Detection of DNA cleavage in apoptotic cells.
At least two discrete deoxyribonuclease activities can be detected during apoptotic death, one that generates 30- to 500-kilobase pair (kbp) domain-sized fragments and another that mediates internucleosomal DNA degradation. The latter nuclease has been identified as the caspase-activated deoxyribonuclease (CAD)/CPAN, a unique enzyme that is normally inhibited by the regulatory subunit ICAD (inhibitor of CAD)/DFF45 (DNA fragmentation factor). In this chapter, techniques widely used to detect DNA cleavage in apoptotic cells, including pulsed-field gel electrophoresis, conventional agarose gel electrophoresis, and terminal transferase-mediated dUTP nick end-labeling (TUNEL), are briefly reviewed. In addition, the use of ICAD to inhibit apoptosis-associated nuclease activity is illustrated. When properly applied, these techniques are widely applicable to the characterization of apoptotic cells.
Kaufmann
S H
SH
Division of Oncology Research, Mayo Clinic, Rochester, Minnesota, USA.
Mesner
P W
PW
Samejima
K
K
Toné
S
S
Earnshaw
W C
WC
D017209:Apoptosis; D002467:Cell Nucleus / Q000502:physiology / Q000648:ultrastructure; D004247:DNA / Q000032:analysis* / Q000187:drug effects; D053938:DNA Fragmentation; D004587:Electrophoresis, Agar Gel / Q000379:methods; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D005047:Etoposide / Q000633:toxicity; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D020287:In Situ Nick-End Labeling / Q000379:methods; D007202:Indicators and Reagents
D007202:Indicators and Reagents; D005047:Etoposide; D004247:DNA