Exit of human diploid cells from low serum quiescence

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.

Section: Discussionmentioning

confidence: 99%

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

Owen

Carter

Whitman

et al. 1990

“…The recovery of some cells to greater than control values of precursor incorporation is due to a n "overshoot" and has been previously described deprivation. Recently, the findings of Augenlicht and Baserga (1974) were not reproduced by Duncan et al (1982) utilizing a slightly different experimental protocol involving weekly refeeding of the serum-deprived cultures. Our results are more consistent with the notion that qualitative differences do exist between cultures that are maintained for prolonged (greater than 3week) periods in quiescence, and those that have been noncycling for shorter periods of time.…”

Section: Discussionmentioning

confidence: 99%

Evidence for differences in the mechanism of cell cycle arrest between senescent and serum‐deprived human fibroblasts: Heterokaryon and metabolic inhibitor studies

Burmer

Rabinovitch

Norwood

1984

It has previously been shown that serum-deprived, early passage quiescent human diploid fibroblastlike (HDFL) cells are able to inhibit cycling cells from entry into DNA synthesis upon cell fusion. We have found that the degree of inhibition of DNA synthesis in the heterokaryon correlates with the duration of serum deprivation, which is consistent with the suggestion that serum-deprived cells may enter progressively deeper stages of G0 as they increase their time in quiescence. In contrast to fusions with senescent cells, in heterokaryons between serum-deprived early passage and cycling young cells transient inhibition of protein synthesis with cycloheximide or inhibition of RNA synthesis with 5-6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB) did not stimulate nuclear [3H]-thymidine incorporation. These results suggest that differences may exist in the mechanisms responsible for inhibiting cell cycle progression in senescent vs early passage quiescent HDFL cells.

“…days after subcultivation will yield the variant ratios for S phase cells, while inclusion of HU at this time point will inhibit DNA synthesis and yield basal or G1 variant ratios. Confluent populations have been described as being in either the G1 or the GO state of the cell cycle (Augenlicht and Baserga, 19743, while quiescent populations incubated with 0.5% serum are described as being in GO (Duncan et al, 1982).…”

Section: Discussionmentioning

confidence: 99%

The effects of in vitro age and culture state on histone variant synthesis in human diploid fibroblasts

Dell’Orco

Worthington

1988

Histone variant synthesis patterns from human diploid fibroblast-like cells of different in vitro ages were determined during exponential growth, at confluence, and during low serum arrest. The results are reported as the ratios of H2A variant synthesis (H2A.1 and H2A.2/H2A.x and H2A.z) and H3 variant synthesis (H3.1 and H3.2/H3.3) that have been used to characterize individual cell cycle states. Hydroxyurea was employed in some experiments to reduce S phase cells. The results indicate that high population doubling level (PDL) cells move through the G1 phase of the division cycle during exponential growth and exist in the G0 cell cycle state at confluence and during low serum arrest. Low PDL cells, however, exist in the G1 cell cycle state at confluence and revert to a G0 state only after maintenance as quiescent populations. This would suggest that when stimulated high PDL cells cannot enter into S phase, they revert to a GO cell cycle state.