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Research Article

Kinetic Stabilization of Microtubule Dynamics by Estramustine Is Associated with Tubulin Acetylation, Spindle Abnormalities, and Mitotic Arrest Renu Mohan and Dulal Panda

School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Mumbai, India

Abstract combination showed a significant increase in the overall survival Estramustine (EM) alone or in combination with other and also a significant reduction in the risk of death (5). In addition, anticancer agents is clinically used for the treatment of EM when used singly was found effective in advanced breast cancer hormone refractory prostate cancer. Furthermore, EM has (6) and in combination with docetaxel was found to increase the been shown to potently inhibit the proliferation of different overall survival and improve the quality of life of patients having types of cancer cells in culture apparently by targeting micro- refractory metastatic breast carcinoma (7). EM is given via the oral tubules; however, the antiproliferative mechanism of action of route in the form of EM phosphate (EMP) with 70% to 75% of the EM is not clear. In this work, we have shown that EM strongly oral dose absorbed. EMP is more soluble than the parent suppressed the dynamic instability of individual microtubules compound but is not active in cells because it does not penetrate in MCF-7 cells by reducing the rates of growing and shorten- the plasma membrane. However, it is rapidly dephosphorylated in ing excursions and increasing the time microtubule spent in the gastrointestinal tract and the dephosphorylated form predom- f the pause state. At its half maximal proliferation inhibitory inates 4 h after ingestion (8). The most important adverse effects of EM are cardiovascular and gastrointestinal toxicities, which can concentration (IC50), EM exerted strong suppressive effects on the dynamics of microtubules in MCF-7 cells without detect- be avoided by careful treatment measures (9). ably affecting either the organization or the polymerized mass Chemically, EM consists of an estradiol moiety linked to nor- nitrogen mustard by a carbamate bridge. Originally, it was designed of microtubules. At relatively high concentrations (5 IC50), EM significantly depolymerized microtubules in the cells. to treat breast cancer based on the notion that the estradiol moiety Furthermore, the microtubules were found highly acetylated, may specifically direct the nor-nitrogen mustard to the breast supporting the conclusion that they were stabilized by the cancer cells, wherein the alkylating activity of the nor-nitrogen drug. EM treatment induced spindle abnormalities in MCF-7 mustard can kill the breast cancer cells (10). However, contrary to cells, and a major population of the arrested mitotic cells was this idea, EM was found to be highly effective in treating prostate multipolar. EM also perturbed the microtubule-kinetochore cancer patients. It inhibits cell proliferation and induces mitotic interaction, thereby activating the spindle assembly check- arrest in many types of cancer cells but is especially active in point and leading to apoptotic cell death. [Cancer Res 2008; prostate cancer cells (11). The high efficacy of EM against prostate 68(15):6181–9] cancer cells is thought to be due to the presence of an EM binding protein in these cells (12). The antitumor activity of EM is thought to be due to its action Introduction on microtubules. EM has been found to bind weakly to Estramustine (EM), a conjugate of nor-nitrogen mustard and microtubule-associated proteins (MAP) and to inhibit microtubule estradiol phosphate, has become one of the most valuable drugs for assembly in vitro (13, 14). EM has been shown to bind to tubulin the treatment of hormone refractory prostate cancer (HRPC). dimers (15, 16) and weakly inhibits the polymerization of MAPs- When used alone to treat HRPC, it has shown response rates free tubulin into microtubules (16). Furthermore, EM has been ranging from 19% to 69% (1). EM has also shown promising activity shown to suppress the dynamic instability of individual MAP-free against HRPC in combination with other drugs and is currently microtubules in vitro (16). The EM binding site on tubulin has been undergoing clinical trials in combination with docetaxel, etoposide, suggested to be distinct from the colchicine and vinblastine sites carboplatin, and vinblastine (2, 3). Fizazi and colleagues (2007) (16) and may partially overlap with the Taxol-binding site in have reported that the overall survival rate for metastatic HRPC is tubulin (15). Interestingly, EMP has been shown to bind to brain significantly increased when EM is combined with docetaxel, MAPs and to depolymerize MAP-rich microtubules in vitro (17). paclitaxel, vinblastine, or ixabepilone rather than used by itself (4). Whereas the antimitotic activity of EM seems to be due to its The promising response of the combined application of EM with actions on microtubules, the mechanism by which it inhibits docetaxel led to the Southwest Oncology Group trial, which was a cell cycle progression and mitosis is poorly understood. In this phase 3–randomized study for evaluating the combination of EM study, we have shown that EM suppresses the dynamic instability and docetaxel in 770 HRPC patients. Patients treated with this of individual microtubules in living MCF-7 cells. The kinetic stabilization of microtubule dynamics occurred in the absence of a significant depolymerization of the microtubules. EM also Note: Supplementary data for this article are available at Cancer Research Online increased the acetylation levels of the interphase microtubules in (http://cancerres.aacrjournals.org/). Requests for reprints: Dulal Panda, School of Biosciences and Bioengineering, the MCF-7 cells, further supporting the idea that EM kinetically Indian Institute of Technology, Bombay, Mumbai 400076, India. Phone: 91-22-2576- stabilizes the microtubules. EM also interfered with the microtu- 7838; Fax: 91-22-2572-3480; E-mail: [email protected]. I2008 American Association for Cancer Research. bule-kinetochore interaction, thereby activating the spindle doi:10.1158/0008-5472.CAN-08-0584 checkpoint leading to apoptosis. www.aacrjournals.org 6181 Cancer Res 2008; 68: (15). August 1, 2008

