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Vol. 19, Issue 10, 4076-4085, October 2008
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: Implication for the G2/M Transition
*Department of Molecular Biosciences and KU Microscopy and Analytical Imaging Laboratory, University of Kansas, Lawrence, KS 66045; and Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232
Submitted January 2, 2008;
Revised June 3, 2008;
Accepted July 8, 2008
Monitoring Editor: Yixian Zheng
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(topo II) was identified as a potential binding partner of APC. Topo II is a critical regulator of G2/M transition. Evidence supporting an interaction between endogenous APC and topo II was obtained by coimmunoprecipitation, colocalization, and Förster resonance energy transfer (FRET). The 15-amino acid repeat region of APC (M2-APC) interacted with topo II when expressed as a green fluorescent protein (GFP)-fusion protein in vivo. Although lacking defined nuclear localization signals (NLS) M2-APC predominantly localized to the nucleus. Furthermore, cells expressing M2-APC displayed condensed or fragmented nuclei, and they were arrested in the G2 phase of the cell cycle. Although M2-APC contains a β-catenin binding domain, biochemical studies failed to implicate β-catenin in the observed phenotype. Finally, purified recombinant M2-APC enhanced topo II activity in vitro. Together, these data support a novel role for APC in the G2/M transition, potentially through association with topo II. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The detection of mutant APC in the earliest stages of polyp development supports the idea that mutation of APC is an initiating event in colon carcinogenesis. The most common form of APC mutation results in elimination of the carboxy-terminal half of the APC protein. Because APC is a large, multidomain protein, APC truncation is predicted to impact several cellular mechanisms, the extent of which we are only beginning to understand.
There is accumulating evidence supporting a role for APC in the regulation of cell cycle. Overexpression of APC in NIH3T3 fibroblasts and colon cancer cell lines leads to G1 cell cycle arrest (Ishidate et al., 2000; Heinen et al., 2002), presumably by repressing transcription of Wnt targets such as cyclin D1. APC may also participate directly in mitosis because it is transiently hyperphosphorylated in the M phase of the cell cycle (Bhattacharjee et al., 1996), accumulates at the microtubule-organizing center (Olmeda et al., 2003), and associates with the kinetochore in dividing cells (Fodde et al., 2001; Kaplan et al., 2001). A role for APC in mitosis might be critical for regulation of genomic stability and proper chromosome segregation. APC stabilized by zinc treatment induces G2/M cell cycle arrest in colon cancer cells (Jaiswal and Narayan, 2004). However, to date, little is known about the underlying mechanism by which APC participates in the G2/M cell cycle transition.
Here, we report identification of topoisomerase II (topo II) as a potential APC-binding protein. Topo II enzyme catalyzes DNA topology changes by introducing double-strand DNA breaks that facilitate DNA strand passage and subsequent DNA religation (Wang, 1996; Champoux, 2001; Fortune and Osheroff, 2001; McClendon and Osheroff, 2007). Topo II has been implicated in several cellular functions such as DNA replication and chromosome condensation (Wang, 1996; Nitiss, 1998; Fortune and Osheroff, 2000; Champoux, 2001; Wang, 2002; McClendon and Osheroff, 2007), and it seems to be essential in the control of the G2/M decatenation checkpoint during cell division (Downes et al., 1994). Topo II was also found to be deregulated in colon cancers, with its expression limited to the proliferative zone in the normal colon, but up-regulated and widespread in colon cancer tissue (Murphy et al., 2001). At primary locations of recurrent malignant colon tumors after chemotherapy, the number of topo II-positive cells is greatly increased compared with primary sites with no recurrence (Lazaris et al., 2002), suggesting that changes in topo II expression occur subsequent to APC mutation. Together, these observations make topo II an attractive candidate for mediating the G2/M cell cycle transition.
Overexpression of an APC fragment that interacts with topo II in various colon cancer cells lines led to abnormal nuclear morphology and cell cycle inhibition in G2. Our data suggest a novel role for nuclear APC in the regulation of cell cycle progression, potentially through an interaction with topo II.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Antibodies and Immunofluorescence Microscopy
Immunostaining was performed as described previously (Neufeld and White, 1997) and the following antibodies were used: anti-APC (ab-7, 1:50; Calbiochem, San Diego, CA), anti-APC (ab-4, 1:2000; Calbiochem), anti-APC (ab-1, 1:100; Calbiochem), anti-topo II (1:100; Research Diagnostics, Flanders, NJ), anti-phospho-histone H3 (1:500; Millipore, Billerica, MA), anti-β-catenin (1:200; BD Biosciences Transduction Laboratories, Lexington, KY), and anti-proliferating cell nuclear antigen (PCNA, 1:1000; BD Biosciences Transduction Laboratories); goat anti-mouse immunoglobulin G (IgG) Alexa 488 (1:1000; Invitrogen, Carlsbad, CA), goat anti-rabbit IgG Alexa 568 (1:1000; Invitrogen), and goat anti-mouse IgG Alexa 610-R-phycoerythrin (1:500; Invitrogen). DNA was labeled with Topro-3 (1:500; Invitrogen), or 4,6-diamidino-2-phenylindole (DAPI) (1:5000; Invitrogen). Two-dimensional (2-D) and three-dimensional (3-D) distributions of immunofluorescent signals were examined using a Yokugawa-type spinning disk confocal microscope equipped with an Olympus 150x objective with a numerical aperture of 1.45 and 1KX1K EMCCD (Olympus and Intelligent Imaging Innovations, Denver, Co.). From >200 cells that were viewed, 50 cells were randomly chosen to be imaged. At least 24 image frames were collected at z-intervals of 100 nm for image sets where 3-D colocalization was examined. No deconvolution was performed. All raw confocal image series were analyzed by ImageJ program (National Institutes of Health, Bethesda, MD) and the JACoP plugin without further processing. Mitotic indices were determined by counting DAPI-stained mitotic cells in a field of 100 cells for each repetition.
Protein Colocalization Analysis
Colocalization coefficients were calculated using ImageJ and the JACoP plugin (Bolte and Cordelieres, 2006). Using Costes' method of automatic thresholding, a Pearson's coefficient was calculated for pixels within all of the calculated regions of interest in an image where Alexa 488 and Alexa 568 fluorescence were each detected at levels significantly above background. Mander's coefficients were also calculated to determine the degree of overlap between the corresponding regions of detected signals.
Förster Resonance Energy Transfer (FRET) Analysis
Evidence of FRET between secondary fluorophores was detected using a Zeiss 510 Meta spectral imaging upright laser scanning confocal microscope (Carl Zeiss, Thornwood, NY). Donor fluorescence, alternatively from Alexa 488 or Alexa 610-R-phycoerythrin was photobleached by passing the beam of the 543-nm laser (at 100% output) over defined regions of interest for 75 iterations or by using 200 passes of the 633-nm laser (also 100% output). Proximity of the two proteins was determined by calculating the mean FRET efficiency between donor and acceptor according to the method of Kenworthy and Edidin (1998), where E = 100(Alexa 488post – Alexa 488pre)/Alexa 488post or E = 100(Alexa 610-R-PEpost – Alexa 610-R-PEpre)/Alexa 610-R-PEpost. All pixels within the photobleached regions were used for these calculations. Error bars indicate the SEM. Three-dimensional reconstructions and projections were also performed with ImageJ.
Immunoprecipitation and Western Immunoblots
Immunoprecipitation (IP) and Western immunoblots (IB) were performed using modified standard protocols. Cells at 90% confluence were lysed in lysis buffer (50 mM Tris, pH 7.5, 0.1% NP-40, 100 mM NaCl, 1 mM MgCl2, 5 mM EDTA, protease inhibitor cocktail [Sigma-Aldrich, St. Louis, MO], and Halt phosphatase inhibitor cocktail [Pierce Chemical, Rockford, IL]) on ice for 30 min. Cell lysates were sonicated for 10 pulses, level 1 with 10% output, three times. Specific antibodies were preincubated with protein A Dynabeads (Invitrogen) for 2 h at room temperature. Dynabeads saturated with antibodies were added to 1 mg of soluble lysate and incubated overnight at 4°C. IP pellets were subject to two washes of 15 min by using lysis buffer and one wash using phosphate-buffered saline (PBS)/Tween 20 at 4°C. The following antibodies were used for IP: anti-topo II sera (a generous gift from Dr. Joe Holden), affinity-purified anti-APC-M2 rabbit polyclonal antibody made against amino acids 1000–1326, and polyclonal anti-GFP (Invitrogen). Immunoblots were probed with the following antibodies: anti-APC (ab-1, 1:100; Calbiochem), anti-APC-M2 polyclonal (1:4000), anti-β-catenin (1:2000; Sigma-Aldrich), anti-topo II (1:1000; Research Diagnostics), anti-topo IIβ (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), and anti--tubulin (1:2000; Calbiochem).
Transfection and Reporter Gene Assay
HCT116βw, SW480, and HCA7 cells were transfected using Lipofectamine 2000 reagent according to the manufacturer's protocol (Invitrogen). For luciferase assays, HCT116βw cells grown in 24-well plates were cotransfected with 2 µg of the GFP-M2-APC or GFP expression construct, 100 ng of the TCF-reporter construct SuperTOP-flash or FOPflash (Millipore), and 50 ng of the pRL-TK Renilla luciferase construct (Promega, Madison, WI) as a control to normalize the transfection efficiency. After 24 h, cells were harvested and luciferase activities were determined using Dual-Luciferase assay system (Promega) and a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). SuperTOP-flash and FOPflash luciferase activities were first normalized by pRL-TK Renilla luciferase, and then the normalized SuperTOP-flash luciferase activity was divided by normalized FOPflash luciferase activity to calculate relative β-catenin activity.
Fluorescence Activated Cell Sorting (FACS) Analysis
Propidium iodide staining of GFP-expressing cells in suspension was performed using a standard protocol as described previously (Lamm et al., 1997). HCT116βw cells at 35% confluence were transfected using Lipofectamine 2000 (Invitrogen). Thirty hours after transfection, cells were fixed with 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) on ice for 1 h, followed by overnight permeabilization by using 70% ethanol in PBS. Rehydrated cells were then stained with 40 µg/ml propidium iodide (Sigma-Aldrich) in PBS for 30 min at 37°C. FACS analysis was performed using an FACScan (BD Biosciences, San Jose, CA).
Topo II Assays and Recombinant Proteins
To generate recombinant S tag fused M2-APC, the corresponding cDNA for APC (amino acids 1000–1326) was subcloned into a pET-30a(+) vector. Both tags (S and His) were fused to the amino terminus of the protein. The expression and purification of S-M2-APC fusion protein was performed as described previously (Azuma et al., 2003). Recombinant human topo II was made as described previously (Worland and Wang, 1989; Kingma et al., 1997). In vitro topo II relaxation and decatenation assays were performed as described previously (Fortune and Osheroff, 2001).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(Neufeld and White, unpublished data).
We verified the APC–topo II interaction in both HCT116 cells and cells derived from this cell line. Although HCT116 cells were initially cultured from human colon cancer tissue, they express full-length APC and maintain a stable karyotype. The original HCT116 cell line possesses one wild-type and one mutant β-catenin allele. The mutant allele encodes a stabilized version of β-catenin, which is not down-regulated by APC. The HCT116βw line we use for most experiments was engineered to eliminate the mutant β-catenin allele; thus, it expresses only wild-type β-catenin protein (Chan et al., 2002). Full-length endogenous APC coimmunoprecipitated with topo II in HCT116 cell lysates (Figure 1A). No full-length APC was precipitated with control rabbit IgG. In reciprocal experiments, topo II coprecipitated with full-length APC by using an affinity-purified APC antibody, but not with preimmune sera (Figure 1B, left). We consistently detected nearly 2% of the total topo II coprecipitated with APC (Figure 1B, right). In contrast, although topoisomerase IIβ is 75% identical to topo II, topo IIβ did not coprecipitate with APC (Figure 1B, left). This apparent binding preference for topo II over topo IIβ increases the likelihood that the APC-topo II interaction is specific.
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further implicated topo II as a binding partner of APC. Endogenous APC was found to colocalize with topo II within single confocal slices taken through the nucleus where both occur as overlapping puncta (Figure 1Ca). When the entire cell thickness was visualized by a series of confocal images captured in the z-plane, areas of overlap were apparent throughout the nuclei (Figure 1Cb). To show the areas of overlap from an entire cell thickness as a single image, overlapping signals in the 3-D data sets were projected onto a 2-D surface and are displayed in yellow (Figure 1Cc). The degree of overlap between the corresponding regions was calculated for each 2-D confocal section and is displayed as the average for the entire imaged volume. On average, 1.9% of the topo II signal coincided with APC, and 10% of the APC signal coincided with topo II (Figure 1Cd).
