pp32/PHAPI determines the apoptosis response of non-small-cell lung cancer.

During malignant transformation, cancer cells have to evade cell-intrinsic tumor suppressor mechanisms including apoptosis, thus acquiring a phenotype that is relatively resistant to clinically applied anticancer therapies. Molecular characterization of apoptotic signal transduction defects may help to identify prognostic markers and to develop novel therapeutic strategies. To this end we have undertaken functional analyses of drug-induced apoptosis in human non-small cell-lung cancer (NSCLC) cells. We found that primary drug resistance correlated with defects in apoptosome-dependent caspase activation in vitro. While cytochrome c-induced apoptosome formation was maintained, the subsequent activation of caspase-9 and -3 was abolished in resistant NSCLC. The addition of recombinant pp32/putative human HLA class II-associated protein (pp32/PHAPI), described as a putative tumor suppressor in prostate cancer, successfully restored defective cytochrome c-induced caspase activation in vitro. Conditional expression of pp32/PHAPI sensitized NSCLC cells to apoptosis in vitro and in a murine tumor model in vivo. Immunohistochemical analyses of tumor samples from NSCLC patients revealed that the expression of pp32/PHAPI correlated with an improved outcome following chemotherapy. These results identify pp32/PHAPI as regulator of the apoptosis response of cancer cells in vitro and in vivo, and as a predictor of survival following chemotherapy for advanced NSCLC.

Lumen geometry of ion channels formed by Vibrio cholerae EL Tor cytolysin elucidated by nonelectrolyte exclusion.

Vibrio cholerae EL Tor cytolysin, a water-soluble protein with a molecular mass of 63 kDa, forms small pores in target cell membranes. In this communication, planar lipid bilayers under voltage clamp conditions were used to investigate the geometric properties of the pores. It was established that all cytolysin channels were inserted into membranes with the same orientation. Sharp asymmetry in the I-V curve of fully open cytolysin channels persisting at high electrolyte concentrations indicated asymmetry in the geometry of the channel lumen. Using the nonelectrolyte exclusion method, evidence was obtained that the cis opening of the channel had a larger diameter (< or = 1.9 nm) than the trans opening (< or = 1.6 nm). The channel lumen appeared constricted, with a diameter of < or = 1.2 nm. Cup-shaped lumen geometry was deduced for both channel openings, which appeared to be connected to each other via a central narrow part. The latter contributed significantly to the total electrical resistance and determined the discontinuous character of channel filling with nonelectrolytes. Comparisons of the properties of pores formed by cytolysins of two V. cholerae biotypes (EL Tor and non-O1) indicated that the two ion channels possessed a similar geometry.

Coupling of cholesterol and cone-shaped lipids in bilayers augments membrane permeabilization by the cholesterol-specific toxins streptolysin O and Vibrio cholerae cytolysin.

Vibrio cholerae cytolysin (VCC) forms oligomeric pores in lipid bilayers containing cholesterol. Membrane permeabilization is inefficient if the sterol is embedded within bilayers prepared from phosphatidylcholine only but is greatly enhanced if the target membrane also contains ceramide. Although the enhancement of VCC action is stereospecific with respect to cholesterol, we show here that no such specificity applies to the two stereocenters in ceramide; all four stereoisomers of ceramide enhanced VCC activity in cholesterol-containing bilayers. A wide variety of ceramide analogs were as effective as D-erythro-ceramide, as was diacylglycerol, suggesting that the effect of ceramide exemplifies a general trend of lipids with a small headgroup to augment the activity of VCC. Incorporation of these cone-shaped lipids into cholesterol-containing bilayers also gave similar effects with streptolysin O, another cholesterol-specific but structurally unrelated cytolysin. In contrast, the activity of staphylococcal alpha-hemolysin, which does not share with the other toxins the requirement for cholesterol, was far less affected by the presence of lipids with a conical shape. The collective data indicate that sphingolipids and glycerolipids do not interact with the cytolysins specifically. Instead, lipids that have a conical molecular shape appear to effect a change in the energetic state of membrane cholesterol that in turn augments the interaction of the sterol with the cholesterol-specific cytolysins.

Interaction of Escherichia coli hemolysin with biological membranes. A study using cysteine scanning mutagenesis.

Escherichia coli hemolysin (HlyA) is a membrane-permeabilizing protein belonging to the family of RTX-toxins. Lytic activity depends on binding of Ca2(+) to the C-terminus of the molecule. The N-terminus of HlyA harbors hydrophobic sequences that are believed to constitute the membrane-inserting domain. In this study, 13 HlyA cysteine-replacement mutants were constructed and labeled with the polarity-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan). The fluorescence emission of the label was examined in soluble and membrane-bound toxin. Binding effected a major blue shift in the emission of six residues within the N-terminal hydrophobic domain, indicating insertion of this domain into the lipid bilayer. The emission shifts occurred both in the presence and absence of Ca2(+), suggesting that Ca2(+) is not required for the toxin to enter membranes. However, binding of Ca2(+) to HlyA in solution effected conformational changes in both the C-terminal and N-terminal domain that paralleled activation. Our data indicate that binding of Ca2(+) to the toxin in solution effects a conformational change that is relayed to the N-terminal domain, rendering it capable of adopting the structure of a functional pore upon membrane binding.

Vibrio cholerae cytolysin: assembly and membrane insertion of the oligomeric pore are tightly linked and are not detectably restricted by membrane fluidity.

Hemolytic strains of Vibrio cholerae secrete a cytolysin that, upon binding as a monomer, forms pentameric pores in animal cell membranes. Pore formation is inhibited at low temperature and in the absence of cholesterol. We here posed the following questions: firstly, can oligomerization be observed in the absence of pore formation? Secondly, is membrane fluidity responsible for the effect of temperature or of cholesterol upon pore formation? The first issue was approached by chemical cross-linking, by electrophoretic heteromer analysis, and by electron microscopy. None of these methods yielded any evidence of a non-lytic pre-pore oligomer. The second question was addressed by the use of two susceptible liposome models, consisting of cholesterol admixed to bovine brain lipids and to asolectin, respectively. The two liposome species clearly differed in membrane fluidity as judged by diphenylhexatriene fluorescence polarization. Nevertheless, their permeabilization by the cytolysin decreased with temperature in a closely parallel fashion, virtually vanishing at 5 degrees C. Omission of cholesterol from the liposomes uniformly led to an increase in membrane fluidity but prevented permeabilization by the cytolysin. The effects of temperature and of cholesterol upon cytolysin activity are thus not mediated by fluidization of the target membrane. The findings of our study distinguish V. cholerae cytolysin from several previously characterized pore-forming toxins.

Oligomerization of Vibrio cholerae cytolysin yields a pentameric pore and has a dual specificity for cholesterol and sphingolipids in the target membrane.

Vibrio cholerae cytolysin permeabilizes animal cell membranes. Upon binding to the target lipid bilayer, the protein assembles into homo-oligomeric pores of an as yet unknown stoichiometry. Pore formation has been observed with model liposomes consisting of phosphatidylcholine and cholesterol, but the latter were much less susceptible to the cytolysin than were erythrocytes or intestinal epithelial cells. We here show that liposome permeabilization is strongly promoted if cholesterol is combined with sphingolipids, whereby the most pronounced effects are observed with monohexosylceramides and free ceramide. These two lipid species are prevalent in mammalian intestinal brush border membranes. We therefore propose that, on its natural target membranes, the cytolysin has a dual specificity for both cholesterol and ceramides. To assess the stoichiometry of the pore, we generated hybrid oligomers of two naturally occurring variants of the toxin that differ in molecular weight. On SDS-polyacrylamide gel electrophoresis, the mixed oligomers formed a pattern of six distinct bands. Ordered by decreasing electrophoretic mobility, the six oligomer species must comprise 0 to 5 subunits of the larger form; the pore thus is a pentamer. Due to both lipid specificity and pore stoichiometry, V. cholerae cytolysin represents a novel prototype in the class of bacterial pore-forming toxins.

