EXPERIMENTAL

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RESEARCH

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Monoclonal Antibodies to Human DNA Topoisomerase I and the Two lsoforms of DNA Topoisomerase II: 170- and 180-kDa lsozymes CLAUDIA

NEGRI, ROBERTO CHIESA, ANTONELLA CERINO, MARCO BESTAGNO, CINZIA SALA, ZINI,* NADIR M. MARALDI,* AND GIULIA C. B. ASTALDI RICOTTI’

NICOLETTA Istituto

di Genetica Biochimica ed Evoluzionktica *Istituto di Citomorfologk Normale

de1 Consiglio Nazion& e Patologica de1 Consiglia

Press. Inc.

INTRODUCTION The nuclear enzymes DNA topoisomerase I and DNA topoisomerase II are of high interest, not only for studies on DNA replication, transcription, and repair [l-3], but also for clinical research. In fact both DNA topoisomerases I and II were found to be targets of autoantibodies present. in sera of patients affected by different autoimmune diseases [4-71; furthermore DNA topoisomerases were identified as targets of some antitumor drugs [8-lo] and antibiotics [ll]. In normal eukaryotic cells DNA topoisomerase I does not show relevant fluctuations across the cell cycle,

0014-4827192

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AND METHODS

Cell cultures. HeLa cells, grown as monolayers, were cultured in F12 medium (GIBCO, Grand Island, NY) supplemented with 10% FCS, 4 mM glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin, trypsinized when confluent (normally every 3-4 days), and splitted 1:lO in fresh medium. For evaluating the presence of the topoisomerase II isoforms in different stages of proliferation, HeLa cells were seeded on Day 0 at 5

be addressed.

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Pavin, Italy

which, on the contrary, have been observed for DNA topoisomerase II [ 11, Two distinct human DNA topoisomerase II isozymes have been described recently [ 12-141. The two isozymes not only show biophysical and biochemical differences, but they are also differentially regulated during cellular proliferation, as suggested by variation in their quantity detectable by polyclonal antibody [ 141. The existence of two forms of DNA topoisomerase II is also suggested indirectly by the incomplete inhibition of the enzyme activity with teniposide [ 151. In order to study with more accuracy the possible variation in quantity and localization of human topoisomerases in the course of cell growth, we produced several monoclonal antibodies (MoAbs) specific to DNA topoisomerase I, to 170-kDa DNA topoisomerase II, and to a very unstable form of 180-kDa DNA topoisomerase II. The latter bands at 150-kDa when the nuclear extract is handled as usual, whereas it bands at 180-kDa when the nuclear proteins are extracted very quickly and in the presence of protease inhibitors. The three groups of MoAbs have been characterized by different assays and their specificity has been proven by immunodeletion of the enzyme activity and by immunoprecipitation of DNA topoisomerases with wellcharacterized polyclonal antisera. The localization of the different types of topoisomerases has been determined by using the obtained MoAbs with immunofluorescence; the ultrastructural localization of the 180-kDa topoisomerase II isoform in cells and in nuclear and subnuclear fractions has been obtained by electron microscope immunocytochemis-

Several monoclonal antibodies of different isotypes specific to human DNA topoisomerase I, to 170- and 180-kDa DNA topoisomerase II isozymes, were produced and characterized. The specificity of monoclonal antibodies was confirmed by comparison with polyclonal antibodies by Western blot, by immunoprecipitation of enzyme activity, and by immunoprecipitation of DNA topoisomerases with characterized polyclonal antisera. Morphological studies performed by immunofluorescence indicate that the three groups of monoclonal antibodies (MoAbs) stain the nucleus with characteristic patterns, which can be compared with those obtained with polyclonal antibodies. In particular the MoAbs to the lOO-kDa DNA topoisomerase I stain the nucleolus and the nucleoplasm; the MoAbs to 170- and 180-kDa DNA topoisomerase II give completely distinct intranuclear patterns: those to the 170-kDa protein stain mainly the nucleoplasm, whereas those to the 180-kDa protein stain only the nucleolus. The two DNA topoisomerase II isozymes clearly exhibit fluctuations in their expression during cell growth: the 170-kDa isozyme is more abundant during the logarithmic phase of growth, while the 180-kDa isozyme is mainly present during the plateau phase of growth. o 1992 Academic

