title: An important role for CDK2 in G1 to S checkpoint activation and DNA damage response in human embryonic stem cells.
abstract: A precise understanding of mechanisms used by human embryonic stem cells (hESCs) to maintain genomic integrity is very important for their potential clinical applications. The G1 checkpoint serves to protect genomic integrity and prevents cells with damaged DNA from entering S-phase. Previously, we have shown that downregulation of cyclin-dependent kinase 2 (CDK2) in hESC causes G1 arrest, loss of pluripotency, upregulation of cell cycle inhibitors p21 and p27 and differentiation toward extraembryonic lineages. In this study, we investigate in detail the role of CDK2 in cellular processes, which are crucial to the maintenance of genomic stability in hESC such as G1 checkpoint activation, DNA repair, and apoptosis. Our results suggest that downregulation of CDK2 triggers the G1 checkpoint through the activation of the ATM-CHK2-p53-p21 pathway. Downregulation of CDK2 is able to induce sustained DNA damage and to elicit the DNA damage response (DDR) as evidenced by the formation of distinct γ-H2.AX and RAD52-BRCA1 foci in hESC nuclei. CDK2 downregulation causes high apoptosis at the early time points; however, this is gradually decreased overtime as the DDR is initiated. Our mass spectrometry analysis suggest that CDK2 does interact with a large number of proteins that are involved in key cellular processes such as DNA replication, cell cycle progression, DNA repair, chromatin modeling, thus, suggesting a crucial role for CDK2 in orchestrating a fine balance between cellular proliferation, cell death, and DNA repair in hESC.
authors.affiliation: Institute of Human Genetics, Newcastle University, International Centre for Life, Newcastle upon Tyne, United Kingdom.
authors.forename: Irina
authors.initials: I
authors.lastname: Neganova
authors.forename: Felipe
authors.initials: F
authors.lastname: Vilella
authors.forename: Stuart P
authors.initials: SP
authors.lastname: Atkinson
authors.forename: Maria
authors.initials: M
authors.lastname: Lloret
authors.forename: João F
authors.initials: JF
authors.lastname: Passos
authors.forename: Thomas
authors.initials: T
authors.lastname: von Zglinicki
authors.forename: José-Enrique
authors.initials: JE
authors.lastname: O'Connor
authors.forename: Deborah
authors.initials: D
authors.lastname: Burks
authors.forename: Richard
authors.initials: R
authors.lastname: Jones
authors.forename: Lyle
authors.initials: L
authors.lastname: Armstrong
authors.forename: Majlinda
authors.initials: M
authors.lastname: Lako
mesh_terms: D017209:Apoptosis; D064007:Ataxia Telangiectasia Mutated Proteins; D019313:BRCA1 Protein / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D016193:G1 Phase; D020868:Gene Silencing; D006657:Histones / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D034741:RNA, Small Interfering; D051721:Rad52 DNA Repair and Recombination Protein / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D019313:BRCA1 Protein; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; C497194:RAD52 protein, human; D034741:RNA, Small Interfering; D051721:Rad52 DNA Repair and Recombination Protein; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2