Quantal entry of diphtheria toxin to the cytosol

The rate-limiting step in diphtheria toxin (DT) intoxication of Vero cells has been determined utilizing cycloheximide as an inhibitor of the intoxication process. Cycloheximide is shown to inhibit the toxin catalyzed ADP-ribosylation of elongation factor 2 (EF-2). The inhibition is blocked by puromycin thus establishing the ribosome as the location of cycloheximide protection. Washing cells free of cycloheximide rapidly reverses the protective effect. The initial rates of protein synthesis inhibition observed after removal of cycloheximide from DT-intoxicated cells are 5 to 12-fold greater than rates observed in unprotected cells and are shown to reflect ADP-ribosylation of EF-2 by cytosolic DT. Ten to thirty minutes after cycloheximide removal, the rate of protein synthesis inhibition abruptly changes to values identical to those of unprotected cells. Both the initial rates and extent of the initial rapid inactivation are directly related to toxin concentration and time of incubation with DT in the presence of cycloheximide. We concluded that: the rate-limiting step in protein synthesis inhibition by DT is not the ADP-ribosylation of EF-2 by cytosolic toxin but rather the earlier entry step of DT into the cytosol. DT enters the cytosol as a bolus of sufficient size to rapidly inactivate all EF-2 in that cell. It is inferred from 1 and 2 that the first order inactivation rate exhibited by DT is the result of the probability of the release of a bolus of toxin to the cytosol of any cell in the population per unit time. Autoradiographic analysis of intoxicated cell populations support this two-population state model. The size of a single bolus or quantum of DT is calculated from data over the range of 10(-11) to 10(-9) M DT and is found to remain constant. We suggest that the cytosolic entry mechanism of DT results from a unique ability of the internalized toxin molecules to destabilize the vesicular membrane resulting in a random release of a bolus of toxin into the cytosol. Because the bolus size remains constant over a 50-fold change in receptor occupancy the possibility is raised that DT undergoes a post-receptor packaging process, package size remaining a constant and package number increasing with receptor occupancy.     

Interaction of diphtheria toxin B subunit with sensitive and insensitive mammalian cells

The recombinant fluorescent derivative of diphtheria toxin (EGFP-SbB) obtained by the replacement of toxin A subunit by enhanced green fluorescent protein (EGFP) has been used for visualization of the interaction of diphtheria toxin (DT) with sensitive and insensitive cells. It was shown that EGFP-SbB could interact with cell surface of both toxin-sensitive monkey cells (Vero cell line) and toxin-resistant mouse cells (3T3 cell line). The affinity of this protein for receptors of Vero cells was three times higher as compared with 3T3 cells. It was demonstrated that fluorescent derivate was able to interact with receptors of both cell lines and to internalize into these cells. Internalization of EGFP-SbB into the cells was inhibited by endocytosis inhibitor phenyl arsine oxide. We suppose that diverse sensitivity to DT of monkey and mouse cells can be explained not only by differences in their receptor affinity for DT but also by the processes that occur after internalization of the toxin into the cells.     

Specific chemical cleavage of diphtheria toxin with hydroxylamine. Purification and characterization of the modified proteins

Specific chemical cleavage of diphtheria toxin with hydroxylamine was performed to remove peptides of 10 and 7 kDa from the carboxyl terminus. The resulting modified proteins of 51 and 48 kDa (HA51DT and HA48DT, respectively) were purified and characterized with respect to structural and biological properties. The 51-kDa toxin binds to ATP-agarose, as does intact diphtheria toxin, while HA48DT does not bind to the nucleotide matrix. Neither modified toxin binds to the membranes of diptheria toxin-sensitive cells, and, consequently, neither is toxic. However, when covalently linked to a membrane binding moiety, both HA51DT and HA48DT are toxic. Cell-killing ability during a short exposure time indicated that concanavalin A (Con A) derivatives of diphtheria toxin and HA51DT are equally toxic, ConA HA48DT being somewhat less toxic, while the conjugate of ConA to A-chain kills a small number of cells only at inordinately high concentration (1 microM). We have thus separated the cell membrane binding function of diphtheria toxin from its membrane permeation function by removing specific small peptides from the carboxyl terminus. These modified toxins may have applications in the preparation of highly potent hybrid toxins.     

