Involvement of Rb family proteins, focal adhesion proteins and protein synthesis in senescent morphogenesis induced by hydrogen peroxide.
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.
Chen
Q M
QM
Department of Pharmacology, University of Arizona, Skaggs Pharmaceutical Science Building, Tucson, AZ 85721, USA. chen@pharmacy.arizona.edu
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V C
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Catania
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Burton
M
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Toussaint
O
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Dilley
T
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D000199:Actins / Q000378:metabolism / Q000648:ultrastructure; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D016922:Cellular Senescence / Q000502:physiology*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009024:Morphogenesis; D009856:Oncogene Proteins, Viral / Q000235:genetics; D027383:Papillomaviridae / Q000235:genetics; D050725:Papillomavirus E7 Proteins; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D012097:Repressor Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D016596:Vinculin / Q000378:metabolism
D000199:Actins; D003598:Cytoskeletal Proteins; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; C496061:PXN protein, human; D050725:Papillomavirus E7 Proteins; D051419:Paxillin; D010750:Phosphoproteins; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; C059731:oncogene protein E7, Human papillomavirus type 16; D016596:Vinculin; D006861:Hydrogen Peroxide