From the Laboratory of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and
University of Ghent, B-9000 Ghent, Belgium
Murine L929 fibrosarcoma cells treated with tumor necrosis factor (TNF) rapidly die in a necrotic way, due to excessive formation of reactive oxygen intermediates. We investigated the
role of caspases in the necrotic cell death pathway. When the cytokine response modifier A
(CrmA), a serpin-like caspase inhibitor of viral origin, was stably overexpressed in L929 cells,
the latter became 1,000-fold more sensitive to TNF-mediated cell death. In addition, TNF
sensitization was also observed when the cells were pretreated with Ac-YVAD-cmk or
zDEVD-fmk, which inhibits caspase-1- and caspase-3-like proteases, respectively. zVAD-fmk
and zD-fmk, two broad-spectrum inhibitors of caspases, also rendered the cells more sensitive,
since the half-maximal dose for TNF-mediated necrosis decreased by a factor of 1,000. The
presence of zVAD-fmk also resulted in a more rapid increase of TNF-mediated production of oxygen radicals. zVAD-fmk-dependent sensitization of TNF cytotoxicity could be completely
inhibited by the oxygen radical scavenger butylated hydroxyanisole. These results indicate an
involvement of caspases in protection against TNF-induced formation of oxygen radicals and
necrosis.
 |
Introduction |
Tumor necrosis factor (TNF) is an important mediator
in many immunological and inflammatory responses,
as well as in a number of pathological conditions. In vitro,
TNF is able to induce cell death, activation of transcription
factors, and proliferation (1). In murine L929 fibrosarcoma cells, TNF induces necrosis, a type of cell death characterized by swelling, finally leading to disruption of the
plasma membrane (4). This cytotoxicity is due to an increase in oxygen radical accumulation; inhibition of this
process by particular radical scavengers blocks TNF-induced cell death (5). On the other hand, L929 cells can be killed by apoptosis when human Fas antigen is expressed and triggered by agonistic antibodies (6, 7). Apoptosis is mainly
characterized by membrane blebbing, DNA fragmentation,
shrinking, and condensation of the cells and their organelles, and subsequent disintegration (8).
Recent research has documented that caspases play an
important role in apoptosis (9). Inhibition of one or
more caspases can block apoptotic cell death induced by
several stimuli; current models implicate caspase-8, and
possibly caspase-10b, as key mediators of TNF- and Fas-mediated apoptosis (12). In this study, we investigated the role of caspases in TNF-mediated necrosis. We found
that treatment of L929 cells with caspase inhibitors sensitize
rather than protect against this mode of cell death.
| |
Materials and Methods |
Cells.
L929 murine fibrosarcoma cells and HeLa H21 cervix
carcinoma cells were cultured in DMEM supplemented with 5%
newborn bovine serum and 5% FCS, penicillin (100 U/ml) streptomycin (0.1 mg/ml), and L-glutamine (0.03%). KYM rhabdomyosarcoma and PC60R55R75 murine T cell hybridoma cells were
cultured in RPMI 1640, supplemented with 10% FCS, penicillin
(100 U/ml), streptomycin (0.1 mg/ml), and L-glutamine (0.03%),
and additionally 2-mercaptoethanol (5 × 10
5 M) and sodium
pyruvate (1 mM) for PC60R55R75 cells.
Cytokines, Antibodies, and Reagents.
Recombinant murine TNF
was produced in our laboratory and was purified to at least 99%
homogeneity. The specific activity was 1.4 × 108 IU/mg, as determined in a standardized cytotoxicity assay on L929 cells. Actinomycin D, butylated hydroxyanisole (BHA),1 diethylmaleate
(DEM), H2O2, and tert-butyl hydroperoxide (tBuOOH) were purchased from Sigma Chemical Co. (St. Louis, MO). Monochlorobimane was supplied by Molecular Probes (Eugene, OR). Dihydrorhodamine 123 (DHR123; Molecular Probes) was prepared as
a 5 mM stock solution in DMSO and was used at 1 µM. Propidium iodide (PI; Becton Dickinson, San Jose, CA) was dissolved at
3 mM in PBS and was used at 30 µM.
The caspase peptide inhibitors zDEVD-fmk, zVAD-fmk, and
zD-fmk were purchased from Enzyme Systems Products (Dublin,
CA). Ac-YVAD-cmk and zAAD-cmk were supplied by Calbiochem-Novabiochem International (San Diego, CA). Anti-cytokine response modifier A (CrmA) antibodies were provided by
D. Pickup (Durham, NC).
Plasmids.
Cowpox CrmA cDNA (a gift from D. Pickup,
Durham, NC) was inserted as an EcoRI fragment into the EcoRI
site of pCAGGS (16). pSV2neo, which contains the neomycin-resistant gene under control of the SV40 early promoter, was
used as a selection marker (17).
Cytotoxicity Assays.
