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DNA Topoisomerase II Immunostaining in Human Leukemia and Rhabdomyosarcoma Cell Lines and Their Responses to Topoisomerase II Inhibitors¹

Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101 [J. S. W., M. K. D., B. G., W. T. B.], and Department of Pharmacology, College of Medicine, University of Tennessee, Memphis, Tennessee 38163 [W. T. B.]

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 (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-ß-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.

¹ This work was supported in part by Research Grants CA-30103 and CA-40570 and Cancer Center Support (CORE) Grant CA-21765, all from the National Cancer Institute, DHHS, and in part by American Lebanese Syrian Associated Charities. B. G. is the recipient of a Fellowship from the Dr. Mildred Scheel Foundation, Federal Republic of Germany.

² To whom requests for reprints should be addressed, at the Department of Pharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38101.

Received 3/ 9/92. Accepted 5/13/92.

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