Endothelin-induced Apoptosis of A375 Human Melanoma Cells
1998; Elsevier BV; Volume: 273; Issue: 20 Linguagem: Inglês
10.1074/jbc.273.20.12584
ISSN1083-351X
AutoresMakoto Okazawa, Takuma Shiraki, Haruaki Ninomiya, Shígeo Kobayashi, Tomoh Masaki,
Tópico(s)melanin and skin pigmentation
ResumoEndothelin-1 (ET-1) inhibited serum-dependent growth of asynchronized A375 human melanoma cells, and the growth inhibitory effect was markedly enhanced when ET-1 was applied to the cells synchronized at G1/S boundary by double thymidine blocks. Flow cytometric analysis revealed that ET-1 did not inhibit the cell cycle progression after the release of the block but caused a significant increase of the hypodiploid cell population that is characteristic of apoptotic cell death. ET-1-induced apoptosis was confirmed by the appearance of chromatin condensation on nuclear staining and DNA fragmentation on gel electrophoresis. The increase in the hypodiploid cell peak was manifest within 16 h of exposure to 5 nm ET-1. Within the same time range, ET-1 caused actin reorganization and drastic morphological changes of the surviving cells from epithelioid to an elongated bipolar shape. These phenotypical changes were preceded by ET-1-induced increase and nuclear accumulation of the tumor suppressor protein p53. All of these effects of ET-1 were mediated by ETB via a pertussis toxin-sensitive G protein. Flow cytometric analysis with fluorescent dye-labeled ET-1 revealed up-regulation of ETB expressed by the cells in G1/early S phases, and overexpression of the receptor protein by cDNA microinjection conferred the responsiveness (both apoptosis and morphological changes) to ET-1 irrespective of the position of the cell in the cell cycle. These results indicated the presence of ETB-mediated signaling pathways to apoptotic cell machinery and cytoskeletal organization. Furthermore, the densities of ETB expressed by individual A375 melanoma cells appeared to be regulated by a cell cycle-dependent mechanism, and the receptor density can be a limiting factor to control the apoptotic and cytoskeletal responses of the cells to ET-1. Although the molecular mechanisms remain to be elucidated, these findings added a new dimension to the diverse biological activities of ETs and also indicated a novel mechanism to control the responsiveness of the cell to the peptides. Endothelin-1 (ET-1) inhibited serum-dependent growth of asynchronized A375 human melanoma cells, and the growth inhibitory effect was markedly enhanced when ET-1 was applied to the cells synchronized at G1/S boundary by double thymidine blocks. Flow cytometric analysis revealed that ET-1 did not inhibit the cell cycle progression after the release of the block but caused a significant increase of the hypodiploid cell population that is characteristic of apoptotic cell death. ET-1-induced apoptosis was confirmed by the appearance of chromatin condensation on nuclear staining and DNA fragmentation on gel electrophoresis. The increase in the hypodiploid cell peak was manifest within 16 h of exposure to 5 nm ET-1. Within the same time range, ET-1 caused actin reorganization and drastic morphological changes of the surviving cells from epithelioid to an elongated bipolar shape. These phenotypical changes were preceded by ET-1-induced increase and nuclear accumulation of the tumor suppressor protein p53. All of these effects of ET-1 were mediated by ETB via a pertussis toxin-sensitive G protein. Flow cytometric analysis with fluorescent dye-labeled ET-1 revealed up-regulation of ETB expressed by the cells in G1/early S phases, and overexpression of the receptor protein by cDNA microinjection conferred the responsiveness (both apoptosis and morphological