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INTRODUCTION |
For completion of cell division, the DNA of replicated chromosomes
must be disentangled to allow the segregation of sister chromatids. In
humans, this is achieved by the unique decatenation activity of DNA
topoisomerase II (topo1 II).
Topo II is essential for normal and neoplastic cellular proliferation,
and several common anti-cancer drugs exert their cytotoxic effects
through this enzyme (1, 2).
Topoisomerase II activity in mammalian cells has been attributed to at
least two isoforms. Topo II
(p170) associates with chromosomes
during prophase and throughout mitosis and is thought to be a major
component of the nuclear scaffold (3, 4). It has a peak of expression
during G2/M of the cell cycle (5). In contrast, the closely
related topo II
(p180) isoform is thought to have a more general
role in DNA metabolism, with expression levels that remain relatively
constant during cell and growth cycles (5). Both isoforms interact with
the C-terminal region of the tumor suppressor protein, p53 (6). p53 is
a component of a multiprotein complex that contains the histone
deacetylase HDAC1 and the corepressor Sin3a (7-11).
HDAC1, and the closely related HDAC2, are both components of two
separate multiprotein complexes. The NuRD/Mi-2 repression complex
contains both nucleosome remodeling and histone deacetylase activities
(12), whereas the Sin3 complex contains only the latter (9). Both
complexes contain the Rb-associated proteins RbAp46 and RbAp48 and
associate with various, sometimes DNA-binding, transcriptional
repressor and corepressor proteins (11). The Xenopus NuRD
complex (which contains homologues of mammalian HDAC1, RbAp48, and the
methyl-CpG-binding protein MBD3) copurifies with DNA topoisomerase II
(13), raising the possibility that mammalian topo II isoforms and HDAC1
may interact in a multiprotein complex.
Here we show that HDAC1 and DNA topoisomerase II isoforms physically
interact both in vivo and in vitro. We also show
that the HDAC inhibitor, TSA, suppresses apoptosis induced by the topo II poison etoposide, but not by the topo I inhibitor camptothecin. Our
results raise the interesting possibility that chromatin remodeling by
a topo II-HDAC-containing complex is involved in topo II-catalyzed DNA
rearrangements and/or generation of etoposide-induced DNA strand breaks
in vivo.
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MATERIALS AND METHODS |
Cells, Reagents, and Materials--
The human cell lines HL-60
(promyelocytic leukemia; p53 null) and HeLa were grown in RPMI 1640 medium containing 8% fetal calf serum. Regions of HDAC1 cDNA were
subcloned into the pGEX3T-4 family of vectors (Amersham Pharmacia
Biotech) and verified by sequencing. GST fusion proteins were
purified essentially as described previously (6). Recombinant human DNA
topoisomerase II
and -
were made in a yeast system and purified
as described previously (14). Characterization and use of rabbit
polyclonal antibodies against topo II
(18511
) and topo II
(18513
) are described elsewhere (15). A polyclonal rabbit antibody
against mammalian HDAC1 was raised against a synthetic peptide
corresponding to amino acid residues 467-482 and affinity-purified as
described previously (16). Antibody against topo I was obtained
commercially (TopoGen, number 2012).
Immunoprecipitations, in Vitro Binding Assays, and Western Blot
Analysis--
HeLa whole cell extract was prepared by lysing cells in
incubation buffer (50 mM Tris, pH 7.5, 150 mM
NaCl, 5 mM EDTA, 5 mM EGTA, 10% (v/v)
glycerol) containing 1.0% (v/v) Nonidet P-40, 0.5% (w/v) sodium
deoxycholate, 0.1% (w/v) SDS, protease inhibitors (0.1 mM
phenylmethylsulfonyl fluoride and "Complete
MiniTM" tablets, Roche Molecular Biochemicals) and
50 units of DNase I (Amersham Pharmacia Biotech) per 108
cells. The lysate was incubated in ice for 10 min, and the clarified supernatant was used in standard immunoprecipitations as described previously (6, 17). To confirm specificity, cognate blocking peptide
(10 µg) was incubated with the antibody for 30 min before the
addition of extract. Preimmune serum and irrelevant antisera were used
as controls. GST pull down experiments used equivalent amounts of GST
fusion proteins prebound to glutathione-Sepharose beads (Amersham
Pharmacia Biotech) as described previously (6). Interactions
with recombinant topo II
were performed in incubation buffer
containing 0.1% (v/v) Nonidet P-40.
Yeast Two-hybrid Assays--
Yeast strains CG-1945 from a
Matchmaker Two-Hybrid System II kit
(CLONTECH) were transformed with appropriate binary
combinations of constructs containing the GAL4 DNA-binding domain and
the GAL4 activation domain, as recommended by the manufacturers.
HIS3 reporter gene expression was assayed on plates
(6), in the presence of 25 mM 3-amino-1,2,4-triazole
to suppress background growth (18).
Enzyme Assays--
Histone deacetylase activity was assayed as
described previously (17). DNA strand-passage assays were
performed on kinetoplast DNA (kDNA) as described previously
(6).
Detection of Apoptosis--
HL-60 cells were grown until in
mid-log phase, then treated with 100 nM (30 ng/ml) TSA for
0.5 h before additional treatments with either 100 µM (59 µg/ml) etoposide or 5.8 µM (2 µg/ml) camptothecin for 1.5 h. Control samples were treated with
the dilution vehicles (0.1% Me2SO and 0.1%
ethanol). All cells were observed in situ with
phase-contrast microscopy to count cells with an apoptotic morphology,
after staining with 10 µM Hoechst 33342 with the addition of 0.1 µM propidium iodide to visualize necrotic cells.
Cells were also labeled with either FITC-annexin V conjugate
(PharMingen) or with the FAM-VAD-FMK reagent provided in the
CaspaTagTM fluorescein caspase activity kit (Intergen).
Labeled cells were detected by indirect fluorescence microscopy (all
cells) or by FACS analysis on a Coulter Epics flow cytometer.
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RESULTS |
Interaction of HDAC1 and DNA Topoisomerase II--
HeLa whole cell
extract was immunoprecipitated with affinity-purified antibody against
mammalian HDAC1 and precipitated material tested for the presence of
topo II
by Western blotting (Fig. 1A). Anti-HDAC1 brought down
easily detectable amounts of topo II
. There was no detectable
immunoprecipitation of topo II
with preimmune serum, and
immunoprecipitation was completely abolished by inclusion in the
incubation mix of the peptide used to raise the anti-HDAC1 antibody
(Fig. 1A). The anti-HDAC1 antibody did not immunoprecipitate
detectable levels of topo II
(data not shown), and antibody to topo
II
brought down only a comparatively small amount of the
isoform
(Fig. 1A). However, antisera against both topo II
and
topo II
immunoprecipitate 6-9% of total deacetylase activity from
HeLa whole cell extract (Fig. 1B). The activity is fully
inhibited by TSA. Negative control immunoprecipitations with preimmune
serum, an irrelevant antibody (anti-CDK7), or an antibody against DNA
topoisomerase I (topo I) did not bring down activity above that of the
no-antibody control.

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Fig. 1.
Evidence for the physical association of topo
II and HDAC1 proteins. A,
Immunoprecipitation of endogenous topo II (molecular mass,
170 kDa), with the indicated antibodies (10 µg of total IgG), from
100 µg of HeLa whole cell extract (WCE). B,
immunoprecipitation of deacetylase activity by antisera against topo
II and topo II (18511 and 18513 ) from HeLa WCE. Deacetylase
activity is inhibited by TSA (10 ng/ml). Activity immunoprecipitated is
expressed as a percentage of total input deacetylase activity, less a
background of nonenzymatic release of [3H]acetate of
1.94%. Percentages are averages of three separate experiments, with
bars representing S.D.
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We performed in vitro pull down experiments of endogenous
protein with GST fusion proteins. Full-length mammalian HDAC1, tagged with a GST moiety, but not GST itself, bound endogenous topo II
in
whole cell extract (Fig. 2A).
