Nitric oxide donors induce large‐scale deletion mutations in human lymphoblastoid cells: Implications for mutations in T‐lymphocytes from arthritis patients

Abstract

Rheumatoid arthritis (RA) is an inflammatory disease in which high levels of reactive nitrogen oxygen species (RNOS) may be present in the affected joints. RNOS are known to produce small-scale mutational events (transitions, transversions, small insertions, and small deletions) but the ability of these compounds to cause deletion of large segments of genomic DNA has not been previously determined. To address this question, a human lymphoblastoid cell line (WIL2-NS) was exposed to nitric oxide (NO)-donating drugs and hypoxanthine phosphoribosyltransferase (hprt)-negative clones were selected and analyzed by multiplex-PCR. Large-scale deletions accounted for 60-80% of hprt mutations arising in drug-treated cultures compared to 12% in untreated cultures (P-values of 0.006 and 0.0001, respectively, in two experiments). Deletion mutations in untreated cultures affected exon 9, whereas 75% of drug-induced deletion mutations affected exons 2, 3, and 9, and the remainder were very large, ranging from 26 to 1200 kbp. To compare this spectrum of NO-induced mutations in a lymphoblastoid line to that arising in vivo in arthritis patients, T-cells from RA patients, osteoarthritis (OA) patients, and controls were cloned and similarly analyzed. We previously showed that the overall frequency of Hprt mutant clones from patients is appreciably elevated compared to that of control subjects. Large-scale hprt deletions (0.5 to >26 kb) were detected in mutant T-cell clones from both RA and OA patients and also from control subjects. A total of 54 mutant clones from 16 RA patients and 19 mutant clones from 6 OA patients were studied. Of these, 6 clones (from 3 RA and 1 OA patient) had suffered large-scale deletions. A total of 9 control subjects were studied and 62 mutant clones were obtained. Of these, 19 had suffered large-scale deletions, arising in 7 of 9 control subjects. In conclusion, (1) RNOS are capable of inducing large-scale deletion mutations in a human lymphoblastoid cell line and (2) large-scale deletion mutations were found in 10-30% of T-cell clones from RA and OA patients and controls, which we hypothesize may be induced by RNOS.

Recent Advances in Nanotheranostics for Treat‐to‐Target of Rheumatoid Arthritis

“…Additionally, nitric oxide (NO) and reactive nitrogen oxygen species (RNOS) also play prominent roles in pathogenesis of RA 14,15. High levels of NO and RNOS in the inflamed sites give rise to cell mutation, genotoxicity, and large deletion of genomic DNA, contributing to the destructive, proliferative synovitis of RA 16. Synovial fibroblasts are involved in the differentiation and activation of osteoclasts, producing a large number of proinflammatory cytokines and high levels of chemokines in the inflamed joints, which incite synovial hyperplasia, massive angiogenesis, cartilage matrix disintegration, and bone destruction ( Figure ) 17,18…”

New insights into the functional role of the rheumatoid arthritis shared epitope

“…NO is a ubiquitous signaling molecule with versatile effects in the immune system. In RA, increased NO levels correlate significantly with inflammatory markers [35] and anti-rheumatic agents have been shown to suppress NO production [35,36]. NO has also been implicated in the pathogenesis of experimental autoimmune models in mice.…”

A role for calreticulin in the pathogenesis of rheumatoid arthritis

“…As mentioned earlier, one of the hallmarks of SE‐induced csCRT‐mediated signaling is the production of NO, a ubiquitous signaling molecule with versatile effects in the immune system. In RA, increased NO levels correlate significantly with inflammatory markers of the disease and anti‐rheumatic agents have been shown to suppress NO production 50–52 . NO has also been implicated in the pathogenesis of experimental autoimmune models in mice.…”

“…In RA, increased NO levels correlate significantly with inflammatory markers of the disease and antirheumatic agents have been shown to suppress NO production. [50][51][52] NO has also been implicated in the pathogenesis of experimental autoimmune models in mice. For example, SJL mice are known for their NO overproduction.…”