HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hirsch, T. Articles by Kroemer, G. Search for Related Content PubMed PubMed Citation Articles by Hirsch, T. Articles by Kroemer, G.

Proteasome Activation Occurs at an Early, Premitochondrial Step of Thymocyte Apoptosis¹

Centre National de la Recherche Scientifique, Unité Propre de Recherche 420, Villejuif, France

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Proteasomes and mitochondrial membrane changes are involved in thymocyte apoptosis. The hierarchical relationship between protease activation and mitochondrial alterations has been elusive. Here we show that inhibition of proteasomes by two specific agents, lactacystin or MG132, prevents all manifestations of thymocyte apoptosis induced by the glucocorticoid receptor agonist dexamethasone or by the topoisomerase II inhibitor etoposide. Lactacystin and MG132 prevent the early disruption of the mitochondrial transmembrane potential (_m), which precedes caspase activation, exposure of phosphatidylserine, and nuclear DNA fragmentation. In contrast, stabilization of the _m using the permeability transition pore inhibitor bongkrekic acid or inhibition of caspases by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone does not prevent the activation of proteasomes, as determined with the fluorogenic substrate N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcoumarin. Thus, proteasome activation occurs upstream from mitochondrial changes and caspase activation. Whereas the proteasome-specific agents lactacystin and MG132 truly maintain thymocyte viability, a number of protease inhibitors that inhibit nuclear DNA fragmentation (acetyl-Asp-Glu-Val-Asp-fluoromethylketone; N-Boc-Asp(OMe)-fluoromethylketone; N-tosyl-L-Phe-chloromethylketone) do not prevent the cytolysis induced by DEX or etoposide. These latter agents fail to interfere with the preapoptotic _m disruption. Altogether, our data indicate that different proteases may be involved in the pre- or postmitochondrial phase of apoptosis. Only those protease inhibitors that interrupt the apoptotic process at the premitochondrial stage can actually preserve cell viability.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Apoptosis may be defined as a form of cell death in which the activation of catabolic hydrolases (proteases and nucleases), within a near-to-intact plasma membrane, contributes to the acquisition of a stereotyped pattern of biochemical and morphologic alterations (1, 2). Overwhelming evidence suggests the involvement of specific cysteine proteases cleaving after aspartic acid (caspases), which catalyze a highly selective pattern of protein degradation, in apoptosis (3, 4, 5, 6, 7). Inhibitors acting on a wide range of caspases such as the Baculovirus protein p35 (8, 9, 10), N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk)³ (11, 12, 13, 14, 15, 16, 17, 18, 19), its truncated analogue Boc-Asp(OMe)-fluoromethylketone (B-D.fmk) (16), or acetyl-Asp-Glu-Val-Asp-fluoromethylketone (Ac-DEVD.fmk) (20) inhibit the acquisition of apoptotic morphology and endonuclease activation in most if not all experimental models. In addition to caspases, different serine proteases, calpains, and proteasomes have been implicated in the apoptotic process (7). Thus, proteasome inhibitors such as lactacystin and MG132 protect thymocytes, T cell hybridomas, and PC12 cells against a number of different apoptosis inducers (glucocorticoids, etoposide, irradiation, anti-CD3, ligation of CD95, withdrawal of nerve cell growth factor) (21, 22, 23, 24). Proteasomes are also involved in the developmentally programmed cell death of the intersegmental muscle of the insect Manduca sexta (25, 26), suggesting that they may play a major role throughout evolution. However, they appear to be less common effectors of the apoptotic pathway than caspases, given that inhibitors of proteasomes are ineffective in some models of apoptosis (27) and actually can induce apoptosis in proliferating cell lines (23, 28, 29, 30, 31).

Apoptosis is also characterized by changes in mitochondrial membrane function. These changes affect the inner membrane, causing a perturbation of the transmembrane potential (_m), which at least in some cases is preceded by an increase in the matrix volume (32, 33). They also affect the outer membrane through which apoptogenic factors leak into the cytosol (32, 33, 34, 35, 36). It has been proposed that the apoptotic process can be subdivided into three phases: 1) a heterogeneous premitochondrial initiation phase during which receptor-mediated stimuli or damage pathways act on the cell; 2) a common mitochondrial effector phase, during which mitochondrial membrane alterations occur; and 3) a postmitochondrial degradation stage, during which apoptogenic factors released from the mitochondrial intermembrane space activate proteases, mainly caspases, and nucleases (32, 37). The mechanisms linking caspase activation and mitochondrial changes appear complex. Upstream caspases activated by CD95 cross-linking during the premitochondrial initiation stage can induce an increase in mitochondrial membrane permeability, both in cells and in isolated mitochondria (38). In addition, at least two different apoptogenic proteins released from mitochondria, cytochrome c and AIF, can activate downstream caspases that participate in the postmitochondrial stage of apoptosis (34, 35, 36, 38).