Cells were seeded on day 
1 at 2 × 104
cells/well in a 96-well plate. The next day, inhibitors and TNF
were added at the given concentrations. Typically, the cells were
incubated with TNF or H2O2 for 18 h, and cell viability was assessed using staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as previously described (18). The percentage of cell survival was calculated as follows: (A595/655 treated
cells A595/655 medium)/(A595/655 untreated cells A595/655 medium) × 100.
Measurement of Oxygen Radical Formation and Cell Death by Flow
Cytometry.
DHR123 was added at the same time as TNF to suspension cultures, obtained by seeding cells in uncoated 24-well
tissue culture plates (Sarstedt, Newton, NC). Cell samples were
taken at different time points and analyzed on a FACScalibur®
flow cytometer equipped with a 488-nm argon ion laser. PI fluorescence was detected at 610 nm and served as a measure for cell
death. Rhodamine 123 fluorescence, as a result of DHR123 oxidation, was analyzed on PI-negative cells and detected at 525 nm.
Relative rhodamine 123 fluorescence is defined as the ratio between emitted fluorescence at a given time point and initial fluorescence for the same condition.
Quantitation of Free Thiol Groups in Cell Lysates.
Cells were seeded
on day 1 at 2 × 104 cells/microwell. The next day, zVAD-fmk
(25 µM) or DEM (3 mM) were added 2 h before or 3 h after the
start of TNF treatment, respectively. After 0.5 h of incubation
with DEM, the supernatants were replaced with 400 µM
monochlorobimane (19) in PBS and incubated for 30 min. After
washing the cells, fluorescence was measured in a spectrofluorometer (CytoFluor 2300; PerSeptive Biosystems, Cambridge,
MA) at 480 nm, using an excitation wavelength of 360 nm.
Fluorogenic Substrate Assay for Caspase Activity.
Cytosolic cell extracts were prepared by lysing the cells in a buffer containing 1%
Nonidet P-40, 200 mM NaCl, 20 mM Tris/HCl, pH 7.4, 10 µg/ml leupeptin, aprotinin (0.27 trypsin inhibitory U/ml), and
100 µM PMSF. Caspase-1- or caspase-3-like activities were determined by incubation of cell lysate (containing 25 µg total protein) with 50 µM of the fluorogenic substrates Ac-YVAD-AMC or Ac-DEVD-AMC (Peptide Institute, Osaka, Japan), respectively, in 200 µl cell-free system buffer, comprising 10 mM
Hepes, pH 7.4, 220 mM mannitol, 68 mM sucrose, 2 mM NaCl,
2.5 mM KH2PO4, 0.5 mM EGTA, 2 mM MgCl2, 5 mM pyruvate, 0.1 mM PMSF, and 1 mM dithiothreitol (12). The release
of fluorescent 7-amino-4-methylcoumarin was measured for 1 h
at 2 min intervals by spectrofluorometry; data are expressed as the
increase in fluorescence as a function of time.
Measurement of Nuclear Factor (NF)-
B Activity.
L929 cells carried a reporter construct consisting of a luciferase gene under
control of the minimal chicken conalbumin promoter preceded
by three NF-B sites (20). Cells were seeded on day 1 at 2 × 104/microwell. The next day, cells were pretreated with different caspase inhibitors for 2 h and stimulated with TNF. After 3 h of
incubation, cells were lysed according to the luciferase assay protocol of Promega Biotec (Madison, WI); luciferin (Duchefa Biochemie, Haarlem, The Netherlands) was added and luciferase activity
was measured on a Topcount Luminometer (Packard Instrument
Co., Meriden, CT).
| |
Results |
Overexpression of CrmA Renders L929 Cells More Sensitive
to TNF-mediated Necrosis.
L929 cells were cotransfected with
cDNA encoding CrmA from cowpox virus and a pSV2neo
selection plasmid. Individual neomycin-resistant clones were
screened for CrmA expression by Western analysis and tested for their sensitivity to TNF-mediated necrosis (Fig.
1). Cells expressing CrmA were up to 1,000 times more
sensitive to TNF as compared to mock-transfected cells
(LD50 of ~0.05 IU/ml, as compared to ~50 IU/ml for
control clones). These results suggest a protective role for
CrmA-sensitive caspases against TNF-induced production
of oxygen radicals.
View larger version (23K):
[in this window]
[in a new window]
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1.
CrmA expression enhances TNF-induced necrosis. (A)
Western blot analysis of transfected L929 clones. Lane 1, control L929
cells transfected with pSV2neo alone; lanes 2-4, different clones cotransfected with pCAGGS CrmA and pSV2neo. Arrowhead, CrmA expression.
(B) Sensitizing effect on TNF-mediated necrosis in L929 cells.  , control;  , clone 2;  , clone 5; and  , clone 12.
|
|
Blocking of Caspases by Oligopeptide Inhibitors Sensitizes
L929 Cells to TNF-mediated Necrosis.
L929 cells were pretreated for 2 h with various caspase inhibitors, and their
sensitivity to TNF was analyzed. When the cells were pretreated with Ac-YVAD-cmk or zDEVD-fmk (100 µM),
which are tetrapeptide inhibitors of caspase-1 and caspase-3
subfamily members, respectively, they became significantly
more sensitive to TNF-mediated cell death (with LD50 of 1 IU/ml as compared to 30 IU/ml for controls; Fig. 2 A).
When Ac-YVAD-cmk and zDEVD-fmk were combined,
no additional sensitization was observed, suggesting that
they act on the same pathway. Two more broad-range
caspase-blocking agents are zVAD-fmk and zD-fmk. When
these inhibitors were added before TNF stimulation at a
concentration of 25 µM, they drastically sensitized the cells
to TNF (LD50 of 0.02 IU/ml). In contrast, zAAD-cmk, an
inhibitor of granzyme B, did not alter TNF sensitivity, excluding nonspecific effects. Taken together, it is evident
that members of the caspase family are responsible for protection against TNF-induced necrosis in L929 cells. Presumably additional caspases besides caspase-1 or caspase-3
are involved in this protective effect, as suggested by the
weak sensitization by Ac-YVAD-cmk and zDEVD-fmk,
compared to the strong effect of zVAD-fmk and zD-fmk.
View larger version (23K):
[in this window]
[in a new window]
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
Sensitizing effect of peptide caspase inhibitors on TNF-induced necrosis in L929 cells, added 2 h before TNF treatment. (A) Without addition
of BHA. , Ac-YVAD-cmk (100 µM); , zDEVD-fmk (100 µM); , Ac-YVAD-cmk + zDEVD-fmk (100 µM each); , zVAD-fmk (25 µM);  ,
zD-fmk (25 µM);  , zAAD-cmk (100 µM); and  , control. (B) With BHA (100 µM) added at the same time as TNF (same symbols as in A).
|
|
Sensitization of TNF-induced Necrosis by Peptide Caspase Inhibitors Is Abrogated by BHA.
Death of L929 cells after incubation with TNF follows excessive production of oxygen radicals in the mitochondria, and scavenging of these
radicals by some antioxidants, such as BHA, protects the
cells (5). When the effect of peptide caspase inhibitors on
TNF-induced necrosis of L929 cells was analyzed in the
presence of BHA, their sensitizing effect was completely
abrogated in the case of zDEVD-fmk or Ac-YVAD-cmk,
and to a great extent, when zVAD-fmk or zD-fmk were
used (Fig. 2 B). This indicates that sensitization by caspase
inhibitors enhances oxygen radical-dependent cytotoxicity.
Enhanced Cytotoxicity in the Presence of zVAD-fmk Is Correlated with Increased Oxygen Radical Accumulation.
Oxygen
radical accumulation was fluorimetrically measured using
DHR123 oxidation as a specific marker. Since rhodamine
123 fluorescence was measured in cells with intact membranes (PI-negative), the influence of PI fluorescence could
be ruled out. As shown in Fig. 3 A, incubation of L929
cells with TNF resulted in a small but significant increase of
oxygen radicals, which could be blocked by BHA. However, when the cells were pretreated with zVAD-fmk, oxygen radical levels raised up to 10-fold after 6 h of treatment with TNF. Again, BHA (100 µM) could strongly inhibit
this radical accumulation. zVAD-fmk alone had no effect
on radical production after 6 h. Fig. 3 B shows cell killing
of the same samples, as measured by PI uptake due to loss
of cell membrane integrity, demonstrating the correlation
between oxygen radical accumulation and cell death.
View larger version (17K):
[in this window]
[in a new window]
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of zVAD-fmk on TNF-induced reactive oxygen formation and cell death. (A) Effect on TNF-induced oxygen radical production
(relative DHR123 fluorescence as compared to untreated cells). , TNF alone (500 IU/ml); , TNF + BHA (100 µM); , TNF + zVAD-fmk (25 µM); , TNF + zVAD-fmk + BHA; and , zVAD-fmk alone. (B) Effect on TNF-induced cell killing determined on the basis of PI-negative cells
(same experiment and symbols as in A).
|
|
Increased Oxygen Radical Accumulation After TNF +
zVAD Treatment Is the Result of Higher Radical Production
Rather than an Impaired Scavenging System.
In the case of TNF-mediated radical production in L929 cells, it was previously
shown that excess radicals are scavenged by the mitochondrial glutathione system (5). As the increased levels of oxygen radicals after TNF + zVAD-fmk treatment may result either from an enhanced production of radicals or an impaired mitochondrial glutathione system, we analyzed cellular thiol concentrations using monochlorobimane fluorescence after treatment with zVAD-fmk in the presence or
absence of TNF. However, no significant decrease in fluorescence could be observed (Fig. 4 A). This suggests that the sensitizing effect of zVAD-fmk on TNF-mediated oxygen radical production is not the result of depleted thiol
pools, such as mitochondrial glutathione.
