Growth failure in zinc-deficient animals is associated with decreased DNA synthesis; zinc deprivation of 3T3 cells, by use of diethylenetrinitrilopentaacetate (DTPA), impairs thymidine incorporation when the cells are stimulated with fetal bovine serum (FBS). The purpose of this study was to determine the step of cell cycle progression that is affected by zinc deprivation. Swiss murine 3T3 cells were cultured for 3 d in complete media and then for 2 d in low serum media. Cells were then placed in serum-free media and stimulated in sequence with platelet-derived growth factor (PDGF; 3 h), epidermal growth factor (EGF; 0.5 h) and insulin-like growth factor-I (IGF-I; 16 h). The combination of growth factors stimulated thymidine incorporation to the same extent as 10% FBS, and DTPA or EDTA (0.6 mmol/L) inhibited thymidine incorporation. Inhibition was prevented by addition of zinc, but not calcium, iron or cadmium (0.4 mmol/L). When DTPA was present during all stages with no addition of zinc, or zinc added during the competency-priming (PDGF and EGF) step, the IGF-I step, or both steps, the zinc effect occurred at the IGF-I step. Zinc addition 4 h before the measurement of thymidine incorporation had no ameliorative effect, but the presence of zinc during the prior 12 h increased incorporation. Thus zinc exerts its major effect on DNA synthesis during the IGF-I stimulatory phase of the cell cycle. The total zinc concentration of 3T3 cells treated with DTPA for 16 h was not different from that of untreated cells; hence only a small compartment of the cell is affected by DTPA.

Depletion of zinc inhibits growth in animals and proliferation of cultured cells. Additionally, zinc can serve as an antioxidant protecting many compounds, including proteins, from oxidation. Regulation of cell division also involves insulin-like growth factor type I (IGF-I) and its receptor, especially during late G1 phase, allowing progression of the cell to S phase with subsequent DNA synthesis. We examined the effects of zinc depletion from the culture media of Swiss 3T3 cells on the cell cycle and IGF-I receptor expression. Cells were exposed to reduced fetal bovine serum concentrations to induce growth arrest, then returned to normal fetal bovine serum concentrations with the divalent cation chelator diethylenetriamine pentaacetic acid. Reducing the fetal bovine serum concentration did not induce quiescence in the cells as previously suggested. Zinc depletion reduced the proliferative fraction (S and G2/M phases) of the cell cycle. The addition of glutathione to the zinc-depleted media partially returned the proliferative fraction to the control level. Fetal bovine serum deprivation reduced IGF-I receptor expression whereas the absence of zinc had little effect on receptor expression. We conclude that depletion of zinc from culture media inhibits 3T3 cell proliferation independent of insulin-like growth factor-I receptor expression, and part of this inhibition is due to the antioxidant capacity of this divalent cation.