 |
INTRODUCTION |
Recent work has demonstrated that a family of cysteine proteases,
now termed caspases (1), related to the Caenorhabditis elegans protease CED-3 and the mammalian
interleukin-1
-converting enzyme becomes activated during apoptosis
and is necessary for several processes within the apoptotic pathway.
Caspases are constitutively expressed and synthesized as zymogens that
require proteolytic cleavage, either through a proteolytic cascade or
by intermolecular autoproteolysis, to become activated (2). Once
activated, caspases cleave their substrates in a highly
sequence-specific fashion with a near absolute requirement for an
aspartic acid in the P1 position (3). For example, caspase-3 cleaves
its substrates at a conserved DXXD motif (2). Many caspase
substrates have been identified and include nuclear proteins such as
poly(ADP-ribose) polymerase
(PARP)1 and retinoblastoma
(RB) as well as structural proteins of the nucleus and cytoskeleton
including lamins and gelsolin (4-7). It is believed that cleavage of
some of the known caspase substrates leads to the characteristic
changes in morphology and biochemistry observed in apoptotic cells (8).
As an example, caspase-3 mediated cleavage of DNA fragmentation factor
results in chromatin condensation and DNA fragmentation during
apoptosis (9, 10).
Although caspases play a major part in the demise of cells that have
been triggered to undergo apoptosis, there is evidence that other
proteases including calpain may also be involved in this process.
Calpain is a family of calcium-dependent cysteine proteases
of which two isozymes, µ- and m-calpain, are ubiquitously expressed
(11). These enzymes are heterodimeric and consist of an 80-kDa
catalytic subunit and a 30-kDa subunit whose function is unclear (11).
In contrast to caspases, calpain does not appear to have strict
sequence requirements for substrate cleavage (11). Under normal
physiological conditions these proteases engage in limited proteolysis
of a number of different substrates found throughout the cell,
including nuclear proteins such as p53 and cyclin D1
(12, 13), as well as proteins associated with the cytoskeleton and
plasma membrane, including talin and
N-methyl-D-aspartate receptors (14, 15). Unlike
the caspases, which function only during apoptosis, calpain has been
implicated in several processes involved in normal cellular metabolism
and physiology (13-17), such as remodeling of the actin
cytoskeleton during cell motility (16).
Uncontrolled or constitutive calpain activity has been observed in
several pathological conditions including Alzheimer's disease, muscular dystrophies, and tumorigenesis (18-21). Excessive calpain activation has also been observed in many instances of cell death (22-24). For example, proteolysis of several calmodulin-binding proteins by calpain occurs in neurons that have been exposed to neurotoxins (24). Other studies have demonstrated that calpain may play
a role in the apoptotic death of a variety of different cell types
(25-31). For instance, calpain-mediated cleavage of the cytoplasmic
domain of the integrin
3 subunit in human umbilical vein
endothelial cells contributes to disruption of signaling and detachment
from the matrix resulting in apoptosis (30).
Previously we have shown that the proapoptotic protein Bax is cleaved
from its native 21-kDa form to an 18-kDa species by calpain during
drug-induced apoptosis of HL-60 cells (32, 33). Here we extend these
findings and show that calpain is activated secondary to caspase
activation during apoptosis. Time course experiments demonstrate that
calpain activation occurs after cleavage of caspase substrates and DNA
fragmentation. Experiments with HL-60 cells revealed that inhibition of
calpain activity by the calpain inhibitor calpeptin failed to block
caspase activity and apoptosis, whereas pretreatment of cells with the
pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone
(Z-VAD-fmk) blocked both caspase and calpain activity and partially
inhibited cell death. Thus, in this apoptosis model there appears to be a hierarchy of protease family activation during the degradation phase
of the apoptotic program.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
HL-60 cells were cultured in 5%
CO2 and 95% humidified air atmosphere at 37 °C in
complete RPMI 1640 medium (Cellgro) containing 10% heat-inactivated
fetal bovine serum (Atlanta Biologicals), 100 units/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 2 mM glutamine (all from Life Technologies, Inc.). Cells were split every 3 days to ensure logarithmic growth.
