ARTICLE |
Correspondence to: Udo Kellner, Dept. of General Pathology, University of Kiel, Michaelisstr. 11, 24105 Kiel, Germany.
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Summary |
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We report five novel monoclonal antibodies (Ki-S1, Ki-S4, Ki-S6, Ki-S7, and Ki-S8) reactive with a proliferation-related nuclear antigen. In immunoprecipitation and Western blot experiments using crude nuclear extracts, they recognized a protein of 170 kD that, after proteolytic digestion of the immunoprecipitate and sequencing of the resulting peptides, was identified as the -isoform of human topoisomerase II. This was confirmed by testing the antibodies on a highly purified enzyme preparation. Crossreactivity with topoisomerase IIß was ruled out by testing the antibodies on crude extracts from yeast cells expressing the ß-isoform exclusively. The antibodies bind the antigen with different affinities and at different epitopes, apparently located within the carboxyl third of the enzyme. All five antibodies are suitable for archival material after adequate antigen retrieval, thereby enabling retrospective studies. This report illustrates the tissue and subcellular distribution of the antigen through the cell cycle by immunohistochemistry and confocal fluorescence microscopy. The antibodies will be useful tools in further analysis of morphological and functional aspects of topoisomerase II and may serve diagnostic purposes, as well as providing prognostic information in tumor pathology. (J Histochem Cytochem 45:251-263, 1997)
Key Words:
human topoisomerase II, monoclonal antibodies, Western blot analysis, immunohistochemistry, confocal laser microscopy, proliferation
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Introduction |
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Deregulated growth is one cardinal feature of malignancy. Because alterations leading to a loss of growth control may occur at many levels of the cell cycle machinery ( 160 kD (
. In addition, four newly produced MAbs directed against the same antigen are presented.
Topoisomerases are highly conserved nuclear enzymes that control and modify the topological states of DNA (reviewed in , exhibits strong fluctuations in its activity and the amount of enzyme as detected by polyclonal antibodies (
is tightly linked to cell proliferation (
emerges not only as a marker for cell proliferation (
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Materials and Methods |
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Cell Preparations and Cultures
All cells were grown in liquid culture under standard conditions at 37C in a humidified atmosphere containing 5% CO2. Peripheral blood lymphocytes (PBLs) were isolated as described before and stimulated with phytohemagglutinin A (PHA) for 12, 24, 36, 48, 72 hr (
Generation of Monoclonal Antibodies
L428 cells were lysed in hypotonic KCl solution and disrupted by pottering. The nuclei were washed and harvested by centrifugation. Subsequently, with incomplete Freund's adjuvant added, they were used for immunization of female Balb/c mice by three IP injections at 10-day intervals. After boostering during 4 successive days, the splenocytes were fused by somatic hybridization with P3x63-Ag.8653 mouse myeloma cells (
Antibody Purification
Separation of the MAbs from cell culture supernatants was carried out in a single step using protein G-Sepharose 4 Fast Flow (Pharmacia LKB; Freiburg, Germany).
A 1 M Tris buffer titrated with HCl to pH 8.0 was used as a starting buffer. Antibody containing supernatant was incubated overnight with protein G-Sepharose at 4C, sedimented, washed with 10 mM Tris buffer, pH 8, and redissolved in this buffer before application to a column. Bound proteins were eluted using 0.2 M glycine-HCl at pH 3. The purified antibodies were adjusted immediately to pH 8 by titration with 10% starting buffer.
Isotyping
Supernatants of all six antibody-producing clones were tested in an enzyme-linked immunoadsorbent assay (ELISA) with isotype-specific goat anti-mouse antibodies (Sigma; München, Germany).
