Understanding CDT1 Degradation Across Distinct Cell Cycle Arrests

The cell cycle is the highly controlled process of genome replication followed by cell duplication. Within G1 of the cell cycle, CDT1 operates as an essential DNA replication factor to promote cellular proliferation. CDT1 is highly regulated as over-expression induces DNA damage and allows for cells to exhibit checkpoint deficiencies if this protein is not properly degraded, leading to tumorigenesis (Hanahan & Weinberg, 2011; Liontos et al., 2007). Various mechanisms are responsible for CDT1 regulation throughout active proliferation including transcriptional regulation and targeted protein degradation. While CDT1 regulation is well- described during proliferation, CDT1 regulation during reversible cell cycle arrest (quiescence or G0) and drug-induced arrest operate via unknown mechanisms. We hypothesized that CDT1 is regulated in cell cycle arrest via specific, targeted protein degradation. CDT1 instability across cell cycle arrest will maintain genome stability by preventing re-replication of the DNA. Cycle arrest was induced in cell line models via two physiological methods: serum starvation (SS), contact inhibition (CI) or treated with drug inhibitors (CDK inhibitors) and we observed CDT1 abundance via Immunoblot. Our data indicated a correlation between CUL3/SPOP (Cullin-dependent) activity and degradation of CDT1 in both cancerous and diploid epithelial cell lines. Furthermore, we observed that different methods of cell cycle arrest were not equivalent as drug-induced arrest demonstrated a Cullin-independent mechanism of CDT1 degradation and the physiological arrest demonstrated Cullin-dependence. The goal of this research was to determine how CDT1 is regulated, which proteins are involved in CDT1 regulation, and what the mechanism controls CDT1 degradation in drug-arrested cells compared to physiologic arrest. Our research seeks to understand cell cycle dynamics to improve possible chemotherapeutic treatments by the eventual manipulation of arrested cancer cells.