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Molecular Cancer Research 1:186-194 (2003)
© 2003 American Association for Cancer Research

Cell Cycle, Cell Death, and Senescence

Expression of Bcl-xS in Xenopus Oocytes Induces BH3-Dependent and Caspase-Dependent Cytochrome c Release and Apoptosis1

Tali Braun1, Shachar Dar1, Dmitry Vorobiov2, Liora Lindenboim1, Nathan Dascal2 and Reuven Stein1

1 Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences and 2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel

Requests for reprints: Reuven Stein, Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, 69978 Tel-Aviv, Israel. Phone: 972-3-640-8608; Fax: 972-3-640-7643. E-mail: reuvens{at}post.tau.ac.il


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The mechanism of action of pro-apoptotic proteins is difficult to study in vivo because of their death effect, which makes it problematic to obtain sufficient homogeneous experimental material for biochemical analysis. We show here that pro-apoptotic genes expressed in Xenopus oocytes constitute a useful in vivo system for studying their mechanism of action. In the present study, we used this system to study the death effects of Bcl-xS, a pro-apoptotic member of the Bcl-2 family. The results showed that expression of Bcl-xS in oocytes induces oocyte death by a caspase-dependent mechanism, which includes BH3-dependent cytochrome c release and is inhibited by co-expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. The release of cytochrome c was found to be dependent on caspase activity. Bcl-xS was localized mainly in the mitochondria, and Bcl-xS transmembrane and BH3 domains were required for its apoptotic effect. These findings suggest that Bcl-xS induces apoptosis in Xenopus oocytes mainly by its presence in the mitochondria, where it induces BH3- and caspase-dependent release of cytochrome c, which leads to oocyte death.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The Bcl-2 family of proteins comprises both anti- and pro-apoptotic members and plays an important role in regulating caspase activation (for reviews, see Refs. 1–3). The homology between members of the Bcl-2 family resides mainly within four conserved domains, designated Bcl-2 homology (BH)3 domains BH1, BH2, BH3, and BH4. All four BH domains are present in some Bcl-2 family members, such as Bcl-2 and Bcl-xL. Others (such as Bax) contain the BH1, BH2, and BH3 domains and lack BH4, whereas others (such as Bid) contain only the BH3 domain (4).

The bcl-xL mRNA encodes a 233-amino-acid protein containing the four BH domains and a transmembrane (TM) region. Bcl-xS, however, as a result of alternative splicing, lacks an internal 63-amino-acid segment that contains the conserved BH1 and BH2 domains. It therefore contains only the BH3 and BH4 domains and the TM region. Previous studies have suggested that Bcl-xS is pro-apoptotic, but its mechanism of action is not completely understood. In stable transfectants, Bcl-xS inhibits the ability of Bcl-2 or Bcl-xL to protect the cells from apoptosis induced by different stimuli (5, 6). In transiently transfected PC12 cells, Bcl-xS induces apoptosis in a caspase-dependent manner (7), whereas in HEK293 cells, it does not induce cell death (8). In addition, previous studies suggested that expression of Bcl-xS in 3T3 cells induces cell death in a caspase-independent manner (9) without cytochrome c release, due to a Bcl-xS-induced depletion of cytochrome c (10). It is not clear, however, how general these Bcl-xS effects are.

An increasing body of evidence suggests that Bcl-xS may be involved in many apoptotic processes. For example, Bcl-xS expression is increased in some apoptotic systems, such as transient forebrain global ischemia (11, 12), apoptosis in involuting mammary epithelial cells (13), apoptosis during hypertensive nephrosclerosis (14), and apoptosis induced by actinomycin D in human gastric cancer TMK-1 cells (15) and in injured carotid arteries (16). Furthermore, infection with an adenoviral vector containing Bcl-xS cDNA specifically induces apoptosis in several cancer cells, suggesting that the targeting of Bcl-xS expression on cancer cells may be a useful therapeutic approach for the specific killing of these cells (17–21).

A major problem in studying the mechanism of action of pro-apoptotic proteins is that it is difficult, because of their death effect, to obtain sufficient quantities of homogeneous experimental material for biochemical analysis. In investigating the function of genes of interest, a common method is to express them, either stably or transiently, in cultured cells. However, genes that encode death-inducing proteins are incompatible with the establishment of stable overexpressing cell lines. Even if such a cell line is established, it is likely that it underwent prior selection against the death activity of the transfected gene. Inducible cell lines may be used to overcome these problems, but they are difficult to establish because in most cases, there is some leakiness of the expressed gene even without induction. Thus, the approach commonly used to investigate the mechanism of action of pro-apoptotic genes in cultured cells is transient transfection (22). Because the transfection efficiency in most existing cell lines is relatively low, however, the experimental material usually contains a substantial background of untransfected cells.

Studies have shown that the biochemical events of mammalian apoptosis can be recapitulated in a cell-free system derived from eggs of the frog Xenopus laevis (23–29) and that microinjection of cytochrome c into X. laevis oocytes induces their death and reduces their membrane potential (30), suggesting that the Xenopus apoptotic machinery has similar properties to those of its mammalian counterpart.

