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Abstract Colorectal cancer is a leading cause of cancer-related death. Glutaminolysis has been suggested as a therapeutic target for cancer. Costunolide is a natural sesquiterpene lactone showing potent antitumor activity. Our studies were aimed at evaluating how costunolide affected glutaminolysis leading to proliferation inhibition in human colorectal cancer cells. Costunolide suppressed viability and proliferation of HCT116 cells concentration-dependently, but did not apparently affect human intestinal epithelial cells. Costunolide at 20 μM reduced viability and proliferation of HCT116 cells time-dependently. Costunolide also repressed phosphorylation of mTOR and its downstream kinases p70S6K and 4E-BP1. Examinations of glutaminolysis metabolites showed that costunolide increased intracellular glutamine levels, but decreased intracellular levels of glutamate, α-ketoglutarate (α-KG), and ATP in HCT116 cells, suggesting costunolide blockade of glutaminolysis. Furthermore, costunolide inhibited promoter activity of glutaminase 1 (GLS1), the first rate-limiting enzyme in glutaminolysis, and reduced mRNA and protein expression of GLS1 in HCT116 cells, The GLS1 inhibitor BPTES, similar to costunolide, significantly reduced intracellular levels of α-KG and ATP and inhibited proliferation in HCT116 cells. Finally, costunolide increased phosphorylation and nuclear translocation of p53 in HCT116 cells. Both p53 inhibitor pifithrin-α and p53 siRNA significantly rescued costunolide suppression of GLS1 promoter activity and expression in HCT116 cells. These data in aggregate suggested that activation of p53 was required for costunolide inhibition of GLS1 resulting in blockade of glutaminolysis and inhibition of proliferation in colorectal cancer cells, which was a novel mechanism underlying the antitumor activity of costunolide against colorectal cancer. Graphical abstract Unlabelled Image Highlights • Costunolide inhibits viability and proliferation in HCT116 cells. • Reduction of GLS1 mediates costunolide blockade of glutaminolysis in HCT116 cells. • Activation of p53 is required for costunolide reduction of GLS1 in HCT116 cells. • GLS1-meidated glutaminolysis is an attractive target for colorectal cancer. [ABSTRACT FROM AUTHOR]

Abstract Myelodysplastic syndromes (MDS) are a varied set of hematologic neoplasms and a high risk of progression to acute myeloid leukemia (AML). 4-Amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative, play an important role in various types of cancer cells as a tumor inhibitor. However, little is known concerning its antitumor effect on MDS. The cell viability and the percentage of apoptotic cells were used to measure MTT, Flow Cytometry and Hoechst 33342/PI staining. In addition, real-time quantitative RT-PCR (qRT-PCR) and western blotting were used to analyzed the expression of p53, as well as the levels of BNIP3, apoptosis proteins of Caspase-3, BAX and BCL-2. After SKM-1 cells were incubated with DAC, ATRA and ATPR, the viability of the SKM-1 cells was inhibited in a dose- and time-dependent manner. Both Hoechst staining and flow cytometry showed the apoptosis of SKM-1 cells was increased. Moreover, SKM-1 cells treated with ATPR unveiled elevated mRNA and protein levels of p53, BNIP3, BAX and Caspase-3 expression and decreased BCL-2 expression. However, silencing p53 suppressed the pro-apoptosis function of ATPR. Consequently, these data provide the first evidence for ATPR increased apoptosis in SKM-1 cells by p53 that is mutually dependent on and obligatorily linked to BNIP3 gene activation. Highlights • DAC, ATRA and ATPR inhibited the metabolic activity of SKM-1 Cells. • DAC, ATRA and ATPR induced the apoptosis of SKM-1 Cells. • p53 induced SKM-1 cells apoptosis via BNIP3, BAX, BCL-2 and Caspase-3. • ATPR up-regulated the protein level of p53 and induced SKM-1 cell apoptosis via p53. [ABSTRACT FROM AUTHOR]

Background and Purpose: Impairment of the tumour suppressor p53 pathway is a major event in human cancers, making p53 activation one of the most attractive therapeutic strategies to halt cancer. Here, we have identified a new selective p53 activator and investigated its potential as an anticancer agent.Experimental Approach: Anti-proliferative activity of the (R)-tryptophanol-derived bicyclic lactam SYNAP was evaluated in a range of human cancer cells with different p53 status. The anticancer activity and mechanism of action of SYNAP was studied in two- and three-dimensional models of human colon adenocarcinoma HCT116 cells with wild-type p53 and corresponding p53-null isogenic derivative cells, alone and in combination with known chemotherapeutic agents.Key Results: SYNAP showed anti-proliferative effect in human cancer cells dependent on p53 status. In HCT116 cells, SYNAP caused p53-dependent growth inhibition, associated with cell cycle arrest and apoptosis, anti-migratory activity and regulation of the expression of p53 transcriptional targets. Data also indicated that SYNAP targeted p53, inhibiting its interaction with its endogenous inhibitors, murine double minute (MDM)2 and MDMX. Moreover, SYNAP sensitized colon cancer cells to the cytotoxic effect of known chemotherapeutic agents. SYNAP did not induce acquired or cross-resistance and re-sensitized doxorubicin-resistant colon cancer cells to chemotherapy. Additionally, SYNAP was non-genotoxic and had low cytotoxicity against normal cells.Conclusion and Implications: SYNAP revealed encouraging anticancer activity, either alone or in combination with known chemotherapeutic agents, in colon cancer cells. Apart from its promising application in cancer therapy, SYNAP may provide a starting point for improved p53 activators. [ABSTRACT FROM AUTHOR]

