Overexpression of heme oxygenase-1 in murine melanoma: increased proliferation and viability of tumor cells, decreased survival of mice

Heme oxygenase-1 (HO-1), a cytoprotective enzyme, can be induced in tumors in response to anti-cancer therapies. We investigated the role of HO-1 in B16(F10), S91, and Sk-mel188 melanoma cells. Overexpression of HO-1 after transduction with adenoviral vectors increased cell proliferation, resistance to oxidative stress generated by H2O2, and angiogenic potential as determined by induction of endothelial cell divisions. Likewise, cells stably transfected with HO-1 cDNA (B16-HO-1) showed higher proliferation, stress resistance, and angiogenic activity than the wild-type line (B16-WT). HO-1 overexpression in tumors significantly shortened survival of mice after subcutaneous injection of cancer cells (38 and 22 days for B16-WT and B16-HO-1, respectively; P=0.017). This also resulted in development of more packed tumors, with more melanoma cells, and reduced inflammatory edemas. Mice injected with B16-HO-1 had lower levels of tumor necrosis factor and higher serum concentrations of its soluble receptor tumor necrosis factor-RI, whereas tumors overexpressing HO-1 displayed augmented vascularization and stronger production of vascular endothelial growth factor. Finally, B16-HO-1 cells injected intravenously formed more metastases in lungs. Thus, HO-1 overexpression increased viability, proliferation, and angiogenic potential of melanoma cells, augmented metastasis, and decreased survival of tumor-bearing mice, suggesting that induction of HO-1 may be detrimental in anti-cancer therapy of melanoma.     

Glutathione S-transferase and microsomal epoxide hydrolase gene polymorphisms and risk of chronic obstructive pulmonary disease in Slovak population

Aim

To determine the risk of chronic obstructive pulmonary disease (COPD) associated with polymorphisms in the glutathione S-transferase (GST) M1, GST T1, and microsomal epoxide hydrolase (EPHX1) genes in a cohort of Slovak population.

Methods

Two hundred and seventeen patients with the diagnosis of COPD and 160 control subjects were enrolled in the study. Blood samples were collected from all subjects and the DNA from peripheral blood lymphocytes was used for subsequent genotyping assays, using polymerase chain reaction and restriction fragment-length polymorphism methods.

Results

In an unadjusted model, an increased risk for COPD was observed in subjects with EPHX1 His113-His113 genotype (odds ratio [OR], 2.32; 95% confidence interval [CI], 1.20-4.69; P = 0.008), compared with the carriers of the Tyr113 allele. However, after the adjustments for age, sex, and smoking status, the risk was not significant (adjusted OR, 1.79; 95% CI, 0.91-3.53; P = 0.093). In a combined analysis of gene polymorphisms, the genotype combination EPHX1 His113-His113/GSTM1 null significantly increased the risk of COPD in both, unadjusted (OR, 5.08; 95% CI, 1.70-20.43; P = 0.001) and adjusted model (OR, 4.87; 95% CI, 1.57-15.13; P = 0.006).