View larger version (16K):
[in this window]
[in a new window]
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of zVAD-fmk on radical scavenging in L929 cells. (A) zVAD-fmk does not alter free thiol concentrations. Cells were treated with
zVAD-fmk (25 µM) for 4 h [zVAD-fmk (-4)] or 2 h [zVAD-fmk (-2)] before TNF addition, or with DEM 3 h after TNF addition. Open bars, without
TNF; filled bars, 1,000 IU/ml TNF. (B) Effect of zVAD-fmk on H2O2- or tBuOOH-induced oxygen radical production (relative DHR123 fluorescence
as compared to untreated cells). , H2O2 (50 µM); , H2O2 + zVAD-fmk (25 µM); , tBuOOH (100 µM); , tBuOOH + zVAD-fmk; and ,
zVAD-fmk alone.
|
|
Therefore, we tested whether zVAD-fmk had any effect
on the accumulation of radicals induced by the addition of
exogenous H2O2 or tBuOOH, which cause lipid peroxidation in the cells. As shown in Fig. 4 B, zVAD-fmk did not
alter radical accumulation. Again, this indicates that the signaling pathway to radical formation, rather than the scavenging capacity of the cells, is affected by caspase inhibition.
TNF Treatment Does Not Result in Detectable Caspase Activity in L929 Cells.
To study whether caspase activity occurs after TNF treatment of L929 cells, lysates were prepared after several incubation periods. Caspase-3- and
caspase-1-like activities were determined with the substrates
Ac-DEVD-AMC and Ac-YVAD-AMC, respectively. As
shown in Table 1, no significant 7-amino-4-methylcoumarin release was detected in L929 lysates. PC60R55R75 cells,
which die in an apoptotic mode after TNF treatment (21),
were used as a control. After 4 h, DEVD cleavage activity
began to appear, peaking at ~6 h. These results suggest that
caspase activity is correlated with apoptotic and not with
necrotic cell death. Furthermore, the sensitization by caspase
inhibitors apparently is due to inhibition of low, constitutive levels of caspases.
TNF-mediated Apoptosis in HeLa H21 and KYM Cells Is
Inhibited by Caspase Inhibitors.
To test whether only TNF-mediated necrosis was enhanced by inhibition of caspases,
the effect of zVAD-fmk was also analyzed in HeLa H21
and KYM cells, which respond to TNF treatment by dying in an apoptotic way. When these cells were pretreated with
zVAD-fmk for 2 h before TNF addition, complete protection against TNF was observed, even at 40,000 IU/ml
TNF (Fig. 5). These results indicate that the antagonistic
role of caspases is specific for TNF-induced reactive oxygen formation leading to necrosis.
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 5.
Inhibitory effect of zVAD-fmk on TNF-mediated apoptosis
in HeLa H21 cells ( and ; 1 µg/ml actinomycin D added) and KYM
cells ( and ). Open symbols, TNF only; closed symbols, 25 µM zVAD-fmk added 2 h before TNF treatment.
|
|
Caspase Inhibitors Do Not Enhance NF-B Activation.
Treatment of L929 cells with TNF also results in activation
of the transcription factor NF-B (22). Using a reporter
construct consisting of two NF-B sites and a minimal promoter linked to the luciferase gene, we checked whether
NF-B activation was affected by caspase inhibitors. Table
2 shows relative luciferase activities after 3 h, as compared
to untreated cells. In contrast to the 1,000-fold sensitization
of TNF-mediated necrosis, the presence of caspase inhibitors does not influence TNF-dependent activation of NF-B.
At 500 IU/ml TNF, luciferase activity in the presence of
zVAD-fmk was even lower than in cells treated with TNF
alone. However, as revealed by microscopic analysis, cells
were already dying at that point. We conclude that the
higher sensitivity to the cytotoxic activity of TNF on L929
cells in the presence of caspase inhibitors is not correlated
with altered NF-B activation.
| |
Discussion |
In this study, we investigated the role of caspases in
TNF-mediated necrosis. First, we used the cowpox CrmA
gene product as an inhibitor of a number of caspases. Surprisingly, expression of CrmA in L929 cells rendered them
far more sensitive to TNF as compared to control cells not
expressing CrmA. Furthermore, blocking of caspases by
peptide inhibitors sensitized the cells to TNF-induced cytotoxicity. zDEVD-fmk and Ac-YVAD-cmk had moderate sensitizing activity, whereas zVAD-fmk and zD-fmk
strongly potentiated TNF-mediated necrosis. In the latter
case, the concentration of TNF required for half-maximal
cytotoxicity decreased 1,000-fold. zDEVD-fmk and Ac-YVAD-cmk have a different specificity pattern, and when
they were combined, they could not synergize with each other, suggesting the possibility that they inhibit consecutively acting caspases. TNF sensitization induced by zVAD-fmk was accompanied by an enhanced production of oxygen radicals, as measured by DHR123 oxidation. Scavenging
of oxygen radicals by BHA completely abrogated the sensitizing effect of zVAD-fmk on TNF-induced necrosis. This indicates that enhanced oxygen radical production is the
main cause of zVAD-fmk-mediated sensitization.
