Inhibition of TRIP1/S8/Hsug1, a Component of the Human 19S Proteasome, Enhances Mitotic Apoptosis Induced by Spindle Poisons

Inhibition of TRIP1/S8/hSug1, a component of the human 19S proteasome, enhances mitotic apoptosis induced by spindle poisons

Hiroshi Y. Yamada and Gary J. Gorbsky inhibitor drugs in trial as anticancer agents target elements of the 20S catalytic subcomplex. Our results Molecular, Cell, and Developmental Biology Research Program, suggest that targeting the ATPase subunits in 19S Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma regulatory complex in the proteasome may enhance the antitumor effects of spindle poisons. [Mol Cancer Ther 2006;5(1):29–38] Abstract Mitotic spindle poisons (e.g., Taxol and vinblastine), used Introduction as chemotherapy drugs, inhibit mitotic spindle function, Spindle poisons (e.g., Taxol and vinblastine) are commonly activate the mitotic spindle checkpoint, arrest cells in used chemotherapy drugs (1–3). In clinically relevant mitosis, and then cause cell death by mechanisms that are doses (e.g., 5–200 nmol/L in Taxol; ref. 4), they inhibit poorly understood. By expression cloning, we identified a mitotic spindle function and activate the mitotic spindle truncated version of human TRIP1 (also known as S8, checkpoint (5–7). The spindle checkpoint causes extended hSug1), an AAA (ATPases associated with diverse cellular mitotic arrest through inhibition of a ubiquitin ligase activities) family ATPase subunit of the 19S proteasome complex called the anaphase-promoting complex/cyclo- regulatory complex, as an enhancer of spindle poison– some and its activator Cdc20. In some cases, mitotic arrest mediated apoptosis. Stable expression of the truncated results in cell death initiated during mitosis (mitotic TRIP1/S8/hSug1 in HeLa cells [OP-TRIP1(88-406)] apoptosis) or apoptosis observed after the cells exit mitosis resulted in a decrease of measurable cellular proteasome abnormally without normal chromosome segregation activity, indicating that OP-TRIP1(88-406) had a domi- (sometimes called adaptation or mitotic slippage; ref. 4). nant-negative effect on proteasome function. OP- The signal transduction pathways by which spindle TRIP1(88-406) revealed an increased apoptotic response poisons and other mitotic inhibitors lead to cell death after treatment with spindle poisons or with proteasome remain to be clarified (8). A few molecules have been inhibitors. The increased apoptosis coincided with a identified to affect spindle poison–mediated cell killing. significant decrease in expression of BubR1, a kinase Mitotic apoptosis is observed on down-regulation of certain required for activation and maintenance of the mitotic kinetochore components, such as Ndc80/Hec1 or Nuf2 by spindle checkpoint in response to treatment with spindle small interfering RNA (siRNA) or conditional promoter poisons. Small interfering RNA (siRNA)–mediated knock- shutoff (9, 10). Taxol treatment activates p38 mitogen- down of TRIP1/S8/hSug1 resulted in a reduction of activated protein kinase, and suppression of p38 by specific general proteasome activity and an increase in mitotic inhibitors suppresses Taxol-mediated cell death (11, 12). index. The siRNA treatment also caused increased cell Spindle checkpoint components Bub1 and BubR1 kinases, if death after spindle poison treatment. These results overexpressed, stimulate the apoptotic response (13). The indicate that inhibition of TRIP1/S8/hSug1 function by hBubR1 protein is reduced during extended spindle expression of a truncated version of the protein or by poison–mediated mitotic arrest at least in part due to a siRNA-mediated suppression enhances cell death in proteasome-dependent degradation, and this reduction response to spindle poison treatment. Current proteasome has been proposed to be part of the link between spindle checkpoint and induction of apoptosis (13). Postmitotic apoptosis is observed if the spindle checkpoint is compromised by repression of Mad2 or BubR1 with siRNA (14, 15) Received 4/26/05; revised 10/24/05; accepted 11/15/05. or expression of a dominant-negative form of the Cdc20 Grant support: U.S. Department of Defense Breast Cancer Research protein (16). Breast cancer cell lines SKBr3 and HCC-1433 Program fellowship DAMD 17-02-1-0532 (H.Y. Yamada) and National and ovarian cancer cell lines A2780 and OVCAR have Institute of General Medical Science grant RO1-GM50412 (G.J. Gorbsky). weakened spindle checkpoint function due to decreased The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked expression of BubR1 and show elevated sensitivity to advertisement in accordance with 18 U.S.C. Section 1734 solely to spindle poisons (17). indicate this fact. The proteasome, a large protease complex that degrades Requests for reprints: Hiroshi Y. Yamada, Oklahoma Medical Research polyubiquitylated cellular proteins, has recently gained Foundation, 825 Northeast 13th Street, MS48, Oklahoma City, OK 73104-5097. Phone: 405-271-2037; Fax: 405-271-7312. prominence as a potential target for cancer therapy (18–21). E-mail: [email protected] Proteasome inhibitors (e.g., bortezomib/Velcade, lactacys- Copyright C 2006 American Association for Cancer Research. tin, and MG132; refs. 18–21) are cytotoxic, but the precise doi:10.1158/1535-7163.MCT-05-0126 mechanism of cell killing remains unclear. Bortezomib/

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Velcade has shown promise for a variety of cancers, morphology and became attached in the presence of including multiple myeloma (19). Regulation of proteolysis nocodazole were retained. DNA from the attached cells is crucial in cellular growth control for normal cells. was recovered with DNAzol reagent used according to the Inappropriate accumulation or reduction of cell cycle manufacturer’s instructions (Invitrogen, Carlsbad, CA). To regulators have been linked to oncogenesis (22), and avoid integration of the plasmids into the COS7 cell regulated proteolysis plays a major role in maintaining genome, we collected plasmid DNA no longer than 40 normal levels of proteins. A large percentage of regulated hours after transfection. Recovered DNA was transformed proteolysis is carried out by ubiquitin-mediated targeting into UltraMax DH5 competent bacteria (Invitrogen). Plas- (23–26). The ubiquitin-mediated proteolysis system mid recovered from the bacteria was used for an additional requires a set of enzymes: an ubiquitin-activating enzyme round of transfection of COS7 cells to enrich plasmids (E1), several ubiquitin-conjugating enzymes (E2), and a with positive activity. After the second round, we isolated large variety of ubiquitin ligases (E3). These enzymes individual plasmids from bacterial colonies and sequenced covalently attach multiple copies of the ubiquitin to the them. target. The resulting polyubiquitin chain on the target is CellCultureandMicroscopicAnalysis recognized by the 26S proteasome. The 26S proteasome is a We cultured and prepared cells as described previously complex of two subcomplexes: a 19S regulatory complex (29). The fixed samples were analyzed with a Zeiss and a 20S catalytic complex. Structural studies show that Axioplan IIi microscope equipped with a Hamamatsu the barrel-shaped 20S catalytic complex is capped by the Orca II camera and Metamorph imaging system (Universal 19S regulatory complex(es) at one or both ends to form the Imaging Corp., Downingtown, PA). For live cell observa- 26S complex (18, 27). The 19S complex is believed to bind tion, a planapochromat Â60 (numerical aperture 1.4) to, refold, and transfer the polyubiquitylated target protein objective (Nikon USA, Melville, NY) was used with a into central cavity of the 20S catalytic core, where the target SenSys CCD camera (Photometrics Ltd., Tucson, AZ) protein is degraded by the protease activity. Consistent connected to a Nikon Diaphot microscope and imaged with the chaperone-like activity required, the 19S regula- with Metamorph software. We used Annexin V FLUOS tory complex contains six ATPase subunits. In yeast, (Roche Biochemicals, Indianapolis, IN) to stain phosphati- conditional mutants in different proteasome subunits dylserine exposed on the cell surface to identify apoptotic show a mitotic arrest phenotype (28). This implies that the cells following the manufacturer’s instructions. Propidium proteasome acts as whole and each component is required iodide (PI)–positive (necrotic) cells were not scored as for activity. It also suggests that mitosis is a particularly apoptotic. We also used Live/Dead Cell Death Assay kit sensitive target when proteasome activity is compromised. (Molecular Probes, Eugene, OR) to assess cell death using We set out to identify factors affecting spindle poison– different markers. mediated cell killing and developed a mammalian gene Stable Cell Line Generation cloning protocol. One of the candidate plasmids, pSC3, The plasmid vector for the cDNA library, pEXP1 encodes a portion of TRIP1/S8/hSug1, an ATPase subunit (Clontech), contains the cytomegalovirus promoter up- of the 19S proteasome. When stably integrated, expression stream of the cloning site for the library cDNA and an of the truncated TRIP1/S8 showed no effect on normal cell internal ribosome entry site that permits the cotranslation growth but enhanced spindle poison–mediated cell death. of a puromycin resistance gene. Thus, if integrants are Similarly, repression of endogenous TRIP1/S8 by siRNA established, nearly all puromycin-positive cells will stably also results in increased cell death in response to spindle express the gene of interest. We transfected HeLa cells poisons. Thus, TRIP1/S8 seems to be a promising target to with individual candidate plasmids and selected cells enhance spindle poison–mediated cell killing and may with puromycin (0.2–0.5 Ag/mL) for 2 to 3 weeks. For each define an additional class of targets to inhibit proteasome plasmid, some 200 to 500 surviving colonies were pooled function. and used for experiments to avoid clonal variation in expression of the integrant and to avoid indirect effects of stable cell generation, such as mutations within the parental Materials and Methods genome induced by integration of the plasmid. For full- Expression Cloning Screen length TRIP1/S8 integrant generation, we used pCMS- We transfected COS7 cells (in two 15-cm plates, f70% EGFP vector (Clontech) and G418 (250 Ag/mL) for selection. confluent) with a human testis cDNA library (Clontech, Fluorescence-Activated Cell Sorting Analysis for Palo Alto, CA). Twenty-four hours later, the cells were Poly(ADP-Ribose) Polymerase ^ Positive Cells extensively washed to remove untransfected DNA and We treated control HeLa cells and OP-TRIP1(88-406) cells dead cells and were treated with nocodazole (100 ng/mL). with nocodazole (100 ng/mL) for 0, 16, or 24 hours. We Rounded mitotic cells were shaken from the plate and suspended the cells by trypsinization and fixed them with collected every hour, up to hour 14, and transferred to 80% ethanol (À20jC) for at least 2 hours. The permeabilized new plates containing nocodazole (100 ng/mL). At 16 cells were rehydrated for 5 to 10 minutes and resuspended hours after the beginning of nocodazole treatment, the in PBS, blocked with 20% boiled normal goat serum in PBS, new plates were rinsed with PBS to remove any still and incubated with rabbit anti-p85 poly(ADP-ribose) rounded mitotic cells, and the cells that had altered their polymerase (PARP) fragment antibody (1:200; Promega,

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Madison, WI) in 5% goat serum for 1.5 to 3 hours. After protease inhibitor cocktail, and 400 nmol/L microcystin LR being rinsed with PBS twice, samples were incubated with and boiled with SDS loading buffer for 6 minutes. Protein secondary antibody (FITC anti-rabbit, 1:400) for at least 1 concentration was measured with the BCA protein assay hour. Samples were then rinsed and treated with RNase kit (Pierce, Rockford, IL) and protein amounts loaded on (0.1 mg/mL), PI (50 Ag/mL), and 0.1% Triton X-100 at the gels were equalized. We used rabbit anti-BubR1 (gift room temperature for at least 3 hours. The samples were from Dr. T. Stukenberg, University of Virginia, Charlottes- analyzed with a FACSCalibur flow cytometers, and the cell ville, VA), mouse anti–cyclin B (BD Transduction Labora- cycle profile was estimated by ModFit software with the tories, San Jose, CA), rabbit anti-PARP p85 fragment, aid of the Flow and Image Cytometry Laboratory (Univer- mouse anti-Cdc27 (BD Transduction Laboratories), rabbit sity of Oklahoma Health Sciences Center). anti-TRIP1/proteasome S8 subunit (Calbiochem), mouse Drug Sensitivity (Colony Formation) Assay anti-Bub3 (BD Transduction Laboratories), rabbit anti- We plated f500 cells in 12-well plates, 1,000 cells in phosphorylated histone H3 (Upstate Biotechnology, Lake 60-mm plates, or 3,000 cells in 10-cm plates. The next day Placid, NY), and mouse anti-h-tubulin (Amersham Bio- (day 0), we added drugs in different concentrations and sciences, Piscataway, NJ). incubated the plates at 37jC. On day 4, half of the medium was replaced and fresh drugs were added. The cells were fixed and stained on day 8 with 0.5% methylene blue in Results 50% ethanol for 20 minutes, rinsed with distilled water, and Isolation of TRIP1/S8/hSug1 Fragment as a Spindle dried (30). Assays were repeated at least thrice and typical Poison ^ Mediated Cell Death Enhancer results are shown as pictures. Cell proliferation was We noted that mitotic cells undergoing apoptosis quantified by imaging plates and then summing the exhibited increased adherence to the culture substratum intensities of stained colonies using Metamorph software. compared with healthy mitotic cells. This allowed us to Proteasome ActivityAssay select for cells containing plasmids whose expression Sample cells were directly extracted or harvested and caused increased apoptosis in cells treated with spindle frozen at À80jC until use. Extracts were prepared by poisons. After screening 3 million cDNAs, we obtained vortexing cells in low-salt buffer [20 mmol/L Tris-HCl 34 candidate plasmids, among which 7 cDNA fragments (pH 7.0), 5 mmol/L ATP, 1 mmol/L DTT, 0.1 mmol/L showed enhanced spindle poison–mediated cell killing EDTA, and 20% glycerol supplemented with 400 nmol/L when overexpressed in HeLa cells. For control purposes, microcystin LR and protease inhibitor cocktail (Sigma, St. we established 48 clones from the cDNA library without Louis, MO)] and were cleared by centrifugation at 15,000 Â selection. None among the 48 colonies showed elevated g for 15 minutes at 4jC. The sample protein concentration sensitivity to spindle poisons. was adjusted to 150 Ag/mL. Samples were incubated at One of the positive clones, pSC3, encoded a proteasome 37jC for 30 or 60 minutes with the fluorogenic proteasome subunit TRIP1/S8/hSug1 (TRIP1/S8, NM_002805) with 87 substrate III (SucLLVY-AMC; Calbiochem, La Jolla, CA) at amino acids truncated from its NH2 terminus [TRIP1(48- 250 nm. Reactions were terminated by the addition of 100 406); Fig. 1A]. The TRIP1/S8 protein is an AAA (ATPases AL ethanol to 50 AL of the reaction mixture. Fluorescence associated with a variety of cellular activities) family was measured in a 96-well plate with a plate reader (Tecan, ATPase subunit in the 19S regulatory complex of the 26S Maennedorf, Switzerland; excitation, 360 nm; emission, proteasome (31–33). A subpopulation of TRIP1/S8 is found 465 nm). Samples added MG132 (10 Amol/L) were used for as a part of the APIS (AAA proteins independent of 20S) negative controls. complex, which may play a role in transcriptional siRNA-Mediated Protein Knockdown regulation independent of the full proteasome (34, 35). We used chemically modified siRNA duplex (Stealth To confirm the effect of the cDNA expression on spindle siRNA, Invitrogen) to knockdown human TRIP1/S8 target- poison–mediated cell killing, we generated a HeLa-based ing 5V-GCTCATCATACGGACTGTACCTTTA-3V.Asa cell line that stably expresses TRIP1(88-406) [OP-TRIP1- negative control, siRNA for green fluorescent protein was (88-406)]. The messenger overexpression was verified by used. Transfection was done with Oligofectamine reagent quantitative PCR (Supplementary Fig. S1).1 In standard (Invitrogen) following the manufacturer’s instructions. culture conditions, the estimated doubling time was 18.1 F Immunoblotting 0.7 hours for OP-TRIP1(88-406) and 18.1 F 1.9 hours for Cells were extracted either in radioimmunoprecipitation control parental HeLa cells; thus, growth rates were assay buffer [1% (w/w) NP40, 1% (w/v) sodium deoxy- comparable. Fluorescence-activated cell sorting analysis cholate, 0.1% SDS, 1% Triton X-100, 10 mmol/L sodium showed no apparent difference in the cell cycle profiles phosphate (pH 7.2), 2 mmol/L EDTA, 150 mmol/L NaCl, comparing cycling HeLa cells and OP-TRIP1(88-406) cells.2 50 mmol/L NaF, and 0.2 mmol/L sodium vanadate] Annexin V labeling indicated no difference in spontaneous supplemented with 10 Amol/L MG132, 5 Ag/mL protease inhibitor cocktail, and 400 nmol/L microcystin LR, or in NP40 lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 1% (w/w) 1 Supplementary material for this article is available at Molecular Cancer NP40, 250 mmol/L NaCl, 10 mmol/L NaF, and 5 mmol/L Therapeutics Online (http://mct.aacrjournals.org/). EDTA] supplemented with 10 Amol/L MG132, 5 Ag/mL 2 Unpublished results.

