Resistance to chemotherapeutic drugs is a major obstacle in the treatment of leukemia and multiple myeloma. We have previously found that myeloma and leukemic cells in transition from low-density log phase conditions to high-density plateau phase conditions export substantial amounts of endogenous topoisomerase II alpha from the nucleus to the cytoplasm. In order for topoisomerase-targeted chemotherapy to function, the topoisomerase target must have access to the nuclear DNA. Therefore, the nuclear export of topoisomerase II alpha may contribute to drug resistance, and defining this mechanism may lead to methods to preclude this avenue of resistance. We have identified nuclear export signals for topoisomerase II alpha at amino acids 1017-1028 and 1054-1066, using FITC-labeled BSA-export signal peptide conjugates microinjected into the nuclei of HeLa cells. Functional confirmation of both signals (1017-1028 and 1054-1066) was provided by transfection of human myeloma cells with plasmids containing the gene for a full-length human FLAG-topoisomerase fusion protein, mutated at hydrophobic amino acid residues in the export signals. Of the six putative export signals tested, the two sites above were found to induce export into the cytoplasm. Export by both signals was blocked by treatment of the cells with leptomycin B, indicating that a CRM-1-dependent pathway mediates export. Site-directed mutagenesis of two central hydrophobic residues in either export signal in full-length human topoisomerase blocked export of recombinant FLAG-topoisomerase II alpha, indicating that both signals may be required for export. Interestingly, this pair of nuclear export signals (1017-1028 and 1054-1066) also defines a dimerization domain of the topoisomerase II alpha molecule.

Summary. The resistance of several leukaemic and myeloma cell lines (CCRF, L1210, HL-60, KG-1a and RPMI 8226) to VP-16 was found to increase with cell density and to be maximal (3´5-to 39-fold) in plateau phase cell cultures, as measured by clonogenic and MTT assays. Non-transformed con¯uent Flow 2000 human ®broblasts and Chinese hamster ovary (CHO) cells were also ®ve-and 15-fold resistant to VP-16 respectively. The transition from log to plateau phase was accompanied by a drastic decrease in topoisomerase (topo) IIa content in CHO cells and human ®broblasts, while the leukaemic cells maintained constant cellular levels of topo IIa and topo IIb. However, the nuclear topo IIa content was found to decrease as a result of translocation of the enzyme to the cytoplasmic compartment in the leukaemic cells. This was con®rmed by subcellular fractionation experiments, Western blotting analyses and immunocytochemistry studies. The quantity of topo IIa in plateau phase cytoplasmic fractions ranged from 18% in L1210 cells to 50% in HL-60 and 8226 cells, as measured by both immunoblotting and quanti®cation of the label in immuno¯uorescent images. The cytoplasmic fraction from plateau phase cells retained topo II catalytic activity, as measured by the decatenation of kinetoplast DNA. The nuclear±cytoplasmic ratio of topo IIa may be critical in determining the sensitivity of leukaemic cells to topo II inhibitors. Cytoplasmic traf®cking of topo IIa was observed in plasma cells obtained from patients with multiple myeloma, and perhaps contributes to drug resistance in this disease.