ing the EGFP-tubulin were selected and maintained in media containing G418. The stable cell lines were treated with different concentrations of EM, and 48 h after the drug addition, the cells were fixed and stained with antibody against acetylated tubulin. Level of tubulin or acetyl tubulin was obtained by measuring the total fluorescence intensity (mean intensity area of the cell) of each of f50 cells observed for each drug concentration. Measurement of microtubule dynamics. GFP-tubulin transfected MCF-7 cells were seeded on to glass coverslips and treated with EM for 24 h. Before recording the dynamics, coverslips were transferred to culture media lacking phenol red in a glass-bottomed culture dish. Time lapse imaging of microtubules was carried out using an FV-500 laser scanning confocal microscope (Olympus) with a 60 water immersion objective. Fifty images of each cell were acquired at 4-s interval using fluoview software. The position of the plus ends of microtubules was tracked by Image J software, and the dynamic variables were calculated using Microsoft Excel. Life history traces were obtained by plotting the lengths of individual microtubules against time. Length changes of z0.5 Am were considered as growth or shortening events and changes of <0.5 Am for a minimum of two scans were considered to be in the pause state (neither growing nor Figure 1. EM inhibited the proliferation of MCF-7 cells by arresting cells at shortening detectably). A transition from a growth (G) or a paused (P) state mitosis. Cells were treated without or with different concentrations of EM to a shortening (S) is called a ‘‘catastrophe,’’ and a transition from a (2–20 Amol/L) for one cell cycle. The inhibition cell proliferation (n) was determined shortening state to a growth or a pause state is called a ‘‘rescue.’’ The o by the SRB assay, and the mitotic index ( ) was measured by staining the catastrophe frequency per unit time was calculated by dividing the number DNA with DAPI. Data are the average of three independent experiments. of catastrophies (transition from G to S and from P to S) by the sum total time spent in G plus P. The rescue frequency was calculated by dividing the numbers of rescues (transition from S to G and from S to P) by the total Materials and Methods time spent in S. The catastrophe and rescue frequencies per unit length Reagents. EM was a kind gift from Dr. Leslie Wilson (University of were determined by dividing the number of transitions by the length the California-Santa Barbara). Paclitaxel, sulforhodamine B (SRB), mouse microtubules have grown or shortened. Thirty microtubules, which were monoclonal anti–a-tubulin antibody, rabbit anti–g-tubulin antibody, visible for z190 s, were analyzed for each condition, and various dynamic alkaline phosphatase (ALP)–conjugated anti-mouse IgG, ALP conjugated variables were calculated as previously described (16, 21, 22). Statistical anti-rabbit IgG, FITC-conjugated anti-rabbit IgG, fetal bovine serum, and significance was calculated by the Student’s t test. bovine serum albumin were purchased from Sigma. Anti-BubR1 antibody Western analysis. The effects of EM on the polymerized mass of and Annexin V were purchased from BD PharMingen. Anti-mouse IgG- microtubules in MCF-7 cells were determined using established protocol Alexa 568 conjugate and 4¶,6-diamidino-2-phenylindole (DAPI) were (23). Western blot analysis was done as described previously (20). The blots purchased from Molecular Probes. All other reagents were of analytic grade. were incubated with mouse monoclonal anti–a-tubulin antibody (1:1,000) Cell culture. Human breast cancer (MCF-7) cells were cultured in or mouse monoclonal anti–acetyl tubulin antibody (1:1,000). Intensity of the MEM (Hi Media) supplemented with 10% FCS, 1.5 g/L sodium bicarbo- blot was determined using the Image J software, and the intensity was nate, 10 Ag/mL of human insulin, and 1% antibiotic-antimycotic solution plotted against the EM concentration. containing streptomycin, amphoterecin B, and penicillin. EM stock solu- Annexin V/propidium iodide staining. MCF-7 cells were grown in the tion was prepared in 100% DMSO and was added to the culture medium absence and presence of different concentrations of EM for 48 h. Annexin (0.1% v/v) 24 h after seeding. DMSO (0.1%) was used as a vehicle control. V/propidium iodide staining of the treated cells were carried out using the Cell proliferation assay. MCF-7 cells (0.8 105/mL) were seeded in Annexin V apoptosis kit, as described recently (24), and the cells were each well in 96-well plates for 24 h. The cells were incubated with different examined under a fluorescence microscope. A flow cytometric analysis concentrations of EM at 37jC for one cell cycle (48 h). The inhibition of cell (FACS Caliber, Becton Dickinson) of live MCF-7 cells stained with Annexin proliferation was measured by a widely used sulforhodamine assay (18, 19). V–FITC, and propidium iodide was performed to examine EM-induced cell Immunofluorescence microscopy. Microtubules, centrosomes, kinet- death (25). The data were analyzed using the Modfit LT program. ochores, BubR1, and DNA were visualized, as described previously (19, 20). Cell cycle analysis. MCF-7 cells without or with different concentrations Briefly, MCF-7 cells (0.6 105 cells/mL) seeded on glass coverslips were of EM were grown for one cell cycle, fixed in 70% (v/v) ethanol, and exposed to different concentrations of EM for one cell cycle at 37jC. Cells incubated with 10 Ag/mL Rnase and 400 Ag/mL propidium iodide at 37jC were stained with the following antibodies: mouse monoclonal anti– for 30 min. DNA content of the cells was quantified in a flow cytometer a-tubulin antibody (1:300), anti-BubR1 antibody (1:1,000), anticentromere (FACS Caliber, Becton Dickinson; ref. 26), and the cell cycle distribution was antibody (1:1,500) kindly provided by Dr. K.F. Sullivan (Scripps Research analyzed using the Modfit LT program. Institute), mouse monoclonal anti–acetyl tubulin (1:600), and rabbit anti–g-tubulin antibody (1:2,000). The secondary antibodies used were FITC-conjugated anti-mouse IgG, FITC-conjugated anti-rabbit IgG, goat Results anti-human–FITC conjugate, and Alexa 568 conjugated sheep antimouse EM inhibited proliferation of MCF-7 cells at mitosis. EM IgG. To visualize nuclei and DNA, cells were stained with 1 Ag/mL DAPI inhibited the proliferation of MCF-7 cells in a concentration- A or Hoechst 33258 (0.8 g/mL). Immunostained cells were examined with dependent manner with a half-maximal inhibitory concentration a Nikon Eclipse 2000-U fluorescence microscope and the images were (IC )of5F 1 Amol/L (Fig. 1). EM arrested the cell cycle analyzed with Image-Pro Plus software. Mitotic index was estimated by 50 progression at mitosis (Fig. 1). For example, 49 F 3% and 63 F 3% determining percentage of mitotic cells in a cell population (19). The colocalization of acetylated tubulin and tubulin upon EM treat- of the MCF-7 cells were arrested at mitosis in the presence of 5 and A ment was analyzed using GFP-tubulin transfected MCF-7 cells. MCF-7 were 10 mol/L EM, respectively, indicating that the inhibition of cell transfected with a plasmid (pEGFP-Tub) encoding a fusion protein con- proliferation by EM occurred in association with the inhibition of sisting of human a-tubulin and a green fluorescent protein (gifted by Prof. cell cycle progression at mitosis. A flow cytometry analysis using Leslie Wilson, University of California-Santa Barbara). Cells stably express- propidium iodide staining showed that EM inhibited MCF-7 cells at