Fusion Protein of Interleukin 4 and Diphtherial Toxin with High Cytotoxicity to Cancer Cells

One means to improve the selectivity of cancer therapyis by directing foreign protein with activity againsttherapeutic targets that displays different expression levelon malignant cells from normal cells. There have hadsubstantial efforts to rationally de…     

Fusion protein of interleukin 4 and diphtherial toxin with high cytotoxicity to cancer cells

Receptor of human interleukin 4 (hlL4R) has been found to be present on many types of cancer, so it may be a good target for cancer therapy. Here, fusion toxin gene DT4H has been constructed by fusing DNA sequence encoding the first 389 amino acids of diphtherial toxin (DT), which can not bind its own receptor, to human interleukin 4 (hIL4) gene. In order to improve the affinity of fusion toxin for hIL4R,a circularly permuted form of hIL4 (cpIL4) was used. The fusion gene was expressed in Escherichia coli where the fusion toxin DT4H was highly expressed. Purified DT4H was very cytotoxic to cancer cell line U251 cells, and moderate cytotoxic to HepG2 and MCF-7 cells. SGC-7901 cells were insensitive to it. The cytotoxic action of DT4H was specific because it was blocked by excess hIL4. These results suggest that DT4H may be a useful agent in the treatment of certain malignancies.     

A genetic method to quantitate induced chromosome breaks using a mouse/human monochromosomal hybrid cell line: identification of potential clastogenic agents

A genetic assay to detect the clastogenic potential of environmental agents is described. This assay is based on the cloning efficiency of cells in a medium that permits the growth of cells following loss of a specific chromosome segment resulting from a chromosome break. For this purpose a mouse/human hybrid cell line R12-2 containing a dominantly marked chromosome 5 as the only human component has been constructed. This chromosome 5 carries two dominant selectable markers, Ecogpt and the gene for sensitivity to diphtheria toxin (DTs). Ecogpt codes for the enzyme xanthine-guanine phosphoribosyltransferase which allows selection for cells containing chromosome 5 or the segment carrying Ecogpt as judged by growth in medium supplemented with mycophenolic acid and xanthine (MX medium). Human cells are sensitive to 10(-13) M DT, whereas mouse cells are resistant to 10(-7) M DT and DTs is expressed as a dominant phenotype. Cultivation of R12-2 cells in the medium containing 10(-13) M DT permit the selection of cells that have lost chromosome 5 or the segment carrying DTs. The presence of two selectable markers on the same chromosome permits the identification and quantitation of cells for the selective loss of a specific chromosome segment. Growth of R12-2 cells in MX medium containing 10(-13) M DT therefore, provides a convenient method to determine the frequency of clastogen induced breaks in chromosome 5. The utility of the proposed genetic assay is assessed using X-irradiation as a model clastogen. Our results clearly show a dose related response that is consistent with cytogenetic observations.     

Diphtheria toxin receptor sites on membranes of cultured cells and erythrocytes demonstrated by fluorescence and electron microscopy

Diphtheria toxin (DT) receptor sites were identified on cell membranes by indirect immunofluorescence and immunoferritin labelling. The results obtained were correlated (1) with the binding of 125I-labelled DT (to the very same cell populations used in the ultrastructural studies), and (2) with toxin sensitivity as shown by inhibition of 14C-amino acid incorporation into protein. Binding and immunocytochemical studies were carried out at 0 degrees C to preclude internalization of DT. Receptor sites for DT were observed on functionally responsive, cultured human diploid (BUD-8) fibroblasts as well as on a small fraction of erythrocytes (RBC) of DT-susceptible species, i.e. human and sheep. On all BUD-8 cell sections DT receptor sites were located in annular patches and not on microvilli as had been observed in the KB epithelial cell line. Immunoferritin label was revealed on only 10% +/- 2.5 (SD) of human RBC in discreet loci along the cell membrane. Specificity of binding of DT to human fibroblast and RBC receptor sites was further demonstrated by competition between 125I-labeled and non-labelled DT. There was no specific binding to DT-treated cultured mouse L929 fibroblasts or to RBC of mouse of rat, species resistant to the toxin. Thus, direct ultrastructural immunocytochemical evidence is provided for the presence of specific cellular DT receptors on cultured cells and erythrocytes of species sensitive to DT, but not on those of species resistant to DT.     