In the converse experiment, a GST fusion protein containing the
C-terminal domain (CTD) of topo II
was able to pull down endogenous
HDAC1 (Fig. 2B). The fusion protein of the CTD of topo II
was also able to pull down small amounts of HDAC1 (Fig. 2B).
Fusion proteins of the CTD of topo II
and topo II
were able to
pull down between 9 and 11% of deacetylase activity (Fig.
2C), comparable with the amounts brought down by immunoprecipitation (Fig. 1B). Whereas GST-topo II
pulls
down less HDAC1, as detected on Western blots, than comparable amounts of GST-topo II
(Fig. 2B), the two different fusion
proteins bring down similar amounts of deacetylase activity (Fig.
2C). A possible explanation for this quantitative
discrepancy is that other deacetylases, in addition to HDAC1, are
preferentially associated with topo II
.

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Fig. 2.
Pull down experiments of endogenous topo II
and HDAC1. A, pull down of endogenous topo II by 5 µg of immobilized (bound) GST-HDAC1, but not by
immobilized GST. Topo II is also depleted from the supernatant
(unbound) by GST-HDAC1. B, pull down of
endogenous HDAC1 (molecular mass, 62 kDa) by immobilized
GST-topo II and topo II , containing the CTD of each isoform.
C, pull down of deacetylase activity with immobilized
GST-topo II CTD and topo II CTD. Bars represent ranges
of two experiments.
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GST fusion proteins containing the
C-terminal domain of HDAC1 interact with recombinant topo II
(Fig.
3). This domain has previously been shown to contain the LXCXE motif
(residues 414-418), that appears to mediate interactions with the
retinoblastoma protein pRb (19). In contrast, an N-terminal HDAC1
fusion protein, containing the catalytic site, showed minimal
interaction with recombinant topo II
(Fig. 3).

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Fig. 3.
In vitro interaction of
recombinant topo II and HDAC1. Regions of
HDAC1 cDNA were subcloned and expressed as GST fusion proteins.
Immobilized fusion proteins (5 µg) were tested for interaction with 1 µg of recombinant topo II .
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A yeast two-hybrid system (18) was used to test for direct in
vivo interaction between topo II and HDAC1. Inserts were
constructed to express the topo II
and topo II
C-terminal domains
(6) and the HDAC1 region 220-482 (Fig. 3). Expression of the
integrated, GAL4-dependent HIS3 reporter gene
was used to detect interactions between "bait" and "prey"
proteins in vivo. Topo II
CTD or topo II
CTD as bait,
together with HDAC1 as prey, allowed growth of large colonies (over 2 mm diameter) on His-selective medium. All three proteins were
ineffective when expressed individually (Fig. 4).

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Fig. 4.
Yeast two-hybrid assay showing that the CTDs
of topo II and topo II
can interact with HDAC1 CTD (amino acids 220-482) in
vivo. Expression of the reporter gene HIS3
in yeast stain CG-1945 (CLONTECH) was determined by
two parallel series of spot assays on selective medium plates lacking
tryptophan, leucine, and histidine ( Trp, Leu,
His), but in the presence of 25 mM
3-amino-1,2,4-triazole to suppress background growth (6, 18). Colony
size was compared with that on plates lacking tryptophan and leucine
( Trp Leu) as control. Strong growth in His medium
occurs only in cells in which the bait and prey proteins physically
interact.
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To explore the biological significance of the topo II-HDAC1
interaction, we tested the ability of full-length recombinant HDAC1 to
modulate the functional properties of recombinant topo II
and -
.
Both of these enzymes can decatenate kinetoplast DNA (kDNA) to
minicircle monomers, a process that requires a double-stranded break in
the kDNA to allow strand passage. The addition of increasing amounts of
HDAC1 to the reaction decreases the decatenation of kDNA by topo II
and -
(Fig. 5). Addition of GST alone
did not affect decatenation by either topo II
and -
.

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Fig. 5.
Functional association of topo II and histone
deacetylase. Decatentation of 1.4 µg of kinetoplast DNA
concatemer (concat.) to monomers of minicircle DNA by
recombinant topo II (0.60 pmol) and topo II (0.22 pmol) is
inhibited by increasing amounts of GST-HDAC1 (1.4-13.9 pmol), but not
by GST alone (4.2-13.9 pmol).
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Suppression of Etoposide-mediated Apoptosis by the HDAC
Inhibitor Trichostatin A--
We tested the effect of the HDAC
inhibitor trichostatin A (20) on apoptosis induced by the
chemotherapeutic agent etoposide (VP-16). Etoposide causes
topoisomerase II-mediated DNA damage by increasing the steady-state
concentration of covalent DNA cleavage complexes (1, 2, 4). Cells
treated with etoposide acquire an apoptotic morphology, notably the
condensation of chromatin at the nuclear periphery and blebbing of the
plasma membrane (2, 21). HL-60 cells displayed apoptotic chromatin
condensation after only 1.5-h treatment with either 100 µM etoposide or 5.8 µM camptothecin, an
inhibitor of topo I (Fig. 6A).
Plasma membrane changes during early apoptosis include the exposure of
phosphatidylserine to the external cellular environment (22). This
change was measured by binding of FITC-conjungated annexin V and
counting of labeled cells by fluorescence microscopy (Fig.
6B). Activation of cysteine aspartyl proteases (caspases)
(21) during the apoptosis of HL-60 cells was assayed with a fluorescent
substrate and FACS analysis of viable cells (Fig. 6C).
Chromatin condensation, membrane changes, and caspase activation all
demonstrated that prior treatment with 100 nM TSA
suppresses the apoptotic effect of etoposide (Fig. 6, A-C).
In contrast, TSA did not affect apoptosis induced by the topo I
inhibitor camptothecin (Fig. 6, A-C). Note that topo I does
not associate with detectable amounts of deacetylase activity (Fig.
1B). An identical anti-apoptotic effect of TSA treatment was
also observed for the human lung adenocarcinoma cell line H1299 and
HeLa cells (data not shown).

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Fig. 6.
TSA suppresses apoptosis induced by topo
II-mediated DNA damage, but not by topo I inhibition, in HL-60
cells. A, cells were treated as indicated (see
"Materials and Methods" for details). Nuclear changes were
visualized by indirect fluorescence microscopy after staining with
Hoechst 33342 with the addition of propidium iodide (PI) to
visualize necrotic cells. The nuclear morphology of cells were scored
for apoptotic cells (PI ; clear bars) and
necrotic cells (PI+; gray bars) and expressed as
percentages of total cells in the field of view. Values are averages of
two separate experiments. B, plasma membrane changes were
detected by the binding of annexin V conjugated to FITC, after
treatment with TSA, etoposide, or camptothecin as indicated (see
"Materials and Methods"). Staining was visualized by indirect
fluorescence microscopy and expressed as percentages of total cells.
Bars represent S.D. values for three separate experiments.
C, broad-spectrum caspase activity was detected by labeling
live cells with the FAM-VAD-FMK fluorescein-conjugated caspase
substrate (see "Materials and Methods") after treatment with
inhibitors, as indicated. Labeled cells were detected by FACS analysis,
and activity is expressed as the percentage of the viable cell
subpopulation that stains positive for the substrate. Values are
averages of two separate experiments.
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DISCUSSION |
The results presented show that the histone deacetylase HDAC1 is
physically associated with each of the two isoforms of human topoisomerase II, topo II
and topo II
. The association occurs in vivo, being detectable by coimmunoprecipitation from
human cell extracts and by yeast two-hybrid assay. It also occurs
in vitro. GST-coupled recombinant topo II
and topo II
pull down significant amounts of HDAC activity from cell extracts,
while recombinant HDAC1 inhibits the in vitro decatenation
activity of recombinant topo II
. Since completion of the work
reported here, Tsai et al. (23) have reported essentially
the same findings for the two very similar deacetylases HDAC1 and
HDAC2. Interestingly, whereas Tsai et al. (23) find evidence
for an interaction between topo II
and various regions of HDAC2,
including N-terminal residues 1-57, we find that only the C-terminal
region of HDAC1 (residues 220-482) interacts with topo II in
vitro. These two deacetylases seem to differ in their mode of
interaction with topo II.
