title: Widdrol activates DNA damage checkpoint through the signaling Chk2-p53-Cdc25A-p21-MCM4 pathway in HT29 cells.
abstract: Widdrol is an odorant compound isolated from Juniperus chinensis. We previously reported that widdrol induces Gap 1 (G1) phase cell cycle arrest and leads to apoptosis in human colon adenocarcinoma HT29 cells. It was also reported that this cell cycle arrest is associated with the induction of checkpoint kinase 2 (Chk2), p53 phosphorylation and cyclin dependent kinase (Cdk) inhibitor p21 expression. In this paper, we investigated the molecular mechanisms of widdrol on the activation of G1 DNA damage checkpoint at early phase when DNA damages occurred in HT29 cells. First of all, we examined that widdrol breaks DNA directly or not. As the results of DNA electrophoresis and formation of phosphorylated histone H2AX (γH2AX) foci in HT29 cells, widdrol generates DNA double-strand breaks directly within 0.5 h both in vitro and in vivo. Based on this result, the change of proteins related in checkpoint pathway was examined over a time course of 0.5-24 h. Treatment of HT29 cells with widdrol elicits the following: (1) phosphorylation of Chk2 and p53, (2) reduction of cell division cycle 25A (Cdc25A) expression, (3) increase of Cdk inhibitor p21 expression, and (4) decrease of the levels of Cdk2 and cyclin E expression in a time-dependent manner. Moreover, only the expression level of mini-chromosome maintenance 4 (MCM4) protein, a subunit of the eukaryotic DNA replicative helicase, is rapidly down-regulated in HT29 cells treated with widdrol over the same time course, but those of the other MCM proteins are unchanged. Overall, our results indicated that widdrol breaks DNA directly in HT29 cells, and this DNA damage results in checkpoint activation via Chk2-p53-Cdc25A-p21-MCM4 pathway and finally cells go to G1-phase cell cycle arrest and apoptosis.
authors.affiliation: Department of Biomaterial Control (BK21 Program), Dongeui University Graduate School, Busan, Korea.
authors.forename: Hee Jung
authors.initials: HJ
authors.lastname: Yun
authors.forename: Sook Kyung
authors.initials: SK
authors.lastname: Hyun
authors.forename: Jung Ha
authors.initials: JH
authors.lastname: Park
authors.forename: Byung Woo
authors.initials: BW
authors.lastname: Kim
authors.forename: Hyun Ju
authors.initials: HJ
authors.lastname: Kwon
mesh_terms: D000230:Adenocarcinoma / Q000201:enzymology* / Q000473:pathology; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001567:Benzocycloheptenes / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000473:pathology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects*; D019073:HT29 Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D051379:Mice; D064148:Minichromosome Maintenance Complex Component 4; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D020687:cdc25 Phosphatases / Q000378:metabolism*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D001567:Benzocycloheptenes; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D009687:Nuclear Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C078670:widdrol; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; C578633:Chek2 protein, mouse; D017346:Protein Serine-Threonine Kinases; C517004:CDC25A protein, human; D020687:cdc25 Phosphatases; C099243:MCM4 protein, human; D064148:Minichromosome Maintenance Complex Component 4