P53 Apoptotic Pathway Research Articles

Bioactive compound 1,8-Cineole selectively induces G2/M arrest in A431 cells through the upregulation of the p53 signaling pathway and molecular docking studies

BackgroundCallistemon citrinus has been traditionally known for its medicinal property. Recently, our research group identified 1,8-Cineole, as one of the predominant compound present in the hexane extract (HE-C), whose leaves have potent anticancer activity. Hypothesis/PurposeThe present study was designed to isolate 1,8-Cineole from Callistemon citrinus plant and to determine their role in anticancer effects in in vitro using skin carcinoma cells. Moreover, the molecular mechanism of apoptosis and molecular docking studies were also investigated. Study design/MethodsIn vitro cytotoxicity test was performed with HE-C fractionates 1F, 2F, and 3F against A431 and HaCaT cell lines. MTT and AB assay demonstrated that 1F was toxic to cancer cells with no adverse effect to non-malignant cells and it was subjected to 1H NMR, 13C NMR spectroscopy and further characterized by FTIR and GC–MS analysis. On the basis of spectroscopic data, the metabolite was confirmed as 1,8-Cineole. ResultsBased on the cytotoxicity results, the well-characterized metabolite 1,8-Cineole was investigated upon to understand the mechanism that caused cancer cell death. In this process, the changes in mitochondrial membrane potential (ΔΨm) were confirmed by Rh-123/DAPI staining; the ultra structure was observed by TEM and quantified by flow cytometric analysis. These results proved that the compound effectively induced the apoptosis and G2/M phase arrest in A431 cells by increasing the expression of p53 and that it was monitored by FACS. Further, the expression of apoptotic proteins, such as Bax/Bcl-2, Cyt-c, caspase-9, and caspase-3 was confirmed by western blot. The molecular docking simulations predicted the hydrophobic interaction between 1,8-cineole with Bcl-2 and PARP1 receptor. Conclusions1,8-Cineole is a potential candidate for skin carcinoma, which is possible by regulating the p53 apoptotic signaling pathway.

Cofilin-mediated Neuronal Apoptosis via p53 Translocation and PLD1 Regulation

Amyloid β (Aβ) accumulation is an early event in the pathogenesis of Alzheimer’s disease (AD), leading to mitochondrial and synaptic dysfunction, tau accumulation, and eventual neuronal death. While the p53 apoptotic pathway has clearly been associated with Aβ deposits and neuronal apoptosis, the critical upstream factors contributing to p53 activation in AD are not well understood. We have previously shown that cofilin activation plays a pivotal role in Aβ-induced mitochondrial and synaptic dysfunction. In this study, we show that activated cofilin (S3A) preferentially forms a complex with p53 and promotes its mitochondrial and nuclear localization, resulting in transcription of p53-responsive genes and promotion of apoptosis. Conversely, reduction of endogenous cofilin by knockdown or genetic deficiency inhibits mitochondrial and nuclear translocation of p53 in cultured cells and in APP/PS1 mice. This cofilin-p53 pro-apoptotic pathway is subject to negative regulation by PLD1 thorough cofilin inactivation and inhibition of cofilin/p53 complex formation. Finally, activated cofilin is unable to induce apoptosis in cells genetically lacking p53. These findings taken together indicate that cofilin coopts and requires the nuclear and mitochondrial pro-apoptotic p53 program to induce and execute apoptosis, while PLD1 functions in a regulatory multi-brake capacity in this pathway.

Blueberry anthocyanin induces apoptosis in HepG-2 cells and the mechanism of the process

The objective of this study was to evaluate the anti-proliferation and inducing apoptosis of human hepatocellular carcinoma HepG-2 cells by anthocyanin. In order to investigate the involvement of apoptotic-related proteins and gene expression, RT-PCR, western blot, transcriptome sequencing, and appropriate biochemical assays were employed. The results indicated that anthocyanin showed significant inhibitory effect on HepG-2 cells (p < 0.05). Anthocyanin induced cell apoptosis in HepG-2 cells, increased in the generation of reactive oxygen species, decreased mitochondrial membrane potential, increased the activity caspase-3, released cytochrome c from the mitochondria into the cytosol, down-regulated the expression of Bcl-2, and up-regulated the expression of Bax. The transcriptome sequencing analysis revealed that anthocyanin activated p38 MAPK signal pathway, p53 signaling pathway and apoptotic signaling pathway in response to endoplasmic reticulum stress and inactivated TGF-beta signaling pathway. Genes involved in apoptosis included GADD45A, GADD45B, DDIT4, CHAC1, ATF-4, CDKN1A, and EPHA2. The present findings indicated that anthocyanin from blueberry can significantly induce HepG-2 cells apoptosis and might be used as an adjuvant ingredient for cancer prevention in the future.

