In the present paper, we report that mitosis is a key step in the cellular response to genotoxic agents in human cells. Cells with damaged DNA recruit γH2AX (phosphorylated histone H2AX), phosphorylate Chk1 (checkpoint kinase 1) and arrest in the G2-phase of the cell cycle. Strikingly, nearly all cells escape the DNA damage checkpoint and become rounded, by a mechanism that correlates with Chk1 dephosphorylation. The rounded cells are alive and in mitosis as measured by low phospho-Tyr15 Cdk1 (cyclin-dependent kinase 1), high Cdk activity, active Plk1 (Polo-like kinase 1) and high phospho-histone H3 signals. This phenomenon is independent of the type of DNA damage, but is dependent on pharmacologically relevant doses of genotoxicity. Entry into mitosis is likely to be caused by checkpoint adaptation, and the HT-29 cell-based model provides a powerful experimental system in which to explore its molecular basis. We propose that mitosis with damaged DNA is a biologically significant event because it may cause genomic rearrangement in cells that survive genotoxic damage.
Edited by Angel NebredaKeywords: Cdk1 Mitosis Tissue culture Protein phosphatase 1Ca a b s t r a c tWe developed a quantitative method to measure the activity of cyclin-dependent kinases (Cdks) by western blotting, without radioisotopes. We prepared a recombinant protein substrate based upon the natural Cdk1 substrate, PP1Ca. By combining this substrate in a western blot method using fluorochrome based antibodies and phospho-imager analysis, we measured the K m of ATP binding to Cdk1 to be 3.5 lM. We then measured Cdk1 activity in cell extracts from interphase or mitotic cells, and demonstrated that previously identified Cdk inhibitors could be detected by this assay. Our data show that we have a safe, reliable assay to identify Cdk1 inhibitors and measure Cdk1 activity. Crown
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