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the G2-M phase (Supplementary Fig. S1). For example, 1.6%, 54.8%, EM reduced the polymerized fraction of cellular tubulin. and 57% of the cells were found to be in the G2-M phase in the At a concentration of 5 Amol/L EM, the mass of polymerized absence and presence of 5 and 10 Amol/L EM, respectively. microtubules was similar to that of control cells (Fig. 2C and D). Effects of EM on MCF-7 cell microtubules. Untreated MCF-7 Relative to the control values, 10 Amol/L EM decreased the cells displayed a regular network of interphase microtubules and a microtubule polymer mass by 15% and 25 Amol/L EM reduced it normal mitotic spindle with proper chromosome alignment at the by 29% (Fig. 2D). These results were in agreement with the metaphase plate (Fig. 2A and B). In the presence of 5 Amol/L EM, depolymerizing effects of EM on interphase microtubules as the interphase microtubules were similar to those in control visualized by immunofluorescence microscopy. cells (Fig. 2A). EM (10 Amol/L, 2 IC50) had a modest depoly- EM induces apoptotic cell death. Control MCF-7 cells merizing effect on the interphase microtubules. EM (25 Amol/L, remained viable after 48 h, as seen by the absence of Annexin V 5 IC50) caused a significant depolymerization of the micro- and propidium iodide staining (Supplementary Fig. S2A). After tubules (Fig. 2A), and the cells exhibited a spherical morphology. 48 h of EM treatment, cells were found at various stages of Disruption of the mitotic spindles in the cells occurred at much apoptosis. After 48 h, 5 Amol/L EM–treated cells were stained lower EM concentrations than those required to cause a significant positive for Annexin V alone, indicating that the cells were in early depolymerization of the interphase microtubule network (Fig. 2B). apoptosis, whereas cells treated with 10 Amol/L EM–treated cells Mitotic cells treated with EM (5 Amol/L) showed abnormal bipolar were stained positive for both Annexin V and propidium iodide, spindles, as well as unipolar and multipolar spindles with indicating them to be in the later apoptotic stages. Cells treated misaligned chromosomes around the spindle. Cells treated with with 25 Amol/L EM–stained positive for propidium iodide alone, 10 Amol/L EM also showed spindle microtubule abnormalities. suggesting that these cells were dead. Furthermore, a flow cyto- Spindles formed had either unipolar or multipolar organization. metric analysis of live MCF-7 cells stained with Annexin V/ At 25 Amol/L EM, a major proportion (>80%) of the mitotic cells propidium iodide confirmed that EM treatment killed MCF-7 cells had multipolar spindles and the chromosomes were not con- through apoptosis (Supplementary Fig. S2B). In the absence of EM, gressed to the metaphase plate. 83% of the cells were live and 4% and 10% of the cells were in early