Time Course of Drug-induced Apoptosis and Inhibitor
Studies--
For the time course experiments, 3 × 105 cells were cultured in 6-well dishes (Nunclon) in 3 ml
of medium for 36 h at 37 °C before the addition of the drug
9-amino-20(S)-camptothecin (9-AC; Ref. 32) or its diluent (20%
dextrose and 0.9% NaCl in sterile water). Cells were harvested at the
appropriate time points, washed with ice-cold PBS, and subjected to
lysis and protein extraction for the purposes of immunoblotting and
in vitro fluorogenic assays. For the inhibitor studies,
cells cultured as described above were pretreated with either 10 µM Z-Leu-Nle-H (calpeptin; Calbiochem) for 30 min or 100 µM Z-VAD-fmk (Enzyme Systems Products) for 2 h at
37 °C. Control cultures were pretreated for the same time periods
with an equivalent volume of inhibitor vehicle (Me2SO
0.1%) or were left untreated. Cells were then challenged with 4.0 µM 9-AC to induce apoptosis, and control cultures were
treated with a corresponding volume of 9-AC diluent or left untreated. Cells were collected at 0 and 20 h after drug treatment, washed with ice-cold PBS, and lysed for protein extraction with subsequent immunoblotting as described below.
Extraction of Proteins, Immunoblot, and Densitometric
Analysis--
At each time point after the addition of 9-AC or 9-AC
diluent, cells were harvested, washed with ice-cold PBS, and lysed in a
buffer containing Triton X-100 and Nonidet P-40 supplemented with
protease inhibitors as described (32). Quantitation of protein was
carried out with the BCA reagent (Pierce). Equal amounts of protein
(25-30 µg) in the presence of 5%
-mercaptoethanol (Bio-Rad) were
electrophoresed on 6% (RB), 8% (PARP), or 12% (Bax, 30-kDa calpain
subunit) SDS-PAGE gels. The gels were subsequently transferred to
polyvinylidene difluoride membranes (Millipore) by electroblotting at
4 °C for 2 h at 90-100 V. Immunoblotting was performed as
described (32) with the following antibodies: human-specific rabbit
anti-Bax polyclonal serum used at 1:1000 (PharMingen); human-specific
mouse anti-PARP monoclonal antibody at 1:2000 (clone C2-10,
PharMingen); a mouse anti-calpain monoclonal antibody used at 1:500
(Chemicon), which recognizes the conserved 30-kDa subunit of both human
µ- and m-calpain; human-specific mouse anti-cytochrome oxidase
(subunit II) (COX II) monoclonal antibody (Molecular Probes) used at
1:100; and human-specific mouse anti-RB monoclonal antibody (clone
G3-245, PharMingen) used at 1:100. Monoclonal sheep anti-mouse IgG or
donkey anti-rabbit IgG horseradish peroxidase-conjugated secondary
antibodies (Pharmacia Biotech) were used at 1:2000. Immunodetection was
carried out with the ECL detection system (Amersham Pharmacia Biotech).
Densitometry was performed using a Hewlett Packard 2C scanner with
analysis by Kodak Digital Science 1D software. To determine the percent autolysis of the 30-kDa calpain subunit and cleavage of Bax, the following calculation was used: (amount of cleavage product(s)/amount of intact protein + amount of cleavage product(s)) × 100.
In Vitro Fluorogenic Caspase-3 Cleavage Assay--
Whole cell
protein lysates (10 µg), generated as described above, from
9-AC-treated cells or diluent-treated control cells were incubated with
25 µM caspase-3 substrate
acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC)
(Calbiochem) (34) for 1 h at 37 °C in the presence of a caspase
reaction buffer containing 100 mM HEPES, pH 7.4, 10%
sucrose, 5 mM dithiothreitol, and 0.1% CHAPS (all from
Sigma) (35). For the inhibition studies, 1.0 µM caspase-3
inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) (Calbiochem)
(34) was added simultaneously with the substrate. All reactions were carried out in 96-well plates (Dynatech) with proteolysis of the fluorescent peptides measured using a fluorescent plate reader (Perkin-Elmer LS50B) with filter settings at 380 nm for excitation and
460 nm for emission. All reactions were accompanied by an AMC standard curve.
