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.

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.

Nuclear c-Myc plays an important role in the cytotoxicity of tumor necrosis factor alpha in tumor cells.

The phosphoprotein c-Myc has the potential to kill cells by apoptosis. To investigate whether c-Myc is involved in tumor necrosis factor alpha (TNF-alpha)-mediated cell killing, we have examined two HeLa cell lines (D98 and H21) which show dramatic differences in their susceptibilities to TNF-alpha cytotoxicity. Northern (RNA) blot analyses showed that there were no significant differences between these cell lines in basal or TNF-alpha-induced mRNA expression for a variety of proteins, including manganous superoxide dismutase, A20 zinc finger protein, plasminogen activator inhibitor type 2, and hsp70, all of which are known to influence the susceptibility of certain cells to TNF-alpha killing. On the other hand, there was a dramatic increase in c-Myc mRNA expression in TNF-alpha-sensitive D98 cells, but not in TNF-alpha-resistant H21 cells, which was only observed when the cells were treated with cycloheximide. Western blot (immunoblot) analyses revealed that even in the absence of TNF-alpha or cycloheximide, c-Myc was detectable only in nuclear extracts of TNF-alpha-sensitive D98 cells, implying a role for preexisting c-Myc in TNF-alpha killing. In support of this interpretation, a c-myc antisense oligonucleotide specifically inhibited the TNF-alpha killing of D98 cells, provided that the oligonucleotide was added 6 h prior to TNF-alpha treatment. Either dexamethasone treatment or transient expression of c-myc antisense cDNA fragments decreased nuclear c-Myc in D98 cells and rendered the cells more resistant to TNF-alpha cytotoxicity. Nuclear c-Myc was also detectable in a TNF-alpha-sensitive human HT-1080 fibrosarcoma cell line, but it was undetectable in a derivative of HT-1080 (SS-HT-1080) known to be resistant to TNF-alpha killing because of overexpression of plasminogen activator inhibitor type 2. HT-1080 cells transfected with antisense c-myc cDNA had significantly less nuclear c-Myc and were resistant to TNF-alpha cytotoxicity. Together, these data indicate that a nuclear transcription factor, c-Myc, plays an important role in sensitizing two different tumor cell types to TNF-alpha cytotoxicity.

Cyclin D3 sensitizes tumor cells to tumor necrosis factor-induced, c-Myc-dependent apoptosis.

c-Myc is an important mediator of apoptosis in cytokine- or serum-deprived cells and sensitizes various cell types to tumor necrosis factor alpha (TNF) cytotoxicity. However, downstream mediators of c-Myc-dependent apoptosis are largely unknown. In this study, we investigated whether one or more cyclins which, like c-Myc, are important regulators of the cell cycle are involved in TNF-induced apoptosis downstream of c-Myc. Cyclin D3 and c-Myc levels in HeLa and fibrosarcoma cells correlated with sensitivity of these cells to TNF-induced apoptosis, as both proteins were highly expressed in TNF-sensitive HeLa D98 cells and HT-1080 fibrosarcoma cells but not in their TNF-resistant counterparts, HeLa H21 and SS-HT-1080 cells, respectively. All other cyclins tested were equally expressed in all tumor cell lines. Reduction in the expression of c-Myc by dexamethasone or inhibition of the transcriptional activity of c-Myc by introduction of a dominant negative form of c-Myc into TNF-sensitive HeLa D98 cells strongly suppressed the expression of cyclin D3 (but none of the other cyclins) and rendered the cells resistant to TNF-induced apoptosis. Conversely, introduction of the c-myc gene into TNF-resistant, c-Myc- and cyclin D3-deficient HeLa H21 cells resulted in enhanced cyclin D3 expression and TNF killing. When cyclin D3 expression in HeLa cells was altered by sense or antisense cyclin D3 cDNA, there was a concomitant alteration in their susceptibility to TNF-induced apoptosis without any change in c-Myc levels. Overall, our results show that cyclin D3 sensitizes tumor cells to TNF-induced apoptosis and indicate that the expression of c-Myc and expression of cyclin D3 in HeLa and in HT-1080 fibrosarcoma cells are closely linked.

Efficient synthesis of mosquitocidal toxins in Asticcacaulis excentricus demonstrates potential of gram-negative bacteria in mosquito control.

The control of mosquitoes with chemical insecticides pollutes the environment and leads to resistance in mosquito populations. Bacterial control of mosquito larvae with Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis, which produce protein toxins, has proved useful, safe, and nonpolluting. These bacteria do, however, suffer from disadvantages, including rapid setting, UV sensitivity, and lack of persistance of spores, proteolysis of toxins, narrow host range, and high production costs. Here we show that the Gram-negative bacterium Asticcacaulis excentricus is a promising host for delivering toxins to mosquito larvae. Plasmid-transformed A. excentricus cells expressing the binary toxin of B. sphaericus exhibited toxicity to Culex and Anopheles mosquito larvae similar to that of the high-toxicity strains of B. sphaericus which produce several toxins. A. excentricus has potential advantages as a larvicide compared with the bacilli, especially persistance in the larval feeding zone, resistance to UV light, lack of toxin-degrading proteases, and low production costs.

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.

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.

Gene from tropical Bacillus sphaericus encoding a protease closely related to subtilisins from Antarctic bacilli.

