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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1407-1417
Temporal and Spatial Distribution of DNA Topoisomerase II Alters
During Proliferation, Differentiation, and Apoptosis in HL-60 Cells
By
Koichi Sugimoto,
Konagi Yamada,
Motoki Egashira,
Yoshio Yazaki,
Hisamaru Hirai,
Akihiko Kikuchi, and
Kazuo Oshimi
From the Department of Hematology, Juntendo University School of
Medicine, Tokyo, Japan; the Third Department of Internal Medicine,
Faculty of Medicine, University of Tokyo, Tokyo, Japan; and the
Laboratory of Medical Mycology, Research Institute of Disease Mechanism
and Control, Nagoya University School of Medicine, Nagoya, Japan.
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ABSTRACT |
We related cellular content of DNA topoisomerase (topo) II and
II with the cell cycle position in proliferating, differentiated, and apoptotic HL-60 cells using two-dimensional flow cytometry. In
logarithmically growing HL-60 cells, topo II increased especially in
late S to G2/M phases, although the topo II level was almost constant throughout the cell cycle. Induction of differentiation by
all-trans retinoic acid dramatically reduced the topo II but not the topo II level. A new G2/M population containing virtually no
topo II appeared during differentiation and was supposed to be alive
and noncycling. Two-dimensional flow cytometry of topo II or II
staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin
nick end-labeling assay showed that one topo II epitope situated at
the C-terminal end decreased specifically in apoptotic HL-60 cells
treated with Ara-C, etoposide, and vincristine. The amounts of a topo
II epitope and another topo II epitope located at a more central
portion were almost equal between apoptotic and nonapoptotic cells.
Western blot analysis confirmed that topo II protein was completely
degraded into smaller fragments and lost its C-terminal end during
apoptosis. On the contrary, a large portion of topo II remained of
its original size, although both topo II and II left from the
nuclear fraction in apoptotic cells. Confocal laser microscopy showed
nuclear localization of topo II and II in growing HL-60 cells.
Although topo II and II were distributed throughout the cell
during mitosis, only topo II was densely concentrated in the mitotic
chromosomes. Both enzymes were dissociated from the genomic DNA even at
an early phase of apoptosis and completely separated from the propidium iodide signal of DNA in the advanced stage. Chromatin condensation process in apoptosis is therefore completely topo II-independent and
obviously differs from the mitotic one.
| |
INTRODUCTION |
DNA TOPOISOMERASE II (topo II) catalyzes
the local changes in DNA topology by passing a double-stranded DNA
helix through a transient double-strand break site and then rejoining
the strand break.1,2 Conditional yeast mutants in the
top2 gene showed that this enzymatic activity is required for
segregation of daughter chromosomes during anaphase.3
Biochemical studies using Xenopus egg extracts showed that topo
II is essential for the condensation of interphase chromatin into
metaphase chromosomes.4 Treatment of mammalian cells with
ICRF-193, which inhibits topo II activity without causing DNA damage,
also leads to incomplete chromosomal condensation and segregation,
resulting in polyploidity.5 Topo II is the direct target of
certain classes of antitumor agents. Etoposide and doxorubicin interact
with topo II to inhibit the religation step of the enzyme, thereby
stabilizing cleavable enzyme-DNA complexes that lead to DNA
double-strand breaks and eventually to cell death.6 Gene
rearrangement in the MLL gene at chromosome 11q23 is frequently
observed in chemotherapy-associated leukemias.7,8 The break
cluster region in this gene has been shown to coincide with the DNA
cleavage sites specifically induced by topo II inhibitors in
vivo.9,10 Although only one topo II is known in yeasts and
Drosophila, two isozymes of topo II have been identified in mammalian cells.1,11,12 These two isozymes, topo II
(170-kD form) and topo II (180-kD form), with striking similarities
in their amino acid sequences, are encoded by different genes. The topo
II staining showed fine punctuate fluorescence all over the nucleus
except the nucleolar domain.13,14 Although topo II was
considered to exist preferentially in the nucleoli,14,15 a
recent report has shown that topo II is completely excluded from
nucleoli.16 The cellular concentration of topo II but not topo II was reported to correlate with mitotic
activity.17-19 A decrease in cellular content of topo II
was previously reported during differentiation and E1A-induced
apoptosis.20-22
Apoptosis is a distinct form of cell death that occurs in response to
various stimuli, including DNA damage, withdrawal of growth factors,
and inappropriate expression of genes that stimulate cell cycle
progression.23,24 Apoptosis begins with condensation of
nuclear chromatin at the nuclear periphery followed by blebbing of the
nuclear and cytoplasmic membranes and culminates in the fragmentation
of residual nuclear structures into discrete apoptotic bodies.23-25 Although the regulation of apoptosis is
complex, substantial evidence indicates that
interleukin-1-converting enzyme (ICE)-like proteases
play a central role in this process.26,27 Several nuclear
proteins essential for DNA metabolism are specifically degraded by the
action of the ICE-like proteases during apoptosis. These include poly
(ADP-ribose) polymerase (PARP), nuclear lamins, DNA-dependent protein
kinase catalytic subunit (DNA-PK cs), DNA topo I and II, NuMA, and RNA
polymerase I upstream binding factor UBF.28-32
In this study, we showed a dramatic increase of topo II but not topo
II content in late S to G2/M phases in logarithmically growing HL-60
cells using two-dimensional flow cytometry. During differentiation, the
majority of the HL-60 cells were confined to G1/G0 position and
simultaneously a new cell population emerged that contained tetraploid
DNA and almost no topo II protein. Two-dimensional flow cytometry
combining topo II or II staining with terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) assay
suggested a decrease of a C-terminal but not a more central epitope of
topo II in apoptotic HL-60 cells. Western blot analysis and
immunostaining showed that both topo II and II were rapidly
dissociated from the chromatin in apoptotic HL-60 cells, although only
topo II was extensively degraded during apoptosis.
