Cell surface sulfhydryls are required for the cytotoxicity of diphtheria toxin but not of ricin in Chinese hamster ovary cells.

A previous study on cleavage of disulfide bonds in endocytosed model compounds had shown that an initial phase of cleavage was totally inhibited by membrane-impermeant sulfhydryl inhibitors and thus was mediated by cell surface sulfhydryls (Feener, E. P., Shen, W.-C., and Ryser, H. J.-P. (1990) J. Biol. Chem. 265, 18780-18785). This paper uses the same inhibitors (5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuriphenylsulfonic acid) to examine the role of surface sulfhydryls in the cytotoxicity of diphtheria toxin (DT). Since the interchain disulfide of endocytosed DT must be cleaved prior to translocation of chain A from endosomes to cytoplasm, it was postulated that surface sulfhydryls might mediate the cleavage of that disulfide bond as well. Both sulfhydryl blockers did indeed markedly inhibit DT cytotoxicity. This effect was not due to inactivation of unbound DT, inhibition of receptor-mediated endocytosis, or impairment of acidification of endosomes. We conclude that cell surface sulfhydryls susceptible to blockage by 5,5'-dithiobis(2-nitro-benzoic acid) and p-chloromercuriphenylsulfonic acid are required for the cytotoxicity of DT and, most likely, for the reductive cleavage of DT's interchain disulfides. Ricin cytotoxicity was not decreased; this is consistent with the view that ricin reaches the cytoplasm from a late endocytic structure and with the finding that endocytosed disulfides are also cleaved in a cell fraction containing elements of the Golgi apparatus (Feener, E. P., Shen, W.-C., and Ryser, H. J.-P. (1990) J. Biol. Chem. 265, 18780-18785).     

Inhibition of farnesylation blocks growth but not differentiation in FRTL-5 thyroid cells.

The cholesterol lowering drug lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, blocks DNA synthesis and proliferation of thyrotropin (TSH) primed FRTL-5 rat thyroid cells. The blockade can be completely prevented and/or reversed by mevalonate and largely prevented and/or reversed by farnesol whereas cholesterol and/or other non-sterol mevalonate derivatives such as ubiquinone, dolichol or isopentenyladenosine are ineffective. TSH-dependent augmentation of cyclic-AMP and cAMP dependent differentiated functions, such as iodide uptake, are unaffected by lovastatin. 3H-Thymidine incorporation into DNA is also decreased by alpha-hydroxyfarnesyl-phosphonic acid, an inhibitor of protein farnesylation which mimicks the effect of lovastatin since it also leaves unaffected TSH stimulated iodide uptake. It is suggested that the HMG-CoA reductase inhibitor lovastatin affects cell proliferation mainly through inhibition of protein farnesylation which results in altered function proteins relevant for proliferation control, notably p21ras and/or other small GTPases.     

Interactions between diphtheria toxin entry and anion transport in Vero cells. II. Inhibition of anion antiport by diphtheria toxin

When cells with surface-bound diphtheria toxin were exposed to pH 4.5, the toxin became shielded against lactoperoxidase-catalyzed radioiodination, indicating that the toxin was inserted into the membrane. Cells thus treated had strongly reduced ability to take up 36Cl-, 35SO4(2-), and [14C]SCN-. The reduction of chloride uptake was strongest at neutral pH, whereas that of sulfate was strongest at acidic pH. Lineweaver-Burk plots indicated that the toxin treatment reduced the Jmax but not the Km for the anions. The toxin also inhibited the NaCl-stimulated efflux of 35SO4(2-), indicating that the toxin inhibits the antiporter. No inhibition was found when toxin-treated cells were not exposed to low pH, whereas exposure to pH 4.5 for 20 s induced close to maximal inhibition. Half-maximal inhibition was obtained after exposure to pH 5.4. The concentration of diphtheria toxin required to obtain maximal inhibition (0.3 micrograms/ml) was sufficient to ensure close to maximal toxin binding to the cells. Even in ATP-depleted cells and in the absence of permeant anions, low pH induced inhibition of anion antiport in toxin-treated Vero cells. There was no measurable inhibition of anion antiport in cells with little or no ability to bind the toxin.     

