Published OnlineFirst April 18, 2014; DOI: 10.1158/1535-7163.MCT-13-0685
Centmitor-1, a Novel Acridinyl-Acetohydrazide, Possesses Similar
Molecular Interaction Field and Antimitotic Cellular Phenotype as
Rigosertib, ON 01910.Na
Jenni H.E. Mäki-Jouppila, Leena J. Laine, Jonathan Rehnberg, et al.
Mol Cancer Ther 2014;13:1054-1066. Published OnlineFirst April 18, 2014.
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Published OnlineFirst April 18, 2014; DOI: 10.1158/1535-7163.MCT-13-0685
Molecular
Cancer
Therapeutics
Small Molecule Therapeutics
Centmitor-1, a Novel Acridinyl-Acetohydrazide, Possesses
Similar Molecular Interaction Field and Antimitotic Cellular
Phenotype as Rigosertib, ON 01910.Na
€ki-Jouppila1,2,3,4, Leena J. Laine1,2, Jonathan Rehnberg1, Elli Narvi1,2, Pekka Tiikkainen1,
Jenni H.E. Ma
Elvira Hukasova6, Pasi Halonen1, Arne Lindqvist6, Lila Kallio1, Antti Poso5, and Marko J. Kallio1,2
Abstract
Mitosis is an attractive target for the development of new anticancer drugs. In a search for novel mitotic
inhibitors, we virtually screened for low molecular weight compounds that would possess similar steric and
electrostatic features, but different chemical structure than rigosertib (ON 01910.Na), a putative inhibitor of
phosphoinositide 3-kinase (PI3K) and polo-like kinase 1 (Plk1) pathways. Highest scoring hit compounds were
tested in cell-based assays for their ability to induce mitotic arrest. We identified a novel acridinyl-acetohydrazide, here named as Centmitor-1 (Cent-1), that possesses highly similar molecular interaction field as
rigosertib. In cells, Cent-1 phenocopied the cellular effects of rigosertib and caused mitotic arrest characterized
by chromosome alignment defects, multipolar spindles, centrosome fragmentation, and activated spindle
assembly checkpoint. We compared the effects of Cent-1 and rigosertib on microtubules and found that both
compounds modulated microtubule plus-ends and reduced microtubule dynamics. Also, mitotic spindle forces
were affected by the compounds as tension across sister kinetochores was reduced in mitotic cells. Our results
showed that both Cent-1 and rigosertib target processes that occur during mitosis as they had immediate
antimitotic effects when added to cells during mitosis. Analysis of Plk1 activity in cells using a Förster resonance
energy transfer (FRET)-based assay indicated that neither compound affected the activity of the kinase. Taken
together, these findings suggest that Cent-1 and rigosertib elicit their antimitotic effects by targeting mitotic
processes without impairment of Plk1 kinase activity. Mol Cancer Ther; 13(5); 1054–66. 2014 AACR.
Introduction
Mitosis has been a target of anticancer therapies for
decades. The earliest mitosis-perturbing drugs were antitubulin agents such as taxanes and vinca alkaloids, and
their derivatives (1, 2). These drugs inhibit microtubule
assembly or disassembly dynamics by targeting tubulin
subunits, the building blocks of microtubules. Microtubules
undergo major rearrangements during mitosis: within a
Authors' Affiliations: 1VTT Health, VTT Technical Research Centre of
Finland; 2Centre for Biotechnology and 3Department of Pharmacology,
Drug Development and Therapeutics, University of Turku, Turku; 4Drug
Research Doctoral Programme and FinPharma Doctoral Program Drug
Discovery; 5School of Pharmacy, University of Eastern Finland, Kuopio,
Finland; and 6Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
€ki-Jouppila and L.J. Laine contributed equally to this article.
J.H.E. Ma
Current address of P. Halonen: Division of Molecular Carcinogenesis, NKI,
1066CX Amsterdam, the Netherlands.
Corresponding Author: Marko J. Kallio, VTT Technical Research Centre of
€inen Pitka
€katu 4 C, 20521 Turku, Finland. Phone: 358Finland, Ita
24788614; Fax: 358-207222840; E-mail: marko.kallio@vtt.fi
doi: 10.1158/1535-7163.MCT-13-0685
2014 American Association for Cancer Research.
1054
short period of time, interphase microtubules are disassembled and reassembled to form the mitotic spindle apparatus that is needed for ordered chromosome segregation
and exit from M phase. Tubulin-targeting drugs interfere
with these processes and impair normal spindle function
and chromosome alignment at the metaphase plate (3, 4).
This leads to persistent activity of the spindle assembly
checkpoint (SAC; ref. 5), typically resulting in a long-lasting
mitotic arrest and ultimately cell death, a phenomenon
that possesses therapeutic value. Since the discovery of
tubulin-targeting agents, several novel antimitotic compounds have been developed to specifically target mitotic
kinases and motor proteins (for reviews see refs. 6, 7). These
agents are expected to reduce side effects, such as neurotoxicity, associated with antitubulin drugs (1, 2) and provide new therapeutic opportunities for the treatment of
cancer pending on their successful clinical validation.
One interesting anticancer compound that is currently in
phase II and III clinical trials for the treatment of blood
malignancies and solid tumors (8–10) is rigosertib (ON
01910.Na) developed by Onconova. Rigosertib functions
by inducing multipolarity, mitotic arrest, and subsequent
cell death (11). The mechanism of action of rigosertib was
initially reported to occur via the inhibition of polo-like
kinase 1 (Plk1; ref. 11), an important mitotic regulator that
is necessary for spindle assembly, chromosome alignment,
Mol Cancer Ther; 13(5) May 2014
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Centmitor-1 Possesses Antimitotic Properties
and cytokinesis (12, 13). Because Plk1 is often overexpressed in cancer cells and its inhibition lowers tumor
cells’ viability (14), it is considered to be a promising drug
target (15). In addition to targeting Plk1, rigosertib has
been reported to show activity against phosphoinositide 3kinase (PI3K) and mitogen-activated protein kinase
(MAPK) pathways (11, 16, 17). However, the mechanism
of action of the compound is not completely understood.
Here, we designed and executed a high-throughput
screen (HTS) to identify low molecular weight (LMW)
compounds that possess similar steric and electrostatic
features but different chemical structure as rigosertib. In
this study, we characterize a novel LMW compound, an
acridinyl-acetohydrazide (C22H16BrN3O3), here termed as
Centmitor-1 (Cent-1). We analyze and compare the effects
of Cent-1 and rigosertib on different aspects of mitosis and
microtubule dynamics.
Materials and Methods
Chemicals
Cent-1 (ChemBridge Corporation; 5676127) was used at
5 mmol/L concentration and rigosertib (ON 01910.Na;
Selleck Chemicals; S1362) at 250 nmol/L concentration
unless stated otherwise. Nocodazole (Sigma; M1404) was
used at 0.5 to 3 mmol/L, taxol (Paclitaxel; Sigma; T7191) at
0.1 to 0.6 mmol/L, thymidine (Sigma; T9250) at 2 mmol/L,
insulin (Sigma; 19278) at 100 ng/mL, vinblastine (Sigma;
V1377) at 3 mmol/L, staurosporine (Sigma;, S5921) at 1
mmol/L, ZM447439 (Tocris Bioscience; 2458) at 5 mmol/L,
MG132 (Sigma; C2211) at 10 to 20 mmol/L, monastrol
(Sigma; M8515) at 100 mmol/L, dimethylenastron (Alexis
Biochemicals; ALX-270-438) at 5 mmol/L, BI2536 (Selleck
Chemicals; S1109) at 200 nmol/L, and wortmannin (Tocris
Bioscience; 1232) at 0.2 to 1 mmol/L concentration.
