* Medizinische Poliklinik and Institute of Pathology, University of Würzburg, Germany; § Department of Human Genetics and
Department of Molecular and Structural Biology, University of Aarhus, Denmark; ¶ Institute of Molecular Medicine, John
Radcliffe Hospital, University of Oxford, United Kingdom; and ** Laboratory of Medical Mycology, Institute for Disease
Mechanism and Control, Nagoya University, Japan
We visualized DNA topoisomerases in A431
cells and isolated chromosomes by isoenzyme-selective
immunofluorescence microscopy. In interphase, topoisomerase I mainly had a homogeneous nuclear distribution. 10-15% of the cells exhibited granular patterns, 30% showed bright intranucleolar patches. Topoisomerase II isoenzymes showed spotted () or reticular (
)
nuclear patterns throughout interphase. In contrast to
topoisomerase II
, topoisomerase II
was completely
excluded from nucleoli. In mitosis, topoisomerase II
diffused completely into the cytosol, whereas topoisomerases I and II
remained chromosome bound.
Chromosomal staining of topoisomerase I was homogeneous, whereas topoisomerase II
accumulated in the long axes of the chromosome arms and in the centriols.
Topoisomerase antigens were 2-3-fold higher in mitosis
than in interphase, but specific activities of topoisomerase I and II were reduced 5- and 2.4-fold, respectively. These changes were associated with mitotic
enzyme hyperphosphorylation. In interphase, topoisomerases could be completely linked to DNA by
etoposide or camptothecin, whereas in mitosis, 50% of
topoisomerase II
escaped poisoning. Refractoriness to
etoposide could be assigned to the salt-stable scaffold
fraction of topoisomerase II
, which increased from
<2% in G1 phase to 48% in mitosis. Topoisomerases I
and II
remained completely extractable throughout
the cell cycle. In summary, expression of topoisomerases increases towards mitosis, but specific activities decrease. Topoisomerase II
is released from the
heterochromatin, whereas topoisomerase I and II
remain chromosome bound. Scaffold-associated topoisomerase II
appears not to be involved in catalytic
DNA turnover, though it may play a role in the replicational cycle of centriols, where it accumulates during M
phase.
In the cell, DNA topology is regulated and controlled
by ubiquitous enzymes known as topoisomerases,
which break and reseal the polyphosphate backbone
of the DNA and pass other strands of DNA through the
transient gaps (16, 19, 39, 40, 59). There are two types
of DNA topoisomerases with differing properties. Type I
topoisomerases can alter the pitch of DNA double helices by cutting one DNA strand and allowing passage of the
complementary strand through the transient nick (19).
Type II topoisomerases require ATP hydrolysis for catalytic activity and alter DNA topology by creating transient
double strand breaks through which a second intact double helix is passed (40). Topoisomerase function is required for replication, transcription (29, 67), recombination (32, 50), and repair (27, 31, 51) of DNA but also for chromosome (de)condensation (1, 20, 66) and sister chromatid segregation (13, 37). To date it is not completely
clear how these multiple functions are assigned to the various types and isoforms of topoisomerases. In particular, it
is unknown why mammals possess two isoforms of type II
topoisomerases, To reconcile these controversial findings, it has been hypothesized that topoisomerase II might have a structural
as well as a diffusible enzymatic role in the formation of
condensed chromosomes (1), and that in mammals, these
two functions could correspond to the two isoforms of topoisomerase II (44). However, the data available on the
nuclear localization of topoisomerase II Cells
Human A431 epidermoid cells (ATTC No. 1555, American Type Culture
Collection, Rockville, MD) were grown in liquid culture (DME with FBS
10% (vol/vol), 10 g/liter penicillin/streptomycin, 1% l-glutamine) in a humidified atmosphere containing 5% (vol/vol) CO2. Cells were routinely
checked to be free of mycoplasms by immunoassays and cultural analysis.
For indirect immunofluorescence, cells were grown on microscope slides.
Cells were trapped in mitosis by treatment with 0.26 µM demecolcine
(Sigma Chemical Co., Deisenhofen, Germany) for 16 h and continued to
grow in a synchronized manner for one cell cyle after washing out demecolcine. Analysis of cell cyle phases was performed on cells detached from
the substratum by trypsinization, fixed with 70% ethanol, and permeabilized with 0.1% Nonidet P-40. DNA was stained with bisbenzimide (0.1 µg/ml), and cellular DNA content was analyzed using a Partec flow cytometer equipped with a xenon lamp and trout erythrocytes as a DNA
standard. Mitotic indices were scored by counting 200 cells stained with bisbenzimide under the fluorescence microscope.
Antibodies and Recombinant Human Topoisomerases
Topoisomerase I.
We used Topoisomerase II Topoisomerase II Indirect Immunofluorescence Microscopy
Cells grown on microscope slides were fixed with formaldehyde (3.7% in
PBS) for 10 min at 5°C and subsequently permeabilized with Nonidet P-40
(0.01% in PBS) for 5 min at 4°C. After washing with PBS, cells were
blocked for 1 h at 20°C with PBS containing 5% standard goat serum and
1% BSA. Subsequently, cells were incubated for 1 h at 20°C with primary
antibodies diluted 1:400 (Ki-S1), 1:2,000 (Scl-70), or 1:250 (3H10) in PBS
containing 1% BSA. After washing, bound antibodies were visualized by
incubation for 1 h at 20°C with goat anti-human, -mouse, or -rabbit fab2fragments, which were labeled with CY3 (Dianova GmbH, Hamburg,
Germany) and diluted 1:1,000 in PBS containing 1% BSA and 1% standard
goat serum. After washing with PBS, DNA was counterstained with 1 µg/ml of bisbenzimide (Hoechst 33258) in PBS for 5 min at 20°C. Stained cells
were mounted in antifade solution (PBS containing 1.5% N-propyl-gallate and 60% glycerol) and examined at 480 or 1200 magnification using a
Leitz DM epifluorescence microscope coupled to a cooled CCD camera
(PM512; Photometrics Ltd., Tuscon, AZ). Camera control and image acquisition were done with an Apple Quadra 800 computer equipped with
imaging software from IPLabSpectrum. Fluorophores were selectively imaged with filters specially prepared as described by Pinkel et al. (43). Signals from bisbenzimide and CY3 were visually distinct and readily identifiable by inspection using appropriate filters.
