17β-estradiol, Progesterone, and Dihydrotestosterone Suppress the Growth of Human Melanoma by Inhibiting Interleukin-8 Production
2001; Elsevier BV; Volume: 117; Issue: 2 Linguagem: Inglês
10.1046/j.1523-1747.2001.01422.x
ISSN1523-1747
AutoresNaoko Kanda, Shinichi Watanabe,
Tópico(s)Mast cells and histamine
ResumoWe studied the effects of 17β-estradiol, progesterone, and dihydrotestosterone on in vitro growth of human metastatic melanoma. Each sex hormone inhibited the growth of melanoma receptor-dependently; 17β-estradiol inhibited 3H-thymidine uptake of estrogen receptor-positive WM266-4 and NM26, but not that of the receptor-negative HS15. Progesterone inhibited 3H-thymidine uptake of progesterone receptor-positive WM266-4 and HS15, but not that of the receptor-negative NM26. Dihydrotestosterone inhibited 3H-thymidine uptake of androgen receptor-positive HS15 and NM26, but not that of the receptor-negative WM266-4. The growth inhibition by each hormone was counteracted by the respective hormone receptor antagonist. The combination of more than two hormones neither gave additive nor synergistic growth inhibition. The growth inhibition by each sex hormone was counteracted by interleukin-8 but not by the other growth factors. Each sex hormone reduced the constitutive interleukin-8 secretion and mRNA levels in the respective receptor-positive melanoma but not in the receptor-negative melanoma. Transient transfection showed that each sex hormone inhibited the constitutive chloramphenicol acetyltransferase expression driven by interleukin-8 promoter in the respective receptor-positive melanoma but not in the receptor-negative melanoma. Transfection with a series of 5′-deleted interleukin-8 promoter/chloramphenicol acetyltransferase reporter constructs demonstrated that the sequences between - 98 and - 63 bp on interleukin-8 promoter may be involved in the transcriptional repression. These data suggest that 17β-estradiol, progesterone, and dihydrotestosterone suppress the growth of melanoma by inhibiting interleukin-8 production in a receptor-dependent manner. We studied the effects of 17β-estradiol, progesterone, and dihydrotestosterone on in vitro growth of human metastatic melanoma. Each sex hormone inhibited the growth of melanoma receptor-dependently; 17β-estradiol inhibited 3H-thymidine uptake of estrogen receptor-positive WM266-4 and NM26, but not that of the receptor-negative HS15. Progesterone inhibited 3H-thymidine uptake of progesterone receptor-positive WM266-4 and HS15, but not that of the receptor-negative NM26. Dihydrotestosterone inhibited 3H-thymidine uptake of androgen receptor-positive HS15 and NM26, but not that of the receptor-negative WM266-4. The growth inhibition by each hormone was counteracted by the respective hormone receptor antagonist. The combination of more than two hormones neither gave additive nor synergistic growth inhibition. The growth inhibition by each sex hormone was counteracted by interleukin-8 but not by the other growth factors. Each sex hormone reduced the constitutive interleukin-8 secretion and mRNA levels in the respective receptor-positive melanoma but not in the receptor-negative melanoma. Transient transfection showed that each sex hormone inhibited the constitutive chloramphenicol acetyltransferase expression driven by interleukin-8 promoter in the respective receptor-positive melanoma but not in the receptor-negative melanoma. Transfection with a series of 5′-deleted interleukin-8 promoter/chloramphenicol acetyltransferase reporter constructs demonstrated that the sequences between - 98 and - 63 bp on interleukin-8 promoter may be involved in the transcriptional repression. These data suggest that 17β-estradiol, progesterone, and