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• W2005929145 abstract "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. 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. endothelin endothelin A receptor endothelin B receptor Endothelins (ET-1, ET-2, ET-3) are paracrine signal peptides of 21 amino acid residues that bind to two highly homologous, G-protein-coupled, heptahelical receptors, ET(A)-R and ET(B)-R (Elshourbagy et al., 1993Elshourbagy N.A. Korman D.R. Wu H.L. et al.Molecular characterization and regulation of the human endothelin receptors.J Biol Chem. 1993; 268: 3873-3879Abstract Full Text PDF PubMed Google Scholar). As a predominant activity in the vascular system, where ETs were first studied, they cause vasoconstriction of smooth muscle cells, but in various nonvascular cells effects on proliferation as well as on differentiation have also been reported (Simonson and Dunn, 1990Simonson M.S. Dunn M.J. Cellular signaling by peptides of the endothelin gene family.FASEB J. 1990; 4: 2989-3000Crossref PubMed Scopus (382) Google Scholar). Normal human melanocytes were shown to bind ET-1 and respond with increased proliferation and pigmentation (Yada et al., 1991Yada Y. Higuchi K. Imokawa G. Effects of endothelins on signal transduction and proliferation in human melanocytes.J Biol Chem. 1991; 266: 18352-18357Abstract Full Text PDF PubMed Google Scholar). According to a recent model, ET-1 secretion of epidermal keratinocytes is upregulated by ultraviolet (UV) irradiation resulting in enhanced melanocyte proliferation and pigment production leading to skin tanning (Imokawa et al., 1992Imokawa G. Yada Y. Miyagishi M. Endothelins secreted from human keratinocytes are intrinsic mitogens for human melanocytes.J Biol Chem. 1992; 267: 24675-24680Abstract Full Text PDF PubMed Google Scholar;Yohn et al., 1993Yohn J.J. Morelli J.G. Walchak S.J. Rundell K.B. Norris D.A. Zamora M.R. Cultured human keratinocytes synthesize and secrete endothelin-1.J Invest Dermatol. 1993; 100: 23-26Abstract Full Text PDF PubMed Google Scholar). Mutation of the ET(B)-R gene is associated with megacolon and pigmentary disorders in human patients indicating its important role also for neural crest cell migration in embryogenesis (Puffenberger et al., 1994Puffenberger E.G. Hosoda K. Washington S.S. Nakao K. deWit D. Yanagisawa M. Chakravart A. A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung's disease.Cell. 1994; 79: 1257-1266Abstract Full Text PDF PubMed Scopus (758) Google Scholar). The study of aberrant gene expression can provide considerable insight into the characteristics of malignant cells. For human melanomas, two-dimensional protein analyses demonstrated downregulation of a high proportion of melanocyte proteins in melanoma cell lines (Eberle et al., 1995aEberle J. Garbe C. Kroumpouzos G. Orfanos C.E. Protein patterns of benign and malignant human melanocytes show consistent changes in gene expression.Recent Results Cancer Res. 1995; 139: 123-135Crossref PubMed Scopus (9) Google Scholar), and comparative hybridization techniques subsequently identified several downregulated genes including the pigmentation genes tyrosinase, TRP-1, and TRP-2 (Eberle et al., 1995bEberle J. Garbe C. Orfanos C.E. Identification of genes specifically regulated in human melanoma cells.Arch Dermatol Res. 1995; 287: 421-427Crossref PubMed Scopus (9) Google Scholar, Eberle et al., 1995cEberle J. Garbe C. Wang N. Orfanos C.E. Incomplete expression of the tyrosinase gene family (tyrosinase, TRP-1, and TRP-2) in human malignant melanoma cells in vitro.Pigment Cell Res. 1995; 8: 307-313Crossref PubMed Scopus (37) Google Scholar) as well as the ET receptors (Eberle et al., 1999Eberle J. Weitmann S. Thieck O. Pech H. Paul M. Orfanos C.E. Downregulation of endothelin B receptor in human melanoma cell lines parallel to differentiation genes.J Invest Dermatol. 