Cell cycle is tightly regulated in eukaryotic cells through the activation of cyclin-dependent kinases (CDKs) by cyclins. Among the cyclin families, cyclin A (cyclin A2) is notable for its dual functions during the cell cycle. While cyclin A is a well-known S-phase promoting factor, its precise role in the G₂-M transition still remains obscure. In retrospect, the use of RNAi approaches in the past limits the complete understanding of the G₂-M-specific function of cyclin A due to several caveats, including slow kinetics of knockdown, potential off-target effects, and failure to control the timing of knockdown. Recently, we have developed a conditional gene inactivation system in HeLa cells by integrating the tetracycline-controlled promoter system and the auxin-inducible degron (AID) sys...[ Read more ]

Cell cycle is tightly regulated in eukaryotic cells through the activation of cyclin-dependent kinases (CDKs) by cyclins. Among the cyclin families, cyclin A (cyclin A2) is notable for its dual functions during the cell cycle. While cyclin A is a well-known S-phase promoting factor, its precise role in the G₂-M transition still remains obscure. In retrospect, the use of RNAi approaches in the past limits the complete understanding of the G₂-M-specific function of cyclin A due to several caveats, including slow kinetics of knockdown, potential off-target effects, and failure to control the timing of knockdown. Recently, we have developed a conditional gene inactivation system in HeLa cells by integrating the tetracycline-controlled promoter system and the auxin-inducible degron (AID) system to enable a dual control of cyclin A at transcriptional and post-translational level, respectively. In this study, AID-cyclin A cell lines were generated and the role of cyclin A during the cell cycle was characterized. In addition to requiring for S phase progression, cyclin A was found to be involved in mitotic entry since cyclin A2^KO cells exhibited a G₂-M delay. The delay was transient, suggesting potential compensation by other proteins. While we confirmed that both cyclin B1 and cyclin A contribute to CDK1 activation, I uncovered another layer of regulation on CDK1 activity modulated by cyclin A through a novel transcriptional downregulation on CDC25A. The essential function of cyclin A in maintaining cell viability was confirmed by clonogenic survival assay. Strikingly, flow cytometry analysis revealed that the cell density was negatively correlated to the degree of G₂-arrest, suggesting a unique cell cycle regulation by cyclin A in a cell density-dependent manner. Use of different conditional mediums suggested that growth stimulant released from high density cells was the cause for this cell density-dependent regulation. These results extend our understanding on the requirement of cyclin A in cancer cells and provide insights on potential therapeutic targets in cyclin A-overexpressed tumors.