Simone Helmig

Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain-induced cardiovascular differentiation

Growing stem cells are subjected to mechanical forces, which may initiate differentiation programs. Mechanical strain stimulated cardiovascular differentiation of mouse embryonic stem (ES) cells as evaluated by quantification of contracting cardiac foci and capillary areas, respectively. Mechanical strain rapidly elevated intracellular reactive oxygen species (ROS). After 24 h up‐regulation of NADPH oxidase subunits p22‐phox, p47‐phox, p67‐phox, and Nox‐4 as well as Nox‐1 and Nox‐4 mRNA was observed. In parallel, mechanical strain increased hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) mRNA and protein as well as MEF2C and GATA‐4 mRNA, which are involved in cardiovascular development. Furthermore, phosphorylation of extracellular‐regulated kinase 1,2 (ERK1,2), p38, and c‐jun N‐terminal kinase (c‐Jun NH2‐terminal kinase (JNK)) was observed. Stimulation of cardiovascular commitment, HIF‐1α, VEGF, and MEF2C expression as well as MAPK activation were abolished by free radical scavengers, whereas GATA‐4 expression was increased. Cardiomyogenesis was inhibited by the p38 inhibitor SB203580, the ERK1,2 inhibitor UO126, and the JNK inhibitor SP600125. Vasculogenesis/angiogenesis was blunted following inhibition of ERK1,2 and JNK, whereas p38 inhibition was ineffective. Our data outline a role of ROS as mechanotransducing molecules in mechanical strain‐stimulated cardiovascular differentiation of ES cells, and point toward a microenvironment of elevated ROS required for signaling cascades initiating cardiovascular differentiation programs.—Schmelter, M., Ateghang, B., Helmig, S., Wartenberg, M., Sauer, H. Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain‐induced cardiovascular differentiation. FASEB J. 20, E294–E306 (2006)

XRCC1 polymorphism and lung cancer risk.

DNA repair plays a critical role in protecting the genome of the cell from the insults of carcinogens or ionizing radiation. Reduced DNA repair capacity can increase the susceptibility to environmental- or occupational-induced cancers. Three coding polymorphisms at codon 194, codon 280 and codon 399 in the x-ray cross complementing group 1 (XRCC1) DNA repair gene have been identified, and it is possible that these polymorphisms may affect DNA repair capacity and thus modulate cancer susceptibility. In this review, we summarize the literature and discuss the relevance of XRCC1 polymorphisms and lung cancer risk. The frequency of genetic polymorphisms is dependent on the ethnic origins of a population. The frequency of the variant allele of codon 194 among Asians is on average 31.2% (95% confidence interval [CI]: 29.6–32.8), which is significantly higher than among Caucasians (6.6%; 95% CI: 5.9–7.4) or Africans (7.3%; 95% CI: 5.7–9.2). The variant allele in codon 399 occurs among Africans at a frequency of 15.5% (95% CI: 13.5–17.7), 34.7% in Caucasians (95% CI: 33.8–35.6) and 26.5% in Asians (95% CI: 25.6–27.4). Results regarding lung cancer risk are inconsistent. The lung cancer risk associated with polymorphisms of the XRCC1 codon 194 demonstrate an odds ratio (OR) of around 1.0. For the XRCC1 codon 280, lung cancer risk varied between ORs of 0.26 and 1.8; and for the XRCC1 codon 399 between 0.32 and 3.25. Only two studies showed significantly elevated risks (OR: 3.25; 95% CI: 1.2–10.7; OR: 1.3; 95% CI: 1.0–1.8, respectively), whereas one study showed a decreased lung cancer risk (OR: 0.60; 95% CI: 0.46–40.80). Lung cancer risk increased with cigarette smoking. A significant association was not observed between the single-nucleotide polymorphisms and tobacco-related cancers. Lung cancer risk increased significantly for the variant XRCC1 -77 genotypes (TC and CC) compared with the TT genotype (OR: 1.46; 95% CI: 1.18–1.82). The risk was more pronounced in smokers (OR: 1.63; 95% CI: 1.20–2.21) than in nonsmokers (OR: 1.28; 95% CI: 0.94–1.76). No association with polymorphisms were found for various histological tumor types. The XRCC1 399 Gln/Gln variant genotype was associated with a higher median survival time.

