Zebrafish embryo, a tool to study tumor angiogenesis

Zebrafish (Danio rerio) represents a powerful model system in cancer research. Recent observations have shown the possibility to exploit zebrafish to investigate tumor angiogenesis, a pivotal step in cancer progression and target for anti-tumor therapies. Experimental models have been established in zebrafish adults, juveniles, and embryos, each one with its own advantages and disadvantages. Novel genetic tools and high resolution in vivo imaging techniques are also becoming available in zebrafish. It is anticipated that zebrafish will represent an important tool for chemical discovery and gene targeting in tumor angiogenesis. This review focuses on the recently developed tumor angiogenesis models in zebrafish, with particular emphasis to tumor engrafting in zebrafish embryos.     

The "chemoinvasion assay": a tool to study tumor and endothelial cell invasion of basement membranes

Several genetic and epigenetic factors, both in the cell and in the host, contribute to the progression of tumors towards metastases. The escape of cancer cells from a primary, localized tumor to distant organs transforms a relatively curable pathology to an almost untreatable one. Metastatic lesions are often resistant to cancer therapy because of the progressive phenotypic changes that they have undergone. In this article we will give a bird's eye view on the features of metastatic cells and potential therapeutic targets. In particular, the invasion of basement membranes represents a fundamental step for cell dispersion. Over seventeen years ago we established the Matrigel "chemoinvasion" assay, a useful tool for studying the mechanisms involved in tumor and endothelial cell invasion of basement membranes and for the screening of anti-invasive agents. We will describe the assay and review some of the major results it enabled to obtain.     

Yeast as a tool to study Bax/mitochondrial interactions in cell death

The budding yeast Saccharomyces cerevisiae has proven to be a powerful tool in investigations of the molecular aspects of the events involved in apoptosis, particularly the steps implicating mitochondria. Yeast does not have obvious homologs of the proteins involved in the regulation of apoptosis, and provides a simplified model system in which the function of these proteins can be unraveled. This review focuses on the interactions of two of the major pro-apoptotic Bcl-2 family members, Bax and Bid, with mitochondria. It is shown that yeast has allowed questioning of several crucial aspects of the function of these two proteins, namely the molecular mechanisms driving their insertion into the mitochondrial outer membrane and those leading to the permeabilization to cytochrome c. More recently, signaling pathways leading to Bax-induced cell death, as well as other forms of cell death, have been identified in yeast. Both 'apoptosis-like' and autophagy-related forms of cell degradation are involved, and mitochondria play a central role in these two signaling pathways.     

Zebrafish as a tool in Alzheimer's disease research

Alzheimer's disease is the most prevalent form of neurodegenerative disease. Despite many years of intensive research our understanding of the molecular events leading to this pathology is far from complete. No effective treatments have been defined and questions surround the validity and utility of existing animal models. The zebrafish (and, in particular, its embryos) is a malleable and accessible model possessing a vertebrate neural structure and genome. Zebrafish genes orthologous to those mutated in human familial Alzheimer's disease have been defined. Work in zebrafish has permitted discovery of unique characteristics of these genes that would have been difficult to observe with other models. In this brief review we give an overview of Alzheimer's disease and transgenic animal models before examining the current contribution of zebrafish to this research area. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

Adenovirus-mediated gene transfer as a tool to study angiogenesis in the chick embryo

Abstract. An adenoviral construct encoding a nuclear-localized beta-galactosidase marker protein was injected into the heart of chick embryos at Hamburger-Hamilton (HH) stage 14-15 (approximately 52-56 h of incubation). Reporter gene expression was determined 48-54 h after injection. Efficient gene transfer into endothelial cells (ECs) of intraembryonic and yolk sac vessels was observed. ECs of vessels in the head region, which undergo massive expansion around the time of injection, were efficiently labeled. However, limb bud vasculature, which starts to develop around stage 16 (HH), carried scarce (wing bud) or no (leg bud) lacZ marker. In contrast, ECs of the allantois, a structure that develops even later (around stage HH 18), expressed lacZ reporter. This observation suggests that EC precursors infected at an earlier time migrated into the allantois. A few non-endothelial cell types were also labeled by the reporter. These results suggest that adenovirus-mediated gene transfer provides a powerful tool to study angiogenesis in the developing chick embryo.     

