Effects of anti-microtubule agents on microtubule organization in cells ...

[Cell Cycle 7:14, 2146-2156; 15 July 2008]; ©2008 Landes Bioscience

Report

Effects of anti-microtubule agents on microtubule organization in cells lacking the kinesin-13 MCAK David G. Hedrick,1 Jane R. Stout2 and Claire E. Walczak2,* 1Department

of Anatomy and Cell Biology; 2Department of Biochemistry and Molecular Biology; Indiana University Medical Sciences; Bloomington, Indiana USA

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suppress dynamic instability at low doses.6,7 Vinblastine is used clinically to treat Hodgkin’s lymphomas and testicular cancers.1 These drugs have proven to be extraordinarily effective at killing cancer cells but have significant drawbacks, including damaging side effects to hematopoetic, gastrointestinal, and skin cells.8,9 In addition, differentiated cells also experience toxic side effects, which can include neuropathies and myalgias.8,9 In normal cells, a precisely regulated mitotic cycle is critically important for the production of new cells and for the maintenance of genomic integrity. The mitotic spindle is composed of nearly 800 proteins that work in concert to ensure proper mitotic spindle function.10 Several classes of mitotic spindle proteins act to regulate proper spindle MT dynamics.11 MCAK, a member of the Kinesin-13 family of molecular motors, is an important MT dynamics regulator and acts to depolymerize MTs in vivo and in vitro.12-15 Its overexpression or inhibition in cells results in disruption of MT dynamics, which leads to improper spindle assembly.16-19 MCAK is also required for amphitelic attachment of MTs to the kinetochore on chromosomes, and defects in MT-chromosome attachment lead to aneuploidy.20-24 Proper MCAK function and regulation may therefore be an important mechanism by which cells prevent aneuploidy. Current research is focusing on manipulating the cellular mitotic machinery to effectively kill cancer cells.25,26 MCAK may represent a novel target because it regulates MT dynamics. Supporting this idea, one low affinity inhibitor that targets MCAK is effective in inhibiting MT depolymerization in vitro and in vivo.27 In addition, MCAK is upregulated in some cancers.28,29 Most notably increased expression of MCAK in gastric cancers has been associated with increased lymphatic invasion, metastasis to lymph nodes and an overall poor prognosis.30 Furthermore, MCAK expression was found at high levels in many breast cancer cell lines, and knockdown of MCAK by RNAi significantly affected the proliferation of several tested breast cancer derived cell lines.29 Therefore, inhibition of MCAK function may be effective in treating cancer, particularly in combination with known drugs that also alter MT dynamics. Here, we investigate MCAK knockdown via RNA interference (RNAi) in a limited number of normal and malignant cell lines. The cell lines chosen were ones in which we could achieve effective MCAK knockdown (>90%) and that had a flattened morphology that was amenable to microscopic observation. In addition, we looked at the effect of combining MCAK knockdown with paclitaxel or vinblastine to identify spindle assembly defects. We found that the

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Dynamic microtubules are necessary for proper mitotic spindle assembly and chromosome segregation during mitosis. Members of the kinesin superfamily of molecular motor proteins are important to spindle function. Of particular interest is the Kinesin-13 family member MCAK, which acts to regulate microtubule dynamics during spindle assembly and to ensure proper attachments of chromosomes to spindle microtubules. The unique ability of MCAK to regulate microtubule dynamics makes it a potential target for development of new drugs that alter spindle function. Here, we knocked down MCAK via RNAi in normal and malignant cell lines and found that the two tested malignant cell lines were acutely sensitive to MCAK knockdown, while the tested normal cells were less sensitive. In addition, we looked at the effect of combining MCAK knockdown and drug treatment with paclitaxel or vinblastine to identify spindle assembly defects. We found that MCAK knockdown increased the morphological defects of the microtubule cytoskeleton in HeLa cells caused by anti-microtubule drugs. Our studies support the idea that MCAK would be a good target for new chemotherapeutic development and may be particularly useful in combination therapies with currently available anti-microtubule agents.

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Key words: mitosis, cell cycle, kinesins, chemotherapeutic target, microtubule dynamics

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Introduction

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Normal mitotic spindle formation is critical for accurate chromosome segregation and relies on the reorganization of the microtubule (MT) cytoskeleton in mitosis. Several drugs that target MTs are effective in halting mitosis by disrupting MT dynamics, which leads to apoptosis and cell death in rapidly dividing cells.1,2 Two prominent classes of anti-MT drugs are the taxanes and the vinca alkaloids. Paclitaxel, the prototypical taxane is used clinically to treat ovarian, breast and non-small cell lung cancer.1,3 It functions to increase MT polymer at high doses and suppress dynamic instability at low doses.1,4,5 Vinblastine, a vinca alkaloid, functions at a site distinct from paclitaxel and acts to decrease MT polymer at high doses and *Correspondence to: Claire E. Walczak; Indiana University; Medical Sciences; 915 E. 3rd St.; Myers Hall 262; Bloomington, Indiana 47405 USA; Tel.: 812.855.5919; Fax: 812.855.6082; Email: [email protected] Submitted: 01/16/08; Accepted: 05/07/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/6239 2146

