1 Department of Pathology, University of Turku, Kiinamyllynkatu 10, FIN-20520
Turku, Finland
2 Turku Graduate School of Biomedical Sciences, Turku, Finland
* Author for correspondence (e-mail: markku.kallajoki{at}utu.fi)
Accepted 15 October 2002
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Summary |
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Key words: NuMA, Lamins, Apoptosis, Caspases, Caspase independent
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Introduction |
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One of the proteins to be degraded during apoptosis is NuMA (nuclear
mitotic apparatus protein), a 238 kDa protein that is a component of the
nuclear matrix during interphase and that redistributes to the spindle poles
in mitosis (Lyderson and Pettyjohn,
1980; Kallajoki et al.,
1992
). Several studies have shown that NuMA is essential for
normal mitosis as an organizer of the mitotic spindle
(Kallajoki et al., 1991
;
Kallajoki et al., 1993
;
Yang and Snyder, 1992
;
Compton and Cleveland, 1993
;
Gaglio et al., 1995
;
Gaglio et al., 1996
;
Merdes et al., 1996
). In the
mitotic spindle, NuMA interacts with the dynein-dynactin complex
(Gaglio et al., 1995
;
Gaglio et al., 1996
;
Merdes et al., 1996
), and it
seems that NuMA and another noncentrosomal protein, the human homologue of the
KIN C motor family (HSET), co-operate in association with dynein to anchor
microtubule minus ends at spindle poles and to support chromosome movement
(Gordon et al., 2001
).
Recently, NuMA has been shown to be a part of a microtubule aster-promoting
activity (APA), a multi-protein complex that induces spindle formation in
mitosis and is regulated by small GTPase Ran and importin ß
(Nachury et al., 2001
;
Wiese et al., 2001
).
The primary function of NuMA during interphase is still unclear. The cDNA
sequence of NuMA shows homology to some structural filament-forming proteins
such as cytokeratins, nuclear lamins and myosin heavy chain
(Compton et al., 1992;
Yang et al., 1992
).
Overexpression studies have shown that overexpression of NuMA lacking the
nuclear localization signal results in cytoplasmic aggregates composed of 5 nm
NuMA filaments (Saredi et al.,
1996
; Gueth-Hallonet et al.,
1998
), whereas overexpression of full-length NuMA leads to a
quasi-hexagonal lattice-like structure in the nucleus
(Gueth-Hallonet et al., 1998
).
Harborth et al. have also shown that NuMA can self assemble into multiarm
oligomers in vitro (Harborth et al.,
1999
). All these results suggest that NuMA can, at least under
some circumstances, form ordered structures and that it might have a
structural function in the interphase nucleus. More recently, this has been
supported by RNA interference (RNAi) studies; silencing of NuMA gene results
in an apoptotic phenotype in HeLa cells suggesting that NuMA is essential for
these cells (Harborth et al.,
2001
).
Several studies have shown that NuMA is specifically degraded in early
apoptotic cell death. Weaver et al. showed that NuMA is cleaved into
approximately 200 and 48 kDa fragments in dexamethasone-treated thymocytes
(Weaver et al., 1996). A
180 kDa form of NuMA was described in HeLa cells treated with
5,6-dichloro-1-ß-D-ribofuranosyl-benzimidatsole and in HL60 cells treated
with camptothecin, staurosporine, cycloheximide and A23187
(Hsu and Yeh, 1996
). Multiple
cleavage products of NuMA have also been reported in Jurkat T cells during
Fas-mediated apoptosis (Casiano et al.,
1996
; Greidinger et al.,
1996
). In hydroxyurea- and staurosporine-treated BHK cells, NuMA
is cleaved between residues 1701 and 1725, releasing the C-terminal tail
domain, which contains a functionally important nuclear localization signal
(Gueth-Hallonet et al., 1997
).
Immunofluorescence analysis has shown that the normal diffuse distribution of
NuMA is changed during apoptosis and NuMA is excluded from the condensed
chromatin (Gueth-Hallonet et al.,
1997
). Hirata et al. have further shown, using isolated nuclei
from HeLa cells and recombinant caspases
(Hirata et al., 1998
), that at
least caspase-3, -4, -6 and -7 degrade NuMA in vitro. Indeed, the cleavage of
NuMA can be inhibited with several protease inhibitors including VEID-CHO,
DMQD-CHO (Hirata et al., 1998
)
and TPCK but not with Ac-YVAD-cmk, TLCK or E-64
(Gueth-Hallonet et al., 1997
).
In addition to caspases, granzyme B has been shown to cleave NuMA
(Andrade et al., 1998
).
