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Department of Molecular, Cellular, and Developmental Biology University of California Santa Barbara, Santa Barbara, California 93106 [T. O., L. W., M. A. J.], and Centre de Recherche Pierre Fabre, 81106 Castres Cedex, France [B. T. H.]
2 To whom requests for reprints should be addressed, at Department of Molecular, Cellular, and Developmental Biology, University of California Santa Barbara, Santa Barbara, CA 93106. Phone: (805) 893-5317; Fax: (805) 893-4724; E-mail: jordan{at}lifesci.ucsb.edu
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Introduction |
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All three of the Vinca alkaloids block mitosis at the metaphase/anaphase transition, leading to apoptosis (7–9). We have hypothesized that suppression of microtubule dynamics during mitosis is responsible for the ability of Vinca alkaloids to inhibit mitotic progression and cell proliferation. Microtubules are intrinsically dynamic polymers, undergoing two kinds of dynamic behaviors, called dynamic "instability" and "treadmilling." Dynamic instability is the stochastic switching of microtubule ends between episodes of prolonged growing and rapid shortening (10). Treadmilling is net growing at microtubule plus ends and net shortening at minus ends (11–13). Both extensive dynamic instability and treadmilling (or flux) occur in mitotic spindles. The rapid dynamics of spindle microtubules play a critical role in the intricate movements of the chromosomes (14, 15) and may play a crucial role in passage through the metaphase/anaphase checkpoint.
Considerable data have suggested that suppression of microtubule dynamics by Vinca alkaloids is responsible for their ability to block mitosis. All three of the Vinca alkaloids suppress both microtubule treadmilling and dynamic instability in vitro with bovine brain microtubules (7). In addition, in living cells, low concentrations of VBL suppress the growing and shortening dynamics of microtubules during interphase, at the same drug concentrations that block mitosis and inhibit cell proliferation (16). The spindle abnormalities induced by the Vinca alkaloids also suggest that the drugs may act to alter microtubule dynamics during mitosis (8). Whereas the Vinca alkaloids act specifically during mitosis, it has not been possible to visualize the dynamics of individual microtubules in mitotic cells.
In this work, we used CENP-B labeled with GFP to mark the plus ends of kinetochore-microtubules to analyze the effects of VFL, VNB, and VBL on the dynamics of centromeres, and their attached spindle microtubules during mitosis. The kinetochores lie just outside and adjacent to the centromeres, which contain CENP-B. After nuclear envelope breakdown, the microtubules emanating from opposite spindle poles become attached at their plus ends to the centromeres at the kinetochores. These kinetochore-microtubules and their associated motor proteins are responsible for the movements of the chromosomes. During metaphase, before passage through the metaphase-anaphase checkpoint, the two tethered centromeres of each pair of sister chromatids with their attached kinetochore-microtubules alternately stretch apart from each other and return to a relaxed position (17). Elastic heterochromatin between the sister centromeres contains proteins involved in maintaining sister-chromatid cohesion during stretching and relaxing.
We find that all three of the drugs suppress centromere dynamics similarly at the concentrations that block mitosis at the metaphase/anaphase transition. They all markedly decrease overall centromere dynamicity, the rate of relaxation (or coming-together), and the duration of stretching (or pulling apart) of the centromeres. Suppression of the stretching and relaxation movements of the centromeres correlates with mitotic block in a drug concentration-dependent manner, suggesting that suppression of centromere dynamic movement may lead directly to invoking the spindle checkpoint.
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Cell Proliferation and Mitotic Index.
