Secretogranin II: a key AP-1-regulated protein that mediates neuronal differentiation and protection from nitric oxide-induced apoptosis of neuroblastoma cells.

Identification of AP-1 target genes in apoptosis and differentiation has proved elusive. Secretogranin II (SgII) is a protein widely distributed in nervous and endocrine tissues, and abundant in neuroendocrine granules. We addressed whether SgII is regulated by AP-1, and if SgII is involved in neuronal differentiation or the cellular response to nitrosative stress. Nitric oxide (NO) upregulated sgII mRNA dependent on a cyclic AMP response element (CRE) in the sgII promoter, and NO stimulated SgII protein secretion in neuroblastoma cells. Upregulation of sgII mRNA, sgII CRE-driven gene expression and SgII protein synthesis/export were attenuated in cells transformed with dominant-negative c-Jun (TAM67), which became sensitized to NO-induced apoptosis and failed to undergo nerve growth factor-dependent neuronal differentiation. Stable transformation of TAM67 cells with sgII restored neuronal differentiation and resistance to NO. RNAi knockdown of sgII in cells expressing functional c-Jun abolished neuronal differentiation and rendered the cells sensitive to NO-induced apoptosis. Therefore, SgII represents a key AP-1-regulated protein that counteracts NO toxicity and mediates neuronal differentiation of neuroblastoma cells.

Critical role for cathepsin B in mediating caspase-1-dependent interleukin-18 maturation and caspase-1-independent necrosis triggered by the microbial toxin nigericin.

The potassium ionophore nigericin induces cell death and promotes the maturation and release of IL-1beta in lipopolysaccharide (LPS)-primed monocytes and macrophages, the latter depending on caspase-1 activation by an unknown mechanism. Here, we investigate the pathway that triggers cell death and activates caspase-1. We show that without LPS priming, nigericin alone triggered caspase-1 activation and IL-18 generation in THP-1 monocytic cells. Simultaneously, nigericin induced caspase-1-independent necrotic cell death, which was blocked by the cathepsin B inhibitor CA-074-Me and other cathepsin inhibitors. Cathepsin B activation after nigericin treatment was determined biochemically and corroborated by rapid lysosomal leakage and translocation of cathepsin B to the cytoplasm. IL-18 maturation was prevented by both caspase-1 and cathepsin B inhibitors in THP-1 cells, primary mouse macrophages and human blood monocytes. Moreover, IL-18 generation was reduced in THP-1 cells stably transformed either with cystatin A (an endogenous cathepsin inhibitor) or antisense cathepsin B cDNA. Collectively, our study establishes a critical role for cathepsin B in nigericin-induced caspase-1-dependent IL-18 maturation and caspase-1-independent necrosis.

NF-kappa B activation mediates doxorubicin-induced cell death in N-type neuroblastoma cells.

Neuroblastoma is the most common extracranial solid tumor of childhood. N-type neuroblastoma cells (represented by SH-SY5Y and IMR32 cell lines) are characterized by a neuronal phenotype. N-type cell lines are generally N-myc amplified, express the anti-apoptotic protein Bcl-2, and do not express caspase-8. The present study was designed to determine the mechanism by which N-type cells die in response to specific cytotoxic agents (such as cisplatin and doxorubicin) commonly used to treat this disease. We found that N-type cells were equally sensitive to cisplatin and doxorubicin. Yet death induced by cisplatin was inhibited by the nonselective caspase inhibitor z-Val-Ala-Asp-fluoromethylketone or the specific caspase-9 inhibitor N-acetyl-Leu-Glu-His-Asp-aldehyde, whereas in contrast, caspase inhibition did not prevent doxorubicin-induced death. Neither the reactive oxygen species nor the mitochondrial permeability transition appears to play an important role in this process. Doxorubicin induced NF-kappa B transcriptional activation in association with I-kappa B alpha degradation prior to loss of cell viability. Surprisingly, the antioxidant and NF-kappa B inhibitor pyrrolidine dithiocarbamate blocked doxorubicin-induced NF-kappa B transcriptional activation and provided profound protection against doxorubicin killing. Moreover, SH-SY5Y cells expressing a super-repressor form of I-kappa B were completely resistant to doxorubicin killing. Together these findings show that NF-kappa B activation mediates doxorubicin-induced cell death without evidence of caspase function and suggest that cisplatin and doxorubicin engage different death pathways to kill neuroblastoma cells.

