| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain;
Division of Immunology and Transplantation Biology, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305; and
Centre National de la Recherche Scientifique, Institut Gustave Roussy, Villejuif, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In a previous work we initiated the study of the biochemical mechanism for granulysin-induced apoptosis (11). We found that granulysin induced sphingomyelinase activity, leading to generation of the apoptotic mediator ceramide. However, ceramide generation seemed not to be essential for granulysin-induced cell death, since it was still able to induce apoptosis in cells depleted of sphingomyelin, implicating additional mechanisms in granulysin-induced cell death. The caspase-3 subfamily inhibitor N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO)3 did not substantially affect cell death, while the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) partially inhibited granulysin-induced apoptosis. These data indicated that granulysin induced apoptosis through both caspase- and ceramide-dependent and -independent pathways.
In the present study we further characterize the apoptotic pathways
initiated by granulysin. The results obtained indicate that granulysin
induces apoptosis through two different pathways, one fast and the
other slow, both of which implicate the participation of mitochondria.
The fast pathway (1–5 h) is independent of ceramide generation and is
associated with a caspase-independent rapid loss of mitochondrial
membrane potential (
m). This
m loss is associated with the translocation
of the mitochondrial apoptosis-inducing factor (AIF) to the nucleus. We
also show that the AIF apoptotic pathway remains active in the presence
of caspase inhibitors. Enforced overexpression of Bcl-2 prevented
granulysin-induced loss of m and AIF
translocation, correlating to the observed protection from cell death.
The slow pathway (
12 h) is associated with ceramide generation, and
although ceramide also mediates cell death through the mitochondrial
pathway, m loss induced by ceramide is
inhibited by Z-VAD-fmk. Previous results has shown that although
cytochrome c is also released from mitochondria upon
granulysin treatment, caspase-9 activation is not detected during
granulysin-induced apoptosis (13). Taken together,
these data point to the release of AIF from mitochondria as an
important apoptotic effector during granulysin-induced cell death.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The Jurkat T cell leukemia (American Type Culture Collection, Manassas, VA; clone E6.1) was used as target in the granulysin- and ceramide-induced cytotoxicity assays. Jurkat cells were cultured in RPMI 1640 medium (Life Technologies, Barcelona, Spain) supplemented with 5% FCS, L-glutamine, and penicillin/streptomycin (hereafter, complete medium) using standard cell culture procedures. Jurkat cell lines stably overexpressing the viral protein CrmA, J-CrmA, and the murine Bcl-2 protein, J-Bcl2, as well as vector-transfected cells, J-vector, were gifts from Dr. D. Johnson (Pittsburgh University, Pittsburgh, PA) and were generated as previously described (14). Transfected cell lines were maintained in complete medium containing 0.5 mg/ml G418 (Life Technologies). All cell lines used were Mycoplasma free, as routinely controlled by PCR.
Materials
MTT, carbonyl cyanide m-chlorophenylhydrazone (CCCP), ceramide type III from bovine brain, and dodecane were products of Sigma (Madrid, Spain). Ac-DEVD-CHO and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) were obtained from Bachem (Bubendorf, Switzerland). 3,3'-Dihexyloxacarbocyanine iodide, 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)), was obtained from Molecular Probes (Leiden, The Netherlands), and [1-14C]palmitic acid was purchased from Amersham (Madrid, Spain). The rabbit anti-mouse AIF polyclonal antiserum, which largely cross-reacts with human AIF, was generated as previously described (15).
Expression and purification of recombinant functional granulysin
Recombinant granulysin was expressed and purified essentially as described previously (10). Briefly, a portion of the initial 519 cDNA, coding for glycine residue 63 through the natural stop codon after leucine residue 145 of P520 (16), was cloned into the pET28a vector (Novagen, Madison, WI) to express polyhistidine-tagged recombinant 9-kDa granulysin in Escherichia coli BL21 (DE3). The recombinant protein was expressed and purified under denaturing conditions according to Novagen’s instructions. After purification, the granulysin was reduced by the addition of 1 mM DTT, allowed to refold in the presence of oxidized DTT, and dialyzed against Tris-buffered saline. The histidine tag was removed by thrombin treatment, and proteins were purified by gel filtration chromatography in a Sephadex G-25 column. Protein content was determined by the Bradford method, with BSA as a standard. Protein purity was always >95%, as assessed by SDS-PAGE in 15% polyacrylamide gels and Coomassie blue staining.
Cytotoxicity assays
For granulysin cytotoxicity testing, 2 x
105 cells/ml (100 µl/well) were resuspended in
a serum-free medium composed of RPMI 1640, DMEM, and Ham’s F-12 medium
(2/1/1, v/v/v), supplemented with BSA (1 mg/ml), sodium selenite (4.3
ng/ml), ethanolamine (1.53 µg/ml), transferrin (10 µg/ml), insulin
(5 µg/ml), glutamine (2 mM) and antibiotics (penicillin, 100 U/ml;
streptomycin, 100 µg/ml) (17). Cells were incubated for
different times (30 min to 16 h) in the presence or the absence of
20–50 µM recombinant granulysin. Natural (C18)
ceramide (50–200 nM) was first dissolved in ethanol-dodecane (98:2,
v/v), and then added to the serum-free culture medium, as indicated
previously (18, 19). At the doses used, ethanol and
dodecane (final concentration of solvent, 0.2–0.5%) had no effect on
cell proliferation. Ac-DEVD-CHO, an inhibitor of caspases of the
caspase-3 subfamily, or Z-VAD-fmk, a general caspase inhibitor
(20), were used at 600 and 100 µM, respectively. Peptide
inhibitors were first dissolved in DMSO, then diluted in culture medium
(final DMSO concentration, 
0.4%, v/v). Cells were preincubated for
1 h with the protease inhibitors before adding the toxic stimuli
to assure a sufficient incorporation by cells. Cell growth was
determined by a modification of the MTT reduction method of Mosmann
(21) and expressed as a percentage of growth in control
cultures. Cell death was determined by microscopic inspection of trypan
blue-stained cells.
Evaluation of mitochondrial membrane potential
(m) by flow cytometry
To evaluate m, the cationic
lipophilic fluorochrome DiOC6(3) was used
(22). Cells (1.5 x 105 in 100
µl) were incubated with 20 nM DiOC6(3) for 15
min at 37°C. DiOC6(3) was prepared from a 40
µM stock solution in DMSO. This solution was diluted with sterile
PBS, pH 7.4, to a 400 nM working solution, followed by a further
dilution with the medium containing cells. As a negative control, cells
were treated in parallel cultures with the protonophore-uncoupling
agent CCCP at a final concentration of 50 µM (stock solution, 10 mM
in ethanol). Cells were diluted with PBS to a final volume of 1 ml and
analyzed by flow cytometry.
Ceramide and sphingomyelin determinations
Ceramide and sphingomyelin levels were assayed essentially as
described previously (11, 19). Jurkat cells (2 x
106 in 10 ml) were labeled for 48 h with 5
µCi [1-14C]palmitic acid bound to fatty
acid-free serum albumin (1:1, molar ratio) in complete medium. Cells
were then harvested, resuspended in serum-free medium, and incubated
with 50 µM granulysin for different periods of time (30 min–16 h).
The cells were harvested and washed with cold RPMI medium. Cell
viability was determined in an aliquot of the corresponding cell
suspensions by the trypan blue exclusion method, and total cell lipids
were extracted at 4°C with chloroform-methanol (2:1, v/v) as
previously described (19). Radioactivity in aliquots from
chloroformic phases was determined by liquid scintillation counting,
and equal amounts of radioactivity for each sample were applied to TLC
silica gel G plates (Scharlau, Barcelona, Spain). Plates were
prewashed with chloroform-methanol (1:1, v/v) and heat activated at
110°C for 1 h. A first development of TLC-loaded samples was
performed with chloroform-methanol-water (60:30:5, v/v/v) up to 10 cm
from the bottom of the plate, and a second development was performed to
its full length with hexane-diethyl ether-acetic acid (80:20:2, v/v/v).
Plates were air-dried, and radiolabeled bands were located by film
autoradiography (Hyperfilm 
-max; Amersham Pharmacia) at room
temperature for 2–3 days. Then, sample lanes were covered with glass,
allowing the marker lanes to be iodine stained. Authentic standards of
ceramide, sphingomyelin, cholesterol, phosphatidylcholine, and
phosphatidylserine (Sigma) were used as markers. The corresponding
radiolabeled lipids were scraped and transferred to vials, to which 4
ml scintillation mixture (Normascint 11; Scharlau) containing 10%
(v/v) methanol was added. Radioactivity in samples was determined by
liquid scintillation counting, and results are expressed as
ceramide-sphingomyelin ratios.
Fluorescence and confocal microscopy
Morphological evaluation of apoptosis was monitored by labeling cell nuclei with oxidized p-phenylenediamine (PPDA) in glycerol and visualized by fluorescence microscopy (23). Briefly, cells were washed with PBS, pH 7.4, and fixed at room temperature with 1% paraformaldehyde in PBS for 15 min. Fixed cells were washed with PBS, centrifuged onto polylysine-coated glass coverslips in wells of a 24-well plate, mounted on a glass slide over a drop of PPDA stain (10 mg PPDA in 1 ml PBS mixed with 9 ml oxidized glycerol) (24), and photographed.
Translocation of AIF from mitochondria to the nucleus during apoptosis was analyzed by confocal microscopy, as indicated previously (15). Briefly, control cells or cells treated with granulysin for the times indicated were collected, washed with PBS, and fixed in a solution of 4% paraformaldehyde in PBS. Cell suspensions were then placed onto round coverglasses previously treated with L-polylysine, in wells of a 24-well plate, and centrifuged 5 min at 1200 rpm. Coverglasses were then washed once with PBS, placed onto a drop of a 0.1% solution of saponin in PBS containing 5% goat serum and a 1/500 dilution of the anti-AIF antiserum, and incubated for 30 min at room temperature in a humidified chamber. Coverglasses were then washed once with a 0.1% solution of saponin in PBS and incubated in the same way with a 1/200 dilution of an anti-rabbit IgG Ab labeled with FITC (Caltag, Barcelona, Spain). Finally, coverglasses were washed sequentially with 0.1% saponin, PBS, and distilled water; mounted onto the glass slides using a drop of Mowiol (Calbiochem, Madrid, Spain); and stored at 4°C in the dark until observation. Preparations were observed in a Zeiss 310 confocal microscope, and analyzed using LSM 3.95 software. Cells were observed in 10 successive focal planes, separated by 1 µm, and adjusted from the bottom to the top of the cell. The pictures shown correspond to the central part of the cell, normally the fifth or sixth focal plane.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Two main pathways of apoptosis have been elucidated to date: one is triggered by death receptors, and the other is the mitochondrial pathway, the main pathway used for stress-induced apoptosis (25, 26). In the first pathway the recruitment of adaptors to engaged receptors leads to activation of apical caspases, such as caspase-8 (27), while in the second pathway, the liberation of apoptogenic factors from mitochondria, such as cytochrome c, leads to the activation of caspase-9 (28). As a final step in both pathways, executioner caspases of the caspase-3 subfamily are activated. The death receptor pathway is blocked by CrmA, a viral serpin that preferentially binds and inhibits caspase-8 (29), while the Bcl-2 protein blocks the mitochondrial apoptotic pathway (30).
To analyze the relative involvement of each pathway in
granulysin-induced cell death, we evaluated the cytotoxic potential of
granulysin overexpressing CrmA or Bcl-2 (14). As shown in
Fig. 1
, CrmA offered some protection from
granulysin-induced cell death (30% protection); the protection offered
by Bcl-2 is more pronounced, although not complete (65% protection).
This result points to a main implication of the mitochondrial apoptotic
pathway in granulysin-induced apoptosis.
|
, CrmA protection was
limited (35% protection), while Bcl-2 protection from ceramide-induced
apoptosis was complete (>90% protection). Similar results were
obtained in previous studies using short chain ceramides
(31, 32, 33). Therefore, the contribution of ceramide
generation to granulysin-induced cell death seems to be strictly
mediated through the mitochondrial apoptotic pathway.
Loss of m induced by granulysin is
independent of caspase activation and ceramide generation
To further asses the role of mitochondria in granulysin-induced
cell death, we tested whether granulysin caused a loss in
m using the cationic lipophilic
fluorochrome DiOC6(3) (22). It has
been reported that this fluorochrome is sensitive to changes not only
in mitochondrial, but also in plasma membrane potential
(34). However, the contribution of plasma membrane
potential to DiOC6(3) fluorescence is small, as
demonstrated in a recent study (35). We have also shown by
fluorescence microscopy that DiOC6(3) staining
follows a punctate cytoplasmic pattern typical of a mitochondrial
localization, with no detectable contribution of the plasma membrane.
Treatment with the protonophore uncoupling agent CCCP or with the
cytotoxic drug doxorubicin induces the complete loss of
DiOC6(3) staining, while incubation in the
presence of 0.15 M KCl, which abolishes plasma membrane potential, does
not affect the fluorescence labeling (data not shown), in agreement
with the results previously shown by flow cytometry (35).
