Role of Endogenous Proteins as Negative Growth Modulators During In Vitro Cellular Aging of Human Diploid Fibroblasts

1991 ◽  
pp. 53-70
Author(s):  
Mary Beth Porter ◽  
James R. Smith
Keyword(s):  
Cellular Aging ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Endogenous Proteins ◽  
Negative Growth

scholarly journals Expression of Senescence-Associated microRNAs and Target Genes in Cellular Aging and Modulation by Tocotrienol-Rich Fraction

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Sharon Gwee Sian Khee ◽  
Yasmin Anum Mohd Yusof ◽  
Suzana Makpol
Keyword(s):  
Cellular Senescence ◽  
Target Genes ◽  
Replicative Senescence ◽  
Primary Cultures ◽  
Ectopic Expression ◽  
Cellular Aging ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Tocotrienol Rich Fraction

Emerging evidences highlight the implication of microRNAs as a posttranscriptional regulator in aging. Several senescence-associated microRNAs (SA-miRNAs) are found to be differentially expressed during cellular senescence. However, the role of dietary compounds on SA-miRNAs remains elusive. This study aimed to elucidate the modulatory role of tocotrienol-rich fraction (TRF) on SA-miRNAs (miR-20a, miR-24, miR-34a, miR-106a, and miR-449a) and established target genes of miR-34a (CCND1, CDK4, and SIRT1) during replicative senescence of human diploid fibroblasts (HDFs). Primary cultures of HDFs at young and senescent were incubated with TRF at 0.5 mg/mL. Taqman microRNA assay showed significant upregulation of miR-24 and miR-34a and downregulation of miR-20a and miR-449a in senescent HDFs (P<0.05). TRF reduced miR-34a expression in senescent HDFs and increased miR-20a expression in young HDFs and increased miR-449a expression in both young and senescent HDFs. Our results also demonstrated that ectopic expression of miR-34a reduced the expression of CDK4 significantly (P<0.05). TRF inhibited miR-34a expression thus relieved its inhibition on CDK4 gene expression. No significant change was observed on the expression of CCND1, SIRT1, and miR-34a upstream transcriptional regulator, TP53. In conclusion tocotrienol-rich fraction prevented cellular senescence of human diploid fibroblasts via modulation of SA-miRNAs and target genes expression.

Transcription factor activity during cellular aging of human diploid fibroblasts

1992 ◽  
Vol 70 (10-11) ◽  
pp. 1064-1072 ◽  
Author(s):  
Karl T. Riabowol
Keyword(s):  
Cell Cycle ◽  
Binding Activity ◽  
Cellular Aging ◽  
Proliferative Potential ◽  
Activator Protein 1 ◽  
Direct Role ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Molecular Events

Human diploid fibroblasts display a limited proliferative life span in vitro, which is directly correlated to the age of the donor from which the cells were explanted. In an effort to identify molecular events that may underlie the loss of proliferative potential in aging fibroblasts, we have determined, at the protein level, the abundance of several cell-cycle-regulated proteins and the activity of the two major members of the activator protein-1 (AP-1) DNA binding complex. We find that cyclin A and p34cdc2 expression is decreased by two- to four-fold in old fibroblasts, but that Fos expression and binding activity are reduced by as much as 95% in old, as opposed to young cells, despite equivalent amounts of p105Rb and Jun proteins being expressed. We have further determined that the composition of the protein complex which binds a consensus (-TGACTCA-) AP-1 site changes dramatically during in vitro aging. Since we have shown previously that AP-1 activity is required for progression through the cell cycle, we propose that the quantitative and qualitative changes seen in AP-1 may play a direct role in the gradual loss of proliferative ability seen as cells approach senescence.Key words: cellular aging, Fos, transcription factors.

Induction of 6-thioguanine-resistant mutants in human diploid fibroblasts in vitro with ethylene oxide

1992 ◽  
Vol 19 (2) ◽  
pp. 93-97 ◽  
Author(s):  
Ada Kolman ◽  
Tatiana Bohušová ◽  
B. Lambert ◽  
J. W. I. M. Simons
Keyword(s):  
Ethylene Oxide ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Resistant Mutants

14-3-3 Facilitates Insulin-Stimulated Intracellular Trafficking of Insulin Receptor Substrate 1

2002 ◽  
Vol 16 (3) ◽  
pp. 552-562 ◽  
Author(s):  
Xiaoqin Xiang ◽  
Mingsheng Yuan ◽  
Ying Song ◽  
Neil Ruderman ◽  
Rong Wen ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Intracellular Trafficking ◽  
High Speed ◽  
Insulin Treatment ◽  
Insulin Receptor Substrate ◽  
Insulin Receptor Substrate 1 ◽  
Integral Role ◽  
Endogenous Proteins

Abstract The appearance of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and PI3K in a high-speed pellet fraction (HSP) is thought to be a key event in insulin action. Conversely, the disappearance of the IRS-1/PI3K complex from this fraction has been linked to insulin desensitization. The present study examines the role of 14-3-3, a specific phospho-serine binding protein, in mediating the disappearance of IRS-1 from the HSP after insulin treatment. An in vitro pull-down assay using recombinant 14-3-3 revealed that insulin enhances the association of 14-3-3 with IRS-1 in cultured adipocytes and that this is completely inhibited by wortmannin. An association of IRS-1 and 14-3-3 was also observed and was maximal after stimulation by insulin, when endogenous proteins were immunoprecipitated. Epidermal growth factor (EGF), 12-O-tetradecanoylphorbol-13-acetate, and okadaic acid, other agents that cause serine/threonine phosphorylation of IRS-1, also stimulated IRS binding to 14-3-3. The enhancement of IRS-1 binding to 14-3-3 by insulin was accompanied by movement of IRS-1 and the p85 subunit of PI3K from the HSP to the cytosol. In keeping with a key role of 14-3-3 in mediating this redistribution of IRS-1, the complexes of IRS-1 and 14-3-3 were found in the cytosol but not in the HSP of insulin-treated cells. In addition, colocalization of IRS-1 and 14-3-3 was observed in the cytoplasm after insulin treatment by confocal microscopy. Finally, the addition of a phosphorylated 14-3-3 binding peptide to an adipocyte homogenate (to remove 14-3-3 from IRS-1) increased the abundance of IRS-1/PI3K complexes in the HSP and decreased their abundance in the cytosol. These findings strongly suggest that 14-3-3 participates in the intracellular trafficking of IRS-1 by promoting the displacement of serine-phosphorylated IRS-1 from particular structures. They also suggest that 14-3-3 proteins could play an integral role in the process of insulin desensitization.

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