Regulation of topoisomerase II (TOPO II) isozymes  and β is influenced by the growth and transformation state of cells. Using HL-60 cells induced to differentiate by all-trans retinoic acid (RA), we have investigated the expression and regulation of TOPO II isozymes as well as the levels of topoisomerase I (TOPO I). During RA-induced differentiation of human leukemia HL-60 cells, levels of TOPO I remained unchanged, whereas the levels and phosphorylation of TOPO II and TOPO IIβ proteins were increased twofold to fourfold and fourfold to eightfold, respectively. The elevation of TOPO II ( and β) protein levels and phosphorylation was apparent at 48 hours of treatment with RA and persisted through 96 hours. The increased level of TOPO IIβ protein was also detected in differentiated cells subsequently cultured for 96 hours in RA-free medium. Pulse chase experiments in cells labeled with 35S-methionine showed that the rate of degradation of TOPO IIβ protein in control cells was about twofold faster than that in the differentiated RA-treated cells. The level of decatenation activity of kDNA was comparable in nuclear extracts from control or RA-treated cells. Whereas etoposide (1 to 10 μmol/L) -induced DNA cleavage was not significantly different, apoptosis was significantly lower (P = .012) in RA-treated versus control cells after exposure to 10 μmol/L etoposide. Consistent with unaltered levels of TOPO I, camptothecin (CPT) -induced DNA cleavage was similar in control or RA-treated cells. However, apoptosis after exposure to 1 to 10 μmol/L CPT was significantly lower (P = .003 to P < .001) in RA-treated versus control cells. Results suggest that TOPO IIβ protein levels are posttranscriptionally regulated and that degradation of TOPO IIβ is decreased during RA-induced differentiation. Furthermore, whereas the total level of TOPO II ( + β) is increased with RA, the level of TOPO II catalytic activity and etoposide-stabilized DNA cleavage activity remains unaltered. Thus, TOPO IIβ may have a specific role in transcription of genes involved in differentiation with RA treatment. © 1998 by The American Society of Hematology.

Topoisomerase II (topo II), an essential enzyme for cell viability, is also the target for clinically important anti-neoplastic agents that stimulate topo II-mediated DNA scission. The role of alterations in topo IIalpha phosphorylation and its effect on drug-induced DNA damage and cytotoxicity were investigated. Following loading of HL-60 cells with the calcium buffer 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetra(acetoxymethyl) ester (BAPTA-AM), which abrogates intracellular Ca2+ transients, a significant decrease in etoposide (VP-16)- or amsacrine (m-AMSA)-stabilized topo II-DNA cleavable complex formation and a corresponding decrease in cytotoxicity was observed. In a cell-free system, nuclear extracts from BAPTA-AM-treated cells exhibited markedly less activity when assayed for VP-16-stabilized topo II-DNA complex formation, but not decatenation of kinetoplast DNA. In contrast, the loading of HL-60 cells with N,N,N', N'-tetrakis-(2-pyridyl)ethylenediamine (TPEN), which binds heavy metals without disturbing calcium or magnesium concentrations, did not significantly affect VP-16-stimulated topo II-DNA cleavable complex formation or cytotoxicity. In HL-60 cells the accumulation of BAPTA, but not TPEN, also led to the hypophosphorylation of topo IIalpha. Tryptic phosphopeptide mapping of topo IIalpha protein from HL-60 cells revealed: (a) eight major phosphorylation sites in untreated cells; (b) hypophosphorylation of two out of eight sites in BAPTA-AM-treated cells; and (c) hypophosphorylation of between two and four out of eight sites in topo II-poison-resistant HL-60 cells. The two hypophosphorylated sites present following BAPTA-AM treatment of wild-type cells were identical with the hypophosphorylated sites in the resistant cells, but were not the same as the sites that are substrates for casein kinase II [Wells, Addison, Fry, Ganapathi and Hickson (1994) J. Biol. Chem. 269, 29746-29751]. In summary, changes in intracellular Ca2+ transients that lead to the site-specific hypophosphorylation of topo IIalpha are possibly involved in regulating the DNA damage caused by and the cytotoxic potential of topo II poisons.

Thrombopoietin stimulated blast colony formation in 11/20 acute myelogenous leukaemia (AML) patients studied. The FAB subtypes of the blasts responding to thrombopoietin were not restricted to those of the megakaryocyte lineage, but also included M1-M5 AML blasts. The morphology of colony cells produced by megakaryocytic blasts showed megakaryocytoid features, whereas colony cells produced by M1-M5 AML blasts remained myeloblasts. An increase in CD41 was observed in the cells of colonies produced by blasts from the megakaryocyte lineage involving leukaemia and chronic myeloid leukaemia in blastic crisis. Thrombopoietin receptor was observed on leukaemic blasts which formed colonies following incubation with thrombopoietin.