抗凋亡因子Survivin研究进展

Survivin是在肿瘤组织及胚胎中发现的一类细胞因子,它是IAPs(inhibitorsofapoptosisprotein)家族的成员之一,具有其独特的分子结构和组织表达特异性,在细胞中参与细胞周期的调控,主要在细胞周期的G2/M期通过抑制caspase-3及caspase-7的活性发挥作用.Survivin在细胞中的活性可能受p53的调节.Survivin也是胚胎发育早期过程中调节细胞分裂分化的一类重要的因子.对Survivin的研究对于肿瘤治疗的研究及揭示胚胎早期的发育机制有重要的意义.     

M期启动调节的普遍机制

细胞周期内发生的事件是细胞正常繁殖所必需的。对于所有的细胞周期来说,有两个事件是主要的:S期和M期。前者是染色体复制时期,后者是复制了的染色体分离并进入两个子细胞的时期。本文讨论细胞周期中M期启动的调控。据现在所知,所有的真核细胞存在一个共同的调控机制,其中心是蛋白激酶P34~(cdc2),它在有丝分裂及减数分裂的M期都被活化。在活化时,该激酶的磷酸化状态需要发生改变,并要和周期素(cyclin)相互作用。周期素是一类在细胞周期过程中水平发生变化的蛋白质。P34~(cdc2)被认为可以     

肝胆肿瘤细胞转录因子Oct-4的表达对Survivin抗凋亡作用的调控

对临床原发性肝癌、胆囊癌、胆管癌标本Oct-4和Survivin的表达进行了免疫组化鉴定;进一步建立肝癌、胆囊癌、胆管癌细胞系EHBH-H1、EH-GB1和EH-CA1,利用腺病毒携带Survivin-shRNA或Oct-4基因感染癌细胞系,并利用流式细胞术观察细胞周期和细胞凋亡的变化,探讨转录因子Oct-4与Survivin之间的相互调控及对癌细胞遗传特性的影响。结果表明,临床原发性肝癌、胆囊癌、胆管癌标本Oct-4阳性率达60.7%,Survivin阳性率达75.0%,且两者之间存在明显的正相关关系;特异性shRNA沉默EH-CA1癌细胞Survivin的表达后,其Oct-4表达没有变化,但诱导细胞周期阻滞和细胞凋亡;EH-GB1癌细胞获得Oct-4表达后,Survivin表达增强,促进细胞周期运行并下调细胞凋亡。实验证实Oct-4可以增强癌细胞Survivin的表达,从而发挥促进细胞周期运行、抑制细胞凋亡的作用,此研究为建立并优化肿瘤基因治疗的策略提供了新的靶点。     

凋亡抑制蛋白Survivin 研究进展

存活素(Survivin)是凋亡抑制蛋白家族成员之一,具有抑制细胞凋亡和调节细胞周期的双重功能,主要表达于胚胎和发育的胎儿组织中,高表达于大多数恶性肿瘤组织,而在终末分化成熟的正常成人组织中无表达或低表达.本文就Survivin的结构、作用机制、组织分布及其在肿瘤治疗中的研究进展作一综述.     

多头绒泡菌类p34^cdc2蛋白的定位研究

目前关于动物和酵母细胞中p34cdc2 的定位研究结果尚存在分歧 ,而关于该蛋白在植物细胞中的定位尚不清楚。以多头绒泡菌 (Physarumpolycephalum)S期、G2早期、G2中期、G2晚期、前期、中期和后末期的原质团和细胞核为材料进行免疫印迹 ,发现原质团和细胞核都含有一种分子量约 34kD的类p34cdc2 蛋白 ,该蛋白在原质团和细胞核中的含量在整个细胞周期进程中基本保持稳定。以抗p34cdc2 单克隆抗体为探针的免疫电镜结果显示 ,类p34cdc2 蛋白既分布于细胞核也分布于细胞质中 ,在细胞核中主要与染色体和核仁结合。经抗p34cdc2 单克隆抗体处理后 ,多头绒泡菌的有丝分裂启始迟滞约 2h。结果表明 ,多头绒泡菌类p34cdc2 蛋白存在于细胞核和细胞质中 ,与细胞有丝分裂密切相关 ,其含量在细胞周期进程中基本保持稳定。     

