Ectopic expression of human topoisomerase IIα fragments and etoposide resistance in mammalian cells

Ernst, Angelika; Soltermann, Alex; Sigrist, Jürg A.; Widmer, Lukas; Gasser, Susan M.; Stahel, Rolf A.

doi:10.1002/1097-0215(20001001)88:1<99::aid-ijc16>3.0.co;2-4

Cited by 9 publications

(4 citation statements)

References 19 publications

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“…This could result from cytoplasmic topo IIα serving as a drug sink, by trapping VP-16 in this compartment (Ernst et al, 2000). This is supported by data demonstrating that the binding of VP-16 to topo II can occur in the absence of DNA (Burden et al, 1996).…”

Section: Discussionmentioning

confidence: 80%

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

Turner

Derderian

et al. 2004

Journal of Cell Science

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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.

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Section: Discussionmentioning

confidence: 80%

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

Turner

Derderian

et al. 2004

Journal of Cell Science

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“…1A) (Mirski et al, , 1999, this truncated TOP2a does contain two additional putative bipartite NLS sequences, NLS 606-676 and NLS 727-743 . Fusion-truncated forms of human TOP2a, which contained these NLS sequences, were excluded from the nucleus, suggesting that these NLS motifs were nonfunctional Ernst et al, 2000). TOP2a/90 was analyzed for additional NLS sequences due to the inclusion of the unique 25-aa C-terminal sequence encoded by a processed intron 19 (i.e., aa 762-786: VNTQSMFPESII-SEIPAESFSKQIW) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In contrast, if dominant-negative TOP2a/90: TOP2a/170 heterodimers form in the nucleus, then this would suggest that the truncated isoform is imported by an uncharacterized NLS sequence. Also, since C-terminal truncated TOP2a isoforms, which lack NLS, are mostly excluded from the nucleus (Vassetzky et al, 1996, Soltermann et al, 1999Ernst et al, 2000), yet can bind to and inhibit etoposide activity (Leroy et al, 2001;Vilain et al, 2003), TOP2a/90 may sequester etoposide prior to nuclear entry. Additional studies are ongoing to investigate these potential mechanisms.…”

Section: Discussionmentioning

confidence: 99%

The Novel C-terminal Truncated 90-kDa Isoform of Topoisomerase IIα (TOP2α/90) Is a Determinant of Etoposide Resistance in K562 Leukemia Cells via Heterodimerization with the TOP2α/170 Isoform

et al. 2018

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DNA topoisomerase II (170 kDa, TOP2/170) is essential in proliferating cells by resolving DNA topological entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated isoform (TOP2/90), detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide. TOP2/90 (786 aa) is the translation product of a TOP2 mRNA that retains a processed intron 19. TOP2/90 lacks the active-site tyrosine-805 required to generate double-strand DNA breaks as well as nuclear localization signals present in the TOP2/170 isoform (1531 aa). Here, we found that TOP2/90, like TOP2/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Coimmunoprecipitation of endogenous TOP2/90 and TOP2/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2/90 in K562 cells suppressed, whereas siRNA-mediated knockdown of TOP2/90 in K/VP.5 cells enhanced, etoposide-mediated DNA strand breaks compared with similarly treated cells transfected with empty vector or control siRNAs, respectively. In addition, forced expression of TOP2/90 in K562 cells inhibited etoposide cytotoxicity assessed by clonogenic assays. qPCR and immunoassays demonstrated TOP2/90 mRNA and protein expression in normal human tissues/cells and in leukemia cells from patients. Together, results strongly suggest that TOP2/90 expression decreases drug-induced TOP2-DNA covalent complexes and is a determinant of chemoresistance through a dominant-negative effect related to heterodimerization with TOP2/170. Alternative processing of TOP2 pre-mRNA, and subsequent synthesis of TOP2/90, may be an important mechanism regulating the formation and/or stability of cytotoxic TOP2/170-DNA covalent complexes in response to TOP2-targeting agents.

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“…Nevertheless, it has been reported that truncation of 366 residues of the COOH‐terminal led to full loss of catalytic activity, ( 31 ) whereas the expression of the NH 2 ‐terminal fragments of topo II alpha are rapidly degraded. ( 32 ) Consequently, if the smaller 2.0 kb mRNA is traduced, it will produce a non‐functional enzyme or an unstable protein.…”

Section: Discussionmentioning

confidence: 99%

Characterization of human NSCLC cell line with innate etoposide‐resistance mediated by cytoplasmic localization of topoisomerase II alpha

Lucio

Manuel

Rodríguez³

2005

Cancer Science

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(1) This enzyme is essential for the viability of cells because it is involved in chromosome segregation and is the major component of the nuclear matrix and mitotic and meiotic chromosomes.(2) Topo II alpha is a major target for a variety of antineoplastic agents. Epipodophyllotoxins (etoposide [VP16] ), teniposide (VM-26), aminocridines (m-AMSA), anthracyclines, anthracenediones, doxorubicin, daunorubicin, mitoxantrone and actinomycin D, are examples of such agents and are collectively referred to as topo II poisons.(3) These agents allow topo II to cleave DNA, and slow the religation steps catalyzed by the enzyme. As a consequence, covalent topo II breaks accumulate in the cell and produce cell death. (4) Resistance to topo II-targeting drugs in drug-selected cell lines has been associated with reduced expression of the topo II alpha enzyme. In addition, a number of point mutations or deletions in topo II alpha have been described in resistant cell lines. These mutations tend to be clustered in either the NH 2 -proximal ATP-binding domain (5)(6)(7)(8) or near to the active site of tyrosine residue in the central DNA binding and religation domain.(9 -12) More recently, a third cluster of mutations and deletions has been described in the COOH-terminal region of the enzyme, all of which result in the aberrant localization of topo II alpha in the cytoplasm. (13 -25) The presence of a cytoplasmic enzyme provides a plausible explanation for the resistance of topo II poisons, as it would result in the formation of fewer topo II/DNA complexes that could be stabilized by topo II-targeting drugs and, hence, reduce drug sensibility.The first description of topo II poison resistance and topo II alpha cytoplasmic localization was found in the small-cell lung cancer (SCLC) cell line H69 / VP with resistance to etoposide, (13,14) and later to several other topo II poison-resistant cell lines representative from different tissues. (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) In all these cell lines, deletions in the TOP2A gene around the nucleotide 4267 conduce to an alternative splicing that is translated into a truncated 160 kDa topo II alpha with cytoplasmic localization. A remarkable finding of these studies is that the truncation affected at least one of the two bipartite nuclear localization signals (NLS) present in the COOHterminal region of topo II alpha. (26) In these cells, topo II alpha retains most of its catalytic activity intact, but a significant reduction in the nuclear enzyme concentration has been used to explain the increase in topo II poison resistance.In the present report, we characterized a non-small-cell lung cancer (NSCLC) cell line with innate resistance to etoposide. This resistance could be associated with the expression of topo II alpha mainly in the cytoplasmic region. INER-37 cells transcribe two topo II alpha mRNAs: 4.8 kb and 2.0 kb. The large transcript appears to be translated into a truncated protein product of 160 kDa, where the more proximal COOH-terminal NLS 1454 −1497 has been lost. ...

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Ectopic expression of human topoisomerase IIα fragments and etoposide resistance in mammalian cells

Cited by 9 publications

References 19 publications

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

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

The Novel C-terminal Truncated 90-kDa Isoform of Topoisomerase IIα (TOP2α/90) Is a Determinant of Etoposide Resistance in K562 Leukemia Cells via Heterodimerization with the TOP2α/170 Isoform

Characterization of human NSCLC cell line with innate etoposide‐resistance mediated by cytoplasmic localization of topoisomerase II alpha

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