We undertook to identify the protease(s) involved in the in vivo degradation of the 100 kDa mosquitocidal toxin (Mtx) from Bacillus sphaericus SSII-1 and isolated a B. sphaericus SSII-1 gene flanked upstream by a typical Shine-Dalgarno ribosome binding site and downstream by a strong rho-independent transcription terminator. The predicted ORF encodes a 432 amino acid protein with significant homology throughout its sequence to two subtilisin-like serine proteases from the Antarctic psychrophilic (cold-adapted) bacilli, TA39 and TA41. The predicted N-terminal sequence suggests that the B. sphaericus protease is related to sfericase, a partially characterized serine protease from B. sphaericus. Only B. sphaericus strains which produce Mtx-degrading protease activity harbour the subtilisin-like protease gene, suggesting that this protease may be responsible for or contribute to the degradation of Mtx in B. sphaericus SSII-1. A 36-kDa protease with Mtx-degrading activity and similar properties to sfericase was also purified from sporulated cultures of B. sphaericus SSII-1. Further studies are needed to determine the relationship of this protease to sfericase and to the predicted product of the subtilisin-like serine protease gene.

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.

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.

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.

The prospects for therapy with tumour necrosis factors and their antagonists.

Tumour necrosis factors (TNFs) alpha and beta are capable of causing dramatic necrosis of solid tumours. TNF-alpha has undergone clinical trials as an anti-cancer drug. Finding a role for TNFs in the treatment of cancer will depend on suppressing severe side effects and targeting the drug. Conversely, due to the participation of TNFs in inflammatory responses, antagonists of TNF-alpha are being considered for the treatment of several conditions, especially septic shock.

A Bacillus sphaericus gene encoding a novel type of mosquitocidal toxin of 31.8 kDa.

A size-fractionated genomic library of Bacillus sphaericus strain SSII-1 was constructed and screened for toxicity against larvae of the mosquito Culex quinquefasciatus (Cq). One toxin-producing clone, pS35, was identified and a 2.7-kb subclone was completely sequenced. An open reading frame of 879 bp encoding a 31.8-kDa protein (designated Mtx2) was identified. Purified, recombinant Mtx2 was toxic to Cq larvae. Mtx2 shows no significant homology to known insecticidal toxins, but has homology to two toxins active against mammalian cells, namely the epsilon-toxin of Clostridium perfringens and the cytotoxin of Pseudomonas aeruginosa. Thus, Mtx2 represents a new type of mosquitocidal toxin.

Expression and processing of human rhinovirus type 14 polypeptide precursors in Escherichia coli maxicells.

Human rhinovirus serotype-14 (HRV-14) cDNA, encompassing 87.9% of the coding region, was subcloned in an Escherichia coli expression vector, generating plasmid pKCC101. HRV-14 polypeptides encoded by pKCC101 were synthesized in E. coli maxicells. Pulse-chase experiments with pKCC110, a smaller derivative of pKCC101 containing the protease 3C coding region, have clearly demonstrated the proteolysis of a 55-kDa precursor to several polypeptides, including a doublet with the expected size of protease 3C (20 kDa). The proteolysis of the 55-kDa precursor polypeptide was prevented by ZnCl2, a known inhibitor of picornavirus 3C proteases. Results with a derivative of pKCC110 (pKCC115) which is partially deleted for the protease 3C sequence, support the idea that the doublet proteins are specified by the protease 3C coding region. Taken together, our investigations indicate that the precursor form of protease 3C must be responsible for its own cleavage.

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.

Efficient and rapid affinity purification of proteins using recombinant fusion proteases.

In the affinity purification of recombinant fusion proteins, the rate-limiting step is usually the efficient proteolytic cleavage and removal of the affinity tail and the protease from the purified recombinant protein. We have developed a rapid, convenient and efficient method of affinity purification which can overcome this limitation. In one example of the method, the protease 3C from a picornavirus (3Cpro), which cleaves specific sequences containing a minimum of 6-7 amino acids, has been expressed as a fusion with glutathione S-transferase. The resultant recombinant 'fusion protease' cleaves fusion proteins bearing (from the amino-terminus) the same affinity tail as the fusion protease, a 3Cpro cleavage recognition site, and the recombinant protein of interest. The recombinant protein is purified in a single chromatographic step which removes both the affinity tail and the fusion protease. The advantages over existing methods include much improved specificity of proteolytic cleavage, complete removal of the protease and the affinity tail in one step, and the option of adding any desired amount of fusion protease to ensure efficient cleavage. The potential flexibility of the method is shown by the use of various affinity tails and alternative fusion proteases.

Mosquitocidal toxins, genes and bacteria: the hit squad.

Certain entomopathogenic species of bacilli and Clostridium produce one or more toxins that kill mosquito larvae even at concentrations in the picomolar range. Altogether, 19 distinct genes are known that encode mosquitocidal toxins, which vary in their potency, species specificity and mode of action. Unlike chemical insecticides, mosquitocidal bacilli used as larvicides are safe for animals and the environment, and do not affect non-pest insects. Mosquitocidal bacteria are effective to varying degrees against Culex, Anopheles and Aedes mosquito larvae, but their rapid sedimentation from the larval feeding zone, UV-light sensitivity and narrow host range have hampered their development. New genetic engineering approaches are being investigated that could overcome these limitations and allow stable expression of broad host range combinations of toxins in UV-resistant, buoyant recombinant bacteria, as discussed here by Alan Porter.