Abstract
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.
Highlights
Apoptosis is a form of programmed cell death leading to the elimination of damaged, harmful, or unwanted cells in a wide range of organisms [1, 2]
We have studied the regulation of the caspase-Activated DNase (CAD) by its inhibitor, ICAD
ICAD-L is a mandatory chaperone that must be present during the translation of CAD, as the CAD polypeptide formed in the absence of ICAD-L is unable to fold into an active conformation [4, 13]
Summary
2.7-kb ICAD open reading frame in DT40 cell lines using a targeting vector containing a 2.1-kb 5Ј-arm and a 3.4-kb 3Ј-arm from the noncoding regions. Flanked by mutant loxP sites (loxPpuro-tk-loxP cassette) was used. This was constructed by cloning the Herpes simplex virus tk gene from the plasmid pBT/SP-TK (gift of Adrian Bird) into the loxP-puroout of ICAD plus CAD in chicken DT40 cells. The (hICAD-L), human ICAD-S (hICAD-S), or both This system loxP-puro-tk-loxP cassette was removed by Cre-recombinase recapitulated the normal regulation of apoptotic DNA frag- expressed from transiently transfected vector pCAGGS-Cre mentation in the knock-out cells. In this system, ICAD-L could efficiently act as an inhibitory 6 –7 days to obtain individual colonies without drug selection. A chaperone and inhibitor, later ICAD-L can be efficiently Isolation of Human ICAD-L and CAD cDNAs—Total RNA replaced by ICAD-S, which works as an inhibitor but not as a from HeLa cells was isolated, and 5 g was used for first strand
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
