W. J. Pledger

Epidermal growth factor (EGF) and somatomedin C regulate G1 progression in competent BALB/c-3T3 cells☆

Abstract The activity in platelet-poor plasma that allowed density-arrested BALB/c-3T3 cells rendered competent by a transient exposure to platelet-derived growth factor (PDGF) to traverse G1 and enter the S phase has been termed progression activity. Epidermal growth factor (EGF) and somatomedin C-supplemented medium was shown to be capable of replacing the progression activity of 5% platelet-poor plasma (PPP) for competent density-inhibited BALB/c-3T3 cells. Exposure of competent cells to medium supplemented with EGF and somatomedin C reduced the 12 h minimum G1 lag time found in plasma-supplemented medium by 2 h. It is suggested that the reduction in the minimum time required for progression through G1 is due to the availability of free, unbound somatomedin C. Complete G1 traverse required both EGF and somatomedin C; however, the traverse of the last 6 h of G1 and entry into the S phase required only somatomedin C. Though EGF and somatomedin C could replace the G1 phase progression activity of plasma, medium supplemented with EGF and somatomedin C did not support complete cell cycle traverse or growth of sparse cultures of BALB/c-3T3 cells.

A model of cell cycle control: Sequential events regulated by growth factors

PDGF is a potent mitogen that initiates the proliferation of quiescent fibroblastic cells. EGF and somatomedin C (or insulin) can replace the requirement for plasma to function synergistically with PDGF to stimulate DNA synthesis. PDGF, EGF and somatomedin C control discrete cellular events in the cell cycle. Cyclic AMP can potentiate the effects of polypeptide mitogens. The down-regulation of EGF receptors by PDGF and cyclic AMP brings about a loss of the requirement for exogenous EGF. The transient treatment of density-arrested fibroblasts with PDGF allows better study of synergistic actions of PDGF and plasma-derived factors. These synergistic interactions are important to understand in determining how multiple growth factors regulate cellular proliferation.

Epidermal growth factor (EGF) is required only during the traverse of early G1 in PDGF stimulated density-arrested BALB/c-3T3 cells☆

Density-arrested BALB/c-3T3 cells that had received a transient exposure to PDGF and were then transferred to medium containing only EGF and somatomedin C (Sm-C) began DNA synthesis after the G0/G1 lag. Supraphysiological concentrations of insulin could be employed to replace the Sm-C requirement. This G0/G1 lag phase was bisected by the requirement for the exogenous presence of EGF. Our data indicated that EGF was required during the traverse of only the first half of G0/G1 phase (6 h) and not during the traverse of late G1. Subphysiological serum concentrations of Sm-C were also necessary to be present with EGF for progression through early G0/G1; however, traverse of the final half of G0/G1 and commitment to DNA synthesis required the presence of Sm-C. It was found that physiological concentrations of Sm-C were required for the traverse late G1. The requirement for Sm-C for G0/G1 traverse of BALB/c-3T3 cells as opposed to human fibroblasts or glial cells may be due to a difference in endogenous synthesis of an insulin-like growth factor. Our data are in close agreement with previous reports that EGF is only required for approximately the first 8 h during traverse of the G0/G1 phase. The requirement for EGF to be present for the first 6 h of G0/G1 could result from a continued or repetitious event or by more than one distinct EGF-requiring event.

Mitogenic response to epidermal growth factor (EGF) modulated by platelet-derived growth factor in cultured fibroblasts

The mitotic effects of epidermal growth factor (EGF) were investigated in two cultured fibroblast lines, BALB/c-3T3 and C3H 10T1/2 cells. EGF (30 ng/ml) added to quiescent 3T3 cells in medium containing either platelet-poor plasma or 10(-5) M insulin caused only minimal increases in the percentage of cells stimulated to initiate DNA synthesis. In contrast, EGF acted synergistically with either insulin or plasma to stimulate DNA synthesis in quiescent cultures of 10T1/2 cells, although the maximum effects of EGF were measured at concentrations several-fold greater than those found in either serum or plasma. In either 3T3 or 10T1/2 cells a transient preexposure to platelet-derived growth factor (PDGF) caused over a 10-fold increase in the sensitivity to the mitogenic effects of EGF. It is therefore possible that a primary action of PDGF is to increase the sensitivity of fibroblasts to EGF, independent of whether EGF alone is found to be mitogenic.

Serum Factor Requirements for the Initiation of Cellular Proliferation

The regulatory events in the G0/G1 portion of the cell cycle are believed to control, at least in part, the rate of cellular proliferation. When quiescent density-arrested fibroblasts are stimulated to initiate proliferation, for example, by the addition of fresh serum, they begin DNA synthesis after a defined G0/G1 phase lag. Such stimulation of density-arrested cells provides a population of cells that can be useful in the investigation to elucidate the biochemical events that regulate the traverse of G0/G1. Serum is a complex heterogeneous mixture of proteins, hormones, growth factors, and other components. This complexity has made it difficult to characterize the necessary events leading to proliferation because other cellular reactions, not required for the control of proliferation, are also stimulated by the addition of serum.