A western blot assay to measure cyclin dependent kinase activity in cells or in vitro without the use of radioisotopes.

We 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, PP1Cα. By combining this substrate in a western blot method using fluorochrome based antibodies and phospho-imager analysis, we measured the Km of ATP binding to Cdk1 to be 3.

Human cells enter mitosis with damaged DNA after treatment with pharmacological concentrations of genotoxic agents.

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.

Characterization of novel checkpoint kinase 1 inhibitors by in vitro assays and in human cancer cells treated with topoisomerase inhibitors.

Aims: We have developed biochemical and cell based assays to characterize small therapeutic molecules that inhibit the DNA damage checkpoint enzyme, Chk1 (Checkpoint kinase 1).

Main Methods: To prepare a screen of large chemical libraries, we purified the full-length and the catalytic domain versions of human Chk1. We characterized their properties and then selected full-length Chk1 as the variant most suitable for screening.