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1

Neutrophil-mediated experimental metastasis is enhanced by VEGFR inhibition in a zebrafish xenograft model  

PubMed Central

Inhibition of VEGF signalling effectively suppresses localized tumour growth but accelerates tumour invasiveness and micrometastasis by unknown mechanisms. To study the dynamic and reciprocal interactions between tumour cells and their microenvironment during these processes, we established a xenograft model by injecting tumour cells into the blood circulation of transparent zebrafish embryos. This reproducibly results in rapid simultaneous formation of a localized tumour and experimental micrometastasis, allowing time-resolved imaging of both processes at single-cell resolution within 1 week. The tumour vasculature was initiated de novo by remodelling of primitive endothelial cells into a functional network. Roles of myeloid cells in critical tumourigenesis steps such as vascularization and invasion were revealed by genetic and pharmaceutical approaches. We discovered that the physiological migration of neutrophils controlled tumour invasion by conditioning the collagen matrix and forming the metastatic niche, as detected by two-photon confocal microscopy and second harmonic generation. Administration of VEGFR inhibitors blocked tumour vascularization and a localized tumour growth but enhanced migration of neutrophils, which in turn promoted tumour invasion and formation of micrometastasis. This demonstrates the in vivo cooperation between VEGF signalling and myeloid cells in metastasis and provides a new mechanism underlying the recent findings that VEGFR targeting can promote tumour invasiveness. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:22374800

He, Shuning; Lamers, Gerda EM; Beenakker, Jan-Willem M; Cui, Chao; Ghotra, Veerander PS; Danen, Erik HJ; Meijer, Annemarie H; Spaink, Herman P; Snaar-Jagalska, B Ewa

2012-01-01

2

Zebrafish Models of Rhabdomyosarcoma  

PubMed Central

Rhabdomyosarcoma, an aggressive malignant neoplasm that shows features of skeletal muscle, is the most common soft tissue tumor of childhood. In children, the major subtypes are embryonal and alveolar. Although localized disease responds to a multimodal treatment, the prognosis for patients with high-risk features and metastasis remains dismal. Several in vivo models of rhabdomyosarcoma have been developed in mouse, human xenografts, zebrafish and Drosophila to better understand the underlying mechanisms governing malignancy. The findings so far have indicated the potential role of skeletal muscle precursor cells in malignant transformation. To better understand histogenesis and different aspects of tumorigenesis in rhabdomyosarcoma, we have previously developed a robust zebrafish model of kRAS-induced rhabdomyosarcoma, which shares morphologic and immunophenotypic features with the human counterpart. Cross-species mircroarray comparisons confirm that conserved genetic pathways drive rhabdomyosarcoma growth. The ease for ex vivo manipulation allows development of different transgenic and co-injection strategies to induce tumor formation in zebrafish. In contrast to other vertebrate model systems, the tumor onset in zebrafish is short, allowing for efficient study of different tumor processes including tumor growth, self-renewal, and maintenance. PMID:21951539

Chen, Eleanor Y.; Langenau, David M.

2013-01-01

3

Catch of the day: zebrafish as a human cancer model.  

PubMed

Zebrafish are making big waves in the field of cancer research. The effect has been widespread and continues to gain speed as more and more cancer researchers ride the wave of zebrafish biology. This has been largely due to the development of transgenic and xenograft models of cancer, which recapitulate many aspects of different human cancers including lymphoblastic T-cell leukemia, pancreatic cancer, melanoma and rhabdomyosarcoma. These models are already being utilized by academia and industry to search for genetic and chemical modifiers of cancer with success. The attention has been further stimulated by the amenability of zebrafish to pharmacological testing and the superior imaging properties of fish tissues that allow visualization of cancer progression and angiogenesis in live animals. This review summarizes the current zebrafish models of cancer and discusses their utility in human cancer research and future directions in the field. PMID:18372910

Stoletov, K; Klemke, R

2008-07-31

4

Hooked! Modeling human disease in zebrafish.  

PubMed

Zebrafish have been widely used as a model system for studying developmental processes, but in the last decade, they have also emerged as a valuable system for modeling human disease. The development and function of zebrafish organs are strikingly similar to those of humans, and the ease of creating mutant or transgenic fish has facilitated the generation of disease models. Here, we highlight the use of zebrafish for defining disease pathways and for discovering new therapies. PMID:22751109

Santoriello, Cristina; Zon, Leonard I

2012-07-01

5

A zebrafish model of hyperammonemia.  

PubMed

Hyperammonemia is the principal consequence of urea cycle defects and liver failure, and the exposure of the brain to elevated ammonia concentrations leads to a wide range of neuro-cognitive deficits, intellectual disabilities, coma and death. Current treatments focus almost exclusively on either reducing ammonia levels through the activation of alternative pathways for ammonia disposal or on liver transplantation. Ammonia is toxic to most fish and its pathophysiology appears to be similar to that in mammals. Since hyperammonemia can be induced in fish simply by immersing them in water with elevated concentration of ammonia, we sought to develop a zebrafish (Danio rerio) model of hyperammonemia. When exposed to 3mM ammonium acetate (NH4Ac), 50% of 4-day old (dpf) fish died within 3hours and 4mM NH4Ac was 100% lethal. We used 4dpf zebrafish exposed to 4mM NH4Ac to test whether the glutamine synthetase inhibitor methionine sulfoximine (MSO) and/or NMDA receptor antagonists MK-801, memantine and ketamine, which are known to protect the mammalian brain from hyperammonemia, prolong survival of hyperammonemic fish. MSO, MK-801, memantine and ketamine all prolonged the lives of the ammonia-treated fish. Treatment with the combination of MSO and an NMDA receptor antagonist was more effective than either drug alone. These results suggest that zebrafish can be used to screen for ammonia-neuroprotective agents. If successful, drugs that are discovered in this screen could complement current treatment approaches to improve the outcome of patients with hyperammonemia. PMID:25069822

Feldman, B; Tuchman, M; Caldovic, L

2014-01-01

6

Zebrafish models of cerebrovascular disease  

PubMed Central

Perturbations in cerebral blood flow and abnormalities in blood vessel structure are the hallmarks of cerebrovascular disease. While there are many genetic and environmental factors that affect these entities through a heterogeneous group of disease processes, the ultimate final pathologic insult in humans is defined as a stroke, or damage to brain parenchyma. In the case of ischemic stroke, blood fails to reach its target destination whereas in hemorrhagic stroke, extravasation of blood occurs outside of the blood vessel lumen, resulting in direct damage to brain parenchyma. As these acute events can be neurologically devastating, if not fatal, development of novel therapeutics are urgently needed. The zebrafish (Danio rerio) is an attractive model for the study of cerebrovascular disease because of its morphological and physiological similarity to human cerebral vasculature, its ability to be genetically manipulated, and its fecundity allowing for large-scale, phenotype-based screens. PMID:24517974

Walcott, Brian P; Peterson, Randall T

2014-01-01

7

Adult zebrafish model for pneumococcal pathogenesis.  

PubMed

Streptococcus pneumoniae (pneumococcus) is a leading cause of community acquired pneumonia, septicemia, and meningitis. Due to incomplete understanding of the host and bacterial factors contributing to these diseases optimal treatment and prevention methods are lacking. In the present study we examined whether the adult zebrafish (Danio rerio) can be used to investigate the pathophysiology of pneumococcal diseases. Here we show that both intraperitoneal and intramuscular injections of the pneumococcal strain TIGR4 cause a fulminant, dose-dependent infection in adult zebrafish, while isogenic mutant bacteria lacking the polysaccharide capsule, autolysin, or pneumolysin are attenuated in the model. Infection through the intraperitoneal route is characterized by rapid expansion of pneumococci in the bloodstream, followed by penetration of the blood-brain barrier and progression to meningitis. Using Rag1 mutant zebrafish, which are devoid of somatic recombination and thus lack adaptive immune responses, we show that clearance of pneumococci in adult zebrafish depends mainly on innate immune responses. In conclusion, this study provides evidence that the adult zebrafish can be used as a model for a pneumococcal infection, and that it can be used to study both host and bacterial factors involved in the pathogenesis. However, our results do not support the use of the zebrafish in studies on the role of adaptive immunity in pneumococcal disease or in the development of new pneumococcal vaccines. PMID:24076065

Saralahti, Anni; Piippo, Hannaleena; Parikka, Mataleena; Henriques-Normark, Birgitta; Rämet, Mika; Rounioja, Samuli

2014-02-01

8

Gaining translational momentum: more zebrafish models for neuroscience research.  

PubMed

Zebrafish (Danio rerio) are rapidly becoming a popular model organism in translational neuroscience and biological psychiatry research. Here we discuss conceptual, practical and other related aspects of using zebrafish in this field ("from tank to bedside"), and critically evaluate both advantages and limitations of zebrafish models of human brain disorders. We emphasize the need to more actively develop zebrafish models for neuroscience research focusing on complex traits. PMID:24593944

Kalueff, Allan V; Echevarria, David J; Stewart, Adam Michael

2014-12-01

9

Zebrafish model for human long QT syndrome.  

PubMed

Long QT syndrome (LQTS) is a disorder of ventricular repolarization that predisposes affected individuals to lethal cardiac arrhythmias. To date, an appropriate animal model of inherited LQTS does not exist. The zebrafish is a powerful vertebrate model used to dissect molecular pathways of cardiovascular development and disease. Because fundamental electrical properties of the zebrafish heart are remarkably similar to those of the human heart, the zebrafish may be an appropriate model for studying human inherited arrhythmias. Here we describe the molecular, cellular, and electrophysiological basis of a zebrafish mutant characterized by ventricular asystole. Genetic mapping and direct sequencing identify the affected gene as kcnh2, which encodes the channel responsible for the rapidly activating delayed rectifier K(+) current (I(Kr)). We show that complete loss of functional I(Kr) in embryonic hearts leads to ventricular cell membrane depolarization, inability to generate action potentials (APs), and disrupted calcium release. A small hyperpolarizing current restores spontaneous APs, implying wild-type function of other ionic currents critical for AP generation. Heterozygous fish manifest overt cellular and electrocardiographic evidence for delayed ventricular repolarization. Our findings provide insight into the pathogenesis of homozygous kcnh2 mutations and expand the use of zebrafish mutants as a model system to study human arrhythmias. PMID:17592134

Arnaout, Rima; Ferrer, Tania; Huisken, Jan; Spitzer, Kenneth; Stainier, Didier Y R; Tristani-Firouzi, Martin; Chi, Neil C

2007-07-01

10

Spermatogenesis in Ferret Testis Xenografts: A New Model  

PubMed Central

Testis xenografting is both a promising tool to study spermatogenesis and a means to preserve the genetic information and reproductive potential of prepubertal male animals. The present study was conducted to evaluate this technique using testis tissue from domestic ferrets, an important biomedical model and a model for the conservation of small carnivore species. Fresh testis tissue from 8-wk-old ferrets was implanted ectopically under the skin on the backs of castrated nude mice and subsequently evaluated for testosterone production and establishment of spermatogenesis at 10, 20, 25, and 30 wk after xenografting. A total of 40% of fresh ferret xenografts were harvested. Seminal vesicles were collected from the recipient mice and weighed as an assay for bioactive testosterone. The weights of seminal vesicles from the mice showed no significant difference from those of uncastrated, control nude mice, indicating that the xenografts were producing physiologically relevant amounts of testosterone. The ferret testis xenografts produced differentiating germ cells and sperm at the same time as did testis from age-matched control ferrets. These data demonstrate the ability of Mustelidae testicular tissue to establish spermatogenesis in nude mice after testis xenografting. PMID:21535925

Gourdon, Jim C; Travis, Alexander J

2011-01-01

11

Adult zebrafish as a model organism for behavioural genetics  

Microsoft Academic Search

Recent research has demonstrated the suitability of adult zebrafish to model some aspects of complex behaviour. Studies of reward behaviour, learning and memory, aggression, anxiety and sleep strongly suggest that conserved regulatory processes underlie behaviour in zebrafish and mammals. The isolation and molecular analysis of zebrafish behavioural mutants is now starting, allowing the identification of novel behavioural control genes. As

William Norton; Laure Bally-Cuif

2010-01-01

12

Zebrafish as an emerging model for studying complex brain disorders  

PubMed Central

The zebrafish (Danio rerio) is rapidly becoming a popular model organism in pharmacogenetics and neuropharmacology. Both larval and adult zebrafish are currently used to increase our understanding of brain function, dysfunction, and their genetic and pharmacological modulation. Here we review the developing utility of zebrafish in the analysis of complex brain disorders (including, for example, depression, autism, psychoses, drug abuse and cognitive disorders), also covering zebrafish applications towards the goal of modeling major human neuropsychiatric and drug-induced syndromes. We argue that zebrafish models of complex brain disorders and drug-induced conditions have become a rapidly emerging critical field in translational neuropharmacology research. PMID:24412421

Kalueff, Allan V.; Stewart, Adam Michael; Gerlai, Robert

2014-01-01

13

Zebrafish models of dyslipidemia: Relevance to atherosclerosis and angiogenesis  

PubMed Central

Lipid and lipoprotein metabolism in zebrafish and in humans are remarkably similar. Zebrafish express all major nuclear receptors, lipid transporters, apolipoproteins and enzymes involved in lipoprotein metabolism. Unlike mice, zebrafish express cetp and the Cetp activity is detected in zebrafish plasma. Feeding zebrafish a high cholesterol diet, without any genetic intervention, results in significant hypercholesterolemia and robust lipoprotein oxidation, making zebrafish an attractive animal model to study mechanisms relevant to early development of human atherosclerosis. These studies are facilitated by the optical transparency of zebrafish larvae and the availability of transgenic zebrafish expressing fluorescent proteins in endothelial cells and macrophages. Thus, vascular processes can be monitored in live animals. In this review article we discuss recent advances in using dyslipidemic zebrafish in atherosclerosis-related studies. We also summarize recent work connecting lipid metabolism with regulation of angiogenesis, the work that considerably benefited from using the zebrafish model. These studies uncovered the role of aibp, abca1, abcg1, mtp, apoB and apoC2 in regulation of angiogenesis in zebrafish and paved the way for future studies in mammals, which may suggest new therapeutic approaches to modulation of excessive or diminished angiogenesis contributing to the pathogenesis of human disease. PMID:24095954

Fang, Longhou; Liu, Chao; Miller, Yury I.

2013-01-01

14

Genomic profiling of patient-derived colon cancer xenograft models.  

PubMed

Recent evidence suggests that patient derived xenograft (PDX) models can maintain certain pathological and molecular features of the original disease. However, these characterizations are limited to immunohistochemistry or by tissue microarray analysis. We conducted a high-throughput sequencing of primary colon tumor and PDX has not been reported yet.Fresh primary colon cancer tissues that originate from surgery were implanted into the subcutaneous space of 6- to 8-week-old female BALB/c nu/nu or NOD/SCID mice and serially passaged in vivo. Ion AmpliSeq Cancer Hotspot Panel v2 (Ion Torrent) was used to detect frequent somatic mutations and similarity of molecular characteristics between the 10 patient tumors and matched PDX.Histologic and immunohistochemical analyses revealed a high degree of pathologic similarity including histologic architecture and expression of CEA, CK7, and CD20 between the patient and xenograft tumors. In 80% cases, all of the somatic mutations detected in primary tumor were concordantly detected in PDX models. However, 2 PDX models showed gained mutations such as PIK3CA or FBWX7 mutation.Ten patient-derived advanced colon cancer xenograft models were established. These models maintained the key characteristic features of the original tumors, suggesting useful tool for preclinical personalized medicine platform. PMID:25526474

Lee, Won-Suk; Kim, Hye-Youn; Seok, Jae Yeon; Jang, Ho Hee; Park, Yeon Ho; Kim, So-Young; Shin, Dong Bok; Hong, Suntaek

2014-12-01

15

Zebrafish as model organisms for studying drug induced liver injury  

PubMed Central

Drug induced liver injury (DILI) is a major challenge in clinical medicine and drug development. New models are needed for predicting which potential therapeutic compounds will cause DILI in humans, and new markers and mediators of DILI still need to be identified. This review will highlight the strengths and weaknesses of using zebrafish as a high throughput in vivo model for studying DILI. Although the zebrafish liver architecture is different to the mammalian liver, the main physiological processes remain similar. Zebrafish metabolize drugs using similar pathways as humans; they possess a wide range of cytochrome P450 enzymes enabling metabolic reactions including hydroxylation, conjugation, oxidation, demethylation and de-ethylation. Following exposure to a range of liver toxic drugs, the zebrafish liver develops histological patterns of injury comparable to mammals and liver injury biomarkers can be quantified in the zebrafish circulation. The zebrafish immune system is similar to mammals, but the zebrafish inflammatory response to DILI is not yet defined. To quantify DILI in zebrafish a wide variety of methods can be used including: visual assessment, quantification of serum enzymes and experimental serum biomarkers and scoring histopathology. With further development, the zebrafish may be a model that complements rodents and may have value for the discovery of new disease pathways and translational biomarkers. PMID:24773296

Vliegenthart, A D B; Tucker, C S; Del Pozo, J; Dear, J W

2014-01-01

16

Using the zebrafish model for Alzheimer’s disease research  

PubMed Central

Rodent models have been extensively used to investigate the cause and mechanisms behind Alzheimer’s disease. Despite many years of intensive research using these models we still lack a detailed understanding of the molecular events that lead to neurodegeneration. Although zebrafish lack the complexity of advanced cognitive behaviors evident in rodent models they have proven to be a very informative model for the study of human diseases. In this review we give an overview of how the zebrafish has been used to study Alzheimer’s disease. Zebrafish possess genes orthologous to those mutated in familial Alzheimer’s disease and research using zebrafish has revealed unique characteristics of these genes that have been difficult to observe in rodent models. The zebrafish is becoming an increasingly popular model for the investigation of Alzheimer’s disease and will complement studies using other models to help complete our understanding of this disease. PMID:25071820

Newman, Morgan; Ebrahimie, Esmaeil; Lardelli, Michael

2014-01-01

17

Adult zebrafish as a model organism for behavioural genetics  

PubMed Central

Recent research has demonstrated the suitability of adult zebrafish to model some aspects of complex behaviour. Studies of reward behaviour, learning and memory, aggression, anxiety and sleep strongly suggest that conserved regulatory processes underlie behaviour in zebrafish and mammals. The isolation and molecular analysis of zebrafish behavioural mutants is now starting, allowing the identification of novel behavioural control genes. As a result of this, studies of adult zebrafish are now helping to uncover the genetic pathways and neural circuits that control vertebrate behaviour. PMID:20678210

2010-01-01

18

Host-Pathogen Interactions Made Transparent with the Zebrafish Model  

PubMed Central

The zebrafish holds much promise as a high-throughput drug screening model for immune-related diseases, including inflammatory and infectious diseases and cancer. This is due to the excellent possibilities for in vivo imaging in combination with advanced tools for genomic and large scale mutant analysis. The context of the embryo’s developing immune system makes it possible to study the contribution of different immune cell types to disease progression. Furthermore, due to the temporal separation of innate immunity from adaptive responses, zebrafish embryos and larvae are particularly useful for dissecting the innate host factors involved in pathology. Recent studies have underscored the remarkable similarity of the zebrafish and human immune systems, which is important for biomedical applications. This review is focused on the use of zebrafish as a model for infectious diseases, with emphasis on bacterial pathogens. Following a brief overview of the zebrafish immune system and the tools and methods used to study host-pathogen interactions in zebrafish, we discuss the current knowledge on receptors and downstream signaling components that are involved in the zebrafish embryo’s innate immune response. We summarize recent insights gained from the use of bacterial infection models, particularly the Mycobacterium marinum model, that illustrate the potential of the zebrafish model for high-throughput antimicrobial drug screening. PMID:21366518

Meijer, Annemarie H; Spaink, Herman P

2011-01-01

19

A bioenergetic model for zebrafish Danio rerio (Hamilton)  

USGS Publications Warehouse

A bioenergetics model was developed from observed consumption, respiration and growth rates for zebrafish Danio rerio across a range (18-32?? C) of water temperatures, and evaluated with a 50 day laboratory trial at 28?? C. No significant bias in variable estimates was found during the validation trial; namely, predicted zebrafish mass generally agreed with observed mass. ?? 2008 The Authors.

Chizinski, C.J.; Sharma, B.; Pope, K.L.; Patino, R.

2008-01-01

20

montalcino, a Zebrafish Model for Variegate Porphyria  

PubMed Central

Objective Inherited or acquired mutations in the heme biosynthetic pathway lead to a debilitating class of diseases collectively known as porphyrias, with symptoms that can include anemia, cutaneous photosensitivity, and neurovisceral dysfunction. In a genetic screen for hematopoietic mutants, we isolated a zebrafish mutant, montalcino (mno), which displays hypochromic anemia and porphyria. The objective of this study was to identify the defective gene and characterize the phenotype of the zebrafish mutant. Methods Genetic linkage analysis was utilized to identify the region harboring the mno mutation. Candidate gene analysis together with RT-PCR was utilized to identify the genetic mutation, which was confirmed via allele specific oligo hybridizations. Whole mount in situ hybridizations and 0-dianisidine staining were used to characterize the phenotype of the mno mutant. mRNA and morpholino microinjections were performed to phenocopy and/or rescue the mutant phenotype. Results Homozygous mno mutant embryos have a defect in the protoporphyrinogen oxidase (ppox) gene, which encodes the enzyme that catalyzes the oxidation of protoporphyrinogen. Homozygous mutant embryos are deficient in hemoglobin, and by 36 hpf are visibly anemic and porphyric. The hypochromic anemia of mno embryos was partially rescued by human ppox, providing evidence for the conservation of function between human and zebrafish ppox. Conclusion In humans, mutations in ppox result in variegate porphyria. At present, effective treatment for acute attacks requires the administration intravenous hemin and/or glucose. Thus, mno represents a powerful model for investigation, and a tool for future screens aimed at identifying chemical modifiers of variegate porphyria. PMID:18550261

Dooley, Kimberly A.; Fraenkel, Paula G.; Langer, Nathaniel B.; Schmid, Bettina; Davidson, Alan J.; Weber, Gerhard; Chiang, Ken; Foott, Helen; Dwyer, Caitlin; Wingert, Rebecca A.; Zhou, Yi; Paw, Barry H.; Zon, Leonard I.

2008-01-01

21

Adult Zebrafish model of streptococcal infection  

PubMed Central

Streptococcal pathogens cause a wide array of clinical syndromes in humans, including invasive systemic infections resulting in high mortality rates. Many of these pathogens are human specific, and therefore difficult to analyze in vivo using typical animal models, as these models rarely replicate what is observed in human infections. This unit describes the use of the zebrafish (Danio rerio) as an animal model for streptococcal infection to analyze multiple disease states. This model closely mimics the necrotizing fasciitis/myositis pathology observed in humans from a Streptococcus pyogenes infection. The use of a zoonotic pathogen, Streptococcus iniae, which replicates systemic infections caused by many streptococcal pathogens, including dissemination to the brain, is also described. Included protocols describe both intraperitoneal and intramuscular infections, as well as methods for histological and quantitative measurements of infection. PMID:19412913

Phelps, Hilary A.; Runft, Donna L.

2009-01-01

22

Xenograft models for preclinical drug testing: implications for adrenocortical cancer.  

PubMed

Adrenocortical carcinoma (ACC) is a very rare but aggressive tumor, whose biological and cellular features and processes underlying the development, progression and metastatic evolution are still obscure. Despite many attempts to use general cytostatic and cytotoxic drugs, the only available drug therapy for advanced ACC is still represented by mitotane (MTT). However, the mechanism of action of this adrenolytic derivative of the pesticide DDT has still been poorly characterized. In this context, the development of more specific drugs for ACC treatment is based on the knowledge of the molecular pathways involved in the tumor growth. Xenograft models for the screening of such drugs at preclinical levels is mandatory. In the first part of this review, we will summarize the "pro" and "con" of the different xenograft models available for anticancer drug testing in different types of tumors in general and in the last part, we will focus on the preclinical evidence obtained so far with the use of such models applied to drug screening for anticancer effects in ACC. PMID:22056412

Luconi, Michaela; Mannelli, Massimo

2012-03-31

23

Development and characterization of xenograft model systems for adenoid cystic carcinoma  

PubMed Central

Adenoid Cystic Carcinoma (ACC) is one of the most common malignancies to arise in human salivary glands, and also arises in glandular tissue of other organ systems. To address the paucity of experimental model systems for this tumor type, we have undertaken a program of transplanting tissue samples of human ACC into immunodeficient nu/nu mice to create xenograft model systems. In 17 of 23 attempts (74%) xenograft tumors were successfully grown. In all cases, the histologic appearance of the donating tumor was recapitulated in the subsequent xenograft. Characterization of a subset of xenograft models by immunohistochemical biomarkers and by RNA transcript microarray analysis showed good fidelity in the recapitulation of gene expression patterns in the xenograft tumors compared to the human donor tumors. Since ACC is known to frequently contain a t(6;9) translocation that fuses the MYB and NFIB genes, fluorescence in situ hybridization (FISH) of twelve ACC xenograft models was performed that assayed MYB locus break-apart and MYB-NFIB locus fusion. 11/12 (92%) xenograft models revealed MYB locus rearrangement and 10/12 (83%) xenograft models showed evidence of fusion of the MYB and NFIB loci. The two related xenograft models (derived from primary and metastatic tumors, respectively, of the same human subject) were karyotyped, showing a t(1;6) translocation, suggesting MYB translocation to a novel fusion partner gene. Overall, our results indicate that ACC is amenable to xenografting and that ACC xenograft models recapitulate the molecular and morphologic characteristics of human tumors, suggesting utility as valid experimental and preclinical model systems for this disease. PMID:21709671

Moskaluk, Christopher A.; Baras, Alexander S.; Mancuso, Stefani; Fan, Hao; Davidson, Robert; Dirks, Dawn; Golden, Wendy; Frierson, Henry F.

2014-01-01

24

Human Tumor Xenograft Models for Preclinical Assessment of Anticancer Drug Development  

PubMed Central

Xenograft models of human cancer play an important role in the screening and evaluation of candidates for new anticancer agents. The models, which are derived from human tumor cell lines and are classified according to the transplant site, such as ectopic xenograft and orthotopic xenograft, are still utilized to evaluate therapeutic efficacy and toxicity. The metastasis model is modified for the evaluation and prediction of cancer progression. Recently, animal models are made from patient-derived tumor tissue. The patient-derived tumor xenograft models with physiological characters similar to those of patients have been established for personalized medicine. In the discovery of anticancer drugs, standard animal models save time and money and provide evidence to support clinical trials. The current strategy for using xenograft models as an informative tool is introduced. PMID:24795792

2014-01-01

25

Fit for consumption: zebrafish as a model for tuberculosis  

PubMed Central

Despite efforts to generate new vaccines and antibiotics for tuberculosis, the disease remains a public health problem worldwide. The zebrafish Danio rerio has emerged as a useful model to investigate mycobacterial pathogenesis and treatment. Infection of zebrafish with Mycobacterium marinum, the closest relative of the Mycobacterium tuberculosis complex, recapitulates many aspects of human tuberculosis. The zebrafish model affords optical transparency, abundant genetic tools and in vivo imaging of the progression of infection. Here, we review how the zebrafish–M. marinum system has been deployed to make novel observations about the role of innate immunity, the tuberculous granuloma, and crucial host and bacterial genes. Finally, we assess how these findings relate to human disease and provide a framework for novel strategies to treat tuberculosis. PMID:24973748

Cronan, Mark R.; Tobin, David M.

2014-01-01

26

The zebrafish as a model for complex tissue regeneration  

PubMed Central

For centuries, philosophers and scientists have been fascinated by the principles and implications of regeneration in lower vertebrate species. Two features have made zebrafish an informative model system for determining mechanisms of regenerative events. First, they are highly regenerative, able to regrow amputated fins, as well as a lesioned brain, retina, spinal cord, heart, and other tissues. Second, they are amenable to both forward and reverse genetic approaches, with a research toolset regularly updated by an expanding community of zebrafish researchers. Zebrafish studies have helped identify new mechanistic underpinnings of regeneration in multiple tissues, and in some cases have served as a guide for contemplating regenerative strategies in mammals. Here, we review the recent history of zebrafish as a genetic model system for understanding how and why tissue regeneration occurs. PMID:23927865

Gemberling, Matthew; Bailey, Travis J.; Hyde, David R.; Poss, Kenneth D.

2013-01-01

27

Zebrafish ( Danio rerio) embryos as a model for testing proteratogens  

Microsoft Academic Search

Zebrafish embryos have been shown to be a useful model for the detection of direct acting teratogens. This communication presents a protocol for a 3-day in vitro zebrafish embryo teratogenicity assay and describes results obtained for 10 proteratogens: 2-acetylaminofluorene, benzo[a]pyrene, aflatoxin B1, carbamazepine, phenytoin, trimethadione, cyclophosphamide, ifosfamide, tegafur and thio-TEPA. The selection of the test substances accounts for differences in

Stefan Weigt; Nicole Huebler; Ruben Strecker; Thomas Braunbeck; Thomas H. Broschard

2011-01-01

28

The emerging use of zebrafish to model metabolic disease  

PubMed Central

The zebrafish research community is celebrating! The zebrafish genome has recently been sequenced, the Zebrafish Mutation Project (launched by the Wellcome Trust Sanger Institute) has published the results of its first large-scale ethylnitrosourea (ENU) mutagenesis screen, and a host of new techniques, such as the genome editing technologies TALEN and CRISPR-Cas, are enabling specific mutations to be created in model organisms and investigated in vivo. The zebrafish truly seems to be coming of age. These powerful resources invoke the question of whether zebrafish can be increasingly used to model human disease, particularly common, chronic diseases of metabolism such as obesity and type 2 diabetes. In recent years, there has been considerable success, mainly from genomic approaches, in identifying genetic variants that are associated with these conditions in humans; however, mechanistic insights into the role of implicated disease loci are lacking. In this Review, we highlight some of the advantages and disadvantages of zebrafish to address the organism’s utility as a model system for human metabolic diseases. PMID:24046387

Seth, Asha; Stemple, Derek L.; Barroso, Inês

2013-01-01

29

Conservation and Early Expression of Zebrafish Tyrosine Kinases Support the Utility of Zebrafish as a Model for Tyrosine Kinase Biology  

PubMed Central

Abstract Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome. PMID:23234507

Challa, Anil Kumar

2013-01-01

30

A zebrafish retinal graded photochemical stress model  

PubMed Central

Introduction In order to develop a model for investigating the genes that contribute to retinal degeneration, we examined the early graded photochemical stress response in the adult zebrafish (Danio rerio) retina and investigated the role of an NMDA inhibitor, thiokynurenate. Methods Following intravitreal injection of rose bengal (6 or 12 mg/mL), light (37×103 or 83×103 lx) was directed onto the central retina with and without 400 nM thiokynurenate. Histologic and electron microscopic analysis was performed at 2 and 4 h and gene expression analysis was carried out at 2, 4 and 6 h. Results Light and electron microscopy demonstrated a graded photochemical response in photoreceptor, nuclear, and ganglion cell layer thickness. Increased vacuolation of the inner plexiform layer was also observed. The inhibitor produced a distinct lesion pattern. Cellular stress genes were elevated in low and high lesions, while some homeobox gene expression was reduced with thiokynurenate. Discussion The phenotypic and genetic changes observed from this model can serve as a basis for understanding the pathology of retinal oxidative and cellular stress. These changes may aid our understanding of aging and macular degeneration. PMID:19269339

Eichenbaum, Joseph W.; Cinaroglu, Ayca; Eichenbaum, Kenneth D.; Sadler, Kirsten C.

2013-01-01

31

Modeling tuberculous meningitis in zebrafish using Mycobacterium marinum  

PubMed Central

Tuberculous meningitis (TBM) is one of the most severe extrapulmonary manifestations of tuberculosis, with a high morbidity and mortality. Characteristic pathological features of TBM are Rich foci, i.e. brain- and spinal-cord-specific granulomas formed after hematogenous spread of pulmonary tuberculosis. Little is known about the early pathogenesis of TBM and the role of Rich foci. We have adapted the zebrafish model of Mycobacterium marinum infection (zebrafish–M. marinum model) to study TBM. First, we analyzed whether TBM occurs in adult zebrafish and showed that intraperitoneal infection resulted in granuloma formation in the meninges in 20% of the cases, with occasional brain parenchyma involvement. In zebrafish embryos, bacterial infiltration and clustering of infected phagocytes was observed after infection at three different inoculation sites: parenchyma, hindbrain ventricle and caudal vein. Infection via the bloodstream resulted in the formation of early granulomas in brain tissue in 70% of the cases. In these zebrafish embryos, infiltrates were located in the proximity of blood vessels. Interestingly, no differences were observed when embryos were infected before or after early formation of the blood-brain barrier (BBB), indicating that bacteria are able to cross this barrier with relatively high efficiency. In agreement with this observation, infected zebrafish larvae also showed infiltration of the brain tissue. Upon infection of embryos with an M. marinum ESX-1 mutant, only small clusters and scattered isolated phagocytes with high bacterial loads were present in the brain tissue. In conclusion, our adapted zebrafish–M. marinum infection model for studying granuloma formation in the brain will allow for the detailed analysis of both bacterial and host factors involved in TBM. It will help solve longstanding questions on the role of Rich foci and potentially contribute to the development of better diagnostic tools and therapeutics. PMID:24997190

van Leeuwen, Lisanne M.; van der Kuip, Martijn; Youssef, Sameh A.; de Bruin, Alain; Bitter, Wilbert; van Furth, A. Marceline; van der Sar, Astrid M.

2014-01-01

32

Usefulness of the nude mouse model in mesothelioma based on a direct patient-xenograft comparison.  

PubMed

A patient with malignant mesothelioma experienced tumor recurrence 3 months after pleuropneumonectomy. Samples of the tumor were transplanted into nude mice to assess chemosensitivity. There was close concordance between the results in xenografts and the clinical outcome in this patient. Both mitomycin and to a lesser extent cisplatin were effective as single agents against the nude mouse xenografts, and the combination of these two drugs produced a complete response both in the patient and in the xenografts. The patient survived 18 months from onset of chemotherapy and 24 months from diagnosis. The duration of clinical complete response to chemotherapy was 14 months, despite the fact that mitomycin, the most effective agent against the xenografts, was discontinued after only two cycles because the patient developed pulmonary toxicity. This direct patient-xenograft correlation further validates the usefulness of the nude mouse model in the search for effective therapies for malignant mesothelioma, a tumor characterized by frequent refractoriness to most available agents. PMID:2065276

Chahinian, A P; Kirschner, P A; Gordon, R E; Szrajer, L; Holland, J F

1991-08-01

33

A jump persistent turning walker to model zebrafish locomotion.  

PubMed

Zebrafish are gaining momentum as a laboratory animal species for the investigation of several functional and dysfunctional biological processes. Mathematical models of zebrafish behaviour are expected to considerably aid in the design of hypothesis-driven studies by enabling preliminary in silico tests that can be used to infer possible experimental outcomes without the use of zebrafish. This study is motivated by observations of sudden, drastic changes in zebrafish locomotion in the form of large deviations in turn rate. We demonstrate that such deviations can be captured through a stochastic mean reverting jump diffusion model, a process that is commonly used in financial engineering to describe large changes in the price of an asset. The jump process-based model is validated on trajectory data of adult subjects swimming in a shallow circular tank obtained from an overhead camera. Through statistical comparison of the empirical distribution of the turn rate against theoretical predictions, we demonstrate the feasibility of describing zebrafish as a jump persistent turning walker. The critical role of the jump term is assessed through comparison with a simplified mean reversion diffusion model, which does not allow for describing the heavy-tailed distributions observed in the fish turn rate. PMID:25392396

Mwaffo, Violet; Anderson, Ross P; Butail, Sachit; Porfiri, Maurizio

2015-01-01

34

Zebrafish as a model system for mitochondrial biology and diseases.  

PubMed

Animal models for studying human disease are essential to the continuing evolution of medicine. Rodent models are attractive for the obvious similarities in development and genetic makeup compared with humans, but have cost and technical limitations. The zebrafish (Danio rerio) represents an ideal alternative vertebrate model of human disease because of its high conservation of genetic information and physiological processes, inexpensive maintenance, and optical clarity facilitating direct observation. This review highlights recent advances in understanding genetic disease states associated with the dynamic organelle, the mitochondrion, using the zebrafish. Mitochondrial diseases that have been replicated in the zebrafish include those affecting the nervous and cardiovascular systems, as well as red blood cell function. Gene silencing techniques, including morpholino knockdown and transcription activator-like (TAL)-effector endonucleases, have been exploited to demonstrate how loss of function can induce human disease-like states in zebrafish. Moreover, modeling mitochondrial diseases has been facilitated greatly by the creation of transgenic fish with fluorescently labeled mitochondria for in vivo visualization of these structures. In addition, behavioral assays have been developed to examine changes in motor activity and sensory responses, particularly in larval stages. Zebrafish are poised to advance our understanding of the pathogenesis of human mitochondrial diseases beyond the current state of knowledge and provide a key tool in the development of novel therapeutic approaches to treat these conditions. PMID:24055494

Steele, Shelby L; Prykhozhij, Sergey V; Berman, Jason N

2014-02-01

35

Zebrafish: A Model for the Study of Addiction Genetics  

PubMed Central

Drug abuse and dependence are multifaceted disorders with complex genetic underpinnings. Identifying specific genetic correlates is challenging and may be more readily accomplished by defining endophenotypes specific for addictive disorders. Symptoms and syndromes, including acute drug response, consumption, preference, and withdrawal, are potential endophenotypes characterizing addiction that have been investigated using model organisms. We present a review of major genes involved in serotonergic, dopaminergic, GABAergic, and adrenoreceptor signaling that are considered to be directly involved in nicotine, opioid, cannabinoid, and ethanol use and dependence. The zebrafish genome encodes likely homologs of the vast majority of these loci. We also review the known expression patterns of these genes in zebrafish. The information presented in this review provides support for the use of zebrafish as a viable model for studying genetic factors related to drug addiction. Expansion of investigations into drug response using model organisms holds the potential to advance our understanding of drug response and addiction in humans. PMID:22207143

Klee, Eric W; Schneider, Henning; Clark, Karl; Cousin, Margot; Ebbert, Jon; Hooten, Michael; Karpyak, Victor; Warner, David; Ekker, Stephen

2013-01-01

36

Using engineered endonucleases to create knockout and knockin zebrafish models.  

PubMed

Over the last few years, the technology to create targeted knockout and knockin zebrafish animals has exploded. We have gained the ability to create targeted knockouts through the use of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR associated system (CRISPR/Cas). Furthermore, using the high-efficiency TALEN system, we were able to create knockin zebrafish using a single-stranded DNA (ssDNA) protocol described here. Through the use of these technologies, the zebrafish has become a valuable vertebrate model and an excellent bridge between the invertebrate and mammalian model systems for the study of human disease. PMID:25408414

Bedell, Victoria M; Ekker, Stephen C

2015-01-01

37

Zebrafish (Danio rerio) embryos as a model for testing proteratogens.  

PubMed

Zebrafish embryos have been shown to be a useful model for the detection of direct acting teratogens. This communication presents a protocol for a 3-day in vitro zebrafish embryo teratogenicity assay and describes results obtained for 10 proteratogens: 2-acetylaminofluorene, benzo[a]pyrene, aflatoxin B(1), carbamazepine, phenytoin, trimethadione, cyclophosphamide, ifosfamide, tegafur and thio-TEPA. The selection of the test substances accounts for differences in structure, origin, metabolism and water solubility. Apart from 2-acetylaminofluorene, which mainly produces lethal effects, all proteratogens tested were teratogenic in zebrafish embryos exposed for 3 days. The test substances and/or the substance class produced characteristic patterns of fingerprint endpoints. Several substances produced effects that could be identified already at 1 dpf (days post fertilization), whereas the effects of others could only be identified unambiguously after hatching at ? 3 dpf. The LC?? and EC?? values were used to calculate the teratogenicity index (TI) for the different substances, and the EC?? values were related to human plasma concentrations. Results lead to the conclusion that zebrafish embryos are able to activate proteratogenic substances without addition of an exogenous metabolic activation system. Moreover, the teratogenic effects were observed at concentrations relevant to human exposure data. Along with other findings, our results indicate that zebrafish embryos are a useful alternative method for traditional teratogenicity testing with mammalian species. PMID:21237239

Weigt, Stefan; Huebler, Nicole; Strecker, Ruben; Braunbeck, Thomas; Broschard, Thomas H

2011-03-15

38

Embryonic Senescence and Laminopathies in a Progeroid Zebrafish Model  

PubMed Central

Background Mutations that disrupt the conversion of prelamin A to mature lamin A cause the rare genetic disorder Hutchinson-Gilford progeria syndrome and a group of laminopathies. Our understanding of how A-type lamins function in vivo during early vertebrate development through aging remains limited, and would benefit from a suitable experimental model. The zebrafish has proven to be a tractable model organism for studying both development and aging at the molecular genetic level. Zebrafish show an array of senescence symptoms resembling those in humans, which can be targeted to specific aging pathways conserved in vertebrates. However, no zebrafish models bearing human premature senescence currently exist. Principal Findings We describe the induction of embryonic senescence and laminopathies in zebrafish harboring disturbed expressions of the lamin A gene (LMNA). Impairments in these fish arise in the skin, muscle and adipose tissue, and sometimes in the cartilage. Reduced function of lamin A/C by translational blocking of the LMNA gene induced apoptosis, cell-cycle arrest, and craniofacial abnormalities/cartilage defects. By contrast, induced cryptic splicing of LMNA, which generates the deletion of 8 amino acid residues lamin A (zlamin A-?8), showed embryonic senescence and S-phase accumulation/arrest. Interestingly, the abnormal muscle and lipodystrophic phenotypes were common in both cases. Hence, both decrease-of-function of lamin A/C and gain-of-function of aberrant lamin A protein induced laminopathies that are associated with mesenchymal cell lineages during zebrafish early development. Visualization of individual cells expressing zebrafish progerin (zProgerin/zlamin A-?37) fused to green fluorescent protein further revealed misshapen nuclear membrane. A farnesyltransferase inhibitor reduced these nuclear abnormalities and significantly prevented embryonic senescence and muscle fiber damage induced by zProgerin. Importantly, the adult Progerin fish survived and remained fertile with relatively mild phenotypes only, but had shortened lifespan with obvious distortion of body shape. Conclusion We generated new zebrafish models for a human premature aging disorder, and further demonstrated the utility for studying laminopathies. Premature aging could also be modeled in zebrafish embryos. This genetic model may thus provide a new platform for future drug screening as well as genetic analyses aimed at identifying modifier genes that influence not only progeria and laminopathies but also other age-associated human diseases common in vertebrates. PMID:21479207

Koshimizu, Eriko; Imamura, Shintaro; Qi, Jie; Toure, Jamal; Valdez, Delgado M.; Carr, Christopher E.; Hanai, Jun-ichi; Kishi, Shuji

2011-01-01

39

Improvement of Radiation-Mediated Immunosuppression of Human NSCLC Tumour Xenografts in a Nude Rat Model  

PubMed Central

Human tumour xenografts in a nude rat model have consistently been used as an essential part of preclinical studies for anticancer drugs activity in human. Commonly, these animals receive whole body irradiation to assure immunosuppression. But whole body dose delivery might be inhomogeneous and the resulting incomplete bone marrow depletion may modify tumour behaviour. To improve irradiation-mediated immunosuppression of human non-small cell lung cancer (NSCLC) xenografts in a nude rat model irradiation (2 + 2 Gy) from opposite sides of animals has been performed using a conventional X-ray tube. The described modification of whole body irradiation improves growth properties of human NSCLC xenografts in a nude rat model. The design of the whole body irradiation mediated immunosuppression described here for NSCLC xenografts may be useful for research applications involving other types of human tumours. PMID:20368799

Tokalov, Sergey V.; Enghardt, Wolfgang; Abolmaali, Nasreddin

2010-01-01

40

Developing an Experimental Model of Vascular Toxicity in Embryonic Zebrafish  

EPA Science Inventory

Developing an Experimental Model of Vascular Toxicity in Embryonic Zebrafish Tamara Tal, Integrated Systems Toxicology Division, U.S. EPA Background: There are tens of thousands of chemicals that have yet to be fully evaluated for their toxicity by validated in vivo testing ...

41

Modeling PTSD in the zebrafish: are we there yet?  

PubMed

Post-traumatic stress disorder is an anxiety disorder that can develop following one or more traumatic events that threaten one's safety or make the victim feel helpless. Currently there are an increasing number of cases in the population in part due to the number of soldiers returning from combat. The disorder is characterized by symptoms that include hypervigilance, sleep disturbances, social and cognitive degradation, and memory flashbacks. Most of the research has been centered on the human and rodent as subjects but recently another viable contender has emerged - the zebrafish (Danio rerio). The zebrafish is a strong comparative model with the ability to exhibit a wide variety of behaviors, complex learning, and neurobiological changes that can be extrapolated to the human condition. The zebrafish is an ideal organism to study pharmacological treatments as well as the neurological underpinnings of the disorder. Here we review a sampling of the human and rodent model literature on post-traumatic stress disorder focusing on symptomology, current treatments, and stress paradigms. We also make the argument for the inclusion of the zebrafish model in future studies investigating the causes, symptoms, and treatments of post-traumatic stress disorder. PMID:24821404

Caramillo, Erika M; Khan, Kanza M; Collier, Adam D; Echevarria, David J

2015-01-01

42

Zebrafish model for allogeneic hematopoietic cell transplantation not requiring preconditioning  

PubMed Central

Recent work on vertebrate hematopoiesis has uncovered the presence of deeply rooted similarities between fish and mammals at molecular and cellular levels. Although small animal models such as zebrafish are ideally suited for genetic and chemical screens, the study of cellular aspects of hematopoietic development in lower vertebrates is severely hampered by the complex nature of their histocompatibility-determining genes. Hence, even when hosts are sublethally irradiated before hematopoietic cell transplantation, stable and long-term reconstitution by allogeneic stem cells often fails. Here, we describe the unexpected observation that transplantation and maintenance of allogeneic hematopoietic stem cells in zebrafish homozygous for the c-mybt25127 allele, carrying a missense mutation (Ile181Asn) in the DNA binding domain can be achieved without prior conditioning. Using this model, we examined several critical parameters of zebrafish hematopoiesis in a near-physiological setting. Limiting dilution analysis suggests that the kidney marrow of adult zebrafish harbors about 10 transplantable hematopoietic stem cells; this tissue also contains thymus-settling precursors that colonize the thymic rudiment within days after transplantation and initiate robust T-cell development. We also demonstrate that c-myb mutants can be stably reconstituted with hematopoietic cells carrying specific genetic defects in lymphocyte development, exemplifying one of the many potential uses of this model in experimental hematology. PMID:23431192

Hess, Isabell; Iwanami, Norimasa; Schorpp, Michael; Boehm, Thomas

2013-01-01

43

A zebrafish model for subgenomic hepatitis C virus replication.  

PubMed

Persistent infection with hepatitis C virus (HCV) is a major risk factor in the development of hepatocellular carcinoma. The elucidation of the pathogenesis of HCV-associated liver disease is hampered by the absence of an appropriate small animal model. Zebrafish exhibits high genetic homology to mammals, and is easily manipulated experimentally. In this study, we describe the use of a zebrafish model for the analysis of HCV replication mechanisms. As the 5' untranslated region (UTR), the core protein, the non-structural protein 5B (NS5B) and the 3'UTR are essential for HCV replication, we constructed a HCV sub-replicon gene construct including the 4 gene sequences and the enhanced green fluorescent protein (EGFP) reporter gene; these genes were transcribed through the mouse hepatocyte nuclear factor 4 (mHNF4) promoter. By microinjection of the subgenomic replicon vector into zebrafish larvae, the virus was easily detected by observing EGFP fluorescence in the liver. The positive core and NS5B signals showed positive expression of the HCV gene construct in zebrafish by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Importantly, the negative strand sequence of the HCV subgenomic RNA was detected by RT-PCR and hybridization in situ, demonstrating that the HCV sub-replicon has positive replication activity. Furthermore, the hybridization signal mainly appeared in the liver region of larvae, as detected by the sense probe of the core protein or NS5B, which confirmed that the sub-replicon amplification occurred in the zebrafish liver. The amplification of the sub-replicon caused alterations in the expression of certain genes, which is similar to HCV infection in human liver cells. To verify the use of this zebrafish model in drug evaluation, two drugs against HCV used in clinical practice, ribavirin and oxymatrine, were tested and these drugs showed significant inhibition of replication of the HCV sub-replicon in the larvae. In conclusion, this zebrafish model of HCV may prove to be a novel and simple in vivo model for the study of the mechanisms of HCV replication and may also prove useful in the disovery of new anti-HCV drugs. PMID:25572289

Ding, Cun-Bao; Zhao, Ye; Zhang, Jing-Pu; Peng, Zong-Gen; Song, Dan-Qing; Jiang, Jian-Dong

2015-03-01

44

Genetic modeling of Li-Fraumeni syndrome in zebrafish  

PubMed Central

SUMMARY Li-Fraumeni syndrome (LFS) is a highly penetrant, autosomal dominant, human familial cancer predisposition. Although a key role for the tumor suppressor p53 has been implicated in LFS, the genetic and cellular mechanisms underpinning this disease remain unknown. Therefore, modeling LFS in a vertebrate system that is accessible to both large-scale genetic screens and in vivo cell biological studies will facilitate the in vivo dissection of disease mechanisms, help identify candidate genes, and spur the discovery of therapeutic compounds. Here, we describe a forward genetic screen in zebrafish embryos that was used to identify LFS candidate genes, which yielded a p53 mutant (p53I166T) that as an adult develops tumors, predominantly sarcomas, with 100% penetrance. As in humans with LFS, tumors arise in heterozygotes and display loss of heterozygosity (LOH). This report of LOH indicates that Knudson’s two-hit hypothesis, a hallmark of human autosomal dominant cancer syndromes, can be modeled in zebrafish. Furthermore, as with some LFS mutations, the zebrafish p53I166T allele is a loss-of-function allele with dominant-negative activity in vivo. Additionally, we demonstrate that the p53 regulatory pathway, including Mdm2 regulation, is evolutionarily conserved in zebrafish, providing a bona fide biological context in which to systematically uncover novel modifier genes and therapeutic agents for human LFS. PMID:20075382

Parant, John M.; George, Stephen A.; Holden, Joseph A.; Yost, H. Joseph

2010-01-01

45

A new model to study visual attention in zebrafish.  

PubMed

The major part of cognitive tasks applied to zebrafish has not fully assessed their attentional ability, a process by which the nervous system learns, organizes sensory input and generates coordinated behaviour. In an attempt to maximize the value of zebrafish as an animal model of cognition, we tested the possibility to apply a modified version of novel object recognition test named virtual object recognition test (VORT) using 2D geometrical shapes (square, triangle, circle, cross, etc.) on two iPod 3.5-inch widescreen displays, located on two opposite walls of the water tank. Each fish was subjected to a familiarization trial (T1), and after different time delays (from 5 min to 96 h) to a novel shape recognition trial (T2). A progressive decrease, across time, of memory performance, in terms of mean discrimination index and mean exploration time, was shown. The predictive validity was tested using cholinergic drugs. Nicotine (0.02 mg/kg intraperitoneally, IP) significantly increased, while scopolamine (0.025 mg/kg IP) and mecamylamine decreased, mean discrimination index. Zebrafish discriminated different movements (vertical, horizontal, oblique) and the discrimination index increased significantly when moving poorly discriminated shapes were presented, thus increasing visual attention. Taken together these findings demonstrate that VORT is a viable, fast and useful model to evaluate sustained attention in zebrafish and for predicting the efficacy of pharmacotherapies for cognitive disorders. PMID:24681194

Braida, Daniela; Ponzoni, Luisa; Martucci, Roberta; Sala, Mariaelvina

2014-12-01

46

A Synthetic dl-Nordihydroguaiaretic acid (Nordy), Inhibits Angiogenesis, Invasion and Proliferation of Glioma Stem Cells within a Zebrafish Xenotransplantation Model  

PubMed Central

The zebrafish (Danio rerio) and their transparent embryos represent a promising model system in cancer research. Compared with other vertebrate model systems, we had previously shown that the zebrafish model provides many advantages over mouse or chicken models to study tumor invasion, angiogenesis, and tumorigenesis. In this study, we systematically investigated the biological features of glioma stem cells (GSCs) in a zebrafish model, such as tumor angiogenesis, invasion, and proliferation. We demonstrated that several verified anti-angiogenic agents inhibited angiogenesis that was induced by xenografted-GSCs. We next evaluated the effects of a synthetic dl-nordihydroguaiaretic acid compound (dl-NDGA or “Nordy”), which revealed anti-tumor activity against human GSCs in vitro by establishing parameters through studying its ability to suppress angiogenesis, tumor invasion, and proliferation. Furthermore, our results indicated that Nordy might inhibit GSCs invasion and proliferation through regulation of the arachidonate 5-lipoxygenase (Alox-5) pathway. Moreover, the combination of Nordy and a VEGF inhibitor exhibited an enhanced ability to suppress angiogenesis that was induced by GSCs. By contrast, even following treatment with 50 µM Nordy, there was no discernible effect on zebrafish embryonic development. Together, these results suggested efficacy and safety of using Nordy in vivo, and further demonstrated that this model should be suitable for studying GSCs and anti-GSC drug evaluation. PMID:24454929

Yu, Shicang; Xu, Chuan; Chen, Guilai; Gu, Ai; Li, Tingting; Cui, Youhong; Zhang, Xia; Bian, Xiuwu

2014-01-01

47

A synthetic dl-nordihydroguaiaretic acid (Nordy), inhibits angiogenesis, invasion and proliferation of glioma stem cells within a zebrafish xenotransplantation model.  

PubMed

The zebrafish (Danio rerio) and their transparent embryos represent a promising model system in cancer research. Compared with other vertebrate model systems, we had previously shown that the zebrafish model provides many advantages over mouse or chicken models to study tumor invasion, angiogenesis, and tumorigenesis. In this study, we systematically investigated the biological features of glioma stem cells (GSCs) in a zebrafish model, such as tumor angiogenesis, invasion, and proliferation. We demonstrated that several verified anti-angiogenic agents inhibited angiogenesis that was induced by xenografted-GSCs. We next evaluated the effects of a synthetic dl-nordihydroguaiaretic acid compound (dl-NDGA or "Nordy"), which revealed anti-tumor activity against human GSCs in vitro by establishing parameters through studying its ability to suppress angiogenesis, tumor invasion, and proliferation. Furthermore, our results indicated that Nordy might inhibit GSCs invasion and proliferation through regulation of the arachidonate 5-lipoxygenase (Alox-5) pathway. Moreover, the combination of Nordy and a VEGF inhibitor exhibited an enhanced ability to suppress angiogenesis that was induced by GSCs. By contrast, even following treatment with 50 µM Nordy, there was no discernible effect on zebrafish embryonic development. Together, these results suggested efficacy and safety of using Nordy in vivo, and further demonstrated that this model should be suitable for studying GSCs and anti-GSC drug evaluation. PMID:24454929

Yang, Xiaojun; Cui, Wei; Yu, Shicang; Xu, Chuan; Chen, Guilai; Gu, Ai; Li, Tingting; Cui, Youhong; Zhang, Xia; Bian, Xiuwu

2014-01-01

48

Establishment and characterization of a murine xenograft model of appendiceal mucinous adenocarcinoma  

PubMed Central

We describe the clinical, pathologic and molecular characteristics of a xenograft model of metastatic mucinous appendiceal adenocarcinoma. Tumours from patients with mucinous appendiceal neoplasms were implanted in nude mice and observed for evidence of intraperitoneal tumour growth. Morphologic and immunohistochemical features, temporal growth characteristics relative to controls, and loss of heterozygosity (LOH) at multiple chromosomal alleles were assessed in a successfully engrafted tumour. Two of seventeen implanted tumours successfully engrafted and only one mucinous adenocarcinoma propagated throughout the course of the study. The successful xenograft is morphologically similar to the original tumour, produces abundant extracellular mucin and exhibits non-invasive growth on peritoneal surfaces. The temporal growth characteristics of the xenograft tumour relative to controls reveal that tumour burden can be followed indirectly by measuring the weight or abdominal girth of engrafted animals. The cytokeratin, mucin core protein, CDX2, Ki-67 and p53 expression patterns are identical in the xenograft and resected tumour and are consistent with the expected pattern of protein expression for mucinous adenocarcinoma of the appendix. LOH was found in 1 of 10 informative chromosomal loci (chromosome 10p23) in xenograft tumour cells. Although we were unable to engraft a low-grade appendiceal mucinous neoplasm, the engrafted adenocarcinoma will be useful for future evaluation of novel therapeutic strategies directed at mucinous appendiceal adenocarcinoma and evaluation of strategies for treating widespread, bulky, mucinous peritoneal surface neoplasms. Xenograft tumour enrichment can facilitate molecular studies of appendiceal epithelial neoplasia. PMID:20586814

Mavanur, Arun A; Parimi, Vamsi; O’Malley, Mark; Nikiforova, Marina; Bartlett, David L; Davison, Jon M

2010-01-01

49

Neuronal regeneration in a zebrafish model of adult brain injury  

PubMed Central

SUMMARY Neural stem cells in the subventricular zone (SVZ) of the adult mammalian forebrain are a potential source of neurons for neural tissue repair after brain insults such as ischemic stroke and traumatic brain injury (TBI). Recent studies show that neurogenesis in the ventricular zone (VZ) of the adult zebrafish telencephalon has features in common with neurogenesis in the adult mammalian SVZ. Here, we established a zebrafish model to study injury-induced neurogenesis in the adult brain. We show that the adult zebrafish brain possesses a remarkable capacity for neuronal regeneration. Telencephalon injury prompted the proliferation of neuronal precursor cells (NPCs) in the VZ of the injured hemisphere, compared with in the contralateral hemisphere. The distribution of NPCs, viewed by BrdU labeling and ngn1-promoter-driven GFP, suggested that they migrated laterally and reached the injury site via the subpallium and pallium. The number of NPCs reaching the injury site significantly decreased when the fish were treated with an inhibitor of ?-secretase, a component of the Notch signaling pathway, suggesting that injury-induced neurogenesis mechanisms are at least partly conserved between fish and mammals. The injury-induced NPCs differentiated into mature neurons in the regions surrounding the injury site within a week after the injury. Most of these cells expressed T-box brain protein (Tbr1), suggesting they had adopted the normal neuronal fate in this region. These results suggest that the telencephalic VZ contributes to neural tissue recovery following telencephalic injury in the adult zebrafish, and that the adult zebrafish is a useful model for regenerative medicine. PMID:22028327

Kishimoto, Norihito; Shimizu, Kohei; Sawamoto, Kazunobu

2012-01-01

50

Systematic Analysis of a Xenograft Mice Model for KSHV+ Primary Effusion Lymphoma (PEL)  

PubMed Central

Kaposi's sarcoma-associated herpesvirus is the causative agent of primary effusion lymphoma (PEL), which arises preferentially in the setting of infection with human immunodeficiency virus (HIV). Even with standard cytotoxic chemotherapy, PEL continues to cause high mortality rates, requiring the development of novel therapeutic strategies. PEL xenograft models employing immunodeficient mice have been used to study the in vivo effects of a variety of therapeutic approaches. However, it remains unclear whether these xenograft models entirely reflect clinical presentations of KSHV+ PEL, especially given the recent description of extracavitary solid tumor variants arising in patients. In addition, effusion and solid tumor cells propagated in vivo exhibit unique biology, differing from one another or from their parental cell lines propagated through in vitro culture. Therefore, we used a KSHV+ PEL/BCBL-1 xenograft model involving non-obese diabetic/severe-combined immunodeficient (NOD/SCID) mice, and compared characteristics of effusion and solid tumors with their parent cell culture-derived counterparts. Our results indicate that although this xenograft model can be used for study of effusion and solid lymphoma observed in patients, tumor cells in vivo display unique features to those passed in vitro, including viral lytic gene expression profile, rate of solid tumor development, the host proteins and the complex of tumor microenvironment. These items should be carefully considered when the xenograft model is used for testing novel therapeutic strategies against KSHV-related lymphoma. PMID:24587336

Dai, Lu; Trillo-Tinoco, Jimena; Bai, Lihua; Kang, Baoli; Xu, Zengguang; Wen, Xiaofei; Valle, Luis Del; Qin, Zhiqiang

2014-01-01

51

Data-driven stochastic modelling of zebrafish locomotion.  

PubMed

In this work, we develop a data-driven modelling framework to reproduce the locomotion of fish in a confined environment. Specifically, we highlight the primary characteristics of the motion of individual zebrafish (Danio rerio), and study how these can be suitably encapsulated within a mathematical framework utilising a limited number of calibrated model parameters. Using data captured from individual zebrafish via automated visual tracking, we develop a model using stochastic differential equations and describe fish as a self propelled particle moving in a plane. Based on recent experimental evidence of the importance of speed regulation in social behaviour, we extend stochastic models of fish locomotion by introducing experimentally-derived processes describing dynamic speed regulation. Salient metrics are defined which are then used to calibrate key parameters of coupled stochastic differential equations, describing both speed and angular speed of swimming fish. The effects of external constraints are also included, based on experimentally observed responses. Understanding the spontaneous dynamics of zebrafish using a bottom-up, purely data-driven approach is expected to yield a modelling framework for quantitative investigation of individual behaviour in the presence of various external constraints or biological assays. PMID:25358499

Zienkiewicz, Adam; Barton, David A W; Porfiri, Maurizio; di Bernardo, Mario

2014-10-31

52

Zebrafish: an in vivo model for nano EHS studies  

PubMed Central

To assure a responsible and sustainable growth of nanotechnology, the environmental health and safety (EHS) aspect of engineered nanomaterials and nano-related products needs to be addressed at a rate commensurate with the expansion of nanotechnology. Zebrafish has been demonstrated as a correlative in vivo vertebrate model for such task, and the current advances of using zebrafish for nano EHS studies are summarized here. In addition to morphological and histopathological observations, the accessibility of gene manipulation would greatly empower such a model for detailed mechanistic studies of any nanoparticles of interest. The potential for establishing high-throughput screening platforms to facilitate the nano EHS studies is highlighted, and a discussion is presented on how toxicogenomics approaches represent a future direction to guide the identification of toxicity pathways. PMID:23208995

Zhao, Yan; Nel, André E.; Lin, Shuo

2014-01-01

53

Continuous Local Delivery of Interferon-? Stabilizes Tumor Vasculature in an Orthotopic Glioblastoma Xenograft Resection Model  

PubMed Central

Background High-grade glioblastomas have immature, leaky tumor blood vessels that impede the efficacy of adjuvant therapy. We assessed the ability of human interferon-beta (hIFN-?) delivered locally via gene transfer to effect vascular stabilization in an orthotopic glioblastoma xenograft resection model. Methods Xenografts were established by injecting three grade IV glioblastoma cell lines (GBM6-luc, MT330-luc, and SJG2-luc) into the cerebral cortex of nude rats. Tumors underwent subtotal resection, and then had gel foam containing an adeno-associated virus vector encoding either hIFN-? or green fluorescence protein (GFP, control) placed in the resection cavity. The primary end point was stabilization of tumor vasculature, as evidenced by CD34, ?SMA, and CA IX staining. Overall survival was a secondary endpoint. Results hIFN-? treatment altered the tumor vasculature of GBM6-luc and SJG2-luc xenografts, decreasing the density of endothelial cells, stabilizing vessels with pericytes, and decreasing tumor hypoxia. The mean survival for rats with these tumors was not significantly improved, however. In rats with MT330-luc xenografts, hIFN-? resulted in tumor regression, with a 6-month survival of 55% (INF-? group) and 9% (control group). Conclusion The use of AAV hIFN-? in our orthotopic glioblastoma resection model stabilized tumor vasculature, and dramatically improved survival in rats with MT330 xenografts. PMID:21878236

Denbo, Jason W.; Williams, Regan F.; Orr, W. Shannon; Sims, Thomas L.; Ng, Catherine Y.; Zhou, Junfang; Spence, Yunyu; Morton, Christopher L.; Nathwani, Amit C.; Duntsch, Christopher; Pfeffer, Lawrence M.; Davidoff, Andrew M.