DNA Fragmentation Assay--
The following protocol was adapted
from McGahon et al. (36). HL-60 cells, cultured as described
above, were treated with either 4.0 µM 9-AC or 9-AC
diluent for the time course experiments, or cells were pretreated with
either 10 µM calpeptin or 100 µM Z-VAD-fmk
and then challenged with 4.0 µM 9-AC or 9-AC diluent for
6 h. At the appropriate time point after the addition of 9-AC or
diluent, cells (5 × 105) were harvested and
centrifuged at 2000 rpm. Supernatants were removed, and the pelleted
cells were resusupended in 20 µl of lysis buffer (20 mM
EDTA, 100 mM Tris, pH 8.0, and 0.8% sodium lauroyl
sarcosine; all from Sigma). After complete resuspension, 10 µl of a 1 mg/ml RNase/T1 mixture mix (Ambion) was added, and the lysed cells were
incubated in a 37 °C water bath for 1.5 h. Upon completion of
this incubation, 10 µl of 20 mg/ml proteinase K (Ambion) was added to
each sample followed by incubation for at least 16 h at 50 °C
with constant rotation. The recovered DNA (10 µl/sample) was
electrophoresed on 1.0% agarose gels containing 1 µg/ml ethidium
bromide in TAE buffer (40 mM Tris acetate, pH 8.0, and 2 mM EDTA) to visualize DNA fragmentation.
Trypan Blue Exclusion Assay--
Loss of membrane integrity was
determined by the inability of cells to exclude the vital dye trypan
blue. For the time course experiments, cells were cultured as described
above and either left untreated or were pretreated with Z-VAD-fmk (100 µM for 2 h), calpeptin (10 µM for 30 min), or both calpeptin and Z-VAD-fmk. Cells were then left untreated,
treated with 9-AC diluent, or challenged with 4 µM 9-AC.
At the appropriate time interval, triplicate samples were removed from
each culture condition, diluted 1:5 with trypan blue, and counted (at
least 50 cells were counted from each sample). The percentage of cells
scoring positive for uptake of the dye was calculated using the
following formula: (number of trypan blue positive cells/number of
trypan blue positive cells + number of trypan blue negative cells) × 100. To photograph the cells, the entire culture volume from each
culture condition at each time point was diluted 5-fold with trypan
blue and allowed to equilibrate for 10 min at 25 °C. Cells were then
washed once with PBS, resuspended in PBS at 1 × 106
cells/ml, and plated into 24-well plates (Nunclon) for light microscopy.
 |
RESULTS |
Time Course of Caspase Activation and DNA Fragmentation with 9-AC
Treatment of HL-60 Cells--
Previous studies suggested that drug
treatment of HL-60 cells activated both caspases and calpain (33). To
delineate well characterized caspase-dependent events during
apoptosis relative to the timing of calpain activation and Bax
cleavage, HL-60 cells were treated with the topoisomerase I inhibitor
9-AC, a camptothecin analog (32), to induce apoptosis. Whole cell
protein lysates were generated for immunoblotting with antibodies
against the caspase substrates PARP and RB, both of which have been
shown to be proteolytically cleaved in HL-60 cells during apoptosis (4, 5). The DNA repair enzyme PARP is a substrate for caspases-2, -3, -7, and -9 (37), which proteolyze it from its full-length form of 116 kDa to an apoptosis-specific 85-kDa fragment (4). Another caspase
target is RB, which has been shown to be cleaved to a 48-kDa fragment
in a caspase-dependent manner in HL-60 cells during
etoposide-induced apoptosis (5). As shown in Fig.
1A, PARP cleavage was detected
by 2 h after the introduction of 9-AC, with complete conversion of
the full-length 116-kDa protein to the 85-kDa fragment by 4 h.
