BBA - Molecular Basis of Disease (v.1772, #9)

Molecular basis of the differences between normal and tumor tissues of gastric cancer by Sanghwa Yang; Jihye Shin; Kyu Hyun Park; Hei-Cheul Jeung; Sun Young Rha; Sung Hoon Noh; Woo Ick Yang; Hyun Cheol Chung (1033-1040).
To be able to describe the differences between the normal and tumor tissues of gastric cancer at a molecular level would be essential in the study of the disease. We investigated the gene expression pattern in the two types of tissues from gastric cancer by performing expression profiling of 86 tissues on 17K complementary DNA microarrays. To select for the differentially expressed genes, class prediction algorithm was employed. For predictor selection, samples were first divided into a training (n  = 58), and a test set (n  = 28). A group of 894 genes was selected by a t-test in a training set, which was used for cross-validation in the training set and class (normal or tumor) prediction in the test set. Smaller groups of 894 genes were individually tested for their ability to correctly predict the normal or tumor samples based on gene expression pattern. The expression ratios of the 5 genes chosen from microarray data can be validated by real time RT-PCR over 6 tissue samples, resulting in a high level of correlation, individually or combined. When a representative predictor set of 92 genes was examined, pathways of ‘focal adhesion’ (with gene components of THBS2, PDGFD, MAPK1, COL1A2, COL6A3), ‘ECM-receptor interaction’ pathway (THBS2, COL1A2, COL6A3, FN1) and ‘TGF-beta signaling’ (THBS2, MAPK1, INHBA) represent some of the main differences between normal and tumor of gastric cancer at a molecular level.
Keywords: cDNA microarray; Gene expression; Gastric cancer; Real-time RT-PCR;

Mitochondrial and cytosolic thiol redox state are not detectably altered in isolated human NADH:ubiquinone oxidoreductase deficiency by Sjoerd Verkaart; Werner J.H. Koopman; Julia Cheek; Sjenet E. van Emst-de Vries; Lambertus W.P.J. van den Heuvel; Jan A.M. Smeitink; Peter H.G.M. Willems (1041-1051).
Isolated complex I deficiency is the most common enzymatic defect of the oxidative phosphorylation (OXPHOS) system, causing a wide range of clinical phenotypes. We reported before that the rates at which reactive oxygen species (ROS)-sensitive dyes are converted into their fluorescent oxidation products are markedly increased in cultured skin fibroblasts of patients with nuclear-inherited isolated complex I deficiency. Using video-imaging microscopy we show here that these cells also display a marked increase in NAD(P)H autofluorescence. Linear regression analysis revealed a negative correlation with the residual complex I activity and a positive correlation with the oxidation rates of the ROS-sensitive dyes 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein and hydroethidine for a cohort of 10 patient cell lines. On the other hand, video-imaging microscopy of cells expressing reduction–oxidation sensitive GFP1 in either the mitochondrial matrix or cytosol showed the absence of any detectable change in thiol redox state. In agreement with this result, neither the glutathione nor the glutathione disulfide content differed significantly between patient and healthy fibroblasts. Finally, video-rate confocal microscopy of cells loaded with C11-BODIPY581/591 demonstrated that the extent of lipid peroxidation, which is regarded as a measure of oxidative damage, was not altered in patient fibroblasts. Our results indicate that fibroblasts of patients with isolated complex I deficiency maintain their thiol redox state despite marked increases in ROS production.
Keywords: Human skin fibroblasts; NADH:ubiquinone oxidoreductase; Thiol redox state; roGFP1; Redox environment; Superoxide; Reactive oxygen species; Glutathione; Lipid peroxidation;

It has been found that β-carotene cleavage products (CarCP), besides having mutagenic and toxic effects on mitochondria due to their prooxidative properties, also initiate spontaneous apoptosis of human neutrophils. Therefore, it was expected that antioxidants such as α-tocopherol would inhibit the stimulation of apoptosis and caspase-3 activity by CarCP. However, we found that α-tocopherol increases caspase-3 up-regulation and stimulation of apoptosis of human neutrophils by CarCP. Ascorbic acid does not alter this caspase-3 up-regulating and proapoptotic effect exerted by α-tocopherol. Both α-tocopherol and ascorbic acid, in the absence of CarCP, decrease intracellular caspase-3 activity and spontaneous apoptosis of neutrophils. Uric acid alone or in combination with CarCP does not exert apparent effects on caspase-3 activity and apoptosis. Up-regulating effect of α-tocopherol is not observed in the presence of retinol that markedly stimulates apoptosis by itself, whereas increase of caspase-3 activity is induced by concomitant addition of α-tocopherol and β-ionone, a cyclohexenyl degradation product of β-carotene with shorter aliphatic chain.
Keywords: Tocopherol; Carotenoid; Neutrophil; Apoptosis; Oxidative stress;

