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From the MRC Toxicology Unit, Leicester, United Kingdom
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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m) and the closely related mitochondrial
permeability transition8,9
have recently been implicated
in the redistribution of cytochrome c. The mitochondrial
permeability transition, which is usually associated with a loss of
m, involves the opening of a channel in the inner
mitochondrial membrane. This channel allows the ingress of water into
the mitochondrial matrix, resulting in osmotic swelling and the
subsequent rupture of the outer membrane.10
Ultrastructural evidence for mitochondrial swelling and discontinuities
in the outer membrane have been reported during the redistribution of
cytochrome c, but, surprisingly, these changes occurred in
the absence of any loss of m.11
The
presence of a specific channel in the outer mitochondrial membrane has
also been suggested as a route for the egress of cytochrome
c10, and such a mechanism may not necessarily
involve mitochondrial swelling. The involvement of the cytoskeleton in the development of apoptosis has also been reported by numerous groups,12-14 and particular emphasis has been placed on changes in actin cleavage/distribution.15,16 Caspase-dependent cleavage of the actin-regulatory protein gelsolin has even been implicated in the release of cytochrome c from mitochondria.17
We have recently reported the presence of discontinuities in the outer
membrane of ultracondensed mitochondria in apoptotic THP.1
cells.18
In the present study we clearly demonstrate that
these ultracondensed mitochondria occurred only in cells exhibiting a
reduced m. Furthermore, we show that both of these
changes, together with all other morphological indicators of apoptosis,
were prevented by the inhibition of caspase activity. The
redistribution of mitochondrial cytochrome c was unaffected
by this inhibition and thus preceded all of the other changes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Media and serum were purchased from Gibco (Paisley, UK). The broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.fmk) was purchased from Enzyme Systems (Dublin, CA), and the protease inhibitors N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) and N-tosyl-L-lysine chloromethyl ketone (TLCK) were from Boehringer-Mannheim (Lewes, UK). The fluorescent probe 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and the mouse monoclonal antibody recognizing mitochondrial membrane-bound cytochrome c oxidase (subunit II) were purchased from Molecular Probes (Eugene, OR). The mouse monoclonal antibody recognizing human cytochrome c was from PharMingen (San Diego, CA). All other chemicals and primary antibodies were obtained from Sigma Chemical Company (Poole, UK).
THP.1 cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum and 2 mmol/L glutamine in an atmosphere of 5% CO2 in air at 37°C.19 Logarithmically growing cells were used for all experiments. To induce apoptosis, 0.5 x 106 cells/ml were incubated in the presence of cycloheximide (25 µmol/L), etoposide (25 µmol/L), or TPCK (75 µmol/L) as previously described.19,20 The proportion of cells undergoing apoptosis was determined by flow cytometry after staining with Hoechst 33342/propidium iodide20 or labeling with Annexin V, as previously described.18 To assess the effects of caspase inhibition on apoptosis, THP.1 cells were treated with Z-VAD.fmk (50 µmol/L) 5 minutes before exposure to the apoptotic stimulus.
Flow Cytometric Analysis of m
Suspensions of 0.5 x 106
cells were incubated
for 20 minutes at 37°C with DiOC6,(3) (50 nmol/L).
Control experiments were performed by incubating cells for a further 10
minutes at 37°C with m-carbonyl cyanide
m-chlorophenyl hydrazone (50 µmol/L), an uncoupling agent
that abolishes the m. Experimental samples were
incubated, for 4 hours, with etoposide or TPCK, in the presence or
absence of Z-VAD.fmk, before being analyzed and sorted, into
subpopulations of cells with normal and decreased m,
in a Becton-Dickinson "Vantage" flow cytometer. To differentiate
between these subpopulations each experiment was calibrated, using
cells treated with the uncoupling agent, to select the subpopulation of
cells (about 80%) with a decreased m.
Electron Microscopy and Immunocytochemistry
Sorted cells from the flow cytometer were collected in 4% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.4), until the volume of the fixative had been doubled by the carrier solution. Samples were collected for 2 hours, combined, and spun down in a swing-out rotor at 3000 x g. Cells in culture (3 x 106) were spun down before the supernatant was gently replaced with 2% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.4). All samples were fixed overnight, at 4°C, and postfixed with 1% osmium tetroxide/1% potassium ferrocyanide overnight at 4°C. Fixed pellets were stained en bloc with 5% aqueous uranyl acetate overnight at room temperature, dehydrated, and embedded in Agar 100 epoxy resin. Sections up to 1 µm were examined unstained by electron spectroscopic imaging with a Zeiss 902A electron microscope. Ultrathin sections were stained with lead citrate and examined in a Jeol 100-CXII electron microscope equipped with a rotating stage/eucentric goniometer. All quantitative assessments were based on counts of at least 500 cells at each treatment/time point.
