Research Article
Delocalization of the Microtubule Motor Dynein from Mitotic Spindles
by the Human Papillomavirus E7 Oncoprotein Is Not Sufficient for
Induction of Multipolar Mitoses
1,2
1
1,2
Christine L. Nguyen, Margaret E. McLaughlin-Drubin, and Karl Münger
1
Infectious Diseases Division, Channing Laboratories, Brigham and Women’s Hospital and 2Committee on Virology,
Harvard Medical School, Boston, Massachusetts
Abstract
Dynein is a minus end–directed microtubule motor that
transports numerous cargoes throughout the cell. During
mitosis, dynein motor activity is necessary for the positioning
of spindle microtubules and has also been implicated in
inactivating the spindle assembly checkpoint. Mutations in
dynein motor and/or accessory proteins are associated with
human disease, including cancer, and the delocalization of
dynein from mitotic spindles has been correlated with an
increased incidence of multipolar spindle formation in some
cancer cells that contain supernumerary centrosomes. The
high-risk human papillomavirus type 16 (HPV16) E7 oncoprotein induces centrosome overduplication and has been shown
to cause multipolar mitotic spindle formation, a diagnostic
hallmark of HPV-associated neoplasias. Here, we show that
HPV16 E7 expression leads to an increased population of
mitotic cells with dynein delocalized from the mitotic spindle.
This function maps to sequences of HPV16 E7 that are distinct
from the region necessary for centrosome overduplication.
However, contrary to previous reports, we provide evidence
that dynein delocalization by HPV16 E7 is neither necessary
nor sufficient to cause the formation of multipolar mitoses.
[Cancer Res 2008;68(21):8715–22]
Introduction
Dynein is a minus end–directed microtubule motor protein that
is composed of several subunits. The approximately 520-kDa heavy
chain subunits are responsible for generating movement along the
microtubule via ATPase activity and the approximately 74-kDa
intermediate chain subunits are thought to anchor dynein to its
cargo (reviewed in ref. 1). Dynein also contains smaller intermediate chain subunits and light chain subunits whose functions are
not clearly understood, although it has been suggested that dynein
light chains may also play an important role in diverse dynein
motor complex independent processes, such as apoptosis, enzyme
regulation, transcriptional regulation, and kidney development
(2). The processivity of dynein is increased upon complex
formation with the dynactin complex and dynactin is therefore
commonly considered an obligatory cofactor (3). The dynein motor
complex aids in the positioning of the Golgi complex and
mitochondria, along with other organelles, and transports cargo
from the endoplasmic reticulum, endosomes, and lysosomes
Requests for reprints: Karl Münger, Channing Laboratories, Brigham and
Women’s Hospital, Room 861, 181 Longwood Avenue, Boston, MA 02115. Phone:
617-525-4282; Fax: 617-525-4283; E-mail: [email protected].
I2008 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-08-1303
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(reviewed in refs. 4–7). Being a cytoplasmic motor complex, it
likely plays an important role in transporting numerous cargoes,
and additionally, dynein is also implicated in axonal transport in
neurons (reviewed in ref. 8). Furthermore, dynein transports
proteins, such as pericentrin and g-tubulin, which are necessary
for centrosome assembly during interphase (9). In mitosis, the
dynein motor complex positions mitotic spindle poles (reviewed in
ref. 10) and is responsible for targeting the nuclear mitotic
apparatus protein 1 (NuMA) to spindle poles, where NuMA focuses
and stabilizes microtubule ends and tethers them to the
centrosomes (11, 12). Dynein is also recruited to kinetochores
where it is reported to play a role in spindle assembly checkpoint
inactivation by transporting checkpoint proteins away from
properly attached kinetochores (13–15).
Likely due to the many integral functions of dynein, mutations in
dynein motors and associated proteins have been linked to human
diseases, including neurodegenerative disease (16) and cancer.
Dynein light chain 1 (DLC1) expression is regulated by estrogen
and associates with and acts as a transcriptional coactivator of the
estrogen receptor (ER; ref. 17). DLC1 is also a substrate of the
p21-activated kinase 1 (PAK1) and PAK1-mediated DLC1 phosphorylation has been linked to proliferation of ER-positive breast
cancer cells (18). DLC1 seems to be frequently overexpressed in
breast cancer and may contribute to accelerated cell cycle
progression due to reduction of nuclear p21CIP1 and increased
cyclin-dependent kinase 2 activity (18, 19). Moreover, a genomewide computer analysis of single nucleotide polymorphisms in
tumoral versus normal tissue identified a cancer-specific DLC1
variant with a glycine to cysteine mutation at amino acid residue
79, which may affect ligand binding (20). Additionally, it was shown
that in some cancer cell lines, there was an increased proportion of
mitotic cells where dynein was delocalized from the mitotic spindle
(21). In these cells, dynein delocalization was correlated with an
increased incidence in multipolarity during mitosis, and therefore,
dynein was purported to play a role in the coalescence of
supernumerary centrosomes. Spindle multipolarity, together with
centrosome amplification, has been observed in many tumor
tissues, including high-risk human papillomavirus (HPV)-associated
lesions and cancers (22–24). Centrosome-associated mitotic
abnormalities likely importantly contribute to the genomic instability that drives carcinogenesis (reviewed in refs. 25–27). Thus, it
is possible that the disruption of microtubule motors by high-risk
HPV may contribute to HPV-associated tumorigenesis.