Transgenic mice expressing a fully nontoxic diphtheria toxin mutant, not CRM197 mutant, acquire immune tolerance against diphtheria toxin

We previously developed a method termed "toxin receptor-mediated cell knockout" (TRECK). By the TRECK method, a single or repeated shot(s) of diphtheria toxin (DT) conditionally ablates a specific cell population from transgenic mice expressing the DT receptor transgene under the control of a cell type-specific promoter. In some cases of TRECK, frequent and high-dose administration of DT is required, raising the concern that these frequent injections of DT could cause production of anti-DT antibody, which would neutralize further DT administration. To solve this problem, we aimed to generate transgenic mice genetically expressing a nontoxic DT mutant, with the expectation that they may naturally acquire immune tolerance to DT. Unexpectedly, the G52E DT mutant, which is well known as the nontoxic DT variant cross reacting material 197 (CRM197), exhibited cytotoxicity in yeast and mammalian cells. Cytotoxicity of CRM197 was abrogated in cells mutated for elongation factor 2 (EF-2), indicating that CRM197 exerts its toxic effects through EF-2, similar to wild-type DT. On the other hand, the K51E/E148K DT mutant exhibited no detectable cytotoxicity. This led us to successfully obtain DT gene transgenic mice, which exhibited no histological abnormalities, and indeed acquired immune tolerance to DT.     

Targeting head and neck squamous cell carcinoma using a novel fusion toxin-diphtheria toxin/HN-1

The current treatment strategies, chemotherapy and radiation therapy being used for the management of cancer are deficient in targeted approach leading to treatment related toxicities and relapse. Contrarily, fusion toxins exhibit remarkable tumor specificity thus emerging as an alternative therapy for the treatment of cancer. Diphtheria toxin-HN-1 peptide (DT/HN-1) is a fusion toxin designed to target the head and neck squamous cell carcinoma (HNSCC). The aim of this study was to construct, characterize, and evaluate the cytotoxicity and specificity of DT/HN-1 fusion toxin against the HNSCC cells. The purified DT/HN-1 fusion toxin was characterized by SDS-PAGE and western blotting. Refolding of purified fusion toxins was monitored by fluorescence spectra and circular dichroism spectra. The activity of DT/HN-1 fusion toxin was demonstrated on various HNSCC cell lines by cell viability assay, cell proliferation assay, protein synthesis inhibition assay, apoptosis and cell cycle analysis. The fusion toxin DT/HN-1 demonstrated remarkably high degree of cytotoxicity specific to the HNSCC cells. The IC₅₀ of DT/HN-1 fusion toxin was ~1 to 5 nM in all the three HNSCC cell lines. The percentage apoptotic cells in DT/HN-1 treated UMB-SCC-745 cells are 16% compared to 4% in untreated. To further demonstrate the specific toxicity of DT/HN-1 fusion toxin towards the HNSCC cells we constructed, characterized and evaluated the efficacy of DT protein. The DT protein coding for only a fragment of diphtheria toxin without its native receptor binding domain failed to exhibit any cytotoxicity on all the cell lines used in this study thus establishing the importance of a ligand in achieving targeted toxicity. To evaluate the translocation ability of HN-1 peptide, an additional construct DTΔT/HN-1 was constructed, characterized and evaluated for its cytotoxic activity. The fusion toxin DTΔT/HN-1 deficient of the translocation domain of diphtheria toxin showed no cytotoxicity on all the cell lines clearly indicating the inability of HN-1 peptide to translocate catalytic domain of the toxin into the cytosol.     