Therefore, DiOC6(3) can be properly used to
estimate the changes in m produced during
apoptosis.
Granulysin treatment of Jurkat cells induced a rapid and progressive
loss of m, already detected after 1 h
of incubation (compare Fig. 2, A and C), in agreement with our previous results
(13). The percentage of cells exhibiting a low
m was about 75% after 12 h of
incubation (Fig. 2G). In Jurkat cells, the loss of
m induced by several apoptotic signals,
like the death receptor Fas/CD95 or the cytotoxic drug doxorubicin
(25, 36), is dependent on the previous activation of
Z-VAD-fmk-sensitive caspases. However, the
m loss induced by granulysin was not
inhibited by Z-VAD-fmk at any time point tested (1 h, Fig. 2D; 5 h, Fig. 2F; or 12 h, Fig. 2H).
|
), we performed a
time-course analysis of granulysin-induced ceramide generation to
estimate whether the observed rapid loss in
m induced by granulysin could be mediated
by ceramide. We did not observe an increase in the Cer/SM ratio until
12 h of incubation with granulysin, in agreement with our previous
study, in which this was the only time point analyzed (Table I)
(11). No increase was observed after 1 or 5 h of
incubation, although at 5 h the cytotoxicity of granulysin was
already significant (Table I). These data do not support a role for
ceramide generation in the rapid (1–5 h) m
loss induced by granulysin. In addition, the prominent loss in
m induced by natural ceramide (compare Fig. 3, A and B) was
almost completely prevented by coincubation with the general caspase
inhibitor Z-VAD-fmk (Fig. 3C). These data and those shown in
Fig. 2 clearly distinguish between the apoptotic mechanisms initiated
by granulysin and ceramide.
|
|
m
Although protection from granulysin-induced cell death by Bcl-2
was significant, it was not as complete as that observed for
ceramide-induced apoptosis (Fig. 1). We further examined the mechanism
of the residual toxicity induced by granulysin in Bcl-2-overexpressing
Jurkat cells (J-Bcl2).
Similar to that observed with Z-VAD-fmk (see Fig. 3C), Bcl-2
overexpression almost completely prevented ceramide-induced
m loss (>90% protection; Fig. 3D). Granulysin-induced m loss
was detectable in J-vector cells after 5 h of incubation (compare
Fig. 4, A and B).
This m loss was also observed in J-Bcl2
cells (Fig. 4E), although to a much lesser extent than in
control cells (
60% protection). The m
loss induced by granulysin in J-vector cells after 12 h was
prominent (Fig. 4C). At this time point the protection from
m loss offered by Bcl-2 overexpression was
higher than that observed at the 5 h point, although it was not
complete (Fig. 4F; 70% protection). Therefore, the
protection offered by Bcl-2 on m loss
correlates with the protection from cell death shown in Fig. 1 for both
ceramide- and granulysin-induced apoptosis.
|
In our previous study (11) we showed that while the
inhibitor of the caspase-3 subfamily Ac-DEVD-CHO had almost no effect
on granulysin-induced cell death, the general caspase inhibitor
Z-VAD-fmk had some protective effect. We also showed that the
processing of caspase-3 was somewhat limited during granulysin-induced
apoptosis. However, granulysin induced the typical nuclear apoptotic
morphology, which in most cases has been described to be dependent on
caspase-3-mediated activation of the caspase-activated DNase
(37). To test whether the nuclear apoptotic morphology
observed was dependent on this subtle caspase-3 activation, we
performed PPDA nuclear staining of cells treated with granulysin in the
presence of Ac-DEVD-CHO and also of Z-VAD-fmk for comparison. As
previously shown, granulysin induced the chromatin condensation and
nuclear fragmentation typical of apoptosis (compare Fig. 5, A and B).
However, this nuclear apoptotic morphology was no longer observed in
cells treated with granulysin in the presence of Ac-DEVD-CHO (Fig. 5C) or Z-VAD-fmk (Fig. 5D). The nuclear
morphology of cells dying in the presence of both caspase inhibitors
was very similar: the chromatin condensed partially and was marginated
to the edges of the nucleus, but there was no sign of nuclear
fragmentation.
|
E). Nevertheless, we were unable to detect any cell with
the typical, fragmented nuclear apoptotic morphology. This abnormal
nuclear morphology was similar to that observed for granulysin-treated
Jurkat cells in the presence of caspase inhibitors (compare Fig. 5, C and D). When J-Bcl2 cells were incubated for
12 h with granulysin, cell death increased to 20%, while the
percentage of cells with apoptotic nuclear morphology increased to a
maximum of 22%. However, at this time point all the apoptotic nuclear
morphologies detected were characterized by extensive chromatin
condensation and nuclear fragmentation (Fig. 5F).