KYM and HeLa H21 cells respond to TNF treatment in
an apoptotic way. When these cells were treated with TNF
in the presence of zVAD-fmk, cell death was inhibited, revealing a fundamental difference between necrosis and apoptosis. In contrast to apoptosis, TNF-induced necrosis of
L929 cells is not dependent on caspase activation; rather,
the results shown here indicate a protective role for a low
level of constitutively active caspase(s) in this mode of cell
death. Alternatively, TNF may induce activation of a
caspase that counteracts or deviates the pathway leading to mitochondrial oxygen radical production; this caspase activity would be at a level below the detection limit obtainable with fluorogenic substrates. TNF-induced cell death is
primarily mediated by the p55 TNF receptor (21), which
contains a death domain (DD) in its intracellular part.
Upon ligand-induced clustering of receptor DDs, other
DD-containing components of the signaling pathway are
recruited, leading to cell death (3, 23). In the case of
TNF-mediated necrosis in L929 cells, the DD of the p55
TNF receptor has been shown to be necessary and sufficient for fully active TNF signaling to necrosis (26).
TRADD, which also has a DD, binds to the DD of clustered p55 TNF receptor, and is in turn necessary for recruiting the DD-containing FADD/MORT1 (25, 27, 28).
The latter was first identified as a factor recruited by Fas,
another DD-containing receptor, upon activation (14). In
the case of the p55 TNF receptor, recruitment of FADD in
the receptor complex has not yet been demonstrated at
physiological receptor numbers. FADD/MORT1 possesses
another domain that connects Fas and the TNF receptor
complex to caspase-8 (12, 13) or the homologues caspase-10 and caspase-10b (15). Caspase-8 contains a COOH-terminal caspase-3-like domain that is proteolytically released
into the cytosol after stimulation of Fas or the p55 TNF receptor (14). It is generally assumed that caspase-8 is the
apex of a pathway leading to apoptosis in which the downstream executors are other caspases. The proteolytic activity of caspase-8 and caspase-10b is inhibited by zVAD-fmk,
zDEVD-fmk, and CrmA, but not by Ac-YVAD-cmk (14,
29, 30). In this study, we show that neither zVAD-fmk,
zDEVD-fmk, nor CrmA block TNF signaling to necrosis,
but, on the contrary, considerably enhance cytotoxicity. Obviously, TNF-mediated necrosis in L929 cells is not dependent on caspase-8/caspase-10, but in fact is attenuated
by one or more caspases.
Our results suggest a new role for caspases as negative
regulators of TNF-induced oxygen radical production and
consequent necrosis. As shown previously, TNF-induced
radical formation in L929 cells depends on an intact electron transport system in the mitochondria, and probably involves O2 ·, H 2O2, and/or lipid hydroperoxides (5). Although evidence for the existence of mitochondrial caspases
has recently been reported (31, 32), a role for caspases in
the electron transport system has not yet been demonstrated. However, since CrmA is probably located in the
cytosol, it is unlikely that mitochondrial caspases are involved. Rather, it seems that one or more caspases interfere
with the signal from the triggered receptor to the mitochondria. Alternatively, the production of oxygen radicals
may be counteracted by caspases at the level of the mitochondria themselves (Fig. 6).
View larger version (13K):
[in this window]
[in a new window]
|
Fig. 6.
Possible mechanisms of action in caspase inhibitor-mediated
sensitization of TNF-induced necrosis in L929 cells. A putative caspase
(CASP-X ), inhibited by CrmA or zVAD-fmk, acts as a negative regulator
of premitochondrial signaling (1) or mitochondrial production of reactive
oxygen intermediates (ROI; 2). Alternatively, damaging of mitochondria
by ROI could impair normal functioning, resulting in an even higher
radical production; normally, the cell possesses a mechanism to remove
these damaged mitochondria by a process involving one or more caspases
(3). Interference with this clean-up process enhances necrosis.
|
|
A third hypothetical model is the following. Degradation
of mitochondrial proteins has been documented both in
physiological and pathological conditions (33). This is especially the case when membrane proteins are damaged by
oxygen radicals. In mitochondria of rat liver cells, increasing the radical production results in enhanced protease activity (34). In addition, oxidative damage to intracellular
proteins increases their susceptibility to proteolysis (35). Although it is known that in some of these turnover processes, mitochondrial and/or cytosolic ATP-dependent
protease complexes play an important role, there is also evidence for involvement of ATP-independent proteases in
mitochondrial catabolism. Possibly, caspases could be key
elements in such an intracellular mitochondrial quality control system. As cells increase their production of oxygen
radicals in the mitochondria after p55 TNF receptor stimulation, oxidative damage of lipids and proteins accumulates;
this results in occasional failure of the electron transport
system, which leads to an amplified radical production. It is
conceivable that such defective mitochondria are recognized and eliminated by a specific cellular mechanism, and
this is where caspases could play a role. Elimination of such
deficient but oxygen radical-producing mitochondria should
then be beneficial for the cell to survive the deadly TNF
signal. By inhibiting cytosolic caspase activity, this "rescue
mechanism" would be impaired, and hence the cells would
accumulate excessive reactive oxygen-producing mitochondria and would be far more sensitive to TNF-induced
necrosis. Whatever the exact mechanism is, a low activity
of caspases is implied, stressing the importance of a stringent control mechanism of caspase activity in healthy cells.
