Assays for 8-hydroxy-2'-deoxyguanosine: a biomarker of in vivo oxidative DNA damage.

HPLC with electrochemical detection (HPLC-EC) is a highly sensitive and a selective method for detecting 8-hydroxy-2'-deoxyguanosine (oh8dG), a biomarker of oxidative DNA damage that is formed from hydroxyl radical attack of guanine residues in DNA. We propose that the noninvasive measurement of oh8dG in urine can be used to estimate in vivo oxidative damage. Application of this assay to urine samples obtained from rats of different ages and various species provide examples of the utility of this assay. The measurement of steady-state levels of oh8dG in DNA combined with the urinary excretion rates of oh8dG and oh8Gua, offer a powerful approach for estimating oxidative DNA damage and its repair. This method will be useful for studies designed to investigate the relationship of oxidative stress in DNA damage and the role of this damage in aging and cancer.     

Age-related increases of 8-hydroxy-2′-deoxyguanosine and DNA–protein crosslinks in mouse organs

Experimental data suggest a possible role of DNA damage in aging, mainly related to oxidative lesions. With the objective of evaluating DNA lesions as molecular biomarkers of aging, we measured 8-hydroxy-2′-deoxyguanosine (8-OH-dG) and DNA–protein crosslinks (DPXL) levels in different organs of mice aged 12 and 24 months. 8-OH-dG was detected by ³²P postlabelling after removing unmodified dG by trifluoracetic acid, which prevented the artificial formation of 8-OH-dG during ³²P labelling procedures. Appreciable 8-OH-dG amounts were detected in 12-month-old mice in liver (1.8±0.7 8-OH-dG/10⁵ normal nucleotides), brain (1.6±0.5) and heart (2.3±0.5). In 24-month-old mice these values were higher in all examined organs (liver, 2.7±0.4; brain, 3.6±1.1; heart, 6.8±2.2 8-OH-dG/10⁵ normal nucleotides). This accounted for a 1.5-fold increase in liver (not significant), 2.3-fold increase in brain (P<0.01), and 3.0-fold increase in heart (P<0.001). A similar trend was observed for DPXL levels, which were the 1.8±0.3%, 1.2±0.2%, and 2.2±0.3% of total DNA in liver, brain, and heart of 12-month-old mice and 1.9±0.4%, 2.0±0.4%, and 3.4±0.5% in 24-month-old mice, with ratios of 1.0, 1.7 (P<0.01), and 1.5 (P<0.001), respectively. Highly significant correlations between 8-OH-dG and DPXL levels were recorded in brain (r=0.619, P<0.001) and heart (r=0.800, P<0.0001), but not in liver (r=0.201, not significant). These data suggest that brain and heart are more severely affected by the monitored age-related DNA lesions than liver, which can be ascribed to certain characteristics of these postmitotic organs, including the low detoxifying capacities, the high oxygen consumption, and the impossibility to replace damaged cells by mitosis. The strong correlation between 8-OH-dG and DPXL supports a possible contribution of oxidative mechanisms to formation of DPXL in those organs, such as brain and heart, which play a primary role in the aging of the whole organism.     

TNF‐α increases αvβ3 integrin expression and migration in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumour with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Tumour necrosis factor (TNF)‐α is a key cytokine involved in inflammation, immunity, cellular homeostasis and tumour progression. Integrins are the major adhesive molecules in mammalian cells and have been associated with metastasis of cancer cells. However, the effects of TNF‐α in migration and integrin expression in chondrosarcoma cells are largely unknown. In this study, we found that TNF‐α increased the migration and the expression of αvβ3 integrin in human chondrosarcoma cells. Activations of MAPK kinase (MEK), extracellular signal‐regulating kinase (ERK) and nuclear factor‐κB (NF‐κB) pathways after TNF‐α treatment were demonstrated, and TNF‐α‐induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of MEK, ERK and NF‐κB cascades. Taken together, our results indicated that TNF‐α enhances the migration of chondrosarcoma cells by increasing αvβ3 integrin expression through the MEK/ERK/NF‐κB signal transduction pathway. J. Cell. Physiol. 226: 792–799, 2011. © 2010 Wiley‐Liss, Inc.     

