沉默Notch1基因促进人乳腺癌MCF-7细胞JNK1和p53磷酸化

 目的：探究沉默Notch1基因对人乳腺癌MCF-7细胞JNK1和p53磷酸化的影响。方法：选取人乳腺癌MCF-7细胞作为研究对象,构建shRNA-Notch1真核表达质粒用于转染MCF-7细胞使Notch1基因沉默。采用Western blotting方法检测MCF-7细胞Notch1、Hes-1、PUMA和NOXA蛋白的表达,JNK1和p53蛋白磷酸化水平以及caspase-3活化水平的改变。应用流式细胞术检测细胞凋亡和线粒体膜电位的变化。结果：人乳腺癌MCF-7细胞Notch1基因被沉默后,Notch1和Hes-1蛋白表达量明显减少(P<0.01),细胞凋亡率显著升高(P<0.01),JNK1和p53的磷酸化水平明显高于对照组(P<0.01),PUMA和NOXA表达量显著升高(P<0.05),cleaved caspase-3蛋白明显多于对照组(P<0.01),线粒体膜电位明显下降(P<0.05)。结论：沉默Notch1基因可能通过激活JNK1信号通路活化p53,促进PUMA和NOXA蛋白表达,进而通过线粒体途径导致人乳腺癌MCF-7细胞凋亡。     

Cullin-3 targets cyclin E for ubiquitination and controls S phase in mammalian cells

Cyclin E is an unstable protein that is degraded in a ubiquitin- and proteasome- dependent pathway. Two factors stimulate cyclin E ubiquitination in vivo: when it is free of its CDK partner, and when it is phosphorylated on threonine 380. We pursued the first of these pathways by using a two-hybrid screen to identify proteins that could bind only to free cyclin E. This resulted in the isolation of human Cul-3, a member of the cullin family of E3 ubiquitin-protein ligases. We found that Cul-3 was bound to cyclin E but not to cyclin E-Cdk2 complexes in mammalian cells, and that overexpression of Cul-3 increased ubiquitination of cyclin E but not other cyclins. Conversely, deletion of the Cul-3 gene in mice caused increased accumulation of cyclin E protein, and had cell-type-specific effects on S-phase regulation. In the extraembryonic ectoderm, in which cells undergo a standard mitotic cycle, there was a greatly increased number of cells in S phase. In the trophectoderm, in which cells go through endocycles, there was a block to entry into S phase. The SCF pathway, which targets cyclins for ubiquitination on the basis of their phosphorylation state, and the Cul-3 pathway, which selects cyclin E for ubiquitination on the basis of its assembly into CDK complexes, may be complementary ways to control cyclin abundance.     

JNK and p53 stress signaling cascades are altered in MCF-7 cells resistant to tumor necrosis factor-mediated apoptosis.

Tumor necrosis factor (TNF) signal transduction is a complex process involving activation of receptor-linked and stress-sensitive signaling cascades that stimulate apoptosis in some tumor cell lines. Initial studies suggested that these signaling events cooperatively induced TNF responses, but recent studies suggest that some of these signals antagonize the apoptotic response or play no discernible role in cell death. As TNF induces cellular stress and activates several stress-sensitive cascades that may play a role in apoptosis, TNF-induced stress signaling was examined in MCF-7 cells and compared with a variant MCF-7 cell line resistant to TNF-mediated apoptosis (MCF-7/3E9). TNF rapidly stimulated both NF-kappaB and JNK activation in MCF-7 and MCF-7/3E9 cells, but JNK activation was significantly reduced (threefold) in apoptotically resistant cells. TNF also stimulated p53, p21WAF1, and Bax accumulation with subsequent PARP cleavage and nucleosomal DNA laddering in MCF-7 cells but did not stimulate these processes in MCF-7/3E9 cells. Importantly, 3E9 cells retained wild-type p53 function, induced p21WAF1 in response to DNA damage, and expressed almost equal sensitivity to other stress stimuli (gamma-radiation, chemotherapeutic agents) as parental MCF-7 cells. These results suggest that selective defects in TNF-activated stress cascades are associated with reduced sensitivity to TNF but not other cell death stimuli. Loss of potent TNF-mediated activation of JNK and p53 cascades may permit tumor cells to evade receptor-mediated apoptosis but have only limited influence on cellular sensitivity to other agents that effectively engage these stress pathways.     

Wild-type p53 reduces radiation hypermutability in p53-mutated human lymphoblast cells

Many studies have shown that an alteration of p53 affects various cellular responses to DNA damage after treatment with ionizing radiation. The human lymphoblast cell WTK1, which contains a mutant p53 (ile237), is 10-fold hypermutable at the thymidine kinase (tk) locus compared with TK6 cells, which are from the same donor but contain wild-type p53. These results implied that the specific p53 mutation found in WTK1 may actively contribute to mutagenesis in a gain of function manner. To further investigate this, the present experiments involved transfecting WTK1 cells with a wild-type p53 vector; this restored p53 activity in WTK1 cells, as evidenced by radiation-induced expression of p21. We compared radiosensitivity, as measured both by clonogenic survival and the induction of apoptosis, as well as mutant fractions (MFs) at the tk locus. WTK1 cells expressing wild-type p53 were more sensitive to gamma-ray-induced toxicity as measured by either clonogenic survival or apoptosis. The mutation assays revealed that both the spontaneous and gamma-ray-induced MFs were significantly decreased in WTK1 cells expressing wild-type p53; the MFs were similar to those observed in p53-null NH32 cells, also derived from the same donor. These results indicate that wild-type p53 can reduce the apparent gain-of-function hypermutable effects of a particular p53 gene mutation and thereby help maintain genomic stability.     

Exonucleolytic degradation of RNA by p53 protein in cytoplasm

p53 in cytoplasm displays an intrinsic 3'-->5' exonuclease activity. To understand the significance of p53 exonuclease activity in cytoplasm, cytoplasmic extracts of various cell lines were examined for exonuclease activity with different single-stranded RNA (ssRNA) substrates. Using an in vitro RNA degradation assay, we observed in cytoplasmic extracts of LCC2 cells, expressing high levels of endogenous wtp53, an efficient 3'-->5' exonuclease activity with RNA substrates, removing the 3'-terminal nucleotides. Interestingly, RNA containing AU-rich sequences (ARE) is the permissive substrate for exonucleolytic degradation. Evidence that exonuclease function with RNA detected in cytoplasmic extracts is attributed to the p53 is supported by several facts: (1) this activity closely parallels with status and levels of endogenous cytoplasmic p53; (2) the endogenous exonuclease exerts identical RNA substrate specificity and excision profile characteristic for purified baculovirus-or bacterially-expressed wtp53s; (3) the exonuclease activity with ARE RNA is competed out by the presence of ss or double-stranded DNA substrate utilized by p53 protein in cytoplasm; (4) immunoprecipitation by specific anti-p53 antibodies markedly reduced the exonuclease activity with both RNA and DNA substrates; and (5) transfection of the wtp53, but not exonuclease-deficient mutant p53-R175H, into p53-null H1299 or HCT116 cells induced high levels of exonuclease activity with ARE RNA substrate in cytoplasm with characteristic excision profile. The efficient ARE RNA degradation correlates with the efficient binding of p53 to ARE RNA in cytoplasm. The possible role of p53 exonuclease activity in ARE-mRNA destabilization in cytoplasm, which may be important for expression of proteins that control cell growth and/or apoptosis is discussed.     

X射线诱导NSCLC组织Axin表达并通过p53或JNK途径促进细胞凋亡

目的研究X-射线对非小细胞肺癌(non-smallcelllungcancer,NSCLC)组织中Axin表达的作用,以及X射线上调Axin表达的机制。方法应用Westernblot、RT-PCR方法检测15例经过X射线照射后NSCLC组织中Axin在蛋白水平及RNA水平的表达变化情况以及caspase-3活化情况。转染Axin及AxinΔp53ΔHIPK2至A549、BE1细胞中,并施加p53或JNK抑制剂,细胞经X线照射后用流式细胞仪(FCM)检测细胞凋亡,研究Axin上调细胞凋亡的机制。结果经X射线照射后,在15例NSCLC肺癌组织中,有8例Axin在RNA水平和蛋白水平上表达上调,与Axin未上调组相比,细胞凋亡显著增加(P<0.05)。转染Axin能使A549、BE1细胞凋亡明显增加,AxinΔp53ΔHIPK2不能上调A549细胞凋亡,p53抑制剂和JNK抑制剂分别抑制A549和BE1细胞凋亡。结论 X射线诱导部分NSCLC组织Axin表达增加并促进细胞凋亡,Axin通过p53或JNK途径促进X射线诱导的细胞凋亡。检测NSCLC组织中是否存在p53基因突变并不能作为判定是否对X射线敏感的指标。     

Antiproliferative activity of cisplatin detected by CFSE in p53-proficient and p53-deficient cells

We have previously developed experimental and data analysis procedures to measure the antiproliferative activity of drugs in continuously proliferating cancer cell lines using carboxyfluorescein diacetate succinimidyl ester (CFSE). The method was applied here to analyze the role of p53 in the effect of the anticancer drug cisplatin, distinguishing events occurring in the first generation of cells from those in the second and subsequent generations. A CFSE-loaded colon carcinoma cell line expressing functional wild-type p53 was treated for 1 with cisplatin in parallel with its p53-deficient counterpart, collecting frequency distributions of DNA and CFSE content up to 72 h after treatment. At a sublethal cisplatin concentration proliferation was temporarily inhibited but then the block was overcome and most cells were able to divide several times. The initial block was stronger in HCTp53-/- cells, resulting in a larger proportion of undivided cells at 24 h. This was confirmed and amplified at a higher, lethal concentration, where undivided G(2)M-blocked p53-deficient cells eventually died by non-apoptotic mechanisms, while p53-proficient cells avoided this with a less stringent block. This gave p53-proficient cells more time to repair and eventually decide on survival or apoptotic death before traversing the cycle into their second generation.     

A mechanism of ubiquitin-independent proteasomal degradation of the tumor suppressors p53 and p73

Protein degradation is an essential and highly regulated process. The proteasomal degradation of the tumor suppressors p53 and p73 is regulated by both polyubiquitination and by an ubiquitin-independent process. Here, we show that this ubiquitin-independent process is mediated by the 20S proteasomes and is regulated by NQO1. NQO1 physically interacts with p53 and p73 in an NADH-dependent manner and protects them from 20S proteasomal degradation. Remarkably, the vast majority of NQO1 in cells is found in physical association with the 20S proteasomes, suggesting that NQO1 functions as a gatekeeper of the 20S proteasomes. We further show that this pathway plays a role in p53 accumulation in response to ionizing radiation. Our findings provide the first evidence for in vivo degradation of p53 and p73 by the 20S proteasomes and its regulation by NQO1 and NADH level.     

Status of p53 in first-trimester cytotrophoblastic cells

p53 has been called the cellular gatekeeper of the genome because it can induce cell-cycle arrest in G1, apoptosis or affect DNA replication in response to DNA damage. As p53 has been observed in first-trimester cytotrophoblastic cells (CTB), but its expression in normal cells is generally not detectable because of its short half-life, p53 could play an important role in cellular differentiation and/or in the control of the invasion of trophoblastic cells; therefore, p53 status was investigated in these cells. Using different antibodies recognizing different epitopes of p53 protein, abundant p53 expression was observed both in nuclear and in cytoplasmic compartments of first-trimester CTB. Whereas p53 was detected in the nuclei of few trophoblastic cells with an antibody recognizing the N-terminal epitope of the protein, high expression level of p53 in the cytoplasm of CTB was detected with an antibody recognizing the middle part of p53. The lack of immunoreactivity of p53 with antibodies recognizing the epitopes located at the N-terminus of p53 and the high level of p53 protein observed in the cytoplasm of CTB suggest that the N-terminus of p53 is involved in the formation of complexes. These cytoplasmic complexes were detected under non-reducing conditions in western blot analysis and had apparent molecular weights (MW) of 195, 167 or 125 kDa. These complexes could prolong the half-life of p53 in the cytoplasm of CTBs. By contrast, in the nuclei of CTBs, p53 seems to be present as a tetramer.     

Sox12 enhances Fbw7-mediated ubiquitination and degradation of GATA3 in Th2 cells

