BMC Cancer 2008, 8:270 http://www.biomedcentral.com/1471-2407/8/270 many). The pathological analysis of the tumors was per- formed by Dr. Robert Cardiff (University of California, Davis) as previously described [13]. Results Comparison of mammary carcinoma and papillary adenocarcinoma tumors: Gene Ontology (GO) analysis of differentially expressed genes Global gene-expression profiling was performed on two types of mammary tumors: the poorly differentiated carci- nomas and the highly differentiated papillary adenocarci- nomas. RNA was extracted and reverse-transcribed from seven individual carcinoma and six individual papillary adenocarcinoma tumors that developed in transgenic mice expressing deregulated Stat5. RNA was also extracted from the mammary glands of three post-estropausal mul- tiparous wild-type females and similarly processed. For comparison of global gene expression in the two types of tumors, the array data were normalized to the median gene-expression levels determined in the wild-type glands. This also relates the tumorigenic expression of an individual gene to its counterpart in the wild-type gland. After normalization and filtration, 2,215 features (1,882 genes) were identified that were differentially expressed in the carcinoma vs. papillary adenocarcinoma tumors at a statistical significance of P < 0.05 (Additional file 1, data sheet B). Principal-component analysis (PCA) and unsu- pervised hierarchical clustering were performed using Genespring to test the degree of similarity between the expression of the deviated genes in the two phenotypes [19]. These analyses assembled the phenotypically differ- ent genes into two sets which separated independently of the transgenic Stat5 variant carried by the host mice (Fig- ure 1). Additional filtration of the genes to include only those exhibiting an over twofold difference narrowed the list to 865 features (773 genes; Additional file 1, data sheet C). PCA and hierarchical clustering of these genes overlapped with that obtained for the broader list of 2,215 features (not shown). A perspective on the cellular components, molecular func- tions and biological processes involved in the divergent gene expression between phenotypes was obtained by GO Slim classification of the set of 865 features (Figure 2 and Additional file 1, data sheets D-F). A high number of dif- ferentially expressed genes were associated with two cellu- lar components: the nucleoplasm and the plasma membrane. In contrast, a relatively low number of these genes were associated with the cytoplasm, most of which were tied to the cytoskeleton. Nucleic-acid binding, cata- lytic activity and activities linked to maintaining cellular structure were the main molecular functions involving the differentially expressed genes. Differences in the expres- sion of genes involved in signal-transduction activity and transporter activity were also noted. The variance among the resulting biological processes was low, but a markedly higher number of genes were found to be involved in "reg- ulation of gene expression, epigenetic". This definition assembles genes in a relatively wide context that involves cytoplasmic processes that are mitotically or meiotically heritable and do not entail a change in DNA sequence. Clustering the differentially expressed genes Additional information on the genes that were differen- tially expressed in the two types of tumors was obtained by applying an unsupervised hierarchical-clustering algo- rithm. It organizes genes according to the similarity or dis- similarity in expression profile, placing the cases with similar expression profiles together as neighboring rows in the clustergram. Based on normalization of each gene value to the median of gene expression in the intact mam- mary gland, this analysis assembled the genes into two cluster pairs (Figure 3 and Additional file 1, data sheets G and H). The analysis is Clusters 1 and 3 contained genes that are highly expressed in papillary adenocarcinomas, while clusters 2 and 4 included genes that are preferen- tially expressed in the carcinoma tumors. Relatively higher levels of gene expression, as compared to the wild-type tis- sue as well, characterized clusters 1 and 3 relative to clus- ters 2 and 4, respectively. This distinction allowed further classification of gene expression within each phenotype. Cluster 1 contained the smallest number of genes: 78 fea- tures (72 genes) with relatively high expression levels in the papillary adenocarcinoma compared to the carcinoma tumors or the wild-type gland. These genes mediate molecular functions of nuclear DNA binding, transcrip- tion-factor activity and oxidoreductase activity. The main biological processes involve DNA binding, transcription- factor activity and mediating responses to DNA damage. Cluster 3 contained 250 features (241 genes) that were also more highly expressed in the papillary adenocarci- noma, although at a lower level than those listed in cluster 1. In the carcinoma tumors, all of these genes were expressed at lower levels than in the wild-type gland. Con- trary to the nuclear activities implicated for most of the genes in cluster 1, the genes in cluster 3 regulate cytoplas- mic and membranal activities such as transporter activity, ion-channel activity and cytoskeletal protein binding. Consequently, a wide set of biological processes were affected, including development, cellular morphogenesis and muscle contraction. Genes with higher expression levels in the carcinoma vs. papillary adenocarcinoma tumors were assembled in clus- ters 2 and 4. In cluster 2 there were 309 features (270 genes) which also showed high expression levels relative to the wild-type mammary gland. Many of these genes encode binding proteins: ion binding (especially calcium- ion binding), receptor binding, cytoskeletal-protein bind- Page 4 of 19 (page number not for citation purposes)