Mutational analysis of Noxa gene in human cancers

There has been mounting evidence that dysregulation of apoptosis is involved in the mechanisms of cancer development and somatic mutations of apoptosis-related genes have been reported in human cancers. Noxa, a Bcl-2 homology 3 (BH3)-only member of the Bcl-2 family, is known to interact with anti-apoptotic Bcl-2 family members and induces apoptosis. The aim of this study was to explore the possibility that the Noxa gene is mutated in human cancers. We have analyzed the entire coding region and all splice sites of the Noxa gene for the detection of somatic mutations in a series of human cancers, including carcinomas from stomach, colon, liver, urinary bladder and lung by polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP), and DNA sequencing. We found one somatic mutation of the Noxa gene in a transitional cell carcinoma (TCC) of the urinary bladder. To evaluate the functional alterations of the mutant in apoptosis, we overexpressed the mutant and wild-type Noxa in 293T and HeLa cells, but could not find any significant difference in cell death between the wild-type and mutant Noxa. These data suggest that Noxa is rarely mutated in human carcinomas and that the contribution of Noxa gene mutation in the pathogenesis of human cancer might not be related to cell death mechanisms.     

气管插管后气囊上液与呼吸机相关性肺炎的病原学相关性分析

目的:探讨气管插管后气囊上液与呼吸机相关性肺炎(ventilator-associated pneumonia,VAP)的病原学相关关系。方法:我院2003年2月~2006年7月行呼吸机治疗者中127例发生VAP,均用气囊封闭气管,对每例气囊上液与下呼吸道分泌物采样行细菌培养,分析病原学结果。结果:气囊上液与下呼吸道分泌物病原菌株呈正相关,差异具有非常显著性意义(P<0.01)。结论:气囊上液细菌感染是引发气管插管患者VAP的主要原因之一,在呼吸机辅助呼吸治疗中,重视清除气囊上液,可防止VAP的发生。     

Higher expression of SIRT1 induced resistance of esophageal squamous cell carcinoma cells to cisplatin

Background

High expression of Sirtuin type 1 (SIRT1) exists in some cancer cells. However, it is still unclear whether SIRT1 affects the sensitivity of esophageal cancer cells to cisplatin. This study was designed to explore the relationship between SIRT1 expression and resistance of esophageal squamous cell carcinoma (ESCC) cells to cisplatin and reveal the underlying mechanism.

Methods

The tissue samples of 68 ESCC patients were collected from Nanjing Drum Tower Hospital, China. All the patients had undergone cisplatin based combination chemotherapy. The expression of SIRT1and Noxa in tissue samples were analyzed by quantitative real-time reverse PCR (qRT-PCR) and Western blot. Human ESCC cell line (ECa9706 cells) was cultured and a cisplatin-resistant subline (ECa9706-CisR cells) was established by continuous exposure to cisplatin at different concentrations. The expression of SIRT1 and Noxa in both cell lines was analyzed by qRT-PCR and Western blot. siRNA technology was utilized to down-regulate the SIRT1 expression in ECa9706-CisR cells. The influence of SIRT1 silence on sensitivity of ECa9706-CisR cells to cisplatin was confirmed using CCK-8 assay and flow cytometry. Furthermore, the level change of Noxa after SIRT1 silence in ECa9706-CisR cells was determined by qRT-PCR and Western blot.

Result

SIRT1 and Noxa expression in chemo-resistant patients was significantly increased and decreased respectively, compared with chemo-sensitive patients. SIRT1 expression in ECa9706-CisR cells was significantly increased with a lower Noxa level, compared with normal ECa9706 cells. Cisplatin 5 µM could cause proliferation inhibition, G2/M phase arrest and apoptosis in ECa9706-CisR cells and these effects could be enhanced dramatically by SIRT1 silencing. Moreover, Noxa expression was increased after treated with SIRT1 siRNA.

Conclusions

Over-expression of SIRT1 may cause resistance of ESCC cells to cisplatin through the mechanism involved with Noxa expression.     