| |
MATERIALS AND METHODS |
Monoclonal antibodies.
Preparations of topo II-specific antibody 8D2 and topo
II-specific antibodies 5A7 and 3G3 were described
previously.22,33 The epitope of 8D2 exists between amino
acids 1260 and 1460 of topo II. The epitopes of 5A7 and 3G3 are
located in amino acids 1583 to 1601 and between amino acids 1260 and
1460 of topo II, respectively.
Cell culture and drug treatment.
The HL-60 human myeloid leukemia cell line was maintained in RPMI 1640 (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal calf serum,
100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L
L-glutamine. The cells were split to keep the cell density
at 2 × 105 to 1 × 106 cells/mL.
To induce cell differentiation, HL-60 cells were treated with 1 µmol/L of all-trans retinoic acid (ATRA; Sigma, St Louis, MO)
for 6 days. Cell density was kept at 2 × 105 to 1 × 106 cells/mL during the treatment. Logarithmically growing
HL-60 cells were treated for the indicated times with cytosine
b-D-arabinofuranose (Ara-C; 4 µmol/L), etoposide (100 µmol/L), or vincristine (0.2 µmol/L) (all reagents were purchased
from Sigma).
Cell fixation.
In brief, 1 × 106 cells were harvested by centrifugation
for 8 minutes at room temperature at 400g, washed once with
phosphate-buffered saline (PBS), and then fixed in 1% formaldehyde in
PBS (pH 7.4) for 15 minutes on ice. After washing in PBS, cells were
resuspended in 70% cold ( 20°C) ethanol and immediately
transferred to the freezer. The cells were stored at 20°C for 1 day before being subject to the indirect immunofluorescence or TUNEL
assay.
Indirect immunofluorescence.
Cells were washed twice in PBS, incubated in 100 µL of PBS containing
0.1% Triton X-100 for 5 minutes at room temperature, and blocked in
100 µL of PBS containing 3% (wt/vol) nonfat dry milk for 30 minutes
at room temperature. To detect topo II and II, cells were
incubated with a 1:30 dilution of 8D2 and 5A7, respectively, in PBS
with 3% nonfat milk for 1.5 hours at room temperature. In some cases,
3G3 was used to detect topo II instead of 5A7. Cells were washed
twice in PBS containing 0.1% Triton X-100 and then incubated in a 1:30
dilution of a fluorescein isothiocyanate (FITC)-conjugated
goat-antimouse IgG (Ortho, Raritan, NJ) in PBS/3% milk solution for 1 hour at room temperature in the dark.
TUNEL assay.
After rehydration in PBS, cells were resuspended in 50 µL of a
cacodylate buffer containing 0.2 mol/L potassium cacodylate, 25 mmol/L
Tris-HCl (pH 6.6), 2.5 mmol/L CoCl2, 0.25 mg/mL bovine serum albumin, 5 U TdT, and 0.5 nmol of biotin-dUTP (all reagents were
purchased from Boehringer Mannheim, Indianapolis, IN). The cells were
incubated in this solution at 37°C for 30 minutes; rinsed in PBS;
resuspended in 100 µL of a solution containing 4× concentrated
saline-sodium citrate buffer, 2.5 µg/mL fluoresceinated avidin
(Boehringer Mannheim), 0.1% Triton X-100, and 5% (wt/vol) nonfat dry
milk; and incubated in this solution for 30 minutes at room temperature
in the dark. This procedure essentially followed the previous report by
Gorczyca et al.34
Flow cytometry.
After incubation in staining buffer, the cells were rinsed in PBS
containing 0.1% Triton X-100 and resuspended in 1 mL of PBS containing
5 µg/mL of propidium iodide (PI) and 200 µg/mL of RNase A (both
from Sigma). Flow cytometry was performed on a CYTRON ABSOLUTE flow
cytometer (Ortho). The orange (PI) and green (fluorescein
isothiocyanate [FITC]) fluorescence emissions from each cell were
separated and measured using the standard optics of the CYTRON
ABSOLUTE. The data from 5 × 104 cells were collected,
stored, and analyzed. The signal of green fluorescence was measured
using linear amplification for topo II and II staining and using
logarithmic amplification for the TUNEL assay.
Two-dimensional flow cytometry of immunostaining and TUNEL assay.
After performing the TUNEL assay protocol described above, cells were
rinsed twice in PBS containing 0.1% Triton X-100 and then resuspended
in PBS/3% milk solution containing the primary antibody, 8D2 or 5A7.
Thereafter, cells were stained with the same procedure for indirect
immunostaining except that 1:50 dilution of phycoerythrin
(PE)-conjugated goat-antimouse IgG (BioSource, Camarillo, CA) was used
as a secondary antibody and that PI staining at the final step was
omitted. In this case, topo II and II signals of orange
fluorescence were measured using linear amplification and the green
fluorescence of TUNEL assay using logarithmic amplification.
Western blot analysis.
Harvested cells were washed once with PBS and suspended in ice-cold
buffer 1 (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol [DTT], 0.05% Triton X-100, and 1 mmol/L
phenylmethylsulfonyl fluoride [PMSF]) at the
concentration of 2 × 107 cells/mL. The suspension was
kept on ice for 20 minutes, vortexed vigorously for 10 seconds to be
lysed, and then spun down at 1,000g for 4 minutes at 4°C. The
supernatant was recovered as a cytoplasmic fraction. The nuclear pellet
was resuspended in the same volume of ice-cold nuclear extraction
buffer 2 (20 mmol/L HEPES, pH 7.9, 400 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT, and 1 mmol/L PMSF), rocked on ice for 30 minutes, and
centrifuged at 13,000g for 10 minutes at 4°C. The supernatant
was recovered as a nuclear fraction. In every experiment in this study,
the nuclear remnant was confirmed to contain essentially no topo II
proteins by immunoblotting. Cytoplasmic and nuclear fractions derived
from 5 × 105 cells were separated on a 7% polyacrylamide
gel. Immunoblotting was performed as described
previously,35 using a 1:200 dilution of 8D2, 5A7, and 3G3.
As a second antibody, alkaline phosphatase-conjugated antimouse Ig
(ProMega, Madison, WI) was used at the dilution of 1:5,000.
Confocal laser microscopy.
The cells immunostained for flow cytometric analysis were rinsed in PBS
containing 0.1% Triton X-100. An aliquot of the cells was resuspended
in 200 µL of PBS containing 200 ng/mL of PI and 200 µg/mL of RNase
A (both from Sigma), resuspended in 100 µL of PBS, and then attached
to a poly-L-lysine coated slide glass. The coverslip was
mounted with 10 µL of antifading mix (50% glycerol, 2.5%
1,4-diazabicyclo[2.2.2]octane [DABCO] in PBS) and sealed with nail
polish. The slides were viewed and photographed through a Bio-Rad
MRC-1024 confocal laser scanning microscope (Bio-Rad, Hercules,
CA).
| |
RESULTS |
The cellular contents of topoisomerase II and II were studied
using monoclonal antibodies 8D2 and 5A7. Specificities of 8D2 to topo
II and 5A7 to topo II were shown previously.22,23 Two-dimensional flow cytometric analysis of cells indirectly
fluorescein-labeled for topo II or II and then counterstained
with PI made it possible to quantify the amounts of topo II or II
and relate them to cellular DNA content, ie, to the cell cycle
position. The DNA content histogram of logarithmically growing HL-60
cells contains two peaks: a large and sharp peak at G1 and the other
small one at G2/M (Fig 1A). S phase cells
distribute between these peaks forming a bridge shape. The cellular
concentration of topo II increases during the cell cycle progression
and a steep increase is prominent from late S to G2/M phases (Fig 1B).
In G1 phase, topo II content varies from almost zero to somewhat
larger than that of early S phase. On the contrary, topo II level
slightly increases in G1 phase and thereafter is not significantly
altered through the cell cycle (Fig 1C). Although the topo II signal appears to increase even during the S phase, subtraction of the nonspecific binding fluorescence of an isotype-matched control antibody
indicates that the topo II content is almost constant. When we
compare the single parameter histograms of the two topo II enzymes, the
range of distribution for topo II signal was much wider than that of
topo II, although these histograms peak at almost the same signal
intensity (Fig 1D and E). These results were representative of five
similar experiments.