1999; 112: 925-932Crossref PubMed Scopus (48) Google Scholar). Expression analyses and receptor binding assays identified ET(B)-R as predominant in melanocytic cells, whereas ET(A)-R was expressed much more weakly. Compared to primary cultures of normal melanocytes and of congenital nevus cells, ET(B)-R mRNA and ET-1 binding was consistently downregulated in 16 of 17 melanoma cell lines analyzed, with the only exception the highly differentiated and pigmented cell line SK-Mel-19. Furthermore, comparison with pigment gene expression revealed a close correlation with ET(B)-R expression whereas ET(A)-R expression was contradictory (Eberle et al., 1999Eberle J. Weitmann S. Thieck O. Pech H. Paul M. Orfanos C.E. Downregulation of endothelin B receptor in human melanoma cell lines parallel to differentiation genes.J Invest Dermatol. 1999; 112: 925-932Crossref PubMed Scopus (48) Google Scholar). In contrast to the consistent cell culture data, immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR) performed on tumor samples very recently suggested an enhanced ET(B)-R expression in melanoma metastases compared to primary tumor and nevus samples (Demunter et al., 2001Demunter A. De Wolf-Peeters C. Degreef H. Stas M. van den Oord J.J. Expression of the endothelin-B receptor in pigment cell lesions of the skin. Evidence for its role as tumor progression marker in malignant melanoma.Virchows Arch. 2001; 438: 485-491Crossref PubMed Scopus (91) Google Scholar). For different cell types it has been shown that ETs are also involved in the regulation of apoptosis. Unlike the situation in endothelial cells and smooth muscle cells, where a pro-survival activity of ET-1 has been reported (Shichiri et al., 1997Shichiri M. Kato H. Marumo F. Hirata Y. Endothelin-1 as an autocrine/paracrine apoptosis survival factor for endothelial cells.Hypertension. 1997; 30: 1198-1203Crossref PubMed Scopus (155) Google Scholar;Wu-Wong et al., 1997Wu-Wong J.R. Chiou W.J. Dickinson R. Opgenorth T.J. Endothelin attenuates apoptosis in human smooth muscle cells.Biochem J. 1997; 328: 733-737Crossref PubMed Scopus (103) Google Scholar), induction of apoptosis by ET-1 was described for the human melanoma cell line A-375, after cell synchronization (Okazawa et al., 1998Okazawa M. Shiraki T. Ninomiya H. Kobayashi S. Masaki T. Endothelin-induced apoptosis of A375 human melanoma cells.J Biol Chem. 1998; 273: 12584-12592Crossref PubMed Scopus (99) Google Scholar). The present study was performed to clarify the role of ET for melanoma cells and to investigate whether downregulation of ET receptors found in melanoma cell lines may be understood as an apoptosis escape of melanoma cells. It turned out, however, that ET-1 results in a decrease rather than an increase of basic apoptotic rates in most melanoma cell lines as in normal melanocytes. Cell transfection experiments were further applied to confirm this finding. Cultures of normal human melanocytes were isolated from human foreskins after trypsin digestion (Eisinger and Marco, 1982Eisinger M. Marco O. Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin.Proc Natl Acad Sci USA. 1982; 79: 2018-2022Crossref PubMed Scopus (449) Google Scholar) and were grown in MCDB 153 (Biochrom, Berlin, Germany) supplemented with 2 mM Ca2+, 5 µg per ml insulin (Sigma, St. Louis, MO), 10 µg per ml human transferrin (Sigma), 0.4% bovine pituitary extract (Life Technologies, Karlsruhe, Germany), 2 ng per ml bovine basic fibroblast growth factor (Boehringer Mannheim, Germany), 1 nM cholera toxin (Calbiochem, La Jolla, CA), and 50 µM hydrocortisone (Serva, Heidelberg, Germany). The use of separated and anonymized foreskin tissue for cultivation of primary cell cultures was approved by the local Ethical Committee of the University Medical Center Benjamin Franklin. Seven human melanoma cell lines were examined in this study: A-375 (Giard et al., 1973Giard D.J. Aaronson S.A. Todaro G.J. Arnstein P. Kersey J.H. Dosik H. Parks W.P. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors.J Natl Cancer Inst. 1973; 51: 1417-1423Crossref PubMed Scopus (1750) Google Scholar), Bro (Lockshin et al., 1985Lockshin A. Giovanella B.C. De Ipolyi P.D. Williams Jr, L.J. Mendoza J.T. Yim S.O. Stehlin J.S.J. Exceptional lethality for nude mice of cells derived from a primary human melanoma.Cancer Res. 1985; 45: 345-350PubMed Google Scholar), M-5 (Liao et al., 1975Liao S.K. Dent P.B. McCulloch P.B. Characterization of human maligant melanoma cell lines. I. Morphology and growth characteristics in culture.J Natl Cancer Inst. 1975; 54: 1037-1044PubMed Google Scholar), Mel-2a (Bruggen et al., 1981Bruggen J. Macher E. Sorg C. Expression of surface antigens and its relation to parameters of malignancy in human malignant melanoma.Cancer Immunol Immunother. 1981; 10: 121-127Google Scholar), MeWo (Bean et al., 1975Bean M.A. Bloom B.R. Herberman R.B. Old L.J. Oettgen H.F. Klein G. Terry W.D. Cell-mediated cytotoxicity for bladder carcinoma: evaluation of a workshop.Cancer Res. 1975; 35: 2902-2913PubMed Google Scholar), SK-Mel-13, and SK-Mel-19 (Carey et al., 1976Carey T.E. Takahashi T. Resnick L.A. Oettgen H.F. Old L.J. Cell surface antigens of human malignant melanoma: mixed hemadsorption assays for humoral immunity to cultured autologous melanoma cells.Proc Natl Acad Sci USA. 1976; 73: 3278-3282Crossref PubMed Scopus (379) Google Scholar). Melanoma cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) (4.5 g per l glucose; Life Technologies) supplemented with 10% fetal bovine serum (FBS) and antibiotics (Biochrom). For routine cultivation, cells were passaged at 70% confluence with 2 × 106 cells per 75 cm2 flask. ET receptors were in general downregulated in melanoma cell lines, as shown in a previous paper (Eberle et al., 1999Eberle J. Weitmann S. Thieck O. Pech H. Paul M. Orfanos C.E. Downregulation of endothelin B receptor in human melanoma cell lines parallel to differentiation genes.J Invest Dermatol. 1999; 112: 925-932Crossref PubMed Scopus (48) Google Scholar). As illustrated in Table I, only SK-Mel-19 showed an expression level of ET(B)-R comparable to normal melanocytes; Mel-2a, SK-Mel-13, and A-375 were characterized by moderate expression of ET(B)-R, MeWo showed weak expression of ET(B)-R, and M-5 showed weak expression of ET(A)-R. ET receptor expression was nearly lost in the melanoma cell line Bro, which was characterized by an approximately 1000-fold decrease of ET binding (Eberle et al., 1999Eberle J. Weitmann S. Thieck O. Pech H. Paul M. Orfanos C.E. Downregulation of endothelin B receptor in human melanoma cell lines parallel to differentiation genes.J Invest Dermatol. 1999; 112: 925-932Crossref PubMed Scopus (48) Google Scholar; Table I).Table IExpression of ET receptors in melanoma cell lines usedaExpression values of the ET receptors were determined by northern blotting and were already reported in a previous study (Eberle et al, 1999).ReceptorNHMbExpression of ET(B)-R in normal melanocytes (NHM) was set to 100%, and expression values of ET(B)-R in melanoma cell lines were calculated in percentage relative to this value.SK-Mee19SK-Mee13Mel-2aA-375MeWoM-5BroET(B)-R1001081922135PCRdPCR is indicated when expression was not detectable by northern blotting; however, RT-PCR was positive.PCRET(A)-RcExpression data for ET(A)-R were put into relation to ET(B)-R due to ET binding experiments, which yielded for M-5 80% of the ET-1 binding capacity determined for MeWo.0.3––––PCR40.1a Expression values of the ET receptors were determined by northern blotting and were already reported in a previous study (Eberle et al., 1999Eberle J. Weitmann S. Thieck O. Pech H. Paul M. Orfanos C.E. Downregulation of endothelin B receptor in human melanoma cell lines parallel to differentiation genes.J Invest Dermatol. 