Báo cáo khoa học: Suppression of microtubule dynamics by benomyl decreases tension across kinetochore pairs and induces apoptosis in cancer cells potx

Suppression of microtubule dynamics by benomyl
decreases tension across kinetochore pairs and induces
apoptosis in cancer cells
K. Rathinasamy and D. Panda
School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India

Keywords
apoptosis; benomyl; bcl2; centrosomes;
microtubule dynamics
Correspondence
D. Panda, School of Biosciences and
Bioengineering, Indian Institute of
Technology Bombay, Powai, Mumbai,
400 076, India
Fax: +91 22 257 23480
Tel: +91 22 257 67838
E-mail:
(Received 9 May 2006, revised 27 June
2006, accepted 11 July 2006)
doi:10.1111/j.1742-4658.2006.05413.x

We found that benomyl, a benzimidazole fungicide, strongly suppressed
the reassembly of cold-depolymerized spindle microtubules in HeLa cells.
Benomyl perturbed microtubule-kinetochore attachment and chromosome
alignment at the metaphase plate. Benomyl also significantly decreased
the distance between the sister kinetochore pairs in metaphase cells and
increased the level of the checkpoint protein BubR1 at the kinetochore
region, indicating that benomyl caused loss of tension across the kinetochores. In addition, benomyl decreased the intercentrosomal distance in
mitotic HeLa cells and blocked the cells at mitosis. Further, we analyzed
the effects of benomyl on the signal transduction pathways in relation to
mitotic block, bcl2 phosphorylation and induction of apoptosis. The results

suggest that benomyl causes loss of tension across the kinetochores, blocks
the cell cycle progression at mitosis and subsequently, induces apoptosis
through the bcl2–bax pathway in a manner qualitatively similar to the
powerful microtubule targeted anticancer drugs like the vinca alkaloids and
paclitaxel. Considering the very high toxicity of the potent anticancer drugs
and the low toxicity of benomyl in humans, we suggest that benomyl could
be useful as an adjuvant in combination with the powerful anticancer drugs
in cancer therapy.

Benomyl is a benzimidazole fungicide that is widely
used in agriculture against a range of fungal diseases
of field crops, fruit trees and ornamentals. It is a broad
spectrum systemic fungicide that is selectively toxic to
microorganisms [1]. The acute toxicity of benomyl is
reported to be very low [median lethal dose (LD50) of
 10 gỈkg)1] in rats [1]. However, other studies suggested that benomyl at a single dose of larger than
100 mgỈkg)1 is capable of inducing testicular toxicity
in experimental animals [2,3]. Chronic administration
of benomyl at doses higher than 500 mgỈkg)1 in mice
was shown to cause impaired liver function and
increased liver weights [4]. Most of the toxicity studies
are performed using very high dosages of benomyl,
which are unlikely to be used at the therapeutic level

in humans. Benomyl and its major metabolite carbendazim have been shown to exhibit diffential sensitivity
against fungal tubulin and mammalian brain tubulin
[5,6]. It is believed that the selective toxicity of benomyl to fungi is due to its higher affinity for fungal
tubulin than for mammalian tubulin [5]. Benomyl is
extensively used as a research tool in fungal genetics
and cell biology [7–9]. Recently, it has been shown that

benomyl inhibits mitosis in mammalian cells, inhibits
polymerization of purified mammalian tubulin into
microtubules and suppresses dynamic instability of
reconstituted bovine brain microtubules in vitro [10].
Benomyl binds to mammalian tubulin with a moderate
affinity and the binding of benomyl to tubulin is
shown to induce conformational changes in tubulin

Abbreviations
CI, congression index; DAPI, 4¢,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; IC50, half-maximal inhibitory concentration;
PARP, poly (ADP ribose) polymerase; SRB, sulforhodamine B.

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K. Rathinasamy and D. Panda

[10]. The binding site of benomyl in tubulin is yet to
be determined. However, it has been suggested that
benomyl binds to tubulin at a site that is distinct from
the colchicine and vinbastine binding sites in tubulin
[10,11]. Although benomyl inhibits mitosis in mammalian cells, its antimitotic mechanism of action is not
clearly understood.
Microtubules are dynamic cytoskeletal polymers
which are present in all eukaryotic cells that play
important roles in various cellular processes such as
cell signaling, cell motility, organelle transport and
maintenance of cell polarity, separation of the duplicated centrosomes, and in cell division and mitosis [12–

19]. At the onset of mitosis, the interphase microtubule
network rapidly disassembles and reorganizes to form
bipolar mitotic spindles [13]. The interactions of spindle microtubules and kinetochores play an important
role in the congression of chromosomes at the metaphase plate [14]. Kinetochores are specialized pairs of
disc shaped structures that are located on either side of
the centromere, through which the chromosomes are
attached to the spindle microtubules [15,16]. The
microtubules attached to the kinetochores are called
kinetochore fibres. Several lines of evidence indicate
that the checkpoint proteins like Mad2, Bub1 and
BubR1 can sense the attachment of microtubules and
kinetochores, and the tension across the sister chromatids [17]. Kinetochores that are not attached to the
microtubules acquire increased concentration of the
motor proteins and the spindle checkpoint proteins
[18]. The interaction of kinetochores with the microtubules, resulting in the formation of the kinetochore
fibre, leads to a reduction in the concentration of the
motor proteins and spindle checkpoint proteins [19].
This reduction of checkpoint proteins is required for
inactivating the spindle checkpoint signal and progression in the cell cycle [20].
The functions of microtubules are thought to be
highly dependent on the assembly dynamics of microtubules [12,13]. It is well established that minor
perturbation of the microtubule dynamics by the
microtubule targeted drugs like the vinca alkaloids,
taxanes, noscopaine, and griseofulvin, arrest the cell
cycle progression at mitosis [13,21–24]. Hence, microtubule dynamics acts as a potential target for most of
the successful anticancer drugs. Cells arrested in the
cell cycle will be eliminated by apoptosis, executed
either through the p53 pathway or bcl2 pathway
depending upon the inducer of apoptosis. The bcl2
pathway involves the bcl2 family of proteins consisting

of the proapoptotic proteins like bax, bad, bid, bak
and the antiapoptotic proteins like bcl2, bcl-XL, bclW, and Bfl1 [25,26]. The bcl2 family of proteins have

Antiproliferative mechanism of action of benomyl

the propensity to form homodimers or heterodimers. It
is believed that the heterodimerization of bcl2 and bax
prevents the cell from undergoing bax mediated apoptosis [27]. Hence, the balance between bcl2–bax heterodimer and bax–bax homodimer determines the fate
of a cell [27,28].
In this study we found that benomyl suppressed the
regrowth of spindle microtubules in HeLa cells and perturbed the attachment of microtubules to kinetochores,
leading to mitotic irregularities in the cells arrested at
mitosis. The relatively nontoxic benomyl was, thus far,
considered to be a systemic fungicide targeting fungal
microtubules. Here, we show that benomyl also targets
mammalian microtubule assembly dynamics, disrupts
the microtubule–kinetochore interactions, decreases the
tension across the sister kinetochores, and activates the
spindle checkpoint protein BubR1 in the mitotically
arrested HeLa cells. We also present evidence indicating
that the cells blocked at mitosis were eliminated by
apoptosis through the bcl2–bax pathway. The results
suggest that the suppression of microtubule dynamics
by benomyl was the cause for the loss of tension across
the kinetochores and activation of the checkpoint proteins and induction of apoptosis.

