Tamiyo Kobayashi

Drug-induced cell cycle modulation leading to cell-cycle arrest, nuclear mis-segregation, or endoreplication

BackgroundCancer cell responses to chemotherapeutic agents vary, and this may reflect different defects in DNA repair, cell-cycle checkpoints, and apoptosis control. Cytometry analysis only quantifies dye-incorporation to examine DNA content and does not reflect the biological complexity of the cell cycle in drug discovery screens.ResultsUsing population and time-lapse imaging analyses of cultured immortalized cells expressing a new version of the fluorescent cell-cycle indicator, Fucci (F luorescent U biquitination-based C ell C ycle I ndicator), we found great diversity in the cell-cycle alterations induced by two anticancer drugs. When treated with etoposide, an inhibitor of DNA topoisomerase II, HeLa and NMuMG cells halted at the G2/M checkpoint. HeLa cells remained there, but NMuMG cells then overrode the checkpoint and underwent nuclear mis-segregation or avoided the checkpoint and entered the endoreplication cycle in a drug concentration dependent manner. In contrast, an inhibitor of Cdk4 led to G1 arrest or endoreplication in NMuMG cells depending upon the initial cell-cycle phase of drug exposure.ConclusionsDrug-induced cell cycle modulation varied not only between different cell types or following treatment with different drugs, but also between cells treated with different concentrations of the same drug or following drug addition during different phases of the cell cycle. By combining cytometry analysis with the Fucci probe, we have developed a novel assay that fully integrates the complexity of cell cycle regulation into drug discovery screens. This assay system will represent a powerful drug-discovery tool for the development of the next generation of anti-cancer therapies.

DNA-binding profiling of human hormone nuclear receptors via fluorescence correlation spectroscopy in a cell-free system

The nuclear hormone receptors (NHRs), a family of transcription factors, bind directly to the hormone response elements (HREs) to regulate gene expression. In this study, we describe a comprehensive NHR–HRE profiling analysis with a new high‐throughput DNA binding assay system utilizing wheat germ cell‐free protein production and fluorescence correlation spectroscopy (FCS). This approach revealed NHR binding to natural response elements and new heterodimeric NHR–HRE bindings. We analyzed 408 possible binding combinations between 34 human NHRs and 12 different HREs, and identified 205 NHR–HRE binding combinations, 124 of which have not been previously reported. Thus, this study provides a novel biochemical classification of the human NHRs, as well as describing a novel approach to the large‐scale analysis of DNA–protein interactions.