Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava, Slovakia1
Department of Biochemistry, Faculty of Sciences, Comenius University, Mlynská dolina CH-I, 84215 Bratislava, Slovakia2
Author for correspondence: Jordan Kolarov. Tel: +421 2 60296 539. Fax: +421 2 60296 452. e-mail: kolarov{at}fns.uniba.sk
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ABSTRACT |
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Keywords: Bcl-2 proteins, oxygen radicals, petite-negative yeast, mitochondrial mutants
Abbreviations: DHR, dihydrorhodamine 123; PI, propidium iodide; ROS, reactive oxygen species; SM, synthetic medium
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INTRODUCTION |
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An intriguing question in the cell-death pathway is the participation of reactive oxygen species (ROS) in the regulation of apoptosis. The observation that Bcl-xL, the antiapoptotic member of the Bcl-2 family of proteins, has an apparent antioxidant function (Hockenbery et al., 1993 ; Kane et al., 1993
) suggested a role of ROS in apoptosis. In some mammalian cells, antioxidants prevented the activation of caspases and cell death, indicating that ROS may act as signal molecules at the start of the death pathway (Maulik et al., 1998
; Tan et al., 1998
). On the other hand, ROS production could be a consequence of mitochondrial damage, or mitochondrial membrane hyperpolarization (Raha & Robinson, 2000
; Aresenijevic et al., 2000
), both observed at the early stage of apoptosis in metazoa (Vander-Heiden et al., 1997
; Green & Reed, 1998
). ROS were shown to accumulate in S. cerevisiae cells in which the apoptotic markers were induced by the defect in cell cycle regulation (cdc48S565G) (Madeo et al., 1997
), Bax expression, oxidative stress (Madeo et al., 1999
), or cell ageing (Laun et al., 2001
). It was suggested that ROS might play a key role in the induction of apoptotic phenotypes and cell death in organisms lacking the major apoptosis regulators, members of the Bcl-2 family of proteins (Madeo et al., 1999
).
In the present work we investigated the effects of Bax and Bcl-xL on ROS formation and cell death of Kluyveromyces lactis. In contrast to S. cerevisiae, which is a petite-positive, facultative anaerobic yeast, K. lactis is a petite-negative, strictly aerobic yeast in which respiration dominates over fermentation. Thus, the energy-yielding mechanism in K. lactis resembles that in mammalian cells, making it a better model for study of apoptosis. In addition, there are specific mutations in MGI genes encoding subunits of mitochondrial F1-ATPase that convert K. lactis into petite-positive yeast (Chen & Clark-Walker, 1993 ; Clark-Walker et al., 2000
). Here we present evidence that the antiapoptotic protein Bcl-xL localizes exclusively to the mitochondria, and prevents the cytotoxic effect of Bax, but does not eliminate the oxidative stress in Bax-expressing cells. We also show that in K. lactis, the cytotoxic effect of Bax, as well as the protective effect of Bcl-xL, does not require functional mitochondrial oxidative phosphorylation.
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METHODS |
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A DNA fragment (EcoRIHindIII) containing the murine Bax gene under the GAL10 promoter from the YEp51-Bax plasmid (provided by J. C. Reed, The Burnham Institute, La Jolla, CA, USA) was cloned in centromere K. lactis shuttle plasmid pCXJ19 (provided by M. Wesolowski-Louvel, University of Lyon, France). A HindIII fragment containing the URA3 gene from the YEp24 plasmid was further inserted into the HindIII site of the above construct. The murine Bcl-xL gene was cloned under the GAL1 promoter in the pCXJ19K vector. The latter was prepared by cloning the PstISmaI fragment containing the LYS2 gene of S. cerevisiae from the YDp-K vector (Berben et al., 1991 ). K. lactis cells were grown at 28 °C in complete medium containing 0·5% (w/v) Bacto yeast extract, 1% (w/v) Bacto-peptone, and 2% (w/v) glucose or 2% (w/v) galactose. Cells were transformed either by electroporation (Sanchez et al., 1993
) or using frozen competent cells (Dohmen et al., 1991
). The transformants were maintained in synthetic medium (SM: 0·67%, w/v, yeast nitrogen base) supplemented with indicated carbon source and nutritional requirements. To induce Bax and Bcl-xL expression, the transformed cells grown to the early exponential phase in SM containing 2% glucose were washed and resuspended in SM containing 2% galactose. In the case of
0 cells, the inducing medium contained 2% galactose plus 1% glucose.
