
	Version PDF

Unstabilized DNA breaks in lymphocytes of patients with systemic sclerosis

European Journal of Dermatology. Volume 16, Number 3, 258-61, May-June 2006, Investigative report

Summary

Author(s) : Franca Majone, Daniela Zamboni, Franco Cozzi, Anna Montaldi, Panagiotis Grypiotis, Roberto Luisetto, Maria Favaro, Marta Tonello, Amelia Ruffatti , Department of Biology, University of Padova, Padova, Italy, Department of Clinical and Experimental Medicine, Division of Rheumatology, University of Padova, Via Giustiniani, 2, 35128 Padova, Italy, Laboratory of Human Genetics, San Bortolo Hospital, Vicenza, Italy.

Summary : The clastogenic effects on DNA, proven by the presence of micronuclei (MN), and the protective cellular mechanisms normally used to stabilize DNA breaks were investigated in patients with systemic sclerosis (SSc). The frequency of micronucleated cells found in cultures of peripheral lymphocytes in patients was significantly higher than in the control group. The patient group with anti-centromere antibodies showed a significantly higher frequency of micronucleated cells than that observed in the patients with anti-topoisomerase I antibodies (4.22% versus 2.34%, p <\; 0.001). Moreover, we attempted to characterize MN for the presence or absence of DNA fragments with free 3’-OH ends by digoxigenin-dUTP (DIG-dUTP) using terminal deoxynucleotidil transferase. It was found that the frequency of MN containing DNA fragments with 3’-OH free ends (unstable fragments) increased in SSc patients compared to that observed in the control group. Moreover, this increase was significantly higher in lymphocytes of the patients with anti-centromere antibodies than in those with anti-topoisomerase I antibodies (35% versus 20.08%, p <\; 0.001). Our results indicate that in SSc patients there is an interference in the protective cellular mechanisms, normally stabilizing DNA breaks.

Keywords : systemic sclerosis, digoxigenin-dUTP, micronuclei, anti-centromere, anti-topoisomerase I

Pictures

ARTICLE

Auteur(s) : Franca Majone¹, Daniela Zamboni², Franco Cozzi², Anna Montaldi³, Panagiotis Grypiotis², Roberto Luisetto¹, Maria Favaro², Marta Tonello², Amelia Ruffatti²

¹Department of Biology, University of Padova, Padova, Italy
²Department of Clinical and Experimental Medicine, Division of Rheumatology, University of Padova, Via Giustiniani, 2, 35128 Padova, Italy
³Laboratory of Human Genetics, San Bortolo Hospital, Vicenza, Italy

accepté le 23 Janvier 2006

A number of studies have demonstrated high levels of spontaneous or induced chromosomal abnormalities in the circulating lymphocytes of subjects affected with scleroderma or systemic sclerosis (SSc) [1-4] and particularly in anti-centromere positive patients. Cytogenetic analysis of lymphocytes of these patients has been carried out using the micronucleus (MN) test [1-4]. MN are small nuclei-like bodies lying outside the main nucleus. They are induced ex vivo due to chromosomal damage by genotoxic compounds with different mechanisms of action and rarely found in normal cells. It is generally accepted that, while clastogenic compounds generate MN containing acentric fragments, aneuploidogenic agents produce MN with whole chromosomes [5, 6]. Through the use of the fluorescent in situ hybridization and incorporation (FISHI) method [7-9], it has also been possible to identify centric fragments within the MN generated by agents capable of inducing both clastogenic and aneuploidogenic effects [7]. It must be remembered that, without an accurate analysis of their content, MN frequency may itself simply indicate that cytogenetic damage of MN has taken place but it cannot qualitatively discriminate the type of damage. The presence of MN in an experimental situation is therefore a minimum requirement for a better understanding of its nature and evolution.A new method has been applied in the study of MN of HTLV-1 Tax-expressing cells [7-9]: MN DNA content is labelled for the presence of unprotected, free, broken DNA ends. Free 3′-OH ends were defined as those DNA ends accessible to in situ addition of digoxigenin-dUTP (DIG-dUTP) via terminal deoxynucleotidyl transferase. DNA breaks with freely detectable 3’-OH ends had previously been shown to be uncapped and liable to degradation, incomplete replication, and loss during cell division [7-9]. On the other hand, in normal situations a DNA break in the cells is sealed by a protective cap normally used to stabilize them [9].The clastogenic effects on the DNA, measured in the presence of MN, and the protective cellular mechanisms normally used to stabilize DNA breaks were evaluated in patients with SSc by DIG-dUTP in situ incorporation.

