Presentation: P02007

Evidence supporting a G1 tetraploidy checkpoint was obtained from different assay methods including flow cytometry, immunoblotting, and microscopy. To benefit cancer research, we demonstrate the ability to gather all this evidence by a single, quantitative, automated assay method: High Content Analysis (HCA). Cells, fluorescently labeled for different cell-cycle associated properties, were automatically imaged, analyzed, and correlated using HCA. 24 hours nocodazole treatment blocked mitosis resulting in small, round tetraploid cells with hyperphosphorylated Rb and histone H3, and small nuclei. Cells remained tetraploid and mononucleated with longer treatments, but other targets reverted to G1 levels, including Rb and histone H3 dephosphorylation and cellular re-spreading. This was accompanied by increasing p53 and p21WAF1 expression levels. The effects accompanying nocodazole-induced mitotic block and the resulting tetraploid cells' reversal to a pseudo G1 state can be automatically measured by HCA, recommending this automated assay method for the large-scale biology challenges of modern cancer drug-discovery.

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