TEMPORAL AND SPATIAL-DISTRIBUTION OF DNA TOPOISOMERASE-II ALTERS DURING PROLIFERATION, DIFFERENTIATION, AND APOPTOSIS IN HL-60 CELLS

We related cellular content of DNA topoisomerase (topo) II alpha and I I beta with the cell cycle position in proliferating, differentiated, and apoptotic HL-60 cells using two-dimensional flow cytometry. In log arithmically growing HL-60 cells, topo II alpha increased especially i n late S to G2/M phases, although the topo II beta level was almost co nstant throughout the cell cycle. Induction of differentiation by all- trans retinoic acid dramatically reduced the topo II alpha but not the topo II beta level. A new G2/M population containing virtually no top o II alpha appeared during differentiation and was supposed to be aliv e and noncycling. Two-dimensional flow cytometry of topo II alpha or I I beta staining and terminal deoxynucleotidyl transferase-mediated dUT P-biotin nick end-labeling assay, showed that one topo II beta 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 II alpha epitope and another topo II beta epitope located at a more central portion were almost equal between apoptotic and nonapo ptotic cells. Western blot analysis confirmed that topo II beta protei n was completely degraded into smaller fragments and lost its C-termin al end during apoptosis. On the contrary, a large portion of topo II a lpha remained of its original size, although both topo II alpha and II beta left from the nuclear fraction in apoptotic cells. Confocal lase r microscopy showed nuclear localization of topo II alpha and II beta in growing HL-60 cells. Although topo II alpha and II beta were distri buted throughout the cell during mitosis, only topo II alpha was dense ly concentrated in the mitotic chromosomes. Both enzymes were dissocia ted from the genomic DNA even at an early phase of apoptosis and compl etely separated from the propidium iodide signal of DNA in the advance d stage. Chromatin condensation process in apoptosis is therefore comp letely topo II-independent and obviously differs from the mitotic one. (C) 1998 by The American Society of Hematology.