Another apoptotic nuclear target is nuclear lamina, the structure
underlying the inner nuclear membrane and supporting the nuclear architecture.
It is composed of four different intermediate filament proteins: lamins A, B1,
B2 and C. Lamins B1 and B2 are encoded by two different genes (LMNB1 and
LMNB2), and they are expressed in all mammalian somatic cells. Lamins A and C
are generated from the LMNA gene by alternative splicing and are absent from
some cell types (Guilly et al.,
1987; Stewart and Burke,
1987
; Röber et al.,
1989
). Lamins were among the first apoptotic target proteins
identified, and the cleavage of lamins has been suggested to play a key role
in the breakdown of nuclear structure
(Lazebnik et al., 1995
). It
seems that lamin B is cleaved by caspase-3
(Slee et al., 2001
) and lamin
A and lamin C are cleaved by caspase-6
(Orth et al., 1996
;
Takahashi et al., 1996
).
Similar to NuMA, Granzymes have been recently shown to degrade lamins
independently of caspases. Both granzyme A and B cleave lamin B, whereas
granzyme A but not granzyme B cleaves lamins A and C
(Zhang et al., 2001
).
In the present study, we studied further the morphological and biochemical changes in NuMA during Fas-receptor-mediated apoptosis especially in relation to other nuclear structures and apoptotic target proteins including nuclear lamins and PARP-1. We describe the changes in the distribution of NuMA and lamins and show that degradation of NuMA is an early process preferentially due to caspase-3 activity since the cleavage is inhibited in the presence of certain caspase inhibitors and NuMA is not cleaved in caspase-3-null MCF-7 human breast cancer cells.
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Materials and Methods |
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Reagents
To induce apoptosis, Jurkat T cells were treated with 100 ng/ml of an
agonistic anti-human Fas receptor IgM antibody (CH-11, MBL Medical &
Biological Laboratories, Nagoya, Japan). To sensitize HeLa cells for
Fas-mediated apoptosis, cells were treated with 100 ng/ml of Fas antibody in
the presence of 30 µM MAPK-kinase inhibitor PD 98059 (Calbiochem, La Jolla,
CA) as described previoulsy (Holmstrom et
al., 1999). To induce apoptosis, MCF-7 cells were treated with 2
µM staurosporine (Sigma).
Benzyloxicarbonyl-Asp(Ome)Glu(Ome)Val-Asp(Ome) fluorometylketone
(z-DEVD-FMK),
Benzyloxicarbonyl-ValGlu(Ome)Ile-Asp(Ome)-fluorometylketone
(z-VEID-FMK) and
Benzyloxicarbonyl-IleGlu(Ome)Thr-Asp(Ome)-fluorometylketone
(z-IETD-FMK) were obtained from R&D Systems (Abingdon, UK) and dissolved
into DMSO. They were added into cell culture coincidently with other reagents
and diluted as shown in Results.
Immunofluorescence microscopy
For immunofluorescence microscopy HeLa and MCF-7 cells were grown on 12 mm
glass coverslips. At the time intervals indicated in the results section,
cells were washed once with PBS (145 mM NaCl, 7.5 mM
Na2HPO4, 2.8 mM NaH2PO4) before
fixing. For Jurkat T cells, 100-200 µl of cell suspension was placed on a
silanized microscope slide for 30 minutes to attach the cells. Additional
medium was then removed with filter paper. All samples were fixed in 3.7%
formaldehyde in PBS for 15 minutes at room temperature, permeabilized with
0.1% Triton X-100 in PBS for 15 minutes and washed with PBS. The coverslips
were treated with 5% normal goat serum (Dako Immunochemicals, Klostrup,
Denmark) in PBS or 5% BSA in PBS (when rabbit anti-goat secondary antibody was
used) for 30 minutes, washed three times with PBS and incubated with primary
antibody/antibodies for 2 hours at room temperature. Mouse monoclonal NuMA
antibody [SPN-3 clone (Kallajoki et al.,
1991; Kallajoki et al.,
1993
)] was used as undiluted culture supernatant and goat
polyclonal lamin B antibody (Santa Cruz Biotechnology, CA), mouse monoclonal
lamin A/C antibody (Novocastra Laboratories, Newcastle upon Tyne, UK), rabbit
polyclonal PARP-1 p85 fragment antibody (Promega, Madison, WI) and cleaved
caspase-3 (Asp 175) antibody (Cell Signaling Technology, Beverly, MA) were
diluted 1:10, 1:20, 1:50 and 1:25 into 1% BSA in PBS, respectively. Coverslips
were washed three times with PBS and then incubated with either
FITC-conjugated goat anti-mouse IgG (Cappel Laboratories, Couchranville, PA,
USA) and TRITC-conjugated goat anti-rabbit IgG (Zymed Laboratories, San
Francisco, CA, USA) or FITC-conjugated rabbit anti-mouse IgG (Zymed) and
TRITC-conjugated rabbit anti-goat IgG (Zymed) secondary antibodies for 1-1.5
hours at room temperature. After washing three times with PBS, samples were
stained for DNA with Hoechst 33258 (1 µg/ml in 25% ethanol/75% PBS) for 5
minutes and embedded in Mowiol 4.88 (Hoechst AG, Frankfurt, Germany). TUNEL
assays were performed using the DeadEnd Fluorometric TUNEL System (Promega).
