Extracellular calcium requirement for promotion of transformation in JB6 cells

Extracellular calcium is required in the induction of neoplastic transformation of preneoplastic mouse JB6 epidermal cells by 12-O-tetradecanoylphorbol-13-acetate (TPA). Depleting extra-cellular calcium by chelation or by use of commercial calcium-depleted medium inhibited TPA-promoted transformation of promotion-sensitive JB6 cells with a half-maximal inhibition at 1.2 mM calcium. Inhibition was reversible by the addition of calcium. The calcium channel blockers lanthanum and nifedipine inhibited promotion of anchorage-independent transformation by TPA maximally at 10.0 microM and 1.0 nM, respectively, suggesting that calcium entry occurs partially via cell channels. None of the above treatments altered expression of transformation in anchorage-independent tumorigenic JB6 cell lines, indicating that the extracellular calcium requirement was at the level of induction, not expression of transformation. The calcium requirement was not merely a requirement for proliferation; calcium concentrations from 0.2 to 1.8 mM had no effect on JB6 cell monolayer growth. Extracellular calcium was required for 7 days for maximal colony induction. The calcium ionophore A23187 was not a promoter and moderately inhibited TPA-promoted transformation, indicating that increases in free cytosolic calcium concentrations are not sufficient for promotion of transformation and may even activate calcium-dependent antipromoting events. The results suggest that a cellular calcium pool supplied by extracellular calcium, but not distinguishable by ionophoretic increases in free cytosolic calcium, is essential in TPA-promoted neoplastic transformation.