Results
Benomyl suppressed the reassembly of spindle
microtubules in HeLa cells
HeLa cells were incubated at 2 °C for 1 h. Then, the

cold media was replaced with warm media containing
different concentrations of benomyl. Subsequently, the
cells were incubated at 37 °C in a CO2 incubator. The
cold treatment caused complete depolymerization of
mitotic spindle microtubules as observed by fluorescence microscopy after immunostaining the HeLa cells
with antia-tubulin IgG (Fig. 1). In the absence of spindle microtubules, centrosomes were located near the
spindle equator and the chromosomes were compactly
aligned at the metaphase plate (Fig. 1). In control
HeLa cells, mitotic spindle microtubules were reassembled within 5 min of incubation at 37 °C (Fig. 1),
while benomyl caused a significant delay in the reassembly of the spindle microtubules. In the presence of
5 lm benomyl, the spindles were only partially reassembled after 5 min of incubation at 37 °C (Fig. 1).
They appeared to be in the initial stage of recovery
near the centrosomes. After 10 min of incubation,
mitotic spindles had considerable amount of microtubules, although their spindle lengths (6.9 ± 1.0 lm*)
were found to be significantly shorter than that of the
control cells (10.1 ± 1.3 lm*). Mitotic cells treated

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K. Rathinasamy and D. Panda

Fig. 1. Benomyl suppressed the reassembly of cold depolymerized spindle microtubules in HeLa cells. Twenty-four hours after seeding, the
cells were cold treated (2 °C) for 1 h to disassemble the spindles. Then, the cells were incubated in a CO2 incubator at 37 °C in the absence
and presence of different concentrations of benomyl. The cells were fixed at the desired time points and stained with antic-tubulin (green),
antia-tubulin (red) IgG and DAPI (blue) to visualize the centrosomes, microtubules and DNA, respectively. Bars, 5 lm.

with 20 lm benomyl did not recover their spindle
microtubules even after 10 min at 37 °C. After 20 min
of incubation, microtubules appeared near the centrosomes and the chromosomes were found to be in
condensed structures (Fig. 1). However, the average
spindle length was measured (3.65 ± 0.64 lm*) and
shown to be considerably smaller than that of the control cells (10.1 ± 1.3 lm*) (*P < 0.01, n ¼ 15 cells).
Benomyl caused disruption of chromosomal
alignment at the metaphase plate
HeLa cells treated with vehicle alone displayed normal
spindle morphology and the mitotic chromosomes
were properly aligned at the metaphase plate (Fig. 2A).
As documented recently [10], the spindle microtubules
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in the cells treated with 5 lm benomyl appeared nearly
normal, but a few chromosomes were unable to congress at the metaphase plate (Fig. 2A). The effects of
microtubule perturbation by benomyl on the chromosomal alignment were assessed by determining the
chromosomal congression index (CI), which is a
measure of the ratio of the width to height of the
chromosomal masses of cells with metaphase type
chromosome alignment [29]. Consistent with the previous report [29], the CI for the control HeLa cells was
found to be 0.31 ± 0.05. Benomyl treatment strongly
increased the CI (Fig. 2B). For example, in the presence of 5 lm benomyl, the CI was increased by 58%
from 0.31 ± 0.05 to 0.49 ± 0.11, and the CI was
increased by 74% in the presence of 10 lm benomyl
(Fig. 2B). Finally, higher concentrations of benomyl

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K. Rathinasamy and D. Panda

Antiproliferative mechanism of action of benomyl

A

Congression Index (CI)

B

0.8

0.6

0.4

0.2

0

0

5.0
Benomyl (μM)

10.0

Fig. 2. Effects of benomyl on the spindle microtubules and chromosome organization. (A) HeLa cells were incubated with different concentrations of benomyl for 20 h. Spindle microtubules (green) and chromosomes (blue) were analyzed as described in Experimental procedures.
Bars, 5 lm. (B) The chromosomal congression index (CI) of the control and drug treated HeLa cells. The CI was calculated as described in

Experimental procedures. Error bars represent SD.

(20 lm) produced clear abnormalities in chromosomal
alignment with nearly 70% of all the mitotic cells having multiple poles with short spindles and the chromosomes were rounded to a ball shaped structure (data
not shown).
Benomyl perturbed microtubule–kinetochore
attachment and caused loss of tension across the
sister kinetochores
All the kinetochores of vehicle treated HeLa cells were
found to be attached to the spindle microtubules and
the chromosomes were properly aligned at the metaphase plate (Fig. 3A). Benomyl treatment perturbed
the attachment of microtubules to kinetochores
(Fig. 3A). In benomyl treated cells, some kinetochore
pairs were found to be attached to kinetochore fibres
from both the poles whereas others were attached to
the kinetochore fibre from one side of the pole, resulting in misalignment of chromosomes at the metaphase
plate.
It has been shown that the tension generated
between kinetochore pairs is finely regulated by the
combined action of microtubule dynamics and motor
proteins [30,31]. The tension across the paired kinetochores is thought to be proportional to the distance
between the sister kinetochore pairs [31–33]. In control
cells the distance between the sister kinetochores was

1.74 ± 0.3 lm* (n ¼ 75). This distance was significantly reduced by 29% and 34% in the presence of
5 and 10 lm benomyl, being equal to 1.25 ± 0.4 lm*
(n ¼ 96) and 1.15 ± 0.4 lm* (n ¼ 98) respectively
(*P < 0.01) (Fig. 3B).
The results suggested that benomyl perturbed
microtubule–kinetochore interactions and caused a

reduction in the tension exerted by microtubules on
kinetochores. This was further investigated by limited
cold treatment of the mitotic spindles and calcium
induced depolymerization of nonkinetochore microtubules. It has been shown that limited cold treatment disassembles most of the spindle microtubules
but keeps the kinetochore fibres intact [34]. HeLa
cells were thus incubated on ice for 10 min in the
absence and presence of benomyl. While the stable
kinetochore microtubules were present in the control cells (Fig. 4), a significant decrease in the
amount of cold stable microtubules was observed in
the presence of 5 lm benomyl (Fig. 4). At 20 lm
benomyl, almost all the spindle microtubules were
disassembled indicating a concentration dependent
perturbation of microtubule–kinetochore interactions
by benomyl (Fig. 4). Benomyl caused a similar
concentration dependent decrease in the quantity of
spindle microtubules in the presence of 1 mm calcium
chloride in a microtubule stabilizing buffer (data not
shown).

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K. Rathinasamy and D. Panda

A

24
Control

20
16
12
8
4
0
0.4

0.8

1.2

1.6

2.0

2.4

Sister Kinetochore Distance (μm)

% Total Pairs Counted

% Total Pairs Counted

B 24

5 μM

Benomyl

20
16
12
8
4
0
0.4

0.8

% Total Pairs Counted

24
20

10 μM
Benomyl

16
12
8
4
0
0.4
0.8
1.2
1.6
2.0

2.4
Sister Kinetochore Distance (μm)

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1.2

1.6

2.0

2.4

Sister Kinetochore Distance (μm)
Fig. 3. Benomyl reduced the tension at the
kinetochores. (A) Immunofluorescent images of mitotic spindle of HeLa cells, centromeres (green), DNA (blue) and merged
images of microtubules (red) and centromeres are shown. HeLa cells were treated
with 5 and 10 lM benomyl and compared
with those of the vehicle treated (control)
cells. Bars, 5 lm; inset bars, 1 lm.
(B) Benomyl decreased the distance
between the sister kinetochores. Sister
kinetochore distances were measured as
described in Experimental procedures.

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K. Rathinasamy and D. Panda

Antiproliferative mechanism of action of benomyl

Fig. 4. Effects of benomyl on the kinetochore–microtubule attachment: Cells were first incubated with different concentrations (0, 5, and
20 lM) of benomyl for 10 min at 37 °C. Then, the warm media was replaced with ice-cold media containing the same concentration of benomyl and incubated on ice for 10 min and subsequently fixed and processed for immunofluorescence using antitubulin IgG (red) and DAPI
(blue). Bars, 5 lm.

Activation of the spindle checkpoint protein
BubR1 by benomyl treatment
To investigate the status of the checkpoint protein
BubR1 following benomyl treatment, we examined
the cellular localization of the checkpoint protein
BubR1 in the benomyl arrested mitotic cells. In the
control treatment, BubR1 was localized to the
kinetochore region in the prometaphase cells and
upon chromosomal alignment the concentration of
BubR1 was decreased at the kinetochore region. In
some control cells a very small amount was detectable
in the kinetochore region of the metaphase chromosomes and in other cells BubR1 was undetectable
after the chromosomal alignment. In the presence of

benomyl (5 and 10 lm), BubR1 was localized in large
quantities at the kinetochores on both the chromosomes that are aligned at the metaphase plate and
those that are not aligned at the metaphase plate
(Fig. 5). The results indicated that the sister kinetochores are not under tension in the presence of benomyl, resulting in the inhibition of degradation of
the checkpoint protein BubR1 at the kinetochore
region.
Benomyl treatment decreased the spindle length
and intercentrosomal distance
The movement of centrosomes towards the opposite
poles is thought to be microtubule dependent and it is

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K. Rathinasamy and D. Panda

Fig. 5. Localization patterns of the checkpoint protein BubR1. Cells treated with 5 lM benomyl for 20 h were compared with the control
metaphase cells. BubR1 (green) and DNA (blue) were stained and visualized as described in Experimental procedures. Bars, 5 lm.

carried out in association with motor proteins and
other cytoskeleton network like actin filaments [35]. In
order to check the effect of benomyl on centrosomes
and the spindle length, the cells were stained to visualize centrosomes and microtubules. The distance
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between the two centrosomes in the mitotic cells that
had achieved a metaphase type of chromosomal alignment was 11.1 ± 1.5 lm* (n ¼ 60) (Fig. 6). Centrosome separation in the mitotic cells was affected
significantly in the presence of benomyl, with an

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K. Rathinasamy and D. Panda

Antiproliferative mechanism of action of benomyl

A

Distance Between the Poles

B

16
12
8
4
0

0

5.0
Benomyl (μM)

10.0

Fig. 6. Benomyl reduced the intercentrosomal distance. (A) Immunofluorescent images showing centrosomes (green), merged images of
microtubules (red) and centrosomes, and merged images of microtubules, centrosomes and DNA (blue) in metaphase HeLa cells. HeLa cells
were treated with 5 and 10 lM benomyl and compared with those of the vehicle treated (control) cells. (B) Benomyl treatment caused a
reduction of intercentrosomal distance. The distance between the intercentrosomes was measured as described in Experimental procedures. Error bars represent SD.

intercentrosomal distance of 7.9 ± 1.5 lm* at 5 lm
(n ¼ 87) (Fig. 6) (29% reduction compared to control),
and 6.9 ± 2.2 lm* at 10 lm (n ¼ 75) (38% reduction
compared to control) (Fig. 6) (*P < 0.01). The intercentrosomal distance was calculated only for those cells
having bipolar centrosomal organization. Cells treated
with 20 lm benomyl showed huge abnormalities in the

centrosomal organization; nearly 40% of all the prophase cells showed three to four centrosomal structures, while the control cells had one centrosome in
interphase and two in prophase, which were separated
by 2–10 lm in different cells.
Benomyl arrests the cells at mitosis, inhibits the
proliferation of HeLa cells and induces apoptosis
Consistent with a previous report [10], we found that
benomyl inhibited HeLa cell proliferation with an IC50
of 5 ± 1 lm (Fig. 7A) and arrested the cell cycle

progression at mitosis in a concentration dependent
fashion. In the present work, we found that the mitotic
block caused by benomyl paralleled its ability to
inhibit cell proliferation. For example, 5 and 20 lm
benomyl blocked 26% and 59% of cells at mitosis and
inhibited cell proliferation by 50% and 70%, respectively (Fig. 7A). In order to find the fate of the cells
arrested at mitosis, cells were incubated with different
concentrations (0–40 lm) of benomyl for 20 or 40 h
and the live and dead ⁄ apoptotic cells were counted
using the combination of hoechst 33342 and propidium
iodide staining. Total cells were counted by hoechst
fluorescence and the dead or apoptotic cells by propidium iodide fluorescence. After 20 h of drug treatment
8% and 23% of dead ⁄ apoptotic cells were detected at
5 and 20 lm benomyl, respectively, whereas, at the
same concentrations 19% and 42% of dead ⁄ apoptotic
cells were detected after 40 h of benomyl treatment
(Fig. 7B). The number of cells undergoing death or

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Antiproliferative mechanism of action of benomyl

75

75

50

50

25

25

0

0
0

B

benomyl from the culture media indicating that the
continued presence of benomyl is required to inhibit
cell proliferation (data not shown).

100

% Mitotic Cells

100

% Inhibition of Cell Proliferation

A

K. Rathinasamy and D. Panda

20
40
Benomyl (μM)

60

Dead cells (20 h)
Dead cells (40 h)

% Dead Cells

40

20

0

0

2

5
20
Benomyl (μM)

40

Fig. 7. Benomyl inhibited HeLa cell proliferation by arresting the
cells at mitosis and induced cell death. (A) Inhibition of proliferation
(s) and mitotic progression (m) in HeLa cells by benomyl. The inhibition of cell proliferation was determined by sulforhodamine B
(SRB) assay after incubating the cells with various concentrations
(0–60 lM) of benomyl for 20 h. The mitotic index was calculated by
DAPI staining method after incubating the cells with different concentrations (0–60 lM) of benomyl for 20 h. The experiment was
performed four times. Data represent mean ± SD. (B) Percentage
of cell death after 20 (light gray) and 40 h (dark gray) of drug incubation. Live and dead ⁄ apoptotic cells were counted after incubating
the HeLa cells with different concentrations (0–40 lM) of benomyl
for 20 h and 40 h as described in Experimental procedures. Error
bars are SD.

apoptosis increased after one cell cycle as shown in
Fig. 7B. The effects of benomyl on the inhibition of
proliferation were found to be reversible. HeLa cells
were incubated with benomyl for 4 h, the cells were
then washed with fresh media, and incubated for an
additional 20 h. Neither mitotic arrest nor inhibition
of cell proliferation was detected after the removal of
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We found that the enzyme poly (ADP ribose) polymerase (PARP) (specifically cleaved in many forms of
programmed cell death [36,37]) was cleaved in HeLa
cells upon benomyl treatment for 40 h (Fig. 8A). The

apoptosis induced by benomyl was also confirmed by
the presence of a DNA laddering pattern (Fig. 8B).
Benomyl caused an increase in the
hyperphosphorylation of bcl2

80

60

Benomyl induced apoptosis in HeLa cells was
confirmed by the cleavage of poly (ADP) ribose
polymerase and the fragmentation of DNA

After 20 h treatment of HeLa cells with benomyl, the
level of phosphorylation of bcl2 increased in a concentration dependent manner as evidenced by the
decreased mobility of the protein (Fig. 9A). At 5 lm
concentration of benomyl, which showed 50% inhibition of proliferation and 26% mitotic block, nearly
45% of the total bcl2 was hyperphosphorylated, while
at 20 lm benomyl, which caused 70% inhibition of
proliferation and 59% mitotic block, nearly 80% of
bcl2 became hyperphosphorylated. Benomyl did not
alter the overall expression of bcl2 as the total amount
of phosphorylated and unphosphorylated bcl2
remained the same as that of the control (Fig. 9A).
Dissociation of bax from bcl2 correlated with the
phosphorylation of bcl2 caused by benomyl
treatment
It has been shown that bcl2 protects cancer cells from
undergoing apoptosis, whereas its dimeric partner
bax induces apoptosis [27,38,39]. Benomyl caused an

increase in the expression of bax minimally (Fig. 9B).
The efficiency of the binding of phosphorylated bcl2 to
bax was tested by coimmunoprecipitation with antibcl2
IgG and subsequent immunoblot analysis with antibax
IgG. In the presence of benomyl, the binding of bcl2
to bax was inhibited considerably (Fig. 9C). For example, cells treated with 20 lm benomyl showed nearly
40% reduction of bax protein in the immunocomplex
precipitated by the antibcl2 IgG.

Discussion
In this study, we found that benomyl at its lower
effective concentration range (at IC50, 5 lm), strongly

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K. Rathinasamy and D. Panda

Antiproliferative mechanism of action of benomyl

A
PARP

116 KD
85 KD

β-Actin

Control

5 μM
20 μM
Benomyl

100 nM
Taxol

B
Fig. 9. Benomyl induced hyperphosphorylation of bcl2 and dissociation of bax from bcl2. HeLa cells were treated with the vehicle
control (lane 1), 5 and 20 lM benomyl (lane 2 and 3), and 100 nM
Taxol (lane 4) for 20 h. Equal amounts of cell lysates were resolved
by SDS ⁄ PAGE followed by immunoblotting with (A) antibcl2 IgG or
(B) antibax IgG. (C) Cell lysates equivalent to 150 lg of total protein
was immunoprecipitated (IP) with antibcl2 IgG, resolved by
SDS ⁄ PAGE (12% gel), and immunoblotted with antibax IgG.

Fig. 8. Benomyl induced apoptosis in HeLa cells. (A) Cleavage of
PARP by benomyl treatment. HeLa cells were treated with the indicated concentrations of drugs for 40 h and equal amounts of cell
lysates were resolved by SDS ⁄ PAGE followed by immunoblotting
with antiPARP IgG, which detects the 116 kDa protein and the
85 kDa fragments. (B) Benomyl caused fragmentation of DNA. The
DNA ladder assay was performed as described in Experimental procedures. Shown are, control after 40 h (lane 1); 20 h after 5 and
20 lM of benomyl treatment (lanes 2 and 3); and 40 h after 5 and
20 lM of benomyl treatment (lanes 4 and 5).

suppressed the reassembly of cold depolymerized spindle microtubules of HeLa cells, suggesting that benomyl perturbs spindle microtubule assembly dynamics.
At higher concentration (20 lm), benomyl suppressed
microtubule nucleation from the centrosomes. Benomyl treatment decreased the interpolar distance in the
mitotic HeLa cells, reduced the distance between sister
kinetochores, caused loss of tension across the kinetochores and activated the checkpoint protein BubR1.

Further, benomyl efficiently arrested the cells at mitosis and induced apoptosis.
The functions of microtubules are shown to be largely determined by their polymerization dynamics. The
frequent transitions between microtubule growth and
shortening are required for the congression of the
chromosomes. During congression the chromosomes

move both toward and away from the poles [30,40,41].
Finally, the chromosomes are positioned at the equatorial center because opposing forces are balanced at
the center of the equator [40]. In this study, we found
that benomyl suppressed the assembly dynamics of
spindle microtubules in HeLa cells (Fig. 1) and perturbed the organization of the chromosomes at the
metaphase plate (Fig. 2A). Benomyl has also been
shown to suppress dynamic instability of purified
microtubules in vitro [10]. Therefore, it is reasonable to
propose that the suppression of microtubule dynamics
by benomyl inhibits spindle microtubules to capture
the chromosomes and align them at the metaphase
plate.
The spindle assembly checkpoint acts as a surveillance mechanism to prevent errors during cell division.
The checkpoints can detect the attachment of microtubules and kinetochores [42,43] and they can also sense
the absence of tension at kinetochores that are
attached to microtubules [44]. The checkpoints block
the metaphase ⁄ anaphase transition until all the kinetochores have successfully attached to the spindle [42]
and sufficient tension is generated across the sister
kinetochores [43]. In the present work, we found that
benomyl decreased the distance between sister kinetochore pairs in the chromosomes aligned at the metaphase plate and those that are not aligned at the
metaphase plate. The checkpoint BubR1 was present
on both the kinetochores of the chromosomes that are
aligned and not aligned at the metaphase plate, indicating that in both cases the chromosomes were not
under tension in the presence of benomyl. The loss

of tension across the kinetochore pairs caused by

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benomyl treatment has caused the sustained activation
of the mitotic spindle checkpoints to induce metaphase
block. The reduction in the distance between sister
kinetochores observed in the presence of benomyl was
found to be comparable with the effects of well known
microtubule targeted agents [23].
It was shown by Ostergren that the poleward force on
a chromosome is proportional to the kinetochore fibre
length [45]. Hays and Salmon have demonstrated that
the net poleward force on a chromosome depends both
on the number of kinetochore microtubules as well as
the distance from the pole [46]. Kinetochore fibres exhibit cold stability [34] and show resistance to calcium
induced depolymerization [47]. We found that benomyl
decreased the quantity of kinetochore microtubules as
observed by the limited cold treatment of HeLa cells
and the calcium induced depolymerization of spindles,
which depolymerized all the microtubules except those
microtubules that are attached to the kinetochores.
At higher concentration of benomyl, the spindle

microtubules are completely depolymerized indicating
that benomyl reduced the microtubule–kinetochore
interactions. Benomyl treatment decreased the spindle
length and the interpolar distance. The decreased interpolar distance together with the reduced number of stable kinetochore microtubules might have decreased the
poleward force on the chromosomes required to segregate the sister chromatids during mitosis.
Cells treated with higher benomyl concentration
(20 lm) caused fragmentation of the centrosomes
resulting in three to four centrosomes in the prophase
cells leading to multipolar mitosis. Following the suppression of microtubule dynamics and abnormalities in
the spindle structure the cells are arrested at mitosis.
We could not detect any anaphase or telophase type
cells in the drug treated (5, 10 and 20 lm) cases. However, there were significant numbers of multinucleate
cells after two cycles of cell division, indicating that a
fraction of the cells might have had an aberrant exit
from the mitotic block without the completion of anaphase. It was observed by the hoechst and propidium
iodide staining that the number of cells undergoing
apoptosis increased after one cell cycle (Fig. 7B) and
paralleled its ability to cause the mitotic block
(Fig. 7A). Thus, the block of cell cycle progression at
mitosis in HeLa cells by benomyl correlates well with
its ability to inhibit the proliferation and induction of
apoptosis ⁄ cell death.
It has been proposed that prolonged mitotic arrest
stimulates the phosphorylation of bcl2, thereby making
it inactive [39,48,49]. Bcl2 in the unphosphorylated
form complexes with bax, and phosphorylation of bcl2
releases bax from the bcl2–bax complex [27,38,39]. It
4124

has been shown in many cells types that bax undergoes

homodimerization in response to a death signal, and
integrates into the mitochondrial membrane, triggering
the release of cytochrome c which causes activation of
the caspases. Caspases initiate the apoptotic DNA
fragmentation by activating certain nucleases [50]. The
116 kDa enzyme PARP is cleaved into a 85 kDa fragment and a 25 kDa fragment in many forms of apoptotic cell death [36]. We observed that benomyl
induced mitotic arrest in HeLa cells was accompanied
by the hyperphosphorylation of bcl2 (Fig. 9A). Benomyl induced phosphorylation of bcl2 released bax from
the bcl2–bax complex (Fig. 9C) indicating that the
physical association of bcl2 and bax was disrupted significantly in the presence of benomyl. The increased
ratio of free bax might have triggered the apoptosis.
The cleavage of PARP and the fragmentation of DNA
confirm the apoptosis induced by benomyl. The results
demonstrate that benomyl induces apoptosis through
the bcl2–bax pathway, in a way similar to that of
paclitaxel induced apoptosis [48]. While benomyl was
used extensively to study the role of checkpoints in
yeast, its actions on mammalian checkpoints and
mechanism of cell death was not studied; we show that
the antitubulin fungicide can activate mammalian
checkpoint and induce apoptosis through the bcl2
pathway by perturbing the dynamics of microtubules
in HeLa cells. One of the major obstacles in the treatment of cancer is the toxicity of anticancer drugs.
Combination of two inducers of apoptosis that work
synergistically at the nontoxic concentrations could be
beneficial in chemotherapy. Similar to taxol and vinblastine, benomyl also induces apoptosis in HeLa cells,
indicating that benomyl may be useful in combination
with other potent anticancer drugs for the treatment of
cancer.

Experimental procedures
Materials
Benomyl was purchased from Sigma-Aldrich (Milwaukee,
WI, USA), paclitaxel, sulforhodamine B (SRB), mouse
monoclonal antibcl2 IgG, mouse monoclonal antia-tubulin
IgG, affinity isolated rabbit antic-tubulin IgG, alkaline
phosphatase-conjugated antimouse IgG, alkaline phosphatase-conjugated antirabbit IgG, fluorescein isothiocyanate
(FITC)-conjugated antirabbit IgG, propidium iodide,
hoechst 33342, fetal bovine serum, BSA, and dithiothreitol
were purchased from Sigma (St. Louis, MO, USA). Mouse
monoclonal antip53 IgG and rabbit polyclonal antiPARP
IgG were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). Antimouse IgG-alexa 568 conjugate and

FEBS Journal 273 (2006) 4114–4128 ª 2006 The Authors Journal compilation ª 2006 FEBS


K. Rathinasamy and D. Panda

4¢,6-diamidino-2-phenylindole (DAPI) were purchased from
Molecular Probes (Eugene, OR, USA). All other reagents
are of analytical grade.

Cell culture, cell proliferation and mitotic index
assays
HeLa cells were grown in minimal essential medium (Hi
media) supplemented with 10% (v ⁄ v) fetal bovine serum
and sodium bicarbonate (1.5 mgỈmL)1) in the presence of
antibiotics (100 units of penicillin, 0.1 mg of streptomycin,
0.25 lg of amphotericin B per mL) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air [10]. Cells were

grown in tissue culture flasks or on poly l-lysine coated
coverslips and treated with benomyl or paclitaxel suspended in 100% dimethylsulfoxide. The final dimethylsulfoxide concentration in all the experiments was 0.1% (v ⁄ v).
The effects of benomyl on cell proliferation were determined in 96-well tissue culture plates by SRB assay
[10,51]. Dimethylsulfoxide alone was used as a vehicle
control. To calculate mitotic index, cells were grown on
poly l-lysine coated coverslips and treated with benomyl
for 20 h and then centrifuged in a Labofuge 400R cytospin (Heraeus, Hanau, Germany) for 10 min (1200 g at
30 °C), fixed with 3.7% (v.v) formaldehyde for 20 min at
room temperature and washed with NaCl ⁄ Pi. Cells were
then stained with DAPI and the mitotic and interphase
cells were counted using the Eclipse TE-2000U microscope
(Nikon, Kanagawa, Japan). At least 600 cells were scored
for each concentration of benomyl and the experiment
was repeated four times.

Immunofluorescence microscopy
Immunofluorescence staining of microtubules and chromosomes was performed as described previously [10]. Cells
were grown on poly l-lysine coated coverslips at a density
of 1 · 105 cellsỈmL)1 in 24-well tissue culture plates. After
20 h of the drug treatment, cells were fixed in 3.7% (v ⁄ v)
formaldehyde for 30 min at 37 °C and then transferred to
cold ()20 °C) methanol for 20 min, and washed in NaCl ⁄ Pi
for 5 min. Nonspecific binding sites were blocked by
incubating with 2% (v ⁄ v) BSA ⁄ NaCl ⁄ Pi at 37 °C for
30 min. Cells were then incubated at 37 °C with mouse
monoclonal antia-tubulin IgG (Sigma) at 1 : 300 dilution
for 2 h, washed with 2% (v ⁄ v) BSA ⁄ NaCl ⁄ Pi at room temperature before incubating with a 1 : 200 dilution of antimouse IgG labelled with Alexa 568 (Molecular Probes) for
1 h. Coverslips were washed with 2% (v ⁄ v) BSA ⁄ NaCl ⁄ Pi
and incubated in DAPI (1 lgỈmL)1) solution for 20 s at
room temperature. Finally, the coverslips were mounted in

80% glycerol in NaCl ⁄ Pi containing 5 mm DABCO (1,4diazabicyclo[2,2,2]octane). The coverslips were observed by
using a 60· water immersion objective in a FV 500 laser
scanning confocal microscope (Olympus, Tokyo, Japan), or

Antiproliferative mechanism of action of benomyl

in an Eclipse TE-2000U microscope (Nikon). The images
were analyzed by using image-pro plus software (Media
Cybernetics, Silver Spring, MD, USA).
The effect of benomyl on chromosome congression was
calculated by measuring the ratio of the width to the height
of the chromosomal mass (congression index) of HeLa cells
with metaphase type chromosome alignment in the absence
and presence of different concentrations of the drug [29].
Chromosomes located outside the metaphase plate were not
included in the calculation.

