Clin. exp. Immunol. (1991) 84, 245-249
ADONIS
000991049100136W
Autoantibodies to DNA topoisomerase II in juvenile rheumatoid arthritis K. L. ZUKLYS, I. S. SZER* & W. SZER Department of Biochemistry, New York University School of Medicine, New York, NY, and *Department of Pediatrics, Tufts University School of Medicine, Boston, MA, USA (Acceptedfor publication 20 November 1990)
SUMMARY Sera from 58 children with juvenile rheumatoid arthritis were examined for the presence of antibodies to DNA topoisomerase II. Eight sera were reactive in immunoblotting with purified human topoisomerase II and a protein encoded by a cloned cDNA expressed in Escherichia coli which represents the carboxy-terminal domain of the human enzyme. In addition, the sera detect topoisomerase II in mitotic chromosomes and chromosome scaffolds. Five of the sera bind to the native enzyme in solution and deplete such solutions of the active enzyme. All eight sera also contain antibodies to nuclear antigens other than topoisomerase II.
Keywords topoisomerase ting
II
juvenile rheumatoid arthritis recombinant protein immunoblot-
INTRODUCTION The presence of circulating antibodies to nuclear antigens (ANA) is a hallmark of rheumatic diseases, and their antigen specificities may serve as markers of the disease. Some ANA are important tools for investigations of intracellular processes and structures (reviewed by Tan, 1989). ANA are frequently found in sera from children with juvenile rheumatoid arthritis (JRA), but little is known about their antigen specificities (Malleson et al., 1989; Pauls et al., 1989; 0stensen et al., 1989; Haber, Osborne & Moore, 1989). Studies with these sera are hindered by their low affinities and the large number of potential antigens detected on immunoblots of nuclear extracts. Except for histones, none of these proteins has been identified. Antigens commonly reactive with sera from patients with other rheumatic diseases, e.g. Sm, RNP, DNA, La, Ro and Scl-70, are very infrequently detected by sera from children with JRA (Lang & Shore, 1990). Here we demonstrate the presence of antibodies to DNA topoisomerase II in some sera from children with JRA. Topoisomerase II, which alters the topological state of DNA, is an essential enzyme in DNA replication and, possibly, in repair, recombination and transcription (reviewed by Wang, 1985). This enzyme is a major component of mitotic chromosomes and chromosome scaffolds (Earnshow et al., 1985; Gasser & Laemmli, 1987). It is a marker of the proliferative state of the K.L.Z. present address: Department of Microbiology, Cornell University Medical College, New York, NY USA. Correspondence: Dr W. Szer, Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, NY 10016 USA.
245
cell (Duguet et al, 1983; Heck & Earnshow, 1986), and appears to be the target of the anti-tumour drugs amsacrine and etoposide (Nelson, Tewey & Liu, 1984; Chen et al., 1984).
PATIENTS AND METHODS Patients and sera Random serum samples were obtained from 58 children (aged 2-15 years) who met the diagnostic criteria for JRA (Cassidy et al., 1986) and attend the Floating Hospital's Pediatric Rheumatology Clinic, Tufts University School of Medicine (pauci-articular onset 44, polyarticular onset 14, systemic onset four). Among the group of children with pauci-articular onset JRA, 15 (34%) had iritis. Within the polyarticular onset, two children had positive rheumatoid factors; neither of these had vasculitis or nodules. Children with systemic onset disease were afebrile at the time of this study. Thirty control sera were obtained from the same clinic (healthy donors seven, dermatomyositis 13, mixed connective tissue disease four, localized scleroderma four, systemic lupus erythematosus two). Rabbit antiserum to recombinant human topoisomerase II was kindly provided by Dr L. F. Liu (Johns Hopkins University). Preparations of antigens for immunoblots Topoisomerase II purified from HeLa cells was a gift from Dr F. B. Dean (Sloan-Kettering Institute). Escherichia coli cell lysates containing a recombinant fragment of human topoisomerase II were prepared according to Hwang et al. (1989). The host strain E. coli BL21 (DE3) and the expression vector pZII06 which carries a cDNA fragment corresponding to the Cterminal one-third of the enzyme were generous gifts from Dr
246
K. L. Zuklys, I. S. Szer & W. Szer (a )
I 2 34 5 6 7 8 9 10
(b)
1
2 3 4 5 67 8 910 11 12 12
200 -
.: t
11693-
69-
93
-
69
-
3
.
.4=
46-
463030-
4-
14
-
Fig. 1. Binding of autoimmune sera and control rabbit anti-topoisomerase serum to topoisomerase II and a cloned recombinant fragment of the enzyme. (a) Immunoblots of purified topoisomerase II: lane 1, rabbit serum; lanes 2-6, examples of JRA sera; lane 7, normal serum; lanes 8, 9 and 10, SLE, dermatomyositis and scleroderma sera, respectively; (b) immunoblots of the recombinant protein: odd-numbered lanes, extracts of non-induced E. coli; even-numbered lanes, extracts of induced bacteria harboring clone pZII06. Lanes 1 and 2, the bacterial extract, Coomassie blue staining; the recombinant protein is indicated by the dot between lanes 1 and 2; lanes 3 and 4, rabbit serum; lanes 5-10, examples of JRA sera positive in experiments (a); lanes 11 and 12, scleroderma serum; (c) immunoblots of micrococcal nuclease and pancreatic DNAse I. Four different human sera were combined, diluted 1/100 and used on blots of micrococcal nuclease (lanes 1 and 2) and DNAse (lane 3).
L. F. Liu. The lysate in buffer A (20 mm Tris-HCl, pH 8-0, 100 NaCl, 1-0 mM EDTA, 3-0 mm CaCl2 and 1-0 mM PMSF) was digested with 100 pg/ml DNAse I (Worthington, Freehold, NJ) and 50 pg/ml micrococcal nuclease (Sigma, St Louis, MO) for I h at 240C to reduce smearing of the enzyme fragment on gels. Host cell lysate from non-induced cells was used as control. Mitotic chromosomes from colcemide-arrested HeLa cells were prepared as described by Gasser & Laemmli (1987). The chromosomes were suspended in buffer A and treated with nucleases as above. Chromosome scaffolds were prepared from nuclease-digested mitotic chromosomes as described (Lewis & Laemmli, 1982).
ml). Incubation was continued for I h and the suspension was centrifuged. The supernatant was tested for its ability to decatanate 500 ng of trypanosome mitochondrial kinetoplast DNA (gift of Dr J. Hurwitz, Sloan-Kettering Institute) at 370C for 30 min in 10 p1 of the standard topoisomerase II reaction buffer (50 mm Tris-HCl, pH 7 5; 60 mm KCI, 0-5 mm ATP, 9 5 mM MgCl2, 05 mM Na EDTA, 0-5 mM DTT). The reaction was stopped by addition of sucrose to 4% and SDS to 0-1% and heating to 650C before analysis on 0-8% agarose gels (Miller, Liu & Englund, 1981).
Immunoblotting Proteins were separated on 7-15% SDS-polyacrylamide gradient gels (Laemmli, 1970) with the following amounts layered per 1-0 cm of gel: mitotic chromosomes 100 pg; scaffold proteins, 8 pg; purified topoisomerase II, 5 pg; bacterial lysate, 40 pg. After electrophoretic transfer (16 h, 60 V) to nitrocellulose membranes (Towbin, Staehelin & Gordon, 1979), the blots were blocked and incubated with patient or control serum (1/ 100 dilution, except rabbit antiserum at 1/1000) overnight at 40C. The strips (3-mm wide) were treated with '25l-labelled protein A (300 x I03 ct/min per ml) for I h and autoradiographed overnight.
Identification of topoisomerase II
mM
Assay of topoisomerase II activity The purified enzyme (12 ng) was incubated with patient or control serum (1/30 dilution) in 30 p1 of phosphate-buffered saline (PBS; 150 mm, pH 7-5) for 2 h at 4 C followed by the addition of 10 p1 of a protein A-Sepharose suspension (30 mg/
RESULTS AND DISCUSSION In an ongoing investigation on the antibody profile of sera from children with JRA, 58 sera were screened by immunoblotting. Using a HeLa cell nuclear sonicate as the source of antigen(s), we observed that some JRA sera react with a protein of about 170 kD, which corresponds to the molecular weight of topoisomerase II (unpublished observations). The identity of the 170kD antigen was determined as follows. First, a purified preparation of topoisomerase II isolated from HeLa cells was used as antigen on immunoblots. As seen in Fig. I a, this antigen is recognized by some JRA sera and by control rabbit antibodies to recombinant human topoisomerase II. Secondly, we have tested blots of extracts from E. coli cells harboring the expression vector pZII-06 containing a cDNA fragment of the human topoisomerase gene (Hwang et al., 1989). After induction, these cells overproduce a recombinant polypeptide which corresponds to the C-terminal one-third of the enzyme (Fig 1 b, lanes 1 and 2). As shown in Fig. Ib, lanes 3-12, JRA sera react
Topoisomerase II antibodies 2
3
v
3 7 8
3
i
-
1
3
k
Fig. 2. Analysis of DNA topoisomerase II decatanation activity after immunoprecipitation with JRA sera. After the removal of immune complexes, several dilutions of the adsorbed enzyme solutions were tested; the 1/40 dilution, which gives up to a 100% decrease in enzyme activity, is shown. Lane 1, input DNA (remains trapped in the well); lane 2, no serum; lane 3, normal serum; lane 4, scleroderma serum; lane 5, rabbit anti-topoisomerase II; lanes 6-13, JRA sera; lane 14, as in lane 2, but ATP is omitted. kt, kinetoplast DNA; mon, monomer DNA circles.
1
2 3 .A
..
