IGFBP-3 inhibits Egr-1 expression independently IGF-1. (A-B) The wild-type 1.2-kb Egr-1 promoter reporter construct (Egr1-A-Luc) was transiently transfected, with or without pBP-3 or pBP-3-ggg, into NSCLC H460 cells. (A) Cells were stimulated by cotransfection of plasmids containing mutants of K-Ras (V12) or H-Ras (V12) or by exposure to hypoxia (1% O₂) or γ-radiation (8 Gy). (B) Cells were stimulated by IGF-1 (50 ng/mL) or FBS (10% and 30%) for 24 hours or cotransfected with plasmids expressing CA MEK. (C) R⁻ (IGF-1R null mouse fibroblasts) and R⁺ (R⁻ cells transfected with IGF-1R) cell lines were cotransfected with Egr1-A-Luc and pBP-3 or pIGFBP-3-ggg and then stimulated by IGF-1 (50 ng/mL) or FBS (10%) for 24 hours. The data are the mean ± SD from 3 independent experiments, with 4 replicates per experiment. *P < .05, **P < .01. (D) In vitro evaluation of the antiangiogenic potential of IGFBP-3. pBP-3–transfected H460 cells show less stimulatory activity for HUVEC proliferation than untransfected H460 cells in a coculture assay system. The values are the mean ± SD from 2 separate experiments, with 3 replicates per experiment. *P < .05, **P < .01, ***P < .001.