p53与肿瘤治疗

p53是目前发现的与人类肿瘤发病相关性最大的抑癌基因之一。野生型p53参与DNA损伤修复、细胞周期调控、细胞凋亡及抑制血管生成等。p53基因的突变会使上述功能丧失,从而导致肿瘤的形成。随着分子生物学技术的发展,对肿瘤抑制基因p53的研究越来越深入。本文综合近年来国内外的研究进展,就p53与肿瘤形成的关系及其在肿瘤治疗中的应用等作一综述。     

p53与细胞死亡

p53是一种重要的肿瘤抑制因子,是迄今发现与人类肿瘤相关性最高的分子之一。超过50%的人类肿瘤含有p53基因突变。因此,p53是肿瘤治疗中的重要分子靶点。p53依赖的细胞凋亡是其抑制肿瘤的重要机制之一。然而,最近研究发现,p53不仅参与细胞凋亡,还与程序性细胞坏死、细胞自噬以及铁诱导的细胞死亡等细胞死亡途径相关。促使肿瘤细胞死亡是肿瘤治疗的重要目标。因此,进一步了解p53与细胞死亡之间的关系,将有助于探索以p53为靶点的肿瘤治疗和p53相关肿瘤细胞耐药机制。     

靶向突变p53蛋白的抗肿瘤药物

p53蛋白是人体内十分重要的肿瘤抑制因子,通过调节细胞周期阻滞、诱导细胞凋亡等作用发挥肿瘤抑制功能。突变后的p53蛋白不仅具有显性负性效应（dominant negative effect,DN）抑制野生型p53蛋白功能,而且还通过功能获得性效应（gain of function,GOF）调节细胞代谢、侵袭、迁移等方式促进肿瘤的发生。p53蛋白在超过50%的肿瘤组织中发生突变,是肿瘤细胞区别于正常细胞的一个特异性药物靶点。因此,针对突变p53蛋白开发新型抗癌药物一直是研究的热点。长期以来,由于突变p53蛋白表面较为光滑,缺乏药物结合口袋,使其被认为是一个不可成药的靶点。随着高通量筛选技术的发展以及对突变p53蛋白结构的深入了解,许多靶向突变p53蛋白的小分子化合物被报道并在体外展现出较好的抗肿瘤活性,多款基于突变p53蛋白研发的化合物已经进入临床试验阶段。本文就靶向p53蛋白治疗肿瘤的直接和间接策略进行综述,重点针对突变p53蛋白重激活剂与降解突变p53蛋白的小分子化合物作用机制进行梳理,以期为后续开发靶向突变p53蛋白药物的创新提供帮助。     

p53在肿瘤发生过程中的功能研究及进展

p53是迄今为止细胞中最为重要的肿瘤抑制因子之一.p53能够响应细胞内外众多的信号刺激,通过抑制细胞生长、诱导细胞凋亡、促进DNA损伤修复等过程抑制肿瘤的发生发展.近年来发现p53在细胞自噬、细胞代谢,尤其在葡萄糖代谢中也发挥重要作用.p53在超过1/2的人类肿瘤中,发生缺失或突变,提示p53突变和肿瘤发生是紧密相关的.本文将主要结合本研究组近十年来围绕p53所开展的相关研究,从p53自身稳定性的调控机制、p53与肿瘤发生、p53与细胞代谢以及p53调控非编码RNA方面进行简要综述,并展望p53研究的新方向.     

穿心莲内酯恢复突变型p53野生型功能的作用及机制研究

目的 p53是人体内重要的肿瘤抑制因子,但在人类肿瘤中因高频突变而失去抑癌功能。突变型p53（mutant p53,mutp53）可促进肿瘤的发生、发展和转移。由于在肿瘤细胞中通常有较高表达,mutp53已成为区别于正常细胞的一个特异性抗肿瘤靶点。本研究旨在探索穿心莲内酯的抗肿瘤作用机制,为寻找靶向mutp53的抗肿瘤化合物提供理论依据。方法 构建可以快速筛选具有恢复mutp53下游转录因子的荧光素酶系统,观察穿心莲内酯对H1299-p53 R273H-PUMA-luciferase和H1299-p53R175H-PUMA-luciferase细胞中PUMA基因的表达情况;采用免疫荧光实验,检测穿心莲内酯对HT29（R273H）和SK-BR-3（R175H）细胞中mutp53的表达影响;采用免疫印迹实验进一步观察穿心莲内酯恢复了mutp53肿瘤细胞中p53下游靶蛋白PUMA、p21、Noxa的表达;随后采用MTT和流式细胞分析,检测穿心莲内酯对肿瘤细胞增殖和凋亡的影响;此外,还通过siRNA敲低Hsp70表达后,研究穿心莲内酯对mutp53下游基因的重激活作用。结果 穿心莲内酯可以增加H1299-p53 R273H-PUMA-luciferase和H1299-p53R175H-PUMA-luciferase细胞中PUMA基因的表达;穿心莲内酯可以降低HT29（R273H）和SK-BR-3（R175H）细胞中mutp53的比例,同时增加野生型p53的比例;穿心莲内酯恢复了mutp53肿瘤细胞中p53下游靶蛋白PUMA、p21、Noxa的表达,进而抑制肿瘤细胞增殖和诱导凋亡;穿心莲内酯可以增加分子伴侣Hsp70的表达,通过siRNA敲低Hsp70后,穿心莲内酯对mutp53下游基因的重激活作用明显受到抑制。结论 穿心莲内酯可能通过影响Hsp70的表达从而激活突变p53下游靶基因而发挥抗肿瘤作用。     

肿瘤细胞中存活蛋白与p53的相互作用

存活蛋白(survivin)作为凋亡抑制蛋白(IAP)家族的最小成员,在肿瘤组织中高表达,且具有严格的细胞周期依赖性,而p53作为细胞周期中的负调节因子,参与了细胞周期调控和细胞凋亡等重要的生物学功能。最近研究表明,存活蛋白与p53的相互作用在肿瘤的发生发展中具有重要作用。该文将从细胞周期与细胞凋亡的角度对存活蛋白/p53通路在肿瘤中的研究进展进行阐明。     

核糖体蛋白参与调控p53诱导活化的分子机制

核转录因子p53是重要的肿瘤抑制因子,具有DNA损伤修复、促细胞凋亡、促细胞分化及增殖抑制等功能,并通过调控细胞周期行进和促进细胞凋亡发挥肿瘤抑制功能。原癌蛋白MDM2为p53的E3泛素化连接酶,MDM2－p53信号轴的功能异常与多种恶性肿瘤的发生发展相关。核糖体蛋白（RP）是蛋白质合成反应的关键调节蛋白,其功能失常与多种疾病相关。近年来的研究发现,RP能通过调节MDM2－p53信号轴在p53相关性肿瘤调控中发挥重要作用。我们根据目前的研究进展,对RP－MDM2－D53信号轴进行简要综述。     

新的抑癌基因ASPP对p53作用的研究进展

p53是一个肿瘤抑制蛋白,它是通过调节相关基因表达,诱导细胞凋亡。p53诱导细胞凋亡的机制多年来一直不太清楚,而最近发现的ASPP(apoptosis stimulating protein of p53)蛋白家族对p53诱导细胞凋亡的机制研究有了新的进展。本文就此作一综述。     

p53家族新成员：p51

p53是一种广谱的肿瘤抑制基因,其新家族成员p51具有同p53相似的DNA结合特笥和相似的功能,同样可以转录激活p53基因的内源性靶分子,如细胞周期抑制基因p21、导致细胞凋亡和生长受抑。本文阐述了它的研究进展。     

The emerging role of p53 in stem cells

Among the hundreds of oncogenes and tumor suppressors that have been identified in the past 50 years, p53 is probably the best characterized; nevertheless, new functions are constantly being discovered. As a tumor suppressor, p53 regulates cellular responses to different stress stimuli by inducing reversible cell cycle arrest and DNA repair, or triggering senescence or apoptosis. Recent findings on the regulation of stem cell (SC) division and reprogramming suggest the intriguing possibility that p53 also carries out its tumor suppression function by regulating SC homeostasis. Specifically, p53 activation may counteract SC expansion by several emerging mechanisms including restriction of self-renewing divisions, inhibition of symmetric division and block of reprogramming of somatic/progenitor cells into SCs.     

Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.     

Monoclonal antibody analysis of p53 expression in normal and transformed cells.

The cellular phosphoprotein p53 binds tightly and specifically to simian virus 40 T antigen and the 58,000-molecular-weight adenovirus E1b protein. Many human and murine tumor cell lines contain elevated levels of the p53 protein even in the absence of these associated viral proteins. Recently the cloned p53 gene, linked to strong viral promoters, has been shown to complement activated ras genes in transformation of primary rodent cell cultures. Overexpression of the p53 gene alone rescues some primary rodent cell cultures from senescence. We isolated three new monoclonal antibodies to the p53 protein, designated PAb242, PAb246, and PAb248, and mapped the epitopes they recognized on p53 in comparison with other previously isolated antibodies. At least five sterically separate epitopes were defined on murine p53. One of the antibodies, PAb246, recognizes an epitope on p53 that is unstable in the absence of bound simian virus 40 T antigen. This effect is demonstrable in vivo and in newly developed in vitro assays of T-p53 complex formation. Using the panel of anti-p53 antibodies and sensitive immunocytochemical methods, we found that p53 has a predominantly nuclear location in established but not transformed cells as well as in the vast majority of transformed cell lines. Several monoclonal antibodies to p53 showed cross-reactions with non-p53 components in immunocytochemical staining.     

Expression of wild-type p53 is not compatible with continued growth of p53-negative tumor cells.

Inactivation of the cellular p53 gene is a common feature of Friend virus-induced murine erythroleukemia cell lines and may represent a necessary step in the progression of this disease. As well, frequent loss or mutation of p53 alleles in diverse human tumors is consistent with the view of p53 as a tumor suppressor gene. To examine the significance of p53 gene inactivation in tumorigenesis, we have attempted to express transfected wild-type p53 in three p53-negative tumor cell lines: murine DP16-1 Friend erythroleukemia cells, human K562 cells, and SKOV-3 cells. We found that aberrant p53 proteins, which differ from wild-type p53 by a single amino acid substitution, were expressed stably in these cells, whereas wild-type p53 expression was not tolerated. The inability of p53-negative tumor cell lines to support long-term expression of wild-type p53 protein is consistent with the view that p53 is a tumor suppressor gene.     

VEGF expression and its reguration by p53 gene transfection in endometrial carcinoma cells

Vascular endothelial growth factor (VEGF) that activates endothelial cell growth induces angiogenesis, which is indispensable to tumor igenesis and tumor progression. On the other hand, tumor suppressor gene p53 has been considered to regulate VEGF expression, but the detailed relationship between them remains unclear. In this study, we aimed to study VEGF expression in endometrial carcinoma cells and the effect of p53 gene transfection on VEGF expression using p53-mutated endometrial carcinoma cell line, HEC-50B. Immunoblotting for detecting VEGF protein, p53 protein and beta-actin was performed using 11 endometrial carcinoma cell lines. Levels of VEGF in the cultured media were measured by Enzyme immunoassay(EIA). Transfection of wild p53 gene was carried out by SuperFect method in HEC-50B cells, which had mutant p53 gene and did not express p53 protein. The results of immunoblotting were analyzed by NIH image and expressed as values. The results of EIA were expressed as the relative value. The VEGF value was 0.8 +/- 0.3 (n = 6) in p53-wild group, whereas in p53-mutant group it was 1.6 +/- 0.8 (n = 5). VEGF expression was correlated significantly with p53 status (P < 0.05). VEGF levels in p53 gene-transfected cells and the conditioned medium were decreased in 48 hours after p53 gene transfection. VEGF expression was down-regulated by p53 in endometrial carcinoma cells.     

Regulation of embryonic and induced pluripotency by aurora kinase-p53 signaling

Many signals must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. However, the exact molecular regulatory mechanisms remain elusive. To unravel the essential internal and external signals required for sustaining the ESC state, we conducted a short hairpin (sh) RNA screen of 104 ESC-associated phosphoregulators. Depletion of one such molecule, aurora kinase A?(Aurka), resulted in compromised self-renewal and consequent differentiation. By integrating global gene expression and computational analyses, we discovered that loss of Aurka leads to upregulated p53 activity?that triggers ESC differentiation. Specifically, Aurka regulates pluripotency through phosphorylation-mediated inhibition of p53-directed ectodermal and mesodermal gene expression. Phosphorylation of p53 not only impairs p53-induced ESC differentiation but also p53-mediated suppression of iPSC reprogramming. Our studies demonstrate an essential role for Aurka-p53 signaling in the regulation of self-renewal, differentiation, and somatic cell reprogramming.