2011-01-01

54

Zebrafish as a model to study live mucus physiology  

PubMed Central

Dysfunctional mucus barriers can result in important pulmonary and gastrointestinal conditions, but model systems to study the underlying causes are largely missing. We identified and characterized five mucin homologues in zebrafish, and demonstrated a strategy for fluorescence labeling of one selected mucin. These tools can be used for in vivo experiments and in pharmacological and genetic screens to study the dynamics and mechanisms of mucosal physiology. PMID:25323747

Jevtov, Irena; Samuelsson, Tore; Yao, Grace; Amsterdam, Adam; Ribbeck, Katharina

2014-01-01

55

Zebrafish as a New Model for Phenotype-based Screening of Positive Inotropic Agents.  

PubMed

The zebrafish-based assay is a widely used animal model system for cardiovascular research. In this study, we investigated the cardiac defects caused by terfenadine and tested the pharmacological response of digoxin to zebrafish with cardiac defects. The study suggested that zebrafish could be a suitable model for phenotype-based screening and evaluation of positive inotropic agents. This phenotype-based heart failure zebrafish model system was then utilized in in-house library screen. Some positive inotropic compound was discovered, which could attenuate the cardiac defects by increasing the flow velocity of caudal artery blood. PMID:24990095

Tang, Chunlei; Xie, Desheng; Feng, Bainian

2015-03-01

56

Zebrafish as a Novel Vertebrate Model To Dissect Enterococcal Pathogenesis  

PubMed Central

Enterococcus faecalis is an opportunistic pathogen responsible for a wide range of life-threatening nosocomial infections, such as septicemia, peritonitis, and endocarditis. E. faecalis infections are associated with a high mortality and substantial health care costs and cause therapeutic problems due to the intrinsic resistance of this bacterium to antibiotics. Several factors contributing to E. faecalis virulence have been identified. Due to the variety of infections caused by this organism, numerous animal models have been used to mimic E. faecalis infections, but none of them is considered ideal for monitoring pathogenesis. Here, we studied for the first time E. faecalis pathogenesis in zebrafish larvae. Using model strains, chosen isogenic mutants, and fluorescent derivatives expressing green fluorescent protein (GFP), we analyzed both lethality and bacterial dissemination in infected larvae. Genetically engineered immunocompromised zebrafish allowed the identification of two critical steps for successful establishment of disease: (i) host phagocytosis evasion mediated by the Epa rhamnopolysaccharide and (ii) tissue damage mediated by the quorum-sensing Fsr regulon. Our results reveal that the zebrafish is a novel, powerful model for studying E. faecalis pathogenesis, enabling us to dissect the mechanism of enterococcal virulence. PMID:24002065

Renshaw, Stephen A.; Ogryzko, Nikolay V.; Foster, Simon J.; Serror, Pascale

2013-01-01

57

Computational Graph Theoretical Model of the Zebrafish Sensorimotor Pathway  

NASA Astrophysics Data System (ADS)

Mapping the detailed connectivity patterns of neural circuits is a central goal of neuroscience and has been the focus of extensive current research [4, 3]. The best quantitative approach to analyze the acquired data is still unclear but graph theory has been used with success [3, 1]. We present a graph theoretical model with vertices and edges representing neurons and synaptic connections, respectively. Our system is the zebrafish posterior lateral line sensorimotor pathway. The goal of our analysis is to elucidate mechanisms of information processing in this neural pathway by comparing the mathematical properties of its graph to those of other, previously described graphs. We create a zebrafish model based on currently known anatomical data. The degree distributions and small-world measures of this model is compared to small-world, random and 3-compartment random graphs of the same size (with over 2500 nodes and 160,000 connections). We find that the zebrafish graph shows small-worldness similar to other neural networks and does not have a scale-free distribution of connections.

Peterson, Joshua M.; Stobb, Michael; Mazzag, Bori; Gahtan, Ethan

2011-11-01

58

Phylogeny of Zebrafish, a "Model Species," within Danio, a "Model Genus".  

PubMed

Zebrafish (Danio rerio) is an important model for vertebrate development, genomics, physiology, behavior, toxicology, and disease. Additionally, work on numerous Danio species is elucidating evolutionary mechanisms for morphological development. Yet, the relationships of zebrafish and its closest relatives remain unclear possibly due to incomplete lineage sorting, speciation with gene flow, and interspecies hybridization. To clarify these relationships, we first constructed phylogenomic data sets from 30,801 restriction-associated DNA (RAD)-tag loci (483,026 variable positions) with clear orthology to a single location in the sequenced zebrafish genome. We then inferred a well-supported species tree for Danio and tested for gene flow during the diversification of the genus. An approach independent of the sequenced zebrafish genome verified all inferred relationships. Although identification of the sister taxon to zebrafish has been contentious, multiple RAD-tag data sets and several analytical methods provided strong evidence for Danio aesculapii as the most closely related extant zebrafish relative studied to date. Data also displayed patterns consistent with gene flow during speciation and postspeciation introgression in the lineage leading to zebrafish. The incorporation of biogeographic data with phylogenomic analyses put these relationships in a phylogeographic context and supplied additional support for D. aesculapii as the sister species to D. rerio. The clear resolution of this study establishes a framework for investigating the evolutionary biology of Danio and the heterogeneity of genome evolution in the recent history of a model organism within an emerging model genus for genetics, development, and evolution. PMID:25415969

McCluskey, Braedan M; Postlethwait, John H

2015-03-01

59

Zebrafish as a Natural Host Model for Vibrio cholerae Colonization and Transmission  

PubMed Central

The human diarrheal disease cholera is caused by the aquatic bacterium Vibrio cholerae. V. cholerae in the environment is associated with several varieties of aquatic life, including insect egg masses, shellfish, and vertebrate fish. Here we describe a novel animal model for V. cholerae, the zebrafish. Pandemic V. cholerae strains specifically colonize the zebrafish intestinal tract after exposure in water with no manipulation of the animal required. Colonization occurs in close contact with the intestinal epithelium and mimics colonization observed in mammals. Zebrafish that are colonized by V. cholerae transmit the bacteria to naive fish, which then become colonized. Striking differences in colonization between V. cholerae classical and El Tor biotypes were apparent. The zebrafish natural habitat in Asia heavily overlaps areas where cholera is endemic, suggesting that zebrafish and V. cholerae evolved in close contact with each other. Thus, the zebrafish provides a natural host model for the study of V. cholerae colonization, transmission, and environmental survival. PMID:24375135

Runft, Donna L.; Mitchell, Kristie C.; Abuaita, Basel H.; Allen, Jonathan P.; Bajer, Sarah; Ginsburg, Kevin; Neely, Melody N.

2014-01-01

60

Zebrafish as a natural host model for Vibrio cholerae colonization and transmission.  

PubMed

The human diarrheal disease cholera is caused by the aquatic bacterium Vibrio cholerae. V. cholerae in the environment is associated with several varieties of aquatic life, including insect egg masses, shellfish, and vertebrate fish. Here we describe a novel animal model for V. cholerae, the zebrafish. Pandemic V. cholerae strains specifically colonize the zebrafish intestinal tract after exposure in water with no manipulation of the animal required. Colonization occurs in close contact with the intestinal epithelium and mimics colonization observed in mammals. Zebrafish that are colonized by V. cholerae transmit the bacteria to naive fish, which then become colonized. Striking differences in colonization between V. cholerae classical and El Tor biotypes were apparent. The zebrafish natural habitat in Asia heavily overlaps areas where cholera is endemic, suggesting that zebrafish and V. cholerae evolved in close contact with each other. Thus, the zebrafish provides a natural host model for the study of V. cholerae colonization, transmission, and environmental survival. PMID:24375135

Runft, Donna L; Mitchell, Kristie C; Abuaita, Basel H; Allen, Jonathan P; Bajer, Sarah; Ginsburg, Kevin; Neely, Melody N; Withey, Jeffrey H

2014-03-01

61

Zebrafish as a systems toxicology model for developmental neurotoxicity testing.  

PubMed

The developing brain is extremely sensitive to many chemicals. Exposure to neurotoxicants during development has been implicated in various neuropsychiatric and neurological disorders, including autism spectrum disorder, attention deficit hyperactive disorder, schizophrenia, Parkinson's disease, and Alzheimer's disease. Although rodents have been widely used for developmental neurotoxicity testing, experiments using large numbers of rodents are time-consuming, expensive, and raise ethical concerns. Using alternative non-mammalian animal models may relieve some of these pressures by allowing testing of large numbers of subjects while reducing expenses and minimizing the use of mammalian subjects. In this review, we discuss some of the advantages of using zebrafish in developmental neurotoxicity testing, focusing on central nervous system development, neurobehavior, toxicokinetics, and toxicodynamics in this species. We also describe some important examples of developmental neurotoxicity testing using zebrafish combined with gene expression profiling, neuroimaging, or neurobehavioral assessment. Zebrafish may be a systems toxicology model that has the potential to reveal the pathways of developmental neurotoxicity and to provide a sound basis for human risk assessments. PMID:25109898

Nishimura, Yuhei; Murakami, Soichiro; Ashikawa, Yoshifumi; Sasagawa, Shota; Umemoto, Noriko; Shimada, Yasuhito; Tanaka, Toshio

2015-02-01

62

Zebrafish as a systems toxicology model for developmental neurotoxicity testing.  

PubMed

The developing brain is extremely sensitive to many chemicals. Exposure to neurotoxicants during development has been implicated in various neuropsychiatric and neurological disorders, including autism spectrum disorder, attention deficit hyperactive disorder, schizophrenia, Parkinson's disease, and Alzheimer's disease. Although rodents have been widely used for developmental neurotoxicity testing, experiments using large numbers of rodents are time-consuming, expensive, and raise ethical concerns. Using alternative non-mammalian animal models may relieve some of these pressures by allowing testing of large numbers of subjects while reducing expenses and minimizing the use of mammalian subjects. In this review, we discuss some of the advantages of using zebrafish in developmental neurotoxicity testing, focusing on central nervous system development, neurobehavior, toxicokinetics, and toxicodynamics in this species. We also describe some important examples of developmental neurotoxicity testing using zebrafish combined with gene expression profiling, neuroimaging, or neurobehavioral assessment. Zebrafish may be a systems toxicology model that has the potential to reveal the pathways of developmental neurotoxicity and to provide a sound basis for human risk assessments. PMID:25174267

Nishimura, Yuhei; Murakami, Soichiro; Ashikawa, Yoshifumi; Sasagawa, Shota; Umemoto, Noriko; Shimada, Yasuhito; Tanaka, Toshio

2014-09-01

63

Toxicity of silver nanoparticles in zebrafish models  

NASA Astrophysics Data System (ADS)

This study was initiated to enhance our insight on the health and environmental impact of silver nanoparticles (Ag-np). Using starch and bovine serum albumin (BSA) as capping agents, silver nanoparticles were synthesized to study their deleterious effects and distribution pattern in zebrafish embryos (Danio rerio). Toxicological endpoints like mortality, hatching, pericardial edema and heart rate were recorded. A concentration-dependent increase in mortality and hatching delay was observed in Ag-np treated embryos. Additionally, nanoparticle treatments resulted in concentration-dependent toxicity, typified by phenotypes that had abnormal body axes, twisted notochord, slow blood flow, pericardial edema and cardiac arrhythmia. Ag+ ions and stabilizing agents showed no significant defects in developing embryos. Transmission electron microscopy (TEM) of the embryos demonstrated that nanoparticles were distributed in the brain, heart, yolk and blood of embryos as evident from the electron-dispersive x-ray analysis (EDS). Furthermore, the acridine orange staining showed an increased apoptosis in Ag-np treated embryos. These results suggest that silver nanoparticles induce a dose-dependent toxicity in embryos, which hinders normal development.

Asharani, P. V.; Lian Wu, Yi; Gong, Zhiyuan; Valiyaveettil, Suresh

2008-06-01

64

Maturation of the developing human fetal prostate in a rodent xenograft model  

PubMed Central

Background Prostate cancer is the most commonly diagnosed non-skin cancer in men. The etiology of prostate cancer is unknown, although both animal and epidemiologic data suggest that early life exposures to various toxicants, may impact DNA methylation status during development, playing an important role. Methods We have developed a xenograft model to characterize the growth and differentiation of human fetal prostate implants (gestational age 12-24 weeks) that can provide new data on the potential role of early life stressors on prostate cancer. The expression of key immunohistochemical markers responsible for prostate maturation was evaluated, including p63, cytokeratin 18, ?-smooth muscle actin, vimentin, caldesmon, Ki-67, prostate specific antigen, estrogen receptor-?, and androgen receptor. Xenografts were separated into epithelial and stromal compartments using laser capture microdissection (LCM), and the DNA methylation status was assessed in >480,000 CpG sites throughout the genome. Results Xenografts demonstrated growth and maturation throughout the 200 days of post-implantation evaluation. DNA methylation profiles of laser capture micro-dissected tissue demonstrated tissue-specific markers clustered by their location in either the epithelium or stroma of human prostate tissue. Differential methylated promoter region CpG-associated gene analysis revealed significantly more stromal than epithelial DNA methylation in the 30 and 90-day xenografts. Functional classification analysis identified CpG-related gene clusters in methylated epithelial and stromal human xenografts. Conclusion This study of human fetal prostate tissue establishes a xenograft model that demonstrates dynamic growth and maturation, allowing for future mechanistic studies of the developmental origins of later life proliferative prostate disease. PMID:24038131

Saffarini, Camelia M.; McDonnell, Elizabeth V.; Amin, Ali; Spade, Daniel J.; Huse, Susan M.; Kostadinov, Stefan; Hall, Susan J.; Boekelheide, Kim

2015-01-01

65

Stromal microenvironment processes unveiled by biological component analysis of gene expression in xenograft tumor models  

Microsoft Academic Search

BACKGROUND: Mouse xenograft models, in which human cancer cells are implanted in immune-suppressed mice, have been popular for studying the mechanisms of novel therapeutic targets, tumor progression and metastasis. We hypothesized that we could exploit the interspecies genetic differences in these experiments. Our purpose is to elucidate stromal microenvironment signals from probes on human arrays unintentionally cross-hybridizing with mouse homologous

Xinan Yang; Younghee Lee; Yong Huang; James L Chen; Rosie H Xing; Yves A Lussier

2010-01-01

66

Chemically induced intestinal damage models in zebrafish larvae.  

PubMed

Several intestinal damage models have been developed using zebrafish, with the aim of recapitulating aspects of human inflammatory bowel disease (IBD). These experimentally induced inflammation models have utilized immersion exposure to an array of colitogenic agents (including live bacteria, bacterial products, and chemicals) to induce varying severity of inflammation. This technical report describes methods used to generate two chemically induced intestinal damage models using either dextran sodium sulfate (DSS) or trinitrobenzene sulfonic acid (TNBS). Methods to monitor intestinal damage and inflammatory processes, and chemical-genetic methods to manipulate the host response to injury are also described. PMID:23448252

Oehlers, Stefan H; Flores, Maria Vega; Hall, Christopher J; Okuda, Kazuhide S; Sison, John Oliver; Crosier, Kathryn E; Crosier, Philip S

2013-06-01

67

A two-scale model for correlation between B cell VDJ usage in zebrafish  

NASA Astrophysics Data System (ADS)

The zebrafish (Danio rerio) is one of the model animals for study of immunology. The dynamics of the adaptive immune system in zebrafish is similar to that in higher animals. In this work, we built a two-scale model to simulate the dynamics of B cells in primary and secondary immune reactions in zebrafish and to explain the reported correlation between VDJ usage of B cell repertoires in distinct zebrafish. The first scale of the model consists of a generalized NK model to simulate the B cell maturation process in the 10-day primary immune response. The second scale uses a delay ordinary differential equation system to model the immune responses in the 6-month lifespan of zebrafish. The generalized NK model shows that mature B cells specific to one antigen mostly possess a single VDJ recombination. The probability that mature B cells in two zebrafish have the same VDJ recombination increases with the B cell population size or the B cell selection intensity and decreases with the B cell hypermutation rate. The ODE model shows a distribution of correlation in the VDJ usage of the B cell repertoires in two six-month-old zebrafish that is highly similar to that from experiment. This work presents a simple theory to explain the experimentally observed correlation in VDJ usage of distinct zebrafish B cell repertoires after an immune response.

Pan, Keyao; Deem, Michael

2011-03-01

68

Generation of Zebrafish Zebrafish Models by CRISPRCRISPR /Cas9Cas9 Genome Editing Genome Editing.  

PubMed

The CRISPR CRISPR /CasCas system identified in archaeaarchaea has been adopted and optimized for genomegenome editing purposes in zebrafishzebrafish . In vitroIn vitro transcribed guide RNAguide RNA and Cas9 Cas9 mRNAmRNA are microinjectedmicroinjected into fertilizedfertilized zebrafish embryosembryos to edit the zebrafish genome. Here, we describe how to design a gRNAgRNA , a fast method for in vitroin vitro transcriptiontranscription of gRNA from oligonucleotidesoligonucleotides , microinjectionmicroinjection into fertilized zebrafish embryos, and a PCRPCR -based restriction fragmentrestriction fragment length assayrestriction fragment length assay to identify mutationsmutations at the gRNA target sitetarget site . PMID:25431076

Hruscha, Alexander; Schmid, Bettina

2015-01-01

69

Toward developmental models of psychiatric disorders in zebrafish  

PubMed Central

Psychiatric disorders are a diverse set of diseases that affect all aspects of mental function including social interaction, thinking, feeling, and mood. Although psychiatric disorders place a large economic burden on society, the drugs available to treat them are often palliative with variable efficacy and intolerable side-effects. The development of novel drugs has been hindered by a lack of knowledge about the etiology of these diseases. It is thus necessary to further investigate psychiatric disorders using a combination of human molecular genetics, gene-by-environment studies, in vitro pharmacological and biochemistry experiments, animal models, and investigation of the non-biological basis of these diseases, such as environmental effects. Many psychiatric disorders, including autism spectrum disorder, attention-deficit/hyperactivity disorder, mental retardation, and schizophrenia can be triggered by alterations to neural development. The zebrafish is a popular model for developmental biology that is increasingly used to study human disease. Recent work has extended this approach to examine psychiatric disorders as well. However, since psychiatric disorders affect complex mental functions that might be human specific, it is not possible to fully model them in fish. In this review, I will propose that the suitability of zebrafish for developmental studies, and the genetic tools available to manipulate them, provide a powerful model to study the roles of genes that are linked to psychiatric disorders during neural development. The relative speed and ease of conducting experiments in zebrafish can be used to address two areas of future research: the contribution of environmental factors to disease onset, and screening for novel therapeutic compounds. PMID:23637652

Norton, William H. J.

2013-01-01

70

Beyond the zebrafish: diverse fish species for modeling human disease.  

PubMed

In recent years, zebrafish, and to a lesser extent medaka, have become widely used small animal models for human diseases. These organisms have convincingly demonstrated the usefulness of fish for improving our understanding of the molecular and cellular mechanisms leading to pathological conditions, and for the development of new diagnostic and therapeutic tools. Despite the usefulness of zebrafish and medaka in the investigation of a wide spectrum of traits, there is evidence to suggest that other fish species could be better suited for more targeted questions. With the emergence of new, improved sequencing technologies that enable genomic resources to be generated with increasing efficiency and speed, the potential of non-mainstream fish species as disease models can now be explored. A key feature of these fish species is that the pathological condition that they model is often related to specific evolutionary adaptations. By exploring these adaptations, new disease-causing and disease-modifier genes might be identified; thus, diverse fish species could be exploited to better understand the complexity of disease processes. In addition, non-mainstream fish models could allow us to study the impact of environmental factors, as well as genetic variation, on complex disease phenotypes. This Review will discuss the opportunities that such fish models offer for current and future biomedical research. PMID:24271780

Schartl, Manfred

2014-02-01

71

Beyond the zebrafish: diverse fish species for modeling human disease  

PubMed Central

ABSTRACT In recent years, zebrafish, and to a lesser extent medaka, have become widely used small animal models for human diseases. These organisms have convincingly demonstrated the usefulness of fish for improving our understanding of the molecular and cellular mechanisms leading to pathological conditions, and for the development of new diagnostic and therapeutic tools. Despite the usefulness of zebrafish and medaka in the investigation of a wide spectrum of traits, there is evidence to suggest that other fish species could be better suited for more targeted questions. With the emergence of new, improved sequencing technologies that enable genomic resources to be generated with increasing efficiency and speed, the potential of non-mainstream fish species as disease models can now be explored. A key feature of these fish species is that the pathological condition that they model is often related to specific evolutionary adaptations. By exploring these adaptations, new disease-causing and disease-modifier genes might be identified; thus, diverse fish species could be exploited to better understand the complexity of disease processes. In addition, non-mainstream fish models could allow us to study the impact of environmental factors, as well as genetic variation, on complex disease phenotypes. This Review will discuss the opportunities that such fish models offer for current and future biomedical research. PMID:24271780

Schartl, Manfred

2014-01-01

72

On the diabetic menu: Zebrafish as a model for pancreas development and function  

PubMed Central

Summary Development of the vertebrate pancreas is a complex stepwise process comprising regionalization, cell differentiation, and morphogenesis. Studies in zebrafish are contributing to an emerging picture of pancreas development in which extrinsic signaling molecules influence intrinsic transcriptional programs to allow ultimate differentiation of specific pancreatic cell types. Zebrafish experiments have revealed roles for several signaling molecules in aspects of this process; for example our own work has shown that Retinoic Acid signals specify the pre-pancreatic endoderm. Time-lapse imaging of live zebrafish embryos has started to provide detailed information about early pancreas morphogenesis. In addition to modeling embryonic development, the zebrafish has recently begun to be used as a model for pancreas regeneration studies. Here we review the significant progress in these areas and consider the future potential of zebrafish as a diabetes research model. PMID:19204986

Kinkel, Mary D.; Prince, Victoria E.

2009-01-01

73

Zebrafish as a model to investigate CNS myelination.  

PubMed

Myelin plays a critical role in proper neuronal function by providing trophic and metabolic support to axons and facilitating energy-efficient saltatory conduction. Myelination is influenced by numerous molecules including growth factors, hormones, transmembrane receptors and extracellular molecules, which activate signaling cascades that drive cellular maturation. Key signaling molecules and downstream signaling cascades controlling myelination have been identified in cell culture systems. However, in vitro systems are not able to faithfully replicate the complex in vivo signaling environment that occurs during development or following injury. Currently, it remains time-consuming and expensive to investigate myelination in vivo in rodents, the most widely used model for studying mammalian myelination. As such, there is a need for alternative in vivo myelination models, particularly ones that can test molecular mechanisms without removing oligodendrocyte lineage cells from their native signaling environment or disrupting intercellular interactions with other cell types present during myelination. Here, we review the ever-increasing role of zebrafish in studies uncovering novel mechanisms controlling vertebrate myelination. These innovative studies range from observations of the behavior of single cells during in vivo myelination as well as mutagenesis- and pharmacology-based screens in whole animals. Additionally, we discuss recent efforts to develop novel models of demyelination and oligodendrocyte cell death in adult zebrafish for the study of cellular behavior in real time during repair and regeneration of damaged nervous systems. GLIA 2015;63:177-193. PMID:25263121

Preston, Marnie A; Macklin, Wendy B

2015-02-01

74

A multi-scale model for correlation in B cell VDJ usage of zebrafish  

NASA Astrophysics Data System (ADS)

The zebrafish (Danio rerio) is one of the model animals used for the study of immunology because the dynamics in the adaptive immune system of zebrafish are similar to that in higher animals. In this work, we built a multi-scale model to simulate the dynamics of B cells in the primary and secondary immune responses of zebrafish. We use this model to explain the reported correlation between VDJ usage of B cell repertoires in individual zebrafish. We use a delay ordinary differential equation (ODE) system to model the immune responses in the 6-month lifespan of a zebrafish. This mean field theory gives the number of high-affinity B cells as a function of time during an infection. The sequences of those B cells are then taken from a distribution calculated by a 'microscopic' random energy model. This generalized NK model shows that mature B cells specific to one antigen largely possess a single VDJ recombination. The model allows first-principle calculation of the probability, p, that two zebrafish responding to the same antigen will select the same VDJ recombination. This probability p increases with the B cell population size and the B cell selection intensity. The probability p decreases with the B cell hypermutation rate. The multi-scale model predicts correlations in the immune system of the zebrafish that are highly similar to that from experiment.

Pan, Keyao; Deem, Michael W.

2011-10-01

75

The Visual System of Zebrafish and its Use to Model Human Ocular Diseases  

PubMed Central

Free swimming zebrafish larvae depend mainly on their sense of vision to evade predation and to catch prey. Hence there is strong selective pressure on the fast maturation of visual function and indeed the visual system already supports a number of visually-driven behaviors in the newly hatched larvae. The ability to exploit the genetic and embryonic accessibility of the zebrafish in combination with a behavioral assessment of visual system function has made the zebrafish a popular model to study vision and its diseases. Here, we review the anatomy, physiology and development of the zebrafish eye as the basis to relate the contributions of the zebrafish to our understanding of human ocular diseases. PMID:21595048

Gestri, Gaia; Link, Brian A; Neuhauss, Stephan CF

2011-01-01

76

Generation and Characterisation of Novel Pancreatic Adenocarcinoma Xenograft Models and Corresponding Primary Cell Lines  

PubMed Central

Pancreatic adenocarcinoma is one of the most lethal cancer types, currently lacking efficient treatment. The heterogeneous nature of these tumours are poorly represented by the classical pancreatic cell lines, which have been through strong clonal selection in vitro, and are often derived from metastases. Here, we describe the establishment of novel pancreatic adenocarcinoma models, xenografts and corresponding in vitro cell lines, from primary pancreatic tumours. The morphology, differentiation grade and gene expression pattern of the xenografts resemble the original tumours well. The cell lines were analysed for colony forming capacity, tumourigenicity and expression of known cancer cell surface markers and cancer stem-like characteristics. These primary cell models will be valuable tools for biological and preclinical studies for this devastating disease. PMID:25148029

Wennerström, Anna B.; Lothe, Inger Marie Bowitz; Sandhu, Vandana; Kure, Elin H.; Myklebost, Ola; Munthe, Else

2014-01-01

77

Emergence of zebrafish models in oncology for validating novel anticancer drug targets and nanomaterials  

PubMed Central

The in vivo zebrafish models have recently attracted great attention in molecular oncology to investigate multiple genetic alterations associated with the development of human cancers and validate novel anticancer drug targets. Particularly, the transparent zebrafish models can be used as a xenotransplantation system to rapidly assess the tumorigenicity and metastatic behavior of cancer stem and/or progenitor cells and their progenies. Moreover, the zebrafish models have emerged as powerful tools for an in vivo testing of novel anticancer agents and nanomaterials for counteracting tumor formation and metastases and improving the efficacy of current radiation and chemotherapeutic treatments against aggressive, metastatic and lethal cancers. PMID:22903142

Mimeault, Murielle; Batra, Surinder K.

2013-01-01

78

First In-Mouse Development and Application of a Surgically Relevant Xenograft Model of Ovarian Carcinoma  

PubMed Central

Purpose Preclinical models of epithelial ovarian cancer have not been exploited to evaluate the clinical standard combination therapy of surgical debulking with follow-up chemotherapy. As surgery is critical to patient survival, here we establish a combined surgical/chemotherapy xenograft model of epithelial ovarian cancer and demonstrate its translational relevance. Experimental Design SKOV-3luc+ ovary cancer cells were injected topically into the ovaries of immunodeficient mice. Disease development and effect of clinical standard treatment including hysterectomy, bilateral salpingoophorectomy and removal of metastasis with follow up chemotherapy (carboplatin 12 mg/kg + paclitaxel 15 mg/kg) was evaluated by clinical parameters. Tumor burden was quantified by bioluminescence imaging (BLI). Results The xenograft ovarian tumors developed were poorly differentiated and multicystic and the disease disseminated into the peritoneal cavity. When compared to the controls with a mean survival time of 4.9 weeks, mice treated with surgery and chemotherapy, surgery or chemotherapy demonstrated significantly improved mean survival of 16.1 weeks (p?=?0.0008), 12.7 weeks (p?=?0.0008), or 10.4 weeks (p?=?0.008), respectively. Conclusion Combined surgical intervention and adjuvant chemotherapy was demonstrated for the first time in an orthotopic xenograft model of ovarian cancer. Similar to observation in human studies the combined approach resulted in the longest medial survival time, advocating application of this strategy in future preclinical therapeutic development for this disease. PMID:24594904

Helland, Øystein; Popa, Mihaela; Vintermyr, Olav K.; Molven, Anders; Gjertsen, Bjørn Tore; Bjørge, Line; McCormack, Emmet

2014-01-01

79

A zebrafish (Danio rerio) model of infectious spleen and kidney necrosis virus (ISKNV) infection  

SciTech Connect

Zebrafish is a model animal for studies of genetics, development, toxicology, oncology, and immunology. In this study, infectious spleen and kidney necrosis virus (ISKNV) was used to establish an infection in zebrafish, and the experimental conditions were established and characterized. Mortality of adult zebrafish infected with ISKNV by intraperitoneal (i.p.) injection exceeded 60%. ISKNV can be passed stably in zebrafish for over ten passages. The ailing zebrafish displayed petechial hemorrhaging and scale protrusion. Histological analysis of moribund fish revealed necrosis of tissue and enlarged cells in kidney and spleen. The real-time RT-PCR analysis of mRNA level confirmed that ISKNV was replicated in zebrafish. Immunohistochemistry and immunofluorescence analyses further confirmed the presence of ISKNV-infected cells in almost all organs of the infected fish. Electron microscope analyses showed that the ISKNV particle was present in the infected tissues. The establishment of zebrafish infection model of ISKNV can offer a valuable tool for studying the interactions between ISKNV and its host.

Xu Xiaopeng; Zhang Lichun; Weng Shaoping; Huang Zhijian; Lu Jing; Lan Dongming [State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen (Zhongshan) University, Guangzhou, 510275 (China); Zhong Xuejun [Guangzhou Kingmed Center for Clinical Laboratory, Guangzhou (China); Yu Xiaoqiang [Division of Cell Biology and Biophysics, School of Biological Science, University of Missouri-Kansas City, Kansas City (United States); Xu Anlong [State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen (Zhongshan) University, Guangzhou, 510275 (China)], E-mail: lssxal@mail.sysu.edu.cn; He Jianguo [State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen (Zhongshan) University, Guangzhou, 510275 (China)], E-mail: lsshjg@mail.sysu.edu.cn

2008-06-20

80

Zebrafish cardiac injury and regeneration models: a noninvasive and invasive in vivo model of cardiac regeneration.  

PubMed

Despite current treatment regimens, heart failure still remains one of the leading causes of morbidity and mortality in the world due to failure to adequately replace lost ventricular myocardium from ischemia-induced infarct. Although adult mammalian ventricular cardiomyocytes have a limited capacity to divide, this proliferation is insufficient to overcome the significant loss of myocardium from ventricular injury. However, lower vertebrates, such as the zebrafish and newt, have the remarkable capacity to fully regenerate their hearts after severe injury. Thus, there is great interest in studying these animal model systems to discover new regenerative approaches that might be applied to injured mammalian hearts. To this end, the zebrafish has been utilized more recently to gain additional mechanistic insight into cardiac regeneration because of its genetic tractability. Here, we describe two cardiac injury methods, a mechanical and a genetic injury model, for studying cardiac regeneration in the zebrafish. PMID:24029953

Dickover, Michael S; Zhang, Ruilin; Han, Peidong; Chi, Neil C

2013-01-01

81

Studies on sensitivity of zebrafish as a model organism for Parkinson's disease: Comparison with rat model  

PubMed Central

Objective: To determine the utility of zebra fish as an animal model for Parkinson's disease (PD) in comparison with rat model. Materials and Methods: MTT assay was performed on rat and zebrafish brain synaptosomal fractions using rotenone as a neurotoxic agent. Quercetin and resveratrol were used as standards to compare anti-apoptotic activity in both organisms. Catalepsy was induced in zebrafish by exposing them to haloperidol (9 ?M) solution. Drug-treated groups were exposed to bromocriptine and pramipexole, 30 min prior to haloperidol exposure at the dose of 2, 5, and 10 ?g/mL. Swimming speed, time spent in the bottom of the tank, and complete cataleptic time were evaluated to assess behavioral changes. In rats, catalepsy was induced using haloperidol (1.25 mg/kg i.p.). Drug-treated groups received bromocriptine (2.5 mg/kg.) and pramipexole (1 mg/kg) orally. Bar test, block test, and locomotor activity were carried out to assess behavioral changes. Results: Resveratrol and quercetin showed comparable inhibition of apoptosis in rats and zebrafish. In anti-cataleptic study, bromocriptine and pramipexole-treated groups showed significant difference (P < 0.05) in behavioral parameters as compared to haloperidol control group in both the experimental organisms. Results obtained from fish model were in correlation with rat model. Conclusion: Findings of the present study revealed that zebrafish model is highly sensitive and can be used for basic screening of drugs against PD. PMID:24554909

Makhija, Dinesh T.; Jagtap, Aarti G.

2014-01-01

82

Parkin Is Protective against Proteotoxic Stress in a Transgenic Zebrafish Model  

PubMed Central

Background Mutations in the gene encoding the E3 ubiquitin ligase parkin (PARK2) are responsible for the majority of autosomal recessive parkinsonism. Similarly to other knockout mouse models of PD-associated genes, parkin knockout mice do not show a substantial neuropathological or behavioral phenotype, while loss of parkin in Drosophila melanogaster leads to a severe phenotype, including reduced lifespan, apoptotic flight muscle degeneration and male sterility. In order to study the function of parkin in more detail and to address possible differences in its role in different species, we chose Danio rerio as a different vertebrate model system. Methodology/Principal Findings We first cloned zebrafish parkin to compare its biochemical and functional aspects with that of human parkin. By using an antisense knockdown strategy we generated a zebrafish model of parkin deficiency (knockdown efficiency between 50% and 60%) and found that the transient knockdown of parkin does not cause morphological or behavioral alterations. Specifically, we did not observe a loss of dopaminergic neurons in parkin-deficient zebrafish. In addition, we established transgenic zebrafish lines stably expressing parkin by using a Gal4/UAS-based bidirectional expression system. While parkin-deficient zebrafish are more vulnerable to proteotoxicity, increased parkin expression protected transgenic zebrafish from cell death induced by proteotoxic stress. Conclusions/Significance Similarly to human parkin, zebrafish parkin is a stress-responsive protein which protects cells from stress-induced cell death. Our transgenic zebrafish model is a novel tool to characterize the protective capacity of parkin in vivo. PMID:20689587

Paquet, Dominik; van Bebber, Frauke; Haass, Christian; Tatzelt, Jörg; Schmid, Bettina; Winklhofer, Konstanze F.

2010-01-01

83

Modeling pegylated liposomal doxorubicin-induced hand-foot syndrome and intestinal mucositis in zebrafish  

PubMed Central

Pegylated liposomal doxorubicin (PLD) has been widely used to treat cancer. The adverse effects of PLD noted in clinical practice, especially hand-foot syndrome (HFS), are regarded as unique, and the management methods for them remain limited. This study was aimed at developing a feasible experimental model for translational medicine to solve this clinical issue by using skin fluorescent transgenic zebrafish. We established an optimal protocol for the administration of Lipo-Dox™, a PLD in current clinical use, to the Tg(k18:dsred) zebrafish line expressing red fluorescence in keratinocytes. We made use of bodyweight, survival rate, gross observation, flssuorescent microscopic assessment, and pathological examination of the zebrafish to assess this model. The consecutive administration protocol of PLD resulted in growth retardation of the zebrafish embryo and survival impairment, indicating establishment of a significant toxicity. We observed fin necrosis and keratinocyte dissociation phenotypes in the PLD-treated fish after consecutive administration. The skin toxicity induced by the Lipo-Dox injection was subsequently reversible, which might be compatible with a clinical course of skin recovery after discontinuation of Lipo-Dox administration. Furthermore, we found that the number of intestinal goblet cells, an important marker of intestinal inflammation, in the Lipo-Dox-injected zebrafish was markedly increased, accompanied by impaired mucosal integrity. The intestinal inflammation induced by Lipo-Dox resembled the intestinal mucositis the clinical patients suffered from after the administration of PLD. In conclusion, we established a zebrafish model for PLD-induced HFS. The intestinal mucositis simultaneously noted in the PLD-treated zebrafish validated the similarity of clinical courses after administration of PLD. This model is easily assessable, efficient, and worthy for use in developing a new therapeutic protocol for prevention or treatment of HFS as well as intestinal mucositis. Further clinical investigations to validate the correlation between human and zebrafish data are warranted. PMID:25061318

Chen, Yau-Hung; Lee, Ya-Ting; Wen, Chi-Chung; Chen, Yun-Chen; Chen, Yu-Jen

2014-01-01

84

Antiangiogenic effects of pazopanib in xenograft hepatocellular carcinoma models: evaluation by quantitative contrast-enhanced ultrasonography  

PubMed Central

Background Antiangiogenesis is a promising therapy for advanced hepatocellular carcinoma (HCC), but the effects are difficult to be evaluated. Pazopanib (GW786034B) is a pan-vascular endothelial growth factor receptor inhibitor, the antitumor effects or antiangiogenic effects haven't been investigated in HCC. Methods In vitro direct effects of pazopanib on human HCC cell lines and endothelial cells were evaluated. In vivo antitumor effects were evaluated in three xenograft nude mice models. In the subcutaneous HCCLM3 model, intratumoral blood perfusion was detected by contrast-enhanced ultrasonography (CEUS), and serial quantitative parameters were profiled from the time-intensity curves of ultrasonograms. Results In vitro proliferation of various HCC cell lines were not inhibited by pazopanib. Pazopanib inhibited migration and invasion and induced apoptosis significantly in two HCC cell lines, HCCLM3 and PLC/PRF/5. Proliferation, migration, and tubule formation of human umbilical vein endothelial cells were inhibited by pazopanib in a dose-dependent manner. In vivo tumor growth was significantly inhibited by pazopanib in HCCLM3, HepG2, and PLC/PRF/5 xenograft models. Various intratumoral perfusion parameters changed over time, and the signal intensity was significantly impaired in the treated tumors before the treatment efficacy on tumor size could be observed. Mean transit time of the contrast media in hotspot areas of the tumors was reversely correlated with intratumoral microvessel density. Conclusions Antitumor effects of pazopanib in HCC xenografts may owe to its antiangiogenic effects, and the in vivo antiangiogenic effects could be evaluated by quantitative CEUS. PMID:21251271

2011-01-01

85

Large-Scale Assessment of the Zebrafish Embryo as a Possible Predictive Model in Toxicity Testing  

PubMed Central

Background In the drug discovery pipeline, safety pharmacology is a major issue. The zebrafish has been proposed as a model that can bridge the gap in this field between cell assays (which are cost-effective, but low in data content) and rodent assays (which are high in data content, but less cost-efficient). However, zebrafish assays are only likely to be useful if they can be shown to have high predictive power. We examined this issue by assaying 60 water-soluble compounds representing a range of chemical classes and toxicological mechanisms. Methodology/Principal Findings Over 20,000 wild-type zebrafish embryos (including controls) were cultured individually in defined buffer in 96-well plates. Embryos were exposed for a 96 hour period starting at 24 hours post fertilization. A logarithmic concentration series was used for range-finding, followed by a narrower geometric series for LC50 determination. Zebrafish embryo LC50 (log mmol/L), and published data on rodent LD50 (log mmol/kg), were found to be strongly correlated (using Kendall's rank correlation tau and Pearson's product-moment correlation). The slope of the regression line for the full set of compounds was 0.73403. However, we found that the slope was strongly influenced by compound class. Thus, while most compounds had a similar toxicity level in both species, some compounds were markedly more toxic in zebrafish than in rodents, or vice versa. Conclusions For the substances examined here, in aggregate, the zebrafish embryo model has good predictivity for toxicity in rodents. However, the correlation between zebrafish and rodent toxicity varies considerably between individual compounds and compound class. We discuss the strengths and limitations of the zebrafish model in light of these findings. PMID:21738604

Ali, Shaukat; van Mil, Harald G. J.; Richardson, Michael K.

2011-01-01

86

The zebrafish swimbladder: A simple model for lung elastin injury and repair.  

PubMed

In this communication we offer data to suggest that the zebrafish swimbladder may provide a simple model of elastin injury and repair which is amenable to genetic analysis and pertinent to lung physiology. In situ hybridization of zebrafish embryos illustrated that elastin gene expression is evident in the developing gut tract prior to swimbladder morphogenesis. Northern blot analysis demonstrated that the major zebrafish elastin mRNA is 2.0 kb which is significantly smaller than its higher vertebrate counterpart. Amino acid analysis of alkali-resistant protein from the anterior chamber of the adult zebrafish swimbladder showed a composition similar to higher vertebrate elastins including significant amounts of desmosine crosslinks. Electron microscopic investigations of the swimbladder wall indicate a simple structure with an inner layer of elastin fibers. Elastase delivery to the swimbladder in vitro resulted in significant fragmentation of elastin in the anterior chamber providing an environment for studying elastin repair within the tissue. PMID:10761635

Perrin, S; Rich, C B; Morris, S M; Stone, P J; Foster, J A

1999-01-01

87

The chick chorioallantoic membrane as an in vivo xenograft model for Burkitt lymphoma  

PubMed Central

Background Burkitt lymphoma (BL) is an aggressive malignancy that arises from B-cells and belongs to the group of Non-Hodgkin lymphomas (NHL). Due to the lack of appropriate in vivo models NHL research is mainly performed in vitro. Here, we studied the use of the chick chorioallantoic membrane (CAM) for the generation of human BL xenograft tumors, which we compared with known characteristics of the human disease. Methods In order to generate experimental BL tumors, we inoculated human BL2B95 and BL2-GFP cells on the CAM. BL2B95 xenograft-tumors were grown for seven days and subsequently analyzed with transmission electron and immunofluorescence microscopy, as well as histological staining approaches. BL2-GFP cells were studied at regular intervals up to seven days, and their metastatic behavior was visualized with intravital immunofluorescence techniques. Results Xenografted BL2B95 cells formed solid tumors in the CAM model with a Ki67-index greater than 90%, preservation of typical tumor markers (CD10, CD19, CD20), a ‘starry sky’ morphology, production of agyrophilic fibers in the stroma, formation of blood and lymphatic vessels and lymphogenic dissemination of BL2B95 to distant sites. We identified macrophages, lymphocytes and heterophilic granulocytes (chick homolog of neutrophils) as the most abundant immune cells in the experimental tumors. BL2-GFP cells could be traced in real-time during their distribution in the CAM, and the first signs for their dissemination were visible after 2-3 days. Conclusions We show that xenografted BL2B95 cells generate tumors in the CAM with a high degree of cellular, molecular and proliferative concord with the human disease, supporting the application of the CAM model for NHL research with a focus on tumor-stroma interactions. Additionally we report that BL2-GFP cells, grafted on the CAM of ex ovo cultured chick embryos, provide a powerful tool to study lymphogenic dissemination in real-time. PMID:24884418

2014-01-01

88

Modeling mucosal candidiasis in larval zebrafish by swimbladder injection.  

PubMed

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level. The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique. The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces. PMID:25490695

Gratacap, Remi L; Bergeron, Audrey C; Wheeler, Robert T

2014-01-01

89

Development of seven new human prostate tumor xenograft models and their histopathological characterization.  

PubMed Central

Seven human prostate tumor models were established by transplanting tumor fragments in NMRI athymic nude mice. Once established, the tumors were serially transplantable in both NMRI and BALB/c nude mice. The xenografts originated from primary prostatic carcinomas (prostatectomy specimens), transurethral resection material, and metastatic lesions (pelvic lymph nodes and scrotal skin). Histological examination revealed that, in the course of several mouse passages (8 to 23), tumors retained their resemblance to the original patient material. The PC-295, PC-310, PC-329, and PC-346 tumors are dependent on androgens for their growth. The PC-324, PC-339, and PC-374 tumors are androgen independent, although growth of PC-374 tumors still seemed androgen sensitive. All tumors are diploid, except for the PC-374, which is tetraploid. The diploid PC-295 tumor has an additional small population of tetraploid cells. All xenografts displayed a heterogeneous expression pattern of the androgen receptor except for the PC-324 and PC-339 tumors in which the androgen receptor could not be detected. Prostatic acid phosphatase and prostate-specific antigen were retained during serial transplantation in all tumors but the PC-324 and PC-339. This panel of permanent human prostate tumor models comprises tumors representing both the androgen-dependent and -independent stages of human prostate cancer with various degrees of differentiation and, therefore, is of great value for the study of many aspects of growth and progression of human prostate cancer. Images Figure 1 Figure 2 PMID:8780407

van Weerden, W. M.; de Ridder, C. M.; Verdaasdonk, C. L.; Romijn, J. C.; van der Kwast, T. H.; Schröder, F. H.; van Steenbrugge, G. J.

1996-01-01

90

Small-sample inference for incomplete longitudinal data with truncation and censoring in tumor xenograft models.  

PubMed

In cancer drug development, demonstrating activity in xenograft models, where mice are grafted with human cancer cells, is an important step in bringing a promising compound to humans. A key outcome variable is the tumor volume measured in a given period of time for groups of mice given different doses of a single or combination anticancer regimen. However, a mouse may die before the end of a study or may be sacrificed when its tumor volume quadruples, and its tumor may be suppressed for some time and then grow back. Thus, incomplete repeated measurements arise. The incompleteness or missingness is also caused by drastic tumor shrinkage (<0.01 cm3) or random truncation. Because of the small sample sizes in these models, asymptotic inferences are usually not appropriate. We propose two parametric test procedures based on the EM algorithm and the Bayesian method to compare treatment effects among different groups while accounting for informative censoring. A real xenograft study on a new antitumor agent, temozolomide, combined with irinotecan is analyzed using the proposed methods. PMID:12229996

Tan, Ming; Fang, Hong-Bin; Tian, Guo-Liang; Houghton, Peter J

2002-09-01

91

An experimental xenograft mouse model of diffuse pontine glioma designed for therapeutic testing  

PubMed Central

The prognosis for diffuse infiltrating pontine gliomas (DIPG) remains extremely poor, with the majority of patients surviving less than 2 years. Here, we have adapted standard xenograft techniques to study glioma growth in the mouse brainstem, and have utilized the mouse model for studying a relevant therapeutic for treating DIPGs. bioluminescence imaging monitoring revealed a progressive increase in signal following the injection of either of two tumor cell types into the brainstem. Mice with orthotopic GS2 tumors, and receiving a single 100 mg/kg dose of temozolomide showed a lengthy period of decreased tumor luminescence, with substantially increased survival relative to untreated mice (P<0.001). A small molecule inhibitor that targets cdk4/6 was used to test AM-38 brainstem xenograft response to treatment. Drug treatment resulted in delayed tumor growth, and significantly extended survival. Our results demonstrate the feasibility of using an orthotopic brainstem tumor model in athymic mice, and for application to testing therapeutic agents in treating DIPG. PMID:22231932

Aoki, Yasuyuki; Hashizume, Rintaro; Ozawa, Tomoko; Banerjee, Anu; Prados, Michael; James, C. David

2013-01-01

92

An experimental xenograft mouse model of diffuse pontine glioma designed for therapeutic testing.  

PubMed

The prognosis for diffuse infiltrating pontine gliomas (DIPG) remains extremely poor, with the majority of patients surviving less than 2 years. Here, we have adapted standard xenograft techniques to study glioma growth in the mouse brainstem, and have utilized the mouse model for studying a relevant therapeutic for treating DIPGs. bioluminescence imaging monitoring revealed a progressive increase in signal following the injection of either of two tumor cell types into the brainstem. Mice with orthotopic GS2 tumors, and receiving a single 100 mg/kg dose of temozolomide showed a lengthy period of decreased tumor luminescence, with substantially increased survival relative to untreated mice (P < 0.001). A small molecule inhibitor that targets cdk4/6 was used to test AM-38 brainstem xenograft response to treatment. Drug treatment resulted in delayed tumor growth, and significantly extended survival. Our results demonstrate the feasibility of using an orthotopic brainstem tumor model in athymic mice, and for application to testing therapeutic agents in treating DIPG. PMID:22231932

Aoki, Yasuyuki; Hashizume, Rintaro; Ozawa, Tomoko; Banerjee, Anu; Prados, Michael; James, C David; Gupta, Nalin

2012-05-01

93

Predictive Markers of Efficacy for an Angiopoietin-2 Targeting Therapeutic in Xenograft Models  

PubMed Central

The clinical efficacy of anti-angiogenic therapies has been difficult to predict, and biomarkers that can predict responsiveness are sorely needed in this era of personalized medicine. CVX-060 is an angiopoietin-2 (Ang2) targeting therapeutic, consisting of two peptides that bind Ang2 with high affinity and specificity, covalently fused to a scaffold antibody. In order to optimize the use of this compound in the clinic the construction of a predictive model is described, based on the efficacy of CVX-060 in 13 cell line and 2 patient-derived xenograft models. Pretreatment size tumors from each of the models were profiled for the levels of 27 protein markers of angiogenesis, SNP haplotype in 5 angiogenesis genes, and somatic mutation status for 11 genes implicated in tumor growth and/or vascularization. CVX-060 efficacy was determined as tumor growth inhibition (TGI%) at termination of each study. A predictive statistical model was constructed based on the correlation of these efficacy data with the marker profiles, and the model was subsequently tested by prospective analysis in 11 additional models. The results reveal a range of CVX-060 efficacy in xenograft models of diverse tissue types (0-64% TGI, median = 27%) and define a subset of 3 proteins (Ang1, EGF, Emmprin), the levels of which may be predictive of TGI by Ang2 blockade. The direction of the associations is such that better efficacy correlates with high levels of target and low levels of compensatory/antagonizing molecules. This effort has revealed a set of candidate predictive markers for CVX-060 efficacy that will be further evaluated in ongoing clinical trials. PMID:24244628

Triana-Baltzer, Gallen; Pavlicek, Adam; Goulart, Ariadne; Huang, Hanhua; Pirie-Shepherd, Steven; Levin, Nancy

2013-01-01

94

Lessons from patient-derived xenografts for better in vitro modeling of human cancer.  

PubMed

The development of novel cancer therapeutics is often plagued by discrepancies between drug efficacies obtained in preclinical studies and outcomes of clinical trials. The inconsistencies can be attributed to a lack of clinical relevance of the cancer models used for drug testing. While commonly used in vitro culture systems are advantageous for addressing specific experimental questions, they are often gross, fidelity-lacking simplifications that largely ignore the heterogeneity of cancers as well as the complexity of the tumor microenvironment. Factors such as tumor architecture, interactions among cancer cells and between cancer and stromal cells, and an acidic tumor microenvironment are critical characteristics observed in patient-derived cancer xenograft models and in the clinic. By mimicking these crucial in vivo characteristics through use of 3D cultures, co-culture systems and acidic culture conditions, an in vitro cancer model/microenvironment that is more physiologically relevant may be engineered to produce results more readily applicable to the clinic. PMID:25305336

Choi, Stephen Yiu Chuen; Lin, Dong; Gout, Peter W; Collins, Colin C; Xu, Yong; Wang, Yuzhuo

2014-12-15

95

Protective effect of fucoidan against AAPH-induced oxidative stress in zebrafish model.  

PubMed

Fucoidan, extracted from Ecklonia cava, has been extensively studied because of its wide biological activities. However, antioxidative activities have not been yet examined. Therefore we evaluated in vitro and in vivo studies on antioxidative activities of E. cava fucoidan (ECF). ECF exhibited more prominent effects in peroxyl radical scavenging activity, compared to the other scavenging activities. Thus, ECF was further evaluated for its protective ability against 2,2'-azobis dihydrochloride induced oxidative stress in Vero cells and ECF strongly reduced the AAPH-induced oxidative damage through scavenging intracellular reactive oxygen species. Furthermore, we evaluated protective effect of ECF against AAPH-induced oxidative stress in zebrafish model. ECF significantly reduced ROS generation, lipid peroxidation and cell death in zebrafish model. These findings indicate that ECF has antioxidant activities in vitro Vero cells and in vivo zebrafish model, even though ECF is not a polyphenol or flavonoid compound and does not contain benzene rings or conjugated structures. PMID:24507271

Kim, Eun-A; Lee, Seung-Hong; Ko, Chang-ik; Cha, Seon-Heui; Kang, Min-Cheol; Kang, Sung-Myung; Ko, Seok-Chun; Lee, Won-Woo; Ko, Ju-Young; Lee, Ji-Hyeok; Kang, Nalae; Oh, Jae-Young; Ahn, Ginnae; Jee, Young Heun; Jeon, You-Jin

2014-02-15

96

Evaluation of zebrafish Danio rerio as a model for enteric septicemia of catfish (ESC).  

PubMed

Zebrafish (also known as zebra danio) Danio rerio were injected intramuscularly with Edwardsiella ictaluri at doses of 6 x 10(3), 6 x 10(4), or 6 x 10(5) colony-forming units per gram (CFU/g) or sterile phosphate-buffered saline (sham) or were not injected. Mortality occurred from 2 to 5 d postinjection (dpi) at rates of 0, 76.6, and 81.3% for the low, medium, and high doses, respectively, and E. ictaluri was isolated from dead fish. Survivors were sampled at 10 dpi and E. ictaluri was not isolated. Sham-injected and noninjected controls did not suffer mortality. Histopathology trials were performed in which zebrafish were injected with 1 x 10(4) CFU/g or sham-injected and sampled at 12, 24, 48, 72, and 96 h postinjection for histological interpretation. Collectively, these zebrafish demonstrated increasing severity of splenic, hepatic, cardiac, and renal interstitial necrosis over time. To evaluate the progression of chronic infection, zebrafish were injected with 1 x 10(2) CFU/g and held for 1 month postinjection. Beginning at 12 dpi and continuing for an additional 2 weeks, zebrafish demonstrated abnormal spiraling and circling swimming behaviors. Histopathology demonstrated necrotizing encephalitis. In immersion trials, zebrafish were exposed to low, medium, and high doses (averaging 1.16 x 10(5), 1.16 x 10(6), and 1.16 x 10(7) CFU/mL of tank water) of E. ictaluri for 2 h. Mortality occurred from 5 to 9 d postexposure at rates of 0, 3.3, and 13.3% for the low, medium, and high doses, respectively; E. ictaluri was isolated from dead fish. Channel catfish Ictalurus punctatus exposed to the medium doses suffered 100% mortality, and E. ictaluri was isolated from these fish. This study demonstrates the potential use of zebrafish as a model for E. ictaluri pathogenesis. PMID:18201056

Petrie-Hanson, L; Romano, C L; Mackey, R B; Khosravi, P; Hohn, C M; Boyle, C R

2007-09-01

97

Translating Discovery in Zebrafish Pancreatic Development to Human Pancreatic Cancer: Biomarkers, Targets, Pathogenesis, and Therapeutics  

PubMed Central

Abstract Experimental studies in the zebrafish have greatly facilitated understanding of genetic regulation of the early developmental events in the pancreas. Various approaches using forward and reverse genetics, chemical genetics, and transgenesis in zebrafish have demonstrated generally conserved regulatory roles of mammalian genes and discovered novel genetic pathways in exocrine pancreatic development. Accumulating evidence has supported the use of zebrafish as a model of human malignant diseases, including pancreatic cancer. Studies have shown that the genetic regulators of exocrine pancreatic development in zebrafish can be translated into potential clinical biomarkers and therapeutic targets in human pancreatic adenocarcinoma. Transgenic zebrafish expressing oncogenic K-ras and zebrafish tumor xenograft model have emerged as valuable tools for dissecting the pathogenetic mechanisms of pancreatic cancer and for drug discovery and toxicology. Future analysis of the pancreas in zebrafish will continue to advance understanding of the genetic regulation and biological mechanisms during organogenesis. Results of those studies are expected to provide new insights into how aberrant developmental pathways contribute to formation and growth of pancreatic neoplasia, and hopefully generate valid biomarkers and targets as well as effective and safe therapeutics in pancreatic cancer. PMID:23682805

Kazi, Abid A.; Yee, Rosemary K.

2013-01-01

98

The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia  

PubMed Central

Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis – a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA. PMID:24812435

Danilova, Nadia; Bibikova, Elena; Covey, Todd M.; Nathanson, David; Dimitrova, Elizabeth; Konto, Yoan; Lindgren, Anne; Glader, Bertil; Radu, Caius G.; Sakamoto, Kathleen M.; Lin, Shuo

2014-01-01

99

ZFIN, the Zebrafish Model Organism Database: increased support for mutants and transgenics  

PubMed Central

ZFIN, the Zebrafish Model Organism Database (http://zfin.org), is the central resource for zebrafish genetic, genomic, phenotypic and developmental data. ZFIN curators manually curate and integrate comprehensive data involving zebrafish genes, mutants, transgenics, phenotypes, genotypes, gene expressions, morpholinos, antibodies, anatomical structures and publications. Integrated views of these data, as well as data gathered through collaborations and data exchanges, are provided through a wide selection of web-based search forms. Among the vertebrate model organisms, zebrafish are uniquely well suited for rapid and targeted generation of mutant lines. The recent rapid production of mutants and transgenic zebrafish is making management of data associated with these resources particularly important to the research community. Here, we describe recent enhancements to ZFIN aimed at improving our support for mutant and transgenic lines, including (i) enhanced mutant/transgenic search functionality; (ii) more expressive phenotype curation methods; (iii) new downloads files and archival data access; (iv) incorporation of new data loads from laboratories undertaking large-scale generation of mutant or transgenic lines and (v) new GBrowse tracks for transgenic insertions, genes with antibodies and morpholinos. PMID:23074187

Howe, Douglas G.; Bradford, Yvonne M.; Conlin, Tom; Eagle, Anne E.; Fashena, David; Frazer, Ken; Knight, Jonathan; Mani, Prita; Martin, Ryan; Moxon, Sierra A. Taylor; Paddock, Holly; Pich, Christian; Ramachandran, Sridhar; Ruef, Barbara J.; Ruzicka, Leyla; Schaper, Kevin; Shao, Xiang; Singer, Amy; Sprunger, Brock; Van Slyke, Ceri E.; Westerfield, Monte

2013-01-01

100

Zebrafish as a genetic model in pre-clinical drug testing and screening.  

PubMed

The traditional drug discovery pipeline for the identification and development of compounds that selectively target specific molecules to ameliorate disease remains a major focus for medical research. However, the zebrafish is increasingly providing alternative strategies for various components of this pipeline. Zebrafish and their embryos are small, easily accessible and relatively low cost, making them applicable to high-throughput, small molecule screening. Zebrafish can also be manipulated by a range of forward and reverse genetics techniques to facilitate gene discovery and functional studies. Moreover, their physiological and developmental complexity provides accurate models of human disease to underpin mechanism of action and in vivo validation studies. Finally, several of these biological characteristics make zebrafish eminently suitable for toxicity testing, including eco-toxicology. Here we review the application of zebrafish to preclinical drug development and toxicity testing, including recent advances in mutant generation, drug screening and toxicology that serve to further enhance the capabilities of this valuable model organism in drug discovery. PMID:23521675

Gibert, Y; Trengove, M C; Ward, A C

2013-01-01

101

Efficacy of protracted temozolomide dosing is limited in MGMT unmethylated GBM xenograft models  

PubMed Central

Background Temozolomide (TMZ) is important chemotherapy for glioblastoma multiforme (GBM), but the optimal dosing schedule is unclear. Methods The efficacies of different clinically relevant dosing regimens were compared in a panel of 7 primary GBM xenografts in an intracranial therapy evaluation model. Results Protracted TMZ therapy (TMZ daily M–F, 3 wk every 4) provided superior survival to a placebo-treated group in 1 of 4 O6-DNA methylguanine-methyltransferase (MGMT) promoter hypermethylated lines (GBM12) and none of the 3 MGMT unmethylated lines, while standard therapy (TMZ daily M–F, 1 wk every 4) provided superior survival to the placebo-treated group in 2 of 3 MGMT unmethylated lines (GBM14 and GBM43) and none of the methylated lines. In comparing GBM12, GBM14, and GBM43 intracranial specimens, both GBM14 and GBM43 mice treated with protracted TMZ had a significant elevation in MGMT levels compared with placebo. Similarly, high MGMT was found in a second model of acquired TMZ resistance in GBM14 flank xenografts, and resistance was reversed in vitro by treatment with the MGMT inhibitor O6-benzylguanine, demonstrating a mechanistic link between MGMT overexpression and TMZ resistance in this line. Additionally, in an analysis of gene expression data, comparison of parental and TMZ-resistant GBM14 demonstrated enrichment of functional ontologies for cell cycle control within the S, G2, and M phases of the cell cycle and DNA damage checkpoints. Conclusions Across the 7 tumor models studied, there was no consistent difference between protracted and standard TMZ regimens. The efficacy of protracted TMZ regimens may be limited in a subset of MGMT unmethylated tumors by induction of MGMT expression. PMID:23479134

Cen, Ling; Carlson, Brett L.; Pokorny, Jenny L.; Mladek, Ann C.; Grogan, Patrick T.; Schroeder, Mark A.; Decker, Paul A.; Anderson, S. Keith; Giannini, Caterina; Wu, Wenting; Ballman, Karla V.; Kitange, Gaspar J.; Sarkaria, Jann N.

2013-01-01

102

Inflammation precedes the development of human malignant mesotheliomas in a SCID mouse xenograft model  

PubMed Central

Asbestos fibers cause chronic inflammation that may be critical to the development of malignant mesothelioma (MM). Two human MM cell lines (Hmeso, PPM Mill) were used in a SCID mouse xenograft model to assess time-dependent patterns of inflammation and tumor formation. After intraperitoneal (IP) injection of MM cells, mice were euthanized at 7, 14, and 30 days, and peritoneal lavage fluid (PLF) was examined for immune cell profiles and human and mouse cytokines. Increases in human MM-derived IL-6, IL-8, bFGF, and VEGF were observed in mice at 7 days postinjection of either MM line, and a striking neutrophilia was observed at all time points. Free-floating tumor spheroids developed in mice at 14 days, and both spheroids and adherent MM tumor masses occurred in all mice at 30 days. Results suggest that inflammation and cytokine production precede and may be critical to the development of MMs. PMID:20716277

Hillegass, Jedd M.; Shukla, Arti; Lathrop, Sherrill A.; MacPherson, Maximilian B.; Beuschel, Stacie L.; Butnor, Kelly J.; Testa, Joseph R.; Pass, Harvey I.; Carbone, Michele; Steele, Chad; Mossman, Brooke T.

2010-01-01

103

A new model system swims into focus: using the zebrafish to visualize intestinal metabolism in vivo  

PubMed Central

Many fundamental questions remain regarding the cellular and molecular mechanisms of digestive lipid metabolism. One major impediment to answering important questions in the field has been the lack of a tractable and sufficiently complex model system. Until recently, most studies of lipid metabolism have been performed in vitro or in mice, yet each approach possesses certain limitations. The zebrafish (Danio rerio) offers an excellent model system in which to study lipid metabolism in vivo, owing to its small size, genetic tractability and optical clarity. Fluorescent lipid dyes and optical reporters of lipid-modifying enzymes are now being used in live zebrafish to generate visible readouts of digestive physiology. Here we review recent advances in visualizing intestinal lipid metabolism in live larval zebrafish. PMID:20174460

Carten, Juliana D; Farber, Steven A

2009-01-01

104

Gene knockdown by morpholino-modified oligonucleotides in the zebrafish model: applications for developmental toxicology  

PubMed Central

Summary The zebrafish (Danio rerio) has long been used as a model for developmental biology, making it an excellent model to use also in developmental toxicology. The many advantages of zebrafish include their small size, prolific spawning, rapid development, and transparent embryos. They can be easily manipulated genetically through the use of transgenic technology and gene knock-down via morpholino-modified antisense oligonucleotides (MOs). Knocking down specific genes to assess their role in the response to toxicant exposure provides a way to further our knowledge of how developmental toxicants work on a molecular and mechanistic level, while establishing a relationship between these molecular events and morphological, behavioral, and/or physiological effects (i.e. phenotypic anchoring). In this chapter we address important considerations for using MOs to study developmental toxicology in zebrafish embryos and provide a protocol for their use. PMID:22669659

Timme-Laragy, Alicia R.; Karchner, Sibel I.; Hahn, Mark E.

2014-01-01

105

The State of the Art of the Zebrafish Model for Toxicology and Toxicologic Pathology Research—Advantages and Current Limitations  

PubMed Central

The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1–2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology. PMID:12597434

Spitsbergen, Jan M.; Kent, Michael L.