The pesticide adjuvant, Toximul™, alters hepatic metabolism through effects on downstream targets of PPARα by Jacqueline Upham; Philip D. Acott; Patrick O'Regan; Christopher J. Sinal; John F.S. Crocker; Laurette Geldenhuys; Mary G. Murphy (1057-1064).
Previous studies demonstrated that chronic dermal exposure to the pesticide adjuvant (surfactant), Toximul™ (Tox), has significant detrimental effects on hepatic lipid metabolism. This study demonstrated that young mice dermally exposed to Tox for 12 days have significant increases in expression of peroxisomal acyl-CoA oxidase (mRNA and protein), bifunctional enzyme (mRNA) and thiolase (mRNA), as well as the P450 oxidizing enzymes Cyp4A10 and Cyp4A14 (mRNA and protein). Tox produced a similar pattern of increases in wild type adult female mice but did not induce these responses in PPARα-null mice. These data support the hypothesis that Tox, a heterogeneous blend of nonionic and anionic surfactants, modulates hepatic metabolism at least in part through activation of PPARα. Notably, all three groups of Tox-treated mice had increased relative liver weights due to significant accumulation of lipid. This could be endogenous in nature and/or a component(s) of Tox or a metabolite thereof. The ability of Tox and other hydrocarbon pollutants to induce fatty liver despite being PPARα agonists indicates a novel consequence of exposure to this class of chemicals, and may provide a new understanding of fatty liver in populations with industrial exposure.
Keywords: Pesticide adjuvant; PPARα; PPARα-null mice; MRNA and protein expression; Cyp4A; Peroxisomal fatty-acid oxidation;

Flavonoids as protectors against doxorubicin cardiotoxicity: Role of iron chelation, antioxidant activity and inhibition of carbonyl reductase by Helena Kaiserová; Tomáš Šimůnek; Wim J.F. van der Vijgh; Aalt Bast; Eva Kvasničková (1065-1074).
Anthracycline antibiotics (e.g. doxorubicin and daunorubicin) are among the most effective and widely used anticancer drugs. Unfortunately, their clinical use is limited by the dose-dependent cardiotoxicity. Flavonoids represent a potentially attractive class of compounds to mitigate the anthracycline cardiotoxicity due to their iron-chelating, antioxidant and carbonyl reductase-inhibitory effects. The relative contribution of various characteristics of the flavonoids to their cardioprotective activity is, however, not known. A series of ten flavonoids including quercetin, quercitrin, 7-monohydroxyethylrutoside (monoHER) and seven original synthetic compounds were employed to examine the relationships between their inhibitory effects on carbonyl reduction, iron-chelation and antioxidant properties with respect to their protective potential against doxorubicin-induced cardiotoxicity. Cardioprotection was investigated in the neonatal rat ventricular cardiomyocytes whereas the H9c2 cardiomyoblast cells were used for cytotoxicity testing. Iron chelation was examined via the calcein assay and antioxidant effects and site-specific scavenging were quantified by means of inhibition of lipid peroxidation and hydroxyl radical scavenging activity, respectively. Inhibition of carbonyl reductases was assessed in cytosol from human liver. None of the flavonoids tested had better cardioprotective action than the reference cardioprotector, monoHER. However, a newly synthesized quaternary ammonium analog with comparable cardioprotective effects has been identified. No direct correlation between the iron-chelating and/or antioxidant effect and cardioprotective potential has been found. A major role of carbonyl reductase inhibition seems unlikely, as the best two cardioprotectors of the series are only weak reductase inhibitors.
Keywords: Anthracyclines; Cardioprotection; Iron chelator; Hydroxyl radical scavenging; Lipid peroxidation; Doxorubicinol;

