ASPP2对p53家族成员诱导凋亡功能的调控作用及其在肿瘤治疗中的意义

ASPP2（p53凋亡激活蛋白2，Apoptosis Stimulating Proteins of p53 2）作为p53家族共同转录激活辅因子能和p53野生型、p63和p73结合，并促进其对下游促凋亡靶基因的转录，促进细胞凋亡。研究表明ASPP2参与细胞生长，凋亡以及损伤应激等一系列的生理反应，对研究肿瘤发生和治疗具有重要意义。     

P53调节蛋白ASPPs的分子生物学特性及其与肿瘤的关系

ASPPs(含有富含脯氨酸结构域、锚蛋白重复区及SH3结构域的蛋白)是一类新发现的凋亡调节蛋白,它们能够特异性地调节P53蛋白家族的凋亡活性。ASPPs由ASPP1、ASPP2和iASPP3个同源性很高的成员组成,其中ASPP1、ASPP2可正向调节P53活性,而iASPP的功能恰好与之相反;越来越多的研究表明,此类蛋白功能多样,可参与多条细胞信号通路。ASPPs的异常调控与肿瘤发生、发展密切相关,有望成为肿瘤治疗的新靶点。     

ASPP2对P53家族的调控及其在肿瘤治疗中的意义

P53凋亡激活蛋白2(apoptosis stimulating proteins of P53 2,ASPP2)作为P53家族共同转录激活辅因子能和p53野生型、p63和p73结合,并促进其对下游促凋亡靶基因的转录,促进细胞凋亡.研究表明ASPP2参与细胞生长,凋亡以及损伤应激等一系列的生理反应,对研究肿瘤发生和治疗具有重要意义.     

TP53BP2/ASPP2通过p53非依赖性方式激活mTOR通路抑制HepG2人肝癌细胞自噬

目的研究肿瘤蛋白p53结合蛋白2/p53凋亡刺激蛋白2(TP53BP2/ASPP2)对HepG2人肝癌细胞自噬的调节作用和机制。方法通过腺病毒、慢病毒感染HepG2细胞上调或下调细胞中ASPP2的表达, HepG2细胞继续在不含胎牛血清的培养基中培养24 h,通过Western blot法检测自噬相关蛋白微管相关蛋白1轻链3(LC3)、 beclin1、 P62、自噬相关基因5(ATG5)、 ATG7和哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白mTOR、磷酸化的mTOR(p-mTOR)、真核转录起始因子4E结合蛋白1(4EBP1)、磷酸化的4EBP1(p-4EBP1)、核糖体蛋白S6、磷酸化的S6(p-S6)、核糖体S6激酶B1(RPS6KB1/p70S6K)、磷酸化的p70S6K(p-p70S6K)的蛋白表达,荧光显微镜检测细胞表达的绿色荧光蛋白-微管相关蛋白1轻链3(GFP-LC3)融合蛋白。同时,使用慢病毒感染不表达p53的Hep3B细胞,敲低细胞中的ASPP2水平, Western blot法检测Hep3B细胞ASPP2和mTOR通路相关蛋白mTOR、 p-mTOR、 4EBP1、 p-4EBP1、 S6、 p-S6、 RPS6KB1/p70S6K、 p-p70S6K的蛋白表达。结果当ASPP2的表达上调时, mTOR复合物1(mTORC1)通路被激活,并且自噬相关蛋白的表达和自噬体的数量减少。在下调ASPP2表达后, mTORC1通路受到抑制,自噬相关蛋白的表达水平和自噬体的数量增加。在p53表达沉默的Hep3B细胞中,当ASPP2下调后, mTORC1途径仍然受到抑制。结论 ASPP2以p53非依赖性方式激活mTOR通路抑制HepG2人肝癌细胞自噬。     

PUMA的研究进展

PUMA(p53 up-regulated modulator of apoptosis)是近年发现的可被内外源性p53快速诱导、具有强大促凋亡作用的Bcl-2蛋白家族一员。它通过BH3结构域与其他Bcl-2家族蛋白相互作用,诱导细胞色素c释放,引起凋亡。PUMA引起的凋亡有两种(p53依赖的和p53不依赖的)调控通路,已有研究表明PUMA敲除可显著抑制细胞凋亡。对于该基因的调控机制、作用机制及在肿瘤治疗中的应用等方面的问题,正待深入研究。     

PUMA的研究进展

PUMA(p53 up-regulated modulator of apoptosis)是近年发现的可被内外源性p53快速诱导、具有强大促凋亡作用的Bcl-2蛋白家族一员。它通过BH3结构域与其他Bcl-2家族蛋白相互作用，诱导细胞色素c释放，引起凋亡。PUMA引起的凋亡有两种(p53依赖的和p53不依赖的)调控通路，已有研究表明PUMA敲除可显著抑制细胞凋亡。对于该基因的调控机制、作用机制及在肿瘤治疗中的应用等方面的问题，正待深入研究。     

非小细胞肺癌中ASPP1和ASPP2基因启动子甲基化的意义

目的 检测非小细胞肺癌(NSCLC)中ASPP1和ASPP2基因启动子CpG岛的甲基化状态和相应的蛋白表达,及癌细胞的凋亡水平和p53蛋白的表达,探讨ASPP基因在NSCLC中的作用及其临床意义.方法 采用甲基化特异性PCR(MSP)检测90例NSCLC组织、25例癌旁肺组织中ASPP1、ASPP2基因启动子的甲基化水平,并测序证实其甲基化位点;免疫组织化学EnVision法检测NSCLC组织中ASPP1、ASPP2和p53蛋白的表达;原位末端转移酶标记检测法(TUNEL)检测癌细胞凋亡,计算其凋亡指数(AI).结果 ASPP1基因启动子甲基化率在NSCLC中为42.2%(38/90),癌旁组织中为16.0%(4/25),二者比较差异有统计学意义(P=0.019);ASPP1基因启动子甲基化与患者年龄、性别、组织类型和分化程度无相关性(均P＞0.05),但与患者的TNM分期和淋巴结转移存在相关性(P=0.031,P=0.030);90例肺癌组织中ASPP1甲基化者其蛋白表达率明显低于ASPP1未甲基化者(P=0.002);ASPP1蛋白阳性组的AI值高于阴性组(P=0.022).90例NSCLC和25例癌旁组织中均未检测到ASPP2基因启动子甲基化;ASPP2蛋白表达阳性组和阴性组之间AI值差异也无统计学意义(P=0.282).ASPP1和p53表达呈负相关,(r=-0.259,P＜0.01),ASPP2和p53的表达状态无关(r=-0.119,P＞0.05).结论 NSCLC中ASPP1基因启动子甲基化可能是ASPP1蛋白表达下调的原因,高甲基化可能与NSCLC的恶性进展有关,ASPP1蛋白的表达能促进细胞凋亡.

Abstract：

Objective To investigate the methylation status of CpG islands in the promoter region and the protein expression of ASPP1 and ASPP2 genes in non-small-cell lung cancer (NSCLC) and their relationship with cellular apoptosis and p53 gene expression. Methods The 5'CpG island methylation patterns of ASPP1 and ASPP2 were evaluated by methylation specific polymerase chain reaction (MSP) followed by confirmation of sequencing. Immunohistochemistry was used to detect the expression of ASPP1,ASPP2 and p53 in lung carcinoma tissue samples ( n= 90) and adjacent non-neoplastic lung tissue samples (n = 25 ) . TUNEL assay was used to detect the apoptotic activity. Results The presence of ASPP1 methylation was significantly higher in NSCLC than that in the adjacent non-neoplastic lung tissue [42. 2%(38/90) vs. 16. 0% (4/25), P =0. 019]. ASPP1 promoter methylation had a close relationship with TNM stage and lymph node metastasis( P =0. 031, P =0. 030), but was not related to the age, sex, histological types and the grades of tumor differentiation (P =0. 389, P =0. 278, P =0. 570, and P =0. 103). Tumors with ASPP1 promoter methylation demonstrated a lower expression of ASPP1 as compared with those without the methylation (P =0. 002). ASPP1 expression was associated with a higher apoptotic index (AI) (P =0. 022) and a decreased p53 expression( r = -0. 259, P ＜ 0. 01 ). Methylation in the promoter region of ASPP2 gene was not detected in lung cancer (n = 90 ) or adjacent non-neoplastic lung tissue (n = 25 ).Expression of ASPP2 protein did not correlate with AI ( P = 0. 282 ) and p53 status in NSCLC. Conclusions High methylation of ASPP1 gene promoter regions is one of the important mechanisms that down-regulate its protein expression in NSCLC. ASPP1 promoter methylation may be associated with the malignant progression of the tumor, and ASPP1 expression promotes cellular apoptosis.     

Puma与肿瘤的相关性研究

 摘 要 p53上调节的细胞凋亡调控因子(p53 up-regulated modulator of apoptosis, Puma)是Bcl-2蛋白家族的促凋亡成员之一,具有显著的促凋亡作用。研究表明,Puma可能与抑制肿瘤发生有关,并可通过多种方式诱导肿瘤细胞凋亡。在肿瘤治疗方面,Puma表达增高有利于化疗,其表达缺失有利于减轻放疗后副作用。近年来,Puma与肿瘤细胞凋亡及肿瘤治疗的相关研究成为热点,本文就Puma与肿瘤的相关性研究进行综述。     