Mode of primary binding to target membranes and pore formation induced by Vibrio cholerae cytolysin (hemolysin).

Vibrio cholerae cytolysin (VCC) is produced by many non-choleratoxigenic strains of V. cholerae, and possibly represents a relevant pathogenicity determinant of these bacteria. The protein is secreted as a pro-toxin that is proteolytically cleaved to yield the active toxin with a molecular mass of approximately 63 kDa. We here describe a simple procedure for preparative isolation of mature VCC from bacterial culture supernatants, and present information on its mode of binding and pore formation in biological membranes. At low concentrations, toxin monomers interact with a high-affinity binding site on highly susceptible rabbit erythrocytes. This as yet unidentified binding site is absent on human erythrocytes, which are less susceptible to the toxin action. At higher concentrations, binding of the toxin occurs to both rabbit and human erythrocytes in a non-saturable manner. Cell-bound toxin monomers oligomerize to form supramolecular structures that are seen in the electron microscope as apparently hollow funnels, and oligomerization correlates functionally with the appearance of small transmembrane pores. Osmotic protection experiments indicate that the toxin channels are of finite size with a diameter of 1-2 nm. The mode of action of VCC closely resembles that of classical pore-forming toxins such as staphylococcal alpha-toxin and the aerolysin of Aeromonas hydrophila.

Potent membrane-permeabilizing and cytocidal action of Vibrio cholerae cytolysin on human intestinal cells.

Many strains of Vibrio cholerae non-O1 and O1 El Tor that cause diarrhea do not harbor genes for a known secretogenic toxin. However, these strains usually elaborate a pore-forming toxin, hitherto characterized as a hemolysin and here designated V. cholerae cytolysin, whose action on intestinal cells has not yet been described. We report that V. cholerae cytolysin binds as a monomer to Intestine 407 cells and then assembles into detergent-stable oligomers that probably represent tetra- or pentamers. Oligomer formation is accompanied by generation of small transmembrane pores that allow rapid flux of K+ but not influx of Ca2+ or propidium iodide. Pore formation is followed by irreversible ATP depletion and cell death. Binding of fewer than 10(4) toxin molecules per cell in vitro is lethal. The possibility is raised that production of this toxin by bacteria that are in close contact with intestinal cells is rapidly cytocidal in vivo, and death of intestinal cells may be a cause of diarrhea.

Characterization of Vibrio cholerae El Tor cytolysin as an oligomerizing pore-forming toxin.

V. cholerae El Tor cytolysin is a secreted, water-soluble protein of M(r) 60,000 that may be relevant to the pathogenesis of acute diarrhea. In this communication, we demonstrate that the toxin binds to and oligomerizes in target membranes to form SDS-stable aggregates of M(r) 200,000-250,000 that generate small transmembrane pores. Pores formed in erythrocytes were approximately 0.7 nm in size, as demonstrated by osmotic protection experiments. Binding was shown to occur in a temperature-independent manner preceding the temperature-dependent oligomerization step. Pores were also shown to be formed in L929 and HEp-2 cells, human fibroblasts and keratinocytes, albeit with highly varying efficacy. At neutral pH and in the presence of serum, human fibroblasts were able to repair a limited number of lesions. The collective data identify V. cholerae El Tor cytolysin as an oligomerizing toxin that damages cells by creating small transmembrane pores.

Spectrophotometric determination of paraoxonase within mouse carrier red blood cells.

