2 PATHWAYS FOR INDUCTION OF APOPTOSIS BY ULTRAVIOLET-RADIATION IN CULTURED HUMAN KERATINOCYTES

Loss of attachment may induce apoptosis in epithelial cells, but it is unclear whether substrate adhesion modulates apoptosis triggered by g enotoxic agents such as ultraviolet radiation (UV). To investigate thi s issue, we plated neonatal human keratinocytes on different substrate s and irradiated them with UVB. DNA strand breaks were nick-labeled to identify apoptotic nuclei, Keratinocytes grown in monolayers were les s susceptible to UV-induced apoptosis than were cells freshly seeded o n glass (ED50 2130 +/- 96 J per m(2), mean +/- SD, versus 131 +/- 96 J per m(2), mean +/- SD, respectively). This phenomenon depended on dif ferences in integrin-mediated adhesion, because blocking of integrin b eta 1 with a monoclonal antibody increased sensitivity of keratinocyte monolayers to UV and an increase in beta 1 integrin receptor occupanc y by plating on fibronectin, type IV collagen, or keratinocyte-derived extracellular matrix diminished the UV-dependent apoptosis, Down-regu lation of p53 with an anti-sense oligonucleotide did not affect apopto sis in glass-plated keratinocytes but effectively suppressed apoptosis in keratinocytes adhering via beta 1 integrin, Thus, in addition to t he known p53-dependent pathway, UV was able to induce a p53-independen t apoptosis that could be blocked by integrin-mediated cell attachment (the integrin-sensitive pathway), The susceptibility to the p53-depen dent apoptosis, but not to the integrin-sensitive process, varied amon g keratinocytes of different clonogenic potential: transit amplifying cells > stem cells > terminally differentiated cells. The p53-independ ent integrin-sensitive apoptotic pathway may provide an additional mec hanism counteracting UV carcinogenesis in the skin.