Also illustrated in Fig. 1A is RB cleavage, which occurred
after 4 h of 9-AC treatment as indicated by the appearance of the
48-kDa cleavage product (5). In addition to caspase activation detected
by proteolysis of PARP and RB, we observed DNA fragmentation by 4 h of 9-AC treatment as illustrated by the characteristic ladder
formation (Fig. 1B). Pretreatment of HL-60 cells with the
pan-caspase inhibitor Z-VAD-fmk (3) inhibited DNA fragmentation in
cells treated with 9-AC, whereas pretreatment with the calpain-specific
inhibitor calpeptin (38) did not inhibit this process (Fig.
1B). These results demonstrate the requirement for
caspase-3-like proteases in the pathway leading to DNA fragmentation in
HL-60 cells treated with 9-AC.

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 1.
Time course of caspase activation and DNA
fragmentation with 9-AC treatment of HL-60 cells. HL-60 cells were
treated with 9-AC or 9-AC diluent (C) for up to 12 h
and harvested at different time intervals, and cell lysates were
prepared for SDS-PAGE and immunoblotting for PARP and RB cleavage.
A, a representative immunoblot showing the time course of
both PARP (upper panel) and RB (lower panel)
cleavage. B, cells were treated with either 9-AC or diluent
(C) for up to 6 h or were pretreated with calpeptin
(Calpt.) or Z-VAD-fmk (zVAD) and then treated
with 9-AC or diluent (C) for 6 h. DNA was extracted and
electrophoresed on 1% agarose gels to visualize DNA fragmentation.
M, molecular weight markers.
|
|
In Vitro Kinetics of Caspase-3-like Activity with 9-AC Treatment of
HL-60 Cells--
The fluorogenic peptide Ac-DEVD-AMC has been shown to
be a suitable substrate for caspase-3 in vitro (34). To
confirm the immunoblot results for PARP cleavage, which demonstrated
caspase-3-like activity after 2 h of 9-AC treatment, we analyzed
the ability of protein extracts, taken from 9-AC or 9-AC
diluent-treated HL-60 cells at successive time points, to hydrolyze
Ac-DEVD-AMC. At 2 h following drug challenge there was a 4-fold
increase in Ac-DEVD-AMC hydrolysis compared with lysates made from
diluent-treated cells (Fig. 2). This
increase in activity coincided with the initiation of PARP cleavage
(Fig. 1A). Peak values for Ac-DEVD-AMC hydrolysis were
achieved by 8 h after introduction of drug to the cultures, at
which point there was a 20-fold increase in caspase-3-like activity
compared with control lysates (Fig. 2). From 8 to 12 h, this
activity progressively declined, although there was still a 15-fold
increase in activity at 12 h compared with control lysates (Fig.
2). The addition of 1.0 µM caspase-3 inhibitor
Ac-DEVD-CHO (34) to the lysates derived from drug-treated cells at each time point reduced the hydrolysis of Ac-DEVD-AMC to levels that were
indistinguishable from those derived from control diluent-treated cells
(Fig. 2).

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 2.
In vitro kinetics of
caspase-3-like activity with 9-AC treatment of HL-60 cells. HL-60
cells were treated with 9-AC or its diluent and harvested at different
time intervals, and the resulting lysates (10 µg) were incubated with
the fluorogenic peptide substrate Ac-DEVD-AMC (25 µg) in the absence
or presence of 1 µM caspase-3 inhibitor Ac-DEVD-CHO for
1 h at 37 °C. The reactions were analyzed with a fluorometer
using a 380-nm excitation wavelength and a 460-nm emission wavelength.
