Analysis of 5-methylcytosine in DNA of breast and colon cancer tissues

5-methylcytosine (m(5)C) can be used as a sensitive marker of progress of the tumor formation induced by the oxidative damage reactions. We have analyzed the amount of m(5)C in DNA of patients with breast and colon cancers. Two dimensional thin layer chromatography (TLC) has been used to monitor 5-methylcytosine level in DNA extracted from cancer tissues. The level of methylation of cytosine at C-5 position in DNA from breast cancer patients correlates well with the malignancy of tumors. Interestingly higher amount of m(5)C in DNA for the breast cancer patients treated with different chemotherapeutics was observed. It suggests an activation of DNA methyltransferase as well as a genomic suppression of the DNA repair genes expression. These differences clearly reflect the health condition of patients and support the global analysis of m(5)C in DNA as a good marker for diagnosis of neoplasia in clinical practice.     

PLA2R1 promotes DNA damage and inhibits spontaneous tumor formation during aging

Although aging is a major risk factor for most types of cancers, it is barely studied in this context. The transmembrane protein PLA2R1 (phospholipase A2 receptor) promotes cellular senescence, which can inhibit oncogene-induced tumor initiation. Functions and mechanisms of action of PLA2R1 during aging are largely unknown. In this study, we observed that old Pla2r1 knockout mice were more prone to spontaneously develop a wide spectrum of tumors compared to control littermates. Consistently, these knockout mice displayed increased Parp1, a master regulator of DNA damage repair, and decreased DNA damage, correlating with large human dataset analysis. Forced PLA2R1 expression in normal human cells decreased PARP1 expression, induced DNA damage and subsequent senescence, while the constitutive expression of PARP1 rescued cells from these PLA2R1-induced effects. Mechanistically, PARP1 expression is repressed by a ROS (reactive oxygen species)-Rb-dependent mechanism upon PLA2R1 expression. In conclusion, our results suggest that PLA2R1 suppresses aging-induced tumors by repressing PARP1, via a ROS–Rb signaling axis, and inducing DNA damage and its tumor suppressive responses.Subject terms: Cancer, Mechanisms of disease, DNA damage and repair, Ageing     

Fast deprotection of synthetic oligodeoxyribonucleotides using standard reagents under microwave irradiation

Fast methods for the removal of permanent amide exo-cyclic protective groups widely used in phosphoramidite-method DNA synthesis are desirable for many genomics and proteomics applications. In this communication, we present a method for the deprotection of a range of N-acyl deoxyribonucleosides (T, dA Bz, dC Bz, dC Ac, dG ibu, dG PAC) and synthetic oligodeoxyribonucleotides, ranging in length from 5-mer to 50-mer. Oligodeoxyribonucleotides were synthesized using standard amide protecting groups (dA Bz, dC Bz, dG ibu) and phosphoramidite chemistry on cis-diol solid phase support. This deprotection method utilizes 29% aqueous ammonia solution at 170 degrees C for 5 minutes under monomode microwave irradiation at a 20-nmole reaction scale. Reaction products were analyzed by TLC, RP-HPLC, CE, ESI-MS, real-time PCR, agarose gel electrophoresis, and by DNA uracil glycosylase (UDG) and phosphodiesterase I (PDE) enzymatic digestions.     

The contribution of endogenous sources of DNA damage to the multiple mutations in cancer

There is increasing evidence that most human cancers contain multiple mutations. By the time a tumor is clinically detectable it may have accumulated tens of thousands of mutations. In normal cells, mutations are rare events occurring at a rate of 10(-10) mutations per nucleotide per cell per generation. We have argued that the mutation rates exhibited by normal human cells are insufficient to account for the large number of mutations found in human cancers, and therefore, that an early event in tumorigenesis is the development of a mutator phenotype. In normal cells, spontaneous and induced DNA damage is balanced by multiple pathways for DNA repair, and most DNA damage is repaired without error. However, in tumor cells this balance may be shifted such that damage overwhelms the repair capacity, resulting in the accumulation of multiple mutations. Our hypothesis is that multiple random mutations occur during carcinogenesis. The sequential mutations that are observed in some human tumors result from selective events required for tumor progression. We consider the possibility that endogenous sources of DNA damage, in particular oxidative DNA damage, may contribute to genomic instability and to a mutator phenotype in some tumors. Endogenous and environmental sources of reactive oxygen species (ROS) are abundant. In tumor cells, antioxidant or DNA repair capacity may be insufficient to compensate for the production of ROS, and these endogenous ROS may be capable of damaging DNA and inducing mutations in critical DNA stability genes. The possibility that oxidative DNA damage could be a significant source of the genomic instability characteristic of human cancers is exciting, because it may be feasible to modulate the extent of oxidative damage through antioxidant therapy. The use of antioxidants to reduce the extent of molecular damage by ROS could delay the progression of cancer.     

