p53 mediates nitric oxide-induced apoptosis in murine neural progenitor cells.

Studies have shown that nitric oxide (NO)-induced apoptosis is mediated by a variety of cellular signaling pathways. However, the information is relatively limited to neural progenitor cells (NPCs). In this study, the role of p53 in the NO-induced apoptosis was examined in an in vitro model of NPCs. Comparisons were made between NPCs derived from either wild type or p53 knockout mice brain stimulated by diethylenetriamine/nitric oxide adduct (DETA/NO), an established NO donor that constantly releases NO through its known first order pharmacological kinetics and prolonged half-life. We found that treatment by DETA/NO both time- and dose-dependently induced a significant increase of apoptosis in wild type NPCs, while p53 knockout NPCs were resistant to the DETA/NO challenge. In addition, the DETA/NO-triggered alteration of mitochondrial membrane permeability, cleavage of caspase-9/3, and expression of pro-apoptotic Bcl-2 family members noxa and puma occurred in wild type NPCs but not in p53 knockout NPCs. Our current results suggest a central role of p53 in the NO-induced apoptotic pathway in NPCs, which may hence provide new insights into the regulation of cell death in NPCs that respond to overproduction of NO in injured brain.

PKCdelta mediates Nrf2-dependent protection of neuronal cells from NO-induced apoptosis.

A chemical inhibitor library of 84 compounds was screened to investigate the signaling pathway(s) leading to activation of Nrf2 in response to nitric oxide (NO). We identified the protein kinase C delta (PKCdelta) inhibitor rottlerin as the only compound that reduced NO-induced ARE-luciferase reporter activity and diminished NO-induced up-regulation of two Nrf2/ARE-regulated proteins - NAD(P)H:quinone oxidoreductase-1 (NQO1) and hemeoxygenase-1 (HO-1) in SH-Sy5y cells. Rottlerin also sensitized neuroblastoma cells and mouse primary cortical neurons to NO-induced apoptosis. Stable over-expression of PKCdelta augmented NO-induced, ARE-dependent gene expression of HO-1 in SH-Sy5y cells, which were more protected from NO killing. Conversely, NO-induced ARE-dependent gene expression was reduced in PKCdelta-knockdown SH-EP cells, which displayed greater sensitivity to apoptosis. PKCdelta(-/-) cortical neurons exhibited increased NO-induced apoptosis and less HO-1 mRNA and protein induction compared with wild type neurons. Hence, PKCdelta is an important positive modulator of NO-induced Nrf2/ARE-dependent signaling that counteracts NO-mediated apoptosis in neuronal cells.

Mammalian sterile 20-like kinase 3 (MST3) mediates oxidative-stress-induced cell death by modulating JNK activation.

MST3 (mammalian sterile 20-like kinase 3) is a sterile 20 kinase reported to have a role in Fas-ligation- and staurosporine-induced cell death by unknown mechanism(s). We found that MST3-deficient cells are resistant to H2O2, which was reversed by reconstituting recombinant MST3. H2O2-induced JNK (c-Jun N-terminal kinase) activation was greatly enhanced in shMST3 cells (a cell line treated with short hairpin RNA against MST3). Suppression of JNK activity by the inhibitor SP600125 or by dominant-negative JNK2 re-sensitized cells to H2O2. Furthermore, c-Jun Ser-63 phosphorylation was augmented in shMST3 cells, whereas JunAA (dominant-negative c-Jun) reduced H2O2 resistance, implicating an AP-1 (activator protein 1) pathway in H2O2-induced survival signalling. Total cytoprotective HO-1 (haem oxygenase 1) expression, which was attenuated by JunAA, was induced up to 5-fold higher in shMST3 cells compared with controls. Zinc protoporphyrin IX, a potent inhibitor of HO reversed the H2O2-resistance of shMST3 cells. Our results reveal that H2O2-induced MST3-mediated cell death involves suppressing both a JNK survival pathway and up-regulation of HO-1.

Focused PCR screen reveals p53 dependence of nitric oxide-induced apoptosis and up-regulation of maspin and plasminogen activator inhibitor-1 in tumor cells.