1999; 112: 925-932Crossref PubMed Scopus (48) Google Scholar).b Expression of ET(B)-R in normal melanocytes (NHM) was set to 100%, and expression values of ET(B)-R in melanoma cell lines were calculated in percentage relative to this value.c Expression data for ET(A)-R were put into relation to ET(B)-R due to ET binding experiments, which yielded for M-5 80% of the ET-1 binding capacity determined for MeWo.d PCR is indicated when expression was not detectable by northern blotting; however, RT-PCR was positive. Open table in a new tab The full-length cDNA of ET(B)-R kindly provided by M. Paul, Department of Clinical Pharmacology and Toxicology, UKBF, Berlin, was subcloned as a BamHI fragment downstream of the CMV promoter in the expression vector pBK-CMV (Stratagene, La Jolla, CA). For better eucaryotic expression, the lactose promoter between CMV promoter and cloning site was further deleted by fusion of the two restriction sites NheI, which separates the two promoters, and SpeI within the cloning site. Plasmid DNA used for transfection was extracted by plasmid purification kits of Qiagen (Hilden, Germany). For melanoma cell transfection, lipofectin (Life Technologies) and DNA were incubated separately for 30 min at room temperature in serum-free DMEM (Life Technologies) and were then mixed and incubated for another 10 min at room temperature. Cells at a confluence of 40%-60%, in 75 cm2 flasks, were washed once and incubated at 37°C with this solution containing 2 µl per ml lipofectin and 1 µg per ml plasmid DNA. After 2 d, cells were washed and were then further incubated with 400 µg per ml geneticin (Life Technologies) for selection. Individual cell clones resistant to the antibiotic were selected by limited dilution in microtiter plates. A detailed protocol for determination of relative numbers of ET-1 binding sites has been described previously (Eberle et al., 1999Eberle J. Weitmann S. Thieck O. Pech H. Paul M. Orfanos C.E. Downregulation of endothelin B receptor in human melanoma cell lines parallel to differentiation genes.J Invest Dermatol. 1999; 112: 925-932Crossref PubMed Scopus (48) Google Scholar). In brief, melanoma cells were seeded in 24-well plates (2 × 105 cells per well). On the following day, when confluence was about 50%-70%, growth medium was exchanged with serum-free DMEM. After 24 h, cells were washed twice with ice-cold binding buffer (50 mM HEPES, 5 mM MgCl2, 0.3% bovine serum albumin, pH 7.4) and incubated at 4°C in 200 µl of this buffer containing 0.125 µCi of 125I ET-1 (2000 Ci per mmol; Amersham, Buckinghamshire, U.K.). After 4 h of moderate shaking, supernatants were discarded and cells were washed twice with cold binding buffer and lyzed for 45 min in 1 M NaOH at 37°C. Non-specific binding was determined in duplicate wells preincubated for 30 min with a 100-fold excess of ET-1 (Sigma). Radioactivity in the cellular fractions was measured with a gamma counter (Packard, Meriden, CT). Values for unspecific binding were subtracted from the total bound counts, and values were further normalized by cell number. Cell proliferation was determined by the thymidine incorporation assay (O'Keefe et al., 1988O'Keefe E.J. Chiu M.L. Payne Jr, R.E. Stimulation of growth of keratinocytes by basic fibroblast growth factor.J Invest Dermatol. 