Conclusion

Although none of the tested gene polymorphisms was significantly related to an increased risk of COPD alone, our results suggest that the homozygous exon 3 mutant variant of EPHX1 gene in the combination with GSTM1 null genotype is a significant predictor of increased susceptibility to COPD in the Slovak population. The findings of the present study emphasize the importance of detoxifying and antioxidant pathways in the pathogenesis of COPD.Chronic obstructive pulmonary disease (COPD) represents a major public health care problem worldwide due to its increasing prevalence, morbidity, and mortality (1). Generally, COPD is characterized by progressive and only partially reversible airflow limitation (2). Although cigarette smoking is the most important risk factor for COPD, only 20%-30% of chronic smokers develop severe impairment of lung function associated with COPD (3). Besides smoking, other environmental and genetic factors and gene-environment interactions influence the development of COPD (4).Severe α-1-antitrypsin deficiency is a well established genetic risk factor for COPD that has provided a basis for the protease-antiprotease hypothesis in the pathogenesis of COPD (5,6). Other candidate genes that might play a role in the development of COPD are involved in endogenous protease/antiprotease imbalance, inflammatory processes, metabolism of mutagens and carcinogens in tobacco smoke, and in mucocilliary clearance (7). Interindividual differences in the polymorphisms of enzymes metabolizing the xenobiotic substances and free radicals contained in the cigarette smoke may play a role in the individual susceptibility to the decrease in lung functions in smokers (8).Microsomal epoxide hydrolase (EPHX1) is generally considered to be a protective enzyme involved in the defense from oxidative damage (9,10). Two common polymorphic sites in the EPHX1 gene that influence the enzyme activity can be detected (11). An exon 3 thymine-to-cytosine mutation changes Tyr residue 113 to His, thus reducing the enzyme activity by about 50%. The second mutation, an adenine-to-guanine transition in exon 4 of the gene, changes His residue 139 to Arg and results in the production of EPHX1 with the activity increased by about 25% (11). The combination of these polymorphisms leads to a formation of several functional phenotypes of EPHX1. The slow metabolizing type of EPHX1 was associated with emphysema and COPD (9). In another study, an association of slow metabolizing EPHX1 phenotype with an accelerated deterioration of lung function in smokers was observed (12). In addition, several studies conducted in different populations have suggested that the EPHX1 genotype may influence individual susceptibility to COPD (9,13-15). Nevertheless, other investigators failed to confirm an association between the EPHX1 gene polymorphisms and COPD (16-18).Glutathione S-transferases (GST) play a role in the detoxification of carcinogenic compounds contained in cigarette smoke and in the antioxidant protection (19,20). Recently, the GSTM1 and the GSTT1 gene polymorphisms have been excessively studied with respect to their potential contribution to the risk of COPD (8,17,21,22). The deficiency in the activity of GSTM1 and GSTT1 enzymes is caused by the inherited homozygous absence of the GSTM1 or GSTT1 gene, respectively (ie, GSTM1 null or GSTT1 null genotype). Previously, the homozygous GSTM1 null genotype has been associated with lung cancer (23), emphysema (21), and reductions in the lung function in Caucasian smokers with non-small-cell lung cancer (22). However, another study conducted in Koreans found no differences in the frequencies of polymorphic genotypes of GSTM1 and GSTT1 genes between patients with COPD and healthy smokers (17).Since current data on the potential associations between an increased COPD risk and genes encoding the enzymes metabolizing xenobiotic substances are inconsistent, the aim of our study was to analyze the relation between COPD and gene polymorphisms of EPHX1, GSTM1, and GSTT1 genes in a sample of Slovak population.     

Nuclear thymidylate synthase expression in sporadic colorectal cancer depends on the site of the tumor

Colorectal carcinoma (CRC) is a heterogeneous disease with specific epidemiological, pathological, molecular, and clinical characteristics that depend on the location of the tumor relative to the splenic flexure. Thymidylate synthase (TS) is a major target of 5-fluorouracil-based chemotherapy for CRC and high expression of this enzyme in tumor cells can influence the effect of therapy. We examined differences in TS protein expression in nuclei of tumor cells between CRCs located proximal and distal to the splenic flexure. Nuclear TS was detected by immunohistochemistry with a TS 106 monoclonal antibody on tissue microarrays constructed from 269 CRCs. The median histological score of nuclear TS expression of all proximal tumors was two times higher (p = 0.0003) and in men three times higher (p = 0.00023) than that found in distal tumors. In multivariate analysis which included age, sex, Astler–Coller stage, histological grade, and site, only proximal location of the tumor was identified as an independent factor associated with higher TS expression (odds ratio 2.46, 95% confidence interval = 1.29–4.70, p = 0.0062). These results demonstrate significant differences in nuclear TS expression between proximal and distal cancers and suggest the potential importance of the site of the tumor for proper stratification of patients for chemotherapy.     

