野生型p53对肺癌细胞生长的抑制作用

目的探讨外源性野生型p53对肺癌细胞生长的抑制作用. 方法通过向肺癌细胞中分别转染携带有荧光蛋白基因的野生型P53基因和突变型P53基因,而后用荧光显微镜观察荧光蛋白表达;中性红和MTT染料摄入法测定细胞生长活性. 结果 p53蛋白与绿色荧光蛋白之融合蛋白表达定位在胞浆和胞膜;经中性红和MTT两种方法测定,外源性野生型p53对肺癌细胞生长具有抑制作用;OD值分别为 0.71 ±0.081(OD540)和0.65±0.059(OD490),均低于对照组(p<0.05). 结论 p53外显子5～8可在肺癌细胞中获得表达并对细胞生长活性有抑制作用.     

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.     

p53对人乳腺癌细胞MCF-7生长及周期相关基因的影响

目的探讨抑癌基因p53对人乳腺癌细胞MCF-7生长以及周期相关基因cyclinD1、cyclinE、CDK2和CDK4表达的影响.方法将正义的p53真核表达载体导人MCF-7细胞,获得稳定表达正义p53的细胞模型,并经PCR和Western blot鉴定.分析了细胞的各种生长特性包括生长曲线、血清依赖、单细胞克隆形成、软琼脂成集落等;利用TdR双阻断法同步化细胞,流式细胞术分析了细胞周期;Western blot检测了其他周期相关基因的表达.结果p53的高表达可以使细胞的生长速度减慢、血清依赖性增加、单细胞克隆成集落能力降低、非锚定依赖性生长能力减弱,S期的进程减缓,一定程度上抑制cyclinD1和CDK2的表达,cyclinE和CDK4的表达未见明显变化.结论抑癌基因p53的表达能够抑制人乳腺癌细胞MCF-7的生长,并可能通过阻抑cyclinD1和CDK2的表达而阻抑细胞周期S期进程,实现其调节功能.     

miR-129-5p靶向调控VCP抑制骨肉瘤细胞体外迁徙侵袭

目的 探讨miR-129-5p是否靶向调控VCP基因抑制骨肉瘤细胞迁徙侵袭.方法 构建miR-129-5p过表达及低表达的慢病毒载体,转染骨肉瘤细胞U2-OS;采用实时荧光定量PCR检测上调及下调miR-129-5p的U2-OS细胞中miR-129-5p的表达量;采用RT-PCR和Western blot技术检测VCP mRNA和蛋白表达;采用划痕实验和Transwell侵袭实验检测细胞迁徙、侵袭情况.结果 实时荧光定量PCR结果显示U2-OS细胞中miR-129-5p表达被明显上调或下调;RT-PCR和Western blot检测结果显示:miR-129-5p上调U2-OS细胞组中VCP mRNA和蛋白表达水平显著低于阴性对照细胞组(阴性慢病毒转染);miR-129-5p下调U2-OS细胞组中VCP mRNA和蛋白表达水平显著高于阴性对照细胞组;miR-129-5p上调U2-OS细胞迁徙和侵袭力显著低于阴性对照细胞,miR-129-5p下调的U2-OS细胞迁徙和侵袭力显著高于阴性对照细胞.结论 miR-129-5p靶向调控VCP的表达而抑制骨肉瘤细胞迁徙和侵袭能力.     

野生型p53对肺癌细胞生长的抑制作用

目的　探讨外源性野生型p5 3对肺癌细胞生长的抑制作用 .方法　通过向肺癌细胞中分别转染携带有荧光蛋白基因的野生型P5 3基因和突变型P5 3基因 ,而后用荧光显微镜观察荧光蛋白表达 ;中性红和MTT染料摄入法测定细胞生长活性 .结果　p5 3蛋白与绿色荧光蛋白之融合蛋白表达定位在胞浆和胞膜 ;经中性红和MTT两种方法测定 ,外源性野生型p5 3对肺癌细胞生长具有抑制作用 ;OD值分别为 0 .71± 0 .0 81(OD540 )和 0 .6 5± 0 .0 5 9(OD490 ) ,均低于对照组 (p <0 .0 5 ) .结论　p5 3外显子 5～ 8可在肺癌细胞中获得表达并对细胞生长活性有抑制作用 .     

