Loss of p53 and MCT-1 Overexpression Synergistically
Promote Chromosome Instability and Tumorigenicity
Ravi Kasiappan,1 Hung-Ju Shih,1,2 Kang-Lin Chu,1 Wei-Ti Chen,1 Hui-Ping Liu,3
Shiu-Feng Huang,1 Chik On Choy,1 Chung-Li Shu,1 Richard Din,4
Jan-Show Chu,5 and Hsin-Ling Hsu1
1
Division of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli County;
Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan, Republic of China;
3
Departments of Cardiovascular and Thoracic Surgery, Chang Gung Memorial Hospital,
Taoyuan, Taiwan, Republic of China; 4School of Public Health, University of California
at Berkeley, Berkeley, California; and 5Department of Pathology, Taipei Medical
University, Taipei Medical University Hospital, Taipei, Taiwan, Republic of China
2
Abstract
Introduction
MCT-1 oncoprotein accelerates p53 degradation by means
of the ubiquitin-dependent proteolysis. Our present data
show that induction of MCT-1 increases chromosomal
translocations and deregulated G2-M checkpoint in response
to chemotherapeutic genotoxin. Remarkably, increases
in chromosome copy number, multinucleation, and
cytokinesis failure are also promoted while MCT-1 is induced
in p53-deficient cells. In such a circumstance, the
Ras–mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase–mitogen-activated protein
kinase signaling activity and the expression of metastatic
molecules are amplified. Given a p53-silencing background,
MCT-1 malignantly transforms normal breast epithelial
cells that are satisfactory for stimulating cell migration/
adhesion and tumorigenesis. Detailed analyses of MCT-1
oncogenicity in H1299 p53-null lung cancer cells have
shown that ectopically expressed MCT-1 advances xenograft
tumorigenicity and angiogenesis, which cannot be
completely suppressed by induction of p53. MCT-1
counteracts mutually with p53 at transcriptional levels.
Clinical validations confirm that MCT-1 mRNA levels are
differentially enriched in comparison between human lung
cancer and nontumorigenic tissues. The levels of p53 mRNA
are comparatively reduced in a subset of cancer specimens,
which highly present MCT-1 mRNA. Our results indicate
that synergistic promotions of chromosomal imbalances
and oncogenic potency as a result of MCT-1 expression
and p53 loss play important roles in tumor development.
(Mol Cancer Res 2009;7(4):536–48)
Cancer pathogenesis is involved in multiple pathways, including inactivation of tumor suppressors, activation of
oncogenes, loss of cell differentiation, augmentation of proliferative activity, alteration of hormone receptor status, and increase
of metastatic potential (1). The fine balance between the
proto-oncogene function and tumor suppressor activity plays a
critical role in regulation of cell growth, cell cycle, and genome
stabilization.
Genome aberrations are the hallmark of tumorigenesis.
Mitotic checkpoint controls the integrity of chromosome
structure (2-5). This checkpoint monitors chromosome alignment during metaphase and prevents premature progression
through mitosis. Specifically, it can stop mitotic cells from
entering anaphase and prohibit chromosomes from moving
toward the spindle poles. Proper chromosome segregation is
mediated by centrosome cycle, mitotic spindle assembly,
and mitotic kinase activation (6-8). Errors that are occurring
at mitosis can result in mitotic catastrophes, characterized by
spindle collapse, multipolar spindles, or cytokinesis failure.
Cells that have been encountered with mitotic catastrophes
can end up either entering apoptosis or exiting from mitosis
with multinucleation or chromosome aberrations. Consequently, the consequential chromosome instability comprises gains
or losses of whole chromosome(s) (aneuploidy), translocations, amplifications/deletions, or breaks (9-11).
Loss of p53 and hyperactivation of Ras–mitogen-activated
protein kinase (MAPK) signaling are implicated in mitotic abnormalities. A deficiency of p53 activity increases in the number of centrosome along with mitotic defects, which leads to
chromosome missegregation (12). In circumstances with little
or no functional p53, cells are highly proliferative and nuclear
structure rapidly disintegrates during aneuploidy development
(13). Conversely, the p53-proficient cells can better escape
from mitotic failures and limit the progress of chromosomal abnormality. Moreover, the mitotic abnormalities coupled with
centrosome amplification are induced by Ras–MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)–MAPK activity, in which cells are greatly at risk of genomic instability
(14). Several studies have indicated that activation of MAPK
signaling is important for cell transformation and genomic destabilization, which are induced by many oncoproteins, including Mos, Ras, and Raf (15-17). Furthermore, activating MAPK
Received 9/9/08; revised 11/18/08; accepted 12/8/08; published online 4/16/09.
Grant support: MG-097-PP-06 (H-L. Hsu) and National Science Council grant
95-2320-B-400-012 (H-L. Hsu).
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.
Note: Supplementary data for this article are available at Molecular Cancer
Research Online (http://mcr.aacrjournals.org/).
Hsin-Ling Hsu, Division of Molecular and Genomic Medicine, National Health
Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 350, Taiwan,
Republic of China. Phone: 886-37-246-166, ext. 35329; Fax: 886-37-586-459.
E-mail: [email protected]
Copyright © 2009 American Association for Cancer Research.
doi:10.1158/1541-7786.MCR-08-0422
536
Mol Cancer Res 2009;7(4). April 2009
MCT-1 Promotes Chromosome Aberration and Tumorigenicity
(ERK1/2) and H-Ras selectively induces cell invasive phenotypes by enhancing matrix metalloproteinase-9 (MMP-9) and
cyclooxygenase-2 expression along with increasing invasion
and migration abilities (18, 19). The chromosome instability
caused by p53 deficiency can be enhanced to a greater extent
by the constitutively active MAPK pathway. This shows that a
p53-dependent checkpoint mechanism can effectively prevent
the oncogene-mediated MAPK activation and chromosomal
instability. In addition, the Ras-MAPK pathway controls cell
cycle progression through G2 phase and affects the critical
checkpoint at G2-M transition following DNA damage (20).
Thus, oncogenic Ras activation and p53 dysfunction can disrupt genomic integrity via a MAPK-dependent pathway.
The resistances to apoptosis and deregulation of DNA damage checkpoints have been identified in the oncoprotein MCT-1
(multiple copies in a T-cell malignancy)–expressing cells
(21-23). MCT-1 gene amplification was firstly recognized in
the human B-cell and T-cell lymphoma (21). Ectopic expression of MCT-1 accelerates p53 protein degradation by activation of the AKT-MDM2-proteosome pathway (23). Genome
abnormalities, including chromosome amplification and translocation, are accumulated as MCT-1 reduces p53 activity.
MCT-1 is a CDC2/MAPK phospho-oncoprotein involved in
cell cycle regulation and DNA damage response (24, 25).
Ectopically expressed MCT-1 activates the cell survival kinase,
AKT, and prevents apoptosis induced by serum starvation (21).
Several lines of evidence show that MCT-1 implicates in the
translational regulation and tumor development. MCT-1 physically associates with eukaryotic ribosomal complexes (26),
interacts with the cap complex through a RNA-binding motif,
and recruits density-regulated protein (DENR/DRP) that contains the SUI1 translation initiation domain (27). Oncogenic
MCT-1 can transform normal breast epithelial cells and enhance xenograft tumorigenicity of MCF-7 breast cancer cells
by enhancing invasiveness and decreasing apoptosis (22, 28).
However, MCT-1 tumorigenic effects underlying chromosome
aberrations are unknown at present.
Our present data provide direct clinical evidence that MCT-1
mRNA levels are induced in human lung cancers. A p53silencing cellular background was used to recapitulate the
MCT-1 oncogenic strength on promoting chromosome instability and mitotic abnormality. Cell migration and adhesion abilities are enhanced and tumorigenesis is established under a
circumstance of MCT-1 expressing/p53 silencing. Further,
increasing MCT-1 levels in the p53-null lung cancer cells can
advance xenograft tumorigenicity and angiogenesis but that
cannot be completely suppressed by induction of p53. These
results are the first to show that MCT-1 oncoprotein and p53
reduction collectively enhance chromosome instability and
tumorigenicity.
Results
Overexpression of MCT-1 Increases Etoposide-Induced
Chromosomal Translocations
In our previous findings, p53 and p21 protein levels are
decreased by MCT-1 overexpression in response to bleomycin (BLM) and etoposide (ETO) genotoxicity, and their reductions are attenuated by the inhibition of MEK signaling
Mol Cancer Res 2009;7(4). April 2009
(23). To further investigate the role of MCT-1 in response
to genotoxic stress, the chemotherapeutic agent ETO was
treated with MCT-1–expressing and vector control MCF10A cells (Fig. 1A). In comparison with controls, the levels
of phospho-Ser 15 p53 and total p53 protein were relatively
reduced by ectopically expressed MCT-1 either before ETO
(0 hour) or after ETO treatment for 3 hours. In the latter case,
the cells were then recultured in an ETO-free medium for 24
hours (3 h→R24). Cytogenetic G-banding analysis subsequently
investigated chromosome deviations after ETO-treated cells recovered for 24 hours. Chromosome aberrations were quantified.
Table 1 indicated the frequencies of diverse and arbitrary
chromosomal abnormalities identified from each sample cohort
(n = 30). Chromosomal exchanges, including reciprocal translocations (symmetrical) and dicentric formations (asymmetrical), were predominantly increased under MCT-1 oncogenic
stress (63%) compared with control group (30%). Chromosome
translocations were evidently increased >2-fold when MCT-1–
expressing cells underwent ETO damage, a topoisomerase II
inhibitor that is widely used in cancer therapy. As well, chromosome deletions were more frequently observed in MCT-1–
expressing cells (20%) than controls (3.3%). Therefore, genome surveillance machinery is impaired in MCT-1 oncogenic
stress. High frequency of chromosomal aberrations that are additionally induced by MCT-1 could eventually enhance the tumor progression.
Consistent with MCT-1 induction diminishing p53 protein
amounts (Fig. 1A), a quantitative reverse transcription-PCR
(Q-RT-PCR) analysis indicated that MCT-1 significantly reduced p53 mRNA production to 0.4-fold (Fig. 1B), revealing
that MCT-1 also down-regulated p53 at transcriptional stage.
To inspect the oncogenic effect of MCT-1 in a p53-deficient
background, p53 short hairpin RNA 2 was delivered into
MCF-10A cells. As examined by Q-RT-PCR, p53 gene expressions were dramatically knocked down in both control
(control-p53) and MCT-1–expressing cells (MCT-1-p53;
Fig. 1B).
To evaluate the possible mechanism of MCT-1 affecting p53
mRNA amounts, the drug that inhibits transcription (actinomycin D, 0.5 μg/mL) was added into culture and then endogenous
p53 mRNA decay was monitored for different time periods
(0-8 hours). Figure 1C data revealed that p53 mRNA turned
over more rapidly under MCT-1 oncogenic stress than that of
p53 mRNA that was comparatively stable in controls. This implicates that MCT-1 activity has promoting effect on p53
mRNA turnover.
Ras Signaling Is Stimulated by MCT-1 Expression and
p53 Deficiency
As we examined cell proliferation kinase by Raf-1 RBD
affinity assay (Fig. 2A), the amounts of active H-Ras were really
elevated in both p53-deficient control (control-p53) and p53deficient MCT-1–expressing cells (MCT-1-p53). After normalizing to internal α-actin, the active H-Ras was determined to be
a 2.2-fold increase in MCT-1-p53 group and a 1.36-fold enrichment in control-p53 sample (Fig. 2B). On DNA damage (+ETO),
H-Ras activity all significantly reduced but still remained at a
1.46-fold induction in MCT-1-p53 group in comparison with
control-p53 sample.
537
538 Kasiappan et al.
FIGURE 1. Constitutive expression of MCT-1 promotes
chromosomal instability. A.
MCF-10A cells were treated with
ETO for 3 h and then cultured in
ETO-free medium for another
24 h (3 h→R24). The total p53
and phospho-p53 (Ser 15 ) proteins are reduced in MCT-1–
expressing cells. B. Quantitative
real-time PCR indicates that
MCT-1 reduces p53 mRNA levels. The p53 short hairpin
RNA 2 effectively inactivates
p53 gene presentation in both
control and MCT-1–expressing
cells. C. MCT-1 expression increases p53 mRNA decay as
monitored by mRNA turnover
rate after actinomycin D blocking
de novo transcription.
Signaling downstream of H-Ras, the phosphorylation of
MEK1/2 and MAPK (ERK1/2) mitogenic cascade was enhanced
in both MCT-1 and MCT-1-p53 cells even in response to genotoxin ETO (Fig. 2C). MEK1/2 phosphorylation in MCT-1-p53 cells
moderately decreased, which was consistent with H-Ras deactivation by ETO treatment (Fig. 2A and B), under which MAPK
phosphorylation remained high. We speculate that H-Ras could
be just one of upstream regulators of MEK1/2 and MAPK.
MAPK response is followed on H-Ras-MEK signaling activity
and that its phospho-alteration could be temporally delayed.
In evaluation of cell proliferation status, equivalent numbers
of the p53-silencing cells were cultured in the medium lacking
sera and growth factors (−EGF/insulin) for 24 hours. Following
incubation in epidermal growth factor (EGF)/insulin-containing
medium (+EGF/insulin) for another 36 hours, the accumulative
cell numbers (Y axis) was indicated by the colorimetric 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay (Fig. 2D). In response to growth stimulation, a 1.8-fold increase of MCT-1-p53 cell populations was identified compared
with control-p53 set. Therefore, p53 loss, along with MCT-1 induction, profoundly induces H-Ras activity and mitogenesis.
