The Expression of a p53 Isoform Δ133p53, in the Cell Cycle

Abstract

Overview: p53 is a principal tumour suppressor protein that regulates the cell cycle. By doing so, p53 prevents damaged cells from undergoing DNA replication and cell division by activating pathways involved in DNA repair, cell death, cell cycle arrest or senescence. However, in over 50% of cancers, p53 loses its tumour suppressive function, allowing cells to proliferate. Yet, the recent discovery of at least 12 different p53 isoforms, some of which have tumour promoting functions, has given insight into the potential mechanisms behind p53 inactivation in cancer. One such isoform family of p53; Δ133p53 (α, β and γ), lack the first 132 amino acids and have been associated with increased cellular proliferation, metastasis and tumorigenesis. However, the expression and mechanism of action of this isoform is unknown, particularly in the cell cycle. Therefore, this thesis aims to investigate the expression and role of Δ133p53 in the cell cycle. Methods: The lung adenocarcinoma cell line; A549 and a modified A549-FUCCI cell line were synchronised to the G1/S phase of the cell cycle using a double thymidine block. Protein and mRNA expression of full-length p53 (FLp53) and the Δ133p53 isoforms were examined using western blot and RT-qPCR at each phase of the cell cycle and an RNAscope® in situ hybridisation assay (ISH) was carried out at the S and M phases of the cell cycle. Then, to confirm the effect of Δ133p53 on the cell cycle, A549-FUCCI cells were treated with a siRNA targeting either Δ133p53 or FLp53 and were tracked using live cell imaging over 46 hours. Also, to determine if the previously described inflammatory function of Δ133p53 is cell cycle-dependent, an RNAscope® ISH assay and RT-qPCR was carried out, where the expression of cytokines were determined at the S and M phases of the cell cycle. Results: Western blot analyses demonstrated that Δ133p53 expression increases during the M/G1 phase of the cell cycle and remains high throughout the G1 phase. In contrast, FLp53 expression peaked during the S and G2 phases of the cell cycle. Consistent with our western blot analyses, a knockdown of Δ133p53 resulted in longer M and G1 phases of the cell cycle. RT-qPCR showed that CCL2 was also upregulated during the M and G1 phases of the cell cycle, similarly to Δ133p53. Cumulatively, this suggested that Δ133p53 promotes proliferation during the M and G1 phases of the cell cycle where it may activate CCL2 to recruit monocytes and activate invasion and metastasis. This illustrates that inhibiting Δ133p53 expression during the M and G1 phases of the cell cycle may be a potential therapeutic target site to reduce cancer cell proliferation and metastasis in patients.