关键词： 肝细胞癌ATF3   MMP-2   maspin   组织芯片  

摘要： 目的:检测ATF3、MMP-2及maspin在肝细胞癌(hepatocellular carcinoma,HCC)组织芯片中的表达情况,探讨ATF3、MMP-2及maspin表达与HCC发生、发展的关系及其意义。方法:对手工制作组织芯片的方法进行改良,构建含HCC、癌旁肝组织、远癌肝组织、正常肝组织及肝内转移灶的组织芯片,应用免疫组织化学和图像分析技术检测ATF3、MMP-2及maspin的相对表达量,并分析其与各临床病理参数的关系及其意义。结果:ATF3及MMP-2在HCC中表达明显高于癌旁肝组织、远癌肝组织及正常肝组织(P<0.01),而maspin在HCC中的表达明显低于癌旁肝组织、远癌肝组织及正常肝组织(P<0.01)。ATF3蛋白在女性HCC患者中的表达量高于男性患者(P<0.01),在转移组中表达高于无转移组(P<0.01);MMP-2蛋白随HCC病理学分级的升高,相对表达量降低(P<0.05),MMP-2蛋白在直径大于3cm的HCC中的相对表迭量高于直径小于3cm的表达量(P<0.05),包膜侵犯组高于无包膜侵犯组(P<0.05),肝内转移灶表达量高于原发灶(P<0.05);ATF3和MMP-2均随着HCC的进展表达量逐渐增高,maspin却随着HCC病理学分级的升高,相对表达量降低(P<0.05)。ATF3蛋白表达与MMP-2表达呈正相关(r_s=0.13,P<0.05),与maspin表达呈负相关(r_s=-0.23,P<0.01);MMP-2蛋白表达与maspm表达呈负相关(r_s=-0.17,P<0.01)。结论:在HCC组织中,ATF3、MMP-2及maspin的表达存在相关关系,ATF3、MMP-2高表达及maspin缺失表达与HCC的发生密切相关,他们可能在HCC发生发展、侵袭转移过程中发挥重要作用。选择性抑制ATF3有可能成为治疗HCC新的靶点。

关键词： 膀胱尿路上皮癌   ATF3   免疫组织化学   RT—PCR  

摘要： 目的探讨膀胱尿路上皮癌中转录因子ATF3的表达及其临床意义。方法采用逆转录聚合酶链反应(RT-PCR)和免疫组织化学方法检测32例膀胱癌组织和14例正常膀胱粘膜中ATF3 mRNA和ATF3蛋白的表达,分析其表达与肿瘤病理分级、临床分期的关系。结果膀胱癌组织中ATF3 mRNA相对表达量稍高于正常膀胱粘膜组织,浸润膀胱癌组织中ATF3 mRNA相对表达量稍高于浅表膀胱癌组织,但差别均无显著性。ATF3蛋白在膀胱癌组织和正常膀胱粘膜中的阳性表达率分别为93.7%(30/32)、21.4%(3/14),差异有显著性。结论ATF3表达参与了膀胱尿路上皮癌的发病过程,进一步研究其功能有可能揭示膀胱癌的发病机制。

关键词： ATF3   shRNA   肾小球系膜细胞  

摘要： 目的:构建针对大鼠ATF3(activating transcription factor3,ATF3)基因的特异性短发卡RNA(small hairpinRNA,sh RNA)真核表达质粒。方法:用DNA重组技术将针对大鼠ATF3基因不同位点所设计的5个shRNA序列克隆到真核表达质粒pGenesil-1中。在酶切分析及序列测定后,用脂质体将5个shRNA分别转染至大鼠肾小球系膜细胞(glomerular mesangial cells,GMC),随后用Western blot检测蛋白的相对表达量来筛选最佳沉默效率的shRNA。结果:限制性酶切及核酸序列分析证明重组质粒构建正确。Western blot证实在重组质粒中,shATF3-1具有最佳的沉默效率。结论:成功构建了针对ATF3基因的shRNA的真核表达质粒,并且该shRNA可以特异性地降低大鼠肾小球系膜细胞ATF3的蛋白表达。

关键词： DNA repair   ATF3   p15(paf)   PCNA   UV  

摘要： After genotoxic stress, normal cells trigger DNA repair or, if unable to repair, undergo apoptosis to eradicate the cells that bear the risk of becoming tumorigenic. Here we show that repression of the transcription factor, activating transcription factor 3 (ATF3), after ultraviolet (UV)-mediated genotoxic stress impairs the DNA repair process. We provide evidence that ATF3 directly regulates the proliferating cell nuclear antigen (PCNA)-associated factor KIAA0101/p15(PAF). We further show that the expressions of ATF3 and p15(PAF) is sufficient to trigger the DNA repair machinery, and that attenuation of their expression alters DNA repair mechanisms. We show that the expression of p15(PAF) compensates for a lack of ATF3 expression, thereby constituting a major effector of ATF3 in the DNA repair process. In addition, we provide evidence that p15(PAF) expression is required for the correct function of PCNA during DNA repair, as prevention of their interaction significantly alters DNA repair mechanisms. Finally, defective DNA repair, because of the downregulation of p15(PAF) expression, rendered the cells more sensitive to UV-induced cell death. Therefore, our results suggest ATF3 and p15(PAF) as novel gatekeepers of genomic integrity after UV exposure.

