Multiparametric cell-based assay for the evaluation of transcription inhibition by high-content imaging.

Loss of normal cell cycle regulation is a hallmark of human cancer. Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and have been actively pursued as promising therapeutic targets. Likewise, members of the CDK family are functionally related to transcriptional modulation, a molecular pathway suitable for therapeutic intervention. We used a set of 2500 compounds in the U2OS cell line to evaluate its effect in the cell division process. Interestingly, out of this analysis, we identified a subpopulation of compounds that are able to inhibit RNA polymerase activity, thus interfering with gene transcription processes. After this finding, we developed, validated, and fully automated a multiparameter high-content imaging (HCI) assay to measure phosphorylation of the RNA polymerase II carboxyl terminal domain (pCTD). Simultaneously, we measured both the DNA content and cell proliferation index in the treated cells. The linear regression analysis comparing the IC50 for pCTD and the 4N EC50 for DNA content or IC50 for cell proliferation showed an excellent agreement (r (2) = 0.84 and r (2) = 0.94, respectively). Our results confirm that this method allows discriminating between cell cycle and transcription inhibition and confirms HCI as a powerful technology for the identification of compounds with an effective and selective pathway phenotype.