Attenuation of drug-stimulated topoisomerase II DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium bufferis correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase II alpha

Topoisomerase II (topo II), an essential enzyme for cell viability, is also the target for clinically important anti-neoplastic agents that stimulate toro II-mediated DNA scission. The role of alterations in toro II alpha pho sphorylation and its effect on drug-induced DNA damage and cytotoxicity wer e investigated. Follow ing loading of HL-60 cells with the calcium buffer 1 ,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetra(acetoxymeth yl) ester (BAPTA-AM), which abrogates intracellular Ca2+ transients, a sign ificant decrease in etoposide (VP-16)- or amsacrine (m-AMSA)-stabilized top o, II-DNA cleavable complex formation and a corresponding decrease in cytot oxicity was observed, In a cell-free system, nuclear extracts from BAPTA-AM -treated cells exhibited markedly less activity when assayed for VP-16-stab ilized topo II-DNA complex formation, but not decatenation of kinetoplast D NA. In contrast, the loading of HL-60 cells with N,N,N',N'-tetrakis-(2-pyri dyl)ethylenediamine (TPEN), which binds heavy metals without disturbing cal cium or magnesium concentrations, did not significantly affect VP-16-stimul ated topo II-DNA cleavable complex formation or cytotoxicity. In HL-60 cell s the accumulation of BAPTA, but not TPEN, also led to the hypophosphorylat ion of toro II alpha. Tryptic phosphopeptide mapping of topo IIa protein fr om HL-60 cells revealed: (a) eight major phosphorylation sites in untreated cells; (b) hypophosphorylation of two out of eight sites in BAPTA-AM-treat ed cells; and (c) hypophosphorylation of between two and four out of eight sites in topo II-poison-resistant HL-60 cells. The two hypophosphorylated s ites 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 su mmary, changes in intracellular Ca2+ transients that lead to the site-speci fic hypophosphorylation of topo II alpha are possibly involved in regulatin g the DNA damage caused by and the cytotoxic potential of topo II poisons.