关键词： Diarylheptanoid   Cell cycle arrest   Apoptosis   p53   ATF3  

摘要： Purpose The aim of the study is to dissect the cytotoxic mechanisms of 1-(4-hydroxy-3-methoxyphenyl)-7-(3,4dihydroxyphenyl)-4E-en-3-heptanone (compound 1) in SH-SY5Y cells and therefore to provide new insight into neuroblastoma chemotherapy. Methods Nine diarylheptanoids were isolated from Alpinia officinarum by chromatography and their cytotoxicity was evaluated by an MTS assay. Flow cytometry, BrdU incorporation assay and fluorescence staining were employed to investigate cytostatic and apoptotic effects induced by the compound 1. In addition, Western blot, qPCR and siRNA techniques were used to elucidate the molecular mechanisms of the cytotoxicity. Results The study to elucidate the cytotoxic mechanisms of compound 1, the most potent diarylheptanoid showed that cell cycle-related proteins, cyclins, CDKs and CDKIs, as well as two main apoptotic related families, caspase and Bcl 2 were involved in S phase arrest and apoptosis in neuroblastoma cell line SH-SY5Y. Furthermore, following the drug treatment, the protein expression of p53, phospho-p53 (Ser20) as well as the p53 transcriptional activated genes ATF3, puma and Apaf-1 were increased dramatically;MDM2 and Aurora A, the two p53 negative regulators were decreased;the p53 protein stability was enhanced, whereas the p53 mRNA expression level slightly decreased and ATF3 mRNA expression apparently increased. In addition, the knockdown of ATF3 gene by siRNA partially suppressed p53, caspase 3, S phase arrest and apoptosis triggered by compound 1. Conclusion These results suggest that compound 1 induces S phase arrest and apoptosis via up regulation of ATF3 and stabilization of p53 in SH-SY5Y cell line. Therefore, compound 1 might be a promising lead structure for neuroblastoma therapy.

摘要： Stress response gene ATF3 plays a pleiotropic role in determining cell fate in response to mitogenic or stress stimuli. An alternate promoter of the human ATF3 gene (designated P1 in this study) has recently been reported, which is located 43.5 kb upstream of the previously reported P2 promoter. We showed here that the P1 promoter is highly conserved between human and mouse and is functional in response to various stimuli, whereas the P1 promoter was dominantly induced by serum and the P2 promoter was more efficiently activated in response to TGF- and oncogenic HRAS. The P1 promoter contains multiple transcriptional start sites, and the different 5-UTRs markedly affected their translation in response to stress. In human prostate and Hodgkin ReedSternberg cancer cells with elevated expression of ATF3, the P1 promoter was constitutively activated and its chromatin structure was modified into active configuration. The differential usage of alternate promoters of the ATF3 gene at both transcriptional and translational level and the modification of chromatin structure may provide a novel mechanism for expressing ATF3 in determining cell fate during stress response and cancer.

摘要： JDP2 is a ubiquitously expressed bZIP repressor protein. JDP2 binds TPA response element and cyclic AMP response element located within various promoters. JDP2 displays a high degree of homology to the immediate early gene ATF3. ATF3 plays a crucial role in the cellular adaptive response to multiple stress insults as well as growth stimuli. We have identified ATF3 as a potential target gene for JDP2 repression. JDP2 regulates the ATF3 promoter potentially through binding to both the consensus ATF/CRE site and a non-consensus ATF3 auto-repression DNA-binding element. Expression of ATF3 protein in wild-type mouse embryo fibroblast (MEF) cells is below the detectable levels, whereas, JDP2 disrupted MEF cells display noticeable level of ATF3 protein. Following either serum or ER stress stimulation, ATF3 expression is potentiated in JDP2-KO fibroblast cells as compared with wild-type cells. Mice with either JDP2 over-expression or JDP2 disruption display undetectable level of ATF3 protein. However, ATF3 induction in response to either growth or stress signals is dependent on JDP2 expression level. ATF3 induction is attenuated in JDP2 over-expressing mice whereas is potentiated in JDP2-KO mice as compared with the corresponding wild-type mice. Collectively, the data presented strongly suggest that JDP2 plays a role in the determination of the ATF3 adaptive cellular threshold response to different stress insults and growth stimuli.