人外周血活化淋巴细胞survivin基因的转录激活和表达相关的信号转导通路研究

Survivin是与细胞增殖和细胞凋亡调控密切相关的重要功能蛋白质,也是肿瘤临床诊断的分子标志物及治疗研究的理想靶标.由于发现人外周血活化淋巴细胞存在Survivin蛋白的显著表达,故以植物血凝素(PHA)和IL-2共刺激培养的正常人外周血单个核细胞为实验材料,采用RT-PCR、蛋白质印迹以及细胞周期分析等实验方法,观察淋巴细胞活化与Survivin蛋白表达之间的相互关系,并利用3种激酶抑制剂探索了淋巴细胞活化所致Survivin蛋白表达相关的信号转导通路.实验结果表明：人外周血单个核细胞在刺激培养36 h前后出现survivin基因的明显转录激活和蛋白质的起始表达,表达产物的水平随培养时间的延长而增加,且具有明显的细胞周期和周期时相依赖性.JAK2的抑制剂AG490阻断细胞周期的运行,且强烈抑制survivin基因的转录激活和蛋白质表达,PI3K和MEK的抑制剂Wortmannin和PD98059也有一定程度的抑制作用.所得实验结论是：survivin基因的转录激活和蛋白质表达与淋巴细胞活化相关联,代表细胞处在增殖状态.JAK2介导的JAK-STAT信号通路是淋巴细胞活化,Survivin蛋白表达必需和最重要的信号转导通路,PI3K和MEK介导的信号通路也具有一定的影响作用.     

"细胞分裂"课件的设计及分析

1　教材分析本章主要讲述细胞分裂的方式、特点和过程。细胞分裂的方式主要有三种:有丝分裂、无丝分裂和减数分裂。本章着重讲述有丝分裂的过程。主要说明有丝分裂的细胞周期、有丝分裂的几个不同分裂时期及各个时期染色体变化。在细胞周期的间期主要完成组成染色体的ＤＮＡ分子的复制和有关蛋白质的合成。这些知识与学习后面有关遗传物质基础知识有紧密联系,有丝分裂的过程和分裂期特点与讲述减数分裂知识有直接关系。所以真核细胞有丝分裂的细胞周期和有丝分裂过程既是重点又是难点。2　课件设计21　导言复习提问:真核细胞的细胞结构分为…     

多头绒泡菌类p34cdc2蛋白的定位研究

目前关于动物和酵母细胞中p34cdc2的定位研究结果尚存在分歧,而关于该蛋白在植物细胞中的定位尚不清楚.以多头绒泡菌( Physarum polycephalum )S期、G2早期、G2中期、G2晚期、前期、中期和后末期的原质团和细胞核为材料进行免疫印迹,发现原质团和细胞核都含有一种分子量约34 kD的类p34cdc2蛋白,该蛋白在原质团和细胞核中的含量在整个细胞周期进程中基本保持稳定.以抗p34cdc2单克隆抗体为探针的免疫电镜结果显示,类p34cdc2蛋白既分布于细胞核也分布于细胞质中,在细胞核中主要与染色体和核仁结合.经抗p34cdc2单克隆抗体处理后,多头绒泡菌的有丝分裂启始迟滞约2 h.结果表明,多头绒泡菌类p34cdc2蛋白存在于细胞核和细胞质中,与细胞有丝分裂密切相关,其含量在细胞周期进程中基本保持稳定.     

细胞周期调控的研究进展

细胞周期是一种非常复杂和精细的调节过程,有大量调节蛋白参与其中。此过程的核心是细胞周期依赖性蛋白激酶（CDKs）。CDKs的激活又依赖于另一类呈细胞周期特异性或时相性表达的细胞周期蛋白（cyclins）,而CDKs调节的关键步骤是细胞周期检查点。PLKs是多种细胞周期检查点的主要调节因子,Aurora蛋白激酶主要在细胞有丝分裂期起作用。本文就上述因素在细胞周期进程中的作用作一综述。     

Survivin小分子抑制剂的研究进展

Survivin是凋亡抑制蛋白家族的一员,在抑制细胞凋亡、调控细胞周期、参与血管形成等方面发挥重要的生物学功能。Survivin在多种肿瘤组织中过量表达,与肿瘤不良预后和耐药性密切相关。Survivin作为一种潜在的肿瘤治疗靶点,其小分子抑制剂用于肿瘤治疗的研究为人们所关注。概述了Survivin的结构、功能及其在肿瘤组织中的特异性表达,综述了目前靶向Survivin的小分子抑制剂的研究进展。     

Elevation of Survivin levels by hematopoietic growth factors occurs in quiescent CD34+ hematopoietic stem and progenitor cells before cell cycle entry

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is overexpressed during G(2)/M phase in most cancer cells. In contrast, we previously reported that Survivin is expressed throughout the cell cycle in normal CD34(+) hematopoietic stem and progenitor cells stimulated by the combination of Thrombopoietin (Tpo), Stem Cell Factor (SCF) and Flt3 ligand (FL). In order to address whether Survivin expression is specifically up-regulated by hematopoietic growth factors before cell cycle entry, we isolated quiescent CD34(+) cells and investigated Survivin expression in response to growth factor stimulation. Survivin is up-regulated in CD34(+) cells with 2N DNA content following growth factor addition, suggesting it becomes elevated during G(0)/G(1). Survivin is barely detectable in freshly isolated umbilical cord blood (UCB) Ki-67(negative) and Cyclin D(negative) CD34(+) cells, however incubation with Tpo, SCF and FL for 20 hrs results in up-regulation without entry of cells into cell cycle. Culture of G(0) CD34(+) cells isolated based on Hoechst 33342/PyroninY staining with Tpo, SCF and FL for 48 hrs, results in significantly elevated Survivin mRNA and protein levels. Moreover, labeling of fresh G(0) CD34(+) cells with 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) before culture with growth factors for up to 72 hrs, revealed that Survivin expression was elevated in CFSE(bright) G(0) CD34(+) cells, indicating that up-regulation occurred before entry into G1. These results suggest that up-regulation of Survivin expression in CD34(+) cells is an early event in cell cycle entry that is regulated by hematopoietic growth factors and does not simply reflect cell cycle progression and cell division.     

Elevation of Survivin Levels by Hematopoietic Growth Factors Occurs in Quiescent CD34+ Hematopoietic Stem and Progenitor Cells Before Cell Cycle Entry

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is over-expressed during G2/M phase in most cancer cells. In contrast, we previously reported that Survivin is expressed throughout the cell cycle in normal CD34+ hematopoietic stem and progenitor cells stimulated by the combination of Thrombopoietin (Tpo), Stem Cell Factor (SCF) and Flt3 ligand (FL). In order to address whether Survivin expression is specifically up-regulated by hematopoietic growth factors before cell cycle entry, we isolated quiescent CD34+ cells and investigated Survivin expression in response to growth factor stimulation. Survivin is up-regulated in CD34+ cells with 2N DNA content following growth factor addition, suggesting it becomes elevated during G0/G1. Survivin is barely detectable in freshly isolated umbilical cord blood (UCB) Ki-67negative and Cyclin Dnegative CD34+ cells, however incubation with Tpo, SCF and FL for 20 hrs results in up-regulation without entry of cells into cell cycle. Culture of G0 CD34+ cells isolated based on Hoechst 33342/PyroninY staining with Tpo, SCF and FL for 48 hrs, results in significantly elevated Survivin mRNA and protein levels. Moreover, labeling of fresh G0 CD34+ cells with 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) before culture with growth factors for up to 72 hrs, revealed that Survivin expression was elevated in CFSEbright G0 CD34+ cells, indicating that up-regulation occurred before entry into G1. These results suggest that up-regulation of Survivin expression in CD34+ cells is an early event in cell cycle entry that is regulated by hematopoietic growth factors and does not simply reflect cell cycle progression and cell division.