2007-01-01

106

Developmental nephrotoxicity of aristolochic acid in a zebrafish model  

SciTech Connect

Aristolochic acid (AA) is a component of Aristolochia plant extracts which is used as a treatment for different pathologies and their toxicological effects have not been sufficiently studied. The aim of this study was to evaluate AA-induced nephrotoxicity in zebrafish embryos. After soaking zebrafish embryos in AA, the embryos displayed malformed kidney phenotypes, such as curved, cystic pronephric tubes, pronephric ducts, and cases of atrophic glomeruli. The percentages of embryos with malformed kidney phenotypes increased as the exposure dosages of AA increased. Furthermore, AA-treated embryos exhibited significantly reduced glomerular filtration rates (GFRs) in comparison with mock-control littermates (mock-control: 100 ± 2.24% vs. 10 ppm AA treatment for 3–5 h: 71.48 ± 18.84% ? 39.41 ± 15.88%), indicating that AA treatment not only caused morphological kidney changes but also induced renal failure. In addition to kidney malformations, AA-treated zebrafish embryos also exhibited deformed hearts, swollen pericardiums, impaired blood circulation and the accumulation(s) of red blood cells. Whole-mount in situ hybridization studies using cmlc2 and wt1b as riboprobes indicated that the kidney is more sensitive than the heart to AA damage. Real-time PCR showed that AA can up-regulate the expression of proinflammatory genes like TNF?, cox2 and mpo. These results support the following conclusions: (1) AA-induced renal failure is mediated by inflammation, which causes circulation dysfunction followed by serious heart malformation; and (2) the kidney is more sensitive than the heart to AA injury. -- Highlights: ? Zebrafish were used to evaluate aristolochic acid (AA)-induced nephrotoxicity. ? AA-treated zebrafish embryos exhibited deformed heart as well as malformed kidney. ? Kidney is more sensitive to AA injury than the heart.

Ding, Yu-Ju; Chen, Yau-Hung, E-mail: yauhung@mail.tku.edu.tw

2012-05-15

107

Modeling human osteosarcoma in mice through 3AB-OS cancer stem cell xenografts.  

PubMed

Osteosarcoma is the second leading cause of cancer-related death for children and young adults. In this study, we have subcutaneously injected-with and without matrigel-athymic mice (Fox1nu/nu) with human osteosarcoma 3AB-OS pluripotent cancer stem cells (CSCs), which we previously isolated from human osteosarcoma MG63 cells. Engrafted 3AB-OS cells were highly tumorigenic and matrigel greatly accelerated both tumor engraftment and growth rate. 3AB-OS CSC xenografts lacked crucial regulators of beta-catenin levels (E-cadherin, APC, and GSK-3beta), and crucial factors to restrain proliferation, resulting therefore in a strong proliferation potential. During the first weeks of engraftment 3AB-OS-derived tumors expressed high levels of pAKT, beta1-integrin and pFAK, nuclear beta-catenin, c-Myc, cyclin D2, along with high levels of hyperphosphorylated-inactive pRb and anti-apoptotic proteins such as Bcl-2 and XIAP, and matrigel increased the expression of proliferative markers. Thereafter 3AB-OS tumor xenografts obtained with matrigel co-injection showed decreased proliferative potential and AKT levels, and undetectable hyperphosphorylated pRb, whereas beta1-integrin and pFAK levels still increased. Engrafted tumor cells also showed multilineage commitment with matrigel particularly favoring the mesenchymal lineage. Concomitantly, many blood vessels and muscle fibers appeared in the tumor mass. Our findings suggest that matrigel might regulate 3AB-OS cell behavior providing adequate cues for transducing proliferation and differentiation signals triggered by pAKT, beta1-integrin, and pFAK and addressed by pRb protein. Our results provide for the first time a mouse model that recapitulates in vivo crucial features of human osteosarcoma CSCs that could be used to test and predict the efficacy in vivo of novel therapeutic treatments. PMID:22688921

Di Fiore, Riccardo; Guercio, Annalisa; Puleio, Roberto; Di Marco, Patrizia; Drago-Ferrante, Rosa; D'Anneo, Antonella; De Blasio, Anna; Carlisi, Daniela; Di Bella, Santina; Pentimalli, Francesca; Forte, Iris M; Giordano, Antonio; Tesoriere, Giovanni; Vento, Renza

2012-11-01

108

Survivin Antisense Oligonucleotides Effectively Radiosensitize Colorectal Cancer Cells in Both Tissue Culture and Murine Xenograft Models  

SciTech Connect

Purpose: Survivin shows a radiation resistance factor in colorectal cancer. In the present study, we determined whether survivin messenger RNA levels in patients with rectal cancer predict tumor response after neoadjuvant radiochemotherapy and whether inhibition of survivin by the use of antisense oligonucleotides (ASOs) enhances radiation responses. Methods and Materials: SW480 colorectal carcinoma cells were transfected with survivin ASO (LY2181308) and irradiated with doses ranging from 0-8 Gy. Survivin expression, cell-cycle distribution, {gamma}H2AX fluorescence, and induction of apoptosis were monitored by means of immunoblotting, flow cytometry, and caspase 3/7 activity. Clonogenic survival was determined by using a colony-forming assay. An SW480 xenograft model was used to investigate the effect of survivin attenuation and irradiation on tumor growth. Furthermore, survivin messenger RNA levels were studied in patient biopsy specimens by using Affymetrix microarray analysis. Results: In the translational study of 20 patients with rectal cancer, increased survivin levels were associated with significantly greater risk of local tumor recurrence (p = 0.009). Treatment of SW480 cells with survivin ASOs and irradiation resulted in an increased percentage of apoptotic cells, caspase 3/7 activity, fraction of cells in the G{sub 2}/M phase, and H2AX phosphorylation. Clonogenic survival decreased compared with control-treated cells. Furthermore, treatment of SW480 xenografts with survivin ASOs and irradiation resulted in a significant delay in tumor growth. Conclusion: Survivin appears to be a molecular biomarker in patients with rectal cancer. Furthermore, in vitro and in vivo data suggest a potential role of survivin as a molecular target to improve treatment response to radiotherapy in patients with rectal cancer.

Roedel, Franz [Department of Radiation Therapy and Oncology, University of Frankfurt/Main, Frankfurt (Germany)], E-mail: franz.roedel@kgu.de; Frey, Benjamin [Department of Radiation Oncology, University of Erlangen, Erlangen (Germany); Leitmann, Werner M.S. [Lilly Deutschland GmbH, Bad Homburg (Germany); Capalbo, Gianni; Weiss, Christian; Roedel, Claus [Department of Radiation Therapy and Oncology, University of Frankfurt/Main, Frankfurt (Germany)

2008-05-01

109

Development of novel combined anticalcification protocols including immunologic modification for prolonged durability of cardiac xenograft: preclinical study using large-animal long-term circulatory models.  

PubMed

Cardiac xenografts are conventionally cross-linked with glutaraldehyde (GA) to impart tissue stability, reduce antigenicity, and maintain tissue sterility. However, GA-fixed xenografts are prone to calcification after long-term implantation in humans, because of phospholipids, free aldehyde groups, and residual antigenicity. We evaluated preclinical safety and efficacy using large-animal long-term circulatory models for our novel combined anticalcification protocol including immunological modification, which had been proven effective in small animal experiments. Bovine/porcine xenografts were treated with decellularization, immunological modification with ?-galactosidase, GA fixation with organic solvent, and detoxification with glycine. Valve conduits made of these xenografts were transplanted into the pulmonary root of goats, and hemodynamic, radiological, immunohistopathological, and biochemical results were obtained for 12 months after implantation. Evaluation of echocardiography and cardiac catheterization demonstrated good hemodynamic status and function of the pulmonary xenograft valves. Durability of the xenografts was well preserved without calcification by specimen radiography and immunohistopathological examination. The calcium concentrations of the explanted xenografts were lower than the control xenografts. This preclinical study using large-animal long-term circulatory models demonstrated that our synergistic and simultaneous employment of multiple anticalcification therapies and novel tissue treatments, including immunological modifications, have promising safety and efficacy and should be examined further in future clinical studies. PMID:25303800

Lim, Hong-Gook; Jeong, Saeromi; Shin, Jun-Seop; Park, Chung-Gyu; Kim, Yong Jin

2015-01-01

110

Intratumoral Heterogeneity of Breast Cancer Xenograft Models: Texture Analysis of Diffusion-Weighted MR Imaging  

PubMed Central

Objective To investigate whether there is a relationship between texture analysis parameters of apparent diffusion coefficient (ADC) maps and histopathologic features of MCF-7 and MDA-MB-231 xenograft models. Materials and Methods MCF-7 estradiol (+), MCF-7 estradiol (-), and MDA-MB-231 xenograft models were made with approval of the animal care committee. Twelve tumors of MCF-7 estradiol (+), 9 tumors of MCF-7 estradiol (-), and 6 tumors in MDA-MB-231 were included. Diffusion-weighted MR images were obtained on a 9.4-T system. An analysis of the first and second order texture analysis of ADC maps was performed. The texture analysis parameters and histopathologic features were compared among these groups by the analysis of variance test. Correlations between texture parameters and histopathologic features were analyzed. We also evaluated the intraobserver agreement in assessing the texture parameters. Results MCF-7 estradiol (+) showed a higher standard deviation, maximum, skewness, and kurtosis of ADC values than MCF-7 estradiol (-) and MDA-MB-231 (p < 0.01 for all). The contrast of the MCF-7 groups was higher than that of the MDA-MB-231 (p = 0.004). The correlation (COR) of the texture analysis of MCF-7 groups was lower than that of MDA-MB-231 (p < 0.001). The histopathologic analysis showed that Ki-67mean and Ki-67diff of MCF-7 estradiol (+) were higher than that of MCF-7 estradiol (-) or MDA-MB-231 (p < 0.05). The microvessel density (MVD)mean and MVDdiff of MDA-MB-231 were higher than those of MCF-7 groups (p < 0.001). A diffuse-multifocal necrosis was more frequently found in MDA-MB-231 (p < 0.001). The proportion of necrosis moderately correlated with the contrast (r = -0.438, p = 0.022) and strongly with COR (r = 0.540, p = 0.004). Standard deviation (r = 0.622, r = 0.437), skewness (r = 0.404, r = 0.484), and kurtosis (r = 0.408, r = 0.452) correlated with Ki-67mean and Ki-67diff (p < 0.05 for all). COR moderately correlated with Ki-67diff (r = -0.388, p = 0.045). Skewness (r = -0.643, r = -0.464), kurtosis (r = -0.581, r = -0.389), contrast (r = -0.473, r = -0.549) and COR (r = 0.588, r = 0.580) correlated with MVDmean and MVDdiff (p < 0.05 for all). Conclusion The texture analysis of ADC maps may help to determine the intratumoral spatial heterogeneity of necrosis patterns, amount of cellular proliferation and the vascularity in MCF-7 and MDA-MB-231 xenograft breast cancer models. PMID:25246820

Yun, Bo La; Li, Mulan; Jang, Min Hye; Park, So Yeon; Kang, Ho Chul; Kim, Bohyoung; Song, In Chan; Moon, Woo Kyung

2014-01-01

111

Modeling mixtures of thyroid gland function disruptors in a vertebrate alternative model, the zebrafish eleutheroembryo  

SciTech Connect

Maternal thyroxine (T4) plays an essential role in fetal brain development, and even mild and transitory deficits in free-T4 in pregnant women can produce irreversible neurological effects in their offspring. Women of childbearing age are daily exposed to mixtures of chemicals disrupting the thyroid gland function (TGFDs) through the diet, drinking water, air and pharmaceuticals, which has raised the highest concern for the potential additive or synergic effects on the development of mild hypothyroxinemia during early pregnancy. Recently we demonstrated that zebrafish eleutheroembryos provide a suitable alternative model for screening chemicals impairing the thyroid hormone synthesis. The present study used the intrafollicular T4-content (IT4C) of zebrafish eleutheroembryos as integrative endpoint for testing the hypotheses that the effect of mixtures of TGFDs with a similar mode of action [inhibition of thyroid peroxidase (TPO)] was well predicted by a concentration addition concept (CA) model, whereas the response addition concept (RA) model predicted better the effect of dissimilarly acting binary mixtures of TGFDs [TPO-inhibitors and sodium-iodide symporter (NIS)-inhibitors]. However, CA model provided better prediction of joint effects than RA in five out of the six tested mixtures. The exception being the mixture MMI (TPO-inhibitor)-KClO{sub 4} (NIS-inhibitor) dosed at a fixed ratio of EC{sub 10} that provided similar CA and RA predictions and hence it was difficult to get any conclusive result. There results support the phenomenological similarity criterion stating that the concept of concentration addition could be extended to mixture constituents having common apical endpoints or common adverse outcomes. - Highlights: • Potential synergic or additive effect of mixtures of chemicals on thyroid function. • Zebrafish as alternative model for testing the effect of mixtures of goitrogens. • Concentration addition seems to predict better the effect of mixtures of goitrogens.

Thienpont, Benedicte; Barata, Carlos [Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA, CSIC), Jordi Girona, 18-26, 08034 Barcelona (Spain); Raldúa, Demetrio, E-mail: drpqam@cid.csic.es [Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA, CSIC), Jordi Girona, 18-26, 08034 Barcelona (Spain); Maladies Rares: Génétique et Métabolisme (MRGM), University of Bordeaux, EA 4576, F-33400 Talence (France)

2013-06-01

112

Zebrafish as a model for the study of the phase II cytosolic sulfotransferases.  

PubMed

Cytosolic sulfotransferases (SULTs) are traditionally known as the Phase II drug-metabolizing or detoxifying enzymes that serve for the detoxification of drugs and other xenobiotics. These enzymes in general catalyze the transfer of a sulfonate group from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to low-molecular weight substrate compounds containing hydroxyl or amino group(s). Despite considerable efforts made in recent years, some fundamental aspects of the SULTs, particularly their ontogeny, cell type/tissue/organ-specific distribution, and physiological relevance, particularly their involvement in drug metabolism and detoxification, still remain poorly understood. To better understand these fundamental issues, we have embarked on developing the zebrafish as a model for studies concerning the SULTs. To date, fifteen zebrafish SULTs have been cloned, expressed, purified, and characterized. These zebrafish SULTs, which fall into four major SULT gene families, exhibited differential substrate specificities and distinct patterns of expression at different stages during embryogenesis, through larval development, and on to maturity. The information obtained, as summarized in this review, provides a foundation for further investigation into the physiological and pharmacological involvement of the SULTs using the zebrafish as a model. PMID:20545621

Liu, Tzu-An; Bhuiyan, Shakhawat; Liu, Ming-Yih; Sugahara, Takuya; Sakakibara, Yoichi; Suiko, Masahito; Yasuda, Shin; Kakuta, Yoshimitsu; Kimura, Makoto; Williams, Frederick E; Liu, Ming-Cheh

2010-07-01

113

Characterization of novel genomic alterations and therapeutic approaches using acute megakaryoblastic leukemia xenograft models  

PubMed Central

Acute megakaryoblastic leukemia (AMKL) is a heterogeneous disease generally associated with poor prognosis. Gene expression profiles indicate the existence of distinct molecular subgroups, and several genetic alterations have been characterized in the past years, including the t(1;22)(p13;q13) and the trisomy 21 associated with GATA1 mutations. However, the majority of patients do not present with known mutations, and the limited access to primary patient leukemic cells impedes the efficient development of novel therapeutic strategies. In this study, using a xenotransplantation approach, we have modeled human pediatric AMKL in immunodeficient mice. Analysis of high-throughput RNA sequencing identified recurrent fusion genes defining new molecular subgroups. One subgroup of patients presented with MLL or NUP98 fusion genes leading to up-regulation of the HOX A cluster genes. A novel CBFA2T3-GLIS2 fusion gene resulting from a cryptic inversion of chromosome 16 was identified in another subgroup of 31% of non–Down syndrome AMKL and strongly associated with a gene expression signature of Hedgehog pathway activation. These molecular data provide useful markers for the diagnosis and follow up of patients. Finally, we show that AMKL xenograft models constitute a relevant in vivo preclinical screening platform to validate the efficacy of novel therapies such as Aurora A kinase inhibitors. PMID:23045605

Thiollier, Clarisse; Lopez, Cécile K.; Gerby, Bastien; Ignacimouttou, Cathy; Poglio, Sandrine; Duffourd, Yannis; Guégan, Justine; Rivera-Munoz, Paola; Bluteau, Olivier; Mabialah, Vinciane; Diop, M’Boyba; Wen, Qiang; Petit, Arnaud; Bauchet, Anne-Laure; Reinhardt, Dirk; Bornhauser, Beat; Gautheret, Daniel; Lecluse, Yann; Landman-Parker, Judith; Radford, Isabelle; Vainchenker, William; Dastugue, Nicole; de Botton, Stéphane; Dessen, Philippe; Bourquin, Jean-Pierre; Crispino, John D.; Ballerini, Paola; Bernard, Olivier A.; Pflumio, Françoise

2012-01-01

114

Imaging Tumor Variation in Response to Photodynamic Therapy in Pancreatic Cancer Xenograft Models  

SciTech Connect

Purpose: A treatment monitoring study investigated the differential effects of orthotopic pancreatic cancer models in response to interstitial photodynamic therapy (PDT), and the validity of using magnetic resonance imaging as a surrogate measure of response was assessed. Methods and Materials: Different orthotopic pancreatic cancer xenograft models (AsPC-1 and Panc-1) were used to represent the range of pathophysiology observed in human beings. Identical dose escalation studies (10, 20, and 40J/cm) using interstitial verteporfin PDT were performed, and magnetic resonance imaging with T2-weighted and T1-weighted contrast were used to monitor the total tumor volume and the vascular perfusion volume, respectively. Results: There was a significant amount of necrosis in the slower-growing Panc-1 tumor using high light dose, although complete necrosis was not observed. Lower doses were required for the same level of tumor kill in the faster-growing AsPC-1 cell line. Conclusions: The tumor growth rate and vascular pattern of the tumor affect the optimal PDT treatment regimen, with faster-growing tumors being relatively easier to treat. This highlights the fact that therapy in human beings shows a heterogeneous range of outcomes, and suggests a need for careful individualized treatment outcomes assessment in clinical work.

Samkoe, Kimberley S., E-mail: samkoe@dartmouth.ed [Thayer School of Engineering, Dartmouth College, Hanover, NH (United States); Chen, Alina [Thayer School of Engineering, Dartmouth College, Hanover, NH (United States); Rizvi, Imran [Thayer School of Engineering, Dartmouth College, Hanover, NH (United States); Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA (United States); O'Hara, Julia A. [Thayer School of Engineering, Dartmouth College, Hanover, NH (United States); Hoopes, P. Jack [Thayer School of Engineering, Dartmouth College, Hanover, NH (United States); Department of Surgery, Dartmouth Medical School, Hanover, NH (United States); Pereira, Stephen P. [Institute of Hepatology, University College London Medical School, London (United Kingdom); Hasan, Tayyaba [Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA (United States); Pogue, Brian W. [Thayer School of Engineering, Dartmouth College, Hanover, NH (United States); Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA (United States); Department of Surgery, Dartmouth Medical School, Hanover, NH (United States)

2010-01-15

115

Characterization of novel genomic alterations and therapeutic approaches using acute megakaryoblastic leukemia xenograft models.  

PubMed

Acute megakaryoblastic leukemia (AMKL) is a heterogeneous disease generally associated with poor prognosis. Gene expression profiles indicate the existence of distinct molecular subgroups, and several genetic alterations have been characterized in the past years, including the t(1;22)(p13;q13) and the trisomy 21 associated with GATA1 mutations. However, the majority of patients do not present with known mutations, and the limited access to primary patient leukemic cells impedes the efficient development of novel therapeutic strategies. In this study, using a xenotransplantation approach, we have modeled human pediatric AMKL in immunodeficient mice. Analysis of high-throughput RNA sequencing identified recurrent fusion genes defining new molecular subgroups. One subgroup of patients presented with MLL or NUP98 fusion genes leading to up-regulation of the HOX A cluster genes. A novel CBFA2T3-GLIS2 fusion gene resulting from a cryptic inversion of chromosome 16 was identified in another subgroup of 31% of non-Down syndrome AMKL and strongly associated with a gene expression signature of Hedgehog pathway activation. These molecular data provide useful markers for the diagnosis and follow up of patients. Finally, we show that AMKL xenograft models constitute a relevant in vivo preclinical screening platform to validate the efficacy of novel therapies such as Aurora A kinase inhibitors. PMID:23045605

Thiollier, Clarisse; Lopez, Cécile K; Gerby, Bastien; Ignacimouttou, Cathy; Poglio, Sandrine; Duffourd, Yannis; Guégan, Justine; Rivera-Munoz, Paola; Bluteau, Olivier; Mabialah, Vinciane; Diop, M'boyba; Wen, Qiang; Petit, Arnaud; Bauchet, Anne-Laure; Reinhardt, Dirk; Bornhauser, Beat; Gautheret, Daniel; Lecluse, Yann; Landman-Parker, Judith; Radford, Isabelle; Vainchenker, William; Dastugue, Nicole; de Botton, Stéphane; Dessen, Philippe; Bourquin, Jean-Pierre; Crispino, John D; Ballerini, Paola; Bernard, Olivier A; Pflumio, Françoise; Mercher, Thomas

2012-10-22

116

Proteomics of early zebrafish embryos  

Microsoft Academic Search

BACKGROUND: Zebrafish (D. rerio) has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. While genetic methods are readily available in zebrafish, protocols for two dimensional (2D) gel electrophoresis and proteomics have yet to be developed. RESULTS: As a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a

Vinzenz Link; Andrej Shevchenko; Carl-Philipp Heisenberg

2006-01-01

117

Adult zebrafish as a model system for cutaneous wound healing research  

PubMed Central

Upon injury, the skin must quickly regenerate to regain its barrier function. In mammals, wound healing is rapid and scar-free during embryogenesis, whereas in adults it involves multiple steps including blood clotting, inflammation, re-epithelialization, vascularization, and granulation tissue formation and maturation, resulting in a scar. We have established a rapid and robust method to introduce full-thickness wounds onto the flank of adult zebrafish, and show that apart from external fibrin clot formation, all steps of adult mammalian wound repair also exist in zebrafish. Wound re-epithelialization is extremely rapid and initiates with no apparent lag-phase, subsequently followed by the immigration of inflammatory cells and the formation of granulation tissue, consisting of macrophages, fibroblasts, blood vessels and collagen. The granulation tissue later regresses, resulting in minimal scar formation. Studies after chemical treatment or with transgenic fish further suggest that wound re-epithelialization occurs independently of inflammation and Fibroblast growth factor (FGF) signaling, whereas both are essential for fibroblast recruitment and granulation tissue formation. Together these results demonstrate that major steps and principles of cutaneous wound healing are conserved among adult mammals and adult zebrafish, making zebrafish a valuable model for studying vertebrate skin repair. PMID:23325040

Richardson, Rebecca; Slanchev, Krasimir; Kraus, Christopher; Knyphausen, Philipp; Eming, Sabine; Hammerschmidt, Matthias

2013-01-01

118

A Zebrafish Model of Myelodysplastic Syndrome Produced through tet2 Genomic Editing.  

PubMed

The ten-eleven translocation 2 gene (TET2) encodes a member of the TET family of DNA methylcytosine oxidases that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) to initiate the demethylation of DNA within genomic CpG islands. Somatic loss-of-function mutations of TET2 are frequently observed in human myelodysplastic syndrome (MDS), which is a clonal malignancy characterized by dysplastic changes of developing blood cell progenitors, leading to ineffective hematopoiesis. We used genome-editing technology to disrupt the zebrafish Tet2 catalytic domain. tet2(m/m) (homozygous for the mutation) zebrafish exhibited normal embryonic and larval hematopoiesis but developed progressive clonal myelodysplasia as they aged, culminating in myelodysplastic syndromes (MDS) at 24 months of age, with dysplasia of myeloid progenitor cells and anemia with abnormal circulating erythrocytes. The resultant tet2(m/m) mutant zebrafish lines show decreased levels of 5hmC in hematopoietic cells of the kidney marrow but not in other cell types, most likely reflecting the ability of other Tet family members to provide this enzymatic activity in nonhematopoietic tissues but not in hematopoietic cells. tet2(m/m) zebrafish are viable and fertile, providing an ideal model to dissect altered pathways in hematopoietic cells and, for small-molecule screens in embryos, to identify compounds with specific activity against tet2 mutant cells. PMID:25512612

Gjini, Evisa; Mansour, Marc R; Sander, Jeffry D; Moritz, Nadine; Nguyen, Ashley T; Kesarsing, Michiel; Gans, Emma; He, Shuning; Chen, Si; Ko, Myunggon; Kuang, You-Yi; Yang, Song; Zhou, Yi; Rodig, Scott; Zon, Leonard I; Joung, J Keith; Rao, Anjana; Look, A Thomas

2015-03-01

119

A novel model of demyelination and remyelination in a GFP-transgenic zebrafish  

PubMed Central

ABSTRACT Demyelinating diseases consist of a variety of autoimmune conditions in which the myelin sheath is damaged due to genetic and/or environmental factors. During clinical treatment, some patients undergo partial remyelination, especially during the early disease stages. However, the mechanisms that regulate demyelination remain unclear. The myelin structure, myelin formation and myelin-related gene expression are highly conserved between mammals and zebrafish. Therefore, the zebrafish is an ideal model organism to study myelination. In this study, we generated a transgenic zebrafish Tg(mbp:nfsB-egfp) expressing a fusion protein composed of enhanced green fluorescent protein (EGFP) and NTR from the myelin basic protein (mbp) promoter. Tg(mbp:nfsB-egfp) expressed NTR-EGFP reproducibly and hereditarily in oligodendrocytes along the spinal cord. Treatment of zebrafish larvae Tg(mbp:nfsB-egfp) with metronidazole (Mtz) resulted in the selective ablation of oligodendrocytes and led to demyelination, accompanied by behavioral changes, including decreased total movement distance, velocity, total movement time and fast movement time. After withdrawal of Mtz for a seven day recovery period, the expression of EGFP and MBP protein was observed again which indicates remyelination. Additionally, locomotor capacity was restored. Collectively, Tg(mbp:nfsB-egfp), a heritable and stable transgenic line, provides a novel, powerful tool to study the mechanisms of demyelination and remyelination. PMID:25527642

Fang, Yangwu; Lei, Xudan; Li, Xiang; Chen, Yanan; Xu, Fei; Feng, Xizeng; Wei, Shihui; Li, Yuhao

2015-01-01

120

Toxicity evaluation of biodegradable chitosan nanoparticles using a zebrafish embryo model  

PubMed Central

Background Although there are a number of reports regarding the toxicity evaluation of inorganic nanoparticles, knowledge on biodegradable nanomaterials, which have always been considered safe, is still limited. For example, the toxicity of chitosan nanoparticles, one of the most widely used drug/gene delivery vehicles, is largely unknown. In the present study, the zebrafish model was used for a safety evaluation of this nanocarrier. Methods Chitosan nanoparticles with two particle sizes were prepared by ionic cross-linking of chitosan with sodium tripolyphosphate. Chitosan nanoparticles of different concentrations were incubated with zebrafish embryos, and ZnO nanoparticles were used as the positive control. Results Embryo exposure to chitosan nanoparticles and ZnO nanoparticles resulted in a decreased hatching rate and increased mortality, which was concentration-dependent. Chitosan nanoparticles at a size of 200 nm caused malformations, including a bent spine, pericardial edema, and an opaque yolk in zebrafish embryos. Furthermore, embryos exposed to chitosan nanoparticles showed an increased rate of cell death, high expression of reactive oxygen species, as well as overexpression of heat shock protein 70, indicating that chitosan nanoparticles can cause physiological stress in zebrafish. The results also suggest that the toxicity of biodegradable nanocarriers such as chitosan nanoparticles must be addressed, especially considering the in vivo distribution of these nanoscaled particles. Conclusion Our results add new insights into the potential toxicity of nanoparticles produced by biodegradable materials, and may help us to understand better the nanotoxicity of drug delivery carriers. PMID:22267920

Hu, Yu-Lan; Qi, Wang; Han, Feng; Shao, Jian-Zhong; Gao, Jian-Qing

2011-01-01

121

Cellular characterization of ultrasound-stimulated microbubble radiation enhancement in a prostate cancer xenograft model.  

PubMed

Tumor radiation resistance poses a major obstacle in achieving an optimal outcome in radiation therapy. In the current study, we characterize a novel therapeutic approach that combines ultrasound-driven microbubbles with radiation to increase treatment responses in a prostate cancer xenograft model in mice. Tumor response to ultrasound-driven microbubbles and radiation was assessed 24 hours after treatment, which consisted of radiation treatments alone (2 Gy or 8 Gy) or ultrasound-stimulated microbubbles only, or a combination of radiation and ultrasound-stimulated microbubbles. Immunohistochemical analysis using in situ end labeling (ISEL) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) revealed increased cell death within tumors exposed to combined treatments compared with untreated tumors or tumors exposed to radiation alone. Several biomarkers were investigated to evaluate cell proliferation (Ki67), blood leakage (factor VIII), angiogenesis (cluster of differentiation molecule CD31), ceramide-formation, angiogenesis signaling [vascular endothelial growth factor (VEGF)], oxygen limitation (prolyl hydroxylase PHD2) and DNA damage/repair (?H2AX). Results demonstrated reduced vascularity due to vascular disruption by ultrasound-stimulated microbubbles, increased ceramide production and increased DNA damage of tumor cells, despite decreased tumor oxygenation with significantly less proliferating cells in the combined treatments. This combined approach could be a feasible option as a novel enhancing approach in radiation therapy. PMID:24487407

Al-Mahrouki, Azza A; Iradji, Sara; Tran, William Tyler; Czarnota, Gregory J

2014-03-01

122

Novel LIMK2 Inhibitor Blocks Panc-1 Tumor Growth in a mouse xenograft model  

PubMed Central

LIM kinases (LIMKs) are important cell cytoskeleton regulators that play a prominent role in cancer manifestation and neuronal diseases. The LIMK family consists of two homologues, LIMK1 and LIMK2, which differ from one another in expression profile, intercellular localization, and function. The main substrate of LIMK is cofilin, a member of the actin-depolymerizing factor (ADF) protein family. When phosphorylated by LIMK, cofilin is inactive. LIMKs play a contributory role in several neurodevelopmental disorders and in cancer growth and metastasis. We recently reported the development and validation of a novel LIMK inhibitor, referred to here as T56-LIMKi, using a combination of computational methods and classical biochemistry techniques. Here we report that T56-LIMKi inhibits LIMK2 with high specificity, and shows little or no cross-reactivity with LIMK1. We found that T56-LIMKi decreases phosphorylated cofilin (p-cofilin) levels and thus inhibits growth of several cancerous cell lines, including those of pancreatic cancer, glioma and schwannoma. Because the most promising in-vitro effect of T56-LIMKi was observed in the pancreatic cancer cell line Panc-1, we tested the inhibitor on a nude mouse Panc-1 xenograft model. T56-LIMKi reduced tumor size and p-cofilin levels in the Panc-1 tumors, leading us to propose T56-LIMKi as a candidate drug for cancer therapy.

Rak, Roni; Haklai, Roni; Elad-Tzfadia, Galit; Wolfson, Haim J.; Carmeli, Shmuel; Kloog, Yoel

2014-01-01

123

Hepatic vitamin A preloading reduces colorectal cancer metastatic multiplicity in a mouse xenograft model.  

PubMed

Previous research in our laboratory showed that retinol inhibited all-trans retinoic acid (ATRA)-resistant human colon cancer cell invasion via a retinoic acid receptor-independent mechanism in vitro. The objective of the current study was to determine if dietary vitamin A supplementation inhibited metastasis of ATRA-resistant colon cancer cells in a nude mouse xenograft model. Female nude mice (BALB/cAnNCr-nu/nu, n = 14 per group) consumed a control diet (2,400 IU retinyl palmitate/kg diet) or a vitamin A supplemented diet (200,000 IU retinyl palmitate/kg diet) for 1 mo prior to tumor cell injection to preload the liver with vitamin A. HCT-116, ATRA-resistant, human colon cancer cells were intrasplenically injected. Mice continued to consume their respective diets for 5 wk following surgery. Consumption of supplemental vitamin A decreased hepatic metastatic multiplicity to 17% of control. Hepatic and splenic retinol and retinyl ester concentrations were significantly higher in the mice supplemented with vitamin A when compared to mice consuming the control diet. Supplemental vitamin A did not decrease body weight, feed intake, or cause toxicity. Thus, supplemental dietary vitamin A may decrease the overall number of hepatic metastasis resulting from colon cancer. PMID:22642873

Park, Eun Young; Pinali, Daniel; Lindley, Krista; Lane, Michelle A

2012-01-01

124

Systematic repurposing screening in xenograft models identifies approved drugs with novel anti-cancer activity.  

PubMed

Approved drugs target approximately 400 different mechanisms of action, of which as few as 60 are currently used as anti-cancer therapies. Given that on average it takes 10-15 years for a new cancer therapeutic to be approved, and the recent success of drug repurposing for agents such as thalidomide, we hypothesized that effective, safe cancer treatments may be found by testing approved drugs in new therapeutic settings. Here, we report in-vivo testing of a broad compound collection in cancer xenograft models. Using 182 compounds that target 125 unique target mechanisms, we identified 3 drugs that displayed reproducible activity in combination with the chemotherapeutic temozolomide. Candidate drugs appear effective at dose equivalents that exceed current prescription levels, suggesting that additional pre-clinical efforts will be needed before these drugs can be tested for efficacy in clinical trials. In total, we suggest drug repurposing is a relatively resource-intensive method that can identify approved medicines with a narrow margin of anti-cancer activity. PMID:25093583

Roix, Jeffrey J; Harrison, S D; Rainbolt, Elizabeth A; Meshaw, Kathryn R; McMurry, Avery S; Cheung, Peter; Saha, Saurabh

2014-01-01

125

Cellular characterization of ultrasound-stimulated microbubble radiation enhancement in a prostate cancer xenograft model  

PubMed Central

Tumor radiation resistance poses a major obstacle in achieving an optimal outcome in radiation therapy. In the current study, we characterize a novel therapeutic approach that combines ultrasound-driven microbubbles with radiation to increase treatment responses in a prostate cancer xenograft model in mice. Tumor response to ultrasound-driven microbubbles and radiation was assessed 24 hours after treatment, which consisted of radiation treatments alone (2 Gy or 8 Gy) or ultrasound-stimulated microbubbles only, or a combination of radiation and ultrasound-stimulated microbubbles. Immunohistochemical analysis using in situ end labeling (ISEL) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) revealed increased cell death within tumors exposed to combined treatments compared with untreated tumors or tumors exposed to radiation alone. Several biomarkers were investigated to evaluate cell proliferation (Ki67), blood leakage (factor VIII), angiogenesis (cluster of differentiation molecule CD31), ceramide-formation, angiogenesis signaling [vascular endothelial growth factor (VEGF)], oxygen limitation (prolyl hydroxylase PHD2) and DNA damage/repair (?H2AX). Results demonstrated reduced vascularity due to vascular disruption by ultrasound-stimulated microbubbles, increased ceramide production and increased DNA damage of tumor cells, despite decreased tumor oxygenation with significantly less proliferating cells in the combined treatments. This combined approach could be a feasible option as a novel enhancing approach in radiation therapy. PMID:24487407

Al-Mahrouki, Azza A.; Iradji, Sara; Tran, William Tyler; Czarnota, Gregory J.

2014-01-01

126

A human cancer xenograft model utilizing normal pancreatic duct epithelial cells conditionally transformed with defined oncogenes.  

PubMed

Pancreatic ductal adenocarcinomas (PDACs) are considered to arise through neoplastic transformation of human pancreatic duct epithelial cells (HPDECs). In order to evaluate the biological significance of genetic and epigenetic alterations in PDACs, we isolated primary HPDECs and established an in vitro carcinogenesis model. Firstly, lentivirus-mediated transduction of KRAS(G12V), MYC and human papillomavirus 16 (HPV16) E6/E7 under the control of a tetracyclin-inducible promoter efficiently immortalized and transformed primary HPDECs, which gave rise to adenocarcinomas subcutaneously in an immune-deficient mouse xenograft model, depending on expression of the four genes. The tumors regressed promptly upon shutting-off the oncogenes, and the remaining tissues showed histological features corresponding to normal ductal structures with simple columnar epithelium. Reexpression of the oncogenes resulted in development of multiple PDACs through pancreatic intraepithelial neoplasia-like structures. We also succeeded in efficient immortalization of primary HPDECs with transduction of mutant CDK4, cyclin D1 and TERT. The cells maintained a normal diploid status and formed duct-like structures in a three-dimensional culture. In combination with p53 silencing, KRAS(G12V) alone was sufficient to fully transform the immortalized HPDECs, and MYC markedly accelerated the development of tumors. Our PDAC model supports critical roles of KRAS mutations, inactivation of the p53 and p16-pRB pathways, active telomerase and MYC expression in pancreatic carcinogenesis and thus recapitulates many features of human PDAC development. The present system with reversible control of oncogene expression enabled de novo development of PDAC from quasinormal human tissues preformed subcutaneously in mice and might be applicable to carcinogenesis models in many organ sites. PMID:24858378

Inagawa, Yuki; Yamada, Kenji; Yugawa, Takashi; Ohno, Shin-ichi; Hiraoka, Nobuyoshi; Esaki, Minoru; Shibata, Tatsuhiro; Aoki, Kazunori; Saya, Hideyuki; Kiyono, Tohru

2014-08-01

127

Establishment of a Lung Metastatic Breast Tumor Xenograft Model in Nude Rats  

PubMed Central

Objective Larger animal models provide relevant tumor burden in the development of advanced clinical imaging methods for non-invasive cancer detection and diagnosis, and are especially valuable for studying metastatic disease. Most available experimental models, however, are based on immune-compromised mice. To lay the foundation for studying spontaneous metastasis using non-invasive magnetic resonance imaging (MRI), this study aims to establish a highly metastatic breast cancer xenograft model in nude rats. Materials and Methods A highly metastatic variant of human adenocarcinoma MDA-MB-231 known as LM2 was inoculated into nude rats. Orthotopic and subcutaneous (flank) sites were compared, with half of the orthotopic injections guided by ultrasound imaging. MRI with gadolinium contrast administration was performed weekly beginning on Day 6 and ending on Day 104. Excised tumors were assessed on histology using hematoxylin and eosin and CD34. Fisher's exact test was used to compare successful tumor induction amongst different inoculation methods. Results Primary LM2 tumors were established orthotopically in all cases under ultrasound-guided injection, and none otherwise (p?=?0.0028). Contrast-enhanced MRI revealed rapidly progressing tumors that reached critical size (15 mm diameter) in 2 to 3 weeks after inoculation. MRI and histology findings were consistent: LM2 tumors were characterized by low vascularity confined to the tumor rim and large necrotic cores with increasing interstitial fluid pressure. Conclusions The metastatic LM2 breast tumor model was successfully established in the mammary fat pads of nude rats, using ultrasound needle guidance as a non-invasive alternative to surgery. This platform lays the foundation for future development and application of MRI to study spontaneous metastasis and different stages throughout the metastatic cascade. PMID:24835641

Nofiele, Joris Tchouala; Cheng, Hai-Ling Margaret

2014-01-01

128

Molecular psychiatry of zebrafish.  

PubMed

Due to their well-characterized neural development and high genetic homology to mammals, zebrafish (Danio rerio) have emerged as a powerful model organism in the field of biological psychiatry. Here, we discuss the molecular psychiatry of zebrafish, and its implications for translational neuroscience research and modeling central nervous system (CNS) disorders. In particular, we outline recent genetic and technological developments allowing for in vivo examinations, high-throughput screening and whole-brain analyses in larval and adult zebrafish. We also summarize the application of these molecular techniques to the understanding of neuropsychiatric disease, outlining the potential of zebrafish for modeling complex brain disorders, including attention-deficit/hyperactivity disorder (ADHD), aggression, post-traumatic stress and substance abuse. Critically evaluating the advantages and limitations of larval and adult fish tests, we suggest that zebrafish models become a rapidly emerging new field in modern molecular psychiatry research. PMID:25349164

Stewart, A M; Ullmann, J F P; Norton, W H J; Parker, M O; Brennan, C H; Gerlai, R; Kalueff, A V

2015-02-01

129

Aggressiveness of human melanoma xenograft models is promoted by aneuploidy-driven gene expression deregulation  

PubMed Central

Melanoma is a devastating skin cancer characterized by distinct biological subtypes. Besides frequent mutations in growth- and survival-promoting genes like BRAF and NRAS, melanomas additionally harbor complex non-random genomic alterations. Using an integrative approach, we have analysed genomic and gene expression changes in human melanoma cell lines (N=32) derived from primary tumors and various metastatic sites and investigated the relation to local growth aggressiveness as xenografts in immuno-compromised mice (N=22). Although the vast majority (>90%) of melanoma models harbored mutations in either BRAF or NRAS, significant differences in subcutaneous growth aggressiveness became obvious. Unsupervised clustering revealed that genomic alterations rather than gene expression data reflected this aggressive phenotype, while no association with histology, stage or metastatic site of the original melanoma was found. Genomic clustering allowed separation of melanoma models into two subgroups with differing local growth aggressiveness in vivo. Regarding genes expressed at significantly altered levels between these subgroups, a surprising correlation with the respective gene doses (>85% accordance) was found. Genes deregulated at the DNA and mRNA level included well-known cancer genes partly already linked to melanoma (RAS genes, PTEN, AURKA, MAPK inhibitors Sprouty/Spred), but also novel candidates like SIPA1 (a Rap1GAP). Pathway mining further supported deregulation of Rap1 signaling in the aggressive subgroup e.g. by additional repression of two Rap1GEFs. Accordingly, siRNA-mediated down-regulation of SIPA1 exerted significant effects on clonogenicity, adherence and migration in aggressive melanoma models. Together our data suggest that an aneuploidy-driven gene expression deregulation drives local aggressiveness in human melanoma. PMID:22535842

Schmidt, Wolfgang M.; Spiegl-Kreinecker, Sabine; Lötsch, Daniela; Heffeter, Petra; Hegedus, Balazs; Grusch, Michael; Kiss, Robert; Berger, Walter

2012-01-01

130

A tissue-engineered humanized xenograft model of human breast cancer metastasis to bone  

PubMed Central

ABSTRACT The skeleton is a preferred homing site for breast cancer metastasis. To date, treatment options for patients with bone metastases are mostly palliative and the disease is still incurable. Indeed, key mechanisms involved in breast cancer osteotropism are still only partially understood due to the lack of suitable animal models to mimic metastasis of human tumor cells to a human bone microenvironment. In the presented study, we investigate the use of a human tissue-engineered bone construct to develop a humanized xenograft model of breast cancer-induced bone metastasis in a murine host. Primary human osteoblastic cell-seeded melt electrospun scaffolds in combination with recombinant human bone morphogenetic protein 7 were implanted subcutaneously in non-obese diabetic/severe combined immunodeficient mice. The tissue-engineered constructs led to the formation of a morphologically intact ‘organ’ bone incorporating a high amount of mineralized tissue, live osteocytes and bone marrow spaces. The newly formed bone was largely humanized, as indicated by the incorporation of human bone cells and human-derived matrix proteins. After intracardiac injection, the dissemination of luciferase-expressing human breast cancer cell lines to the humanized bone ossicles was detected by bioluminescent imaging. Histological analysis revealed the presence of metastases with clear osteolysis in the newly formed bone. Thus, human tissue-engineered bone constructs can be applied efficiently as a target tissue for human breast cancer cells injected into the blood circulation and replicate the osteolytic phenotype associated with breast cancer-induced bone lesions. In conclusion, we have developed an appropriate model for investigation of species-specific mechanisms of human breast cancer-related bone metastasis in vivo. PMID:24713276

Thibaudeau, Laure; Taubenberger, Anna V.; Holzapfel, Boris M.; Quent, Verena M.; Fuehrmann, Tobias; Hesami, Parisa; Brown, Toby D.; Dalton, Paul D.; Power, Carl A.; Hollier, Brett G.; Hutmacher, Dietmar W.

2014-01-01

131

A Tale of Two Models: Mouse and Zebrafish as Complementary Models for Lymphatic Studies  

PubMed Central

Lymphatic vessels provide essential roles in maintaining fluid homeostasis and lipid absorption. Dysfunctions of the lymphatic vessels lead to debilitating pathological conditions, collectively known as lymphedema. In addition, lymphatic vessels are a critical moderator for the onset and progression of diverse human diseases including metastatic cancer and obesity. Despite their clinical importance, there is no currently effective pharmacological therapy to regulate functions of lymphatic vessels. Recent efforts to manipulate the Vascular Endothelial Growth Factor-C (VEGFC) pathway, which is arguably the most important signaling pathway regulating lymphatic endothelial cells, to alleviate lymphedema yielded largely mixed results, necessitating identification of new targetable signaling pathways for therapeutic intervention for lymphedema. Zebrafish, a relatively new model system to investigate lymphatic biology, appears to be an ideal model to identify novel therapeutic targets for lymphatic biology. In this review, we will provide an overview of our current understanding of the lymphatic vessels in vertebrates, and discuss zebrafish as a promising in vivo model to study lymphatic vessels. PMID:24854860

Kim, Jun-Dae; Jin, Suk-Won

2014-01-01

132

Early detection of antiangiogenic treatment responses in a mouse xenograft tumor model using quantitative perfusion MRI.  

PubMed

Angiogenesis plays a major role in tumor growth and metastasis, with tumor perfusion regarded as a marker for angiogenesis. To evaluate antiangiogenic treatment response in vivo, we investigated arterial spin labeling (ASL) magnetic resonance imaging (MRI) to measure tumor perfusion quantitatively. Chronic and 24-h acute treatment responses to bevacizumab were assessed by ASL and dynamic-contrast-enhanced (DCE) MRI in the A498 xenograft mouse model. After the MRI, tumor vasculature was assessed by CD34 staining. After 39 days of chronic treatment, tumor perfusion decreased to 44.8 ± 16.1 mL/100 g/min (P < 0.05), compared to 92.6 ± 42.9 mL/100 g/min in the control group. In the acute treatment study, tumor perfusion in the treated group decreased from 107.2 ± 32.7 to 73.7 ± 27.8 mL/100 g/min (P < 0.01; two-way analysis of variance), as well as compared with control group post dosing. A significant reduction in vessel density and vessel size was observed after the chronic treatment, while only vessel size was reduced 24 h after acute treatment. The tumor perfusion correlated with vessel size (r = 0.66; P < 0.005) after chronic, but not after acute treatment. The results from DCE-MRI also detected a significant change between treated and control groups in both chronic and acute treatment studies, but not between 0 and 24 h in the acute treatment group. These results indicate that tumor perfusion measured by MRI can detect early vascular responses to antiangiogenic treatment. With its noninvasive and quantitative nature, ASL MRI would be valuable for longitudinal assessment of tumor perfusion and in translation from animal models to human. PMID:24403176

Rajendran, Reshmi; Huang, Wei; Tang, Annie Mei Yee; Liang, Jie Ming; Choo, Stephanie; Reese, Torsten; Hentze, Hannes; van Boxtel, Susan; Cliffe, Adam; Rogers, Keith; Henry, Brian; Chuang, Kai Hsiang

2014-02-01

133

Effects of aurothiomalate treatment on canine osteosarcoma in a murine xenograft model.  

PubMed

Osteosarcoma is a highly fatal cancer, with most patients ultimately succumbing to metastatic disease. The purpose of this study was to evaluate the effects of the antirheumatoid drug aurothiomalate on canine and human osteosarcoma cells and on canine osteosarcoma growth and metastasis in a mouse xenograft model. We hypothesized that aurothiomalate would decrease osteosarcoma cell survival, tumor cellular proliferation, tumor growth, and metastasis. After performing clonogenic assays, aurothiomalate or a placebo was administered to 54 mice inoculated with canine osteosarcoma. Survival, tumor growth, embolization, metastasis, histopathology, cell proliferation marker Ki67, and apoptosis marker caspase-3 were compared between groups. Statistical analysis was carried out using the Kaplan-Meier method with the log-rank test and one-way analysis of variance with the Tukey's test or Dunn's method. Aurothiomalate caused dose-dependent inhibition of osteosarcoma cell survival (P<0.001) and decreased tumor growth (P<0.001). Pulmonary macrometastasis and Ki67 labeling were reduced with low-dose aurothiomalate (P=0.033 and 0.005, respectively), and tumor emboli and pulmonary micrometastases were decreased with high-dose aurothiomalate (P=0.010 and 0.011, respectively). There was no difference in survival, tumor development, ulceration, mitotic indices, tumor necrosis, nonpulmonary metastases, and caspase-3 labeling. Aurothiomalate treatment inhibited osteosarcoma cell survival and reduced tumor cell proliferation, growth, embolization, and pulmonary metastasis. Given aurothiomalate's established utility in canine and human medicine, our results suggest that this compound may hold promise as an adjunctive therapy for osteosarcoma. Further translational research is warranted to better characterize the dose response of canine and human osteosarcoma to aurothiomalate. PMID:24304691

Scharf, Valery F; Farese, James P; Siemann, Dietmar W; Abbott, Jeffrey R; Kiupel, Matti; Salute, Marc E; Milner, Rowan J

2014-03-01

134

siRNA targeting Livin decreases tumor in a xenograft model for colon cancer  

PubMed Central

AIM: To evaluate the effect of silencing Livin gene expression with siRNA to apoptosis and proliferation in a colon cancer cell line. METHODS: To investigate the anticancer effect of silencing Livin gene expression, we established an siRNA transfected cell line using the HCT116 colon cancer cell line. After confirming the successful transfection, MTT assay, flow cytometry and annexin V staining were employed to evaluate the antiapoptotic effect. To confirm the in vivo effect of Livin-siRNA, different doses of Livin-siRNA were injected into xenografted tumors in BALB/c nude mice model. RESULTS: Livin expression was dramatically decreased after siRNA transfection, especially at 25 ?mol/L of siRNA, but this suppression was not dose-dependent. The cell count at 18 h after transfection was significantly reduced as compared with controls (P < 0.01), but tended not to decrease proportionally depending on transfected dose or time. MTT assay revealed that silencing the Livin gene suppressed cellular proliferation at 18 h after transfection (P = 0.04); however, the inhibitory effect disappeared thereafter. Also, there was no significant difference in cellular proliferation depending on siRNA dose. The rate of apoptosis also increased with silencing of the Livin gene. In vivo, the tumor size significantly decreased after Livin-siRNA injection at 20 ?mol/L concentration (P = 0.03). There were no significant body weight changes of mice after siRNA injection. Histologic examination revealed no significant toxic reaction in kidney, liver and brain of mice. CONCLUSION: siRNA-mediated downregulation of Livin expression can induce apoptosis in colon cancer in vitro and in vivo, which suggests the possibility of new cancer therapeutics using siRNA. PMID:21633662

Oh, Bo-Young; Lee, Ryung-Ah; Kim, Kwang Ho

2011-01-01

135

Carbohydrate restriction and lactate transporter inhibition in a mouse xenograft model of human prostate cancer  

PubMed Central

OBJECTIVES To determine if a no-carbohydrate ketogenic diet (NCKD) and lactate transporter inhibition can exert a synergistic effect on delaying prostate tumour growth in a xenograft mouse model of human prostate cancer. MATERIALS AND METHODS 120 nude athymic male mice (aged 6–8 weeks) were injected s.c. in the flank with 1.0 x 105 LAPC-4 prostate cancer cells. Mice were randomized to one of four treatment groups: Western diet (WD, 35% fat, 16% protein, 49% carbohydrate) and vehicle (Veh) treatment; WD and mono-carboxylate transporter-1 (MCT1) inhibition via ?-cyano-4-hydroxycinnamate (CHC) delivered through a mini osmotic pump; NCKD (84% fat, 16% protein, 0% carbohydrate) plus Veh ; or NCKD and MCT1 inhibition. Mice were fed and weighed three times per week and feed was adjusted to maintain similar body weights. Tumour size was measured twice weekly and the combined effect of treatment was tested via Kruskal – Wallis analysis of all four groups. Independent effects of treatment (NCKD vs. WD and CHC vs. Veh) on tumour volume were tested using linear regression analysis. All mice were killed on Day 53 (conclusion of pump ejection), and serum and tumour sections were analysed for various markers. Again, combined and independent effects of treatment were tested using Kruskal – Wallis and linear regression analysis, respectively. RESULTS There were no significant differences in tumour volumes among the four groups (P=0.09). When testing the independent effects of treatment, NCKD was significantly associated with lower tumour volumes at the end of the experiment (P=0.026), while CHC administration was not (P=0.981). However, CHC was associated with increased necrotic fraction (P<0.001). CONCLUSIONS Differences in tumour volumes were observed only in comparisons between mice fed a NCKD and mice fed a WD. MCT1 inhibition did not have a significant effect on tumour volume, although it was associated with increased necrotic fraction. PMID:22394625

Kim, Howard S.; Masko, Elizabeth M.; Poulton, Susan L.; Kennedy, Kelly M.; Pizzo, Salvatore V.; Dewhirst, Mark W.; Freedland, Stephen J.

2012-01-01

136

DMU-212 inhibits tumor growth in xenograft model of human ovarian cancer.  

PubMed

DMU-212 has been shown to evoke a mitochondrial apoptotic pathway in transformed fibroblasts and breast cancer. However, recently published data indicated the ability of DMU-212 to evoke apoptosis in both mitochondria- and receptor-mediated manner in two ovarian cancer cell lines, namely A-2780 and SKOV-3, which showed varied sensitivity to the compound tested. The pronounced cytotoxic effects of DMU-212 observed in A-2780 cells were related to the execution of extracellular apoptosis pathway and cell cycle arrest in G2/M phase. In view of the great anticancer potential of DMU-212 against A-2780 cell line, the aim of the current study was to assess antiproliferative activity of DMU-212 in xenograft model of ovarian cancer. To evaluate in vitro metabolic properties of cells that were to be injected into SCID mice, uptake and decline of DMU-212 in A-2780 ovarian cancer cell line was investigated. It was found that the concentration of the test compound in A-2780 cells was growing within first eight hours, and then the gradual decline was observed. A-2780 cells stably transfected with pcDNA3.1/Zeo(-)-Luc vector were subcutaneously inoculated into the right flanks of SCID mice. After seven days of the treatment with DMU-212 (50mg/kg b.w), tumor growth appeared to be suppressed in the animals treated with the compound tested. At day 14 of the experiment, tumor burden in mice treated with DMU-212 was significantly lower, as compared to untreated controls. Our findings suggest that DMU-212 might be considered as a potential anticancer agent used in ovarian cancer therapy. PMID:24768110

Piotrowska, Hanna; Myszkowski, Krzysztof; Abraszek, Joanna; Kwiatkowska-Borowczyk, Eliza; Amarowicz, Ryszard; Murias, Marek; Wierzchowski, Marcin; Jodynis-Liebert, Jadwiga

2014-05-01

137

Histamine, leukotriene C4 and interleukin-2 increase antibody uptake into a human carcinoma xenograft model.  

PubMed Central

Systemically administered radiolabelled anti-tumour antibody is ineffective in treating the majority of patients with liver metastasis from colorectal carcinoma. We have assessed whether agents which increase capillary permeability can increase tumour uptake of antibody isotope conjugate. We developed a xenograft model of colorectal carcinoma using an antibody directed against carcinoembryonic antigen (CEA). Tumours were grown subcutaneously in the hind limbs of athymic rats to derive their circulation from the femoral artery. Cannulae were placed in the common iliac artery and iliolumbar vein. Antibody was delivered systemically, regionally and regionally with histamine, leukotriene C4 and interleukin-2. Regionally administered anti-CEA antibody resulted in a significantly greater (P = 0.004) tumour to normal tissue ratio (1.66, s.d. = 0.68) compared to systematically administered antibody (1.25, s.d. = 0.73). The addition of vasoactive drugs produced an approximately 3-fold increase with an increase to a mean tumour:liver ratio of 3.24 (s.d. = 1.39) for histamine (P less than 0.001 compared to systemic delivery), 3.21 (s.d. = 1.13, P less than 0.001) for leukotriene C4 and 3.80 (s.d. = 1.53, P less than 0.001) for interleukin-2. The addition of histamine significantly (P = 0.004) increased the mean tumour to liver ratio (1.73, s.d. = 0.44) of non-specific antibody uptake compared with either systemic (1.12, s.d. = 0.24) or regional delivery (1.25, s.d. = 0.54) of non-specific antibody alone. Increasing tumour capillary permeability can produce a significant clinically useful increase in tumour uptake of antibody-isotope conjugate. Images Figure 1 PMID:1931608

Hennigan, T. W.; Begent, R. H.; Allen-Mersh, T. G.

1991-01-01

138

Zebrafish as an alternative model for hypoxic-ischemic brain damage  

PubMed Central

Acute cerebral ischemia is one of the leading causes of mortality and chronic disability. Animal models provide an essential tool for understanding the complex cellular and molecular pathophysiology of hypoxic-ischemia and for testing novel neuroprotective drugs in the pre-clinical setting. In this study we tested zebrafish as a novel model for hypoxic-ischemic brain damage. We built an air-proof chamber where water inside had a low oxygen concentration (0.6-0.8 mg/L) proximate to complete hypoxia. Each zebrafish was placed individually in the hypoxia chamber and was subjected to hypoxia treatment until it became motionless, lying on its side on the bottom of the chamber (time to hypoxia = 679.52 ± 90 seconds, mean ± SD, n =23), followed by transferring into a recovery beaker. Overall, 60.87% of subjects did not recover from hypoxia while 39% survived. The size and distribution of brain injury were determined by triphenyltetrazolium chloride (TTC) staining. Bilateral, moderate to complete TTC decoloration or demarcation of the infarct after 10 minutes of hypoxic treatment was clearly visible in the optic tectum of the optic lobe. The size of the infarct expanded to the deep structure of the optic lobe with longer hypoxic treatments. The zebrafish that survived hypoxia experienced initial twitching followed by unbalanced erratic movements until they regained coordinated, balanced swimming ability. These data indicate that zebrafish are susceptible to hypoxic attack and suggest that the model we present in this study can be used as an alternative model to evaluate hypoxia-induced brain damage. PMID:21760967

Yu, Xinge; Li, Yang V

2011-01-01

139

Novel Dedifferentiated Liposarcoma Xenograft Models Reveal PTEN Down-Regulation as a Malignant Signature and Response to PI3K Pathway Inhibition  

PubMed Central

Liposarcoma is a type of soft tissue sarcoma that exhibits poor survival and a high recurrence rate. Treatment is generally limited to surgery and radiation, which emphasizes the need for better understanding of this disease. Because very few in vivo and in vitro models can reproducibly recapitulate the human disease, we generated several xenograft models from surgically resected human dedifferentiated liposarcoma. All xenografts recapitulated morphological and gene expression characteristics of the patient tumors after continuous in vivo passages. Importantly, xenograftability was directly correlated with disease-specific survival of liposarcoma patients. Thus, the ability for the tumor of a patient to engraft may help identify those patients who will benefit from more aggressive treatment regimens. Gene expression analyses highlighted the association between xenograftability and a unique gene expression signature, including down-regulated PTEN tumor-suppressor gene expression and a progenitor-like phenotype. When treated with the PI3K/AKT/mTOR pathway inhibitor rapamycin alone or in combination with the multikinase inhibitor sorafenib, all xenografts responded with increased lipid content and a more differentiated gene expression profile. These human xenograft models may facilitate liposarcoma research and accelerate the generation of readily translatable preclinical data that could ultimately influence patient care. PMID:23416162

Smith, Kathleen B.; Tran, Linh M.; Tam, Brenna M.; Shurell, Elizabeth M.; Li, Yunfeng; Braas, Daniel; Tap, William D.; Christofk, Heather R.; Dry, Sarah M.; Eilber, Fritz C.; Wu, Hong

2014-01-01

140

Perspectives on Zebrafish Models of Hallucinogenic Drugs and Related Psychotropic Compounds  

PubMed Central

Among different classes of psychotropic drugs, hallucinogenic agents exert one of the most prominent effects on human and animal behaviors, markedly altering sensory, motor, affective, and cognitive responses. The growing clinical and preclinical interest in psychedelic, dissociative, and deliriant hallucinogens necessitates novel translational, sensitive, and high-throughput in vivo models and screens. Primate and rodent models have been traditionally used to study cellular mechanisms and neural circuits of hallucinogenic drugs’ action. The utility of zebrafish (Danio rerio) in neuroscience research is rapidly growing due to their high physiological and genetic homology to humans, ease of genetic manipulation, robust behaviors, and cost effectiveness. Possessing a fully characterized genome, both adult and larval zebrafish are currently widely used for in vivo screening of various psychotropic compounds, including hallucinogens and related drugs. Recognizing the growing importance of hallucinogens in biological psychiatry, here we discuss hallucinogenic-induced phenotypes in zebrafish and evaluate their potential as efficient preclinical models of drug-induced states in humans. PMID:23883191

2013-01-01

141

Use of TSH?:EGFP transgenic zebrafish as a rapid in vivo model for assessing thyroid-disrupting chemicals  

SciTech Connect

Accumulating evidence indicates that a wide range of chemicals have the ability to interfere with the hypothalamic–pituitary–thyroid (HPT) axis. Novel endpoints should be evaluated in addition to existing methods in order to effectively assess the effects of these chemicals on the HPT axis. Thyroid-stimulating hormone subunit ? (TSH?) plays central regulatory roles in the HPT system. We identified the regulatory region that determines the expression level of zebrafish TSH? in the anterior pituitary. In the transgenic zebrafish with EGFP driven by the TSH? promoter, the similar responsive patterns between the expression levels of TSH?:EGFP and endogenous TSH? mRNA in the pituitary are observed following treatments with goitrogen chemicals and exogenous thyroid hormones (THs). These results suggest that the TSH?:EGFP transgenic reporter zebrafish may be a useful alternative in vivo model for the assessment of chemicals interfering with the HPT system. Highlights: ? The promoter of zebrafish TSH? gene has been identified. ? The stable TSH?:EGFP transgenic zebrafish reporter germline has been generated. ? The EGFP in the transgenic fish recapitulated the pattern of pituitary TSH? mRNA. ? The transgenic zebrafish may be an in vivo model for EDC assessment.

Ji, Cheng [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China) [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Graduate University of Chinese Academy of Sciences, Beijing (China); Jin, Xia; He, Jiangyan [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China)] [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Yin, Zhan, E-mail: zyin@ihb.ac.cn [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China)] [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China)

2012-07-15

142

Natural Variability of Kozak Sequences Correlates with Function in a Zebrafish Model  

PubMed Central

In eukaryotes, targeting the small ribosomal subunit to the mRNA transcript requires a Kozak sequence at the translation initiation site. Despite the critical importance of the Kozak sequence to regulation of gene expression, there have been no correlation studies between its natural variance and efficiency of translation. Combining bioinformatics analysis with molecular biology techniques, and using zebrafish as a test case, we identify Kozak sequences based on their natural variance and characterize their function in vivo. Our data reveal that while the canonical Kozak sequence is efficient, in zebrafish it is neither the most common nor the most efficient translation initiation sequence. Rather, the most frequent natural variation of the Kozak sequence is almost twice as efficient. We conclude that the canonical Kozak sequence is a poor predictor of translation efficiency in different model organisms. Furthermore, our results provide an experimental approach to testing and optimizing an important tool for molecular biology. PMID:25248153

Grzegorski, Steven J.; Chiari, Estelle F.; Robbins, Amy; Kish, Phillip E.; Kahana, Alon

2014-01-01

143

Embryonic fate map of first pharyngeal arch structures in the sox10: kaede zebrafish transgenic model.  