dihydrotestosterone suppress the growth of melanoma by inhibiting interleukin-8 production in a receptor-dependent manner. androgen receptor basic fibroblast growth factor chloramphenicol acetyl transferase dihydrotestosterone 17 β-estradiol estrogen receptor progesterone receptor growth-regulated protein platelet-derived growth factor It has long been suggested that sex hormones modulate the growth of malignant melanoma. Mutually conflicting data are reported, however; melanomas seemed to metastasize more slowly in women than in men (Shaw et al., 1978Shaw H.M. Milton G.W. Farrago G. McCarthy W.H. Endocrine influences on survival from malignant melanoma.Cancer. 1978; 42: 669-677Crossref PubMed Scopus (120) Google Scholar), and survival after metastasis was longer in women than in men (Rampen, 1980Rampen F. Malignant melanoma: sex differences in survival after evidence of distant metastasis.Br J Cancer. 1980; 42: 52-57Crossref PubMed Scopus (30) Google Scholar). These data indicate the growth inhibition by estrogen or progesterone and/or growth stimulation by androgens on melanoma. In contrast, for stage II melanoma, a lower survival rate was observed in pregnant women than in nonpregnant women with melanoma (Shiu et al., 1976Shiu M.H. Schottenfeld D. Maclean B. Fortner J.G. Adverse effect of pregnancy on melanoma. A reappraisal.Cancer. 1976; 37: 181-187Crossref PubMed Scopus (118) Google Scholar). Early studies also reported the occurrence, rapid enlargement, or frequent metastasis of melanoma during pregnancy (Pack and Scharnagel, 1951Pack G.T. Scharnagel M. The prognosis for malignant melanoma in the pregnant woman.Cancer. 1951; 4: 324-334Crossref PubMed Scopus (161) Google Scholar). These reports suggest the growth stimulation by estrogen and/or progesterone on melanoma; however, it is still controversial if pregnancy affects the growth of melanoma.Holly, 1986Holly E.A. Melanoma and pregnancy.Recent Results Cancer Res. 1986; 102: 118-126Crossref PubMed Scopus (15) Google Scholar summarized the etiologic studies and reported that 10 of 11 studies showed no survival difference between women with melanoma associated with pregnancy and those with no association, indicating few deleterious effects of pregnancy on the survival.Wong et al., 1989Wong J.H. Sterns E.H. Kopald K.H. Nizze J.A. Morton D.L. Prognostic significance of pregnancy in stage I melanoma.Arch Surg. 1989; 124: 1227-1231Crossref PubMed Scopus (78) Google Scholar,Mackie et al., 1991Mackie R.M. Bufalino R. Morabito A. Sutherland C. Cascinelli N. For the world health organization melanoma programme: Lack of effect of pregnancy on the outcome of melanoma.Lancet. 1991; 337: 653-655Abstract PubMed Scopus (141) Google Scholar, andDriscoll et al., 1993Driscoll M.S. Grin-Jorgensen C.M. Grant-Kels J.M. Does pregnancy influence the prognosis of malignant melanoma?.J Am Acad Dermatol. 1993; 29: 6119-6630Google Scholar also suggested that pregnancy may have no effects on the outcome of patients with melanoma. Various experimental results for the hormonal growth regulation on melanoma are confusing and inconclusive; estrogen in vivo inhibited the growth of hamster HM-1 melanoma (Schleicher et al., 1987Schleicher R.L. Hitsberger M.H. Beattie C.W. Inhibition of hamster growth by estrogen.Cancer Res. 1987; 47: 453-459PubMed Google Scholar) or human melanoma UISO-MEL-2 (Feucht et al., 1988Feucht K.A. Walker M.J. Das Gupta T.K. Beattie C.W. Effect of 17 β-estradiol on the growth of estrogen receptor-positive human melanoma in vitro and in athymic mice.Cancer Res. 1988; 48: 7093-7101PubMed Google Scholar) grafted in athymic mice. Melanoma B16 grew more slowly in normal female mice than oophorectomized female or