An engineered mouse embryonic stem cell model with survivin as a molecular marker and EGFP as the reporter for high throughput screening of embryotoxic chemicals in vitro

Embryonic stem cell test (EST) is the only generally accepted in vitro method for assessing embryotoxicity without animal sacrifice. However, the implementation and application of EST for regulatory embryotoxicity screening are impeded by its technical complexity, long testing period, and limited endpoint data. In this study, a high throughput embryotoxicity screening based on mouse embryonic stem cells (mESCs) expressing enhanced green fluorescent protein (EGFP) driven by a human survivin promoter and a human cytomegalovirus promoter, respectively, was developed. These EGFP expressing mESCs were cultured in three-dimensional (3D) fibrous scaffolds in microbioreactors on a multiwell plate with EGFP fluorescence signals as cell responses to chemicals monitored noninvasively in a high throughput manner. Nine chemicals with known developmental toxicity were used to validate the survivin-based embryotoxicity assay, which showed that strongly embryotoxic compounds such as 5-fluorouracil, retinoic acid, and methotrexate downregulated survivin expression by more than 50% in 3 days, while weakly embryotoxic compounds such as boric acid, methoxyacetic acid, and tetracyclin showed modest downregulation effect and nonembryotoxic saccharin, penicillin G, and acrylamide had negligible downregulation effect on survivin expression, confirming that survivin can be used as a molecular endpoint for high throughput screening of embryotoxicants. The potential developmental toxicity of three Chinese herbal medicines were also evaluated using this assay, demonstrating its application in in vitro developmental toxicity test for drug safety assessment.     

抗凋亡蛋白survivin研究与癌治疗新策略的进展

survivin是凋亡抑制蛋白家族中的一个新成员,在近乎所有的人类肿瘤中都显示高表达,而在终末分化的正常组织中未能检测出表达。survivin表达的另一个独特性质是,其表达受细胞周期调控,在G2／M期有异常高的特异性表达。survivin的功能意义在于参与细胞凋亡调控和细胞分裂调控。引人兴趣的是,新近癌生物学基础与应用研究则更关注它是一个枢纽癌基因。本文将重点讨论与survivin在细胞死亡和细胞分裂功能相联系的、当前一些新的癌治疗策略的进展。     

Expression of survivin mRNA and protein in gastric cancer cell line (MKN-45) during cisplatin treatment

Survivin is a member of the inhibitor of apoptosis protein (IAP) family. Survivin has been reported to be expressed in many cancers, but not in differentiated normal tissue. Recent studies revealed that survivin correlated with the chemo-resitance of cancer cells. In the present study, the changes in expression levels of survivin messenger RNA (mRNA) and survivin protein in a gastric cancer cell line (MKN-45) during cisplatin (CDDP) treatment were analyzed and compared with the occurrence of apoptotic cell death. Cell growth was inhibited even with a low dose CDDP (0.1 or 1 g/ml) 1 hr treatment. However, the percentage of apoptotic cells did not change after 48 hr incubation with low dose CDDP. Only with high dose CDDP (10 g/ml), did the percentage of apoptotic cells explosively increase between 12 and 24 hr treatment. Relative expression levels of survivin mRNA and survivin protein increased after CDDP treatment. The cell expression rates of survivin mRNA after 48 hr treatment with 0.1 and 1 g/ml of CDDP were 2 to 6 fold higher than that of the survivin mRNA of untreated cells. Also, the relative cell expression level of survivin protein after 24 hr treatment with 0.1 or 1 g/ml of CDDP was 3 to 6.5 fold higher than that of the survivin protein of untreated cells. These results indicate that survivin expression may correlate with the chemo-resistance of malignant cells.     

COX inhibitors modulate bFGF-induced cell survival in MCF-7 breast cancer cells

Basic fibroblast growth factor (bFGF) serves as a modulator of survival in breast cancer cells. The mechanisms by which bFGF transduces the anti-apoptotic signal and interacts with COX inhibitors were investigated. bFGF reduced apoptosis in MCF-7 breast cancer cells and up-regulated the expression of mitocondrial Bcl-2, whereas COX inhibitors meloxicam (selective COX-2) and aspirin (non-selective), induced apoptosis. bFGF up-regulated survivin protein expression and induced cdc-2 phosphorylation moderately at early (2-6 h), and substantially at late (24 h), time-points. Survivin mRNA expression was up-regulated only at the later time-point. COX inhibitors prevented up-regulation of survivin protein expression at both 2 and 24 h and prevented early modest increases in cdc-2 phosphorylation. Up-regulation of survivin mRNA was not found to be modulated by the COX-2 inhibitor meloxicam. bFGF regulation of survivin expression was found to be ERK1/2 kinase dependent and bFGF-induced phosphorylation of c-raf was prevented by the COX-2 inhibitor. bFGF was, however, unable to induce COX-2 protein expression or modulate COX-2 activity in MCF-7 cells as evidenced by unaltered PGE(2) production. These results indicate that bFGF regulates survivin expression in MCF-7 breast cancer cells by signaling through an ERK1/2 dependent pathway. COX-2 inhibitors can modulate bFGF-induced survivin expression in a COX-2 independent manner.     

