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Differences in the Regulation of Intracellular Calcium in Normal and Neoplastic Keratinocytes Are Not Caused by ras Gene Mutations¹

Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland 20892 [F. H. K., H. H., S. H. Y.], and Johns Hopkins Oncology Center, Baltimore, Maryland 21205 [R. W. T.]

The development of resistance to terminal differentiation is an early event in epidermal neoplasia. Altered differentiation can be detected in vitro since normal epidermal cells are induced to differentiate in medium with Ca²⁺ > 0.1 mM while neoplastic epidermal cells and keratinocytes transduced with a v-ras^Ha gene are resistant to Ca²⁺. In normal epidermal cells, the elevation of extracellular Ca²⁺ (Ca_o) from 0.05 to 1.2 mM causes a biphasic intracellular Ca²⁺ (Ca_i) response in which a transient (10 min) peak of 4–5-fold over basal values is followed by a sustained (>25 h) 2-fold increase in steady-state Ca_i. The transient peak in Ca_i is dependent on a serum component and independent of Ca_o, while the sustained plateau is directly dependent on Ca_o. The transient peak responding to a serum factor is lost in normal cells after 24 h in 1.2 mM Ca²⁺, a time when these cells are differentiating. Two neoplastic keratinocyte cell lines, SP-1 and 308, which produce benign tumors in vivo, also have a biphasic Ca_i response to an increase in Ca_o. In these cells, the transient peak is also serum dependent and amplified to 10-fold over basal values. However, the plateau value is not sustained and returns to basal values by 8 h, independent of Ca_o. Furthermore, 308 cells remain sensitive to the serum-induced Ca_i transient after 24 h in 1.2 mM Ca²⁺. To determine whether the activating c-ras^Ha mutation in 308 and SP-1 cells was responsible for the altered Ca_i regulation, a v-ras^Ha gene was introduced into normal keratinocytes by a defective retrovirus. This also produces the papilloma phenotype in vivo. Recipient cells were resistant to Ca²⁺-induced terminal differentiation although they did not proliferate in 1.2 mM Ca²⁺. The Ca_i profile in response to 1.2 mM Ca²⁺ was identical in normal and v-ras^Ha keratinocytes, and these cells lost the serum-induced transient Ca_i peak after 24 h. Thus, the activation of the c-ras^Ha gene in 308 or SP-1 cells is probably not solely responsible for the altered Ca_i response in neoplastic cell lines. Sustained physiological elevation of Ca_i may be relevant to the loss of proliferative potential in both normal and v-ras^Ha keratinocytes in 1.2 mM Ca²⁺. In addition, v-ras^Ha-mediated or activated c-ras^Ha-mediated changes in a complementary pathway may contribute to the block in terminal differentiation in neoplastic cells.

¹ This research was supported in part by a grant from the Proctor and Gamble Company.

² Present address: Avon Products, Inc., Division Street, Toxicology Department, Suffern, NY 10901.

³ To whom requests for reprints should be addressed, at Room 3B25, Building 37, Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Cancer Etiology, National Cancer Institute, 9000 Rockville Pike, Bethesda, MD 20892.

Received 1/17/91. Accepted 5/31/91.