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Molecular Biology and Genetics |
Department of Medical Genetics, Biomedical Research Center, Osaka University Medical School, and CREST, Japan Science and Technology Corporation, Suita 565-0871, Japan [Y. E., S. S., Y. T.], and IDUN Pharmaceuticals, Inc., La Jolla, California 92037 [A. S., K. J. T.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent studies have suggested that mitochondria play an essential role in apoptotic signal transduction (8 , 9) . Various apoptotic stimuli have been shown to induce mitochondrial changes (10, 11) , resulting in release of apoptogenic factors such as mitochondrial cytochrome c (12, 13, 14) and apoptosis-inducing factor (15 , 16) into the cytoplasm, which are observed in the early phase of apoptosis. The release of these proteins is prevented by mitochondrial antiapoptotic proteins, Bcl-2 (17, 18, 19) and Bcl-xL (20) , for which the C. elegans homologue is Ced-9 (21) , via an unknown mechanism (8) .
Biochemical studies have revealed that cytochrome c interacts with a cytoplasmic factor called Apaf-1 (22) , the mammalian homologue of the C. elegans death regulator Ced-4, and the complex recruits procaspase 9 (Apaf-3) (23) to induce its oligomerization (7) . The formation of the ternary complex has been shown to proceed in a dATP (or ATP)-dependent manner in vitro (23) . Procaspase 9 is autoactivated on oligomerization, which in turn activates caspase 3 (23) . In addition to the prevention of release of mitochondrial apoptogenic factors to the cytoplasm, Bcl-2/Bcl-xL has also been shown to inhibit caspase 9 activation via forming ternary complex called apoptosome, comprising Bcl-2/Bcl-xL-Apaf-1-procaspase 9 (24 , 25) . The activation of effector caspases such as caspase 3 results in proteolytic cleavage of various cellular substrates (3 , 4) , including DFF45(ICAD) (26 , 27) , which is an inhibitor of CAD (DFF40), a DNase required for oligonucleosomal DNA fragmentation (28 , 29) .
Apoptotic signals triggered by Fas (Apo1/CD95) antigen, the death receptor, were recently reported to be transmitted by two pathways depending on the type of cell (30)
. In type I cells, Fas stimulation activates caspase 8 by rapid formation of the DISC3
(31)
, followed by activation of caspase 3, and apoptosis as well as the activation of these caspases are not blocked by Bcl-2. In type II cells, DISC formation is less prominent and mitochondrial changes as assessed by mitochondrial membrane potential (
m) precede the activation of caspases 8 and 3, with overexpression of Bcl-2 inhibiting both apoptosis and activation of these caspases. Thus, death signals in Fas-mediated apoptosis are transmitted by either mitochondria-dependent or -independent pathways. Fas-mediated apoptosis involving the mitochondria-dependent pathway seems to share the machinery for transmission of apoptotic signals with other cell death stimuli, including calcium ionophore, 
-irradiation, chemotherapy drugs, and reactive oxygen species (8
, 9)
.
We and others have recently shown that depletion of the intracellular ATP completely blocks Fas-mediated apoptosis (32 , 33) and that ATP-dependent steps exist both upstream and downstream of caspase 3-like protease activation during apoptotic signal transduction in Jurkat and HeLa cells (32) , both of which are classified as type II cells. We also suggested that the ATP level is a determinant of cell death by apoptosis or necrosis (32 , 33) . Because apoptotic death signals are probably transmitted from the cytoplasm to the nucleus, one of the ATP-dependent steps functioning downstream of the activation of caspase 3 might be an active nuclear transport mechanism, which requires ATP hydrolysis (34 , 35) . Indeed, we have previously shown that active nuclear transport is essential for apoptotic changes of the nucleus to occur (36) .
In the present study, we examined the ATP-dependent steps of Fas-mediated apoptotic signal transduction in type I cells, which are likely to be specific for death receptor-mediated apoptosis. We also studied ATP-dependent steps in type II cells, which involve the mitochondria and, therefore, are likely to be related to apoptosis in general. Our results indicated that the ATP-dependent step(s) of Fas-mediated apoptosis in type I cells are located downstream of the activation of caspase 3, whereas the activation of caspase 9 is the most upstream ATP-dependent step in type II cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Death-inducing Treatments.
