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Arsenic Trioxide Induces Abnormal Mitotic Spindles Through a PIP4KIIγ/Rho Pathway

Arsenic Trioxide Induces Abnormal Mitotic Spindles Through a PIP4KIIγ/Rho Pathway Abstract Arsenite-induced spindle abnormalities result in mitotic cell apoptosis in several cancer cell lines, but how arsenite induces these effects is not known. Evidence to date has revealed that arsenite activates Rho guanosine triphosphatases (GTPases). Because Rho GTPases regulate spindle orientation, chromosome congression, and cytokinesis, we therefore examined the involvement of Rho GTPases and their modulators in arsenite-induced mitotic abnormalities. We demonstrated that arsenic trioxide (ATO) disrupted the positioning of bipolar mitotic spindles and induced centrosome and spindle abnormalities. ATO increased the level of the active guanosine triphosphate-bound form of Rho. Inhibition of Rho-associated protein kinases (ROCKs) by Y-27632 ameliorated ATO-induced spindle defects, mitotic arrest, and cell death. These results indicate that ATO may induce spindle abnormalities and mitotic cell death through a Rho/ROCK pathway. In addition, screening of a human kinase and phosphatase shRNA library to select genes that mediate ATO induction of spindle abnormalities resulted in the identification of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), a phosphatidylinositol 4,5-biphosphate (PIP2) synthesis enzyme that belongs to the phosphatidylinositol phosphate kinase (PIPK) family. Sequestration of PIP2 by ectopic overexpression of the pleckstrin homology domain of phospholipase C-δ1 protected cells from ATO-induced cell death. Furthermore, depletion of PIP4KIIγ, but not other isoforms of the PIPK family, not only reduced Rho GTPase activation in ATO-treated cells but also alleviated ATO-induced spindle defects, mitotic arrest, and mitotic cell apoptosis. Thus, our results imply that ATO induces abnormalities in mitotic spindles through a PIP4KIIγ/Rho pathway, leading to apoptosis of mitotic cells. arsenic trioxide, mitotic spindle, apoptosis, PIP4KIIγ, Rho Arsenic trioxide (ATO) has potent antineoplastic effects in vitro and in vivo. ATO cures acute promyelocytic leukemia by initiating degradation of the oncogenic fusion protein PML/RARA (Jeanne et al., 2010). ATO also induces apoptosis and suppresses the growth of PML/RARA-negative malignant cells (Gazitt and Akay, 2005; Miller et al., 2002). Despite extensive research, the precise mechanisms of action of ATO in malignant cells are not well understood. Trivalent arsenic compounds induce centrosome amplification and disrupt mitotic spindles, inducing mitotic arrest and consequently triggering mitotic arrest–mediated apoptosis in a variety of cancer cells (Cai et al., 2003; Huang et al., 2000; Ling et al., 2002; McCollum et al., 2005; McNeely et al., 2008; States et al., 2002; Taylor et al., 2008; Yih et al., 2005, 2006). These reports reveal a tight link between arsenite-induced apoptosis and aberrant mitotic spindles in cancer cells. However, how arsenite induces spindle abnormalities remains elusive. Mitotic spindles, the machines driving chromosome segregation during mitosis, adopt a bipolar configuration to divide the chromosomes into two equivalent sets through precise modulation of microtubule dynamics (Tanenbaum and Medema, 2010). The Rho guanosine triphosphatases (GTPases), including Rho, Rac, and Cdc42, are key regulators of cytoskeletal dynamics and affect many cellular processes, including cell polarity, migration, vesicle trafficking, and cell cycle progression and cytokinesis. The Rho GTPases can control the reorganization of actin and microtubule cytoskeletal structures during mitosis (Hollenbeck, 2001). They have been demonstrated to modulate centrosome positioning and spindle orientation (Bakal et al., 2005; Rosenblatt et al., 2004), proper attachment between kinetochores and microtubules (Yasuda et al., 2004), and cytokinesis (Piekny et al., 2005). In addition, Rho-associated protein kinases (ROCKs), the downstream effectors of Rho, associate with nucleophosmin and may thus modulate the number of centrosomes (Ferretti et al., 2010; Ma et al., 2006). P21-activated kinases, the effectors of Cdc42 and Rac1, may control centrosome maturation and spindle formation through phosphorylation of polo-like kinase 1 (Maroto et al., 2008). LIM kinases, when activated by the ROCKs or P21-activated kinases, colocalize with γ-tubulin at centrosomes early during mitosis and may play a role in mitotic spindle assembly (Chakrabarti et al., 2007). These studies demonstrate the critical roles of the Rho GTPases and their downstream effectors in regulating the mitotic machinery and imply that perturbation of Rho GTPase signaling might disrupt the assembly of mitotic spindles and alter mitotic progression. The Rho GTPases and their effectors modulate cytoskeleton-dependent cellular events by regulating the targeting and activation of phosphatidylinositol phosphate kinases (PIPKs) and generation of phosphatidylinositol-4,5-bisphosphate (PIP2) (Doughman et al., 2003; van den Bout and Divecha, 2009). The activation of the Rho GTPases and their targeting to specific cell compartments are also regulated by PIPKs and PIP2 (Chao et al., 2010; Zheng et al., 1996). The spatiotemporal cross-regulation of the Rho GTPases and PIPKs thus may be critical for the positioning and assembly of mitotic spindles. ATO reportedly activates the Rho GTPases in diverse cell systems. Rac is activated by ATO in leukemia cells and might negatively regulate ATO-induced apoptosis (Verma et al., 2002). Rho is activated by ATO in chronic myelogenous leukemia cells and mediates the activation of c-Jun N-terminal kinase, which leads to apoptosis (Potin et al., 2007). Rho is also activated by ATO in macrophages and mediates the activation of reduced NADPH oxidase, the production of reactive oxygen species, and secretion of cytokines (Lemarie et al., 2008). Cdc42 is activated by ATO in endothelial cells and mediates the activation of NADPH oxidase, production of reactive oxygen species, and cell migration (Qian et al., 2005). In addition, inhibition of Rac1 blocks ATO-stimulated differentiation and dysfunction of liver sinusoidal endothelial cells (Straub et al., 2008). These results indicate that the Rho GTPases are critical mediators that control diverse cellular responses to ATO. Because the Rho GTPases play critical roles in regulating mitotic spindle assembly and can be activated by ATO, we therefore investigated whether these GTPases and their possible modulators are involved in the induction of ATO-induced spindle abnormalities. MATERIALS AND METHODS Cell culture and reagents. HeLa-S3 cells were obtained from the American Type Culture Collection (Manassas, VA). CGL2 cells (a HeLa cell/normal human fibroblast hybrid) (Stanbridge et al., 1981) were kindly provided by Dr E. J. Stanbridge (University of California-Irvine). Cells were routinely maintained in Dulbecco's Modified Eagle's Medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen), 0.37% sodium bicarbonate, 100 U/ml of penicillin, and 100 μg/ml of streptomycin at 37°C in an humidified incubator in air and 10% CO2 and were passaged twice per week. ATO (Sigma-Aldrich, St Louis, MO) was freshly dissolved in 0.1 N NaOH to make a 10 mM stock solution before use. Y-27632 (Calbiochem, Merck KGaA, Darmstadt, Germany) was dissolved in dimethyl sulfoxide to make a 10 mM stock and stored in aliquots at –20°C. Immunofluorescence staining of mitotic spindles and spindle positioning assay. HeLa-S3 or CGL2 cells (1 × 105) seeded on glass coverslips were incubated for 20 h at 37°C with or without drugs, washed twice with PBS, and fixed in situ with 90% methanol at –20°C for 10 min. The cells were then immunostained for mitotic spindles with an antibody against human α-tubulin (Sigma) and for centrosomes with an antibody against human γ-tubulin (Sigma) as described (Yih et al., 2006). Nuclei were simultaneously counterstained with 0.1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI; Sigma). After thorough rinsing with PBS containing 0.2% (vol/vol) Tween 20, the cells were mounted using VECTASHIELD (Vector Laboratories, Inc., Burlingame, CA) and examined under a fluorescence microscope (Axioplan 2 Imaging MOT; Carl Zeiss MicroImaging, Oberkochen, Germany). The positions of mitotic spindles were evaluated by measuring the distance between the spindle center and the cell center. Cells were fixed, immunostained for mitotic spindles, and photographed (Axioplan 2). The distance was measured by MetaMorph COMPLETE software (Molecular Devices, Sunnyvale, CA). A total of 100 cells from three independent experiments were examined, and the distances in ATO-arrested mitotic cells were compared with those measured in untreated mitotic cells. To determine the percentage of mitotic cells with abnormal spindles, mitotic cells that contained distorted or disorganized mitotic spindles, multipolar spindles, or mitotic spindles with aggregated spindle fibers or with elongated polar distance were classified as mitotic cells with abnormal mitotic spindles (Wu et al., 2009). At least 500 mitotic cells from three independent experiments were analyzed. Time-lapse fluorescence analysis. HeLa-S3 cells were transfected with pEYFP-tubulin Vector (Clontech, Mountain View, CA). Cells stably expressing enhanced YFP (EYFP)-tubulin fusion protein were established and plated on a 60-mm Petri dish 20 h before ATO treatment. Cells were treated with 3 μM ATO for up to 40 h, during which time the dish was placed on the objective stage set up in a humidified chamber with 10% CO2 in air at 37°C. Fluorescence images were collected every 15 min using an inverted fluorescence microscope (Axiovert 200M; Carl Zeiss) equipped with an oil immersion objective lens (×63, plan Apo NA 1.4) and a MetaMorph COMPLETE-controlled CCD camera. The acquired images were processed by MetaMorph COMPLETE software. The time-lapse recordings were repeated for two and four times for untreated and ATO-treated cells, respectively. Rho-guanosine triphosphate pull-down assay. The level of guanosine triphosphate (GTP)-bound Rho, Rac, or Cdc42 was measured using the Rho or Rac1/Cdc42 activation assay kits from Millipore (Temecula, CA). After treatments, cells were rapidly lysed at 4°C in buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.5), 250 mM NaCl, 1% NP-40, 10 mM MgCl2, 1 mM EDTA, 2% glycerol, 10 μg/ml leupeptin, 25 mM sodium fluoride, and 1 mM sodium orthovanadate. After centrifugation (14,000 × g for 5 min at 4°C), the clear lysate was incubated with the affinity beads conjugated with Rhotekin Rho binding domain or P21-activated kinase-1 p21-binding domain to specifically pull-down the GTP-bound Rho or Rac/Cdc42, respectively. After thorough washing, Rho, Rac, or Cdc42 levels were assessed by immunoblotting with the corresponding antibody (Millipore). Briefly, each of the bead/protein complexes in Laemmli buffer containing 0.1 M dithiothreitol was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (12% acrylamide) and transferred to an Immobilon polyvinylidene fluoride membrane (Millipore). After blocking in PBS containing 5% (wt/vol) skim milk powder and 0.2% Tween 20 for 1 h, each membrane was incubated with the corresponding antibody overnight at 4°C. Antigen-antibody complexes were visualized using horseradish peroxidase-conjugated secondary antibodies followed by ECL (SuperSignal West Pico; Pierce, Rockford, IL). Lentiviral transduction and ATO selection. A pseudotyped lentivirus-based human kinase