Duplicate pellets were fixed with 4% formaldehyde (pH 7.4), freshly
made up from paraformaldehyde in Dulbecco's phosphate-buffered saline
(PBS), for 1 hour at room temperature. They were rinsed in PBS,
dehydrated in ethanol, and infiltrated with Unicryl resin from British
Biocell International (Cardiff, Wales). The resin was polymerized with
UV radiation (
360 nm) at 4°C, according to the manufacturer's
instructions.
Ultrathin sections were blocked with normal goat serum and diluted 1:50
in PBS containing 1% bovine serum albumin and 1% Tween 20, for 4
hours at room temperature. They were incubated in primary antibody, a
mouse IgG1 monoclonal antibody (clone AC-15) raised against a slightly
modified ß-cytoplasmic actin N-terminal peptide from Sigma Chemical
Company, diluted 1:10 in PBS containing 1% normal goat serum, 1%
bovine serum albumin, and 1% Tween 20 (PBSGAT), for 18 hours at 4°C.
In control incubations the primary antibody was replaced with mouse
IgG1 from Dako (Ely, UK). Thorough washing in PBSGAT was followed by
incubation in a goat-derived anti-mouse IgG from British Biocell
International (Cardiff, Wales), which had been absorbed against human
serum proteins and conjugated with 20 nm colloidal gold, diluted 1:50
in PBSGAT, for 18 hours at 4°C. Serial sections were incubated with a
range of dilutions of antibodies to 
-actin, tubulin, and vimentin
before treatment with the appropriate colloidal gold-conjugated IgG.
Ultrathin sections were examined unstained or after staining with
uranyl acetate and lead citrate.
Preparation of Cytosolic Extracts
Cytosolic extracts were isolated as described previously.18,21 Briefly, 50 x 106 THP.1 cells were washed twice in ice-cold PBS and resuspended in 200 µl of extraction buffer (220 mmol/L mannitol, 68 mmol/L sucrose, 50 mmol/L piperazine-N,N'-bis(2-ethanesulfonic acid/KOH, pH 7.5, 50 mmol/L KCl, 5 mmol/L EGTA, 2 mmol/L MgCl2, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml aprotinin). They were incubated, on ice, for 30 minutes and then homogenized, using a glass Dounce homogenizer, with 40 strokes of the "B" pestle. Homogenates were centrifuged at 14,000 x g, for 15 minutes at 4°C, and the resulting supernatants were used as the cytosolic extracts.
Western Blot Analysis
Cellular proteins were resolved on 10% sodium dodecyl sulfate polyacrylamide gels and blotted onto "Hybond-C" nitrocellulose membranes from Amersham International (Little Chalfont, UK). Cytochrome c was detected using a mouse monoclonal antibody (7H8.2C12) to human cytochrome c, and parallel samples were checked for mitochondrial contamination, using the antibody (12C4-F12) to cytochrome c oxidase.18 Actin was detected in samples of 0.2 x 106 cells, prepared as previously described,21 with both the monoclonal (AC-15) antibody that was used for immunocytochemistry and with a rabbit-derived polyclonal antibody to the C-terminal actin fragment, attached to Multiple Antigen Peptide. The bound primary antibodies were visualized with an enhanced chemiluminescence kit from Amersham International, using an appropriate secondary antibody conjugated with horseradish peroxidase from Sigma Chemical Company.
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Results |
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Untreated THP.1 cells were large (12–16 µm in diameter) and
irregular in outline, with many short, irregular microvilli and a
multilobed nucleus (Figure 1a)
. The
diffuse perinuclear and perinucleolar heterochromatin was weakly
differentiated from the euchromatin, but the nucleolus was clearly
visible in most sections. Several mitochondria, with irregular
transverse or obliquely arranged cristae, were evident in cell
profiles. Treatment of cells for 4 hours with cycloheximide (25
µmol/L), an inhibitor of protein synthesis, resulted in several of
the cells showing the characteristic features of apoptosis, including
shrinkage, loss of microvilli, increased cytosolic density,
vacuolation, and perinuclear clumping of condensed chromatin (Figure 1b)
. Small ultracondensed mitochondria were present within most of
these cells, but this change was also observed in a few cells that
showed no other morphological signs of apoptosis (Figure 1b)
. The
cytoplasm of apoptotic cells also included spheroidal inclusions
(1.0–2.5 µm in diameter) of a fine amorphous material (Figure 1b)
and clusters of small vesicles (Figure 1c)
. These clusters were found
in 5–10% of treated cells; usually only one cluster was visible in a
cell profile, but six were found in one cell. A few of these clusters
were adjacent to the cell membrane (Figure 1c)
, but most were
restricted to the perinuclear region. The majority of clusters
consisted of a large proportion (>90%) of remarkably uniform vesicles
35–45 nm in diameter; the remaining vesicles were larger and oblate. A
few clusters were closely associated with distorted membranous
cisternae, some of which resembled the trans elements of the
Golgi apparatus. Combinations of two or more of these morphological
changes were usually evident, even in the absence of an overall
increase in cytoplasmic staining density. Cytoplasmic and nuclear
changes similar to those observed after cycloheximide were also
detected after the treatment of cells, for 4 hours, with etoposide, a
DNA topoisomerase II inhibitor (Figure 1d)
. The spheroidal cytoplasmic
inclusions of fine amorphous material were often closely associated
with the cell membrane, and some were observed within cellular
protrusions or apoptotic buds. A few of these membrane-limited
structures, containing spheroidal inclusions, were found free in the
extracellular medium (Figure 2a)
. In
agreement with previous results,19
the morphological
changes (Figure 2b)
and the incidence of apoptosis (Figure 3)
were particularly pronounced in cells
after treatment, for 4 hours, with a combination of the trypsin-like
protease inhibitor TLCK (100 µmol/L) with cycloheximide (25
µmol/L). The chymotrypsin-like protease inhibitor TPCK (75 µmol/L)
was also a potent inducer of apoptosis (Figure 3)
and resulted in
cytoplasmic changes indistinguishable from those observed after
etoposide, cycloheximide, or cycloheximide and TLCK (Figure 2c)
.
Despite these similarities, however, treatment with TPCK for 4 hours
resulted in different nuclear changes, with dispersal of the nucleolar
dense fibrillar component and small clumps of partially condensed
chromatin throughout the nucleoplasm. This morphology has been
correlated with the formation of large kilobase pair fragments of DNA,
in the absence of internucleosomal cleavage.20
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The development of all of the detectable morphological changes
induced by the three agents (Figures 1 and 2)
was prevented by
Z-VAD.fmk (50 µmol/L). Cells treated with this broad-spectrum caspase
inhibitor before incubation with etoposide (Figure 4a)
or TPCK (Figure 4b)
were
morphologically indistinguishable from controls, thus demonstrating the
key role of caspase activation in these structural changes. Both
etoposide and TPCK induced a time-dependent increase in the cytosolic
level of cytochrome c, which was first clearly detected
after 2 hours of treatment (Figure 5
,
lane 4). Cytochrome oxidase subunit II, a marker for mitochondrial
contamination, was not detected in any of these samples (data not
shown). Treatment with Z-VAD.fmk did not block the increase in
cytosolic cytochrome c induced by either stimulus (Figure 5
,
lane 7). Thus, as Z-VAD.fmk blocked the morphological changes without
inhibiting this increase, the release of mitochondrial cytochrome
c is upstream, or independent of caspase activation.
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Ultracondensed mitochondria were the most consistent feature of
cells treated, for 4 hours, with cycloheximide, etoposide, or TPCK.
Most of these ultracondensed mitochondria appeared in sections as
uniform short rods or circles, 200–600 nm in diameter (Figures 1b, 1d, 2a, and 4c)
, with an increased electron density that was entirely
restricted to the matrix of the inner compartment (Figure 4d)
. The
organelles were rarely more than 1.5 µm long, but in a few instances
bizarre elongated forms, over 4 µm long, were observed (Figure 2b)
.