Cellular transcription modulator SMARCE1 binds to HBV core promoter containing naturally occurring deletions and represses viral replication by Hong Pan; Dan Dan Niu; Huixing Feng; Lisa F.P. Ng; Ee Chee Ren; Wei Ning Chen (1075-1084).
Suppression of hepatitis B virus (HBV) replication, a causative agent for chronic hepatitis, is an effective approach to controlling disease progression. Host factors have a significant effect on viral replication efficiency and need to be better characterized. We have reported association between clinical virus load and deletions in HBV viral promoter. We showed here that HBV genome with such deletions led to decreased replication compared with wild type virus. Consistently, the promoter with deletion showed lower activity. A cellular transcription regulator recognizing the promoter with deletion was revealed in gel shift assay and subsequently identified as SMARCE 1 through DNA–protein array assay. The ability of SMARCE 1 in modulating the replication efficiency of HBV was further demonstrated. Taken together, our studies show a direct dependence of HBV on a host factor to modulate its replication efficiency, and provided a new platform for molecular characterization of mechanisms of disease outcome as a result of binding of new transcription factors to rearranged promoter sequences.
Keywords: HBV; Core promoter and deletion; Replication; DNA–protein interactions; SMARCE1;

We have investigated the role of two polymorphic sites (R190W and N192K) on the binding and activation of the formyl peptide receptor (FPR) by viral and formyl peptides. WEDWVGWI, a peptide with antiviral activity derived from the membrane proximal region of feline immunodeficiency virus, binds with high affinity to FPR. The three tryptophans in the peptide are all essential for FPR binding, just as they were essential for antiviral activity [S. Giannecchini, A. Di Fenza, A.M. D'Ursi, D. Matteucci, P. Rovero, M. Bendinelli, Antiviral activity and conformational features of an octapeptide derived from the membrane-proximal ectodomain of the feline immunodeficiency virus transmembrane glycoprotein, J. Virol. 77 (2003) 3724]. Formyl-NleWEDWVGWI behaved as a weak partial agonist with FPR W190/N192 but a stronger partial agonist with FPR R190/K192 and FPR R190/N192. Formyl-NleNleWEDWVGWI behaved as a full agonist toward all three FPRs but exhibited a much higher EC50 with W190/N192 FPR (300 ± 45 nM) than for R190/K192 FPR (40 ± 3 nM) or R190/N192 (60 ± 8 nM). Formyl-MYKWPWYVWL preferentially activated R190/K192 and R190/N192 FPRs by > 5 fold compared to W190/N192 FPR. Formyl-MFEDAVAWF, a peptide derived from a protein in Mycobacterium avium subsp. paratuberculosis and formyl-MFTFEPFPTN, a peptide derived from the N-terminus of chemotaxis inhibitory protein of Staphylococcus aureus with an added N-terminal formyl-methionine exhibited the greatest selectivity for R190/K192 and R190/N192 FPRs with ∼ 10 fold lower EC50s than that observed with FPR W190/N192. Thus, individuals with the W190 polymorphism may display a reduced ability to detect certain formyl peptides.
Keywords: Formyl peptides; Signal Transduction; G protein-coupled receptor; polymorphism; Feline immunodeficiency virus; Chemotaxis inhibitory protein of Staphylococcus aureus;

Heterogeneity in retinoic acid signaling in neuroblastomas: Role of matrix metalloproteinases in retinoic acid-induced differentiation by Suchitra Joshi; Rakeshwar S. Guleria; Jing Pan; Donald DiPette; Ugra S. Singh (1093-1102).
Causes of retinoid resistance often observed in neuroblastomas are unknown. We studied all trans-retinoic acid (RA) signaling in neuroblastoma cells differing in N-myc levels in terms of neurite formation, expression of tissue transglutaminase, neuronal marker proteins, matrix metalloproteinases (MMPs), and activation of Rac1 and Cdc42. Poor invasiveness observed in SH-SY5Y, LA-N-5, and SMS-KCNR cells was associated with RA-induced neurite formation, Cdc42 activation and N-myc down regulation; expression of constitutively active Cdc42 down regulated N-myc expression and reduced invasion in RA-resistant SK-N-BE(2) and IMR32 cells. RA treatment for 24 h transiently increased invasion and expression of MMP9 in SH-SY5Y, LA-N-5 and MMP2 in SMS-KCNR cells. MMP inhibition prevented RA-induced neurite formation indicating a role in differentiation. Variation in RA signaling thus follows a defined pattern and relates to invasive potential. A defective RA signaling might result in retinoid resistance and unpredictable clinical outcome observed in some neuroblastomas.
Keywords: Neuroblastoma; Retinoic acid; Tissue-transglutaminase; Matrix metallo-proteinases; Rho GTPases;