Reassembly of spindle microtubules after cold
treatment
Cultured cells were grown on glass coverslips for 24 h and
then incubated at 2 °C for 1 h. After cold treatment, the
cold medium was replaced with warm medium containing
different concentrations (0, 5 and 20 lm) of benomyl and
incubated at 37 °C. Cells were then fixed at different time
points (0, 5, 10 and 20 min) with 3.7% (v ⁄ v) formaldehyde
at room temperature for 20 min. The fixed cells were then
processed with rabbit antic-tubulin IgG, mouse monoclonal
antia-tubulin IgG and DAPI to visualize the centrosomes,
spindle microtubules and DNA, respectively. The secondary
antibodies used were FITC-conjugated antirabbit IgG and

Alexa 568-conjugated antimouse IgG. The average length
of the spindles at different time points was calculated by
measuring the distance between the poles using the imagepro plus software.

Measurement of sister kinetochore distance
Cells grown on glass coverslips were treated with vehicle or
benomyl (5 and 10 lm) for 20 h and then fixed with 3.7%
(v ⁄ v) formaldehyde, permeabilized with 0.2% (v ⁄ v) Triton
X-100 ⁄ NaCl ⁄ Pi [23], and processed with primary and secondary antibodies as described above. Antibodies raised
against the centromere were kindly provided by KF Sullivan (Scripps Research Institute, La Jola, CA, USA) and
were used at 1 : 1500 dilution, monoclonal antia-tubulin
IgG was used to identify the mitotic cells and to visualize
the attachment of spindle microtubules at the kinetochores.
The secondary antibodies used were goat antihuman-FITC
conjugate and antimouse IgG-Alexa 568 conjugate. The sister kinetochores that lie in the same focal plane were selected and the distance between them was measured as the
distance between the centre of one sister kinetochore to the
other using the image-pro plus software.

Visualization of kinetochore microtubules and
BubR1
Kinetochore microtubules were detected as described previously [34]. Briefly, cells grown on coverslips were first

FEBS Journal 273 (2006) 4114–4128 ª 2006 The Authors Journal compilation ª 2006 FEBS

4125


Antiproliferative mechanism of action of benomyl

K. Rathinasamy and D. Panda

incubated with different concentrations (0, 5, and 20 lm) of
benomyl for 10 min at 37 °C. Then, the warm media was
replaced with ice-cold media containing the same concentrations of benomyl and incubated on ice for 10 min and
subsequently fixed and processed for immunofluorescence
microscopy using antibodies raised against tubulin, and
DAPI, to stain kinetochore microtubules and DNA,
respectively. In an another experiment, nonkinetochore
microtubules were depolymerized by incubating the cells in
a calcium containing buffer [1 mm CaCl2, 100 mm Pipes,
1 mm MgCl2, 0.2% (v ⁄ v) Triton X-100] for 2 min at 37 °C
followed by fixation in the same buffer containing 3.7%
(v ⁄ v) formaldehyde [47] and processed for immunofluorescence using antibodies raised against a-tubulin, and DAPI.
To visualize the spindle checkpoint protein BubR1, HeLa
cells were grown in the absence and presence of benomyl,
fixed and permeabilized with 0.4% (v ⁄ v) Triton X-100 ⁄
NaCl ⁄ Pi for 5 min and processed for indirect immunofluorescence. Antibodies raised against BubR1 were used at
1 : 1000 dilutions in 2% (v ⁄ v) BSA ⁄ NaCl ⁄ Pi. The secondary antibody was FITC conjugated antimouse IgG used at
1 : 500 dilution.

Measurement of intercentrosomal distance
Cells were grown on glass coverslips and treated with vehicle or benomyl (5 and 10 lm) for 20 h, and then fixed with
3.7% (v ⁄ v) formaldehyde. The cells were then processed
with antibodies raised in rabbit against c-tubulin, mouse
monoclonal a-tubulin, and DAPI, to visualize the centrosomes, spindle microtubules and DNA, respectively. The
secondary antibodies used were antirabbit-FITC conjugate
and antimouse-Alexa 568 conjugate. The distance between
the centrosomes was measured by using the image-pro
plus software.

Live ⁄ dead cell counting using hoechst/propidium
iodide staining
HeLa cells were plated at a density of 5 · 104 cellsỈmL)1
and grown in 24-well tissue culture plates 24 h before the
addition of benomyl. Cells were then grown in the absence
and presence of different concentrations of benomyl for an
additional 20 or 40 h. Subsequently, cells were incubated
with hoechst 33342 (1 lgỈmL)1) and propidium iodide at
37 °C in a CO2 incubator for 20 min. After incubation,
cells were centrifuged in a Labofuge 400R cytospin at
1200 g for 10 min at 30 °C, washed twice with NaCl ⁄ Pi,
fixed with chilled ()20 °C) methanol and incubated at
)20 °C for 10 min. Coverslips were then washed in
NaCl ⁄ Pi and mounted on clean glass slides. Total cells were
counted using the hoechst fluorescence and dead ⁄ apoptotic
cells were counted using the propidium iodide fluorescence
under the fluorescence microscope.

4126

Western analysis
HeLa cells were seeded at 1.5 · 105 cellsỈmL)1 in tissue culture flasks. After 24 h of incubation, the media was
removed and fresh media containing different concentrations of benomyl or paclitaxel were added and incubated
for another 20 or 40 h. Paclitaxel (100 nm) was used as a
positive control to monitor the hyper phosphorylated form
of bcl2 and cleavage of PARP [36,48]. Both the floating
and attached cells were harvested with the help of a cell
scraper and collected by centrifugation. The cells were
washed twice in NaCl ⁄ Pi and lysed in lysis buffer [50 mm
Tris, pH 7.4, 150 mm NaCl, 0.1% (v ⁄ v) Triton X-100,

0.2% (v ⁄ v) Nonidet P-40, 4 mm EDTA, 50 mm NaF, and
1 mm dithiothreitol] containing protease inhibitors [48,49].
The lysed cell suspension was centrifuged at 750 g for
20 min and the resulting supernatants were used as cell
lysates. Protein concentration of the extract was determined
by Bradford assay [52] and cell lysates equivalent to 50 lg
of protein was separated by SDS ⁄ PAGE [10% or 12%
(w ⁄ v) acrylamide gel] and electroblotted onto a nitrocellulose membrane. The blot was blocked with Tris buffered
saline containing 0.05% (v ⁄ v) Tween 20 (TBST) and 5%
(w ⁄ v) nonfat skim milk for 2 h at room temperature. The
blots were then incubated with fresh blocking solution with
an appropriate dilution of mouse antihuman bcl2 IgG,
mouse antip53 IgG, rabbit polyclonal antibax IgG or rabbit
polyclonal antiPARP IgG. The blot was washed three times
with TBST and incubated for 1 h with an alkaline phosphatase conjugated antirabbit or antimouse IgG for 1 h at
room temperature. After three washes with TBST the membrane was developed using bromo-chloro-indolyl-phosphate ⁄ nitro blue tetrazolium substrate.