Data are expressed as fluorescence units (FU)
released/min/mg of protein. Results shown are representative of three
independent experiments. , 9-AC diluent; , 9-AC; ,
9-AC/Ac-DEVD-CHO.
|
|
Time Course of Calpain Activation with 9-AC Treatment of HL-60
Cells--
Under normal conditions calpain activation is a controlled
process leading to limited cleavage of its substrates (11, 39). Autolysis of the 80- and 30-kDa calpain subunits is believed to be an
irreversible process that results in activation of calpain but is
thought to occur in vivo only under extreme conditions such
as during necrosis and apoptosis (39). Autolysis of the 30-kDa subunit
has been used previously to indicate calpain activation during
apoptosis (26). As shown in Fig. 3, the
30-kDa subunit of calpain underwent autolysis beginning at 8-10 h,
with a significant increase in the amount of calpain subunit breakdown
products observed at 12 and 24 h. In HL-60 cells, Bax has been
shown to be cleaved by calpain from its native 21-kDa form to an 18-kDa
fragment upon treatment with 9-AC (32, 33). As illustrated in Fig. 3,
Bax cleavage was initiated at 10 h, and by 24 h more than
50% of the 21-kDa Bax had been converted to the 18-kDa form as
determined by densitometric analysis. Bax normally resides in the
cytosol, but upon treatment of cells with apoptotic stimuli, it rapidly translocates to the mitochondria where it inserts into the outer membrane as a 42 kDa homodimer (40-42). We have shown that calpain activity within mitochondria-enriched fractions is responsible for Bax
cleavage (33). To determine whether Bax cleavage at the mitochondria is
the result of a selective proteolytic event as opposed to a general
degradative process occurring during the late stages of apoptosis, we
examined the status of another mitochondrial membrane protein, the
electron transport chain component COX II (43). As shown in Fig. 3, COX
II remained intact over the 24-h time interval of drug treatment,
suggesting that Bax cleavage is not the result of a bulk degradation of
mitochondrial membrane proteins. Together with the results presented in
Fig. 1, these experiments show that caspase-3-like proteases become
activated after 2 h of 9-AC treatment and attain peak activity by
8 h, the time at which calpain autolysis/activation is first
detected.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 3.
Time course of calpain activation with 9-AC
treatment of HL-60 cells. HL-60 cells were treated with 9-AC or
diluent for up to 24 h. Cells were harvested at different time
intervals, and cell lysates were made and subjected to SDS-PAGE
and immunoblotting with antibodies against the 30-kDa subunit of
calpain (upper panel), Bax (middle panel),
and COX II (lower panel).
|
|
The Pan-caspase Inhibitor Z-VAD-fmk Blocks the Activation of Both
Caspases and Calpain--
Because we have demonstrated that calpain
appeared to be active downstream of the caspases, we sought to
determine whether calpain activity was dependent upon caspase
activation. The pan-caspase inhibitor Z-VAD-fmk blocks the processing
of caspases-2, -3, -6, and -7, suggesting that its target is a
caspase(s) at or near the initial stage of the apoptotic program (3).
When HL-60 cells were pretreated with this caspase inhibitor at 100 µM prior to 9-AC treatment, PARP cleavage was effectively
blocked (Fig. 4A). Autolysis
of the 30-kDa calpain subunit was also completely blocked, whereas
cleavage of Bax was inhibited by more than 80% in the presence of
Z-VAD-fmk (Fig. 4A). In contrast, pretreatment with the
calpain inhibitor calpeptin at 10 µM did not block PARP cleavage (Fig. 4B). Although calpeptin was unable to
completely inhibit autolysis of the 30-kDa calpain subunit, it did
inhibit calpain activation by ~60% (Fig. 4B). Calpeptin
pretreatment also resulted in a 65% reduction in Bax cleavage (Fig.
4B). Despite the fact that calpeptin was highly effective at
blocking calpain activation and Bax cleavage, it did not provide any
protection against 9-AC-induced apoptosis (see Fig.
5). As illustrated in Fig. 4C,
pretreatment with a combination of calpeptin and Z-VAD-fmk gave similar
results to cultures treated with Z-VAD-fmk alone. The ability of
Z-VAD-fmk to effectively block caspase function as well as calpain
activation suggests that calpain activation may be a caspase-mediated
event.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 4.
Z-VAD-fmk blocks the activation of both
caspases and calpain. HL-60 cells were either left untreated
(None) or pretreated with 100 µM Z-VAD-fmk
(zVAD) for 2 h (A), 10 µM
calpeptin (Calpt.) for 30 min (B), or calpeptin
and Z-VAD-fmk (Calpt./zVAD) (C). Cells
were then left untreated, treated with 9-AC diluent, or challenged with
9-AC. Lysates were made from cells harvested at 0 and 20 h of
treatment and subjected to SDS-PAGE and immunoblotting with antibodies
against PARP (upper panel), the 30 kDa calpain subunit
(middle panel), and Bax (lower panel).
