Defective cytochrome c release and the resulting loss of caspase-3 activation was recently shown to be essential for the susceptibility of human melanoma cells to CD95/Fas-induced apoptosis. Cytochrome c release from mitochondria is regulated by the relative amounts of apoptosis-promoting and apoptosis-inhibiting Bcl-2 proteins in the outer membrane of these organelles. The assignment of Bax/Bcl-2 ratios by quantitative Western blotting in 11 melanoma cell populations revealed a relation to the susceptibility to CD95-mediated apoptosis. We could show that a low Bax/Bcl-2 ratio was characteristic for resistant cells and a high Bax/Bcl-2 ratio was characteristic for sensitive cells. Low Bax expression was not a consequence of mutations in the p53 coding sequence. The Bax/Bcl-2 ratio was also in clear correlation with sensitivity to another cell death inducer, N-acetylsphingosine. Furthermore, Bcl-2 overexpression abolished apoptosis triggered by both apoptotic stimuli, confirming the critical role of the Bax/Bcl-2 ratio as a rheostat that determines the susceptibility to apoptosis in melanoma cells by regulating mitochondrial function. Interestingly, some chemotherapeutics lead to the activation of death pathways by CD95L upregulation, ceramide generation, direct activation of upstream caspases, or upregulation of proapoptotic genes. Taken together, these signals enter the apoptotic pathway upstream of mitochondria, resulting in activation of this central checkpoint. We therefore assumed that apoptosis deficiency of malignant melanoma can be circumvented by drugs directly influencing mitochondrial functions. For this purpose we used betulinic acid, a cytotoxic agent selective for melanoma, straightly perturbing mitochondrial functions. In fact, betulinic acid induced mitochondrial cytochrome c release and DNA fragmentation in both CD95-resistant and CD95-sensitive melanoma cell populations, independent of the Bax/Bcl-2 ratio.

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.

Sebaceous glands may be involved in a pathway conceptually similar to that of the hypothalamic-pituitary-adrenal (HPA) axis. Such a pathway has been described and may occur in human skin and lately in the sebaceous glands because they express neuropeptide receptors. Corticotropin-releasing hormone (CRH) is the most proximal element of the HPA axis, and it acts as central coordinator for neuroendocrine and behavioral responses to stress. To further examine the probability of an HPA equivalent pathway, we investigated the expression of CRH, CRH-binding protein (CRH-BP), and CRH receptors (CRH-R) in SZ95 sebocytes in vitro and their regulation by CRH and several other hormones. CRH, CRH-BP, CRH-R1, and CRH-R2 were detectable in SZ95 sebocytes at the mRNA and protein levels: CRH-R1 was the predominant type (CRH-R1͞CRH-R2 ‫؍‬ 2). CRH was biologically active on human sebocytes: it induced biphasic increase in synthesis of sebaceous lipids with a maximum stimulation at 10 ؊7 M and up-regulated mRNA levels of 3␤-hydroxysteroid dehydrogenase͞⌬ 5-4 isomerase, although it did not affect cell viability, cell proliferation, or IL-1␤-induced IL-8 release. CRH, dehydroepiandrosterone, and 17␤-estradiol did not modulate CRH-R expression, whereas testosterone at 10 ؊7 M down-regulated CRH-R1 and CRH-R2 mRNA expression at 6 to 24 h, and growth hormone (GH) switched CRH-R1 mRNA expression to CRH-R2 at 24 h. Based on these findings, CRH may be an autocrine hormone for human sebocytes that exerts homeostatic lipogenic activity, whereas testosterone and growth hormone induce CRH negative feedback. The findings implicate CRH in the clinical development of acne, seborrhea, androgenetic alopecia, skin aging, xerosis, and other skin disorders associated with alterations in lipid formation of sebaceous origin.

Since their introduction 15 years ago, retinoids have been increasingly used for topical and systemic treatment of psoriasis and other hyperkeratotic and parakeratotic skin disorders, keratotic genodermatoses, severe acne and acne-related dermatoses, and also for therapy and/or chemoprevention of skin cancer and other neoplasia. Oxidative metabolites of vitamin A (retinol) are natural retinoids present at low levels in the peripheral blood. Synthetic retinoids are classified into 3 generations including nonaromatic, monoaromatic and polyaromatic compounds. They are detectable in plasma 30-60 minutes after systemic administration, and reach maximum concentrations 2 to 4 hours later. Elimination half-life is 10 to 20 hours for isotretinoin, 80 to 175 days for etretinate and 2 to 4 days for, trans-acitretin; the latter, however, partially converts into etretinate. Retinoid concentrations in skin are rather low in contrast to subcutaneous fat tissue. Intracellularly, retinoids interact with cytosolic proteins and specific nuclear receptors. Two classes of nuclear receptors have been suggested to mediate retinoid activity at the molecular level, RARs and RXRs. The expression of retinoid receptors is tissue specific; skin mainly espresses RAR gamma and RXR alpha. Retinoids affect epidermal cell growth and differentiation as well as sebaceous gland activity and exhibit immunomodulatory and anti-inflammatory properties. Current retinoid research targets the development of receptor-selective retinoids for tailoring and/or improving their therapeutic profile. Currently, tretinoin is used systemically for acute promyelocytic leukaemia, etretinate and acitretin for psoriasis and related disorders, as well as other disorders of keratinisation and isotretinoin for seborrhoea, severe acne, rosacea and acneiform dermatoses. Systemic retinoids are also applied for chemoprevention of epithelial skin cancer and cutaneous T cell lymphoma. The major adverse effect of retinoids is teratogenicity; all other adverse effects are dose-dependent and controllable. Contraception is, therefore, essential during retinoid treatment in women of child-bearing age. Clinical monitoring requires physical examination for adverse effects every 3 to 4 weeks and proper laboratory investigations, also including analysis of retinoid bioavailability in selected cases. Topical retinoids are rapidly developing at present and seem promising for the future; their clinical application includes acne, aging, photodamage, precanceroses, skin cancer and disorders of skin pigmentation. The development of receptor-specific retinoids for topical treatment of psoriasis and/or acne may lead to interesting new compounds based on our current concepts of retinoid function.