SKW6.4 and Jurkat cells (106 cells/ml) with or without 1 h of pretreatment to deplete intracellular ATP, as described above, were incubated with 0.3 µg/ml and 0.2 µg/ml, respectively, of the agonistic antihuman Fas monoclonal antibody CH-11 (MBL, Nagoya, Japan) for various periods. The concentration of anti-Fas antibody was determined to induce apoptosis in SKW6.4 and Jurkat cells in a similar kinetics (see Fig. 2
). Apoptotic and necrotic cells were identified using fluorescence microscopy with Hoechst 33342 and propidium iodide, as described previously (38)
. For quantitative analysis, more than 1000 cells were counted.
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Immunohistochemical Analysis of Subcellular Localization of Cytochrome c.
SKW6.4 and Jurkat cells were treated with anti-Fas antibody under various conditions for 2 h. Cells were then harvested, washed with PBS, and fixed with 7% formaldehyde in PBS for 10 min at RT. Fixed cells (105 cells) were applied to slide glasses, dried, washed twice with PBS, and permeabilized with 0.5% Triton X-100 in PBS for 10 min at RT. After washing with PBS, the cells were incubated with antibovine cytochrome c mouse monoclonal antibody (7H8; Pharmingen) and antibovine F1-ATPase rabbit polyclonal antibody, which recognize human cytochrome c and human F1-ATPase, respectively, at 4°C for 24 h. After washing with PBS, cells were then incubated with RITC-conjugated antimouse IgG antibody and FITC-conjugated antirabbit IgG antibody for 15 min at RT. Cells were washed with PBS and then observed under a fluorescent microscope (BX50; Olympus, Tokyo, Japan) with excitation at 480 nm for FITC and at 530 nm for RITC.
Biochemical Subcellular Fractionation of Apoptotic Cells.
SKW6.4 and Jurkat cells were treated with anti-Fas antibody, as described above. At the indicated times, cells were harvested, washed with PBS and incubated with 10 µM digitonin (Sigma Chemical Co.) in mitochondrial isolation buffer [0.3 M mannitol, 0.2 mM EDTA/K, 0.1% fatty acid-free BSA, 10 mM Hepes-KOH (pH 7.4), with 1 µg/ml each of chymostatin, leupeptin, antipain, and pepstatin] at 107 cells/ml at 37°C for 3 min. This treatment resulted in disruption of the cell membrane, but not the mitochondrial membrane. Conditions were chosen to achieve only 50% cell lysis, which was assessed by the release of lactate dehydrogenase (data not shown), to minimize damage to the mitochondria during fractionation. After centrifugation at 3,000 rpm for 2 min at 4°C, aliquot of the supernatant (cytoplasmic fraction) and the precipitate (containing mitochondria) corresponding to 105 cells each were analyzed by Western blotting using antibovine cytochrome c mouse monoclonal antibody (7H8; Pharmingen). Immunoreactive bands were visualized using horseradish peroxidase-conjugated antimouse IgG antibody and enhanced chemiluminescence detection reagents.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. ATP depletion did not alter Fas-antigen expression on the cell surface (32)
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, whereas many apoptotic cells appeared when ATP was supplied by glycolysis after the addition of glucose or supplied by the mitochondria after omitting oligomycin from the glucose-free medium (Fig. 2B)
, confirming our previous observations (32)
. Similarly, ATP depletion greatly reduced the induction of apoptotic nuclear changes in SKW6.4 cells (Fig. 2A)
. The large number of apoptotic cells in cultures where ATP was supplied either by the mitochondria or by glycolysis (Fig. 2)
argues against the possibility that apoptosis might be glucose-dependent or oligomycin-sensitive, but not ATP-dependent. The number of necrotic cells was not increased significantly during incubation for 4 h under ATP-depleted conditions, but longer incubation increased the occurrence of necrosis (data not shown). From these results, we concluded that Fas-mediated apoptotic nuclear changes were ATP-dependent in both type I and type II cells.
Activation of Caspases in SKW6.4 Cells under ATP-depleted Conditions.
To define how far Fas-mediated apoptotic signal transduction could proceed under ATP-depleted conditions, we examined whether or not various caspases were activated by analyzing the cleavage of procaspases. We selected caspases 3, 8, and 9, because the activation of caspase 3 seems likely to be one of the last steps in the caspase cascade, whereas activation of caspase 8 by DISC and activation of caspase 9 by Apaf-1 and cytochrome c are likely to be the initial steps of the caspase cascade in type I and type II cells, respectively.