Monensin blocks the transport of diphtheria toxin to the cell cytoplasm

Lysosomotropic amines are believed to inhibit the transport of diphtheria toxin to the cell cytoplasm by raising the pH within intracellular vesicles. If so, then other drugs that dissipate intracellular proton gradients should have a similar effect on toxin transport. We found that monensin, a proton ionophore unrelated to lysosomotropic amines, is a potent inhibitor of the cytotoxic effect of diphtheria toxin. Monensin appears to block the escape of endocytosed toxin from a vesicle to the cytoplasm. Monensin fails to protect cells from the effects of diphtheria toxin that is bound to the cell surface and exposed to acidic medium, suggesting that the step normally blocked by the drug is circumvented under these conditions. The inhibition of toxin transport caused by monensin could not be relieved when monensin was replaced by ammonium chloride, nor when ammonium chloride was again replaced by monensin. This suggests that both drugs block the same step of toxin transport. The effect of monensin on the transport of diphtheria toxin to the cytoplasm is consistent with the proposal (Draper and Simon. 1980. J. Cell Biol. 87:849-854; Sandvig and Olsnes. 1980. J. Cell Biol. 87:828-832) that the toxin is endocytosed and then, in response to an acidic environment, penetrates through the membrane of an intracellular vesicle to reach the cytoplasm.     

An antibody that inhibits the binding of diphtheria toxin to cells revealed the association of a 27-kDa membrane protein with the diphtheria toxin receptor

A monoclonal antibody that blocks the binding of diphtheria toxin to Vero cells was isolated by immunizing mice with Vero cell membrane. The antibody inhibits the binding of diphtheria toxin and also CRM197, a mutant form of diphtheria toxin, to Vero cells, and consequently inhibits the cytotoxicity of diphtheria toxin. This antibody does not directly react with the receptor molecule of diphtheria toxin (DTR14.5). Immunoprecipitation and immunoblotting studies revealed that this antibody binds to a novel membrane protein of 27 kDa (DRAP27). When diphtheria toxin receptor was passed through an affinity column made with this antibody, the receptor was trapped only in the presence of DRAP27. These results indicate that DRAP27 and DTR14.5 closely associate in Vero cell membrane and that the inhibition of the binding of diphtheria toxin to the receptor is due to the binding of the antibody to the DRAP27 molecule. Binding studies using 125I-labeled antibody showed that there are many more molecules of DRAP27 on the cell surface than diphtheria toxin-binding sites. However, there is a correlation between the sensitivity of a cell line to diphtheria toxin and the number of DRAP27 molecules on the cell surface, suggesting that DRAP27 is involved in the entry of diphtheria toxin into the target cell.     

Effects of BCL-2 overexpression on the sensitivity of MCF-7 breast cancer cells to ricin, diphtheria and Pseudomonas toxin and immunotoxins

Immunotoxins are presently being evaluated as novel agents for cancer therapy. The direct mechanism by which immunotoxins kill cancer cells is inhibition of protein synthesis, but cytotoxicity due to induction of apoptosis has also been observed with these agents. Some cancers that express high levels of BCL-2 are relatively resistant to apoptosis inducing agents. It is therefore important to determine to what degree the toxicity of ricin, diphtheria toxin, Pseudomonas exotoxin and Pseudomonas exotoxin derived immunotoxins towards cancer cells can be attributed to inhibition of protein synthesis, and to what degree to subsequent induction of apoptosis. We compared the sensitivity of MCF-7 breast cancer cells that were stably transfected with a BCL-2 expression plasmid and thus protected against apoptosis and of MCF-7 cells transfected with a control plasmid towards ricin, diphtheria and Pseudomonas toxin, a Pseudomonas toxin-derived immunotoxin (LMB-7) and tumour necrosis factor (TNF). We found that BCL-2 mediated inhibition of apoptosis renders the cells almost completely resistant (1000-fold) to tumour necrosis factor, but the same cells were only 3–10 fold more resistant to cytotoxicity induced by immunotoxin LMB-7 as well as Pseudo-monas exotoxin, diphtheria toxin and ricin. We next studied several leukaemia cell lines with variable levels of BCL-2 expression and found them quite sensitive to a Pseudomonas exotoxin containing immunotoxin independent of the level of BCL-2. Our data indicate that although BCL-2 overexpression can have a modest effect on sensitivity to an immunotoxin, cell lines derived from patients are still very sensitive to immunotoxins.     

Monensin intercalation in liposomes: effect on cytotoxicities of ricin,Pseudomonas exotoxin A and diphtheria toxin in CHO cells

