p53功能调节的新进展

p53的结构和功能异常与肿瘤的发生密切相关。研究表明 ,p53的磷酸化和 或乙酰化可直接影响p53的稳定性及活性 ,多种蛋白包括癌基因蛋白 (如Mdm2 ,p3 3 ING1,E1a ,c Myc等 )亦通过不同途径影响p53的功能。了解p53功能的调节将助于进一步认识其在肿瘤发生发展中的作用。     

磷酸化修饰对p53线粒体作用的影响以及与卵巢癌顺铂耐药的关系

目的探讨磷酸化修饰对p53直接线粒体转移的影响以及在顺铂诱导的卵巢癌细胞凋亡中的作用以及与卵巢癌顺铂耐药的关系。方法应用Western Blot检测顺铂作用前后卵巢癌顺铂敏感细胞OV2008、A2780s和顺铂耐药细胞C13＊、A2780cp的线粒体和细胞浆中Smac蛋白含量以及线粒体和全细胞中磷酸化p53（P-p53）的含量,并应用Hoechst染色法检测卵巢癌细胞的凋亡率。结果顺铂能引起卵巢癌敏感细胞OV2008、A2780s线粒体释放Smac至胞浆并引起细胞凋亡,但在C13＊和A2780cp中无此反应。顺铂处理后进入卵巢癌敏感细胞线粒体中的p53蛋白为磷酸化的p53。结论顺铂能引起p53转移至线粒体并导致卵巢癌敏感细胞线粒体释放Smac至胞浆,促进卵巢癌细胞的凋亡,并且磷酸化修饰作用可以影响p53的直接线粒体功能,与卵巢癌化疗耐药有关。     

IL-6经MEK/ERK途径促进ERα-Ser118磷酸化影响卵巢癌细胞他莫昔芬耐药

目的研究IL-6信号通路对ERα的Ser118位点磷酸化影响、对ERα转录活性的影响及IL-6诱导卵巢癌TAM耐药的机制。方法脂质体转染法获得内源性过表达IL-6的人卵巢癌A2780细胞株和内源性抑制IL-6表达的人卵巢癌CAOV3细胞株,Western blot法检测内源性和外源性IL-6对ERK和ERα的Ser118位点磷酸化影响,MTT法检测IL-6的MEK/ERK信号通路对A2780细胞TAM耐药性的影响,荧光素酶活性检测IL-6的MEK/ERK信号通路对ERα转录活性的影响。结果过表达IL-6能上调A2780细胞中ERα-Ser118的磷酸化水平,而抑制IL-6表达下调CAOV3细胞中ERα-Ser118的磷酸化水平;外源性IL-6上调A2780细胞中ERK的磷酸化水平,而MEK1/2特异性抑制剂PD98059则可以逆转IL-6介导的ERK及ERα-Ser118的磷酸化;PD98059可明显降低IL-6导致的A2890细胞对TAM的耐药性;IL-6明显促进ERα转录活性,而PD98059可逆转这一作用。结论 IL-6经MEK/ERK通路磷酸化ERα的Ser118位点促进ERα转录活性而激活ER途径,诱导OVCA细胞对TAM的耐药性。     

糖原合成酶激酶3在阿尔茨海默病中的作用

糖原合成酶激酶-3(glycogen synthase kinase-3,GSK-3)作为体内主要的蛋白激酶之一,广泛地参与了生长发育、凋亡衰老和肿瘤形成等重大生命过程.GSK-3β-Ser9和GSK-3α-Ser21位点的磷酸化修饰是机体对GSK-3活性调控的关键过程,GSK-3参与了tau蛋白过度磷酸化修饰、β淀粉样蛋白异常聚集和神经元凋亡等过程,因此对GSK-3活性调控的研究是阿尔茨海默病等神经退行性疾病的发病机制和相关治疗研究中的焦点问题之一.     