Western Blot and Immuno-band Depletion Assay
Cells were cultured with or without inhibitors, followed by trypsinization
(also in the presence or absence of inhibitors), sedimentation of detached
cells (1,000 g, 5 min, 4°C), subsequent lysis in 1% SDS for 5 min at 90°C,
and mechanical DNA shearing with a syringe. Samples equivalent to 5 × 105 cells were subjected to SDS-polyacrylamide (8%) gel electrophoresis. Proteins in the gel were electrophoretically transferred to PVDF membranes (Immobilon P; Millipore Corp., Bedford, MA) by the semi-dry method using 70 mM CAPS buffer, pH 11. Immunostaining of immobilized proteins with various topoisomerase antibodies was carried out at
room temperature for 1 h using peroxidase-labeled goat secondary antibodies (Dianova GmbH), and the ECL system. Migration distances of immunostained protein bands were compared to those of rabbit muscle myosin
(212 kD), Determination of Extractable Nuclear
Topoisomerase Activities
Cells were detached by trypsinization, and cell nuclei were isolated and
extracted with 350 mM NaCl, as described in (5). For activity assays, nuclear extracts were serially diluted into a final volume of 40 µl of 10 mM
BisTrisPropane, pH 7.9, containing 10 mM MgCl2, 10 mM KCl, 0.5 mM
DTT, 0.5 mM EDTA, and 0.03 mg/ml BSA. Topoisomerase I activity was assessed by relaxation of 250 ng pBR 322 plasmid DNA in the presence of
1 mM Na3VO4. Under these conditions, topoisomerase II activity was
completely inhibited, as demonstrated by the absence of DNA-unknotting
activity. Topoisomerase II activity was measured by unknotting of 250 ng
of bacteriophage P4 knotted plasmid DNA in the presence of 1 mM ATP
(23). Incubation at 37°C for 30 min was terminated by addition of 1%
SDS. Samples were then digested with 1 mg/ml proteinase K at 37°C for
30 min. Gel electrophoresis was performed at 0.4 V/cm for 12 h in 1% agarose gels with TAE buffer. Gels were stained with 0.5 µg/ml ethidium bromide after electrophoresis. Fluorescence of ethidium bromide in the gels
(excitation 302 nm, emission >600 nm) was documented by Polaroid photography. The relative amounts of relaxed and supercoiled pBR322 DNA
or unknotted P4-DNA were determined by video-amplified fluorescence
intensity measurements of the respective DNA bands in each lane of the
gel using a video-densitometer. One unit of topoisomerase I was defined
as the amount of enzyme that relaxes 250 ng of pBR 322 DNA by 50% under the given conditions. One unit of topoisomerase II was defined as the
amount of enzyme catalyzing a halfmaximal increase in the band of unknotted P4 DNA.
Determination of Nuclear Topoisomerase
Antigen Levels
5 × 105 nuclei purified from synchronized cells in G1, S, or G2 phase,
cells trapped in mitosis by demecolcine, or a normal logarithmic cell culture were lysed with hot SDS, sheared with a syringe, and subjected to
Western blotting. Blots were probed with antibodies specific for human
topoisomerase I, II Preparation of Metaphase Chromosomes
Cells were treated with colcemid (0.1 µg/ml) for 1 h, harvested, sedimented for 5 min at 500 g, swelled with 0.075 M KCl for 10 min at 7°C, and
sedimented again for 5 min at 500 g. Cells were fixed 3 times in freshly
prepared methanol/acetic acid (3:1, vol/vol) for 3 min, spotted onto microscope slides, and air dried. Immunostaining was done as described for
whole cells.
Phosphatase Treatment and Analysis of Molecular
Weight Shifts
Nuclei from 5 × 107 cells trapped in mitosis by demecolcine, or from a
normal logarithmic cell culture were extracted with 800 mM NaCl. Extracts were precipitated with 3 M ammonium sulfate. Precipitates were
renatured with 200 mM diethanolamine, pH 9.9, containing 2 mM MgCl2
and 1 mM PMSF, and incubated with 30 U of alkaline phosphatase from
calf intestine (Boehringer Mannheim GmbH, Mannheim, Germany) for
90 min at 37°C. Controls were incubated with an equivalent amount of
phosphatase storage buffer without phosphatase. Subsequently, proteins
were precipitated with 15% (wt/vol) of trichloroacetic acid for 10 min at
37°C. Precipitates were washed with an equal volume of acetone cooled to
Specificity Controls of Topoisomerase Antibodies
Isoenzyme specific detection of human topoisomerases I,
II
Topoisomerase I was detected by peptide antibodies directed against the termini of the enzyme and by human
Scl-70 autoantibodies. In Western blots both peptide antibodies as well as the Scl-70 autoantibodies reacted with
purified recombinant topoisomerase I (Fig. 1, lanes 2-4),
and in lysates of whole A431 cells, a single band was
stained (Fig. 1, lanes 5-7) which had a size identical to that
of purified human topoisomerase I visualized by Coomassie blue staining (Fig. 1, lane 1). Immunofluorescent
images of A431 cells stained with Scl-70 autoantibodies
(Fig. 2 a) could be completely blocked by preincubating
the antibodies with 1 µg of purified and heat-inactivated
human topoisomerase I (Fig. 2 b).
Topoisomerase II Topoisomerase II In summary, the data shown in Figs. 1 and 2 prove that
we obtained selective and specific visualization of topoisomerases I, II Cellular Localization of Topoisomerases
Fluorescent images of A431 cells in logarithmic growth
stained with antibodies specific for topoisomerases I (Fig.
3 a), II
During interphase, the bulk
of all three topoisomerases was localized in the nucleoplasm, whereas the cytosol was not significantly stained
by any of the antibodies. Topoisomerase I mostly showed a diffuse, homogeneous distribution in the nucleoplasm
throughout interphase (Fig. 3 a). In S phase, at least 30%
of the cells exhibited spotted patterns (Fig. 4 a, top cell),
and 10% showed bright, intranucleolar patches (Fig. 4 a,
bottom cell). Intranucleolar localization of topoisomerase
I was most prominent in S phase cells, but also detectable
in G1 and G2 phase (not shown). Both type II topoisomerases showed a highly nonhomogeneous distribution in
the nucleoplasm, which did not significantly change from
G1 through G2 phase. Fig. 3, b and c shows representative
examples of nonsynchronized cells in logarithmic growth.
Topoisomerase II During mitosis, all of topoisomerase I remained attached
to mitotic chromosomes. It showed a diffuse distribution in
the chromosomal fibers (Fig. 4 d). Topoisomerase II Nuclear Enzyme Levels and Specific Activities
Our observation that during mitosis topoisomerase II Table I.
Activity of Topoisomerases in Mitotic and Interphase
A431 Cells. Mean Results of Three Independent Experiments
To differentiate between these possibilities, we compared the amounts of topoisomerase antigens present in
the nuclei of interphase and metaphase cells with the extractable catalytic activities. The results are summarized in
Table I. Antigen levels of all three topoisomerases were
higher (2.2-fold for topoisomerase I, 1.5-fold for topoisomerase II
Topoisomerase II activity extractable from metaphase
cells was reduced 1.7-fold as compared to interphase.
Western blot analysis of the nuclear remainder digested
with DNase after extraction showed that extractability of
topoisomerase II Noncatalytic Role of Topoisomerase II As shown in Figs. 4 e and 6 b, the whole cellular complement of topoisomerase II As cells procede from S phase to mitosis, topoisomerase
II Taken together, these observations strongly support the
notion that in mitotic chromosomes, at least 48% of topoisomerase II Altered Phosphorylation of Topoisomerases
during Mitosis
In mitotic cells, topoisomerase I and II
In this study we present data on the localization and activity of type I and II DNA topoisomerases in human A431
epidermoid cells during mitosis and interphase. The cell
line A431 was chosen for the investigation because it expresses unusually high levels of human topoisomerases,
can be synchronized, and allows a clear morphological
characterization of nuclei and cytoplasm after fixation. A431
cells are hypertriploid, which may in part explain their overexpression of topoisomerases. Moreover, these cells do
not stop logarithmic growth or undergo differentiation in
postconfluent culture. Thus, our data are representative
for cells in cell cycle but not necessarily for quiescent cells.
The cell line was chosen because we wanted to avoid mixed
effects of cell cycle arrest and differentiation induction,
which are to be expected in cells able to induce differentiation genes upon growth arrest. Despite these restrictions,
our results fit into a pattern emerging from accumulated
knowledge about the general role of DNA topoisomerases in chromosome condensation and chromatin disjunction
during the cell cycle (44), although some of our results
conflict with those previously published on the cellular localization of DNA topoisomerases in other mammalian
tumor cells (42, 68, 69).