No information is available on the relationship between noncaspase proteases and mitochondrial function. We therefore decided to study the temporal and functional relationship between proteasome activation and mitochondrial changes in a model of thymocyte apoptosis, namely apoptosis induced by glucocorticoids or etoposide. Our results indicate that proteasomes act upstream of mitochondria, whereas proteases inhibited by Ac-DEVD.fmk, B-D.fmk), or N-tosyl-L-Phe-chloromethylketone (TPCK) act downstream of the _m disruption. In the models that we have studied, only those protease inhibitors that act upstream of mitochondria can actually preserve cell viability.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Inhibition and induction of apoptosis

Thymocytes from female 4- to 6-wk-old BALB/c mice were cultured in RPMI 1640 medium supplemented with 10% FCS, L-glutamine, and antibiotics. Cells (1–5 x 10⁶/ml) were cultured in a humidified atmosphere containing 5% CO₂ at 37°C during the indicated interval (5 to 12 h), alone or with combinations of the following reagents: dexamethasone (DEX; 1 µM, Sigma, St. Louis, MO); etoposide (VP-16, 10 µM, Sigma); MG132 (CBZ-leucyl-leucyl-leucinal, 30 µM, Peptides International, Louisville, KY); lactacystin (30 µM, purchased from Dr. M. R. Corey, Harvard University, Cambridge, MA); N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLnL, 30 µM, also called "calpain inhibitor I; Bachem, Basel, Switzerland); N-tosyl-L-Phe-chloromethylketone (TPCK, 10 µM, Sigma); N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk, 50 µM, Enzyme Systems, Dublin, CA); Boc-Asp(OMe)-fluoromethylketone (B-D.fmk, 50 µM, Enzyme Systems); acetyl-Asp-Glu-Val-Asp-fluoromethylketone (Ac-DEVD.fmk, 50 µM, Enzyme Systems); and/or bongkrekic acid (BA, 50 µM, provided by Dr. Duine, Delft University, Delft, The Netherlands). Preliminary experiments were performed with variable doses of these inhibitors (1 to 200 µM), and results are shown for optimal nontoxic concentrations of each agent.

Determination of apoptosis-associated parameters

In accord with published protocols (39, 40, 41), the following fluorochromes were used to determine different apoptosis-associated changes: 3,3'-dihexyloxacarbocyanine iodide (DiOC₆(3), 20 nM, 15 min, 37°C) for _m quantification; hydroethidine (HE, 4 µM, 15 min, 37°C) for the determination of superoxide anion generation causing oxidation of HE to ethidium (Eth); annexin V-FITC (1 µg/ml, 10 min, 4°C, Nexins Research, Hoeven, The Netherlands) for determination of phosphatidylserine (PS) exposure on the outer plasma membrane; and finally ethidium bromide (EthBr, 200 ng/ml, 5 min, room temperature) or propidium iodide (PI, 2 µg/ml, 5 min, 37°C), which both are vital dyes. These dyes were used alone (PI) or in combination (DiOC₆(3) + HE; annexin V-FITC + EthBr). Cytofluorometry was performed on a Coulter Elite II analyzer. Fluorescence was registered for all cells (large and apoptotic), while excluding debris, in FL1 (DiOC₆(3), annexin V-FITC) or FL3 (HE, EthBr, PI). The frequency of subdiploid cells was determined by PI staining of ethanol-permeabilized cells (41).

Determination of proteasome activity

Proteasome activity was determined by means of the cell-permeable fluorogenic substrate N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido4-methylcoumarin (Bachem), following published protocols (42). Cells (4 x 10⁶ in 200 µl of PBS, pH 7.4) were incubated during 30 min at 37°C with this substrate (100 µM), and the 7-amino-4-methylcoumarin fluorescence generated by its cleavage was measured in a Kontron SFM 25 spectrofluorometer (Kontron AG, Zurich, Switzerland). The excitation wavelength was set at 380 nm, and the emission wavelength was set at 460 nM. Background values of nonstimulated cells were not reduced by the proteasome inhibitors MG132 (30 µM) or lactacystin (30 µM) and thus were subtracted from the experimental values.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Inhibition of proteasomes prevents thymocyte apoptosis at the premitochondrial level

The glucocorticoid receptor agonist DEX and the topoisomerase type II inhibitor etoposide both induce apoptosis in thymocytes. Simultaneously, they cause an increase in proteasome activity, as determined by means of the fluorescent substrate N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcoumarin (Fig. 1). Several different proteasome inhibitors, which have been used at nontoxic doses, reduce this enzymatic activity. This applies to the specific proteasome inhibitors MG132 and lactacystin (21), as well as to LLnL, an inhibitor of proteasomes and calpain (21, 42) (Fig. 1). We have evaluated the effect of these inhibitors on several apoptosis-associated parameters quantified by cytofluorometry (Fig. 2). The mitochondrial transmembrane potential (_m) was measured using the potential-sensitive cationic lipophilic dye DiOC₆(3), which incorporates into the mitochondrial matrix as a function of the Nernst equation, correlating with the _m (43) (Fig. 2A). The overproduction of superoxide anion by the uncoupled respiratory chain was quantified by the oxidative conversion of nonfluorescent, cell-permeable HE into fluorescent Eth, which is retained in the cell due to its hydrophily (39) (Fig. 2A). Moreover, we measured, among viable (i.e., EthBr-excluding) cells, the apoptosis-associated exposure of PS on the outer leaflet of the plasma membrane using the PS-specific protein annexin V conjugated to FITC (Fig. 2B). The endonuclease-driven loss of nuclear DNA leading to hypoploidy was measured with the DNA-intercalating dye PI, after permeabilizatin of cells with ethanol. MG132, lactacystin, and LLnL inhibit the dissipation of the _m induced by DEX and etoposide (Fig. 2A), in addition to suppressing the generation of superoxide anion (Fig. 2A), the loss of nuclear DNA leading to hypoploidy (numbers in black circles in Fig. 2A), and the exposure of PS (Fig. 2B). The cytoprotective effect of these agents correlates with the inhibition of proteasome enzymatic activity (Figs. 1 and 2). Collectively, these results indicate that proteasome inhibition interrupts the apoptotic process at an early step, upstream of mitochondria.