Human influenza virus (IAV) are among the most common pathogens to cause human respiratory infections. A better understanding on interplay between IAV and host factors may provide clues for disease prevention and control. While many viruses are known to downregulate p53 upon entering the cell to reduce the innate host antiviral response, IAV infection is unusual in that it activates p53. However, it has not been clear whether this process has proviral or antiviral effects. In this study, using human isogenic p53 wild-type and p53null A549 cells generated from the CRISPR/Cas9 technology, we observed that p53null cells exhibit significantly reduced viral propagation when infected with influenza A virus (strain A/Puerto Rico/8/1934 H1N1). Genome-wide microarray analysis revealed that p53 regulates the expression of a large set of interferon-inducible genes, among which the interferon-induced transmembrane family members IFITM1, IFITM2, and IFITM3 were most significantly downregulated by the expression of p53. Knockdown of interferon-induced transmembrane proteins (IFITMs) by short interfering RNAs enhanced influenza virus infectivity in p53null A549 cells, while overexpressed IFITMs in A549 cells blocked virus entry. Intriguingly, regulation of IFITMs by p53 is independent of its transcriptional activity, as the p53 short isoform Δ40p53 recapitulates IFITM regulation. Taken together, these data reveal that p53 activation by IAV is an essential step in maintaining its infectivity. This novel association between human p53 and the broad spectrum antiviral proteins, the IFITMs, demonstrates a previous mechanism employed by influenza virus to enhance its propagation via p53 inhibition of IFITMs. [ABSTRACT FROM AUTHOR]

Oxidative stress (OS)-induced retinal pigment epithelium (RPE) cell apoptosis is critically implicated in the pathogenesis of age-related macular degeneration (AMD), a leading cause of blindness in the elderly. Heterochromatin, a compact and transcriptional inert chromatin structure, has been recently shown to be dynamically regulated in response to stress stimuli. The functional mechanism of heterochromatin on OS exposure is unclear, however. Here we show that OS increases heterochromatin formation both in vivo and in vitro, which is essential for protecting RPE cells from oxidative damage. Mechanistically, OS-induced heterochromatin selectively accumulates at p53-regulated proapoptotic target promoters and inhibits their transcription. Furthermore, OS-induced desumoylation of p53 promotes p53-heterochromatin interaction and regulates p53 promoter selection, resulting in the locus-specific recruitment of heterochromatin and transcription repression. Together, our findings demonstrate a protective function of OS-induced heterochromatin formation in which p53 desumoylation-guided promoter selection and subsequent heterochromatin recruitment play a critical role. We propose that targeting heterochromatin provides a plausible therapeutic strategy for the treatment of AMD. [ABSTRACT FROM AUTHOR]

Abstract: This study was done in order to investigate time‐dependent effect of AFB1 on expression of genes involving in cell cycle check point machinery at G, S, and M phases. For this purpose, 24 mature male Swiss albino mice were randomly divided into control and test groups. The animals in test group subdivided into three groups, which received the AFB1 at a daily dose of 20 µg/kg body weight, through intraperitoneal (i.p.) route, for 7, 14, and 21 days. The p21, p53, cyclin D1, CDK4, and ERα expressions at both mRNA and protein level were analyzed by using reverse transcription PCR (RT‐PCR) and immunohistochemistry, respectively. Moreover, the tubular differentiation (TDI) and spermiogenesis (SPI) indices were analyzed. Finally, the testicular DNA fragmentation was assessed by using DNA Ladder test. Observations revealed that the AFB1 remarkably (P < .05) reduced cyclin D1, Cdk4, and ERα expression at both mRNA and protein levels. Up‐regulated p21 and p53 expression was revealed in AFB1‐received animals, which developed time dependently. Histological examinations exhibited a significant reduction in TDI and SPI indices. Finally, the AFB1 resulted in severe DNA fragmentation. Our data showed that the AFB1 by down‐regulating the cyclin D1, Cdk4, and ERα expression adversely affects cyclin D1/Cdk4 and cyclin D1/ERα interactions. Moreover, the AFB1‐induced overexpression of p21 (as a kinase inhibitor), in turn results in cell cycle arrest via inhibiting the Cdk4 interaction with cyclin D1. Finally, the AFB1‐induced DNA damage triggers the p53‐dependent apoptosis pathway independent to p21 overexpression. [ABSTRACT FROM AUTHOR]

The transcription factors p53 and p73 are critical to the induction of apoptotic cell death, particularly in response to cell stress that activates c-Jun N-terminal kinase (JNK). Mutations in the DNA-binding domain of p53, which are commonly seen in cancers, result in conformational changes that enable p53 to interact with and inhibit p73, thereby suppressing apoptosis. In contrast, wild-type p53 reportedly does not interact with p73. We found that JNK-mediated phosphorylation of Thr81 in the proline-rich domain (PRD) of p53 enabled wild-type p53, as well as mutant p53, to form a complex with p73. Structural algorithms predicted that phosphorylation of Thr81 exposes the DNA-binding domain in p53 to enable its binding to p73. The dimerization of wild-type p53 with p73 facilitated the expression of apoptotic target genes [such as those encoding p53-up-regulated modulator of apoptosis (PUMA) and Bcl-2-associated X protein (BAX)] and, subsequently, the induction of apoptosis in response to JNK activation by cell stress in various cells. Thus, JNK phosphorylation of mutant and wild-type p53 promotes the formation of a p53/p73 complex that determines cell fate: apoptosis in the context of wild-type p53 or cell survival in the context of the mutant. These findings refine our current understanding of both the mechanistic links between p53 and p73 and the functional role for Thr81 phosphorylation. [ABSTRACT FROM AUTHOR]

The transcription factor gene MYC is important in breast cancer, and its mRNA is maintained at a high level even in the absence of gene amplification. The mechanism(s) underlying increased MYC mRNA expression is unknown. Here, we demonstrate that MYC mRNA was stabilized upon estrogen stimulation of estrogen receptor-positive breast cancer cells via SRC-dependent effects on a recently described RNA-binding protein, IMP1 with an N-terminal deletion (ΔN-IMP1). We also show that loss of the tumor suppressor p53 increased MYC mRNA levels even in the absence of estrogen stimulation. However, in cells with wild-type p53, SRC acted to overcome p53-mediated inhibition of estrogen-stimulated cell cycle entry and progression. SRC thus promotes cell proliferation in two ways: by stabilizing MYC mRNA and by inhibiting p53 function. Since estrogen receptor-positive breast cancers typically express wild-type p53, these studies establish a rationale for p53 status to be predictive for effective SRC inhibitor treatment in this subtype of breast cancer. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the mechanism of p53 in regulating colistin-induced autophagy in PC-12 cells. Importantly, cells were treated with 125µg/ml colistin for 12 and 24 h after transfection with p53 siRNA or recombinant plasmid. The hallmarks of autophagy and apoptosis were examined by real-time PCR and western blot, fluorescence/immunofluorescence microscopy, and electron microscopy. The results showed that silencing of p53 leads to down-regulation of Atg5 and beclin1 for 12 h while up-regulation at 24 h and up-regulation of p62 noted. The ratio of LC3-II/I and autophagic vacuoles were significantly increased at 24 h, but autophagy flux was blocked. The cleavage of caspase3 and PARP (poly ADP-ribose polymerase) were enhanced, while PC-12-sip53 cells exposed to 3-MA showed down-regulation of apoptosis. By contrast, the expression of autophagy-related genes and protein reduced in p53 overexpressing cells following a time dependent manner. Meanwhile, there was an increase in the expression of activated caspase3 and PARP, condensed and fragmented nuclei were evident. Conclusively, the data supported that silencing of p53 promotes impaired autophagy, which acts as a pro-apoptotic induction factor in PC-12 cells treated with colistin for 24 h, and overexpression of p53 inhibits autophagy and accelerates apoptosis. Hence, it has been suggested that p53 could not act as a neuro-protective target in colistin-induced neurotoxicity. [ABSTRACT FROM AUTHOR]

Developing a sensitive and selective sensing platform for the p53 gene and its mutation analysis is essential and may aid in early cancer screening and assessment of prognosis. Here, we developed a highly sensitive and selective p53 gene assay based on the coupling of a triple-helix magnetic probe (THMP) to a fluorescent liposome hybridization assembly, a process initiated by rolling circle amplification (RCA). In the presence of p53, the THMP unfolds and activates an enzymatic cleavage reaction, thus releasing the RCA primer and initiating the RCA product-assisted fluorescent liposome hybridization assembly. The resultant double-stranded DNA structures bind the intercalating SG dye from the fluorescent liposomes, thus dramatically enhancing the fluorescence signal. In the absence of p53, the THMP remains intact and blocks the trigger release and fluorescent liposome assembly, thus resulting in a low background signal. The THMPs were designed with integrated target recognition by Watson–Crick base-pairing, site-specific cleavage by an endonuclease and background signal elimination by magnetic isolation, thus avoiding the need to design multiple probes. Moreover, the use of fluorescent liposome assembly and magnetic isolation helps in avoiding sample matrix interference and nonspecific staining. Through cooperative amplification coupling with enzyme cleavage recycling, the RCA-assisted fluorescent liposome assembly and magnetic isolation improved the sensitivity, with a detection limit of 0.07 fM. The excellent capacity of the THMP to specifically detect the involved targets and the precise site-specific endonuclease cleavage ensured remarkable selectivity for p53 against single-base mismatches. This proposed approach worked well in biological samples, thus demonstrating great potential for biomedical and clinical diagnosis applications. [ABSTRACT FROM AUTHOR]