Ionizing radiation but not anticancer drugs causes cell cycle arrest and failure to activate the mitochondrial death pathway in MCF-7 breast carcinoma cells.

There is considerable evidence that ionizing radiation (IR) and chemotherapeutic drugs mediate apoptosis through the intrinsic death pathway via the release of mitochondrial cytochrome c and activation of caspases -9 and -3. Here we show that MCF-7 cells that lack caspase-3 undergo a caspase-dependent apoptotic cell death in the absence of DNA fragmentation and alpha-fodrin cleavage following treatment with etoposide or doxorubicin, but not after exposure to IR. Re-expression of caspase-3 restored DNA fragmentation and alpha-fodrin cleavage following drug treatment, but it did not alter the radiation-resistant phenotype of these cells. In contrast to the anticancer drugs, IR failed to induce the intrinsic death pathway in MCF-7/casp-3 cells, an event readily observed in IR-induced apoptosis of HeLa cells. Although IR-induced DNA double-strand breaks were repaired with similar efficiencies in all cell lines, cell cycle analyses revealed a persistent G2/M arrest in the two MCF-7 cell lines, but not in HeLa cells. Together, our data demonstrate that caspase-3 is required for DNA fragmentation and alpha-fodrin cleavage in drug-induced apoptosis and that the intrinsic death pathway is fully functional in MCF-7 cells. Furthermore, they show that the radiation-resistant phenotype of MCF-7 cells is not due to the lack of caspase-3, but is caused by the failure of IR to activate the intrinsic death pathway. We propose (1) different signaling pathways are induced by anticancer drugs and IR, and (2) IR-induced G2/M arrest prevents the generation of an apoptotic signal required for the activation of the intrinsic death pathway.

Caspase-8/FLICE functions as an executioner caspase in anticancer drug-induced apoptosis.

Caspase-8 plays an essential role in apoptosis triggered by death receptors. Through the cleavage of Bid, a proapoptotic Bcl-2 member, it further activates the mitochondrial cytochrome c/Apaf-1 pathway. Because caspase-8 can be processed also by anticancer drugs independently of death receptors, we investigated its exact role and order in the caspase cascade. We show that in Jurkat cells either deficient for caspase-8 or overexpressing its inhibitor c-FLIP apoptosis mediated by CD95, but not by anticancer drugs was inhibited. In the absence of active caspase-8, anticancer drugs still induced the processing of caspase-9, -3 and Bid, indicating that Bid cleavage does not require caspase-8. Overexpression of Bcl-x(L) prevented the processing of caspase-8 as well as caspase-9, -6 and Bid in response to drugs, but was less effective in CD95-induced apoptosis. Similar responses were observed by overexpression of a dominant-negative caspase-9 mutant. To further determine the order of caspase-8 activation, we employed MCF7 cells lacking caspase-3. In contrast to caspase-9 that was cleaved in these cells, anticancer drugs induced caspase-8 activation only in caspase-3 transfected MCF7 cells. Thus, our data indicate that, unlike its proximal role in receptor signaling, in the mitochondrial pathway caspase-8 rather functions as an amplifying executioner caspase.

Expression analysis of the human caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopolysaccharide and interferon-gamma.

Based on high sequence homology, there are six members in the caspase-1 subfamily: caspases 1, 4, 5, and 13 in humans and caspases 1, 11, and 12 in mice. Only caspase-1 is known to activate interleukin-1beta and interleukin-18, and caspase-11 activates pro-caspase-1 in vivo. Almost nothing is known about caspases 4, 5, and 13. Here we report a sensitive and specific polymerase chain reaction system to analyze closely related genes. We employed this system to analyze the gene expression and regulation of human caspases 1, 4, 5, and 13, demonstrating that they have different expression patterns in normal tissues and cell lines. Interferon-gamma strongly induced CASP1 and CASP5 but not CASP4 or CASP13 gene expression in HT-29 colon carcinoma cells. In contrast to the mRNA, interferon-gamma up-regulated caspase-1 but not caspase-5 protein. In the monocytic cell line THP-1, CASP1 mRNA and caspase-1 protein are expressed constitutively, and their levels were not increased by lipopolysaccharide, whereas both CASP5 mRNA and caspase-5 protein were induced by lipopolysaccharide. Caspase-1 subfamily members displayed different in vitro activities toward pro-caspases 1 and 3 and pro-interleukin-1beta. Our results demonstrate that caspase-1 and caspase-5 levels are modulated by interferon-gamma and lipopolysaccharide, respectively, and suggest that caspase-1 subfamily members are differentially regulated and may have distinct functions.