Additional Aneuploidy Is Occurring in MCT-1 Oncogenic
Cells with p53 Silencing
Cytogenetic G-banding analysis was again to inspect the
chromosomal abnormalities that happened in a p53-deficient
background (Fig. 3A). To our surprise, chromosome copy
numbers were increased notably as p53 silenced in MCT-1–
expressing cells (MCT-1-p53). However, chromosome amplification did not show in p53-proficient MCF-10A (control) and
was infrequently detected in p53-deficient controls (controlp53). Only increasing MCT-1 levels (MCT-1) also cannot dramatically alter chromosome numbers. In analysis of aneuploid
and diploid populations after p53 loss, statistics (P < 0.001)
were acquired from at least 150 individual metaphase spreads
(Table 2). Comparatively, many more aneuploidies were detected in MCT-1-p53 sample (42.8%) but less recognized in
control-p53 group (13.8%).
To study if p53 induction can effectively prevent polyploidy
generation, the p53-silencing MCF-10A was further restored
with p53 wild-type gene (Table 2). Surprisingly, ∼98.8% of
p53-restored MCT-1-p53 cells (MCT-1-p53 + p53) exhibited aneuploidy but no significant change of aneuploidy populations
(15.5%) in p53-restored control-p53 sample (control-p53 +
p53). Thus, p53 renovation cannot suppress but it actually advances aneuploidy; this could address the dark side of p53 in
progressing chromosome instability under MCT-1-p53 situation.
Flow cytometry analysis further confirmed that the polyploidy frequencies were enhanced significantly in MCT-1-p53 cells
(57.2%) compared with control-p53 cells (3.9%; Fig. 3B). The
populations with DNA content more than 4N (polyploidy) were
even further increased when MCT-1-p53 cells (96.8%) were
Mol Cancer Res 2009;7(4). April 2009
MCT-1 Promotes Chromosome Aberration and Tumorigenicity
Table 1. ETO-Induced Chromosomal Aberrations
Control cells
Deletion
Breakage
Translocation
MCT-1–inducing cells
Deletion
Breakage
Translocation
Events
Ratio
12
2, 5, 5, 10, 17, 17, 21, 22, der(9), del(1)
t(2;?), t(6;10), t(7;11), dic(1;13), dic(2;4), dic(4;6), dic(der(9);X), dic(10;18), dic(X;16)
1
10
9
3.3%
33.3%
30%
1, 5, 6, der(9), 18, 2
dup(1), i(8), 4, 2, 1, 7, 11, X
t(2;?), t(5;11), t(6;22), t(6;22), t(7;8), t(7;22), t(10;22), t(18;21), t(X;13), t(X;20), t(2;7),
dic(del(1);15), dic(5;6), dic(5;19), dic(8;14), dic(10;22), dic(2;12), dic(2;17), dic(2;X)
6
8
19
20%
26%
63%
NOTE: In comparison with controls, MCT-1 overexpression greatly induces translocations and deletions.
Abbreviations: t, translocation; del, deletion; dic, dicentric; dup, duplication; der, derivative.
treated with the radiomimetic agent BLM for 24 hours. On
BLM treatment, however, 47.7% of control-p53 cells were
mainly arrested at G2-M phase and have no major increase in
the polyploidy populations. Inhibition of MEK activity by
UO126 (20 μmol/L) during BLM treatment (BLM + UO126)
led to a moderate reduction of MCT-1-p53 polyploidy, 96.8%
to 80.3%, indicating that UO126 modestly decreased 16.5% of
polyploidy development.
In concordance with chromosomal instability, DNA damage–induced γ-H2AX foci that emerged before BLM treatment
(−BLM) were particularly found in the MCT-1-p53 group
(Fig. 3C). Along with promoting intrinsic γ-H2AX foci (nonBLM induced), the phospho-MAPK (p44/42) was greatly enhanced (Fig. 3D), in which MEK-MAPK signaling coincided
with the progress of chromosomal instability in MCT-1-p53.
However, no dramatic MAPK phosphorylation has been ob-
FIGURE 2. Ras-MEK-MAPK
signaling and cell proliferation
are stimulated by MCT-1 inducing/p53 silencing. A. H-Ras activity was analyzed by Raf-1
RBD affinity assay. Compared
with p53 knockdown controls
(control-p53), the active H-Ras
is comparatively promoted as
MCT-1–expressing cells abrogate p53 expression (MCT-1p53). B. On ETO treatment,
H-Ras activity still increases in
MCT-1-p53 cells. C. H-Ras
downstream targets, phosphoMEK1/2 (pMEK1/2) and phospho-MAPK (pMAPK; ERK1/2),
are also further activated in
MCT-1 and MCT-1-p53 cells.
D. MTT assays indicate the
accumulative number of cells
(Y axis) after stimulation with
EGF and insulin.
Mol Cancer Res 2009;7(4). April 2009
served in BLM-untreated MCT-1–expressing cells, which indicated that γ-H2AX formation before BLM treatment could be
independent of MAPK activation.
To further confirm the effect of G2-M checkpoint in response to BLM, ATM-CHK1-CDC25c signaling activation
was examined (Fig. 3E). Compared with control-p53 sample,
the remarkable decrease in the phosphorylation of ATM
(Ser1981), CHK1 (Ser317), and CDC25c (Ser216) was quite
matched with G 2 -M checkpoint impairment identified in
MCT-1-p53 cells. No significant alterations in CDC25c phosphorylation in BLM-treated or BLM-untreated cells could be
due to loss of p53 inhibitory effect on CDC25c activation.
Cells cotreated with UO126 and BLM treatment (BLM +
UO126) have only diminished phosphorylation on CHK1
but not ATM and CDC25c. Thus, MAPK hyperphosphorylation and ATM signaling down-regulation happen together
539
540 Kasiappan et al.
FIGURE 3. Polyploidy is promoted by p53 silencing/MCT-1 expressing. A. G-banding analysis shows an increase of chromosomal copy numbers while
p53 silencing and MCT-1 expressing. B. Flow cytometry data reveal that the polyploidy predominantly occurs in MCT-1-p53 cells either before (−BLM) or after
BLM (+BLM) treatment. Adding MEK inhibitor throughout BLM treatment (+BLM + UO126) can moderately decrease polyploidy. C. Even lacking BLM, the
intrinsic DNA damage γ-H2AX foci are increased in MCT-1-p53 sample. D. In response to BLM, phospho-MAPK and γ-H2AX are stimulated in MCT-1-p53
cells. E. Phosphorylation of ATM-CHK1-CDC25c is comparatively reduced in MCT-1-p53 cells that associate with deregulated G2-M checkpoint.
with polyploidy and γ-H2AX increments, particularly in a
MCT-1-p53 context.
More Mitotic and Nuclear Abnormalities Happen as MCT1–Expressing Cells Lose p53
In examination of nuclear morphology and mitotic process,
the majority of p53 knockdown controls (control-p53) have
completed chromosome segregation followed on chromosomes
uniformly aligned on the metaphase plate (Fig. 4A). However,
the cellular chromosomes of p53-silencing MCT-1 cells (MCT1-p53) aligned at the spindle equator but kept oscillating with
low amplitude around the metaphase plate (Fig. 4B). Desegregated chromosome bridges were observed frequently during
anaphase. In analysis of 250 mitotic cells, the percentages of
Table 2. Aneuploidy Frequencies Are Increased in MCT-1-p53 and MCT-1-p53+p53 Conditions
Control-p53
Samples
A
B
C
D
E
Mean ± SD
MCT-1-p53
Control-p53 + p53
MCT-1-p53 + p53
Diploid (%)
Aneuploid (%)
Diploid (%)
Aneuploid (%)
Diploid (%)
Aneuploid (%)
Diploid (%)
Aneuploid (%)
86
90
90
82
83
86.2 ± 3.4
14
10
10
18
17
13.8 ± 3.4
58
62
52
60
54
57.2 ± 3.7
42
38
48
40
46
42.8 ± 3.7
84
86
82
86
ND
84.5 ± 1.5
16
14
18
14
ND
15.5 ± 1.5
0
0
2
0
ND
1.25 ± 0.4
99
99
98
99
ND
98.8 ± 0.4
NOTE: From independent experiments, the frequencies of diploidy and aneuploidy were evaluated between p53-deficient control (control-p53), p53-deficient MCT-1
(MCT-1-p53), and p53-deficient cells that reconstituted with p53 gene (control-p53 + p53 and MCT-1-p53 + p53).
Abbreviation: ND, not determined.
Mol Cancer Res 2009;7(4). April 2009
MCT-1 Promotes Chromosome Aberration and Tumorigenicity
lagging chromosomes in anaphase (mitotic abnormalities) were
more detected in MCT-1-p53 (30.97%) than those identified in
control-p53 (13.08%; Fig. 4C). Further probing nuclear integrity in telophase (Fig. 4D), 17.39% of MCT-1-p53 cells exhibited binuclei, trinuclei, or micronuclei (Fig. 4E). Conversely,
these dramatic nuclear aberrations were barely detected in
p53-deficient controls (2.34%). Therefore, abnormal cytokinesis can cause intrinsic alterations of chromosome copy numbers
in MCT-1-p53 cells.
To more characterize nuclear activity of the multinucleate
cells, the nuclear mitotic apparatus protein (NuMA) was coimmunostained with the endogenous MCT-1 (Fig. 4F). The numbers of multinucleate MCT-1-p53 cells (top) were greatly
increased over control-p53 sample (bottom). We found three
interphase nuclei (arrow), a mitotic nucleus with chromosome
alignment at the metaphase plate (star), and some micronuclei
(asterisk) that were all enclosed within a single cell. This
proves that unsynchronized cell cycle progression at different
FIGURE 4. Mitotic aberrations happen in MCT-1-p53 cellular context. A. The p53-deficient control cells have intact nuclear structure and regular mitosis
progression. Scale bars, 15 μm. B. MCT-1-p53 cells exhibit lagging chromosomes in anaphase. C. The mitotic abnormalities are compared between controlp53 and MCT-1-p53. D. At telophase, MCT-1-p53 cells show cytokinesis failure, leading to multinucleation or nuclear disintegration. Scale bars, 10 μm. E.
The numbers of cells with cytokinesis incompletion and nuclear aberrations are quantified. Columns, average of three independent experiments; bars, SD. F.
NuMA immunostaining shows irregular nuclear morphology of the multinucleated MCT-1-p53 cells. Unsynchronized cell cycle is progressing in a multinuclear
cell (denoted by arrow, asterisk, and star). Scale bars, 15 μm.
Mol Cancer Res 2009;7(4). April 2009
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542 Kasiappan et al.
FIGURE 5. Metastatic potential and tumorigenesis induced by p53 silencing/MCT-1 expressing. HIF-1α mRNA (A), MMP-9 mRNA (B), and integrin β4
mRNA levels (C) are highly presented in MCT-1 and MCT-1-p53 cells compared with control and control-p53 cells. In concordance with promoting metastatic
potential, MCT-1 and MCT-1-p53 cells adhering to laminin-coated plate (D) and migrating through polyethylene terephthalate–coated membrane (E) increase
more than control and control-p53 cells do. F. In comparison with normal lung tissue (set as 1), 10 of 11 (90.9%) human lung tumor specimens show evidence
of MCT-1 mRNA increments as determined by Q-RT-PCR. MCT-1 mRNA is rarely detected in the nontumorigenic tissues. G. The levels of p53 mRNA in lung
tumors were compared with the relative normal tissues (set as 1). Low p53 mRNA levels are detected in a tumor sample (11), which highly expresses MCT-1.
MCT-1 is undetectable in the tumor with p53 abundance (1). Columns, mean from three different assays; bars, SD.
nuclear compartments of a multinuclear cell perpetuates the rate
of aneuploidy.
MCT-1 Induction and p53 Silence Collectively Promote
Tumorigenesis
The metastatic potential was examined in nontumorigenic
MCF-10A (Fig. 5A-C). A 1.96-fold increase in hypoxia-inducible factor-1α (HIF-1α) mRNA was identified in MCT-1-p53
cells (Fig. 5A). Moreover, MMP-9 mRNA was found to be a
1.99-fold enhancement in MCT-1-p53 circumstance (Fig. 5B).
MCT-1-p53 cells also presented a 1.7-fold amplification in integrin β4 mRNA relative to control-p53 sample (Fig. 5C). In
fact, oncogenic MCT-1 itself has already greatly elevated the
transcripts of HIF-1α (1.74-fold), MMP-9 (1.43-fold), and integrin β4 (1.93-fold) in comparison with comparative controls.
Thus, stimulations in HIF-1α, MMP-9, and integrin β gene
activation are p53 independent. When cell adhesion ability in
a serum-free condition was analyzed with a laminin-coated
96-well plate, MCT-1 and MCT-1-p53 cells showed a dramatic
adhesion activity compared with control or control-p53 cells
(Fig. 5D). Moreover, by using 5% horse serum as a chemoattractant, MCT-1 and MCT-1-p53 cells revealed high migratory
ability through a Boyden chamber (Fig. 5E). The elevation of
HIF-1α, MMP-9, and integrin-β4 mRNAs has implicated that
MCT-1 up-regulates several genes that promote cell survival,
angiogenesis, migration, invasion, and adhesion to extracellular
matrix proteins (29-31).