Fig. 6 illustrates alternative mechanisms for possible interference by caspases in TNF-induced mitochondrial production of reactive oxygen intermediates.
The results reported here prompt us to add a cautionary
note. Indeed, caspases have already been shown to be essential mediators in illness-related cell death, such as neuronal damage following hypoxic-ischemic insult (36) or
fulminant liver destruction after anti-Fas injection (37), and
evidence exists for the implication of caspases in amyotrophic lateral sclerosis (38) and Alzheimer's disease (39). In
the first two indications, inhibition of caspases by tripeptide
derivatives protects treated mice against injury and death.
However, considering the 1,000-fold sensitization of TNF-induced necrotic cell death by inhibitors of caspases, one should be cautious in cases where reactive oxygen-mediated necrosis may be involved, such as neutrophil-induced
endothelial cell necrosis in the systemic inflammatory response syndrome (40); liver necrosis after reperfusion, alcoholic liver disease, or hemochromatosis (iron overload) and
Wilson's disease (copper overload) (41); and myocardial ischemia and reperfusion injury (42). It is not excluded that
in these indications, administration of caspase inhibitors
may rather have an adverse effect. Therefore, the mechanism leading to cell death should be taken into account
when the use of caspase inhibitors would be considered as
disease treatment.
Address correspondence to P. Vandenabeele, Laboratory of Molecular Biology, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium. Phone: 32-9-264-51-31; Fax: 32-9-264-53-48; E-mail: petervda{at}lmb.rug.ac.be
The authors thank W. Burm, A. Meeus, and M. Van den Hemel for technical assistance. They are indebted
to Dr. D. Pickup for donating CrmA cDNA and antiserum.
| 1.
|
Vassalli, P..
1992.
The pathophysiology of tumor necrosis factors.
Annu. Rev. Immunol.
10:
411-452
[Medline].
|
| 2.
| Fiers, W. 1995. Biologic therapy with TNF: preclinical studies. In Biologic Therapy of Cancer. 2nd ed. V.T. DeVita, Jr.,
S. Hellman, and S.A. Rosenberg, editors. J.B. Lippincott,
Philadelphia. 295-327.
|
| 3.
|
Wallach, D.,
M. Boldin,
E. Varfolomeev,
R. Beyaert,
P. Vandenabeele, and
W. Fiers.
1997.
Cell death induction by
receptors of the TNF family: towards a molecular understanding.
FEBS Lett.
410:
96-106
[Medline].
|
| 4.
|
Grooten, J.,
V. Goossens,
B. Vanhaesebroeck, and
W. Fiers.
1993.
Cell membrane permeabilization and cellular collapse,
followed by loss of dehydrogenase activity: early events in tumour necrosis factor-induced cytotoxicity.
Cytokine.
5:
546-555
[Medline].
|
| 5.
|
Goossens, V.,
J. Grooten,
K. De Vos, and
W. Fiers.
1995.
Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity.
Proc. Natl. Acad. Sci. USA.
92:
8115-8119
[Abstract/Free Full Text].
|
| 6.
|
Schulze-Osthoff, K.,
A.C. Bakker,
B. Vanhaesebroeck,
R. Beyaert,
W.A. Jacob, and
W. Fiers.
1992.
Cytotoxic activity
of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation.
J. Biol. Chem.
267:
5317-5323
[Abstract/Free Full Text].
|
| 7.
|
Vercammen, D.,
P. Vandenabeele,
R. Beyaert,
W. Declercq, and
W. Fiers.
1997.
Tumour necrosis factor-induced necrosis
versus anti-Fas-induced apoptosis in L929 cells.
Cytokine.
9:
801-808
[Medline].
|
| 8.
|
Kroemer, G.,
P. Petit,
N. Zamzami,
J.-L. Vayssière, and
B. Mignotte.
1995.
The biochemistry of programmed cell death.
FASEB J.
9:
1277-1287
[Abstract].
|
| 9.
|
Henkart, P.A..
1996.
ICE family proteases: mediators of all
apoptotic cell death?
Immunity.
4:
195-201
[Medline].
|
| 10.
|
Martins, L.M., and
W.C. Earnshaw.
1997.
Apoptosis: alive
and kicking in 1997.
Trends Cell Biol.
7:
111-114
.
|
| 11.
|
Nagata, S..
1997.
Apoptosis by death factor.
Cell.
88:
355-365
[Medline].
|
| 12.
|
Boldin, M.P.,
T.M. Goncharov,
Y.V. Goltsev, and
D. Wallach.
1996.
Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced
cell death.
Cell.
85:
803-815
[Medline].
|
| 13.
|
Muzio, M.,
A.M. Chinnaiyan,
F.C. Kischkel,
K. O'Rourke,
A. Shevchenko,
J. Ni,
C. Scaffidi,
J.D. Bretz,
M. Zhang,
R. Gentz, et al
.
1996.
FLICE, a novel FADD homologous ICE/
CED-3-like protease, is recruited to the CD95 (Fas/Apo-1)
death-inducing signaling complex.
Cell.
85:
817-827
[Medline].
|
| 14.
|
Medema, J.P.,
C. Scaffidi,
F.C. Kischkel,
A. Shevchenko,
M. Mann,
P.H. Krammer, and
M.E. Peter.
1997.
FLICE is activated by association with the CD95 death-inducing signaling
complex (DISC).
EMBO (Eur. Mol. Biol. Organ.) J.
16:
2794-2804
[Medline].
|
| 15.
|
Vincenz, C., and
V.M. Dixit.
1997.
Fas-associated death domain protein interleukin-1 -converting enzyme 2 (FLICE2),
an ICE/Ced-3 homologue, is proximally involved in CD95-
and p55-mediated death signaling.
J. Biol. Chem.
272:
6578-6583
[Abstract/Free Full Text].
|
| 16.
|
Niwa, H.,
K. Yamamura, and
J. Miyazaki.
1991.
Efficient selection for high-expression transfectants with a novel eukaryotic vector.
Gene.
108:
193-200
[Medline].
|
| 17.
|
Southern, P.J., and
P. Berg.
1982.
Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter.
J. Mol. Appl.
Genet.
1:
327-341
[Medline].
|
| 18.
|
Tada, H.,
O. Shiho,
K. Kuroshima,
M. Koyama, and
K. Tsukamoto.
1986.
An improved colorimetric assay for interleukin 2.
J. Immunol. Methods.
93:
157-165
[Medline].
|
| 19.
|
Shrieve, D.C.,
E.A. Bump, and
G.C. Rice.
1988.
Heterogeneity of cellular glutathione among cells derived from a murine fibrosarcoma or a human renal cell carcinoma detected
by flow cytometric analysis.
J. Biol. Chem.
263:
14107-14114
[Abstract/Free Full Text].
|
| 20.
|
Kimura, A.,
A. Israël,
O. Le Bail, and
P. Kourilsky.
1986.
Detailed analysis of the mouse H-2Kb promoter: enhancer-like sequences and their role in the regulation of class I gene
expression.
Cell.
44:
261-272
[Medline].
|
| 21.
|
Vandenabeele, P.,
W. Declercq,
R. Beyaert, and
W. Fiers.
1995.
Two tumour necrosis factor receptors: structure and
function.
Trends Cell Biol.
5:
392-399
.
[Medline] |
| 22.
|
Zhang, Y.,
J.-X. Lin, and
J. Vil ek.
1990.
Interleukin-6 induction by tumor necrosis factor and interleukin-1 in human
fibroblasts involves activation of a nuclear factor binding to a
B-like sequence.
Mol. Cell. Biol.
10:
3818-3823
[Abstract/Free Full Text].
|
| 23.
|
Tartaglia, L.A.,
T.M. Ayres,
G.H.W. Wong, and
D.V. Goeddel.
1993.
A novel domain within the 55 kd TNF receptor signals cell death.
Cell.
74:
845-853
[Medline].
|
| 24.
|
Song, H.Y.,
J.D. Dunbar, and
D.B. Donner.
1994.
Aggregation of the intracellular domain of the type 1 tumor necrosis
factor receptor defined by the two-hybrid system.
J. Biol.
Chem.
269:
22492-22495
[Abstract/Free Full Text].
|
| 25.
|
Boldin, M.P.,
I.L. Mett,
E.E. Varfolomeev,
I. Chumakov,
Y. Shemer-Avni,
J.H. Camonis, and
D. Wallach.
1995.
Self-association of the "death domains" of the p55 tumor necrosis factor (TNF) receptor and Fas/APO1 prompts signaling for TNF
and Fas/APO1 effects.
J. Biol. Chem.
270:
387-391
[Abstract/Free Full Text].
|
| 26.
|
Vandevoorde, V.,
G. Haegeman, and
W. Fiers.
1997.
Induced expression of trimerized intracellular domains of the
human tumor necrosis factor (TNF) p55 receptor elicits TNF
effects.
J. Cell Biol.
137:
1627-1638
[Abstract/Free Full Text].
|
| 27.
|
Chinnaiyan, A.M.,
K. O'Rourke,
M. Tewari, and
V.M. Dixit.
