Abstract
Rapid and extensive target cell DNA fragmentation is a unique characteristic of CTL-mediated killing. We studied the role of the granule pore-forming protein (PFP/perforin/cytolysin) of CTL in mediating lysis and DNA fragmentation of target cells. Perforin was isolated from murine CTL by sequential application of perforin-enriched granule fractions to four chromatographic columns: DEAE-Sepharose, Q- Sepharose, Polyanion SI, and Superose 12. Purified perforin was eluted as a single band of 70 kD in SDS-PAGE. While purified perforin produced potent lysis of a variety of target cells tested, it did not induce any measurable amount of DNA fragmentation. In parallel experiments, intact CTL produced marked DNA fragmentation of the same target cell populations. Our results suggest that perforin alone is not responsible for the DNA fragmentation observed during CTL-mediated killing and that other, as yet unknown, mediators or mechanisms are likely to be involved in the induction of target cell nuclear damage.
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- 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]
- Blakely A., Gorman K., Ostergaard H., Svoboda K., Liu C. C., Young J. D., Clark W. R. Resistance of cloned cytotoxic T lymphocytes to cell-mediated cytotoxicity. J Exp Med. 1987 Oct 1;166(4):1070–1083. doi: 10.1084/jem.166.4.1070. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Duke R. C., Sellins K. S., Cohen J. J. Cytotoxic lymphocyte-derived lytic granules do not induce DNA fragmentation in target cells. J Immunol. 1988 Oct 1;141(7):2191–2194. [PubMed] [Google Scholar]
- Gromkowski S. H., Brown T. C., Masson D., Tschopp J. Lack of DNA degradation in target cells lysed by granules derived from cytolytic T lymphocytes. J Immunol. 1988 Aug 1;141(3):774–778. [PubMed] [Google Scholar]
- Hameed A., Olsen K. J., Lee M. K., Lichtenheld M. G., Podack E. R. Cytolysis by Ca-permeable transmembrane channels. Pore formation causes extensive DNA degradation and cell lysis. J Exp Med. 1989 Mar 1;169(3):765–777. doi: 10.1084/jem.169.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Jiang S. B., Persechini P. M., Zychlinsky A., Liu C. C., Perussia B., Young J. D. Resistance of cytolytic lymphocytes to perforin-mediated killing. Lack of correlation with complement-associated homologous species restriction. J Exp Med. 1988 Dec 1;168(6):2207–2219. doi: 10.1084/jem.168.6.2207. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liu C. C., Steffen M., King F., Young J. D. Identification, isolation, and characterization of a novel cytotoxin in murine cytolytic lymphocytes. Cell. 1987 Nov 6;51(3):393–403. doi: 10.1016/0092-8674(87)90635-0. [DOI] [PubMed] [Google Scholar]
- Martz E., Howell D. M. CTL: virus control cells first and cytolytic cells second? DNA fragmentation, apoptosis and the prelytic halt hypothesis. Immunol Today. 1989 Mar;10(3):79–86. doi: 10.1016/0167-5699(89)90231-4. [DOI] [PubMed] [Google Scholar]
- Persechini P. M., Young J. D. The primary structure of the lymphocyte pore-forming protein perforin: partial amino acid sequencing and determination of isoelectric point. Biochem Biophys Res Commun. 1988 Oct 31;156(2):740–745. doi: 10.1016/s0006-291x(88)80905-7. [DOI] [PubMed] [Google Scholar]
- 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]
- Young J. D., Cohn Z. A. Cellular and humoral mechanisms of cytotoxicity: structural and functional analogies. Adv Immunol. 1987;41:269–332. doi: 10.1016/s0065-2776(08)60033-4. [DOI] [PubMed] [Google Scholar]