and phosphatase (HKP) shRNA library that contains 6300 shRNA clones (corresponding to 1260 genes) from the National RNAi Core Facility Platform (Institute of Molecular Biology/Genomic Research Center, Academia Sinica, Taipei, Taiwan) was used to select for genes related to ATO toxicity. One day before transduction, CGL2 cells were seeded at 1 × 106 in a 100-mm Petri dish. Cells were transduced with HKP pooled lentiviral supernatants (multiplicity of infection = 0.3) in growth medium supplemented with 10 μg/ml polybrene. At 24 h post-transduction, 2 μg/ml puromycin was added to culture medium to eliminate nontransduced cells. The transduced cells were then treated with 0.5 μM ATO in the presence of 1 μg/ml verapamil for 2 weeks. Cells surviving ATO treatment were collected and their genomic DNA isolated. The shRNA was recovered by PCR amplification, cloned, and then sequenced. Oligonucleotides encoded in the lentiviral vector pLKO.1 were used as PCR primers for the identification and sequencing of inserts: 5′-tacaaaatacgtgacgtag-3′ (forward primer) and 5′-ctgttgctattatgtctac-3′ (reverse primer). Sequestration of cellular PIP2. Cellular PIP2 levels can be reduced by overexpressing the pleckstrin homology domain of phospholipase C-δ1 (PHPLCδ1) in cells to specifically bind and sequester PIP2 (Field et al., 2005). To generate the plasmid encoding PHPLCδ1, the PHPLCδ1 complementary DNA (cDNA) was amplified by PCR using primers 5′-GAAGATCTGCGCTGCTGAAGGGCAGC-3′ and 5′-CCCAAGCTTAGTGGATGATCTTGTGCAGCCC-3′ according to the sequence from GenBank (accession no. U09117), digested with BglII and HindIII, and subcloned into the BglII and HindIII sites of the pmCherry-C1 vector (Clontech). CGL2 cells (1 × 105 per well in a 24-well plate) were transfected with 2 μg/ml empty vector or 0.5 or 2 μg/ml PHPLCδ1 with FuGENE HD (Roche, Mannheim, Germany). After 5 h, cells were replated and assessed for cytotoxicity. Depletion of cellular PIPKs. Depletion of specific PIPK isoforms was achieved by transfecting cells with small interference RNAs (siRNAs) or transducing cells with VSV-G-pseudotyped lentivirus-based shRNA. siRNAs specific to each PIPK isoform were obtained from Thermo Scientific Dharmacon (On-Target plus SMART pool; Lafayette, CO). HeLa-S3 cells were plated at a density of 0.5–1 × 105 per 35-mm dish one day before transfection, then were transfected with each siRNA at a final concentration of 50 nmol using Oligofectamine (Invitrogen). At 24-h post-transfection, the medium was replaced with fresh medium, and the cells were cultured for another 24 or 48 h. A double-stranded RNA targeting luciferase (5′-cguacgcggaauacuucgadTdT-3′) was used as the control. For stable depletion of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), PIP4KIIγ-specific shRNA (TRCN37719) was obtained from the National RNAi Core Facility Platform. CGL2 cells were transduced with shRNA-containing virus (multiplicity of infection = 3) in growth medium supplemented with 10 μg/ml polybrene. At 24-h post-transduction, 2 μg/ml puromycin was added to culture medium to select for stable clones (CGL2-sh2C). The expression level of each isoform was examined by reverse transcription (RT)-PCR or immunoblotting. Reverse transcription-PCR. Cellular RNA was extracted with Trizol (Invitrogen) reagent, and 5 μg was reverse transcribed with Superscript III (Invitrogen) and oligo-dT (Promega, Madison, WI). PIPK isoforms were analyzed by PCR with primers flanking consecutive exon sequences. For each product, a linear range of cDNA input was determined empirically via a twofold dilution series in 26–28 cycle reactions in the presence of Fermentas DreamTaq DNA polymerase (Thermo Scientific). PCR products were run on 1% Tris/borate/EDTA-agarose gels (Ogden and Adams, 1987), stained with SYBR Gold (Invitrogen), and quantified with GeneTools (Syngene, Cambridge, U.K.). β-Actin was used as an internal control. The primer sets were as follows: phosphatidylinositol-4-phosphate 5-kinase type-1 (PIP5KI) α, 5′-ACAACGAGAGCCCTTAAGCA-3′ and 5′-GAACCGTTCAGCGTAGAAGC-3′; PIP5KIβ, 5′-AAGGATGAGAAGCGGGATTT-3′ and 5′-TGGAAGGTAACCCTTTGCTG-3′; PIP5KIγ, 5′-AGAAGCCACTACAGCCTCCA-3′ and 5′-TCTTTGGGGACCACAATCTC-3; PIP4KIIα, 5′-GTCTGCTGGTGGGAATTCAT-3′ and 5′-TCCTAGGCGAGTTTTCATGG-3′; PIP4KIIβ, 5′-GACGTTGAGTTCTTGGCACA-3′ and 5′-CAAAGAACCGAGGAAAGCTG-3′; PIP4KIIγ, 5′-GTGCAGCTGAAGATCATGGA-3′ and 5′-TGAGGCCCATGAAGTAGACC-3′; and β-actin, 5′-GCACTCTTCCAGCCTTCC-3′ and 5′-GCGCTCAGGAGGAGCAAT-3′. Cytotoxicity assay. Cells were plated at a density of 4 × 105 per well in a six-well plate or 1 × 105 per well in a 24-well plate one day before siRNA or plasmid transfection. At 5 h post-transfection, the cells were seeded in a 24-well plate (3 × 104 per well); 24 h later, they were treated with drugs for 24–72 h. Cytotoxicity was then determined by assaying viable cell numbers using methylthiazole tetrazolium (WST-8) (Cell Count Kit 8; Dojindo Molecular Technologies, Inc., Gaithersburg, MD) or by colony-forming efficiency assay as described (Kakadiya et al., 2011; Yih et al., 2005). Analysis of cell cycle distribution and apoptosis. Cell cycle progression and apoptosis were monitored using flow cytometry. DNA was stained with propidium iodide, and mitotic cells were quantified by measuring the expression of the mitosis-specific marker phospho-histone H3; further, apoptotic cells were identified by measuring the level of cleaved poly(adenosine diphosphate-ribose) polymerase (PARP) as described (Wu et al., 2010; Yih et al., 2006). In brief, the cells were fixed with ice-cold 70% ethanol for 16 h, then simultaneously immunostained with mouse antihuman phospho-histone H3 (H3 phosphorylated on serine 10; Cell Signaling Technology, Beverly, MA) and rabbit antibody against human cleaved PARP (Cell Signaling Technology), followed by incubation with fluorescein isothiocyanate-conjugated goat anti-mouse IgG and allophycocyanin-conjugated goat anti-rabbit IgG (Invitrogen). Cellular DNA was counterstained with propidium iodide. Levels of phospho-histone H3, cleaved PARP, and propidium iodide of individual cells were analyzed using a flow cytometer (FACSCanto II; BD Biosciences, San Jose, CA), and the cell cycle distribution and the percentage of cells with phospho-histone H3 and cleaved PARP were determined using a computer program provided by BD Biosciences. RESULTS ATO Disrupts the Positioning of Mitotic Spindles Consistent with the results of previous studies, treatment of HeLa-S3 cells with 3 μM ATO induced distorted and disorganized mitotic spindles (Fig. 1B) or multipolar spindles (Fig. 1C) in arrested mitotic cells. The effect of ATO on spindle positioning, as visualized by the expression of EYFP-tubulin in HeLa-S3 cells, was examined using time-lapse fluorescence microscopy. In untreated mitotic cells, the spindle was stably positioned near the geometric center of the cell; the two spindle poles could be clearly visualized in the same focal plane during mitosis, indicating that the spindle axis was horizontal and parallel to the basement plane (Fig. 1D). In ATO-arrested mitotic cells, however, the spindle rotated and moved randomly in the cytoplasm; the spindle was positioned away from the center of the cell and was tilted with one pole frequently out of the focal plane (Fig. 1E). Formation of an extra pole or aster-like structure was also observed during the prolonged arrest at mitosis. Subsequently, these aberrant mitotic cells underwent apoptosis as revealed by the formation of membrane blebs. FIG. 1. Open in new tabDownload slide ATO induces spindle abnormalities in HeLa-S3 cells. (A–C) Representative immunofluorescence images of normal (A) and ATO-arrested mitotic cells (B and C). (A) Normal bipolar spindle. (B) Abnormal spindle with distorted and aggregated microtubules. (C) Multipolar spindle. HeLa-S3 cells were left untreated or treated with 3 μM ATO for 20 h and then fixed and immunostained with anti-α-tubulin to detect mitotic spindles (red) and with anti-γ-tubulin to detect centrosomes (green) and counterstained with DAPI to detect chromosomes (blue). The three fluorescence images were merged. Scale bar, 10 μm. (D and E) Representative live-cell still images of untreated (D) or ATO-treated (E) EYFP-tubulin HeLa cells. Cells stably expressing EYFP-tubulin (green) were left untreated or treated with 3 μM ATO. Hoechst 33258 (0.3 μM) was included in the culture medium of untreated cells. Fluorescence images were collected every 15 min and processed as described in the Materials and Methods section. Numbers in (D) indicate the time (hour:min) after the first frame and in (E) the time after ATO addition. #, *, and + symbols denote three different abnormal mitotic cells at different times in the ATO-treated culture. FIG. 1. Open in new tabDownload slide ATO induces spindle abnormalities in HeLa-S3 cells. (A–C) Representative immunofluorescence images of normal (A) and ATO-arrested mitotic cells (B and C). (A) Normal bipolar spindle. (B) Abnormal spindle with distorted and aggregated microtubules. (C) Multipolar spindle. HeLa-S3 cells were left untreated or treated with 3 μM ATO for 20 h and then fixed and immunostained with anti-α-tubulin to detect mitotic spindles (red) and with anti-γ-tubulin to detect centrosomes (green) and counterstained with DAPI to detect chromosomes (blue). The three fluorescence images were merged. Scale bar, 10 μm. (D and E) Representative live-cell still images of untreated (D) or ATO-treated (E) EYFP-tubulin HeLa cells. Cells stably expressing EYFP-tubulin (green) were left untreated or treated with 3 μM ATO. Hoechst 33258 (0.3 μM) was included in the culture medium of untreated cells. Fluorescence images were collected every 15 min and processed as described in the Materials and Methods section. Numbers in (D) indicate the time (hour:min) after the first frame and in (E) the time after ATO addition. #, *, and + symbols denote three different abnormal mitotic cells at different times in the ATO-treated culture. The effect of ATO on spindle positioning was also assessed in CGL2 cells by measuring the distance between the midpoint of the spindle and the cell center in the horizontal view. In untreated cultures, mitotic spindles were positioned near the center of the cells (Fig. 2A) with the distance ranging from 0.45 to 1.66 μm (Fig. 2D). However, the spindles in ATO-arrested mitotic cells were not at the cell center (Figs. 2B and C); the distance ranged from 0.46 to 4.86 μm (Fig. 2D), which was significantly longer than that in the untreated mitotic cells. This indicated that mitotic spindle was mispositioned in ATO-arrested cells. FIG. 2. Open in new tabDownload slide ATO disrupts spindle positioning. (A–C) Representative immunofluorescence images of untreated (A) and ATO-arrested mitotic cells (B and C). CGL2 cells were treated with 1 μM ATO for 20 h and immunostained for mitotic spindles as in Figure 1. The red line indicates the spindle pole-to-pole axis, and the yellow dot indicates the geometric center of the cell. Each yellow dashed line indicates a cell border. Positioning of the mitotic spindle was evaluated by measuring the distance between the cell center and the midpoint of the spindle axis. (D) Scatter plot of the distance from the cell center to midpoint of the spindle axis. Red dots indicate the average values and error bars indicate the standard deviation of 100 mitotic cells from three independent experiments. *p < 0.01 compared with untreated cells according to Student’s t-test. FIG. 2. Open in new tabDownload slide ATO disrupts spindle positioning. (A–C) Representative immunofluorescence