Increased cell volume, accumulation of lipid droplets in the cytoplasm, and nuclear degeneration are phenomena indicating terminal differentiation of human sebocytes followed by holocrine secretion and cell death. The molecular pathways of natural and induced sebocyte elimination are still unknown, however. In this study, SZ95 sebocytes were found to exhibit DNA fragmentation after a 6 h culture followed by increased lactate dehydrogenase release after 24 h, indicating cell damage. With the help of morphologic studies and using Oil Red detection of cellular lipids, cell enlargement, accumulation of lipid droplets in the cytoplasm, and nuclear fragmentation could be observed under treatment with arachidonic acid. Staurosporine, a potent inhibitor of phospholipid Ca2+ -dependent protein kinase, increased externalized phosphatidylserine levels on SZ95 sebocytes, detected by annexin V/propidium iodide flow cytometry, as early as after 1 h, whereas dose-dependent reduction of bcl-2 mRNA and protein expression, enhanced DNA fragmentation, and increased caspase 3 levels, detected by caspase 3 inhibitor/propidium iodide flow cytometry, were found after 6 h of treatment. SZ95 sebocyte death was detected as early as after 6 h of SZ95 sebocyte treatment with high staurosporine concentrations (10(-6) -10(-5) M). 5alpha-Dihydrotestosterone (10(-8) -10(-5) M) did not affect externalized phosphatidylserine levels and DNA fragmentation in SZ95 sebocytes but slightly decreased lactate dehydrogenase cell release. Neither acitretin nor 13-cis retinoic acid (10(-8) -10(-5) M) affected externalized phosphatidylserine levels, DNA fragmentation, and lactate dehydrogenase cell release, despite the increased caspase 3 levels under treatment with 13-cis retinoic acid. The combined staurosporine and 13-cis retinoic acid treatment enhanced DNA fragmentation in SZ95 sebocytes to the same magnitude as in cells only treated with staurosporine. In conclusion, SZ95 sebocytes in vitro undergo apoptosis, which can be enhanced by the terminal differentiation inductor arachidonic acid or by staurosporine and leads to cell death. 5alpha-Dihydrotestosterone inhibits SZ95 sebocyte death without involving apoptotic pathways, and retinoids did not affect the programmed death of human sebocytes. The latter result fits well with the currently reported inability of normal skin cells to undergo apoptosis after treatment with retinoids, in contrast to their malignant counterparts.

Human sebocytes obtained as explants after in vitro culture of isolated sebaceous glands were recently shown to maintain in part a sebocytic differentiation. The aim of this study was to further identify markers of sebocytic differentiation in vitro. Therefore, the morphology of cultured human sebocytes, and their differentiation with lipid storing and expression of cellular proteins were investigated by microscopy, electron microscopy, study of cell kinetics, cytochemistry and immunocytochemistry, and were compared to cultured human keratinocytes obtained from the same skin specimens. At first, sebocytes in all stages of sebocytic differentiation were detected in vitro. Abundant cytoplasmic lipids and the absence of desmosomes were identified as their ultrastructural characteristics. Secondly, an increasing number of sebocytes storing lipids was detected during cell proliferation. Sebocytes contained up to 4 times more lipids than keratinocytes in vitro. Squalene and increased quantities of wax/sterol esters could be extracted from secondary sebocyte cultures. Thirdly, the monoclonal antibodies 6B10 (keratin 4), RPN1162 (keratin 7), and OM-1 labeled only sebocytes in vitro. Furthermore, sebocytes presented a marked expression of keratin 19 in comparison to keratinocytes, as detected with CK 4.62, and a lack of RPN1161 (keratins 1 and 2) expression, which was typically found to be expressed in cultured keratinocytes. The culture of human sebocytes possessing several characteristics of sebocytic differentiation in vivo offers unique possibilities in investigating direct effects on sebaceous cell growth, differentiation and their regulation.