Zebrafish as a tool in Alzheimer's disease research

Alzheimer's disease is the most prevalent form of neurodegenerative disease. Despite many years of intensive research our understanding of the molecular events leading to this pathology is far from complete. No effective treatments have been defined and questions surround the validity and utility of existing animal models. The zebrafish (and, in particular, its embryos) is a malleable and accessible model possessing a vertebrate neural structure and genome. Zebrafish genes orthologous to those mutated in human familial Alzheimer's disease have been defined. Work in zebrafish has permitted discovery of unique characteristics of these genes that would have been difficult to observe with other models. In this brief review we give an overview of Alzheimer's disease and transgenic animal models before examining the current contribution of zebrafish to this research area. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

RNAi as a tool to study cell biology: building the genome-phenome bridge

In the few short years since its discovery, RNA interference (RNAi) has revolutionized the functional analysis of genomes: both technical and conceptual approaches to the investigation of gene function are being transformed as a result of this new technology. Genome-scale RNAi analyses have already been performed in the model organisms Caenorhabditis elegans (in vivo) and Drosophila melanogaster (in cell lines), ushering in a new era of RNAi-based approaches to probing the inner workings of the cell. The transformation of complex phenotypic data into mineable 'digitized' formats is fostering the emergence of a new area of bioinformatics related to the phenome.     

Zebrafish xenografts as a tool for in vivo studies on human cancer

The zebrafish has become a powerful vertebrate model for genetic studies of embryonic development and organogenesis and increasingly for studies in cancer biology. Zebrafish facilitate the performance of reverse and forward genetic approaches, including mutagenesis and small molecule screens. Moreover, several studies report the feasibility of xenotransplanting human cells into zebrafish embryos and adult fish. This model provides a unique opportunity to monitor tumor-induced angiogenesis, invasiveness, and response to a range of treatments in vivo and in real time. Despite the high conservation of gene function between fish and humans, concern remains that potential differences in zebrafish tissue niches and/or missing microenvironmental cues could limit the relevance and translational utility of data obtained from zebrafish human cancer cell xenograft models. Here, we summarize current data on xenotransplantation of human cells into zebrafish, highlighting the advantages and limitations of this model in comparison to classical murine models of xenotransplantation.     

The quartz crystal microbalance as a continuous monitoring tool for the study of endothelial cell surface attachment and growth

The quartz crystal microbalance (QCM) was used to monitor endothelial cell (EC) adhesion on the gold surface of an oscillating quartz crystal contained in a QCM device. A number of parameters were investigated. First, we observed differential QCM O-ring toxicities for ECs. Second, appropriate conditions for cell culture and QCM cell environment were identified that can eliminate large-scale frequency oscillations in the measurements. These artifacts are not due to added cells but originate in the time-dependent evaporation of water. Having eliminated these artifacts, we then demonstrated that the measured steady-state crystal frequency shift, Delta f, and motional resistance shift, DeltaR, were determined by the number of firmly attached ECs requiring trypsinization from the crystal surface. Last, following steady-state attachment of ECs, the EC growth stimulation by fibroblast growth factor was monitored in a continuous fashion by measuring f and R values over a 72 h. period. We observed the Delta f values to increase in a way that reflected the increase in EC number bound to the QCM surface. Following addition of ECs to the QCM, the time-dependent increase in DeltaR can be interpreted in terms of increase by the ECs of the energy dissipation properties of the solution at the solution-gold surface interface. This effect is due to their rapid surface attachment and the elaboration of their cytoskeletal properties. These results indicate that the QCM technique can be used for the study of EC attachment and growth and suggest its potential for the real time study of per unit surface area cell mass distribution dynamics and viscoelastic properties and the cells' responses to stresses or perturbations brought about using biologically active molecules.     

Transformed cell clones as a tool to study T-DNA integration mediated by Agrobacterium tumefaciens