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MCAK and anti-microtubule agents

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Figure 1. Effects of MCAK knockdown on interphase cells and progression through the cell cycle in HeLa cells. Interphase cells were scored based on their nuclear and cytoskeleton morphology. (A) Micrographs of DNA (blue) and MT (green) staining of control cells (Normal Interphase) or MCAK RNAi cells (Multinucleate Interphase and Dead/Blebbing). Normal interphase cells are defined by smooth regular edges and a single nucleus, multinucleate cells contain more than one nucleus, and dead/blebbing cells have abnormal nuclei and rough edges. Cytoplasmic blebs are indicated by arrowheads. Inset is an enlargement of one Dead/Blebbing cell, with contrast enhanced to demonstrate more clearly the aberrant morphology. Scale bar = 20 μm. (B) Quantification of interphase defects seen upon MCAK knockdown. Increased multinucleate interphase cells as well as a dramatic increase in dead/blebbing cells are seen in cells depleted of MCAK. (C) Quantification of progression through mitosis in control and MCAK depleted cells. MCAK depleted cells are delayed in prometaphase (Promet) as compared to control cells.

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tested malignant cells were acutely sensitive to MCAK knockdown, while the tested normal cells were less sensitive. We also found that MCAK knockdown increased the morphological defects of the MT cytoskeleton in cells caused by anti-MT drugs. Our studies support the idea that MCAK would be a good target for new chemotherapeutic development.

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Results

MCAK knockdown in HeLa cells causes morphological changes in interphase cells. To determine the effects of MCAK depletion on both interphase and mitotic cells, RNAi was used to knockdown MCAK in HeLa cells. MCAK protein was reduced by over 90% as assayed by immunoblot, and MCAK staining of mitotic cells was also significantly reduced (Suppl. Fig. S1A). We found a 3-fold reduction in normal interphase cells and a ~2-fold increase in multinucleate cells after MCAK RNAi (Fig. 1A and B). Most notably there was a 9-fold increase in cells that appeared to be dead or blebbing after MCAK RNAi (Fig. 1B). Blebbing cells were marked by the presence www.landesbioscience.com

of a pyknotic and fragmented nucleus and large cytoplasmic blebs erupting from the cell cortex (Fig. 1A arrowheads). It should be noted that these cells were not significantly increased in RNase free water treated or luciferase RNAi controls. As well positive controls using Eg5 or Tog siRNA yielded the expected phenotypes as described previously but did not result in an increase in dead/ blebbing cells (data not shown).16,19 Despite morphological changes in interphase cells, there was no change in mitotic index (Fig. 1B). However, a significant change in the stage of mitosis occurred after MCAK RNAi (Fig. 1C), resulting in a 1.4 fold increase in prometaphase cells with a concomitant decrease in prophase and telophase cells, consistent with earlier studies.19,23 These results suggest that loss of MCAK in HeLa cells disrupts interphase cellular morphology as well as changes mitotic progression. MCAK knockdown increases MT polymer and induces lagging chromosomes in HeLa cells. To look more closely at potential mitotic defects, mitotic cells were analyzed for MT, spindle, and chromosome organization. Consistent with previous studies,

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Figure 2. MCAK knockdown induces spindle morphology and chromosome segregation defects in mitosis in HeLa cells. (A) MCAK depleted prometaphase cells demonstrate the hairy prometaphase phenotype typified by a disrupted central spindle and increased number of long MTs emanating toward the cell cortex. MTs are in green and chromosomes are in blue for (A and C). (B) Quantification of spindle phenotypes seen in prometaphase. (C) Anaphase cells depleted of MCAK often display lagging chromosomes. One such chromosome, stranded in the center of the dividing cell, is indicated by an arrowhead. (D) Quantification of anaphase cells. Scale bar = 10 μm for (A and C).