Figure 2. Effects of EM on cellular microtubules. MCF-7 cells were incubated in the absence and presence of different concentrations (5–25 Amol/L) of EM for 48 h. Microtubules (red) stained with anti–a-tubulin antibody and DNA (blue) stained with DAPI were analyzed as described in Materials and Methods. Scale bar, 10 Am. A, effect of EM on the interphase microtubules. B, effect of EM on the spindle microtubules. C, MCF-7 cells were treated without or with different concentrations (5, 10, and 25 Amol/L) of EM for 48 h. Polymeric and soluble tubulin fraction were isolated as explained in Materials and Methods, and equal amounts of polymer and soluble tubulin fractions were resolved by SDS-PAGE followed by immunoblotting with anti–a-tubulin antibody. D, polymer fraction of tubulin was measured from the intensity of the blot, which was plotted against the EM concentration. Columns, mean; bars, SD.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2008 American Association for Cancer Research. Cancer Research and late apoptosis, respectively. About 2.6% of the cells had tubules (Fig. 3B and C); it altered each of the dynamic instability undergone death. EM treatment increased the percentage of cells variables in a concentration-dependent manner (Table 1). EM that were apoptotic/dead. For example, at 10 Amol/L EM, there was (2 Amol/L), which is less then half of its IC50 value in MCF-7 cells, an increase in the percentage of cells that were in late apoptosis suppressed the mean growth rate by 23% (from 14.7 F 5.1 to (17.3%) and the dead cells (16.8%). At 25 Amol/L EM, 22.5% of the 11.3 F 2.2 Am/min) and the mean shortening rate by 25% (from cells were dead. 20.1 F 5.1 to 15 F 4.3 Am/min). The rescue frequency, which EM strongly suppressed the dynamic instability of individual was calculated based on time, was increased by 20%, whereas the microtubules in MCF-7 cells. Consistent with the previous time-based catastrophe frequency was reduced by 15% by 2 Amol/L studies (21, 27), control microtubules (vehicle treated) were highly EM. Notably, 2 Amol/L EM increased the length based rescue dynamic, alternating between phases of growth, shortening, and and catastrophe frequencies significantly. Dynamicity, which is the pause (Fig. 3A). EM clearly suppressed the dynamics of the micro- total length grown and shortened during the measured life span of the microtubules, was reduced by 46% in the presence of 2 Amol/L EM (Table 1). A EM (5 mol/L, IC50) strongly stabilized the dynamics of the microtubules. EM (5 Amol/L) reduced the rates of growing and shortening by 25% and 32%, respectively. The mean growth length and the mean shortening length were also strongly reduced (>60%) at this concentration. Interestingly, 5 Amol/L EM showed a high increase in the length-based catastrophe and rescue frequencies. EM (5 Amol/L) also greatly increased the percentage of time microtubules spent in the pause state (72%, which was twice that of the control microtubules) and reduced the overall dynamicity by 72% (Table 1). EM increased the level of acetylated tubulin in MCF-7 cells. Tubulin acetylation is considered to be a marker of microtubule stabilization (28, 29). We performed three different experiments to show that EM increases microtubule acetylation. An indirect immunofluorescence experiment using an antibody against acetylated a-tubulin showed an increase in the level of acetylated tubulin in microtubules of EM-treated cells compared with control cells, which had very low levels of acetylated tubulin (Fig. 4A). The colocalization of microtubules and acetylated tubulin was examined using MCF-7 cells transfected with GFP-tubulin and an antibody against acetylated a-tubulin (Fig. 4B). In control cells, only a few microtubules were acetylated, whereas in the EM- treated cells there was a concentration-dependent increase in the level of acetylated tubulin. The ratio of acetylated tubulin to the GFP-tubulin for the cells treated in the absence and presence of 10 and 25 Amol/L EM were 0.92 F 0.1, 2.2 F 0.8, and 2.5 F 0.5, respectively (Supplementary Fig. S3). The level of tubulin acetylation was also determined by Western blot analysis in MCF-7 cell extracts containing the polymerized tubulin fraction (Fig. 4C). When probed with antibody against a-tubulin, the percentage of polymerized tubulin in cells treated with EM was found to decrease in a concentration-dependent manner. To examine whether the polymerized microtubules that remained after EM treatment had been stabilized by acetylation, we quantified the intensity of the two blots (Supplementary Fig. S4). The ratio of acetylated polymerized tubulin to total polymerized tubulin in the absence and presence of 5, 10, and 25 Amol/L EM were 0.9 F 0.1, 1.4 F 0.2, 1.9 F 0.3, and 2.9 F 0.3, respectively. The relative acetyl tubulin intensity was increased by 46%, 77%, and 138% when the cells were treated with 5, 10, and 25 Amol/L EM, indicating that the majority of the tubulin in the polymerized fraction of the EM- treated cells were acetylated. Together, the results showed that EM treatment caused an increase in the acetylation levels of microtubules in MCF-7 cells and indicated that the microtubules in EM- treated cells had been stabilized by the drug. Figure 3. EM strongly suppressed the dynamic instability of individual EM induced multipolarity in MCF-7 cells. Control mitotic microtubules in MCF-7 cells. Life history plots of the individual microtubules in living MCF-7 cells, measured in the absence (A) and presence of cells had two centrosomes at the poles of the bipolar spindle. Cells 2 Amol/L EM (B) and 5 Amol/L EM (C). treated with 10 and 25 Amol/L EM showed large abnormalities in

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Table 1. EM suppresses dynamic instability of interphase microtubules in live MCF-7 cells

Control 2 Amol/L EM 5 Amol/L EM

c Growth rate (Am/min) 14.7 F 5.1 11.3 F 2.2* 11.0 F 2.7 b c Growth length (Am) 3.0 F 1.8 1.4 F 0.6 1.0 F 0.3 c c Shortening rate (Am/min) 20.1 F 5.1 15.0 F 4.3 13.8 F 4.6 c Shortening length (Am) 4.8 F 3.7 3.0 F 2.0x 1.8 F 0.9 c c % Time spent in growing 38.2 F 1.7 25.4 F 1.3 14.3 F 6.1 b c % Time spent in shortening 21.8 F 7.8 18.3 F 8.6 13.0 F 5.8 c c % Time spent in pause 36.0 F 2.656 .0 F 5.4 72.3 F 9.4 c c Dynamicity (Am/min) 11.2 F 3.8 6.0 F 2.7 3.1 F 1.4 b c Rescue frequency (events/min) 5.6 F 3.0 6.7 F 3.6 8.7 F 3.0 b Catastrophe frequency (events/min) 1.7 F 0.8 1.4 F 0.8 1.2 F 0.4* c Rescue frequency (events/Am) 0.3 F 0.2 0.5 F 0.3x 0.7 F 0.4 c c Catastrophe frequency (events/Am) 0.2 F 0.1 0.5 F 0.3 0.8 F 0.4

NOTE: Data are the mean F SD. *P < 0.01. cP < 0.001. bStatistically not significant. xP < 0.05.