200126-
9369-
46-
30-
14-
Fig. 3. Reactivity of autoimmune sera and control rabbit antitopoisomerase II serum with mitotic chromosomes (lanes 1-12) and chromosome scaffolds (lanes 13 and 14). Lane I, rabbit serum; lanes 2-4, examples of JRA sera positive in experiments of Fig. 1; lanes 5-7, JRA sera negative in these experiments; lanes 8 and 9, normal sera; lanes 10, I I and 12, MCTD, scleroderma and SLE sera, respectively; lanes 13 and 14, positive JRA sera reacted with chromosome scaffolds. Open arrows indicate
positions
of micrococcal nuclease
antigens (see Fig.
b and
c).
fragment which is also recognized by the rabbit sera, including those from healthy indiscleroderma patients, react with several antigens
with the cloned
antibodies. Human viduals and
present
in both induced and non-induced
bacterial extracts
the rabbit anti-topoisomerase II serum (Fig. la, lanes 3 and 4). Surprisingly, most of these antigens (open arrows, Fig. Ib and c) are polypeptides detected by human sera on blots of micrococcal nuclease (Fig. lc, lanes which are not detected
by
247
1-3), the enzyme used to treat the bacterial lysate prior to SDSPAGE. The experiments shown in Fig. I identify the 170-kD antigen and demonstrate that at least some epitopes recognized by JRA sera are located in the C-terminal part of the protein. Eight sera from children with JRA (eight out of 58, 14%) react with purified topoisomerase II as well as with the recombinant protein; none of the 30 control sera is positive. The ability of topoisomerase II to decatanate mitochondrial DNA kinetoplast networks and liberate monomer DNA circles is a specific assay of the enzyme (Miller et al., 1981). The immunoadsorption procedure has been used to test the ability of the JRA sera to bind active enzyme in solution. When five out of the eight immunoblot-positive sera are added to a solution of purified enzyme followed by protein A-Sepharose and the removal of immune complexes, a decrease in enzyme activity is observed (Fig. 2, lanes 6, 9, 10, 12 and 13). The rabbit antitopoisomerase II serum is also active in this assay (Fig. 2, lane 5). Control experiments using several sera from healthy children and those from children with scleroderma were negative (e.g. Fig. 2, lanes 3 and 4). It should be noted that the five positive JRA sera were the strongest in the immunoblot assay. Generally, sera from children with JRA have low antigen affinities and are more often reactive in the immunoblot assay than in immunoprecipitation (unpublished observation). Detection of topoisomerase II in chromosomes The presence of topoisomerase II in mitotic chromosomes and chromosome scaffolds is well established (Wang, 1985; Earnshow et al., 1985; Gasser & Laemmli, 1987). As seen in Fig. 3, lanes 1-4, the JRA sera as well as anti-topoisomerase II antibodies raised in rabbits detect the 1 70-kD antigen on blots of isolated mitotic chromosomes. However, the detection of a 170-kD antigen on blots of mitotic chromosomes does not by itself identify topoisomerase II. In addition to the eight positive sera, six JRA sera which are negative in the assays of Fig. 1 react with high mol. wt proteins similar in size to topoisomerase II (e.g. Fig. 3, lanes 6 and 7). Such proteins are known to be present in chromosomal preparations (Hoffmann et al., 1989). Also note from Fig. 3 that human sera react with antigens other than topoisomerase II; the micrococcal nuclease proteins are indicated by open arrows (Fig. I b). When the nuclease-digested mitotic chromosomes are treated with 2-0 M NaCl and extensively washed to purify scaffold proteins (Lewis & Laemmli, 1982), the only antigen seen on blots is the 1 70-kD polypeptide (Fig. 3, lanes 13 and 14). Some of the JRA sera and a control scleroderma serum react on blots of mitotic chromosomes with antigens of about 100 kD corresponding in molecular weight to topoisomerase I (Fig. 3, lanes 2, 4-6 and 1 1). When tested with Scl-70 as antigen, none of these sera (out of 18), except for the control scleroderma serum, was found positive (not shown). Anti-topoisomerase II antibodies could be useful tools for investigations on the function and distribution of this enzyme. Although all eight anti-topoisomerase II sera contain antibodies to other nuclear antigens (Table 1), immunopurification using the recombinant protein provides a simple method for obtaining antibodies monospecific for topoisomerase II. The immunochemical and clinical characteristics of the eight children whose sera react with topoisomerase II are summarized in Table 1. Seven of the eight have pauci-articular onset and only one has active iritis. Although all eight children have relatively
K. L. Zuklys, I. S. Szer & W. Szer
248
Table 1. Clinical and immunological data from anti-topoisomerase II-positive (immunoblotting) children with (JRA) juvenile rheumatoid arthritis
Age of onset (years)
JRA onset subtype
Disease duration (years)
Indirect immunofluorescence titre/pattern
Binding in solution*
Other antigenst
M
+
H H H
M -
+
1:80
H
M
+
2 2
1:40 1:40
H H
M -
+
2
1:640
H
M
+
45, 60, 80, 90, 100, 120 and > 120 kD 80 kD 38 and 90 kD 45, 60, 100 and > 120 kD 60,80,100,120 and > 120 kD 120kD 60,80,90,100 and > 120 kD Histone Hi, 60, 80, 100 and 195 kD
Patient
Sex
I
M
7
Pauci
0 25
1:320
H
2 3 4
F F F
58 85 2 25
Pauci Pauci Pauci
1-5 35 0 25
1:160 1:40 1:160
5
F
1
Poly
10
6 7
M F
5 4
Pauci Pauci
8
F
10
Pauci
Iritist
Activity of arthritis (time of study)
-
Mild
+ -
Moderate Mild Mild
-
Absent
-
Absent Mild
-
Moderate
H, homogeneous; M, also stains condensed chromosomes of metaphase cells. *See Fig. 2 and text. tMol. wt of unidentified antigens, other than 170 kD, detected on blots of the HeLa cells nuclear sonicate (unpublished data). J Active iritis at time of study.