Deficiency of 53BP1 inhibits the radiosensitivity of colorectal cancer.

The present study aimed to observe the influence of p53 binding protein 1 (53BP1) silencing on the radiosensitivity of colorectal cancer (CRC) cells and investigated the potential underlying mechanisms. The differences in radiosensitivity among four CRC cells were detected by the clone formation assay, while the expression of their 53BP1 was detected by the western blot analysis. HCT116 cells with relatively high expression of 53BP1 were selected to silence the expression of 53BP1 by shRNA intervention. The influence on proliferation, apoptosis, and cell cycle distribution was detected by immunofluorescent staining of Ki-67 and flow cytometry. The expression of relevant proteins in the apoptotic pathway ATM-CHK2-p53 was further analyzed by western blot analysis. The expression of 53BP1 was found to be closely related to the radiosensitivity of the CRC cells. Decreased expression of 53BP1 led to the tolerance of HCT116 cells to radiation. The detection of tumor proliferation, apoptosis, and cell cycle showed that decreased expression of 53BP1 resulted in an increased S-phase percentage of HCT116 cells, an increased proliferating rate, and a decreased apoptotic rate after radiation. The analysis of the molecular pathway showed that the reduced expression of 53BP1 decreased the protein expression of ATM, CHK2, and the phosphorylated products associated with the p53 apoptotic pathway. In conclusion, decreased expression of 53BP1 leads to radiotolerance of CRC cells, and the underlying mechanism is probably related to the decreased expression of relevant proteins in the ATM-CHK2-p53 pathway, which affects cell cycle, apoptosis and proliferation.

Suppressing activity of tributyrin on hepatocarcinogenesis is associated with inhibiting the p53-CRM1 interaction and changing the cellular compartmentalization of p53 protein.

Hepatocellular carcinoma (HCC), an aggressive and the fastest growing life-threatening cancer worldwide, is often diagnosed at intermediate or advanced stages of the disease, which substantially limits therapeutic approaches for its successful treatment. This indicates that the prevention of hepatocarcinogenesis is probably the most promising approach to reduce both the HCC incidence and cancer-related mortality. In previous studies, we demonstrated a potent chemopreventive effect of tributyrin, a butyric acid prodrug, on experimental hepatocarcinogenesis. The cancer-inhibitory effect of tributyrin was linked to the suppression of sustained cell proliferation and induction of apoptotic cell death driven by an activation of the p53 apoptotic signaling pathway. The goal of the present study was to investigate the underlying molecular mechanisms linked to tributyrin-mediated p53 activation. Using in vivo and in vitro models of liver cancer, we demonstrate that an increase in the level of p53 protein in nuclei, a decrease in the level of cytoplasmic p53, and, consequently, an increase in the ratio of nuclear/cytoplasmic p53 in rat preneoplastic livers and in rat and human HCC cell lines caused by tributyrin or sodium butyrate treatments was associated with a marked increase in the level of nuclear chromosome region maintenance 1 (CRM1) protein. Mechanistically, the increase in the level of nuclear p53 protein was associated with a substantially reduced binding interaction between CRM1 and p53. The results demonstrate that the cancer-inhibitory activity of sodium butyrate and its derivatives on liver carcinogenesis may be attributed to retention of p53 and CRM1 proteins in the nucleus, an event that may trigger activation of p53-mediated apoptotic cell death in neoplastic cells.

Human papillomavirus confers radiosensitivity in cancer cervix: a hypothesis toward a possible restoration of apoptotic pathways based on clinical outcomes.

