Apoptosis induced with different cycle-perturbing agents produces differential changes in the fluorescence lifetime of DNA-bound ethidium bromide.
Fluorescence lifetime analysis was used in combination with conventional flow cytometric analysis to monitor changes in residual chromatin in apoptotic HL-60 cell populations following treatment with camptothecin, cycloheximide, genistein, H7, and gamma radiation. Data presented show that all of these metabolic inhibitors, which act through different signaling cascades, produce apoptotic subpopulations with decreased but different lifetimes for DNA-bound ethidium bromide (EB). Additionally, 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-G0/G1 peak in the DNA histogram. A lifetime value of 21.15 +/- 0.12 ns was obtained for EB bound to nonapoptotic cells, while values for EB bound to the apoptotic subpopulations following treatment with the different agents were: camptothecin, 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 electrophoresis or morphology assessment, revealed no significant differences among apoptotic subpopulations induced by treatment with these agents. Our data suggest that the mode of action of the various agents induces structural changes in chromatin organization that differentially alter accessibility of DNA to endonuclease digestion. Subsequent fluorescence lifetime analysis appears sensitive to the resulting differences in the residual chromatin in apoptotic cells following DNA cleavage. Results presented indicate that lifetime analysis, used in conjunction with conventional flow cytometry, can be useful for early detection of apoptosis-induced chromatin changes and may also potentially provide new information on the effects of different apoptosis-inducing agents.
Sailer
B L
BL
Los Alamos National Laboratory, Life Sciences Division, New Mexico 87545, USA.
Valdez
J G
JG
Steinkamp
J A
JA
Crissman
H A
HA
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000528:radiation effects; D002166:Camptothecin / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D002843:Chromatin; D003513:Cycloheximide / Q000494:pharmacology; D004273:DNA, Neoplasm / Q000032:analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004996:Ethidium; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes; D005720:Gamma Rays; D019833:Genistein / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology
D002843:Chromatin; D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D011500:Protein Synthesis Inhibitors; C080501:1-(5-isoquinolinylsulfonyl)-3-methylpiperazine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D003513:Cycloheximide; D019833:Genistein; D004996:Ethidium; D002166:Camptothecin