From the a Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom, the b Division of Oncology Research, Mayo Clinic, Rochester, Minnesota 55905, c Athena Neurosciences, Inc., South San Francisco, California 94080, the h Jefferson Cancer Institute, Philadelphia, Pennsylvania 19107-5541, the g Centre Hospitalier de l'Université Laval Research Center and Laval University, Sainte-Foy, Quebec G1V 4G2, Canada, and the d Biomolecular Structure Center and the Departments of e Biological Structure and i Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195
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ABSTRACT |
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Previous studies have demonstrated that
topoisomerase I is cleaved late during apoptosis, but have not
identified the proteases responsible or examined the functional
consequences of this cleavage. Here, we have shown that treatment of
purified topoisomerase I with caspase-3 resulted in cleavage at
DDVD146 Eukaryotic DNA topoisomerase I (topo
I),1 an abundant nuclear
enzyme (105-106 copies/nucleus) involved in the
regulation of DNA topology and the control of gene expression, is
emerging as a protein of considerable medical significance. The enzyme
is an important target of camptothecin and related antineoplastic
agents (1-5). These agents slow the resealing steps of the topo I
catalytic cycle (6), thereby increasing the number of covalent topo
I·DNA complexes within cells (7) and setting into motion events that
result in target cell apoptosis (8). In addition, topo I is an
important autoantigen in rheumatic disease (9-11). First identified as
an ~70-kDa autoantigen termed Scl-70 that reacts with sera from
scleroderma patients (12), topo I is recognized by sera from 25% of
patients with scleroderma (13) as well as by sera from mice with the
TSK (tight skin) model for this disease (14).
Although the origin and significance of autoantibodies in rheumatic
disease remain controversial, it has been proposed that, at least in
systemic lupus erythematosus, autoantibodies may arise as a result of
abnormalities in the pathway of cell death by apoptosis (15).
Apoptosis is essential for morphogenesis, tissue homeostasis, and
host defense against viruses (16-19). Although the precise biochemical
pathways involved in mammalian cell death continue to receive intense
scrutiny, it is now clear that cysteine-dependent aspartate-directed proteases (caspases (20)) play important roles in
the initiation and execution phases of apoptotic death (19, 21, 22).
These proteases have been shown to cleave a wide variety of cellular
polypeptides located in both the cytoplasm and the nucleus (reviewed in
Refs. 21 and 23-27).
Like a number of other autoantigens (28, 29), topo I is cleaved during
apoptosis. However, a contradictory picture has emerged from previous
studies of topo I degradation during apoptotic execution. Initial
studies indicated that topo I levels markedly diminished during
etoposide-induced apoptosis of HL-60 cells without production of a
discrete cleavage fragment (8). Compared with apoptotic cleavage of
other caspase targets, e.g. poly(ADP-ribose) polymerase (30)
or lamin B1 (31), topo I cleavage appeared to be a later
event and was typically incomplete (8, 28). In contrast to this result,
topo I fragments of 70 kDa have been reported in HeLa cells exposed to
UV-B irradiation (28), HL-60 cells treated with etoposide (32), and
Jurkat cells exposed to anti-CD95 antibody (29, 32). In this last model
system, topo I proteolysis was inhibited by preincubation of cells with the broad spectrum caspase inhibitor benzyloxycarbonyl-VAD fluoromethyl ketone (10 µM) for 30 min before addition of anti-CD95
antibody. In contrast, during tumor necrosis factor-induced apoptosis
in C3HA fibroblasts (33) and necrosis of HL-60 or Jurkat cells induced
by HgCl2, ethanol, H2O2, or heat
(32), topo I was degraded into small fragments.
Several questions about the apoptotic cleavage of topo I remain
unanswered. 1) Which proteases are responsible for topo I cleavage
during apoptosis? 2) Where are the cleavage sites located within the
topo I molecule? 3) Are the topo I fragments generated during apoptosis
enzymatically active? 4) Are the same topo I fragments invariably
generated in different cells undergoing apoptosis? In this study, we
have mapped the sites at which caspase-3 and caspase-6 cleave topo I,
compared the resulting fragments with those generated in
situ in several cell types undergoing apoptosis, demonstrated that
the major topo I cleavage fragment retains enzymatic activity, and
probed the location of the cleaved fragment within apoptotic cells. The
results of this study not only identify topo I as a caspase substrate,
but also provide an explanation for its slow cleavage relative to other
apoptotic events and demonstrate its variable cleavage in different
apoptotic cell types.
Materials--
Reagents were obtained from the following
suppliers: YVAD-cmk from Bachem (Essex, United Kingdom); Hybond-C
membranes, peroxidase-coupled anti-mouse and anti-human secondary
antibodies, and ECL enhanced chemiluminescence reagents from Amersham
International (Buckinghamshire, UK); agonistic anti-Fas (Fas is the
cell-surface death receptor also known as CD95 and Apo-1) antibody
CH-11 from Kamiya Biomedical Co. (Seattle, WA); etoposide, paclitaxel
(Taxol®), and 5-fluoro-2'-deoxyuridine from Sigma; and
polyvinylidene difluoride membrane from Bio-Rad (Hertfordshire, UK).
The following antibodies were used to detect topo I by immunoblotting:
C-21 murine monoclonal IgM (34) directed against an epitope in the C-terminal 67 kDa of topo I (a kind gift of Y.-C. Cheng, Yale University Medical School, New Haven, CT) and human autoantiserum 3094 specific for topo I (9). All other reagents were obtained as described
previously (35).
Cleavage of Purified Topoisomerase I with Recombinant
Caspases--
Recombinant human topo I was expressed in Sf9
cells; purified as described (36); and stored at 5 mg/ml in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, and 50% (v/v) glycerol at Microsequencing--
Topoisomerase I (5 µg) cleaved as
described above was subjected to SDS-PAGE on a gel containing 7.5%
acrylamide and transferred to a polyvinylidene difluoride membrane.
After staining with Coomassie Blue, putative cleavage products were
excised and sequenced by automated Edman degradation using an ABI 476A
protein sequencer.
Cell Culture and Induction of Apoptosis--
Jurkat cells
(kindly provided by Drs. C. M. Eischen and P. J. Leibson,
Mayo Medical Center) and K562 cells (American Type Culture Collection,
Rockville, MD) were cultured in RPMI 1640 medium containing 5% (v/v)
heat-inactivated fetal bovine serum, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 2 mM glutamine (medium A) at
concentrations below 1 × 106 cells/ml to ensure
logarithmic growth. A549 human lung cancer cells and MCF-7 breast
cancer cells (from American Type Culture Collection) were passaged in
medium A (A549) or in minimal essential medium containing Earle's
salts, 10% (v/v) fetal bovine serum, nonessential amino acids, 1 mM sodium pyruvate, and 10 µg/ml insulin (MCF-7).
MDA-MB-468 human breast cancer cells (kindly provided by Dr. Nancy
Davidson, Johns Hopkins Oncology Center, Baltimore, MD) were cultured
in improved minimal essential medium (Biofluids, Inc., Rockville, MD)
supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 2 mM
glutamine. All cell lines were maintained in a humidified atmosphere
containing 5% (v/v) CO2 and passaged at least twice weekly.
To induce apoptosis, Jurkat cells were treated with 500 ng/ml anti-Fas
antibody CH-11 or 68 µM etoposide for 0-4 h as recently described (35). A549 cells treated with 68 µM etoposide
or 0.2 µM topotecan for 48 h were separated into
adherent (nonapoptotic) and nonadherent (apoptotic) cell populations.
Likewise, adherent and nonadherent populations were isolated from
MDA-MB-468 cells treated with 100 µM
5-fluoro-2'-deoxyuridine (41) or from MDA-MB-468 cells, MCF-7 cells,
and A549 cells treated with 100 nM paclitaxel (42) for
24 h, followed by a 24-h incubation in drug-free medium. All cells
were washed with ice-cold serum-free RPMI 1640 medium containing 10 mM HEPES (pH 7.4) and lysed under denaturing conditions in
solubilization buffer consisting of 6 M guanidine
hydrochloride, 250 mM Tris-HCl (pH 8.5 at 21 °C), 10 mM EDTA, 1 mM freshly added Fractionation of Apoptotic Cells--
To induce more complete
cleavage of topo I, Jurkat cells were treated with 17 µM
etoposide or 100 ng/ml CH-11 antibody for 14 h. After the cells
were sedimented at 200 × g for 10 min, all further
steps were performed at 4 °C. Cells were washed once with RPMI 1640 medium containing 10 mM HEPES (pH 7.4) followed by
phosphate-buffered saline, incubated for 20 min in nuclear isolation
buffer (10 mM NaCl, 10 mM Tris-HCl (pH 7.4),
and 5 mM MgSO4 containing freshly added 1 mM Immunolocalization of Topoisomerase I in Apoptotic
Cells--
After treatment with anti-Fas antibody CH-11 or etoposide
as described above, Jurkat cells were sedimented onto coverslips, air-dried, fixed in 3.7% (w/v) formaldehyde, and permeabilized with
0.2% (w/v) Nonidet P-40 (44). Topoisomerase I was visualized by
indirect immunofluorescence using anti-topo I antibody C-21 and
rhodamine-labeled affinity-purified anti-mouse IgM (Kirkegaard & Perry
Laboratories, Gaithersburg, MD) as described previously (44). After the
final series of washes, samples were incubated for 3 min with 1 µg/ml
Hoechst 33258 in phosphate-buffered saline prior to mounting in
Vectashield (Vector Laboratories, Inc., Burlingame, CA). Samples were
visualized using a DeltaVision microscope (Applied Precision, Issaquah, WA).
Cleavage of Topoisomerase I in Jurkat Cells Undergoing
Apoptosis--
Initial studies were performed to confirm that topo I
is cleaved to a detectable fragment during apoptosis and to assess the timing of this cleavage relative to other proteolytic events. After
addition of the agonistic anti-Fas antibody CH-11 to Jurkat cells, proteolytic cleavage of procaspase-7 (Fig.
1D) and procaspase-3 (data not
shown) was detected within 60 min, the same time frame in which we have
previously demonstrated caspase activation using catalytic assays for
DEVD-aminotrifluoromethylcoumarin cleavage and affinity labeling (35).
Cleavage of poly(ADP-ribose) polymerase at DEVD Cleavage of Topoisomerase I by Cloned Human
Caspases--
Examination of the topo I sequence (46) did not reveal
any perfect matches to the preferred cleavage sites of the known caspases (47, 48). Accordingly, there were two formal possibilities that could explain the delayed cleavage of topo I. First, topo I might
be cleaved by a non-caspase protease that was activated downstream of
the caspases. Several groups have presented indirect evidence for such
proteases (49-51), two of which have recently been identified (52,
53). Second, it was possible that the delay reflected a slow rate of
cleavage by one or more caspases acting at a catalytically less
favorable site. This second hypothesis was consistent with the results
of the experiments described below.
Although 12 human caspases have been identified to date, caspase-3 and
caspase-6 are the most widely studied with respect to the cleavage of
target proteins during apoptosis. To examine the possibility that
caspase-3 and/or caspase-6 can cleave topoisomerase I in
vitro, purified recombinant human topo I was incubated with extracts prepared from Sf9 cells infected with baculoviruses
expressing either human caspase-3 or caspase-6. Each of these caspases
cleaved topoisomerase I at two sites (Fig.
2A). Caspase-3 generated an 80-kDa major fragment and a 76-kDa minor fragment. In contrast, caspase-6 predominantly generated the 76-kDa fragment plus a minor fragment of 82 kDa. Interestingly, neither cleavage reaction was particularly efficient, with ~30 or 50% of the topoisomerase I remaining intact after the 2-h incubation with caspase-3 or caspase-6, respectively. This is to be contrasted with the quantitative cleavage of poly(ADP-ribose) polymerase by caspase-3 within minutes under similar conditions (54). Cleavage of topo I by each caspase was
completely inhibited by preincubation of extracts with the caspase
inhibitor YVAD-cmk at 100 µM (data not shown), consistent with the view that the cleavages were caspase-mediated.
To confirm that the slow rate of caspase-mediated topo I cleavage is an
intrinsic property of the enzyme-substrate interaction and is not due
to other factors present either in vivo or in Sf9 cell extracts, the cleavage reaction was reconstituted from purified components. Purified bovine poly(ADP-ribose) polymerase and human topo
I were incubated with purified recombinant human caspase-3 in the same
tube for various times at 37 °C, denatured in SDS-PAGE sample
buffer, and analyzed by immunoblotting (Fig. 2B). Under these conditions, caspase-3 cleaved all of the poly(ADP-ribose) polymerase within 15 min. In contrast, topo I cleavage was undetectable at 30 min, but became evident at later time points.
Collectively, the observations in Fig. 2 (A and
B) establish several points. First, topo I is cleaved by
both caspase-3 and caspase-6, making it one of a small number of
polypeptides that are reportedly cleaved by both caspases (55, 56).
Second, caspase-mediated topo I cleavage is relatively inefficient
compared with other substrates.
Mapping the Caspase Cleavage Sites in Topoisomerase I--
To
further explore the caspase-mediated cleavage of topo I, the amino acid
sequences of the two cleaved fragments generated by caspase-3 and the
two cleaved fragments generated by caspase-6 were determined (Fig.
2C). The analysis was simplified by the fact that, in each
case, caspase cleavage liberated a long carboxyl-terminal fragment with
a free N terminus. Thus, N-terminal sequencing of each fragment was
sufficient to identify the cleavage site. The four cleaved fragments
yielded three distinct cleavage sites that were defined in two cases by
eight amino acids of peptide sequence and in one case by seven amino
acids of sequence (underlined in Fig. 2C). This
analysis revealed sites of cleavage adjacent to Asp123,
Asp146, and Asp170. Cleavage occurred in the
sequences PEDD123
Of the three sequences, only DDVD146
The present finding that PEDD123 Topoisomerase I Cleavage in Various Cell Types--
All previous
studies demonstrating apoptotic cleavage of human topo I were performed
using leukemia cell lines. To determine whether topo I cleavage is
limited to this cell type, we examined several different cell types
undergoing apoptosis in response to various stimuli. Topoisomerase I
cleavage products were observed not only in Jurkat (Fig.
3A, lane 5) and
K562 (data not shown; identical to Jurkat cells) leukemia cells, but
also in A549 lung (lanes 8 and 10) and
MDA-MB-468 breast (lanes 13 and 15)
cancer cells undergoing apoptosis in response to previously described proapoptotic stimuli (31, 41, 42, 59). Interestingly, different
patterns of topo I cleavage were observed in these model systems. In
the Jurkat, K562, and A549 cell lines, the single topo I cleavage
product comigrated with the major fragment (b) generated by
caspase-3 in vitro (cf. Fig. 3A,
lanes 2, 5, 8, and 10). In contrast, apoptotic MDA-MB-468 cells contained two
topo I cleavage products, a larger fragment (b) that
comigrated with the major caspase-3 cleavage product and a smaller
fragment (c) that comigrated with the major caspase-6
cleavage product.
To determine whether these apparent differences reflect differences
between the cell lines as opposed to differences in the apoptotic
stimuli, three different cell lines were treated with 100 nM paclitaxel (Fig. 3B). Consistent with the
results in Fig. 3A, two topo I fragments were evident in
apoptotic MDA-MB-468 cells (Fig. 3B, lane 3). As
was the case for etoposide and topotecan treatment, one fragment
predominated in A549 cells (Fig. 3B, lane 9).
When the exposure of the blot was increased in order to begin to bring
up the background, a faint smear was seen just beneath the major
cleavage product (Fig. 3B, lane 9'). Although
this is the region of the gel in which fragment c
runs, this smear did not appear upon close inspection to correspond to
a discrete band. Thus, there is a clear (although slight) difference in
the pattern of topo I cleavage between MDA-MB-468 and A549 cells in
response to a single initiating response. An even more dramatic
difference was observed when topo I processing in apoptotic MCF-7 cells
was examined. Topoisomerase I cleavage was entirely absent in these cells (Fig. 3B, lane 6), which lack caspase-3
(60). This indicates that topo I cleavage is mediated by caspase-3 or a
caspase that lies downstream of caspase-3.
Localization of Topoisomerase I Cleavage Products in
Apoptosis--
Recent results indicate that topo II dissociates
from the chromatin during the course of apoptosis (61). To determine
whether topo I likewise undergoes relocalization, we examined the
distribution of intact topo I and its fragment in apoptotic cells.
Topoisomerase I has four predicted nuclear localization signals:
Lys59-Glu65,
Lys150-Asp156,
Lys174-AspD180, and
Lys192-Glu198 (Fig. 2C) (62).
Although cleavage at DDVD146
Several approaches were used to examine the localization of topo I and
its major fragment during apoptosis. In initial experiments, normal and
apoptotic Jurkat cells were examined by indirect immunofluorescence using monoclonal anti-topo I antibody according to protocols previously described (44). In these studies (data not shown), a decrease in topo I
staining was observed in many of the apoptotic cells. It appeared that
the low levels of remaining detectable antigen were frequently (but not
invariably) excluded from condensed apoptotic bodies. We could not,
however, rule out the possibility that changes in chromatin structure
were masking the topo I epitope in the apoptotic bodies, nor could we
ascertain whether the topo I antibody (which detects both full-length
polypeptide and cleaved fragments (Figs. 1-3)) was providing
information about the topo I that remained intact in the apoptotic
cells as opposed to the topo I cleavage fragment.
To circumvent these difficulties, the distributions of topo I and its
cleavage product were compared by subcellular fractionation. Jurkat
cells were induced to undergo apoptosis by treatment with low levels of
agonistic anti-Fas antibody or etoposide for 14 h, lysed by
homogenization, and subjected to differential sedimentation to produce
a sedimentable fraction (containing nuclei and nuclear fragments) and
cytosol. Immunoblotting revealed that both intact and fragmented
topo I were recovered exclusively in the sedimentable fraction (Fig.
4A, upper panel).
In contrast, procaspase-2 was exclusively cytoplasmic (Fig.
4A, lower panel); and the shuttling protein B23
was found predominantly in the nucleus, but with low levels detected in
the cytoplasm (middle panel). Based on these results, it
appears that topo I remains associated with nuclei during
apoptosis.
The Major Topoisomerase I Cleavage Fragment Remains
Catalytically Active--
To further examine the subcellular location
of the topo I fragment as well as assess its catalytic function, a band
depletion assay was performed. The basis of this assay is described in
detail by Kaufmann et al. (43). Under normal conditions, the
catalytic intermediate that contains the active-site Tyr723
covalently linked to a 3'-phosphate of the substrate DNA has a short
half-life. Recent crystallographic data suggest that the presence of
the plant alkaloid camptothecin perturbs the structure of this
intermediate such that the free 5'-hydroxyl of the DNA might be
displaced by ~4.5 Å from the phosphate group that would be the site
of attack for religation (63). As a consequence, the religation of this
intermediate is slowed; and topo I·DNA covalent complexes accumulate.
If cells containing these covalent topo I·DNA intermediates are lysed
under denaturing conditions and subjected to SDS-PAGE, the topo I
trapped in these complexes migrates as a smear with reduced mobility,
resulting in a reduction in the signal for topo I at
Mr ~ 100,000.
This band depletion assay is illustrated in Fig. 4B
(lanes 1-5). Treatment of control Jurkat cells
with increasing concentrations of the topo I poison topotecan resulted
in progressive loss of the topo I signal at Mr ~ 100,000. Control experiments revealed that the signal for topo I
could be restored within 2 min by exposing the cells to conditions that
shift the cleavage-religation equilibrium of the enzyme in favor of
free topo I (data not shown) (43). Application of the same assay to
Jurkat cells induced to undergo apoptosis by treatment with anti-Fas
antibody (Fig. 4B, lanes 6-10) or
etoposide (data not shown) revealed that signals for topo I and the
cleavage product were both attenuated as the concentration of topotecan
increased. These observations indicate not only that the topo I
fragment remains in the vicinity of DNA, but also that the fragment
remains catalytically active in situ, a result that is
consistent with the previous finding that the amino-terminal ~200
amino acids are dispensable for topo I activity (36, 46).
Conclusions--
Recent results indicate that treatment of HeLa
cells with topo I poisons results in down-regulation of topo I
polypeptide levels as a result of ubiquitin-mediated proteolysis (64,
65). In contrast, we observed topo I cleavage to one or two relatively stable fragments in cells undergoing apoptosis after treatment with a
variety of stimuli, including the topo I poison topotecan (Fig. 4). The
more abundant of these fragments retains the capacity to cleave DNA and
to bind topotecan (Fig. 4). These observations provide evidence for an
alternative pathway of topo I degradation. Moreover, the lack of topo I
cleavage in MCF-7 cells, which lack caspase-3, indicates that topo I
cleavage in situ is mediated by caspase-3 or a protease
downstream of caspase-3, e.g. caspase-6 (66).
In complementary experiments, we demonstrated that topo I is a
substrate of two different caspases, caspase-3 and caspase-6 (Fig.
2A). Although two other polypeptides have recently been reported to be dual substrates of these two caspases (55, 56), topo I
is the first substrate for which the cleavage sites have been
identified. Two of the three cleavages mediated by caspase-3 and
caspase-6 occurred at sequences that differed from those reportedly preferred by these enzymes (Fig. 2C). These results have
several implications. On the one hand, the demonstration of cleavage at disfavored sequences indicates that cleavage site predictions based
solely on examination of tetrapeptide substrates might not identify all
caspase cleavage sites in native protein substrates. On the other hand,
the use of kinetically less favorable sites might also explain the
relatively slow rate of topo I cleavage by recombinant caspases
in vitro (Fig. 2, A and B) and by
apoptotic proteases in situ (Fig. 1). In addition, these
results raise the possibility that topo I cleavage might be used as a
possible indicator of the duration of caspase activation within cells.
Previous studies have demonstrated that the nuclear protein
poly(ADP-ribose) polymerase is cleaved relatively soon after activation
of caspase-3-like proteases in cells (37, 67-70). Hence
poly(ADP-ribose) polymerase cleavage indicates that caspases have been
activated, but does not provide an indication of how long they have
been activated. In contrast, because of its slow cleavage, topo I might
be useful as an indicator of how long caspases have been active in cells.
Y and EEED170
G, whereas
treatment with caspase-6 resulted in cleavage at
PEDD123
G and EEED170
G. After treatment of
Jurkat T lymphocytic leukemia cells with anti-Fas antibody or A549 lung
cancer cells with topotecan, etoposide, or paclitaxel, the
topoisomerase I fragment comigrated with the product that resulted from
caspase-3 cleavage at DDVD146
Y. In contrast, two
discrete topoisomerase I fragments that appeared to result from
cleavage at DDVD146
Y and EEED170
G were
observed after treatment of MDA-MB-468 breast cancer cells with
paclitaxel. Topoisomerase I cleavage did not occur in apoptotic MCF-7
cells, which lack caspase-3. Cell fractionation and band depletion
studies with the topoisomerase I poison topotecan revealed that the
topoisomerase I fragment remains in proximity to the chromatin and
retains the ability to bind to and cleave DNA. These observations
indicate that topoisomerase I is a substrate of caspase-3 and possibly
caspase-6, but is cleaved at sequences that differ from those
ordinarily preferred by these enzymes, thereby providing a potential
explanation why topoisomerase I cleavage lags behind that of classical
caspase substrates such as poly(ADP-ribose) polymerase and lamin
B1.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
20 °C.
Recombinant human caspase-3 and caspase-6 were expressed in Sf9
cells as described (37). Sf9 cell extracts containing recombinant caspases were preincubated at 37 °C for 15 min with 100 µM YVAD-cmk (added from a 10 mM stock in
Me2SO) or diluent. Purified topo I was then added. After a
2-h incubation at 37 °C, samples were boiled in SDS sample buffer
for 5 min, subjected to SDS-PAGE (38) on gels containing 7.5 or 10%
acrylamide, and transferred to nitrocellulose for immunoblotting. For
some experiments, histidine-tagged caspase-3 (39) was expressed in
Escherichia coli and purified by nickel chelate
chromatography (40).
-phenylmethylsulfonyl fluoride, and 1% (v/v)
-mercaptoethanol as
described previously (43). After sonication, samples were prepared for
SDS-PAGE as described (43). Aliquots containing 50 µg of protein were
subjected to SDS-PAGE on gels containing 7% acrylamide, transferred to
nitrocellulose, and subjected to immunoblotting. Quantitation of
polypeptides on the resulting blots was performed by comparison with
serial dilutions of untreated cells as described (43).
-phenylmethylsulfonyl fluoride and 100 units/ml
aprotinin), and homogenized in a tight-fitting Dounce homogenizer.
Nuclei and nuclear fragments were sedimented at 15,000 × g for 15 min and dissolved in solubilization buffer. The
post-nuclear supernatant was sedimented at 105,000 × g
for 60 min. Protein in the supernatant from this second centrifugation
step (cytosol) was precipitated with 10% trichloroacetic acid, washed
once with 10% trichloroacetic acid and three times with
20 °C
acetone, and dissolved in solubilization buffer.
RESULTS AND DISCUSSION
G (30) to yield a
characteristic 89-kDa fragment likewise was detected at 60 min (Fig.
1B). Cleavage of lamin B1 (manifested in Fig.
1C as a decrease in the intensity of the 67-kDa full-length polypeptide) began between 60 and 80 min after addition of CH-11 antibody to the cells. By 180 min, half of the poly(ADP-ribose) polymerase and 75% of the lamin B1 molecules were cleaved
in these cells. In contrast, the earliest cleavage of topo I to a
detectable fragment was not evident until 140 min after addition of
CH-11 antibody to the cells (Fig. 1A). Moreover, <5% of
the topo I was cleaved under these conditions. A similar disparity
between cleavage of topo I and the other two caspase substrates
was observed after treatment of the Jurkat cells with etoposide (data
not shown). These results not only confirmed that topo I was cleaved to
a detectable fragment during apoptosis initiated by triggering two discrete pathways in Jurkat cells (35, 45), but also indicated that
topo I cleavage was delayed relative to other cleavages.
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Fig. 1.
Comparison of cleavages of selected
polypeptides during anti-Fas antibody-induced apoptosis. Jurkat
cells were treated with 500 ng/ml anti-Fas antibody CH-11 for the
indicated lengths of time and then solubilized for SDS-PAGE followed by
immunoblotting with antibodies that recognize topo I (A),
poly(ADP-ribose) polymerase (PARP; B), lamin
B1 (C), procaspase-7 (D), and
procaspase-2 (E). Control cells were harvested prior to
addition of anti-Fas antibody. Each lanewas loaded with 50 µg of
protein, and the equality of loading was confirmed by blotting with
anti-procaspase-2 (E) and anti-B23 (data not shown)
antibodies. Frag, 80-kDa topo I fragment or 89-kDa PARP
fragment.
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Fig. 2.
Topoisomerase I is cleaved by caspases
in vitro. A, purified topo I from
Sf9 cells was incubated in MDB buffer (71) (lane 1),
caspase-3 extracts (lane 2), or caspase-6 extracts
(lane 3). After 2 h at 37 °C, the reaction mixture
was subjected to 10% SDS-PAGE and immunoblotted with human
autoantiserum containing anti-topo I antibodies. B,
topoisomerase I is cleaved more slowly than poly(ADP-ribose) polymerase
by caspase-3. Topoisomerase I purified from Sf9 cells (800 ng)
was incubated with purified bovine poly(ADP-ribose) polymerase
(PARP; 400 ng) and purified recombinant caspase-3 (60 ng) in
MDB buffer at 37 °C. At the indicated time points, aliquots were
removed, boiled in SDS sample buffer, divided in half, subjected to
10% SDS-PAGE as duplicate sets of time points in a single gel, and
immunoblotted with either human autoantiserum containing anti-topo I
antibodies or monoclonal antibody C-II-10 against mammalian
poly(ADP-ribose) polymerase (72). The positions of relevant molecular
weight markers are indicated on the left. C, diagram of topo
I showing the three caspase cleavage sites. Purified topo I was
incubated with either caspase-3 or caspase-6 for 2 h at 37 °C,
subjected to SDS-PAGE on a 7.5% acrylamide gel, and transferred to a
polyvinylidene difluoride membrane. Cleaved fragments stained with
Coomassie Blue were excised and subjected to sequential Edman
degradation (the resulting peptide sequences are
underlined). The four putative nuclear localization signals
(NLS) Lys59-Glu65,
Lys150-Asp156,
Lys174-Asp180, and
Lys192-Glu198 (open circles); the
active-site Tyr723; and the domain structure of topo I are
also indicated.
G (caspase-6), DDVD146
Y
(caspase-3), and EEED170
G (caspase-3 and caspase-6).
Although this analysis cannot rule out the possibility that additional
caspase-mediated cleavages occur in the N-terminal domain, our kinetic
analysis revealed no proteolyzed species corresponding to cleavages in
this domain. Therefore, if such cleavages do occur, they either must
take place after the cleavages described above or must produce species
that are further processed rapidly to yield those species.
Y corresponds to a
canonical caspase-3 cleavage site. The other two sequences are unusual and would not be predicted based on current understanding of caspase cleavage specificities. A recent comprehensive study employing a
positional scanning substrate combinatorial library (48) demonstrated that the optimal tetrapeptide recognition sequences for caspase-3 and
caspase-6 were DEVD and VEHD, respectively, in remarkable agreement
with the previously mapped cleavage sites in the endogenous substrates
poly(ADP-ribose) polymerase (DEVD
G) (30) and lamin A (VEID
N) (54,
57). The combinatorial approach indicated that glutamic acid and
proline were significantly disfavored at the P4 position by caspase-3
and caspase-6, respectively (48). The use of these disfavored sites
provides a potential explanation for the slow and incomplete cleavage
of topo I (Figs. 1 and 2).
G and
EEED170
G are in fact used when caspases cleave
topoisomerase I not only provides evidence that factors in addition to
primary sequence are important in cleavage site selection, but also
suggests that studies of caspase cleavage site specificity using small
peptides might need be interpreted with caution when drawing
conclusions about the cleavage of native protein substrates. This is
consistent with recent results showing that caspase cleavage of
poly(ADP-ribose) polymerase was significantly increased following
phosphatase treatment of apoptotic extracts, whereas cleavage of
tetrapeptide substrates was unaffected (58).
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Fig. 3.
Topoisomerase I is cleaved to similar
fragments both in vitro and in
vivo. A, comparison of topo I cleavage
in vitro and in cells. Lane 1,
purified topo I; lanes 2 and 3, purified topo I
cleaved by caspase-3 and caspase-6, respectively, in vitro;
lanes 4 and 5, Jurkat cells treated
with medium lacking or containing 100 ng/ml anti-Fas antibody CH-11 for
14 h, respectively; lanes 6-10, A549 cells untreated
(lane 6) and treated with 68 µM etoposide
(lanes 7 and 8) or 0.2 µM topotecan (TPT; lanes
9 and 10) (lanes 7 and
9 contain adherent (nonapoptotic) cells, whereas
lanes 8 and 10 contain nonadherent
(apoptotic) cells); lanes 11-15, MDA-MB-468
cells untreated (lane 11) and treated with 100 µM 5-fluoro-2'-deoxyuridine (5FUdR;
lanes 12 and 13) or 100 nM
paclitaxel (lanes 14 and 15)
(lanes 12 and 14 contain adherent
(nonapoptotic) cells, whereas lanes 13 and
15 contain nonadherent (apoptotic) cells). All samples were
subjected to SDS-PAGE on the same 7% (w/v) acrylamide gel, transferred
to nitrocellulose, and probed with monoclonal anti-topo I antibody
C-21. Because of differences in the optimal exposure time for various
lanes, two different fluorographs of the same blot have been spliced to
compose this figure. A, adherent cells; F,
floating (nonadherent) cells. B, comparison of topo I
cleavage after treatment of several cell lines with paclitaxel.