Association of Transforming Growth Factor β1 Gene Polymorphisms and Asbestos-Induced Fibrosis and Tumors

Aim Inhaled asbestos fibers are known to cause progressive lung or pleural fibrosis and malignancies such as lung cancer or diffuse malignant mesothelioma. Transforming growth factor β1 (TGF-β1), a multifunctional cytokine, regulates the proliferation and differentiation of cells. Transforming growth factor β1 is known to promote the pathogenesis of lung fibrosis and acts as a tumor suppressor in normal cells. Two genetic polymorphisms in codons 10 (Leu10Pro) and 25 (Arg25Pro) of the TGF-β1 gene are suggested to be associated with a different TGF-β1 protein production. Therefore, we examined an association between the 2 TGF-β1 gene polymorphisms and asbestos-induced lung fibrosis and lung cancer. Methods Detection of the 2 polymorphisms was performed by rapid capillary polymerase chain reaction, with melting curve analysis, using fluorescence-labeled hybridization probes. To investigate the association between TGF-β1 gene polymorphisms in codons 10 and 25 and the susceptibility to asbestos-induced diseases, association studies were performed with healthy control subjects (n = 83), patients with pulmonary fibrosis (n = 591), and patients with bronchial carcinoma (n = 147). Results Compared with a healthy control group, odds ratio (OR) analysis revealed an inverse relationship for the proline allele at codon 10 or 25 with pulmonary fibrosis (higher risk) and lung cancer (lower risk). The proline allele at codon 10 or 25 is significantly associated with a higher risk for fibrotic lung diseases (ORcrude, 1.46; 95% confidence interval [CI], 1.01-2.11; P = 0.045 and ORadjusted, 1.76; 95% CI, 1.14-2.72; P = 0.011, respectively, for codon 10; OR, 2.13; 95% CI, 1.33-3.99; P = 0.019 and ORadjusted, 2.27; 95% CI, 1.14-4.52; P = 0.02, respectively, for codon 25) when compared with patients with lung cancer. A significant association for the proline allele is also revealed when comparing patients with asbestosis (ORcrude, 3.01; 95% CI, 1.44-6.29; P = 0.003 and ORadjusted, 3.72; 95% CI, 1.56-8.85; P = 0.011) with patients with asbestos-induced lung cancer. Conclusions In summary, the results confirm the hypothesis that TGF-β1 polymorphisms are associated with asbestos-induced fibrotic or malignant lung diseases in whites.

RNA expressions of AHR, ARNT and CYP1B1 are influenced by AHR Arg554Lys polymorphism.

AIM The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that together with Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) controls the expression of Xenobiotic metabolising enzymes (XME) such as CYP1B1. In the absence of exogenous ligands, AHR is supposed to be involved in promotion of cell cycle progression. Polymorphisms of the AHR gene are suggested to be associated with susceptibility to cancer. Because of its critical role in xenobiotic induced toxicity and carcinogenesis as well as its ligand independent relevance we investigated the effects of AHR Arg554Lys Polymorphism on gene expression level of the AHR, ARNT and CYP1B1. METHODS Detection of the AHR Arg554Lys polymorphism of the AHR gene was performed by rapid capillary PCR with melting curve analysis. The quantitative Real-Time PCR (qRT-PCR) of AHR, ARNT and CYP1B1 mRNAs was carried out in white blood cells from 287 Caucasians. Calculations of expression were made with the 2(-ΔΔCT) method. RESULTS The relative AHR mRNA expression revealed significant differences between the two homozygote AHR genotypes Arg554Arg (11.0±1.0; n=228) and Lys554Lys (0.6±0.4; n=3; p<0.001). Also significant differences were seen between the heterozygote genotype Arg554Lys (13.0±3.0; n=40) and the homozygote Lys554Lys genotype (0.6±0.4; n=3; p<0.001). These differences above were replicated significantly in the relative mRNA expression of ARNT and CYP1B1. Comparing the determined CT-values, a correlation coefficient of R=0.748 for AHR and ARNT, R=0.626 for ARNT and CYP1B1 as well as R=0.533 for AHR and CYP1B1 was calculated. CONCLUSION Our findings suggest that the homozygote variant genotype of AHR Lys554Lys is associated with a significantly lower AHR, ARNT and CYP1B1 mRNA expression.