Monoclonal antibodies specific for poly(dG) X poly(dC) and poly(dG) X poly(dm5C)

Most duplex DNAs that are in the "B" conformation are not immunogenic. One important exception is poly(dG) X poly(dC), which produces a good immune response even though, by many criteria, it adopts a conventional right-handed helix. In order to investigate what features are being recognized, monoclonal antibodies were prepared against poly(dG) X poly(dC) and the related polymer poly(dG) X poly(dm5C). Jel 72, which is an immunoglobulin G, binds only to poly(dG) X poly(dC), while Jel 68, which is an immunoglobulin M, binds approximately 10-fold more strongly to poly(dG) X poly(dm5C) than to poly(dG) X poly(dC). For both antibodies, no significant interaction could be detected with any other synthetic DNA duplexes including poly[d(Gm5C)] X poly[d(Gm5C)] in both the "B" and "Z" forms, poly[d(Tm5Cm5C)] X poly[d(GGA)], and poly[d(TCC)] X poly[d(GGA)], poly(dI) X poly(dC), or poly(dI) X poly(dm5C). The binding to poly(dG) X poly(dC) was inhibited by ethidium and by disruption of the DNA duplex, confirming that the antibodies were not recognizing single-stranded or multistranded structures. Furthermore, Jel 68 binds significantly to phage XP-12 DNA, which contains only m5C residues and will precipitate this DNA in the absence of a second antibody. The results suggest that (dG)n X (dm5C)n sequences in natural DNA exist in recognizably distinct conformations.     

Base composition of DNA from symbiotic dinoflagellates: a tool for phylogenetic classification

DNA of eight endosymbiotic dinoflagellates (zooxanthellae) from seven different host species has been analyzed as to its thermal characteristics and base composition by means of spectrophotometry and high performance liquid chromatography. All algae under investigation contain both methylcytosine and hydroxymethyluracil in addition to the bases typical of nuclear DNA. As a result, melting temperatures are decreased, suggesting lower contents of guanine plus cytosine than actually present. True percentages of guanine plus cytosine plus methylcytosine range from about 43 to 54 mol%. They are unique for the symbionts from different hosts, indicating phylogenetic separation of the taxa comparised within the genus Symbiodinium.Abbreviations dA deoxyadenosine - dC deoxycytidine - dG deoxyguanosine - dT deoxythymidine - m5dC 5-methyldeoxycytidine - hmdU 5-hydroxymethyldeoxyuridine - rC ribocytidine - Br8G bromine-80guanosine - A adenine - C cytosine - G guanine - T thymine - m5C 5-methylcytosine - hmU 5-hydroxymethyluracil - G+C guanine plus cytosine plus 5-methylcytosine - HPLC high performance liquid chromatography - T _m temperature at the midpoint of hyperchromic shift - CTAB N-cetyl-N,N,N-trimethyl-ammonium bromide - EDTA ethylenediamine-tetraacetic acid, disodium salt - TRIS tris-(hydroxymethyl)-aminomethane - 1×SSC standard saline citrate (0.15 M NaCl+0.015 M trisodium citrate, pH 7.0)     

Conformational analysis of the B and Z forms of the d(m5C-G)3 and d(br5C-G)3 hexamers in solution. A 300-MHz and 500-MHz NMR study

The B and the Z forms of the DNA hexamers d(m5C-G)3 and d(br5C-G)3 were investigated by means of NMR spectroscopy. It is demonstrated that the low-salt form of d(m5C-G)3 is a B DNA structure. The form, which becomes increasingly predominant when increasing amounts of MgCl2 and/or methanol are added to the solution, has Z DNA characteristics. It is shown that the major geometrical features of the Z form of d(m5C-G)3 in the crystal structure are maintained in solution, with the dC residues S sugar conformation, gamma + and the base in the anti orientation and the dG residues N (except the 3'-terminal residue), gamma t and syn. Neither the Z form of the methylated nor that of the brominated compound resembles the Z' form, in which the deoxy guanosine sugar rings adopt a C1'-exo conformation. Substitution of m5C by br5C causes no perceptible conformational changes in either the B or in the Z forms.     

