A drug-resistant variant of topoisomerase II alpha in human HL-60 cells exhibits alterations in catalytic pH optimum, DNA binding and sub-nuclear distribution

Abstract

Anion-exchange chromatography of partially purified human HL-60 topoisomerase II resolves the known alpha (170 kDa) and beta (180 kDa) isoenzymes at 150 mM NaCl and 230 mM NaCl, respectively. An additional topoisomerase II fraction was eluted by > 300 mM NaCl. It could be identified by Western blotting as a late-eluting variant of topoisomerase II alpha, which is functionally altered as compared to the early-eluting form, having the following properties: a shift in the catalytic optimum to pH 9; increased stability in DNA complex formation; approximately 100-fold resistance to orthovanadate; approximately 1000-fold resistance to the cytostatic substances N-[4-(9-acridinylamino)-3-methoxyphenyl]-methanesulphonamide (amsacrine) and the podophyllotoxin etoposide (VP 16). 80% of the late-eluting topoisomerase II alpha could be captured by SDS on calf thymus DNA without further enhancement by drugs. In contrast, the early-eluting topoisomerase II alpha exhibits 10% complex formation with SDS alone, and an increase to 90% complex formation in the presence of drugs. A HL-60 subline (HL-60/R), approximately 1000-fold resistant to etoposide and amsacrine, has equivalent proportions of topoisomerase II alpha and topoisomerase II beta and similar levels of both isoenzymes, as compared to the drug-sensitive HL-60/WT cells. However, determination of the cellular levels of the early-eluting and late-eluting forms of topoisomerase II alpha revealed that the HL-60/R cell line contains approximately 80% of the late-eluting topoisomerase II alpha, whereas the sensitive HL-60/WT cell line contains only 15-20% of this form. The nuclear distribution of the two forms also differs. Sensitive HL-60/WT cells show a diffuse nuclear distribution but in resistant cells the distribution is localized in the nucleoli. Apparently two functionally distinct subforms of topoisomerase II alpha coexist in drug-sensitive and drug-resistant HL-60 cells and changes in their relative levels affect the cellular sensitivity to topoisomerase-II-targeting drugs.