AMD3100 and T140 inhibit the SDF-1-induced chemotaxis of myeloid leukemia cells, and trigger the internalization of surface CXCR4. (A) Four-hour transmigration of MO7e cells towards SDF-1 (200 ng/mL). AMD3100 and T140 were added at concentrations of 10^-5 M and 10^-6 M, respectively. ^a)P<0.05, compared to the control. (B) U937 cells were incubated with or without 10^-5 M AMD3100 and 10^-6 M T140, respectively, for 3 hr and subsequently subjected to flow cytometry.

AMD3100 stimulates the proliferation of myeloid leukemia cells. Cells were incubated in the presence of AMD3100 (A) or T140 (B) for up to 72 hr. (C) Bone marrow CD34⁺ cells from 3 each patients with AML and CML were incubated in the presence of AMD3100 (10^-5 M) for 72 hr. (D) U937 cells were incubated with AMD3100 (10^-5 M). As indicated, the cells were pretreated with pertussis toxin (PTX) for 2 hr before the incubation. (E) U937 cells were incubated in X-VIVO medium with or without AMD3100 (10^-5 M) and subsequently subjected to cell cycle analysis. (F) U937 cells were incubated in RPMI-1640 medium without serum in the presence or absence of AMD3100 (10^-5 M) for 72 hr, stained with annexin V and propidium iodide (PI), and subjected to flow cytometry. Representative flow cytometry profiles are shown in the left panel. In the right panel, the percentages of the annexin V-positive apoptotic cells are presented ^a)P<0.05, compared with the control.

AMD3100 induces the phosphorylation of SDF-1-linked signaling molecules. (A) MO7e cells were incubated with AMD3100 (10^-5 M) and subsequently subjected to western blot analysis. Representative results of 3 experiments are shown. (B) U937 cells were treated with 10^-5 M AMD3100, 10 µM LY294002, 100 nM wortmannin, 50 µM PD98059, 10 µM AG490, 50 nM rapamycin, and 10 µM SB203580 for 3 days.

Survival of leukemia cells in an extended period of culture. (A) HL-60 cells (a), K562 cells (b), KG1a cells (c), and U937 cells (d) were incubated with AMD3100 (10^-7 M to 10^-5 M) for up to 14 days. Data are representative results of 3 or more independent experiments showing similar results. (B) Effects of AMD3100 on the formation of tumor cell colonies were examined using K562 cells. Representative profiles of the colony assay are shown.

Myeloid leukemia cells express CXCR7 on the cell surface, which is internalized by AMD3100. (A) Representative profiles of CXCR7 expression on the cell surface. (B) U937 cells were incubated with 10^-5 M AMD3100 and 10^-5 M T140, respectively, for 3 hr and subsequently subjected to flow cytometry.

Knockdown of CXCR7, but not CXCR4, delays the proliferation enhancement induced by AMD3100. U937 cells were transfected with 25 nM CXCR4 siRNA or 5 nM CXCR7 siRNA and were further incubated in serum-free medium in the presence or absence of AMD3100 for up to 3 days. (A) Representative profiles of the decrease in cell surface expression of CXCR4 and CXCR7 induced by siRNA. (B) Representative results of proliferation of the CXCR4 siRNA-transfected cells among 3 or more independent experiments showing similar results. (C) Representative results of the proliferation of the CXCR7 siRNA-transfected cells among 3 or more independent experiments showing similar results.