Figure 4

MCT-1 decreases p53 promoter function and protein expression. (A) The primers of p53 promoter region (-420 to -84) were hybridized with the ChIP complex. The p53 promoter DNA specifically contained in the IP complex of MCT-1 Ab but not normal IgG. (B) Luciferase activity of the pGL3-p53 promoter was depressed by ectopic MCT-1 (MCT-1) compared with control H1299 (control). (C) The pGL3-p53 promoter was conversely activated while MCT-1 silencing in H1299 context. (D) The half-life of exogenic p53 mRNA was decreased in ectopic MCT-1 group (MCT-1 + p53) relative to the corresponding control (control + p53) (P < 0.0001). (E) The overall p53 mRNA productions in the p53-restored H1299 cells were declined evidently in ectopic MCT-1 background (MCT-1 + p53). (F) Upon etoposide (ETO) exposure for 4 h, ectopic MCT-1 reduced the p53 functional activation of p21 (lanes 3 vs. 4). (G) The shRNA interference of intrinsic MCT-1 in MCF-10A cells conversely stimulated p53 and p21 proteins upon ETO treatment for 30 min. (H) The effects of wild-type MCT-1 (WT) and MCT-1 mutants that were directly mutated on Serine 118 residue (S118) to Alanine (S118A), Aspartic acid (S118D), and Glutamic acid (S118E) were studied. The dephosphorylation mutant (S118A) significantly improved the pGL3-p53 promoter function, but pGL3-p53 promoter was still repressed by cells expressing the phosphorylation-mimetic MCT-1 (S118D and S118E) and wild-type MCT-1. Statistical data was assessed with the Student's t test. **p < 0.002; ***p < 0.0001.