Establishment and Characterization of an Immortalized Human Sebaceous Gland Cell Line (SZ95)1

Abstract

Human facial sebaceous gland cells were transfected with a PBR-322-based plasmid containing the coding region for the Simian virus-40 large T antigen. The resulting proliferating cell cultures have been passaged over 50 times to date, have been cloned, and show no signs of senescence after 4&DF;1 2 y in vitro, whereas normal human sebocytes can only be grown for three to six passages. The immortalized transfected cells, termed SZ95, expressed the Simian virus-40 large T antigen and presented an hyper-diploid-aneuploid karyotype with a modal chromosome number of 64.5. The SZ95 cell line exhibited epithelial, polymorphous characteristics with different cell sizes of up to 3.25-fold during proliferation and 6-fold at confluence, showing numerous cytoplasmic lipid droplets. The cells showed large cytoplasm profiles with abundant organelles, including vacuoles and myelin figures which indicated lipid synthesis. Lack of or only few desmosomal areas were observed. SZ95 cells expressed molecules typically associated with human sebocytes, such as keratins 7, 13, and 19, and several proteins of the polymorphous epithelial mucin family. Functional studies revealed synthesis of the sebaceous lipids squalene and wax esters as well as of triglycerides and free fatty acids, even after 25-40 passages; active lipid secretion; population doubling times of 52.4 +/- 1.6 h; reduced growth but maintenance of lipid synthesis under serum-free conditions; and retrieval of cell proliferation after addition of 5alpha-dihydrotestosterone. Retinoids significantly inhibited proliferation of certain SZ95 cell clones in the expected magnitude 13-cis-retinoic acid > all-trans-retinoic acid > > acitretin. Thus SZ95 is an immortalized human sebaceous gland cell line that shows the morphologic, phenotypic and functional characteristics of normal human sebocytes.

Corticotropin-releasing hormone: An autocrine hormone that promotes lipogenesis in human sebocytes

“…Immortalized human facial SZ95 sebocytes, which have been shown to conserve the major characteristics of normal sebocytes (9), and a dysplastic oral keratinocyte cell line (European Collection of Cell Cultures ref. no.…”

“…CRH-R1 has been detected in epidermal and follicular keratinocytes, melanocytes, and dermal fibroblasts. CRH and CRH-R have been recently detected in human sebaceous glands by in situ experiments (8); therefore, we engaged a currently developed reproducible human sebocyte culture system (9) to perform a comprehensive analysis of CRH, CRH-BP, and CRH-R1͞ CRH-R2 expression and function on human sebocytes in vitro. The expression of CRH͞CRH-R system was analyzed by reverse transcription (RT)-PCR, real time quantitative RT-PCR, and immunocytochemistry under basic conditions and after stimulation by several hormones.…”

Indian hedgehog and β-catenin signaling: Role in the sebaceous lineage of normal and neoplastic mammalian epidermis

“…SZ95 are a line of human facial skin sebocytes that have been immortalized by transfection of simian virus 40 large T antigen (37) and were cultured in Sebomed medium (Biochrom, Berlin) containing 10% FCS (Sera-Lab, Crawley Down, Sussex, U.K.), 3 ng͞ml keratinocyte growth factor (PeproTech, Rocky Hill, NJ), and 20 ng͞ml epidermal growth factor (PeproTech). SZ95 cells were retrovirally infected by using supernatant of AM12 amphotropic producer cells as described (38) and selected in 1.5 g͞ml puromycin.…”

Age‐specific hormonal decline is accompanied by transcriptional changes in human sebocytes in vitro

“…Immortalized human SZ95 sebocytes (Zouboulis et al, 1999) were maintained in Sebomed® medium supplemented with 10% steroid-free fetal calf serum (cc pro, Neustadt, Germany), 5 ng mL −1 human recombinant epidermal growth factor, 1 µM retinol, 0.1 µM linoleic acid, 1 mg mL −1 human protease-free albumin, 1 mM Ca 2+ and 50 µg mL −1 gentamicin (Invitrogen, Karlsruhe, Germany) in culture flasks in a humidified atmosphere of 5% CO 2 at 37 °C. The cells were incubated with a mixture of hormones (Sigma, Munich, Germany) at concentrations representing the median serum values in healthy 20-and 60-year-old women (f20 and f60), respectively (Table 4).…”

“…In order to gain greater insights into the molecular mechanisms involved in aging and to what extent hormones may play a role, human sebaceous gland cells (SZ95 sebocytes) (Zouboulis et al ., 1999) were treated with 17 β -estradiol, progesterone, testosterone, dehydro-epiandrosterone (DHEA), growth hormone (GH) and insulin-like growth factor-I (IGF-I) at levels corresponding to the average serum levels for females from 20 (f20) to 60 (f60) years of age. The cells behaved biologically at f60 in a manner concomitant with their aging phenotype in vivo .…”