E1A specifically enhances sensitivity to topoisomerase IIalpha targeting anticancer drug by up-regulating the promoter activity

DNA topoisomerases I and II (topo I and II) are nuclear enzymes involved in cellular replication and are targets for several anticancer drugs. We showed previously that E1A gene transfer enhanced the sensitivity of Ewing's sarcoma cells to the topo IIalpha targeting agents etoposide and Adriamycin in vitro and in vivo. To determine whether this effect was specific for topo IIalpha, we investigated the effect of E1A gene transfer on cell sensitivity to agents that target topo I and IIbeta. Transfecting TC71 human Ewing's sarcoma cells with an adenoviral vector containing the E1A gene enhanced their sensitivity to the topo IIalpha targeting agents etoposide (16-fold) and Adriamycin (8-fold). By contrast, E1A gene transfer did not affect cellular sensitivity to either amsacrine or camptothecin. Western blot analysis indicated that topo IIalpha protein levels increased 3.1-fold after E1A gene transfer, but topo I and IIbeta protein levels did not change. A plasmid containing topo IIalpha gene promoter with luciferase reporter gene was constructed to determine the effects of E1A gene transfer on the activity of the topo IIalpha promoter. E1A increased the activity of the topo IIalpha gene promoter by 3.5-fold relative to that of cells transfected with Ad-beta-gal. These results suggest that elevated topo IIalpha protein levels and enhanced sensitivity to topo IIalpha targeting agents were secondary to a direct effect of E1A on the topo IIalpha promoter. Combining E1A gene therapy with topo IIalpha targeting anticancer drugs may therefore have therapeutic benefit by increasing tumor cell sensitivity.     

Interaction between glucose-regulated destruction domain of DNA topoisomerase IIalpha and MPN domain of Jab1/CSN5

DNA topoisomerase (topo) IIalpha, an essential enzyme for cell proliferation, is targeted to a proteasome-dependent degradation pathway when human tumor cells are glucose-starved. Here we show that the topo IIalpha destabilization depends on the newly identified domain, GRDD (glucose-regulated destruction domain), which was mapped to the N-terminal 70-170 amino acid sequence. Indeed, the deletion of GRDD conferred a stable feature on topo IIalpha, whereas the fusion of GRDD rendered green fluorescent protein unstable under glucose starvation conditions. Nuclear localization was a prerequisite for GRDD function, because the inhibition of nuclear translocation resulted in the suppression of GRDD-mediated topo IIalpha degradation. Further, GRDD was identified as an interactive domain for Jab1/CSN5, which promoted the degradation of topo IIalpha in a manner dependent on the MPN (Mpr1p/Prd1p N terminus) domain. Depleting Jab1/CSN5 by antisense oligonucleotide and treating cells with the CSN-associated kinase inhibitor, curcumin, inhibited topo IIalpha degradation induced by glucose starvation. These findings demonstrate that GRDD can act as a stress-activated degron for regulating topo IIalpha stability, possibly through interaction with the MPN domain of Jab1/CSN5.     

The cytoplasmic trafficking of DNA topoisomerase IIalpha correlates with etoposide resistance in human myeloma cells

In this study we have investigated the role of topoisomerase (topo) IIalpha trafficking in cellular drug resistance. To accomplish this, it was necessary to separate the influence of cell cycle, drug uptake, topo protein levels, and enzyme trafficking on drug sensitivity. Thus, we developed a cell model (called accelerated plateau) using human myeloma H929 cells that reproducibly translocates topo IIalpha to the cytoplasm. Compared to log-phase cells, the cytoplasmic redistribution of topo IIalpha in plateau-phase cells correlated with a 10-fold resistance to VP-16 and a 40-60% reduction in the number of drug-induced double-strand DNA breaks. In addition, 7-fold more VP-16 was necessary to achieve 50% topo IIalpha band depletion, suggesting that there are fewer drug-induced topo-DNA complexes formed in quiescent cells than in log-phase cells. The total cellular amount of topo IIalpha and topo IIbeta protein in log- and plateau-phase cells was similar as determined by Western blot analysis. There was a 25% reduction in S-phase cell number in plateau cells (determined by bromodeoxyuridine (BrdU) incorporation), while there was no significant difference in the equilibrium concentrations of [(3)H]-VP-16 when log cells were compared with plateau cells. Furthermore, the nuclear/cytoplasmic ratio of topo IIalpha is increased 58-fold in accelerated-plateau H929 cells treated with leptomycin B (LMB) when compared to untreated cells. It appears that the nuclear-cytoplasmic shuttling of topo IIalpha, which decreases the amount of nuclear target enzyme, is a major mechanism of drug resistance to topo II inhibitors in plateau-phase myeloma cells.     