Cells were seeded on poly-L-lysine-treated (50 mg/ml, 2 h, 37°C, washed once with sterile water) sterile glass coverslips in six-well plates at 1 x 105 cells/2 ml/well. One day later the medium was replaced with fresh medium containing VFL, VBL, or VNB (1 nM-1 µM), and additionally incubated for one cell cycle (28 h). Cells after such treatment and untreated (control) cells were harvested by combining floating cells with attached cells that had been released by trypsinization [0.5 mg/ml in PBS: 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, and 0.5 mM EDTA (pH 7.2)] for 5 min (37°C), and live cells were counted using a hemacytometer. Trypan blue dye was used to distinguish living from dead cells. Inhibition of cell proliferation was calculated from the difference in cell number for control cultures during the same time period. To evaluate mitotic indices, cells were grown for 20 h in the absence or presence of drug. Mitotic indices were determined by microscopic examination of chromosomes and GFP-CENP-B centromeres in cells that were fixed in formalin/methanol (described below), stained with 4,6-diamidino-2-phenylindole, and imaged using a Nikon Eclipse E800 microscope (Nikon, Melville, NY) with x60 and x100 (numerical aperture 1.4 for both) objectives. Results are the mean and SE of four independent experiments in which a minimum of 1000 cells was counted for each condition in each experiment. IC50s were determined by linear regression of double-reciprocal plots of proliferation or mitotic index versus drug concentration.
Immunofluorescence Microscopy.
Immunofluorescence localization of microtubules and chromosomes was performed on cells that were fixed in 10% formalin in PBS (20 min at 25°C) followed by 10 min in methanol (4°C), washed three times with PBS, and incubated for 30 min with 1% normal donkey serum (Jackson ImmunoResearch Laboratories, Ltd., West Grove, PA) to avoid nonspecific binding. Cells were incubated with rabbit anti-
-tubulin (Cytoskeleton, Denver, CO; 1:1000 dilution) and mouse antihuman histone monoclonal antibodies (Abcam Limited, Cambridge, United Kingdom; 1 h, 37°C). Cells were rinsed three times in PBS containing 1% BSA and incubated with donkey antirabbit rhodamine-conjugated secondary antibody and CY5-conjugated antimurine antibody (both from Jackson ImmunoResearch Laboratories, Ltd.; 1:1000 dilution) for 1 h at 37°C. Cells were rinsed three times in PBS-BSA and mounted with VectaShield (Vector, Burlingame, CA). Images were captured with a Nikon Diaphot 200 (Nikon) inverted confocal microscope with a x100 (numerical aperture 1.4) Nikon PlanApo lens at 3–10% laser power and on a Nikon Eclipse E800 (Nikon) microscope with x60 and x100 (numerical aperture 1.4 for both) objectives.
Imaging of Centromeres in Living Cells.
After incubation with or without drugs for 4 h to allow attainment of an equilibrium drug concentration in the cells (8), poly-lysine-coated coverslips with live cells attached were mounted in a Dvorak-Stotler chamber (Nicholson Precision Instruments, Gaithersburg, MD) in the medium in which they had been cultured (with or without drug) and maintained on the microscope stage at a temperature of 37°C in a Lucite box. Images of live cells were collected on a Nikon Diaphot 200 inverted confocal microscope with a x100 Nikon PlanApo lens with a x7 zoom at 3–10% laser power. Pairs of fluorescent centromeres were easily identified because as the cells enter mitosis, the centromere pairs are oriented perpendicular to the Z axis of the microscope. Each time course consisted of a series of 120 single images (2 Kalman images each) taken at 5-s intervals (total time 10 min), at slow scan speed and collected at 128 x 128 pixel box size (each pixel was 0.1 µm). In each time course several centromere pairs could be distinguished and followed.
Image Processing and Quantitative Motility Analysis.
Time-lapse image sequences were viewed as movies using Bio-Rad Confocal Assistant Software 4.01 to identify centromere pairs that could be tracked through the sequence for at least 5 min (60 frames). Image stacks were imported into MetaMorph (version 4.0) imaging software (Universal Imaging Corp., Downington, PA) for analysis. The x-y position assigned to a centromere was determined by the brightest pixel at the center of the fluorescent signal and its x-y position, and the distance from the sister centromere was recorded on a spreadsheet (Microsoft Excel; Microsoft Corporation, Redmond, WA). Three independent determinations were made of the position of each centromere in each frame of the movie and the results were averaged. The series of separation distances was used to determine rates of separation and relaxing, the durations of these movements, and the frequencies of transition from stretching to relaxing and vice versa.
