PUMA Promotes Bax Translocation by Both Directly Interacting with Bax and by Competitive Binding to Bcl-XL during UV-induced Apoptosis

Cell apoptosis induced by UV irradiation is a highly complex process in which different molecular signaling pathways are involved. p53 up-regulated modulator of apoptosis (PUMA) has been proposed as an important regulator in UV irradiation-induced apoptosis. However, the molecular mechanism through which PUMA regulates apoptosis, especially how PUMA activates Bcl-2-associated X protein (Bax) in response to UV irradiation is still controversial. In this study, by using real-time single-cell analysis and fluorescence resonance energy transfer, we investigated the tripartite nexus among PUMA, Bax, and Bcl-X_L in living human lung adenocarcinoma cells (ASTC-a-1) to illustrate how PUMA promotes Bax translocation to initiate apoptosis. Our results show that the interaction between PUMA and Bax increased gradually, with Bax translocating to mitochondria and colocalizing with PUMA after UV irradiation, indicating PUMA promotes Bax translocation directly. Simultaneously, the interaction increased markedly between PUMA and Bcl-X_L and decreased significantly between Bcl-X_L and Bax after UV treatment, suggesting PUMA competitively binds to Bcl-X_L to activate Bax indirectly. The above-mentioned results were further confirmed by coimmunoprecipitation experiments. In addition, pifithrin-α (a p53 inhibitor) and cycloheximide (a protein synthesis inhibitor) could inhibit PUMA-mediated Bax translocation and cell apoptosis. Together, these studies create an important conclusion that PUMA promotes Bax translocation by both by directly interacting with Bax and by competitive binding to Bcl-X_L in UV-induced apoptosis.     

Bax conformational change is a crucial step for PUMA-mediated apoptosis in human leukemia

The BH3-only protein, PUMA, plays an important role in p53-mediated apoptosis. The apoptotic effect of PUMA on the mitochondria was studied using a p53-negative, human leukemia K562 cell line. Overexpression of PUMA was accompanied by an increased Bax expression, Bax conformational change, and translocation to mitochondria. A PUMA-BH3 peptide can induce Bax conformational change, cytochrome c release, and reduction in the mitochondrial membrane potential (DeltaPsi(m)) in isolated K562 mitochondria and can be inhibited by Bcl-XL. The homo-dimer of Bax/Bax was also weakly shown after mitochondria were treated with PUMA-BH3 peptide but may not be lethal for PUMA-induced apoptosis in K562 cells. Our results suggest that PUMA-induced Bax conformational change and Bax translocation to mitochondria can be separate events and the conformational change in Bax is crucial for PUMA-induced mitochondrial dysfunction.     

p73 Induces apoptosis via PUMA transactivation and Bax mitochondrial translocation

p73, an important developmental gene, shares a high sequence homology with p53 and induces both G(1) cell cycle arrest and apoptosis. However, the molecular mechanisms through which p73 induces apoptosis are unclear. We found that p73-induced apoptosis is mediated by PUMA (p53 up-regulated modulator of apoptosis) induction, which, in turn, causes Bax mitochondrial translocation and cytochrome c release. Overexpression of p73 isoforms promotes cell death and bax promoter transactivation in a time-dependent manner. However, the kinetics of apoptosis do not correlate with the increase of Bax protein levels. Instead, p73-induced mitochondrial translocation of Bax is kinetically compatible with the induction of cell death. p73 is localized in the nucleus and remains nuclear during the induction of cell death, indicating that the effect of p73 on Bax translocation is indirect. The ability of p73 to directly transactivate PUMA and the direct effect of PUMA on Bax conformation and mitochondrial relocalization suggest a molecular link between p73 and the mitochondrial apoptotic pathway. Our data therefore indicate that PUMA-mediated Bax mitochondrial translocation, rather than its direct transactivation, correlates with cell death. Finally, human DeltaNp73, an isoform lacking the amino-terminal transactivation domain, inhibits TAp73-induced as well as p53-induced apoptosis. The DeltaNp73 isoforms seem therefore to act as dominant negatives, repressing the PUMA/Bax system and, thus, finely tuning p73-induced apoptosis. Our findings demonstrate that p73 elicits apoptosis via the mitochondrial pathway using PUMA and Bax as mediators.     

A role for the deubiquitinating enzyme USP28 in control of the DNA-damage response

The Chk2-p53-PUMA pathway is a major regulator of DNA-damage-induced apoptosis in response to double-strand breaks in vivo. Through analysis of 53BP1 complexes we have discovered a new ubiquitin protease, USP28, which regulates this pathway. Using a human cell line that faithfully recapitulated the Chk2-p53-PUMA pathway, we show that USP28 is required to stabilize Chk2 and 53BP1 in response to DNA damage. In this cell line, both USP28 and Chk2 are required for DNA-damage-induced apoptosis, and they accomplish this in part through regulation of the p53 induction of proapoptotic genes like PUMA. Our studies implicate DNA-damage-induced ubiquitination and deubiquitination as a major regulator of the DNA-damage response for Chk2, 53BP1, and a number of other proteins in the DNA-damage checkpoint pathway, including several mediators, such as Mdc1, Claspin, and TopBP1.     