In an attempt to study in vivo the mechanism of action of the pro-apoptotic gene Bcl-xS, and to overcome the abovementioned difficulties, we used an experimental system in which the biochemical features of Bcl-xS are studied in X. laevis oocytes that received injections of Bcl-xS cRNA. The advantage of this system is that the gene is expressed by all the oocytes that received injections, and thus sufficient material for biochemical analysis can be easily obtained in one experiment. The expression of Bcl-xS in Xenopus oocytes was found to induce death by an apoptotic process. The cell death was found to be mediated by a caspase-dependent mechanism, which included BH3-dependent cytochrome c release and was inhibited by co-expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Interestingly, the release of cytochrome c was found to be dependent on caspase activity, suggesting that caspases of the oocytes play an important role in this release. In addition, our findings show that expression of pro-apoptotic genes in Xenopus oocytes provides a very useful in vivo system for studying the mechanism of action of death genes.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Expression of Bcl-xS in Xenopus Oocytes Induces Their Death
Stage 6 Xenopus oocytes received injections of increasing concentrations of Bcl-xS cRNA. The oocytes that received injections expressed Bcl-xS proteins in a dose-dependent (Fig. 1A) manner and exhibited the typical morphological changes characteristic of dead Xenopus oocytes (Fig. 1B). These include the appearance of irregular mottled patterns on their cell surface and the blurring of the sharp border between the animal (dark or pigmented) and the vegetal (white) pole (Fig. 1B). We did not observe these morphological changes after injecting the oocytes with equivalent volumes of distilled water (Fig. 1B) or with 0.5 ng cRNA of the G protein-gated inward rectifier K+ channel (data not shown).



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  		FIGURE 1. Bcl-xS expression in Xenopus oocytes induces their death. Groups of 25 stage 6 Xenopus oocytes received injections of the indicated concentrations of Bcl-xS cRNA. After 24 h, the oocytes were collected, mechanically disrupted, and crude extracts were prepared from the different treatments as described in "Materials and Methods." A. Expression of Bcl-xS protein in the extracts was determined by SDS-PAGE followed by immunoblotting using rabbit polyclonal anti-Bcl-x antibody. The morphology of oocytes that received injections of Bcl-xS cRNA (1 ng) or double-distilled water (Control) was observed under a binocular microscope. B. Representative images of control and Bcl-xS-injected oocytes taken 24 h after injection.

 
In an attempt to confirm that the morphology exhibited by Bcl-xS cRNA-injected oocytes indeed represents dead oocytes, we recorded the membrane potential of these oocytes to assess the integrity of their plasma membrane. Oocytes that received injections of Bcl-xS cRNA showed a substantial depolarization of their membrane potential [from -39.7 ± 9 mV (n=49) to -6.9 ± 7 mV (n=38)] (P < 0.01), indicating damage to their membranes and confirming that oocytes exhibiting the characteristic morphology of dying oocytes are indeed not viable and that morphological assessment is a reliable method of determining oocyte viability. Therefore, in the following experiments, assessment of oocyte morphology was used as a viability assay.

To further characterize the effect of Bcl-xS on the viability of the oocytes that received injections, we injected increasing concentrations of Bcl-xS cRNA into the oocytes and determined their viability after different periods of time. Bcl-xS induced death of these oocytes in a dose-dependent and time-course-dependent manner (Fig. 2). However, both the dose response and the time course of this Bcl-xS effect varied between different experiments, and different batches of oocytes varied in their susceptibility to Bcl-xS. Thus, some batches were very sensitive and died rapidly, whereas others were less sensitive and took longer to die. In all cases, however, the oocytes died in a dose-dependent and time-course-dependent manner, whereas the control oocytes did not die.



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  		FIGURE 2. Bcl-xS expression in Xenopus oocytes induces dose-dependent and time-course-dependent cell death. Groups of Stage 6 Xenopus oocytes received injections of double-distilled water (con) or the indicated concentrations of Bcl-xS cRNA and then incubated for 12, 24, 36, 48, or 60 h. At each time point, the viability of oocytes in each group was determined microscopically by their morphology as described in Fig. 1. The data shown are from a representative experiment (one of four independent experiments with similar results).

 
Mechanism of Bcl-xS-Induced Cell Death
In an attempt to unravel the mechanism whereby Bcl-xS expression induces oocyte death, we first examined the subcellular distribution of Bcl-xS. Oocytes were fractionated into cytosolic and membranous fractions by differential centrifugation. This procedure yields a membranous fraction that is highly enriched with mitochondria, as indicated by the presence of the mitochondrial protein porin in this fraction but not in the cytosolic fraction (data not shown). Examination of the presence of Bcl-xS in the cytosolic and membranous fractions and the percentage of oocyte death at different times revealed that soon after injection of Bcl-xS cRNA (after 24 h in the case of the experiments presented in Fig. 3), Bcl-xS protein was present mainly in the cytosolic fraction, where it caused a relatively low level of oocyte death (35%). After a longer period (42 h for the experiments presented in Fig. 3), Bcl-xS protein was found mainly in the membranous fraction, where it caused massive oocyte death (84%). These results thus suggest that following its synthesis, cytosolic Bcl-xS translocates to the mitochondria, and that this translocation correlates with the intensity of its death-inducing effect. Different experiments differed in the time taken for Bcl-xS to be located mainly in the membranous fraction, but in most of these experiments, the percentage of oocyte death correlated with the amount of Bcl-xS protein present in the membranous fraction.



