Induction of DNA synthesis in BALB/c 3T3 cells by serum components: Reevaluation of the commitment process

Abstract

Serum contains a growth factor derived from platelets and also growth factors derived from platelet-poor plasma. Extracts of heated (1000) human platelets function synergistically with platelet-poor plasma to induce DNA synthesis in quiescent, density-inhibited BALB/c 3T3 cells. Platelet-poor plasma alone did not induce DNA synthesis. Cells exposed to platelet extracts became competent to enter the cell cycle, but the rate of entry into the S phase depended upon the concentration of platelet-poor plasma. The time required for the induction of this competent state was a function of the concentration of the platelet extract. A 2-hr exposure to 100',g of the platelet extract at 370 caused the entire cell population to become competent to enter the S phase. At 40 or 250 the cells did not become competent to synthesize DNA. The platelet extract-induced competent state was stable for at least 13 hr after removal of the platelet extract; however, in the absence of platelet-poor plasma, these competent cells did not progress through the cell cycle. The addition of an optimal concentration of platelet-poor plasma (5%) to these competent cells initiated cell cycle traverse with a ra id, first-order entry of cells into the S phase beginning 12 hr after addition of the plasma. The addition of a suboptimal concentration of the plasma (0.25%) did not increase the rate of cell entry into the S phase. Thus, the induction of DNA synthesis in quiescent BALB/c 3T3 cells can be resolved into at least two phases, controlled by different serum components: (i) competence, induced by the plateletderived growth factor; and (ii) progression of competent cells into the cell cycle, mediated by factors in platelet-poor plasma.The growth of 3T3 cells (1), diploid fibroblasts (2), and smooth muscle cells (3) in vitro is controlled by the concentration of serum in the medium. Serum can be separated into two sets of components which control different cell functions. One set maintains cell viability (4), while the other stimulates replication (5). A heat-stable (1000) cationic growth factor (6) derived from. platelets (7) is released into serum during the clotting process (8, 9). Human serum contains about 770 pg of this polypeptide growth factor per mg of protein, as demonstrated by radioimmunoassay (7). Defibrinogenated platelet-poor plasma, a fraction prepared from unclotted blood, contains only low levels of the growth factor and does not stimulate the replication of diploid fibroblasts or BALB/c 3T3 cells (3, 7-9). Platelet-poor plasma does, however, contain the factors that maintain cell viability (3).The process by which resting cells become committed to enter the growth cycle remains unclear. Smith and Martin (10) have proposed that the commitment of quiescent cells to synthesize DNA is a random event characterized by a first-order rate constant, the transition probability. According to thisThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ...

Connecting signaling and cell cycle progression in growth factor-stimulated cells

“…Using subsaturating concentrations of growth factors, Pledger, Stiles, Antoniades, and Sher demonstrated that in Balb/c 3T3s cell cycle progression required the input of two di erent types of factors (Pledger et al, 1977(Pledger et al, , 1978Stiles et al, 1979b). Growth factors such as PDGF or FGF made the cells competent, but did not drive them into S phase.…”

Cell cycle: Regulatory events in G1 → S transition of mammalian cells

“…As the first step towards proliferation quiescent cells initially require growth factors (see Fig. 11, such as platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF), for initiating early regulatory processes, referred to as reaching "competence" [Pledger et al, 1977;Scher et al, 19791. In the presence of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I), competent cells can progress to the next stage in G1, termed the "V" point [Leof et al, 19831. Cells at the "V" point exhibit a rapid rate of protein synthesis [Rossow et al, 19791 and soon reach the "restriction" point "R" [Pardee, 19741. In the presence of IGF-I or insulin, these cells will then commence DNA synthesis without any additional requirement for growth factors, tran-P rog ressi o n { Factors IGF-Vinsulin IL-2, etc.…”

A cell cycle and mutational analysis of anchorage-independent growth: cell adhesion and TGF-beta 1 control G1/S transit specifically

“…Mitogenic peptide growth factors, such as platelet-derived growth factor, insulin like growth factor-I, and EGF, are among the best studied examples of such external cell cycle regulators (Pardee, 1989;Sporn and Roberts, 1990). Although the exact roles of these growth factors seem to be somewhat variable in different fibroblast cell lines, it is clear that they are required to stimulate entry of quiescent (G0-synchronized) fibroblasts into the cell cycle and progression of the cells through most of G1 (Pledger et al, 1977;Pardee, 1989). Transit from late G1 into S is independent of mitogenic growth factor action (Pledger et al, 1977;Yen and Pardee, 1978;Rossow et al, 1979).…”

“…Although the exact roles of these growth factors seem to be somewhat variable in different fibroblast cell lines, it is clear that they are required to stimulate entry of quiescent (G0-synchronized) fibroblasts into the cell cycle and progression of the cells through most of G1 (Pledger et al, 1977;Pardee, 1989). Transit from late G1 into S is independent of mitogenic growth factor action (Pledger et al, 1977;Yen and Pardee, 1978;Rossow et al, 1979). When compared to the growth factors discussed above, the effects of TGF-/$1 on fibroblast proliferation seem quite distinct.…”

“…To determine if there are additional attachment-dependent cell cycle transitions, we prepared parallel cultures of adherent and nonadherent NRK and NIH-3T3 fibroblasts that had been synchronized at the G1/S interface by exposure to hydroxyurea (Draetta and Beach, 1988;Pledger et al, 1977;DeCaprio et al, 1989). (Based on our previous studies, we reasoned that an hydroxyurea treatment would synchronize the cells immediately after the START-like block to anchorage-independent growth.)…”