Department of Physiology and Biochemistry, University of Malta, Msida MSD 06, Malta
Correspondence
Rena Balzan
rena.balzan{at}um.edu.mt
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ABSTRACT |
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INTRODUCTION |
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The apoptotic phenotype observed in the cell-cycle mutant cdc48S565G of S. cerevisiae is associated with accumulation of reactive oxygen species (ROS) (Madeo et al., 1999). ROS also accumulate in yeast cells undergoing apoptosis induced by mammalian Bax (Ligr et al., 1998
). Furthermore, apoptosis is induced by growth of a gsh1 deletion mutant in the absence of glutathione and by exposure of yeast cells to low concentrations of H2O2. These findings have led to the conclusion that production of ROS is a necessary and sufficient condition for the induction of apoptosis in yeast (Madeo et al., 1999
).
Aspirin is known to scavenge ROS (Saini et al., 1998), yet it induces apoptosis in cancer cell lines (Elder et al., 1996
; Castano et al., 1999
; Goel et al., 2003
). In the present work, we studied the effect of aspirin on yeast cells with differential protection against ROS, as obtained with wild-type, cytoplasmic copper,zinc superoxide dismutase (CuZnSOD, SOD1)-deficient and mitochondrial manganese superoxide dismutase (MnSOD, SOD2)-deficient strains, and with differential production of ROS as obtained with growth on fermentable and non-fermentable carbon sources. In general, we found that aspirin induced apoptosis in MnSOD-deficient cells growing on ethanol as the non-fermentable carbon source. Our results suggest that generation of ROS may be a relatively late event and not the initial cause of the observed cell death process.
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METHODS |
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Measurement of cell growth and cell viability.
Cell growth was measured as the optical density at 600 nm. Cultures with OD600 values greater than 1·0 were diluted as necessary. The dilutions were in the range 1 in 10 to 1 in 20. Since a viable cell is defined as one that can form a colony on rich YEPD medium, cell viability was measured by plating serial dilutions of the treated and untreated yeast cultures onto YEPD plates to find the number of cells (300) to be plated, as determined by counting colonies (Longo et al., 1996
). The percentage of colony-forming units (c.f.u.) of cells treated with aspirin was obtained by relating the c.f.u. counts of treated cells to those of untreated cells, which were considered to be 100 %.
Detection of apoptosis or necrosis.
Quantification of apoptosis by propidium iodide (PI) staining and fluorescence-activated cell sorting (FACS) analysis, using a Bio-Rad BRYTE HS flowcytometer, was performed as described previously (Sazer & Sherwood, 1990). Annexin-V-Fluorescein (Annexin-V-Fluos Staining Kit; Roche Diagnostics) and PI staining were essentially performed as described previously (Madeo et al., 1997
), with some modifications, and FACS analysis was carried out. Yeast cells (1x107) were washed in sorbitol buffer (1·2 M sorbitol, 0·5 mM MgCl2, 35 mM potassium phosphate, pH 6·8), incubated with Tris/DTT buffer (100 mM Tris/Cl, pH 9·4, 10 mM DTT) for 15 min at 30 °C with gentle shaking to break cysteine bridges, washed again in sorbitol buffer, digested with Zymolase-20T (Seikagu Corporation) [5 mg (g wet weight of cells)-1] in sorbitol buffer for 2 h at 30 °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 100 µl of binding buffer/sorbitol. Annexin-V-Fluos (10 µl) and 10 µl PI (500 µg ml-1) were added and the cells were incubated for 20 min at room temperature. The cells were harvested, suspended in 0·5 ml of binding buffer and analysed on the Bio-Rad BRYTE HS flowcytometer using 488 nm excitation and a 515 nm bandpass filter for fluorescein detection and a filter >560 nm for PI detection.
Measurement of intracellular oxidation level.
Intracellular ROS were detected by using the oxidant-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate (DCDHF-DA) (Molecular Probes). One millilitre of yeast cell culture (107 cells), to which was added 2 µl of DCDHF-DA from a fresh 5 mM stock solution in ethanol, was incubated at 28 °C for 20 min. The cells were then washed twice in sterile distilled water and resuspended in 1 ml of 50 mM Tris/Cl buffer, pH 7·5. Two drops of chloroform and 1 drop of 0·1 % (w/v) SDS were added and the cells were vortexed for 20 s and allowed to stand for 15 min to allow the dye to diffuse into the buffer. The cells were pelleted and the fluorescence of the supernatant was measured using a Bio-Tek Instruments fluorometer with excitation at 490 nm and emission at 518 nm.
