Human topoisomerase IIα nuclear export is mediated by two CRM-1-dependent nuclear export signals

Abstract

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.

Topoisomerase IIα mRNA and protein expression in ovarian carcinoma: correlation with clinicopathological factors and prognosis

“…In earlier cell culture studies, it was reported that nuclear export of Top IIa is mediated by CRM1, a mechanism, which could be blocked by the CRM1 inhibitor lepotomycin B (LMB). [33][34][35] Our data reveal an in vivo implication of nuclear export mechanisms in Top IIa cytoplasmic localization.…”

ABCG2 expression, function, and promoter methylation in human multiple myeloma

“…cDNA (2 L/well) was assayed and the QPCR performed as previously described. 31 The ABCG2 expression data were found to be log normally distributed. Consequently, the geometric means were used to average both within and between patient data.…”

“…We have shown previously that myeloma cells grown at different densities exhibit specific characteristics, including drug resistance to topoisomerase I and II inhibitors that depends on the nuclear to cytoplasmic trafficking of topoisomerases. 31,33,34 Myeloma cell lines (8226, H929, 8226MR) grown at 2 ϫ 10 5 cells/mL media were defined as low density (log phase), and cells grown at 2 ϫ 10 6 cells/mL were defined as high density (plateau phase). Cell lines were placed at log and plateau density conditions and grown for 24 hours at 37°C in 5% CO 2 .…”

Cancerous multi-drug resistance is reduced by Leptomycin B treatment in CCRF-CEM/Taxol cellsCancerous multi-drug resistance is reduced by Leptomycin B treatment in CCRF-CEM/Taxol cells

“…The second class consists of secondary active transporters, including the small MDR superfamily, the multidrug and toxic compound extrusion family, the resistance-nodulation-cell division family, and the major facilitator superfamily [2]. In addition, intracellular nuclear export of either tumor suppressive agents or drug targets (proteins, mRNAs, or DNAs) result in drug resistance due to overexpression of nucleo-cytoplasmic transporters, including human major vault proteins, represented by Lung Resistance-related Protein (LRP) [3], and nuclear export signal (NES) mediated nuclear export proteins, represented by Chromosomal region maintenance 1 (CRM1) [4,5].…”

“…As a specific inhibitor, LMB covalently binds to the individual binding-domain sulphydryl group of Cys-529 in CRM1 via it's α, β-unsaturated δ-lactone [11]. More recently, some studies reported that LMB exhibits antitumor activities by inhibiing nuclear export of topoisomerase Ⅱα in human myeloma cells, which enhances the cancerous cells sensitive to the topoisomerase Ⅱα-targeted anti-cancer drugs, such as etoposide (VP-16) [4,5]. Some other studies suggested that LMB also causes cell cycle G 1 arrest [13] and induces apoptosis by sequestrating NES-containing p53 or BCR-ABL in the nucleus [14,18].…”