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  		FIGURE 3. Bcl-xS is translocated to the membranous fraction. Stage 6 Xenopus oocytes received injections of 1 ng of Bcl-xS cRNA. At the indicated time points, groups of 25 oocytes were collected and the viability of the oocytes in each group was determined by their morphology as described in Fig. 1. The oocytes were then mechanically disrupted and membranous (MF) and cytosolic (Cyt) fractions were prepared as described in "Materials and Methods." Control (Con) is composed of oocytes that did not receive injections. Equal volumes (each representing three oocytes) of these fractions were subjected to SDS-PAGE and immunoblotting as described in Fig. 1. Percentage survival denotes the number of oocytes with viable morphology expressed as a percentage of the total number of oocytes in each treatment. The data shown are from a representative experiment (one of five independent experiments with similar results).

 
Previous studies have shown that the anti-apoptotic members of the Bcl-2 family, Bcl-2 and Bcl-xL, inhibit Bcl-xS-induced apoptosis in PC12 cells (7), whereas in SKL cells, Bcl-xS-induced apoptosis is inhibited only by Bcl-xL (31). It was therefore of interest to examine whether these anti-apoptotic proteins would inhibit Bcl-xS-induced oocyte death. Bcl-2 and Bcl-xL cRNAs were co-injected with Bcl-xS cRNA and the viability of the oocytes was determined. Both Bcl-2 and Bcl-xL were highly effective in preventing the death of Bcl-xS-induced cell death (Fig. 4).



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  		FIGURE 4. Bcl-2 and Bcl-xL inhibit oocyte death induced by Bcl-xS expression. Groups of 25 stage 6 Xenopus oocytes received injections of 1 ng of Bcl-xS cRNA alone or together with 1 ng Bcl-xL cRNA or 1 ng of Bcl-2 cRNA. Oocyte viability at each of the indicated time points was determined by their morphology as described in Fig. 1. The results are from a representative experiment (one of three independent experiments with similar results).

 
Next, we investigated the role of caspases in the Bcl-xS-induced oocyte death. The ability of Bcl-xS to induce caspase activity was examined by measurement of the cleavage of the fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-7-AMC) (DEVDase activity) in the cytosolic fraction. As shown in Fig. 5, DEVDase activity was detected in the cytosolic fractions prepared from Bcl-xS-injected oocytes. This activity increased over time from injection of Bcl-xS cRNA, and its onset and extent were found to be approximately correlated with the onset and extent of oocyte death. In the membranous fractions of control oocytes, DEVDase activity was very low, and was not substantially increased by Bcl-xS expression (data not shown). These results thus suggest that expression of Bcl-xS in the oocytes induces caspase activity in the cytosol. To demonstrate that this caspase activity is needed for Bcl-xS-induced oocyte death, we examined the effect of the broad-spectrum caspase inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoro-methylketone (Z-DEVD-FMK; intracellular concentration, 100 µM) on the viability of Bcl-xS-injected oocytes. Injection of Bcl-xS cRNA (1 ng) caused massive (84%) death of oocytes after 12 h, whereas there was no evidence of death among oocytes that received co-injections of Z-DEVD-FMK and Bcl-xS at that time. That the injected Z-DEVD-FMK did indeed inhibit caspase activity in the oocytes was indicated by the reduction in DEVDase activity in extracts prepared from oocytes that received injections of Bcl-xS and Z-DEVD-FMK compared to that from oocytes that received injections of Bcl-xS only (Fig. 6). Taken together, these results suggest that caspases are activated and needed for the cytotoxic effect of Bcl-xS on the oocytes.



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  		FIGURE 5. Expression of Bcl-xS induces DEVDase activity in the cytosolic fraction of Xenopus oocytes. Groups of 50 stage 6 Xenopus oocytes received injections of 2.5 ng of Bcl-xS cRNA. At the indicated time points, the viability of the oocytes in each group was determined by their morphology as described in Fig. 1. The oocytes were then collected, and membranous and cytosolic fractions from each group were prepared as described in "Materials and Methods." DEVDase activity was determined in equal volumes (each equivalent to three oocytes) of the cytosolic fraction of each treatment, as described in "Materials and Methods." The data show the results of a representative experiment (one of six independent experiments).

 


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  		FIGURE 6. Bcl-xS-induced oocyte death needs caspase activity. Groups of stage 6 Xenopus oocytes (each group containing 25 oocytes) received injections of 1 ng of Bcl-xS cRNA and the caspase inhibitor Z-DEVD-FMK (DEVD-FMK) (final concentration, 100 µM), of the vehicle DMSO alone (final concentration of 0.005%), or did not receive injections (Con). After 12 h, the viability of the oocytes was determined by their morphology as described in Fig. 1, and the oocytes were collected and the cytosolic fractions from each group prepared as described in "Materials and Methods." DEVDase activity in the cytosolic fraction was determined as described in Fig. 5. The data show the results of a representative experiment (one of four independent experiments).