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Stimulation of proteasome activity by etoposide or DEX. Thymocytes were cultured for 5 h in the presence of the indicated agents (gray areas), followed by addition of the proteasome substrate N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcoumarin and the determination of 7-amino-4-methylcoumarin fluorescence. This experiment was repeated twice yielding similar results.

View larger version (63K): [in this window] [in a new window]   FIGURE 2. Effects of three proteasome inhibitors (MG132, lactacystin, and LLnL) on thymocyte apoptosis induced by DEX or etoposide. Cells were cultured for 5 h in the presence of the indicated combination of agents, followed by staining with DiOC6(3) and HE (A) or FITC-annexin V and EthBr (B), as described in Materials and Methods. Numbers refer to percentage of cells found in each quadrant. In addition, aliquots of cells were fixed and permeabilized with ethanol and stained with PI to determine the frequency of hypoploid nuclei (numbers in black circles in A). Results are representative of three independent experiments.

Proteasome activation occurs upstream of _m dissipation and caspase activation

We have used BA, a ligand of the adenine nucleotide translocator and an inhibitor of the mitochondrial megachannel (also called permeability transition pore) (40, 44, 45, 46), and Z-VAD.fmk, a broad spectrum inhibitor of caspases (11, 12, 13, 14, 15, 16, 17, 18, 19), to evaluate the impact of the megachannel and caspases on proteasome function. As shown in Figure 3A, neither of these two agents prevents the activation of proteasomes induced by etoposide or DEX. In parallel control experiments, BA stabilizes the _m (measured by means of the potential-sensitive dye DiOC₆(3)) of thymocytes treated with DEX or etoposide (40, 46) (Fig. 3B). Moreover, it prevents the postmitochondrial manifestations of apoptosis including superoxide anion generation (measured by the oxidative conversion of nonfluorescent HE into fluorescent Eth) (Fig. 3B), DNA fragmentation (measured by PI staining of ethanol-permeabilized cells) (Fig. 3C), PS exposure (measured by means of annexin V-FITC conjugate) (Fig. 3C), and maintains cell viability (measured with EthBr) (Fig. 3C) (40), thus confirming that opening of the megachannel is a critical event of the apoptotic process. Z-VAD.fmk blocks most of the apoptosis-associated changes (complete _m disruption, superoxide anion generation, PS exposure, nuclear apoptosis) (Fig. 3, B and C), with the notable exception of the initial _m dissipation (19) (Fig. 3B). Thus, both BA and Z-VAD.fmk interrupt the apoptotic process downstream of proteasome activation. These data support the idea that proteasome activation occurs upstream of and independently from _m dissipation and caspase activation.

View larger version (36K): [in this window] [in a new window]   FIGURE 3. Effect of BA and the caspase inhibitor Z-VAD.fmk on proteasome activation and apoptosis. Thymocytes were cultured during 5 h in the presence of the indicated agent, followed by determination of proteasome activity (A), as in Figure 1, or the indicated apoptosis-associated parameters (B, C), as in Figure 2. Circles in C indicate the percentage of hypoploid cells.

In addition to proteasome inhibitors, a number of protease inhibitors have been reported to prevent thymocyte apoptosis (7, 11, 16, 19). We therefore have evaluated the effect of different protease inhibitors not acting on proteasomes on several parameters of thymocyte apoptosis. Two inhibitors of caspases, B-D.fmk and Ac-DEVD.fmk, which do not inhibit proteasomes (Refs. 21 and 22, Fig. 3A, and data not shown) largely prevent the PI-detectable loss of nuclear DNA (Fig. 4B) and concomitantly prevent oligonucleosomal DNA fragmentation (Ref. 16 and data not shown). Similarly, the chymotryptic inhibitor TPCK, which does not inhibit caspases (47), prevents DNA fragmentation in this model (Fig. 4B). However, these inhibitors are relatively inefficient in preventing the initial step of _m dissipation (Fig. 4A), as has been observed for Z-VAD.fmk (19) (Fig. 3B). Thus, in contrast to proteasome inhibitors (see above), B-D.fmk, Ac-DEVD.fmk, and TPCK act on a postmitochondrial rather than a premitochondrial step of the apoptotic process. On prolonged culture of thymocytes (12 h), B-D.fmk, Ac-DEVD.fmk, and TPCK fail to maintain the viability of cells, as assessed with PI. In the same conditions, however, the proteasome inhibitors MG132 and lactacystin do maintain cell viability (Fig. 5). These results support the contention that only protease inhibitors capable of interrupting the process of apoptosis at the premitochondrial stage (lactacystin, MG132) but not those acting at the postmitochondrial stage (B-D.fmk, Ac-DEVD.fmk, TPCK) are truly cytoprotective.

