Enforced expression of hMgcRacGAP inhibits proliferation and induces macrophage differentiation of human myeloid leukemia HL-60 cells.

(A) Proliferation of HL-60 cells overexpressing hMgcRacGAP. The Flag-tagged hMgcRacGAP cDNAs were transduced into HL-60 cells using retrovirus vector pMX-IRES-EGFP. GFP⁺ HL-60 cells infected with these viruses were sorted on FACS 2 days after virus infection (HL-60/pMX-MgcRacGAP-IRES-EGFP cells). GFP⁺ HL-60 cells transduced with a blank pMX-IRES-EGFP vector were similarly sorted and used as a negative control (HL-60/pMX-IRES-EGFP cells). Both transfectants were counted at the indicated time points after the sorting. The results shown are the averages ± SD of triplicate cultures. (B) Enforced expression of the full-length form of hMgcRacGAP induces macrophage differentiation of HL-60 cells. Differentiation of parental HL-60, HL-60/pMX-IRES-EGFP, and HL-60/pMX-MgcRacGAP-IRES-EGFP cells was evaluated using flow cytometric analysis. The x-axis indicates forward scatter. The y-axis indicates side scatter. (C) CD14 expression of HL-60/pMX-IRES-EGFP cells and HL-60/pMX-MgcRacGAP-IRES-EGFP cells. The cells were stained with PE-conjugated mouse antihuman CD14 antibody or PE-conjugated isotype-matched control mouse IgG_2a. (D) Morphologic changes in HL-60 cells overexpressing hMgcRacGAP. May-Grunwald-Giemsa staining profiles of HL-60/pMX-IRES-EGFP and HL-60/pMX-MgcRacGAP-IRES-EGFP cells are shown. HL-60 cells treated with TPA (16 nmol/L) for 2 days are also shown as a positive control. Cells were centrifuged onto glass slides and stained with May-Grunwald-Giemsa stain. Photographs were taken at 400 × magnification.