In brief, cells grown on coverslips were first fixed and permeabilized as
described above. Samples were pre-equilibrated with equilibrate buffer for 10
minutes at room temperature and then incubated with buffer containing
equilibrating buffer, nucleotide mix and TdT enzyme for 1 hour at 37°C
protected from light. The reaction was terminated by immersing the samples in
2xSSC (1xSSC; 0.15 M NaCl, 0.015 M trisodium citrate) for 15
minutes at room temperature. Samples were washed three times in PBS for 5
minutes and finally stained for DNA with Hoechst 33258. All samples were
analyzed using Olympus BX 50 fluorescence microscope (Olympus Optical Co. LTD,
Tokyo, Japan) and AnalySIS software (Soft Imaging Systems) or Leica confocal
scanning laser microscope. To determine the amounts of apoptotic, atypical
apoptotic and NuMA-negative cells on the coverslips, 800-1200 cells from at
least eight randomly selected areas were counted for each sample. The mean
values and the standard deviations were determined after three parallel
experiments.
Electrophoresis and immunoblotting
HeLa and MCF-7 cells grown on 55 mm petri dishes were first scraped into
the medium. Cell suspensions were then centrifuged at 1000 g
for 5 minutes, washed with PBS and finally pelleted at 12,000
g for 5 minutes. Cell numbers were counted with a
haemocytometer. Cell pellets were resuspended directly into hot SDS-PAGE
electrophoresis sample buffer at a concentration equivalent to 107
cells/ml and sonicated for 5 seconds. Samples were loaded on 5% polyacrylamide
gels for NuMA, on 5% or 10% gels for PARP-1, on 10% gels for lamins and on
10%, 12% or 14% gels for caspases. Two parallel gels were run: one for
Coomassie brilliant blue staining to control equal loading and another for
immunoblotting. Proteins were transferred electrophoretically to
nitrocellulose (Schleicher & Schuell, Dassel, Germany) in a buffer
containing 25 mM Tris, 192 mM glycine, 0.05% SDS and 10% methanol at 300 mA
constant current for 1.5 hours. The transfer was controlled by Ponseau red
staining. The filters were preincubated in 4% bovine serum albumin (BSA) in
0.2% Tween 20 in TBS (Tris-buffered saline: 20 mM Tris-HCl, pH 7.4, 0.15 mM
NaCl) overnight and incubated with NuMA antibody (SPN-3), lamin A/C antibody
(Novocastra), lamin B antibody (Santa Cruz Biotechnology), mouse monoclonal
PARP-1 antibody (clone C-2-10, Sigma), rabbit polyclonal caspase-8 antibody
(NeoMarkers, Fremont, CA, USA), rabbit polyclonal caspase-3 antibody (BD
Pharmingen) or mouse monoclonal caspase-7 antibody (BD Pharmingen) diluted in
1% BSA, 0.2% Tween 20 in TBS for 2-3 hours at room temperature. The filters
were washed three times with 0.2% Tween 20 in TBS and incubated for 1-1.5
hours at room temperature with peroxidase-labeled sheep anti-mouse IgG
(Amersham, Buckinghamshire, UK), donkey anti-rabbit IgG (Amersham) or rabbit
anti-goat IgG (Zymed) diluted 1:1500-10000 in 1% BSA, 0.2% Tween 20 in TBS.
After three washes with 0.2% Tween 20 in TBS the immunoreactivity was detected
by using enhanced chemiluminescence reaction (ECL Western blotting detection
system, Amersham). When incubated with another primary antibody, filters were
first washed with 0.2% Tween 20 in TBS, then incubated with +50°C
stripping buffer (2% SDS, 100 mM ß-mercaptoethanol, 63 mM Tris, pH 6.8)
for 1 hour and washed four times with 0.2% Tween 20 in TBS.
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Results |
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|
To analyze the biochemical processing of NuMA during apoptosis and to
compare it to the other known apoptotic target proteins, western blotting was
used (Fig. 2). NuMA antibody
detected one approximately 240 kDa protein in control cells. In apoptotic
cells, NuMA was cleaved into two forms of approximately 180 and 190 kDa after
60 minutes. The latter is likely to represent an intermediate cleavage product
of NuMA, since the 180 kDa fragment became more evident at later time points.
In addition, a weaker 160 kDa form of NuMA was observed after 90 and 120
minutes. When compared to other apoptotic target proteins, the cleavage of
NuMA is an early process, which takes place simultaneously with the cleavage
of lamin B and poly(ADP-ribose) polymerase-1 (PARP-1), a well characterized
early apoptotic target protein cleaved by caspases-3 and -7
(Tewari et al., 1995
;
Germain et al., 1999
;
Slee et al., 2001
). The lamin
A/C antibody detected neither lamin A nor lamin C in Jurkat T cells, although
both were detected in control HeLa cells.
|
Changes in NuMA and nuclear lamins in apoptotic HeLa cells
As the small size of Jurkat T cells sets limits to closer morphological
analysis we continued with HeLa cells sensitized to Fas-mediated apoptosis
with mitogen-activated protein kinase (MAPK) kinase inhibitor PD 98059 [a
specific inhibitor of the EKR1/2 pathway by inhibiting MKK1
(Alessi et al., 1995)] as
described previously (Holmstrom et al.,
1999
). The distribution of NuMA was compared to changes in lamin
B, lamins A/C and to DNA stain. When treated with 100 ng/ml of a Fas receptor
antibody in the presence of 30 µM MAPK-kinase inhibitor, early
morphological signs of apoptosis were noticed using phase contrast microscopy
8-12 hours after the induction of apoptosis. After 24 hours approximately
40-50% of the cells were detached into the culture medium. These cells were
apoptotic as judged by immunofluorescence microscopy (data not shown). On
glass coverslips, normal cells, early phases of apoptosis and cells with
typical apoptotic bodies were seen when stained for DNA
(Fig. 3D,I). Approximately 30%
of adherent cells were apoptotic (see later
Fig. 8). NuMA was found to
encircle the fragmented nuclei in the apoptotic bodies
(Fig. 3A,F). To ensure that the
morphological changes described were truly apoptotic, samples were double
stained with NuMA antibody and antibodies detecting either the cleaved p85
fragment of PARP-1 or the cleaved caspase-3 (Asp175).
Fig. 3A-E shows that the cells
in which NuMA is excluded from the condensed chromatin stain with the p85
fragment specific PARP-1 antibody, whereas normal cells do not. An antibody
against cleaved caspase-3 first showed cytoplasmic bright spots in the cells
with shrunken nuclei and partially condensed NuMA and chromatin (data not
shown). At the later stage, cleaved caspase-3 located diffusively in the whole
cell excluding the nuclear fragments (Fig.
3F-J).
|
|
When viewed with confocal microscopy, NuMA staining was granular excluding nucleoli in normal interphase cells (Fig. 4A). In early apoptotic cells NuMA started to concentrate in the center of the nucleus and chromatin condensed close to the nuclear rim (Fig. 4F-J). At the end of apoptosis, NuMA clearly redistributed around the NuMA-negative fragmented nuclei in the apoptotic bodies (Fig. 4K-O). Lamin B staining showed that the normal circle-like staining pattern changes into a folded distribution in the beginning of apoptosis (Fig. 4G). Later, Lamin B condensed and seemed to encircle NuMA and the fragmented nuclei (Fig. 4L). The double staining with both NuMA and lamin B antibodies revealed that these proteins do not usually colocalize (Fig. 4C,H,M). Lamins A and C seemed to behave in a same way as lamin B (Fig. 4P), and in double staining these proteins colocalized in apoptotic nuclei (Fig. 4R).
|
NuMA is cleaved differently in Fas-treated HeLa cells
For western blot analysis both detached and adherent cells were harvested.
