The antiapoptotic protein Bcl-xL prevents the cytotoxic effect of Bax, but not Bax-induced formation of reactive oxygen species, in Kluyveromyces lactis

Daniela Poliaková1, Barbora Sokolíková1, Jordan Kolarov1,2 and L’udmila Sabova1

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


   ABSTRACT
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INTRODUCTION
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The murine proapoptotic protein Bax was expressed in Kluyveromyces lactis to investigate its effect on cell survival and production of reactive oxygen species (ROS). Bax expression decreased the number of cells capable of growing and forming colonies, and it increased the number of cells producing ROS, as detected by both dihydrorhodamine 123 fluorescence and the intracellular content of SH groups. Mutation in the ß-subunit of F1-ATPase, or mitochondrial deficiency resulting from deletion of mtDNA ({rho}0 mutant), increased the sensitivity to Bax, indicating that Bax cytotoxicity does not require mitochondrial respiratory-chain functions. The antiapoptotic protein Bcl-xL, when co-expressed with Bax, localized to the mitochondria and prevented Bax cytotoxicity. However, this co-expression did not prevent the production of ROS. These data suggest that in K. lactis cells expressing Bax, ROS are not the sine qua non of cell death and that the antiapoptotic function of Bcl-xL is not limited to its antioxidant property.

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


   INTRODUCTION
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe, were used to demonstrate that characteristics of apoptotic cells, found in multicellular organisms, are present in unicellular eukaryotes (Sato et al., 1994 ; Hanada et al., 1995 ; Ink et al., 1997 ). In S. cerevisiae the appearance of specific apoptotic markers, such as DNA breakage, chromatin condensation, loss of asymmetric distribution of plasma-membrane phosphatidylserine, and growth arrest preceding cell death, was observed after exposure to oxidative stress (Madeo et al., 1999 ), in cells with a defective cell cycle (Madeo et al., 1997 ) and in old mother cells (Laun et al., 2001 ), as well as in cells expressing the proapoptotic members of the Bcl-2 family (Bax, Bak) (Ligr et al., 1998 ; Madeo et al., 1999 ). The onset of apoptosis induced by the latter two proteins was successfully prevented by co-expression of the antiapoptotic members of the Bcl-2 family (Bcl-2, Bcl-xL), suggesting that both types of mammalian proteins function in yeast similarly as in metazoan cells (for a review see Matsuyama et al., 1999 ). Experimental data indicate that the cell-death-regulating activity of the Bcl-2 family members in mammalian cells depends on their ability to localize into mitochondrial membranes and to influence their functions. Bax expressed in yeast cells also localizes to mitochondria (Priault et al., 1999 ; Gross et al., 2000 ). Its expression induces mitochondrial dysfunctions (Manon et al., 1997 ; Kiova et al., 2000 ), probably due to the formation of a channel in the outer mitochondrial membrane (Pavlov et al., 2001 ). There are contradictory results in both mammalian and yeast cells as to what extent, if at all, the mitochondrial functions are required for apoptosis. It was shown, for instance, that human cells lacking mtDNA ({rho}0 cells) were resistant to in vitro induction of apoptosis by staurosporin (Dey & Moraes, 2000 ), suggesting that respiratory-chain function is required for apoptosis. Conversely, increased in vivo apoptosis in cells lacking mtDNA expression was recently observed (Wang et al., 2001 ). Furthermore, S. cerevisiae mutants deficient in oxidative phosphorylation functions (Matsuyama et al., 1998 ; Marzo et al., 1998 ; Harris et al., 2000 ), or wild-type cells grown under fermentative conditions (Priault et al., 1999 ), showed reduced Bax-induced toxicity and cell death (Gross et al., 2000 ). On the other hand, it was demonstrated (Kiova et al., 2000 ) that the wild-type cells, as well as the mutants with defective oxidative phosphorylation due to mutations in either nuclear DNA, or mtDNA, needed 2–3 cell divisions in the presence of Bax before the growth arrest and cell death occurred.

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.


   METHODS
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INTRODUCTION
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Yeast strains, plasmids and growth conditions.
The parental wild-type K. lactis strain CK56-16C ({alpha} ade1 lysA1 uraA1) and the strain with a mutation in the ß subunit of F1-ATPase, CK56-7A ({alpha} ade1 lysA1 uraA1 mgi1-1), were kindly provided by X. J. Chen (Australian National University, Canberra). Cells lacking mitochondrial DNA ({rho}0) were prepared by ethidium bromide mutagenesis from the mgi1-1 mutant strain as described by Chen & Clark-Walker (1993) .