Rare copy number variation in cerebral palsy

Recent studies have established the role of rare copy number variants (CNVs) in several neurological disorders but the contribution of rare CNVs to cerebral palsy (CP) is not known. Fifty Caucasian families having children with CP were studied using two microarray designs. Potentially pathogenic, rare (<1% population frequency) CNVs were identified, and their frequency determined, by comparing the CNVs found in cases with 8329 adult controls with no known neurological disorders. Ten of the 50 cases (20%) had rare CNVs of potential relevance to CP; there were a total of 14 CNVs, which were observed in <0.1% (<8/8329) of the control population. Eight inherited from an unaffected mother: a 751-kb deletion including FSCB, a 1.5-Mb duplication of 7q21.13, a 534-kb duplication of 15q11.2, a 446-kb duplication including CTNND2, a 219-kb duplication including MCPH1, a 169-kb duplication of 22q13.33, a 64-kb duplication of MC2R, and a 135-bp exonic deletion of SLC06A1. Three inherited from an unaffected father: a 386-kb deletion of 12p12.2-p12.1, a 234-kb duplication of 10q26.13, and a 4-kb exonic deletion of COPS3. The inheritance was unknown for three CNVs: a 157-bp exonic deletion of ACOX1, a 693-kb duplication of 17q25.3, and a 265-kb duplication of DAAM1. This is the first systematic study of CNVs in CP, and although it did not identify de novo mutations, has shown inherited, rare CNVs involving potentially pathogenic genes and pathways requiring further investigation.     

Ductus venosus Doppler measurement during labor

OBJECTIVES: To assess ductus venosus (DV) indices during the first stage of labor and the effect of ruptured membranes, meconium stained liquor and epidural analgesia (EDA). METHODS: Prospective cross-sectional study. Eighty-one women with low-risk singleton term pregnancies participated, 51 had normal labor (Group 1), and 30 experienced ruptured membranes and/or stained liquor (Group 2). Of the latter group 14 received EDA. The effect of various interventions and application of EDA on the ductus venosus index (DVI) and pulsatility index for veins (DV PIV) were tested. RESULTS: The feasibility rate was 94%. A significant increase of DV indices (DVI, DV PIV) was found in group 2 (P<0.001 and P<0.0005, respectively). The A-velocity was also significantly lower in group 2 (P<0.02). A markedly significant increase of DV indices (P<0.0001) among participants receiving EDA was observed in group 2. The mean+/-SD indices were: 0.53+/-0.10 for the DVI and 0.68+/-0.14 for the DV PIV in those women. There was a significant positive correlation of DV indices with the duration of amniorrhea in group 2 (PIV: r=0.66; P<0.002; DVI: r=0.68; P<0.001). CONCLUSIONS: Long-term amniorrhea seems to affect the fetal venous circulation reflected in increased DV waveform indices.     

Improved spectral resolution and high reliability of in vivo 1H MRS at 7 T allow the characterization of the effect of acute exercise on carnosine in skeletal muscle

The aims of this study were to observe the behavior of carnosine peaks in human soleus (SOL) and gastrocnemius (GM) muscles following acute exercise, to determine the relaxation times and to assess the repeatability of carnosine quantification by ¹H MRS at 7 T. Relaxation constants in GM and SOL were measured by a stimulated echo acquisition mode (STEAM) localization sequence. For T₁ measurement, an inversion recovery sequence was used. The repeatability of the measurement and the absolute quantification of carnosine were determined in both muscles in five healthy volunteers. For absolute quantification, an internal water reference signal was used. The effect of acute exercise on carnosine levels and resonance lines was tested in eight recreational runners/cyclists. The defined carnosine measurement protocol was applied three times – before and twice after (approximately 20 and 40 min) a 1‐h submaximal street run and additional toe‐hopping. The measured T₁ relaxation times for the C2‐H carnosine peak at 7 T were 2002 ± 94 and 1997 ± 259 ms for GM and SOL, respectively, and the T₂ times were 95.8 ± 9.4 and 81.0 ± 21.8 ms for GM and SOL, respectively. The coefficient of variation of the carnosine quantification measurement was 9.1% for GM and 6.3% for SOL, showing high repeatability, and the intraclass correlation coefficients (ICCs) of 0.93 for GM and 0.98 for SOL indicate the high reliability of the measurement. Acute exercise did not change the concentration of carnosine in the muscle, but affected the shape of the resonance lines, in terms of the shifting and splitting into doublets. Carnosine measurement by ¹H MRS at 7 T in skeletal muscle exhibits high repeatability and reliability. The observed effects of acute exercise were more prominent in GM, probably as a result of the larger portion of glycolytic fibers in this muscle and the more pronounced exercise‐induced change in pH. Our results support the application of the MRS‐based assessment of carnosine for pH measurement in muscle compartments. © 2015 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.     