野生型p53基因诱导肝癌细胞凋亡及p21（WAF1／CIP …

目的：研究野生型ｐ５３基因对人肝癌细胞系细胞凋亡的作用。方法：通过脂质体转染方法将野生型ｐ５３基因（ｗｔ－ｐ５３）导入ｐ５３缺陷的ＨＣＣ－９２０４细胞系中，并获稳定表达。结果：转染ｗｔ－ｐ５３后细胞生长缓慢，Ｇ１期细胞数量由转基因前的４８．５％增加到７８．０％，并有较多细胞逐渐死亡。电镜观察和ＤＮＡ分析证实细胞死亡方式主要是细胞凋亡。免疫组化结果显示细胞转染ｗｔ－ｐ５３后，ｐ２１ＷＡＦ１／ＣＩＰ１     

口腔鳞状细胞癌组织中p14ARF蛋白表达及临床意义

目的 研究细胞周期抑制蛋白p14ARF 在口腔鳞状细胞癌组织中的表达及意义.方法 应用免疫组织化学S-P方法检测70 例口腔鳞状细胞癌组织标本中p14ARF蛋白的表达,同时以20 例口腔正常粘膜组织作对照.结果 正常口腔粘膜组织中p14ARF的表达率为90 %,口腔鳞状细胞癌组织中p14ARF的阳性表达率为41.43% ( P< 0.01) ; 在口腔鳞状细胞癌组织中其表达与口腔鳞状细胞癌的病理分级密切相关,中、低分化的口腔鳞状细胞癌组织其p14ARF表达明显低于高分化口腔癌组织,其表达随T分期的增加而降低.结论 口腔鳞状细胞癌组织中p14ARF蛋白表达具有明显的组织病理学特征,抑癌基因p14ARF在口腔鳞状细胞癌的进展中可能具有重要作用.     

p38丝裂原活化蛋白激酶信号转导通路在紫外线诱导表皮细胞凋亡中的作用

大量流行病学调查及分子生物学实验研究表明：紫外线可通过诱导活性氧及细胞因子产生DNA损伤和基因表达的改变导致细胞凋亡,这个过程非常复杂,有多个信号转导途径参与.由于细胞凋亡与皮肤癌的发生密切相关,近年来UVB诱导表皮细胞凋亡引起了人们广泛的关注.p38丝裂原活化蛋白激酶（p38MAPK）是细胞内重要的信号传导系统之一,参与细胞生长、发育、分化和凋亡等一系列生理、病理过程.p38MAPK可被紫外线激活,并在紫外线诱发的细胞应答过程中发挥重要的作用.     

p63基因在肺癌组织中的表达

目的研究肺鳞状细胞癌、腺癌、大细胞肺癌、小细胞肺癌以及淋巴结或肺内转移性肿瘤标本组织中p63基因的mRNA转录因子和蛋白表达水平,探讨p63基因表达与其定位在3q 27-q29区域改变的关系.方法采用cDNA微阵列技术同时检测72例不同病理组织学类型的原发性肺癌(包括鳞状细胞癌、腺癌、大细胞癌、小细胞癌)和肺癌肺内转移及淋巴结转移灶内的p63基因mRNA水平.另外,利用组织芯片技术构建150例原发性肺癌石蜡包埋标本组织芯片,采用免疫组织化学LSAB法检测p63基因蛋白表达情况.同时应用比较基因组杂交(CGH)技术对70例原发性肺癌标本进行了3号染色体长臂改变的分析.结果p63mRNA转录因子在肺鳞状细胞癌标本表达明显增强,与腺癌、大细胞癌、小细胞癌相比增多10倍以上.肺癌转移灶内p63基因mRNA表达水平明显高于其相对应的原发性灶,差异有统计学意义(P＜0.001).免疫组织化学染色结果显示肺鳞状细胞癌阳性表达率为94.64%;腺癌是1.79%;4例大细胞肺癌中2例为阳性染色;但所有小细胞肺癌标本免疫组织化学染色均为阴性.pT1分期肺癌的p63基因蛋白表达阳性率与pT2分期肺癌相比有统计学差异(P＜0.05).比较基因组杂交结果发现肺鳞状细胞癌3号染色体长臂27-29区域有显著的扩增,腺癌表现为缺失.鳞状细胞癌p63基因的表达增强与3q27-q29区域的显著扩增有明显正相关性(P＜0.000 1),说明定位于3号染色体长臂27-29区域的p63基因的拷贝有明显的扩增.结论p63mRNA转录因子和蛋白在肺鳞状细胞癌较其他肿瘤表达显著增高,转移灶高于原发灶;肺鳞癌p63表达增强与3号染色体长臂3q27-q29区域的扩增显著相关.     