Monensin, a car☐ylic ionophore was intercalated in liposomes (liposomal monensin) and its effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells was studied. Intercalation of monensin in liposomal bilayer is found to have no effect on its stability and interaction with cells. Liposomal monensin)(1 nM) substantially enhance the cytotoxicities of ricin (62-fold) and Pseudomonas exotoxin A (11.5-fold) while it has no effect on diphtheria toxin. This observed effect is highly dependent on the liposomal lipid composition. The potentiating ability of monensin (1 nM) in neutral vesicles is significantly higher (2.2-fold) as compared to negatively charged vesicles. This ability is drastically reduced by incorporation of stearylamine in liposomes and is found to be dependent on the density of stearylamine as well as on the concentration of serum in the medium. Monensin in liposomes containing 24 mol% stearylamine has a very marginal effect on the cytotoxicity of ricin (7.5-fold) which is further reduced (1.5-fold) in the presence of 20% serum. The uptake of ¹²⁵I-gelonin from neutral vesicles is significantly higher (∼ 2.0-fold) than that from the negative vesicles. The uptake from positive vesicles is highly dependent on the concentration of stearylamine. The reduction in the lag period (30 min) of ricin action by monensin in neutral and negative vesicle is comparable with free monensin. However, monensin in positive vesicle has no effect on it. These studies have suggested that liposomes could be used as a delivery vehicle for monensin for selective elimination of tumor cells in combination with hybrid toxins.     

Mutant with diphtheria toxin receptor and acidification function but defective in entry of toxin

A mutant of Chinese hamster ovary cells, GE1, that is highly resistant to diphtheria toxin was isolated. The mutant contains 50% ADP-ribosylatable elongation factor 2, but its protein synthesis was not inhibited by the toxin even at concentrations above 100 μg/ml. ¹²⁵I-labeled diphtheria toxin was associated with GE1 cells as well as with the parent cells but did not block protein synthesis of GE1 cells even when the cells were exposed to low pH in the presence or absence of NH₄Cl. The infections of GE1 cells and the parent cells by vesicular stomatitis virus were similar. GE1 cells were cross-resistant to Pseudomonas aeruginosa exotoxin A and so were about 1000 times more resistant to this toxin than the parent cells. Hybrids of GE1 cells and the parent cells or mutant cells lacking a functional receptor were more sensitive to diphtheria toxin than GE1 cells. These results suggest that entry of diphtheria toxin into cells requires a cellular factor(s) in addition to those involved in receptor function and acidification of endosomes and that GE1 cells do not express this cellular factor. This character is recessive in GE1 cells.     

Monensin intercalation in liposomes: effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells.

Monensin, a carboxylic ionophore was intercalated in liposomes (liposomal monensin) and its effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells was studied. Intercalation of monensin in liposomal bilayer is found to have no effect on its stability and interaction with cells. Liposomal monensin (1 nM) substantially enhance the cytotoxicities of ricin (62-fold) and Pseudomonas exotoxin A (11.5-fold) while it has no effect on diphtheria toxin. This observed effect is highly dependent on the liposomal lipid composition. The potentiating ability of monensin (1 nM) in neutral vesicles is significantly higher (2.2-fold) as compared to negatively charges vesicles. This ability is drastically reduced by incorporation of stearylamine in liposomes and is found to be dependent on the density of stearylamine as well as on the concentration of serum in the medium. Monensin in liposomes containing 24 mol% stearylamine has a very marginal effect on the cytotoxicity of ricin (7.5-fold) which is further reduced (1.5-fold) in the presence of 20% serum. The uptake of 125I-gelonin from neutral vesicles is significantly higher (approximately 2.0-fold) than that from the negative vesicles. The uptake from positive vesicles is highly dependent on the concentration of stearylamine. The reduction in the lag period (30 min) of ricin action by monensin in neutral and negative vesicle is comparable with free monensin. However, monensin in positive vesicle has no effect on it. These studies have suggested that liposomes could be used as a delivery vehicle for monensin for selective elimination of tumor cells in combination with hybrid toxins.     

Interactions between diphtheria toxin entry and anion transport in Vero cells. IV. Evidence that entry of diphtheria toxin is dependent on efficient anion transport

Entry of prebound diphtheria toxin at low pH occurred rapidly in the presence of isotonic NaCl, NaBr, NaSCN, NaI, and NaNO3, but not in the presence of Na2SO4, 2-(N-morpholino)ethanesulfonic acid neutralized with Tris, or in buffer osmotically balanced with mannitol. SCN- was the most efficient anion to facilitate entry. Uptake studies with radioactively labeled anions showed that SCN- was transported into cells 3 times faster than Cl-, while the entry of SO2-4 occurred much more slowly. The anion transport inhibitors 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and piretanide inhibited entry at low pH even in the presence of permeant anions. When cells with bound toxin were exposed to low pH in the absence of permeant anions, then briefly exposed to neutral pH and subsequently exposed to pH 4.5 in the presence of isotonic NaCl, toxin entry was induced. The data indicate that efficient anion transport at the time of exposure to low pH is required for entry of surface-bound diphtheria toxin into the cytosol. Since insertion of diphtheria toxin into the membrane occurs even in the absence of permeant anions, the results indicate that low pH is required not only for insertion of fragment B into the membrane, but also for the subsequent entry of fragment A into the cytosol.     