SOX14 is a member of the SOX family of transcription factors mainly involved in the regulation of neural development. Recently, it became evident that SOX14 is one of four hypermethylated genes in cervical carcinoma, considered as a tumor suppressor candidate in this type of malignancy. In this paper we elucidated the role of SOX14 in the regulation of malignant properties of cervical carcinoma cells in vitro. Functional analysis performed in HeLa cells revealed that SOX14 overexpression decreased viability and promoted apoptosis through altering the expression of apoptosis related genes. Our results demonstrated that overexpression of SOX14 initiated accumulation of p53, demonstrating potential cross-talk between SOX14 and the p53 signaling pathway. Further analysis unambiguously showed that SOX14 triggered posttranslational modification of p53 protein, as detected by the significantly increased level of phospho-p53 (Ser-15) in SOX14-overexpressing HeLa cells. Moreover, the obtained results revealed that SOX14 activated p53 protein, which was confirmed by elevated p21Waf1/Cip1, a well known target gene of p53. This study advances our understanding about the role of SOX14 and might explain the molecular mechanism by which this transcription factor could exert tumor suppressor properties in cervical carcinoma. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSC) are promising cells for medical therapy. In in vitro expansion, MSC can give rise to progeny with genomic and epigenomic alterations, resulting in senescence, loss of terminal differentiation, and transformation to cancer. However, MSC genome protects its genetic instability by a guardian function of the P53 tumor suppressor gene and epigenetic balance system during MSC culture. Mutations of P53 and epigenetic alterations have been reported to disrupt the quality and quantity of MSC and initiate tumorigenesis. We monitor P53 and epigenetic changes in MSC derived from amniotic fluid (AF-MSC), amnion membrane (AM-MSC), endometrium (EM-MSC), and Wharton's jelly (WJ-MSC) by the missense mutation analysis of the P53 gene and the expression levels of P53, and epigenetic insulin-like growth factor 2 ( IGF2) and H19-imprinted genes. Our work demonstrates a variation of P53 expression among different MSC types. AF-MSC has a high P53 expression level with retaining a stability of P53 expression throughout a long culture period, whereas EM-MSC and WJ-MSC showed variation of P53 gene expression during culture. Epigenetic analysis showed a stable H19 expression pattern in AF-MSC, AM-MSC, and EM-MSC culture, whereas H19 expression fluctuated in WJ-MSC culture. We conclude that gene instability can be found during in vitro MSC expansion. With awareness to MSC quality and safety in MSC transformation risk, P53 mutation and IGF2 and H19-imprinted gene analysis should be applied to monitor in therapeutic-grade MSC. We also demonstrated that AF-MSC is one of the most interesting MSC for medical therapy because of its high genomic stability and epigenetic fidelity. [ABSTRACT FROM AUTHOR]

p53 is a gene that codes for a protein that regulates the cell cycle and hence functions as a tumor suppression. p53 plays an important role in cell cycle control and apoptosis. Defective p53 could allow abnormal cells to proliferate, resulting in cancer. Recently, much attention has been devoted to developing procedures to detect the presence of p53 protein at very low concentrations in a physiological environment. This article reviews DNA biosensors (genosensors) reported for the qualitative and quantitative determination of p53. We discuss critical aspects of genosensor design with particular emphasis on analytical characteristics and analysis of real samples. We assess the design and the construction of electrochemical, electrochemiluminescence, optical, and photoelectrochemical genosensors, describing the contributions so far in the p53 field and the analytical challenges involved in this research area. [ABSTRACT FROM AUTHOR]

The prognostic impact of biallelicATMabnormalities (ATMmutation and concurrent 11q deletion) remains unknown. We studiedATM,BIRC3,SF3B1, andNOTCH1genes in 118 treatment-naïve CLL patients at diagnosis. Patients with biallelicATMalteration had a similar time to first treatment (TTFT) and shorter overall survival (OS) compared with patients with isolated 11q deletion and shorter TTFT and OS when compared to patients with wild-typeATM.Furthermore, biallelicATMalteration (HR: 6.4;p ≤ 0.007) was significantly associated with an increased risk of death similar to p53 deletion (HR: 6.1;p ≤ 0.004), superior to 11q deletion alone (HR: 2.8;p ≤ 0.022) and independent of other significant parameters such as age, advanced clinical stage, and complex karyotype. Our results suggest the identification ofATMmutations in CLL patients with 11q deletion at diagnosis is clinically relevant and predicts disease progression, poor response to the treatment, and reduced OS independent of other molecular prognostic factors. [ABSTRACT FROM AUTHOR]

