The IL-2 diphtheria toxin fusion protein denileukin diftitox modulates the onset of diabetes in female nonobese diabetic animals in a time-dependent manner and breaks tolerance in male nonobese diabetic animals.

Denileukin diftitox, also known as DAB(389)IL-2 or Ontak, is a fusion protein toxin consisting of the full-length sequence of the IL-2 protein and as toxophore the truncated diphtheria toxin. As a consequence, it delivers the toxic agent to CD25-bearing cells, whereby CD25 represents the high-affinity α-subunit of the IL-2 receptor. Initially it was developed for the treatment of patients with cutaneous T cell lymphoma. Meanwhile, denileukin diftitox is also used as an adjuvant in other tumor therapies and neoplastic disorders. In this study, to our knowledge we report for the first time that denileukin diftitox has also dramatic effects regarding the pathology of type 1 diabetes using the NOD mouse model. Repeated injections of denileukin diftitox into female NOD mice at 12 wk of age led to a clear acceleration of disease onset, whereas injection at 7 wk of age did not. Using male NOD mice, which are much less susceptible to diabetes, we demonstrate that the injection of denileukin diftitox leads to a dramatic development of type 1 diabetes within days after injection, thereby obviously breaking pre-existing tolerance mechanisms. This is accompanied by an increased IFN-γ production of autoreactive splenic cells and a decreased presence of regulatory CD4(+)CD25(+)Foxp3(+) T cells. In contrast, transfer of CD4(+)CD25(+)Foxp3(+) T cells could correct the defect after denileukin diftitox treatment. Furthermore, whereas IFN-γ production was increased in the pancreata of treated animals, insulin expression was strongly reduced. These finding should be considered when denileukin diftitox is used for the treatment of patients suffering from tumors and/or autoimmune disorders.

Mouse models demonstrating the role of stem/progenitor cells in gastric carcinogenesis.

Advances have been made in identifying the gastric epithelial stem cells and their immediate descendents which act as uncommitted or committed progenitors giving rise to cell lineages producing the various contents of the gastric juice and several hormones. New research suggests that these epithelial stem/progenitor cells also play an important role in the development of gastric cancer. In this review, we summarize results of examining three genetically manipulated mouse models in which the biological features and differentiation program of the gastric stem/progenitor cells were altered by three different approaches: 1) knockout of the trefoil factor 1 gene which is expressed initially in the partially committed pre-pit cell progenitors known to produce both mucus- and acid-secreting cell lineages, 2) expression of Simian virus 40 large T antigen gene in the acid-secreting parietal cell lineage, and 3) ablation of the parietal cells by using the attenuated diphtheria toxin A fragment. Systematic analysis of these animal models provided some clues to the role played by gastric stem/progenitor cells during carcinogenesis and to the cellular origin of gastric cancer.

In vitro expression of the diphtheria toxin A-chain gene under the control of human chorionic gonadotropin gene promoters as a means of directing toxicity to ovarian cancer cell lines.

OBJECTIVE: Our goal was to determine whether toxicity of the diphtheria toxin A-chain gene regulated by the human chorionic gonadotropin promoter can be directed to malignant ovarian cell lines. STUDY DESIGN: Plasmids containing diphtheria toxin A-chain gene linked to the regulatory elements of the metalloergothioneine and human chorionic gonadotropin promoters were transfected into the cell lines. Expression of diphtheria toxin A-chain gene was determined by the inhibition of a cotransfected luciferase reporter gene. RESULTS: Cytotoxicity of the diphtheria toxin A-chain gene is shown in a dose-responsive manner. Transfection of a plasmid expressing the diphtheria toxin A-chain gene controlled by a constitutive promoter readily inhibits protein synthesis. Specific inhibition of luciferase protein synthesis occurs in ovarian cancer cells transfected with the diphtheria toxin A-chain gene under the control of the human chorionic gonadotropin promoters when compared with normal ovarian epithelial cells or fibroblasts. CONCLUSIONS: These data demonstrate the preferential expression of the diphtheria toxin A-chain gene, regulated by the human chorionic gonadotropin promoter, to ovarian cancer cell lines. This provides an avenue for targeting such cells for suicide, toxin, or cytokine genes.

Membrane translocation of diphtheria toxin fragment A exploits early to late endosome trafficking machinery.

After reaching early endosomes by receptor-mediated endocytosis, diphtheria toxin (DT) molecules have two possible fates. A large pool enters the degradative pathway whereas a few molecules become cytotoxic by translocating their catalytic fragment A (DTA) into the cytosol. Impairment of DT degradation by microtubule depolymerization does not block DT cytotoxicity. Therefore, DTA membrane translocation into the cytosol occurs from an endocytic compartment located upstream of late endosomes. Comparisons between early endosomes and endocytic carrier vesicles in a cell-free translocation assay have demonstrated that early endosomes are the earliest endocytic compartment from which DTA translocates. DTA translocation is ATP-dependent, requires early endosomal acidification, and is increased by the addition of cytosol. Cytosol-dependent DTA translocation is GTP gamma S-insensitive but is blocked by anti-beta COP antibodies.

Diphtheria toxin as a molecular tool in the study of acidic fibroblast growth factor signalling.

