Tripeptidyl Peptidase II Is Required for c-MYC-Induced Centriole Overduplication and a Novel Therapeutic Target in c-MYC-Associated Neoplasms.

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Genes & Cancer
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DOI: 10.1177/1947601910389605
2010 1: 883 originally published online 16 November 2010Genes & Cancer
Münger
Stefan Duensing, Sebastian Darr, Rolando Cuevas, Nadja Melquiot, Anthony G. Brickner, Anette Duensing and Karl
Associated Neoplasms−Therapeutic Target in c-MYC
Induced Centriole Overduplication and a Novel−Tripeptidyl Peptidase II Is Required for c-MYC
 
 
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Introduction
The c-MYC proto-oncogene encodes a transcription factor
that is overexpressed in a wide range of human cancers.
c-MYC contributes to tumorigenesis by altering a spectrum
of cellular functions such as proliferation, differentiation,
and sensitivity to apoptosis. c-MYC is rapidly induced in
response to mitogenic stimuli and contributes to the regula-
tion of normal cell cycle progression.1 Several lines of evi-
dence suggest that c-MYC plays a role in both structural
and numerical chromosomal instability by inducing DNA
damage2-5 as well as centrosome aberrations,6 which can
result in cell division errors and aneuploidy.7 Besides
c-MYC, the related N-MYC protein as well as other basic
helix-loop-helix transcription factors have previously been
implicated in centrosome abnormalities.8,9
Centrosomes are the major microtubule organizing cen-
ters in most mammalian cells and orchestrate cell division
through various functions.10,11 The single centrosome of a
cell normally duplicates precisely once prior to mitosis, thus
assuring bipolarity of the cell division process and proper
chromosome segregation. During this process, the 2 pre-
existing (maternal) centrioles disengage, and each nucleates
the formation of a single new centriole (daughter centriole).12
It has recently been discovered that certain oncogenic stimuli
can override this “one-and-only-one” rule of centriole dupli-
cation and stimulate the formation of more than one daughter
at a single maternal centriole (centriole multiplication).11,13
Several key players have been identified that are involved in
centriole multiplication including cyclin E/CDK2 com-
plexes,6,13 hSAS-6,14 and PLK4.15-18
There is compelling evidence that centriole duplication
control is regulated by proteolysis, and inhibitors of certain
protein degradation systems have been found to rapidly cause
1Cancer Virology Program, University of Pittsburgh Cancer Institute,
Pittsburgh, PA, USA
2Department of Microbiology and Molecular Genetics, University of
Pittsburgh School of Medicine, Pittsburgh, PA, USA
3Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical
School, Boston, MA, USA
4Institute of Virology, Hannover Medical School, Hannover, Germany
5Departments of Medicine and Immunology, University of Pittsburgh
Cancer Institute, University of Pittsburgh School of Medicine,
Pittsburgh, PA, USA
6Department of Pathology, University of Pittsburgh School of Medicine,
Pittsburgh, PA, USA
*These authors contributed equally.
Corresponding Authors:
Stefan Duensing, Cancer Virology Program, University of Pittsburgh
Cancer Institute, 5117 Centre Avenue, Pittsburgh, PA 15213
Email: duensing@pitt.edu
Karl Münger, Channing Laboratory, Brigham and Women’s Hospital,
Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115
Email: kmunger@rics.bwh.harvard.edu
Tripeptidyl Peptidase II Is Required for
c-MYC–Induced Centriole Overduplication
and a Novel Therapeutic Target in
c-MYC–Associated Neoplasms
Stefan Duensing1,2,*, Sebastian Darr1,3,4,*, Rolando Cuevas1,*, Nadja Melquiot3, Anthony G. Brickner5,
Anette Duensing1,6, and Karl Münger3
Submitted 08-Sep-2010; revised 12-Oct-2010; accepted 12-Oct-2010
Abstract
Centrosome aberrations are frequently detected in c-MYC–associated human malignancies. Here, we show that c-MYC–induced centrosome and centriole
overduplication critically depend on the protease tripeptidyl peptidase II (TPPII). We found that TPPII localizes to centrosomes and that overexpression
of TPPII, similar to c-MYC, can disrupt centriole duplication control and cause centriole multiplication, a process during which maternal centrioles
nucleate the formation of more than a single daughter centriole. We report that inactivation of TPPII using chemical inhibitors or siRNA-mediated protein
knockdown effectively reduced c-MYC–induced centriole overduplication. Remarkably, the potent and selective TPPII inhibitor butabindide not only
potently suppressed centriole aberrations but also caused significant cell death and growth suppression in aggressive human Burkitt lymphoma cells with
c-MYC overexpression. Taken together, these results highlight the role of TPPII in c-MYC–induced centriole overduplication and encourage further studies
to explore TPPII as a novel antineoplastic drug target.
