Kinetic Stabilization of Microtubule Dynamics by Estramustine Is

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
Estramustine (EM) alone or in combination with other
anticancer agents is clinically used for the treatment of
hormone refractory prostate cancer. Furthermore, EM has
been shown to potently inhibit the proliferation of different
types of cancer cells in culture apparently by targeting microtubules; however, the antiproliferative mechanism of action of
EM is not clear. In this work, we have shown that EM strongly
suppressed the dynamic instability of individual microtubules
in MCF-7 cells by reducing the rates of growing and shortening excursions and increasing the time microtubule spent in
the pause state. At its half maximal proliferation inhibitory
concentration (IC50), EM exerted strong suppressive effects on
the dynamics of microtubules in MCF-7 cells without detectably affecting either the organization or the polymerized mass
of microtubules. At relatively high concentrations (5 IC50),
EM significantly depolymerized microtubules in the cells.
Furthermore, the microtubules were found highly acetylated,
supporting the conclusion that they were stabilized by the
drug. EM treatment induced spindle abnormalities in MCF-7
cells, and a major population of the arrested mitotic cells was
multipolar. EM also perturbed the microtubule-kinetochore
interaction, thereby activating the spindle assembly checkpoint and leading to apoptotic cell death. [Cancer Res 2008;
68(15):6181–9]
Introduction
Estramustine (EM), a conjugate of nor-nitrogen mustard and
estradiol phosphate, has become one of the most valuable drugs for
the treatment of hormone refractory prostate cancer (HRPC).
When used alone to treat HRPC, it has shown response rates
ranging from 19% to 69% (1). EM has also shown promising activity
against HRPC in combination with other drugs and is currently
undergoing clinical trials in combination with docetaxel, etoposide,
carboplatin, and vinblastine (2, 3). Fizazi and colleagues (2007)
have reported that the overall survival rate for metastatic HRPC is
significantly increased when EM is combined with docetaxel,
paclitaxel, vinblastine, or ixabepilone rather than used by itself (4).
The promising response of the combined application of EM with
docetaxel led to the Southwest Oncology Group trial, which was a
phase 3–randomized study for evaluating the combination of EM
and docetaxel in 770 HRPC patients. Patients treated with this
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
Requests for reprints: Dulal Panda, School of Biosciences and Bioengineering,
Indian Institute of Technology, Bombay, Mumbai 400076, India. Phone: 91-22-25767838; Fax: 91-22-2572-3480; E-mail: [email protected].
I2008 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-08-0584
www.aacrjournals.org
combination showed a significant increase in the overall survival
and also a significant reduction in the risk of death (5). In addition,
EM when used singly was found effective in advanced breast cancer
(6) and in combination with docetaxel was found to increase the
overall survival and improve the quality of life of patients having
refractory metastatic breast carcinoma (7). EM is given via the oral
route in the form of EM phosphate (EMP) with 70% to 75% of the
oral dose absorbed. EMP is more soluble than the parent
compound but is not active in cells because it does not penetrate
the plasma membrane. However, it is rapidly dephosphorylated in
the gastrointestinal tract and the dephosphorylated form predominates f4 h after ingestion (8). The most important adverse effects
of EM are cardiovascular and gastrointestinal toxicities, which can
be avoided by careful treatment measures (9).
Chemically, EM consists of an estradiol moiety linked to nornitrogen mustard by a carbamate bridge. Originally, it was designed
to treat breast cancer based on the notion that the estradiol moiety
may specifically direct the nor-nitrogen mustard to the breast
cancer cells, wherein the alkylating activity of the nor-nitrogen
mustard can kill the breast cancer cells (10). However, contrary to
this idea, EM was found to be highly effective in treating prostate
cancer patients. It inhibits cell proliferation and induces mitotic
arrest in many types of cancer cells but is especially active in
prostate cancer cells (11). The high efficacy of EM against prostate
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
on microtubules. EM has been found to bind weakly to
microtubule-associated proteins (MAP) and to inhibit microtubule
assembly in vitro (13, 14). EM has been shown to bind to tubulin
dimers (15, 16) and weakly inhibits the polymerization of MAPsfree tubulin into microtubules (16). Furthermore, EM has been
shown to suppress the dynamic instability of individual MAP-free
microtubules in vitro (16). The EM binding site on tubulin has been
suggested to be distinct from the colchicine and vinblastine sites
(16) and may partially overlap with the Taxol-binding site in
tubulin (15). Interestingly, EMP has been shown to bind to brain
MAPs and to depolymerize MAP-rich microtubules in vitro (17).
