【作者】 张亚飞；

【导师】 樊代明； 时永全；

【作者基本信息】 第四军医大学 ， 内科学， 2009， 博士

【摘要】 p53的失活是肿瘤转化过程中最常见的事件之一。人类大部分肿瘤的发生和发展同p53的变异和缺失密切相关。p53在人类肿瘤中突变率很高，平均达到50％；尽管如此，仍有约50％的肿瘤表达野生型p53（wt-p53），其中多数因细胞内降解代谢或下游信号通路的改变而致p53正常功能缺失。MDM2在调节p53浓度及活性中起关键作用。在正常细胞中，MDM2通过对p53蛋白持续而又快速的降解使p53活性保持在一个适当的水平，这是生物体维持正常生长的一种平衡调控手段。MDM2本身是p53的转录靶基因，因而两者构成负反馈环路，调节p53的稳定性。但在一些细胞中，由于基因扩增或其它原因导致MDM2水平的升高，从而导致对p53产生构成性的、非正常的抑制作用，并且不可逆地促使其降解，使p53对细胞正常的调控平衡被破坏，造成细胞生长失控，因而变得具有肿瘤细胞的特性。将p53从被MDM2的抑制中释放出来或阻断MDM2与其它基础转录因子的相互作用以恢复p53的正常功能，即可抑制癌的发生或发展。已有的研究显示，核糖体蛋白L23（RPL23）是细胞内调节MDM2-p53反馈环的一个重要分子。通过与MDM2的直接结合，RPL23抑制MDM2对p53的泛素化和降解而间接促使wt-p53功能的活化，进而诱导p53依赖的细胞周期阻滞和/或凋亡。此外，研究显示外源性表达的RPL23在胞核和胞浆内均可与MDM2结合，提示RPL23蛋白可以复合物的形式将MDM2阻滞于胞核或胞浆，进而间接抑制MDM2与p53的结合及对p53的泛素化和降解。基于此，我们设想以RPL23基因为靶点的肿瘤基因治疗是一可行策略。目的：探讨腺病毒介导的RPL23基因转染是否可通过对MDM2-p53相互作用的抑制诱导胃癌细胞内源性wt-p53蛋白的积聚活化并进而抑制其生长。方法：1、利用免疫组织化学染色的方法观察p53和MDM2在胃癌组织中的表达情况；2、构建rAd5-RPL23重组腺病毒，并利用Western Blot、免疫沉淀及免疫荧光染色等方法鉴定其对胃癌细胞MDM2-p53反馈环的调节；3、应用Ad-RPL23重组腺病毒转染wt-p53型胃癌细胞系AGS和MKN45，观察其对胃癌细胞周期和/或凋亡的影响，并进行裸鼠体内成瘤实验，进一步验证Ad-RPL23重组腺病毒对p53野生型胃癌的生长抑制作用。结果：1、p53和MDM2在胃癌组织中的表达及意义我们利用免疫组化染色检测了p53和MDM2在94例胃癌原发灶中的表达。结果显示：p53（突变型）的检出率为36％（34/94），此外，在这些可以检出突变型p53蛋白表达的胃癌原发灶中并非所有癌细胞均是p53阳性表达，其阳性率差异比较大，从10％-99％，均数约30％，提示在胃癌原发灶中有更多的癌细胞为p53野生型；在62．8％（59/94）的胃癌原发灶中可以检测到MDM2表达，定位于胞浆和胞核，并且呈异质性表达；在我们的研究中亦发现胃癌淋巴结转移灶MDM2表达阳性率显著高于原发灶，提示MDM2的水平增高可能与胃癌细胞的转移密切相关。2、Ad-RPL23重组腺病毒的构建及其对MDM2-p53反馈环的调节本研究首先利用武汉晶赛公司Adenovirus Expression System重组腺病毒构建系统成功构建了rAd5-RPL23（Ad-RPL23），随后利用Western Blot、免疫共沉淀和免疫荧光等方法证实在胃癌细胞系MKN45中外源性RPL23蛋白可与内源性MDM2蛋白相互结合并抑制其E3泛素酶活性，进而诱导wt-p53蛋白的积聚活化。进一步实验，我们证实Ad-RPL23诱导的p53活化源于其对MDM2降解p53过程的抑制。3、Ad-RPL23转染对胃癌细胞细胞周期和/或凋亡的影响Ad-RPL23转染后，p53野生型人胃癌细胞系MKN45和AGS出现G₁-S期阻滞，Western Blot结果显示，Ad-RPL23转染的MKN45和AGS细胞p53蛋白表达水平与MDM2蛋白表达水平具有一致性，显示外源性RPL23表达可抑制MDM2-p53相互作用并引起p53积聚活化，进而诱导p21的表达。此外，在MKN45细胞中，Ad-RPL23转染可以诱导明显的细胞凋亡，Western Blot结果显示，作为p53转录的靶基因，Bax和PUMA，两个已知的凋亡相关分子，表达均明显上调。在p53突变型胃癌细胞系MGC803中，Ad-RPL23转染对细胞周期及凋亡未有影响，显示外源性的RPL23是通过p53途径发挥其抑癌效应的。裸鼠体内成瘤实验进一步证实了上述结果。总之，本实验提供的数据表明，在野生型p53的胃癌细胞系中，外源性RPL23对MDM2-p53相互作用的抑制可以诱导p53的积聚活化，并进而诱导相应的细胞周期阻滞和/或凋亡。结论：外源性RPL23诱导的p53积聚活化是一种胃癌治疗的新策略。更多还原