Influence of the Cyp1B1 L432V gene polymorphism and exposure to tobacco smoke on Cyp1B1 mRNA expression in human leukocytes.

Cytochrome P450 1B1 (CYP1B1), a phase I enzyme, is involved in the activation of a broad spectrum of procarcinogens. An association of the Cyp1B1 L432V polymorphism with diverse types of cancer, as well as an impact on the catalytic activity of the enzyme, has been described. To show the functional impact of the allelic variant Cyp1B1*3, we investigated the quantitative Cyp1B1 mRNA expression in a population of smokers, nonsmokers, and ex-smokers and determined their genotypes. Detection of the L432V polymorphism in exon 3 of the Cyp1B1 gene was performed by rapid capillary polymerase chain reaction (PCR) with melting curve analysis. For quantitative comparison of Cyp1B1 mRNA levels, real-time PCR was performed using SYBR Green fluorescence in a LightCycler system. Calculations of expression were made with the 2–ΔΔCT method. In comparing relative Cyp1B1 mRNA expression, highly significant differences between the two homozygote genotypes *1/*1 and *3/*3 (0.185 ± 0.027, n = 118 versus 0.071 ± 0.013, n = 56; p = 0.000), as well as between the heterozygote genotype *1/*3 and the homozygote genotype *3/*3 (0.178 ± 0.025, n = 171 versus 0.071 ± 0.013, n = 56; p = 0.000), were revealed. Significant differences between the genotypes were also detected within the subgroups of smokers, nonsmokers, and ex-smokers. No significant differences were determined in comparing the relative Cyp1B1 mRNA expression with regard to tobacco smoke exposure. Our results suggest that genotypes carrying the C allele (*1/*1 and *1/*3) at Cyp1B1 L432V polymorphism have a significantly higher Cyp1B1 mRNA expression compared with the genotype without the C allele (*3/*3). Gene expression of Cyp1B1 mRNA cannot be used as a biomarker for exposure of tobacco smoke.

Interleukin-6 Contributes to the Paracrine Effects of Cardiospheres Cultured from Human, Murine and Rat Hearts

Cardiosphere‐derived cells (CDCs) were cultured from human, murine, and rat hearts. Diluted supernatant (conditioned‐medium) of the cultures improved the contractile behavior of isolated rat cardiomyocytes (CMCs). This effect is mediated by the paracrine release of cytokines. The present study tested the hypothesis, that the cardiovascular state of the donor’s heart influences this effect on CMCs and tries to identify the responsible factors. CDCs were cultured from human tissue samples of cardiac surgery and from murine and rat hearts. The supernatants of cultured CDCs from hypertensive humans and rats showed a higher improvement of the contractile behavior of CMCs compared to CDCs of normotensive origin. Subsequently, the cytokine profile of the supernatants was analyzed. Among the cytokines elevated in supernatants originating from hypertensive humans or rats was Interleukin‑6. CDCs were also generated from Interleukin‑6−/−‐mice and their wildtype littermates. The supernatant of the cultured Interleukin‑6−/−‐CDCs had no effect on the contractile behavior, whereas the supernatant of the Interleukin‑6+/+‐CDCs showed a positive effect. To confirm the hypothesis that Interleukin‑6 contributes to the paracrine effects, CMCs were incubated with Interleukin‑6. It improved the contractile function in a concentration dependent way. Finally, the effect of the supernatant of cultured CDCs derived from a hypertensive human sample could be abolished by simultaneous incubation with a specific Interleukin‑6 antibody. CDCs release cytokines that improve the contractile behavior of CMCs. This effect is more intense in CDCs from hypertensive donors. Interleukin‑6 is involved in this phenomenon. J. Cell. Physiol. 229: 1681–1689, 2014.

