1 Experimental Therapeutics, Departments of Interdisciplinary Oncology and Biochemistry and Molecular Biology, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, 12902 Magnolia Drive, Tampa, FL 33612, USA
2 Molecular Oncology Programs, Departments of Interdisciplinary Oncology and Biochemistry and Molecular Biology, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, 12902 Magnolia Drive, Tampa, FL 33612, USA
Author for correspondence (e-mail: sullivad{at}moffitt.usf.edu)
Accepted 9 February 2004
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Summary |
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Key words: Topoisomerase II alpha (topo II), Nuclear export signal (NES), CRM-1, Site-directed mutagenesis, Microinjection
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Introduction |
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In general, protein import occurs when the transport receptors, importin- and importin-ß, form a complex with the protein-NLS and escort the protein cargo across the NPC into the nucleus (Yoneda et al., 1999
). The protein cargo is released into the nucleus when importin-
binds Ran-GTP. Protein export occurs when Ran-GTP and the nuclear export receptor, CRM-1, bind to a protein bearing a NES and transports this protein cargo into the cytosol (Fukuda et al., 1997
; Fornerod et al., 1997
; Ossareh-Nazari et al., 1997
). The protein cargo is released into the cytoplasm when Ran-GTP is hydrolyzed by Rna1p, a small GTPase-activating protein. Ran-GDP is then transported back into the nucleus by a small protein called Ntf2 (Ribbeck et al., 1998
). A guanine nucleotide exchange factor then swaps out Ran-GDP for GTP. In this manner, continued nuclear-cytoplasmic shuttling occurs by maintaining a gradient of Ran-GTP in the nucleus and Ran-GDP in the cytoplasm (Gorlich, 1998
).
DNA topoisomerases (topo) are nuclear enzymes that maintain the topology of DNA during cellular processes such as DNA replication, transcription and genetic recombination (reviewed by Wang, 2002). Different topo enzymes have been identified in humans that are distinguished by differences in their gene locus, molecular mass, catalytic requirements, mechanism of catalysis, and biological roles. Type I enzymes (topo I) are active as monomers and catalyze transient single strand DNA breaks without ATP hydrolysis. The amino-terminal region of topo I has an important role in regulating the cellular localization of topo I because it contains the nuclear localization signal and nucleolin binding site (Mo et al., 2000
). Type II enzymes include topo II
(170 kDa) and topo IIß (180 kDa). The type II enzymes form dimers and couple double strand DNA breaks with ATP hydrolysis. Topo II
is targeted to the nucleus via a bipartite NLS located in the carboxyl terminus, whereas topo IIß contains two NLS comparable to the region in topo II
and a third weaker NLS (Cowell et al., 1998
; Mirski et al., 1999
).
DNA topoisomerases are also the principal targets of commonly used chemotherapeutic agents, including topotecan, doxorubicin, CPT-11 and etoposide. Generally, topo poisons convert the enzyme into a lethal DNA damaging agent, such that the more active enzyme that is present in the nucleus the more effective the poison (reviewed by Engel et al., 2003). Protein degradation and altered subcellular localization of topo have been postulated as two potential mechanisms that contribute to cellular drug resistance by attenuating the amount of drug target in the nucleus (Valkov et al., 1997
; Valkov et al., 2000
; Sullivan et al., 1987
; Kang and Chung, 2002
; Engel et al., 2004
). For example, adhesion of human myelomonocytoid U937 cells to fibronectin by
1 integrin protects cells against mitoxantrone and etoposide-mediated DNA damage, and is accompanied by an altered sub-nuclear relocalization of topo IIß to the nucleolus (Hazelhurst et al., 2001
). Thus, changes in the nuclear localization or binding properties of the nuclear pool of topo IIß protein may have a role in cellular drug resistance to etoposide and mitoxantrone in these cells. Furthermore, several cell lines that have been selected for resistance to topo poisons have been characterized for mechanisms of cellular drug resistance and are often shown to express a truncated topo II
enzyme that has lost its C-terminal NLS so that it remains in the cytoplasm (Wessel et al., 1997
; Harker et al., 1995
). A truncated topo IIß has also been reported to associate, in vivo, with the mtDNA genome in bovine mitochondria (Low et al., 2003
).