Background Motion.
To determine how much of the observed centromere movement was attributable to simple diffusion or to electronic noise rather than to microtubule dynamics and/or motor proteins (which require microtubules for their activity), we measured centromere dynamics in the absence of microtubules. U2OS cells were incubated with 10 µM VBL for 4 h to completely depolymerize all of the microtubules. The mean center-to-center separation between sister centromeres (8 pairs in 4 cells) in the absence of microtubules was 0.58 ± 0.02 µm, and the average rates of stretching and relaxing were 0.60 ± 0.01 µm/min and 0.56 ± 0.02 µm/min, respectively. We determined the total distance that a centromere moved toward and away from its sister for the total time. Mean distance moved plotted against length of interval was 0.34 µm/min. Thus, 0.34 µm/min is the mean rate of movement in the absence of microtubules. Any movement less than or equal to this was considered to be background or diffusional movement and was classified as a "pause."
Criteria for Selection of Centromere Pairs for Measurement of Dynamics.
For determination of centromere dynamics, only cells in which the majority of chromosomes had congressed to a well-formed and distinct metaphase plate were measured and only centromere pairs of congressed chromosomes were included. We included only bipolar spindles for measurement in all of the experiments. At high drug concentration (50 nM) 
80% of spindles were bipolar, and many centromeres had not congressed.
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Spindle and Microtubule Organization, and Localization of GFP-CENP-B.
The arrangement of GFP-CENP-B, microtubules, and chromosomes in the absence and presence of VFL (4 h incubation) are shown in fixed and immunostained cells in Fig. 2, A–F. In untreated cells at metaphase (Fig. 2A), chromosomes were completely congressed to the metaphase plate, and all of the centromere pairs were oriented parallel to the spindle axis. The GFP-labeled centromeres appeared as round or elongated green spots of approximately 300–400 nm diameter. During anaphase, sister chromatids and their associated centromeres separated and the centromeres appeared as single, spherical dots (Fig. 2B). After incubation with low concentrations of the Vinca alkaloids, as shown with 5 nM VFL (Fig. 2C) and 18 nM VFL (Fig. 2D), a few spindles appeared relatively normal and bipolar with all of the chromosomes congressed to the metaphase plate. GFP-centromere pairs were similar to those in untreated cells, except that they were slightly larger and more diffuse. After incubation with 50 nM VFL, many metaphase cells had one or more chromosomes that remained uncongressed and were located at one or both spindle poles (Fig. 2E), and some spindles were tripolar or multipolar (VFL, 19.8 ± 4.3%; VNB, 18.3 ± 5.1%; VBL, 23.6 ± 4.8%; Fig. 2F).
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4 h (8). From the data presented in Figs. 6–8, it is evident that, in general, all three of the Vinca alkaloids suppressed centromere dynamics over the concentration range that blocked mitosis and inhibited cell proliferation (5–50 nM). The suppressive effects increased with increasing drug concentration in this range. For example, as shown diagrammatically in Fig. 5 for 50 nM VFL, the rate of stretching decreased only slightly (by 10%), but significantly, from 1.0 ± 0.4 µm/min in controls to 0.9 ± 0.1 µm/min at 50 nM VFL, whereas the rate of relaxation decreased significantly and strongly (by 54%), from 1.5 ± 0.4 µm/min in controls to 0.7 ± 0.2 µm/min. The durations of stretching and relaxation were both reduced strongly and significantly by 50 nM VFL (Figs. 5 and 6). VNB and VBL suppressed stretching and relaxation similarly to VFL with the exception that VNB had little effect on the duration of stretching (Fig. 6). The intercentromere distance is an indicator of tension on centromeres (20). Interestingly, 5 nM VFL (a concentration that did not induce mitotic block), increased the mean intercentromere distance by 31%, from 0.68 ± 0.09 µm in controls to 0.89 ± 0.09 µm (Fig. 9), suggesting that this low VFL concentration actually increased tension. At 18 nM VFL the distance was almost equal to that in controls, and at 50 nM VFL it was reduced by 13% to 0.59 µm (Fig. 9) indicating that 50 nM VFL reduced tension. We also calculated the mean maximal and mean minimal separation distances for each centromere pair ("Materials and Methods") and found that, as with the mean separation distance, these distances were increased by 5 nM VFL (Fig. 9). At 5 nM VNB and VBL (concentrations that induced mitotic block), the mean separation distance was not appreciably altered (by only 4–7%), but at 50 nM VBL it was strongly reduced (by 40%). Interestingly, all of the concentrations of the three Vinca alkaloids significantly increased the minimal separation distance except for 50 nM VBL (Fig. 9).
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Which Effects of the Drugs on Centromere Dynamics Might Be Responsible for Blocking Mitosis?
The most prominent effects of all three of the drugs were on the overall dynamicity, the transition frequencies, and the percentages of time spent in the stretching, relaxation, and pause phases (Table 1; Figs. 7 and 8). For example, at the concentrations closest to the IC50s for mitotic arrest (18 nM VFL, 5 nM VNB, and 5 nM VBL), the three drugs reduced the dynamicity by 44%, 25%, and 26%, they increased the time spent paused by 63%, 52%, and 36%, and they reduced the transition frequencies by 25%, 14%, and 27%, respectively.
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It is noticeable that change in the mean separation distance (Table 1; Fig. 9) was small for all three of the drugs at their IC50s, but additional analysis of the data (see "Discussion") indicates that the separation distance may contribute importantly to signaling passage through the metaphase/anaphase checkpoint.
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Discussion |
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18 nM for VFL, 5 nM for VNB, and 5 nM for VBL), centromere dynamicity was reduced by 44%, 25%, and 26%, respectively, and the amount of time the centromere pairs remained in a paused state increased by 63%, 52%, and 36%, respectively (Table 1). In addition, at their IC50s the three drugs all suppressed the relaxation rate, decreased the stretching duration, and decreased the transition frequencies. They did not greatly or consistently alter the stretching rate or the relaxation duration. In addition, the drugs induced relatively small changes in the mean separation distance. Suppression of the stretching and relaxation movements correlated strongly with inhibition of mitosis at prometaphase/metaphase suggesting that suppression of centromere dynamic movements is important in the metaphase/anaphase checkpoint.
The Spindle Checkpoint and Spindle Tension.
The metaphase checkpoint ensures that chromosomes properly attach to the mitotic spindle and align at the metaphase plate before anaphase onset. Passage through the checkpoint appears to depend on tension at the kinetochores and/or on occupancy of the kinetochores by a sufficient number of microtubules (22–24). Whereas all three of the Vinca alkaloids affected specific dynamics parameters somewhat differently, they all reduced centromere dynamicity significantly, and to similar degrees, at the concentrations that induced mitotic accumulation suggesting that the important parameters resulting in mitotic block are those of which the combined effects reduce tension at the kinetochores.
Cell Cycle Progression at the Mitotic Checkpoint Is Sensitive to Moderate Suppression of Centromere Dynamics.
Fifty nM VNB induced maximal mitotic block in U2OS cells; yet, at this concentration there was only minimal or moderate suppression of many of the parameters of centromere dynamics (Figs. 6–9). Fifty nM VNB induced only 18% reduction in the rate of stretching, 38% reduction in the rate of relaxation, 4% reduction in the mean separation, 44% reduction in the transition frequency, and 46% reduction in dynamicity. Thus, such changes appear to be sufficient to nearly completely inhibit the transition from metaphase into anaphase.
The Effects of the Vinca Alkaloids on Centromere Dynamics Correlate Well with Their Effects on Microtubule Dynamics in Vitro.
