Inorganic phosphate is necessary for the stimulation of DNA synthesis in swiss 3T3 cells by pure mitogenic agents

Abstract

We compared the ability of dialysed fetal bovine serum and of combinations of purified growth-promoting factors such as insulin, epidermal growth factor (EGF), vasopressin, fibroblast-derived growth factor and antitubulin agents to stimulate DNA synthesis in 3T3 cells maintained in the absence or presence of inorganic phosphate (P i ). When DNA synthesis was stimulated by serum in the absence of P i the level induced was 70% of that observed in P i -containing medium. In contrast, combinations of growth-promoting factors in the absence of P i stimulated less than 8% of the DNA synthesis which they induced in complete medium. Addition of as little as 50 μM P i fully restored the ability of the factors to stimulate DNA synthesis. Cells stimulated by purified mitogens in the absence of P i became blocked in early G1, and for up to 48 h the block was reversible by readdition of p i . The effectiveness of dialysed serum to stimulate DNA in the absence of P i suggest that dialysed serum might contain a component capable of supplying P i to support DNA synthesis. Indeed, delipidization of serum by solvent extraction resulted in loss of ability to stimulate DNA synthesis in the absence of p i , but delipidized serum stimulated DNA synthesis virtually, as well as dialysed serum in the presence of P i . Previous conclusions suggesting that P i is not essential for DNA synthesis appear to require re-evaluation.