1′,2′-Dideacetylboronolide, an α-pyrone from Iboza riparia

The structural elucidation of 1′,2′-dideacetylboronolide, 5,6-dihydro-6-(3′-acetoxy-1′,2′-dihydroxyheptyl)2-pyrone, a new α-pyrone isolated from the leaves of Iboza riparia has been performed. Additionally, three sterols, sitosterol, stigmasterol and campesterol, have been identified in this species.     

1α,25-Dihydroxyvitamin D3-induced changes in intracellular pH in osteoblast-like cells modulate gene expression

1α,25-Dihydroxyvitamin D₃ exerts rapid nongenomic effects on rat osteoblast-like cells independent of the classic nuclear receptor. These effects include changes in phospholipid metabolism and cell calcium. Intracellular calcium itself has been proposed to regulate intracellular pH in osteoblast cell lines. The purpose of this study was to determine the effect of 1α,25-dihydroxyvitamin D₃ on intracellular pH, the relationship of changes in calcium to changes in pH, and the role of pH changes in genomic activation. 1α,25-Dihydroxyvitamin D₃ increased intracellular pH within 10 min in rat osteoblast-like cells, an effect that was inhibited by removal of extracellular sodium and by the biologically inactive epimer 1β,25-dihydroxyvitamin D₃. The hormone increased intracellular calcium in Quin 2 loaded cells in the presence and absence of extracellular sodium. The 1α,25-dihydroxyvitamin D₃-induced increments in osteocalcin and osteopontin mRNA levels were abolished in sodium-free medium. The results indicate that 1α,25-dihydroxyvitamin D₃-induced increments in cellular calcium precede cell alkalinization and that these changes in intracellular pH may modulate steady-state mRNA levels of genes induced by vitamin D.     

HSP70 interacts with TRAF2 and differentially regulates TNFα signalling in human colon cancer cells

Members of tumour necrosis factor (TNF) family usually trigger both survival and apoptotic signals in various cell types. Heat shock proteins (HSPs) are conserved proteins implicated in protection of cells from stress stimuli. However, the mechanisms of HSPs in TNFα‐induced signalling pathway have not been fully elucidated. We report here that HSP70 over‐expression in human colon cancer cells can inhibit TNFα‐induced NFκB activation but promote TNFα‐induced activation of c‐Jun N‐terminal kinase (JNK) through interaction with TNF receptor (TNFR)‐associated factor 2 (TRAF2). We provide evidence that HSP70 over‐expression can sequester TRAF2 in detergent‐soluble fractions possibly through interacting with TRAF2, leading to reduced recruitment of receptor‐interacting protein (RIP1) and IκBα kinase (IKK) signalosome to the TNFR1–TRADD complex and inhibited NFκB activation after TNFα stimuli. In addition, we found that HSP70–TRAF2 interaction can promote TNFα‐induced JNK activation. Therefore, our study suggests that HSP70 may differentially regulate TNFα‐induced activation of NFκB and JNK through interaction with TRAF2, contributing to the pro‐apoptotic roles of HSP70 in TNFα‐induced apoptosis of human colon cancer cells.     

Fast quantification of the urinary marker of oxidative stress 8-hydroxy-2′-deoxyguanosine using solid-phase extraction and high-performance liquid chromatography with triple-stage quadrupole mass detection

Exogenous and endogenous oxidants constantly cause oxidative damage to DNA. Since the reactive oxidants itself are not suitable for analysis, oxidized bases like 8-hydroxy-2′-deoxyguanosine (8OHdG) are used as biomarkers for oxidative stress, either in cellular DNA or as elimination product in urine. A simple, fast and robust analytical procedure is described for urinary 8OHdG as an indicator of oxidative damage in humans. The adduct was purified from human urine by applying a single solid-phase extraction step on LiChrolut EN^®. After evaporation of the eluate, the residue was resolved and an aliquote was injected into a HPLC system with a triple quadrupole mass spectrometer. The limit of detection was 0.2 ng ml⁻¹ (7 fmol absolute) when using one product ion as quantifier and two further product ions as qualifier. The coefficient of variation was 10.1% (n=5 at 2.8 ng ml⁻¹ urine). The sample throughput was about 50 samples a day. Thus, this method is more sensitive and much faster than the common method using HPLC with electrochemical detection. The results of a study with nine volunteers investigated at six time-points each over 5 days are presented. The mean excretion of 8OHdG was 2.1 ng mg⁻¹ creatinine (range 0.17–5.9 ng mg⁻¹ creatinine; 4 of 53 samples were below the LOD). A relatively large intra- (relative SD 66%) and inter-individual (relative SD 71%) variation in urinary 8OHdG excretion rates was found.     