We found previously that the major effects of VFL and VNB on dynamic instability of microtubules assembled from purified brain tubulin in vitro differed significantly from those of VBL. The two newer Vinca alkaloids slowed growth rates, increased growth durations, and reduced shortening durations but in contrast with VBL, did not reduce shortening rates or increase the percentage of time the microtubules spent in an attenuated state (7). The differential effects of the three drugs on microtubule shortening rates in vitro correlate well with their effects on centromere stretching rates. The stretching rate must reflect the shortening rate of the kinetochore-attached microtubules. In mitotic cells, 50 nM VFL or VNB only minimally reduced the stretching rate (by 11–18%; Fig. 6). Similarly, 400 nM VFL or VNB did not affect the microtubule shortening rate in vitro. In contrast, 400 nM VBL strongly reduced the shortening rate in vitro (by 44%; Ref. 7) and 50 nM VBL reduced the stretching rate by 49% (Fig. 6). Centromere stretching may also involve tension exerted on the centromere pairs by poleward treadmilling or flux of the kinetochore microtubules. VBL inhibits the rate of treadmilling 7 times more strongly than VNB and 28 times more strongly than VFL (7). Thus, the effects of the three drugs on centromere stretching in living cells correlate well with their inhibitory effects both on microtubule treadmilling and dynamic instability in vitro.
It is likely that centromere relaxation involves the kinetochore-microtubules growing toward and pushing against the centromeres (21). Microtubule growing rates in vitro and centromere relaxation rates in mitotic cells were suppressed similarly by all three of the Vinca alkaloids (Ref. 7; Fig. 6).
Interestingly, the differences between the effects of the Vinca alkaloids on the durations of microtubule growth and shortening excursions observed previously in vitro (7) did not correlate with the durations of centromere stretching and relaxation excursions in cells. Specifically, 400 nM VFL and VNB increased the growth duration in vitro by >50%, but 400 nM VBL had little effect on it (7). Similarly, VFL and VNB reduced the shortening duration in vitro, but VBL increased it (7). In contrast, VFL and VBL affected the durations of relaxing and stretching similarly in cells, whereas VNB had little effect (Fig. 6). This lack of correlation is perhaps not surprising. In vitro, the microtubule plus ends are unconstrained and transition freely between phases of growth and shortening. However, in cells, the plus ends of kinetochore-microtubules are constrained because they are tethered at the kinetochores (22) and because they are connected to the opposite microtubule bundle through the elastic -satellite DNA that connects the sister chromatids. Thus, the durations of the centromere stretching and relaxation movements are probably determined by external factors and may not be as susceptible to drug action as microtubules in vitro.
At first glance, it may appear contradictory that the magnitude of the effects of the Vinca alkaloids at a concentration of 400 nM on microtubule dynamicity in vitro (suppression by 21% to 32%) were weaker than that exerted by a concentration of 50 nM on centromere dynamicity (suppression by 46% to 75%). However, this is readily explained, because Vinca alkaloids are concentrated between 130- and 430-fold in cells (8). Thus, it is likely that in U2OS cells the intracellular concentrations of the drugs after incubation with 50 nM drug were significantly higher than the 400 nM drug used in vitro.
Why Does VFL Inhibit Cell Proliferation and Block Mitosis Less Potently Than VNB or VBL?
VNB and VBL reduced cell proliferation by 50% at concentrations of 5.7 nM and 1.0 nM, respectively, whereas VFL was significantly less potent (IC50, 40 nM). Similarly, the IC50s for mitotic accumulation induced by the drugs also differed significantly, with the IC50 of VFL (18.8 nM) being much higher than those of VNB (7.3 nM) and VBL (6.1 nM). Therefore, it is surprising that many of the centromere dynamics parameters were affected similarly by similar concentrations of the three drugs. The principal exception was the centromere separation distance, which reflects the amount of tension exerted by the two tethered kinetochores. At 5 nM VFL the mean, maximal, and minimal separation distances were all increased significantly, indicating that VFL significantly increases tension. In contrast, 5 nM VNB or VBL had little effect on the separation distance. Thus, 5 nM VFL increased the tension on the kinetochore proteins, which would be predicted to facilitate passage through the mitotic checkpoint, as was observed. On the other hand, VNB and VBL had little or opposite effects, consistent with a reduction of tension and their increased ability to block mitosis. These data support the hypothesis that the centromere separation distance may play an important role in determining passage through the metaphase/anaphase checkpoint.
