Endonuclease V Regulates Atherosclerosis Through C-C Motif Chemokine Ligand 2-Mediated Monocyte Infiltration.

Background In cardiovascular diseases, atherosclerotic disorder are the most frequent and important with respect to morbidity and mortality. Inflammation mediated by immune cells is central in all parts of the atherosclerotic progress, and further understanding of the underlying mechanisms is needed. Growing evidence suggests that deamination of adenosine-to-inosine in RNA is crucial for a correct immune response; nevertheless, the role of adenosine-to-inosine RNA editing in atherogenesis has barely been studied. Several proteins have affinity for inosines in RNA, one being ENDOV (endonuclease V), which binds and cleaves RNA at inosines. Data on ENDOV in atherosclerosis are lacking. Methods and Results Quantitative polymerase chain reaction on ENDOV mRNA showed an increased level in human carotid atherosclerotic plaques compared with control veins. Inosine-ribonuclease activity as measured by an enzyme activity assay is detected in immune cells relevant for the atherosclerotic process. Abolishing EndoV in atherogenic apolipoprotein E-deficient (ApoE-/-) mice reduces the atherosclerotic plaque burden, both in size and lipid content. In addition, in a brain stroke model, mice without ENDOV suffer less damage than control mice. Finally, lack of EndoV reduces the recruitment of monocytes to atherosclerotic lesions in atherogenic ApoE-/- mice. Conclusions ENDOV is upregulated in human atherosclerotic lesions, and data from mice suggest that ENDOV promotes atherogenesis by enhancing the monocyte recruitment into the atherosclerotic lesion, potentially by increasing the effect of CCL2 activation on these cells.

Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe4S4] Cluster Nitrosylation.

Here we characterize the [Fe4S4] cluster nitrosylation of a DNA repair enzyme, endonuclease III (EndoIII), using DNA-modified gold electrochemistry and protein film voltammetry, electrophoretic mobility shift assays, mass spectrometry of whole and trypsin-digested protein, and a variety of spectroscopies. Exposure of EndoIII to nitric oxide under anaerobic conditions transforms the [Fe4S4] cluster into a dinitrosyl iron complex, [(Cys)2Fe(NO)2]-, and Roussin's red ester, [(µ-Cys)2Fe2(NO)4], in a 1:1 ratio with an average retention of 3.05 ± 0.01 Fe per nitrosylated cluster. The formation of the dinitrosyl iron complex is consistent with previous reports, but the Roussin's red ester is an unreported product of EndoIII nitrosylation. Hyperfine sublevel correlation (HYSCORE) pulse EPR spectroscopy detects two distinct classes of NO with 14N hyperfine couplings consistent with the dinitrosyl iron complex and reduced Roussin's red ester. Whole-protein mass spectrometry of EndoIII nitrosylated with 14NO and 15NO support the assignment of a protein-bound [(µ-Cys)2Fe2(NO)4] Roussin's red ester. The [Fe4S4]2+/3+ redox couple of DNA-bound EndoIII is observable using DNA-modified gold electrochemistry, but nitrosylated EndoIII does not display observable redox activity using DNA electrochemistry on gold despite having a similar DNA-binding affinity as the native protein. However, direct electrochemistry of protein films on graphite reveals the reduction potential of native and nitrosylated EndoIII to be 127 ± 6 and -674 ± 8 mV vs NHE, respectively, corresponding to a shift of approximately -800 mV with cluster nitrosylation. Collectively, these data demonstrate that DNA-bound redox activity, and by extension DNA-mediated charge transport, is modulated by [Fe4S4] cluster nitrosylation.

Expression of DNA repair protein: MYH, NTH1, and MTH1 in colorectal cancer.

BACKGROUND/AIMS: Oxidative stress is one of the causative agents of carcinogenesis. The DNA repair protein: MYH, NTH1 and MTH1 eliminate the oxidative DNA damage. In various tumors, overexpression of MTH1 has been reported. This is the first report demonstrating the expression of those proteins in colorectal cancer. METHODOLOGY: We investigated the expression of MYH, NTH1 and MTH1 by immunohistochemical typing methods in colorectal cancers obtained from consecutive patients undergoing surgery at the University of Tokyo hospital. RESULTS: High MYH immunoreactivity was detected in 57% of cases (46/81), which showed a significant correlation with the depth of tumor (p=0.04). Cytoplasmic expression of NTH1 was detected in 35% of cases (28/81), which showed a significant correlation with lymph node metastasis (p=0.001), histological grade according to the Dukes' Classification (p=0.005), and disease-free survival (p=0.04). High MTH1 immunoreactivity was detected in 84% of cases (68/81), which showed a significant correlation with tumor location (p=0.04). CONCLUSIONS: The expression pattern of DNA repair protein for oxidative DNA damage showed a significant correlation with aggressive features of colorectal cancer such as the depth of tumor, and lymph node metastasis, as well as with Dukes' classification and disease-free survival.

Evidence of alterations in base excision repair of oxidative DNA damage during spontaneous hepatocarcinogenesis in Long Evans Cinnamon rats.

The Long-Evans Cinnamon (LEC) rat, an animal model for Wilson's disease, is an inbred mutant strain, which because of the genetic copper metabolism disorder develops hepatitis approximately 4 months after birth, followed by chronic hepatitis later in life, and eventually all of the surviving animals from liver injury and hepatitis develop spontaneous hepatocellular carcinomas. This animal model also shows that the generation of reactive oxygen species and the accumulation of oxidative damage in the liver DNA has significantly increased over the lifetime of LEC versus the wild-type Long-Evans Agouti (LEA) rats. Thus, the LEC rats having this genetically induced oxidative condition are proved to be very useful model for the study of endogenous DNA lesions and their relation to spontaneous carcinogenesis. In this study, we tested the hypothesis that differences do exist between these two rat strains in respect to their capacity to repair oxidative DNA base modification, which could explain the elevation of endogenous oxidative damage in the LEC rat liver DNA. We found that both the activity and expression at the protein and RNA levels of major DNA glycosylases, endonuclease III and 8-oxoguanine DNA-glycosylase, which initiate the excision and repair of oxidized bases, were significantly altered during the acute (16-18 weeks) and early chronic (24 weeks) phases of hepatitis. Enzyme levels were restored in the later period of chronic hepatitis (week 40) in the LEC rat liver as compared with the age-matched LEA rats. This early reduction in the capacity to repair oxidative DNA base damage could have contributed to the accumulation of mutagenic adducts in liver DNA. These findings show for the first time in an animal model that acute hepatitis impairs the repair of oxidative DNA base damage and strongly suggest that the repair of endogenous DNA adducts plays a critical role in the development of spontaneous hepatocellular carcinoma in LEC rats.