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MCAK knockdown disrupted mitotic spindles (Fig. 2A).16,18,19 Prometaphase cells often had an increase in MTs that emanated toward the cell cortex and appeared to originate from the poles, a phenotype we refer to as “hairy” spindles (Fig. 2A). In addition MTs that make up the central spindle appeared more disorganized and were diminished in intensity. This phenotype was seen in 62% of MCAK RNAi treated cells as opposed to only 16% of control cells (Fig. 2B). Normal appearing central spindles were seen in metaphase cells after MCAK RNAi; however there was a 3-fold increase in the number of metaphase cells with increased astral polymer (data not shown). In addition to the defects in spindle MT polymer, we also noted that there was an 8-fold increase in cells with lagging chromosomes during anaphase/telophase after MCAK knockdown (Fig. 2C and D). Lagging chromosomes were defined as chromosomes that were either segregating behind the bulk of the chromosome mass (Fig. 2C) or that were left behind in the midzone at telophase. These data show that MCAK is necessary to regulate proper MT

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organization and distribution within the mitotic spindle and that loss of MCAK in HeLa cells specifically increased astral MTs, delayed mitotic progression, and reduced the ability of cells to accurately segregate chromosomes. MCAK knockdown increases sensitivity to anti-MT drugs. Because MCAK knockdown alone can alter interphase cellular morphology and interfere with normal MT organization, the consequences of treating control and MCAK knockdown cells with anti-MT drugs were analyzed to see if cell sensitivity to these drugs would increase. Two clinically utilized drugs were chosen, paclitaxel and vinblastine, which interfere with normal MT dynamics, albeit by different mechanisms.1 Three concentrations of paclitaxel were chosen based on published effects on mitotic spindle morphology and MT dynamics31 in combination with our own experimental evaluation of multiple drug concentrations. The lowest dose of paclitaxel (0.1 nM) showed no phenotypic effect on either control RNAi or MCAK RNAi cells and demonstrated that this treatment

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itself would not alter cell morphology (Suppl. Fig. S2A and B). Control RNAi cells treated with an intermediate concentration of paclitaxel (10 nM) showed a dramatic increase in multinucleate interphase cells, but there was no increase in the mitotic index or in the percentage of dead/blebbing cells over that of non-drug treated control RNAi cells (Fig. 3A). Interestingly, MCAK RNAi cells treated with 10 nM paclitaxel had a nearly 3-fold greater mitotic index than MCAK RNAi or paclitaxel treatments alone. In addition, MCAK RNAi cells treated with paclitaxel had a small but statistically significant increase in the percentage of dead/blebbing cells as compared to those with MCAK RNAi alone (Fig. 3A). Also, the number of multinucleate interphase cells was dramatically reduced in MCAK RNAi cells treated with paclitaxel relative to those cells treated with paclitaxel alone. Together these data show that MCAK RNAi can exacerbate the effect of paclitaxel to increase the percentage of dead/blebbing and to block progression through mitosis. Mitotic cells in both control and MCAK RNAi in the presence of 10 nM paclitaxel were almost entirely halted in prometaphase (data not shown). As shown above, MCAK RNAi caused a significant increase in hairy prometaphase cells. However, in paclitaxel treated cells and in cells treated with both MCAK RNAi and paclitaxel, the majority of the cells had multipolar spindles, as judged by the MT morphology (Fig. 3B and C) and by the observation that the multiple poles were stained by gamma-tubulin antibodies (data not shown). In the cells treated with paclitaxel alone, these multipolar spindles consisted of numerous spindle-like arrays interacting with individual populations of chromosomes within the cell (Fig. 3B). In contrast, the multipolar spindles in the MCAK RNAi cells treated with paclitaxel appeared far more disorganized and had reduced interactions with chromosomes. The chromosomes were often positioned centrally in the cell, with multiple organizing centers surrounding them but with MTs radiating out, away from the chromosomes and toward the cell cortex (Fig. 3B). In addition to the multipolar spindles in cells treated with paclitaxel alone, there was a small population of

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Figure 3. Combination of MCAK knockdown and 10 nM paclitaxel delays cells in mitosis and increases the number of dead/blebbing cells in HeLa cells. (A) Quantification of interphase and mitotic cells in the presence of MCAK knockdown and/or 24 hours of 10 nM paclitaxel treatment. (B) Mitotic cells in the presence of 10 nM paclitaxel demonstrate a multipolar phenotype. Combination of MCAK knockdown and 10 nM paclitaxel results in an increase in complex multipolar cells in which the chromosomes (blue) are not clearly attached to MTs (green). Scale bar = 10 μm. (C) Quantification of mitotic phenotypes in control or MCAK RNAi cells treated with DMSO or 10 nM paclitaxel diluted in DMSO.

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of cells with high vinblastine concentrations caused morphological defects that predominated over the effects MCAK RNAi. Our findings show that a combination of MCAK RNAi and vinblastine treatment induced increased chromosome and spindle morphology defects over either treatment alone, and therefore HeLa cells that lack MCAK are likely to be hypersensitive to treatment with vinblastine. Malignant versus non-malignant cells are differentially affected by MCAK knockdown. The morphological defects analysis combining MCAK knockdown and anti-MT drugs was performed in HeLa cells, a cervical carcinoma cell line used extensively in studies on mitosis. We also wanted to compare the effects of knockdown in other cancer derived cell lines as well as a non tumor-derived control. For our non-tumor derived cell line, we chose the telomerase immortalized cell line hTERT-RPE1, which have a normal diploid complement of chromosomes and remain flat during mitosis, making it easier to analyze morphological defects in the spindle.33 We were able to obtain more than 90% MCAK knockdown as judged by immunoblot and confirmed localization loss by immunofluorescence (Suppl. Fig. S1B). Surprisingly we found no significant cell cycle defects or mitotic defects with MCAK RNAi in these cells (Fig. 5A and B). There was a small but significant increase in prometaphase cells with disrupted spindles and excess astral MT polymer (Fig. 6A and B); however not nearly to the extent seen in HeLa cells. In addition, there were no other spindle abnormalities and no significant increase in anaphase cells with lagging chromosomes (Fig. 5B). These results show that MCAK knockdown has a greatly diminished effect on this non-tumor derived human cell line and suggest that either MCAK plays a less important role or its loss can be compensated for in normal cells. To determine whether the effects of MCAK knockdown were also prevalent in other tumor derived cells, we analyzed the human osteosarcoma cell line U2OS, another commonly used cell line for studying mitosis.17 We were able to knockdown MCAK from U2OS cells by more than 90% as judged by immunoblot and immunofluorescence (Suppl. Fig. S1C). MCAK knockdown in U2OS cells caused similar cell cycle defects as those seen in HeLa cells (Fig. 5C and D). U2OS MCAK RNAi cells displayed >2-fold increase in the percentage of multinucleate cells and >3-fold increase in the percentage of dead/blebbing cells. There was no significant increase in mitotic index (Fig. 5C), but there were morphological defects in the spindle. There was a 1.2-fold increase in the percentage of prometaphase cells upon MCAK knockdown as well as a 2.4-fold increase in cells displaying excess astral polymer in prometaphase (Fig. 6C and D). Surprisingly, MCAK RNAi alone in U2OS cells caused an increase in multipolar prometaphase cells with two-thirds of those cells displaying the complex multipolar phenotype seen in HeLa cells with a combination MCAK knockdown and paclitaxel treatment. In addition, MCAK knockdown caused an increase in the percentage of anaphase cells with lagging chromosomes (Fig. 5D). These results are consistent with the idea that cancer cells are more sensitive to MCAK loss than normal cells.

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prometaphase cells that displayed a “stuck-at-poles” phenotype, in which bipolar spindles had chromosomes distributed radially around one or both poles (Fig. 3C). This phenotype was reduced in the MCAK RNAi cells that were treated with 10 nM paclitaxel. At this concentration of paclitaxel, combination of MCAK RNAi and drug induced more deleterious effects than either treatment alone. Cells treated with 100 nM paclitaxel, the highest concentration used, had phenotypes typical of that induced by paclitaxel whether or not MCAK was knocked down (Suppl. Fig. S2C and D). These phenotypes included a strong mitotic block, predominated by multipolar cells as well as an increase in dead/blebbing cells. Thus at high concentration paclitaxel treatment, the paclitaxel effects predominated. Together the data shows that HeLa cells are differentially sensitive to the effects of paclitaxel treatment and that different concentrations of the drug result in morphological changes in the spindle that are sensitive to the presence or absence of MCAK. Another commonly used anti-neoplastic agent is the anti-MT drug vinblastine. Based on other published results32 as well as our own experimental evaluation of multiple drug concentrations, three vinblastine concentrations were used to analyze the effects of this drug individually and in combination with MCAK RNAi. When cells were treated with 0.1 nM vinblastine, there were no obvious morphological defects in either control or MCAK RNAi cells, demonstrating that treatment alone does not alter cell morphology (Suppl. Fig. S3A and B). MCAK RNAi cells treated with 3.5 nM vinblastine caused an ~1.5-fold increase in cells with multiple nuclei as compared to MCAK RNAi alone and a 2-fold increase over vinblastine treatment alone (Fig. 4A). Vinblastine treatment also caused a small but significant increase in mitotic index in both control and MCAK RNAi cells over non-drug treated controls. The percentage of cells that were dead/blebbing was unchanged by 3.5 nM vinblastine treatment (Fig. 4A). Mitotic cells were analyzed after treatment with 3.5 nM vinblastine to observe any morphological defects in mitotic spindle organization. Nearly all cells accumulated in prometaphase, similar to cells treated with paclitaxel (data not shown). Vinblastine treatment reduced the percentage of prometaphase cells that were hairy (over both control and MCAK RNAi alone), consistent with its action as a MT depolymerizing agent (Fig. 4A). In control cells treated with vinblastine, most mitotic cells formed bipolar spindles that exhibited either MT or chromosomal abnormalities (Fig. 4B). Nearly 40% of prometaphase vinblastine treated cells displayed the stuck-at-poles phenotype as compared to only 2% in control cells (Fig. 4B and C). Additionally, over one third of vinblastine treated prometaphase cells were anastral (Fig. 4B and C). MCAK knockdown rescued this anastral phenotype in vinblastine-treated cells, consistent with the idea that MCAK inhibition and vinblastine treatment have antagonistic effects on MT polymer levels in mitotic cells. Notably, treatment of MCAK RNAi cells with vinblastine caused a significant increase in the number of cells with multipolar spindles, suggesting that this combination alters MT dynamics to result in more severe spindle defects. Finally, cells treated with 20 nM vinblastine had a significant increase in mitotic index as well as an increase in dead/blebbing cells in both control and MCAK RNAi (Suppl. Fig. S3C and D). In addition, this vinblastine concentration caused an almost complete depletion of MTs in both interphase and mitotic cells (Suppl. Fig. S3D). Similar to our findings with paclitaxel treatment, treatment 2150

