Differentiation and Apoptosis in Human Immortalized Sebocytes

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Differentiation and Apoptosis in Human Immortalized Sebocytes Anna Wróbel, Holger Seltmann, Sabine Fimmel, Karin Müller-Decker, Miki Tsukada, Birgit Bogdanoff, Nathalie Mandt, Ulrike Blume- Peytavi, Constantin E. Orfanos, Christos C. Zouboulis Journal of Investigative Dermatology Volume 120, Issue 2, Pages 175-181 (February 2003) DOI: 10.1046/j.1523-1747.2003.12029.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Spontaneous apoptosis and necrosis in SZ95 sebocytes. (A) Significant, time-dependent DNA fragmentation in SZ95 sebocytes. (B) Increased LDH activity released from the cytoplasm of damaged SZ95 sebocytes into the supernatant after 24 h in culture. The values represent mean±standard deviation of three consecutive experiments. *p<0.05, **p<0.01, ***p<0.001. Journal of Investigative Dermatology 2003 120, 175-181DOI: (10.1046/j.1523-1747.2003.12029.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Lipid synthesis and fragmentation of nuclei in SZ95 sebocytes after treatment with arachidonic acid. Visualization of lipids in SZ95 sebocytes by Oil Red staining. Cells were treated with vehicle solution as control (A, B) or with 100 μM arachidonic acid for 48 h (C, D). Nuclei were counterstained with hematoxylin. Note markedly Oil Red positive cells as well as cells with fragmented nuclei in arachidonic-acid-treated cultures (*). Magnifications: (A) 160×; (B, C) 630×; (D) 1260×. Journal of Investigative Dermatology 2003 120, 175-181DOI: (10.1046/j.1523-1747.2003.12029.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Enhancement of sebocyte apoptosis by staurosporine in a time- and dose-dependent manner. (A) The highest levels of DNA fragmentation under staurosporine treatment in SZ95 sebocytes were detected after 6 h of incubation. (B) Staurosporine exhibited a cytotoxic effect on SZ95 sebocytes at concentrations of 10−6 and 10−5 M after 6–24 h of incubation as detected by the LDH assay. The values represent the y-fold increase of the mean±standard deviation (where y=the value of the yaxis) of three consecutive experiments, whereas control=1. *p<0.05, **p<0.01, ***p<0.001. Journal of Investigative Dermatology 2003 120, 175-181DOI: (10.1046/j.1523-1747.2003.12029.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effects of 5α-DHT on DNA fragmentation and LDH release by SZ95 sebocytes. 5α-DHT did not affect DNA fragmentation (A) but significantly inhibited LDH release of SZ95 sebocytes compared to controls after 6 h and especially after 24 h of treatment (B). The values represent the y-fold increase of the mean±standard deviation (where y=the value of the y axis) of three consecutive experiments, whereas control=1. *p<0.05, **p<0.01. Journal of Investigative Dermatology 2003 120, 175-181DOI: (10.1046/j.1523-1747.2003.12029.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effects of combined 13cRA/staurosporine treatment on DNA fragmentation and LDH release by SZ95 sebocytes. 13cRA (10−8 M and 10−7 M) and staurosporine (10−7 M) induced DNA fragmentation in SZ95 sebocytes to the same magnitude as SZ95 sebocytes treated with staurosporine alone (n=3) (A) but did not affect LDH release (B). The values represent the y-fold increase of the mean±standard deviation (where y=the value of the y axis) of three consecutive experiments, whereas control=1. **p<0.01, ***p<0.001. Journal of Investigative Dermatology 2003 120, 175-181DOI: (10.1046/j.1523-1747.2003.12029.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effects of retinoids, 5α-DHT, and staurosporine on phosphatidylserine externalization in SZ95 sebocytes. Staurosporine (10−7 and 3×10−7 M) enhanced phosphatidylserine externalization, and consequently apoptosis, in SZ95 sebocytes as assessed by annexin V detection using flow cytometry. In contrast, 13cRA (10−7 M), acitretin (10−7 M), and 5α-DHT (10−7 M) induced detectable annexin V rates were similar to those in control cultures. The values represent mean±standard deviation of three consecutive experiments. ***p<0.001. Journal of Investigative Dermatology 2003 120, 175-181DOI: (10.1046/j.1523-1747.2003.12029.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Effects of 13cRA and staurosporine on caspase 3 expression in SZ95 sebocytes. Caspase 3 expression in SZ95 sebocytes was analyzed by flow cytometry; marker 1 (M1) represents caspase 3 positive dead cells and marker 2 (M2) caspase 3 positive vital cells [100 – (M1+M2): caspase negative cells]. Dead cells were detected by staining with propidium iodide. Data are representative of three experiments. FL1, excitation 488 nm, emission 530 nm. Journal of Investigative Dermatology 2003 120, 175-181DOI: (10.1046/j.1523-1747.2003.12029.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 bax and bcl-2 mRNA expression under 13cRA and staurosporine treatment.bax and bcl-2 mRNA expression in SZ95 sebocytes under 13cRA (10−7 M) or staurosporine (10−7 M) treatment for 6 h was detected by RT-PCR and evaluated in comparison to β-actin. bcl-2 mRNA expression was downregulated by staurosporine but not by 13cRA. Journal of Investigative Dermatology 2003 120, 175-181DOI: (10.1046/j.1523-1747.2003.12029.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 bax/bcl-2 ratio after flow cytometry of bax and bcl-2 protein expression under 13cRA and staurosporine treatment. The bcl-2/bax ratio was increased by staurosporine and the combined staurosporine (10−7 M) (n=3) and 13cRA (10−7 M) treatment (n=2) at similar levels, whereas it was not affected by 13cRA (n=3). The values represent the y-fold increase of the mean±standard deviation (where y=the value of the y axis) of the subsequent experiments, whereas control=1. *p<0.05, **p<0.01. Journal of Investigative Dermatology 2003 120, 175-181DOI: (10.1046/j.1523-1747.2003.12029.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions