Skip to main content
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1995 Mar 1;181(3):1037–1046. doi: 10.1084/jem.181.3.1037

Synergistic roles of granzymes A and B in mediating target cell death by rat basophilic leukemia mast cell tumors also expressing cytolysin/perforin

PMCID: PMC2191929  PMID: 7869027

Abstract

We have studied the cytotoxic activity of rat basophilic leukemia (RBL) cells transfected with cDNAs for the cytotoxic T lymphocyte (CTL) granule components, cytolysin (perforin), granzyme A, and granzyme B. With red cell targets, cytolysin expression conferred potent hemolytic activity, which was not influenced by coexpression of granzymes. With tumor targets, RBL cells expressing cytolysin alone were weakly cytotoxic, but both cytolytic and nucleolytic activity were enhanced by coexpression of granzyme B. RBL cells expressing all three CTL granule components showed still higher cytotoxic activities, with apoptotic target death. Analysis of the cytotoxic activity of individual transfectant clones showed that cytolytic and nucleolytic activity correlated with granzyme expression but was independent of cytolysin expression within the range examined. A synergism between granzymes A and B was apparent when the triple transfectant was compared with RBL cells expressing cytolysin and one granzyme. These data implicate granzymes as the major mediators of tumor target damage by cytotoxic lymphocytes.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Chang H. L., Zaroukian M. H., Esselman W. J. T200 alternate exon use in murine lymphoid cells determined by reverse transcription-polymerase chain reaction. J Immunol. 1989 Jul 1;143(1):315–321. [PubMed] [Google Scholar]
  2. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  3. Garcia-Sanz J. A., MacDonald H. R., Jenne D. E., Tschopp J., Nabholz M. Cell specificity of granzyme gene expression. J Immunol. 1990 Nov 1;145(9):3111–3118. [PubMed] [Google Scholar]
  4. Gershenfeld H. K., Hershberger R. J., Shows T. B., Weissman I. L. Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1184–1188. doi: 10.1073/pnas.85.4.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hayes M. P., Berrebi G. A., Henkart P. A. Induction of target cell DNA release by the cytotoxic T lymphocyte granule protease granzyme A. J Exp Med. 1989 Sep 1;170(3):933–946. doi: 10.1084/jem.170.3.933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Henkart P. A. Mechanism of lymphocyte-mediated cytotoxicity. Annu Rev Immunol. 1985;3:31–58. doi: 10.1146/annurev.iy.03.040185.000335. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Heusel J. W., Wesselschmidt R. L., Shresta S., Russell J. H., Ley T. J. Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells. Cell. 1994 Mar 25;76(6):977–987. doi: 10.1016/0092-8674(94)90376-x. [DOI] [PubMed] [Google Scholar]
  9. Izumi M., Miyazawa H., Kamakura T., Yamaguchi I., Endo T., Hanaoka F. Blasticidin S-resistance gene (bsr): a novel selectable marker for mammalian cells. Exp Cell Res. 1991 Dec;197(2):229–233. doi: 10.1016/0014-4827(91)90427-v. [DOI] [PubMed] [Google Scholar]
  10. Jacobson M. D., Burne J. F., Raff M. C. Programmed cell death and Bcl-2 protection in the absence of a nucleus. EMBO J. 1994 Apr 15;13(8):1899–1910. doi: 10.1002/j.1460-2075.1994.tb06459.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jenne D. E., Masson D., Zimmer M., Haefliger J. A., Li W. H., Tschopp J. Isolation and complete structure of the lymphocyte serine protease granzyme G, a novel member of the granzyme multigene family in murine cytolytic T lymphocytes. Evolutionary origin of lymphocyte proteases. Biochemistry. 1989 Sep 19;28(19):7953–7961. doi: 10.1021/bi00445a060. [DOI] [PubMed] [Google Scholar]
  12. Kägi D., Ledermann B., Bürki K., Seiler P., Odermatt B., Olsen K. J., Podack E. R., Zinkernagel R. M., Hengartner H. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature. 1994 May 5;369(6475):31–37. doi: 10.1038/369031a0. [DOI] [PubMed] [Google Scholar]
  13. Liu F. T., Bohn J. W., Ferry E. L., Yamamoto H., Molinaro C. A., Sherman L. A., Klinman N. R., Katz D. H. Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization. J Immunol. 1980 Jun;124(6):2728–2737. [PubMed] [Google Scholar]
  14. Martz E. Mechanism of specific tumor-cell lysis by alloimmune T lymphocytes: resolution and characterization of discrete steps in the cellular interaction. Contemp Top Immunobiol. 1977;7:301–361. doi: 10.1007/978-1-4684-3054-7_9. [DOI] [PubMed] [Google Scholar]
  15. Miura M., Zhu H., Rotello R., Hartwieg E. A., Yuan J. Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell. 1993 Nov 19;75(4):653–660. doi: 10.1016/0092-8674(93)90486-a. [DOI] [PubMed] [Google Scholar]
  16. Morgan B. P. Complement membrane attack on nucleated cells: resistance, recovery and non-lethal effects. Biochem J. 1989 Nov 15;264(1):1–14. doi: 10.1042/bj2640001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nakajima H., Henkart P. A. Cytotoxic lymphocyte granzymes trigger a target cell internal disintegration pathway leading to cytolysis and DNA breakdown. J Immunol. 1994 Feb 1;152(3):1057–1063. [PubMed] [Google Scholar]
  18. Nishioka W. K., Welsh R. M. Inhibition of cytotoxic T lymphocyte-induced target cell DNA fragmentation, but not lysis, by inhibitors of DNA topoisomerases I and II. J Exp Med. 1992 Jan 1;175(1):23–27. doi: 10.1084/jem.175.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pasternack M. S., Bleier K. J., McInerney T. N. Granzyme A binding to target cell proteins. Granzyme A binds to and cleaves nucleolin in vitro. J Biol Chem. 1991 Aug 5;266(22):14703–14708. [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. Perez P., Hoffman R. W., Shaw S., Bluestone J. A., Segal D. M. Specific targeting of cytotoxic T cells by anti-T3 linked to anti-target cell antibody. Nature. 1985 Jul 25;316(6026):354–356. doi: 10.1038/316354a0. [DOI] [PubMed] [Google Scholar]
  22. Russell J. H. Internal disintegration model of cytotoxic lymphocyte-induced target damage. Immunol Rev. 1983;72:97–118. doi: 10.1111/j.1600-065x.1983.tb01074.x. [DOI] [PubMed] [Google Scholar]
  23. Sellins K. S., Cohen J. J. Cytotoxic T lymphocytes induce different types of DNA damage in target cells of different origins. J Immunol. 1991 Aug 1;147(3):795–803. [PubMed] [Google Scholar]
  24. Shi L., Kam C. M., Powers J. C., Aebersold R., Greenberg A. H. Purification of three cytotoxic lymphocyte granule serine proteases that induce apoptosis through distinct substrate and target cell interactions. J Exp Med. 1992 Dec 1;176(6):1521–1529. doi: 10.1084/jem.176.6.1521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shi L., Nishioka W. K., Th'ng J., Bradbury E. M., Litchfield D. W., Greenberg A. H. Premature p34cdc2 activation required for apoptosis. Science. 1994 Feb 25;263(5150):1143–1145. doi: 10.1126/science.8108732. [DOI] [PubMed] [Google Scholar]
  26. Shiver J. W., Henkart P. A. A noncytotoxic mast cell tumor line exhibits potent IgE-dependent cytotoxicity after transfection with the cytolysin/perforin gene. Cell. 1991 Mar 22;64(6):1175–1181. doi: 10.1016/0092-8674(91)90272-z. [DOI] [PubMed] [Google Scholar]
  27. Shiver J. W., Su L., Henkart P. A. Cytotoxicity with target DNA breakdown by rat basophilic leukemia cells expressing both cytolysin and granzyme A. Cell. 1992 Oct 16;71(2):315–322. doi: 10.1016/0092-8674(92)90359-k. [DOI] [PubMed] [Google Scholar]
  28. Trapani J. A., Klein J. L., White P. C., Dupont B. Molecular cloning of an inducible serine esterase gene from human cytotoxic lymphocytes. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6924–6928. doi: 10.1073/pnas.85.18.6924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ucker D. S., Obermiller P. S., Eckhart W., Apgar J. R., Berger N. A., Meyers J. Genome digestion is a dispensable consequence of physiological cell death mediated by cytotoxic T lymphocytes. Mol Cell Biol. 1992 Jul;12(7):3060–3069. doi: 10.1128/mcb.12.7.3060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Velotti F., Palmieri G., D'Ambrosio D., Piccoli M., Frati L., Santoni A. Differential expression of granzyme A and granzyme B proteases and their secretion by fresh rat natural killer cells (NK) and lymphokine-activated killer cells with NK phenotype (LAK-NK). Eur J Immunol. 1992 Apr;22(4):1049–1053. doi: 10.1002/eji.1830220426. [DOI] [PubMed] [Google Scholar]
  31. Yuan J., Shaham S., Ledoux S., Ellis H. M., Horvitz H. R. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell. 1993 Nov 19;75(4):641–652. doi: 10.1016/0092-8674(93)90485-9. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES