Sci. p53. The mitochondrial p53/Bad complex promotes apoptosis via activation and oligomerization of Bak. Removal of Bad expression by RNA interference notably attenuates apoptosis induced by etoposide. Hence, our collective data provide the first evidence that Bad plays dual functions in both p53 transcription-dependent and -impartial pathways. Bad (Bcl-2 antagonist of cell death), a member of the Bcl-2 protein family, is a positive regulator of cell death and is localized in the cytoplasm under physiological conditions. Like other BH3-only proteins, Bad selectively displaces Bax to heterodimerize with Bcl-2 or Bcl-xL while promoting apoptosis (5, 25). The apoptotic activity of Bad is determined largely by its phosphorylation status at serine 112 (Ser112), Ser136, and Ser155 (5). In unstressed cells, Bad is usually hyperphosphorylated by several Fgfr2 protein kinases, including protein kinase A and protein kinase B (AKT), and as a result is usually held onto by 14-3-3 (7, 11). However, LY2608204 in response to apoptotic stimuli, Bad is usually rapidly dephosphorylated and migrates to the mitochondria, where it induces cell death. The p53 tumor suppressor is usually a pivotal mediator of cell cycle LY2608204 arrest and apoptosis in response to diverse stress stimuli. It transactivates numerous target genes, including those for p21waf1 (9), Bax (16), p53DINP1 (18), Bid (20), Puma (17), and many other proteins (21, 24). However, a steady input of evidence for the transcription-independent function of p53 in apoptosis has been accumulating recently (13, 15). Although a detailed mechanism has not yet been decided, recent data have revealed that upon an apoptotic stimulus, p53 translocates into mitochondria to interact directly with Bcl-2 or Bcl-xL to displace and activate proapoptotic Bax signaling (6, 15). Similarly, disruption of the association complex between Bak/Mcl-1 or Bak/Bcl-2 by p53 is also related to p53-dependent but transcription-independent apoptosis (14). In this study, we have recognized a p53-responsive element located approximately 6.6 kb upstream of the ATG translational start codon of the Bad gene and have shown that p53 transcriptionally activates Bad expression through binding to this region. We provide evidence that Bad is a part of a negative opinions loop control system serving to maintain the equilibrium level of Bad by reducing p53 nuclear access. Excess Bad binds to p53 within the cytosol, preventing p53 from entering the nucleus for further transcription of (R&D Systems, Inc., Minneapolis, MN); anti–actin (Abcam, Cambridge, United Kingdom); anti-OxPhosComplex IV subunit IV antibody (COX IV; Molecular Probes, Eugene, Oregon); and anti–tubulin (Santa Cruz) and polyclonal antibody anti-Max (Santa Cruz). Hoechst 33342 and etoposide were purchased from Sigma, and 1,6-bismaleimidohexane (BMH) was purchased from Pierce Biotechnology (Rockford, IL). RNA interference (RNAi) and apoptosis assays. A549 (p53+/+) cells LY2608204 were cultured in 12-well plates to subconfluence (3 LY2608204 105 cells) and transiently transfected with small interfering RNA (siRNA) against Bad (Cell Signaling Technology, Danvers, Massachusetts) by using Oligofectamine (Invitrogen Cergy Pontoise, France). At 24 h posttransfection, the culture medium was replaced with fresh medium, followed by addition LY2608204 of etoposide at 50 g/ml. After 24 h and 48 h of incubation, both floating and adherent cells were stained with Hoechst 33342 (2 g/ml). Microscopic fields were selected randomly, and 10 fields were observed for each slide at a magnification of 400. The percentages of apoptotic cells among the total quantity of cells were counted based on the numbers of apoptotic cells characteristic for DNA fragmentation and/or chromatin condensation under fluorescence microscopy. The data collected are the averages and.