TTV infection and its relation to serum transaminases in apparently healthy blood donors and in patients with clotting disorders who have been investigated previously for hepatitis C virus and GBV-C/HGV infection in Belgium

A novel DNA virus, TT virus (TTV), has been proposed as a possible etiologic agent for non A-E hepatitis. The aim of the present study was to determine the prevalence of TTV infection using PCR in healthy blood donors and in patients with clotting disorders who have been investigated previously for GBV-C/HGV and HCV infection in Belgium. In this study, PCR using primers proposed by Takahashi et al. [(1998) Hepatology Research 12:233-239] proved far more sensitive than those used by Okamoto et al. [(1998) Journal of Medical Virology 56:128-132]. The sequence of the PCR products showed 87% identity to the published sequence. TTV was present in 29.7% of healthy blood donors, a figure intermediate between the low rate of infection observed in Scotland and the high rates in the Far East. TTV was detected in 46.5% of 127 patients studied with clotting disorders as compared to 79.5% for HCV and 11.8% for GBV-C/HGV infection. However, there was no impact on the level of serum transaminases. Treatment with interferon for HCV infection co-infected with TTV suppressed temporarily serum TTV DNA. Therefore, it was concluded that TTV DNA is detected frequently in serum of healthy blood donors in Belgium and more often in patients with clotting disorders. TTV does not cause liver disease or contribute to the severity of liver disease.     

Responses of calves to acute stress: individual consistency and relations between behavioral and physiological measures

The present study examined the consistency over time of individual differences in behavioral and physiological responsiveness of calves to intuitively alarming test situations as well as the relationships between behavioral and physiological measures. Twenty Holstein Friesian heifer calves were individually subjected to the same series of two behavioral and two hypothalamo-pituitary-adrenocortical (HPA) axis reactivity tests at 3, 13 and 26 weeks of age. Novel environment (open field, OF) and novel object (NO) tests involved measurement of behavioral, plasma cortisol and heart rate responses. Plasma ACTH and/or cortisol response profiles were determined after administration of exogenous CRH and ACTH, respectively, in the HPA axis reactivity tests. Principal component analysis (PCA) was used to condense correlated measures within ages into principal components reflecting independent dimensions underlying the calves' reactivity. Cortisol responses to the OF and NO tests were positively associated with the latency to contact and negatively related to the time spent in contact with the NO. Individual differences in scores of a principal component summarizing this pattern of inter-correlations, as well as differences in separate measures of adrenocortical and behavioral reactivity in the OF and NO tests proved highly consistent over time. The cardiac response to confinement in a start box prior to the OF test was positively associated with the cortisol responses to the OF and NO tests at 26 weeks of age. HPA axis reactivity to ACTH or CRH was unrelated to adrenocortical and behavioral responses to novelty. These findings strongly suggest that the responsiveness of calves was mediated by stable individual characteristics. Correlated adrenocortical and behavioral responses to novelty may reflect underlying fearfulness, defining the individual's susceptibility to the elicitation of fear. Other independent characteristics mediating reactivity may include activity or coping style (related to locomotion) and underlying sociality (associated with vocalization).