male mice (Proctor et al., 1976Proctor J.W. Auclair B.G. Stokowski L. Brief communication: Endocrine factors and the growth and spread of B16 melanoma.J Natl Cancer Inst. 1976; 57: 1197-1198Crossref PubMed Scopus (40) Google Scholar). These data support the inhibitory effect of estrogen on the in vivo growth of melanoma. Some data denied the growth-regulatory effect of estrogen, however; estrogen did not alter the in vitro growth of S91 mouse melanoma B line cells or human melanoma cell lines UISO-MEL-1, 2, and 4 (Cobb and McGrath, 1974Cobb J.P. McGrath A. Brief communication: In vitro effects of melanocyte-stimulating hormone, adrenocorticotropic hormone, 17β-estradiol, or testosterone propionate on Cloudman S91 melanoma cells.J Natl Cancer Inst. 1974; 52: 567-570Crossref PubMed Scopus (19) Google Scholar;Feucht et al., 1988Feucht K.A. Walker M.J. Das Gupta T.K. Beattie C.W. Effect of 17 β-estradiol on the growth of estrogen receptor-positive human melanoma in vitro and in athymic mice.Cancer Res. 1988; 48: 7093-7101PubMed Google Scholar), and there were no differences in the growth rate of B16 between pregnant and nonpregnant female mice (Proctor et al., 1976Proctor J.W. Auclair B.G. Stokowski L. Brief communication: Endocrine factors and the growth and spread of B16 melanoma.J Natl Cancer Inst. 1976; 57: 1197-1198Crossref PubMed Scopus (40) Google Scholar). On the other hand, several studies suggest the growth-stimulatory effects of estrogen; estrogen enhanced in vivo growth and lung metastasis of B16 in mice in a concentration-dependent manner (Lopez et al., 1978Lopez R.E. Bhakoo H. Paolini N.S. Rosen F. Holyoke E.D. Goldrosen M.H. Effect of estrogen on the growth of B-16 melanoma.Surg Forum. 1978; 29: 153-154PubMed Google Scholar). Melanoma grew larger in normal female hamsters than in oophorectomized females (Rosenberg et al., 1963Rosenberg J.C. Assimacopoulos C. Rosenerg S.A. The malignant melanoma of hamsters: III. Effects of sex and castration on the growth of the transplanted tumor.Ann N Y Acad Sci. 1963; 100 (296): 297PubMed Google Scholar). Thus the hormonal growth regulation on melanoma seems highly complicated and may involve a variety of elements in melanoma itself and tumor-surrounding or distant tissues or organs. One of such elements is the presence or absence of the respective hormone receptors. The incidence of estrogen receptor (ER) is reported to be 9–79% (Walker et al., 1987Walker B.J. Beattie C.W. Patel M.K. Ronan S.M. Das Gupta T.K. Estrogen receptor in malignant melanoma.J Clin Oncol. 1987; 5: 1256-1261Crossref PubMed Scopus (65) Google Scholar;Cohen et al., 1990Cohen C. DeRose P.B. Campbell W.G. Schlosnagle D.C. Sgoutas D. Estrogen receptor status in malignant melanoma.Am J Dermatopathol. 1990; 12: 562-564Crossref PubMed Scopus (42) Google Scholar) and that of progesterone receptor (PR) is 21–44% (Neifeld and Lippman, 1980Neifeld J.P. Lippman M.E. Steroid hormone receptors and melanoma.J Invest Dermatol. 1980; 74: 379-381Crossref PubMed Scopus (61) Google Scholar;Karakosis et al., 1980Karakosis C.P. Lopez R.E. Bhakoo H.S. Rosen F. Moore R. Carison M. Estrogen and progesterone receptors and tamoxifen in malignant melanoma.Cancer Treat Rep. 1980; 64: 819-827PubMed Google Scholar), and that of androgen receptor (AR) is 15–17% (Neifeld and Lippman, 1980Neifeld J.P. Lippman M.E. Steroid hormone receptors and melanoma.J Invest Dermatol. 1980; 74: 379-381Crossref PubMed Scopus (61) Google Scholar) in melanoma by radiolabeled ligand binding assays using dextran-coated charcoal. Owing to the high false positivity of the binding assays (Cohen et al., 1990Cohen C. DeRose P.B. Campbell W.G. Schlosnagle D.C. Sgoutas D. Estrogen receptor status in malignant melanoma.Am J Dermatopathol. 