Cell culture
HeLa cells (ATCC CCL-2, obtained 2006) were grown in
Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, penicillin–streptomycin (0.1 mg/mL),
L-glutamine (2 mmol/L), sodium pyruvate (1 mmol/L),
HEPES (20 mmol/L), and nonessential amino acids
(0.1 mmol/L). HeLa cells expressing H2B-GFP and
mCherry tubulin were a kind gift from Stephan Geley’s
laboratory (Medical University, Innsbruck, Austria), obtained in 2012. Their growth medium was supplied with
250 mg/mL of G418. A549 cells (ATCC CCL-185, obtained
2005) expressing GFP-tubulin were cultured in RPMI 1640
(Gibco) supplemented with 10% FBS, penicillin–streptomycin (0.1 mg/mL), and L-glutamine (2 mmol/L). U2OS
cells stably expressing a Förster resonance energy transfer
(FRET)-based Plk1 reporter (obtained in 2010 from the
laboratory of Rene Medema, the Netherlands Cancer
Institute, Amsterdam, the Netherlands), described in 18,
were cultured in DMEM with GlutaMAX (Invitrogen)
supplemented with 6% heat-inactivated FBS and 1% penicillin–streptomycin. No authentication of the cell lines
was done by the authors.
www.aacrjournals.org
For analyzing SAC activity by Western blot, HeLa cells
were synchronized with thymidine to G1–S border (cells
were incubated with 2 mmol/L thymidine for 19 hours,
followed by 8-hour release and second thymidine block for
16 hours) and released into 5 mmol/L Cent-1 or 0.1 mmol/L
taxol for 12 hours. ZM447439 (20 mmol/L) was added to
mitotic cells for 2 hours before cell harvest for Western
blotting. For analyzing PI3K/AKT pathway, HeLa cells
were starved in serum-free medium for 16 hours, treated
with dimethyl sulfoxide (DMSO), 5 mmol/L Cent-1,
250 nmol/L rigosertib or 1 mmol/L wortmannin for a total
of 3 hours and 100 ng/mL insulin for 30 minutes.
Virtual HTS
Compound structures for the virtual screen were collected from four different vendors: Chembridge (ChemBridge Corporation), Chemical Diversity, Tripos (Tripos
International), and Micro Source. Altogether 65,000 compound structures were used in the virtual HTS. Brutus
and Almond softwares were used for the superimposition
of the compound library against the template compound
rigosertib, and for similarity searches using three-dimensional (3D) structure and molecular interaction fields as
search criteria. All compounds were built using the vendor-provided sdf files and Sybyl ligand-preparation utilities with default settings.
Cell-based screen
HeLa cells were plated on 384-well plates with Multidrop Combi (Thermo Fisher Scientific) and screen compounds were added to the cells 24 hours after plating using
Hamilton Microlab Star robotics (Hamilton) at 0.2 or 5
mmol/L final concentrations. Eg5 inhibitor monastrol was
used as a positive control compound that induces mitotic
arrest, and DMSO served as a negative control. Cells were
imaged live with phase contrast optics at 6 and 24 hours
after treatment followed by fixation and DNA staining.
Immunofluorescence
Cells were fixed for 15 minutes with 2% paraformaldehyde in 60 mmol/L Pipes, 25 mmol/L Hepes, 10 mmol/L
EGTA, 4 mmol/L MgSO4 (PHEM) containing 0.5% TritonX-100. For imaging microtubules, 0.2% glutaraldehyde
was included in the fix. For EB1 immunofluorescence,
cells were fixed in ice-cold MeOH for 10 minutes, and
rehydrated in PBS. Cells were washed with 10 mmol/L
MOPS, 150 mmol/L NaCl, and 0.05% Tween 20 (MBST),
blocked in MBST containing 20% boiled normal goat
serum for 1 hour at room temperature, and stained with
antibodies for 1 hour at room temperature. Primary antibodies included mouse anti-Bub1 (Upstate; 05-899), rabbit
anti-BubR1 (Proteinatlas), mouse anti-BubR1 (Abcam;
ab4637), mouse anti-cenpA (Abcam; ab13939), mouse
anti-CETN3 (Abnova; H00001070-M01), human autoimmune serum (Crest; Antibodies Inc.), mouse-anti-EB1
EA3 (kind gift from Prof. Gary Gorbsky, Oklahoma Medical Research Foundation, Oklahoma City, OK), mouse
anti-NuMA (kind gift from Prof. Markku Kallajoki,
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University of Jyväskylä, Jyväskylä, Finland), mouse antiPlk1 (Abcam; 17057), rabbit anti-pericentrin (Abcam;
ab4448), rat anti–a-tubulin YL-1/2 (Abcam; ab6160),
mouse anti–a-tubulin DM1A (Abcam; ab7291), and
mouse anti–g-tubulin (Abcam; ab11316). Secondary antibodies were Alexa Fluor 488, 555, and 647 dyes against
mouse, rabbit, rat, and human antigens (Invitrogen). DNA
was stained with DAPI (40 ,6-diamidino-2-phenylindole).
Coverslips were washed with H2O and mounted on
microscope slides with Vectashield mounting medium
(Vector Laboratories; H-1000).
Microscopy
Zeiss inverted 200M microscope (Zeiss GmbH)
equipped with MetaMorph software (Molecular Devices)
was used for analyzing 384-well plates and fixed cells on
coverslips. Kinetochore intensities were quantified with
Metamorph from maximum projections created from a
Z-stack of images acquired every 0.5 mm. Zeiss Axiovert
200M microscope equipped with spinning disk CSU22
confocal scanner (Yokogawa) and SlideBook 5.0 software
(Intelligent Imaging Innovations, Inc.) was used for microtubule dynamics measurements and for the analysis of
kinetochore distances. ImageJ was used for image processing and quantification of pole fragmentation. An area
was drawn around each centrosome so that all centrosome
fragments that were seen adjacent to main centrosomes
were included inside the region of interest. Only bipolar
cells were chosen for the analysis. The parameters measured were area, aspect ratio (AR ¼ major axis divided by
minor axis), and roundness (formulas available at http://
rsbweb.nih.gov/ij/docs/menus/analyze.html).
In vitro tubulin polymerization assay
Fluorescence-based tubulin polymerization assay (Cytoskeleton Inc.; BK011P) was used to determine the effects of
Cent-1 on tubulin polymerization in vitro. The assay was
performed as described by the manufacturer. DMSO, taxol,
and vinblastine were used as controls. Tubulin polymeri-
zation was recorded every minute for 60 minutes by Victor
1420 Multilabel HTS Counter (PerkinElmer).
Determination of microtubule dynamicity
Microtubule dynamicity measurements were performed in live A549 cells stably expressing EGFP-a-tubulin (BD Biosciences; Clontech) using the spinning disk
confocal microscope with 100 oil objective. Measurements were performed 1 to 2 hours after compound
addition on cells. Z-stacks containing four focus levels
with 0.3-mm step size were acquired every 10 seconds
during a filming session that lasted for 250 seconds. The
plus-ends of microtubules were digitally marked in the
recorded time-lapse movies to allow measurement of their
dynamicity. Dynamicity refers here to the combined
growth and shrinkage velocities of the microtubules,
which were determined as a measurement of the distance
that the microtubule plus-end traveled in a specific period
of time. Ten to 20 microtubules per cell and 5 to 8 cells per
sample were analyzed in each of three independent
experiments. Microtubule dynamics data were analyzed
using SlideBook 5.0 and Graph Pad Prism 4 (GraphPad
Software, Inc.).
FRET for analysis of Plk1 kinase activity
U2OS cells stably expressing a Plk1-responsible FRETbased probe (18) were monitored on a DeltaVision Spectris Imaging system (Applied Precision), using a NA 0.75
20 air objective. Cells were imaged at 37 C in Liebowitz15 medium supplemented with 6% heat-inactivated FBS
and 1% penicillin-streptomycin. Images were acquired
every 20 minutes and processed using ImageJ (http://rsb.
info.nih.gov/ij/). Imaging and quantification of FRET
ratio was performed as described previously (19).
Results
Discovery of Cent-1, an antimitotic LMW compound
To identify small-molecule analogs of rigosertib
(Fig. 1A), we performed a ligand-based virtual HTS, in
Figure 1. Comparison of rigosertib and Cent-1 structures. A, structure of the template compound rigosertib (ON 01910.Na). B, hit compound N0 -(3-bromo-4hydroxybenzylidene)-2-(9-oxo-10(9H)-acridinyl)acetohydrazide named Cent-1. C, Brutus-aligned structures of rigosertib (blue ball-and-stick model) and
Cent-1 (gray/turquoise stick model) are shown. Brutus score of 1.62 indicates exceptionally high level of similarity in 3D structure and electrostatic potential
between the two compounds. Oxygens are colored red and sulfur yellow.
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Molecular Cancer Therapeutics
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Centmitor-1 Possesses Antimitotic Properties
which 65,000 LMW compounds were screened for those
that have similar interaction fields and 3D structures as
the template compound. Virtual screening was conducted
using Almond and Brutus (20–22), field-based molecular
alignment, and virtual screening tools that take into
account both steric and electrostatic features of the molecules. One criterion was that the compounds should not
have similar chemical structures as the template compound. Therefore, compounds having similar backbone
structures as rigosertib were omitted from the hit list. The
200 top-ranking compounds from each hit list were purchased and subjected to secondary cell-based screens, in
which HeLa cells were treated with the compounds and
analyzed for mitotic phenotypes. In total, 4 hit compounds
resulted in a notable increase in mitotic index and were
investigated further.
One of the highest scoring compounds was an acridinyl-acetohydrazide (C22H16BrN3O3) that we named as
Cent-1 (Fig. 1B) due to its effects on centrosome morphology. The field-based similarity score of Cent-1 in comparison with rigosertib was 1.62, which is very high (the
maximum is 2.0, but less than 0.01% of random compound
pairs will reach a value higher than 1.5 according to inhouse tests), indicating that these molecules closely
resemble one another in their 3D structure and charge
distribution. The superimposition image of Cent-1 and
rigosertib is shown in Fig. 1C. Besides the high similarity
of Cent-1 to rigosertib in 3D comparisons, the compound
induced a strong antimitotic phenotype that resembled
the cellular effects of rigosertib. For these reasons, Cent-1
was selected for more detailed analyses.
Cent-1 induces a transient mitotic arrest followed by
abnormal exit from M phase or cell death
To analyze the impact of Cent-1 on cell-cycle progression, the compound (5 mmol/L) was applied on cycling
HeLa cells that were subsequently filmed with IncuCyte
live-cell imager using 1-hour image capture interval
(Fig. 2A). The fates of 60 individual mitotic cells were
determined for each treatment condition (Fig. 2B). DMSO
served as a negative control, and nocodazole and taxol as
mitotic arrest inducing positive controls. DMSO-treated
cells progressed normally through mitosis (the average
duration of mitosis was 1.5 1.8 hours). All nocodazoleand taxol-treated cells arrested in mitosis for several hours
before they underwent cell death (average time spent in
mitosis before cell death was 14.3 5.4 and 16.7 5.9
hours, respectively). Almost half of Cent-1–treated cells
(48.3%) exhibited a long M-phase arrest followed by cell
death (average time spent in mitosis before cell death was
14.3 5.9 hours). Rest of the cells exhibited a mitotic delay
before they exited mitosis with (38.3%) or without (13.3%)
cytokinesis. Cells often died postmitotically soon after the
aberrant exit from mitosis (result not shown). For comparison, we determined the fate of rigosertib–treated cells.
The majority of rigosertib-treated HeLa cells died in
mitosis (76.7%), whereas a smaller proportion exited M
phase with (16.7%) or without (6.7%) cytokinesis (Sup-
www.aacrjournals.org
plementary Fig. S1A). We also determined mitotic and cell
death indices from the same samples at 0-, 12-, 24-, and 36hour time points after exposing the cells to the compounds
(Fig. 2C). In Cent-1–treated populations, mitotic index
peaked at 24 hours (49.4%) and cell death index was the
highest at 36 hours (50.8%). Rigosertib-treated cells
caused a similar accumulation of cells into mitosis
followed by increase in cell death index (Supplementary
Fig. S1B).
As rigosertib was earlier shown to cause apoptosis (11),
we analyzed the potential apoptotic effects of Cent-1 on
HeLa cells using Annexin V-FITC and propidium iodide
staining followed by flow cytometry, and Western blotting with an antibody recognizing cleaved PARP at 12-,
24-, and 48-hour time points after adding the compound.
Cent-1 caused the accumulation of cells to G2–M-phase
and an increase in the sub-G1 cell fraction in comparison
with DMSO control (Supplementary Fig. S2A). After 48hour Cent-1 treatment, 32.8% of cells were in early apoptosis and 14.3% in late apoptosis or necrosis measured by
Annexin V-FITC and propidium iodide staining. In comparison, after DMSO treatment, 10.4% of cells were early
apoptotic and 8.6% late apoptotic or necrotic (Supplementary Fig. S2B). Furthermore, Cent-1 caused a slight
increase in the level of cleaved PARP (Supplementary
Fig. S2C).
Cent-1 induces multipolarity and centrosome
fragmentation
Next, the mitotic effects of Cent-1 were investigated
using a HeLa cell line stably expressing H2B-GFP and
mCherry-tubulin. Cells were imaged live immediately
after Cent-1 or DMSO was added to the culture medium
(Supplementary Movies S1 and S2). All Cent-1–treated
cells exhibited a long prometaphase delay that was followed by death or abnormal exit from mitosis. Typically,
cells assembled multipolar spindles immediately upon
nuclear envelope breakdown (NEB) and underwent multipolar anaphase after spending several hours in prometaphase (Supplementary Movie S1). Alternatively, a number of cells formed bipolar spindles through fusion of
multiple poles during the prometaphase delay (Supplementary Movie S1) or established a bipolar spindles upon
entry into mitosis. Many chromosomes in these cells
congressed to the metaphase plate, indicating that spindle
fibers and kinetochores were capable of establishing functional connections. However, cells always exhibited a
number of chromosomes that remained unaligned near
the spindle poles (Supplementary Movie S1). Control cells
established a bipolar spindle and divided normally (Supplementary Movie S2).
To confirm the live-cell imaging findings, DMSO- or
Cent-1–treated HeLa cells were fixed and immunostained
with various antibodies. Cent-1 treatment caused heterogeneous anomalies that ranged from cells having bipolar
spindles with a few unaligned chromosomes to cells
having multipolar spindles and fully misaligned chromosomes (Fig. 3A). The proportion of multipolar cells was
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Figure 2. Cent-1 treatment induces
mitotic arrest followed by cell death
or forced mitotic exit. A,
representative still images from the
time-lapse films of HeLa cells
treated with the indicated
compounds. In DMSO, cells
undergo normal cell division. In the
presence of nocodazole (Noc) and
taxol, cells accumulate to mitosis
and die after a long arrest. Cent-1–
treated cells first arrest in mitosis
for several hours and then either die
from the arrest or undergo a forced
exit from M phase with or without
cytokinesis. Cells were imaged
with IncuCyte after the addition of
DMSO, 5 mmol/L Cent-1, 3 mmol/L
Noc, or 0.6 mmol/L taxol. Numbers,
hours starting from compound
addition. Arrows mark example
mitotic cells. Scale bar, 100 mm. B,
graphs show the length of mitosis
and fates of individual cells (n ¼ 60)
after the indicated treatments. C,
graphs show mitotic and cell death
indices that were determined from
the time-lapse films at different
time points following the addition
of the indicated compounds. Data,
mean SD from three film frames.
