Skip to main content
NCBI home page
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
. 1984 Dec;81(24):7932–7936. doi: 10.1073/pnas.81.24.7932

Necrosin: purification and properties of a cytotoxin derived from a murine macrophage-like cell line.

F C Kull Jr, P Cuatrecasas
PMCID: PMC392267  PMID: 6595667

Abstract

An acidic proteinaceous cytotoxin was isolated from the serum-free, cell-free supernatants of J774.1 (a murine macrophage-like line) cells that had been treated with bacterial endotoxin. Cytotoxic activity was routinely monitored using the sensitive murine tumorigenic fibroblast line L-M. The toxin was purified 8000-fold by ion-exchange and electrophoretic procedures. It was purified to greater than 90% homogeneity, as assessed by photometric scanning of silver-stained NaDodSO4/polyacrylamide gels. The specific activity of the purified toxin was 32,000 units/microgram. The toxin was composed of self-aggregating non-sulfhydryl-linked multimers of a Mr 15,000 subunit. The pI of the monomer was 4.6. The active multimeric forms, as assessed by gel filtration and assayed using L-M cells, were of Mr 70,000 and 55,000. These forms were identical to those observed both in crude supernatants and in purified fractions that had not been subjected to denaturing agents. We call these forms "holotoxins" and conclude that they are aggregates of the Mr 15,000 protein. The purified toxin (1-1000 pM, 0.06-60 ng/ml) was active against a random assortment of tumorigenic and normal cell lines of murine, bovine, and human origin. For example, the diploid bovine endothelial line CPAE was nearly as sensitive as the L-M line. Similarities to other toxic macrophage products, lymphotoxin, and tumor necrosis factor are discussed.

Full text

PDF
7932

Images in this article

7934Image
on p.7934
7934Image
on p.7934
7935Image
on p.7935

Selected References

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

  1. Adams D. O., Kao K. J., Farb R., Pizzo S. V. Effector mechanisms of cytolytically activated macrophages. II. Secretion of a cytolytic factor by activated macrophages and its relationship to secreted neutral proteases. J Immunol. 1980 Jan;124(1):293–300. [PubMed] [Google Scholar]
  2. Aggarwal B. B., Moffat B., Harkins R. N. Human lymphotoxin. Production by a lymphoblastoid cell line, purification, and initial characterization. J Biol Chem. 1984 Jan 10;259(1):686–691. [PubMed] [Google Scholar]
  3. Aune T. M., Pierce C. W. Identification and initial characterization of a nonspecific suppressor factor (macrophage-SF) produced by soluble immune response suppressor (SIRS)-treated macrophages. J Immunol. 1981 Nov;127(5):1828–1833. [PubMed] [Google Scholar]
  4. Bearden J. C., Jr Quantitation of submicrogram quantities of protein by an improved protein-dye binding assay. Biochim Biophys Acta. 1978 Apr 26;533(2):525–529. doi: 10.1016/0005-2795(78)90398-7. [DOI] [PubMed] [Google Scholar]
  5. Carswell E. A., Old L. J., Kassel R. L., Green S., Fiore N., Williamson B. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3666–3670. doi: 10.1073/pnas.72.9.3666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clark I. A., Virelizier J. L., Carswell E. A., Wood P. R. Possible importance of macrophage-derived mediators in acute malaria. Infect Immun. 1981 Jun;32(3):1058–1066. doi: 10.1128/iai.32.3.1058-1066.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Currie G. A. Activated macrophages kill tumour cells by releasing arginase. Nature. 1978 Jun 29;273(5665):758–759. doi: 10.1038/273758a0. [DOI] [PubMed] [Google Scholar]
  8. Green S., Dobrjansky A., Carswell E. A., Kassel R. L., Old L. J., Fiore N., Schwartz M. K. Partial purification of a serum factor that causes necrosis of tumors. Proc Natl Acad Sci U S A. 1976 Feb;73(2):381–385. doi: 10.1073/pnas.73.2.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Haidaris C. G., Haynes J. D., Meltzer M. S., Allison A. C. Serum containing tumor necrosis factor is cytotoxic for the human malaria parasite Plasmodium falciparum. Infect Immun. 1983 Oct;42(1):385–393. doi: 10.1128/iai.42.1.385-393.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Helson L., Green S., Carswell E., Old L. J. Effect of tumour necrosis factor on cultured human melanoma cells. Nature. 1975 Dec 25;258(5537):731–732. doi: 10.1038/258731a0. [DOI] [PubMed] [Google Scholar]
  11. Hunkapiller M. W., Lujan E., Ostrander F., Hood L. E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. doi: 10.1016/s0076-6879(83)91019-4. [DOI] [PubMed] [Google Scholar]
  12. Kull F. C., Jr, Cuatrecasas P. Estimation of cell number by neutral red content. Applications for proliferative and survival assays. Appl Biochem Biotechnol. 1983 Apr;8(2):97–103. doi: 10.1007/BF02778090. [DOI] [PubMed] [Google Scholar]
  13. Kull F. C., Jr, Cuatrecasas P. Possible requirement of internalization in the mechanism of in vitro cytotoxicity in tumor necrosis serum. Cancer Res. 1981 Dec;41(12 Pt 1):4885–4890. [PubMed] [Google Scholar]
  14. Kull F. C., Jr, Cuatrecasas P. Preliminary characterization of the tumor cell cytotoxin in tumor necrosis serum. J Immunol. 1981 Apr;126(4):1279–1283. [PubMed] [Google Scholar]
  15. 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]
  16. Matthews N. Tumour-necrosis factor from the rabbit. V. Synthesis in vitro by mononuclear phagocytes from various tissues of normal and BCG-injected rabbits. Br J Cancer. 1981 Sep;44(3):418–424. doi: 10.1038/bjc.1981.200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Matthews N., Watkins J. F. Tumour-necrosis factor from the rabbit. I. Mode of action, specificity and physicochemical properties. Br J Cancer. 1978 Aug;38(2):302–309. doi: 10.1038/bjc.1978.202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Menkin V. STUDIES ON THE ISOLATION OF THE FACTOR RESPONSIBLE FOR TISSUE INJURY IN INFLAMMATION. Science. 1943 Feb 12;97(2511):165–167. doi: 10.1126/science.97.2511.165. [DOI] [PubMed] [Google Scholar]
  19. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  20. Männel D. N., Moore R. N., Mergenhagen S. E. Macrophages as a source of tumoricidal activity (tumor-necrotizing factor). Infect Immun. 1980 Nov;30(2):523–530. doi: 10.1128/iai.30.2.523-530.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nathan C. F., Silverstein S. C., Brukner L. H., Cohn Z. A. Extracellular cytolysis by activated macrophages and granulocytes. II. Hydrogen peroxide as a mediator of cytotoxicity. J Exp Med. 1979 Jan 1;149(1):100–113. doi: 10.1084/jem.149.1.100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Playfair J. H., de Souza J. B., Taverne J. Endotoxin induced 'tumour-necrosis serum' kills a subpopulation of normal lymphocytes in vitro. Clin Exp Immunol. 1982 Mar;47(3):753–755. [PMC free article] [PubMed] [Google Scholar]
  23. Ralph P., Nakoinz I. Phagocytosis and cytolysis by a macrophage tumour and its cloned cell line. Nature. 1975 Oct 2;257(5525):393–394. doi: 10.1038/257393a0. [DOI] [PubMed] [Google Scholar]
  24. Reidarson T. H., Granger G. A., Klostergaard J. Inducible macrophage cytotoxins. II. Tumor lysis mechanism involving target cell-binding proteases. J Natl Cancer Inst. 1982 Oct;69(4):889–894. [PubMed] [Google Scholar]
  25. Ruff M. R., Gifford G. E. Purification and physico-chemical characterization of rabbit tumor necrosis factor. J Immunol. 1980 Oct;125(4):1671–1677. [PubMed] [Google Scholar]
  26. Stewart W. E., 2nd, Chudzio T., Lin L. S., Wiranowska-Stewart M. Interferoids: in vitro and in vivo conversion of native interferons to lower molecular weight forms. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4814–4818. doi: 10.1073/pnas.75.10.4814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Taverne J., Depledge P., Playfair J. H. Differential sensitivity in vivo of lethal and nonlethal malarial parasites to endotoxin-induced serum factor. Infect Immun. 1982 Sep;37(3):927–934. doi: 10.1128/iai.37.3.927-934.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yip Y. K., Barrowclough B. S., Urban C., Vilcek J. Molecular weight of human gamma interferon is similar to that of other human interferons. Science. 1982 Jan 22;215(4531):411–413. doi: 10.1126/science.6173921. [DOI] [PubMed] [Google Scholar]
  29. Zacharchuk C. M., Drysdale B. E., Mayer M. M., Shin H. S. Macrophage-mediated cytotoxicity: role of a soluble macrophage cytotoxic factor similar to lymphotoxin and tumor necrosis factor. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6341–6345. doi: 10.1073/pnas.80.20.6341. [DOI] [PMC free article] [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