To further examine the association between APC and topo II, we analyzed FRET between the two immunolabeled proteins in fixed, permeabilized HCT116βw cells (Figure 1D). Given the nature of FRET, it is estimated that energy transfer would be detected only if two antibody-labeled proteins are <30 nm apart. Detection of an average energy transfer efficiency (E) of 20.8% between immunolabeled Alexa 488-APC and Alexa 568-topo II within photobleached regions of interest indicates that endogenous APC and topo II are in proximity and is consistent with a direct interaction in colon epithelial cells. This value is significantly more positive than measurements performed on cells in which photobleaching was omitted (E = –0.9%). Likewise, cells transiently expressing GFP showed no energy transfer between endogenous topo II and exogenous GFP (E = –6.6%), even though GFP was abundant in the nucleus (see, e.g., Figure 5A).
The 15-Amino Acid Repeat Region of APC Colocalizes with Topo II, Alters Nuclear Morphology, and Causes Cell Cycle Arrest in G2
APC is a 310-kDa protein with several distinct protein binding domains (Figure 2A). To identify potential topo II binding domains in APC, we expressed five GFP-fused APC fragments (Figure 2A) in HCT116βw cells and compared their localization with that of endogenous topo II (Figure 2B). As expected, GFP fused with either NT-APC, M1-APC, or CT-APC predominantly localized to the cytoplasm (Figure 2B). M3-APC (amino acids 1211–2075) contains all 20-amino acid repeats and two nuclear localization signals (NLSs) (Zhang et al., 2000) and appeared to be located in the nucleus when fused to GFP. GFP fused with M2-APC (amino acids 959-1338) showed the most prominent overlap with topo II in the nucleus. This region of APC contains all four 15-amino acid β-catenin binding repeats and one 20 amino acid repeat but no defined NLS.
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The Abnormal Nuclear Morphology after Expression of the 15-Amino Acid Repeat of APC Is Not Due to Altered β-Catenin
M2-APC comprises all four of the 15-amino acid β-catenin binding repeats and one of the 20-amino acid repeats involved in β-catenin down-regulation (Figure 2A). Activated Wnt signaling resulting from stabilized β-catenin was recently reported to contribute to chromosome instability (Aoki et al., 2007) and lead to G2 arrest (Olmeda et al., 2003). Thus, we predicted that the abnormal nuclear morphology seen in cells expressing M2-APC was dependent on M2-APC association with and stabilization of nuclear β-catenin. We found β-catenin expression and localization identical in HCT116βw cells expressing M2-APC, GFP, or nontransfected (Figure 5A). Furthermore, immunoblots from total lysates demonstrated comparable levels of β-catenin in M2-APC– and GFP-expressing cells (Figure 5D). Moreover, <1% of the total β-catenin coprecipitated with GFP-M2-APC (Figure 5B). In contrast, >10% of the total β-catenin was precipitated along with endogenous full-length APC in parallel experiments under the same experimental conditions (Figure 5C). Finally, β-catenin activity measured in M2-APC–expressing cells was comparable with that in GFP-expressing cells (Figure 5E). Thus, it seems unlikely that the abnormal nuclear morphology and the G2 cell cycle arrest observed in M2-APC–expressing cells result from β-catenin sequestration by M2-APC.