Materials and methods

Patients and controls. We examined 30 patients affected with SSc (29 women and 1 man with mean age of 51.5 years ± 11.7 SD; 14 had a limited and 16 a diffuse clinical form. All the patients fulfilled the American Rheumatism Association criteria for SSc [10]. Raynaud’s phenomenon, disease duration (months) and the presence of lung, heart and esophagus involvement were evaluated in our scleroderma patients according to previously described criteria [11]. Renal involvement was investigated by the following parameters: hypertension (minimum blood pressure > 100 mmHg), renal failure (serum creatinine > 1.3 mg/dL), proteinuria (> 0.5 g/24 h) and hematuria. Clinical information for 4 anti-centromere positive patients was not available. None of the patients was taking, or had taken during the 6 months before the study, potentially genotoxic drugs or had been in contact with other forms of oxidative stress influencing micronuclei formation (e.g. UV). According to the antinuclear antibody (ANA) profile, each patient was assigned to one of the following groups: anti-centromere positive or anti-topoisomerase I positive group. We considered 15 healthy age and sex matched subjects as controls.

Detection of ANA. Sera were screened for ANA by indirect immunofluorescence on HEp-2 cells (Immunoconcepts) and anti-centromere antibodies were detected by the centromere fluorescence pattern. Precipitating antibodies to topoisomerase I were investigated using an in-house counter-immunoelectrophoresis method.

Peripheral lymphocyte cultures. Blood samples were collected with heparin as an anticoagulant. Blood was diluted 1:1 with phosphate-buffered saline (PBS) and layered onto histopaque at two parts diluted blood to 1 part histopaque. After centrifugation at 500 × g for 45 min plasma was removed and discarded. The mononuclear cell (MNC) layer was then removed and added to 20 mL RPMI. Following centrifugation at 400 × g for 15 min the cells were resuspended in 20 mL RPMI and after another centrifugation they were put into 5 mL RPMI. The cells were then counted, centrifugated and resuspended in an appropriate volume of RPMI. Lymphocyte cultures were set up in a medium containing phytohemagglutinin and incubated in a 5% CO₂ for 69 hours at 37 °C. At the 44th hour, cytochalasin B (3 μg/mL, Sigma) was added. Lymphocytes were harvested and fixed for 15 min in paraformaldehyde (1% in PBS) for in situ incorporation analysis.

Micronuclei assay. Interphase preparations were obtained following the procedures previously described [7-9]. Treatment with cytochalasin B blocks the cytodieresis of the cells leading to formation of binucleate cells used for scoring MN. The frequency of MN was expressed as the number of micronucleated cells (containing one or more MN) per 100 binucleate lymphocytes; 3,000 cells were counted from each experimental point, using almost two slides for every point. MN are visible as small nuclei near the main nucleus in the cytoplasm of interphase cells that have completed at least one cell cycle.