Tripartite-motif protein family member 65 (TRIM65) is an important protein involved in white matter lesion. However, the role of TRIM65 in human cancer remains less understood. Through the Cancer Genome Atlas (TCGA) gene alteration database, we found that TRIM65 is upregulated in a significant portion of non-small cell lung carcinoma (NSCLC) patients. Our cell growth assay revealed that TRIM65 overexpression promotes cell proliferation, while knockdown of TRIM65 displays opposite effect. Mechanistically, TRIM65 binds to p53, one of the most critical tumor suppressors, and serves as an E3 ligase toward p53. Consequently, TRIM65 inactivates p53 through facilitating p53 poly-ubiquitination and proteasome-mediated degradation. Notably, chemotherapeutic reagent cisplatin induction of p53 is markedly attenuated in response to ectopic expression of TRIM65. Cell growth inhibition by TRIM65 knockdown is more significant in p53 positive H460 than p53 negative H1299 cells, and knockdown of p53 in H460 cells also shows compromised cell growth inhibition by TRIM65 knockdown, indicating that p53 is required, at least in part, for TRIM65 function. Our findings demonstrate TRIM65 as a potential oncogenic protein, highly likely through p53 inactivation, and provide insight into development of novel approaches targeting TRIM65 for NSCLC treatment, and also overcoming chemotherapy resistance. [ABSTRACT FROM AUTHOR]

DNAJB9 is a recently isolated member of the molecular chaperone gene family, whose precise function is largely unknown. In the present study, we have identified DNAJB9 as an inducible gene of the tumor suppressor p53. DNAJB9 expression was induced by p53 or genotoxic stress in a p53-dependent manner, which was mediated by the Ras/Raf/ERK pathway. In addition, depletion of DNAJB9 by using siRNAs greatly increased genotoxic stress/p53-induced apoptosis, suggesting that DNAJB9 inhibits the pro-apoptotic function of p53. We also found that DNAJB9 physically interacts with p53 through its J domain, through which it inhibits the pro-apoptotic function of p53. Moreover, DNAJB9 colocalized with p53 in both cytoplasm and nucleus under genotoxic conditions. Together, these results demonstrate that DNAJB9 is a downstream target of p53 that belongs to the group of negative feedback regulators of p53. [ABSTRACT FROM AUTHOR]

The transcription factor p53 regulates the expression of genes crucial for biological processes such as cell proliferation, metabolism, cell repair, senescence and apoptosis. Activation of p53 also suppresses neoplastic transformations, thereby inhibiting the growth of mutated and/or damaged cells. p53-binding proteins, such as mouse double minute 2 homolog (MDM2), inhibit p53 activation and thus regulate p53-mediated stress responses. Here, we found that long glucocorticoid-induced leucine zipper (L-GILZ), a recently identified isoform of GILZ, activates p53 and that the overexpression of L-GILZ in p53+/+ HCT116 human colorectal carcinoma cells suppresses the growth of xenografts in mice. In the presence of both p53 and MDM2, L-GILZ binds preferentially to MDM2 and interferes with p53/MDM2 complex formation, making p53 available for downstream gene activation. Consistent with this finding, L-GILZ induced p21 and p53 upregulated modulator of apoptosis (PUMA) expression only in p53+/+ cells, while L-GILZ silencing reversed the anti-proliferative activity of dexamethasone as well as expression of p53, p21 and PUMA. Furthermore, L-GILZ stabilizes p53 proteins by decreasing p53 ubiquitination and increasing MDM2 ubiquitination. These findings reveal L-GILZ as a regulator of p53 and a candidate for new therapeutic anti-cancer strategies for tumors associated with p53 deregulation. [ABSTRACT FROM AUTHOR]

Histone deacetylase 2 (HDAC2) regulates biological processes by deacetylation of histones and non-histone proteins. HDAC2 is overexpressed in numerous cancer types, suggesting general cancer-relevant functions of HDAC2. In human tumors the TP53 gene encoding p53 is frequently mutated and wild-type p53 is often disarmed. Molecular pathways inactivating wild-type p53 often remain to be defined and understood. Remarkably, current data link HDAC2 to the regulation of the tumor suppressor p53 by deacetylation and to the maintenance of genomic stability. Here, we summarize recent findings on HDAC2 overexpression in solid and hematopoietic cancers with a focus on mechanisms connecting HDAC2 and p53 in vitro and in vivo. In addition, we present an evidence-based model that integrates molecular pathways and feedback loops by which p53 and further transcription factors govern the expression and the ubiquitin-dependent proteasomal degradation of HDAC2 and of p53 itself. Understanding the interactions between p53 and HDAC2 might aid in the development of new therapeutic approaches against cancer. [ABSTRACT FROM AUTHOR]

Background: The induction of hepatic stellate cell (HSC) apoptosis has potential as a potent strategy to diminish the progression of liver fibrosis. Previous studies have demonstrated the ability of soluble egg antigens (SEA) from schistosomes to inhibit HSC activation and to induce apoptosis in vitro. In this study, we aimed to explore the mechanism of SEA-induced apoptosis in HSCs. Methodology/Principal Findings: In this study, we found that SEA could upregulate p53 and DR5 and downregulate the p-Akt. The apoptosis of HSCs induced by SEA could be reduced in HSCs that were treated with p53-specific siRNA and in HSCs that were treated with DR5-specific shRNA. In addition, GW501516, which enhances the expression of Akt, could also decrease the SEA-induced HSC apoptosis. We also found that the increased expression of p53 and DR5 induced by SEA through Mdm2 were reduced by GW501516. Conclusions/Significance: Our data suggest that SEA can induce HSC apoptosis by downregulating Akt expression and upregulating p53-dependent DR5 expression. [ABSTRACT FROM AUTHOR]

