【作者】 赵兰君；

【导师】 罗瑛；

【作者基本信息】 昆明理工大学 ， 环境生物学， 2013， 博士

【摘要】 肿瘤是严重威胁人类生命与健康的一类疾病。p53作为重要的抑癌基因,它是人类肿瘤中最常发生突变的基因之一,大约有半数肿瘤都有p53的突变。有趣的是,p53并不像其他大部分抑癌基因一样发生缺失或基因的截短,它的74%的突变都是点突变；在这些点突变中,97%发生在p53的DNA结合域。但是,研究发现,不同突变位点的p53突变蛋白在功能缺失和癌基因功能获得方面都有很大差异。因此,研究清楚不同p53突变蛋白的功能可以帮助我们更透彻地了解p53在肿瘤发生发展中的作用。在前期研究中,我们在几株ALT肿瘤细胞株中发现了同一个p53突变基因p53N236S(在人类中是p53N239S)的高表达,并且在体外实验中发现它以功能获得的形式具有癌基因潜能并与H-RasG12V协同促进肿瘤形成。根据IARC p53数据库,p53N239S的突变频率约为0.16%。虽然不是热点突变,但是它广泛存在于各类肿瘤中,在人类肿瘤的发生发展中发挥极其重要的作用。在国家自然基金的资助下,我们建立了p53N236S敲入小鼠模型。本文研究的一个方向就是探明p53N236S敲入小鼠中突变p53S在体内的功能缺失和功能获得。对p53S敲入小鼠的研究发现：p53S敲入小鼠中p53S突变蛋白不但丧失了调控细胞周期抑制和细胞凋亡的功能,而且还能促进肿瘤的转移,获得了新的癌基因功能。本研究从小鼠整体的存活情况,肿瘤生成情况和p53S敲入小鼠中p53S的功能两方面来展开。1)我们统计分析了p53S小鼠的生存曲线,肿瘤发生率,肿瘤谱和肿瘤转移率,并与p53敲除小鼠模型相比较。研究发现：①p53s/s小鼠与p53/-小鼠的生存曲线并无明显差异。同p53/小鼠一样,p53s/s小鼠在大约三个月左右就会罹患肿瘤,到十个月时,小鼠几乎全部死亡。在所解剖的40例p53s/s小鼠中,只有两例未发现明显的肿瘤。这提示我们,p53S已经丧失了调控细胞周期抑制和细胞凋亡的功能,不能抑制肿瘤的发生。另外,p53s/+小鼠的存活曲线与p53+/-小鼠的存活曲线也无明显差异。因为野生型p53的存在,大大延缓了小鼠的发病时间,延长了存活寿命。②通过对比p53S小鼠和p53敲除小鼠罹患的肿瘤类型,我们发现,p53S小鼠和p53敲除小鼠罹患的肿瘤都以淋巴瘤和肉瘤为主,并无明显差异。但是,与p53敲除小鼠不同的是,在p53s/+罹患的肉瘤中,我们发现约有40%的肿瘤存在转移,转移率明显高于p53+/-小鼠。并且在所检测的16例肿瘤中,只有两例发现有LOH。这提示我们,p53S可以在小鼠体内促进肿瘤的转移,获得了新的癌基因特性。有趣的是,之前研究者对p53热点突变R172H敲入小鼠分析发现：虽然R172H小鼠与p53敲除小鼠的存活曲线和肿瘤谱差异不大,但是R172H杂合小鼠的骨肉瘤等较易发生转移。这提示我们,虽然不是肿瘤突变热点,p53S小鼠与热点突变R172H小鼠的肿瘤生成情况有很强的相似性。鉴于以上结果,可能需要我们重新审视p53的非突变热点在肿瘤发生过程中的重要性。2)我们利用p53S小鼠和p53S MEF细胞研究分析了p53S的功能,结果发现：①在体内实验中,我们对p53s/s小鼠进行电离辐射,然后分析小鼠胸腺组织细胞凋亡和周期抑制相关蛋白的诱导表达情况。实时荧光定量PCR的结果显示：p53眺小鼠已经丧失了转录调控p21,PUMA等p53下游分子的能力。同时,在体外实验中,对p53s/s MEF细胞用阿霉素处理,然后分析细胞凋亡和周期抑制相关蛋白的诱导表达情况。Western blot的结果显示p53s/s MEF细胞也丧失了转录调控p21蛋白的能力,验证了体内实验的结果。这提示我们,p53S丧失了调控细胞周期抑制和细胞凋亡的能力。②在体内实验中,我们对p53s/+小鼠进行电离辐射,结果发现,p53s/+小鼠还具有调控p21,PUMA等p53下游分子的能力。同时,在体外实验中,对p53s/+MEF细胞用阿霉素处理,结果发现,p53s/+MEF细胞也还具有调控p21蛋白的能力。这提示我们,p53S杂合子中仍具有野生型p53的功能,在辐射或阿霉素导致的DNA损伤应激中,p53S并没有通过负显性抑制来影响其中野生型p53的功能。③我们对p53S MEF细胞和p53S小鼠肿瘤组织中的蛋白表达情况进行分析。结果显示：在p53s/+的MEF细胞中并未见p53蛋白的累积。但是p53蛋白在p53s/+小鼠的肿瘤组织中大量累积,与之前报道的李氏综合征病人的肿瘤组织中的情况是一样的。④我们通过划痕实验和transwell实验对p53S MEF细胞的迁移能力进行了分析。结果发现,与野生型的MEF细胞细胞相比,p53s/+和p53s/sMEF细胞的迁移能力增强。总结我们的实验结果,并结合之前对p53的研究结果发现：p53的突变与肿瘤的发生、发展和预后有着密切的关系。由于突变p53在肿瘤细胞中通常有较高表达,从而成为区别于正常细胞的一个特异性抗肿瘤靶点。在肿瘤细胞中重新激活野生型p53,使突变p53恢复野生型p53的功能；或通过无毒性天然药物靶向降解突变p53,降低突变p53促进肿瘤形成的作用,将是未来肿瘤治疗中的一个重要方向。针对突变p53的药物开发已经取得了一些成果,有一些药物已经进入了临床实验阶段。但是更多的天然植物资源等待我们去开发和研究,以期获得更多的疗效显著,毒副作用小的新药物。利用云南省特有的资源普洱茶,我们选择遗传背景清楚的,带有p53S的肿瘤细胞为筛选体系,并利用野生型MEF细胞来排除非特异的细胞毒性,研究普洱茶靶向调控突变p53S的抗肿瘤功效。对普洱茶抗肿瘤的研究发现：普洱茶可以通过下调突变p53S来抑制肿瘤细胞的生长。本研究主要得到以下发现：1)普洱茶在不影响野生型MEF细胞的浓度下,可以明显的抑制携带p53突变的肿瘤细胞系SCID22-3B-1, p53-/-+S+Ras的生长,对不携带p53突变的肿瘤细胞p53-/-+Ras的生长也有一定的抑制作用。2)为了进一步研究普洱茶对肿瘤细胞或野生型细胞生长的影响,我们通过流式细胞术分析了加茶前后野生型MEF细胞,p53-/-+Ras,p53-/-+S+Ras肿瘤细胞株的细胞周期。结果发现,p53-/-+Ras加普洱茶处理后,处于S期的细胞明显减少,处于G2期的细胞明显增多,发生了G2期阻滞。有意思的是,p53-/-+S+Ras加普洱茶处理后,处于S期的细胞明显减少,处于G1期的细胞明显增多,发生了G1期阻滞。但是同样浓度的普洱茶对野生型MEF细胞的细胞周期并无明显影响。3)为了研究普洱茶对肿瘤细胞生长抑制的分子机理,检测普洱茶是否靶向调控突变p53S。我们通过Western blot分析了加茶前后p53-/-+Ras,p53-/-+S+Ras细胞中p53等相关蛋白的表达情况。