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• W2064034345 abstract "Interleukin (IL)-6 is a pleiotropic cytokine that has been shown to inhibit the growth of early stage and to promote the proliferation of advanced stage melanoma cells in vitro. In patients with metastasizing melanomas, highly increased IL-6 blood levels correlate with a poor response to chemotherapy and a worse overall prognosis, suggesting that IL-6 promotes melanoma progression in vivo. Here, we analyzed the role of IL-6 in melanoma development and progression in a transgenic mouse model. We bred IL-6-deficient mice with MT-ret transgenic animals predisposed for melanomas. While MT-ret transgenic animals develop severe melanosis of the skin and subcutis and subsequent melanomas at an incidence of 80% during their first year of life, MT-ret mice devoid of IL-6 developed preneoplastic melanosis and consecutive melanomas significantly less frequently (47%; P < 0.05). Moreover, the tumors were significantly smaller in the groups of MT-ret mice lacking one (P < 0.05) or both (P < 0.01) copies of the IL-6 gene. Immunoblot analysis revealed that ret transgene expression was not reduced in the skin of mice lacking IL-6, indicating that the observed decrease of melanoma incidence and of tumor sizes was not because of a down-regulation of transgene expression. Taken together, these results indicate that IL-6 enhances both the development of melanoma precursor lesions and the subsequent growth of the resulting tumors in the MT-ret model of melanoma development. Interleukin (IL)-6 is a pleiotropic cytokine that has been shown to inhibit the growth of early stage and to promote the proliferation of advanced stage melanoma cells in vitro. In patients with metastasizing melanomas, highly increased IL-6 blood levels correlate with a poor response to chemotherapy and a worse overall prognosis, suggesting that IL-6 promotes melanoma progression in vivo. Here, we analyzed the role of IL-6 in melanoma development and progression in a transgenic mouse model. We bred IL-6-deficient mice with MT-ret transgenic animals predisposed for melanomas. While MT-ret transgenic animals develop severe melanosis of the skin and subcutis and subsequent melanomas at an incidence of 80% during their first year of life, MT-ret mice devoid of IL-6 developed preneoplastic melanosis and consecutive melanomas significantly less frequently (47%; P < 0.05). Moreover, the tumors were significantly smaller in the groups of MT-ret mice lacking one (P < 0.05) or both (P < 0.01) copies of the IL-6 gene. Immunoblot analysis revealed that ret transgene expression was not reduced in the skin of mice lacking IL-6, indicating that the observed decrease of melanoma incidence and of tumor sizes was not because of a down-regulation of transgene expression. Taken together, these results indicate that IL-6 enhances both the development of melanoma precursor lesions and the subsequent growth of the resulting tumors in the MT-ret model of melanoma development. Interleukin (IL)-6 is a pleiotropic cytokine that induces the acute phase response, stimulates B- and T-lymphocytes, and regulates the growth, differentiation, and death of several cell populations including neurons and melanocytes.1Marz P Otten U Rose-John S Neuronal activities of IL-6-type cytokines often depend on soluble cytokine receptors.Eur J Neurosci. 1999; 11: 2995-3004Crossref PubMed Scopus (110) Google Scholar, 2Kamimura D Ishihara K Hirano T IL-6 signal transduction and its physiological roles: the signal orchestration model.Rev Physiol Biochem Pharmacol. 2003; 149: 1-38Crossref PubMed Google Scholar, 3Kallen KJ The role of transsignalling via the agonistic soluble IL-6 receptor in human diseases.Biochim Biophys Acta. 2002; 1592: 323-343Crossref PubMed Scopus (173) Google Scholar IL-6 promotes the development and progression of plasmacytomas4Kovalchuk AL Kim JS Park SS Coleman AE Ward JM Morse III, HC Kishimoto T Potter M Janz S IL-6 transgenic mouse model for extraosseous plasmacytoma.Proc Natl Acad Sci USA. 2002; 99: 1509-1514Crossref PubMed Scopus (110) Google Scholar and gliomas4Kovalchuk AL Kim JS Park SS Coleman AE Ward JM Morse III, HC Kishimoto T Potter M Janz S IL-6 transgenic mouse model for extraosseous plasmacytoma.Proc Natl Acad Sci USA. 2002; 99: 1509-1514Crossref PubMed Scopus (110) Google Scholar, 5Weissenberger J Loeffler S Kappeler A Kopf M Lukes A Afanasieva TA Aguzzi A Weis J IL-6 is required for glioma development in a mouse model.Oncogene. 2004; 23: 3308-3316Crossref PubMed Scopus (156) Google Scholar in vivo. It is also a growth-promoting factor for human basal cell carcinoma, Kaposi's sarcoma, and prostate cancer cells in vitro.3Kallen KJ The role of transsignalling via the agonistic soluble IL-6 receptor in human diseases.Biochim Biophys Acta. 2002; 1592: 323-343Crossref PubMed Scopus (173) Google Scholar, 6Steiner H Godoy-Tundidor S Rogatsch H Berger AP Fuchs D Comuzzi B Bartsch G Hobisch A Culig Z Accelerated in vivo growth of prostate tumors that up-regulate interleukin-6 is associated with reduced retinoblastoma protein expression and activation of the mitogen-activated protein kinase pathway.Am J Pathol. 2003; 162: 655-663Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar The IL-6 receptor system consists of IL-6 receptor α (IL-6Rα), the primary IL-6 receptor, and the ubiquitous gp130 signal-transducing receptor subunit. Binding of IL-6 to its receptor complex leads to the activation of janus kinases (Jaks) and subsequent phosphorylation, dimerization, and nuclear translocation of signal transducer and activator of transcription 3 (STAT3).2Kamimura D Ishihara K Hirano T IL-6 signal transduction and its physiological roles: the signal orchestration model.Rev Physiol Biochem Pharmacol. 2003; 149: 1-38Crossref PubMed Google Scholar, 7Heinrich PC Behrmann I Haan S Hermanns HM Muller-Newen G Schaper F Principles of interleukin (IL)-6-type cytokine signalling and its regulation.Biochem J. 2003; 374: 1-20Crossref PubMed Scopus (2479) Google Scholar STAT3 promotes tumor progression by regulating the expression of genes involved in growth control such as c-myc, anti-apoptosis [Bcl-x(L) and Mcl-1] and angiogenesis (vascular endothelial growth factor).8Catlett-Falcone R Landowski TH Oshiro MM Turkson J Levitzki A Savino R Ciliberto G Moscinski L Fernandez-Luna JL Nunez G Dalton WS Jove R Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells.Immunity. 1999; 10: 105-115Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar, 9Bromberg J Signal transducers and activators of transcription as regulators of growth, apoptosis and breast development.Breast Cancer Res. 2000; 2: 86-90Crossref PubMed Scopus (99) Google Scholar, 10Niu G Wright KL Huang M Song L Haura E Turkson J Zhang S Wang T Sinibaldi D Coppola D Heller R Ellis LM Karras J Bromberg J Pardoll D Jove R Yu H Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis.Oncogene. 2002; 21: 2000-2008Crossref PubMed Scopus (1011) Google Scholar, 11Kiuchi N Nakajima K Ichiba M Fukada T Narimatsu M Mizuno K Hibi M Hirano T STAT3 is required for the gp130-mediated full activation of the c-myc gene.J Exp Med. 1999; 189: 63-73Crossref PubMed Scopus (331) Google Scholar In a recent study, STAT3 was found to be constitutively activated in some, but not all human melanoma cell lines and tumor specimens analyzed.12Niu G Bowman T Huang M Shivers S Reintgen D Daud A Chang A Kraker A Jove R Yu H Roles of activated Src and Stat3 signaling in melanoma tumor cell growth.Oncogene. 2002; 21: 7001-7010Crossref PubMed Scopus (370) Google Scholar However, blocking experiments revealed that STAT3 activation in these cell lines was apparently mainly caused by Src tyrosine kinase activity and not by Jak activation.12Niu G Bowman T Huang M Shivers S Reintgen D Daud A Chang A Kraker A Jove R Yu H Roles of activated Src and Stat3 signaling in melanoma tumor cell growth.Oncogene. 2002; 21: 7001-7010Crossref PubMed Scopus (370) Google Scholar In vitro growth of early stage melanoma cells has been shown to be inhibited by IL-6 in several studies.13Swope VB Abdel-Malek Z Kassem LM Nordlund JJ Interleukins 1 alpha and 6 and tumor necrosis factor-alpha are paracrine inhibitors of human melanocyte proliferation and melanogenesis.J Invest Dermatol. 1991; 96: 180-185Abstract Full Text PDF PubMed Google Scholar, 14Florenes VA Lu C Bhattacharya N Rak J Sheehan C Slingerland JM Kerbel RS Interleukin-6 dependent induction of the cyclin dependent kinase inhibitor p21WAF1/CIP1 is lost during progression of human malignant melanoma.Oncogene. 1999; 18: 1023-1032Crossref PubMed Scopus (71) Google Scholar, 15Fontaine V Mahieu M Content J Interferon-gamma and interleukin-6 inhibit proliferation in human melanoma cells by different signalling pathways.Melanoma Res. 1998; 8: 24-30Crossref PubMed Scopus (8) Google Scholar, 16Lu C Vickers MF Kerbel RS Interleukin 6: a fibroblast-derived growth inhibitor of human melanoma cells from early but not advanced stages of tumor progression.Proc Natl Acad Sci USA. 1992; 89: 9215-9219Crossref PubMed Scopus (168) Google Scholar In the A375 cell line, this inhibition is mediated by IL-6-induced STAT activation.17Kortylewski M Heinrich PC Mackiewicz A Schniertshauer U Klingmuller U Nakajima K Hirano T Horn F Behrmann I Interleukin-6 and oncostatin M-induced growth inhibition of human A375 melanoma cells is STAT-dependent and involves upregulation of the cyclin-dependent kinase inhibitor p27/Kip1.Oncogene. 1999; 18: 3742-3753Crossref PubMed Scopus (123) Google Scholar, 18Bohm M Schulte U Funk JO Raghunath M Behrmann I Kortylewski M Heinrich PC Kues T Luger TA Schwarz T Interleukin-6-resistant melanoma cells exhibit reduced activation of STAT3 and lack of inhibition of cyclin E-associated kinase activity.J Invest Dermatol. 2001; 117: 132-140Crossref PubMed Google Scholar Cells derived from advanced melanomas at metastatic stages often lose this anti-proliferative response to IL-6.16Lu C Vickers MF Kerbel RS Interleukin 6: a fibroblast-derived growth inhibitor of human melanoma cells from early but not advanced stages of tumor progression.Proc Natl Acad Sci USA. 1992; 89: 9215-9219Crossref PubMed Scopus (168) Google Scholar, 19Lu C Kerbel RS Interleukin-6 undergoes transition from paracrine growth inhibitor to autocrine stimulator during human melanoma progression [published erratum appears in J Cell Biol 1993 Apr; 121(2): following 477].J Cell Biol. 1993; 120: 1281-1288Crossref PubMed Scopus (234) Google Scholar, 20Silvani A Ferrari G Paonessa G Toniatti C Parmiani G Colombo MP Down-regulation of interleukin 6 receptor alpha chain in interleukin 6 transduced melanoma cells causes selective resistance to interleukin 6 but not to oncostatin M.Cancer Res. 1995; 55: 2200-2205PubMed Google Scholar In fact, anti-sense oligonucleotides blocking IL-6 gene expression inhibited the growth of these cell lines, suggesting that IL-6 promotes advanced stage melanoma cell growth by an autocrine mechanism.19Lu C Kerbel RS Interleukin-6 undergoes transition from paracrine growth inhibitor to autocrine stimulator during human melanoma progression [published erratum appears in J Cell Biol 1993 Apr; 121(2): following 477].J Cell Biol. 1993; 120: 1281-1288Crossref PubMed Scopus (234) Google Scholar In support of this hypothesis, melanoma cells, and especially cells from advanced tumors, have been shown to express both IL-6 and IL-6 receptor α.19Lu C Kerbel RS Interleukin-6 undergoes transition from paracrine growth inhibitor to autocrine stimulator during human melanoma progression [published erratum appears in J Cell Biol 1993 Apr; 121(2): following 477].J Cell Biol. 1993; 120: 1281-1288Crossref PubMed Scopus (234) Google Scholar, 21Lee JD Sievers TM Skotzko M Chandler CF Morton DL McBride WH Economou JS Interleukin-6 production by human melanoma cell lines.Lymphokine Cytokine Res. 1992; 11: 161-166PubMed Google Scholar, 22Francis GM Krohn EG Woods KV Buzaid AC Grimm EA Interleukin-6 production and secretion in human melanoma cell lines: regulation by interleukin-1.Melanoma Res. 1996; 6: 191-201Crossref PubMed Scopus (19) Google Scholar, 23Candi E Knight RA Spinedi A Guerrieri P Melino G A possible growth factor role of IL-6 in neuroectodermal tumours.J Neurooncol. 1997; 31: 115-122Crossref PubMed Scopus (19) Google Scholar, 24Mattei S Colombo MP Melani C Silvani A Parmiani G Herlyn M Expression of cytokine/growth factors and their receptors in human melanoma and melanocytes.Int J Cancer. 1994; 56: 853-857Crossref PubMed Scopus (232) Google Scholar, 25Moretti S Pinzi C Spallanzani A Berti E Chiarugi A Mazzoli S Fabiani M Vallecchi C Herlyn M Immunohistochemical evidence of cytokine networks during progression of human melanocytic lesions.Int J Cancer. 1999; 84: 160-168Crossref PubMed Scopus (88) Google Scholar, 26Ciotti P Rainero ML Nicolo G Spina B Garre C Casabona F Santi PL Bianchi-Scarra G Cytokine expression in human primary and metastatic melanoma cells: analysis in fresh bioptic specimens.Melanoma Res. 1995; 5: 41-47Crossref PubMed Scopus (41) Google Scholar Moreover, IL-6 is probably produced in significant amounts as a paracrine stimulator by stromal cells and by cells in the invaded tissue in the vicinity of melanomas including cells of reactive inflammatory infiltrates. In addition, ultraviolet light alters the expression of many genes in keratinocytes with a consequent release of cytokines such as IL-1α, IL-6, and IL-12 in vitro,27Eberlein-Konig B Jager C Przybilla B Ultraviolet B radiation-induced production of interleukin 1alpha and interleukin 6 in a human squamous carcinoma cell line is wavelength-dependent and can be inhibited by pharmacological agents.Br J Dermatol. 1998; 139: 415-421Crossref PubMed Scopus (20) Google Scholar suggesting that exposure to sunlight might increase IL-6 levels in skin. Supporting the hypothesis that IL-6 promotes late-stage melanoma progression, elevated IL-6 serum levels have been found in patients with metastatic melanoma; IL-6 elevation was associated with larger tumor burden, resistance to chemo- and immunotherapy, and shorter median survival rate.28Mouawad R Khayat D Merle S Antoine EC Gil-Delgado M Soubrane C Is there any relationship between interleukin-6/interleukin-6 receptor modulation and endogenous interleukin-6 release in metastatic malignant melanoma patients treated by biochemotherapy?.Melanoma Res. 1999; 9: 181-188Crossref PubMed Scopus (24) Google Scholar, 29Mouawad R Benhammouda A Rixe O Antoine EC Borel C Weil M Khayat D Soubrane C Endogenous interleukin 6 levels in patients with metastatic malignant melanoma: correlation with tumor burden.Clin Cancer Res. 1996; 2: 1405-1409PubMed Google Scholar, 30Tartour E Dorval T Mosseri V Deneux L Mathiot C Brailly H Montero F Joyeux I Pouillart P Fridman WH Serum interleukin 6 and C-reactive protein levels correlate with resistance to IL-2 therapy and poor survival in melanoma patients.Br J Cancer. 1994; 69: 911-913Crossref PubMed Scopus (120) Google Scholar Taken together, IL-6 effects on melanomas appear to be variable, depending on the tumor cell lines studied and on the stage of the disease. To elucidate the functional role of IL-6 in the pathogenesis of melanomas in vivo, we made use of a mouse model that reflects the full range of progressive conversions of nonneoplastic melanocytes to melanoma cells. MT-ret transgenic mice express the human cellular Ret oncogene under the control of a mouse metallothionein-I (MT) promotor-enhancer.31Iwamoto T Takahashi M Ito M Hamatani K Ohbayashi M Wajjwalku W Isobe K Nakashima I Aberrant melanogenesis and melanocytic tumour development in transgenic mice that carry a metallothionein/ret fusion gene.EMBO J. 1991; 10: 3167-3175PubMed Google Scholar Metallothionein is expressed in mouse and human skin.32van den Oord JJ De Ley M Distribution of metallothionein in normal and pathological human skin.Arch Dermatol Res. 1994; 286: 62-68Crossref PubMed Scopus (48) Google Scholar, 33Karasawa M Nishimura N Nishimura H Tohyama C Hashiba H Kuroki T Localization of metallothionein in hair follicles of normal skin and the basal cell layer of hyperplastic epidermis: possible association with cell proliferation.J Invest Dermatol. 1991; 97: 97-100Abstract Full Text PDF PubMed Google Scholar, 34Miles AT Hawksworth GM Beattie JH Rodilla V Induction, regulation, degradation, and biological significance of mammalian metallothioneins.Crit Rev Biochem Mol Biol. 2000; 35: 35-70Crossref PubMed Scopus (396) Google Scholar In MT-ret transgenic animals, the c-Ret protein is expressed in the skin, mainly in the inner and outer root sheaths of hair follicles.35Kato M Takeda K Kawamoto Y Tsuzuki T Dai Y Nakayama S Toriyama K Tamada Y Takahashi M Nakashima I RET tyrosine kinase enhances hair growth in association with promotion of melanogenesis.Oncogene. 2001; 20: 7536-7541Crossref PubMed Scopus (22) Google Scholar Highest expression levels are found soon after birth.35Kato M Takeda K Kawamoto Y Tsuzuki T Dai Y Nakayama S Toriyama K Tamada Y Takahashi M Nakashima I RET tyrosine kinase enhances hair growth in association with promotion of melanogenesis.Oncogene. 2001; 20: 7536-7541Crossref PubMed Scopus (22) Google Scholar Homozygous MT-ret mice die in utero.36Kato M Takahashi M Akhand AA Liu W Dai Y Shimizu S Iwamoto T Suzuki H Nakashima I Transgenic mouse model for skin malignant melanoma.Oncogene. 1998; 17: 1885-1888Crossref PubMed Scopus (176) Google Scholar Heterozygous MT-ret (MT-ret+/−) transgenic 304BL/6 mice are viable and develop severe systemic melanosis of the skin and the subcutis; melanocytic skin tumors develop from these precursor lesions and progress to malignant melanomas.36Kato M Takahashi M Akhand AA Liu W Dai Y Shimizu S Iwamoto T Suzuki H Nakashima I Transgenic mouse model for skin malignant melanoma.Oncogene. 1998; 17: 1885-1888Crossref PubMed Scopus (176) Google Scholar The development of these malignant melanomas, most of which are phenotypically related to small cell melanomas in humans, resembles malignant transformation of human giant congenital melanocytic nevus and neurocutaneous melanosis. To analyze the contribution of IL-6 to the development and progression of melanomas in MT-ret mice, we crossed these mice with IL-6-deficient animals.37Kopf M Baumann H Freer G Freudenberg M Lamers M Kishimoto T Zinkernagel R Bluethmann H Kohler G Impaired immune and acute-phase responses in interleukin-6-deficient mice.Nature. 1994; 368: 339-342Crossref PubMed Scopus (1503) Google Scholar We report here that ablation of IL-6 leads to a reduction in tumor incidence and tumor size in this model of spontaneous melanoma. MT-ret transgenic mice36Kato M Takahashi M Akhand AA Liu W Dai Y Shimizu S Iwamoto T Suzuki H Nakashima I Transgenic mouse model for skin malignant melanoma.Oncogene. 1998; 17: 1885-1888Crossref PubMed Scopus (176) Google Scholar were crossed with IL-6-deficient mice.37Kopf M Baumann H Freer G Freudenberg M Lamers M Kishimoto T Zinkernagel R Bluethmann H Kohler G Impaired immune and acute-phase responses in interleukin-6-deficient mice.Nature. 1994; 368: 339-342Crossref PubMed Scopus (1503) Google Scholar All mice had been backcrossed for at least 10 generations to the C57BL/6 background. To produce MT-ret+/−/IL-6+/− and MT-ret+/−/IL-6−/− mice, MT-ret+/− mice were crossed with IL-6−/− mice and heterozygous MT-ret+/−/IL-6+/− offspring was crossed again to yield the MT-ret+/−/IL-6−/− genotype. Genotyping was performed by polymerase chain reaction of DNA isolated from tail biopsies (MT-ret+/− sense, 5′TCC CTT TTT GAT CAT ATC TAC ACC A3′ and MT-ret+/− anti-sense, 5′AAT CCA TGT GGA AGG GAG GGC TCG A3′, IL-6−/− 5′TTC CATC CAG TTG CCT TCT TGG3′, and 5′TTC TCA TTT CCA CGA TTT CCC AG′3, or neo 5′-CCG GAG AAC CTG CGT GCA ATC C3′). Details of the genotyping procedure are available on request. Tail DNA of all mice was stored at 4°C for regenotyping. The mice were monitored daily. Five weeks after the first detection of a visible or palpable tumor, the animals were killed using CO2. The carcasses were dissected and inspected carefully. The number of tumor nodules was counted. The diameters of all tumors in whole mount sections of the tumor-infiltrated areas were measured using a microscopic scale bar. Animals that did not develop a tumor were killed at 65 weeks of age. Tumor incidence and acceleration was monitored in Kaplan-Meier curves. These results were analyzed statistically using Wilcoxon's signed-rank test. The extent of tumor necrosis in each group was determined by counting the number of tumor nodules containing necrotic areas. These data were evaluated statistically using the χ2Kamimura D Ishihara K Hirano T IL-6 signal transduction and its physiological roles: the signal orchestration model.Rev Physiol Biochem Pharmacol. 2003; 149: 1-38Crossref PubMed Google Scholar test. All experiments were performed according to the laws of the State of Bern governing the welfare of animals. Normal skin biopsies, preneoplastic and neoplastic skin lesions, metastases, and all major organs were fixed in 4% buffered paraformaldehyde overnight at room temperature and embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin (H&E) and analyzed histologically to assess tumor morphology, spread, vascularization, and interactions with adjacent tissues. In addition, unfixed material was snap-frozen in liquid nitrogen and stored at −80°C for cryostat sectioning and for molecular biological and enzymatic studies. Immunostainings were performed using a polyclonal antiserum against S100 protein (DAKO, Glostrup, Denmark) and polyclonal antisera against STAT3 (Santa Cruz Biotechnology, Santa Cruz, CA) as well as activated, tyrosine 705 phosphorylated pY-STAT3 (Cell Signaling Technology, Beverly, MA). Consecutive 3- to 5-μm paraffin sections and cryostat sections were subjected to immunostaining using the alkaline phosphatase technique. Tissue sections were pretreated by boiling either in a pressure cooker for 5 minutes in citrate buffer at pH 6.0 (S100, STAT3) or in a microwave oven for 3 × 5 minutes in 5% urea buffer (pY-STAT3) and then washed for 5 minutes in Tris-buffered saline. Sections were then incubated for 30 minutes at 37°C with 10% normal goat serum before overnight incubation at 4°C with the primary antibody diluted 1:300 in 10% normal goat serum. Bound antibody was visualized using biotinylated goat anti-rabbit IgG or goat anti-mouse IgG (DAKO), alkaline phosphatase-conjugated streptavidin (DAKO), and new fuchsin-naphtol, followed by counterstaining with Mayer's hematoxylin. To ensure the specificity of the primary antibodies, consecutive tissue sections were incubated either in the absence of primary antibody or with IgG of a nonimmunized rabbit. In these control sections no immunostaining was detected (not shown). Positive controls for the STAT3 and pY-STAT3 immunostaining included paraffin sections of 1) sections of brains of transgenic mice overexpressing the v-src oncogene in astrocytes under the control of the GFAP promoter, thereby constitutively activating STAT3;5Weissenberger J Loeffler S Kappeler A Kopf M Lukes A Afanasieva TA Aguzzi A Weis J IL-6 is required for glioma development in a mouse model.Oncogene. 2004; 23: 3308-3316Crossref PubMed Scopus (156) Google Scholar 2) human glioblastomas, which overexpress STAT3 and accumulate activated pY-STAT3;5Weissenberger J Loeffler S Kappeler A Kopf M Lukes A Afanasieva TA Aguzzi A Weis J IL-6 is required for glioma development in a mouse model.Oncogene. 2004; 23: 3308-3316Crossref PubMed Scopus (156) Google Scholar, 38Rahaman SO Harbor PC Chernova O Barnett GH Vogelbaum MA Haque SJ Inhibition of constitutively active Stat3 suppresses proliferation and induces apoptosis in glioblastoma multiforme cells.Oncogene. 2002; 21: 8404-8413Crossref PubMed Scopus (315) Google Scholar and 3) mouse dorsal root ganglion neurons, which constitutively express IL-6 and IL-6Rα.39Thier M Marz P Otten U Weis J Rose-John S Interleukin-6 (IL-6) and its soluble receptor support survival of sensory neurons.J Neurosci Res. 1999; 55: 411-422Crossref PubMed Scopus (84) Google Scholar In addition, staining with Bandeiraea simplicifolia lectin (Sigma, St. Louis, MO) was used to label lymphocytes as described.40Sowalsky RA Fox BS Pattern of lectin binding to murine T lymphocytes.Immunology. 1992; 75: 92-98PubMed Google Scholar Lymph nodes of MT-ret mice served as positive controls. Paraffin sections from seven tumor-bearing mice of each group were stained using the lectin. The total number of tumor nodules as well as the number of nodules containing labeled cells was counted. A cell line (Mel25) was generated from a primary neck melanoma of a 29-week-old MT-ret+/− mouse. Tumor cells were dissociated by sieving through cell strainers (Falcon Becton Dickinson, Basel, Switzerland) and cultured in Dulbecco's modified Eagle's medium supplemented with 6% bovine calf serum and 6% horse serum. The cultures were treated with 100 μg/ml of cis-hydroxyproline (Sigma) as described41Kao WW Prockop DJ Proline analogue removes fibroblasts from cultured mixed cell populations.Nature. 1977; 266: 63-64Crossref PubMed Scopus (91) Google Scholar until complete removal of fibroblasts. The cell line has now been cultured for more than 40 passages and is morphologically stable. It is immunoreactive for S100 protein, a melanoma cell marker (Figure 1h), and shows overexpression of Ret (Figure 3a). To determine whether Mel 25 cells express IL-6 and IL-6 receptor, reverse transcriptase-polymerase chain reaction was performed using the following primer sequences: mu IL-6 for 5′AGT TGC CTT CTT GGG ACT GA3′ and mu IL-6 reverse 5′CAG AAT TGC CAT TGC ACA AC3′. Mu IL-6R for 5′AAG CTT GGT TCC GAT TTC CT3′ and mu IL-6R reverse 5′TTC GCC TGA AGT CCT GAG AT3′. The β-actin gene served as a control: mu β-actin for 5′GCT ACA GCT TCA CCA CCA CA3′ and mu β-actin reverse 5′AAG GAA GGC TGG AAA AGA GC3′.Figure 3a: Analysis of ret expression in the skin of adult MT-ret+/− mice by two independent immunoblots. Ret protein levels are rather low in the skin of MT-ret+/− mice, and there is no detectable decrease in ret expression in mice lacking one (IL-6+/−) or both (IL-6−/−) copies of the IL-6 gene. Melanomas of an MT-ret+/− and an MT-ret+/−/IL-6+/− mouse express larger amounts of ret protein, as does the Mel25 cell line. As expected, the B16-F0 cell line does not show any ret expression. b: Immunoblot analysis of MT-ret+/− melanomas. Tumors derived from IL-6+/+ and IL-6−/− mice show similar ret protein expression. Wt: skin from wild-type BL/6 mouse. c: No increase in ret protein expression in immunoblots of Mel25 cells after stimulation with H-IL-6 for 15 minutes to 72 hours.