School of Science Division of Life Science 37 Targeting Mitotic Regulators in Cancer Cells for Potential Treatment Supervisor: POON Randy Yat Choi / LIFS Student: TANG Chun Hei / BCB Course: UROP 1100, Fall PTTG1 (securin) playing an important role in cell cycle regulation and sister chromatid during the mitosis especially on the onset of anaphase. PTTG1 is responsible to be the inhibitory protein to regulate separase which mainly used to cleave the cohesin complexes during metaphase. Different findings shows that down regulation of PTTG1 in tumour cell lines and tumours result in suppression of tumour growth, suggesting its important role in tumorigenesis. However, it is unclear if PTTG1 plays a role in premature separation of chromosomes during prolonged metaphase. In this project, we studied how turning off different APC/C subunits, PTTG1, and the accumulation of cyclin B affect premature sister chromatids separation. Using livecell imaging analysis, we found that there were occurrence of premature separation phenotype after clear metaphase plate. These results indicate that prolonged mitotic arrest can promote genome instability and have implications in cancer therapies. Targeting Mitotic Regulators in Cancer Cells for Potential Treatment Supervisor: POON Randy Yat Choi / LIFS Student: WONG Meng Hwei / BCB Course: UROP 1100, Summer The auxin-inducible degron 2 (AID2) system, an improved version of AID, was developed by introducing a site-directed mutation in TIR1, resulting in OsTIR1 (F74G). AID2 system uses a lower ligand 5-Ph-IAA concentration, effectively eliminating basal degradation and enabling rapid protein depletion. We evaluated the AID2 system in HeLa cells with stable expression of OsTIR1 (F74G) by monitoring mAID-CDK2 degradation via Western blot analysis. Compared to control cells containing wild type OsTIR1, all clones of AID2 showed highly efficient and near-complete depletion of mAID-CDK2 upon treatment with 1 mM of 5-Ph-IAA. We performed fluorescence-activated cell sorting (FACS) to isolate cells with optimal OsTIR1 (F74G) expression and degradation efficiency. A clone (clone 5) was selected to establish a stable cell line. The resulting cell line, HtTA2, will be able to serves as a robust platform for future AID2-based protein-depletion studies. Targeting Mitotic Regulators in Cancer Cells for Potential Treatment Supervisor: POON Randy Yat Choi / LIFS Student: ZHUANG Boyi / BCB-IRE Course: UROP 2100, Spring During mitotic arrest, cancer cells go through a variety of cell fate decision, whether to go through cell death or slippage. Our study suggested that the depletion of a ROS metabolizing protein in the mitochondria, SOD2, cause HeLa cells to not only proceed with cell death quicker during mitotic arrest, but also reduced the chance of slippage. During the analysis of the LCI footage, we also discovered unique cell death phenotype if SOD2 depletion and caspase inhibition is simultaneously enacted during mitotic arrest, and it is believed that this may be a form of cell death called ferroptosis. However, there haven’t been conclusive results yet, and further investigation is required.
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