mild arthritis, the presence of this autoantibody does not differentiate between the variety of heterogeneous disorders classified as JRA. In addition, serum from each child reacts with other, mostly unidentified antigens. Antibodies which react with enzymes such as aminoacyltRNA synthetases and DNA topoisomerase I have been found in patients with inflammatory myopathies and systemic sclerosis (Weiner et al., 1988; Targoff & Arnett, 1990). Furthermore, the presence of these antibodies seems to correspond to particular clinical patterns of disease expression (Yoshida et al., 1983). Until now antibodies recognizing human topoisomerase II have only been described in systemic lupus erythematosus and in cryptogenic fibrosing alveolitis (Hoffmann et al., 1989; Meliconi et al., 1989). Here we document the presence of anti-topoisomerase II antibodies in eight children with mild JRA, whose autoantibody profile does not suggest a distinct diagnostic entity.
ACKNOWLEDGMENTS We thank Dr H. Sierakowska for helpful discussions, Dr L. F. Liu (Johns Hopkins University) for the gift of rabbit serum to human topoisomerase II and plasmid pZII-06 and Drs J. Hurwitz and F. B. Dean (Sloan-Kettering Institute) for purified topoisomerase II and trypanosome mitochondrial kinetoplast DNA. This work was supported by National Institutes of Health grants GM23705-17 and CA16239-16.
REFERENCES CASSIDY, J.T., LEVINSON, J.E., BASS, J.C., BAUM, J., BREWER, E.J. JR, FINK, C.W., HANSON, V., JACOBS, J.C., MASI, A.T., SCHALLER, J.G., FRIES, J.F., MCSHANE, D. & YOUNG, D. (1986) A study of classification criteria for a diagnosis of juvenile rheumatoid arthritis. Arthritis Rheum. 29, 274.
CHEN, G.L., YANG, L., ROWE, T.C., HALLIGAN, B.D., TEWEY, K.M. & Liu, L.F. (1984) Nonintercalative antitumor drugs interfere with breakage-reunion reaction of mammalian DNA topoisomerase II. J. biol. Chem. 259, 13,560. DUGUET, M., LAVENOT, C., HARPER, F., MIRAMBEAU, G. & DE ROCONDO, A.M. (1983) DNA topoisomerases from rat liver: physiological variations. Nucleic Acids Res. 11, 1059. EARNSHOW, W.C., HALLIGAN, B., COOKE, C.A., HECK, M.M.S. & Liu, L.F. (1985) Topoisomerase II is a structural component of chromosome scaffolds. J. cell. Biol. 100, 1706. GASSER, S.M. & LAEMMLI, U.K. (1987) Improved methods for the isolation of individual and clustered mitotic chromosomes. Exp. cell. Res. 173, 85. HABER, P.L., OSBORN, T.G. & MOORE, T.L. (1989) Antinuclear antibody in juvenile rheumatoid arthritis sera reacts with 40-50 kD antigen(s) found in HeLa nuclear extracts. J. Rheumatol. 16, 949. HECK, M.M.S. & EARNSHOW, W.C. (1986) Topoisomerase II: a specific marker for cell proliferation. J. cell. Biol. 103, 2569. HOFFMANN, A., HECK, M.M.S., BORDWELL, B.J., ROTHFIELD, N.F. & EARNSHOW, W.C. (1989) Human antibody to topoisomerase II. Exp. cell. Res. 180, 409. HWANG, J., SHYY, S., CHEN, A.Y., JUAN, C.C. & WHANG-PENG, J. (1989) Studies of topoisomerase-specific antitumor drugs in human lymphocytes using rabbit antisera against recombinant human topoisomerase II polypeptide. Cancer Res. 49, 958. LAEMMLI, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680. LANG, B.A. & SHORE, A. (1990) A review of current concepts on the pathogenesis of Juvenile Rheumatoid Arthritis. J. Rheumatol. (Suppl. 21) 17, 1. LEWIS, C.D. & LAEMMLI, U.K. (1982) Higher order metaphase chromosome structure: evidence for metalloprotein interactions. Cell, 29, 171. MALLESON P., PETTY, R.E., FUNG, M. & CANDIDO, E.P.M. (1989) Reactivity of antinuclear antibodies with histones and other antigens in juvenile rheumatoid arthritis. Arthritis Rheum. 32, 919. MELICONI, R., BESTAGNO, M., STURANI, C., NEGRI, C., GALAVOTTI, B., SALA, C., FACCHINI, A., CIARROCCHI, G., GASBARRINI, G. & ASTALDI
