Altered Proliferation, Synthetic Activity, and Differentiation of Cultured Human sebocytes in the Absence of Vitamin A and Their Modulation by Synthetic Retinoids

Human sebocytes maintained in medium containing delipidized serum were studied for ultrastructural characteristics, cell proliferation, lipid synthesis, immunophenotype, and keratin expression before and after the addition of the synthetic retinoids isotretinoin and acitretin (10-8 - 10-5 M).Compared to the properties of sebocytes cultured in normal sebocyte medium (1–2 × 10-7 M vitamin A), the use of delipidized serum (undetectable amounts of vitamin A) resulted in prominent decrease of i) proliferation; ii) number of intracellular lipid droplets and synthesis of total lipids, especially triglycerides, squalene, and wax esters; and iii) labeling with monoclonal antibodies identifying progressive and late-stage sebocyte differentiation. Intercellular spaces narrowed and cell-to-cell contacts were established by abundant desmosomes. Lanosterol was induced. Keratins 14, 16, 17, and 18 were upregulated and the keratin 16: keratin 4 ratio, negatively correlating with sebocyte differentiation, increased.Addition of isotretinoin and acitretin exerted a biphasic effect. At concentrations ≤ 10-7 M, both compounds enhanced sebocyte proliferation and synthesis of total lipids, especially triglycerides and cholesterol, and decreased Ianosterol, keratin 16, and the keratin 16: keratin 4 ratio. In contrast, retinoid concentrations > 10-7 M inhibited sebocyte proliferation in a dose-dependent manner.Our findings indicate that vitamin A is essential for proliferation, synthetic activity, and differentiation of human sebocytes in vitro. Synthetic retinoids partially reinstate the altered functions of sebocytes maintained in medium containing delipidized serum. In contrast to the previously shown isotretinoin-specific response of cultured sebocytes in the presence of vitamin A, similar effects of isotretinoin and acitretin were obtained in its absence. This suggests different interactions of synthetic retinoids with vitamin A, possibly influencing their efficacy on the sebacceous gland

Selective Induction of Apoptosis in Melanoma Cells by Tyrosinase Promoter-Controlled CD95 Ligand Overexpression

Induction of apoptosis has been demonstrated previously by overexpression of CD95 ligand (CD95L) in cultured human melanoma cells. For in vivo approaches based on CD95L, however, targeted expression is a prerequisite and tyrosinase promoters have been considered for selection. Luciferase reporter gene assays performed for a representative panel of melanoma cell lines characterized by strong (SK-Mel-19), moderate (SK-Mel-13, MeWo), weak (A-375), and missing expression (M-5) of endogenous tyrosinase revealed high tyrosinase promoter activities in SK-Mel-19, SK-Mel-13, and MeWo, but only weak activities in A-375 and M-5 as well as in non-melanoma cell lines. After transfection of a CMV promoter CD95L expression construct, melanoma cells were found highly sensitive, as compared with non-melanoma cells. By applying a tyrosinase promoter CD95L construct, apoptosis was selectively induced in SK-Mel-19, SK-Mel-13, MeWo as well as in A-375, which was characterized by high CD95 surface expression and high sensitivity to agonistic CD95 activation. M5 and non-melanoma cell lines remained uninfluenced. Also, resistance to agonistic CD95 activation seen in MeWo characterized by weak CD95 surface expression was overcome by overexpression of CD95L. Our investigations provide evidence that tyrosinase promoter CD95L constructs may be of value for selective induction of apoptosis in therapeutic strategies for melanoma

The Bax/Bcl-2 Ratio Determines the Susceptibility of Human Melanoma Cells to CD95/Fas-Mediated Apoptosis

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

Bcl-2 antagonizes apoptotic cell death induced by two new ceramide analogues

AbstractCeramides which arise in part from the breakdown of sphingomyelin comprise a class of antiproliferative lipids and have been implicated in the regulation of programmed cell death better known as apoptosis. In the present study, two new synthetic ceramide analogues, N-thioacetylsphingosine and FS-5, were used in Molt4 cells to induce cell death. Besides their cytotoxic effects at concentrations ≥14 μM the data obtained clearly show that both analogues induced apoptosis at concentrations below this critical concentration as assessed by trypan blue exclusion and cleavage of the death substrate poly-(ADP-ribose) polymerase (PARP). Additional experiments in bcl-2-transfected Molt4 cells revealed that the apoptotic but not the lytic effects of the analogues were antagonized by the apoptosis inhibitor Bcl-2. Furthermore, neither N-thio-acetylsphingosine nor FS-5 induced PARP cleavage in bcl-2-transfected Molt4 cells indicating that the induction of apoptotic cell death by cell permeable ceramides is not due to unspecific disturbance of the cell membrane

Topical all- trans retinoic acid (RA) induces an early, coordinated increase in RA-inducible skin-specific gene/psoriasin and cellular RA-binding protein II mRNA levels which precedes skin erythema