In experiments to assess the biological significance of the topo
II-HDAC interaction, we analyzed the effect of the deacetylase inhibitor TSA on processes known to require topo II activity. The most
striking effect so far has been on the ability of the topo II poison
etoposide to drive cells into apoptosis. We show that treatment
with TSA prior to the addition of etoposide suppresses apoptosis in a
variety of cell lines. The effect is seen even with HL60 cells, in
which apoptosis is detectable within less than 1 h, a finding that
minimizes the probability that inhibition of apoptosis is due to
pleiotropic effects of TSA, such as its ability to alter cell cycle
progression. The inhibitory effect of TSA was detected in several
p53-null cell lines, so the interaction between HDAC1 and p53 (7)
cannot be responsible. Microscopically detectable chromatin remodeling
is a diagnostic characteristic of cells in the later stages of
apoptosis, and recent reports indicate that both topo II and histone
acetylation play a role in this process (24). However, our results
indicate that this is not the stage at which TSA exerts the inhibitory
effect reported here. We have shown that TSA inhibition is detectable
even when using an assay that measures one of the earliest changes of
apoptosis, namely the alteration in membrane phospholipids detected by
binding of annexin V (22). These findings argue that TSA is acting at a
relatively early stage in apoptosis, prior to the onset of major changes in nuclear ultrastructure. It remains possible that TSA also
effects more subtle chromatin changes, possibly those determining expression of genes required for progression through apoptosis (25).
These effects are not mutually exclusive. Indeed, recent results
indicate that both topo II and changes in acetylation act at various
stages in the pathways by which cells progress through apoptosis (24,
26-28).
In attempting to explain the effect of TSA on etoposide-induced
apoptosis, it is important to note that etoposide is a topo II poison
that blocks the enzyme after DNA cleavage but prior to strand passage
(4). Covalent topoisomerase-DNA cleavage complexes accumulate in the
presence of such poisons. DNA replication, transcription, or helicase
activity all disrupt these complexes, releasing the DNA double-strand
breaks that can precipitate apoptosis (1, 4). Reducing either the
accumulation of topo II-DNA complexes, or their breakdown, will both
reduce DNA damage and hence apoptosis. A possible explanation for the
results presented here is that HDAC-dependent chromatin
remodeling is necessary for the initiation of topo II-catalyzed DNA
rearrangement or for dissociation of topo II-DNA complexes, or both. If
this were the case, then HDAC inhibitors such as TSA would be expected
to prevent the appearance of DNA damage in the presence of topo II
poisons, and consequent progression into apoptosis, exactly as we have found. Crucially, TSA has no effect on apoptosis induced by the topo I
inhibitor camptothecin. We show here that topo I is not associated with
HDAC1. Further support for these ideas comes from the recent results of
Tsai et al. (23), who show that topo II is associated not
only with HDAC1/2, but also with MTA2, a protein that is part of the
NuRD chromatin remodeling complex. The NuRD complex contains both
HDAC1/2 and Mi-2, a protein with ATPase/helicase activity (12).
We and others (23) find that HDAC1 can inhibit the catalytic activity
of topo II in vitro. This is an important confirmation of
the ability of topo II and HDAC1 to interact, but is not, at first
sight, consistent with the proposition outlined above that HDAC
activity facilitates topo II catalysis, or its consequences. This can
be resolved by noting that the GST-HDAC1 construct used to inhibit topo
II in vitro is catalytically inactive, presumably because it lacks essential protein partners such as RbAp46/48 (8, 11).
It would be wrong to assume that catalytically active HDAC1,
in the context of a multiprotein complex that includes both HDAC and
chromatin remodeling activities (23), is also inhibitory. It might even
be the case that, in vivo, topo II activity is inhibited
only by association with HDAC rendered catalytically inactive by
inhibitors such as TSA. Such an effect would complement the suppression
of topo II activity brought about by inhibition of chromatin remodeling.