Topoisomerase II antibodies Ricor, G.C.B. (1989) Autoantibodies to DNA topoisomerase II in cryptogenic fibrosing alveolitis and connective tissue disease. Clin. exp. Immunol. 76, 184. MILLER, K.G., Liu, L.F. & ENGLUND, P.T. (1981) A homogeneous type II DNA topoisomerase from HeLa cell nuclei. J. biol. Chem. 256, 9334. NELSON, E.M., TEWEY, A.M. & Liu, L.F. (1984) Mechanism of antitumor drug action: poisoning of mammalian topoisomerase II by 4'-(9-acridinylamino)-methanesulfon-m-anisidide. Proc. natl Acad. Sci. USA, 81, 1361. OSTENSEN, M., FREDRIKSEN, K., KASS, E. & REKWIG, O.-P. (1989) Identification of antihistone antibodies in subsets of juvenile chronic arthritis. Ann. rheum. Dis. 48, 14. PAULS, J.D., SILVERMAN, E., LAXER, R.M. & FRITZLER, M.J. (1989) Antibodies to histones HI and H5 in sera of patients with juvenile rheumatoid arthritis. Arthritis Rheum. 32, 877. TAN, E.M. (1989) Antinuclear antibodies: diagnostic markers for
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autoimmune diseases and probes for cell biology. Adv. Immunol. 44, 93. TARGOFF, I.N. & ARNETT, F.C. (1990) Clinical manifestations in patients with antibody to PL-12 antigen (alanyl-tRNA synthetase). Am. J. Med. 88, 241. TOWBIN, H., STAEHELIN, T. & GORDON, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. natl Acad. Sci. USA, 76, 4350. WANG, J.C. (1985) DNA topoisomerases. Annu. Rev. Biochem. 54,665. WEINER, E.S., EARNSHOW, W.C., SENECAL, J.-L., BORDWELL, B., JOHNSON, P. & ROTHFIELD, N.F. (1988) Clinical associations of anticentromere antibodies and antibodies to topoisomerase I. Arthritis Rheum. 31, 378. YOSHIDA, S., AKIZUKI, M., MIMORI, T., YAMAGATA, H., INADA, S. & HOMMA, M. (1983) The precipitating antibody to an acidic nuclear protein antigen, the Jo-1, in connective tissue diseases. Arthritis Rheum. 26, 604.
ADONIS
000991049100136W
Autoantibodies to DNA topoisomerase II in juvenile rheumatoid arthritis K. L. ZUKLYS, I. S. SZER* & W. SZER Department of Biochemistry, New York University School of Medicine, New York, NY, and *Department of Pediatrics, Tufts University School of Medicine, Boston, MA, USA (Acceptedfor publication 20 November 1990)
SUMMARY Sera from 58 children with juvenile rheumatoid arthritis were examined for the presence of antibodies to DNA topoisomerase II. Eight sera were reactive in immunoblotting with purified human topoisomerase II and a protein encoded by a cloned cDNA expressed in Escherichia coli which represents the carboxy-terminal domain of the human enzyme. In addition, the sera detect topoisomerase II in mitotic chromosomes and chromosome scaffolds. Five of the sera bind to the native enzyme in solution and deplete such solutions of the active enzyme. All eight sera also contain antibodies to nuclear antigens other than topoisomerase II.
Keywords topoisomerase ting
II
juvenile rheumatoid arthritis recombinant protein immunoblot-
INTRODUCTION The presence of circulating antibodies to nuclear antigens (ANA) is a hallmark of rheumatic diseases, and their antigen specificities may serve as markers of the disease. Some ANA are important tools for investigations of intracellular processes and structures (reviewed by Tan, 1989). ANA are frequently found in sera from children with juvenile rheumatoid arthritis (JRA), but little is known about their antigen specificities (Malleson et al., 1989; Pauls et al., 1989; 0stensen et al., 1989; Haber, Osborne & Moore, 1989). Studies with these sera are hindered by their low affinities and the large number of potential antigens detected on immunoblots of nuclear extracts. Except for histones, none of these proteins has been identified. Antigens commonly reactive with sera from patients with other rheumatic diseases, e.g. Sm, RNP, DNA, La, Ro and Scl-70, are very infrequently detected by sera from children with JRA (Lang & Shore, 1990). Here we demonstrate the presence of antibodies to DNA topoisomerase II in some sera from children with JRA. Topoisomerase II, which alters the topological state of DNA, is an essential enzyme in DNA replication and, possibly, in repair, recombination and transcription (reviewed by Wang, 1985). This enzyme is a major component of mitotic chromosomes and chromosome scaffolds (Earnshow et al., 1985; Gasser & Laemmli, 1987). It is a marker of the proliferative state of the K.L.Z. present address: Department of Microbiology, Cornell University Medical College, New York, NY USA. Correspondence: Dr W. Szer, Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, NY 10016 USA.