结果发现,p53-/-+S+Ras加茶处理后p53蛋白和HSP90蛋白的表达量降低,而p53-/-+Ras加茶处理后HSP90未见明显变化。这提示我们,普洱茶可能通过靶向调控突变p53S来抑制肿瘤细胞的生长。为了进一步验证这个结论,我们又通过Western blot检测了加茶前后p53s/s,p53s/s+Ras细胞中p53等蛋白的表达情况。结果发现,p53s/s,p53s/s+Ras细胞加茶处理后p53蛋白和HSP90蛋白的表达量均降低,验证了普洱茶靶向调控突变p53S这一观点。同时,我们也用Western blot和qRT-PCR检测了加茶前后野生型细胞中相应基因和蛋白表达。结果发现,普洱茶并未引起野生型细胞中p53的应激表达。有意思的是,普洱茶也降低了野生型细胞中HSP90的表达。4)那么,普洱茶是在转录水平还是在蛋白降解水平调控突变p53S的呢?为了解答这个问题,首先我们通过qR-T-PCR检测了加茶前后p53-/-+S+Ras细胞中p53mRNA的水平。结果发现,p53-/-+S+Ras细胞加茶后p53mRNA的水平降低。进一步,我们用蛋白酶体抑制剂MG132和普洱茶共同处理p53-/-+S+Ras细胞,通过Western blot检测处理前后细胞中p53蛋白的表达情况。结果发现,用MG132处理细胞可以部分抑制普洱茶对突变p53S的降低。这提示我们,普洱茶在转录水平和蛋白降解水平共同调控突变p53S的表达。5)普洱茶是一种成分复杂的复合物,我们期待分离纯化普洱茶抗肿瘤活性的有效部位或成分。首先我们通过四种方法初步分离出16种组分进行活性测定。通过组分对p53-/-+S+Ras细胞的作用分析,我们发现其中的茶组分B4,D1对细胞的杀伤效果与普洱茶相当。有趣的是,通过不同方法分离出来的B4和D1化学性质相似,表明我们的活性跟踪方法是可靠的。对活性部位的进一步的纯化分离和活性鉴定正在进行中。综上所述,在小鼠体内,p53N236S不但丧失了调控细胞周期抑制和细胞凋亡的功能,而且还能促进小鼠体内肿瘤的转移,获得了新的癌基因活性。而且普洱茶在不影响野生型细胞的前提下,通过靶向降低p53N236S来抑制肿瘤细胞的生长。对p53N236S敲入小鼠和普洱茶的研究不但为靶向调控突变p53的个性化治疗提供了理论依据,而且还为普洱茶作为低毒、高效、廉价的抗肿瘤剂的应用提供了线索。更多还原

【Abstract】 Cancer is a serious threat to human life and health. And p53is the most commonly mutated tumor suppressor in human cancers with a mutation frequency about50%. Unlike most tumor suppressor genes, which typically undergo biallelic inactivation during carcinogenesis by deletions or truncating mutations, TP53is frequently (74%) inactivated by a single monoallelic missense mutation. And about97%of missense mutations are mapped to the DNA binding domain. However, it has been shown that p53mutant proteins resulted from different site of p53point mutations vary dramatically in terms of their loss of function and gain of function. Thus, understanding of the function of different p53mutants will help us to better understand the role of p53in tumorigenesis.In our previous studies, a p53mutant p53N236S(p53N239S in human, p53S) was found highly expressed in several ALT tumor cell lines. And our previous in vitro studies indicated that the gain of function of p53S by cooperating with H-RasG12V and explained the oncogenic effect of p53mutant p53S in promoting tumorigenesis. According to the IARC p53database, p53N239S is widely present in various types of human tumors and plays an important role in the development of cancer. Supported by NSFC grants, we established the p53N236S knock-in mouse model. The first part of this thesis is to study the loss and gain of function of p53S in p53S knock-in mouse.1, During the study of p53S knock-in mouse, we have observed that p53S not only lost transcriptional regulatory function in both cell cycle arrest and apoptotic pathways, but also gained new function in promoting tumor metastasis in vivo. Tumor development, survival of p53S knock-in mice and the function of p53S in p53S knock-in mice have been analyzed.1) we analyzed the survival curves, the incidence of tumor, tumor spectrum and tumor metastasis comparing p53knock out and p53S knock-in mice.①It was found that the survival curves comparing p53-/-and p53s/s mice were identical. Like p53-/-mice, p53s/s mice were highly susceptible to onset spontaneous tumor formation when they were three months old. By ten months, all p53s/s mice had died or had to be sacrificed due to tumorigenesis. Necropsy revealed obvious tumors in38of40p53s/s mice. These results suggested that p53S lost the regulatory function in both cell cycle arrest and apoptotic pathways, thus lost the function of inhibiting tumorigenesis. Besides, the survival curves of p53s/+and p53+/-mice also mostly overlapped and showed no statistical difference. Due to the presence of wild type p53,p53s/+mice have a greatly delayed rate of tumorigenesis compared with p53s/s mice.②In addition, the most common tumor types seen in p53knock out and p53S knock-in mice were lymphomas and sarcomas. The comparison of p53mutant (p53S) and null alleles showed no differences in the kinds of tumors that these mice developed. However, the tumor spectrum of p53s/+mice showed one difference. That is, sarcomas that developed in p53s/+mice frequently metastasized(40%). In contrast,metastasis is rare in tumors of p53+/-mice.Loss of heterozygosity studies of tumors indicated loss of heterozygosity in only2of16tumors.These data suggested that p53S gained new function in promoting tumor metastasis in vivo.Interestingly, in previous studies,it was found that although p53+/172H mice displayed a similar tumor spectrum and survival curve as p53+/-mice,tumors from/p53+/172H metastasized with high frequency. Our data showed that the in vivo tumorigenesis was very similar in p53N236S and p53R172H mice.We might need to re-evaluate the role of low frequency mutation in the progress of human tumors.2) using p53S knock-in mice and p53S MEF cells, the function of p53S was analyzed.①For in vivo experiments, p53s/s mice were exposure to irradiation, the RNA was extracted from their thymus and real time PCR were performed to detect the activation of cell cycle arrest and apoptosis pathways after irradiation. We found that p53S lost its function in regulating its downstream factors, like p21and PUMA in vivo. In vitro, the protein was extracted from p53S MEF after treatment with doxorubicin and Western blot were performed. Like in vivo experiments, p53S also was found lost its regulatory function for p21protein in vitro.The results showed that p53S lost its regulatory function in both cell cycle arrest and apoptotic pathways.②Similar experiments were performed in p53s/+mice after exposure to irradiation. We found that p53protein from p53s/+mice still had the capacity to regulate its downstream factors, like p21and PUMA. The protein was extracted from p53s/+MEF after treatment with doxorubicin and Western blot were performed. Like in vivo experiments,p53protein from p53s/+MEFs also retained its regulatory function for p21protein. These results indicated that p53S did not act in a dominant negative manner to interfere with the function of the wild type p53allele.③Further more, Western blot was performed to detect the protein expression in p53S MEF and tumor samples derived from p53S mice. It was found that mutant p53protein was accumulated in tumor samples derived from p53s/+mice, but not p53s/+MEF cells.④Finally, wound healing assay and transwell assay were performed to test the invasive power of p53S MEF. It was revealed that p53s/+and p53s/s MEF showed increased invasive properties comparing with wild type MEF.Previous studies and our data showed that the p53mutants were closely related to the occurrence, development and prognosis of human cancer. As mutant p53proteins were frequently accumulated in tumor cells, which was different from nomal cells.It became a specific target for anti-cancer strategy. Elevating wild type p53activity, rescuing mutant p53protein to the wild type conformation or down-regulating mutant p53with non-toxic natural chemicals will be an important focus in cancer therapy.A few compounds that targeted p53in tumors had already advanced to clinical investigation. In order to search for more novel anti-cancer drugs with high activities and low side effects, we needed to screen more natural botanic sources for leading compounds with anti-cancer activity. By using Pu-erh tea produced in the Yunnan province and several mouse tumor cell lines with mutant p53expressed and clear genetic background, we tried to find the anti-tumor action of Pu-erh tea by targeting mutant p53.In addition,we used the wild type MEFs as a control to test toxicity of drugs.2, Pu-erh tea inhibited tumor cell growth by down-regulating mutant p53. The main results of this study were listed as follows:1)Pu-erh tea could effectively inhibit the growth of the SCID22-3B-1and p53-/-+S+Ras tumor cells. And Pu-erh tea could also inhibit cell growth of the p53-/-+Ras tumor cells, but not as dramatically as the p53-/-+S+Ras tumor cells. But the same concentration of Pu-erh tea did not affect the growth of the wild type cells. 2)To further understand the action of tumor cell or wild type cell growth by Pu-erh tea, flow cytometry was performed to analyze the cell cycle regulation in p53-/-+Ras, p53-/-+S+Ras tumor cells and wild type cells after Pu-erh tea treatment. The results showed that in Pu-erh tea treated p53-’-+Ras cells, the proportion of cells in S phase was significantly decreased and that in G2phase was significantly increased, suggesting a G2arrest induced by Pu-erh tea treatment in p53-/-+Ras cells. Interestingly, in p53-/-+S+Ras cells, Pu-erh tea caused a significant decrease of cells in S phase and an increase of cells in G1phase, suggesting a G1arrest. No obvious cell cycle arrest were observed in wild type