Regulation of p53 expression by thymidylate synthase

Previous studies showed that thymidylate synthase (TS), as an RNA binding protein, regulates its own synthesis by impairing the translation of TS mRNA. In this report, we present evidence that p53 expression is affected in a similar manner by TS. For these studies, we used a TS-depleted human colon cancer HCT-C cell that had been transfected with either the human TS cDNA or the Escherichia coli TS gene. The level of p53 protein in transfected cells overexpressing human TS was significantly reduced when compared with its corresponding parent HCT-C cells. This suppression of p53 expression was the direct result of decreased translational efficiency of p53 mRNA. Similar results were obtained upon transfection of HCT-C cells with pcDNA 3.1 (+) containing the E. coli TS gene. These findings provide evidence that TS, from diverse species, specifically regulates p53 expression at the translational level. In addition, TS-overexpressing cells with suppressed levels of p53 are significantly impaired in their ability to arrest in G1 phase in response to exposure to a DNA-damaging agent such as gamma-irradiation. These studies provide support for the in vivo biological relevance of the interaction between TS and p53 mRNA and identify a molecular pathway for controlling p53 expression.     

Regulation of p53 stability and p53-dependent apoptosis by NADH quinone oxidoreductase 1

The tumor suppressor gene wild-type p53 encodes a labile protein that accumulates in cells after different stress signals and can cause either growth arrest or apoptosis. One of the p53 target genes, p53-inducible gene 3 (PIG3), encodes a protein with significant homology to oxidoreductases, enzymes involved in cellular responses to oxidative stress and irradiation. This fact raised the possibility that cellular oxidation-reduction events controlled by such enzymes also may regulate the level of p53. Here we show that NADH quinone oxidoreductase 1 (NQO1) regulates p53 stability. The NQO1 inhibitor dicoumarol caused a reduction in the level of both endogenous and gamma-irradiation-induced p53 in HCT116 human colon carcinoma cells. This reduction was prevented by the proteasome inhibitors MG132 and lactacystin, suggesting enhanced p53 degradation in the presence of dicoumarol. Dicoumarol-induced degradation of p53 also was prevented in the presence of simian virus 40 large T antigen, which is known to bind and to stabilize p53. Cells overexpressing NQO1 were resistant to dicoumarol, and this finding indicates the direct involvement of NQO1 in p53 stabilization. NQO1 inhibition induced p53 degradation and blocked wild-type p53-mediated apoptosis in gamma-irradiated normal thymocytes and in M1 myeloid leukemic cells that overexpress wild-type p53. Dicoumarol also reduced the level of p53 in its mutant form in M1 cells. The results indicate that NQO1 plays an important role in regulating p53 functions by inhibiting its degradation.     

Myocardial Regulation of p300 and p53 by Doxorubicin Involves Ubiquitin Pathways

Background Doxorubicin (Dox) depletes p300 from cardiac myocytes and induces apoptosis of these cells. p300 protein possesses ubiquitin ligase activity for the p53 tumor suppressor gene product, catalyzes p53 polyubiqutination, and facilitates p53 degradation in an ubiquitin-dependent manner. The present study investigated the ubiquitin-dependent regulation of p53 by Dox and p300 in cardiac myocytes. Methods and Results Primary cardiac myocytes from neonatal rats were exposed to a proteasome inhibitor, MG132, in culture. MG132 increased both p300 and p53 protein levels in these cells, suggesting that ubiquitin-dependent degradation is involved in the homeostasis of these proteins. Notably, treatment of cardiac myocytes with Dox decreased the protein levels of p300 but markedly increased those of p53. By immunoprecipitation-Western blotting, it was shown that treatment with Dox decreased poly-ubiquitinated p53 but increased that of p300 in cardiac myocytes. Finally, the overexpression of p300 in cardiomyocytes suppressed the Dox-mediated increase in the p53 level in addition to inhibiting Dox-induced apoptosis. Conclusion Dox reciprocally regulates p300 and p53 through ubiquitin-dependent pathways and that p300, by its ubiquitin ligase activity, is partially involved in the ubiquitin-dependent degradation of p53 in cardiac myocytes. (Circ J 2008; 72: 1506 - 1511).     

Regulation of p53 by protein kinase C during multi-stage carcinogenesis

Conclusions The hypothesis that we are currently trying to demonstrate is that of initial cells start the process of carcinogenesis by inducing G1-cyclin-dependent RB phosphorylation, E2F release and an increase of glycolysis, with the consequent sustained diacylglycerol production: PKC is down-regulated and p53 is maintained in its latent, inactive form; genomic damage progressively accumulates and the chromosomal aberrations may activate other oncogenes or deactivate tumor suppressors, leading to progression towards malignancy. This model agrees with the abnormally high level of diacylglycerol found in a variety of malignant tumors (Mills et al. 1993, Hendickse et al. 1995; Casamassima et al. 1996) (see Fig. 1B). To take an extreme view, not only are the presence, the loss, or the mutations sufficient screening data for the status of p53 in malignancy, but also the presence or the absence of phosphate groups at the carboxyl ends, as revealed by Pab421 antibody.The Journal Cancer Research and Clinical Oncology occasionally publishes Editorials and Guest editorials on current and controversial problems in experimental and clinical oncology. These papers reflect the personal opinions of the authors. Readers should send any comments directly to the authors.     

