p53-p21/p27通路调节人核糖核苷酸还原酶和吉西他滨抗癌耐药机制的研究
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  • 英文题名:Study on Regulation of Human Ribonucleotide Reductase by p53-p21/p27 Pathway and the Mechanism of Gemcitabine Resistance
  • 作者:薛丽君
  • 论文级别:博士
  • 学科专业名称:生理学
  • 学位年度:2004
  • 导师:金中初
  • 学科代码:071003
  • 学位授予单位:浙江大学
  • 论文提交日期:2004-05-01
摘要
癌症是严重威胁人类生命的常见病和多发病。由于核糖核苷酸还原酶(Ribonucleotide reductase,RR)是催化核苷经脱氧还原为脱氧核苷(dNTP)的专一酶类,而dNTP为DNA损伤修复及复制所需的重要原料,因而RR成为癌症治疗的重要靶点之一,阐明该酶的功能及其调节机制将对肿瘤的发生和防治等研究领域产生重要的影响。
     随着分子生物学技术的发展,RR的调节通路正在不断地揭示,但主要集中在阐明RR单一亚基及其作用的规律,而功能性的RR是由大小亚基组成的α2β2形式,其中任一亚基单独存在均不具备RR活性。另外,已经证明RR的小亚基hRRM2的过度表达与抗癌药吉西他滨(Gemcitabine)等抗肿瘤耐药密切相关,但对其耐药机制的认识还十分肤浅。
     越来越多的研究提示,RR的大小亚基在细胞静息状态下是相互分离的,只有它们彼此特定结合才能行使RR功能。遗传毒作用可激活RR功能,使后者参与受损DNA修复和维持遗传稳定性。另一方面,生物在长期的进化过程中,发展了一套细胞应对遗传毒损伤的保护性机制,即细胞周期检查点(checkpoint)的监控作用。P53是迄今发现的最为重要的检查点蛋白之一,P21基因是P53下游的作用靶子,DNA损伤引起的细胞生长抑制,相当部分是通过P53蛋白介导的P21蛋白来实现的。P21及其同源蛋白P27可与CDKs和Cyclin-CDK复合物
    
    浙江大学博士学位论文
    结合并抑制它们的活性,使细胞周期从G,向S期过渡时受阻停顿,以便修复受损的DNA或
    最终导致细胞的凋亡。P21介导DNA修复和复制的抑制机制之一是通过耗竭dNTP库。最近
    研究又发现P53R2是p53实现DNA修复功能极其重要的执行者之一。本研究预实验初步显示,
    p53R2、hRRMZ两者与p53蛋白存在相互结合。这些研究提示P53检查点通路与核糖核普酸
    还原酶功能紧密关联。
     细胞蛋白质活动场所的变更是调节蛋白质功能的另一重要方式,RR也不例外。关于RR
    代谢相关的dNDP在细胞的哪个部位合成一直来存在争议。二十世纪八十年代,Reddy和
    Pardee等人提出“复制酶体”(replitase)之说,强调RR制造dNDP是在核内进行。随后
    另一研究小组用免疫荧光技术发现,hRRMZ和hRRMI在静息细胞均位于细胞浆,因而又否定
    了“复制酶体”之说。然而,最近的研究发现DNA损伤后RR的另一小亚基 p53R2位于细胞
    核,而与它构成RR全酶的结合伙伴hRRMI在静息细胞位于细胞浆。这些研究暗示在RR活性
    的调节上有可能存在细胞浆与细胞核间RR亚基的浆一核穿梭机制。
    根据以上研究和本研究预实验结果,假设细胞周期检查点蛋白P53等可能通过诱导RR
    亚基的结合和分离或空间位置的改变等转录后调节因素来调变RR的功能。
     此外,基于以下认识:蛋白功能的正常有赖于其编码基因转录、蛋白翻译和蛋白翻译后
    因素的协同配合和整体结合。RR在接受转录后调控的同时,也接受转录的调节。结合文献
    君}预实验提示,作为RR经典的小亚基,hRRMZ转录表达的调节机制尽管已有所认识,但对
    hRRMZ转录表达偶联的吉西他滨(Gemcitabine,Gem)抗癌耐药机制的了解还不够深入和全
    面。Gem为dCTP的类似物,在耐Gem的人肿瘤细胞株,hRRMZ表达比亲代细胞要高得多。所
    以hRRMZ的转录表达增高在Gem肿瘤耐药上可能起着关键性作用。
     因此,本研究采用蛋白免疫沉淀印迹、激光共聚焦、质粒转染、基因微阵分析、凝胶迁
    移和超迁移试验等现代分子生物学技术,从蛋白质间交互作用角度,观察了RR四聚体形成
    及其形成时空间场所变更的规律,以及p53、p21和p27对RR四聚体形成的调节影响。同时,
    还深入研究了抗癌药Gem与hRRMZ转录相关的耐药机制。本研究旨在深入阐明,RR全酶调
    变及其相关通路的分子机制和抗癌药Gem耐药的分子机制,并为RR酶作为癌症防治分子生
    物靶提供科学依据和新的思路。
    
    浙江大学博士学位论文
    本研究结果的创新性
    首次发现p53与RR亚基进行蛋白间交互作用的新型调节规律。静息条件下p53与
    p53R2和hRRMZ结合;应答遗传毒损伤时与p53R2和hRRMZ解离,并促使它们结合
    大亚基。刷新了p53对RR的调控仅靠转录诱导RR小亚基p53R2的模式;同时也为
    生命科学后基因组时代研究蛋白质之间相互作用关系提供新的思路与启示。
    首次提出并证实RR亚基通过细胞浆与细胞核间浆一核穿梭作用参与DNA修复的机
    制;问答了RR研究史上久而未决的关于dNDP在细胞内何处生成的定位问题。
    首次观察到在细胞对DNA损伤的反应上p21/p27下调RR活性,结合p53对RR的调
    节作用,提出p21/p27与p53有可能相互拮抗调节RR四聚体的形成,及其通过精
    细地调节dNTP库以确保DNA修复和复制时遗传稳定的理论假设。这为p53和
    p21/p27蛋白抑制细胞增殖一癌变的发生诊释了新的含义。
    首次阐明了hRRMZ近端启动子上CC从T盒经由NF一Y激活的转录上调可能与吉西他
    滨抗癌耐药有关。该机制的阐明将为克服吉西他滨及其类似物抗癌耐药提供新思
    路。
    第一部分:
    野生型p53通过与p53R2和hR朋2进行蛋白一蛋白间的交互作用调节人核糖核普酸还原酶的
    功能
     核糖核普酸还原酶(RR)是人体唯一负责催化核糖核昔(NDP)为脱氧核糖核营(dNDP)的
    酶,为DNA的修复和复制提?
Cancer is a common disease that hazards human health. Since ribonucleotide reductase (RR) is the only enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides (dNDP), which are precursors for DNA repair and replication, RR has become one of the most important targets for cancer therapy. Clarifying RR function and its regulation mechanism will take an important impact on research fields of carcinogenesis, cancer prevention and cancer therapy.
    With development of molecular biology, more and more RR regulation pathways have been revealed. However, most of studies focus on elucidating regulatory rules of one subunit of RR. Now we know, the functional RR holoenzyme (form: a2p2) consists of two large subunits (hRRMl) and two small subunits (hRRM2 and p53R2). No RR activity will be shown with any subunit alone. Furthermore, overexpression of human ribonucleotide reductase subunit M2 (HRRM2) has been shown to well related to Gemcitabine resistance, but the regulatory mechanisms of functional RR holoenzyme and Gemcitabine resistance are still not clear.
    Lots of studies indicate that RR subunits are separated with each other at quiescent cells. There is no RR function until large and small subunits specifically bind together. Genotoxins activate RR, which would involve in DNA repair and maintain genomic fidelity. On the other
    
    
    hand, in response to genotoxins, eukaryotes activate pathways of cell cycle checkpoint to prevent genomic instability. So far, it is known that p53 is one of the most important checkpoint proteins, and p21 is down stream target of p53. Following DNA damage, quite a number of inhibitions of cell growth are induced by p53-dependent p21 activation. Both p21 and p27 interact with, and inhibit, cyclin-cdk complexes, resulting in retarding S-phase entry from G1 for repairing damaged DNA or initiating apoptosis. One mechanism identified for inhibition of DNA repair or replication by p21 is depletion of dNTP pools. Recently, one research group found that p53R2 is one of the most important participators to carry out p53's repair function in the damage response. Our pilot study showed that RR subunits p53R2 and hRRM2 bind with p53 protein. These results indicate p53 check-point pathway correlates tightly with RR function.
    Like other proteins, one of the important ways to regulate RR function is to alter RR subunits' localizations. In the field of RR research, there are two opposing opinions about where deoxyribonucleotide synthesis. On the 20th century eighty years, Reddy and Pardee et al. brought forward "replitase" model, suggesting deoxyribonucleotides are synthesized by ribonucleotide reductase in nucleus. However, subsequent immunocytochemical studies of both hRRMl and hRRM2 showing localization in the cytoplasm of mammalian cells led other groups to argue against the "replitase" model. Additional evidence has recently shown that the hRRM2 homologue, p53R2, was found in nucleus after DNA damage, but that p53R2' s binding partner hRRMl remained in the cytoplasm of resting cells. These findings indicated that the RR subunits might traffic between cytoplasm and nucleus in response to DNA damage.
    Based on above studies, we hypothesized that checkpoint proteins such as p53, p21 and p27 might post-transcriptionally regulate RR through inducing RR subunits binding and separating with each other or altering their cellular localization.
    Moreover, it is well known that cooperation between gene transcription, protein translation and modification after translation is required for one protein to display normal function. RR is subject to not only post-transcriptional regulation, but also transcriptional regulation. As a classic small subunit of RR, hRRM2 has been well studied and some transcriptional mechanisms have been recognized. However, the mechanism of hRRM2 transcription related to Gemcitabine
    
    
    resistance is still not clear. Gemcitabine is a nucleoside dCTP analogue. The hRRM2 expression level has been reported to be much higher in selected Gemcitabin
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