM Saikumar P Patel Y Protection of ATP-depleted cells by impermeant strychnine derivatives: implications for glycine cytoprotection.Am J Pathol. 2001; 158: 1021-1028Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar ATP values were expressed as nmole per mg cell protein. Protein was quantified with the bicinchoninic acid (BCA) reagent purchased from Pierce. Data were expressed as means ± SE (n ≥4). Statistical differences between means were determined using analysis of variance by two-tailed tests. P < 0.05 was considered to reflect significant differences. An important consideration for study of apoptosis sensitivity of hypoxic cells was to minimize cell injury caused by hypoxic incubation per se. Removal of hypoxic injury was necessary for establishing a "clean" model to reveal apoptosis that was specifically induced by other insults. Because energy deprivation is a main cause of acute cell damage under hypoxia, we reasoned that facilitation of anaerobic ATP generation via glycolysis might assist the cells to maintain viability. To facilitate glycolysis, we provided 5.5 mmol/L glucose in the hypoxic incubation buffer. As shown in Figure 1A, in the absence of glucose, hypoxia led to rapid decreases in cellular ATP. At the end of 2 hours of hypoxic incubation, ATP levels were less than 10% of control (Figure 1A, −Glucose). When glucose was provided, cellular ATP was maintained at substantial levels. As a result, cellular ATP was 65% of control after 6 hours of hypoxic incubation (Figure 1A, +Glucose). Of significance, provision of glucose diminished cell injury and death caused by hypoxic incubation for a period of time. As shown in Figure 1B, less than 5% apoptosis was triggered by 6 hours of hypoxic incubation, when glucose was present (+Glucose). In sharp contrast, in the absence of glucose, over 50% of cells underwent apoptosis (Figure 1B, −Glucose). Amelioration of acute hypoxic injury by glucose provided us a relatively clean model to compare staurosporine-induced apoptosis under hypoxia and under normal oxygen in subsequent experiments. Staurosporine (STA) is a broad-spectrum inhibitor of protein kinases, which is also a potent inducer of apoptosis. Apoptosis induced by STA has been widely documented in diverse types of cells. Thus, we chose STA as the apoptosis inducer to examine the effects of hypoxia on cellular sensitivity to apoptosis. The kinetics of apoptosis development was monitored following the addition of STA, under normal oxygen tension (21% O2) or severe hypoxia (near 0% O2) (Figure 2A). In cells with normal oxygen (Figure 2A, +O2), significant amounts of apoptosis were induced after 4 hours of STA incubation. The rate of apoptosis increased to ∼50% by the end of 6 hours of STA exposure. In sharp contrast, apoptosis was significantly less when STA was added to hypoxic cells. For example, only 11% and 15% apoptosis was induced by 4 and 6 hours of STA exposure, respectively (Figure 2A, −O2). These morphological observations were consistent with biochemical analyses. As shown in Figure 2B, STA activated caspases in cells under normal oxygen tensions and this activation was significantly suppressed in hypoxic cells. We further examined the breakdown of endogenous caspase substrates during STA treatment. As shown in Figure 2C, STA induced proteolysis of lamin B and gelsolin, releasing characteristic fragments of apoptosis.23Earnshaw WC Martins LM Kaufmann SH Mammalian caspases: structure, activation, substrates, and functions during apoptosis.Annu Rev Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2463) Google Scholar Again, fragmentation of these proteins was significantly ameliorated under hypoxia. Together, these results indicate clearly that hypoxic cells, compared with normoxic ones, were more resistant to apoptosis. Our results have demonstrated apoptosis resistance of hypoxic cells (Figure 2). Although our previous studies showed IAP-2 induction in these cells and suggested a role for IAP-2 in apoptosis resistance, hypoxia also activates other pathways for cell survival. For example, a recent study has documented hypoxic activation of the survival pathway of Akt/PI3K.11Alvarez-Tejado M Naranjo-Suarez S Jimenez C Carrera AC Landazuri MO del Peso L Hypoxia induces the activation of the phosphatidylinositol 3-kinase/Akt cell survival pathway in PC12 cells: protective role in apoptosis.J Biol Chem. 2001; 276: 22368-22374Crossref PubMed Scopus (214) Google Scholar Thus, to further examine the molecular basis of the acquired apoptosis resistance, we determined the critical steps in the apoptotic cascade that were affected by hypoxia. Our first experiments analyzed the translocation of Bax to mitochondria by immunoblotting. Bax is a pro-apoptotic member of Bcl-2 family proteins. Accumulation of Bax in mitochondria has been recognized as an early event of apoptosis, which leads to permeabilization of the mitochondrial outer membrane, followed by the release of apoptogenic factors including cyt. c.24Adams JM Cory S The Bcl-2 protein family: arbiters of cell survival.Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4841) Google Scholar, 25Budihardjo I Oliver H Lutter M Luo X Wang X Biochemical pathways of caspase activation during apoptosis.Annu Rev Cell Dev Biol. 1999; 15: 269-290Crossref PubMed Scopus (2292) Google Scholar, 26Green DR Apoptotic pathways: the roads to ruin.Cell. 1998; 94: 695-698Abstract Full Text Full Text PDF PubMed Scopus (1116) Google Scholar, 27Gross A McDonnell JM Korsmeyer SJ BCL-2 family members and the mitochondria in apoptosis.Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3280) Google Scholar, 28Martinou JC Green DR Breaking the mitochondrial barrier.Nat Rev Mol Cell Biol. 2001; 2: 63-67Crossref PubMed Scopus (855) Google Scholar As shown in Figure 3A, in control cells the majority of Bax was detected in the cytosolic fraction, with limited amounts in the membrane-bound organellar fraction (lane 1). After 2 hours of STA incubation, significant amounts of Bax accumulated in the membrane fraction, which was accompanied by the loss of Bax from the cytosol (lane 2). Bax tra
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