images of untreated (A) and ATO-arrested mitotic cells (B and C). CGL2 cells were treated with 1 μM ATO for 20 h and immunostained for mitotic spindles as in Figure 1. The red line indicates the spindle pole-to-pole axis, and the yellow dot indicates the geometric center of the cell. Each yellow dashed line indicates a cell border. Positioning of the mitotic spindle was evaluated by measuring the distance between the cell center and the midpoint of the spindle axis. (D) Scatter plot of the distance from the cell center to midpoint of the spindle axis. Red dots indicate the average values and error bars indicate the standard deviation of 100 mitotic cells from three independent experiments. *p < 0.01 compared with untreated cells according to Student’s t-test. Inhibition of ROCKs Ameliorates ATO-induced Spindle Abnormalities and Cell Death To understand whether Rho GTPases were involved in ATO induction of spindle abnormalities, the effect of ATO on activation of the Rho GTPases was examined. ATO increased the level of the active GTP-bound form of Rho but not that of Rac or Cdc42 (Fig. 3A). ROCKs are a major target of Rho-GTP and a key-signaling molecule involved in cytoskeleton regulation (Amano et al., 2010). The effect of Y-27632, a specific inhibitor of ROCKs, on ATO-induced mitotic cells was thus examined. Y-27632 alone (5 or 10 μM) did not significantly affect mitotic spindles or cell viability in CGL2 cells (Figs. 3B–E). But treatment of the cells with 20 μM Y-27632 resulted in the formation of disorganized mitotic spindles and induced cell death (data not shown), consistent with the results of a previous report (Rosenblatt et al., 2004). In ATO-treated cultures, up to 70% of the ATO-arrested mitotic cells contained disorganized or multipolar spindles (Fig. 3B). In addition, the mitotic index was significantly increased and the number of cells in cytokinesis decreased compared with untreated cultures (Figs. 3C and D), indicating that these ATO-induced aberrant cells were stuck in mitosis. Cotreatment of cells with ATO and Y-27632 considerably reduced the induction of spindle abnormalities (Fig. 3B), decreased the mitotic index (Fig. 3C), and increased the number of cells in cytokinesis (Fig. 3D), indicating that inhibition of ROCKs might prevent the formation of abnormal mitotic spindles and thereby release the arrest of mitotic cells in ATO-treated cultures. In addition, cotreatment with Y-27632 enhanced the viability of ATO-treated cells (Fig. 3E). These results indicated that ATO might induce defects in mitotic spindles and cause cell death by activating a Rho/ROCK pathway. FIG. 3. Open in new tabDownload slide Y-27632 ameliorates ATO-induced spindle abnormalities and cell death. (A) ATO activates Rho GTPases. CGL2 cells were untreated or treated with 3 μM ATO for 3 h and then the GTP-bound fraction of Rho, Rac, or Cdc42 (the active form) was pulled down as described in the Materials and Methods section. The pulled-down complexes and whole-cell lysates were analyzed by immunoblotting to detect the level of Rho, Rac, or Cdc42. (B–D) Y-27632 reduces ATO-induced spindle abnormalities, mitotic arrest, and blockade of cytokinesis. CGL2 cells were treated with 1 μM ATO plus 5 or 10 μM Y-27632 for 20 h and immunostained for mitotic spindles as described in Figure 1 (B and D) or analyzed for cell cycle distribution (C). The percentage of mitotic cells with spindle defects was calculated using data acquired from at least 500 mitotic cells in three independent experiments. The percentage of cells in cytokinesis was calculated using data acquired from at least 1000 cells in three independent experiments. (E) Y-27632 reduces ATO-induced cell death. CGL2 cells were treated with 1 μM ATO plus 5 or 10 μM Y-27632 for 72 h and then cell viability was determined by the WST-8 assay. Y-27632 alone showed no significant cytotoxicity. #p < 0.01 compared with untreated cells. *p < 0.05 and **p < 0.01 compared with ATO treatment alone according to Student’s t-test. FIG. 3. Open in new tabDownload slide Y-27632 ameliorates ATO-induced spindle abnormalities and cell death. (A) ATO activates Rho GTPases. CGL2 cells were untreated or treated with 3 μM ATO for 3 h and then the GTP-bound fraction of Rho, Rac, or Cdc42 (the active form) was pulled down as described in the Materials and Methods section. The pulled-down complexes and whole-cell lysates were analyzed by immunoblotting to detect the level of Rho, Rac, or Cdc42. (B–D) Y-27632 reduces ATO-induced spindle abnormalities, mitotic arrest, and blockade of cytokinesis. CGL2 cells were treated with 1 μM ATO plus 5 or 10 μM Y-27632 for 20 h and immunostained for mitotic spindles as described in Figure 1 (B and D) or analyzed for cell cycle distribution (C). The percentage of mitotic cells with spindle defects was calculated using data acquired from at least 500 mitotic cells in three independent experiments. The percentage of cells in cytokinesis was calculated using data acquired from at least 1000 cells in three independent experiments. (E) Y-27632 reduces ATO-induced cell death. CGL2 cells were treated with 1 μM ATO plus 5 or 10 μM Y-27632 for 72 h and then cell viability was determined by the WST-8 assay. Y-27632 alone showed no significant cytotoxicity. #p < 0.01 compared with untreated cells. *p < 0.05 and **p < 0.01 compared with ATO treatment alone according to Student’s t-test. PIP4KIIγ Is Involved in ATO Cytotoxicity Because ATO induces CGL2 cell death mainly via induction of abnormal mitotic spindles and mitotic cell apoptosis (Yih et al., 2005), a pseudotyped lentivirus-based HKP shRNA library was screened for genes that are related to ATO induction of spindle abnormalities in CGL2 cells. After transduction and ATO selection, the shRNA was recovered from cells surviving ATO treatment by genomic PCR using the primers for the lentiviral vector pLKO.1, i.e., only the inserts containing the vector sequences at both ends could be detected. In total, 387 clones were verified by sequencing; among them, 45 contained shRNA sequences that corresponded to the same gene, namely PIP4K2C, which encodes PIP4KIIγ, a PIP2 synthesis enzyme belonging to the PIPK family. This result indicated that depletion of PIP4KIIγ induced cell resistance to ATO and implied that PIP4KIIγ and PIP2 might play important roles in ATO induction of spindle abnormalities. To confirm the role of PIP2 in ATO-treated cells, CGL2 cells were transfected with an expression vector encoding PHPLCδ1, which can specifically bind and sequester cellular PIP2 (Field et al., 2005). Expression of PHPLCδ1 significantly enhanced the viability of ATO-treated cells (Fig. 4), indicating that ATO might induce cytotoxicity through generation of PIP2. PIP2 is synthesized via the action of two distinct but related PIPKs, PIP5KI and PIP4KII. Three isoforms, α, β, and γ, have been identified in each subfamily (Doughman et al., 2003). We thus examined whether depletion of individual isoforms could affect the cytotoxicity of ATO. As HeLa-S3 cells exhibit high transfection efficiency and are also susceptible to ATO-induced spindle defects and mitotic apoptosis (Huang et al., 2000), HeLa-S3 cells were transfected with isoform-specific siRNA. The specificity and efficiency of each siRNA were confirmed by RT-PCR and at least 80% downregulation of each isoform was achieved (Fig. 5A). Depletion of PIP4KIIγ, but not other isoforms, significantly reduced ATO cytotoxicity as determined by the cell viability assay (Fig. 5B) or colony-forming efficiency assay (Fig. 5C). These results confirmed that PIP4KIIγ is involved in ATO cytotoxicity. FIG. 4. Open in new tabDownload slide Sequestration of cellular PIP2 reduces ATO cytotoxicity. CGL2 cells were transfected with 2 μg empty vector or with 0.5 or 2 μg PHPLCδ1 plasmid and then replated and treated with 1 or 2 μM ATO for 72 h. Cell viability was measured by the WST-8 assay. *p < 0.01 compared with cells transfected with empty vector and treated with the same ATO concentration according to Student’s t-test. FIG. 4. Open in new tabDownload slide Sequestration of cellular PIP2 reduces ATO cytotoxicity. CGL2 cells were transfected with 2 μg empty vector or with 0.5 or 2 μg PHPLCδ1 plasmid and then replated and treated with 1 or 2 μM ATO for 72 h. Cell viability was measured by the WST-8 assay. *p < 0.01 compared with cells transfected with empty vector and treated with the same ATO concentration according to Student’s t-test. FIG. 5. Open in new tabDownload slide Depletion of PIP4KIIγ reduces ATO cytotoxicity. (A) Specificity and depletion efficiency of PIPK isoform-specific siRNA. HeLa-S3 cells were transfected with control siRNA (C) or PIPK isoform-specific siRNA. After 48 h, RNA was extracted and the expression level of each PIPK isoform was evaluated by RT-PCR. The numbers below each gel are the relative levels of each PIPK in cells transfected with a specific siRNA compared with that in control siRNA-transfected cells. (B and C) Depletion of PIP4KIIγ induces cell resistance to ATO. HeLa-S3 cells were transfected with 50 nmol PIPK isoform-specific siRNA or control siRNA and then the cells were replated and treated with ATO for 72 h for the viability assay (B) and for 24 h for the colony-forming efficiency analysis (C). *p <0.01 compared with cells transfected with control siRNA and treated with the same ATO concentration according to Student’s t-test. FIG. 5. Open in new tabDownload slide Depletion of PIP4KIIγ reduces ATO cytotoxicity. (A) Specificity and depletion efficiency of PIPK isoform-specific siRNA. HeLa-S3 cells were transfected with control siRNA (C) or PIPK isoform-specific siRNA. After 48 h, RNA was extracted and the expression level of each PIPK isoform was evaluated by RT-PCR. The numbers below each gel are the relative levels of each PIPK in cells transfected with a specific siRNA compared with that in control siRNA-transfected cells. (B and C) Depletion of PIP4KIIγ induces cell resistance to ATO. HeLa-S3 cells were transfected with 50 nmol PIPK isoform-specific siRNA or control siRNA and then the cells were replated and treated with ATO for 72 h for the viability assay (B) and for 24 h for the colony-forming efficiency analysis (C). *p <0.01 compared with cells transfected with control siRNA and treated with the same ATO concentration according to Student’s t-test. Depletion of PIP4KIIγ Reduces Activation of Rho GTPases and Induction of Spindle Abnormalities by ATO and Protects Cells from ATO Cytotoxicity To understand the role of PIP4KIIγ on ATO induction of spindle abnormalities, we established a cell line, CGL2-sh2C, that was stably depleted of PIP4KIIγ by transduction of CGL2 cells with pseudotyped lentivirus-based shRNA. The proliferation rate of CGL2-sh2C cells was relatively equal to that of parental CGL2 cells (Fig. 6A). However, in untreated CGL2-sh2C cells, the percentage of mitotic cells with spindle abnormalities was slightly, but significantly, increased as compared with CGL2 cells (Fig. 6B), indicating that PIP4KIIγ might play certain roles in the formation of bipolar mitotic spindles. CGL2-sh2C cells were more resistant to ATO as compared with the parental cells (Fig. 6A), consistent with the results from siRNA (Figs. 5B and C). In addition, ATO-induced activation of Rho was negligible in CGL2-sh2C cells (Fig. 6C), indicating that ATO might activate Rho through PIP4KIIγ-mediated generation of PIP2. Furthermore, CGL2 cells treated with ATO for 24 h were arrested in mitosis (mitotic index > 55, Fig. 6D left), and up to 80% of the arrested mitotic cells contained spindle abnormalities (Fig. 6B). Thereafter, the cells underwent apoptosis, i.e., the mitotic index decreased and apoptotic cells increased with only a limited increase in G1 cells (Fig. 6D left). In CGL2-sh2C cells, in addition to a significant decrease in spindle abnormalities after ATO treatment (Fig. 6B), the number of cells arrested at mitosis was reduced, mitotic cell apoptosis was also decreased, and G1 cells gradually increased (Fig. 6D right). These results indicated that depletion of PIP4KIIγ could prevent ATO induction of Rho activation and spindle abnormalities and hence reduce mitotic cell apoptosis. FIG. 6. Open in new tabDownload slide Depletion of PIP4KIIγ reduces ATO-induced Rho GTPase activation and alleviates ATO-induced spindle defects, mitotic arrest, and mitotic cell apoptosis. (A) ATO is less cytotoxic to CGL2 cells depleted of PIP4KIIγ (CGL2-sh2C) than to parental CGL2 cells. CGL2 and CGL2-sh2C cells were treated with ATO at the indicated concentrations for 72 h and then cell viability was analyzed with the WST-8 assay. (B) ATO-induced spindle damage is decreased in CGL2-sh2C cells. CGL2 and CGL2-sh2C cells were treated with 1 μM ATO for 24 h and immunostained for mitotic spindles. The percentage of mitotic cells with spindle defects was calculated using data acquired from at least 500 mitotic cells in three independent experiments. (C) ATO-induced Rho GTPase activation is reduced in CGL2-sh2C cells. CGL2 and CGL2-sh2C cells were treated with ATO, and Rho GTPase activation was analyzed as in Figure 3. (D) ATO-induced mitotic arrest and apoptosis are decreased in CGL2-sh2C cells. CGL2 and CGL2-sh2C cells were treated with 1 μM ATO for 24–72 h. The cells were fixed and analyzed for cell cycle distribution and apoptosis. #p < 0.01 compared with untreated parental CGL2 cells. *p < 0.01 compared with parental CGL2 cells treated with the same ATO concentration according to Student’s t-test. FIG. 6. Open in new tabDownload slide Depletion of PIP4KIIγ reduces ATO-induced Rho GTPase activation and alleviates ATO-induced spindle defects, mitotic arrest, and mitotic cell apoptosis. (A) ATO is less cytotoxic to CGL2 cells depleted of PIP4KIIγ (CGL2-sh2C) than to parental CGL2 cells. CGL2 and CGL2-sh2C cells were treated with ATO at the indicated concentrations for 72 h and then cell viability was analyzed with the WST-8 assay. (B) ATO-induced spindle damage is decreased in CGL2-sh2C cells. CGL2 and CGL2-sh2C cells were treated with 1 μM ATO for 24 h and immunostained for mitotic spindles. The percentage of mitotic cells with spindle defects was calculated using data acquired from at least 500 mitotic cells in three independent experiments. (C) ATO-induced Rho GTPase activation is reduced in CGL2-sh2C cells. CGL2 and CGL2-sh2C cells were treated with ATO, and Rho GTPase activation was analyzed as in Figure 3. (D) ATO-induced mitotic arrest and apoptosis are decreased in CGL2-sh2C cells. CGL2 and CGL2-sh2C cells were treated with 1 μM ATO for 24–72 h. The cells were fixed and analyzed for cell cycle distribution and apoptosis. #p < 0.01 compared with untreated parental CGL2 cells. *p < 0.01 compared with parental CGL2 cells treated with the same ATO concentration according to Student’s t-test. DISCUSSION Evidence to date has revealed that induction of spindle defects with consequent mitotic arrest is one of the major mechanisms for arsenite-induced apoptosis in cancer cells (Huang et al., 2000; Ling et al., 2002; States et al., 2002; Yih et al., 2005). The effects of arsenite on tubulin polymerization have been investigated previously in several studies but there is disagreement among the reported results. Arsenite has been demonstrated to inhibit tubulin polymerization in vitro (Carre et al., 2002; Li and Broome, 1999). Li and Broome (1999) proposed that arsenite may bind to the vicinal cysteines (Cys12 and Cys213) near the GTP-binding site of tubulin, cross-link the tubulin molecule, and hamper GTP access to the GTP-binding pocket. Because polymerization of tubulin requires GTP binding, arsenite therefore can inhibit tubulin polymerization and mitosis. It has also been shown that arsenite can bind to the free thiol of tubulin, hence alter tubulin conformation and antagonize taxol-induced tubulin polymerization, mitotic arrest, and apoptosis (Carre et al., 2002). However, the reduction of taxol-induced mitotic arrest and apoptosis by arsenite has been demonstrated to be primarily resulting from delayed S phase progression (Duan et al., 2009). Arsenite has also been demonstrated to promote tubulin polymerization in vitro (Huang and Lee, 1998; Ling et al., 2002). It has been shown that arsenite induces thickening and increased density of microtubules in interphase cells (Ling et al., 2002) and aggregated and lengthened spindle fibers in mitotic cells (Huang and Lee, 1998; Taylor et al., 2008). Huang and Lee (1998) demonstrated that arsenite might alter the function of mitotic spindle by attenuating the dynamic instability of mitotic spindle instead of disrupting spindle formation directly. Furthermore, arsenite has been reported to induce abnormal mitotic spindles independent of tubulin polymerization (Taylor et al., 2008) and to synergistically enhance taxol-induced mitotic arrest and apoptosis (Duan et al., 2010). The reason for the discrepancy among previous studies is not clear. Nonetheless, these results imply that, in addition to microtubules, arsenite might target other cellular component(s) to induce spindle abnormalities. We previously demonstrated that centrosome amplification is induced during mitotic arrest and consequently leads to spindle multipolarity and mitotic cell apoptosis (Yih et al., 2006), indicating that ATO might alter the behavior of centrosomes and/or spindle poles when cells enter mitosis. In the present study, our time-lapse recordings demonstrated that, upon entering mitosis, the spindle poles of ATO-arrested mitotic cells were mispositioned and the mitotic spindle moved randomly around the cell and then became multipolar. This indicated that ATO might first target the machinery regulating mitotic spindle positioning and then led to the formation of abnormal mitotic spindles. Our results showed that ATO activated Rho and that inhibition of ROCKs could reduce ATO induction of spindle abnormalities and cell death. Microtubules can be regulated by Rho GTPases and their effectors (Hollenbeck, 2001). ROCKs were identified as a centrosomal component required for centrosome positioning (Chevrier et al., 2002). Activated Rho may target ROCK at aster microtubules and the cell cortex for centrosome separation and spindle orientation in prometaphase (Narumiya and Yasuda, 2006). In addition, constitutive activation of the receptors known to mediate Rho signaling promotes centrosome amplification and chromosome instability in a Rho/ROCK-dependent manner (Fukasawa, 2011). Treatment of cells with Y-27632 interferes with centrosome separation after nuclear envelope breakdown and results in aberrant spindle phenotypes (Rosenblatt et al., 2004). These results indicate the involvement of Rho signaling in the regulation of centrosome separation and spindle positioning and imply that alteration of Rho signaling would lead to spindle abnormalities. Thus, ATO may induce defects in mitotic spindles by activating a Rho/ROCK pathway. Previous studies have demonstrated that PIPKs can regulate the activation of Rho GTPases. For example, PIP2 has been shown to stimulate GDP dissociation from Rho A and consequently GTP/GDP exchange, hence activating Rho GTPases (Zheng et al., 1996). PIP5KIα has been demonstrated to modulate cell migration or morphology by regulating Rac1 targeting and activation (Chao et al., 2010). Rho GTPases can also modulate cytoskeleton-dependent events by recruiting and activating PIPKs. Generation of PIP2 thus potentiates the effects of Rho GTPases on cytoskeletal remodeling (Paris et al., 1997; Randazzo, 1997). Thus, Rho GTPases and PIPKs are spatiotemporally cross-regulated. Our results showed that depletion of PIP4KIIγ not only prevented ATO-induced Rho activation but also reduced the induction of spindle abnormalities, mitotic arrest, and cell death. Thus, ATO might induce spindle abnormalities through PIP4KIIγ-mediated activation of Rho/ROCK pathway. PIP2 is synthesized from phosphatidylinositol-4-phosphate by PIP5KI and from phosphatidylinositol-5-phosphate by PIP4KII. PIP5KIs modulate many cellular processes, including motility, focal adhesion assembly/disassembly, and vesicular trafficking (van den Bout and Divecha, 2009). In contrast to PIP5KIs, little is known about the physiological functions of PIP4KIIs. PIP4KIIs are functionally nonredundant with PIP5KIs (Kunz et al., 2000) and therefore may modulate a set of cellular processes distinct from those modulated by PIP5KIs. PIP2 localizes primarily to the plasma membrane but is also found on secretory vesicles, lysosomes, the endoplasmic reticulum, Golgi, the cytokinesis cleavage furrow, and in the nucleus. PIP2 can either bind to intracellular proteins and directly modulate their subcellular localization and activity or act as a precursor for the generation of other second messengers, hence directly or indirectly regulating a plethora of cellular processes (Doughman et al., 2003; Kwiatkowska, 2010). Because the isoforms of PIP5KI (PIP5KIα, PIP5KIβ, and PIP5KIγ) have specific and distinct subcellular localizations, it has been postulated that local increases in PIP2 concentration by specific PIPK isoforms mediate its diverse signaling functions (Doughman et al., 2003; van den Bout and Divecha, 2009). Our results show that, in cells depleted of PIP4KIIγ, the frequency of mitotic cells with abnormal spindles was significantly increased as compared with that in the non-depleted control cells therefore imply a specific role of PIP4KIIγ at the spindle apparatus. Furthermore, we also demonstrated that sequestration of cellular PIP2 could reduce ATO-induced cell death and that depletion of PIP4KIIγ could prevent ATO induction of spindle abnormalities and mitotic cell apoptosis. It has been demonstrated that modulation of the activity or localization of PPK-1, a PIP2 synthesis enzyme of Caenorhabditis elegans, controls spindle movement by regulating the pulling forces on astral microtubules at the one-cell embryo stage (Panbianco et al., 2008), indicating that PIP2 may regulate the positioning and assembly of mitotic spindles. PLC-γ1, a PIP2 hydrolyzing enzyme, has been reported to interact with β-tubulin and subsequently increase its prevalence on spindle poles during mitosis (Chang et al., 2005), also indicating a role for PIP2 in mitotic spindle positioning and assembly. These results suggest that precise regulation of PIP2 level is required for accurate positioning and assembly of bipolar mitotic spindles. Therefore, our results imply that PIP4KIIγ might mediate PIP2 generation required for positioning and assembly of bipolar spindles and alteration of PIP4KIIγ function by ATO might thus lead to spindle abnormalities. FUNDING Academia Sinica; National Science Council (NSC), Taiwan (NSC99-2320-B-001-008-MY3). 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Chem. 1996 271 23815 23819 Google Scholar Crossref Search ADS PubMed WorldCat © The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected] http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Toxicological Sciences Oxford University Press

Arsenic Trioxide Induces Abnormal Mitotic Spindles Through a PIP4KIIγ/Rho Pathway

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Oxford University Press
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© The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected]
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Molecular Toxicology
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1096-6080
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DOI
10.1093/toxsci/kfs129
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22496355
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Abstract

Abstract Arsenite-induced spindle abnormalities result in mitotic cell apoptosis in several cancer cell lines, but how arsenite induces these effects is not known. Evidence to date has revealed that arsenite activates Rho guanosine triphosphatases (GTPases). Because Rho GTPases regulate spindle orientation, chromosome congression, and cytokinesis, we therefore examined the involvement of Rho GTPases and their modulators in arsenite-induced mitotic abnormalities. We demonstrated that arsenic trioxide (ATO) disrupted the positioning of bipolar mitotic spindles and induced centrosome and spindle abnormalities. ATO increased the level of the active guanosine triphosphate-bound form of Rho. Inhibition of Rho-associated protein kinases (ROCKs) by Y-27632 ameliorated ATO-induced spindle defects, mitotic arrest, and cell death. These results indicate that ATO may induce spindle abnormalities and mitotic cell death through a Rho/ROCK pathway. In addition, screening of a human kinase and phosphatase shRNA library to select genes that mediate ATO induction of spindle abnormalities resulted in the identification of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), a phosphatidylinositol 4,5-biphosphate (PIP2) synthesis enzyme that belongs to the phosphatidylinositol phosphate kinase (PIPK) family. Sequestration of PIP2 by ectopic overexpression of the pleckstrin homology domain of phospholipase C-δ1 protected cells from ATO-induced cell death. Furthermore, depletion of PIP4KIIγ, but not other isoforms of the PIPK family, not only reduced Rho GTPase activation in ATO-treated cells but also alleviated ATO-induced spindle defects, mitotic arrest, and mitotic cell apoptosis. Thus, our results imply that ATO induces abnormalities in mitotic spindles through a PIP4KIIγ/Rho pathway, leading to apoptosis of mitotic cells. arsenic trioxide, mitotic spindle, apoptosis, PIP4KIIγ, Rho Arsenic trioxide (ATO) has potent antineoplastic effects in vitro and in vivo. ATO cures acute promyelocytic leukemia by initiating degradation of the oncogenic fusion protein PML/RARA (Jeanne et al., 2010). ATO also induces apoptosis and suppresses the growth of PML/RARA-negative malignant cells (Gazitt and Akay, 2005; Miller et al., 2002). Despite extensive research, the precise mechanisms of action of ATO in malignant cells are not well understood. Trivalent arsenic compounds induce centrosome amplification and disrupt mitotic spindles, inducing mitotic arrest and consequently triggering mitotic arrest–mediated apoptosis in a variety of cancer cells (Cai et al., 2003; Huang et al., 2000; Ling et al., 2002; McCollum et al., 2005; McNeely et al., 2008; States et al., 2002; Taylor et al., 2008; Yih et al., 2005, 2006). These reports reveal a tight link between arsenite-induced apoptosis and aberrant mitotic spindles in cancer cells. However, how arsenite induces spindle abnormalities remains elusive. Mitotic spindles, the machines driving chromosome segregation during mitosis, adopt a bipolar configuration to divide the chromosomes into two equivalent sets through precise modulation of microtubule dynamics (Tanenbaum and Medema, 2010). The Rho guanosine triphosphatases (GTPases), including Rho, Rac, and Cdc42, are key regulators of cytoskeletal dynamics and affect many cellular processes, including cell polarity, migration, vesicle trafficking, and cell cycle progression and cytokinesis. The Rho GTPases can control the reorganization of actin and microtubule cytoskeletal structures during mitosis (Hollenbeck, 2001). They have been demonstrated to modulate centrosome positioning and spindle orientation (Bakal et al., 2005; Rosenblatt et al., 2004), proper attachment between kinetochores and microtubules (Yasuda et al., 2004), and cytokinesis (Piekny et al., 2005). In addition, Rho-associated protein kinases (ROCKs), the downstream effectors of Rho, associate with nucleophosmin and may thus modulate the number of centrosomes (Ferretti et al., 2010; Ma et al., 2006). P21-activated kinases, the effectors of Cdc42 and Rac1, may control centrosome maturation and spindle formation through phosphorylation of polo-like kinase 1 (Maroto et al., 2008). LIM kinases, when activated by the ROCKs or P21-activated kinases, colocalize with γ-tubulin at centrosomes early during mitosis and may play a role in mitotic spindle assembly (Chakrabarti et al., 2007). These studies demonstrate the critical roles of the Rho GTPases and their downstream effectors in regulating the mitotic machinery and imply that perturbation of Rho GTPase signaling might disrupt the assembly of mitotic spindles and alter mitotic progression. The Rho GTPases and their effectors modulate cytoskeleton-dependent cellular events by regulating the targeting and activation of phosphatidylinositol phosphate kinases (PIPKs) and generation of phosphatidylinositol-4,5-bisphosphate (PIP2) (Doughman et al., 2003; van den Bout and Divecha, 2009). The activation of the Rho GTPases and their targeting to specific cell compartments are also regulated by PIPKs and PIP2 (Chao et al., 2010; Zheng et al., 1996). The spatiotemporal cross-regulation of the Rho GTPases and PIPKs thus may be critical for the positioning and assembly of mitotic spindles. ATO reportedly activates the Rho GTPases in diverse cell systems. Rac is activated by ATO in leukemia cells and might negatively regulate ATO-induced apoptosis (Verma et al., 2002). Rho is activated by ATO in chronic myelogenous leukemia cells and mediates the activation of c-Jun N-terminal kinase, which leads to apoptosis (Potin et al., 2007). Rho is also activated by ATO in macrophages and mediates the activation of reduced NADPH oxidase, the production of reactive oxygen species, and secretion of cytokines (Lemarie et al., 2008). Cdc42 is activated by ATO in endothelial cells and mediates the activation of NADPH oxidase, production of reactive oxygen species, and cell migration (Qian et al., 2005). In addition, inhibition of Rac1 blocks ATO-stimulated differentiation and dysfunction of liver sinusoidal endothelial cells (Straub et al., 2008). These results indicate that the Rho GTPases are critical mediators that control diverse cellular responses to ATO. Because the Rho GTPases play critical roles in regulating mitotic spindle assembly and can be activated by ATO, we therefore investigated whether these GTPases and their possible modulators are involved in the induction of ATO-induced spindle abnormalities. MATERIALS AND METHODS Cell culture and reagents. HeLa-S3 cells were obtained from the American Type Culture Collection (Manassas, VA). CGL2 cells (a HeLa cell/normal human fibroblast hybrid) (Stanbridge et al., 1981) were kindly provided by Dr E. J. Stanbridge (University of California-Irvine). Cells were routinely maintained in Dulbecco's Modified Eagle's Medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen), 0.37% sodium bicarbonate, 100 U/ml of penicillin, and 100 μg/ml of streptomycin at 37°C in an humidified incubator in air and 10% CO2 and were passaged twice per week. ATO (Sigma-Aldrich, St Louis, MO) was freshly dissolved in 0.1 N NaOH to make a 10 mM stock solution before use. Y-27632 (Calbiochem, Merck KGaA, Darmstadt, Germany) was dissolved in dimethyl sulfoxide to make a 10 mM stock and stored in aliquots at –20°C. Immunofluorescence staining of mitotic spindles and spindle positioning assay. HeLa-S3 or CGL2 cells (1 × 105) seeded on glass coverslips were incubated for 20 h at 37°C with or without drugs, washed twice with PBS, and fixed in situ with 90% methanol at –20°C for 10 min. The cells were then immunostained for mitotic spindles with an antibody against human α-tubulin (Sigma) and for centrosomes with an antibody against human γ-tubulin (Sigma) as described (Yih et al., 2006). Nuclei were simultaneously counterstained with 0.1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI; Sigma). After thorough rinsing with PBS containing 0.2% (vol/vol) Tween 20, the cells were mounted using VECTASHIELD (Vector Laboratories, Inc., Burlingame, CA) and examined under a fluorescence microscope (Axioplan 2 Imaging MOT; Carl Zeiss MicroImaging, Oberkochen, Germany). The positions of mitotic spindles were evaluated by measuring the distance between the spindle center and the cell center. Cells were fixed, immunostained for mitotic spindles, and photographed (Axioplan 2). The distance was measured by MetaMorph COMPLETE software (Molecular Devices, Sunnyvale, CA). A total of 100 cells from three independent experiments were examined, and the distances in ATO-arrested mitotic cells were compared with those measured in untreated mitotic cells. To determine the percentage of mitotic cells with abnormal spindles, mitotic cells that contained distorted or disorganized mitotic spindles, multipolar spindles, or mitotic spindles with aggregated spindle fibers or with elongated polar distance were classified as mitotic cells with abnormal mitotic spindles (Wu et al., 2009). At least 500 mitotic cells from three independent experiments were analyzed. Time-lapse fluorescence analysis. HeLa-S3 cells were transfected with pEYFP-tubulin Vector (Clontech, Mountain View, CA). Cells stably expressing enhanced YFP (EYFP)-tubulin fusion protein were established and plated on a 60-mm Petri dish 20 h before ATO treatment. Cells were treated with 3 μM ATO for up to 40 h, during which time the dish was placed on the objective stage set up in a humidified chamber with 10% CO2 in air at 37°C. Fluorescence images were collected every 15 min using an inverted fluorescence microscope (Axiovert 200M; Carl Zeiss) equipped with an oil immersion objective lens (×63, plan Apo NA 1.4) and a MetaMorph COMPLETE-controlled CCD camera. The acquired images were processed by MetaMorph COMPLETE software. The time-lapse recordings were repeated for two and four times for untreated and ATO-treated cells, respectively. Rho-guanosine triphosphate pull-down assay. The level of guanosine triphosphate (GTP)-bound Rho, Rac, or Cdc42 was measured using the Rho or Rac1/Cdc42 activation assay kits from Millipore (Temecula, CA). After treatments, cells were rapidly lysed at 4°C in buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.5), 250 mM NaCl, 1% NP-40, 10 mM MgCl2, 1 mM EDTA, 2% glycerol, 10 μg/ml leupeptin, 25 mM sodium fluoride, and 1 mM sodium orthovanadate. After centrifugation (14,000 × g for 5 min at 4°C), the clear lysate was incubated with the affinity beads conjugated with Rhotekin Rho binding domain or P21-activated kinase-1 p21-binding domain to specifically pull-down the GTP-bound Rho or Rac/Cdc42, respectively. After thorough washing, Rho, Rac, or Cdc42 levels were assessed by immunoblotting with the corresponding antibody (Millipore). Briefly, each of the bead/protein complexes in Laemmli buffer containing 0.1 M dithiothreitol was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (12% acrylamide) and transferred to an Immobilon polyvinylidene fluoride membrane (Millipore). After blocking in PBS containing 5% (wt/vol) skim milk powder and 0.2% Tween 20 for 1 h, each membrane was incubated with the corresponding antibody overnight at 4°C. Antigen-antibody complexes were visualized using horseradish peroxidase-conjugated secondary antibodies followed by ECL (SuperSignal West Pico; Pierce, Rockford, IL). Lentiviral transduction and ATO selection. A pseudotyped lentivirus-based human kinase and phosphatase (HKP) shRNA library that contains 6300 shRNA clones (corresponding to 1260 genes) from the National RNAi Core Facility Platform (Institute of Molecular Biology/Genomic Research Center, Academia Sinica, Taipei, Taiwan) was used to select for genes related to ATO toxicity. One day before transduction, CGL2 cells were seeded at 1 × 106 in a 100-mm Petri dish. Cells were transduced with HKP pooled lentiviral supernatants (multiplicity of infection = 0.3) in growth medium supplemented with 10 μg/ml polybrene. At 24 h post-transduction, 2 μg/ml puromycin was added to culture medium to eliminate nontransduced cells. The transduced cells were then treated with 0.5 μM ATO in the presence of 1 μg/ml verapamil for 2 weeks. Cells surviving ATO treatment were collected and their genomic DNA isolated. The shRNA was recovered by PCR amplification, cloned, and then sequenced. Oligonucleotides encoded in the lentiviral vector pLKO.1 were used as PCR primers for the identification and sequencing of inserts: 5′-tacaaaatacgtgacgtag-3′ (forward primer) and 5′-ctgttgctattatgtctac-3′ (reverse primer). Sequestration of cellular PIP2. Cellular PIP2 levels can be reduced by overexpressing the pleckstrin homology domain of phospholipase C-δ1 (PHPLCδ1) in cells to specifically bind and sequester PIP2 (Field et al., 2005). To generate the plasmid encoding PHPLCδ1, the PHPLCδ1 complementary DNA (cDNA) was amplified by PCR using primers 5′-GAAGATCTGCGCTGCTGAAGGGCAGC-3′ and 5′-CCCAAGCTTAGTGGATGATCTTGTGCAGCCC-3′ according to the sequence from GenBank (accession no. U09117), digested with BglII and HindIII, and subcloned into the BglII and HindIII sites of the pmCherry-C1 vector (Clontech). CGL2 cells (1 × 105 per well in a 24-well plate) were transfected with 2 μg/ml empty vector or 0.5 or 2 μg/ml PHPLCδ1 with FuGENE HD (Roche, Mannheim, Germany). After 5 h, cells were replated and assessed for cytotoxicity. Depletion of cellular PIPKs. Depletion of specific PIPK isoforms was achieved by transfecting cells with small interference RNAs (siRNAs) or transducing cells with VSV-G-pseudotyped lentivirus-based shRNA. siRNAs specific to each PIPK isoform were obtained from Thermo Scientific Dharmacon (On-Target plus SMART pool; Lafayette, CO). HeLa-S3 cells were plated at a density of 0.5–1 × 105 per 35-mm dish one day before transfection, then were transfected with each siRNA at a final concentration of 50 nmol using Oligofectamine (Invitrogen). At 24-h post-transfection, the medium was replaced with fresh medium, and the cells were cultured for another 24 or 48 h. A double-stranded RNA targeting luciferase (5′-cguacgcggaauacuucgadTdT-3′) was used as the control. For stable depletion of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), PIP4KIIγ-specific shRNA (TRCN37719) was obtained from the National RNAi Core Facility Platform. CGL2 cells were transduced with shRNA-containing virus (multiplicity of infection = 3) in growth medium supplemented with 10 μg/ml polybrene. At 24-h post-transduction, 2 μg/ml puromycin was added to culture medium to select for stable clones (CGL2-sh2C). The expression level of each isoform was examined by reverse transcription (RT)-PCR or immunoblotting. Reverse transcription-PCR. Cellular RNA was extracted with Trizol (Invitrogen) reagent, and 5 μg was reverse transcribed with Superscript III (Invitrogen) and oligo-dT (Promega, Madison, WI). PIPK isoforms were analyzed by PCR with primers flanking consecutive exon sequences. For each product, a linear range of cDNA input was determined empirically via a twofold dilution series in 26–28 cycle reactions in the presence of Fermentas DreamTaq DNA polymerase (Thermo Scientific). PCR products were run on 1% Tris/borate/EDTA-agarose gels (Ogden and Adams, 1987), stained with SYBR Gold (Invitrogen), and quantified with GeneTools (Syngene, Cambridge, U.K.). β-Actin was used as an internal control. The primer sets were as follows: phosphatidylinositol-4-phosphate 5-kinase type-1 (PIP5KI) α, 5′-ACAACGAGAGCCCTTAAGCA-3′ and 5′-GAACCGTTCAGCGTAGAAGC-3′; PIP5KIβ, 5′-AAGGATGAGAAGCGGGATTT-3′ and 5′-TGGAAGGTAACCCTTTGCTG-3′; PIP5KIγ, 5′-AGAAGCCACTACAGCCTCCA-3′ and 5′-TCTTTGGGGACCACAATCTC-3; PIP4KIIα, 5′-GTCTGCTGGTGGGAATTCAT-3′ and 5′-TCCTAGGCGAGTTTTCATGG-3′; PIP4KIIβ, 5′-GACGTTGAGTTCTTGGCACA-3′ and 5′-CAAAGAACCGAGGAAAGCTG-3′; PIP4KIIγ, 5′-GTGCAGCTGAAGATCATGGA-3′ and 5′-TGAGGCCCATGAAGTAGACC-3′; and β-actin, 5′-GCACTCTTCCAGCCTTCC-3′ and 5′-GCGCTCAGGAGGAGCAAT-3′. Cytotoxicity assay. Cells were plated at a density of 4 × 105 per well in a six-well plate or 1 × 105 per well in a 24-well plate one day before siRNA or plasmid transfection. At 5 h post-transfection, the cells were seeded in a 24-well plate (3 × 104 per well); 24 h later, they were treated with drugs for 24–72 h. Cytotoxicity was then determined by assaying viable cell numbers using methylthiazole tetrazolium (WST-8) (Cell Count Kit 8; Dojindo Molecular Technologies, Inc., Gaithersburg, MD) or by colony-forming efficiency assay as described (Kakadiya et al., 2011; Yih et al., 2005). Analysis of cell cycle distribution and apoptosis. Cell cycle progression and apoptosis were monitored using flow cytometry. DNA was stained with propidium iodide, and mitotic cells were quantified by measuring the expression of the mitosis-specific marker phospho-histone H3; further, apoptotic cells were identified by measuring the level of cleaved poly(adenosine diphosphate-ribose) polymerase (PARP) as described (Wu et al., 2010; Yih et al., 2006). In brief, the cells were fixed with ice-cold 70% ethanol for 16 h, then simultaneously immunostained with mouse antihuman phospho-histone H3 (H3 phosphorylated on serine 10; Cell Signaling Technology, Beverly, MA) and rabbit antibody against human cleaved PARP (Cell Signaling Technology), followed by incubation with fluorescein isothiocyanate-conjugated goat anti-mouse IgG and allophycocyanin-conjugated goat anti-rabbit IgG (Invitrogen). Cellular DNA was counterstained with propidium iodide. Levels of phospho-histone H3, cleaved PARP, and propidium iodide of individual cells were analyzed using a flow cytometer (FACSCanto II; BD Biosciences, San Jose, CA), and the cell cycle distribution and the percentage of cells with phospho-histone H3 and cleaved PARP were determined using a computer program provided by BD Biosciences. RESULTS ATO Disrupts the Positioning of Mitotic Spindles Consistent with the results of previous studies, treatment of HeLa-S3 cells with 3 μM ATO induced distorted and disorganized mitotic spindles (Fig. 1B) or multipolar spindles (Fig. 1C) in arrested mitotic cells. The effect of ATO on spindle positioning, as visualized by the expression of EYFP-tubulin in HeLa-S3 cells, was examined using time-lapse fluorescence microscopy. In untreated mitotic cells, the spindle was stably positioned near the geometric center of the cell; the two spindle poles could be clearly visualized in the same focal plane during mitosis, indicating that the spindle axis was horizontal and parallel to the basement plane (Fig. 1D). In ATO-arrested mitotic cells, however, the spindle rotated and moved randomly in the cytoplasm; the spindle was positioned away from the center of the cell and was tilted with one pole frequently out of the focal plane (Fig. 1E). Formation of an extra pole or aster-like structure was also observed during the prolonged arrest at mitosis. Subsequently, these aberrant mitotic cells underwent apoptosis as revealed by the formation of membrane blebs. FIG. 1. Open in new tabDownload slide ATO induces spindle abnormalities in HeLa-S3 cells. (A–C) Representative immunofluorescence images of normal (A) and ATO-arrested mitotic cells (B and C). (A) Normal bipolar spindle. (B) Abnormal spindle with distorted and aggregated microtubules. (C) Multipolar spindle. HeLa-S3 cells were left untreated or treated with 3 μM ATO for 20 h and then fixed and immunostained with anti-α-tubulin to detect mitotic spindles (red) and with anti-γ-tubulin to detect centrosomes (green) and counterstained with DAPI to detect chromosomes (blue). The three fluorescence images were merged. Scale bar, 10 μm. (D and E) Representative live-cell still images of untreated (D) or ATO-treated (E) EYFP-tubulin HeLa cells. Cells stably expressing EYFP-tubulin (green) were left untreated or treated with 3 μM ATO. Hoechst 33258 (0.3 μM) was included in the culture medium of untreated cells. Fluorescence images were collected every 15 min and processed as described in the Materials and Methods section. Numbers in (D) indicate the time (hour:min) after the first frame and in (E) the time after ATO addition. #, *, and + symbols denote three different abnormal mitotic cells at different times in the ATO-treated culture. FIG. 1. Open in new tabDownload slide ATO induces spindle abnormalities in HeLa-S3 cells. (A–C) Representative immunofluorescence images of normal (A) and ATO-arrested mitotic cells (B and C). (A) Normal bipolar spindle. (B) Abnormal spindle with distorted and aggregated microtubules. (C) Multipolar spindle. HeLa-S3 cells were left untreated or treated with 3 μM ATO for 20 h and then fixed and immunostained with anti-α-tubulin to detect mitotic spindles (red) and with anti-γ-tubulin to detect centrosomes (green) and counterstained with DAPI to detect chromosomes (blue). The three fluorescence images were merged. Scale bar, 10 μm. (D and E) Representative live-cell still images of untreated (D) or ATO-treated (E) EYFP-tubulin HeLa cells. Cells stably expressing EYFP-tubulin (green) were left untreated or treated with 3 μM ATO. Hoechst 33258 (0.3 μM) was included in the culture medium of untreated cells. Fluorescence images were collected every 15 min and processed as described in the Materials and Methods section. Numbers in (D) indicate the time (hour:min) after the first frame and in (E) the time after ATO addition. #, *, and + symbols denote three different abnormal mitotic cells at different times in the ATO-treated culture. The effect of ATO on spindle positioning was also assessed in CGL2 cells by measuring the distance between the midpoint of the spindle and the cell center in the horizontal view. In untreated cultures, mitotic spindles were positioned near the center of the cells (Fig. 2A) with the distance ranging from 0.45 to 1.66 μm (Fig. 2D). However, the spindles in ATO-arrested mitotic cells were not at the cell center (Figs. 2B and C); the distance ranged from 0.46 to 4.86 μm (Fig. 2D), which was significantly longer than that in the untreated mitotic cells. This indicated that mitotic spindle was mispositioned in ATO-arrested cells. FIG. 2. Open in new tabDownload slide ATO disrupts spindle positioning. (A–C) Representative immunofluorescence images of untreated (A) and ATO-arrested mitotic cells (B and C). CGL2 cells were treated with 1 μM ATO for 20 h and immunostained for mitotic spindles as in Figure 1. The red line indicates the spindle pole-to-pole axis, and the yellow dot indicates the geometric center of the cell. Each yellow dashed line indicates a cell border. Positioning of the mitotic spindle was evaluated by measuring the distance between the cell center and the midpoint of the spindle axis. (D) Scatter plot of the distance from the cell center to midpoint of the spindle axis. Red dots indicate the average values and error bars indicate the standard deviation of 100 mitotic cells from three independent experiments. *p < 0.01 compared with untreated cells according to Student’s t-test. FIG. 2. Open in new tabDownload slide ATO disrupts spindle positioning. (A–C) Representative immunofluorescence images of untreated (A) and ATO-arrested mitotic cells (B and C). CGL2 cells were treated with 1 μM ATO for 20 h and immunostained for mitotic spindles as in Figure 1. The red line indicates the spindle pole-to-pole axis, and the yellow dot indicates the geometric center of the cell. Each yellow dashed line indicates a cell border. Positioning of the mitotic spindle was evaluated by measuring the distance between the cell center and the midpoint of the spindle axis. (D) Scatter plot of the distance from the cell center to midpoint of the spindle axis. Red dots indicate the average values and error bars indicate the standard deviation of 100 mitotic cells from three independent experiments. *p < 0.01 compared with untreated cells according to Student’s t-test. Inhibition of ROCKs Ameliorates ATO-induced Spindle Abnormalities and Cell Death To understand whether Rho GTPases were involved in ATO induction of spindle abnormalities, the effect of ATO on activation of the Rho GTPases was examined. ATO increased the level of the active GTP-bound form of Rho but not that of Rac or Cdc42 (Fig. 3A). ROCKs are a major target of Rho-GTP and a key-signaling molecule involved in cytoskeleton regulation (Amano et al., 2010). The effect of Y-27632, a specific inhibitor of ROCKs, on ATO-induced mitotic cells was thus examined. Y-27632 alone (5 or 10 μM) did not significantly affect mitotic spindles or cell viability in CGL2 cells (Figs. 3B–E). But treatment of the cells with 20 μM Y-27632 resulted in the formation of disorganized mitotic spindles and induced cell death (data not shown), consistent with the results of a previous report (Rosenblatt et al., 2004). In ATO-treated cultures, up to 70% of the ATO-arrested mitotic cells contained disorganized or multipolar spindles (Fig. 3B). In addition, the mitotic index was significantly increased and the number of cells in cytokinesis decreased compared with untreated cultures (Figs. 3C and D), indicating that these ATO-induced aberrant cells were stuck in mitosis. Cotreatment of cells with ATO and Y-27632 considerably reduced the induction of spindle abnormalities (Fig. 3B), decreased the mitotic index (Fig. 3C), and increased the number of cells in cytokinesis (Fig. 3D), indicating that inhibition of ROCKs might prevent the formation of abnormal mitotic spindles and thereby release the arrest of mitotic cells in ATO-treated cultures. In addition, cotreatment with Y-27632 enhanced the viability of ATO-treated cells (Fig. 3E). These results indicated that ATO might induce defects in mitotic spindles and cause cell death by activating a Rho/ROCK pathway. FIG. 3. Open in new tabDownload slide Y-27632 ameliorates ATO-induced spindle abnormalities and cell death. (A) ATO activates Rho GTPases. CGL2 cells were untreated or treated with 3 μM ATO for 3 h and then the GTP-bound fraction of Rho, Rac, or Cdc42 (the active form) was pulled down as described in the Materials and Methods section. The pulled-down complexes and whole-cell lysates were analyzed by immunoblotting to detect the level of Rho, Rac, or Cdc42. (B–D) Y-27632 reduces ATO-induced spindle abnormalities, mitotic arrest, and blockade of cytokinesis. CGL2 cells were treated with 1 μM ATO plus 5 or 10 μM Y-27632 for 20 h and immunostained for mitotic spindles as described in Figure 1 (B and D) or analyzed for cell cycle distribution (C). The percentage of mitotic cells with spindle defects was calculated using data acquired from at least 500 mitotic cells in three independent experiments. The percentage of cells in cytokinesis was calculated using data acquired from at least 1000 cells in three independent experiments. (E) Y-27632 reduces ATO-induced cell death. CGL2 cells were treated with 1 μM ATO plus 5 or 10 μM Y-27632 for 72 h and then cell viability was determined by the WST-8 assay. Y-27632 alone showed no significant cytotoxicity. #p < 0.01 compared with untreated cells. *p < 0.05 and **p < 0.01 compared with ATO treatment alone according to Student’s t-test. FIG. 3. Open in new tabDownload slide Y-27632 ameliorates ATO-induced spindle abnormalities and cell death. (A) ATO activates Rho GTPases. CGL2 cells were untreated or treated with 3 μM ATO for 3 h and then the GTP-bound fraction of Rho, Rac, or Cdc42 (the active form) was pulled down as described in the Materials and Methods section. The pulled-down complexes and whole-cell lysates were analyzed by immunoblotting to detect the level of Rho, Rac, or Cdc42. (B–D) Y-27632 reduces ATO-induced spindle abnormalities, mitotic arrest, and blockade of cytokinesis. CGL2 cells were treated with 1 μM ATO plus 5 or 10 μM Y-27632 for 20 h and immunostained for mitotic spindles as described in Figure 1 (B and D) or analyzed for cell cycle distribution (C). The percentage of mitotic cells with spindle defects was calculated using data acquired from at least 500 mitotic cells in three independent experiments. The percentage of cells in cytokinesis was calculated using data acquired from at least 1000 cells in three independent experiments. (E) Y-27632 reduces ATO-induced cell death. CGL2 cells were treated with 1 μM ATO plus 5 or 10 μM Y-27632 for 72 h and then cell viability was determined by the WST-8 assay. Y-27632 alone showed no significant cytotoxicity. #p < 0.01 compared with untreated cells. *p < 0.05 and **p < 0.01 compared with ATO treatment alone according to Student’s t-test. PIP4KIIγ Is Involved in ATO Cytotoxicity Because ATO induces CGL2 cell death mainly via induction of abnormal mitotic spindles and mitotic cell apoptosis (Yih et al., 2005), a pseudotyped lentivirus-based HKP shRNA library was screened for genes that are related to ATO induction of spindle abnormalities in CGL2 cells. After transduction and ATO selection, the shRNA was recovered from cells surviving ATO treatment by genomic PCR using the primers for the lentiviral vector pLKO.1, i.e., only the inserts containing the vector sequences at both ends could be detected. In total, 387 clones were verified by sequencing; among them, 45 contained shRNA sequences that corresponded to the same gene, namely PIP4K2C, which encodes PIP4KIIγ, a PIP2 synthesis enzyme belonging to the PIPK family. This result indicated that depletion of PIP4KIIγ induced cell resistance to ATO and implied that PIP4KIIγ and PIP2 might play important roles in ATO induction of spindle abnormalities. To confirm the role of PIP2 in ATO-treated cells, CGL2 cells were transfected with an expression vector encoding PHPLCδ1, which can specifically bind and sequester cellular PIP2 (Field et al., 2005). Expression of PHPLCδ1 significantly enhanced the viability of ATO-treated cells (Fig. 4), indicating that ATO might induce cytotoxicity through generation of PIP2. PIP2 is synthesized via the action of two distinct but related PIPKs, PIP5KI and PIP4KII. Three isoforms, α, β, and γ, have been identified in each subfamily (Doughman et al., 2003). We thus examined whether depletion of individual isoforms could affect the cytotoxicity of ATO. As HeLa-S3 cells exhibit high transfection efficiency and are also susceptible to ATO-induced spindle defects and mitotic apoptosis (Huang et al., 2000), HeLa-S3 cells were transfected with isoform-specific siRNA. The specificity and efficiency of each siRNA were confirmed by RT-PCR and at least 80% downregulation of each isoform was achieved (Fig. 5A). Depletion of PIP4KIIγ, but not other isoforms, significantly reduced ATO cytotoxicity as determined by the cell viability assay (Fig. 5B) or colony-forming efficiency assay (Fig. 5C). These results confirmed that PIP4KIIγ is involved in ATO cytotoxicity. FIG. 4. Open in new tabDownload slide Sequestration of cellular PIP2 reduces ATO cytotoxicity. CGL2 cells were transfected with 2 μg empty vector or with 0.5 or 2 μg PHPLCδ1 plasmid and then replated and treated with 1 or 2 μM ATO for 72 h. Cell viability was measured by the WST-8 assay. *p < 0.01 compared with cells transfected with empty vector and treated with the same ATO concentration according to Student’s t-test. FIG. 4. Open in new tabDownload slide Sequestration of cellular PIP2 reduces ATO cytotoxicity. CGL2 cells were transfected with 2 μg empty vector or with 0.5 or 2 μg PHPLCδ1 plasmid and then replated and treated with 1 or 2 μM ATO for 72 h. Cell viability was measured by the WST-8 assay. *p < 0.01 compared with cells transfected with empty vector and treated with the same ATO concentration according to Student’s t-test. FIG. 5. Open in new tabDownload slide Depletion of PIP4KIIγ reduces ATO cytotoxicity. (A) Specificity and depletion efficiency of PIPK isoform-specific siRNA. HeLa-S3 cells were transfected with control siRNA (C) or PIPK isoform-specific siRNA. After 48 h, RNA was extracted and the expression level of each PIPK isoform was evaluated by RT-PCR. The numbers below each gel are the relative levels of each PIPK in cells transfected with a specific siRNA compared with that in control siRNA-transfected cells. (B and C) Depletion of PIP4KIIγ induces cell resistance to ATO. HeLa-S3 cells were transfected with 50 nmol PIPK isoform-specific siRNA or control siRNA and then the cells were replated and treated with ATO for 72 h for the viability assay (B) and for 24 h for the colony-forming efficiency analysis (C). *p <0.01 compared with cells transfected with control siRNA and treated with the same ATO concentration according to Student’s t-test. FIG. 5. Open in new tabDownload slide Depletion of PIP4KIIγ reduces ATO cytotoxicity. (A) Specificity and depletion efficiency of PIPK isoform-specific siRNA. HeLa-S3 cells were transfected with control siRNA (C) or PIPK isoform-specific siRNA. After 48 h, RNA was extracted and the expression level of each PIPK isoform was evaluated by RT-PCR. The numbers below each gel are the relative levels of each PIPK in cells transfected with a specific siRNA compared with that in control siRNA-transfected cells. (B and C) Depletion of PIP4KIIγ induces cell resistance to ATO. HeLa-S3 cells were transfected with 50 nmol PIPK isoform-specific siRNA or control siRNA and then the cells were replated and treated with ATO for 72 h for the viability assay (B) and for 24 h for the colony-forming efficiency analysis (C). *p <0.01 compared with cells transfected with control siRNA and treated with the same ATO concentration according to Student’s t-test. Depletion of PIP4KIIγ Reduces Activation of Rho GTPases and Induction of Spindle Abnormalities by ATO and Protects Cells from ATO Cytotoxicity To understand the role of PIP4KIIγ on ATO induction of spindle abnormalities, we established a cell line, CGL2-sh2C, that was stably depleted of PIP4KIIγ by transduction of CGL2 cells with pseudotyped lentivirus-based shRNA. The proliferation rate of CGL2-sh2C cells was relatively equal to that of parental CGL2 cells (Fig. 6A). However, in untreated CGL2-sh2C cells, the percentage of mitotic cells with spindle abnormalities was slightly, but significantly, increased as compared with CGL2 cells (Fig. 6B), indicating that PIP4KIIγ might play certain roles in the formation of bipolar mitotic spindles. CGL2-sh2C cells were more resistant to ATO as compared with the parental cells (Fig. 6A), consistent with the results from siRNA (Figs. 5B and C). In addition, ATO-induced activation of Rho was negligible in CGL2-sh2C cells (Fig. 6C), indicating that ATO might activate Rho through PIP4KIIγ-mediated generation of PIP2. Furthermore, CGL2 cells treated with ATO for 24 h were arrested in mitosis (mitotic index > 55, Fig. 6D left), and up to 80% of the arrested mitotic cells contained spindle abnormalities (Fig. 6B). Thereafter, the cells underwent apoptosis, i.e., the mitotic index decreased and apoptotic cells increased with only a limited increase in G1 cells (Fig. 6D left). In CGL2-sh2C cells, in addition to a significant decrease in spindle abnormalities after ATO treatment (Fig. 6B), the number of cells arrested at mitosis was reduced, mitotic cell apoptosis was also decreased, and G1 cells gradually increased (Fig. 6D right). These results indicated that depletion of PIP4KIIγ could prevent ATO induction of Rho activation and spindle abnormalities and hence reduce mitotic cell apoptosis. FIG. 6. Open in new tabDownload slide Depletion of PIP4KIIγ reduces ATO-induced Rho GTPase activation and alleviates ATO-induced spindle defects, mitotic arrest, and mitotic cell apoptosis. (A) ATO is less cytotoxic to CGL2 cells depleted of PIP4KIIγ (CGL2-sh2C) than to parental CGL2 cells. CGL2 and CGL2-sh2C cells were treated with ATO at the indicated concentrations for 72 h and then cell viability was analyzed with the WST-8 assay. (B) ATO-induced spindle damage is decreased in CGL2-sh2C cells. CGL2 and CGL2-sh2C cells were treated with 1 μM ATO for 24 h and immunostained for mitotic spindles. The percentage of mitotic cells with spindle defects was calculated using data acquired from at least 500 mitotic cells in three independent experiments. (C) ATO-induced Rho GTPase activation is reduced in CGL2-sh2C cells. CGL2 and CGL2-sh2C cells were treated with ATO, and Rho GTPase activation was analyzed as in Figure 3. (D) ATO-induced mitotic arrest and apoptosis are decreased in CGL2-sh2C cells. CGL2 and CGL2-sh2C cells were treated with 1 μM ATO for 24–72 h. The cells were fixed and analyzed for cell cycle distribution and apoptosis. #p < 0.01 compared with untreated parental CGL2 cells. *p < 0.01 compared with parental CGL2 cells treated with the same ATO concentration according to Student’s t-test. FIG. 6. Open in new tabDownload slide Depletion of PIP4KIIγ reduces ATO-induced Rho GTPase activation and alleviates ATO-induced spindle defects, mitotic arrest, and mitotic cell apoptosis. (A) ATO is less cytotoxic to CGL2 cells depleted of PIP4KIIγ (CGL2-sh2C) than to parental CGL2 cells. CGL2 and CGL2-sh2C cells were treated with ATO at the indicated concentrations for 72 h and then cell viability was analyzed with the WST-8 assay. (B) ATO-induced spindle damage is decreased in CGL2-sh2C cells. CGL2 and CGL2-sh2C cells were treated with 1 μM ATO for 24 h and immunostained for mitotic spindles. The percentage of mitotic cells with spindle defects was calculated using data acquired from at least 500 mitotic cells in three independent experiments. (C) ATO-induced Rho GTPase activation is reduced in CGL2-sh2C cells. CGL2 and CGL2-sh2C cells were treated with ATO, and Rho GTPase activation was analyzed as in Figure 3. (D) ATO-induced mitotic arrest and apoptosis are decreased in CGL2-sh2C cells. CGL2 and CGL2-sh2C cells were treated with 1 μM ATO for 24–72 h. The cells were fixed and analyzed for cell cycle distribution and apoptosis. #p < 0.01 compared with untreated parental CGL2 cells. *p < 0.01 compared with parental CGL2 cells treated with the same ATO concentration according to Student’s t-test. DISCUSSION Evidence to date has revealed that induction of spindle defects with consequent mitotic arrest is one of the major mechanisms for arsenite-induced apoptosis in cancer cells (Huang et al., 2000; Ling et al., 2002; States et al., 2002; Yih et al., 2005). The effects of arsenite on tubulin polymerization have been investigated previously in several studies but there is disagreement among the reported results. Arsenite has been demonstrated to inhibit tubulin polymerization in vitro (Carre et al., 2002; Li and Broome, 1999). Li and Broome (1999) proposed that arsenite may bind to the vicinal cysteines (Cys12 and Cys213) near the GTP-binding site of tubulin, cross-link the tubulin molecule, and hamper GTP access to the GTP-binding pocket. Because polymerization of tubulin requires GTP binding, arsenite therefore can inhibit tubulin polymerization and mitosis. It has also been shown that arsenite can bind to the free thiol of tubulin, hence alter tubulin conformation and antagonize taxol-induced tubulin polymerization, mitotic arrest, and apoptosis (Carre et al., 2002). However, the reduction of taxol-induced mitotic arrest and apoptosis by arsenite has been demonstrated to be primarily resulting from delayed S phase progression (Duan et al., 2009). Arsenite has also been demonstrated to promote tubulin polymerization in vitro (Huang and Lee, 1998; Ling et al., 2002). It has been shown that arsenite induces thickening and increased density of microtubules in interphase cells (Ling et al., 2002) and aggregated and lengthened spindle fibers in mitotic cells (Huang and Lee, 1998; Taylor et al., 2008). Huang and Lee (1998) demonstrated that arsenite might alter the function of mitotic spindle by attenuating the dynamic instability of mitotic spindle instead of disrupting spindle formation directly. Furthermore, arsenite has been reported to induce abnormal mitotic spindles independent of tubulin polymerization (Taylor et al., 2008) and to synergistically enhance taxol-induced mitotic arrest and apoptosis (Duan et al., 2010). The reason for the discrepancy among previous studies is not clear. Nonetheless, these results imply that, in addition to microtubules, arsenite might target other cellular component(s) to induce spindle abnormalities. We previously demonstrated that centrosome amplification is induced during mitotic arrest and consequently leads to spindle multipolarity and mitotic cell apoptosis (Yih et al., 2006), indicating that ATO might alter the behavior of centrosomes and/or spindle poles when cells enter mitosis. In the present study, our time-lapse recordings demonstrated that, upon entering mitosis, the spindle poles of ATO-arrested mitotic cells were mispositioned and the mitotic spindle moved randomly around the cell and then became multipolar. This indicated that ATO might first target the machinery regulating mitotic spindle positioning and then led to the formation of abnormal mitotic spindles. Our results showed that ATO activated Rho and that inhibition of ROCKs could reduce ATO induction of spindle abnormalities and cell death. Microtubules can be regulated by Rho GTPases and their effectors (Hollenbeck, 2001). ROCKs were identified as a centrosomal component required for centrosome positioning (Chevrier et al., 2002). Activated Rho may target ROCK at aster microtubules and the cell cortex for centrosome separation and spindle orientation in prometaphase (Narumiya and Yasuda, 2006). In addition, constitutive activation of the receptors known to mediate Rho signaling promotes centrosome amplification and chromosome instability in a Rho/ROCK-dependent manner (Fukasawa, 2011). Treatment of cells with Y-27632 interferes with centrosome separation after nuclear envelope breakdown and results in aberrant spindle phenotypes (Rosenblatt et al., 2004). These results indicate the involvement of Rho signaling in the regulation of centrosome separation and spindle positioning and imply that alteration of Rho signaling would lead to spindle abnormalities. Thus, ATO may induce defects in mitotic spindles by activating a Rho/ROCK pathway. Previous studies have demonstrated that PIPKs can regulate the activation of Rho GTPases. For example, PIP2 has been shown to stimulate GDP dissociation from Rho A and consequently GTP/GDP exchange, hence activating Rho GTPases (Zheng et al., 1996). PIP5KIα has been demonstrated to modulate cell migration or morphology by regulating Rac1 targeting and activation (Chao et al., 2010). Rho GTPases can also modulate cytoskeleton-dependent events by recruiting and activating PIPKs. Generation of PIP2 thus potentiates the effects of Rho GTPases on cytoskeletal remodeling (Paris et al., 1997; Randazzo, 1997). Thus, Rho GTPases and PIPKs are spatiotemporally cross-regulated. Our results showed that depletion of PIP4KIIγ not only prevented ATO-induced Rho activation but also reduced the induction of spindle abnormalities, mitotic arrest, and cell death. Thus, ATO might induce spindle abnormalities through PIP4KIIγ-mediated activation of Rho/ROCK pathway. PIP2 is synthesized from phosphatidylinositol-4-phosphate by PIP5KI and from phosphatidylinositol-5-phosphate by PIP4KII. PIP5KIs modulate many cellular processes, including motility, focal adhesion assembly/disassembly, and vesicular trafficking (van den Bout and Divecha, 2009). In contrast to PIP5KIs, little is known about the physiological functions of PIP4KIIs. PIP4KIIs are functionally nonredundant with PIP5KIs (Kunz et al., 2000) and therefore may modulate a set of cellular processes distinct from those modulated by PIP5KIs. PIP2 localizes primarily to the plasma membrane but is also found on secretory vesicles, lysosomes, the endoplasmic reticulum, Golgi, the cytokinesis cleavage furrow, and in the nucleus. PIP2 can either bind to intracellular proteins and directly modulate their subcellular localization and activity or act as a precursor for the generation of other second messengers, hence directly or indirectly regulating a plethora of cellular processes (Doughman et al., 2003; Kwiatkowska, 2010). Because the isoforms of PIP5KI (PIP5KIα, PIP5KIβ, and PIP5KIγ) have specific and distinct subcellular localizations, it has been postulated that local increases in PIP2 concentration by specific PIPK isoforms mediate its diverse signaling functions (Doughman et al., 2003; van den Bout and Divecha, 2009). Our results show that, in cells depleted of PIP4KIIγ, the frequency of mitotic cells with abnormal spindles was significantly increased as compared with that in the non-depleted control cells therefore imply a specific role of PIP4KIIγ at the spindle apparatus. Furthermore, we also demonstrated that sequestration of cellular PIP2 could reduce ATO-induced cell death and that depletion of PIP4KIIγ could prevent ATO induction of spindle abnormalities and mitotic cell apoptosis. It has been demonstrated that modulation of the activity or localization of PPK-1, a PIP2 synthesis enzyme of Caenorhabditis elegans, controls spindle movement by regulating the pulling forces on astral microtubules at the one-cell embryo stage (Panbianco et al., 2008), indicating that PIP2 may regulate the positioning and assembly of mitotic spindles. PLC-γ1, a PIP2 hydrolyzing enzyme, has been reported to interact with β-tubulin and subsequently increase its prevalence on spindle poles during mitosis (Chang et al., 2005), also indicating a role for PIP2 in mitotic spindle positioning and assembly. These results suggest that precise regulation of PIP2 level is required for accurate positioning and assembly of bipolar mitotic spindles. Therefore, our results imply that PIP4KIIγ might mediate PIP2 generation required for positioning and assembly of bipolar spindles and alteration of PIP4KIIγ function by ATO might thus lead to spindle abnormalities. FUNDING Academia Sinica; National Science Council (NSC), Taiwan (NSC99-2320-B-001-008-MY3). 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Journal

Toxicological Sciences – Oxford University Press

Published: Jul 1, 2012

Keywords: arsenic trioxide mitotic spindle apoptosis PIP4KIIγ Rho

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