A DNA fragment (EcoRI–HindIII) 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 PstI–SmaI 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 {rho}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 (300–500) of cells were plated in parallel on glucose- and galactose-containing SM plates. After 5–7 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 106–107 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-{alpha}-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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Expression of Bax and Bcl-xL in K. lactis
Expression of mammalian Bcl-2 proteins in mitochondrial mutants of S. cerevisiae has resulted in a number of conflicting reports with regard to the mitochondrial functions required for their cytotoxic effect. We have shown that the reduced growth of cells with impaired mitochondrial functions on galactose is, at least in part, responsible for the reduced cytotoxicity of Bax in these cells (Kiova et al., 2000 ). To avoid the undesirable effect of carbon source on the growth of mitochondrial mutants, we expressed Bax and Bcl-xL in K. lactis. An important advantage of this yeast is that the mitochondrial respiratory-deficient mutant ({rho}0) metabolizes galactose in the presence of glucose (Fig. 1). The evident lack of glucose repression thus allows an efficient induction of genes under the control of GAL1–GAL10 promoters under respiratory-deficient conditions. This is not the case for S. cerevisiae, where most respiratory-deficient mutants grow poorly on galactose (Donnini et al., 1992 ). As shown in Fig. 2, the wild-type and the mutant strains transformed with the control plasmids formed colonies with similar efficiency on both, glucose and galactose. Expression of Bax resulted in a reduction of plating efficiency of wild-type and mgi1-1 mutant cells (to 60% and 20%, respectively). The strongest effect of Bax expression was observed in the mitochondrial respiratory-deficient ({rho}0) mutant. The plating efficiency of these cells was almost completely inhibited by Bax expression (Fig. 2). Similarly as in S. cerevisiae (Hanada et al., 1995 ), co-expression of the antiapoptotic protein Bcl-xL reversed Bax-induced growth arrest in all three K. lactis strains. To determine the viability of cells expressing Bax and Bcl-xL, we stained the cells with PI (see Fig. 4 and Table 1). About 95% of the Bax-expressing respiratory-deficient ({rho}0) cells grown for 24 h in the inducing medium containing 2% galactose plus 1% glucose were stained with PI. The number of PI-stained cells was lower (62%) in the mgi1-1 mutant as well as in the wild-type (see below, Table 1). Co-expression of Bcl-xL prevented the Bax-induced PI staining in all three strains, suggesting that preventive effect of Bcl-xL, like the toxic effect of Bax, did not require functional oxidative phosphorylation.



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Fig. 1. Induction of ß-galactosidase activity in K. lactis mgi1-1 and {rho}0 mutant cells. (a) The mgi1-1 mutant strain [similar results were obtained with the parental (CK56-16C) strain (data not shown)] and (b) the corresponding mitochondrial ({rho}0) mutant, grown in SM with 2% glucose, were washed and transferred to fresh SM containing 2% glucose ({blacksquare}), 2% galactose plus 1% glucose ({blacktriangleup}), or 2% galactose ({bullet}) as carbon source. Aliquots were withdrawn at the times indicated and the ß-galactosidase activity was assayed in crude cell extracts as described by Guarente (1983) .

 


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Fig. 2. Plating efficiency of wild-type and mutant K. lactis cells expressing Bcl-2 proteins. Equal numbers of wild-type, mgi1-1 and {rho}0 mutant cells transformed with two control plasmids (white bars), Bax-containing (black bars), or Bax and Bcl-xL containing plasmids (hatched bars) were plated on solid SM containing 2% glucose or 2% galactose. In the case of the {rho}0 mutant the galactose medium was supplemented with 1% glucose. The numbers of colonies were scored after 7 days growth at 28  °C. The number of cells able to form colonies under inducing (galactose, or galactose plus glucose for {rho}0 cells) conditions is expressed as a percentage of the number of cells able to form colonies under non-inducing (glucose) conditions. The error bars represent standard deviations.

 


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Fig. 4. ROS accumulation in K. lactis cells expressing Bcl-2 proteins. Wild-type (a), mgi1-1 (b) and {rho}0 (c) cells were grown under inducing conditions for 6 h. Cells transformed with control plasmids (1), Bax- and Bcl-xL-containing plasmids (2), or control and Bax-containing plasmids (3) were grown under inducing conditions. Aliquots were withdrawn, stained with either DHR or PI, and analysed by fluorescence microscopy using an appropriate filter. Data for DHR staining of Bax-expressing cells are not shown due to the non-specific staining of dead cells. VIS, examined under visible light.

 

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Table 1. Viability and ROS production, determined by PI and DHR staining, of cells expressing Bax and co-expressing Bax+Bcl-xL after 24 h growth under inducing conditions

 
Bcl-xL protein localizes to the mitochondria of K. lactis
It has recently been shown that Bax protein expressed in S. cerevisiae is localized in mitochondria (Priault et al., 1999 ; Gross et al., 2000 ) and influences the mitochondrial metabolism (Manon et al., 1997 ; Kiova et al., 2000 ). We examined whether Bcl-xL was localized in the same organelles. The Western-immunoblotting analysis of crude mitochondrial and cytosol fractions of Bcl-xL-expressing K. lactis cells demonstrated that Bcl-xL protein was localized in the mitochondrial fraction, as soon as 6 h after induction in galactose medium and was still present there 48 h after induction (Fig. 3).



