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Proteinases are involved in both DNA fragmentation and membrane damage during CTL-mediated target cell killing
C D Helgason1, E A Atkinson, M J Pinkoski
1Department of Biochemistry, University of Alberta, Edmonton, Canada.
Abstract:
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.
Insights
Proteinases play a dual role in cytotoxic T lymphocyte (CTL)-mediated cell death. Inhibitors show proteinases initiate DNA fragmentation and cause ongoing membrane damage during CTL lysis.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Cytotoxic T lymphocytes (CTLs) induce target cell death through complex mechanisms.
- Proteinases are implicated in CTL-mediated lysis, but their specific roles remain unclear.
Purpose of the Study:
- To investigate the involvement and specific roles of proteinases in CTL-mediated target cell lysis.
- To differentiate the functions of proteinases in DNA fragmentation and membrane damage.
Main Methods:
- Utilized various proteinase inhibitors with different specificities (ATEE, BAEE, DCI) to probe CTL mechanisms.
- Measured chromium release (membrane damage) and DNA fragmentation in target cells.
- Assessed the impact of inhibitor timing on lytic processes.
Main Results:
- N-Acetyl-L-tyrosine ethyl ester (ATEE) and 3,4-dichloroisocoumarin (DCI) inhibited both DNA fragmentation and chromium release.
- DCI was more potent in inhibiting chromium release than DNA fragmentation.
- DCI's effect on DNA fragmentation was abolished when added later in the lytic cycle, unlike its effect on lysis.
Conclusions:
- Proteinases are crucial for initiating DNA fragmentation in CTL-mediated target cell death.
- Proteinases also play a continuous role in causing membrane damage during the lytic cycle.
- A dual-role model for proteinases in CTL-mediated cytotoxicity is proposed.