Abstract 5644: Activation of p53 in prolonged prometaphase

Abstract

Abstract The tumor suppressor p53 plays a critical role in protecting cells from aberrant divisions in the face of DNA damage, and while its role in DNA damage checkpoints have been extensively studied, its role in the mitotic checkpoint is less well characterized. The mitotic checkpoint prevents completion of mitosis until chromosomes have made bipolar kinetochore attachments to microtubules. Previous studies have shown that cells that experience a prolonged prometaphase(>1.5 h) following treatment with microtubule destabilizing drugs will arrest in G1 following eventual exit from mitosis. Given its role in DNA damage checkpoints, p53 appears to be a likely candidate for involvement in this arrest. The goal of the current study is to investigate the role of p53 in this G1 arrest following prolonged prometaphase. Three cells lines were utilized:MCF10A, MCF10A/αp53 which overexpressed shRNA to p53, and MCF10A/OD which overexpresses the p53 oligomerization domain. Cells were treated with 9 μM RO-3306 for 18 h to synchronize cells at the G2/M transition. The RO-3306 was then removed and cells were treated with a microtubule destabilizing drug, nocodazole, at a concentration of 0.08 μM for 1.5 or 2.5 h to cause prometaphase arrest. The cells were then washed to remove nocodazole, and the cells were harvested at various time points and analyzed via flow cytometry and Western blot. Flow cytometry analysis in in all three cell lines showed an accumulation of cells in G1 at 12 hours in for both treatment times (1.5 and 2.5 h), with subsequent increases in sub-G1 populations by 24 hours. Western blot analysis revealed activation of p53 by phosphorylation of ser20 and activation of p21 in the MCF10A and MCF10A/OD, but not in the MCF10A/αp53 for both treatment times. Taken together, these results suggest that activation of p53 alone is not sufficient to elicit the G1 arrest, as the MCF10A/αp53 cells arrested in G1 despite the absence of p53 protein. Citation Format: Megan Anderson, Aime A. Levesque. Activation of p53 in prolonged prometaphase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5644.