The Snail transcription factor plays a key role in regulating diverse developmental processes but is not thought to play a role in mammalian neural precursors. Here, we have examined radial glial precursor cells of the embryonic murine cortex and demonstrate that Snail regulates their survival, self-renewal, and differentiation into intermediate progenitors and neurons via two distinct and separable target pathways. First, Snail promotes cell survival by antagonizing a p53-dependent death pathway because coincident p53 knockdown rescues survival deficits caused by Snail knockdown. Second, we show that the cell cycle phosphatase Cdc25b is regulated by Snail in radial precursors and that Cdc25b coexpression is sufficient to rescue the decreased radial precursor proliferation and differentiation observed upon Snail knockdown. Thus, Snail acts via p53 and Cdc25b to coordinately regulate multiple aspects of mammalian embryonic neural precursor biology. [ABSTRACT FROM AUTHOR]

Cadmium and cadmium compounds are contaminants of the environment, food, and drinking water and are important constituents of cigarette smoke. Cd exposure has also been associated with airborne particulate CdO and with Cd-containing quantum dots in medical therapy. Adverse cadmium effects reported in the literature have stimulated during recent years an ongoing discussion to better elucidate cadmium outcomes at cell and molecular level. The present work is designed to gain an insight into the mechanism of p53 impairment at gene and protein level to understand Cd-induced resistance to apoptosis. We used a hepatoma cell line (HepG2) derived from liver, known to be metal responsive. At genotoxic cadmium concentrations no cell cycle arrest was observed. The p53 at gene and protein level was not regulated. Fluorescence images showed that p53 was correctly translocated into the nucleus but that the p21Cip1/WAF-1, a downstream protein of p53 network involved in cell cycle regulation, was not activated at the highest cadmium concentrations used. The miRNAs analysis revealed an upregulation of mir-372, an miRNA able to affect p21Cip1/WAF-1 expression and promote cell cycle progression and proliferation. The role ofmetallothioneins and possible conformational changes of p53 are discussed. [ABSTRACT FROM AUTHOR]

Among the main causes of cancer cell resistance to chemotherapy are p53 mutation and hypoxic tumor microenvironment. However, the effect of hypoxia can be very different from one cell type to the other. We studied the effect of hypoxia on the etoposide-induced cell death in two cancer cell lines, HepG2 and A549 cells. Hypoxia decreased etoposide-induced apoptosis in HepG2 cells but not in A549 cells. Here, we evidenced two pathways, known to play important roles in cancer cell resistance, that are differently affected by hypoxia in these two cell types. First, in HepG2 cells, hypoxia decreased p53 protein level and activity by acting post-transcriptionally and independently of HIF-1. The results suggest an effect of hypoxia on p53 translation. On the other hand, in A549 cells, no effect of hypoxia was observed on p53 level. Secondly, hypoxia decreased DNA damage response in HepG2 cells while this was not the case in A549 cells. Indeed, a decrease in the phosphorylation level of CHK2 and H2AX with a decrease in ATM activity was observed. Importantly, these results evidenced that hypoxia can prevent cancer cell apoptosis by acting at different levels in the cell and that these effects are strongly cell-type dependent. J. Cell. Physiol. 228: 2365-2376, 2013. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

To gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53, X-ray crystallographic structures of four p53 core-domain variants were determined. These include an oncogenic mutant, V157F, two single-site suppressor mutants, N235K and N239Y, and the rescued cancer mutant V157F/N235K/N239Y. The V157F mutation substitutes a smaller hydrophobic valine with a larger hydrophobic phenylalanine within strand S4 of the hydrophobic core. The structure of this cancer mutant shows no gross structural changes in the overall fold of the p53 core domain, only minor rearrangements of side chains within the hydrophobic core of the protein. Based on biochemical analysis, these small local perturbations induce instability in the protein, increasing the free energy by 3.6 kcal mol−1 (15.1 kJ mol−1). Further biochemical evidence shows that each suppressor mutation, N235K or N239Y, acts individually to restore thermodynamic stability to V157F and that both together are more effective than either alone. All rescued mutants were found to have wild-type DNA-binding activity when assessed at a permissive temperature, thus pointing to thermodynamic stability as the critical underlying variable. Interestingly, thermodynamic analysis shows that while N239Y demonstrates stabilization of the wild-type p53 core domain, N235K does not. These observations suggest distinct structural mechanisms of rescue. A new salt bridge between Lys235 and Glu198, found in both the N235K and rescued cancer mutant structures, suggests a rescue mechanism that relies on stabilizing the β-sandwich scaffold. On the other hand, the substitution N239Y creates an advantageous hydrophobic contact between the aromatic ring of this tyrosine and the adjacent Leu137. Surprisingly, the rescued cancer mutant shows much larger structural deviations than the cancer mutant alone when compared with wild-type p53. These suppressor mutations appear to rescue p53 function by creating novel intradomain interactions that stabilize the core domain, allowing compensation for the destabilizing V157F mutation. [ABSTRACT FROM AUTHOR]