Targeting glioblastoma multiforme with an IL-13/diphtheria toxin fusion protein in vitro and in vivo in nude mice

Fusion proteins composed of tumor binding agents and potent catalytic toxins show promise for intracranial therapy of brain cancer and an advantage over systemic therapy. Glioblastoma multiforme (GBM) is the most common form of brain cancer and overexpresses IL-13R. Thus, we developed an interleukin-13 receptor targeting fusion protein, DT(390)IL13, composed of human interleukin-13 and the first 389 amino acids of diphtheria toxin. To measure its ability to inhibit GBM, DT(390)IL13 was tested in vitro and found to inhibit selectively the U373 MG GBM cell line with an IC(50) around 12 pmol/l. Cytotoxicity was neutralized by anti-human-interleukin-13 antibody, but not by control antibodies. In vivo, small U373 MG glioblastoma xenografts in nude mice completely regressed in most animals after five intratumoral injections of 1 microg of DT(390)IL13 q.o.d., but not by the control fusion protein DT(390)IL-2. DT(390)IL13 was also tested against primary explant GBM cells of a patient's excised tumor and the IC(50) was similar to that measured for U373 MG. Further studies showed a therapeutic window for DT(390)IL13 of 1-30 microg/injection and histology studies and enzyme measurements showed that the maximum tolerated dose of DT(390)IL13 had little effect on kidney, liver, spleen, lung and heart in non-tumor-bearing immunocompetent mice. Together, these data suggest that DT(390)IL13 may provide an important, alternative therapy for brain cancer.     

Lineage-specific cell disruption in living mice by Cre-mediated expression of diphtheria toxin A chain

We have established a transgenic mouse line in which floxed neomycin resistant cassette was inserted between the CAG promoter and EGFP. When these transgenic mice were mated with Cre-expressing transgenic animals, the offspring obtained were fluorescent green. We then established a transgenic mouse line in which EGFP in the above construct was replaced by diphtheria toxin A chain (DT). When the latter transgenic mice were mated with mice expressing Cre restricted to germ cells, we obtained healthy but sterile offspring due to a disruption of germ line cells by DT expression. We predict that this strategy will be useful for the construction of new animal models for human diseases, featuring a variety of missing cell lineages produced by disruption with DT.     

Control of cellular phosphatidylinositol 4,5-bisphosphate levels by adhesion signals and rho GTPases in NIH 3T3 fibroblasts involvement of both phosphatidylinositol-4-phosphate 5-kinase and phospholipase C.

The involvement of small GTPases of the Rho family in the control of phosphoinositide metabolism by adhesion signals was examined in NIH 3T3 fibroblasts. Abrogation of adhesion signals by detachment of cells from their substratum resulted in a time-dependent decrease in the cellular level of PtdIns(4,5)P2 by approximately 50%. This effect could be mimicked by treatment of adherent cells with Clostridium difficile toxin B and toxin B-1470, which inhibit specific subsets of Rho and Ras GTPases. Detachment of cells that had been pretreated with the clostridial toxins did not cause a further reduction in PtdIns(4,5)P2 levels, suggesting that the target GTPases are integrated into the control of phosphoinositide levels by adhesion signals. The reduction in PtdIns(4,5)P2 levels could be attributed to reduced activity of the major PtdIns(4, 5)P2-producing enzyme, PtdIns4P 5-kinase. Unexpectedly, both cell detachment and toxin treatment resulted in a twofold to threefold increase in inositol phosphate production in intact cells. In lysates of these cells, in vitro phospholipase C activity was found to be elevated by 30-50%. The effects of cell detachment and toxin treatment on inositol phosphate formation could be mimicked by expression of dominant-negative N17 Rac1. Taken together, these data suggest that adhesion-controlled small GTPases of the Rho family are involved in the regulation of the cellular PtdIns(4,5)P2 levels in NIH 3T3 fibroblasts, by controlling the activities of both PtdIns4P 5-kinase and phospholipase C.     