We note that the relationship between the IC50s for inhibition of proliferation and for mitotic block differ for the three drugs. Thus, the IC50 for mitotic accumulation is half the IC50 for inhibition of proliferation for VFL, equal to the IC50 for VNB, and 6-fold higher for VBL. This relationship suggests that interesting differences exist among the drugs in the permanence of the mitotic block they produce.
Why Does 5 nM VFL Increase the Intercentromere Distance? What Is the Role of Motor Proteins in Centromere Dynamics?
The magnitude of the intercentromere distance must depend, in part, on microtubule shortening and/or treadmilling, and/or motor proteins. VFL suppresses treadmilling in vitro significantly less potently than VNB or VBL (7); this difference may be responsible for its inability to suppress the intercentromere stretching distance at 5 nM drug. The affinities of the three drugs for tubulin differ, and VFL has fewer binding sites on tubulin than VNB, indicating that a clear difference exists in the interactions of these Vinca alkaloids with tubulin (25–28). The increased intercentromere distance induced by 5 nM VFL may result from complex interactions between the drug and kinetochore motor proteins, as well as through differential drug effects at both microtubule ends (29). The lack of a strong suppression of centromere dynamics by 5 nM VFL might facilitate the activity of motors acting at the microtubule surface, perhaps leading to a motor-driven increase in the intercentromere distance.
Taken together, the data show that reduced tension on kinetochores/centromeres as a result of suppression of microtubule dynamics by the Vinca alkaloids plays a major role in their ability to block mitosis and inhibit tumor cell growth. The results, as well as similar results with taxol (30), indicate a central role for microtubule treadmilling and dynamic instability in regulating cell cycle progression through the spindle checkpoint.
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Acknowledgments |
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Footnotes |
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2 The abbreviations used are: VBL, vinblastine; VFL, vinflunine; VNB, vinorelbine; CENP-B, centromere-binding protein B; GFP, green fluorescent protein; U2OS, human osteosarcoma cell line.
Received 10/16/02; revised 12/16/02; accepted 2/19/03.
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References |
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-satellite DNA domains visualized in situ in living human cells.J. Cell Biol.
,
135:545
–557,1996
./ß-tubulin dimer of vinorelbine and vinflunine revealed by nuclear magnetic resonance analyses.Biochem. Pharmacol.
64:733
,2002
.[CrossRef][Medline]
Jordan, M. A. Mechanism of action of antitumor drugs that interact with microtubules and tubulin.Curr. Med. Chem.–Anti-Cancer Agents
,
2:1
–17,2002
.
Panda, D., Jordan, M. A., Chin, K., and Wilson, L. Differential effects of vinblastine on polymerization and dynamics at opposite microtubule ends.J. Biol. Chem.
,
271:29807
–29812,1996
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), VNB (
), or VBL (
). Cell proliferation was determined by counting live cells at the time of drug addition and 28 h later. The IC50 for inhibition of proliferation for VFL was 40 nM, for VNB was 5.7 nM, and for VBL was <1.0 nM. Accumulation of cells in mitosis was determined by counting cells by immunofluorescence microscopy after fixation and staining of microtubules and chromatin. The IC50 for mitotic accumulation for VFL was 18.8 nM, for VNB was 7.3 nM, and for VBL was <6.1 nM. Values are mean of four independent experiments; bars, ±SE. Where error bars are not visible, they were smaller than the symbol.
