EGF activates transcription of EGR-1 expression in endothelial cell lines. (A) Constitutive expression of EGF in the human Burkitt Raji B lymphoma cells and the human MDA-MB-231 breast carcinoma cells, as shown by RT-PCR analysis. (B) bEnd.3 cells were treated with vehicle alone or 10 ng/mL epidermal growth factor (EGF) for 1 hour. Positive control consisted of bEnd.3 cells incubated with 50 ng/mL PMA for 1 hour. Total RNA was extracted and subjected to RT-PCR to quantify EGR-1 expression. (C) 293 cells were cotransfected with the pGL3-EGR-1 promoter construct and the pRL-TK Renilla luciferase as described in “Transient transfections and luciferase assays.” After 24 hours, the 293 cells were preincubated for 1 hour with blocking mAbs specific for epidermal growth factor (EGFR) or control mouse immunoglobulin G (IgG). Thereafter, the murine 267 T lymphoma cells (3 × 10⁶ cells/well), the human Burkitt Raji B lymphoma cells (3 × 10⁶ cells/well), and the human MDA-MB-231 breast carcinoma cells (5 × 10⁵ cells/well) were added to adherent cells for 16 hours. Cells were collected, and luciferase activity was measured. Controls included 293 cells without tumor cells. The firefly luciferase was normalized to Renilla luciferase activity. Results are representative of 3 independent experiments performed in triplicate. Bars indicate SD; *P ≤ .05, significantly different from the control mouse IgG treatment.