28.5% after 8-hour treatment with the drug (Fig. 3B) and
increased during longer incubation times (37.5% at 24
hours and 44% at 48 hours). Similarly, rigosertib treatment
resulted in a mixture of bipolar and multipolar cells
(Supplementary Fig. S3).
Rigosertib was earlier shown to reduce the centrosomeassociated pool of g-tubulin and cause centrosome fragmentation (11). To evaluate the possible effects of Cent-1
on centrosomes, cells were stained with antibodies against
g-tubulin, pericentrin, and centrin after 6-hour Cent-1
treatment. In these cells, g-tubulin was greatly diminished
at spindle poles (Fig. 3C). Moreover, based on pericentrin
staining, the centrosomes were disintegrated (Fig. 3D).
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Using morphometric analysis, we detected significant
differences (P < 0.01–P < 0.001) in the following centrosome parameters: area, roundness, and AR between Cent-1
and DMSO, and between rigosertib and DMSO treatments (Fig. 3E). Furthermore, Cent-1–induced multipolar
cells exhibited a-tubulin–positive foci that were often
negative for pericentrin and centrin, suggesting that these
foci lack normal centrosome constituents (Fig. 3F). The
same observation was made in rigosertib–induced multipolar cells (data not shown). In addition to having
centrosome defects, Cent-1- and rigosertib-treated cells
exhibited shorter spindle length compared with control
(Fig. 3G). In control cells treated with DMSO, the average
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Centmitor-1 Possesses Antimitotic Properties
Figure 3. Cent-1 treatment causes chromosome alignment defects, multipolarity, and centrosome fragmentation. A, representative micrographs of DMSOand Cent-1–treated HeLa cells showing bipolar and multipolar mitotic phenotype and abnormal chromosome orientations. B, graphs showing the
quantification of spindle pole number (based on a-tubulin staining) at different time points after Cent-1 addition. DMSO treatment was for 8 hours. Data, mean
SD from two separate experiments. A total of 100 cells were analyzed per time point in both experiments. C and D, representative micrographs of DMSOand Cent-1–treated HeLa cells after staining with g-tubulin or pericentrin antibodies showing reduced g-tubulin staining and spindle pole fragmentation.
E, graph shows centrosome area, roundness, and AR according to the quantification of pericentrin-stained foci. Data, mean SD of 40 cells from two
separate experiments. F, micrographs show a representative multipolar cell after Cent-1 treatment stained with antibodies against a-tubulin, pericentrin, and
centrin. The arrow points to an a-tubulin–positive pole that is negative for pericentrin and centrin. G, the graph shows quantification of spindle length
from bipolar cells stained for pericentrin after the indicated treatments. Data, mean SD from two separate experiments (n ¼ 40 cells). H, micrographs show
NuMA localization in cells treated with DMSO, Cent-1, or rigosertib. Cells were incubated in the presence of DMSO, 5 mmol/L Cent-1, or 250 nmol/L rigosertib
for 6 hours unless stated otherwise. High-magnification views of one centrosome are shown in C, D, and F. Asterisks, statistically significant differences
between the controls and indicated compound treatments ( , P < 0.01; , P < 0.001). Scale bars, 10 mm.
spindle length was 10.4 1.0 mm, whereas Cent-1 reduced
the spindle length to 8.9 1.4 mm (P < 0.001) and rigosertib
to 8.0 0.9 mm (P < 0.001).
One mitotic protein contributing to the formation of
spindle poles and maintenance of normal spindle archi-
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tecture is the nuclear mitotic apparatus protein 1 ( NuMA;
ref. 23). Interestingly, depletion of NuMA causes defects
in chromosome alignment (24), resembling the phenotype
caused by Cent-1 and rigosertib. Treatment of cells with
Cent-1 or rigosertib caused a prominent redistribution of
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NuMA from the spindle poles to several new foci throughout the cytosol in comparison with control (Fig. 3H).
Collectively, these data show that Cent-1 and rigosertib
induce multipolarity and cause the disintegration of the
centrosomes.
Mitotic checkpoint is active in Cent-1–treated cells
To investigate why cells arrest in mitosis after Cent-1
treatment, we analyzed the levels of proteins involved
in SAC signaling. The kinetochore occupancy of SAC
proteins BubR1 and Bub1 at the unaligned chromosomes is a common marker for an active SAC (25,
26). To analyze SAC activity, HeLa cells were incubated
in the presence of Cent-1, or control compounds (taxol
or DMSO) for 6 hours. After fixation and immunofluorescence staining, BubR1 and Bub1 levels were quantified at the kinetochores of unaligned chromosomes
near the spindle poles. Only bipolar cells were chosen
for the analysis, because it was more feasible to categorize aligned/unaligned chromosomes in comparison
with multipolar cells. Similarly to control metaphase
cells, BubR1 and Bub1 were mostly undetectable at the
kinetochores of metaphase-aligned chromosomes in
Cent-1–treated cells (Fig. 4A and B). However, in the
same cells, the levels of both proteins remained elevated
at the kinetochores of unaligned chromosomes, indicating continued SAC activity (Fig. 4C and D). Also in
rigosertib-treated cells the kinetochores of unaligned
chromosomes were positive for BubR1 and Bub1 (Supplementary Fig. S4).
In another approach, we used an Aurora B kinase
inhibitor ZM447439 that is known to cause premature
SAC inactivation, leading to forced exit from M phase (27).
Hela cells were accumulated into mitosis by treatment
with Cent-1 or taxol, collected and further incubated with
or without ZM447439 for 2 hours in the continued presence of Cent-1 or taxol. If mitotic arrest is Aurora B and
SAC-mediated, cells should exit mitosis within the 2-hour
incubation period. This was the case for both Cent-1 and
taxol; the levels of mitotic markers cyclin B1 and phosphohistone H3 were greatly reduced upon ZM447439 treatment (Fig. 4E).
Cent-1 and rigosertib modulate microtubule
dynamics and microtubule plus-ends
It is well established that interference with microtubules leads to defects in spindle assembly, resulting in a
SAC-mediated mitotic arrest (28). To test whether Cent-1
impairs microtubule functions, we first investigated
whether the compound directly affects tubulin polymerization in vitro. The polymerization rate of tubulin was
monitored in the presence of various concentrations of
Cent-1. Vinblastine that induces tubulin depolymerization and taxol that increases polymerization rate served as
controls. Both reference drugs affected the rate of tubulin
polymerization as expected, whereas the addition of Cent1 at 1 or 5 mmol/L concentrations had no effect on the
reaction (Fig. 5A). Higher concentrations (10 and 20
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Mol Cancer Ther; 13(5) May 2014
mmol/L) of Cent-1 resulted in a slight increase in tubulin
polymerization rate (Fig. 5A). It was previously reported
that rigosertib does not affect the rate of tubulin polymerization in vitro although only one concentration (5 mmol/L)
was tested (11).
