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CDNA microarray profiles of canine mammary tumour cell lines reveal deregulated pathways pertaining to their phenotype

ABSTRACT: Mammary cancer is the most common type of cancer in female dogs with a lifetime risk of over 24% when dogs are not spayed. The elucidation of the complete canine genome opens new areas for development of cancer therapies. These should be tested first by in vitro models such as cell lines. However, to date, no canine mammary cell lines have been characterized by expression profiling. In this study, canine mammary tumour cell lines with histologically distinct primary tumours of origin were characterized using a newly developed canine cDNA microarray. Comparisons of gene expression profiles showed enrichment for distinct biological pathways and were related to biological properties of the cell lines such as growth rate and in vitro tumourigenicity. Additionally, gene expression profiles of cell lines also showed correspondence to their tumour of origin. Major differences were found in Wnt, cell cycle, cytokine/Rho-GTPase, alternative complement and integrin signalling pathways. Because these pathways show an overlap at the molecular level with those found in human breast cancer, the expression profiling of spontaneous canine mammary cancer may also function as a biological sieve to identify conserved gene expression or pathway profiles of evolutionary significance that are involved in tumourigenesis. These results are the basis for further characterization of canine mammary carcinomas and development of new therapies directed towards specific pathways. In addition these cell lines can be used to further investigate identified deregulated pathways and characterize until now unannotated genes. Keywords: cell line type comparision Three canine mammary tumor cell lines (CMT) originating from benign mixed tumor (CMT-U229), primary mammary osteosarcoma (CMT-U335) and primary mammary anaplastic carcinoma (P114) were compared directly to each other in this study. Total RNA was isolated from cells grown to near confluence. In vitro transcription followed by labeling and hybridization to cDNA microarray was carried out. In a loop design of hybridization, labeled cRNA from cell lines were hybridized against each other. Statistical analysis of the log transformed normalized data was done using SAM (significance analysis of microarray) and differentially expressed genes from each experimental subset (comparison of two cell lines) were identified. Dye swaps and at least one biological replicates were included under each experimental subset (each comparision).

ORGANISM(S): Canis lupus familiaris

SUBMITTER: Nagesha Rao

PROVIDER: E-GEOD-7659 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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cDNA microarray profiles of canine mammary tumour cell lines reveal deregulated pathways pertaining to their phenotype.

Rao N A S NA van Wolferen M E ME van den Ham R R van Leenen D D Groot Koerkamp M J A MJ Holstege F C P FC Mol J A JA

Animal genetics 20080506 4

Mammary cancer is the most common type of cancer in female dogs with a lifetime risk of over 24% when dogs are not spayed. The elucidation of the complete canine genome opens new areas for development of cancer therapies. These should be tested first by in vitro models such as cell lines. However, to date, no canine mammary cell lines have been characterized by expression profiling. In this study, canine mammary tumour cell lines with histologically distinct primary tumours of origin were charac ...[more]

PMID: 18462483

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Project description:Background. Gene expression microarrays have been intensively applied to screen for genes involved in specific biological processes of interest such as diseases or responses to environmental stimuli. For mammalian species, cataloging of the global gene expression profiles in large tissue collections under normal conditions have been focusing on human and mouse genomes but is lacking for the pig genome. Methodology/Principal Findings. Here we present the results from a large-scale porcine study establishing microarray cDNA expression profiles of approximately 20.000 genes across 23 healthy tissues. As expected, a large portion of the genes show tissue specific expression in agreement with mappings to gene descriptions, Gene Ontology terms and KEGG pathways. Two-way hierarchical clustering identified expected tissue clusters in accordance with tissue type and a number of cDNA clusters having similar gene expression patterns across tissues. For one of these cDNA clusters, we demonstrate that possible tissue associated gene function can be inferred for previously uncharacterized genes based on their shared expression patterns with functionally annotated genes. We show that gene expression in common porcine tissues is similar to the expression in homologous tissues of human. Conclusions/Significance. The results from this study constitute a valuable and publicly available resource of basic gene expression profiles in normal porcine tissues and will contribute to the identification and functional annotation of porcine genes. Keywords: Microarray, Gene expression survey, Large-scale, Normal Healthy tissues, cDNA, Whole genome, Transcriptome The microarray experiment was carried out using a two-channel common reference design with two independent RNA extractions from each tissue sample. For each total-RNA extraction, 20 μg was labeled with Alexa 594 and 20 μg with Alexa 488 using SuperScript Indirect cDNA labeling System from Invitrogen. The RNA extracts labeled with Alexa 488 was collected from all tissue samples, mixed and used as reference sample referred to here as a the common reference. The RNA extracts labeled with Alexa 594 were used individually as labeled tissue samples. The labeled RNA extracts were used in two batches of 23 hybridizations corresponding to the two independent RNA extraction batches. One labeled tissue sample RNA (Alexa 594) and one labeled common reference sample RNA (Alexa 488) was thus hybridized to each of 46 microarrays. The same common reference sample was thus used in all the 46 (2x23) hybridizations. Two rounds of hybridizations on a Discovery XT hybridization station from Ventana were carried out corresponding to the two RNA batches. The hybridized microarrays were scanned and converted into TIFF images using Scanner and ScanArray Express software from Perkin Elmer.

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CDNA microarray profiles of canine mammary tumour cell lines reveal deregulated pathways pertaining to their phenotype

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cDNA microarray profiles of canine mammary tumour cell lines reveal deregulated pathways pertaining to their phenotype.

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