Stefan Fronhoffs

Induction of immediate-early genes by angiotensin II and endothelin-1 in adult rat cardiomyocytes.

Objective: Few molecular signals for induction of myocardial hypertrophy have been identified. This study was carried out to investigate the action of angiotensin II and endothelin on the growth- and differentiation-related genes Egr-1 (early growth response gene 1) and c-fos in isolated adult rat cardiomyocytes. Methods: Cardiac myocytes from male Wistar-Kyoto rats were isolated and incubated with angiotensin II and endothelin-1 in Dulbeccos modified Eagles medium. RNA was isolated and blotted, and densitometric analysis was performed. All experiments were repeated at least three times. Results Endothelin-1 (10-7mmol/l) induced a 20-25-fold rise in Egr-1 messenger RNA within 15min. This effect was dose-dependent, c-fos was induced 10-20-fold within 15 min with similar dose-response characteristics. Angiotensin II also induced Egr-1 and c-fos with kinetics similar to endothelin but a cofactor from fetal calf serum was needed for full c-fos expression. The protein kinase C activator phorbol 12-myristate 13-acetate also induced Egr-1. Conclusions: The results identify Egr-1 and c-fos as target genes for the action of endothelin and angiotensin II in the adult myocardium suggesting that induction of the genes may be part of the signal transduction pathway for angiotensin II and endothelin in the myocardium.

Stromal cell‐derived factor 1α (SDF‐1α) induces gene‐expression of early growth response‐1 (Egr‐1) and VEGF in human arterial endothelial cells and enhances VEGF induced cell proliferation

Abstract. Stromal cell‐derived factor‐1 (SDF‐1), mainly known as a chemotactic factor for haematopoietic progenitor cells, also provides angiogenetic potency. Since the intracellular signalling of SDF‐1‐induced neovascularization remains unclear, we studied in human umbilical arterial endothelial cells (HUAEC) the influence of SDF‐1α on induction of the genes of early growth response‐1 (Egr‐1) and VEGF, as well as the activation of extracellular regulated kinases (ERK) 1/2, which are all known to be involved in endothelial cell proliferation. We found a time‐dependent induction of Egr‐1 and VEGF mRNA expression and phosphorylation of ERK1/2 by SDF‐1α. Furthermore, we demonstrated that Egr‐1 expression is dependent on ERK 1/2 activation. Finally, we tried to confirm the relevance of the induced gene expression by detecting the [3H]thymidine incorporation as a marker for cell proliferation in HUAEC after stimulation with SDF‐1α alone or together with VEGF. This particular test showed, that SDF‐1α alone has no effect, but is able to significantly enhance VEGF induced DNA synthesis. In summary, SDF‐1α is involved in different steps of endothelial cell proliferation, but, since Egr‐1 and VEGF offer different functions, it may also play a so far undefined role on other conditions of the endothelium.

High Percentage of False-Positive Results of Cytokeratin 19 RT-PCR in Blood: A Model for the Analysis of Illegitimate Gene Expression

Cytokeratin 19 (CK19) RT-PCR is widely used in order to detect circulating tumor cells in peripheral blood and bone marrow. However, increasing amounts of information support the fact that it is also associated with a high percentage of false-positive results. In our study, we not only managed to demonstrate the significant limitations of this method, but were also able to clarify the reasons behind these limitations. We developed a completely novel RT-PCR for CK19 and sequenced an intron at nucleotide (nt) 980 of the CK19 mRNA to exclude DNA contamination. Tumor dilution experiments were performed in order to analyze the specificity and sensitivity of the method. Control experiments using the blood of healthy donors were performed. Tumor cell dilution experiments gave a detection limit of one tumor cell. If tumor cells were mixed with an equal volume of pure mononuclear cells, the detection limit was 1 tumor cell in 105 mononuclear cells. RT-PCR of mononuclear cells from healthy blood donors gave false-positive results in 29% of the cases. We conclude that a significant decrease in the sensitivity of CK19 RT-PCR occurs if it is performed in blood cells and that the illegitimate CK19 gene expression in normal cells can lead to false-positive results. These limitations have to be taken into account if RT-PCR is to be used for the detection of tumor cells either in blood or in bone marrow in clinical practice.

Identification of syntenin and other TNF-inducible genes in human umbilical arterial endothelial cells by suppression subtractive hybridization

Endothelial cells play an important regulatory role in inflammatory responses by upregulating various proinflammatory gene products including cytokines and adhesion molecules. A highly potent mediator of this process is tumor necrosis factor‐α (TNF). In the present study, the suppression subtractive hybridization (SSH) method was employed to identify rarely transcribed TNF‐inducible genes in human umbilical arterial endothelial cells. Following mRNA isolation of non‐stimulated and TNF‐stimulated cells, cDNAs of both populations were prepared and subtracted by suppression PCR. Sequencing of the enriched cDNAs identified 12 genes differentially expressed including vascular cell adhesion molecule‐1, monocyte chemoattractant protein‐1, interleukin‐8 and IκBα, an inhibitor of the transcription factor nuclear factor‐κB. Interestingly, also syntenin, a PDZ motif‐containing protein which binds to the cytoplasmic domain of syndecans, was identified by SSH. Time course studies using RT‐PCR analysis confirmed that all genes were differentially expressed and rapidly induced by TNF. Our data reveal that SSH is a powerful technique of high sensitivity for the detection of differential gene expression in primary arterial endothelial cells.