Next, we investigated the effects of Cent-1, rigosertib,
and control drugs nocodazole and taxol on the microtubule network of interphase cells using immunofluorescence (Fig. 5B). HeLa cells were treated with the compounds for 6 hours, fixed and immunostained with an
antibody against a-tubulin to visualize microtubules. As
expected, nocodazole disrupted the microtubule network
in comparison with DMSO, whereas taxol caused microtubule bundling (Fig. 5B). Cent-1 treatment caused very
minor, if any, changes compared with the DMSO control;
the mass and shape of interphase cell microtubules
seemed normal (Fig. 5B). Only slight curving of microtubules was observed in some cells. Similarly, rigosertib
incubation induced slight changes to the microtubule
network of interphase cells at 250 nmol/L concentration
(Fig. 5B). It was previously published that at high concentration (2.5 mmol/L), rigosertib depolymerizes microtubules (29) and our experiments confirmed this finding,
most microtubules were lost after 6-hour incubation with
2.5 mmol/L rigosertib (Supplementary Fig. S5).
Next, we fixed and immunostained Cent-1- or rigosertib-treated cells with an antibody against end-binding
protein 1 (EB1), which is a highly conserved microtubule
plus-end protein that is needed for normal microtubule
dynamics (30, 31). In DMSO-treated control cells, EB1
showed a characteristic comet-like staining pattern at the
tips of growing microtubule plus-ends (Fig. 5C). Although
Cent-1 and rigosertib did not have major effects on the
microtubule filaments in cells at low concentrations (Fig.
5B), the same treatments caused notable changes in the
localization of EB1 in interphase cells; EB1 comets were
much shorter and seemed fragmented (Fig. 5C).
This prompted us to investigate whether microtubule
dynamics was affected by the compounds in cells. The
model used was a human A549 lung carcinoma cell line
stably expressing EGFP-a-tubulin that we have earlier
validated for the measurement of microtubule dynamics
(32). In A549 EGFP-a-tubulin cells, 6-hour treatment with
5 mmol/L Cent-1 caused a similar phenotype as in HeLa
cells; the cells showed chromosome misalignment and
multipolarity, and exhibited a long mitotic delay (Supplementary Fig. S6A and S6B). Moreover, 5 mmol/L Cent1 had only minor effects on interphase cell microtubules
(Supplementary Fig. S6C). Interestingly, these cells were
much more sensitive to rigosertib treatment than HeLa
cells, and treatment with 250 nmol/L concentration of
rigosertib completely abolished microtubules (data not
shown), whereas 50 nmol/L concentration did not eliminate microtubules but was sufficient to cause mitotic
arrest (Supplementary Fig. S6A–S6C). For determining
microtubule dynamics, A549 EGFP-a-tubulin cells were
treated with DMSO, 5 mmol/L Cent-1, or 50 nmol/L
rigosertib for 1 to 2 hours. The cells were then imaged
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Centmitor-1 Possesses Antimitotic Properties
Figure 4. Mitotic checkpoint is
active in Cent-1–treated cells. A
and B, representative micrographs
showing BubR1 and Bub1 staining
in control prometaphase and
metaphase cells, and in
compound-treated cells. HeLa
cells were treated with DMSO, 0.1
mmol/L taxol, or 5 mmol/L Cent-1 for
6 hours before fixation and
immunostaining. Scale bars, 10
mm. C and D, micrographs showing
quantification of kinetochore signal
intensities of BubR1 and Bub1
immunostainings normalized
against the Crest signals in cells
treated as indicated. Data, mean SD from three separate
experiments. Twenty kinetochores
per cell were quantified from 15
cells in one experiment, except in
Cent-1–treated cells, in which
kinetochores of all unaligned
chromosomes (4–20 kinetochores
per cell) were quantified. E,
Western blot showing cyclin B,
phospho-histone H3, and
glyceraldehyde-3-phosphate
dehydrogenase levels in taxol- or
Cent-1–treated cell extracts before
and after the addition of Aurora B
inhibitor ZM447439 that causes an
override of SAC-mediated mitotic
arrest.
live and microtubule plus-ends were tracked manually
to determine average microtubule dynamicity (the distance a microtubule plus-end travels in a set time) for
each treatment. In control cells (n ¼ 21), the average
microtubule dynamicity was 0.096 0.036 mm/s whereas
in Cent-1 (n ¼ 20) and rigosertib-treated cells (n ¼ 18)
overall microtubule dynamicity was reduced to 0.051 0.019 (P < 0.001) and 0.039 0.014 (P < 0.001) mm/s,
respectively (Fig. 5D). Representative time-lapse movies
of Cent-1-, rigosertib-, and DMSO-treated cells are available as Supplementary material (Supplementary Movies
S3–S5). On the basis of these results, Cent-1 and rigosertib
modulate microtubule plus-ends and microtubule dynamicity in interphase cells.
Cent-1 and rigosertib decrease interkinetochore
tension by targeting mitotic substrates other than
Plk1
To extend the analysis of Cent-1 and rigosertib effects
on microtubules to mitotic cells, we measured interkine-
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tochore distances in HeLa cells using Cenp-A as an inner
kinetochore marker (33). Generation of tension across
sister kinetochores stabilizes microtubule–kinetochore
interactions and is required for SAC inactivation and
normal mitotic progression (5). Cells were treated with
Cent-1, rigosertib, or controls (DMSO, nocodazole, or
taxol) for 6 hours. Interkinetochore distances were measured separately for chromosomes that had aligned at the
metaphase plate and chromosomes that remained
unaligned near the spindle poles in Cent-1- and rigosertib-treated cells. Only bipolar cells were selected for the
analysis. Results show that the average interkinetochore
distance of control cells at metaphase plate was 1.51 0.09
mm, whereas treatment with Cent-1 and rigosertib
reduced the distance to 1.07 0.09 mm (P < 0.001) and
1.06 0.04 mm (P < 0.001), respectively (Fig. 6A and B). The
average interkinetochore distances of unaligned chromosomes were at similar range in control prometaphase cells
(0.89 0.07 mm), in Cent-1–treated cells (0.85 0.02 mm),
and in rigosertib-treated cells (0.85 0.04 mm). As
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Figure 5. Cent-1 and rigosertib reduce microtubule dynamicity and cause mislocalization of EB1 in cells. A, the graph showing in vitro tubulin polymerization in
the presence of Cent-1 or indicated control compounds. B, representative micrographs of drug-treated interphase HeLa cells immunostained with a-tubulin
antibody. Cent-1 and rigosertib induce mild effects on the microtubule network, whereas the positive control drugs induce strong microtubule depolymerizing
(Noc) and stabilizing (taxol) effects. C, representative micrographs of drug-treated interphase cells stained with EB1 antibody. HeLa cells were treated
with DMSO, 5 mmol/L Cent-1, 250 nmol/L rigosertib, 0.5 mmol/L nocodazole (Noc), or 0.1 mmol/L taxol for 6 hours. The morphology of the EB1 comets was
notably altered by all compound treatments compared with the DMSO control. Scale bars, 10 mm. D, the graph showing changes in microtubule dynamicity
upon Cent-1 and rigosertib treatments compared with DMSO. Microtubule dynamicity (the distance a microtubule plus-end travels in a set time) was
measured in A549 cells expressing EGFP-a-tubulin after 1-hour incubation with DMSO, 5 mmol/L Cent-1, or 50 nmol/L rigosertib. Each dot in the graph
represents the average microtubule plus-end velocity per analyzed cell (total number of cells analyzed per treatment was 18–21 from three experiments). For
each analyzed cell, we recorded 10 to 20 microtubules. Line, the mean microtubule dynamicity in each treatment category. Asterisks, statistically significant
differences between the controls and indicated treatments ( , P < 0.001).
anticipated, the shortest distances were measured for the
control drugs nocodazole (0.77 0.05 mm) and taxol (0.78
0.05 mm) that diminish tension to minimum by interfering directly with tubulin polymerization. These results
indicate that although many chromosomes were able to
move to the metaphase plate in Cent-1- and rigosertibtreated cells, the kinetochores were not under normal
tension, suggesting that Cent-1 and rigosertib influence
mitotic spindle forces.
