Unknown,Transcriptomics,Genomics,Proteomics

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C3A Cells: Exposure vs Control [one-channel arrays]

ABSTRACT: Gene expression profiles comparing FCCP, CCCP, Dinoterb, Benzoapyrene, TCP, Medium control, 0,1% DMSO in C3A cells C3A cells were exposed for 2 hours to substances, 6 replicate wells per concentration were pooled. Total RNA was extracted using RNeasy Mini Kit (QUIAGEN, Product 74104, Hombrechtikon, Switzerland). Quality of extracted RNA was checked via the Nanodrop 1000 spectraphotomoeter V3.7, as well as running the samples on a 1.5% agarose gel. cDNA was generated and labeled with Cy3 or Cy5-CTP using the low RNA input linear amplification kit (Perkin-Elmer). Total RNA was reverse transcribed to cDNA using RNasinÂ® Plus RNAse Inhibitor (N2611), M-MLV Reverse transcriptase (RNase H Minus, Point Mutant) (M3682), 5x Buffer (M3682) and random primer (C1181) from Promega AG (product number in brackets) (Promega AG, DÃ¼bendorf, Switzerland). For Microarray analysis Agilent SurePrint G3 Human Gene Expression Microarrays (8x60K) were used. Each treatment consisted of two biological replicates, the control consisted of three biological replicates.

ORGANISM(S): Homo sapiens

SUBMITTER: Jessica Legradi

PROVIDER: E-GEOD-75783 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Tumor cell invasion and metastasis are hallmarks of malignancy. Despite recent advances in the understanding of lymphatic spread, the mechanisms by which tumors metastasize to sentinel/distant lymph nodes and beyond are poorly understood. To gain new insights into this complex process, we established a highly metastatic melanoma cell line (B16F1-variant) by in vivo passaging the B16 parental cell line through the lymphatic system. Here, we characterized morphology, rate of cell proliferation, colony formation, migration, tumorogenicity, lymph flow, and capacities to induce tumor- and sentinel lymph node- lymphangiogenesis. Furthermore, microarray-based comparative analysis bewteen parental and passaged cell lines was performed to identify specific gene expression profiles. The most differentially expressed gene was SPP (osteopontin), a secreted glycophosphoprotein which is known to be involved in cancer metastasis. Overexpression of osteopontin in B16 F1-variant was confirmed by Western blot and quantitative RT-PCR. Treatment of cultured lymphatic endothelial cells (LEC) with osteopontin promoted cell migration mediated by the integrin α9 pathway. Our results identify osteopontin as a novel lymphangiogenic factor. B16 and B16 variants were cultured in DMEM supplemented with 20% fetal bovine serum (FBS; Invitrogen, Grand Island, NY) supplemented with antibiotic-antimycotic solution. Every B16 variant was cultured in duplicates. Total cellular RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA) extracted with chloroform, precipitated with isopropanol, washed with 70% ethanol, and dissolved in DNase-free/RNase-free distilled water. The concentration of RNA was measured using NanoDrop ND-1000 spectrophotometer (Witec AG, Littau, Switzerland) and RNA quality was assessed using 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Digoxigenin-UTP–labeled cRNA was generated and amplified from 500ng of total RNA using the NanoAmp RT-IVT Labeling Kit (Applied Biosystems, Foster City, CA) following the manufacturer's protocol, and was hybridized to Applied Biosystems Mouse Genome Survey Microarrays V2.0. Chemiluminescence detection, image acquisition, and analysis were performed using the Chemiluminescence Detection Kit (Applied Biosystems) and the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer following the manufacturer's protocol. A total of two biological replicates were generated for each B16 variant and each cell line data consisted of pooled RNA of two diffferent passage numbers.

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C3A Cells: Exposure vs Control [one-channel arrays]

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