Cell density, negative proliferation control, and phosphorylation of retinoblastoma protein.

Cell density negative control (CDNC) of normal human fibroblast proliferation occurs after stimulation by mitogens with different signal transduction mechanism. Delayed exposure to agents that interfere with CDNC, such as double-stranded RNA and vanadate, reveals the existence of a biochemical event, involved in CDNC, that occurs 5-8 hr after the beginning of mitogenic stimulation. This is earlier than the point of "mitogenic commitment," defined by the duration of mitogen exposure required for cell cycle entry (8-18 hr). Phosphorylation of the retinoblastoma gene product (pRB) begins 8-10 hr after mitogen stimulation and is nearly complete at 18 hr, just as the first cells enter S-phase. CDNC prevents pRB phosphorylation. Interferon beta delays pRB phosphorylation by up to 20 hr but has little effect on the timing of mitogenic commitment. Thus mitogenic commitment is located in time between CDNC and pRB phosphorylation. When agents that cause a release from CDNC are applied to dense, negatively controlled cultures after 18 hr of EGF stimulation, pRB phosphorylation occurs 6-8 hr after release. This suggests that the negatively controlled cells process the mitogenic signal but accumulate at a restriction point. The relatively early timing of CDNC-related events in the prereplicative phase raises the possibility that pRB phosphorylation is a consequence rather than a prerequisite for release from cell density negative control.