[CANCER RESEARCH 52. 3011-3014, June I, 1992] Stimulation of Anchorage-independent Cell Growth by Endothelin in NRK 49F Cells1 Masatoshi Kusuhara,2 Ken Yamaguchi, Masaru Kuranami, Ai Suzaki, Shiro Ishikawa, Hanlim Moon, Isamu Adachi, Shingo Hori, and Shunnosuke Handa Growth Factor Division, National Cancer Center Research Institute [M. Kus, K. Y., M. Kur, A. S., S. I., H. M.j and Department of Medicine, National Cancer Center Hospital [I. A.], Tsukiji 5-1-1, Chuo-ku, Tokyo 104; and Department of Medicine fS. Ho., S. Ha.], Keio University School of Medicine, Shinanomachi 35, Shinjuku- ku, Tokyo 160, Japan ABSTRACT Endothelin (ET) is a vasoconstrictor peptide originally isolated from vascular endothelial cells. Recent studies have revealed that ET has many biological functions including growth factor-like activity. The present study aims to clarify whether ET-1 possesses the ability to stimulate anchorage-independent cellular growth, an indicator of factors with trans forming activity. We found that NRK 49F cells possess a large number of high-affinity ET-1 receptors; labeled '"I-ET-1 binding was displaced by unlabeled ET-2 in a similar dose response, but in the case of ET-3, 100-fold more was required. Specific '"I-ET-3 binding was undetectable in NRK 49F cells, indicating that ET receptors in NRK 49F cells are 11-1112 selective. NRK 49F is a cell line which is most commonly used to assay for anchorage-independent cellular growth. Therefore, we explored whether ETs promote anchorage-independent cellular growth in this cell line. II- 1 and ET-2 stimulated NRK colony formation dose dependent!)' in the presence of 1 MMepidermal growth factor (EGF). In contrast, ET-3 did not have colony-stimulating ability. In the presence of EGF, the maximal effect of ET-1 was approximately 90% of that of transforming growth factor-/?. Moreover, in the presence of maximal stimulating concentra tions of EGF and transforming growth factor-/?, ET-1 additionally induced colony formation. These results indicate that ET-1 and -2 possess trans forming growth factor-like activity for NRK 49F cells. Since ET-1 and -2 increased intracellular calcium levels, this ion may participate in signal transduction pathways by which ET-1 and -2 promote colony formation. INTRODUCTION ET-13 was isolated originally as a factor possessing vasocon- strictive activity from culture media conditioned by primary- cultured endothelial cells (1). Three types of ET, ET-1, ET-2, and ET-3, are now known to compose the ET family, and their production and secretion have been demonstrated in many tissues (2). Recent research has revealed that ET-1 also has a wide range of pharmacological effects in tissues other than blood vessels, such as an inotropic effect on cardiac myocytes (3), a neurotransmitter effect on the central nerve system (4), and modulation of endocrine secretions (5-8). Several studies, including ours, demonstrated that ET-1 stim ulates DNA synthesis in various types of cultured cells, includ ing Swiss 3T3 (9, 10), vascular smooth muscle (11, 12), and Received 6/19/91; accepted 3/20/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This investigation was supported in part by a research grant from the Princess Takamatsu Cancer Research Fund, by a Grant-in-Aid from the Ministry of Health and Welfare for the Comprehensive 10-Year Strategy of Cancer Control, and by Grants-in-Aid for Cancer Research (1-5 and 2-9) from the Ministry of Health and Welfare. 2To whom requests for reprints should be addressed, at Growth Factor Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104, Japan. 'The abbreviations used are: ET, endothelin; DMEM, Dulbecco's modified Eagle medium; CS, calf serum; TGF, transforming growth factor; EGF, epidermal growth factor; [Ca2*]¡, intracellular calcium ion concentration; A'd, dissociation constant. renal mesangial (13). However, how ET-1 influences cellular growth is poorly understood. Our recent observations that cancer cells frequently produce and secrete ET-1 and that primary-cultured human fibroblasts possess ET-1 receptors sug gest that ET-1 produced by cancer cells could act as a paracrine growth factor on mesenchymal cells (14). In the present study, we explored whether ET-1 promotes anchorage-independent cell growth. For these experiments, NRK 49F was selected as an indicator cell line, which is most commonly used to assay for anchorage-independent cell growth (15, 16). MATERIALS AND METHODS Cell Culture and Materials. NRK 49F cells were provided by the Japanese Cancer Research Resources Bank (Tokyo, Japan). They were grown in DMEM (Nissui Seiyaku, Tokyo, Japan) containing 5% CS (GIBCO Laboratories, Grand Island, NY), 100 Mgof streptomycin/ml, and 100 units of penicillin G/ml at 37°Cin a fully humidified atmos phere of 5% COz in air. Confluent cells were further cultured in DMEM containing 1% CS for 24 h to minimize the effect of serum. Synthetic ET-1, -2, and -3 were purchased from the Peptide Institute (Osaka, Japan); recombinant TGF-/3 and human EGF from Earth Chemical Co., Ltd. (Hyogo, Japan); bovine serum albumin (Conn fraction V) from Dai-ichi Pure Chemicals Co., Ltd. (Osaka, Japan); '"I-ET-1 (specific activity, 74 TBq/mmol) and '"I-ET-3 (specific activ ity, 74 TBq/mmol) from Amersham International (Buckinghamshire, United Kingdom); and Fura-2 acetoxymethyl ester from Molecular Probes, Inc. (Eugene, OR). Binding Assay. The ET-1 binding assay was performed as previously described, with slight modification (17). NRK 49F cells were grown to confluence in a 48-well plate and further cultured for 24 h in DMEM containing 1% CS. Hanks' balanced salt solution containing 20 m\i N- (2-hydroxyethyl)piperazine-yV'-(2-ethanesulfonic acid) and 0.1 % bovine serum albumin (pH 7.4) was used as assay buffer. After these cells were washed, they were incubated at 37°Cfor 60 min with 0.12 nM '"I-ET- 1 plus various concentrations of unlabeled ET-1, -2, and -3. They were washed three times and solubilized with l N NaOH. Cell-bound radio activity was determined in a gamma spectrometer. Cell numbers were estimated by counting other wells which were treated in the same manner as those of the binding studies. Specific binding was calculated as total binding minus binding in the presence of 1 pM unlabeled ET-1. Since specific '"l-ET-3 binding could not be detected under the above conditions, the binding assay was done by using a larger number of NRK 49F cells. NRK 49F cells in 60-mm dishes were incubated with 0.3 nM '"I-ET-3 in the presence or absence of a 5000-fold excess of unlabeled ET-3. Soft Agar Colony Formation Assay. To assess anchorage-independent cell growth, a soft agar colony formation assay using NRK 49F cells was performed as previously described, with slight modification (18). NRK 49F cells (20,000/dish) were suspended in 0.9 ml of culture medium containing 0.3% agar and spread over a prehardened feeder layer comprising 0.9 ml of the same medium, but containing 0.5% agar, in a 35-mm Petri dish. Test materials dissolved in 0.2 ml of culture medium were placed on the top of the prehardened agar layers. After 14 days of incubation at 37°C in a humidified CO2 incubator, colonies 3011 on July 15, 2015. © 1992 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from