FISHI. It was carried out using the terminal deoxynucleotidyltransferase (TdT) which catalyses the addition of deoxyribonucleotide triphosphates to the 3’-OH ends of the single or double-stranded DNA. Digoxigenin-11-dUTP (the digoxigenin is bound to position 5 of the pyrimidine by an arm of 11 carbon atoms) was added to the 3’-OH ends to the substrates of the TdT. Antibody diction of DIG-dUTP labelling employed a specific antibody linked to fluorescein isothiocyanate (FITC), a fluorochrome which, stimulated in the light of a 494m wavelength, emits a green signal (λ = 523 nm). The experimental protocol for FISHI use 2 washes with HBS (NaCl 280 mM, Na₂PO₄ × 7H₂O 1.5 mM, Hepes 50 mM). The TdT incorporation reaction of DIG-11-dUTP required the following: 10 μL of a solution (Boheringer) containing potassium cocodylate 1M, Tri-HCl 125 mM (pH 6.6, 4 °C), bovine serum albumin (BSA) 1.25 mg/mL, Ca Cl₂ 10 mM ; 0.2 μL of a solution (Boheringer) containing TdT (25 units/μL), EDTA 1 mM, 2 mercaptoethanol 4 mM, glicerol 50% (v/v) (pH 6.6, 4 °C); 1 μL of DIG-11-dUTP (1 mM) mixture (Boheringer). Distilled water was added to a final volume of 50 μL. The cells were incubated in this solution at 37 °C for 1 hour in an HBS-moist environment. At the end of the incubation the slides were immersed in 0.1% Triton X-100 and 0.5% BSA in HBS to equilibrate them with anti-DIG-11-dUTP (1:50) labelled with FITC (Boheringer). Equilibration was conducted at room temperature for 30 minutes in an HBS moist environment. The slides were subsequently washed 3 times for 5 minutes with the same HBS solution, and then counterstained with propidium iodide (0.3 μg/mL). Over 3,000 cells were counted for each data point.

Statistical analysis. Statistical comparison of the frequencies of cytogenetic effects in the different groups was performed by the G test [12].

Results

ANA were present in all scleroderma sera, with a centromere pattern in 15 cases and diffuse grainy staining in 15. A centromere fluorescent pattern was found in 13 patients with limited SSc and in 2 with diffuse SSc. Anti-topoisomerase I antibody was detected by counter-immunoelectroforesis in all sera with a diffuse grainy pattern, 1 of which had limited SSc and 14 diffuse SSc. All patients presented Raynaud’s phenomenon. Information on disease duration and esophageal, lung, heart, and renal involvement was available in 26/30 patients (11 anti-centromere and 15 anti-topoisomerase I positive). In the anti-centromere positive group, disease duration was 151.7 months ± 95.7 SD and 7/11 (63.6%) of the patients presented esophageal involvement, 8/11 (72.7%) lung involvement, 2/11 (18.2%) heart involvement, and 1/11 (9%) renal involvement. In the anti-topisomerase I positive group, disease duration was 133.5 months ± 54.2 SD and 9/15 (60%) of the patients presented esophageal involvement, 11/15 (73.3%) lung involvement, 1/15 (6.6%) heart involvement and none renal involvement.

The results of cytogenetic analysis carried out on peripheral lymphocytes of scleroderma patients and healthy controls are shown in (Table 1). Groups of scleroderma patients showing anti-centromere and anti-topoisomerase I antibodies were chosen. The mean frequency of micronucleated cells found in both patient groups was statistically higher compared that in the control group (4.22% and 2.34% versus 0.82%, p < 0.001). The first group (patients with anti-centromere antibody) showed a higher frequency of micronucleated cells than that in the second group (with anti-topoisomerase I antibody) (4.22% versus 2.34%, p < 0.001).

The nature of chromosome damage within MN (figure 1) was examined in the lymphocytes of patients and controls by the in situ DIG-dUTP incorporation method using TdT [7, 8]. Table 1 shows that the frequency of unstable fragments, which have incorporated DIG-dUTP (figures 1A, C), was significantly higher in both patient groups compared with the control group (35% and 20.08% versus 1.18%, p < 0.001). Moreover, the increase was significantly higher in the lymphocytes of patients with anti-centromere antibodies than in those with anti-topoisomerase I antibodies (35% versus 20.08%, p < 0.001).
Table 1 Results from digoxigenin-dUTP in situ incorporation analysis in lymphocytes of scleroderma patients and healthy controls

	N°	Micronuclei (%)	Micronuclei (%) with DIG-dUTP signals
Healthy controls	15	0.82 ± 0.05	1.18 ± 0.48
Anti-centromere positive patients	15	4.22 ± 0.30	35.00 ± 1.92
Anti-topoisomerase I positive patients	15	2.34 ± 0.3	20.08 ± 2.12

Discussion

In our study a cytogenetic analysis was performed on the peripheral lymphocytes of 30 patients affected with SSc and on 15 healthy control subjects in order to investigate the presence of the clastogenic effect. We then evaluated for the first time whether the protective cellular mechanisms normally used to stabilize DNA breaks [7-9] are active in scleroderma patients. The first part of this investigation confirms the results of previous studies in which the frequency of MN in SSc patients was significantly higher than that in the control group [1, 2]. The results reported here once again emphasize the strong genetic instability in patients affected with SSc. Moreover, in agreement with these reports [1, 2] it was found that patients with anti-centromere antibodies, presented a higher frequency of MN cells than patients with anti-topoisomerase I antibodies.