Caspase-3 is essential for procaspase-9 processing and cisplatin-induced apoptosis of MCF-7 breast cancer cells.

In this study, we sought to investigate in more detail the role of caspase-3 in apoptotic processes in cultured cells and in cell-free extracts of breast cancer cells. We present evidence that apoptosis of caspase-3-deficient MCF-7 breast cancer cells is defective in response to cisplatin treatment, as determined by chromatin condensation, nuclear fragmentation, DNA fragmentation, and release of cytochrome c from the mitochondria. Reconstitution of MCF-7 cells by stable transfection of CASP-3 cDNA restores all these defects and results in an extensive apoptosis after cisplatin treatment. We further show that in extracts from caspase-3-deficient MCF-7 cells, procaspase-9 processing is strongly impaired after stimulation with either cytochrome c or recombinant caspase-8. Reconstitution of MCF-7 cell extracts with procaspase-3 corrects this defect, resulting in an efficient and complete processing of procaspase-9. Together, our data define caspase-3 as an important integrator of the apoptotic process in MCF-7 breast cancer cells and reveal an essential function of caspase-3 for procaspase-9 processing.

Oral meperidine, atropine, and pentobarbital for pediatric conscious sedation.

PURPOSE: To document the effectiveness and safety of using a combination of oral meperidine, atropine, and pentobarbital for pediatric conscious sedation. METHOD: Sixty-three patients ages 6 months to 23 years (M = 5 years) undergoing outpatient cardiac catheterization were given an oral premedication consisting of meperidine, atropine, and pentobarbital for sedation. The effectiveness of the medication in providing sedation and the complication of over sedation when using a standard dose based upon weight were analyzed. RESULTS: Seventy six percent of the patients were deemed sedated on arrival to the catheterization laboratory. Forty eight percent did not require any further sedation during the procedure. Need for further sedation was related to the length of the procedure. Three patients (4.8%) incurred complications of sedation; all three had received additional sedation during the procedure. CONCLUSIONS: The combination of meperidine, atropine, and pentobarbital is a safe and effective premedication for cardiac catheterization when administered in standard dosage based upon body weight.

Diagnosis and catheter treatment of innominate artery stenosis following stage I Norwood procedure.

Four infants aged 20-115 days (mean, 57.8 days) who had undergone stage I Norwood procedure for hypoplastic left heart syndrome came to early cardiac catheterization (6-112; mean, 47.3 days) following surgery because of significant arterial desaturation (pulse oximetry indicating oxygen saturations consistently in the 40%-70% range). Cardiac catheterization demonstrated a significant systolic pressure gradient between the ascending aorta and innominate artery (30-65; mean, 51 mm Hg) as the likely cause of diminished pulmonary blood flow in these patients. Routine angiography by itself was not conclusive in identifying a discrete area of obstruction, but selective angiography coupled with a knowledge of the obstruction did reveal the stenosis. All patients were successfully treated with balloon dilatation of the stenotic area, with the pressure gradient being reduced to 7-25 (mean, 17 mm Hg) immediately following dilatation. On follow-up catheterization in three patients, the systolic gradients were 3, 6, and 9 mm Hg. Arterial oxygen saturations rose from 63.5% predilatation to 77.3% immediately postdilatation and 81% on follow-up evaluation. In conclusion, innominate artery stenosis is an important cause of diminished blood flow through a modified right Blalock-Taussig shunt. Routine angiography will often miss the diagnosis. Pressure gradients and selective angiograms are necessary in order to make the diagnosis, although careful noninvasive assessment should also be diagnostic of this problem. Catheter dilatation is therapeutic in this situation and can be performed early after surgery in the absence of a fresh suture line.

NF-kappaB/Rel proteins are required for neuronal differentiation of SH-SY5Y neuroblastoma cells.