PubMed

Cranial neural crest cells follow stereotypic patterns of migration to form craniofacial structures. The zebrafish is a powerful vertebrate genetic model where transgenics with reporter proteins under the transcriptional regulation of lineage-specific promoters can be generated. Numerous studies demonstrate that the zebrafish ethmoid plate is embryologically analogous to the mammalian palate. A fate map correlating embryonic cranial neural crest to defined jaw structures would provide a useful context for the morphogenetic analysis of craniofacial development. To that end, the sox10:kaede transgenic was generated, where sox10 provides lineage restriction to the neural crest. Specific regions of neural crest were labeled at the 10-somite stage by photoconversion of the kaede reporter protein. Lineage analysis was carried out during pharyngeal development in wild-type animals, after miR140 injection, and after estradiol treatment. At the 10-somite stage, cranial neural crest cells anterior of the eye contributed to the median ethmoid plate, whereas cells medial to the eye formed the lateral ethmoid plate and trabeculae and a posterior population formed the mandible. miR-140 overexpression and estradiol inhibition of Hedgehog signaling resulted in cleft development, with failed migration of the anterior cell population to form the median ethmoid plate. The sox10:kaede transgenic line provides a useful tool for neural crest lineage analysis. These studies illustrate the advantages of the zebrafish model for application in morphogenetic studies of vertebrate craniofacial development. PMID:22948622

Dougherty, Max; Kamel, George; Shubinets, Valeriy; Hickey, Graham; Grimaldi, Michael; Liao, Eric C

2012-09-01

144

Spotlight on Zebrafish: Translational Impact  

PubMed Central

In recent years, the zebrafish has emerged as an increasingly prominent model in biomedical research. To showcase the translational impact of the model across multiple disease areas, Disease Models & Mechanisms has compiled a Special Issue that includes thought-provoking reviews, original research reporting new and important insights into disease mechanisms, and novel resources that expand the zebrafish toolkit. This Editorial provides a summary of the issue’s contents, highlighting the diversity of zebrafish disease models and their clinical applications. PMID:24973741

Patton, E. Elizabeth; Dhillon, Paraminder; Amatruda, James F.; Ramakrishnan, Lalita

2014-01-01

145

Spotlight on zebrafish: translational impact.  

PubMed

In recent years, the zebrafish has emerged as an increasingly prominent model in biomedical research. To showcase the translational impact of the model across multiple disease areas, Disease Models & Mechanisms has compiled a Special Issue that includes thought-provoking reviews, original research reporting new and important insights into disease mechanisms, and novel resources that expand the zebrafish toolkit. This Editorial provides a summary of the issue's contents, highlighting the diversity of zebrafish disease models and their clinical applications. PMID:24973741

Patton, E Elizabeth; Dhillon, Paraminder; Amatruda, James F; Ramakrishnan, Lalita

2014-07-01

146

Pazopanib, a Receptor Tyrosine Kinase Inhibitor, Suppresses Tumor Growth through Angiogenesis in Dedifferentiated Liposarcoma Xenograft Models123  

PubMed Central

INTRODUCTION: The rarity of dedifferentiated liposarcoma (DDLPS) and the lack of experimental DDLPS models limit the development of novel therapeutic strategies. Pazopanib (PAZ) is a tyrosine kinase inhibitor that is approved for the treatment of non-adipocytic advanced soft tissue sarcoma. The activity of this agent has not yet been properly explored in preclinical liposarcoma models nor in a randomized phase ? clinical trial in this entity. The aim of the present study was to investigate whether PAZ had antitumor activity in DDLPS models in vivo. MATERIAL AND METHODS: We established two patient-derived DDLPS xenograft models (UZLX-STS3 and UZLX-STS5) through implantation of tumor material from sarcoma patients in athymic nude NMRI mice. An animal model of the SW872 liposarcoma cell line was also used. To investigate the efficacy of PAZ in vivo, mice bearing tumors were treated for 2 weeks with sterile water, doxorubicin (1.2 mg/kg, intraperitoneally, twice per week), PAZ [40 mg/kg, orally (p.o.), twice per day], or PAZ plus doxorubicin (same schedules as for single treatments). RESULTS: Patient-derived xenografts retained the histologic and molecular features of DDLPS. PAZ significantly delayed tumor growth by decreasing proliferation and inhibited angiogenesis in all models tested. Combining the angiogenesis inhibitor with an anthracycline did not show superior efficacy. CONCLUSION: These results suggest that PAZ has potential antitumor activity in DDLPS primarily through antiangiogenic effects and therefore should be explored in clinical trials. PMID:25500074

Li, Haifu; Wozniak, Agnieszka; Sciot, Raf; Cornillie, Jasmien; Wellens, Jasmien; Van Looy, Thomas; Vanleeuw, Ulla; Stas, Marguerite; Hompes, Daphne; Debiec-Rychter, Maria; Schöffski, Patrick

2014-01-01

147

Zebrafish hatching  

NSDL National Science Digital Library

Zebrafish larvae from eggs cleaned of microorganisms sometimes need help hatching out of their chorionic sac. Here one can see that the microorganisms might soon rupture the sac so the zebrafish can swim free.

Mildred Hoover (Salem State College; Biology Department)

2008-07-19

148

Effect of carbon dioxide pneumoperitoneum on human renal cell carcinoma proliferation and metastasis in an orthotropic xenograft nude mouse model  

PubMed Central

Introduction This study aimed to explore the effect of carbon dioxide (CO2) pneumoperitoneum on tumor proliferation and metastasis in an orthotropic xenograft nude mice model of human renal cell carcinoma (RCC) and evaluate the safety of CO2 pneumoperitoneum laparoscopy for treating RCC. Material and methods RCC 786-0 cells were injected to establish an orthotropic xenograft model. Fifty nude mice were given orthotropic inoculations and randomized to five groups: group A (control); group B (CO2 pneumoperitoneum for 2 h); group C (CO2 pneumoperitoneum for 4 h); group D (CO2 pneumoperitoneum for 4 h and 24 h after waking); group E (CO2 pneumoperitoneum for 4 h and 48 h after waking). The proliferation status was observed in RCC specimens by immunohistochemical staining for Ki67. The protein levels of hypoxia-inducible factor-1? (HIF-1?) and vascular endothelial growth factor (VEGF) were examined by western blotting. Results All groups showed similar Ki67-positive staining in RCC samples (p > 0.05). The relative expression of HIF-1? and VEGF gradually increased in both group B and group C, as compared with group A, but only the difference between group C and group A reached statistical significance (p < 0.05). The protein levels of HIF-1? and VEGF decreased in both group D and group E, as compared with group B and group C; however, the differences between group D, group E, and group A did not reach statistical significance (p > 0.05). Conclusions In an orthotropic xenograft nude mice model of RCC, CO2 pneumoperitoneum has no effect on expression of the cellular proliferation marker Ki67. However, CO2 pneumoperitoneum rapidly induces transient expression of HIF-1? and VEGF. Thus, CO2 pneumoperitoneum laparoscopy may be a safe method for treating RCC. PMID:25395958

Chen, Yuan-Zhuo; Xu, Yun-Fei

2014-01-01

149

Mind the fish: zebrafish as a model in cognitive social neuroscience  

PubMed Central

Understanding how the brain implements social behavior on one hand, and how social processes feedback on the brain to promote fine-tuning of behavioral output according to changes in the social environment is a major challenge in contemporary neuroscience. A critical step to take this challenge successfully is finding the appropriate level of analysis when relating social to biological phenomena. Given the enormous complexity of both the neural networks of the brain and social systems, the use of a cognitive level of analysis (in an information processing perspective) is proposed here as an explanatory interface between brain and behavior. A conceptual framework for a cognitive approach to comparative social neuroscience is proposed, consisting of the following steps to be taken across different species with varying social systems: (1) identification of the functional building blocks of social skills; (2) identification of the cognitive mechanisms underlying the previously identified social skills; and (3) mapping these information processing mechanisms onto the brain. Teleost fish are presented here as a group of choice to develop this approach, given the diversity of social systems present in closely related species that allows for planned phylogenetic comparisons, and the availability of neurogenetic tools that allows the visualization and manipulation of selected neural circuits in model species such as the zebrafish. Finally, the state-of-the art of zebrafish social cognition and of the tools available to map social cognitive abilities to neural circuits in zebrafish are reviewed. PMID:23964204

Oliveira, Rui F.

2013-01-01

150

Tanshinone IIA Exhibits Anticonvulsant Activity in Zebrafish and Mouse Seizure Models  

PubMed Central

Danshen or Chinese red sage (Salvia miltiorrhiza, Bunge) is used by traditional Chinese medicine (TCM) practitioners to treat neurological, cardiovascular, and cerebrovascular disorders and is included in some TCM formulations to control epileptic seizures. In this study, acetonic crude extracts of danshen inhibited pentylenetetrazol (PTZ)-induced seizure activity in zebrafish larvae. Subsequent zebrafish bioassay-guided fractionation of the extract resulted in the isolation of four major tanshinones, which suppressed PTZ-induced activity to varying degrees. One of the active tanshinones, tanshinone IIA, also reduced c-fos expression in the brains of PTZ-exposed zebrafish larvae. In rodent seizure models, tanshinone IIA showed anticonvulsive activity in the mouse 6-Hz psychomotor seizure test in a biphasic manner and modified seizure thresholds in a complex manner for the mouse i.v. PTZ seizure assay. Interestingly, tanshinone IIA is used as a prescription drug in China to address cerebral ischemia in patients. Here, we provide the first in vivo evidence demonstrating that tanshinone IIA has anticonvulsant properties as well. PMID:23937066

2013-01-01

151

Antitumor effect of para-toluenesulfonamide against lung cancer xenograft in a mouse model  

PubMed Central

Background Conventional chemotherapy and radiation therapy against non-small cell lung cancer (NSCLC) are relatively insensitive and unsatisfactory. Para-toluenesulfonamide (PTS), a unique antitumor drug for local intratumoral injection, shows an efficacy of severely suppressing solid tumor growth with mild side effects in clinical trials. The aim of this study was to investigate the effect of PTS on lung cancer H460 cells in vivo in nude mice and its underlying mechanisms in vitro. Methods A lung cancer model for in vivo experiment was established in BALB/c nude mice using H460 cells to examine the effect of local injection of PTS on tumor suppression. We also assessed the injury to the normal tissue by subcutaneous injection of PTS. In vitro, PTS was diluted into different doses for study on its antitumor mechanisms. We evaluated the necrotic effect of PTS on H460 cells by PI and Hoechst 33342 staining. Cell viability and membrane permeability were also determined by using CCK-8 and LDH assays respectively. All these tests were conducted in comparison with traditional local injection of anhydrous ethanol. Results PTS was shown to significantly inhibit the growth of H460 tumor xenografts in nude mice by inducing necrosis of the tumor histologically. Its effect on tumor growth was significantly stronger than that of anhydrous ethanol. By contrast, the injured normal tissue by PTS injection was less than that by ethanol. In vitro, PTS still demonstrated excellent necrotizing effect on H460 cells when diluted to a lower concentration. Detailed analysis of PTS on H460 cells indicated that PTS had a better effect on attenuating the cell viability and increasing the cell membrane permeability than ethanol at the same level. Conclusions PTS exhibits excellent inhibition effect on the growth of lung cancer by necrotizing tumor in vivo and in vitro, reducing tumor cell viability and augmenting the membrane permeability in vitro, with only mild injury to normal tissue. The antitumor effect of PTS on lung cancer in vivo and in vitro is stronger than that of ethanol. PMID:23991305

Gao, Yang; Gao, Yonghua; Guan, Weijie; Huang, Liyan; Xu, Xiaoming; Zhang, Chenting; Chen, Xiuqing; Wu, Yizhuang; Zeng, Guangqiao

2013-01-01

152

Pseudotyped AAV Vector-Mediated Gene Transfer in a Human Fetal Trachea Xenograft Model: Implications for In Utero Gene Therapy for Cystic Fibrosis  

PubMed Central

Background Lung disease including airway infection and inflammation currently causes the majority of morbidities and mortalities associated with cystic fibrosis (CF), making the airway epithelium and the submucosal glands (SMG) novel target cells for gene therapy in CF. These target cells are relatively inaccessible to postnatal gene transfer limiting the success of gene therapy. Our previous work in a human-fetal trachea xenograft model suggests the potential benefit for treating CF in utero. In this study, we aim to validate adeno-associated virus serotype 2 (AAV2) gene transfer in a human fetal trachea xenograft model and to compare transduction efficiencies of pseudotyping AAV2 vectors in fetal xenografts and postnatal xenograft controls. Methodology/Principal Findings Human fetal trachea or postnatal bronchus controls were xenografted onto immunocompromised SCID mice for a four-week engraftment period. After injection of AAV2/2, 2/1, 2/5, 2/7 or 2/8 with a LacZ reporter into both types of xenografts, we analyzed for transgene expression in the respiratory epithelium and SMGs. At 1 month, transduction by AAV2/2 and AAV2/8 in respiratory epithelium and SMG cells was significantly greater than that of AAV2/1, 2/5, and 2/7 in xenograft tracheas. Efficiency in SMG transduction was significantly greater in AAV2/8 than AAV2/2. At 3 months, AAV2/2 and AAV2/8 transgene expression was >99% of respiratory epithelium and SMG. At 1 month, transduction efficiency of AAV2/2 and AAV2/8 was significantly less in adult postnatal bronchial xenografts than in fetal tracheal xenografts. Conclusions/Significance Based on the effectiveness of AAV vectors in SMG transduction, our findings suggest the potential utility of pseudotyped AAV vectors for treatment of cystic fibrosis. The human fetal trachea xenograft model may serve as an effective tool for further development of fetal gene therapy strategies for the in utero treatment of cystic fibrosis. PMID:22937069

Leung, Alice; Katz, Anna B.; Lim, Foong-Yen; Habli, Mounira; Jones, Helen N.; Wilson, James M.; Crombleholme, Timothy M.

2012-01-01

153

Exploring alternative models of rostral-caudal patterning in the zebrafish neurectoderm with computer simulations.  

PubMed

The StarLogo and NetLogo programming environments allow developmental biologists to build computer models of cell-cell interactions in an epithelium and visualize emergent properties of hypothetical genetic regulatory networks operating in the cells. These environments were used to explore alternative models that show how a posteriorizing morphogen gradient might define gene-expression domains along the rostral-caudal axis in the zebrafish neurectoderm. The models illustrate how a hypothetical genetic network based on auto-activation and cross-repression could lead to establishment of discrete non-overlapping gene-expression domains. PMID:15261658

Chitnis, Ajay B; Itoh, Motoyuki

2004-08-01

154

Novel xenograft model expressing human hepatocyte growth factor shows ligand-dependent growth of c-Met-expressing tumors.  

PubMed

c-Met, a receptor tyrosine kinase responsible for cellular migration, invasion, and proliferation, is overexpressed in human cancers. Although ligand-independent c-Met activation has been described, the majority of tumors are ligand dependent and rely on binding of hepatocyte growth factor (HGF) for receptor activation. Both receptor and ligand are attractive therapeutic targets; however, preclinical models are limited because murine HGF does not activate human c-Met. The goal of this study was to develop a xenograft model in which human HGF (hHGF) is produced in a controllable fashion in the mouse. Severe combined immunodeficient mice were treated with adenovirus encoding the hHGF transgene (Ad-hHGF) via tail vein injection, and transgene expression was determined by the presence of hHGF mRNA in mouse tissue and hHGF in serum. Ad-hHGF administration to severe combined immunodeficient mice resulted in hHGF production that was (a) dependent on quantity of virus delivered; (b) biologically active, resulting in liver hypertrophy; and (c) sustainable over 40 days. In this model, the ligand-dependent human tumor cell line SW1417 showed enhanced tumor growth, whereas the ligand-independent cell lines SW480 and GTL-16 showed no augmented tumor growth. This novel xenograft model is ideal for investigating c-Met/HGF-dependent human tumor progression and for evaluating c-Met targeted therapy. PMID:17431125

Francone, Todd D; Landmann, Ron G; Chen, Chin-Tung; Sun, Mark Y; Kuntz, Eleanor J; Zeng, Zhaoshi; Dematteo, Ronald P; Paty, Philip B; Weiser, Martin R

2007-04-01

155

Zebrafish provide a sensitive model of persisting neurobehavioral effects of developmental chlorpyrifos exposure: Comparison with nicotine and pilocarpine effects and relationship to dopamine deficits  

Microsoft Academic Search

Chlorpyrifos (CPF) an organophosphate pesticide causes persisting behavioral dysfunction in rat models when exposure is during early development. In earlier work zebrafish were used as a complementary model to study mechanisms of CPF-induced neurotoxicity induced during early development. We found that developmental (first five days after fertilization) chlorpyrifos exposure significantly impaired learning in zebrafish. However, this testing was time and

Donnie Eddins; Daniel Cerutti; Paul Williams; Elwood Linney; Edward D. Levin

2010-01-01

156

MEDI3617, a human anti-angiopoietin 2 monoclonal antibody, inhibits angiogenesis and tumor growth in human tumor xenograft models.  

PubMed

Angiopoietin 2 (Ang2) is an important regulator of angiogenesis, blood vessel maturation and integrity of the vascular endothelium. The correlation between the dynamic expression of Ang2 in tumors with regions of high angiogenic activity and a poor prognosis in many tumor types makes Ang2 an ideal drug target. We have generated MEDI3617, a human anti-Ang2 monoclonal antibody that neutralizes Ang2 by preventing its binding to the Tie2 receptor in vitro, and inhibits angiogenesis and tumor growth in vivo. Treatment of mice with MEDI3617 resulted in inhibition of angiogenesis in several mouse models including: FGF2-induced angiogenesis in a basement extract plug model, tumor and retinal angiogenesis. In xenograft tumor models, treatment with MEDI3617 resulted in a reduction in tumor angiogenesis and an increase in tumor hypoxia. The administration of MEDI3617 as a single agent to mice bearing human tumor xenografts resulted in tumor growth inhibition against a broad spectrum of tumor types. Combining MEDI3617 with chemotherapy or bevacizumab resulted in a delay in tumor growth and no body weight loss was observed in the combination groups. These results, combined with pharmacodynamic studies, demonstrate that treatment of tumor-bearing mice with MEDI3617 significantly inhibited tumor growth as a single agent by blocking tumor angiogenesis. Together, these data show that MEDI3617 is a robust antiangiogenic agent and support the clinical evaluation and biomarker development of MEDI3617 in cancer patients. PMID:22327175

Leow, Ching Ching; Coffman, Karen; Inigo, Ivan; Breen, Shannon; Czapiga, Meggan; Soukharev, Serguei; Gingles, Neill; Peterson, Norman; Fazenbaker, Christine; Woods, Rob; Jallal, Bahija; Ricketts, Sally-Ann; Lavallee, Theresa; Coats, Steve; Chang, Yong

2012-05-01

157

Activin type IB receptor signaling in prostate cancer cells promotes lymph node metastasis in a xenograft model  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer ActRIB signaling induces Snail and S100A4 expressions in prostate cancer cells. Black-Right-Pointing-Pointer The prostate cancer cell lines expressing an active form of ActRIB were established. Black-Right-Pointing-Pointer ActRIB signaling promotes EMT and lymph node metastasis in xenograft model. -- Abstract: Activin, a member of the transforming growth factor-{beta} family, has been known to be a growth and differentiating factor. Despite its pluripotent effects, the roles of activin signaling in prostate cancer pathogenesis are still unclear. In this study, we established several cell lines that express a constitutive active form of activin type IB receptor (ActRIBCA) in human prostate cancer cells, ALVA41 (ALVA-ActRIBCA). There was no apparent change in the proliferation of ALVA-ActRIBCA cells in vitro; however, their migratory ability was significantly enhanced. In a xenograft model, histological analysis revealed that the expression of Snail, a cell-adhesion-suppressing transcription factor, was dramatically increased in ALVA-ActRIBCA tumors, indicating epithelial mesenchymal transition (EMT). Finally, mice bearing ALVA-ActRIBCA cells developed multiple lymph node metastases. In this study, we demonstrated that ActRIBCA signaling can promote cell migration in prostate cancer cells via a network of signaling molecules that work together to trigger the process of EMT, and thereby aid in the aggressiveness and progression of prostate cancers.

Nomura, Masatoshi, E-mail: nomura@med.kyushu-u.ac.jp [Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)] [Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Tanaka, Kimitaka; Wang, Lixiang; Goto, Yutaka; Mukasa, Chizu; Ashida, Kenji; Takayanagi, Ryoichi [Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)] [Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

2013-01-04

158

New insights into the pathogenesis of tuberculosis revealed by Mycobacterium marinum: the zebrafish model from the systems biology perspective.  

PubMed

Tuberculosis remains a worldwide health concern, largely due to the emergence of multi-drug-resistant (MDR) and extensive-drug-resistant (XDR) Mycobacterium tuberculosis co-infection with HIV. The exact mechanism of Mycobacterium virulence, pathogenesis, and persistence is not fully understood. The hallmark of tuberculosis, granulomas are promoted by Mycobacterium virulence factors, and they have long been considered a structural advantage to the host. However, this traditional view has been challenged recently, largely due to the evidence originating from the M. marinum-zebrafish model. As a genetically tractable model, zebrafish provide unprecedented opportunities to address the pathogenesis of tuberculosis from a systems biology perspective. The latest data from this model are summarized in this review, special attention is given to the shared pathway and network between zebrafish and humans. This research serves to deepen our understanding of this complex process and to promote the discovery of better countermeasures against tuberculosis. PMID:22181703

Deng, Wanyan; Tang, Xiemei; Hou, Manmei; Li, Chunmei; Xie, Jianping

2011-01-01

159

Zebrafish Heart Failure Models for the Evaluation of Chemical Probes and Drugs  

PubMed Central

Abstract Heart failure is a complex disease that involves genetic, environmental, and physiological factors. As a result, current medication and treatment for heart failure produces limited efficacy, and better medication is in demand. Although mammalian models exist, simple and low-cost models will be more beneficial for drug discovery and mechanistic studies of heart failure. We previously reported that aristolochic acid (AA) caused cardiac defects in zebrafish embryos that resemble heart failure. Here, we showed that cardiac troponin T and atrial natriuretic peptide were expressed at significantly higher levels in AA-treated embryos, presumably due to cardiac hypertrophy. In addition, several human heart failure drugs could moderately attenuate the AA-induced heart failure by 10%–40%, further verifying the model for drug discovery. We then developed a drug screening assay using the AA-treated zebrafish embryos and identified three compounds. Mitogen-activated protein kinase kinase inhibitor (MEK-I), an inhibitor for the MEK-1/2 known to be involved in cardiac hypertrophy and heart failure, showed nearly 60% heart failure attenuation. C25, a chalcone derivative, and A11, a phenolic compound, showed around 80% and 90% attenuation, respectively. Time course experiments revealed that, to obtain 50% efficacy, these compounds were required within different hours of AA treatment. Furthermore, quantitative polymerase chain reaction showed that C25, not MEK-I or A11, strongly suppressed inflammation. Finally, C25 and MEK-I, but not A11, could also rescue the doxorubicin-induced heart failure in zebrafish embryos. In summary, we have established two tractable heart failure models for drug discovery and three potential drugs have been identified that seem to attenuate heart failure by different mechanisms. PMID:24351044

Monte, Aaron; Cook, James M.; Kabir, Mohd Shahjahan; Peterson, Karl P.

2013-01-01

160

The Zebrafish Brain in Research and Teaching: A Simple in Vivo and in Vitro Model for the Study of Spontaneous Neural Activity  

ERIC Educational Resources Information Center

Recently, the zebrafish ("Danio rerio") has been established as a key animal model in neuroscience. Behavioral, genetic, and immunohistochemical techniques have been used to describe the connectivity of diverse neural circuits. However, few studies have used zebrafish to understand the function of cerebral structures or to study neural circuits.…

Vargas, R.; Johannesdottir, I. P.; Sigurgeirsson, B.; Porsteinsson, H.; Karlsson, K. AE.

2011-01-01

161

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme  

PubMed Central

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality. PMID:23051742

Baumann, Brian C.; Dorsey, Jay F.; Benci, Joseph L.; Joh, Daniel Y.; Kao, Gary D.

2012-01-01

162

A zebrafish model of manganism reveals reversible and treatable symptoms that are independent of neurotoxicity.  

PubMed

Manganese (manganese ion; referred to as Mn) is essential for neuronal function, yet it is toxic at high concentrations. Environmental and occupational exposure to high concentrations of Mn causes manganism, a well-defined movement disorder in humans, with symptoms resembling Parkinson's disease (PD). However, manganism is distinct from PD and the neural basis of its pathology is poorly understood. To address this issue, we generated a zebrafish model of manganism by incubating larvae in rearing medium containing Mn. We find that Mn-treated zebrafish larvae exhibit specific postural and locomotor defects. Larvae begin to float on their sides, show a curved spine and swim in circles. We discovered that treatment with Mn causes postural defects by interfering with mechanotransduction at the neuromasts. Furthermore, we find that the circling locomotion could be caused by long-duration bursting in the motor neurons, which can lead to long-duration tail bends in the Mn-treated larvae. Mn-treated larvae also exhibited fewer startle movements. Additionally, we show that the intensity of tyrosine hydroxylase immunoreactivity is reversibly reduced after Mn-treatment. This led us to propose that reduced dopamine neuromodulation drives the changes in startle movements. To test this, when we supplied an external source of dopamine to Mn-treated larvae, the larvae exhibited a normal number of startle swims. Taken together, these results indicate that Mn interferes with neuronal function at the sensory, motor and modulatory levels, and open avenues for therapeutically targeted studies on the zebrafish model of manganism. PMID:25261567

Bakthavatsalam, Subha; Das Sharma, Shreya; Sonawane, Mahendra; Thirumalai, Vatsala; Datta, Ankona

2014-11-01

163

A genetic model of amyotrophic lateral sclerosis in zebrafish displays phenotypic hallmarks of motoneuron disease  

PubMed Central

SUMMARY Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that, for ?80% of patients, is fatal within five years of diagnosis. To better understand ALS, animal models have been essential; however, only rodent models of ALS exhibit the major hallmarks of the disease. Here, we report the generation of transgenic zebrafish overexpressing mutant Sod1. The construct used to generate these lines contained the zebrafish sod1 gene and ?16 kb of flanking sequences. We generated lines expressing the G93R mutation, as well as lines expressing wild-type Sod1. Focusing on two G93R lines, we found that they displayed the major phenotypes of ALS. Changes at the neuromuscular junction were observed at larval and adult stages. In adulthood the G93R mutants exhibited decreased endurance in a swim tunnel test. An analysis of muscle revealed normal muscle force, however, at the end stage the fish exhibited motoneuron loss, muscle atrophy, paralysis and premature death. These phenotypes were more severe in lines expressing higher levels of mutant Sod1 and were absent in lines overexpressing wild-type Sod1. Thus, we have generated a vertebrate model of ALS to complement existing mammal models. PMID:20504969

Ramesh, Tennore; Lyon, Alison N.; Pineda, Ricardo H.; Wang, Chunping; Janssen, Paul M. L.; Canan, Benjamin D.; Burghes, Arthur H. M.; Beattie, Christine E.

2010-01-01

164

Drift-Diffusion Analysis of Neutrophil Migration during Inflammation Resolution in a Zebrafish Model.  

PubMed

Neutrophils must be removed from inflammatory sites for inflammation to resolve. Recent work in zebrafish has shown neutrophils can migrate away from inflammatory sites, as well as die in situ. The signals regulating the process of reverse migration are of considerable interest, but remain unknown. We wished to study the behaviour of neutrophils during reverse migration, to see whether they moved away from inflamed sites in a directed fashion in the same way as they are recruited or whether the inherent random component of their migration was enough to account for this behaviour. Using neutrophil-driven photoconvertible Kaede protein in transgenic zebrafish larvae, we were able to specifically label neutrophils at an inflammatory site generated by tailfin transection. The locations of these neutrophils over time were observed and fitted using regression methods with two separate models: pure-diffusion and drift-diffusion equations. While a model hypothesis test (the F-test) suggested that the datapoints could be fitted by the drift-diffusion model, implying a fugetaxis process, dynamic simulation of the models suggested that migration of neutrophils away from a wound is better described by a zero-drift, "diffusion" process. This has implications for understanding the mechanisms of reverse migration and, by extension, neutrophil retention at inflammatory sites. PMID:22899935

Holmes, Geoffrey R; Dixon, Giles; Anderson, Sean R; Reyes-Aldasoro, Constantino Carlos; Elks, Philip M; Billings, Stephen A; Whyte, Moira K B; Kadirkamanathan, Visakan; Renshaw, Stephen A

2012-01-01

165

Distinct choline metabolic profiles are associated with differences in gene expression for basal-like and luminal-like breast cancer xenograft models  

PubMed Central

Background Increased concentrations of choline-containing compounds are frequently observed in breast carcinomas, and may serve as biomarkers for both diagnostic and treatment monitoring purposes. However, underlying mechanisms for the abnormal choline metabolism are poorly understood. Methods The concentrations of choline-derived metabolites were determined in xenografted primary human breast carcinomas, representing basal-like and luminal-like subtypes. Quantification of metabolites in fresh frozen tissue was performed using high-resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS). The expression of genes involved in phosphatidylcholine (PtdCho) metabolism was retrieved from whole genome expression microarray analyses. The metabolite profiles from xenografts were compared with profiles from human breast cancer, sampled from patients with estrogen/progesterone receptor positive (ER+/PgR+) or triple negative (ER-/PgR-/HER2-) breast cancer. Results In basal-like xenografts, glycerophosphocholine (GPC) concentrations were higher than phosphocholine (PCho) concentrations, whereas this pattern was reversed in luminal-like xenografts. These differences may be explained by lower choline kinase (CHKA, CHKB) expression as well as higher PtdCho degradation mediated by higher expression of phospholipase A2 group 4A (PLA2G4A) and phospholipase B1 (PLB1) in the basal-like model. The glycine concentration was higher in the basal-like model. Although glycine could be derived from energy metabolism pathways, the gene expression data suggested a metabolic shift from PtdCho synthesis to glycine formation in basal-like xenografts. In agreement with results from the xenograft models, tissue samples from triple negative breast carcinomas had higher GPC/PCho ratio than samples from ER+/PgR+ carcinomas, suggesting that the choline metabolism in the experimental models is representative for luminal-like and basal-like human breast cancer. Conclusions The differences in choline metabolite concentrations corresponded well with differences in gene expression, demonstrating distinct metabolic profiles in the xenograft models representing basal-like and luminal-like breast cancer. The same characteristics of choline metabolite profiles were also observed in patient material from ER+/PgR+ and triple-negative breast cancer, suggesting that the xenografts are relevant model systems for studies of choline metabolism in luminal-like and basal-like breast cancer. PMID:20716336

2010-01-01

166

Antitumor activity of motesanib alone and in combination with cisplatin or docetaxel in multiple human non–small-cell lung cancer xenograft models  

PubMed Central

Background Non–small-cell lung cancer (NSCLC) is categorized into various histologic subtypes that play an important role in prognosis and treatment outcome. We investigated the antitumor activity of motesanib, a selective antagonist of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3, platelet-derived growth factor receptor, and Kit, alone and combined with chemotherapy in five human NSCLC xenograft models (A549, Calu-6, NCI-H358, NCI-H1299, and NCI-H1650) containing diverse genetic mutations. Results Motesanib as a single agent dose-dependently inhibited tumor xenograft growth compared with vehicle in all five of the models (P?xenografts compared with either single agent alone (P?xenografts compared with either single agent alone (P?xenografts, motesanib with and without cisplatin significantly decreased tumor blood vessel area (P?xenograft models containing diverse genetic mutations, and that it had enhanced activity when combined with cisplatin or docetaxel. These effects appeared to be mediated primarily by antiangiogenic mechanisms. PMID:22992329

2012-01-01

167

Binding difference of fipronil with GABAARs in fruitfly and zebrafish: insights from homology modeling, docking, and molecular dynamics simulation studies.  

PubMed

Fipronil, which targets GABAA receptors (GABAARs), is the first phenylpyrazole insecticide widely used in crop protection and public hygiene. However, its high toxicity on fishes greatly limited its applications. In the present study, a series of computational methods including homology modeling, docking, and molecular dynamics simulation studies were integrated to explore the binding difference of fipronil with GABAARs from fruitfly and zebrafish systems. It was found that, in the zebrafish system, the H-bond between 6'Thr and fipronil exerted key effects on the recognition of fipronil, which was absent in the fruitfly system. On the other hand, in the fruitfly system, strong electrostatic interaction between 2'Ala and fipronil was favorable to the binding of fipronil but detrimental to the binding in the zebrafish system. These findings marked the binding difference of fipronil with different GABAARs, which might be helpful in designing selective insecticides against pests instead of fishes. PMID:25302733

Zheng, Nan; Cheng, Jiagao; Zhang, Wei; Li, Weihua; Shao, Xusheng; Xu, Zhiping; Xu, Xiaoyong; Li, Zhong

2014-11-01

168

Use of the Zebrafish Larvae as a Model to Study Cigarette Smoke Condensate Toxicity  

PubMed Central

The smoking of tobacco continues to be the leading cause of premature death worldwide and is linked to the development of a number of serious illnesses including heart disease, respiratory diseases, stroke and cancer. Currently, cell line based toxicity assays are typically used to gain information on the general toxicity of cigarettes and other tobacco products. However, they provide little information regarding the complex disease-related changes that have been linked to smoking. The ethical concerns and high cost associated with mammalian studies have limited their widespread use for in vivo toxicological studies of tobacco. The zebrafish has emerged as a low-cost, high-throughput, in vivo model in the study of toxicology. In this study, smoke condensates from 2 reference cigarettes and 6 Canadian brands of cigarettes with different design features were assessed for acute, developmental, cardiac, and behavioural toxicity (neurotoxicity) in zebrafish larvae. By making use of this multifaceted approach we have developed an in vivo model with which to compare the toxicity profiles of smoke condensates from cigarettes with different design features. This model system may provide insights into the development of smoking related disease and could provide a cost-effective, high-throughput platform for the future evaluation of tobacco products. PMID:25526262

Ellis, Lee D.; Soo, Evelyn C.; Achenbach, John C.; Morash, Michael G.; Soanes, Kelly H.

2014-01-01

169

Zebrafish erythropoiesis and the utility of fish as models of anemia  

PubMed Central

Erythrocytes contain oxygen-carrying hemoglobin to all body cells. Impairments in the generation of erythrocytes, a process known as erythropoiesis, or in hemoglobin synthesis alter cell function because of decreased oxygen supply and lead to anemic diseases. Thus, understanding how erythropoiesis is regulated during embryogenesis and adulthood is important to develop novel therapies for anemia. The zebrafish, Danio rerio, provides a powerful model for such study. Their small size and the ability to generate a large number of embryos enable large-scale analysis, and their transparency facilitates the visualization of erythroid cell migration. Importantly, the high conservation of hematopoietic genes among vertebrates and the ability to successfully transplant hematopoietic cells into fish have enabled the establishment of models of human anemic diseases in fish. In this review, we summarize the current progress in our understanding of erythropoiesis on the basis of zebrafish studies and highlight fish models of human anemias. These analyses could enable the discovery of novel drugs as future therapies. PMID:23257067

2012-01-01

170

Graphene-based anticancer nanosystem and its biosafety evaluation using a zebrafish model.  

PubMed

In this paper, a facile strategy to develop graphene-based delivery nanosystems for effective drug loading and sustained drug release was proposed and validated. Specifically, biocompatible naphthalene-terminated PEG (NP) and anticancer drugs (curcumin or doxorubicin (DOX)) were simultaneously integrated onto oxidized graphene (GO), leading to self-assembled, nanosized complexes. It was found that the oxidation degree of GO had a significant impact on the drug-loading efficiency and the structural stability of nanosystems. Interestingly, the nanoassemblies resulted in more effective cellular entry of DOX in comparison with free DOX or DOX-loaded PEG-polyester micelles at equivalent DOX dose, as demonstrated by confocal microscopy studies. Moreover, the nanoassemblies not only exhibited a sustained drug release pattern without an initial burst release, but also significantly improved the stability of formulations which were resistant to drug leaking even in the presence of strong surfactants such as aromatic sodium benzenesulfonate (SBen) and aliphatic sodium dodecylsulfonate (SDS). In addition, the nanoassemblies without DOX loading showed negligible in vitro cytotoxicity, whereas DOX-loaded counterparts led to considerable toxicity against HeLa cells. The DOX-mediated cytotoxicity of the graphene-based formulation was around 20 folds lower than that of free DOX, most likely due to the slow DOX release from complexes. A zebrafish model was established to assess the in vivo safety profile of curcumin-loaded nanosystems. The results showed they were able to excrete from the zebrafish body rapidly and had nearly no influence on the zebrafish upgrowth. Those encouraging results may prompt the advance of graphene-based nanotherapeutics for biomedical applications. PMID:23286342

Liu, Chen-Wei; Xiong, Feng; Jia, Hui-Zhen; Wang, Xu-Li; Cheng, Han; Sun, Yong-Hua; Zhang, Xian-Zheng; Zhuo, Ren-Xi; Feng, Jun

2013-02-11

171

The genetic heterogeneity and mutational burden of engineered melanomas in zebrafish models  

PubMed Central

Background Melanoma is the most deadly form of skin cancer. Expression of oncogenic BRAF or NRAS, which are frequently mutated in human melanomas, promote the formation of nevi but are not sufficient for tumorigenesis. Even with germline mutated p53, these engineered melanomas present with variable onset and pathology, implicating additional somatic mutations in a multi-hit tumorigenic process. Results To decipher the genetics of these melanomas, we sequence the protein coding exons of 53 primary melanomas generated from several BRAFV600E or NRASQ61K driven transgenic zebrafish lines. We find that engineered zebrafish melanomas show an overall low mutation burden, which has a strong, inverse association with the number of initiating germline drivers. Although tumors reveal distinct mutation spectrums, they show mostly C?>?T transitions without UV light exposure, and enrichment of mutations in melanogenesis, p53 and MAPK signaling. Importantly, a recurrent amplification occurring with pre-configured drivers BRAFV600E and p53-/- suggests a novel path of BRAF cooperativity through the protein kinase A pathway. Conclusion This is the first analysis of a melanoma mutational landscape in the absence of UV light, where tumors manifest with remarkably low mutation burden and high heterogeneity. Genotype specific amplification of protein kinase A in cooperation with BRAF and p53 mutation suggests the involvement of melanogenesis in these tumors. This work is important for defining the spectrum of events in BRAF or NRAS driven melanoma in the absence of UV light, and for informed exploitation of models such as transgenic zebrafish to better understand mechanisms leading to human melanoma formation. PMID:24148783

2013-01-01

172

In vivo antimetastatic effects of uPAR retargeted measles virus in syngeneic and xenograft models of mammary cancer  

PubMed Central

Purpose The urokinase receptor (uPAR) plays a critical role in breast cancer (BC) progression and metastases, and is a validated target for novel therapies. The current study investigates the effects of MV-uPA, an oncolytic measles virus fully retargeted against uPAR in syngeneic and xenograft BC metastases models. Methods In vitro replication and cytotoxicity of MVs retargeted against human (MV-h-uPA) or mouse (MV-m-uPA) uPAR were assessed in human and murine cancer and non-cancer mammary epithelial cells. The in vivo effects of species-specific uPAR retargeted MVs were assessed in syngeneic and xenograft models of experimental metastases, established by intravenous administration of luciferase expressing 4T1 or MDA-MD-231 cells. Metastases progression was assessed by in vivo bioluminescence imaging. Tumor targeting was evaluated by qRT-PCR of MV-N, rescue of viable viral particles and immunostaining of MV particles in lungs from tumor bearing mice. Results In vitro, MV-h-uPA and MV-m-uPA selectively infected, replicated and induced cytotoxicity in cancer compared to non-cancer cells in a species-specific manner. In vivo, MV-m-uPA delayed 4T1 lung metastases progression and prolonged survival. These effects were associated with identification of viable viral particles, viral RNA and detection of MV-N by immunostaining from lung tissues in treated mice. In the human MDA-MB-231 metastases model, intravenous administration of MV-h-uPA markedly inhibited metastases progression and significantly improved survival, compared to controls. No significant treatment related toxicity was observed in treated mice. Conclusions The above preclinical findings strongly suggest that uPAR retargeted measles virotherapy is a novel and feasible systemic therapy strategy against metastatic breast cancer. PMID:25519042

Jing, Yuqi; Bejarano, Marcela Toro; Zaias, Julia; Merchan, Jaime R.

2014-01-01

173

Modeling Nociception in Zebrafish: A Way Forward for Unbiased Analgesic Discovery  

PubMed Central

Acute and chronic pain conditions are often debilitating, inflicting severe physiological, emotional and economic costs and affect a large percentage of the global population. However, the development of therapeutic analgesic agents based primarily on targeted drug development has been largely ineffective. An alternative approach to analgesic development would be to develop low cost, high throughput, untargeted animal based behavioral screens that model complex nociceptive behaviors in which to screen for analgesic compounds. Here we describe the development of a behavioral based assay in zebrafish larvae that is effective in identifying small molecule compounds with analgesic properties. In a place aversion assay, which likely utilizes supraspinal neuronal circuitry, individually arrayed zebrafish larvae show temperature-dependent aversion to increasing and decreasing temperatures deviating from rearing temperature. Modeling thermal hyperalgesia, the addition of the noxious inflammatory compound and TRPA1 agonist allyl isothiocyanate sensitized heat aversion and reversed cool aversion leading larvae to avoid rearing temperature in favor of otherwise acutely aversive cooler temperatures. We show that small molecules with known analgesic properties are able to inhibit acute and/or sensitized temperature aversion. PMID:25587718

Curtright, Andrew; Rosser, Micaela; Goh, Shamii; Keown, Bailey; Wagner, Erinn; Sharifi, Jasmine; Raible, David W.; Dhaka, Ajay

2015-01-01

174

Zebrafish as a model system for environmental health studies in the grade 9-12 classroom.  

PubMed

Developing zebrafish embryos were used as a model system for high school students to conduct scientific investigations that reveal features of normal development and to test how different environmental toxicants impact the developmental process. The primary goal of the module was to engage students from a wide range of socio-economic backgrounds, with particular focus on underserved inner-city high schools, in inquiry-based learning and hands-on experimentation. In addition, the module served as a platform for both teachers and students to design additional inquiry-based experiments. In this module, students spawned adult zebrafish to generate developing embryos, exposed the embryos to various toxicants, then gathered, and analyzed data obtained from control and experimental embryos. The module provided a flexible, experimental framework for students to test the effects of numerous environmental toxicants, such as ethanol, caffeine, and nicotine, on the development of a model vertebrate organism. Students also observed the effects of dose on experimental outcomes. From observations of the effects of the chemical agents on vertebrate embryos, students drew conclusions on how these chemicals could impact human development and health. Results of pre-tests and post-tests completed by participating students indicate statistically significant changes in awareness of the impact of environmental agents on fish and human beings In addition, the program's evaluator concluded that participation in the module resulted in significant changes in the attitude of students and teachers toward science in general and environmental health in particular. PMID:24941301

Tomasiewicz, Henry G; Hesselbach, Renee; Carvan, Michael John; Goldberg, Barbara; Berg, Craig A; Petering, David H

2014-08-01

175

Novel mechanism of macrophage-mediated metastasis revealed in a zebrafish model of tumor development.  

PubMed

Cancer metastasis can occur at early stages of tumor development due to facilitative alterations in the tumor microenvironment. Although imaging techniques have considerably improved our understanding of metastasis, early events remain challenging to study due to the small numbers of malignant cells involved that are often undetectable. Using a novel zebrafish model to investigate this process, we discovered that tumor-associated macrophages (TAM) acted to facilitate metastasis by binding tumor cells and mediating their intravasation. Mechanistic investigations revealed that IL6 and TNF? promoted the ability of macrophages to mediate this step. M2 macrophages were particularly potent when induced by IL4, IL10, and TGF?. In contrast, IFN?-lipopolysaccharide-induced M1 macrophages lacked the capability to function in the same way in the model. Confirming these observations, we found that human TAM isolated from primary breast, lung, colorectal, and endometrial cancers exhibited a similar capability in invasion and metastasis. Taken together, our work shows how zebrafish can be used to study how host contributions can facilitate metastasis at its earliest stages, and they reveal a new macrophage-dependent mechanism of metastasis with possible prognostic implications. Cancer Res; 75(2); 306-15. ©2014 AACR. PMID:25492861

Wang, Jian; Cao, Ziquan; Zhang, Xing-Mei; Nakamura, Masaki; Sun, Meili; Hartman, Johan; Harris, Robert A; Sun, Yuping; Cao, Yihai

2015-01-15

176

Modeling nociception in zebrafish: a way forward for unbiased analgesic discovery.  

PubMed

Acute and chronic pain conditions are often debilitating, inflicting severe physiological, emotional and economic costs and affect a large percentage of the global population. However, the development of therapeutic analgesic agents based primarily on targeted drug development has been largely ineffective. An alternative approach to analgesic development would be to develop low cost, high throughput, untargeted animal based behavioral screens that model complex nociceptive behaviors in which to screen for analgesic compounds. Here we describe the development of a behavioral based assay in zebrafish larvae that is effective in identifying small molecule compounds with analgesic properties. In a place aversion assay, which likely utilizes supraspinal neuronal circuitry, individually arrayed zebrafish larvae show temperature-dependent aversion to increasing and decreasing temperatures deviating from rearing temperature. Modeling thermal hyperalgesia, the addition of the noxious inflammatory compound and TRPA1 agonist allyl isothiocyanate sensitized heat aversion and reversed cool aversion leading larvae to avoid rearing temperature in favor of otherwise acutely aversive cooler temperatures. We show that small molecules with known analgesic properties are able to inhibit acute and/or sensitized temperature aversion. PMID:25587718

Curtright, Andrew; Rosser, Micaela; Goh, Shamii; Keown, Bailey; Wagner, Erinn; Sharifi, Jasmine; Raible, David W; Dhaka, Ajay

2015-01-01

177

MYBPC1 mutations impair skeletal muscle function in zebrafish models of arthrogryposis  

PubMed Central

Myosin-binding protein C1 (MYBPC1) is an abundant skeletal muscle protein that is expressed predominantly in slow-twitch muscle fibers. Human MYBPC1 mutations are associated with distal arthrogryposis type 1 and lethal congenital contracture syndrome type 4. As MYBPC1 function is incompletely understood, the mechanism by which human mutations result in contractures is unknown. Here, we demonstrate using antisense morpholino knockdown, that mybpc1 is required for embryonic motor activity and survival in a zebrafish model of arthrogryposis. Mybpc1 morphant embryos have severe body curvature, cardiac edema, impaired motor excitation and are delayed in hatching. Myofibril organization is selectively impaired in slow skeletal muscle and sarcomere numbers are greatly reduced in mybpc1 knockdown embryos, although electron microscopy reveals normal sarcomere structure. To evaluate the effects of human distal arthrogryposis mutations, mybpc1 mRNAs containing the corresponding human W236R and Y856H MYBPC1 mutations were injected into embryos. Dominant-negative effects of these mutations were suggested by the resultant mild bent body curvature, decreased motor activity, as well as impaired overall survival compared with overexpression of wild-type RNA. These results demonstrate a critical role for mybpc1 in slow skeletal muscle development and establish zebrafish as a tractable model of human distal arthrogryposis. PMID:23873045

Ha, Kyungsoo; Buchan, Jillian G.; Alvarado, David M.; Mccall, Kevin; Vydyanath, Anupama; Luther, Pradeep K.; Goldsmith, Matthew I.; Dobbs, Matthew B.; Gurnett, Christina A.

2013-01-01

178

A model of glucose-6-phosphate dehydrogenase deficiency in the zebrafish  

PubMed Central

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common genetic defect and enzymopathy worldwide, affecting approximately 400 million people and causing acute hemolysis in persons exposed to prooxidant compounds such as menthol, naphthalene, anti-malarial drugs, and fava beans. Mouse models have not been useful because of a lack of significant response to oxidative challenge. We turned to zebrafish (Danrio rerio) embryos, which develop ex utero and are transparent, allowing visualization of hemolysis. We designed morpholinos to zebrafish g6pd that were effective in reducing gene expression as shown by Western blot and G6PD enzyme activity, resulting in a brisk hemolysis and pericardial edema secondary to anemia. Titration of the g6pd knockdown allowed us to generate embryos that displayed no overt phenotype until exposed to the prooxidant compounds 1-naphthol, menthol, or primaquine, after which they developed hemolysis and pericardial edema within 48–72 hours. We were also able to show that g6pd morphants displayed significant levels of increased oxidative stress compared with controls. We anticipate that this will be a useful model of G6PD deficiency to study hemolysis as well as oxidative stress that occurs after exposure to prooxidants, similar to what occurs in G6PD-deficient persons. PMID:23603363

Patrinostro, Xiaobai; Carter, Michelle L.; Kramer, Ashley C.; Lund, Troy C.

2013-01-01

179

Polymethoxy-1-alkenes from Aphanizomenon ovalisporum Inhibit Vertebrate Development in the Zebrafish (Danio rerio) Embryo Model  

PubMed Central

Cyanobacteria are recognized producers of a wide array of toxic or otherwise bioactive secondary metabolites. The present study utilized the zebrafish (Danio rerio) embryo as an aquatic animal model of vertebrate development to identify, purify and characterize lipophilic inhibitors of development (i.e., developmental toxins) from an isolate of the freshwater cyanobacterial species, Aphanizomenon ovalisporum.Bioassay-guided fractionation led to the purification, and subsequent chemical characterization, of an apparent homologous series of isotactic polymethoxy-1-alkenes (1–6), including three congeners (4–6) previously identified from the strain, and two variants previously identified from other species (2 and 3), as well as one apparently novel member of the series (1). Five of the PMAs in the series (1–5) were purified in sufficient quantity for comparative toxicological characterization, and toxicity in the zebrafish embryo model was found to generally correlate with relative chain length and/or methoxylation. Moreover, exposure of embryos to a combination of variants indicates an apparent synergistic interaction between the congeners. Although PMAs have been identified previously in cyanobacteria, this is the first report of their apparent toxicity. These results, along with the previously reported presence of the PMAs from several cyanobacterial species, suggest a possibly widespread distribution of the PMAs as toxic secondary metabolites and warrants further chemical and toxicological investigation. PMID:23170087

Jaja-Chimedza, Asha; Gantar, Miroslav; Gibbs, Patrick D. L.; Schmale, Michael C.; Berry, John P.

2012-01-01

180

MYBPC1 mutations impair skeletal muscle function in zebrafish models of arthrogryposis.  

PubMed

Myosin-binding protein C1 (MYBPC1) is an abundant skeletal muscle protein that is expressed predominantly in slow-twitch muscle fibers. Human MYBPC1 mutations are associated with distal arthrogryposis type 1 and lethal congenital contracture syndrome type 4. As MYBPC1 function is incompletely understood, the mechanism by which human mutations result in contractures is unknown. Here, we demonstrate using antisense morpholino knockdown, that mybpc1 is required for embryonic motor activity and survival in a zebrafish model of arthrogryposis. Mybpc1 morphant embryos have severe body curvature, cardiac edema, impaired motor excitation and are delayed in hatching. Myofibril organization is selectively impaired in slow skeletal muscle and sarcomere numbers are greatly reduced in mybpc1 knockdown embryos, although electron microscopy reveals normal sarcomere structure. To evaluate the effects of human distal arthrogryposis mutations, mybpc1 mRNAs containing the corresponding human W236R and Y856H MYBPC1 mutations were injected into embryos. Dominant-negative effects of these mutations were suggested by the resultant mild bent body curvature, decreased motor activity, as well as impaired overall survival compared with overexpression of wild-type RNA. These results demonstrate a critical role for mybpc1 in slow skeletal muscle development and establish zebrafish as a tractable model of human distal arthrogryposis. PMID:23873045

Ha, Kyungsoo; Buchan, Jillian G; Alvarado, David M; McCall, Kevin; Vydyanath, Anupama; Luther, Pradeep K; Goldsmith, Matthew I; Dobbs, Matthew B; Gurnett, Christina A

2013-12-15

181

INDUCED AND SPONTANEOUS NEOPLASIA IN ZEBRAFISH.  

EPA Science Inventory

To address the potential of zebrafish as a cancer model, it is important to determine the susceptibility of zebrafish to tumors, and to compare zebrafish tumors with human tumors. To determine whether the commonly-used germ line mutagen, ethylnitrosourea (ENU) induces tumors, we ...

182

Vascular Development in the Zebrafish  

PubMed Central

The zebrafish has emerged as an excellent vertebrate model system for studying blood and lymphatic vascular development. The small size, external and rapid development, and optical transparency of zebrafish embryos are some of the advantages the zebrafish model system offers. Multiple well-established techniques have been developed for imaging and functionally manipulating vascular tissues in zebrafish embryos, expanding on and amplifying these basic advantages and accelerating use of this model system for studying vascular development. In the past decade, studies performed using zebrafish as a model system have provided many novel insights into vascular development. In this article we discuss the amenability of this model system for studying blood vessel development and review contributions made by this system to our understanding of vascular development. PMID:22553495

Gore, Aniket V.; Monzo, Kathryn; Cha, Young R.; Pan, Weijun; Weinstein, Brant M.

2012-01-01

183

Local delivery of cannabinoid-loaded microparticles inhibits tumor growth in a murine xenograft model of glioblastoma multiforme.  

PubMed

Cannabinoids, the active components of marijuana and their derivatives, are currently investigated due to their potential therapeutic application for the management of many different diseases, including cancer. Specifically, ?(9)-Tetrahydrocannabinol (THC) and Cannabidiol (CBD) - the two major ingredients of marijuana - have been shown to inhibit tumor growth in a number of animal models of cancer, including glioma. Although there are several pharmaceutical preparations that permit the oral administration of THC or its analogue nabilone or the oromucosal delivery of a THC- and CBD-enriched cannabis extract, the systemic administration of cannabinoids has several limitations in part derived from the high lipophilicity exhibited by these compounds. In this work we analyzed CBD- and THC-loaded poly-?-caprolactone microparticles as an alternative delivery system for long-term cannabinoid administration in a murine xenograft model of glioma. In vitro characterization of THC- and CBD-loaded microparticles showed that this method of microencapsulation facilitates a sustained release of the two cannabinoids for several days. Local administration of THC-, CBD- or a mixture (1:1 w:w) of THC- and CBD-loaded microparticles every 5 days to mice bearing glioma xenografts reduced tumour growth with the same efficacy than a daily local administration of the equivalent amount of those cannabinoids in solution. Moreover, treatment with cannabinoid-loaded microparticles enhanced apoptosis and decreased cell proliferation and angiogenesis in these tumours. Our findings support that THC- and CBD-loaded microparticles could be used as an alternative method of cannabinoid delivery in anticancer therapies. PMID:23349970

Hernán Pérez de la Ossa, Dolores; Lorente, Mar; Gil-Alegre, Maria Esther; Torres, Sofía; García-Taboada, Elena; Aberturas, María Del Rosario; Molpeceres, Jesús; Velasco, Guillermo; Torres-Suárez, Ana Isabel

2013-01-01

184

A Novel Zebrafish Model to Provide Mechanistic Insights into the Inflammatory Events in Carrageenan-Induced Abdominal Edema  

PubMed Central

A suitable small animal model may help in the screening and evaluation of new drugs, especially those from natural products, which can be administered at lower dosages, fulfilling an urgent worldwide need. In this study, we explore whether zebrafish could be a model organism for carrageenan-induced abdominal edema. The research results showed that intraperitoneal (i.p.) administration of 1.5% ?-carrageenan in a volume of 20 µL significantly increased abdominal edema in adult zebrafish. Levels of the proinflammatory proteins tumor necrosis factor-? (TNF-?) and inducible nitric oxide synthase (iNOS) were increased in carrageenan-injected adult zebrafish during the development of abdominal edema. An associated enhancement was also observed in the leukocyte marker, myeloperoxidase (MPO). To support these results, we further observed that i.p. methylprednisolone (MP; 1 µg), a positive control, significantly inhibited carrageenan-induced inflammation 24 h after carrageenan administration. Furthermore, i.p. pretreatment with either an anti-TNF-? antibody (1?5 dilution in a volume of 20 µL) or the iNOS-selective inhibitor aminoguanidine (AG; 1 µg) inhibited carrageenan-induced abdominal edema in adult zebrafish. This new animal model is uncomplicated, easy to develop, and involves a straightforward inducement of inflammatory edema for the evaluation of small volumes of drugs or test compounds. PMID:25141004

Huang, Shi-Ying; Feng, Chien-Wei; Hung, Han-Chun; Chakraborty, Chiranjib; Chen, Chun-Hong; Chen, Wu-Fu; Jean, Yen-Hsuan; Wang, Hui-Min David; Sung, Chun-Sung; Sun, Yu-Min; Wu, Chang-Yi; Liu, Wangta; Hsiao, Chung-Der; Wen, Zhi-Hong

2014-01-01

185

Optimized cell transplantation using adult rag2 mutant zebrafish  

PubMed Central

Cell transplantation into adult zebrafish has lagged behind mouse due to the lack of immune compromised models. Here, we have created homozygous rag2E450fs mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft zebrafish muscle, blood stem cells, and cancers. rag2E450fs mutant zebrafish are the first immune compromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer. PMID:25042784

Tang, Qin; Abdelfattah, Nouran S.; Blackburn, Jessica S.; Moore, John C.; Martinez, Sarah A.; Moore, Finola E.; Lobbardi, Riadh; Tenente, Inês M.; Ignatius, Myron S.; Berman, Jason N.; Liwski, Robert S.; Houvras, Yariv; Langenau, David M.

2014-01-01

186

From Omics to Drug Metabolism and High Content Screen of Natural Product in Zebrafish: A New Model for Discovery of Neuroactive Compound  

PubMed Central

The zebrafish (Danio rerio) has recently become a common model in the fields of genetics, environmental science, toxicology, and especially drug screening. Zebrafish has emerged as a biomedically relevant model for in vivo high content drug screening and the simultaneous determination of multiple efficacy parameters, including behaviour, selectivity, and toxicity in the content of the whole organism. A zebrafish behavioural assay has been demonstrated as a novel, rapid, and high-throughput approach to the discovery of neuroactive, psychoactive, and memory-modulating compounds. Recent studies found a functional similarity of drug metabolism systems in zebrafish and mammals, providing a clue with why some compounds are active in zebrafish in vivo but not in vitro, as well as providing grounds for the rationales supporting the use of a zebrafish screen to identify prodrugs. Here, we discuss the advantages of the zebrafish model for evaluating drug metabolism and the mode of pharmacological action with the emerging omics approaches. Why this model is suitable for identifying lead compounds from natural products for therapy of disorders with multifactorial etiopathogenesis and imbalance of angiogenesis, such as Parkinson's disease, epilepsy, cardiotoxicity, cerebral hemorrhage, dyslipidemia, and hyperlipidemia, is addressed. PMID:22919414

Hung, Ming Wai; Zhang, Zai Jun; Li, Shang; Lei, Benson; Yuan, Shuai; Cui, Guo Zhen; Man Hoi, Pui; Chan, Kelvin; Lee, Simon Ming Yuen

2012-01-01

187

Zebrafish as a correlative and predictive model for assessing biomaterial nanotoxicity.  

PubMed

The lack of correlative and predictive models to assess acute and chronic toxicities limits the rapid pre-clinical development of new therapeutics. This barrier is due in part to the exponential growth of nanotechnology and nanotherapeutics, coupled with the lack of rigorous and robust screening assays and putative standards. It is a fairly simple and cost-effective process to initially screen the toxicity of a nanomaterial by using invitro cell cultures; unfortunately it is nearly impossible to imitate a complimentary invivo system. Small mammalian models are the most common method used to assess possible toxicities and biodistribution of nanomaterials in humans. Alternatively, Daniorerio, commonly known as zebrafish, are proving to be a quick, cheap, and facile model to conservatively assess toxicity of nanomaterials. PMID:19389433

Fako, Valerie E; Furgeson, Darin Y

2009-06-21

188

Towards a comprehensive eye model for zebrafish retinal imaging using full range spectral domain optical coherence tomography  

NASA Astrophysics Data System (ADS)

In regenerative medicine, the zebrafish is a prominent animal model for studying degeneration and regeneration processes, e.g. of photoreceptor cells in the retina. By means of optical coherence tomography (OCT), these studies can be conducted over weeks using the same individual and hence reducing the variability of the results. To allow an improvement of zebrafish retinal OCT imaging by suitable optics, we developed a zebrafish eye model using geometrical data obtained by in vivo dispersion encoded full range OCT as well as a dispersion comprising gradient index (GRIN) lens model based on refractive index data found in the literature. Using non-sequential ray tracing, the focal length of the spherical GRIN lens (diameter of 0.96 mm) was determined to be 1.22 mm at 800 nm wavelength giving a Matheissen's ratio (ratio of focal length to radius of the lens) of 2.54, which fits well into the range between 2.19 and 2.82, found for various fish lenses. Additionally, a mean refractive index of 1.64 at 800 nm could be retrieved for the lens to yield the same focal position as found for the GRIN condition. With the aid of the zebrafish eye model, the optics of the OCT scanner head were adjusted to provide high-resolution retinal images with a field of view of 30° x 30°. The introduced model therefore provides the basis for improved retinal imaging with OCT and can be further used to study the image formation within the zebrafish eye.

Gaertner, Maria; Weber, Anke; Cimalla, Peter; Köttig, Felix; Brand, Michael; Koch, Edmund

2014-03-01

189

Targeting Plasminogen Activator Inhibitor-1 Inhibits Angiogenesis and Tumor Growth in a Human Cancer Xenograft Model  

PubMed Central

Cancers of the urinary bladder result in aggressive and highly angiogenic tumors for which standard treatments have only limited success. Patients with advanced disease have a 5-year survival rate of less than 20%, and no new anticancer agent has been successfully introduced into the clinic armamentarium for the treatment of bladder cancer in more than 20 years. Investigations have identified plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, as being highly expressed in several malignancies, including bladder cancer, in which high expression is associated with a poor prognosis. In this study, we evaluated PAI-1 as a potential therapeutic target for bladder cancer. PAI-1 expression was manipulated in a panel of cell lines and functional inhibition was achieved using the small molecule tiplaxtinin. Reduction or inhibition of PAI-1 resulted in the reduction of cellular proliferation, cell adhesion, and colony formation, and the induction of apoptosis and anoikis in vitro. Treatment of T24 xenografts with tiplaxtinin resulted in inhibition of angiogenesis and induction of apoptosis, leading to a significant reduction in tumor growth. Similar results were obtained through evaluation of the human cervical cancer HeLa cell line, showing that PAI-1–mediated effects are not restricted to tumor cells of bladder origin. Collectively, these data show that targeting PAI-1 may be beneficial and support the notion that novel drugs such as tiplaxtinin could be investigated as anticancer agents. PMID:24072883

Gomes-Giacoia, Evan; Miyake, Makito; Goodison, Steve; Rosser, Charles J.

2015-01-01

190

Antitumor effects of flavopiridol on human uterine leiomyoma in vitro and in a xenograft model.  

PubMed

Dysregulated cyclin-dependent kinases (CDKs) are considered a potential target for cancer therapy. Flavopiridol is a potent CDK inhibitor. In this study, the antiproliferative effect of the flavonoid compound flavopiridol and its mechanism in human uterine leiomyoma cells were investigated. The present study focused on the effect of flavopiridol in cell proliferation and cell cycle progression in primary cultured human uterine leiomyoma cells. Cell viability and cell proliferation assays were conducted. Flow cytometry was performed to determine the effect of flavopiridol on cell cycle. The expression of cell cycle regulatory-related proteins was evaluated by Western blotting. Cell viability and proliferation of uterine leiomyoma cells were significantly reduced by flavopiridol treatment in a dose-dependent manner. Flow cytometry results showed that flavopiridol induced G1 phase arrest. Flavopiridol-induced growth inhibition in uterine leiomyoma cells was associated with increased expression of p21(cip/wafl) and p27(kip1) in a dose-dependent manner. Downregulation of CDK2/4 and Cyclin A with a concomitant increase in dephosphorylation of retinoblastoma was observed. This study demonstrates that flavopiridol inhibits cell proliferation by initiating G1 cell cycle arrest in human uterine leiomyoma. We also found that flavopiridol is effective in inhibiting xenografted human uterine leiomyoma growth. These results indicate that flavopiridol could prove to be a promising chemopreventive and therapeutic agent for human uterine leiomyoma. PMID:24572052

Lee, Hyun-Gyo; Baek, Jong-Woo; Shin, So-Jin; Kwon, Sang-Hoon; Cha, Soon-Do; Park, Won-Jin; Chung, Rosa; Choi, Eun-Som; Lee, Gun-Ho; Cho, Chi-Heum

2014-09-01

191

WaveCNV: allele-specific copy number alterations in primary tumors and xenograft models from next-generation sequencing  

PubMed Central

Motivation: Copy number variations (CNVs) are a major source of genomic variability and are especially significant in cancer. Until recently microarray technologies have been used to characterize CNVs in genomes. However, advances in next-generation sequencing technology offer significant opportunities to deduce copy number directly from genome sequencing data. Unfortunately cancer genomes differ from normal genomes in several aspects that make them far less amenable to copy number detection. For example, cancer genomes are often aneuploid and an admixture of diploid/non-tumor cell fractions. Also patient-derived xenograft models can be laden with mouse contamination that strongly affects accurate assignment of copy number. Hence, there is a need to develop analytical tools that can take into account cancer-specific parameters for detecting CNVs directly from genome sequencing data. Results: We have developed WaveCNV, a software package to identify copy number alterations by detecting breakpoints of CNVs using translation-invariant discrete wavelet transforms and assign digitized copy numbers to each event using next-generation sequencing data. We also assign alleles specifying the chromosomal ratio following duplication/loss. We verified copy number calls using both microarray (correlation coefficient 0.97) and quantitative polymerase chain reaction (correlation coefficient 0.94) and found them to be highly concordant. We demonstrate its utility in pancreatic primary and xenograft sequencing data. Availability and implementation: Source code and executables are available at https://github.com/WaveCNV. The segmentation algorithm is implemented in MATLAB, and copy number assignment is implemented Perl. Contact: lakshmi.muthuswamy@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24192544

Holt, Carson; Losic, Bojan; Pai, Deepa; Zhao, Zhen; Trinh, Quang; Syam, Sujata; Arshadi, Niloofar; Jang, Gun Ho; Ali, Johar; Beck, Tim; McPherson, John; Muthuswamy, Lakshmi B.