The cellular response evoked by antiproliferating osmium(VI) nitrido compounds of general formula OsN(N⋀N)Cl3 (N⋀N = 2,2'-bipyridine 1, 1,10-phenanthroline 2, 3,4,7,8-tetramethyl-1,10-phenanthroline 3, or 4,7-diphenyl-1,10-phenanthroline 4) can be tuned by subtle ligand modifications. Complex 2 induces DNA damage, resulting in activation of the p53 pathway, cell cycle arrest at the G2/M phase, and caspase-dependent apoptotic cell death. In contrast, 4 evokes endoplasmic reticulum (ER) stress leading to the upregulation of proteins of the unfolded protein response pathway, increase in ER size, and p53-independent apoptotic cell death. To the best of our knowledge, 4 is the first osmium compound to induce ER stress in cancer cells. [ABSTRACT FROM AUTHOR]

Purpose: We studied whether immunohistochemical expression of p53 in Wilms tumors correlates with tumor aggressiveness. We also examined whether preoperative chemotherapy results in any alteration of p53 expression. Materials and Methods: A total of 18 patients underwent preoperative chemotherapy and 30 underwent immediate surgery for Wilms tumor. All children were younger than 10 years and had histologically confirmed disease. Patients with a bilateral tumor or a syndrome related to Wilms tumor were excluded. All pathology slides were uniformly stained for p53 protein, and p53 staining density and intensity were scored. The p53 scoring was then compared to the clinical behavior of the Wilms tumor, ie unfavorable tumor staging, and survival and recurrence rates. Results: In the direct surgery and the preoperatively treated groups p53 positivity correlated with unfavorable Wilms tumor staging (p = 0.007). In addition, a positive p53 correlation predicted poorer survival (p = 0.017). Interestingly patients who underwent preoperative chemotherapy had an increased intensity of p53 staining compared to the direct surgery group (p <0.001). Conclusions: This study provides preliminary evidence that a higher score for immunohistochemical p53 expression correlates with unfavorable Wilms tumor staging and predicts poorer survival. This test could become a useful addition to the current histopathological analysis of Wilms tumor. [Copyright &y& Elsevier]

Cellular senescence both protects multicellular organisms from cancer and contributes to their ageing. The pre-eminent tumour suppressor p53 has an important role in the induction and maintenance of senescence, but how it carries out this function remains poorly understood. In addition, although increasing evidence supports the idea that metabolic changes underlie many cell-fate decisions and p53-mediated tumour suppression, few connections between metabolic enzymes and senescence have been established. Here we describe a new mechanism by which p53 links these functions. We show that p53 represses the expression of the tricarboxylic-acid-cycle-associated malic enzymes ME1 and ME2 in human and mouse cells. Both malic enzymes are important for NADPH production, lipogenesis and glutamine metabolism, but ME2 has a more profound effect. Through the inhibition of malic enzymes, p53 regulates cell metabolism and proliferation. Downregulation of ME1 and ME2 reciprocally activates p53 through distinct MDM2- and AMP-activated protein kinase-mediated mechanisms in a feed-forward manner, bolstering this pathway and enhancing p53 activation. Downregulation of ME1 and ME2 also modulates the outcome of p53 activation, leading to strong induction of senescence, but not apoptosis, whereas enforced expression of either malic enzyme suppresses senescence. Our findings define physiological functions of malic enzymes, demonstrate a positive-feedback mechanism that sustains p53 activation, and reveal a connection between metabolism and senescence mediated by p53. [ABSTRACT FROM AUTHOR]

Background Inactivation of wild type P53 by its main cellular inhibitors (MDM2 and MDMX) is a well recognised feature of tumour formation in liposarcomas. MDM2 over-expression has been detected in approximately 80% of liposarcomas but only limited information is available about MDMX over-expression. To date, we are not aware of any study that has described the patterns of MDM2 and MDMX co-expression in liposarcomas. Such information has become more pertinent as various novel MDM2 and / or MDMX single and dual affinity antagonist compounds are emerging as an alternative approach for potential targeted therapeutic strategies. Methods We analysed a case series of 61 fully characterized liposarcomas of various sub-types by immunohistochemistry, to assess the expression levels of P53, MDM2 and MDMX, simultaneously. P53 sequencing was performed in all cases that expressed P53 protein in 10% or more of cells to rule out mutation-related over-expression. Results 50 cases over-expressed MDM2 and 42 of these co-expressed MDMX at varying relative levels. The relative expression levels of the two proteins with respect to each other were subtype-dependent. This apparently affected the detected levels of P53 directly in two distinct patterns. Diminished levels of P53 were observed when MDM2 was significantly higher in relation to MDMX, suggesting a dominant role for MDM2 in the degradation of P53. Higher levels of P53 were noted with increasing MDMX levels suggesting an interaction between MDM2 and MDMX that resulted in a reduced efficiency of MDM2 in degrading P53. Of the 26 cases of liposarcoma with elevated P53 expression, 5 were found to have a somatic mutation in the P53 gene. Conclusions The results suggest that complex dynamic interactions between MDM2 and MDMX proteins may directly affect the cellular levels of P53. This therefore suggests that careful characterization of both these markers will be necessary in tumours when considering in vivo evaluation of novel blocker compounds for MDM proteins, as a therapeutic strategy to restore wild type P53 function. [ABSTRACT FROM AUTHOR]

