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The p53 family of proteins contains two members that have been implicated in sensitization of cells and organisms to genotoxic stress, i.e., p53 itself and p73. In vitro, lack of either p53 or p73 can protect certain cell types in the adult organism against death upon exposure to DNA damaging agents. The present study was designed to assess the rel...
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Hmgb1 (high mobility group box-1; amphoterin) is highly expressed in brain during early development of vertebrate and nonvertebrate species. However, its role in brain development remains elusive. Here we have cloned the zebrafish Hmgb1 and specifically manipulated Hmgb1 expression using injection of morpholino antisense oligonucleotides or Hmgb1 c...
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... Both alterations together lead to a strong activation of the transcription of p53 target genes and result in cell cycle arrest and cell death [1]. This p53-dependent DNA damage response is also observed in zebrafish where ionising radiation leads to a strong increase in the mortality of zebrafish that is reduced after inactivation of p53 [21]. ...
... Due to the absence of p53, p53 -/zebrafish should be less sensitive to ionising radiation [21]. To test this prediction, we irradiated WT and p53 -/zebrafish embryos at 24 hpf with increasing doses of ionising radiation and monitored their survival. ...
... Exposure of wild-type and p53 -/zebrafish embryos to ionising irradiation resulted in a significantly better survival of the p53 -/embryos compared to their wild-type siblings. This result is in line with an earlier described radioprotection by mutated p53 or after suppression of wild-type p53 with antisense morpholino-oligonucleotides [8,21]. ...
p53 is well-known for its tumour-suppressive activity. However, in the past decade it became clear that p53 is also involved in other processes including stem cell proliferation, differentiation and animal development. To investigate the role of p53 in early embryonic development, we targeted p53 by CRISPR/Cas9 to make a p53 knock-out zebrafish (Danio rerio). Our data show developmental and behavioural effects in p53-deficient zebrafish embryos and larvae. Specifically, we found that early development of zebrafish was clearly delayed in the absence of p53. However, after 1 day (1 dpf), the p53-deficient embryos appeared to recover, as evidenced by a similar level of pigmentation at 26 hpf, similar size of the eye at 4 dpf and only a minor difference in body size at 4 dpf compared to p53 wild-type siblings. The recovery of development after 1 dpf in p53-deficient embryos could be due to a compensatory mechanism involving other p53 family members. p63 and p73 were found over-expressed with respect to wild-type siblings. However, despite this adaptation, the hatching time remained delayed in p53-/- zebrafish. In addition to differences in development, p53-null zebrafish embryos also showed differences in behaviour. We observed an overall reduced activity and a reduced travel distance under non-stressed conditions and after exposing the larvae to vibration. We also observed a longer latency until the larvae started to move after touching with a needle. Overall, these data indicate that p53 is involved in early development and locomotion activities.
... Interestingly, PFTa-a common P53 inhibitor (Komarov et al. 1999) significantly reduced TPEN-induced apoptosis. This observation suggests that P73 might also be a target for PFTa Davidson et al. 2008. Of note, this inhibitory action of PFTa on P73 could not be disclosed in cells wherein P53 is functional (e.g. ...
Chronic myeloid leukemia (CML) is a hematologic disorder characterized by the constitutive expression of BCR-ABL tyrosine kinase. Although successful implementation of tyrosine kinase inhibitors for the treatment of CML remain a traditional choice for molecularly targeted therapy, some patients present primary or secondary resistance to such therapy. Therefore, alternative therapeutic strategies are required to treat resistant CML cells. Accordingly, new anti-proliferative and/or pro-apoptotic compounds would be needed for clinical treatment. In the present investigation, we demonstrate that TPEN (e.g. 3 μM), a lipid-soluble metal chelator, induces apoptosis in K562 cells via a molecular cascade involving H2O2 ≫ JNK, NF-κB > c-JUN, P73 > PUMA, BAX > loss of ΔΨm > CASPASE-3 > nuclei/DNA fragmentation. Fragmentation of the nuclei and DNA are indicative of cell death by apoptosis. Remarkably, the antioxidant N-acetyl-cysteine, and inhibitors of the transcription factors CASPASE 3 and (JNK) kinase, decreased oxidative stress (OS) and cell death in these cells. This is evidenced by fluorescence microscopy, flow cytometry and immunocytochemistry for OS markers (e.g. generation of H2O2 and DJ 1 oxidation) and nuclear expression of apoptotic markers (e.g. activated caspase 3 and JNK kinase). In addition, TPEN causes no detectable damage in human peripheral blood lymphocyte cells (hPBLCs). We conclude that TPEN selectively induces apoptosis in K562 cells via an OS-mechanism. Our findings may provide insight into more effective CML anticancer therapies.