The fourth dimension in immunological space: how the struggle for nutrients selects high-affinity lymphocytes

Lymphocyte activation via the antigen receptor is associated with radical shifts in metabolism and changes in requirements for nutrients and cytokines. Concomitantly, drastic changes occur in the expression of pro-and anti-apoptotic proteins that alter the sensitivity of lymphocytes to limiting concentrations of key survival factors. Antigen affinity is a primary determinant for the capacity of activated lymphocytes to access these vital resources. The shift in metabolic needs and the variable access to key survival factors is used by the immune system to eliminate activated low-affinity cells and to generate an optimal high-affinity response. In this review, we focus on the control of apoptosis regulators in activated lymphocytes by nutrients, cytokines, and costimulation. We propose that the struggle among individual clones that leads to the formation of high-affinity effector cell populations is in effect an 'invisible' fourth signal required for effective immune responses.     

胃癌组织中SIRT1、Noxa的表达及其临床意义

目的探讨SIRT1与Noxa在胃癌组织中的表达水平相关性,分析两者表达与患者临床病理学特征及预后的关系。方法应用实时荧光定量PCR法和免疫组化EnVision两步法检测106例胃癌及癌周正常组织中SIRT1及Noxa的mRNA和蛋白表达,分析两者相关性及其与患者性别、年龄、肿瘤部位、肿瘤大小、浸润深度及转移等临床病理特征的关系。结果实时荧光定量PCR结果显示,胃癌组织中SIRT1 mRNA表达较高,Noxa mRNA表达较低。免疫组化结果显示,SIRT1在胃癌组织中的阳性率高于正常胃组织(79.2%vs 41.5%),Noxa在胃癌组织中的阳性率低于正常胃组织(43.3%vs 71.6%),差异有统计学意义(P0.05)。SIRT1表达与肿瘤分化程度、肿瘤直径、浸润深度、淋巴结和远隔器官转移情况相关(P均0.05),Noxa表达与肿瘤直径、浸润深度、淋巴结和远隔器官转移情况相关(P均0.05)。胃癌组织中SIRT1与Noxa表达呈负相关。SIRT1阳性、Noxa阴性的患者2年生存率较低,其是预后较差的独立危险因素。结论 SIRT1和Noxa在胃癌的发生、发展中可能具有协同作用,联合检测两者的表达有助于判断患者的临床表现和预后。     

bcl-2家族BH3-only蛋白在依托泊苷诱导胃腺癌细胞凋亡中的作用

目的研究bcl-2家族BH3-only蛋白Noxa、Bim和PUMA在依托泊苷诱导胃腺癌细胞凋亡中的作用。方法Annexin V-FITC染色,流式细胞仪(FCM)检测细胞凋亡率,半定量RT-PCR检测细胞中BH3-only蛋白的mRNA表达量。结果依托泊苷诱导胃腺癌细胞凋亡具有剂量、时间依赖性,SGC-7901细胞系比BGC-823更加敏感,100μM的依托泊苷作用48小时后SGC-7901的凋亡率达到50%,而BGC-823的凋亡率只有20%。BH3-only蛋白Noxa的mRNA表达明显上调,Bim少量上调与依托泊苷诱导的细胞凋亡有关系;相反,PUMA的mRNA表达量在依托泊苷治疗前后没有变化。结论依托泊苷诱导的胃腺癌细胞凋亡与BH3-only蛋白Noxa和Bim表达上调有关联。     

电针治疗血管性痴呆对海马CAl区Noxa、Caspase-3表达的影响

为探讨电针对血管性痴呆（Vascular dementia．VD）大鼠的学习记忆能力的影响及其作用的分子机制。采用双侧颈总动脉永久性结扎法制作VD大鼠模型，Morro水迷宫检测大鼠平均逃避潜伏期和搜索策略，HE染色观察海马的病理变化，免疫荧光染色观测海马神经元细胞Noxa、Caspase-3的表达。结果：电针治疗改善VD大鼠的学习记忆能力．减轻VD大鼠海马病理损害，减少VD大鼠的海马CAl区Noxa、Caspase-3表达阳性细胞数（P〈0．01）。说明电针治疗改善海马神经元的凋亡状况．从而促进智能恢复．