To evaluate if high baseline local human papillomavirus (HPV) titer confers radiosensitivity in cancer cervix. A hypothesis is proposed to explain the clinical outcomes. 121 serial HPV titers from cervical smears of 21 patients were estimated during radiotherapy (RT) and correlated with RT dose-response curves, local response and local disease-free survival (LDFS). Local response (p = 0.04) and LDFS (p = 0.06) were better in high HPV than low HPV baseline group. On multivariate analysis, RT doses for 50% tumor regression and baseline HPV titer were the only predictors for LDFS. Serial reductions of HPV titers following RT could restore the HPV induced temporarily downregulated p53 and pRb apoptotic pathways resulting in radiosensitivity of these tumors.

The nucleoprotein of influenza A virus induces p53 signaling and apoptosis via attenuation of host ubiquitin ligase RNF43.

The interplay between influenza virus and host factors to support the viral life cycle is well documented. Influenza A virus (IAV) proteins interact with an array of cellular proteins and hijack host pathways which are at the helm of cellular responses to facilitate virus invasion. The multifaceted nature of the ubiquitination pathway for protein regulation makes it a vulnerable target of many viruses including IAV. To this end we conducted a yeast two-hybrid screen to search for cellular ubiquitin ligases important for influenza virus replication. We identified host protein, RING finger protein 43 (RNF43), a RING-type E3 ubiquitin ligase, as a novel interactor of nucleoprotein (NP) of IAV and an essential partner to induce NP-driven p53-mediated apoptosis in IAV-infected cells. In this study, we demonstrate that IAV leads to attenuation of RNF43 transcripts and hence its respective protein levels in the cellular milieu whereas in RNF43 depleted cells, viral replication was escalated several folds. Moreover, RNF43 polyubiquitinates p53 which further leads to its destabilization resulting in a decrease in induction of the p53 apoptotic pathway, a hitherto unknown process targeted by NP for p53 stabilization and accumulation. Collectively, these results conclude that NP targets RNF43 to modulate p53 ubiquitination levels and hence causes p53 stabilization which is conducive to an enhanced apoptosis level in the host cells. In conclusion, our study unravels a novel strategy adopted by IAV for utilizing the much conserved ubiquitin proteasomal pathway.

Highly specific in vivo gene delivery for p53-mediated apoptosis and genetic photodynamic therapies of tumour.

Anticancer therapies are often compromised by nonspecific effects and challenged by tumour environments’ inherent physicochemical and biological characteristics. Often, therapeutic effect can be increased by addressing multiple parameters simultaneously. Here we report on exploiting extravasation due to inherent vascular leakiness for the delivery of a pH-sensitive polymer carrier. Tumours’ acidic microenvironment instigates a charge reversal that promotes cellular internalization where endosomes destabilize and gene delivery is achieved. We assess our carrier with an aggressive non-small cell lung carcinoma (NSCLC) in vivo model and achieve >30% transfection efficiency via systemic delivery. Rejuvenation of the p53 apoptotic pathway as well as expression of KillerRed protein for sensitization in photodynamic therapy (PDT) is accomplished. A single administration greatly suppresses tumour growth and extends median animal survival from 28 days in control subjects to 68 days. The carrier has capacity for multiple payloads for greater therapeutic response where inter-individual variability can compromise efficacy.

P14ARF enhances chemosensitivity to cisplatin in human osteosarcoma U2OS cells through p53 apoptotic pathway

To explore the effects of tumor suppressor p14ARF on chemosensitivity of human osteosarcoma U2OS cells to cisplatin and elucidate its molecular mechanism. U2OS cells expressing no p14ARF and U2OS-ARF cells expressing p14ARF stably through stable transfection were treated with cisplatin. Cell viability and IC50 were assayed with methyl thiazolyl tetrazolium (MTT). Apoptosis was examined by fluorescence-activated cell sorting and Hoechst33258 staining. The expressions of p53, Bax, p21, Mdm2 and Fas were detected by Western blot. And colorimetry was used to determine the activities of caspase-3, caspase-8 and caspase-9. The viability was 84.8% ± 4.4%, 86.9% ± 5.0% and 66.7% ± 4.6% respectively in U2OS, U2OS-vec and U2OS-ARF cells. The values of IC50 were (15.8 ± 0.9) µmol/L, (16.3 ± 0.6) and (8.9 ± 0.8) µmol/L respectively in U2OS, U2OS-vec and U2OS-ARF cells. The levels of viability and IC50 obviously decreased in U2OS-ARF cells in response to cisplatin (P < 0.05). There were higher apoptotic rate and more obvious apoptotic morphological changes in U2OS-ARF cells than U2OS and U2OS-vec cells. The basal levels of p53, Mdm2 and p21 in U2OS-ARF cells were slightly higher than those in U2OS-vec cells. Cisplatin up-regulated p53, Mdm2 and p21 in both cell lines. However, the up-regulation was more pronounced in U2OS-ARF cells. Cisplatin did not change the levels of Bax and Fas in U2OS-vec cells. Bax protein was up-regulated in U2OS-ARF cells while the level of Fas remained constant. p14ARF also enhanced the activities of caspase-9 and caspase-3 in response to cisplatin. p14ARF enhances the chemosensitivity to cisplatin in human osteosarcoma U2OS cells through p53 apoptotic pathway. And intrinsic mitochondrial apoptosis is involved.

Oxazoloisoindolinones with in vitro antitumor activity selectively activate a p53-pathway through potential inhibition of the p53–MDM2 interaction

One of the most appealing targets for anticancer treatment is the p53 tumor suppressor protein. In half of human cancers, this protein is inactivated due to endogenous negative regulators such as MDM2. Actually, restoring the p53 activity, particularly through the inhibition of its interaction with MDM2, is considered a valuable therapeutic strategy against cancers with a wild-type p53 status. In this work, we report the synthesis of nine enantiopure phenylalaninol-derived oxazolopyrrolidone lactams and the evaluation of their biological effects as p53–MDM2 interaction inhibitors. Using a yeast-based screening assay, two oxazoloisoindolinones, compounds 1b and 3a, were identified as potential p53–MDM2 interaction inhibitors. The molecular mechanism of oxazoloisoindolinone 3a was further validated in human colon adenocarcinoma HCT116 cells with wild-type p53 (HCT116 p53+/+) and in its isogenic derivative without p53 (HCT116 p53−/−). Indeed, using these cells, we demonstrated that oxazoloisoindolinone 3a exhibited a p53-dependent in vitro antitumor activity through induction of G0/G1-phase cell cycle arrest and apoptosis. The selective activation of a p53-apoptotic pathway by oxazoloisoindolinone 3a was further supported by the occurrence of PARP cleavage only in p53-expressing HCT116 cells. Moreover, oxazoloisoindolinone 3a led to p53 protein stabilization and to the up-regulation of p53 transcriptional activity with increased expression levels of several p53 target genes, as p21(WAF1/CIP1), MDM2, BAX and PUMA, in p53+/+ but not in p53−/− HCT116 cells. Additionally, the ability of oxazoloisoindolinone 3a to block the p53–MDM2 interaction in HCT116 p53+/+ cells was confirmed by co-immunoprecipitation. Finally, the molecular docking analysis of the interactions between the synthesized compounds and MDM2 revealed that oxazoloisoindolinone 3a binds to MDM2. Altogether, this work adds, for the first time, the oxazoloisoindolinone scaffold to the list of chemotypes activators of a wild-type p53-pathway with promising antitumor activity. Moreover, it may open the way to the development of a new class of p53–MDM2 interaction inhibitors.

High dose ionizing radiation regulates micro RNA and gene expression changes in human peripheral blood mononuclear cells.

