Features of cell death, mitochondrial activation and caspase dependence of rabbit anti-T-lymphocyte globulin signaling in lymphoblastic Jurkat cells are distinct from classical apoptosis signaling of CD95.
Rabbit anti-T-lymphocyte-globulin (ATG) is used for immunosuppression in organ and stem cell transplantation. The aim of this study was to investigate ATG-induced cell death compared to CD95-signaling of apoptosis. We measured features of cell death at the cell membrane, mitochondria, nuclei and caspase-3 cleavage. We used the following inhibitors: the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (O-Me)-fluoromethyl ketone (zVAD-fmk), the serine protease inhibitors 3,4 dichloroisocoumarin (DCI) and N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and the reducing agent N-acetycysteine (NAC). ATG-induced cellular changes were rapid, included mitochondrial membrane permeability (MMP) induction and annexin V/propidium iodide (PI) positivity but little caspase-3 activation and nuclear morphology changes. MMP was not sensitive to caspase inhibition, serine protease inhibition with DCI moderately reduced MMP. These findings were in contrast to CD95-signaling. Interestingly, TLCK massively augmented CD95-induced MMP which could be abrogated by NAC. In conclusion, ATG-signaling differs in features and kinetics from CD95-induced apoptosis with caspase-independent mechanisms involved in MMP.
a Department of Medical Oncology , National Center for Tumor Diseases, Heidelberg University Hospital , Heidelberg , Germany.|b Department of Hematology and Oncology , Albert Ludwigs-University Medical Center Freiburg , Freiburg , Germany.|c Department of Hematology and Oncology , Charité Medical Center Berlin , Campus Virchow Klinik, Berlin, Germany.
Christian
C
Ziegler
b Department of Hematology and Oncology , Albert Ludwigs-University Medical Center Freiburg , Freiburg , Germany.
Jürgen
J
Finke
a Department of Medical Oncology , National Center for Tumor Diseases, Heidelberg University Hospital , Heidelberg , Germany.|b Department of Hematology and Oncology , Albert Ludwigs-University Medical Center Freiburg , Freiburg , Germany.
Carsten
C
Grüllich
D000818:Animals; D000961:Antilymphocyte Serum / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D020169:Caspases / Q000378:metabolism*; D002463:Cell Membrane Permeability; D006801:Humans; D019169:Jurkat Cells; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects*; D019014:fas Receptor / Q000378:metabolism*
D000961:Antilymphocyte Serum; D019014:fas Receptor; D053148:Caspase 3; D020169:Caspases; D020782:Matrix Metalloproteinases
CD95; Caspase; Jurkat; rabbit anti-T-lymphocyte-globulin (ATG); reactive oxygen species (ROS); serine protease