We have previously reported that topo II resides in the cytoplasm of several plateau-phase human hematological cell lines (8226, CCRF, H929, HL-60), and that the cytoplasmic translocation paralleled a decrease in sensitivity to etoposide (Valkov et al., 2000
; Engel et al., 2004
). No difference in the concentration or molecular mass of topo I, II
or IIß was observed, indicating that the cytoplasmic location was not a result of protein degradation or of a truncated enzyme. These data may also be clinically relevant, in that topo II
has been found to have a cytoplasmic distribution in malignant plasma cells from bone marrow aspirates from patients with multiple myeloma (Valkov et al., 2000
). In summary, these data suggest that topo II
may be translocated from the nucleus to the cytoplasm under specific cellular conditions, and this may result in altered drug sensitivity.
Although there is evidence that topo II can be transported between the nucleus and cytoplasm (Valkov et al., 2000
; Oloumi et al., 2000
), the majority of data are limited to describing the import of topo into the nucleus from the cytosol. In fact, there has been only one study that has addressed the mechanism of the nuclear export of topo (Mirski et al., 2003
). The results of this study were restricted to predicting the existence of a NES in topo II
and topo IIß from peptide studies, and did not define a functional NES in the full-length protein (Mirski et al., 2003
). The results of our study demonstrate that human topo II
contains two functional LMB-sensitive NESs in the full-length protein.
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Materials and Methods |
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Putative NES peptides
The complete amino acid sequence for human topo II (accession number NP 001058) was downloaded from the National Center for Biotechnology Information database and searched for matches to the NES consensus sequence from Table 1. Six amino acid sequences in topo II
matched the NES consensus sequence (Table 2), and were synthesized as native (nt) or mutated (
) peptides. The mutated peptides contain alanine in place of those hydrophobic residues suspected of being critical for nuclear export (leucine, isoleucine, or valine). To facilitate conjugation with preactivated SMCC-BSA, the NES-peptides were designed with a cysteine residue at the amino terminus. The peptides obtained from the Biopeptide Company (San Diego, California) were as follows: (NES80-91), C80GLYKIFDEILVN91; (mutated NES80-91), C
80GAYKAFDEAAAN91; (NES230-241), C230SLDKDIVALMVR241; (mutated NES230-241), C
230SADKDAAAAMAR241; (NES467-476), C467TLAVSGLGVVG477; (mutated NES467-477), C
467TAAASGAGAAG477; (NES1017-1028), C1017DILRDFFELRLK1028; (mutated NES1017-1028), C
1017CDIARDAFEARAK1028. Peptides (NES569-580), C569FLEEFITPIVKV580; (mutated NES569-580), C
569AAEEAATPAAKA580; (NES1054-1066), C1054FILEKIDGKIIIE1066; and (mutated NES1054-1066); C
1054FIAEKADGKAIAE1066 were obtained from the University of Florida Protein Chemistry Core Facility (Gainesville, FL, USA). All peptides were HPLC purified to >95% and analyzed by mass spectroscopy. In addition, peptide sequences and purity were confirmed by Rick Feldhoff, PhD at the University of Louisville, School of Medicine, Department of Biochemistry (Louisville, KY, USA).
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Generation of BSA-peptide-FITC conjugates
Peptides were crosslinked to BSA and FITC as previously described (Stommel et al., 1999). A total of 2 mg Imject® Maleimide activated sulfosuccinmidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate bovine serum albumin (Sulfo-SMCC BSA) (Pierce, Rockford, IL, USA) were reconstituted in 200 µl distilled water. Then, a molar excess (1-2 mg) of native or mutated topo II
NES peptide in 400 µl of conjugation buffer (83 mM sodium phosphate buffer, 0.1 M EDTA, 0.9 M NaCl, 0.002% sodium azide, pH 7.2) was mixed with the Sulfo-SMCC BSA and reacted for 30 minutes at room temperature. The reaction was quenched by adding 40 mM cysteine solution in deionized water to the peptide-SMCC-BSA solution to obtain a molar excess of cysteine to peptide sample (approximately 7 nmoles cysteine/nmole of SMCC-BSA-peptide). The conjugates were purified by size exclusion chromatography at room temperature using the Pharmacia P-500 FPLC system with LKB control Unit UV-1. The high resolution column (10 mm inner diameter and 30 cm length) (Amersham Pharmacia, Piscataway, NJ, USA) was packed at 2.0 ml/minute with Superdex 200 prep grade (Amersham Pharmacia) in filtered and degassed PBS, pH 7.4. The peptide conjugates were loaded onto the column using a 500 µl Superloop and were run at 0.5 ml/minute in degassed dH2O. The 500 µl fractions were collected with a Fraction-100 collector (Pharmacia Biotech) and stored at 4°C overnight. Total protein was estimated in peak samples by measuring the absorbance at 562 nm using the Fisherbrand Protein Assay. Approximately 25 µg of protein from eluted fractions were loaded onto a 10% SDS-PAGE gel and electrophoresed at 7 W for 2-3 hours. Peptide conjugation was confirmed by silver stain analysis. Similar fractions of crosslinked BSA-peptide were pooled and concentrated on a Microsep 30 k filter by centrifuging at 5000 g in an SS-34 rotor until dry. The samples were eluted with 400 µl of PBS, pH 7.4 to obtain approximately 2 mg/ml peptide-conjugate solution. FITC was solubilized in DMSO at 1 mg/ml and added to the peptide sample in four 5 µl aliquots until a total of 20 µl of FITC (Sigma) were added. FITC was reacted with the peptides for 6 hours at 4°C, and then 23 µl of 1 M NH4Cl in PBS, pH 7.4 were added to the sample and incubated for 2 hours at 4°C. FITC-BSA-peptide conjugates were separated from unincorporated label by FPLC as described above. The samples were concentrated in a SpeedVac and the ratio of fluorescein to protein was determined by measuring the absorbance at 495 nm and 280 nm. The nuclear control, tetramethylrhodamine-bovine serum albumin (TRITC-BSA), was obtained from Sigma.
Microinjection
To promote cell adherence, Fisherbrand glass coverslips were pretreated with 1 N HCl for a minimum of 4 hours at 50°C and then rinsed extensively with deionized water. Coverslips were washed in 100% ethanol and dried between pieces of Whatman paper. Subconfluent HeLa cells were plated onto the center of glass coverslips in NUNC brand Petri dishes and incubated at 37°C for 24-48 hours preceding microinjection. Prior to microinjection, cells were gently rinsed with sterile PBS warmed to 37°C and replaced with Leibovitz's L-15 medium containing no phenol red.
The peptide samples from above were centrifuged at 13,000 g for 30 minutes at 4°C, and the supernatant then loaded into Eppendorf Femptotips (diameter of 0.5 µm ± 0.2 µm). All cells were injected using the semi-automated Eppendorf Injectman NI2 and Femtojet microinjector on a Nikon TE 2000 inverted microscope under exactly the same conditions (injection pressure (Pi), 100 hPa; compensation pressure (Pc), 30 hPa; injection time (It) 0.2 seconds, atmospheric conditions). Following injection the cells were incubated at 37°C for up to 90 minutes, washed with Leibovitz's L-15 medium, and then fixed with 4% paraformaldehyde for 3 minutes at room temperature, rinsed in PBS and mounted on Shandon microscope slides with mounting medium containing DAPI. Fluorescence was observed with a Leitz Orthoplan 2 microscope and images were captured using a CCD camera with Smart Capture program (Vysis, Downers Grove, IL, USA).
Topoisomerase II cloning and site-directed mutagenesis
Topo II primers were designed that contained the eight amino acid FLAG peptide preceded by a start codon and a Kozak motif (5'-ATG GAC TAC AAA GAC GAT GAC GAC AAG GAA GTG TCA CCA TTG CAG CCT GTA AAT GAA AAT ATG-3' forward primer, 5'-ATG CGG CCG CTT AAA ACA GAT CAT CTT CAT CTG ACT CTT C-3' reverse primer). Amplification was performed with an enzyme mixture of Taq and Pyrococcus species GB-D thermostable DNA polymerases (Elongase, Invitrogen), in 60 mM Tris-SO4 (pH 9.1), 18 mM (NH4)2SO4, 2 mM MgSO4, 200 µM dNTP mixture and 200 nM each primer. Forty cycles were performed (94°C for 30 seconds, 60°C for 30 seconds and 5.5 minutes at 68°C) and the PCR products were agarose gel-purified and ligated to a pcDNA3.1 vector (CMV promoter) using a TOPO-TA cloning system (Invitrogen). The 5' end of the new FLAG-topo II
fusion protein vector was sequenced to ensure that the DNA was in-frame. Site-directed mutagenesis was performed using a Quickchange XL site-directed mutagenesis kit (Stratagene). Briefly, 100 ng of template dsDNA were mixed with 125 ng of each oligonucleotide primer, 2.5 units of PfuTurbo DNA polymerase, in a reaction mixture containing 2 mM dNTPs. Eighteen cycles were performed (95°C for 50 seconds, 60°C for 50 seconds and 68°C for 20 minutes), after which the parental plasmid was digested with methylation-specific enzyme Dpn-I. Ultra-competent cells were transformed with mutated plasmid and clones were sequenced to determine the presence of desired mutations. DNA sequencing was performed at the H. Lee Moffitt Cancer Center Molecular Biology Core Facility. Primers containing mutant sequences are listed in Fig. 1.
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Transfection protocol
Human myeloma H929 and HL-60 cells (ATCC) were plated at log phase density (2x105 cells/ml) 2 days prior to transfection. Transfection was performed as previously described (Van den Hoff et al., 1992). Briefly, 40 µg of wild-type or mutated topo II
plasmid in 300 µl of a solution containing 10 mM Tris-HCl, 1 mM EDTA, pH 8.0, were precipitated by the addition of 30 µl of 5 M NaCl and two volumes of 95% ethanol on ice for 10 minutes. Plasmid was pelleted by centrifugation for 15 minutes at 20,000 g at 4°C, washed with 75% ethanol and re-centrifuged. All remaining ethanol was removed using a pipette and the DNA immediately resuspended in 50 µl of cytomix buffer containing 120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4, 25 mM Hepes, 2 mM EGTA, 5 mM MgCl2, 2 mM ATP and 5 mM glutathione, and the pH was adjusted to 7.6 by the addition of KOH. ATP and glutathione were made fresh and added prior to each transfection (Van den Hoff et al., 1992
). Two days prior to transfection, human myeloma H929 cells and leukemia HL-60 cells were placed in fresh growth medium (RPMI/10% FBS/pen-strep) at a concentration of 2x105 cells/ml. Cells were collected and 1.6x107 cells pelleted by centrifugation at 1500 g for 5 minutes. Cell pellets were washed twice in 10 ml of sterile PBS, resuspended in 350 µl of cytomix buffer (4°C), mixed with prepared DNA and placed in a 4 mm electroporation cuvette. Electroporation was at 250V/750 capacitance, after which cells were split into even groups and plated at log (2x105 cells/ml) and plateau (2x106 cells/ml) growth conditions for 20 hours in a 5% CO2 incubator at 37°C with RPMI medium containing 5% FBS.
Immunofluorescence
Twenty hours post-transfection, viable H929 and HL-60 cells were isolated by centrifugation at 2000 g for 20 minutes at 20°C on a Ficoll gradient and washed with PBS. Transfected cells were plated on a glass microscope slide using cytospin funnels and fixed with 4% paraformaldehyde at 20°C for ten minutes. The fixation was stopped by washing in PBS and cells were permeabilized for 24 hours in a solution containing 1% glycine and 0.25% Triton X-100 in PBS. Slides were stained with anti-FLAG M2 monoclonal antibody-FITC conjugate (Sigma) diluted 1:100 with 0.1% NP-40 and 1% BSA in PBS, and incubated for 1 hour at room temperature. Slides were washed in PBS, dried briefly and counterstained with Vectashield mounting medium antifade/DAPI (1:1) (Vector Laboratories Inc., Burlingame, CA, USA). Immunofluorescence was observed with a Leitz Orthoplan 2 fluorescent microscope and images were captured by a CCD camera with Smart Capture program (Vysis, Downers Grove, IL). Quantitation of FITC fluorescence was performed using the Adobe Photoshop 7.0 program.
Western blot analysis
HeLa cells grown in RPMI medium containing 5% FBS were transfected directly on 100 cm2 tissue culture plates. Plasmid DNA (10 µg) was mixed with 60 µl of Superfect transfection reagent (Qiagen) in 300 µl of serum-free medium for 10 minutes, followed by 600 µl of serum containing medium, and the entire mixture was then added directly to cell culture plates. Transfection was allowed to proceed for 3 hours at 37°C in a 5% CO2 incubator and terminated by the removal of transfection solution and the addition of 15 ml of 5% FBS-containing RPMI medium. After incubation for 24 hours, the cells were harvested by the addition of 0.53 mM EDTA, washed with cold PBS, and lysed in SDS buffer (2% SDS, 10% glycerol, 0.06 M Tris, pH 6.8). Protein from 2x105 cells per lane was separated on an 8% SDS-PAGE gels and electroblotted (Biorad) onto nitrocellulose membranes (Amersham). The blots were blocked for 1 hour at ambient temperature in a blocking buffer containing 0.1 M Tris-HCl-buffered saline, 0.5% Tween 20, and 5% non-fat milk. Blots were stained by the direct addition of anti-FLAG M2 (Sigma) antibody and incubated overnight at 4°C. Membranes were washed three times for 10 minutes with 0.1 M Tris-HCl-buffered saline and incubated with anti-mouse IgG antibody (Sigma) in 0.1 M Tris-HCl buffered saline, 0.5% Tween 20 and 5% non-fat milk for 60 minutes at room temperature. Antibody binding was visualized by ECL (Amersham) on autoradiography film (Kodak).
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Results |
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LMB blocks NES1054-1066 and NES1017-1028 mediated nuclear export
Leptomycin B (LMB) (Hamamoto et al., 1983a; Hamamoto et al., 1983b
; Hamamoto et al., 1985
) is a specific inhibitor of CRM-1-mediated nuclear export of proteins (Nishi et al., 1994
). To determine if the nuclear export of BSA conjugated to peptides NES1054-1066 and NES1017-1028 was CRM-1 dependent, LMB-pretreated HeLa cells were microinjected in the presence of 2 ng/ml LMB in ethanol. Fluorescence microscopy demonstrated that LMB blocked the export of BSA conjugated to peptides NES1054-1066 and NES1017-1028 (Fig. 2). BSA-NES1054-1066 had a strong perinuclear staining, suggesting that the protein cargo was docking at the NPC (Siomi et al., 1997
; Arlucea et al., 1998
).
Although the NES defined by microinjection are sufficient to transport a non-shuttled protein to the cytoplasm, these leucine rich sequences may or may not serve a role in exporting topo II. To determine if these NES are necessary for topo II
export, we observed the trafficking of FLAG-topo II
expressed in human myeloma cell lines in the accelerated-plateau cell system.
Topoisomerase II cloning, site directed mutagenesis, and gene expression
We first attempted to study full-length topo II protein trafficking using a green fluorescent protein-topoisomerase II alpha (GFP-topo II
) fusion protein expression vector. However, we found that expression of GFP-topo II
recombinant protein was cytotoxic, inducing apoptosis in all cell lines tested (HeLa, HL-60, H992, 8226, MCF-7, Chinese hamster ovary) 16-48 hours after transfection. In addition, translocation of GFP-topo II
to the cytoplasm in plateau density cells was extremely limited when compared to endogenous topo II
(data not shown). GFP-topo II
plasmid containing site-directed mutations of the six putative nuclear export signals listed in Fig. 1, showed minimal change in export when compared to wild-type GFP-topo II
(data not shown). Additionally, we produced a GFP-topo II
fusion plasmid in which the active-site, tyrosine 805, was mutated to an alanine. GFP-topo II
A805 gave similar negative results; it was lethal to the transfected cells and minimally exported to the cytoplasm in plateau density cells (contrary to endogenous topo II
). We made one additional plasmid with a destabilized GFP-topo II
fusion protein, theorizing that the lethality of the fusion protein may be due to accumulation of GFP-topo II
in the transfected cells. A destabilized GFP fusion protein is turned over every 4 hours in transfected cells, thus not allowing cellular accumulation of recombinant protein to cytolytic levels. Destabilized GFP-topo II
protein was also cytotoxic and did not translocate to the cytoplasm in plateau density cells.
The lack of success with GFP led us to look for alternative topo II fusion proteins, such as the FLAG peptide. FLAG peptide is an eight amino acid protein (NYKNNNNK) that does not occur in nature. FLAG does not contain any putative nuclear export signals and its small size limits any secondary protein structure problems. HeLa cells transfected with FLAG-topo II
plasmid vectors express full-length (170 kDa) topo II
recombinant proteins (Fig. 3).
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Since our primary interest was human myeloma cells, we encountered another technical problem inherent with these cells, which is difficulty in transfecting them. In an attempt to transfect human myeloma cell lines, we tried a number of commercially available transfection reagents and numerous protocols, with no success. The only technique that yielded positive transfectants was a method described by Van den Hoff et al. (Van den Hoff et al., 1992). This method utilizes a cytomix buffer made to approximate the intracellular environment (see Materials and Methods). In addition, this buffer contains ATP and glutathione to promote the rapid repair of cellular membranes. We were able to transfect cells with a high degree of efficiency for these cell lines (2-20%) using this method.
FLAG-topoisomerase II immunofluorescence
BSA-peptide microinjection data indicated that the putative nuclear export sites at 1017-1028 and 1054-1066 may function to signal export of topo II. To confirm these data with a full-length topo II
protein, H929 human myeloma cells were transfected with FLAG-topo II
expression vectors possessing mutated hydrophobic residues in the nuclear export sites at 1017-1028 and 1054-1066 (Fig. 1). Twenty hours post-transfection, viable cells were isolated by centrifugation on a Ficoll-paque gradient and plated on glass microscope slides using cytospin funnels. After fixing and permeabilization, slides were stained with anti-FLAG M2 monoclonal antibody-FITC conjugate and counterstained with mounting medium containing DAPI to show the location of the nuclei. Images were acquired using a fluorescence microscope (Fig. 4), with quantitation of FITC fluorescence using Adobe Photoshop 7.0 (data presented in Fig. 5). Fig. 4 establishes that the wild-type (non-mutated) FLAG-topo II
protein is present in the nucleus of the cells plated at log density, whereas FLAG-topo II
protein is located in the cytoplasm in cells plated at plateau density. Quantitation of fluorescence revealed a statistically significant shift (P=0.00001) for log cells with a nuclear:cytoplasmic ratio of 5.9:1, to a ratio of 0.42:1 in plateau cells (Fig. 5) when using the wild-type FLAG-topo II
plasmid. When the putative export signals at either 1017-1028 (Fig. 4) or 1054-1066 were mutated, export to the cytoplasm was abrogated. Quantitative analysis of fluorescence of both mutant proteins (1017-1028 or 1054-1066) revealed no statistically significant change in the levels of nuclear or cytoplasmic FLAG-topo II
in log or plateau density cell cultures. Even though export to the cytoplasm of mutant 1054-1066 was abrogated, qualitatively it appeared that the mutated FLAG-topo II
protein was localized predominantly at the nuclear membrane as compared to mutant 1017-1028. The putative signal at 467-476 was similar to wild-type (Fig. 4) as were putative signals at amino acid 80-91, 230-241 and 569-580 (data not shown).
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Peptide NES1054-1066 and NES1017-1028 are conserved
To determine if peptides NES1054-1066 and NES1017-1028 are conserved, a BLAST search of the SWISS-PROT database was performed to identify homologous sequences in topo II. Tables 3 and 4 summarize a list of representative species containing homologous topo II
sequences. The data show that the characteristic spacing of hydrophobic residues in peptides NES1054-1066 and NES1017-1028 are highly conserved in a broad range of species. For example, leucine residues appearing in human topo II
NES are often substituted with the hydrophobic amino acids isoleucine or valine. Furthermore, Phe1054 and Ile1055 in peptide NES1054-1066 are highly conserved from mammals to the most primitive eukaryotic organism, Giardia lamblia, unlike Leu1056. This suggests that the presence of phenylalanine and isoleucine are critical for nuclear export of this peptide, and thus an omission of these two hydrophobic amino acids from the peptide sequence could explain why a previous report failed to identify NES1054-1066 as a nuclear export signal (Mirski et al., 2003
).
