Knockdown of CDK2AP1 in Primary Human Fibroblasts Induces p53 Dependent Senescence

Abstract

Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1) is known to be a tumor suppressor that plays a role in cell cycle regulation by sequestering monomeric CDK2, and targeting it for proteolysis. A reduction of CDK2AP1 expression is considered to be a negative prognostic indicator in patients with oral squamous cell carcinoma and also associated with increased invasion in human gastric cancer tissue. CDK2AP1 overexpression was shown to inhibit growth, reduce invasion and increase apoptosis in prostate cancer cell lines. In this study, we investigated the effect of CDK2AP1 downregulation in primary human dermal fibroblasts. Using a short-hairpin RNA to reduce its expression, we found that knockdown of CDK2AP1in primary human fibroblasts resulted in reduced proliferation and in the induction of senescence associated beta-galactosidase activity. CDK2AP1 knockdown also resulted in a significant reduction in the percentage of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Immunocytochemical analysis also revealed that the CDK2AP1 knockdown significantly increased the percentage of cells that exhibited γ-H2AX foci, which could indicate presence of DNA damage. CDK2AP1 knockdown also resulted in increased mRNA levels of p53, p21, BAX and PUMA and p53 protein levels. In primary human fibroblasts in which p53 and CDK2AP1 were simultaneously downregulated, there was: (a) no increase in senescence associated beta-galactosidase activity, (b) decrease in the number of cells in the G1-phase and increase in number of cells in the S-phase of the cell cycle, and (c) decrease in the mRNA levels of p21, BAX and PUMA when compared with CDK2AP1 knockdown only fibroblasts. Taken together, this suggests that the observed phenotype is p53 dependent. We also observed a prominent increase in the levels of ARF protein in the CDK2AP1 knockdown cells, which suggests a possible role of ARF in p53 stabilization following CDK2AP1 knockdown. Altogether, our results show that knockdown of CDK2AP1 in primary human fibroblasts reduced proliferation and induced premature senescence, with the observed phenotype being p53 dependent.

Dinaciclib potently suppresses MCL-1 and selectively induces the cell death in human iPS cells without affecting the viability of cardiac tissue

“…p53 specific sequence and control shRNAs were obtained from Addgene (Cambridge, MA, USA). Lentiviral particles were produced using the second-generation packaging system obtained from Addgene as reported previously 41 . In short, 10 μg of p53 or control shRNA plasmid, 7.5 μgs of packaging plasmid (psPAX2 plasmid) and 2.5 μg of envelope plasmid (pMD2.G plasmid) were transfected in HEK293t cells using FUGENE 6 (Promega, Fitchburg, WI, USA) transfection reagent according to manufacturer’s instructions.…”

“…Protein extracts were centrifuged at 12,000 rpm at 4 °C for 5 minutes and supernatant was collected for Western Blot analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting analysis were carried out as previously described 41 . The membrane was photographed by the chemiluminescence imager (LAS 4000; GE Healthcare).…”

Human Dermal Stem/Progenitor Cell-Derived Conditioned Medium Improves Senescent Human Dermal Fibroblasts

“…The tumor suppressor p53 regulates cell cycle arrest, apoptosis, and cellular senescence [ 2 , 3 , 4 , 5 ]. We thus investigated whether hDSPC-CM could inhibit the p53 signaling pathway.…”

“…According to previous reports, cell cycle regulators and DNA damage response pathways are strongly implicated in the onset of senescence [ 1 , 2 , 3 , 5 , 6 , 20 ]. For example, increased p53 phosphorylation forces cells into a state of premature senescence [ 2 , 3 , 4 ]; as cells enter G0, p21, a well-known target gene of p53, increases in a p53-dependent manner.…”

“…Various harmful signals, including oxidative stress, DNA damage, and telomere shortening, have been reported to promote cellular senescence [ 2 , 3 ]. This cellular status is characterized by growth arrest, enlarged cell size, increased expression of senescence-associated β-galactosidase (SA-β-Gal), and accumulation of the tumor suppressors p53 and p21, which participate in cell cycle arrest [ 4 , 5 ]. Cellular senescence is closely related to the aging process; the number of senescent cells gradually increases with age, and this accumulation contributes to tissue aging and the development of age-related diseases [ 6 ].…”