Nucleolar DNA Topoisomerases
We found that topoisomerase II Localization and Activity of Topoisomerases
in Mitotic Cells
A number of studies show that topoisomerase II is an
abundant protein of the chromosomal scaffold which links
the bases of the radial chromosomal DNA loops to the central protein core of the chromosomal fiber (12). In extracted or expanded human chromosomes, topoisomerase
II antibodies identify a series of foci along the center of
the long axis of the chromatid arms (13, 53). Similar data
were obtained in indian muntiac cells (48). Until now, localization of Our observation that topoisomerase II In interphase cells, topoisomerase II The majority of topoisomerase II Topoisomerase I, like topoisomerase II In agreement with previous publications (10, 25, 47), we
observed expressional upregulation of all three topoisomerases during the cell cycle. The enzymes were phosphorylated during interphase as well as mitosis but during mitosis phosphorylation appeared to be enhanced or altered, as
could be deduced from a significant increase in phosphatase-sensitive electrophoretic retardation of the enzyme
bands in SDS-polyacrylamide gels (Fig. 10). A large body of
evidence suggests that phosphorylation increases catalytic
activity of topoisomerase I as well as topoisomerase II in
vitro (9, 19). Thus, we were puzzled by our observation that specific activity of topoisomerase I and II decreased
in metaphase cells, although the enzymes were apparently
more phosphorylated than in interphase cells. It is widely
assumed that phosphorylation of topoisomerases in vivo
has the same stimulatory effect as phosphorylation in vitro
and that consequently topoisomerases should be most active during mitosis where phosphate incorporation is highest. However, previous comparisons of topoisomerase activities in nuclear extract from S phase and mitotic cells
did ambiguously support this concept (15, 55). Moreover,
it has been observed in cells resistant to etoposide, that hyperphosphorylation can inactivate topoisomerase II (54)
suggesting that in vivo regulation of topoisomerase activity by phosphorylation might actually be biphasic, stimulating enzyme activity during S and G2 phase but inhibiting
it in mitosis due to phosphorylation of additional or novel
sites. On the other hand, it might be that during mitosis,
catalytic activity of topoisomerases is modulated by additional factors, such as and
, which are encoded by separate
genes (24, 56), whereas insects and fungi have only one
isoform (62). Topoisomerase II has been identified as the
major nonhistone protein which links the basis of the radial chromatin loops to the central axis of the mitotic chromosome fiber (44). In mammals, the enzyme is concentrated in the long axes of the chromosomes (48, 53),
whereas in insects it is distributed uniformly in the whole
chromosomal fiber. Moreover, in insects, 70% of the enzyme dissociates from the chromosomes during the mitotic
cycle and diffuses into the cytosol (52). It has also been
shown that the bulk of topoisomerase II can be extracted
from reconstituted chromosomes of Xenopus laevis oocytes without disrupting their morphology (20).
and
in mammalian cells equivocally support this concept. One set of
studies carried out with polyclonal peptide antibodies directed against unique epitopes of the two isoenzymes
showed that both type II topoisomerases diffused into the
cytosol during mitosis, whereas mitotic chromosomes were
not stained (42). Other studies carried out with monoclonal antibodies claimed that topoisomerase II
is exclusively located in the nucleoli and belongs to the structural
elements of the insoluble nucleolar remnant, thus excluding its role in diffusible topoisomerase II activity during
mitotic chromosome condensation (68, 69). However, the
monoclonal antibodies used in the latter studies for detecting topoisomerase II
, as we observed, did not actually
bind to recombinant purified human topoisomerase II
, as
will be discussed later. This observation led us to reinvestigate the subcellular localization of human topoisomerase
isoenzymes using a new set of monoclonal and polyclonal antibodies directed against human DNA-topoisomerases
I, II
, and II
, different from those used in previous studies. Making use of purified recombinant human topoisomerases produced in Saccharomyces cerevisiae, we
could clearly establish the specificity of these antibodies.
Moreover, similar results were obtained with monoclonal antibodies and peptide antibodies targetting the same
topoisomerase by different epitopes. Our results confirm
and complete previous reports regarding the localization
of topoisomerase I and II
(12, 14). However, the nuclear
localization of topoisomerase II
observed here is different
from that previously reported (42, 68, 69). We find that topoisomerase II
is not located inside the nucleoli but is
exclusively present in the extranucleolar nucleoplasm
throughout interphase. In mitosis it is not part of the chromosomal scaffold but becomes released from the chromatin, whereas the whole cellular complement of topoisomerase II
localizes to the central axis of the chromosomes.
In contrast to previous reports on the cellular localization
of topoisomerase II isoenzymes, our findings are in good
agreement with concepts currently held on the differential
role of topoisomerase II
and
in mitotic cells (44).
Materials and Methods
-globuline fractions of human Scl-70 auto-antibodies (Dunn Laboratories, Asbach, Germany) and rabbit antibodies
generated against peptides homologous to the NH2 (residues 1-17) or
COOH terminus (residues 745-765) of human topoisomerase I (Genosys Biotechnologies, Cambridge, UK). Human topoisomerase I was heterologously expressed in S. cerevisiae and purified, as described previously (7).
.
We have previously established that the mouse
monoclonal antibody directed against the proliferation-associated nuclear
antigen Ki-S1 is specific for a COOH-terminal epitope of human topoisomerase II
and does not cross-react with topoisomerase II
(6). In the
same study we have demonstrated that peptide antibodies generated against unique NH2- (residues 1-15) or COOH-terminal peptides (residues 1512-1530) of human topoisomerase II
are specific for human topoisomerase II
and do not cross-react with human topoisomerase II
.
Heterologous production of human topoisomerase II
was also described
in 6.
.
Production and characterization of the mouse monoclonal antibody 3H10 has recently been described in (26). Rabbit antibodies raised against peptides homologous to a unique COOH-terminal sequence (residues 1611-1621) of human topoisomerase II
have previously been characterized (6). Recombinant human topoisomerase II
purified
from S. cerevisiae was a kind gift (Dr. Ole Westergaard, Department of
Molecular and Structural Biology, University of Aarhus, DK).
2-macroglobulin from bovine plasma (170 kD),
-galactosidase
from Escherichia coli (116 kD), human transferrin (76 kD), and bovine
liver glutamic dehydrogenase (53 kD). Band-de/repletion phenomena were
quantified by comparing the optical densities (determined with a video
densitometer; Froebel, Wasserburg, Germany) of immunobands located
on the same gel and film after subtracting the background value of the
x-ray film.
, or II
, as described under Western blot and Immuno-band Depletion Assay. For quantitative determination of protein
expression, the intensities of the immunoreactive bands were measured by
video-densitometry of the x-ray films using a video densitometer (Froebel) and compared to those obtained with defined amounts of purified recombinant topoisomerases analyzed in the same gel and on the same x-ray film.
20°C, dissolved in SDS loading buffer, and finally subjected to SDS-gel
electrophoresis in 5.5% polyacrylamide gels and Western blotting.
Results
, and II
by Western blotting and indirect immunofluorescence microscopy was established using purified recombinant human topoisomerases produced in yeast as test
antigens. Fig. 1 compares Western blots of purified enzymes and of whole A431 cell lysates probed with antibodies directed against various epitopes of topoisomerases I,
II
, or II
. Fig. 2 shows specificity controls of immunostaining of topoisomerases I, II
, or II
in fixed and
permeabilized cells, using heat-inactivated (60°C, 5 min),
purified recombinant human topoisomerases for preabsorbtion of the antibodies.