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NES1054-1066 and NES1017-1028 reside within a putative coiled-coil domain
We were interested in predicting the structural features of the region containing NES1017-1028 and NES1054-1066. Each topo II monomer can be divided into three domains, an N-terminal domain that contains the ATP-binding region, the central domain containing the active site tyrosine residue, and the C-terminal domain that contains the nuclear localization sequences (Watt and Hickson, 1994). Both of the NES are situated upstream of the bipartite NLS (Fig. 6) and downstream of the active-site tyrosine residue (Tyr805). Furthermore, several CK-2 phosphorylation sites downstream of both NES have been identified in vitro and could be important for regulating the subcellular localization of topo II
. Although NES1017-1028 alone is predicted to form an
-helix (Mirski et al., 2003
), we were interested in predicting the motif of the complete amino acid sequence stretching from NES1017-1028 to NES1054-1066. According to EMBOSS and Predict Protein, two programs designed to predict protein motifs, amino acids 1017-1066 are characterized by a high potential to form
-helices and also contain five 4-3 hydrophobic repeats, a typical feature of a coiled-coil motif. Such a repeating pattern of hydrophobic amino acids has been shown to form a hydrophobic core, which is critical for dimerization (Sodek et al., 1972
). In this manner, the hydrophobic amino acids are predicted to align on the same interface that facilitates DNA binding or protein-protein interactions. Interestingly, amino acids 1013-1056 in topo II
have previously been shown to form a stable two-stranded
-helical coiled-coil in solution (Frere et al., 1995
; Frere-Gallois et al., 1997
; Bjergbeck et al., 1999
). Perhaps more importantly than the predictive data above, the crystal structure of topo II from Saccharomyces cervisiae was shown to have primary and secondary dimerization domains (Fass et al., 1999
). The primary dimerization region is highly conserved and corresponds to amino acids 1013-1056 in human topo II
(Frere et al., 1995
).
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Discussion |
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One of the potential consequences of exporting a pool of topo II to the cytoplasm is a decrease in sensitivity to topo poisons. This could result from cytoplasmic topo II
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
). For this to occur, the amount of drug binding to topo II
would need to be sufficient to result in a decrease in drug-induced DNA damage (Burden et al., 1996
). Another possibility is that the shuttling of topo II
to the cytoplasm results in a decrease in the amount of nuclear enzyme available to form enzyme-drug-DNA ternary complexes. In either case, blocking the export of topo II
with drugs such as LMB may sensitize cells to topo poisons by maintaining the amount of drug target in the nucleus. Although LMB has been shown to have undesirable cytotoxic effect in clinical trials, synthetic derivatives of LMB have become available and may be promising alternatives to LMB therapy (Kalesse et al., 2001
; Koster et al., 2003
) in hematological malignancies.
Resistance to chemotherapeutic drugs is a major obstacle in the treatment of leukemia and myeloma. We have previously reported that resistance to topoisomerase II poisons such as VP-16 was found to increase dramatically with concurrent increases in cell density (Valkov et al., 2000
; Engel et al., 2004
). Myeloma and leukemic cells in transition from low-density log phase conditions to high-density plateau phase conditions exhibited a substantial export of endogenous topoisomerase II
from the nucleus to the cytoplasm. In order for topoisomerase-targeted chemotherapy to function, the topoisomerase target must have access to the nuclear DNA. Thus, the nuclear export of topoisomerase II
must be added to the list of potential mechanisms of resistance to topo poisons. It is unique in that it does not require drug exposure and may mimic the high cell density microenvironment seen in the bone marrow of patients with multiple myeloma. Further defining this mechanism, and possibly modulating export, may lead to methods to preclude this avenue of resistance.
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Acknowledgments |
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Footnotes |
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References |
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