 
In most experiments, the effects of Z-DEVD-FMK on survival as well as on inhibition of caspase activity were transient, suggesting that at the concentration and experimental conditions used in this study, this inhibitor was effective for a short time only. Nevertheless, while this inhibitor was active, it had a protective effect.

Bcl-xS Expression Induces Cytochrome c Release in a Caspase-Dependent Manner
Caspase activation and apoptosis are induced by pro-apoptotic members of the Bcl-2 family via a mechanism that apparently involves cytochrome c release from the mitochondria (for recent reviews see, e.g., Refs. 1, 32, 33). In view of the present results suggesting that in Xenopus oocytes Bcl-xS translocates to the mitochondria and induces caspase activation, it was of interest to examine whether Bcl-xS expression induces cytochrome c released in the oocytes, and if so, to examine the relationship between this release and caspase activation.

Oocytes received injections of Bcl-xS cRNA and the cytosolic fraction was then examined for the presence of cytochrome c. As shown in Fig. 7A, the expression of Bcl-xS increased with time after the injection and induced a time-course-dependent increase in the appearance of cytochrome c in the cytosolic fractions (Fig. 7B), indicating that Bcl-xS expression induces cytochrome c release. Examination of these cytosolic fractions showed that Bcl-xS expression also induced a time-course-dependent increase in DEVDase activity (Fig. 7C), and that the rate of the Bcl-xS-induced release of cytochrome c was similar to that of the induction of DEVDase activity.



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  		FIGURE 7. Bcl-xS expression in oocytes induces cytochrome c release concomitantly with DEVDase activation. Groups of stage 6 Xenopus oocytes (each group containing 50 oocytes) received injections of 1 ng of Bcl-xS cRNA or did not receive injections (Con). At the indicated time points, the viability of the oocytes in each treatment was determined by their morphology as described in Fig. 1. The oocytes were then collected and subjected to subcellular fractionation as described in Fig. 3. Equal volumes (each equivalent to three oocytes) of each of the different cytosolic fractions were subjected to SDS-PAGE and immunoblotting using rabbit polyclonal anti-Bcl-x antibody (A) or mouse anti-cytochrome c antibodies (B), or subjected to DEVDase assay (C) as described in Fig. 5. The data show the results of a representative experiment (one of six independent experiments).

 
Cytochrome c release has been shown to be mediated in a caspase-independent manner in many systems, but there are other systems in which the release of cytochrome c is caspase dependent (e.g., Refs. 34–37). We therefore examined whether the Bcl-xS-induced cytochrome c release in the Xenopus oocyte system is dependent on caspase activity. Oocytes received co-injections of Bcl-xS cRNA and caspase inhibitors, and cytochrome c release was determined by examining for the presence of cytochrome c in the cytosolic fraction. As shown in Fig. 8A, the caspase inhibitor Z-DEVD-FMK completely abolished the Bcl-xS-induced release of cytochrome c, at least at the level that can be detected by Western blot assay. This effect of Z-DEVD-FMK is probably mediated by its caspase-inhibitory activity, as hardly any DEVDase activity was detected in the cytosolic fraction obtained from Z-DEVD-FMK-injected oocytes (Fig. 8B). Similar results were obtained with another caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoro-methylketone (Z-VAD-FMK) (data not shown). These results suggest that the apparent release of cytochrome c induced by the expression of Bcl-xS in Xenopus oocytes is mediated in a caspase-dependent manner.



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  		FIGURE 8. Bcl-xS-induced cytochrome c release requires caspase activity. Groups of stage 6 Xenopus oocytes (each group containing 25 oocytes) received injections of 1 ng of Bcl-xS cRNA and the caspase inhibitor Z-DEVD-FMK (DEVD-FMK) (final concentration, 100 µM), of the vehicle DMSO alone (final concentration 0.005%), or did not receive injections (Con). After 18 h, the oocytes in each group were collected and fractionated. The cytosolic fraction from each group was subjected to SDS-PAGE and immunoblotting using mouse anti-cytochrome c antibodies (A) or to DEVDase assay (B). The data show the results of a representative experiment (one of four independent experiments).

 
The Mechanism Whereby Anti-Apoptotic Proteins Inhibit Bcl-xS-Induced Oocyte Death
The results described above showed that Bcl-xS induced oocyte death by a mechanism involving caspase activation and cytochrome c release, and that this death was inhibited by the anti-apoptotic proteins Bcl-xL and Bcl-2. It was therefore of interest to identify the step at which the cytotoxic effect of Bcl-xS is inhibited by anti-apoptotic proteins such as Bcl-xL. To that end, we examined the effects of Bcl-xL on translocation of Bcl-xS to the mitochondria, caspase activation, and cytochrome c release after co-injection of Bcl-xS cRNA with Bcl-xL cRNA. As shown in Fig. 9A, both Bcl-xL and Bcl-xS were expressed in the oocytes that received injections. Expression of Bcl-xL did not prevent the translocation of Bcl-xS to the membranous fraction (Fig. 9A) but completely abolished both Bcl-xS-induced caspase activation (Fig. 9B) and cytochrome c release (Fig. 9C). Interestingly, although the amounts of Bcl-xL and Bcl-xS cRNAs we injected into the oocytes were equal, the amount of Bcl-xL expressed was greater than that of Bcl-xS, and Bcl-xL was present in both the cytosolic and the membranous oocyte fractions. Taken together, these results suggest that Bcl-xL acts not by preventing translocation of Bcl-xS to the mitochondria, but rather by inhibiting the more downstream effects of Bcl-xS.