P53翻译后修饰研究进展

肿瘤抑制基因p53在肿瘤发生中发挥着重要作用。P53蛋白的翻译后修饰及其与多种细胞蛋白间相互作用使P53蛋白呈现功能多样性。P53的翻译后修饰不是单个位点的修饰而是包括磷酸化、乙酰化、泛素化及SUMO化作用的多位点修饰。翻译后修饰对P53功能至关重要,更可能与某些肿瘤的发生密切相关。发生在蛋白质水平上的P53的功能性灭活是没有发生p53基因突变肿瘤发生的重要机制之一。     

HSP90在乳腺癌细胞增殖中的作用与机制研究

目的： 应用热休克蛋白90(HSP90)功能特异性抑制剂格尔德霉素(GA)研究HSP90在乳腺癌细胞增殖中的作用，进而探讨相关机制。方法: 培养人乳腺癌细胞株MDA-MB-435s，采用MTT法检测GA对MDA-MB-435s细胞的生长抑制作用，流式细胞术分析细胞周期，应用Western免疫印迹法检测细胞内Stat3磷酸化状态和突变型p53表达变化。结果: 不同浓度GA对乳腺癌MDA-MB-435s细胞有生长抑制作用，呈时效量效依赖关系，GA作用后细胞呈现G2/M期阻滞。400 nmol/L GA作用48h后，细胞内Stat3磷酸化水平明显低于对照组，同时突变型p53表达明显低于对照组。结论: 抑制HSP90功能可明显抑制乳腺癌MDA-MB-435s细胞的增殖，此与下调细胞内Stat3磷酸化水平和突变型p53表达有关。     

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。     

NUCB2 siRNA对肝细胞胰岛素信号通路及细胞凋亡的影响

目的探讨靶向NUCB2基因特异性siRNA对大鼠肝细胞IAR20 NUCB2基因沉默效应、对大鼠肝细胞胰岛素信号通路及细胞凋亡的影响。方法构建合成靶向NUCB2基因的siRNA,转染IAR20细胞。Western blot检测PEPCK、G-6-Pase、InsR、IRS-1、Akt的蛋白含量及其磷酸化水平。采用流式细胞术检测细胞凋亡情况,使用RT-PCR检测p53及caspase3 mRNA水平。结果转染siRNA 48 h后,IAR20细胞NUCB2表达明显降低(P均0.05)。PEPCK、G-6-Pase蛋白及mRNA表达量明显增加,同时IR、IRS-1、AKT的磷酸化表达降低(P0.01或P0.05)。IAR20细胞凋亡明显增加,p53及caspase 3 mRNA表达及cleaved caspase 3明显增加(P0.01或P0.05)。结论 NUCB2特异性siRNA能通过下调IR/IRS-1/Akt信号通路加重大鼠肝细胞胰岛素抵抗,并能够通过上调caspase 3及p53表达增加细胞凋亡。     

p53通过诱导miR199a-3p抑制SOCS7,从而抑制UUO中的STAT3活化和肾纤维化

<正>近来p53在肾纤维化中的作用被研究较多,但其功能仍然存在争议,其潜在的机制尚不清楚。Yang等的研究显示,在药理学和基因上阻断p53可减轻单侧输尿管梗阻(unilateral urethral obstruction,UUO)小鼠的肾间质性纤维化、凋亡和炎症。有趣的是,p53的阻断与miR-215-5p、miR-199a-5p3p和STAT3的抑制有关。在培养的人肾小管上皮细胞(HK-2)中,TGF-β1处理导致纤维化改变,包括胶原蛋白I(collagen I)和波形蛋白(vimentin)表达,且与p53积聚、p53 Ser15磷酸化和miR-199a-3p表     

p53/Rb细胞转导通路相关基因和蛋白在衰老大鼠睾丸组织的表达

目的 通过检测p53、Rb、p16、p19、p21在衰老大鼠睾丸组织的表达,探讨p53/Rb细胞转导通路相关基因和蛋白在睾丸组织衰老中的意义. 方法 选用8周龄雄性SD大鼠30只,体重180～220g,随机分为正常对照组和模型组,每组15只.模型组大鼠采用D-半乳糖连续腹腔注射建立亚急性衰老大鼠模型.采用RT-PCR方法 检测p53、Rb基因在大鼠睾丸组织的表达;Western blotting法检测大鼠睾丸磷酸化Rb、p16、p19、p21蛋白的表达.结果 模型组睾丸组织p53、Rb基因表达明显高于正常对照组(P<0.01);模型组睾丸组织与正常组比较:p16、p19、p21蛋白的表达明显升高,而磷酸Rb蛋白表达显著下降(P<0.01). 结论 Rb/p53细胞转导通路相关基因及蛋白在衰老大鼠睾丸组织发生明显改变,Rb/p53细胞转导通路阻滞可能在睾丸组织的衰老过程中起着一定作用.     

