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Vol. 16, Issue 12, 5857-5865, December 2005
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Department of Pathology, University of Pennsylvania, Philadelphia, PA 19104
Submitted August 1, 2005;
Revised September 20, 2005;
Accepted September 29, 2005
Monitoring Editor: Gerard Evan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-tubulin. In addition, overexpression of UXT disrupts centrosome structure. Furthermore, abrogation of UXT protein expression by small interfering RNA knockdown leads to cell death. Together, our findings suggest that UXT is a component of centrosome and is essential for cell viability. We propose that UXT may facilitate transformation by corrupting regulated centrosome functions. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-tubulin ring complex disposed within the centrosome matrix is required for both microtubule nucleation at the centrosome (Zheng et al., 1995
) and organization of centrioles (Ruiz et al., 1999).
The normal G1 stage cell contains a single centrosome, which is duplicated as DNA replicates during the S phase. The precisely duplicated centrosomes separate during the G2 phase, each localized to the spindle pole during the M phase of the cell cycle. Abnormalities of composition of the centrosomes or aberrant numbers of centrosomes have been detected in a variety of tumors (Lingle et al., 2002; Jiang et al., 2003; Yamamoto et al., 2004). The notion that abnormal centrosomes contribute to the progressive malignant phenotype by causing incorrect chromosome segregation and aneuploidy is gaining acceptance (Nigg, 2002).
Molecular chaperones play critical roles in many fundamental cellular processes (Hartl and Hayer-Hartl, 2002), including centrosome behavior. For example, Hsp90 is a core centrosomal component and is essential for certain centrosome functions (Lange et al., 2000). In addition, the tailless complex polypeptide 1 (TCP-1) family of chaperones is also localized to the centrosome (Brown et al., 1996) and facilitates -tubulin maturation (Melki et al., 1993). Delivery of newly synthesized -tubulin to TCP-1 requires a member of the prefoldin (Gim-complex, genes involved in microtubule biogenesis) family of chaperones (Vainberg et al., 1998). Notably, the characterized prefoldin is a hexameric molecular chaperone complex built from two related classes of subunits (Siegert et al., 2000) and is proposed to play a general role in de novo protein folding (Lundin et al., 2004). Loss of prefoldin impairs the function of the centrosome (Le Bot et al., 2003) and can affect cell viability (Maeda et al., 2001).
Cdc14A is a human homologue of the multifunctional protein phosphatase Cdc14 that can antagonize cyclin-dependent kinase activities (Simons et al., 2004). It is localized to the centrosome and seems to be important for centrosome separation (Simons et al., 2004). In this study, we have discovered that UXT is a binding partner of Cdc14A through yeast two-hybrid screens.
Because of its abundance in tumor samples, UXT has been thought to be involved in tumorigenesis. For example, a majority of the expressed sequence tag (EST) clones corresponding to UXT are derived from a variety of tumor cell lines, and UXT seems to be overexpressed in these tumor tissues (Schroer et al., 1999). Here, we demonstrate that UXT is a novel component of the centrosome and is associated with -tubulin. In addition, overexpression of UXT causes loss of pericentriolar material. Furthermore, knockdown of the expression of UXT promotes p53-independent cell death. We propose that UXT may contribute to tumorigenesis by corrupting centrosome activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-tubulin antibody, the monoclonal anti--tubulin, anti-FLAG, and anti-green fluorescent protein (GFP) antibodies were purchased from Sigma-Aldrich (St. Louis, MO).
Cell Culture and Treatments
The human osteoblast sarcoma U2 (U2OS) cell line and the human embryonic kidney 293T (HEK293T) cell line were obtained from the American Type Culture Collection (Manassas, VA). These cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) and 1% antibiotics at 37°C with 5% CO2. All transfections were carried out using FuGENE 6 (Roche Diagnostics, Indianapolis, IN) following the manufacturer's instructions.
Yeast Two-Hybrid Screening
The Matchmaker GAL4 two-hybrid system 3 (BD Biosciences Clontech, Palo Alto, CA) was used to perform yeast two-hybrid screening. The Gal4-fused full-length Cdc14A expressed from pGBTK7 plasmid was used as bait. Transformants (2 x 106) from a HeLa cDNA library (pGAD-GH plasmid; BD Biosciences Clontech) were screened in the yeast strain AH109 (BD Biosciences Clontech) and 11 colonies were identified as positive clones. Among these 11 positive colonies, seven were revealed to encode UXT.
Vectors
To generate pcDNA3-FLAG:UXT, full-length UXT was subcloned from the clone isolated from yeast two-hybrid screening by digestion with EcoRI and XbaI and ligation to the pcDNA3 vector (Invitrogen) that contains two FLAG tag sequences between the HindIII/EcoRI sites. The pEGFP:UXT was constructed by inserting UXT at the EcoRI/Xho I site into pEFGPC1 (BD Biosciences Clontech). Enhanced green fluorescent protein (EGFP):Cdc14A was constructed by inserting Cdc14A amplified by PCR from a plasmid containing full-length Cdc14A (a gift from Dr. H. Yu, University of Texas Southwestern Medical Center, Dallas, TX) into pEGFPC1 at the EcoRI/XhoI sites. The -tubulin/green fluorescence protein (TGFP)-expressing plasmid was kindly provided by Dr. Alexey Khodjakov (Wadsworth Center, New York State Department of Health, Albany, NY). All the plasmid sequences were verified by automated DNA sequencing.
Coimmunoprecipitation
The HEK293T cells (2 x 106) were transfected with various combinations of expression plasmids (4 µg each) by using FuGENE 6. After 48 h, cells were harvested, washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in 800 µl of lysis buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, and 0.5% Nonidet P-40) containing 1 mM sodium orthovanadate, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1% protease inhibitor cocktail (Sigma-Aldrich) for 15 min on ice. After lysis, cells were centrifuged at 13,000 x g for 10 min at 4°C, and the protein content was determined using the bicinchoninic acid assay (Pierce Chemical, Rockford, IL). Total cell lysate (500 µg) was precleared with protein G agarose beads and mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for 1 h. FLAG:UXT, EGFP-UXT, FLAG-Cdc14A, or EGFP-Cdc14A, -tubulin-GFP was then immunoprecipitated using the anti-GFP or anti-FLAG monoclonal antibody (mAb) combined with the protein G agarose beads. The immune complex was then washed four times with the washing buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Nonidet P-40) and subjected to SDS-PAGE. The proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), and the blots were probed with different antibodies. The secondary antibody coupled with horseradish peroxidase (Santa Cruz Biotechnology) was detected by enhanced chemiluminescence reagents (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).
Stable Cell Line
The human U2OS osteoblast sarcoma cell line was transfected using the FuGENE 6 reagent. The G418-resistant clones were screened for expression of GFP fusion proteins using a Nikon fluorescent microscope equipped with a xenon lamp power supply and a GFP filter set. Fluorescent positive clones were further analyzed by Western blot using the anti-GFP mAb or the mouse anti-UXT antibody.
Indirect Immunofluorescence
Cells were cultured, fixed either in methanol or in a paraformaldehyde/Triton X-100 solution, and immunostained following a standard protocol. Controls using equivalent amounts of preimmune IgG or without primary antibody were included in each experiment. The samples were stained with 4,6-diamino-2-phenylindole (DAPI), mounted on slides using Vectashield (Vector Laboratories, Burlingame, CA), and imaged by using Laser Scanning Microscope 410 (Carl Zeiss, Thornwood, NY).
Electron Microscope
U2OS cells transfected with EGFP or EGFP:UXT were washed with PBS and fixed with 2.5% glutaraldehyde for 60 min at room temperature. After post-fixation with 1% OsO4 for 60 min, the samples were dehydrated through an ethanol series and embedded in Epon-Araldite plastic. Thin sections were cut on a LKB Nova ultramicrotome and stained with uranyl acetate and lead citrate.
Generation and Transfection of Small Interfering RNA (siRNA)
We used the following target sequences for UXT siRNA: 434UAC AAG GCC UGC AGA AUU U452 and 362GCA ACA GCC UCA CCA AGG A580. We obtained similar results using these two oligonucleotides. The data presented here were obtained using the latter oligonucleotide. U2OS cells were transfected with siRNAs in six-well dishes by using 3 µl of Oligofectamine reagent (Invitrogen) and diluted in serum- and antibiotic-free Opti-MEM in a final volume of 0.2 ml and containing the following amounts of siRNA: 5 µl of 20 µM solution per transfection for the siRNA. The plating medium volume was 1300 µl per transfection. After 4 h of transfection in serum-free Opti-MEM, 500 µl of Opti-MEM plus 30% FBS were added to each transfection. The final siRNA concentration was 50 nM. We used the nonspecific control siRNA duplexes (Dharmacon, Chicago, IL) as the control.
Flow Cytometry and Cell Cycle Analysis
U2OS cells were transfected with siRNA duplexes. At indicated times, we processed floating and adherent cells for flow cytometry analysis. Briefly, cells were suspended in NP-40 solution (0.1% sodium citrate, 0.0564% NaCl, 0.03% NP-40) with 200 µg ml-1 RNase and stained with 20 µg ml-1 propidium iodide. Cells were analyzed for DNA content. We evaluated dead cells as the percentage of subG1 cell population calculated with MultiCycle software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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To verify that the interaction between Cdc14A and UXT also occurs in mammalian cells, we cotransfected EGFP: Cdc14A and a FLAG-tagged UXT form into HEK293T cells and performed coimmunoprecipitation. As shown in Figure 1A, the 98-kDa EGFP:Cdc14A fusion protein was coprecipitated with the FLAG:UXT by the anti-FLAG antibody. Conversely, the FLAG:UXT was coprecipitated with EGFP: Cdc14A, but not GFP (Figure 1B). Examination of the UXT-Cdc14A interactions at the endogenous level by the same approach must await new reagents because available antibodies against UXT and hCdc14A are not effective in coimmunoprecipitation analysis.
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-tubulin (Figure 2A). Immunostaining of U2OS stably expressing EGFP:UXT with anti-
-tubulin antibodies indicated that EGFP:UXT localized to the spindle poles during mitosis (Figure 2B). In comparison, the EGFP protein alone is homogenously distributed in U2OS or HeLa cells (our unpublished data). To further eliminate the possibility that the centrosomal localization was caused by the EGFP tag, we created a FLAG-tagged UXT construct. Coimmunostaining of -tubulin and the FLAG epitope confirmed that the ectopic FLAG-tagged UXT was targeted to the centrosome (Figure 2C). Thus, we conclude that UXT is localized to the centrosome. Notably, nocodazole treatment of the cells has no effect on the centrosomal localization of GFP-tagged or FLAG-tagged UXT (our unpublished data), indicating that UXT is an integral component of the centrosome.
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). The interaction between prefoldins and their substrates depends on coiled-coil motifs. The organization of centrosomal pericentriolar material also involves multiple coiled-coil proteins (Walton and Sousa, 2004). We therefore created two point mutations (L50P and L59P) in this region that presumably disrupted the coiled-coil domain. Indeed, we found that mutagenesis of the two leucine residues caused the dislocation of UXT from the centrosome (Figure S2).
As shown in Figure 3A, UXT contains five -helixes. We created five deletion mutants of UXT based on the predicted secondary structure and determined their localization in U2OS cells. The results of the localization study are summarized in Figure 3B. The confocal microscopy images of these mutants were shown as supplemental data (Figure S1). We found that the three centrosomal localized mutants (GFP: UXT1-148, GFP:UXT9-157, and GFP:UXT9-148) encompass all the five -helixes. Deletion of the N-terminal (GFP: UXT25-157) or C-terminal (GFP:UXT1-129) -helix abolished centrosomal localization (Figure S2). These data suggest that although the distal amino acid segments at each end of the UXT polypeptide are not essential for centrosomal localization, both the N-terminal and C-terminal -helix as well as the coiled-coil region are required for targeting UXT to the centrosome.
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Overexpression of UXT in Some Human Tumors
It has been proposed that UXT may be involved in tumorigenesis because an EST database survey indicated UXT is abundantly expressed in a variety of tumor tissues (Schroer et al., 1999). There are no clear data to support such a hypothesis. The DiscoverLight tissue lysate arrays (Pierce Chemical) provide a convenient method for protein expression screening. 38 tissue samples (corresponding to 19 pairs of human normal and tumor tissue lysates) were examined with the UXT-specific antibody 1B2. Our data indicate that UXT protein levels were elevated in several tumor tissues, including bladder, breast, ovary, and thyroid, but not in the matching normal tissues (Figure 5A). These results were verified by Western blot analyses using DiscoverLight tissue lysate sets (Figure 5B).
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UXT Is Associated with -Tubulin
We performed a yeast two-hybrid screen of a Hela cDNA library with the full-length UXT as bait and isolated the cDNA encoding amino acids from 322 to 451 of -tubulin. The specificity of the interaction between UXT and -tubulin was confirmed by the yeast two-hybrid assay (our unpublished data). To test whether UXT was also a physiological -tubulin binding partner, we performed coimmunoprecipitation assays. High-affinity and specific antibodies are not available for immunoprecipitation of endogenous UXT or -tubulin. We used the seminative coimmunoprecipitation approach. GFP-tagged -tubulin or FLAG-tagged UXT was introduced into HEK293T cells, respectively. The anti-FLAG antibody was used to immune precipitate FLAG-tagged UXT. As shown in Figure 6A, endogenous -tubulin was coimmunoprecipitated with FLAG-tagged UXT. Conversely, the anti-GFP antibody was used to immune precipitate GFP-tagged -tubulin. As shown in Figure 6B, endogenous UXT was coimmunoprecipitated with GFP-tagged -tubulin. Endogenous -tubulin was not coimmunoprecipitated with UXT (our unpublished data), eliminating the possibility that -tubulin was coimmunoprecipitated with UXT because of its abundance in the cells.
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-tubulin antibodies. Intriguingly, 95% of U2OS cells with high EGFP:UXT expression, which is defined as formation of more than two aggregates in the cytosol, showed loss or dramatic reduction of -tubulin staining (Figure 7A). As a control, only 8% of EGFP-overexpressing cells showed reduced -tubulin staining (our unpublished data). Consistent with the centrosome activity in microtubule nucleation and stabilization, over expression of UXT also reduced the microtubule number and affected the distribution of the microtubule network. Microtubule network irradiates from the cell center (centrosome) in untransfected cells but not UXT-overexpressed cells (Figure S3). To determine whether the alteration of -tubulin localization reflects any structural changes of the centrosome, we used electron microscopy to examine the centrosome under higher resolution. In U2OS cells transfected with EGFP (control), the centrosomes were typically intact (Figure 7B, top). In contrast, the centrosomes in EGFP:UXT-transfected cells were almost completely dis-assembled (Figure 7B, bottom).
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Knockdown of UXT with siRNA Causes p53-independent Cell Death
To further explore the function of UXT, we performed siRNA knockdown of UXT in U2OS cells using UXT siRNA duplex. We used the following nonoverlapping target sequences for UXT siRNA: 362GCA ACA GCC UCA CCA AGG A580 and 434UAC AAG GCC UGC AGA AUU U452. Similar results using both of these two oligonucleotides were obtained. The data presented here were obtained using the first oligonucleotide. The data obtained using the latter oligonucleotide are shown as supplemental data (Figure S4). To assess the potency of the siRNA for UXT, we transfected the siRNA into the U2OS cells stably expressing EGFP:UXT. It was clear that the GFP signal was knocked down in a dose-dependent manner when the siRNA concentration for UXT increased (our unpublished data). Seventy-two hours after transfection, the UXT siRNA oligo caused significant, but incomplete, reduction of UXT protein expression (Figure 8A).
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To test whether p53 plays any role in the cell death induced by UXT siRNA treatment, we transfected UXT siRNA into the p53 deficient HCT116 (p53-/-) and the parental HCT116 (p53+/+) cell line. Massive cell death was observed in both cell lines (Figure 8D). Thus, it seems that the cell death caused by UXT knockdown is not dependent on the p53 status.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-tubulin. Consistent with a role of UXT in the centrosome, ectopic expression of the protein can alter -tubulin localization. Furthermore, we provided evidence that UXT is essential for cell viability. Immunofluorescent staining of the endogenous UXT protein revealed strong signals at the centrosome. Interestingly, the two anti-UXT antibodies generate different centrosomal staining patterns. One antibody detects UXT in the centrosome in the interphase, whereas the other only recognizes UXT disposed in the mitotic centrosome. A plausible explanation for this observation is the dynamic UXT protein disposition during the cell cycle in which UXT epitopes become obscured by interactions with other centrosomal molecules. Alternatively, UXT may be subject to posttranslational modification in a cell cycle-dependent manner, which alters the structure of UXT and thus the epitopes for the antibodies. Although further experiments are needed to distinguish these possibilities, we think that the observed change of UXT staining pattern may be used as a hallmark for the centrosome duplication events.
Because of its extensive sequence similarity to prefoldin, it is possible that UXT is a component of a protein chaperone complex. Indeed, UXT can bind to the prefoldin subunit 2 (Gstaiger et al., 2003). In eukaryotes and archaea, the characterized prefoldin stoichiometry is a hexameric molecular chaperone complex built from two related classes of subunits (Siegert et al., 2000). In comparison, the prefoldinlike chaperone of Escherichia coli consists of a homotrimer of the Skp protein (Salisbury, 2003). We have recently purified UXT using size exclusion chromatography. The UXT protein, whose predicted molecular weight is 20 kDa, migrated as a single peak with a calculated molecular weight of 120 kDa (our unpublished data). We suggest that UXT forms a homohexamer similar to other prefoldin complexes.
We have demonstrated that UXT is essential for cell viability in human cells by siRNA knockdown studies. It is noteworthy that the UXT homologue in Caenorhabditis elegans, the H20J04.5 protein, has also been identified as an essential gene product during the recent genome-wide gene function analysis using siRNA (Lange et al., 2000; Maeda et al., 2001). The prefoldin complex is essential for the proper folding of cytoskeletal proteins (Lundin et al., 2004). Inactivation of prefoldin subunits in C. elegans by siRNA abrogates pronuclear migration during embryogenesis, which indicates that prefoldin is required for microtubule function (Le Bot et al., 2003). Thus, the current data support the notion that UXT plays a crucial role in normal centrosomal biogenesis and cell survival, probably by ensuring proper folding of proteins and preventing protein aggregation in the crowded environment of the centrosome.
Our data indicate that over-expression of UXT in U2OS cells causes dislocation of the pericentriolar protein -tubulin. Electron microscopic studies revealed disorganized centrosomes in UXT-overexpressing cells. Thus, it seems that overexpression of UXT causes not only dispersion of -tubulin but also loss of structural integrity in the centrosome. Because UXT can directly bind to the essential centrosomal protein -tubulin, overexpression of UXT may interfere with the -tubulin function, thereby impairing the organization of the centrosome. Another possibility is that, because UXT can bind to the prefoldin subunit 2 (Gstaiger et al., 2003), overexpressed UXT may cause disorganization of the centrosome by sequestering this subunit and disrupting the prefoldin complex that is required for the assembly of core centrosomal components, including -tubulin.
The current study provides additional evidence to support the hypothesis that UXT overexpression may correlate with or contribute to tumorigenesis. UXT was originally identified as a novel gene mapped to the chromosomal region Xp11, a locus linked to a variety of disease (Schroer et al., 1999). Because UXT is prevalent and abundant in various human tumor tissues, it was suspected to be involved in tumor development (Schroer et al., 1999). In our study, we have demonstrated that UXT expression is elevated in some forms of human cancers, including bladder, breast, ovarian, and thyroid cancers. In contrast to our study, it has been reported that UXT is a coactivator for the androgen receptor (Markus et al., 2002) and that the expression of UXT in prostate cancer is less than that in normal tissue (Taneja et al., 2004). In our initial screening experiment using the DicoverLight tissue lysate sets, UXT expression level is similar between the normal prostate tissue and prostate tumor. Thus, we have not checked the expression levels UXT in prostate cancers by Western blotting. It is possible that the effects of UXT abnormality on transformation are tissue specific. It is also possible that either up-regulation or down-regulation of UXT can contribute to oncogenesis. It should also be noted that the "discrepancy" may arise from the difference between the antibody and the assays used in our study and those used in the previous study. In the study of Taneja et al. (2004), a polyclonal antibody preparation was used for immunostaining of cancerous or normal tissues, which may account for their results. In our study, we used a monoclonal anti-UXT antibody to detect UXT protein levels by Western blot. We think that this is a more stringent assay because it is based on both the binding of the antibody and the size of the molecule detected. Although additional experiments are necessary to substantiate the linkage between UXT levels and human cancer, we predict that UXT may be used as a marker for malignant transformation in at least some human cancers. Moreover, based on our findings that UXT is localized to the centrosome and that over-expression of UXT led to dislocation of centrosomal -tubulin and centrosome organization, we propose that UXT abnormality may cause dysfunction of the centrosome, thereby resulting in defects in chromosome separation that ultimately lead to aneuploidy and malignant transformation.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-tubulin and GFP, and Bert Vogelstein for HCT116 p53+/+ and p53-/- isogenic human colon cancer cells. This work was supported by grants from the National Institutes of Health, National Cancer Institute, and the Abramson Family Cancer Research Institute. |
Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Mark I. Greene (greene{at}reo.med.upenn.edu).
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Gstaiger, M., Luke, B., Hess, D., Oakeley, E. J., Wirbelauer, C., Blondel, M., Vigneron, M., Peter, M., and Krek, W. ((2003). ). Control of nutrient-sensitive transcription programs by the unconventional prefoldin URI. Science 302, , 1208-1212.
Hartl, F. U., and Hayer-Hartl, M. ((2002). ). Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295, , 1852-1858.
Jiang, F., Caraway, N. P., Sabichi, A. L., Zhang, H. Z., Ruitrok, A., Grossman, H. B., Gu, J., Lerner, S. P., Lippman, S., and Katz, R. L. ((2003). ). Centrosomal abnormality is common in and a potential biomarker for bladder cancer. Int. J. Cancer 106, , 661-665.[CrossRef][Medline]
Lange, B. M., Bachi, A., Wilm, M., and Gonzalez, C. ((2000). ). Hsp90 is a core centrosomal component and is required at different stages of the centrosome cycle in Drosophila and vertebrates. EMBO J. 19, , 1252-1262.[CrossRef][Medline]
Le Bot, N., Tsai, M. C., Andrews, R. K., and Ahringer, J. ((2003). ). TAC-1, a regulator of microtubule length in the C. elegans embryo. Curr. Biol. 13, , 1499-1505.[CrossRef][Medline]
Lingle, W. L., Barrett, S. L., Negron, V. C., D'Assoro, A. B., Boeneman, K., Liu, W., Whitehead, C. M., Reynolds, C., and Salisbury, J. L. ((2002). ). Centrosome amplification drives chromosomal instability in breast tumor development. Proc. Natl. Acad. Sci. USA 99, , 1978-1983.
Lundin, V. F., Stirling, P. C., Gomez-Reino, J., Mwenifumbo, J. C., Obst, J. M., Valpuesta, J. M., and Leroux, M. R. ((2004). ). Molecular clamp mechanism of substrate binding by hydrophobic coiled-coil residues of the archaeal chaperone prefoldin. Proc. Natl. Acad. Sci. USA 101, , 4367-4372.
Maeda, I., Kohara, Y., Yamamoto, M., and Sugimoto, A. ((2001). ). Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Curr. Biol. 11, , 171-176.[CrossRef][Medline]
Melki, R., Vainberg, I. E., Chow, R. L., and Cowan, N. J. ((1993). ). Chaperonin-mediated folding of vertebrate actin-related protein and -tubulin. J. Cell Biol. 122, , 1301-1310.
Markus S. M., Taneja S. S., Logan S. K., Li W., Ha S., Hittelman A. B., Rogatsky, I., and Garabedian, M. J. ((2002). ). Identification and characterization of ART-27, a novel coactivator for the androgen receptor N terminus. Mol. Biol. Cell 13, , 670-682.
Nigg, E. A. ((2002). ). Centrosome aberrations: cause or consequence of cancer progression? Nat. Rev. Cancer 2, , 815-825.[CrossRef][Medline]
Ruiz, F., Beisson, J., Rossier, J., and Dupuis-Williams, P. ((1999). ). Basal body duplication in Paramecium requires -tubulin. Curr. Biol. 9, , 43-46.[CrossRef][Medline]
Salisbury, J. L. ((2003). ). Centrosomes: coiled-coils organize the cell center. Curr. Biol. 13, , 88-90.
Schroer, A., Schneider, S., Ropers, H., and Nothwang, H. ((1999). ). Cloning and characterization of UXT, a novel gene in human Xp11, which is widely and abundantly expressed in tumor tissue. Genomics 56, , 340-343.[CrossRef][Medline]
Siegert, R., Leroux, M. R., Scheufler, C., Hartl, F. U., and Moarefi, I. ((2000). ). Structure of the molecular chaperone prefoldin: unique interaction of multiple coiled coil tentacles with unfolded proteins. Cell 103, , 621-632.[CrossRef][Medline]
Simons, C. T., Staes, A., Rommelaere, H., Ampe, C., Lewis, S. A., and Cowan, N. J. ((2004). ). Selective contribution of eukaryotic prefoldin subunits to actin and tubulin binding. J. Biol. Chem. 279, , 4196-4203.
Taneja, S. S., Ha, S., Swenson, N. K., Torra, I. P., Rome, S., Walden, P. D., Huang, H. Y., Shapiro, E., Garabedian, M. J., and Logan, S. K. ((2004). ). ART-27, an androgen receptor coactivator regulated in prostate development and cancer. J. Biol. Chem. 279, , 13944-13952.
Vainberg, I. E., Lewis, S. A., Rommelaere, H., Ampe, C., Vandekerckhove, J., Klein, H. L., and Cowan, N. J. ((1998). ). Prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin. Cell 93, , 863-873.[CrossRef][Medline]
Walton, T. A., and Sousa, M. C. ((2004). ). Crystal structure of Skp, a prefoldinlike chaperone that protects soluble and membrane proteins from aggregation. Mol. Cell 15, , 367-374.[CrossRef][Medline]
Yamamoto, Y., Matsuyama, H., Furuya, T., Oga, A., Yoshihiro, S., Okuda, M., Kawauchi, S., Sasaki, K., and Naito, K. ((2004). ). Over expression of oncogenic STK15/BTAK/Aurora A kinase in human pancreatic cancer. Clin. Cancer Res. 10, , 6449-6455.
Zheng, Y., Wong, M. L., Alberts, B., and Mitchison, T. ((1995). ). A g-tubulin ring complex from the unfertilized egg of Xenopus laevis can nucleate microtubule assembly in vitro. Nature 378, , 578-583.[CrossRef][Medline]
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-tubulin antibody (bottom). The protein levels of UXT were specifically reduced after 72-h treatment with siRNA for UXT. (B) UXT knockdown inhibits cell proliferation. U2OS cells were treated with nonspecific siRNA (left) or siRNA for UXT (right). Pictures were taken 72 h later. (C) UXT knockdown leads to cell death. U2OS cells were treated with nonspecific RNA or the UXT siRNA oligos. Seventy-two hours later, all the cells were collected and used for propidium iodide staining of DNA content by fluorescence-activated cell sorting. (D) p53 is not required for cell death caused by UXT knockdown. The HCT116 (p53+/+) or p53-negative HCT116 (p53-/-) cells were treated with either the control siRNA or the UXT siRNA. Seventy-two hours after transfection, the percentage of cell death was assessed by the trypan blue assay. The diagram shows the representative results of three experiments.