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Journal Article 2014; 35(Suppl 2): 141-148 PubMed PMID: 25638378 Citation  Keywords:  Animals, Electron Transport Complex II:drug effects, Formazans:metabolism, Interferon-gamma:pharmacology, Mice, Mice, Inbred C57BL, Peritoneum:cytology, Pyrimidines:pharmacology, Spleen:cytology,.   

OBJECTIVES: Formation of formazan is a commonly used measure of cytotoxicity of compounds. It is a product of reduction of tetrazolium salts such as 4-[3- (4-iodophenyl)-2- (4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride. The extent of substrates reduction reflects the activity of enzymes succinate dehydrogenase (SDH; respiratory complex II) and lactate dehydrogenase (LDH), respectively. The aim of present study was a) to investigate formazan formation under the conditions of in vitro stimulation of cells with interferon-γ (IFN-γ) and lipopolysaccharide (LPS), and b) to analyse possible interference of pyrimidine analogues with formazan production.

METHODS: Peritoneal cells and splenocytes were obtained from C57BL/6 mice. They were cultured at 37 degrees C, 5% CO2 in humidified incubator. Levels of formazan were determined at the interval of 24 h of culture using the WST-1 and LDH assays. Nitric oxide (NO) was activated by IFN-γ plus LPS and assayed by Griess reagent 24 h afterwards. Pyrimidines were applied concomitantly with immunostimulatory agents.

RESULTS: IFN-γ enhanced concentration of SDH-produced formazan by macrophages (not by splenocytes) by approximately 50%. The activity of LDH remained unaffected. LPS was ineffective in both cases. While pyrimidines with NO-inhibitory properties suppressed the IFN-γ-enhanced levels of SDH-produced formazan, they did not change the LDH-dependent formazan production.

CONCLUSION: IFN-γ augments the SDH-produced formazan by macrophages. It does not change the LDH-dependent formazan formation. The enhancing effect may have a significant impact upon the appropriate interpretation of cytotoxic properties of drugs investigated under the conditions of immune stimulation of cells.

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Journal Article 2002; 23(4): 325-328 PubMed PMID: 12195235 Citation  Keywords:  Animals, Antineoplastic Agents:pharmacology, Astrocytes:cytology, Cerebral Cortex:cytology, DNA-Binding Proteins:metabolism, Gene Expression:drug effects, Glioma, Growth Inhibitors:pharmacology, Interferon-gamma:pharmacology, Interleukin-6:pharmacology, L.   

OBJECTIVES: The purpose of this study was to compare the effects of neurotrophic cytokines on the JAK/STAT transcription factors and the immediate early gene, c-fos, in C6 glioma cells and in primary astrocyte cultures.

METHODS: In this study we compared the effects of interleukin 6, leukemia inhibitory factor and interferon gamma on C6 glioma cells and on primary astrocyte cultures using electrophoretic mobility shift assay and quantitative solution hybridization/Northern Blot analysis.

RESULTS: We show that interleukin 6, leukemia inhibitory factor and interferon gamma treatment induce the nuclear STAT3 and STAT1 proteins to bind to the sis inducible element of c-fos in both C6 glioma cells and primary cortical astrocytes. Furthermore, quantitative solution hybridization and Northern blot analysis show the differential regulation of c-fos mRNA levels by interleukin-6 (8.1 and 4.0 folds) and leukemia inhibitory factor (5.4 and 3.2 folds) in C6 and astrocyte cultures (respectively). However, interferon gamma increases in c-fos mRNA levels (2.9 fold in both C6 and astrocyte cultures) were not significant in a one way ANOVA.

CONCLUSION: This study suggests that two initial cytokine signaling pathways are present and functional in C6 glioma cells and in primary astrocyte cultures....

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