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RESULTS |
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The mode of cell death was also investigated in EG110 cells growing in YEPD medium in the presence of 15 mM aspirin. After 24 h, 94 % of these cells had high Annexin and high PI staining, and thus were necrotic (Fig. 4b, right panel). The percentage of aspirin-treated cells showing apoptosis was very low, less than 2 % (Fig. 4a, b
, right panels). As expected, untreated cells showed no characteristics of apoptosis or necrosis (Fig. 4a, b
, left panels). Thus, in the presence of aspirin, MnSOD-deficient cells appear to die through an early necrotic process in the presence of glucose and through a late apoptotic process in the presence of ethanol.
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DISCUSSION |
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In non-fermentable ethanol medium, MnSOD-deficient cells that were not treated with aspirin showed a small amount of late apoptosis (Fig. 3a). This was not unexpected because mitochondrial electron transport in non-fermentatively growing cells is a major source of ROS production (Longo et al., 1996
). Moreover, MnSOD-deficient cells are not protected against ROS generated in the mitochondria. In fact, van Loon et al. (1986)
found a yeast mutant lacking MnSOD to be hypersensitive to 100 % oxygen. Ethanol is expected to increase the mitochondrial production of ROS in yeast cells (Costa et al., 1997
). However, it would appear that the condition of MnSOD deficiency, combined with non-fermentative growth on ethanol, did not induce significant apoptosis unless the cells also were treated with aspirin (Fig. 3
). The possibility that aspirin caused increased generation of ROS was excluded by the lack of effect of the antioxidants N-acetylcysteine and vitamin E on growth or apoptosis. Furthermore, aspirin was found to act as an antioxidant until the appearance of apoptosis, when there was a moderately significant increase in the intracellular oxidation level (Fig. 5
). Generation of ROS as a relatively late event, with the ROS not acting as effectors of cell death but possibly as late signals of the apoptotic process, has been observed in neural cells (Schulz et al., 1996
). In myeloid leukaemia cells, Cai & Jones (1998)
observed that increased cellular oxidation levels occurred in parallel with caspase activation after mitochondrial release of cytochrome c. These findings can be extended, with caution, to yeast cells. The involvement of mitochondria in yeast cell apoptosis is evidenced by a decrease in the membrane potential, dysfunction of the mitochondrial proton pump, and release of cytochrome c to the cytoplasm in cell death mediated by deletion of the histone chaperone ASF1/CIA1 (Yamaki et al., 2001
). These findings have been confirmed in stationary phase yeast cells that are committed to apoptosis by acetic acid (Ludovico et al., 2002
).
The observed antioxidant effect of aspirin does not support a primary role for ROS in the apoptosis of MnSOD-deficient cells growing on ethanol medium. A drastic fall in the viability of these cells, as measured by the ability to form new colonies (Fig. 2a), occurred much earlier than apoptosis when the antioxidant effect of aspirin (Fig. 5
) was at its highest. It would seem that most of the cells were irrevocably committed to death before the actual characteristics typical of apoptosis, such as DNA cleavage, had set in.
Our finding that the presence of MnSOD in S. cerevisiae cells, such as EG103 and EG118, protected the cells from growth inhibition suggests a mitochondrial target for aspirin. Cytoplasmic CuZnSOD is expected to compensate to some extent for the absence of mitochondrial MnSOD in cellular protection against ROS (Longo et al., 1996). However, the presence of CuZnSOD in EG110 cells did not protect the cells from commitment to apoptosis. This throws further doubt on a primary role of ROS in the process. Because of growth inhibition culminating in apoptosis, the question arises as to whether MnSOD-deficient cells were less able to maintain the intracellular level of NADPH needed for reductive biosynthesis, apart from energy requirements, when grown on ethanol than when grown on glycerol or acetate, which appeared to provide full protection against growth inhibition by aspirin. We propose to address this question experimentally in future work.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 18 June 2003;
revised 18 September 2003;
accepted 30 September 2003.
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