Osteosarcoma is an aggressive malignancy and represents the most frequent primary bone malignancy of dogs and humans. Prognostic factors reported for osteosarcoma include tumor size, presence of metastatic disease, and serum alkaline phosphatase (ALP) concentration at the time of diagnosis. To date, there have been no studies to determine whether the behavior of osteosarcoma cells differ based on serum ALP concentration. Here we report on the generation of six canine osteosarcoma cell lines from osteosarcoma-bearing dogs with differences in serum ALP concentration. To determine whether in vitro behavior differs between primary osteosarcoma cell lines generated from patients with normal or increased serum ALP assays were performed to evaluate proliferation, migration, invasion, and chemosensitivity. There were no significant differences in cell proliferation, migration, invasion, or chemosensitivity between cell lines associated normal or increased serum ALP concentration.
Osteosarcoma (OSA) is the most frequently occurring malignant primary bone tumour in dogs and children and arises from cells of the osteoblast lineage. Inappropriate Wnt signalling activity has been implicated in human OSA. Altered expression of β-catenin, an integral member of the Wnt signalling pathway, has been associated with numerous human cancers, including OSA. In this study, 30 of the 37 primary canine OSA tissues and 2 of the 3 metastatic OSAs were positive for β-catenin expression as determined by immunohistochemistry, whereas 2 normal bones stained negative for β-catenin. No mutations were identified in exon 3 of β-catenin in the three OSA cases in which DNA sequencing was performed. Finally, there was no relationship between β-catenin expression and overall survival time or disease-free interval. Our results indicate β-catenin is frequently expressed within the cytoplasm of neoplastic cells in canine OSA but contains no detectable mutations in exon 3, similar to human OSA.
In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1-to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.
Background Mutations in the Wnt signalling pathway molecule β-catenin are associated with liver cancer. Aims Our aim was to confirm the effects of stabilized β-catenin on liver growth, identify whether those effects were reversible and cell autonomous or non-cell autonomous and to model β-catenin-induced liver cancer in mice. Methods Using a liver-specific inducible promoter, we generated transgenic mice in which the expression of mutant β-catenin can be induced or repressed within hepatocytes in mice of different ages. Results Similar to other models, the hepatic expression of mutant β-catenin in our model beginning in utero or induced in quiescent adult liver resulted in a two-fold liver enlargement and development of disease with a latency of 1–5 months, and mice displayed elevated blood ammonia and altered hepatic gene expression. Our model additionally allowed us to discover that molecular and phenotypic abnormalities were reversible following the inhibition of transgene expression. Hepatocyte transplant studies indicated that mutant β-catenin could not increase the growth of transgene-expressing foci in either growth-permissive or -restrictive hepatic environments, but still directly altered hepatocyte gene expression. Mice with continuous but focal transgene expression developed hepatic neoplasms after the age of 1 year. Conclusions Our findings indicate that hepatocyte gene expression is directly affected by mutant β-catenin in a cell autonomous manner. However, hepatomegaly associated with diffuse hepatocyte-specific expression of mutant β-catenin is secondary to liver functional alteration or non-cell autonomous. Both phenotypes are reversible. Nevertheless, some foci of transgene-expressing cells progressed to carcinoma, confirming the association of mutant β-catenin with liver cancer.
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