Immunocytochemistry and Immunohistochemistry
PBLs were harvested and processed as described below, immediately or on Day 1, 2, or 3 after PHA stimulation. Cytospin preparations were fixed in acetone at room temperature (RT) for 10 min and incubated with the primary antibodies (Ki-S1, Ki-S4, Ki-S6, Ki-S7, and Ki-S8, as well as affinity-purified polyclonal anti-topoisomerase II rabbit antibody) for 30 min. The immunoreaction was enhanced by the APAAP method and the slides were briefly counterstained with Mayer's hematoxylin. Freshly isolated peripheral blood monocytes (
) served as negative controls. Frozen sections of an exhaustive spectrum of normal human tissues were immunohistochemically examined with all six MAbs to survey their distribution patterns and possible crossreactivities. Their tissue reaction sites were detected as described elsewhere (
Immunoprecipitation
A total of 107 L428 cells were labeled with [35S]-methionine overnight; all further steps were carried at 4C. After intensive rinses with ice-cold PBS, the cells were lysed for 5 min with 2% Triton X-100, 1 mM EDTA, 1 µM PMSF, in PBS at pH 7.4. Nuclei were separated by centrifugation through 50% sucrose. The nuclei were then disintegrated by resuspension in extraction buffer and dropwise addition of 5 M NaCl to a final concentration of 0.35 M. After lysis for 30 min at 4C, nuclear membranes and DNA were sedimented (15,000 x g for 10 min) and discarded. The supernatant was used for immunoprecipitation with the MAbs Ki-S1, Ki-S4, and Ki-S8 coupled to rabbit anti-mouse IgG-conjugated protein A-Sepharose (Biochrom; Berlin, Germany). After brief sedimentation, the precipitate was washed with low salt (4% Triton X-100 in PBS) and with high salt buffer (PBS/4% Triton X-100/500 mM NaCl) in alternate steps. Finally, the pellet was dissolved in 50 µl low salt buffer admixed with 50 µl loading buffer (
Antibodies and Western Blot Experiments
In addition to the MAbs described above, we used a rabbit polyclonal antiserum against topoisomerase II purchased from ICI (Cambridge, UK), and a polyclonal antibody from BioTrend (Köln, Germany) raised against the C-terminal peptide EEDDVDFAMFN of the ß-enzyme (
(TopoGEN; Columbus, OH) as an antigen. The purity of the preparation (>90%) was verified by India Ink staining (
Expression of Human Topoisomerase II in Yeast Cells
Strains, plasmids and the experimental procedure were as previously described ( plasmid pHT212 and untagged human topoisomerase IIß pHT400. The expression of the enzyme is under the control of the moderate constitutive triose phosphate isomerase promoter. For Western blot experiments we used crude extracts prepared by vortexing at 4C for 30 min in the presence of 2 vol of extraction buffer [50 mM Tris (pH 7.8), 1 M NaCl, 1 mM phenylmethylsulfonylfluoride], and 1 vol of acid-washed glass beads (425-600 µm; Sigma). After centifugation at 4000 x g for 10 min and at 15,000 x g for 30 min, the supernatant was filtered (0.65-µm pore size filters). Samples of the extract were mixed with 10 x Laemmli buffer before submission to 6% SDS-PAGE. Electrophoresis and staining were performed as described before, except that gold-labled secondary antibodies were used (Amersham). Bands were finally visualized by silver enhancement (Amersham).
Protein Sequencing
A total of 109 unlabeled L428 cells were immunoprecipitated as described above. After SDS-PAGE, the proteins were blotted to PVDF membranes (Millipore) and stained with Coomassie Brilliant Blue R 250 (Biorad; München, Germany). After destaining with 50% methanol, the antigen was excised and further processed for sequencing. The proteins were digested with trypsin and the proteolytic fragments separated by narrow-pore HPLC (130 Å; Applied Biosystems) on a reverse-phase column (Vydac C4, 300 Å pore size, 5 µm particle size; 2.1 x 125 mm). Peptides were eluted with a linear gradient (0-80% b over 50 min; Solvent A, 0.1% TFA/H2O; Solvent B, 0.09% TFA/70% acetoneitrile) at a flow rate of 200 µl/min. Peptide-containing fractions detected at 214 nm were collected manually into siliconized Eppendorf tubes and frozen immediately. Protein sequences were determined by Standard Edman degradation on an automatic sequenator (473 A; Applied Biosystems).