View larger version (51K): [in this window] [in a new window]   FIGURE 4. Effect of diverse protease inhibitors on mitochondrial alterations and nuclear apoptosis induced by DEX or etoposide. After 5 h of culture in the presence of the indicated agent, cells were subjected to the determination of the m and superoxide anion generation (A), or determination of hypoploidy (B). Results are representative of three independent experiments.

View larger version (52K): [in this window] [in a new window]   FIGURE 5. Effect of different protease inhibitors on thymocyte viability. After 12 h of culture in the presence of different combinations of protease inhibitors and apoptosis inducers, cells were stained with PI, and the frequency of cells having completely lost the capacity of PI exclusion (PI++) or of cells with a partial disruption of membrane function (PI+) was determined. Representative staining profile of thymocytes obtained after 12 h of culture in the presence or absence of etoposide and MG132 are shown in A. Asterisks in B denote significant inhibitory effects of MG132 and lactacystin (three independent experiments, p < 0.001, paired Student t test).

In the models of apoptosis studied herein, inhibition of proteasomes interrupts the process at an early, premitochondrial step. Since proteasomes and mitochondria are usually not found in close association (48), it is unlikely that proteasomes would have a direct effect on mitochondria. At present, it is not known which is/are the critical protein(s) that must be ubiquinated and then degraded by the proteasome to facilitate apoptosis induction. However, it appears plausible to assume that the proteasome participates in one or several signaling pathways during the premitochondrial initiation phase of apoptosis. In accord with this speculation, we found that inhibition of the mitochondrial megachannel and inhibition of caspase activation do not interfere with the activation of proteasomes, whereas MG132 and lactacystin, two specific inhibitors of proteasomes, prevent the _m dissipation induced by etoposide and DEX (Figs. 2 and 3A). Concomitantly, MG132 and lactacystin inhibit all extramitochondrial manifestations of apoptosis including nuclear DNA fragmentation, plasma membrane PS exposure, and cytolysis (Figs. 2 and 3). In strict contrast with these proteasome inhibitors, caspase inhibitors (B-D.fmk, Ac-DEVD.fmk) or the chymotryptic inhibitor TPCK fail to prevent the _m dissipation (Fig. 4A) and later cytolysis (Fig. 5B), although they do prevent DNA fragmentation (Fig. 4B). These latter agents thus act at a postmitochondrial step and ultimately are incapable of preventing lytic cell death.

The data contained in this work suggest that the manifestation of the mitochondrial changes have a higher predictive value for cell death than the activation of catabolic enzymes including caspases and nucleases. Indeed, in a number of models of apoptosis, the broad spectrum caspase inhibitor Z-VAD.fmk can prevent caspase and consequent nuclease activation yet fails to prevent the disruption of the _m and cytolysis. This applies to cell death induced by overexpression of Bax (17), opening of the mitochondrial megachannel by protoporphyrin IX (45), ligation of the glucocorticoid receptor, or topoisomerase II inhibition (19). Moreover, cross-linking of CD45 or CD99 can cause a type of thymocyte death that is preceded by _m disruption but does not involve the activation of caspases and nucleases (49, 50). Conversely, activation of caspases can occur without cell death, as this has been demonstrated for activated T cells (51), T cell hybridoma cells transfected with ALG-2 (52), and CD95-stimulated Jurkat cells overexpressing Bcl-X_L (53). Altogether these findings support the idea that, at least in some models, mitochondrial changes are more important in determining cell death than caspase activation. It remains to be clarified, however, which changes in cellular physiology finally account for caspase-independent cytolysis. On theoretical grounds, the mitochondrial perturbations occurring during apoptosis should provoke an insufficiency in energy metabolism, ion homeostasis, and/or redox balance that altogether compromise plasma membrane integrity. However, the exact mechanisms linking mitochondrial dysfunction and cell lysis await further characterization.

In conclusion, our data support a scenario in which proteasomes act upstream of mitochondria to regulate cell death, whereas some caspases act downstream of mitochondria to participate in the acquisition of the apoptotic phenotype, beyond the point of no return leading to cell death.

	Acknowledgments

 
We thank Dr. Duine (Delft University, Delft, The Netherlands) for the gift of BA.

	Footnotes

 
¹ This work was supported by grants from Association pour la Recherche sur le Cancer, Agence Nationale de Recherches sur le SIDA, Centre National de la Recherche Scientifique, Fondation pour la Recherche Médicale, Hoechst-Marion-Roussel, Institut National de la Santé et de la Recherche Médicale, La Lutte Contre le Cancre, and Sidaction (to G.K.). S.A.S. and I.M. received fellowships from the European Commission and the Spanish Ministry of Sciences, respectively.

² Address correspondence and reprint requests to Dr. Guido Kroemer, 19 rue Guy Môquet, Bôite Postale 8, F-94801 Villejuif, France. E-mail address:

³ Abbreviations used in this paper: Z-VAD.fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; Ac-DEVD.fmk, acetyl-Asp-Glu-Val-Asp-fluoromethylketone; B-D.fmk, Boc-Asp(OMe)-fluoromethylketone; _m, mitochondrial transmembrane potential; DEX, dexamethasone; Eth, ethidium; DiOC₆(3), 3,3'-dihexyloxacarbocyanine iodide; HE, hydroethidine; LLnL, N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (= calpain inhibitor I); PI, propidium iodide; PS, phosphatidylserine; TPCK, N-tosyl-L-Phe-chloromethylketone; BA, bongkrekic acid.