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| Fig 1.
Alterations in topo II and II levels in
logarithmically growing HL-60 cells as a function of DNA content, ie,
the cell cycle position. (A) DNA histogram showing the cell cycle
distribution of logarithmically growing HL-60 cells. (B and C)
Two-dimensional flow cytometric analyses of DNA content and topo II
and II signals, respectively. Isotype-matched negative controls are
depicted as contour maps. (D and E) Histograms of topo II and II
contents, respectively. Isotype-matched control fluorescence curves are the most proximal to the Y-axis.
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Two-dimensional flow cytometric analysis on differentiating HL-60 cells
showed alterations in the cell cycle distribution and changes in topo
II and II levels at each cell cycle position. We induced
differentiation of HL-60 cells with the addition of 1 µmol/L of ATRA
to the culture medium for 6 days. More than 90% of the cells were
confirmed to express CD11b on the cell surface by day 4 (data not
shown). During differentiation, the cell population belonging to S and
G2/M phases gradually decreased, and only a small portion of the cells
were found in S phase at day 6 (Fig 2A through
D). By day 2, the amount of topo II as a
function of cell cycle position showed similar pattern to that of the
nontreated cells, although the topo II signal decreased slightly
(Fig 2E and F). At day 4, a large portion of the cells were confined to G1 and a considerable part of these G1 cells no longer expressed topo
II enzyme (Fig 2C and G). A new cell population that belongs to G2/M
phase and simultaneously contains almost no topo II appeared at this
stage, and this cell group became more prominent at day 6 (Fig 2G and
H). Because a portion of the G0/G1 cells had a relatively high level of
topo II signal, as shown in Fig 2H, if the G2/M population were
constituted of the clumped G0/G1 cells, some cells of this population
should also contain a high level of topo II signal. Actually, even
when we increased the detection gain or the numbers of cells analyzed,
the G2/M population in Fig 2H showed no upward tail, which corresponds
to a cell group containing a rather high level of topo II signal.
Furthermore, we clearly detected this G2/M cell population by
two-dimensional flow cytometry still after the gating to eliminate the
clumped G0/G1 cells. In contrast with topo II, the signal of topo
II decreased a little at day 2 and essentially kept this level until
day 6 (Fig 2I through L). At days 4 and 6, there appeared a sub-G1
population that contained almost no topo II (Fig 2K, L, S, and T).
The results of TUNEL assay suggested that these cells were apoptotic
(data not shown), which agrees with the results described below showing
that 5A7 epitope of topo II specifically decreases during apoptosis.
The single-parameter histograms confirmed that topo II level
decreased steeply and the peak shifted to the position of almost no
topo II signal at day 4 (Fig 2M through P). Topo II level was not so much altered during differentiation (Fig 2Q through T). Similar results were observed in three independent studies.
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| Fig 2.
Topo II level decreases dramatically and a new G2/M
cell population expressing almost no topo II emerges during the
ATRA-induced differentiation. Logarithmically growing HL-60 cells are
treated with 1 µmol/L of ATRA, before the treatment (A, E, I, M, and
Q), for 2 days (B, F, J, N, and R), for 4 days (C, G, K, O, and S), and
for 6 days (D, H, L, P, and T). DNA histograms with insets showing the
percentage of cells in each phase of the cell cycle (A through D).
Two-dimensional flow cytometric analyses of DNA contents and topo II
and II signals (E through H and I through L, respectively).
Histograms of topo II and II contents (M through P and Q through
T, respectively).
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We next examined topo II and II levels and related them with the
cell cycle position in apoptotic HL-60 cells treated with antitumor
drugs. The extent of DNA strand breaks, one of the hallmarks of
apoptosis, was also correlated to the cell cycle position using the
TUNEL assay combined with PI staining. We used three antitumor drugs
with different mechanisms of action: pyrimidine analogue antimetabolite
Ara-C, topo II inhibitor etoposide, and vinca alkaloid antimitotic
agent vincristine.36 Based on the results of previous reports,31,37 we experimentally determined the doses of
Ara-C (4 µmol/L) and etoposide (100 µmol/L) that induce apoptosis
in 50% to 80% of rapidly growing HL-60 cells in 6 to 8 hours (data not shown). Measured as an apoptotic cell percentage, 0.05 µmol/L of
vincristine had essentially the same effect as that of 4 µmol/L (data
not shown). We therefore treated HL-60 cells with 0.2 µmol/L of
vincristine. At this concentration, it took about 18 hours to induce
apoptosis in more than 50% of the treated cells.
