RESUMEN
Vanadate and peroxovanadium derivatives are potent inhibitors of protein tyrosine phosphatases (PTPs) and exhibit insulinomimetic activities in several cell systems. We have found that in 293 and 293T cells, intercellular adhesion molecule-1 (ICAM-1) gene transcription is activated by bpV(Pic), a picolinic acid-stabilized peroxovanadium derivative. To identify the bpV(Pic)-responsive element(s), several deletion and site-specific mutants of the ICAM-1 gene promoter cloned upstream from the firefly luciferase reporter gene were transiently transfected into both cell lines. Deletion or site-specific mutation of the NF-kappa B site did not affect bpV(Pic) responsiveness, whereas deletion or mutation of the palindromic interferon-gamma-responsive element (pI gamma RE)/gamma-interferon activated sequence site greatly decreased bpV(Pic) responsiveness in both cell types. bpV(Pic) synergistically co-operated with interferon-gamma to increase the transcriptional activity of the ICAM-1 promoter. Electrophoretic mobility-shift assays showed that bpV(Pic) induces signal transducers and activators of transcription (STAT)-1 binding to the ICAM-1 pI gamma RE/GAS in 293T cells, suggesting that the peroxovanadium compound specifically inhibits the phosphatase(s) required to regulate the JAK/STAT signal-transduction pathway.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Molécula 1 de Adhesión Intercelular/genética , FN-kappa B/metabolismo , Compuestos Organometálicos/farmacología , Transactivadores/metabolismo , Transcripción Genética/efectos de los fármacos , Secuencia de Bases , Western Blotting , Línea Celular Transformada , Cartilla de ADN , Citometría de Flujo , Humanos , Interferón gamma/farmacología , Regiones Promotoras Genéticas , Unión Proteica , Factor de Transcripción STAT1 , TransfecciónRESUMEN
Cell viability and phagocytic activity of coelomocytes from the gastrointestinal tract of Lumbricus terrestris were examined by flow cytometry after in vitro exposure to heavy metals. Control coelomocytes were incubated for 18 h at 15 degrees C, 5% CO2, in Ca(++)-containing LBSS medium with 10(-4)-10(-9) M mercury chloride, methylmercury, cadmium chloride, zinc chloride, lead chloride or lead acetate. Heterogeneity of coelomocyte population was demonstrated by forward scatter (FSC) analysis and cytometric profile showing two different populations of type I/small (60%) and type-II/large (40%) cells. Exposure to either form of Hg, Cd and Zn was relatively highly toxic and affected both cell viability and phagocytosis, whereas Pb was relatively well tolerated by the coelomocytes. A fraction of cells within large coelomocyte population was exceptionally sensitive to the Hg-induced cytotoxicity, which did not affect, however, the relative phagocytic activity of the remaining cells. Overall, at least three different patterns of metal-specific toxicity, affecting both viability and phagocytic functions of earthworm coelomocytes, were confirmed in our in vitro studies. Further characterisation of both the target cells from heterogeneous coelomocyte population and the specific interaction of target cell-xenobiotic can possibly reduce biomonitoring problems in earthworm toxicology and immunotoxicology.