P53 and P16 gene mutations in non-small cell lung cancer

The aim of this study was to assess prospectively the occurrence of p53 and p16 mutations (considered separately and together) in NSCLC in terms of their clinical and prognostic relevance. Study group included 87 patients who underwent pulmonary resection for cure. p53 and p16 mutations were found in 22 (25%) and 14 (16%) cases, respectively. In eight patients (9%) both mutations were present, and the tendency for their common occurrence was significant (p = 0.02). There was no relation between mutation and clinical characteristics. Median survival in the entire group was 17 months and the 3-year survival probability--41%. There was no correlation between the occurrence of any mutation (considered separately or together) and survival. These results indicate that p53 and p16 gene mutations tend to occur together in NSCLC, however these alterations seem not to have noteworthy clinical and prognostic significance.     

P53与妊娠滋养细胞肿瘤的增殖和凋亡

目的探讨P53在妊娠滋养细胞肿瘤(GTT)中的表达意义及其与GTT中细胞增殖、凋亡的关系。方法采用免疫组化SP法及核酸原位末端标记(TUNEL)法,检测妊娠滋养细胞肿瘤中P53和PCNA的表达率及细胞凋亡指数(AI)。结果在正常早期绒毛(NP)、完全性葡萄胎(CM)、侵袭性葡萄胎(IM)、绒癌(CCA)中,P53表达率(P53-Ⅰ)分别为4.12%、21.68%、39.61%和27.39%;PCNA表达率(PI)分别为10.40%、20.76%、53.60%和51.95%;凋亡指数(AI)分别为1.88%、2.59%、6.45%和1.26%;各组间比较均P<0.001。P53-Ⅰ和PI呈显著正相关(r=0.587,P<0.001),而与AI无明确的相关性。结论GTT的发生可能是由于机体的细胞增殖与凋亡调节系统失调所致;P53可能促进GTT中的细胞增殖活性,与GTT的发生、发展密切相关;未发现P53与GTT细胞凋亡的相关性。     

Novel protective properties of IGFBP-3 result in enhanced pericyte ensheathment, reduced microglial activation, increased microglial apoptosis, and neuronal protection after ischemic retinal injury

This study was conducted to determine the perivascular cell responses to increased endothelial cell expression of insulin-like growth factor binding protein-3 (IGFBP-3) in mouse retina. The contribution of bone marrow cells in the IGFBP-3-mediated response was examined using green fluorescent protein-positive (GFP(+)) adult chimeric mice subjected to laser-induced retinal vessel occlusion injury. Intravitreal injection of an endothelial-specific IGFBP-3-expressing plasmid resulted in increased differentiation of GFP(+) hematopoietic stem cells (HSCs) into pericytes and astrocytes as determined by immunohistochemical analysis. Administration of IGFBP-3 plasmid to mouse pups that underwent the oxygen-induced retinopathy model resulted in increased pericyte ensheathment and reduced pericyte apoptosis in the developing retina. Increased IGFBP-3 expression reduced the number of activated microglial cells and decreased apoptosis of neuronal cells in the oxygen-induced retinopathy model. In summary, IGFBP-3 increased differentiation of GFP(+) HSCs into pericytes and astrocytes while increasing vascular ensheathment of pericytes and decreasing apoptosis of pericytes and retinal neurons. All of these cytoprotective effects exhibited by IGFBP-3 overexpression can result in a more stable retinal vascular bed. Thus, endothelial expression of IGFBP-3 may represent a physiologic response to injury and may represent a therapeutic strategy for the treatment of ischemic vascular eye diseases, such as diabetic retinopathy and retinopathy of prematurity.     

胃癌中P53蛋白转录调控活性的分析及其临床意义

目的 探讨P53蛋白转录调控活性在胃癌发生发展中的作用.方法 应用双荧光素酶报告基因测试(dual-luciferase reporter assay)检测人类胃癌细胞和正常细胞中p53转录调控活性,并通过逆转录-PCR对细胞中p53全长进行扩增,构建pMD18-T-p53重组质粒测序,检测p53突变对其活性的影响.应用免疫组织化学的方法 联合检测P21WAF1/Cip1,Gadd45α,Mdm2三种蛋白在76例胃癌及癌旁组织中表达水平,通过分析它们与肿瘤的临床病理特征及各个分子之间的相关性来间接反应P53在组织中的转录调控活性.结果 双荧光素酶报告基因显示p53转录调控活性在正常细胞中明显高于肿瘤细胞,在p53基因发生突变的细胞系MKN28中P53蛋白转录活性最低.免疫组化结果显示P21WAF1/Cip1,Gadd45α,Mdm2在癌旁组织中的表达明显高于肿瘤组织(P<0.05),且随着肿瘤TNM分期的增高有表达降低的趋势(P<0.05).P21WAF1/Cip1,Gadd45α,Mdm2之间的相关性分析结果显示3个分子在胃癌组织和癌旁组织中的表达呈正相关(P<0.05).结论 P53蛋白转录调控活性的变化在胃癌的发生发展中起着重要的作用,p53突变可以影响其转录调控活性.P21WAF1/Cip1,Gadd45α,Mdm2联合检测可以作为间接反映P53蛋白转录调控活性的效应分子.     

Proteasome inhibitors induce p53-independent apoptosis in human cancer cells

Proteasome inhibitors are used against human cancer, but their mechanisms of action are not entirely understood. For example, the role of the tumor suppressor p53 is controversial. We reevaluated the role of p53 in proteasome inhibitor-induced apoptosis by using isogenic human cancer cell lines with different p53 status. We found that well-known proteasome inhibitors such as MG132 and bortezomib, as well as the recently discovered proteasome inhibitor thiostrepton, induced p53-independent apoptosis in human cancer cell lines that correlated with p53-independent induction of proapoptotic Noxa but not Puma protein. In addition, these drugs inhibited growth of several cancer cell lines independently of p53 status. Notably, thiostrepton induced more potent apoptosis in HepG2 cells with p53 knockdown than in parental cells with wild-type p53. Our data confirm that proteasome inhibitors generally induce p53-independent apoptosis in human cancer cells.     

Bcl-2 constitutively suppresses p53-dependent apoptosis in colorectal cancer cells

To dissect apoptotic genes governing the survival of colorectal carcinoma cells, we employed RNAi to silence Bcl-2 and Bcl-x(L) in isogenic clones of p53+/+ and p53-/- cells, and of Bax+/- and Bax-/- cells. We identify a novel proapoptotic function of p53 that does not require activation by genotoxic agents and that appears to be constitutively suppressed by Bcl-2. Silencing of Bcl-2 induced massive p53-dependent apoptosis. The "Bcl-2/p53 axis" requires Bax and caspase 2 as essential apoptotic mediators. This newly discovered Bcl-2/p53 functional interface represents a key regulator of apoptosis which can be activated by targeting Bcl-2 in colorectal carcinoma cells.     

氧化应激和P53参与波动高糖诱导的肾小管上皮细胞凋亡

目的: 探讨波动性高糖引起的肾小管上皮细胞凋亡的机制。方法: 体外培养人肾小管上皮细胞 (HK-2),给予稳定高糖或波动高糖干预,并使用抗氧化剂和P53特异性抑制剂验证氧化应激和P53在波动高糖诱导的肾小管上皮细胞凋亡中的作用;此外,SD大鼠随机分为正常对照组(A)、糖尿病稳定高血糖组(B)和糖尿病波动高血糖组(C)。采用链脲佐菌素(STZ)65 mg/kg腹腔注射诱发糖尿病,血糖波动组每天定时腹腔注射速效胰岛素,并错时给予葡萄糖,造成一天中血糖浓度大幅波动模型。采用比色法检测超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,免疫组化法和Western blotting检测NADPH氧化酶4(Nox4)和P53蛋白表达,流式细胞术和原位缺口末端标记法(TUNEL)检测肾脏细胞凋亡。结果: (1)与稳定高糖比较,波动高糖引起更加明显的HK-2细胞凋亡,P53蛋白表达明显上调,同时培养上清液中MDA含量增加和SOD活性下降,抗氧化剂和P53抑制剂可明显抑制磷酸化P53蛋白表达和细胞凋亡。(2)在体动物实验制模12周后,与B组比较,C组肾组织Nox4表达明显增加,肾小管磷酸化P53蛋白表达上调,细胞凋亡数增加。结论: 氧化应激和P53参与波动性高糖诱导的肾小管上皮细胞凋亡。     

EGCG通过调控SIRT1-P53通路诱导人卵巢癌SKOV-3细胞凋亡

 目的: 研究表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)调控人卵巢癌SKOV-3细胞活力和凋亡的分子机制。方法: SKOV-3细胞给予EGCG(0~50 μmol/L)、SIRT1激动剂SRT1720(1 μmol/L)和SIRT1抑制剂EX527(1 μmol/L)处理后,用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,real-time PCR检测Bax和Bcl-2 mRNA的表达水平;采用SIRT1去乙酰化酶活性检测试剂盒检测SIRT1酶活性;使用Western blot法检测SIRT1、乙酰化P53和P53的蛋白表达变化。结果: 与正常对照组相比,单独给予EGCG或EX527处理之后SKOV-3细胞活力下降,凋亡率增加;SIRT1的酶活性和蛋白表达水平均明显下降;P53的乙酰化水平显著增加。与EGCG组相比,SRT1720预处理组的细胞活力上升,凋亡率下降,Bax/Bcl-2的相对比值及激活型caspase-3的蛋白水平明显下降,并且SIRT1的酶活性和蛋白表达水平显著增加,P53的乙酰化水平下降。结论: EGCG可通过调控SIRT1-P53通路抑制卵巢癌SKOV-3细胞活力并诱导其凋亡。     

P53在急性肾损伤小鼠肾脏的表达及其与细胞凋亡的关系

 目的：探讨缺血再灌注致急性肾损伤（AKI）小鼠肾脏不同部位P53的表达及其与细胞凋亡的关系。方法：采用随机对照动物实验方法,将18只小鼠随机分为假手术组、AKI组及P53抑制剂pifithrin-alpha（PFT-α）组,每组6只。采用双侧肾蒂夹闭45 min后松开动脉夹的方法建立小鼠AKI模型,PFT-α组于建模前5 min腹腔注射PFT-α  2.2 mg/kg,并于建模后48 h采血检测尿素氮和肌酐,取肾脏组织行HE染色观察肾组织病理学变化,采用Western blotting法测定P53蛋白含量,免疫荧光法确定肾脏不同部位P53的表达,TUNEL法检测肾脏细胞凋亡,免疫组化方法检测肿瘤坏死因子受体（TNFR）、caspase-3及Bcl-2蛋白水平 。结果：（1）PFT-α组和AKI组小鼠血尿素氮和肌酐水平均明显高于假手术组,而PFT-α组与AKI组比较血尿素氮和肌酐水平明显降低（P<0.05）;肾组织HE染色显示假手术组肾组织细胞形态完整,排列整齐,无明显病理改变,AKI组肾小管上皮细胞刷状缘脱落、空泡及滴状变性,皮髓质间有明显淤血带;PFT-α组肾小管上皮部分刷状缘脱落消失,空泡及滴状变性减轻,皮髓质间无明显淤血带;（2）假手术组小鼠肾脏有少量P53表达且未检测到凋亡细胞,而AKI组小鼠缺血再灌注48 h后P53蛋白水平及凋亡细胞明显增加（P<0.05）,并且均主要定位在肾皮质,PFT-α组较AKI组小鼠肾脏P53蛋白含量及凋亡细胞指数均减少（P<0.05）;（3）与假手术组相比,AKI组小鼠肾脏TNFR蛋白及caspase-3蛋白水平升高（P<0.05）,Bcl-2蛋白水平下降（P<0.05）,PFT-α组较AKI组小鼠肾脏TNFR蛋白及caspase-3蛋白水平降低（P<0.05）,Bcl-2蛋白水平升高（P<0.05）。结论：急性肾损伤时,肾组织P53表达增加,主要定位于皮质,通过调控凋亡蛋白Bcl-2和TNFR的水平,促进caspase-3的释放,从而介导细胞凋亡。     

iASPP对表达野生型p53基因的乳腺癌细胞株MCF-7凋亡的影响

目的： P53凋亡刺激蛋白（ASPP）家族成员能够调节P53诱导的细胞凋亡。其中ASPP家族抑制成员(iASPP)主要通过特异性地和P53蛋白结合抑制肿瘤细胞的凋亡,本实验通过构建iASPP的RNAi质粒,研究在体内外抑制iASPP基因后对表达野生型p53基因的乳腺癌细胞MCF-7凋亡的影响。方法: 采用分子生物学方法构建iASPP的腺病毒RNAi质粒pAd-iASPP-RNAi;体外转染MCF-7细胞,同时建立MCF-7细胞的裸鼠移植瘤模型;分别采用RT-PCR和Western blotting检测转染后瘤细胞iASPP mRNA和蛋白质表达的变化;采用流式细胞术检测转染前后瘤细胞凋亡的变化。结果: 转染iASPP RNAi后,MCF-7细胞的iASPP mRNA及蛋白表达量降低（抑制率分别为95.4%和96.8%,P<0.01）;MCF-7细胞的凋亡百分率、坏死百分率与对照组相比具有显著差异（P<0.01）。裸鼠移植瘤模型经pAd-iASPP-RNAi处理后,iASPP mRNA及蛋白表达量也明显降低（抑制率分别为87.4%和89.2%,P<0.01）;移植瘤细胞的凋亡百分率和坏死百分率显著上升（P<0.01和P<0.05）。结论: 在体内外抑制iASPP基因的表达能够通过p53途径诱导表达野生型p53的乳腺癌细胞凋亡。     