Binding Site Determination
An approximately 60-kD fusion peptide of the carboxyl terminus of human topoisomerase II expressed in E. coli (DH5
) using the p Flag expression system (IBI, Zürich, Switzerland; the vector and the integrated cDNA were a generous gift from S. Gasser, ISREC, Lausanne, Switzerland) was electrophoretically run in a 5-15% polyacrylamide gel and stained with all five MAbs. Cambridge polyclonal antibody served as a positive control.
To map the antibody binding sites, 60 µg of nuclear extracts from Hl-60 cells was submitted to proteolytic fragmentation using 0.6 µg (30 µU) S. aureus V8 endoproteinase (EC 3.4.21.19; Boehringer) for 5, 15, 30, and 480 min at 25C, pH 7.8. The proteolytic fragments were separated by SDS-PAGE (5-20% gels) as described above and analyzed by Western blotting with the antibodies Ki-S1, Ki-S4, Ki-S6, Ki-S7, and Ki-S8, as well as the rabbit polyclonal antiserum (ICI). The latter is directed against the utmost C-terminal KPIKYLEESDEDDLF sequence of the molecule.
Binding Affinity of Monoclonal Antibodies
Aliquots (3000 mU to 46.9 mU) of purified human topoisomerase II (TopoGEN) were transferred onto PVDF membranes and incubated with 1 µg/ml of each antibody (purified as described above) and with 1 µg/ml rabbit polyclonal antibody. Visualization of immunoreactive peptides was obtained by peroxidase-coupled secondary antibodies and a chemiluminescence kit (Amersham) used according to the supplier's instructions. Densitometry was performed with a Vilber Lourmat densitometer. The intensities of different dilutions were plotted and the regression was estimated. The antigen concentration at a medium staining intensity was estimated in three independent experiments, and the standard deviation and mean standard error were calculated.
Confocal Fluorescence Microscopy
Cells growing on slides were fixed in ice-cold acetone for 10 min and incubated at RT with Ki-S1 or Ki-S4 as the primary antibody (ascites) at a dilution of 1:30,000 for 1 hr. After three rinses in PBS, DATF-conjugated anti-mouse Ig from goat (Dako; Hamburg, Germany) was applied as a second antibody at a concentration of 1:100 for 1 hr. After washing in PBS slides were mounted with Histogel (Camon; Wiesbaden, Germany) and sealed with Eukitt. Control samples were treated identically except that the primary antibody was omitted.
Optical sections of 0.6 µm thickness were examined on a Zeiss LSM 10 confocal microscope equipped with an argon/krypton laser and filters for FITC detection. Images were recorded with a Zeiss Apoplan 63 x/1.4 or a 100 x/1.3 lens using zoom factors up to 4. They were stored on an optical disc (Mitsubishi MW-5U1) and photographed from a high-resolution flat screen (FVM 1702; Lucius & Baer, München, Germany). Grading of the antigen expression was done by two independent observers.
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Results |
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Immunoreactivity and Antigen Distribution
Ki-S1 was found to be IgG2a, whereas all other antibodies were of the IgG1 isotype.