Fig. 1.
Reactivity of topoisomerase antibodies in Western
blotting. 50 ng of purified recombinant human topoisomerase I
(lanes 2-4), II (lanes 9 and 10), or II
(lanes 14 and 15) or 5 × 105 A431 cells lysed in hot SDS (lanes 5-7, 11, 12, 16, and 17)
were separated by SDS-PAGE and blotted onto Immobilon-P
membranes. Lanes 1, 8, and 13 show Coomassie blue staining of
300 ng of purified topoisomerases I, II
, and II
, respectively.
Blot membranes were probed with antibodies, as follows. Lanes 2 and 5: Scl-70, 1:2,000; lanes 3 and 6: rabbit anti-peptide directed
against human topoisomerase I-COOH terminus, 1:5,000; lanes 4 and 7: rabbit anti-peptide directed against human topoisomerase
I-NH2 terminus, 1:1,000; lanes 9 and 11: Mouse monoclonal antibody Ki-S1, 1:500; lanes 10 and 12: rabbit anti-peptide antibody
directed against human topoisomerase II
-COOH terminus, 1:
5,000; lanes 14 and 16: Mouse monoclonal antibody 3H10, 1:800;
lanes 15 and 17: Rabbit anti-peptide antibody directed against
human topoisomerase II
-COOH terminus, 1:5,000. Strips represent the whole running distance of the gel, excluding the stacking
portion.
[View Larger Version of this Image (30K GIF file)]
Fig. 2.
Specificity controls of indirect immunofluorescence microscopy of topoisomerases. A431 cells were grown on microscope
slides, fixed, permeabilized, and double stained with topoisomerase antibodies and bisbenzimide (Hoechst Frankfurt, Germany). Images of immunofluorescence (top) and DNA (bottom) are paired. (a) Immunostaining of topoisomerase I with Scl-70 autoantibodies;
(b) as a, but Scl-70 autoantibodies were preincubated with 1 µg of heat-inactivated (60°C, 5 min) human topoisomerase I for 1 h at 20°C;
(c) Immunostaining of topoisomerase II with Ki-S1 mouse monoclonal antibody; (d) as c, but Ki-S1 antibody was preincubated with 1 mg of heat-inactivated (60°C, 5 min) human topoisomerase II
for 1 h at 20°C; (e) as c, but Ki-S1 antibody was preincubated with 1 µg
of heat-inactivated (60°C, 5 min) human topoisomerase II
for 1 h at 20°C; (f) immunostaining of topoisomerase II
with 3H10 mouse
monoclonal antibody; (g) as f, but 3H10 antibody was preincubated with 1 µg of heat-inactivated (60°C, 5 min) human topoisomerase
II
for 1 h at 20°C; (h) as f, but 3H10 antibody was preincubated with 1 mg of heat-inactivated (60°C, 5 min) human topoisomerase II
for 1 h at 20°C.
[View Larger Version of this Image (69K GIF file)]
was detected by peptide antibodies
directed against the COOH terminus of the enzyme and by
the mouse monoclonal antibody Ki-S1 also recognizing a
unique COOH-terminal epitope of human topoisomerase
II
(6). In Western blots, peptide antibodies and the Ki-S1
monoclonal antibody reacted with recombinant human topoisomerase II
produced in S. cerevisiae (Fig. 1, lanes
9 and 10). In lysates of whole A431 cells, both antibodies
stained a single band of 170 kD (Fig. 1, lanes 11 and 12)
identical in size to purified human topoisomerase II
visualized by Coomassie blue staining (Fig. 1, lane 8). Immunofluorescent images of A431 cells stained with Ki-S1 antibody
(Fig. 2 c) could be completely blocked by preincubating
the antibodies with 1 µg of purified and heat-inactivated topoisomerase II
(Fig. 2 d), whereas the signal was not
diminished by preincubation with heat-inactivated topoisomerase II
(Fig. 2 e). Similar fluorescent images of
A431 cells were obtained with peptide antibodies recognizing a unique COOH-terminal epitope of topoisomerase
II
. These could be blocked by preincubating the antibodies with 0.1 µg of the immunogenic peptide or with 1 µg of
heat-inactivated recombinant topoisomerase II
but not with topoisomerase II
(not shown).
was detected by peptide antibodies
directed against the COOH terminus of the enzyme previously described in (6) and by the mouse monoclonal antibody 3H10. Fig. 1 attests to the ability of the peptide antibody (lane 14) and the monoclonal antibody 3H10 (lane
15) to react in Western blots with purified human topoisomerase II
and to stain uniformly a band of 180 kD in
blots of whole A431 cell lysates (Fig. 1, lanes 16 and 17), similar in size to that of purified human topoisomerase II
detected by Coomassie blue staining (Fig. 1, lane 13). Crossreactions with purified topoisomerase II
(not shown) or
with topoisomerase II
present in the cell lysates (Fig. 1,
lanes 16 and 17), could be excluded. Fluorescent images of
A431 cells stained with monoclonal antibody 3H10 (Fig. 2 f)
could be completely blocked by preincubating the antibody with 1 µg of purified and heat-inactivated human topoisomerase II
(Fig. 2 g), whereas the signal was not
diminished by preincubation with heat inactivated topoisomerase II
(Fig. 2 h). We also tested the specificity of
the monoclonal antibody 8F8 used in previous studies for
characterizing the cellular localization of topoisomerase II
(36, 68, 69). We observed that 8F8 antibody does not bind
to Western blots of recombinant human topoisomerase II
purified from S. cerevisiae. Moreover, the nucleolar
staining of 8F8 antibody could not be blocked by preabsorption with purified human topoisomerase II
. The
Western blot signal obtained with the 8F8 antibody in whole
cell lysates had a different size (150 kD) than that obtained with peptide antibodies directed against a COOHterminal epitope of human topoisomerase II
(180 kD; 6). Finally, the intensity of the 150-kD band stained by 8F8
antibody did not correlate to the level of topoisomerase
II
-specific mRNA when several cell lines with different
expression levels were compared, whereas the 180-kD band
stained by the peptide antibody showed a clear correlation
(63). We assume that these differences in immune-reactivity between 8F8 and the antibodies used in this study for labeling topoisomerase II
may explain some of the discrepancies between this and previous studies.
, and II
by Western blot and indirect
immunofluorescence microscopy. Subsequently, we used
these techniques for investigating the subcellular localization of topoisomerases in human epidermoid A431 cells.
(Fig. 3 b), or II
(Fig. 3 c) were bright, clearly different from each other, and also different from the DNApatterns obtained by simultaneous staining with bisbenzimide (Hoechst 33258) which is shown for comparison.
The overviews shown in Fig. 3 are representative for the
whole cell population. Fig. 4 shows enlarged images of representative cells in interphase (4, a-c) or mitosis (4, d-f). Fig. 5 relates localization of topoisomerases to the
nucleoli of interphase cells, whereas Fig. 6 shows localization of topoisomerases in isolated mitotic chromosomes.
Fig. 3.
Fluorescent images of topoisomerases in human A431
cells. Monolayers of A431 cells grown on micoscopic slides were
fixed, permeabilized, and doublestained with bisbenzimide (Hoechst;
right) and topoisomerase antibodies (left). (a) Immunostaining of
topoisomerase I with Scl-70 autoantibodies. (b) Immunostaining
of topoisomerase II with Ki-S1 mouse monoclonal antibody. (c)
Immunostaining of topoisomerase II
with 3H10 mouse monoclonal antibody.