Plating efficiency and viability assay.
Fresh colonies of transformed K. lactis cells were resuspended in sterile water and equal numbers (300500) of cells were plated in parallel on glucose- and galactose-containing SM plates. After 57 days the colonies were counted and the number of colonies on glucose plates was taken as 100%. The number of cells with compromised cell membrane permeability was determined after staining with 30 µM propidium iodide (PI) using an Olympus BX-50 fluorescent microscope equipped with the corresponding filter.
Determination of intracellular ROS and SH groups.
ROS were detected after incubating 106107 cells in 500 µl growth medium containing 50 µM dihydrorhodamine 123 (DHR, Molecular Probes) for 15 min at room temperature. The samples were viewed with a fluorescent microscope equipped with a rhodamine optical filter.
SH groups in glutathione and soluble cellular proteins were assayed using 2,2-dithiobisnitrobenzoic acid (Hu et al., 1994 ). Cells grown under inducing conditions were harvested, washed, resuspended in 0·25 M Tris/HCl, 0·02 M EDTA, pH 8·2, and broken by vortexing with glass beads. Homogenates were centrifuged at 4000 g for 5 min and the supernatant fractions were used to determine the SH groups and protein content. Total SH groups were calculated and normalized for protein content.
Western blots.
Transformed cells grown under inducing conditions were harvested, washed and resuspended in 0·6 M sorbitol, 0·05 M EDTA, 0·05 M Tris/HCl, pH 7·5, supplemented with a cocktail of protein inhibitors (0·025 mM N--p-tosyl-L-lysine chloromethane, 0·5 mM N-tosyl-L-phenylalanine chloromethane, 1 mM phenylmethylsulfonyl fluoride, 5 mM aprotinin; Sigma). Cells were broken by vortexing with glass beads, the homogenates were centrifuged at 4000 g for 5 min and supernatants were collected. The supernatants were centrifuged at 10000 g for 10 min to obtain a pellet fraction enriched in mitochondria and the supernatant fractions representing the cytosol. TCA-precipitated proteins from both the cytosol and mitochondrial fractions were solubilized in electrophoresis sample buffer and analysed by immunoblotting using Bcl-xL (Santa Cruz Biotechnology) or ADP/ATP carrier antibodies (provided by I. Hapala, Institute of Animal Biochemistry and Genetics, Slovakia) and ECL-based detection.
Reproducibility of the results.
All experiments were repeated at least three times. Data reporting plating efficiency and viability of cells are means values with standard deviations. The data for SH content are reported as a percentage of the SH value in cells transformed with control plasmids.
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RESULTS |
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To determine whether antioxidants can have a protective effect on Bax-triggered inhibition of growth and viability of K. lactis cells, glutathione was included in the induction medium. Within the concentration range 125 mM glutathione we did not observe a protective effect either on the plating efficiency or on the viability of Bax-expressing wild-type and mutant cells (data not shown).
The observed effects of Bcl-2 proteins on ROS formation in K. lactis cells were further confirmed by assessing another indicator of oxidative stress, the intracellular pool of SH groups. As shown in Fig. 5(a), H2O2 and antimycin A, which are well-known inductors of ROS formation in many different cell types, decreased the intracellular SH content in K. lactis to different extents depending on the time of exposure. Bax expression in wild-type and ATPase (mgi1-1) mutant cells had a similar effect to H2O2 and antimycin A, resulting in a decrease of the intracellular pool of SH groups as compared to the cells transformed with control plasmids (Fig. 5b
). In agreement with the results obtained by staining, the oxidative stress induced by Bax was stronger in the mgi1-1 mutant than in the wild-type, and more importantly, it was not suppressed by Bcl-xL. The cellular SH content in the mitochondrial respiratory-deficient
0 mutant remained unchanged after expression of Bax alone, or of Bax and Bcl-xL together (Fig. 5b
). It should be noted that the expression of Bcl-xL alone had no effect either on DHR fluorescence, or on the intracellular SH content, in any of the tested strains (data not shown).