2014-01-01

192

Targeting of histone deacetylases to reactivate tumour suppressor genes and its therapeutic potential in a human cervical cancer xenograft model.  

PubMed

Aberrant histone acetylation plays an essential role in the neoplastic process via the epigenetic silencing of tumour suppressor genes (TSGs); therefore, the inhibition of histone deacetylases (HDAC) has become a promising target in cancer therapeutics. To investigate the correlation of histone acetylation with clinicopathological features and TSG expression, we examined the expression of acetylated H3 (AcH3), RAR?2, E-cadherin, and ?-catenin by immunohistochemistry in 65 cervical squamous cell carcinoma patients. The results revealed that the absence of AcH3 was directly associated with poor histological differentiation and nodal metastasis as well as reduced/negative expression of RAR?2, E-cadherin, and ?-catenin in clinical tumour samples. We further demonstrated that the clinically available HDAC inhibitors valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA), in combination with all-trans retinoic acid (ATRA), can overcome the epigenetic barriers to transcription of RAR?2 in human cervical cancer cells. Chromatin immunoprecipitation analysis showed that the combination treatment increased the enrichment of acetylated histone in the RAR?2-RARE promoter region. In view of these findings, we evaluated the antitumor effects induced by combined VPA and ATRA treatment in a xenograft model implanted with poorly differentiated human squamous cell carcinoma. Notably, VPA restored RAR?2 expression via epigenetic modulation. Additive antitumour effects were produced in tumour xenografts by combining VPA with ATRA treatment. Mechanistically, the combination treatment reactivated the expression of TSGs RAR?2, E-cadherin, P21 (CIP1) , and P53 and reduced the level of p-Stat3. Sequentially, upregulation of involucrin and loricrin, which indicate terminal differentiation, strongly contributed to tumour growth inhibition along with partial apoptosis. In conclusion, targeted therapy with HDAC inhibitors and RAR?2 agonists may represent a novel therapeutic approach for patients with cervical squamous cell carcinoma. PMID:24260446

Feng, Dingqing; Wu, Jiao; Tian, Yuan; Zhou, Hu; Zhou, Ying; Hu, Weiping; Zhao, Weidong; Wei, Haiming; Ling, Bin; Ma, Chunhong

2013-01-01

193

Antitumor effect of FP3 in combination with cetuximab on patient-derived tumor tissue xenograft models of primary colon carcinoma and related lymphatic and hepatic metastases.  

PubMed

FP3 is an engineered protein which contains the extracellular domain 2 of vascular endothelial growth factor (VEGF) receptor 1 (Flt-1) and the extracellular domain 3 and 4 of VEGF receptor 2 (Flk-1, KDR) fused to the Fc portion of human immunoglobulin G1. Previous studies have demonstrated its antiangiogenic effects in vitro and in vivo, and its antitumor activity in vivo. Cetuximab is a monoclonal antibody against epidermal growth factor (EGF) receptor. Combined inhibition of VEGF and EGF signaling may act additively or synergistically. In this study, patient-derived tumor tissue (PDTT) xenograft models of primary colon carcinoma and lymphatic and hepatic metastases were established for assessment of the antitumor activity of FP3 in combination with cetuximab. Xenografts were treated with FP3 and cetuximab, alone or in combination. After tumor growth was confirmed, volume and microvessel density in tumors were evaluated. Levels of VEGF, EGFR and PCNA in the tumor were examined by immunohistochemical staining, and levels of related cell signaling pathway proteins were examined by western blotting. FP3 in combination with cetuximab showed significant antitumor activity in three xenograft models (primary colon carcinoma, lymphatic metastasis and hepatic metastasis). The microvessel density in tumor tissues treated with FP3 in combination with cetuximab was lower compared to that of the control. Antitumor activity of FP3 in combination with cetuximab was significantly higher than that of each agent alone in two xenograft models (colon carcinoma lymphatic metastasis and hepatic metastasis). This study indicated that addition of FP3 to cetuximab significantly improved tumor growth inhibition in the PDTT xenograft models of colon carcinoma lymphatic and hepatic metastases. Combination anti-VEGF (FP3) and anti-EGFR (cetuximab) therapies may represent a novel therapeutic strategy for the management of metastatic colon carcinoma. PMID:22505231

Dong, Xiaofang; Jin, Ketao; Hu, Xiaoyan; Du, Fangmin; Lan, Huanrong; Han, Na; Ma, Zhaosheng; Xie, Bojian; Cui, Binbin; Teng, Lisong; Cao, Feilin

2012-07-01

194

Effect of Melatonin on Tumor Growth and Angiogenesis in Xenograft Model of Breast Cancer  

PubMed Central

As neovascularization is essential for tumor growth and metastasis, controlling angiogenesis is a promising tactic in limiting cancer progression. Melatonin has been studied for their inhibitory properties on angiogenesis in cancer. We performed an in vivo study to evaluate the effects of melatonin treatment on angiogenesis in breast cancer. Cell viability was measured by MTT assay after melatonin treatment in triple-negative breast cancer cells (MDA-MB-231). After, cells were implanted in athymic nude mice and treated with melatonin or vehicle daily, administered intraperitoneally 1 hour before turning the room light off. Volume of the tumors was measured weekly with a digital caliper and at the end of treatments animals underwent single photon emission computed tomography (SPECT) with Technetium-99m tagged vascular endothelial growth factor (VEGF) C to detect in vivo angiogenesis. In addition, expression of pro-angiogenic/growth factors in the tumor extracts was evaluated by membrane antibody array and collected tumor tissues were analyzed with histochemical staining. Melatonin in vitro treatment (1 mM) decreased cell viability (p<0.05). The breast cancer xenografts nude mice treated with melatonin showed reduced tumor size and cell proliferation (Ki-67) compared to control animals after 21 days of treatment (p<0.05). Expression of VEGF receptor 2 decreased significantly in the treated animals compared to that of control when determined by immunohistochemistry (p<0.05) but the changes were not significant on SPECT (p>0.05) images. In addition, there was a decrease of micro-vessel density (Von Willebrand Factor) in melatonin treated mice (p<0.05). However, semiquantitative densitometry analysis of membrane array indicated increased expression of epidermal growth factor receptor and insulin-like growth factor 1 in treated tumors compared to vehicle treated tumors (p<0.05). In conclusion, melatonin treatment showed effectiveness in reducing tumor growth and cell proliferation, as well as in the inhibition of angiogenesis. PMID:24416386

Jardim-Perassi, Bruna Victorasso; Arbab, Ali S.; Ferreira, Lívia Carvalho; Borin, Thaiz Ferraz; Varma, Nadimpalli R. S.; Iskander, A. S. M.; Shankar, Adarsh; Ali, Meser M.; de Campos Zuccari, Debora Aparecida Pires

2014-01-01

195

Emerging applications of zebrafish as a model organism to study oxidative mechanisms and their role in inflammation and vascular accumulation of oxidized lipids  

PubMed Central

With the advent of genetic engineering, zebrafish (Danio rerio) were recognized as an attractive model organism to study many biological processes. Remarkably, the small size and optical transparency of zebrafish larvae enable high-resolution imaging of live animals. Zebrafish respond to various environmental and pathological factors with robust oxidative stress. In this article, we provide an overview of molecular mechanisms involved in oxidative stress and antioxidant response in zebrafish. Existing applications of generically-encoded fluorescent sensors allow imaging, in real time, production of H2O2 and studying its involvement in inflammatory responses, as well as activation of oxidation-sensitive transcription factors HIF and NRF2. Oxidative stress, combined with hypelipidemia, leads to oxidation of lipoproteins, the process that contributes significantly to development of human atherosclerosis. Recent work found that feeding zebrafish a high-cholesterol diet results in hypercholesterolemia, vascular lipid accumulation and extreme lipoprotein oxidation. Generation of a transgenic zebrafish expressing a GFP-tagged human antibody to malondialdehyde (MDA)-modified LDL makes possible in vivo visualization of MDA epitopes in the vascular wall and testing the efficacy of antioxidants and dietary interventions. Thus, using zebrafish as a model organism provides important advantages in studying the role of ROS and lipid oxidation in basic biologic and pathologic processes. PMID:22906686

Fang, Longhou; Miller, Yury I.

2012-01-01

196

Quantitative Phenotyping-Based In Vivo Chemical Screening in a Zebrafish Model of Leukemia Stem Cell Xenotransplantation  

PubMed Central

Zebrafish-based chemical screening has recently emerged as a rapid and efficient method to identify important compounds that modulate specific biological processes and to test the therapeutic efficacy in disease models, including cancer. In leukemia, the ablation of leukemia stem cells (LSCs) is necessary to permanently eradicate the leukemia cell population. However, because of the very small number of LSCs in leukemia cell populations, their use in xenotransplantation studies (in vivo) and the difficulties in functionally and pathophysiologically replicating clinical conditions in cell culture experiments (in vitro), the progress of drug discovery for LSC inhibitors has been painfully slow. In this study, we developed a novel phenotype-based in vivo screening method using LSCs xenotransplanted into zebrafish. Aldehyde dehydrogenase-positive (ALDH+) cells were purified from chronic myelogenous leukemia K562 cells tagged with a fluorescent protein (Kusabira-orange) and then implanted in young zebrafish at 48 hours post-fertilization. Twenty-four hours after transplantation, the animals were treated with one of eight different therapeutic agents (imatinib, dasatinib, parthenolide, TDZD-8, arsenic trioxide, niclosamide, salinomycin, and thioridazine). Cancer cell proliferation, and cell migration were determined by high-content imaging. Of the eight compounds that were tested, all except imatinib and dasatinib selectively inhibited ALDH+ cell proliferation in zebrafish. In addition, these anti-LSC agents suppressed tumor cell migration in LSC-xenotransplants. Our approach offers a simple, rapid, and reliable in vivo screening system that facilitates the phenotype-driven discovery of drugs effective in suppressing LSCs. PMID:24454867

Zhang, Beibei; Shimada, Yasuhito; Kuroyanagi, Junya; Umemoto, Noriko; Nishimura, Yuhei; Tanaka, Toshio

2014-01-01

197

Transient Exposure to Ethanol during Zebrafish Embryogenesis Results in Defects in Neuronal Differentiation: An Alternative Model System to Study FASD  

PubMed Central

Background The exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS). In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines. Methodology/Principal Findings In order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification. Conclusion Our investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s) of ethanol-induced developmental toxicity at very early stages of embryonic development. PMID:25383948

Joya, Xavier; Garcia-Algar, Oscar; Vall, Oriol; Pujades, Cristina

2014-01-01

198

Evaluation of zebrafish brain development using optical coherence tomography.  

PubMed

The zebrafish is a well-established model system used to study and understand various human biological processes. The present study used OCT to investigate growth of the adult zebrafish brain. Twenty zebrafish were studied, using their standard lengths as indicators of their age. Zebrafish brain aging was evaluated by analyzing signal attenuation rates and texture features in regions of interest (ROIs). Optical scattering originates from light interaction with biological structures. During development, the zebrafish brain gains cells. Signal attenuation rate, therefore, increases with increasing zebrafish brain age. This study's analyses of texture features could not identify aging in zebrafish brain. These results, therefore, indicated that the OCT signal attenuation rate can indicate zebrafish brain aging, and its analysis provides a more effective means of observing zebrafish brain aging than texture features analysis. Using OCT system could further increase the technique's potential for recognition and monitoring of zebrafish brain development. PMID:22961725

Lin, Yu-Sheng; Chu, Chin-Chou; Tsui, Po-Hsiang; Chang, Chien-Cheng

2013-09-01

199

Xenograft and Transgenic Mouse Models of Epithelial Ovarian Cancer and Non Invasive Imaging Modalities to Monitor Ovarian Tumor Growth In situ -Applications in Evaluating Novel Therapeutic Agents  

PubMed Central

Epithelial ovarian cancer (EOC) is the most commonly fatal gynecologic malignancy in developed countries. Most EOC patients are diagnosed at advanced stage when disease has spread beyond the ovary. While many patients initially respond to surgery and chemotherapy, the long term prognosis is generally unfavorable, with recurrence and development of drug resistant disease. There is a critical need to identify new therapeutic agents that prolong disease-free intervals and effectively manage recurrent disease. Murine models of ovarian carcinoma are excellent models to study tumor biology in the search for new treatments for EOC. Described in this unit are methods for establishing xenograft or allograft models of EOC using ovarian carcinoma cell lines, in vivo imaging strategies for detection and quantification of EOC in transgenic and in xenograft/allograft models, and procedures for necropsy and pathological evaluation of experimental animals. PMID:20634901

Connolly, Denise C.; Hensley, Harvey H.

2009-01-01

200

Intravital Fluorescence Imaging of Small Interfering RNA–Mediated Gene Repression in a Dual Reporter Melanoma Xenograft Model  

PubMed Central

Development of RNA interference (RNAi)-based therapeutics has been hampered by the lack of effective and efficient means of delivery. Reliable model systems for screening and optimizing delivery of RNAi-based agents in vivo are crucial for preclinical research aimed at advancing nucleic acid-based therapies. We describe here a dual fluorescent reporter xenograft melanoma model prepared by intradermal injection of human A375 melanoma cells expressing tandem tomato fluorescent protein (tdTFP) containing a small interfering RNA (siRNA) target site as well as enhanced green fluorescent protein (EGFP), which is used as a normalization control. Intratumoral injection of a siRNA specific to the incorporated siRNA target site, complexed with a cationic lipid that has been optimized for in vivo delivery, resulted in 65%±11% knockdown of tdTFP relative to EGFP quantified by in vivo imaging and 68%±10% by reverse transcription–quantitative polymerase chain reaction. No effect was observed with nonspecific control siRNA treatment. This model provides a platform on which siRNA delivery technologies can be screened and optimized in vivo. PMID:23098239

Hickerson, Robyn P.; Gonzalez-Gonzalez, Emilio; Vlassov, Alexander V.; Li, Mu; Lara, Maria Fernanda; Contag, Christopher H.

2012-01-01

201

Using Zebrafish to Study Renal Regeneration  

PubMed Central

Over the past several decades, the zebrafish has become one of the major vertebrate model organisms used in biomedical research. In this arena, the zebrafish has emerged as an applicable system for the study of kidney diseases and renal regeneration. The relevance of the zebrafish model for nephrology research has been increasingly appreciated as the understanding of zebrafish kidney structure, ontogeny, and the response to damage has steadily expanded. Recent studies have documented the amazing regenerative characteristics of the zebrafish kidney, which include the ability to replace epithelial populations after acute injury and to grow new renal functional units, termed nephrons. Here we discuss how nephron composition is conserved between zebrafish and mammals, and highlight how recent findings from zebrafish studies utilizing transgenic technologies and chemical genetics can complement traditional murine approaches in the effort to dissect how the kidney responds to acute damage and identify therapeutics that enhance human renal regeneration. PMID:24183931

McCampbell, Kristen K.; Wingert, Rebecca A.

2013-01-01

202

Mapping of Zebrafish Research: A Global Outlook  

PubMed Central

Abstract On the basis of analysis of 17,151 records on zebrafish identified from Zebrafish Information Network: the zebrafish model organism database and Web of Science, the research performance on this model organism has been evaluated. The earliest research work on zebrafish as reflected in the databases goes back to 1951. After a rather slow growth till the 1980s, research on zebrafish gained momentum in the 1990s. Analysis shows a rapid and consistent increase in the publication output with 226 publications in the year 1996, to 1929 publications in the year 2012. The prominent areas of zebrafish research, journals, and leading authors as reflected from the research output have been identified. USA is the most productive country with 8196 articles. The most frequently used keywords were also determined to gain insights about the research trends and some of the commonly used keywords other than zebrafish and Danio rerio are development, retina, and gene expression. PMID:24131434

Mahesh, Gopalakrishnan; Panwar, Yatish

2013-01-01

203

Focused chemical genomics using zebrafish xenotransplantation as a pre-clinical therapeutic platform for T-cell acute lymphoblastic leukemia.  

PubMed

Cancer therapeutics is evolving to precision medicine, with the goal of matching targeted compounds with molecular aberrations underlying a patient's cancer. While murine models offer a pre-clinical tool, associated costs and time are not compatible with actionable patient-directed interventions. Using the paradigm of T-cell acute lymphoblastic leukemia, a high-risk disease with defined molecular underpinnings, we developed a zebrafish human cancer xenotransplantation model to inform therapeutic decisions. Using a focused chemical genomic approach, we demonstrate that xenografted cell lines harboring mutations in the NOTCH1 and PI3K/AKT pathways respond concordantly to their targeted therapies, patient-derived T-cell acute lymphoblastic leukemia can be successfully engrafted in zebrafish and specific drug responses can be quantitatively determined. Using this approach, we identified a mutation sensitive to ?-secretase inhibition in a xenograft from a child with T-cell acute lymphoblastic leukemia, confirmed by Sanger sequencing and validated as a gain-of-function NOTCH1 mutation. The zebrafish xenotransplantation platform provides a novel cost-effective means of tailoring leukemia therapy in real time. PMID:25281505

Bentley, Victoria L; Veinotte, Chansey J; Corkery, Dale P; Pinder, Jordan B; LeBlanc, Marissa A; Bedard, Karen; Weng, Andrew P; Berman, Jason N; Dellaire, Graham

2015-01-01

204

Focused chemical genomics using zebrafish xenotransplantation as a pre-clinical therapeutic platform for T-cell acute lymphoblastic leukemia  

PubMed Central

Cancer therapeutics is evolving to precision medicine, with the goal of matching targeted compounds with molecular aberrations underlying a patient’s cancer. While murine models offer a pre-clinical tool, associated costs and time are not compatible with actionable patient-directed interventions. Using the paradigm of T-cell acute lymphoblastic leukemia, a high-risk disease with defined molecular underpinnings, we developed a zebrafish human cancer xenotransplantation model to inform therapeutic decisions. Using a focused chemical genomic approach, we demonstrate that xenografted cell lines harboring mutations in the NOTCH1 and PI3K/AKT pathways respond concordantly to their targeted therapies, patient-derived T-cell acute lymphoblastic leukemia can be successfully engrafted in zebrafish and specific drug responses can be quantitatively determined. Using this approach, we identified a mutation sensitive to ?-secretase inhibition in a xenograft from a child with T-cell acute lymphoblastic leukemia, confirmed by Sanger sequencing and validated as a gain-of-function NOTCH1 mutation. The zebrafish xenotransplantation platform provides a novel cost-effective means of tailoring leukemia therapy in real time. PMID:25281505

Bentley, Victoria L.; Veinotte, Chansey J.; Corkery, Dale P.; Pinder, Jordan B.; LeBlanc, Marissa A.; Bedard, Karen; Weng, Andrew P.; Berman, Jason N.; Dellaire, Graham

2015-01-01

205

Using heterogeneity of the patient-derived xenograft model to identify the chemoresistant population in ovarian cancer  

PubMed Central

A cornerstone of preclinical cancer research has been the use of clonal cell lines. However, this resource has underperformed in its ability to effectively identify novel therapeutics and evaluate the heterogeneity in a patient's tumor. The patient-derived xenograft (PDX) model retains the heterogeneity of patient tumors, allowing a means to not only examine efficacy of a therapy, but also basic tenets of cancer biology in response to treatment. Herein we describe the development and characterization of an ovarian-PDX model in order to study the development of chemoresistance. We demonstrate that PDX tumors are not simply composed of tumor-initiating cells, but recapitulate the original tumor's heterogeneity, oncogene expression profiles, and clinical response to chemotherapy. Combined carboplatin/paclitaxel treatment of PDX tumors enriches the cancer stem cell populations, but persistent tumors are not entirely composed of these populations. RNA-Seq analysis of six pair of treated PDX tumors compared to untreated tumors demonstrates a consistently contrasting genetic profile after therapy, suggesting similar, but few, pathways are mediating chemoresistance. Pathways and genes identified by this methodology represent novel approaches to targeting the chemoresistant population in ovarian cancer PMID:25209969

Dobbin, Zachary C.; Katre, Ashwini A.; Steg, Adam D.; Erickson, Britt K.; Shah, Monjri M.; Alvarez, Ronald D.; Conner, Michael G.; Schneider, David; Chen, Dongquan; Landen, Charles N.

2014-01-01

206

The isothiocyanate erucin abrogates telomerase in hepatocellular carcinoma cells in vitro and in an orthotopic xenograft tumour model of HCC.  

PubMed

In contrast to cancer cells, most normal human cells have no or low telomerase levels which makes it an attractive target for anti-cancer drugs. The small molecule sulforaphane from broccoli is known for its cancer therapeutic potential in vitro and in vivo. In animals and humans it was found to be quickly metabolized into 4-methylthiobutyl isothiocyanate (MTBITC, erucin) which we recently identified as strong selective apoptosis inducer in hepatocellular carcinoma (HCC) cells. Here, we investigated the relevance of telomerase abrogation for cytotoxic efficacy of MTBITC against HCC. The drug was effective against telomerase, independent from TP53 and MTBITC also blocked telomerase in chemoresistant subpopulations. By using an orthotopic human liver cancer xenograft model, we give first evidence that MTBITC at 50 mg/KG b.w./d significantly decreased telomerase activity in vivo without affecting enzyme activity of adjacent normal tissue. Upon drug exposure, telomerase decrease was consistent with a dose-dependent switch to anti-survival, cell arrest and apoptosis in our in vitro HCC models. Blocking telomerase by the specific inhibitor TMPyP4 further sensitized cancer cells to MTBITC-mediated cytotoxicity. Overexpression of hTERT, but not enzyme activity deficient DNhTERT, protected against apoptosis; neither DNA damage nor cytostasis induction by MTBITC was prevented by hTERT overexpression. These findings imply that telomerase enzyme activity does not protect against MTBITC-induced DNA damage but impacts signalling processes upstream of apoptosis execution level. PMID:25256442

Herz, Corinna; Hertrampf, Anke; Zimmermann, Stefan; Stetter, Nadine; Wagner, Meike; Kleinhans, Claudia; Erlacher, Miriam; Schüler, Julia; Platz, Stefanie; Rohn, Sascha; Mersch-Sundermann, Volker; Lamy, Evelyn

2014-12-01

207

The isothiocyanate erucin abrogates telomerase in hepatocellular carcinoma cells in vitro and in an orthotopic xenograft tumour model of HCC  

PubMed Central

In contrast to cancer cells, most normal human cells have no or low telomerase levels which makes it an attractive target for anti-cancer drugs. The small molecule sulforaphane from broccoli is known for its cancer therapeutic potential in vitro and in vivo. In animals and humans it was found to be quickly metabolized into 4-methylthiobutyl isothiocyanate (MTBITC, erucin) which we recently identified as strong selective apoptosis inducer in hepatocellular carcinoma (HCC) cells. Here, we investigated the relevance of telomerase abrogation for cytotoxic efficacy of MTBITC against HCC. The drug was effective against telomerase, independent from TP53 and MTBITC also blocked telomerase in chemoresistant subpopulations. By using an orthotopic human liver cancer xenograft model, we give first evidence that MTBITC at 50 mg/KG b.w./d significantly decreased telomerase activity in vivo without affecting enzyme activity of adjacent normal tissue. Upon drug exposure, telomerase decrease was consistent with a dose-dependent switch to anti-survival, cell arrest and apoptosis in our in vitro HCC models. Blocking telomerase by the specific inhibitor TMPyP4 further sensitized cancer cells to MTBITC-mediated cytotoxicity. Overexpression of hTERT, but not enzyme activity deficient DNhTERT, protected against apoptosis; neither DNA damage nor cytostasis induction by MTBITC was prevented by hTERT overexpression. These findings imply that telomerase enzyme activity does not protect against MTBITC-induced DNA damage but impacts signalling processes upstream of apoptosis execution level. PMID:25256442

Herz, Corinna; Hertrampf, Anke; Zimmermann, Stefan; Stetter, Nadine; Wagner, Meike; Kleinhans, Claudia; Erlacher, Miriam; Schüler, Julia; Platz, Stefanie; Rohn, Sascha; Mersch-Sundermann, Volker; Lamy, Evelyn

2014-01-01

208

The Zebrafish- Danio rerio – Is a Useful Model for Measuring the Effects of Small-molecule Mitigators of Late Effects of Ionizing Irradiation  

PubMed Central

Background/Aim Use of zebrafish models may decrease the cost of screening new irradiation protectors and mitigators. Materials and Methods Zebrafish (Danio rerio) models were tested for screening water-soluble radiation protectors and mitigators. Irradiation of embryos and monitoring survival, and measuring fibrosis of the caudal musculature of adults allowed for testing of acute and late effects, respectively. Results Incubation of zebrafish embryos either before or after irradiation in ethyl pyruvate (1 mM) increased survival. Irradiation of adults to 15 to 75 Gy, delivered in single-fraction at 13 Gy/min, showed dose-dependent fibrosis at 30 days, quantitated as physiological decrease in swimming tail movement, and histopathological detection of collagen deposition in the dorsal musculature. Continuous administration of small-molecule radioprotector drugs in the water after irradiation reduced both acute and chronic injuries. Conclusion The zebrafish is cost-effective for screening new radiation countermeasures. PMID:23160669

EPPERLY, MICHAEL W.; BAHARY, NATHAN; QUADER, MUBINA; DEWALD, VALERIE; GREENBERGER, JOEL S.

2013-01-01

209

Analysis of RNAseq datasets from a comparative infectious disease zebrafish model using GeneTiles bioinformatics.  

PubMed

We present a RNA deep sequencing (RNAseq) analysis of a comparison of the transcriptome responses to infection of zebrafish larvae with Staphylococcus epidermidis and Mycobacterium marinum bacteria. We show how our developed GeneTiles software can improve RNAseq analysis approaches by more confidently identifying a large set of markers upon infection with these bacteria. For analysis of RNAseq data currently, software programs such as Bowtie2 and Samtools are indispensable. However, these programs that are designed for a LINUX environment require some dedicated programming skills and have no options for visualisation of the resulting mapped sequence reads. Especially with large data sets, this makes the analysis time consuming and difficult for non-expert users. We have applied the GeneTiles software to the analysis of previously published and newly obtained RNAseq datasets of our zebrafish infection model, and we have shown the applicability of this approach also to published RNAseq datasets of other organisms by comparing our data with a published mammalian infection study. In addition, we have implemented the DEXSeq module in the GeneTiles software to identify genes, such as glucagon A, that are differentially spliced under infection conditions. In the analysis of our RNAseq data, this has led to the possibility to improve the size of data sets that could be efficiently compared without using problem-dedicated programs, leading to a quick identification of marker sets. Therefore, this approach will also be highly useful for transcriptome analyses of other organisms for which well-characterised genomes are available. PMID:25503064

Veneman, Wouter J; de Sonneville, Jan; van der Kolk, Kees-Jan; Ordas, Anita; Al-Ars, Zaid; Meijer, Annemarie H; Spaink, Herman P

2015-03-01

210

Characterization of Antiestrogenic Activity of the Chinese Herb, Prunella vulgaris, Using In Vitro and In Vivo (Mouse Xenograft) Models1  

PubMed Central

Prunella vulgaris (PV), a commonly used Chinese herb, also known as Self-heal, has a wide range of reported medicinal activities. By screening multiple herbs using the endometrial cancer cell line, ECC-1, and an alkaline phosphatase detection assay, we found that PV displayed significant antiestrogenic activity. We investigated the possible usefulness of antiestrogenic activity using both in vitro and in vivo models of endometrial function. Using the well-differentiated, hormone-responsive endometrial cell line, ECC-1, PV extract, at concentrations that were not toxic to the cells, significantly reduced alkaline phosphatase activity and cell proliferation in response to estrogen in a dose-dependent manner. The expression of CYR61, an estrogen-induced protein, was blocked in ECC-1 cells by both the antiestrogen ICI 182?780 and PV extract. Interestingly, PV extract did not appear to directly inhibit estrogen signaling. Rather, we found that its activities were probably related to an ability to function as an aryl hydrocarbon receptor (AHR) agonist in ECC-1 cells. In support of this hypothesis, we noted that PV induced CYP1A1, CYP1B1, and AHR repressor expression in a dose-dependent manner—responses that were blocked by small interfering RNA treatment to reduce AHR and specific AHR antagonists. Ovariectomized immunodeficient RAG-2/gamma(c) knockout mice implanted with human endometrial xenografts developed implants only when treated with estrogen. Mice treated with estrogen and PV tea in their drinking water had fewer and smaller xenograft implants compared with their estrogen-treated counterparts that drank only water (P < 0.05). Analysis of the resulting implants by immunohistochemistry demonstrated persistent estrogen receptor (ER), but reduced proliferation and CYR61 expression. Mouse uterine tissue weight in PV-treated mice was not different from controls, and cycle fecundity of intact C57 female mice was unaffected by PV tea treatment. PV, or Self-heal, exhibits significant antiestrogenic properties, both in vitro and in vivo. This activity is likely due to the ability of PV-activated AHR to interfere with estrogen. This herb may be useful as an adjunct for the treatment of estrogen-dependent processes like endometriosis and breast and uterine cancers. Full characterization of this herb will likely provide new insights into the crosstalk between AHR and ESR1, with potential for therapeutic applications in women. PMID:18923163

Collins, Nancy H.; Lessey, Elizabeth C.; DuSell, Carolyn D.; McDonnell, Donald P.; Fowler, Lindsay; Palomino, Wilder A.; Illera, Maria J.; Yu, Xianzhong; Mo, Bilan; Houwing, Angela M.; Lessey, Bruce A.

2008-01-01

211

Characterization of antiestrogenic activity of the Chinese herb, prunella vulgaris, using in vitro and in vivo (Mouse Xenograft) models.  

PubMed

Prunella vulgaris (PV), a commonly used Chinese herb, also known as Self-heal, has a wide range of reported medicinal activities. By screening multiple herbs using the endometrial cancer cell line, ECC-1, and an alkaline phosphatase detection assay, we found that PV displayed significant antiestrogenic activity. We investigated the possible usefulness of antiestrogenic activity using both in vitro and in vivo models of endometrial function. Using the well-differentiated, hormone-responsive endometrial cell line, ECC-1, PV extract, at concentrations that were not toxic to the cells, significantly reduced alkaline phosphatase activity and cell proliferation in response to estrogen in a dose-dependent manner. The expression of CYR61, an estrogen-induced protein, was blocked in ECC-1 cells by both the antiestrogen ICI 182,780 and PV extract. Interestingly, PV extract did not appear to directly inhibit estrogen signaling. Rather, we found that its activities were probably related to an ability to function as an aryl hydrocarbon receptor (AHR) agonist in ECC-1 cells. In support of this hypothesis, we noted that PV induced CYP1A1, CYP1B1, and AHR repressor expression in a dose-dependent manner--responses that were blocked by small interfering RNA treatment to reduce AHR and specific AHR antagonists. Ovariectomized immunodeficient RAG-2/gamma(c) knockout mice implanted with human endometrial xenografts developed implants only when treated with estrogen. Mice treated with estrogen and PV tea in their drinking water had fewer and smaller xenograft implants compared with their estrogen-treated counterparts that drank only water (P < 0.05). Analysis of the resulting implants by immunohistochemistry demonstrated persistent estrogen receptor (ER), but reduced proliferation and CYR61 expression. Mouse uterine tissue weight in PV-treated mice was not different from controls, and cycle fecundity of intact C57 female mice was unaffected by PV tea treatment. PV, or Self-heal, exhibits significant antiestrogenic properties, both in vitro and in vivo. This activity is likely due to the ability of PV-activated AHR to interfere with estrogen. This herb may be useful as an adjunct for the treatment of estrogen-dependent processes like endometriosis and breast and uterine cancers. Full characterization of this herb will likely provide new insights into the crosstalk between AHR and ESR1, with potential for therapeutic applications in women. PMID:18923163

Collins, Nancy H; Lessey, Elizabeth C; DuSell, Carolyn D; McDonnell, Donald P; Fowler, Lindsay; Palomino, Wilder A; Illera, Maria J; Yu, Xianzhong; Mo, Bilan; Houwing, Angela M; Lessey, Bruce A

2009-02-01

212

The Addition of A Pregnenolone Pendant Group Enhances the Anticancer Properties of Titanocene Dichloride in a MCF-7 Xenograft Model  

PubMed Central

Background/Aim Titanocene dichloride held great promise as a chemotherapeutic compound in preclinical studies. However, subsequent clinical trials revealed hepatoxicity and nephrotoxicity, which limited its use in clinical applications. Therefore, we used steroid pendant groups to improve the targeting of titanocene in the MCF-7 breast cancer cell line, and demonstrated a 10-fold lower effective dose compared to titanocene in in vitro assays. The aim of the present study was to test the efficacy of a titanocene functionalized with pregnenolone (Ti-Preg) in an in vivo breast cancer model. Materials and Methods Xenografts from the MCF7 breast cancer cell line were implanted into athymic nu/nu mice to evaluate the potential of Ti-Preg as an anti-breast cancer agent. Results Ti-Preg demonstrated a significant inhibition of MCF-7 tumor growth when compared to vehicle and to titanocene controls. Conclusion Our findings demonstrate the potential of steroid pendent groups for targeting chemotherapeutics to steroid hormone-dependent cancer. PMID:24692689

Ramos, Gladiany; Loperena, Yaliz; Ortiz, Giovanni; Reyes, Fiorella; Szeto, Ada; Vera, Jose; Velez, Javier; Morales, Jessica; Morrero, Deborah; Castillo, Linnette; Dharmawardhane, Surangani; Melendez, Enrique; Washington, A. Valance

2014-01-01

213

Antitumor effects of TRAIL-expressing mesenchymal stromal cells in a mouse xenograft model of human mesothelioma.  

PubMed

Malignant mesothelioma (MM) remains a highly deadly malignancy with poor treatment option. The MM cells further promote a highly inflammatory microenvironment, which contributes to tumor initiation, development, severity and propagation. We reasoned that the anti-inflammatory actions of mesenchymal stromal cells (MSCs) and further antitumor effects of MSCs engineered to overexpress tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein (MSC-TRAIL) would effectively inhibit mesothelioma growth. Using a mouse xenograft model of intraperitoneal human mesothelioma, native mouse (mMSCs) or human (hMSC) MSCs were administered either systemically (intravenously or intraperitoneally) at various times following tumor inoculation. Both mMSCs and hMSCs localized at the sites of MM tumor growth in vivo and decreased local inflammation. Further, a trend towards decrease in tumor burden was observed. Parallel studies of in vitro exposure of nine primary human mesothelioma cell lines to mMSCs or hMSCs demonstrated reduced tumor cell migration. MSC-TRAIL exposure induced apoptosis of TRAIL-sensitive MM cells in vitro, and both mouse and human MSC-TRAIL significantly reduced the inflammatory tumor environment in vivo. Moreover, human MSC-TRAIL administration significantly reduced peritoneal tumor burden in vivo and increased tumor cell apoptosis. These proof-of-concept studies suggest that TRAIL-expressing MSCs may be useful against malignant mesothelioma. PMID:25525034

Lathrop, M J; Sage, E K; Macura, S L; Brooks, E M; Cruz, F; Bonenfant, N R; Sokocevic, D; MacPherson, M B; Beuschel, S L; Dunaway, C W; Shukla, A; Janes, S M; Steele, C; Mossman, B T; Weiss, D J

2015-01-01

214

Selective in vivo targeting of human liver tumors by optimized AAV3 vectors in a murine xenograft model.  

PubMed

Current challenges for recombinant adeno-associated virus (rAAV) vector-based cancer treatment include the low efficiency and the lack of specificity in vivo. rAAV serotype 3 (rAAV3) vectors have previously been shown to be ineffective in normal mouse tissues following systemic administration. In the present study, we report that rAAV3 vectors can efficiently target and transduce various human liver cancer cells in vivo. Elimination of specific surface-exposed serine and threonine residues on rAAV3 capsids results in further augmentation in the transduction efficiency of these vectors, without any change in the viral tropism and cellular receptor interactions. In addition, we have identified a potential chemotherapy drug, shikonin, as a multifunctional compound to inhibit liver tumor growth as well as to significantly enhance the efficacy of rAAV vector-based gene therapy in vivo. Furthermore, we also document that suppression of tumorigenesis in a human liver cancer xenograft model can be achieved through systemic administration of the optimized rAAV3 vectors carrying a therapeutic gene, and shikonin at a dose that does not lead to liver damage. Our research provides a novel means to achieve not only targeted delivery but also the potential for gene therapy of human liver cancer. PMID:25296041

Ling, Chen; Wang, Yuan; Zhang, Yuanhui; Ejjigani, Anila; Yin, Zifei; Lu, Yuan; Wang, Lina; Wang, Meng; Li, Jun; Hu, Zhongbo; Aslanidi, George V; Zhong, Li; Gao, Guangping; Srivastava, Arun; Ling, Changquan

2014-12-01

215

Comparison of effects of anti-angiogenic agents in the zebrafish efficacy–toxicity model for translational anti-angiogenic drug discovery  

PubMed Central

Background Anti-angiogenic therapy in certain cancers has been associated with improved control of tumor growth and metastasis. Development of anti-angiogenic agents has, however, been saddled with higher attrition rate due to suboptimal efficacy, narrow therapeutic windows, or development of organ-specific toxicities. The aim of this study was to evaluate the translational ability of the zebrafish efficacy–toxicity model to stratify anti-angiogenic agents based on efficacy, therapeutic windows, and off-target effects to streamline the compound selection process in anti-angiogenic discovery. Methods The embryonic model of zebrafish was employed for studying angiogenesis and toxicity. The zebrafish were treated with anti-angiogenic compounds to evaluate their effects on angiogenesis and zebrafish-toxicity parameters. Angiogenesis was measured by scoring the development of subintestinal vessels. Toxicity was evaluated by calculating the median lethal concentration, the lowest observed effect concentration, and gross morphological changes. Results of efficacy and toxicity were used to predict the therapeutic window. Results In alignment with the clinical outcomes, the zebrafish assays demonstrated that vascular endothelial growth factor receptor (VEGFR) inhibitors are the most potent anti-angiogenic agents, followed by multikinase inhibitors and inhibitors of endothelial cell proliferation. The toxicity assays reported cardiac phenotype in zebrafish treated with VEGFR inhibitors and multikinase inhibitors with VEGFR activity suggestive of cardiotoxic potential of these compounds. Several other pathological features were reported for multikinase inhibitors suggestive of off-target effects. The predicted therapeutic window was translational with the clinical trial outcomes of the anti-angiogenic agents. The zebrafish efficacy–toxicity approach could stratify anti-angiogenic agents based on the mechanism of action and delineate chemical structure-driven biological activity of anti-angiogenic compounds. Conclusion The zebrafish efficacy–toxicity approach can be used as a predictive model for translational anti-angiogenic drug discovery to streamline compound selection, resulting in safer and efficacious anti-angiogenic agents entering the clinics. PMID:25170251

Chimote, Geetanjali; Sreenivasan, Jayasree; Pawar, Nilambari; Subramanian, Jyothi; Sivaramakrishnan, Hariharan; Sharma, Somesh

2014-01-01

216

Ovarian Tumor Attachment, Invasion, and Vascularization Reflect Unique Microenvironments in the Peritoneum: Insights from Xenograft and Mathematical Models  

PubMed Central

Ovarian cancer relapse is often characterized by metastatic spread throughout the peritoneal cavity with tumors attached to multiple organs. In this study, interaction of ovarian cancer cells with the peritoneal tumor microenvironment was evaluated in a xenograft model based on intraperitoneal injection of fluorescent SKOV3.ip1 ovarian cancer cells. Intra-vital microscopy of mixed GFP-red fluorescent protein (RFP) cell populations injected into the peritoneum demonstrated that cancer cells aggregate and attach as mixed spheroids, emphasizing the importance of homotypic adhesion in tumor formation. Electron microscopy provided high resolution structural information about local attachment sites. Experimental measurements from the mouse model were used to build a three-dimensional cellular Potts ovarian tumor model (OvTM) that examines ovarian cancer cell attachment, chemotaxis, growth, and vascularization. OvTM simulations provide insight into the relative influence of cancer cell–cell adhesion, oxygen availability, and local architecture on tumor growth and morphology. Notably, tumors on the mesentery, omentum, or spleen readily invade the “open” architecture, while tumors attached to the gut encounter barriers that restrict invasion and instead rapidly expand into the peritoneal space. Simulations suggest that rapid neovascularization of SKOV3.ip1 tumors is triggered by constitutive release of angiogenic factors in the absence of hypoxia. This research highlights the importance of cellular adhesion and tumor microenvironment in the seeding of secondary ovarian tumors on diverse organs within the peritoneal cavity. Results of the OvTM simulations indicate that invasion is strongly influenced by features underlying the mesothelial lining at different sites, but is also affected by local production of chemotactic factors. The integrated in vivo mouse model and computer simulations provide a unique platform for evaluating targeted therapies for ovarian cancer relapse. PMID:23730620

Steinkamp, Mara P.; Winner, Kimberly Kanigel; Davies, Suzy; Muller, Carolyn; Zhang, Yong; Hoffman, Robert M.; Shirinifard, Abbas; Moses, Melanie; Jiang, Yi; Wilson, Bridget S.

2013-01-01

217

Microgavage of Zebrafish Larvae  

PubMed Central

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe interactions17-25. Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results.We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system. PMID:23463135

Cocchiaro, Jordan L.; Rawls, John F.

2013-01-01

218

Modeling Stress and Anxiety in Zebrafish Jonathan M. Cachat, Peter R. Canavello, Marco F. Elegante,  

E-print Network

behavior, preda- tor stress, endocrine response, endocrine signaling, behavioral phenotyping, drug their potential for studying disorders such as Parkin- son's, Alzheimer's, anxiety, and depression (12). While complex neuropsychiatric disorders are difficult to reproduce in zebrafish, analogous brain mechanisms may

Kalueff, Allan V.

219

Circulating Tumor Cells as a Biomarker of Response to Treatment in Patient-Derived Xenograft Mouse Models of Pancreatic Adenocarcinoma  

PubMed Central

Circulating tumor cells (CTCs) are cells shed from solid tumors into circulation and have been shown to be prognostic in the setting of metastatic disease. These cells are obtained through a routine blood draw and may serve as an easily accessible marker for monitoring treatment effectiveness. Because of the rapid progression of pancreatic ductal adenocarcinoma (PDAC), early insight into treatment effectiveness may allow for necessary and timely changes in treatment regimens. The objective of this study was to evaluate CTC burden as a biomarker of response to treatment with a oral phosphatidylinositol-3-kinase inhibitor, BKM120, in patient-derived xenograft (PDX) mouse models of PDAC. PDX mice were randomized to receive vehicle or BKM120 treatment for 28 days and CTCs were enumerated from whole blood before and after treatment using a microfluidic chip that selected for EpCAM (epithelial cell adhesion molecule) positive cells. This microfluidic device allowed for the release of captured CTCs and enumeration of these cells via their electrical impedance signatures. Median CTC counts significantly decreased in the BKM120 group from pre- to post-treatment (26.61 to 2.21 CTCs/250 µL, p?=?0.0207) while no significant change was observed in the vehicle group (23.26 to 11.89 CTCs/250 µL, p?=?0.8081). This reduction in CTC burden in the treatment group correlated with tumor growth inhibition indicating CTC burden is a promising biomarker of response to treatment in preclinical models. Mutant enriched sequencing of isolated CTCs confirmed that they harbored KRAS G12V mutations, identical to the matched tumors. In the long-term, PDX mice are a useful preclinical model for furthering our understanding of CTCs. Clinically, mutational analysis of CTCs and serial monitoring of CTC burden may be used as a minimally invasive approach to predict and monitor treatment response to guide therapeutic regimens. PMID:24586805

Torphy, Robert J.; Tignanelli, Christopher J.; Kamande, Joyce W.; Moffitt, Richard A.; Herrera Loeza, Silvia G.; Soper, Steven A.; Yeh, Jen Jen

2014-01-01

220

Targeted Hsp70 expression combined with CIK-activated immune reconstruction synergistically exerts antitumor efficacy in patient-derived hepatocellular carcinoma xenograft mouse models.  

PubMed

The patient-derived tumor xenograft (PDTX) models can reproduce a similar natural genetic background and similar biological behaviors to tumor cells in patients, which is conducive to the assessment of personalized cancer treatment. In this study, to verify the targeting and effectiveness of the therapeutic strategy using a Survivin promoter-regulated oncolytic adenovirus expressing Hsp70, the PDTX models of hepatocellular carcinoma (HCC) were established in nude mice and the cytokine-induced killer (CIK) cells were intravenously infused into mice to partially reconstruct the mouse immune function. The results demonstrated that, either the immune anti-tumor effect caused by CIK cell infusion or the oncolytic effect generated by oncolytic adenovirus replication was very limited. However, the synergistic tumor inhibitory effect was significantly enhanced after treatments with oncolytic adenovirus expressing Hsp70 combined with CIK cells. Oncolytic adenovirus mediated the specific expression of Hsp70 in cancer tissues allowed the CIK chemotaxis, and induce the infiltration of CD3+ T cells in tumor stroma, thereby exhibiting anti-tumor activity. The anti-tumor effect was more effective for the highly malignant tumor xenografts with highly Survivin expression. This strategy can synergistically activate multiple anti-tumor mechanisms and exert effective anti-tumor activities that have a significant inhibitory effect against the growth of HCC xenografts. PMID:25473902

Hu, Huanzhang; Qiu, Yinghe; Guo, Minggao; Huang, Yao; Fang, Lin; Peng, Zhangxiao; Ji, Weidan; Xu, Yang; Shen, Shuwen; Yan, Yan; Huang, Xuandong; Zheng, Junnian; Su, Changqing

2014-11-26

221

Sqstm1 knock-down causes a locomotor phenotype ameliorated by rapamycin in a zebrafish model of ALS/FTLD.  

PubMed

Mutations in SQSTM1, encoding for the protein SQSTM1/p62, have been recently reported in 1-3.5% of patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS/FTLD). Inclusions positive for SQSTM1/p62 have been detected in patients with neurodegenerative disorders, including ALS/FTLD. In order to investigate the pathogenic mechanisms induced by SQSTM1 mutations in ALS/FTLD, we developed a zebrafish model. Knock-down of the sqstm1 zebrafish ortholog, as well as impairment of its splicing, led to a specific phenotype, consisting of behavioral and axonal anomalies. Here, we report swimming deficits associated with shorter motor neuronal axons that could be rescued by the overexpression of wild-type human SQSTM1. Interestingly, no rescue of the loss-of-function phenotype was observed when overexpressing human SQSTM1 constructs carrying ALS/FTLD-related mutations. Consistent with its role in autophagy regulation, we found increased mTOR levels upon knock-down of sqstm1. Furthermore, treatment of zebrafish embryos with rapamycin, a known inhibitor of the mTOR pathway, yielded an amelioration of the locomotor phenotype in the sqstm1 knock-down model. Our results suggest that loss-of-function of SQSTM1 causes phenotypic features characterized by locomotor deficits and motor neuron axonal defects that are associated with a misregulation of autophagic processes. PMID:25410659

Lattante, Serena; de Calbiac, Hortense; Le Ber, Isabelle; Brice, Alexis; Ciura, Sorana; Kabashi, Edor

2014-11-19

222

Zebrafish Models for Ectopic Mineralization Disorders: Practical Issues from Morpholino Design to Post-Injection Observations  

PubMed Central

Zebrafish (ZF, Danio rerio) has emerged as an important and popular model species to study different human diseases. Key regulators of skeletal development and calcium metabolism are highly conserved between mammals and ZF. The corresponding orthologs share significant sequence similarities and an overlap in expression patterns when compared to mammals, making ZF a potential model for the study of mineralization-related disorders and soft tissue mineralization. To characterize the function of early mineralization-related genes in ZF, these genes can be knocked down by injecting morpholinos into early stage embryos. Validation of the morpholino needs to be performed and the concern of aspecific effects can be addressed by applying one or more independent techniques to knock down the gene of interest. Post-injection assessment of early mineralization defects can be done using general light microscopy, calcein staining, Alizarin red staining, Alizarin red-Alcian blue double staining, and by the use of transgenic lines. Examination of general molecular defects can be done by performing protein and gene expression analysis, and more specific processes can be explored by investigating ectopic mineralization-related mechanisms such as apoptosis and mitochondrial dysfunction. In this paper, we will discuss all details about the aforementioned techniques; shared knowledge will be very useful for the future investigation of ZF models for ectopic mineralization disorders and to understand the underlying pathways involved in soft tissue calcification. PMID:23760765

Hosen, Mohammad Jakir; Vanakker, Olivier M.; Willaert, Andy; Huysseune, Ann; Coucke, Paul; De Paepe, Anne

2013-01-01

223

Zebrafish models for ectopic mineralization disorders: practical issues from morpholino design to post-injection observations.  

PubMed

Zebrafish (ZF, Danio rerio) has emerged as an important and popular model species to study different human diseases. Key regulators of skeletal development and calcium metabolism are highly conserved between mammals and ZF. The corresponding orthologs share significant sequence similarities and an overlap in expression patterns when compared to mammals, making ZF a potential model for the study of mineralization-related disorders and soft tissue mineralization. To characterize the function of early mineralization-related genes in ZF, these genes can be knocked down by injecting morpholinos into early stage embryos. Validation of the morpholino needs to be performed and the concern of aspecific effects can be addressed by applying one or more independent techniques to knock down the gene of interest. Post-injection assessment of early mineralization defects can be done using general light microscopy, calcein staining, Alizarin red staining, Alizarin red-Alcian blue double staining, and by the use of transgenic lines. Examination of general molecular defects can be done by performing protein and gene expression analysis, and more specific processes can be explored by investigating ectopic mineralization-related mechanisms such as apoptosis and mitochondrial dysfunction. In this paper, we will discuss all details about the aforementioned techniques; shared knowledge will be very useful for the future investigation of ZF models for ectopic mineralization disorders and to understand the underlying pathways involved in soft tissue calcification. PMID:23760765

Hosen, Mohammad Jakir; Vanakker, Olivier M; Willaert, Andy; Huysseune, Ann; Coucke, Paul; De Paepe, Anne

2013-01-01

224

Effects of chitosan on xenograft models of melanoma in C57BL/6 mice and hepatoma formation in SCID mice.  

PubMed

According to the World Health Organization, Complementary and alternative medicine (CAM) is a comprehensive term referring to traditional medical treatments and various forms of indigenous medicines, also known as indigenous or folk medicine. Cancer patients often use CAM in the form of nutritional supplements, psychological techniques and natural medical approaches in the place of or in parallel to conventional medicine. The present study aimed to determine if Chitosan can inhibit lung metastasis and hepatoma formation, by studying xenograft of B16F10 melanoma cells in C57BL/6 mice and of Smmu 7721 cells in SCID mice, respectively. For the lung metastasis model, after a five-week treatment, the survival rates of B6 mice were 15% for the control group and 35%, 20%, 45% and 40% for the 320,000 kDa, 173,000 kDa, 86,000 kDa and 8,000 kDa molecular-weight treatment groups, respectively. Chitosan treatment dramatically increased lifespan and inhibited tumor metastasis especially in treatment groups of the low-molecular weight compound. For the hepatoma growth model, the size of the liver tumor mass was approximately >14 mm in the control group. In comparison to the control group, the tumor mass grew slowly with Chitosan treatment, especially at the low-molecular weight treatment group. Chitosan slowed-down the rate of tumor growth but did not inhibit tumor formation. Data presented herein demonstrate that Chitosan has anticancer effects and thus further study of the substance is warranted to examine for mechanisms of action and optimal dosage. PMID:24222124

Yeh, Ming-Yang; Wu, Ming-Fang; Shang, Hung-Sheng; Chang, Jin-Biou; Shih, Yung-Luen; Chen, Yung-Liang; Hung, Hsiao-Fang; Lu, Hsu-Feng; Yeh, Chun; Wood, W Gibson; Hung, Fang-Ming; Chung, Jing-Gung

2013-11-01

225

Zebrafish orthologs of human muscular dystrophy genes  

PubMed Central

Background Human muscular dystrophies are a heterogeneous group of genetic disorders which cause decreased muscle strength and often result in premature death. There is no known cure for muscular dystrophy, nor have all causative genes been identified. Recent work in the small vertebrate zebrafish Danio rerio suggests that mutation or misregulation of zebrafish dystrophy orthologs can also cause muscular degeneration phenotypes in fish. To aid in the identification of new causative genes, this study identifies and maps zebrafish orthologs for all known human muscular dystrophy genes. Results Zebrafish sequence databases were queried for transcripts orthologous to human dystrophy-causing genes, identifying transcripts for 28 out of 29 genes of interest. In addition, the genomic locations of all 29 genes have been found, allowing rapid candidate gene discovery during genetic mapping of zebrafish dystrophy mutants. 19 genes show conservation of syntenic relationships with humans and at least two genes appear to be duplicated in zebrafish. Significant sequence coverage on one or more BAC clone(s) was also identified for 24 of the genes to provide better local sequence information and easy updating of genomic locations as the zebrafish genome assembly continues to evolve. Conclusion This resource supports zebrafish as a dystrophy model, suggesting maintenance of all known dystrophy-associated genes in the zebrafish genome. Coupled with the ability to conduct genetic screens and small molecule screens, zebrafish are thus an attractive model organism for isolating new dystrophy-causing genes/pathways and for use in high-throughput therapeutic discovery. PMID:17374169

Steffen, Leta S; Guyon, Jeffrey R; Vogel, Emily D; Beltre, Rosanna; Pusack, Timothy J; Zhou, Yi; Zon, Leonard I; Kunkel, Louis M

2007-01-01

226

Evaluation of a CXCR4 antagonist in a xenograft mouse model of inflammatory breast cancer  

Microsoft Academic Search

CXCL12\\/CXCR4 signaling, being important in the homing of cancer cells to lungs, bone and other organs, is a promising therapeutic\\u000a target. Our purpose was to determine whether a peptide-based antagonist of CXCR4 would reduce primary tumor growth and\\/or\\u000a metastasis in a preclinical mouse model of inflammatory breast cancer. We improved an existing model of inflammatory breast\\u000a cancer for this study

Balraj Singh; Kendra R. Cook; Cecilia Martin; Eugene H. Huang; Kailash Mosalpuria; Savitri Krishnamurthy; Massimo Cristofanilli; Anthony Lucci

2010-01-01

227

Laser-targeted ablation of the zebrafish embryonic ventricle: A novel model of cardiac injury and repair????  

PubMed Central

Background While the adult zebrafish (Danio rerio) heart demonstrates a remarkable capacity for self-renewal following apical resection little is known about the response to injury in the embryonic heart. Methods Injury to the beating zebrafish embryo heart was induced by laser using a transgenic zebrafish expressing cardiomyocyte specific green fluorescent protein. Changes in ejection fraction (EF), heart rate (HR), and caudal vein blood flow (CVBF) assessed by video capture techniques were assessed at 2, 24 and 48 h post-laser. Change in total and mitotic ventricular cardiomyocyte number following laser injury was also assessed by counting respectively DAPI (VCt) and Phospho-histone H3 (VCm) positive nuclei in isolated hearts using confocal microscopy. Results Laser injury to the ventricle resulted in bradycardia and mild bleeding into the pericardium. At 2 h post-laser injury, there was a significant reduction in cardiac performance in lasered-hearts compared with controls (HR 117 ± 11 vs 167 ± 9 bpm, p ? 0.001; EF 14.1 ± 1.8 vs 20.1 ± 1.3%, p ? 0.001; CVBF 103 ± 15 vs 316 ± 13?ms? 1, p ? 0.001, respectively). Isolated hearts showed a significant reduction in VCt at 2 h post-laser compared to controls (195 ± 15 vs 238 ± 15, p ? 0.05). Histology showed necrosis and apoptosis (TUNEL assay) at the site of laser injury. At 24 h post-laser cardiac performance and VCt had recovered fully to control levels. Pretreatment with the cell-cycle inhibitor, aphidicolin, significantly inhibited functional recovery of the ventricle accompanied by a significant inhibition of cardiomyocyte proliferation. Conclusions Laser-targeted injury of the zebrafish embryonic heart is a novel and reproducible model of cardiac injury and repair suitable for pharmacological and molecular studies. PMID:23871347

Matrone, Gianfranco; Taylor, Jonathan M.; Wilson, Kathryn S.; Baily, James; Love, Gordon D.; Girkin, John M.; Mullins, John J.; Tucker, Carl S.; Denvir, Martin A.

2013-01-01

228

Metformin displays anti-myeloma activity and synergistic effect with dexamethasone in in vitro and in vivo xenograft models.  

PubMed

Epidemiologic studies and meta-analyses have suggested that patients with type 2 diabetes mellitus (T2DM) have a higher incidence of malignancies, including myeloma. Metformin is a widely prescribed antidiabetic drug. Recently, researchers have shown that metformin has direct anticancer activity against many tumor cell lines, mainly through activating AMP-activated protein kinase (AMPK) or reducing the blood insulin level. In the present study, we investigated whether metformin exerts an anti-myeloma effect in in vitro and in vivo xenograft models and explored the underlying mechanism. We found that metformin can inhibit proliferation of MM cells by inducing apoptosis and cell cycle arrest in the G0/G1 phase. Western blot showed that metformin activated caspase 3, caspase 9, PARP-1, Bak, and p21 and inactivated Mcl-1, HIAP-1, cyclin D1, CDK4, and CDK6. Metformin inhibited the expression of insulin growth factor-I receptor (IGF-IR), and phosphatidyl inositol 3-kinase (PI3K), protein kinase B (PKB/AKT) and the downstream mammalian target of rapamycin (mTOR). IGF-I blocked metformin-induced MM cell apoptosis and reactivation of the PI3K/AKT/mTOR signaling pathway. Metformin also demonstrated synergistic activity with dexamethasone but not bortezomib to eradicate MM cells in vitro and in vivo, especially in MM.1S cells. We conclude that metformin inhibits MM cell proliferation through the IGF-1R/PI3K/AKT/mTOR signaling pathway. Metformin and dexamethasone combination therapy may be an option for MM treatment. PMID:25305450

Zi, Fu-Ming; He, Jing-Song; Li, Yi; Wu, Cai; Yang, Li; Yang, Yang; Wang, Li-Juan; He, Dong-Hua; Zhao, Yi; Wu, Wen-Jun; Zheng, Gao-Feng; Han, Xiao-Yan; Huang, He; Yi, Qing; Cai, Zhen

2015-01-28

229

5-fluorouracil enhances the antitumor effect of sorafenib and sunitinib in a xenograft model of human renal cell carcinoma  

PubMed Central

Sorafenib and sunitinib are multi-kinase inhibitors with antitumor activity in patients with advanced renal cell carcinoma (RCC). Several studies have evaluated the effect of sorafenib/sunitinib in combination with chemotherapeutic agents in different types of tumor. However, few studies have addressed the activity of fluorinated pyrimidine in combination with sorafenib/sunitinib. In this study, we examined the potential of combination therapy with 5FU and sorafenib/sunitinib in human RCC cell lines. Three human RCC cell lines, ACHN, Caki-1 and Caki-2, were used to assess sensitivity to 5-fluorouracil (5FU), sorafenib and sunitinib alone or in combination using an in vitro cell survival assay. Caki-2 cells demonstrated significantly higher resistance to 5FU and sorafenib as compared to ACHN and Caki-1. Additive antitumor effects of the combination therapy were observed in the in vitro study. There was a tendency for a positive correlation between the sensitivity to sunitinib and platelet-derived growth factor ? (PDGFR-?) expression levels, which were examined by reverse transcription polymerase chain reaction. Caki-1 xenograft models were prepared by inoculating cells subcutaneously into nude mice, which were divided randomly into six groups: control, 5FU (8 mg/kg/day, intraperitoneally), sorafenib (15 mg/kg/day, orally), sunitinib (20 mg/kg/day, orally), and 5FU with sorafenib or sunitinib. The treatments were administered on 5 days each week, and tumor growth was monitored for 42 days following inoculation of cells. Synergistic antitumor effects of the combination therapy were observed in an in vivo study. The resected tumors were evaluated using the Ki-67 labeling index and microvessel density. Both the Ki-67 labeling index and microvessel density were decreased in tumors treated with the combination therapy compared to those treated with sorafenib/sunitinib alone. These findings suggest that the combination therapy of 5FU with sorafenib/sunitinib may be an effective therapeutic modality for advanced RCC patients. PMID:22783417

MIYAKE, MAKITO; ANAI, SATOSHI; FUJIMOTO, KIYOHIDE; OHNISHI, SAYURI; KUWADA, MASAOMI; NAKAI, YASUSHI; INOUE, TAKESHI; TOMIOKA, ATSUSHI; TANAKA, NOBUMICHI; HIRAO, YOSHIHIKO

2012-01-01

230

Naltrindole Inhibits Human Multiple Myeloma Cell Proliferation In Vitro and in a Murine Xenograft Model In Vivo  

PubMed Central

It has been demonstrated previously that immune cell activation and proliferation were sensitive to the effects of naltrindole, a nonpeptidic ?-opioid receptor-selective antagonist; therefore, we hypothesized that human multiple myeloma (MM) would be a valuable model for studying potential antineoplastic properties of naltrindole. [3H]naltrindole exhibited saturable, low-affinity binding to intact human MM cells; however, the pharmacological profile of the binding site differed considerably from the properties of ?-, ?-, and ?-opioid receptors, and opioid receptor mRNA was not detected in MM cells by reverse transcriptase-polymerase chain reaction. Naltrindole inhibited the proliferation of cultured human U266 MM cells in a time- and dose-dependent manner with an EC50 of 16 ?M. The naltrindole-induced inhibition of U266 cell proliferation was not blocked by a 10-fold molar excess of naltrexone, a nonselective opioid antagonist. Additive inhibition of MM cell proliferation was observed when using a combination of naltrindole with the histone deacetylase inhibitor sodium valproate, the proteasome inhibitor bortezomib, the glucocorticoid receptor agonist dexamethasone, and the HMG CoA reductase inhibitor simvastatin. Treatment of U266 cells with naltrindole significantly decreased the level of the active, phosphorylated form of the kinases, extracellular signal-regulated kinase and Akt, which may be related to its antiproliferative activity. The antiproliferative activity of naltrindole toward MM cells was maintained in cocultures of MM and bone marrow-derived stromal cells, mimicking the bone marrow microenvironment. In vivo, naltrindole significantly decreased tumor cell volumes in human MM cell xenografts in severe combined immunodeficient mice. We hypothesize that naltrindole inhibits the proliferation of MM cells through a nonopioid receptor-dependent mechanism. PMID:22537770

Mundra, Jyoti Joshi; Terskiy, Alexandra

2012-01-01

231

Glial Cell Development and Function in Zebrafish.  

PubMed

The zebrafish is a premier vertebrate model system that offers many experimental advantages for in vivo imaging and genetic studies. This review provides an overview of glial cell types in the central and peripheral nervous system of zebrafish. We highlight some recent work that exploited the strengths of the zebrafish system to increase the understanding of the role of Gpr126 in Schwann cell myelination and illuminate the mechanisms controlling oligodendrocyte development and myelination. We also summarize similarities and differences between zebrafish radial glia and mammalian astrocytes and consider the possibility that their distinct characteristics may represent extremes in a continuum of cell identity. Finally, we focus on the emergence of zebrafish as a model for elucidating the development and function of microglia. These recent studies have highlighted the power of the zebrafish system for analyzing important aspects of glial development and function. PMID:25395296

Lyons, David A; Talbot, William S

2014-11-13

232

snow white, a Zebrafish Model of Hermansky-Pudlak Syndrome Type 5  

PubMed Central

Hermansky-Pudlak Syndrome (HPS) is a set of genetically heterogeneous diseases caused by mutations in one of nine known HPS genes. HPS patients display oculocutaneous hypopigmentation and bleeding diathesis and, depending on the disease subtype, pulmonary fibrosis, congenital nystagmus, reduced visual acuity, and platelet aggregation deficiency. Mouse models for all known HPS subtypes have contributed greatly to our understanding of the disease, but many of the molecular and cellular mechanisms underlying HPS remain unknown. Here, we characterize ocular defects in the zebrafish (Danio rerio) mutant snow white (snw), which possesses a recessive, missense mutation in hps5 (hps5I76N). Melanosome biogenesis is disrupted in snw/hps5 mutants, resulting in hypopigmentation, a significant decrease in the number, size, and maturity of melanosomes, and the presence of ectopic multi-melanosome clusters throughout the mutant retina and choroid. snw/hps5I76N is the first Hps5 mutation identified within the N-terminal WD40 repeat protein–protein binding domain. Through in vitro coexpression assays, we demonstrate that Hps5I76N retains the ability to bind its protein complex partners, Hps3 and Hps6. Furthermore, while Hps5 and Hps6 stabilize each other’s expression, this stabilization is disrupted by Hps5I76N. The snw/hps5I76N mutant provides a valuable resource for structure–function analyses of Hps5 and enables further elucidation of the molecular and cellular mechanisms underlying HPS. PMID:23893484

Daly, Christina M. S.; Willer, Jason; Gregg, Ronald; Gross, Jeffrey M.

2013-01-01

233

snow white, a zebrafish model of Hermansky-Pudlak Syndrome type 5.  

PubMed

Hermansky-Pudlak Syndrome (HPS) is a set of genetically heterogeneous diseases caused by mutations in one of nine known HPS genes. HPS patients display oculocutaneous hypopigmentation and bleeding diathesis and, depending on the disease subtype, pulmonary fibrosis, congenital nystagmus, reduced visual acuity, and platelet aggregation deficiency. Mouse models for all known HPS subtypes have contributed greatly to our understanding of the disease, but many of the molecular and cellular mechanisms underlying HPS remain unknown. Here, we characterize ocular defects in the zebrafish (Danio rerio) mutant snow white (snw), which possesses a recessive, missense mutation in hps5 (hps5I76N). Melanosome biogenesis is disrupted in snw/hps5 mutants, resulting in hypopigmentation, a significant decrease in the number, size, and maturity of melanosomes, and the presence of ectopic multi-melanosome clusters throughout the mutant retina and choroid. snw/hps5I76N is the first Hps5 mutation identified within the N-terminal WD40 repeat protein-protein binding domain. Through in vitro coexpression assays, we demonstrate that Hps5I76N retains the ability to bind its protein complex partners, Hps3 and Hps6. Furthermore, while Hps5 and Hps6 stabilize each other's expression, this stabilization is disrupted by Hps5I76N. The snw/hps5I76N mutant provides a valuable resource for structure-function analyses of Hps5 and enables further elucidation of the molecular and cellular mechanisms underlying HPS. PMID:23893484

Daly, Christina M S; Willer, Jason; Gregg, Ronald; Gross, Jeffrey M

2013-10-01

234

Microcephaly models in the developing zebrafish retinal neuroepithelium point to an underlying defect in metaphase progression  

PubMed Central

Autosomal recessive primary microcephaly (MCPH) is a congenital disorder characterized by significantly reduced brain size and mental retardation. Nine genes are currently known to be associated with the condition, all of which encode centrosomal or spindle pole proteins. MCPH is associated with a reduction in proliferation of neural progenitors during fetal development. The cellular mechanisms underlying the proliferation defect, however, are not fully understood. The zebrafish retinal neuroepithelium provides an ideal system to investigate this question. Mutant or morpholino-mediated knockdown of three known MCPH genes (stil, aspm and wdr62) and a fourth centrosomal gene, odf2, which is linked to several MCPH proteins, results in a marked reduction in head and eye size. Imaging studies reveal a dramatic rise in the fraction of proliferating cells in mitosis in all cases, and time-lapse microscopy points to a failure of progression through prometaphase. There was also increased apoptosis in all the MCPH models but this appears to be secondary to the mitotic defect as we frequently saw mitotically arrested cells disappear, and knocking down p53 apoptosis did not rescue the mitotic phenotype, either in whole retinas or clones. PMID:24153002

Novorol, Claire; Burkhardt, Janina; Wood, Kirstin J.; Iqbal, Anila; Roque, Claudio; Coutts, Nicola; Almeida, Alexandra D.; He, Jie; Wilkinson, Christopher J.; Harris, William A.

2013-01-01

235

Neural and Synaptic Defects in slytherin a Zebrafish Model for Human Congenital Disorders of Glycosylation  

SciTech Connect

Congenital disorder of glycosylation type IIc (CDG IIc) is characterized by mental retardation, slowed growth and severe immunodeficiency, attributed to the lack of fucosylated glycoproteins. While impaired Notch signaling has been implicated in some aspects of CDG IIc pathogenesis, the molecular and cellular mechanisms remain poorly understood. We have identified a zebrafish mutant slytherin (srn), which harbors a missense point mutation in GDP-mannose 4,6 dehydratase (GMDS), the rate-limiting enzyme in protein fucosylation, including that of Notch. Here we report that some of the mechanisms underlying the neural phenotypes in srn and in CGD IIc are Notch-dependent, while others are Notch-independent. We show, for the first time in a vertebrate in vivo, that defects in protein fucosylation leads to defects in neuronal differentiation, maintenance, axon branching, and synapse formation. Srn is thus a useful and important vertebrate model for human CDG IIc that has provided new insights into the neural phenotypes that are hallmarks of the human disorder and has also highlighted the role of protein fucosylation in neural development.

Y Song; J Willer; P Scherer; J Panzer; A Kugath; E Skordalakes; R Gregg; G Willer; R Balice-Gordon

2011-12-31

236

Analysis of Movement Behavior of Zebrafish (DANIO RERIO) Under Chemical Stress Using Hidden Markov Model  

NASA Astrophysics Data System (ADS)

Based on computer vision techniques, the movement tracks of an indicator species (zebrafish) were continuously observed in two dimensions before and after the treatments with a toxic chemical (formaldehyde, 2.5 ppm). Behavioral patterns based on the shape of movement segments were regarded as states, while linear and angular speeds measured from the movement segments were used as observed events for training with a hidden Markov model (HMM). The state sequences were estimated by HMM based on transition and emission probability matrices, and observed events. The movement tracks were further reconstructed based on behavior state sequences generated by HMM. Subsequently, permutation entropy and fractal dimension were calculated to monitor behavioral changes before and after the treatments. Both parameters based on the real and reconstructed data significantly decreased after the treatments, and individual variability was minimized with the parameters obtained from the reconstructed tracks. The parameter extraction based on optimal state sequence by HMM was suitable for resolving the problem of variability in behavioral data, and would be an effective means of monitoring chemical stress in the environment.

Li, Yan; Lee, Jang-Myung; Chon, Tae-Soo; Liu, Yuedan; Kim, Hungsoo; Bae, Mi-Jung; Park, Young-Seuk

2013-01-01

237

Indole Alkaloids from Fischerella Inhibit Vertebrate Development in the Zebrafish (Danio rerio) Embryo Model  

PubMed Central

Cyanobacteria are recognized producers of toxic or otherwise bioactive metabolite associated, in particular, with so-called “harmful algal blooms” (HABs) and eutrophication of freshwater systems. In the present study, two apparently teratogenic indole alkaloids from a freshwater strain of the widespread cyanobacterial genus, Fischerella (Stigonemataceae), were isolated by bioassay-guided fractionation, specifically using the zebrafish (Danio rerio) embryo, as a model of vertebrate development. The two alkaloids include the previously known 12-epi-hapalindole H isonitrile (1), and a new nitrile-containing variant, 12-epi-ambiguine B nitrile (2). Although both compounds were toxic to developing embryos, the former compound was shown to be relatively more potent, and to correlate best with the observed embryo toxicity. Related indole alkaloids from Fischerella, and other genera in the Stigonemataceae, have been widely reported as antimicrobial compounds, specifically in association with apparent allelopathy. However, this is the first report of their vertebrate toxicity, and the observed teratogenicity of these alkaloids supports a possible contribution to the toxicity of this widespread cyanobacterial family, particularly in relation to freshwater HABs and eutrophication. PMID:25533520

Walton, Katherine; Gantar, Miroslav; Gibbs, Patrick D. L.; Schmale, Michael C.; Berry, John P.

2014-01-01

238

Rargb regulates organ laterality in a zebrafish model of right atrial isomerism  

PubMed Central

Developmental signals determine organ morphology and position during embryogenesis. To discover novel modifiers of liver development, we performed a chemical genetic screen in zebrafish and identified retinoic acid as a positive regulator of hepatogenesis. Knockdown of the four RA receptors revealed that all receptors affect liver formation, however specific receptors exert differential effects. Rargb knockdown results in bilateral livers but does not impact organ size, revealing a unique role for Rargb in conferring left–right positional information. Bilateral populations of hepatoblasts are detectable in rargb morphants, indicating Rargb acts during hepatic specification to position the liver, and primitive endoderm is competent to form liver on both sides. Hearts remain at the midline and gut looping is perturbed in rargb morphants, suggesting Rargb affects lateral plate mesoderm migration. Overexpression of Bmp during somitogenesis similarly results in bilateral livers and midline hearts, and inhibition of Bmp signaling rescues the rargb morphant phenotype, indicating Rargb functions upstream of Bmp to regulate organ sidedness. Loss of rargb causes biliary and organ laterality defects as well as asplenia, paralleling symptoms of the human condition right atrial isomerism. Our findings uncover a novel role for RA in regulating organ laterality and provide an animal model of one form of human heterotaxia. PMID:22982668

Garnaas, Maija K.; Cutting, Claire C.; Meyers, Alison; Kelsey, Peter B.; Harris, James M.; North, Trista E.; Goessling, Wolfram

2013-01-01

239

Indole Alkaloids from Fischerella Inhibit Vertebrate Development in the Zebrafish (Danio rerio) Embryo Model.  

PubMed

Cyanobacteria are recognized producers of toxic or otherwise bioactive metabolite associated, in particular, with so-called "harmful algal blooms" (HABs) and eutrophication of freshwater systems. In the present study, two apparently teratogenic indole alkaloids from a freshwater strain of the widespread cyanobacterial genus, Fischerella (Stigonemataceae), were isolated by bioassay-guided fractionation, specifically using the zebrafish (Danio rerio) embryo, as a model of vertebrate development. The two alkaloids include the previously known 12-epi-hapalindole H isonitrile (1), and a new nitrile-containing variant, 12-epi-ambiguine B nitrile (2). Although both compounds were toxic to developing embryos, the former compound was shown to be relatively more potent, and to correlate best with the observed embryo toxicity. Related indole alkaloids from Fischerella, and other genera in the Stigonemataceae, have been widely reported as antimicrobial compounds, specifically in association with apparent allelopathy. However, this is the first report of their vertebrate toxicity, and the observed teratogenicity of these alkaloids supports a possible contribution to the toxicity of this widespread cyanobacterial family, particularly in relation to freshwater HABs and eutrophication. PMID:25533520

Walton, Katherine; Gantar, Miroslav; Gibbs, Patrick D L; Schmale, Michael C; Berry, John P

2014-01-01

240

Zyflamend, a combination of herbal extracts, attenuates tumor growth in murine xenograft models of prostate cancer.  

PubMed

Prostate cancer (PrC) is the second deadliest cancer of males in the United States Hormone deprivation therapy (HDT), a common therapy for advanced forms of the disease, results in tumor regression; unfortunately, tumors inevitably become castrate-resistant. Diet is not an appropriate primary therapy for refractory forms of the disease; however, diet may be effective as an adjuvant to HDT, potentially extending the latency period and delaying relapse and/or inhibiting refractory growth. Zyflamend® is a combination of extracts from multiple herbs, each with reported anticancer properties. Zyflamend can inhibit growth of various PrC cell lines, but no studies have investigated its potential use in vivo using a model of castrate-resistant PrC. In this study, oral doses of Zyflamend at human equivalent doses inhibited androgen-dependent and castrate-resistant tumor growth in a mouse model that mimics advanced stages of the disease, and reduced the expression of a number of biomarkers linked to PrC progression including pAKT, prostate specific antigen, histone deacetylases, and androgen receptor. In summary, this is the first article to report that Zyflamend, when provided at human equivalent doses, can potentiate the effects of hormone deprivation on tumor regression and growth inhibition of androgen-dependent and castrate-resistant PrC tumors in vivo. PMID:22663543

Huang, E-Chu; McEntee, Michael F; Whelan, Jay

2012-01-01

241

Multicolor Fluorescent Intravital Live Microscopy (FILM) for Surgical Tumor Resection in a Mouse Xenograft Model  

PubMed Central

Background Complete surgical resection of neoplasia remains one of the most efficient tumor therapies. However, malignant cell clusters are often left behind during surgery due to the inability to visualize and differentiate them against host tissue. Here we establish the feasibility of multicolor fluorescent intravital live microscopy (FILM) where multiple cellular and/or unique tissue compartments are stained simultaneously and imaged in real time. Methodology/Principal Findings Theoretical simulations of imaging probe localization were carried out for three agents with specificity for cancer cells, stromal host response, or vascular perfusion. This transport analysis gave insight into the probe pharmacokinetics and tissue distribution, facilitating the experimental design and allowing predictions to be made about the localization of the probes in other animal models and in the clinic. The imaging probes were administered systemically at optimal time points based on the simulations, and the multicolor FILM images obtained in vivo were then compared to conventional pathological sections. Our data show the feasibility of real time in vivo pathology at cellular resolution and molecular specificity with excellent agreement between intravital and traditional in vitro immunohistochemistry. Conclusions/Significance Multicolor FILM is an accurate method for identifying malignant tissue and cells in vivo. The imaging probes distributed in a manner similar to predictions based on transport principles, and these models can be used to design future probes and experiments. FILM can provide critical real time feedback and should be a useful tool for more effective and complete cancer resection. PMID:19956597

Thurber, Greg M.; Figueiredo, Jose L.; Weissleder, Ralph

2009-01-01

242

Hypoxically preconditioned human peripheral blood mononuclear cells improve blood flow in hindlimb ischemia xenograft model.  

PubMed

Transplantation of peripheral blood mononuclear cells (PBMNCs) is a promising therapeutic approach for the treatment of hindlimb ischemia. However, insufficient angiogenesis in ischemic hindlimb after cell transplantation reduces the importance and practicality of this approach. Previously, we demonstrated using mouse models that hypoxic preconditioning augmented the cellular functions of rodent PBMNCs, such as increased cell adhesion capacity and accelerated neovascularization in ischemic hindlimb. To test the clinical application of this therapeutic strategy in this study, we investigated whether the protocol of hypoxic preconditioning, which was established in a condition of 2% O2 for 24 h, can be made available for human PBMNCs (hPBMNCs). In addition, we grafted preconditioned hPBMNCs in a hindlimb ischemia mouse model. Hypoxic preconditioning enhanced cell adhesion capacity and oxidative stress resistance in hPBMNCs. We also observed an up-regulation of platelet endothelial cell adhesion molecule-1 (PECAM-1) in hPBMNCs by hypoxic preconditioning. Furthermore, preconditioned hPBMNCs significantly recovered limb blood flow in ischemic mice after transplantation. These results indicate that our established preconditioning protocol is available for hPBMNCs to effectively reinforce multiple cellular functions. Taken together with our series of study, we believe that this simple but powerful therapeutic strategy will be helpful in curing patients with severe hindlimb ischemia. PMID:25360221

Kudo, Tomoaki; Kubo, Masayuki; Katsura, Shunsaku; Nishimoto, Arata; Ueno, Koji; Samura, Makoto; Fujii, Yasuhiko; Hosoyama, Tohru; Hamano, Kimikazu

2014-01-01

243

The in ovo CAM-assay as a xenograft model for sarcoma.  

PubMed

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions. This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products. PMID:23892612

Sys, Gwen M L; Lapeire, Lore; Stevens, Nikita; Favoreel, Herman; Forsyth, Ramses; Bracke, Marc; De Wever, Olivier

2013-01-01

244

Zebrafish: a model animal for analyzing the impact of environmental pollutants on muscle and brain mitochondrial bioenergetics.  

PubMed

Mercury, anthropogenic release of uranium (U), and nanoparticles constitute hazardous environmental pollutants able to accumulate along the aquatic food chain with severe risk for animal and human health. The impact of such pollutants on living organisms has been up to now approached by classical toxicology in which huge doses of toxic compounds, environmentally irrelevant, are displayed through routes that never occur in the lifespan of organisms (for instance injecting a bolus of mercury to an animal although the main route is through prey and fish eating). We wanted to address the effect of such pollutants on the muscle and brain mitochondrial bioenergetics under realistic conditions, at unprecedented low doses, using an aquatic model animal, the zebrafish Danio rerio. We developed an original method to measure brain mitochondrial respiration: a single brain was put in 1.5 mL conical tube containing a respiratory buffer. Brains were gently homogenized by 13 strokes with a conical plastic pestle, and the homogenates were immediately used for respiration measurements. Skinned muscle fibers were prepared by saponin permeabilization. Zebrafish were contaminated with food containing 13 ?g of methylmercury (MeHg)/g, an environmentally relevant dose. In permeabilized muscle fibers, we observed a strong inhibition of both state 3 mitochondrial respiration and cytochrome c oxidase activity after 49 days of MeHg exposure. We measured a dramatic decrease in the rate of ATP release by skinned muscle fibers. Contrarily to muscles, brain mitochondrial respiration was not modified by MeHg exposure although brain accumulated twice as much MeHg than muscles. When zebrafish were exposed to 30 ?g/L of waterborne U, the basal mitochondrial respiratory control ratio was decreased in muscles after 28 days of exposure. This was due to an increase of the inner mitochondrial membrane permeability. The impact of a daily ration of food containing gold nanoparticles of two sizes (12 and 50 nm) was investigated at a very low dose for 60 days (40 ng gold/fish/day). Mitochondrial dysfunctions appeared in brain and muscle for both tested sizes. In conclusion, at low environmental doses, dietary or waterborne heavy metals impinged on zebrafish tissue mitochondrial respiration. Due to its incredible simplicity avoiding tedious and time-consuming mitochondria isolation, our one-pot method allowing brain respiratory analysis should give colleagues the incentive to use zebrafish brain as a model in bioenergetics. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy. PMID:22842533

Bourdineaud, Jean-Paul; Rossignol, R; Brèthes, D

2013-01-01

245

213 dioxin significantly enhanced the tumor mass formation in a xenografted mouse model transplanted with bg-1 ovarian cancer cells.  

PubMed

Previous studies suggest that environmental factors, such as high levels of meat consumption, caffeine, cigarette smoking, and endocrine disrupting chemicals (EDC), may enhance a high risk of ovarian cancer. Cytochrome P450 (CYP) 1A1 may play a major role in metabolic activation of procarcinogens to carcinogens. For example, polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (HAHs) occur by pyrolysis of fossil fuels and flow into the body by organic matter, such as tobacco leaves and contaminated water by pesticides. 2,3,7,8-tetrachlorodibenzo-?-dioxin (TCDD) is a commonplace pollutant and promoter of carcinogenesis as the most potent substance. In this study, we examined the effects of 17?-oestradiol (E2), TCDD, and TCDD in the presence of E2 on the expressions of CYP1A1, CYP1B1, and aryl hydrocarbon receptor (AhR) by RT-PCR analysis and Western blot analysis. In addition, the cell viability by TCDD and E2 were examined in BG-1 human ovarian cancer cells by MTT assay. To evaluate the cell viability, BG-1 cells were cultured with a negative control (0.1% DMSO), E2 (1×10(-9)M) or TCDD (10(-6)-10(-8)M). E2 markedly increased BG-1 cell viability ~5 times, and TCDD also induced BG-1 cell viability highest at the concentration of 1×10(-8)M compared to a control (0.1% DMSO) (P<0.05). When respective treatment was co-treated with ICI 182780, an ER antagonist, BG-1 cell viability was reversed to the level of a negative control. Although the mRNA expression of CYP1B1 was not altered by E2, TCDD, or TCDD plus E2, the transcriptional level of CYP1A1 appeared to be increased by E2 and TCDD in a time-dependent manner. When the cells were treated with TCDD plus E2, the mRNA level of CYP1A1 was more greatly increased than by only TCDD or E2 treatment. In xenograft mouse models transplanted with BG-1 cells, E2 treatment significantly increased the tumour mass formation ~6 times, and TCDD also induced tumour formation ~4 times compared to a vehicle (0.1% DMSO in PBS) during 8 weeks in this xenograted mouse model. In addition, TCDD plus E2 treatment also greatly induced ovarian tumour formation compared to only E2 treatment in this mouse model. Taken together, these results indicate that TCDD may induce ovarian cancer cell growth via CYP1A1 gene expression and have disruptive effect in oestrogen receptor (ER) expressing cells or tissues. PMID:25472261

Go, R-E; Choi, K-C

2014-12-01

246

Combined magnetic resonance, fluorescence, and histology imaging strategy in a human breast tumor xenograft model  

PubMed Central

Applications of molecular imaging in cancer and other diseases frequently require combining in vivo imaging modalities, such as magnetic resonance and optical imaging, with ex vivo optical, fluorescence, histology, and immunohistochemical (IHC) imaging, to investigate and relate molecular and biological processes to imaging parameters within the same region of interest. We have developed a multimodal image reconstruction and fusion framework that accurately combines in vivo magnetic resonance imaging (MRI) and magnetic resonance spectroscopic imaging (MRSI), ex vivo brightfield and fluorescence microscopic imaging, and ex vivo histology imaging. Ex vivo brightfield microscopic imaging was used as an intermediate modality to facilitate the ultimate link between ex vivo histology and in vivo MRI/MRSI. Tissue sectioning necessary for optical and histology imaging required generation of a three-dimensional (3D) reconstruction module for 2D ex vivo optical and histology imaging data. We developed an external fiducial marker based 3D reconstruction method, which was able to fuse optical brightfield and fluorescence with histology imaging data. Registration of 3D tumor shape was pursued to combine in vivo MRI/MRSI and ex vivo optical brightfield and fluorescence imaging data. This registration strategy was applied to in vivo MRI/MRSI, ex vivo optical brightfield/fluorescence, as well as histology imaging data sets obtained from human breast tumor models. 3D human breast tumor data sets were successfully reconstructed and fused with this platform. PMID:22945331

Jiang, Lu; Greenwood, Tiffany R.; Amstalden van Hove, Erika R.; Chughtai, Kamila; Raman, Venu; Winnard, Paul T.; Heeren, Ron; Artemov, Dmitri; Glunde, Kristine

2014-01-01

247

Tumor endothelin-1 enhances metastatic colonization of the lung in mouse xenograft models of bladder cancer  

PubMed Central

Many patients with advanced bladder cancer develop lethal metastases to the lung. The vasoconstricting protein endothelin-1 (ET-1) has been implicated in this process, although the mechanism(s) by which it promotes metastasis remains unclear. Here, we have evaluated whether tumor ET-1 expression can serve as a biomarker for lung metastasis and whether it is required for metastatic disease. Evaluation of ET-1 mRNA and protein expression in four patient cohorts revealed that levels of ET-1 are higher in patients with muscle-invasive bladder cancers, which are associated with higher incidence of metastasis, and that high ET-1 levels are associated with decreased disease-specific survival. Consistent with its proinflammatory activity, we found that tumor-derived ET-1 acts through endothelin-1 receptor A (ETAR) to enhance migration and invasion of both tumor cells and macrophages and induces expression of inflammatory cytokines and proteases. Using human and mouse cancer cells depleted of ET-1 and pharmacologic blockade of ET receptors in lung metastasis models, we found that tumor ET-1 expression and ETAR activity are necessary for metastatic lung colonization and that this process is preceded by and dependent on macrophage infiltration of the lung. In contrast, tumor ET-1 expression and ETAR activity appeared less important in established primary or metastatic tumor growth. These findings strongly suggest that ETAR inhibitors might be more effective as adjuvant therapeutic agents than as initial treatment for advanced primary or metastatic disease. PMID:21183790

Said, Neveen; Smith, Steven; Sanchez-Carbayo, Marta; Theodorescu, Dan

2010-01-01

248

Uptake of verteporfin by orthotopic xenograft pancreas models with different levels of aggression  

NASA Astrophysics Data System (ADS)

Pancreatic cancer is an aggressive disease with a poor prognosis, usually treated with chemoradiation therapy. Interstitial photodynamic therapy is a potentially effective adjuvant treatment that is under development. In the current study, two orthotopic pancreatic cancer models (AsPC-1 and Panc-1), have been characterized with respect to growth rates, morphology and liposomal drug (Verteporfin) uptake and distribution in SCID mice. Fluorescence of Verteporfin was measured in liver and tumor in vivo using a PDT fluorescence dosimeter with measurements taken before and up to one hour after tail vein injection. Fluorescence reached a plateau by about 15 minutes and did not decrease over the first hour. At time points from 15 minutes to 24 hrs, the internal organs (kidney, spleen, pancreas, tumor, muscle, lung, liver, and skin were excised and scanned on a Typhoon imager. The ratio of fluorescence in tumor versus normal tissues was analyzed with image processing, calculated at each time point and compared to in vivo results. Tissue distribution of Verteporfin in relation to functional vasculature marked by DiOc7 was carried out on frozen sections. Final analysis will result in determination of the ideal time point to administer light to achieve maximum tumor destruction while preserving normal tissue.

O'Hara, Julia; Samkoe, Kimberley S.; Chen, Alina; Hoopes, P. Jack; Rizvi, Imran; Hasan, Tayyaba; Pogue, Brian W.

2009-06-01

249

Novel Vitamin K analogs suppress seizures in zebrafish and mouse models of epilepsy.  

PubMed

Epilepsy is a debilitating disease affecting 1-2% of the world's population. Despite this high prevalence, 30% of patients suffering from epilepsy are not successfully managed by current medication suggesting a critical need for new anti-epileptic drugs (AEDs). In an effort to discover new therapeutics for the management of epilepsy, we began our study by screening drugs that, like some currently used AEDs, inhibit histone deacetylases (HDACs) using a well-established larval zebrafish model. In this model, 7-day post fertilization (dpf) larvae are treated with the widely used seizure-inducing compound pentylenetetrazol (PTZ) which stimulates a rapid increase in swimming behavior previously determined to be a measurable manifestation of seizures. In our first screen, we tested a number of different HDAC inhibitors and found that one, 2-benzamido-1 4-naphthoquinone (NQN1), significantly decreased swim activity to levels equal to that of valproic acid, 2-n-propylpentanoic acid (VPA). We continued to screen structurally related compounds including Vitamin K3 (VK3) and a number of novel Vitamin K (VK) analogs. We found that VK3 was a robust inhibitor of the PTZ-induced swim activity, as were several of our novel compounds. Three of these compounds were subsequently tested on mouse seizure models at the National Institute of Neurological Disorders and Stroke (NINDS) Anticonvulsant Screening Program. Compound 2h reduced seizures particularly well in the minimal clonic seizure (6Hz) and corneal-kindled mouse models of epilepsy, with no observable toxicity. As VK3 affects mitochondrial function, we tested the effects of our compounds on mitochondrial respiration and ATP production in a mouse hippocampal cell line. We demonstrate that these compounds affect ATP metabolism and increase total cellular ATP. Our data indicate the potential utility of these and other VK analogs for the prevention of seizures and suggest the potential mechanism for this protection may lie in the ability of these compounds to affect energy production. PMID:24291671

Rahn, J J; Bestman, J E; Josey, B J; Inks, E S; Stackley, K D; Rogers, C E; Chou, C J; Chan, S S L

2014-02-14

250

Host immune response and acute disease in a zebrafish model of francisella pathogenesis  

USGS Publications Warehouse

Members of the bacterial genus Francisella are highly virulent and infectious pathogens. New models to study Francisella pathogenesis in evolutionarily distinct species are needed to provide comparative insight, as the mechanisms of host resistance and pathogen virulence are not well understood. We took advantage of the recent discovery of a novel species of Francisella to establish a zebrafish/Francisella comparative model of pathogenesis and host immune response. Adult zebraflsh were susceptible to acute Francisella-induced disease and suffered mortality in a dose-dependent manner. Using immunohistochemical analysis, we localized bacterial antigens primarily to lymphoid tissues and livers of zebraflsh following infection by intraperitoneal injection, which corresponded to regions of local cellular necrosis. Francisella sp. bacteria replicated rapidly in these tissues beginning 12 h postinfection, and bacterial titers rose steadily, leveled off, and then decreased by 7 days postinfection. Zebraflsh mounted a significant tissue-specific proinflammatory response to infection as measured by the upregulation of interleukin-l?? (IL-1??), gamma interferon, and tumor necrosis factor alpha mRNA beginning by 6 h postinfection and persisting for up to 7 days postinfection. In addition, exposure of zebraflsh to heat-killed bacteria demonstrated that the significant induction of IL-?? was highly specific to live bacteria. Taken together, the pathology and immune response to acute Francisella infection in zebraflsh share many features with those in mammals, highlighting the usefulness of this new model system for addressing both general and specific questions about Francisella host-pathogen interactions via an evolutionary approach. Copyright ?? 2009, American Society for Microbiology. All Rights Reserved.

Vojtech, L.N.; Sanders, G.E.; Conway, C.; Ostland, V.; Hansen, J.D.

2009-01-01

251

Tumour bed irradiation of human tumour xenografts in a nude rat model using a common X-ray tube  

Microsoft Academic Search

Studies that investigate the radiation of human tumour xenografts require an appropriate radiation source and highly standardized\\u000a conditions during radiation. This work reports on the design of a standardized irradiation device using a commercially available\\u000a X-ray tube with a custom constructed lead collimator with two circular apertures and an animal bed plate, permitting synchronous\\u000a irradiation of two animals. Dosimetry and

S. V. Tokalov; W. Enghardt; N. Abolmaali

2010-01-01

252

Viral Diseases in Zebrafish: What Is Known and Unknown  

PubMed Central

Naturally occurring viral infections have the potential to introduce confounding variability that leads to invalid and misinterpreted data. Whereas the viral diseases of research rodents are well characterized and closely monitored, no naturally occurring viral infections have been characterized for the laboratory zebrafish (Danio rerio), an increasingly important biomedical research model. Despite the ignorance about naturally occurring zebrafish viruses, zebrafish models are rapidly expanding in areas of biomedical research where the confounding effects of unknown infectious agents present a serious concern. In addition, many zebrafish research colonies remain linked to the ornamental (pet) zebrafish trade, which can contribute to the introduction of new pathogens into research colonies, whereas mice used for research are purpose bred, with no introduction of new mice from the pet industry. Identification, characterization, and monitoring of naturally occurring viruses in zebrafish are crucial to the improvement of zebrafish health, the reduction of unwanted variability, and the continued development of the zebrafish as a model organism. This article addresses the importance of identifying and characterizing the viral diseases of zebrafish as the scope of zebrafish models expands into new research areas and also briefly addresses zebrafish susceptibility to experimental viral infection and the utility of the zebrafish as an infection and immunology model. PMID:23382345

Crim, Marcus J.; Riley, Lela K.

2013-01-01

253

Combined Bcl-2/mTOR Inhibition Leads to Enhanced Radiosensitization via Induction of Apoptosis and Autophagy in Non-Small-Cell Lung Tumor Xenograft Model  

PubMed Central

Purpose Radiotherapy has a central role in the treatment of non-small-cell lung cancer. Effectiveness of this modality, however, is often limited as resistance results from defects in cell death. Experimental Design We investigated whether simultaneous upregulation of apoptosis, via Bcl-2 inhibitor ABT-737, and autophagy, via mTOR inhibitor rapamycin, can be used to enhance radiosensitivity of H460 cells in vitro and growth delay in a xenograft model. Results In vitro studies confirmed that ABT-737 and rapamycin induce apoptosis and autophagy, respectively. ABT-737 induced cleaved caspase-3, a marker of apoptosis, and rapamycin correlated with an increase in punctate localization of GFP-LC3, characteristic of autophagy. The combination ABT-737/rapamycin markedly enhanced sensitivity of H460 cells to radiation (DER=2.47, p=0.002) in clonogenic assay. In addition, the combination ABT-737/rapamycin/radiation showed a dramatic tumor growth delay in a mouse xenograft model. In vivo immunohistochemistry staining showed that combination therapy yielded over a 100% increase in caspase-3 activity (apoptosis) and a 6-fold decrease in p62 protein level (indicative of autophagic flux) as compared to radiation alone control group. Moreover, cell proliferation (Ki67 staining) was reduced by 77% (p=0.001) and vascular density (vWF staining) by 67.5% (p=0.09) compared to radiation alone. Additional in vitro studies in human umbilical endothelial cells indicated that combined therapy also significantly decrease tubule formation. Conclusion These results suggest that concurrent induction of apoptosis and autophagy enhances radiation therapy both in vitro and in lung cancer xenograft models. Further investigations are warranted to assess the clinical potential of such strategy in lung cancer patients. PMID:19773376

Kim, Kwang Woon; Moretti, Luigi; Mitchell, Lauren Rhea; Jung, Dae Kwang; Lu, Bo

2009-01-01

254

Anticancer effects of residual powder from Barley-Shochu distillation remnants against the orthotopic xenograft mouse models of hepatocellular carcinoma in vivo.  

PubMed

Barely-Shochu is a traditional Japanese liquor distilled from fermented barley with Saccharomyces cerevisiae. Barely-Shochu distillation remnants (SDR) are by-products in the manufacturing process of barley-Shochu. We have already reported on valuable powder from Shochu distillation remnants (PSDR) including antioxidative compounds such as polyphenols. In this study, we investigated the therapeutic effects of barely-PSDR against orthotopic xenograft mouse models of hepatocellular carcinoma (HCC) in vivo. We constructed a mouse model of HCC by orthotopical inoculation of HepG2 cells into the liver of SCID mice. Barely-PSDR (2250 mg/kg) was orally treated once each day for 21 d after the inoculation of HepG2 cells. The livers were removed from anaesthetized mice after the treatment with barely-PSDR and fixed in formalin. The liver sections were analyzed by hematoxylin and eosin (HE) staining and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) methods. Remarkably high reduction of tumorigenesis was obtained in the mouse models of HCC after the oral administration of barely-PSDR in vivo. Induction of apoptosis in the liver section on the mouse models treated with barely-PSDR was observed. Furthermore, prolonged survival was obtained. Thus, therapeutic effects of barely-PSDR without side effects on the orthotopic xenograft mouse models were revealed for the first time. PMID:22687544

Ohgidani, Masahiro; Ichihara, Hideaki; Goto, Koichi; Matsumoto, Yoko; Ueoka, Ryuichi

2012-01-01

255

Axon degeneration and PGC-1?-mediated protection in a zebrafish model of ?-synuclein toxicity.  

PubMed

?-synuclein (aSyn) expression is implicated in neurodegenerative processes, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In animal models of these diseases, axon pathology often precedes cell death, raising the question of whether aSyn has compartment-specific toxic effects that could require early and/or independent therapeutic intervention. The relevance of axonal pathology to degeneration can only be addressed through longitudinal, in vivo monitoring of different neuronal compartments. With current imaging methods, dopaminergic neurons do not readily lend themselves to such a task in any vertebrate system. We therefore expressed human wild-type aSyn in zebrafish peripheral sensory neurons, which project elaborate superficial axons that can be continuously imaged in vivo. Axonal outgrowth was normal in these neurons but, by 2 days post-fertilization (dpf), many aSyn-expressing axons became dystrophic, with focal varicosities or diffuse beading. Approximately 20% of aSyn-expressing cells died by 3 dpf. Time-lapse imaging revealed that focal axonal swelling, but not overt fragmentation, usually preceded cell death. Co-expressing aSyn with a mitochondrial reporter revealed deficits in mitochondrial transport and morphology even when axons appeared overtly normal. The axon-protective protein Wallerian degeneration slow (WldS) delayed axon degeneration but not cell death caused by aSyn. By contrast, the transcriptional coactivator PGC-1?, which has roles in the regulation of mitochondrial biogenesis and reactive-oxygen-species detoxification, abrogated aSyn toxicity in both the axon and the cell body. The rapid onset of axonal pathology in this system, and the relatively moderate degree of cell death, provide a new model for the study of aSyn toxicity and protection. Moreover, the accessibility of peripheral sensory axons will allow effects of aSyn to be studied in different neuronal compartments and might have utility in screening for novel disease-modifying compounds. PMID:24626988

O'Donnell, Kelley C; Lulla, Aaron; Stahl, Mark C; Wheat, Nickolas D; Bronstein, Jeff M; Sagasti, Alvaro

2014-05-01

256

l-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome.  

PubMed

Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (?-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite ?-KIC can partially rescue development in zebrafish models for CdLS. PMID:25378554

Xu, Baoshan; Sowa, Nenja; Cardenas, Maria E; Gerton, Jennifer L

2014-11-01

257

Unique and conserved aspects of gut development in zebrafish  

Microsoft Academic Search

Although the development of the digestive system of humans and vertebrate model organisms has been well characterized, relatively little is known about how the zebrafish digestive system forms. We define developmental milestones during organogenesis of the zebrafish digestive tract, liver, and pancreas and identify important differences in the way the digestive endoderm of zebrafish and amniotes is organized. Such differences

Kenneth N Wallace; Michael Pack

2003-01-01

258

Castration induces up-regulation of intratumoral androgen biosynthesis and androgen receptor expression in an orthotopic VCaP human prostate cancer xenograft model.  

PubMed

Androgens are key factors involved in the development and progression of prostate cancer (PCa), and PCa growth can be suppressed by androgen deprivation therapy. In a considerable proportion of men receiving androgen deprivation therapy, however, PCa progresses to castration-resistant PCa (CRPC), making the development of efficient therapies challenging. We used an orthotopic VCaP human PCa xenograft model to study cellular and molecular changes in tumors after androgen deprivation therapy (castration). Tumor growth was monitored through weekly serum prostate-specific antigen measurements, and mice with recurrent tumors after castration were randomized to treatment groups. Serum prostate-specific antigen concentrations showed significant correlation with tumor volume. Castration-resistant tumors retained concentrations of intratumoral androgen (androstenedione, testosterone, and 5?-dihydrotestosterone) at levels similar to tumors growing in intact hosts. Accordingly, castration induced up-regulation of enzymes involved in androgen synthesis (CYP17A1, AKR1C3, and HSD17B6), as well as expression of full-length androgen receptor (AR) and AR splice variants (AR-V1 and AR-V7). Furthermore, AR target gene expression was maintained in castration-resistant xenografts. The AR antagonists enzalutamide (MDV3100) and ARN-509 suppressed PSA production of castration-resistant tumors, confirming the androgen dependency of these tumors. Taken together, the findings demonstrate that our VCaP xenograft model exhibits the key characteristics of clinical CRPC and thus provides a valuable tool for identifying druggable targets and for testing therapeutic strategies targeting AR signaling in CRPC. PMID:24949550

Knuuttila, Matias; Yatkin, Emrah; Kallio, Jenny; Savolainen, Saija; Laajala, Teemu D; Aittokallio, Tero; Oksala, Riikka; Häkkinen, Merja; Keski-Rahkonen, Pekka; Auriola, Seppo; Poutanen, Matti; Mäkelä, Sari

2014-08-01

259

Liver immune responses to inflammatory stimuli in a diet-obesity model of zebrafish.  

PubMed

Obesity and metabolic syndrome-related diseases are becoming important medical challenges for the western world. Non-Alcoholic Fatty Liver Disease (NAFLD) is a manifestation of these altered conditions in the liver, and inflammation appears to be a factor that is tightly connected to its evolution. In this study, we used a Diet-Induced Obesity approach in zebrafish (Danio rerio) based on overfeeding to analyze liver transcriptomic modulation in the disease and to determine how obesity affects the immune response against an acute inflammatory stimulus such as lipopolysaccharide (LPS). Overfed zebrafish developed an obese phenotype, showed signs of liver steatosis and its modulation profile resembled that observed in humans, with overexpression of tac4, col4a3, col4a5, lysyl oxidases and genes involved in retinoid metabolism. In response to LPS, healthy fish exhibited a typical host defense reaction comparable to that which occurs in mammals, whereas there was no significant gene modulation when comparing expression in the livers of LPS-stimulated and non-stimulated obese zebrafish at the same statistical level. The stimulation of obese fish represents a double-hit to the already damaged liver and can help understand the evolution of the disease. Finally, a comparison of the differential gene activation between stimulated healthy and obese zebrafish revealed the expected difference in the metabolic state between healthy and diseased livers. The differentially modulated genes are currently being studied as putative new pathological markers in NAFLD-stimulated livers in humans. PMID:25371540

Forn-Cuní, Gabriel; Varela, Mónica; Fernández, Conrado M; Figueras, Antonio; Novoa, Beatriz

2014-11-01

260

Ethanol exposure alters zebrafish development: A novel model of fetal alcohol syndrome  

Microsoft Academic Search

Prenatal exposure to alcohol has been shown to produce the overt physical and behavioral symptoms known as fetal alcohol syndrome (FAS) in humans. Also, it is believed that low concentrations and\\/or short durations of alcohol exposure can produce more subtle effects. The purpose of this study was to investigate the effects of embryonic ethanol exposure on the zebrafish (Danio rerio)

Joseph Bilotta; Jalynn A. Barnett; Laura Hancock; Shannon Saszik

2004-01-01

261

MOLECULAR CLONING, BACULOVIRUS EXPRESSION, AND TISSUE DISTRIBUTION OF THE ZEBRAFISH ALDEHYDE DEHYDROGENASE 2  

E-print Network

MOLECULAR CLONING, BACULOVIRUS EXPRESSION, AND TISSUE DISTRIBUTION OF THE ZEBRAFISH ALDEHYDE zebrafish (Danio rerio) as a model for ethanol metabolism by cloning, expressing, and characterizing the zebrafish ALDH2. The zebrafish ALDH2 cDNA was cloned and found to be 1892 bp in length and encoding

Tullos, Desiree

262

Long-term effects of ionizing radiation on gene expression in a zebrafish model.  

PubMed

Understanding how initial radiation injury translates into long-term effects is an important problem in radiation biology. Here, we define a set of changes in the transcription profile that are associated with the long-term response to radiation exposure. The study was performed in vivo using zebrafish, an established radiobiological model organism. To study the long-term response, 24 hour post-fertilization embryos were exposed to 0.1 Gy (low dose) or 1.0 Gy (moderate dose) of whole-body gamma radiation and allowed to develop for 16 weeks. Liver mRNA profiles were then analyzed using the Affymetrix microarray platform, with validation by quantitative PCR. As a basis for comparison, 16-week old adults were exposed at the same doses and analyzed after 4 hours. Statistical analysis was performed in a way to minimize the effects of multiple comparisons. The responses to these two treatment regimes differed greatly: 360 probe sets were associated primarily with the long-term response, whereas a different 2062 probe sets were associated primarily with the response when adults of the same age were irradiated 4 hours before exposure. Surprisingly, a ten-fold difference in radiation dose (0.1 versus 1.0 Gy) had little effect. Analysis at the gene and pathway level indicated that the long-term response includes the induction of cytokine and inflammatory regulators and transcription and growth factors. The acute response includes the induction of p53 target genes and modulation of the hypoxia-induced transcription factor-C/EBP axis. Results help define genes and pathways affected in the long-term, low and moderate dose radiation response and differentiate them from those affected in an acute response in the same tissue. PMID:23936019

Jaafar, Lahcen; Podolsky, Robert H; Dynan, William S

2013-01-01

263

Relative developmental toxicities of pentachloroanisole and pentachlorophenol in a zebrafish model (Danio rerio).  

PubMed

Pentachloroanisole (PCA) and pentachlorophenol (PCP) are chlorinated aromatic compounds that have been found in the environment and in human populations. The objective of this study is to characterize the effects of PCA in comparison to those of PCP on development at environmental relevant levels using a fish model. Zebrafish embryos were exposed to 0.1, 1, 10, 100, 500, 1000?g/L PCA and PCP respectively for 96h. Malformation observation, LC50 testing for survival rate at 96 hours post fertilization (hpf) and EC50 testing for hatching rate at 72 hpf indicated that the developmental toxicity of PCP was about 15 times higher than that of PCA. PCP exposure at 10?g/L resulted in elevated 3, 3', 5-triiodothyronine (T3) levels and decreased thyroxine (T4) levels, whereas PCA had no effects on T3 or T4 levels. PCP and PCA exposure at 1 and 10?g/L showed possible hyperthyroid effects similar to that of T3, due to increased relative mRNA expression of synapsin I (SYN), iodothyronine deiodinase type III (Dio3), thyroid hormone receptor alpha a (THR?a) and thyroid hormone receptor beta (THR?), and decreased expression of iodothyronine deiodinase type II (Dio2). The results indicate that both PCA and PCP exposure can cause morphological deformities, possibly affect the timing and coordination of development in the central nervous system, and alter thyroid hormone levels by disrupting thyroid hormone regulating pathways. However, the developmental toxicity of PCA is at least ten times lower than that of PCP. Our results on the relative developmental toxicities of PCA and PCP and the possible underlying mechanisms will be useful to support interpretation of envrionmental concentrations and body burden levels observed in human populations. PMID:25463847

Cheng, Yan; Ekker, Marc; Chan, Hing Man

2015-02-01

264

Correlative light and electron microscopy imaging of autophagy in a zebrafish infection model.  

PubMed

High-resolution imaging of autophagy has been used intensively in cell culture studies, but so far it has been difficult to visualize this process in detail in whole animal models. In this study we present a versatile method for high-resolution imaging of microbial infection in zebrafish larvae by injecting pathogens into the tail fin. This allows visualization of autophagic compartments by light and electron microscopy, which makes it possible to correlate images acquired by the 2 techniques. Using this method we have studied the autophagy response against Mycobacterium marinum infection. We show that mycobacteria during the progress of infection are frequently associated with GFP-Lc3-positive vesicles, and that 2 types of GFP-Lc3-positive vesicles were observed. The majority of these vesicles were approximately 1 ?m in size and in close vicinity of bacteria, and a smaller number of GFP-Lc3-positive vesicles was larger in size and were observed to contain bacteria. Quantitative data showed that these larger vesicles occurred significantly more in leukocytes than in other cell types, and that approximately 70% of these vesicles were positive for a lysosomal marker. Using electron microscopy, it was found that approximately 5% of intracellular bacteria were present in autophagic vacuoles and that the remaining intracellular bacteria were present in phagosomes, lysosomes, free inside the cytoplasm or occurred as large aggregates. Based on correlation of light and electron microscopy images, it was shown that GFP-Lc3-positive vesicles displayed autophagic morphology. This study provides a new approach for injection of pathogens into the tail fin, which allows combined light and electron microscopy imaging in vivo and opens new research directions for studying autophagy process related to infectious diseases. PMID:25126731

Hosseini, Rohola; Lamers, Gerda Em; Hodzic, Zlatan; Meijer, Annemarie H; Schaaf, Marcel Jm; Spaink, Herman P

2014-10-01

265

Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin.  

PubMed

Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin) or TSC2 (encodes tuberin) genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242) in the tsc2 gene. This tsc2(vu242) allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2(vu242) is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2(vu242/vu242) homozygous zebrafish, and is moderately increased in tsc2(vu242/+) heterozygotes. Forebrain neurons are poorly organized in tsc2(vu242/vu242) homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2(vu242/vu242) mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC. PMID:20959633

Kim, Seok-Hyung; Speirs, Christina K; Solnica-Krezel, Lilianna; Ess, Kevin C

2011-03-01

266

Modeling Stress and Anxiety in Zebrafish Jonathan M. Cachat, Peter R. Canavello, Marco F. Elegante, Brett K. Bartels, Salem I. Elkhayat,  

E-print Network

1 Modeling Stress and Anxiety in Zebrafish Jonathan M. Cachat, Peter R. Canavello, Marco F be investigated using such models [31]. Stress and anxiety have been studied extensively using various animal for anxiety research due to their robust cortisol stress response [2], behavioral strain differences [14

Kalueff, Allan V.

267

Vorinostat, an HDAC inhibitor attenuates epidermoid squamous cell carcinoma growth by dampening mTOR signaling pathway in a human xenograft murine model  

SciTech Connect

Histone deacetylase (HDAC) inhibitors are potent anticancer agents and show efficacy against various human neoplasms. Vorinostat is a potent HDAC inhibitor and has shown potential to inhibit growth of human xenograft tumors. However, its effect on the growth of skin neoplasm remains undefined. In this study, we show that vorinostat (2 ?M) reduced expression of HDAC1, 2, 3, and 7 in epidermoid carcinoma A431 cells. Consistently, it increased acetylation of histone H3 and p53. Vorinostat (100 mg/kg body weight, IP) treatment reduced human xenograft tumor growth in highly immunosuppressed nu/nu mice. Histologically, the vorinostat-treated tumor showed features of well-differentiation with large necrotic areas. Based on proliferating cell nuclear antigen (PCNA) staining and expression of cyclins D1, D2, E, and A, vorinostat seems to impair proliferation by down-regulating the expression of these proteins. However, it also induced apoptosis. The mechanism by which vorinostat blocks proliferation and makes tumor cells prone to apoptosis, involved inhibition of mTOR signaling which was accompanied by reduction in cell survival AKT and extracellular-signal regulated kinase (ERK) signaling pathways. Our data provide a novel mechanism-based therapeutic intervention for cutaneous squamous cell carcinoma (SCC). Vorinostat may be utilized to cure skin neoplasms in organ transplant recipient (OTR). These patients have high morbidity and surgical removal of these lesions which frequently develop in these patients, is difficult. -- Highlights: ? Vorinostat reduces SCC growth in a xenograft murine model. ? Vorinostat dampens proliferation and induces apoptosis in tumor cells. ? Diminution in mTOR, Akt and ERK signaling underlies inhibition in proliferation. ? Vorinostat by inhibiting HDACs inhibits epithelial–mesenchymal transition.

Kurundkar, Deepali; Srivastava, Ritesh K.; Chaudhary, Sandeep C. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States)] [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States); Ballestas, Mary E. [Department of Pediatrics Infectious Disease, Children's of Alabama, School of Medicine, University of Alabama at Birmingham, AL (United States)] [Department of Pediatrics Infectious Disease, Children's of Alabama, School of Medicine, University of Alabama at Birmingham, AL (United States); Kopelovich, Levy [Division of Cancer Prevention, National Cancer Institute, 6130 Executive Blvd., Suite 2114, Bethesda, MD 20892 (United States)] [Division of Cancer Prevention, National Cancer Institute, 6130 Executive Blvd., Suite 2114, Bethesda, MD 20892 (United States); Elmets, Craig A. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States)] [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States)] [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States)

2013-01-15

268

Honokiol Crosses BBB and BCSFB, and Inhibits Brain Tumor Growth in Rat 9L Intracerebral Gliosarcoma Model and Human U251 Xenograft Glioma Model  

PubMed Central

Background Gliosarcoma is one of the most common malignant brain tumors, and anti-angiogenesis is a promising approach for the treatment of gliosarcoma. However, chemotherapy is obstructed by the physical obstacle formed by the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB). Honokiol has been known to possess potent activities in the central nervous system diseases, and anti-angiogenic and anti-tumor properties. Here, we hypothesized that honokiol could cross the BBB and BCSFB for the treatment of gliosarcoma. Methodologies We first evaluated the abilities of honokiol to cross the BBB and BCSFB by measuring the penetration of honokiol into brain and blood-cerebrospinal fluid, and compared the honokiol amount taken up by brain with that by other tissues. Then we investigated the effect of honokiol on the growth inhibition of rat 9L gliosarcoma cells and human U251 glioma cells in vitro. Finally we established rat 9L intracerebral gliosarcoma model in Fisher 344 rats and human U251 xenograft glioma model in nude mice to investigate the anti-tumor activity. Principal Findings We showed for the first time that honokiol could effectively cross BBB and BCSFB. The ratios of brain/plasma concentration were respectively 1.29, 2.54, 2.56 and 2.72 at 5, 30, 60 and 120 min. And about 10% of honokiol in plasma crossed BCSFB into cerebrospinal fluid (CSF). In vitro, honokiol produced dose-dependent inhibition of the growth of rat 9L gliosarcoma cells and human U251 glioma cells with IC50 of 15.61 µg/mL and 16.38 µg/mL, respectively. In vivo, treatment with 20 mg/kg body weight of honokiol (honokiol was given twice per week for 3 weeks by intravenous injection) resulted in significant reduction of tumor volume (112.70±10.16 mm3) compared with vehicle group (238.63±19.69 mm3, P?=?0.000), with 52.77% inhibiting rate in rat 9L intracerebral gliosarcoma model, and (1450.83±348.36 mm3) compared with vehicle group (2914.17±780.52 mm3, P?=?0.002), with 50.21% inhibiting rate in human U251 xenograft glioma model. Honokiol also significantly improved the survival over vehicle group in the two models (P<0.05). Conclusions/Significance This study provided the first evidence that honokiol could effectively cross BBB and BCSFB and inhibit brain tumor growth in rat 9L intracerebral gliosarcoma model and human U251 xenograft glioma model. It suggested a significant strategy for offering a potential new therapy for the treatment of gliosarcoma. PMID:21559510

Zhang, Xiaoyan; Deng, Linyu; Zheng, Hao; Deng, Chongyang; Wen, Jiaolin; Wang, Ning; Peng, Cheng; Zhao, Xia; Wei, Yuquan; Chen, Lijuan

2011-01-01

269

Zebrafish Behavioral Response to Chronic Drug  

E-print Network

study the effects of withdrawal from addictive substances to produce anxiogenic-like responses in zebrafish. Key words: Zebrafish, Withdrawal, Cortisol, Anxiety, Stress, Novel Tank test, Addiction #12;3 | P as ideal model species for analysis of genetic and behavioral mechanisms of drug addiction because

Kalueff, Allan V.

270

Zebrafish transgenic Enhancer TRAP line database (ZETRAP)  

Microsoft Academic Search

BACKGROUND: The zebrafish, Danio rerio, is used as a model organism to study vertebrate genetics and development. An effective enhancer trap (ET) in zebrafish using the Tol2 transposon has been demonstrated. This approach could be used to study embryogenesis of a vertebrate species in real time and with high resolution. DESCRIPTION: The information gathered during the course of systematic investigation

Benjamin GH Choo; Igor Kondrichin; Sergey Parinov; Alexander Emelyanov; William Go; Wei-chang Toh; Vladimir Korzh

2006-01-01

271

A zebrafish model for Waardenburg syndrome type IV reveals diverse roles for Sox10 in the otic vesicle  

PubMed Central

SUMMARY In humans, mutations in the SOX10 gene are a cause of the auditory-pigmentary disorder Waardenburg syndrome type IV (WS4) and related variants. SOX10 encodes an Sry-related HMG box protein essential for the development of the neural crest; deafness in WS4 and other Waardenburg syndromes is usually attributed to loss of neural-crest-derived melanocytes in the stria vascularis of the cochlea. However, SOX10 is strongly expressed in the developing otic vesicle and so direct roles for SOX10 in the otic epithelium might also be important. Here, we examine the otic phenotype of zebrafish sox10 mutants, a model for WS4. As a cochlea is not present in the fish ear, the severe otic phenotype in these mutants cannot be attributed to effects on this tissue. In zebrafish sox10 mutants, we see abnormalities in all otic placodal derivatives. Gene expression studies indicate deregulated expression of several otic genes, including fgf8, in sox10 mutants. Using a combination of mutant and morphant data, we show that the three sox genes belonging to group E (sox9a, sox9b and sox10) provide a link between otic induction pathways and subsequent otic patterning: they act redundantly to maintain sox10 expression throughout otic tissue and to restrict fgf8 expression to anterior macula regions. Single-cell labelling experiments indicate a small and transient neural crest contribution to the zebrafish ear during normal development, but this is unlikely to account for the strong defects seen in the sox10 mutant. We discuss the implication that the deafness in WS4 patients with SOX10 mutations might reflect a haploinsufficiency for SOX10 in the otic epithelium, resulting in patterning and functional abnormalities in the inner ear. PMID:19132125

Dutton, Kirsten; Abbas, Leila; Spencer, Joanne; Brannon, Claire; Mowbray, Catriona; Nikaido, Masataka; Kelsh, Robert N.; Whitfield, Tanya T.

2009-01-01

272

The N-terminal region of centrosomal protein 290 (CEP290) restores vision in a zebrafish model of human blindness.  

PubMed

The gene coding for centrosomal protein 290 (CEP290), a large multidomain protein, is the most frequently mutated gene underlying the non-syndromic blinding disorder Leber's congenital amaurosis (LCA). CEP290 has also been implicated in several cilia-related syndromic disorders including Meckel-Gruber syndrome, Joubert syndrome, Senor-Loken syndrome and Bardet-Biedl syndrome (BBS). In this study, we characterize the developmental and functional roles of cep290 in zebrafish. An antisense oligonucleotide [Morpholino (MO)], designed to generate an altered cep290 splice product that models the most common LCA mutation, was used for gene knockdown. We show that cep290 MO-injected embryos have reduced Kupffer's vesicle size and delays in melanosome transport, two phenotypes that are observed upon knockdown of bbs genes in zebrafish. Consistent with a role in cilia function, the cep290 MO-injected embryos exhibited a curved body axis. Patients with LCA caused by mutations in CEP290 have reduced visual perception, although they present with a fully laminated retina. Similarly, the histological examination of retinas from cep290 MO-injected zebrafish revealed no gross lamination defects, yet the embryos had a statistically significant reduction in visual function. Finally, we demonstrate that the vision impairment caused by the disruption of cep290 can be rescued by expressing only the N-terminal region of the human CEP290 protein. These data reveal that a specific region of the CEP290 protein is sufficient to restore visual function and this region may be a viable gene therapy target for LCA patients with mutations in CEP290. PMID:21257638

Baye, Lisa M; Patrinostro, Xiaobai; Swaminathan, Svetha; Beck, John S; Zhang, Yan; Stone, Edwin M; Sheffield, Val C; Slusarski, Diane C

2011-04-15

273

The N-terminal region of centrosomal protein 290 (CEP290) restores vision in a zebrafish model of human blindness  

PubMed Central

The gene coding for centrosomal protein 290 (CEP290), a large multidomain protein, is the most frequently mutated gene underlying the non-syndromic blinding disorder Leber's congenital amaurosis (LCA). CEP290 has also been implicated in several cilia-related syndromic disorders including Meckel–Gruber syndrome, Joubert syndrome, Senor–Loken syndrome and Bardet–Biedl syndrome (BBS). In this study, we characterize the developmental and functional roles of cep290 in zebrafish. An antisense oligonucleotide [Morpholino (MO)], designed to generate an altered cep290 splice product that models the most common LCA mutation, was used for gene knockdown. We show that cep290 MO-injected embryos have reduced Kupffer's vesicle size and delays in melanosome transport, two phenotypes that are observed upon knockdown of bbs genes in zebrafish. Consistent with a role in cilia function, the cep290 MO-injected embryos exhibited a curved body axis. Patients with LCA caused by mutations in CEP290 have reduced visual perception, although they present with a fully laminated retina. Similarly, the histological examination of retinas from cep290 MO-injected zebrafish revealed no gross lamination defects, yet the embryos had a statistically significant reduction in visual function. Finally, we demonstrate that the vision impairment caused by the disruption of cep290 can be rescued by expressing only the N-terminal region of the human CEP290 protein. These data reveal that a specific region of the CEP290 protein is sufficient to restore visual function and this region may be a viable gene therapy target for LCA patients with mutations in CEP290. PMID:21257638

Baye, Lisa M.; Patrinostro, Xiaobai; Swaminathan, Svetha; Beck, John S.; Zhang, Yan; Stone, Edwin M.; Sheffield, Val C.; Slusarski, Diane C.

2011-01-01

274

Decorin Protein Core Affects the Global Gene Expression Profile of the Tumor Microenvironment in a Triple-Negative Orthotopic Breast Carcinoma Xenograft Model  

PubMed Central

Decorin, a member of the small leucine-rich proteoglycan gene family, exists and functions wholly within the tumor microenvironment to suppress tumorigenesis by directly targeting and antagonizing multiple receptor tyrosine kinases, such as the EGFR and Met. This leads to potent and sustained signal attenuation, growth arrest, and angiostasis. We thus sought to evaluate the tumoricidal benefits of systemic decorin on a triple-negative orthotopic breast carcinoma xenograft model. To this end, we employed a novel high-density mixed expression array capable of differentiating and simultaneously measuring gene signatures of both Mus musculus (stromal) and Homo sapiens (epithelial) tissue origins. We found that decorin protein core modulated the differential expression of 374 genes within the stromal compartment of the tumor xenograft. Further, our top gene ontology classes strongly suggests an unexpected and preferential role for decorin protein core to inhibit genes necessary for immunomodulatory responses while simultaneously inducing expression of those possessing cellular adhesion and tumor suppressive gene properties. Rigorous verification of the top scoring candidates led to the discovery of three genes heretofore unlinked to malignant breast cancer that were reproducibly found to be induced in several models of tumor stroma. Collectively, our data provide highly novel and unexpected stromal gene signatures as a direct function of systemic administration of decorin protein core and reveals a fundamental basis of action for decorin to modulate the tumor stroma as a biological mechanism for the ascribed anti-tumorigenic properties. PMID:23029096

Owens, Rick T.; Iniguez, Leonardo A.; Purkins, George; Vadigepalli, Rajanikanth; Evans, Barry; Schaefer, Liliana; Peiper, Stephen C.; Wang, Zi-Xuan; Iozzo, Renato V.

2012-01-01

275

Anti-CD45 pretargeted radioimmunotherapy using bismuth-213: high rates of complete remission and long-term survival in a mouse myeloid leukemia xenograft model  

PubMed Central

Pretargeted radioimmunotherapy (PRIT) using an anti-CD45 antibody (Ab)–streptavidin (SA) conjugate and DOTA-biotin labeled with ?-emitting radionuclides has been explored as a strategy to decrease relapse and toxicity. ?-emitting radionuclides exhibit high cytotoxicity coupled with a short path length, potentially increasing the therapeutic index and making them an attractive alternative to ?-emitting radionuclides for patients with acute myeloid leukemia. Accordingly, we have used 213Bi in mice with human leukemia xenografts. Results demonstrated excellent localization of 213Bi-DOTA-biotin to tumors with minimal uptake into normal organs. After 10 minutes, 4.5% ± 1.1% of the injected dose of 213Bi was delivered per gram of tumor. ?-imaging demonstrated uniform radionuclide distribution within tumor tissue 45 minutes after 213Bi-DOTA-biotin injection. Radiation absorbed doses were similar to those observed using a ?-emitting radionuclide (90Y) in the same model. We conducted therapy experiments in a xenograft model using a single-dose of 213Bi-DOTA-biotin given 24 hours after anti-CD45 Ab-SA conjugate. Among mice treated with anti-CD45 Ab-SA conjugate followed by 800 ?Ci of 213Bi- or 90Y-DOTA-biotin, 80% and 20%, respectively, survived leukemia-free for more than 100 days with minimal toxicity. These data suggest that anti-CD45 PRIT using an ?-emitting radionuclide may be highly effective and minimally toxic for treatment of acute myeloid leukemia. PMID:21613259

Kenoyer, Aimee L.; Bäck, Tom; Hamlin, Donald K.; Wilbur, D. Scott; Fisher, Darrell R.; Park, Steven I.; Frayo, Shani; Axtman, Amanda; Orgun, Nural; Orozco, Johnnie; Shenoi, Jaideep; Lin, Yukang; Gopal, Ajay K.; Green, Damian J.; Appelbaum, Frederick R.; Press, Oliver W.