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Loss of plasma membrane integrity is
attenuated by Z-VAD-fmk but not by calpeptin. Survival of HL-60
cells was assessed by measuring loss of membrane integrity with the
uptake of the vital dye trypan blue. Cells were left untreated
(None) or were pretreated with 100 µM
Z-VAD-fmk for 2 h (zVAD), 10 µM calpeptin
for 30 min (Calpt.), or calpeptin plus Z-VAD-fmk
(Calpt./zVAD). Following preincubation with these
inhibitors, cells were challenged with 9-AC or its diluent. At
different time intervals, triplicate samples were removed from each
culture condition, diluted with trypan blue, and counted. The data
presented are representative of three independent experiments.
|
|
Loss of Membrane Integrity Is Partially Inhibited by Z-VAD-fmk but
Not by Calpeptin--
The culmination of apoptosis in HL-60 cells
treated with 9-AC is the breakdown of the plasma membrane reflected by
the inability of cells to exclude the vital dye trypan blue. As shown
in Fig. 5, control cells and cells treated with protease inhibitors
alone displayed a small amount (~5%) of membrane integrity loss that remained essentially unchanged over the 20-h observation period. Cells
treated with 9-AC alone remained impermeable to the dye for up to
6 h, at which point less than 10% of the cells were stained (Fig.
5). However, after 6 h there was a steady progression in the
number of cells that exhibited plasma membrane damage such that by
20 h, over 70% of the cells stained positive for the dye (Fig.
5). Cells that were pretreated with calpeptin before the addition of
9-AC showed virtually the same kinetics for loss of membrane integrity
as cells treated with 9-AC alone (Fig. 5). Similar to cells treated
with 9-AC alone, less than 10% of the cells pretreated with Z-VAD-fmk
displayed loss of membrane integrity up to 6 h (Fig. 5). Although
Z-VAD-fmk-pretreated cells became permeable to the dye over the next
several hours, the overall levels of membrane integrity loss were
reduced relative to cells treated with 9-AC. Indeed, by 20 h there
was a 40% reduction in the number of Z-VAD-fmk-treated cells that had
lost membrane integrity (Fig. 5). Cells pretreated with a combination
of Z-VAD-fmk and calpeptin displayed essentially the same kinetics and
levels of membrane integrity loss as cells treated with Z-VAD-fmk alone (Fig. 5). These results indicate that the effects of Z-VAD-fmk plus
calpeptin were not additive in terms of inhibiting cell death and that
Z-VAD-fmk alone afforded considerable protection from cell death even
up to 20 h of 9-AC treatment.
Pretreatment with Z-VAD-fmk, but Not Calpeptin, Results in
Nonapoptotic Death--
The breakdown of the plasma membrane in
9-AC-treated cells was preceded by a defined series of morphological
changes associated with apoptosis. Control, diluent-treated cells (Fig.
6 A-C) and cells
treated with calpeptin, Z-VAD-fmk, or calpeptin plus Z-VAD-fmk in the
absence of 9-AC displayed no change in morphology or plasma membrane
integrity throughout the 20-h time course interval (data not shown). In
contrast, treatment of cells with 9-AC resulted in an early onset (2 h)
of membrane blebbing that progressed to cellular condensation and
formation of apoptotic bodies by 6 h (Fig. 6, D and
E). These changes were followed several hours (12 h) later
by disruption of the plasma membrane as measured by uptake of trypan
blue (Fig. 6F). Pretreatment with calpeptin was ineffective at inhibiting any of these 9-AC-mediated morphological changes (data
not shown). HL-60 cells pretreated with Z-VAD-fmk displayed little
plasma membrane blebbing and did not condense in size or form apoptotic
bodies in the presence of 9-AC (Fig. 6, G and H). The cells that eventually became permeable to trypan blue dye were
similar in size and shape to the surrounding viable cells (Fig.
6I). Cells pretreated with a combination of calpeptin and Z-VAD-fmk showed identical results as cells pretreated with Z-VAD-fmk alone (data not shown). In summary, 9-AC treatment of HL-60 cells leads
to a series of events beginning with membrane blebbing, progressing to
cellular condensation with formation of apoptotic bodies, and ending
with the breakdown of the plasma membrane, all of which are insensitive
to calpeptin. In contrast, cells cultured in the presence of Z-VAD-fmk
and 9-AC exhibit some blebbing at later time points, but do not
condense in size. However, these cells eventually lose membrane
integrity and become permeable to trypan blue. Taken together these
results suggest that the 9-AC-induced apoptotic death observed in HL-60
cells with or without calpeptin differs from the 9-AC-mediated death
observed in the presence of Z-VAD-fmk.

View larger version (165K):
[in this window]
[in a new window]
|
Fig. 6.
Pretreatment with Z-VAD-fmk, but not
calpeptin, results in nonapoptotic death. HL-60 cells were
treated with either 9-AC diluent (A-C) or 9-AC
(D-F) or pretreated with 100 µM Z-VAD-fmk for
2 h followed by 9-AC (G-I) for up to 12 h. At
2 h (A, D, and G), 6 h
(B, E, and H), and 12 h
(C, F, and I) of treatment, trypan
blue was added to each culture, and the cells were subsequently washed,
resuspended in PBS, and analyzed by light microscopy. Note that the
trypan blue-stained cells at 12 h in cultures treated with
Z-VAD-fmk and 9-AC (I) have not condensed in size compared
with stained cells from cultures treated with 9-AC alone
(F). Magnification, ×400.
|
|
 |
DISCUSSION |
Previous reports have demonstrated a role for calpain in the
apoptotic death of several cell types including primary cultures of
thymocytes, neurons, human umbilical vein endothelial cells, and
neutrophils as well as in permanently established cell lines (25-31,
35, 44). Recent data obtained in our laboratory indicated that calpain
activity was present in HL-60 cells triggered to undergo apoptosis with
the chemotherapeutic agent 9-AC (33). In this study we wanted to
determine the timing of calpain activation relative to that of the
caspases. Second, we wanted to determine what role calpain may play in
our model of drug-induced apoptosis of HL-60 cells. The data presented
here indicate a sequential relationship between activation of caspases,
the primary death effector proteases, and activation of calpain. Our
data suggest that there is a caspase-dependent activation
of calpain during the degradation stage of apoptosis.
HL-60 cells treated with the topoisomerase I inhibitor 9-AC exhibited
caspase-3-like activity as early as 2 h after treatment, with peak
activity at 8 h (Figs. 1 and 2). This initiation of caspase
activity was coincident with membrane blebbing (Fig. 6D), but preceded DNA fragmentation (Fig. 1B) and condensation in
cell size (Fig. 6, E and F), all of which
occurred in cells still displaying intact plasma membranes (Fig. 5). In
comparison with the early activation of caspases, calpain activation
and subsequent cleavage of Bax took place several hours after the
initiation of morphological changes and DNA fragmentation (Fig. 3).
Indeed, the kinetics of calpain activity paralleled the initiation and
progressive loss of membrane integrity (Figs. 3 and 5). These results
taken together indicate a sequential activation of proteases consisting
of an early activation of caspases followed by a 6-h increase in
caspase function, which at its apex coincided with calpain activation.
