Bl. Sailer et al., APOPTOSIS INDUCED WITH DIFFERENT CYCLE-PERTURBING AGENTS PRODUCES DIFFERENTIAL CHANGES IN THE FLUORESCENCE LIFETIME OF DNA-BOUND ETHIDIUM-BROMIDE, Cytometry, 31(3), 1998, pp. 208-216
Fluorescence lifetime analysis was used in combination with convention
al flow cytometric analysis to monitor changes in residual chromatin i
n apoptotic HL-60 cell populations following treatment with camptothec
in, cycloheximide, genistein, H7, and gamma radiation, Data presented
show that all of these metabolic inhibitors, which act through differe
nt signaling cascades, produce apoptotic subpopulations with decreased
but different lifetimes for DNA-bound ethidium bromide (EB). Addition
ally,, treatment with certain agents reduced the fluorescence lifetime
in the apoptotic cells prior to extensive endonuclease degradation of
DNA and the appearance of the typical sub-G(0)/G(1) peak in the DNA h
istogram, A Lifetime value of 21.15 +/- 0.12 ns was obtained for EB bo
und to nonapoptotic cells, while values for EB bound to the apoptotic
subpopulations following treatment with the different agents were: cam
ptothecin, 19.87 +/- 0.08 ns; cycloheximide, 19.39 +/- 0.02 ns; H7, 19
.77 +/- 0.03 ns; genistein, 20.04 +/- 0.04 ns; and gamma radiation, 19
.67 +/- 0.03 ns. Traditional methods of analysis, including gel electr
ophoresis or morphology assessment, revealed no significant difference
s among apoptotic subpopulations induced by treatment with these agent
s, Our data suggest that the mode of action of the various agents indu
ces structural changes in chromatin organization that differentially a
lter accessibility of DNA to endonuclease digestion. Subsequent fluore
scence lifetime analysis appears sensitive to the resulting difference
s in the residual chromatin in apoptotic cells following DNA cleavage,
Results presented indicate that lifetime analysis, used in conjunctio
n with conventional flow cytometry, can be useful for early detection
of apoptosis-induced chromatic changes and may also potentially provid
e new information on the effects of different apoptosis-induced agents
. (C) 1998 Wiley-Liss, Inc.