A method has been developed to continuously measure paraoxonase activity spectrophotometrically in carrier red blood cells (RBCs) containing paraoxonase. This enzyme has a broad substrate specificity that includes parathion, paraoxon, soman, sarin, di-isopropyl fluorophosphate and many other organophosphorus compounds. Paraoxon is hydrolysed by paraoxonase to the less toxic 4-nitrophenol and diethyl phosphate. Determination of enzymic activity was based on the liberation of 4-nitrophenol in the presence of mouse RBCs. Paraoxonase was encapsulated within murine RBCs by hypotonic dialysis with subsequent resealing and annealing. The enzyme within resealed RBCs actively hydrolyses paraoxon in biological fluids to its less toxic metabolites. Paraoxonase incorporated within RBCs, like other enzymes, was found to be quite stable once encapsulated into RBCs and this formed the basis for this spectrophotometric method. Increasing absorbance at 400 nm indicated paraoxon hydrolysis and was the basis employed to determine enzymic activity. The Km of the enzyme within erythrocytes was 0.04 mM. This method offers a convenient, rapid and continuous way to monitor paraoxonase activity inside the carrier cell.

Entero-cytolysin (EC) from Vibrio cholerae non-O1 (some properties and pore-forming activity).

A thermolabile extracellular entero-cytolysin (EC) from Vibrio cholerae non-O1 was purified by ammonium sulphate fractionation, DE-52 cellulose ion exchange chromatography, gel-filtration on Ultrogel AcA-44 and high performance liquid chromatography on a Mono Q. The purified EC had a molecular weight of 63 kD and an isoelectric point of 6.2. It was not inactivated by cholesterol or 5,5'-dithio-bis(2-nitrobenzoic acid), nor activated by dithiothreitol. EC had no immunological cross-reactivity with cholera toxin. The EC caused fluid accumulation in the intestines of infant rabbits, death of mice by intravenous injection, and increased vascular permeability in the paw oedema test in mice. V. cholerae non-O1 EC lysed erythrocytes from various species and cultured cells (CHO, L-929, L-41, HEp-2, Vero, MDCK and BHK-21). In contrast to cholera toxin, EC caused crude destruction of target cells. The EC caused hemolysis by a colloid-osmotic mechanism due to the formation of hydrophilic pores of 1.8-2.0 nm diameter in the cell membrane. This EC also was able to open pores in lipid membranes. The induced channels were anion-selective and had a diameter of 1.8-2.0 nm.

The mode of action of Vibrio cholerae cytolysin. The influences on both erythrocytes and planar lipid bilayers.

The interaction with erythrocytes of cholera cytolysin (CC) obtained from a non-01 Vibrio cholerae strain results in the osmotic rupture of target cells upon formation by CC of the waterfilled pores in their membranes. The aggregation of several toxin monomers is required for the formation of one CC channel with a radius of 0.9-1.0 nm. The investigations using planar bilayer lipid membranes suggest that the CC-induced pore is an interprotein anion selective channel carrying a fixed positive charge. The role of the charge was supported by the influence of pH on the selectivity, single conductance and voltage gating of the CC channels. The ability of the CC to modify both model and natural membranes has a maximum at pH 6.0-7.0. It was found that CC channels insert into the membrane asymmetrically. The effect of proteolytic treatment of the channel by papain also indicates that the two entrances of the channel protrude from the plane of the membrane into the solution for different distances. It is proposed that the biological effects of the non-01 V. cholera cytolysin are based on its channel-forming activity.

Antagonism of the lethal effects of cyanide with resealed erythrocytes containing rhodanese and thiosulfate.

A new concept has been presented for the antagonism of cyanide and possibly other chemical toxicants. Until now, only a half dozen truly specific "antidotes" were known. There are many other "antidotes" which merely prevent the absorption or enhance the elimination of a toxic compound rather than specifically destroying the substance to prevent its toxic effect. This new approach has considerable conceptual significance in toxicology, as it suggests the encapsulating other enzymes to degrade various other chemical toxicants. There are many chemical toxicants for which there are no specific antidotes, and the conceptual approach of employing erythrocyte-encapsulated enzyme provides an innovative, specific approach to antagonize the toxic and lethal effects of these chemicals.