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Turner, Joel G.
Human topoisomerase II alpha nuclear export is mediated by two Crm-1 dependent nuclear export signals
h [electronic resource] /
by Joel G. Turner.
[Tampa, Fla.] :
University of South Florida,
Thesis (M.S.)--University of South Florida, 2004.
Includes bibliographical references.
Text (Electronic thesis) in PDF format.
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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 exhibit a substantial export 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. In the current report, we have defined 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.
Co-adviser: Daniel M. Sullivan
Co-adviser: James R. Garey
nuclear export signal.
topoisomerase II alpha.
t USF Electronic Theses and Dissertations.
Human Topoisomerase II Alpha Nuclear Export Is Mediated by Two Crm-1 Dependent Nuclear Export Signals by Joel G. Turner A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Biology College of Arts and Sciences University of South Florida Co-Major Professor: Daniel M. Sullivan, MD Co-Major Professor: James R. Garey, Ph.D. Richard S. Pollenz, Ph.D. Date of Approval: March 19, 2004 Keywords: topoisomerase II alpha, nuclear export signal, Crm-1, site-directed mutagenesis, microinjection. Copyright 2004, Joel G. Turner
Acknowledgements I would like to express my gratitude to my mentor Dr. Daniel Sullivan. I would like to acknowledge the Moffitt Cancer Center Analytic Microscopy and Molecular Biology Cores for their expertise and assistance. I would also like to thank Dr. Scott Kaufman and Dr. Richard Pollenz for their valuable technical advice. In addition, I would like to thank and acknowledge the contributions by Roxane Engel who produced the FITC labeled BSA-export signal peptide conjugates and who also contributed to the preparation of this document, and the microinjections that were performed by Jennifer Derderian.
Table of Contents List of Tablesi List of Figuresii Abstractiii Introduction1 Topoisomerase II alpha structure and function1 Therapeutic agents that target topoisomerase II alpha3 Etoposide and topoisomerase II alpha3 Etoposide mechanism of inhibition4 Etoposide and drug resistance5 Rational drug design based on etoposide molecular structure6 Nuclear import and export: overview7 Cellular requirements for Crm1 mediated export9 Cancer and disregulation of nuclear export10 Drug resistance via nuclear export of topoisomerase II alpha12 Materials and Methods16 Cell Culture and Accelerated-plateau Cell Model16 Putative NES Peptides16 Generation of BSA-peptide-FITC Conjugates18 Microinjection20 Topoisomerase II alpha Cloning and Site Directed Mutagenesis21 Transfection Protocol22 Immunofluorescence23 Western Blot24 Results 25 Peptides NES1054-1066 and NES1017-1028 Signal the Nuclear Export of BSA-FITC25 LMB Blocks NES1054-1066 and NES1017-1028 Mediated Nuclear Export25 Topo II alpha Cloning, Site Directed Mutagenesis, and Gene Expression27 FLAG-topoisomerase II alpha Immunofluorescence30 Peptide NES1054-1066 and NES1017-1028 are Conserved33 NES1054-1066 and NES1017-1028 Reside Within a Putative Coiled-coil Domain33 Discussion36 Future Directions40 Crm1 proof of principle40
High-throughput assay41 Phosphorylation of topoisomerase II alpha41 Other questions42 Summary43 References44
i List of Tables Table I. Determination of a Consensus Sequence from Established NES17 Table II. Putative NES in Human Topo II alpha18 Table III: Site-directed Mutagenesis.22 Table IV. Sequence Alignment of Topo II alpha NES1017-102834 Table V. Sequence Alignment of DNA Topo II alpha NES1054-106635
ii List of Figures Figure 1.Domain map of human topoisomerase II alpha2 Figure 2Immunofluorescent microscopy staining for topoisomerase II 13 Figure 3Immunofluorescent staining for topo II, histones and merged image15 Figure 4Microinjection of BSA-FITC Conjugates26 Figure 5Western blot of FLAG-topo II alpha Plasmid Expression29 Figure 6Nuclear Export of FLAG-topo II alpha Protein31 Figure 7FLAG-topo II alpha Immunofluorescence32 Figure 8Complete Amino Acid Sequence of Topo II alpha38
iii Human Topoisomerase II Alpha Nuclear Export Is Mediated by Two Crm-1 Dependent Nuclear Export Signals Joel G. Turner 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 exhibit a substantial export 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. In the current report, we have defined 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
iv 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.
1 INTRODUCTION Topoisomerase II alpha Structure and Function The fundamental problem with developing therapies against cancer is that cancer cells are extremely similar to normal cells both immunologically and physiologically. This similarity makes it difficult to design therapies that can selectively destroy the cancer without collateral damage to healthy cells. One strategy to accomplish this is to selectively target rapidly dividing cells. This selective targeting is the basic principle involved when using topoisomerases as targets for cancer chemotherapy. Topoisomerases are highly expressed in proliferating cells and are absolutely essential for cell survival. Without topoisomerases, replication of DNA, transcription of RNA and chromatin assembly would be impossible. It is appropriate to first briefly explain the normal structure and function of topoisomerase II in cells in order to address how various agents inhibit topoisomerase II and induce cell death (Bakshi, et al ., 2001). The primary function of topoisomerase II is to catalyze the breaking and rejoining of double-stranded DNA in order to relax supercoiled structures, and resolve nucleic acid knots and tangles. Topoisomerase II is composed of three domains; an amino-terminal ATPase domain, a central catalytic domain which includes an active tyrosine site, and a carboxy-terminus domain which contains the nuclear localization signal (NLS) and other potential phosphorylation sites (Figure 1).
2 Two topoisomerase molecules form a homodimer that creates a transient break (gate) in a duplex DNA, transports a second strand through the gate, and then religates the cleaved strand (Liu, et al ., 1980). To provide a stable structure during Figure 1 : Domain map of human topoisomerase II alpha transport of the second strand, the active site tyrosines of topoisomerase form a covalent bond with the 5' termini of the DNA gate. Topoisomerase II has the ability to recognize and bind to topological structures such as negative and positive supercoiled nucleic acids. After binding to the DNA, and in the presence of a divalent cation, the enzyme forms double-stranded nicks on opposite strands of the double helix leaving a four base 5' overhang on each cleaved strand (Lui, et al ., 1983). Formation of covalent linkages does not require ATP because the enzymecleaved DNA complex conserves the chemical energy of the phosphodiester bond (transesterification). However, ATP binds as a co-factor and triggers a conformational change that allows strand passage through the gate. After strand passage and ligation of the DNA break, ATP hydrolysis is required for the opening of the molecule to release the nucleic acid products. ATPase domain breakage/reunion domain carboxy-terminal domain Catalytic site Tyr 805 NLS1454-1497C NSer 1524 Ser 1392Ser 1342Ser 1360Ser 1376 Ser 1212Ser 29Ser 1246Ser 1353
3 Therapeutic Agents That Target Topoisomerase II alpha Drugs that target topoisomerase II consist of two types, topoisomerase poisons, and catalytic inhibitors (Andoh and Ishida, 1998). Topoisomerase poisons cause the enzyme itself to produce great numbers of double-stranded breaks in the DNA, whereas catalytic inhibitors act as antagonists, which inhibit enzyme activity. There are currently six anti-neoplastic drugs the FDA has approved for clinical use that act on topoisomerase II ; etoposide, teniposide, doxorubicin, daunorubicin, idarubicin, and mitoxantrone (Burden and Osheroff, 1998). All drugs presently in clinical (non-investigational) use are topoisomerase poisons. Etoposide and Topoisomerase II alpha Remarkably, etoposide containing plant derivatives have been used for centuries to treat skin cancers in India (Slevin, 1991). Etoposide was first isolated in 1966, received FDA approval for treatment of cancer in 1983, but it wasnÂt until 1984 that a connection between etoposide and topoisomerase II inhibition was made. Etoposide does not block topoisomerase catalytic functioning; instead, it increases the concentration of DNA cleavage complexes in proliferating cells. Etoposide specifically binds to topoisomerase II Etoposide binding prevents strand passage and traps the enzyme in a conformation possessing a double stranded DNA break with the active tyrosine groups covalent-linked to each separated strand. When replicating machinery or helicases attempt to traverse this topoisomerase roadblock, permanent double-stranded breaks in the DNA are made. The DNA breaks become involved in recombination events generating large deletions and
4 insertions resulting in genomic instability in dividing cells, and eventually lead to cell death by apoptosis (Hande, 1998). Etoposide Mechanism of Inhibition Etoposide has a relatively weak affinity for DNA and functions by preventing the DNA religation step of topoisomerases II by directly binding the enzyme. Although etoposide has been used to treat cancer for many years, it is still unclear as to how it binds its target enzyme. A recent study has given a strong indication how etoposide works as a topoisomerase poison (Leroy, et al ., 2001). Etoposide binding was examined in the absence of DNA using radiolabeled etoposide and recombinant proteins from the N-terminal ATPase (aa 1-266) and catalytic (aa 430-1214) domains of topoisomerase II Affinities (Kd values) of etoposide for the ATPase and catalytic subunits of human topoisomerase II were found to be 20 and 9M respectively. ATP was found to displace etoposide from the N-terminal domain and full-length topoisomerase, but not from the catalytic core domain. In addition, a computer generated molecular model of the N-terminal domain of topoisomerase II was produced and molecular docking analysis of etoposide was performed. Using the molecular models, amino acids that were predicted to come in contact with etoposide were mutated. The mutated N-terminal domain proteins had significantly reduced drug binding efficiencies, which strongly suggests that etoposide can interact with the ATP binding pocket of eukaryotic topoisomerase II in vitro Two possible models would be consistent with these results, either the N-terminus and catalytic sites cooperate to form a
5 single binding unit or the sites bind in a separate manner and compete for the drug (Leroy, et al ., 2001). Etoposide and Drug Resistance Cancer chemotherapy with anti-topoisomerase II drugs can lose its effectiveness when cells develop resistance to the action of the drug. Drug resistance in cancer patients to etoposide can be produced in multiple ways. Upregulated expression of p-glycoprotein and breast cancer resistance protein has been found in some drug resistant human cell lines (Litman, et al ., 2001). These cell lines are normally derived from cancer patients and selected in vitro for drug resistance with chronic levels of drug. Breast cancer resistance protein and p-glycoprotein are essentially molecular pumps that increase the rate of drug efflux from the cells and thereby confer resistance. An atypical means of drug resistance is the development of mutant topoisomerases in cancer cells (Okada, 2001), (Mao, et al ., 1999). Interestingly, the particular mutations which confer resistance are found in the ATPase domain and in the catalytic domain of topoisomerase, sites very similar to those predicted for etoposide binding. These mutations also decrease the catalytic efficiency of topoisomerase II Additional mutated topoisomerases have been reported in the carboxy terminus region of topoisomerase II (Yu, et al ., 1997). This type of mutation produces a cytoplasmic form of topoisomerase II because the nuclear localization signal (NLS) has been truncated (Figure 1). For topoisomerase II to cause lethal breaks in the DNA it must be in the nucleus, therefore cells expressing a cytoplasmic variety would not be affected by etoposide. It has been speculated that the cytoplasmic topoisomerase II provides a
6 cytoplasmic buffer or sink that will bind up etoposide before it can reach the nucleus, and subsequently result in drug resistance (Yu, et al ., 1997). Rational Drug Design Based On Etoposide Molecular Structure Most chemotherapeutic agents originate from natural biological sources such as plants and bacteria. This type of drug development is very difficult, labor intensive, and, at best, is a hit-or-miss proposition. A more logical methodology would be to alter the molecular structures of drugs already known to be effective. Molecular alterations could be designed to improve drug action, counter drug resistance, increase uptake, or reduce toxicity. In order to enhance the cytotoxic effect of etoposide to topoisomerase II a glycoside moiety of etoposide was substituted by an aminoalkyl group to produce a new drug, TOP-53 (Byl, et al ., 2001). Compared to etoposide, TOP-53 was more effective in killing cancer cells, generated more chromosomal breaks, and had improved cellular uptake in animal studies. The in vitro effective dose was seven fold lower than that of the parent drug. TOP-53 was also found to be effective against mutant topoisomerases that were resistant to etoposide. Early TOP-53 experimental results clearly demonstrate the important role of the particular modified side group in mediating drug-enzyme interactions. This opens the door for additional potential modifications of the same side group. Etoposide has an additional problem in that it is relatively insoluble in water. Very large fluid volumes are required to administer the drug to patients. To address this problem, an etoposide methyl side group was replaced by a phosphate moiety (Hande, 1998). The new drug, etoposide phosphate, was found to be more easily administered to
7 patients, had less toxic side effects, and increased uptake, while maintaining the clinical effectiveness of etoposide. Nuclear Import and Export: Overview In nuclear-cytoplasmic trafficking, there are three key components: 1) the nuclear pore complex, 2) transport receptor molecules and related components, and 3) the protein or RNA cargo itself. All nucleocytoplasmic trafficking events proceed through the nuclear pore complex (NPC). The NPC is one of the largest macromolecular assemblies in the eukaryotic cell, which serves as a portal in the double membrane of the nuclear envelope. Recent investigations have elucidated several molecular pathways for the nuclear import and export of proteins (Kau and Silver, 2003; Weis, 2003) across transport passageways or nuclear pore complexes (NPC) (Dreger, 2003). The NPC is a large (125 MDa) multimeric protein structure that perforates the nuclear envelope and channels proteins greater than 60 kDa into or out of the nucleus. The constituents of the NPC have been described in yeast (Rout, et al ., 2000) and mammalian cells (Cronshaw, et al ., 2002). Nuclear export/import receptor proteins, which are specifically bound to cargo proteins, interact with FG-nucleoporins (F-phenylalanine, G-glycine) on the NPC. FGnucleoporins line the NPC channel and are thought to act as "step-stepping stones" as the receptor complex binds to successive FG-repeats and thereby makes its way through the NPC channel. The nucleoporins form a hydrophobic mesh that is thought to restrict proteins that are not complexed with receptor molecules. There are three types of transport receptors which have been identified: 1) the importin -like proteins, also known as karyopherins (including Imp transportin, Crm1,
8 and CAS), 2) the nuclear transport factor 2 (NTF2/p10) homodimer (imports GTPase Ran into the nucleus), and 3) the Tap/Mex67 family (transports mRNA). Transport receptors move between the nucleus and cytoplasm by interacting with nucleoporin complexes in the NPC, specifically those that have phenylalanine/glycine (FG)-rich motifs (Weis, 2002). The protein cargo is released into the cytoplasm when Ran-GTP is hydrolyzed to Ran-GDP by a GTPase-accelerating protein (Ran-GAP). Ran-GDP is then transported back into the nucleus and guanine nucleotide-exchange factor then swaps out Ran-GDP for GTP (Ribbeck, et al ., 1998). 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). RanGTP is formed exclusively in the nucleus because the Ran guanine nucleotide exchange factor (RanGEF) is bound to the DNA via histone complexes, whereas RanGDP is exclusive to the cytoplasm because the Ran GTPaseactivating protein (RanGAP) is located in the cytoplasm. Exclusive localization of RanGAP to the cytoplasm and RanGEF to the DNA maintains a gradient even during cellular division. Translocation through the NPC itself does not require energy and is reversible. The concentration gradient provided by the RanGTP cycle provides the energy input required to produce transport in importin-exportin mediated transport. The RanGTP gradient also creates a specific directionality by regulation of the substrate binding and release reactions. Proteins targeted for receptor-mediated transport across the NPC must either contain a nuclear localization signal (NLS) or a nuclear export signal (NES). Protein NLS are typically short clusters of basic amino acids, often preceded by an acidic amino acid or proline residue. However, a NLS may also consist of bipartite clusters of basic
9 amino acids separated by a spacer region of approximately ten amino acids, often flanked by a neutral or acidic amino acid. Previously described NLS are annotated in SWISSProt (Bairoch and Apweiler, 2000) and PIR (Wu, et al ., 2002), and can be retrieved at the NLS database located at the Predict NLS server (Cokol, et al ., 2000). Protein NES are hydrophobic rich sequences that have a characteristic spacing of leucine, isoleucine, valine, and/or phenylalanine. To date, approximately 75 experimentally validated protein-NES have been identified and compiled in the NESbase version 1.0 database (La Cour, et al ., 2000). In general, protein import occurs when the transport receptors, importinand importin, 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 importinbinds Ran-GTP. Protein export occurs when Ran-GTP and the nuclear export receptor, CRM-1, binds to a protein bearing an NES and transports the protein cargo into the cytosol (Fukuda, et al ., 1997; Fornerod, et al ., 1997; Ossareh, et al ., 1997). Topoisomerase II is targeted to the nucleus via a bipartite NLS located in the carboxyl-terminus, whereas topoisomerase II contains two NLS comparable to the region in topoisomerase II and a third weaker NLS (Cowell, et al ., 1998; Mirski, et al ., 1999). Cellular Requirements for Crm1 Mediated Export Using a recombinant and fluorescently conjugated Crm1 protein which was microinjected into cells, it was found that Crm1 import is not temperature-dependant (Zhang, et al ., 2003). More importantly, Crm1 import into the cell nucleus does not
10 require ATP, Ran, or Ran-dependent GTP hydrolysis. Nuclear import of Crm1 is not by passive diffusion, but via an active translocation process that is blocked by NPC binding by wheat germ agglutinin. Crm1 nuclear import is blocked by an excess of importin implying that the two molecules compete with one another and, therefore, possibly interact with the same target site on the NPC. However, export of Crm1 is stopped by ATP depletion in vivo The ATP requirement is not indirectly related to production of GTP because export did not occur in the presence of RanGTP without the addition of ATP also (Zhang, et al ., 2003). Restoration of Crm1 export by the addition of ATP suggests that an ATP-consuming step is needed for Crm1 nuclear export. Cancer and Disregulation of Nuclear Export In order for a neoplasm to develop, many changes must take place genetically and/or epigenetically. The hallmarks of cancer include, uncontrolled proliferation, insensitivity to negative growth regulation, evasion of apoptosis, lack of senescence, invasion and metastasis, angiogenesis, and genomic elasticity (Hanahan and Weinberg, 2000). Many of the genes and proteins that control these hallmarks are regulated by nuclear-cytoplasmic trafficking. Nuclear localization is essential to protein function. Transcription factors, activators or repressors must have access to genomic DNA in order perform their respective functions. Nuclear transport can be regulated in at least three ways; 1) modification of the cargo, 2) the transporters, or 3) the nuclear pore complex itself. Modifications of the cargo could be via phosphorylation, ubiquitinylation, glycosylation, sumoylation, or methylation. Mutations or deletions of nuclear localization signals and/or nuclear export
11 peptide sequences would prevent binding by transporter proteins and subsequently prevent nuclear-cytoplasmic shuttling. In the same manner, masking of signals by additional proteins or by protein folding could disrupt trafficking. Some binding partners are necessary for nuclear localization; therefore, alterations in binding partners could also disregulate trafficking. The tumor suppressor BRCA1 must disengage from its binding partner (BARD1) in order to be exported (Baer and Ludwig, 2002). Mutations in the transporter proteins or other essential transporter related proteins such as RanGTP could cause disregulation of nuclear shuttling. The nuclear pore proteins could be altered in such a way to either limit or promote transport (export or import) of specific proteins such as oncogenic or tumor suppressive proteins. Most of the research done on tumor suppressor genes has been directed towards genetic or epigenetic knockout of specific genes. However, defects in nuclear shuttling of tumor suppressive factors, such as APC, p53, VHL, and BRCA1, can circumvent their respective tumor suppressive effect (Fabbro and Henderson, 2000). A similar mechanism may exist in the trafficking of topoisomerases out of the nucleus into the cytoplasm. The proto-oncogene and transcriptional activator NFB is an example of misregulation of cellular trafficking (Kau, et al ., 2004). NFB promotes cell proliferation, is anti-apoptotic, and is involved in resistance to anticancer therapies. Regulation of NFB translocation is performed by its binding to I B, blocking its NLS and keeping it in the cytoplasm. NFB is released when I B is phosphorylated and subsequently degraded, exposing the NFB NLS and allowing nuclear import. Acetylation of NFB by p300 inhibits dimerization with I B and prevents any subsequent nuclear export. NFB is almost exclusively located in the nucleus in many
12 types of cancer, for example, lymphoma, leukemia, breast, ovarian, colon, pancreatic and thyroid tumors. Defects that cause aggressive phosphorylation of I B or hyperacetylation of NFB by p300 are mechanisms that promote NFB nuclear localization and its subsequent tumor promotion. Another molecule that is regulated by nucleocytoplasmic transport is the signaling molecule AKT. Constituent activation of AKT signaling can result in uncontrolled cellular proliferation and tumorogenesis (Kau, et al ., 2004). In normal cells AKT activity is regulated by PTEN, which inhibits phosphorylation of AKT by phosphatidylinositol 3kinase (PI3K). In cancer cells that lack PTEN activity, AKT is activated by PI3K and phosphorylates the tumor suppressors FOXO and p27, leading to their export from the nucleus. FOXO and p27 must be in the nucleus to inhibit E2F activation. E2F is a transcription factor that promotes cell-cycle progression by directing the expression of a number of cell cycle genes include various cyclins and cyclin dependent kinases (CDK). Drug Resistance via Nuclear Export of Topoisomerase II alpha Protein degradation and altered subcellular localization of topoisomerase 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, et al ., 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 topoisomerase II to the nucleolus (Hazelhurst, et al ., 2001). Similar changes in cellular localization were found in multiple myeloma cells
13 that adhered to endothelial cells (Figure 2). Thus, changes in the nuclear localization or binding properties of the nuclear pool of topoisomerase II protein may have a role in cellular drug resistance to etoposide and mitoxantrone in these cells. Figure 2: Immunofluorescent microscopy staining for topoisomerase II alpha. H929 multiple myeloma cell adhesion to endothelial cells causes translocation of topoisomerase II to the cytoplasm. B) Non-adherent H929 cells. (Sullivan Lab, unpublished data).
14 As previously reported, topoisomerase 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) (Figure 3)). No difference in the concentration or molecular mass of topoisomerase I, II or II 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 topoisomerase 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 topoisomerase 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 topoisomerase 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 topoisomerase into the nucleus from the cytosol. In fact, there has been only one study that has addressed the mechanism of the nuclear export of topoisomerase (Mirski, et al ., 2003). The results of this study were restricted to predicting the existence of an NES in topoisomerase II and topoisomerase II from peptide studies, and did not define a functional NES in the full-length protein (Mirski, et al ., 2003). The results of this study demonstrate that human topoisomerase II contains two functional LMB-sensitive NES in the full-length protein.
15 Figure 3: Immunofluorescent staining for topo II (A, D, G and J), histones (B, E, H and K) and merged images (C, F, I and L) in log and plateau phase CCRF and 8226 cell lines. Log phase 8226 cells (A-C) were at a density of 2x105 cells/ml; plateau phase 8226 cells (D-F) at 9x105 cells/ml; log phase CCRF cells (G-I) at 2x105 cells/ml; and plateau phase CCRF cells (J-L) at 1.6x106 cells/ml (Valkov et al., 2000).
16 MATERIALS AND METHODS Cell Culture and Accelerated-plateau Cell Model HeLa cells were grown in Alpha Minimal Essential Medium (Gibco) containing 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 100 U/ml penicillin, 100 g/ml streptomycin and 10% FBS (Hyclone). HL-60 leukemia cells and H929 myeloma cells were grown in RPMI media containing 100 U/ml penicillin, 100 g/ml streptomycin and 10% FBS (Hyclone). The nuclear-cytoplasmic trafficking of topoisomerase II was examined by plating cells in an accelerated-plateau cell model previously described in this laboratory (Valkov, et al ., 2000; Engel, et al ., 2004). For HL-60 and H929 cells, logphase conditions are defined by growing cells at 2.0 x 105 cells/ml and plateau-phase cells at 2.0 x 106 cells/ml. Both log and plateau phase cells were grown in fresh media in a 5% CO2 incubator at 37C for 24 hours prior to experiments. Putative NES Peptides The complete amino acid sequence for human topoisomerase 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 I. Six amino acid sequences in topoisomerase II matched the NES consensus sequence (Table II), 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-
17 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), C80GAYKA FDEAAA N91; (NES230-241), C230SLDKDIVALMVR241; (mutated NES230-241), C230SADKDAA AA MA R241; (NES467-476), C467TLAVSGLGVVG477; (mutated NES467-477), C467T AAA SGA GAA G477; (NES1017-1028), C1017DILRDFFELRLK1028; (mutated NES1017-1028), C1017CDIARDA FEA RA K1 028. Peptides (NES569-580), C569FLEEFITPIVKV580; (mutated NES569-580), C569AAEEAA TPAA KA 580; (NES1054-1066), C1054FILEKIDGKIIIE1066; and (mutated NES1054-1066); C1054FIAEKA DGKA IA E1066 were obtained from the University of Florida Protein Chemistry Core Facility (Gainesville, FL). 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). -actin (Wada, et al. 1998)A L PHA I MR L D L A -actin (Wada, et al. 1998) A L PHA I LR L D L A TFIIIA (F ride l l, et al. 1996)L -PV L EN L T L PKI (We n, et al. 1995) E L ALK L AG L D I N MAPKK (Fukuda, et al. 1996)A L QKK L EE L E L D RevHIV-1 (Fischer, et al. 1996)Q L -PP L ER L T L D Ran BP-1 (Zolotukhin and Felber, 1997)K V AEK L EA L S V R C-Abl (Taagepera, et al. 1998)L ESN L RE L Q I C hZ yxin (Nix and Beckerle, 1997) L TMKE V EE L E L L MdM2 (Roth, et al. 1998)S L S F DESL A L C P53 (Strommel, et al. 1999) F RE L NEA L E L KD NES Consensus HX1-4HX2-3HXH X = L eu, Ile, Val, or Phe Table I. Determination of a Co nsensus Sequence from Established NES
18 Generation of BSA-peptide-FITC Conjugates The generation of BSA-peptide -FITC conjugates was performed by Roxane Engel. Peptides were crosslinked to BSA and FITC as previously described (Stommel et al ., 1999). A total of 2 mg Imject Maleimide activated sulfosuccinmidyl 4-(Nmaleimidomethyl) cyclohexane-1-carboxylate bovine serum albumin (Sulfo-SMCC BSA) (Pierce, Rockford, IL) were reconstituted in 200 l distilled water. Then, a molar excess (1-2 mg) of native or mutated topoisomerase II NES peptide in 400 l 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). F D I I I K G D I K E L I (1054-1065) K L R L E F F D R L I (1017-1028) V K V I P T I F E E L F (569-580) V V G L G S V A L T (467-476) R V M L A V I D K D L S (230-241) V L I E D F I K Y L G (80-90) Table II Putative NES in Human Topo II
19 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) was packed at 2.0 ml/min 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 l Superloop and were run at 0.5 ml/min in degassed dH2O. The 500 l fractions were collected with a Fraction-100 collector (Pharmacia Biotech) and stored at 4C overnight. Total protein was estimated in peak samples by measuring the absorbance at 562 nm using the Fisherbrand Protein Assay. Approximately 25 g of protein from eluted fractions were loaded onto a 10% SDS-page gel and electrophoresed with 7 watts for 2-3 h. Peptide conjugation was confirmed by silver stain analysis. Similar fractions of crosslinked BSA-peptide were pooled and concentrated on a Microsep 30k filter by centrifuging at 5,000 x g in an SS-34 rotor until dry. The samples were eluted with 400 l of PBS, pH 7.4 to obtain approximately 2 mg/ml peptideconjugate solution. FITC was solubilized in DMSO at 1 mg/ml and added to the peptide sample in four 5 l aliquots until a total of 20 l of FITC (Sigma) were added. FITC was reacted with the peptides for 6 h at 4C, and then 23 l of 1 M NH4Cl in PBS, pH 7.4 were added to the sample and incubated for 2 h at 4C. 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.
20 Microinjection Microinjections were performed by Jennifer Derderian. To promote cell adherence, Fisherbrand glass coverslips were pretreated with 1N HCl for a minimum of 4h at 50C 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 37C for 24-48 hours preceding microinjection. Prior to microinjection, cells were gently rinsed with sterile PBS warmed to 37C and replaced with LeibovitzÂs L-15 Medium containing no phenol red. The peptide samples from above were centrifuged at 13,000 x g for 30 min at 4C, and the supernatant then loaded into Eppendorf Femptotips (diameter of 0.5 m 0.2 m). 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 sec, atmospheric conditions). Following injection, the cells were incubated at 37C for up to 90 min, washed with LeibovitzÂs L-15 Medium, and then fixed with 4% paraformaldehyde for 3 minutes at room temperature, rinsed in PBS and mounted onto 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).
21 Topoisomerase II Cloning and Site Directed Mutagenesis Topoisomerase II primers were designed that contained the eight amino acid FLAG peptide preceded by a start codon and a Kozak motif (5Â-ATG GAC TAC AAA GAC GAT GAC GAC AAG GAA GTG TCA CCA TTG CAG CCT GTA AAT GAA AAT ATG-3Â forward primer, 5Â-ATG CGG CCG CTT AAA ACA GAT CAT CTT CAT CTG ACT CTT C-3Â 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 M dNTP mixture and 200 nM each primer. Forty cycles were performed (94C for 30s, 60C for 30s, and 5.5 min at 68C), 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 5Â 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 (95C for 50 s, 60C for 50 s, and 68C for 20 min), 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 Table III.
22 Transfection Protocol Human myeloma H929 and HL-60 cells (ATCC) were plated at log phase density (2x105 cells/ml) two days prior to transfection. Transfection was performed as previously described (Van den Hoff, et al ., 1992). Briefly, 40 g of wild-type or mutated topoisomerase II plasmid in 300 l of a solution containing 10 mM Tris-HCl, 1 mM EDTA, pH 8.0, were precipitated by the addition of 30 l of 5 M NaCl and two volumes of 95% ethanol on ice for ten minutes. Plasmid was pelleted by centrifugation for fifteen minutes at 20,000 x g at 4C, washed with 75% ethanol and re-centrifuged. All remaining ethanol was removed by pipet and the DNA immediately resuspended in 50 l 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, pH 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 media (RPMI/10% FBS/pen-strep) at a concentration of 2 x105 cells/ml. Cells were collected
23 and 1.6x107 cells pelleted by centrifugation at 1500 x g for 5 minutes. Cell pellets were washed twice in 10 ml of sterile PBS, resuspended in 350 l of cytomix buffer (4C), 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 (2 x105 cells/ml) and plateau (2 x106 cells/ml) growth conditions for twenty hours in a 5 % CO2 incubator at 37C with RPMI medium containing 5% FBS. Immunofluorescence Twenty hours post-transfection, viable H929 and HL-60 cells were isolated by centrifugation at 2000 x g for 20 minutes at 20C 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 20oC for ten minutes. The fixation was stopped by washing in PBS and cells were permeabilized for twenty-four hours in a solution containing 1% glycine and 0.25% Triton X-100 in PBS. Slides were stained with antiFLAG M2 monoclonal antibody-FITC conjugate (Sigma) diluted 1:100 with 0.1% NP-40 and 1% BSA in PBS, and incubated one hour at room temperature. Slides were washed in PBS, dried briefly and counterstained with Vectashield mounting media antifade/DAPI (1:1) (Vector Laboratories Inc., Burlingame, CA). Immunofluorescence was observed with a Leitz Orthoplan 2 fluorescent microscope and images were captured by a CCDcamera with Smart Capture program (Vysis, Downers Grove, IL Quantitation of FITC fluorescence was performed using the Adobe Photoshop 7.0 program.
24 Western Blot HeLa cells grown in RPMI media containing 5 % FBS were transfected directly on 100 cm2 tissue culture plates. Plasmid DNA (10 g) was mixed with 60 l of Superfect transfection reagent (Qiagen) in 300 l of serum-free media for 10 minutes, followed by 600 l of serum containing media, and the entire mixture was then added directly to cell culture plates. Transfection was allowed to proceed for three hours at 37C in a 5 % CO2 incubator, and terminated by the removal of transfection solution and the addition of 15 ml of 5% FBS containing RPMI media. After incubation for twentyfour 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 one 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 4C. Membranes were washed three times for ten 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% tween20, and 5% non-fat milk for sixty minutes at room temperature. Antibody binding was visualized by ECL (Amersham) on autoradiography film (Kodak).
25 RESULTS Peptides NES1054-1066 and NES1017-1028 Signal the Nuclear Export of BSA-FITC Of the six putative NES identified in topoisomerase II two peptides, NES10541066 and NES1017-1028, signaled the export of BSA into the cytoplasm when microinjected into the nuclei of HeLa cells (Figure 4). BSA-NES1054-1066 showed strong cytoplasmic staining and was seen in the cytoplasm within 15 minutes of microinjection, as compared to TRITC-BSA alone (not shown), or the mutated BSA-NES1054-1066 conjugate. BSANES1017-1028 also appeared cytoplasmic within 15 minutes of being microinjected into the nucleus, but complete nuclear clearing (seen with BSA-NES1054-1066) was not observed even after 90 minutes. The mutated BSA-NES1017-1028 was nuclear in all cells even 90 minutes after microinjection. BSA-NES80-90 (Figure 4), mutated BSA-NES80-90 (Figure 4), BSA-NES230-241, mutated BSA-NES230-241, and BSA-NES467-476, BSA-NES569-580, and mutated BSA-NES569-580 all remained in the nucleus even 4 h after microinjection (data not shown). LMB Blocks NES1054-1066 and NES1017-1028 Mediated Nuclear Export 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 NES1054-1066and 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
26 Figure 4: HeLa cells microinjected with either wild-type (left column) or mutated (right column) peptide-BSA-FITC conjugates (green), and then counterstained with DAPI (blue). A total of 20-50 cells were successfully microinjected per peptide and similar results were seen in all cells. The bottom of the figure presents HeLa cells that were microinjected with wild-type peptide-BSA-FITC conjugates in the presence of 2 ng/ml NES1054-66 NES1017-28 NES80-91 Native Mutated LMB NE S 1 0 17-2 8 LMB Â… NES1054-66
27 LMB. Mutated peptide BSA-NES 1054-66 is at a higher magnification than the other micrographs. LMB blocked the export of BSA conjugated to peptides NES1054-1066 and NES1017-1028(Figure 4). BSA-NES1054-1066 had a strong perinuclear staining, suggesting that the protein cargo is 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 topoisomerase II To determine if these NES are necessary for topoisomerase II export, I 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 I first attempted to study full-length topoisomerase II protein trafficking using a green fluorescent protein-topoisomerase II alpha (GFP-topo II ) fusion protein expression vector. However, expression of GFP-topo II recombinant protein was found to be cytotoxic, inducing apoptosis in all cell lines tested (HeLa, HL-60, H992, 8226, MCF-7, Chinese hamster ovary) 16-48 hours after transfection. The GFP also inhibited translocation of the wildtype topoisomerase-GFP fusion protein from the nucleus to the cytoplasm in plateau density cells (data not shown). Additionally, a GFP-topo II fusion plasmid in which the active-site tyrosine 805 was mutated to an alanine, GFP-topo II A805, also produced similar negative results, being lethal to the transfected cells and
28 minimally exported to the cytoplasm in plateau density cells, (again contrary to endogenous topoisomerase II ). A final GFP plasmid was produced 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. Destabilized GFP fusion protein is turned over every four hours in transfected cells, thus not allowing cellular accumulation of recombinant protein to cytolytic levels. The 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 fusion proteins led to an investigation into alternative topoisomerase 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) topoisomerase II recombinant proteins (Figure 5). Human myeloma cells are comparatively small and very difficult to transfect with plasmid DNA. In an attempt to transfect human myeloma cell lines, a number of commercially available transfection reagents and numerous protocols were tried with no success. The only technique that yielded positive transfectants was a method described by Van den Hoff, et al ., 1992. This method utilizes a cytomix buffer made to approximate the intracellular environment (see Methods). In addition, this buffer contains ATP and glutathione to promote the rapid repair of cellular membranes. I was able to transfect myeloma cells with a high degree of efficiency for these cell lines (2-20 %) using the Van den Hoff cytomix buffer.
29 Figure 5: Western blot of full-length FLAG-topo II protein. HeLa cells were transfected with plasmid containing FLAG-topo II plasmid via cationic lipid, and harvested after twenty hours. Protein extracts from 2x105 cells per lane were separated on a SDS-PAGE gel, blotted onto nitrocellulose and probed with FLAG M2 antibody. Lane A is protein from cells transfected with non-mutated FLAG-topo II plasmid, lane B is FLAG-topo II plasmid 1054-1066, lane C transfected with FLAG-topo II plasmid 1017-1028, and lane D is a non-transfected control.cells were transfected with FLAGtopo II expression vectors possessing mutated hydrophobic residues in the nuclear export sites at 1017-1028 and 1054-1066 (Table III). 117 kDa A B C D207 kDa
30 FLAG-topo 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 topoisomerase II To confirm these data with a full-length topoisomerase II protein, H929 human myeloma Twenty hours post-transfection, viable cells were isolated by centrifugation on a Ficollpaque gradient and plated on glass microscope slides using cytospin funnels. After fixing and permeabilization, slides were stained with anti-FLAG M2 monoclonal antibodyFITC conjugate and counterstained with mounting media containing DAPI to show the location of the nuclei. Images were acquired using a fluorescent microscope (Figure 6), with quantitation of FITC fluorescence using Adobe Photoshop 7.0 (data expressed in Figure 7). Figures 6 and 7 establish 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 (Figure 6) when using the wild-type FLAG-topo II plasmid. When the putative export signals at either 1017-1028 (Figure 7) 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
31 was similar to wild-type (Figure 7) as were putative signals at amino acid 80-91, 230241, and 569-580 (data not shown). Figure 6: Nuclear export of wild-type and mutant FLAG-topo II plasmids. Human myeloma H929 transfected cells (n=20) stained with anti-FLAG M2 monoclonal antibody-FITC conjugate were assayed for nuclear and cytoplasmic immunofluorescence. Quantitation of FITC fluorescence was performed using Adobe Photoshop 7.0 program. Wild-type FLAG-topo II was exported to the cytoplasm in cells at plateau density (p=0.00001), whereas topoisomerase II mutated at the putative export sites, 1016-1027 and 1053-1066, did not demonstrate statistically significant levels of export to the cytoplasm. 0 25 50 75 100FLAG-Topo II alpha protein (% nuclear vs cytoplasmic)Wild-type log Wild-type plateau 1016-1027 log 1016-1027 plateau 1053-1066 log 1053-1066 plateau Cytoplasmic Nuclear *p=0.00001
32 Figure 7: FLAG-topo II Immunofluorescence. Human multiple myeloma H929 cells were transfected by electroporation with full-length wild-type and mutated topo II and plated for twenty hours at log and plateau cell densities. Cytospins containing fixed cells were stained with FITC labeled anti-FLAG M2 antibody (green), counterstained with DAPI for nuclear staining, and assayed by immunofluorescent microscopy. FITC DAPI Merged FITC DAPI Merged Log Plateau Wild-type Mutated NES 467-77 Mutated NES 1017-28 Mutated NES 1054-66
33 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 topoisomerase II Tables IV and V summarize a list of representative species containing homologous topoisomerase II sequences. The data show that the characteristic spacing of hydrophobic residues in peptides NES1054-1066 and NES1017-1028are highly conserved in a broad range of species. For example, leucine residues appearing in human topoisomerase II NES are often substituted with the hydrophobic amino acids isoleucine or valine. Furthermore, Phe1054 and Ile1055 in peptide NES1054-1066are 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). NES1054-1066 and NES1017-1028 Reside Within a Putative Coiled-coil Domain Predicting the structural features of the region containing NES1017-1028 and NES1054-1066 was of particular interest. Each topoisomerase II monomer can be divided into three domains, 1) an N-terminal domain that contains the ATP-binding region, 2) the central domain containing the active site tyrosine residue, and 3) the C-terminal domain that contains the nuclear localization sequences (Figure 1) (Watt and Hickson, 1994). Both of the NES are situated upstream of the bipartite NLS (Figure 8) and downstream of the active-site tyrosine residue (Tyr805). Furthermore, several CK-2 phosphorylation
34 Table IV. Sequence Alignment of Topo II NES1017-1028Homo sapiens (human) NES1017-1028D I L RD F FE L R L K Sus scrofa (pig), Mus musculus (mouse), Rattus norvegicus (rat) D I L RD F FE L R L K Cricetulus griseus (Chinese hamster)D I L *D F FE L R L K Gallus gallus (chicken)D I L ** F FE L R L Saccharomyces cerevisiae (yeast)* I L ** F ** V R L Aspergillus niger (fungi), penicillium citrinum (fungi) D I L ** F F* V R L K Nicotiana tabacum (tobacco plant)D I L ** F ** V R L Encephalitozoon cuniculi (protozoa)* I L ** F ** V R L Bombyx mori (silkmoth)* I L R* F ** L R V sites downstream of both NES have been identified in vitro and could be important for regulating the subcellular localization of topoisomerase II Although NES1017-1028 alone is predicted to form an -helix (Mirski, et al ., 2003), I was interested in predicting the motif of the complete amino acid sequence stretching from NES1017-1028 to NES1 054-1066.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,
35 Table V. Sequence Alignment of DNA Topo II NES1054-1066Homo sapiens (human) NES1054-1066 F I L EK I DG K I I I E Cricetulus griseus (Chinese hamster), Sus scrofa (pig) F I L EK I DG K I I I E Gallus gallus (chicken), Mus musculus (mouse), Rattus norvegicus (rat) F I L EK I DG K I V I E Caenorhabditis elegans (nematode) F I L *K I ** I V L E Saccharomyces cerevisiae (yeast) F I ** I ** L V Candida glabrata (yeast) F I ** I ** L I V Aspergillus candidus (fungi), Trichophyton rubrum (fungi) F V ** I *G L V V Giardia lamblia (protist) F I * I ** L I 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 topoisomerase 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 topoisomerase II from S. 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 topoisomerase II (Frere, et al ., 1995).
36 DISCUSSION Topoisomerase II is an essential enzyme involved in the normal processing of DNA and is expressed at high levels in rapidly dividing cells. These qualities make it a perfect target for chemotherapeutic agents in treatment of various cancers. Human topoisomerase I (Mo, et al ., 2000), II and II (Mirski, et al ., 1999) have NLS that target their movement into the nucleus, but only one report has begun to address the mechanism of nuclear export of topoisomerase II and topoisomerase II (Mirski, et al ., 2003). The nuclear export of topoisomerase I in response to topotecan or camptothecin exposure has been reported (Danks, et al ., 1996), as has the redistribution of topoisomerase I from the nucleus to the nucleoli (Buckwalter, et al ., 1996). The lack of data describing the nuclear-cytoplasmic shuttling of topoisomerase enzymes may be because topoisomerase is usually found to occur in the nucleus of cells, and a cytoplasmic distribution of topoisomerase II has only been attributed to the expression of a truncated protein that has lost its C-terminal NLS. However, proteins that appear predominately nuclear may still shuttle between the nucleus and the cytoplasm, if the rate of nuclear import is greater than the rate of nuclear export. Thus, demonstrating that a protein shuttles between the nucleus and cytoplasm requires defining the specific conditions that will shift the steady state kinetics toward nuclear export. Many conditions have been shown to alter the shuttling of proteins between the nucleus and cytoplasm, including changes in the cell cycle and oxidative stress (reviewed in Damelin, et al ., 2002). For example, in the accelerated-plateau cell model (Valkov, et al ., 2000; Engel, et
37 al ., 2004) used in the present experiments, it is likely that intensive cell-cell contact initiates a signal that induces the export of topoisomerase II to the cytoplasm (Nix and Beckerle, 1997; Gottardi, et al ., 1996). This is supported by the findings of others that a cytoplasmic distribution of topoisomerase II occurs in the outer-proliferating cells of multi-cell spheroids in Xenograft tumors when compared to monolayers formed by these cells (Oloumi, et al ., 2000). Phosphorylation has also been shown to be important in regulating the subcellular localization of many proteins and could have a role in topoisomerase II trafficking. This is suggested by the finding that the cells of multi-cell spheroids contain a cytoplasmic pool of topoisomerase II and have a 10-fold decrease in the phosphorylation state of the enzyme when compared to monolayers (Oloumi, et al ., 2000). Since CK-2 has been shown to phosphorylate topoisomerase II on several serine and threonine residues near the NES or NLS (Ackerman, et al ., 1985), CK-2 is a logical candidate to investigate for modulating topoisomerase II trafficking. One of the potential consequences of exporting a pool of topoisomerase II to the cytoplasm is a decrease in sensitivity to topoisomerase poisons. This could result from cytoplasmic topoisomerase 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 topoisomerase II can occur in the absence of DNA (Burden, et al ., For this to occur, the amount of drug binding to topoisomerase 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 topoisomerase 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 topoisomerase II with drugs such as
38 1 MEVSPLQPVN ENMQVNKIKK NEDAKKRLSV ERIYQKKTQL EHILLRPDTY IGSVELVTQQ 61 MWVYDEDVGI NYREVTFVPG LYKIFDEILV NAADNKQRDP KMSCIRVTID PENNLISIWN 121 NGKGIPVVEH KVEKMYVPAL IFGQLLTSSN YDDDEKKVTG GRNGYGAKLC NIFSTKFTVE 181 TASREYKKMF KQTWMDNMGR AGEMELKPFN GEDYTCITFQ PDLSKFKMQS LDKDIVALMV 241 RRAYDIAGST KDVKVFLNGN KLPVKGFRSY VDMYLKDKLD ETGNSLKVIH EQVNHRWEVC 301 LTMSEKGFQQ ISFVNSIATS KGGRHVDYVA DQIVTKLVDV VKKKNKGGVA VKAHQVKNHM 361 WIFVNALIEN PTFDSQTKEN MTLQPKSFGS TCQLSEKFIK AAIGCGIVES ILNWVKFKAQ 421 VQLNKKCSAV KHNRIKGIPK LDDANDAGGR NSTECTLILT EGDSAKTLAV SGLGVVGRDK 481 YGVFPLRGKI LNVREASHKQ IMENAEINNI IKIVGLQYKK NYEDEDSLKT LRYGKIMIMT 541 DQDQDGSHIK GLLINFIHHN WPSLLRHRFL EEFITPIVKV SKNKQEMAFY SLPEFEEWKS 601 STPNHKKWKV KYYKGLGTST SKEAKEYFAD MKRHRIQFKY SGPEDDAAIS LAFSKKQIDD 661 RKEWLTNFME DRRQRKLLGL PEDYLYGQTT TYLTYNDFIN KELILFSNSD NERSIPSMVD 721 GLKPGQRKVL FTCFKRNDKR EVKVAQLAGS VAEMSSYHHG EMSLMMTIIN LAQNFVGSNN 781 LNLLQPIGQF GTRLHGGKDS ASPR Y IFTML SSLARLLFPP KDDHTLKFLY DDNQRVEPEW 841 YIPIIPMVLI NGAEGIGTGW SCKIPNFDVR EIVNNIRRLM DGEEPLPMLP SYKNFKGTIE 901 ELAPNQYVIS GEVAILNSTT IEISELPVRT WTQTYKEQVL EPMLNGTEKT PPLITDYREY 961 HTDTTVKFVV KMTEEKLAEA ERVGLHKVFK LQTSLTCNSM VLFDHVGCLK KYDTVL DILR 1021 DFFELRLK YY GLRKEWLLGM LGAESAKLNN QAR FILEKID GKIIIE NKPK KELIKVLIQR 1081 GYDSDPVKAW KEAQQKVPDE EENEE S DNEK ETEKSDSVTD SGPTFNYLLD MPLWYLTKEK 1141 KDELCRLRNE KEQELDTLKR KSPSDLWKED LATFIEELEA VEAKEKQDEQ VGLPGKGGKA 1201 KGKKTQMAEV LPSPRGQRVI PRITIEMKAE AEKKNKKKIK NENTEGSPQE DGVELEGLKQ 1261 RLEKKQKREP GTKTKKQTTL AFKPIKKGKK RNPWSDSESD RSSDESNFDV PPRETEPRRA 1321 ATKTKFTMDL DSDEDFSDFD EK T DDEDFVP SDASPPKTKT SPKLSNKELK PQKSVV S DLE 1381 ADDVKGSVPL SSSPPATHFP DETEITNPVP KKNVTVKKTA AKSQSSTSTT GAKKRAAPKG 1441 TKRDPALNSG VSQKPDPAKT KNRRKRKP S T SDDSDSNFEK IVSKAVTSKK SKGESDDFHM 1501 DFDSAVAPRA KSVRAKKPIK YLEE S DEDDL F Figure 8: The complete amino acid sequence of human DNA topoisomerase II ( accession number NP 001058). The active-site tyrosine residue (805) is indicated ( ): NES1017-1028, C1017DILRDFFELRLK1028 and NES1054-1066, C1054FILEKIDGKIIIE1066are shaded ; bipartite NLS is single underlined; ser/thr CK2 phosphorylation sites are italicized ; the predicted coiled-coil region is outlined with a box.1996).
39 LMB may sensitize cells to topoisomerase 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.
40 FUTURE DIRECTIONS Crm1 Proof of Principle In this paper I relied primarily on the Crm1 inhibitory activity of LMB to prove that topoisomerase export is Crm1 mediated, however I was unable to show that blocking export would make cancer cells more sensitive to etoposide or mitoxantrone chemotherapy because LMB itself is so toxic. It may be possible to block Crm1 mediated export by other more specific means. One way in which this could be done is by using an antisense phosphorothioate oligonucleotide or siRNA specifically directed against Crm1. This might work to inhibit expression of Crm1 without the cytotoxic effects of LMB. Once Crm1 expression is knocked out, or at least substantially reduced, it may be possible to investigate an increased sensitivity to topoisomerase II directed chemotherapy ( i.e., etoposide and mitoxantrone). It is interesting to note that the dimerization region of topoisomerase II contains both NES amino acid sequences. As a proof of principle experiment, a plasmid coding for the dimerization region could be produced and transfected into cells. If it is determined that nuclear export of topoisomerase II is blocked, I could then proceed to determine whether the cells are more sensitive to chemotherapy. It is unknown whether this peptide would block all nuclear export or only export of topoisomerase II Specificity could be determined is by using a plasmid containing a GFP/MAPKK NES fusion as a control for the specificity of export blocking (Kanwal, et al ., 2002). If a recombinant protein possessing the dimerization region was effective, peptidomimetics could be produced,
41 and possibly small molecules developed based on the peptide binding properties that could block nuclear export. High-Throughput Assay New compounds are being developed to block nuclear export as a means to treat cancer. Using multiple myeloma cell lines I can easily test inhibition of nuclear export using the log vs. plateau system developed in this laboratory. Potential export blockers could be tested in cell culture in both log and plateau cell density conditions. Immunofluorescent staining for topoisomerase II could be performed, and pixel intensities determined to give a relative measure of nuclear export. Phosphorylation of Topoisomerase II alpha Even though I have found the NES responsible for nuclear export, and that it is Crm1 mediated, the exact mechanism that is inducing nuclear export of topoisomerase in cancer cells has not yet been determined. A likely candidate is phosphorylation of the topoisomerase molecule itself. Heretofore it has not been determined whether phosphorylation is necessary for nuclear export of topoisomerase II Caseine kinase II has been shown to phosphorylate topoisomerase II on several serine and threonine residues near the NES or NLS (Ackerman, et al ., 1985), and therefore is a logical candidate to investigate for modulating topoisomerase II trafficking. There are many ways in which phosphorylation could induce nuclear export, phosphorylation may affect topoisomerase dimerization, and it could reveal or mask nuclear localization or nuclear export signals by changing the molecular conformation of topoisomerase II .
42 If it is discovered that phosphorylation in necessary for nuclear export, there are a number of phosphorylation inhibitors that I could test in the high-throughput assay. Figure 1 maps out the potential phosphorylation sites on the topoisomerase II peptide. Specific inhibitors of casein kinase 2, protein kinase C, p34cdc2, or MAP kinase could be tested. In addition, as a proof of principle experiment, antisense phosphorothioate oligonucleotides or siRNA directed at each kinase could be investigated to see if blocking topoisomerase II phosphorylation negatively regulates nuclear export. Other Questions Another question that could be addressed in future experiments is whether topoisomerase II is exported as a monomer or a dimer. It is possible that a topoisomerase II monomer would likely reveal export signals for CRM-1 binding. Dimerization and phosphorylation status may be interdependent of each other. Also, it is very likely that nuclear export that is caused by plateau cell densities may be mediated by a signaling pathway, microarray analysis may provide some clue as to what pathway(s) may be involve. The signaling pathway may provide additional targets for chemotherapy.
43 SUMMARY Resistance to chemotherapeutic drugs is a major obstacle in the treatment of leukemia and myeloma. This lab has 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 topoisomerase 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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