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.
Highlights
Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1) is a cell cycle regulator that controls the G1-S phase transition by negatively regulating CDK2 [1]
Based on qPCR analysis, we were able to achieve 90% knockdown of CDK2AP1, with statistical analysis indicating significant differences (p
Following the generation of CDK2AP1 knockdown primary human fibroblasts, we examined the effect of the knockdown on cell proliferation
Summary
CDK2AP1 is a cell cycle regulator that controls the G1-S phase transition by negatively regulating CDK2 [1]. In vitro studies focused on overexpression of CDK2AP1 in prostate cancer cell lines results in a decrease in levels of CDK2 and its kinase activity, leading to an accumulation of cells in the G1 phase and a reduction in cells that are in the S phase of the cell cycle [2]. This outcome has been reasoned to be mediated by either the sequestration of monomeric CDK2 or by targeting it for proteolysis. When xenograft formation was used to examine in vivo tumorigenesis, CKD2AP1downregulation was found to inhibit tumor growth [7]
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