title: DNA topoisomerase II immunostaining in human leukemia and rhabdomyosarcoma cell lines and their responses to topoisomerase II inhibitors.
abstract: DNA topoisomerase II is an enzyme that affects nuclear structure and function and is the target of a number of anticancer drugs in clinical use, including teniposide (VM-26). We have used our polyclonal antisera that recognize both the M(r) 170,000 and 180,000 forms of topoisomerase II to examine the nuclear distribution of topoisomerase II in cytospin preparations of drug-sensitive (CEM) and VM-26-resistant (CEM/VM-1 and CEM/VM-1-5) human leukemic lymphoblasts. We have also examined the nuclear distribution of topoisomerase II in monolayer cultures of a human rhabdomyosarcoma (Rh30) cell line. In the absence of drug, we observed a focal "patchy" staining of nuclear topoisomerase II in all cell lines, that was especially notable in the lymphoblastic cells. Treatment of CEM and Rh30 cells with VM-26 under conditions that increase the number of covalent topoisomerase II-DNA complexes increased both the intensity and the homogeneity of nuclear topoisomerase II staining in a subpopulation of cells; focal staining was less evident after treatment with drug. These responses were roughly proportional to the concentration of VM-26 used and required only brief (approximately 25-min) incubation with drug. We also found that treatment of CEM cells with 4'-(9-acridinylamino)methanesulfon-m-anisidide similarly increased the intensity and homogeneity of nuclear topoisomerase II immunostaining. In contrast, 4'-(9-acridinylamino)methanesulfon-o-anisidide and 1-beta-D-arabinofuranosylcytosine, agents that do not inhibit topoisomerase II, did not produce this effect. Finally, the VM-26-mediated alteration in topoisomerase II staining intensity and distribution was attenuated in proportion to the degree of VM-26 resistance in the CEM/VM-1 and CEM/VM-1-5 sublines. These results appear to be related to the ability of the drug to stabilize DNA-topoisomerase covalent ("cleavable") complexes in intact cells. Our findings indicate that anti-topoisomerase II drugs, such as VM-26, have profound effects on the ability to detect topoisomerase II in the nucleus and provide a novel way of examining drug-stabilized DNA topoisomerase II complexes in intact single tumor cells.
authors.affiliation: Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
authors.forename: J S
authors.initials: JS
authors.lastname: Wolverton
authors.forename: M K
authors.initials: MK
authors.lastname: Danks
authors.forename: B
authors.initials: B
authors.lastname: Granzen
authors.forename: W T
authors.initials: WT
authors.lastname: Beck
mesh_terms: D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004250:DNA Topoisomerases, Type II / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D007700:Kinetics; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000201:enzymology*; D012208:Rhabdomyosarcoma / Q000201:enzymology*; D013713:Teniposide / Q000494:pharmacology*; D013997:Time Factors; D059005:Topoisomerase II Inhibitors; D014407:Tumor Cells, Cultured
chemical_list: D059005:Topoisomerase II Inhibitors; D013713:Teniposide; D004250:DNA Topoisomerases, Type II