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

Abstract

Bisbenzimides (Hoechst 33342 and Hoechst 33258) are cell-permeable, adenine-thymine-specific dyes that bind to the minor groove of DNA and stain DNA. Hoechst 33342 induces apoptosis in BC3H-1 myocytes and hepatoma cells. To determine if Hoechst 33342 or Hoechst 33258 induces apoptosis in human promyelocytic leukemia cells (HL-60) and inhibits topoisomerase I activity. A variety of methods were used to detect apoptosis: cell viability (trypan blue exclusion), nuclear fluorescence staining (Hoechst 33342 or Hoechst 33258 stained for 10 minutes), flow cytometric quantitation of annexin binding to phosphatidylserine, and DNA fragmentation (agarose gel electrophoresis). Topoisomerase I activity was determined by a plasmid unwinding assay. A large teaching hospital and research laboratories. None. None. Apoptosis is characterized by decreased cell viability, condensation of nuclear chromatin, increased phosphatidylserine translocation, and DNA fragmentation into oligonucleosomes composed of multiples 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. Hoechst 33342, but not Hoechst 33258, induced apoptosis in the HL-60 cells in a time- and dose-dependent manner. Endogenous nuclear topoisomerase I activity in HL-60 cells was inhibited by treatment with Hoechst 33342 but not Hoechst 33258. Hoechst 33342-induced HL-60 cell apoptosis may be related to the dye's inhibition of topoisomerase I activity.