Functional and molecular characterisation of mammary side population cells

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

An economic model of long-term use of celecoxib in patients with osteoarthritis

<p>Abstract</p> <p>Background</p> <p>Previous evaluations of the cost-effectiveness of the cyclooxygenase-2 selective inhibitor celecoxib (Celebrex, Pfizer Inc, USA) have produced conflicting results. The recent controversy over the cardiovascular (CV) risks of rofecoxib and other coxibs has renewed interest in the economic profile of celecoxib, the only coxib now available in the United States. The objective of our study was to evaluate the long-term cost-effectiveness of celecoxib compared with nonselective nonsteroidal anti-inflammatory drugs (nsNSAIDs) in a population of 60-year-old osteoarthritis (OA) patients with average risks of upper gastrointestinal (UGI) complications who require chronic daily NSAID therapy.</p> <p>Methods</p> <p>We used decision analysis based on data from the literature to evaluate cost-effectiveness from a modified societal perspective over patients' lifetimes, with outcomes expressed as incremental costs per quality-adjusted life-year (QALY) gained. Sensitivity tests were performed to evaluate the impacts of advancing age, CV thromboembolic event risk, different analytic horizons and alternate treatment strategies after UGI adverse events.</p> <p>Results</p> <p>Our main findings were: 1) the base model incremental cost-effectiveness ratio (ICER) for celecoxib versus nsNSAIDs was $31,097 per QALY; 2) the ICER per QALY was$19,309 for a model in which UGI ulcer and ulcer complication event risks increased with advancing age; 3) the ICER per QALY was $17,120 in sensitivity analyses combining serious CV thromboembolic event (myocardial infarction, stroke, CV death) risks with base model assumptions.</p> <p>Conclusion</p> <p>Our model suggests that chronic celecoxib is cost-effective versus nsNSAIDs in a population of 60-year-old OA patients with average risks of UGI events.</p

Sequence variants of the Axin gene in breast, colon, and other cancers: an analysis of mutations that interfere with GSK3 binding

Axin is a recently discovered component of a multiprotein complex containing APC, beta-catenin, GSK3, and PP2A, which functions in the degradation of the beta-catenin protein. As part of WNT signal transduction, the function of the Axin complex is inhibited, leading to the accumulation of beta-catenin. The inappropriate stabilization of beta-catenin has been implicated in a range of human tumors. Two oncogenic mechanisms leading to beta-catenin stabilization are the loss of the APC tumor suppressor protein and the mutational activation of beta-catenin, such that the Axin/APC complex can no longer regulate it. Studies in Drosophila and mammalian tissue culture showed loss of Axin function interfered with beta-catenin turnover and activated beta-catenin/TCF-dependent transcription. Based on these observations, Axin was screened for mutations in a range of human tumor cell lines and primary breast tumor samples. We identified two sequence variants causing amino acid substitutions in four colon cancer cell lines, a Ser-to-Leu at residue 215 in LS513 and a Leu-to-Met at residue 396 in HCT-8, HCT-15, and DLD-1. The Axin L396M mutation was selected for further study since it lay within a region that was shown to interact with glycogen synthase kinase-3. Biochemical and functional studies showed that the L396M change interfered with Axin's ability to bind GSK3. Interestingly, this mutation and a neighboring L392M change differentially altered Axin's ability to interfere with two upstream activators of TCF-dependent transcription, Frat1 and Disheveled

Identification of the Axin and Frat binding region of glycogen synthase kinase-3

Glycogen synthase kinase-3 (GSK-3) is a key component of several signaling pathways including those regulated by Wnt and insulin ligands. Specificity in GSK-3 signaling is thought to involve interactions with scaffold proteins that localize GSK-3 regulators and substrates. This report shows that GSK-3 forms a low affinity homodimer that is disrupted by binding to Axin and Frat. Based on the crystal structure of GSK-3, we have used surface-scanning mutagenesis to identify residues that differentially affect GSK-3 interactions. Mutations that disrupt Frat and Axin cluster at the dimer interface explaining their effect on homodimer formation. Loss of the Axin binding site blocks the ability of dominant negative GSK-3 to cause axis duplication in Xenopus embryos. The Axin binding site is conserved within all GSK-3 proteins, and its loss affects both cell motility and gene expression in the nonmetazoan,Dictyostelium. Surprisingly, we find no genetic interaction between a non-Axin-binding GSK-3 mutant and T-cell factor activity, arguing that Axin interactions alone cannot explain the regulation of T-cell factor-mediated gene expression