【Abstract】 Functional integrity of the p53 gene is an essential cellular defense againstneoplastic transformation. In fact, nearly all of the different kinds of humanmalignancies analyzed thus far were shown to contain mutations of the p53 geneor alternations in the p53 regulating pathway. Mutations in the p53 gene arefound in about 50% of all human tumors. Even so, half of human tumors retainwild-type p53, which are thought to have inadequate p53 function due toabnormalities in p53 regulation or defective signaling in the p53 pathway.Murine double minute 2 （MDM2）, is an oncogene which encodes a proteinbest characterized for its role in the inactivation and degradation of wt p53. Innormal cells, steady-state levels of wt p53 are maintained at very low levels bycontinuous degradation by MDM2, which is essential for cells to hold p53function in check during normal development. The MDM2 gene itself is a downstream target of p53, thus forming a tight autoregulatory feedback loop.Consistent with this notion, gene amplification and over-expression of MDM2have been shown in a variety of tumors, which results in the inactivation of wtp53 protein, with an effect similar to that of mutations in the p53 gene.Interfering with the MDM2-p53 feedback loop leads to p53 （wt） activation,ultimately preventing neoplasia. Ribosomal proteins L23 （RPL23） was reportedto be able to inhibit MDM2-mediated p53 ubiquitination through direct bindingto MDM2, and subsequently induce p53 levels as well as its activity. In addition,RPL23 could interact with MDM2 in both the nucleus and the cytoplasm,which meant MDM2 was retained in the cytoplasm and the nucleus as acomplex, and this complex formation represented one more mechanism bywhich RPL23 indirectly inhibited the MDM2-p53 binding. Based on the knownmolecular properties of the RPL23 protein, we proposed a rationale for usingRPL23 as a target for cancer gene therapy.Objective:To investigate whether by disrupting the interaction between MDM2 andp53, adenovirus-mediated delivery of human ribosomal protein L23 （Ad-RPL23）stabilizes p53 protein, specifically activates the p53 pathway, and has in vitroand in vivo antitumor activity against gastric cancer harboring wt-p53.Methods:1. The expression of p53 and MDM2 in gastric cancer was determined byimmunohistochemical staining; 2. Adenovirus expressing a his-tagged humanribosomal protein L23 （Ad-RPL23） was constructed, and immunoblotting,immunoprecipitation, and immunofluorescence staining were applied to verifythe inhibition of MDM2-p53 interaction by Ad-RPL23; 3. Using an in vitrosystem with cultured gastric cancer MKN45 and AGS cells carrying wt-p53, the effects of Ad-RPL23 transfection on cell cycle and apoptosis were investigated,and studies using nude mice were applied further to ascertain the antitumoreffect of Ad-RPL23 in vivo.Results:1. Expression of p53 and MDM2 in gastric cancer94 primary gastric adenocarcinomas were assessed for MDM2 and p53expression using immunohistochemistry. It was found that p53 protein （mt-p53）was detected in 36% （34/94） of primary gastric cancer, and the percentage ofp53-positive cells was highly variable ranging from 10% to 99% （median, 30%）,indicating that more cancer cells in gastric cancer express wt-p53; MDM2protein was located in the cytoplasm and nucleus of gastric cancer cells, and thepositive rate of MDM2 expression in primary gastric cancer was 62.8% （59/94）.In addition, immunohistochemical staining for MDM2 revealed that expressionof MDM2 in lymph node metastasis was significantly higher than that inprimary gastric focus, indicating that overexpression of MDM2 were related tometastasis of gastric cancer.2. Construction and verification of Ad-RPL23Adenoviruses expressing a his-tagged human ribosomal protein L23 （Ad-RPL23）was constructed using the adenovirus expression system of WuhanGenesil Biotechnology Co., Ltd. Consequently, by immunoblotting,immunoprecipitation, and immunofluorescence staining, we showed that p53was accumulated in gastric cancer MKN45 cells through disruption of theinteraction between MDM2 and p53 by adenovirus delivered L23. Further, weprovided evidence that the accumulation of p53 induced by Ad-RPL23 waslargely attributable to decreased degradation of the protein.3. The growth inhibitory effect of Ad-RPL23 on in vitro and in vivo human gastric cancer cell linesIn MKN45 and AGS cells harboring wt-p53, inhibition of MDM2-p53interaction by Ad-RPL23 stabilized p53 and thus resulted in substantial cellcycle arrest at G₁-S checkpoint, which was mediated largely by up-regulation ofp21, a transcriptional target of p53. We also showed that in MKN45 cells withwt-p53, inhibition of MDM2-p53 interaction by Ad-RPL23 resulted insignificant apoptotic cell death, which was associated with up-regulation of twowell-established transcriptional targets of p53, the proapoptotic proteins Bax andPUMA. Of note, no effects of Ad-RPL23 on cell cycle progression or apoptosiswere found in MGC803 cells with mt-p53, indicating the specificity ofexogenous RPL23 in activation of the p53 pathway. Encouraging results werealso obtained in the Ad-RPL23 treatment for subcutaneous tumors of MKN45gastric cancer. In conclusion, the data presented in this study verified thatinhibition of MDM2-p53 interaction by Ad-RPL23 could stabilize p53 andinduce cell cycle arrest and apoptosis in gastric cancer cells carrying wt-p53.Conclusion:Exogenous RPL23 induced wt-p53 stabilization and activation may be anovel therapeutic approach for patients with gastric cancer.更多还原