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

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.