Investigation Title Transcription profiling of mouse day 0 versus day 1 involuting mammary gland Comment[Submitted Name] Expression analysis of Day 0 versus Day 1 involuting mouse mammary gland Experimental Design unknown_experiment_design_type transcription profiling by array Experimental Design Term Source REF EFO Comment[ArrayExpressReleaseDate] 2008-06-13 Comment[SecondaryAccession] GSE5317 Comment[AEMIAMESCORE] 2 Comment[ArrayExpressAccession] E-GEOD-5317 Comment[MAGETAB TimeStamp_Version] 2010-08-06 15:44:56 Last Changed Rev: 13058 Experimental Factor Name Experimental Factor Type Experimental Factor Term Source REF Person Last Name Myal Person First Name Yvonne Person Mid Initials Person Email myal@cc.umanitoba.ca Person Phone Person Fax Person Address Dr. Yvonne Myal, Pathology, University of Manitoba, 770 Bannatyne Ave., Winnipeg, R3E0W3, Canada Person Affiliation University of Manitoba Person Roles submitter Person Roles Term Source REF Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2008-06-13 PubMed ID 17127459 Publication DOI 17127459 Publication Author List Anne Blanchard, Robert Shiu, Stephanie Booth, Garrett Sorensen, Nicole DeCorby, Andreea Nistor, Paul Wong, Etienne Leygue, Yvonne Myal Publication Title Gene expression profiling of early involuting mammary gland reveals novel genes potentially relevant to human breast cancer. Publication Status journal_article Publication Status Term Source REF The MGED Ontology Experiment Description Mammary gland involution represents one of the most dramatic examples of programmed cell death/apoptosis and tissue regression during development, yet large gaps still exist in the understanding of the mechanisms involved, and the key factors that trigger involution, are not yet identified. With the focus on identifying “novel” genes associated with mammary gland regression, we used microarray analysis to examine differentially expressed genes during early mammary gland involution in the mouse. We believe that such analysis may facilitate the identification of genes that report on proliferative changes in the breast, particularly with regards to growth arrest and growth inhibition. We further reasoned that genes that inhibit mammary gland proliferation might have relevance to breast tumorigenesis since cancer is generally associated with a defect in apoptosis and cell growth arrest. Gene expression from day 0 (D0) of involution was compared to day 1 (D1, 24 hr post weaning) to identify genes exhibiting differential expression. A total of 5,826 genes were identified as being differentially expressed. Changes in gene expression were confirmed by quantitative real time PCR analysis of 21 randomly selected genes. Following the selection of a number of highly regulated mouse genes within this cohort, database searches were then employed to identify genes associated with human tumorigenesis. With an ultimate goal being the identification of genes, which under normal physiological conditions, inhibit breast proliferation, and therefore have the potential to impact on the clinical management of breast cancer, we further examined their relevance to human tumorigenesis. Among genes identified as having human homologues, and with previously established links to human tumorigenesis, were several known genes among the top 200 differentially expressed genes including EGF, EGFR, TGFß2, PTHLH, IGFBP1, that were down regulated. Upregulated known genes included ANNEXIN A1 and A3, THYMOSIN BETA 10, IGFBP5, LIF, BID, BCL2, JUN, KIT, and CLAUDIN 1. CLAUDIN 1 was the most highly upregulated gene in our dataset. Moreover, we identified a number of genes not previously implicated or not well characterized in human breast cancer including ANGPTL4, SLC34A2, GOS2, STEAP, SOX4, TNFRSF12A and SAA2. The expression levels of these genes in human breast cancer were confirmed in breast cancer cell lines and breast tumor tissues. For some of these genes, expression was consistently down regulated in a small panel of human breast tumor specimens. We believe that these genes now warrant further investigation to determine their relevance to human breast tumorigenesis and their potential utility as therapeutic targets for human breast cancer. This pilot study was used to demonstrate proof of principle that through the analysis of gene expression during mammary gland