In the second part of the study we analysed the nature of chromosome damage present within the observed MN. To this end, the in situ DIG-dUTP incorporation method using the TdT enzyme was used. This method uncovers free, uncapped DNA ends, which are an indicator of deep genetic instability [7, 8]. In fact DNA breaks incorporate DIG-dUTP only if they are not capped. Breaks of this kind may induce degradation or fusion giving rise to new aberrations [7-9]. Identification of DNA breaks of this kind within the MN may provide a direct, precise indication of the unstable fragments present within the MN. The results obtained by this method suggest that scleroderma patients have a significantly higher percentage of unprotected DNA breaks with respect to normal control subjects. In addition, unprotected DNA breaks were significantly more frequent in patients with anti-centromere antibodies than in those with antitopoisomerase I antibodies. It is interesting to note that research on mammalian cells cultivated in vitro and knocked out for genes involved in the production of proteins, which are important in the non homologous end joining (NHEJ) eukaryote repair system, such as Ku80, reveals an increase in unstable DNA breaks [9, 13]; so indicating the importance of these proteins in protection of broken DNA ends [14, 15]. Anti-Ku antibodies have been observed in SSc patients [16] indicating that in this illness, there may be an interference in the NHEJ repair system by lack of important proteins protecting DNA breaks. In agreement with the present results it should be remembered that a telomere reduction, which may cause chromosomal instability, was observed in scleroderma [17]. Telomeric repetitions may be added at broken DNA ends, leading to their stabilization and blocking any evolution towards inappropriate fusions [18].

Until now the chromosome damage observed in patients with scleroderma has been related to the presence of a clastogenic factor in the plasma of these patients and different classes of substances with clastogenic activity have been identified [19-21]. Moreover a possible interference of different classes of antibodies with the clastogenic factor has also been suggested [19].

This work confirms the presence of clastogenic events made known by MN in the lymphocytes of scleroderma patients. Moreover, it was found that if a DNA break occurs in scleroderma, it remains unstable, without any protection, leading to the constitution of new chromosome aberrations. Until the present it was unknown if the unstable breaks in scleroderma patients were due to the telomere reductions or could be related to an interference by the protective Ku protein. It should be remembered that unstable DNA breaks are present in cells expressing HTLV-I Tax protein [7-9] and in different types of adrenal tumors (data not shown). These results provide interesting prospects for understanding the relationships between the control mechanisms of genome stability and retrovirus mediated oncogenesis and tumorgenesis [8, 9]. The significance of this particular type of genetic instability in SSc still remains to be understood.

Acknowledgements

We thank Claudio Friso and Renzo Mazzaro (Department of Biology) for the technical assistance and Savio De Souza for the preparation of this manuscript.

References

1 Porciello G, Scarpato R, Storino F, Cagetti F, Marcolongo R, Migliore L, Ferri C, Galeazzi M. The high frequency of spontaneous micronuclei observed in lymphocytes of systemic sclerosis patients: preliminary results. Reumatismo 2002; 34: 36-9.

2 Porciello G, Scarpato R, Ferri C, Storino F, Cagetti F, Morozzi G, Bellisai F, Migliore L, Marcolongo R, Galeazzi M. Spontaneous chromosome damage (micronuclei) in systemic sclerosis and Raynaud’s phenomenon. J Rheumatol 2003; 30: 1244-7.

3 Porciello G, Scarpato R, Storino F, Cagetti F, Bellisai F, Morozzi G, Marcolongo R, Migliore L, Ferri C, Galeazzi M. Chromosome aberrations in Raynaud’s phenomenon. Eur J Dermatol 2004; 14: 327-31.