[View Larger Version of this Image (66K GIF file)]
Fig. 4.
Localization of topoisomerases in interphase and mitosis. Close-up pictures of representative cells in interphase (a-c) or mitosis (d-f) immunostained for topoisomerase I (a and d), topoisomerase II (b and e), or topoisomerase II
(c and f). The
left of each pair of images represents immunostaining. The right
shows the corresponding DNA pattern (Hoechst).
[View Larger Version of this Image (83K GIF file)]
Fig. 5.
Colocalization of topoisomerases with DNA in interphase cells. Pseudo-color coded
fluorescent images of topoisomerases (red) were stacked
with corresponding patterns of
bisbenzimide-stained DNA
(blue). (Middle) immunostaining of topoisomerase I with Scl70 (a), topoisomerase II with
Ki-S1(b), and topoisomerase II
with 3H10 (c). (Left) Corresponding image of bisbenzimide-stained DNA (Hoechst).
(Right) Stacked image of immunostaining (red) and DNA (blue). Arrows indicate the positions of nucleoli.
[View Larger Version of this Image (52K GIF file)]
Fig. 6.
Localization of topoisomerases in isolated chromosomes. Isolated chromosomes
were doublestained for DNA
and topoisomerases I (a), II
(b), or II
(c), as in Fig. 3.
Paired images of immunofluorescence (left) and corresponding DNA pattern (right) are
shown.
[View Larger Version of this Image (64K GIF file)]
appeared to be concentrated in numerous spots located extranucleolarly in the nucleus (Fig.
4 b, top cell). In addition, a less intensive homogeneous
background staining of the nucleoplasm was observed, which was more pronounced inside nucleoli (Fig. 4 b,
bottom cell). Topoisomerase II
exhibited a patchy reticular distribution, markedly different from topoisomerase
II
. It was most dense in peri-nucleolar regions, but it was
clearly always excluded from the interior of the nucleoli
(Fig. 4 c). This finding, which is in contrast to previous reports (42, 68, 69), can be more clearly seen in the pseudocolored overlay picture of DNA and immunofluorescent images shown in Fig. 5. Nucleoli could be discriminated as
spherical areas within the chromatin, which were not
stained by Hoechst (Fig. 5, a-c, left, arrows). In the Hoechst-
negative intranucleolar space, immunostaining of topoisomerase II
was also negative. Consequently, in the
stacked picture (Fig. 5 c, right), all of the red topoisomerase II
-specific signal (Fig. 5 c, middle) turns violet, due
to colocalization with the blue Hoechst signal (Fig. 5 c,
left). In contrast, stacked pictures of topoisomerase II
and DNA (Fig. 5 b, right) or topoisomerase I and DNA
(Fig. 5 a, right) show prominent red immunosignals inside
the Hoechst-negative nucleolar regions, which are not
shifted to blue and can be distinguished from the violet
signals obtained for enzymes colocalized with DNA in the
adjacent chromatin. It can also be seen that topoisomerase II
exhibited a homogeneous intranucleolar staining only
slightly more prominent than in the surrounding nucleoplasm, whereas topoisomerase I showed very prominent
granular patterns inside the nucleoli, which are clearly
brighter than the signal in the surrounding chromatin.
was
also completely bound to the mitotic chromatin (Fig. 4 e),
but in contrast to topoisomerase I it appeared to be highly
concentrated in threadlike structures along the chromosome arms and in addition in the centriols (Fig. 4 e and Fig. 3
b). Topoisomerase II
diffused completely into the cytosol
and was not detectable at all in the condensed chromatin (Fig. 4 f). Immunostaining of isolated chromosomes (Fig. 6)
confirmed these observations. Topoisomerase I showed a
diffuse, grainy pattern and was localized in the whole chromosomal fibers (Fig. 6 a), whereas topoisomerase II
was
localized in numerous spots along the longitudinal axes of
the chromosome arms (Fig. 6 b). Isolated chromosomes
were not stained at all by topoisomerase II
antibodies (Fig. 6 c), confirming that during mitosis the bulk of the
enzyme is not attached to the chromatin.
was not bound to the DNA but diffused into the cytosol
could either indicate that (a) the enzyme becomes inactivated, or (b) its activity remains unaltered, but binding to
the condensed chromatin is inhibited sterically. Activity
might also be modulated during mitosis in such a way that
the equilibrium between bound and free enzyme becomes
shifted towards the free form. Investigating these possibilities, we first compared the ability of all three topoisomerases to enzymatically interact with genomic DNA during
mitosis: We treated A431 cells in logarithmic growth containing <1% mitoses and cultures arrested in metaphase
containing >90% mitoses (see Table I) with camptothecin or etoposide, which stabilize the covalent complexes of
DNA and topoisomerase I or II, respectively. Drug-
induced covalent DNA linkage of catalytically active enzymes was monitored by immuno-band depletion, i.e., the
inability of the covalently DNA-linked enzymes to enter
SDS-polyacrylamide gels, resulting in a loss of the respective protein band in Western blots of whole cell lysates.
Drug-induced band depletion was quantified by comparative densitometry of the immuno-blots (Table I). As
shown in Fig. 7, a complete immuno-band depletion of topoisomerase II
was similarly observed in log-phase A431
cells and in cultures arrested in metaphase, indicating that all topoisomerase II
molecules, although mainly located
in the cytosol, are capable of interacting with the condensed metaphase chromatin. In contrast, we observed that
15% of topoisomerase I and 50% of topoisomerase II
could not be linked to the chromatin of mitotic cells by
camptothecin or etoposide, respectively, whereas a complete band depletion was obtained for both enzymes in
log-phase cells (Fig. 7 and Table I). This might indicate
that during mitosis, either the specific activity of these enzymes is largely reduced or a certain fraction of the enzymes is inactive or only partially capable of interacting
enzymatically with the DNA.
Fig. 7.
Drug-induced topoisomerase band-depletion in mitosis
and interphase. Cells in logarithmic growth (Log) or blocked in
metaphase by demecolcine (M) were treated with 30 µM camptothecin (line 1, +Drug) or 200 µM etoposide (lines 2 and 3,
+Drug) for 1 h at 37°C. Controls were incubated without drug.
Subsequently, the cells were harvested and lysed with hot SDS,
and lysate equivalent of 5 × 105 cells was loaded onto each lane.
Western blots were probed with rabbit peptide antibodies directed against the COOH termini of human topoisomerase I (top),
II (middle), or II
(bottom). This is a representative example of
at least three identical experiments with similar outcome. A
quantitative analysis of band intensities is given in Table I. The
observed differences in band intensity between treated and untreated cells were significant on the 0.01 level (Wilcoxon's signed
rank test).
[View Larger Version of this Image (55K GIF file)]
, and 2.0-fold for topoisomerase II
) in cells
locked in metaphase than in log phase cells, which is due
to an increase in enzyme expression in S, G2, and M phase, as shown in Fig. 8. These data are in close agreement with
previous findings (2, 17, 28, 65). However, when measuring topoisomerase enzyme activities extractable from the
nuclei, we obtained an unexpected result (Table I): in
metaphase cells, activity of topoisomerase I extractable
from each nucleus was reduced 2.2-fold, as compared to
interphase cells. This decrease could be due to a lesser
extractability of topoisomerase I during mitosis. We investigated this possibility by digesting the nuclear remnant with DNase after salt extraction, applying it to Western
blotting, and comparing it to an equivalent number of nonextracted nuclei. As shown in Fig. 9 a, topoisomerase I antigen was extracted by >90% both from inter- and metaphase nuclei. Thus, the metaphase-associated decrease in
topoisomerase I activity can not be explained by differences in extractability, but must be due to a decrease in
specific catalytic activity. We calculated a 4.7-fold reduction of specific topoisomerase I activity in mitotic cells,
taking into account that mitotic cells contain 2.2-fold more
topoisomerase I antigen than interphase cells (Fig. 8). The
significant decrease in specific catalytic activity may account for the slightly reduced drug sensitivity of topoisomerase I during mitosis (Fig. 7).
