However, this level of progression back into G1 is at a slower rate compared to control cells

involvement of ATM. The DNA damage was observed as early as 3 h after Cuc B treatment suggesting that it is an early event after Cuc B incubation. Generally, ATM activates Chk2 by phosphorylation it on Thr-68, Neuromedin N chemical information PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19645759 while ATR activates Chk1. However, Chk1 could be activated by both ATR and ATM on Ser-345. In this study, a significant increase of phosphorylation of Chk1 on Ser-345 after Cuc B exposure was observed, whereas the phosphorylation of Chk2 on Thr-68 was not affected. To establish the role of ATM in Cuc Bmediated G2/M phase arrest in A549, ATM was knocked down by transfection with ATM siRNA. Cuc B-mediated G2/M phase arrest was dramatically reversed by ATM siRNA transfection. Cuc B caused Chk1 phosphorylation is also blocked by ATM siRNA. Similarly, Chk1 knocked down reversed Cuc B induced G2/M phase arrest. Thus, these results illustrated that Cuc B induced G2/M phase arrest in A549 cells via ATM-Chk1 pathway. ATM-activated Chk1 can phosphorylate Cdc25C, contributing to G2/M phase checkpoints. Cdc25C is essential for promoting mitosis though dephosphorylating Tyr-15 on Cdk1. Phosphorylation of Cdc25C on Ser-216 is an inactive state of Cdc25C, which made a binding site for proteins of the 14-3-3-s. The binding of phosphorylated Cdc25C with 14-3-3-s in the cytoplasm prevents Cdc25C from dephosphorylating the cyclingdependent kinase Cdk1, resulting in cells arrest in G2/M phase. Our results showed that Cuc B induced phosphorylation Cdc25C on Ser-216 in a dose-dependent manner, which could be blocked by ATM siRNA and Chk1 siRNA suggesting that Cdc25C was another downstream effector in Cuc B induced DNA damage response. Additionally, DNA damage could induce ATM to activate p53 through phosphorylating it directly on Ser15 and/or on Ser-20 via Chk1/Chk2. We found that Cuc B exposure induced p53 phosphorylation on Ser-15 but not on Cucurbitacin B Induced DNA Damage Causes G2/M Arrest 9 Cucurbitacin B Induced DNA Damage Causes G2/M Arrest Ser-20 illustrating that ATM directly activated p53 by phosphorylation on Ser-15. This contributes primarily to enhance the activity of p53 as a transcription factor. The 14-3-3-s, a gene directly regulated by p53, is induced by DNA damage and is required for G2/M phase arrest. Our results showed that the expression of 14-3-3-s was increased after Cuc B treatment. Furthermore, the increased p53 phosphorylation on Ser-15 and 14-3-3-s expression by Cuc B were reversed by ATM siRNA. In addition, the binding of 14-3-3-s with Cdc25C phosphorylation on Ser-216 increased after Cuc B treatment. Thus, an ATM-p5314-3-3-s branch pathway might exist in Cuc B induced DNA damage response. When Cdc25C is in inactive status, Cdk1 keeps an inhibitory phosphorylation on Tyr-15. Cell phase progression from G2 to M phase is highly dependent upon the activity of the Cyclin B/Cdk1 complex which is inactivated via inhibitory phosphorylation of conserved Thr-14 and Tyr-15 residues of Cdk1. We detected the effect of Cuc B on the phosphorylation of Cdk1 on Tyr-15. Cuc B dose-dependently increased phosphorylation of Cdk1 on Tyr-15, which could be inhibited by ATM siRNA and Chk1 siRNA. Furthermore, Cuc B induced G2/M phase arrest in A549 cells could be significantly reversed by ATM siRNA and Chk1 siRNA. All the results showed that Cuc B induced DNA damage and G2/M checkpoint in A549 cells through ATM activated Chk1-Cdc25C-Cdk1 and p53-14-33-s pathways. Previous studies reported that Cuc B induced high level of intracellular ROS format

34 thoughts on “However, this level of progression back into G1 is at a slower rate compared to control cells”

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