阿糖胞苷诱导肺腺癌细胞凋亡与p73激活相关

目的 体外观察阿糖胞苷(1-β-D-arabinofuranosylcytosine, Ara-C)对肺腺癌A549细胞凋亡的诱导作用,探讨p53、p73基因在此凋亡过程中的调控作用.方法 Ara-C体外作用于A549细胞,TUNEL法检测A549细胞的凋亡;透射电镜观察A549细胞凋亡的典型超微结构;免疫印迹法检测A...     

Wip1在肺癌细胞中的作用机制初探

目的: 检测Wip1及相关蛋白在肺癌细胞中的表达情况,探讨Wip1在肺癌发生中的作用机制。方法: Western blotting检测各种细胞(肺腺癌细胞A549、肺鳞癌细胞NCI-1299、大细胞肺癌细胞 NCI-H460和正常支气管上皮细胞HBE)中Wip1、 p53、p-p53、p38 MAPK和p-p38 MAPK蛋白的表达。结果: A549、NCI-1299和H460细胞中Wip1蛋白的表达均高于HBE细胞(P<0.05);各种肺癌细胞间Wip1蛋白的表达无明显差异(P>0.05);p-p53和p-p38 MAPK蛋白在肺癌细胞中的表达低于HBE细胞(P<0.05);肺癌细胞中Wip1表达增加,p-p38 MAPK和p-p53表达降低,存在着Wip1-p38 MAPK-p53信号通路的失衡。结论: 肺癌细胞(A549、NCI-1299、H460)中存在Wip1-p38 MAPK-p53信号通路的失衡。这可能是Wip1在肺癌中的致癌机制之一。     

Wip1相关研究进展

正Fiscella等~([1])首次通过γ射线或UV射线诱导发现了野生型p53诱导的磷酸酶1(wild-type p53-induced phosphatase 1,Wip1),并且证实Wip1的表达依赖于p53。编码Wip1蛋白的基因称为PPM1D,定位于人染色体17q23和小鼠第11号染色体上,PPM1D编码的mRNA在很多器官组织包括睾丸和     

原癌基因Wip1在室管膜瘤中表达增加并与P53相关

目的 探讨野生型P53-诱导的蛋白磷酸酶1(Wip1)在室管膜瘤中的表达及其与P53相关性.方法 用免疫组织化学、反转录聚合酶链反应(RT-PCa)和Western blot 方法检测31例室管膜瘤及10例正常脑组织中Wip1mRNA、蛋白表达.并用免疫组化检测P53.同时,回顾性分析临床病理因素与Wipl表达之间的相...     

Wip1基因沉默对结肠癌细胞化疗敏感性的影响

目的:观察靶向Wip1基因的特异性siRNA序列对结肠癌细胞Wip1基因的抑制效应,探讨Wip1基因沉默对结肠癌细胞化疗敏感性的影响。方法:将Wip1-811 siRNA转染入Wip1高表达的RKO结肠癌细胞株,采用real-time PCR法检测Wip1 mRNA的表达,采用Western blotting方法检测Wip1蛋白表达,采用MTS方法检测结肠癌细胞活力,流式细胞术检测细胞凋亡及细胞周期。结果:Wip1-811 siRNA明显抑制Wip1的表达。抑制Wip1基因表达后,转染组RKO结肠癌细胞对抗肿瘤药物的敏感性增加,5-氟尿嘧啶处理组RKO结肠癌细胞活力由(89.4±6.6)%降至(74.7±3.9)%(P0.05),奥沙利铂处理组细胞活力由(77.9±2.4)%降至(66.7±2.9)%(P0.05)。转染Wip1-811 siRNA后,5-氟尿嘧啶处理组细胞凋亡比例由(7.7±0.5)%升至(12.3±3.2)%(P0.05);奥沙利铂处理组细胞凋亡比例由(14.7±2.1)%升至(34.0±2.1)%(P0.05)。结论:沉默Wip1基因表达能增强结肠癌细胞的化疗敏感性。     

Expression of Epstein-Barr virus BZLF1 immediate-early protein induces p53 degradation independent of MDM2, leading to repression of p53-mediated transcription

