Damage to nuclear DNA induced by Shiga toxin 1 and ricin in human endothelial cells.
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Ribosome-inactivating proteins (RIPs) remove a specific adenine from 28S rRNA leading to inactivation of ribosomes and arrest of translation. Great interest as to a possible second physiological substrate for RIPs came from the observation that in vitro RIPs remove adenine from DNA. This paper addresses the problem of nuclear lesions induced by RIPs in human endothelial cells susceptible to the bacterial RIP Shiga toxin 1 and the plant RIP ricin. With both toxins, nuclear DNA damage as evaluated by two independent techniques (alkaline-halo assay and alkaline filter elution) appears early, concomitant with (ricin) or after (Shiga toxin 1) the inhibition of protein synthesis. At this time, the annexin V binding assay, caspase 3 activity, the formation of typical < or = 50 Kb DNA fragments, and changes in morphology associated with apoptosis were negative. Furthermore, a block of translation comparable to that induced by RIPs, but obtained with cycloheximide, did not induce nuclear damage. Such damage is consistent with the enzymatic activity (removal of adenine) of RIPs acting in vitro on RNA-free chromatin and DNA. The results unequivocally indicate that RIPs can damage nuclear DNA in whole cells by means that are not secondary to ribosome inactivation or apoptosis. (+info)
Retrograde transport of protein toxins under conditions of COPI dysfunction.
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Retrograde transport dependent on coat protein I (COPI) was impaired using two different approaches and the effects on the retrograde transport of protein toxins were investigated. One approach was to study ldlF cells that express a temperature-sensitive defect in the epsilon-COP subunit of COPI. The second approach was to treat cells with 1,3-cyclohexanebis(methylamine) (CBM), a drug that interferes with the binding of COPI to Golgi membranes. With both approaches, cells remained sensitive to a variety of protein toxins regardless of whether the toxins contained a KDEL motif. Moreover, cholera toxin, which contains a KDEL sequence, was observed by immunofluorescence microscopy to enter the endoplasmic reticulum of Vero cells in the presence of CBM. These data support published evidence indicating the presence in cells of a COPI- and KDEL receptor-independent pathway of retrograde transport from the Golgi complex to the endoplasmic reticulum. In addition, the results suggest that certain toxins containing a KDEL motif may use either the COPI-dependent or COPI-independent pathway of retrograde transport. (+info)
Comparison of the apoptosis-inducing abilities of various protein synthesis inhibitors in U937 cells.
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We compared the abilities of ricin, diphtheria toxin, cycloheximide, and anisomycin to induce apoptosis, using human myeloid leukemia U937 cells at the concentration of each toxin at which almost complete protein synthesis inhibition was attained within 3 h. Among these toxins, anisomycin was found to be the most potent apoptosis inducer. After a 6-h exposure to anisomycin (1 microg/ml), nearly 95% of the cells had apoptotic nuclear morphological changes, while 53%, 30%, and 10% of the cells showed apoptotic changes after exposure to ricin (0.1 microg/ml), diphtheria toxin (10 microg/ml), and cycloheximide (10 microg/ml), respectively. Furthermore, a rapid increase in caspase-3-like activity was observed in anisomycin-treated cells. A similar increase in caspase-3-like activity was also observed in ricin-treated cells on a slower time schedule. However, only a slight increase in the protease activity was induced by diphtheria toxin or cycloheximide even after 6 h of incubation. Since both ricin and anisomycin are known to act on 28S ribosomal RNA, our results suggest that this action mechanism may be responsible for their potent apoptosis induction, and protein synthesis inhibition alone is not sufficient to induce apoptosis. (+info)
Ribosome inactivation by the toxic lectins abrin and ricin. Kinetics of the enzymic activity of the toxin A-chains.
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A sensitive test system for toxin-treated ribosomes was worked out by treating rabbit reticulocyte ribosomes with abrin A-chain, ricin A-chain or ricinus agglutinin A-chain, adding neutralizing amounts of specific antitoxins and testing for polyphenylalanine-synthesizing activity in a system where the concentration of elongation factors and ribosomes were varied. The strongest inhibition was obtained in the presence of low concentrations of elongation factor (EF-2). The activity of the ribosomes decreased with time of incubation with the toxin A-chains. Addition of anti-toxins stopped further inactivation. In systems containing untreated and toxin-treated ribosomes the ability to polymerize phenylalanine was proportional to the concentration of untreated ribosomes. There was a linear relationship between toxin A-chain concentration and the number of ribosomes inactivated per minute. The inactivation rate increased with temperature, and the estimated activation energy was 10.6 kcal (44.3 kJ). Linewaver-Burk plots of the data obtained by incubating various ribosome concentrations with toxins indicated a molecular activity of about 1500 ribosomes/minute for abrin and ricin A-chains and 100 ribosomes/minute for ricinus agglutinin A-chain. The apparent Michaelis constant was 0.1-0.2 muM for all three A-chains. The activity of the A-chains in the intact cell is discussed. (+info)
A Phase I study with an anti-CD30 ricin A-chain immunotoxin (Ki-4.dgA) in patients with refractory CD30+ Hodgkin's and non-Hodgkin's lymphoma.