1990; 12: 562-564Crossref PubMed Scopus (42) Google Scholar), however, different methods have been recently used for the detection of hormone receptors; western blotting (Swami et al., 2000Swami S. Krishnan A.V. Feldman D. 1a,25-dihydroxyvitamin D3 down-regulates estrogen receptor abundance and suppresses estrogen actions in MCF-7 human breast cancer cells.Clin Cancer Res. 2000; 6: 3371-3379PubMed Google Scholar), immunohistochemical methods (Cohen et al., 1990Cohen C. DeRose P.B. Campbell W.G. Schlosnagle D.C. Sgoutas D. Estrogen receptor status in malignant melanoma.Am J Dermatopathol. 1990; 12: 562-564Crossref PubMed Scopus (42) Google Scholar;Duncan et al., 1994Duncan L.M. Travers R.L. Koerner F.C. Mihm Jr, M.C. Sober A.J. Estrogen and progesterone receptor analysis in pregnancy-associated melanoma: absence of immunohistochemically detectable hormone receptors.Hum Pathol. 1994; 25: 36-41Abstract Full Text PDF PubMed Scopus (40) Google Scholar), or enzyme immunoassays (Kuenen-Boumeester et al., 1996Kuenen-Boumeester V. van der Kwast T.H. Claassen C.C. Look M.P. Liem G.S. Klijn J.G.M. Henzen-Logmans S.C. The clinical significance of androgen receptors in breast cancer and their relation to histological and cell biological parameters.Eur J Cancer. 1996; 32: 1560-1565Abstract Full Text PDF Scopus (106) Google Scholar). Even with the improved methods, however, different papers still report different results on the incidence of hormone receptor, possibly because different laboratories may use different anti-hormone receptor antibodies, or because the presence or absence of hormone receptor may depend on tumor location in the whole body or tumor activation status.Ferno et al., 1987Ferno M. Borg A. Ingvar C. Jonsson P.E. Estrogen receptor and binding site for estramustine in metastatic malignant melanoma.Anticancer Res. 1987; 7: 741-744PubMed Google Scholar reported that ER was positive in 56% of metastatic melanoma by enzyme immunoassay, whereasCohen et al., 1990Cohen C. DeRose P.B. Campbell W.G. Schlosnagle D.C. Sgoutas D. Estrogen receptor status in malignant melanoma.Am J Dermatopathol. 1990; 12: 562-564Crossref PubMed Scopus (42) Google Scholar showed that ER was negative in all 33 primary and metastatic melanoma by immunohistochemistry. Previous studies support that metastatic melanoma constitutively produces a variety of growth factors and thus regulates its own growth in an autocrine manner (Herlyn, 1990Herlyn M. Human melanoma: Development and progression.Cancer Metastasis Rev. 1990; 9: 101-112Crossref PubMed Scopus (155) Google Scholar). The mRNA or protein expression in melanoma is reported for basic fibroblast growth factor (bFGF), interleukin (IL) -8, melanocyte growth stimulatory activity/growth-regulated protein (GRO) -α platelet-derived growth factor (PDGF), etc. (Herlyn, 1990Herlyn M. Human melanoma: Development and progression.Cancer Metastasis Rev. 1990; 9: 101-112Crossref PubMed Scopus (155) Google Scholar), and the pattern of the expression is heterogeneous among various melanoma cells. It is thus hypothesized that sex hormones may upregulate or downregulate the autocrine production of growth factors in certain melanoma cells and thus regulate their growth. In this study, we aimed to examine the growth-regulatory effects of sex hormones, 17β-estradiol (E2), progesterone, and dihydrotestosterone (DHT) in human metastatic melanoma cell lines. We further examined the mechanism for the growth-regulatory effects of these sex hormones, focusing on their effects on autocrine production of growth factors. Metastatic melanoma cell line WM266-4 obtained from a female patient was purchased from Dainippon Pharmaceutical (Osaka, Japan). HS15 and NM26 were removed from metastatic lymph nodes of male patients, and were established by a monolayer system as described (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;Baker et al., 1986Baker F.L. Spitzer G. Ajani J.A. et al.Drug and radiation sensitivity measurements of successful primary monolayer culturing of human tumor cells using cell-adhesive matrix and supplemented medium.Cancer Res. 1986; 46: 1263-1274PubMed Google Scholar). Briefly, biopsy specimens were dissected free of adherent normal tissues, and finely minced. The resulting cell suspensions were washed, resuspendend in culture medium, then inoculated into 35 mm diameter dishes, cultured at 37°C in a humidified atmosphere of 5% CO2, and fed twice weekly. Confluent culture was trypsinized, and expanded into larger dishes or flasks. The subculture was performed once a week. Human breast cancer MCF-7 cells were purchased from Dainippon. These cell lines were cultured in Dulbecco's modified Eagle medium (Gibco-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco-BRL), 1% nonessential amino acids, 1 mM sodium pyruvate (ICN Biomedicals, Aurora, OH), and 100 U per ml penicillin G, 100 μg per ml streptomycin, 0.25 μg per ml amphotericin B (Gibco-BRL). Human prostate cancer LNCaP cells were purchased from Dainippon, and were maintained in RPMI 1640 (GIBCO-BRL) supplemented with 10% fetal bovine serum. E2, 17α-estradiol, progesterone, pregnenolone, DHT, and β-dihydrotestosterone were purchased from Sigma (St Louis, MO). ICI 182,780, bicalutamide were obtained from Zeneca Pharmaceuticals (Macclesfield, U.K.). RU486 was from Schering AG (Berlin, Germany). These agents were dissolved in ethanol as 10 mM stock solution and were kept in the dark until used. Recombinant human IL-8 was from Sigma. Recombinant human GRO-α was from Pepro Tech EC (London, U.K.). Mouse IgG monoclonal anti-human IL-8 antibody was from BioSource International (Camarillo, CA), and was specific to natural and recombinant human IL-8, with no cross-reactivity to human or mouse GRO-α, GRO-β, or GRO-γ. Human recombinant PDGF-AB was from Carbiochem-Novabiochem Corp. (San Diego CA). Recombinant human IL-6 was from Boehringer Mannheim (Indianapolis, IN). Recombinant human bFGF was from Becton Dickinson (San Jose, CA). Control mouse IgG was from Dako Corp. (Carpinteria, CA). Melanoma cells were harvested and lyzed. The cytosolic fractions were separated, and used for radiolabeled ligand binding assays as described (Feucht et al., 1988Feucht K.A. Walker M.J. Das Gupta T.K. Beattie C.W. Effect of 17 β-estradiol on the growth of estrogen receptor-positive human melanoma in vitro and in athymic mice.Cancer Res. 1988; 48: 7093-7101PubMed Google Scholar;Pottratz et al., 1994Pottratz S.T. Bellido T. Mocharia H. Crabb D. Manolagas S.C. 17β-estradiol inhibits expression of human interleukin-6 promoter-receptor constructs by a receptor-dependent mechanism.J Clin Invest. 1994; 93: 944-950Crossref PubMed Scopus (244) Google Scholar). The protein concentration of cytosolic preparation was determined using a Bio-Rad protein assay kit (Hercules, CA). Aliquots of the fractions were incubated at 4°C for 3 h with 0.05–1.0 nM 3H-E2 (Amersham Corp., Arlington Heights, IL), 3H-R5020 (New England Nuclear, Cambridge, MA), or 3H-R1881 (New England Nuclear) for ER, PR, or AR measurements, respectively. One hundred-fold excess of nonradioactive hormone was used to correct for nonspecific binding. Bound and free hormones were separated using dextran-coated charcoal, and specific binding was determined as described (Feucht et al., 1988Feucht K.A. Walker M.J. Das Gupta T.K. Beattie C.W. Effect of 17 β-estradiol on the growth of estrogen receptor-positive human