changes) to ET-1 irrespective of the position of the cell in the cell cycle. These results indicated the presence of ETB-mediated signaling pathways to apoptotic cell machinery and cytoskeletal organization. Furthermore, the densities of ETB expressed by individual A375 melanoma cells appeared to be regulated by a cell cycle-dependent mechanism, and the receptor density can be a limiting factor to control the apoptotic and cytoskeletal responses of the cells to ET-1. Although the molecular mechanisms remain to be elucidated, these findings added a new dimension to the diverse biological activities of ETs and also indicated a novel mechanism to control the responsiveness of the cell to the peptides. The endothelin (ET) 1The abbreviations used are: ET, endothelin; ETB, endothelinB; BSA, bovine serum albumin; [Ca2+] i, intracellular calcium concentration; FCS, fetal calf serum; GFP, green fluorescent protein; G protein, guanyl nucleotide-binding regulatory protein; GPCR, G protein-coupled receptor; hETB, human endothelinB; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; PI, propidium iodide; DMEM, Dulbecco's modified Eagle's medium; PTX, pertussis toxin; SSTR3, somatostatin receptor type 3.1The abbreviations used are: ET, endothelin; ETB, endothelinB; BSA, bovine serum albumin; [Ca2+] i, intracellular calcium concentration; FCS, fetal calf serum; GFP, green fluorescent protein; G protein, guanyl nucleotide-binding regulatory protein; GPCR, G protein-coupled receptor; hETB, human endothelinB; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; PI, propidium iodide; DMEM, Dulbecco's modified Eagle's medium; PTX, pertussis toxin; SSTR3, somatostatin receptor type 3.family of peptides include three isoforms, ET-1, -2, and -3 (1Inoue A. Yanagisawa M. Kimura S. Kasuya Y. Miyauchi T. Goto K. Masaki T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2863-2867Crossref PubMed Scopus (2554) Google Scholar) and exert their effects by binding to specific G protein-coupled receptor (GPCR) subtypes, ETA and ETB (2Arai H. Hori S. Aramori S. Ohkubo H. Nakanishi S. Nature. 1990; 348: 730-732Crossref PubMed Scopus (2501) Google Scholar, 3Sakurai T. Yanagisawa M. Takuwa H. Miyazaki H. Kimura S. Goto K. Masaki T. Nature. 1990; 348: 732-735Crossref PubMed Scopus (2353) Google Scholar). Subsequent to the identification of ET-1 as a potent vasoconstrictive peptide (4Yanagisawa M. Kurihara H. Kimura S. Tomobe Y. Kobayashi M. Mitsui Y. Yazaki Y. Goto K. Masaki T. Nature. 1988; 332: 411-415Crossref PubMed Scopus (10163) Google Scholar), numerous studies described their diverse biological effects on target cells (reviewed in Ref. 5Masaki T. Yanagisawa M. Goto K. Med. Res. Rev. 1992; 12: 391-421Crossref PubMed Scopus (253) Google Scholar), and from these studies has emerged the recognition of ETs as one of the growth factors on definite cell lineages. ET-1 has been reported to stimulate DNA synthesis and proliferation of a variety of cells such as smooth muscle cells, glomerular mesangial cells, osteoblasts, fibroblasts, and mature melanocytes (reviewed in Ref. 6Rubanyi G.M. Polokoff M.A. Pharmacol. Rev. 1994; 46: 325-415PubMed Google Scholar). Because of the predominant expression of ETA by these cell types and also because of the mitogenic signaling by ETA in transfected cells (7Sugawara F. Ninomiya H. Okamoto Y. Miwa S. Mazda O. Katsura Y. Masaki T. Mol. Pharmacol. 1996; 49: 447-457PubMed Google Scholar), it is generally accepted that ETA transmits a positive signal to the cell growth. In contrast, despite the diverse distribution of ETB both in tissues and cell lines, there are only a few reports in the literature that provided circumstantial evidence for the mitogenic signaling transmitted by ETB (8Takagi Y. Fukase M. Tanaka S. Yoshimi H. Tokunaga O. Fujita T. Biochem. Biophys. Res. Commun. 