cells at this concentration.3) To understand the molecular basis of Pu-erh tea action on tumor cell cycle arrest and to test whether mutant p53in the molecular target for Pu-erh tea action. Western blot was performed to check the protein expression in p53-/-+Ras and p53-/-+S+Ras tumor cells after Pu-erh tea treatment. Our data showed that mutant p53and HSP90protein level were down-regulated in p53-/-+S+Ras tumor cells after Pu-erh tea treatment. But in p53-/-+Ras tumor cells, we did not observe obvious change of HSP90after Pu-erh tea treatment. The results implied that Pu-erh tea might inhibit tumor cell growth by targeting mutant p53.To further confirm the Pu-erh tea action on p53S,Western blot was also performed to check the protein expression in p53s/s+Ras and p53s/s cells after Pu-erh tea treatment. It was found that the p53S and HSP90protein also decreased in both p53s/s and p53s/s+Ras cells after Pu-erh tea treatment. Besides, our data showed that Pu-erh tea treatment did not up-regulate the expression of wild type p53at both mRNA and protein levels. Interestingly, HSP90protein levels decrease in the wild type cells.4) So, Pu-erh tea down-regulated mutant p53by inhibiting the p53transcription or promoting degradation of p53? To answer this question, real-time PCR was performed to detect p53mRNA in p53-/-+S+Ras cells after Pu-erh tea treatment first. It was found that mutant p53mRNA level decreased after Pu-erh tea treatment. Moreover, Western blot was performed to detect the p53protein level after Pu-erh tea and MG132treatment. Our data showed that MG132could rescue the down-regulating of mutant p53by Pu-erh tea. The results implied that Pu-erh tea down-regulated mutant p53both by inhibiting the p53transcription and promoting degradation of p53. 5) As Pu-erh tea is a compound with complex components, we aim to purify the active fractions or compounds in Pu-erh tea.16components were initially purified through four methods and the anti-tumor action of16components on p53-/-+S+Ras tumor cells were analyzed. It was found that anti-tumor action comparing components B4, D1and Pu-erh tea were identical. Interestingly, components B4and D1separated by different methods displayed similar chemical property, suggesting that our way for following active fraction is reliable. The further isolation of fractions with anti-tumor activity is still going on.In conclusion, p53N236S not only lost transcriptional regulatory function in both cell cycle arrest and apoptotic pathways, but also gained new function in promoting tumor metastasis in vivo. Pu-erh tea inhibited tumor cell growth by down-regulating mutant p53N236S without affecting wild type cells. Our studies not only provide the basis for personalized treatment aiming different p53mutants, but also shed light on the application of Pu-erh tea as an anti-tumor agent with low side effects.更多还原