melanoma in vitro and in athymic mice.Cancer Res. 1988; 48: 7093-7101PubMed Google Scholar). Binding constants, Kd and Bmax, were calculated according to the method of Scatchard (Scatchard, 1949Scatchard G. The attractions of proteins for small molecules and ions.Ann N Y Acad Sci. 1949; 51: 660-672Crossref Scopus (17561) Google Scholar). Aliquots (50 μg protein) of the extracts from melanoma, MCF-7, and LNCaP cells were resolved by sodium dodecyl sulfate–polyacrylamide (8%) gel electrophoresis under nonreducing conditions. The proteins were electrotransferred on to nitrocellulose membranes as described (Zhu et al., 1999Zhu W. Smith A. Young Y.F. A nonsteroidal anti-inflammatory drug, flufenamic acid, inhibits the expression of the androgen receptor in LNCaP cells.Endocrinology. 1999; 140: 5451-5454Crossref PubMed Google Scholar;Swami et al., 2000Swami S. Krishnan A.V. Feldman D. 1a,25-dihydroxyvitamin D3 down-regulates estrogen receptor abundance and suppresses estrogen actions in MCF-7 human breast cancer cells.Clin Cancer Res. 2000; 6: 3371-3379PubMed Google Scholar). After blocking with 5% nonfat dry milk, membranes were probed for 2 h with 10 μg per sheet primary antibodies: mouse monoclonal IgG1 anti-ERα C-314 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal IgG anti-PR H-190 (Santa Cruz Biotechnology), or rabbit polyclonal IgG anti-AR N-20 (Santa Cruz Biotechnology). The blots were washed and incubated for 60 min with second antibodies: peroxidase-conjugated goat anti-mouse IgG or anti-rabbit IgG (Pierce, Rockford, IL). After three washes, immunoreactive bands were detected using an enhanced chemi luminescence kit (Amersham). WM266-4, HS15, and NM26 cells were plated at 5 × 103 cells per well in triplicate in flat-bottomed 96-well plates in 100 μl of culture medium and adhered for 18 h. The medium was discarded and the plates were washed with phosphate-buffered saline three times, then incubated with sex hormones and/or recombinant cytokines at the indicated concentrations in 100 μl per well of serum-free, phenol red-free Dulbecco's modified Eagle medium for 20 h. Then 0.5 μCi per well of 3H-thymidine (Amersham) was added and the cells were incubated for an additional 4 h prior to harvest. The incorporation of 3H-thymidine was assayed by liquid scintillation. To measure the secretion of cytokines, melanoma cells were plated in triplicate at 5 × 104 per well in 24-well plates, adhered overnight, washed, and incubated with sex hormones in 1 ml per well of phenol red-free, serum-free medium for 24 h, and the culture supernatants were harvested and stored at -70°C until used. The activity of IL-6 and IL-8 in the supernatants was measured by an enzyme-linked immunosorbent assay (ELISA) kit (Biosource), that of GRO-α and bFGF was measured by ELISA kits (R&D Systems, Minneapolis, MN), and that of PDGF-AB was measured by ELISA kit (Genzyme Techne, Minneapolis, MN), according to the manufacturers' instructions. The sensitivity of the assay for IL-6, IL-8, GRO-α, bFGF, or PDGF-AB was 2, 5, 10, 3, or 8.4 pg per ml, respectively. For the analysis of cytokine mRNA expression, northern blot was performed as described (Zachariae et al., 1991Zachariae C.O.C. Thestrup-Pedersen K. Matsushima K. Expression and secretion of leukocyte chemotactic cytokines by normal human melanocytes and melanoma cells.J Invest Dermatol. 1991; 97: 593-599Abstract Full Text PDF PubMed Google Scholar). Melanoma cells were incubated with sex hormones as above for 8 h, and were harvested. Total RNA was extracted from the harvested cells by guanidium isothiocyanate method (Ultraspec, Houston, TX). RNA (25 μg) was electrophoresed in a 1% agarose gel and was transferred to nitrocellulose membranes. Blots were hybridized with a 32P-labeled cDNA probe for human IL-8, which was 0.45 kb EcoRI–EcoRI fragment (Mukaida et al., 1989Mukaida N. Shiroo M. Matsushima K. Genomic structure of the human monocyte-derived neutrophil chemotactic factor IL-8.J Immunol. 