To study whether Cent-1 and rigosertib elicit their antimitotic effects through targeting a protein that is operating
during M phase, we applied the compounds on mitotic
post-NEB HeLa cells and monitored their fate using IncuCyte at 15-minute intervals for 24 hours. Proteasome
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Mol Cancer Ther; 13(5) May 2014
inhibitor MG132 and kinesin Eg5 inhibitors monastrol
(34) and dimethylenastron were used as controls. As
expected, MG132 induced a mitotic arrest followed by cell
death due to the inhibition of protein degradation essential for anaphase onset and ordered exit from M phase (35).
In agreement with the known early mitotic function of
Eg5 in centrosome separation, monastrol and dimethylenastron had no effect when applied to post-NEB mitotic
cells (34, 36). In sharp contrast, both Cent-1 and rigosertib
caused an immediate effect in mitotic cells; most cells
arrested to M phase for several hours before they died
(Fig. 6C). On the basis of these results, the target protein of
Cent-1 and rigosertib is present in mitotic cells in which it
contributes to microtubule-mediated processes.
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Centmitor-1 Possesses Antimitotic Properties
Figure 6. Cent-1 and rigosertib cause a reduction in interkinetochore distance, and have immediate antimitotic effects without influencing Plk1 kinase activity.
A, representative micrographs of control- and drug-treated mitotic cells stained for inner kinetochore marker CenpA (red) and centromere marker Crest
(green). HeLa cells were incubated with DMSO, 5 mmol/L Cent-1, 250 nmol/L rigosertib, 0.5 mmol/L nocodazole (Noc), or 0.1 mmol/L taxol for 6 hours before
fixation and immunocytochemistry. Images are maximum Z-projections of 5 to 7 layers acquired at 0.5-mm step interval. Insets, higher-magnification views of
selected sister kinetochore pairs. Scale bar, 10 mm. B, the graph shows quantification of interkinetochore distances from control- and drug-treated
cells. Data, mean SD from three experiments. A total of 100 kinetochore pairs were measured from 10 cells in each experiment, except for unaligned
kinetochore pairs in Cent-1 and rigosertib treatments (for these, 1–10 kinetochores were measured per cell). Asterisks, statistically significant differences
between the controls and indicated treatments ( , P < 0.001). C, mitotic HeLa cells respond to Cent-1 and rigosertib treatments. Cells in mitosis (n ¼ 22–36)
were treated with 5 mmol/L Cent-1, 250 nmol/L rigosertib, 10 mmol/L MG132, 100 mmol/L monastrol (Mon), or 5 mmol/L dimethylenastron (DMA) and
their fate was recorded using time-lapse filming at 15-minute intervals. Graph, the percentage of mitotic cells exhibiting the indicated fates. D and E,
U2OS cells expressing a FRET-based sensor for measuring Plk1 activity were treated with 200 nmol/L BI2536, 5 or 10 mmol/L Cent-1, or 500 nmol/L rigosertib.
Each dot represents one cell, n ¼ 20 cells/condition. D, duration of mitosis. Note that many of the BI2536-treated cells were still in mitosis when filming ended,
and therefore the data for BI2536 underrepresent the actual duration of mitosis. E, quantification of YFP–YFP/CFP–YFP emission ratio of mitotic cells
in D. Plk1 activity is absent in interphase and peaks in mitosis.
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Plk1 localization at the kinetochores of individual chromosomes changes upon their congression to the cell
equator; the protein accumulates to unaligned kinetochores but is removed upon chromosome alignment to
the metaphase plate (37). It has been reported that failure
to remove active Plk1 from the kinetochores of chromosomes at the metaphase plate leads to decreased interkinetochore tension (38). To determine whether Plk1 is
affected by Cent-1 or rigosertib, we analyzed Plk1 localization in compound-treated cells. Microscopic analysis
did not reveal any changes from the normal dynamic
behavior of Plk1 at mitotic kinetochores (Supplementary
Fig. S7A).
We next measured the effects of Cent-1 and rigosertib
on Plk1 kinase activity using U2OS cells stably expressing
a FRET-based probe (18). Cent-1 and rigosertib caused a
mitotic delay in U2OS cells (Fig. 6D). As expected, Plk1
activity increased when cells progressed from interphase
to mitosis. The mitotic increase in Plk1 activity was efficiently blocked by BI2536 (Fig. 6E), a known Plk1 inhibitor
(13). However, in cells treated with Cent-1 or rigosertib,
Plk1 activity stayed at the normal mitotic level (Fig. 6E).
Taken together, our results suggest that Cent-1 and ribosertib do not act through Plk1 but instead target another
mitotic protein(s).
Finally, we analyzed whether Cent-1 and rigosertib
affect PI3K activity in cells as rigosertib has been
reported to inhibit PI3K in vitro (17). HeLa cells were
starved in serum-free medium and the PI3K/AKT pathway was activated by the addition of insulin (39). On the
basis of the analysis of AKT phosphorylation at Ser473,
a marker of PI3K activity (40), the addition of insulin
activated the kinase in the presence of DMSO, Cent-1,
and rigosertib, whereas a known PI3K inhibitor wortmannin completely abolished AKT phosphorylation
(Supplementary Fig. S7B and S7C). This result suggests
that Cent-1 and rigosertib do not function by directly
inhibiting PI3K in HeLa cells.
Discussion
In this study, we described the identification and characterization of Cent-1, a novel LMW compound that
possesses similar steric and electrostatic features and
antimitotic phenotype as the template compound rigosertib, which is a multikinase inhibitor with claimed
activity against PI3K and Plk1 pathways (11, 16, 17). The
template compound has shown cytotoxic effects in human
tumor cell lines and tumor growth suppression in xenograft models (11). At the moment, rigosertib is clinically
evaluated for the treatment of hematopoietic malignancies and solid tumors (9, 10, 41). We analyzed the effects of
Cent-1 in cells side by side with rigosertib and relevant
control compounds. Cent-1 was found to cause multipolarity and chromosome misalignment (Fig. 3) that triggered a SAC-mediated M-phase arrest (Fig. 4) followed by
mitotic cell death or aberrant exit from cell division (Fig. 2).
Multipolarity was often associated with the formation of
acentrosomal poles that were negative for pericentrin and
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Mol Cancer Ther; 13(5) May 2014
centrin (Fig. 3). Furthermore, we detected other spindle
and microtubule defects such as reduced microtubule
dynamics in interphase cells (Fig. 5) and reduced interkinetochore tension in mitotic cells (Fig. 6). Treatment of
cells with rigosertib induced a similar pleiotropic antimitotic phenotype.
The template compound rigosertib was originally proposed to target Plk1 via an allosteric mechanism of action
(11), but that notion has been challenged by others
(29, 42, 43). Our results showing that cells exhibit strong
antimitotic phenotypes upon treatment with Cent-1 or
rigosertib without any inhibition of Plk1 activity (Fig. 6)
support the view that Plk1 inhibition is not the mechanism
by which these compounds function. Moreover, whereas
Cent-1 and rigosertib treatments lead to multipolarity,
specific Plk1 inhibition with small molecules causes
monopolarity (13). More recently, rigosertib was shown
to inhibit the PI3K/AKT pathway (16, 17). In our analysis,
we did not observe significant changes in the phosphorylation status of AKT Ser473, a downstream target epitope
of PI3K (40) upon Cent-1 or rigosertib treatment in insulinstimulated cells (Supplementary Fig. S7). This does not,
however, exclude the possibility that the compounds
could perturb downstream elements of the PI3K/AKT
pathway, or that differences in the experimental setup
could explain the discrepancy between the results.
Importantly, because both compounds induced immediate M-phase arrest when applied to early mitotic cells,
at least one of their target proteins must be present in
mitotic cells.
We made three particularly interesting observations in
mitotic cells that may shed some light to the mechanism of
action of Cent-1 and rigosertib during M phase. First, both
compounds caused centrosome fragmentation and
reduced the amount of centrosome-associated g-tubulin
(Fig. 3). Second, they significantly retarded microtubule
dynamics in interphase cells (Fig. 5), shortened spindle
length in mitosis (Fig. 3), and decreased tension across
sister kinetochores in mitotic chromosomes (Fig. 6). Third,
the compounds caused delocalization of NuMA (Fig. 3)
and EB1 (Fig. 5) in mitotic cells. Together, these data imply
that Cent-1 and rigosertib impair microtubule-mediated
processes during M phase. It is noteworthy that acentrosomal spindle poles (44), reduced interkinetochore tension
(45), mislocalization of NuMA and EB1 (46–48), and chromosome misalignment (45) are all consequences of treatment of cells with low doses of microtubule drugs, which
strengthens the notion that the compounds may modulate
microtubule dynamics. The compounds do not apparently
perturb microtubule polymerization in vitro at low-range
concentrations (Fig. 5; ref. 11). Also, the current data do not
allow to conclude whether their impact on microtubules in
cells is direct or indirect. Cent-1 and rigosertib may impair
microtubule functions in cells by having an impact on
microtubule-associated proteins. At the moment, the identity of such proteins remains unknown but it is noteworthy
that depletion of EB1 has been reported to slow down
microtubule plus-end dynamics and reduce spindle
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Centmitor-1 Possesses Antimitotic Properties
length (30, 49, 50). It is therefore plausible to hypothesize
that EB1 mislocalization by Cent-1 and rigosertib can
contribute to the observed microtubule defects in cells.
In summary, our results provide evidence that ligandbased in silico HTS is a feasible method for the identification of novel antimitotic compounds when a known
inhibitor is used as a template. Cent-1 is a new mitotic
inhibitor that possesses similar steric and electrostatic
features as the template compound rigosertib, which
shows clinical anticancer potency. In cells, both compounds induced various defects in mitotic processes that
involve dynamic microtubules. However, the precise
mechanism of action of Cent-1 and rigosertib remains to
be clarified.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: J.H.E. M€aki-Jouppila, L.J. Laine, J. Rehnberg,
P. Tiikkainen, M.J. Kallio
Development of methodology: J. Rehnberg, E. Narvi, P. Tiikkainen,
P. Halonen
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): J.H.E. M€aki-Jouppila, J. Rehnberg, E. Narvi,
E. Hukasova, P. Halonen
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.H.E. M€aki-Jouppila, L.J. Laine,
J. Rehnberg, E. Narvi, P. Tiikkainen, E. Hukasova, P. Halonen, A. Poso,
M.J. Kallio
Writing, review, and/or revision of the manuscript: J.H.E. M€aki-Jouppila,
L.J. Laine, E. Narvi, A. Poso, M.J. Kallio
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.H.E. M€aki-Jouppila
Study supervision: L.J. Laine, A. Lindqvist, L. Kallio, M.J. Kallio
Acknowledgments
The authors thank Jouko Sandholm for the help with flow cytometry
that was performed at the Cell Imaging Core, Turku Centre for Biotechnology. They also thank Stephan Geley for providing the HeLa H2B-GFP
mCherry-tubulin cell line, and professors Markku Kallajoki and Gary
Gorbsky for providing antibodies.
Grant Support
This study was supported by Academy of Finland grants 120804 (to M.J.
Kallio) and 134272 (to L.J. Laine), Marie Curie EXT grant 002697 (to M.J.
Kallio and J. Rehnberg), the Academy of Finland Centre of Excellence for
Translational Genome-Scale Biology funding (for M.J. Kallio, P. Tiikkainen, P. Halonen, E. Narvi, and L. Kallio), the Finnish Cancer Organisations
grants (to M.J. Kallio and J.H.E. M€aki-Jouppila), FinPharma Doctoral
Program funding (for J.H.E. M€aki-Jouppila), and Finnish Cultural Foundation grant (to J.H.E. M€aki-Jouppila). A. Poso was funded by the University of Eastern Finland, School of Pharmacy. E. Hukasova and A.
Lindqvist were funded by the Swedish Research Council, the Swedish
Cancer Society, and the Swedish Foundation for Strategic Research.
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.
Received August 16, 2013; revised February 5, 2014; accepted February
25, 2014; published OnlineFirst April 18, 2014.
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Supplementary Figures
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2
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Supplementary Figure Legends
Supplementary Figure S1. The majority of rigosertib treated HeLa cells exhibit long mitotic
arrest followed by cell death. (A) Graph shows the duration of mitosis and fates of individual
compound treated cells (n=60). (B) Mitotic (MI) and cell death indices (CDI) were calculated from
IncuCyte films at different time points following the addition of 250 nM rigosertib. Data is mean ±
SD from three film frames.
Supplementary Figure S2. Centmitor-1 induces mitotic arrest followed by apoptosis. (A) Flow
cytometry profiles of HeLa cells treated with DMSO, 5 µM Cent-1, or 100 nM taxol showing the
accumulation of cells into M phase by Cent-1 and taxol treatments. The histogram depicts the
percentages of cells in different cell cycle phases from three biological replicates. (B) Annexin VFITC and propidium iodide staining of HeLa cells followed by flow cytometry showing viable (V),
early apoptotic (EA) and late apoptotic or necrotic (LA/N) cell populations after DMSO, Cent-1 and
taxol treatments. The histogram shows the percentages of cells in each population from three
biological replicates. (C) Western blot showing whole-cell lysates of HeLa cells treated with
DMSO, 1 µM staurosporine (St), 5 µM Cent-1, or 100 nM taxol for the indicated times before the
harvest and probed with antibodies against the cleaved form of PARP (cPARP) and GAPDH.
Supplementary Figure S3. Rigosertib treatment induces chromosome misalignment and
multipolarity in HeLa cells. Representative micrographs of rigosertib treated HeLa cells showing
bipolar and multipolar mitotic phenotypes, and chromosome orientations. The cells were incubated
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in the presence of DMSO or 250 nM rigosertib for 6 h before fixation and immunostaining with tubulin antibody. Scale bar equals 10 µm.
Supplementary Figure S4. SAC proteins BubR1 and Bub1 localize to the kinetochores of
unaligned chromosomes in rigosertib treated HeLa cells. (A, B) Representative micrographs
showing BubR1 and Bub1 staining in control prometaphase and metaphase cells, and in rigosertib
treated cells. HeLa cells were incubated with DMSO or 250 nM rigosertib for 6 h before fixation
and immunostaining. Scale bars equal 10 µm.
Supplementary
Figure
S5.
High
concentration
of
rigosertib
induces
microtubule
depolymerisation in HeLa cells. Images showing -tubulin stained HeLa cells after treatment with
DMSO or 2.5 µM rigosertib for 6 h. Scale bar equals 10 µm.
Supplementary Figure S6. Impact of Cent-1 and rigosertib on microtubules in A549 cells
expressing EGFP- -tubulin. (A) Representative micrographs of Cent-1 and rigosertib treated
mitotic A549 EGFP- -tubulin cells showing unaligned chromosomes and multiple
-tubulin
positive foci. (B) Graph shows the quantification of mitotic index in A549-EGFP- -tubulin cells
treated with DMSO, Cent-1, or rigosertib. (C) Representative micrographs of A549-EGFP- -tubulin
cells showing interphase microtubule networks after exposure to DMSO, Cent-1, or rigosertib. Cells
were incubated in the presence of DMSO, 5 µM Cent-1, or 50 nM rigosertib for 6 h before fixation
and immunostaining. Scale bars equal 10 µm.
Supplementary Figure S7. Analysis of Plk1 localization and PI3K activity in Cent-1 and
rigosertib treated HeLa cells. (A) Representative micrographs showing Plk1 staining in control
prometaphase and metaphase cells and in Cent-1 and rigosertib exposed early mitotic cells. HeLa
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cells were treated with DMSO, 5 µM Cent-1, or 250 nM rigosertib for 6 h before fixation and
immunostaining. Scale bar equals 10 µm. (B) Western blot analysis of phospho-AKT levels in
HeLa cells grown in normal growth medium in the presence of serum (Ctrl 1), serum-starved
overnight (Ctrl 2) and treated with DMSO, 5 µM Cent-1, 250 nM rigosertib (Rigo), or 1 µM
wortmannin (Wort) for 2.5 h followed by the addition of 100 ng/ml insulin for 30 min. (C)
Quantification shows phospho-AKT from two replicate experiments (bars indicate Western blot
signal intensity ± SD).
Supplementary Movies
Supplementary Movie S1. Cent-1 treated HeLa H2B-GFP mCherry-tubulin cells arrest in
mitosis for several hours with misaligned chromosomes, followed by cell death or abnormal
exit from M phase. The cell on the left hand side forms a multipolar spindle after nuclear envelope
breakdown (NEB) and exhibits a long mitotic arrest followed by cell division into three daughter
cells (NEB to anaphase time is 5 h 15 min). The cell on the right hand side initially forms a tri-foci
spindle (time point 01:15), then two microtubule emanating foci fuse together (time point 01:35) to
form a bipolar spindle. The cell exhibits a long mitotic arrest followed by cell death (NEB to cell
death time is 10 h 30 min). During the arrest most chromosomes congress to the metaphase plate
but some remain misaligned near the spindle poles. The cells were treated with 5 µM Cent-1 for 1 h
before time-lapse filming started with 5 min frame capture interval. The numbers indicate time in
h:min. H2B-GFP is coloured red and mCherry tubulin green.
Supplementary Movie S2. Control HeLa H2B-GFP mCherry-tubulin cells undergo normal cell
division. The representative cell forms a bipolar spindle and exhibits normal chromosome
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congression to the metaphase plate followed by cell division into two daughter cells (nuclear
envelope breakdown to anaphase time is 38 min). The cell was treated with DMSO for 1 h before
time-lapse filming started with 2 min frame capture interval. The numbers indicate time in h:min.
H2B-GFP is coloured red and mCherry tubulin green.
Supplementary Movie S3. Cent-1 treatment decreases microtubule dynamicity in human lung
carcinoma A549 cells. A549 cells stably expressing EGFP- -tubulin were treated with 5 µM Cent1 for 1 h before the analysis of microtubule dynamicity using 10 s frame capture interval for a total
of 4 min. For the analysis, microtubule plus-ends were marked (blue dots) and the total growth and
shrinkage velocities of individual microtubules were measured.
Supplementary Movie S4. Rigosertib treatment decreases microtubule dynamicity in human
lung carcinoma A549 cells. A549 cells stably expressing EGFP- -tubulin were treated with 50 nM
rigosertib for 1 h before the analysis of microtubule dynamicity using 10 s frame capture interval
for a total of 4 min. For the analysis, microtubule plus-ends were marked (blue dots) and the total
growth and shrinkage velocities of individual microtubules were measured.
Supplementary Movie S5. Microtubule dynamicity in a control A549 cell stably expressing
EGFP- -tubulin. Cells were treated with DMSO for 1 h before the analysis of microtubule
dynamicity using 10 s frame capture interval for a total of 4 min. For the analysis, microtubule plusends were marked (blue dots) and the total growth and shrinkage velocities of individual
microtubules were measured.
10
Supplementary methods
Live cell imaging
Live cell imaging of HeLa cells stably expressing histone-H2B-GFP and mCherry-tubulin was
performed with Zeiss LSM780 confocal microscope using Zen 2010B software. Normal culture
conditions were maintained with Incubation system S composed of Pecon XLmulti S1 heating unit
with heating insert P and CO2 control (PeCon GmbH, Erbach, Germany). Histone-H2B-GFP and
mCherry-tubulin were exited with 488 and 543 nm laser lines, and emissions were collected at 494556 and 560-648 nm, respectively. Live cells were also imaged using IncuCyte live-cell imager
(Essen Instruments Ltd., Hertfordshire, UK).
Flow cytometry
HeLa cells for flow cytometric cell cycle analysis were harvested by trypsinization and washed with
PBS. Samples were resuspended in citrate buffer (40 mM Na-citrate, 0.3 % Triton X-100)
containing propidium iodide (50 µg/ml, P3566, Molecular Probes). The cells were incubated for 15
min at RT and kept protected from light. For measuring apoptosis, cells were harvested by
trypsinization and Annexin V-FITC apoptosis detection kit (Abcam, ab14085) with propidium
iodide was used according to manufacturer’s instructions. Flow cytometric data was collected using
BD FACSCalibur (BD Biosciences, San Jose, USA) and CellQuest Pro software (BD Biosciences,
San Jose, USA) and analyzed with the Flowing Software ver. 2.5.1 (Mr. Perttu Terho, Turku Centre
for Biotechnology, Turku, Finland, www.flowingsoftware.com).
11
Western blotting
HeLa cells were centrifuged and washed once with cold PBS before preparation of extract or
freezing the pellets in liquid nitrogen. For the preparation of extracts, cells were lysed in 20 mM
Tris-HCl (pH 7.7), 100 mM KCl, 50 mM sucrose, 1 mM MgCl2, 0.1 mM CaCl2, 0.5 % TX-100
(APC-buffer) containing protease inhibitor cocktail (Roche, 04693132001) and phosphatase
inhibitor PhosSTOP (Roche, 4906837001) for 7 min on ice, and cell lysates were cleared by
centrifugation. Equal amounts of samples were loaded and run on 4-20 % gradient gel. Membranes
were incubated in blocking solution (5 % milk, Odyssey blocking buffer or BSA in TBS) for 1 h,
primary antibody for 1 h at RT or overnight at +4°C, and secondary antibody for 1 h in TBST at
RT. Primary antibodies included rabbit-anti-phospho AKT (Ser473, Cell Signaling, 4060), rabbitanti-AKT (Cell Signaling 9272), mouse anti-cyclin B1 (BD Bioscience–Pharmingen, 554178),
mouse anti-GAPDH (Advanced ImmunoChemical Inc., mAb 6C5), rabbit anti-phospho-histone H3
Ser10 (Upstate, 06-570) and mouse anti-cleaved PARP (Cell Signaling, 9546). Secondary
antibodies included IR Dye® Conjugated anti-mouse 800 and anti-rabbit 800 (Rockland
Immunochemicals Inc.) and Alexa Fluor® anti-mouse 680 and anti-rabbit 680 (Invitrogen). Signals
were detected using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, USA).
Statistical analysis
Statistical analysis was performed using IBM SPSS Statistics (IBM Corp., Armonk, USA) or Graph
Pad Prism 4 (GraphPad Software, Inc., La Jolla, USA) software. One-way ANOVA followed by
Tukey’s HSD t-test was performed assuming equal variances. Independent experiments were taken
into account as random factors. Values of ***p<0.001, **p<0.01 and *p<0.05 were considered
statistically significant. Data are presented as mean ± standard deviation (SD).
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