In concert with the high-risk HPV E6 oncoprotein, the high-risk
HPV E7 oncoprotein is necessary for the establishment and
maintenance of the transformed phenotype of HPV-associated
neoplasias. High-risk HPV E6 and E7 disrupt the p53 and pRB
tumor suppressor pathways, respectively. Additional mutations to
the host genome are required for the malignant progression of
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HPV-associated premalignant lesions, however, and each of the
viral oncoproteins actively contributes to the destabilization of the
host genome through different mechanisms. The expression of
high-risk HPV16 E7 results in the deregulation of the centrosome
duplication cycle, allowing for excess rounds of centrosomal
duplication to occur during one round of cell division (24, 28, 29).
The accumulation of supernumerary centrosomes increases the
frequency of aberrant mitoses and often causes the formation of
multiple mitotic spindle poles. In fact, multipolar mitoses are a
diagnostic hallmark used by pathologists to identify high-risk HPVassociated dysplasias (30). Due to the correlation between dynein
delocalization and spindle multipolarity (21) and our observation
that the recruitment of g-tubulin to centrosomes, a dyneindependent process (9), is altered in HPV16 E7–expressing cells (31),
we were interested in determining whether HPV16 E7 affects
dynein dynamics. Here, we show that the expression of HPV16 E7
in cells leads to an increased population of mitotic cells where
dynein is delocalized from the mitotic spindle. This function of
HPV16 E7 is independent of its ability to cause centrosome
overduplication and deregulate g-tubulin dynamics, however, and
maps to the carboxyl (COOH) terminus of HPV16 E7. Importantly,
using primary human foreskin fibroblasts (HFF) and human
foreskin keratinocytes (HFK) as well as immortalized mouse
embryo fibroblasts, we did not observe a correlation between
dynein delocalization and the incidence of multipolar mitoses
upon HPV16 E7 expression. Nevertheless, because dynein is
involved in many cellular processes, it is possible that HPV16
E7–mediated dynein delocalization may contribute to the many
other functions of HPV16 E7, including cell cycle deregulation and
abrogation of the spindle assembly checkpoint.
Materials and Methods
Cells. NIH 3T3 mouse embryo fibroblasts, HPV-negative cervical cancer
cells C33a, and HPV-positive cervical cancer cells CaSki, SiHa, and HeLa
were obtained from the American Type Culture Collection (ATCC) and
maintained in DMEM (Invitrogen) supplemented with 10% calf serum,
penicillin (50 units/mL), and streptomycin (50 Ag/mL). HEK293 human
embryonic kidney cells were also obtained from ATCC and were maintained
in DMEM supplemented with 10% fetal bovine serum, penicillin (50 units/
mL), and streptomycin (50 Ag/mL). Normal oral keratinocytes (NOK)
immortalized by human telomerase (hTERT) F HPV16 E7 were previously
described (32) and were maintained in keratinocyte serum-free medium
supplemented with human recombinant epidermal growth factor 1-53,
bovine pituitary extract (Life Technologies Invitrogen), penicillin (50 units/
mL), streptomycin (50 Ag/mL), gentamicin (10 Ag/mL), and amphotericin B
(0.75 Ag/mL).
NIH 3T3 cells with stable expression of empty vector (3T3-poz), COOHterminally FLAG- and HA-tagged HPV16 E7 (3T3-CE7), or COOH-terminally
FLAG- and HA-tagged HPV16 E7D21-24 (3T3-D21-24) were previously
described (31). NIH 3T3 cells with stable expression of COOH-terminally
FLAG- and HA-tagged HPV16 E7H2G, E7H2P, E7D6-10, E7C24G, E7E26G,
E7E46A, E7CVQ68-70AAA (E7CVQ), E7D79-83, and E7C91S [all in the poz-C
vector (33, 34)] were made via cotransfections with pBudCE4.1 (Invitrogen)
at a 5:1 ratio after selection with 1 mg/mL zeocin (Invitrogen) as previously
described (31).
