Ku80 functions as a tumor suppressor in hepatocellular carcinoma by inducing S-phase arrest through a p53-dependent pathway
Ku80 is a component of the protein complex called DNA-dependent protein kinase, which is involved in DNA double-strand break repair and multiple other functions. Previous studies revealed that Ku80 haplo-insufficient and poly (adenosine diphosphate-ribose) polymerase-null transgenic mice developed hepatocellular carcinoma (HCC) at a high frequency. The role of Ku80 has never been investigated in human HCC. Ku80 expressions in HCC and adjacent liver tissue were investigated by using immunohistochemical staining and western blot. Ku80 was transfected into a Ku80-deficient HCC cell line SMMC7721 cells, and the growth features of the Ku80-expressing cells and vector-transfected cells were studied both in vitro and in vivo. Cell cycle analysis and RNA interference were employed to investigate the mechanisms underlying the growth regulation associated with Ku80 expression. Ku80 was found frequently downregulated in HCC compared with adjacent liver tissue. Ku80 downregulation was significantly correlated with elevated hepatitis B virus-DNA load and severity of liver cirrhosis. Overexpression of Ku80 in SMMC7721 cells significantly suppressed cell proliferation in vitro and in vivo. Ku80 overexpression caused S-phase cell cycle arrest and was associated with upregulation of p53 and p21CIP1/WAF1, and the inhibition of p53 or p21CIP1/WAF1 expression by RNA interference overcame the growth suppression and S-phase arrest in the Ku80-expressing cells. A novel mechanism was revealed that Ku80 functions as a tumor suppressor in HCC by inducing S-phase arrest through a p53-dependent pathway.
Carcinogenesis vol.33 no.3 pp.538–547, 2012
Advance Access publication January 5, 2012
Ku80 functions as a tumor suppressor in hepatocellular carcinoma by inducing S-phase
arrest through a p53-dependent pathway
, Min Xiong
, Da-qian Zhan
Bin-yong Liang, Yang-yang Wang, David H.Gutmann
and Xiao-ping Chen
Research Laboratory and Hepatic Surgical Center, Department of Surgery and
Department of Gynecology, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, 1095 Jie Fang Da Dao,
Wuhan, China 430030 and
Department of Neurology, Washington University
School of Medicine, St Louis, MO 63110, USA
To whom correspondence should be addressed. Tel: þ86 27 83665392;
Fax: þ86 27 83803209;
Correspondence may also be addressed to Xiao-ping Chen.
Tel: þ86 27 83665392; Fax: þ86 27 83662851;
Ku80 is a component of the protein complex called DNA-dependent
protein kinase, which is involved in DNA double-strand break
repair and multiple other functions. Previous studies revealed that
Ku80 haplo-insufﬁcient and poly (adenosine diphosphate-ribose)
polymerase-null transgenic mice developed hepatocellular
carcinoma (HCC) at a high frequency. The role of Ku80 has never
been investigated in human HCC. Ku80 expressions in HCC and
adjacent liver tissue were investigated by using immunohistochem-
ical staining and western blot. Ku80 was transfected into a
Ku80-deﬁcient HCC cell line SMMC7721 cells, and the growth
features of the Ku80-expressing cells and vector-transfected cells
were studied both in vitro and in vivo. Cell cycle analysis and RNA
interference were employed to investigate the mechanisms
underlying the growth regulation associated with Ku80 expression.
Ku80 was found frequently downregulated in HCC compared with
adjacent liver tissue. Ku80 downregulation was signiﬁcantly
correlated with elevated hepatitis B virus-DNA load and severity
of liver cirrhosis. Overexpression of Ku80 in SMMC7721 cells
signiﬁcantly suppressed cell proliferation in vitro and in vivo.
Ku80 overexpression caused S-phase cell cycle arrest and was
associated with upregulation of p53 and p21
inhibition of p53 or p21
expression by RNA interference
overcame the growth suppression and S-phase arrest in the
Ku80-expressing cells. A novel mechanism was revealed that
Ku80 functions as a tumor suppressor in HCC by inducing S-phase
arrest through a p53-dependent pathway.
