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 IIα 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 IIα. Tryptic phosphopeptide mapping of topo IIα 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 IIα are possibly involved in regulating the DNA damage caused by and the cytotoxic potential of topo II poisons.
Attenuation of drug-stimulated topoisomerase II–DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIα
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Masako AOYAMA, Dale R. GRABOWSKI, George R. DUBYAK, Andreas I. CONSTANTINOU, Lisa A. RYBICKI, Ronald M. BUKOWSKI, Mahrukh K. GANAPATHI, Ian D. HICKSON, Ram GANAPATHI; Attenuation of drug-stimulated topoisomerase II–DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIα. Biochem J 15 December 1998; 336 (3): 727–733. doi: https://doi.org/10.1042/bj3360727
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