title: Interleukin-2 activated NK cells do not use the CD95L- and TRAIL-pathways in the rapid induction of apoptosis of rat colon carcinoma CC531s cells.
abstract: Natural Killer (NK) cells can induce apoptosis in target cells in at least four ways: by secretion of granzyme B/perforin (GrB/P) and via the CD95L, TRAIL and TNF-alpha pathways. In this study we examined the pathways used by interleukin-2 activated rat NK (A-NK) cells to induce apoptosis in the rat colon carcinoma cell line CC531s. Co-incubation of A-NK cells with CC531s cells for three hours resulted in 70% apoptosis in the latter. Addition of the GrB/P pathway-inhibitor concanamycin A reduced the number of apoptotic cells to 54%. Blockade of the CD95L, TRAIL and TNF-alpha pathways by specific antibodies hardly had an additional effect. However, co-incubation with transfected MEC cells that expressed CD95L or 2PK3-cells that expressed TRAIL did induce apoptosis in CC531s cells. Furthermore the A-NK cells contained CD95L and TRAIL. However, comparison of non- and permeabilized cells revealed that the majority of TRAIL was present in the cytosol of A-NK cells and was not available for induction of apoptosis. The presence of elevated levels of bcl-2 in CC531 cells reduced the sensitivity towards induction of apoptosis both by A-NK cells as well as the CD95L and TRAIL expressing cell lines. Using the caspase-inhibitors ac-IEPD-CHO, ac-DEVD-CHO and zVAD-fmk, it was shown that inhibition of the effector caspase-3 prevented A-NK cell induced apoptosis in CC531-bcl-2 cells, but not in CC531s cells. In conclusion, A-NK cells kill by secretion of GrB/P and not by the CD95L, TRAIL or TNF pathways albeit both CD95L and TRAIL are produced by the A-NK cells.
authors.affiliation: Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands. velthius@lacdr.leidenuniv.nl
authors.forename: Jurjen H L
authors.initials: JH
authors.lastname: Velthuis
authors.forename: Hans J G M
authors.initials: HJ
authors.lastname: de Bont
authors.forename: Jan-Paul
authors.initials: JP
authors.lastname: Medema
authors.forename: Peter J K
authors.initials: PJ
authors.lastname: Kuppen
authors.forename: Gerard J
authors.initials: GJ
authors.lastname: Mulder
authors.forename: J Fred
authors.initials: JF
authors.lastname: Nagelkerke
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D002277:Carcinoma / Q000378:metabolism / Q000473:pathology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology; D003602:Cytotoxicity, Immunologic / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D053804:Granzymes; D006801:Humans; D015151:Immunoblotting; D007376:Interleukin-2 / Q000378:metabolism; D007694:Killer Cells, Natural / Q000502:physiology*; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D012697:Serine Endopeptidases / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; C505662:Faslg protein, rat; D007376:Interleukin-2; D008562:Membrane Glycoproteins; D019253:Proto-Oncogene Proteins c-bcl-2; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; C505654:Tnfsf10 protein, mouse; C505655:Tnfsf10 protein, rat; D014409:Tumor Necrosis Factor-alpha; C509213:GZMB protein, human; D053804:Granzymes; C509214:Gzmb protein, mouse; C509215:Gzmb protein, rat; D012697:Serine Endopeptidases; D020169:Caspases