Fig. 8.
Cell cycle-coupled
expression of topoisomerases. Cells were synchronized by demecolcine treatment and harvested at the
indicated cell cycle stages (G1,
S, G2/M). Mitotic cells (M)
were harvested directly after
treatment with demecolcine
for 16 h. Log-phase cells not
treated with demecolcine
(Log) served as controls. 5 × 105 nuclei isolated from the cells
were lysed with hot SDS and applied to each lane. Western blots
were probed with rabbit anti-peptide antibodies directed against
the COOH termini of human topoisomerases I (top), II (middle), or II
(bottom), respectively. This is a representative example of at least three identical experiments with similar outcomes.
A quantitative analysis of band intensities is given in Table I.
[View Larger Version of this Image (59K GIF file)]
Fig. 9.
Cell cycle-coupled changes in DNA extractability of topoisomerases. (a) Nuclei were isolated from cells in logarithmic growth (Log) or blocked in metaphase by demecolcine (M). Nuclei were either lysed in hot SDS (N) or were first extracted with 350 mM NaCl,
and the nuclear remnant (R) was lysed in hot SDS after digestion with DNase I (50 U/106 nuclei) for 20 min at 37°C. 7 × 105 nuclei (N)
or an equivalent amount of DNase-digested nuclear remnant (R) was applied to Western blotting. Blots were probed with rabbit peptide
antibodies directed against the COOH terminus of human topoisomerase I (lanes 1-4), II (lanes 5-8), or II
(lanes 9-12). This is a representative example of three identical experiments with similar outcomes. (b) The procedure described in a was performed with synchronized cells harvested in G1, S, or G2 phase and with cells blocked in metaphase by demecolcine (M). The relative amounts of topoisomerases I, II
, and II
that were nonextractable by 350 mM NaCl as compared to total nuclear content, were determined by
comparative videodensitometry of the immunoblots. Mean values of three identical experiments are plotted. Bars represent standard
errors of the mean. (c) Nuclei were isolated from cells blocked in metaphase by demecolcine and extracted with 350 mM NaCl. Nonextractable nuclear remnants were dissolved in an equal volume of 3.5-fold diluted extraction buffer and incubated with (lane 2) and without (lane 1) 200 µM etoposide in the presence of 1 mM ATP. Extracts were diluted 3.5-fold and incubated with (lane 4) and without
(lane 3) 200 µm etoposide in the presence of 1 mM ATP and 4 µg calf thymus DNA. After 30 min the reaction was stopped by addition
of hot SDS. Samples were sheared with a syringe and applied to Western blotting, and blots were probed with peptide antibodies directed against the COOH terminus of human topoisomerase II
. A representative example of three identical experiments with similar
outcomes is shown.
[View Larger Versions of these Images (46 + 29 + 21K GIF file)]
was almost complete (88%) in interphase cells (Fig. 9 a, lanes 5 and 6), whereas in metaphase
cells a substantial fraction (48%) escaped extraction (Fig.
9 a, lanes 7 and 8). In contrast, extractability of topoisomerase II
was >95% in interphase and in metaphase
(Fig. 8 a, lanes 9-12). As we were not able to determine
the individual contributions of
- and
-isoenzymes to
overall topoisomerase II activity in the extracts, we can not
decide whether the decrease in extractable topoisomerase
II activity is solely due to reduced extractability of the
-isoenzyme or, in addition, to a decrease in specific activity of topoisomerase II
and/or of the extractable fraction of topoisomerase II
. However, it seems reasonable to assume that the dissociation of topoisomerase II
from the
mitotic chromosomes is associated with an altered catalytic activity of the enzyme.
in Mitotic Cells
becomes concentrated in the
long axes of the condensed chromosomes. In this situation,
50% of the enzyme molecules are not capable of interacting with the DNA in a catalytic manner, as can be deduced
from their reduced drug sensitivity shown in Fig. 7. Drug
insensitivity of topoisomerase II
in metaphase cannot be
due to a reduced accessibility of topoisomerase II cleavage sites in the condensed chromatin, because the
-isoenzyme under the same conditions is capable of getting completely DNA linked in the presence of etoposide (Fig. 7).
Thus, it must be due to a decrease in the catalytic activity
of topoisomerase II
or physical separation of the active
site of the enzyme from the DNA.
gets increasingly recruited to the nuclear scaffold fraction not extractable by 350 mM NaCl, whereas topoisomerase I and II
remain extractable by >90% throughout the cell cycle (Fig. 9 b). In metaphase, at least 48% of
topoisomerase II
can not be extracted by 350 mM NaCl
(Fig. 9, a and b and Table I). When comparing the drug sensitivity (i.e., the ability to covalently bind to DNA in
the presence of etoposide) of extractable and nonextractable fractions of topoisomerase II
in metaphase cells
(Fig. 8 c), it becomes apparent that actually the nonextractable portion is the one that does not interact with the
DNA in a catalytic manner and consequently is refractory
to etoposide treatment, whereas most of the extractable enzyme in the presence of etoposide (and ATP) forms a
covalent complex with exogenously added calf thymus
DNA, which is electrophoretically less mobile than the
free enzyme (Fig. 9 c).
serves a role not involving catalytic DNA
turnover. Furthermore, in mitotic cells, topoisomerase II
is not only located in the chromosomal scaffold but also in
the centriols (Figs. 3 b and 4 e). This observation implies
that the enzyme might be one of the as yet unidentified
components of the centrosome. As centrosomes of quiescent cells are not stained by topoisomerase II
antibodies (28, 49) and only one pair of centriols/centrosomes is
stained in mitotic cells, it appears likely that the enzyme
could play a functional role in the replication cycle and/or
the maturation of the centriols (30).
exhibited an increase in apparent molecular weight (Fig. 8, lane 4), which
has previously been assigned to M phase specific phosphorylation of these enzymes (10, 25, 26). When desalting nuclear extracts thoroughly by sequential precipitation with
trichloroacetic acid and acetone and separating them in
5.5% polyacrylamide gels, which allow enhanced resolution in the 100-300-kD region, we observed that topoisomerase II
also exhibited a mitotic increase in apparent
molecular weight. As shown in Fig. 10 (compare lanes 1 and 3), during mitosis, topoisomerase I was shifted by 2 kD
from 102 to 104 kD. Topoisomerase II
was shifted by 5 kD
from 170 to 175 kD, whereas topoisomerase II
showed a
much larger shift (by 10 kD) from 180 to 190 kD. When extensively treating the extracts with alkaline phosphatase
prior to electrophoresis, smaller and sharper immunoreactive bands were created in all cases (Fig. 10, lanes 2 and 4).
For topoisomerase I, phosphatase treatment gave rise to a
band of 101 kD, which was identical in interphase and
mitotic cells and slightly smaller than the normal band
position in interphase cells not treated with phosphatase
(102 kD). Topoisomerase II
was shifted upon dephosphorylation to a band of 168 kD, which was similar in size in interphase and mitotic cells and significantly smaller
than the normal band position of interphase cells not
treated with phosphatase (170 kD). In the case of topoisomerase II
, phosphatase treatment in interphase decreased the size by 2 kD (from 180 to 178 kD) , whereas in
mitotic extracts it decreased by 8 kD (from 190 to 182 kD).
However, even after extensive phosphatase treatment (50 U, 120 min, 37°C) there remained a size difference of 4 kD between extracts of interphase and mitotic cells (Fig. 10,
lanes 2 and 4). These data indicate that all three topoisomerases are phosphorylated during interphase as well as
during mitosis, since phosphatase treatment induces a mobility shift in both cell cycle phases. However, during mitosis, phosphatase-induced mobility shifts are three to fourfold larger than in interphase, indicating that the enzymes
are phosphorylated more or in a different way during mitosis. In the case of topoisomerase I and II
, the mitotic mobility shifts can be solely explained by an altered phosphorylation, since phosphatase treatment gives rise to bands
of similar size in mitosis and interphase. In the case of topoisomerase II
, phosphatase treatment did not produce
bands of similar size in mitosis and interphase. We concluded, that in addition to a phosphorylation-related increase in apparent molecular weight, there appears to exist a phosphatase-resistant shift of 4 kD, which could either
be due to an unusual (phosphatase-resistant) type of phosphorylation of topoisomerase II
not present on the other
topoisomerases, or, more likely, due to the presence of an
additional posttranslational modification, which we have
not yet been able to identify.
Fig. 10.
Treatment of nuclear extracts with alkaline
phosphatase. Nuclei of mitotic (lanes 3 and 4) and interphase cells (lanes 1 and 2)
were extracted with 800 mM
NaCl. Extracts were precipitated with 3 M ammonium
sulfate, renatured with diethanolamine buffer, pH 9.8, and
incubated with 30 U of alkaline phosphatase at 37°C for
90 min (lanes 2 and 4). Controls (lanes 1 and 3) were incubated with an equivalent amount of phosphatase storage
buffer without enzyme. Extracts were subsequently precipitated
with 15% trichloroacetic acid. Precipitates were washed with acetone, dissolved in SDS sample buffer containing 5 M urea, separated on 5.5% polyacrylamide gels with a 3% stacking portion,
transferred to Immobilon P membranes, and probed with peptide antibodies specific for the COOH-terminal portions of topoisomerase I (row 1), II (row 2), or II
(row 3). Molecular
weight values of enzyme specific protein bands indicated on the
right margin were derived from Rf-plots of migration distances of marker proteins (see Materials and Methods) run in the same gel. The coefficient of variance of these molecular weight values was <20% in a run of three similar experiments, of which this one is a representative example. The observed differences in apparent molecular weight were significant on the 0.05 level (Wilcoxon's signed rank test).
[View Larger Version of this Image (52K GIF file)]
Discussion
is excluded from nucleoli
during all stages of the cell cycle. This finding is in striking controversy to previous immunohistochemical studies,
where the enzyme was mainly found inside the nucleoli (42,
68, 69). The intranuclear localization of topoisomerase II
and the increased expression during S phase (42; Fig. 8)
and cellular differentiation (64, 65) have led to the hypothesis that topoisomerase II
mainly has a function in the
transcription process of the heavily transcribed rRNA
genes. However, this hypothesis is not supported by any
direct biochemical evidence. In contrast, intranucleolar localization of topoisomerase I seen here and also reported
before (33, 34, 36, 38) is supported by multiple biochemical observations implying a functional role of the enzyme
in rRNA metabolism, such as preferred cleavage in regions flanking rRNA genes (8, 34), interaction with nucleolin (3), and phosphorylation of rRNA-splicing factors
(46). Studies in yeast suggest that both type I and type II
topoisomerases are required for the folding of rDNA into
the functional organization of nucleolar genes, as cofactors
of RNA polymerase I, and for the topological organization of
the nucleolar chromatin (21). Our data suggest that in rapidly proliferating mammalian cells, which contain two isoforms of type II topoisomerases, this function is reserved
to the
-isoenzyme which appeared to accumulate inside
nucleoli of most interphase cells, whereas the
-isoenzyme
was completely excluded from the nucleoli throughout interphase. We observed similar patterns in HeLa cells and
stimulated human peripheral lymphocytes, whereas in primary cultures of nondividing cells, which do not express the
-form of topoisomerase II, topoisomerase II
appeared to be mainly localized in the nucleoplasm but also
in the nucleoli. Most recently, Turley et al. (57) performed
an extensive immunohistochemical analysis of the distribution of the two isoforms of DNA-topoisomerase II in
various normal and neoplastic human tissues also using the 3H10 monoclonal antibody for visualizing topoisomerase
II
. They showed that normal, quiescent tissues expressed
only topoisomerase II
, which was localized in the nucleoplasm and the nucleoli. Expression of topoisomerase II
was restricted to the proliferative compartment of normal
tissues and to tumors, where it exhibited a nucleolarly enhanced localization. From these observations it can be hypothesized that topoisomerase II
has a higher affinity to
the nucleolar chromatin than topoisomerase II
and that
the extranucleolar localization of topoisomerase II
in
A431 cells observed here could be due to the high levels of
topoisomerase II
expressed in these cells (Table I), which
may compete with topoisomerase II
for binding to the
nucleolar chromatin. Thus, extranucleolar localization of
topoisomerase II
should be a feature of rapidly proliferating cells expressing high levels of topoisomerase II
. It
could be imagined that the differences between this study and previous studies on postconfluent Chinese hamster fibroblasts (42) reporting localization of topoisomerase II
in the nucleoplasm and in the nucleoli are due to the fact
that A431 cells are a more pure model for rapidly cycling
cells than postconfluent Chinese hamster fibroblast. However, reports on an exclusively nucleolar localization of topoisomerase II
in rapidly proliferating HeLa cells (68, 69)
are clearly in contrast to our findings and all other available studies and are most likely due to differences in the
specificity of the immunostaining methods. We believe that the antibodies and fixation procedures used here allow a
more specific and selective labeling of topoisomerase II
in situ than those used in these other studies. Having purified recombinant human topoisomerases available in sufficient amounts, we could support our data by appropriate
controls of specificity and isoenzyme selectivity of the immunological techniques used. Moreover, we could monitor localization of type I and II topoisomerases through the complete cell cycle and even in isolated mitotic chromosomes.
