Resistance of senescent keratinocytes to UV-induced apoptosis

Irradiation with ultraviolet (UV) triggers programmed cell death (apoptosis) in keratinocytes. This process is believed to protect against skin carcinogenesis since the cells with damaged DNA are selectively removed, limiting the likelihood of the development of a malignant keratinocyte clone. The p53 protein is able to detect mutation-bearing DNA fragments and is thus indispensable for the UV-induced apoptosis in the epidermis. Since age is a risk factor for the development of skin tumors we investigated whether ultraviolet induces apoptosis and p53 activation in senescent keratinocytes. Cultured senescent keratinocytes were irradiated with broad-band ultraviolet, apoptosis was assessed using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) technique and the p53 activation pattern was determined with Western blotting and immunofluorescent staining with a panel of anti-p53 antibodies recognising different conformational forms of the protein (PAb 122, PAb 240, DO-7). In senescent keratinocytes arrested in the G1 phase of cell cycle, ultraviolet irradiation (100-2000 J/m2) caused accumulation and nuclear translocation of p53. However, in contrast to young cells where UV induces apoptotic cell death in G1, apoptosis was not detected in senescent cells. There were subtle differences in the p53 activation pattern between senescent keratinocytes and known patterns in young keratinocytes and other cell types. In senescent keratinocytes a constitutional nuclear expression of p53 (conformational form recognized by PAb 240) was present and the p53 induction in response to ultraviolet radiation was rapid. Suppression of apoptosis in senescent keratinocytes may be an important mechanism responsible for enhanced skin carcinogenesis in old age.