Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Nov;83(21):8258–8262. doi: 10.1073/pnas.83.21.8258

Genetic construction, expression, and melanoma-selective cytotoxicity of a diphtheria toxin-related alpha-melanocyte-stimulating hormone fusion protein.

J R Murphy, W Bishai, M Borowski, A Miyanohara, J Boyd, S Nagle
PMCID: PMC386907  PMID: 3095831

Abstract

The structural gene for diphtheria toxin, tox, has been modified at its Sph I site by the introduction of an oligonucleotide linker encoding a unique Pst I restriction endonuclease site and a synthetic oligonucleotide encoding alpha-melanocyte-stimulating hormone (alpha-MSH). The resulting fusion gene directs the expression of a diphtheria toxin-related alpha-MSH hybrid protein in which the diphtheria toxin receptor-binding domain has been replaced with alpha-MSH sequences. The chimeric toxin has been partially purified from periplasmic extracts of recombinant Escherichia coli K-12 and has been found to be selectively toxic for alpha-MSH receptor-positive human malignant melanoma NEL-M1 cells in vitro.

Full text

PDF
8259

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bacha P., Murphy J. R., Moynihan M. Toxicity of diphtheria toxin-related proteins produced by suppression of nonsense mutations. J Biol Chem. 1980 Nov 25;255(22):10658–10662. [PubMed] [Google Scholar]
  2. Bacha P., Murphy J. R., Reichlin S. Thyrotropin-releasing hormone-diphtheria toxin-related polypeptide conjugates. Potential role of the hydrophobic domain in toxin entry. J Biol Chem. 1983 Feb 10;258(3):1565–1570. [PubMed] [Google Scholar]
  3. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  4. Buck G. A., Groman N. B. Identification of deoxyribonucleic acid restriction fragments of beta-converting corynebacteriophages that carry the gene for diphtheria toxin. J Bacteriol. 1981 Oct;148(1):153–162. doi: 10.1128/jb.148.1.153-162.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Cawley D. B., Simpson D. L., Herschman H. R. Asialoglycoprotein receptor mediates the toxic effects of an asialofetuin-diphtheria toxin fragment A conjugate on cultured rat hepatocytes. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3383–3387. doi: 10.1073/pnas.78.6.3383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chang T. M., Dazord A., Neville D. M., Jr Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. II. Biochemical and biologic properties of diphtheria toxin fragment A-S-S-human placental lactogen. J Biol Chem. 1977 Feb 25;252(4):1515–1522. [PubMed] [Google Scholar]
  9. Costa J. J., Michel J. L., Rappuoli R., Murphy J. R. Restriction map of corynebacteriophages beta c and beta vir and physical localization of the diphtheria tox operon. J Bacteriol. 1981 Oct;148(1):124–130. doi: 10.1128/jb.148.1.124-130.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DiSorbo D. M., Harris N. A., Nathanson L. Suppression of DNA synthesis in NEL-M1 human melanoma cells by triamcinolone acetonide. Cancer Res. 1985 Apr;45(4):1633–1637. [PubMed] [Google Scholar]
  12. Eberle A., Schwyzer R. Hormone-receptor interactions. The message sequence of alpha-melanotropin: demonstration of two active sites. Clin Endocrinol (Oxf) 1976;5 (Suppl):41S–48S. doi: 10.1111/j.1365-2265.1976.tb03814.x. [DOI] [PubMed] [Google Scholar]
  13. Gilliland D. G., Collier R. J., Moehring J. M., Moehring T. J. Chimeric toxins: toxic, disulfide-linked conjugate of concanavalin A with fragment A from diphtheria toxin. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5319–5323. doi: 10.1073/pnas.75.11.5319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goff S. A., Goldberg A. L. Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes. Cell. 1985 Jun;41(2):587–595. doi: 10.1016/s0092-8674(85)80031-3. [DOI] [PubMed] [Google Scholar]
  15. Greenfield L., Bjorn M. J., Horn G., Fong D., Buck G. A., Collier R. J., Kaplan D. A. Nucleotide sequence of the structural gene for diphtheria toxin carried by corynebacteriophage beta. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6853–6857. doi: 10.1073/pnas.80.22.6853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. JENSEN F. C., GIRARDI A. J., GILDEN R. V., KOPROWSKI H. INFECTION OF HUMAN AND SIMIAN TISSUE CULTURES WITH ROUS SARCOMA VIRUS. Proc Natl Acad Sci U S A. 1964 Jul;52:53–59. doi: 10.1073/pnas.52.1.53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kaczorek M., Delpeyroux F., Chenciner N., Streeck R. E., Murphy J. R., Boquet P., Tiollais P. Nucleotide sequence and expression of the diphtheria tox228 gene in Escherichia coli. Science. 1983 Aug 26;221(4613):855–858. doi: 10.1126/science.6348945. [DOI] [PubMed] [Google Scholar]
  18. Kao F., Chasin L., Puck T. T. Genetics of somatic mammalian cells. X. Complementation analysis of glycine-requiring mutants. Proc Natl Acad Sci U S A. 1969 Dec;64(4):1284–1291. doi: 10.1073/pnas.64.4.1284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Leong D., Coleman K. D., Murphy J. R. Cloned diphtheria toxin fragment A is expressed from the tox promoter and exported to the periplasm by the SecA apparatus of Escherichia coli K12. J Biol Chem. 1983 Dec 25;258(24):15016–15020. [PubMed] [Google Scholar]
  22. Leong D., Coleman K. D., Murphy J. R. Cloned fragment A of diphtheria toxin is expressed and secreted into the periplasmic space of Escherichia coli K12. Science. 1983 Apr 29;220(4596):515–517. doi: 10.1126/science.6403984. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Moynihan M. R., Pappenheimer A. M., Jr Kinetics of adenosinediphosphoribosylation of elongation factor 2 in cells exposed to diphtheria toxin. Infect Immun. 1981 May;32(2):575–582. doi: 10.1128/iai.32.2.575-582.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oeltmann T. N., Heath E. C. A hybrid protein containing the toxic subunit of ricin and the cell-specific subunit of human chorionic gonadotropin. I. Synthesis and characterization. J Biol Chem. 1979 Feb 25;254(4):1022–1027. [PubMed] [Google Scholar]
  26. Oeltmann T. N. Synthesis and in vitro activity of a hormone-diphtheria toxin fragment A hybrid. Biochem Biophys Res Commun. 1985 Dec 17;133(2):430–435. doi: 10.1016/0006-291x(85)90924-6. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Ratti G., Rappuoli R., Giannini G. The complete nucleotide sequence of the gene coding for diphtheria toxin in the corynephage omega (tox+) genome. Nucleic Acids Res. 1983 Oct 11;11(19):6589–6595. doi: 10.1093/nar/11.19.6589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Robb M., Nichols J. C., Whoriskey S. K., Murphy J. R. Isolation of hybridoma cell lines and characterization of monoclonal antibodies against cholera enterotoxin and its subunits. Infect Immun. 1982 Oct;38(1):267–272. doi: 10.1128/iai.38.1.267-272.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sandvig K., Olsnes S. Entry of the toxic proteins abrin, modeccin, ricin, and diphtheria toxin into cells. II. Effect of pH, metabolic inhibitors, and ionophores and evidence for toxin penetration from endocytotic vesicles. J Biol Chem. 1982 Jul 10;257(13):7504–7513. [PubMed] [Google Scholar]
  31. Sandvig K., Olsnes S., Pihl A. Inhibitory effect of ammonium chloride and chloroquine on the entry of the toxic lectin modeccin into HeLa cells. Biochem Biophys Res Commun. 1979 Sep 27;90(2):648–655. doi: 10.1016/0006-291x(79)91284-1. [DOI] [PubMed] [Google Scholar]
  32. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schnaitman C. A. Effect of ethylenediaminetetraacetic acid, Triton X-100, and lysozyme on the morphology and chemical composition of isolate cell walls of Escherichia coli. J Bacteriol. 1971 Oct;108(1):553–563. doi: 10.1128/jb.108.1.553-563.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tweten R. K., Collier R. J. Molecular cloning and expression of gene fragments from corynebacteriophage beta encoding enzymatically active peptides of diphtheria toxin. J Bacteriol. 1983 Nov;156(2):680–685. doi: 10.1128/jb.156.2.680-685.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Varga J. M., Moellmann G., Fritsch P., Godawska E., Lerner A. B. Association of cell surface receptors for melanotropin with the Golgi region in mouse melanoma cells. Proc Natl Acad Sci U S A. 1976 Feb;73(2):559–562. doi: 10.1073/pnas.73.2.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  39. Vitetta E. S., Krolick K. A., Miyama-Inaba M., Cushley W., Uhr J. W. Immunotoxins: a new approach to cancer therapy. Science. 1983 Feb 11;219(4585):644–650. doi: 10.1126/science.6218613. [DOI] [PubMed] [Google Scholar]
  40. Yamaguchi T., Kato R., Beppu M., Terao T., Inoue Y., Ikawa Y., Osawa T. Preparation of concanavalin A-ricin A-chain conjugate and its biologic activity against various cultured cells. J Natl Cancer Inst. 1979 Jun;62(6):1387–1395. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES