[Show abstract][Hide abstract] ABSTRACT: Myc is a pleiotropic transcription factor that is involved in many cellular activities relevant to carcinogenesis including hepatocarcinogenesis. The zebrafish has been increasingly used to model human diseases and it is particularly valuable to help identify common and conserved molecular mechanisms in vertebrates. Here we generated a liver tumor model in transgenic zebrafish by liver-specific expression of mouse Myc using a Tet-on system. Dosage-dependent induction of Myc expression specifically in the liver was observed in our Myc transgenic zebrafish, TO(Myc), and the elevated Myc expression caused liver hyperplasia which was progressed to hepatocellular adenoma and carcinoma with prolonged induction. Next generation sequencing-based transcriptomic analyses indicated that ribosome proteins were overwhelmingly up-regulated in the Myc-induced liver tumors. Cross species analyses showed that the zebrafish Myc model correlated well with Myc transgenic mouse models for liver cancers. The Myc-induced zebrafish liver tumors also possessed molecular signatures highly similar to human hepatocellular carcinoma. Finally, we found that a small Myc target gene set of 16 genes that could be used to identify liver tumors due to Myc up-regulation. Thus, our zebrafish model demonstrated the conserved role of Myc in promoting hepatocarcinogenesis in all vertebrate species.
[Show abstract][Hide abstract] ABSTRACT: Abstract
Myc is a pleiotropic transcription factor that is involved in many cellular activities relevant to carcinogenesis including hepatocarcinogenesis. The zebrafish has been increasingly used to model human diseases and it is particularly valuable to help identify common and conserved molecular mechanisms in vertebrates. Here we generated a liver tumor model in transgenic zebrafish by liver-specific expression of mouse Myc using a Tet-on system. Dosage-dependent induction of Myc expression specifically in the liver was observed in our Myc transgenic zebrafish, TO(Myc), and the elevated Myc expression caused liver hyperplasia which was progressed to hepatocellular adenoma and carcinoma with prolonged induction. Next generation sequencing-based transcriptomic analyses indicated that ribosome proteins were overwhelmingly up-regulated in the Myc-induced liver tumors. Cross species analyses showed that the zebrafish Myc model correlated well with Myc transgenic mouse models for liver cancers. The Myc-induced zebrafish liver tumors also possessed molecular signatures highly similar to human hepatocellular carcinoma. Finally, we found that a small Myc target gene set of 16 genes that could be used to identify liver tumors due to Myc up-regulation. Thus, our zebrafish model demonstrated the conserved role of Myc in promoting hepatocarcinogenesis in all vertebrate species.
Disease Models and Mechanisms 10/2012; · 4.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although the underlying molecular mechanism of hepatocellular carcinoma remains unclear, signalling pathways essential in cell survival and growth are altered, including the Raf-MEK-MAPK pathway. This pathway can be activated by hepatitis B or C virus infections and the ectopic expression of the Raf-1 oncogene is frequently seen in hepatocellular carcinomas. In addition, the Raf-MEK-MAPK pathway was also shown to be deregulated in zebrafish liver tumours. Based on the genetic conservation between zebrafish and human liver tumours, the zebrafish was used as an animal model to better understand the molecular basis of hepatocellular carcinoma. Here we establish an inducible oncogenic zebrafish cell model, in which oncogenic human Raf-1(ΔRaf1) can be post-transcriptionally activated in zebrafish liver cells by administration of 4-hydroxytamoxifen (4HT). The ΔRaf1 activation resulted in the hyperactivation of the zebrafish MEK-ERK cascade, promoted cell growth and proliferation, and inhibited apoptosis. The mitogenic transformation of the ZFL-ΔRaf1-ER cells was confirmed by in vivo allo-transplantation and in silico microarray analyses. Gene expression profiling of cells treated with 4HT and a MEK-inhibitor identified a Raf-MEK-dependent signature set. This transcriptome response was compared to zebrafish and human liver cancer transcriptomes. We identified, and validated by quantitative PCR, a set of genes transcriptionally regulated by hyperactive MAPK signalling in ZFL-ΔRaf1-ER cells, zebrafish liver tumours and human liver tumours, suggesting that the in vitro zebrafish liver cell model can be used for further study of the molecular basis of human hepatocellular carcinoma. The molecular targeting of the commonly regulated hepatocellular carcinoma genes using the ZFL-ΔRaf1-ER cell model can be applied for high-throughput preclinical target discovery.
The Journal of Pathology 09/2011; 225(1):19-28. DOI:10.1002/path.2936 · 7.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Liver cancer, mainly hepatocellular carcinoma, is a major malignancy and currently there are no effective treatment protocols due to insufficient understanding of hepatocarcinogenesis. As a potentially high-throughput and cost-effective experimental model, the zebrafish is increasingly recognized for disease studies. Here, we aim at using the zebrafish to generate a convenient hepatocellular carcinoma model.
Using the Tet-on system for liver-specific expression of fish oncogene xmrk, a hyperactive version of epidermal growth factor receptor homolog, we have generated transgenic zebrafish with inducible development of liver cancer.
Liver tumors were rapidly induced with 100% penetrance in both juvenile and adult xmrk transgenic fish. Histological examination indicated that they all showed features of hepatocellular carcinoma. The induced liver tumors regressed rapidly upon inducer withdrawal. During the tumor induction stage, we detected increased cell proliferation and activation of Xmrk downstream targets Erk and Stat5, which were important for liver tumorigenesis as proved by inhibition experiments. When tumors regressed, there were decreased phosphorylated Erk and Stat5 accompanied with an increase in apoptosis.
Our zebrafish model demonstrates the potential of a hyperactivated epidermal growth factor receptor pathway in initiating heptocarcinogenesis. It provides clear evidence for the requirement of only a single oncogene for HCC initiation and maintenance and is thus a convenient model for further investigation of oncogene addiction and future anti-cancer drug screening.
Journal of Hepatology 08/2011; 56(2):419-25. DOI:10.1016/j.jhep.2011.07.025 · 11.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA methylation is an epigenetic mechanism associated with regulation of gene expression and it is modulated during chemical carcinogenesis. The zebrafish is increasingly employed as a human disease model; however there is a lack of information on DNA methylation in zebrafish and during fish tumorigenesis.
A novel CpG island tiling array containing 44,000 probes, in combination with immunoprecipitation of methylated DNA, was used to achieve the first comprehensive methylation profiling of normal adult zebrafish liver. DNA methylation alterations were detected in zebrafish liver tumors induced by the environmental carcinogen 7, 12-dimethylbenz(a)anthracene. Genes significantly hypomethylated in tumors were associated particularly with proliferation, glycolysis, transcription, cell cycle, apoptosis, growth and metastasis. Hypermethylated genes included those associated with anti-angiogenesis and cellular adhesion. Of 49 genes that were altered in expression within tumors, and which also had appropriate CpG islands and were co-represented on the tiling array, approximately 45% showed significant changes in both gene expression and methylation.
The functional pathways containing differentially methylated genes in zebrafish hepatocellular carcinoma have also been reported to be aberrantly methylated during tumorigenesis in humans. These findings increase the confidence in the use of zebrafish as a model for human cancer in addition to providing the first comprehensive mapping of DNA methylation in the normal adult zebrafish liver.
[Show abstract][Hide abstract] ABSTRACT: The zebrafish (Danio rerio) has been increasingly recognized as a promising animal model for cancer research. Zebrafish tumors can be generated by treatment
with chemical carcinogens or by genetic approaches. The liver has been a main target organ for tumorigenesis after carcinogen
treatment while many other tissue-specific tumors have been generated by tissue-specific expression of proven oncogenes. We
have used both the chemical and transgenic approaches to generate liver tumors. By comparative analyses of transcriptome profiles
between human liver tumors and carcinogen-induced zebrafish liver tumors, we have demonstrated a remarkable similarity in
the molecular hallmarks during liver tumorigenesis between humans and zebrafish, thus validating the zebrafish model for human
cancer studies. Recently, we have also generated stable transgenic zebrafish lines overexpressing the c-Myc and kras
in the liver using two different inducible gene expression systems. In both cases, we found that tumors can be reproducibly
induced in the liver, and histopathological examination confirmed the production of liver neoplasia including heptocellular
carcinoma. Thus, we have successfully established transgenic zebrafish models for liver cancers and these models will be further
characterized in order to understand the molecular and genetic mechanisms of liver carcinogenesis as well as for anti-cancer
Molecular Genetics of Liver Neoplasia, 12/2010: pages 197-218;
[Show abstract][Hide abstract] ABSTRACT: A retrovirus homologue gene of cellular cyclin D₁, walleye dermal sarcoma virus rv-cyclin gene (orf A or rv-cyclin), was expressed in the livers of zebrafish under the control of liver fatty acid-binding protein (lfabp) promoter. To prevent possible fatality caused by overexpression of the oncogene, the GAL4/upstream activation sequence (GAL4/UAS) system was used to maintain the transgenic lines. Thus, both GAL4-activator [Tg(lfabp:GAL4)] and UAS-effector [Tg(UAS:rvcyclin)] lines were generated, and the rv-cyclin gene was activated in the liver after crossing these two lines. Since no obvious neoplasia phenotypes were observed in the double-transgenic line, cancer susceptibility of the transgenic fish expressing rv-cyclin was tested by carcinogen treatment. Unexpectedly, transgenic fish expressing rv-cyclin gene (rvcyclin+) were more resistant to the carcinogen than siblings not expressing this gene (rvcyclin-). Lower incidences of multiple and malignant liver tumors were observed in rvcyclin+ than in rvcyclin- fish, and the liver tumors in the rvcyclin+ group appeared later and were less malignant. These results suggest that expression of rv-cyclin protects the fish liver from carcinogen damage and delays onset of malignancy. These findings indicate that transgenic fish models are powerful systems for investigating mechanisms of inhibition and regression of liver tumors.
[Show abstract][Hide abstract] ABSTRACT: Walleye dermal sarcoma (WDS) is a benign tumor of walleye fish that develops and completely regresses seasonally. The retrovirus associated with this disease, walleye dermal sarcoma virus, encodes three accessory genes, two of which, rv-cyclin (orfA) and orfb, are thought to play a role in tumor development. In this study, we attempted to recapitulate WDS development by expressing rv-cyclin in chimeric and stable transgenic zebrafish. Six stable transgenic lines expressing rv-cyclin from the constitutive CMVtk promoter were generated. Immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction demonstrate that rv-cyclin is widely expressed in different tissues in these fish. These lines were viable and histologically normal for up to 2 years. No increase in tumors or tissue proliferation was observed following N-ethyl N-nitrosourea exposure or following tail wounding and subsequent tissue regeneration compared to controls. These data indicate that rv-cyclin is not independently sufficient for tumor induction in zebrafish.
[Show abstract][Hide abstract] ABSTRACT: A stable Tg(UAS:GFP) zebrafish line was generated and crossed with Tg(hsp70:GAL4) line, in which the GAL4 gene is under the control of an inducible zebrafish promoter derived from the heat shock 70 protein gene (hsp70). The dynamic green fluorescent protein (GFP) expression in early zebrafish embryos in the GAL4/UAS binary system was then investigated. We found that, at early developmental stages, expression of GFP effector gene was restricted and required a long recovery time to reach a detectable level. At later developmental stage (after 2 days postfertilization), GFP could be activated in multiple tissues in a shorter time, apparently due to a higher level of GAL4 messenger RNA induction. It appears that the type of tissues expressing GFP was dependent on whether they had been developed at the time of heat shock. Therefore, the delayed and restricted transgene expression should be taken into consideration when GAL4/UAS system is used to study transgene expression in early developmental stages.
[Show abstract][Hide abstract] ABSTRACT: For decades scientists have observed that chronic inflammation is associated with increased neoplasia in the inflamed tissue. Many human and animal cancers are associated with chronic infection by parasites, bacteria, viral agents, or with chronic inflammation due to genetic disease. Medical scientists have also observed increased lymphoma and leukemia of the lymphoid lineage, and more recently in the myelomonocytic lineage, in humans and animals with chronic infection. Our goal in this research was to develop a reliable method to induce a high incidence of intestinal neoplasia in zebrafish to allow better understanding of the molecular pathogenesis of these neoplasms. In our carcinogenesis studies with wild-type lines of zebrafish at Oregon State University, the incidence of intestinal neoplasia never exceeded 16%. Therefore, we chose to study mutant lines of zebrafish given bath treatment as fry with the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA). To maximize intestinal neoplasia, we examined the influence of infection with the intestinal nematode Pseudocapillaria tomentosa on the incidence of intestinal neoplasia. Using the uma mutant line in a TL genetic background given a single treatment with DMBA prior to parasite infection, the incidence of intestinal neoplasia did not exceed 10% by 6-10 months of age, despite parasite infection. The uma line develops myelodysplastic syndrome (MDS) and leukemia unrelated to carcinogen treatment. However, variable penetrance of this hemopoietic neoplasia has made study of this blood neoplasia difficult. In our studies with Pseudocapillaria infection, the incidence of MDS/leukemia in fish of the uma line not infected with parasites was 1/101 (0.9%), regardless of carcinogen treatment. In contrast, the incidence of MDS/leukemia in fish of the uma line infected with parasites was 8/60 (13%; P< 0.05, chi-square test). Studies conducted with the Singapore (SING) line of zebrafish infected with Pseudocapillaria early in life, then given bath exposure to DMBA at 3 and 5 weeks post-fertilization showed incidences of intestinal neoplasia greater than 50% by 1 year post-treatment. Although different lines of fish were used in the different protocols, these experiments suggest that the optimal protocol for maximizing intestinal neoplasia will infect fish with parasites prior to carcinogen treatment.
8th Internaional Conference on Zebrafish Development and Genetics, Madison, Wisconsin; 06/2008
[Show abstract][Hide abstract] ABSTRACT: Although the transgenic technology has been successfully used to generate fluorescent zebrafish and medaka for ornamental purposes, the practicability of the technology has not been demonstrated in other ornamental fish species. In the present study, we have tested the transgenic technology in a bona fide ornamental fish species, the white skirt tetra (Gymnocorymbus ternetzi). First, its embryonic development was briefly described. Second, we successfully introduced an rfp (red fluorescent protein) gene construct driven by a strong muscle-specific mylz2 promoter from the zebrafish into the white skirt tetra and demonstrated muscle-specific expression of the RFP reporter protein. Importantly, the vivid red fluorescent color was prominently visible in adult transgenic founders under the normal daylight, like the currently marketed red fluorescent transgenic zebrafish. Thus, our current study demonstrated the feasibility of using the well-characterized zebrafish mylz2 promoters to produce useful fluorescent ornamental fish in other fish species by the transgenic technology.
[Show abstract][Hide abstract] ABSTRACT: The zebrafish (Danio rerio) has been long advocated as a model for cancer research, but little is known about the real molecular similarities between zebrafish and human tumors. Comparative analysis of microarray data from zebrafish liver tumors with those from four human tumor types revealed molecular conservation at various levels between fish and human tumors. This approach provides a useful strategy for identifying an expression signature that is strongly associated with a disease phenotype.