Primary HFKs were isolated from anonymous newborn circumcisions
and cultured as described previously (35). HFK cells stably expressing
HPV16 E7 were created by transfecting primary HFK populations with
p1435, a human h-actin HPV16 E7 expression plasmid, or p1318, the
parental plasmid (36), as a control, together with pcDNA3.1 (Invitrogen)
using the Amaxa Human Keratinocyte Nucleofector kit (Amaxa Biosystems)
per the manufacturer’s instructions. G418 (0.2 mg/mL) was then used to
select for transfected cells.
Cancer Res 2008; 68: (21). November 1, 2008
Primary HFFs were also isolated from anonymous newborn circumcisions. After the epidermis was separated from the dermis, the dermis was
minced and incubated in a 2 mg/mL collagenase solution in DMEM and
trypsin (4:1) for 2 to 4 h at 37jC. HFFs were maintained in DMEM
supplemented with 10% calf serum, penicillin (50 units/mL), and
streptomycin (50 Ag/mL). HFF cells stably expressing wild-type or mutant
HPV16 E7 were generated from primary HFF populations infected with the
following pBABE-puromycin–based retroviral constructs: pBABE-puromycin (pBABE), pBABE-puromycin HPV16 E7 (pBABE-E7), pBABE-puromycin
HPV16 E7D21-24 (pBABE-E7D21-24), and HPV16 E7D79-83 (pBABE-E7D7983). Recombinant retroviruses were produced as previously described (32).
Infections of 50% confluent HFFs were performed with a mixture of 2 mL
viral supernatant, 8 Ag/mL polybrene, and 2 mL DMEM for 6 h. The cells
were maintained in DMEM supplemented with 10% calf serum, penicillin
(50 units/mL), and streptomycin (50 Ag/mL) following selection with
1 Ag/mL puromycin.
Immunofluorescence. Cells were plated onto coverslips and extracted,
fixed, and stained as previously described (31). Primary antibodies used in
these studies were anti-dynein (MAB1618, Chemicon International), anti-atubulin (ab18251, Abcam), and anti-g-tubulin (H-183, Santa Cruz Biotechnology). Secondary antibodies used in these studies were Alexa Fluor 488
and Rhodamine Red-X (Invitrogen) and nuclei were counterstained with
Hoechst 33258 dye. Immunofluorescence images were taken with a Nikon
TE-300 inverted microscope equipped with phase and fluorescence optics.
Images were collected digitally using a progressive-scan, interline-cooled,
charge-coupled device camera (Hamamatsu Corp.), analyzed with MetaMorph software (Molecular Devices), and processed for presentation using
Photoshop (Adobe Systems).
Western blotting. Whole-cell lysates were produced using ML buffer
[20 mmol/L Tris (pH 8), 1 mmol/L EDTA, 300 mmol/L NaCl, 0.5% NP40,
protease inhibitors (Complete EDTA-free tablets; Roche Diagnostics)] and
cleared by centrifugation (4jC, 20 min, 16,000 g ). Aliquots (100 Ag) of
whole-cell lysates were run on SDS 12% polyacrylamide gels. After
electrophoresis, proteins were electrotransferred to a polyvinylidene
difluoride membrane (Millipore). The membrane was probed with antiactin (MAB1501, Chemicon International), anti-E7 (ED19), and anti-HA
(A190-108A, Bethyl Laboratories, Inc.) antibodies. Secondary antibodies
used were horseradish peroxidase–linked anti-mouse or anti-rabbit IgG (GE
Healthcare). Proteins were visualized using Western Lightning Chemiluminescence Reagent Plus (Perkin-Elmer Life Sciences, Inc.) or SuperSignal
West Dura Extended Duration Substrate (Thermo Scientific) and exposed
on Kodak BioMax XAR film or electronically acquired with a Kodak Image
Station 4000R equipped with Kodak Imaging Software, version 4.0.
Results
HPV16 E7 expression leads to the delocalization of dynein
from mitotic spindles. It was previously shown that the
microtubule motor protein dynein is delocalized from mitotic
spindles in some cancer cell lines and this was correlated to an
increased incidence of multipolar metaphases in cells containing
supernumerary centrosomes due to the abrogation of centrosome
coalescence (21). Furthermore, dynein is responsible for the
recruitment of g-tubulin to centrosomes (9). Therefore, because
HPV16 E7–expressing cells have increased levels of multipolar
metaphases (37) and the recruitment of g-tubulin to centrosomes
is altered in the presence of HPV16 E7 (31), we wanted to
determine if dynein is delocalized from mitotic spindles in HPV16
E7–expressing cells. Immunofluorescent staining for dynein, as well
as the mitotic spindle marker a-tubulin, was performed on mitotic
NIH 3T3 cells that stably express control vector, 3T3-poz, or COOHterminally HA/FLAG-tagged HPV16 E7, 3T3-CE7. In mitotic cells,
dynein localizes to mitotic spindles and, therefore, stains microtubules that emanate from mitotic spindle poles (Fig. 1A).
Abnormal dynein localization, however, seemed punctate and did
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HPV16 E7 and Dynein
Figure 1. Dynein is delocalized from mitotic spindles in HPV16 E7–expressing cells. A, immunofluorescent staining of dynein (green ) and a-tubulin (red ). An example
of normal dynein staining, on mitotic spindles, and abnormal dynein staining, punctate and away from spindles, is shown for both NIH 3T3 and NOK-hTERT cells.
For each cell type, the example of normal dynein localization is an image of a control cell, whereas the abnormal example is an image of an HPV16 E7–expressing cell.
Normal and abnormal dynein localization was seen in both cell populations. B, bar graphs showing the percentage of mitotic cells with abnormal dynein localization
in either NIH 3T3 cells stably expressing empty vector (Control ) or COOH-terminally HA/FLAG-tagged HPV16 E7 (CE7 ) or NOK-hTERT cells, HFFs, or HFKs
stably expressing empty vector or HPV16 E7, as indicated. For the NIH 3T3 cells, the results represent averages from two independent experiments where >100 mitotic
cells were counted per experiment. For the NOK-hTERT cells, the results represent averages from three independent experiments where 100 to 225 mitotic cells
were counted per experiment. For HFF cells, the results represent averages from two independent experiments where >110 mitotic cells were counted per experiment.
For HFK cells, the results represent averages from three independent experiments where >100 mitotic cells were counted per experiment. Bars, SE.
not largely localize to mitotic spindles (Fig. 1A). Mitotic cells that
showed questionable, intermediate staining results were not
included in this analysis. The incidence of dynein delocalization
in the mitotic 3T3-CE7 cells was f2.5-fold higher than in the 3T3poz cells (21.6% compared with 8.6%; P < 0.02, Student’s t test;
Fig. 1B). Furthermore, NOKs immortalized by hTERT or primary
HFFs or HFKs, each with the stable expression of HPV16 E7 at
levels similar to HPV16-positive cervical carcinoma cells (data not
shown) or control vector, were examined to determine if this
phenotype was also observed in human non–cancer-derived cells.
Although the parental cells each had different base levels of
abnormal dynein localization, HPV16 E7 expression in each of
these cells resulted in an f2-fold increase in the fraction of mitotic
cells with delocalized dynein [2.2-fold in NOK cells, 23.4% compared with 10.5% (P < 0.02); 2.1-fold in HFF cells, 15.2% compared
with 7.2% (P < 0.01); and 1.7-fold in HFK cells, 21.4% compared with
12.3% (P < 0.02); Fig. 1B].
Dynein delocalization occurs in HPV-positive cervical
cancer cells. To determine if dynein delocalization was observed
under conditions of biologically relevant HPV16 E7 expression, we
compared the percentage of dynein delocalization in HPV16positive cervical carcinoma cell lines CaSki and SiHa to HEK293
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cells, which were previously shown to have predominantly normal
dynein localization during mitosis (21), and to primary HFKs. We
observed an increased incidence of delocalized dynein in the
mitotic HPV-positive cervical cancer cells, 19.1% and 23.7% for
CaSki and SiHa, respectively, compared with 8.5% and 6.1% seen in
mitotic HEK293 and HFK cells, respectively (Fig. 2). Together, these
results suggest that HPV16 E7 expression results in an increased
incidence of dynein delocalized from the mitotic spindles and that
a considerable fraction of mitotic HPV16-positive cancer cells
contain delocalized dynein.
HPV16 E7–induced dynein delocalization is not dependent
on HPV16 E7–mediated centrosome overduplication. The
expression of HPV16 E7 causes centrosome overduplication in
primary human cells (24, 38) and HPV16 E7 associates with and
deregulates g-tubulin (31); both of these activities rely on amino
acid residues 21 to 24 of HPV16 E7 (31, 39). To determine whether
abnormal dynein localization as a result of HPV16 E7 expression was
related to the ability of HPV16 E7 to induce aberrant centrosome
duplication and/or to subvert g-tubulin dynamics, we generated a panel of stable NIH 3T3–based cell lines to map the domain(s)
of E7 necessary for dynein delocalization. The NIH 3T3 cell line was
chosen because it is an immortalized non–cancer-derived cell line
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Figure 2. Dynein is delocalized from mitotic spindles in HPV-positive cervical
carcinoma cells. Bar graph showing the percentage of mitotic cells with abnormal
dynein localization in HPV-negative cells, HEK293 and primary HFKs, or HPVpositive cervical cancer cells, CaSki and SiHa. Columns, average from two
independent experiments where >100 mitotic cells were counted per experiment;
bars, SE.
with a low level of abnormal dynein localization during mitosis
(Fig. 1B). Furthermore, as reported previously (31), HPV16 E7
expression in NIH 3T3 cells induces a 3.3-fold increase in cells with
supernumerary centrosomes, whereas expression of the HPV16
E7D21-24 mutant does not. Therefore, NIH 3T3 cells with the stable
expression of various previously characterized HPV16 E7 mutants,
each containing COOH-terminal HA/FLAG tags, were established
and analyzed for the incidence of abnormal dynein localization as
well as for the incidence of supernumerary centrosomes. As was
previously shown (31, 39), HPV16 E7D21-24 was unable to cause
centrosome overduplication (Fig. 3A). Interestingly, however,
expression of HPV16 E7D21-24 induced dynein delocalization with
a similar efficiency as wild-type E7 (2.2-fold versus 2.5-fold,
respectively, over control cells). In contrast, the COOH-terminal
mutants E7CVQ68-70AAA (hereby called E7CVQ), E7D79-83, and
E7C91S each displayed a statistically significant (1.6-, 2.0-, and
1.8-fold, respectively; P < 0.05, Student’s t test) decrease in the ability
to cause dynein delocalization compared with wild-type HPV16 E7.
Interestingly, although the E7CVQ, E7D79-83, and E7C91S mutants
also display a decreased ability to cause centrosome overduplication compared with wild-type HPV16 E7 (1.9-fold, P = 0.02; 1.5-fold,
P = 0.04; and 1.8-fold, P = 0.02, respectively), their capacity to induce
supernumerary centrosomes was still significantly higher compared
with the E7D21-24 mutant (1.8-fold, P = 0.03; 2.4-fold, P = 0.0001; and
2.0-fold, P = 0.02, respectively; Fig. 3A). Hence, the ability of HPV16
E7 to disrupt dynein localization in mitotic cells maps to the COOH
terminus of HPV16 E7.
Although the expression levels of the wild-type and mutant
HPV16 E7–expressing cells differed somewhat, expression of
HPV16 E7 in the various cell lines was detectable via Western
blotting (Fig. 3A), and more importantly, variations in expression
levels did not seem to directly influence the phenotypes examined.
Nevertheless, to confirm that the observed phenotypes are not a
consequence of expression levels, we generated single-cell clones
from the 3T3-poz, 3T3-CE7, 3T3-D21-24, and 3T3-D79-83 cell lines
and examined clones that expressed the E7 constructs at similarly
low levels [3T3-CE7(1), 3T3-D21-24(3), and 3T3-D79-83(8)]; the 3T3poz clone 3T3-poz(8) was chosen at random (Fig. 3B). The
expression of HPV16 E7 in the 3T3-CE7(1) cell line was quantified
and is 4-fold lower than that of the pooled 3T3-CE7 cell line (data
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not shown). Again, the same pattern of activity is observed in the
cells expressing wild-type HPV16 E7 or the E7D21-24 and E7D79-83
mutants. The expression of wild-type HPV16 E7 in the 3T3-CE7(1)
clonal cell line results in a 2.4-fold increase in cells with supernumerary centrosomes (P = 0.01) and a 1.4-fold increase in mitotic
cells with delocalized dynein (P = 0.05; Fig. 3B). We suspect that the
more modest fold increase in dynein delocalization observed with
the clonal cell line expressing HPV16 E7 compared with the pooled
population (Fig. 3A) is due to a slightly higher basal level of dynein
delocalization detected in the 3T3-poz(8) cell line, which may be
inherent to the specific clone; this supports the notion that pooled
populations of cell lines are likely more reliable because variations
among single cells are averaged out over the entire population.
Regardless, in comparison with the control 3T3-poz(8) cells, the
3T3-E7D21-24(3) cell line displayed a 1.5-fold increase in the
amount of mitotic cells with delocalized dynein (P = 0.1), whereas
an increase in cells with supernumerary centrosomes was not
observed (0.9-fold, P = 0.6; Fig. 3B). Conversely, in comparison with
the control 3T3-poz(8) cells, the 3T3-E7D79-83(8) cell line did not
display an increase in the amount of mitotic cells with delocalized
dynein (0.8-fold, P = 0.2) yet contained a 3.4-fold increased amount
of cells with supernumerary centrosomes (P = 0.02; Fig. 3B). The
fact that the same mapping pattern was observed with pooled
populations that contained varied expression of the HPV16 E7
constructs compared with clonal populations expressing similar
levels of HPV16 E7 provides further support for the notion that the
observed phenotypes are not consequences of the levels of HPV16
E7 expression.
Additionally, to ensure that wild-type HPV16 E7 as well as the
E7D21-24 and E7D79-83 mutants displayed these observed
activities in human cells, we examined primary HFFs with stable
expression of control vector, wild-type HPV16 E7, or the E7D21-24
or E7D79-83 mutants. Once again, the same pattern of activity is
observed whereby the HFF cells expressing wild-type HPV16 E7
display a 2.2-fold (P = 0.02) increase in cells with supernumerary
centrosomes and a 1.4-fold (P = 0.02) increase in mitotic cells
where dynein is delocalized from the mitotic spindle (Fig. 3C). In
cells expressing the HPV16 E7D21-24 mutant, a 1.6-fold (P = 0.05)
increase in mitotic cells with delocalized dynein is observed,
whereas there is no detectable increase in cells with supernumerary centrosomes (1-fold, P = 0.9; Fig. 3C). In cells expressing the
HPV16 E7D79-83 mutant, a 2.5-fold (P = 0.07) increase in cells with
supernumerary centrosomes is observed, yet there is no appreciable increase in the amount of mitotic cells with delocalized dynein
(1.1-fold, P = 0.2; Fig. 3C). These data confirm our finding that the
ability of HPV16 E7 to disrupt dynein localization in mitotic cells
maps to a separate region of HPV16 E7 as that responsible for the
deregulation of centrosome duplication.
Dynein delocalization in HPV16 E7–expressing cells does
not clearly correlate with induction of multipolar metaphases.
As mentioned above, the delocalization of dynein from mitotic
spindles in some cancer cells has been associated with an
increased incidence of multipolar mitoses (21). To investigate
whether HPV16 E7–mediated dynein delocalization contributes to
the formation of multipolar mitoses in HPV16 E7–expressing cells,
we used NIH 3T3 cells and primary HFFs and HFKs. HPV16 E7
has previously been shown to induce centrosome overduplication
in NIH 3T3 cells and in primary cells (31, 38), as confirmed here
(Fig. 3C); in the primary HFKs used in the following experiment,
the stable expression of HPV16 E7 resulted in a 2.2-fold increase in
cells with supernumerary centrosomes (P = 0.0004). Furthermore,
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HPV16 E7 and Dynein
HPV16 E7 has been implicated in the induction of multipolar
mitoses in U2OS osteosarcoma cells (24). To determine whether
there was a correlation between multipolar mitoses and dynein
delocalization, it was first necessary to investigate whether
there was an increased incidence of multipolar metaphases in
HPV16 E7–expressing NIH 3T3 cells, HFFs, or HFKs. We examined
at least 200 metaphases each for control and HPV16 E7–expressing
cells. In the stable NIH 3T3 cells, we did not detect a noticeable
difference between control 3T3-poz (4 of 200; 2%) and 3T3-CE7
cells (5 of 200; 2.5%; Fig. 4A). With HFFs, we again did not observe a
single case of multipolar mitosis in either control or HPV16 E7–
expressing cells. In the HFK cells, the expression of HPV16 E7
resulted in a slight increase in the incidence of multipolar mitoses
(4 of 225; 1.78%) compared with the empty vector control (2 of 225;
0.89%); however, this difference was not statistically significant
(P = 0.7, Fisher’s exact test). Moreover, at least some of the
previously observed NOK cells as well as the primary HFK cells
undergoing multipolar mitoses exhibited normal mitotic spindleassociated dynein staining (Fig. 4B; data not shown), suggesting
that dynein delocalization induced as a consequence of HPV16 E7
expression is not sufficient to generate multipolar mitoses and,
conversely, that multipolar mitoses can arise in cells with normal
dynein localization. Taken together, these results suggest that
multipolar mitoses do not automatically ensue in cells with
Figure 3. HPV16 E7–mediated dynein delocalization
and centrosome overduplication are separate events.
A, bar graph showing the fold increase in either dynein
delocalization (black columns ) or supernumerary
centrosomes (gray columns ) in NIH 3T3 cells with
stable expression of the indicated HPV16 E7 plasmids
compared with the empty vector control. For dynein
delocalization counts, the results represent averages
from two independent experiments where >125 cells
were counted per experiment. For supernumerary
centrosome counts, the results represent averages
from three independent experiments where >150 cells
were counted per experiment. Bars, SE. Western blots
provided beneath the graph show the expression
levels of the various cell lines, where HA antibody
detects the HA/FLAG-tagged E7 proteins; actin is
shown as a loading control. B and C, as described in
A, cell lines analyzed are clonal populations of the
3T3-poz, 3T3-CE7, 3T3-D21-24, and 3T3-D79-83 cell
lines or primary HFFs stably expressing empty vector
(ctrl), wild-type E7, or the E7D21-24 or E7D79-83
mutants, as indicated. For dynein delocalization
counts, the results represent averages from two
independent experiments where >100 cells were
counted per experiment. For supernumerary
centrosome counts, the results represent averages
from two independent experiments where >200 cells
were counted per experiment. Bars, SE. Western
blots are provided to show expression levels of the
various HPV16 E7 constructs; actin is shown as a
loading control.
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Figure 4. HPV16 E7–mediated dynein delocalization does not correlate with an
increased incidence of multipolarity. A, bar graph depicting the percentage of
multipolar mitoses in either control or HPV16 E7–expressing NIH 3T3, HFF, or
HFK cells, as indicated. For NIH 3T3 and HFF cells, 200 mitotic cells each
were analyzed, and for HFK cells, 225 mitotic cells were analyzed. B,
immunofluorescent staining of dynein (green ) and g-tubulin (red ) in a multipolar
mitotic NOK-hTERT+16E7 cell.
supernumerary centrosomes that exhibit increased dynein delocalization during mitosis.
Discussion
Recently, the deregulation of dynein localization was implicated
in the formation of multipolar mitoses (21). Additionally, the
recruitment of g-tubulin onto centrosomes is mediated by dynein
(9). In light of these observations and given that we have previously
shown that the expression of HPV16 E7 leads to centrosome
overduplication and mitotic abnormalities associated with multipolar metaphases (24, 37, 38), and that HPV16 E7 expression alters
the recruitment of g-tubulin to centrosomes (31), we investigated
whether dynein was delocalized from mitotic spindles in HPV16
E7–expressing cells. After examining various cell types, including
primary and immortalized cells, we conclude that the expression of
HPV16 E7 leads to a higher frequency of mitotic cells with dynein
delocalized from mitotic spindles compared with the empty vector
control. Furthermore, the capacity to delocalize dynein from
mitotic spindles maps to the COOH terminus of HPV16 E7, which
is distinct from the amino-terminal domain that has previously
been identified as a major determinant for the induction of
supernumerary centrosomes (24, 38) and subversion of g-tubulin
dynamics (31). Interestingly, however, HPV16 E7–induced dynein
delocalization does not correlate with multipolar spindle formation,
Cancer Res 2008; 68: (21). November 1, 2008
even in cell lines that display supernumerary centrosomes; this
unexpected result will be discussed further below.
Dynein is a microtubule motor that is clearly important for
numerous cellular processes, therefore making it difficult to
determine what, if any, is the primary purpose behind the
deregulation of dynein localization by HPV16 E7. One of the key
roles of HPV16 E7 in the viral life cycle is the establishment of
a DNA-replication–competent cellular milieu, which is achieved
via the deregulation of the cell cycle at various stages. HPV16
E7–mediated pRB degradation promotes S-phase entry and is also
predicted to disrupt mitotic checkpoints through aberrant Mad2
expression (40). However, because dynein is implicated in the
inactivation of the spindle assembly checkpoint at kinetochores
(13–15), it would seem that dynein delocalization may lead to a
prolonged mitotic checkpoint. Nonetheless, one could argue that
allowing cells to aberrantly proceed into anaphase could be
detrimental to the host cells and, thus, to the virus. Many viruses
must maintain a sensitive balance within a cell whereby they use
numerous cellular components but ensure that the cell has
sufficient resources to survive for the duration of infection.
Therefore, the delocalization of dynein by HPV16 E7 may help
dampen the abrogation of the mitotic spindle assembly checkpoint
by HPV16 E7 (41).
Because dynein is a minus end–directed motor, it is responsible
for transporting many proteins and/or complexes to centrosomes,
where the minus ends of microtubules are anchored. Centrosomes
have previously been suggested to act as a scaffold for proteasomal
machinery (42, 43) and the dynein complex transports aggregates
of misfolded proteins to the centrosome to facilitate degradation
(44). The centrosome-associated proteasome is not limited to the
degradation of misfolded proteins (43), however, and it is therefore
likely that other cargoes of the dynein motor complex are also
degraded at the centrosome. Interestingly, it was shown that
preventing the degradation of mps1 at the centrosome causes
centrosome reduplication (45), suggesting that degradation of
proteins specifically at the centrosome may be an important step in
various tightly coordinated cellular processes. It is interesting to
note that the COOH-terminal mutants have a slightly decreased
ability to cause centrosome overduplication compared with wildtype HPV16 E7, and it will be interesting to determine whether
dynein delocalization also contributes at least partially to HPV16
E7–mediated centrosome overduplication. Furthermore, it remains
possible that the delocalization of dynein interferes with the
degradation of other proteins that are important to cell cycle
regulation and, thus, contributes to HPV16 E7–mediated disruption of the cell cycle.
The delocalization of dynein was shown to correlate with the
increased incidence of multipolar mitoses in certain cancer cell
lines (21). It was purported that dynein was somehow involved in
the process of centrosome coalescence (21), whereby supernumerary centrosomes are clustered together to ensure for the formation of a bipolar spindle during mitosis (46, 47). Therefore, we
hypothesized that the delocalization of dynein in HPV16 E7–
expressing cells may contribute to disruption of centrosomal
coalescence and to the emergence of abnormal, multipolar mitoses
in HPV16 E7–expressing cells. An increased incidence of multipolar
metaphases was previously detected in U2OS osteosarcoma cells
expressing HPV16 E7, and it was shown that this was an immediate
effect as evidenced by the fact that multipolarity was increased in
cells 48 h after expression of HPV16 E7 (24). In both primary
human cells and immortalized mouse cells expressing HPV16 E7,
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HPV16 E7 and Dynein
however, we did not notice a significantly increased frequency of
multipolar mitotic cells (Fig. 4A). The reason for the differences in
our observations may be due to the fact that we analyzed cells that
have not been in culture for extended periods of time (after HPV16
E7 expression) and, therefore, have not acquired necessary
downstream alterations that may play a role in the formation of
multipolar spindles. Although it was shown that HPV16 E7 could
increase the frequency of multipolar metaphases 48 h after
transfection (24), this experiment was performed in U2OS
osteosarcoma cells that may already have a compromised cellular
environment that is permissive to multipolar spindle formation. In
fact, preliminary experiments suggested that U2OS cells have a
higher population of mitotic cells with delocalized dynein than
HEK293 cells (data not shown) and we therefore decided not to use
these cells for studies regarding the effect of HPV16 E7 expression
on dynein localization. Accordingly, our results agree with the
observation that HPV16 E7 expression in primary HFKs does not
result in a dramatic increase of multipolar mitotic cells (24).
Importantly, our data show that, in cells containing supernumerary
centrosomes, the emergence of multipolar metaphases is not an
obligatory result of dynein delocalization and that multipolarity
can occur even in the presence of properly localized dynein. As
such, this finding is consistent with results reported by Rebacz and
colleagues (48) who showed that griseofulvin, a compound isolated
from Penicillium, inhibited centrosome coalescence without
affecting the localization of dynein to mitotic spindles.
Overall, our results do not rule out the possibility that the
increased delocalization of dynein from mitotic spindles by HPV16
E7 plays a role in HPV16 E7–induced spindle multipolarity but
suggest that it may need to work in concert with other cellular
alterations that occur after extended cell divisions. Additionally, the
fact that there seemed to be a synergistic effect on spindle
multipolarity when both HPV16 E6 and E7 were expressed in
primary HFKs (24) suggests that the effects of HPV16 E7 on dynein
delocalization may need to compile with the effects of HPV16 E6
expression to induce abnormal multipolar metaphases. For
example, if HPV16 E7–mediated dynein delocalization indeed
contributes to multipolar spindle formation, the expression of
HPV16 E6 may be necessary to disrupt cellular checkpoints that
would normally render a cell intolerant to forming and/or
maintaining multiple mitotic spindle poles. Furthermore, HPV16
E6–mediated cytokinesis defects may cause a cell to have extra
cellular factors or chromosomal DNA that would somehow alter
cellular signals and, together with HPV16 E7–mediated dynein
delocalization, would now allow for multipolar spindle formation.
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Received 4/7/2008; revised 8/13/2008; accepted 8/17/2008.
Grant support: USPHS grants T32CA009031 (C.L. Nguyen), R01 CA066980,
CA081135 (K. Münger), and ACS PF-07-072-01-MBC (M.E. McLaughlin-Drubin).
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.
We thank Hiroyuki Hayakawa for assistance with preliminary experiments and for
helpful discussions.
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Research.
Delocalization of the Microtubule Motor Dynein from Mitotic
Spindles by the Human Papillomavirus E7 Oncoprotein Is
Not Sufficient for Induction of Multipolar Mitoses
Christine L. Nguyen, Margaret E. McLaughlin-Drubin and Karl Münger
Cancer Res 2008;68:8715-8722.
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