1990; 168: 537-543Crossref PubMed Scopus (58) Google Scholar, 9Vigne P. Marsault R. Breittmayer J.P. Frelin C. Biochem. J. 1990; 266: 415-420Crossref PubMed Scopus (134) Google Scholar). Furthermore, Mallat et al. (10Mallat A. Fouassier L. Préaux A.-M. Gal C.S.-L. Raufaste D. Rosenbaum J. Dhumeaux D. Jouneax C. Mavier P. Lotersztajn S. J. Clin. Invest. 1995; 96: 42-49Crossref PubMed Scopus (113) Google Scholar) revealed ETB-mediated growth suppression of hepatic Ito cells in their myofibroblastic phenotype, demonstrating the ability of ETB to transmit a signal to the cell growth that is completely opposite to that transmitted by ETA. Gene targeting revealed the critical role of ETB in embryonic development of melanocytes (11Hosoda K. Hammer R.E. Richardson J.A. Baynash A.G. Chenug J.C. Gialid A. Yanagisawa M. Cell. 1994; 79: 1267-1276Abstract Full Text PDF PubMed Scopus (882) Google Scholar). Subsequent studies on primary cultured melanocyte precursor cells revealed multiple roles of ETB in growth/differentiation of the cells that apparently differ between the differentiation stages of the cells (12Reid K. Turnley A.M. Maxwell G.D. Kurihara Y. Kurihara H. Barlett P.F. Murphy M. Development. 1996; 122: 3911-3919PubMed Google Scholar). In keeping with these findings, various human melanoma cell lines have been shown to express this receptor subtype (13Yohn J. Smith C. Stevens T. Hoffman T.A. Morelli J.G. Hurt D.L. Yanagisawa M. Kane M.A. Zamora M.R. Biochem. Biophys. Res. Commun. 1994; 201: 449-457Crossref PubMed Scopus (55) Google Scholar, 14Kikuchi K. Nakagawa H. Kadono T. Etoh T. Byers H.R. Mihm M.C. Tamaki K. Biochem. Biophys. Res. Commun. 1996; 219: 734-739Crossref PubMed Scopus (38) Google Scholar), which indicates the possibility that each melanoma is derived from the fully differentiated cell, melanocyte, and retains the characteristics of undifferentiated melanocyte-precursor cells. Recently, two GPCRs for peptide ligands, namely angiotensin II receptor type 2 (ATII2) and somatostatin receptor 3 (SSTR3), have been shown to transmit growth inhibitory effects associated with programmed cell death or apoptosis of the target cells (15Yamada T. Horiuchi M. Dzau V.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 156-160Crossref PubMed Scopus (663) Google Scholar, 16Srikant C.B. Biochem. Biophys. Res. Commun. 1995; 209: 400-407Crossref PubMed Scopus (100) Google Scholar, 17Sharma K. Patel Y.C. Srikant B. Mol. Endocrinol. 1996; 10: 1688-1696Crossref PubMed Scopus (254) Google Scholar). Because of the critical involvement of apoptosis in the cellular differentiation (18Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2383) Google Scholar), given the multiple roles of ETB on growth/differentiation of melanocyte-precursor cells and the heterogeneity of melanoma cell lines, we postulated that ETB may cause a growth inhibitory effect on some of the melanoma cell lines. In the initial screening on the three human melanoma cell lines A375, MeWo and HM3KO, we found that ET-1 caused a growth inhibitory effect only on A375. Therefore, the current study was conducted to characterize the growth inhibitory effect of ET-1 on this cell line. Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were from Life Technologies Inc. (Tokyo, Japan); synthetic human ET-1 and ET-3, BQ123, and BQ788 were from the Peptide Institute (Osaka, Japan); 125I-ET-1 (74 TBq/mmol), [γ-32P]ATP (220 TBq/mmol), FluoroLinkTM Cy2 Reactive Dye-Pack, and Cy2-avidin were from Amersham Pharmacia Biotech (Buckinghamshire, UK); EASYTAGTM Express Protein labeling mix 35S (43.5 TBq/mmol) was from NEN Life Science Products (Tokyo, Japan); pertussis toxin (PTX) was from Funakoshi Co. (Tokyo, Japan); Texas Red-phalloidin was from Molecular Probes (Eugene, OR); mouse monoclonal anti-α-tubulin antibody was from MONOSAN (Uden, Netherlands); mouse monoclonal anti-p53 antibody (DO-1) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All other chemicals were of reagent grade and were obtained commercially. A375 human melanoma cells were obtained from the American Type Culture Collection (Rockville, MD). The cells were routinely maintained in DMEM, 10% FCS at 37 °C in a humidified atmosphere containing 5% CO2. They were passaged twice a week at the ratio of 1:5 to keep an exponentially growing state. The substratum was cell culture grade plasticware (Costar, Cambridge, MA) except for the use of poly-l-lysine-coated glass-bottom culture dishes (MatTek Corp., Ashland, MA) for immunocytochemical experiments. Cell cycle synchronization was achieved by two sequential thymidine blocks according to the procedures described by Steinet al. (19Stein G.S. Stein J.L. Lian J.B. Last T.J. Owen T. McCabe L. Studzinski G.P. Cell Growth and Apoptosis. Oxford University Press, New York1995: 193-203Google Scholar). In brief, cells in the exponential growth phase were exposed to 2.5 mm thymidine in DMEM, 10% FCS for 16 h and then incubated in the fresh medium for 10 h. The cells were once again exposed to 2.5 mm thymidine for 16 h, and the block was released by replacing the medium to fresh DMEM, 1% FCS. Where indicated, the cells were exposed to 400 ng/ml PTX during the last 2 h of the second thymidine block. As an alternative method of cell synchronization, a plant amino acid mimosine was used according to the procedures described by Krek and DeCaprio (20Krek W. DeCaprio J.A. Methods Enzymol. 1995; 254: 114-124Crossref PubMed Scopus (87) Google Scholar). In brief, cells were exposed to 0.1 mm mimosine in DMEM, 10% FCS for 18 h, and the block was released by replacing the medium with fresh DMEM, 1% FCS. Binding assays on attached cells were done according to the procedures described (3Sakurai T. Yanagisawa M. Takuwa H. Miyazaki H. Kimura S. Goto K. Masaki T. Nature. 1990; 348: 732-735Crossref PubMed Scopus (2353) Google Scholar) with slight modifications. In brief, for saturation isotherms, cells at ∼50% confluence in 48-well plates were washed with ice-cold phosphate-buffered saline (PBS) and then incubated at 4 °C for 60 min in 0.25 ml of PBS, 0.2% BSA containing increasing concentrations (5–300 pm) of 125I-ET-1. After washing the cells with PBS, the radioactivity associated with the cells was recovered in 0.1 n NaOH and counted in a γ-counter. Nonspecific binding was defined as the binding in the presence of 100 nm unlabeled ET-1 and was always less than 10% of the total binding capacity. For displacement experiments, the cells were incubated with 100 pm125I-ET-1 and increasing concentrations (100 pm to 1 μm) of an unlabeled peptide. Asynchronous or phase-synchronized cells at ∼50% confluence in 96-well plates were incubated for 24 h in DMEM, 1% FCS containing increasing concentrations of ET-1. The number of viable cells in each well was estimated by measurement of the rate of mitochondrial metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) using a cell proliferation assay kit (Promega, Madison, WI) according to the manufacturer's instructions. For cell cycle analysis, cells were washed with PBS, harvested by trypsinization, fixed in 70% ethanol, and then labeled with propidium iodide (PI) by incubation for 30 min at room temperature in PBS containing 50 μg/ml PI and 1 mg/ml ribonuclease A. The DNA content per nucleus was analyzed by a FACScan flow cytometer (Becton Dickinson Co., Mansfield, MA). For p53 immunostaining, the harvested cells were fixed first in 1% paraformaldehyde and then in 70% ethanol. The cells were labeled with anti-p53 antibody followed by staining with fluorescein isothiocyanate-labeled goat anti-mouse IgG and then subjected to flow cytometry. For Cy2-ET-1 binding, cells were harvested by scraping and fixed in 100% methanol. After washing with PBS, 0.2% BSA, the cells were incubated for 30 min at room temperature in the same medium containing Cy2-ET-1 at the indicated concentrations. The cells were washed with PBS, 0.2% BSA, and the fluorescence associated with each cell was analyzed. Cy2-ET-1 was prepared by using FluoroLinkTM Cy2 ReactiveDye-Pack (Amersham Pharmacia Biotech) as described (21Okamoto Y. Ninomiya H. Tanioka M. Sakamoto A. Miwa S. Masaki T. J. Biol. Chem. 1997; 272 (21586): 21589Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). For Texas Red phalloidin/anti-α-tubulin double staining, cells in poly-l-lysine-coated glass-bottom dishes were washed with PBS, fixed in 3.7% formaldehyde/PBS at room temperature for 10 min, and permeabilized in acetone at −20 °C for 10 min. The following procedures were done at room temperature. After blocking in PBS, 3% BSA, Texas Red-phalloidin and anti-α-tubulin antibody were simultaneously applied for 30 min in the same medium. The cells were further treated with biotinylated anti-mouse IgG followed by Cy2-avidin and then mounted in fluoromount G (Southern Biotechnology Associates, Inc., Birmingham, AL). Fluorescent images of the cells were obtained with a MRC1024 laser-scanning confocal microscope (Bio-Rad, Osaka, Japan). For p53 immunostaining, fixed and permeabilized cells were stained with anti-p53 antibody in PBS for 30 min and then processed in exactly the same way. DNA staining with PI of the fixed cells were done as described above for the flow cytometry. Isolation of DNA and analysis by agarose gel electrophoresis were done as described (22Ramachandra S. Studzinski G.P. Studzinski G.P. Cell Growth and Apoptosis. Oxford University Press, New York1995: 119-142Google Scholar). In brief, cells were lysed by incubation at 50 °C for 12 h in the lysis buffer (10 mm Tris-HCl, pH 8.0, 100 mm NaCl, 25 mm EDTA, 0.5% SDS, and 0.2 mg/ml proteinase K). DNA was extracted once with phenol/chloroform/isoamyl alcohol and twice with chloroform/isoamyl alcohol. After ethanol precipitation and resuspension, 10 μg of DNA samples were subjected to 1.2% agarose gel electrophoresis and visualized under UV light. During the last 7 h of the second thymidine block, the cells in 60-mm dishes were washed with PBS and, after a preincubation in methionine/cysteine (Met/Cys)-free DMEM for 20 min, were pulse-labeled for 1.5 h with [35S]Met/Cys (50 μCi/ml) in the same medium supplemented with 10% FCS. The cells were then chased for 4.5 h in the same medium. All the medium used contained 2.5 mmthymidine to keep the cell cycle blocked. The cells were then incubated in DMEM, 1% FCS with or without ET-1. At the indicated time, the cells were washed in PBS and then harvested in 0.25 ml of the lysis buffer (20 mm Tris-HCl, pH 7.8, 137 mm NaCl, 5 mm EGTA, 1 mm EDTA, 2 mm DTT, 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml pepstatin A, 1% Nonidet P-40, and 1% SDS). p53 in the cell lysate was recovered by immunoprecipitation with anti-p53 antibody and protein A-Sepharose 4B (Zymed, South San Francisco). The recovered proteins were dissolved on 10% SDS-polyacrylamide gel electrophoresis, and the autoradiograph was developed by BAS2000 image analyzer. The entire coding sequence of human ETB (hETB) (23Sakamoto A. Yanagisawa M. Sakurai T. Takuwa Y. Yanagisawa H. Masaki T. Biochem. Biophys. Res. Commun. 