Whereas the antimitotic activity of EM seems to be due to its
actions on microtubules, the mechanism by which it inhibits
cell cycle progression and mitosis is poorly understood. In this
study, we have shown that EM suppresses the dynamic instability
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
increased the acetylation levels of the interphase microtubules in
the MCF-7 cells, further supporting the idea that EM kinetically
stabilizes the microtubules. EM also interfered with the microtubule-kinetochore interaction, thereby activating the spindle
checkpoint leading to apoptosis.
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Figure 1. EM inhibited the proliferation of MCF-7 cells by arresting cells at
mitosis. Cells were treated without or with different concentrations of EM
(2–20 Amol/L) for one cell cycle. The inhibition cell proliferation (n) was determined
by the SRB assay, and the mitotic index (o) was measured by staining the
DNA with DAPI. Data are the average of three independent experiments.
Materials and Methods
Reagents. EM was a kind gift from Dr. Leslie Wilson (University of
California-Santa Barbara). Paclitaxel, sulforhodamine B (SRB), mouse
monoclonal anti–a-tubulin antibody, rabbit anti–g-tubulin antibody,
alkaline phosphatase (ALP)–conjugated anti-mouse IgG, ALP conjugated
anti-rabbit IgG, FITC-conjugated anti-rabbit IgG, fetal bovine serum, and
bovine serum albumin were purchased from Sigma. Anti-BubR1 antibody
and Annexin V were purchased from BD PharMingen. Anti-mouse IgGAlexa 568 conjugate and 4¶,6-diamidino-2-phenylindole (DAPI) were
purchased from Molecular Probes. All other reagents were of analytic grade.
Cell culture. Human breast cancer (MCF-7) cells were cultured in
MEM (Hi Media) supplemented with 10% FCS, 1.5 g/L sodium bicarbonate, 10 Ag/mL of human insulin, and 1% antibiotic-antimycotic solution
containing streptomycin, amphoterecin B, and penicillin. EM stock solution was prepared in 100% DMSO and was added to the culture medium
(0.1% v/v) 24 h after seeding. DMSO (0.1%) was used as a vehicle control.
Cell proliferation assay. MCF-7 cells (0.8 105/mL) were seeded in
each well in 96-well plates for 24 h. The cells were incubated with different
concentrations of EM at 37jC for one cell cycle (48 h). The inhibition of cell
proliferation was measured by a widely used sulforhodamine assay (18, 19).
Immunofluorescence microscopy. Microtubules, centrosomes, kinetochores, BubR1, and DNA were visualized, as described previously (19, 20).
Briefly, MCF-7 cells (0.6 105 cells/mL) seeded on glass coverslips were
exposed to different concentrations of EM for one cell cycle at 37jC. Cells
were stained with the following antibodies: mouse monoclonal anti–
a-tubulin antibody (1:300), anti-BubR1 antibody (1:1,000), anticentromere
antibody (1:1,500) kindly provided by Dr. K.F. Sullivan (Scripps Research
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
anti-human–FITC conjugate, and Alexa 568 conjugated sheep antimouse
IgG. To visualize nuclei and DNA, cells were stained with 1 Ag/mL DAPI
or Hoechst 33258 (0.8 Ag/mL). Immunostained cells were examined with
a Nikon Eclipse 2000-U fluorescence microscope and the images were
analyzed with Image-Pro Plus software. Mitotic index was estimated by
determining percentage of mitotic cells in a cell population (19).