Regulation of arthritis by p53: critical role of adaptive immunity

OBJECTIVE: The p53 tumor-suppressor protein is expressed in rheumatoid arthritis synovium, and loss of p53 function through somatic mutation can occur in longstanding disease. Previous studies demonstrated that p53 is protective in murine collagen-induced arthritis (CIA). To determine if adaptive immune responses or synovial effector functions are responsible for this effect, passive models of arthritis were studied in p53 wild-type and knockout mice. METHODS: Models of passive CIA, passive K/BxN serum transfer arthritis, and active CIA were induced in DBA/1 p53(-/-) or p53(+) mice. Hind paws were evaluated for histologic evidence of inflammation and joint destruction. Synovial interleukin-6 and matrix metalloproteinases 3 and 13 gene expression was analyzed by real-time quantitative polymerase chain reaction. To evaluate T cell function in p53(-/-) mice, draining lymph node (LN) cells from mice immunized with type II collagen (CII) were evaluated in vitro. RESULTS: Increased disease severity in p53(-/-) mice was confirmed in the standard CIA model. However, clinical arthritis, joint destruction, and synovial gene expression in the passive CIA and K/BxN serum transfer arthritis models were similar in p53(-/-) and p53(+) mice. To determine if the p53 effect was related to T cell function, LN cells from CII-immunized mice were isolated and stimulated with antigen in vitro. CII-stimulated T cell proliferation and interferon-gamma production were significantly higher in p53(-/-) mice. An independent assessment of Th1 function using the cutaneous delayed-type hypersensitivity model confirmed that p53(-/-) mice have enhanced T cell responses in vivo. CONCLUSION: Adaptive immune responses, rather than antibody-mediated responses, in p53(-/-) mice account for increased disease severity in the active CIA model.     

Regulation of p53 by macrophage migration inhibitory factor in inflammatory arthritis

OBJECTIVE: To study the capacity of macrophage migration inhibitory factor (MIF) to regulate proliferation, apoptosis, and p53 in an animal model of rheumatoid arthritis (RA) and in fibroblast-like synoviocytes (FLS) from humans with RA. METHODS: Antigen-induced arthritis (AIA) was induced in MIF(-/-) mice and littermate controls. FLS were obtained from patients with RA. Western blotting and immunohistochemistry were used to measure p53 in cells and tissues. Apoptosis was detected in cells by flow cytometry using TUNEL and annexin V/propidium iodide labeling. Apoptosis in tissue was detected using TUNEL. Proliferation was assessed in cultured cells and tissue by (3)H-thymidine incorporation and Ki-67 immunostaining, respectively. RESULTS: MIF inhibited p53 expression in human RA FLS. Levels of p53 were correspondingly increased in MIF(-/-) mouse tissues and cells. Spontaneous and sodium nitroprusside-induced apoptosis were significantly increased in MIF(-/-) cells. In vitro exposure of FLS to MIF reduced apoptosis and significantly induced FLS proliferation. Synoviocyte proliferation in MIF(-/-) mice was correspondingly reduced. A decrease in the severity of AIA in MIF(-/-) mice was associated with an increase in p53 and apoptosis in synovium. Evidence of in situ proliferation was scant in this model, and no difference in in situ proliferation was detectable in MIF(-/-) mice compared with wild-type mice. CONCLUSION: These results indicate a role for MIF in the regulation of p53 expression and p53-mediated events in the inflamed synovium and support the hypothesis that MIF is of critical importance in the pathogenesis of RA.     

Augmented DNA-binding activity of p53 protein encoded by a carboxyl-terminal alternatively spliced mRNA is blocked by p53 protein encoded by the regularly spliced form.

DNA-binding activity of the wild-type p53 is central to its function in vivo. However, recombinant or in vitro translated wild-type p53 proteins, unless modified, are poor DNA binders. The fact that the in vitro produced protein gains DNA-binding activity upon modification at the C terminus raises the possibility that similar mechanisms may exist in the cell. Data presented here show that a C-terminal alternatively spliced wild-type p53 (ASp53) mRNA expressed by bacteria or transcribed in vitro codes for a p53 protein that efficiently binds DNA. Our results support the conclusion that the augmented DNA binding activity of an ASp53 protein is probably due to attenuation of the negative effect residing at the C terminus of the wild-type p53 protein encoded by the regularly spliced mRNA (RSp53) rather than acquisition of additional functionality by the alternatively spliced C' terminus. In addition, we found that ASp53 forms a complex with the non-DNA-binding RSp53, which in turn blocks the DNA-binding activity of ASp53. Interaction between these two wild-type p53 proteins may underline a mechanism that controls the activity of the wild-type p53 protein in the cell.     