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Fig. 3. Western blots demonstrating that Bcl-xL is localized in the mitochondrial fractions. Wild-type cells co-transformed with Bax- and Bcl-xL-containing plasmids were grown in liquid SM with 2% galactose and at the times indicated aliquots of cells were removed, homogenized and fractioned to yield the cytosolic (C) and mitochondrial (M) fractions. The proteins of both fractions were analysed by Western blotting using (a) Bcl-xL antibody and (b) antibody against mitochondrial ADP/ATP carrier (AAC).

 
Expression of Bax leads to accumulation of ROS and a decrease of intracellular SH content
To follow Bax-induced intracellular accumulation of ROS, we stained the Bax-transformed cells with DHR, which is oxidized to the fluorescent rhodamine 123 by peroxides. As shown in Fig. 4(a, b) and Table 1, most of the wild-type and ATPase (mgi1-1) mutant cells simultaneously expressing Bax and Bcl-xL were stained with DHR, but not with PI, indicating that Bcl-xL suppressed cell death though it did not suppress ROS formation in these cells. DHR also stained similar proportions of Bax-expressing wild-type and ATPase (mgi1-1) mutant cells (not shown), but it should be taken into account that dead cells became non-specifically stained with DHR and contributed to the apparent numbers of DHR-positive cells. Nevertheless, the percentages of positive cells in this case were still no greater than with cells expressing Bax+Bcl-xL, consistent with Bcl-xL not eliminating the oxidative stress in these cells. In contrast to the above two respiratory-competent strains, the respiratory-deficient ({rho}0) cells protected from Bax-induced death by expression of Bcl-xL were scarcely stained by either PI or DHR (Fig. 4c, Table 1).

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 1–25 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 {rho}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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Fig. 5. Oxidative stress and Bax expression affect the intracellular content of SH groups. (a) Wild-type cells grown in SM with 2% galactose were treated with either 5 mM H2O2 (white bars) or 7·5 µM antimycin A (black bars). At the indicated times aliquots of cells were removed, homogenized, and the content of SH groups was determined. The intracellular SH contents are shown as a percentage of the SH content of untreated control cells. (b) Cells of the indicated genotypes co-transformed either with control plus Bax-encoding plasmids (white bars), or with Bax- plus Bcl-xL-encoding plasmids (black bars) were grown to the exponential phase in liquid SM containing glucose. The cells were then transferred to galactose-containing medium and the intracellular SH content was determined after 6 h. The values are reported as a percentage of the SH content in cells growing under non-inducing conditions. [The absolute values for SH content corresponding to 100% were: 0·28±0·008 mmol (mg protein)-1 for wild-type, 0·20±0·009 mmol (mg protein)-1 for the mgi1-1 strain and 0·18±0·004 mmol (mg protein)-1 for the {rho}0 strain]. Error bars represent standard deviations.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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In this work we investigated the response of K. lactis cells to two proteins, Bax and Bcl-xL, that are regulators of apoptosis in metazoa. K. lactis possess several advantages for investigation of the role of Bcl-2 proteins in apoptosis. Like most mammalian cells, K. lactis are strictly aerobic, with an intensive mitochondrial oxidative metabolism. There are mutant strains of K. lactis (Clark-Walker et al., 2000 ), including the mgi1-1 mutant used in this work, in which the mtDNA can be deleted to produce mitochondrial respiratory-deficient ({rho}0) mutants. It is also important that the low glucose repression in the {rho}0 mutant (Fig. 1) allowed the cells to metabolize galactose in the presence of glucose; therefore GAL promoters can be efficiently used for gene expression under respiratory-deficient conditions.

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 {rho}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 ; Kiova et al., 2000 ), whereas K. lactis mitochondrial mutants do not grow on galactose at all. As mentioned above, K. lactis {rho}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 {rho}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 {rho}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 {rho}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 {rho}0 cells from Bax-induced growth arrest and death. However, in contrast to the wild-type only a few {rho}0 cells simultaneously expressing Bax and Bcl-xL were stained by DHR. In addition, the intracellular SH contents of {rho}0 cells did not show any significant change after Bax and/or Bcl-xL expression. Although a limited ROS generation in mammalian {rho}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 {rho}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).


   ACKNOWLEDGEMENTS
 
We are grateful to Dr K. Luciakova for critical reading of the manuscript and to G. Huskova for technical assistance. This work was supported by Howard Hughes Medical Institute grant no. 75195-547301 and by grant no. 7285 from the Slovak grant agency (VEGA).


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RESULTS
DISCUSSION
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Received 8 March 2002; revised 7 May 2002; accepted 14 May 2002.



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