The nuclear morphology observed in Fig. 5, C–E, is
reminiscent of that induced by AIF, a protein released from
mitochondria in addition to cytochrome c during apoptosis
which exerts its proapoptotic activity in a caspase-independent manner
(15, 38).
AIF implication in granulysin-induced apoptosis
Therefore, we tested the possible involvement of AIF in
granulysin-induced cell death. During apoptosis induction by a variety
of stimuli, AIF is released from mitochondria and rapidly translocates
to the nucleus, where it induces peripheral chromatin condensation and
large-scale (50-kbp) DNA fragmentation (15). This
translocation can be followed by confocal microscopy using a specific
antiserum. As shown in Fig. 6A, AIF was almost exclusively
localized in the cytoplasm of control Jurkat cells. Given the small
size of the cytoplasm in these cells, it was difficult to distinguish
the punctate staining corresponding to a typical mitochondrial
localization. However, when cells were treated with granulysin for
5 h (Fig. 6B) or 12 h (Fig. 6C), the
massive translocation of AIF to the nucleus was evident. Although
Z-VAD-fmk partially inhibited granulysin-induced cell death, as
previously reported (11), the percentage of AIF
translocation correlated well with the extent of cell death observed in
the presence of the caspase inhibitors Ac-DEVD-CHO (Fig. 6D)
and Z-VAD-fmk (Fig. 6E). This is in agreement with the
observed caspase independence in granulysin-induced
m loss (Fig. 2) and with the AIF-like
nuclear morphology of granulysin-treated cells in the presence of
caspase inhibitors (Fig. 5).
|
F). In most J-Bcl2 cells, AIF remained localized in the
cytoplasm after 12 h of incubation with granulysin, while there
was a small percentage of cells in which AIF was translocated into the
nucleus. In Fig. 6G, a field was chosen in which both types
of AIF distribution in granulysin-treated J-Bcl2 cells could be
observed. In the experiment corresponding to the photograph shown in
Fig. 6G, of a total of 300 J-Bcl2 cells counted in different
aleatory fields, only 20% of the cells showed nuclear staining for
AIF. This correlated well with the observed protection from cell death
(Fig. 1) and m loss (Fig. 4) as a result of
Bcl-2 overexpression. These data clearly demonstrate a role for AIF in granulysin-induced apoptosis and suggest that this is the apoptotic pathway remaining active in the presence of caspase inhibitors.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). We showed previously that
sphingomyelinase activation and ceramide generation were involved in
granulysin-induced apoptosis (11), but the present data
indicate that this pathway is only active at late time points (12 h),
while granulysin toxicity is detectable at much earlier time points.
This agrees with the observation that granulysin toxicity is maintained
in cells depleted of sphingomyelin (SM) and ceramide by prolonged
incubation with the ceramide synthase inhibitor fumonisin
B1 (11). Together these data
indicate the existence of a "fast," ceramide-independent pathway of
granulysin toxicity (Fig. 7).
|
1 h) m loss, which is caspase
independent (Fig. 2). Granulysin-induced m
loss is associated with the release of cytochrome c from
mitochondria (13) and also, as demonstrated here, with AIF
translocation from mitochondria to nucleus (Fig. 6). The means by which
granulysin induces this caspase-independent, rapid
m loss remains unclear. In a cell-free
system, it has been shown that granulysin is unable to directly disrupt
m of isolated mitochondria
(13), indicating that m loss
induced by granulysin is due to the action of a cytoplasmic mediator.