2011-01-01

276

Differential response to EGFR- and VEGF-targeted therapies in patient-derived tumor tissue xenograft models of colon carcinoma and related metastases.  

PubMed

Heterogeneity in primary tumors and related metastases may result in failure of antitumor therapies, particularly in targeted therapies for the treatment of cancer. In this study, patient-derived tumor tissue (PDTT) xenograft models of colon carcinoma with lymphatic and hepatic metastases were used to evaluate the response to EGFR- and VEGF-targeted therapies. Our results showed that primary colon carcinoma and its corresponding lymphatic and hepatic metastases have a different response rate to anti-EGFR (cetuximab) and anti-VEGF (bevacizumab) therapies. However, the underlying mechanism of these types of phenomenon is still unclear. To investigate whether such phenomena may result from the heterogeneity in primary colon carcinoma and related metastases, we compared the expression levels of cell signaling pathway proteins using immunohistochemical staining and western blotting, and the gene status of KRAS using pyrosequencing in the same primary colon carcinoma and its corresponding lymphatic and hepatic metastatic tissues which were used for establishing the PDTT xenograft models. Our results showed that the expression levels of EGFR, VEGF, Akt/pAkt, ERK/pERK, MAPK/pMAPK, and mTOR/pmTOR were different in primary colon carcinoma and matched lymphatic and hepatic metastases although the KRAS gene status in all cases was wild-type. Our results indicate that the heterogeneity in primary colon carcinoma and its corresponding lymphatic and hepatic metastases may result in differences in the response to dual-inhibition of EGFR and VEGF. PMID:22581265

Jin, Ketao; Lan, Huanrong; Cao, Feilin; Han, Na; Xu, Zhenzhen; Li, Guangliang; He, Kuifeng; Teng, Lisong

2012-08-01

277

Functions of the Wnt/?-catenin pathway in an anemia-induced zebrafish model of cardiomyopathy are location dependent.  

PubMed

Recent evidence that the heart is not a terminally-differentiated organ has provided more credence to investigations of pathways involved in inducing cardiomyocyte (CM) hyperplasia as a therapy for heart disease. Here, we leveraged zebrafish as a novel vertebrate model of cardiomyopathy to explore the therapeutic potential based on the Wnt/?-catenin signaling. In the anemia-induced zebrafish model of cardiomyopathy (tr265), we detected differently regulated CM hyperplasia and CM hypertrophy in the compact region and the trabecular region. To assess the effects of the Wnt/?-catenin pathway on these two regions, the anemia line was crossed with heat shock-inducible transgenic fish to upregulate or downregulate the pathway. Upregulation resulted in increased cardiomyocyte hyperplasia in the heart and increased cardiomyocyte hypertrophy in the trabecular region, while downregulation resulted in reduced cardiomyocyte hyperplasia in the heart and reduced cardiomyocyte hypertrophy in the trabecular region. Importantly, upregulation of the pathway resulted in improved fish survival, while downregulation decreased it. In summary, our data suggested that (1) the compact region and the trabecular region respond differently during cardiac remodeling; (2) activation of the Wnt/?-catenin pathway might exert a cardioprotective function via promoting cardiomyocyte hyperplasia. PMID:22056559

Hoage, Tiffany; Sun, Xiaojing; Xu, Xiaolei

2011-11-25

278

Functional activity of the membrane-associated complement inhibitor CD59 in a pig-to-human in vitro model for hyperacute xenograft rejection.  

PubMed Central

Hyperacute rejection triggered by activation of the recipient's complement system represents the major barrier to successful xenotransplantation. Transfer of human membrane-associated complement regulators to donor organs has been suggested as one strategy to interfere with complement-mediated hyperacute xenograft rejection. Pigs are discussed as potential organ donors. We therefore investigated a putative protective function of the membrane-bound complement inhibitor CD59 in a pig-to-human in vitro model of hyperacute xenograft rejection. Aortic porcine endothelial cells were transfected with human CD59 cDNA. Expression of human CD59 was demonstrated by cytofluorimetric and RNA analysis. Removal of CD59 from the cell surface by phosphatidylinositol-specific phospholipase C (PI-PLC) demonstrated its production as a glycosyl phosphatidylinositol (GPI)-anchored protein. Functional activity of the transfected CD59 was tested by a lactate dehydrogenase (LDH) release assay for complement-mediated lysis. Porcine endothelial cells expressing human CD59 were significantly protected from lysis by human serum complement compared with CD59- cells. The protective effect was abolished by preincubating the cells with anti-CD59 antibodies or PI-PLC. We calculated by Scatchard analysis that the established CD59+ cell line expressed a CD59 level comparable to that of human endothelial cells. Our results recommend the production of pigs transgenic for CD59. Images Fig. 3 PMID:8536377

Heckl-Ostreicher, B; Binder, R; Kirschfink, M

1995-01-01

279

Metformin impairs Rho GTPase signaling to induce apoptosis in neuroblastoma cells and inhibits growth of tumors in the xenograft mouse model of neuroblastoma  

PubMed Central

Metformin has been shown to inhibit tumor growth in xenograft rodent models of adult cancers, and various human clinical trials are in progress. However, the precise molecular mechanisms of metformin action are largely unknown. In the present study we examined the anti-tumor activity of metformin against neuroblastoma, and determined the underlying signaling mechanisms. Using human neuroblastoma xenograft mice, we demonstrated that oral administration of metformin (100 and 250 mg/kg body weight) significantly inhibited the growth of tumors. The interference of metformin in spheroid formation further confirmed the anti-tumor activity of metformin. In tumors, the activation of Rac1 (GTP-Rac1) and Cdc42 (GTP-Cdc42) was increased while RhoA activation (GTP-RhoA) was decreased by metformin. It also induced phosphorylation of JNK and inhibited the phosphorylation of ERK1/2 without affecting p38 MAP Kinase. Infection of cells by adenoviruses expressing dominant negative Rac1 (Rac1-N17), Cdc42 (Cdc42-N17) or constitutively active RhoA (RhoA-V14), or incubation of cells with pharmacological inhibitors of Rac1 (NSC23766) or Cdc42 (ML141) significantly protected neuroblastoma cells from metformin-induced apoptosis. Additionally, inhibition of JNK activity along with Rac1 or Cdc42 attenuated cytotoxic effects of metformin. These studies demonstrated that metformin impairs Rho GTPases signaling to induce apoptosis via JNK pathway. PMID:25365944

Kumar, Ambrish; Al-Sammarraie, Nadia; DiPette, Donald J.; Singh, Ugra S.

2014-01-01

280

Metformin impairs Rho GTPase signaling to induce apoptosis in neuroblastoma cells and inhibits growth of tumors in the xenograft mouse model of neuroblastoma.  

PubMed

Metformin has been shown to inhibit tumor growth in xenograft rodent models of adult cancers, and various human clinical trials are in progress. However, the precise molecular mechanisms of metformin action are largely unknown. In the present study we examined the anti-tumor activity of metformin against neuroblastoma, and determined the underlying signaling mechanisms. Using human neuroblastoma xenograft mice, we demonstrated that oral administration of metformin (100 and 250 mg/kg body weight) significantly inhibited the growth of tumors. The interference of metformin in spheroid formation further confirmed the anti-tumor activity of metformin. In tumors, the activation of Rac1 (GTP-Rac1) and Cdc42 (GTP-Cdc42) was increased while RhoA activation (GTP-RhoA) was decreased by metformin. It also induced phosphorylation of JNK and inhibited the phosphorylation of ERK1/2 without affecting p38 MAP Kinase. Infection of cells by adenoviruses expressing dominant negative Rac1 (Rac1-N17), Cdc42 (Cdc42-N17) or constitutively active RhoA (RhoA-V14), or incubation of cells with pharmacological inhibitors of Rac1 (NSC23766) or Cdc42 (ML141) significantly protected neuroblastoma cells from metformin-induced apoptosis. Additionally, inhibition of JNK activity along with Rac1 or Cdc42 attenuated cytotoxic effects of metformin. These studies demonstrated that metformin impairs Rho GTPases signaling to induce apoptosis via JNK pathway. PMID:25365944

Kumar, Ambrish; Al-Sammarraie, Nadia; DiPette, Donald J; Singh, Ugra S

2014-11-30

281

A zebrafish model of CLN2 disease is deficient in tripeptidyl peptidase 1 and displays progressive neurodegeneration accompanied by a reduction in proliferation.  

PubMed

Tripeptidyl peptidase 1 (TPP1) deficiency causes CLN2 disease, late infantile (or classic late infantile neuronal ceroid lipofuscinosis), a paediatric neurodegenerative disease of autosomal recessive inheritance. Patients suffer from blindness, ataxia, epilepsy and cognitive defects, with MRI indicating widespread brain atrophy, and profound neuron loss is evident within the retina and brain. Currently there are no effective therapies for this disease, which causes premature death in adolescence. Zebrafish have been successfully used to model a range of neurological and behavioural abnormalities. The aim of this study was to characterize the pathological and functional consequences of Tpp1 deficiency in zebrafish and to correlate these with human CLN2 disease, thereby providing a platform for drug discovery. Our data show that homozygous tpp1(sa0011) mutant (tpp1(sa0011)(-/-)) zebrafish display a severe, progressive, early onset neurodegenerative phenotype, characterized by a significantly small retina, a small head and curved body. The mutant zebrafish have significantly reduced median survival with death occurring 5 days post-fertilization. As in human patients with CLN2 disease, mutant zebrafish display storage of subunit c of mitochondrial ATP-synthase, hypertrophic lysosomes as well as localized apoptotic cell death in the retina, optic tectum and cerebellum. Further neuropathological phenotypes of these mutants provide novel insights into mechanisms of pathogenesis in CLN2 disease. Secondary neurogenesis in the retina, optic tectum and cerebellum is impaired and axon tracts within the spinal cord, optic nerve and the posterior commissure are disorganized, with the optic nerve failing to reach its target. This severe neurodegenerative phenotype eventually results in functional motor impairment, but this is preceded by a phase of hyperactivity that is consistent with seizures. Importantly, both of these locomotion phenotypes can be assayed in an automated manner suitable for high-throughput studies. Our study provides proof-of-principle that tpp1(sa0011)(-/-) mutants can utilize the advantages of zebrafish for understanding pathogenesis and drug discovery in CLN2 disease and other epilepsies. PMID:23587805

Mahmood, Fahad; Fu, Sonia; Cooke, Jennifer; Wilson, Stephen W; Cooper, Jonathan D; Russell, Claire

2013-05-01

282

Investigational drug MLN0128, a novel TORC1/2 inhibitor, demonstrates potent oral antitumor activity in human breast cancer xenograft models.  

PubMed

Aberrant activation of the mammalian target of rapamycin (mTOR) signaling plays an important role in breast cancer progression and represents a potential therapeutic target for breast cancer. In this study, we report the impact of the investigational drug MLN0128, a potent and selective small molecule active-site TORC1/2 kinase inhibitor, on tumor growth and metastasis using human breast cancer xenograft models. We assessed in vitro antiproliferative activity of MLN0128 in a panel of breast cancer cell lines. We next evaluated the impact of MLN0128 on tumor growth, angiogenesis and metastasis using mammary fat pad xenograft models of a non-VEGF (ML20) and a VEGF-driven (MV165) MCF-7 sublines harboring PIK3CA mutations. MLN0128 potently inhibited cell proliferation in various breast cancer cell lines harboring PIK3CA (IC(50): 1.5-53 nM), PTEN (IC(50): 1-149 nM), KRAS, and/or BRAF mutations (IC(50): 13-162 nM), and in human endothelial cells (IC(50): 33-40 nM) in vitro. In vivo, MLN0128 decreased primary tumor growth significantly in both non-VEGF (ML20; p = 0.05) and VEGF-driven MCF-7 (MV165; p = 0.014) xenograft models. MLN0128 decreased the phosphorylation of Akt, S6, 4E-BP1, and NDRG1 in both models. In contrast, rapamycin increased Akt activity and failed to reduce the phosphorylation of 4E-BP1, PRAS40, and NDRG1. VEGF-induced lung metastasis in MV165 is inhibited by MLN0128 and rapamycin. In conclusion, MLN0128 inhibits TORC1/2-dependent signaling in preclinical models of breast cancer. MLN0128 appears to be superior in blocking mTORC1/2 signaling in contrast to rapamycin. Our findings support the clinical research of MLN0128 in patients with breast cancer and metastasis. PMID:23085766

Gökmen-Polar, Yesim; Liu, Yi; Toroni, Rachel A; Sanders, Kerry L; Mehta, Rutika; Badve, Sunil; Rommel, Christian; Sledge, George W

2012-12-01

283

Lenvatinib, an angiogenesis inhibitor targeting VEGFR/FGFR, shows broad antitumor activity in human tumor xenograft models associated with microvessel density and pericyte coverage  

PubMed Central

Background Lenvatinib is an oral inhibitor of multiple receptor tyrosine kinases (RTKs) targeting vascular endothelial growth factor receptor (VEGFR1-3), fibroblast growth factor receptor (FGFR1-4), platelet growth factor receptor ? (PDGFR ?), RET and KIT. Antiangiogenesis activity of lenvatinib in VEGF- and FGF-driven angiogenesis models in both in vitro and in vivo was determined. Roles of tumor vasculature (microvessel density (MVD) and pericyte coverage) as biomarkers for lenvatinib were also examined in this study. Method We evaluated antiangiogenesis activity of lenvatinib against VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. Effects of lenvatinib on in vivo angiogenesis, which was enhanced by overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells, were examined in the mouse dorsal air sac assay. We determined antitumor activity of lenvatinib in a broad panel of human tumor xenograft models to test if vascular score, which consisted of high MVD and low pericyte coverage, was associated with sensitivity to lenvatinib treatment. Vascular score was also analyzed using human tumor specimens with 18 different types of human primary tumors. Result Lenvatinib inhibited VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. In vivo angiogenesis induced by overexpressed VEGF (KP-1/VEGF transfectants) or FGF (KP-1/FGF transfectants) was significantly suppressed with oral treatments of lenvatinib. Lenvatinib showed significant antitumor activity in KP-1/VEGF and five 5 of 7 different types of human tumor xenograft models at between 1 to 100?mg/kg. We divided 19 human tumor xenograft models into lenvatinib-sensitive (tumor-shrinkage) and relatively resistant (slow-growth) subgroups based on sensitivity to lenvatinib treatments at 100?mg/kg. IHC analysis showed that vascular score was significantly higher in sensitive subgroup than relatively resistant subgroup (p?

2014-01-01

284

Combined Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor (EGFR) Blockade Inhibits Tumor Growth in Xenograft Models of EGFR Inhibitor Resistance  

PubMed Central

Purpose The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) gefitinib and erlotinib benefit some non–small cell lung cancer (NSCLC) patients, but most do not respond (primary resistance) and those who initially respond eventually progress (acquired resistance). EGFR TKI resistance is not completely understood and has been associated with certain EGFR and K-RAS mutations and MET amplification. Experimental Design We hypothesized that dual inhibition of the vascular endothelial growth factor (VEGF) and EGFR pathways may overcome primary and acquired resistance. We investigated the VEGF receptor/EGFR TKI vandetanib, and the combination of bevacizumab and erlotinib in vivo using xenograft models of EGFR TKI sensitivity, primary resistance, and three models of acquired resistance, including models with mutated K-RAS and secondary EGFR T790M mutation. Results Vandetanib, gefitinib, and erlotinib had similar profiles of in vitro activity and caused sustained tumor regressions in vivo in the sensitive HCC827 model. In all four resistant models, vandetanib and bevacizumab/erlotinib were significantly more effective than erlotinib or gefitinib alone. Erlotinib resistance was associated with a rise in both host and tumor-derived VEGF but not EGFR secondary mutations in the KRAS mutant-bearing A549 xenografts. Dual inhibition reduced tumor endothelial proliferation compared with VEGF or EGFR blockade alone, suggesting that the enhanced activity of dual inhibition is due at least in part to antiendothelial effects. Conclusion These studies suggest that erlotinib resistance may be associated with a rise in both tumor cell and host stromal VEGF and that combined blockade of the VEGFR and EGFR pathways can abrogate primary or acquired resistance to EGFR TKIs. This approach merits further evaluation in NSCLC patients. PMID:19447865

Naumov, George N.; Nilsson, Monique B.; Cascone, Tina; Briggs, Alexandra; Straume, Oddbjorn; Akslen, Lars A.; Lifshits, Eugene; Byers, Lauren Averett; Xu, Li; Wu, Hua-kang; Jänne, Pasi; Kobayashi, Susumu; Halmos, Balazs; Tenen, Daniel; Tang, Xi M.; Engelman, Jeffrey; Yeap, Beow; Folkman, Judah; Johnson, Bruce E.; Heymach, John V.

2010-01-01

285

Enhanced anti-tumor activity of the glycoengineered type II CD20 antibody obinutuzumab (GA101) in combination with chemotherapy in xenograft models of human lymphoma.  

PubMed

Obinutuzumab (GA101) is a novel glycoengineered type II CD20 antibody in development for non-Hodgkin lymphoma. We compared the anti-tumor activity of obinutuzumab and rituximab in preclinical studies using subcutaneous Z138 and WSU-DLCL2 xenograft mouse models. Obinutuzumab and rituximab were assessed alone and in combination with bendamustine, fludarabine, chlorambucil, doxorubicin and cyclophosphamide/vincristine. Owing to strong single-agent efficacy in these models, suboptimal doses of obinutuzumab were applied to demonstrate a combination effect. Obinutuzumab plus bendamustine achieved superior tumor growth inhibition versus rituximab plus bendamustine and showed a statistically significant effect versus the respective single treatments. Combinations of obinutuzumab with fludarabine, chlorambucil or cyclophosphamide/vincristine demonstrated significantly superior activity to rituximab-based treatment. Obinutuzumab monotherapy was at least as effective as rituximab plus chemotherapy in vivo, and obinutuzumab plus chemotherapy was superior to the respective monotherapies. These data support further clinical investigation of obinutuzumab plus chemotherapy. PMID:24304419

Herting, Frank; Friess, Thomas; Bader, Sabine; Muth, Gunter; Hölzlwimmer, Gabriele; Rieder, Natascha; Umana, Pablo; Klein, Christian

2014-09-01

286

Enhanced anti-tumor activity of the glycoengineered type II CD20 antibody obinutuzumab (GA101) in combination with chemotherapy in xenograft models of human lymphoma  

PubMed Central

Obinutuzumab (GA101) is a novel glycoengineered type II CD20 antibody in development for non-Hodgkin lymphoma. We compared the anti-tumor activity of obinutuzumab and rituximab in preclinical studies using subcutaneous Z138 and WSU-DLCL2 xenograft mouse models. Obinutuzumab and rituximab were assessed alone and in combination with bendamustine, fludarabine, chlorambucil, doxorubicin and cyclophosphamide/vincristine. Owing to strong single-agent efficacy in these models, suboptimal doses of obinutuzumab were applied to demonstrate a combination effect. Obinutuzumab plus bendamustine achieved superior tumor growth inhibition versus rituximab plus bendamustine and showed a statistically significant effect versus the respective single treatments. Combinations of obinutuzumab with fludarabine, chlorambucil or cyclophosphamide/vincristine demonstrated significantly superior activity to rituximab-based treatment. Obinutuzumab monotherapy was at least as effective as rituximab plus chemotherapy in vivo, and obinutuzumab plus chemotherapy was superior to the respective monotherapies. These data support further clinical investigation of obinutuzumab plus chemotherapy. PMID:24304419

Herting, Frank; Friess, Thomas; Bader, Sabine; Muth, Gunter; Hölzlwimmer, Gabriele; Rieder, Natascha; Umana, Pablo

2014-01-01

287

Intratracheally Administered 5-Azacytidine Is Effective Against Orthotopic Human Lung Cancer Xenograft Models and Devoid of Important Systemic Toxicity  

PubMed Central

Introduction Hypermethylation of key tumor suppressor genes plays an important role in lung carcinogenesis. The purpose of this study is to explore the therapeutic potential of regional administration (via the airways) of the demethylating agent 5-azacytidine (5-Aza) for the treatment of early lung cancer. Patients and Methods We administered 5-Aza solution directly into the trachea in imprinting control region (ICR) mice (to study its toxicity) and in nude mice bearing orthotopic human lung cancer xenografts (to assess its antitumor activity). Results In vitro, 5-Aza inhibited the growth of human lung cancer cell lines H226, H358, and H460 in a dose-dependent manner. The concentrations to inhibit cell growth by 50% (IC50) were about 0.6-4.9 ?g/mL. 5-Azacytidine reversed hypermethylation in the promoter of tumor suppressor gene RASSF1a in the H226 cells at a 6000-fold lower concentration than its IC50. In animal studies, intratracheal (I.T.) administration of 90 mg/kg 5-Aza (the maximum tolerated dose of 5-Aza intravenous injection [I.V.]) resulted in moderate pulmonary toxicity and 5-fold reduced myelosuppression compared with the same dose of I.V. 5-Aza. Using an optimized multiple dose schedule, I.T. 5-Aza was about 3-fold more effective than I.V. 5-Aza in prolonging the survival of mice bearing orthotopic H460 and H358 xenografts, and did not cause any detectable toxicity. Conclusion 5-Azacytidine can reverse the hypermethylation in the human lung cancer cell lines at a nontoxic dose. Regional administration to the airways enhances the therapeutic index of 5-Aza by 75-fold. The potential of regional administration of 5-Aza (including by aerosolization) for the treatment of advanced bronchial premalignancy deserves further investigation. PMID:21062731

Mahesh, Sameer; Saxena, Ashish; Qiu, Xuan; Perez-Soler, Roman; Zou, Yiyu

2014-01-01

288

In vivo anti-metastatic effects of uPAR retargeted measles virus in syngeneic and xenograft models of mammary cancer.  

PubMed

The urokinase receptor (uPAR) plays a critical role in breast cancer (BC) progression and metastases and is a validated target for novel therapies. The current study investigates the effects of MV-uPA, an oncolytic measles virus fully retargeted against uPAR in syngeneic and xenograft BC metastases models. In vitro replication and cytotoxicity of MVs retargeted against human (MV-h-uPA) or mouse (MV-m-uPA) uPAR were assessed in human and murine cancer and non-cancer mammary epithelial cells. The in vivo effects of species-specific uPAR retargeted MVs were assessed in syngeneic and xenograft models of experimental metastases, established by intravenous administration of luciferase expressing 4T1 or MDA-MD-231 cells. Metastases progression was assessed by in vivo bioluminescence imaging. Tumor targeting was evaluated by qRT-PCR of MV-N, rescue of viable viral particles, and immunostaining of MV particles in lungs from tumor bearing mice. In vitro, MV-h-uPA and MV-m-uPA selectively infected, replicated, and induced cytotoxicity in cancer compared to non-cancer cells in a species-specific manner. In vivo, MV-m-uPA delayed 4T1 lung metastases progression and prolonged survival. These effects were associated with identification of viable viral particles, viral RNA, and detection of MV-N by immunostaining from lung tissues in treated mice. In the human MDA-MB-231 metastases model, intravenous administration of MV-h-uPA markedly inhibited metastases progression and significantly improved survival, compared to controls. No significant treatment-related toxicity was observed in treated mice. The above preclinical findings strongly suggest that uPAR retargeted measles virotherapy is a novel and feasible systemic therapy strategy against metastatic breast cancer. PMID:25519042

Jing, Yuqi; Bejarano, Marcela Toro; Zaias, Julia; Merchan, Jaime R

2015-01-01

289

In Silico cancer cell versus stroma cellularity index computed from species-specific human and mouse transcriptome of xenograft models: towards accurate stroma targeting therapy assessment  

PubMed Central

Background The current state of the art for measuring stromal response to targeted therapy requires burdensome and rate limiting quantitative histology. Transcriptome measures are increasingly affordable and provide an opportunity for developing a stromal versus cancer ratio in xenograft models. In these models, human cancer cells are transplanted into mouse host tissues (stroma) and together coevolve into a tumour microenvironment. However, profiling the mouse or human component separately remains problematic. Indeed, laser capture microdissection is labour intensive. Moreover, gene expression using commercial microarrays introduces significant and underreported cross-species hybridization errors that are commonly overlooked by biologists. Method We developed a customized dual-species array, H&M array, and performed cross-species and species-specific hybridization measurements. We validated a new methodology for establishing the stroma vs cancer ratio using transcriptomic data. Results In the biological validation of the H&M array, cross-species hybridization of human and mouse probes was significantly reduced (4.5 and 9.4 fold reduction, respectively; p < 2x10-16 for both, Mann-Whitney test). We confirmed the capability of the H&M array to determine the stromal to cancer cells ratio based on the estimation of cellularity index of mouse/human mRNA content in vitro. This new metrics enable to investigate more efficiently the stroma-cancer cell interactions (e.g. cellularity) bypassing labour intensive requirement and biases of laser capture microdissection. Conclusion These results provide the initial evidence of improved and cost-efficient analytics for the investigation of cancer cell microenvironment, using species-specificity arrays specifically designed for xenografts models. PMID:25079962

2014-01-01

290

In vivo molecular imaging of gastric cancer in human-murine xenograft models with confocal laser endomicroscopy using a tumor vascular homing peptide.  

PubMed

The early detection of premalignant lesions and cancers are very important for improving the survival of patients with gastric malignancies. Confocal laser endomicroscopy (CLE) is a novel imaging tool for achieving real-time microscopy during the ongoing endoscopy at subcellular resolution. In the present study, to evaluate the feasibility of real-time molecular imaging of GEBP11 by CLE in gastric cancer, CLE was performed on two types of tumor-bearing mice models, as well as surgical specimens of patients with gastric cancer, after the application of GEBP11. A whole-body fluorescent imaging device was first used to screen for the strongest specific fluorescent signal in xenograft models. Next, the tumor sites, as well as human tissues, were scanned with CLE. After this, targeted specimens were obtained for fluorescence microscopy and histology. We confirmed that GEBP11 could specifically bind to co-HUVECs by means of CLE in cell experiments. Thereafter, a specific signal was observed in both subcutaneous and orthotopic xenograft models in vivo after the injection of FITC-GEBP11 via tail vein, whereas the group injected with FITC-URP showed no fluorescent signals. In human tissues, a specific signal of GEBP11 was observed in 26/28 neoplastic specimens and in 8/28 samples of non-neoplastic specimens from the patients (p?

Liu, Lijuan; Yin, Jipeng; Liu, Changhao; Guan, Guofeng; Shi, Doufei; Wang, Xiaojuan; Xu, Bing; Tian, Zuhong; Zhao, Jing; Nie, Yongzhan; Wang, Biaoluo; Liang, Shuhui; Wu, Kaichun; Ding, Jie

2015-01-28

291

A New Zebrafish Model of Oro-Intestinal Pathogen Colonization Reveals a Key Role for Adhesion in Protection by Probiotic Bacteria  

PubMed Central

The beneficial contribution of commensal bacteria to host health and homeostasis led to the concept that exogenous non-pathogenic bacteria called probiotics could be used to limit disease caused by pathogens. However, despite recent progress using gnotobiotic mammal and invertebrate models, mechanisms underlying protection afforded by commensal and probiotic bacteria against pathogens remain poorly understood. Here we developed a zebrafish model of controlled co-infection in which germ-free zebrafish raised on axenic living protozoa enabled the study of interactions between host and commensal and pathogenic bacteria. We screened enteric fish pathogens and identified Edwardsiella ictaluri as a virulent strain inducing a strong inflammatory response and rapid mortality in zebrafish larvae infected by the natural oro-intestinal route. Using mortality induced by infection as a phenotypic read-out, we pre-colonized zebrafish larvae with 37 potential probiotic bacterial strains and screened for survival upon E. ictaluri infection. We identified 3 robustly protective strains, including Vibrio parahaemolyticus and 2 Escherichia coli strains. We showed that the observed protective effect of E. coli was not correlated with a reduced host inflammatory response, nor with the release of biocidal molecules by protective bacteria, but rather with the presence of specific adhesion factors such as F pili that promote the emergence of probiotic bacteria in zebrafish larvae. Our study therefore provides new insights into the molecular events underlying the probiotic effect and constitutes a potentially high-throughput in vivo approach to the study of the molecular basis of pathogen exclusion in a relevant model of vertebrate oro-intestinal infection. PMID:22911651

Bégaud, Evelyne; Herbomel, Philippe; Levraud, Jean-Pierre; Ghigo, Jean-Marc

2012-01-01

292

Insights from Molecular Dynamics Simulations: Structural Basis for the V567D Mutation-Induced Instability of Zebrafish Alpha-Dystroglycan and Comparison with the Murine Model  

PubMed Central

A missense amino acid mutation of valine to aspartic acid in 567 position of alpha-dystroglycan (DG), identified in dag1-mutated zebrafish, results in a reduced transcription and a complete absence of the protein. Lacking experimental structural data for zebrafish DG domains, the detailed mechanism for the observed mutation-induced destabilization of the DG complex and membrane damage, remained unclear. With the aim to contribute to a better clarification of the structure-function relationships featuring the DG complex, three-dimensional structural models of wild-type and mutant (V567D) C-terminal domain of alpha-DG from zebrafish were constructed by a template-based modelling approach. We then ran extensive molecular dynamics (MD) simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability. A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish. Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein. A biochemical analysis of the murine alpha-DG mutant I591D confirmed a pronounced instability of the protein. Taken together, the computational and biochemical analysis suggest that the V567D/I591D mutation, belonging to the G beta-strand, plays a key role in inducing a destabilization of the alpha-DG C-terminal Ig-like domain that could possibly affect and propagate to the entire DG complex. The structural features herein identified may be of crucial help to understand the molecular basis of primary dystroglycanopathies. PMID:25078606

Pirolli, Davide; Sciandra, Francesca; Bozzi, Manuela; Giardina, Bruno; Brancaccio, Andrea; De Rosa, Maria Cristina

2014-01-01

293

Functional Ginger Extracts from Supercritical Fluid Carbon Dioxide Extraction via In Vitro and In Vivo Assays: Antioxidation, Antimicroorganism, and Mice Xenografts Models  

PubMed Central

Supercritical fluid carbon dioxide extraction technology was developed to gain the active components from a Taiwan native plant, Zingiber officinale (ginger). We studied the biological effects of ginger extracts via multiple assays and demonstrated the biofunctions in each platform. Investigations of ginger extracts indicated antioxidative properties in dose-dependant manners on radical scavenging activities, reducing powers and metal chelating powers. We found that ginger extracts processed moderate scavenging values, middle metal chelating levels, and slight ferric reducing powers. The antibacterial susceptibility of ginger extracts on Staphylococcus aureus, Streptococcus sobrinus, S. mutans, and Escherichia coli was determined with the broth microdilution method technique. The ginger extracts had operative antimicroorganism potentials against both Gram-positive and Gram-negative bacteria. We further discovered the strong inhibitions of ginger extracts on lethal carcinogenic melanoma through in vivo xenograft model. To sum up, the data confirmed the possible applications as medical cosmetology agents, pharmaceutical antibiotics, and food supplements. PMID:23983624

Lee, Chih-Chen; Chiou, Li-Yu; Wang, Jheng-Yang; Chou, Sin-You; Lan, John Chi-Wei; Huang, Tsi-Shu; Huang, Kuo-Chuan

2013-01-01

294

Thymoquinone overcomes chemoresistance and enhances the anticancer effects of bortezomib through abrogation of NF-?B regulated gene products in multiple myeloma xenograft mouse model  

PubMed Central

Multiple myeloma (MM) is a B cell malignancy characterized by clonal proliferation of plasma cells in the bone marrow. With the advent of novel targeted agents, the median survival rate has increased to 5?7 years. However, majority of patients with myeloma suffer relapse or develop chemoresistance to existing therapeutic agents. Thus, there is a need to develop novel alternative therapies for the treatment of MM. Thus in the present study, we investigated whether thymoquinone (TQ), a bioactive constituent of black seed oil, could suppress the proliferation and induce chemosensitization in human myeloma cells and xenograft mouse model. Our results show that TQ inhibited the proliferation of MM cells irrespective of their sensitivity to doxorubicin, melphalan or bortezomib. Interestingly, TQ treatment also resulted in a significant inhibition in the proliferation of CD138+ cells isolated from MM patient samples in a concentration dependent manner. TQ also potentiated the apoptotic effects of bortezomib in various MM cell lines through the activation of caspase-3, resulting in the cleavage of PARP. TQ treatment also inhibited chemotaxis and invasion induced by CXCL12 in MM cells. Furthermore, in a xenograft mouse model, TQ potentiated the antitumor effects of bortezomib (p < 0.05, vehicle versus bortezomib + TQ; p < 0.05, bortezomib versus bortezomib + TQ), and this correlated with modulation of various markers for survival and angiogenesis, such as Ki-67, vascular endothelial growth factor (VEGF), Bcl-2 and p65 expression. Overall, our results demonstrate that TQ can enhance the anticancer activity of bortezomib in vitro and in vivo and may have a substantial potential in the treatment of MM. PMID:24504138

Siveen, Kodappully Sivaraman; Mustafa, Nurulhuda; Li, Feng; Kannaiyan, Radhamani; Ahn, Kwang Seok; Kumar, Alan Prem; Chng, Wee-Joo; Sethi1, Gautam

2014-01-01

295

Antitumor activity of (R,R')-4-methoxy-1-naphthylfenoterol in a rat C6 glioma xenograft model in the mouse.  

PubMed

(R,R')-4-methoxy-1-naphthylfenoterol (MNF) inhibits cancer cell proliferation in vitro through cell-type specific modulation of ?2-adrenergic receptor and/or cannabinoid receptor function. Here, we report an investigation into antitumor activity of MNF in rat C6 glioma cells. The potent antiproliferative action of MNF in these cells (IC50 of ?1 nmol/L) was refractory to pharmacological inhibition of ?2-adrenergic receptor while a synthetic inverse agonist of cannabinoid receptor 1 significantly blocked MNF activity. The antitumor activity of MNF was then assessed in a C6 glioblastoma xenograft model in mice. Three days after subcutaneous implantation of C6 cells into the lower flank of nude mice, these animals were subjected to i.p. injections of saline or MNF (2 mg/kg) for 19 days and tumor volumes were measured over the course of the experiment. Gene expression analysis, quantitative RT-PCR and immunoblot assays were performed on the tumors after treatment. Significant reduction in mean tumor volumes was observed in mice receiving MNF when compared with the saline-treated group. We identified clusters in expression of genes involved in cellular proliferation, as well as molecular markers for glioblastoma that were significantly downregulated in tumors of MNF-treated mice as compared to saline-injected controls. The efficacy of MNF against C6 glioma cell proliferation in vivo and in vitro was accompanied by marked reduction in the expression of cell cycle regulator proteins. This study is the first demonstration of MNF-dependent chemoprevention of a glioblastoma xenograft model and may offer a potential mechanism for its anticancer action in vivo. PMID:25505565

Bernier, Michel; Paul, Rajib K; Dossou, Katina S S; Wnorowski, Artur; Ramamoorthy, Anuradha; Paris, Arnaud; Moaddel, Ruin; Cloix, Jean-François; Wainer, Irving W

2013-12-01

296

Lorvotuzumab mertansine, a CD56-targeting antibody-drug conjugate with potent antitumor activity against small cell lung cancer in human xenograft models.  

PubMed

Lorvotuzumab mertansine (LM) is an antibody-drug conjugate composed of a humanized anti-CD56 antibody, lorvotuzumab, linked via a cleavable disulfide linker to the tubulin-binding maytansinoid DM1. CD56 is expressed on most small cell lung cancers (SCLC), providing a promising therapeutic target for treatment of this aggressive cancer, which has a poor five-year survival rate of only 5-10%. We performed immunohistochemical staining on SCLC tumor microarrays, which confirmed that CD56 is expressed at high levels on most (~74%) SCLC tumors. Conjugation of lorvotuzumab with DM1 did not alter its specific binding to cells and LM demonstrated potent target-dependent cytotoxicity against CD56-positive SCLC cells in vitro. The anti-tumor activity of LM was evaluated against SCLC xenograft models in mice, both as monotherapy and in combination with platinum/etoposide and paclitaxel/carboplatin. Dose-dependent and antigen-specific anti-tumor activity of LM monotherapy was demonstrated at doses as low as 3 mg/kg. LM was highly active in combination with standard-of-care platinum/etoposide therapies, even in relatively resistant xenograft models. LM demonstrated outstanding anti-tumor activity in combination with carboplatin/etoposide, with superior activity over chemotherapy alone when LM was used in combinations at significantly reduced doses (6-fold below the minimally efficacious dose for LM monotherapy). The combination of LM with carboplatin/paclitaxel was also highly active. This study provides the rationale for clinical evaluation of LM as a promising novel targeted therapy for SCLC, both as monotherapy and in combination with chemotherapy. PMID:24492307

Whiteman, Kathleen R; Johnson, Holly A; Mayo, Michele F; Audette, Charlene A; Carrigan, Christina N; LaBelle, Alyssa; Zukerberg, Lawrence; Lambert, John M; Lutz, Robert J

2014-01-01

297

Thymoquinone overcomes chemoresistance and enhances the anticancer effects of bortezomib through abrogation of NF-?B regulated gene products in multiple myeloma xenograft mouse model.  

PubMed

Multiple myeloma (MM) is a B cell malignancy characterized by clonal proliferation of plasma cells in the bone marrow. With the advent of novel targeted agents, the median survival rate has increased to 5 -7 years. However, majority of patients with myeloma suffer relapse or develop chemoresistance to existing therapeutic agents. Thus, there is a need to develop novel alternative therapies for the treatment of MM. Thus in the present study, we investigated whether thymoquinone (TQ), a bioactive constituent of black seed oil, could suppress the proliferation and induce chemosensitization in human myeloma cells and xenograft mouse model. Our results show that TQ inhibited the proliferation of MM cells irrespective of their sensitivity to doxorubicin, melphalan or bortezomib. Interestingly, TQ treatment also resulted in a significant inhibition in the proliferation of CD138+ cells isolated from MM patient samples in a concentration dependent manner. TQ also potentiated the apoptotic effects of bortezomib in various MM cell lines through the activation of caspase-3, resulting in the cleavage of PARP. TQ treatment also inhibited chemotaxis and invasion induced by CXCL12 in MM cells. Furthermore, in a xenograft mouse model, TQ potentiated the antitumor effects of bortezomib (p<0.05, vehicle versus bortezomib + TQ; p<0.05, bortezomib versus bortezomib + TQ), and this correlated with modulation of various markers for survival and angiogenesis, such as Ki-67, vascular endothelial growth factor (VEGF), Bcl-2 and p65 expression. Overall, our results demonstrate that TQ can enhance the anticancer activity of bortezomib in vitro and in vivo and may have a substantial potential in the treatment of MM. PMID:24504138

Siveen, Kodappully Sivaraman; Mustafa, Nurulhuda; Li, Feng; Kannaiyan, Radhamani; Ahn, Kwang Seok; Kumar, Alan Prem; Chng, Wee-Joo; Sethi, Gautam

2014-02-15

298

Anti-CD45 Pretargeted Radioimmunotherapy using Bismuth-213: High Rates of Complete Remission and Long-Term Survival in a Mouse Myeloid Leukemia Xenograft Model  

SciTech Connect

Pretargeted radioimmunotherapy (PRIT) using an anti-CD45 antibody (Ab)-streptavidin (SA) conjugate and DOTA-biotin labeled with beta-emitting radionuclides has been explored as a strategy to decrease relapse and toxicity. Alpha-emitting radionuclides exhibit high cytotoxicity coupled with a short path-length, potentially increasing the therapeutic index and making them an attractive alternative to beta-emitting radionuclides for patients with Acute Myeloid Leukemia (AML). Accordingly, we have used 213Bi in mice with human leukemia xenografts. Results demonstrated excellent localization of 213Bi-DOTA-biotin to tumors with minimal uptake into normal organs. After 10 minutes, 4.5 ± 1.1% of the injected dose of 213Bi was delivered per gram of tumor. Alpha imaging demonstrated uniform radionuclide distribution within tumor tissue 45 minutes after 213Bi-DOTA-biotin injection. Radiation absorbed doses were similar to those observed using a beta-emitting radionuclide (90Y) in the same model. We conducted therapy experiments in a xenograft model using a single-dose of 213Bi-DOTA-biotin given 24 hours after anti-CD45 Ab-SA conjugate. Among mice treated with anti-CD45 Ab-SA conjugate followed by 800 ?Ci of 213Bi- or 90Y-DOTA-biotin, 80% and 20%, respectively, survived leukemia-free for >100 days with minimal toxicity. These data suggest that anti-CD45 PRIT using an alpha-emitting radionuclide may be highly effective and minimally toxic for treatment of AML.

Pagel, John M.; Kenoyer, Aimee L.; Back, Tom; Hamlin, Donald K.; Wilbur, D. Scott; Fisher, Darrell R.; Park, Steven I.; Frayo, Shani; Axtman, Amanda; Orgun, Nural; Orozoco, Johnnie; Shenoi, Jaideep; Lin, Yukang; Gopal, Ajay K.; Green, Damian J.; Appelbaum, Frederick R.; Press, Oliver W.

2011-07-21

299

A Small Molecule Inhibitor of ETV1, YK-4-279, Prevents Prostate Cancer Growth and Metastasis in a Mouse Xenograft Model  

PubMed Central

Background The erythroblastosis virus E26 transforming sequences (ETS) family of transcription factors consists of a highly conserved group of genes that play important roles in cellular proliferation, differentiation, migration and invasion. Chromosomal translocations fusing ETS factors to promoters of androgen responsive genes have been found in prostate cancers, including the most clinically aggressive forms. ERG and ETV1 are the most commonly translocated ETS proteins. Over-expression of these proteins in prostate cancer cells results in a more invasive phenotype. Inhibition of ETS activity by small molecule inhibitors may provide a novel method for the treatment of prostate cancer. Methods and Findings We recently demonstrated that the small molecule YK-4-279 inhibits biological activity of ETV1 in fusion-positive prostate cancer cells leading to decreased motility and invasion in-vitro. Here, we present data from an in-vivo mouse xenograft model. SCID-beige mice were subcutaneously implanted with fusion-positive LNCaP-luc-M6 and fusion-negative PC-3M-luc-C6 tumors. Animals were treated with YK-4-279, and its effects on primary tumor growth and lung metastasis were evaluated. YK-4-279 treatment resulted in decreased growth of the primary tumor only in LNCaP-luc-M6 cohort. When primary tumors were grown to comparable sizes, YK-4-279 inhibited tumor metastasis to the lungs. Expression of ETV1 target genes MMP7, FKBP10 and GLYATL2 were reduced in YK-4-279 treated animals. ETS fusion-negative PC-3M-luc-C6 xenografts were unresponsive to the compound. Furthermore, YK-4-279 is a chiral molecule that exists as a racemic mixture of R and S enantiomers. We established that (S)-YK-4-279 is the active enantiomer in prostate cancer cells. Conclusion Our results demonstrate that YK-4-279 is a potent inhibitor of ETV1 and inhibits both the primary tumor growth and metastasis of fusion positive prostate cancer xenografts. Therefore, YK-4-279 or similar compounds may be evaluated as a potential therapeutic tool for treatment of human prostate cancer at different stages. PMID:25479232

Rahim, Said; Minas, Tsion; Hong, Sung-Hyeok; Justvig, Sarah; Çelik, Haydar; Kont, Yasemin Saygideger; Han, Jenny; Kallarakal, Abraham T.; Kong, Yali; Rudek, Michelle A.; Brown, Milton L.; Kallakury, Bhaskar; Toretsky, Jeffrey A.; Üren, Aykut

2014-01-01

300

Role of Chd7 in Zebrafish: A Model for CHARGE Syndrome  

PubMed Central

CHARGE syndrome is caused by mutations in the CHD7 gene. Several organ systems including the retina, cranial nerves, inner ear and heart are affected in CHARGE syndrome. However, the mechanistic link between mutations in CHD7 and many of the organ systems dysfunction remains elusive. Here, we show that Chd7 is required for the organization of the neural retina in zebrafish. We observe an abnormal expression or a complete absence of molecular markers for the retinal ganglion cells and photoreceptors, indicating that Chd7 regulates the differentiation of retinal cells and plays an essential role in retinal cell development. In addition, zebrafish with reduced Chd7 display an abnormal organization and clustering of cranial motor neurons. We also note a pronounced reduction in the facial branchiomotor neurons and the vagal motor neurons display aberrant positioning. Further, these fish exhibit a severe loss of the facial nerves. Knock-down of Chd7 results in a curvature of the long body axis and these fish develop irregular shaped vertebrae and have a reduction in bone mineralization. Chd7 knockdown also results in a loss of proper segment polarity illustrated by flawed efnb2a and ttna expression, which is associated with later vascular segmentation defects. These critical roles for Chd7 in retinal and vertebral development were previously unrecognized and our results provide new insights into the role of Chd7 during development and in CHARGE syndrome pathogenesis. PMID:22363697

Zaouter, Charlotte; Drapeau, Pierre; Albertson, R. Craig; Moldovan, Florina

2012-01-01

301

Noncell-autonomous photoreceptor degeneration in a zebrafish model of choroideremia.  

PubMed

Choroideremia is an X-linked hereditary retinal degeneration resulting from mutations in the Rab escort protein-1 (REP1). The Rep1 protein facilitates posttranslational modification of Rab proteins, which regulate intracellular trafficking in the retinal pigment epithelium (RPE) and photoreceptors and are likely involved in the removal of outer segment disk membranes by the RPE. A critical question for potential treatment of choroideremia is whether photoreceptor degeneration results from autonomous defects in opsin transport within the photoreceptor or as a nonautonomous and secondary consequence of RPE degeneration. To address this question, we have characterized the retinal pathology in zebrafish rep1 mutants, which carry a recessive nonsense mutation in the REP1 gene. Zebrafish rep1 mutants exhibit degeneration of the RPE and photoreceptors and complete loss of visual function as measured by electroretinograms. In the mutant RPE, photoreceptor outer segment material was not effectively eliminated, and large vacuoles were observed. However, opsin trafficking in photoreceptors occurred normally. Mosaic analysis revealed that photoreceptor degeneration was nonautonomous and required contact with the mutant RPE as mutant photoreceptors were rescued in wild-type hosts and wild-type photoreceptors degenerated in mutant hosts. We conclude that mutations in REP1 disrupt cellular processes in the RPE, which causes photoreceptor death as a secondary consequence. These results suggest that therapies that correct the RPE may successfully rescue photoreceptor loss in choroideremia. PMID:17360570

Krock, Bryan L; Bilotta, Joseph; Perkins, Brian D

2007-03-13

302

Skeletal myogenesis in the zebrafish and its implications for muscle disease modelling.  

PubMed

Current evidence indicates that post-embryonic muscle growth and regeneration in amniotes is mediated almost entirely by stem cells derived from muscle progenitor cells (MPCs), known as satellite cells. Exhaustion and impairment of satellite cell activity is involved in the severe muscle loss associated with degenerative muscle diseases such as Muscular Dystrophies and is the main cause of age-associated muscle wasting. Understanding the molecular and cellular basis of satellite cell function in muscle generation and regeneration (myogenesis) is critical to the broader goal of developing treatments that may ameliorate such conditions.Considerable knowledge exists regarding the embryonic stages of amniote myogenesis. Much less is known about how post-embryonic amniote myogenesis proceeds, how adult myogenesis relates to embryonic myogenesis on a cellular or genetic level. Of the studies focusing on post-embryonic amniote myogenesis, most are post-mortem and in vitro analyses, precluding the understanding of cellular behaviours and genetic mechanisms in an undisturbed in vivo setting. Zebrafish are optically clear throughout much of their post-embryonic development, facilitating their use in live imaging of cellular processes. Zebrafish also possess a compartment of MPCs, which appear similar to satellite cells and persist throughout the post-embryonic development of the fish, permitting their use in examining the contribution of these cells to muscle tissue growth and regeneration. PMID:25344666

Gurevich, David; Siegel, Ashley; Currie, Peter D

2015-01-01

303

Antibody Directed against Human YKL-40 Increases Tumor Volume in a Human Melanoma Xenograft Model in Scid Mice  

PubMed Central

Induced overexpression of the secretory protein YKL-40 promotes tumor growth in xenograft experiments. We investigated if targeting YKL-40 with a monoclonal antibody could inhibit tumor growth. YKL-40 expressing human melanoma cells (LOX) were injected subcutenously in Balb/c scid mice. Animals were treated with intraperitoneal injections of anti-YKL-40, isoptype control or PBS. Non-YKL-40 expressing human pancreatic carcinoma cell line PaCa 5061 served as additional control. MR imaging was used for evaluation of tumor growth. Two days after the first injections of anti-YKL-40, tumor volume had increased significantly compared with controls, whereas no effects were observed for control tumors from PaCa 5061 cells lacking YKL-40 expression. After 18 days, mean tumor size of the mice receiving repeated anti-YKL-40 injections was 1.82 g, >4 times higher than mean tumor size of the controls (0.42 g). The effect of anti-YKL-40 on the increase of tumor volume started within hours after injection and was dose dependent. Intratumoral hemorrhage was observed in the treated animals. The strong effect on tumor size indicates important roles for YKL-40 in melanoma growth and argues for a careful evaluation of antibody therapy directed against YKL-40. PMID:24752554

Salamon, Johannes; Hoffmann, Tatjana; Elies, Eva; Peldschus, Kersten; Johansen, Julia S.; Lüers, Georg; Schumacher, Udo; Wicklein, Daniel

2014-01-01

304

Thymoquinone Inhibits Tumor Growth and Induces Apoptosis in a Breast Cancer Xenograft Mouse Model: The Role of p38 MAPK and ROS  

PubMed Central

Due to narrow therapeutic window of cancer therapeutic agents and the development of resistance against these agents, there is a need to discover novel agents to treat breast cancer. The antitumor activities of thymoquinone (TQ), a compound isolated from Nigella sativa oil, were investigated in breast carcinoma in vitro and in vivo. Cell responses after TQ treatment were assessed by using different assays including MTT assay, annexin V-propidium iodide staining, Mitosox staining and Western blot. The antitumor effect was studied by breast tumor xenograft mouse model, and the tumor tissues were examined by histology and immunohistochemistry. The level of anti-oxidant enzymes/molecules in mouse liver tissues was measured by commercial kits. Here, we show that TQ induced p38 phosphorylation and ROS production in breast cancer cells. These inductions were found to be responsible for TQ’s anti-proliferative and pro-apoptotic effects. Moreover, TQ-induced ROS production regulated p38 phosphorylation but not vice versa. TQ treatment was found to suppress the tumor growth and this effect was further enhanced by combination with doxorubicin. TQ also inhibited the protein expression of anti-apoptotic genes, such as XIAP, survivin, Bcl-xL and Bcl-2, in breast cancer cells and breast tumor xenograft. Reduced Ki67 and increased TUNEL staining were observed in TQ-treated tumors. TQ was also found to increase the level of catalase, superoxide dismutase and glutathione in mouse liver tissues. Overall, our results demonstrated that the anti-proliferative and pro-apoptotic effects of TQ in breast cancer are mediated through p38 phosphorylation via ROS generation. PMID:24098377

Woo, Chern Chiuh; Hsu, Annie; Kumar, Alan Prem; Sethi, Gautam; Tan, Kwong Huat Benny

2013-01-01

305

Toxicity to embryo and adult zebrafish of copper complexes with two malonic acids as models for dissolved organic matter  

SciTech Connect

The toxicity to embryo and adult zebrafish (Danio rerio) of Cu complexes with two substituted malonic acids, benzyl- and n-hexadecyl-, chosen as models for low-molecular-weight natural dissolved organic matter, were investigated. Toxicity test solutions at pH 6.5 {+-} 0.1 with the required Cu ion-specific electrode. In the absence of malonic acids, concentrations of Cu{sup 2+} up to 1.13 {mu}mol/L increased the embryo hatching time from approx. 2 d in control solutions (no Cu or malonic acid) and solutions containing malonic acids without Cu to approx. 8 d. The Cu-benzylmalonic acid complex in the presence of inorganic Cu species did not delay hatching beyond that attributable to Cu{sup 2+}. In contrast, 0.60 {mu}mol/L Cu-n-hexadecylmalonic complexes delayed hatching by 5.5 d in excess of that attributable to 1.13 {mu}mol/L Cu{sup 2+}, assuming that the hatching delays caused by the different Cu species were additive, possibly because of Cu entry into the embryo as the lipophilic Cu-n-hexadecylmalonic complex. None of the Cu-malonic acid complexes was acutely toxic to adult zebrafish at concentrations up to 1.4 {mu}mol/L, possibly because Cu was removed from the Cu-malonic acid complexes by stronger chelating groups at the gill surface. Substituted malonic acids with similar proton and Cu association constants can be readily prepared with a variety of simple substituents, radiolabeled if required. Their results show that these acids could be useful ligands for investigating intracellular transport and metabolism of metal-organic complexes.

Palmer, F.B.; Evans, C.W. [Univ. of Auckland (New Zealand); Butler, C.A. [Woodward Clyde NZ Ltd., Auckland (New Zealand); Timperley, M.H. [National Inst. of Water and Atmospheric Research, Auckland (New Zealand)

1998-08-01

306

Utility of 3’-[18F]fluoro-3’-deoxythymidine as a PET tracer to monitor response to gene therapy in a xenograft model of head and neck carcinoma  

PubMed Central

Noninvasive imaging methodologies are needed to assess treatment responses to novel molecular targeting approaches for the treatment of squamous cell carcinoma of the head and neck (SCCHN). Computer tomography and magnetic resonance imaging do not effectively distinguish tumors from fibrotic tissue commonly associated with SCCHN tumors. Positron emission tomography (PET) offers functional non-invasive imaging of tumors. We determined the uptake of the PET tracers 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and 3’-[18F]Fluoro-3’-deoxythymidine ([18F]FLT) in several SCCHN xenograft models. In addition, we evaluated the utility of [18F]FLT microPET imaging in monitoring treatment response to an EGFR antisense approach targeted therapy that has shown safety and efficacy in a phase I trial. Two of the 3 SCCHN xenograft models tested demonstrated no appreciable uptake or retention of [18F]FDG, but consistent accumulation of [18F]FLT. The third tumor xenograft SCCHN model (Cal33) demonstrated variable uptake of both tracers. SCCHN xenografts (1483) treated with EGFR antisense gene therapy decreased tumor volumes in 4/6 mice. Reduced uptake of [18F]FLT was observed in tumors that responded to epidermal growth factor antisense (EGFRAS) gene therapy compared to non-responding tumors or tumors treated with control sense plasmid DNA. These findings indicate that [18F]FLT PET imaging may be useful in monitoring SCCHN response to molecular targeted therapies, while [18F]FDG uptake in SCCHN xenografts may not be reflective of the level of metabolic activity characteristic of human SCCHN tumors. PMID:23342298

Mason, Neale S; Lopresti, Brian J; Ruszkiewicz, James; Dong, Xinxin; Joyce, Sonali; Leef, George; Sen, Malabika; Wahed, Abdus S; Mathis, Chester A; Grandis, Jennifer R; Thomas, Sufi M

2013-01-01

307

Marked therapeutic efficacy of a novel polyethylene glycol-SN38 conjugate, EZN-2208, in xenograft models of B-cell non-Hodgkin’s lymphoma  

PubMed Central

Examination of the clinical utility of SN38 (10-hydroxy-7-ethyl-camptothecin), the active metabolite of CPT-11, has not been possible to date due to poor solubility of SN38. Here we evaluated the activity of EZN-2208, a water-soluble polyethyleneglycol-SN38 conjugate, in pre-clinical models of Burkitt’s non-Hodgkin’s lymphoma (NHL) (Raji and Daudi), and follicular NHL (DoHH2). In vitro, the IC50 of EZN-2208 ranged from 3–24 nM, which was 30- to 45-fold lower than CPT-11 or 2.5- to 3.5-fold higher than SN38. In both an early-disease Raji model and an advanced-disease Daudi model, treatment with multiple doses of EZN-2208 resulted in 90% and 100% cures of animals, respectively (cure defined as no sign of tumors by gross observations at the termination of study). The activity of EZN-2208 was dramatically superior to that of CPT-11 in all three models. The excellent therapeutic efficacy of EZN-2208 in several B-cell NHL xenograft models merits its evaluation in the clinic for lymphoid malignancies. PMID:19794091

Sapra, Puja; Kraft, Patricia; Mehlig, Mary; Malaby, Jennifer; Zhao, Hong; Greenberger, Lee M.; Horak, Ivan D.

2009-01-01

308

Marked therapeutic efficacy of a novel polyethylene glycol-SN38 conjugate, EZN-2208, in xenograft models of B-cell non-Hodgkin's lymphoma.  

PubMed

Examination of the clinical utility of SN38 (10-hydroxy-7-ethyl-camptothecin), the active metabolite of CPT-11, has not been possible to date due to poor solubility of SN38. Here we evaluated the activity of EZN-2208, a water-soluble polyethyleneglycol-SN38 conjugate, in pre-clinical models of Burkitt's non-Hodgkin's lymphoma (NHL) (Raji and Daudi), and follicular NHL (DoHH2). In vitro, the IC50 of EZN-2208 ranged from 3-24 nM, which was 30- to 45-fold lower than CPT-11 or 2.5- to 3.5-fold higher than SN38. In both an early-disease Raji model and an advanced-disease Daudi model, treatment with multiple doses of EZN-2208 resulted in 90% and 100% cures of animals, respectively (cure defined as no sign of tumors by gross observations at the termination of study). The activity of EZN-2208 was dramatically superior to that of CPT-11 in all three models. The excellent therapeutic efficacy of EZN-2208 in several B-cell NHL xenograft models merits its evaluation in the clinic for lymphoid malignancies. PMID:19794091

Sapra, Puja; Kraft, Patricia; Mehlig, Mary; Malaby, Jennifer; Zhao, Hong; Greenberger, Lee M; Horak, Ivan D

2009-10-01

309

Irradiation-Dependent Effects on Tumor Perfusion and Endogenous and Exogenous Hypoxia Markers in an A549 Xenograft Model  

SciTech Connect

Purpose: Hypoxia is a major determinant of tumor radiosensitivity, and microenvironmental changes in response to ionizing radiation (IR) are often heterogenous. We analyzed IR-dependent changes in hypoxia and perfusion in A549 human lung adenocarcinoma xenografts. Materials and Methods: Immunohistological analysis of two exogenously added chemical hypoxic markers, pimonidazole and CCI-103F, and of the endogenous marker Glut-1 was performed time dependently after IR. Tumor vessels and apoptosis were analyzed using CD31 and caspase-3 antibodies. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and fluorescent beads (Hoechst 33342) were used to monitor vascular perfusion. Results: CCI-103F signals measuring the fraction of hypoxic areas after IR were significantly decreased by approximately 50% when compared with pimonidazole signals, representing the fraction of hypoxic areas from the same tumors before IR. Interestingly, Glut-1 signals were significantly decreased at early time point (6.5 h) after IR returning to the initial levels at 30.5 h. Vascular density showed no difference between irradiated and control groups, whereas apoptosis was significantly induced at 10.5 h post-IR. DCE-MRI indicated increased perfusion 1 h post-IR. Conclusions: The discrepancy between the hypoxic fractions of CCI-103F and Glut-1 forces us to consider the possibility that both markers reflect different metabolic alterations of tumor microenvironment. The reliability of endogenous markers such as Glut-1 to measure reoxygenation in irradiated tumors needs further consideration. Monitoring tumor microvascular response to IR by DCE-MRI and measuring tumor volume alterations should be encouraged.

Fokas, Emmanouil, E-mail: emmanouil.fokas@yahoo.d [Department of Radiotherapy and Radiation Oncology, Medical Faculty of Philipps University, Marburg (Germany); Haenze, Joerg [Department of Internal Medicine, Justus Liebig University, Giessen (Germany); Kamlah, Florentine [Department of Radiotherapy and Radiation Oncology, Medical Faculty of Philipps University, Marburg (Germany); Eul, Bastian G.; Lang, Nico [Department of Internal Medicine, Justus Liebig University, Giessen (Germany); Keil, Boris; Heverhagen, Johannes T. [Department of Radiology, Medical Faculty of Philipps University, Marburg (Germany); Engenhart-Cabillic, Rita; An Hanxiang [Department of Radiotherapy and Radiation Oncology, Medical Faculty of Philipps University, Marburg (Germany); Rose, Frank, E-mail: rosef@med.uni-marburg.d [Department of Radiotherapy and Radiation Oncology, Medical Faculty of Philipps University, Marburg (Germany)

2010-08-01

310

5?-Reductase Inhibition Suppresses Testosterone-Induced Initial Regrowth of Regressed Xenograft Prostate Tumors in Animal Models  

PubMed Central

Androgen deprivation therapy (ADT) is the standard treatment for patients with prostate-specific antigen progression after treatment for localized prostate cancer. An alternative to continuous ADT is intermittent ADT (IADT), which allows recovery of testosterone during off-cycles to stimulate regrowth and differentiation of the regressed prostate tumor. IADT offers patients a reduction in side effects associated with ADT, improved quality of life, and reduced cost with no difference in overall survival. Our previous studies showed that IADT coupled with 5?-reductase inhibitor (5ARI), which blocks testosterone conversion to DHT could prolong survival of animals bearing androgen-sensitive prostate tumors when off-cycle duration was fixed. To further investigate this clinically relevant observation, we measured the time course of testosterone-induced regrowth of regressed LuCaP35 and LNCaP xenograft tumors in the presence or absence of a 5ARI. 5?-Reductase inhibitors suppressed the initial regrowth of regressed prostate tumors. However, tumors resumed growth and were no longer responsive to 5?-reductase inhibition several days after testosterone replacement. This finding was substantiated by bromodeoxyuridine and Ki67 staining of LuCaP35 tumors, which showed inhibition of prostate tumor cell proliferation by 5ARI on day 2, but not day 14, after testosterone replacement. 5?-Reductase inhibitors also suppressed testosterone-stimulated proliferation of LNCaP cells precultured in androgen-free media, suggesting that blocking testosterone conversion to DHT can inhibit prostate tumor cell proliferation via an intracrine mechanism. These results suggest that short off-cycle coupled with 5?-reductase inhibition could maximize suppression of prostate tumor growth and, thus, improve potential survival benefit achieved in combination with IADT. PMID:23671262

Masoodi, Khalid Z.; Ramos Garcia, Raquel; Pascal, Laura E.; Wang, Yujuan; Ma, Hei M.; O'Malley, Katherine; Eisermann, Kurtis; Shevrin, Daniel H.; Nguyen, Holly M.; Vessella, Robert L.; Nelson, Joel B.; Parikh, Rahul A.

2013-01-01

311

Meta-[{sup 211}At]astatobenzylguanidine (MABG): In vivo evaluation in an athymic mouse human neuroblastoma xenograft model  

SciTech Connect

Because of the short range and high linear energy transfer of {sup 211}At {alpha}-particles, the MIBG analogue MABG might be useful for the therapy of micrometastatic neuroblastoma and previous in vitro studies have demonstrated that under single-cell conditions, the cytotoxicity of MABG is > 1000 times higher than [{sup 131}I]MIBG. A paired label protocol was used to compare the tissue distribution of MABG and [{sup 131}I]MIBG in athymic mice bearing subcutaneous SK-N-SH human neuroblastoma xenografts from 1-24 hr after injection. In tumor, significantly higher (p < 0.05) uptake was observed for MABG (3.8 {plus_minus} 0.8%ID/g vs 3.1 {plus_minus} 0.7%ID/g at 8 hr). Pretreatment with desipramine reduced tumor uptake of MABG by 43%, suggesting that accumulation was related to the uptake-1 mechanism. Significantly higher uptake of MABG also was observed in normal tissue targets. For example, at 8 hr, heart uptake of MABG was 6.0 {plus_minus} 0.9 % ID/g compared with 4.5 {plus_minus} 0.8%ID/g for [{sup 131}I]MIBG. Two strategies were investigated to increase the tumor-to-hear uptake ratio. Pretreatment of mice with unlabeled MIBG (4 mg/kg) increased MABG tumor uptake by 1.5-fold while reducing uptake in several normal tissues including heart. The vesicular uptake blocker tetrabenazine (TBZ; 20 mg/kg), reduced MABG hear uptake by 30% of control values with not significant decrease in tumor levels. We conclude that MABG deserves further evaluation as a potential agent for the treatment of neuroblastoma, particularly in combination with strategies to minimize radiation dose to normal target tissues.