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delle Ricer&he Via Abbintegrasso 207, 27100, Nazionule de& Richer& c/o I.O.R., Bologna,

452

MoAbs

TO

HUMAN

DNA

X 10’ cell/ml. At different days, aliquots of 1.25 X lo5 cells were removed and lysed in sodium dodecyl sulfate (SDS). The crude extract from the lysed cells was electrophoresed and electroblotted as described below. Polyclonul antibodies. Rabbit antiserum to recombinant 170-kDa topoisomerase II was kindly given by Dr. L. F. Liu (Johns Hopkins Medical School, Baltimore, MD); rabbit antisera to synthetic dodecapeptides, as predicted by 170- and 180-kDa DNA topoisomerase II isozymes partial cDNA clones obtained from a human Raji HNz cDNA library [12], were kindly given by Dr. F. H. Drake (Smith Kline Beechman Pharmaceutical, King of Prussia, PA). Scl ‘IO/DNA topoisomerase I positive serum from a patient affected by progressive systemic sclerosis (PSS) was kindly provided by Professor A. Facchini (Department Internal Medicine, University of Bologna, Italy). These antibodies were used diluted from 1:250 to 1:2500. Purification of DNA topoisomerases. To obtain DNA topoisomerases from HeLa cells the procedure of Miller et al. [16] with minor modifications has been followed: final steps were hydroxylapatite chromatography followed by gel filtration on a 1.5-m Bio-Gel A column and DNA-cellulose chromatography. This preparation was frozen and stored in liquid nitrogen before being utilized to immunize mice. Preparation of monoclonal antibodies to topoisomerases. Female Balb/c mice, 8 to 12 weeks old, were injected intraperitoneum twice at 40-day intervals with 100 pg of the previously described topoisomerase preparation in Freund’s adjuvant. The third injection consisted of 100 pg of the same preparation in the absence of adjuvant and was given 40 days after the second injection. Three days later, the splenocytes were fused with the myeloma line Sp2/O/Ag14 and cultured in modified RPM1 1640 and supplemented with human endothelial culture supernatant as described [17, 181. Screening techniques. The supernatants of hybridomas were screened using the previously described topoisomerase preparation by ELISA [19, 201. The positive clones were expanded in massive culture and tested against total protein cellular extract by Western blot [21] to select the clones that recognize only single protein bands corresponding to DNA topoisomerases I and two isoforms of topoisomerase II. The supernatants of hybridomas containing the different MoAbs were used diluted up to 1:5000. Alternatively the MoAbs were used after affinity purification. The MoAb isotypes were determined using the “EIA Mouse Typer” (mouse subisotyping panel) from Bio-Rad (Richmond, CA). In order to obtain cellular extracts containing nondegraded 180kDa DNA topoisomerase II, HeLa cells were washed, immediately after harvesting, with cold phosphate buffer saline (PBS) containing protease inhibitors [5 fig/ml pepstatin-A, 1 mM phenylmethylsulfonyl fluoride, 10 pg/ml soybean trypsin inhibitor, and 1 mM benzamidine; Sigma, St. Louis, MO]. The cellular pellet was lysed with 2% SDS in PBS plus protease inhibitors at 70°C for 5 min [12,14], loaded on cold 7.5% polyacrylamide gel, and electrophoresed following Laemmli conditions [22]. The proteins were transferred as described by Towbin et al. [23] onto nitrocellulose membrane; the membrane was washed and incubated overnight with PBS, 10% fetal calf serum (GIBCO), 0.2% Tween 20 to prevent nonspecific bindings. The membrane (either whole or cut into strips) was then incubated for 3 h at 20°C with MoAbs and polyclonai rabbit antisera to 170- and 180-kDa DNA topoisomerase II isozymes. Bound immunoglobulins were detected by an alkaline phosphatase-conjugated anti-mouse or rabbit immunoglobulins secondary antibody (Bio-Rad). Visualization of immunoreactive peptides was obtained by p-nitrotetrazolium blue, 5bromo-4-chloro-3-indolyl phosphate (Sigma). Immunoprecipitation of DNA topoisomerase Z and II relaxation activity. Eight micrograms of DNA topoisomerase I or II was mixed in 100 ~1 of different dilutions of monoclonal antibodies for 2 h at room temperature, then overnight at 4°C. After incubation, the sam-