1989; 143: 1366-1371PubMed Google Scholar). To control for differences in RNA sample loading and transfer, the blots were also hybridized with a 32P-labeled, 1.8 kb HindIII–HindIII fragment of the gene encoding human β-actin. The membranes were exposed to X-ray films (Hyperfilm MP; Amersham) for 17 h at -80°C. Autoradiograms were scanned using a Molecular Dynamics computing densitometer (model 300 A; Molecular Devices, Menlo Park, CA). IL-8 mRNA levels were normalized to those of β-actin. pCAT3-basic vector carrying two SV40 poly(A) signals, one downstream of the chloramphenicol acetyl transferase (CAT) reporter gene, and the other upstream of the multicloning site was purchased from Promega (Madison, WI). The HincII–HindIII fragment of the genomic IL-8 DNA, which spans nucleotides -546 to +44 bp relative to the transcriptional start site (Mukaida et al., 1989Mukaida N. Shiroo M. Matsushima K. Genomic structure of the human monocyte-derived neutrophil chemotactic factor IL-8.J Immunol. 1989; 143: 1366-1371PubMed Google Scholar), was subcloned into PUC19, treated with appropriate restriction endonucleases and further subcloned into pCAT3-basic vector (Dorn and Derse, 1988Dorn P.L. Derse D. cis- and trans-acting regulation of gene expression of equine infectious anemia virus.J Virol. 1988; 62: 3522-3526Crossref PubMed Google Scholar;Mukaida et al., 1989Mukaida N. Shiroo M. Matsushima K. Genomic structure of the human monocyte-derived neutrophil chemotactic factor IL-8.J Immunol. 1989; 143: 1366-1371PubMed Google Scholar). cDNA encoding human ER (1.8 kb) (Green et al., 1986Green S. Walter P. Kumar V. Krust A. Bornert J.-M. Argos P. Chambon P. Human oestrogen receptor cDNA. sequence, expression and homology to v-erb-A.Nature. 1986; 320: 134-139Crossref PubMed Scopus (1913) Google Scholar), PR (4.4 kb) (Kastner et al., 1990Kastner P. Krust A. Turcotte B. Stropp U. Tora L. Gronemeyer H. Chambon P. Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B.EMBO J. 1990; 9: 1603-1614Crossref PubMed Scopus (1282) Google Scholar), and AR (3.2kb) (Brinkmann et al, 1989) were subcloned into EcoRI site of pSG5 (Stratagene, La Jolla, CA), downstream of T7 promoter as described (Green et al., 1988Green S. Issemann I. Sheer E. A versatile in vivo eukaryotic expression vector for protein engineering.Nucleic Acids Res. 1988; 16: 369Crossref PubMed Scopus (540) Google Scholar) to produce expression plasmids for ER, PR, and AR, respectively. The carboxyl-terminal-truncated mutant plasmids for ER, PR, and AR were generated by cutting the wild-type plasmids with restriction enzymes, inserting oligonucleotides with translation stop codons, and religating the vectors as described (Kastner et al., 1990Kastner P. Krust A. Turcotte B. Stropp U. Tora L. Gronemeyer H. Chambon P. Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B.EMBO J. 1990; 9: 1603-1614Crossref PubMed Scopus (1282) Google Scholar;Simental et al., 1991Simental J.A. Sar M. Lane M.V. French F.S. Wilson E.M. Transcriptional activation and nuclear targeting signals of the human androgen receptor.J Biol Chem. 1991; 266: 510-518Abstract Full Text PDF PubMed Google Scholar;Stein and Yang, 1995Stein B. Yang M.X. Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-kB and C/EBPβ.Mol Cell Biol. 1995; 15: 4971-4979Crossref PubMed Google Scholar). Transfection of melanoma cells was carried out by the calcium coprecipitation method using CellPhect transfection kit (Amersham) according to the manufacturer's instruction. Briefly, 1 d before the experiment, confluent cultures of melanoma cells were trypsinized, and cells were seeded at 1 × 106 cells per 100 mm diameter dish and incubated overnight at 37°C. The cultures were replenished with fresh medium and kept at 37°C for 4 h before transfection. The cells were incubated for an additional 4 h with the DNA-calcium precipitate containing 10 μg pIL-8 CAT. In some experiments, the cells were cotransfected with either 5 μg of empty vector pSG5 or 5 μg of wild-type or mutant hormone receptor expression plasmid together with pIL-8 CAT. The cultures were then glycerol shocked and replenished with fresh medium. After 3 h, the cells were trypsinized and subdivided into 24-well plates at 5 × 104 cells per well and incubated in 1 ml per well of culture medium overnight. This procedure eliminates differences in transfection efficiency as the same construct is used for the transfection of separate cultures. Then the medium was discarded and the cells were washed with phosphate-buffered saline, and maintained in serum-free, phenol red-free medium in the presence or absence of sex hormones at indicated concentrations. After 24 h, the cells were harvested and lyzed by three freeze/thaw cycles. The cell lysate was centrifuged and supernatant was assayed for CAT expression by CAT-ELISA (Roche Diagnostics, Tokyo, Japan) according to the manufacturer's instructions. Total protein amount was measured by a Bio-Rad protein assay kit. The CAT expression was presented as picograms of CAT enzyme synthesized per microgram of total protein. pCAT3-control vector (Promega) containing SV40 early promoter and enhancer sequences was used as a positive control, and promoterless pCAT3-basic vector was used as a negative control. The expression of wild-type or mutant hormone receptors was analyzed by Western blot using cell lysate and specific antibodies. The hormone-binding activity of the expressed receptors was analyzed by radiolabeled ligand binding assays. One-way analysis of variance with Dunnet's multiple comparison test was used for the data in Figure 2. One-way analysis of variance with Scheffe's multiple comparison test was used for the data in Figure 3, Figure 5, Figure 7, and Figure 8, and Table II.Figure 2The inhibitory effects of E2, progesterone, and DHT on the proliferation of melanoma cell lines. (a, d) E2, (b, e) progesterone, (c, f) DHT. WM266-4, HS15, and NM26 cells were incubated with E2, progesterone, or DHT at the indicated concentrations and were pulsed with 3H-thymidine before harvesting as described in Materials and Methods. The data are shown as percentage vs 3H-thymidine uptake of control cultures with medium alone. In parallel experiments, 17α-estradiol (a), pregnenolone (b), or β-dihydrotestosterone (c) was added to the melanoma cells, and 3H-thymidine uptake was analyzed. In (d–f), melanoma cells were treated with E2 (10-9 M) in the presence or absence of indicated concentrations of ICI 182,780, or treated with progesterone (10-9 M) in the presence or absence of indicated concentrations of RU486, or treated with DHT (10-8 M) in the presence or absence of indicated concentrations of bicaltamide. Results represent the mean ± SEM of four separate experiments. *p < 0.05 vs control cultures with medium alone, by analysis of variance with Dunnet's multiple comparison test in (a–c). *p < 0.05 vs cultures with E2, progesterone, or DHT alone, by analysis of variance with Dunnet's multiple comparison test in (d–f). The values of 3H-thymidine uptake in control cultures with medium alone were mean ± SEM (n = 4) 18,536 ± 1523, 15,231 ± 1324, and 10,324 ± 1094 cpm for WM266
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