Alterations of Expression of the Cytoskeleton after Immortalization of Human Fibroblasts.

Abstract

ABSTRACT. Human diploid fibroblasts (HDF) have a limited in vitro lifespan of population doubling level (PDL) 50-70. The molecular mechanismunderlying cellular senescence and immortalization is not thoroughly understood. It has been reported that the cytoskeleton has diverse functions and may have a role in growth regulation through association with other cellular components.To shed light on the relationship between functions of the cytoskeleton and senescence or immortalization, we investigated the alterations in gene expression after immortalization and measuredthe amountsof mRNAs for talin, vinculin, a-actinin, tropomyosin 1 (TM1), vimentin, lamin A and C, and a-tubulin by slot blot and Northern blot analyses. Wefound that the mRNAs for vinculin and vimentin were reduced and the mRNA for lamin A was increased in immortalized cells. Wealso studied the cytoskeletal protein levels and their intracellular distributions by Western blot analysis and immunostaining. Most of the proteins studied behaved in a way similar to the mRNAs through senescence and immortalization. Vinculin, tropomyosins and vimentin showed their altered distributions in immortalized The physical end of eukaryotic chromosomes is called telomere and is comprised of simple repeat sequences of 5-15 kb. The unit sequence of it is 5'-TTA GGG-3' in mammals and is highly conserved throughout evolution (4). Although DNApolymerase is incapable of the complete replication of linear DNA molecules, the specialized telomere structures and the ribonucleoprotein enzyme, telomerase, allow for the complete replication of chromosomeends (4). In agreement with this notion, telomerase activity has been detected in unicellular eukaryotes, the germ line cells and humancancer cells (21, 33 Olovnikov and others proposed the telomere hypothesis of cellular ageing, arguing that absence of telomerase and shortening of telomere due to incomplete DNAreplication are the cause of cellular senescence (21, 37). One would expect alterations of gene and gene expression on the basis of the "programme" theory of senescence. In an attempt to find out genes involved in the eel-

Senescence and cytoskeleton: overproduction of vimentin induces senescent-like morphology in human fibroblasts

“…Thus, the senescent fibroblasts could express a high level of vimentin protein. In fact, the increased vimentin-expression in senescent cells observed in the present study is consistent with that reported with SV40-transformed, but senescent, human fibroblasts (SV40-transformed immortal counterparts on the contrary exhibit a reduced expression of vimentin) (Satoh et al 1994;Kaneko et al 1995). Similarly, an increase in vimentin has been reported with Sertoli cells in testis tubules of elderly men (de Miguel et al 1997) and with old rats' astrocytes, which contain bundles of hypertrophic intermediate filaments (Berciano et al 1995).…”

Inhibition of Anchorage-independent Growth of Transformed NIH3T3 Cells by Epithelial Protein Lost in Neoplasm (EPLIN) Requires Localization of EPLIN to Actin Cytoskeleton

“…Loss of cytoskeletal architecture is thought to be a contributing factor, rather than a consequence, of neoplastic transformation (Janmey and Chaponnier, 1995;BenZe'ev, 1997;Pawlak and Helfman, 2001). Expression of several actin-binding proteins, including tropomyosin, vinculin, ␣-actinin, and gelsolin has shown to be downregulated in many transformed cells (Kaneko et al, 1995). Restoring these proteins can reverse the malignant phenotype in several different experimental models of transformation (Gluck et al, 1993;Braverman et al, 1996;Kwon et al, 1997).…”

Critical pathways in cellular senescence and immortalization revealed by gene expression profiling

“…Supporting evidence for the involvement of cytoskeletal genes in senescence comes from several laboratories that have shown an increase in expression of cytoskeletal genes, such as vimentin and fibronectin, in senescent human fibroblasts (Murano et al ., 1991; Satoh et al ., 1994; Kaneko et al ., 1995). Among the genes that were overexpressed in senescent Werner syndrome fibroblasts, several are cytoskeletal related including fibronectin, PAI-1 and thrombospondin (Murano et al ., 1991; Lecka-Czernik et al ., 1996).…”