BackgroundHigh dose ionizing radiation (IR) induces potent toxic cell effects mediated by either direct DNA damage or the production of reactive oxygen species (ROS). IR-induced modulations in multiple biological processes have been proposed to be partly regulated by radiosensitive microRNA (miRNA). In order to gain new insights into the role of miRNAs in the regulation of biological processes after IR, we have investigated changes in mRNA and miRNA expression after high dose IR.ResultsIR induced changes in the mRNA and miRNA profiles of human peripheral blood mononuclear cells (PBMCs). When comparing non-irradiated and irradiated samples, we detected a time-dependent increase in differentially expressed mRNAs and miRNAs, with the highest differences detectable 20 hours after exposure. Gene ontology analysis revealed that very early events (up to 4 hours) after irradiation were specifically associated with p53 signaling and apoptotic pathways, whereas a large number of diverse cellular processes were deregulated after 20 hours. Transcription factor analysis of all up-regulated genes confirmed the importance of p53 in the early post-irradiation phase. When analyzing miRNA expression, we found 177 miRNAs that were significantly regulated in the late post-irradiation phase. Integrating miRNA and target gene expression data, we found a significant negative correlation between miRNA-mRNA and identified hepatic leukemia factor (HLF) as a transcription factor down-regulated in the response to IR. These regulated miRNAs and the HLF target genes were involved in modulating radio-responsive pathways, such as apoptosis, the MAKP signaling pathway, endocytosis, and cytokine-cytokine interactions.ConclusionUsing a large dataset of mRNA and miRNA expression profiles, we describe the interplay of mRNAs and miRNAs in the regulation of gene expression in response to IR at a posttranscriptional level and their involvement in the modulation of radiation-induced biological pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-814) contains supplementary material, which is available to authorized users.

Urokinase-type plasminogen activator receptor regulates apoptotic sensitivity of colon cancer HCT116 cell line to TRAIL via JNK-p53 pathway

The urokinase-type plasminogen activator receptor (uPAR) serves not only as an anchor for urokinase-type plasminogen activator but also participates in intracellular signal transduction events. In this study, we investigated whether uPAR could modulate TRAIL-induced apoptosis in human colon cancer cells HCT116. Using an antisense strategy, we established a stable HCT116 cell line with down-regulated uPAR. The sensitivity to TRAIL-induced apoptosis was evaluated by FACS analysis. Our results show that the inhibition of uPAR could sensitize HCT116 to TRAIL-induced apoptosis. uPAR inhibition changed the expression of mitochondrial apoptotic pathway proteins, including Bcl-2, Bax, Bid and p53, in a pro-apoptotic manner. We also found that the inhibition of uPAR down-regulated the phosphorylation of FAK, ERK and JNK. The inhibition of p53 by RNA interference rescued cells from enhanced apoptosis, thus indicating that p53 is critical for enhancing TRAIL-induced apoptosis. Furthermore, JNK, but not ERK, inhibition involved in the up-regulation of p53. JNK negatively regulated p53 protein level. Overall, our results show that uPAR inhibition can sensitize colon cancer cells HCT116 to TRAIL-induced apoptosis via active p53 and mitochondrial apoptotic pathways that JNK inhibition is involved.

Taurine protects HK-2 cells from oxidized LDL-induced cytotoxicity via the ROS-mediated mitochondrial and p53-related apoptotic pathways

Oxidized LDL (oxLDL) induces a pro-oxidative environment and promotes apoptosis, causing the progression of renal diseases in humans. Taurine is a semi-essential amino acid in mammals and has been shown to be a potent endogenous antioxidant. The kidney plays a pivotal role in maintaining the balance of taurine. However, the mechanisms underlying the protective effects of taurine against oxLDL-induced injury in renal epithelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effects of taurine on human proximal tubular epithelial (HK-2) cells exposed to oxLDL and explored the related mechanisms. We observed that oxLDL increased the contents of ROS and of malondialdehyde (MDA), which is a lipid peroxidation by-product that acts as an indicator of the cellular oxidation status. In addition, oxLDL induced cell death and apoptosis in HK-2 cells. Pretreatment with taurine at 100μM significantly attenuated the oxLDL-induced cytotoxicity. We determined that oxLDL triggered the phosphorylation of ERK and, in turn, the activation of p53 and other apoptosis-related events, including calcium accumulation, destabilization of the mitochondrial permeability and disruption of the balance between pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins. The malfunctions induced by oxLDL were effectively blocked by taurine. Thus, our results suggested that taurine exhibits potential therapeutic activity by preventing oxLDL-induced nephrotoxicity. The inhibition of oxLDL-induced epithelial apoptosis by taurine was at least partially due to its anti-oxidant activity and its ability to modulate the ERK and p53 apoptotic pathways.