centrosomal organization (Fig. 5A). In EM-treated cells, multiple EM treatment caused an increase in the level of BubR1 in centrosomes with short spindles were seen around the abnormal blocked mitotic cells. The outer domain of the kinetochore centrosomes. In the multipolar spindles, the poles were stained complex contains several mitotic spindle checkpoint proteins, with g-tubulin, which indicated that the poles were associated with including Mad1, Mad2, Bub1, BubR1, and Bub3 (23). These centrosomes (Fig. 5A, right). Unipolar spindles were also observed checkpoint proteins can sense the attachment of microtubules to in EM-treated cells. The occurrence of different types of spindles in kinetochores and the tension across the sister chromatids. To MCF-7 cells upon EM treatment was quantified in f500 cells. In investigate the status of checkpoint proteins after EM treatment, the absence of EM, f80% of the mitotic cells showed bipolar the cellular localization of the checkpoint protein BubR1 was spindles, whereas only 7% and 13% of the mitotic cells in the examined in EM arrested mitotic cells (Fig. 5C). In control cells, absence of EM showed multipolar and unipolar spindle morphol- BubR1 was localized to the kinetochore region of mitotic cells. Only ogies, respectively (Supplementary Fig. S5). EM treatment induced a very small amount of BubR1 was detectable in the kineto- spindle multipolarity in a concentration-dependent manner. For chore region of the metaphase chromosomes. In the EM-treated example, 56%, 68%, and 72% of the mitotic cells were multipolar in cells, large quantities of BubR1 were localized at the kinetochores the presence of 5, 10, and 15 Amol/L EM, respectively. The of chromosomes that were not aligned at the metaphase plate occurrence of unipolar spindles was less frequent than that of (Fig. 5C). The increased level of the checkpoint protein, BubR1, may multipolar spindles. At 15 Amol/L EM, 72% of the mitotic cells were lead to the mitotic block in the EM-treated cells. multipolar whereas only f28% of the mitotic cells showed unipolar spindle organization. The results suggest that EM treatment induces spindle abnormalities associated with defects Discussion in centrosomal organization. Several of the microtubule-interacting EM alone or in combination with other anticancer agents is drugs, like Taxol, nocodazole, vinblastine, and podophyllotoxin, clinically used for the treatment of HRPC. In addition, EM was also have been shown to affect centrosome organization (30). proved to be highly effective in metastatic breast carcinoma (6, 7). EM perturbed the microtubule-kinetochore attachment in We found that low concentrations of EM strongly suppressed mitotic MCF-7 cells. The kinetochores of control metaphase the dynamic instability of microtubules in human breast cancer spindles were attached to the spindle microtubules and the chro- (MCF-7) cells without detectably altering the mass of polymerized mosomes were properly aligned at the metaphase plate (Fig. 5B). microtubules. The microtubules, which became stabilized upon EM When EM was used at its IC50 concentration (5 Amol/L) or greater treatment, were highly acetylated. The stabilization of microtubule than its IC50 concentration, the arrangement of the kineto- dynamics by EM inflicted spindle abnormalities, such as the chores was perturbed (Fig. 5B). In the case of cells treated with perturbation of microtubule-kinetochore attachment, centrosomal 5, 10, and 25 Amol/L EM, kinetochore organization was completely organization, and multipolarity, which in turn activated the spindle disrupted resulting in misalignment of chromosomes at the meta- checkpoint protein, BubR1. Mitotic defects induced by EM phase plate. At these concentrations of EM, microtubules were ultimately forced the cell to undergo apoptosis. completely depolymerized and the centromeres were scattered Western blot analysis of the polymerized microtubules suggested in the cell so that the sister kinetochores were not identifiable. that high concentrations of EM had a modest depolymerizing effect The results suggest that EM perturbs microtubule-kinetochore on the microtubules in MCF-7 cells. Although low concentrations interactions and reduces the tension exerted by microtubules (IC50 or 2 IC50) of EM had no discernible effect on the interphase on kinetochores. microtubules, high concentrations (5 IC50) of EM significantly www.aacrjournals.org 6185 Cancer Res 2008; 68: (15). August 1, 2008

Figure 4. EM increased microtubule acetylation. A, MCF-7 cells stained with antibody against acetylated tubulin. B, GFP-tubulin transfected MCF-7 cells were incubated with EM (10 and 25 Amol/L) for 48 h and stained for acetylated microtubules (red) and chromosomes (blue), as described in Materials and Methods. Scale bar, 10 Am. C, EM increases acetylation of polymer fraction of cellular tubulin. MCF-7 cells were treated with the vehicle control (0), 5, 10, and 25 Amol/L EM for 48 h. Polymeric and soluble tubulin fraction were isolated as explained in Materials and Methods and immunoblotted with anti–a-tubulin antibody or anti–acetyl tubulin antibody.

