Qin M. Chen

Human diploid fibroblasts (HDFs) inevitably undergo replicative senescence in culture after serial passages. Recent work indicates that early passage HDFs undergo irreversible growth arrest and...

Early passage human diploid fibroblasts develop senescent morphology prematurely within a week after a 2-hour pulse treatment with low or mild dose H2O2. We test here the role of cell cycle checkpoints, cytoskeletal proteins and de novo protein synthesis in senescent morphogenesis following H2O2 treatment. H2O2 treatment causes transient elevation of p53 protein and prolonged inhibition of Rb hyperphosphorylation. Expression of human papillomaviral E6 gene prevented elevation of p53 but did not affect senescent morphogenesis. Expression of human papillomaviral E7 gene reduced the level of Rb protein and prevented induction of senescent morphology by H2O2. The mutants of the E7 gene, in which the Rb family protein binding site was destroyed, could not reduce Rb protein or prevent H2O2 from inducing senescent morphology. Senescent-like cells showed enhanced actin stress fibers. In untreated cells, vinculin and paxillin preferentially distributed along the edge of the cells. In contrast, vinculin and paxillin distributed randomly and sporadically throughout senescent-like cells. E7 expression prevented enhancement of actin filament formation and redistribution of vinculin or paxillin. Neither wild-type nor E7 cells showed changes in the protein level of actin, vinculin or paxillin measured by western blot after H2O2 treatment. Finally, depletion of methionine in the culture medium after H2O2 treatment prevented senescent morphogenesis without affecting dephosphorylation of Rb protein. Our results suggest that senescent morphology likely develops by a program involving activated Rb family proteins, enhancement of actin stress fibers, redistribution of focal adhesion proteins and de novo protein synthesis.

Abstract: Normal human diploid fibroblasts (HDFs) undergo replicative senescence inevitably in tissue culture after a certain number of cell divisions. A number of molecular changes observed in replicative senescent cells occur in somatic cells during the process of aging. Genetic studies on replicative senescence indicate the control of tumor suppression mechanisms. Despite the significance of replicative senescence in aging and cancer, little is known about the central cause of the complex changes observed in replicative senescent cells. The interest in the phenomenon has intensified in recent years, since damaging agents, certain oncogenes and tumor suppressor genes have been found to induce features of senescence in early passage young HDFs or in immortalized tumor cells. The reported features of senescence are summarized here in order to clarify the concept of replicative senescence or premature senescence. The experimental results of extending the replicative life span by reducing ambient oxygen tension or by N‐tert‐butyl‐alpha‐phenylnitrone (PBN) argue a role of oxidative damage in replicative senescence. By inducing premature senescence with a pulse treatment of H₂O₂, we can study the role of the cell cycle checkpoint proteins p53, p21, p16 and Rb in gaining each feature of senescence. Although p53 and Rb control G1 arrest and Rb appears to control cell enlargement, activation of the senescent associate β‐galactosidase, loss of cell replication and multiple molecular changes observed in premature senescent or replicative senescent cells are likely controlled by mechanisms beyond the cell cycle checkpoints.