An ordered sequence of events is required before BALB/c-3T3 cells become committed to DNA synthesis

Abstract

An ordered sequence of events must be completed before cells become committed to synthesize DNA. A platelet-derived growth factor (PDGF), present in heated (100 degrees ) extracts of human platelets, induces density-inhibited BALB/c-3T3 cells to become competent to proliferate. Platelet-poor plasma induces these competent cells to leave the competence point, progress through G(0)/G(1), and enter the S phase. Treatment of G(0)-arrested, incompetent cells with plasma, before the addition of PDGF, did not shorten the latent period for DNA synthesis or increase the rate of entry into the S phase. Growth arrest points in the plasma-dependent progression sequence were detected in G(0)/G(1). PDGF-treated competent cells were exposed to an optimal concentration of plasma (5%) for various lengths of time and were then transferred to medium lacking plasma; the subsequent readdition of plasma stimulated the cells to enter the S phase. The lag period until DNA synthesis, in such experiments, was dictated by the length of the initial exposure to plasma. PDGF-treated competent cells that were incubated with plasma for 5 hr during the initial exposure did not leave the competence point; they began DNA synthesis 12 hr after the readdition of plasma. However, a population of cells treated with plasma for 10 hr became arrested at a point 6 hr before DNA synthesis, whereas a population treated with plasma for 12-15 hr became arrested at a point immediately before DNA synthesis. Cells remained arrested at this latter point for as long as 24 hr, and these arrested cells were not committed to DNA synthesis. The addition of plasma induced immediate entry into the S phase with an apparent first-order rate of entry being determined by the plasma concentration. This plasma-dependent commitment (transition) to DNA synthesis was blocked by cycloheximide but not by hydroxyurea. Removal of the hydroxyurea allowed cells to enter the S phase synchronously in the absence of plasma.