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Updated: May 15, 2026
High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes
Published on: March 3, 2015
Multiparametric cell-based assay for the evaluation of transcription inhibition by high-content imaging
Raquel Torres-Guzmán1, Shaoyou Chu, Juan A Velasco
1Translational Sciences & Technologies, Lilly Research Laboratories, Eli Lilly & Company, Lilly Research Laboratories, Alcobendas, Madrid, Spain.
Abstract:
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.
Insights
Researchers screened 2500 compounds and found some that inhibit RNA polymerase activity, affecting gene transcription. A high-content imaging assay was developed to distinguish between cell cycle and transcription inhibition, proving effective for identifying targeted compounds.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Cancer is characterized by disrupted cell cycle regulation.
- Cyclin-dependent kinases (CDKs) are key cell cycle regulators and therapeutic targets.
- CDK family members also influence transcriptional modulation, offering another therapeutic avenue.
Purpose of the Study:
- To screen compounds for effects on cell division and identify those inhibiting gene transcription.
- To develop and validate a high-content imaging (HCI) assay for measuring RNA polymerase II activity and cell proliferation.
- To differentiate between cell cycle and transcription inhibition mechanisms.
Main Methods:
- Screening of 2500 compounds in U2OS cells to assess effects on cell division.
- Development of an automated, multiparameter HCI assay to measure RNA polymerase II C-terminal domain phosphorylation (pCTD), DNA content, and proliferation.
- Linear regression analysis to compare IC50 values for pCTD inhibition with DNA content and proliferation inhibition.
Main Results:
- A subset of compounds was identified that inhibit RNA polymerase activity, impacting gene transcription.
- The developed HCI assay demonstrated excellent agreement (r² = 0.84 for DNA content, r² = 0.94 for proliferation) in distinguishing inhibition types.
- The assay successfully discriminated between compounds affecting cell cycle progression versus those inhibiting transcription.
Conclusions:
- The HCI assay is a powerful tool for identifying compounds with selective pathway phenotypes.
- This method enables the discrimination between cell cycle and transcription inhibition.
- The findings support the potential of targeting transcriptional modulation in cancer therapy.