Cell shrinkage as a signal to apoptosis in NIH 3T3 fibroblasts

Cell shrinkage is a hallmark of the apoptotic mode of programmed cell death, but it is as yet unclear whether a reduction in cell volume is a primary activation signal of apoptosis. Here we studied the effect of an acute elevation of osmolarity (NaCl or sucrose additions, final osmolarity 687 mosmol l⁻¹) on NIH 3T3 fibroblasts to identify components involved in the signal transduction from shrinkage to apoptosis. After 1.5 h the activity of caspase-3 started to increase followed after 3 h by the appearance of many apoptotic-like bodies. The caspase-3 activity increase was greatly enhanced in cells expressing a constitutively active G protein, Rac (RacV₁₂A₃ cell), indicating that Rac acts upstream to caspase-3 activation. The stress-activated protein kinase, p38, was significantly activated by phosphorylation within 30 min after induction of osmotic shrinkage, the phosphorylation being accelerated in fibroblasts overexpressing Rac. Conversely, the activation of the extracellular signal-regulated kinase (Erk1/2) was initially significantly decreased. Subsequent to activation of p38, p53 was activated through serine-15 phosphorylation, and active p53 was translocated from the cytosol to the nucleus. Inhibition of p38 in Rac cells reduced the activation of both p53 and caspase-3. After 60 min in hypertonic medium the rate constants for K⁺ and taurine efflux were increased, particular in Rac cells. We suggest the following sequence of events in the cell shrinkage-induced apoptotic response: cellular shrinkage activates Rac, with activation of p38, followed by phosphorylation and nuclear translocation of p53, resulting in permeability increases and caspase-3 activation.     

Phosphorylation of a C-SRC-related protein in macrophages activated in vitro with lymphokine

Treatment of proteose peptone elicited peritoneal macrophages from C3H/HeN mice or the macrophage cell line B6MP102 with a T-cell lymphokine preparation induces cytotoxicity for SV3T3 tumor cells. The Triton X-100 (TX-100) insoluble fractions from activated macrophages possessed kinase activity for an endogenous 53 kDa phosphoprotein (pp53) which was markedly greater than extracts from untreated macrophages. Addition of the tyrosine phosphatase inhibitor, Na₃,VO₄ to the cytotoxicity assay also enhanced tumor cell lysis and Na₃VO₄ treated macrophages showed increased phosphorylation of pp53. Moreover, addition of Na₃VO₄ to the cytoskeleton kinase assay enhanced the phosphorylation of pp53 in a dose dependent manner. Pp53 was immunoprecipitated from the in vitro phosphorylated TX-100 insoluble fraction with monoclonal antibody to pp60^v-src. Anti-pp60^v-src also precipitated a 53 and a 60 kDa phosphoprotein from whole cell extracts and from TX-100 cytoskeleton extracts of macrophages phosphorylated as viable intact cells. Addition of a known tyrosine kinase inhibitor, quercetin, to the macrophage cytoskeleton kinase assay inhibited phosphorylation of pp53, and the in vitro phosphorylated pp53 was resistant to 1 N NaOH hydrolysis, indicating phosphorylation of tyrosine residues. Immune complex kinase assays of anti-pp60^c-src precipitated TX-100 insoluble macrophage fractions revealed strong phosphorylation for -casein which was inhibited by quercetin. These data suggest that macrophage pp53 is a c-src-related gene product that is inducible by stimuli that activate macrophages to cytotoxicity.     

Promoter methylations of p16INK4a and p14ARF genes in early and advanced gastric cancer. Correlations of the modes of their occurrence with histologic type

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16^INK4a and p14^ARF. These share an exon using different reading frames, and act through Rb and p53 pathways. Recently, it has been found that silencing of p16^INK4a and p14^ARF expressions by aberrant methylation of the CpG islands in the promoter regions is an alternative mechanism that inactivates possible tumor suppressor functions in various tumors. To clarify the features of gastric cancers with promoter methylation of p16^INK4a and p14^ARF, we investigated the methylation status in gastric cancer cell lines and primary gastric cancers using methylation-specific PCR (MSP), and correlated the methylation status with microsatellite instability (MSI), DNA ploidy pattern, p53 immunohistochemistry, and various clinicopathologic factors, paying attention to the correlations with the histologic types. Of 10 cell lines studied, silencing of the expression of p16^INK4a and p14^ARF due to promoter methylation was detected by MSP and RT-PCR in six (60%) and two (20%) cell lines, respectively. p14^ARF silencing was detected only in cell lines derived from gastric cancer of the diffuse type, while p16^INK4a silencing was found in cell lines derived from both diffuse and intestinal types. In 59 primary gastric cancers, promoter methylation of p16^INK4a and p14^ARF was found in 10 (17%) and 14 (24%) of the tumors independently, there being an association with DNA diploidy, but not with p53 immunohistochemistry. p16^INK4a methylation was found irrespective of tumor stages and histology. Whereas p14^ARF methylation was found more frequently in intestinal type cancers in an early stage and in diffuse type cancers in an advanced stage, MSI tended to be related especially to p14^ARF methylation in cancers of the intestinal type. Thus, the significance of p14^ARF methylation differed between intestinal and diffuse types, while such a difference was not observed in p16^INK4a methylation.     

