Regulation of apoptosis in the thyroid epithelium

Sammanfattning: Apoptosis or programmed cell death allows the multicellular organism to remove cells that are in excess or potentially dangerous. Apoptosis has to be strictly controlled, yet inappropriately activated or suppressed apoptototic may contribute to a variety of diseases, including cancer, autoimmune disorders and neurodegenerative conditions. Due to the generation of hydrogen peroxide (H2O2) required for biosynthesis of thyroid hormone the thyroid epithelial cells are constantly subjected to large amounts of reactive oxygen species (ROS), which are capable of inducing apoptosis in many cell types. However, the mechanism by which ROS induce apoptosis has not been fully understood. The aim of this thesis was to elucidate the mechanism of apoptosis induced by H2O2, staurosporine and transforming growth factor-beta (TGF-beta1) in primary cultured thyroid cells. The enzymatic activity of caspase-3 was measured by monitoring cleavage of the exogenous peptide substrat Ac-DEVD-AMC (papers I, II and IV). Analysis of nuclear morphology and DNA fragmentation was accomplished by DAPI staining followed by fluorescence microscopy and gel electrophoresis (paper I). Immunofluorescence, confocal microscopy and electron microscopy were performed for subcellular localization studies (papers II-IV). Protein identification was performed by immunoblotting (paper IV). Using an experimental model of the thyroid follicular epithelium in which thyrocytes are grown as a tight monolayer on filter in a bicameral chamber, the effects of H2O2, staurosporine and TGF-beta1 on the integrity of the thyroid epithelium were studied (papers III and IV). The present findings provide evidence of a caspase-3-dependent mechanism of apoptosis in H2O2-, -staurosporine- and TGF-beta1-treated thyroid epithelial cells. A characteristic fragmentation pattern commonly referred to as DNA laddering was found in cells treated with staurosporine. H2O2, at micromolar concentrations, and TGF-beta1 caused a more diffuse degradation of DNA. Selenium-substituted cells with a high glutathione peroxidase (GPx) activity had a much better capacity to withstand H2O2 and effectively protect against proapoptotic signals than cells with low GPx activity. Localization studies demonstrated translocation of cytochrome c (cyt-c) from mitochondria to the cytoplasm in staurosporine-treated cells. During H2O2-mediated thyroid cell apoptosis, the cyt-c release from mitochondria to the cytosol was restricted. Instead, H2O2-treated cells showed aggregation and fragmentation of mitochondria and shrunken and highly condensed nuclie. Caspase and calpain inhibitors modified the apoptotic response to H2O2, suggesting cooperation of these proteolytic systems in the execution of apoptosis. The thyroid epithelial barrier is rapidly disrupted by H2O2. Cells with ordinary GPx activity do not have a better capacity to prevent H2O2-induced epithelial dysfunction than cells with a low GPx activity. However, selenium yet exerts a protective effect on epithelial integrity. Staurosporine and TGF-beta1 induce loss of barrier function, but this occur independently of caspase-3 activation and apoptosis.

  Denna avhandling är EVENTUELLT nedladdningsbar som PDF. Kolla denna länk för att se om den går att ladda ner.