Project description:PURPOSE: Tissue culture is traditionally performed at atmospheric oxygen concentration (21%), which induces hyperoxic stress, as endogenous physiologic oxygen tension found in tissues varies between 2% and 9%. This discrepancy may lead to misinterpretation of results and may explain why effects observed in vitro cannot always be reproduced in vivo and vice versa. Only a few studies have been conducted in low physiologic oxygen conditions to understand the development and differentiation of cells from the eye. METHODS: The aim of this study was to investigate the growth and gene expression profile of melanocytes from the choroid permanently exposed to 21% (hyperoxic) or 3% (physiologic) oxygen with proliferation assays and DNA microarray. The cellular behavior of the melanocytes was then compared to that of cancer cells. RESULTS: The gross morphology and melanin content of choroidal melanocytes changed slightly when they were exposed to 3% O2, and the doubling time was statistically significantly faster. There was an increase in the percentage of choroidal melanocytes in the active phases of the cell cycle as observed by using the proliferation marker Ki67. The caveolin-1 senescence marker was not increased in choroidal melanocytes or uveal melanoma cells grown in hyperoxia. In comparison, the morphology of the uveal melanoma cells was similar between the two oxygen levels, and the doubling time was slower at 3% O2. Surprisingly, gene expression profiling of the choroidal melanocytes did not reveal a large list of transcripts considerably dysregulated between the two oxygen concentrations; only the lactate transporter monocarboxylate transporter (MCT4) was statistically significantly upregulated at 3% O2. CONCLUSIONS: This study showed that the oxygen concentration must be tightly controlled in experimental settings, because it influences the subsequent cellular behavior of human choroidal melanocytes.
Project description:Study the RNA expression differences in uveal melanoma cells and normal melanocytes, providing new insights into the tumorgenesis of uveal melanoma.
Project description:Genome wide DNA methylation profiling of primary uveal melanoma cells, normal uveal melanocytes, neural crest stem cells, embryonic stem cells and uveal melanoma cell lines. The Illumina Infinium 27k Human DNA methylation Beadchip Rev B was used to obtain DNA methylation profiles across approximately 27,000 CpGs in the samples. Samples included 58 primary UM, 3 NUM and NCSC controls and 2 cell lines. Bisulphite converted DNA from the 63 samples were hybridised to the Illumina Infinium 27k Human Methylation Beadchip Rev B
Project description:Genome wide DNA methylation profiling of primary uveal melanoma cells, normal uveal melanocytes, neural crest stem cells, embryonic stem cells and uveal melanoma cell lines. The Illumina Infinium 27k Human DNA methylation Beadchip Rev B was used to obtain DNA methylation profiles across approximately 27,000 CpGs in the samples. Samples included 58 primary UM, 3 NUM and NCSC controls and 2 cell lines.
Project description:Purpose: Epigenetic mechanisms and alterations in uveal melanoma (UM) development are still not well understood. In this pilot study, histone posttranslational modifications (PTMs), which are epigenetic mechanisms regulating gene expression, were analyzed in UM formalin-fixed paraffin-embedded (FFPE) tissues and control tissue as well as in UM cell lines and healthy melanocytes to provide a deeper insight into the pathogenesis of UM and the potentially prognostic relevance of these molecular markers. Methods: FFPE tissue of UM (n=24) and normal choroid (n=4) as well as human UM cell lines (n=7), human skin melanocytes (n=6) and uveal melanocytes (n=2), were analyzed by a quantitative mass spectrometry (MS) approach.
Project description:G protein alpha q and 11 are mutated in 80% of uveal melanoma. We observed that treatment with the BRD4 inhibitor JQ1 resulted in different phenotypic responses in G-protein mutant uveal melanoma cell lines and wild type uveal melanoma cell lines. We used microarrarys to profile the gene expression changes occuring in wild type and mutant cell lines in response to treament with JQ1 Uveal melanoma cells were profiled in triplicate on Affymetrix Human Genome U133A 2.0 Array arrays per manufacturer's instructions
Project description:Uveal melanoma is the most common cancer of the eye arising from melanocytes within the choroid, ciliary bodies and iris. Almost half of uveal melanomas metastasize hematogenously to distant organs, most often the liver, where the disease becomes fatal. One of the first genetic alterations to be identified in primary uveal melanomas was monosomy 3, which was found to be strongly associated with metastasis. We used gene expression profiling to identify two phenotypically distinct classes of uveal melanomas: class 1 tumors with low-grade morphology and low metastatic risk; and class 2 tumors with aggressive morphology and high metastatic risk. Our initial studies suggested that gene expression profiling was a better predictor of metastasis than monosomy 3. For this study, BAC-array comparative genomic hybridization was used to assay genomic DNA isolated from fresh frozen primary uveal melanoma samples of known molecular class based on gene expression profiling. Independent samples were sent to the aCGH cores at the University of California, San Francisco and the Roswell Park Cancer Institute for hybridization, and log2 ratios were reported. Total genomic DNA was obtained from tumor samples that were collected at the time of treatment. Samples were analyzed by the Microarray Shared Resource at the Comprehensive Cancer Center, University of California, San Francisco or by the Microarray and Genomics Facility of the Roswell Park Cancer Institute.