Similarly to amebapore and NK-lysin, bioactive granulysin seems to
exert its effects by association and insertion into lipidic domains of
the plasma membrane (39). While the amebapore structure
favors a pore-forming mechanism of action, granulysin and NK-lysin
structures favor a membrane perturbation mechanism of action
(39). In fact, it has been postulated that NK-lysin gets
inserted into membranes affecting just the outer leaflet of the
bilayer, but that this perturbation is sufficient to alter ionic
equilibrium and the traffic of small molecules through membranes
(40). Given the structural similarity between granulysin
and NK-lysin (39), it is possible that granulysin exerts
its effects on the plasma membrane by a similar mechanism
(13). Previous results from our group demonstrated that
granulysin is able to lyse negatively charged liposomes and to
immediately induce cytoplasmic calcium concentration spikes in treated
Jurkat cells (13). The increase in cytoplasmic calcium
concentration is known to induce m loss
(41, 42) and apoptotic cell death through caspase-9 and
caspase-3 activation (43, 44, 45). In consequence, the rapid
m loss induced by granulysin could be
mediated by the cytoplasmic calcium concentration increase due to
plasma membrane perturbation induced by granulysin insertion.
Although granulysin induces the rapid release of cytochrome
c from mitochondria, the activation of caspase-9 has not be
detected during granulysin-induced apoptosis (13).
However, a somewhat limited caspase-3 activation, more easily
detectable at early time points, was detected during granulysin-induced
apoptosis (11, 13). It is possible that unprocessed
procaspase 9, when complexed with cytochrome c and dATP, can
activate downstream caspases, as previously proposed (46),
or, alternatively, that the observed activation of caspase-3 may occur
by a caspase-9-independent mechanism. In agreement with the described
pathway for nuclear fragmentation (37), caspase
inhibition by Ac-DEVD-CHO or Z-VAD-fmk prevents the typical apoptotic
nuclear morphology (Fig. 5). In both cases the nuclear morphology
detected resembled that induced by AIF in the absence of caspase and
caspase-activated DNase activation (15, 38). However,
Z-VAD-fmk treatment resulted in a partial, although significant,
protection from cell death, which was not the case for Ac-DEVD-CHO
treatment (11). This additional protection by Z-VAD-fmk
should then be ascribed to the inhibition of the ceramide-induced
apoptotic pathway (see Fig. 7 and below).
This is in agreement with the fact that Z-VAD-fmk protection from
granulysin-induced cell death was no longer observed in
sphingomyelin-depleted cells, in which granulysin does not induce
ceramide generation (11). In addition, we have observed
that Z-VAD-fmk does not protect from granulysin-induced cell death at
early time points (from 1–5 h; data not shown).
It has been previously shown in other models of apoptosis
induction that AIF, once released from mitochondria, translocates
directly to the nucleus and exerts a particular effect on chromatin
organization, characterized by a limited condensation and margination
on the nuclear edges (15, 38, 47). This could represent an
early event during nuclear apoptosis that is accompanied by DNA
fragmentation in high m.w. fragments (50 kb) (15).
Later, caspase-mediated caspase-activated DNase activation would result
in the final, typical apoptotic nuclear morphology, characterized by
generalized chromatin condensation, nuclear fragmentation, and
oligonucleosomal DNA laddering (47). Hence, the particular
nuclear phenotype induced by AIF can only be observed if caspase
activity is inhibited, which is the case for granulysin-induced cell
death. However, the role of caspase activation in granulysin-induced
cell death is rather limited, contributing only to nuclear
fragmentation, indicating that the main apoptotic effector could
be AIF.
On the other hand, the slow pathway of granulysin-induced
apoptosis is associated with SMase activation and ceramide generation.
This is probably related to the ability of granulysin to activate
SMases after its insertion into the plasma membrane, given its homology
with saposins, known activators of SMases and glycosylceramidases
(10, 11). Ceramide-induced cell death is also mediated by
the mitochondrial pathway of apoptosis and is associated with
m loss. However, and contrary to the fast
pathway of granulysin-induced apoptosis, the
m loss induced by natural
(C18) ceramide is completely prevented by
Z-VAD-fmk (Fig. 3). This places a still unidentified caspase upstream
of mitochondrial permeability transition during ceramide-induced
apoptosis, in accord with the complete protection from ceramide-induced
cell death, at least in Jurkat cells, by the general caspase inhibitor
Z-VAD-fmk (11). The role of ceramide generation as part of
the apoptotic execution machinery is controversial (48).
Although ceramide generation is normally observed during apoptotic cell
death, it is usually a late event that could constitute a safeguard
mechanism to assure the completion of the apoptotic process (49, 50). This could also be the case for granulysin-induced
apoptosis, since, similar to Fas-induced apoptosis, both apoptotic
processes can take place in the absence of ceramide generation
(11, 50). A recent study points to a role for ceramide
generation more related to the changes that take place in the plasma
membrane during apoptosis and linked to phosphatidylserine exposure in
the outer leaflet of the plasma membrane and the formation of apoptotic
blebs (51).