Influence of cell cycle and oncogene activity upon topoisomerase IIalpha expression and drug toxicity

The cell cycle, oncogenic signaling, and topoisomerase (topo) IIalpha levels all influence sensitivity to anti-topo II drugs. Because the cell cycle and oncogenic signaling influence each other as well as topo IIalpha levels, it is difficult to assess the importance of any one of these factors independently of the others during drug treatment. Such information, however, is vital to an understanding of the cellular basis of drug toxicity. We, therefore, developed a series of analytical procedures to individually assess the role of each of these factors during treatment with the anti-topo II drug etoposide. All studies were performed with asynchronously proliferating cultures by the use of time-lapse and quantitative fluorescence staining procedures. To our surprise, we found that neither oncogene action nor the cell cycle altered topo IIalpha protein levels in actively cycling cells. Only a minor population of slowly cycling cells within these cultures responded to constitutively active oncogenes by elevating topo IIalpha production. Thus, it was possible to study the effects of the cell cycle and oncogene action on drug-treated cells while topo IIalpha levels remained constant. Toxicity analyses were performed with two consecutive time-lapse observations separated by a brief drug treatment. The cell cycle phase was determined from the first observation, and cell fate was determined from the second. Cells were most sensitive to drug treatment from mid-S phase through G(2) phase, with G(1) phase cells nearly threefold less sensitive. In addition, the presence of an oncogenic src gene or microinjected Ras protein increased drug toxicity by approximately threefold in actively cycling cells and by at least this level in the small population of slowly cycling cells. We conclude that both cell cycle phase and oncogenic signaling influence drug toxicity independently of alterations in topo IIalpha levels.     

Human topoisomerase IIalpha: targeting to subchromosomal sites of activity during interphase and mitosis

Mammalian topoisomerase IIalpha (topo IIalpha) plays a vital role in the removal of topological complexities left on DNA during S phase. Here, we developed a new assay to selectively identify sites of catalytic activity of topo IIalpha with subcellular resolution. We show that topo IIalpha activity concentrates at replicating heterochromatin in late S in a replication-dependent manner and at centric heterochromatin during G2 and M phases. Inhibitor studies indicate that this cell cycle-dependent concentration over heterochromatin is sensitive to chromatin structure. We further show that catalytically active topo IIalpha concentrates along the longitudinal axis of mitotic chromosomes. Finally, we found that catalytically inert forms of the enzyme localize predominantly to splicing speckles in a dynamic manner and that this pool is differentially sensitive to changes in the activities of topo IIalpha itself and RNA polymerase II. Together, our data implicate several previously unsuspected activities in the partitioning of the enzyme between sites of activity and putative depots.     

Topoisomerase II binds importin alpha isoforms and exportin/CRM1 but does not shuttle between the nucleus and cytoplasm in proliferating cells

Resistance to anticancer drugs that target DNA topoisomerase II (topo II) isoforms alpha and/or beta is associated with decreased nuclear and increased cytoplasmic topo IIalpha. Earlier studies have confirmed that functional nuclear localization and export signal sequences (NLS and NES) are present in both isoforms. In this study, we show that topo II alpha and beta bind and are imported into the nucleus by importin alpha1, alpha3, and alpha5 in conjunction with importin beta. Topo IIalpha also binds exportin/CRM1 in vitro. However, wild-type topo IIalpha has only been observed in the cytoplasm of cells that are entering plateau phase growth. This suggests that topo IIalpha may shuttle between the nucleus and the cytoplasm with the equilibrium towards the nucleus in proliferating cells but towards the cytoplasm in plateau phase cells. The CRM1 inhibitor Leptomycin B increases the nuclear localization of GFP-tagged topo IIalpha with a mutant NLS, suggesting that its export is being inhibited. However, homokaryon shuttling experiments indicate that fluorescence-tagged wild-type topo II alpha and beta proteins do not shuttle in proliferating Cos-1 or HeLa cells. We conclude that topo II alpha and beta nuclear export is inhibited in proliferating cells so that these proteins do not shuttle.     

Dynamics of human DNA topoisomerases IIalpha and IIbeta in living cells

DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (alpha and beta) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIalpha and IIbeta behaved similarly in interphase but differently in mitosis, where only topo IIalpha was chromosome associated to a major part. During interphase, both isozymes joined in nucleolar reassembly and accumulated in nucleoli, which seemed not to involve catalytic DNA turnover because treatment with teniposide (stabilizing covalent catalytic DNA intermediates of topo II) relocated the bulk of the enzymes from the nucleoli to nucleoplasmic granules. Photobleaching revealed that the entire complement of both isozymes was completely mobile and free to exchange between nuclear subcompartments in interphase. In chromosomes, topo IIalpha was also completely mobile and had a uniform distribution. However, hypotonic cell lysis triggered an axial pattern. These observations suggest that topo II is not an immobile, structural component of the chromosomal scaffold or the interphase karyoskeleton, but rather a dynamic interaction partner of such structures.     

Sequence determinants of nuclear localization in the alpha and beta isoforms of human topoisomerase II.

The alpha and beta isoforms of DNA topoisomerase II (topo II) are targets for several widely used chemotherapeutic agents, and resistance to some of these drugs may be associated with reduced nuclear localization of the alpha isoform. Human topo IIalpha contains a strong bipartite nuclear localization signal (NLS) sequence between amino acids 1454 and 1497 (alphaNLS(1454-1497)). In the present study, we show that human topo IIalpha tagged with green fluorescence protein is still detectable in the nucleus when alphaNLS(1454-1497) has been disrupted. Seven additional regions in topo IIalpha containing overlapping potential bipartite NLSs were evaluated for their nuclear targeting abilities using a beta-galactosidase reporter system. A moderately functional NLS was identified between amino acids 1259 and 1296. When human topo IIbeta was examined in a similar fashion, it was found to contain two strongly functional sequences betaNLS(1522-1548) and betaNLS(1538-1573) in the region of topo IIbeta comparable to the region in topo IIalpha that contains the strongly functional alphaNLS(1454-1497). The third, betaNLS(1294-1332), although weaker than the other two beta sequences, is significantly stronger than the analogous alphaNLS(1259-1296). Differences in the NLS sequences of human topo II isoforms may contribute to their differences in subnuclear localization.     

Substrate specificity plays an important role in uncoupling the catalytic and scaffolding activities of rat testis DNA topoisomerase IIalpha

Topoisomerase II (topo II) is a dyadic enzyme found in all eukaryotic cells. Topo II is involved in a number of cellular processes related to DNA metabolism, including DNA replication, recombination and the maintenance of genomic stability. We discovered a correlation between the development of postnatal testis and increased binding of topo IIalpha to the chromatin fraction. We used this observation to characterize DNA-binding specificity and catalytic properties of purified testis topo IIalpha. The results indicate that topo IIalpha binds a substrate containing the preferred site with greater affinity and, consequently, catalyzes the conversion of form I to form IV DNA more efficiently in contrast to substrates lacking such a site. Interestingly, topo IIalpha displayed high-affinity and cooperativity in binding to the scaffold associated region. In contrast to the preferred site, however, high-affinity binding of topo IIalpha to the scaffold-associated region failed to result in enhanced catalytic activity. Intriguingly, competition assays involving scaffold-associated region revealed an additional DNA-binding site within the dyadic topo IIalpha. These results implicate a dual role for topo IIalpha in vivo consistent with the notion that its sequestration to the chromatin might play a role in chromosome condensation and decondensation during spermatogenesis.     