1991; 178: 656-663Crossref PubMed Scopus (247) Google Scholar) was subcloned into a mammalian expression vector pcDNA3 (Invitrogen, San Diego) to give pEhETB. pEGFP-N1, an expression plasmid of green fluorescent protein (GFP), was obtained fromCLONTECH (Palo Alto, CA). The plasmid DNAs were prepared at 100 ng/μl in PBS and injected into the cell nucleus using a Zeiss microinjection system (Carl Zeiss, Tokyo, Japan). The relative ET-1 binding capacities of cDNA-injected cells were estimated by Cy5-ET-1 binding assay (21Okamoto Y. Ninomiya H. Tanioka M. Sakamoto A. Miwa S. Masaki T. J. Biol. Chem. 1997; 272 (21586): 21589Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) followed by a densitometric analysis of the fluorescent images. Cy5-ET-1 was used instead of Cy2-ET-1 to discriminate from the GFP fluorescence. In brief, 5 h after the injection, the cells were incubated at 4 °C for 2 h in the binding buffer (140 mm NaCl, 4 mm KCl, 1 mm CaCl2, 1 mmNa2HPO4, 1 mm MgCl2, 25 mm HEPES, pH 7.4, 11.7 mm glucose, 0.1% BSA) containing 10 nm Cy5-ET-1. Unbound ligand was washed, and the fluorescent images (a series of 10 horizontal sections from the cell top to the bottom) were obtained with MRC1024. On each section, the region of interest was set to include individual cells, and the Cy5 fluorescent intensities (256 degrees in each pixel) associated with each cells were summed up to give a parameter that represented the Cy5-ET-1 binding capacities of individual cells. Where necessary, statistical analysis was done by analysis of variance. Melanoma cell lines are classified according to the differentiation stages assessed by morphology, expression of cell-surface marker antigens, and pigmentation (24Houghton A.N. Real F.X. Davis L.J. Cardo C.C. Old L.J. J. Exp. Med. 1987; 164: 812-829Crossref Scopus (140) Google Scholar), and A375 cell line belongs to a relatively undifferentiated class because of the epithelioid shape, expression of HLA-DR antigen, lack of gp100 antigen, and lack of pigmentation. 2M. Okazawa and H. Ninomiya, unpublished observations.Saturation isotherms with 125I-ET-1 revealed that, under the routine culture conditions, the cells express ET-1 binding sites with K d values of 31 ± 1 pm andB max values of 3.2 ± 0.1 × 103 per cell (means ± S.E., n = 3) (Fig. 1 a). The complete and partial displacements of the binding by BQ788 (an ETB-selective antagonist; Ref. 25Ishikawa K. Ihara M. Noguchi K. Mase N. Saeki T. Fukuroda T. Fukami T. Ozaki S. Nagase T. Nishikibe M. Yano M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4892-4896Crossref PubMed Scopus (603) Google Scholar) and BQ123 (an ETA-selective antagonist; Ref. 26Ihara M. Noguchi K. Saeki T. Fukuroda T. Tsuchida S. Kimura S. Fukami T. Ishikawa K. Nishikibe M. Yano M. Life Sci. 1992; 50: 247-255Crossref PubMed Scopus (888) Google Scholar), respectively, indicated the predominant expression of ETB (Fig. 1 b). From the biphasic displacement curve of ET-3 (whose affinity to ETA is lower than that of ET-1), the ratio of the densities of ETA and ETB was roughly estimated to be 1:4 (Fig. 1 b). Initial examination of the effect of ET-1 on A375 cell growth was done by measurement of the mitochondrial metabolism of the dye MTT which depends both on the number and viability of the cells. When applied to asynchronous cells in serum-free DMEM, ET-1 neither stimulates nor inhibits the cell growth within 24 h incubation (data not shown), but it caused a weak but significant inhibition of the FCS (1%)-dependent cell growth (Fig. 2 a). In light of the cell cycle dependence of growth inhibition by somatostatin (16Srikant C.B. Biochem. Biophys. Res. Commun. 