With the Ara-C treatment, the G2/M peak disappeared and a small peak at
sub-G1 position emerged (Fig 3B). As a function of the cell cycle
position, the topo II level decreased a little in these cells (Fig
3F). On the contrary, Ara-C-treated cells were divided into two populations with nearly normal and very small
topo II contents (Fig 3J). TUNEL-positive cells were distributed in
sub-G1 to S phases, suggesting a partial loss of DNA stainability in
apoptotic cells (Fig 3N). Most of the nonapoptotic cells were restricted in G1 phase. Comparison between Fig 3J and N suggests a
possibility that the topo II signal should decrease specifically in
apoptotic cells.
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| Fig 3.
Antitumor drugs alter the cell cycle distribution and
topo II and II contents of HL-60 cells. DNA histograms (A through D), two-dimensional flow cytometric analyses of DNA-topo II contents (E through H), DNA-topo II contents (I through L), and DNA
content-TUNEL assay (M through P) of control and Ara-C-, etoposide-,
and vincristine-treated HL-60 cells.
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Etoposide-treated cells also lost the G2/M population and the G1 peak
had a broader shoulder at sub-G1 side (Fig 3C). The topo II level
was somewhat decreased at any position in the cell cycle (Fig 3G). Only
part of the G1 cells contained a normal amount of topo II and all of
the remaining cells had a decreased topo II signal (Fig 3K). TUNEL
assay showed that only a portion of G1 cells were free from apoptosis
(Fig 3O). Therefore, a decrease of the topo II signal also seemed to
correlate with apoptosis in etoposide-treated HL-60 cells.
Treatment with vincristine confined HL-60 cells to late S and G2/M
phases (Fig 3D). As a result, most of the cells expressed a higher
level of topo II than normal control (Fig 3H). As for topo II,
these cells were divided into two populations with intact and decreased
enzyme levels (Fig 3L). Both apoptotic and nonapoptotic cells were in
late S to G2/M phases (Fig 3P).
To address the possible relationship between topo II level and
apoptosis more directly, cells were labeled by the TUNEL assay, stained
for topo II or topo II, and then analyzed by two-dimensional flow
cytometry. In Ara-C-treated HL-60 cells, a large portion of the
apoptotic cells contained approximately the same amount of topo II
as the nonapoptotic ones (Fig 4B). On the
contrary, the apoptotic cells apparently contained less topo II,
with apoptotic and nonapoptotic populations essentially nonoverlapping
as for the topo II level (Fig 4F). When we used etoposide, the
apoptotic and nonapoptotic HL-60 cells expressed almost equal amounts
of topo II (Fig 4C). However, the topo II level clearly separated these two populations (Fig 4G). Also, in vincristine-treated cells, although the apoptotic and nonapoptotic cells contained a similar level
of topo II, they differed sharply in their content of topo II
(Fig 4D and H). Every result shown in Fig 3 and 4 was reproducively obtained in at least three separate experiments. Because the three antitumor drugs used in this study have apparently different mechanisms of action, these observations indicate that a decrease in the topo
II level is not a drug-specific event but a more general phenomenon
accompanying the drug-induced apoptosis.
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| Fig 4.
Topo II but not topo II signal decreases
specifically in apoptotic HL-60 cells treated with antitumor drugs.
Two-dimensional flow cytometric analyses of topo II and II
contents and TUNEL assay (A through D and E through H, respectively).
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Because we used the monoclonal antibody 5A7 specific to the C-terminal
portion of topo II (amino acids 1583 to 1601) in the experiments
described above, we could not distinguish the two possibilities that
the full molecule or only the N-terminal portion of topo II was lost
during apoptosis. To investigate this question, we determined the topo
II level of the Ara-C-treated HL-60 cells using a monoclonal
antibody 3G3, which recognizes a more central epitope of topo II
(between amino acids 1260 and 1460) than that of 5A7. Flow cytometry
using 5A7 as a primary antibody clearly separated Ara-C-treated HL-60
cells into two populations with almost normal and decreased levels of
topo II signal as shown above (Fig 5B).On the contrary, 3G3 did not discriminate between the apoptotic and
nonapoptotic cells, both of which showed an almost normal level of topo
II signal (Fig 5D). Similar results were obtained in three
independent experiments. Etoposide-treated HL-60 cells also divided
into apoptotic and nonapoptotic populations by 5A7 but not by 3G3 (data
not shown). These results indicate that the 5A7 epitope at the
C-terminal portion of topo II should be degraded or modified during
apoptosis, although a more central 3G3 epitope of topo II was
preserved.
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| Fig 5.
5A7 but not 3G3 separates apoptotic HL-60 cells from
nonapoptotic ones. Two-dimensional flow cytometric analyses of DNA-topo II contents are performed on logarithmically growing (A and C) and
Ara-C-treated HL-60 cells (B and D) using topo II-specific monoclonal antibodies, 5A7 (A and B) and 3G3 (C and D).