1995.
FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis.
Cell.
81:
505-512
[Medline].
|
| 28.
|
Hsu, H.,
J. Xiong, and
D.V. Goeddel.
1995.
The TNF receptor 1-associated protein TRADD signals cell death and
NF-B activation.
Cell.
81:
495-504
[Medline].
|
| 29.
|
Srinivasula, S.M.,
M. Ahmad,
T. Fernandes-Alnemri,
G. Litwack, and
E.S. Alnemri.
1996.
Molecular ordering of the
Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a
CrmA-inhibitable protease that activates multiple Ced-3/
ICE-like cysteine proteases.
Proc. Natl. Acad. Sci. USA.
93:
14486-14491
[Abstract/Free Full Text].
|
| 30.
|
Zhou, Q.,
S. Snipas,
K. Orth,
M. Muzio,
V.M. Dixit, and
G.S. Salvesen.
1997.
Target protease specificity of the viral
serpin CrmA. Analysis of five caspases.
J. Biol. Chem.
272:
7797-7800
[Abstract/Free Full Text].
|
| 31.
|
Susin, S.A.,
N. Zamzami,
M. Castedo,
T. Hirsch,
P. Marchetti,
A. Macho,
E. Daugas,
M. Geuskens, and
G. Kroemer.
1996.
Bcl-2 inhibits the mitochondrial release of an apoptogenic protease.
J. Exp. Med.
184:
1331-1341
[Abstract/Free Full Text].
|
| 32.
|
Zamzami, N.,
P. Marchetti,
M. Castedo,
C. Zanin,
J.-L. Vayssière,
P.X. Petit, and
G. Kroemer.
1995.
Reduction in mitochondrial potential constitutes an early irreversible step of
programmed lymphocyte death in vivo.
J. Exp. Med.
181:
1661-1672
[Abstract/Free Full Text].
|
| 33.
|
Rep, M., and
L.A. Grivell.
1996.
The role of protein degradation in mitochondrial function and biogenesis.
Curr. Genet.
30:
367-380
[Medline].
|
| 34.
|
Dean, R.T., and
J.K. Pollak.
1985.
Endogenous free radical
generation may influence proteolysis in mitochondria.
Biochem. Biophys. Res. Commun.
126:
1082-1089
[Medline].
|
| 35.
|
Davies, K.J.A.,
S.W. Lin, and
R.E. Pacifici.
1987.
Protein
damage and degradation by oxygen radicals. IV. Degradation
of denatured protein.
J. Biol. Chem.
262:
9914-9920
[Abstract/Free Full Text].
|
| 36.
|
Hara, H.,
R.M. Friedlander,
V. Gagliardini,
C. Ayata,
K. Fink,
Z. Huang,
M. Shimizu-Sasamata,
J. Yuan, and
M.A. Moskowitz.
1997.
Inhibition of interleukin 1 converting
enzyme family proteases reduces ischemic and excitotoxic
neuronal damage.
Proc. Natl. Acad. Sci. USA.
94:
2007-2012
[Abstract/Free Full Text].
|
| 37.
|
Rodriguez, I.,
K. Matsuura,
C. Ody,
S. Nagata, and
P. Vassalli.
1996.
Systemic injection of a tripeptide inhibits the intracellular activation of CPP32-like proteases in vivo and fully
protects mice against Fas-mediated fulminant liver destruction and death.
J. Exp. Med.
184:
2067-2072
[Abstract/Free Full Text].
|
| 38.
|
Friedlander, R.M.,
R.H. Brown,
V. Gagliardini,
J. Wang, and
J. Yuan.
1997.
Inhibition of ICE slows ALS in mice.
Nature.
388:
31
[Medline].
|
| 39.
|
Kim, T.-W.,
W.H. Pettingell,
Y.-K. Jung,
D.M. Kovacs, and
R.E. Tanzi.
1997.
Alternative cleavage of Alzheimer-associated presenilins during apoptosis by a caspase-3 family protease.
Science.
277:
373-376
[Abstract/Free Full Text].
|
| 40.
|
Wang, J.H.,
H.P. Redmond,
R.W.G. Watson,
S. Duggan,
J. McCarthy,
M. Barry, and
D. Bouchier-Hayes.
1996.
Mechanisms involved in the induction of human endothelial cell
necrosis.
Cell. Immunol.
168:
91-99
[Medline].
|
| 41.
|
Rosser, B.G., and
G.J. Gores.
1995.
Liver cell necrosis: cellular mechanisms and clinical implications.
Gastroenterology.
108:
252-275
[Medline].
|
| 42.
|
Maxwell, S.R.J., and
G.Y.H. Lip.
1997.
Reperfusion injury:
a review of the pathophysiology, clinical manifestations and
therapeutic options.
Int. J. Cardiol.
58:
95-117
[Medline].
|
Top
Abstract
Introduction
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
