Christos C. Zouboulis, Holger Seltmann, Constantin E. Orfanos

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Presentation transcript:

Establishment and Characterization of an Immortalized Human Sebaceous Gland Cell Line (SZ95)1 Christos C. Zouboulis, Holger Seltmann, Constantin E. Orfanos Journal of Investigative Dermatology Volume 113, Issue 6, Pages 1011-1020 (December 1999) DOI: 10.1046/j.1523-1747.1999.00771.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Detection of the SV-40 large T antigen. Cytocentrifuge preparations of (a) SZ95 cells and (b) HMEC-1 cells positively labeled with a MoAb against the human SV-40 large T antigen. Both cells types mostly reveal strong nuclear staining, whereas some cells also exhibit cytoplasmic labeling. Scale bar: 3 μm. (c) Western blot analysis of SV-40 large T antigen expression in non-transfected human sebocytes (lane 1), human keratinocytes (lane 2), SZ95 cells (34th subculture) (lane 3), SZ95/K6 cells (lane 4), SK95/K7 cells (lane 5), and SZ95/K28 cells (lane 6). A 94 kDa band, compatible for the SV-40 large T antigen, was detected in the SZ95 cell line and its clones. Journal of Investigative Dermatology 1999 113, 1011-1020DOI: (10.1046/j.1523-1747.1999.00771.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Cell morphology. Subconfluent cultures of (a) human sebocytes of the donor prior to transfection experiments (second subculture), (b) SZ95 cells (first subculture), and (c) SZ95/K7 cells (50th subculture) exhibit a similar epithelial, polymorphous appearance with cells of different sizes. Scale bar: 20 μm. Journal of Investigative Dermatology 1999 113, 1011-1020DOI: (10.1046/j.1523-1747.1999.00771.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Electron microscopy. SZ95 cells show (a) microvillous transformation of their cell membranes and (a, b) large cytoplasm profiles with abundant organelles, especially Golgi membranes and ellipsoid vacuoles. Lack of or only few desmosomal areas are detected. (c) Several inclusion bodies, round and ellipsoid vacuoles as well as (d) myelin figures of various types are seen in the cytoplasm of SZ95 cells, indicating lipid synthesis. Scale bar: 0.47 μm. Journal of Investigative Dermatology 1999 113, 1011-1020DOI: (10.1046/j.1523-1747.1999.00771.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Detection of sebocyte proteins. Cytocentrifuge preparations of (a) SZ95 cells and (b) human keratinocytes labeled with the OM-1 MoAb against the sebaceous gland antigen. SZ95 cells, but not keratinocytes, shown positive cytoplasm staining. Scale bar: 3 μm. (c) Western blot analysis of SZ95 cells (34th subculture) (lane 1), SZ95/K6 cells (lane 2), SK95/K7 cells (lane 3), SZ95/K28 cells (lane 4), and human keratinocytes (lane 5) for expression of human epithelial sialo-mucin (ESM), human milk fat globulin-1 (HMFG-1), human milk fat globulin-2 (HMFG-2), mucin-like carcinoma-associated antigen (MCA), epithelial membrane antigen (EMA), Thomsen–Friedenreich antigen (TF-antigen), keratins 7, 13, and 19, and 5α-reductase type 1 (5α-Red 1). The SZ95 cell line and its clones expressed all investigated proteins, whereas keratinocytes only expressed keratin 13 and 5α-reductase type 1. Journal of Investigative Dermatology 1999 113, 1011-1020DOI: (10.1046/j.1523-1747.1999.00771.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Synthesis of lipids. SZ95 cells positively labeled with (a) Oil Red dye and (b) Nile Red fluorescence dye identifying neutral lipids. Scale bars: 10 μm. (c) Storage imaging of HPTLC fractionated lipids synthesized in SZ95/K6 (lanes 3 and 4) and SZ95/K7 cells (lanes 5 and 6) been pulsed with 0.5 μCi [2-14C]-acetic acid, sodium salt per ml (45–60 mCi per mmol) for 24 h. The cells (lanes 3–5) synthesize several fractions of neutral lipids, among them the sebaceous lipids squalene (Sq) and wax esters (WE) as well as triglycerides (Tg), cholesterol (Cho), cholesterol esters (ChE), diglycerides (Dg), lanosterol (Lan), and free fatty acids (FFA). Cholesterol and triglycerides were mainly isolated from human keratinocytes (lane 7), whereas wax esters were not identified. Neutral lipids were also found to a lesser extent in the supernatants (SN; lanes 6 and 8). Lane 1, lipid standards, lane 2, human sebum lipids, lane 4, fatty acids (FA) extracted from the SZ95/K6 cells Journal of Investigative Dermatology 1999 113, 1011-1020DOI: (10.1046/j.1523-1747.1999.00771.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Cell kinetics. Proliferation of SZ95/K7 cells over 18 d. Seeding cell density influenced the duration of logarithmic proliferation. Logarithmic proliferation occurred from 2 to 14 d and population doubling times was 54.3 h at 500 cells per well, 2–10 d and 51.7 h at 1000 cells per well, 2.6 d and 51.3 h at 2000 cells per well, respectively. No logarithmic growth could be detected at a seeding density of 4000 cells per well. The log of the mean values ± SD of sextuplicate evaluations are presented. Journal of Investigative Dermatology 1999 113, 1011-1020DOI: (10.1046/j.1523-1747.1999.00771.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Effect of 5α-DHT on cell proliferation. Proliferation of SZ95/K7 cells (2000 cells per well) over 18 d in serum-free medium (control) and in serum-free medium supplemented with 10-6 M 5α-DHT. From day 8, 5α-DHT significant increased the proliferation of SZ95 cells, exhibiting population doubling times of 136 h (control) and 53.7 h (5α-DHT-treated cells). The log of the mean values ± SD of sextuplicate evaluations are presented. *p < 0.05; **p < 0.01 Journal of Investigative Dermatology 1999 113, 1011-1020DOI: (10.1046/j.1523-1747.1999.00771.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Effects of retinoids on cell proliferation. Addition of all-trans-retinoic acid, 13-cis-retinoic acid or acitretin (all 10-7 M) to SZ95/K6, SZ95/K7, SZ95/K28 cells and human keratinocytes maintained in serum-free medium (seeding density 1000 cells per well). SZ95/K6 and SZ95/K7 cell growth was significantly inhibited by retinoids in the magnitude 13-cis-retinoic acid > all-trans-retinoic acid > > acitretin, whereas SZ95/K28 cells and keratinocytes responded to retinoids with stimulation of cell proliferation. The mean values ± SD of sextuplicate evaluations at treatment day 9 are presented. Comparison has been made with untreated controls (100%). *p < 0.05, **p < 0.01, ***p < 0.001. Journal of Investigative Dermatology 1999 113, 1011-1020DOI: (10.1046/j.1523-1747.1999.00771.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions