Inhibitors of cyclin-dependent kinases induce features of replicative senescence in early passage human diploid fibroblasts

Foci of phosphorylated histone H2AX and ATM are the surrogate markers of DNA double strand breaks. We previously reported that the residual foci increased their size after irradiation, which amplifies DNA damage signals. Here, we addressed whether amplification of DNA damage signal is involved in replicative senescence of normal human diploid fibroblasts. Large phosphorylated H2AX foci (>1.5 μm diameter) were specifically detected in presenescent cells. The frequency of cells with large foci was well correlated with that of cells positive for senescence-associatedβ-galactosidase staining. Hypoxic cell culture condition extended replicative life span of normal human fibroblast, and we found that the formation of large foci delayed in those cells. Our immuno-FISH analysis revealed that large foci partially localized at telomeres in senescent cells. Importantly, large foci of phosphorylated H2AX were always colocalized with phosphorylated ATM foci. Furthermore, Ser15-phosphorylated p53 showed colocalization with the large foci. Since the treatment of senescent cells with phosphoinositide 3-kinase inhibitor, wortmannin, suppressed p53 phosphorylation, it is suggested that amplification of DNA damage signaling sustains persistent activation of ATM-p53 pathway, which is essential for replicative senescence.

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Effects of Glucose Restriction on Replicative Senescence of Human Diploid Fibroblasts IMR-90

Cellular Physiology and Biochemistry ◽

10.1159/000350090 ◽

2013 ◽

Vol 31 (4-5) ◽

pp. 718-727 ◽

Cited By ~ 4

Author(s):

Junhua Jin ◽

Tiemei Zhang

Keyword(s):

Replicative Senescence ◽

Diploid Fibroblasts ◽

Human Diploid Fibroblasts ◽

Glucose Restriction

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Features of replicative senescence induced by direct addition of antennapedia-p16INK4A fusion protein to human diploid fibroblasts

FEBS Letters ◽

10.1016/s0014-5793(98)00426-8 ◽

1998 ◽

Vol 427 (2) ◽

pp. 203-208 ◽

Cited By ~ 61

Author(s):

Daishiro Kato ◽

Kazuhiro Miyazawa ◽

Marugarida Ruas ◽

Maria Starborg ◽

Ikuo Wada ◽

...

Keyword(s):

Fusion Protein ◽

Replicative Senescence ◽

Diploid Fibroblasts ◽

Human Diploid Fibroblasts ◽

Direct Addition

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

Oxidative Medicine and Cellular Longevity ◽

10.1155/2014/725929 ◽

2014 ◽

Vol 2014 ◽

pp. 1-12 ◽

Cited By ~ 7

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.

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