The DNA of annexin V-binding apoptotic cells is highly fragmented

Jurkat leukemia cells induced to undergo apoptosis by treatment with an ant ibody against the Pas receptor have two annexin V (AV)-binding subpopulatio ns: (a) single-positive cells that bind AV but not propidium iodide Bn; and (b) double-positive cells that bind AV and PI. The single-positive populat ion is thought to represent an early stage of apoptosis. We have examined t he relationship between AV binding and a classical characteristic of apopto sis, DNA fragmentation. Time course studies with Jurkat cells treated for 1 , 2, or 4 h with anti-Fas indicated that the proportion of AV-binding cells was increased after 2 h, A significant increase in DNA fragmentation was o bserved only at 4 h as measured by the mean tail moment determined with the alkaline single cell gel electrophoresis (comet) assay. This correlation s uggests a temporal relationship between the two parameters, but does not pr ovide direct evidence of what happens in individual cells. We developed a m ethod to measure fluorescent markers of cellular structure or function with a laser scanning cytometer and then perform the comet assay on the same ce lls. Cells in each AV-binding subpopulation were re-examined before and aft er electrophoresis, Most AV(-)/PI- cells had no DNA damage, although a few cells showed a pattern of damage characteristic for apoptosis, Double-posit ive cells all had damaged DNA; approximately half had the apoptotic pattern , and the rest had a pattern typical for necrosis, Nearly all of the single -positive cells had damaged DNA with the apoptotic pattern, Both AV-positiv e populations contained cells with little or no detectable DNA after electr ophoresis, indicating that the DNA was highly fragmented, These results ind icate that AV binding is an excellent marker for apoptotic cells, but that these cells already have fragmented DNA.