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To assess the response of Mel25 cells to exogenous IL-6 and IL-6Rα, melanoma cells were starved for 2 hours in serum-free medium and then cultured in Dulbecco's modified Eagle's medium for 15 and 30 minutes in the presence or absence of 10 to 100 ng/ml of recombinant IL-6 (R&D Systems, Abingdon, UK) or incubated for 0 minutes, 15 minutes, 1 hour, 6 hours, 24 hours, and 72 hours with 100 ng/ml of hyper-IL-6 (H-IL-6), a fusion protein of sIL-6R and IL-6, linked by a flexible peptide chain42Rakemann T Niehof M Kubicka S Fischer M Manns MP Rose-John S Trautwein C The designer cytokine hyper-interleukin-6 is a potent activator of STAT3-dependent gene transcription in vivo and in vitro.J Biol Chem. 1999; 274: 1257-1266Crossref PubMed Scopus (55) Google Scholar (generous gift by Prof. S. Rose-John, University of Kiel, Kiel, Germany). After incubation with H-IL-6, the survival and proliferation of Mel25 cells was analyzed using the MTT assay as described.43Thier M Simon R Kruttgen A Rose John S Heinrich PC Schroder JM Weis J Site-directed mutagenesis of human CNTF: functional analysis of recombinant variants.J Neurosci Res. 1995; 40: 826-835Crossref PubMed Scopus (12) Google Scholar The melanoma cell lines A375 (human) and B16-F0 (mouse, both obtained from American Type Culture Collection, Rockville, MD) served as controls. The pharmacological blockers, LY294002 and PD90059, were purchased from Sigma and Calbiochem (San Diego, CA), respectively. Using the MTT assay and these blockers, we determined survival-promoting effects of the PI3K/Akt and of the MAPK pathways, respectively, in Mel25 cells under full serum conditions. Mouse tissues were snap-frozen in liquid nitrogen immediately on removal and weighed. Tissues were homogenized in ice-cold buffer (10% w/v) containing 0.32 mol/L sucrose, 0.5% Nonidet P-40, 0.5% sodium deoxycholate plus NaVO4, and Complete Protease Inhibitor Cocktail (Roche Diagnostics, Rotkreuz, Switzerland). Cultured melanoma cells were lysed on the plate with RIPA lysis buffer, Complete Protease Inhibitor Cocktail, and NaVO4. The lysates were cleared by centrifugation at 14,000 × g. Protein concentration was determined by use of the BCA kit (Pierce, Rockford, IL) using bovine serum albumin as a standard. Equal amounts of protein on each lane were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. The samples were then transferred (wet) on nitrocellulose paper. After blocking with 5% skim milk, blots were incubated with polyclonal antisera against Ret and STAT3 (both Santa Cruz Biotechnology), tyrosine-705 phosphorylated pY-STAT3 (Cell Signaling Technology), polyclonal anti-phospho-Akt (phospho-Ser 473) (Cell Signaling Technology); polyclonal anti-Akt 1:1000 (Cell Signaling Technology); monoclonal anti-phospho-p44/42 MAPK (Cell Signaling Technology) at 1:2000; monoclonal anti-MAPK at 1" @default.
• W2064034345 created "2016-06-24" @default.
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• W2064034345 date "2005-03-01" @default.
• W2064034345 modified "2023-10-13" @default.
• W2064034345 title "Interleukin-6 Gene Ablation in a Transgenic Mouse Model of Malignant Skin Melanoma" @default.
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