A large number of tobacco SR1 cell clones transformed by the wild-type Agrobacterium C58 have been analysed for the presence of screenable markers such as tumour morphology, opine synthesis and hormone dependence. Distinct phenotypic classes were observed depending upon whether the cell clones were isolated from primary tumours or were obtained via cocultivation of protoplasts. These classes of tobacco SR1-C58 transformants appear to arise from errors in the Ti plasmid (T-DNA) transfer and integration mechanism itself rather than from subsequent T-DNA rearrangements, since 900 subclones, obtained by recloning a wild-type SR1-C58-transformed cell clone, yielded no variation in the phenotypes. A detailed genomic T-DNA analysis showed the presence of characteristic, abnormally short T-DNAs in the teratoma-forming, Acs- class and also in the Nos- class. The abnormal right border in two Nos- clones ends close to a sequence that resembles the normal T-DNA terminus and lies adjacent to the nos promoter, suggesting that this sequence could have functioned as a recognition site directing these particular T-DNA transfers. On the basis of the phenotypic and genomic blotting data it is clear that the short T-DNAs are characteristic of the cocultivation method. Other phenomena causing phenotypic variation, such as the loss of the T-DNA, and the gradual repression of T-DNA gene expression by methylation, are the main causes of aberrations in primary tumours. Moreover, the physical data suggest that early in the transformation cycle of Agrobacterium a replication step of a preselected T-DNA occurs before integration into the plant genome.     

肿瘤新生血管相关内皮细胞受体研究进展

血管生成对肿瘤的生长,存留和转移是至关重要的,增殖的肿瘤血管内皮细胞不同于静止期内皮细胞,增生血管内皮细胞上特异或高表达的许多受体目前已被鉴定,它们是肿瘤抗血管生成疗法的理想靶点,简要综述了其中主要的几种受体,如血管内皮细胞生长因子（VEGF）受体家族,Tie受体族,整合素受体αvβ3,ATP合成酶等。     

Chick embryo chorioallantoic membrane model systems to study and visualize human tumor cell metastasis

Since their introduction almost a century ago, chick embryo model systems involving the technique of chorioallantoic grafting have proved invaluable in the in vivo studies of tumor development and angiogenesis and tumor cell dissemination. The ability of the chick embryo’s chorioallantoic membrane (CAM) to efficiently support the growth of inoculated xenogenic tumor cells greatly facilitates analysis of human tumor cell metastasis. During spontaneous metastasis, the highly vascularized CAM sustains rapid tumor formation within several days following cell grafting. The dense capillary network of the CAM also serves as a repository of aggressive tumor cells that escaped from the primary tumor and intravasated into the host vasculature. This spontaneous metastasis setting provides a unique experimental model to study in vivo the intravasation step of the metastatic cascade. During experimental metastasis when tumor cells are inoculated intravenously, the CAM capillary system serves as a place for initial arrest and then, for tumor cell extravasation and colonization. The tissue composition and accessibility of the CAM for experimental interventions makes chick embryo CAM systems attractive models to follow the fate and visualize microscopically the behavior of grafted tumor cells in both spontaneous and experimental metastasis settings.     

Fluorescence polarization as a tool to study lectin-sugar interaction

The binding ofRicinus communis agglutinin andAbrus agglutinin to 4-methylumbelliferyl β-D-galactopyranoside was studied by equilibrium dialysis, fluo-rescence quenching and fluorescence polarization. The number of binding sites and the association constant value obtained by fluorescence polarization for bothRicinus communis agglutinin andAbrus agglutinin are in close agreement with those obtained by the other methods. This indicates the potential of ligand-fluorescence polarization measurements in the investigation of lectin-sugar interactions.     

IL-12 inhibition of endothelial cell functions and angiogenesis depends on lymphocyte-endothelial cell cross-talk

In vivo IL-12-dependent tumor inhibition rests on the ability of IL-12 to activate a CD8-mediated cytotoxicity, inhibit angiogenesis, and cause vascular injury. Although in vivo studies have shown that such inhibition stems from complex interactions of immune cells and the production of IFN-gamma and other downstream angiostatic chemokines, the mechanisms involved are still poorly defined. Here we show that IL-12 activates an anti-angiogenic program in Con A-activated mouse spleen cells (activated spc) or human PBMC (activated PBMC). The soluble factors they release in its presence arrest the cycle of endothelial cells (EC), inhibit in vitro angiogenesis, negatively modulate the production of matrix metalloproteinase-9, and the ability of EC to adhere to vitronectin and up-regulate ICAM-1 and VCAM-1 expression. These effects do not require direct cell-cell contact, yet result from continuous interaction between activated lymphoid cells and EC. We used neutralizing Abs to show that the IFN-inducible protein-10 and monokine-induced by IFN-gamma chemokines are pivotal in inducing these effects. Experiments with nu/nu mice, nonobese diabetic-SCID mice, or activated spc enriched in specific cell subpopulations demonstrated that CD4(+), CD8(+), and NK cells are all needed to mediate the full anti-angiogenetic effect of IL-12.