Keywords
c-MYC, TPPII, centrosome, butabindide, cancer
Original Article
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884 Genes & Cancer / vol 1 no 9 (2010)
centriole multiplication.6,13 Components of the 26S protea-
some as well as several proteases have been found to localize
to the centrosome and contribute to centriole duplication
control.19-21 Besides microtubule organization, the centro-
some has been suggested to play an important role in MHC
class I–mediated antigen presentation.22 This function
requires a specific peptide length for MHC loading, which
has been suggested to depend on the concerted proteolytic
action of the proteasome and, under certain conditions, ami-
nopeptidases such as tripeptidyl peptidase II (TPPII).23-25
TPPII is a serine peptidase of the subtilisin family that
sequentially removes tripeptides from the amino-terminal
end of oligopeptides released by the proteasome. TPPII has
also been reported to possess endoproteolytic activity, but
this function remains poorly characterized. TPPII subunits
can assemble into higher order, spindle-shaped giant pepti-
dase complexes of 6 MDa, thus forming the largest known
protease complex in eukaryotic cells.26 TPPII has been impli-
cated in cancer and has been shown to be upregulated in
c-MYC–associated Burkitt lymphoma cells.27
We have recently shown that c-MYC–overexpressing
cells contain aberrant centrosome numbers.6,28 Here, we
extend these findings by showing that c-MYC can stimulate
centriole multiplication in a process that critically depends on
TPPII. We show that TPPII localizes to the centrosome and
that its overexpression, like c-MYC, stimulates centriole
multiplication. Moreover, we demonstrate that inactivation of
TPPII with either chemical inhibitors or by siRNA-mediated
protein knockdown effectively suppresses c-MYC–induced
centriole overduplication. Remarkably, the potent and selec-
tive TPPII inhibitor butabindide, which has originally been
designed to control appetite and overeating,29 showed pro-
nounced proapoptotic and growth-suppressive activities in
highly aggressive c-MYC–driven Burkitt lymphoma cells.
Our findings therefore highlight the role of TPPII in c-MYC–
induced centriole overduplication and suggest targeting
TPPII with the potent and selective inhibitor butabindide as a
novel antineoplastic strategy.
Results
Overexpression of c-MYC induces centriole multiplication.
The c-MYC oncogene has previously been implicated in
centrosome duplication control.6,28 We found that transient
overexpression of c-MYC led to an increase of cells with
abnormal centrosome numbers (more than 2 per cell) in U-2
OS cells (Fig. 1A) from 4.9% in control-transfected cells to
11.5% in c-MYC–transfected cells (P ≤ 0.005) (Fig. 1B) as
well as to an increase of cells with abnormal centriole num-
bers (more than 4 per cell with no specific arrangement of
supernumerary centrioles) from 4.6% in controls to 12% in
c-MYC–transfected U-2 OS/centrin-GFP cells (P ≤ 0.0005)
(Fig. 1B). c-MYC was also found to stimulate abnormal
centrosome numbers in nonimmortalized normal human
keratinocytes from 2.6% in controls to 7.2% in c-MYC–
transfected cells (P ≤ 0.05) (Fig. 1B). To corroborate our
results, we engineered U-2 OS/centrin-GFP cells to stably
overexpress empty control vector or c-MYC. Stable over-
expression led to a statistically significant 2.8-fold increase
of cells with abnormal centriole numbers from 6.3% in con-
trols to 17.4% in c-MYC–expressing cells (P ≤ 0.05) (Fig.
1C).
Remarkably, we found that a proportion of transiently
c-MYC–transfected cells with abnormal centriole numbers
showed a phenotype in which single maternal centrioles
organize the concurrent formation of more than one daughter
(referred to as centriole multiplication in contrast to centriole
overduplication with no specific centriole arrangement) (Fig.
1D). At 24 hours posttransfection, the proportion of cells
with centriole multiplication was significantly increased
from 1% in controls to 4.3% in c-MYC–transfected cells
(P ≤ 0.0005). These results confirm and extend previous
findings by showing that c-MYC overexpression rapidly dis-
rupts centriole duplication control, which in part involves
centriole multiplication.
c-MYC–induced abnormal centriole duplication requires TPPII
activity. There is compelling evidence that proteolysis plays a
crucial role in centriole duplication control.6,13 Cells overex-
pressing c-MYC, in particular, Burkitt lymphoma cells, have
previously been reported to have impaired ubiquitin-
proteasome activity and to rely more on alternative proteolytic
pathways, in particular, tripeptidyl peptidase II (TPPII)–
mediated proteolysis.27,30,31 We therefore tested whether 2
inhibitors of TPPII activity, Ala-Ala-Phe-chloromethylke-
tone (AAF-CMK) and butabindide, can interfere with
c-MYC–induced abnormal centriole duplication (Fig. 2).
Whereas AAF-CMK covalently binds to TPPII, butabindide
is a selective and competitive inhibitor of TPPII.29 Treatment
of c-MYC–transfected U-2 OS/centrin-GFP cells with 1 µM
AAF-CMK for 24 hours resulted in a suppression of c-
MYC–induced centriole overduplication from 12.4% in
c-MYC–transfected, DMSO-treated controls to 8.9% in
c-MYC–transfected, AAF-CMK–treated cells (P ≤ 0.05)
(Fig. 2A). Treatment with increasing amounts of the potent
and selective TPPII inhibitor butabindide led to a complete
abrogation of c-MYC–induced centriole overduplication
from 10.2% in untreated, c-MYC–transfected U-2 OS/
centrin-GFP cells to 4% in c-MYC–transfected treated cells
with 10 µM butabindide for 24 hours (P ≤ 0.001), which is
similar to empty vector–transfected controls (4.6%) (Fig. 2B).
These results show that c-MYC–induced centriole overdupli-
cation is effectively abrogated by TPPII inhibitors.
Knockdown of TPPII suppresses c-MYC–induced centriole
overduplication. To further corroborate the role of TPPII in
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c-MYC, TPPII, and centriole overduplication / Duensing et al. 885
c-MYC–induced centriole overduplication, we performed a
series of siRNA and shRNA experiments (Fig. 3). c-MYC–
induced centriole overduplication (10.6%) was signifi-
cantly suppressed when TPPII was depleted (3.9%; P ≤
0.0001) (Fig. 3A and 3B), whereas control siRNA duplexes
had no effect (Fig. 3B). A similar significant inhibition of
c-MYC–induced centriole overduplication was detected
when TPPII was depleted by shRNA from 12.8% in
c-MYC–transfected cells to 6.4% in c-MYC–transfected
and TPPII-depleted cells (P ≤ 0.0001) (Fig. 3C and 3D).
In both siRNA and shRNA experiments, we also ectopi-
cally expressed TPPII, which led to a significant increase of
cells with centriole overduplication from 5.4% in empty
vector controls to 11.5% (P ≤ 0.0001) (Fig. 3B) and 4.9% to
11% (P ≤ 0.0001) (Fig. 3D), respectively. siRNA- or shRNA-
mediated knockdown of TPPII was found to efficiently
Figure 1. Overexpression of c-MYC induces abnormal duplication of centrosomes and centrioles. (A) Immunofluorescence microscopic analysis of U-2
OS cells for centrosome aberrations using an anti-γ-tubulin antibody. Note the abnormal centrosome numbers in a U-2 OS cell transiently transfected
with c-MYC (right panel: arrowhead, inset) in comparison to an empty vector–transfected cell (left panel). Farnesylatable GFP is used as a transfection
marker. Nuclei stained with DAPI. Scale bar indicates 10 µm. (B) Quantification of abnormal centrosome numbers in U-2 OS cells (left panel), abnormal
centriole numbers in U-2 OS/centrin-GFP cells (middle panel), and abnormal centrosome numbers in normal human keratinocytes (NHKs) (right
panel) following transient transfection (48 hours) with either empty vector (control) or c-MYC. Each bar indicates average ± standard error of at
least 3 independent experiments with at least 100 cells counted per experiment. Asterisks indicate statistically significant differences and P values. (C)
Quantification of abnormal centriole numbers in U-2 OS/centrin-GFP cells manipulated to stably express either empty vector (control) or c-MYC. Each
bar indicates average ± standard error of at least 3 independent counts of at least 100 cells. Asterisk indicates statistically significant differences and
P value. (D) Fluorescence microscopic analysis of U-2 OS/centrin-GFP cells transiently transfected (24 hours) with empty vector (control) or c-MYC.
Note the presence of 2 daughter centrioles (arrows) at a single maternal centriole (arrowhead) in the c-MYC–expressing cell (centriole multiplication).
Quantification of centriole multiplication in U-2 OS/centrin-GFP cells after transient transfection (24 hours) of empty vector (control) or c-MYC (right
panel). Each bar indicates average ± standard error of 5 independent experiments with at least 100 cells counted per experiment. Asterisks indicate
statistically significant differences and P values.
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886 Genes & Cancer / vol 1 no 9 (2010)
abrogate the increase of cells with centriole overduplication
(Fig. 3B and 3D).
However, we were not able to substantiate an increase of
TPPII mRNA in cells transfected with c-MYC by quantita-
tive real-time PCR, whereas a significant increase of orni-
thine decarboxylase (ODC), a known transcriptional target of
c-MYC, was readily detectable (data not shown). As expected
from these results, we did not find an increase of TPPII
expression on the protein level (data not shown). Hence,
although TPPII overexpression clearly stimulates centriole
overduplication, c-MYC does not seem to induce its upregu-
lation on the transcriptional or protein level. Nonetheless,
c-MYC strictly requires TPPII for centriole overduplication,
and our results therefore support the idea that additional
c-MYC–associated oncogenic mechanisms may be involved
in c-MYC–induced centriole overduplication.
TPPII is a centrosomal protein and rapidly subverts centriole
duplication control when overexpressed. Having shown that
TPPII is critically required for c-MYC–induced centriole over-
duplication and can stimulate centriole overduplication itself,
we next investigated whether TPPII localizes to the centro-
some. Immunofluorescence microscopic analysis of U-2 OS/
centrin-GFP cells showed that TPPII is present at the centro-
some throughout the cell division cycle (Fig. 4A). Based on
Figure 2. Inhibitors of TPPII suppress c-MYC–induced centriole
overduplication. (A and B) Quantification of numerical centriole
abnormalities in U-2 OS/centrin-GFP cells transiently transfected with
empty vector (control) or c-MYC for 24 hours and either treated with 0.1%
DMSO or 1 µM of the TPPII inhibitor Ala-Ala-Phe-chloromethylketone
(AAF-CMK) for an additional 24 hours (A). (B) Cells were treated
with the TPPII inhibitor butabindide for 24 hours at the indicated
concentrations. Each bar indicates average ± standard error of at least 3
independent experiments with at least 100 cells counted per experiment.
Asterisks indicate statistically significant differences between DMSO and
AAF-CMK–treated cells (A), control- and c-MYC–transfected cells (B),
and c-MYC–transfected cells in comparison to c-MYC–transfected cells
treated with butabindide (B) and P values.
Figure 3. Knockdown of TPPII inhibits c-MYC–induced centriole
overduplication. (A) Immunoblot analysis of TPPII expression following
siRNA-mediated knockdown in U-2 OS/centrin-GFP cells in comparison
to control siRNA-transfected cells (siCtrl). Immunoblot for actin shows
protein loading. (B) Quantification of U-2 OS/centrin-GFP cells with
centriole overduplication following transient overexpression (24 hours)
of empty vector (control) or c-MYC alone or c-MYC in combination with
control siRNA duplexes (siControl) or siRNA duplexes targeting TPPII
(siTPPII). Centriole overduplication following transient overexpression
(24 hours) of TPPII alone or in combination with control siRNA
(siControl) or siRNA targeting TPPII (siTPPII) is also shown. Each bar
indicates average ± standard error of 3 independent experiments with
at least 100 cells counted per experiment. Asterisks indicate statistically
significant differences and P values. (C) Immunoblot analysis of TPPII
expression following shRNA-mediated knockdown in U-2 OS/centrin-
GFP cells in comparison to control shRNA-transfected cells (shCtrl).
Immunoblot for actin shows protein loading. (D) Quantification of U-2
OS/centrin-GFP cells with centriole overduplication following transient
overexpression (48 hours) of empty vector (control) or c-MYC alone or
c-MYC in combination with an shRNA construct targeting TPPII (shTPPII).
Centriole overduplication following transient overexpression (48 hours)
of TPPII alone or in combination with shRNA targeting TPPII (shTPPII) is
also shown. Each bar indicates average ± standard error of 3 independent
experiments with at least 100 cells counted per experiment. Asterisks
indicate statistically significant differences and P values.
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c-MYC, TPPII, and centriole overduplication / Duensing et al. 887
the fact that daughter centrioles are commonly smaller in size
compared to maternal centrioles, we observed a staining pat-
tern suggestive of selective association with daughter centri-
oles in late mitosis (anaphase) and in the unduplicated state,
whereas TPPII was detected mostly between mother and
daughter centrioles during the G2 phase (Fig. 4A).
We next found that ectopic TPPII expression was sufficient
to cause centriole multiplication (Fig. 4B) similar to c-MYC
overexpression. The proportion of cells with centriole overdu-
plication (i.e., more than 4 centrioles without the characteristic
arrangement of 2 or more daughters at single maternal centri-
oles) was 1.9% following TPPII overexpression, whereas
Figure 4. TPPII localizes to the centrosome and stimulates centriole multiplication. (A) Immunofluorescence microscopic analysis of TPPII in U-2 OS/
centrin-GFP cells. Note the presence of TPPII at centrioles throughout the cell division cycle. Nuclei stained with DAPI. Scale bar indicates 10 µm.
(B) Fluorescence microscopic analysis of centrioles in a U-2 OS/centrin-GFP cells transiently transfected with empty vector (control) or TPPII. Note the
presence of centriole multiplication in the TPPII-transfected cell as evidenced by the concurrent presence of 2 daughter centrioles (arrows) at a single
maternal centriole (arrowhead). (C) Quantification of centriole overduplication (gray bars) and centriole multiplication (black bars) in U-2 OS/centrin-
GFP cells transiently transfected with empty vector (control) or TPPII. Each bar indicates average ± standard error of 3 independent experiments with at
least 100 cells counted per experiment. Asterisks indicate statistically significant differences and P values.
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888 Genes & Cancer / vol 1 no 9 (2010)
5.5% of cells showed centriole multiplication (Fig. 4B) com-
pared to 0.1% of cells with centriole overduplication (P ≤
0.001) and 1% of cells with centriole multiplication (P ≤
0.0001) in empty vector–transfected control cells (Fig. 4C).
These results extend a previous report that has implicated
TPPII in centrosome overduplication32 by showing that TPPII
upregulation causes a genuine centriole duplication defect and
not merely centrosome accumulation, a possibility that has not
rigorously been excluded in that previous study.32 In combina-
tion with our data that TPPII is required for c-MYC–induced
centriole multiplication, these findings further support the
notion that c-MYC and TPPII may function in the same path-
way to induce centriole multiplication but cannot rule out that
additional c-MYC–associated oncogenic mechanisms may be
involved.
Butabindide suppresses centriole overduplication and malig-
nant growth in Burkitt lymphoma cells. We next sought to
explore whether the TPPII inhibitor butabindide may sup-
press centrosome aberrations in cells that constitutively
overexpress c-MYC. We first compared the baseline levels
of numerical centrosome aberrations between 2 low-grade
malignant B cell lines U266 (multiple myeloma with unde-
tectable c-MYC mRNA expression) and HBL2 (mantle cell
lymphoma) and 3 EBV-negative Burkitt lymphoma cell
lines with c-MYC overexpression (BJAB, DG75, BL2),
respectively. Overall, cell lines with c-MYC overexpres-
sion showed a trend towards increased proportions of cells
with numerical centriole aberrations (Fig. 5A). We next
treated BJAB, DG75, and BL2 cells with 1 µM or 10 µM
butabindide for up to 4 days. All drug treatments led to a
significant reduction of the proportion of cells with centri-
ole overduplication (Fig. 5B). In addition, treatment of each
of the 3 Burkitt lymphoma cell lines with 10 µM butabin-
dide caused a significant increase of cell death at 96 hours
as evidenced by an increase in cells with sub-G1 DNA con-
tent by flow cytometry (Fig. 5C).
