DOI: 10.1126/science.1104905
, 127 (2005);307Science
et al.Nicholas J. Quintyne
Spindle Multipolarity Is Prevented by Centrosomal Clustering
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on the manuscript. Supported by Grant-in-Aid for
Specially Promoted Research from the Ministry of
Education, Culture, Science, and Technology.
Supporting Online Material
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Figs. S1 and S2
References
3 August 2004; accepted 10 November 2004
10.1126/science.1103631
Spindle Multipolarity Is Prevented
by Centrosomal Clustering
Nicholas J. Quintyne,1
Janet E. Reing,1
Diane R. Hoffelder,1
Susanne M. Gollin,2
William S. Saunders1
*
Most tumor cells are characterized by increased genomic instability and
chromosome segregational defects, often associated with hyperamplification
of the centrosome and the formation of multipolar spindles. However, extra
centrosomes do not always lead to multipolarity. Here, we describe a process
of centrosomal clustering that prevented the formation of multipolar spindles
in noncancer cells. Noncancer cells needed to overcome this clustering
mechanism to allow multipolar spindles to form at a high frequency. The
microtubule motor cytoplasmic dynein was a critical part of this coalescing
machinery, and in some tumor cells overexpression of the spindle protein
NuMA interfered with dynein localization, promoting multipolarity.
Hyperamplification of the centrosome has
been observed in many tumor tissues and cell
lines and is linked with both aneuploidy and
tumorigenesis (1–4). The extent of genomic
instability is generally correlated with the
degree of centrosomal abnormalities (3–5).
Furthermore, centrosome abnormalities are
more severe in high-grade and recurrent tumors
and in cell lines that show aggressive malignant
phenotypes (1–4). In mitosis, supernumerary
centrosomes can lead to an increase in spindle
poles, and multipolar spindles are found in
many cancer cell types (6).
Although centrosome amplification is clearly
important for multipolar spindles, the presence
of extra centrosomes does not always lead
to multipolar spindle formation. Certain cell
types can apparently suppress multipolarity
and form a bipolar spindle during mitosis even
though the centrosomes are amplified (7–9).
Furthermore, some centrosomal defects induce
centrosomal amplification without multipolarity
(10). To suppress multipolar spindles, the
cell could functionally silence the extra centrosomes,
preventing them from forming a
spindle pole and leaving only two centrosomes
active. Alternatively, a cell could coalesce
the extra centrosomes into only two functional
spindle poles (7, 11, 12).
The spindle protein NuMA has been shown
to be critical for spindle assembly (13–17),
and the NUMA1 gene maps to one of the most
frequently amplified chromosomal segments in
cancer cells (18, 19). We examined two cell
lines that exhibited relatively high amounts of
NuMA expression (Fig. 1A) and that had
È20% multipolar spindles (Fig. 1, B and D).
If the overexpression of NuMA is driving
multipolarity, then reduction of NuMA by
small interfering RNA (siRNA) should lead
to a return of bipolar spindles. Three days
after a single siRNA transfection (20), NuMA
amounts were reduced (Fig. 1C and fig. S1),
whereas amounts of the associated proteins
dynein and dynactin were unchanged (fig. S2)
(16). To examine only cells that received
siRNA (È50%), we labeled the siRNA duplex
with a fluorescein marker. In both the
UPCI:SCC103 and UPCI:SCC078 oral cancer
cell lines, transfection with the NuMA siRNA
nearly eliminated multipolar spindles (Fig. 1,
B and D). Similar results were observed for
the SK-HEP-1 liver adenocarcinoma cell line
(Fig. 1D). When the NuMA level was allowed
to recover 10 days after a single siRNA treatment,
the frequency of multipolarity returned
to that of untreated cells. Thus, overexpression
of NuMA perturbs the ability of these
cells to coalesce supernumerary centrosomes
into a single pole. NuMA provides a cohesive
force in maintaining spindle microtubules
around a single centrosome (13, 16),
but these results suggest a previously unknown
role for elevated amounts of NuMA
as an inhibitor of centrosome coalescence in
cells with supernumerary centrosomes.
1
Department of Biological Sciences, 2
Department of
Human Genetics and the University of Pittsburgh
Cancer Institute, University of Pittsburgh, 4249 Fifth
Avenue, Pittsburgh, PA 15260, USA.
*To whom correspondence should be addressed.
E-mail: wsaund@pitt.edu
Fig. 1. Reduction of NuMA decreases spindle multipolarity. (A) Immunoblots of whole-cell extracts
from the indicated cell lines were probed with antibodies to NuMA, dynein intermediate chain, the
dynactin subunit p150Glued, and HSET. Only NuMA and dynein showed an increase in expression in
cancer cells when compared to normal oral keratinocytes (UP3) (24). (B) UPCI:SCC103 cells were
stained with antibodies to centrosomal g-tubulin (red), fluorescein-labeled siRNA (green), and the
DNA dye 4¶,6¶-diamidino-2-phenylindole (DAPI) (blue) before and after siRNA transfection. (C)
NuMA protein reduction after transfection. (D) Decrease in multipolar spindles in metaphase cells
after siRNA to NuMA. In each case, the decrease in multipolar spindles was matched by an increase in
bipolar spindles. (E) UPCI:SCC103 cells stained with antibodies to centrin-2 (green) and g-tubulin
(red) and with DAPI (blue). Magnified views of spindle poles are shown at bottom at 3Â magnification.
Asterisks match the magnified images with the larger image. Bars indicate 10 mm in all figures.
www.sciencemag.org SCIENCE VOL 307 7 JANUARY 2005 127
R E P O R T S
CORRECTED 25 MARCH 2005; SEE LAST PAGE
Does NuMA act directly on the spindle
or exert its effect on other spindle proteins?
Dynein (13, 16), its associated activator
dynactin (21), and the kinesin motor HSET
(22) all localized along the spindle microtubules
in noncancer HEK293 cells (Fig. 2)
(22, 23). Whereas HSET and dynactin
localized normally in the oral cancer cells,
spindle dynein immunoreactivity was absent
or sharply reduced (Fig. 2), although
dynein intermediate chain expression was
elevated in these cells (Fig. 1A) (24). Punctate
dynein staining was still visible in the
cancer cells, probably representing nonspindle
vesicle-associated motor molecules.
Reduction of NuMA did not interfere with
dynein localization at the centrosomes of
interphase cancer cells. A survey of various
cancer cell types showed a similar loss of
spindle dynein, but not dynactin, immunolabeling
from nearly all cultured cancer cell
lines tested (table S1).
Because NuMA overexpression led to
multipolar spindle formation and dynein
plays a critical role in spindle formation
and maintenance, we tested whether the
overexpression of NuMA was the cause of
the change in spindle-associated dynein.
When NuMA amounts were reduced in the
UPCI:SCC103 oral cancer cell line (or
UPCI:SCC078), dynein was visible on nearly
all of the spindles (Fig. 3A, image e). More
than 90% of the spindles in UPCI:SCC103
cells lacked dynein staining before treatment,
whereas after siRNA treatment 980%
of the transfected cells had visible dynein
staining (Fig. 4A). Thus, overexpression of
NuMA induced both a change in spindle
dynein and multipolarity in these cancer cell
lines. Similar results were observed for the
SK-HEP-1 liver adenocarcinoma cell line
(table S1).
However, spindle dynein delocalization
in other cancer cell lines is not dependent on
NuMA. In the UPCI:SCC070 cells, which do
not have NUMA1 amplification (19), reduction
of NuMA had no effect on spindle multipolarity
and did not restore dynein staining (Fig. 3A,
image f). Similar observations were made in
other tumor cell lines (table S1). Although a
reduction of spindle dynein staining seems to
be a common feature of many cancer cell lines,
only some cell types achieve this change by
overexpression of NuMA.
Could a change in spindle dynein account
for the failure of centrosome clustering in
cancer cells? We first checked for a correlation
between the frequency of multipolar
spindles and centrosomal amplification or
dynein localization in various cancer cell
lines. Of the tested lines, only those with
centrosomal amplification and no dynein
immunolabeling on the mitotic spindle were
able to induce multipolar spindles in 910%
of the metaphase cells (table S2). However,
other unknown variables could also account
for the differences between these different
cell lines.
To confirm that these two factors were
critical for spindle multipolarity, we determined
whether inducing extra centrosomes
and/or inhibiting dynein function would
result in multipolarity in noncancer cells.
We treated HEK293 cells with the microtubule
inhibitor Colcemid (Irvine Scientific,
Santa Ana, CA) for 28 to 36 hours, and the
frequency of cells with extra centrosomes
increased from G5% to È80% (Fig. 3B,
images a and a_, and fig. S3). The increase
in spindle multipolarity was limited to
between È8% and È20% of the metaphase
cells (Fig. 4, A and B; this would still be
relatively high for the untreated cancer
cells). Thus, centrosome amplification alone
leads to only a limited increase in multipolarity
in this nontumor cell line. To inhibit
dynein, we transfected the HEK293 cells with
either plasmids expressing NuMA (14) to mimic
the overexpression seen in the oral cancer
cells or a plasmid expressing the dyneinbinding
fragment of p150Glued, CC1 (25).
Overexpression of NuMA was able to displace
dynein from the spindle of È50% of the
HEK293 cells, reproducing the change we
saw in cancer cells with NUMA1 amplification,
but by itself did not increase the frequency
of multipolar spindles (Figs. 3B, images
b and b_ and 4, B and C) (21). Similarly, expression
of CC1 reduced dynein on the spindle
as expected but only marginally elevated spindle
multipolarity (Fig. 4, B and C). Thus, inhibition
of dynein or amplification of centrosomes
alone was sufficient to induce only a modest
increase in multipolar spindles. However, when
cells were treated with both Colcemid and
overexpression of either NuMA or CC1, multipolar
spindle frequency increased to È60 to
70% of metaphase cells (Figs. 3B, images c
and c_ and 4, B and C).
We also tried two other methods of
amplifying centrosomes in conjunction with
inhibition of dynein. HEK293 cells were
transfected with a plasmid expressing the
centrosomal kinase hMps1 (26). Centrosomal
amplification rose from 6.1% to 36% of
the cells, but multipolarity only increased
when cells were cotransfected with plasmids
expressing NuMA or CC1 (fig. S4, A and B).
Similarly, the preexisting centrosomal coalescence
of N1E-115 cells (8, 9) was
eliminated by NuMA or CC1 overexpression,
and spindle multipolarity jumped from
È5% to È80%. Another human oral cancer
cell line (UPCI:SCC114) was found to possess
similar, but less pronounced, centrosomal
Fig. 2. Dynein is depleted from
the spindle in oral cancer cells.
HEK293 (images a to e) and
UPCI:SCC103 (images a’ to e’
and a’’ to e’’) cells were stained
with antibodies to dynein, the
p150Glued or Arp 1 subunits of
dynactin, or HSET (green) and
with DAPI (blue). Spindles in
UPCI:SCC103 cells lacked visible
dynein but were positive for
dynactin and HSET. Antibodies
to two different subunits of the
dynein macromolecular complex
gave similar results, indicating
that the loss of immunoreactivity
was not likely to be caused by
epitope masking.
7 JANUARY 2005 VOL 307 SCIENCE www.sciencemag.org128
R E P O R T S
clustering like that of N1E-115. Unlike the
other human cancer lines we tested that did
not show centrosomal clustering, expression of
either NuMA or CC1 alone in UPCI:SCC114
markedly elevated spindle multipolarity in the
absence of Colcemid exposure or hMps1
overexpression (fig. S4, C to E).
It appears that spindle multipolarity arises
via two distinct steps, an increase in centrosome
number and an inhibition of centrosomal
coalescence (7, 11, 12). Centrosome
hyperamplification has been described in
numerous tumor types (1–4). However, a
second change leading to a loss of centrosome
coalescence was required to manifest
the multipolar phenotype. Thus, clustering
may be an important mechanism for preserving
genomic stability in noncancer cells.
Overcoming centrosomal clustering appears
to involve a change in spindle dynein, either
a reduction of dynein amounts or a change to
a more diffuse position within the spindle
such that the strong fluorescent signal is not
seen. It is unlikely that dynein is completely
inhibited in the spindle of cancer cells, because
we did not see the splaying of spindle
poles observed after injection of antibodies
to dynein (21) and we observed NuMA
labeling on the spindle, which requires transport
by dynein (27). Apparently, enough
dynein activity remains to prevent these
phenotypes. However, these results show that
dynein plays an important role in maintaining
the coalescing mechanism to prevent multipolar
spindles.
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28. The authors thank D. Compton for antibodies to
NuMA and HSET; A. Merdes for the pGW-NUMA1
plasmid; T. Schroer for the CC1 plasmid and DLIC
and Arp1 antibodies; M. Winey for the hMps1
plasmid; Z. Yu for culturing the UP3 cells; and
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discussions. The work was supported by NIH
grant DE016086 and American Cancer Society
grant RSG-96-016-06 to W.S.S. and the Oral
Cavity Cancer Center suppported by NIH grant
P60DE13059.
Supporting Online Material
www.sciencemag.org/cgi/content/full/307/5706/127/
DC1
Materials and Methods
Figs. S1 to S4
Tables S1 and S2
7 September 2004; accepted 3 November 2004
10.1126/science.1104905
Fig. 4. Inhibition of dynein or NuMA overexpression
stimulated formation of multipolar
spindles only in cells with amplified
centrosomes. (A) Indicated cell lines were
scored for the presence or absence of
dynein from the metaphase spindle. FB, cultured
human skin fibroblast. HEK293 metaphase
cells were scored for dynein depletion
from the spindle (B) and frequency of multipolarity
(C) after treatment with Colcemid,
transfection with plasmids to NuMA or CC1,
or double treated. ox, overexpression
Fig. 3. Dynein is restored to spindles after siRNA-mediated knockdown of NuMA. (A) Cells stained
with antibodies to dynein intermediate chain (green) and DAPI (blue). Normal human oral
keratinocytes (UP3) (image a) and skin fibroblasts (image b) showed the expected spindle-associated
dynein. Untransfected metaphase UPCI:SCC103 showed little or no detectable dynein on multipolar
(image c) or bipolar spindles (image d). Dynein returned to spindles in UPCI:SCC103 cultures transfected
with NuMA siRNA (red, image e) but not for UPCI:SCC070 (image f). (B) Inhibition of dynein or NuMA
overexpression stimulated formation of multipolar spindles only in cells with amplified centrosomes.
HEK293 cells were treated with Colcemid (a and a’), or overexpression of NuMA (images b and b’), or
both (images c and c’). Antibodies used in (A) images and (B) images a to c were anti-dynein LIC (green)
and in (B) images a’ to c’ were anti-g-tubulin (red). DAPI, blue. Insets, 3Â magnifications.
www.sciencemag.org SCIENCE VOL 307 7 JANUARY 2005 129
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1www.sciencemag.org SCIENCE Erratum post date 25 MARCH 2005
post date 25 March 2005
ERRATUM
C O R R E C T I O N S A N D C L A R I F I C A T I O N S
RReeppoorrttss:: “Spindle multipolarity is prevented by centrosomsal clustering” by N.
J. Quintyne et al. (7 Jan. 2005, p. 127). There was an error in Fig. 3. Panel B, a
was mistakenly printed twice, with the second printing slightly overlapping
panel B, a′.The corrected figure is shown here.