1995; 209: 400-407Crossref PubMed Scopus (100) Google Scholar), we tested several synchronization procedures and found that the inhibitory effect of ET-1 was markedly enhanced when ET-1 was applied to the cells synchronized at G1/S boundary by double thymidine blocks (Fig. 2 a). The growth inhibitory effect of ET-1 (5 nm) was completely blocked by BQ788 (1 μm) but not by BQ123 (1 μm) (Fig.2 a). Flow cytometric analysis revealed that ET-1 (5 nm) did not inhibit the FCS (1%)-dependent cell cycle progression after the release of the block up to 24 h, but it caused a significant increase of the hypodiploid cell peak (Fig.2 b) that is a characteristic feature of cells undergoing apoptosis (27Darzynkiewicz Z. Bruno S. Bino G.D. Gorczyca W. Hotz M.A. Lassota P. Traganos F. Cytometry. 1992; 13: 795-808Crossref PubMed Scopus (1897) Google Scholar). Time course analysis showed a 16-h time lag for ET-1 to cause a significant increase in the hypodiploid peak (Fig.2 c). ET-1-induced apoptosis was confirmed by the appearance of chromatin condensation on nuclear staining (Fig.3 a) and that of DNA fragmentation on gel electrophoresis (Fig. 3 b). All of these effects of ET-1 (5 nm) were blocked by BQ788 (1 μm) but not by BQ123 (1 μm) (Figs.2 d and 3 b). Treatment of the cells with PTX prior to the stimulation (400 ng/ml for 2 h) completely abolished the effect of ET-1 (Figs. 2 d and 3 b). In separate experiments using asynchronous cells, flow cytometric analysis failed to detect the hypodiploid peak regardless of the presence or absence of ET-1 (data not shown). Collectively, these results demonstrated that, when A375 cells were synchronized at G1/S boundary and then allowed to proceed the cell cycle, ET-1 inhibited the cell growth by inducing apoptosis and that the effect was transmitted by ETB via a PTX-sensitive G protein.Figure 3ET-1-induced chromatin condensation and DNA fragmentation. a, nuclear staining with PI. Phase-synchronized cells in glass-bottom dishes were incubated for 16 h in DMEM, 1% FCS with or without 5 nm ET-1. The cells were fixed and stained with PI, and fluorescent images were obtained by confocal microscopy. Arrowheads indicate cells with condensed chromatin. b, DNA fragmentation. Phase-synchronized cells were incubated for 16 h in DMEM, 1% FCS. The medium contained no drug (lane 1), 5 nm ET-1 (lane 2), 5 nm ET-1 with 1 μmBQ123 (lane 3), or BQ788 (lane 4). In separate experiments, the cells were treated with PTX (400 ng/ml) during the last 2 h of the second thymidine block and then were incubated for 24 h in DMEM, 1% FCS with 5 nm ET-1 (lane 5). The cellular DNA was extracted and separated on 1.2% agarose gel as described under "Experimental Procedures." Molecular sizes are given on the right (in kilobase pairs). Shown are the representative results from three independent determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Microscopic observation of the culture bed revealed that, when applied to the synchronized cells, ET-1 induced distinct morphological changes of the surviving cells. Within 12 h of stimulation with ET-1 (5 nm), more than 70% of the cells began to extend cytoplasmic processes that contained bundles of filamentous actin (stained with Texas Red-phalloidin) (Fig.4 b). After 24 h stimulation, the cells were apparently bipolar and elongated in shape and lost the bundles of filamentous actin (Fig. 4 c). In contrast, a mesh-like organization of microtubules radiating from the centrosome in control cells (stained with anti-αtubulin antibody) was apparently maintained in ET-1-stimulated cells (Fig. 4,d–f). These effects of ET-1 on the actin reorganization were blocked by BQ788 but not by BQ123 and were also blocked by pretreatment of the cells with PTX (data not shown). In separate experiments using asynchronous cells, less than 5% of the cells in the culture bed underwent the morphological changes in response to ET-1 (5 nm for 24 h) (not shown). Because of the well documented role of the tumor suppressor protein p53 in apoptotic cell death (28Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6673) Google Scholar) and the somatostatin-induced increase of p53 in SSTR3-expressing cells (17Sharma K. Patel Y.C. Srikant B. Mol. Endocrinol. 