TOPOISOMERASES

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II

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ples were centrifuged at 13,000g for 20 min at 4°C and the supernatants tested for the presence of enzyme activity [19]. The standard assay mixture (20 11) for DNA topoisomerase I contained 50 n&f Tris-HCI, pH 7.9,120 m&f KCI, 10 mA4 D’M’, 0.5 m&f EDTA, 30 rglml BSA, 40 ng of naturally supercoiled plasmid PAT 153, and an aliquot of DNA topoisomerase I, purified according to Liu and Miller [24], sufficient to relax 100% of the substrate. The standard assay mixture (20 ~1) for DNA topoisomerase II contained 50 mM Tris-HCl, pH 7.9,120 mM KCl, 10 mM DTT, 0.5 n&f EDTA, 30 pg/ml BSA, 0.5 mM ATP, 40 ng of naturally supercoiled plasmid pAT 153, and an aliquot of DNA topoisomerase II sufficient to relax 100% of the substrate. After incubation at 37°C for 30 min, the reactions were stopped by the addition of 2 ~1 of 5% (w/v) SDS, 50% (v/v) glycerol, and 0.25 mg/ml of bromophenol blue. Reaction mixtures were loaded onto 1% agarose gels, made in TAE buffer (40 mh4Tris-acetate, 2 mMEDTA, 18 m&f NaCl, pH 8.0). Gels were run at 6 V/cm for 2 h. Gels were stained for 30 min with 1 @g/ml ethidium bromide and washed twice for 15 min with HzO. Photographs were taken under uv 312 nm light with Polaroid 55 film. Recognition of the DNA topoisomerases, after immurwprecipitation with characterized polyclonal antisera, by monoclonal antibodies. Seventy micrograms crude nuclear extract was added to 2 ~1 ofi (1) serum of PSS patient (anti DNA topoisomerase I positive); (2) rabbit antiserum to synthetic dodecapeptide as predicted by 170-kDa topoisomerase II isozyme partial DNA clone; (3) rabbit antiserum to synthetic dodecapeptide as predicted by 180-kDa topoisomerase II isozyme partial cDNA clone, incubated for 2 h at room temperature and overnight at 4°C. Protein A-Sepharose beads in 0.1 M potassium phosphate buffer (KPB), pH 8.0, were added to each tube and the samples were incubated 1 h at room temperature with shaking. The supernatant was collected to control if the enzyme was no longer detectable by the corresponding monoclonal, but still detectable by the other two monoclonals. The beads were washed three times in KPB. The immunoprecipitates were resuspended in SDS-sample buffer, incubated for 5 min at 100°C, and loaded onto SDS-polyacrylamide gel and then transferred onto nitrocellulose membrane for blotting analysis with the three groups of MoAbs and processed as reported above. Zmmunofluorescence studies. HeLa cells were cultured on glass coverslips, rinsed with PBS, fixed, and permeabilized with 100% acetone for 1 min at 20°C. After this treatment the cells were washed with PBS and incubated with the supernatants of hybridomas containing MoAbs diluted up to 1:50 for 30 min at room temperature and then, with the second antibody [F(ab), fragment, DAKO, Copenhagen, Denmark], conjugated with fluorescein isothiocyanate, for 30 min at room temperature and washed for 1 h in PBS. Immunofluorescence microscopy was carried out using a Leitz Orthoplan equipped with 63X objective.

RESULTS Characterization of the Preparation of the Topoisomerases Used for Obtaining the MoAbs The topoisomerase preparation, analyzed by SDSpolyacrylamide gel, presents major bands corresponding to topoisomerase II isoforms and a minor one corresponding to topoisomerase I (Fig. 1). This preparation, when used under standard assay conditions (see Immunoprecipitation of DNA Topoisomerase I and II Relaxation Activity), is able to relax 100% of supercoiled DNA.