In a few cells, reorganization of the inner membrane resulted in the
formation of discrete globular profiles within the poorly defined
outline of the outer membrane (Figures 2d and 4d)
. This membrane often
disappeared into obliquity, but discontinuities were identified where
it could not be traced, even by tilting the section or by electron
spectroscopic imaging of thick (1-µm) sections. Time-course studies
showed that less than 5% of cells fixed after 1 hour of treatment with
cycloheximide, etoposide, or TPCK contained ultracondensed
mitochondria. No significant increase in the incidence of
ultracondensed mitochondria was detected after 2 hours of treatment,
but most (55–60%) cells examined after 3 hours contained these
organelles. Only at this time did a few cells contain both noncondensed
and ultracondensed mitochondria (Figure 4c)
together with some rare,
possibly transitional forms, in which a slight increase in density was
evident despite the irregular disposition of many of the cristae
(Figure 4d)
. Thus most cells were devoid of any recognizable
transitional form and contained homogeneous populations of either
noncondensed or ultracondensed mitochondria. The presence of only small
numbers of untreated THP.1 cells with ultracondensed mitochondria is
consistent with the low background incidence of apoptosis in these
cells. The virtual absence of mixed populations of noncondensed and
ultracondensed mitochondria, or of intermediate forms, indicates that a
rapid transition probably occurs throughout all of the mitochondria
within each affected cell. To establish the significance of these
ultrastructural changes, cells were examined by flow cytometry, so that
the incidence of apoptosis, as indicated by staining with Hoechst
33342/propidium iodide (Figure 3)
, Annexin V, and DiOC6(3),
could be quantified. Treatment with either etoposide or TPCK resulted
in marked increases in the percentage of apoptotic cells, as assessed
by these three independent methods. This etoposide- and TPCK-induced
apoptosis was abolished by Z-VAD.fmk (Table 1)
. Staining with DiOC6(3)
was also used to monitor changes in m. Many of the
cells with low m were smaller than the control cells,
as indicated by a decrease in forward light scatter (Figure 6)
, another characteristic of apoptotic
cells. As these cells constituted only a small proportion of the total
cell population, any structural changes could not be correlated
directly with these particular cells. The cells were therefore sorted
and then examined by electron microscopy to establish whether
ultracondensed mitochondria were present in either or both of the
subpopulations.
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m
Almost all of the cells (>95%) isolated because of their reduced
m were apoptotic and contained ultracondensed
mitochondria. The incidence of other signs of apoptosis was also
greatly enhanced in these cells, particularly the cytoplasmic
inclusions of amorphous material. Many of the sorted cells had
chromatin condensed against the inner membrane of the nuclear envelope
and only a few had normal, intact Golgi stacks. Although sorted for
their low m, they exhibited a wide range of
morphologies (Figure 7a)
, ranging from
cells with a dense, shrunken cytoplasm to others showing signs of
lysis. In contrast, the cells sorted for normal m did
not exhibit any morphological signs of apoptosis. The mitochondria of
most of these cells (>80%) had a normal appearance, and those present
in the remaining cells were swollen rather than ultracondensed (Figure 7b)
. Thus these results demonstrate that a reduction in
m is definitely not dependent on the development of
swollen mitochondria and, in this system, involves their
ultracondensation.
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The cytoplasmic inclusions of amorphous material were readily
apparent in nonosmicated samples that had been embedded in acrylic
resin (Figure 7c)
. These inclusions were not labeled by antibodies to
-actin, tubulin, or vimentin (data not shown), but they were heavily
labeled by the antibody to the ß-cytoplasmic actin N-terminal peptide
(Figure 7, c and d)
. In untreated THP.1 cells, diffuse labeling with
this antibody was observed throughout the cytoplasm, particularly
against the inner leaflet of the cell membrane (data not shown).