Vaidyanathan, G.; Friedman, H.S.; Keir, S.T. [Duke Univ. Medical Center, Durhamk, NC (United States)] [and others

1996-05-01

312

Characterization and assessment of the sensitivity and resistance of a newly established human gastrointestinal stromal tumour xenograft model to treatment with tyrosine kinase inhibitors  

PubMed Central

Background Acquired resistance to tyrosine kinase inhibitors (TKIs) in gastrointestinal stromal tumours (GISTs) is most commonly caused by secondary KIT or PDGFRA mutations. In this study we characterize a newly established GIST xenograft model, UZLX-GIST9, and evaluate the in vivo response of the model to standard TKIs (imatinib, sunitinib, and regorafenib). Methods Tumour fragments from a metastatic lesion of a GIST patient clinically progressing after treatment with imatinib, sunitinib and regorafenib were engrafted in a nude, immunodeficient mouse. Upon sequential passaging from mouse to mouse, tumour fragments were collected for histopathological and molecular characterization. The sensitivity of the model to treatment with TKIs was evaluated in 28 mice [passage 2 (n?=?8), passage 4 (n?=?20), 41 tumours]. Mice were grouped as follows: control (untreated), imatinib (50 mg/kg/BID), imatinib (100 mg/kg/BID), sunitinib (40 mg/kg/QD), and regorafenib (30 mg/kg/QD). After three weeks of oral treatment, tumours were collected for subsequent analysis. The efficacy of treatment was assessed by tumour volume, histopathology and Western immunoblotting. Results UZLX-GIST9 maintains the same typical morphological features and immunohistochemical characteristics as the original patient biopsy and expresses CD117 and DOG1. The KIT mutational profile (p.P577del + W557LfsX5+ D820G) remains the same as the original tissue sample originating from an intraspinal metastatic site. Three week treatment with different TKIs showed that the model is resistant to imatinib. Sunitinib induces tumour growth delay and regorafenib reduces the tumour burden by 30% as compared to control animals. While none of the TKIs had a significant effect on cell proliferation or cell survival, a remarkable increase of necrosis and significant reduction of microvessel density was observed under sunitinib and regorafenib. Western immunoblotting showed a mild reduction in KIT and AKT activation only in regorafenib treated tumours. Conclusions We established a novel human GIST xenograft, UZLX-GIST9, harbouring KIT exon 11 and 17 mutations and maintaining the pheno-and genotype of the original tumour. UZLX-GIST9 shows different levels of response to standard TKIs. This model will help to study TKI resistance and to explore novel treatment approaches for patients with TKI-resistant GIST. PMID:25132955

2014-01-01

313

Current Status of Sperm Cryopreservation in Biomedical Research Fish Models: Zebrafish, Medaka, and Xiphophorus*  

PubMed Central

Aquarium fishes are becoming increasingly important because of their value in biomedical research and the ornamental fish trade, and because many have become threatened or endangered in the wild. This review summarizes the current status of sperm cryopreservation in three fishes widely used in biomedical research: zebrafish, medaka, and live-bearing fishes of the genus Xiphophorus, and will focus on the needs and opportunities for future research and application of cryopreservation in aquarium fish. First, we summarize the basic biological characteristics regarding natural habitat, testis structure, spermatogenesis, sperm morphology, and sperm physiology. Second, we compare protocol development of sperm cryopreservation. Third, we emphasize the importance of artificial fertilization in sperm cryopreservation to evaluate the viability of thawed sperm. We conclude with a look to future research directions for sperm cryopreservation and the application of this technique in aquarium species. PMID:18691673

Yang, Huiping; Tiersch, Terrence R.

2009-01-01

314

A zebrafish model of glucocorticoid resistance shows serotonergic modulation of the stress response  

PubMed Central

One function of glucocorticoids is to restore homeostasis after an acute stress response by providing negative feedback to stress circuits in the brain. Loss of this negative feedback leads to elevated physiological stress and may contribute to depression, anxiety, and post-traumatic stress disorder. We investigated the early, developmental effects of glucocorticoid signaling deficits on stress physiology and related behaviors using a mutant zebrafish, grs357, with non-functional glucocorticoid receptors (GRs). These mutants are morphologically inconspicuous and adult-viable. A previous study of adult grs357 mutants showed loss of glucocorticoid-mediated negative feedback and elevated physiological and behavioral stress markers. Already at 5 days post-fertilization, mutant larvae had elevated whole body cortisol, increased expression of pro-opiomelanocortin (POMC), the precursor of adrenocorticotropic hormone (ACTH), and failed to show normal suppression of stress markers after dexamethasone treatment. Mutant larvae had larger auditory-evoked startle responses compared to wildtype sibling controls (grwt), despite having lower spontaneous activity levels. Fluoxetine (Prozac) treatment in mutants decreased startle responding and increased spontaneous activity, making them behaviorally similar to wildtype. This result mirrors known effects of selective serotonin reuptake inhibitors (SSRIs) in modifying glucocorticoid signaling and alleviating stress disorders in human patients. Our results suggest that larval grs357 zebrafish can be used to study behavioral, physiological, and molecular aspects of stress disorders. Most importantly, interactions between glucocorticoid and serotonin signaling appear to be highly conserved among vertebrates, suggesting deep homologies at the neural circuit level and opening up new avenues for research into psychiatric conditions. PMID:23087630

Griffiths, Brian B.; Schoonheim, Peter J.; Ziv, Limor; Voelker, Lisa; Baier, Herwig; Gahtan, Ethan

2012-01-01

315

Defects of protein production in erythroid cells revealed in a zebrafish Diamond–Blackfan anemia model for mutation in RPS19  

PubMed Central

Diamond–Blackfan anemia (DBA) is a rare congenital red cell aplasia that classically presents during early infancy in DBA patients. Approximately, 25% of patients carry a mutation in the ribosomal protein (RP) S19 gene; mutations in RPS24, RPS17, RPL35A, RPL11, and RPL5 have been reported. How ribosome protein deficiency causes defects specifically to red blood cells in DBA has not been well elucidated. To genetically model the predominant ribosome defect in DBA, we generated an rps19 null mutant through the use of TALEN-mediated gene targeting in zebrafish. Molecular characterization of this mutant line demonstrated that rps19 deficiency reproduced the erythroid defects of DBA, including a lack of mature red blood cells and p53 activation. Notably, we found that rps19 mutants' production of globin proteins was significantly inhibited; however, globin transcript level was either increased or unaffected in rps19 mutant embryos. This dissociation of RNA/protein levels of globin genes was confirmed in another zebrafish DBA model with defects in rpl11. Using transgenic zebrafish with specific expression of mCherry in erythroid cells, we showed that protein production in erythroid cells was decreased when either rps19 or rpl11 was mutated. L-Leucine treatment alleviated the defects of protein production in erythroid cells and partially rescued the anemic phenotype in both rps19 and rpl11 mutants. Analysis of this model suggests that the decreased protein production in erythroid cells likely contributes to the blood-specific phenotype of DBA. Furthermore, the newly generated rps19 zebrafish mutant should serve as a useful animal model to study DBA. Our in vivo findings may provide clues for the future therapy strategy for DBA. PMID:25058426

Zhang, Y; Ear, J; Yang, Z; Morimoto, K; Zhang, B; Lin, S

2014-01-01

316

Molecular control of neurogenesis in the regenerating central nervous system of the adult zebrafish   

E-print Network

In contrast to mammals, adult zebrafish display cellular regeneration of lost motor neurons and achieve functional recovery following a complete spinal cord transection. Using adult zebrafish as a model to study how key ...

Dias, Tatyana Beverly

2012-11-30

317

A Novel 99mTc-Labeled Molecular Probe for Tumor Angiogenesis Imaging in Hepatoma Xenografts Model: A Pilot Study  

PubMed Central

Introduction Visualization of tumor angiogenesis using radionuclide targeting provides important diagnostic information. In previous study, we proved that an arginine-arginine-leucine (RRL) peptide should be a tumor endothelial cell specific binding sequence. The overall aim of this study was to evaluate whether 99mTc-radiolabeled RRL could be noninvasively used for imaging of malignant tumors in vivo, and act as a new molecular probe targeting tumor angiogenesis. Methods The RRL peptide was designed and radiosynthesized with 99mTc by a one-step method. The radiolabeling efficiency and radiochemical purity were then characterized in vitro. 99mTc-RRL was injected intravenously in HepG2 xenograft-bearing BALB/c nude mice. Biodistribution and in vivo imaging were performed periodically. The relationship between tumor size and %ID uptake of 99mTc-RRL was also explored. Results The labeling efficiencies of 99mTc-RRL reached 76.9%±4.5% (n?=?6) within 30–60 min at room temperature, and the radiochemical purity exceeded 96% after purification. In vitro stability experiment revealed the radiolabeled peptide was stable. Biodistribution data showed that 99mTc-RRL rapidly cleared from the blood and predominantly accumulated in the kidneys and tumor. The specific uptake of 99mTc-RRL in tumor was significantly higher than that of unlabeled RRL blocking and free pertechnetate control test after injection (p<0.05). The ratio of the tumor-to-muscle exceeded 6.5, tumor-to-liver reached 1.98 and tumor-to-blood reached 1.95. In planar gamma imaging study, the tumors were imaged clearly at 2–6 h after injection of 99mTc-RRL, whereas the tumor was not imaged clearly in blocking group. The tumor-to-muscle ratio of images with 99mTc-RRL was comparable with that of 18F-FDG PET images. Immunohistochemical analysis verified the excessive vasculature of tumor. There was a linear relationship between the tumor size and uptake of 99mTc-RRL with R2?=?0.821. Conclusion 99mTc-RRL can be used as a potential candidate for visualization of tumor angiogenesis in malignant carcinomas. PMID:23573294

Zhao, Qian; Yan, Ping; Wang, Rong Fu; Zhang, Chun Li; Li, Ling; Yin, Lei

2013-01-01

318

Effect of tumour necrosis factor on the uptake of specific and control monoclonal antibodies in a human tumour xenograft model.  

PubMed Central

The investigations reported in this paper aim to exploit tumour necrosis factor (TNF)-induced vascular changes in an attempt to increase the tumour uptake of specific monoclonal antibody. The vascular permeability to monoclonal antibody of a human tumour xenograft increased 2.6-fold by 1 h post injection of 2.5 x 10(3) U of TNF, although this effect was lost by 3 h. The normal tissues also demonstrated increased vascular permeability to IgG, but to a lesser extent. Liver permeability increased 1.5-fold at 1 h but returned to the control value by 6 h. Lung permeability increased 1.4-fold at 1 h post injection and returned to normal by 3 h. Muscle values were not significantly increased compared with controls. The blood activity was cleared more quickly in the TNF-treated mice (t1/2 beta = 101 h, compared with 121 h in control mice). This was probably due to the increased vascular permeability in normal organs of treated mice. At 1 day and 3 days post injection, the tumour uptake of the specific, but not the control, antibody was significantly increased by 25% and 29% respectively. This resulted in an increase in the area under the tumour activity curve, and therefore tumour radiation dose, of 25% in treated compared with control mice. In addition, a consequence of the faster blood clearance of the isotope in the TNF-treated mice was a reduction in the area under the blood activity curve of 12%, thereby reducing systemic toxicity. The increase in vascular permeability to IgG following TNF injection resulted in both specific and control antibodies having improved access to the tumour antigens, and a transient increase in uptake was observed. Only in the case of the specific antibody was the increase maintained, since this antibody binds to the available antigenic sites, whereas the control antibody was cleared from the tumour without binding. No evidence of tumour necrosis was observed at the TNF doses given, nor was there any toxicity to the mice. PMID:7710925

Rowlinson-Busza, G.; Maraveyas, A.; Epenetos, A. A.

1995-01-01

319

A novel zebrafish model of hyperthermia-induced seizures reveals a role for TRPV4 channels and NMDA-type glutamate receptors  

PubMed Central

Febrile seizures are the most common seizure type in children under the age of five, but mechanisms underlying seizure generation in vivo remain unclear. Animal models to address this issue primarily focus on immature rodents heated indirectly using a controlled water bath or air blower. Here we describe an in vivo model of hyperthermia-induced seizures in larval zebrafish aged 3 to 7 days post-fertilization (dpf). Bath controlled changes in temperature are rapid and reversible in this model. Acute electrographic seizures following transient hyperthermia showed age-dependence, strain independence, and absence of mortality. Electrographic seizures recorded in the larval zebrafish forebrain were blocked by adding antagonists to the transient receptor potential vanilloid (TRPV4) channel or N-methyl-d-aspartate (NMDA) glutamate receptor to the bathing medium. Application of GABA, GABA re-uptake inhibitors, or TRPV1 antagonist had no effect on hyperthermic seizures. Expression of vanilloid channel and glutamate receptor mRNA was confirmed by quantitative PCR analysis at each developmental stage in larval zebrafish. Taken together, our findings suggest a role of heat-activation of TRPV4 channels and enhanced NMDA receptor-mediated glutamatergic transmission in hyperthermia-induced seizures. PMID:22735490

Hunt, Robert F.; Hortopan, Gabriela A.; Gillespie, Anna; Baraban, Scott C.

2012-01-01

320

A vertebrate model for the study of lipid binding/transfer protein function: conservation of OSBP-related proteins between zebrafish and human.  

PubMed

Oxysterol-binding protein (OSBP) and OSBP-related (ORP) or OSBP-like (OSBPL) proteins constitute a family of lipid-binding/transfer proteins (LTPs) present in eukaryotes from yeast to man. The mechanisms of ORP function have remained incompletely understood. However, several ORPs are present at membrane contact sites and act as either lipid transporters or sensors that control lipid metabolism, cell signaling, and vesicle transport. Zebrafish, Danio rerio, has gained increasing popularity as a model organism in developmental biology, human disease, toxicology, and drug discovery. However, LTPs in the fish are thus far unexplored. In this article we report a series of bioinformatic analyses showing that the OSBPL gene family is highly conserved between the fish and human. The OSBPL subfamily structure is markedly similar between the two organisms, and all 12 human genes have orthologs, designated osbpl and located on 11 chromosomes in D. rerio. Interestingly, osbpl2 and osbpl3 are present as two closely related homologs (a and b), due to gene duplication events in the teleost lineage. Moreover, the domain structures of the distinct ORP proteins are almost identical between zebrafish and man, and molecular modeling in the present study suggests that ORD liganding by phosphatidylinositol-4-phosphate (PI4P) is a feature conserved between yeast Osh3p, human ORP3, and zebrafish Osbpl3. The present analysis identifies D. rerio as an attractive model to study the functions of ORPs in vertebrate development and metabolism. PMID:24326072

Zhou, You; Wohlfahrt, Gerd; Paavola, Jere; Olkkonen, Vesa M

2014-04-11

321

Frankincense essential oil prepared from hydrodistillation of Boswellia sacra gum resins induces human pancreatic cancer cell death in cultures and in a xenograft murine model  

PubMed Central

Background Regardless of the availability of therapeutic options, the overall 5-year survival for patients diagnosed with pancreatic cancer remains less than 5%. Gum resins from Boswellia species, also known as frankincense, have been used as a major ingredient in Ayurvedic and Chinese medicine to treat a variety of health-related conditions. Both frankincense chemical extracts and essential oil prepared from Boswellia species gum resins exhibit anti-neoplastic activity, and have been investigated as potential anti-cancer agents. The goals of this study are to identify optimal condition for preparing frankincense essential oil that possesses potent anti-tumor activity, and to evaluate the activity in both cultured human pancreatic cancer cells and a xenograft mouse cancer model. Methods Boswellia sacra gum resins were hydrodistilled at 78°C; and essential oil distillate fractions were collected at different durations (Fraction I at 0–2 h, Fraction II at 8–10 h, and Fraction III at 11–12 h). Hydrodistillation of the second half of gum resins was performed at 100°C; and distillate was collected at 11–12 h (Fraction IV). Chemical compositions were identified by gas chromatography–mass spectrometry (GC-MS); and total boswellic acids contents were quantified by high-performance liquid chromatography (HPLC). Frankincense essential oil-modulated pancreatic tumor cell viability and cytotoxicity were determined by colorimetric assays. Levels of apoptotic markers, signaling molecules, and cell cycle regulators expression were characterized by Western blot analysis. A heterotopic (subcutaneous) human pancreatic cancer xenograft nude mouse model was used to evaluate anti-tumor capability of Fraction IV frankincense essential oil in vivo. Frankincense essential oil-induced tumor cytostatic and cytotoxic activities in animals were assessed by immunohistochemistry. Results Longer duration and higher temperature hydrodistillation produced more abundant high molecular weight compounds, including boswellic acids, in frankincense essential oil fraactions. Human pancreatic cancer cells were sensitive to Fractions III and IV (containing higher molecular weight compounds) treatment with suppressed cell viability and increased cell death. Essential oil activated the caspase-dependent apoptotic pathway, induced a rapid and transient activation of Akt and Erk1/2, and suppressed levels of cyclin D1 cdk4 expression in cultured pancreatic cancer cells. In addition, Boswellia sacra essential oil Fraction IV exhibited anti-proliferative and pro-apoptotic activities against pancreatic tumors in the heterotopic xenograft mouse model. Conclusion All fractions of frankincense essential oil from Boswellia sacra are capable of suppressing viability and inducing apoptosis of a panel of human pancreatic cancer cell lines. Potency of essential oil-suppressed tumor cell viability may be associated with the greater abundance of high molecular weight compounds in Fractions III and IV. Although chemical component(s) responsible for tumor cell cytotoxicity remains undefined, crude essential oil prepared from hydrodistillation of Boswellia sacra gum resins might be a useful alternative therapeutic agent for treating patients with pancreatic adenocarcinoma, an aggressive cancer with poor prognosis. PMID:23237355

2012-01-01

322

Enhanced antitumor activity and mechanism of biodegradable polymeric micelles-encapsulated chetomin in both transgenic zebrafish and mouse models  

NASA Astrophysics Data System (ADS)

Chetomin is a promising molecule with anti-tumor activities in the epipolythiodioxopiperazine family of fungal secondary metabolites; however, strong hydrophobicity has limited its further applications. In this work, chetomin was encapsulated into polymeric micelles to obtain an aqueous formulation, and the chetomin loaded micelles (Che-M) exhibited small particle size and high encapsulation efficiency. When the concentration of copolymer was higher than the critical gelation concentration, the Che-M could form a thermosensitive hydrogel (Che-H), which was free-flowing sol at ambient temperature and converted into a non-flowing gel at body temperature. The molecular modeling study has indicated that chetomin interacted with PCL as a core, which was embraced by PEG as a shell. Che-M showed equal cytotoxicity with free chetomin, but the apoptosis inducing effects of Che-M were more significant. Besides, Che-M could increase the GSSG level, decrease the GSH level, and increase the ROS in CT26 cells. Furthermore, stronger inhibitory effects of Che-M were observed on embryonic angiogenesis, tumor-induced angiogenesis and tumor growth in transgenic zebrafish models. In addition, Che-M was effective in inhibiting tumor growth and prolonging survival in a subcutaneous CT26 tumor model. In a colorectal peritoneal carcinomatosis model, both Che-M and Che-H showed excellent therapeutic effects, but Che-H was more effective. In conclusion, Che-M and Che-H may serve as candidates for cancer therapy.

Wu, Qinjie; Li, Guoyou; Deng, Senyi; Ouyang, Liang; Li, Ling; Liu, Lei; Luo, Na; Song, Xiangrong; He, Gu; Gong, Changyang; Wei, Yuquan

2014-09-01

323

RNA sequencing of FACS-sorted immune cell populations from zebrafish infection models to identify cell specific responses to intracellular pathogens.  

PubMed

The zebrafish (Danio rerio) is increasingly used as a model for studying infectious diseases. This nonmammalian vertebrate host, which is transparent at the early life stages, is especially attractive for live imaging of interactions between pathogens and host cells. A number of useful fluorescent reporter lines have recently been developed and significant advances in RNA sequencing technology have been made, which now make it possible to apply the zebrafish model for investigating changes in transcriptional activity of specific immune cell types during the course of an infection process.Here we describe how to sequence RNA extracted from fluorescently labeled macrophages obtained by cell-sorting of 5-day-old zebrafish larvae of the transgenic Tg(mpeg1:Gal4-VP16);Tg(UAS-E1b:Kaede) line. This technique showed reproducible results and allowed to detect specific expression of macrophage markers in the mpeg1 positive cell population, whereas no markers specific for neutrophils or lymphoid cells were detected. This protocol has been also successfully extended to other immune cell types as well as cells infected by Mycobacterium marinum. PMID:25172286

Rougeot, Julien; Zakrzewska, Ania; Kanwal, Zakia; Jansen, Hans J; Spaink, Herman P; Meijer, Annemarie H

2014-01-01

324

Using zebrafish models to explore genetic and epigenetic impacts on evolutionary developmental origins of aging.  

PubMed

Can we reset, reprogram, rejuvenate, or reverse the organismal aging process? Certain genetic manipulations could at least reset and reprogram epigenetic dynamics beyond phenotypic plasticity and elasticity in cells, which can be manipulated further into organisms. However, in a whole complex aging organism, how can we rejuvenate intrinsic resources and infrastructures in an intact and noninvasive manner? The incidence of diseases increases exponentially with age, accompanied by progressive deteriorations of physiological functions in organisms. Aging-associated diseases are sporadic but essentially inevitable complications arising from senescence. Senescence is often considered the antithesis of early development, but yet there may be factors and mechanisms in common between these 2 phenomena to rejuvenate over the dynamic process of aging. The association between early development and late-onset disease with advancing age is thought to come from a consequence of developmental plasticity, the phenomenon by which one genotype can give rise to a range of physiologically and/or morphologically adaptive states based on diverse epigenotypes in response to intrinsic or extrinsic environmental cues and genetic perturbations. We hypothesized that the future aging process can be predictive based on adaptivity during the early developmental period. Modulating the thresholds and windows of plasticity and its robustness by molecular genetic and chemical epigenetic approaches, we have successfully conducted experiments to isolate zebrafish mutants expressing apparently altered senescence phenotypes during their embryonic and/or larval stages ("embryonic/larval senescence"). Subsequently, at least some of these mutant animals were found to show a shortened life span, whereas others would be expected to live longer into adulthood. We anticipate that previously uncharacterized developmental genes may mediate the aging process and play a pivotal role in senescence. On the other hand, unexpected senescence-related genes might also be involved in the early developmental process and its regulation. The ease of manipulation using the zebrafish system allows us to conduct an exhaustive exploration of novel genes, genotypes, and epigenotypes that can be linked to the senescence phenotype, which facilitates searching for the evolutionary and developmental origins of aging in vertebrates. PMID:24239812

Kishi, Shuji

2014-02-01

325

The Zebrafish Models to Explore Genetic and Epigenetic Impacts on Evolutionary Developmental Origins of Aging  

PubMed Central

Can we reset, reprogram, rejuvenate or reverse the organismal aging process? Certain genetic manipulations could at least reset and reprogram epigenetic dynamics beyond phenotypic plasticity and elasticity in cells, which can be further manipulated into organisms. However, in a whole complex aging organism, how can we rejuvenate intrinsic resources and infrastructures in an intact/noninvasive manner? The incidence of diseases increases exponentially with age, accompanied by progressive deteriorations of physiological functions in organisms. Aging-associated diseases are sporadic but essentially inevitable complications arising from senescence. Senescence is often considered the antithesis of early development, but yet there may be factors and mechanisms in common between these two phenomena to rejuvenate over the dynamic process of aging. The association between early development and late-onset disease with advancing age is thought to come from a consequence of developmental plasticity, the phenomenon by which one genotype can give rise to a range of physiologically and/or morphologically adaptive states based on diverse epigenotypes, in response to intrinsic or extrinsic environmental cues and genetic perturbations. We hypothesized that the future aging process can be predictive based on adaptivity during the early developmental period. Modulating the thresholds and windows of plasticity and its robustness by molecular genetic and chemical epigenetic approaches, we have successfully conducted experiments to isolate zebrafish mutants expressing apparently altered senescence phenotypes during their embryonic and/or larval stages (“embryonic/larval senescence”). Subsequently, at least some of these mutant animals were found to show shortened lifespan, while some others would be expected to live longer in adulthoods. We anticipate that previously uncharacterized developmental genes may mediate the aging process and play a pivotal role in senescence. On the other hand, unexpected senescence-related genes might also be involved in the early developmental process and its regulation. The ease of manipulation using the zebrafish system allows us to conduct an exhaustive exploration of novel genes/genotypes and epigenotype that can be linked to the senescence phenotype, and thereby facilitates searching for the evolutionary and developmental origins of aging in vertebrates. PMID:24239812

Kishi, Shuji

2014-01-01

326

Deriving Cell Lines from Zebrafish Embryos and Tumors  

PubMed Central

Abstract Over the last two decades the zebrafish has emerged as a powerful model organism in science. The experimental accessibility, the broad range of zebrafish mutants, and the highly conserved genetic and biochemical pathways between zebrafish and mammals lifted zebrafish to become one of the most attractive vertebrate models to study gene function and to model human diseases. Zebrafish cell lines are highly attractive to investigate cell biology and zebrafish cell lines complement the experimental tools that are available already. We established a straightforward method to culture cells from a single zebrafish embryo or a single tumor. Here we describe the generation of fibroblast-like cell lines from wild-type and ptenb?/? embryos and an endothelial-like cell line from a tumor of an adult ptena+/?ptenb?/? zebrafish. This protocol can easily be adapted to establish stable cell lines from any mutant or transgenic zebrafish line and the average time to obtain a pro-stable cell line is 3–5 months. PMID:23672287

Choorapoikayil, Suma; Overvoorde, John

2013-01-01

327

Advances in zebrafish chemical screening technologies  

PubMed Central

Due to several inherent advantages, zebrafish are being utilized in increasingly sophisticated screens to assess the physiological effects of chemical compounds directly in living vertebrate organisms. Diverse screening platforms showcase these advantages. Morphological assays encompassing basic qualitative observations to automated imaging, manipulation, and data-processing systems provide whole organism to subcellular levels of detail. Behavioral screens extend chemical screening to the level of complex systems. In addition, zebrafish-based disease models provide a means of identifying new potential therapeutic strategies. Automated systems for handling/sorting, high-resolution imaging and quantitative data collection have significantly increased throughput in recent years. These advances will make it easier to capture multiple streams of information from a given sample and facilitate integration of zebrafish at the earliest stages of the drug-discovery process, providing potential solutions to current drug-development bottlenecks. Here we outline advances that have been made within the growing field of zebrafish chemical screening. PMID:23043478

Mathias, Jonathan R; Saxena, Meera T; Mumm, Jeff S

2013-01-01

328

Synergistic induction of AHR regulated genes in developmental toxicity from co-exposure to two model PAHs in zebrafish  

PubMed Central

Polycyclic aromatic hydrocarbons (PAHs) are pollutants created by the incomplete combustion of carbon, and are increasing in the environment largely due to the burning of fossil fuels. PAHs occur as complex mixtures, and some combinations have been shown to cause synergistic developmental toxicity in fish embryos, characterized by pericardial edema and craniofacial malformations. Previous studies have indicated that in the zebrafish model, this toxicity is mediated by the aryl hydrocarbon receptor 2 (AHR2), and enhanced by inhibition of CYP1A activity. In this study, we further examined this interaction of the model PAH and AHR agonist ?-naphthoflavone (BNF) with and without the AHR partial agonist/antagonist and CYP1A inhibitor ?-naphthoflavone (ANF) to determine 1) whether ANF was acting as an AHR antagonist, 2) what alterations BNF and ANF both alone and in combination had on mRNA expression of the AHR regulated genes cytochrome P450 (cyp) 1a, 1b1, and 1c1, and the AHR repressor (ahrr2) prior to vs. during deformity onset, and 3) compare CYP1A enzyme activity with mRNA induction. Zebrafish embryos were exposed from 24–48 or 24–96 hpf to BNF, 1–100 ?g/L, ANF, 1–150 ?g/L, a BNF+ANF co-exposure (1 ?g/L + 100 ?g/L), or a DMSO solvent control. RNA was extracted and examined by quantitative real time PCR. Both BNF and ANF each individually resulted in a dose dependent increase CYP1A, CYP1B1, CYP1C1, and AHRR2 mRNA, confirming their activities as AHR agonists. In the BNF+ANF co-exposures prior to deformity onset, expression of these genes was synergistic, and expression levels of the AHR regulated genes resembled the higher doses of BNF alone. Gene induction during deformities was also significantly increased in the co-exposure, but to a lesser magnitude than prior to deformity onset. EROD measurements of CYP1A activity showed ANF inhibited activity induction by BNF in the co-exposure group; this finding is not predicted by mRNA expression, which is synergistically induced in this treatment. This suggests that inhibition of CYP1A activity may alter metabolism and/or increase the half-life of the AHR agonist(s), allowing for increased AHR activation. This study furthers a mechanistic understanding of interactions underlying PAH synergistic toxicity. PMID:17964672

Timme-Laragy, Alicia. R.; Cockman, Crystal. J.; Matson, Cole. W.; Di Giulio, Richard. T.

2007-01-01

329

Purpurin inhibits adipocyte-derived leucine aminopeptidase and angiogenesis in a zebrafish model.  

PubMed

Adipocyte-derived leucine aminopeptidase (A-LAP) is a novel member of the M1 family of zinc metallopeptidases, which has been reported to play a crucial role in angiogenesis. In the present study, we conducted a target-based screening of natural products and synthetic chemical libraries using the purified enzyme to search novel inhibitors of A-LAP. Amongst several hits isolated, a natural product purpurin was identified as one of the most potent inhibitors of A-LAP from the screening. In vitro enzymatic analyses demonstrated that purpurin inhibited A-LAP activity in a non-competitive manner with a Ki value of 20 M. In addition, purpurin showed a strong selectivity toward A-LAP versus another member of M1 family of zinc metallopeptidase, aminopeptidase N (APN). In angiogenesis assays, purpurin inhibited the vascular endothelial growth factor (VEGF)-induced invasion and tube formation of human umbilical vein endothelial cells (HUVEC). Moreover, purpurin inhibited in vivo angiogenesis in zebrafish embryo without toxicity. These data demonstrate that purpurin is a novel specific inhibitor of A-LAP and could be developed as a new anti-angiogenic agent. PMID:24928393

Park, Hyomi; Shim, Joong Sup; Kim, Beom Seok; Jung, Hye Jin; Huh, Tae-Lin; Kwon, Ho Jeong

2014-07-18

330

Mycobacteria Counteract a TLR-Mediated Nitrosative Defense Mechanism in a Zebrafish Infection Model  

PubMed Central

Pulmonary tuberculosis (TB), caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb), is a major world health problem. The production of reactive nitrogen species (RNS) is a potent cytostatic and cytotoxic defense mechanism against intracellular pathogens. Nevertheless, the protective role of RNS during Mtb infection remains controversial. Here we use an anti-nitrotyrosine antibody as a readout to study nitration output by the zebrafish host during early mycobacterial pathogenesis. We found that recognition of Mycobacterium marinum, a close relative of Mtb, was sufficient to induce a nitrosative defense mechanism in a manner dependent on MyD88, the central adaptor protein in Toll like receptor (TLR) mediated pathogen recognition. However, this host response was attenuated by mycobacteria via a virulence mechanism independent of the well-characterized RD1 virulence locus. Our results indicate a mechanism of pathogenic mycobacteria to circumvent host defense in vivo. Shifting the balance of host-pathogen interactions in favor of the host by targeting this virulence mechanism may help to alleviate the problem of infection with Mtb strains that are resistant to multiple drug treatments. PMID:24967596

van Hensbergen, Vincent; Schutz, Esther; Redd, Michael J.; Murayama, Emi; Spaink, Herman P.; Meijer, Annemarie H.

2014-01-01

331

Larval Zebrafish Model for FDA-Approved Drug Repositioning for Tobacco Dependence Treatment  

PubMed Central

Cigarette smoking remains the most preventable cause of death and excess health care costs in the United States, and is a leading cause of death among alcoholics. Long-term tobacco abstinence rates are low, and pharmacotherapeutic options are limited. Repositioning medications approved by the U.S. Food and Drug Administration (FDA) may efficiently provide clinicians with new treatment options. We developed a drug-repositioning paradigm using larval zebrafish locomotion and established predictive clinical validity using FDA-approved smoking cessation therapeutics. We evaluated 39 physician-vetted medications for nicotine-induced locomotor activation blockade. We further evaluated candidate medications for altered ethanol response, as well as in combination with varenicline for nicotine-response attenuation. Six medications specifically inhibited the nicotine response. Among this set, apomorphine and topiramate blocked both nicotine and ethanol responses. Both positively interact with varenicline in the Bliss Independence test, indicating potential synergistic interactions suggesting these are candidates for translation into Phase II clinical trials for smoking cessation. PMID:24658307

Cousin, Margot A.; Ebbert, Jon O.; Wiinamaki, Amanda R.; Urban, Mark D.; Argue, David P.; Ekker, Stephen C.; Klee, Eric W.

2014-01-01

332

Zebrafish screen identifies novel compound with selective toxicity against leukemia  

PubMed Central

To detect targeted antileukemia agents we have designed a novel, high-content in vivo screen using genetically engineered, T-cell reporting zebrafish. We exploited the developmental similarities between normal and malignant T lymphoblasts to screen a small molecule library for activity against immature T cells with a simple visual readout in zebrafish larvae. After screening 26 400 molecules, we identified Lenaldekar (LDK), a compound that eliminates immature T cells in developing zebrafish without affecting the cell cycle in other cell types. LDK is well tolerated in vertebrates and induces long-term remission in adult zebrafish with cMYC-induced T-cell acute lymphoblastic leukemia (T-ALL). LDK causes dephosphorylation of members of the PI3 kinase/AKT/mTOR pathway and delays sensitive cells in late mitosis. Among human cancers, LDK selectively affects survival of hematopoietic malignancy lines and primary leukemias, including therapy-refractory B-ALL and chronic myelogenous leukemia samples, and inhibits growth of human T-ALL xenografts. This work demonstrates the utility of our method using zebrafish for antineoplastic candidate drug identification and suggests a new approach for targeted leukemia therapy. Although our efforts focused on leukemia therapy, this screening approach has broad implications as it can be translated to other cancer types involving malignant degeneration of developmentally arrested cells. PMID:22490804

Ridges, Suzanne; Heaton, Will L.; Joshi, Deepa; Choi, Henry; Eiring, Anna; Batchelor, Lance; Choudhry, Priya; Manos, Elizabeth J.; Sofla, Hossein; Sanati, Ali; Welborn, Seth; Agarwal, Archana; Spangrude, Gerald J.; Miles, Rodney R.; Cox, James E.; Frazer, J. Kimble; Deininger, Michael; Balan, Kaveri; Sigman, Matthew; Müschen, Markus; Perova, Tatiana; Johnson, Radia; Montpellier, Bertrand; Guidos, Cynthia J.; Jones, David A.

2012-01-01

333

Biodegradable polymeric micelle-encapsulated quercetin suppresses tumor growth and metastasis in both transgenic zebrafish and mouse models.  

PubMed

Quercetin (Que) loaded polymeric micelles were prepared to obtain an aqueous formulation of Que with enhanced anti-tumor and anti-metastasis activities. A simple solid dispersion method was used, and the obtained Que micelles had a small particle size (about 31 nm), high drug loading, and high encapsulation efficiency. Que micelles showed improved cellular uptake, an enhanced apoptosis induction effect, and stronger inhibitory effects on proliferation, migration, and invasion of 4T1 cells than free Que. The enhanced in vitro antiangiogenesis effects of Que micelles were proved by the results that Que micelles significantly suppressed proliferation, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs). Subsequently, transgenic zebrafish models were employed to investigate anti-tumor and anti-metastasis effects of Que micelles, in which stronger inhibitory effects of Que micelles were observed on embryonic angiogenesis, tumor-induced angiogenesis, tumor growth, and tumor metastasis. Furthermore, in a subcutaneous 4T1 tumor model, Que micelles were more effective in suppressing tumor growth and spontaneous pulmonary metastasis, and prolonging the survival of tumor-bearing mice. Besides, immunohistochemical and immunofluorescent assays suggested that tumors in the Que micelle-treated group showed more apoptosis, fewer microvessels, and fewer proliferation-positive cells. In conclusion, Que micelles, which are synthesized as an aqueous formulation of Que, possess enhanced anti-tumor and anti-metastasis activity, which can serve as potential candidates for cancer therapy. PMID:24165931

Wu, Qinjie; Deng, Senyi; Li, Ling; Sun, Lu; Yang, Xi; Liu, Xinyu; Liu, Lei; Qian, Zhiyong; Wei, Yuquan; Gong, Changyang

2013-12-21

334

Biodegradable polymeric micelle-encapsulated quercetin suppresses tumor growth and metastasis in both transgenic zebrafish and mouse models  

NASA Astrophysics Data System (ADS)

Quercetin (Que) loaded polymeric micelles were prepared to obtain an aqueous formulation of Que with enhanced anti-tumor and anti-metastasis activities. A simple solid dispersion method was used, and the obtained Que micelles had a small particle size (about 31 nm), high drug loading, and high encapsulation efficiency. Que micelles showed improved cellular uptake, an enhanced apoptosis induction effect, and stronger inhibitory effects on proliferation, migration, and invasion of 4T1 cells than free Que. The enhanced in vitro antiangiogenesis effects of Que micelles were proved by the results that Que micelles significantly suppressed proliferation, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs). Subsequently, transgenic zebrafish models were employed to investigate anti-tumor and anti-metastasis effects of Que micelles, in which stronger inhibitory effects of Que micelles were observed on embryonic angiogenesis, tumor-induced angiogenesis, tumor growth, and tumor metastasis. Furthermore, in a subcutaneous 4T1 tumor model, Que micelles were more effective in suppressing tumor growth and spontaneous pulmonary metastasis, and prolonging the survival of tumor-bearing mice. Besides, immunohistochemical and immunofluorescent assays suggested that tumors in the Que micelle-treated group showed more apoptosis, fewer microvessels, and fewer proliferation-positive cells. In conclusion, Que micelles, which are synthesized as an aqueous formulation of Que, possess enhanced anti-tumor and anti-metastasis activity, which can serve as potential candidates for cancer therapy.

Wu, Qinjie; Deng, Senyi; Li, Ling; Sun, Lu; Yang, Xi; Liu, Xinyu; Liu, Lei; Qian, Zhiyong; Wei, Yuquan; Gong, Changyang

2013-11-01

335

VEGFR tyrosine kinase inhibitor II (VRI) induced vascular insufficiency in zebrafish as a model for studying vascular toxicity and vascular preservation.  

PubMed

In ischemic disorders such as chronic wounds and myocardial ischemia, there is inadequate tissue perfusion due to vascular insufficiency. Besides, it has been observed that prolonged use of anti-angiogenic agents in cancer therapy produces cardiovascular toxicity caused by impaired vessel integrity and regeneration. In the present study, we used VEGFR tyrosine kinase inhibitor II (VRI) to chemically induce vascular insufficiency in zebrafish in vivo and human umbilical vein endothelial cells (HUVEC) in vitro to further study the mechanisms of vascular morphogenesis in these pathological conditions. We also explored the possibility of treating vascular insufficiency by enhancing vascular regeneration and repair with pharmacological intervention. We observed that pretreatment of VRI induced blood vessel loss in developing zebrafish by inhibiting angiogenesis and increasing endothelial cell apoptosis, accompanied by down-regulation of kdr, kdrl and flt-1 genes expression. The VRI-induced blood vessel loss in zebrafish could be restored by post-treatment of calycosin, a cardiovascular protective isoflavone. Similarly, VRI induced cytotoxicity and apoptosis in HUVEC which could be rescued by calycosin post-treatment. Further investigation of the underlying mechanisms showed that the PI3K/AKT/Bad cell survival pathway was a main contributor of the vascular regenerative effect of calycosin. These findings indicated that the cardiovascular toxicity in anti-angiogenic therapy was mainly caused by insufficient endothelial cell survival, suggesting its essential role in vascular integrity, repair and regeneration. In addition, we showed that VRI-induced blood vessel loss in zebrafish represented a simple and effective in vivo model for studying vascular insufficiency and evaluating cancer drug vascular toxicities. PMID:25234792

Li, Shang; Dang, Yuan Ye; Oi Lam Che, Ginny; Kwan, Yiu Wa; Chan, Shun Wan; Leung, George Pak Heng; Lee, Simon Ming Yuen; Hoi, Maggie Pui Man

2014-11-01

336

Gecko Crude Peptides Induce Apoptosis in Human Liver Carcinoma Cells In Vitro and Exert Antitumor Activity in a Mouse Ascites H22 Xenograft Model  

PubMed Central

Aim. To investigate the anti-tumor effects and mechanisms of gecko crude peptides (GCPs) in vitro and in vivo. Methods. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay was applied to measure the effects of GCPs on the HepG2 cell viability. Fluorescence morphology was used to identify apoptotic cells. A xenograft H22 liver cancer model was established in Kunming mice. The tumor-bearing mice were treated with daily intraperitoneal injections of normal saline (NS group) or GCPs (80, 40 or 20?mg/kg) for 10 days, or once per two days with 2?mg/kg doxorubicin (ADR group; n = 10 each). Serum tumor necrosis factor (TNF-?) and interleukin (IL)-6 were quantified using ELISA assay. Results. GCPs significantly inhibited the growth of HepG2 cells and induced typical apoptotic morphological features through increasing bcl-2/bax ratio in a dose- and time-dependent manner in vitro. The tumor weights of the ADR group, GCPs (H) group, GCPs (M) group, GCPs (L) group were smaller compared to the NS group. While the white blood cell count, thymus index, spleen index were higher in the high dose GCPs group than the NS group (P < 0.05), the VEGF expression in tumor tissue and serum TNF-? and IL-6 levels in the GCPs groups were lower than the NS group (P < 0.05). PMID:23093861

Song, Ying; Wang, Jian-Gang; Li, Rui-Fang; Li, Yan; Cui, Zhao-Chu; Duan, Leng-Xin; Lu, Fei

2012-01-01

337

C7a, a Biphosphinic Cyclopalladated Compound, Efficiently Controls the Development of a Patient-Derived Xenograft Model of Adult T Cell Leukemia/Lymphoma  

PubMed Central

Adult T-cell leukemia/lymphoma (ATLL) is a highly aggressive disease that occurs in individuals infected with the human T lymphotropic virus type 1 (HTLV-1). Patients with aggressive ATLL have a poor prognosis because the leukemic cells are resistant to conventional chemotherapy. We have investigated the therapeutic efficacy of a biphosphinic cyclopalladated complex {Pd2 [S(?)C2, N-dmpa]2 (?-dppe)Cl2}, termed C7a, in a patient-derived xenograft model of ATLL, and investigated the mechanism of C7a action in HTLV-1-positive and negative transformed T cell lines in vitro. In vivo survival studies in immunocompromised mice inoculated with human RV-ATL cells and intraperitoneally treated with C7a led to significantly increased survival of the treated mice. We investigated the mechanism of C7a activity in vitro and found that it induced mitochondrial release of cytochrome c, caspase activation, nuclear condensation and DNA degradation. These results suggest that C7a triggers apoptotic cell death in both HTLV-1 infected and uninfected human transformed T-cell lines. Significantly, C7a was not cytotoxic to peripheral blood mononuclear cells (PBMC) from healthy donors and HTLV-1-infected individuals. C7a inhibited more than 60% of the ex vivo spontaneous proliferation of PBMC from HTLV-1-infected individuals. These results support a potential therapeutic role for C7a in both ATLL and HTLV-1-negative T-cell lymphomas. PMID:21994769

Guimaraes-Correa, Ana B.; Crawford, Lindsey B.; Figueiredo, Carlos R.; Gimenes, Karina P.; Pinto, Lorena A.; Rios Grassi, Maria Fernanda; Feuer, Gerold; Travassos, Luiz R.; Caires, Antonio C.F.; Rodrigues, Elaine G.; Marriott, Susan J.

2011-01-01

338

A xenograft mouse model coupled with in-depth plasma proteome analysis facilitates identification of novel serum biomarkers for human ovarian cancer  

PubMed Central

Proteomics discovery of novel cancer serum biomarkers is hindered by the great complexity of serum, patient-to-patient variability, and triggering by the tumor of an acute-phase inflammatory reaction. This host response alters many serum protein levels in cancer patients, but these changes have low specificity as they can be triggered by diverse causes. We addressed these hurdles by utilizing a xenograft mouse model coupled with an in-depth 4-D protein profiling method to identify human proteins in the mouse serum. This strategy ensures identified putative biomarkers are shed by the tumor, and detection of low-abundance proteins shed by the tumor is enhanced because the mouse blood volume is more than a thousand times smaller than that of a human. Using TOV-112D ovarian tumors, more than 200 human proteins were identified in the mouse serum, including novel candidate biomarkers and proteins previously reported to be elevated in either ovarian tumors or the blood of ovarian cancer patients. Subsequent quantitation of selected putative biomarkers in human sera using label-free multiple reaction monitoring (MRM) mass spectrometry (MS) showed that chloride intracellular channel 1, the mature form of cathepsin D, and peroxiredoxin 6 were elevated significantly in sera from ovarian carcinoma patients. PMID:22032327

Tang, Hsin-Yao; Beer, Lynn A.; Chang-Wong, Tony; Hammond, Rachel; Gimotty, Phyllis; Coukos, George; Speicher, David W.

2011-01-01

339

Combination of the c-Met Inhibitor Tivantinib and Zoledronic Acid Prevents Tumor Bone Engraftment and Inhibits Progression of Established Bone Metastases in a Breast Xenograft Model  

PubMed Central

Bone is the most common metastatic site for breast cancer. There is a significant need to understand the molecular mechanisms controlling the engraftment and growth of tumor cells in bone and to discover novel effective therapeutic strategies. The aim of this study was to assess the effects of tivantinib and Zoledronic Acid (ZA) in combination in a breast xenograft model of bone metastases. Cancer cells were intracardially implanted into immunodeficient mice and the effects of drugs alone or in combination on bone metastasis were evaluated by in vivo non-invasive optical and micro-CT imaging technologies. Drugs were administered either before (preventive regimen) or after (therapeutic regimen) bone metastases were detectable. In the preventive regimen, the combination of tivantinib plus ZA was much more effective than single agents in delaying bone metastatic tumor growth. When administered in the therapeutic schedule, the combination delayed metastatic progression and was effective in improving survival. These effects were not ascribed to a direct cytotoxic effect of the combined therapy on breast cancer cells in vitro. The results of this study provide the rationale for the design of new combinatorial strategies with tivantinib and ZA for the treatment of breast cancer bone metastases. PMID:24260160

Previdi, Sara; Scolari, Federica; Chilà, Rosaria; Ricci, Francesca; Abbadessa, Giovanni; Broggini, Massimo

2013-01-01

340

Shikonin suppresses tumor growth and synergizes with gemcitabine in a pancreatic cancer xenograft model: Involvement of NF-?B signaling pathway.  

PubMed

Although gemcitabine is currently the best chemotherapeutic agent available for the treatment of advanced pancreatic cancer, eventual failure of response is a significant clinical problem. Therefore, novel therapeutic approaches against this disease are highly needed. The aim of this study was to evaluate whether shikonin, a naphthoquinone derivative, has potential in the treatment of pancreatic cancer when used either alone or in combination with gemcitabine. Our in vitro results showed that shikonin inhibited the proliferation of three different human pancreatic cancer cell lines and potentiated the cytotoxic effect of gemcitabine, which correlated with the down-regulation of constitutive as well as gemcitabine-induced activation of NF-?B and NF-?B-regulated gene products. Most importantly, using a xenograft model of human pancreatic cancer, we found shikonin alone significantly suppressed tumor growth and argumented the antitumor activity of gemcitabine. These effects also correlated with the down-regulation of NF-?B activity and its target genes, decreased proliferation (PCNA and Ki-67), decreased microvessel density (CD31), and increased apoptosis (TUNEL) in tumor remnants. Collectively, our results suggest that shikonin can suppress the growth of human pancreatic tumors and potentiate the antitumor effects of gemcitabine through the suppression of NF-?B and NF-?B-regulated gene products. PMID:24522113

Wang, Yongwei; Zhou, Yinan; Jia, Guang; Han, Bing; Liu, Ji; Teng, Yueqiu; Lv, Jiachen; Song, Zengfu; Li, Yilong; Ji, Liang; Pan, Shangha; Jiang, Hongchi; Sun, Bei

2014-04-01

341

Novel Mouse Xenograft Models Reveal a Critical Role of CD4+ T Cells in the Proliferation of EBV-Infected T and NK Cells  

PubMed Central

Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic herpesvirus, ectopically infects T or NK cells to cause severe diseases of unknown pathogenesis, including chronic active EBV infection (CAEBV) and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). We developed xenograft models of CAEBV and EBV-HLH by transplanting patients' PBMC to immunodeficient mice of the NOD/Shi-scid/IL-2R?null strain. In these models, EBV-infected T, NK, or B cells proliferated systemically and reproduced histological characteristics of the two diseases. Analysis of the TCR repertoire expression revealed that identical predominant EBV-infected T-cell clones proliferated in patients and corresponding mice transplanted with their PBMC. Expression of the EBV nuclear antigen 1 (EBNA1), the latent membrane protein 1 (LMP1), and LMP2, but not EBNA2, in the engrafted cells is consistent with the latency II program of EBV gene expression known in CAEBV. High levels of human cytokines, including IL-8, IFN-?, and RANTES, were detected in the peripheral blood of the model mice, mirroring hypercytokinemia characteristic to both CAEBV and EBV-HLH. Transplantation of individual immunophenotypic subsets isolated from patients' PBMC as well as that of various combinations of these subsets revealed a critical role of CD4+ T cells in the engraftment of EBV-infected T and NK cells. In accordance with this finding, in vivo depletion of CD4+ T cells by the administration of the OKT4 antibody following transplantation of PBMC prevented the engraftment of EBV-infected T and NK cells. This is the first report of animal models of CAEBV and EBV-HLH that are expected to be useful tools in the development of novel therapeutic strategies for the treatment of the diseases. PMID:22028658

Arai, Ayako; Nakazawa, Atsuko; Kawano, Fuyuko; Ichikawa, Sayumi; Shimizu, Norio; Yamamoto, Naoki; Morio, Tomohiro; Ohga, Shouichi; Nakamura, Hiroyuki; Ito, Mamoru; Miura, Osamu; Komano, Jun; Fujiwara, Shigeyoshi

2011-01-01

342

Polygenic Sex Determination System in Zebrafish  

PubMed Central

Background Despite the popularity of zebrafish as a research model, its sex determination (SD) mechanism is still unknown. Most cytogenetic studies failed to find dimorphic sex chromosomes and no primary sex determining switch has been identified even though the assembly of zebrafish genome sequence is near to completion and a high resolution genetic map is available. Recent publications suggest that environmental factors within the natural range have minimal impact on sex ratios of zebrafish populations. The primary aim of this study is to find out more about how sex is determined in zebrafish. Methodology/Principal Findings Using classical breeding experiments, we found that sex ratios across families were wide ranging (4.8% to 97.3% males). On the other hand, repeated single pair crossings produced broods of very similar sex ratios, indicating that parental genotypes have a role in the sex ratio of the offspring. Variation among family sex ratios was reduced after selection for breeding pairs with predominantly male or female offspring, another indication that zebrafish sex is regulated genetically. Further examinations by a PCR-based “blind assay" and array comparative genomic hybridization both failed to find universal sex-linked differences between the male and female genomes. Together with the ability to increase the sex bias of lines by selective breeding, these data suggest that zebrafish is unlikely to utilize a chromosomal sex determination (CSD) system. Conclusions/Significance Taken together, our study suggests that zebrafish sex is genetically determined with limited, secondary influences from the environment. As we have not found any sign for CSD in the species, we propose that the zebrafish has a polygenic sex determination system. PMID:22506019

Liew, Woei Chang; Bartfai, Richard; Lim, Zijie; Sreenivasan, Rajini; Siegfried, Kellee R.; Orban, Laszlo

2012-01-01

343

Modelling of human Wiskott–Aldrich syndrome protein mutants in zebrafish larvae using in vivo live imaging  

PubMed Central

Summary Wiskott–Aldrich syndrome (WAS) and X-linked neutropenia (XLN) are immunodeficiencies in which the function of several haematopoietic cell lineages is perturbed as a result of mutations in the actin regulator WASp. From in vitro cell biology experiments, and biochemical and structural approaches, we know much about the functional domains of WASp and how WASp might regulate the dynamic actin cytoskeleton downstream of activators such as Cdc42, but in vivo experiments are much more challenging. In patients, there is a correlation between clinical disease and genotype, with severe reductions in WASp expression or function associating with complex multilineage immunodeficiency, whereas specific mutations that cause constitutive activation of WASp result in congenital neutropenia. Here, we take advantage of the genetic tractability and translucency of zebrafish larvae to first characterise how a null mutant in zfWASp influences the behaviour of neutrophils and macrophages in response to tissue damage and to clearance of infections. We then use this mutant background to study how leukocyte lineage-specific transgenic replacement with human WASp variants (including normal wild type and point mutations that either fail to bind Cdc42 or cannot be phosphorylated, and a constitutively active mutant equivalent to that seen in XLN patients) alter the capacity for generation of neutrophils, their chemotactic response to wounds and the phagocytic clearance capacity of macrophages. This model provides a unique insight into WASp-related immunodeficiency at both a cellular and whole organism level. PMID:23868979

Jones, Rebecca A.; Feng, Yi; Worth, Austen J.; Thrasher, Adrian J.; Burns, Siobhan O.; Martin, Paul

2013-01-01

344

Transgenic zebrafish model to study translational control mediated by upstream open reading frame of human chop gene  

PubMed Central

Upstream open reading frame (uORF)-mediated translational inhibition is important in controlling key regulatory genes expression. However, understanding the underlying molecular mechanism of such uORF-mediated control system in vivo is challenging in the absence of an animal model. Therefore, we generated a zebrafish transgenic line, termed huORFZ, harboring a construct in which the uORF sequence from human CCAAT/enhancer-binding protein homologous protein gene (huORFchop) is added to the leader of GFP and is driven by a cytomegalovirus promoter. The translation of transgenic huORFchop-gfp mRNA was absolutely inhibited by the huORFchop cassette in huORFZ embryos during normal conditions, but the downstream GFP was only apparent when the huORFZ embryos were treated with endoplasmic reticulum (ER) stresses. Interestingly, the number and location of GFP-responsive embryonic cells were dependent on the developmental stage and type of ER stresses encountered. These results indicate that the translation of the huORFchop-tag downstream reporter gene is controlled in the huORFZ line. Moreover, using cell sorting and microarray analysis of huORFZ embryos, we identified such putative factors as Nrg/ErbB, PI3K and hsp90, which are involved in huORFchop-mediated translational control under heat-shock stress. Therefore, using the huORFZ embryos allows us to study the regulatory network involved in human uORFchop-mediated translational inhibition. PMID:21873270

Lee, Hung-Chieh; Chen, Yi-Jiun; Liu, Yu-Wei; Lin, Kai-Yen; Chen, Shaio-Wen; Lin, Cheng-Yung; Lu, Yi-Chin; Hsu, Pei-Chun; Lee, Sheng-Chung; Tsai, Huai-Jen

2011-01-01

345

Expression of H-RASV12 in a zebrafish model of Costello syndrome causes cellular senescence in adult proliferating cells  

PubMed Central

Summary Constitutively active, ‘oncogenic’ H-RAS can drive proliferation and transformation in human cancer, or be a potent inducer of cellular senescence. Moreover, aberrant activation of the Ras pathway owing to germline mutations can cause severe developmental disorders. In this study we have generated transgenic zebrafish that constitutively express low levels, or can be induced to express high levels, of oncogenic H-RAS. We observed that fish carrying the integrated transgene in their germline display several hallmarks of Costello syndrome, a rare genetic disease caused by activating mutations in the gene H-RAS, and can be used as a model for the disease. In Costello-like fish, low levels of oncogenic H-RAS expression are associated with both reduced proliferation and an increase in senescence markers in adult progenitor cell compartments in the brain and heart, together with activated DNA damage responses. Overexpression of H-RAS through a heat-shock-inducible promoter in larvae led to hyperproliferation, activation of the DNA damage response and tp53-dependent cell cycle arrest. Thus, oncogene-induced senescence of adult proliferating cells contributes to the development of Costello syndrome and provides an alternative pathway to transformation in the presence of widespread constitutively active H-RAS expression. PMID:19132118

Santoriello, Cristina; Deflorian, Gianluca; Pezzimenti, Federica; Kawakami, Koichi; Lanfrancone, Luisa; d’Adda di Fagagna, Fabrizio; Mione, Marina

2009-01-01

346

Small Molecule Suppressors of Drosophila Kinesin Deficiency Rescue Motor Axon Development in a Zebrafish Model of Spinal Muscular Atrophy  

PubMed Central

Proximal spinal muscular atrophy (SMA) is the most common inherited motor neuropathy and the leading hereditary cause of infant mortality. Currently there is no effective treatment for the disease, reflecting a need for pharmacologic interventions that restore performance of dysfunctional motor neurons or suppress the consequences of their dysfunction. In a series of assays relevant to motor neuron biology, we explored the activities of a collection of tetrahydroindoles that were reported to alter the metabolism of amyloid precursor protein (APP). In Drosophila larvae the compounds suppressed aberrant larval locomotion due to mutations in the Khc and Klc genes, which respectively encode the heavy and light chains of kinesin-1. A representative compound of this class also suppressed the appearance of axonal swellings (alternatively termed axonal spheroids or neuritic beads) in the segmental nerves of the kinesin-deficient Drosophila larvae. Given the importance of kinesin-dependent transport for extension and maintenance of axons and their growth cones, three members of the class were tested for neurotrophic effects on isolated rat spinal motor neurons. Each compound stimulated neurite outgrowth. In addition, consistent with SMA being an axonopathy of motor neurons, the three axonotrophic compounds rescued motor axon development in a zebrafish model of SMA. The results introduce a collection of small molecules as pharmacologic suppressors of SMA-associated phenotypes and nominate specific members of the collection for development as candidate SMA therapeutics. More generally, the results reinforce the perception of SMA as an axonopathy and suggest novel approaches to treating the disease. PMID:24023935

Gassman, Andrew; Hao, Le T.; Bhoite, Leena; Bradford, Chad L.; Chien, Chi-Bin; Beattie, Christine E.; Manfredi, John P.

2013-01-01

347

An automated predator avoidance task in zebrafish  

PubMed Central

Zebrafish are becoming increasingly popular in behavioral neuroscience as investigators have started to realize the benefits of sophisticated genetic tools specifically developed for this species along with the pharmacological tools already available for other laboratory model organisms. The zebrafish has been proposed as an in vivo tool for the analysis of vertebrate fear responses as well as human psychopathological conditions such as anxiety. We have been developing behavioral tasks for zebrafish that could be utilized for screening mutation or drug induced changes in fear responses. In this paper we present a modified version of a previously developed predator avoidance paradigm that now allows the induction and quantification of avoidance reactions that we previously could not elicit. Most importantly, in the current paradigm zebrafish are now shown to respond to the appearance of a moving image of a sympatric predator, the Indian leaf fish, by increasing their distance from the image, a robust reaction that is easy to quantify in an automated manner. Unexpectedly, however, another fear response, the “diving” response, was seen robustly only at the beginning of the test but not in response to the predator stimulus. We discuss the implications of these results and conclude that although zebrafish fear responses are complex and context dependent, the current paradigm is a significant step towards high throughput screening for alterations in fear responses of zebrafish. PMID:20674614

Ahmed, Omar; Seguin, Diane; Gerlai, Robert

2010-01-01

348

A spatio-temporal model of Notch signalling in the zebrafish segmentation clock: conditions for synchronised oscillatory dynamics.  

PubMed

In the vertebrate embryo, tissue blocks called somites are laid down in head-to-tail succession, a process known as somitogenesis. Research into somitogenesis has been both experimental and mathematical. For zebrafish, there is experimental evidence for oscillatory gene expression in cells in the presomitic mesoderm (PSM) as well as evidence that Notch signalling synchronises the oscillations in neighbouring PSM cells. A biological mechanism has previously been proposed to explain these phenomena. Here we have converted this mechanism into a mathematical model of partial differential equations in which the nuclear and cytoplasmic diffusion of protein and mRNA molecules is explicitly considered. By performing simulations, we have found ranges of values for the model parameters (such as diffusion and degradation rates) that yield oscillatory dynamics within PSM cells and that enable Notch signalling to synchronise the oscillations in two touching cells. Our model contains a Hill coefficient that measures the co-operativity between two proteins (Her1, Her7) and three genes (her1, her7, deltaC) which they inhibit. This coefficient appears to be bounded below by the requirement for oscillations in individual cells and bounded above by the requirement for synchronisation. Consistent with experimental data and a previous spatially non-explicit mathematical model, we have found that signalling can increase the average level of Her1 protein. Biological pattern formation would be impossible without a certain robustness to variety in cell shape and size; our results possess such robustness. Our spatially-explicit modelling approach, together with new imaging technologies that can measure intracellular protein diffusion rates, is likely to yield significant new insight into somitogenesis and other biological processes. PMID:21386903

Terry, Alan J; Sturrock, Marc; Dale, J Kim; Maroto, Miguel; Chaplain, Mark A J

2011-01-01

349

Axonal Transport Defects in a Mitofusin 2 Loss of Function Model of Charcot-Marie-Tooth Disease in Zebrafish  

PubMed Central

Charcot-Marie-Tooth disease (CMT) represents a group of neurodegenerative disorders typically characterised by demyelination (CMT1) or distal axon degeneration (CMT2) of motor and sensory neurons. The majority of CMT2 cases are caused by mutations in mitofusin 2 (MFN2); an essential gene encoding a protein responsible for fusion of the mitochondrial outer membrane. The mechanism of action of MFN2 mutations is still not fully resolved. To investigate a role for loss of Mfn2 function in disease we investigated an ENU-induced nonsense mutation in zebrafish MFN2 and characterised the phenotype of these fish at the whole organism, pathological, and subcellular level. We show that unlike mice, loss of MFN2 function in zebrafish leads to an adult onset, progressive phenotype with predominant symptoms of motor dysfunction similar to CMT2. Mutant zebrafish show progressive loss of swimming associated with alterations at the neuro-muscular junction. At the cellular level, we provide direct evidence that mitochondrial transport along axons is perturbed in Mfn2 mutant zebrafish, suggesting that this is a key mechanism of disease in CMT. The progressive phenotype and pathology suggest that zebrafish will be useful for further investigating the disease mechanism and potential treatment of axonal forms of CMT. Our findings support the idea that MFN2 mutation status should be investigated in patients presenting with early-onset recessively inherited axonal CMT. PMID:23840650

Chapman, Anna L.; Bennett, Ellen J.; Ramesh, Tennore M.; De Vos, Kurt J.; Grierson, Andrew J.

2013-01-01

350

Ontogeny and nutritional control of adipogenesis in zebrafish (Danio rerio)  

PubMed Central

The global obesity epidemic demands an improved understanding of the developmental and environmental factors regulating fat storage. Adipocytes serve as major sites of fat storage and as regulators of energy balance and inflammation. The optical transparency of developing zebrafish provides new opportunities to investigate mechanisms governing adipocyte biology, however zebrafish adipocytes remain uncharacterized. We have developed methods for visualizing zebrafish adipocytes in vivo by labeling neutral lipid droplets with Nile Red. Our results establish that neutral lipid droplets first accumulate in visceral adipocytes during larval stages and increase in number and distribution as zebrafish grow. We show that the cellular anatomy of zebrafish adipocytes is similar to mammalian white adipocytes and identify peroxisome-proliferator activated receptor ? and fatty acid binding protein 11a as markers of the zebrafish adipocyte lineage. By monitoring adipocyte development prior to neutral lipid deposition, we find that the first visceral preadipocytes appear in association with the pancreas shortly after initiation of exogenous nutrition. Zebrafish reared in the absence of food fail to form visceral preadipocytes, indicating that exogenous nutrition is required for adipocyte development. These results reveal homologies between zebrafish and mammalian adipocytes and establish the zebrafish as a new model for adipocyte research. PMID:19366995

Flynn, Edward J.; Trent, Chad M.; Rawls, John F.

2009-01-01

351