Received for publication December 23, 1997. Accepted for publication February 25, 1998.

	References

Top Abstract Introduction Materials and Methods Results and Discussion References

 

1. Cohen, J. J.. 1993. Apoptosis. Immunol. Today 14:126.[Medline]
2. Thompson, C. B.. 1995. Apoptosis in the pathogenesis and treatment of disease. Science 267:1456.[Abstract/Free Full Text]
3. Kumar, S., M. F. Lavin. 1996. The ICE family of cysteine proteases as effectors of cell death. Cell Death Differ. 3:255.
4. Henkart, P. A.. 1996. ICE family proteases: mediators of all apoptotic cell death?. Immunity 4:195.[Medline]
5. Chinnaiyan, A. M., V. M. Dixit. 1996. The cell-death machine. Curr. Biol. 6:555.[Medline]
6. Fraser, A., G. Evan. 1996. A license to kill. Cell 85:781.[Medline]
7. Zhivotovsky, B., D. H. Burgess, D. M. Vanags, S. Orrenius. 1997. Involvement of cellular proteolytic machinery in apoptosis. Biochem. Biophys. Res. Commun. 230:481.[Medline]
8. Bump, N. J., M. Hackett, M. Hugunin, S. Seshagiri, K. Brady, P. Chen, C. Ferenz, S. Franklin, T. Ghayur, P. Li, P. Licari, J. Mankovich, L. F. Shi, A. H. Greenberg, L. K. Miller, W. W. Wong. 1995. Inhibition of ICE family proteases by baculovirus antiapoptotic protein p35. Science 269:1885.[Abstract/Free Full Text]
9. Robertson, N. M., J. Zangrilli, T. Fernandesalnemri, P. D. Friesen, G. Litwack, E. S. Alnemri. 1997. Baculovirus P35 inhibits the glucocorticoid-mediated pathway of cell death. Cancer Res. 57:43.[Abstract/Free Full Text]
10. Datta, R., H. Kojima, D. Banach, N. J. Bump, R. V. Talanian, E. S. Alnemri, R. R. Weichselbaum, W. W. Wong, D. W. Kufe. 1997. Activation of a CrmA-insensitive, p35-sensitive pathway in ionizing radiation-induced apoptosis. J. Biol. Chem. 272:1965.[Abstract/Free Full Text]
11. Fearnhead, H. O., A. J. Rivett, D. Dinsdale, G. M. Cohen. 1995. A pre-existing protease is a common effector of thymocyte apoptosis mediated by diverse stimuli. FEBS Lett. 357:242.[Medline]
12. Zhu, H. J., H. O. Fearnhead, G. M. Cohen. 1995. An ICE-like protease is a common mediator of apoptosis induced by diverse stimuli in human monocytic THP.1 cells. FEBS Lett. 374:303.[Medline]
13. Cain, K., S. H. Inayathussain, C. Couet, G. M. Cohen. 1996. A cleavage-site-directed inhibitor of interleukin 1 beta-converting enzyme-like proteases inhibits apoptosis in primary cultures of rat hepatocytes. Biochem. J. 314:27.
14. Slee, E. A., H. J. Zhu, S. C. Chow, M. Macfarlane, D. W. Nicholson, G. M. Cohen. 1996. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.fmk) inhibits apoptosis by blocking the processing of CPP32. Biochem. J. 315:21.
15. Jacobson, M. D., M. Weil, M. C. Raff. 1996. Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death. J. Cell Biol. 133:1041.[Abstract]
16. Sarin, A., M. L. Wu, P. A. Henkart. 1996. Different interleukin-1 beta converting enzyme (ICE) family protease requirements for the apoptotic death of T lymphocytes triggered by diverse stimuli. J. Exp. Med. 184:2445.[Abstract/Free Full Text]
17. Xiang, J., D. T. Chao, S. J. Korsmeyer. 1996. Bax-induced cell death may not require interleukin 1beta-converting enzyme-like proteases. Proc. Natl. Acad. Sci. USA 93:14559.[Abstract/Free Full Text]
18. McCarthy, N. J., M. K. B. Whyte, C. S. Gilbert, G. I. Evan. 1997. Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. J. Cell Biol. 136:215.[Abstract/Free Full Text]
19. Hirsch, T., P. Marchetti, S. A. Susin, B. Dallaporta, N. Zamzami, I. Marzo, M. Geuskens, G. Kroemer. 1997. The apoptosis-necrosis paradox: apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene 15:1573.[Medline]
20. Dubrez, L., I. Savoy, A. Hamman, E. Solary. 1996. Pivotal role of a DEVD-sensitive step in etoposide-induced and Fas-mediated apoptotic pathways. EMBO J. 15:5504.[Medline]
21. Grimm, L. M., A. L. Goldberg, G. G. Poirier, L. M. Schwartz, B. A. Osborne. 1996. Proteasomes play an essential role in thymocyte apoptosis. EMBO J. 15:3835.[Medline]
22. Sadoul, R., P. A. Fernandez, A. L. Quiquerez, I. Martinou, M. Maki, M. Schroter, J. D. Becherer, M. Irmler, J. Tschopp, J. C. Martinou. 1996. Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons. EMBO J. 15:3845.[Medline]
23. Cui, H. L., K. Matsui, S. Omura, S. L. Schauer, R. A. Matulka, G. E. Sonnenshein, S. T. Ju. 1997. Proteasome regulation of activation-induced T cell death. Proc. Natl. Acad. Sci. USA 94:7515.[Abstract/Free Full Text]
24. Matsui, K., S. Omura, H. L. Cui, S. L. Schauer, G. E. Sonenshein, S. T. Ju. 1997. Proteasome regulation of Fas ligand cytotoxicity. Eur. J. Immunol. 27:2269.[Medline]
25. Jones, M. E. E., M. F. Haire, P. M. Kloetzel, D. L. Mykles, L. M. Schwartz. 1995. Changes in the structure and function of the multicatalytic proteinase (proteasome) during programmed cell death in the intersegmental muscles of the hawkmoth, Manduca sexta. Dev. Biol. 169:436.[Medline]
26. Low, P., K. Bussell, S. P. Daxson, M. A. Billet, R. J. Mayer, S. E. Reynolds. 1997. Expression of a 26S proteasome ATPase subunit, MS73, in muscles that undergo developmentally programmed cell death, and its control by ecdysteroid hormones in the insect Manduca sexta. FEBS Lett. 400:345.[Medline]
27. Shimizu, T., Y. Pommier. 1996. DNA fragmentation induced by protease activation in p53-null human leukemia HL60 cells undergoing apoptosis following treatment with the topoisomerase I inhibitor camptothecin: cell-free system studies. Exp. Cell Res. 226:292.[Medline]