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1 and Isotype of Antitopoisomerase TABLE

Specificity

MoAbs Isotype

MoAb

specific to lOO-kDa (DNA topoisomerase 6C2 6B5

protein I)

MoAbs specific to 150-kDa protein (180-kDa DNA topoisomerase 9 HlO 8D7 12E2 5B2 12E7 8F8

A FIG. preparation isomerases

B

1. SDS-polyacrylamide

gel electrophoresis of the protein used for obtaining the MoAbs against the DNA topo(B) and MW markers (A) stained with Coomassie blue.

Specificity of Mono&ma1 Antibodies

We obtained MoAbs which react with three different peptides when tested on total cellular proteins by Western blot. Figure 2 shows that: (1) one group of MoAbs is specific for a protein of apparent molecular weight of 170-kDa (lane B), which is also recognized by rabbit antiserum to recombinant 170-kDa DNA topoisomerase II (lane A); (2) the second group of MoAbs reacts with a protein of 150-kDa (lane C); (3) the third group of MoAbs (lane D) recognizes a protein of apparent molec-

ABC

DE

FIG. 2. Binding of different antibodies to DNA topoisomerase by Western blot, using an antigen crude cellular extract. Lane A, rabbit antiserum to recombinant DNA topoisomerase II. Lane B, 6F3 MoAb to the 170-kDa protein (1:4500). Lane C, SD7 MoAb to the 150-kDa protein (1:50). Lane D, 6B5 MoAb to the lOO-kDa protein (1:200). Lane E, serum from a Scl ‘IO/DNA topoisomerase I positive patient.

specific to 170-kDa protein (170-kDa DNA topoisomerase 6F3 6G2 7E6 llH12 lF6 9ElO 4A12 1OGl

IgM IgG2a

K K

IgGl IgG2a IgM IgGl IgGl IgG2a

K K X K K K

IgGl IgGl IgGl IgM IgGl IgGl IgGl IgGl

K K K K K K K K

II)

MoAbs

II)

ular weight of lOO-kDa, which is also stained by serum from Scl70/DNA topoisomerase I positive PSS patient (lane E). Table 1 summarizes the isotype and the specificity of obtained MoAbs. Immunoprecipitation Relaxation Activity

of DNA Topoisomerase I and II

To test whether the proteins recognized by the three groups of MoAbs have DNA topoisomerase activity, we used MoAbs to immunoprecipitate purified enzyme preparations. Figure 3 (top) shows that three different dilutions of the two monoclonal antibodies to the lOOkDa protein immunodelete DNA topoisomerase I activity (lanes 4-9), whereas an irrilevant MoAb (lanes lo12) and the medium in which MoAbs were cultured (lane 3) did not affect DNA topoisomerase I activity. Figure 3 (bottom) shows that three different dilutions of a MoAb to the 170-kDa protein as well as a MoAb to the 150-kDa protein immunodelete DNA topoisomerase II activity (lanes 5-7 and lanes 11-13, respectively). When the two MoAbs were mixed together a stronger reduction of the DNA topoisomerase II activity was observed (lanes 8-10). On the contrary, neither the culture medium (lane 3) nor an irrilevant MoAb (lane 4) affect the DNA topoisomerase II activity. All the MoAbs to 170and 150-kDa proteins (listed in Table 1) are able to precipitate DNA topoisomerase II activity (data not shown).