When SKW6.4 cells were treated with agonistic anti-Fas antibody under ATP-depleted conditions, procaspase 3 was cleaved similarly to under ATP-replete conditions (Fig. 3A)
, indicating that activation of caspase 3 by Fas stimulation did not require the presence of intracellular ATP in type I cells. As expected from the ATP-independent activation of caspase 3, upstream caspase 8 was rapidly cleaved irrespective of the intracellular ATP level (Fig. 3B)
, indicating that activation of caspase 8 by DISC was independent of ATP. As suggested by the previous observation that caspase 9 was probably activated by caspase 3 in type I cells (30)
, caspase 9 was also activated irrespective of the intracellular ATP level (Fig. 3C)
. Consistently, the cleavage of DFF45 (ICAD), an inhibitor of CAD (DFF40) and one of the substrates for caspase 3, was not affected by depletion of intracellular ATP in SKW6.4 cells (Fig. 3D)
. These results indicated that activation of caspases including caspase 8 was not ATP-dependent in SKW6.4 cells, in which rapid DISC formation has been reported (30)
, and that ATP-dependent steps could only exist downstream of the activation of caspase 3 in these cells.
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. As expected from this result, cleavage of the downstream procaspase, procaspase 3, was also inhibited by ATP depletion (Fig. 4A)
. The results were consistent with our previous observation that an increase in caspase 3 activity assessed by hydrolysis of the specific substrate DEVD-MCA was inhibited by intracellular ATP depletion in Jurkat and HeLa cells (32)
. Cleavage of DFF45 (ICAD) and caspase 8, which was also suggested to be catalyzed by activated caspases in type II cells, was also inhibited by intracellular ATP depletion in Jurkat cells (Fig. 4, B and D)
. The results indicated that ATP-dependent steps were present upstream of the activation of caspase 9 in Jurkat cells.
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. To examine the possibility that ATP depletion affects cytochrome c release, we determined the subcellular localization of cytochrome c after treatment with anti-Fas antibody by fluorescent microscopy and by biochemical fractionation of cells.
Fluorescent microscopy revealed that cytochrome c was colocalized with F1-ATPase (a protein found on the mitochondrial inner membrane) in a compact and granular fashion in the absence of Fas stimulation, regardless of the intracellular ATP level in both SKW6.4 and Jurkat cells (Fig. 5)
, indicating the mitochondrial localization of cytochrome c. When cells were treated with anti-Fas antibody under ATP-replete conditions, fluorescent signals for cytochrome c became diffuse compared with those for F1-ATPase, which remained granular, indicating the release of cytochrome c into the cytoplasm (Fig. 5)
. Even in the absence of intracellular ATP, cytochrome c became diffuse after Fas treatment of both SKW6.4 and Jurkat cells (Fig. 5)
, suggesting that Fas-mediated cytochrome c release was not affected by ATP depletion in these cell types.
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, consistent with the results of immunostaining shown in Fig. 5
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate the apoptotic signal transduction, we analyzed the ATP-dependence of the well-characterized Fas-mediated process of apoptosis, which has recently been reported to occur via two distinct signaling pathways depending on cell type (30) . Type I cells are characterized by rapid formation of DISC and activation of caspase 8 after Fas stimulation, followed by activation of caspase 3 and release of cytochrome c from the mitochondria into the cytoplasm. In these cells, Fas-mediated apoptosis is not inhibited by overexpression of Bcl-2, suggesting that mitochondrial dysfunction and cytochrome c release are not essential for Fas-mediated apoptosis, but are rather a consequence of caspase activation. On the other hand, type II cells show little detectable DISC formation and relatively slower activation of caspases than type I cells. Fas-mediated apoptosis of type II cells is inhibited by Bcl-2 overexpression and has been suggested to involve the mitochondria in its signal transduction pathway and to share its main machinery with other apoptotic stimuli.
In this study, we showed that caspase 3 was fully activated independent of intracellular ATP in type I cells treated with anti-Fas antibody and the ATP-dependent step was downstream of the activation of caspase 3. On the other hand, the activation of caspase 9, which occurs just downstream of the release of cytochrome c, seemed to be the most upstream ATP-dependent step in type II cells (Fig. 7)
. Because apoptosis other than that induced by death receptor stimulation is generally mediated by mitochondrial changes, which are prevented by overexpression of Bcl-2 and Bcl-xL (8, 9, 10, 11)
, the activation of caspase 9 is suggested to be one of the major common ATP-dependent steps in apoptosis. In both type I and II cells, active nuclear transport should be one of the common downstream ATP-dependent steps in apoptotic signal transduction. It is possible that a machinery of nuclear changes requires ATP, as well. These observations on ATP dependence also help to define the existence of two distinct pathways for Fas-mediated apoptotic signal transduction.
