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. 1989 Mar 1;169(3):765–777. doi: 10.1084/jem.169.3.765

Cytolysis by Ca-permeable transmembrane channels. Pore formation causes extensive DNA degradation and cell lysis

PMCID: PMC2189290  PMID: 2538546

Abstract

This study investigates the effect of the purified membrane pore formers, staphylococcal alpha-toxin and CTL perforin, on target cell lysis as measured by 51Cr release and on nuclear damage as measured by DNA degradation and 125IUdR release. Both pore formers cause dose- dependent cell lysis, which is accompanied by DNA release. The ratio of DNA/Cr release depends on the nature of target cell and shows the same pattern as the ratio of release of the two markers reported for CTL- mediated lysis of the same targets. DNA degradation is dependent on the presence of intracellular Ca in the target cell and is totally blocked if Ca is chelated by Quin 2 intracellularly and EGTA extracellularly. DNA degradation, in addition, is inhibited by the lysosomotropic agents NH4Cl, chloroquine, and monensin. rTNF doubles the degree of DNA degradation mediated by alpha-toxin in 3-h assays. We conclude that pore formers alone can mediate DNA degradation. In addition, they may promote the uptake of other factors and thereby accelerate their time course of action. DNA degradation by pore formers requires active target participation in a pathway that is dependent on intracellular Ca and lysosomes. These aspects of target lysis resemble CTL- and NK cell- mediated cytolysis.

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Selected References

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  1. Allbritton N. L., Verret C. R., Wolley R. C., Eisen H. N. Calcium ion concentrations and DNA fragmentation in target cell destruction by murine cloned cytotoxic T lymphocytes. J Exp Med. 1988 Feb 1;167(2):514–527. doi: 10.1084/jem.167.2.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bhakdi S., Füssle R., Tranum-Jensen J. Staphylococcal alpha-toxin: oligomerization of hydrophilic monomers to form amphiphilic hexamers induced through contact with deoxycholate detergent micelles. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5475–5479. doi: 10.1073/pnas.78.9.5475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blumenthal R., Millard P. J., Henkart M. P., Reynolds C. W., Henkart P. A. Liposomes as targets for granule cytolysin from cytotoxic large granular lymphocyte tumors. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5551–5555. doi: 10.1073/pnas.81.17.5551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carney D. F., Koski C. L., Shin M. L. Elimination of terminal complement intermediates from the plasma membrane of nucleated cells: the rate of disappearance differs for cells carrying C5b-7 or C5b-8 or a mixture of C5b-8 with a limited number of C5b-9. J Immunol. 1985 Mar;134(3):1804–1809. [PubMed] [Google Scholar]
  5. Cohen J. J., Duke R. C. Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J Immunol. 1984 Jan;132(1):38–42. [PubMed] [Google Scholar]
  6. Criado M., Lindstrom J. M., Anderson C. G., Dennert G. Cytotoxic granules from killer cells: specificity of granules and insertion of channels of defined size into target membranes. J Immunol. 1985 Dec;135(6):4245–4251. [PubMed] [Google Scholar]
  7. Dennert G., Landon C., Nowicki M. Cell-mediated and glucocorticoid-mediated target cell lysis do not appear to share common pathways. J Immunol. 1988 Aug 1;141(3):785–791. [PubMed] [Google Scholar]
  8. Dennert G., Podack E. R. Cytolysis by H-2-specific T killer cells. Assembly of tubular complexes on target membranes. J Exp Med. 1983 May 1;157(5):1483–1495. doi: 10.1084/jem.157.5.1483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Duke R. C., Chervenak R., Cohen J. J. Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6361–6365. doi: 10.1073/pnas.80.20.6361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duke R. C., Cohen J. J., Chervenak R. Differences in target cell DNA fragmentation induced by mouse cytotoxic T lymphocytes and natural killer cells. J Immunol. 1986 Sep 1;137(5):1442–1447. [PubMed] [Google Scholar]
  11. Füssle R., Bhakdi S., Sziegoleit A., Tranum-Jensen J., Kranz T., Wellensiek H. J. On the mechanism of membrane damage by Staphylococcus aureus alpha-toxin. J Cell Biol. 1981 Oct;91(1):83–94. doi: 10.1083/jcb.91.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gromkowski S. H., Brown T. C., Cerutti P. A., Cerottini J. C. DNA of human Raji target cells is damaged upon lymphocyte-mediated lysis. J Immunol. 1986 Feb 1;136(3):752–756. [PubMed] [Google Scholar]
  13. Henkart P. A., Millard P. J., Reynolds C. W., Henkart M. P. Cytolytic activity of purified cytoplasmic granules from cytotoxic rat large granular lymphocyte tumors. J Exp Med. 1984 Jul 1;160(1):75–93. doi: 10.1084/jem.160.1.75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Howell D. M., Martz E. The degree of CTL-induced DNA solubilization is not determined by the human vs mouse origin of the target cell. J Immunol. 1987 Jun 1;138(11):3695–3698. [PubMed] [Google Scholar]
  15. Li C. B., Gray P. W., Lin P. F., McGrath K. M., Ruddle F. H., Ruddle N. H. Cloning and expression of murine lymphotoxin cDNA. J Immunol. 1987 Jun 15;138(12):4496–4501. [PubMed] [Google Scholar]
  16. Lichtenheld M. G., Olsen K. J., Lu P., Lowrey D. M., Hameed A., Hengartner H., Podack E. R. Structure and function of human perforin. Nature. 1988 Sep 29;335(6189):448–451. doi: 10.1038/335448a0. [DOI] [PubMed] [Google Scholar]
  17. Lichtenstein A. K., Ganz T., Nguyen T. M., Selsted M. E., Lehrer R. I. Mechanism of target cytolysis by peptide defensins. Target cell metabolic activities, possibly involving endocytosis, are crucial for expression of cytotoxicity. J Immunol. 1988 Apr 15;140(8):2686–2694. [PubMed] [Google Scholar]
  18. Lowrey D. M., Hameed A., Lichtenheld M., Podack E. R. Isolation and characterization of cytotoxic granules from human lymphokine (interleukin 2) activated killer cells. Cancer Res. 1988 Aug 15;48(16):4681–4688. [PubMed] [Google Scholar]
  19. Ostergaard H., Clark W. R. The role of Ca2+ in activation of mature cytotoxic T lymphocytes for lysis. J Immunol. 1987 Dec 1;139(11):3573–3579. [PubMed] [Google Scholar]
  20. Pasternack M. S., Eisen H. N. A novel serine esterase expressed by cytotoxic T lymphocytes. 1985 Apr 25-May 1Nature. 314(6013):743–745. doi: 10.1038/314743a0. [DOI] [PubMed] [Google Scholar]
  21. Podack E. R., Dennert G. Assembly of two types of tubules with putative cytolytic function by cloned natural killer cells. 1983 Mar 31-Apr 6Nature. 302(5907):442–445. doi: 10.1038/302442a0. [DOI] [PubMed] [Google Scholar]
  22. Podack E. R., Konigsberg P. J. Cytolytic T cell granules. Isolation, structural, biochemical, and functional characterization. J Exp Med. 1984 Sep 1;160(3):695–710. doi: 10.1084/jem.160.3.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Podack E. R., Penichet K. O., Lin B. Y., Chiao J. W., Mittelman A. Increased cytotoxicity and CD16 (Leu 11) expression in long-term, IL-2-activated human peripheral blood mononuclear cells. Blood Cells. 1987;13(1-2):117–122. [PubMed] [Google Scholar]
  24. Podack E. R., Young J. D., Cohn Z. A. Isolation and biochemical and functional characterization of perforin 1 from cytolytic T-cell granules. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8629–8633. doi: 10.1073/pnas.82.24.8629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Russell J. H., Dobos C. B. Mechanisms of immune lysis. II. CTL-induced nuclear disintegration of the target begins within minutes of cell contact. J Immunol. 1980 Sep;125(3):1256–1261. [PubMed] [Google Scholar]
  26. Russell J. H., Masakowski V. R., Dobos C. B. Mechanisms of immune lysis. I. Physiological distinction between target cell death mediated by cytotoxic T lymphocytes and antibody plus complement. J Immunol. 1980 Mar;124(3):1100–1105. [PubMed] [Google Scholar]
  27. Schmid D. S., Powell M. B., Mahoney K. A., Ruddle N. H. A comparison of lysis mediated by Lyt 2+ TNP-specific cytotoxic-T-lymphocyte (CTL) lines with that mediated by rapidly internalized lymphotoxin-containing supernatant fluids: evidence for a role of soluble mediators in CTL-mediated killing. Cell Immunol. 1985 Jun;93(1):68–82. doi: 10.1016/0008-8749(85)90389-2. [DOI] [PubMed] [Google Scholar]
  28. Schmid D. S., Tite J. P., Ruddle N. H. DNA fragmentation: manifestation of target cell destruction mediated by cytotoxic T-cell lines, lymphotoxin-secreting helper T-cell clones, and cell-free lymphotoxin-containing supernatant. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1881–1885. doi: 10.1073/pnas.83.6.1881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sellins K. S., Cohen J. J. Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes. J Immunol. 1987 Nov 15;139(10):3199–3206. [PubMed] [Google Scholar]
  30. Shinkai Y., Takio K., Okumura K. Homology of perforin to the ninth component of complement (C9). Nature. 1988 Aug 11;334(6182):525–527. doi: 10.1038/334525a0. [DOI] [PubMed] [Google Scholar]
  31. Shipley W. U., Baker A. R., Colten H. R. DNA degradation in mammalian cells following complement-mediated cytolysis. J Immunol. 1971 Feb;106(2):576–579. [PubMed] [Google Scholar]
  32. Tsien R. Y., Pozzan T., Rink T. J. Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol. 1982 Aug;94(2):325–334. doi: 10.1083/jcb.94.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wyllie A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature. 1980 Apr 10;284(5756):555–556. doi: 10.1038/284555a0. [DOI] [PubMed] [Google Scholar]
  34. Wyllie A. H., Kerr J. F., Currie A. R. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251–306. doi: 10.1016/s0074-7696(08)62312-8. [DOI] [PubMed] [Google Scholar]
  35. Young J. D., Hengartner H., Podack E. R., Cohn Z. A. Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity. Cell. 1986 Mar 28;44(6):849–859. doi: 10.1016/0092-8674(86)90007-3. [DOI] [PubMed] [Google Scholar]
  36. Young J. D., Podack E. R., Cohn Z. A. Properties of a purified pore-forming protein (perforin 1) isolated from H-2-restricted cytotoxic T cell granules. J Exp Med. 1986 Jul 1;164(1):144–155. doi: 10.1084/jem.164.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zalman L. S., Brothers M. A., Chiu F. J., Müller-Eberhard H. J. Mechanism of cytotoxicity of human large granular lymphocytes: relationship of the cytotoxic lymphocyte protein to the ninth component (C9) of human complement. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5262–5266. doi: 10.1073/pnas.83.14.5262. [DOI] [PMC free article] [PubMed] [Google Scholar]

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