Molecular circuits of apoptosis regulation and cell division control: the survivin paradigm

The coupling of cell proliferation to cell death is thought to function as a pivotal crossroad, essential to preserve normal homeostasis and to eliminate dangerous cells before they divide. Survivin is a prototype molecule at this crossroad, intercalated in protection against mitochondrial cell death and orchestrating various aspects of cell division. Dramatically exploited in cancer and an unfavorable gene signature for disease outcome, the survivin pathway has now provided tangible opportunities for targeted, rational cancer therapy.     

Aging brain microenvironment decreases hippocampal neurogenesis through Wnt-mediated survivin signaling

Accumulating evidence suggests that adult hippocampal neurogenesis relies on the controlled and continued proliferation of neural progenitor cells (NPCs). With age, neurogenesis decreases through mechanisms that remain unclear but are believed to involve changes in the NPC microenvironment. Here, we provide evidence that NPC proliferation in the adult brain is in part regulated by astrocytes via Wnt signaling and that this cellular cross-talk is modified in the aging brain, leading to decreased proliferation of NPCs. Furthermore, we show that astrocytes regulate the NPC cell cycle by acting on the expression levels of survivin, a known mitotic regulator. Among cell cycle genes found down-regulated in aged NPCs, survivin was the only one that restored NPC proliferation in the aged brain. Our results provide a mechanism for the gradual loss of neurogenesis in the brain associated with aging and suggest that targeted modulation of survivin expression directly or through Wnt signaling could be used to stimulate adult neurogenesis.     

人eRF3a与survivinSE互作用的结构域分析

第二类肽链释放因子eRF3（eukaryotic polypeptide release factor）是一种GTPase,它促进第一类肽链释放因子eRFl的释放活性,并与细胞周期调控、细胞骨架组装、细胞凋亡和肿瘤形成等过程相关。哺乳动物细胞中eRF3有两种——eRF3a和eRF3b,分别由GSPTl和GSPT2（G1 to Sphase transition 1／2）基因编码。生存素（survivin）是迄今发现的最强有力的凋亡抑制因子,具有独特的结构和复杂的功能,不仅可以抑制细胞凋亡,还参与细胞有丝分裂、血管的生成等过程。eRF3和survivin都与细胞周期和细胞凋亡的调控相关。该实验室的前期研究表明,eRF3和survivin具有相互作用关系。该研究进一步对eRF3a进行截短突变。采用酵母双杂交和pull．down两种分析方法依次验证eRF3a（1．72aa）和eRF3a（1—36aa）与survivin的相互作用关系。结果表明,eRF3a（1．72aa）和eRF3a（1—36aa）均可以与survivin相互作用,由此确定eRF3a与survivinf相互作用的最小结构域位于其N末端1-36aa之间,从而为进一步证实eRF3a的N端结构域与survivin协同作用参与细胞周期和细胞凋亡的调控提供了数据支持。     

Regulation of survivin by retinoic acid and its role in paclitaxel-mediated cytotoxicity in MCF-7 breast cancer cells

The chemotherapeutic drug paclitaxel induces microtubular stabilization and mitotic arrest associated with increased survivin expression. Survivin is a member of the inhibitor of apoptosis (iap) family which is highly expressed in during G2/M phase where it regulates spindle formation during mitosis. It is also constitutively overexpressed in most cancer cells where it may play a role in chemotherapeutic resistance. MCF-7 breast cancer cells stably overexpressing the sense strand of survivin (MCF-7(survivin-S) cells) were more resistant to paclitaxel than cells depleted of survivin (MCF-7(survivin-AS) despite G2/M arrest in both cell lines. However, survivin overexpression did not protect cells relative to control MCF-7(pcDNA3) cells. Paclitaxel-induced cytotoxicity can be enhanced by retinoic acid and here we show that RA strongly reduces survivin expression in MCF-7 cells and prevents paclitaxel-mediated induction of survivin expression. Mitochondrial release of cytochrome c after paclitaxel alone or in combination with RA was weak, however robust Smac release was observed. While RA/paclitaxel-treated MCF-7 (pcDNA3) cultures contained condensed apoptotic nuclei, MCF-7(survivin-S) nuclei were morphologically distinct with hypercondensed disorganized chromatin and released mitochondrial AIF-1. RA also reduced paclitaxel-associated levels of cyclin B1 expression consistent with mitotic exit. MCF-7(survivin-S) cells displayed a 30% increase in >2N/<4N ploidy while there was no change in this compartment in vector control cells following RA/paclitaxel. We propose that RA sensitizes MCF-7 cells to paclitaxel at least in part through survivin downregulation and the promotion of aberrant mitotic progression resulting in apoptosis. In addition we provide biochemical and morphological data which suggest that RA-treated MCF-7(survivin-S) cells can also undergo catastrophic mitosis when exposed to paclitaxel.