LAMTOR1 depletion induces p53-dependent apoptosis via aberrant lysosomal activation

Lysosomal regulation is a poorly understood mechanism that is central to degradation and recycling processes. Here we report that LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) downregulation affects lysosomal activation, through mechanisms that are not solely due to mTORC1 inhibition. LAMTOR1 depletion strongly increases lysosomal structures that display a scattered intracellular positioning. Despite their altered positioning, those dispersed structures remain overall functional: (i) the trafficking and maturation of the lysosomal enzyme cathepsin B is not altered; (ii) the autophagic flux, ending up in the degradation of autophagic substrate inside lysosomes, is stimulated. Consequently, LAMTOR1-depleted cells face an aberrant lysosomal catabolism that produces excessive reactive oxygen species (ROS). ROS accumulation in turn triggers p53-dependent cell cycle arrest and apoptosis. Both mTORC1 activity and the stimulated autophagy are not necessary to this lysosomal cell death pathway. Thus, LAMTOR1 expression affects the tuning of lysosomal activation that can lead to p53-dependent apoptosis through excessive catabolism.     

The p53-inducible E3 ubiquitin ligase p53RFP induces p53-dependent apoptosis

Recently, it has been shown that really interesting new gene (RING)-in between ring finger (IBR)-RING domain-containing proteins, such as Parkin and Parc, are E3 ubiquitin ligases and are involved in regulation of apoptosis. In this report, we show that p53-inducible RING-finger protein (p53RFP), a p53-inducible E3 ubiquitin ligase, induces p53-dependent but caspase-independent apoptosis. p53RFP contains an N-terminal RING-IBR-RING domain and an uncharacterized, evolutionally highly conserved C-terminal domain. p53RFP interacts with E2 ubiquitin-conjugating enzymes UbcH7 and UbcH8 but not with UbcH5, and this interaction is mediated through the RING-IBR-RING domain of p53RFP. Interestingly, the conserved C-terminal domain of p53RFP is required and sufficient for p53RFP-mediated apoptosis, suggesting p53RFP-mediated apoptosis does not require its E3 ubiquitin ligase activity. Together with a recent report showing that p53RFP is involved in ubiquitination and degradation of p21, a p53 downstream protein promoting growth arrest and antagonizing apoptosis, our findings suggest that p53RFP is involved in switching a cell from p53-mediated growth arrest to apoptosis.     

CCN1 induces apoptosis in esophageal adenocarcinoma through p53-dependent downregulation of survivin

Many cancer drugs have been developed to control tumor growth by inducing cancer cell apoptosis. However, several intracellular barriers could fail this attempt. One of these barrier is high expression of survivin. Survivin can interfere caspase activation and thereby abort apoptosis. In this study, we found that CCN1 suppressed the survivin expression in tumor cells of esophageal adenocarcinoma (EAC) and thus allowed apoptosis to finish. Furthermore, we demonstrated that this downregulation was dependent on p53 phosphorylation at Ser20, and CCN1 induced EAC cell apoptosis through the activation of p53.     

Knockdown of human p53 gene expression in 293-T cells by retroviral vector-mediated short hairpin RNA

RNA interference （RNAi） is an evolutionarily conserved process of gene silencing in multiple organisms, which has become a powerful tool for investigating gene function by reverse genetics. Recently, many groups have reported to use synthesized oligonucleotides or siRNA encoding plasmids to induce RNAi in mammalian cells by transfection, but this is still limited in its application, especially when it is necessary to generate long-term gene silencing in vivo. To circumvent this problem, retrovirus- or lentivirus-delivered RNAi has been developed. Here, we described two retroviral systems for delivering short hairpin RNA （shRNA） transcribed from the H1 promoter. The results showed that retroviral vector-mediated RNAi can substantially downregulate the expression of human p53 in 293-T cells. Furthermore, the retroviral vectormediated RNAi in our transduction system can stably inactivate the p53 gene for a long time. Compared to shRNAs transcribed from the U6 promoter, HI-driven shRNA also dramatically reduced the expression of p53. The p53 downregulation efficiencies of H1- and U6-driven shRNAs were almost identical. The results indicate that retroviral vector-delivered RNAi would be a useful tool in functional genomics and gene therapy.     

The can and can't dos of p53 RNA

The p53 protein, like any other protein, cannot be made in the cell without RNA. And even once made, the p53 protein will be more rapidly degraded without the p53 RNA. Furthermore, the p53 RNA helps deciding which p53 isoform should be produced and under which cell conditions. Mutant p53 mRNA codes for an unstable and inactive protein. These matters are discussed in this article as well as the recent reports on p53 RNA mutations, interacting-proteins, 3′ processing and 5′–3′ loop.     

The antiproliferative activity of aloe-emodin is through p53-dependent and p21-dependent apoptotic pathway in human hepatoma cell lines

The aim of this study is to investigate the anticancer effect of aloe-emodin in two human liver cancer cell lines, Hep G2 and Hep 3B. We observed that aloe-emodin inhibited cell proliferation and induced apoptosis in both examined cell lines, but with different the antiproliferative mechanisms. In Hep G2 cells, aloe-emodin induced p53 expression and was accompanied by induction of p21 expression that was associated with a cell cycle arrest in G1 phase. In addition, aloe-emodin had a marked increase in Fas/APO1 receptor and Bax expression. In contrast, with p53-deficient Hep 3B cells, the inhibition of cell proliferation of aloe-emodin was mediated through a p21-dependent manner that did not cause cell cycle arrest or increase the level of Fas/APO1 receptor, but rather promoted aloe-emodin induced apoptosis by enhancing expression of Bax. These findings suggest that aloe-emodin may be useful in liver cancer prevention.     

The magnitude of methylmercury-induced cytotoxicity and cell cycle arrest is p53-dependent

BACKGROUND: Methylmercury (MeHg), a ubiquitous environmental contaminant, is a known potent teratogen selectively affecting the developing central nervous system. While a definitive mechanism for MeHg-induced developmental neurotoxicity remains elusive, in utero exposure has been associated with reduced brain weight and reduction in cell number. This suggests early toxicant interference with critical molecular signaling events controlling cell behavior, i.e., proliferation. METHODS: To examine the role of p53, a major regulator of the G(1)/S and G(2)/M cell cycle checkpoints, in MeHg toxicity, we isolated GD 14 primary embryonal fibroblasts from homozygous wild-type p53 (p53+/+) and homozygous null p53 (p53-/-) mice. Cells were treated at passages 4-7 for 24 or 48 hr with 0, 1.0, or 2.5 microM MeHg and analyzed for effects on viability, cell cycle progression (using BrdU-Hoechst flow cytometric analysis), and apoptosis via annexin V-FITC and propidium iodide (PI) staining. RESULTS: The p53+/+ cells are more sensitive than p53-/- cells to MeHg-induced cytotoxicity, cell cycle inhibition, and induction of apoptosis: at 24 hr, 2.5 microM MeHg reduced p53+/+ cell viability to 72.6% +/- 3.2%, while p53-/- viability was 94.6% +/- 0.4%. The p53-/- cells underwent less necrosis and less apoptosis following MeHg treatment. MeHg (2.5 microM) also halted all cycling in the p53+/+ cells, while 42.6% +/- 7.2% of p53-/- cells were able to reach a new G(0)/G(1) in 48 hr. Time- and dose-dependent accumulation of cells in G(2)/M phase (1.0 and 2.5 microM MeHg) was observed independent of the p53 genotype; however, the magnitude of change was p53-dependent. CONCLUSIONS: These studies suggest that MeHg-induced cell cycle arrest occurs via both p53-dependent and -independent pathways in our model system; however, cell death resulting from MeHg exposure is highly dependent on p53.     

Ellipticine induces apoptosis through p53-dependent pathway in human hepatocellular carcinoma HepG2 cells

Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole), one of the simplest naturally occurring alkaloids, was isolated from the leaves of the evergreen tree Ochrosia elliptica Labill (Apocynaceae). Here, we reported that ellipticine inhibited the cell growth of human hepatocellular carcinoma cell line HepG2 and provided molecular understanding of this effect. The XTT assay results showed that ellipticine decreased the cell viability of HepG2 cells in a dose- and time-dependent manner, and the IC50 value was 4.1 microM. Furthermore, apoptosis induction by ellipticine in HepG2 cells was verified by the appearance of DNA fragmentation and annexin V-FITC/propidium iodide (PI) staining assay. Ellipticine treatment was found to result in the upregulation of p53, Fas/APO-1 receptor and Fas ligand. Besides, ellipticine also initiated mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, alteration of mitochondrial membrane potential (DeltaPsim), and activation of caspase-9 and caspase-3. Taken together, ellipticine decreased the cell growth and induced apoptosis in HepG2 cell.