involution, it may be indeed possible to identify “novel” genes relevant to the growth arrest in the mammary gland and relevant to human breast cancer. Experiment Overall Design: Pooled RNA from D0 mice and from D1 mice, (five mice per time point), was compared for differential gene expression by microarray analysis conducted by Paradigm Array Labs (Research Triangle Park, North Carolina, USA). The Affymetrix GeneChip Mouse Genome 430 Plus 2.0 Array (Affymetrix, Santa Clara CA. USA) was used. The array contained 45,100 probe sets (The Affymetrix probe-set identificator) and represented 39,000 transcripts. The Affymetrix Statistical Algorithms, implemented in the Affymetrix Microarray Suite 5.0 software, were used in the expression analysis of GeneChip probe arrays. Protocol Name P-G5317-5 P-G5317-2 P-G5317-4 P-G5317-1 P-G5317-3 Protocol Type specified_biomaterial_action specified_biomaterial_action grow grow nucleic_acid_extraction Protocol Description Female CD1 mice, 6 weeks of age, were purchased from Charles River Breeding Laboratory (Wilmington, MA) and housed at the University of Manitoba Animal Care Facility. All procedures were approved by the Animal Care Committee of the University of Manitoba, under Canadian Council for Animal Care Guidelines. Virgin female mice were mated with CD1 males. Litter sizes were normalized to six pups 1-2 days after delivery, with the delivery day considered as day 0 (D0) of lactation. At day ten (D10) of lactation, when lactation was fully established, the pups were forcibly weaned. This time point was deemed day 0 (D0) of involution. Animals were sacrificed and mammary glands were dissected from D0 and D1, involuting mice. Dissected tissue was immediately frozen on dry ice and stored at ?70oC. Female CD1 mice, 6 weeks of age, were purchased from Charles River Breeding Laboratory (Wilmington, MA) and housed at the University of Manitoba Animal Care Facility. All procedures were approved by the Animal Care Committee of the University of Manitoba, under Canadian Council for Animal Care Guidelines. Virgin female mice were mated with CD1 males. Litter sizes were normalized to six pups 1-2 days after delivery, with the delivery day considered as day 0 (D0) of lactation. At day ten (D10) of lactation, when lactation was fully established, the pups were forcibly weaned. This time point was deemed day 0 (D0) of involution. Animals were sacrificed and mammary glands were dissected from D0 and D1, involuting mice. Dissected tissue was immediately frozen on dry ice and stored at â€"70oC. Female CD1 mice, 6 weeks of age, were purchased from Charles River Breeding Laboratory (Wilmington, MA) and housed at the University of Manitoba Animal Care Facility. All procedures were approved by the Animal Care Committee of the University of Manitoba, under Canadian Council for Animal Care Guidelines. Virgin female mice were mated with CD1 males. Litter sizes were normalized to six pups 1-2 days after delivery, with the delivery day considered as day 0 (D0) of lactation. At day ten (D10) of lactation, when lactation was fully established, the pups were forcibly weaned. This time point was deemed day 0 (D0) of involution. Animals were sacrificed and mammary glands were dissected from D0 and D1, involuting mice. Dissected tissue was immediately frozen on dry ice and stored at ?70oC. Female CD1 mice, 6 weeks of age, were purchased from Charles River Breeding Laboratory (Wilmington, MA) and housed at the University of Manitoba Animal Care Facility. All procedures were approved by the Animal Care Committee of the University of Manitoba, under Canadian Council for Animal Care Guidelines. Virgin female mice were mated with CD1 males. Litter sizes were normalized to six pups 1-2 days after delivery, with the delivery day considered as day 0 (D0) of lactation. At day ten (D10) of lactation, when lactation was fully established, the pups were forcibly weaned. This time point was deemed day 0 (D0) of involution. Animals were sacrificed and mammary glands were dissected from D0 and D1, involuting mice. Dissected tissue was immediately frozen on dry ice and stored at â€"70oC. Trizol Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF SDRF File E-GEOD-5317.sdrf.txt Term Source Name The MGED Ontology ArrayExpress EFO The MGED Ontology Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version