Temporal and Spatial Distribution of DNA Topoisomerase II Alters During Proliferation, Differentiation, and Apoptosis in HL-60 Cells

Abstract

AbstractWe related cellular content of DNA topoisomerase (topo) IIα and IIβ with the cell cycle position in proliferating, differentiated, and apoptotic HL-60 cells using two-dimensional flow cytometry. In logarithmically growing HL-60 cells, topo IIα increased especially in late S to G2/M phases, although the topo IIβ level was almost constant throughout the cell cycle. Induction of differentiation by all-trans retinoic acid dramatically reduced the topo IIα but not the topo IIβ level. A new G2/M population containing virtually no topo IIα appeared during differentiation and was supposed to be alive and noncycling. Two-dimensional flow cytometry of topo IIα or IIβ staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay showed that one topo IIβ epitope situated at the C-terminal end decreased specifically in apoptotic HL-60 cells treated with Ara-C, etoposide, and vincristine. The amounts of a topo IIα epitope and another topo IIβ epitope located at a more central portion were almost equal between apoptotic and nonapoptotic cells. Western blot analysis confirmed that topo IIβ protein was completely degraded into smaller fragments and lost its C-terminal end during apoptosis. On the contrary, a large portion of topo IIα remained of its original size, although both topo IIα and IIβ left from the nuclear fraction in apoptotic cells. Confocal laser microscopy showed nuclear localization of topo IIα and IIβ in growing HL-60 cells. Although topo IIα and IIβ were distributed throughout the cell during mitosis, only topo IIα was densely concentrated in the mitotic chromosomes. Both enzymes were dissociated from the genomic DNA even at an early phase of apoptosis and completely separated from the propidium iodide signal of DNA in the advanced stage. Chromatin condensation process in apoptosis is therefore completely topo II-independent and obviously differs from the mitotic one.