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DISCUSSION |
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We expressed the murine Bax and Bcl-xL genes in the parental wild-type K. lactis strain, in the mgi1-1 mutant with a defect in the ß subunit of F1-ATPase, and in the corresponding 0 mutant, and measured the growth and viability of transformed cells. As demonstrated by plating and PI exclusion assays, K. lactis cells responded to Bax expression by growth arrest and cell death, similar to the response in S. cerevisiae. Bcl-xL, when co-expressed with Bax, localizes in mitochondria and almost completely prevents the cytotoxic effect of Bax in K. lactis. In contrast to results pointing to reduced Bax cytotoxicity in cells with nonfunctional mitochondrial oxidative phosphorylation (Harris et al., 2000
; Gross et al., 2000
; Matsuyama et al., 1998
; Priault et al., 1999
), the mitochondrial mutants of K. lactis were even more sensitive to Bax than the corresponding wild-type strain. Therefore, our results suggest that the cytotoxic effect of Bax, as well as the preventive action of Bcl-xL, do not require functional mitochondrial oxidative phosphorylation. One possible explanation for the discrepancy between S. cerevisiae and K. lactis could relate to the fact that the mitochondrial mutants of S. cerevisiae have a reduced ability to metabolize and to grow on galactose (Donnini et al., 1992
; Ki
ova et al., 2000
), whereas K. lactis mitochondrial mutants do not grow on galactose at all. As mentioned above, K. lactis
0 cells lacked glucose repression, as evidenced by the ability of galactose to induce ß-galactosidase activity in the presence of glucose. If we take into account that Bax protein is targeted to the yeast mitochondria (Priault et al., 1999
; Gross et al., 2000
), the high sensitivity of K. lactis
0 mutant to Bax could be due to an additive mitotoxic effect (Skulachev et al., 2000
) in this petite-negative species. The higher Bax cytotoxicity in
0 cells as compared to the corresponding parental strain might be also attributed to the presence of glucose in the inducing medium. However, the undisturbed growth of
0 cells transformed with either control or Bax+Bcl-xL-containing plasmids indicates that this is not the case.
As seen in mammalian cells, Bcl-xL, when expressed in K. lactis, was largely localized to the mitochondria and efficiently prevented Bax-induced growth arrest and PI staining. However, Bcl-xL did not suppress ROS formation in these cells, indicating that its protective effect does not necessarily include an antioxidant action, or activation of antioxidant defence. Bcl-xL also protected 0 cells from Bax-induced growth arrest and death. However, in contrast to the wild-type only a few
0 cells simultaneously expressing Bax and Bcl-xL were stained by DHR. In addition, the intracellular SH contents of
0 cells did not show any significant change after Bax and/or Bcl-xL expression. Although a limited ROS generation in mammalian
0 cells was reported (Jacobson et al., 1993
; Shimizu et al., 1995
), so far we have no explanation for the observation that the K. lactis
0 cells, regarding ROS formation and the cellular SH content, responded differently from the respiratory-competent cells as regards the expression of Bcl-xL.
ROS are considered to be a potent trigger of apoptosis in some experimental systems (Liu et al., 1996 ; Maulik et al., 1998
; Tan et al., 1998
) and it was suggested that Bcl-xL plays an antioxidative role, possibly by protecting the cellular constituents from oxidative damage (Hockenbery et al., 1993
; Longo et al., 1997
). The apoptotic phenotypes of S. cerevisiae induced by different factors, including Bax, are also accompanied by formation of ROS within the cells (Madeo et al., 1999
; Laun et al., 2001
). Our finding that the cytotoxic effect of Bax in K. lactis, but not Bax-induced ROS formation, is inhibited by Bcl-xL indicates that the ROS are not in the main pathway of Bax-induced death of K. lactis cells and that the antiapoptotic effect of Bcl-xL protein includes, besides the antioxidative properties, also other mitochondrially related mechanism(s).
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ACKNOWLEDGEMENTS |
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Received 8 March 2002;
revised 7 May 2002;
accepted 14 May 2002.
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