Noxa在凋亡中的作用及机制

Noxa是内源性途径凋亡中Bc l-2家族促凋亡的BH3-on ly亚家族成员之一,在诱导细胞凋亡中起重要作用。它通过定位于线粒体,引起线粒体释放细胞色素C以形成凋亡体,启动caspase级联反应而诱导细胞发生凋亡。Noxa的表达主要受p53调控,但是多种刺激因子可以通过p53或者不依赖p53等多种途径诱导Noxa表达上调,最后由Noxa来诱导细胞发生凋亡。     

细胞自噬的调控与自噬程序性死亡的研究进展

自噬(autophagy)是一种溶酶体依赖性降解途径,涉及细胞内长寿蛋白和受损伤细胞器的降解,其既是细胞保守的自我防御机制,又是一种程序性细胞死亡机制(PCD),与机体的多种疾病有密切关系.自噬具有独特的形态改变和特有的调控通路,自噬的调控涉及到多种机制、如翻译后修饰等.凋亡是研究最清楚的程序性细胞死亡机制,凋亡与细胞自噬程序性死亡之间存在着复杂的关系.对哺乳动物细胞自噬的分子调控机制,自噬程序性细胞死亡过程及其与凋亡的关系等方面进行探讨很有意义.     

The Lats2 tumor suppressor augments p53-mediated apoptosis by promoting the nuclear proapoptotic function of ASPP1

Apoptosis is an important mechanism to eliminate potentially tumorigenic cells. The tumor suppressor p53 plays a pivotal role in this process. Many tumors harbor mutant p53, but others evade its tumor-suppressive effects by altering the expression of proteins that regulate the p53 pathway. ASPP1 (apoptosis-stimulating protein of p53-1) is a key mediator of the nuclear p53 apoptotic response. Under basal conditions, ASPP1 is cytoplasmic. We report that, in response to oncogenic stress, the tumor suppressor Lats2 (large tumor suppressor 2) phosphorylates ASPP1 and drives its translocation into the nucleus. Together, Lats2 and ASPP1 shunt p53 to proapoptotic promoters and promote the death of polyploid cells. These effects are overridden by the Yap1 (Yes-associated protein 1) oncoprotein, which disrupts Lats2-ASPP1 binding and antagonizes the tumor-suppressing function of the Lats2/ASPP1/p53 axis.     

ASPP2 is a haploinsufficient tumor suppressor that cooperates with p53 to suppress tumor growth

ASPP2 stimulates the apoptotic function of the p53 family in vivo. We show here that ASPP2-/- pups died before weaning. This postnatal lethality was significantly enhanced in p53+/- background and both deletions are synthetic lethal. ASPP2+/- mice developed spontaneous tumors. The tumor onset was accelerated by gamma-irradiation or in p53+/- background. Tumors derived from ASPP2+/- mice retained wild-type ASPP2 allele even though some of them lost p53. These provide the first genetic evidence that ASPP2 is a haploinsufficient tumor suppressor that shares overlapping function(s) with p53 in mouse development and tumor suppression.     

Helicobacter pylori infection and expressions of apoptosis-related proteins p53, ASPP2 and iASPP in gastric cancer and precancerous lesions

Aims

There has been limited information about the relations between Helicobacter pylori infection and expressions of apoptosis-related proteins p53, ASPP and iASPP in gastric cancer and precancerous lesions.

Methods

H. pylori in gastric mucosa were identified by W-S staining and rapid urease test. Expression of apoptosis-related proteins P53, ASPP2 and iASPP in the gastric tissues were determined by immunohistochemistry.

Results

The concentrations of H. pylori and expressions of p53 and iASPP in gastric carcinoma group and precancerous lesion group were higher than in benign gastric diseases group (P < 0.05). The expressions of ASPP2 in gastric carcinoma and precancerous lesion group were lower than in benign gastric diseases group (P < 0.05). The expressions of p53 and iASPP in H. pylori positive group were higher than in H. pylori negative group (P < 0.05), whereas ASPP2 in H. pylori positive group were lower than in H. pylori negative group (P < 0.05).

Conclusion

There was a higher rate H. pylori infection, an increased expression of apoptosis inhibitor iASPP, and decreased expression of apoptosis stimulator ASPP2 in gastric cancer or precancerous tissues. These results suggest that H. pylori may cause gastric cancer by up-regulating iASPP and down-regulating ASPP2.     

肿瘤中INK4a/ARF和ASPP基因异常甲基化的研究进展

人类许多肿瘤的发生与P53功能失活有关,其中约有50%具有野生型P53,此种情况下,P53抑癌功能的丧失是由INK4a、ARF、ASPP等抑癌基因的异常引起,5′CpG岛异常甲基化是INK4a/ARF功能失活的主要途径,也是ASPP失活的方式之一。逆转DNA异常甲基化有望重新激活这些抑癌基因的表达,为肿瘤的治疗提供新途径。