28. Shinohara, K., M. Tomioka, H. Nakano, S. Tone, H. Ito, S. Kawashima. 1996. Apoptosis induction resulting from proteasome inhibition. Biochem. J. 317:385.
29. Tanimoto, Y., Y. Onishi, S. Hashimoto, H. Kizaki. 1997. Peptidyl aldehyde inhibitors of proteasome induce apoptosis rapidly in mouse lymphoma RVC cells. J. Biochem. 121:542.[Abstract/Free Full Text]
30. Lopes, U. G., P. Erhardt, R. J. Yao, G. M. Cooper. 1997. p53-dependent induction of apoptosis by proteasome inhibitors. J. Biol. Chem. 272:12893.[Abstract/Free Full Text]
31. Drexler, H. C. A.. 1997. Activation of the cell death program by inhibition of proteasome function. Proc. Natl. Acad. Sci. USA 94:855.[Abstract/Free Full Text]
32. Kroemer, G., N. Zamzami, S. A. Susin. 1997. Mitochondrial control of apoptosis. Immunol. Today 18:44.[Medline]
33. vander Heiden, M. G., N. S. Chandal, E. K. Williamson, P. T. Shcumacker, C. B. Thompson. 1997. Bcl-XL regulates the membrane potential and volume homeostasis of mitochondria. Cell 91:627.[Medline]
34. Liu, X., C. N. Kim, J. Yang, R. Jemmerson, X. Wang. 1996. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome. c. Cell 86:147.
35. Yang, J., X. Liu, K. Bhalla, C. N. Kim, A. M. Ibrado, J. Cai, T.-I. Peng, D. P. Jones, X. Wang. 1997. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275:1129.[Abstract/Free Full Text]
36. Kluck, R. M., E. Bossy-Wetzel, D. R. Green, D. D. Newmeyer. 1997. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275:1132.[Abstract/Free Full Text]
37. Ellerby, H. M., S. J. Martin, L. M. Ellerby, S. S. Naiem, S. Rabizadeh, G. S. Salvese, C. A. Casiano, N. R. Cashman, D. R. Green, D. E. Bredesen. 1997. Establishment of a cell-free system of neuronal apoptosis: comparison of premitochondrial, mitochondrial, and postmitochondrial phases. J. Neurosci. 17:6165.[Abstract/Free Full Text]
38. Susin, S. A., N. Zamzami, M. Castedo, E. Daugas, H.-G. Wang, S. Geley, F. Fassy, J. Reed, G. Kroemer. 1997. The central executioner of apoptosis: multiple links between protease activation and mitochondria in Fas/Apo-1/CD95- and ceramide-induced apoptosis. J. Exp. Med. 186:25.[Abstract/Free Full Text]
39. Zamzami, N., P. Marchetti, M. Castedo, D. Decaudin, A. Macho, T. Hirsch, S. A. Susin, P. X. Petit, B. Mignotte, G. Kroemer. 1995. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J. Exp. Med. 182:367.[Abstract/Free Full Text]
40. Marchetti, P., M. Castedo, S. A. Susin, N. Zamzami, T. Hirsch, A. Haeffner, F. Hirsch, M. Geuskens, G. Kroemer. 1996. Mitochondrial permeability transition is a central coordinating event of apoptosis. J. Exp. Med. 184:1155.[Abstract/Free Full Text]
41. Kroemer, G., B. Lisardo, P. Zamzami, S. Hortelano, and C. Martinez-A. 1997. Detection of apoptosis and apoptosis associated alterations. In The Immunology Methods Manual, Ch. 14.2. R. Lefkovitz, ed. Academic Press, New York, pp. 1111–1125.
42. Reidlinger, J., A. M. Pike, P. J. Savory, R. Z. Murray, A. J. Rivett. 1997. Catalytic properties of 26 S and 20 S proteasomes and radiolabeling of MB1, LMP7, and C7 subunits associated with trypsin-like and chymotrypsin-like activities. J. Biol. Chem. 272:24899.[Abstract/Free Full Text]
43. Zamzami, N., P. Marchetti, M. Castedo, C. Zanin, J.-L. Vayssière, P. X. Petit, G. Kroemer. 1995. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181:1661.[Abstract/Free Full Text]
44. Zoratti, M., I. Szabò. 1995. The mitochondrial permeability transition. Biochim. Biophys. Acta 1241:139.[Medline]
45. Marchetti, P., T. Hirsch, N. Zamzami, M. Castedo, D. Decaudin, S. A. Susin, B. Masse, G. Kroemer. 1996. Mitochondrial permeability transition triggers lymphocyte apoptosis. J. Immunol. 157:4830.[Abstract]
46. Zamzami, N., S. A. Susin, P. Marchetti, T. Hirsch, I. Gómez-Monterrey, M. Castedo, G. Kroemer. 1996. Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183:1533.[Abstract/Free Full Text]
47. Dong, Z., P. Saikumar, J. M. Weinberg, M. A. Vekatachalam. 1997. Internucleosomal DNA cleavage triggered by plasma membrane damage during necrotic cell death: involvement of serine but not cysteine proteases. Am. J. Pathol. 151:1205.[Abstract]
48. Palmer, A., A. J. Rivett, S. Thomson, K. B. Hendil, G. W. Butcher, G. Fuertes, E. Knecht. 1996. Subpopulations of proteasomes in rat liver nuclei, microsomes and cytosol. Biochem. J. 316:401.
49. Lesage, S., A.-M. Steff, F. Philippoussis, M. Pagé, S. Trop, V. Mateo, P. Hugo. 1997. CD4⁺CD8⁺ thymocytes are preferentially induced to die following CD45 cross-linking, through a novel apoptotic pathway. J. Immunol. 159:4762.[Abstract]
50. Bernard, G., J. P. Breittmayer, M. Dematteis, P. Trampont, P. Hofman, A. Senik, A. Bernard. 1997. Apoptosis of immature thymocytes mediated by E2/CD99. J. Immunol. 158:2543.[Abstract]
51. Miossec, C., V. Dutilleul, F. Fassy, A. Diu-Hercend. 1997. Evidence for CPP32 activation in the absence of apoptosis during T lymphocyte stimulation. J. Biol. Chem. 272:13459.[Abstract/Free Full Text]
52. Lacana, E., J. K. Ganjei, P. Vito, L. Dadamio. 1997. Dissociation of apoptosis and activation of IL-1 beta-converting enzyme/Ced-3 proteases by ALG-2 and the truncated Alzheimer’s gene ALG-3. J. Immunol. 158:5129.[Abstract]
53. Boise, L. H., C. B. Thompson. 1997. Bcl-XL can inhibit apoptosis in cells that have undergone Fas-induced protease activation. Proc. Natl. Acad. Sci. USA 94:3759.[Abstract/Free Full Text]