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We then investigated possible cleavages of topo II and II during
apoptosis by Western blot analysis using 8D2 and 5A7/3G3, respectively.
The flow cytometric TUNEL analysis showed that approximately 50% of
HL-60 cells underwent apoptosis after 5 hours of incubation with 4 µmol/L of Ara-C (data not shown). In 10 hours, more than 95% of the
treated cells were positive for the TUNEL assay (data not shown). We
separated rapidly growing and Ara-C-treated HL-60 cells into
cytoplasmic and nuclear fractions with 0.05% Triton X-100. Each
fraction was then subjected to immunoblotting. In logarithmically
growing HL-60 cells, both topo II and II were detected at their
expected size (170 kD and 180 kD, respectively) exclusively in the
nuclear fraction (0 hours; Fig 6A, B, and
C). Although distribution of topo II was
completely changed from the nuclear to cytoplasmic fractions during the
course of apoptosis, a large portion of topo II remained of its
original size even in apoptotic cells (Fig 6A). Only a small amount of
degraded topo II fragments were detected in the cytoplasmic fraction
of the apoptotic cells. When we used 5A7 to detect topo II, the
180-kD band in the nuclear fraction became faint in 5 hours and no
bands were detected in either the cytoplasmic or nuclear
fraction after 10 hours of incubation (Fig 6B). Another topo
II-specific antibody 3G3 showed the appearance of several smaller
fragments of 125 to 160 kD in the cytoplasmic fraction besides a
proportional reduction in the amount of the 180-kD band in the nuclear
fraction after 5 hours of Ara-C treatment (0 and 5 hours; Fig 6C). The
smaller fragments in the cytoplasmic fraction became more prominent and the intact 180-kD band disappeared in 10 hours (10 hours; Fig 6C),
indicating that topo II was completely degraded into these smaller
fragments. Essentially the same results were obtained in three
independent experiments and also in etoposide-treated cells with a
slightly shorter time course (about 7 to 8 hours for complete
apoptosis; data not shown). These results confirmed that the C-terminal
5A7 epitope is lost and a more central 3G3 epitope is preserved in the
apoptotically degraded topo II fragments. The Western blot analysis
thus shows that a large portion of topo II remains of its original
size even in apoptotic HL-60 cells, although intact topo II is lost
at an early phase of apoptosis.
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| Fig 6.
Topo II but not topo II is extensively degraded
during the Ara-C-induced apoptosis. Western blot analyses of
logarithmically growing HL-60 cells (0 hours) and those treated with 4 µmol/L of Ara-C for 5 and 10 hours (5 and 10 hr, respectively) with
topo II-specific 8D2 (A) and topo II-specific 5A7 and 3G3
monoclonal antibodies (B and C, respectively).
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Topo II and II (fragments) moved completely from the nuclear to
cytoplasmic fractions in apoptotic cells. Because the fractionation procedure was biochemical, the change in the distribution of topo II
enzymes may have merely reflected a collapse of nuclear integrity. To
investigate a probable change in the cellular localization of topo
II and II during apoptosis more directly, we immunostained intact
and Ara-C-treated apoptotic HL-60 cells using 8D2 and 3G3 and then
examined them with confocal laser microscopy (Fig
7). Topo II-specific 8D2 and topo
II-specific 3G3 signals are visualized as green color and PI
counterstained DNA red. In logarithmically growing cells, topo II
and II were localized in the nucleus showing a fine granular pattern
except for the nucleoli (Fig 7A and C, respectively). Both topo II
enzymes were distributed throughout the cell during mitosis (Fig 7A and
D). Only topo II signal was concentrated in the mitotic chromosomes
with merged intense yellow color. This observation was confirmed by the
comparison of topo II signals in the chromosomes and in the mitotic
cytoplasm (data not shown). When we stained HL-60 cells treated with
Ara-C for 5 hours, some nuclei showed chromatin condensation at the
nuclear periphery and others showed a typical apoptotic pattern with
discrete apoptotic bodies. Topo II signal was dissociated from the
chromatin at an early phase of apoptosis and completely separated from
the bright red signal of DNA in an advanced stage (Fig 7B, upper and lower cells, respectively). Topo II was also segregated from the
chromatin even at an early stage of apoptosis (Fig 7E). These results
were representative of three independent experiments conducted under
similar conditions.
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| Fig 7.
Topo II and II are dissociated from the chromatin
during apoptosis. Logarithmically growing (A, C, and D) and apoptotic HL-60 cells treated with Ara-C for 5 hours (B and E) are immunostained with topo II-specific 8D2 (A and B) and topo II-specific 3G3 monoclonal antibodies (C through E). Topo II and II signals are
green arising from the FITC-conjugated secondary antibody, and PI
counterstaining for DNA is red.
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| |
DISCUSSION |
In this study, we showed temporal and spatial changes in topo II and
II distributions in proliferating, differentiated, and apoptotic
HL-60 cells using two-dimensional flow cytometry, Western blot
analysis, and confocal laser microscopy. At first, we related topo
II and II levels with the cell cycle position in logarithmically
growing HL-60 cells. Although a previous study determined the contents
of topo II and II in synchronized cells at 2-hour intervals for a
total of 28 hours, the cells were not so well restricted to narrow
positions in the cell cycle, especially several hours after the release
from serum starvation.18 Treatment such as serum starvation
might also influence the cell viability or topo II levels. Another
study, in which cell size was regarded to reflect the cell cycle
position, fractionated an asynchronous cell population by centrifugal
elutriation and then measured the topo II level of each
fraction.19 We believe that the two-dimensional flow
cytometry determines topo II and II levels more precisely as
functions of the cell cycle position. Our results clearly showed the
steep increase of topo II level in late S to G2/M phases, which
correlates well with the known topo II function in chromosome condensation and segregation.1-4 Some of the G1 cells
expressed a larger amount of topo II than the early S cells. This
observation supports the previous hypothesis that topo II should be
degraded from anaphase to early G1 phase until the topo II level
becomes quite low.19,22 Although both topo II and II
antigens were recently reported to be twofold to threefold higher in
mitosis than in interphase, careful examination of the report's data
showed that topo II band intensity increases only from G1 to S
phases and thereafter is not significantly altered.16 This
agrees well with our observation that the topo II content is almost
constant after G1 phase. We suppose that the topo II content
decreases to 50% after cell division and returns to the previous level
during G1 phase.
Two-dimensional flow cytometry showed the appearance of a new cell
population containing tetraploid DNA and essentially no topo II
during differentiation. Because microscopic examination confirmed that
less than 0.3% of the cells were in mitosis at day 6 of the ATRA
treatment (data not shown), the new population should be in G2 phase.
This cell group was not detected in logarithmically growing cells.
These G2 cells are presumed to be noncycling and alive for the
following reasons. First, they do not seem to proceed along the cell
cycle further, because a sizable amount of topo II is necessary for
the initiation of chromatin condensation in early M
phase.4,5 Second, this population increased in cell number
from day 4 to day 6 of the ATRA treatment, although the cell influx
from the S phase must have decreased. This indicates that these cells
really stayed at the same stage in the cell cycle. Third, the results
from the TUNEL assay on the same differentiated HL-60 samples showed
that the apoptotic population was small and restricted to the sub-G1
position (data not shown). We therefore believe that the
two-dimensional analysis of our system first clearly detected a
G2-arrested nonapoptotic population during the course of
differentiation. Because G2 arrest has mainly been studied as a
cellular response to DNA damage,38 little is known about
the differentiation-induced G2 arrest. Apigenin, a flavone, was
reported to cause both G2 arrest and morphologic differentiation in rat
neuronal cells.39 Another report showed that even
irradiation-induced G2 arrest leads to  light chain gene expression,
a sign of differentiation, in 70Z/3 pre-B-cell line.40
Therefore, G2 arrest seems to induce differentiation in some kinds of
cells. Because ATRA does not directly block G2/M transition, our result
indicates that cell differentiation itself induces G2 arrest. It seems
interesting to determine whether differentiation-induced G2 arrest is a
general phenomenon. Cell growth and differentiation are tightly coupled in hematopoietic cells of myeloid lineage, and the half-life of peripheral granulocytes is only a few days.41 ATRA
treatment induces differentiation and subsequent spontaneous cell death even in acute promyelocytic leukemia (APL) cells.42 Because G2-arrested HL-60 cells are terminally differentiated, they are supposed to undergo apoptosis in a few days.
Two-dimensional flow cytometric analysis of topo II staining (5A7 or
3G3) and TUNEL assay indicated that only the C-terminal portion but not
the entire molecule of topo II should be degraded in apoptotic
cells. Western blot analysis of Ara-C-treated HL-60 cells using 3G3
clearly showed the proteolytic cleavage of topo II during apoptosis.
