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Cell Growth & Differentiation Vol. 13, 325-333, July 2002
© 2002 American Association for Cancer Research

Both {alpha} and ß Isoforms of Mammalian DNA Topoisomerase II Associate with Chromosomes in Mitosis1

Allison P. Null2, Joanna Hudson and Gary J. Gorbsky3

Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104


   Abstract
TOP
 Abstract
Introduction
 Results and Discussion
 Materials and Methods
 References
 
Two isoforms of DNA topoisomerase II, {alpha} and ß, coded byseparate genes, are expressed in actively cycling vertebratecells. Some previous studies have suggested that only topoisomerase II{alpha} remains associated with chromosomes at mitosis. Here, the distributions of topoisomerase II{alpha} and ß in mitosis were studied by subcellular fractionation and by immunolocalization. Both isoforms of topoisomerase II were found to remain associated with mitotic chromatin. Topoisomerase II{alpha} was distributed along chromosome arms throughout mitosis and was highly concentrated at centromeres until mid-anaphase, particularly in some cell types. Topoisomerase IIß showed weak concentration at centromeres in early mitosis in some cell types and was distributed along chromosome arms at every stage of mitosis through telophase. These studies suggest that in most cells both the major topoisomerase II isoforms may play roles in chromatin remodeling during M phase.


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Materials and Methods
 References
 
DNA topoisomerase II catalyzes the ATP-dependent passage of one double-stranded DNA molecule through a transient break in another. Topoisomerase II has been implicated in many aspects of DNA metabolism, including transcription, replication, recombination, and segregation (reviewed in Refs. 1, 2, 3 ). Many lines of evidence reveal essential functions for topoisomerase II in M phase. Topoisomerase II mutants in yeast fail to separate their chromosomes at mitosis (4 , 5) . In meiotic extracts of Xenopus eggs, immunodepletion of topoisomerase II inhibits chromosome condensation (6) . In mammalian cells, chemical inhibitors of topoisomerase II block both chromosome condensation and segregation (7, 8, 9, 10, 11) . In addition to its enzymatic roles, topoisomerase II has been proposed to be a structural component of the interphase nuclear matrix (12, 13, 14) and of the M-phase chromosome scaffold (15 , 16) . Topoisomerase II is the target of a number of clinically approved or experimental antineoplastic agents (reviewed in Refs. 17, 18, 19 ).

In nonvertebrate eukaryotes, such as yeast and Drosophila, a single isoform of topoisomerase II is present. In mammals, two major isoforms of the enzyme, encoded by different genes, have been identified: topoisomerase II{alpha} (Mr 170,000) and topoisomerase IIß (Mr 180,000; Ref. 20 ). Topoisomerase II isoforms show differences in cell cycle expression. Topoisomerase II{alpha} is preferentially expressed in proliferating cells, whereas topoisomerase IIß is expressed in both actively dividing cells and cells that have withdrawn from the cell cycle (1 , 21, 22, 23, 24, 25) . In most studies, expression of topoisomerase II{alpha} has been found to increase during S phase, peak at G2-M, and be diminished during G1. In contrast, topoisomerase IIß is expressed at relatively constant levels through the cell cycle. Some cell lines that exhibit greatly reduced expression of active topoisomerase IIß have been reported (26 , 27) . In addition, mice in which the topoisomerase IIß gene has been ablated survive through embryogenesis but die at birth with neurological and neuromuscular defects (28) . These studies indicate that expression of topoisomerase IIß is not an absolute cellular requirement for mitosis and cell division. Nonetheless, where it is expressed in most cells, topoisomerase IIß may be an important contributor to the proper maintenance and segregation of the mitotic chromosomes. Screening of cDNAs by Petruti-Mot and Earnshaw (29) recently identified several differentially spliced versions of topoisomerase II{alpha} and ß. These findings suggest that additional complexity may exist among isoforms in their roles in interphase and mitosis.

Considerable diversity of opinion exists regarding the apparent cellular localization of the topoisomerase II{alpha} and ß both in interphase and in M phase. In interphase cells, some reports indicate that topoisomerase II{alpha} is found both generally within the nucleoplasm and within the nucleolus (14 , 30) , whereas others suggest that topoisomerase II{alpha} is excluded from the nucleolus (24 , 31) . In M phase, topoisomerase II{alpha} has been described as diffusely distributed along the chromosome arms (10 , 24 , 31) or concentrated in axial core structures of the mitotic chromosomes (32 , 33) . In addition to its presence in the chromosome arms, several studies show that topoisomerase II{alpha} is concentrated at the centromeres of mitotic chromosomes (8 , 10 , 33 , 34) . Distribution along the chromosome arms and at the centromeres is consistent with enzymatic and/or structural roles for topoisomerase II in chromosome condensation and segregation.

The localization of topoisomerase IIß is yet more controversial. Early reports suggested that in interphase cells, topoisomerase IIß was restricted to the nucleolus (35 , 36) . Other reports suggested that it was both found both in the nucleolus and in the surrounding nucleoplasm (14 , 30) . Finally, some studies suggested that the enzyme was distributed within the nucleus but excluded from the nucleolus (24) . Previous immunolabeling studies have led researchers to conclude that topoisomerase IIß dissociates from the chromatin during mitosis (24 , 30 , 31) . In contrast, previous immunoblotting studies of isolated mouse chromosomes suggested that a significant proportion of topoisomerase IIß remains associated with the condensed mitotic chromosomes (33) .

Immunolocalization studies are complicated because fixation and permeabilization methods lead to redistribution of intracellular proteins (37) . The high density of the nuclear matrix and the condensed mitotic chromatin may make antigen accessibility problematic. To assess the distribution of topoisomerase II isoforms, particularly at mitosis, we prepared a novel antibody to topoisomerase IIß and used it in conjunction with commercial antibody to topoisomerase II{alpha}. Consistent with most previous reports, we find that topoisomerase II{alpha} is abundantly expressed only at certain stages of cell cycle, whereas topoisomerase IIß is present at all times. However in contrast to previous immunolocalization studies, we find that significant amounts of topoisomerase IIß remains associated with condensed mitotic chromosomes. The lack of detection of topoisomerase IIß in mitotic chromosomes by immunolabeling in previous studies may have resulted from the masking of antigenic sites caused by chromosome condensation and by fixation and labeling protocols.


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Materials and Methods
 References
 
Characterization of Antibodies to Topoisomerase II Isoforms.
To examine the specificity of the topoisomerase II antibodies to topoisomerase II{alpha} and ß, whole cell extracts from cycling populations of HeLa cells and Ptk1 cells were analyzed by Western blotting (Fig. 1A)Citation . In HeLa cell extracts, antibodies to topoisomerase II{alpha}, both monoclonal and polyclonal, recognized a single band of Mr 170,000. The monoclonal antibody to topoisomerase II{alpha} recognizes only protein from primate cells, so it is not useful for Ptk1 cells. The polyclonal anti-topoisomerase II{alpha} antibody identified a Mr 170,000 protein in the Ptk1 cell extract. The antibody to topoisomerase IIß recognized a band with mobility of Mr 180,000 in both HeLa cell and Ptk1 cell extracts. This band corresponds to full-length topoisomerase IIß. In HeLa cell extracts, the anti-topoisomerase IIß antibody labeled another band of Mr 150,000. The presence of a major band at Mr 150,000, immunoreactive with antibody to topoisomerase IIß in human cell extracts, has been reported by previous researchers (21 , 36) . The Mr 150,000 breakdown product was not detected in extracts of Ptk1 cells (Fig. 1A)Citation , nor was it present in extracts from several other cell lines tested (data not shown). The labeling of cell extracts with a mixture of antibodies to topoisomerase II{alpha} and ß confirmed that each antibody recognized distinct protein bands. To test whether that the antipeptide antibody we prepared to topoisomerase IIß was capable of recognizing topoisomerase IIß in native form, we carried out immunoprecipitation experiments with extracts prepared by lysing cells in a high salt buffer containing nonionic detergent. Both the crude immune serum and the affinity purified antibody could immunoprecipitate topoisomerase IIß from the cell extracts (Fig. 1B)Citation .



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  		Fig. 1. A, characterization of antibodies to topoisomerase II{alpha} and ß by Western blotting of whole cell extracts from HeLa cells (left panel) and Ptk1 cells (right panel). Cells were dissolved in SDS sample buffer, electrophoresed on 5–20% polyacrylamide gels, and transferred to PVDF paper. They were then probed with antibodies to topoisomerase II{alpha}, with antibody to topoisomerase IIß or with a mixture of antibodies to topoisomerase II{alpha} and ß. The antibody to topoisomerase II recognizes a single band of Mr 170,000. The antibody to topoisomerase IIß recognizes a major band of Mr 180,000. In the HeLa extract, but not the Ptk1 extract, another band of Mr 150,000, thought to be a breakdown product of topoisomerase IIß, is also identified. In the final lane of the HeLa panel, preincubation of anti-topoisomerase IIß with an excess of the peptide used for immunization blocks the labeling of topoisomerase IIß. The peptide inhibited binding of anti-topoisomerase IIß to the Mr 180,000 band and the putative breakdown product. B, immunoprecipitation of topoisomerase IIß from mitotic cell extracts by anti-topoisomerase IIß peptide antibody. Mitotic cell extracts were precipitated with crude immune serum or with affinity-purified anti-topoisomerase IIß antibody linked to protein A beads. The beads were washed extensively, and attached proteins were dissolved in SDS sample buffer. Samples were run on 4–12% polyacrylamide gels, transferred to PVDF paper, and probed with anti-topoisomerase IIß antibody. One major specific band runs at Mr ~185,000 corresponding to the mobility of mitotic topoisomerase IIß. The dense band at Mr ~55,000 represents IgG heavy chain of the precipitating antibody. Mock immunoprecipitations in the absence of added cell extract reveal nonspecific background bands in the crude serum.

 
Both {alpha} and ß Isoforms of Topoisomerase II Are Components of Chromosomes Isolated from HeLa Cells Arrested at M Phase.
Mitotic chromosomes were isolated from HeLa cells arrested in M phase by treatment with Colcemid. When analyzed by Western blotting, antibodies to both topoisomerase II{alpha} and ß labeled bands of the appropriate molecular weight (Fig. 2A)Citation . Coincubation of anti-topoisomerase IIß with a 100-fold molar excess of the peptide to which it was produced eliminated the binding of the antibody to topoisomerase IIß. Nonspecific peptides caused no inhibition of labeling. Cell fractions obtained during the chromosome isolation protocol were also analyzed by Western blotting of samples loaded with equal protein (Fig. 2B)Citation . Both the {alpha} and ß isoforms of topoisomerase II were concentrated in the chromosome fraction, in the slow speed centrifugation pellet (containing interphase nuclei), and in the medium speed centrifugation pellet (containing fragments of nuclei and clumps of chromosomes). The cytoplasmic fraction (containing small cellular organelles and soluble cytoplasmic components) showed no topoisomerase II{alpha} but did reveal a small amount of topoisomerase IIß, suggesting that some topoisomerase IIß was released in the cytoplasm during M phase. Indirect immunofluorescent labeling of chromosomes with antibodies to topoisomerase II{alpha} and ß also confirmed that both isoforms are present in isolated chromosomes (Fig. 2C)Citation .



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  		Fig. 2. Mitotic chromosomes isolated from HeLa cells contain both topoisomerase II{alpha} and ß. A, isolated chromosomes were analyzed by Western blotting with antibody to topoisomerase II{alpha}, antibody to topoisomerase IIß, antibody to topoisomerase IIß preincubated with blocking peptide, antibody to topoisomerase IIß preincubated with nonspecific peptide, and a mixture of antibodies to topoisomerase II{alpha} and ß. B, fractions prepared during the isolation of mitotic chromosomes were analyzed by Western blotting for the presence of topoisomerase II{alpha} and ß. The lanes show the final isolated chromosomes, the cytoplasm remaining in the supernatant after pelleting of the chromosomes, the low-speed pellet containing large pieces of cellular debris and clumps of interphase nuclei, and nuclei containing contaminating interphase nuclei and aggregates of mitotic chromosomes. Topoisomerase II{alpha} was present in all lanes that contain either chromosomes or interphase nuclei but was not detected in the cytoplasm. Topoisomerase IIß was present at high concentration in all lanes that contain either chromosomes or interphase nuclei. A small amount of topoisomerase IIß was detected in the cytoplasmic extract, suggesting that either some topoisomerase IIß was not associated with the chromatin or that it was released during cell fractionation. C, isolated mitotic chromosome was triple labeled to reveal topoisomerase II{alpha}, topoisomerase IIß, and the distribution of DNA. Antibodies to both topoisomerase II isoforms label along the chromosome arms.

 
Immunolocalization of Topoisomerase II{alpha} and ß in Whole Cells.
To further analyze the distribution of topoisomerase II isoforms during the cell cycle, we prepared cultured cells for immunofluorescence microscopy. To avoid artifactual redistribution of proteins during specimen preparation, cells were fixed by immersion in methanol at -20°C. Direct fixation with cold methanol has been shown to result in the least redistribution of internal cellular proteins (37) . We examined the expression of topoisomerase II{alpha} and ß by double antibody labeling of two human cell types, HeLa cells and early passage cultures of normal human keratinocytes (Fig. 3)Citation . Identical results were obtained with both cell types. As expected, topoisomerase II{alpha} was expressed at different levels in the nuclei of interphase cells but was strongly expressed in all M-phase cells where it was associated with the mitotic chromosomes. Topoisomerase IIß was expressed in the nuclei of all interphase cells. In mitotic cells, much topoisomerase IIß localized on the mitotic chromosomes, although a certain degree of diffuse cytoplasmic fluorescence was also detected.



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  		Fig. 3. Triple labeling of HeLa cells (A–C) and human keratinocytes (D–F) with antibody to topoisomerase II{alpha} (A, D) antibody to topoisomerase IIß (B, E) and with the DNA stain DAPI (C, F). Among the interphase cells in both cultures, all nuclei are labeled with antibody to topoisomerase IIß. A subset of the interphase cells (open arrows) does not show labeling for topoisomerase II{alpha}. Cells in M phase (closed arrows) show labeling of mitotic chromosomes with antibodies to topoisomerase II{alpha} and topoisomerase IIß. Bar, 5 µm.

 
To examine the distributions of topoisomerase II isoforms in more detail, Ptk1 cells derived from rat kangaroo kidney were labeled. These cells contain relatively few, large chromosomes and remain more flattened during passage through mitosis than most other cell types. Because our mouse monoclonal antibody to topoisomerase II{alpha} recognizes only protein from primate cells and because the polyclonal antibodies to topoisomerase II{alpha} and ß were produced in the same species, rabbit, parallel cultures were separately labeled to track the two isoforms. Cells were routinely colabeled with human scleroderma autoimmune sera to identify centromeres. The presence of doubled centromere spots in interphase cells was a marker to allow unambiguous identification of cells in late G2 phase. Cells in various stages of mitosis were identified by the morphology and arrangement of their chromosomes.

As was the case with HeLa cells and keratinocytes, the nuclei of interphase Ptk1 cells labeled with anti-topoisomerase II{alpha} showed the expected gradation of fluorescent labeling ranging from undetectable to intense. Invariably, cells in late G2 phase were strongly labeled (Fig. 4)Citation . Some cells with single centromere spots were also labeled, likely representing cells in S phase or early G2. All of the cells that lacked detectable anti-topoisomerase II labeling contained centromeres present as single unpaired spots, indicating that likely these cells were in early interphase, possibly G1. In prophase cells, individual chromosome arms could be distinguished by anti-topoisomerase II{alpha} labeling, and as reported in some previous studies (8 , 10) , the protein was concentrated at the centromeres (arrows in Fig. 4Citation ). This labeling pattern was most pronounced at prometaphase after nuclear envelope breakdown. The chromosome arms and centromeres remained labeled at metaphase. However, during anaphase, the enhanced concentration of label at the centromeres diminished, and the topoisomerase II{alpha} distribution along the chromosomes became more uniform.



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  		Fig. 4. Triple labeling of Ptk1 cells with autoimmune antibody to detect centromeres (A, D, G, J, M, and P), with antibody to topoisomerase II{alpha} (B, E, H, K, N, and Q) and with the DNA stain DAPI (C, F, I, L, O, and R). Many interphase cells exhibit low or undetectable expression of topoisomerase II{alpha} (A–C). Cells in late G2 phase as evidenced by double centromere spots invariably show strong expression of topoisomerase II{alpha} (D–F). Prophase cells showed diffuse labeling of the chromosome arms with anti-topoisomerase II{alpha} and a concentration at the centromeres (arrows; G–I). During prometaphase, topoisomerase II{alpha} was concentrated at the centromeres (arrows) and along the central axis of the chromosome arms (arrowhead; J–L). Metaphase cells showed strong labeling of centromeres (arrows) and chromosome arms (M–O). At late anaphase, the topoisomerase II{alpha} was no longer highly concentrated at centromeres but was present on chromosome arms (P–R). Bar, 5 µm.

 
A similar analysis of Ptk1 cells was carried out with the anti-topoisomerase IIß antibody (Fig. 5)Citation . Labeling was evident in the nuclei of all interphase cells. All interphase cells were labeled whether they contained with single or doubled centromere spots. In prophase cells, topoisomerase IIß appeared inhomogeneously distributed within the nucleus and appeared to be preferentially associated with the condensing chromatin. After nuclear envelope breakdown at prometaphase, most anti-topoisomerase IIß label was associated with the chromosome arms, although some was detectable more diffusely in the cytoplasm. In metaphase, the label continued to be associated with the chromosome arms. Topoisomerase IIß did not concentrate at centromeres as strongly as did topoisomerase II{alpha}. However, enhanced labeling of centromeres with anti-topoisomerase IIß antibody was sometimes evident on chromosomes of late prometaphase and metaphase cells (arrow in Fig. 5Citation ). The protein remained bound to chromosome arms in anaphase and was concentrated in the reforming nuclei at telophase.



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  		Fig. 5. Triple labeling of Ptk1 cells with human autoimmune serum to identify centromeres (A, D, G, J, M, P, and S) with anti-topoisomerase IIß antibody (B, E, H, K, N, Q, and T) and with DAPI for DNA (C, F, I, L, O, R, and U). Topoisomerase IIß was expressed in all interphase cells, whether they had single (A–C) or doubled (D–F) centromere spots. Prophase cells showed inhomogeneous nuclear labeling with anti-topoisomerase IIß with concentration near sites of chromosome condensation (G–I). By prometaphase, topoisomerase IIß was predominately detected closely associated with the mitotic chromosomes (J–L). At metaphase, topoisomerase IIß was found on chromosomes with only a slight tendency to concentrate at centromeres (arrows; M–O). At anaphase, topoisomerase IIß was distributed along chromosome arms (P–R). In telophase cells, topoisomerase IIß remained associated with chromosomes during chromosome decondensation (S–U). Bar, 5 µm.

 
To evaluate the distributions of the two isoforms in cell lines that remain relatively flat in mitosis but allow simultaneous visualization of topoisomerase II{alpha} and ß, we examined the distribution of the two isoforms in mitotic cells of that Bsc-1 line, derived from African green monkey kidney, and in a derivative of the porcine line LLC-Pk, developed in our laboratory that stably expresses green fluorescent protein-topoisomerase II{alpha} (38) . In both studies, we saw codistribution of topoisomerase II{alpha} and ß in association with chromosome arms in mitotic cells (Figs. 6Citation and 7Citation ).



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  		Fig. 6. Triple labeling of mitotic Bsc-1 cells with antibody to topoisomerase II{alpha} (A, D), with antibody to topoisomerase IIß (B, E), and with the DNA stain DAPI (C, F). Cells exhibit labeling of mitotic chromosome arms with antibodies to both topoisomerase II{alpha} and topoisomerase IIß. Bar, 5 µm.

 


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  		Fig. 7. Labeling of mitotic LLC-Pk cells stably expressing green fluorescent protein-topoisomerase II{alpha} (A, D) with antibody to topoisomerase IIß (B, E) and with the DNA stain DAPI (C, F). The prophase cell (A–C) was fixed in room temperature methanol. The anaphase cell (D–F) was lysed with detergent and fixed in formaldehyde. Before immunolabeling, both cells were treated with DNase to partially digest the DNA and better expose topoisomerase IIß antigen.

 
Association of Topoisomerase IIß with Mitotic Chromosomes.
Certain previous reports have indicated that although topoisomerase II{alpha} remains associated with chromatin during mitosis, topoisomerase IIß does not (24 , 30 , 31 , 39) . We have re-examined the question of the association of topoisomerase IIß with mitotic chromosomes and have drawn different conclusions. Our evidence, based on cell fractionation and immunolabeling, suggests that topoisomerase IIß is a component of the mitotic chromosomes. Some time ago, we found that topoisomerase IIß was present in isolated mitotic chromosomes prepared from mouse P388D1 lymphoid cells (33) . Subsequent to this work, many published studies from other laboratories, based primarily on immunolabeling, have suggested that topoisomerase IIß diffuses from the chromatin at M phase. Because the topoisomerase IIß-specific antibody used in our own initial study recognized only denatured antigen, it was not adaptable for immunolocalization. We prepared a new antibody, useful for both Western blotting and immunolabeling. With this antibody we determined that topoisomerase IIß was abundantly represented in cell fractions enriched in mitotic chromosomes. The antibody also labeled chromosomes in mitotic cells fixed to optimally preserve native protein localization. The discrepancies between our immunolabeling data and those previously reported cannot be entirely reconciled. We suggest that the previous failures to detect topoisomerase IIß in chromosomes by immunolocalization may have been caused by problems in antigen accessibility. Inaccessibility may have been caused by the physiological compaction of chromatin during mitotic chromosome condensation or by fixation and labeling protocols, resulting in protein relocation and/or antigen masking. In one study, Meyer et al. (24) , based on immunofluorescent labeling of whole cells and isolated chromosomes from human A431 cells, concluded that topoisomerase IIß diffused away from the chromosomes at mitosis. However, in Western blots shown in this same report, approximately equal levels of topoisomerase IIß were detected in the nuclear fractions (presumably low-speed centrifugation pellets), prepared from cycling cells, and cells were blocked in M phase with Colcemid. A subsequent study from the same group examined the distribution of topoisomerase II isoforms in fractions prepared from human HL60 cells (39) . In this case, although some topoisomerase IIß sedimented with the mitotic chromosomes, most was found to be in the cytoplasmic fraction. In contrast, using HeLa cells, Kimura et al. (40) reported that most topoisomerase IIß remained in the insoluble fraction when cells were disrupted in buffer containing 1% Triton X-100 detergent. Reported differences in the solubility of topoisomerase IIß in mitotic cell extracts may reflect properties of the different cell lines used for fractionation or perhaps differences in cell lysis and extraction procedures. However, clearly, under reasonably mild extraction conditions, considerable amounts of topoisomerase IIß are found associated with mitotic chromosomes.

Topoisomerase IIß Mitotic Chromosomes, Mechanisms of Association and Possible Roles.
It is possible that topoisomerase IIß overlaps in function in M phase with that of topoisomerase II{alpha}. Both {alpha} and ß isoforms of topoisomerase II undergo mitosis-specific phosphorylation (33 , 40, 41, 42, 43, 44, 45) . The in vivo role of mitotic phosphorylation of either isoform of topoisomerase II on the localization or on enzyme activity remains unresolved (reviewed in Refs. 1 , 22 ). Topoisomerase II is essential in M phase for resolving DNA catenations that would inhibit chromosome condensation in prophase and for facilitating chromatid separation at anaphase. Topoisomerase II may also contribute to the structure of an axial mitotic chromosome scaffold composed of nonhistone protein (15 , 16) . Functional redundancy of topoisomerase II{alpha} and ß is suggested because human topoisomerase IIß rescues a temperature-sensitive yeast topoisomerase II mutant strain at the restrictive temperature (46) . However, some findings suggest that topoisomerase IIß may not fully overlap in mitotic function with topoisomerase II{alpha}. For example, the distribution of the two isoforms is not entirely overlapping. The ß isoform is more easily extracted from mitotic chromosomes at lower salt concentration (39 , 40) . In this study, we show that, in certain cell lines, topoisomerase II{alpha} is highly concentrated at mitotic centromeres through early anaphase. In those same cells, topoisomerase IIß exhibits, at most, a very weak concentration at centromeres.

Why do mammalian cells contain two genes for topoisomerase II? Does topoisomerase IIß have important roles in cell cycle progression, particularly at M phase? Most previous studies suggest that topoisomerase II{alpha} plays a dominant if not exclusive role in mitosis. In cell lines that express low levels of topoisomerase IIß, or in mice in which the gene has been ablated, cell division does take place (26, 27, 28) . The mouse embryos lacking topoisomerase IIß die shortly after birth because of impaired breathing. Although this evidence suggests that topoisomerase IIß is not absolutely required for cell division, it does not preclude a mitotic role for the enzyme where it is expressed, nor does it eliminate the possibility that topoisomerase IIß may play a specific accessory role, preventing chromatin damage and facilitating accurate chromosome segregation in mitosis. Recent evidence has been presented suggesting that certain forms of lissencephaly may result from defects in embryonic mitoses (47) . It is possible that the cell divisions of neuronal precursor cells may have more stringent requirements during development than those of other cell types.

Definitive demonstration of possible differential roles of topoisomerase II{alpha} and ß in mitosis and other stages of the cell cycle are not yet available. The potential that differentially spliced variants of each isoform also exist adds additional complexity (29) . In this study, we show, in contrast to widespread opinion, that both topoisomerase IIß and topoisomerase II{alpha} are associated with mitotic chromosomes. The prominent role played by topoisomerase II as a target for cancer therapy emphasizes the importance of further study in understanding the regulation and function of both isoforms in normal and neoplastic cell division.


   Materials and Methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
References
 
Cell Culture.
HeLa S3 suspension cells were cultured in 1-liter spinner flasks in HEPES-buffered Dulbecco’s modified minimal essential medium (Life Technologies, Inc., Grand Island, NY) supplemented with 5% bovine calf serum (HyClone Laboratories, Logan, UT), 0.1 mM nonessential amino acids (Life Technologies, Inc.), 1 mM sodium pyruvate, 0.05% pluronic F68, and either 20 g/ml gentamicin sulfate or 100 g/ml streptomycin plus 60 g/ml penicillin. Adherent HeLa cells were grown in the same medium lacking pluronic F68. The Ptk1 (rat kangaroo kidney) and Bsc-1 (African green monkey kidney) cell lines were grown in monolayer on tissue culture dishes in MEM (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 20 mM HEPES buffer, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate (Sigma, St. Louis, MO), and 100 µg/ml streptomycin plus 60 µg/ml penicillin. Normal human epidermal keratinocytes were obtained from Clonetics Corp. (San Diego, CA) as frozen primary cultures from foreskin. Keratinocytes were grown in defined keratinocyte-SFM medium (Life Technologies, Inc.) with 100 µg/ml streptomycin and 60 µg/ml penicillin.

Antibodies.
Polyclonal anti-topoisomerase II{alpha} antibody was purchased from Topogen, Inc. (Columbus, OH). Monoclonal anti-topoisomerase II{alpha} antibody was a generous gift from R. G. Robinson and S. A. Coughlin of Sterling-Winthrop Pharmaceuticals (Collegeville, PA). Antibody to topoisomerase IIß was prepared under contract with Quality Controlled Biochemicals, Inc., (Hopkinton, MA) by injecting rabbits with a peptide corresponding to amino acids 1488–1504 (VEAVNSDSDSEFGIPK) of topoisomerase IIß that was synthesized with an additional COOH-terminal cysteine and coupled via the sulfhydryl to keyhole limpet hemocyanin. Specific antibody was affinity purified from immune serum on peptide-linked matrix (Sulfolink gel; Pierce, Rockford, IL). The peptide matrix was rinsed with phosphate buffer [50 mM NaH2PO4 (pH 6.5)], and 5 ml of immune serum was incubated with the gel overnight at 4°C with agitation. The gel and serum were poured into a column, washed with high salt phosphate buffer [50 mM NaH2PO4, 0.5 M NaCl (pH 6.5)], and eluted with glycine buffer [100 mM Glycine-HCl (pH 2.5)]. Fractions were neutralized with 1 M Tris-HCl (pH 9.5). Pooled antibody was dialyzed against phosphate buffer [10 mM NaH2PO, 20 mM NaCl (pH 7.0)]. The Sulfolink peptide matrix was stored in high salt phosphate buffer containing 0.05% sodium azide.

Immunoprecipitation.
Adherent HeLa cells were blocked overnight in 0.15 µg/ml Colcemid. Rounded mitotic cells were shaken off the dishes and collected by centrifugation at 250 x g for 5 min. They were resuspended in 7 mM Na2HPO4, 1.5 mM KH2PO4 (pH 7.2), 137 mM NaCl, and 2.7 mM KCl at 37°C, and washed twice by centrifugation and resuspension. The cells were counted, aliquoted to microfuge tubes, and briefly centrifuged. Excess buffer was removed, and the cell pellets were snap frozen in liquid nitrogen and stored at -80°C. The mitotic index was generally 90–95%. To prepare extracts for immunoprecipitation, cell pellets were thawed and lysed in a buffer containing 100 mM Tris (pH 8.0), 0.75 M NaCl, 2 mM MgCl2, 0.75% NP40, and 5 µg/ml of each of the protease inhibitors Pefabloc SC, leupeptin, and pepstatin A. The extracts were immunoprecipitated for 2 h at room temperature with protein A beads that had been previously conjugated with anti-topoisomerase IIß immune serum or with affinity purified anti-topoisomerase IIß antibody. The protein A beads were washed extensively with buffer and then treated with SDS-gel electrophoresis sample buffer. Mock immunoprecipitations were carried out simultaneously with antibody-conjugated beads that were not exposed to cell extract.

Cell Fractionation and Preparation of Mitotic Chromosomes.
HeLa S3 cells in suspension at 5 x 105 cells/ml were blocked with 0.15 µg/ml Colcemid for 16–18 h. The mitotic index was routinely 70–95%. Cells were collected by centrifugation at 250 x g for 5 min, resuspended in 7 mM Na2HPO4, 1.5 mM KH2PO4 (pH 7.2), 137 mM NaCl, and 2.7 mM KCl at 37°C, and centrifuged once more. Cells were then resuspended in swelling buffer [10 mM HEPES, 40 mM KCl, 5 mM EGTA, 4 mM MgSO4 (pH 7.4)] and collected by centrifugation at 200 x g twice. Swollen cells were incubated on ice for 5 min and then lysed in extraction buffer [1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid in 60 mM Pipes, 25 mM HEPES (pH 6.9), 10 mM EGTA, and 4 mM MgSO4] with 1 mM DTT, 200 nM microcystin, 5 µg/ml of each of the protease inhibitors Pefabloc SC, leupeptin, and pepstatin A. The extract was centrifuged at 200 x g at 4°C for 5 min, and the pellet, consisting of large pieces of cellular debris and clumps of interphase nuclei, was collected. The supernatant was then centrifuged at 400 x g for 5 min. The pellet from this centrifugation contained dispersed nuclei from contaminating interphase cells and some clumps of mitotic chromosomes. The supernatant was then centrifuged at 1600 x g for 10 min, and the pellet, containing the isolated chromosomes, was collected. The supernatant from this centrifugation contained cytoplasm consisting of organelles other than nuclei and chromosomes as well as soluble components.

Western Blotting.
Whole cell extracts were prepared by first rinsing monolayer cells with 7 mM Na2HPO4, 1.5 mM KH2PO4 (pH 7.2), 137 mM NaCl, and 2.7 mM KCl at room temperature, lysing in 2x Laemmli SDS sample buffer with 10% ß-mercaptoethanol, and boiling for 5 min. Electrophoresis was carried out under denaturing conditions with 6% polyacrylamide gels or with 5–20% polyacrylamide gradient gels. Proteins were transferred from the gels to PVDF4 paper (Millipore, Bedford, MA) in a Genie electroblotting apparatus (Idea Scientific, Minneapolis, MN) by blotting at 12 V for 2 h in ice-cold, modified Towbin transfer buffer [25 mM Tris (Sigma), 192 mM Glycine (Sigma), and 0.005% SDS (Amresco, Solon, OH)]. Nonspecific binding was blocked by incubation with 5% BSA (Sigma) in blotting buffer [10 mM Tris (pH 8.0), 150 mM NaCl, and 0.5% Tween-20], either overnight at 4°C or 1 h at room temperature. The blot was washed once with blotting buffer and then incubated with rabbit polyclonal anti-topoisomerase II{alpha} at 1:1000 dilution, mouse monoclonal anti-topoisomerase II{alpha} at 1:1000 dilution, or with the anti-topoisomerase IIß affinity-purified rabbit antipeptide antibody at 1.5 µg/ml for 1.5 h. Blots were washed three times for 5 min with blotting buffer and then incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) at 1:20,000 for 1 h. Blots were washed three times in blotting buffer and visualized with a chemiluminescence kit (Pierce) according to instructions from the manufacturer.

Immunofluorescence.
Ptk1 cells were cultured on 18- or 22-mm square coverslips. When cells were ~75% confluent, the coverslips were rinsed in 100 mM MOPS (pH 7.2) with 4 mM MgSO4 and 10 mM EGTA, and cells were prepared by fixation in methanol at -20°C for 15 min. Coverslips were washed once in MBST, and nonspecific binding was blocked with a 30-min preincubation in 20% boiled, normal goat serum (Life Technologies, Inc.) in MBS. After a brief MBST rinse, coverslips were incubated for 45 min in 5% boiled, normal goat serum in MBS with rabbit polyclonal antibody to topoisomerase II{alpha} at a dilution of 1:100, mouse monoclonal anti-topoisomerase II{alpha} at a dilution of 1:50, or affinity-purified anti-topoisomerase II{alpha} at 1.5 µg/ml. In some cases, coverslips were colabeled with human autoimmune scleroderma serum containing antibodies to mammalian centromeres. This serum was generously provided by Dr. J. B. Rattner (University of Calgary, Calgary, Alberta, Canada) or purchased from Cortex Biochem (San Leandro, CA) and used at a 1:500 dilution. After labeling with primary antibody, the coverslips were washed three times for 5 min with MBST. They were then incubated for 45 min with appropriate Cy3-conjugated and fluorescein-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, Inc.) at 1:400 dilutions in MBS containing 5% boiled normal goat serum. Cells were washed three more times in MBST and treated with the DNA dye DAPI in MBST at 0.1 g/ml. Coverslips were rinsed with H2O and mounted on glass slides with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) that had been supplemented to 10 mM MgCl2. The edges of the coverslips were sealed with nail polish, allowed to dry at room temperature, and stored at -20°C. Images were obtained with a Nikon Diaphot microscope equipped with an intensified charge-coupled device camera (Dage-MTI, Michigan City, IN) and captured using Image 1 software (Universal Imaging, Media, PA).


   Acknowledgments
 
We thank Dr. Ronald Robinson and Dr. Susan A. Coughlin for generously providing monoclonal antibody to topoisomerase II{alpha}.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by grants from the American Cancer Society. Back

2 Present address: Virginia Commonwealth University, Department of Chemistry, P. O. Box 842006, Richmond, VA 23284. Back

3 To whom requests for reprints should be addressed, at BRC 266, 975 N. E. 10th Street, University of Oklahoma Health Science Center, Oklahoma City, OK 73104. Phone: (405) 271-3486; Fax: (405) 271-7158; E-mail: gary-gorbsky{at}ouhsc.edu Back

4 The abbreviations used are: PVDF, polyvinylidene difluoride; MOPS, 4-morpholinepropanesulfonic acid; MBST, 10 mM MOPS (pH 7.4), 150 mM NaCl, 0.5% Tween-20; MBS, 10 mM MOPS (pH 7.4) and 150 mM NaCl; DAPI, 4',6-diamidino-2-phenylindole. Back

Received for publication 4/12/02. Revision received 1/ 2/02. Accepted for publication 6/ 3/02.