Immunoprecipitation
Cell lysates equivalent to 150 lg of protein was immunoprecipitated with 1 lL of antibcl2 IgG and protein-A
agarose for 3 h at 4 °C. The immunoadsorbed pellets were
washed four times with lysis buffer and finally resuspended
in SDS sample buffer. The immunoprecipitates were subjected to SDS ⁄ PAGE [12% (w ⁄ v) acrylamide gel], transferred
to a nitrocellulose membrane and probed with antibax IgG.

Detection of apoptosis by DNA ladder assay
HeLa cells (3 · 105 cellsỈmL)1) were incubated in the
absence or presence of 5 and 20 lm benomyl for either 20
or 40 h. DNA was isolated according to the standard protocol with minor modification [53]. All reagents, pipette tips
and centrifuge tubes used were autoclaved. Cells were harvested and washed in NaCl ⁄ Pi. Cells were incubated with
ice-cold lysis buffer [10 mm Tris pH 7.4, 1 mm EDTA,

0.2% (v ⁄ v) Triton X-100] for 30 min on ice. The cells

FEBS Journal 273 (2006) 4114–4128 ª 2006 The Authors Journal compilation ª 2006 FEBS


K. Rathinasamy and D. Panda

lysates were then centrifuged at 14 000 g for 15 min and
the supernatants were incubated with 0.2 mgỈmL)1 of proteinase K in a digestion buffer containing 150 mm NaCl,
10 mm Tris ⁄ Hcl pH 8.0, 40 mm EDTA and 1% (w ⁄ v) SDS,
for 4 h at 37 °C. DNA was extracted from the digested cell
lysates with phenol ⁄ chloroform and pelleted using 100%
ethanol. DNA pellets were dissolved in 10 mm Tris ⁄ HCl
pH 8.0, 1 mm EDTA buffer and treated with DNase-free
RNase for 2 h at 37 °C. Finally, the samples were electrophoressed in 1% (w ⁄ v) agarose gel containing 0.5 lgỈmL)1
ethidium bromide at a constant voltage of 50 V and visualized under UV illumination.

Acknowledgements

Antiproliferative mechanism of action of benomyl

10

11

12
13

We thank Dr K. F. Sullivan for providing the anticentromere antibody. We also thank Dr P. Curmi for critically reading the manuscript. This work is supported
by a grant from the Department of Biotechnology,

Government of India. D.P. is a Swarnajayanti Fellow.

14

References

16

1 WHO (1993) Environmental Health Criteria Number
148: Benomyl World Health Organization, Geneva.
2 Hess RA, Moore BJ, Forrer J, Linder RE & Abuel-Atta
AA (1991) The fungicide benomyl (methyl 1-(butylcarbomyl)-2-benzimidazolecarbamate) causes testicular dysfunction by inducing the sloughing of germ cells and
occlusion of efferent ductules. Fundam Appl Toxicol 17,
733–745.
3 Lim J & Miller MG (1997) The role of the benomyl
metabolite carbendazim in benomyl-induced testicular
toxicity. Toxicol Appl Pharmacol 142, 401–410.
4 McCarroll NE, Protzel A, Ioannou Y, Frank Stack
HF, Jackson MA, Waters MD & Dearfield KL (2002)
A survey of EPA ⁄ OPP and open literature on selected
pesticide chemicals. III. Mutagenicity and carcinogenicity of benomyl and carbendazim. Mutat Res 512,
1–35.
5 Kilmartin JV (1981) Purification of yeast tubulin by
self-assembly in vitro. Biochemistry 20, 3629–3633.
6 Davidse LC & Flach W (1977) Differential binding of
methyl benzimidazol-2-yl carbamate to fungal tubulin as
a mechanism of resistance to this antimitotic agent in
mutant strains of Aspergillus nidulans. J Cell Biol 72,
174–193.
7 Hoyt MA, Totis L & Roberts BT (1991) S. cerevisiae

genes required for cell cycle arrest in response to loss of
microtubule function. Cell 66, 507–517.
8 Li R & Murray AW (1991) Feedback control of mitosis
in budding yeast. Cell 66, 519–531.
9 Richards KL, Anders KR, Nogales E, Schwartz K,
Downing KH & Botstein D (2000) Structure-function

15

17

18

19

20

21

22

23

24

relationships in yeast tubulins. Mol Biol Cell 11, 1887–
1903.
Gupta K, Bishop J, Peck A, Brown J, Wilson L &
Panda D (2004) Antimitotic antifungal compound benomyl inhibits brain microtubule polymerization and
dynamics and cancer cell proliferation at mitosis, by

binding to a novel site in tubulin. Biochemistry 443,
6645–6655.
Downing KH (2000) Structural basis for the interaction
of tubulin with proteins and drugs that affect microtubule dynamics. Annu Rev Cell Dev Biol 16, 89–111.
Desai A & Mitchison TJ (1997) Microtubule polymerization dynamics. Annu Rev Cell Dev Biol 13, 83–117.
Jordan MA & Wilson L (2004) Microtubules as a target
for anticancer drugs. Nat Rev Cancer 4, 253–265.
Skibbens RV, Skeen VP & Salmon ED (1993) Directional instability of kinetochore motility during chromosome congression and segregation in mitotic newt lung
cells: a push-pull mechanism. J Cell Biol 122, 859–875.
Kapoor TM & Compton DA (2002) Searching for the
middle ground: Mechanisms of chromosome alignment
during mitosis. J Cell Biol 157, 551–556.
Mitchison TJ (1989) Mitosis: Basic concepts. Curr Opin
Cell Biol 1, 67–74.
Zhou J, Yao J & Joshi HC (2002) Attachment and tension in the spindle assembly checkpoint. J Cell Sci 115,
3547–3555.
Canman JC, Sharma N, Straight A, Shannon KB, Fang
G & Salmon ED (2002) Anaphase onset does not require
the microtubule-dependent depletion of kinetochore and
centromere-binding proteins. J Cell Sci 115, 3787–3795.
Hoffman DB, Pearson CG, Yen TJ, Howell BJ & Salmon ED (2001) Microtubule-dependent changes in
assembly of microtubule motor proteins and mitotic
spindle checkpoint proteins at PtK1 kinetochores. Mol
Biol Cell 12, 1995–2009.
Howell BJ, Hoffman DB, Fang G, Murray AW &
Salmon ED (2000) Visualization of Mad2 dynamics at
kinetochores, along spindle fibers, and at spindle poles
in living cells. J Cell Biol 150, 1233–1250.
Toso RJ, Jordan MA, Farrell KW, Matsumoto B &
Wilson L (1993) Kinetic stabilization of microtubule

dynamic instability in vitro by vinblastine. Biochemistry
32, 1285–1293.
Derry WB, Wilson L & Jordan MA (1995) Substoichiometric binding of taxol suppresses microtubule
dynamics. Biochemistry 34, 2203–2211.
Zhou J, Panda D, Landen JW, Wilson L & Joshi HC
(2002) Minor Alteration of microtubule dynamics causes
loss of tension across kinetochore pairs and activates
the spindle checkpoint. J Biol Chem 277, 17200–17208.
Panda D, Rathinasamy K, Santra MK & Wilson L
(2005) Kinetic suppression of microtubule dynamic
instability by griseofulvin: Implications for its possible

