u-0126 and 1-10-phenanthroline

It was originally shown by Woerner and Schrenk [Woerner, W., Schrenk, D., 1998. 2,3,7,8-Tetrachlorodibenzo-p-dioxin suppresses apoptosis and leads to hyperphosphorylation of p53 in rat hepatocytes. Environ. Toxicol. Pharmacol. 6, 239-247] that TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) acts as an antagonist against the action of UV-irradiation to induce apoptosis in rat primary hepatocytes. Since prevention of apoptosis has been shown to promote carcinogenesis, we have decided to investigate this phenomenon in a human mammary gland epithelial cell line, MCF10A. We found that, in this cell line, TCDD can antagonize apoptosis that was induced by a variety of treatments, such as UV- and gamma-irradiation, growth factor starvation and trypsinization, or by the addition of H(2)O(2), TGFbeta, and staurosporine. Furthermore, other agents that are known to elicit defensive cellular responses, such as LPS, Fe(3+), nitric oxide and hypoxia could also antagonize UV induced apoptosis just as in the case of TCDD. In addition, we found that, in this cell line, such anti-apoptotic action of TCDD resembles that of exogenously added EGF or TGF alpha. To study the basic mechanism of such an action of TCDD, we tested a variety of diagnostic agents to reverse the effect of TCDD. Antagonists of TCDD which were found to be effective in this way were (a) inhibitors of c-Src kinase, such as PP-2 and CGP77675, (b) those known to block the action of TGF alpha, such as anti-TGF alpha antibody, and alpha(1)-antitrypsin, (c) PD98059, a specific inhibitor of ERK activation, but not SB202190 (an inhibitor of p38 MAPK activation) or SP600125 (a JNK inhibitor) and (d) Ah receptor antagonists, alpha-naphthoflavone and 1, 10-phenanthroline. These results support the notion that TCDD acts as an anti-apoptotic agent by mimicking the action of EGF through activation of the c-Src/ERK signaling pathway.

Topics: Adaptation, Physiological; alpha 1-Antitrypsin; Apoptosis; bcl-2-Associated X Protein; Benzoflavones; Butadienes; Cell Line; DNA Fragmentation; Epidermal Growth Factor; Epithelial Cells; Female; Ferric Compounds; Flavonoids; Glutathione; Humans; Hydrogen Peroxide; Lipopolysaccharides; Mitogen-Activated Protein Kinases; Nitriles; Nitro Compounds; Phenanthrolines; Polychlorinated Dibenzodioxins; Pyrimidines; Pyrroles; Quaternary Ammonium Compounds; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transforming Growth Factor alpha; Ultraviolet Rays

The membrane-permeable intracellular heavy metal chelator, 1,10-phenanthroline, which prevents progesterone-induced germinal vesicle breakdown (GVBD), would be expected to regulate phosphorylation (activation) of the MAP kinase (MAPK) cascade in Xenopus oocytes. Here, our experiments show that 1,10-phenanthroline itself results in the phosphorylation of MAPK in both oocytes and a cell-free system. In contrast, 1,7-phenanthroline, the nonchelating analogue, had no effect. A supplement of zinc (as a heavy metal) given to 1,10-phenanthroline-loaded oocytes suppressed the stimulatory effects of 1,10-phenanthroline, while 1,10-phenanthroline withdrawal caused dephosphorylation of activated MAPK. Further, treatment with a MEK (a MAPK kinase) inhibitor, PD 098059 or U0126, suppressed 1,10-phenanthroline-stimulated MAPK phosphorylation, indicating that 1,10-phenanthroline can phosphorylate MAPK in a MEK-dependent fashion. Our results suggest that phosphorylation of MAPK by 1,10-phenanthroline depends on the interaction of MEK. Thus, the intracellular heavy metal (zinc) regulates MAPK phosphorylation and 1,10-phenanthroline can serve as a unique tool for investigating MAPK phosphorylation mechanism.

Topics: Animals; Blotting, Western; Butadienes; Cell-Free System; Chromones; Colforsin; Cycloheximide; Enzyme Inhibitors; Female; Flavonoids; Indoles; Kinetics; Maleimides; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Morpholines; Nitriles; Oocytes; Pentetic Acid; Phenanthrolines; Phosphorylation; Progesterone; Threonine; Tyrosine; Xenopus laevis; Zinc Sulfate

Hemin is present in intracranial hematomas in high micromolar concentrations and is a potent, lipophilic oxidant. Growing evidence suggests that heme-mediated injury may contribute to the pathogenesis of CNS hemorrhage. Extracellular signal-regulated kinases (ERKs) are activated by oxidants in some cell types, and may alter cellular vulnerability to oxidative stress. In this study, the effect of hemin on ERK activation was investigated in cultured murine cortical astrocytes, and the consequence of this activation on cell viability was quantified. Hemin was rapidly taken up by astrocytes, and generated reactive oxygen species (ROS) within 30 min. Increased immunoreactivity of dually phosphorylated ERK1/2 was observed in hemin-treated cultures at 30-120 min, without change in total ERK. Surprisingly, ERK activation was not attenuated by concomitant treatment with antioxidants (U74500A or 1,10-phenanthroline) at concentrations that blocked ROS generation. Cell death commenced after 2 h of hemin exposure and was reduced by antioxidants and by the caspase inhibitor Z-VAD-FMK. Cytotoxicity was also attenuated by MEK inhibition with PD98059 or U0126 at concentrations that were sufficient to prevent ERK activation. Whereas the effect of Z-VAD-FMK on cell survival was transient, the effect of MEK inhibitors was long-lasting. MEK inhibitors had no effect on cellular hemin uptake or subsequent ROS generation. The present results suggest that hemin activates ERK in astrocytes via a mechanism that is independent of ROS generation. This activation sensitizes astrocytes to hemin-mediated oxidative injury.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antioxidants; Astrocytes; Blood Proteins; Butadienes; Cell Survival; Cells, Cultured; Cerebral Cortex; Cysteine Proteinase Inhibitors; Enzyme Activation; Flavonoids; Free Radicals; Hemin; MAP Kinase Kinase Kinase 1; Mice; Mitogen-Activated Protein Kinases; Nitriles; Oxidative Stress; Phenanthrolines; Pregnatrienes; Protease Inhibitors; Protein Serine-Threonine Kinases; Reactive Oxygen Species; Signal Transduction