   References
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 

  1. Larsen A. K., Skladanowski A., Bojanowski K. The roles of DNA topoisomerase II during the cell cycle. Prog. Cell Cycle Res., 2: 229-239, 1996.[Medline]
  2. Burden D. A., Osheroff N. Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme. Biochim. Biophys. Acta, 1400: 139-154, 1998.[Medline]
  3. Bakshi R. P., Galande S., Muniyappa K. Functional and regulatory characteristics of eukaryotic type II DNA topoisomerase. Crit. Rev. Biochem. Mol. Biol., 36: 1-37, 2001.[Medline]
  4. Holm C., Stearns T., Botstein D. DNA topoisomerase II must act at mitosis to prevent nondisjunction and chromosome breakage. Mol. Cell. Biol., 9: 159-168, 1989.[Abstract/Free Full Text]
  5. Uemura T., Ohkura H., Adachi Y., Morino K., Shiozaki K., Yanagida M. DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe. Cell, 50: 917-925, 1987.[Medline]
  6. Adachi Y., Luke M., Laemmli U. K. Chromosome assembly in vitro: topoisomerase II is required for condensation. Cell, 64: 137-148, 1991.[Medline]
  7. Andoh T., Sato M., Narita T., Ishida R. Role of DNA topoisomerase II in chromosome dynamics in mammalian cells. Biotechnol. Appl. Biochem., 18: 165-174, 1994.
  8. Gorbsky G. J. Cell cycle progression and chromosome segregation in mammalian cells cultured in the presence of the topoisomerase II inhibitors ICRF-187 ((+)-1, 2-bis(3, 5-dioxopiperazinyl-1-yl)propane; ADR-529) and ICRF-159 (razoxane). Cancer Res., 54: 1042-1048, 1994.[Abstract/Free Full Text]
  9. Iwai M., Hara A., Andoh T., Ishida R. ICRF-193, a catalytic inhibitor of DNA topoisomerase II, delays the cell cycle progression from metaphase, but not from anaphase to the G1 phase in mammalian cells. FEBS Lett., 406: 267-270, 1997.[Medline]
  10. Rattner J. B., Hendzel M. J., Furbee C. S., Muller M. T., Bazett-Jones D. P. Topoisomerase II{alpha} is associated with the mammalian centromere in a cell cycle- and species-specific manner and is required for proper centromere/kinetochore structure. J. Cell Biol., 134: 1097-1107, 1996.[Abstract/Free Full Text]
  11. Roberge M., Anderson H. Topoisomerase II inhibitors affect entry into mitosis and chromosome condensation in BHK cells. Cell Growth Differ., 7: 83-90, 1996.[Abstract]
  12. Berrios M., Osheroff N., Fisher P. A. In situ localization of DNA topoisomerase II, a major polypeptide component of the Drosophila nuclear matrix fraction. Proc. Natl. Acad. Sci. USA, 82: 4142-4146, 1985.[Abstract/Free Full Text]
  13. Fernandes D. J., Catapano C. V. Nuclear matrix targets for anticancer agents. Cancer Cells, 3: 134-140, 1991.[Medline]
  14. Zini N., Santi S., Ognibene A., Bavelloni A., Neri L. M., Valmori A., Mariani E., Negri C., Astaldi-Ricotti G. C., Maraldi N. M. Discrete localization of different DNA topoisomerases in HeLa and K562 cell nuclei and subnuclear fractions. Exp. Cell Res., 210: 336-348, 1994.[Medline]
  15. Earnshaw W. C., Halligan B., Cooke C. A., Heck M. M., Liu L. F. Topoisomerase II is a structural component of mitotic chromosome scaffolds. J. Cell Biol., 100: 1706-1715, 1985.[Abstract/Free Full Text]
  16. Gasser S. M., Laroche T., Falquet J., Boy de la Tour E., Laemmli U. K. Metaphase chromosome structure involvement of topoisomerase II. J. Mol. Biol., 188: 613-629, 1986.[Medline]
  17. Andoh T., Ishida R. Catalytic inhibitors of DNA topoisomerase II. Biochim. Biophys. Acta, 1400: 155-171, 1998.[Medline]
  18. Fortune J. M., Osheroff N. Topoisomerase II as a target for anticancer drugs: when enzymes stop being nice. Prog. Nucleic Acid Res. Mol. Biol., 64: 221-253, 2000.[Medline]
  19. Wang H., Mao Y., Zhou N., Hu T., Hsieh T. S., Liu L. F. ATP-bound topoisomerase II as a target for antitumor drugs. J. Biol. Chem., 276: 15990-15995, 2001.[Abstract/Free Full Text]
  20. Chung T. D., Drake F. H., Tan K. B., Per S. R., Crooke S. T., Mirabelli C. K. Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes. Proc. Natl. Acad. Sci. USA, 86: 9431-9435, 1989.[Abstract/Free Full Text]
  21. Bauman M. E., Holden J. A., Brown K. A., Harker W. G., Perkins S. L. Differential immunohistochemical staining for DNA topoisomerase II {alpha} and ß in human tissues and for DNA topoisomerase II ß in non-Hodgkin’s lymphomas. Mod. Pathol., 10: 168-175, 1997.[Medline]
  22. Isaacs R. J., Davies S. L., Sandri M. I., Redwood C., Wells N. J., Hickson I. D. Physiological regulation of eukaryotic topoisomerase II. Biochim. Biophys. Acta, 1400: 121-137, 1998.[Medline]
  23. Kimura K., Saijo M., Ui M., Enomoto T. Growth state- and cell cycle-dependent fluctuation in the expression of two forms of DNA topoisomerase II and possible specific modification of the higher molecular weight form in the M phase. J. Biol. Chem., 269: 1173-1176, 1994.[Abstract/Free Full Text]
  24. Meyer K. N., Kjeldsen E., Straub T., Knudsen B. R., Hickson I. D., Kikuchi A., Kreipe H., Boege F. Cell cycle-coupled relocation of types I and II topoisomerases and modulation of catalytic enzyme activities. J. Cell Biol., 136: 775-788, 1997.[Abstract/Free Full Text]
  25. Woesssner R. D., Mattern M. R., Mirabelli C. K., Johnson R. K., Drake F. H. Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells. Cell Growth Differ., 2: 209-214, 1991.[Abstract]
  26. Dereuddre S., Frey S., Delaporte C., Jacquemin-Sablon A. Cloning and characterization of full-length cDNAs coding for the DNA topoisomerase II ß from Chinese hamster lung cells sensitive and resistant 9-OH-ellipticine. Biochim. Biophys. Acta, 1264: 178-182, 1995.[Medline]
  27. Harker W. G., Slade D. L., Parr R. L., Feldhoff P. W., Sullivan D. M., Holguin M. H. Alterations in the topoisomerase II {alpha} gene, messenger RNA, and subcellular protein distribution as well as reduced expression of the DNA topoisomerase II ß enzyme in a mitoxantrone-resistant HL-60 human leukemia cell line. Cancer Res., 55: 1707-1716, 1995.[Abstract/Free Full Text]
  28. Yang X., Li W., Prescott E. D., Burden S. J., Wang J. C. DNA topoisomerase IIß and neural development. Science (Wash. DC), 287: 131-134, 2000.[Abstract/Free Full Text]
  29. Petruti-Mot A. S., Earnshaw W. C. Two differentially spliced forms of topoisomerase II{alpha} and ß mRNAs are conserved between birds and humans. Gene (Amst.), 258: 183-192, 2000.[Medline]
  30. Petrov P., Drake F. H., Loranger A., Huang W., Hancock R. Localization of DNA topoisomerase II in Chinese hamster fibroblasts by confocal and electron microscopy. Exp. Cell Res., 204: 73-81, 1993.[Medline]
  31. Chaly N., Chen X., Dentry J., Brown D. L. Organization of DNA topoisomerase II isotypes during the cell cycle of human lymphocytes and HeLa cells. Chromosome Res., 4: 457-466, 1996.[Medline]
  32. Earnshaw W. C., Heck M. M. Localization of topoisomerase II in mitotic chromosomes. J. Cell Biol., 100: 1716-1725, 1985.[Abstract/Free Full Text]
  33. Taagepera S., Rao P. N., Drake F. H., Gorbsky G. J. DNA topoisomerase II{alpha} is the major chromosome protein recognized by the mitotic phosphoprotein antibody MPM-2. Proc. Natl. Acad. Sci. USA, 90: 8407-8411, 1993.[Abstract/Free Full Text]
  34. Sumner A. T. The distribution of topoisomerase II on mammalian chromosomes. Chromosome Res., 4: 5-14, 1996.[Medline]
  35. Zini N., Martelli A. M., Sabatelli P., Santi S., Negri C., Ricotti G. C. B. A., Maraldi N. M. The 180-kDa isoform of topoisomerase II is localized in the nucleolus and belongs to the structural elements of the nucleolar remnant. Exp. Cell Res., 200: 460-466, 1992.[Medline]
  36. Negri C., Chiesa R., Cerino A., Bestagno M., Sala C., Zini N., Maraldi N. M., Ricotti G. C. B. A. Monoclonal antibodies to human DNA topoisomerase I and the two isoforms of DNA topoisomerase II: 170- and 180-kDa isozymes. Exp. Cell Res., 200: 452-459, 1992.[Medline]
  37. Melan M. A., Sluder G. Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. J. Cell Sci., 101: 731-743, 1992.[Abstract/Free Full Text]
  38. Tavormina P. A., Côme M-G., Hudson J. R., Mo Y-Y., Beck W. T., Gorbsky G. J. Rapid exchange of mammalian topoisomerase II{alpha} at kinetochores and chromosome arms in mitosis. J. Cell Biol., : in press 2002.
  39. Grue P., Grasser A., Sehested M., Jensen P. B., Uhse A., Straub T., Ness W., Boege F. Essential mitotic functions of DNA topoisomerase II{alpha} are not adopted by topoisomerase IIß in human H69 cells. J. Biol. Chem., 273: 33660-33666, 1998.[Abstract/Free Full Text]
  40. Kimura K., Nozaki N., Enomoto T., Tanaka M., Kikuchi A. Analysis of M phase-specific phosphorylation of DNA topoisomerase II. J. Biol. Chem., 271: 21439-21445, 1996.[Abstract/Free Full Text]
  41. Burden D. A., Goldsmith L. J., Sullivan D. M. Cell-cycle-dependent phosphorylation and activity of Chinese-hamster ovary topoisomerase II. Biochem. J., 293: 297-304, 1993.
  42. Daum J. R., Gorbsky G. J. Casein kinase II catalyzes a mitotic phosphorylation on threonine 1342 of human topoisomerase II{alpha}, which is recognized by the 3F3/2 phosphoepitope antibody. J. Biol. Chem., 273: 30622-30629, 1998.[Abstract/Free Full Text]
  43. Heck M. M., Hittelman W. N., Earnshaw W. C. In vivo phosphorylation of the 170-kDa form of eukaryotic DNA topoisomerase II. Cell cycle analysis. J. Biol. Chem., 264: 15161-15164, 1989.[Abstract/Free Full Text]
  44. Saijo M., Ui M., Enomoto T. Growth state and cell cycle dependent phosphorylation of DNA topoisomerase II in Swiss 3T3 cells. Biochemistry, 31: 359-363, 1992.[Medline]
  45. Wells N. J., Hickson I. D. Human topoisomerase II {alpha} is phosphorylated in a cell-cycle phase-dependent manner by a proline-directed kinase. Eur. J. Biochem., 231: 491-497, 1995.[Medline]
  46. Meczes E. L., Marsh K. L., Fisher L. M., Rogers M. P., Austin C. A. Complementation of temperature-sensitive topoisomerase II mutations in Saccharomyces cerevisiae by a human TOP2 ß construct allows the study of topoisomerase II ß inhibitors in yeast. Cancer Chemother. Pharmacol., 39: 367-375, 1997.[Medline]
  47. Vallee R. B., Tai C., Faulkner N. E. LIS1: cellular function of a disease-causing gene. Trends Cell Biol., 11: 155-160, 2001.[Medline]



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