The expression, cellular localization, and activation of the NF-kappaB/Rel transcription factors are altered during neuronal differentiation, but the significance is unclear. Here we investigate the requirement for NF-kappaB/Rel proteins in neuronal differentiation. SH-SY5Y neuroblastoma cells were induced to differentiate with retinoic acid (RA) or 12-O-tetradecanoylphorbol 13-acetate (TPA), and differentiation was demonstrated by morphological criteria and the enhanced expression of Bcl-2. NF-kappaB was transiently activated after the addition of the differentiation inducers before the morphological signs of differentiation and the enhanced Bcl-2 synthesis. The onset of NF-kappaB activation coincided with a significant reduction in the amount of only one of four NF-kappaB-inhibitory proteins examined (I-kappaBbeta). In contrast, NF-kappaB activation and the reduction in I-kappaBbeta failed to occur in SH-SY5Y cells transformed with I-kappaBalphaM, a dominant-negative inhibitor of NF-kappaB/Rel proteins. These I-kappaBalphaM-expressing cells failed to differentiate into neuronal cell types when treated with RA or TPA, and the increased Bcl-2 synthesis was blocked. Therefore, NF-kappaB/Rel proteins are required for neuronal differentiation of SH-SY5Y neuroblastoma cells.

Protein translocation in apoptosis.

In programmed cell death (apoptosis), receptor-generated or other signals are transmitted to all cellular compartments, resulting in an apoptotic cell with extensive cytoplasmic and nuclear alterations. Protein translocation is now recognized as being crucial in the induction, amplification and regulation of this process. Diverse mechanisms trigger protein translocation to and from the plasma membrane, mitochondrion and nucleus during apoptosis. This review discusses where, why and how the various protein-translocation events take place and highlights their importance in the execution and regulation of apoptosis.

Crystallization and preliminary X-ray diffraction studies of the 51 kDa protein of the mosquito-larvicidal binary toxin from Bacillus sphaericus.

Certain strains of Bacillus sphaericus produce a highly toxic mosquito-larvicidal protein during sporulation which is active against vectors of dengue, encephalitis and malaria. This toxin is initially expressed as 51 and 42 kDa proteins and is converted to 43 and 39 kDa proteins, respectively, which form the active heterodimer complex. For a better understanding of the toxicity mechanism at the molecular level, the 51 kDa protein of the binary toxin of B. sphaericus strain 2297 was expressed as a glutathione-S-transferase fusion protein and purified by affinity chromatography. Protein crystals were grown from an amorphous precipitate in five months using the hanging-drop vapor-diffusion method. The protein crystals were dissolved and were found to be composed of a proteolytically modified 45.2 kDa derivative similar to the active form of this protein. The crystals form in space group P43212 (or P41212) and diffract to 2.6 A, with unit-cell dimensions a = b = 133.48, c = 69. 76 A.

Molecular cloning and characterization of two novel pro-apoptotic isoforms of caspase-10.

Caspase-10/a (Mch4) and caspase-10/b (FLICE2) are related death effector domain-containing cysteine aspartases presumed to be at or near the apex of apoptotic signaling pathways. We report the cloning and characterization of two novel proteins that are splice isoforms of the caspase-10 family. Caspase-10/c is a truncated protein that is essentially a prodomain-only form of the caspase that lacks proteolytic activity in vitro but efficiently induces the formation of perinuclear filamentous structures and cell death in vivo. Caspase-10/c mRNA is specifically up-regulated upon TNF stimulation, suggesting a potential role of this isoform in amplifying the apoptotic response to extracellular stimuli such as cytokines. Caspase-10/d is a hybrid of the known caspases Mch4 and FLICE2, as it is identical to FLICE2 except for the small (p12) catalytic subunit, which is identical to Mch4. Caspase-10/d is proteolytically active in vitro and also induces cell death in vivo, although it is less active than Mch4. The mRNAs for all known isoforms of caspase-10 are abundantly expressed in fetal lung, kidney, and skeletal muscle but are very poorly expressed or absent in these tissues in the adult, implying a possible role for the caspase-10 family in fetal development.

Emerging roles of caspase-3 in apoptosis.

