Involvement of Rb family proteins, focal adhesion proteins and protein synthesis in senescent morphogenesis induced by hydrogen peroxide

Abstract

Early passage human diploid fibroblasts develop senescent morphology prematurely within a week after a 2-hour pulse treatment with low or mild dose H(2)O(2). We test here the role of cell cycle checkpoints, cytoskeletal proteins and de novo protein synthesis in senescent morphogenesis following H(2)O(2) treatment. H(2)O(2) 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 H(2)O(2). The mutants of the E7 gene, in which the Rb family protein binding site was destroyed, could not reduce Rb protein or prevent H(2)O(2) 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 H(2)O(2) treatment. Finally, depletion of methionine in the culture medium after H(2)O(2) 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.

Design and Development of an HBT-Based Ratiometric Fluorescent Probe to Monitor Stress-Induced Premature Senescence

“…Although the signal was weak in the green channel, this might be able to detect acid lysosomal β-Gal. The change in cell morphology because of cellular senescence is characterized by an enlarged, flattened, and irregular shape . These morphological changes were observed in many cells after the treatment using H 2 O 2 (Figure S9).…”

Analysis of senescence-responsive stress fiber proteome reveals reorganization of stress fibers mediated by elongation factor eEF2 in HFF-1 cells

“…Along with these responses, interestingly, a significant thickening of individual SFs is induced in senescent cells. It remains, however, controversial whether senescence leads to thicker or thinner SFs ( Chen et al. , 2000 ; Nishio and Inoue, 2005 ), despite their inherent connection to cell morphology and migration ( Hotulainen and Lappalainen, 2006 ; Meng and Takeichi, 2009 ; Lin et al.…”

Pancreatic endocrine‐like cells differentiated from human umbilical cords Wharton’s jelly mesenchymal stem cells using small molecules

“…The cell cycle analysis revealed that TSA at 100 nM/L concentration caused an arrest in cell cycle at G 0 /G 1 phase (Figure 4b), possibly through the activation of p53-and p21mediated pathways (Figure 4e-g). The observed morphological changes are reported to be typically associated with cellular senescence (Chen et al, 2000), and hence, we assumed that the inhibition of cell proliferation and changes in cell shape may precede cell senescence. Therefore, we extended our treatment period up to 5 days and did not find any increase in SA-β-gal activity, confirming there was no occurrence of cellular senescence (Figure 4d-g).…”