PRIMA-1 induces apoptosis by inhibiting JNK signaling but promoting the activation of Bax

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 40% of breast cancers and are indicative of tumor resistance to chemotherapeutic agents. Recently, there has been a high degree of interest in pharmacological approaches for restoring the normal function to mutant p53. The low molecular weight compound p53 reactivation and induction of massive apoptosis (PRIMA-1) was shown to induce cytotoxic effects and apoptosis in human tumor cells with mutant p53. Here, we studied the molecular mechanisms of PRIMA-1-induced apoptosis in human breast cancer cells with p53 mutations such as MDA-231 and GI-101A as compared to MCF-7 cells. We show that PRIMA-1 selectively induces apoptosis in human breast cancer cells MDA-231 and GI-101A compared to the MCF-7. This effect was paralleled by an increase in total p53 level in the nucleus and the induction of its phosphorylation at Ser-15 site. Using the chromatin immunoprecipitation (ChIP) assays, we show that PRIMA-1 restored p53 DNA binding activity to the promoters of the proapoptotic genes such as Bax and PUMA, but inhibited the binding activity to the promoters of the MAP4K4 gene. Knockdown of p53 protein in breast cancer cells using siRNA followed by PRIMA-1 treatment resulted in decline of Bax and PUMA proteins expression. Cell incubation with either PRIMA-1 or SP600125 (c-Jun NH2-terminal kinase inhibitor) resulted in the abrogation of adriamycin-induced c-Jun NH2-terminal kinase (JNK) activation, whereas Bax activation was not inhibited. We conclude that both Bax and PUMA but not JNK signaling are involved in PRIMA-1-induced apoptosis in breast cancer cells with p53 mutation.     

P53/PUMA are potential targets that mediate the protection of brain-derived neurotrophic factor (BDNF)/TrkB from etoposide-induced cell death in neuroblastoma (NB)

The over-expressions of brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor TrkB have been reported to induce chemo-resistance in neuroblastoma (NB) cells. In this study, we investigated the roles of P53 and BCL2 family members in the protection of BDNF/TrkB from etoposide-induced NB cell death. TB3 and TB8, two tetracycline (TET)-regulated TrkB-expressing NB cell lines, were utilized. The expressions of P53 and BCL2 family members were detected by Western blot or RT-PCR. Transfection of siRNAs was used to knockdown P53 or PUMA. Activated lentiviral was used to over-express PUMA. Cell survival was performed by MTS assay, and the percentage of cell confluence was measured by IncuCyte ZOOM. Our results showed that etoposide treatment induced significant and time-dependent increase of P53, which could be blocked by pre-treatment with BDNF, and knockdown P53 by transfecting siRNA attenuated etoposide-induced TrkB-expressing NB cell death. PUMA was the most significantly changed BCL2 family member after treatment with etoposide, and pre-treatment with BDNF blocked the increased expression of PUMA. Transfection with siRNA inhibited etoposide-induced increased expression of PUMA, and attenuated etoposide-induced NB cell death. We also found that over-expression of PUMA by infection of activated lentiviral induced TrkB-expressing NB cell death in the absence of etoposide, and treatment of BDNF protected NB cells from PUMA-induced cell death. Our results suggested that P53 and PUMA may be potential targets that mediated the protection of BDNF/TrkB from etoposide-induced NB cell death.     

Release of cytochrome c from isolated mitochondria by etoposide

The efficacy of chemotherapeutic agents on tumor cells has been shown to be modulated by tumor suppressor gene p53 and its target genes such as Bcl-2 family members (Bax, Noxa, and PUMA). However, various chemotherapeutic agents can induce cell death in tumor cells that do not express the functional p53, suggesting that some chemotherapeutic agents may induce cell death in a p53-independent pathway. Here we showed that etoposide can induce the similar degree of cell death in p53-deficient HCT 116 cells, whereas 5'-FU-mediated cell death is strongly dependent on the existence of functional p53 in HCT 116 cells. Further, we provide the evidence that etoposide can induce the cytochrome c release from isolated mitochondria, and etoposide-induced cytochrome c release is not accompanied with the large amplitude swelling of mitochondria. These data suggest that etoposide can directly induce the mitochondrial dysfunction irrespective of p53 status, and it may, at least in part, account for the p53-independent pathway in cell death induced by chemotherapeutic agents.     

N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome C release and apoptotic cell death

Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase-activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N-terminus, generating a potent proapoptotic 18-kDa fragment (Bax/p18). Both the calpain-mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane-enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and fragmentation of DNA. Unlike the full-length Bax, Bax/p18 did not interact with the antiapoptotic Bcl-2 protein in the mitochondrial fraction of drug-treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and caspase-3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase-3-mediated apoptosis that was not blocked by overexpression of Bcl-2 protein. Therefore, Bax/p18 has a cytochrome c-releasing activity that promotes cell death independent of Bcl-2. Finally, Bcl-2 overexpression inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution.     

mtCLIC/CLIC4, an organellular chloride channel protein, is increased by DNA damage and participates in the apoptotic response to p53

mtCLIC/CLIC4 (referred to here as mtCLIC) is a p53- and tumor necrosis factor alpha-regulated cytoplasmic and mitochondrial protein that belongs to the CLIC family of intracellular chloride channels. mtCLIC associates with the inner mitochondrial membrane. Dual regulation of mtCLIC by two stress response pathways suggested that this chloride channel protein might contribute to the cellular response to cytotoxic stimuli. DNA damage or overexpression of p53 upregulates mtCLIC and induces apoptosis. Overexpression of mtCLIC by transient transfection reduces mitochondrial membrane potential, releases cytochrome c into the cytoplasm, activates caspases, and induces apoptosis. mtCLIC is additive with Bax in inducing apoptosis without a physical association of the two proteins. Antisense mtCLIC prevents the increase in mtCLIC levels and reduces apoptosis induced by p53 but not apoptosis induced by Bax, suggesting that the two proapoptotic proteins function through independent pathways. Our studies indicate that mtCLIC, like Bax, Noxa, p53AIP1, and PUMA, participates in a stress-induced death pathway converging on mitochondria and should be considered a target for cancer therapy through genetic or pharmacologic approaches.