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, an essential regulator of the G2/M transition prompted us to examine whether M2-APC could interact with topo II and possibly mediate the G2 arrest through this interaction. HCT116βw cells transiently expressing M2-APC had increased levels of total topo II (Figure 6A) and the topo II coprecipitated with GFP-M2-APC (Figure 6B). In contrast, no topo II precipitated with the GFP in cells expressing GFP alone. Because M2-APC and topo II interact, it is possible that M2-APC can influence the activity of topo II. To test this directly, in vitro DNA relaxation and decatenation assays were performed using purified recombinant proteins topo II and M2-APC. Purified M2-APC stimulated topo II activity in both DNA relaxation (Figure 6C) and decatenation assays (data not shown). This enhancement was apparent when purified M2-APC was provided at equal molar concentration to the topo II and was further pronounced with 4 times more M2-APC in the reaction (Figure 6C). In the absence of topo II, purified M2-APC did not relax the DNA (Figure 6C) or bind to the DNA slowing its migration (data not shown). Using a higher concentration of recombinant topo II, M2-APC still enhanced topo II activity (Figure 6D). In contrast, bovine serum albumin (BSA) protein at an equal molar ratio slightly inhibited topo II activity (Figure 6D). These in vitro assays provide additional support for a functional interaction between APC and topo II. Together, our data implicate topo II as a mediator of abnormal DNA morphology and G2 arrest associated with exogenous M2-APC expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. This interaction was demonstrated by colocalization, reciprocal coimmunoprecipitation, FRET, and functional assays. Topo II interacts with the 15-amino acid repeat region of APC (M2-APC, amino acids 959-1338). When overexpressed in colon cancer cell lines, M2-APC located predominantly to the nucleus. Cells expressing M2-APC displayed abnormal nuclear morphology and were inhibited in the G2 phase of the cell cycle. Although M2-APC could bind β-catenin, we found no evidence supporting a role for β-catenin in the observed phenotypes. Our data suggest a novel role for nuclear APC in the regulation of cell cycle progression, potentially through an interaction between topo II and the 15-amino acid repeat region of APC.
The GFP–M2-APC protein was not expected to locate predominantly to the nucleus. Although endogenous full-length APC exists in both cytoplasm and nucleus (Neufeld and White, 1997; Anderson et al., 2002), the classic NLSs that are thought to facilitate nuclear localization of APC (Zhang et al., 2000) are not present in the M2-APC fragment. The M2-APC region is retained in most truncated forms of APC associated with colorectal cancer and these truncated APC proteins are capable of nucleocytoplasmic shuttling (Fagman et al., 2003). However, the nuclear import ability of these truncated APC proteins has been attributed to the armadillo repeat region (amino acids 334–625), not the 15-amino acid repeat region (Galea et al., 2001). With an estimated molecular weight of 
70 kDa, GFP-M2-APC is too large to enter the nucleus by diffusion, which has a size limitation of 30–50 kDa (Schulz and Peters, 1987; Mattaj and Englmeier, 1998; Moroianu, 1999). Therefore, M2-APC likely contains a nonstandard NLS or is carried into the nucleus by a nuclear binding partner. Because here we show that M2-APC binds to the abundant nuclear protein topo II, it is possible that nuclear entry of M2-APC is facilitated in part by topo II. APC has also been reported to bind PCNA through the M2 region (Narayan et al., 2005), and we have confirmed that PCNA coimmunoprecipitates with M2-APC (data not shown). We reason that other nuclear proteins, such as PCNA, might contribute to the nuclear entry of M2-APC as well.
As an abundant nuclear protein, topo II is involved in several processes throughout the cell cycle, including transcription, DNA replication, chromatin recombination and organization, and regulation of the G2 decatenation checkpoint (Wang, 1996; Nitiss, 1998; Fortune and Osheroff, 2000; Champoux, 2001; Wang, 2002; McClendon and Osheroff, 2007). It is possible that the topo II we consistently found associated with endogenous APC and with the M2-APC fragment (Figures 1B and 6B) corresponds to a specific pool of topo II involved in regulation of the G2/M transition. Other pools of topo II might be positioned near replicating heterochromatin during S phase (Agostinho et al., 2004), tightly associated with DNA to aid chromosome assembly at the G2/M transition (Earnshaw and Heck, 1985; Gasser et al., 1986; Swedlow and Hirano, 2003), responsible for transcription regulation at G1/S (Huang et al., 2007), or involved in chromosome segregation at the exit of mitosis (Hirano and Mitchison, 1993; Downes et al., 1994; Grue et al., 1998). One explanation for the rather modest amount of topo II that coprecipitated with endogenous APC is that the interaction between APC and topo II is transient, only occurring at a specific point of the cell cycle.
APC is not the first tumor suppressor protein to interact with topo II. BRCA1 (Yamane et al., 2003; Lou et al., 2005), RB (Bhat et al., 1999), and p53 (Cowell et al., 2000) each bind to topo II and thereby effect topo II functions. A recent report also links topo II to the oncoprotein β-catenin and its transcription cofactor TCF (Huang et al., 2007). Overexpression of topo II led to enhanced β-catenin/TCF transcription activity, whereas expression of a constitutively active β-catenin led to increased topo II activity measured in a whole cell lysate. We predict that β-catenin/TCF associates with a topo II pool that is involved in transcription regulation at G1/S. It is possible that this interaction is indirect and mediated by APC. APC has been found in association with the enhancer region of genes regulated by β-catenin/TCF (Sierra et al., 2006). In contrast, we suspect that the APC/topo II interaction we described here is direct. Purified recombinant human M2-APC enhanced the plasmid relaxation activity of purified topo II in our in vitro assays which lacked other potential linker proteins (Figure 6, C and D). Moreover, M2-APC does not seem to bind directly to DNA as determined by gel shift assays (data not shown). We propose that the pool of topo II that interacts with APC to impact G2 cell cycle transition is separate from the pool involved in β-catenin mediated transcription.
We report that the topo II protein level was higher in cells expressing GFP-M2-APC than in GFP-expressing cells (Figure 6A). This observation is consistent with previous reports that topo II levels peak in G2 (Heck et al., 1988). We also report that purified M2-APC enhances topo II activity in an in vitro relaxation assay (Figure 6, C and D). Cell cycle progression from G2 to M is likely dependent on the maintenance of topo II activity at a precise level. Indeed, Epstein-Barr virus kinase BGLF4 both stimulates topo II activity and induces premature chromosome condensation similar to the abnormal DNA morphology we report with M2-APC expression (Lee et al., 2007). Furthermore, protein kinase C
regulates topo II activity specifically during S phase with aberrant activation of topo II by protein kinase C leading to apoptosis (Yoshida et al., 2006). At the simplest level, we propose that endogenous APC regulates topo II activity and thereby facilitates G2-M cell cycle progression.
Topo II activity is essential for the chromatin decatenation required before mitosis (Downes et al., 1994). APC locates near centrosomes during early and late stages of mitosis and has also been implicated in maintenance of chromatin structure (Olmeda et al., 2003; Huang et al., 2007). Therefore, we cannot exclude the possibility that both APC and topo II provide multiple functions throughout the G2/M transition. We propose that a functional interaction between APC and topo II results in regulation of topo II activity. Without this regulation, cell cycle progression would be affected. Given that truncated APC found in most colorectal cancers includes the M2-APC region, it is possible that topo II regulation is maintained in these cancers. In this scenario, retention of the topo II regulatory domain in truncated APC might be essential for cell viability. Alternatively, truncated APC proteins associated with cancer might behave more like the M2-APC fragment and be unable to effectively regulate topo II activity. The resulting stimulation of DNA cleavage by topo II would be predicted to induce chromosome instability, a hallmark of cancer.
Together, we propose a novel function for the tumor suppressor APC in regulation of the G2-M cell cycle transition, potentially through interaction with topo II. Future investigation will be needed to determine the precise underlying mechanism by which M2-APC promotes G2 cell cycle arrest. Such studies will potentially reveal a novel activity of APC in tumor suppression. This expanded role of the tumor suppressor APC has obvious implications in explaining the deregulation of topo II in colon cancer tissue (Murphy et al., 2001) and for the use of topo II inhibitors as chemotherapeutic agents to treat colorectal cancer.
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ACKNOWLEDGMENTS |
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sera, Robert Coffey (Vanderbilt University) for providing reagents and reviewing the manuscript, and Jo Ann Byl (Vanderbilt University) for providing technical support with the topoisomerase activity assays. This work was funded by National Institutes of Health grants R01 CA-10922 (to Y. W. and K.L.N.) and GM-33944 (to N. O.) and by Higuchi Biosciences Center J.R. & Inez Jay Award (to Y. W., Y. A., and K.L.N.). |
Footnotes |
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Address correspondence to: Kristi L. Neufeld (klneuf{at}ku.edu)
Abbreviations used: APC, adenomatous polyposis coli; FRET, Förster resonance energy transfer; NLS, nuclear localization signal; topo II, topoisomerase II; FACS, fluorescence activated cell sorting; GFP, green fluorescent protein.
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