ABSTRACT: Similar to the bone marrow, the mammary gland contains a distinct population of Hoechst-effluxing side population cells, MG-SPs. To better characterize MG-SPs, their microarray gene profiles were compared to the remaining cells, which retain Hoechst dye (MG-NSPs). For analysis, gene ontology (GO) that describes genes in terms of biological processes and ontology traverser (OT) that performs enrichment analysis were utilized. OT showed that MG-SP specific genes were enriched in the GO categories of cell cycle regulation and checkpoints, multi-drug resistant transporters, organogenesis, and vasculogenesis. The MG-NSP upregulated genes were enriched in the GO category of cellular organization and biogenesis which includes basal epithelial markers, p63, smooth muscle actin (SMA), myosin, ï?¡6 integrin, cytokeratin (CK) 14, as well as luminal markers, CK8 and CD24. Additional studies showed that a higher percentage of MG-SPs exist in the G1 phase of the cell cycle compared to the MG-NSPs. G1 cell cycle block of MG-SPs may be explained by higher expression of cell cycle negative regulatory genes such as TGF-ï?¢2 (transforming growth factor-ï?¢2), IGFBP-5 (insulin like growth factor binding protein-5), P18 INK4C and Wnt-5a (wingless-5a). Accordingly, a smaller percentage of MG-SPs expressed nuclear b-catenin, possibly as a consequence of the higher expression of Wnt-5a. In conclusion, microarray gene profiling suggests that MG-SPs are a lineage deficient mammary gland sub-population expressing key genes involved in cell cycle regulation, development and angiogenesis. Supplemental File Descriptions:; Table 1 is a list of 1632 Genes differentially expressed by MG-SP and MG-NSP; Criteria for comparison included 1.2-fold difference in expression levels, false discovery rate (FDR) 9.4%, P value less than 0.05. Table 2 is a list of 771 Genes differentially expressed by MG-SP and MG-NSP; Criteria for comparison included 1.5-fold difference in expression levels, FDR 3.4%, P value less than 0.05. Table 3 is a list of 335 Genes differentially expressed by MG-SP and MG-NSP; Criteria for comparison included 2-fold difference in expression levels, FDR 0%, P value less than 0.05. Table 4 is a list of 90 Genes differentially expressed by MG-SP and MG-NSP; Criteria for comparison included 2-fold difference in expression levels, FDR 0%, P value less than 0.01. Experiment Overall Design: Gene expression profiles were obtained by hybridizing amplified RNA from four replicate MG-SP and MG-NSP samples to Affymetrix 430 2.0 microarray chips. To isolate MG-SP and MG-NSPs, mammary gland cells were stained using the Hoechst dye 33342 and fluorescence displayed at two wavelength emissions, blue and red. The MG-SP and MG-NSP regions were indicated by trapezoids on the left (R1) and right (R2), respectively. We and others have previously shown that the R1 region is composed of side population cells since verapamil blocks their appearance. The cells in each region (R1 and R2) were sorted, their RNA isolated and amplified by two rounds of in vitro amplification and applied to Affymetrix chips. Hybridization, scanning, and production of raw data files were performed according to the Affymetrix standard protocols. Normalization and model-based expression measurements were performed with dChip. Two of the chips were eliminated from further analysis due to the high percentage of probe sets called an array outlier after model based expression analysis by dChip (Not included). The genes were filtered to eliminate those with very low expression values in most samples. From 45,000 probe sets, 16,744 probe sets were retained and used for further analysis. For normalization purposes and to allow for data comparison with other centers, universal mouse reference RNA (Stratagene) was hybridized to two chips. All other arrays were normalized to one of the universal mouse reference RNA arrays expressing average intensity closest to the median intensity of all chips (Stratagene Ref 2).