2-(3′-Hydroxypropylidene)-1α-hydroxy-19-norvitamin D compounds with truncated side chains

Our recent studies with 2-(3′-hydroxypropylidene) analogs of 1α,25-dihydroxy-19-norvitamin D₃ showed that this 2-substituent creates compounds with very potent biological activity. In the continuing search for vitamin D compounds with selective activity profiles, we prepared a series of 1α-hydroxy-19-norvitamin D analogs characterized by the presence of a 3′-hydroxypropylidene substituent at C-2 and a truncated side chain. These vitamin D compounds were efficiently prepared using convergent syntheses. The C,D-fragments, namely the Grundmann ketones 19, 20, 27, 36 and 37 were synthesized from the known 8β-benzoyloxy-22-aldehydes 12 and 29. These hydrindanones were subjected to Lythgoe type Wittig–Horner coupling with phosphine oxide 21, prepared by us previously, and after hydroxyl deprotection the set of 19-norvitamins 7–11 was successfully obtained. According to our expectations, all analogs (with an exception of the 20R-compound 7) have pronounced in vitro activity. When compared to the natural hormone 1α,25-(OH)₂D₃ (1), they show the same or only slightly reduced affinity for the vitamin D receptor while being similarly effective as 1 in differentiation of HL-60 cells into monocytes.     

Blood Platelets Contain and Secrete Laminin-8 (α4β1γ1) and Adhere to Laminin-8 via α6β1 Integrin

Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin γ1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to γ1 and β1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin β1 antibody–Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to β1 and γ1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin α4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20–35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to β1 and α6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (α4β1γ1) and that the cells adhere to the protein by using α6β1 integrin.     

Effects of the vitamin D3 analog 1α,25-dihydroxyvitamin D3-3β-bromoacetate on rat osteosarcoma cells: Comparison with 1α,25-dihydroxyvitamin D3

The actions of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1α,25-(OH)₂D₃ in many cell types including osteoblastic cells. We have compared the effects of 1α,25-(OH)₂D₃ and a novel 1α,25-(OH)₂D₃ bromoester analog (1,25-(OH)₂-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 × 10⁻⁸ M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)₂-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE, intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 × 10⁻⁸ M, both 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)₂-BE are similar to those of 1α,25-(OH)₂D₃, and since 1,25-(OH)₂-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors. © 1996 Wiley-Liss, Inc.     

α-, β-, and γ-Tubulin Polymerization in Response to DNA Damage

Microtubules provide structural support for a cell and play key roles in cell motility, mitosis, and meiosis. They are also the targets of several anticancer agents, indicating their importance in maintaining cell viability. We have investigated the possibility that alterations in microtubule structure and tubulin polymerization may be part of the cellular response to DNA damage. In this report, we find that γ-radiation stimulates the production and polymerization of α-, β-, and γ- tubulin in hematopoeitic cell lines (Ramos, DP16), leading to visible changes in microtubule structures. We have found that this microtubule reorganization can be prevented by caffeine, a drug that concomitantly inhibits DNA damage-induced cell cycle arrest and apoptosis. Our results support the idea that microtubule polymerization is an important facet of the mammalian response to DNA damage.     