This article has been cited by other articles:

				Q. Miao, Y. Sun, T. Wei, X. Zhao, K. Zhao, L. Yan, X. Zhang, H. Shu, and F. Yang Chymotrypsin B Cached in Rat Liver Lysosomes and Involved in Apoptotic Regulation through a Mitochondrial Pathway J. Biol. Chem., March 28, 2008; 283(13): 8218 - 8228. [Abstract] [Full Text] [PDF]

				C. Yang, V. Kaushal, S. V. Shah, and G. P. Kaushal Mcl-1 is downregulated in cisplatin-induced apoptosis, and proteasome inhibitors restore Mcl-1 and promote survival in renal tubular epithelial cells Am J Physiol Renal Physiol, June 1, 2007; 292(6): F1710 - F1717. [Abstract] [Full Text] [PDF]

				G. Kroemer, L. Galluzzi, and C. Brenner Mitochondrial Membrane Permeabilization in Cell Death Physiol Rev, January 1, 2007; 87(1): 99 - 163. [Abstract] [Full Text] [PDF]

				D. Sohn, G. Totzke, F. Essmann, K. Schulze-Osthoff, B. Levkau, and R. U. Janicke The proteasome is required for rapid initiation of death receptor-induced apoptosis. Mol. Cell. Biol., March 1, 2006; 26(5): 1967 - 1978. [Abstract] [Full Text] [PDF]

				R. Auger, I. Motta, K. Benihoud, D. M. Ojcius, and J. M. Kanellopoulos A Role for Mitogen-activated Protein KinaseErk1/2 Activation and Non-selective Pore Formation in P2X7 Receptor-mediated Thymocyte Death J. Biol. Chem., July 29, 2005; 280(30): 28142 - 28151. [Abstract] [Full Text] [PDF]

				S. Lepine, B. Lakatos, M.-P. Courageot, H. Le Stunff, J.-C. Sulpice, and F. Giraud Sphingosine Contributes to Glucocorticoid-Induced Apoptosis of Thymocytes Independently of the Mitochondrial Pathway J. Immunol., September 15, 2004; 173(6): 3783 - 3790. [Abstract] [Full Text] [PDF]

