Successful separation of apoptosis and necrosis pathways in HaCaT keratinocyte cells induced by UVB irradiation

UVB irradiation can induce apoptotic, necrotic, and differentiation pathway s in normal human keratinocytes. The present study was undertaken to determ ine at what dose of UVB each of these pathways is induced and whether these pathways are distinct or overlapping. We have observed that UVB induces fr agmentation of DNA in human HaCaT keratinocytes, in a bimodal manner. Low d oses of UVB, 5-20 mJ/cm(2), increase the levels of apoptosis as shown by in creased levels of fragmented DNA, Fas, PARP, and FasL protein, and the numb er of apoptotic cells as assessed by FACS analysis. At higher doses of UVB (20 and 30 mJ/cm(2)) the number of apoptotic cells becomes reduced, as does the amount of Fas, PARP, and FasL protein. At these higher doses, cell via bility is decreased as measured by DNA synthesis (BrdU labeling) neutral re d uptake, which represents an increasing necrotic phenotype. Expression of markers of keratinocyte differentiation, involucrin, keratin K1, and kerati n K10, are also observed to decrease with increasing UVB dose. These change s are accompanied by a further increase in DNA fragmentation. We conclude t hat low doses of UVB (5-20 mJ/cm(2)) induced an apoptotic pathway, whereas increasing doses (greater than 20 mJ/cm(2)) of UVB produce a direct necroti c effect and inhibit terminal differentiation.