1988; 90: 767-769Abstract Full Text PDF PubMed Google Scholar). Cells were seeded in 96-well plates with 104 cells per well in appropriate growth medium. After 24 h, cells were washed once and were incubated with growth medium containing the intended concentration of FBS (0%, 3%, or 10%). After another 24 h, medium was exchanged again and serum concentration was maintained. ET-1 was added in a concentration of 10 nM or 100 nM, whereas the controls received the same volume of 50 mM acetic acid, the solution in which ET-1 was dissolved. After 20 h, cells were incubated for another 4 h with 2.5 µCi per ml 3H-thymidine (68.5 Ci per mmol). Cells were harvested with a cell harvester, and tritium incorporation was measured in a scintillation counter. Synchronization of melanoma cells and normal melanocytes was performed according to a protocol described byOkazawa et al., 1998Okazawa M. Shiraki T. Ninomiya H. Kobayashi S. Masaki T. Endothelin-induced apoptosis of A375 human melanoma cells.J Biol Chem. 1998; 273: 12584-12592Crossref PubMed Scopus (99) Google Scholar. Growth medium for cell synchronization was always prewarmed to 37°C and growth plates were handled outside the cell incubator only for short periods of time in order to avoid cell cycle interruption. Melanoma cells were seeded in 24-well plates (10,000–30,000 cells per well). The seeding density was dependent on the proliferation and cell shape of the respective cell line; a seeding density was chosen such that cells were still subconfluent at the end of the experiment. Normal melanocytes were seeded in six-well plates (2 × 105 cells per well). The utmost care was taken over seeding really equal cell numbers. On the following day, synchronization was started by addition of thymidine to a final concentration of 2.5 mM. The thymidine block was released after 16 h by washing the cells and further incubation with growth medium containing 25 µM deoxycytidine. The second thymidine block was started after another 9 h by washing the cells and again treatment with 2.5 mM thymidine. The second block was released also after 16 h by washing the cells and incubation in growth medium containing 1% FBS (melanoma cells) and 25 µM deoxycytidine. Treated cells received ET-1 whereas control cells received the same volume of ET solution buffer. Apoptosis rates were measured of synchronized cells (described above) as well as of nonsynchronized cells 24 h after stimulation with ET-1 by using the cell death detection enzyme-linked immunosorbent assay (ELISA) (Roche Diagnostics, Mannheim, Germany), which detects mononucleosomes and oligonucleosomes formed in apoptotic cells; the protocol has been described earlier (Wieder et al., 1998Wieder T. Orfanos C.E. Geilen C.C. Induction of ceramide-mediated apoptosis by the anticancer phospholipid analog hexadecylphosphocholine.J Biol Chem. 1998; 273: 11025-11031Crossref PubMed Scopus (143) Google Scholar). Nonsynchronized cells were seeded in 24-well and six-well plates, respectively, as described above for synchronized cells. ELISA values of melanoma cells were normalized by confluence of the cultures, and ELISA values of normal melanocytes were normalized by cell numbers that had been determined in parallel dishes. Relative apoptotic rates were calculated as the ratio of the normalized values from treated and control cells. For statistical analysis, individual values (single wells) of a given experiment were normalized by the mean value of untreated cells in this experiment. Thus, values of independent experiments could be combined in groups. To demonstrate statistical significance of enhanced cell proliferation or reduced apoptosis due to ET-1 treatment, Student's t test was applied (unpaired and heteroskedastic). To investigate the effects of ET-1 on cell proliferation, thymidine incorporation was determined in normal melanocytes as well as in melanoma cell lines after incubation with 10 nM and 100 nM ET-1, respectively. As shown in Table II, a strong proliferation increase after treatment with 10 nM ET-1 was observed for two independent cultures of normal human melanocytes (6.3-fold ± 0.3 and 3.9-fold ± 0.8, respectively); application of 100 nM ET-1 did not further increase