Association of human DNA topoisomerase IIalpha with mitotic chromosomes in mammalian cells is independent of its catalytic activity.

DNA topoisomerase (topo) II is an essential nuclear enzyme that plays an important role in DNA metabolism and chromosome organization. In the present study, we expressed human topo IIalpha in mammalian cells by fusion to an enhanced green fluorescent protein (EGFP). Decatenation assays indicated that the EGFP-topo IIalpha is catalytically active in vitro. Assays for band depletion, growth inhibition, and cytotoxicity by topo II inhibitors suggested that the fusion protein is also functional in vivo. By following its subcellular localization throughout the cell cycle in living cells, we found that the fusion protein is localized to the nucleus and nucleolus at interphase, and it is bound to chromosomal DNA at every stage of mitosis. Of importance, a mutant EGFP-topo IIalpha, in which the active Tyr 805 is replaced by Phe (Y805F) and is catalytically inactive, still binds to chromosomal DNA throughout the cell cycle like the wild-type enzyme. Together, our results suggest that the ability of topo IIalpha to bind to chromosomal DNA in the cell, a presumed requirement for its structural role, can be separated from its catalytic activity.     

The cytochrome b5 tail anchors and stabilizes subdomains of human DNA topoisomerase II alpha in the cytoplasm of retrovirally infected mammalian cells.

DNA topoisomerase II (topo II) is the target of many anticancer drugs and is often altered in drug-resistant cell lines. In some tumor cell lines truncated isoforms of topo IIalpha are localized to the cytoplasm. To study the localization and function of individual enzyme domains, we have epitope-tagged several fragments of human topo IIalpha and expressed them by retroviral infection of rodent and human cells. We find that fusion of the topo II fragments to the hydrophobic tail of human liver cytochrome b5 anchors the fusion protein to the outer face of cytoplasmic membranes, as determined by colocalization with calnexin and selective detergent permeabilization. Moreover, whereas the minimal ATPase domain (aa 1-266) is weakly and diffusely expressed, addition of the cytb5 anchor (1-266-b5) increases its steady-state level 16-fold with no apparent toxicity. Similar results are obtained with the complete ATPase domain (aa 1-426). A C-terminal domain (aa 1030-1504) of human topo IIalpha containing an intact dimerization motif is stably expressed and accumulates in the nucleus. Fusion to the cytb5 anchor counteracts the nuclear localization signal and relocalizes the protein to cytoplasmic membranes. In conclusion, we describe a technique that stabilizes and targets retrovirally expressed proteins such that they are exposed on the cytoplasmic surface of cellular membranes. This approach may be of general use for regulating the nuclear accumulation of drugs or proteins in living cells.     

Modulation of human DNA topoisomerase IIalpha function by interaction with 14-3-3epsilon

Human DNA topoisomerase IIalpha (topo II), a ubiquitous nuclear enzyme, is essential for normal and neoplastic cellular proliferation and survival. Several common anticancer drugs exert their cytotoxic effects through interaction with topo II. In experimental systems, altered topo II expression has been associated with the appearance of drug resistance. This mechanism, however, does not adequately account for clinical cases of resistance to topo II-directed drugs. Modulation by protein-protein interactions represents one mechanism of topo II regulation that has not been extensively defined. Our laboratory has identified 14-3-3epsilon as a topo II-interacting protein. In this study, glutathione S-transferase co-precipitation, affinity column chromatography, and immunoprecipitations confirm the authenticity of these interactions. Three assays evaluate the impact of 14-3-3epsilon on distinct topo II functional properties. Using both a modified alkaline comet assay and a DNA cleavage assay, we demonstrate that 14-3-3epsilon negatively affects the ability of the chemotherapeutic, etoposide, to trap topo II in cleavable complexes with DNA, thereby preventing DNA strand breaks. By electrophoretic mobility shift assay, this appears to be due to reduced DNA binding activity. The association of topo II with 14-3-3 proteins does not extend to all 14-3-3 isoforms. No protein interaction or disruption of topo II function was observed with 14-3-3final sigma.