Human Surfactant Protein D Alters Oxidative Stress and HMGA1 Expression to Induce p53 Apoptotic Pathway in Eosinophil Leukemic Cell Line

Surfactant protein D (SP-D), an innate immune molecule, has an indispensable role in host defense and regulation of inflammation. Immune related functions regulated by SP-D include agglutination of pathogens, phagocytosis, oxidative burst, antigen presentation, T lymphocyte proliferation, cytokine secretion, induction of apoptosis and clearance of apoptotic cells. The present study unravels a novel ability of SP-D to reduce the viability of leukemic cells (eosinophilic leukemic cell line, AML14.3D10; acute myeloid leukemia cell line, THP-1; acute lymphoid leukemia cell lines, Jurkat, Raji; and human breast epithelial cell line, MCF-7), and explains the underlying mechanisms. SP-D and a recombinant fragment of human SP-D (rhSP-D) induced G2/M phase cell cycle arrest, and dose and time-dependent apoptosis in the AML14.3D10 eosinophilic leukemia cell line. Levels of various apoptotic markers viz. activated p53, cleaved caspase-9 and PARP, along with G2/M checkpoints (p21 and Tyr15 phosphorylation of cdc2) showed significant increase in these cells. We further attempted to elucidate the underlying mechanisms of rhSP-D induced apoptosis using proteomic analysis. This approach identified large scale molecular changes initiated by SP-D in a human cell for the first time. Among others, the proteomics analysis highlighted a decreased expression of survival related proteins such as HMGA1, overexpression of proteins to protect the cells from oxidative burst, while a drastic decrease in mitochondrial antioxidant defense system. rhSP-D mediated enhanced oxidative burst in AML14.3D10 cells was confirmed, while antioxidant, N-acetyl-L-cysteine, abrogated the rhSP-D induced apoptosis. The rhSP-D mediated reduced viability was specific to the cancer cell lines and viability of human PBMCs from healthy controls was not affected. The study suggests involvement of SP-D in host’s immunosurveillance and therapeutic potential of rhSP-D in the eosinophilic leukemia and cancers of other origins.

Evaluation of Zinc (II) chelators for inhibiting p53-mediated apoptosis

In a previous study, we reported that sodium orthovanadate (vanadate) is the first known inhibitor that is capable of protecting mice from death from the radiation-induced gastrointestinal syndrome via its ability to block both transcription-dependent and transcription-independent p53 apoptotic pathways. In this paper, we report that vanadate has a unique activity for inducing the denaturation of p53 relative to other known radioprotective p53 inhibitors, pifithrin-α (PFTα) and pifithrin-µ (PFTµ). This potent radioprotective effect of vanadate prompted us to undertake a more extensive search for p53 inhibitors that can induce p53 denaturation. Based on the fact that p53 denaturation can be induced by the dissociation of a zinc ion, which is used as a structural factor of p53, we screened some zinc (II) chelators for the suppression of the DNA binding activity of p53 in vitro and the inhibition of radiation-induced p53-dependent apoptosis in MOLT-4 cells. The findings indicate that two of five zinc (II) chelators also suppressed apoptosis. Among the inhibitors tested, Bispicen (N,N'-Bis(2-pyridylmethyl)-1,2-ethanediamine) had the highest inhibition activity. A mechanistic study using cells bearing different p53 status or functions (i.e., p53-knockdown MOLT-4 transformant and its revertants, p53 mutant cells, p53-null cells), and p53-independent apoptotic stimuli revealed that the suppressive effect of Bispicen on apoptosis is specifically mediated through p53. Moreover, Bispicen, similar to vanadate, induces the denaturation of p53 as well as the blocking of both transcription-dependent and -independent apoptotic pathways. Our findings indicate that the use of zinc (II) chelators represent a new approach for protecting against radiation-induced p53-dependent apoptosis through the inhibition of p53-dependent apoptotic pathways.

The chemopreventive activity of the butyric acid prodrug tributyrin in experimental rat hepatocarcinogenesis is associated with p53 acetylation and activation of the p53 apoptotic signaling pathway

