BMC Cancer 2008, 8:270 http://www.biomedcentral.com/1471-2407/8/270 higher in the papillary adenocarcinomas (Figure 5C). However, the expression of its ligands, Jagged 2 and Dll1, was higher in the carcinoma tumors. Interestingly, the papillary adenocarcinoma also exhibited a higher expres- sion level of Fringe – a β-1,3-N-acetylglucosaminyltrans- ferase that modifies Notch receptors and alters their ligand-binding specificity towards the Dll ligands [35,36]. Thus, the papillary adenocarcinoma may preferentially adopt Dll-Notch signaling over that of Jagged. While cyto- plasmic Notch 1 is more highly expressed in the papillary adenocarcinoma, the members of the γ-secretase complex, PSN1 and nicastrin (NCSTN), which release its NICD, were highly expressed in the carcinoma tumors. Nuclear activity of the NICD depends on its binding RBP-Jk, which was also highly expressed in the carcinomas. The TGF-β super-family comprises TGF-βs, bone morpho- genetic protein (BMP) and activin. TGF-β or activin bind type II serine/threonine kinase-coupled receptor to further recruit and phosphorylate the type I receptor. In turn, the type I receptor recruits and phosphorylates the R-Smads (for example Smad3) which dissociate and interact with the collaborating Smad, Smad4, to affect gene expression [37,38]. BMP ligands have a higher affinity to the extracel- lular domain of BMPRI, which then assembles BMPRII. In the current study, TGF-β1 and TGF-βRII were found to be highly expressed in the carcinoma tumors along with the downstream Smad3 (Figure 5D). Activin and its Type I receptor were also highly expressed in these poorly differ- entiated tumors. In contrast, Smad4, which oligomerizes with Smad3 to form a trimeric protein complex, was more highly expressed in the papillary adenocarcinomas and may enhance the effect of Smad3 in these tumors. DNA damage or abnormally structured DNA triggers mul- tiple checkpoint pathways that arrest cell-cycle progres- sion [39]. Failure to arrest cell-cycle progression before or at mitosis causes aberrant chromosomal segregation, asymmetric division, aneuploidy and cancers [40]. The mitotic catastrophes influence regulatory protein expres- sion. In the papillary adenocarcinoma tumors, proteins encoded by genes involved in the early steps of the response to DNA damage were more highly expressed than in the carcinomas (Figure 5E). This included the Atr which functions to recognize DNA damage and histone H2afx – a downstream effector involved in DNA repair via chromatin modeling [39]. The papillary adenocarcinoma also exhibited higher expression of BRCA1 and BRCA2, which are involved in DNA repair, Chk1 and Chk2 – highly mobile messengers which are capable of "globally" spreading the DNA-damage-induced signal throughout the nucleus [41], and the cyclin-dependent kinase inhibi- tor p18(INK4c) [42]. G2/M – a later checkpoint, prevents cells from entering into mitosis via inhibition of Cdc2/ cyclin B. Interestingly, three key proteins that regulate this checkpoint are encoded by genes that were highly expressed in the carcinoma tumors: p21, which inhibits Cdc2 directly, 14-3-3σ, which anchors Cdc2 in the cyto- plasm where it cannot induce mitosis, and GAdd45, which specifically dissociates Cdc2 from cyclin B1 by co- association with Cdc2 [39,41]. p21 regulates also G1/M, but its involvement also in this later step together with GAdd45 and 14-3-3σ could be indicatory. Glycosylation is an important post-translational modifi- cation of many biologically relevant molecules. A change in the structure of the glycans added to glycoproteins and glycolipids is a common feature of malignancy [43]. O- glycosylation is initiated by the transfer of GalNAcα- to threonine or serine residues of the polypeptide backbone, where eight O-glycan core structures can then be synthe- sized [44]. In the current study, the genes encoding the N- acetylgalactosaminyltransferases that are involved in the generation of core structures 1, 5, 6, 7 and 8 were highly expressed in the carcinoma tumors (Figure 7 and Addi- tional file 1, data sheet I). The extension of core 1 to form the disalyl-T antigen is mediated by sialyltransferase (Siat5). The expression of the gene encoding this enzyme was also higher in the carcinoma cells, as were the expres- sions of Siat7d and Siat9, which mediate sialylated glyco- conjugates involved in sphingolipid metabolism (not shown). Validation of Affymetrix data by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) To confirm the differences in gene-expression profiles measured by gene-array analysis in the poorly differenti- ated carcinoma tumors vs. the highly differentiated papil- lary adenocarcinomas, semi-quantitative RT-PCR analysis was performed (Figure 8). We amplified selected genes' expressions in individual RNA samples extracted from normal mammary glands of aged multiparous females, as well as from tumors of the different phenotypes. Compa- rable relative expressions of these genes in carcinoma and papillary adenocarcinoma tumors were calculated for the RT-PCR and array analyses. Discussion The number of clearly differentiated phenotypes observed among breast tumors suggests that diversity is an inherent feature of the disease, involving complex but quantifiable parameters. A considerable number of the genes involved in the diversity between the poorly differentiated carci- noma and highly differentiated papillary adenocarcinoma tumors are associated with the nucleoplasm and the plasma membrane. Thus, nuclear dictates expressed by DNA- and RNA-binding activities, and communication processes between cells and their microenvironment, are involved in determining the phenotypical differences between the tumor types. The diversity in genes mediating Page 13 of 19 (page number not for citation purposes)