Genetic analysis of the short splice variant of the inhibitor of caspase-activated DNase (ICAD-S) in chicken DT40 cells.
We have studied the regulation of the caspase-Activated DNase (CAD) by its inhibitor, ICAD. To study the role of ICAD short and long splice forms ICAD-S and ICAD-L, respectively, in vivo, we constructed chicken DT40 cell lines in which the entire coding regions of ICAD alone or ICAD plus CAD were deleted. ICAD and ICAD/CAD double knock-outs lacked both DNA fragmentation and nuclear fragmentation after the induction of apoptosis. We constructed a model humanized system in which human ICAD-L and CAD proteins expressed in DT40 ICAD/CAD double knock-out cells could rescue both DNA fragmentation and stage II chromatin condensation. ICAD-S could not replace ICAD-L as a chaperone for folding active CAD in these cells. However, a modified version of ICAD-S, in which the two caspase-3 cleavage sites were replaced with two tobacco etch virus (TEV) protease cleavage sites (ICAD-S(2TEV)) and which was therefore resistant to caspase cleavage, did inhibit CAD activation upon induction of apoptosis in vivo. Moreover, ICAD-L(2TEV) was functional as a chaperone for the production of active CAD in DT40 cells. In extracts prepared from these cells, we were able to activate CAD by cleavage of ICAD-L(2TEV) with TEV protease under non-apoptotic conditions. Thus, ICAD appears to be the only functional inhibitor of CAD activation in these cell-free extracts. Taken together, these observations indicate that ICAD-S may function together with ICAD-L as a buffer to prevent inappropriate CAD activation, particularly in cells where ICAD-S is the dominant form of ICAD protein.
Ageichik
Alexander V
AV
Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Swann Building, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, United Kingdom and the.
Samejima
Kumiko
K
Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Swann Building, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, United Kingdom and the.
Kaufmann
Scott H
SH
Department of Molecular Pharmacology and Division of Oncology Research, Mayo Clinic College of Medicine, Rochester, Minnesota 55905.
Earnshaw
William C
WC
Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Swann Building, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, United Kingdom and the. Electronic address: Bill.Earnshaw@ed.ac.uk.
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D003851:Deoxyribonucleases / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D012326:RNA Splicing
D051017:Apoptosis Regulatory Proteins; C109664:caspase-activated DNase inhibitor; D004247:DNA; D003851:Deoxyribonucleases; C109663:caspase-activated deoxyribonuclease