Actin Cleavage
To determine whether the ultrastructural changes were the result
of actin cleavage, cellular proteins from both controls and treated
cells were examined by Western blotting. The presence of intact actin
was indicated by the presence of a ~42-kd band that reacted with both
the monoclonal antibody to the ß-cytoplasmic actin N-terminal peptide
and confirmed with the polyclonal antibody to the C-terminal residues
of actin. This polyclonal antibody also detected several subsidiary
bands, but no differences could be detected between samples from
control and treated cells (Figure 8)
and
thus no sign of actin cleavage was detected.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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m
Ultracondensed mitochondria, the most striking ultrastructural
feature of the apoptotic THP.1 cells studied in our experiments, have
been reported previously, but their significance was not fully
established.18
Fluorescence-activated cell sorting has now
enabled us to demonstrate, unequivocally, that these organelles are
only present in cells exhibiting a low m. The
uniformity of the cytoplasmic response to the three disparate agents
used in our study is consistent with these changes being part of a
common apoptotic pathway.2
The ultracondensed mitochondria described in the present study are
distinct from those of the condensed conformation.22
Highly condensed mitochondria have been reported as a short-term
response to cell injury.23,24
In these early studies,
however, condensation was followed by a rapid return to the orthodox
configuration, followed by the development of the high-amplitude
swelling and flocculent densities that are normally associated with
oncosis. Unlike this transient response to injury, the changes observed
in the present study resemble the "pleomorphic
micromitochondriosis" described in apoptotic nodal
myocytes25
and the "mitochondrial pyknosis" or
"hypercondensation" observed during apoptosis in a colon carcinoma
cell line.26
Interestingly, both of these studies also
reported mitochondrial proliferation, and the latter detected a
reduction in m. Other studies have described
"ultracondensed" mitochondria in apoptotic lymphoblastic leukemic
cells and implicated them in the signaling of the apoptotic
process.27,28
The ultracondensed mitochondria observed in
THP.1 cells were not transient, as they were present at all stages of
apoptosis and in cells undergoing secondary necrosis. They were not an
artifact of the sorting procedure, as they were absent from cells
sorted for normal m (Figure 7b)
. Strikingly, all of
the mitochondria in cells sorted for low m were
ultracondensed (Figure 7a)
, whereas those in cells with normal
m were not condensed. Thus, in THP.1 cells, a
reduction in m accompanied the development of
ultracondensed mitochondria. Reduction in m has
been proposed as a critical early step in the development of lymphocyte
apoptosis,8
but in the present study, as in most models of
apoptosis, this change is preceded by the redistribution of cytochrome
c.10
Redistribution of Mitochondrial Cytochrome c
A failure of electrical and osmotic homeostasis, resulting in
swelling and subsequently in rupture of the outer mitochondrial
membrane, has been proposed as a mechanism for the release of
cytochrome c into the cytosol.11
The
mitochondrial permeability transition is usually associated with
swollen mitochondria9,10
and with loss of
m.10
The predominance of ultracondensed
mitochondria, in apoptotic THP.1 cells, indicated that overdistension
of mitochondria may be discounted as the mechanism for cytochrome
c release in the present study.
The presence of Bcl-xL, an anti-apoptotic pore-forming member of the Bcl-2 family, may prevent osmotic disruption of the outer mitochondrial membrane,11 but other studies have suggested that a pore formed by Bax, a pro-apoptotic member of this family, may make possible the release of cytochrome c.10 The presence of such a pore may account for the absence of any detectable morphological changes in the mitochondria of apoptotic THP.1 cells, before the redistribution of cytochrome c.
The prevention of all of the ultrastructural changes by Z-VAD.fmk
(Figure 4, a and b)
, without blocking the release of mitochondrial
cytochrome c (Figure 5)
, clearly shows that this release
precedes ultracondensation. Although Z-VAD.fmk does not inhibit the
release of mitochondrial cytochrome c during
chemical-induced apoptosis, it does prevent this redistribution during
receptor-mediated apoptosis.29
Thus activation of
caspases, which is critical for the initiation of receptor-mediated
apoptosis, may occur after commitment to cell death during
chemical-induced apoptosis. In chemical-induced apoptosis, the increase
in cytosolic cytochrome c (Figure 5)
, in the presence of
Apaf-1, activates caspase-9, which, in turn, activates the
"effector" caspases-3 and -7. These enzymes are responsible for
most of the biochemical and morphological changes associated with the
apoptotic phenotype.30,31
The caspase-dependent ultracondensation of mitochondria and, particularly, the attendant discontinuities in the outer mitochondrial membrane may result in the liberation of more cytochrome c and other caspase activators, including apoptosis-inducing factor, thus amplifying the initial apoptotic stimulus.
This interpretation is consistent with the divergent morphologies that
occurred in cells sorted for reduced m. Despite the
consistent presence of ultracondensed mitochondria, these cells
represented many stages of the apoptotic process, thus reflecting the
delay between the activation of the caspase cascade and the instant of
fixation for each cell.