Caspases are crucial mediators of programmed cell death (apoptosis). Among them, caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins. However, the specific requirements of this (or any other) caspase in apoptosis have remained largely unknown until now. Pathways to caspase-3 activation have been identified that are either dependent on or independent of mitochondrial cytochrome c release and caspase-9 function. Caspase-3 is essential for normal brain development and is important or essential in other apoptotic scenarios in a remarkable tissue-, cell type- or death stimulus-specific manner. Caspase-3 is also required for some typical hallmarks of apoptosis, and is indispensable for apoptotic chromatin condensation and DNA fragmentation in all cell types examined. Thus, caspase-3 is essential for certain processes associated with the dismantling of the cell and the formation of apoptotic bodies, but it may also function before or at the stage when commitment to loss of cell viability is made.

Activation of caspase-1 in the nucleus requires nuclear translocation of pro-caspase-1 mediated by its prodomain.

The interleukin-1beta-converting enzyme-like protease precursor, pro-caspase-1, has an N-terminal prodomain that is removed during cleavage activation of the protease. Here we show that tumor necrosis factor treatment of HeLa cells induced apoptosis without detectable proteolytic activation of caspase-1 in the cytosol. Instead, tumor necrosis factor induced the translocation of pro-caspase-1 to the nucleus where it was proteolytically activated, releasing the intact prodomain. We identified a nuclear localization signal in the prodomain, which was required for translocation of both pro-caspase-1 as well as its prodomain to the nucleus. Surprisingly, transfected MCF-7 carcinoma or embryonic kidney 293T cells expressing the prodomain alone underwent apoptosis. These results show that death signal-induced nuclear targeting is a novel activity of a caspase prodomain and indicate that caspase-1 and its prodomain may have hitherto unsuspected nuclear functions in apoptosis.

Caspase-3 is required for alpha-fodrin cleavage but dispensable for cleavage of other death substrates in apoptosis.

Although the commonly activated death protease caspase-3 appears not to be essential for apoptosis during development except in the brain, it was not shown whether substrates known to be cleaved by caspase-3 are still proteolyzed in its absence. We have addressed this question with MCF-7 breast carcinoma cells that we recently showed lack caspase-3 owing to the functional deletion of the CASP-3 gene. Tumor necrosis factor- or staurosporine-induced apoptosis of caspase-3-deficient MCF-7 cells resulted in cleavage of the death substrates PARP, Rb, PAK2, DNA-PKcs, gelsolin, and DFF-45, but not alpha-fodrin. In contrast, all these substrates including alpha-fodrin were cleaved in apoptotic HeLa cells expressing caspase-3. Introduction of CASP-3 cDNA, but not CASP-10 cDNA, into MCF-7 cells restored alpha-fodrin cleavage. In addition, tumor necrosis factor- or staurosporine-induced apoptosis of MCF-7 cells stably expressing pro-caspase-3 also resulted in alpha-fodrin cleavage. Although the specific caspase inhibitory peptides Z-VAD-fmk and Z-DEVD-fmk prevented apoptosis of MCF-7 cells, we were unable to detect activation of caspases 2 and 7, which are known to be inhibited by Z-DEVD-fmk. Together our results suggest that caspase-3 is essential for cleavage of alpha-fodrin, but dispensable for the cleavage of PARP, Rb, PAK2, DNA-PKcs, gelsolin, and DFF-45 and imply that one or more caspases other than caspases 2, 3, and 7 is activated and plays a crucial role in the cleavage of these substrates in MCF-7 cells.

Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis.

Interleukin 1beta-converting enzyme-like proteases (caspases) are crucial components of cell death pathways. Among the caspases identified, caspase-3 stands out because it is commonly activated by numerous death signals and cleaves a variety of important cellular proteins. Studies in caspase-3 knock-out mice have shown that this protease is essential for brain development. To investigate the requirement for caspase-3 in apoptosis, we took advantage of the MCF-7 breast carcinoma cell line, which we show here has lost caspase-3 owing to a 47-base pair deletion within exon 3 of the CASP-3 gene. This deletion results in the skipping of exon 3 during pre-mRNA splicing, thereby abrogating translation of the CASP-3 mRNA. Although MCF-7 cells were still sensitive to tumor necrosis factor (TNF)- or staurosporine-induced apoptosis, no DNA fragmentation was observed. In addition, MCF-7 cells undergoing cell death did not display some of the distinct morphological features typical of apoptotic cells such as shrinkage and blebbing. Introduction of the CASP-3 gene into MCF-7 cells resulted in DNA fragmentation and cellular blebbing following TNF treatment. These results indicate that although caspase-3 is not essential for TNF- or staurosporine-induced apoptosis, it is required for DNA fragmentation and some of the typical morphological changes of cells undergoing apoptosis.