The reversibility of non-genotoxic phenotypic alterations has been explored in order to develop novel preventive and therapeutic approaches for cancer control. Previously, it has been demonstrated that histone deacetylase (HDAC) inhibitor tributyrin, a butyric acid prodrug, to have chemopreventive effects on rat hepatocarcinogenesis. The goal of this study was to determine molecular mechanisms associated with the chemopreventive activity of tributyrin. Male Wistar rats were allocated randomly to untreated control group and two experimental groups. Rats in the experimental group 1 were treated with maltodextrin (3g/kg body wt), and rats in experimental group 2 were treated with tributyrin (2g/kg body wt) daily for 8 weeks. Two weeks after treatment initiation, rats from experimental groups were subjected to a 'resistant hepatocyte' model of hepatocarcinogenesis. Treatment with tributyrin resulted in lower HDAC activity and Hdac3 and Hdac4 gene expression, and an increase of histone H3 lysine 9 and 18 and histone H4 lysine 16 acetylation as compared with the experimental group 1. In addition to the increase in histone acetylation, tributyrin caused an increase in the acetylation of the nuclear p53 protein. These changes were accompanied by a normalization of the p53-signaling network, particularly by the upregulation of pro-apoptotic genes, and a consequent increase of apoptosis and autophagy in the livers of tributyrin-treated rats. These results indicate that the chemopreventive activity of tributyrin may be related to an increase of histone and p53 acetylation, which could lead to the induction of the p53 apoptotic pathway.

Erratum: FOXO3 signalling links ATM to the p53 apoptotic pathway following DNA damage

Erratum: FOXO3 signalling links ATM to the p53 apoptotic pathway following DNA damage

Signaling by Unfolding

Biochemistry In response to DNA damage, nuclear p53, a tumor suppressor, induces expression of the p53 up-regulated modulator of apoptosis (PUMA). PUMA in turn regulates the activity of cytosolic p53. In the cytosol, p53 is sequestered by the anti-apoptotic protein BCL-xL, a member of the BCL-2 family of anti-apoptotic proteins. PUMA disrupts this interaction and frees p53 to initiate apoptosis. BCL-2 proteins are composed of BCL-2 homology (BH) domains, and PUMA is a single–BH domain protein. Follis et al. used NMR spectroscopy and biophysical methods to show that PUMA is intrinsically unstructured, but that its BH3 domain folds upon binding to a hydrophobic groove in BCL-xL. NMR spectroscopy and x-ray crystallography showed that PUMA binding caused partial unfolding of two helices in BCL-xL. Although PUMA forms complexes with other anti-apoptotic complexes, it did not induce similar structural perturbations in these proteins. Structural data suggested that a π-stacking interaction between Trp-71 of PUMA and His-113 of BCL-xL was key to the destabilization of regions of the BCL-xL structure. Thus, the p53 apoptotic pathway is activated by regulated unfolding of an anti-apoptotic protein. Nat. Chem. Biol. 10.1038/NCHEMBIO.1166 (2013).

Microrna-34a As a Marker for Fludarabine Resistance and Impairment of p53-Pathway in Chronic Lymphocytic Leukemia