Ischemic preconditioning enhances scavenging activity of reactive oxygen species and diminishes transmural difference of infarct size

Reactive oxygen species (ROS) enhance myocardial ischemia-reperfusion (I/R) injury. Ischemic preconditioning (PC) provides potent cardioprotective effects in I/R. However, it has not been elucidated whether PC diminishes ROS stress in I/R and whether PC protects the myocardium from ROS stress transmurally and homogeneously. Isolated rabbit hearts perfused with Krebs-Henseleit buffer underwent 30 min of ischemia and 60 min of reperfusion. Hemodynamic changes and myocardial damage extent were analyzed in four groups. The control group underwent I/R alone. The H2O2 group underwent I/R with H2O2 infusion (50 microM) in the first minute of reperfusion to enhance oxidative stress. The PC and H2O2+PC groups underwent 5 min of PC before control and H2O2 protocols, respectively. Extracted myocardial DNA was analyzed for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative DNA damage, with the use of the HPLC-electrochemical detection method. Glutathione peroxidase (GPX) activity and the reduced form of GSH were measured by spectrophotometric assays. The myocardial infarct size was significantly reduced in the PC group (19 +/- 2%) compared with the control group (37 +/- 4%; P < 0.05), particularly in the subendocardium. H2O2 transmurally increased the infarct size by 59 +/- 4% (P < 0.05), which was significantly diminished in the H2O2+PC group (31 +/- 4%; P < 0.01). The GSH levels, but not GPX activity, were well preserved transmurally in protocols with PC. The 8-OHdG levels were significantly decreased in PC and were significantly enhanced in H2O2 (P < 0.01). These changes in oxidative DNA damage were effectively diminished by PC. In conclusion, PC enhanced the scavenging activity of GSH against ROS transmurally, reduced myocardial damage, particularly in the subendocardium, and diminished the transmural difference in myocardial infarct size.     

Synthesis and properties of oligodeoxynucleotides containing 5-carboxy-2'-deoxycytidines

5-Carboxy-2'-deoxycytidine (dC(COO-)) was synthesized as an anion-carrier to seek a new possibility of modified oligodeoxynucleotides capable of stabilization of duplexes and triplexes. The base pairing properties of this compound were evaluated by use of ab initio calculations. These calculations suggest that the Hoogsteen-type base pair of dC(COO-)-G is less stable than that of the canonical C+-G pair and the Watson-Crick-type base pair of dC(COO-)-G is slightly more stable than the natural G-C base pair. The modified cytosine base showed a basicity similar to that of cytosine (pKa 4.2). It turned out that oligodeoxynucleotides 13mer and 14mer incorporating dC(COO-) could form duplexes with the complementary DNA oligomer, which were more stable than the unmodified duplex. In contrast, it formed a relatively unstable triplex with the target ds DNA.     

The mechanisms of oxidative DNA damage and apoptosis induced by norsalsolinol, an endogenous tetrahydroisoquinoline derivative associated with Parkinson's disease

Tetrahydroisoquinoline (TIQ) derivatives are putative neurotoxins that may contribute to the degeneration of dopaminergic neurons in Parkinson's disease. One TIQ, norsalsolinol (NorSAL), is present in dopamine-rich areas of human brain, including the substantia nigra. Here, we demonstrate that NorSAL reduces cell viability and induces apoptosis via cytochrome c release and caspase 3 activation in SH-SY5Y human neuroblastoma cells. Cytochrome c release, caspase 3 activation, and apoptosis induction were all inhibited by the antioxidant N -acetylcysteine. Thus, reactive oxygen species (ROS) contribute to apoptosis induced by NorSAL. Treatment with NorSAL also increased levels of oxidative damage to DNA, a stimulus for apoptosis, in SH-SY5Y. To clarify the mechanism of intracellular DNA damage, we examined the DNA damage caused by NorSAL using ³²P-5'-end-labeled isolated DNA fragments. NorSAL induced DNA damage in the presence of Cu(II). Catalase and bathocuproine, a Cu(I) chelator, inhibited this DNA damage, suggesting that ROS such as the Cu(I)-hydroperoxo complex derived from the reaction of H₂O₂ with Cu(I), promote DNA damage by NorSAL. In summary, NorSAL-generated ROS induced oxidative DNA damage, which led to caspase-dependent apoptosis in neuronal cells.     