丙型肝炎病毒NS5A对p53活性调控机制研究

目的 研究HCV NS5A对抑癌蛋白p53活性的抑制作用及其作用机制。 方法 采用荧光素酶报告基因系统观察pwwp-luc,pwwp-mut-luc,pc53-NS3及pCNS5A分别转染或共同转染HepG2、Huh7细胞的荧光素酶活性。应用凝胶滞留试验观察HCV NS5A是否影响p53与其特异DNA序列的结合。 结果 内源性p53能激活p21启动子转录功能,使荧光素酶活性明显增加(3.49×10~5与0.60×10~5,t=5.92,P<0.01)。外源性p53也能激活p21启动子转录功能,荧光素酶活性为5.63×10~5,与对照组(0.47×10~5)比较差异具有显著性(t=10.12,P<0.01)。HCV NS5A能抑制内源性和外源性p53对p21启动子的激活作用,并呈剂量依赖性(F≥20.71,P<0.01)。凝胶滞留试验显示HCV NS5A能阻碍p53与其特异DNA序列的结合。 结论 HCV NS5A能抑制p53的反式激活功能使其目的基因p21启动子的转录功能下降,其机制是HCV NS5A能阻碍p53与其特异DNA序列的结合。     

Regulation of p53 is critical for vertebrate limb regeneration

Extensive regeneration of the vertebrate body plan is found in salamander and fish species. In these organisms, regeneration takes place through reprogramming of differentiated cells, proliferation, and subsequent redifferentiation of adult tissues. Such plasticity is rarely found in adult mammalian tissues, and this has been proposed as the basis of their inability to regenerate complex structures. Despite their importance, the mechanisms underlying the regulation of the differentiated state during regeneration remain unclear. Here, we analyzed the role of the tumor-suppressor p53 during salamander limb regeneration. The activity of p53 initially decreases and then returns to baseline. Its down-regulation is required for formation of the blastema, and its up-regulation is necessary for the redifferentiation phase. Importantly, we show that a decrease in the level of p53 activity is critical for cell cycle reentry of postmitotic, differentiated cells, whereas an increase is required for muscle differentiation. In addition, we have uncovered a potential mechanism for the regulation of p53 during limb regeneration, based on its competitive inhibition by ΔNp73. Our results suggest that the regulation of p53 activity is a pivotal mechanism that controls the plasticity of the differentiated state during regeneration.Unlike mammals, which exhibit limited regenerative abilities, the urodele amphibians—or salamanders—are capable of regenerating an extraordinary range of body structures, including ocular tissues, tail, sections of the heart, parts of the nervous system, and entire limbs (1). In salamanders, such as the newt and axolotl, limb regeneration depends on the formation of a blastema, a mound of progenitor cells of restricted potential that arises after amputation (2–4). Following a period of proliferation, blastema cells redifferentiate and restore the structures of the limb.Extensive evidence indicates that limb regeneration depends on reprogramming of cells in mature limb tissues. Upon amputation, muscle, cartilage, and connective tissue cells underneath the injury site lose their differentiated characteristics and re-enter the cell cycle to give rise to the blastema (5–8). This mechanism has also been observed during zebrafish heart and fin regeneration (9, 10). In contrast, reversals of the differentiated state are rarely observed in mammalian tissues, which led to the suggestion that inability to undergo dedifferentiation could contribute to the failure of regeneration in mammals (11). Despite their significance, the mechanisms underlying regulation of the differentiated state during vertebrate regeneration remain poorly understood.Recently, the tumor suppressor p53, whose best-characterized functions are in the maintenance of genome stability (12), has been implicated in the suppression of artificial cell reprogramming to pluripotency (13–17) and the promotion of differentiation pathways in mammals (18). In addition, it has been observed that inhibiting p53 disrupts limb regrowth in salamanders (19), although its role in this context has remained unknown. It is possible that p53 could play a role in the regulation of dedifferentiation and redifferentiation events intrinsic to vertebrate regeneration. Our results demonstrate that the regulation of p53 activity is critical for limb regeneration by controlling key cell fate decisions throughout this process.     

Regulation of the proapoptotic factor Bax by Ku70-dependent deubiquitylation

The DNA end-joining protein Ku70 is one of several proteins that inhibit apoptosis by sequestering the proapoptotic factor Bax from the mitochondria. However, the molecular mechanism underlying Ku70-dependent inhibition of Bax is not fully understood. Here, we show that the absence of Ku70 results in the accumulation of ubiquitylated Bax. Under normal growth conditions, Bax ubiquitylation promotes its degradation. Upon induction of apoptosis in wild-type cells, a significant reduction in the levels of ubiquitylated Bax was observed, whereas in Ku70(-/-) cells, the ubiquitylated Bax was robustly accumulated. Addition of recombinant Ku70 into a protein extract of Ku70(-/-) cells resulted in a decrease in the levels of ubiquitylated Bax, even in the presence of proteasome inhibitors. Moreover, an in vitro deubiquitylation assay demonstrated that recombinant Ku70 hydrolyzed polyubiquitin chains into monoubiquitin units. Thus, Ku70 regulates apoptosis by sequestering Bax from the mitochondria and mediating Bax deubiquitylation. These results shed light on the role of proteasome inhibitors as tumor suppressors.