The Epstein-Barr virus (EBV) lytic program elicits ATM-dependent DNA damage response, resulting in phosphorylation of p53 at N-terminus, which prevents interaction with MDM2. Nevertheless, p53-downstream signaling is blocked. We found here that during the lytic infection p53 was actively degraded in a proteasome-dependent manner even with a reduced level of MDM2. BZLF1 protein enhanced the ubiquitination of p53 in SaOS-2 cells. The degradation of p53 was observed even in the presence of Nutlin-3, an inhibitor of p53-MDM2 interaction, and also in mouse embryo fibroblasts lacking mdm2 gene, indicating that the BZLF1 protein-induced degradation of p53 was independent of MDM2. Furthermore, Nutlin-3 increased the level of p53 in the latent phase of EBV infection but not in the lytic phase. Although p53 level is regulated by MDM2 in the latent phase, it might be mediated by the BZLF1 protein-associated E3 ubiquitin ligase in the lytic phase for efficient viral propagation.     

p53 expression in CMV-infected cells: association with the alternative expression of the p53 transactivated genes p21/WAF1 and MDM2

The p53 tumour suppressor gene is a cell cycle regulator, able to induce cell cycle arrest to allow DNA repair or apoptosis. The molecular mechanisms underlying p53 action imply transactivation of p53 dependent genes such as WAF1 (for wild type p53 associated fragment 1) and the murine double minute (MDM2) gene. In some cases, inactivation of the p53 gene results from p53 gene mutations leading to p53 protein accumulation, but in others it may results from mechanisms other than mutation, such as interaction with viral or cellular proteins. The expression of p53 protein and p53 transactivated gene proteins p21/WAF1 and MDM2, combined with in situ detection of apoptosis, was studied in specimens of CMV-infected patients as an in vivo model of p53 alteration not due to point mutation. p53 positivity was found in CMV + cells in different tissues, in cells with typical inclusion bodies, and in in situ hybridization and immunohistochemistry CMV + cells without inclusions (hidden infection). Although this p53 reactivity was accompanied by the expression of MDM2 and p21/WAF1 proteins, the patterns of MDM2 and p21/WAF1 protein expression were mutually exclusive, and were associated with the presence or absence of inclusion bodies. Nuclei bearing inclusion bodies were usually MDM2 +, p21/WAF1?, while hidden infected cells were usually MDM2?, p21/WAF1 +. Apoptosis was not detected in any tissue section from CMV-infected patients. Two alternative patterns were found in CMV-infected tissues: p53 +, p21/WAF1 +, MDM2?, or p53 +, p21/WAF1?, MDM2 + protein expression. These may represent examples of p53 dependent alternative effects in the course of CMV infection. Early stages are represented by CMV + cells without inclusion bodies, which display p53 and p21/WAF1 expression, suggesting that p53 could be acting as a growth suppressor protein. Late CMV infection is represented by cells harbouring inclusion bodies. These cells showed a p53 +, p21/WAF1?, MDM2 + profile, consistent with MDM2 mediated p53 inactivation. The absence of p21/WAF1 expression and lack of apoptosis suggest that the p53 protein expressed by MDM2 + cells could be functionally inactivated in CMV-infected cells with inclusion bodies. Previous studies have suggested that p53 inactivation by MDM2 over-expression occurs in sarcomas and lymphomas. Our observations seem to indicate that this mechanism of MDM2 mediated p53 inactivation may play a role in the late phase of CMV infection.     

Activation of p53 tumor suppressor by hepatitis C virus core protein.

In addition to being a structural protein that packages the viral genomic RNA, hepatitis C virus (HCV) core protein possesses regulatory functions. In this report, we demonstrate that the HCV core protein could enhance the gene transactivation activity of the tumor suppressor p53, regardless of whether p53 was derived from an exogenous or an endogenous gene. The activation of p53 by the HCV core protein was supported by the observation that the HCV core protein could enhance the expression of p21(waf1/Cip1), a downstream effector gene of p53, in a p53-dependent manner. Further studies indicated that the HCV core protein could also suppress hepatocellular growth via p53. The HCV core protein and p53 could bind to each other in vitro, which was evidenced by the coimmunoprecipitation, the GST pull-down, and the Far-Western blot assays. The deletion-mapping analysis indicated that the carboxy-terminal sequence of p53 located between amino acids 366 and 380 was required for the core protein binding. These results raised the possibility that the HCV core protein might activate p53 through direct physical interaction. The persistent perturbation of p53 activity by the HCV core protein during chronic infection may have important consequences in HCV pathogenesis.