Fig. 5 shows that NuMA is
cleaved into an approximately 180 kDa product in apoptotic HeLa cells. In
addition, the 190 kDa intermediate fragment was observed in the
beginning of apoptosis but neither
160 kDa fragment nor other smaller
fragments were detected even 36 hours after induction of apoptosis although
100% of the cells were apoptotic and no full-length NuMA was detected in
western blots. The cleavage of NuMA was an early process beginning 8 hours
after induction of apoptosis, and it was highly coincident with the cleavage
of PARP-1 and lamin B into typical apoptotic 85 kDa and 46kDa fragments,
respectively. Surprisingly, the antibody detecting both 70 kDa lamin A and 60
kDa lamin C detected no major degradation products. Only a minimal amount of
approximately 50 kDa product was seen after 16 hours. The amounts of lamins A
and C were not diminished, suggesting that lamins A and C were not
significantly cleaved during apoptosis. To ensure that caspases are truly
activated in Fas-ligated HeLa cells, immunoblotting with caspase-8, -3 and -7
antibodies was done. Fig. 5
shows that the amounts of 55/57 kDa proform of caspase-8, 32 kDa proform of
caspase-3 and 35 kDa proform of caspase-7 were diminished, apparently owing to
their activation. In addition, the cleaved 42/44 kDa and
25 kDa fragments
of caspase-8 were detected after 16 hours.
|
The cleavage of NuMA is inhibited in the presence of caspase
inhibitors
To find out whether degradation of NuMA is really due to caspase activity
and to find out which caspase cleaves NuMA in vivo we induced apoptosis in the
presence of different caspase inhibitors. Three peptide-based, cell-permeable
and irreversible caspase inhibitors were used: z-DEVD-FMK, z-VEID-FMK and
z-IETD-FMK. These are suggested to inhibit caspases-3, -6 and -8,
respectively. They all act as substrates for the caspases, bind to the active
site, form irreversible bond with the enzyme and block the caspase from
further action. Fig. 6 shows
that when used in a 100 µM concentration for 24 hours, all these inhibitors
managed to inhibit the cleavage of NuMA, lamins, PARP-1 and proforms of
caspase-8, -3 and -7. Actually all these inhibitors had an identical
dose-dependent effect when compared to each other. In a 10 µM concentration
the cleavage of NuMA and PARP-1 was only partly inhibited whereas the cleavage
of lamins was still effectively inhibited. The 1 µM concentration had only
little and the 0.1 µM concentration practically no effect on the cleavage
of the proteins tested.
|
To examine the effects of the caspase inhibitors on the morphology of the cells treated, immunofluorescence analysis was done. When treated with Fas antibody and PD in the presence of 100 µM z-DEVD-FMK, z-VEID-FMK or z-IETD-FMK for 24 hours all cells were adherent and only single apoptotic cells were seen. Interestingly, a population of cells with altered apoptotic nuclear morphology was also seen (Fig. 7). With Hoechst staining these cells had heavily convoluted nuclei with slightly condensed chromatin structure (Fig. 7B,D). Moreover, these nuclei seemed to be negative or weakly positive when stained with NuMA (Fig. 7A), lamin A/C (Fig. 7C) or lamin B (data not shown) antibodies. In the presence of 30 µM z-DEVD-FMK even more cells with atypical and typical apoptotic nuclei existed when incubated for 24 hours. In addition, cells with an abnormal distribution of nuclear NuMA were found (Fig. 7E). The normal diffuse distribution of NuMA had changed and NuMA had condensed into multiple areas in the nucleus. Hoechst stain shows that these cells do not have shrunken nuclei, the nucleoli are still visible but the nuclei are slightly convoluted and they have altered chromatin structure (Fig. 7F).
|
We next determined the amounts of apoptotic, atypical apoptotic and NuMA-negative cells on the coverslips in different samples. Fig. 8 shows that in the presence of Fas antibody, PD and 100 µM z-DEVD-FMK, z-VEID-FMK or z-IETD-FMK approximately 0-2% and 4-10% of the cells showed apoptotic and atypical apoptotic nuclei, respectively, whereas in the presence of only Fas antibody and PD, 25-35% of the cells were apoptotic but no atypical apoptotic cells were found. The amount of NuMA-negative cells also correlated with the amount of atypical apoptotic cells as shown in Fig. 8. To find out whether this atypical apoptotic morphology was due to the effect of caspase inhibitors only, HeLa cells were treated with 100 µM z-DEVD-FMK for 24 hours. However, neither apoptotic nor atypical apoptotic cells were found in the samples.
To investigate whether atypical apoptotic morphology described above was due to caspase activation, cells treated with Fas antibody and PD in the presence of 30 µM z-DEVD-FMK for 24 hours were stained with cleaved caspase-3 and p85 fragment of PARP-1 antibodies. Since the atypical apoptotic cells also showed partial chromatin condensation, we performed a TUNEL assay to reveal possible DNA cleavage into oligonucleosomal 180-200 bp fragments. Fig. 9 shows that typical apoptotic cells stain with both antibodies and incorporate fluorescein-12-dUTP, whereas atypical apoptotic cells do not. According to this data, it seems that caspase-3 is not activated in atypical apoptotic cells but rather this is an early morphological change owing to upstream caspases or another proteinases independent of effector caspases.
|
NuMA or lamins are not cleaved in staurosporine treated
caspase-3-null MCF-7 cells
Since caspases-3 and -7 were the most probable candidates responsible for
the cleavage of NuMA, we tested the fate of NuMA in MCF-7 human breast cancer
cell line known to lack caspase-3
(Jänicke et al., 1998b).
MCF-7 cells undergo apoptosis lacking several morphological features of
apoptosis like cytoplasmic blebbing and formation of apoptotic bodies (e.g.
Jänicke et al., 1998b
).
When treated with 2 µM staurosporine for 12 hours as described previously
(Johnson et al., 2000
), the
whole cells and the nuclei shrank and a part of the cells detached
(Fig. 10F-O). DNA staining
showed shrunken nuclei and partially condensed chromatin
(Fig. 10I,N). In a few cells
chromatin was even concentrated to the nuclear periphery (data not shown).
When stained with NuMA antibody (Fig.
10F,K), all cells were NuMA positive but NuMA was only seldom
separated from the chromatin. Cytoplasmic blebbing and formation of apoptotic
bodies were not observed. In western blotting, NuMA antibody detected the
full-length NuMA but, interestingly, no typical apoptotic cleavage products
after 12 hours of incubation with 2 µM staurosporine
(Fig. 11). Similarly, when
immunoblotted with the lamin A/C and lamin B antibodies, no cleavage products
were noticed, suggesting that lamins were not cleaved. Interestingly, PARP-1
antibody showed the appearance of apoptotic p85 fragment in
staurosporine-treated cells. This was supported by immunofluorescence studies;
when stained with a specific p85 fragment of PARP-1 antibody, a population of
apoptotic cells were positive with this antibody
(Fig. 10K-O). Previously
caspase-7 has been shown to cleave PARP-1 in apoptotic MCF-7 cells
(Germain et al., 1999
). In
agreement with this, immunoblotting with caspase-7 antibody showed that the
amount of procaspase-7 was diminished presumably owing to its
cleavage/activation. A partial cleavage of 55/57 kDa procaspase-8 into 42/44
kDa fragments was also detected in staurosporine-treated cells but the active
fragment was not detected. The 32kDa procaspase-3 was not detected in MCF-7
cells although it was detected in control Jurkat T cells (data not shown). In
summary, these results further suggest that although both caspase-3 and -7
cleave NuMA in vitro, caspase-3 is the main effector caspase to cleave NuMA in
vivo.
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![]() |
Discussion |
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The role of caspase-4 remained unclear in this study but since caspase-4 is
not among the effector caspases in Fas-mediated apoptosis it is unlikely that
it would play a significant role in the cleavage of NuMA in Fas-treated Jurkat
T or HeLa cells. Caspase-7, however, was activated
(Fig. 5) and cleaves NuMA
similarly to caspase-3 in vitro (Hirata et
al., 1998). Thus, it is possible that both caspase-3 and -7 cleave
NuMA in Fas-treated HeLa cells. In staurosporine-treated caspase-3-deficient
MCF-7 cells pro-caspase-7 and -8 and PARP-1 were cleaved, whereas NuMA and
lamins were not (Fig. 11).
Indeed, there is evidence that caspase-7 is activated in staurosporine- and
TNF/cycloheximide-treated MCF-7 cells
(Germain et al., 1999
),
although this was not completely supported
(Jänicke et al., 1998a
).
Taken together, these results indicate that caspase-3 is necessary for the
cleavage of NuMA and lamin B in vivo, whereas PARP-1 can be cleaved also in
the absence of caspase-3, probably by caspase-7.
The amino-acid sequence of NuMA does not contain any specific caspase
cleavage site when scanned for enzymatic cleavage sites with Exspasy
Peptidecutter (Swiss Institute of Bioinformatics, University of Geneva and
University of Lausanne, Switzerland). According to the molecular weight of the
fragments the predicted caspase cleavage site of NuMA is DSLD-L between
residues 1726-1727 (Nicholson and
Thornberry, 1997). Caspases-3 and -7 prefer this sequence (DxxD)
in a target protein. However, Gueth-Hallonet et al. showed that at least one
cleavage site is located between residues 1701-1725
(Gueth-Hallonet et al., 1997
).
This sequence contains three Asp residues at positions 1705, 1719 and 1723,
all of which can serve as a potential cleavage site for caspases. In their
studies, changing D to G at position 1705 did not prevent the cleavage of the
molecule. To determine the specific cleavage site(s) of NuMA, N-terminal
sequencing or mass spectrometry analysis of the cleavage fragments is
required.
The changes in NuMA and lamins during apoptosis
The time schedule/kinetics of cleavage of NuMA compared to other nuclear
caspase substrates examined revealed that in both cell lines NuMA was cleaved
coincidently with PARP-1 and lamin B. In Jurkat T-cells this time point was 60
minutes, which is exactly the same as shown previously
(Casiano et al., 1996).
Greidinger et al. noticed a minimal cleavage of PARP-1 and NuMA already 30 and
50 minutes after induction of apoptosis, respectively
(Greidinger et al., 1996
).
However, all these studies show that NuMA is among the first substrates
cleaved in Fas-mediated apoptosis. In both cell lines studied, the amount of
apoptotic cells correlated with the amount of cleaved NuMA in western
analysis: in Jurkat T and HeLa cells
26% and
65% of all cells in the
sample were apoptotic after 120 minutes and 24 hours, respectively. The
immunofluorescence and confocal microscope analysis of apoptotic HeLa cells
(Figs 3 and
4) revealed that the apoptotic
breakdown of the nucleus is a rapid process since relatively few early
apoptotic cells were found at a certain time point. The normal distribution of
NuMA changed simultaneously with the chromatin condensation approximately
12-16 hours after the induction of apoptosis. On the other hand, a small
amount of cleaved NuMA was detected by western blotting already after 8 hours.
Therefore, it is possible that cleavage of NuMA detaches it from the condensed
chromatin. Since apoptotic Jurkat T and HeLa cells showed similar nuclear
morphology in immunofluorescence analysis we propose that the cleavage of NuMA
into an 160 kDa fragment does not play a significant role in apoptosis.
Our results concerning the fate of nuclear lamins during apoptosis showed
that different caspases cleave lamin A/C and B, since lamin B but not lamin
A/C was cleaved in Fas-treated HeLa cells. This is consistent with the earlier
studies (Orth et al., 1996;
Takahashi et al., 1996
;
Slee et al., 2001
). Caspase-3
and -6 are the main candidates for cleaving lamin B and A/C, respectively
(Orth et al., 1996
;
Slee et al., 2001
). Our
results support this. Interestingly, Fas-treated HeLa cells showed typical
apoptotic features, although lamins A and C were not degraded. This shows
clearly that cleavage of lamins A and C is not a prerequisite of the apoptotic
breakdown of the nucleus. The fact that lamins A and C are not expressed in
all nucleated cell types (Guilly et al.,
1987
; Stewart and Burke,
1987
; Röber et al.,
1989
) favor the idea that lamin B but not lamins A and C is
essential for nuclear structure. Indeed, in agreement with Slee et al.
(Slee et al., 2001
) we were
not able to detect any lamin A/C in Jurkat T cells by immunoblotting,
suggesting that this cell type lacks lamin A/C. Finally, recent RNA
interference studies have confirmed this hypothesis: silencing of lamin B1 or
B2 gene results in an apoptotic phenotype in HeLa cells, whereas silencing of
the lamin A/C gene has no effect on the viability of the cells
(Harborth et al., 2001
).
The effects of caspase inhibitors
Previously, the inhibition of NuMA cleavage has been reported from studies
using a few caspase inhibitors
(Gueth-Hallonet et al., 1997;
Hirata et al., 1998
). In the
present study, three different peptide-based caspase inhibitors, z-DEVD-FMK,
z-VEID-FMK and z-IETD-FMK, were tested. When used in a concentration of 100
µM, all these inhibitors managed to inhibit the cleavage of NuMA, lamins,
PARP and proforms of caspase-3, -6 and -8 either directly or by blocking the
caspase pathway and activation of downstream effector caspases
(Fig. 6). The latter is highly
probable especially in the case of z-IETD-FMK since it inhibits caspase-8,
which is the main initiator caspase in Fas-mediated apoptosis. Although the
inhibitors are designed to be specific for a certain caspase they also inhibit
other caspases especially when used in higher concentrations (e.g.
Hirata et al., 1998
).
z-DEVD-FMK, for example, inhibits caspase-3 but also partly inhibits
caspases-7 and -8 (Sigma, manufacturers' data). z-VEID-FMK, which inhibits
caspase-6, was also able to abolish the cleavage of pro-caspase-8, although
caspase-6 is activated downstream of caspase-8 in Fas-mediated apoptosis
(Fig. 6). Therefore, it seems
clear that these inhibitors serve as general caspase inhibitors by inhibiting
all caspases in a 100 µM concentration. To find out the possible
differences between the effects of the inhibitors, lower inhibitor
concentrations were tested. However, the results were similar when used at the
same concentration.
