Subcellular distribution of the α and β topoisomerase II-DNA complexes stabilized by VM-26

Abstract

Studies were done to determine (a) the subcellular distribution of the α (170 kDa) and β (180 kDa) isozymes of topoisomerase II, and (b) the extent to which each isozyme forms complexes with DNA in tumor cells incubated with and without VM-26. Western blotting revealed that topoisomerase IIβ was highly unstable during cell fractionation. However, preincubation of human CEM leukemia cells with 5–100 μM VM-26 for 30 min protected the β isozyme from degradation by progressively increasing the amount of this isoform bound to DNA. The amount of topoisomerase IIβ detected in nuclei of CEM cells incubated for 30 min with 25 μM VM-26 was 7-fold greater than in nuclei from untreated control cells. VM-26 also had a protective effect on topoisomerase IIβ in HL-60 leukemia and WiDR colon carcinoma cells. In contrast, the intercalating agents mitoxantrone and m-AMSA did not protect topoisomerase IIβ from degradation during cell fractionation. The stabilization of topoisomerase IIβ by VM-26 allowed subsequent studies of the subcellular distribution of the topoisomerase II isozymes. Both isozymes were detected in the nonmatrix (high salt-soluble) fraction of nuclei from CEM cells, but only topoisomerase IIα was present in the nuclear matrix. VM-26 stabilized binding of the α and β topoisomerase II isoenzymes to nonmatrix DNA and topoisomerase IIα to matrix DNA. The differences observed in the subnuclear distribution and DNA binding pattern of the topoisomerase II isozymes support the hypotheses that each isozyme has a distinct cellular function, and that both the α and β isozymes are potential targets for VM-26 in intact cells. In addition, the results demonstrated that pretreatment of various cell lines with VM-26is a useful way to stabilize topoisomerase IIβ during cell fractionation.