The colocalization of acetylated tubulin and tubulin upon EM treatment was analyzed using GFP-tubulin transfected MCF-7 cells. MCF-7 were
transfected with a plasmid (pEGFP-Tub) encoding a fusion protein consisting of human a-tubulin and a green fluorescent protein (gifted by Prof.
Leslie Wilson, University of California-Santa Barbara). Cells stably express-
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
shortening detectably). A transition from a growth (G) or a paused (P) state
to a shortening (S) is called a ‘‘catastrophe,’’ and a transition from a
shortening state to a growth or a pause state is called a ‘‘rescue.’’ The
catastrophe frequency per unit time was calculated by dividing the number
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
time spent in S. The catastrophe and rescue frequencies per unit length
were determined by dividing the number of transitions by the length the
microtubules have grown or shortened. Thirty microtubules, which were
visible for z190 s, were analyzed for each condition, and various dynamic
variables were calculated as previously described (16, 21, 22). Statistical
significance was calculated by the Student’s t test.
Western analysis. The effects of EM on the polymerized mass of
microtubules in MCF-7 cells were determined using established protocol
(23). Western blot analysis was done as described previously (20). The blots
were incubated with mouse monoclonal anti–a-tubulin antibody (1:1,000)
or mouse monoclonal anti–acetyl tubulin antibody (1:1,000). Intensity of the
blot was determined using the Image J software, and the intensity was
plotted against the EM concentration.
Annexin V/propidium iodide staining. MCF-7 cells were grown in the
absence and presence of different concentrations of EM for 48 h. Annexin
V/propidium iodide staining of the treated cells were carried out using the
Annexin V apoptosis kit, as described recently (24), and the cells were
examined under a fluorescence microscope. A flow cytometric analysis
(FACS Caliber, Becton Dickinson) of live MCF-7 cells stained with Annexin
V–FITC, and propidium iodide was performed to examine EM-induced cell
death (25). The data were analyzed using the Modfit LT program.
Cell cycle analysis. MCF-7 cells without or with different concentrations
of EM were grown for one cell cycle, fixed in 70% (v/v) ethanol, and
incubated with 10 Ag/mL Rnase and 400 Ag/mL propidium iodide at 37jC
for 30 min. DNA content of the cells was quantified in a flow cytometer
(FACS Caliber, Becton Dickinson; ref. 26), and the cell cycle distribution was
analyzed using the Modfit LT program.
Results
EM inhibited proliferation of MCF-7 cells at mitosis. EM
inhibited the proliferation of MCF-7 cells in a concentrationdependent manner with a half-maximal inhibitory concentration
(IC50) of 5 F 1 Amol/L (Fig. 1). EM arrested the cell cycle
progression at mitosis (Fig. 1). For example, 49 F 3% and 63 F 3%
of the MCF-7 cells were arrested at mitosis in the presence of 5 and
10 Amol/L EM, respectively, indicating that the inhibition of cell
proliferation by EM occurred in association with the inhibition of
cell cycle progression at mitosis. A flow cytometry analysis using
propidium iodide staining showed that EM inhibited MCF-7 cells at
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Estramustine Suppresses Microtubule Dynamics
the G2-M phase (Supplementary Fig. S1). For example, 1.6%, 54.8%,
and 57% of the cells were found to be in the G2-M phase in the
absence and presence of 5 and 10 Amol/L EM, respectively.
Effects of EM on MCF-7 cell microtubules. Untreated MCF-7
cells displayed a regular network of interphase microtubules and a
normal mitotic spindle with proper chromosome alignment at the
metaphase plate (Fig. 2A and B). In the presence of 5 Amol/L EM,
the interphase microtubules were similar to those in control
cells (Fig. 2A). EM (10 Amol/L, 2 IC50) had a modest depolymerizing effect on the interphase microtubules. EM (25 Amol/L,
5 IC50) caused a significant depolymerization of the microtubules (Fig. 2A), and the cells exhibited a spherical morphology.
