The purpose of this project will be to better understand the mechanism of action of CF in human keratinocytes.In a preliminary phase we will try to identify the site of interaction of CF with cell compartments, using a specific antibody (Chen et al, 1998; Bradford et al, 2000). After that, we will study the possible interactions of CF with distinct cellular compartments. We will first try to evaluate whether CF directly interfere with the generation of reactive oxygen species both per se and after UVB irradiation of human keratinocytes. We will add CF to the cells either alone or in combination with UVB irradiation. In addition, we will also pre-load CF to the cells (24 hrs) as opposed to administering CF immediately before UVB irradiation. This will give us an indirect clue as to whether CF interfere with the anti-oxidizing machinery and the cell death pathway. The apoptotic process will be evaluated by TUNEL staining. In addition, the involvement of mitochondria will be assessed by the mitochondrial membrane potential by flow cytometry. Several apoptotic stimuli induce the release of cytochrome C from the mitochondria into the cytosol (Slee et al, 2000; Von Ahsen et al, 2000), which is considered to be a pivotal event in the initiation of apoptosis. Therefore, the involvement of mitochondria will also be confirmed by the release of cytochrome C by western blot. Finally, the activation of several caspases will be analyzed by western blot analysis. The involvement of individual caspases, and in particular the different caspase pathways will also be evaluated by the use of caspase specific inhibitors
|Titolo:||Effects of Carboxyfullerenes on the apoptotic machinery in human keratinocytes|
|Autori:||C. Pincelli; A. Marconi|
|Data di pubblicazione:||2003|
|Appare nelle tipologie:||Partecipazione a progetti di ricerca|
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