title: 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 CDK2AP1 in 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.
authors.affiliation: Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, United States of America; King Abdullah International Medical Research Center, King Saud bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia.
authors.forename: Khaled N
authors.initials: KN
authors.lastname: Alsayegh
authors.affiliation: Integrated Life Sciences Program, Virginia Commonwealth University, Richmond, Virginia, United States of America.
authors.forename: Venkat S
authors.initials: VS
authors.lastname: Gadepalli
authors.affiliation: Center for the Study of Biological Complexity, Life Sciences Program, Virginia Commonwealth University, Richmond, Virginia, United States of America; Department of Chemical and Life Sciences Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America.
authors.forename: Shilpa
authors.initials: S
authors.lastname: Iyer
authors.affiliation: Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, United States of America; Integrated Life Sciences Program, Virginia Commonwealth University, Richmond, Virginia, United States of America; Center for the Study of Biological Complexity, Life Sciences Program, Virginia Commonwealth University, Richmond, Virginia, United States of America; Department of Chemical and Life Sciences Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America; Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia, United States of America.
authors.forename: Raj R
authors.initials: RR
authors.lastname: Rao
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics; D049109:Cell Proliferation; D016922:Cellular Senescence / Q000235:genetics*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics; D004249:DNA Damage; D005347:Fibroblasts / Q000166:cytology*; D055785:Gene Knockdown Techniques; D006801:Humans; D011518:Proto-Oncogene Proteins / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D025803:Tumor Suppressor Protein p14ARF / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000172:deficiency* / Q000235:genetics*; D051028:bcl-2-Associated X Protein / Q000235:genetics
chemical_list: D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; C096151:CDK2AP1 protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D051028:bcl-2-Associated X Protein