Disruption of the mitotic spindles in the cells occurred at much
lower EM concentrations than those required to cause a significant
depolymerization of the interphase microtubule network (Fig. 2B).
Mitotic cells treated with EM (5 Amol/L) showed abnormal bipolar
spindles, as well as unipolar and multipolar spindles with
misaligned chromosomes around the spindle. Cells treated with
10 Amol/L EM also showed spindle microtubule abnormalities.
Spindles formed had either unipolar or multipolar organization.
At 25 Amol/L EM, a major proportion (>80%) of the mitotic cells
had multipolar spindles and the chromosomes were not congressed to the metaphase plate.
EM reduced the polymerized fraction of cellular tubulin.
At a concentration of 5 Amol/L EM, the mass of polymerized
microtubules was similar to that of control cells (Fig. 2C and D).
Relative to the control values, 10 Amol/L EM decreased the
microtubule polymer mass by 15% and 25 Amol/L EM reduced it
by 29% (Fig. 2D). These results were in agreement with the
depolymerizing effects of EM on interphase microtubules as
visualized by immunofluorescence microscopy.
EM induces apoptotic cell death. Control MCF-7 cells
remained viable after 48 h, as seen by the absence of Annexin V
and propidium iodide staining (Supplementary Fig. S2A). After
48 h of EM treatment, cells were found at various stages of
apoptosis. After 48 h, 5 Amol/L EM–treated cells were stained
positive for Annexin V alone, indicating that the cells were in early
apoptosis, whereas cells treated with 10 Amol/L EM–treated cells
were stained positive for both Annexin V and propidium iodide,
indicating them to be in the later apoptotic stages. Cells treated
with 25 Amol/L EM–stained positive for propidium iodide alone,
suggesting that these cells were dead. Furthermore, a flow cytometric analysis of live MCF-7 cells stained with Annexin V/
propidium iodide confirmed that EM treatment killed MCF-7 cells
through apoptosis (Supplementary Fig. S2B). In the absence of EM,
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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and late apoptosis, respectively. About 2.6% of the cells had
undergone death. EM treatment increased the percentage of cells
that were apoptotic/dead. For example, at 10 Amol/L EM, there was
an increase in the percentage of cells that were in late apoptosis
(17.3%) and the dead cells (16.8%). At 25 Amol/L EM, 22.5% of the
cells were dead.
EM strongly suppressed the dynamic instability of individual
microtubules in MCF-7 cells. Consistent with the previous
studies (21, 27), control microtubules (vehicle treated) were highly
dynamic, alternating between phases of growth, shortening, and
pause (Fig. 3A). EM clearly suppressed the dynamics of the micro-
Figure 3. EM strongly suppressed the dynamic instability of individual
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
2 Amol/L EM (B ) and 5 Amol/L EM (C ).
Cancer Res 2008; 68: (15). August 1, 2008
tubules (Fig. 3B and C); it altered each of the dynamic instability
variables in a concentration-dependent manner (Table 1). EM
(2 Amol/L), which is less then half of its IC50 value in MCF-7 cells,
suppressed the mean growth rate by 23% ( from 14.7 F 5.1 to
11.3 F 2.2 Am/min) and the mean shortening rate by 25% ( from
20.1 F 5.1 to 15 F 4.3 Am/min). The rescue frequency, which
was calculated based on time, was increased by 20%, whereas the
time-based catastrophe frequency was reduced by 15% by 2 Amol/L
EM. Notably, 2 Amol/L EM increased the length based rescue
and catastrophe frequencies significantly. Dynamicity, which is the
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).
EM (5 Amol/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 EMtreated 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 EMtreated 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 EMtreated cells had been stabilized by the drug.
