We have reported that an inhibitor of DNA methylation, 5-azacytidine, makes cloned, antigenspecific CD4+ T cells autoreactive, and that procainamide and hydralazine mimic this effect. Those results suggested that procainamide and hydralazine may induce autoimmunity by inhibiting DNA methylation and causing T cell autoreactivity. We report now that Nacetylprocainamide, a procainamide derivative that does not induce lupus, is also a DNA methylation inhibitor, but it is 100 times less potent than procainamide in inducing T cell autoreactivity.DNA methylation inhibitors induce expression of genes normally suppressed by mechanisms associated with cytosine methylation (1) and, in certain systems, can change cellular phenotype and alter differentiation (2,3). We have previously reported that 5-azacytidine (5-azaC), a DNA methylation inhibitor, made 4 cloned antigen-reactive T cells respond to autologous class I1 major histocompatibility complex (MHC) determinants without specific antigen, presumably by inducing expression of suppressed genes (4). This response was termed autoreactivity.In those studies, we demonstrated that the 5- azaC-treated T cells responded to autologous but not allogeneic macrophages, and that monoclonal antibodies to class I1 but not class I MHC determinants would inhibit activation (4). Those results suggested the possibility that similar inhibition of T cell DNA methylation in vivo could lead to an autoimmune disease.To determine if inhibitors of DNA methylation could produce an autoimmune disease, we tested whether 2 drugs known to induce a lupus-like syndrome inhibit DNA methylation and induce T cell autoreactivity. We found that hydralazine (Hyd) and procainamide (Pca) mimicked the effects of 5-azaC on T cells by inducing T cell autoreactivity in these cloned lines and decreasing deoxymethylcytidine (d"C) content in T cell DNA, thus supporting the hypothesis that DNA methylation inhibitors may induce autoimmune disease (5).To further test this hypothesis, we compared Pca and Hyd with structural analogs, using the assays previously developed. N-acetylprocainamide (Napa) is a procainamide derivative that rarely induces lupus ( 6 4 , and hydralazine is a phthalazine (Phth) derivative containing a hydrazine side chain (l-hydrazinophthalazine) (9,lO). Previously, investigators speculated that the hydrazine side chain of hydralazine may be responsible for the lupus-like syndrome (9); therefore, we compared Pca with Napa and Hyd with Phth, for the ability to induce T cell autoreactivity and inhibit DNA methylation. MATERIALS AND METHODST cell cultures. CD4 + , cloned, tetanus toxoid (TTh reactive, interleukin-2 (IL-2tdependent T cell lines were generated and maintained as previously described (4,ll).

Inhibitors of DNA methylation, such as 5-azacytidine, induce gene expression. We have previously reported that cloned T cells treated with 5-azacytidine lose the requirement for Ag and can be activated by autologous HLA-D molecules alone, thus becoming auto-reactive. This phenomenon could potentially mediate an autoimmune disease in vivo. Inasmuch as several drugs are known to cause autoimmune disease, we asked whether they exert the same effects on T cells as 5-azacytidine. We report that hydralazine and procainamide, two drugs associated with a lupus-like autoimmune disease, also inhibit DNA methylation and induce self-reactivity in cloned T cell lines. These results suggest that drug-induced autoimmune disease may be due to activation of as yet unidentified genes through mechanisms involving DNA methylation.