Figure 7.
Abolition of p53-mediated transcription and apoptosis induction by inhibition of p38 MAPK. (A) Effect of chemical p38 MAPK inhibitors on Env-induced p53-mediated transcription within syncytia. A p53-inducible GFP reporter gene was transfected into HeLa Env/CD4 cells 24 h before coculture for 36h, resulting in expression of GFP mainly within syncytia, but not single cells (left panel). The effect of the p38 MAPK inhibitors SB203580 and SB202190 (as well as that of the ineffective control compound SB202474) is shown in the right panel. (B) Effect of DN p38. HeLa Env/CD4 cells were transfected with a p53-inducible luciferase reporter construct (p53-Luc), a noninducible luciferase construct (pTA-Luc), with p38 DN or its empty vector, 24 h before coculture, followed by coincubation of HeLa Env and HeLa CD4 cells for 36 h and determination of luciferase activity. (C and D) Effect of pharmacological p38 MAPK inhibiton (C) or DN p38 (D) on the immunofluorescence-detectable exposure of the Bax or Bak NH2 termini, the mitochondrial release of cytochrome c, and Hoechst 33342-detectable chromatin condensation 36 h after syncytium formation. Note that these parameters are largely negative in single cells (<5%). (E) Effect of p38 MAPK knock-down by siRNA. Cells were subjected to siRNA 24 h before coculture for 36 h (syncytia, Syn) or separate coculture for 36 h (single cells, SC), and the expression of p38 MAPK, PUMA, and Bax was monitored by immunoblot. The frequency of apoptotic cells was determined among single cells and syncytia by Hoechst 33312 staining and fluorescence microscopy.