245
cell (Duguet et al, 1983; Heck & Earnshow, 1986), and appears to be the target of the anti-tumour drugs amsacrine and etoposide (Nelson, Tewey & Liu, 1984; Chen et al., 1984).
PATIENTS AND METHODS Patients and sera Random serum samples were obtained from 58 children (aged 2-15 years) who met the diagnostic criteria for JRA (Cassidy et al., 1986) and attend the Floating Hospital's Pediatric Rheumatology Clinic, Tufts University School of Medicine (pauci-articular onset 44, polyarticular onset 14, systemic onset four). Among the group of children with pauci-articular onset JRA, 15 (34%) had iritis. Within the polyarticular onset, two children had positive rheumatoid factors; neither of these had vasculitis or nodules. Children with systemic onset disease were afebrile at the time of this study. Thirty control sera were obtained from the same clinic (healthy donors seven, dermatomyositis 13, mixed connective tissue disease four, localized scleroderma four, systemic lupus erythematosus two). Rabbit antiserum to recombinant human topoisomerase II was kindly provided by Dr L. F. Liu (Johns Hopkins University). Preparations of antigens for immunoblots Topoisomerase II purified from HeLa cells was a gift from Dr F. B. Dean (Sloan-Kettering Institute). Escherichia coli cell lysates containing a recombinant fragment of human topoisomerase II were prepared according to Hwang et al. (1989). The host strain E. coli BL21 (DE3) and the expression vector pZII06 which carries a cDNA fragment corresponding to the Cterminal one-third of the enzyme were generous gifts from Dr
246
K. L. Zuklys, I. S. Szer & W. Szer (a )
I 2 34 5 6 7 8 9 10
(b)
1
2 3 4 5 67 8 910 11 12 12
200 -
.: t
11693-
69-
93
-
69
-
3
.
.4=
46-
463030-
4-
14
-
Fig. 1. Binding of autoimmune sera and control rabbit anti-topoisomerase serum to topoisomerase II and a cloned recombinant fragment of the enzyme. (a) Immunoblots of purified topoisomerase II: lane 1, rabbit serum; lanes 2-6, examples of JRA sera; lane 7, normal serum; lanes 8, 9 and 10, SLE, dermatomyositis and scleroderma sera, respectively; (b) immunoblots of the recombinant protein: odd-numbered lanes, extracts of non-induced E. coli; even-numbered lanes, extracts of induced bacteria harboring clone pZII06. Lanes 1 and 2, the bacterial extract, Coomassie blue staining; the recombinant protein is indicated by the dot between lanes 1 and 2; lanes 3 and 4, rabbit serum; lanes 5-10, examples of JRA sera positive in experiments (a); lanes 11 and 12, scleroderma serum; (c) immunoblots of micrococcal nuclease and pancreatic DNAse I. Four different human sera were combined, diluted 1/100 and used on blots of micrococcal nuclease (lanes 1 and 2) and DNAse (lane 3).
L. F. Liu. The lysate in buffer A (20 mm Tris-HCl, pH 8-0, 100 NaCl, 1-0 mM EDTA, 3-0 mm CaCl2 and 1-0 mM PMSF) was digested with 100 pg/ml DNAse I (Worthington, Freehold, NJ) and 50 pg/ml micrococcal nuclease (Sigma, St Louis, MO) for I h at 240C to reduce smearing of the enzyme fragment on gels. Host cell lysate from non-induced cells was used as control. Mitotic chromosomes from colcemide-arrested HeLa cells were prepared as described by Gasser & Laemmli (1987). The chromosomes were suspended in buffer A and treated with nucleases as above. Chromosome scaffolds were prepared from nuclease-digested mitotic chromosomes as described (Lewis & Laemmli, 1982).
ml). Incubation was continued for I h and the suspension was centrifuged. The supernatant was tested for its ability to decatanate 500 ng of trypanosome mitochondrial kinetoplast DNA (gift of Dr J. Hurwitz, Sloan-Kettering Institute) at 370C for 30 min in 10 p1 of the standard topoisomerase II reaction buffer (50 mm Tris-HCl, pH 7 5; 60 mm KCI, 0-5 mm ATP, 9 5 mM MgCl2, 05 mM Na EDTA, 0-5 mM DTT). The reaction was stopped by addition of sucrose to 4% and SDS to 0-1% and heating to 650C before analysis on 0-8% agarose gels (Miller, Liu & Englund, 1981).