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  		FIGURE 9. Effects of Bcl-xL expression on Bcl-xS-induced mitochondria translocation, DEVDase activity, and cytochrome c release. Groups of 50 stage 6 Xenopus oocytes received injections of 2.5 ng of Bcl-xS cRNA, with or without 2.5 ng Bcl-xL cRNA, or did not receive injections. At the indicated time points, the viability of the oocytes in each group was determined by their morphology as described in Fig. 1, and oocytes from each group were then collected and fractionated into membranous (MF) and cytosolic (Cyt) fractions. The expression of Bcl-xS and Bcl-xL in the membranous and cytosolic fraction was determined (A) as described in Fig. 1. DEVDase activity (B) and cytochrome c release (C) were determined in the cytosolic fractions as described in Fig. 7. The data show the results of a representative experiment (one of four independent experiments).

 
One mechanism suggested to explain the protective effect of anti-apoptotic members of the Bcl-2 family is that they bind to pro-apoptotic proteins and neutralize their pro-apoptotic activity. We therefore examined whether Bcl-xS can interact with Bcl-2 in Xenopus oocytes. Bcl-xS was co-expressed with Bcl-2, and its ability to form heterocomplexes with Bcl-2 was examined by the co-immunoprecipitation assay. As a negative control, the oocytes received injections of Bcl-xL alone. Oocyte lysates were immunoprecipitated with anti-Bcl-x antibody and immunoblotted with anti-Bcl-2 antibody. Given the capacity of non-ionic detergents to enable the dimerization of Bcl-2 family members, the immunoprecipitation assay was performed in the absence of detergent or in the presence of 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS), a detergent that does not promote artificial Bax oligomerization (38). Bcl-xS or Bcl-xL was immunoprecipitated by the anti-Bcl-x antibody (Fig. 10). In addition, Bcl-2 was co-precipitated from oocyte extract that received co-injections of Bcl-xS and Bcl-2 (both in the absence and in the presence of CHAPS), but not from oocyte extract that received injections of Bcl-xL only. These results thus suggest that Bcl-xS is capable of forming heterocomplexes with Bcl-2 in Xenopus oocytes.



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  		FIGURE 10. Bcl-xS co-immunoprecipitate with Bcl-2. Groups of 50 stage 6 Xenopus oocytes received injections of 1 ng Bcl-xL cRNA, 1 ng of Bcl-xS cRNA, or 1 ng Bcl-xS, and 1 ng Bcl-2 cRNAs. After 24 h, oocytes from each group were collected and crude extracts were prepared from each treatment. The extracts were immunoprecipitated with anti-Bcl-x antibodies in the presence or absence of the detergent CHAPS (1%), as described in "Materials and Methods," and the immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-Bcl-2 or anti-Bcl-x antibodies. The data show the results of a representative experiment (one of three independent experiments). WB, Western blot; IP, immunoprecipitation.

 
The TM and the BH3 Domains, But Not the BH4 Domain, Are Needed for Bcl-xS-Induced Oocyte Death and Cytochrome c Release
Previous work from our laboratory, conducted in PC12 cells, showed that the apoptotic effect of Bcl-xS requires the COOH-terminal hydrophobic domain [also termed the transmembrane (TM) domain] and the BH3 domain, but not the BH4 domain (39). To further study the mechanism of Bcl-xS-induced apoptosis in oocytes and to examine whether these domains are needed for Bcl-xS-induced cytochrome c release, we examined the ability of FLAG-tag fusion proteins of Bcl-xS to induce oocyte death and cytochrome c release. The proteins tested were Bcl-xS wild type, Bcl-xS {Delta}BH4 (in which the BH4 domain is deleted), Bcl-xS {Delta}GD (in which the two well-conserved amino acids, glycine and aspartic acid, are deleted from the BH3 domain), and Bcl-xS {Delta}TM (in which the TM domain is deleted). First, we examined the subcellular distribution of these Bcl-xS mutants. The Bcl-xS {Delta}TM mutant was mainly present in the cytosolic fraction, whereas the Bcl-xS {Delta}GD and {Delta}BH4 mutants and to a lesser extent the Bcl-xS wild type were present in both the membranous and the cytosolic fractions (Fig. 11A). The Bcl-xS {Delta}TM and {Delta}GD mutants were ineffective in inducing oocyte death and cytochrome c release, whereas Bcl-xS {Delta}BH4 and Bcl-xS wild type were effective (Fig. 11B). Interestingly, Bcl-xS {Delta}BH4 was more toxic than the Bcl-xS wild type, suggesting that the BH4 domain has anti-apoptotic properties, as previously shown for the BH4 domain of Bcl-2 (40). The {Delta}TM and {Delta}GD Bcl-xS mutants were expressed in the oocytes in amounts that exceeded those of wild-type Bcl-xS, suggesting that the lack of effectiveness of these mutants in the oocytes is attributable not to their lack of expression but rather to their mutation. These findings suggest that the Bcl-xS-induced death of Xenopus oocytes and release of cytochrome c depend on both the presence of Bcl-xS in the mitochondria and the activity of the Bcl-xS BH3 domain. It should, however, be noted that the ability of Bcl-xS {Delta}TM and {Delta}GD mutants to promote oocyte death was not completely abolished, and that in some experiments, expression of these mutants over a longer period (48–60 h) induced death activity, albeit at a very low level (data not shown). Thus, deletion of the TM domain or of the GD amino acids in the BH3 domain largely reduced but did not completely abolish the death-promoting effect of Bcl-xS.



