Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation

J Cell Biol. 1992 Nov;119(3):493-501. doi: 10.1083/jcb.119.3.493.

Abstract

Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy-transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Apoptosis / physiology*
  • Cell Nucleus / metabolism*
  • Cell Nucleus / ultrastructure
  • Cells, Cultured
  • DNA / analysis
  • DNA / isolation & purification
  • DNA / metabolism*
  • Dexamethasone / pharmacology
  • Electrophoresis, Agar Gel
  • Female
  • Humans
  • Ileum / cytology
  • Intestine, Large / cytology
  • Kinetics
  • Male
  • Organ Specificity
  • Ovarian Follicle / cytology
  • Rats
  • Rats, Inbred Strains
  • Skin / cytology
  • Thymus Gland / cytology*
  • Thymus Gland / ultrastructure

Substances

  • Dexamethasone
  • DNA