Multiple pathways regulating the anti-apoptotic protein clusterin in breast cancer by Melissa K. Ranney; Ikhlas S.A. Ahmed; Kelly R. Potts; Rolf J. Craven (1103-1111).
Cancer chemotherapy inhibits tumor growth, in part, by triggering apoptosis, and anti-apoptotic proteins reduce the effectiveness of chemotherapy. Clusterin, a chaperone-like protein that binds to apoptotic and DNA repair proteins, is induced by chemotherapy and promotes tumor cell survival. Histone deacetylase inhibitors (HDIs) such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA) are pharmacological agents that induce differentiation and apoptosis in cancer cells by altering chromatin structure, and we have found that combinations of chemotherapeutic drugs such as doxorubicin and HDIs efficiently induce apoptosis, even though they paradoxically induce high levels of clusterin. The hyper-expressed form of clusterin localizes to mitochondria, inhibits cytochrome c release, and is inhibited by the proteasome. When HDIs are used as single agents, clusterin suppresses cytochrome c release and apoptosis. However, doxorubicin/HDI-induced apoptosis is not inhibited by clusterin, and clusterin-resistant apoptosis corresponds with markers of the extrinsic/receptor-mediated apoptotic pathway. Thus, chemotherapy–HDI combinations are capable of overcoming an innate anti-apoptotic pathway of tumor cells, suggesting that chemotherapy–HDI combinations have potential for treating advanced stage breast cancer.
Keywords: Clusterin; Doxorubicin; Breast cancer; Apoptosis; Caspase; PARP; Histone deacetylase; Calpain; Proteasome;

Myocardial cytochrome oxidase activity is decreased following carbon monoxide exposure by Kelechi N. Iheagwara; Stephen R. Thom; Clifford S. Deutschman; Richard J. Levy (1112-1116).
Carbon monoxide (CO) inhalation often leads to cardiac dysfunction, dysrhythmias, ischemia, infarction, and death. However, the underlying mechanism of CO toxicity is poorly understood. We hypothesize that inhaled CO interrupts myocardial oxidative phosphorylation by decreasing the activity of myocardial cytochrome oxidase (CcOX), the terminal oxidase of the electron transport chain. Male C57Bl6 mice were exposed to either 1000 ppm (0.1%) CO or air for 3 h. Cardiac ventricles were harvested and mitochondria were isolated. CcOX kinetics and heme aa3 content were measured. V max, K m, and turnover number were determined. Levels of CcOX subunit I message and protein were evaluated. Carboxyhemoglobin (COHb) levels were measured and tissue hypoxia was assessed with immunohistochemistry for pimonidazole hydrochloride. CO significantly decreased myocardial CcOX activity and V max without altering K m. Heme aa3 content and CcOX I protein levels significantly decreased following CO exposure while enzyme turnover number and CcOX I mRNA levels remained unchanged. CO exposure increased COHb levels without evidence of tissue hypoxia as compared to sham and hypoxic controls. Decreased CcOX activity following CO inhalation was likely due to decreased heme aa3 and CcOX subunit I content. Importantly, myocardial CcOX impairment could underlie CO induced cardiac dysfunction.
Keywords: Cytochrome oxidase; Carbon monoxide; Heart; Inhibition;

Corrigendum to “Quantification of chloride channel 2 (CLCN2) gene isoforms in normal versus lesion- and epilepsy-associated brain tissue” [Biochim. Biophys. Acta 1772 (2007) 15–20] by Matteo Bertelli; Stefano Cecchin; Cristina Lapucci; Paola de Gemmis; Daniela Danieli; Emanuele S.G. d'Amore; Luciano Buttolo; Filippo Giunta; Pietro Mortini; Massimo Pandolfo (1117).