The mechanism of action of virginiamycin M on the binding of aminoacyl-tRNA to ribosomes directed by elongation factor Tu

Mitochondrial DNA from Ustilago cynodontis has been investigated in several of its properties. Its dG + dC content is equal to 33.5%; its buoyant density (1.698 g/cm3) is higher, by 5 mg/cm3, and its melting temperature (82.5 degrees C) is lower than expected for a bacterial DNA having the same base composition; the first derivative of its melting curve indicates a large compositional heterogeneity, its molarity of elution from hydroxyapatite is high, 0.28 M phosphate, and allows its partial separation from nuclear DNA. Degradation by micrococcal nuclease indicates that about 25% of the DNA is formed by stretches having no more than 15% dG + dC. Finally, the unit size of mitochondrial genome is about 50 X 10(6). In most of its properties, the mitochondrial genome of U. cynodontis presents strong analogies with that of Saccharomyces cerevisiae. A parallel investigation on mitochondrial DNA from Acanthamoeba castellanii which has as genome unit size of only 27 X 10(6), has shown that this shares with the former the dG + dC content (32.9%), the melting temperature (82.5 degrees C), a large compositional heterogeneity and a very similar pattern of micrococcal nuclease degradation; its buoyant density (1.692 g/cm3) and its molarity of elution from hydroxyapatite (0.25 M phosphate) are, however, normal, probably because of a different short-sequence pattern and the fact that its dA + dT-rich stretches are shorter, on the average.     

Identification of high-confidence somatic mutations in whole genome sequence of formalin-fixed breast cancer specimens

The utilization of archived, formalin-fixed paraffin-embedded (FFPE) tumor samples for massive parallel sequencing has been challenging due to DNA damage and contamination with normal stroma. Here, we perform whole genome sequencing of DNA isolated from two triple-negative breast cancer tumors archived for >11 years as 5 μm FFPE sections and matched germline DNA. The tumor samples show differing amounts of FFPE damaged DNA sequencing reads revealed as relatively high alignment mismatch rates enriched for C · G > T · A substitutions compared to germline samples. This increase in mismatch rate is observable with as few as one million reads, allowing for an upfront evaluation of the sample integrity before whole genome sequencing. By applying innovative quality filters incorporating global nucleotide mismatch rates and local mismatch rates, we present a method to identify high-confidence somatic mutations even in the presence of FFPE induced DNA damage. This results in a breast cancer mutational profile consistent with previous studies and revealing potentially important functional mutations. Our study demonstrates the feasibility of performing genome-wide deep sequencing analysis of FFPE archived tumors of limited sample size such as residual cancer after treatment or metastatic biopsies.     

Distribution of 5-methyldeoxycytidine in products of staphylococcal nuclease digestion of nuclei and purified DNA

We have compared the distribution of 5-methyldeoxycytidine (m5dC) between staphylococcal nuclease (SN) sensitive and resistant regions of human diploid fibroblast chromatin to the corresponding distribution in purified DNA. After SN digestion of fibroblast nuclei or purified DNA, nuclease-resistant products were separated from sensitive products by perchloric acid or ethanol precipitation; the radioactively labeled nucleosides were then fractionated by high-performance liquid chromatography and quantitated. Our results indicate that m5dC is preferentially associated with SN-resistant regions of both chromatin and purified DNA. The magnitudes of these preferences in fibroblast chromatin and DNA are similar; we find that the enrichment of m5dC content in SN-resistant fractions of nuclei and DNA relative to the corresponding sensitive fractions is approximately 2-3-fold. Therefore, highly methylated regions of DNA have an intrinsic resistance to digestion by SN that is of sufficient magnitude to explain the high degree of nuclease resistance of chromatin containing highly methylated DNA.     

Impact of benzo[a]pyrene-2'-deoxyguanosine lesions on methylation of DNA by SssI and HhaI DNA methyltransferases

DNA damage caused by the binding of the tumorigen 7R,8S-diol 9S,10R-epoxide (B[a]PDE), a metabolite of bezo[a]pyrene, to guanine in CpG dinucleotide sequences could affect DNA methylation and, thus, represent a potential epigenetic mechanism of chemical carcinogenesis. In this work, we investigated the impact of stereoisomeric (+)- and (-)-trans-anti-B[a]P-N(2)-dG adducts (B(+) and B(-)) on DNA methylation by prokaryotic DNA methyltransferases M.SssI and M.HhaI. These two methyltransferases recognize CpG and GCGC sequences, respectively, and transfer a methyl group to the C5 atom of cytosine (C). A series of 18-mer unmethylated or hemimethylated oligodeoxynucleotide duplexes containing trans-anti-B[a]P-N(2)-dG adducts was generated. The B(+) or B(-) residues were introduced either 5' or 3' adjacent or opposite to the target 2'-deoxycytidines. The B[a]PDE lesions practically produced no effect on M.SssI binding to DNA but reduced M.HhaI binding by 1-2 orders of magnitude. In most cases, the benzo[a]pyrenyl residues decreased the methylation efficiency of hemimethylated and unmethylated DNA by M.SssI and M.HhaI. An absence of the methylation of hemimethylated duplexes was observed when either the (+)- or the (-)-trans-anti-B[a]P-N(2)-dG adduct was positioned 5' to the target dC. The effects observed may be related to the minor groove conformation of the bulky benzo[a]pyrenyl residue and to a perturbation of the normal contacts of the methyltransferase catalytic loop with the B[a]PDE-modified DNA. Our results indicate that a trans-anti-B[a]P-N(2)-dG lesion flanking a target dC in the CpG dinucleotide sequence on its 5'-side has a greater adverse impact on methylation than the same lesion when it is 3' adjacent or opposite to the target dC.     

Oxidative DNA base modifications and polycyclic aromatic hydrocarbon DNA adducts in squamous cell carcinoma of larynx

Tobacco smoke, recognized as a major etiological factor for cancers of the upper aerodigestive tract, represents an abundant source of reactive oxygen species (ROS), which are believed to play a significant role in mutagenesis and carcinogenesis. An additional source of ROS in tissues exposed to tobacco smoke may be metabolic oxidation of polycyclic aromatic hydrocarbons (PAH). To investigate the relationships between oxidative DNA lesions and aromatic DNA adducts, six modified DNA bases 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine and the total level of PAH-related DNA adducts were measured in cancerous and the surrounding normal larynx tissues (68 subjects), using gas chromatography/isotope-dilution mass spectroscopy with selected ion monitoring and the 32 P-postlabeling-HPLC assay, respectively. The levels of oxidative DNA lesions in cancerous and adjacent tissue were comparable; the differences between the two types of tissue were significant only for 5-hydroxypyrimidines (slightly higher levels were observed in the adjacent tissue). Comparable levels of DNA lesions in cancerous and the surrounding normal tissues observed in the larynx tumors support a field cancerization theory. The surrounding tissues may still be recognized as normal by histological criteria. However, molecular alterations resulting from the chronic tobacco smoke exposure, which equally affects larynx epithelia, may lead to multiple premalignant lesions. Thus, a demonstration of similar levels of DNA damage in cancerous and the adjacent tissue could explain a frequent formation of secondary tumors in the larynx and the frequent recurrence in this type of cancer. A weak, but distinct effect of tumor grading and metastatic status was observed in both kinds of tissue in the case of 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine. This effect was displayed as a gradual shift in the data distribution toward high values from G1 through G2-G3 and from non-metastatic to metastatic tumors. Since the levels of oxidative DNA base modifications tended to increase with the tumor aggressiveness, we postulate that the oxidative DNA lesions increase genetic instability and thus contribute to tumor progression in laryngeal cancer. No associations between aromatic adduct levels and oxidative DNA lesions were present, suggesting that the metabolism of PAH does not contribute significantly to the oxidative stress in larynx tissues, remaining the tobacco smoke ROS as a major source of oxidative DNA damage in the exposed tissue.     

A chemical and physical method for determining the complete base composition of plant DNA.

Two physical methods are routinely used to determine the base composition of DNA. One measures the temperature corresponding to the midpoint of the absorbance rise (TM) and relates it to base composition with the equation, TM = 41 (dG + dC) + 69, the other measures buoyant density (rho) and relates it to base composition rho = 0.098(dG + dC) + 1.6535. The base composition of DNA from various sources was first determined by a chemical method and these values compared to those determined by the physical methods. Higher plants contained up to 7 mol% 5-methyldeoxycytidine in their DNA and in all cases tested deoxyguanosine = deoxycytidine + 5-methyldeoxycytidine. After determining that TM was unaffected by the amount of 5-methyldeoxycytidine in DNA, the mol% of dA, dT, dG, and the total of dC plus 5-methyldeoxycytidine for any DNA could be calculated. Buoyant density on the other hand, was lowered 0.004 g . cm-3 for every 6.3 mol% 5-methyldeoxycytidine. Therefore, both physical parameters were related to the mole fraction of 5-methyldeoxycytidine by the following equation: (see article). With a value of r 5-methyldeoxycytidine an estimation of deoxycytidine was made. The resultant values agreed with the chromatographic determinations.     

Oxidative DNA Base Modifications and Polycyclic Aromatic Hydrocarbon DNA Adducts in Squamous Cell Carcinoma of Larynx

Tobacco smoke, recognized as a major etiological factor for cancers of the upper aerodigestive tract, represents an abundant source of reactive oxygen species (ROS), which are believed to play a significant role in mutagenesis and carcinogenesis. An additional source of ROS in tissues exposed to tobacco smoke may be metabolic oxidation of polycyclic aromatic hydrocarbons (PAH). To investigate the relationships between oxidative DNA lesions and aromatic DNA adducts, six modified DNA bases 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine and the total level of PAH-related DNA adducts were measured in cancerous and the surrounding normal larynx tissues (68 subjects), using gas chromatography/isotope-dilution mass spectroscopy with selected ion monitoring and the 32 P-postlabeling-HPLC assay, respectively. The levels of oxidative DNA lesions in cancerous and adjacent tissue were comparable; the differences between the two types of tissue were significant only for 5-hydroxypyrimidines (slightly higher levels were observed in the adjacent tissue). Comparable levels of DNA lesions in cancerous and the surrounding normal tissues observed in the larynx tumors support a field cancerization theory. The surrounding tissues may still be recognized as normal by histological criteria. However, molecular alterations resulting from the chronic tobacco smoke exposure, which equally affects larynx epithelia, may lead to multiple premalignant lesions. Thus, a demonstration of similar levels of DNA damage in cancerous and the adjacent tissue could explain a frequent formation of secondary tumors in the larynx and the frequent recurrence in this type of cancer. A weak, but distinct effect of tumor grading and metastatic status was observed in both kinds of tissue in the case of 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine. This effect was displayed as a gradual shift in the data distribution toward high values from G1 through G2-G3 and from non-metastatic to metastatic tumors. Since the levels of oxidative DNA base modifications tended to increase with the tumor aggressiveness, we postulate that the oxidative DNA lesions increase genetic instability and thus contribute to tumor progression in laryngeal cancer. No associations between aromatic adduct levels and oxidative DNA lesions were present, suggesting that the metabolism of PAH does not contribute significantly to the oxidative stress in larynx tissues, remaining the tobacco smoke ROS as a major source of oxidative DNA damage in the exposed tissue.     