To further investigate whether TPPII inhibition restrains
Burkitt lymphoma growth, we cultured the human Burkitt
lymphoma cell line GA-10 in the presence of the protea-
some inhibitor clastolactacystin β-lactone (CLBL; 1 µM) or
0.1% DMSO, respectively (Fig. 5D). Continuous culture in
the presence of the proteasome inhibitor CLBL promotes
the use of components of alternative proteolytic pathways,
in particular, TPPII, to maintain a certain level of intracel-
lular proteolysis.33 The GA-10 cell line was derived from a
highly aggressive t(8;14)-positive, Epstein-Barr virus–
negative Burkitt lymphoma with mutated p53 and was
resistant to conventional chemotherapy.34 After exposure to
CLBL, we first observed a marked decrease of cell viabil-
ity, but proteasome inhibitor–resistant cells emerged within
approximately 10 days (Fig. 5D). As expected from previ-
ous studies,33,35 GA-10 cells adapted to grow in the presence
of CLBL exhibited increased TPPII protein levels (Fig. 5E).
The levels of centrosome abnormalities did not differ sig-
nificantly between parental GA-10 cells (18.7%), GA-10
cells mock adapted in DMSO (19.9%), and CLBL-adapted
cells (19.4%), respectively. Importantly, however, adapted
GA-10 cells were more sensitive to TPPII inhibition by but-
abindide compared to their nonadapted counterparts (Fig.
5F). Moreover, 10 µM butabindide reduced soft agar colony
formation of the GA-10/adapted cell line by 6.5-fold (data
not shown). To explore whether the growth-inhibitory effect
of butabindide in adapted GA-10 cells may be related to
interference with centrosome duplication, we specifically
analyzed mitotic cells for the number of γ-tubulin–positive
(i.e., centrosome associated) mitotic spindle poles. We did
not observe a significant increase of monopolar spindles
in butabindide-treated adapted GA-10 cells compared to
vehicle-treated cells (4.6% v. 3.1%, respectively). However,
we observed an increase of cells with multinucleation and
enlarged nuclei, suggestive of abortive mitoses (data not
shown) in line with previous results.32 Taken together, these
results suggest potent antineoplastic activities of the TPPII
inhibitor butabindide in aggressive, c-MYC–associated
Burkitt lymphomas.
Discussion
The c-MYC proto-oncogene has been implicated in the
induction of genomic instability through a number of mech-
anisms including aberrant centrosome duplication.6 Here,
we show that c-MYC can rapidly stimulate centriole multi-
plication, a recently identified pathway of centriole overdu-
plication, in which a single maternal centriole nucleates the
concurrent formation of 2 or more daughters.13-15 We found
that c-MYC–induced centriole overduplication critically
depends on the giant protease TPPII. We identified TPPII as
a centriolar protein that by itself can stimulate centriole
multiplication when overexpressed. Furthermore, we show
that siRNA-mediated knockdown of TPPII as well as treat-
ment with the potent and selective TPPII inhibitor butabin-
dide abolish c-MYC–induced centriole overduplication. To
test butabindide as a potential anticancer agent, we treated
highly aggressive, c-MYC–associated Burkitt lymphoma
cells with butabindide and found not only suppression of
centriole overduplication but also potent growth-suppressive
and proapoptotic activities.
Our results go beyond the findings by Stavropoulou
et al.32 in several ways. We not only identify centriole mul-
tiplication as the cellular mechanism of centrosome aberra-
tions in the context of overexpressed TPPII but also
demonstrate that a fraction of TPPII has a centrosomal
localization. In addition, we provide evidence that TPPII
overexpression phenocopies the centriole duplication defect
detected in c-MYC–overexpressing cells and demonstrate
the potential use of the potent and selective TPPII inhibitor
butabindide as an antineoplastic agent.
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c-MYC, TPPII, and centriole overduplication / Duensing et al. 889
Figure 5. The TPPII inhibitor butabindide suppresses centriole aberrations and has proapoptotic and growth-suppressive activities in Burkitt lymphoma
cells. (A) Quantification of centriole abnormalities in 2 low-grade malignant B cell lines (U266 and HBL2) and 3 Burkitt lymphoma cell lines (BJAB, DG75,
and BL2). Each bar indicates average ± standard error of 3 independent experiments with at least 100 cells counted per experiment. (B) Quantification
of centriole abnormalities in BJAB, DG75, and BL2 Burkitt lymphoma cells either left untreated (c) or treated with butabindide diluted in sterile water at
the concentrations indicated. Each bar indicates average ± standard error of 3 independent experiments with at least 100 cells counted per experiment.
Asterisks indicate statistically significant differences compared to the corresponding untreated controls (c) and P values. (C) Flow cytometric analysis
of BJAB, DG75, and BL2 cells treated with 10 µM butabindide for the time intervals indicated. Note the significant increase of apoptotic cells (sub-G1
population) after 24 hours and 96 hours. (D) Analysis of the proportion of viable GA-10 Burkitt lymphoma cells grown in the presence of 0.1% DMSO
or 1 µM clastolactacystin β-lactone (CLBL). Number of viable cells was determined by counting trypan blue–negative cells in a hemocytometer on days
in culture indicated. (E) Immunoblot analysis of TPPII and c-MYC expression in GA-10 cells adapted to grow in the presence of 0.1% DMSO or 1 µM
clastolactacystin β-lactone (CLBL). Note the induction of TPPII in adapted cells. (F) Analysis of cell growth of GA-10 cells mock adapted to 0.1% DMSO
and GA-10 cells adapted to 1 µM CLBL (GA-10/adapted) either untreated (black squares) or treated with butabindide as indicated (open symbols).
Number of viable cells was determined by counting trypan blue–negative cells in a hemocytometer on days in culture indicated.
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890 Genes & Cancer / vol 1 no 9 (2010)
The c-MYC oncogene has been suggested to transcrip-
tionally regulate a large fraction of human genes. We were
not able to substantiate an increase of TPPII expression in
cells transfected with c-MYC by quantitative real-time
PCR and immunoblot analysis (data not shown). Hence,
c-MYC requires TPPII for centriole overduplication, but it
is conceivable that other c-MYC–induced oncogenic mech-
anisms may also contribute to this phenotype.
The selective and potent TPII inhibitor butabindide had
initially been designed to target a membrane-bound isoform
of TPPII involved in cholecystokinin inactivation with the
intention to control appetite and food intake.29 No major
toxicities have been reported in rodents.29 Here, we show
that this compound has promising antineoplastic activities
in clinically aggressive Burkitt lymphoma. Although we
show that butabindide suppresses centriole overduplication
in Burkitt lymphoma cells, we do not have evidence that
this activity is the cause of the pronounced apoptotic cell
death in butabindide-treated Burkitt lymphoma cells. None-
theless, we did find morphological signs of failed mitoses
(data not shown) in accordance with previous experiments
in which TPPII was knocked down in Burkitt lymphoma
cells.32
The precise role of TPPII in aberrant centriole biogene-
sis remains to be determined, but there is convincing evi-
dence that key regulators of centriole multiplication such as
PLK4 are substrates for proteases.36 Our results further-
more warrant preclinical experiments to explore the use of
butabindide as a novel anticancer or chemoprevention agent
in tumors with c-MYC overexpression.
Materials and Methods
Cell culture, transfections, and inhibitor treatments. U-2 OS
and U-2 OS/centrin-GFP cells (plasmid kindly provided by
M. Bornens, Institut Curie, Paris, France)37 were main-
tained as previously described.13 Stable cell populations
were generated as previously described.38 Normal human
keratinocytes (NHKs) were isolated from neonatal fore-
skins and cultured as previously described.39 BJAB, DG75,
BL2 (kindly provided by Elliott Kieff, Channing Labora-
tory, Brigham and Women’s Hospital, Boston, MA), U266,
and HBL2 cells were maintained in RPMI 1640 medium
supplemented with 10% fetal bovine serum, 50 U/mL peni-
cillin, and 50 µg/mL streptomycin. GA-10 cells were
obtained from the American Type Culture Collection
(Manassas, VA) and maintained according to the supplier’s
recommendations. Proteasome inhibitor–adapted GA-10
cells were obtained by the culture of cells in the presence of
1 µM clastolactacystin β-lactone (CLBL; Sigma-Aldrich,
St. Louis, MO), giving rise to the GA-10/ad cell line,
whereas control GA-10 cells were grown in 0.1% DMSO.
Expression plasmids used were encoding c-MYC (kindly
provided by P. Leder, Harvard Medical School, Boston,
MA) or TPP2 (obtained from OriGene, Rockville, MD).
Empty vector controls were included in all experiments. In
all transient overexpression experiments, cells were
cotransfected with either farnesylatable green fluorescent
protein (GFP) or DsRed fluorescent protein (Clontech,
Mountain View, CA) as the transfection marker. The TPPII
inhibitor H-Ala-Ala-Phe-chloromethylketone (AAF-CMK;
Sigma-Aldrich) was dissolved in DMSO, whereas butabin-
dide (Tocris Cookson, Bristol, UK) was diluted in sterile
water and used at the concentrations indicated. Appropriate
solvent controls (DMSO or water) were included in all
experiments.
Immunological methods. Immunoblot analysis was per-
formed as described previously.13 Antibodies used were
directed against c-MYC (9E10; Covance, Princeton, NJ),
TPPII (Proteintech Group, Chicago, IL), or β-actin (Milli-
pore/Chemicon, Billerica, MA). For immunofluorescence
analysis, cells were either grown on coverslips, or cytospin
preparations were made from cells growing in suspension.
For visualization of centrosomes, cells were fixed in 4%
paraformaldehyde in PBS and permeabilized with 1%
Triton-X 100 in PBS for 10 minutes each at room tempera-
ture. Cells were then blocked with 10% normal donkey
serum (Jackson Immunoresearch, West Grove, PA) and
incubated with a mouse monoclonal anti-γ-tubulin antibody
(GTU-88; Sigma-Aldrich) at a 1:1,000 dilution overnight
at 4°C followed by a donkey anti-mouse rhodamine red–
conjugated secondary antibody (Jackson Immunoresearch)
at a 1:100 dilution in PBS. For visualization of centrioles,
cells were stained with an anti-centrin mouse monoclonal
antibody (Abcam, Cambridge, MA) at a 1:200 dilution for 45
minutes at 37°C, followed by a donkey anti-mouse FITC-
labeled secondary antibody (Invitrogen, Carlsbad, CA) at a
1:500 dilution. For visualization of TPPII, cells were pre-
extracted with 1% Triton-X 100 in PBS for 5 minutes, fixed
in 4% paraformaldehyde/PBS for 10 minutes at room tem-
perature, and rinsed 3 times in PBS. Cells were then blocked
as described above and incubated with an anti-TPP2 anti-
body at a 1:500 dilution (Proteintech Group) overnight at
37ºC. After 3 rinses in PBS, cells were incubated with sec-
ondary anti-rabbit antibody at a 1:1,000 dilution (Jackson
Immunoresearch) for 1 hour at 37ºC and subsequently rinsed
3 times with PBS and stained with 4′,6′-diamidino-2-phenyl-
indole (DAPI; Vector, Burlingame, CA). Cells were analyzed
using a Leica (Wetzlar, Germany) or Olympus (Tokyo,
Japan) epifluorescence microscope equipped with a digital
camera system. Pictures were transferred to Adobe Photo-
shop (San Jose, CA) for printout.
siRNA and shRNA. U-2 OS cells were grown on coverslips
to 60% confluence and transfected with the indicated plasmids
(c-MYC or TPP2) together with siRNA duplexes targeting
TPPII or a shRNA plasmid targeting TPPII (Thermo
at MHH-Bibliothek on January 2, 2011gan.sagepub.comDownloaded from

c-MYC, TPPII, and centriole overduplication / Duensing et al. 891
Scientific, Auburn, AL) and a DsRed-encoding plasmid as
transfection marker using FuGENE 6 Transfection Reagent
(Roche, Mannheim, Germany). Herring sperm DNA was used
to normalize the total quantity of DNA transfected. After 24
hours, cells were stained for centrioles, and the percentage of
cells displaying supernumerary centrioles was assessed.
Flow cytometry. DNA content of cells was analyzed by
propidium iodide staining followed by flow cytometry
(FACSCalibur; Becton Dickinson, Franklin Lakes, NJ).
Data were acquired and analyzed using the CellQuest soft-
ware (Becton Dickinson).
Statistical analysis. Statistical significance was assessed
using the 2-tailed Student t test for independent samples.
Acknowledgments
The authors are grateful to Michel Bornens, Elliott Kieff, and Philip
Leder for sharing important reagents. They also thank Alyce Chen
for critical reading of the paper.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with
respect to the authorship and/or publication of this article.
Funding
This work was supported by PHS grant R01 CA112598 and a
Research Scholar Grant from the American Cancer Society (to S.
Duensing), PHS grant R01 CA066980 and a grant from AstraZeneca
(to K. Münger), PHS grant R01 CA118880 and grants from
Gabrielle’s Angel Foundation for Cancer Research and The
Pittsburgh Foundation (to A.G. Brickner), a grant from the Deutsche
Forschungsgemeinschaft (to N. Melquiot), and grants from the
Studienstiftung des Deutschen Volkes and the Biomedical Exchange
Program of the International Academy of Life Sciences (to S. Darr).
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