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Stimulation of Anchorage-independent Cell Growth by Endothelin in NRK 49F Cells¹

Growth Factor Division, National Cancer Center Research Institute [M. Kus, K. Y., M. Kur, A. S., S. I., H. M.] and Department of Medicine, National Cancer Center Hospital [I. A.], Tsukiji 5-1-1, Chuo-ku, Tokyo 104; and Department of Medicine [S. Ho., S. Ha.], Keio University School of Medicine, Shinanomachi 35, Shinjukuku, Tokyo 160, Japan

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 transforming activity.

We found that NRK 49F cells possess a large number of high-affinity ET-1 receptors; labeled ¹²⁵I-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 ¹²⁵I-ET-3 binding was undetectable in NRK 49F cells, indicating that ET receptors in NRK 49F cells are ET-1/ET-2 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. ET-1 and ET-2 stimulated NRK colony formation dose dependently in the presence of 1 nM epidermal 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-ß. Moreover, in the presence of maximal stimulating concentrations of EGF and transforming growth factor-ß, ET-1 additionally induced colony formation. These results indicate that ET-1 and -2 possess transforming 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.

¹ This 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.

² To 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.

Received 6/19/91. Accepted 3/20/92.