Induction of apoptosis in human HaCaT keratinocytes

Abstract

Although cell death by apoptosis has been recognized as an important control mechanism in the maintenance of tissue homeostasis and in the elimination of cells with damaged DNA, information on the induction and characteristics of apoptosis in keratinocytes is rather scarce. Apoptotic mechanisms may play an important role in normal and disturbed homeostasis of the skin. In the present study, we therefore investigated the effects of several potential inducers of apoptosis in the human keratinocyte cell line HaCaT. Apoptosis was assessed with respect to morphological changes by light and electron microscopic examinations and to DNA integrity by a specific ELISA. UVB irradiation induced the morphology and internucleosomal DNA fragmentation characteristic of apoptosis in a dose- and time-dependent manner. Interferon-γ caused DNA cleavage at the linker regions without producing morphological features consistent with apoptotic cell death. In contrast, treatment with dithranol and NP-40 resulted in necrotic alterations in the keratinocytes. Treatment with the calcium ionophore A23187 caused morphological changes which were similar to the characteristics of ‘nonapoptotic programmed cell death’. Dexamethasone, tumor necrosis factor-α, transforming growth factor-β, TPA, retinoic acid, the podophyllin derivative etoposide, the thromboxane A2 analogue U46619, cycloheximide, and the nitric oxide donors sodium nitroprusside and S-nitroso-glutathione, which are all known to induce apoptosis in other cell types, did not affect HaCaT keratinocytes. These results demonstrate that apoptosis can be induced in keratinocytes in vitro but the apoptosis differs from that in other cell types, such as haematopoietic cells, with regard to the type of inducer and/or the sensitivity of the target cells. Since keratinocytes are affected by numerous external and internal stimuli, they might posses several protective mechanisms to prevent apoptosis and to ensure the structural integrity of the outermost barrier of the body.