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Figure 1. Expression of truncated TRIP1/S8 sensitizes cells to spindle poison. A, schematic presentation of TRIP1/S8/hSug1 and the pSC3 product TRIP1(88-406) that lacks 87 amino acids at the NH2 terminus. TRIP1/S8 is an ATPases associated with a variety of cellular activities (AAA domain) family ATPase subunit found in the 19S regulatory complex of the proteasome. B, stable expression of a fragment of the TRIP1/S8 proteasome protein increases cell sensitivity to nocodazole and Taxol in a colony formation assay. The OP-TRIP1(88-406) line, which stably expresses the fragment of the TRIP1/S8 protein (Supplementary Fig. S1),1 shows decreased cell proliferation compared with the control cells when challenged with nocodazole or Taxol at low concentration for 8 d. Left, representative plates. Cell proliferation was quantified and presented as percentages normalized to zero drug dose. Black columns, controls; gray columns, OP-TRIP1(88-406) cell line. It is likely that the truncated form of TRIP1/S8 acts in a dominant-negative fashion because overproduction of the full-length TRIP1/S8 did not significantly affect spindle poison sensitivity (see Supplementary Fig. S2).1

apoptotic death rate in standard culture conditions (<2.5%). lead to increased sensitivity to challenge by sublethal However, in colony growth assays, OP-TRIP1(88-406) concentrations of proteasome inhibitor in a growth assay. cells were significantly more sensitive than controls to We treated OP-TRIP1(88-406) with MG132 or ALLN, drugs the microtubule-depolymerizing drug nocodazole and the that inhibit proteasome function in assays in cultured cells microtubule-stabilizing drug Taxol (Fig. 1B). OP-TRIP1(88- (18). OP-TRIP1(88-406) cells showed reduced cell prolifer- 406) cells did not show heightened sensitivity to the ation when treated with low concentrations of MG132 or topoisomerase II inhibitor VM26 (teniposide) 4V-dimethyl- ALLN (Fig. 2B). To distinguish if the truncated protein was epipodophyllotoxin or to the DNA-damaging drug bleo- functioning as a dominant negative, we generated a HeLa- mycin (data not shown). based cell line that overexpressed the full-length TRIP1/S8 Proteasome Activity Is Compromised in OP-TRIP1 protein (Supplementary Fig. S2).1 We observed an increase (88-406) Cells in proteasome activity in the extract, but the sensitivity to The construct isolated with our screen encodes a protein spindle poisons showed little difference from control. This fragment that lacks the NH2-terminal 87 amino acids supports that the truncated protein is dominant-negative of TRIP1/S8. We speculated that it may function in a form. dominant-negative manner and interfere with normal Spindle Poisons Enhance Mitotic Apoptosis in OP- proteasome activity. To test this hypothesis, we compared TRIP1(88-406) proteasome activities in extracts from control cells and OP- To study further the spindle poison sensitivity of OP- TRIP1(88-406) cells using the fluorogenic proteasome TRIP1(88-406) cells, we observed the cellular responses to substrate (SucLLVY-AMC; Fig. 2A; refs. 36, 37). OP- spindle poison treatment over shorter time ranges by live TRIP1(88-406) cells showed a 34% reduction in proteasome cell microscopy. Used at moderate doses (25–200 ng/mL), activity compared with control HeLa cells in asynchronous nocodazole causes mitotic arrest of most cultured cells culture and a 20% reduction in cells arrested with within one cell cycle without immediate cytotoxicity. nocodazole. These results suggest that proteasome function Normally, we and other researchers use nocodazole at is partially compromised in OP-TRIP1(88-406) cells. Pro- 100 ng/mL to accumulate living cells in M phase. We teasome activity is required at several stages of the cell reasoned that direct microscopic observation of the effects cycle (e.g., to degrade mitotic cyclins and allow cell cycle of expression of TRIP1(88-406) would provide information progression). We reasoned that lower proteasome activity about the mechanism of enhanced toxicity to nocodazole. associated with expression of the TRIP1/S8 fragment might We synchronized HeLa and OP-TRIP1(88-406) cells in early

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S phase with a double block in aphidicolin (an inhibitor of exposure of phosphatidylserine detected by Annexin V. In DNA polymerase), released the cells into medium contain- all, these experiments indicate that expression of TRIP1(88- ing nocodazole, and examined them by phase-contrast 406) facilitates mitotic apoptosis under spindle poison microscopy (Fig. 3A and B). Control HeLa cells entered challenge. mitosis f6 hours after release and remained arrested in Both compromised spindle checkpoint function and mitosis. After 14 hours, a small proportion of control cells elevated apoptosis in response to challenge with spindle began to exhibit membrane blebbing, characteristic of poisons have been associated with deregulation of the apoptosis during mitotic arrest. OP-TRIP1(88-406) cells expression of spindle checkpoint components (13–17, 38). entered mitosis f6 to 8 hours after release from S-phase We tested nocodazole-treated HeLa and OP-TRIP1(88-406) arrest and remained arrested in mitosis. However, a larger cells to compare whether mitotic and/or apoptotic marker proportion of OP-TRIP1(88-406) cells became apoptotic proteins behave differently and whether the levels of soon after mitotic arrest. Apoptotic cells were counted and expression of spindle checkpoint proteins are affected. We the apoptotic percentage in the total cells is plotted as the treated cells with nocodazole (100 ng/mL) for 4 hours, dark gray regions in Fig. 3A. Light gray regions represent collected the arrested mitotic cells, incubated the mitotic percentage of normal mitotic cells. Frames from a time- cells further with nocodazole for the indicated amount of lapse video of the apoptotic phenotype of OP-TRIP1(88- time up to 20 hours, and prepared samples for immuno- 406) cells are shown in Fig. 3B, revealing conversion of blotting (Fig. 3C). Cyclin B levels remained high in both the normal rounded mitotic cells in the first panel to the control and OP-TRIP1(88-406), suggesting that the cells advanced apoptotic cells in the last panel. We also remained arrested in mitosis with high Cdk1/cyclin B examined whether the membrane blebbing phenotype kinase activity. The PARP cleavage fragment, a marker of correlated with apoptosis. About 50% of the blebbing cells apoptosis generated by caspase-3 and caspase-7, appeared were Annexin V positive compared with <3% of cells with after 16 hours of nocodazole treatment in control smooth membranes, suggesting that the membrane bleb- HeLa cells. In contrast, in OP-TRIP1(88-406) cells, the bing is an early sign of apoptosis, preceding cell surface PARP fragment was apparent at 4 hours and increased

Figure 2. Stable expression of a NH2-terminal truncation of the proteasome subunit TRIP1/S8 results in decreased proteasome activity and elevated sensitivity to proteasome inhibitors. A, proteasome activity is reduced in OP-TRIP1(88-406) cells in both asynchronous culture and cells treated with nocodazole. Cell extracts were prepared and incubated with fluorogenic proteasome substrate III (SucLLVY-AMC) for 30 and 60 min in 37jC, and fluorescence was measured. The conversion rate was calculated and proteasome activity in extracts from OP-TRIP1(88-406) cells was compared with extracts from control HeLa cells in both normal cultures (left) and cultures treated with nocodazole (100 ng/mL, 16 h; right). B, OP-TRIP1(88-406) cells show markedly decreased cell proliferation compared with the parental line when treated with the proteasome inhibitor MG132 (top) or ALLN (bottom) for 8 d. Experiments were repeated at least thrice. Right, quantification of cell proliferation. Cell proliferation was normalized to zero drug dose. Black columns, control HeLa cells; gray columns, OP-TRIP1(88-406) cells.

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Figure 3. OP-TRIP1(88-406) cells undergo accelerated mitotic apoptosis in the presence of spindle poisons. A, OP-TRIP1(88-406) cells show increased apoptosis when examined after treatment with nocodazole. HeLa and OP-TRIP1(88-406) cells were synchronized in early S phase with a double aphidicolin block and released into medium containing nocodazole (100 ng/mL). Cell morphology was monitored by phase-contrast microscopy at the indicated time points. Rounded cells with smooth surfaces were scored as normal mitotic cells. Cells with membrane protrusions (blebs) were scored as apoptotic (dark gray). B, an example of OP-TRIP1(88-406) cells undergoing apoptosis. Cells were synchronized and released into nocodazole as in A and filmed with time-lapse video microscopy. Video recording was initiated 3 h after the cells under observation had entered mitosis in the presence of nocodazole (time 3:00). Most OP-TRIP1(88-406) cells arrested in mitosis for 3.5 to 5.8 h and then initiated membrane blebbing. Most parental HeLa cells remained arrested in mitosis without showing blebbing for at least 10 h (data not shown). Bar, 20 Am. C, apoptosis was initiated during nocodazole-mediated mitotic arrest. HeLa and OP-TRIP1(88-406) cells were treated with 100 ng/mL nocodazole for 4 h. Rounded mitotic cells were collected and further cultured in medium containing nocodazole (100 ng/mL). At the indicated times, cell extracts were prepared and proteins were separated by SDS-PAGE and analyzed by immunoblotting. Equal amounts of protein were loaded in each lane. OP-TRIP1(88-406) cells show accelerated cleavage of PARP, dephosphorylation and loss of the Cdc27 protein, and loss of BubR1 protein, whereas cyclin B levels remained high during continued incubation in nocodazole. D, comparison of fluorescence-activated cell sorting profiles revealed an increase in PARP fragment – positive cells with G2-M DNA content in nocodazole-treated OP-TRIP1(88-406) cells compared with controls. We treated control and OP-TRIP1(88-406) cells without or with nocodazole (100 ng/mL) for 16 or 24 h and collected samples for fluorescence-activated cell sorting analysis. Top row, DNA content of all sample cells (All cells). Samples are also labeled with anti-PARP fragment antibody and then with FITC secondary antibody. Insets, percentage of cells with G2-M DNA content.

with time. Cdc27 is a component of the anaphase- whereas in OP-TRIP1(88-406) both phosphorylation and promoting complex/cyclosome. In mitosis, Cdc27 is total amount of Cdc27 protein decreased, consistent with multiply phosphorylated and undergoes a large mobility increased apoptosis in OP-TRIP1(88-406) cells. Expression shift on SDS-PAGE gels. Cdc27 is also a caspase-3-like of the spindle checkpoint component BubR1 in OP- protease target and is degraded during apoptosis (39). TRIP1(88-406) was of particular interest because the level With time in nocodazole, the mitotic hyperphosphoryla- of this protein has been proposed to serve as a link tion of Cdc27 remained high in control HeLa cells, between spindle checkpoint and spindle poison–mediated

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cell killing (13). The BubR1 expression level was compa- When used alone, each drug induced low-level production rable with or slightly higher than that of control cells at of the PARP fragment. However, when cells were incubated 4 hours. However, with time, BubR1 levels diminished in either nocodazole or Taxol, cotreatment with MG132 more rapidly in the OP-TRIP1(88-406) cells. showed much higher generation of the PARP fragment. We interpreted the results above as indicating that Initiation of cell death during mitosis with perturbed the OP-TRIP1(88-406) cells initiate apoptosis directly from proteasome function led us to investigate whether the mitosis after spindle poison treatment. To verify this modulatory effect of proteasome inhibition on the cytotoxic interpretation, we asked in which phases of the cell cycle response to spindle poisons occurred during M phase or phase PARP fragments, an apoptotic marker, were during another phase of the cell cycle. We tested whether generated in response to spindle poison. We collected proteasome inhibition would increase apoptosis in cells control and OP-TRIP1(88-406) cells with or without nocodazole treatment, labeled PARP fragment–positive cells by immunofluorescence, and monitored the cell cycle by fluorescence-activated cell sorting (Fig. 3D). Without nocodazole, the cell cycle profiles of control and OP- TRIP1(88-406) were indistinguishable, and a small number of apoptotic cells (PARP fragment–positive cells) were observed preferentially in the population with sub-G1 DNA content in both cell lines. With nocodazole treatment, both cell types arrested in G2-M. The degree of G2-M arrest seemed to be higher in OP-TRIP1(88-406) cells (G2-M: 59% after 16 hours in nocodazole) than in controls (G2-M: 40% after 16 hours in nocodazole). Monitoring nocodazole-treated, PARP-positive cells, we observed two peaks in sub-G1 and G2-M in both cell lines, indicating a population of PARP fragment–positive cells has G2-M DNA content (asterisks on right). The ratio of PARP-positive G2-M cells is consistently higher in OP-TRIP1(88-406) cells than in controls as indicated in Fig. 3D (inset). Together, the results in Fig. 3 suggest that that cells expressing truncated TRIP1 are arrested in mitosis more readily than controls in response to spindle poisons, and cells expressing truncated TRIP1 are more prone to apoptosis during mitosis than are control cells. Drug-Mediated Inhibition of the Proteasome In- creases Cytotoxicity and Apoptosis in Cells Treated with Spindle Poisons The identification of a truncated proteasome subunit as a spindle poison–sensitizing factor and the observed decrease in proteasome activity in OP-TRIP1(88-406) (Fig. 2A) suggested that inhibition of proteasome activity might generally enhance the cytotoxicity induced by Figure 4. Treatment of HeLa cells with spindle poisons and a spindle poisons. To test this idea using an independent proteasome inhibitor increases cytotoxicity and apoptosis. A, MG132 approach, we treated asynchronous HeLa cell cultures enhances cell death due to spindle poisons. HeLa cells were treated with nocodazole (100 ng/mL), MG132 (10 Amol/L), or Taxol (20 nmol/L) alone with nocodazole or Taxol for 16 hours with or without or in combination with MG132 (10 Amol/L) for 16 h. Cell death was scored cotreatment with the proteasome inhibitor, MG132. The using fluorescent markers of membrane permeability (Live/Dead Cell percentage of dead cells was assessed microscopically Death Assay kit). B, immunoblot for an apoptosis marker, PARP fragment. using the Live/Dead Cell Death Assay kit, which HeLa cells were treated with nocodazole [100 ng/mL (Noc100) or 50 ng/ mL (Noc50)] alone, MG132 [1 Amol/L (MG1)] alone, Taxol [20 nmol/L determines the fraction of cells with disrupted plasma (Tax20)or10nmol/L(Tax10)] alone, nocodazole and MG132 membranes. A combination of MG132 with either noco- (Noc100+MG1 or Noc50+MG1), or Taxol and MG132 (Tax20+MG1 or dazole or Taxol showed higher cytotoxicity than any of Tax10+MG1) simultaneously for 16 h and monitored for generation of PARP fragment. h-Tubulin is shown as a loading control. C, inactivation of the drugs alone. The combination of MG132 with the proteasome after mitotic arrest in HeLa cells does not significantly nocodazole seemed to show synergistic effects, whereas increase the level of apoptosis as monitored by PARP fragmentation. HeLa the combination of MG132 and Taxol was additive (Fig. 4A). cells were treated with nocodazole (100 ng/mL) or Taxol (1 Amol/L) for To examine the effects on a biochemical marker of 4 h and mitotic cells were collected by shaking the plate. Mitotic cells were then incubated in the same microtubule drug for an additional 4 and 8 h apoptosis, we collected samples and monitored generation with or without the addition of MG132 (10 Amol/L). Extracts were ofthePARPcleavagefragmentbyimmunoblot(Fig.4B). prepared and blotted for PARP fragmentation.

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that were already in M phase. We treated HeLa cell with medium containing nocodazole (100 ng/mL) for a cultures for 4 hours with nocodazole (100 ng/mL) or Taxol further 8 hours. We then observed the effects by phase- (1 Amol/L) and collected the arrested mitotic cells. We then contrast microscopy (Fig. 5A). The results of these treat- treated each population for an additional 4 or 8 hours ments were quantified in Fig. 5B. In the absence of with spindle poisons in the presence or absence of MG132 nocodazole, cells treated with TRIP1/S8 siRNA were viable (10 Amol/L). After this time, we collected samples and but exhibited a higher mitotic index and a higher monitored PARP cleavage fragment production by immu- percentage of Annexin V–positive (apoptotic) cells. The noblotting (Fig. 4C). Surprisingly, treatment with MG132 number of PI-positive cells (necrotic or terminal stage in did not substantially increase PARP fragment production apoptosis) was approximately equal to that of the control. in cells that were already arrested in M phase with both Nocodazole treatment led to an increase in the number spindle poisons. Thus, sequential inactivation of the of mitotic cells and Annexin V–positive cells, the normal proteasome after mitotic arrest did not seem to enhance response to spindle poison. When TRIP1/S8 siRNA-treated mitotic apoptosis. This finding suggests that the target of cells were incubated in nocodazole, the number of cells proteasome inhibition may function before M phase. that were Annexin V or PI positive increased, whereas siRNA-Mediated TRIP1/S8 Protein Knockdown the number of healthy mitotic cells was decreased. We Resulted in Mitotic Cell Accumulation, Enhanced Cell interpret this reduction of healthy mitotic cells as the result Killing with Spindle Poison Treatment, and Compro- of increased apoptosis initiated during mitotic arrest. mised Proteasome Function We prepared extracts from duplicate cultures and The studies above led us to investigate further the monitored the amount for TRIP1/S8, PARP fragment, function of endogenous TRIP1/S8 and cellular responses Cdc27, phosphorylated histone H3, BubR1, and h-tubulin to spindle poisons by siRNA-mediated inhibition of (Fig. 5C). The siRNA treatment produced a 50% to 70% TRIP1/S8. We transfected siRNA into HeLa cells, and reduction of TRIP1/S8 protein expression in both cycling 40 hours later, we treated the cells with normal medium or cells and cells incubated with nocodazole. In siRNA-treated

Figure 5. siRNA-mediated TRIP1/S8 repression results in mitotic cell accumulation, enhanced cell killing with spindle poison treatment, and compromised proteasome function. A, cellular phenotype. HeLa cells were transfected with siRNA against green fluorescent protein (Control) or TRIP1/S8 (TRIP1). Forty h later, cells were incubated with or without 100 ng/mL nocodazole, cultured further for 8 h, and observed by phase-contrast microscopy. Mitotic cells have smooth round morphology. Bottom right, cells (TRIP1 siRNA, nocodazole positive) show many apoptotic cells with membrane blebbing. Bar, 20 Am. B, phenotype quantification. Cells treated as in A were stained with fluorescent Annexin V and PI and categorized into three phenotypes: Annexin V positive (black area, early apoptotic cells), mitotic (gray area, rounded, healthy mitotic cells), and necrotic (PI positive; striped area, necrotic cells and apoptotic cells in terminal stages). The sum of three categories represents the total rounded cells. C, immunoblots. Cell extracts were prepared from the treated cultures and monitored for TRIP1/S8, PARP fragment (apoptosis marker), Cdc27, phosphorylated histone H3 (mitotic marker), and h-tubulin (loading control). Ct, control; Tr, TRIP1. D, in TRIP1/S8 siRNA-treated cells, proteasome activity was reduced by 60% compared with control. HeLa cells were transfected with siRNA against green fluorescent protein (negative control; black column) or TRIP1/S8 (gray column). Forty-eight h after transfection, cell extracts were prepared and, after equalizing the protein amount, incubated with fluorogenic proteasome substrate III (SucLLVY- AMC) at 37jC for 30 and 60 min. The conversion rate was calculated and normalized to control transfectants.

