Proliferation inhibition assays on 3 AML cell lines. Results of inhibition assays are shown for TF1-vRaf (A), ML1 (B), and Monomac 6 (C) using DT₃₈₈GMCSF (▴), DTU2GMCSF (▾), and DTU2GMCSF plus exogenous pro-uPA (▪). The x-axis represents the log of the molar drug concentration and the y-axis represents cell viability expressed as percent control of ³H-thymidine incorporation in counts per minute. TF1-vRaf w as sensitive to DTU2GMCSF (IC₅₀ = 3.14 pM); the sensitivity was enhanced by the addition of exogenous pro-uPA (IC₅₀ = 0.26 pM) and became similar to that of DT₃₈₈GMCSF (IC₅₀ = 0.64 pM) (A). ML1 was not sensitive to DTU2GMCSF unless exogenous pro-uPA was added (IC₅₀ = 30 pM). The IC₅₀ for DT₃₈₈GMCSF was 22 pM (B). Monomac 6 was not sensitive to DTU2GMCSF even when exogenous pro-uPA was added (C). (D) Blocking assay. The proliferation inhibition assay on TF1-vRaf with DTU2GMCSF (▴), DTU2GMCSF plus anti-uPA (▪), and DTU2GMCF plus anti-GM-CSF (▾) is shown. On the x-axis is the log molar drug concentration, on the y-axis is the percentage of control ³H-thymidine incorporation. Both anti-GM-CSF and anti-uPA greatly decreased DTU2GMCSF efficacy (IC₅₀ = 400 pM and 0.67 μM, respectively) compared to an IC₅₀ = 2.3 pM for DTU2GMCSF alone, thus demonstrating the dual specificity of DTU2GMCSF, which requires the expression of both GM-CSFR and the uPA/uPAR protease system.