FEBS Journal 273 (2006) 4114–4128 ª 2006 The Authors Journal compilation ª 2006 FEBS

4127


Antiproliferative mechanism of action of benomyl

25

26
27

28

29

30

31
32

33

34

35

36

37

38
39

40

K. Rathinasamy and D. Panda

use in the treatment of cancer. Proc Natl Acad Sci USA
102, 9878–9883.
Farrow SN & Brown R (1996) New members of the
bcl-2 family and their protein partners. Curr Opin Genet
Dev 6, 45–49.
Kroemer G (1997) The proto-oncogene bcl-2 and its
role in regulating apoptosis. Nature Med 3, 614–620.
Oltvai ZN, Milliman CL & Korsmeyer SJ (1993) Bcl-2
heterodimerizes in vivo with a conserved homolog, bax,
that accelerates programmed cell death. Cell 74, 609–619.

Gross A, Jockel J, Wei MC & Korsmeyer SJ (1998)
Enforced dimerization of bax results in its translocation,
mitochondrial dysfunction and apoptosis. EMBO J 17,
3878–3885.
Green RA & Kaplan KB (2003) Chromosome instability in colorectal tumor cells is associated with defects in
microtubule plus-end attachments caused by a dominant
mutation in APC. J Cell Biol 163, 949–961.
Cassimeris L, Rieder CL & Salmon ED (1994) Microtubule assembly and kinetochore directional instability in
vertebrate monopolar spindles: Implications for the
mechanism of chromosome congression. J Cell Sci 107,
285–297.
Nicklas RB (1997) How Cells Get the Right Chromosomes. Science 275, 632–637.
Rieder CL & Salmon ED (1998) The vertebrate cell
kinetochore and its roles during mitosis. Trends Cell
Biol 8, 310–318.
Shelby RD, Hahn KM & Sullivan KF (1996) Dynamic
elastic behavior of alpha-satellite DNA domains visualized in situ in living human cells. J Cell Biol 135, 545–
557.
DeLuca JG, Moree B, Hickey JM, Kilmartin JV & Salmon ED (2002) hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell
death in HeLa cells. J Cell Biol 159, 549–555.
Uzbekov R, Kireyev I & Prigent C (2002) Centrosome
separation: respective role of microtubules and actin
filaments. Biol Cell 94, 275–288.
Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE
& Poirier GG (1993) Specific proteolytic cleavage of poly
(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res 53, 3976–3985.
Kaufmann SH (1989) Induction of endonucleolytic
DNA cleavage in human acute myelogenous leukemia
cells by etoposide, camptothecin, and other cytotoxic
anticancer drugs: a cautionary note. Cancer Res 49,

5870–5878.
Haldar S, Basu A & Croce CM (1997) Bcl2 is the guardian of microtubule integrity. Cancer Res 57, 229–233.
Haldar S, Jena N & Croce CM (1995) Inactivation of
bcl-2 by phosphorylation. Proc Natl Acad Sci USA 92,
4507–4511.
Rieder CL, Davison EA, Jensen LC, Cassimeris L &
Salmon ED (1986) Oscillatory movements of mono-

4128

41

42

43

44
45
46

47

48

49

50

51

52

53

oriented chromosomes and their position relative to the
spindle pole result from the ejection properties of the
aster and half-spindle. J Cell Biol 103, 581–591.
Ault JG, DeMarco AJ, Salmon ED & Rieder CL (1991)
Studies on the ejection properties of asters: astral microtubule turnover influences the oscillatory behavior and
positioning of mono-oriented chromosomes. J Cell Sci
99, 701–710.
Rieder CL, Schultz A, Cole R & Sluder G (1994) Anaphase onset in vertebrate somatic cells is controlled by a
checkpoint that monitors sister kinetochore attachment
to the spindle. J Cell Biol 127, 1301–1310.
Waters JC, Chen RH, Murray AW & Salmon ED
(1998) Localization of Mad2 to kinetochores depends
on microtubule attachment, not tension. J Cell Biol 141,
1181–1191.
Li X & Nicklas RB (1995) Mitotic forces control a cellcycle checkpoint. Nature 373, 630–632.
Ostergren G (1950) Consideration of some elementary
features of mitosis. Hereditas 36, 1–19.
Hays TS & Salmon ED (1990) Poleward force at the
kinetochore in metaphase depends on the number of
kinetochore microtubules. J Cell Biol 110, 391–404.
Mitchison T, Evans L, Schulze E & Kirschner M (1986)
Sites of microtubule assembly and disassembly in the
mitotic spindle. Cell 45, 515–527.
Srivastava RK, Srivastava AP, Korsmeyer SJ, Nesterova M, Cho-Chung YS & Longo DL (1998) Involvement of microtubules in the regulation of bcl2
phosphorylation and apoptosis through cyclic AMPdependent protein kinase. Mol Cell Biol 18, 3509–
3517.

Scatena CD, Stewart ZA, Mays D, Tang LJ, Keefer CJ,
Leach SD & Pietenpol JA (1998) Mitotic phosphorylation of bcl-2 during normal Cell cycle progression and
taxol-induced growth arrest. J Biol Chem 273, 30777–
30784.
Mukae N, Enari M, Sakahira H, Fukuda Y, Inazawa J,
Toh H & Nagata S (1998) Molecular cloning and characterization of human caspase-activated DNase. Proc
Natl Acad Sci USA 95, 9123–9128.
Skehan P, Storeng R, Scudiero D, Monks A, McMahon
J, Vistica D, Warren JT, Bokesch H, Kenney S & Boyd
MR (1990) New colorimetric cytotoxicity assay for
anticancer-drug screening. J Natl Cancer Inst 82, 1107–
1112.
Bradford MM (1976) A rapid and sensitive method for
the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem
72, 248–254.
Ramoneda BM & Perez-Tomas R (2002) Activation of
protein kinase C for protection of cells against apoptosis induced by the immunosuppressor prodigiosin.
Biochem Pharmacol 63, 463–469.

FEBS Journal 273 (2006) 4114–4128 ª 2006 The Authors Journal compilation ª 2006 FEBS