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Induction of Anchorage-independent Growth of JB6 Mouse Epidermal Cells by 1,25-Dihydroxyvitamin D₃¹

Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108 [J. H., T. K.]; Department of Biochemistry, School of Dentistry, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142, Japan [E. A., T. S.]; and Cell Biology Section, Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, Maryland 20892 [N. C.]

1,25-Dihydroxyvitamin D₃ [1,25(OH)₂D₃], a hormonally active form of vitamin D₃, was shown previously to enhance chemically induced transformation of BALB 3T3 cells and Syrian hamster embryo cells. This report demonstrates that 1,25(OH)₂D₃, like phorbol ester tumor promoters, induces anchorage-independent growth of mouse JB6 epidermal cells. When plated on agar plates containing 1,25(OH)₂D₃ at concentrations higher than 0.05 ng/ml or 0.12 nM, JB6 cells formed colonies on the surface of agar plates dose dependently. This anchorageindependent growth was further confirmed by stimulation of DNA synthesis after liquefying the agar layer with NaI. A phorbol-ester resistant variant of JB6 cells was also resistant to 1,25(OH)₂D₃ in terms of induction of anchorage independency. Induction of anchorage-independent growth was specific for 1,25(OH)₂D₃: other derivatives of vitamin D₃ also induced colony formation on agar plates but only at a higher concentration (500 ng/ml) and to much less extent than did 1,25(OH)₂D₃. JB6 cells were found to contain a receptor specific for 1,25(OH)₂D₃ with a K_d of 55.7 pM and N_max of 102.5 fmol/mg protein, suggesting a receptor-mediated mechanism of the induction. The clone that was resistant to 1,25(OH)₂D₃ also contained the receptor. DNA-cellulose chromatography showed that a 1,25(OH)₂D₃-receptor complex interacted with DNA. In contrast to 1,25(OH)₂D₃, retinoic acid did not induce anchorage-independent growth of JB6 cells, but it inhibited the induction by 1,25(OH)₂D₃ when applied with it.

¹ Supported in part by a Grant-in-Aid for Special Project Research, Cancer Bioscience from the Ministry of Education, Science, and Culture, Japan.

² To whom requests for reprints should be addressed.

Received 8/19/85. Revised 4/ 7/86. Revised 7/28/86. Accepted 7/30/86.