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. 1984 Feb 1;98(2):466–472. doi: 10.1083/jcb.98.2.466

Entry of diphtheria toxin into cells: possible existence of cellular factor(s) for entry of diphtheria toxin into cells was studied in somatic cell hybrids and hybrid toxins

PMCID: PMC2113112  PMID: 6693491

Abstract

Ehrlich ascites tumor cells were found to be very insensitive to diphtheria toxin. We formed 37 hybrids from Ehrlich tumor cells and diphtheria toxin-sensitive human fibroblasts. The effects of diphtheria toxin on protein synthesis in those hybrids were examined. The hybrids were divided into three groups on the basis of toxin sensitivity. Group A hybrids were as sensitive to diphtheria toxin as human fibroblasts, Group C were as resistant as Ehrlich tumor cells, and Group B had intermediate sensitivity. Group A hybrids had diphtheria toxin-binding sites but Group B and C had no detectable binding sites. Elongation factor-2 of all the hybrids was susceptible to ADP-ribosylation by fragment A of diphtheria toxin. Cells of Group A and B became more sensitive to CRM 45 (cross-reacting material 45 of diphtheria toxin) after they were exposed to low pH (pH = 4.5). The resistance of Group C to CRM 45 was not affected by the same treatment. Group A and B hybrids and human fibroblasts had similar sensitivities to a hybrid toxin composed of wheat germ agglutinin and fragment A of diphtheria toxin, but Group C and Ehrlich tumor cells were resistant to this hybrid toxin. All the hybrids and Ehrlich tumor cells were more sensitive to a hybrid toxin composed of wheat germ agglutinin and subunit A of ricin than were human fibroblasts. On subcloning of Group B hybrids, one Group C hybrid was obtained, but no Group A hybrid. These facts suggest that Ehrlich ascites tumor cells differ from human fibroblasts in the expression of a factor(s) that is involved in entry of fragment A of diphtheria toxin into the cytoplasm after the toxin binds to its surface receptors.

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Selected References

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  1. Boquet P., Silverman M. S., Pappenheimer A. M., Jr, Vernon W. B. Binding of triton X-100 to diphtheria toxin, crossreacting material 45, and their fragments. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4449–4453. doi: 10.1073/pnas.73.12.4449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cawley D. B., Herschman H. R., Gilliland D. G., Collier R. J. Epidermal growth factor-toxin A chain conjugates: EGF-ricin A is a potent toxin while EGF-diphtheria fragment A is nontoxic. Cell. 1980 Nov;22(2 Pt 2):563–570. doi: 10.1016/0092-8674(80)90366-9. [DOI] [PubMed] [Google Scholar]
  3. Creagan R. P., Chen S., Ruddle F. H. Genetic analysis of the cell surface: association of human chromosome 5 with sensitivity to diphtheria toxin in mouse-human somatic cell hybrids. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2237–2241. doi: 10.1073/pnas.72.6.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DeLuca C., Brown J. A., Shows T. B. Lysosomal arylsulfatase deficiencies in humans: chromosome assignments for arylsulfatase A and B. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1957–1961. doi: 10.1073/pnas.76.4.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Didsbury J. R., Moehring J. M., Moehring T. J. Binding and uptake of diphtheria toxin by toxin-resistant Chinese hamster ovary and mouse cells. Mol Cell Biol. 1983 Jul;3(7):1283–1294. doi: 10.1128/mcb.3.7.1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Donovan J. J., Simon M. I., Draper R. K., Montal M. Diphtheria toxin forms transmembrane channels in planar lipid bilayers. Proc Natl Acad Sci U S A. 1981 Jan;78(1):172–176. doi: 10.1073/pnas.78.1.172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Draper R. K., Chin D., Eurey-Owens D., Scheffler I. E., Simon M. I. Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin. J Cell Biol. 1979 Oct;83(1):116–125. doi: 10.1083/jcb.83.1.116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gill D. M., Pappenheimer A. M., Jr, Brown R., Kurnick J. T. Studies on the mode of action of diphtheria toxin. VII. Toxin-stimulated hydrolysis of nicotinamide adenine dinucleotide in mammalian cell extracts. J Exp Med. 1969 Jan 1;129(1):1–21. doi: 10.1084/jem.129.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hayakawa S., Uchida T., Mekada E., Moynihan M. R., Okada Y. Monoclonal antibody against diphtheria toxin. Effect on toxin binding and entry into cells. J Biol Chem. 1983 Apr 10;258(7):4311–4317. [PubMed] [Google Scholar]
  10. Honjo T., Nishizuka Y., Hayaishi O. Diphtheria toxin-dependent adenosine diphosphate ribosylation of aminoacyl transferase II and inhibition of protein synthesis. J Biol Chem. 1968 Jun 25;243(12):3553–3555. [PubMed] [Google Scholar]
  11. Iglewski B. H., Kabat D. NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin,. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2284–2288. doi: 10.1073/pnas.72.6.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Keen J. H., Maxfield F. R., Hardegree M. C., Habig W. H. Receptor-mediated endocytosis of diphtheria toxin by cells in culture. Proc Natl Acad Sci U S A. 1982 May;79(9):2912–2916. doi: 10.1073/pnas.79.9.2912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kim K., Groman N. B. In vitro inhibition of diphtheria toxin action by ammonium salts and amines. J Bacteriol. 1965 Dec;90(6):1552–1556. doi: 10.1128/jb.90.6.1552-1556.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Leppla S., Dorland R. B., Middlebrook J. L. Inhibition of diphtheria toxin degradation and cytotoxic action by chloroquine. J Biol Chem. 1980 Mar 25;255(6):2247–2250. [PubMed] [Google Scholar]
  15. Mekada E., Uchida T., Okada Y. Methylamine stimulates the action of ricin toxin but inhibits that of diphtheria toxin. J Biol Chem. 1981 Feb 10;256(3):1225–1228. [PubMed] [Google Scholar]
  16. Mekada E., Uchida T., Okada Y. Modification of the cell surface with neuraminidase increases the sensitivities of cells to diphtheria toxin and Pseudomonas aeruginosa exotoxin. Exp Cell Res. 1979 Oct 1;123(1):137–146. doi: 10.1016/0014-4827(79)90430-0. [DOI] [PubMed] [Google Scholar]
  17. Middlebrook J. L., Dorland R. B. Response of cultured mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae: differential cytotoxicity. Can J Microbiol. 1977 Feb;23(2):183–189. doi: 10.1139/m77-026. [DOI] [PubMed] [Google Scholar]
  18. Miyake Y., Kim J., Okada Y. Effects of cytochalasin D on fusion of cells by HVJ (Sendai virus). Exp Cell Res. 1978 Oct 1;116(1):167–178. doi: 10.1016/0014-4827(78)90073-3. [DOI] [PubMed] [Google Scholar]
  19. Moehring J. M., Moehring T. J. Characterization of the diphtheria toxin-resistance system in Chinese hamster ovary cells. Somatic Cell Genet. 1979 Jul;5(4):453–468. doi: 10.1007/BF01538880. [DOI] [PubMed] [Google Scholar]
  20. Nicolson G. L., Blaustein J. The interaction of Ricinus communis agglutinin with normal and tumor cell surfaces. Biochim Biophys Acta. 1972 May 9;266(2):543–547. doi: 10.1016/0005-2736(72)90109-5. [DOI] [PubMed] [Google Scholar]
  21. Okada Y., Murayama F. Requirement of calcium ions for the cell fusion reaction of animal cells by HVJ. Exp Cell Res. 1966 Nov-Dec;44(2):527–551. doi: 10.1016/0014-4827(66)90458-7. [DOI] [PubMed] [Google Scholar]
  22. Olsnes S., Refsnes K., Pihl A. Mechanism of action of the toxic lectins abrin and ricin. Nature. 1974 Jun 14;249(458):627–631. doi: 10.1038/249627a0. [DOI] [PubMed] [Google Scholar]
  23. Robbins A. R., Peng S. S., Marshall J. L. Mutant Chinese hamster ovary cells pleiotropically defective in receptor-mediated endocytosis. J Cell Biol. 1983 Apr;96(4):1064–1071. doi: 10.1083/jcb.96.4.1064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Roth J. Methods for assessing immunologic and biologic properties of iodinated peptide hormones. Methods Enzymol. 1975;37:223–233. doi: 10.1016/s0076-6879(75)37018-3. [DOI] [PubMed] [Google Scholar]
  25. Sandvig K., Olsnes S. Diphtheria toxin entry into cells is facilitated by low pH. J Cell Biol. 1980 Dec;87(3 Pt 1):828–832. doi: 10.1083/jcb.87.3.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tanaka K., Sekiguchi M., Okada Y. Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus). Proc Natl Acad Sci U S A. 1975 Oct;72(10):4071–4075. doi: 10.1073/pnas.72.10.4071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Uchida T., Gill D. M., Pappenheimer A. M., Jr Mutation in the structural gene for diphtheria toxin carried by temperate phage . Nat New Biol. 1971 Sep 1;233(35):8–11. doi: 10.1038/newbio233008a0. [DOI] [PubMed] [Google Scholar]
  28. Uchida T., Kim J., Yamaizumi M., Miyake Y., Okada Y. Reconstitution of lipid vesicles associated with HVJ (Sendai virus) sikes. Purification and some properties of vesicles containing nontoxic fragment A of diphtheria toxin. J Cell Biol. 1979 Jan;80(1):10–20. doi: 10.1083/jcb.80.1.10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Uchida T., Pappenheimer A. M., Jr, Greany R. Diphtheria toxin and related proteins. I. Isolation and properties of mutant proteins serologically related to diphtheria toxin. J Biol Chem. 1973 Jun 10;248(11):3838–3844. [PubMed] [Google Scholar]
  30. Yamaizumi M., Uchida T., Okada Y. Macromolecules can penetrate the host cell membrane during the early period of incubation with HVJ (Sendai virus). Virology. 1979 May;95(1):218–221. doi: 10.1016/0042-6822(79)90418-5. [DOI] [PubMed] [Google Scholar]
  31. van Someren H., Beijersbergen van Henegouwen H., Los W., Wurzer-Figurelli E., Doppert B., Vervloet M., Meera Khan P. Enzyme electrophoresis on cellulose acetate gel. II. Zymogram patterns in man-Chinese hamster somatic cell hybrids. Humangenetik. 1974;25(3):189–201. doi: 10.1007/BF00281426. [DOI] [PubMed] [Google Scholar]

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