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

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

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

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 gÆkg)1] in rats [1] However, other studies

sugges-ted that benomyl at a single dose of larger than

100 mgÆkg)1 is capable of inducing testicular toxicity

in experimental animals [2,3] Chronic administration

of benomyl at doses higher than 500 mgÆkg)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 carbend-azim have been shown to exhibit diffential sensitivity against fungal tubulin and mammalian brain tubulin [5,6] It is believed that the selective toxicity of beno-myl 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

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: panda@iitb.ac.in

(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 kineto-chores 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

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.

[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

mamma-lian 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

duplica-ted 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

spin-dle microtubules and kinetochores play an important

role in the congression of chromosomes at the

meta-phase 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

chrom-atids [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

micro-tubules, 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

progres-sion in the cell cycle [20]

The functions of microtubules are thought to be

highly dependent on the assembly dynamics of

micro-tubules [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,

micro-tubule 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,

bcl-W, and Bfl1 [25,26] The bcl2 family of proteins have

the propensity to form homodimers or heterodimers It

is believed that the heterodimerization of bcl2 and bax prevents the cell from undergoing bax mediated apop-tosis [27] Hence, the balance between bcl2–bax het-erodimer 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 per-turbed 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 pro-teins and induction of apoptosis

Results

Benomyl suppressed the reassembly of spindle microtubules in HeLa cells

HeLa cells were incubated at 2C for 1 h Then, the cold media was replaced with warm media containing different concentrations of benomyl Subsequently, the cells were incubated at 37C in a CO2incubator The cold treatment caused complete depolymerization of mitotic spindle microtubules as observed by fluores-cence microscopy after immunostaining the HeLa cells with antia-tubulin IgG (Fig 1) In the absence of spin-dle 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 reassem-bled within 5 min of incubation at 37C (Fig 1), while benomyl caused a significant delay in the reas-sembly of the spindle microtubules In the presence of

5 lm benomyl, the spindles were only partially reas-sembled after 5 min of incubation at 37C (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 micro-tubules, 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

with 20 lm benomyl did not recover their spindle

microtubules even after 10 min at 37C After 20 min

of incubation, microtubules appeared near the

centro-somes and the chromocentro-somes 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

con-trol cells (10.1 ± 1.3 lm*) (*P < 0.01, n¼ 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

in the cells treated with 5 lm benomyl appeared nearly normal, but a few chromosomes were unable to con-gress at the metaphase plate (Fig 2A) The effects of microtubule perturbation by benomyl on the chromo-somal 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 previ-ous 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 pres-ence 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

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.

(20 lm) produced clear abnormalities in chromosomal

alignment with nearly 70% of all the mitotic cells

hav-ing multiple poles with short spindles and the

chromo-somes 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

meta-phase 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,

result-ing 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

kineto-chores 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* (n¼ 75) This distance was signifi-cantly reduced by 29% and 34% in the presence of

5 and 10 lm benomyl, being equal to 1.25 ± 0.4 lm* (n¼ 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 micro-tubules It has been shown that limited cold treat-ment 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 con-trol 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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Fig 2 Effects of benomyl on the spindle microtubules and chromosome organization (A) HeLa cells were incubated with different concen-trations 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.

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Fig 3 Benomyl reduced the tension at the kinetochores (A) Immunofluorescent ima-ges of mitotic spindle of HeLa cells, cen-tromeres (green), DNA (blue) and merged images of microtubules (red) and centrom-eres are shown HeLa cells were treated with 5 and 10 l M 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.

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

chromo-somes 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 chromo-somes 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 kineto-chores are not under tension in the presence of beno-myl, 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

Fig 4 Effects of benomyl on the kinetochore–microtubule attachment: Cells were first incubated with different concentrations (0, 5, and

20 l M ) of benomyl for 10 min at 37 C Then, the warm media was replaced with ice-cold media containing the same concentration of beno-myl and incubated on ice for 10 min and subsequently fixed and processed for immunofluorescence using antitubulin IgG (red) and DAPI (blue) 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

visual-ize centrosomes and microtubules The distance

between the two centrosomes in the mitotic cells that had achieved a metaphase type of chromosomal align-ment was 11.1 ± 1.5 lm* (n¼ 60) (Fig 6) Centro-some separation in the mitotic cells was affected significantly in the presence of benomyl, with an Fig 5 Localization patterns of the checkpoint protein BubR1 Cells treated with 5 l M 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.

intercentrosomal distance of 7.9 ± 1.5 lm* at 5 lm

(n¼ 87) (Fig 6) (29% reduction compared to control),

and 6.9 ± 2.2 lm* at 10 lm (n¼ 75) (38% reduction

compared to control) (Fig 6) (*P < 0.01) The

interc-entrosomal 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

pro-phase cells showed three to four centrosomal

struc-tures, 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%, respect-ively (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 propi-dium 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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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 l M 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 proce-dures Error bars represent 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

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

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

We found that the enzyme poly (ADP ribose) poly-merase (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 After 20 h treatment of HeLa cells with benomyl, the level of phosphorylation of bcl2 increased in a concen-tration dependent manner as evidenced by the decreased mobility of the protein (Fig 9A) At 5 lm concentration of benomyl, which showed 50% inhibi-tion of proliferainhibi-tion 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 exam-ple, 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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Dead cells (20 h)

Dead cells (40 h)

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

inhi-bition of cell proliferation was determined by sulforhodamine B

(SRB) assay after incubating the cells with various concentrations

(0–60 l M ) of benomyl for 20 h The mitotic index was calculated by

DAPI staining method after incubating the cells with different

con-centrations (0–60 l M ) 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

incu-bation Live and dead ⁄ apoptotic cells were counted after incubating

the HeLa cells with different concentrations (0–40 l M ) of benomyl

for 20 h and 40 h as described in Experimental procedures Error

bars are SD.

suppressed the reassembly of cold depolymerized

spin-dle microtubules of HeLa cells, suggesting that

beno-myl perturbs spindle microtubule assembly dynamics

At higher concentration (20 lm), benomyl suppressed

microtubule nucleation from the centrosomes

Beno-myl treatment decreased the interpolar distance in the

mitotic HeLa cells, reduced the distance between sister

kinetochores, caused loss of tension across the

kineto-chores and activated the checkpoint protein BubR1

Further, benomyl efficiently arrested the cells at

mito-sis and induced apoptomito-sis

The functions of microtubules are shown to be

lar-gely 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 equa-torial 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 per-turbed 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 surveil-lance mechanism to prevent errors during cell division The checkpoints can detect the attachment of microtu-bules 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 kineto-chores 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 kineto-chore pairs in the chromosomes aligned at the meta-phase 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, indica-ting 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

A

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85 KD

Control

Benomyl Taxol

5 μM 20 μM 100 n M

β-Actin

Fig 8 Benomyl induced apoptosis in HeLa cells (A) Cleavage of

PARP by benomyl treatment HeLa cells were treated with the

indi-cated 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

pro-cedures Shown are, control after 40 h (lane 1); 20 h after 5 and

20 l M of benomyl treatment (lanes 2 and 3); and 40 h after 5 and

20 l M of benomyl treatment (lanes 4 and 5).

Fig 9 Benomyl induced hyperphosphorylation of bcl2 and dissoci-ation of bax from bcl2 HeLa cells were treated with the vehicle control (lane 1), 5 and 20 l M benomyl (lane 2 and 3), and 100 n M 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.