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Swiss 3T3 fibroblasts can be weakly stimulated to grow by bombesin, epidermal growth factor or ceruloplasmin when cells are maintained in Dulbecco's Modified Essential Medium (DMEM), the pH of which is 7.75. Addition of insulin synergizes with the other mitogens. However, only ceruloplasmin promotes DNA synthesis in Minimum Essential Medium (MEM). The pH in this medium is 7.0. All the other growth factors synergize with the ceruloplasmin effects, but such synergism is not evident with insulin. If the pH in MEM is increased to 7.25 or 7.75 by supplementation with HEPES or NaHCO3, respectively, the results are similar to those found in DMEM. Since the oxidation of iron is increased at alkaline pH, the reoxidation of iron at the cell surface may facilitate growth at alkaline pH. We propose that iron reoxidation is limiting for cell growth and that part of the ceruloplasmin effect is mediated by its action as a terminal oxidase for ferrous iron on the cell surface. Observations consistent with this explanation include: 1) combinations of insulin with bombesin or epidermal growth factors do not promote cell proliferation at pH 7.0; 2) fetal calf serum, which has ferroxidase activity, and ceruloplasmin plus or minus other growth factors stimulate cell proliferation at pH 7.0; and 3) alkaline pH also restores the mitogenic effect of growth factors.

The synthesis of DNA in CCl 39 cells is inhibited by the presence of the Fe2+ chelator bathophenanthroline disulfonate (BPS) when growth is stimulated by thrombin EGF plus insulin, but not by fetal calf serum. The presence of transferrin and Fe3+ in fetal calf serum can be the basis for lack of BPS effect with serum. The impermeable Fe3+ chelator Tiron does not, by itself, inhibit growth factor induced DNA synthesis, but it induces together with BPS inhibition on fetal calf serum induced DNA synthesis. The combined effect of BPS and Tiron is similar to inhibition of DNA synthesis by impermeable polyvalent DTPA which can chelate both Fe2+ and Fe3+ but does not inhibit ribonucleotide reductase in intact cells. Ferrous iron that bind BPS can relieve the inhibition at stoichiometric concentration. Ferric iron also prevents the inhibition even though it does not bind BPS. BPS does not inhibit DNA synthesis in HeLa cells. BPS reacts with iron from CCl 39 cells but not from HeLa cells. Data show that iron available for impermeable external chelators is in the ferrous state, and that exogenous iron should be reduced before it reverses the inhibition.

We previously showed that thrombin-stimulated DNA synthesis in CCL 39 cells was inhibited by hydroxyurea (HU) and bathophenanthroline disulfonate (BPS) (Proc. Natl. Acad. Sci. USA, in press). A clear difference exists between these two inhibitors. Inhibition mediated by HU was immediate and must be present in the culture medium. BPS was equally effective when it was present in the medium or after preincubation, but it required at least 12 h to achieve maximal effect. The permeable form 1,10 phenanthroline had the same inhibitory effect in short-term incubations that BPS. Moreover, 1,10 phenanthroline was cytotoxic in long-term incubations indicating that the site of BPS inhibition was outside the cell. Further, long-term incubations with HU did not affect the ability of the cell to reinitiate DNA synthesis after removal of the chelator.