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 IIalpha.
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.
Aoyama
M
M
Experimental Therapeutics Program, Taussig Cancer Center, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
Grabowski
D R
DR
Dubyak
G R
GR
Constantinou
A I
AI
Rybicki
L A
LA
Bukowski
R M
RM
Ganapathi
M K
MK
Hickson
I D
ID
Ganapathi
R
R
D000951:Antigens, Neoplasm / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D002021:Buffers; D002118:Calcium; D002614:Chelating Agents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004317:Doxorubicin / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005029:Ethylenediamines / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D010449:Peptide Mapping; D010641:Phenotype; D010766:Phosphorylation
D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D002021:Buffers; D002614:Chelating Agents; D004268:DNA-Binding Proteins; D005029:Ethylenediamines; D007527:Isoenzymes; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D004317:Doxorubicin; D004247:DNA; D004250:DNA Topoisomerases, Type II; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; D002118:Calcium