1996; 10: 1688-1696Crossref PubMed Scopus (254) Google Scholar), we examined the effect of ET-1 on the expression level and subcellular localization of this transcription factor in A375 cells. A375 cells have been reported to express only the wild-type p53 (29Kroumpouzos G. Eberle J. Garbe C. Orfanos C.E. Pigm. Cell Res. 1994; 7: 348-353Crossref PubMed Scopus (20) Google Scholar). When applied to the phase-synchronized cells, ET-1 caused a significant increase in p53 level as revealed by flow cytometry, and time course analysis showed that at least a 12-h exposure was required to cause the increase (Fig.5 a). The ET-1-induced increase was blocked by BQ788 and also by pretreatment of the cells with PTX (Fig. 5 b). Immunocytochemical analysis revealed ET-1-induced nuclear accumulation of p53 (Fig. 5 c) that was blocked by BQ788 and also by pretreatment of the cells with PTX (data not shown). Time course analysis showed that at least 6 h incubation was required for ET-1 to cause the translocation of p53 (data not shown). In an attempt to detect earlier changes in p53 metabolism, we conducted metabolic labeling and immunoprecipitation of the p53 protein. In accordance with the results of flow cytometry, there was no detectable increase in p53 protein level in ET-1-stimulated samples within 3 h incubation (Fig. 5 d). After 2 h incubation, however, there appeared high molecular weight forms of the p53 most likely due to ubiquitination of the molecule (30Shkedy D. Gonen H. Bercovich B. Ciechanover A. FEBS Lett. 1994; 348: 126-130Crossref PubMed Scopus (39) Google Scholar, 31Maki C.G. Huibregtse J.M. Howley P.M. Cancer Res. 1996; 56: 2649-2654PubMed Google Scholar), and ET-1 caused apparent reduction of the high molecular weight forms (Fig. 5 d). Despite repeated trials, we failed to demonstrate an increased survival of 35S-labeled p53 after a long term treatment with ET-1 (16 and 24 h), most likely due to the ET-1-induced apoptosis and hence the decrease of the total number of the cells in a well (data not shown). In separate experiments using asynchronous cells, ET-1 failed to affect the expression level and subcellular localization of p53 regardless of the presence or absence of FCS (data not shown). All of the effects of ET-1 on A375 cells described above were rather specific for the cells that were synchronized at G1/S boundary by double thymidine blocks and then allowed to undergo the cell cycle. In an attempt to explore the cell cycle-dependent modulation of ETB signaling, we examined the differences in receptor densities between the phases of the cell cycle. For this purpose, the receptor densities on individual cells were assessed by the binding of Cy2-labeled ET-1 and flow cytometry. Cy2-ET-1 binding capacities were highest in cells arrested at the G1/S boundary and gradually decreased as the cells underwent the cycle after the release of the block (Fig. 6 a) to reach the level of those in asynchronous cells (not shown) at 24 h. The average Cy2 fluorescent intensities from the phase-synchronized cells were 5.2 ± 1.4-fold (mean ± S.E., n = 3) higher than those from asynchronous cells. This increase in Cy2-ET-1 binding capacities reflected an increase of ETB because the bindi
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