赵　健

论文题目：肝癌细胞中p53基因结构和转录功能的研究&E1B55kDa缺陷型腺病毒对肝癌细胞的杀伤研究

作者简介：赵健，女，1968年03月出生，1996年09月师从于第二军医大学郭亚军教授，于1999年06月获博士学位。

 

 

摘  要

 

p53基因是一种肿瘤抑制基因，通过转录激活细胞周期和细胞凋亡调节基因，而促进DNA损伤的细胞进行修复或发生凋亡，对维持正常组织平衡起重要作用。本研究通过对6株人肝癌细胞株p53基因结构和p53蛋白转录活性的检测，进一步揭示了p53基因结构和功能的相互关系。在此基础上，利用腺病毒早期蛋白与p53蛋白的相互作用，构建了选择性杀伤p53功能异常的肿瘤细胞的缺陷型腺病毒，为肿瘤生物治疗提供了新途径。

一、           p53基因结构和功能的相互关系

用DNA直接测序的方法检测肝癌细胞的p53基因结构，在6株新建系的肝癌细胞中有4株发生p53基因突变，其中2种突变发生在p53-DNA相互作用结构域内，2种在结构域外。将含有p53反式激活元件的报告质粒mdm2-luc导入肝癌细胞，通过检测荧光素酶的表达水平，检测肝癌细胞中p53蛋白的转录活性。结果显示，发生在p53-DNA相互作用结构域以外的基因突变不影响p53蛋白的转录活性，而发生在p53-DNA相互作用结构域之内的基因突变则大大降低p53蛋白的转录活性。研究结果证明了p53是一种DNA结合蛋白，通过与特定的DNA序列结合而发挥其转录调节的功能。

    HBV病毒的HBx等癌蛋白可与p53蛋白结合并破坏p53的功能。基因转染实验结果显示2株p53基因序列正常的肝癌细胞，其p53蛋白的转录激活能力大大降低。免疫原位杂交结果显示，这两株肝癌细胞的p53蛋白表达被阻抑在细胞浆中，不能进入细胞核。结果表明，p53蛋白是一种核蛋白，在细胞核内通过与DNA结合而发挥作用。p53蛋白与病毒癌蛋白（如HBx）的结合，可使p53蛋白被阻抑在细胞浆中，从而降低p53蛋白的转录激活功能。

    上述研究结果表明，p53是一种DNA结合蛋白，p53-DNA相互作用域内的基因突变和p53蛋白阻抑在细胞浆内，均可降低p53蛋白的转录活性，破坏其维持组织平衡的功能。

二、           缺陷型腺病毒对肝癌细胞的杀伤作用

    腺病毒的E1B55kDa蛋白在细胞内与p53蛋白结合，通过抑制p53蛋白的功能而有利于腺病毒在细胞内的复制和繁殖。约50%的肿瘤细胞存在p53基因突变或功能丧失，因此，设想E1B55kDa缺陷型腺病毒只可在p53基因突变或功能缺失的肿瘤细胞内繁殖，而不能在p53基因正常的细胞中繁殖，是一种新型的肿瘤生物治疗手段。

通过对一系列p53基因和功能状态各异的肝癌细胞的病毒杀伤实验，发现缺陷型腺病毒对p53基因突变或缺失同时p53蛋白转录活性降低的肝癌细胞的杀伤作用最明显。进一步的分子杂交和免疫杂交结果显示，腺病毒的细胞毒性作用是通过上调一些细胞凋亡和细胞周期调节相关基因（如Bax-like和p21基因），而诱导细胞凋亡和细胞周期阻滞。并首次发现在发生细胞毒性反应的肝癌细胞中，三种Bax-like细胞凋亡相关蛋白表达水平显著升高，可能直接参与了腺病毒的细胞毒性作用，从分子水平阐明了腺病毒细胞毒性作用的机理。

上述研究结果表明腺病毒的细胞毒性作用是一种潜在的肿瘤生物治疗手段，利用腺病毒早期蛋白与细胞周期和细胞凋亡调节蛋白的相互作用，可以构建选择性杀伤肿瘤细胞的缺陷型腺病毒作为杀伤肿瘤细胞的工具。

 

Abstract

p53, a tumor suppressor gene, induce DNA repairing or apoptosis in DNA damaged cells through activating cell cycle and apoptosis regulate genes at transcription level, and play a important role in maintaining normal tissue homeostasis. In this study, we analyse the relationship between p53 gene structure and p53 function by detecting p53 gene and transcriptional activity status in 6 human hepatocellular carcinoma cell lines. We further construct a attenuated adenovirus that can kill tumor cells with p53 dysfunction selectively, based on the interaction of adenovirus early proteins and p53. This kind of adenovirus provide new strategy for cancer bio-therapy.

1.      p53 gene structure and p53 protein activity

p53 gene mutations were found in 4 out of 6 newly established human hepatocellular carcinoma cell lines by direct DNA sequencing. Two of the mutations were withing p53-DNA interface, and the other two were outside the interface. P53 protein transcription activities were analysed by transfer report plasmids mdm2-luc which could be trans-activated by p53, and p53 transcription activities were evaluated by detecting the expression level of luciferase. Mutations within p53-DNA interface damage p53 transcription activity obviously, whereas, mutations outside p53-DNA interface do not influence p53 activity. This result indicates p53 is a DNA binding protein and execute its transcription regualtion through binding with specific DNA sequence.

P53 transcription activity can also be damage by binding with onco-protein, like HBx antigen in HBV virus. P53 transcription activities were greatly reduced in 2 HCC cell lines containing wild type p53. Immunostaining showed that p53 proteins were blocked in cytoplasm. This result indicates that p53 a nuclear, and plays its role through binding with DNA in nucleus. Blockage of p53 protein in cytoplasm induced by binding with virus onco-protein will damage p53 transcription activity.

Taken together, p53 is a DNA binding protein. Both p53 mutation inside p53-DNA interface and blockage of p53 protein in cytoplasm will inactivate p53 transcription activity.

 

2.      The cytotoxic effect of mutant adenovirus on HCC cell lines

    E1B55kDa protein of adenovirus binds to cellular p53 protein and inhibits its activity to facilitate the replication of adenovirus in host cells. About 50% tumor cells contain p53 gene mutation or p53 dysfunction. Thus we suggest E1B55kDa deleted adenovirus will only replicate in cells with mutant p53 or p53 dysfunction, but not in cells with wild type p53. E1B55kDa delete adenovirus might be a potent tool for cancer bio-therapy.

    HCC cell lines with mutant or delete p53 gene and reduced transcription activity were more susceptible to mutant adenovirus induced cytolysis, after detecting the cytotoxic effect of adenovirus on a serial of HCC cell lines with difficult p53 gene and function status. Further molecular and immune blotting showed that the cytotoxic effect of adenovirus was induced by cell cycle blockage and apoptosis through upregulating genes involved in cell cycle and apoptosis regulation, like p21 and Bax-like genes. We found, for the first time, three Bax-like proteins were greatly induced in cytotoxic HCC cells. These genes might involve in adenovirus induced cytolysis directly. Our results explain the mechanism of adenovirus induced cytotoxic effect on molecular lever.

    Taken together, our results indicate the cytotoxic effect induced by adenovirus could be used to kill tumor cells potentially. Various mutant adenovirus could be constructed on the basis of adenovirus early proteins interference with cell cycle and apoptosis regulation proteins, and used as cancer therapy strategy.