proliferation. We investigated the proliferation effect in SK-Mel-19 characterized by undiminished ET(B)-R expression and in SK-Mel-13 characterized by moderate expression of ET(B)-R. Both melanoma cell lines showed nearly no response when cultured in standard growth medium containing 10% FBS. As normal melanocytes were routinely grown serum-free, we further investigated the influence of FBS on the growth effects caused by ET. The growth effects were slightly increased to 1.2-fold ± 0.1 (SK-Mel-19) and to 1.09-fold ± 0.05 (SK-Mel-13) when cells were incubated with 3% FBS for 24 h before starting ET treatment, but further decrease of FBS concentration to 0% did not enhance the growth effect. Also, preincubation of normal melanocytes with growth medium containing 10% FBS did not abolish the growth response of normal melanocytes.Table IIEffects of ET-1 on cell proliferationaProliferation rates were determined by thymidine incorporation; values represent the ratio of treated versus untreated cells (mean values of a minimum of three experiments and standard deviation).CellsbTwo independent cultures of normal human melanocytes (NHM-A and NHM-B) and four melanoma cell lines (SK-Mel-19, SK-Mel-13, MeWo, Bro) were investigated.%FBScStandard growth medium of melanoma cells contained 10% FBS whereas standard melanocyte growth medium was serum-free. To investigate whether the missing response of melanoma cells to ET was due to the concentration of FBS in the growth medium, melanoma cells were also grown with 3% FBS and serum-free, and NHM-B were also grown with 10% FBS.10 nM ET-1100nM ET-1MeanSDp-valuedp-values were calculated according to Student's t test, and are given when increased thymidine incorporation after ET-1 treatment was statistically significant (NHM, SK-Mel-19, SK-Mel-13).MeanSDp-valuedp-values were calculated according to Student's t test, and are given when increased thymidine incorporation after ET-1 treatment was statistically significant (NHM, SK-Mel-19, SK-Mel-13).NHM-A06.270.28<10-116.710.55<10-NHM-B03.870.82<10-134.010.95<10-12NHM-B102.130.27< 0.0031.710.10< 0.03SKM1901.110.060.850.05< 0.008SKM1931.200.10< 0.0050.860.04< 0.05SKM19101.000.020.880.06SKM1301.070.021.030.22SKM1331.090.05< 0.021.030.01SKM13101.070.050.960.06Bro31.040.021.020.05MeWo31.020.021.010.03a Proliferation rates were determined by thymidine incorporation; values represent the ratio of treated versus untreated cells (mean values of a minimum of three experiments and standard deviation).b Two independent cultures of normal human melanocytes (NHM-A and NHM-B) and four melanoma cell lines (SK-Mel-19, SK-Mel-13, MeWo, Bro) were investigated.c Standard growth medium of melanoma cells contained 10% FBS whereas standard melanocyte growth medium was serum-free. To investigate whether the missing response of melanoma cells to ET was due to the concentration of FBS in the growth medium, melanoma cells were also grown with 3% FBS and serum-free, and NHM-B were also grown with 10% FBS.d p-values were calculated according to Student's t test, and are given when increased thymidine incorporation after ET-1 treatment was statistically significant (NHM, SK-Mel-19, SK-Mel-13). Open table in a new tab The effects of FBS alone on proliferation of melanoma cells were investigated for SK-Mel-13. Cells that had been propagated with 10% FBS received a medium exchange with 0%, 3%, or 10% FBS, and tritium incorporation was determined after 48 h, as had been performed in the proliferation experiments. Reduction of FBS concentration from 10% to 3% resulted in a reduction of thymidine incorporation to 72% (p <0.002), and reduction from 10% to 0% resulted in reduced thymidine incorporation of 57% (p <0.0001). Other cell lines and normal melanocytes showed a comparable tendency. Two cell lines characterized by weak expression of