depolymerized them (Fig. 2). In contrast to its weaker depolyme- (33, 34). Separation of pericentreolar material without centriolar rizing effects on the interphase microtubules, EM showed relatively duplication may also cause multipolar spindle formation (35). stronger depolymerizing effects on the spindle microtubules. A Interference with the functions of the spindle protein NUMA pronounced effect was the formation of multipolar spindles at and/or the motor protein dynein involved in the coalescence of the higher concentrations of EM. A normal bipolar spindle has two centrosomes can also lead to multipolarity (34, 36). It is possible centrosomes and the centrosome duplication is considered to be a that EM treatment might alter any of these processes required to tightly regulated process (31). A failure to regulate the centrosome produce a normal bipolar spindle. number leads to the formation of multipolar spindles and defects Monopolar spindles were also observed in the EM-treated cells. in chromosome distribution during mitosis (32). EM treatment Monopolar spindles may be formed by the failure of centrosome resulted in the formation of multipolar spindles containing centro- separation so that the microtubules nucleate from a single nuclea- somes that stained with g-tubulin. Multiple centrosomes can be tion center. Alternatively, monopolar spindles may be formed if formed either by fragmentation of centrosomes into multiple EM directly or indirectly interferes with the function or the expres- microtubule organizing centers or by amplification of centrosomes sion of the kinesin Eg5 (37) or Kif2A (38), which are crucial for the

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2008 American Association for Cancer Research. Estramustine Suppresses Microtubule Dynamics organization of centrosomes. Monastrol is known to produce uni- kinetochores together with the accumulation of BubR1 strongly polar spindles by inhibiting Eg5 (39). Normally cancerous mitotic suggested that the microtubules were not properly attached to cells having multipolar spindles continue to progress through the the kinetochores in the EM-treated cells and the tension at the cell cycle due to a failure to arrest at the mitotic checkpoint and kinetochores was not maintained properly. EM suppressed the form multinucleated interphase cells. In the EM-treated interphase dynamics of interphase microtubules. Therefore, it is logical to cells, multinucleated cells were not observed, indicating that assume that the dynamics of spindle microtubules will be severely there was no mitotic exit. EM treatment resulted in a robust suppressed by EM treatment so that these microtubules are unable mitotic arrest, which activated the mitotic checkpoint and induced to attach properly to the kinetochores and generate sufficient apoptotic cell death. tension so as to facilitate proper chromosome segregation. Many EM at its low effective concentration (IC50) disrupted microtu- drugs that suppress microtubule dynamics are found to reduce the bule-kinetochore attachment and relatively higher concentrations tension between the sister kinetochores and to activate the spindle of EM completely disrupted kinetochore organization, thereby assembly checkpoint (42). generating pronounced effects on the important mitotic func- EM strongly inhibited the dynamic instability of microtubules in tions dependent upon proper association of microtubules with the MCF-7 cells (Fig. 3; Table 1). EM also strongly suppressed the kinetochores (Fig. 5B). It is believed that proper kinetochore- growing and shortening rates of reconstituted MAP-free micro- microtubule interaction causes a reduction in the concentration tubules in vitro (16). The suppressive effects of EM on the dynamics of spindle checkpoint proteins (40). A single unattached or impro- of individual of microtubules of MCF-7 cells were found to be perly attached kinetochore can cause an accumulation of the qualitatively similar to that of the reconstituted brain microtu- spindle checkpoint protein BubR1, preventing the cell cycle prog- bules in vitro (16). However, EM exerted comparatively stronger ression to anaphase (41). BubR1 accumulation was seen in all the effect on the overall dynamicity (72% by 5 Amol/L EM) and the mitotic cells treated with EM (Fig. 5C). The disorganization of percentage of time microtubules spent in the attenuation phase

Figure 5. Effects of EM on centrosomes, kinetochores, and BubR1 in MCF-7 cells. A, MCF-7 cells were treated without or with 5, 10, and 25 Amol/L EM and stained with g-tubulin antibody (to visualize the centrosomes) and a-tubulin (to visualize the spindle). B, effects of EM on kinetochore-microtubule attachment. Immunofluorescent images of mitotic MCF-7 cells treated with 5, 10, and 25 Amol/L EM and compared with those of the vehicle-treated (control) cells. C, EM activated the checkpoint protein BubR1. MCF-7 cells were treated with different concentrations of EM (5–25 Amol/L) for 48 h. Scale bar, 10 Am. www.aacrjournals.org 6187 Cancer Res 2008; 68: (15). August 1, 2008