4 Migliore L, Caterina B, Scarpato R. Cytogenetic study and FISH analysis in lymphocytes of systemic lupus erythematosus (SLE) and systemic sclerosis (SS) patients. Mutagenesis 1999; 14: 227-31.

5 Majone F, Brunetti R, Fumagalli O, Gabriele M, Levis AG. Induction of micronuclei by mitomycin C and colchicine in the marine mussel Mytilus galloprovincialis. Mutat Res 1990; 224: 147-51.

6 Majone F, Tonetto S, Soligo C, Panozzo M. Identification of kinetochores and DNA synthesis in micronuclei induced by mitomycin C and colchicine in Chinese hamster ovary cells. Teratogen Carcinogen Mutagen 1992; 12: 155-66.

7 Majone F, Jeang KT. Clastogenic effect of the Human T-cell Leukemia Virus Type I Tax oncoprotein correlates with unstabilized DNA breaks. J Biol Chem 2000; 275: 32906-10.

8 Jeang KT, Giam C, Majone F, Aboud M. Life, death and Tax: role of HTLV-I oncoprotein in genetic instability and cellular transformation. J Biol Chem 2004; 279: 31991-4.

9 Majone F, Luisetto R, Zamboni D, Twanaga Y, Jeang KT. Ku protein as a potential human T-cell leukemia virus type 1 (HTLV-1) Tax target in clastogenic chromosomal instability of mammalian cells. Retrovirology 2005; 2: 45.

10 Subcommittee for Scleroderma Criteria of the American Rheumatism association Diagnostic and Therapeutic criteria Committee. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum 1980; 23: 581-90.

11 Medsger Jr. TA, Silman AJ, Steen VD, Black CM, Akesson A, Bacon PA, Harris CA, Jablonska S, Jayson MI, Jimenez SA, Krieg T, Leroy EC, Maddison PJ, Russell ML, Schachter RK, Wollheim FA, Zacharaie H. A disease severity scale for systemic sclerosis: development and testing. J Rheumatol 1999; 26: 2159-67.

12 Sokal P, Rohlf FJ. Biometry. San Francisco: Freeman, 1991.

13 Bailey SM, Meyne J, Cornforth M, McConnell TS, Goodwin HS. A new method for detecting pericentric inversions using COD-FISH. Cytogenet Cel Genet 1996; 75: 248-53.

14 Martin SG, Laroche T, Suka N, Grunstein M, Gasser SM. Relocalization of telomeric Ku and SIR proteins in response to DNA strands breaks in yeast. Cell 1999; 97: 621-33.

15 Matsumoto T, Fukui K, Niwa O, Sugawara M, Szostak JD, Yanagida M. Identification of healed terminal DNA fragments in linear minichromosomes of Schizosaccharomyces pombe. Mol Cell Biol 1987; 7: 4424-30.

16 Franceschini F, Cavazzana I, Generali D. Anti Ku in connective tissue disease: clinical and serological evalutation of 14 patients. J Rheumatol 2002; 29: 1393-7.

17 Artlett CM, Black CM, Briggs DC, Stevens CO, Welsh KI. Telomere reduction in scleroderma patients: a possible cause for chromosomal instability. Br J Rheumato 1996; 35: 732-7.

18 Wilkie AOM, Lamb J, Harris PC, Finney RD, Higgs DR. A truncated human chromosome 16 associated with α thalassaemia is stabilized by addition of telomeric repeat (TTAGGG)_n. Nature 1990; 346: 868-71.

19 Emerit I, Filipe P, Meunier P, Auclair G, Freitas G, Derouassant J, Gouyette A, Fernandes A. Clastogenic activity in the plasma of scleroderma patients: a biomarker of oxidative stress. Dermatology 1997; 194: 140-6.

20 Kasai H. Analysis of a form of oxidative DNA damage, 8-hydroxy-2’-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutat Res 1997; 387: 147-63.

21 Kawanishi S, Hiraku Y, Oikawa S. Mechanism of guanine-specific DNA damage by oxidative stress and its role in carcinogenesis and aging. Mutat Res 2000; 488: 65-76.