AbstractAbstract 3883 Background.MicroRNAs (miRNAs) are known to be involved in the pathogenesis of chronic lymphocytic leukemia (CLL) and affect its clinical course (Calin et al. NEJM, 2005; Mraz et al. Blood, 2012). Moreover, we and others have shown that several miRNAs are down-regulated in the aggressive CLL subtype harboring p53 aberration (Mraz et al. Leukemia, 2009). The role of miRNAs in primary or acquired resistance to therapy in CLL, however, is poorly understood. Aim.In this study, we screened for miRNAs that are induced by fludarabine-mediated apoptosis in vitro, and we suggested that differences in the expression of one of the identified miRNAs (miR-34a) can be used to distinguish patients with impairment of the p53-apoptotic pathway. Results.Ten primary CLL B-cell samples (purity>95% of CD5+19+ cells) were treated in vitro with fludarabine dose (IC50 dose of 3.5 ug/mL, 48hrs), and the expression of 750 miRNAs was subsequently profiled (TaqMan miRNA Cards, ABI). In comparison with untreated control samples, 15 miRNAs were induced by fludarabine (fold change>1.5, SAM FDR<0.05). The most prominently up-regulated miRNA was miR-34a (fold change 3.7, P=0.003), which is a known p53 down-stream target (He et al. Nature, 2007). We then compared miR-34a up-regulation post fludarabine treatment to the decrease in cell viability (wt-p53 samples, N=20). This revealed that miR-34a induction was significantly higher in CLL samples more sensitive to fludarabine and suggested its role in the apoptotic effects of fludarabine in B-cells. Moreover, the up-regulation of miR-34a was also observed in vivo in samples obtained from fifty FCR-treated CLL patients (fold change 2.2, P<0.0001, analyzed at day 0 and 3 of FCR). These data encouraged us to develop an assay for absolute quantification of miR-34a which would allow determining the copy numbers of miR-34a, defining precise cut-offs, and comparing miR-34a levels during the course of the disease in one patient. We designed synthetic RNA oligos that were used to construct standard curves for both miR-34a and a normalization gene (RNU48). Using this assay, we profiled the expression of miR-34a in a cohort of CLL patients (N=200) to define a cut-off value that would discriminate therapy resistant cases. The distribution of miR-34a expression in the cohort ranged from 1 to 81820 molecules (per 10e6 copies of RNU48). Significantly, miR-34a levels below 2500 copies (N=47) were correlated with shorter overall survival (9.6 years vs. not-reached, HR 2.2 [CI 1.1–4.5], P=0.03). Subsequently, the expression of miR-34a was compared in samples stratified by known prognostic markers: chromosomal aberrations (del17p13, del11q23, tris.12, and del13q14), IgHV status, expression of CD38, CD49d, age, gender, and Rai stage. The lower miR-34a levels were only associated with the deletion of 17p13 locus that includes the p53 gene (N=18, fold change −3.4, P=0.003). Remarkably, CLL samples with sole p53 mutation not accompanied by p53 deletion (N=13) also expressed low levels of miR-34a compared to wt-p53 (P=0.005, fold change −2.7). Most notably, 77% (10/13) of these samples had miR-34a levels below the cut-off of 2500 copies. We further validated our observations and assay by analyzing miR-34a expression in paired samples from 12 CLL cases that acquired p53 aberration during the course of the disease. This emphasized that miR-34a expression decreased in all cases after occurrence of p53 mutation (P=0.0008, fold change −6.1). Additionally, the effect of miR-34a up-regulation on therapy response is currently being investigated in a cohort of FCR-treated patients (N=50). Conclusions.Our data provide complex evidence for the use of miR-34a as a marker of fludarabine-resistant disease. MicroRNA-34a quantification can identify p53 mutated cases that would not be recognized by FISH (mutation not accompanied by del17p13), and miR-34a down-regulation can be used as a sensor for acquisition of p53 abnormality during the course of the disease. This can be accomplished without treatment of cells with gamma-irradiation, which was previously used to identify functional impairment of the p53-pathway (Pettitt et al. Blood, 2001; Mous et al. Leukemia, 2009; Lin et al. CCR, 2012). The described assay for absolute quantification of miR-34a encourages further inter-laboratory validation.IGA MZCR NT11218–6/2010, CZ.1.07/2.3.00/20.0045, CZ.1.05/1.1.00/02.0068 Disclosures:No relevant conflicts of interest to declare.

Loss of zygotic NUP107 protein causes missing of pharyngeal skeleton and other tissue defects with impaired nuclear pore function in zebrafish embryos.

The Nup107-160 multiprotein subcomplex is essential for the assembly of nuclear pore complexes. The developmental functions of individual constituents of this subcomplex in vertebrates remain elusive. In particular, it is unknown whether Nup107 plays an important role in development of vertebrate embryos. Zebrafish nup107 is maternally expressed and its zygotic expression becomes prominent in the head region and the intestine from 24 h postfertilization (hpf) onward. In this study, we generate a zebrafish mutant line, nup107(tsu068Gt), in which the nup107 locus is disrupted by an insertion of Tol2 transposon element in the first intron and as a result it fails to produce normal transcripts. Homozygous nup107(tsu068Gt) mutant embryos exhibit tissue-specific defects after 3 days postfertilization (dpf), including loss of the pharyngeal skeletons, degeneration of the intestine, absence of the swim bladder, and smaller eyes. These mutants die at 5-6 days. Extensive apoptosis occurs in the affected tissues, which is partially dependent on p53 apoptotic pathways. In cells of the defective tissues, FG-repeat nucleoporins are disturbed and nuclear pore number is reduced, leading to impaired translocation of mRNAs from the nucleus to the cytoplasm. Our findings shed new light on developmental function of Nup107 in vertebrates.