We investigated p53-dependent gene expression in nitric oxide (NO)-induced apoptosis of two tumor cell types. Seventy-seven putative p53-regulated genes were screened for NO-mediated expression changes. Twenty-four genes were up-regulated and three genes were down-regulated significantly by NO in human neuroblastoma cells. Genes known to be involved in apoptosis, which were up-regulated by > or = 2-fold, included FAS, CASP-1, BIK, PUMA, DR4 and the serpins maspin (SERPINB5), and plasminogen activator inhibitor-1 (PAI-1). Real-time PCR confirmed maspin and PAI-1 mRNAs exhibited the greatest NO-induced induction, which occurred in a p53-dependent manner. The substantial NO-mediated up-regulation of these serpins mRNAs correlated with large increases in their protein levels, which occurred before or coinciding with apoptosis. p53-deficient neuroblastoma cells were largely resistant to NO killing and showed much reduced maspin and PAI-1 mRNA and protein levels after NO treatment. p53 was activated by NO mainly in the nuclei of neuroblastoma cells. p53(-/-) HCT116 colon carcinoma cells were strongly resistant to NO-induced apoptosis and failed to up-regulate maspin and PAI-1 (in contrast to p53(+/+) HCT116 cells). Our results suggest that both apoptosis and induction of the two serpins by NO require the transcriptional activity of p53. Because maspin is a tumor suppressor and PAI-1 can promote senescence and regulate cell death, it will now be worth investigating whether their p53-mediated expression contributes to the NO-induced p53-dependent death of tumor cells.

Caspase-4 interacts with TNF receptor-associated factor 6 and mediates lipopolysaccharide-induced NF-kappaB-dependent production of IL-8 and CC chemokine ligand 4 (macrophage-inflammatory protein-1 ).

Human caspase-4 does not have a corresponding mouse ortholog. Caspase-4 falls within the class of "inflammatory caspases," being homologous with human caspases 1 and 5 and mouse caspases 1, 11, and 12. To address the function of caspase-4, we generated caspase-4-deficient human THP1 monocytic cell lines which exhibited substantially reduced LPS-induced secretion of several chemokines and cytokines, including IL-8 (CXCL8), CCL4 (macrophage-inflammatory protein-1beta), CCL20 (macrophage-inflammatory protein-3alpha), and IL-1beta. The LPS-induced expression of the mRNAs encoding these cytokines was correspondingly reduced in the caspase-4-deficient clones. Because a specific NF-kappaB inhibitor blocked LPS-induced IL-8 and CCL4 mRNA expression as well as IL-8 and CCL4 secretion in THP1 cells, we investigated the role of caspase-4 in NF-kappaB signaling. LPS-induced NF-kappaB nuclear translocation and activation were inhibited in all caspase-4-deficient clones. LPS stimulation led to the interaction of endogenous caspase-4 and TNFR-associated factor 6 (TRAF6) via a TRAF6-binding motif (PPESGE), which we identified in caspase-4. Mutation of this site in caspase-4 resulted in the loss of the TRAF6-caspase-4 interaction. Similar TRAF6-binding motifs are known to be functionally important for TRAF6 interactions with other molecules including caspase-8, and for mediating NF-kappaB activation in various immune and nonimmune cell types. Our data suggest that the TRAF6-caspase-4 interaction, triggered by LPS, leads to NF-kappaB-dependent transcriptional up-regulation and secretion of important cytokines and chemokines in innate immune signaling in human monocytic cells.

Protein/DNA arrays identify nitric oxide-regulated cis-element and trans-factor activities some of which govern neuroblastoma cell viability.

Toxic nitric oxide (NO) levels can regulate gene expression. Using a novel protein/DNA array, we show that toxic NO levels regulate the binding of trans-factors to various cis-elements in neuroblastoma cells, including CRE and those recognized by the transcription factors AP1, AP2, Brn-3a, EGR, E2F1 and SP1. Functionality of some of the cis-elements was confirmed by electro mobility shift and reporter assays. Interestingly, CREB, AP-1, Brn-3a, EGR and E2F1 can control mammalian cell viability. NO induced the anti-apoptotic Bcl-2 protein and its mRNA prior to the onset of death of 30-60% of the cells. Promoter analysis of the bcl-2 gene confirmed the involvement of a CRE in NO-dependent bcl-2 transcription. Neuroblastoma cells over-expressing bcl-2 became much more resistant to NO-induced apoptosis; conversely, Bcl-2 knockdown cells were rendered markedly more sensitive to NO. Together these results suggest that Bcl-2 counteracts NO-induced apoptosis in a fraction of the cell population. Thus, NO stimulates the binding of many trans-factors to their cognate cis-elements, some of which can regulate cell viability through transcriptional activation of target genes. Our results emphasize that a DNA/protein array approach can reveal novel, global transcription factor activities stimulated by cell death-regulating molecules.

Reoxygenation-specific activation of the antioxidant transcription factor Nrf2 mediates cytoprotective gene expression in ischemia-reperfusion injury.

Tissue reoxygenation following hypoxia is associated with ischemia-reperfusion injury (IRI) and may signal the development of ischemic preconditioning, an adaptive state that is protective against subsequent IRI. Here we used microarray RNA analysis of in vivo and in vitro models of IRI to delineate the underlying molecular mechanisms. Microarray analysis of renal tissue after ischemia-reperfusion revealed a number of highly up-regulated antioxidant genes including aldehyde dehydrogenases (ALDH1A1 and ALDH1A7), glutathione S-transferases (GSTM5, GSTA2 and GSTP1), and NAD(P)H quinone oxidoreductase (NQO1). The transcription factor NF-E2-related factor-2 (Nrf2), a master regulator of this antioxidant response, is also elevated in IRI. Furthermore, microarray analysis of renal epithelial cells exposed to hypoxia/reoxygenation identified Nrf2 to be up-regulated on reoxygenation. We also reveal a reoxygenation-specific nuclear accumulation of Nrf2 protein and subsequent activation of a NQO1 promoter reporter construct. Attenuating reactive oxygen species (ROS) in reoxygenation using the antioxidant N-acetyl cysteine results in inhibition of Nrf-2 activation. mRNA levels for Nrf2-dependent genes were detected in human liver biopsy 1 h after transplantation. These results indicate that reoxygenation-dependent Nrf-2 activity facilitates ischemic preconditioning through the induction of antioxidant gene expression and that ROS may be critical in signaling this event.

Does apoptosis-inducing factor (AIF) have both life and death functions in cells?

Apoptosis-inducing factor (AIF) is expelled from mitochondria after some apoptotic stimuli and translocates to the nucleus, which may contribute to DNA and nuclear fragmentation in some non-physiological mammalian cell deaths. Conversely, the requirement for mitochondrial AIF in oxidative phosphorylation and energy generation provides a plausible explanation for the embryonic lethality or neurodegeneration that has been found in different AIF-deficient mouse models. These findings may help illuminate the ability of mitochondrial AIF to suppress cytoplasmic stress granule formation and to promote the tumorigenic growth of cancer cells. AIF is ideally located in the mitochondrion to perform a vital normal function in energy production. Once it translocates to the nucleus, however, the cell might die either of energy failure or nuclear fragmentation (or both). We propose that the main function of AIF is to support energy production in both normal and transformed cell physiology, whereas nuclear-translocated AIF might contribute to stress-induced or pathological cell death in certain scenarios.

Mifepristone induces growth arrest, caspase activation, and apoptosis of estrogen receptor-expressing, antiestrogen-resistant breast cancer cells.

PURPOSE: A major clinical problem in the treatment of breast cancer is the inherent and acquired resistance to antiestrogen therapy. In this study, we sought to determine whether antiprogestin treatment, used as a monotherapy or in combination with antiestrogen therapy, induced growth arrest and active cell death in antiestrogen-resistant breast cancer cells. EXPERIMENTAL DESIGN: MCF-7 sublines were established from independent clonal isolations performed in the absence of drug selection and tested for their response to the antiestrogens 4-hydroxytamoxifen (4-OHT) and ICI 182,780 (fulvestrant), and the antiprogestin mifepristone (MIF). The cytostatic (growth arrest) effects of the hormones were assessed with proliferation assays, cell counting, flow cytometry, and a determination of the phosphorylation status of the retinoblastoma protein. The cytotoxic (apoptotic) effects were analyzed by assessing increases in caspase activity and cleavage of poly(ADP-ribose) polymerase. RESULTS: All of the clonally derived MCF-7 sublines expressed estrogen receptor and progesterone receptor but showed a wide range of antiestrogen sensitivity, including resistance to physiological levels of 4-OHT. Importantly, all of the clones were sensitive to the antiprogestin MIF, whether used as a monotherapy or in combination with 4-OHT. MIF induced retinoblastoma activation, G(1) arrest, and apoptosis preceded by caspase activation. CONCLUSIONS: We demonstrate that: (a) estrogen receptor(+)progesterone receptor(+), 4-OHT-resistant clonal variants can be isolated from an MCF-7 cell line in the absence of antiestrogen selection; and (b) MIF and MIF plus 4-OHT combination therapy induces growth arrest and active cell death of the antiestrogen-resistant breast cancer cells. These preclinical findings show potential for a combined hormonal regimen of an antiestrogen and an antiprogestin to combat the emergence of antiestrogen-resistant breast cancer cells and, ultimately, improve the therapeutic index of antiestrogen therapy.

Apoptosis initiated by dependence receptors: a new paradigm for cell death?

A distinct group of receptors including DCC, UNC5, RET and Ptc1 is known to function in ligand-dependent neuronal growth and differentiation or axon guidance. Acting as "dependence receptors", they may also regulate neuronal cell survival by inducing apoptosis in the absence of cognate ligand. Receptor-initiated apoptosis requires proteolytic (caspase) cleavage and exposure of a pro-apoptotic region in the cytoplasmic domains of the receptors. In contrast, classical apoptosis induced by growth factor or cytokine deprivation involves loss of survival signaling without receptor cleavage. DCC, UNC5, RET and Ptc1 are downregulated or mutated in diverse cancers, and show properties characteristic of tumor suppressors, consistent with their ability to promote neuronal cell death. Dysfunctional dependence receptors have been linked to the loss of specific neurons in certain inherited and neurodegenerative diseases. Dependence receptor-initiated apoptosis represents a novel paradigm for the controlled removal of specific cells during neural development and elimination of malignant cells that have strayed beyond regions of ligand availability.

Topoisomerase inhibitor camptothecin sensitizes mouse hepatocytes in vitro and in vivo to TNF-mediated apoptosis.

Topoisomerases are nuclear enzymes that maintain and modulate DNA structure. Inhibitors of topoisomerases like camptothecin (CPT), etoposide, and others are widely used antitumor drugs that interfere with transcription, induce DNA strand breaks, and trigger apoptosis preferentially in dividing cells. Because transcription inhibitors (actinomycin D, galactosamine, alpha-amanitin) sensitize primary hepatocytes to the cytotoxic action of tumor necrosis factor (TNF), we reasoned whether topoisomerase inhibitors would act similarly. CPT alone was not toxic to primary cultured murine hepatocytes. When incubated with CPT, murine hepatocytes displayed an inhibition of protein synthesis and were thereby rendered sensitive to apoptosis induction by TNF. Apoptosis was characterized by morphology (condensed/fragmented nuclei, membrane blebbing), caspase-3-like protease activity, fragmentation of nuclear DNA, and late cytolysis. Hepatocytes derived from TNF receptor-1 knockout mice were resistant to CPT/TNF-induced apoptosis. CPT treatment completely abrogated the TNF-induced NF-kappa B activation, and mRNA expression of the antiapoptotic factors TNF-receptor associated factor 2, FLICE-inhibitory protein, and X-linked inhibitor of apoptosis protein was also inhibited by CPT. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-chloromethylketone (zDEVD-fmk), as well as depletion of intracellular ATP by fructose prevented CPT/TNF-induced apoptosis. In vivo, CPT treatment sensitized mice to TNF-induced liver damage. In conclusion, the combination of topoisomerase inhibition and TNF blocks survival signaling and elicits a type of hepatocyte death similar to actinomycin D/TNF or galactosamine/TNF. During antitumor treatment with topoisomerase inhibitors, an impaired immune function often results in opportunistic infections, a situation where the systemic presence of TNF might be critical for the hepatotoxicity reported in clinical topoisomerase inhibitor studies.

Nitric oxide-induced transcriptional up-regulation of protective genes by Nrf2 via the antioxidant response element counteracts apoptosis of neuroblastoma cells.

Nitric oxide (NO) is a signaling molecule that in excess causes cell death. Here we report a mechanism of NO-induced transcriptional up-regulation of genes encoding detoxifying enzymes and protective proteins and their role in counteracting NO-induced apoptosis of neuroblastoma cells. Promoter analysis using reporter assays identified the antioxidant response element (ARE) located in the promoter region of NAD(P)H:quinone oxidoreductase 1 (Nqo1) and other detoxifying enzyme genes as responsible for NO-mediated gene induction. The transcription factors NF-E2-related factor 2 (Nrf2) and small maf proteins were detected in NO-induced nuclear protein-ARE complexes. Nrf2 augmented NO-induced, ARE-dependent gene expression, which was blocked by dominant-negative Nrf2 (DN-Nrf2) lacking the transcriptional activation domain. Consistent with these results, Nrf2 was localized in the cytoplasm in unstimulated cells, and NO triggered its rapid nuclear accumulation. Neuroblastoma cells were stably transfected with DN-Nrf2, which repressed both the expression of protective genes and their induction by NO. These DN-Nrf2 cells exhibited reduced NQO1 enzymatic activity and were sensitized to NO-induced apoptosis. Similar results were obtained when Nrf2 expression was blocked by RNA interference. Conversely, stable cells expressing higher levels of Nrf2 protein had elevated NQO1 activity and were protected from NO. Finally, NO-mediated ARE-dependent gene induction occurred well before apoptosis as judged by caspase activation. These results together suggest that NO signals the transcriptional up-regulation of NQO1 and other detoxifying enzyme and protective genes through Nrf2 via the ARE to counteract NO-induced apoptosis of neuroblastoma cells.