EM induced multipolarity in MCF-7 cells. Control mitotic
cells had two centrosomes at the poles of the bipolar spindle. Cells
treated with 10 and 25 Amol/L EM showed large abnormalities in
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Estramustine Suppresses Microtubule Dynamics
Table 1. EM suppresses dynamic instability of interphase microtubules in live MCF-7 cells
Growth rate (Am/min)
Growth length (Am)
Shortening rate (Am/min)
Shortening length (Am)
% Time spent in growing
% Time spent in shortening
% Time spent in pause
Dynamicity (Am/min)
Rescue frequency (events/min)
Catastrophe frequency (events/min)
Rescue frequency (events/Am)
Catastrophe frequency (events/Am)
Control
2 Amol/L EM
5 Amol/L EM
F
F
F
F
F
F
F
F
F
F
F
F
11.3 F 2.2*
b
1.4 F 0.6
c
15.0 F 4.3
3.0 F 2.0x
c
25.4 F 1.3
b
18.3 F 8.6
c
56.0 F 5.4
c
6.0 F 2.7
b
6.7 F 3.6
b
1.4 F 0.8
0.5 F 0.3x
c
0.5 F 0.3
11.0 F 2.7
c
1.0 F 0.3
c
13.8 F 4.6
c
1.8 F 0.9
c
14.3 F 6.1
c
13.0 F 5.8
c
72.3 F 9.4
c
3.1 F 1.4
c
8.7 F 3.0
1.2 F 0.4*
c
0.7 F 0.4
c
0.8 F 0.4
14.7
3.0
20.1
4.8
38.2
21.8
36.0
11.2
5.6
1.7
0.3
0.2
5.1
1.8
5.1
3.7
1.7
7.8
2.6
3.8
3.0
0.8
0.2
0.1
c
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
centrosomes with short spindles were seen around the abnormal
centrosomes. In the multipolar spindles, the poles were stained
with g-tubulin, which indicated that the poles were associated with
centrosomes (Fig. 5A, right). Unipolar spindles were also observed
in EM-treated cells. The occurrence of different types of spindles in
MCF-7 cells upon EM treatment was quantified in f500 cells. In
the absence of EM, f80% of the mitotic cells showed bipolar
spindles, whereas only 7% and 13% of the mitotic cells in the
absence of EM showed multipolar and unipolar spindle morphologies, respectively (Supplementary Fig. S5). EM treatment induced
spindle multipolarity in a concentration-dependent manner. For
example, 56%, 68%, and 72% of the mitotic cells were multipolar in
the presence of 5, 10, and 15 Amol/L EM, respectively. The
occurrence of unipolar spindles was less frequent than that of
multipolar spindles. At 15 Amol/L EM, 72% of the mitotic cells were
multipolar whereas only f28% of the mitotic cells showed
unipolar spindle organization. The results suggest that EM
treatment induces spindle abnormalities associated with defects
in centrosomal organization. Several of the microtubule-interacting
drugs, like Taxol, nocodazole, vinblastine, and podophyllotoxin,
have been shown to affect centrosome organization (30).
EM perturbed the microtubule-kinetochore attachment in
mitotic MCF-7 cells. The kinetochores of control metaphase
spindles were attached to the spindle microtubules and the chromosomes were properly aligned at the metaphase plate (Fig. 5B).
When EM was used at its IC50 concentration (5 Amol/L) or greater
than its IC50 concentration, the arrangement of the kinetochores was perturbed (Fig. 5B). In the case of cells treated with
5, 10, and 25 Amol/L EM, kinetochore organization was completely
disrupted resulting in misalignment of chromosomes at the metaphase plate. At these concentrations of EM, microtubules were
completely depolymerized and the centromeres were scattered
in the cell so that the sister kinetochores were not identifiable.
The results suggest that EM perturbs microtubule-kinetochore
interactions and reduces the tension exerted by microtubules
on kinetochores.