The distribution of the antigen in normal human tissues was restricted to sites known to harbor actively dividing cells, e.g., the basal and suprabasal cell layers of stratified epithelium, the bottoms of crypts in the gastrointestinal mucosa, proliferating endometrium, the dark zone of the germinal centers in lymphoid tissue (Figure 1A), and the immature precursor cells of granulopoiesis and erythropoiesis in the bone marrow. A particularly intense immunoreaction was observed in normal fertile testicular tissue. The majority of spermatogonia and spermatocytes were strongly stained, but neither spermatids nor spermatozoa expressed the antigen (Figure 1B). Occasional positive cells could be observed in the myometrium and the smooth muscle layer of gastrointestinal organs. Connective tissue displayed occasional immunoreactivity in widely scattered fibroblasts or capillary endothelial cells. Quiescent tissues, notably skeletal and cardiac muscle and neuronal tissue, showed no nuclear antigen expression. Nonspecific crossreactivities with cytoplasmic or matrical antigens were not observed, except for staining of splenic sinusoidal fibers with Ki-S4. Immunohistochemical analysis of a wide variety of malignant tumors revealed labeling indices ranging from 10% to >90%. Numeric differences were not observed with different antibodies, but marked differences in the staining intensity were occasionally apparent. Immunostaining of frozen material and paraffin sections yielded identical results.
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On immunocytochemical examination, only the nuclei of proliferating cells were reactive with our antibodies. Cytospin preparations of unstimulated PBLs, which represent G0 cells, did not show any reactivity. After stimulation with PHA, a constant increase in the number of stained cells could be observed. After 72 hr about 80-90% of the nuclei were stained (Figure 1C-H). The intensity of nuclear labeling increased slightly through S-phase and was maximal in G2/M. Identical results were obtained with all six antibodies.
Whereas preparations of quiescent cells (unstimulated PBLs and M) yielded no immunoreaction, up to 90% of the cells of permanent lines (L428 and HL-60) were labeled (not shown). The staining was essentially confined to the nucleoplasm; only on rare occasions was weak cytoplasmic staining observed. Nucleoli were particularly highlighted by Ki-S1 and Ki-S4, whereas they were unconspicuous in the immunoreaction with the other antibodies. Serum deprivation considerably reduced the number of positive cells in permanent cell lines. On refeeding with 10% FCS, antigen expression gradually increased and reached control levels after 48 hr. Both myeloid and monocytic differentiation of HL-60 cells induced with DMSO or TPA led to a virtual disappearance of the antigen. Whereas in exponentially growing cells the percentage of labeled cells was practically invariable with all five antibodies, variations in staining intensity were occasionally noted (data not shown).
Molecular Mass of the Antigen and Specificity of the Antibodies
Immunoprecipitation of radiolabeled nuclear lysates of L428 cells with Ki-S1 and subsequent SDS-PAGE evaluated by autoradiography yielded a strong protein band at 170 kD. Weak bands at 180 and 133 kD (less than 4% of the signal intensity of the main band at 170 kD) were co-precipitated in this experiment (Figure 2). By contrast, in Western blot experiments using crude nuclear extracts, all five MAbs exclusively stained the 170-kD protein detected by rabbit polyclonal antibody to topoisomerase II, whereas A10 antiserum raised against both topoisomerase II isoforms recognized a double band at 170 and 180 kD (Figure 3). L-428 cells growing at different densities expressed 40-60% of topoisomerase IIß compared to the
-form (Figure 3). Because Ki-S1 precipitated a 180-kD band, the specificity of the five MAbs was further tested on human topoisomerase II
and ß differentially expressed in yeast cells. In Western blot experiments using extracts from these cells, the 180-kD protein was visualized only by an antibody specific for topoisomerase IIß, whereas all five of our antibodies exclusively recognized the
-isoform (Figure 4). Immunoreactive antigens were enriched by immunoprecipitation with Ki-S1, separated by SDS-PAGE, and transferred by Western blot to PVDF membranes. The Coomassie Brilliant Blue-labeled protein was excised and used for protein sequencing. Because sequencing of the intact protein was unsuccessful due to blocking of the N-terminus, the antigen was digested by trypsin. From the obtained proteolytic fragments, the sequence of eight peptides of 10 to 26 amino acids could be determined. All of these eight peptides exhibited complete identity (100%) with the protein predicted by the cDNA sequence of human topoisomerase II (Figure 5). Six of them shared 68-84% sequence similarity with topoisomerase IIß, and two differed only by one amino acid from topoisomerase IIß (90-92% identity; first two sequences).