MoAbs

TO

HUMAN

DNA

RL. SC

123

4

5

67

6910111213

FIG. 3. Analysis on agarose gel of human DNA topoisomerase I (top) and topoisomerase II (bottom) relaxing activity after immunoprecipitation. (top) Lane 1, DNA substrate alone. Lane 2, DNA topoisomerase I without previous preincubation. Lane 3, enzyme preincubated with culture medium. Lanes 4-6, enzyme preincubated with 1:32,1:8, and 1:2 dilutions of 8C2 MoAb. Lanes 7-9, enzyme preincubated with 1:64, 1:32, and 1:8 dilutions of 6B5 MoAb. Lanes 10-12, enzyme preincubated with three different dilutions of an irrilevant MoAb (1:32, 1:8, and 1:2 dilutions of 9A6 MoAb specific to hnRNP Al). (bottom) Lane 1, DNA substrate alone. Lane 2, DNA topoisomerase II without previous preincubation. Lane 3, enzyme preincubated with culture medium. Lane 4, enzyme preincubated with 9A6 MoAb to hnRNP Al (1:4). Lanes 5-7, enzyme preincubated with 1:2, 1:4, and 1:8 dilutions of 6F3 MoAb. Lanes 8-10, enzyme preincubated with 1:2,1:4, and 1:8 dilutions of a pool of 6F3 and 8F8 MoAbs. Lanes 11-13, enzyme preincubated with 1:2, 1:4, and 1:8 dilutions of 8F8 MoAb. RL, relaxed topoisomers of plasmid PAT 153; SC, supercoiled form of the same DNA.

Recognition of the DNA Topoisomerases, after Immunoprecipitation with Characterized Polyclonal Antisera, by Monoclonal Antibodies

To further investigate whether the proteins recognized by the three groups of MoAbs correspond to the topoisomerases recognized by well-characterized polyclonal antisera, we immunoprecipitated DNA topoisomerases from crude nuclear extract with these antisera and detected the enzymes with MoAbs by Western blot. Figure 4A shows that, after immunoprecipitation with the serum of the PSS patient (anti DNA topoisomerase I positive), the MoAb to the lOO-kDa protein does not recognize DNA topoisomerase I in the nuclear extract (lane 3), while the MoAbs to 170-kDa (lane 1) and 150-kDa (lane 2) proteins still recognize the proteins to which they are specific. On the contrary, the immunoprecipitate is recognized by the MoAb to the lOO-kDa protein (lane 4), suggesting once more that this MoAb is specific to DNA topoisomerase I. Lane 5 refers to the nontreated nuclear extract stained by MoAb to the lOO-kDa protein. Similar results were found with MoAbs to the 170kDa protein. Figure 4B shows that nuclear extract immunodeleted with rabbit antiserum to synthetic dodecapeptide of 170-kDa DNA topoisomerase II is recog-

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nized by MoAbs to the 150-kDa protein (lane 2) and to the lOO-kDa protein (lane 3), but not by MoAb to the 170-kDa protein (lane 1). Again, the immunoprecipitate is recognized by MoAb to the 170-kDa protein (lane 4). Lane 5 refers to the nontreated nuclear extract stained with MoAb to the 170-kDa protein. Figure 4C shows the results obtained immunoprecipitating the nuclear extract with the rabbit antiserum to synthetic dodecapeptide of 180-kDa DNA topoisomerase II (lanes l-3). MoAb to the 150-kDa protein does not stain the nuclear extract any longer (lane 2), but stains the immunoprecipitate (lane 4). MoAbs to the lOO-kDa protein (lane 3) and to the 170-kDa protein (lane 1) recognize their antigens in spite of the fact that l&JO-kDa DNA topoisomerase II was immunoprecipitated. Lane 5 refers to nontreated nuclear extract stained with MoAb to the 150-kDa protein. These results indicate that the MoAbs to loo-, 170-, and 150-kDa proteins are specific, respectively, to DNA topoisomerase I, 170-kDa DNA topoisomerase II isozyme, and 180-kDa DNA topoisomerase II isozyme. The 150-kDa Protein Is a Degradation Product of a 180-kDa Protein

Since the MoAbs to the 150-kDa protein as well as rabbit antiserum to synthetic dodecapeptide of the 180kDa isozyme stain nucleoli by immunofluorescence (see Immunofluorescence Studies), and because the 180kDa isozyme has been described as an extremely unstable protein [14], we investigated whether the 150-kDa protein, which has DNA topoisomerase II activity, could be a degradation product of the 180-kDa topoisomerase II, by using, instead of cellular extract obtained by traditional methods [6, 211 and stored at -2O”C, freshly prepared cellular extracts (see Screening Techniques). The same MoAb stains a 150-kDa protein only, from a stored cellular extract (Fig. 5, lane C and Fig. 2, lane C) and also a 180-kDa protein (Fig. 5, lane D) from freshly prepared cellular extract. The latter protein is also stained by rabbit antiserum to a synthetic dodecapeptide of 180-kDa DNA topoisomerase II isozyme (Fig. 5, lane E). This suggests that the 150-kDa protein, which has DNA topoisomerase II activity and recognizes the same protein immunoprecipitated by rabbit antiserum to the 180-kDa isozyme, is a degradation product of the 180-kDa DNA topoisomerase II isozyme described by Drake et al. [12-141. This is further supported by the fact that all the monoclonal antibodies to the 150-kDa isoform also recognize the 180-kDa isoform. Furthermore the polyclonal antiserum to the 180-kDa isoform weakly recognizes also the 150-kDa protein, thus suggesting that the 15~ kDa protein is a degradation product of the 180-kDa protein and that the antibodies present in the polyclonal antiserum are mainly directed against epitopes