Prevention of tumor necrosis factor (TNF)-mediated induction of p21WAF1/CIP1 sensitizes MCF-7 carcinoma cells to TNF-induced apoptosis.

The MCF-7 breast carcinoma and MRC-5 lung fibroblast cell lines are sensitive and resistant to tumor necrosis factor (TNF)-induced apoptosis, respectively. As the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) is involved in cell cycle regulation and has been implicated in apoptosis, we studied the influence of p21 on growth of MRC-5 cells and on growth and apoptosis in MCF-7 cells. TNF induced p21 mRNA and protein in both cell types. p21 induction by > 0.5 ng/ml TNF in MRC-5 and MCF-7 cells correlated with the inhibition of cell growth. In contrast, < 0.1 ng/ml TNF stimulated MRC-5 (but not MCF-7) cell growth without reduction in p21 levels. TNF-induced apoptosis in MCF-7 cells was first detected after the TNF-mediated increase in p21 and growth arrest had occurred. MCF-7 cells stably transfected with antisense p21 cDNA became more sensitive to TNF-induced apoptosis. Thus, TNF-induced p21 accompanied by growth arrest may counteract or delay TNF cytotoxicity in MCF-7 cells.

A novel Arabidopsis thaliana protein protects tumor cells from tumor necrosis factor-induced apoptosis.

Recently we have cloned and characterized a novel, oxidative stress-induced Arabidopsis thaliana gene (oxy5), and showed that expression of oxy5 protects bacterial cells from death caused by oxidative stress. As oxidative stress is one pathway of TNF cytotoxicity, we investigated whether the encoded protein could also protect human tumor cells from TNF killing. We stably transfected the oxy5 gene into TNF-sensitive HeLa D98 cells (D98/O.5), and found that all examined transfectants were highly TNF-resistant in the absence of the protein synthesis inhibitor cycloheximide. The acquired TNF resistance of these clones was accompanied by a sharp decrease in the intracellular formation of reactive oxygen species, suggesting the activation of antioxidant enzymes like superoxide dismutases (SODs). Indeed, D98/O.5 clones showed an increased manganous superoxide dismutase (MnSOD) mRNA and protein expression in the absence or presence of TNF stimulation, whereas the expression of the Cu/ZnSOD was not affected. Furthermore, the elevated MnSOD expression in the D98/O.5 clones correlated well with an increased antioxidative activity, which was specifically due to MnSOD as measured by the suppression of xanthine oxidase. Our results demonstrate a novel role for a plant-derived protein in resistance to TNF cytotoxicity, and that the Arabidopsis thaliana protein Oxy5 can exert its protective function across evolutionary boundaries through activation of antioxidant enzymes like MnSOD.

Mutations which abolish phosphorylation of the TRAF-binding domain of TNF receptor 2 enhance receptor-mediated NF-kappa B activation.

We demonstrate that a 41 amino acid region (amino acids 379 to 419) in the cytoplasmic domain of tumor necrosis factor receptor 2 (TNFR2) is phosphorylated by unidentified kinase(s) both in vitro and in vivo. This domain (denoted x1c) corresponds almost exactly to the previously identified TRAF-binding domain and is by itself sufficient as a substrate for phosphorylation. In addition, the x1c domain is also crucial for TNFR2-mediated NF-kappa B activation. The cytoplasmic domain of TNFR2 lacks tyrosines, and conversion of all 12 potential serine and threonine phosphorylation targets in x1c to alanines either had no effect on NF-kappa B activation or resulted in enhanced NF-kappa B activity, depending on the structural context of x1c. The results show that while the TRAF-binding domain of TNFR2 is a major target of kinases, its phosphorylation is not required for NF-kappa B activation. Our data moreover suggest that phosphorylation of x1c negatively regulates the activation of NF-kappa B.