				Y. YANG and X. YU Regulation of apoptosis: the ubiquitous way FASEB J, May 1, 2003; 17(8): 790 - 799. [Abstract] [Full Text] [PDF]

				B.-J. Kroesen, S. Jacobs, B. J. Pettus, H. Sietsma, J. W. Kok, Y. A. Hannun, and L. F. M. H. de Leij BcR-induced Apoptosis Involves Differential Regulation of C16 and C24-Ceramide Formation and Sphingolipid-dependent Activation of the Proteasome J. Biol. Chem., April 18, 2003; 278(17): 14723 - 14731. [Abstract] [Full Text] [PDF]

				H.-K. Lin, S. Altuwaijri, W.-J. Lin, P.-Y. Kan, L. L. Collins, and C. Chang Proteasome Activity Is Required for Androgen Receptor Transcriptional Activity via Regulation of Androgen Receptor Nuclear Translocation and Interaction with Coregulators in Prostate Cancer Cells J. Biol. Chem., September 20, 2002; 277(39): 36570 - 36576. [Abstract] [Full Text] [PDF]

				R. Z. Orlowski, G. W. Small, and Y. Y. Shi Evidence That Inhibition of p44/42 Mitogen-activated Protein Kinase Signaling Is a Factor in Proteasome Inhibitor-mediated Apoptosis J. Biol. Chem., July 26, 2002; 277(31): 27864 - 27871. [Abstract] [Full Text] [PDF]

				K.-O. Chay, S. S. Park, and J. F. Mushinski Linkage of Caspase-mediated Degradation of Paxillin to Apoptosis in Ba/F3 Murine Pro-B Lymphocytes J. Biol. Chem., April 19, 2002; 277(17): 14521 - 14529. [Abstract] [Full Text] [PDF]

				J. Chandra, E. Mansson, V. Gogvadze, S. H. Kaufmann, F. Albertioni, and S. Orrenius Resistance of leukemic cells to 2-chlorodeoxyadenosine is due to a lack of calcium-dependent cytochrome c release Blood, January 15, 2002; 99(2): 655 - 663. [Abstract] [Full Text] [PDF]

				H. Hilbi, R. J. Puro, and A. Zychlinsky Tripeptidyl Peptidase II Promotes Maturation of Caspase-1 in Shigella flexneri-Induced Macrophage Apoptosis Infect. Immun., October 1, 2000; 68(10): 5502 - 5508. [Abstract] [Full Text] [PDF]

				P. Masdehors, H. Merle-Beral, K. Maloum, S. Omura, H. Magdelenat, and J. Delic Deregulation of the ubiquitin system and p53 proteolysis modify the apoptotic response in B-CLL lymphocytes Blood, July 1, 2000; 96(1): 269 - 274. [Abstract] [Full Text] [PDF]

				C. L. Mann, F. M. Hughes Jr., and J. A. Cidlowski Delineation of the Signaling Pathways Involved in Glucocorticoid-Induced and Spontaneous Apoptosis of Rat Thymocytes Endocrinology, February 1, 2000; 141(2): 528 - 538. [Abstract] [Full Text] [PDF]

				N. Canu, C. Barbato, M. T. Ciotti, A. Serafino, L. Dus, and P. Calissano Proteasome Involvement and Accumulation of Ubiquitinated Proteins in Cerebellar Granule Neurons Undergoing Apoptosis J. Neurosci., January 15, 2000; 20(2): 589 - 599. [Abstract] [Full Text] [PDF]

				R. A. Hastings, I. Eyheralde, S. P. Dawson, G. Walker, S. E. Reynolds, M. A. Billett, and R. J. Mayer A 220-kDa Activator Complex of the 26 S Proteasome in Insects and Humans. A ROLE IN TYPE II PROGRAMMED INSECT MUSCLE CELL DEATH AND CROSS-ACTIVATION OF PROTEASOMES FROM DIFFERENT SPECIES J. Biol. Chem., September 3, 1999; 274(36): 25691 - 25700. [Abstract] [Full Text] [PDF]

				B. B. Wolf and D. R. Green Suicidal Tendencies: Apoptotic Cell Death by Caspase Family Proteinases J. Biol. Chem., July 16, 1999; 274(29): 20049 - 20052. [Full Text] [PDF]

				G. Tang and S. H. Leppla Proteasome Activity Is Required for Anthrax Lethal Toxin To Kill Macrophages Infect. Immun., June 1, 1999; 67(6): 3055 - 3060. [Abstract] [Full Text] [PDF]

				B. Dallaporta, P. Marchetti, M. A. de Pablo, C. Maisse, H.-T. Duc, D. Metivier, N. Zamzami, M. Geuskens, and G. Kroemer Plasma Membrane Potential in Thymocyte Apoptosis J. Immunol., June 1, 1999; 162(11): 6534 - 6542. [Abstract] [Full Text] [PDF]

				C. Leveille, H. Zekki, R. Al-Daccak, and W. Mourad CD40- and HLA-DR-mediated cell death pathways share a lot of similarities but differ in their use of ADP-ribosyltransferase activities Int. Immunol., May 1, 1999; 11(5): 719 - 730. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager