Download Topoisomerase II and tubulin inhibitors both induce the formation of

3139
Topoisomerase II and tubulin inhibitors both induce
the formation of apoptotic topoisomerase I
cleavage complexes
Olivier Sordet, Abby Goldman, and Yves Pommier
Laboratory of Molecular Pharmacology, Center for Cancer
Research, National Cancer Institute, NIH, Bethesda, Maryland
Abstract
Topoisomerase I (Top1) is a ubiquitous enzyme that
removes DNA supercoiling generated during transcription
and replication. Top1 can be trapped on DNA as cleavage
complexes by the anticancer drugs referred to as Top1
inhibitors as well as by alterations of the DNA structure.
We reported recently that Top1 cleavage complexes
(Top1cc) are trapped during apoptosis induced by arsenic
trioxide and staurosporine. In the present study, we
generalize the occurrence of apoptotic Top1cc in response
to anticancer drugs, which by themselves do not directly
interact with Top1: the topoisomerase II inhibitors etoposide, doxorubicin, and amsacrine, and the tubulin inhibitors vinblastine and Taxol. In all cases, the Top1cc form
in the early phase of apoptosis and persist throughout
the apoptotic process. Their formation is prevented by
the caspase inhibitor benzyloxycarbonyl-Val-Ala- DL Asp(OMe)-fluoromethylketone and the antioxidant
N-acetyl-L-cysteine. We propose that the trapping of
Top1cc is a general process of programmed cell death,
which is caused by alterations of the DNA structure
(oxidized bases and strand breaks) induced by caspases
and reactive oxygen species. [Mol Cancer Ther 2006;
5(12):3139 – 44]
Introduction
Topoisomerase I (Top1) is an essential enzyme in higher
eukaryotes as it removes DNA supercoiling generated
during transcription and replication (1 – 3). Top1 relaxes
DNA supercoiling by forming DNA single-strand breaks
that are produced as Top1 forms a covalent bond between
its active site tyrosine (Y723) and a 3¶-DNA phosphate.
Received 8/4/06; revised 10/5/06; accepted 10/27/06.
Grant support: Intramural Research Program of the NIH, National Cancer
Institute, Center for Cancer Research.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for reprints: Yves Pommier, Laboratory of Molecular
Pharmacology, Center for Cancer Research, National Cancer Institute,
NIH, Building 37, Room 5068, Bethesda, MD 20892-4255.
Phone: 301-496-5944; Fax: 301-402-0752. E-mail: [email protected]
Copyright C 2006 American Association for Cancer Research.
doi:10.1158/1535-7163.MCT-06-0463
These Top1-linked breaks allow controlled rotation of the
broken DNA around the intact strand (2). Immediately
after the DNA is relaxed, Top1 spontaneously religates
the breaks and restores intact duplex DNA. Under normal
conditions, the covalent Top1-cleaved DNA intermediates,
referred to as Top1 cleavage complexes (Top1cc), are
constitutively transient and almost undetectable because
the DNA religation (closing) step is much faster than
the DNA cleavage (nicking) step. A variety of common
DNA alterations, including base oxidation, methylation,
mismatches, carcinogenic adducts, abasic sites, and strand
breaks, can trap Top1cc by misaligning the DNA ends
within the Top1cc and preventing DNA religation (4, 5).
The alkaloid camptothecin and its therapeutic derivatives
topotecan and irinotecan also trap Top1 by intercalating
specifically inside the Top1cc (2, 6).
Recently, we and others showed the stabilization of
Top1cc in cells undergoing apoptosis (7 – 10). These apoptotic Top1cc have been detected in various human cell
types exposed to arsenic trioxide (10), staurosporine (8), or
UV irradiation (7). The Top1cc formed during apoptosis
likely participate in the cell death program because downregulation of Top1 by small interfering RNA reduces
apoptotic DNA fragmentation induced by arsenic trioxide
(10) and staurosporine (8). In the present study, we asked
whether the formation of Top1cc is a general process of
apoptotic cell death induced by anticancer chemotherapeutic agents. We tested the formation of Top1cc during
apoptosis induced by topoisomerase II (Top2) and tubulin
inhibitors, which by themselves do not directly interact with
Top1. Top2 inhibitors initiate apoptosis by trapping Top2
cleavage complexes (Top2cc), thereby generating DNA
double-strand breaks (11 – 13). Top2 inhibitors, such as
etoposide (VP-16), doxorubicin, and amsacrine (m-AMSA),
are among the most potent inducers of apoptosis and are
commonly prescribed drugs to treat a variety of cancers
(14). The tubulin inhibitors vinblastine and Taxol (paclitaxel) are anticancer drugs that initiate apoptosis by binding
specifically at the interface of the tubulin heterodimer,
thereby interfering with microtubule growth (6, 15, 16).
The present study shows that VP-16, doxorubicin,
m-AMSA, and vinblastine induce Top1cc that form in
the early phase of apoptosis and persist throughout the
apoptotic process. In response to VP-16, the formation of
apoptotic Top1cc is prevented by the caspase inhibitor
benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketone (Z-VAD-fmk) and the antioxidant N-acetyl-L-cysteine.
We propose that the trapping of Top1cc is a general process
of apoptotic cell death, which is caused by alterations of the
DNA structure (oxidized bases and strand breaks) induced
by caspases and reactive oxygen species (ROS).
Mol Cancer Ther 2006;5(12). December 2006
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3140 Apoptotic Top1cc
Materials and Methods
Cell Culture, Drugs, and Chemical Reagents
The human leukemia (CEM and HL-60) and colon
carcinoma (HCT116) cell lines (American Type Culture
Collection, Manassas, VA) were cultured as described (8).
VP-16, doxorubicin, m-AMSA, vinblastine, and N-acetyl-Lcysteine were obtained from Sigma (St. Louis, MO). The
caspase peptide inhibitor z-VAD-fmk was obtained from
Bachem (Torrance, CA). Taxol (paclitaxel) was a kind gift
from Dr. Tito Fojo (National Cancer Institute, NIH,
Bethesda, MD). [2-14C]thymidine was from Perkin-Elmer
Life Sciences (Boston, MA).
Detection of Cellular Topoisomerase Cleavage
Complexes
Topoisomerase cleavage complexes were detected
using the in vivo complex of enzyme bioassay as described
(8, 10, 17). Briefly, cells were lysed in 1% Sarkosyl and
homogenized. The cell lysates were centrifuged on cesium
chloride step gradients at 165,000 g for 20 h at 20jC.
Twenty fractions (0.5 mL) were collected and diluted (v/v)
into 25 mmol/L potassium phosphate buffer (pH 6.6). The
DNA-containing fractions (fractions 7 – 11) were pooled
(except in Fig. 1D) and applied to polivinylidene difluoride
membranes (Immobilon-P, Millipore, Billerica, MA) using a
slot-blot vacuum manifold. Topoisomerase cleavage complexes were detected by immunoblotting using the C21
Top1 mouse monoclonal antibody (1:1,000 dilution; a kind
gift from Dr. Yung-Chi Cheng, Yale University, New
Haven, CT) or a Top2a mouse monoclonal antibody (clone
Ki-S1; 1:1,000 dilution) from Chemicon International
(Temecula, CA).
DNA Fragmentation Assay
Apoptotic DNA fragmentation was quantified by filter
elution assay as described (18). Briefly, cells were incubated
with [2-14C]thymidine (0.02 ACi/mL) for 2 days and chased
overnight in radioisotope-free medium. After drug treatment, cells were loaded onto a protein-absorbing filter
(Metricel membrane filter, 0.8 Amol/L pore size, 25 mm
diameter; Pall Corp., East Hills, NY), washed with PBS, and
lysed in 0.2% sodium Sarkosyl, 2 mol/L NaCl, and 0.04
mol/L EDTA (pH 10.0). The filters were then washed with
0.02 mol/L EDTA (pH 10.0). DNA was depurinated by
incubation of filters in 1 mol/L HCl at 65jC and then
released from the filters with 0.4 mol/L NaOH at room
temperature. Radioactivity was counted by liquid scintillation spectrometry in each fraction (wash, lysis, EDTA wash,
and filter). DNA fragmentation was measured as the
fraction of disintegrations per min in the lysis fraction plus
EDTA wash relative to the total intracellular disintegrations
per min.
Caspase-3 Activity
Caspase-3 activity was measured as described (19).
Briefly, cells were incubated in lysis buffer [150 mmol/L
NaCl, 50 mmol/L Tris-HCl (pH 8.0), 0.1% SDS, 1% NP40,
Figure 1. VP-16 induces Top1cc as cells undergo
apoptosis. A, CEM cells were treated with 200
Amol/L VP-16 for the indicated times. Top, apoptotic
DNA fragmentation. Columns, mean of triplicate
samples; bars, SD. Middle and bottom, detection
of Top1cc and Top2cc. The DNA-containing fractions
were pooled and probed at three concentrations
(10, 3, and 1 Ag DNA) by immunoblotting with
antibodies against Top1 or Top2a. B, top, DEVDAFC peptide cleavage (caspase-3) activity. Columns,
mean of triplicate samples; bars, SD. Middle and
bottom, Western blotting analyses of procaspase-3,
caspase-3, and poly(ADP-ribose) polymerase (PARP ).
Twenty microgram proteins from whole-cell extracts
were loaded in each well. Tubulin was used as a
loading control. Numbers are molecular masses in
kilodaltons. CL, cleavage products. C, apoptotic DNA
fragmentation was quantified in HCT116 cells treated
with 200 Amol/L VP-16 for the indicated times.
Columns, mean of triplicate samples; bars, SD. D,
detection of Top1cc in the DNA-containing fractions
(see Materials and Methods).
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Molecular Cancer Therapeutics
Figure 2. Induction of apoptotic Top1cc by the Top2 inhibitors, VP-16,
m-AMSA, and doxorubicin. A, CEM cells were treated with 200 Amol/L
VP-16 for 4 h, washed, and cultured in VP-16-free medium for the
indicated times. Top, apoptotic DNA fragmentation. Columns, mean of
triplicate samples; bars, SD. Middle and bottom, Top2cc reverse,
whereas Top1cc persist following VP-16 removal. B, human leukemia
HL-60 cells were treated with 1 Amol/L doxorubicin or 1 Amol/L m-AMSA
for 20 h. Top, apoptotic DNA fragmentation. Columns, mean of triplicate
samples; bars, SD. Bottom, Top1cc are induced by both doxorubicin and
m-AMSA. The DNA-containing fractions were pooled and probed at three
concentrations (10, 3, and 1 Ag DNA) by immunoblotting with antibodies
against Top1 or Top2a.
0.5% sodium deoxycholate] for 30 min at 4jC and
centrifuged (10,000 g, 20 min, 4jC). Twenty micrograms
of protein from the resulting supernatant were incubated in
buffer assay [100 mmol/L HEPES (pH 7.0), 1 mmol/L
EDTA, 0.1% CHAPS, 10% glycerol, 20 mmol/L DTT] in
the presence of 100 Amol/L of the fluorogenic peptide
substrate Z-DEVD-AFC (Calbiochem, EMD Biosciences,
La Jolla, CA). 7-Amino-4-trifluoromethylcoumarin (AFC)
released from the substrate was excited at 400 nm to
measure emission at 505 nm. Fluorescence was monitored
continuously at 37jC for 20 min in a dual-luminescence
fluorometer (SPECTRAmax Gemini XS, Molecular Devices,
Sunnyvale, CA). Caspase-3 activity was determined as
initial velocities expressed as relative intensity per min per
milligram.
Western Blotting
Western blotting analyses on whole-cell extracts were
done as described (8) using the rabbit antihuman caspase-3
(1:5,000 dilution; BD PharMingen, San Diego, CA) and
poly(ADP-ribose) polymerase (1:5,000 dilution; Roche,
Indianapolis, IN) antibodies.
Results and Discussion
The Top2 Inhibitors VP-16, Doxorubicin, and m-AMSA
Induce Top1cc During Apoptosis
Trapped Top1cc and Top2cc can be detected in genomic
DNA after cesium chloride gradient centrifugation and
immunoblotting with Top1 and Top2 antibodies (8, 10,
17, 20), respectively. In human leukemia CEM cells exposed
to VP-16, we detected high levels of Top1cc (Fig. 1A,
middle) with kinetics that coincided with the occurrence of
apoptosis measured by DNA fragmentation (Fig. 1A, top).
Apoptotic DNA fragmentation and Top1cc were both
detected after 4 h of VP-16 exposure. Similar levels of
Top1cc persisted throughout the apoptotic process as most
of the cellular DNA became fragmented (Fig. 1A). The
induction of Top1cc induced by VP-16 was coincident with
the activation of caspase-3, as shown by the ability of cell
lysates to cleave DEVD-AFC, a fluorogenic substrate that
mimics the target site of caspase-3 and closely related
caspases (Fig. 1B, top), and the decrease of the 32-kDa
proform of caspase-3 with the appearance of its cleaved
19- and 17-kDa active fragments (Fig. 1B, middle). Kinetics
of caspase-3 activation was further confirmed by the
caspase-dependent cleavage of poly(ADP-ribose) polymerase (Fig. 1B, bottom). VP-16-induced Top1cc were also
observed in the human colon carcinoma HCT116 cells
undergoing apoptosis (Fig. 1C and D). Thus, the induction
of Top1cc by VP-16 can be observed in different human cell
types as they undergo apoptosis.
VP-16 is a specific Top2 inhibitor (21). It is well
established that VP-16 does not trap directly Top1cc.
Accordingly, short exposure to VP-16 (for up to 2 h)
trapped only Top2cc (Fig. 1A, bottom). However, Top1cc
were induced by 4 h of VP-16 exposure (Fig. 1A, middle).
These Top1cc persisted after the removal of VP-16 as cells
were committed to apoptosis, whereas trapping of Top2cc
was only transient (Fig. 2A; ref. 21). Thus, we conclude
that trapping of Top2cc by VP-16 initiates apoptosis and
subsequently induces the formation of Top1cc. Consistently,
two other Top2 inhibitors, doxorubicin and m-AMSA,
also induced Top1cc in apoptotic leukemia HL-60 cells
(Fig. 2B).
Figure 3.
The caspase inhibitor Z-VAD-fmk and the antioxidant
N -acetyl-L-cysteine (NAC ) prevent VP-16-induced apoptosis and Top1cc.
CEM cells were preincubated with z-VAD-fmk (100 Amol/L for 30 min) or
N -acetyl-L-cysteine (30 mmol/L for 30 min) before the addition of
200 Amol/L VP-16 for 8 h. Top, apoptotic DNA fragmentation. Columns,
mean of triplicate samples; bars, SD. Bottom, detection of Top1cc. The
DNA-containing fractions were pooled and probed at three concentrations
(10, 3, and 1 Ag DNA) by immunoblotting with an antibody against Top1.
Mol Cancer Ther 2006;5(12). December 2006
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3141
3142 Apoptotic Top1cc
Figure 4. Induction of apoptotic Top1cc by vinblastine and Taxol. A, CEM cells were treated with 22 nmol/L vinblastine for the indicated times. Last
lane, preincubated with z-VAD-fmk (100 Amol/L for 30 min) before the addition of vinblastine for 24 h. Top, apoptotic DNA fragmentation. Columns,
mean of triplicate samples; bars, SD. Bottom, detection of Top1cc. B, CEM cells were treated with 200 nmol/L Taxol for the indicated times. Top,
apoptotic DNA fragmentation. Columns, mean of triplicate samples; bars, SD. Bottom, detection of Top1cc. C and D, apoptotic CEM cells following
treatment for 24 h with either 22 nmol/L vinblastine (C) or 200 nmol/L Taxol (D ) fail to form detectable Top2cc. VP-16 (200 Amol/L for 1 h) was used as a
positive control for Top2cc. The DNA-containing fractions were pooled and probed at three concentration (10, 3, and 1 Ag DNA) by immunoblotting with
antibodies against Top1 or Top2a.
To further examine the relationship between Top1cc and
apoptosis, we investigated whether inactivating the apoptotic pathways would affect the generation of Top1cc by
VP-16. Inhibition of apoptotic DNA fragmentation by the
caspase peptide inhibitor z-VAD-fmk (Fig. 3, top) prevented the induction of Top1cc by VP-16 (Fig. 3, bottom).
Together, these findings show the appearance of high
levels Top1cc in the early phase of apoptosis induced by
Top2 inhibitors.
VP-16-Induced Top1cc Are Linked to the Generation
of Oxygen Radicals
Because Top1cc can be induced by ROS (22) and
oxidative DNA lesions (4, 8 – 10) and because VP-16 and
doxorubicin generate ROS and oxidative DNA lesions
(23, 24), we tested whether the apoptotic Top1cc induced
by Top2 inhibitors resulted from oxidative lesions. Quenching ROS with the antioxidant N-acetyl-L-cysteine prevented
VP-16-induced Top1cc and apoptosis (Fig. 3). Similarly, the
induction of apoptotic Top1cc by arsenic trioxide and
staurosporine has been shown to involve oxidative DNA
lesions (8 – 10). Thus, we propose that Top2 inhibitors
induce ROS that produce oxidative DNA lesions (oxidized
bases and strand breaks), thereby generating Top1cc in
apoptotic cells. These ROS can be generated by permeabilized mitochondria during apoptosis (9).
The Tubulin Inhibitors Vinblastine and Taxol Also
Induce Apoptotic Top1cc
Next, we examined the occurrence of Top1cc during
apoptosis induced by the anticancer drugs vinblastine
and Taxol, which induce apoptosis following inhibition of
microtubule growth (6, 15, 16). In CEM cells undergoing
apoptosis, both vinblastine (Fig. 4A) and Taxol (Fig. 4B)
also induced Top1cc. Inhibition of apoptosis by the caspase
inhibitor z-VAD-fmk prevented the formation of Top1cc in
vinblastine-treated cells (Fig. 4A).
Although it has been reported that Top2a participates in
chromatin condensation (25) and in the excision of DNA
loops (26) during apoptosis, we did not detect Top2cc in
apoptotic cells following exposure to vinblastine (Fig. 4C)
and Taxol (Fig. 4D). Similarly, apoptotic Top1cc formed in
the absence of Top2cc in response to arsenic trioxide (10).
It is therefore possible that the catalytic activity of Top2a
(rather than its stabilization on DNA as Top2cc) contributes
to chromatin fragmentation. However, we cannot exclude
that the levels of Top2cc formed during apoptosis are
below the threshold of detection of our assays.
Conclusion
Altogether, our findings indicate that the formation of
Top1cc is a general process of apoptotic cell death. Table 1
summarizes the various agents that have been identified as
producing apoptotic Top1cc. In addition to Top2 and
Figure 5.
Proposed mechanism for the induction of apoptotic Top1cc
by Top2 and tubulin inhibitors. Top2 or tubulin inhibitors initiate
apoptosis by activating and producing ROS, which leads to the formation
of Top1cc. Top1cc might participate in apoptosis execution by generating DNA strand breaks [1] and by engaging the apoptotic machinery in
trans [2].