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Ki-4.dgA is an anti-CD30 immunotoxin (IT) constructed by coupling the monoclonal antibody Ki-4 via a sterically hindered disulfide linker to deglycosylated ricin A-chain. This IT was efficacious in vitro and in SCID mice with disseminated human Hodgkin's lymphoma. Accordingly, a Phase I trial in patients (pts) with Hodgkin's lymphoma was designed. The objectives of this Phase I trial were to determine the maximum tolerated dose, the dose-limiting toxicities, pharmacokinetics, and antitumor activity. Seventeen pts with relapsed CD30+ lymphoma were treated with escalating doses (5, 7.5, or 10 mg/m(2)/cycle) of the IT as four bolus infusions on days 1, 3, 5, and 7 for one to three cycles. All of the pts had progressive disease and were heavily pretreated. Nine had primary progressive disease and 14 had advanced disease with massive tumor burdens. The mean age was 35 years (24-52 years). Peak serum concentrations of the intact IT varied from 0.23 to 1.1 microg/ml. Side effects and dose-limiting toxicities were related to vascular leak syndrome, i.e., decreases in serum albumin, edema, weight gain, hypotension, tachycardia, myalgia, and weakness. The maximum tolerated dose was 5 mg/m(2). Seven of 17 (40%) pts made human antiricin antibodies (> or =1.0 microg/ml), and 1 pt developed human antimouse antibodies (> or =1.0 microg/ml). Clinical response in the 15 evaluable pts included 1 partial remission, 1 minor response, and 2 stable diseases. In conclusion, the IT was less well tolerated than other ITs of this type. This might be because of the low number of CD30+ peripheral blood mononuclear cells, and in part because of binding of the IT to soluble CD30 antigen and the resulting circulation of IT/sCD30 complexes. (+info)
Exogenous peptides delivered by ricin require processing by signal peptidase for transporter associated with antigen processing-independent MHC class I-restricted presentation.
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In this study we demonstrate that a disarmed version of the cytotoxin ricin can deliver exogenous CD8(+) T cell epitopes into the MHC class I-restricted pathway by a TAP-independent, signal peptidase-dependent pathway. Defined viral peptide epitopes genetically fused to the N terminus of an attenuated ricin A subunit (RTA) that was reassociated with its partner B subunit were able to reach the early secretory pathway of sensitive cells, including TAP-deficient cells. Successful processing and presentation by MHC class I proteins was not dependent on proteasome activity or on recycling of MHC class I proteins, but rather on a functional secretory pathway. Our results demonstrated a role for signal peptidase in the generation of peptide epitopes associated at the amino terminus of RTA. We showed, first, that potential signal peptide cleavage sites located toward the N terminus of RTA can be posttranslationally cleaved by signal peptidase and, second, that mutation of one of these sites led to a loss of peptide presentation. These results identify a novel MHC class I presentation pathway that exploits the ability of toxins to reach the lumen of the endoplasmic reticulum by retrograde transport, and suggest a role for endoplasmic reticulum signal peptidase in the processing and presentation of MHC class I peptides. Because TAP-negative cells can be sensitized for CTL killing following retrograde transport of toxin-linked peptides, application of these results has direct implications for the development of novel vaccination strategies. (+info)
Current strategies of antibody-based treatment in Hodgkin's disease.
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Many new approaches involving antibody-based agents have given promising results in experimental Hodgkin's disease (HD) models. Clinical trials with monoclonal antibodies, immunotoxins, bispecific constructs and radioimmunoconjugates have demonstrated some clinical efficacy in patients with advanced refractory HD. Although it seems unlikely that resistant patients with larger tumor masses will be cured by either of these approaches, it might be feasible to treat bulky disease by conventional therapy and then administer biological agents to kill residual Hodgkin and Reed-Sternberg cells. Future phase III trials will have to prove a possible superior effect of this combined immunochemotherapy. Currently, the evaluation of the most promising approaches continues. (+info)
Membrane ruffling and macropinocytosis in A431 cells require cholesterol.
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Cholesterol is important for the formation of caveolea and deeply invaginated clathrin-coated pits. We have now investigated whether formation of macropinosomes is dependent on the presence of cholesterol in the plasma membrane. Macropinocytosis in A431 cells was induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, a potent activator of protein kinase C (PKC). When cells were pretreated with methyl-beta-cyclodextrin to extract cholesterol, the phorbol ester was unable to induce the increased endocytosis of ricin otherwise seen, although PKC could still be activated. Electron microscopy revealed that extraction of cholesterol inhibited the formation of membrane ruffles and macropinosomes at the plasma membrane. Furthermore, cholesterol depletion inhibited the phorbol ester-induced reorganization of filamentous actin at the cell periphery, a prerequisite for the formation of membrane ruffles that close into macropinosomes. Under normal conditions the small GTPase Rac1 is activated by the phorbol ester and subsequently localized to the plasma membrane, where it induces the reorganization of actin filaments required for formation of membrane ruffles. Cholesterol depletion did not inhibit the activation of Rac1. However, confocal microscopy showed that extraction of cholesterol prevented the phorbol ester-stimulated localization of Rac1 to the plasma membrane. Thus, our results demonstrate that cholesterol is required for the membrane localization of activated Rac1, actin reorganization, membrane ruffling and macropinocytosis. (+info)