Hoechst 33342 induces apoptosis in HL-60 cells and inhibits topoisomerase I in vivo

Context-Bisbenzimides (Hoechst 33342 and Hoechst 33258) are cell-permeable, adenine-thymine-specific dyes that bind to the minor groove of DNA and sta in DNA. Hoechst 33342 induces apoptosis in BC3H-1 myocytes and hepatoma cel ls. Objective.-To determine if Hoechst 33342 or Hoechst 33258 induces apoptosis in human promyelocytic leukemia cells (HL-60) and inhibits topoisomerase I activity. Design.-A variety of methods were used to detect apoptosis: cell viability (trypan blue exclusion), nuclear fluorescence staining (Hoechst 33342 or Ho echst 33258 stained for 10 minutes), flow cytometric quantitation of annexi n binding to phosphatidylserine, and DNA fragmentation (agarose gel electro phoresis). Topoisomerase I activity was determined by a plasmid unwinding a ssay. Setting,A large teaching hospital and research laboratories. Patients.-None. Intervention.-None. Main Outcome Measurements.-Apoptosis is characterized by decreased cell via bility, condensation of nuclear chromatin, increased phosphatidylserine tra nslocation, and DNA fragmentation into oligonucleosomes composed of multipl es of 180 to 200 base pairs. Inhibition of endogenous nuclear topoisomerase I is detected by the absence of plasmid unwinding from a tightly coiled to relaxed form. Results.-Hoechst 33342, but not Hoechst 33258, induced apoptosis in the HL- 60 cells in a time- and dose-dependent manner. Endogenous nuclear topoisome rase I activity in HL-60 cells was inhibited by treatment with Hoechst 3334 2 but not Hoechst 33258. Conclusion.-Hooechs 33342-induced HL-60 cell apoptosis may be related to th e dye's inhibition of topoisomerase I activity.