Evidence that density‐dependent growth arrest is a two‐stage process in WI‐38 cells

Abstract

It was the goal of this study to determine whether during long-term quiescence WI-38 cells gradually lose labile components which then need to be resynthesized before a stimulated cell can progress through G-1 and enter S. The metabolic and molecular status of WI-38 cells was systematically analyzed as they entered and were maintained for an extended period of time in a state of density-dependent growth arrest. Our results indicate that growth arrest in WI-38 cells can be divided into two stages. The first, which we call "early" growth arrest, occurs during the first 7-10 days following cessation of DNA synthesis and mitosis. It is characterized by few biochemical changes compared to actively proliferating cells. During this period of early growth arrest cells do not exhibit a prolongation of the prereplicative stage following serum stimulation. In contrast, WI-38 cells growth arrested for 10-20 days exhibit a number of changes at the molecular and biochemical level (i.e., a twofold decrease in total protein and total RNA content, and decreased levels of most proteins, but an increased amount of fibronectin and collagen). Also, quiescent WI-38 cells stimulated at any time during "later" or "deep" growth arrest do exhibit a prolonged prereplicative phase. Although changes were also observed in the patterns of expression of ten representative growth-associated genes (i.e., histone H-3, p53, c-Ha-ras, 2A9/calcyclin, 4F1/vimentin, LDL-receptor, insulin receptor, collagen, and fibronectin), these occurred mostly at the time when the cells ceased synthesis of DNA and mitosis and became quiescent. No changes in the steady-state levels of the growth-associated transcripts analyzed occurred while the cells were maintained in the growth-arrested state. Thus, these experiments show that although WI-38 cells do cease to incorporate thymidine and divide under crowded culture conditions, the "quiescent" cells continue to undergo changes, are metabolically active, and certainly do not grossly deteriorate.

Diverse gene sequences are overexpressed in werner syndrome fibroblasts undergoing premature replicative senescence.

“…Strong inverse relationships between expression of one or more of these genes and cellular proliferation have been reported in a variety of systems undergoing the growth arrest that accompanies differentiation or high-density contact inhibition, including osteoblasts (72), endothelial cells (48), myoblasts (61), chondroblasts (78), fibroblasts (57), and smooth muscle cells (73). Indeed, production of fibronectin and collagen, essential ECM elements involved in development, cell adhesion, blood clotting, and maintenance and repair of virtually all tissues, is often coordinately linked, as appeared to be the case here (Fig.…”

Regulation of human ornithine decarboxylase expression following prolonged quiescence: Role for the c‐Myc/Max protein complex

“…As described previously (Owen et al, 1990;Soprano, 1994), density-dependent growth arrest in WI-38 cells can be divided into two stages: the first, which occurs during the first 7-10 days after plating, is called early or short-term quiescence and exhibits no prolongation of the prereplicative stage. The second stage, called prolonged or long-term quiescence, occurs 11 days or more following growth arrest (or 18 days after plating).…”

Epidermal growth factor receptors lose ligand binding ability as WI‐38 cells progress from short‐term to long‐term quiescence

“…We have previously shown that several matrix proteins continued to accumulate during growth arrest. For example, fibronectin and collagen increased more than fourfold from shortto long-term quiescence (Owen et al, 1990). However, we have found that EGF binding increases to normal levels after 3 hours of serum stimulation.…”

“…Long-term, growth-arrested WI-38 cells have only approximately half the total protein of cycling cells (Owen et al, 1990) and exhibit differences in the levels of a variety of mRNAs. The half-life of EGF receptor protein has been reported t o be 10-11 hours in the absence of EGF (Stoscheck and Carpenter, 1984;Stoscheck, 1985).…”

“…Finally, the observation that long-term, growth-arrested WI-38 cells internalize EGF at a slower rate may explain, at least in part, the characteristic prolongation of the pre-replicative phase that occurs following stimulation (Cosenza et al, 1988;Owen et al, 1990). It is tempting to speculate that a critical level of EGF must be bound and internalized in order to initiate the signal transduction pathway regulating GI progression.…”