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FIG. 4. Blots with MoAbs to different DNA topoisomerases immunoprecipitated with polyclonal antisera. (A) Lanes l-3, nuclear extract supernatant after immunoprecipitation with human anti DNA topoisomerase I antiserum and stained with 1:4500 dilution of 6F3 MoAb to the 170-kDa protein (lane l), 1:50 dilution of 8D7 MoAb to the 150-kDa protein (lane 2), and 1:200 dilution of 6B5 MoAb to the lOO-kDa protein (lane 3); lane 4, immunoprecipitate of DNA topoisomerase I stained with 6B5 MoAb to the 100-kDa protein, (1:200); lane 5, nontreated nuclear extract stained with 6B5 MoAb to the lOO-kDa protein (1:200). (B) Lanes 1-3, nuclear extract supernatant after immunoprecipitation with rabbit antiserum to synthetic dodecapeptide of 170-kDa DNA topoisomerase II and stained with 1:4500 dilution of 6F3 MoAb to the 170-kDa protein (lane l), 1:50 dilution of 8D7 MoAb to the 150-kDa protein (lane 2), and 1:200 dilution of 6B5 MoAb to the lOO-kDa protein (lane 3); lane 4, immunoprecipitated 170-kDa DNA topoisomerase II stained with 6F3 MoAb to the 170-kDa protein (1:4500); lane 5, nontreated nuclear extract stained with 6F3 MoAb to the 170-kDa protein (1:4500). (C) Lanes 1-3, nuclear extract supernatant after immunoprecipitation with rabbit antiserum to synthetic dodecapeptide of 180-kDa DNA topoisomerase II and stained with 1:4500 dilution of 6F3 MoAb to the 170-kDa protein (lane l), 1:50 dilution of 8D7 MoAb to the 150-kDa protein (lane 2), and 1:200 dilution of 6B5 MoAb to the 100-kDa protein (lane 3); lane 4, immunoprecipitated 180-kDa DNA topoisomerase II stained with 8D7 MoAb to the 150-kDa protein, (1:50); lane 5, nontreated nuclear extract stained with 8D7 MoAb to the 150-kDa protein (1:50).

different from the ones recognized by the monoclonal antibodies; this is probably due to the fact that the rabbit polyclonal antiserum is directed to a synthetic dodecapeptide predicted by a 180-kDa DNA topoisomerase II isoform partial cDNA clone obtained from a human Raji-HN, cDNA library [12], whereas the monoclonal antibodies are directed to the native human DNA topoisomerases purified from HeLa cells. Therefore, the

ABC

DE

FIG. 5. Binding of different antibodies to DNA topoisomerase II by Western blot, using an antigen rapidly purified crude cellular extract (lanes A, B, D, E) and stored crude cellular extract (lane C). Lane A, 7E6 MoAb to the 170-kDa protein (1:2500). Lane B, rabbit antiserum to the 170-kDa isozyme. Lanes C and D, 8D7 MoAb to the 150/180-kDa protein (1:200). Lane E, rabbit antiserum to a dodecapeptide derived from the 180-kDa isozyme.

group of MoAbs against the 180-kDa topoisomerase II isoform will be referred to as anti-150/180-kDa MoAbs. Presence of 170- and 150/180-kDa Proteins in Cells at Different Stages of Proliferation

To investigate whether the 170- and 150/180-kDa proteins, recognized by the MoAbs, behave as the 170and 180-kDa DNA topoisomerase II isozymes during changes in HeLa cell proliferation rate [14], we tested the expression of the two proteins in cells cultured for different periods of time. Figure 6A shows that the ex-

2

3

4

5

2

FIG. 6. Binding of monoclonal antibodies ase II isozymes during cell cycle by Western with 8F8 MoAb to the 150/180-kDa protein extract from cell cultures from Days 2 to 5. 6G2 MoAb to the 170-kDa protein (1:3000) from cell cultures from Days 2 to 5.

