Functional domains of pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli

Hwang, Jaulang; FitzGerald, David J.; Pastan, Ira

doi:10.1016/0092-8674(87)90363-1

Cited by 411 publications

(244 citation statements)

References 21 publications

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“…To measure the enzymatic activity of DTLIL3, the ADP ribosylation assay modified from Collier and Kandel 18 by Hwang and colleagues 19 was employed. 18,19 Varying amounts of transferrin-CRM107 20 or DTLIL3 in 480 l DTEB buffer consisting of 0.2% BSA, 40 mM DTT, 1 mM EDTA, and 50 mM Tris pH 8.0 were mixed with 10 l 14 C-NAD (NEN-DuPont, NEC743, 588 Ci/mmol, 10 Ci/ml) and 10 l wheat germ extract (Promega, L418A). Samples were incubated at 37°C for 30 min, and protein precipitated for 15 min with 4°C 0.5 ml 12% TCA.…”

Section: Characterization Of Dtlil3mentioning

confidence: 99%

Diphtheria toxin fused to human interleukin-3 is toxic to blasts from patients with myeloid leukemias

et al. 2000

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Leukemic blasts from patients with acute phase chronic myeloid leukemic and refractory acute myeloid leukemia are highly resistant to a number of cytotoxic drugs. To overcome multi-drug resistance, we engineered a diphtheria fusion protein by fusing human interleukin-3 (IL3) to a truncated form of diphtheria toxin (DT) with a (G 4 S) 2 linker (L), expressed and purified the recombinant protein, and tested the cytotoxicity of the DTLIL3 molecule on human leukemias and normal progenitors. The DTLIL3 construct was more cytotoxic to interleukin-3 receptor (IL3R) bearing human myeloid leukemia cell lines than receptor-negative cell lines based on assays of cytotoxicity using thymidine incorporation, growth in semi-solid medium and induction of apoptosis. Exposure of mononuclear cells to 680 pM DTLIL3 for 48 h in culture reduced the number of cells capable of forming colonies in semi-solid medium (colony-forming units leukemia) у10-fold in 4/11 (36%) patients with myeloid acute phase chronic myeloid leukemia (CML) and 3/9 (33%) patients with acute myeloid leukemia (AML). Normal myeloid progenitors (colony-forming unit granulocytemacrophage) from five different donors treated and assayed under identical conditions showed intermediate sensitivity with three-to five-fold reductions in colonies. The sensitivity to DTLIL3 of leukemic progenitors from a number of acute phase CML patients suggests that this agent could have therapeutic potential for some patients with this disease. Leukemia (2000) 14, 576-585.

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Section: Characterization Of Dtlil3mentioning

confidence: 99%

Diphtheria toxin fused to human interleukin-3 is toxic to blasts from patients with myeloid leukemias

et al. 2000

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“…It inhibits protein synthesis by the ADP ribosylation of elongation factor 2 (EF2) (6). PE38, the fragment of PE used to make LMB-2, is missing the cell blinding domain of PE, and therefore should not be toxic to liver cells.…”

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confidence: 99%

Inhibition of TNF-α Produced by Kupffer Cells Protects Against the Nonspecific Liver Toxicity of Immunotoxin Anti-Tac(Fv)-PE38, LMB-2

Onda

Willingham

Wang

et al. 2000

The Journal of Immunology

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LMB-2 (anti-Tac(Fv)-PE38) is a recombinant immunotoxin composed of the Fv fragment of the anti-Tac Ab fused to a 38-kDa form of Pseudomonas exotoxin A. Recent clinical trials showed that LMB-2 is a promising agent for the treatment of patients with Tac-positive leukemia or lymphoma. One major side effect that needs to be overcome is nonspecific liver toxicity. In the current study, we have analyzed the mechanism of this toxicity using a mouse model. Mice that were injected with a lethal dose of LMB-2 showed severe hepatic necrosis. Immunohistochemistry revealed that LMB-2 accumulated in Kupffer cells in the liver, suggesting that the damage to the hepatocytes was indirect. When we examined the effects of LMB-2 on peritoneal macrophages, cells in the same lineage as Kupffer cells, we found that LMB-2 induced the production of TNF-α by these cells. Following LMB-2 administration to mice, the levels of TNF-α in the liver increased to very high levels, whereas the rise in serum levels was modest. In addition, the LMB-2-induced liver toxicity was blocked by a specific TNF binding protein (TNFsRp55). Liver toxicity was also blocked by indomethacin, which also blocked the rise of TNF-α in the liver. Both TNFsRp55 and indomethacin treatment protected mice against a lethal dose of LMB-2. These data indicate that TNF-α produced in the liver by Kupffer cells has an important causal role in the nonspecific liver toxicity of LMB-2. These findings have important clinical implications for the use of immunotoxins in the therapy of patients with cancer.

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“…6 The 66 kDa exotoxin A consists of three functional domains defined by crystal structure and deletion analysis. 8,9 Upon cell binding via the N-terminal cell recognition domain (Ia) and receptor-mediated internalization, ETA is cleaved by a cellular protease within the internal domain II (translocation domain) resulting in an N-terminal 28 kDa and a C-terminal 37 kDa fragment. After reduction of a disulfide bond, the C-terminal fragment containing the enzymatic domain III translocates to the cytoplasm where it ADP-ribosylates and inactivates eukaryotic elongation factor 2.…”

Section: Introductionmentioning

confidence: 99%

A chimeric fusion protein containing transforming growth factor-α mediates gene transfer via binding to the EGF receptor

1998

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Fusion proteins engineered to incorporate distinct functions binding to EGF receptors. Complexes of the chimeric prowhich co-operate in mediating the cell-type specific uptake tein and plasmid DNA carrying a luciferase reporter gene, and intracellular delivery of DNA present an attractive after condensation with poly-L-lysine resulted in an up to approach for the development of self-assembling vector 150-fold increase in reporter gene expression in EGF systems for targeted gene transfer. Here we have chosen receptor expressing cells in comparison to poly-L-lysinethe EGF receptor overexpressed in many human tumors DNA complexes alone. While in COS-1 cells no of epithelial origin as a target for a novel modular fusion additional endosome escape activity was required, in protein. We have fused a cDNA fragment of the human A431 cells gene delivery was dependent on the simul-EGF receptor ligand TGF-␣ to sequences encoding the taneous presence of the endosome destabilizing translocation domain of Pseudomonas exotoxin A as an reagent chloroquine indicating that cell-type specific facendosome escape activity, and the DNA-binding domain of tors such as different intracellular routing of protein-DNA the yeast GAL4 transcription factor. Upon bacterial complexes greatly influence transfection efficiency. expression, this TEG fusion protein displayed specific

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Functional domains of pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli

Cited by 411 publications

References 21 publications

Diphtheria toxin fused to human interleukin-3 is toxic to blasts from patients with myeloid leukemias

Diphtheria toxin fused to human interleukin-3 is toxic to blasts from patients with myeloid leukemias

Inhibition of TNF-α Produced by Kupffer Cells Protects Against the Nonspecific Liver Toxicity of Immunotoxin Anti-Tac(Fv)-PE38, LMB-2

A chimeric fusion protein containing transforming growth factor-α mediates gene transfer via binding to the EGF receptor

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