... Therefore, we hypothesized the potential involvement of p53 in the ability of HDAC inhibition to induce AQP3 expression. To examine this idea, mouse keratinocytes were treated with or without SAHA (2.5µM) in the presence or absence of an inhibitor of p53/p73 (Davidson et al., 2008), pifithrin, for 24h and harvested for Western analysis. As shown in Figure 5a, pifithrin (30µM) significantly inhibited SAHA-induced AQP3 protein expression, while exerting little or no effect on AQP3 levels alone, suggesting the possibility that HDACs regulate AQP3 levels through their ability to modulate the transcriptional activity of one or more p53 family members. ...
... Our results using two different approaches supported the involvement of p53 in SAHA-increased AQP3 levels. The p53 inhibitor used in one approach, pifithrin, can also inhibit p73 at higher concentrations (Codelia et al., 2010, Davidson et al., 2008. However, using two doses of pifithrin, with the lower dose (10µM) expected to inhibit only p53 and not p73, we observed similar inhibition of SAHA-induced AQP3 protein expression. ...
Aquaporin-3 (AQP3), a water and glycerol channel, plays an important role in epidermal function, with studies demonstrating its involvement in keratinocyte proliferation, differentiation and migration and epidermal wound healing and barrier repair. Increasing speculation about the use of histone deacetylase (HDAC) inhibitors to treat skin diseases led us to investigate HDAC's role in the regulation of AQP3. The broad-spectrum HDAC inhibitor, suberolyanilide hydroxamic acid (SAHA) induced AQP3 mRNA and protein expression in a dose- and time-dependent manner in normal keratinocytes. The SAHA-induced increase in AQP3 levels resulted in enhanced [(3)H]glycerol uptake in normal but not in AQP3 knockout keratinocytes, confirming that the expressed AQP3 was functional. Utilization of HDAC inhibitors with different specificities limited our exploration of the responsible HDAC member to HDAC1, HDAC2 or HDAC3. Cre-recombinase-mediated knockdown and overexpression of HDAC3 suggested a role for HDAC3 in suppressing AQP3 expression basally. Further investigation implicated p53 as a transcription factor involved in regulating HDAC inhibitor-induced AQP3 expression. Thus, our study supports the regulation of AQP3 expression by HDAC3 and p53. Since SAHA is already approved to treat cutaneous T-cell lymphoma, it could potentially be used as a novel therapy for skin diseases like psoriasis, where AQP3 is abnormally expressed.
... In the recent past, zebrafish, a tropical minnow, have been widely used as a model for understanding basic pathophysiology and also for screening of drugs for a variety of diseases [18]. Some of the important features which make zebrafish an attractive model includes rapid development, transparent early developmental stages, ease of maintenance and high fecundity [19]. The present study was undertaken to investigate whether a reversible hypometabolic state renders protection against radiation exposure in a whole organism model and the following issues were addressed using zebrafish, a non-hibernating vertebrate organism model: Does SA-like state, if induced prior to lethal doses of ionizing radiation exposure, confer any advantage or protection in terms of damage induction and survival? ...
... The inhibitor, pifithrin α, directly binds the p53 DNA-binding domain, though there may be off-target effects on related tp63 and tp73 because of sequence similarity (Levrero et al., 2000). The concentration of pifithrin α used (5 µM) was similar to effective concentrations previously used and was a maximum tolerable NOAEL concentration (Davidson et al., 2014; Duffy and Wickstrom, 2014). In the present study, neither method of p53 suppression, morpholino antisense knockdown nor pifithrin α inhibition provided any beneficial effect against the tamoxifen induced caudal fin lesion at any observed time point (Fig. 5). ...
The zebrafish is a powerful alternative model used to link phenotypes with molecular effects to discover drug mode of action. Using a zebrafish embryo-larval toxicity bioassay, we evaluated the effects of tamoxifen - a widely used anti-estrogen chemotherapeutic. Zebrafish exposed to ≥10μM tamoxifen exhibited a unique necrotic caudal fin phenotype that was rapidly induced regardless of developmental life-stage when treatment was applied. To define tamoxifen's bioactivity resulting in this phenotype, targeted gene expression was used to evaluate 100 transcripts involved in tissue remodeling, calcium signaling, cell cycle and cell death, growth factors, angiogenesis and hypoxia. The most robustly misregulated transcripts in the tail were matrix metalloproteinases mmp9 and mmp13a, induced 127 and 1145 fold, respectively. Expression of c-fos, c-jun, and ap1s1 were also moderately elevated (3-7 fold), consistent with AP-1 activity - a transcription factor that regulates MMP expression. Immunohistochemistry confirmed high levels of induction for MMP13a in affected caudal fin skin epithelial tissue. The necrotic caudal fin phenotype was significantly attenuated or prevented by three functionally unique MMP inhibitors: EDTA (metal chelator), GM 6001 (broad MMP inhibitor), and SR 11302 (AP-1 transcription factor inhibitor), suggesting MMP-dependence. SR 11302 also inhibited induction of mmp9, mmp13a, and a putative MMP target, igfbp1a. Overall, our studies suggest that tamoxifen's effect is the result of perturbation of the MMP system in the skin leading to ectopic expression, cytotoxicity, and the necrotic caudal fin phenotype. These studies help advance our understanding of tamoxifen's non-classical mode of action and implicate a possible role for MMPs in tissues such as skin.
... 19 In addition, it was reported that PFT-α could prevent the p73 increase and apoptosis induced by a genotoxic agent. 20 As shown in Figure 3c, the percentage of empty vector-transduced cells was unchanged 4 days after the treatment, and there was no effect of PFT-α on their proportion, indicating that empty vector-transduced cells, which are GFP positive, didn't have any growth advantage, nor did they undergo p53/p73-mediated apoptosis. Cells transduced with HBZ were almost equivalent at day ( Figure 3d). ...
Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that induces a fatal T-cell malignancy, adult T-cell leukemia (ATL). Among several regulatory/accessory genes in HTLV-1, HTLV-1 bZIP factor (HBZ) is the only viral gene constitutively expressed in infected cells. Our previous study showed that HBZ functions in two different molecular forms, HBZ protein and HBZ RNA. In this study, we show that HBZ protein targets retinoblastoma protein (Rb), which is a critical tumor suppressor in many types of cancers. HBZ protein interacts with the Rb/E2F-1 complex and activates the transcription of E2F-target genes associated with cell cycle progression and apoptosis. Mouse primary CD4(+) T cells transduced with HBZ show accelerated G1/S transition and apoptosis, and importantly, T cells from HBZ transgenic (HBZ-Tg) mice also demonstrate enhanced cell proliferation and apoptosis. To evaluate the functions of HBZ protein alone in vivo, we generated a new transgenic mouse strain that expresses HBZ mRNA altered by silent mutations but encoding intact protein. In these mice, the numbers of effector/memory and Foxp3(+) T cells were increased, and genes associated with proliferation and apoptosis were upregulated. This study shows that HBZ protein promotes cell proliferation and apoptosis in primary CD4(+) T cells through activation of the Rb/E2F pathway, and that HBZ protein also confers onto CD4(+) T-cell immunophenotype similar to those of ATL cells, suggesting that HBZ protein has important roles in dysregulation of CD4(+) T cells infected with HTLV-1.Oncogene advance online publication, 25 January 2016; doi:10.1038/onc.2015.510.
... 23 The presence of PFT-α (10 μM) itself somewhat caused slower cell proliferation and cell death of HSCs (data not shown and Figure 5i), probably due to its inhibitory activity toward other signaling pathways such as heat shock response, glucocorticoid signaling and p73 pathway. 24,25 Nonetheless, treatment with PFT-α significantly blocked upregulation of p21 and Bax (Figure 5h) as well as cell death (Figure 5i) induced by RCAD loss. These results suggest that aberrant p53 activation may cause apoptosis and cell cycle arrest in RCAD-deficient HSCs. ...
The Ufm1 conjugation system is a novel ubiquitin-like modification system, consisting of Ufm1, Uba5 (E1), Ufc1 (E2) and poorly characterized E3 ligase(s). RCAD/Ufl1 (also known as KIAA0776, NLBP and Maxer) was reported to function as a Ufm1 E3 ligase in ufmylation (Ufm1-mediated conjugation) of DDRGK1 and ASC1 proteins. It has also been implicated in estrogen receptor signaling, unfolded protein response (UPR) and neurodegeneration, yet its physiological function remains completely unknown. In this study, we report that RCAD/Ufl1 is essential for embryonic development, hematopoietic stem cell (HSC) survival and erythroid differentiation. Both germ-line and somatic deletion of RCAD/Ufl1 impaired hematopoietic development, resulting in severe anemia, cytopenia and ultimately animal death. Depletion of RCAD/Ufl1 caused elevated endoplasmic reticulum stress and evoked UPR in bone marrow cells. In addition, loss of RCAD/Ufl1 blocked autophagic degradation, increased mitochondrial mass and reactive oxygen species, and led to DNA damage response, p53 activation and enhanced cell death of HSCs. Collectively, our study provides the first genetic evidence for the indispensable role of RCAD/Ufl1 in murine hematopoiesis and development. The finding of RCAD/Ufl1 as a key regulator of cellular stress response sheds a light into the role of a novel protein network including RCAD/Ufl1 and its associated proteins in regulating cellular homeostasis.Cell Death and Differentiation advance online publication, 8 May 2015; doi:10.1038/cdd.2015.51.
... Due to their high degree of similarity, it is possible that the p53 inhibitor pifithrin-a could have an effect on these homologues. Pifithrin-a was shown to act on p73 in zebra fish embryo [34]; however, its effect on p63 and p73 in mammalian cells is not known. We cannot rule out the possibility that pifithrin-a is inhibiting not only p53, but also p63 and p73. ...
Purpose: Despite clinical progress, mechanisms involved in cellular responses to low and high doses of hyperthermia are not entirely clear. This study investigates the role of Bcl-2 family proteins in control of the mitochondrial pathway of apoptosis during hyperthermia at 42–43 °C and the protective effect of a low dose adaptive survival response, mild thermotolerance induced at 40 °C. Materials and methods: Levels of Bcl-2 family proteins were detected in HeLa cells by western blotting, caspase activation by spectrofluorimetry and apoptosis by chromatin condensation. Results: Hyperthermia (42–43 °C) decreased total and mitochondrial expression of anti-apoptotic proteins Bcl-2 and Bcl-xL, while expression of pro-apoptotic proteins Bax, Bak, Puma and Noxa increased. Hyperthermia perturbed the equilibrium between these anti- and pro-apoptotic Bcl-2 family proteins in favour of pro-apoptotic conditions. Hyperthermia also caused activation of caspases-9 and -3, and chromatin condensation. Disruption of the balance between Bcl-2 family proteins was reversed in thermotolerant (40 °C) cells, thus favouring cell survival. Bcl-2/Bcl-xL inhibitor ABT-737 sensitised cells to apoptosis, which indicates that Bcl-2 family proteins play a role in hyperthermia-induced apoptosis. The adaptive response of mild thermotolerance (40 °C) was still able to protect cells against hyperthermia (42–43 °C) when Bcl-2/Bcl-xL were inhibited. Conclusions: These results improve knowledge about the role of Bcl-2 family proteins in cellular apoptotic responses to hyperthermia (42–43 °C), as well as the adaptive survival response induced by exposure to mild stresses, such as a fever temperature (40 °C). This study could provide rationale to explore the manipulation of Bcl-2 family proteins for increasing tumour sensitivity to hyperthermia.
... Of note, PFT-α has been shown to inhibit the transcriptional activity of p73 [98]. Thus, it could be argued that the impairment of DQA-induced apoptosis by this drug is an off-target effect mediated by the inhibition of p73. ...
Current frontline therapies have improved overall survival in acute promyelocytic leukemia (APL) patients to exceptional rates; however, relapse is still a problem among high-risk and old patients. Therefore, the development of better and safer therapies continues to be a goal in the treatment of this disease. In the present work, we examined three different pathways that hinder cell death in the APL cell line NB4, shedding light on the mechanisms that underlie resistance to apoptosis in these cells and that might help provide them with a proliferative advantage. We found that the proteasome inhibitor MG-132 specifically induces in NB4 cells an Nrf2-mediated antioxidant response which counteracts mitochondria-dependent apoptosis induced by the lipophilic cation dequalinium. More importantly, we also demonstrated that high basal autophagy levels and the gain-of-function of mutant p53 are intrinsic mechanisms of resistance to apoptosis in this cell line. According to our results, the pharmacological inhibition of autophagy and p53 mutants are useful tools to explore resistance to apoptosis in APL and other types of cancer and could be the bases of new therapeutic approaches that improve the efficiency and allow dose reduction of the current treatments.
... Zebrafish p73 is 70 to 95% identical to p73 of other vertebrates (Pan et al., 2003). It seems to be essential for the first 24 hours during zebrafish development (Davidson et al., 2008). In contrast to other observations, where p73 expression was already observed at the one-cell stage (Pan et al., 2003), the expression of p73 could not be detected until midgastrulation onwards by others. ...
Compared to the adult mammalian brain, the brain of the adult zebrafish Danio rerio exhibits a very high proliferative and regenerative potential. The adult mammalian brain in contrast has a very limited neurogenic capacity mainly restricted to two zones, the subventricular zone of the lateral telencephalic ventricles and the subgranular zone of the dentate gyrus of the hippocampus. In contrast, the zebrafish brain harbours 16 proliferation zones distributed all over the brain. The zebrafish has thus become a model for the study of adult neurogenesis and regeneration of nervous tissue. I characterized the expression of the two transcription factors p53 and p73 in the adult zebrafish brain. Both p53 and p73 were shown to play crucial roles in mammalian adult neurogenesis: p53 suppresses the self-renewal of adult neural stem cells and is involved in apoptotic death of neurons following damage. p73 is relevant for the survival of neurons, self-renewal and maintenance of neural stem cells as well as differentiation of precursor cells. It was thus of interest whether these genes have similar roles in the adult zebrafish brain.
I established a detailed map of the expression pattern of p53 and p73 mRNA and p53 protein in the adult zebrafish brain. p53 and p73 mRNA expression overlaps in many regions including neurogenic zones. The p53 protein is expressed in most of these regions indicating that the mRNA expression reflects the protein expression. The p53 protein is expressed in mature neurons, Type I cells (non-dividing radial glial cells) and Type IIIa and Type IIIb cells (neuroblasts) in the adult zebrafish telencephalon. In cells of the oligodendrocyte lineage and in Type II cells (dividing radial glial cells) an expression of the p53 protein is not detectable. After stab injury of the adult zebrafish telencephalon both p53 and p73 genes are up-regulated. p53 is up-regulated in Type I cells. In contrast to the uninjured brain, p53 is expressed in cells of the oligodendrocyte lineage following injury. Furthermore, target genes of p53 are up-regulated and apoptosis is induced after stab injury. These results suggest a role for p53 in constitutive and regenerative neurogenesis. However, tp53M214K mutant zebrafish do not show any phenotype. The structurally related p73 is expressed in a very similar pattern as p53 in the uninjured and injured zebrafish brain. Therefore, redundancy between p53 and p73 may occlude the manifestation of a phenotype in the p53 mutant. Taken together, the analysis of expression of both p53 and p73 in the adult zebrafish brain suggests a role of these genes during constitutive and regenerative neurogenesis. The future elucidation of the precise function of the two genes in these processes requires, however, double mutant analysis.