- and
-isoenzymes in mitotic chromosomes
has not been studied directly. Thus, it was not completely
clear to which of the two isoenzymes the structural role is
assigned. The observation that activity of the
-isoenzyme
is selectively inhibited by AT-rich oligonucleotides (11)
suggested it as a candidate for binding to the chain of ATrich sequences delineating the core of the chromatid fibers
(48). Moreover, immunoprecipitates of the MPM-2 antibody directed against a phosphoepitope of mitotic scaffold
proteins contained mainly the
-form of topoisomerase II
(53). However, from studies in insect cells, which have
only one form of topoisomerase II, it became apparent
that only 30% of the enzyme is tightly linked to the core of
the chromosome, whereas the rest diffuses away during
mitosis (52). The concept of distinct populations of topoisomerase II, which either serve structural functions in the
nuclear scaffold or are active in transcribed regions of the
chromatin in a diffusible manner, is supported by the finding that in vivo, there exist two main classes of cleavage
sites mapping either to scaffold adhesion sequences or to
sites of transcriptionally active chromatin (44). The data
presented here indicate that at least in human A431 cells, two populations of topoisomerase II can be discriminated
morphologically and can indeed be assigned to the two
isoforms
and
. We show that the
-isoenzyme is tightly
linked to mitotic chromosomes and exhibits a threadlike
pattern along the long axis of the chromosome arms. It
does not notably diffuse into the cytosol during mitosis. Moreover, as the cells progress from S phase to mitosis, topoisomerase II
gets increasingly recruited to the salt-stable scaffold protein fraction (Fig. 9, a and b), whereas the
-isoenzyme remains completely extractable through the
whole cell cycle. In agreement with these observations, it
has recently been reported that in mitotic human HeLa
cells, topoisomerase II
is extracted by much lower salt concentrations than topoisomerase II
(26). Taken together, these observations strongly suggest that the
-isoenzyme
is essential in chromosome condensation and disjunction,
whereas the
-isoenzyme plays a minor role. This notion is
also supported by preliminary results, showing that topoisomerase II
does not complement topoisomerase II
in
NIH3T3 fibroblasts, expressing topoisomerase II
-antisense RNA (Andoh, T., S. Toji, and M. Kaneko. 1995. The
Sixth Conference on Topoisomerases in Therapy: 28a.). The
data shown in Fig. 9 c suggest that the salt-stable fraction
of topoisomerase II (i.e., of the
-isoenzyme) in majority does not catalytically interact with the surrounding heterochromatin, as it is not getting DNA-linked in significant quantities, when exposed to high concentrations of
etoposide (Fig. 9 c). In contrast, diffusible fractions of topoisomerase II (i.e., the extractable fraction of topoisomerase II
and topoisomerase II
) are getting completely DNA linked when exposed to etoposide. Thus, it
appears that the diffusable and not the scaffold-linked topoisomerase II is the major drug target. These observations seem to be in contradiction to recent results by Gromova et al., demonstrating that treatment of salt-extracted nuclei with topoisomerase II poisons resulted in a similar
pattern of long-range cleavage of genomic DNA cleavage as
treatment of nonextracted nuclei (18). However, it should
be noted that judging drug-induced covalent DNA linkage
of topoisomerase II by immuno-band depletion, as done
here, is insensitive to rare interactions involving <10% of
the enzyme molecules, whereas measuring drug-induced
DNA-cleavage, as done by Gromova et al., (18) does not
indicate how large a fraction of the topoisomerase II molecules is involved in the process. Taken together, our results and the data of Gromova et al. indicate that a small
fraction of scaffold-bound topoisomerase II
is catalytically active and consequently targeted by topoisomerase II
poisons, whereas the majority is catalytically inactive.
localizes to the
centrioles of dividing cells provides a novel finding, further
indicating that functions of topoisomerase II
extend beyond DNA turnover. It has been shown that centrioles
replicate in a semiconservative manner, independent of
DNA and protein synthesis (41). Interestingly, in quiescent cells, centrosomes are not stained by topoisomerase II
specific antibodies (28, 49), indicating that the localization of topoisomerase II
to the centrioles is restricted to
cycling cells. The functional role of topoisomerase II
in the
replication cycle of centrioles remains to be established.
appears to be
highly condensed in various confined areas located within
the nucleoplasm, but outside the nucleoli. These spots were
more frequent in late S and G2 phase. Moreover, we previously observed formation of similar clusters of topoisomerase II
in the nuclei of peripheral human lymphocytes
upon stimulation with phytohemagglutinin 2 (28). It has
been suggested that multimerization of topoisomerase II
could play a role in formation of the nuclear scaffold of mitotic chromosomes (58). Thus, it might be imagined that
the clusters observed here actually represent such multimers
of topoisomerase II
forming at defined organization centers within the euchromatin of interphase cells, from which
condensation of chromosomes originates, as the cell prepares to enter mitosis. In this context, it is interesting to
note that recently it has been shown that a fraction of topoisomerase II
clusters at centromeric regions of the chromatin as cells prepare to enter mitosis and the enzyme is
required for proper centromere/kinetochore structure (45).
clearly does not have
a structural role in mitotic chromosomes as it diffuses
away from the heterochromatin. This notion is supported
by the observation that salt extractability of the
-isoenzyme does not change during the cell cycle. The data
shown in Table I suggest that diffusional loss from the heterochromatin could be at least partially due to a decrease
in catalytic activity, which might shift the DNA-binding equilibrium of the enzyme towards the unbound form.
However, all of the enzyme molecules remain active and
capable of catalytically interacting with the DNA during
mitosis, as shown by their undiminished susceptibility to
etoposide. This is in clear contrast to the partial refractoriness of the
-isoenzyme. Recently, it has been shown that
topoisomerase II
and
can form low amounts of heterodimers in vivo, as well in yeast as in human HeLa cells
(4). Here we observed a more or less complete spatial dissociation of topoisomerase II
and
antigens in mitotic
A431 cells, which seems to argue against the existence of
significant levels of heterodimers in these cells. The discrepancy could be due to the fact that heterodimers were
present in too small amounts to be detected. Heterodimers might also be specific for interphase cells and not present
during mitosis. In agreement with our data, it has previously been observed that immunoprecipitates of mitotic
scaffold phosphoproteins of mouse P388D1 lymphocytic
cells obtained with the MPM-2 antibody contained only
traces of topoisomerase II
, which were shown not to be
due to
/
-heterodimerization (53).
, remains attached to mitotic chromosomes and does not notably diffuse
into the cytosol. It has been reported before that topoisomerase I is enriched at the centromeres before anaphase
(33). However, the mitotic pattern and the immunostaining of isolated chromosomes shown here (Figs. 4 d and 6 a)
rather argue in favor of a diffuse distribution in the periphery of the chromosomes, which is in clear contrast to the
thread-like pattern of topoisomerase II
. Since salt extractability of topoisomerase I did not change in mitosis as
significantly as that of topoisomerase II
(Fig. 9 b), it
seems unlikely that the type I enzyme has a structural role
in chromosomes similar to that of topoisomerase II
.
-glycerophosphate, a component of
mitotic extracts, which in vitro has a marked inhibitory effect on the enzymatic activity of topoisomerase II and
stimulates its multimerization (58). The data shown in Fig.
10 give some indication of an additional covalent posttranslational modification of topoisomerase II
during mitosis, which increases the apparent molecular size of the
enzyme by 3.8 kD in a phosphatase-insensitive fashion.
We do not yet know what causes this phosphatase-insensitive size shift. However, topoisomerase II
is exported to
the cytosol during mitosis. In A431 cells, enzyme levels
drop significantly from M to G1 phase (Fig. 8). This seems
to indicate a rapid postmitotic degradation of topoisomerase II
, although in HeLa cells the protein moiety of topoisomerase II
appears not to be degraded as the cells go
from mitosis to G1 phase (26). In light of recent findings emphasizing the important role of protein ubiquitination
in the degradation of cell cycle related proteins at the end
of mitosis (22, 35), it is tempting to speculate that the mitotic export of topoisomerases II
to the cytosol, and possibly also its subsequent degradation, might be triggered
by ubiquitination or some other posttranslational modification independent of phosphorylation.
K. Meyer and E. Kjeldsen contributed equally to this publication.
Received for publication 22 July 1996 and in revised form 15 October 1996.
Purified recombinant human topoisomerase IIThis work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 172, B12, and Kr 849/4-2, and in part by the Danish Cancer Society, 95-100-40 and 78-5000, the Danish Research Council, the Danish Center for Human Genome Research, and the Danish Centre for Molecular Gerontontology. Fritz Boege gratefully acknowledges an educational stipend from the Deutsche Krebshilfe, Dr. Mildred Scheel-Stiftung.