To examine if putting additional MCT-1 oncogenic effects
into the p53-silencing condition are sufficient for tumorigenesis, the p53-silencing MCF-10A cells at cultivated p27 and
Mol Cancer Res 2009;7(4). April 2009
MCT-1 Promotes Chromosome Aberration and Tumorigenicity
p38 were s.c. injected into nude mice. Symptoms of tumor
development were only found in 25% of xenografts, which
were injected with the late passage of MCT-1-p53 cells
(p38; Table 3). On the contrary, no sign of tumor development was detected in the mice inoculated with MCT-1–expressing or p53-silencing control cells. This shows that
genomic and mitotic aberrations that accumulated in MCT1 overexpression/p53 loss are enough for malignancy and
tumorigenesis.
To obtain direct clinical evidence, 11 pairs of non–small cell
lung cancer tissue along with adjacent nonneoplastic lung tissues were examined. Among them, four samples were squamous cell carcinoma (4, 5, 10, 11) and eight specimens were
adenocarcinoma (1-3, 6-9), respectively. All of them had done
EGF receptor (EGFR) mutation analysis, and four of the eight
adenocarcinoma tumor tissues had EGFR mutations (1, 6, 8,
12). Q-RT-PCR analysis indicated that MCT-1 gene was differentially induced in nearly all human lung cancer samples
(90.9%) in comparison with relative nontumorigenic lung tissues that expressed extremely low levels of MCT-1 (Fig. 5F).
In an attempt to find the correspondence between MCT-1 and
p53 presentations, p53 mRNA levels were analyzed in lung
cancer samples in parallel with their comparative normal tissues
(Fig. 5G). MCT-1 mRNA presentation was greatly amplified in
a lung tumor that has comparatively lower amounts of p53
mRNA (11). In contrast with that, two of cancer samples
(1, 5) presented low MCT-1 mRNA levels but have high
amount of p53 transcripts. These clinical data reveal that
MCT-1 status could play an important role in the etiology of
human lung cancer.
MCT-1 Induction Promotes Tumor Growth in a p53-Null
Background
To recapitulate whether MCT-1 tumorigenic outcomes can
be augmented in p53-null lung cancer cells, the vector control
H1299 (control/H1299) and MCT-1–expressing H1299
(MCT-1/H1299) were s.c. injected into nude mice. Consequently, larger tumors with more neovascularization emerged
in MCT-1/H1299 xenografts than those in control/H1299 xenograft tumors (Fig. 6A). A 20-fold increase in tumor weights
was found in MCT-1/H1299 xenografts compared with control/H1299 xenografts. The incidence of tumors that developed from MCT-1/H1299 was 4-fold higher than from
control/H1299 (Table 4). Hemoglobin amounts in these tumors were measured by the QuantiChrom hemoglobin assay.
Compared with the hemoglobin standard and normalized with
Table 3. Tumorigenesis Is Caused by MCT-1 Overexpression
Along with p53 Loss
Cell Types
Tumor Incidence (%)
Control-p53 (p27)
0
MCT-1-p53 (p27)
0
Control-p53 (p38)
0
MCT-1-p53 (p38)
25
Total injection times, 2.5 mo
Tumor Weight (g)
ND
ND
0
0.36
NOTE: The p53-silencing MCF-10A cells premixed with Matrigel were injected
into nude mice for 45 d. Only 25% of MCT-1-p53 xenografts emerge tumors.
Mol Cancer Res 2009;7(4). April 2009
tumor weights, MCT-1/H1299 tumors have a 10-fold increment in hemoglobin quantities than control/H1299 tumors.
Angiogenic effect was further characterized by CD31 immunohistochemical analysis that corresponded to microvessel
density. The data show that MCT-1/H1299 tumors have more
than 2.5 times increase in vascular counts relative to those
control/H1299 tumors.
MCT-1 mRNA levels were further evaluated among normal and tumor tissues from nude mice by RT-PCR analysis
(Supplementary Fig. S1). The internal 18S rRNA levels were
constitutively expressed among samples (denoted with asterisks). MCT-1 transcripts (indicated with arrows) were particularly elevated in MCT-1/H1299 tumors (M1 and M2) but
undetectable in normal tissues and control/H1299 tumors
(C1 and C2). Detailed quantitative analysis indicated that
the xenograft tumors that emerged from MCT-1/H1299 presented ∼20-fold MCT-1 mRNA levels in comparison with
control/H1299 xenograft tumors (Fig. 6C). Consistent with
angiogenesis stimulation, both H-Ras and HIF-1α mRNA levels were significantly enriched in MCT-1/H1299 xenograft
tumors as assessed with Q-RT-PCR analysis (Supplementary
Fig. S2). These also correspond with the enhanced H-Ras signaling, proliferation, and migration/adhesion ability observed
in MCF-10A cells with MCT-1 expressing/p53 silencing
(Figs. 2 and 5D and E).
Knock-in p53 Gene Cannot Completely Suppress MCT-1
Tumorigenic Effects
To examine if the exogenous p53 can functionally compromise MCT-1 tumorigenic activity, H1299 cells that were primarily transfected with pLXSN vector or MCT-1 oncogene
were further introduced with pLHCX vector or wild-type p53
gene. Similar to Fig. 1C data, p53 mRNA was in a 0.4-fold
reduction in MCT-1 + p53 cells compared with control + p53
sample (Fig. 6D).
Subsequently, the p53-reconstituted H1299 cells (MCT-1 +
p53 and control + p53) were s.c. injected into nude mice.
Unexpectedly, the ectopic MCT-1 + p53 H1299-injected mice
still developed larger tumors (4.3-fold increase) contiguous
with more hemoglobin amounts (2.3-fold increase) than those
evolving from the control + p53 cells (Table 5). In comparison with Table 4 data, p53 restoration certainly reduced the
extent of tumor burdens and hemoglobin amounts caused by
MCT-1/H1299. As reported by Q-RT-PCR study in H1299
tumors, MCT-1 mRNA expression was a 0.4-fold decrease
when p53 was renovated (compared between MCT-1 and
MCT-1 + p53 xenografts; Fig. 6F). As well, H1299 cells that
ectopically coexpressed MCT-1 and p53 (MCT-1 + p53) have
more than a 0.43-fold decrement in MCT-1 mRNA amounts
than MCT-1/H1299 (Fig. 6G). These data suggest that p53
might act as a transcriptional factor that negatively regulates
MCT-1 gene expression. Even if p53 induction is capable of
suppressing MCT-1 mRNA production, no complete regression in tumorigenicity was found. Therefore, MCT-1 induction enhancing H1299 tumorigenicity is not simply because
of p53 deletion, but it also synergizes with other p53-independent oncogenic factors. In conclusion, MCT-1 overexpression reinforces tumorigenicity and metastaticity while p53 is
missing.
543
544 Kasiappan et al.
FIGURE 6. MCT-1 promotes tumor development in a p53-null background. A. The tumor incidences and burdens are significantly enhanced in MCT-1/
H1299 xenografts compared with control/H1299 xenografts. B. MCT-1 overexpression stimulates the angiogenesis pathway because of increase in hemoglobin amounts and the endothelial marker CD31 highlight microvessels (arrows). C. MCT-1 transcripts are qualitatively enhanced in MCT-1/H1299 tumors.