Immunoblotting Proteins were separated on 7-15% SDS-polyacrylamide gradient gels (Laemmli, 1970) with the following amounts layered per 1-0 cm of gel: mitotic chromosomes 100 pg; scaffold proteins, 8 pg; purified topoisomerase II, 5 pg; bacterial lysate, 40 pg. After electrophoretic transfer (16 h, 60 V) to nitrocellulose membranes (Towbin, Staehelin & Gordon, 1979), the blots were blocked and incubated with patient or control serum (1/ 100 dilution, except rabbit antiserum at 1/1000) overnight at 40C. The strips (3-mm wide) were treated with '25l-labelled protein A (300 x I03 ct/min per ml) for I h and autoradiographed overnight.
Identification of topoisomerase II
mM
Assay of topoisomerase II activity The purified enzyme (12 ng) was incubated with patient or control serum (1/30 dilution) in 30 p1 of phosphate-buffered saline (PBS; 150 mm, pH 7-5) for 2 h at 4 C followed by the addition of 10 p1 of a protein A-Sepharose suspension (30 mg/
RESULTS AND DISCUSSION In an ongoing investigation on the antibody profile of sera from children with JRA, 58 sera were screened by immunoblotting. Using a HeLa cell nuclear sonicate as the source of antigen(s), we observed that some JRA sera react with a protein of about 170 kD, which corresponds to the molecular weight of topoisomerase II (unpublished observations). The identity of the 170kD antigen was determined as follows. First, a purified preparation of topoisomerase II isolated from HeLa cells was used as antigen on immunoblots. As seen in Fig. I a, this antigen is recognized by some JRA sera and by control rabbit antibodies to recombinant human topoisomerase II. Secondly, we have tested blots of extracts from E. coli cells harboring the expression vector pZII-06 containing a cDNA fragment of the human topoisomerase gene (Hwang et al., 1989). After induction, these cells overproduce a recombinant polypeptide which corresponds to the C-terminal one-third of the enzyme (Fig 1 b, lanes 1 and 2). As shown in Fig. Ib, lanes 3-12, JRA sera react
Topoisomerase II antibodies 2
3
v
3 7 8
3
i
-
1
3
k
Fig. 2. Analysis of DNA topoisomerase II decatanation activity after immunoprecipitation with JRA sera. After the removal of immune complexes, several dilutions of the adsorbed enzyme solutions were tested; the 1/40 dilution, which gives up to a 100% decrease in enzyme activity, is shown. Lane 1, input DNA (remains trapped in the well); lane 2, no serum; lane 3, normal serum; lane 4, scleroderma serum; lane 5, rabbit anti-topoisomerase II; lanes 6-13, JRA sera; lane 14, as in lane 2, but ATP is omitted. kt, kinetoplast DNA; mon, monomer DNA circles.
1
2 3 .A
..
200126-
9369-
46-
30-
14-
Fig. 3. Reactivity of autoimmune sera and control rabbit antitopoisomerase II serum with mitotic chromosomes (lanes 1-12) and chromosome scaffolds (lanes 13 and 14). Lane I, rabbit serum; lanes 2-4, examples of JRA sera positive in experiments of Fig. 1; lanes 5-7, JRA sera negative in these experiments; lanes 8 and 9, normal sera; lanes 10, I I and 12, MCTD, scleroderma and SLE sera, respectively; lanes 13 and 14, positive JRA sera reacted with chromosome scaffolds. Open arrows indicate
positions
of micrococcal nuclease
antigens (see Fig.
b and
c).
fragment which is also recognized by the rabbit sera, including those from healthy indiscleroderma patients, react with several antigens
with the cloned
antibodies. Human viduals and
present
in both induced and non-induced
bacterial extracts
the rabbit anti-topoisomerase II serum (Fig. la, lanes 3 and 4). Surprisingly, most of these antigens (open arrows, Fig. Ib and c) are polypeptides detected by human sera on blots of micrococcal nuclease (Fig. lc, lanes which are not detected
by
247
1-3), the enzyme used to treat the bacterial lysate prior to SDSPAGE. The experiments shown in Fig. I identify the 170-kD antigen and demonstrate that at least some epitopes recognized by JRA sera are located in the C-terminal part of the protein. Eight sera from children with JRA (eight out of 58, 14%) react with purified topoisomerase II as well as with the recombinant protein; none of the 30 control sera is positive. The ability of topoisomerase II to decatanate mitochondrial DNA kinetoplast networks and liberate monomer DNA circles is a specific assay of the enzyme (Miller et al., 1981). The immunoadsorption procedure has been used to test the ability of the JRA sera to bind active enzyme in solution. When five out of the eight immunoblot-positive sera are added to a solution of purified enzyme followed by protein A-Sepharose and the removal of immune complexes, a decrease in enzyme activity is observed (Fig. 2, lanes 6, 9, 10, 12 and 13). The rabbit antitopoisomerase II serum is also active in this assay (Fig. 2, lane 5). Control experiments using several sera from healthy children and those from children with scleroderma were negative (e.g. Fig. 2, lanes 3 and 4). It should be noted