Subcellular distribution of the alpha and beta topoisomerase II-DNA complexes stabilized by VM-26.
Studies were done to determine (a) the subcellular distribution of the alpha (170 kDa) and beta (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 II beta was highly unstable during cell fractionation. However, preincubation of human CEM leukemia cells with 5-100 microM VM-26 for 30 min protected the beta isozyme from degradation by progressively increasing the amount of this isoform bound to DNA. The amount of topoisomerase II beta detected in nuclei of CEM cells incubated for 30 min with 25 microM VM-26 was 7-fold greater than in nuclei from untreated control cells. VM-26 also had a protective effect on topoisomerase II beta in HL-60 leukemia and WiDR colon carcinoma cells. In contrast, the intercalating agents mitoxantrone and m-AMSA did not protect topoisomerase II beta from degradation during cell fractionation. The stabilization of topoisomerase II beta 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 II alpha was present in the nuclear matrix. VM-26 stabilized binding of the alpha and beta topoisomerase II isoenzymes to nonmatrix DNA and topoisomerase II alpha 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 alpha and beta isozymes are potential targets for VM-26 in intact cells. In addition, the results demonstrated that pretreatment of various cell lines with VM-26 is a useful way to stabilize topoisomerase II beta during cell fractionation.
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38101.
M K
MK
Danks
J
J
Qiu
C V
CV
Catapano
C A
CA
Schmidt
W T
WT
Beck
D J
DJ
Fernandes
D000677:Amsacrine / Q000494:pharmacology; D000951:Antigens, Neoplasm; D002467:Cell Nucleus / Q000201:enzymology; D004247:DNA / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015530:Nuclear Matrix / Q000201:enzymology; D013347:Subcellular Fractions / Q000378:metabolism*; D013713:Teniposide / Q000494:pharmacology*; D059005:Topoisomerase II Inhibitors; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D059005:Topoisomerase II Inhibitors; D000677:Amsacrine; D004247:DNA; D013713:Teniposide; D003850:Deoxyribonuclease I; D004250:DNA Topoisomerases, Type II