Mol Cancer Ther 2006;5(12). December 2006
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Molecular Cancer Therapeutics
Table 1. Agents known to induce apoptotic Top1cc
Agents that induce
apoptotic Top1cc
VP-16
Doxorubicin
m-AMSA
Vinblatine
Taxol
Colcemid
TRAIL
Fas ligand
TNF-a
Antimycin
BH3I-2¶
Arsenic trioxide
Staurosporine
UV irradiation
Cellular target(s)
References
Stabilization of Top2cc, probably by intercalating within the Top2cc
Stabilization of Top2cc, probably by intercalating within the Top2cc
Stabilization of Top2cc, probably by intercalating within the Top2cc
Binding at the interface of the tubulin heterodimer
Binding at the interface of the tubulin heterodimer
Inhibits tubulin polymerization
Binds to and activates the plasma membrane receptors DR4 and DR5
Binds to and activates the plasma membrane receptor Fas
Binds to and activates the plasma membrane receptor TNFR1
BH3 mimetic that bind to and inhibit the antiapoptotic
effect of BCL-XL at the mitochondrial level
BH3 mimetic that bind to and inhibit the antiapoptotic
effect of BCL-XL at the mitochondrial level
Induces the intracellular accumulation of ROS
Inhibitor of protein kinases: Chk1, Chk2, PDK1, and PKC
Generation of pyrimidine dimers and 4,6-photoproducts
Present study
Present study
Present study
Present study
Present study
(28)
(27)
(27)
(28)
(27)
(27)
(10)
(8, 29)
(7)
Abbreviations: TNF-a, tumor necrosis factor; TRAIL, tumor necrosis factor – related apoptosis ligand; DR4, death receptor 4, DR5, death receptor 5; TNFR1,
tumor necrosis factor receptor 1; BH3, BCL-2 homology domain-3; PDK1, phosphoinositide-dependent kinase 1; PKC, protein kinase C; Chk1, checkpoint
kinase 1; Chk2, checkpoint kinase 2.
tubulin inhibitors, we reported in a meeting abstract (27)
the occurrence of Top1cc during apoptosis induced by
tumor necrosis factor – related apoptosis ligand, Fas ligand,
and the BCL-2 homology domain-3 mimetics antimycin A
and BH3I-2¶.1 During the preparation of this article,
Rockstroh et al. (28) also reported the formation of
apoptotic Top1cc in response to tumor necrosis factor-a
and colcemid (Table 1), and Sen et al. (29) showed the
conservation of apoptotic Top1cc in the parasite Leishmania
donovani.
The formation of Top1cc depends on caspase activation
and ROS. It is well established that caspases activate
endonucleases during apoptosis (30). The DNA breaks
produced by apoptotic nucleases, such as CAD/DFF40
and endonuclease G, (30) may contribute to the trapping of
Top1cc as Top1 can be directly trapped by DNA breaks
(5, 31). In addition, caspases are also involved in the
generation of ROS (32, 33). In fact, z-VAD-fmk prevented
VP-16-induced oxidative DNA lesions (23). Activation of
caspases could therefore serve to generate the ROS that
lead to apoptotic Top1cc.
It is therefore plausible that Top1, which is ubiquitous in
mammalian cells (34), participates in apoptosis by generating Top1-associated DNA breaks. These breaks may
contribute to the cleavage of DNA in high molecular
weight fragments (9, 28). Top1cc could also engage the
apoptotic machinery in trans as Top1cc are potent initiators
of apoptosis (35). Apoptotic Top1cc could therefore serve to
amplify the apoptotic process engaged by Top2cc and
tubulin inhibition (see Fig. 5) as well as by a variety of other
agents, including arsenic trioxide (10), staurosporine (8, 29),
1
O. Sordet et al., submitted for publication.
UV irradiation (7), tumor necrosis factor-a (28), tumor
necrosis factor – related apoptosis ligand, Fas ligand, and
BCL-2 homology domain-3 mimetics (Table 1; ref. 27). The
amount of Top1 associated with chromatin may determine
the intensity of apoptotic and eventually therapeutic
responses to anticancer drugs, such as Top2 and tubulin
inhibitors.
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Topoisomerase II and tubulin inhibitors both induce the
formation of apoptotic topoisomerase I cleavage complexes
Olivier Sordet, Abby Goldman and Yves Pommier
Mol Cancer Ther 2006;5:3139-3144.
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