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Both the Apaf and DISC systems involve the recruitment of precursor forms of the initiator caspases via their CARD and death effector domains, respectively, to induce activation of the caspases. Despite this similarity at the entry step into the caspase cascade, the DISC system was shown to be ATP-independent and the Apaf system was shown to be ATP-dependent. The physiological significance of the ATP dependency of the Apaf system is now unclear, but it might distinguish the physiological release of cytochrome c, which should lead to apoptosis, from accidental release which might be induced by necrosis where intracellular ATP is often lost through disruption of the cell membrane. Death receptor-mediated cell death seems to be a special form of apoptosis, in a sense that it bypasses the mitochondria and directly activates the caspase cascade. The significance of the ATP-dependence of the Apaf system should be elucidated by further investigations.
It was recently suggested that proapoptotic Bcl-2 family members, including Bax and Bid, might transmit apoptotic signals to the mitochondria, based on the observations that Bax and caspase-cleaved Bid are transferred to the mitochondria from the cytoplasm in response to apoptosis-inducing stimuli (39, 40, 41, 42, 43) and can induce cytochrome c release in isolated mitochondria (42, 43, 44, 45) . The apoptosis induced by overexpression of Bax has been reported to be prevented through inhibition of F0F1-ATPase by oligomycin (46) . As described here and in previous studies, inhibition of F0F1-ATPase by oligomycin does not necessarily prevent various types of apoptosis, including Fas-mediated apoptosis (32 , 33 , 47) . Assuming that all proapoptotic Bcl-2 family members act on the mitochondria in the same way as Bax, apoptotic signal transmission to the mitochondria in oligomycin-insensitive apoptosis is likely to be mediated by molecules other than proapoptotic Bcl-2 family members that do not require functional F0F1-ATPase in vivo. Thus, although mitochondrial changes induced by proapoptotic Bcl-2 family members might be involved in apoptotic signal transduction in certain circumstances, this does not seem to sufficiently explain the general mechanism of apoptosis in vivo. Further investigations on signal transmission to the mitochondria by methods that do not depend on F0F1-ATPase is necessary.
A recent study showed that Fas-mediated apoptosis of Jurkat cells does not require intracellular ATP, whereas apoptosis induced by other stimuli requires ATP, suggesting that Fas-mediated apoptosis and that caused by other stimuli can be classified by their ATP requirement (48) . However, the culture medium used contained 1% nondialyzed serum, which may have a trace amount of glucose to support glycolysis. Because Fas-mediated apoptosis of Jurkat cells requires much less intracellular ATP than apoptosis induced by other stimuli (33) , the condition used by Ferrari et al. (48) might provide sufficient ATP to transmit Fas-mediated apoptotic signals, but not to support apoptosis induced by chemotherapy drugs.
In conclusion, we defined the most upstream ATP-dependent step of apoptotic signal transduction as the activation of caspase 9 in the Apaf system, except for Fas-mediated apoptosis of type I cells, although we cannot exclude the possible involvement of kinases with a very low Km to ATP that might function upstream of the activation of caspase 9.
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FOOTNOTES |
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1 Supported in part by Grants-in-Aid for Scientific Research on Priority Areas and for COE Research from the Ministry of Education, Science, Sports and Culture, Japan. 
2 To whom requests for reprints should be addressed, at Department of Medical Genetics, Osaka University Medical School, B8, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3363; Fax: 81-6-6879-3369;E-mail: tsujimot{at}gene.med.osaka-u.ac.jp
3 The abbreviations used are: DISC, death-inducing signaling complex; RITC, rhodamine B isothiocyanate; RT, room temperature.
Received 2/15/99. Accepted 3/ 5/99.
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, incubation with 10 µM oligomycin in glucose-free DMEM (ATP-depleted); 
, incubation with 10 µM oligomycin in 0.35% glucose-containing DMEM (ATP-replete); 
, incubation in glucose-free DMEM without the addition of oligomycin (ATP-replete).