Summary: N 6-methyladenosine (m6A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m6A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m6A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m6A modification has fundamental and broad functions in mammalian cells. [Copyright &y& Elsevier]

DNA strand breakage and perturbation of cell-cycle progression contribute to gene amplification events that can drive cancer. In cells lacking p53, DNA damage does not trigger an effective cell-cycle arrest and in this setting promotes gene amplification. This is also increased in cells harboring oncogenic Ras, in which cell-cycle arrest is perturbed and ROS levels that cause DNA single strand breaks are elevated. This study focused on the effects of v-K-ras and p53 on Methotrexate (MTX)-mediated DHFR amplification. Rat lung epithelial cells expressing v-K-ras or murine lung cancer LKR cells harboring active K-ras continued cell-cycle progression when treated with MTX. However, upon loss of p53, amplification of DHFR and formation of MTX-resistant colonies occurred. Expression levels of cyclin A, Geminin, and Cdt1 were increased in v-K-ras transfectants. Geminin was sufficient to prevent the occurrence of multiple replications via interaction with Cdt1 after MTX treatment, and DHFR amplification proceeded in v-K-ras transfectants that possess a functional p53 in the absence of geminin. Taken together, our findings indicate that p53 not only regulates cell-cycle progression, but also functions through geminin to prevent DHFR amplification and protect genomic integrity. [ABSTRACT FROM AUTHOR]

TP53INP1 (tumor protein 53-induced nuclear protein 1) is a tumor suppressor, whose expression is downregulated in cancers from different organs. It was described as a p53 target gene involved in cell death, cell-cycle arrest and cellular migration. In this work, we show that TP53INP1 is also able to interact with ATG8-family proteins and to induce autophagy-dependent cell death. In agreement with this finding, we observe that TP53INP1, which is mainly nuclear, relocalizes in autophagosomes during autophagy where it is eventually degraded. TP53INP1-LC3 interaction occurs via a functional LC3-interacting region (LIR). Inactivating mutations of this sequence abolish TP53INP1-LC3 interaction, relocalize TP53INP1 in autophagosomes and decrease TP53INP1 ability to trigger cell death. Interestingly, TP53INP1 binds to ATG8-family proteins with higher affinity than p62, suggesting that it could partially displace p62 from autophagosomes, modifying thereby their composition. Moreover, silencing the expression of autophagy related genes (ATG5 or Beclin-1) or inhibiting caspase activity significantly decreases cell death induced by TP53INP1. These data indicate that cell death observed after TP53INP1-LC3 interaction depends on both autophagy and caspase activity. We conclude that TP53INP1 could act as a tumor suppressor by inducing cell death by caspase-dependent autophagy. [ABSTRACT FROM AUTHOR]

The article presents a study discussing the role of methylation status of genes such as p14ARF, TMS1/ASC, and DAPK, in activation of the p53-Bax mitochondrial apoptosis pathway and its clinical significance in cholangiocarcinoma. It is found that frequency of methylation in cholangiocarcinoma tissue was much higher than the frequency of methylation in normal adjacent tissue. It is also mentioned that the 38.9 percent cholangiocarcinoma cases had combined p53 mutation and DAPK, p14ARF, and ASC methylation, and these patients had a poorer pathologic biology and prognosis than the other patients.

Hepatocellular carcinoma (HCC) is increasing in incidence, and the associated mortality rate remains among the highest. For advanced HCC, sorafenib has been shown to slightly prolong survival, and regorafenib and nivolumab, both recently approved by the United States Food and Drug Administration (FDA), may produce clinical benefits to a limited extent. Systemic chemotherapy has been shown to produce a modest response, but there is no clinically valid biomarker that can be used to predict which patients may benefit. In this case study, we present two patients with metastatic HCC, they received systemic treatment using capecitabine, oxaliplatin, and either bevacizumab or sorafenib. The tumor response to treatment was determined by the progression-free survival (PFS). Molecular profiling of the tumors showed differential expression of biochemical markers and different mutational status of the TP53 and β-catenin (CTNNB1) genes. We hypothesize that the PFS correlates with the tumor molecular profiles, which may be predictive of the therapeutic response to systemic chemotherapy. Further investigation is indicated to correlate tumor biomarkers and treatment responses, with the objective of personalizing the therapies for patients with advanced HCC. [ABSTRACT FROM AUTHOR]

An introduction is presented in which the editor discusses various reports within the issue including the Clustered, Regularly Interspaced, Short Palindromic Repeats technology, microRNA, and p53 transcription factors.

The author reflects on the functional role of murine double minute 2 (MDM2) to the negative regulation of tumor suppressor p53. He mentions an article by G. L. Bond and colleagues wherein a single nucleotide polymorphism (SNP) in the MDM2 promoter (SNP309;rs2279744) led in higher MDM2 production with subsequent p53 inactivation. He also talks on the relationship of MDMW SNP309 polymorphism with prostate cancer development or progression.