ET(B)-R (MeWo) or extremely weak expression of ET(A)-R (Bro) were also examined with 3% FBS, and showed no significant growth response. Unlike normal melanocytes, incubation with 100 nM ET-1 had no growth effect on SK-Mel-13, MeWo, and Bro but resulted in growth inhibition for SK-Mel-19 (Table II). The influence of ET-1 on basic apoptotic rates was investigated in normal human melanocytes and in seven melanoma cell lines characterized by different expression levels of ET receptors (Table I). A first series of experiments performed with SK-Mel-19 revealed no significant difference between treatment with 10 nM and 100 nM ET-1 (data not shown); for the following experiments, 100 nM ET was applied. As proapoptotic effects by ET had been reported earlier for the melanoma cell line A-375 only after cell synchronization (Okazawa et al., 1998Okazawa M. Shiraki T. Ninomiya H. Kobayashi S. Masaki T. Endothelin-induced apoptosis of A375 human melanoma cells.J Biol Chem. 1998; 273: 12584-12592Crossref PubMed Scopus (99) Google Scholar), we investigated both nonsynchronized melanoma cells and normal melanocytes as well as cells after synchronization. To rule out the influence of possibly increased cell numbers on the apoptosis values, we determined cell numbers of SK-Mel-19 and of normal melanocytes in parallel with the apoptosis assays. It turned out that cell numbers of normal melanocytes were significantly increased after treatment with ET-1 for 24 h, whereas there was no change in cell numbers of SK-Mel-19 melanoma cells (Table III). Consequently, we normalized all apoptosis values of normal melanocytes by cell numbers. For SK-Mel-19, normalization to actual cell numbers did not lead to any change in the apoptosis values obtained. As other melanoma cell lines responded even less to ET-1 with respect to proliferation (Table II), normalization to cell numbers was not necessary for melanoma cells.Table IIIDecreased cell numbers for normal melanocytes after ET-1 treatmentCellsaCell numbers after ET-1 treatment were determined for six independent melanocyte cultures (NHM) as well as for the melanoma cell line SK-MEL-19 characterized by undiminished expression of ET(B)-R.Synchr.bCells were investigated both without (–) as well as after cell synchronization (+).Cell numbercCell numbers were counted after treatment with 100 nM ET-1 and were compared to untreated cells. Mean values are given of the ratio of treated versus untreated cells (+/–).SDdStandard deviation (SD)p-valueseIncreased cell numbers in NHM cultures after ET-1 treatment were statistically significant; p-values were calculated according to Student's t test.NHM–1.350.28< 0.02NHM+1.220.19< 0.003SKM-191.000.04SKM-19+0.980.07a Cell numbers after ET-1 treatment were determined for six independent melanocyte cultures (NHM) as well as for the melanoma cell line SK-MEL-19 characterized by undiminished expression of ET(B)-R.b Cells were investigated both without (–) as well as after cell synchronization (+).c Cell numbers were counted after treatment with 100 nM ET-1 and were compared to untreated cells. Mean values are given of the ratio of treated versus untreated cells (+/–).d Standard deviation (SD)e Increased cell numbers in NHM cultures after ET-1 treatment were statistically significant; p-values were calculated according to Student's t test. Open table i" @default.
• W2005929145 created "2016-06-24" @default.
• W2005929145 creator A5002457623 @default.
• W2005929145 creator A5029406176 @default.
• W2005929145 creator A5034016144 @default.
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• W2005929145 date "2002-09-01" @default.
• W2005929145 modified "2023-09-23" @default.
• W2005929145 title "Endothelin-1 Decreases Basic Apoptotic Rates in Human Melanoma Cell Lines" @default.
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