6 research outputs found
LY2624587 induces CXCR4 receptor internalization and down-regulation of receptor density on the cell surface.
<p>LY2624587 was labeled with Alexa 488. The labeled antibody was then used to treat MDA-MB-435/CXCR4 stably transfected cells for 1 or 2 hours at 37Ā°C. After these treatments, the cells were examined under a fluorescent microscope for localization of receptor. In one condition, cells were incubated with labeled LY2624587 for 1 or 2 hours first, then fixed with 2% formaldehyde for 10 min (A). In another condition, the cells was fixed with 2% formaldehyde for 10 min first, then incubated with Alexa 488-labeled LY2624587 for 1 or 2 hours (B). The scale bar is 10 nm. C. LY2624587 induces CXCR4 receptor down-regulation of Namalwa cells. D. Peptide antagonist LY2510924 does not induce CXCR4 receptor down-regulation. NHL Namalwa cells were treated by LY2624587 (C) or LY2510924 (D) for 4 days, and analyzed by flow cytometry with a PE-conjugated anti-CXCR4 antibody that is not competing with LY2624587 or LY2510924 for CXCR4 binding. Grey color was isotype IgG control, green color was no drug treatment; and red color was treated by LY2624587 (C) or LY2510924 (D).</p
<i>In vitro</i> activities of anti-CXCR4 monoclonal antibody LY2624587.
<p>A. Inhibition of [<sup>125</sup>I] SDF-1Ī± binding to CXCR4 by LY2624587 (square) or control IgG (triangle). Ligand binding assay was developed with CCRF-CEM cells, and the CCPM in the Y axis stands for corrected counts per minute. B. Dose dependent inhibition of SDF-1 induced GTP binding by LY2624587 (solid circle) or control IgG (triangle). GTP binding assay was developed with CCRF-CEM membrane. C. Inhibition of SDF-1 induced cell migration (chemotaxis) in U937 cells by LY2624587. D. Inhibition of SDF-1 induced cell migration (chemotaxis) in CCRF-CEM cells by LY2624587. E and F. LY2624587 has no apparent agonist activity. LY2624587 was tested in GTPĪ³S35 binding assay (E) or cell migration assay (F) in agonist mode. The GTPĪ³S35 binding assay was developed with CCRF-CEM cell membrane, and the SDF-1-induced cell migration assay was developed with U937 cells as described under āMaterials and Methods.ā</p
LY2624587 inhibits SDF-1 and CXCR4-mediated cell signaling in tumor cells.
<p>Human leukemia CCRF-CEM (A) and NHL Namalwa cells (B)were treated with 100 ng/ml SDF-1 for 10 min in the presence of different concentrations of LY2624587 (CXCR4AB) or control isotype IgG (hIgG4). Western blot analysis of phospho-ERK, phospho-AKT, total ERK or actin was conducted as described under the āMaterials and Methods.ā</p
Anti-tumor growth activities of LY2624587 in NHL Namalwa xenograft models developed with SCID mice.
<p>A. NHL Namalwa cells were subcutaneously implanted in the rear flank side and grown as solid tumors. B. NHL Namalwa cells were intravenously injected into SCID mice via tail vein, and grown as disseminated lymphomas as described under the āMaterials and Methods.ā</p
LY2624587 induces apoptosis of hematologic tumor cells <i>in vitro</i>.
<p>A. Annexin V analysis by flow cytometry in NHL Namalwa cells. Namalwa cells were treated with isotype IgG (hIgG4) or LY2624587 (CXCR4 AB 93ā6) for 48 h, then subjected to flow cytometry analysis utilizing FITC-conjugated annexin V. B. DNA fragmentation and cleaved caspase 3 analysis by cellomics analysis in NHL Namalwa cells. The cells were treated for 48 h in the growth medium with 10 Ī¼g/ml control IgG or LY2624587 (CXCR4 ab). C. DNA fragmentation and cleaved caspase 3 analysis by cellomics analysis in leukemia CCRF-CEM cells. The cells were treated for 96 h in growth medium with 10 Ī¼g/ml control IgG or LY2624587 (CXCR4 ab). The inserts are amplified single cells to show DNA fragmentation or caspase 3 staining.</p
CXCR4 expression in human primary chronic lymphocytic leukemia (CLL) cells and LY2624587 binds to the CXCR4 of CD19+ cells of CCL patients.
<p>The blood samples of 4 CLL patients, EL-1279, EL-1308, EL-1289 and EL-1278 were freshly collected from Methodist Hospital, Indiana University at Indianapolis. 20 ml of fresh blood sample from each patient was collected and subjected to flow cytometry analysis with a PE-conjugated CXCR4 antibody and a FITC-conjugated CD19 antibody. A. CXCR4 surface expression in CD19+ cells from a CLL patient. B. CXCR4 intensities in tumor cells of four CCL patients. Three patients express high levels of CXCR4, and one patient has relatively low CXCR4 expression. C-F. LY2624587 (93ā6) binds to the CXCR4 of CD19+ cells from CLL patient of EL-1279 (B), EL-1308 (C), EL-1289 (D), and EL-1278 (E). Competitive binding of LY2624587 to CXCR4 was determined using 16 nM of a PE-conjugated CXCR4 antibody from R&D Systems as a tracer. This tracer CXCR4 antibody binds to the same CXCR4 epitope of LY2624587.</p