Generation of a colon cancer cell line HCT116 expressing a non-degradable form of CDT1
Loading...
Date
2024-07-04
Authors
Σαμουηλίδου, Θωμαή
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
DNA replication is a tightly regulated procedure, ensuring genome duplication once per cell cycle, thereby safeguarding genomic fidelity. However, maintaining genomic integrity is challenging as cells are exposed to numerous extrinsic and intrinsic stressors in a course of the cell cycle. Under these conditions, cells may acquire aberrations that, if left unrepaired and allowed to accumulate, can severely compromise genomic integrity. Consequently, cells have developed numerous well-orchestrated mechanisms for preventing such catastrophic events. One of these mechanisms involves restraining DNA replication licensing in G1 phase, by controlling CDT1 levels throughout the cell cycle. CDT1 is an essential element of the pre-replication complex, as it recruits an inactive form of the MCM2-7 helicase onto replication origins. Activation of MCM2-7, initiates DNA unwinding thus permitting firing of a subset of potential replication origins. The active and firing origins during S-phase are characterized as early, middle and late- S replicating origins, according to their efficiency as well as their spatiotemporal firing program. It has been demonstrated that the aberrant expression of CDT1 in vitro and in vivo, triggers genomic instability, one of the primary drivers of carcinogenesis. Furthermore, the mRNA levels of CDT1 have been found elevated in many types of malignant neoplasms. Therefore, CDT1 activity is tightly regulated during the cell cycle by two E3 ubiquitin ligases CRL1Skp2 and CRL4-DDB1Cdt2 and Geminin during the S-phase.
In this thesis we set the foundation for the development of a novel tool for studying the implications of both CDT1 overexpression and ectopic expression on replication dynamics and genomic instability. We generated the lentivirus “LV-1279” carrying a non-degradable form of CDT1 “CDT1-A6Cy”, after a series of successful molecular cloning procedures. We transduced HCT-116 to assess the persistence of CDT1-A6Cy in S-phase, as well as its role in the replication dynamics of transduced cells. Our results from selectively analyzed data from transduced cells of a heterogeneous population demonstrated that the non-degradable form of CDT1, “CDT1-A6Cy,” persists in S-phase of HCT-116 cells. Cells harboring CDT1-A6Cy displayed a slightly altered replication profile, skewed towards early-S phase. This preliminary data provides fundamental insights on the role of CDT1 ectopic expression on replication dynamics. Therefore, replicating these results in a monoclonal HCT-116_CDT1-A6Cy cell line will increase the credibility of this data, by utilizing this novel tool for studying the implications of CDT1 ectopic expression on genomic instability and carcinogenesis.
Description
Keywords
Replication licensing, Genomic instability, Replication dynamics, Colon cancer, CDT1, HCT116