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  		FIGURE 11. Effects of the transmembrane, BH3, and BH4 domains of Bcl-xS on Bcl-xS-induced death, Bcl-xS localization, DEVDase activity, and cytochrome c release. Groups of 20 stage 6 Xenopus oocytes received injections of 1 ng of cRNA from each of the FLAG-tag Bcl-xS mutants Bcl-xS {Delta}BH4 ({Delta}BH4), Bcl-xS {Delta}GD ({Delta}GD), or Bcl-xS {Delta}TM ({Delta}TM), or with 1 ng of FLAG-tag wild-type Bcl-xS (WTBcl-xS), or did not receive injections. The viability of the oocytes in each group was determined, 36 h after injection, by their morphology as described in Fig. 1, and the oocytes from each treatment were then collected and fractionated into membranous (MF) and cytosolic (Cyt) fractions. Expression of the different Bcl-xS variants was determined (A) as described in Fig. 1 for anti-Bcl-x antibody ({alpha}-Bcl-x), except that Bcl-xS {Delta}BH4 was detected with anti-FLAG antibody ({alpha}-Flag). The Bcl-xS {Delta}BH4 mutant cannot be detected by the S-18 anti-Bcl-x antibody because it lacks the epitope that the S-18 antibody recognizes. The amounts of cytochrome c in the cytosolic fractions of each of the different treatments (B) were determined as described in Fig. 7. The data show the results of a representative experiment (one of three independent experiments).

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
In this study, we employed the system of cRNA-injected Xenopus oocytes to investigate the mechanism of action of the pro-apoptotic protein Bcl-xS. Our results showed that direct activation of the oocytes' apoptotic pathway by expression of Bcl-xS kills the oocytes by a mechanism that involves the BH3-domain-dependent and caspase-dependent release of cytochrome c. This is the first demonstration that the in vivo expression of a pro-apoptotic member of the Bcl-2 family induces apoptosis in Xenopus oocytes and that the apoptotic pathway of Bcl-xS may involve caspase-mediated cytochrome c release. Death of the Bcl-xS-expressing oocytes was indicated by the appearance of the typical morphology of dying oocytes as well as by a reduction in their membrane potential (an indication of the disruption of membrane integrity). Similar morphological changes and reduction of membrane potential have been reported in Xenopus oocytes that received injections of cytochrome c (30). Although the morphology of the Bcl-xS-expressing dying oocytes did not have the typical features of apoptosis (such as membrane blebbing, bubbling, and cell fragmentation), the biochemical features of this death process resembled apoptotic cell death. We therefore conclude that the Bcl-xS-induced oocyte death is mediated by caspases, involves cytochrome c release, and is inhibited by Bcl-2 and Bcl-xL, the anti-apoptotic members of the Bcl-2 protein family.

Studies have shown that in 3T3 cells, Bcl-xS induces caspase-independent cell death, which results from Bcl-xS-induced depletion of active cytochrome c from these cells (9, 10). In the present study, however, cytochrome c was observed in the cytosolic fraction, and caspase activation (indicated by DEVDase activity) was evident in extracts of Bcl-xS-expressing oocytes. Our results also showed that the death of these oocytes requires caspase activity, because death was inhibited in the presence of the caspase inhibitors Z-DEVD-FMK (Fig. 6) and Z-VAD-FMK (data not shown). The discrepancy between our results and the findings in 3T3 cells may be attributable to differences in the cellular systems used in the two studies. It should, however, be pointed out that the need for caspases is not exclusive to the effect of Bcl-xS on the Xenopus oocytes, but could be more general, as Bcl-xS-induced apoptosis in transiently transfected PC12 and 293 cells also requires caspase activity (7, 31). Nevertheless, we cannot rule out the possibility that Bcl-xS-induced oocyte death is also mediated, at least in part, by a caspase-independent pathway, because the caspase inhibitors did not completely abolish the death of these oocytes. The nature of such death, if it exists, remains to be determined.