MDA-MB-468 cells (lanes 1-3), MCF-7 cells
(lanes 4-6), or A549 cells (lanes
7-9) were treated with paclitaxel followed by drug-free
medium as described under "Experimental Procedures."
Lanes 1, 4, and 7,
diluent-treated adherent cells ( ); lanes 2,
5, and 8, adherent (nonapoptotic)
paclitaxel-treated cells; lanes 3, 6,
and 9, nonadherent (apoptotic) cells that accumulated during
the 24 h after paclitaxel removal. Morphological examination after
staining with Hoechst 33258 confirmed that the vast majority of cells
in these nonadherent populations were apoptotic. Electrophoresis and
blotting were performed as described for A. Lanes
1-9, single exposure of one blot; lane 9',
longer exposure of lane 9.
Y would remove one of
these potential nuclear localization signals, the resulting
carboxyl-terminal fragment would be expected to retain its nuclear
targeting function. In fact, a previous study has indicated that
Lys192-Glu198 is apparently sufficient to
direct the nuclear transport of topo I (36).
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Fig. 4.
Examination of topo I subcellular
distribution and ability to form covalent enzyme·DNA complexes.
A, Jurkat cells incubated without (lanes
1, 4, and 7) or with 100 ng/ml
anti-Fas antibody CH-11 ( Fas; lanes
2, 5, and 8) or 17 µM
etoposide (Etop; lanes 3,
6, and 9) for 14 h were separated into fractions
containing nuclei and nuclear fragments (lanes
4-6) or cytosol (lanes 7-9),
subjected to SDS-PAGE, and blotted with monoclonal anti-topo I antibody
C-21 (upper panel), polyclonal anti-B23 antibody
(middle panel), or monoclonal anti-procaspase-2 antibody
(lower panel). Gels were loaded with 50 µg of
protein/lane. Note that procaspase-2 partitioned exclusively with the
cytosol fraction, whereas topo I partitioned exclusively with the
nuclei and nuclear fragments. B, control Jurkat cells
(lanes 1-5) or Jurkat cells treated with 500 ng/ml anti-Fas antibody CH-11 for 4 h (lanes
6-10) were incubated for 45 min at 37 °C with 0 (lanes 1 and 6), 3 (lanes
2 and 7), 10 (lanes 3 and 8), 30 (lanes 4 and 9),
or 100 (lanes 5 and 10)
µM topotecan. At the completion of the incubation,
samples were sedimented at 3200 × g for 1 min and
immediately lysed under denaturing conditions as recently described
(43). After SDS-PAGE and transfer to nitrocellulose, blots were probed
with monoclonal anti-topo I antibody C-21 (upper panel) or
monoclonal anti-poly(ADP-ribose) polymerase (PARP) antibody
C-II-10. Lanes 6'-10' are longer
exposures of lanes 6-10 so that effect of topotecan on the
topo I fragment can be better discerned. Note that the 80-kDa topo I
fragment (Frag) behaved just like full-length topo I in this
assay. In additional experiments, incubation of cells at 48 °C for 2 min prior to lysis completely restored the topo I signal (data not
shown).
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ACKNOWLEDGEMENTS |
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We thank D. J. McCormick and B. J. Madden of the Mayo Clinic Protein Core Laboratory for assistance with protein sequencing; Y.-C. Cheng for the kind gift of anti-topo I antibody C-21; and C. M. Eischen, P. J. Leibson, and N. E. Davidson for kind gifts of cell lines.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants AG13487 (to E. S. A.), CA69008 (to S. H. K. and W. C. E), and GM49156 (to L. S. and J. J. C.) and by a principal research fellowship from the Wellcome Trust (to W. C. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
f Present address: Emerald BioStructures Inc., 7865 Northeast Day Rd. W., Bainbridge Island, WA 98110.
j Leukemia Society of America Scholar.
k These authors contributed equally to this work.
l Principal Research Fellow of the Wellcome Trust. To whom correspondence should be addressed: Inst. of Cell and Molecular Biology, University of Edinburgh, Michael Swann Bldg., The King's Bldgs., Mayfield Rd., Edinburgh EH9 3JR, Scotland, UK. Tel.: 44-131-650-7101; Fax: 44-131-650-7100; E-mail bill.earnshaw{at}ed.ac.uk.
The abbreviations used are: topo I, DNA topoisomerase I; YVAD-cmk, acetyltyrosinylvalinylalanylaspartyl chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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