Hepatitis C virus triggers mitochondrial permeability transition with production of reactive oxygen species, leading to DNA damage and STAT3 activation

Hepatitis C virus (HCV) infection is frequently associated with the development of hepatocellular carcinomas and non-Hodgkin's B-cell lymphomas. Previously, we reported that HCV infection causes cellular DNA damage and mutations, which are mediated by nitric oxide (NO). NO often damages mitochondria, leading to induction of double-stranded DNA breaks (DSBs) and accumulation of oxidative DNA damage. Here we report that HCV infection causes production of reactive oxygen species (ROS) and lowering of mitochondrial transmembrane potential (DeltaPsi(m)) in in vitro HCV-infected cell cultures. The changes in membrane potential could be inhibited by BCL-2. Furthermore, an inhibitor of ROS production, antioxidant N-acetyl-L-cysteine (NAC), or an inhibitor of NO, 1,400W, prevented the alterations of DeltaPsi(m). The HCV-induced DSB was also abolished by a combination of NO and ROS inhibitors. These results indicated that the mitochondrial damage and DSBs in HCV-infected cells were mediated by both NO and ROS. Among the HCV proteins, core, E1, and NS3 are potent ROS inducers: their expression led to DNA damage and activation of STAT3. Correspondingly, core-protein-transgenic mice showed elevated levels of lipid peroxidation and oxidatively damaged DNA. These HCV studies thus identified ROS, along with the previously identified NO, as the primary inducers of DSBs and mitochondrial damage in HCV-infected cells.     

Enhanced ADP-ribosylation and its diminution by lipoamide after ischemia-reperfusion in perfused rat heart

Poly-ADP-ribose polymerase (PARP) is considered to play an important role in oxidative cell damage. We assumed that ischemia-reperfusion resulting from the increasing reactive oxygen species (ROS) can lead to the activation of endogenous mono- and poly-ADP-ribosylation reactions and that the reduction of ROS level by lipoamide, a less known antioxidant, can reverse these unfavorable processes. Experiments were performed on isolated Langendorff hearts subjected to 60-min ischemia followed by reperfusion. ROS, malondialdehyde, deoxyribonucleic acid (DNA) breaks, and NAD+ content were assayed in the hearts, and the ADP-ribosylation of cytoplasmic and nuclear proteins were determined by Western blot assay. Ischemia-reperfusion caused a moderate (30.2 +/- 8%) increase in ROS production determined by the dihydrorhodamine 123 method and significantly increased the malondialdehyde production (from < 1 to 23 +/- 2.7 nmol/ml), DNA damage (undamaged DNA decreased from 71 +/- 7% to 23.1 +/- 5%), and NAD+ catabolism. In addition, ischemia-reperfusion activated the mono-ADP-ribosylation of GRP78 and the self-ADP-ribosylation of the nuclear PARP. The perfusion of hearts with lipoamide significantly decreased the ischemia-reperfusion-induced cell membrane damage determined by enzyme release (LDH, CK, and GOT), decreased the ROS production, reduced the malondialdehyde production to 5.5 +/- 2.4 nmol/ml, abolished DNA damage, and reduced NAD+ catabolism. The ischemia-reperfusion-induced activation of poly- and mono-ADP-ribosylation reactions were also reverted by lipoamide. In isolated rat heart mitochondria, dihydrolipoamide was found to be a better antioxidant than dihydrolipoic acid. Ischemia-reperfusion by ROS overproduction and increasing DNA breaks activates PARP leading to accelerated NAD+ catabolism, impaired energy metabolism, and cell damage. Lipoamide by reducing ROS levels halts PARP activation and membrane damage and improves the recovery of postischemic myocardium.     

Endogenous formation and repair of oxidatively induced G[8-5 m]T intrastrand cross-link lesion

Exposure to reactive oxygen species (ROS) can give rise to the formation of various DNA damage products. Among them, d(G[8-5 m]T) can be induced in isolated DNA treated with Fenton reagents and in cultured human cells exposed to γ-rays, d(G[8-5m]T) can be recognized and incised by purified Escherichia coli UvrABC nuclease. However, it remains unexplored whether d(G[8-5 m]T) accumulates in mammalian tissues and whether it is a substrate for nucleotide excision repair (NER) in vivo. Here, we found that d(G[8-5 m]T) could be detected in DNA isolated from tissues of healthy humans and animals, and elevated endogenous ROS generation enhanced the accumulation of this lesion in tissues of a rat model of Wilson’s disease. Additionally, XPA-deficient human brain and mouse liver as well as various types of tissues of ERCC1-deficient mice contained higher levels of d(G[8-5 m]T) but not ROS-induced single-nucleobase lesions than the corresponding normal controls. Together, our studies established that d(G[8-5 m]T) can be induced endogenously in mammalian tissues and constitutes a substrate for NER in vivo.