Variable Expression of Apoptotic Phenotype in Keratinocytes Treated with Ultraviolet Radiation, Ceramide, or Suspended in Semisolid Methylcellulose
Robert Gniadecki, Barbara Gajkowska, Jacek Bartosik, Michael Hansen, Hans Christian Wulf
Apoptosis is a form of cellular suicide and is activated in various cells, including keratinocytes, in response to physiological and pathological stimuli. A current hypothesis holds that apoptotic cells in a concerted manner express characteristic phenotypic features comprising cytoplasmic budding, pyknosis, chromatin condensation, karyorhexis and internucleosomal DNA fragmentation. In this study we investigated the effects of different potential inducers of apoptosis on cultured human keratinocytes. Viability was determined with vital dyes (ethidium bromide, trypan blue), cell morphology was investigated with electron microscopy, and nuclear and DNA integrity was assessed with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) or DNA gel electrophoresis. Irradiation with 50 mJ/cm2 ultraviolet B (UVB), treatment with C8 ceramide, or suspension in a semi-solid medium caused apoptosis which was ultrastructurally different from necrotic induced by very high (300 mJ/cm2) doses of UVB. However, the phenotype of dying cells did not exhibit all typical features of apoptosis and cell morphology depended on the method used to induce apoptosis. Cells irradiated with ultraviolet B or treated with C8 ceramide developed large and small budding and DNA nicks but not chromatin condensation or classical karyorhexis. In UVB-irradiated cells a novel form of karyorhexis was observed manifested by formation of a few very small chromatin fragments in nuclear periphery. Cells suspended in methylcellulose developed DNA nicks and pyknosis, but not budding or karyorhexis. In neither case could the typical internucleosomal DNA fragments be detected by gel electrophoresis. These morphological results indicate that in keratinocytes induction of effect or death mechanisms is not concerted but depends on the stimulus inducing apoptosis.