VARIABLE EXPRESSION OF APOPTOTIC PHENOTYPE IN KERATINOCYTES TREATED WITH ULTRAVIOLET-RADIATION, CERAMIDE, OR SUSPENDED IN SEMISOLID METHYLCELLULOSE

Apoptosis is a form of cellular suicide and is activated in various ce lls, including keratinocytes, in response to physiological and patholo gical stimuli, A current hypothesis holds that apoptotic cells in a co ncerted manner express characteristic phenotypic features comprising c ytoplasmic 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 bro mide, trypan blue), cell morphology was investigated with electron mic roscopy, and nuclear and DNA integrity was assessed with TUNEL (termin al deoxynucleotidyl transferase-mediated dUTP nick end labelling) or D NA gel electrophoresis. Irradiation with 50 mJ/cm(2) ultraviolet B (UV B), treatment with C8 ceramide, or suspension in a semisolid medium ca used apoptosis which was ultrastructurally different from necrotic ind uced by very high (300 mJ/cm(2)) doses of UVB, However, the phenotype of dying cells did not exhibit all typical features of apoptosis and c ell morphology depended on the method used to induce apoptosis, Cells irradiated with ultraviolet B or treated with C8 ceramide developed la rge and small budding and DNA nicks but not chromatin condensation or classical karyorhexis. In UVB-irradiated cells a novel form of karyorh exis was observed manifested by formation of a few very small chromati n fragments in nuclear periphery. Cells suspended in methylcellulose d eveloped DNA nicks and pyknosis, but not budding or karyorhexis, In ne ither case could the typical internucleosomal DNA fragments be detecte d by gel electrophoresis. These morphological results indicate that in keratinocytes induction of effect or death mechanisms is not concerte d but depends on the stimulus inducing apoptosis.