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cells incubated in nocodazole, we detected enhanced It seems that in OP-TRIP1(88-406) cells spindle poison– production of the PARP fragment and diminished levels mediated cell death is initiated from mitotic arrest rather and phosphorylation of the Cdc27 protein. However, we than after the exit of mitosis. The simultaneous finding of did not observe significant differences in BubR1 expression. high cyclin B accumulation, high Cdc27 phosphorylation, To test whether TRIP1/S8 knockdown resulted in and appearance of the PARP apoptosis marker fragment reduction of general proteasome activity, we transfected in OP-TRIP1(88-406) cells after only 4 hours of nocodazole HeLa cells with TRIP1/S8 siRNA, prepared cell extracts, treatment (Fig. 3C) is consistent with this interpretation. and measured proteasome activity (Fig. 5D). We observed a Cell cycle analysis of PARP fragment–positive cells also 60% reduction of proteasome activity in extracts with from supports this view (Fig. 3D). Because BubR1 reduction TRIP1/S8 siRNA-treated cultures (Fig. 5D, gray column) seems to take place after initiation of apoptosis, it is compared with that in extracts from control siRNA trans- unlikely to be the direct cause of apoptosis, although the fections (black column). We also obtained the same reduction may accelerate apoptosis. This conclusion is reduction in proteasome activity using the 293T cell line supported by results from TRIP1/S8 siRNA-treated cells, in (data not shown). Thus, repression of TRIP1/S8 leads to which BubR1 amount was not significantly affected when proteasome inhibition. apoptosis occurred (Fig. 5B). We did cell cycle analysis using the TRIP1 knockdown Our results are notable given recent interest in the cells along with controls (Supplementary Fig. S3).1 proteasome and proteasome inhibitors in cancer chemo- Although the siRNA transfection procedure produced therapy (e.g., refs. 18–21). Our identification of a protea- higher numbers of apoptotic cells in the subG1 DNA some subunit in this screen suggests that the proteasome content region, we observed that TRIP1 knockdown cells may be a promising target in multidrug strategies with also showed a population of apoptotic cells with a G2-M spindle poisons. Consistent with this idea, we also show DNA content when treated with spindle poisons, particu- that simultaneous treatment of cultured cells with spindle larly nocodazole. However, the level of G2-M apoptotic poisons and a proteasome inhibitor caused enhanced cell cells in the siRNA-treated cell population was less than that death (Fig. 4A and B). Studies by others indicated that obtained from expression of the truncated TRIP1 protein bortezomib (also known as Velcade or PS341), a protea- shown in Fig. 3D. some inhibitor with clinical potential (18–21), enhances the cytotoxic activity of spindle poisons docetaxel (40) and Taxol (41) against tumor xenografts. The precise molecular Discussion mechanisms by which inhibition of the proteasome affects Mammalian cancer cells treated with clinically relevant mitotic apoptosis and the reaction to spindle poisons doses of spindle poison eventually undergo apoptosis remain uncertain. Based on our evidence, we suggest that either directly from mitosis (mitotic apoptosis) or after an cells with deficient proteasome activity may enter mitosis abnormal mitotic exit. Given the current routine use of inadequately prepared perhaps through insufficient re- spindle poisons (e.g., Taxol/paclitaxel and vinblastine) for moval of a cell cycle inhibitor. This inappropriate state may cancer chemotherapy, identification of cellular factors that trigger an apoptotic pathway. Alternatively, recent study modulate the cell death response is critical in elucidating indicates that the proteasome plays a key role in transcrip- the mechanisms involved. tional regulation (42). Indeed, some evidence implicates We report the identification of a cDNA fragment, TRIP1/S8 in direct regulation of transcription either within pSC3, expressing a NH2-terminal truncation of the 19S or apart from its role in the 19S proteasome (34, 35). proteasome component TRIP1/S8 from an expression Transcriptional errors may result in altered expression of screen aimed at isolating enhancers of spindle poison– proteins that play important roles in mitosis, and it may mediated cell killing. Expression of truncated TRIP1/S8 leave cells more prone to apoptosis during mitosis. This protein seems to work as a dominant negative for interpretation is consistent with our analysis that the proteasome function. Decreased proteasome activity sensi- window for enhancing mitotic apoptosis of spindle poisons tizes HeLa cells to spindle poisons and proteasome with proteasome inhibitor is before not during M phase inhibitors in growth assays. Microscopic analysis indicated (Fig. 4C). Another but not mutually exclusive possibility is that it caused increased mitotic apoptosis. siRNA-mediated that proteasome inhibition alters the state of apoptotic knockdown of TRIP1/S8 also resulted in compromised machinery and makes cells prone to apoptosis. Further proteasome function and enhanced cell death with spindle investigation will be required to test these possibilities. poisons. In contrast, overproduction of full-length TRIP1/ Overall, our results suggest that inhibition of TRIP1/S8 S8 resulted in an increase in proteasome activity and and/or the proteasome alters cellular physiology and in subtle resistance to spindle poisons (Supplementary leaves cells prone to apoptosis when they are arrested in Fig. S2).1 These results suggest that TRIP1/S8 function is mitosis with spindle poisons. Our results suggest that linked to proteasome activity and that proteasome activity targeting TRIP1/S8 or other components of the 19S plays critical role in spindle poison–mediated cell death. proteasome may be useful in anticancer therapy either These findings also suggest that the TRIP1/S8 may be valid alone or in combination with spindle poisons. Known target for enhancing spindle poison–mediated cell killing proteasome inhibitors [e.g., peptide boronate (bortezomib/ in cancer therapy. Velcade), lactacystin, and peptide aldehyde (MG132 and

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Downloaded from mct.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Inhibition of TRIP1/S8/hSug1, a component of the human 19S proteasome, enhances mitotic apoptosis induced by spindle poisons

Hiroshi Y. Yamada and Gary J. Gorbsky

Mol Cancer Ther 2006;5:29-38.

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