TIMP-3 and endocrine therapy of breast cancer: an apoptosis connection emerges

The tissue inhibitors of metalloproteinases (TIMPs) control the activities of matrix metalloproteinases (MMPs) and as such, they have been recognized as potential suppressors of angiogenesis and tumour invasion and metastasis. However, TIMP-3 has several unique properties that set it apart from other TIMPs, including its ability to bind to extracellular matrix, inhibit related adamalysin metalloproteinases (ADAMs), and induce apoptosis. New data suggest that high levels of TIMP-3 mRNAs in human breast tumours are associated with success of adjuvant endocrine therapy, but not chemotherapy.     

Expression of minichromosome maintenance-2 in human malignant fibrous histiocytomas: Correlations with Ki-67 and P53 expression,and apoptosis

This study examined the clinicopathological significance of minichromosome maintenance-2 (MCM2) expression in 38 human malignant fibrous histiocytomas (MFHs) and 36 benign fibrohistiocytic tumors (BFHTs) immunohistochemically, and in 9 human sarcoma or carcinoma cell lines, as well as 7 surgical specimens by Western blotting. MCM2 was detected in all the cell lines and surgical specimens as a single band at 120 kDa, while P53 expression was variable. Nuclear expression of MCM2 was noted in tumor but not mitotic cells of all the MFHs and 26 (72.2%) of the BFHTs, the labeling indices (LIs) being 62.0% in the 28 ordinary types, 38.5% in the 10 myxoid types, and 11.2% in the BFHTs with significant difference. Moreover, the LI was significantly higher for MCM2 than that for Ki-67 in the MFHs of both types (p<0.05). No correlation was noted between the MCM2-LI and P53 expression or apoptotic indices, which were significantly higher in the MFHs than BFHTs (p<0.01). These results indicate that MCM2 would correlate with cell proliferation rather than apoptosis in MFHs, and the expression is ubiquitous in proliferating cells, regardless of the expression of P53. Thus, MCM2 might be a reliable marker of proliferating cells in human MFH.     

P53/MDM2 overexpression in metastatic endometrial cancer: correlation with clinicopathological features and patient outcome

Several studies have reported that p53/mdm2 distortions play a pivotal role in the development and progression of various human malignancies. However, the number of reports having evaluated simultaneously the components of the P53-pathway alterations in advanced-stage human endometrial carcinomas (EC) is low. In this study, we examined the expression of P53/MDM2 proteins in primary and metastatic ECs, and analyzed the clinicopathological characteristics as well as the survival outcome of patients in relation to P53/MDM2 overexpression. The study group comprised 36 patients with advanced EC, whose primary and metastatic tumor slides were sufficient for analysis. Immunohistochemical assessment was made by applying anti-human P53 and MDM2 antibodies and a highly sensitive EnVision⁺/HPR visualization system. Nuclear P53 overexpression was seen in 11 (31%) primary ECs and 12 (33%) metastatic tumors. There was a significant correlation between P53 overexpression (in primary cancers and metastatic tumors) and MDM2 overexpression in metastatic tumors. Nuclear MDM2 overexpression was noted in 42% (15/36) of primary carcinomas and in 47% (17/36) of metastatic tumors. A significant association existed between MDM2 overexpression and histological grading (G1 + G2 versus G3, P = 0.043). P53/MDM2 overexpression occurred simultaneously in 7 out of 36 (19%) primary ECs and in 9 out of 36 (25%) metastatic lesions. Concomitant overexpression of these proteins was reported in 7 out of 36 (19%) cases and tended to be higher in tumors showing VSI compared to neoplasms lacking vascular space invasion (P = 0.051). P53 overexpression, either in primary ECs (P < 0.0001) or metastatic lesions (P < 0.0001), was significantly associated with poor survival in univariate analysis. Moreover, the log-rank test demonstrated that simultaneous P53/MDM2 overexpression was also correlated with decreased length of survival. There was no correlation between MDM2 overexpression and patient survival. Multivariate Cox regression analysis revealed that only P53 overexpression is an independent predictor of survival. In conclusion, our data support the view that patients with P53 overexpression are significantly associated with an unfavorable outcome, whereas MDM2 overexpression is not related to decreased survival length in women operated on for advanced-stage EC.     

Long-term hypoxia exposure enhanced IGFBP-3 protein synthesis and secretion resulting in cell apoptosis in H9c2 myocardial cells

Abstract

Myocardial infarction (MI) usually results in myocardial ischemia, remodeling and hypoxia that lead to cell death. To date, the insulin-like growth factor binding protein-3 (IGFBP3) is known to play an important role in insulin growth factor (IGF) bioavailability. Previous studies have found that hypoxia results in cell apoptosis. However, the detailed mechanism and roles of IGFBP3 in long-term hypoxia (LTH) regulated heart cell apoptosis remains unknown. In this study H9c2 cardiomyoblast cells were treated with investigated long-term hypoxic exposure with the possible mechanisms involved. The results showed that LTH enhanced IGFBP3 protein synthesis and induced its secretion. The accumulated IGFBP3 sequestered Insulin growth factor 1 (IGF-1) away from the type I IGF receptor (IGF-1?R), which blocked the IGF1R/PI3K/Akt survival signaling pathway, resulting in cell apoptosis. According to our findings, IGFBP3 could be a valuable target for developing treatments for cardiac diseases in long-term hypoxia exposure patients.     

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。     

硫化氢对人PMN凋亡及P53/Bcl-2表达的影响

目的:研究硫化氢(H2S)对人中性粒细胞(PMN)凋亡及P53、Bcl-2表达的影响,以进一步探讨H2S抗脂多糖性肺损伤作用的机制。方法:采用体外分离和培养人血PMN。实验分3组,对照组:培养基中加入无菌生理盐水10mL/L;脂多糖(LPS)组:培养基中加入LPS100μg/L;LPS+硫氢化钠(NaHS)组:先LPS前1h向培养基中加入(1×10-5、1×10-4、1×10-3)mol/LNaHS。各组于24h后采用脱氧核苷酸末端转移酶介导的DNA原位末端标记技术(TUNEL)检测PMN凋亡情况;采用流式细胞术检测PMN中P53及Bcl-2蛋白的表达。结果:与对照组比较,LPS组PMN凋亡百分率和DNA断裂百分率均显著减少(均P0.05);与LPS组比较,LPS+NaHS(1×10-5)组PMN凋亡百分率和DNA断裂百分率均有所升高,但无显著差异(均P0.05),LPS+NaHS(1×10-4)组及LPS+NaHS(1×10-3)PMN凋亡百分率和DNA断裂百分率均明显增加,并呈剂量依赖性(均P0.01)。与对照组比较,LPS组P53蛋白表达量显著减少(P0.05);与LPS组比较,LPS+NaHS(1×10-5)组P53蛋白表达量无明显变化(P0.05),LPS+NaHS(1×10-4)组及LPS+NaHS(1×10-3)组P53蛋白表达量明显增多,并呈剂量依赖性(均P0.01);各组Bcl-2蛋白表达量无明显变化(P0.05)。PMN中P53蛋白表达量与凋亡百分率的相关分析结果显示二者具有明显正相关关系(r=0.75,P0.01)。结论:NaHS促进PMN凋亡的机制可能与其上调P53蛋白的表达有关,而与Bcl-2蛋白无关。     

非小细胞肺癌中P53表达的不同亚细胞定位及功能意义的研究

目的:探讨非小细胞肺癌(NSCLC)中不同亚细胞定位P53蛋白及其可能的功能意义。方法:以免疫组化及流式细胞分析术研究了43例NSCLC病人肿瘤组织P53表达定位与G₁/S检查点功能及DNA含量的关系。结果:发现P53蛋白不同的亚细胞定位与病人的病程密切相关,同时,在P53核表达定位的病人肿瘤组织细胞S期比例明显增多而G₀－G₁期比例明显较胞浆表达病人减少,G₂－M期比例及DNA指数在两组间无明显差异。结论:不同亚细胞定 位的P53蛋白具有不同的G₁/S检查点调节功能,胞浆P53蛋白仍有部分G₁/S检查点调控功能,因此出现较少的SPF及较多的G₀－G₁细胞,代表了恶性度较低的细胞亚群;而核表达阳性P53蛋白具有更差的G₁/S检查点调控功能,代表了恶性度较高的肿瘤亚群,这与肿瘤进展可能有关。