3

4

5

to DNA topoisomerblot. Blot A was probed (1:lOOO) tested on crude Blot B was probed with tested on crude extract

MoAbs

TO HUMAN

DNA TOPOISOMERASES

pression of the 150/180-kDa protein is low at the beginning of the culture whereas it increases when cells reach the plateau phase (Days 4 and 5). On the contrary (Fig. 6B) the expression of 170-kDa protein is high when cells are in logarithmic growth (Days 2 and 3) and decreases when cells reach the plateau phase. These results indicate that the expression of the 150/180-protein and of the 170-kDa protein in cells at different stages of proliferation is similar to the expression described for 180and 170-kDa DNA topoisomerase II isozymes, using polyclonal antibodies [ 141. Immunofluorescence

Studies

When the three groups of MoAbs were tested on HeLa cells by immunofluorescence, three completely different patterns were observed: MoAbs specific to the lOO-kDa protein show a homogeneous staining of the nucleolus and a uniform finely punctate staining of the nucleoplasm (Fig. 7A), similar to that obtained using the serum of a Scl 70/DNA topoisomerase I positive PSS patient in which, however, the nucleoplasmic staining has a more coarse punctation (Fig. 7B). MoAbs specific to the 170-kDa protein give fine punctate fluorescence all over the nucleoplasm except the nucleolar domain (Fig. 7C,), as indicated by a comparison with a phase contrast image (Fig. 7CJ; this pattern is similar to that given by a rabbit antiserum specific to recombinant 170-kDa DNA topoisomerase II, in which a more intense cytoplasmic labeling is also present (Fig. 7D). MoAbs specific to the 150/180-kDa protein stain the nucleoli only (Fig. 7E). The rabbit antiserum specific to synthetic dodecapeptide of 180-kDa topoisomerase II isozyme strongly stains the nucleolus, though a punctate fluorescence is present also in the nucleoplasm (Fig. 7F). Similar results were obtained using phytohemoagglutinin (PHA)-stimulated peripheral blood lymphocytes obtained from healthy volunteer donors (data not shown). DISCUSSION

The lack of suitable specific monoclonal antibodies has made it difficult to analyze the content and stability of DNA topoisomerases during the cell cycle in autoimmune patients or in patients affected by malignancies or in aged donors and the mechanism of action of antitumor drugs specific to DNA topoisomerases. In the present paper we describe several monoclonal antibodies of different isotypes which recognize different forms of DNA topoisomerase I and II, both in HeLa cells and in normal PHA-stimulated human lymphocytes. These specific antibodies recognize three different

I AND II

proteins and have allowed us to identify isomerases by the following criteria:

457 them as topo-

(a) The first group of MoAbs recognizes the same protein band at lOO-kDa, stained by anti-SC1 ‘IO/DNA topoisomerase I serum by Western blot, immunoprecipitates DNA topoisomerase I activity, and recognizes the immunoprecipitate obtained with a characterized polyclonal antiserum. Moreover, they behave in immunofluorescence like anti-SC1 70 serum, showing a diffuse fine punctate fluorescence over the nucleoplasm and a homogeneous staining of the nucleolus, as reported previously [4, 25, 261. It is therefore likely that these MoAbs are specific to DNA topoisomerase I. (b) The second group of MoAbs: (1) recognizes the same protein band stained by two different polyclonal antisera specific to 170-kDa DNA topoisomerase II, by Western blot; (2) immunoprecipitates DNA topoisomerase II activity; (3) recognizes the immunoprecipitate of the characterized rabbit antiserum; (4) stains the nucleoplasm but not the nucleoli, like rabbit antiserum to 170-kDa DNA topoisomerase II and with a pattern similar to the one described in literature [27]; (5) is more abundant in rapidly proliferating cells, like it was described for the 170-kDa DNA topoisomerase II [14]. (c) The third group of MoAbs stains a 150-kDa protein, which is apparently a degradation product of a very labile 180-kDa protein, and stains exclusively the nucleoli. This group of MoAbs recognizes a protein that conceivably, corresponds to the 180-kDa DNA topoisomerase II isozyme described by Drake et al. [13, 141 and is know to be very unstable [12,14] because: (1) it recognizes in Western blot a protein band stained by polyclonal antiserum specific to a synthetic dodecapeptide of 180-kDa DNA topoisomerase II isozyme; (2) it immunoprecipitates DNA topaisomerase II activity; (3) it stains the immunoprecipitate of the characterized rabbit antiserum; (4) it stains almost exclusively the nucleolus in immunofluorescence, in a manner that is more specific than that exhibited by the polyclonal antiserum against the 180-kDa isozyme; (5) it increases in amount as cells reach the plateau phase of growth, as described for the 180-kDa DNA topoisomerase II [14]. The immunofluorescence patterns obtained with the MoAbs against topoisomerase I and the different forms of topoisomerase II are similar to those obtained by polyclonal antibodies, but the staining patterns are more specific. In fact, the polyclonal antibodies always give rise, besides a principal fluorescence pattern, also to a diffuse fluorescence in other districts; for example, the rabbit antiserum to 170-kDa topoisomerase II shows a partial positivity also for the nucleolus, which is completely negative in the samples stained with the anti170-kDa MoAbs and the polyclonal antibody against the 180-kDa isoform also shows a diffuse fluorescence over the nucleoplasm, which is absent in the samples

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FIG. 7. Immunofluorescence on HeLa cells using different antibodies to DNA topoisomerases: (A) 6B5 MoAb to the lOO-kDa protein (1:5); (B) Scl70/DNA topoisomerase I antiserum; (C,) 6G2 MoAb to the 170-kDa protein (1:5); (C,) phase contrast of the same field; (D) rabbit antiserum to recombinant 170-kDa DNA topoisomerase II; (E) SD7 MoAb to the 150/180-kDa protein (1:5); (F) rabbit antiserum to a dodecapeptide derived from the X30-kDa DNA topoisomerase II isozyme. Bar, 10 pm.

MoAbs

TO

HUMAN

DNA

TOPOISOMERASES

stained with the anti-150/180-kDa MoAbs. This indicates that the used MoAbs present an higher specificity with respect to the polyclonal antibodies. All the monoclonal antibodies belonging to the same group behave in the same way in the experiments presented in this paper, indicating that they all recognize their own specific protein, and therefore they will be useful to study in detail the epitopes of DNA topoisomerase I and of the two isoforms of DNA topoisomerase II, for example to analyze the sites of binding of antitumors drugs to these enzymes or alterations of the enzymes in drug resistant cells. Further evidence of the fine nucleolar localization of the 150/180-kDa isoform and of its involvement in the structural arrangement of the nucleolar remnant are presented in the accompanying paper [28]. The research is supported by grant PF Biotecnologie e Biostrumentazioni from Consiglio Nazionale delle Ricerche (C.N.R.), Rome, Italy. Claudia Negri is a recipient of a fellowship from Buzzati Traverso Foundation, Rome, Italy. We wish to express our gratitude to: Dr. L. F. Liu for generously providing antiserum to recombinant DNA topoisomerase II; Dr. F. H. Drake for generously providing antisera to 170- and 180-kDa DNA topoisomerase II isozymes; Professor A. Facchini for providing a Scl 70/DNA topoisomerase serum of a PSS patient; Dr. G. Ciarrocchi for helpful discussion and, together with Drs. M. Fontana and C.S., for having provided us with partially purified DNA topoisomerase II preparation; Mrs. Chiesa for technical help; Dr. A. Scovassi for criticism of the manuscript; Mrs. D. Tavarne for skilful preparation of the manuscript; and Mr. A. Valmori for the photographic work.

8.

10. 11.

13.

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