Key Words:

Survivin, Cord blood, CD34+ cells, Cell cycle     

Functional analysis of Survivin in spindle assembly in Xenopus egg extracts

Survivin is a member of the inhibitor of apoptosis (IAP) protein family that serves critical roles in mitosis and cytokinesis. Many studies have suggested Survivin's involvement in spindle regulation, but direct biochemical evidence for this has been lacking. Using the cell-free system of Xenopus egg extracts, we tested whether Survivin was necessary for the assembly of metaphase spindles. Removal or inhibition of Xenopus Survivin causes the disruption in the formation of metaphase spindles. In particular, we observe the generation of microtubule (MT) asters or poorly formed shortened spindle structures. In the latter phenotype the spindle structures display a decrease pole-to-pole length and a reduction of MTs around the chromatin indicating that Survivin may promote the stabilization of MT-chromatin interactions. In addition, function analysis of Survivin's conserved phosphorylation site Thr34 (Thr43 in Xenopus) and tubulin-binding domain was also assessed in regulating spindle assembly. Treatment of Xenopus egg extracts with a recombinant Survivin mutant that contained an alanine residue substitution at Thr43 (SURT43A mutant) or that was missing the C-terminal tubulin-binding domain (SURCL mutant) produced an increased frequency of MT asters and shorten abnormal spindle structures in Xenopus egg extracts. Interestingly, a phosphomimetic mutation made at residue Thr43 of Survivin (SURT43E mutant) generated a high frequency of MT asters implying that premature 'activation' of Survivin may interfere with an early stage of spindle assembly. Taken together, we propose that Survivin is a necessary component of the mitotic spindle and its phosphorylation at residue Thr43 is important for Survivin function in spindle assembly.     

Aurora B kinase exists in a complex with survivin and INCENP and its kinase activity is stimulated by survivin binding and phosphorylation

Aurora B regulates chromosome segregation and cytokinesis and is the first protein to be implicated as a regulator of bipolar attachment of spindle microtubules to kinetochores. Evidence from several systems suggests that Aurora B is physically associated with inner centromere protein (INCENP) in mitosis and has genetic interactions with Survivin. It is unclear whether the Aurora B and INCENP interaction is cell cycle regulated and if Survivin physically interacts in this complex. In this study, we cloned the Xenopus Survivin gene, examined its association with Aurora B and INCENP, and determined the effect of its binding on Aurora B kinase activity. We demonstrate that in the Xenopus early embryo, all of the detectable Survivin is in a complex with both Aurora B and INCENP throughout the cell cycle. Survivin and Aurora B bind different domains on INCENP. Aurora B activity is stimulated >10-fold in mitotic extracts; this activation is phosphatase sensitive, and the binding of Survivin is required for full Aurora B activity. We also find the hydrodynamic properties of the Aurora B/Survivin/INCENP complex are cell cycle regulated. Our data indicate that Aurora B kinase activity is regulated by both Survivin binding and cell cycle-dependent phosphorylation.     

Human survivin is a kinetochore-associated passenger protein

Survivin, a dimeric baculovirus inhibitor of apoptosis repeat (BIR) motif protein that is principally expressed in G2 and mitosis, has been associated with protection against apoptosis of cells that exit mitosis aberrantly. Mammalian survivin has been reported to associate with centrosomes and with the mitotic spindle. We have expressed a human hemagglutinin-tagged survivin plasmid to determine its localization, and find instead that it clearly acts as a passenger protein. In HeLa cells, survivin first associates with the kinetochores, and then translocates to the spindle midzone during anaphase and, finally, to the midbody during cell cleavage. Its localization is similar to that of TD-60, a known passenger protein. Both a point mutation in the baculovirus IAP repeat motif (C84A) and a COOH-terminal deletion mutant (Delta106) of survivin fail to localize to either kinetochores or midbodies, but neither interferes with cell cleavage. The interphase localization of survivin is cell cycle regulated since in permanently transfected NIH3T3 cells it is excluded from the nuclei until G2, where it localizes with centromeres. Survivin remains associated with mitotic kinetochores when microtubule assembly is disrupted and its localization is thus independent of microtubules. We conclude that human survivin is positioned to have an important function in the mechanism of cell cleavage.     