Activation of both caspases and calpain has been reported in one other
lymphoid cell line, U937, during tumor necrosis factor-induced apoptosis (35). Similar to the results described here, calpain in U937
cells appeared to be active following the initiation of caspase
activity (35). However, in U937 cells, calpeptin proved to be more
effective than Z-VAD-fmk at inhibiting several late apoptotic events
including DNA and nuclear fragmentation, plasma membrane blebbing, and
formation of apoptotic bodies (35). Unlike HL-60 and U937 cells, the
neuroblastoma cell line SH-SY5Y was found to simultaneously activate
caspases and calpain during staurosporine-induced apoptosis (26,
44). Interestingly, in SH-SY5Y cells caspases and calpain cleave some
of the same target substrates, such as nonerythroid
-spectrin
(
-fodrin) and calcium/calmodulin-dependent protein
kinase IV, to produce caspase-specific and calpain-specific fragments
(26, 44). This situation is not observed in HL-60 cells because
-spectrin is cleaved early during apoptosis in a
caspase-dependent
fashion,2 whereas Bax is
cleaved by calpain late in apoptosis strengthening the notion of a
temporal relationship between the activation of the two types of
proteases in our model system. The differences reported between cell
lines regarding the timing of calpain activation and the ability of
calpain to effect some apoptotic changes may reflect cell
lineage-specific requirements for calpain activity during apoptosis.
As we noted, caspase activity was at its highest at 8 h after 9-AC
treatment, which was coincident with the onset of calpain activation.
It is tempting to speculate that in some cell types, such as lymphoid
cell lines, calpain activity may be dependent upon caspase function.
Under normal circumstances calpain activity is modulated by the
presence of its endogenous inhibitor calpastatin (11). Calpastatin is
generally thought to reside in the cytosol where it complexes with
calpain, resulting in its inactivation (11). Cleavage of calpastatin by
caspases has been observed in both Fas-induced apoptosis of Jurkat
cells and in staurosporine-induced apoptosis of SH-SY5Y cells,
resulting in a reduction of this inhibitor's effectiveness in
vitro (45). At present, we have been unable to identify
calpastatin in HL-60 cells by Western blotting thereby precluding a
determination of whether caspase-mediated cleavage of calpastatin
occurs in this cell line. However, the reduction in the activity of
calpastatin by caspase cleavage could provide a mechanism for the
uncontrolled calpain activity that is observed during the latter stages
of apoptosis.
Pretreatment of HL-60 cells with the calpain inhibitor calpeptin did
not prevent caspase-3-like function, DNA fragmentation, morphological
changes, or plasma membrane damage associated with 9-AC-induced
apoptosis. In marked contrast, pretreatment with the pan-caspase
inhibitor Z-VAD-fmk resulted in the inhibition of caspase-3-like
function, DNA fragmentation, and morphological changes. In addition,
Z-VAD-fmk treatment reduced cell death by 40% (Fig. 5). However, HL-60
cells pretreated with Z-VAD-fmk eventually died in a process that by
morphological criteria did not resemble the apoptotic changes observed
in cells treated with 9-AC alone (Fig. 6). These results are similar to
previously reported data indicating that inhibition of caspase activity
with Z-VAD-fmk results in a nonapoptotic, necrotic-like death (46-49).
In addition to blocking caspase activation and function, calpain
activation measured by the autolysis of the 30-kDa calpain subunit and
Bax cleavage was also essentially eliminated by Z-VAD-fmk pretreatment. Thus it appears that in the presence of Z-VAD-fmk and 9-AC, cells will
undergo a delayed, nonapoptotic death involving plasma membrane damage and cell lysis, which is essentially independent of both caspase
and calpain activity, as noted previously (46).
The data presented here allow us to construct a scenario that can
account for calpain involvement in apoptosis as well as in normal cell
physiology. During nonapoptotic conditions calpain activity would be
controlled by its inhibitor, calpastatin. Then, perhaps because of a
brief, localized calcium influx, calpain would be activated at cellular
membranes in a transient manner (50). Under these conditions only a
small portion of the total calpain in a cell would be active, resulting
in limited cleavage of calpain substrates, allowing for normally
functioning cellular physiology (13-17, 50). In contrast, under
apoptotic conditions uncontrolled calpain activation might be the
result of a massive and sustained influx of calcium that, together with
caspase activation and the potential for caspase-mediated inactivation
of calpastatin, could lead to a larger fraction of active calpain
engaged in excessive proteolysis of many substrates, including Bax (45,
50). Excessive or uncontrolled calpain activity may play a role that is
downstream and distinct from caspases in the degradation phase of apoptosis.