Golgi Fragmentation in Apoptotic Cells
The appearance of the clusters of cytoplasmic vesicles in apoptotic THP.1 cells is very similar to that reported in mitotic cells and confirmed as the transitional form of the Golgi apparatus.32,33 The suggested origin of the clusters is supported by their absence from THP.1 cells with an intact Golgi apparatus. The presence of intact Golgi cisternae in some cells with ultracondensed mitochondria indicates that the clusters probably develop after the mitochondrial changes. Despite the retention of the nuclear membrane, there is a profound reorganization of the cell architecture during apoptosis, and so modification of the Golgi apparatus in these cells is unsurprising if, to our knowledge, unreported.
Actin Redistribution in Apoptotic Cells
The presence of numerous cytoplasmic inclusions of actin was particularly surprising, as little attention has been given to the redistribution of this protein during apoptosis, despite the large literature concerning its cleavage. Inhibitors of calpain may prevent actin proteolysis during apoptosis, but the extent of this actin fragmentation is very low.16,34 Actin cleavage, by caspase-1 and -3, has been demonstrated in cell-free systems,15,35,36 but its degradation in intact human cells is still controversial.37 Cleavage of actin by caspase-3 results in a "fractin" fragment that can be identified in the cell processes of apoptotic neurones.37 We cannot exclude the possibility that a similar fragment was present in our samples, as an antibody similar to that used in the current study did not detect "fractin."38 Similarly, Guenal et al39 reported caspase-dependent actin cleavage and, using immunofluorescence microscopy, suggested that the resulting fragments accumulated in apoptotic bodies. The present study provides conclusive evidence that actin-rich bodies are extruded from the cytoplasm of THP.1 cells and that these bodies consist of intact ß-actin. The absence of significant fragmentation was confirmed by our immunoblotting results, with the nonspecific antibody used previously to detect actin cleavage in cell extracts.15 Caspase-dependent disorganization of the actin cytoskeleton has been attributed to the cleavage of ß-catenin rather than actin itself,13 and similar effects have been attributed to the depletion of ezrin/radixin/moesin proteins after activation of the caspase cascade.40 Surprisingly, disruption of the cytoskeleton has also been associated with changes in the permeability of the mitochondrial outer membrane.41 Actin redistribution, including the formation of cytoplasmic aggregations, has also been observed, by light microscopy, during the apoptosis of several cell types.12,42-45 Recently, Jaunin et al14 have shown aggregates of ß-actin in transfected HeLa cells overexpressing the death-domain-containing protein MyD88, but not in nontransfected cells. Colocalization of this protein with the ß-actin of apoptotic cells, like that of the "apoptosis-specific protein" reported in several examples of apoptosis,46 may indicate a direct, rather than passive role for actin in cell death.
The release of cellular fragments containing aggregations of actin may thus be one feature of a common process involving the redistribution of the cytoskeleton during apoptosis. The phagocytosis of such fragments by neighboring cells could result in the subsequent proteolysis of actin in secondary lysosomes. Variations in the extent of this process, particularly between facultative and obligate phagocytes, may provide an explanation for the diversity of observations regarding actin proteolysis in cultures of apoptotic cells.
This study has shown that the release of mitochondrial cytochrome c into the cytosol of apoptotic THP.1 cells is normally followed by striking morphological changes in the cytoskeleton, Golgi apparatus, and mitochondria. All of these changes, unlike the release of cytochrome c, are prevented by the inhibition of caspase activity. Our results indicate that cytochrome c release is not the result of the onset of the mitochondrial permeability transition or the loss of the inner mitochondrial membrane potential. The development of ultracondensed mitochondria, which occurs after the release of cytochrome c, is currently the earliest morphologically detected hallmark of apoptosis in THP.1 cells.
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Acknowledgements |
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Footnotes |
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Accepted for publication April 30, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-catenin. J Cell Biol 1997, 139:759-771This article has been cited by other articles:
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J. A. Sanchez-Alcazar, E. Schneider, M. A. Martinez, P. Carmona, I. Hernandez-Munoz, E. Siles, P. De la Torre, J. Ruiz-Cabello, I. Garcia, and J. A. Solis-Herruzo Tumor Necrosis Factor-alpha Increases the Steady-state Reduction of Cytochrome b of the Mitochondrial Respiratory Chain in Metabolically Inhibited L929 Cells J. Biol. Chem., April 28, 2000; 275(18): 13353 - 13361. [Abstract] [Full Text] [PDF]
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) or the absence
(
) of the caspase
inhibitor Z-VAD.fmk (50
µmol/L). Apoptotic cells were stained with
Hoechst 33342 and detected by flow cytometry.