There are two possible explanations for the present finding that the Bcl-xS-induced cytochrome c release in the Xenopus oocytes depends on caspase activity. Bcl-xS might stimulate caspase activation directly, and the activated caspases might then directly or indirectly induce cytochrome c release. This explanation is supported by the recent findings that in some cell lines, caspase-2 is required for stress-induced apoptosis and that it acts upstream of cytochrome c release (41). Alternatively, Bcl-xS may directly stimulate cytochrome c release from the mitochondria in amounts which, in the presence of caspase inhibitors, are too low to be detected by Western blot analysis. This cytochrome c might then activate caspases via the apoptosome, with resulting induction by the activated caspases of abundant release of cytochrome c via an amplification loop. The latter explanation is supported by studies using a cell-free system derived from Xenopus egg extracts, which show that the pro-apoptotic members of the Bcl-2 family, Bax and Bid, directly cause the release of cytochrome c from isolated mitochondria (42), and that the cytochrome c-activated cytosolic fraction contains activated caspases, which in turn induce cytochrome c release (24, 43). If the latter explanation is correct, the question arises: How does Bcl-xS induce the initial cytochrome c release? It is possible that Bcl-xS acts like the typical BH3-only proteins, which do not induce apoptosis by themselves, but promote apoptosis by suppressing the defensive action of Bcl-2/Bcl-xL, or by promoting activation of the multidomain conserved pro-apoptotic family members Bax or Bak (44, 45), or both. Alternatively, Bcl-xS may induce cytochrome c release by itself. We favor the former explanation, because our findings show that Bcl-xS can interact with Bcl-2.

In a previous study from our laboratory, it was shown that the TM and BH3 domains of Bcl-xS are needed for Bcl xS-induced death of PC12 cells (39). The results presented here show that these domains are also needed for Bcl-xS-induced apoptosis in oocytes and further suggest that the Bcl-xS-induced apoptotic effect requires localization of Bcl-xS to the mitochondria, where it induces BH3-dependent induction of cytochrome c release. They suggest, moreover, that Bcl-xL does not prevent translocation of Bcl-xS to the membranous fraction, but rather acts by inhibiting Bcl-xS-induced cytochrome c release and caspase activation. The exact mechanism by which anti-apoptotic proteins inhibit the cytotoxic effect of Bcl-xS remains to be determined. One possible mechanism is via their interaction with Bcl-xS. The present results indeed show that Bcl-2 and Bcl-xS can interact in the oocytes.

Cell-free systems have proved to be very useful models for studying biochemical processes of apoptosis. The system derived from Xenopus egg extract was shown to be a very useful model system for studying apoptosis in vitro (23, 26, 27, 29, 46, 47). Obviously, however, in vitro studies are not sufficient and must be supplemented by studies in vivo. The results presented here show that the in vivo expression of apoptotic proteins in Xenopus oocytes offers the advantage of an in vivo system, enabling efficient biochemical analysis of the mechanism of action of pro-apoptotic proteins. The potential usefulness of this system is further supported by preliminary studies showing that expression of other pro-apoptotic members of the Bcl-2 family, such as Bax and t-Bid, also induces oocyte death.2


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Reagents
Z-VAD-FMK and Z-DEVD-FMK were purchased from Enzyme Systems (Dublin, CA), and were resuspended as 50 or 20 mM stock solutions, respectively, in DMSO (Merck, Darmstadt, Germany). Ac-DEVD-7-AMC was purchased from Biomol Research Laboratories (Plymouth Meeting, PA). Unless otherwise stated, all other reagents were purchased from Sigma (St. Louis, MO).

Preparation of Oocytes
Oocytes were prepared as previously described (48). Briefly, X. laevis females, maintained at 20 ± 2°C on an 11 h/13 h light/dark cycle, were anesthetized in a 0.15% solution of procaine methanesulfonate (MS222), and portions of ovary were removed through a small incision on the abdomen. The incision was sutured and the animal was returned to a separate tank until it had fully recovered from the anesthesia. It was then returned to a large tank in which all the frogs were kept for at least 4 weeks until the next surgery. The oocytes were defolliculated by collagenase treatment and placed in NDE-96 solution [96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 2.5 mM sodium pyruvate, 50 µg ml-1 gentamicin, 5 mM HEPES (pH 7.5)]. After 16–18 h, the oocytes received injections of 50 nl of an aqueous solution of the cRNAs at the concentrations recorded in the figure legends, in the absence or presence of the caspase inhibitors (intracellular concentration, 100 µM) or in the presence of the vehicle DMSO (intracellular concentration, 0.002% for Z-VAD-FMK or 0.005% for Z-DEVD-FMK). Oocytes were inspected 1–1.5 h after cRNA injection and oocytes that were damaged by the injection were discarded. The rest of the oocytes were placed in fresh NDE-96 solution and incubated at 20–22°C. At various time points, their viability was determined by morphological assessment under a binocular microscope (magnification, x20–25).

