TY - JOUR
AU - Liu, Xiangjun
AB - Abstract In recent years, it has been shown that yeast, a unicellular organism, undergoes apoptosis in response to various factors. Here we demonstrate that the highly effective anticancer agent arsenic induces apoptotic process in yeast cells. Reactive oxygen species (ROS) production was observed in the process. Moreover, mitochondrial membrane potential decreased after arsenic treatment. Resistance of the rho0 mutant strain (lacking mtDNA) to arsenic provides further evidence that this death process involves mitochondria. In addition, hypersensitivity of Δ sod1 to arsenic suggests the critical role of ROS. Cell death and DNA fragmentation decreased in a Δ yca1 deletion mutant, indicating the participation of yeast caspase-1 protein in apoptosis. The implications of these findings for arsenic-induced apoptosis are discussed. apoptosis, Saccharomyces cerevisiae, arsenic, mitochondria, caspase Introduction Apoptosis is a genetically regulated process that plays an important role in tissue homeostasis and embryonic development. Defects in apoptosis are often associated with diseases, such as cancer, neurodegenerative disorders, or stroke. Apoptosis is widely accepted as an essential process in multicellular organisms, but was generally considered to be unnecessary for single-cell organisms that do not form multicellular structures. However, recent studies support the notion that unicellular organisms can also undergo apoptosis. Saccharomyces cerevisiae has been used as a model organism for apoptosis research. Like mammalian cells, yeast cells undergo apoptosis, showing characteristic markers such as DNA fragmentation, phosphatidyl serine externalization, and cytochrome c release from mitochondria (Madeo et al., 1997; Manon et al., 1997). Further research demonstrated that in yeast, apoptosis can be induced by different exogenous and intrinsic stresses such as H2O2 (Madeo et al., 1999), acetic acid (Ludovico et al., 2001), aspirin (Balzan et al., 2004), osmotin (Narasimhan et al., 2001), cell aging (Laun et al., 2001), and pheromones (Severin & Hyman, 2002). As in metazoan apoptosis, reactive oxygen species (ROS) act as central regulators of yeast apoptosis (Madeo et al., 1999). The finding of yeast orthologs of a caspase (Madeo et al., 2002), a proapoptotic serine protease HtrA2/Omi (Fahrenkrog et al., 2004) and the transkingdom Bax inhibitor BI-1 (Chae et al., 2003) further underlines the similarity between yeast and metazoan apoptosis. Mitochondria play a central role in mammalian cell death, both as essential organelles targeted for destruction and as perpetrators of the death pathway. Recently, Ludovico (2002) found mitochondrial involvement in yeast apoptosis induced by acetic acid. Arsenic is a toxic metalloid with a long history of usage as a therapeutic agent. It was used to treat the plague, malaria, and cancer, and to promote sweating, throughout the 18th century (Miller et al., 2002). During the last decade, the efficacy of arsenic trioxide in both newly diagnosed and relapsed patients with acute promyelocytic leukemia has been established (Shen et al., 1997). Recently, numerous reports have shown that an important mechanism of this ancient remedy is its proapoptotic effects on mammalian malignant cells. Arsenic inhibits the growth of yeast at moderate concentrations (Samokhvalov et al., 2003). Previous studies have investigated the mechanisms of arsenic detoxification, which is mediated by a cluster of three genes (ARR1, ARR2, ARR3). However, the process by which the yeast dies when injured by arsenic is unknown. In the present study, we demonstrated that arsenic is also able to trigger S. cerevisiae into an apoptotic process associated with characteristic markers. Further evidence supporting the involvement of mitochondria and yeast caspase-1 protein (Yca1p) in arsenic-induced apoptosis is presented. Materials and methods Microorganisms and growth conditions Saccharomyces cerevisiae cells were grown in YPD medium containing 1% yeast extract, 2% peptone and 2% glucose at 30°C. All strains used in this study were in a BY4742 (MATa his 3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) background. Growth and cell survival Growth was monitored by plate assays. Cultures were adjusted to identical OD600 nm, and additional 10-fold dilutions were made. Then, 1.5 μL of each diluted yeast culture was spotted onto yeast peptone dextrose (YPD) plates with or without arsenite, and growth was monitored after 4 days of growth at 30°C. For survival tests, the yeast culture was adjusted to an identical OD600 nm and diluted to the same cell density in distilled water, and about 200 cells were spread onto YPD plates. The number of surviving colonies was determined after 2 days of incubation at 30°C. TdT mediated dUTP nick end labelling (TUNEL) DNA strand breaks were demonstrated by TUNEL with the In Situ Cell Death Detection Kit (Fluorescein, Roche Diagnostics). Yeast cells were fixed with 3.7% (v/v) formaldehyde as described by Madeo (1999), and cell walls were digested with snailase. Cells were washed with phosphate-buffered saline (PBS), incubated in permeabilization solution (0.1% v/v Triton X-100 and 0.1% w/v sodium citrate) for 2 min on ice, washed twice with PBS, and incubated with 10 μL of TUNEL reaction mixture, containing terminal deoxynucleotidyl transferase and fluorescein isothiocyanate (FITC) – dUTP, for 60 min at 37°C. Finally, the cells were washed three times with PBS and analyzed under a fluorescence microscope (Zess Axioskop). Annexin V staining Exposed phosphatidylserine was measured by annexin V labeling as described previously (Madeo et al., 1997). Yeast cells were washed in sorbitol buffer (1.2 M sorbitol, 0.5 mM MgCl2, 35 mM potassium phosphate, pH 6.8), digested with 15 U mL−1 lyticase in sorbitol buffer for 1 h at 28°C, harvested, washed in binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) containing 1.2 M sorbitol, harvested, and resuspended in binding buffer/sorbitol. Two microliters of annexin-FITC (CLONETECH Laboratories Inc.) and 1 μL of propidium iodide (PI, 500 μg mL−1) were added to 40 μL of cell suspension, and then incubated for 20 min at room temperature. Cells were washed, resuspended in binding buffer/sorbitol, and then observed under the fluorescence microscope. Assessment of mitochondrial transmembrane potential (Δψm) Cells were collected and resuspended in 20 mM HEPES buffer (pH 7.4) containing 50 mM glucose. Then, 1 mL of the cell suspension was loaded with 2 μM rhodamine 123 (Rh123) for 30 min, centrifuged, washed, and resuspended in 100 μL of PBS. Δψm was expressed as a fluorescence intensity of Rh123, which was analyzed by flow cytometry with excitation at 488 nm. ROS production This assay was performed as described previously (Madeo et al., 1999). Briefly, dihydrorhodamine 123 (1 μL of 2.5 mg mL−1 stock solution in ethanol) was added to 0.1 mL of cell suspension in treated medium and incubated at 30°C for 30 min. Dihydroethidium (1 μL of 5 mg mL−1 aqueous stock solution) was added to 0.1 mL of cell suspension in treated medium, and incubated at 30°C for 10 min. The production of free intracellular radicals was analyzed using a fluorescence activated cell sorter (FACS) Calibur (Becton Dickinson) at low flow rate with excitation at 488 nm. Results Arsenite induces apoptotic cell death in yeast Exposure of S. cerevisiae strain BY4742 to arsenic at different concentration (1–7 mM) resulted in cell death (Fig. 1c). The percentage of survival, estimated by CFU counts after 24 h of treatment, decreased with increasing concentrations of arsenite in the medium. In order to assess whether the cell death induced