Discussion Importance of MCAK in mitosis is cell type and system dependent. We found that MCAK knockdown in human cell lines results in a disruption of normal spindle morphology with an increase in astral MTs and lagging chromosomes during anaphase/telophase.

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Figure 4. Combination of MCAK knockdown and 3.5 nM vinblastine reduces normal interphase cells and increases spindle morphology defects in HeLa cells. (A) Quantification of interphase and mitotic cells in the presence of MCAK knockdown and/or 24 hours of 3.5 nM vinblastine treatment. (B) Morphological analysis of chromosomes (blue) and MTs (green) depicting the morphological defects quantified in (C). Scale bar = 10 μm. (C) Quantification of mitotic phenotypes in control or MCAK RNAi cells treated with diluent or 3.5 nM vinblastine.


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The cells with the most dramatic spindle disruptions also had errors in the segregation of chromosomes. Previous studies have indicated MCAK function in accurately segregating chromosomes across many cell types;17,22,23 however, we did not find lagging chromosomes in the hTERT-RPE1 cells. It is possible the greater than diploid complement of chromosomes in HeLa and U2OS puts a higher burden on the segregation machinery to accurately segregate the chromosomes,36,37 compared to the predominantly diploid hTERT-RPE1 cells.38 Additionally, checkpoint abnormalities in malignant cells may interfere with efficient recognition of defects such as malattached kinetochores, which would also result in increased segregational errors. MCAK knockdown as a complement to current chemotherapeutic treatments. MCAK knockdown in HeLa cells increased sensitivity to anti-MT chemotherapeutics. In these cells the effects of vinblastine and paclitaxel treatments were mostly consistent with those that have been described previously.1 However, when analyzing the effects of vinblastine on HeLa cells, we found that low doses of drug result in a decrease in MTs especially astral MTs. This contrasts with a previous study that indicated low doses of vinblastine resulted in an increase in these MTs.39 Because individual cell types can have slightly different spindle morphologies, it would not be surprising that individual drugs may affect different cell lines in slightly different ways. We find the increase in mitotic spindle defects seen with the drugs individually is further enhanced by MCAK Figure 5. MCAK knockdown causes deleterious effects in malignant cells but not in normal knockdown. One possibility is that the effect on MT cells. (A and B) Quantification of interphase and mitotic cell morphogologies or anaphase dynamics by MCAK knockdown enhances the effects defects, in hTERT-RPE1 cells after MCAK knockdown. (C and D) Quantification of interphase and mitotic cell morphologies and anaphase defects in U2OS cells after MCAK knockdown. on dynamics by the drugs themselves. We think that this is unlikely because in our studies in both HeLa cells and in PtK cells, inhibition of MCAK results Previous studies of MCAK knockdown have shown a mixture of in a significant increase in MT polymer, most notably in astral MTs spindle assembly effects with some studies indicating an increase in (this study and Rizk et al., in preparation). This effect is distinct from MT polymer during mitosis,16,19,34 and others showing little effect that caused by paclitaxel treatment, which increases MT polymer on MT polymer.17,23,35 The differences in findings may result from with a more dramatic increase in spindle MT polymer (Rizk et al., cell-type specific functions of MCAK. The most dramatic spindle in preparation). On this note, it is interesting that MCAK RNAi irregularities occurred in HeLa and U2OS cell lines, which are derived can partially suppress the loss of astral MTs caused by vinblastine from malignancies and likely have many disruptions in normal regu- treatment, suggesting that at times the drug-treatment and MCAK latory mechanisms. Perhaps MCAK is expressed at differing levels in knockdown can act antagonistically to each other. We favor the idea each cell line, which alters the response to MCAK loss. Consistent that MCAK knockdown may cause the cells to be hypersensitive to with this idea, we found that MCAK protein level relative to tubulin any treatment that perturbs the cytoskeleton or cell cycle progression. levels in a population of cells is increased approximately 3-fold in Since the two cancer cell lines studied seem more sensitive to MCAK HeLa and U2OS cells over that seen in hTERT-RPE1 cells (Suppl. depletion alone relative to the noncancerous cell line, it would not be Fig. S1D). However, given that we are able to substantially decrease surprising if cancer lines in general would turn out to be more sensiMCAK levels via RNAi in all cell lines tested, the remaining levels of tive to the combination of drug treatment and MCAK depletion, MCAK are likely not significantly different. While the number of cells regardless of which mechanism causes this sensitivity. MCAK as a novel target for drug development. Our data support with spindle defects and increased astral MTs in the hTERT-RPE1 was lower than in the other cell lines, these cells have higher levels of the idea that MCAK would be a viable target for new chemoastral MTs in control cells so any subtle increase in the quantity of therapeutic development. Inhibition of MCAK caused an increased astral MTs after MCAK RNAi may not appear as dramatic and thus sensitivity of HeLa cells to the tested anti-MT agents. Ideally this analysis should be expanded to include additional tumor-derived and may have not been scored in our visual analysis. 2152

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Figure 6. MCAK knockdown disrupts spindle morphology in cells, most strongly in malignant cell lines. (A) Micrographs of luciferase RNAi (top) or MCAK RNAi (bottom) in hTERT-RPE1 cells that were stained to visualize DNA (blue) or MTs (green). (B) Quantification of spindle morphology in hTERT-RPE1 prometaphase cells. Cell scoring (C) Micrographs of luciferase RNAi (top) or MCAK RNAi (bottom) in U2OS cells that were stained to visualize DNA (blue) or MTs (green). (D) Quantification of spindle morphology in U2OS prometaphase cells. Scale Bar = 10 μm for (A and C).

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non-tumor derived cell lines. To date we have investigated the effects of MCAK knockdown on six additional cell lines, including a graded series of breast cells (tumor and non-tumor) as well as two additional immortalized RPE cells. We were unable to achieve knockdown of greater than 75% in these other cell lines, which made our studies inconclusive. We did not find any striking defects in the effects of anti-mitotic drug treatment in combination with MCAK inhibition, but this could be either because there was no effect or more likely because the MCAK knockdown levels were not sufficient to disrupt cell function. Consistent with this idea, we showed previously that partial MCAK depletion gave more modest defects in MT morphology of spindles assembled in Xenopus egg extracts.40 Certainly the availability of a small molecule inhibitor for MCAK would simplify such an analysis. In addition resistance to paclitaxel and vinblastine has been shown to be conferred in cancer cell lines by the upregulation of the MDR/P-glycoprotein genes.41 Drug development has focused on new drugs that are resistant to the function of this transporter,42 and perhaps synthesis of MCAK inhibitors would alleviate this problem. In vitro studies have also found that mutations in tubulin subunits cause cell lines to have conferred a resistance to paclitaxel43,44 and vinblastine.45,46 MCAK may function at a site distinct from paclitaxel and vinblastine on the MT, and so its www.landesbioscience.com

inhibitory effects may not be altered by these mutations, an idea that remains to be tested in the laboratory. Our findings that MCAK inhibition alone and in combination with low levels of paclitaxel causes an increase in the percentage of cells that appear to be blebbing or dying would also be a beneficial feature for an anti-mitotic agent. Consistent with this idea, it was recently reported that RNAi-mediated knockdown of MCAK in two different breast cancer cell lines significantly affected both their growth rate and colony formation ability.29 This supports the idea that inhibition of MCAK affects the growth rate of cells, although this may be cell-type dependent as inhibition of MCAK had only a modest effect on the proliferation of human leukemic cells.34 It is currently not clear how or even whether MCAK knockdown affects cell death. We explored whether knockdown of MCAK caused an increase in apoptosis, and there was no increase in a sub-2N population of cells after MCAK knockdown either alone or in combination with intermediate concentrations of paclitaxel or vinblastine (Fig. S4). It is possible that the cells die via necrosis rather than by apoptosis. Alternatively it is possible that MCAK knockdown causes cell morphology changes, such as membrane blebbing, due to defects in MT interactions with the cell cortex. In either situation, a detailed analysis of the effects of MCAK inhibition on multiple cell

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Cell culture. HeLa cells were cultured at 37°C, 5% CO2 in Opti-Mem I (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 units/ ml streptomycin, and 2 mM L-glutamine or 2 mM Glutamax (Invitrogen, Carlsbad, CA). hTERT-RPE1 and U2OS cells were cultured in D-MEM medium; all were supplemented as above. Oligonucleotide design and transfection. SiRNA oligonucleotides to human MCAK (GAUCCAACGCAGUAAUGGUdTdT) and to luciferase were synthesized by Dharmacon (Lafayette, CO). Initial experiments used RNase free water as a negative control. Later experiments used a luciferase siRNA for a negative control, and no difference in results for control experiments were found. In addition, we also performed two positive controls using primers to Tog16 and Eg5.19 All of the defects reported in this paper are specific to the siRNA-mediated knockdown of MCAK. Cells were plated onto 6-well plates at a density of 6000 cells/well for HeLas and at 10,000 cells/well for U2OS and hTERT-RPE1 cells. When needed, poly-L-lysine coverslips were included in the wells for subsequent immunofluorescence analysis. Approximately 36–40 h after plating, cells were transfected in appropriate culture medium with 10% FBS and 2 mM L-glutamine but without antibiotics, designated as transfection media. To transfect cells, for each well, 200 pmol of oligonucleotide was diluted in 185 μL of transfection media. Three microliters of oligofectamine (Invitrogen, Carlsbad, CA) and 12 μL of transfection media were incubated for 5 minutes prior to addition of siRNA mix. After 20 min, 800 uL of transfection media was added, and the mixture was added to a single well. 24 hours post transfection 1.5 ml of transfection media, with or without drugs, was added to each well. Cells were harvested or processed for immunofluorescence 48 hours post-transfection. Generation of hsMCAK specific antibody. Amino acids 2–154 of human MCAK (hsMCAK) were expressed as a Glutathione S-transferase (GST) fusion protein for rabbit antibody production (Covance Inc., Denver, PA). A 6-His-GFP fusion protein was coupled to Affi-gel (Bio-Rad, Hercules, CA) to affinity purify the antibody according to published procedures.47 Drug treatment. Paclitaxel and vinblastine (Sigma-Aldrich, St. Louis, MO) were made as concentrated stocks in cell culture grade DMSO or RNase free water, respectively. Working stock solutions were made at concentrations sufficient to add the smallest volume of drug possible, which was a maximum of 0.1% of the total volume of media. Control treatment for both drugs used the diluent at an equal volume. All drug treatments lasted for 24 h, and drugs were always added to cells 24 h post-transfection of the siRNA. Immunofluorescence. Cells were processed for immunofluorescence as described previously.22 Cells were fixed for 20 min in PHEM (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, pH 6.9) plus 4% formaldehyde and 0.1% glutaraldehyde for MT visualization or 2% formaldehyde for MCAK visualization. For MT fluorescence, DM1α (Sigma-Aldrich, St. Louis, MO) was used at 1/500 or 1/1000 dilution. Anti-hsMCAK-NT-154 was used at 1 μg/ml. Anti-Centromere antibody (ACA) (Antibodies Incorporated, Davis, CA) was diluted 1/50 when co-staining for MTs and 1/100

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Materials and Methods

when co-staining for MCAK. Fluorescent-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were used at 1/50 for MTs, and 1/50 for MCAK, and 1 ug/ml antihuman Alexa 488 or Alexa 594 (Invitrogen, Carlsbad, CA) was used for ACA. DNA was stained with 2 μg/ml Hoechst in TBS-TX. Immunoblotting. Cell extracts were prepared as described previously.48 Cells were counted and brought to equal cell concentration and then diluted with 2X Sample Buffer.47 Cell extracts were electrophoresed through 7.5% SDS-PAGE and transferred to nitrocellulose. Blots were blocked with 5% non-fat dry milk (NFDM) in TBST (20 mM Tris, 150 mM NaCl, pH 7.5, 0.1% Tween 20) plus 0.1% NaN3 for 60 minutes or overnight. Blots were probed with primary antibodies, 1 μg/ml hsMCAK-NT-154 and 1/5000 DM1α, diluted into Ad-Dil (2% BSA in TBST + 0.1% NaN3) for 60 min followed by incubation with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ) in NFDM block for 60 min and then developed with SuperSignal West Pico chemiluminescent substrates (Pierce, Rockford, IL) and exposed to film (Hyperfilm™ ECL, GE Healthcare, Piscataway, NJ). Densitometry measurements were done using Image J (NIH, Bethesda, MD). MCAK and tubulin levels were calculated by subtracting background values from protein band values. MCAK levels were normalized to the pixel intensity of the corresponding tubulin control levels within each lane. When comparing the amount of MCAK knockdown in a given cell line, all samples from that experiment were processed on the same blot to be able to normalize intensities. When comparing the expression level of MCAK between different cell lines, samples from all the cell lines were run simultaneously on one blot so that tubulin levels could be normalized between cell lines. All calculations were done using Microsoft Excel (Redmund, WA). Microscopy, image acquisition and quantification. Cells were imaged on a Nikon 90i microscope with a 100x/1.3 NA Plan Fluor oil objective (Nikon, Melville, NY). Digital images were collected with a Photometrics Coolsnap HQ cooled CCD camera (Roper Scientific, Trenton, NJ). Camera, shutters, and filter wheels were controlled by MetaMorph software (Molecular Devices, Downington, PA). For MCAK localization, cells were imaged with Z-series optical sections through prophase cells at 0.5-μm steps. Localization comparisons were taken from same experiment dates with identical acquisition times. For interphase and spindle morphology, cells were imaged with Z-series optical sections at 0.2-μm steps at exposure times that best represented the images seen in the microscope. Z-series images were deconvolved using AutoDeblur software (Media Cybernetics, Silver Spring, MD) and presented as reconstructed maximally projected images. Micrographs were assembled in Adobe Photoshop CS (Adobe Systems Inc., San Jose, CA) for equivalent contrast enhancement. Cell counting and statistical analysis. Three hundred cells were counted per condition and categorized as either normal, multinucleate, mitotic or dead/blebbing. For mitotic stage, approximately 100 mitotic cells were counted per condition and categorized based upon the individual phenotypes. Data represents mean +/- SD from at least three independent experiments. An F-test variance analysis was done independently for each data set and used to determine the appropriate Student’s t-test. t-tests were performed and significant differences determined when p-values were < 0.05. Flow cytometry. HeLa cells were seeded into 100 mm plates at 250,000 cells per plate approximately 24 hours pre-transfection and

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types with regards to proliferation is an important avenue of future investigation.

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We thank Susan Kline for U2OS cells and general help with cell culture, Alexei Mikhailov for hTERT-RPE1 cells and Jennifer DeLuca for suggestions on the transfection protocol. We also thank Chantal LeBlanc and Stephanie Ems-McClung for comments on the manuscript and all members of the Walczak lab for suggestions on this work. We thank Bill Saunders for suggestions and discussion throughout the course of this work. This work was supported by an ACS Scholar Award #RSG CSM-106128 to CEW and in part by the Indiana METACyt Initiative of Indiana University, funded in part through a major grant from the Lilly Endowment, Inc.

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BMC Cell Biol 2006; 7:26. 20. Walczak CE, Gan EC, Desai A, Mitchison TJ, Kline-Smith SL. The microtubule-destabilizing kinesin XKCM1 is required for chromosome positioning during spindle assembly. Curr Biol 2002; 12:1885-9. 21. Andrews PD, Ovechkina Y, Morrice N, Wagenbach M, Duncan K, Wordeman L, Swedlow JR. Aurora B regulates MCAK at the mitotic centromere. Dev Cell 2004; 6:253-68. 22. Kline-Smith SL, Khodjakov A, Hergert P, Walczak CE. Depletion of centromeric MCAK leads to chromosome congression and segregation defects due to improper kinetochore attachments. Mol Biol Cell 2004; 15:1146-59. 23. Maney T, Hunter AW, Wagenbach M, Wordeman L. Mitotic centromere-associated kinesin is important for anaphase chromosome segregation. J Cell Biol 1998; 142:787-801. 24. Lan W, Zhang X, Kline-Smith SL, Rosasco SE, Barrett-Wilt GA, Shabanowitz J, Hunt DF, Walczak CE, Stukenberg PT. 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into 35 mm wells at 30,000 cells per well along with coverslips to test for knockdown by IF. The siRNA transfection was scaled up and performed as described previously. At 24 post-transfection, media with or without drug treatment was added. At 48 h, post-transfection, the coverslips were processed for IF as described previously, and the 100 mm plates were harvested by trypsinization, saving all media and rinses. The cells were pelleted at 300 x g for 20 min, then rinsed in 1 ml cold PBS, repelleted, and resuspended in 500 μl PBS. A 50 μl aliquot was saved for cell counts, and the rest were fixed with 5 mls ice cold absolute ethanol and stored at -20°C. Two million cells from each condition were pelleted as above, rinsed with cold PBS, then stained with 500 μl of 20 μg/ml propidium iodide in PBS containing 0.1% Triton X-100 and 200 μg/ml RNAse A for 15 minutes in the dark.49,50 Samples were then stored on ice, and analyzed with FacsCaliber (Becton Dickinson) flow cytometer. The data acquisition software was CellQuestPro and then analyzed on Modfit LTv3.0 (Verity Software House) for sub 2N population, as well as for the percentage of cells within G1, G2/M and S-phases. The histograms were generated using WinMDI 2.9 software (Joe Trotter, The Scripps Institute, Flow Cytometry Core Facility).

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Supplementary materials can be found at: www.landesbioscience.com/supplement/HedrickCC7-14-Sup.pdf References

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