In eukaryotic cells proteins are translocated across a number of cellular membranes into various intracellular organelles such as the endoplasmatic reticulum, mitochondria, peroxisomes and chloroplasts. In all these cases the proteins are translocated away from the cytosol. However, certain proteins are also translocated in the opposite direction, from the exterior to the cytosol. Well established examples are some bacterial and plant protein toxins, that exert their effect in the cytosol. A common property of protein toxins with intracellular action is that they contain two functionally different moieties, in many cases consisting of two. disulfide-linked polypeptides. Relatively little is known about how these proteins cross the membrane. The translocation process is best understood in the case of diphtheria toxin, which binds to cell surface receptors, is then taken up by endocytosis and is subsequently translocated to the cytosol, where it inactivates elongation factor 2. Recently it has been recognized that diphtheria toxin as well as a few other protein toxins can be used to carry passenger peptides or proteins into cells (in addition to other usefull roles which the toxins have begun to play in understanding many cellular processes and in certain prophylactic and therapeutic purposes). Here, the approach of using diphtheria toxin as a translocation vehicle in the study of new aspects of signal transduction mechanisms activated by acidic fibroblast growth factor is discussed and the possibility that some proteins have distinct functions in more than one cellular compartment is considered. Finally, this article focuses on the role of the toxins as tools in cell biology and experimental medicine.

Contribution of plasminogen activator urokinase to in vitro cytotoxicity of diphtheria toxin.

Nicking of diphtheria toxin (DT), i.e. proteolytic cleavage at an arginine-rich region within the first disulphide loop, is a prerequisite to the intoxication process. We show that protease(s) required in this process was synthesized and secreted by the sensitive cells and that antibodies against plasminogen activator urokinase (uPA) decreased the in vitro cytotoxicity of DT on Vero cells. Our results demonstrate that uPA secreted by Vero cells cultured in vitro is one of the cellular proteases involved in the cleavage and activation of diphtheria toxin.

Structure-function relationships in diphtheria toxin channels: I. Determining a minimal channel-forming domain.

Diphtheria Toxin (DT) is a 535 amino acid exotoxin, whose active form consists of two polypeptide chains linked by an interchain disulphide bond. DT's N-terminal A fragment kills cells by enzymatically inactivating their protein synthetic machinery; its C-terminal B chain is required for the binding of toxin to sensitive cells and for the translocation of the A fragment into the cytosol. This B fragment, consisting of its N-terminal T domain (amino acids 191-386) and its C-terminal R domain (amino acids 387-535) is responsible for the ion-conducting channels formed by DT in lipid bilayers and cellular plasma membranes. To further delineate the channel-forming region of DT, we studied channels formed by deletion mutants of DT in lipid bilayer membranes under several pH conditions. Channels formed by mutants containing only the T domain (i.e., lacking the A fragment and/or the R domain), as well as those formed by mutants replacing the R domain with Interleukin-2 (IL-2), have single channel conductances and selectivities essentially identical to those of channels formed by wild-type DT. Furthermore, deleting the N-terminal 118 amino acids of the T domain also has minimal effect on the single channel conductance and selectivity of the mutant channels. Together, these data identify a 61 amino acid stretch of the T domain, corresponding to the region which includes alpha-helices TH8 and TH9 in the crystal structure of DT, as the channel-forming region of the toxin.

Structure function relationships in diphtheria toxin channels: II. A residue responsible for the channel's dependence on trans pH.

Ion-conducting channels formed in lipid bilayers by diphtheria toxin are highly pH dependent. Among other properties, the channel's single channel conductance and selectivity depend on proton concentrations on either side of the membrane. We have previously shown that a 61 amino acid fragment of DT is sufficient to form a channel having the same pH-dependent single channel properties as that of the intact toxin. This region corresponds to an alpha-helical hairpin in the recently published crystal structure of DT in solution; the hairpin contains two alpha-helices, each long enough to span a membrane, connected by a loop of about nine residues. This paper reports on the single channel effects of mutations which alter the two negatively charged residues in this loop. Changing Glutamate 349 to neutral glutamine or to positive lysine has no effect on the DT channel's single channel conductance or selectivity. In contrast, mutations of Aspartate 352 to neutral asparagine (DT-D352N) or positive lysine (DT-D352K) cause progressive reductions in single channel conductance at pH 5.3 cis/7.2 trans (in 1 M KCl), consistent with this group interacting electrostatically with ions in the channel. The cation selectivity of these mutant channels is also reduced from that of wild-type channels, a direction consistent with residue 352 influencing permeant ions via electrostatic forces. When both sides of the membrane are at pH 4, the conductance difference between wild-type and DT-D352N channels is minimal, suggesting that Asp 352 (in the wild type) is neutral at this pH. Differences observed between wild-type and DT-D352N channels at pH 4.0 cis/7.2 trans (with a high concentration of permeant buffer in the cis compartment) imply that residue 352 is on or near the trans side of the membrane. Comparing the conductances of wild-type and DT-D352K channels at large (cis) positive voltages supports this conclusion. The trans location of position 352 severely constrains the number of possible membrane topologies for this region.

Structure-function relationships in diphtheria toxin channels: III. Residues which affect the cis pH dependence of channel conductance.

The conductance of channels formed by diphtheria toxin (DT) in lipid bilayer membrane depends strongly on pH. We have previously shown that a 61 amino acid region of the protein, denoted TH8-9, is sufficient to form channels having the same pH-dependent conductance properties as those of whole toxin channels. One residue in this region, Aspartate 352, is responsible for all the dependence of single channel conductance on trans pH, whereas another, Glutamate 349, has no effect. Here, we report that of the seven remaining charged residues in the TH8-9 region, mutations altering the charge on H322, H323, H372, and R377 have minimal effects on single channel conductance; mutations of Glutamates 326, 327, or 362, however, significantly affect single channel conductance as well as its dependence on cis pH. Moreover, Glutamate 362 is titratable from both the cis and trans sides of the membrane, suggesting that this residue lies within the channel; it is more accessible, however, to cis than to trans protons. These results are consistent with the membrane-spanning topology previously proposed for the TH8-9 region, and suggest a geometric model for the DT channel.