In the presence of caspase inhibitors a cell population with an altered
nuclear morphology was observed (Figs
7 and
9). It is possible that in
these cells the apoptotic shrinkage and the degradation of the nucleus is
delayed and/or this morphology is due to another caspase-independent cell
death pathway. The latter is supported by several studies and, indeed, it has
recently been shown that the Fas receptor can initiate another
caspase-8-independent cell death pathway in Jurkat T cells at least in the
presence of pan-caspase inhibitor z-VAD-FMK
(Holler et al., 2000). In the
present study, the presence of z-DEVD-FMK, z-VEID-FMK and z-IETD-FMK resulted
in a similar morphology (Figs 7
and 9). In western blot
analysis we did not detect cleavage of any pro-caspase or target protein
studied and atypical apoptotic cells did not stain with cleaved caspase-3 or
p85 fragment of PARP-1 antibodies, suggesting that caspases were not
activated. Moreover, these cells did not stain with NuMA or lamin antibodies,
which is different from that seen in the nuclei of typical apoptotic cells.
Cells were also TUNEL-negative, indicating that DNA was not cleaved into
oligonucleosomal fragments, and ICAD/DFF-45 [inhibitor of caspase activated
DNAse (Enari et al., 1998
)] was
not cleaved by caspase-3 in these cells. Therefore, we conclude that this
change is truly an atypical feature of apoptosis or secondary necrosis, which
takes place independently of caspases. A similar morphology was also seen in
tumor necrosis factor (TNF)-induced WEHI-S fibrosarcoma cells
(Foghsgaard et al., 2001
). In
their study, cathepsin B, a noncaspase proteinase, resulted in a cell death
with apoptotic features in the presence of pan-caspase inhibitor. Whether or
not cathepsin B played a role in atypical apoptotic morphology seen in the
present study remained unclear.
Atypical apoptotic cells were usually NuMA, lamin A/C and lamin B negative or only small NuMA-containing particles were observed in the nucleus (Figs 7 and 9). However, in the presence of 100 µM caspase inhibitors, cleaved form of NuMA was not observed in western blots (Fig. 6), suggesting that NuMA is not cleaved in these cells. A few possibilities have to be discussed: firstly, it is possible that this cell population is too small to detect their cleaved NuMA but this is very unlikely since up to 10% of the cells showed this morphology. Secondly, it is possible that NuMA is cleaved in the area at the epitope recognized by the antibody (amino acids 255-267). Thirdly, it is possible that NuMA and lamins are not cleaved but degraded by another unspecific protease or set of proteases. Neither can we exclude the possibility that the negative phenotype is due to inaccessibility of the antibodies to the nuclear proteins in these circumstances. This seems unlikely because we did not find any problems in staining of the other cells in the same sample.
The significance of degradation of NuMA
Although NuMA has been used as a marker to indicate the breakdown of the
nuclear matrix (Hirata et al.,
1998; Sodja et al.,
1998
), the significance of the cleavage of NuMA still remains
unclear. NuMA is a component of the nuclear matrix, which can form ordered
structures in the interphase nucleus, and it binds to defined DNA sequences
called matrix-associated regions (MARs) in vitro
(Luderus et al., 1994
). Thus,
the degradation of NuMA could result in the breakdown of normal nuclear
architecture. Granzyme B also cleaves NuMA directly with similar efficiency to
caspase-3, which highlights the importance of degradation of NuMA in
caspase-independent apoptotic cell death (Andrade, 1998). It is also known
that NuMA is degraded in necrotic HL-60 cells
(Bortul et al., 2001
). By
contrast, it seems that certain cell types lack NuMA, and NuMA is
preferentially expressed in the nuclei of proliferating cells
(Merdes and Cleveland, 1998
;
Sanghavi et al., 1998
;
Taimen et al., 2000
). This
suggests that NuMA is not, at least in all cells, needed for the formation of
the nuclear structure. Moreover, apoptotic human neutrophils lacking NuMA show
caspase-3 activation and lamin B cleavage, which suggests that the cleavage
products of NuMA are not required in neutrophil apoptosis
(Sanghavi et al., 1998
). If
the early change of chromatin structure showed here in atypical apoptotic
cells truly results in the disappearance of NuMA from the nucleus, it is clear
that the cleavage of NuMA is not essentially needed for the structural
breakdown of the cell nucleus.
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Acknowledgments |
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