The data on the significant protection from granulysin-induced
cell death by Bcl-2 reinforce the central involvement of the
mitochondrial pathway of apoptosis in this CTL effector mechanism. It
is noteworthy that, although important (75%), Bcl-2 protection from
granulysin-induced apoptosis is not complete. This is in contrast with
Bcl-2 protection from ceramide-induced cell death, which is complete
(Fig. 1). The residual toxicity of granulysin on J-Bcl2 cells (25%)
correlates well to the extent of protection from
m loss (Fig. 4) and from AIF release (Fig. 6), indicating that the fraction of J-Bcl2 cells that remain sensitive
to granulysin lose their m and release AIF
from mitochondria. The reason why protection from ceramide-induced
apoptosis is more efficient than protection from granulysin should be
related to the different mechanisms by which both apoptosis inducers
cause m loss. Ceramide-induced
m loss is mediated by a Z-VAD-sensitive
caspase, while granulysin is able to induce
m loss through a caspase-independent
pathway, possibly mediated by an increase in the intracellular calcium
concentration. A caspase-mediated mechanism for
m loss has been characterized in some
apoptotic processes, such as that mediated by Fas ligation. In this
case, once caspase-8 is activated by recruitment via FADD to the
engaged receptors, Bid, a proapoptotic molecule from the Bcl-2 family,
is cleaved, and the fragments generated are able to insert in the
mitochondria outer membrane, producing by themselves, or in association
with other molecules, mitochondrial permeability transition and
m loss (52, 53). It would be
tempting to place caspase-8 as the Z-VAD-sensitive caspase responsible
for ceramide-induced m loss through Bid
cleavage. However, this possibility is not supported by the poor
protection offered by CrmA on ceramide-induced apoptosis, since the
main target of CrmA antiapoptotic effect is caspase-8
(29). In the case of granulysin, the induction of
mitochondrial permeability transition by disruption of the cellular
ionic equilibrium seems to be rapid and efficient, probably because it
does not need additional molecular intermediates. This could be the
reason for the relative inefficiency of Bcl-2 protection
from this pathway of m disruption. The
presence of a population of J-Bcl2 cells that is still sensitive to
granulysin-induced apoptosis does not seem to be due to the existence
of two populations of J-Bcl2 cells with different level of Bcl-2
expression, as analyzed by flow cytometry in permeabilized J-Bcl2 cells
(data not shown). However, it seems that in the small percentage of
J-Bcl2 cells that are still sensitive to granulysin-induced apoptosis,
the typical apoptotic process is somewhat delayed. In these
circumstances, AIF seems to be released before cytochrome c
from mitochondria, and its particular effect on the nucleus is the only
detected at early time points (Fig. 5E). At later time
points, and presumably in the same cells in which AIF was previously
released from the mitochondria, the caspase pathway should be activated
at least in part, resulting in the typical nuclear fragmentation (Fig. 5F). This is in agreement with the recent observation that
AIF translocation to the nucleus precedes temporally cytochrome
c release during staurosporine-induced apoptosis
(38).
In conclusion, granulysin-induced cell death may constitute an additional molecular mechanism mediated by CTL degranulation against tumor development. Knowledge of the biochemical mechanisms of apoptosis induction activated by granulysin should be useful in future cancer therapies using granulysin or granulysin-derived bioactive peptides (54).
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Alberto Anel, Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, E-50009 Zaragoza, Spain. E-mail address: anel{at}posta.unizar.es
3 Abbreviations used in this paper: Ac-DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp aldehyde; Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; m, mitochondrial transmembrane potential; DiOC6(3), 3,3'-dihexyloxacarbocyanine iodide; CCCP, carbonyl cyanide m-chlorophenylhydrazone; AIF, apoptosis-inducing factor; PPDA, p-phenylenediamine; SM, sphingomyelin.