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These results were further confirmed for all MAbs by Western blot experiments using homogeneously purified human topoisomerase II. The purity of the enzyme preparation was verified by India ink staining, yielding a neat single band at 170 kD. Affinity-purified preparations of the five MAbs antibodies stained this protein (Figure 6), which demonstrates that the antigen recognized by the antibodies actually represents topoisomerase II
. Weak bands of a lower molecular weight were also stained.
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Proteolytic digestion of crude nuclear extracts with V8-protease for 5-15 min yielded a neat set of proteolytic fragments as visualized by Coomassie Brilliant Blue staining. Western blot analysis with the antibodies Ki-S1, Ki-S4, Ki-S6, and Ki-S8 displayed four different staining patterns, whereas no signal was obtained with the ICI antiserum and Ki-S7 (Figure 7). All of these antibodies reacted with the 60-kD C-terminal fusion peptide (Figure 8). However, the binding affinity of Ki-S1 was weak, so that a threefold protein loading was necessary to obtain a signal intensity comparable to that of the other antibodies.
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Binding Affinity of Monoclonal Antibodies to Topoisomerase II
Evaluation of the antigen-antibody complex formation with various concentrations of purified human topoisomerase II demonstrated that all five antibodies bound to the enzyme with different affinities. The strongest reaction was observed with Ki-S1 (set to 100%), of which even 1 µg/ml antibody yielded a detectable signal with 93.8 mU/20 mm2 (4.7 mU/mm2). Compared with Ki-S1, decreasing affinities were observed (in that order) with the antibodies: ICI antiserum, 17.7%; Ki-S4, 8%; Ki-S7, 7.5%; Ki-S8, 5.4%; and Ki-S6, 1.7% (Figure 9).
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Confocal Immunofluorescence Microscopy
In both MCF7 and HeLa cells, the immunoreactivity of the MAbs was restricted to the nucleus of interphase cells (Figure 10). Serial sections of individual nuclei showed that fluorescence was concentrated in discrete dots throughout the nucleoplasm. In cells with high antigen expression, fluorescence was distributed more or less homogeneously in the nucleus. In low-proliferating MCF7 cells, immunoreactivity was less intense, leaving many nuclei completely unstained. Only about 15% of the cells were strongly positive, whereas in HeLa cells 90% were strongly labeled. In control samples, omission of the first antibody reduced fluorescence below the limits of detection (data not shown).
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During chromosome organization at the beginning of mitosis, HeLa cells displayed a finely punctate fluorescence pattern. In L428 cells, Ki-S1 and Ki-S7 distinctly stained the centromere of mitotic chromosomes and weakly marked the chromosomal scaffold. In many cells, the immunoreactive protein appeared to be condensed along the nuclear membrane in a rim-like pattern. In addition, in both interphase cells and at the onset of mitosis, one or two clusters with strong reactivity were found in the majority of cells. These more intensely staining regions were particularly highlighted by the antibody Ki-S4.
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Discussion |
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The five MAbs described here were selected for their reactivity with a nuclear antigen expressed by proliferating cells. This antigen could be identified as the -isoform of human topoisomerase II by the following cri teria. (a) Determination of the molecular mass revealed a 170-kD protein recognized by all five antibodies. This is also the molecular weight of the
isoform of human topoisomerase II (
Nevertheless, immunoprecipitation with Ki-S1 yielded an additional protein of 180 kD, which is the size of topoisomerase IIß. The staining intensity of this band was about 4% that of the 170-kD signal. Despite the constant absence of such a band in Western blot experiments with our antibodies, this finding could have been difficult to interpret except on the basis of a crossreactivity with the ß-isoform. The recent work of Biersack and associates (1996) finally provides a different and plausible explanation. Indeed, in yeast cells expressing both human isoenzymes, these were found to form heterodimers at a substantial rate. Moreover, the presence of such heterodimers could also be demonstrated in (human) HeLa cells (-isoform, which corresponds to 10 times the relative concentration of the co-precipitated topoisomerase. We can therefore assume that topoisomerase II heterodimers are also present in L-428 cells and account for the 180-kD band in our immunoprecipitation experiment.
The binding affinity to topoisomerase II in vitro differs for the antibodies, with Ki-S1 showing the highest affinity (100%), followed (in decreasing order) by Ki-S4 (8%), Ki-S7 (7.5%), Ki-S8 (5.4%), and Ki-S6 (1.7%). Because this observation, together with slight differences in the intensity and subcellular distribution of immunostaining, suggested binding to different epitopes, we performed a Western blot experiment with proteolytic fragments of purified topoisomerase II
. The proteolytic digestion using V8-protease poses a problem insofar as this enzyme cuts at the carboxy side of glutamate residues, corresponding to 123 potential dissections of the topoisomerase II
molecule. Therefore, a complete proteolysis would have yielded minute fragments undetectable by Western blot analysis. By limiting the time of the enzymatic reaction to 5-15 min, we were able to generate peptides in the range of approximately 25-125 kD that were readily detected by immunoblotting. Each of our antibodies, with the exception of Ki-S7, revealed a distinctive pattern of protein bands indicative of different binding sites. Because the signal detectable with ICI antiserum also disappeared after 5-min digestion, it can be speculated that this antibody and Ki-S7 bind to neighboring epitopes at the extreme C-terminus (Figure 5). This is consistent with a 13-amino-acid binding site at the extreme C-terminus containing three V8-protease-sensitive residues that are likely to be rapidly lysed by the enzyme. A complete epitope mapping for all five antibodies is the subject of current investigations.
One year ago we provided evidence that the antigen recognized by Ki-S1 is identical to human topoisomerase II ( (corresponding to the 527 C-terminal residues) was probed in Western blot analysis with our five antibodies, and rabbit polyclonal antibody as a positive control. The polyclonal antiserum yielded a strong signal at approximately 60 kD that was visualized by all of our antibodies. However, of all five antibodies, Ki-S1 bound with the lowest avidity, whereas its affinity to the whole molecule was by far the strongest. These results suggest that Ki-S1 is likely to recognize several epitopes with sequence similarities, of which a lesser percentage is located in the C-terminus of topoisomerase II. Likewise, it seems improbable that this binding site should be located within the 1501-1530 region, which corresponds to the utmost C-terminal sequence of topoisomerase II
, since the Ki-S1 binding site was conserved even after proteolysis for 15 min. The fact that all five antibodies are directed to C-terminal sequences is in itself not surprising. The recently disclosed crystal structure of yeast topoisomerase II reveals that the C-terminus (residues 990-1202) contains several
-helices and two ß-sheets exposed at the surface of the folded molecule (
exists, the
-helical structure and/or the free accessibility of these sites may be responsible for their antigenic potential.
The results of confocal fluorescence microscopy are in line with those described for topoisomerase II in previous investigations ( specifically expressed in the fibrillar component of the nucleolus.
The tissue distribution of the immunoreaction underscores the proliferation specificity of topoisomerase II. Accordingly, the immunohistochemical assessment of Ki-S1 labeling indices turned out to be of high prognostic relevance in breast cancer (
content in various human malignancies. Because the expression levels of the enzyme seem to correlate with the susceptibility of tumor cells to anti-topoisomerase agents (
.
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Acknowledgments |
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Supported in part by a grant from the Deutsche Forschungsgemeinschaft KE 556/S=1.
We thank S. Gasser (IBI, Zurich, Switzerland) for donating the expression rector, for the C-terminus of topo II.
Received for publication June 28, 1996; accepted October 14, 1996.
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