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EM treatment caused an increase in the level of BubR1 in
blocked mitotic cells. The outer domain of the kinetochore
complex contains several mitotic spindle checkpoint proteins,
including Mad1, Mad2, Bub1, BubR1, and Bub3 (23). These
checkpoint proteins can sense the attachment of microtubules to
kinetochores and the tension across the sister chromatids. To
investigate the status of checkpoint proteins after EM treatment,
the cellular localization of the checkpoint protein BubR1 was
examined in EM arrested mitotic cells (Fig. 5C). In control cells,
BubR1 was localized to the kinetochore region of mitotic cells. Only
a very small amount of BubR1 was detectable in the kinetochore region of the metaphase chromosomes. In the EM-treated
cells, large quantities of BubR1 were localized at the kinetochores
of chromosomes that were not aligned at the metaphase plate
(Fig. 5C). The increased level of the checkpoint protein, BubR1, may
lead to the mitotic block in the EM-treated cells.
Discussion
EM alone or in combination with other anticancer agents is
clinically used for the treatment of HRPC. In addition, EM was also
proved to be highly effective in metastatic breast carcinoma (6, 7).
We found that low concentrations of EM strongly suppressed
the dynamic instability of microtubules in human breast cancer
(MCF-7) cells without detectably altering the mass of polymerized
microtubules. The microtubules, which became stabilized upon EM
treatment, were highly acetylated. The stabilization of microtubule
dynamics by EM inflicted spindle abnormalities, such as the
perturbation of microtubule-kinetochore attachment, centrosomal
organization, and multipolarity, which in turn activated the spindle
checkpoint protein, BubR1. Mitotic defects induced by EM
ultimately forced the cell to undergo apoptosis.
Western blot analysis of the polymerized microtubules suggested
that high concentrations of EM had a modest depolymerizing effect
on the microtubules in MCF-7 cells. Although low concentrations
(IC50 or 2 IC50) of EM had no discernible effect on the interphase
microtubules, high concentrations (5 IC50) of EM significantly
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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 depolymerizing effects on the interphase microtubules, EM showed relatively
stronger depolymerizing effects on the spindle microtubules. A
pronounced effect was the formation of multipolar spindles at
higher concentrations of EM. A normal bipolar spindle has two
centrosomes and the centrosome duplication is considered to be a
tightly regulated process (31). A failure to regulate the centrosome
number leads to the formation of multipolar spindles and defects
in chromosome distribution during mitosis (32). EM treatment
resulted in the formation of multipolar spindles containing centrosomes that stained with g-tubulin. Multiple centrosomes can be
formed either by fragmentation of centrosomes into multiple
microtubule organizing centers or by amplification of centrosomes
Cancer Res 2008; 68: (15). August 1, 2008
(33, 34). Separation of pericentreolar material without centriolar
duplication may also cause multipolar spindle formation (35).
Interference with the functions of the spindle protein NUMA
and/or the motor protein dynein involved in the coalescence of the
centrosomes can also lead to multipolarity (34, 36). It is possible
that EM treatment might alter any of these processes required to
produce a normal bipolar spindle.
Monopolar spindles were also observed in the EM-treated cells.
Monopolar spindles may be formed by the failure of centrosome
separation so that the microtubules nucleate from a single nucleation center. Alternatively, monopolar spindles may be formed if
EM directly or indirectly interferes with the function or the expression of the kinesin Eg5 (37) or Kif2A (38), which are crucial for the
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Estramustine Suppresses Microtubule Dynamics
organization of centrosomes. Monastrol is known to produce unipolar spindles by inhibiting Eg5 (39). Normally cancerous mitotic
cells having multipolar spindles continue to progress through the
cell cycle due to a failure to arrest at the mitotic checkpoint and
form multinucleated interphase cells. In the EM-treated interphase
cells, multinucleated cells were not observed, indicating that
there was no mitotic exit. EM treatment resulted in a robust
mitotic arrest, which activated the mitotic checkpoint and induced
apoptotic cell death.
