Binding of circulating autoantibodies in breast cancer to native and peroxynitrite-modified RNA

Abstract

Peroxynitrite (ONOO(-)) is a powerful oxidant and nitrosative agent and has in vivo existence. The half life of ONOO(-) at physiological pH is less than 1 s. It can react with nucleic acids, proteins, lipoproteins, saccharides, cardiolipin, etc., and can modify their native structures. Action of ONOO(-), synthesized in the authors' laboratory by a rapid quenched flow process, on structural changes of commercially available RNA was studied by ultraviolet (UV), fluorescence, and agarose gel electrophoresis. Compared to native RNA, the ONOO(-)-modified RNA showed hyperchromicity at 260 nm. Furthermore, the ethidium bromide (EtBr) assisted emission intensities of ONOO(-)-modified RNA samples were found to be lower than the emission intensity of native RNA-EtBr complex. Agarose gel electrophoresis of ONOO(-)-modified RNA showed a gradual decrease in band intensities compared to native RNA, an observation clearly due to the poor intercalation of EtBr with ONOO(-)-modified RNA. Native and ONOO(-)-modified RNA samples were used as an antigen to detect autoantibodies in sera of patients with clinically defined breast cancer. Both direct binding and inhibition enzyme-linked immunosorbent assay (ELISA) confirmed the prevalence of native and 0.8 mmol/L ONOO(-)-modified RNA specific autoantibodies in breast cancer patients. Moreover, the progressive retardation in the mobility of immune complexes formed with native or 0.8 mmol/L ONOO(-)-modified RNA and affinity purified immunoglobulin G (IgG) from sera of breast cancer patients supports the findings of the direct binding and inhibition ELISAs. The peroxynitrite treatment to RNA at a higher concentration appears to have damaged or destroyed the typical epitopes on RNA and thus there was a sharp decrease in autoantibodies binding to 1.4 mmol/L ONOO(-)-modified RNA. It may be interpreted that cellular nitrosative stress can modify and confer immunogenicity on RNA molecules. Higher concentrations of nitrogen reactive species can be detrimental to RNA. However, the emergence of native as well as 0.8 mmol/L ONOO(-)-modified RNA as a novel antigen/substrate for autoantibodies in breast cancer patients indicates that, in future, these molecules might find a place on the panel of antigens for early diagnosis of breast cancer.

Fig. 1

Emission profile of native RNA, ONOO⁻, ethidium bromide (EtBr), RNA mixed with EtBr and RNA samples treated with 0.8, 1.4, and 2.0 mmol/L ONOO⁻

Fig. 2

Agarose gel electrophoresis of native RNA and RNA modified by different amounts of peroxynitrite Lane 1: native RNA; Lanes 2, 3, and 4: 0.8, 1.4, and 2.0 mmol/L ONOO⁻-RNA

Fig. 3

Direct binding ELISA of breast cancer sera (1:100 dilution) with native RNA, 0.8 and 1.4 mmol/L ONOO⁻-modified RNA Microtitre wells were coated with respective antigens. NHS: normal human serum

Fig. 4

Mobility shift assay of native RNA (a), 0.8 mmol/L ONOO⁻-RNA (b), and 1.4 mmol/L ONOO⁻-RNA (c) in presence of varying amounts of breast cancer IgG 4 μg of RNA or 0.8/1.4 mol/L ONOO⁻-RNA (Lane 1) was mixed with 10, 20, 30, 40, and 50 μg of IgG (Lanes 2 to 6) and electrophoresed on 0.8% agarose gel after necessary incubation