Effect of AG490 on PEL cell lines.

The tyrphostin AG490 inhibits constitutive activation of STAT3 and DNA synthesis in PEL cell lines. (A) Inhibition of constitutively activated STAT3 by AG490. BC-1 cells were incubated with 100 μM AG490 or DMSO (solvent) for 24 hours, and equal amounts of nuclear extracts (2 μg per lane) were incubated with a ³²P-labeled oligonucleotide probe corresponding to the IL-6 response element and analyzed by EMSA. BAF130 cells were stimulated with 1 μg/mL MBP–v-IL-6 at 37°C for 10 minutes before lysis. Excess cold oligonucleotide competition (× 50) completely eliminated this binding activity (data not shown). Shown is a representative experiment of 3 performed. (B) Supershift assay was performed by adding an isotype control, anti-STAT1 and/or anti-STAT3 Abs, to a nuclear extract from BC-1 cells. (C) TF-1 cells (1 × 10⁴ cells per well) were incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) (1 ng/mL) or human IL-6 (50 ng/mL) in the presence of AG490 for 24 hours, including a final 6-hour pulse with [³H]-thymidine. K562 and PEL cells were cultured under the same conditions but without exogenous cytokines. DMSO concentration was adjusted to a final 0.1% in all cultures. DMSO at this concentration did not affect the growth of these cell lines. The assay was performed in triplicate cultures. The results represent the mean percentage (± SD) of radioactivity compared with cultures in DMSO alone: SDs at most points were within 2% of each mean value. Shown is a representative experiment of 4 performed. (D) AG490 induction of apoptosis in PEL cells. Cells were treated with AG490 or 0.1% DMSO for 48 hours, stained with annexin V–FITC, and analyzed for apoptosis by flow cytometry. Marker positioning is based on fluorescence of controls, and data shown are from a representative experiment of 3 performed. The percentages of annexin V–FITC–binding cells are shown.