Expression of cell cycle-dependent genes in young and senescent WI-38 fibroblasts.

Abstract

We studied the expression of 11 cell cycledependent genes in senescent WI-38 fibroblasts and compared the results to those obtained in WI-38 cells from early passages (young cells). Every gene we examined is expressed in the senescent cells at levels similar to those in the young cells, including two genes maximally expressed at the G1/S phase boundary-genes for thymidine kinase and histone H3. The results clearly show that senescent, noncycling WI-38 cells are not similar to quiescent cells. Rather, such senescent WI-38 cells may be blocked just prior to the onset of DNA synthesis.The human diploid fibroblast line WI-38 has a limited lifespan in culture (1, 2) and has been used frequently as a model of cellular senescence. Cultures are capable of a prolonged period of high proliferative activity followed by a gradual decrease in growth rate (1) and an increase in the fraction of cells arrested in the nonreplicative phase (3). The in vitro aging ofthese cells is characterized by an exponential decline in the percentage of the population capable of synthesizing DNA and an increase in cell cycle time (4). The primary increase in cell cycle time occurs in the G1 phase, which progressively lengthens until the cells eventually become arrested (4). Several lines of evidence suggest that senescent cells are blocked in late G1 rather than in Go phase, as are quiescent cells. First, the level of thymidine kinase (TK) activity in senescent, slowly proliferating cultures is similar to that ofyoung, rapidly dividing populations (5). Second, the expansion of the thymidine triphosphate pool prior to DNA synthesis is similar in young and senescent cells (6). Finally, the nuclear fluorescence pattern of senescent cells after staining with quinacrine dihydrochloride is typical of cells that are blocked in late G1 or at the G1/S boundary (7,8).There are several genes whose expression is known to be cell cycle-dependent, the term expression being used here in one of its accepted usages-i.e., as levels of cytoplasmic mRNAs. Cell cycle-dependent genes include the oncogenes c-myc (9), c-fos (10-13), and c-ras (14-16); the gene encoding the cellular tumor antigen p53 (17); genes isolated as cDNA clones in our (18) and in other (19-21) laboratories on the basis of their cell cycle dependency of expression; and genes coding for well-characterized cellular proteins, such as ornithine decarboxylase (ODCase) (22, 23), P-actin (24,14), thymidine kinase (TK) (25), and histones (26)(27)(28). Most of these genes are induced in different cell types stimulated by different mitogens (29), and their mRNA levels can be used as markers of a cell's progression through G1 and S phases (29).In this paper, we report our investigation of the expression of several of these cell cycle genes in senescent WI-38 cells and comparison of the results to those obtained in young cells. These experiments are similar to those performed with temperature-sensitive (ts) mutants of the cell cycle (25,28,30) in which the expression or lack of expression of cell c...

Murine fibroblasts lacking p21 undergo senescence and are resistant to transformation by oncogenic Ras

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Mitogen Stimulation Cooperates with Telomere Shortening To Activate DNA Damage Responses and Senescence Signaling

“…Once arrested, the senescent cells cannot be stimulated to enter S phase by any known combination of physiological mitogens. Many genes, including several proto-oncogenes, remain mitogen inducible in senescent cells (41,47). Thus, senescent cells fail to proliferate not because of a loss of growth factor signal transduction but rather because of selective repression of a few positively acting, growth regulatory genes whose expression is essential for G 1 -phase progression and DNA synthesis (41,47).…”

Insulin-like Growth Factor-I Extends in VitroReplicative Life Span of Skeletal Muscle Satellite Cells by Enhancing G1/S Cell Cycle Progression via the Activation of Phosphatidylinositol 3′-Kinase/Akt Signaling Pathway

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