Separation of specific and nonspecific “irritant” effects of topical all- trans retinoic acid (RA) is a key to understanding the mechanism of retinoid action in skin. Cellular RA-binding protein (CRABP) II has been found to be a marker of RA activity in human skin. We have also previously identified a skin-specific gene (RIS-1/psoriasin) which is rapidly induced in human skin treated with RA. Here we compared the kinetics and time-course of RIS-1 and CRABP II gene activation by RA, sodium lauryl sulfate (SLS), a classical irritant, and their vehicle (VH), using a quantitative reverse transcription polymerase chain reaction. RIS-1 and CRABP II were both expressed at very low levels in untreated normal human skin, and in RA-treated skin the kinetics and time course of RIS-1 and CRABP II mRNA induction were similar. Relative to VH-treated skin, RA induced RIS-1 mRNA levels within 6 h, which further increased to 6.4-fold by 24 h ( n = 4). Similarly, CRABP II mRNA levels increased from 2.6-fold at 6 h to 7.8-fold after 24 h. At 48 h the relative mRNA levels for both genes decreased towards the steady-state levels. Relative to SLS-treated skin, RIS-1 mRNA increased by 3.2-fold after 6 h and by 5.1-fold after 12 h ( n = 3). Also, a 2.6-fold higher CRABP II mRNA observed after 6 h increased to 6-fold after 12 h. After 24 and 48 h RA treatment the relative mRNA levels for both genes decreased towards the steady-state levels. RA-induced skin erythema was not obvious until 24 to 48 h. We conclude, therefore, that induction of RIS-1 and CRABP II mRNA levels by topical RA in human skin are early, coordinated molecular events which precede the clinical cutaneous erythematous response to RA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42209/1/403-288-11-664_62880664.pd

Endothelin-1 Decreases Basic Apoptotic Rates in Human Melanoma Cell Lines

Normal human melanocytes respond to endothelin-1 with induced proliferation and differentiation. Whereas in cultured melanoma cells the predominant endothelin receptor, ET(B)-R, is consistently downregulated, ET(B)-R upregulation was recently reported for melanoma tumors. Contrary to the pro-survival activity described for endothelin in vascular cells, a proapoptotic activity of endothelin-1 has been reported for melanoma cells, in previous studies. We therefore investigated the role of endothelin for melanoma cells with respect to apoptosis and proliferation. Treatment with 10 nM endothelin-1 was a strong mitogenic signal for normal human melanocytes, which responded with a 4–6-fold increase of thymidine incorporation, whereas the response was only 1.2-fold for SK-Mel-19, the melanoma cell line characterized by the highest ET(B)-R expression, and it was even less in other cell lines. Determination of the apoptotic rates revealed that endothelin-1 significantly reduced basic apoptotic rates to 75% both in SK-Mel-19 and in normal melanocytes. After cell synchronization, an antiapoptotic effect of endothelin-1 was seen in five of seven cell lines investigated. In the cell line Bro, which showed no response and which lacks ET(B)-R expression, responsibility could be restored by overexpression of ET(B)-R after stable transfection, indicating that the effectors of the endothelin-1 signal cascade were active in these cells, and that the antiapoptotic effect of endothelin-1 is mediated in a receptor-specific way. This antiapoptotic activity of endothelin for melanoma cells combined with upregulation of endothelin receptors in the tumor may be a crucial step for melanoma progression

RT-PCR for Tyrosinase-mRNA-Positive Cells in Peripheral Blood: Evaluation Strategy and Correlation with Known Prognostic Markers in 123 Melanoma Patients

Reverse transcriptase polymerase chain reaction for the detection of tyrosinase-mRNA-positive cells in peripheral blood of melanoma patients, as a possible marker of hematogenous dissemination, has demonstrated varying detection rates. This study examined the sensitivity and reproducibility of the technique using a protocol of multiple polymerase chain reaction to determine circulating melanocytic cells. For each of the 123 melanoma patients included in this study, four nested polymerase chain reactions were performed from two blood specimens requiring both polymerase chain reactions from at least one blood sample to be positive to consider a patient as positive. Thus, a definitive result was obtained in 98% of the cases, whereas only 1.6% lacked conclusive findings. Thus, we found a correlation between the tyrosinase detection rate and the clinical stage. Circulating tyrosinase-mRNA-positive cells were detected in 13% of patients with primary tumor, 17% with regional skin/lymph node metastasis, and 44% with distant metastasis. Positivity also correlated with known melanoma progression markers such as gender, tumor thickness, and histologic type. Positive results were obtained more frequently in (i) men compared with women, (ii) patients with thick primary melanomas (>4 mm: 38%) compared with those with thinner tumors (1.1–4 mm, 22%;≤1 mm, 5%), and (iii) patients with nonclassifiable (38%), nodular (34%), and occult primary melanomas (30%) compared with those with acrolentiginous (17%), superficial spreading (9%), or lentigo maligna melanoma (0%). These findings suggest that detection of tyrosinase-mRNA-positive cells in peripheral blood is not an adequate marker for identifying melanoma patients with distant metastasis. Reverse transcriptase polymerase chain positivity in early melanoma stages, however, as corresponding to other prognostic parameters, may indicate increased risk for the development of hematogenous metastasis and may be of value as a progression marker