Endogenous CSE/H2S system mediates TNF‐α‐induced insulin resistance in 3T3‐L1 adipocytes

Tumour necrosis factor‐α (TNF‐ α)is a major contributor to the pathogenesis of insulin resistance associated with obesity and type 2 diabetes. It has been found that endogenous hydrogen sulfide (H₂S) contributes to the pathogenesis of diabetes. We have hypothesized that TNF‐α‐induced insulin resistance is involved in endogenous H₂S generation. The aim of the present study is to investigate the role of endogenous H₂S in TNF‐α‐induced insulin resistance by studying 3T3‐L1 adipocytes. We found that treatment of 3T3‐L1 adipocytes with TNF‐α leads to deficiency in insulin‐stimulated glucose consumption and uptake and increase in endogenous H₂S generation. We show that cystathionine γ‐lyase (CSE) is catalysed in 3T3‐L1 adipocytes to generate H₂S and that CSE expression and activity are upregulated by TNF‐α treatment. Inhibited CSE by its potent inhibitors significantly attenuates TNF‐α‐induced insulin resistance in 3T3‐L1 adipocytes, whereas H₂S treatment of 3T3‐L1 adipocytes impairs insulin‐stimulated glucose consumption and uptake. These data indicate that endogenous CSE/H₂S system contributes to TNF‐α‐caused insulin resistance in 3T3‐L1 adipocytes. Our findings suggest that modulation of CSE/H₂S system is a potential therapeutic avenue for insulin resistance. Copyright © 2012 John Wiley & Sons, Ltd.     

Parallel solid-phase synthesis of 3β-peptido-3α-hydroxy-5α-androstan-17-one derivatives for inhibition of type 3 17β-hydroxysteroid dehydrogenase

Type 3 17β-hydroxysteroid dehydrogenase (17β-HSD), a key steroidogenic enzyme, transforms 4-androstene-3,17-dione (Δ⁴-dione) into testosterone. In order to produce potential inhibitors, we performed solid-phase synthesis of model libraries of 3β-peptido-3α-hydroxy-5α-androstan-17-ones with 1, 2, or 3 levels of molecular diversity, obtaining good overall yields (23–58%) and a high average purity (86%, without any purification steps) using the Leznoff's acetal linker. The libraries were rapidly synthesized in a parallel format and the generated compounds were tested as inhibitors of type 3 17β-HSD. Potent inhibitors were identified from these model libraries, especially six members of the level 3 library having at least one phenyl group. One of them, the 3β-(N-heptanoyl- -phenylalanine- -leucine-aminomethyl)-3α-hydroxy-5α-androstan-17-one (42) inhibited the enzyme with an IC₅₀ value of 227 nM, which is twice as potent as the natural substrate Δ⁴-dione when used itself as an inhibitor. Using the proliferation of androgen-sensitive (AR⁺) Shionogi cells as model of androgenicity, the compound 42 induced only a slight proliferation at 1 μM (less than previously reported type 3 17β-HSD inhibitors) and, interestingly, no proliferation at 0.1 μM.     

Binding characteristics of a membrane receptor that recognizes 1α25-dihydroxyvitamin D3 and its epimer, 1β,25-dihydroxyvitamin D3

The Steroid hormon 1α, @5-Dihydroxyvitamin D₃ has been shown to expert rapid effect (15 s to 5 min) in osteoblast. These occur in osteoblast-like cells lacking the nuclear vitamin D receptor, ROS 24/1, suggesting that a separate signalling system mediates the rapid action. These non-genomic action include rapid activation of phospholipase C and opening of calcium channels, pointing to a membrane localization of this signalling system. Previous studies have shown that the 1β epimer of 1α25-dihydroxyvitamina D₃ can block these rapid action, indicating that the 1β epimer may bind to the recptor responsible for the rapid action sin a competative manner. We have assessed the displacement of ³H-1α,25dihydroxyvitamin D₃ by vitamin D compounds, as well as the apparent dissociation constant of 1α25-dihydroxyvitamin D₃ and its 1β epimer for the memberane receptor in membrane prepration from ROS 24/1 cells. Increasing concentrations of 1α25-dihydroxyvitamin D₃, 7.25 nM to 725 nM, displaced ³H-1α25-dihydrxyvitamin D₃ from the membranes with 725 nM of the hormone displacing 40–49% of the radioactivity. Similarly, 1β,25-dihydroxyvitamin D₃, 7.25 nM and 72.5 nM, displaced 1α25-dihydroxyvitamin D₃ binding while 25-hydroxyvitamin D₃, 7.25 nM, did not. The apparent dissociation constant (K_D) for 1α25-dihydroxyvitamin D₃ was detrermined from displacement of ³H-1α25-dihydroxyvitamin D₃ yielding a value of 8.1 × 10^?7 M by Scatchard analysis. The K_D for the 1β epimer determine from displacement of ³H-1α25-dihydroxyvitamin D₃ was 4.8 × 10^?7 M. The data suggest the presence of a receptor on the membrane of ROS 24/1 cells that reconize 1α25-dihydroxyvitamin D₃ and its 1β epimer, but not 25-dihydroxyvitamin D₃. Its ability to reconize the 1β epimer which appears to be a specific anagonist of the rapid effect of the hormone suggests that these studies may be the initial steps in the isolation and characterization of the signalling system mediating the rapid action of vitamin D.     

