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Apc and p53 interaction in DNA damage and genomic instability in hepatocytes.

ABSTRACT: Disruption of Apc (adenomatous polyposis coli) within hepatocytes activates Wnt signalling, perturbs differentiation and ultimately leads to neoplasia. Apc negatively regulates Wnt signalling but is also involved in organizing the cytoskeleton and may have a role in chromosome segregation. In vitro studies have implicated Apc in the control of genomic stability. However, the relevance of this data has been questioned in vivo as Apc is lost earlier than the onset of genomic instability. Here we analyse the relationship between immediate loss of Apc and the acquisition of genomic instability in hepatocytes. We used Cre-lox technology to inactivate Apc and in combination with p53 in vivo, to define the consequences of gene loss on cell cycle regulation, proliferation, death and aneuploidy. We show that, although Apc loss leads to increased proliferation, it also leads to increased apoptosis, the accumulation of p53, p21 and markers of double-strand breaks and DNA repair. Flow cytometry revealed an increased 4N DNA content, consistent with a G2 arrest. Levels of anaphase bridges were also elevated, implicating failed chromosome segregation. This was accompanied by an increase in centrosome number, which demonstrates a role for Apc in maintaining euploidy. To address the role of p53 in these processes, we analysed combined loss of Apc and p53, which led to a further increase in proliferation, cell death, DNA damages and repair and a bypass of G2 arrest than was observed with Apc loss. However, we observed only a marginal effect on anaphase bridges and centrosome number, which could be due to increased cell death. Our data therefore establishes, in an in vivo setting, that APC loss leads to a DNA damage signature and genomic instability in the liver and that additional loss of p53 leads to an increase in the DNA damage signal but not to an immediate increase in the genomic instability phenotype.

SUBMITTER: Meniel V

PROVIDER: S-EPMC4351894 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Apc and p53 interaction in DNA damage and genomic instability in hepatocytes.

Méniel V V Megges M M Young M A MA Cole A A Sansom O J OJ Clarke A R AR

Oncogene 20141027 31

Disruption of Apc (adenomatous polyposis coli) within hepatocytes activates Wnt signalling, perturbs differentiation and ultimately leads to neoplasia. Apc negatively regulates Wnt signalling but is also involved in organizing the cytoskeleton and may have a role in chromosome segregation. In vitro studies have implicated Apc in the control of genomic stability. However, the relevance of this data has been questioned in vivo as Apc is lost earlier than the onset of genomic instability. Here we a ...[more]

PMID: 25347740

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Project description:BackgroundThe DNA damage response (DDR) is a mechanism that protects cells against radiation-induced oxidative DNA damage by causing cell cycle arrest and apoptosis. TP63 is a member of the tumour suppressor TP53 gene family, and ΔNp63α, a TP63 splicing variant, is constitutively expressed in the stem cell-containing basal layer of stratified epithelial tissues, including the mammary gland, where it plays a critical role in stemness and tissue development. ΔNp63α has been reported to transcriptionally inhibit the tumour suppression protein p53. This p53-repressive activity may cause genomic instability in epithelial stem cells exposed to radiation. In this study, we analysed the inhibitory effect of ΔNp63α on radiation-induced DDR.MethodsTo elucidate the role of the p53-repressive effect of ΔNp63α in radiation response, we performed a p63-siRNA knockdown experiment using human mammary epithelial cells (HMECs) expressing ΔNp63α and then performed ectopic and entopic expression experiments using human induced pluripotent stem cells (hiPSCs). After irradiation, the expression of DDR-related genes and proteins in ΔNp63α-expressing and control cells was analysed by RT-qPCR, Western blotting, and flow cytometry.ResultsThe mRNA/protein expression levels of BAX and p21 were significantly increased in p63-siRNA-treated HMECs (sip63) after X-ray irradiation (4 Gy, 0.7 Gy/min) but not in scramble-siRNA treated HMECs (scr). Transcriptomic analysis showed decreased RNA expression of cell cycle-related genes and increased expression of programmed cell death-related genes in sip63 cells compared to scr cells. Furthermore, flow cytometric analysis revealed an increase in apoptotic cells and a decrease in 5-ethynyl-2´-deoxyuridine uptake in sip63 cells compared to scr cells. On the other hand, both the ectopic and entopic expression of ΔNp63α in apoptosis-sensitive hiPSCs reduced the expression levels of BAX after irradiation and significantly decreased the number of apoptotic cells induced by radiation.ConclusionTaken together, these results indicate that ΔNp63α represses p53-related radiation-induced DDR, thereby potentially causing genomic instability in epithelial stem cells.

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Apc and p53 interaction in DNA damage and genomic instability in hepatocytes.

Publications

Apc and p53 interaction in DNA damage and genomic instability in hepatocytes.

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