EM at its low effective concentration (IC50) disrupted microtubule-kinetochore attachment and relatively higher concentrations
of EM completely disrupted kinetochore organization, thereby
generating pronounced effects on the important mitotic functions dependent upon proper association of microtubules with
kinetochores (Fig. 5B). It is believed that proper kinetochoremicrotubule interaction causes a reduction in the concentration
of spindle checkpoint proteins (40). A single unattached or improperly attached kinetochore can cause an accumulation of the
spindle checkpoint protein BubR1, preventing the cell cycle progression to anaphase (41). BubR1 accumulation was seen in all the
mitotic cells treated with EM (Fig. 5C). The disorganization of
kinetochores together with the accumulation of BubR1 strongly
suggested that the microtubules were not properly attached to
the kinetochores in the EM-treated cells and the tension at the
kinetochores was not maintained properly. EM suppressed the
dynamics of interphase microtubules. Therefore, it is logical to
assume that the dynamics of spindle microtubules will be severely
suppressed by EM treatment so that these microtubules are unable
to attach properly to the kinetochores and generate sufficient
tension so as to facilitate proper chromosome segregation. Many
drugs that suppress microtubule dynamics are found to reduce the
tension between the sister kinetochores and to activate the spindle
assembly checkpoint (42).
EM strongly inhibited the dynamic instability of microtubules in
the MCF-7 cells (Fig. 3; Table 1). EM also strongly suppressed the
growing and shortening rates of reconstituted MAP-free microtubules in vitro (16). The suppressive effects of EM on the dynamics
of individual of microtubules of MCF-7 cells were found to be
qualitatively similar to that of the reconstituted brain microtubules in vitro (16). However, EM exerted comparatively stronger
effect on the overall dynamicity (72% by 5 Amol/L EM) and the
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.
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Cancer Research
(101% by 5 Amol/L EM) of cellular microtubules than the corresponding values on microtubules produced in vitro ( for example,
50 Amol/L EM reduced overall dynamicity by 61% and percentage
of time microtubules spent in the attenuation phase by 29%). The
most significant effect of EM on microtubule dynamics in vivo was
the increase in the distance-based rescue and catastrophe
frequencies and the increase in the time microtubules spent in
the attenuated state. EM treatment caused a significant increase in
the pause state suggesting that the microtubules grew or shortened
much shorter distances in the presence of EM than the control
microtubules. EM perturbed the above-mentioned variables of
dynamic instability strongly compared with that of epothilone (21)
or vinblastine (27) at their IC50 concentrations. The overall dynamicity of microtubules in the presence of EM is lower than that of
epothilone (21), paclitaxel (22), vinblastine (27), or nocodazole (43)
at their IC50 concentrations. EM significantly altered the lengthbased catastrophe and rescue frequencies, which are related to the
loss or gain of stabilizing GTP caps at the ends of the microtubule.
The significant (4-fold) increase in the length-based catastrophe
frequency suggested that a large number of microtubule ends
lost their GTP caps in the presence of EM. This study showed that
in vivo microtubule dynamics is extremely sensitive to EM treatment
even at concentrations that do not induce microtubule depolymerization, suggesting that EM induces mitotic block mainly by
suppressing microtubule dynamics. Many of the processes associated with the formation of a proper bipolar spindle are affected
strongly by the suppressive effect of EM on microtubule dynamics.
EM treatment caused a strong increase in the level of tubulin
acetylation (Fig. 4). The increase in the acetylation level of
microtubules in EM-treated cells was found to be associated with
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
example, nocodazole or colchicine-resistant microtubules have
been found to contain more acetylated a-tubulin than the sensitive
cells (28). Furthermore, EM-resistant E4 cells were found to contain
nearly 2-fold higher acetylated a-tubulin than their parent DU145
cells (45). N q-lysine acetylation is a reversible posttranslational
process mediated by the opposing activities of acetyl transferases
and deacetylases. Histone deacetylase 6 (HDAC6; ref. 46) and
sirtuin type 2 (SIRT2; ref. 47) are the known tubulin-specific
deacetylases. Overexpression of both HDAC6 and SIRT2 are shown
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Received 2/15/2008; revised 5/31/2008; accepted 6/2/2008.
Grant support: Department of Science and Technology, Government of India
(D. Panda) and University Grant Commission Fellowship, Government of India
(R. Mohan).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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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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