Increase in Gs and cyclic AMP generation in HIT cells. Evidence that the 45-kDa alpha-subunit of Gs has greater functional activity than the 52-kDa alpha-subunit

Cyclic AMP accumulation in response to forskolin, cholera toxin, or isoproterenol is dramatically increased in HIT T-15 cells, a clonal cell line of Syrian hamster pancreatic islet beta cells, as a function of passage number. Forskolin and cholera toxin elevate cyclic AMP levels 5- to 10-fold higher in later passages (87-100) than in earlier passages (70-80). A similar phenomenon is observed with isoproterenol (10 microM) which increases cyclic AMP levels 56-fold in older HIT cells (passage 94), whereas only marginally stimulating cyclic AMP production in younger cells (passage 70-82). To determine whether a change in the stimulatory or inhibitory guanine nucleotide regulatory proteins, Gs or Gi, was responsible for these observations, ADP-ribosylation of HIT cell membranes with cholera toxin and pertussis toxin was examined. All passages contained two cholera toxin substrates at 52 and 45 kDa. The amount of 52 kDa did not appear to change with passage number, but the amount of 45 kDa increased in the later passages (89 and 94). The ratio of 45 to 52 kDa cholera toxin substrate, as determined by densitometric analysis, increased from 0.1 in passages 70, 75, and 82 to 0.45 at passage 89. No passage related changes in a 40-kDa pertussis toxin substrate were observed. An increase in the amount of the 45-kDa alpha-subunit of Gs was confirmed on immunoblots using antisera specific for the alpha-subunits of Gs. The amount of functional Gs present in various HIT cell passages was examined by determining the extent to which extracts from HIT cell membranes reconstituted guanine nucleotide-sensitive adenylyl cyclase in S49 cyc- membranes. Extracts derived from passage 94 reconstituted three to four times more adenylyl cyclase activity in cyc- membranes than extracts from passages 70, 75, and 82. These data indicate that an increase in functional Gs in later passages may be the underlying cause for the increased responsiveness to isoproterenol and forskolin in later passages. These data also suggest that functional differences exist between the Gs alpha-subunits, with the smaller 45-kDa subunit being more efficacious in coupling to cyclic AMP synthesis than the larger 52-kDa subunit. This is a departure from the commonly held view that the two subunits have similar efficacies in stimulating adenylyl cyclase.     

Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor.

A monkey cDNA (pDTS) encoding a diphtheria toxin (DT) sensitivity determinant was isolated by expression cloning in mouse L-M cells. Mouse cells are naturally resistant to DT, because they lack functional cell surface receptors for the toxin. Unlike wild-type L-M cells, pDTS-transfected mouse cells are extremely toxin sensitive and specifically bind radioiodinated DT. Intoxication of the transfected cells requires receptor-mediated endocytosis of the bound toxin. The cDNA is predicted to encode an integral membrane protein that is identical to the precursor of a heparin-binding EGF-like growth factor. The DT sensitivity protein is thus a growth factor precursor that DT exploits as a receptor.     

Effect of diphtheria toxin on the viability of phagocytes and B-lymphocytes in animals sensitive and insensitive to it

It is well known, that mechanism of diphtheria toxin (DT) action triggers only if toxin penetrates into acid endosome after binding with specific receptor--heparin-binding epidermal grows factor like grows factor (HB-EGF) on the cell surface. We have suggested that DT is capable to penetrate either into B-lymphocytes, which have specific immunoglobulin receptors for DT or into phagocytes, which are able to phagocytosis of DT, because in both of these cases toxin get in endosome with conditions suitable for its activation. To check this hypothesis the comparative studies with insensitive to DT mice lacking specific receptor for DT, and with sensitive to DT guinea pigs were performed. Influence of DT on vitality of phagocytes and B-cells with different specificity from mice and guinea pigs was studied. B-cells were obtained from animals immunized by control antigen--ovalbumine and recombinant diphtheria toxoid--DT without N-terminal 28 aminoacid residues responsible for toxic effect. The results obtained have showed that DT can penetrate into phagocytes and B-cells specific to DT and kill these cells even if they lack classic receptor for DT. This fact evidences that DT is potentially able to inhibit self-directed antibody response and keep from participation of phagocytes in the protection of organism from infection.     

Nasal cholera toxin elicits IL-5 and IL-5 receptor alpha-chain expressing B-1a B cells for innate mucosal IgA antibody responses

In this study, we examine whether native cholera toxin (nCT) as a mucosal adjuvant can support trinitrophenyl (TNP)-LPS-specific mucosal immune responses. C57BL/6 mice were given nasal TNP-LPS in the presence or absence of nCT. Five days later, significantly higher levels of TNP-specific mucosal IgA Ab responses were induced in the nasal washes, saliva, and plasma of mice given nCT plus TNP-LPS than in those given TNP-LPS alone. High numbers of TNP-specific IgA Ab-forming cells were also detected in mucosal tissues such as the nasal passages (NPs), the submandibular glands (SMGs), and nasopharyngeal-associated lymphoreticular tissue of mice given nCT. Flow cytometric analysis showed that higher numbers of surface IgA+, CD5+ B cells (B-1a B cells) in SMGs and NPs of mice given nasal TNP-LPS plus nCT than in those given TNP-LPS alone. Furthermore, increased levels of IL-5R alpha-chain were expressed by B-1a B cells in SMGs and NPs of mice given nasal TNP-LPS plus nCT. Thus, CD4+ T cells from these mucosal effector lymphoid tissues produce high levels of IL-5 at both protein and mRNA levels. When mice were treated with anti-IL-5 mAb, significant reductions in TNP-specific mucosal IgA Ab responses were noted in external secretions. These findings show that nasal nCT as an adjuvant enhances mucosal immune responses to a T cell-independent Ag due to the cross-talk between IL-5Ralpha+ B-1a B cells and IL-5-producing CD4+ T cells in the mucosal effector lymphoid tissues.     

Selective depletion of mouse kidney proximal straight tubule cells causes acute kidney injury

The proximal straight tubule (S3 segment) of the kidney is highly susceptible to ischemia and toxic insults but has a remarkable capacity to repair its structure and function. In response to such injuries, complex processes take place to regenerate the epithelial cells of the S3 segment; however, the precise molecular mechanisms of this regeneration are still being investigated. By applying the ??toxin receptor mediated cell knockout?? method under the control of the S3 segment-specific promoter/enhancer, Gsl5, which drives core 2 ??-1,6-N-acetylglucosaminyltransferase gene expression, we established a transgenic mouse line expressing the human diphtheria toxin (DT) receptor only in the S3 segment. The administration of DT to these transgenic mice caused the selective ablation of S3 segment cells in a dose-dependent manner, and transgenic mice exhibited polyuria containing serum albumin and subsequently developed oliguria. An increase in the concentration of blood urea nitrogen was also observed, and the peak BUN levels occurred 3?C7?days after DT administration. Histological analysis revealed that the most severe injury occurred in the S3 segments of the proximal tubule, in which tubular cells were exfoliated into the tubular lumen. In addition, aquaporin 7, which is localized exclusively to the S3 segment, was diminished. These results indicate that this transgenic mouse can suffer acute kidney injury (AKI) caused by S3 segment-specific damage after DT administration. This transgenic line offers an excellent model to uncover the mechanisms of AKI and its rapid recovery.     

The 27-kD diphtheria toxin receptor-associated protein (DRAP27) from vero cells is the monkey homologue of human CD9 antigen: expression of DRAP27 elevates the number of diphtheria toxin receptors on toxin- sensitive cells

Diphtheria toxin (DT) receptor associates with a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A human-mouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.