Association between head and neck cancer and microsomal epoxide hydrolase genotypes

Abstract. Tobacco-associated carcinogens are catalyzed by microsomal epoxide hydrolase (mEH). Combinations of the Y113H and H139R polymorphic EPHX1 variants have been assumed to alter the enzyme activity and thus the risk of squamous cell head and neck cancer (SCCHN). Based on in vitro data, a putative low, medium and high mEH activity has been associated with combinations of these genotypes, and the respective activity categories have been frequently used in the estimation of risks for smoking-related cancers. We investigated the SCCHN risk for EPHX1 genotypes among 280 cases and 289 controls. We could not detect main effects of the EPHX1 genotypes, but a smaller risk of the 139HR genotype in smokers (odds ratio, OR, 0.57; 95% confidence interval, CI, 0.34–0.95). We could not confirm an increase of the SCCHN risk for genotype combinations according to a putative medium and high enzyme activity (OR 1.28, 95% CI 0.84–1.96; OR 0.98, 95% CI 0.58–1.64, respectively), but a significant heterogeneity of the estimated risks for the singular genotypes within these categories among smokers (P=0.02). Further, p53 mutations among smoking cases were less frequent in the group with a putative high enzyme activity, although insignificant due to small numbers (OR 0.54, 95% CI 0.13–2.17). This supports uncertainties in categorizing genotypes with respect to limited enzyme activity data, especially when taken from in vitro experiments.

Gemcitabine and paclitaxel in metastatic or recurrent squamous cell carcinoma of the head and neck: a phase I-II study.

The purpose of this study was to determine the maximum tolerated dose, toxicity profile and anti-tumor activity of paclitaxel in combination with gemcitabine when administered to patients with unresectable locally recurrent or metastatic squamous cell carcinoma of the head and the neck (SCCHN). Twenty-seven patients were treated in a phase I–II study with gemcitabine at a dose of 800 mg/m2 on days 1 and 8, escalating to a dose of 1000 mg/m2, plus escalating doses of paclitaxel (100, 135 and 175 mg/m2) on day 2. Treatment consisted of 6 cycles repeated every 3 weeks. The main toxicity was myelosuppression. Other toxicities were mild and manageable. Due to grade 4 neutropenia at higher doses the recommended dose level of the gemcitabine/paclitaxel combination was 1000/135 mg/m2. Four patients achieved a partial response and no patient had a complete remission, giving an overall response rate of 14.8%. The median time of survival was 24 weeks. We conclude that the combination of paclitaxel and gemcitabine is tolerated, but shows insufficient clinical activity in patients with recurrent and/or metastatic SCCHN to warrant further testing.

Cholesterol enhances contractile responses in isolated small mesenteric arteries of normotensive and spontaneously hypertensive rats.

OBJECTIVE In order to examine possible mechanisms by which hypercholesterolemia may contribute to the development of cardiovascular disease, we investigated the effect of cholesterol enrichment on contractility in isolated small rat mesenteric arteries. DESIGN Contractile responses of cholesterol-enriched isolated small mesenteric arteries of normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were compared with control groups. METHODS First- to second-order mesenteric arteries (327-349 microm internal lumen diameter) were dissected from the mesenteric bed of 10-20-week-old male WKY rats and SHR, and incubated in cholesterol-free and cholesterol-rich (150 microg/ml) medium. Isolated arteries were mounted on a Mulvany-Halpern myograph for measurement of isometric tension. RESULTS Cholesterol significantly increased active wall tension and active wall pressure in WKY rat arteries and active wall tension in SHR arteries in response to potassium chloride, norepinephrine and serotonin (P < 0.05). In addition, contractile responses to all agonists were significantly higher in cholesterol-enriched SHR arteries compared with cholesterol-enriched WKY rat vessels (P < 0.05). CONCLUSIONS These findings suggest that elevated cholesterol content enhances agonist-stimulated contractility in small mesenteric resistance arteries, providing a possible mechanism by which hypercholesterolemia may contribute to the development of hypertension.

Parallel Genotyping of Different Genes: A Rapid Real-Time PCR Approach

Much progress has been made in identifying clinically relevant point mutations in genomic DNA. As a result, procedures that allow fast, accurate and easy analysis of known point mutations are needed for diagnosis. To substantially increase the throughput of sample analysis and/or to analyze individual gene mutation patterns, it would be helpful to establish the analysis of different parameters in a parallel procedure.