Conversely, the induction of Dkk-1 in the mixed osteoblastic/osteolytic prostate malignancy C4-2B cell line results in experimental bone metastases with an osteolytic phenotype 21-derived, recombinant human Dkk-1 (rhDkk-1) (R&D System, Minneapolis, MN, USA), diluted in phosphate-buffered saline (PBS), pH 7.4 (137?mM NaCl, 3.3?mM KCl, 8?mM Na2HPO4, 1.47?mM KH2PO4, pH 7.4) and incubated for 2?h at room temperature. that this inhibition of Dkk-1 expression in osteolytic PC-3 prostate malignancy cells induced osteoblastic activity in particular phosphatase alkaline activity and mineralisation in murine bone marrow stromal cells. Conversely, the induction of Dkk-1 in the mixed osteoblastic/osteolytic prostate malignancy C4-2B cell collection results in experimental bone metastases with an osteolytic phenotype 21-derived, recombinant human Dkk-1 (rhDkk-1) (R&D System, Minneapolis, MN, USA), diluted in phosphate-buffered saline (PBS), pH 7.4 (137?mM NaCl, 3.3?mM KCl, 8?mM Na2HPO4, 1.47?mM KH2PO4, pH 7.4) and incubated for 2?h at room temperature. Plates were then incubated for 2?h at room temperature with PBS, and 4% BSA (Euromedex, Souffelweyersheim, France). After five cycles of washing with PBS (pH 7.4), 1% BSA, and 0.1% Tween 20 (washing buffer), rhDkk-1 used as standard (R&D System), culture supernatant, bone marrow aspirate or serum were incubated for 2?h at room temperature. After washing, the biotinylated form of the anti-human Dkk-1 antibody was added for 1?h at room temperature. StreptavidinChorseradish peroxidase (Jackson ImmunoResearch Laboratories, Cambridgeshire, UK) was then added with the substrate answer of 3,3,5,5-tetramethylbenzidine (TMB) (Euromedex). Finally, the reaction was stopped by the addition of 100?axis). The axis shows the optical density (OD) obtained for each concentration of the standard (B) expressed as a percentage of the OD value observed with the highest concentration of Dkk-1 (B0). DoseCdilution curves of serum samples from a healthy post-menopausal woman and from a post-menopausal woman with breast malignancy and bone metastases were parallel to the standard curve. Inset: Western blot indicating that the polyclonal antibody used in the ELISA recognises human Dkk-1 at the expected 37?kDa mass (arrow), mw=molecular excess weight markers. Western blot analysis for Dkk-1 RhDkk-1 (0.1?Whole body radiographs (MIN-R2000 films; Kodak, Rochester, NY, USA) of anaesthesised animals were taken 30 days after tumour cell inoculation using an MX-20 cabinet X-ray system (Faxitron X-ray Corporation, Wheeling, IL, USA). The area of osteolytic lesions of the whole body was measured using a Morpho computerised image analysis system (Altavista, France), and the extent of bone destruction per animal was expressed in square millimetres, as explained previously (Peyruchaud Hind limbs from animals were fixed and embedded in methylmethacrylate. Seven-micrometre sections of undecalcified long bones were stained with Goldner’s trichrome. Histological analyses were performed on longitudinal medial sections of tibial metaphases using a Morpho computerised image analysis system (Altavista, France), as explained previously (Peyruchaud 9.32.1?ng?ml?1, 0.220.35?ng?ml?1, and higher serum levels are associated with the presence of bone metastases in patients with breast malignancy. Using real-time quantitative RTCPCR and quantitative ELISA, we exhibited that Dkk-1 was expressed and secreted in large amount only by cultured human breast malignancy cell lines (MCF-7, MDA-MB-231, and MDA-B02) known to induce osteolytic bone metastases in mice. Our findings extend those recently reported by Schwaninger (2007), showing that Dkk-1 mRNA was expressed in the osteolytic MDA-MB-231 breast cancer collection em in vitro /em , whereas osteoblastic breast malignancy cell lines T47D and ZR-75-1 did not express Dkk-1. Moreover, we found high Dkk-1 protein levels in the bone marrow of tumour-bearing legs from mice inoculated with MDA-B02 breast malignancy cells. Our data are in agreement with the observation that PC-3 prostate malignancy cells, which induce osteolytic lesions in animals, secrete Dkk-1 and that osteoblastic lesions caused by C4-2B prostate Eprinomectin malignancy cells revert to an osteolytic phenotype upon transfection of C4-2B cells with a plasmid encoding for Dkk-1 (Hall em et al /em , 2005). Because Eprinomectin the antibody we used crossreacts with human and murine Dkk-1, we could not discriminate between tumour-derived and bone marrow-derived Dkk-1. Because Dkk-1 could not be detected in the bone marrow of naive animals, this suggests that MDA-B02 tumour cells were the major source of Dkk-1 in the bone marrow. However, because both tibias of mice inoculated with MDA-B02 cells presented with bone metastatic lesions, we could not analyse Dkk-1 levels in non-tumour-bearing legs TSPAN10 from inoculated mice, which would have been the optimal control. Additional mechanisms, such as the activation by bone-residing breast malignancy cells of Dkk-1 expression by bone marrow cells, may also be involved in the high local production of Dkk-1. Such a possibility merits further investigation. The relevance of our preclinical findings in humans is usually suggested by the increased levels of Dkk-1 in the serum of Eprinomectin patients with breast malignancy and bone metastases measured by a new sensitive and reproducible assay that we developed. Although.