Tumour necrosis factor-α gene polymorphisms in asbestos-induced diseases

Background: Tumour necrosis factor (TNF)-α influences the pathogenesis of lung fibrosis and carcinogenesis in normal cells. Polymorphisms of this gene have been suggested to be associated with susceptibility to lung diseases. Methods: Association studies were performed in German subjects, using control subjects (n = 177), pulmonary fibrosis patients (n = 612) and bronchial carcinoma patients (n = 374). Results: Compared with a healthy (control) group, a significant result could be obtained for the asbestosis (patient) group (crude odds ratio (ORcrude) = 1.57; 95% confidence interval (CI) 1.05–2.36; p = 0.03), especially with severe lung asbestosis (ORcrude = 4.15; 95% CI 1.06–16.16; p = 0.04). A significant association was revealed when comparing asbestosis patients (ORcrude = 4.08; 95% CI 1.53–10.54; p = 0.004 and ORadjusted = 3.89; 95% CI 1.49–10.17; p = 0.006) with asbestos-induced lung cancer patients. Conclusion: The results confirm the hypothesis that TNF-α polymorphisms are associated with asbestos-induced fibrotic or malignant lung diseases in Germans.

TNF-α −308 genotypes are associated with TNF-α and TGF-β1 mRNA expression in blood leucocytes of humans

AIM Tumor necrosis factor α (TNF-α) influences the pathogenesis of lung-fibrosis and carcinogenesis in normal cells. Polymorphisms of this gene are suggested to be associated with susceptibility to lung-diseases. Additionally TNF-α is postulated to play a significant role in regulating. Transforming growth factor (TGF-β₁) expression Therefore we investigated if the TNF-α or TGF-β₁ gene expression level is different within the -308 TNF-α genotypes. METHODS Quantitative Real-time PCR of TNF-α and TGF-β₁ was performed in 178 Germans. Calculations of expression were made with the 2(-ΔΔCT) method. Detection of the -308 promoter polymorphism of the TNF-α gene was performed by rapid capillary PCR with melting curve analysis. RESULTS The relative TNF-α mRNA expression revealed significant differences between the TNF-α -308 homozygote wild-type G/G (0.00079±0.00011; n=113) and the heterozygote genotype G/A (0.0005±0.00008; n=52; p=0.030) as well as between homozygote wild-type G/G and the homozygote mutant A/A (0.00029±0.00009; n=5; p=0.004). The relative TGF-β mRNA expression showed, similar to TNF-α, the highest mRNA expression was seen within the TNF-α -308 homozygote wild-types, while the lowest mRNA expression lay within the homozygote mutant-types. CONCLUSION Our findings suggest that the G-allele of TNF-α -308 is associated with a significantly higher TNF-α mRNA expression compared to the A-allele and that this also reflects in TGF-β expression. Therefore we support the thesis that TGF-β is regulated by TNF-α.

Oncogene and tumor-suppressor gene products as serum biomarkers in occupational-derived lung cancer.

Since lung cancer is the most frequent occupational cancer and one of the leading causes of cancer mortality in the world, it is one of the biggest challenges for research. In the literature, there are inconsistent results regarding the utility of the serum biomarkers p53, anti-p53 antibodies, EGF receptor or Ras. Based on the published results, routine use of these biomarkers for detection of occupationally derived lung carcinomas is not currently recommended. In this review, we summarize the literature and discuss the relevance of these oncogene and tumor-suppressor gene products as serum biomarkers in occupational-derived lung cancer.

TNF-α mRNA expression correlates with TGF-β mRNA expression in vivo.

TNF-α is postulated to play a significant role in regulating TGF-β1 expression. In lung fibroblasts, for example, TNF-α is supposed to induce TGF-β1 via AP-1 activation. TNF-α receptor, knock-out mice are resistant to induced fibrosis and over-expression of TNF-α causes increased TGF-β1 production in mice. Therefore, we investigated whether TNF-α mRNA levels are associated with the TGF-β1 mRNA levels of blood leucocytes in humans. Quantitative real-time PCR of TNF-α and TGF-β1 was performed in 118 Germans. Calculations of expression were made with the 2−ΔΔCT method. When the investigated population was divided in two groups (TNF-α low and TNF-α high) by the median of the determined TNF-α expression, highly significant (p < 0.0001) differences of TGF-β1 mRNA expression were revealed. Additionally, dividing the investigated population into quartiles of the determined TNF-α expression showed significantly different TGF-β1 mRNA expressions. Comparing the determined CT-values of TNF-α in context with these of TGF-β1, a coefficient of determination R2 = 0.4635 was calculated. In this study we demonstrated in vivo a significant association of the relative TNF-α/B2M mRNA expression and the relative TGF-β1/B2M mRNA expression in 118 Germans.