Engagement of the TcR/CD3 complex stimulates p59 activity: Detection of associated proteins at 72 and 120–130 kD

Engagement of the T cell antigen-receptor complex (TcR/CD3) induces the rapid tyrosine phosphorylation of a spectrum of substrates whose modification is crucial to the activation process. Although CD4-associated p56^lck and TcR/CD3-associated p59^fyn(T) could account for this cascade, TcR/CD3 driven stimulation of p59^fyn(T) activity has not been demonstrated. In this study, we confirm in Brij 96 based buffers that p59^fyn(T) can be co-purified in association with the TcR/CD3 complex, and further demonstrate that antibody-induced cross-linking of TcR/CD3 on the cell surface results in a dramatic increase in the detection of receptor associated kinase activity. This results in an increased phosphorylation and detection of TcR/CD3-p59^fyn(T) associated ζ (16–21 kD), p72 (72 kD) and p120/130 (120–130 kD) chains. A distinction between increased recruitment and/or activity of p59^fyn(T) was not possible due to the fact that receptor associated p59^fyn(T) could not be detected by immunoblotting. However, an alternative approach using membrane vesicles demonstrated an anti-CD3 mediated induced increase (2–5-fold) in the phosphorylation of the fyn kinase. Augmented catalytic activity was accompanied by p59^fyn(T) labelling at the autophosphorylation site Tyr420, consistent with stimulated fyn catalytic activity, as well as the phosphorylation of polypeptides at 18–20 (TcRζ), 31, 90 and 130 kD. Stimulation of fyn activity implicates this kinase as a mediator of the tyrosine phosphorylation events originating from the TcR/CD3 complex.     

Expression of p21WAF1/CIP1 in colorectal adenomas and adenocarcinomas and its correlation with p53 protein expression

The expression of p53-Inducible cylln-dependent kinase Inhibitor, p21^WAF1/CIP1 in non-neopiastic mucosa, adenoma and adenocarclnoma of the colorectum was examined by immunohistochemistry and western bootting and Its relation with the expression of p53 protein was analyzed. Non-neoplastic epithelial cells at the surface area showing no proitferative activity expressed p21^WAF1/CIP1.The expression of p21^WAF1/CIP1 was lmmunohistochemlcally detected in 55% (206/377 of the adenomas and 66% (190/289) of the adenocarcinomas, respectively. The lncldence of strongly positive cases was significantly higher In the adenocarcinomas (27%) than In the adenomas (18%) ( P< .05). The incidence of cases wtth strong p21^WAF1/CIP1 expression was higher In stages 0,1 and 2 carcinomas than in stages 3 and 4 carcinomas ( P <0.05). A decrease in the incidence of cases with strong expression was detected in carclnomas Invading deeper than muscularis propria. The influence of strongly positive cases was signiflcantly lower in carcinomas with lymph node metastasis than those without metastasls ( P <0.05). The expression of p21 as well as p53 detected by western blotting was compatlble with the results of lmmunohistochemlstry in most cases examined. However, there was no significant correlatlon between the expression of p21^WAF1/CIP1 and the abnormal accumulation of p53. These findlngs overall suggest that: (i) the physiological expression of p21^WAF1/CIP1 may be associated with cellular senescence of colorectal mucosa; (ii) reduced expression of p21^WAF1/CIP1 participate in the progression of colorectal carcinoma; and (iii) p53-Independent paulway may be considerably Involved In the inductions of p21^WAF1/CIP1.     