Cyclic strain induces reorganization of integrin α5β1 and α2β1 in human umbilical vein endothelial cells

Cyclic strain has been shown to modulate endothelial cell (EC) morphology, proliferation, and function. We have recently reported that the focal adhesion proteins focal adhesion kinase (pp125^FAK) and paxillin, are tyrosine phosphorylated in EC exposed to strain and these events regulate the morphological change and migration induced by cyclic strain. Integrins are also localized on focal adhesion sites and have been reported to induce tyrosine phosphorylation of pp125^FAK under a variety of stimuli. To study the involvement of different integrins in signaling induced by cyclic strain, we first observed the redistribution of α and β integrins in EC subjected to 4 h cyclic strain. Human umbilical vein endothelial cells (HUVEC) seeded on either fibronectin or collagen surfaces were subjected to 10% average strain at a frequency 60 cycles/min. Confocal microscopy revealed that β₁ integrin reorganized in a linear pattern parallel with the long axis of the elongated cells creating a fusion of focal adhesion plaques in EC plated on either fibronectin (a ligand for α₅β₁) or collagen (a ligand for α₂β₁) coated plates after 4 h exposure to cyclic strain. β₃ integrin, which is a vitronectin receptor, did not redistribute in EC exposed to cyclic strain. Cyclic strain also led to a reorganization of α₅ and α₂ integrins in a linear pattern in HUVEC seeded on fibronectin or collagen, respectively. The expression of integrins α₅, α₂, and β₁ did not change even after 24 h exposure to strain when assessed by immunoprecipitation of these integrins. Cyclic strain-induced tyrosine phosphorylation of pp125^FAK occurred concomitant with the reorganization of β₁ integrin. We concluded that α₅β₁ and α₂β₁ integrins play an important role in transducing mechanical stimuli into intracellular signals. J. Cell. Biochem. 64:505–513. © 1997 Wiley-Liss, Inc.     

Synthesis and function of 8α,25-dihydroxy-3-oxoneocholecalciferol in liver

25-Hydroxycholecalciferol (25-OHD₃) is converted to 8α,25-dihydroxy-3-oxoneocholecalciferol [8,25-(OH)₂-3-oxone-D₃] by liver microsomes, alveolar macrophages and myeloid leukemia cells. The characteristics of this reaction in liver microsomes have been determined. Omission of an NADPH-generating system or NADH resulted in a >75% reduction in the production of 8,25-(OH)₂-3-oxone-D₃. In the absence of the cytosolic fraction, 25-OHD₃ was converted to products that comigrated with 8,25-(OH)₂-3-oxoneo-D₃ on a silica column developed with hexane-isopropanol, thereby preventing quantitation. Production of 8,25-(OH)₂-3-oxoneo-D₃ was unaffected by EDTA and was stimulated by N,N′-diphenyl-p-phenylenediamine. Both progesterone and pregnenolone inhibited production of 8,25-(OH)₂-3-oxoneo-D₃; inhibition by progesterone was greater than that by pregnenolone. 8,25-(OH)₂-3-Oxoneo-D₃ did not bind the thymus receptor for 1,25-dihydroxycholecalciferol [1,25-(OH)₂D₃] at concentrations 10-fold higher than that of 1,25-(OH)₂D₃. The lack of affinity of 8,25-(OH)₂-3-oxoneo-D₃ for the 1,25-(OH)₂D₃ receptor suggests that this metabolite is a degradative product of 25-OHD₃, which might be produced when 25-OHD₃ concentrations in the liver are excessive. Synthesis of this metabolite in the liver may be catalyzed by enzymes that also metabolize other steroids.