(101% by 5 Amol/L EM) of cellular microtubules than the cor- to destabilize microtubules (46, 47). Furthermore, microtubules are responding values on microtubules produced in vitro (for example, hyperacetylated and stabilized when HDAC6and SIRT2 are 50 Amol/L EM reduced overall dynamicity by 61% and percentage knocked down in cells (46, 47). Both HDAC6 and SIRT2 deacetylate of time microtubules spent in the attenuation phase by 29%). The tubulin at the lysine-40 of the a-tubulin subunit. Additionally, most significant effect of EM on microtubule dynamics in vivo was North and colleagues reported that SIRT2 colocalizes and interacts the increase in the distance-based rescue and catastrophe with HDAC6(47). Recently, it has been suggested tubulin does not frequencies and the increase in the time microtubules spent in bind to either HDAC6or SIRT2, but it binds only to the SIRT2- the attenuated state. EM treatment caused a significant increase in HDAC6complex (48). Although it is not yet clarified whether the the pause state suggesting that the microtubules grew or shortened acetylation is the cause or the result of the stabilization of much shorter distances in the presence of EM than the control microtubules, it has been suggested that tubulin acetylation is a microtubules. EM perturbed the above-mentioned variables of consequence of microtubule stability (49). In addition, the finding dynamic instability strongly compared with that of epothilone (21) that the overexpression of MAPs, such as MAP1B, MAP2, or H or vinblastine (27) at their IC50 concentrations. The overall dyna- increases tubulin acetylation supports the idea that tubulin micity of microtubules in the presence of EM is lower than that of acetylation is one of the consequences of microtubule stability epothilone (21), paclitaxel (22), vinblastine (27), or nocodazole (43) (50). We believe that the kinetic stabilization of microtubule at their IC50 concentrations. EM significantly altered the length- dynamics by EM caused an increase in the acetylation level of based catastrophe and rescue frequencies, which are related to the microtubules. However, it is not clear whether EM has any direct loss or gain of stabilizing GTP caps at the ends of the microtubule. role in increasing the level of tubulin acetylation; EM might reduce The significant (4-fold) increase in the length-based catastrophe the interaction of these deacetylases to tubulin by inducing frequency suggested that a large number of microtubule ends conformational change in tubulin or it might inhibit the functional lost their GTP caps in the presence of EM. This study showed that activity of the HDAC6or SIRT2. in vivo microtubule dynamics is extremely sensitive to EM treatment Collectively, the data reveal that the antiproliferative activity of even at concentrations that do not induce microtubule depoly- EM results from its strong suppressive effects on microtubule merization, suggesting that EM induces mitotic block mainly by dynamics. Modulation of microtubule dynamics by EM, in turn, suppressing microtubule dynamics. Many of the processes associ- affected the formation of a normal bipolar spindle, arresting the ated with the formation of a proper bipolar spindle are affected cells at mitosis. Most of the EM-blocked mitotic cells were either strongly by the suppressive effect of EM on microtubule dynamics. multipolar or unipolar. The study has provided a significant insight EM treatment caused a strong increase in the level of tubulin for the antimitotic mechanism of action of EM, which might help acetylation (Fig. 4). The increase in the acetylation level of to develop a sophisticated chemotherapeutic strategy for the microtubules in EM-treated cells was found to be associated with treatment of the HRPC and breast cancer. the suppression of microtubule dynamics. Several studies have provided evidence indicating that the acetylation of tubulin is linked with the drug resistance in many cell types (28, 44, 45). For Disclosure of Potential Conflicts of Interest example, nocodazole or colchicine-resistant microtubules have No potential conflicts of interest were disclosed. been found to contain more acetylated a-tubulin than the sensitive cells (28). Furthermore, EM-resistant E4 cells were found to contain Acknowledgments nearly 2-fold higher acetylated a-tubulin than their parent DU145 q Received 2/15/2008; revised 5/31/2008; accepted 6/2/2008. cells (45). N -lysine acetylation is a reversible posttranslational Grant support: Department of Science and Technology, Government of India process mediated by the opposing activities of acetyl transferases (D. Panda) and University Grant Commission Fellowship, Government of India and deacetylases. Histone deacetylase 6 (HDAC6; ref. 46) and (R. Mohan). The costs of publication of this article were defrayed in part by the payment of page sirtuin type 2 (SIRT2; ref. 47) are the known tubulin-specific charges. This article must therefore be hereby marked advertisement in accordance deacetylases. Overexpression of both HDAC6and SIRT2 are shown with 18 U.S.C. Section 1734 solely to indicate this fact.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2008 American Association for Cancer Research. Kinetic Stabilization of Microtubule Dynamics by Estramustine Is Associated with Tubulin Acetylation, Spindle Abnormalities, and Mitotic Arrest

Renu Mohan and Dulal Panda

Cancer Res 2008;68:6181-6189.

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