Comparison of the 5A7 and 3G3 blots confirms that the cleaved topo
II fragments retained a central portion but lost the C-terminal 5A7
epitope. On the contrary, a large portion of topo II remained of its
original size even in an advanced stage of apoptosis. The consistency
between the results of flow cytometric analysis and Western blotting
argues against a possibility that changes in chromatin structure and
topo II conformation during apoptosis could affect the topo II and
II stainabilities in the flow cytometric analysis. A previous report
showed degradation of topo II enzymes during CD95 (Fas/APO-1) -mediated
T-cell apoptosis using a rabbit antibody reactive to both
isoforms.32 A closer look at its data shows that topo II
disappears at an early phase of apoptosis and that topo II remained
at its original size even in the advanced stage, although its band
became rather faint. The sizes of the topo II degradation products in
this report were very similar to those of topo II fragments detected
in our study. Another report showed a relatively earlier loss of topo
II than topo II during drug-induced apoptosis in HL-60 and KG1A,
although topo II degradates were not detected.31 Therefore,
we believe that degradation of topo II but not of topo II is a
specific and relatively early event in the drug-induced apoptosis.
Both topo II and II were dissociated from the chromatin at an
early phase of apoptosis and completely separated from the genomic DNA
in an advanced stage. The degraded topo II fragments, which lost the
C-terminal portion, specifically left the nuclear fraction and
dissociated from the chromatin in apoptotic HL-60 cells. This suggests
the possible cause-and-effect relationship between the two events.
Indeed, some reports indicate that the C-terminal domain itself or its
phosphorylation is important for the stability of topo II-DNA
interaction.43,44 Topo II was also dissociated from the
chromatin at an early phase of apoptosis, although a large portion of
the enzyme seemed intact, at least by Western blot analysis. This
observation suggests that alternative mechanisms might be operating to
release topo II and maybe also topo II from the chromatin.
Several nuclear proteins, including nuclear lamin, PARP, and DNA-PKcs,
are inactivated by the degradation of their catalytic sites during
apoptosis. Topo II and II are unique in that not the apoptotic
proteolysis of their catalytic sites, which reside in the first 1,400 amino acids,1,2 but their release from the chromatin
abolishes the topo II enzyme activity during apoptosis.
Confocal microscopic study confirmed topo II and II distribution
in the nucleus except the nucleoli during interphase of growing HL-60
cells. In mitotic HL-60 cells, both topo II isozymes were distributed
throughout the cells and topo II signal was densely concentrated in
the chromosomes, which coincides well with the notion that at least
topo II is not only a necessary enzyme for the chromatin
condensation but also a structural component of the mitotic
chromosomes.45-47 Our observation agrees with a recent
report on the point that topo II is not preferentially localized in
the nucleoli.16 Although the report further indicated that
topo II is completely excluded from the chromosomes during mitosis,
we detected topo II signal not only in the mitotic cytoplasm but
also in the chromosomes. Monoclonal antibody 5A7 besides 3G3 confirmed
that a portion of topo II is localized in the mitotic chromosomes
(data not shown). Topo II has furthermore been shown to be present
in the isolated chromosomes, albeit in smaller quantities than topo
II.47 We believe that topo II is at least partially distributed in the mitotic chromosomes. In Ara-C-treated HL-60 cells,
topo II and II were dissociated from the chromatin even at an
early phase of apoptosis and were completely excluded from the
condensed apoptotic bodies. These observations indicate that dramatic
chromatin condensation during apoptosis is entirely topo II-independent. An essential difference must therefore exist between mitotic and apoptotic chromatin condensation.
Differentiation and apoptosis are the two principal cell fates that
follow proliferation after cells exit from the cell cycle. Using the
HL-60 human leukemia cell line as a model, we have shown the specific
loss of topo II during differentiation and the degradation of topo
II even at an early phase of apoptosis. We believe that this study
has clarified the different behavior of two topo II isozymes. As
previously proposed,17-21 our results suggest that topo
II plays an essential role in cell proliferation, especially during
late S to M phases. In contrast, topo II might be necessary for cell
survival because it exists at a substantial level even in the
differentiated cells and is degraded early and specifically during
apoptosis. Because hematologic malignancies are currently treated by
inducing apoptosis or differentiation, monitoring the topo II and
II levels in human leukemia samples may be useful to evaluate the
effects of cytotoxic and differentiation therapies.
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ACKNOWLEDGEMENT |
The authors thank Drs Tetsuya Nakamoto and Tokiharu Takahashi (the
Third Department of Internal Medicine, Faculty of Medicine, University
of Tokyo, Tokyo, Japan) and Dr Katsuhiko Kitsugi (Ortho Clinical
Diagnostics, Tokyo, Japan) for their technical advice and
Dr Masahiro Kizaki (Division of Hematology, Keio University School of
Medicine, Tokyo, Japan) for providing us with HL-60 human myeloid
leukemia cell line.
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FOOTNOTES |
Submitted May 7, 1997;
accepted October 16, 1997.
Supported by Grants-in-Aid for Cancer Research from the Ministry of
Health and Welfare and from the Ministry of Education, Science and
Culture in Japan.
Address reprint requests to Koichi Sugimoto, MD, Department of
Hematology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
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REFERENCES |
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Abstract
Introduction
Abstract