Contamination of milk with drugs, pesticides and other xenotoxins can pose a major health risk to breast-fed infants and dairy consumers. Here we show that the multidrug transporter BCRP (encoded by ABCG2) is strongly induced in the mammary gland of mice, cows and humans during lactation and that it is responsible for the active secretion of clinically and toxicologically important substrates such as the dietary carcinogen PhIP, the anticancer drug topotecan and the antiulcerative cimetidine into mouse milk.

The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) was initially identified as a key regulator of insulin-dependent glycogen synthesis. GSK-3 was subsequently shown to function in a wide range of cellular processes including differentiation, growth, motility and apoptosis. Aberrant regulation of GSK-3 has been implicated in a range of human pathologies including Alzheimer's disease, non-insulin-dependent diabetes mellitus (NIDDM) and cancer. As a consequence, the regulation of GSK-3 and the therapeutic potential of GSK-3 inhibitors have become key areas of investigation. This review will focus on the mechanisms of GSK-3 regulation, with emphasis on modulation by upstream signals, control of substrate specificity and GSK-3 localisation. The details of these mechanisms will be discussed in the context of specific signalling pathways.

Glycogen synthase kinase 3 beta (GSK3 beta) plays a key role in insulin and Wnt signaling, phosphorylating downstream targets by default, and becoming inhibited following the extracellular signaling event. The crystal structure of human GSK3 beta shows a catalytically active conformation in the absence of activation-segment phosphorylation, with the sulphonate of a buffer molecule bridging the activation-segment and N-terminal domain in the same way as the phosphate group of the activation-segment phospho-Ser/Thr in other kinases. The location of this oxyanion binding site in the substrate binding cleft indicates direct coupling of P+4 phosphate-primed substrate binding and catalytic activation, explains the ability of GSK3 beta to processively hyperphosphorylate substrates with Ser/Thr pentad-repeats, and suggests a mechanism for autoinhibition in which the phosphorylated N terminus binds as a competitive pseudosubstrate with phospho-Ser 9 occupying the P+4 site.

Axin promotes the phosphorylation of beta-catenin by GSK-3beta, leading to beta-catenin degradation. Wnt signals interfere with beta-catenin turnover, resulting in enhanced transcription of target genes through the increased formation of beta-catenin complexes containing TCF transcription factors. Little is known about how GSK-3beta-mediated beta-catenin turnover is regulated in response to Wnt signals. We have explored the relationship between Axin and Dvl-2, a member of the Dishevelled family of proteins that function upstream of GSK-3beta. Expression of Dvl-2 activated TCF-dependent transcription. This was blocked by co-expression of GSK-3beta or Axin. Expression of a 59 amino acid GSK-3beta-binding region from Axin strongly activated transcription in the absence of an upstream signal. Introduction of a point mutation into full-length Axin that prevented GSK-3beta binding also generated a transcriptional activator. When co-expressed, Axin and Dvl-2 co-localized within expressing cells. When Dvl-2 localization was altered using a C-terminal CAAX motif, Axin was also redistributed, suggesting a close association between the two proteins, a conclusion supported by co-immunoprecipitation data. Deletion analysis suggested that Dvl-association determinants within Axin were contained between residues 603 and 810. The association of Axin with Dvl-2 may be important in the transmission of Wnt signals from Dvl-2 to GSK-3beta.

Background: Breast cancer is thought to arise in mammary epithelial stem cells. However, the identity of these stem cells is unknown. Methods:Studies in the haematopoetic and muscle systems show that stem cells have the ability to efflux the dye Hoechst 33342. Cells with this phenotype are referred to as the side population (SP). We have adapted the techniques from the haematopoetic and muscle systems to look for a mammary epithelial SP. Results:Of mammary epithelial cells isolated from both the human and mouse mammary epithelia, 0.2-0.45% formed a distinct SP. The SP was relatively undifferentiated but grew as typical differentiated epithelial clones when cultured. Transplantation of murine SP cells at limiting dilution into cleared mammary fat pads generated epithelial ductal and lobuloalveolar structures.Conclusion: These data demonstrate the existence of an undifferentiated SP in human and murine mammary epithelium. Purified SP cells are a live single-cell population that retain the ability to differentiate in vitro and in vivo. Studies of haematopoetic cells have suggested that the SP phenotype constitutes a universal stem cell marker. This work therefore has implications for mammary stem cell biology.