The accumulation of PUMA following MG132 treatment was p53 dependent, as such the accumulation was not observed in p53-deficient cells (Fig. 4, 6, 7); (d) activation of c-Jun NH2-terminal kinase (8); (e) accumulation of the pro-death proteins Bik and Bim (9, 10); and (f) induction of the mitochondria pathway (9–14). The antiapoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL, and Mcl-1) promote cell survival. p53: the attractive tumor suppressor in the cancer research field. Another apoptotic gene, PERP, is induced in response to DNA damage. Citicoline Protects Auditory Hair Cells Against Neomycin-Induced Damage. The Bcl-2 family proteins play important roles in the regulation of apoptosis by targeting to the mitochondria to exert their proapoptotic or antiapoptotic effects (16). Lack of Bax markedly decreases the cytotoxicity of proteasome inhibitors (12). Manage instrument use, information, and service, Spectroscopy, Elemental & Isotope Analysis, Preclinical to Companion Diagnostic Development, Chromatography Columns, Resins, & Spin Filters, Antigen Processing and Presentation by MHCs, Intrinsic and Extrinsic Pathways of Apoptosis, Cytokine, Chemokine, Growth Factor Pathways, Hematopoiesis from Multipotent Stem Cells, Hematopoiesis from Pluripotent Stem Cells, IL-2 Gene Expression in Activated and Quiescent T-Cells, VEGF Family Ligands and Receptor Interactions, p53 promotion of apoptosis via extrinsic pathway, p53 and the intrinsic pathway of apoptosis, Convergence of intrinsic and extrinsic pathways for apoptosis.

*, a nonspecific band. Taken together, these data suggest that ROS did not seem to play a significant role in MG132-induced Bax activation and apoptosis in colon cancer cells. We then asked whether p53 and PUMA would contribute to proteasome inhibition–induced Bax activation and apoptosis. [Mol Cancer Ther 2007;6(3):1062–9].

ROS do not play a role in MG132-induced apoptosis and Bax activation in HCT116 cells. Noxa encodes a BH3-only protein and hence is likely to contribute to p53-mediated apoptosis in a similar manner to PUMA and BAX, although this is yet to be demonstrated. Inhibition of proteasome function resulted in the blockage of normal degradation of p53 and in turn up-regulated PUMA. Results were expressed as the fold changes over the control samples. Akt also plays an important role in regulating p53 pathway under growth/survival conditions and under stress signals. Apoptotic cells were quantified by Hoechst 33342 staining.

Briefly, following a designated treatment, cells were incubated with 2.5 μmol/L dihydroethidium for 30 min at 37°C. Recent evidence has suggested the existence of a transcription-independent pathway of p53-mediated apoptosis. Consistently, inhibition of translation by cycloheximide could also effectively abolish the accumulation of p53 and PUMA and suppress MG132-induced Bax activation and apoptosis. In response to stress activation, BAX forms a homodimer and releases CytoC from the mitochondria.

Chk2 can phosphorylate p53 on Ser20, which prevents MDM2 binding and results in p53 stabilization. S4B and D). NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. Columns, mean; bars, SD.
Genetic deletion of PUMA or p53 in HCT116 cells significantly reduced mitochondrial release of cytochrome c and Smac and the activation of caspase-3 following MG132 treatment despite the presence of Bax (Fig. However, the successful induction of apoptosis by MG132 in these cells indicated that these antiapoptotic proteins must have been antagonized to allow the activation of Bax.


Cycloheximide could effectively inhibit Bax translocation based on both subcellular fractionation study and GFP-Bax assay (Fig. 3A 4C). PUMA couples the nuclear and cytoplasmic proapoptotic function of p53. Bax-deficient HCT116 cells (2 × 105) were transfected with green fluorescent protein (GFP)–Bax a generous gift from Dr. Richard Youle; NIH, Bethesda, MD) using Effectene according to the supplier's protocol (Qiagen, Valencia, CA). Columns, mean; bars, SD. S3).

One important BH3-only molecule is PUMA (23, 28). BioProbes Journal of Cell Biology Application—Stay up-to-date with highlights of the latest breakthroughs and get information about new technologies and products. The identification of this mechanism in colon cancer cells may have therapeutic implications for this type of cancer. Nonphosphorylated p53 is ubiquitylated by the MDM2 (Mouse Double Minute-2) ubiquitin ligase. Although cycloheximide could also suppress the accumulation of prosurvival molecules, such as Mcl-1 or Bcl-xL, the protective effect of cycloheximide against MG132 at the proper dose and time frame probably indicates that the pro-death mechanisms are mostly affected. Consistently, we found that MG132 induced the release of cytochrome c only in Bax-positive parental cells or in Bax-deficient cells stably reconstituted with GFP-Bax (Fig. Induction of apoptosis in various cancer cells by the inhibition of proteasome has been well documented (2, 3), in which the mitochondria apoptotic pathway can play important roles (9–14). To examine whether deletion of PUMA or p53 would affect MG132-induced Bax activation, we did immunoprecipitation assay using an antibody that specifically recognized Bax in the activated conformation (clone 6A7; BD Biosciences; ref. Anti-Bax (N-20) and p53 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Gen TP53 (Tumor protein p53) patří mezi důležité tumor supresorové geny.Jeho produkt – protein p53 funguje jako transkripční faktor a v buňce má funkci senzoru poškození DNA.Proteinu p53 se přezdívá "strážce genomu", právě pro jeho klíčovou roli v reakci na poškození genomu. Family members are classified on the basis of structural similarity to the BH (Bcl2 Homology) domains (BH1, BH2, BH3, and BH4) and a transmembrane domain. 16). BAX thus participates in the death response as an indirect target of p53 through PUMA. To address this issue, we would like to first confirm the finding using different proteasome inhibitors. In PUMA-deficient cell line, despite that there was an accumulation of p53 following MG132 treatment, there was no PUMA expression. Get the latest research from NIH: https://www.nih.gov/coronavirus. In humans, PUMA encodes two BH3-domain-containing proteins–PUMA-Alpha and PUMA-Beta. Certain DNA tumour viruses, such as the human adenovirus and the papillomavirus, bind to and inactivate the protein. In contrast, reactive oxygen species did not seem to be induced by MG132 or bortezomib and antioxidants had no effects on MG132-induced apoptosis. PUMA expression is mainly controlled by p53 (22, 23), which is normally rapidly degraded via the proteasomes (24).

We further revealed that the accumulation of proapoptotic proteins, p53 and PUMA, promoted Bax activation and the subsequent mitochondria activation and apoptosis. The ubiquitin proteasome system plays an important role in regulating apoptosis and cell cycle by controlling the degradation of important molecules, such as p53, I-κB, and cyclins. Collectively, these effects of cycloheximide contributed to the suppression of MG132-induced apoptosis (Fig. We propose that when p53 accumulates in the cytosol, it can function analogously to the BH3-only subset of proapoptotic Bcl-2 proteins to activate Bax and trigger apoptosis. Superoxide anion was detected as described previously (18). Both of these activities are induced by p53 (12-13). 5D). Notably, only those cells with punctated GFP-Bax went into apoptosis based on the morphology of the nuclei (Fig. C, the same treated cells were counterstained with MitoTracker Red (10 nmol/L) and examined by fluorescence microscopy. The ability of p53 to elicit diverse regulatory functions is likely to depend on its phosphorylation pattern, which is conformation dependent.

The p53 mediated apoptosis pathway is one of the major apoptosis signaling pathways involving the stimulation of both the extrinsic and intrinsic pathways by the p53 protein.

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