Abstract

Animal studies have suggested that apoptosis induced by UV irradiation is an important protective mechanism that eliminates epidermal cells with irreparable DNA mutations and thereby can prevent development of non-melanoma skin cancer. Apoptosis induced by UV irradiation is a complex process with activation of several different pathways, and one of these pathways is initiated through death receptor activation. The death receptor Fas (CD95) is present on human epidermal cells together with the corresponding ligand FasL. In this dissertation we investigated how the expression of Fas and FasL on human epidermal cells was affected by in vivo irradiation with either a single dose UVB or UVA I. By immunohistochemistry we found that a single dose of UVB or UVA I irradiation increased the Fas expression on human epidermal cells. The epidermal FasL expression was temporary down regulated in response to a single dose of UVB irradiation. Using histological sections we could not demonstrate any association between Fas expression and the number of apoptotic epidermal cells. However, on a single cell level, using flow cytometry, we demonstrated that apoptosis was restricted to the Fas positive epidermal cell population indicating that Fas receptor activation could be involved in initiating the apoptotic process. This was supported by studies using confocal laser scanning microscopy where we demonstrated that the Fas molecules, present in human epidermis, form clusters in response to UVB irradiation. Simultaneous clustering and co-localisation of the adaptor protein FADD demonstrated a possible functional significance of the UVB-induced Fas clustering.

Initiation of the different death pathways converges towards activation of the proteases that execute apoptosis by degradation of different cellular components. A family of cysteine proteases called caspases plays a central role in the execution of UVB-induced apoptosis. However, we demonstrated that non-caspase proteases could be involved in the execution of UVB-induced apoptosis in keratinocytes and HeLa cells. In an attempt to identify these non-caspase proteases we performed experiments with inhibitors of cathepsins and calpains, but we could not demonstrate a protective role of these inhibitors in UVB-induced apoptosis. Further studies are needed to identify these non-caspase proteases. In conclusion we have shown that in vivo activation of the Fas signalling pathway could be an essential parting of UVB-induced human epidermal cell apoptosis. Moreover, we have demonstrated that non-caspase proteases in human epidermal cells and HeLa cells are likely to be involved in the execution of UVB-induced cell death.