Regulation of P53 stability in p53 mutated human and mouse hepatoma cells

Abstract

The tumor suppressor p53 is frequently mutated in cancer. We have investigated the regulation of P53 in p53 wild type mouse hepatoma cells (line 55.1c), in p53 heterozygeously mutated cells (56.1b) and in p53 defective cells (lines 56.1d, 70.4 and HUH7) under various experimental settings. The basal levels of P53 were low in 55.1c cells, but nuclear accumulation occurred upon UVirradiation. Similarly, UV-exposure induced stabilization of P53 in the heterozygeously p53 mutated 56.1b hepatoma cells. By contrast, the 3 hepatoma lines, which lack transcriptionally active P53, demonstrated high basal nuclear concentrations of P53 protein and, unexpectedly, showed loss of P53 upon UV-irradiation. Expression of p53 mRNA was also decreased in p53 defective cells after 24 hr post UV-irradiation, which may be linked to induction of apoptosis of the irradiated cells under these conditions. Other stressors like H 2 O 2 also mediated a decrease in P53 concentration in p53 defective cells. This effect occurred at very low concentrations and was already detectable 1-2 hr after exposure of cells. There were no signs of apoptosis of H 2 O 2 -exposed cells at this time point and no significant changes in p53 mRNA or MDM2 level. These unexpected findings indicate a new aspect related to regulation of P53 stability in cells with a defect in the tumor suppressor protein. ' 2007 Wiley-Liss, Inc.Key words: p53 mutation; hepatoma cells; MDM2; DFX; UV irradiation; ROS The p53 tumor suppressor protein is a powerful cellular caretaker, which protects cells from malignant transformation by means of transcriptional upregulation of proapoptotic, DNA repair and cell cycle arrest related proteins. In unstressed cells the P53 level is kept low by the E3 ubiquitin ligase MDM2, which transfers activated ubiquitin residues to P53. 1 Polyubiquitination of P53 leads to a rapid proteasomal degradation of the tumor suppressor, preventing P53 from affecting the cells' viability. P53 itself transcriptionally upregulates MDM2 expression, forming a negative feedback loop.Stress signals, caused by UV-light, DNA-damaging agents or hypoxia lead to an activation of several kinases, of which the most prominent members are the ATM (ataxia-telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related), DNA-dependent protein kinase and casein kinase II, which phosphorylate the P53 protein at different sites (for review see Ref.2). Polyphosphorylation at the N-terminus of P53 reduces MDM2-dependent degradation, leading to an accumulation of the P53 protein in the cell followed by upregulation of apoptosis or cell cycle arrest related proteins. Exposure of cells to UV light correlates with post-translational modifications of P53, such as phosphorylation of serine 15 (mouse serine 18) and serine 20 (mouse serine 23). Desferrioxamine (DFX) treatment, which mimics hypoxia, results in phosphorylation of serine 15. Both sites are known to reduce MDM2-binding when phosphorylated. 3

Cyproheptadine, an antihistaminic drug, inhibits proliferation of hepatocellular carcinoma cells by blocking cell cycle progression through the activation of P38 MAP kinase

“…CHK2 has been found to be dispensable for p53-mediated cell cycle arrest [ 23 , 24 ]. We were interested in exploring a p53-independent role for CHK2 in inducing cell cycle arrest because the tumor suppressor p53 is frequently mutated in cancer cells and the Huh-7 HCC cell line used in this study is p53 defective [ 25 ]. Using antibodies specific for CHK2, Thr68-phosphorylated CHK2, p53, and Ser20-phosphorylated p53, we detected a time-dependent increase in the level of Thr68-phosphorylated CHK2 and no change in the level of total CHK2 in Huh-7 cells (Figure 5 , right panel set).…”

Hepatitis C Virus Sensitizes Host Cells to TRAIL-Induced Apoptosis by Up-Regulating DR4 and DR5 via a MEK1-Dependent Pathway

“…6A). Importantly, the Huh7 cell line carries a single p53 mutation (A:T→G:C at codon 220) and is p53 defective [44], [45]. Thus, it is possible that other transcription factors are involved in the HCV-mediated up-regulation of DR4, and further research is needed to identify the transcription factors involved in HCV-regulated DR4 transcription.…”

Differential network analysis for the identification of condition-specific pathway activity and regulation

“…HCC progression involves alterations in many signaling pathways, such as EGF-Ras-MAPK, AKT-mTOR, Jak-Stat and NF-kB cascades (Llovet et al , 2008). In addition, inactivating mutations of the tumor suppressor p53, or p53 loss of expression, are among the most frequent genetic events associated with hepatocyte transformation (Bressac et al , 1990; Hinds et al , 1987), and the dysregulation of p53-dependent genes have been observed in HCC (Hailfinger et al , 2007; Hsu et al , 1993). …”