that the five positive JRA sera were the strongest in the immunoblot assay. Generally, sera from children with JRA have low antigen affinities and are more often reactive in the immunoblot assay than in immunoprecipitation (unpublished observation). Detection of topoisomerase II in chromosomes The presence of topoisomerase II in mitotic chromosomes and chromosome scaffolds is well established (Wang, 1985; Earnshow et al., 1985; Gasser & Laemmli, 1987). As seen in Fig. 3, lanes 1-4, the JRA sera as well as anti-topoisomerase II antibodies raised in rabbits detect the 1 70-kD antigen on blots of isolated mitotic chromosomes. However, the detection of a 170-kD antigen on blots of mitotic chromosomes does not by itself identify topoisomerase II. In addition to the eight positive sera, six JRA sera which are negative in the assays of Fig. 1 react with high mol. wt proteins similar in size to topoisomerase II (e.g. Fig. 3, lanes 6 and 7). Such proteins are known to be present in chromosomal preparations (Hoffmann et al., 1989). Also note from Fig. 3 that human sera react with antigens other than topoisomerase II; the micrococcal nuclease proteins are indicated by open arrows (Fig. I b). When the nuclease-digested mitotic chromosomes are treated with 2-0 M NaCl and extensively washed to purify scaffold proteins (Lewis & Laemmli, 1982), the only antigen seen on blots is the 1 70-kD polypeptide (Fig. 3, lanes 13 and 14). Some of the JRA sera and a control scleroderma serum react on blots of mitotic chromosomes with antigens of about 100 kD corresponding in molecular weight to topoisomerase I (Fig. 3, lanes 2, 4-6 and 1 1). When tested with Scl-70 as antigen, none of these sera (out of 18), except for the control scleroderma serum, was found positive (not shown). Anti-topoisomerase II antibodies could be useful tools for investigations on the function and distribution of this enzyme. Although all eight anti-topoisomerase II sera contain antibodies to other nuclear antigens (Table 1), immunopurification using the recombinant protein provides a simple method for obtaining antibodies monospecific for topoisomerase II. The immunochemical and clinical characteristics of the eight children whose sera react with topoisomerase II are summarized in Table 1. Seven of the eight have pauci-articular onset and only one has active iritis. Although all eight children have relatively
K. L. Zuklys, I. S. Szer & W. Szer
248
Table 1. Clinical and immunological data from anti-topoisomerase II-positive (immunoblotting) children with (JRA) juvenile rheumatoid arthritis
Age of onset (years)
JRA onset subtype
Disease duration (years)
Indirect immunofluorescence titre/pattern
Binding in solution*
Other antigenst
M
+
H H H
M -
+
1:80
H
M
+
2 2
1:40 1:40
H H
M -
+
2
1:640
H
M
+
45, 60, 80, 90, 100, 120 and > 120 kD 80 kD 38 and 90 kD 45, 60, 100 and > 120 kD 60,80,100,120 and > 120 kD 120kD 60,80,90,100 and > 120 kD Histone Hi, 60, 80, 100 and 195 kD
Patient
Sex
I
M
7
Pauci
0 25
1:320
H
2 3 4
F F F
58 85 2 25
Pauci Pauci Pauci
1-5 35 0 25
1:160 1:40 1:160
5
F
1
Poly
10
6 7
M F
5 4
Pauci Pauci
8
F
10
Pauci
Iritist
Activity of arthritis (time of study)
-
Mild
+ -
Moderate Mild Mild
-
Absent
-
Absent Mild
-
Moderate
H, homogeneous; M, also stains condensed chromosomes of metaphase cells. *See Fig. 2 and text. tMol. wt of unidentified antigens, other than 170 kD, detected on blots of the HeLa cells nuclear sonicate (unpublished data). J Active iritis at time of study.
mild arthritis, the presence of this autoantibody does not differentiate between the variety of heterogeneous disorders classified as JRA. In addition, serum from each child reacts with other, mostly unidentified antigens. Antibodies which react with enzymes such as aminoacyltRNA synthetases and DNA topoisomerase I have been found in patients with inflammatory myopathies and systemic sclerosis (Weiner et al., 1988; Targoff & Arnett, 1990). Furthermore, the presence of these antibodies seems to correspond to particular clinical patterns of disease expression (Yoshida et al., 1983). Until now antibodies recognizing human topoisomerase II have only been described in systemic lupus erythematosus and in cryptogenic fibrosing alveolitis (Hoffmann et al., 1989; Meliconi et al., 1989). Here we document the presence of anti-topoisomerase II antibodies in eight children with mild JRA, whose autoantibody profile does not suggest a distinct diagnostic entity.
ACKNOWLEDGMENTS We thank Dr H. Sierakowska for helpful discussions, Dr L. F. Liu (Johns Hopkins University) for the gift of rabbit serum to human topoisomerase II and plasmid pZII-06 and Drs J. Hurwitz and F. B. Dean (Sloan-Kettering Institute) for purified topoisomerase II and trypanosome mitochondrial kinetoplast DNA. This work was supported by National Institutes of Health grants GM23705-17 and CA16239-16.
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