Reduced oxygen tension of 6% O2 induces increases in both surface and total CXCR4 expression in AML cell lines and patient samples. Reoxygenation with 21% O2 induces loss of CXCR4 on hypoxic samples. (A) Histogram of representative experiment showing CXCR4 expression of OCI-AML3 cells under 21% (black line), 6% (dark gray), and reoxygenation with 21% O2 of at least 1 hour (of cells adjusted to hypoxia; black dotted line). Surface expression is shown relative to an irrelevant IgG control (C, light gray). (B) Up-regulation and loss of CXCR4 surface expression depends on O2 in AML cell lines OCI-AML3 and U937. (C) In OCI-AML3 and U937 cell lines, physiologic hypoxia increases total amount of CXCR4 as shown by Western blotting, which is lost when cells are reexposed to 21% O2. (D) Samples from patients with AML (n = 10) show a concordant pattern: using flow cytometry, hypoxia leads to a statistically significant increase in the MFI of CXCR4 expression (n = 6). Increase of O2 from 6% to 21% leads to a significant loss in MFI (n = 10). Western blotting also shows the changes (1.6-fold increase in OD at 6% O2 and 0.7-fold decrease after reoxygenation) in total protein in 1 primary AML sample. Graphs show mean values and SD.