Anthrax toxin receptor 2 determinants that dictate the pH threshold of toxin pore formation

The anthrax toxin receptors, ANTXR1 and ANTXR2, act as molecular clamps to prevent the protective antigen (PA) toxin subunit from forming pores until exposure to low pH. PA forms pores at pH approximately 6.0 or below when it is bound to ANTXR1, but only at pH approximately 5.0 or below when it is bound to ANTXR2. Here, structure-based mutagenesis was used to identify non-conserved ANTXR2 residues responsible for this striking 1.0 pH unit difference in pH threshold. Residues conserved between ANTXR2 and ANTXR1 that influence the ANTXR2-associated pH threshold of pore formation were also identified. All of these residues contact either PA domain 2 or the neighboring edge of PA domain 4. These results provide genetic evidence for receptor release of these regions of PA as being necessary for the protein rearrangements that accompany anthrax toxin pore formation.     

Recombinant Hep G2 cells that express alcohol dehydrogenase and cytochrome P450 2E1 as a model of ethanol-elicited cytotoxicity

HepG2 cells were transfected with recombinant plasmids, one carrying the murine alcohol dehydrogenase (ADH) gene and the other containing the gene encoding human cytochrome P450 2E1 (CYP2E1). One of recombinant clones called VL-17A exhibited ADH and CYP2E1 specific activities comparable to those in isolated rat hepatocytes. VL-17A cells oxidized ethanol and generated acetaldehyde, the levels of which depended upon the initial ethanol concentration. Compared with unexposed VL-17A cells, ethanol exposure increased the cellular redox (lactate:pyruvate ratio) and caused cell toxicity, indicated by increased leakage of lactate dehydrogenase into the medium,. Exposure of VL-17A cells to 100mM ethanol significantly elevated caspase 3 activity, an indicator of apoptosis, but this ethanol concentration did not affect caspase 3 activity in parental HepG2 cells. Because ethanol consumption causes a decline in hepatic protein catabolism, we examined the influence of ethanol exposure on proteasome activity in HepG2, VL-17A, E-47 (CYP2E1(+)) and VA-13 (ADH(+)) cells. Exposure to 100mM ethanol caused a 25% decline in the chymotrypsin-like activity of the proteasome in VL-17A cells, but the enzyme was unaffected in the other cell types. This inhibitory effect on the proteasome was blocked when ethanol metabolism was blocked by 4-methyl pyrazole. We conclude that recombinant VL-17A cells, which express both ADH and CYP2E1 exhibit hepatocyte-like characteristics in response to ethanol. Furthermore, the metabolism of ethanol by these cells via ADH and CYP2E1 is sufficient to bring about an inhibition of proteasome activity that may lead to apoptotic cell death.     

Evidence that membrane phospholipids and protein are required for binding of diphtheria toxin in Vero cells

Treatment with phospholipase C strongly protected monkey kidney (Vero) cells against diphtheria toxin and reduced the ability of the cells to bind 125I-labelled toxin. Treatment with phospholipase D and with trypsin also protected the cells, although to a lesser extent. Phospholipase A2 had no protective effect. Phospholipase C also protected fetal hamster kidney cells against the toxin. After removal of the enzymes, as well as after treatment of the cells with 4-acetamide 4'-isothiocyanostilbene 2,2'-disulfonic acid, diphtheria toxin binding capability was restored slowly, apparently by a process requiring protein synthesis, since cycloheximide blocked the restoration. The data indicate that both phospholipids and protein are involved in the binding sites for diphtheria toxin.     

Inhibition by ricin of protein synthesis in vitro. Ribosomes as the target of the toxin

1. Ricin (a toxic protein from the seeds of Ricinus communis) is a powerful inhibitor of the poly(U)-directed incorporation of phenylalanine into polypeptides catalysed by isolated rat liver ribosomes and elongation factors 1 and 2 (EF 1 and EF 2). The inhibition can be largely overcome by increasing the concentration of ribosomes. 2. The toxin does not affect the binding of phenylalanyl-tRNA to ribosomes catalysed by EF 1, nor does it inhibit the puromycin reaction used as a test for peptide-bond formation catalysed by ribosomes. 3. Ricin inhibits the ribosome-linked GTP hydrolysis catalysed by EF 2. 4. Ribosomes treated with ricin and washed through sucrose gradients containing 0.6m-NH(4)Cl are functionally inactive in those assay systems that are sensitive to the presence of added toxin. 5. It is suggested that ricin brings about an irreversible modification of ribosomes which impairs their ability to interact with EF 2. Since ricin inhibits at a molar concentration much lower than that of ribosomes it probably acts catalytically. No added cofactor is necessary for the inhibitory action of the toxin.