Detection of DNA cleavage in apoptotic cells

Methods Enzymol. 2000:322:3-15. doi: 10.1016/s0076-6879(00)22003-x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Apoptosis*
  • Cell Nucleus / physiology
  • Cell Nucleus / ultrastructure
  • DNA / analysis*
  • DNA / drug effects
  • DNA Fragmentation
  • Electrophoresis, Agar Gel / methods
  • Electrophoresis, Gel, Pulsed-Field / methods
  • Etoposide / toxicity
  • HL-60 Cells
  • HeLa Cells
  • Humans
  • In Situ Nick-End Labeling / methods
  • Indicators and Reagents

Substances

  • Indicators and Reagents
  • Etoposide
  • DNA