Allergic asthma that is caused by inhalation of house dust mites (HDMs) is mainly mediated by Th2 cells. Recently, the roles of Sox (SRY-related high-mobility-group (HMG)-box) family members in various immune responses have been investigated. However, the roles of Sox12, a member of the SoxC group, in Th2 cell differentiation and allergic airway inflammation, remain unknown. We showed that Sox12 mRNA was significantly increased during Th2 cell differentiation. In vivo, HDM-induced eosinophil infiltration into the lung and Th2 cell differentiation were exacerbated in Sox12^−/− mice compared with those in control Sox12^+/− mice. In vitro, Sox12^−/− CD4⁺ T cells that were cultured under Th2 conditions had increased production of Th2 cytokines and GATA3 protein compared with those of control Sox12^+/− CD4⁺ T cells. Importantly, forced expression of Sox12 decreased the protein levels of GATA3 in CD4⁺ T cells under Th2 conditions without affecting mRNA expression. Furthermore, Sox12 induced degradation of GATA3 through the proteasome pathway in CD4⁺ T cells. Consistently, Sox12 enhanced ubiquitination of GATA3, which was mediated by the E3 ligase Fbw7. Finally, we found that Fbw7 knockdown partly abrogated Sox12-mediated GATA3 suppression in CD4⁺ T cells. Taken together, these results suggest that Sox12 suppresses Th2 cell differentiation by accelerating Fbw7-mediated GATA3 degradation, and attenuates HDM-induced allergic inflammation.     

诱导多能干细胞与p53基因的研究

背景：诱导多能干细胞可以绕过胚胎干细胞存在的伦理学问题,已成为目前干细胞研究的热点问题。 目的：探讨诱导多能干细胞的研究进展以及需要解决的问题。 方法：回顾分析诱导多能干细胞的发现,近年来的研究过程,目前存在的问题。检索汤森路透Web of Science数据库关于诱导多能干细胞和p53基因研究相关的文献进行分析。 结果与结论：近年来国内外学者对诱导多能干细胞进行大量研究。例如,日本正在筹备建立干细胞库,将为视网膜等疾病治疗提供基础。但是,在诱导多能干细胞能广泛应用之前,还面临许多安全问题,其中p53相关的功能性研究是必不可少的紧迫问题,p53基因中的其他成员p73也参与诱导多能干细胞的产生和分化也需要进一步深入。关于p53功能的发现将伴随着干细胞研究,促进再生医学的深入和发展。     

HBxAg增加p53蛋白在肝癌细胞内积聚

目的 进一步研究ＨＢＶｘ基因产物ＨＢｘＡｇ与ｐ５３蛋白的相互关系,探讨其在原发性肝癌发生中的作用机制。方法 以肝癌细胞株Ｈｅｐ３Ｂ为靶细胞,应用薄层层析法做报道基因氯霉素乙酰转移酶（ＣＡＴ）试验与ｐ５３和ＨＢｘ基因共转染,并构建了地塞米松诱导表达的ＨＢｘ基因质粒,用免疫荧光法观察ＨＢｘＡｇ表达对细胞内ｐ５３蛋白的影响。结果 应用ＨＢｘ基因与ｐＧ１３ＣＡＴ共转染,随ＨＢｘ量的增加而ＣＡＴ信号增强,免     

SIAH1介导了TRB3的泛素化和降解

目的 利用酵母回转实验和免疫共沉淀实验验证SIAH1和TRB3之间的相互作用并探讨其功能相关性..方法 将全长形式的TRB3基因和SIAH1基因分别克隆入酵母表达载体pDBLeu和pPC86中,共转化至MaV203酵母感受态细胞,验证其相互作用,然后分别构建至真核表达载体pCMV-Myc和pFLAG-CMV-2中,采用...     

Comparison of in vitro micronucleus and gene mutation assay results for p53-competent versus p53-deficient human lymphoblastoid cells

The high frequency of false or irrelevant positive results in in vitro mammalian cell genotoxicity tests is a critical concern for regulators. Here, we tested whether such results may be due to the mammalian cells used in the tests being deficient in p53, which is involved in the maintenance of genomic stability. We compared the in vitro responses of two human lymphoblastoid cell lines derived from the same progenitor cell-p53-competent (TK6) and p53-deficient (WTK-1) cells-in a micronucleus (MN) test and a thymidine kinase gene (TK) mutation assay. We tested 14 chemicals including three mutagens and 11 clastogens and spindle poisons. The three mutagens evoked clear positive responses in both assays in both cell lines. The responses to the clastogens and spindle poisons, on the other hand, depended on the assay endpoint and/or the cell line. Most of clastogens and spindle poisons were positive in the MN test in both cell lines. In the TK mutation assay, on the other hand, WTK-1 cells but not TK6 cells detected spindle poisons, which may have been due to the disturbance of the spindle checkpoint and lack of apoptosis in the p53-deficient cells. Some chemicals that induced chromosome aberrations in rodent cells were negative in both TK6 and WTK-1 cells, indicating that a species-specific factor rather than p53 status was associated with the response. In conclusion, the p53 status did not seriously influence the MN test results but it did influence the TK mutation assay results.