Proteinases are involved in both DNA fragmentation and membrane damage during CTL-mediated target cell killing.
A number of inhibitors with different specificities were used to probe the involvement of proteinases in the mechanism of cytotoxic T lymphocyte (CTL)-mediated lysis. N-Acetyl-L-tyrosine ethyl ester (ATEE) and N-benzoyl-L-arginine ethyl ester (BAEE) are reversible substrate inhibitors of proteinases with trypsin-like and chymotrypsin-like specificities, respectively. BAEE did not prevent either the chromium release or DNA fragmentation induced in mouse tumor target cells by a mixed lymphocyte population. In contrast, ATEE inhibited both processes. The irreversible proteinase inhibitor 3,4-dichloroisocoumarin (DCI) also blocked both chromium release and DNA fragmentation, but at significantly lower concentrations than ATEE. More importantly, chromium release was more susceptible to inhibition by DCI than DNA fragmentation. Addition of a combination of the endonuclease inhibitor aurintricarboxylic acid plus DCI resulted in virtually complete inhibition of both DNA fragmentation and chromium release when the drugs were added at the beginning of the incubation period. In contrast addition of DCI 15 or 30 min following initiation of the lytic cycle abolished the affect of DCI on fragmentation, but not lysis. A model which suggests a dual role for the proteinases in CTL-mediated target cell death is presented. First, proteinases are involved in the initiation of DNA fragmentation. Second, they have an ongoing function in membrane damage.
Helgason
C D
CD
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Atkinson
E A
EA
Pinkoski
M J
MJ
Bleackley
R C
RC
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D002478:Cells, Cultured; D003374:Coumarins / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic; D004247:DNA / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D049934:Isocoumarins; D007700:Kinetics; D012515:Mast-Cell Sarcoma; D051379:Mice; D008807:Mice, Inbred BALB C; D011480:Protease Inhibitors / Q000494:pharmacology*; D013379:Substrate Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives / Q000494:pharmacology
D003374:Coumarins; D049934:Isocoumarins; D011480:Protease Inhibitors; D014443:Tyrosine; D001312:Aurintricarboxylic Acid; C046235:3,4-dichloroisocoumarin; C007651:ethyl N-alpha-acetyl-tyrosinate; D004247:DNA; D001120:Arginine; C009727:benzoylarginine ethyl ester; D010450:Endopeptidases