【作者】 宋珊珊；

【导师】 贺福初；

【作者基本信息】 北京协和医学院 ， 遗传学， 2012， 博士

【摘要】 抑癌基因p53是人类肿瘤中最常见的突变基因之一,大约在50%肿瘤中可以检测到p53的突变。p53蛋白作为序列特异性转录因子,能通过调节大量靶基因的表达介导不同的下游功能,参与细胞周期阻滞、凋亡、老化和DNA损伤修复等诸多细胞事件。在非刺激条件下,p53通过快速的蛋白酶体途径降解维持极低的表达水平。当细胞受到刺激时,p53表达被稳定并从抑制状态释放。这种活化主要通过两种方式实现：(1)翻译后修饰；(2)与不同蛋白发生相互作用。在以往的研究中,已经发现部分蛋白在不同的条件下,通过与p53发生相互作用从而选择性调控p53下游靶基因的表达。但是,由p53介导的细胞周期负调控机制并不清楚。为了寻找p53新的调控分子,我们利用高通量的蛋白芯片筛选新的p53相互作用蛋白。发现MPG (3-methyladenine glycosylase)能在肿瘤细胞中直接与p53相互作用并选择性抑制p53介导的细胞周期阻滞。MPG是DNA碱基切除修复途径(base excision repair, BER)中的关键限速酶。当细胞在自身代谢和受到外界损伤性刺激,产生碱基损伤时,MPG能识别多种损伤碱基,与其结合、切除,产生AP位点,启动BER,维持基因组的稳定性。在肿瘤治疗中常用的烷化剂产生的DNA损伤是MPG识别的主要底物。我们的研究发现,MPG的N端34-79位氨基酸(该区域与MPG的糖苷酶活性无关)能与野生型p53的DNA结合区直接结合,特异性抑制p53介导的细胞周期阻滞,但对p53介导的细胞凋亡没有影响。在p53野生型的肿瘤细胞中,给予烷化剂刺激,能特异的使MPG与p53相互作用解离,MPG对p53的抑制作用消失,转而主要发挥损伤碱基的修复功能,从而降低这些肿瘤细胞对烷化剂的敏感性。相反,在p53突变型的肿瘤细胞中,烷化剂造成的损伤难以完全修复,对细胞的杀伤效应高于p53野生型细胞。因此,MPG通过与p53的结合和解离,协调了自身糖苷酶活性依赖和非糖苷酶活性在DNA损伤修复中的作用。在乳腺癌,肺癌和结肠癌中,MPG在癌组织中的阳性率分别为38.7%,43.4%和25.3%,在所有癌旁组织中MPG表达均为阴性。提示在肿瘤中,p53和MPG的状态共同决定了肿瘤细胞对烷化剂的敏感性。综上所述,本论文首次发现了一个DNA损伤修复酶MPG作为p53选择性调控因子的直接证据,并阐述了其调控的具体机制。这一研究为进一步探讨MPG与p53在肿瘤治疗中的功能联系提供了新的启示。更多还原

【Abstract】 p53is one of the most studied tumor suppressors in the cancer research field. Extensive mutation searches demonstrated that over50%of human tumors carry p53mutations. It is widely accepted that p53is a sequence-specific transcription factor and plays a pivotal role in the regulation of cell cycle progression, apoptosis and DNA repair in response to diverse stress signals. In unstressed conditions, p53is kept at an extremely low level due to rapid proteasomal degradation. When under stress, however, p53is stabilized and released from suppression. There are at least two ways in which p53is activated:posttranslational modifications and interactions with various proteins. A few proteins have been identified that associate with p53to selectively regulate the expression of p53downstream targets. In unstressed conditions, the regulation of p53-mediated cell cycle arrest, particularly the negative regulation, is still not fully understood.To search for novel regulators of p53, we used high-density protein microarrays. Among the novel potential p53-interacting proteins, we found that N-methylpurine DNA glycosylase (MPG, also called AAG or ANPG), the first identified enzyme in the base excision repair (BER) pathway, can bind to p53and selectively repress p53-mediated cell cycle arrest in cancer cells under unstressed situations.As a core enzyme in BER, MPG has been shown to recognize and excise a broad range of modified bases, in addition to normal bases, in DNA. The removal of bases leaves repair intermediates, abasic (apurinic/apyrimidic) AP sites, which are cytotoxic and mutagenic, which makes it apparent that the removal of these BER intermediates is crucial. Alkylating agents have been frequently used in the treatment of human cancers.The major DNA damage produced by alkylating agents is recognized and repaired by MPG.Our current findings show that the MPG N-terminus plays an important role for p53binding and regulation. MPG specifically inhibits p53-mediated cell cycle arrest but not apoptosis. In response to alkylation damage, in p53wild-type tumor cells, MPG dissociated from p53, resulting in the release of p53and cell cycle arrest to repair damaged bases. Then, high MPG combination with wild-type p53in certain tumor cells led to insensitivity to alkylating agents. By contrast, in p53-mutated cells, the AP sites were repaired with low efficacy and the killing effects were higher than the p53wild-type cells. Therefore, MPG coordinates its glycosylase and non-glycosylase modules to participate in the DNA damage repair. Also, the p53status coordinates with MPG to play a pivotal role in determination of cancer sensitivity to alkylating drugs. To our knowledge, this is the first direct evidence to show that a DNA repair enzyme functions as a selective regulator of p53, and these findings provide new insights into functional linkage between MPG and p53in the cancer therapy.更多还原