* significantly different from WT. associated with disease. The cytokine Interleukin 8 (IL-8/CXCL8) is able to directly stimulate osteoclastogenesis and bone resorption in mouse models of breast cancer bone metastasis. In this study, we determined whether circulating levels of IL-8 were associated with increased bone resorption and breast cancer bone metastasis in patients, and investigated IL-8 action and in mice. Using breast cancer patient plasma (36 patients), we identified significantly elevated IL-8 levels in bone metastasis patients compared with patients lacking bone metastasis (p 0.05), as well as a correlation between plasma IL-8 and increased bone resorption (p 0.05), as measured by NTx levels. In a total of 22 Glucagon-Like Peptide 1 (7-36) Amide ER+ and 15 ER? primary invasive ductal carcinomas, all Glucagon-Like Peptide 1 (7-36) Amide cases examined stained positive for IL-8 expression. was confirmed when transgenic mice expressing human IL-8 were examined Glucagon-Like Peptide 1 (7-36) Amide and found to have a profound osteopenic phenotype, with elevated bone resorption and inherently low bone mass. Collectively, these data suggest that IL-8 plays an important role in breast cancer osteolysis and that anti-IL-8 therapy may be useful in the treatment of the skeletal related events associated with breast cancer. and were obtained from R&D Systems. Patient Samples Archival breast cancer patient plasma was obtained from 36 patients (18 with and 18 without bone metastasis) for the measurement of IL-8. Analysis of the archival plasma samples was approved by the University of Arkansas for Medical Sciences and Pennsylvania State University Institutional Review Boards. The clinical assessment of bone metastasis was based on patient bone scan, x-ray evidence of Glucagon-Like Peptide 1 (7-36) Amide bone metastasis, and elevated blood N-Telopeptide (NTx) levels, a clinical marker of bone resorption [19]. The serum NTx levels of all patients were used to help discern the presence or absence of bone metastasis. The women ranged in age from 49C92 years with a median age of 70 in the bone metastasis group and 67 in the no bone metastasis group. A power analysis was conducted to confirm that the size of the sample was sufficient to provide a statistical power of more than 80%. In addition, a series of archival formalin-fixed SCC3B paraffin embedded tumor tissue samples from 22 ER+ (expressing 2+ ? 3+ positivity in 50% cells) and 15 ER- invasive ductal breast carcinomas, irrespective of grade and stage of disease, were also selected for evaluation. Unstained sections were immunostained for IL-8 expression (anti-IL-8 antibody, dilution 1:200, R&D Systems, Minneapolis, MN) with appropriate positive Glucagon-Like Peptide 1 (7-36) Amide and negative controls. The intensity of staining for IL-8 was graded on a scale of 0 to 3+ with 0 representing no detectable staining and 3+ representing the strongest staining. Two independent observers examined each slide. Cell Lines and Culture Conditions The MDA-MB-231 cells (MDA-231), MDA-MET cell lines and transfected variants (sense and anti-sense) were maintained in DMEM, supplemented with 10% fetal bovine serum at 37C in sterile culture dishes [9]. Highly bone metastatic MDA-MET cells were derived from a weakly osteolytic MDA-231 variant by selection [9]. MDA-MET cells secrete full length IL-8 and produce osteolytic lesions (100%) within 4 weeks of inoculation in the circulation or tibia of athymic nude mice [20] and grow effectively in the mammary fat pad [21] compared with MDA-231 cells that produce little full-length IL-8 [20]. MDA-231-IL8 and MDA-MET-AS cells were generated by stable transfection of expression vectors (pcDNA3 Invitrogen, Carlsbad, California) expressing full length hIL-8 or anti-sense hIL-8 cDNA as a fragment by calcium phosphate precipitation. The pcDNA3/IL-8 sense or antisense (AS) DNA transfected cells were grown and single clones isolated by limiting dilution in the presence of the selective marker, G418 (Sigma Chemical Co., St. Louis, Missouri, USA). Clones were screened by measuring the amount of secreted hIL-8 (in sense expressing MDA-231 or the loss of IL-8 in MDA-MET antisense cells) in serum-free 48-h conditioned media. Clones with significantly increased and decreased IL-8 levels respectively were selected for further study. HEK-293 human embryonal.