Received for publication February 6, 2001. Accepted for publication May 21, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
m loss, caspase-9 activity, and apoptosis in Jurkat cells. Exp. Cell Res. 258:223.[Medline]
This article has been cited by other articles:
|
|
 |
|
  H. Zhang, C. Zhong, L. Shi, Y. Guo, and Z. Fan Granulysin Induces Cathepsin B Release from Lysosomes of Target Tumor Cells to Attack Mitochondria through Processing of Bid Leading to Necroptosis J. Immunol., June 1, 2009; 182(11): 6993 - 7000. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Mader, N. Mookherjee, R. E.W. Hancock, and R. C. Bleackley The Human Host Defense Peptide LL-37 Induces Apoptosis in a Calpain- and Apoptosis-Inducing Factor-Dependent Manner Involving Bax Activity Mol. Cancer Res., May 1, 2009; 7(5): 689 - 702. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Nakashima, A. Shiozaki, S. Myojo, M. Ito, M. Tatematsu, M. Sakai, Y. Takamori, K. Ogawa, K. Nagata, and S. Saito Granulysin Produced by Uterine Natural Killer Cells Induces Apoptosis of Extravillous Trophoblasts in Spontaneous Abortion Am. J. Pathol., September 1, 2008; 173(3): 653 - 664. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Bras, V. J. Yuste, G. Roue, S. Barbier, P. Sancho, C. Virely, M. Rubio, S. Baudet, J. E. Esquerda, H. Merle-Beral, et al. Drp1 Mediates Caspase-Independent Type III Cell Death in Normal and Leukemic Cells Mol. Cell. Biol., October 15, 2007; 27(20): 7073 - 7088. [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]
|
|
|
|
|
|
L. P. Huang, S.-C. Lyu, C. Clayberger, and A. M. Krensky Granulysin-Mediated Tumor Rejection in Transgenic Mice J. Immunol., January 1, 2007; 178(1): 77 - 84. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Liu, S. Liu, X. Qu, and J. Liu Construction of a eukaryotic expression system for granulysin and its protective effect in mice infected with Mycobacterium tuberculosis. J. Med. Microbiol., October 1, 2006; 55(Pt 10): 1389 - 1393. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Pardo, C. Urban, E. M. Galvez, P. G. Ekert, U. Muller, J. Kwon-Chung, M. Lobigs, A. Mullbacher, R. Wallich, C. Borner, et al. The mitochondrial protein Bak is pivotal for gliotoxin-induced apoptosis and a critical host factor of Aspergillus fumigatus virulence in mice J. Cell Biol., August 14, 2006; 174(4): 509 - 519. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Dong, Q. Li, S.-c. Lyu, A. M. Krensky, and C. Clayberger A novel apoptosis pathway activated by the carboxyl terminus of p21 Blood, February 1, 2005; 105(3): 1187 - 1194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Kabelitz, D. Wesch, E. Pitters, and M. Zoller Characterization of Tumor Reactivity of Human V{gamma}9V{delta}2 {gamma}{delta} T Cells In Vitro and in SCID Mice In Vivo J. Immunol., December 1, 2004; 173(11): 6767 - 6776. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Kelly, N. J. Waterhouse, E. Cretney, K. A. Browne, S. Ellis, J. A. Trapani, and M. J. Smyth Granzyme M Mediates a Novel Form of Perforin-dependent Cell Death J. Biol. Chem., May 21, 2004; 279(21): 22236 - 22242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Sasson, E. Rimon, A. Dantes, T. Cohen, V. Shinder, A. Land-Bracha, and A. Amsterdam Gonadotrophin-induced gene regulation in human granulosa cells obtained from IVF patients. Modulation of steroidogenic genes, cytoskeletal genes and genes coding for apoptotic signalling and protein kinases Mol. Hum. Reprod., May 1, 2004; 10(5): 299 - 311. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. C. Alano, W. Ying, and R. A. Swanson Poly(ADP-ribose) Polymerase-1-mediated Cell Death in Astrocytes Requires NAD+ Depletion and Mitochondrial Permeability Transition J. Biol. Chem., April 30, 2004; 279(18): 18895 - 18902. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. C. Jung, W. S. Park, H. J. Kim, E. Y. Choi, M.-C. Kook, H.-W. Lee, and Y. Bae TCR-Independent and Caspase-Independent Apoptosis of Murine Thymocytes by CD24 Cross-Linking J. Immunol., January 15, 2004; 172(2): 795 - 802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Okada, Q. Li, J. C. Whitin, C. Clayberger, and A. M. Krensky Intracellular Mediators of Granulysin-Induced Cell Death J. Immunol., September 1, 2003; 171(5): 2556 - 2562. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Arnoult, P. Parone, J.-C. Martinou, B. Antonsson, J. Estaquier, and J. C. Ameisen Mitochondrial release of apoptosis-inducing factor occurs downstream of cytochrome c release in response to several proapoptotic stimuli J. Cell Biol., December 23, 2002; 159(6): 923 - 929. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