Columns, mean; bars, SD from three independent assays. D. MCT-1 still diminishes p53 mRNA production after p53 gene transferring into H1299. E.
Nevertheless, p53 induction cannot fully repress tumorigenicity and hemoglobin amounts caused by MCT-1-p53. MCT-1 mRNA levels are found to be reduced by p53 restoration either in H1299 xenografts (control, MCT-1, control + p53, and MCT-1 + p53; F) or in H1299 cells (G).
Discussion
Our previous work has identified that overexpression
of MCT-1 significantly reduces p53 function, deregulates
DNA damage responses, and increases the incidence of
chromosomal abnormalities (22, 23). We speculate that the
oncogenic chromosome aberrations and tumorigenicity will
enormously amplify while putting MCT-1 oncogenic stress
in already p53-null or p53-deficient cells, which cannot
Mol Cancer Res 2009;7(4). April 2009
MCT-1 Promotes Chromosome Aberration and Tumorigenicity
Table 4. Tumor Development and Angiogenesis Are
Promoted by Increasing MCT-1 in a p53 Null
H1299
(p53 Null)
Xenograft
Tumor
Incidence (%)
Tumor
Weights (g)
Hemoglobin
(mg/g Tissue)
Capillary
Density
Control
MCT-1
20 (1)
80 (4)
0.06 (1)
1.29 (21.5)
0.33 (1)
3.43 (10.4)
1.7 (1)
4.3 (2.5)
NOTE: MCT-1 overexpression stimulates the angiogenesis pathway because
increase in hemoglobin amounts and the endothelial marker CD31 highlight
microvessels.
properly maintain genomic integrity and monitor mitotic
checkpoint. Our current data show that MCT-1 counteracts
p53 function and further promotes chromosomal translocations, deletions, and amplifications in the absence of p53
(Figs. 1 and 3).
The hyperactive Ras-MEK-MAPK signaling cascade is related to centrosome amplification, multipolar spindles, and
chromosome bridges (14). Silencing of p53 in MCT-1–inducing cells (MCT-1-p53) can stimulate Ras-MEK-MAPK activity (Fig. 2), explaining that cell proliferation is less reliant
on sera and growth factors. We are exploring every possibility
of suppressing the progress of polyploidization induced
by oncogenic MCT-1 and p53 deficiency. The polyploidy incidences are reduced moderately by a MEK inhibitor, UO126
(Fig. 3B), revealing that the MEK signaling is rationally
link to chromosomal aberrations that have taken place in a
MCT-1-p53 condition. The deregulated cell growth and mitogenesis could produce not only multinuclei but also micronuclei. Micronucleation can further cause the cells to lose large
segments or to generate small fragments of chromosomes
via breakage of the anaphase bridge. Circumstances of the induction of γ-H2AX foci that represented several intrinsic
DNA breaks are incompletely repaired in a MCT-1-p53 context before encountering with the genotoxin (Fig. 3D and E).
Cytokinesis failure also contributes to chromosomal missegregation and increases the risk of micronuclei, giant nuclei, or
multinuclei (Fig. 4B-F), by which MCT-1 induction further
increases aneuploidy in a p53 knockdown background
(Fig. 3A and B; Table 2). Furthermore, unsynchronized cell
cycle progression in a multinucleate MCT-1-p53 cell can be
another major cause of chromosome inequality (Fig. 4F). In
summary, MCT-1 confers its oncogenic influences by affecting many signaling pathways, which collectively enhance genomic mutations after p53 loss. Significant oncogenic
outcomes are not from a single cause but rather from a confluence of factors
provoked by MCT-1 oncogenicity and p53
absence. The chromosomal instabilities identified in a MCT-1p53 situation are the collective consequences of deregulation
of DNA damage checkpoints and mitotic aberrations. The fundamental mechanism by which MCT-1 concomitantly exacerbates mitotic and nuclear aberrations is under investigation.
Given a MCT-1–inducing/p53-eradicating circumstance,
nontumorigenic MCF-10A cells are malignantly transformed
and provide evidence for tumor development in the xenograft
nude mice. All these in vitro and in vivo findings show for
the first time that MCT-1 induction, along with p53 deficiency, is sufficient for provoking tumorigenesis in the mammals
Mol Cancer Res 2009;7(4). April 2009
(Table 3). MCT-1 overexpression itself may be not only directly oncogenic but could also induce a constellation of invasive and metastatic factors (e.g., HIF-1α, MMP-9, and
integrin β4) by its transcriptional or translational regulation
function. The enhancing H-Ras gene expression followed
by an extremely active Ras-MEK-MAPK signaling cascade
reinforces tumorigenic outcomes and angiogenic effects (18,
19, 32). Oncogenic MCT-1 participates in stimulating the
Ras-MEK-MAPK cascade (Fig. 2A-C) as well activates
HIF-1α, MMP-9, and integrin β4 gene expression
(Fig. 5A-C), which are overexpressed in diverse malignancies. These also speak to MCT-1–promoting abilities in the
cell migration and invasion (Fig. 5D and E).
Ras/Raf/MAPK signaling cascade has a biochemical link
with p53 transcription and p53 activity. Ras inhibition increases
p53 mRNA levels, indicative of a Ras-dependent mechanism
that regulates p53 transcriptional activation (33). Because
the MEK inhibitor (UO126) can partly attenuate p53 and p21 reductions (23), this suggests that MCT-1 could deregulate p53
transcription by constitutively activating Ras-Raf-MAPK.
Our data indicate that by escalating MCT-1 levels in a p53-null
background (H1299) that carried chromosomal inequality and
oncogenic mutations, the tumorigenicity of MCT-1 is manifestly
reinforced. As tumor growth and angiogenesis are synergistically promoted in MCT-1/H1299 xenografts (Fig. 6A; Table 4),
these could relate to stimulation in H-Ras and HIF-1α (Supplementary Fig. S2) and provide in vivo confirmation for MCT-1
oncogenicity predominantly in a p53-null background. Our data
also show that by means of p53 gene transferring cannot totally
repress MCT-1/H1299 (p53 null) tumorigenicity (compare Fig.
6B with Fig. 6E; Tables 4 and 5). Previous studies have indicated
that MCT-1–expressing MCF-7 (p53 proficient) also can promote angiogenesis in xenografting tumorigenicity (28). Reduction of MCT-1 mRNA is identified in p53-reconstituted H1299
cells and xenograft tumors (Fig. 6F and G), implying that p53
could antagonize mutually with MCT-1 at a transcriptional level.
Reciprocally, MCT-1 overexpression works against p53 function
and promotes advanced tumorigenicity in the absence of p53,
besides that MCT-1 has other tumorigenic functions that are
p53 independent.
A very important finding is that increasing MCT-1 transcripts are identified in the majority of human lung cancer specimens examined at this point (Fig. 5F), implicating that MCT1 hyperactivation could associate with the cancer susceptibility.
The clinical evidence might disclose the counteracting relation
between MCT-1 and p53, in which p53 mRNA levels are comparatively low in a sample that highly expresses MCT-1
Table 5. Restoration of p53 Cannot Suppress MCT-1
Tumorigenic Effects
H1299
(p53 Add-Back)
Xenograft
Tumor
Weights (g)
Hemoglobin
(mg/g Tissue)
Capillary
Density
Control + p53
MCT-1 + p53
0.44 (1)
1.88 (4.3)
0.18 (1)
0.41 (2.3)
2.7 (1)
5.4 (2.0)
NOTE: p53 induction cannot fully repress tumorigenicity and hemoglobin
amounts caused by MCT-1.
545
546 Kasiappan et al.
(Fig. 5G, 11). Vice versa, MCT-1 is barely detectable in the tumor sample that is p53 abundant (Fig. 5G, 1).
Our current findings also open some intriguing questions, including how MCT-1 can induce tumorigenesis in a p53-null or
in a p53-preserve backgrounds, how does MCT-1 stimulate metastatic potential after p53 deficiency, whether oncogenic MCT1 and p53 loss synergistically up-regulate the metastatic molecules via a posttranscriptional mechanism as recently reported
(34), and whether oncogenic MCT-1 turns tumor suppressor
p53 into the dark side of contributing to tumorigenesis (35). Advance knowledge in MCT-1 function could illuminate the fundamental mechanism of tumorigenesis and help the design of
therapeutic target.
Materials and Methods
Antibodies and Reagents
Antibodies recognizing p53 and actin were acquired from
Santa Cruz Biotechnology; phospho-CHK1 (Ser317), phospho-CDC25c (Ser216), phospho-p53 (Ser15), phospho-MAPK
(p42/p44), and ERK1/2 were from Cell Signaling Technology;
phospho-ATM (Ser1981), γ-H2AX, and H-Ras were from Upstate Biotechnology; α-actin or β-actin was from Abcam; and
V5 epitope antibody was from Invitrogen. BLM, ETO, and actinomycin D were acquired from Sigma and UO126 was from
Cell Signaling Technology.
MCT-1 Overexpression and p53 Knockdown
MCF-10A cells were transfected with pLXSN MCT-1-V5
using viral supernatants collected from PT67 cells and subsequently transfected with pMKO.1 puro p53 short hairpin
RNA 2 (Addgene) using Lipofectamine 2000 (Invitrogen).
Four cohorts of transfectants (mass culture) were established,
including pLXSN-control (control), MCT-1–overexpressing
cells (MCT-1), p53-silencing control (control-p53), and
MCT-1–expressing/p53-silencing cells (MCT-1-p53). These
cells were cultured in DMEM/F12 medium containing 5%
horse serum, 20 ng/mL EGF, 10 μg/mL insulin, 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 100 units/mL penicillin,
100 μg/mL streptomycin, and selection antibiotic (100 μg/mL
G418 or 0.5 μg/mL puromycin).
Raf-1 RBD Affinity Assay for Active H-Ras
Exponentially growing MCF-10A cells were cultured with
40 μmol/L ETO for 1 h. Active Ras was isolated on Raf-1
RBD-glutathione agarose according to the manufacturer's instructions (Upstate Biotechnology). Cells were extracted with
magnesium-containing lysis buffer and precleared with glutathione beads. Raf-1 RBD agarose (5-10 μg of protein) was incubated with 500 μg to 1 mg of lysates and gently rotated at 4°
C for 30 min. The beads were washed thrice with magnesiumcontaining lysis buffer. Raf-1 RBD-associated H-Ras was resolved by SDS-PAGE and immunoblotting.
Tumor Xenografts and Hemoglobin Assay
Non–small cell lung cancer cells (H1299) were overexpressed in MCT-1 and cultured as previously described (23).
Eight-week-old female BALB/c nude mice were injected with
MCT-1–expressing H1299 or the vector control H1299. Each
mouse was injected with 2 × 106 cells suspended in 100 μL
RPMI 1640 at both s.c. sites. The p53-silencing MCF-10A cells
(2 × 106) were premixed with equal volume of ice-cold BD
Matrigel matrix (10 mg/mL; BD Biosciences) before injecting
into nude mice. When tumor size had reached approximately
4 to 6 mm, tumors were resected, weighted, and processed
for immunohistochemistry or Q-RT-PCR analysis.
Hemoglobin amounts in the tumors were measured by spectrophotometry using the QuantiChrom hemoglobin assay kit
(BioAssay Systems). Tumors excised from mice were immediately washed with PBS, immersed in 1 mL sterile distilled
water, and incubated on an orbital shaker for 10 min at room
temperature. Following centrifugation at 14,000 rpm for
15 min, 50 μL of the supernatant were incubated with 200
μL of the hemoglobin assay reagent for 5 min at room temperature. Hemoglobin concentrations were calculated based on
absorbance absorption at 400 nm. Compared with the standard,
hemoglobin quantities were indicated as absorbance sample/
absorbance standard = mg/g wet tissue.
Human Lung Cancer Tissues and Quantitative Real-time
RT-PCR Analysis
Eleven pairs of surgically resected fresh frozen non–small
cell lung cancer tissues along with adjacent nonneoplastic lung
tissues were obtained from the tissue bank of Chang-Gung
Memorial Hospital (Taoyuan, Taiwan), which were approved
by the Institutional Review Board of Chang-Gung Memorial
Hospital.
Total RNA was extracted from the tissues using Trizol reagent (Invitrogen). cDNA was synthesized from 2 μg of total
RNA using an oligo(dT)12-18 primer and SuperScript II reverse
transcriptase (Invitrogen). The Q-RT-PCR was done with
nucleotide 61 MCT-1 sense (5′-GAGCGGAAGTAGTCAGATTT-3′) and nucleotide 431 MCT-1 antisense (5′TGTTCATGGCATCGGACTAT-3′) primers. Reaction
mixtures (20 μL) contained 150 ng cDNA, 2 μmol/L primers,
and 1× SYBR Green Master Mix (Applied Biosystems). Reactions were run on the ABI Prism 7900 Fast Real-Time PCR
System in triplicate. The reaction was conducted as follows:
95°C for 10 min followed by 45 cycles of a 15-s denaturing
at 95°C and 1-min annealing at 60°C. The RT-PCR products
were resolved by 2% agarose gel electrophoresis and stained
with ethidium bromide. The mRNA levels were calculated. Cycle threshold (ΔCt) = Ct target gene (MCT-1) − Ct endogenous
control (18S rRNA gene). H-Ras, HIF-1α, MMP-9, integrin
β4, and p53 specific primers were respectively designed by
Primer Express software to ensure a single 69-, 76-, 54-, 114-,
and 72-bp amplicon. The probes were labeled with NFQ
(quencher) and FAM (reporter) and synthesized by Integrated
DNA Technologies (Applied Biosystems). The standard Taqman assays were analyzed after normalizing to β-actin.
Cell Proliferation MTT Assay
MCF-10A cells were subcultured in the basal DMEM/F12
medium without growth factors and horse sera. Fifty microliters
of cell suspension (1 × 105 cells/mL) were seeded into each 96well plate and incubated at 37°C for 24 h. After starvation, cells
were supplemented with 50 μL DMEM/F12 medium containing growth factors (40 ng/mL EGF and 20 μg/mL insulin) for
36 h. Cell Proliferation Kit I (Roche) was used as follows: cells
Mol Cancer Res 2009;7(4). April 2009
MCT-1 Promotes Chromosome Aberration and Tumorigenicity
were incubated with 10 μL MTT labeling reagent for 4 h and
then incubated with 100 μL solubilization solution for 24 h at
37°C. The purple formazan crystals were analyzed at an absorbance of 595 nm using a 96-well plate spectrophotometer
(SpectroMAX Plus, Molecular Devices).
Immunofluorescence Microscopy
Cells were fixed with 3.5% formaldehyde in PBS for
15 min at room temperature and then permeabilized with
ice-cold acetone for 3 min at −20°C. The samples were incubated with primary antibody for 2 h followed by washing
with PBS. Alexa Fluor 488–coupled goat anti-mouse or Alexa
Fluor 543–conjugated goat anti-rabbit secondary antibodies
(Invitrogen, Molecular Probes) were incubated and counterstained with 4′,6-diamidino-2-phenylindole for 1 h in the
dark. Images were analyzed by a Nikon Optiphot-2 upright
fluorescence microscope at a 100× objective or a Leica
TCS NT confocal microscope at a 63× objective. Data shown
were representative of three independent experiments.
Genotoxin Treatment, Immunoblotting, and Flow Cytometry
Analysis
Genotoxic agents (6 milliunits BLM or 40 μmol/L ETO)
were used to induce double-strand DNA breaks. Whole-cell
extracts were prepared using CytoBuster protein extraction
reagent (Novagen) by incubating on ice for 15 min. Extracts
were cleared by centrifugation at 4°C for 15 min. Protein samples (60 μg) were heat denatured with NuPAGE lithium dodecyl sulfate sample buffer and NuPAGE reducing agent
(Invitrogen), resolved by 4% to 12% Bis/Tris NuPAGE gel
(Invitrogen), transferred onto Hybond-C extramembrane
(Amersham Biosciences), and immunoblotted with the indicated antibodies as described previously (23).
Genotoxin-treated cells were harvested, washed with PBS,
fixed with 70% ethanol for 2 h, or stored at −20°C. The
fixed samples were resuspended in PBS with 10 μg/mL DNase-free RNase A (Sigma) and 10 μg/mL propidium iodide
(Sigma) at 4°C. Cell cycle profiling was analyzed by BD
FACSCalibur flow cytometer (Becton Dickinson) and processed with ModFit software, version 2.0 (Verity Software
House). Polyploidy populations were quantified with WinMDI software, version 2.8.
Cell Adhesion Assay
Ninety-six–well plates were coated with laminin (10 μg/mL)
and incubated at room temperature for 1 h, subsequently
washed with PBS, and blocked with 1% bovine serum albumin
at 37°C for 1 h. MCF-10A cells (2 × 106/mL) were suspended
in DMEM/F12 serum-free medium. Cell suspensions (100 μL)
were added into each well and incubated in 5% CO2 at 37°C for
1 h. The nonadherent cells were removed by PBS washing.
Adherent cells were fixed with methanol for 15 min and then
stained with 0.2% crystal violet in 2% ethanol for another
30 min. Adherent cells were rinsed, lysed with 0.2% Triton
for 30 min, and measured by spectrophotometer at 595-nm
absorbance.
Cell Migration Assay
Polyethylene terephthalate–coated membrane (8-μm pore
size, HTS FluoroBlok Insert, Falcon) was placed onto 24-well
Mol Cancer Res 2009;7(4). April 2009
plates. The preequilibrated medium (800 μL) containing 5%
horse serum was used as a chemoattractant. MCF-10A cells
(5 × 104) were suspended in 100 μL of serum-free medium
and added into polyethylene terephthalate–coated insert and
then incubated in 5% CO2 at 37°C for 1 h. The unattached
and unmigrated cells were rinsed off and gently wiped off with
a cotton swab. The membranes with migratory cells were fixed
in 70% methanol for 30 min, washed with PBS, and stained
with 10× Mayer's hematoxylin (DakoCytomation). Six randomly selected fields per membrane were analyzed. The experiments were conducted in triplicate.
Cytogenetic Analysis
MCF-10A cells were treated with ETO (10 μmol/L) for
3 h and then cultured in ETO-free medium for 24 h. The cells
were arrested with colcemid (0.1 μg/mL; Invitrogen) for 4 h.
Both detached and adherent cells were harvested and gently
suspended in hypotonic solution (0.075 mol/L KCl) at 37°C
for 8 to 10 min. Subsequently, five drops of Carnoy's fixative
(methanol and glacial acetic acid at 3:1 ratio) were added and
then centrifuged at 1,200 rpm for 5 min. Following fixation for
three times and kept at 4°C for at least 1 h, cells were suspended in fresh fixative solution. Metaphase spreads were
dropped onto ice-cold wet slides and dried at 100°C for
25 min. G-banding analysis was conducted with 0.04% trypsin
for 45 s and stained for 1 min in 0.075% Wright's eosin methylene blue diluted with Hank's buffer. Slides were washed,
mounted, and photographed.
Immunohistochemistry Study
Immunohistochemical staining was done on 4-μm-thick archival formalin-fixed paraffin-embedded tissue sections. Sections were deparaffinized twice in xylene for 10 min and
twice in ethanol for 2 min and then placed in 100 mmol/L Tris,
50 mmol/L EDTA (pH 6.0) buffer and heated at 92°C for
15 min. Samples were washed and the endogenous peroxidase
activity was blocked by 30% H2O2 for 5 min. The CD31 monoclonal antibody (BD Pharmingen) was incubated for 2 h at
room temperature, washed with PBS, and then incubated with
the Dako LSAB2+ HRP System (DakoCytomation Denmark
A/S) and counterstained with hematoxylin. Capillary density
indicated by CD31 staining was detected by a Nikon Optiphot-2 upright microscope at 20× objective and analyzed using
Northern Eclipse software (Empix Imaging).
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Dr. Ronald B. Gartenhaus for much help and Dr. Ning-Hsing Yeh for
the gift of NuMA monoclonal antibody.
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