by arsenic is apoptotic, some characteristic markers of apoptosis were assayed. The exposure of phosphatidylserine on the plasma membrane is an early event in apoptosis, and can be detected by annexin V, a protein with strong affinity for phosphatidylserine. Treatment of S. cerevisiae with 3 mM arsenite resulted in 35.7% of cells being annexin V positive, whereas 2.7% of control cells were annexin V-positive, showing phosphatidylserine translocation to the outer membrane during arsenic treatment (Fig. 1b). In addition, TUNEL-positive cells displaying nuclear green fluorescence were detected after exposure to arsenic, indicating the occurrence of DNA fragmentation (Fig. 1a). The percentage of cells displaying positive TUNEL staining increased with the arsenic concentration, reaching a maximum at 3 mM (Fig. 1d). Untreated cells exhibited negative TUNEL staining. 1 View largeDownload slide Fig. 1. Arsenite induction of yeast cell death showing apoptotic markers. (a) TUNEL reaction (fluorescence and the corresponding bright-field display) after 24 h of treatment of Saccharomyces cerevisiae with 0, 1 or 3 mM arsenite. (b) Annexin V-PI staining of Saccharomyces cerevisiae exposed to 0 or 3 mM arsenite. (c) Relative survival of Saccharomyces cerevisiae in the presence of different concentrations of arsenite. (d) Dose-dependent relationship between yeast cell survival and apoptotic cell response of Saccharomyces cerevisiae determined by TUNEL staining. 1 View largeDownload slide Fig. 1. Arsenite induction of yeast cell death showing apoptotic markers. (a) TUNEL reaction (fluorescence and the corresponding bright-field display) after 24 h of treatment of Saccharomyces cerevisiae with 0, 1 or 3 mM arsenite. (b) Annexin V-PI staining of Saccharomyces cerevisiae exposed to 0 or 3 mM arsenite. (c) Relative survival of Saccharomyces cerevisiae in the presence of different concentrations of arsenite. (d) Dose-dependent relationship between yeast cell survival and apoptotic cell response of Saccharomyces cerevisiae determined by TUNEL staining. ROS are produced during arsenic-induced apoptosis ROS have been reported to regulate some apoptotic processes in yeast (Madeo et al., 1999). To investigate whether oxygen stress plays an important role in arsenic-induced apoptosis, we measured the ROS production of yeast strain BY4742 treated with arsenic by flow cytometry. Dihydrorhodamine 123, which can be oxidized by ROS to become the fluorescent chromophore Rh123, was mixed with yeast cells. Flow cytometric analysis showed that after 12 h of treatment, 24.4% of cells displayed significant ROS production (Fig. 2b). The arsenic-treated cells also showed increased fluorescence after incubation with dihydroethidium (Fig. 2c). We further tested the role of the ROS-scavenging enzyme CuZnSOD (SOD1) in this process. We reasoned that if ROS play a key role in arsenic-induced apoptotic-like cell death, then the deletion of the gene for CuZnSOD may enhance the effect of arsenic. We found that the CuZnSOD-disrupted strain Δ sod1 was, as expected, hypersensitive to arsenic as compared with the wild-type strain (Fig. 2d). 2 View largeDownload slide Fig. 2. Arsenite induction of both ROS production and Δψm reduction. (a) Saccharomyces cerevisiae BY4742 cells were treated or not treated with 3 mM As(III) for 12 h, and Δψm was measured using Rh123 by flow cytometry. (b) ROS accumulation of yeast cells in the absence or presence of arsenic was measured using dihydrorhodamine 123. (c) ROS accumulation of yeast cells in the absence or presence of arsenic was analyzed with FACS after 10 min of incubation with dihydroethidium. (d) Growth of rho0 and Δ sod1 mutants as compared to wild-type cells in the presence of arsenite was monitored by drop assays. 2 View largeDownload slide Fig. 2. Arsenite induction of both ROS production and Δψm reduction. (a) Saccharomyces cerevisiae BY4742 cells were treated or not treated with 3 mM As(III) for 12 h, and Δψm was measured using Rh123 by flow cytometry. (b) ROS accumulation of yeast cells in the absence or presence of arsenic was measured using dihydrorhodamine 123. (c) ROS accumulation of yeast cells in the absence or presence of arsenic was analyzed with FACS after 10 min of incubation with dihydroethidium. (d) Growth of rho0 and Δ sod1 mutants as compared to wild-type cells in the presence of arsenite was monitored by drop assays. Mitochondria are involved in apoptosis induced by arsenic Some studies on mammalian apoptosis reported that the dissipation of Δψm is an early event in the cell death process (Zamzami et al., 1995; Kim et al., 2003). We thus tested whether arsenic-induced apoptosis is also accompanied by Δψm disruption. It was observed that 45.8% of yeast cells lost Δψm when treated with arsenic, in contrast to very little (5.4%) Δψm disruption in wild-type cells (Fig. 2a). The requirement for mitochondrial function in apoptosis induced by arsenic was analyzed by the study of the mutant strain lacking the entire mitochondrial genome (rho0) and its isogenic wild type BY4742. The results showed that rho0 was more resistant to death induced by arsenite, as compared to the wild-type strain (Fig. 2d). Arsenic-induced apoptotic cell death is dependent on Yca1p Yca1p was reported to regulate apoptosis in yeast (Madeo et al., 2002). To investigate whether Yca1p is involved in arsenic-induced cell death, the YCA1-disrupted strain Δ yca1 and its isogenic wild type BY4742 were tested for cell survival after arsenic treatment. The YCA1-disrupted strain showed a 91.3% survival rate, much higher than that of the wild type (50.3%), after arsenic treatment (Fig. 3b). Furthermore, the occurrence of DNA fragmentation determined by TUNEL staining (1.5%) was strongly reduced as compared with the wild type (34.3%) (Fig. 3a). 3 View largeDownload slide Fig. 3. Yeast caspase 1 involvement in apoptotic cell death induced by arsenite. (a) TUNEL reaction of Δ yca1 and its isogenic wild type BY4742 treated with 3 mM arsenite. (b) Relative survival of Δ yca1 and its isogenic wild type BY4742 in the presence of different concentrations of arsenite. 3 View largeDownload slide Fig. 3. Yeast caspase 1 involvement in apoptotic cell death induced by arsenite. (a) TUNEL reaction of Δ yca1 and its isogenic wild type BY4742 treated with 3 mM arsenite. (b) Relative survival of Δ yca1 and its isogenic wild type BY4742 in the presence of different concentrations of arsenite. Discussion Saccharomyces cerevisiae has been successfully used as a model for apoptotic research (Matsuyama et al., 1999; Madeo et al., 2002). It undergoes apoptosis in response to different stimuli, including H2O2, acetic acid, aspirin, and pheromones (Madeo et al., 1999; Ludovico et al., 2001; Severin & Hyman, 2002; Balzan et al., 2004). In the present study, we demonstrated that cell death induced by arsenic in yeast is apoptotic, based on the observation of apoptotic characteristics, including DNA fragmentation, phosphatidylserine exposure, mitochondrial membrane permeabilization, and increased ROS production. ROS are generated during normal processes of mitochondrial oxidative phosphorylation. It has been suggested that mitochondrial ROS play a critical role in yeast and mammalian apoptosis (Hildeman et al., 1999; Madeo et al., 1999). We observed an increase in ROS production in S. cerevisiae BY4742 cells exposed to arsenic. Moreover, we found that deletion of the ROS-scavenging enzyme CuZnSOD enhanced the effect of arsenic, indicating that ROS play important roles in this process. Mitochondria have been shown to serve a central role in mammalian apoptosis (Wang, 2001). Whether mitochondria are necessary for yeast apoptosis is still controversial. Previous studies indicated mitochondrial involvement in yeast apoptosis (Ludovico et al., 2002), whereas others have reported that this process does not necessarily involve mitochondria (Kissova et al., 2000). Our results support the argument for mitochondrial involvement. Absence of the mitochondrial genome in the rho0 strain enhances the cell's resistance to arsenite, indicating that mitochondria are required for that process. Moreover, reduction of mitochondrial membrane potential, being accompanied by increased ROS production in yeast cells treated with arsenic, demonstrates functional mitochondrial alterations during arsenic-induced apoptosis. Similar alterations were also detected in apoptosis induced by arsenic in mammalian cells. Many reports suggest that arsenic-induced apoptosis in malignant mammalian cell lines is associated with a loss of mitochondrial transmembrane potential and ROS production (Chen et al., 1998; Zheng et al., 2004). YCA1 encodes a metacaspase in yeast that undergoes proteolytic processing similar to that undergone by mammalian caspases. It plays an important role in the apoptotic death of yeast under different stress conditions (Madeo et al., 2002; Reiter et al., 2005). Here we show that deletion of YCA1 significantly improved the survival rate in the presence of arsenic. Furthermore, the occurrence of apoptotic marker DNA fragmentation was strongly reduced in comparison with the wild type, suggesting a general role for Yca1p in arsenic-induced apoptosis. Arsenic, a widespread metalloid, is not only a human carcinogen, but is also an effective anticancer agent. Arsenic-induced apoptosis is considered to contribute to its anticancer effect. The present study demonstrates that, as is the case with mammalian cells, the apoptosis induced by arsenic in S. cerevisiae is caspase-dependent involves mitochondria. Therefore, the genetically tractable yeast may prove to be an attractive system with which to unravel apoptotic mechanisms and to identify candidate genes associated with arsenic. Acknowledgements We are grateful to Mrs J. Wang for technical assistance. This work was supported by the Key Project of Chinese Ministry of Education (No. 104232), Trans-Century Training Program Foundation for the Talents by the Ministry of Education, and Tsinghua-Yue-Yuen Medical Sciences Fund. References Balzan R Sapienza K Galea DR Vassallo N Frey H Bannister WH ( 2004) Aspirin commits yeast cells to apoptosis depending on carbon source. Microbiology  150: 109– 115. Google Scholar CrossRef Search ADS PubMed  Chae HJ Ke N Kim HR Chen S Godzik A Dickman M Reed JC ( 2003) Evolutionarily conserved cytoprotection provided by Bax Inhibitor-1 homologs from animals, plants, and yeast. Gene  323: 101– 113. Google Scholar CrossRef Search ADS PubMed  Chen YC Lin-Shiau SY Lin JK ( 1998) Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis. J Cell Physiol  177: 324– 333. Google Scholar CrossRef Search ADS PubMed  Fahrenkrog B Sauder U Aebi U ( 2004) The S. cerevisiae HtrA-like protein Nma111p is a nuclear serine protease that mediates yeast apoptosis. J Cell Sci  117: 115– 126. Google Scholar CrossRef Search ADS PubMed  Hildeman DA Mitchell T Teague TK Henson P Day BJ Kappler J Marrack PC ( 1999) Reactive oxygen species regulate activation-induced T cell apoptosis. Immunity  10: 735– 744. Google Scholar CrossRef Search ADS PubMed  Kim JS He L Lemasters JJ ( 2003) Mitochondrial permeability transition, a common pathway to necrosis and apoptosis. Biochem Biophys Res Commun  304: 463– 470. Google Scholar CrossRef Search ADS PubMed  Kissova I Polcic P Kempna P Zeman I Sabova L Kolarov J ( 2000) The cytotoxic action of Bax on yeast cells does not require mitochondrial ADP/ATP carrier but may be related to its import to the mitochondria. FEBS Lett  471: 113– 118. Google Scholar CrossRef Search ADS PubMed  Laun P Pichova A Madeo F Fuchs J Ellinger A Kohlwein S Dawes I Frohlich K Breitenbach M ( 2001) Aged mother cells of Saccharomyces cerevisiae show markers of oxidative stress and apoptosis. Mol Microbiol  39: 1166– 1173. Google Scholar CrossRef Search ADS PubMed  Ludovico P Rodrigues F Almeida A Silva MT Barrientos A Corte-Real M ( 2002) Cytochrome c release and mitochondria involvement in programmed cell death induced by acetic acid in Saccharomyces cerevisiae. Mol Biol Cell  13: 2598– 2606. Google Scholar CrossRef Search ADS PubMed  Ludovico P Sousa MJ Silva MT Leao C Corte-Real M ( 2001) Saccharomyces cerevisiae commits to a programmed cell death process in response to acetic acid. Microbiology  147: 2409– 2415. Google Scholar CrossRef Search ADS PubMed  Madeo F Fröhlich E Fröhlich KU ( 1997) A yeast mutant showing diagnostic markers of early and late apoptosis. J Cell Biol  139: 729– 734. Google Scholar CrossRef Search ADS PubMed  Madeo F Fröhlich E Ligr M Grey M Sigrist SJ Wolf DH Fröhlich KU ( 1999) Oxygen stress, a regulator of apoptosis in yeast. J Cell Biol  145: 757– 767. Google Scholar CrossRef Search ADS PubMed  Madeo F Herker E Maldener C et al.   . ( 2002) A caspase-related protease regulates apoptosis in yeast. Mol Cell  9: 911– 917. Google Scholar CrossRef Search ADS PubMed  Manon S Chaudhuri B Guerin M ( 1997) Release of cytochrome c and decrease of cytochrome c oxidase in Bax-expressing yeast cells, and prevention of these effects by coexpression of Bcl-xL. FEBS Lett  415: 29– 32. Google Scholar CrossRef Search ADS PubMed  Matsuyama S Nouraini S Reed JC ( 1999) Yeast as a tool for apoptosis research. Curr Opin Microbiol  2: 618– 623. Google Scholar CrossRef Search ADS PubMed  Miller WH Jr Schipper HM Lee JS Singer J Waxman S ( 2002) Mechanisms of action of arsenic trioxide. Cancer Res  62: 3893– 3903. Google Scholar PubMed  Narasimhan ML Damsz B Coca MA Ibeas JI Yun DJ Pardo JM Hasegawa PM Bressan RA ( 2001) A plant defense response effector induces microbial apoptosis. Mol Cell  8: 921– 930. Google Scholar CrossRef Search ADS PubMed  Reiter J Herker E Madeo F Schmitt MJ ( 2005) Viral killer toxins induce caspase-mediated apoptosis in yeast. J Cell Biol  3: 353– 358. Google Scholar CrossRef Search ADS   Samokhvalov VA Museikina NY Mel'nikov GV Ignatov VV ( 2003) Arsenite as an inducer of lipid peroxidation in Saccharomyces cerevisiae cells. Microbiology  72: 266– 269. Google Scholar CrossRef Search ADS   Severin FF Hyman AA ( 2002) Pheromone induces programmed cell death in S. cerevisiae. Curr Biol  12: R233– R235. Google Scholar CrossRef Search ADS PubMed  Shen ZX Chen GQ Ni JH et al.   . ( 1997) Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): II. Clinical efficacy and pharmacokinetics in relapsed patients. Blood . 89: 3354– 3360. Google Scholar PubMed  Wang X ( 2001) The expanding role of mitochondria in apoptosis. Genes Dev  15: 2922– 2933. Google Scholar PubMed  Zamzami N Marchetti P Castedo M Zanin C Vayssiére J-L Petit PX Kroemer G ( 1995) Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med  181: 1661– 1672. Google Scholar CrossRef Search ADS PubMed  Zheng YH Shi Y Tian C Jiang C Jin H Chen J Almasan A Tang H Chen Q ( 2004) Essential role of the voltage-dependent anion channel (VDAC) in mitochondrial permeability transition pore opening and cytochrome c release induced by arsenic trioxide. Oncogene  23: 1239– 1247. Google Scholar CrossRef Search ADS PubMed  © 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
TI - Arsenic induces caspase- and mitochondria-mediated apoptosis in Saccharomyces cerevisiae
JF - FEMS Yeast Research
DO - 10.1111/j.1567-1364.2007.00274.x
DA - 2007-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/arsenic-induces-caspase-and-mitochondria-mediated-apoptosis-in-IPRJBz0IEJ
SP - 860
EP - 865
VL - 7
IS - 6
DP - DeepDyve
ER -