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 (α) or reticular (β) nuclear patterns throughout interphase. In contrast to topoisomerase IIα, topoisomerase IIβ was completely excluded from nucleoli. In mitosis, topoisomerase IIβ diffused completely into the cytosol, whereas topoisomerases I and IIα remained chromosome bound. Chromosomal staining of topoisomerase I was homogeneous, whereas topoisomerase IIα 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 IIα escaped poisoning. Refractoriness to etoposide could be assigned to the salt-stable scaffold fraction of topoisomerase IIα, which increased from <2% in G1 phase to 48% in mitosis. Topoisomerases I and IIβ remained completely extractable throughout the cell cycle. In summary, expression of topoisomerases increases towards mitosis, but specific activities decrease. Topoisomerase IIβ is released from the heterochromatin, whereas topoisomerase I and IIα remain chromosome bound. Scaffold-associated topoisomerase IIα 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, 59–61). 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, α and β, 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).
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 IIα and β 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 IIβ 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 IIβ, as we observed, did not actually bind to recombinant purified human topoisomerase IIβ, 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, IIα, and IIβ, 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 IIα (12, 14). However, the nuclear localization of topoisomerase IIβ observed here is different from that previously reported (42, 68, 69). We find that topoisomerase IIβ 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 IIα 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 IIα and β in mitotic cells (44).
Materials And Methods
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
We used γ-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 IIα and does not cross-react with topoisomerase IIβ (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 IIα are specific for human topoisomerase IIα and do not cross-react with human topoisomerase IIβ. Heterologous production of human topoisomerase IIα 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 IIβ have previously been characterized (6). Recombinant human topoisomerase IIβ purified from S. cerevisiae was a kind gift (Dr. Ole Westergaard, Department of Molecular and Structural Biology, University of Aarhus, DK).
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), α2-macroglobulin from bovine plasma (170 kD), β-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.
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, IIα, or IIβ, 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.
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 −20°C, dissolved in SDS loading buffer, and finally subjected to SDS-gel electrophoresis in 5.5% polyacrylamide gels and Western blotting.
Specificity Controls of Topoisomerase Antibodies
Isoenzyme specific detection of human topoisomerases I, IIα, and IIβ 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, IIα, or IIβ. Fig. 2 shows specificity controls of immunostaining of topoisomerases I, IIα, or IIβ in fixed and permeabilized cells, using heat-inactivated (60°C, 5 min), purified recombinant human topoisomerases for preabsorbtion of the antibodies.
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 IIα 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 IIα (6). In Western blots, peptide antibodies and the Ki-S1 monoclonal antibody reacted with recombinant human topoisomerase IIα 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 IIα 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 IIα (Fig. 2,d), whereas the signal was not diminished by preincubation with heat-inactivated topoisomerase IIβ (Fig. 2 e). Similar fluorescent images of A431 cells were obtained with peptide antibodies recognizing a unique COOH-terminal epitope of topoisomerase IIα. 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 IIα but not with topoisomerase IIβ (not shown).
Topoisomerase IIβ 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 IIβ 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 IIβ detected by Coomassie blue staining (Fig. 1, lane 13). Crossreactions with purified topoisomerase IIα (not shown) or with topoisomerase IIα 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 IIβ (Fig. 2,g), whereas the signal was not diminished by preincubation with heat inactivated topoisomerase IIα (Fig. 2 h). We also tested the specificity of the monoclonal antibody 8F8 used in previous studies for characterizing the cellular localization of topoisomerase IIβ (36, 68, 69). We observed that 8F8 antibody does not bind to Western blots of recombinant human topoisomerase IIβ purified from S. cerevisiae. Moreover, the nucleolar staining of 8F8 antibody could not be blocked by preabsorption with purified human topoisomerase IIβ. 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 IIβ (180 kD; 6). Finally, the intensity of the 150-kD band stained by 8F8 antibody did not correlate to the level of topoisomerase IIβ–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 IIβ may explain some of the discrepancies between this and previous studies.
In summary, the data shown in Figs. 1 and 2 prove that we obtained selective and specific visualization of topoisomerases I, IIα, and IIβ by Western blot and indirect immunofluorescence microscopy. Subsequently, we used these techniques for investigating the subcellular localization of topoisomerases in human epidermoid A431 cells.
Cellular Localization of Topoisomerases
Fluorescent images of A431 cells in logarithmic growth stained with antibodies specific for topoisomerases I (Fig. 3,a), IIα (Fig. 3,b), or IIβ (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.
Localization in Interphase.
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 IIα 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 IIβ exhibited a patchy reticular distribution, markedly different from topoisomerase IIα. 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 IIβ was also negative. Consequently, in the stacked picture (Fig. 5,c, right), all of the red topoisomerase IIβ-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 IIα 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 IIα 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.
Localization in Mitotic Cells and Isolated Chromosomes.
During mitosis, all of topoisomerase I remained attached to mitotic chromosomes. It showed a diffuse distribution in the chromosomal fibers (Fig. 4,d). Topoisomerase IIα 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 IIβ 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 IIα 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 IIβ antibodies (Fig. 6 c), confirming that during mitosis the bulk of the enzyme is not attached to the chromatin.
Nuclear Enzyme Levels and Specific Activities
Our observation that during mitosis topoisomerase IIβ 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 IIβ was similarly observed in log-phase A431 cells and in cultures arrested in metaphase, indicating that all topoisomerase IIβ 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 IIα 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.
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 IIα, and 2.0-fold for topoisomerase IIβ) 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).
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 IIα 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 IIβ was >95% in interphase and in metaphase (Fig. 8 a, lanes 9–12). As we were not able to determine the individual contributions of α- and β-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 α-isoenzyme or, in addition, to a decrease in specific activity of topoisomerase IIβ and/or of the extractable fraction of topoisomerase IIα. However, it seems reasonable to assume that the dissociation of topoisomerase IIβ from the mitotic chromosomes is associated with an altered catalytic activity of the enzyme.
Noncatalytic Role of Topoisomerase IIα in Mitotic Cells
As shown in Figs. 4,e and 6 b, the whole cellular complement of topoisomerase IIα 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 IIα in metaphase cannot be due to a reduced accessibility of topoisomerase II cleavage sites in the condensed chromatin, because the β-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 IIα or physical separation of the active site of the enzyme from the DNA.
As cells procede from S phase to mitosis, topoisomerase IIα gets increasingly recruited to the nuclear scaffold fraction not extractable by 350 mM NaCl, whereas topoisomerase I and IIβ remain extractable by >90% throughout the cell cycle (Fig. 9,b). In metaphase, at least 48% of topoisomerase IIα 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 IIα 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).
Taken together, these observations strongly support the notion that in mitotic chromosomes, at least 48% of topoisomerase IIα serves a role not involving catalytic DNA turnover. Furthermore, in mitotic cells, topoisomerase IIα 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 IIα 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).
Altered Phosphorylation of Topoisomerases during Mitosis
In mitotic cells, topoisomerase I and IIβ 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 IIα 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 IIα was shifted by 5 kD from 170 to 175 kD, whereas topoisomerase IIβ 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 IIα 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 IIβ, 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 IIα, 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 IIβ, 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 IIβ 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.
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 IIβ 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 IIβ and the increased expression during S phase (42; Fig. 8) and cellular differentiation (64, 65) have led to the hypothesis that topoisomerase IIβ 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 α-isoenzyme which appeared to accumulate inside nucleoli of most interphase cells, whereas the β-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 α-form of topoisomerase II, topoisomerase IIβ 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 IIβ. They showed that normal, quiescent tissues expressed only topoisomerase IIβ, which was localized in the nucleoplasm and the nucleoli. Expression of topoisomerase IIα 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 IIα has a higher affinity to the nucleolar chromatin than topoisomerase IIβ and that the extranucleolar localization of topoisomerase IIβ in A431 cells observed here could be due to the high levels of topoisomerase IIα expressed in these cells (Table I), which may compete with topoisomerase IIβ for binding to the nucleolar chromatin. Thus, extranucleolar localization of topoisomerase IIβ should be a feature of rapidly proliferating cells expressing high levels of topoisomerase IIα. It could be imagined that the differences between this study and previous studies on postconfluent Chinese hamster fibroblasts (42) reporting localization of topoisomerase IIβ 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 IIβ 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 IIβ 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.
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 (12–14). 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 α- and β-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 α-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 α-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 α and β. We show that the α-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 IIα gets increasingly recruited to the salt-stable scaffold protein fraction (Fig. 9, a and b), whereas the β-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 IIβ is extracted by much lower salt concentrations than topoisomerase IIα (26). Taken together, these observations strongly suggest that the α-isoenzyme is essential in chromosome condensation and disjunction, whereas the β-isoenzyme plays a minor role. This notion is also supported by preliminary results, showing that topoisomerase IIβ does not complement topoisomerase IIα in NIH3T3 fibroblasts, expressing topoisomerase IIα-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 α-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 IIα and topoisomerase IIβ) 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 IIα is catalytically active and consequently targeted by topoisomerase II poisons, whereas the majority is catalytically inactive.
Our observation that topoisomerase IIα localizes to the centrioles of dividing cells provides a novel finding, further indicating that functions of topoisomerase IIα 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 IIα specific antibodies (28, 49), indicating that the localization of topoisomerase IIα to the centrioles is restricted to cycling cells. The functional role of topoisomerase IIα in the replication cycle of centrioles remains to be established.
In interphase cells, topoisomerase IIα 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 IIα in the nuclei of peripheral human lymphocytes upon stimulation with phytohemagglutinin 2 (28). It has been suggested that multimerization of topoisomerase IIα 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 IIα 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 IIα clusters at centromeric regions of the chromatin as cells prepare to enter mitosis and the enzyme is required for proper centromere/kinetochore structure (45).
The majority of topoisomerase IIβ 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 β-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 α-isoenzyme. Recently, it has been shown that topoisomerase IIα and β 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 IIα and β 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 IIβ, which were shown not to be due to α/β-heterodimerization (53).
Topoisomerase I, like topoisomerase IIα, 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 IIα. Since salt extractability of topoisomerase I did not change in mitosis as significantly as that of topoisomerase IIα (Fig. 9 b), it seems unlikely that the type I enzyme has a structural role in chromosomes similar to that of topoisomerase IIα.
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 β-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 IIβ 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 IIβ 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 IIβ, although in HeLa cells the protein moiety of topoisomerase IIβ 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 IIβ to the cytosol, and possibly also its subsequent degradation, might be triggered by ubiquitination or some other posttranslational modification independent of phosphorylation.
This 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.
Purified recombinant human topoisomerase IIβ was kindly provided by Dr. Ole Westergaard (Department of Structural and Molecular Biology, University of Aarhus, Denmark). We would also like to thank Dr. Westergaard for helpful discussions. We are grateful to Ms. Berit Hornstrup for helping us prepare the manuscript.
K. Meyer and E. Kjeldsen contributed equally to this publication.