抑癌基因p53在特定高温下作用研究

Tumor repressor p53 is activated in response to DNA damage stresses. Activated p53 triggers cell cycle arrest in cells with less DNA damage to repair damage and apoptosis in cells with severe DNA damage. Thus it ensures genomic stability. p53 is mutated in more than 50% of human cancer cells. Mutations in p53 not only predispose an organism to cancer, but also lead these tumors to respond poorly to radio- or chemo-therapy. .There are three main types of programmed cell death including: apoptosis, programmed necrosis and autophagy. Autophagy is a cellular process that involves degradation and recycling long-lived proteins and organelles, in lysosomes. It is a cell survival mechanism to maintain homeostasis, quality control, defense against intra- and extracellular insults, and the cellular energetic balance. If autophagic activity is insufficient, cells will accumulate long-lived proteins and defective organelles which finally leads cell apoptosis. However, if the proteins and organelles destroyed by autophagy are beyond a certain threshold, autophagic cell death will occur. Degradation of proteins and organelles by lysosomes can be achieved by at least three different mechanisms: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). CMA is responsible for the degradation of a subset of cytosolic proteins carrying a consensus motif in their amino acid sequence, similar to KFERQ, in a stress condition such as nutrient deprivation, however, little is known about the consequence of excessive CMA activation. .p53 plays a dual role in the process of macroautophagy, nuclear p53 promotes macroautophagy by regulating gene expression, whereas cytoplasmic p53 suppresses macroautophagy. However, whether p53 plays a role in CMA is unclear. .It was known that p53 is also activated in response to heat shock stress. To investigate what role p53 plays in high temperature conditions, we incubated zebrafish p53M214K mutant and wild type (WT) embryos at a high temperature for continuously growing. Very interestingly, we found that WT embryos (still with about 45% viability at 28 hour post treatment hpt for instance) were much more tolerance to the high temperature than p53M214K mutant embryos (with 100% mortality at 28 hpt) at the high temperature (unpublished). The results were further confirmed in human cell lines(unpublished data). The aims of this project are as follow:.1. To identify in what mechanisms for p53 to promote cell survival in the high temperature stress;.2. To investigate whether the high temperaturre treatment is able to suppress tumor growth of p53-deficient xenografts by transplantation of human cancer cells in immunocompromised mice..The discoveries of this research will not only be the first one to reveal a new function of p53 to promote cell survival at a high temperature and molecular mechanisms behind of it, but also be highly appropriate to adopt thermotherapy to treat p53-deficient tumors/cancers.

细胞程序死亡主要有三种形式：细胞凋亡、细胞自噬和细胞程序坏死。抑癌基因p53可被DNA损伤激活，诱导损伤的细胞停止分裂或走向凋亡，从而确保遗传物质的稳定性。p53突变不仅容易导致肿瘤发生，而且此种癌细胞对于化疗和放疗极不敏感。p53还会被高温激活，但高温下p53起着怎样作用还未有报道。我们在用特定高温处理野生型和p53突变体斑马鱼胚胎时，惊奇发现野生型在此温度下的成活率远远高于突变体，并且在人类细胞系中获得类似的结果（未发表）。在此项目中我们将：.1）利用斑马鱼和人类细胞系，揭示p53在此高温下通过抑制哪种死亡方式来促进细胞成活及其分子机理；.2）利用人类癌细胞系在裸鼠中移植成瘤体系，探讨高温特异性抑制p53突变的癌细胞生长的作用。.研究结果不但首次揭示高温激活p53起着促进细胞成活的作用及其作用机制，并且为癌细胞中p53突变的患者提供一个崭新的治疗策略。

抑癌基因p53是一个转录因子，在50%人类癌细胞中发生变异，在正常情况下并不活跃，但在DNA损伤、高温等多种胁迫被激活。激活后可以主导大量基因表达和沉默，这些基因不仅与控制细胞周期和诱导细胞凋亡相关，而且有些可直接参与损伤修复，损伤比较轻的细胞，将停止细胞分裂，进行损伤修复，损伤严重的细胞p53将促使它走向凋亡，从而确保机体遗传物质的稳定性，所以p53在损伤反应途径中处在一个非常中心的位置。p53突变不仅容易导致肿瘤发生，而且p53突变的癌细胞对于化疗和放疗极不敏感。.40oC对于大多数生命来说，是一个非常关键的温度，当人体核心温度高于40oC时就会引发中暑，这是由于过热会引起细胞死亡，从而导致中枢神经和器官衰竭。已发表的文献显示p53在41.5oC以上高温下，起着诱导细胞凋亡的作用，但p53在40oC及以下次高温中起什么作用还不得而知。该项报道利用斑马鱼和人的细胞系两种模式体系，研究发现在40oC下，与诱导细胞凋亡功能相反，p53通过抑制过度热激反应来促进细胞存活，并揭示在两种不同高温下p53行使相反功能的分子机制。.在40oC次高温下，细胞启动热激反应，热激反应转录因子Hsf1提高Hsc70和Hsp90的表达，一方面Hsc70通过伴侣蛋白介导的细胞自噬(CMA)，来促进错误折叠或多余蛋白的降解;另一方面，Hsp90通过蛋白互作，但不磷酸化，来提高p53的稳定性，从而激活p53，没有磷酸化的p53与Hsf1启动子结合抑制该基因的表达，降低Hsc70介导的蛋白降解，这样维持细胞稳态，促进细胞存活。如果在此种温度下，p53失去功能，那么就会产生过激的热激反应，就会积累过多的Hsf1和Hsc70蛋白，这样就会造成过多Hsc70介导的蛋白降解，细胞就会死亡；在43oC超高温度下，诱发DNA损伤反应，ATM被激活，ATM通过对p53 S37位丝氨酸磷酸化，来稳定p53蛋白并激活其转录功能，磷酸化p53不与Hsf1的启动子结合，仅与细胞凋亡蛋白Bax基因的启动子结合，促进其表达，诱导细胞凋亡。.此外，小鼠肿瘤移植实验显示，40oC处理能够显著p53-/-肿瘤生长，但对p53+/+细胞没有多大影响，并且这种处理对于小鼠的正常组织也没有明显的影响。这项研究对于p53-/-癌症病人的治疗提供了新思路。