JNK-dependent phosphorylation of c-Jun on serine 63 mediates nitric oxide-induced apoptosis of neuroblastoma cells.

c-Jun NH2-terminal kinases (JNKs) potentiate transcriptional activity of c-Jun by phosphorylating serines 63 and 73. Moreover, JNK and c-Jun can modulate apoptosis. However, an involvement of nitric oxide (NO)-induced phosphorylation of c-Jun on Ser-63 and Ser-73 in apoptosis has not been explored. We report that in SH-Sy5y neuroblastoma cells, NO induced apoptosis following JNK activation and phosphorylation of c-Jun almost exclusively on Ser-63. Importantly, NO-induced apoptosis and caspase-3 activity were inhibited in cells stably transformed with dominant-negative c-Jun in which Ser-63 is mutated to alanine (S63A), but not in cells transformed with dominant-negative c-Jun (S73A). Ser-63 of c-Jun (but not Ser-73) was required for NO-induced, c-Jun-dependent transcriptional activity. NO-induced apoptosis, Ser-63 phosphorylation of c-Jun, and caspase-3 activity were all inhibited in SH-Sy5y cells transformed with dominant-negative jnk. A caspase-3 inhibitor prevented apoptosis but not c-Jun phosphorylation. In a different neuroblastoma cell line, NO-induced Ser-63 phosphorylation of c-Jun and apoptosis were blocked by a specific JNK inhibitor. We conclude that NO-inducible apoptosis is mediated by JNK-dependent Ser-63 phosphorylation of c-Jun upstream of caspase-3 activation in neuroblastoma cells.

Green tea polyphenol targets the mitochondria in tumor cells inducing caspase 3-dependent apoptosis.

Induction of apoptosis by green tea polyphenols has been observed in various tumor cell systems, but whether green tea polyphenol-induced apoptosis requires caspase 3 for execution has not been confirmed. We previously reported that green tea polyphenol-induced apoptosis involved Apaf-1 accumulation and caspase 3 activation in the cytosol. In the current study, tumor cells either with deleted caspase 3 gene or expressing wild-type caspase 3 were treated with increasing concentrations of green tea polyphenol(s), followed by morphological analysis and caspase 3 activity assay. The caspase 3 null parental cell line was further examined in comparison with a well-characterized, caspase 3 wild type oral carcinoma cell line by MTT assay and BrdU incorporation assay. The results demonstrated that, while the mitochondrial function gradually declined to insignificant levels, caspase 3 null cells did not undergo apoptosis, which suggested that green tea polyphenol-induced apoptosis is a mitochondria-targeted, caspase 3-executed mechanism.

Neuronal differentiation and protection from nitric oxide-induced apoptosis require c-Jun-dependent expression of NCAM140.

c-Jun, a crucial component of the dimeric transcription factor activating protein 1 (AP-1), can regulate apoptosis induced by oxidative stress and has been implicated in neuronal differentiation, but the mechanisms are largely unknown. We found that specific inhibition of transcription or stable transfection with cDNA encoding dominant-negative c-Jun sensitized SH-SY5Y neuroblastoma cells (TAM-67 cells) to apoptosis induced by the nitric oxide (NO) donor sodium nitroprusside or SIN-1. TAM-67 cells also became refractory to nerve growth factor (NGF)-induced neuronal differentiation. Dominant-negative c-Jun abolished expression of a 140-kDa neural cell adhesion molecule (NCAM140) and dramatically enhanced the expression of NCAM180 in TAM-67 cells. Inhibition of c-Jun in TAM-67 cells also resulted in a corresponding decrease in the amount of NCAM140 mRNA and an increase in the amount of NCAM180 mRNA. Reexpression of NCAM140 in TAM-67 cells restored NGF-induced neuronal differentiation and resistance to NO-induced apoptosis. Our results show that c-Jun/AP-1, through up-regulation of NCAM140, plays an important role in both NGF-induced neuronal differentiation and resistance to apoptosis induced by NO in neuroblastoma cells. As NCAM140 and NCAM180 are translated from differentially spliced mRNAs transcribed from the same gene, alternative splicing of NCAM pre-mRNA (and consequently the synthesis of the smaller NCAM140 species) appears to be regulated by c-Jun/AP-1.