Preparation of cRNAs
The cDNAs of the various genes and Bcl-xS mutants used in this study were subcloned into the pGEMHJ vector (49). Generation of the Bcl-xS mutants Bcl-xS {Delta}BH4, Bcl-xS {Delta}GD, and Bcl-xS {Delta}TM has been described elsewhere (39). The pGEMHJ vector provides the 5'- and 3'-untranslated regions of Xenopus-globin RNA (50), ensuring a high level of protein expression in the oocytes. The pGEMHJ Bcl-xS, pGEMHJ Bcl-2, and pGEMHJ bcl-xL plasmids were generated by subcloning different cDNA fragments in frame into the EcoRI site of pGEMHJ. Bcl-xS, Bcl-xL, Bcl-2, and Bax cDNAs were generated by one-step PCR using pBluescript SK(+)Bcl-xS, pBluescript SK(+)Bcl-xL, pCMV-Bcl-2, or pcDNA3-HamuBax, respectively, as a template and 5'-GATCCATGAGAATTCTAGATGTCTCAGAGC (for Bcl-xS/L), 5'-GATCCATGAGAATTCTAGATGGCGCAAGCC (for Bcl-2), or 5'-GATCCATGAGAATTCTAGATGGACGGGTCC (for Bax) as the forward primers and 5'-TTCCATGAGAATTCTCACTTCCGACTG (for Bcl-xS/L), 5'-TTCCATGAGAATTCTCACTTGTGGCC (for Bcl-2), or 5'-TTCCAGTAGAATTCTCAGCCCATCTTC (for Bax) as the reverse primers. The pGEMHJ Bcl-xS mutant plasmids were generated by inserting BamHI-XbaI fragments, containing FLAG-tag in frame with Bcl-xS cDNA, into the BamHI-XbaI sites of pGEMHJ. The BamHI-XbaI fragments were obtained from pcDNA3FLAG-Bcl-xS {Delta}BH4, pcDNA3FLAG-Bcl-xS {Delta}GD, pcDNA3FLAG-Bcl-xS {Delta}TM, or pcDNA3FLAG-Bcl-xS wild-type expression vectors (39). The DNA plasmids were linearized and then used as templates to transcribe the corresponding cRNAs by T7 RNA polymerase in vitro, using standard procedures.

Subcellular Fractionation of Oocytes
Stage 6 Xenopus oocytes received injections of the desired cRNA. At different time points, groups of 20–50 oocytes were collected into 150 µl homogenization buffer containing 250 mM sucrose, 10 mM HEPES (pH 7.5), 2.5 mM MgCl2, and 1 mM DTT, as well as the protease inhibitors aprotinin (0.15 unit/ml), leupeptin (5 µg/ml), and phenylmethylsulfonyl fluoride (1 mM), and mechanically disrupted. Crude extract was then prepared by low-speed centrifugation (1000 x g, 10 min) to remove the yolk. The soluble fractions were removed and subjected to high-speed centrifugation (21,000 x g, 30 min). The resulting pellets (membranous fraction, representing intracellular membranous organelles such as mitochondria) were dissolved in a volume equivalent to the supernatant (cytosolic fractions).

Electrophysiological Measurements
Oocytes were placed in a 500-ml chamber constantly perfused with the ND-96 solution (same as NDE-96 but without pyruvate and antibiotics), and the resting potential was monitored for 1–3 min until stabilization (recovery from electrode impalement). The membrane potential was measured using an OC-725B oocyte clamp amplifier (Warner Instruments, Hamden, CT) with agarose cushion electrodes (51).

Immunoprecipitation and Western Blotting
Groups of 25 oocytes were collected 24 h after injection and lysed in 150 µl of cold lysis buffer [20 mM Tris (pH 7.5), 5 mM EDTA, 5 mM EGTA, 100 mM NaCl, with or without 1% CHAPS] supplemented with protease inhibitor cocktail (Calbiochem, San Diego, CA). After removal of cellular debris by centrifugation (20,800 x g, 10 min), immunoprecipitation was carried out as follows: lysates were pre-cleared for 30 min at 4°C with anti-rabbit IgG-agarose beads (Sigma) and incubated with 10 µg/ml anti-Bcl-xS S-18 rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4°C and then for a further 45 min with anti-rabbit IgG-agarose beads.

Protein samples were separated by 12.5% SDS-PAGE and electroblotted onto supported nitrocellulose. Equal volumes (each representing three oocytes) from each subcellular fraction were loaded into each lane. Uniformity of sample loading was verified by Ponceau staining of the blots. Each blot was blocked for 30 min in 10 mM Tris base, 150 mM NaCl containing 5% fat-free milk, and then incubated for 16–18 h at 4°C with the primary antibody. Rabbit anti-Bcl-xL/S S-18 (1:1000), mouse anti-Bcl-2 (Santa Cruz Biotechnology), mouse anti-cytochrome c (1:500) (PharMingen, San Diego, CA), or M5 mouse monoclonal anti-FLAG antibody (Sigma) (1:1000) was used as primary antibody. Goat anti-rabbit (1:10,000) or goat anti-mouse IgG peroxidase conjugate (1:5000) was used as second antibody. The blots were developed using the Enhanced Chemiluminescence Kit (Amersham, Arlington Heights, IL).

Assay for DEVDase Activity
Caspase activity was measured in terms of assayed DEVDase activity. From each cytosolic or membranous fraction (corresponding to the content of two to three oocytes), 10 µl were assayed for DEVDase activity using the fluorescent synthetic peptide Ac-DEVD-7-AMC (50 µM) in 500 µl of reaction buffer [50 mM Tris-HCl (pH 7.4), 1 mM DTT, and 2 mM MgCl2]. Fluorescence at 360 nm for excitation and at 460 nm for emission was measured after incubation for 30 min at 37°C.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We are grateful to Shirley Smith for excellent editorial assistance.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 United States-Israel Binational Science Foundation (96-00224). Back

2 Braun and Stein, unpublished results. Back

Received May 14, 2002; revised November 14, 2002; accepted November 27, 2002.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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