Figure 10.

A positive feedback loop between Pim-1 and HBP1 contributes to premature senescence and apoptosis induced by H₂O₂. A, the protein levels of Pim-1, HBP1, and phosphoserine (phospho-HBP1 (p-HBP1)) increase with time progression in tumor cells with initial H₂O₂ treatment. U2OS and HeLa cells were treated with 150 μm H₂O₂ for different lengths of time (0, 12, 24, 36, 48, 60, and 72 h). The protein levels of Pim-1, HBP1, Bax, and phosphoserine (phospho-HBP1 (p-HBP1)) were determined by Western blotting. B, the protein levels of Pim-1 and HBP1 increase with time progression in normal fibroblasts with initial H₂O₂ treatment. 2BS cells were treated with 20 μm H₂O₂ for different lengths of time (0, 2, 4, 6, 8, and 10 days). The protein levels of Pim-1, HBP1, p16, and GAPDH (as loading control) were determined by Western blotting. C, H₂O₂ enhanced transactivation of endogenous HBP1 on Pim-1 promoter. HeLa cells transfected with Luc-Pim-1 were treated with H₂O₂ for different lengths of time (0, 12, 24, 36, 48, 60, and 72 h). The luciferase activity measurements of the group treated with H₂O₂ were normalized to luciferase activity of the group without H₂O₂. Error bars represent S.D. *, p < 0.05; **, p < 0.01. D, H₂O₂ enhanced transactivation of exogenous HBP1 on Pim-1 promoter. HeLa cells transfected with Luc-Pim-1 were treated with H₂O₂ with or without HBP1. Error bars represent S.D. *, p < 0.05; **, p < 0.01. E, HeLa cells were treated with H₂O₂ or transfected with Pim-1 expression plasmid, and then nuclear and cytoplasmic HBP1 protein levels were determined by Western blotting. F, the positive feedback loop between Pim-1 and HBP1 regulates the expression of senescence markers (DNMT1 and p16) and apoptosis marker (Bax). HeLa and 2BS cells were stably transfected with vector, shHBP1, shPim-1, or shHBP1 + shPim-1. After 4 weeks of selection, HeLa cells were treated with 150 μm H₂O₂ for 48 h, and 2BS cells were treated with 20 μm H₂O₂ for 1 week. Then the cells were harvested for Western blotting using anti-Pim-1, anti-HBP1, anti-Bax, anti-DNMT1, anti-p16, or anti-GAPDH antibodies (as loading control). G and H, the positive feedback loop between Pim-1 and HBP1 promotes premature senescence and apoptosis in response to H₂O₂ induction. HeLa or 2BS cells were treated as in F, HeLa cells were stained with Annexin V-FITC and PI, and cell apoptosis was analyzed by FACS (G). 2BS cells were stained for SA-β-gal (H, left), and the percentage of cells positive for SA-β-gal in 2BS cells was determined in three independent experiments (H, right). Error bars represent S.D. *, p < 0.05; **, p < 0.01.