CRM1-mediated nuclear export determines the cytoplasmic localization of the antiapoptotic protein Survivin

Survivin is a member of the inhibitor of apoptosis (IAP) family of negative regulators of programmed cell death that is frequently overexpressed in human tumors. Survivin is not only involved in the regulation of apoptosis, but is also known to play a role in the control of cell cycle progression at the G2/M phase. Survivin is a predominantly cytoplasmic protein expressed in a cell cycle-dependent manner, but the mechanism(s) that determine its nuclear-cytoplasmic localization have not been described. In this study, we report that Survivin is a nuclear shuttling protein that is actively exported from the nucleus via the CRM1-dependent pathway. Nuclear export of Survivin is independent of the export of other shuttling proteins that control the G2/M phase transition, such as cyclin B1 and cdc25. The carboxy-terminal domain of Survivin is both necessary and sufficient for its nuclear export, although this region does not contain a functional leucine-rich nuclear export signal. Differences in the amino acid sequence of this region determine the dramatically different localization of Survivin (in the cytoplasm) and its splicing variant Survivin-DeltaEx3 (in the nucleus). The carboxy-terminal end of Survivin-DeltaEx3 contains a bipartite nuclear localization signal, not present in Survivin, which mediates its strong nuclear accumulation. These data suggest that active transport between the nucleus and cytoplasm may constitute an important regulatory mechanism for Survivin function.     

Survivin withdrawal by nuclear export failure as a physiological switch to commit cells to apoptosis

Apoptosis is a tightly controlled process regulated by many signaling pathways; however, the mechanisms and cellular events that decide whether a cell lives or dies remain poorly understood. Here we showed that when a cell is under apoptotic stress, the prosurvival protein Survivin redistributes from the cytoplasm to the nucleus, thus acting as a physiological switch to commit the cell to apoptosis. The nuclear relocalization of Survivin is a result of inefficient assembly of functional RanGTP–CRM1–Survivin export complex due to apoptotic RanGTP gradient collapse. Subsequently, Survivin undergoes ubiquitination, which not only physically prevents its diffusion back to the cytoplasm but also facilitates its degradation. Together, this spatial and functional regulation of Survivin abolishes its cytoprotective effect toward the apoptotic executors and thus commits a cell to apoptosis. Our data indicate that the withdrawal of Survivin is a novel and active physiological regulatory mechanism that tilts the survival balance and promotes the progression of apoptosis.     

Activated H-Ras regulates hematopoietic cell survival by modulating Survivin

Survivin expression and Ras activation are regulated by hematopoietic growth factors. We investigated whether activated Ras could circumvent growth factor-regulated Survivin expression and if a Ras/Survivin axis mediates growth factor independent survival and proliferation in hematopoietic cells. Survivin expression is up-regulated by IL-3 in Ba/F3 and CD34+ cells and inhibited by the Ras inhibitor, farnesylthiosalicylic acid. Over-expression of constitutively activated H-Ras (CA-Ras) in Ba/F3 cells blocked down-modulation of Survivin expression, G0/G1 arrest, and apoptosis induced by IL-3 withdrawal, while dominant-negative (DN) H-Ras down-regulated Survivin. Survivin disruption by DN T34A Survivin blocked CA-Ras-induced IL-3-independent cell survival and proliferation; however, it did not affect CA-Ras-mediated enhancement of S-phase, indicating that the anti-apoptotic activity of CA-Ras is Survivin dependent while its S-phase enhancing effect is not. These results indicate that CA-Ras modulates Survivin expression independent of hematopoietic growth factors and that a CA-Ras/Survivin axis regulates survival and proliferation of transformed hematopoietic cells.