ABSTRACT
In order to investigate signal transduction pathways and related changes of actin cytoskeleton organization in cellular senescence, H-ras double mutants--V12S35, V12G37, and V12C40--were constitutively expressed in human foreskin fibroblast (HDF). Senescent HDF cells as well as the H-ras mutant expressers accumulated p-Erk1/2 in the cytoplasm with increased MEK activity and failed to translocate it to nuclei on EGF stimulation. Senescent HDF cells, V12S35 and V12G37 expressers, revealed a failure to export actin fiber from nucleus to cytoplasm and also to form stress fibers. Perinuclear expression of Rac1 was prominent in the HDF cells and V12C40 expresser; however, in the V12S35 expresser, translocation of Rac1 from perinucleus to nucleus and strong expression of RhoA were obvious. In summary, the H-ras double mutant expressers induced premature senescence through the MEK pathway, accompanied by nuclear accumulation of actin and Rac1 proteins, cytoplasmic retention of p-Erk1/2, and marked induction of RhoA expression, suggesting the translocational inefficiency of the intracellular proteins in the senescent HDF cells.
Subject(s)
Active Transport, Cell Nucleus , Cellular Senescence/physiology , Cytoskeleton/physiology , Fibroblasts/cytology , Genes, ras , MAP Kinase Kinase Kinase 1 , MAP Kinase Signaling System , 3T3 Cells/drug effects , 3T3 Cells/ultrastructure , Actins/metabolism , Animals , Blood Proteins/metabolism , Cell Nucleus/metabolism , Cell Surface Extensions , Cyclin-Dependent Kinase Inhibitor p16/physiology , Cytoplasm/metabolism , Cytoskeletal Proteins/metabolism , Cytoskeleton/ultrastructure , Diploidy , Fibroblasts/metabolism , Genes, p16 , Genes, p53 , Humans , Male , Membrane Proteins/metabolism , Mice , Microfilament Proteins/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Phosphoproteins/metabolism , Protein Isoforms/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins p21(ras)/physiology , Stress Fibers/metabolism , Tumor Suppressor Protein p53/physiology , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolismABSTRACT
In order to investigate the role of signal transduction and the related changes of actin cytoskeleton organization in the process of cellular senescence, H-ras double mutants--V12S35, V12G37 and V12C40--proteins were expressed constitutively in human diploid fibroblast (HDF) cells by retrovirus infection at PD26. Constitutive expression of V12S35, V12G37 and V12C40 proteins induced premature senescence at PD38, PD47 and PD50, respectively, in contrast to the control cells at PD59. Premature senescence was evidenced by the slow cellular growth rate and SA-beta-galactosidase expression accompanied by morphological changes such as flat and large cell shape. Senescent HDF cells as well as the H-ras mutant expressers accumulated p-Erk1/2 in the cytoplasm with increased MEK activity and failed to translocate it to nuclei on EGF stimulation. Senescent HDF cells as well as V12S35 and V12G37 expressers were unable to export actin fibers from nucleus to cytoplasm, form stress fibers through the MAPK and Ral.GDS pathways. Perinuclear expression of Racl was prominent in the HDF cells and V12C40 expresser, while translocation of Racl from perinucleus to nucleus and strong expression of RhoA were observed in the V12S35 expresser. In summary, the induced premature senescence by H-ras double mutants were accompanied by nuclear accumulation of actin and Racl proteins, cytoplasmic retention of p-Erk1/2 and marked induction of RhoA expression mainly through dysregulation of the MEK pathway.
Subject(s)
Actins/metabolism , Cellular Senescence/physiology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Biological Transport , Cell Nucleus/metabolism , Cells, Cultured , Child , Cytoplasm/metabolism , Diploidy , Enzyme Activation , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Male , Mitogen-Activated Protein Kinase 3 , Mutagenesis , Proto-Oncogene Proteins p21(ras)/metabolism , Retroviridae/physiology , rac1 GTP-Binding Protein/biosynthesis , rhoA GTP-Binding Protein/biosynthesisABSTRACT
Alloplastic implants are known to be inert for many years, though complications are infrequently reported many years after their insertion. We report the case of a patient who had undergone a blow-out fracture repair five years before the discovery of a hematic cyst. He had been free of symptoms for the first five years after his orbital floor repair but then developed pain on eyeball movement and persistent vertical diplopia, which finally led to surgical intervention. At surgery, a hematic cyst was found to have formed around the implanted silastic plate. When alloplastic material is used in orbital fracture repair, we should be alert for late complications which may occur many years after surgery.
