Increased DNA strand breaks in mononuclear cells from patients with rheumatoid arthritis.

Abstract

Immune dysfunction is linked with lymphocyte DNA metabolism. In particular, DNA damage may impair lymphocyte function and induce increased cell turnover; such changes are of relevance to the pathogenesis of rheumatoid arthritis. The rate of DNA unwinding in alkaline solution was used as a measure of the number of DNA strand breaks in mononuclear cells freshly isolated from peripheral blood. The rate of DNA unwinding was significantly increased in celis from patients with rheumatoid arthritis compared with those from healthy subjects and from patients with other autoimmune and connective tissue diseases. These findings support the hypothesis that DNA damage is increased in patients with rheumatoid arthritis and it is one factor contributing to immune dysfunction in this disease. Immune dysfunction is a characteristic of rheumatoid arthritis (RA). The immunological changes include autoimmunity, lymphocytic infiltration of the synovium, diffuse lymphadenopathy, and raised immunoglobulin levels.' Decreased responses of both T and B lymphocytes to mitogens2 and incomplete progression towards lymphocyte activation3 have also been described. In addition, patients with RA have an increased risk of sepsis and death due to infection.4 Some of these observations could be explained by metabolic changes in lymphocytes.DNA damage, including strand breaks, invariably occurs in nucleated cells but is usually repaired. For lymphocytes in particular, DNA metabolism is fundamental to proliferation and hence immune function. Evidence for this comes from studies of lymphocyte depletion in patients with severe combined immunodeficiency, associated with the deficiency of adenosine deaminase (adenosine aminohydrolase, E.C. 3.5.4.4) or purine nucleoside phosphorylase (E.C. 2.4.2.1). DNA strand breaks in circulating mononuclear cells were determined using a fluorimetric method for measuring the rate of DNA unwinding,8 modified from the procedure of Birnboim and Jevcak.9 All steps were carried out under ordinary light. Fresh mononuclear cells were isolated using density centrifugation on Histopaque-1077 (Sigma, Poole, UK). Mononuclear cells were counted and suspended in an isotonic solution (0 25 M mesoinositol, 10 mM sodium phosphate, 1 mM magnesium chloride, pH 7'2) to give a cell concentration of 2x 106/ml, (typically 8-12 ml). The suspension was mixed with an equal volume of lysis buffer (9 M urea, 10 mM sodium hydroxide solution, 2-5 mM cyclohexanediaminetetraacetate, 0-1% (w/v) sodium dodecyl sulphate) and incubated at 0°C foi 10 min. Aliquots (0-4 ml) of this suspension were placed in eight disposable plastic tubes (40x9 mm inner diameter), four Increased DNA strand breaks in rheumatoid arthritis designated total fluorescence and four partial fluorescence. For the background determination the suspension (1-6 ml) was undivided.A 0-1 ml aliquot of alkaline solution 1 (AS 1; 45% v/v lysis buffer in 0-2 M sodium hydroxide) and 0a1 ml of alkaline solution 2 (AS2; 40% v/v lysis buffer in 0-2 M sodium hydroxide) were very gently l...

The Therapeutic Potential of Poly(ADP-Ribose) Polymerase Inhibitors

“…Nevertheless, in a human study analyzing DNA single-strand breakage in peripheral mononuclear cells from arthritic patients, a significant elevation was found when compared with healthy volunteers (Bhusate et al, 1992). Studies conducted approximately a decade ago in humans reported increased frequency of circulating antibodies against PARP, chiefly in patients with systemic lupus erythematosus as well as rheumatoid arthritis (Negri et al, 1990;Lee and Axelrod, 1995;Decker et al, 1998).…”

Inhibition of Caspase-3-mediated Poly(ADP-ribose) Polymerase (PARP) Apoptotic Cleavage by Human PARP Autoantibodies and Effect on Cells Undergoing Apoptosis

“…On the other hand, deposition of antibodies to Ro/SSA ribonucleoprotein in fetal cardiac tissues is thought to be associated with congenital heart block (14,15), and autoantibody subsets to ribosomal P proteins have been shown to be able to penetrate into live cells and cause cellular dysfunction (16). Because elevated rates of spontaneous and induced chromosomal damage have been reported in certain autoimmune rheumatic diseases (17,18), deficient DNA repair was observed in chronic ulcerative colitis (19) and SLE (20,21), and defects in poly(ADP-ribose) synthesis have been described in lupus patients and their healthy biological relatives (22,23), we studied the possibility that autoantibodies to F1 and F2 zinc fingers inhibit the PARP catalytic activity and cause the observed pathogenic effects. We report here that, contrary to our first expectations, PARP antibodies from autoimmune patients do not significantly affect PARP activity but efficiently prevent caspase-3-mediated PARP cleavage during apoptosis and prolong cell survival, which can have important deleterious effects.…”

A Simple Colorimetric Method for Naked‐Eye Detection of Circulating Cell‐Free DNA Using Unlabelled Gold Nanoparticles

“…DNA strand breaks are earlier reported in RA and endogenous nucleases have been frequently referred for leading to this effect . These fragmented cfDNA in RA plasma thereby may not be available for primer annealing and extension steps in the qPCR and hence may have led to the observed failure of qPCR (Figure and Figure 8).…”

“…Immune system dysfunction has been a major and a well known cause for progression of RA . An elevated oxidative stress in RA patients may cause increased cell death and DNA damage in immune cells, moreover proteins required for reversing the DNA strand breaks and repairing them are reported to be down regulated in RA patients . It is thus proposed that restoration of DNA repair function can act as a potent therapeutic target in RA .…”