Phosphorylation and functional regulation of CIC-2 chloride channels expressed in Xenopus oocytes by M cyclin-dependent protein kinase

Many dramatic alterations in various cellular processes during the cell cycle are known to involve ion channels. In ascidian embryos and Caenorhabditis elegans oocytes, for example, the activity of inwardly rectifying Cl⁻ channels is enhanced during the M phase of the cell cycle, but the mechanism underlying this change remains to be established. We show here that the volume-sensitive Cl⁻ channel, ClC-2 is regulated by the M-phase-specific cyclin-dependent kinase, p34^cdc2/cyclin B. ClC-2 channels were phosphorylated by p34^cdc2/cyclin B in both in vitro and cell-free phosphorylation assays. ClC-2 phosphorylation was inhibited by olomoucine and abolished by a ⁶³²Ser-to-Ala (S632A) mutation in the C-terminus, indicating that ⁶³²Ser is a target of phosphorylation by p34^cdc2/cyclin B. Injection of activated p34^cdc2/cyclin B attenuated the ClC-2 currents but not the S632A mutant channel currents expressed in Xenopus oocytes. ClC-2 currents attenuated by p34^cdc2/cyclin B were increased by application of the cyclin-dependent kinase inhibitor, olomoucine (100 μ m ), an effect that was inhibited by calyculin A (5 n m ) but not by okadaic acid (5 n m ). A yeast two-hybrid system revealed a direct interaction between the ClC-2 C-terminus and protein phosphatase 1. These data suggest that the ClC-2 channel is also counter-regulated by protein phosphatase 1. In addition, p34^cdc2/cyclin B decreased the magnitude of ClC-2 channel activation caused by cell swelling. As the activities of both p34^cdc2/cyclin B and protein phosphatase 1 vary during the cell cycle, as does cell volume, the ClC-2 channel could be regulated physiologically by these factors.     

Apocrine carcinomas of the breast in Turkish women: hormone receptors, c-erbB-2 and p53 immunoexpression

The aims of this study were twofold: (i) to determine the occurrence frequency of apocrine carcinoma of the breast (ApBCa) in Turkish breast cancer (BCa) patients; and (ii) to evaluate the expression of estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), gross cystic disease protein-15 (GCDFP-15), c-erbB-2, and p53 in these cases. Six hundred and twenty-six cases of BCa were studied immunohistochemically (streptoavidin-biotin horseradish peroxidase method). The results of ApBCa were compared with those of invasive ductal carcinoma not otherwise specified type (IDC-NOS) cases of similar grade. Thirteen cases of ApBCa were encountered, accounting for 2.1% of all BCa cases. Immunohistochemically, ApBCa positivity was as follows: GCDFP-15 (100%), ER (39%), PR (8%), AR (54%), p53 (39%), and c-erbB-2 (85%). In the IDC-NOS group, GCDFP-15* was expressed in less than 50% of the tumors. The occurrence frequencies of the other markers were as follows: ER (69%), PR (69%)*, AR (46%), c-erbB-2 (0%)*, and p53 (31%), (*) indicating significant differences between the two groups.

For Turkish BCa patients, (i) the occurrence rate of ApBCa (2.1%) was high; and (ii) the following combination would allow for an immunohistochemical identification of ApBCa: GCDFP-15⁺, c-erbB-2⁺, and PR⁻.     

Tumor Necrosis Factor-α-Associated Mechanisms Affecting the Embryonic Response to Cyclophosphamide

Problem  We have previously shown that TNF-α^−/− embryos are more sensitive to the exposure to cyclophosphamide (CP) compared with TNF-α^+/+ embryos; however, the underlying mechanisms are not fully understood. Thus, in our present study, we tried to identify those molecules that might be responsible for the protective effect of the cytokine.
Method of study  CP-treated TNF-α^−/− and TNF-α^+/+ embryos were analyzed for changes in apoptosis by TUNEL and flow cytometry, while cell proliferation was analyzed by BrdU incorporation. The expression of Bax, bcl-2, p53, the p65 subunit of NF-κB and IκBα was assessed by Western blotting and immunohistochemistry.
Results  CP-treated TNF-α^−/− embryos exhibited a more profound decrease in their weight, which was accompanied by an earlier appearance of cellular damage and apoptotic cells and an earlier decrease in cell proliferation in the embryonic brain compared with TNF-α^+/+ embryos. Also, an increased percentage of Bax-positive cells and a decreased percentage of bcl-2-positive cells were detected in TNF-α^−/− embryos 48 hr after exposure, which were accompanied by a decreased percentage of p53-positive cells.
Conclusion  Our data implicate TNF-α to be involved in the protection of the embryo against CP teratogenicity, possibly via alteration in Bax, bcl-2 or p53 expression.