Hepatocellular carcinoma (HCC) is the third most deadly cancer
worldwide, with .500 000 new cases emerging annually. Most
HCC patients are diagnosed at advanced stages that preclude the
optimal surgical treatment (1). For the 20–30% of patients with re-
sectable tumors, the 5 years survival rates are only 30–40% (2). To
develop potential effective treatments for HCC, recent efforts have
focused on exploring the molecular mechanisms underlying the path-
ogenesis of HCC. HCC is closely linked to persistent infection with
hepatitis B virus (HBV) or hepatitis C virus (HCV), the intake of
a-ﬂatoxin B-contaminated food and excessive alcohol consumption
(3). Approximately 90–95% of HCC cases result from the biological
consequences of persistent HBV and HCV infection (4). Many studies
have indicated that HCC develops through a multistage process re-
sulting from the accumulation of genetic changes (5), including p53,
phosphatase and tensin homolog, Rb and E- and T-cadherin inactiva-
tion (6–10) as well as transforming growth factor-b, vascular endo-
thelial growth factor and Ras activation (11–13). However, the
detailed molecular mechanism for the pathogenesis of HCC is still
Ku80 is well known for its critical role in the repair of double-strand
breaks (DSBs) (14). There are two pathways for DSB repair: homolo-
gous recombination and non-homologous end joining (NHEJ). NHEJ is
the predominant mechanism of DSBs repair in higher eukaryotes,
whereas single-cell organisms rely more heavily on homologous re-
combination. Ku80 was found to maintain genome stability by repair-
ing DSBs through NHEJ (15). Misrepaired DSBs are the major DNA
lesions that lead to chromosomal aberration, mutation or carcinogene-
sis. Apart from its important role in DNA repair, Ku80 has been im-
plicated by many studies to be involved in other cellular processes, such
as telomere maintenance, apoptosis and tumor suppression (16,17).
Ku80 has been suggested to be a multifunctional caretaker gene that
suppresses chromosomal aberrations and malignant transformation
(18). Ku80 deletion increased the incidence of cancer in mice that
carried mutations in additional genes, such as p53 (19). Approximately
20% of Ku80
mice developed a broad spectrum of cancers,
including lymphoma, osteosarcoma and leiomyosarcoma by 40 weeks
of age, and 100% of Ku80
mice developed pro-B-cell lym-
phoma by 16 weeks of age. Nevertheless, studies have also indicated
that overexpression of Ku80 may be associated with bladder cancer,
cervical carcinoma, pancreatic cancer and gastric cancer (20–22). Once
Ku80 was silenced by RNA interference (RNAi), the proliferation of
cervical carcinoma and esophageal squamous carcinoma cells was in-
hibited (23,24). These studies suggested that Ku80 played diverse roles
in a number of cancer types. Using a transgenic mouse model, the study
of Tong et al. (25) indicated that Ku80 haplo-insufﬁciency (Ku80
in poly (adenosine diphosphate-ribose) polymerase-1 null (PARP-1
mice promoted the development of hepatocellular adenoma and HCC
without the need for any carcinogen administration. This study sug-
gested that the deletion of DNA damage repair molecules, Ku80 and
PARP-1, played important roles in the development of HCC in mice;
however, PARP-1 was found to increase activation rather than inacti-
vation in human HCC compared with adjacent liver tissue (26). The
role of Ku80 in the pathogenesis and development of human HCC
In this study, Ku80 expression status was investigated in human HCC
and adjacent liver tissue. The growth features of the Ku80-expressing
clones and vector-transfected cells were compared both in vitro and
in vivo. The potential mechanisms of growth regulation associated with
Ku80 expression were further investigated.
Materials and methods
Antibodies and reagents
PcDNA3.1(þ)-myc-his-Ku80 plasmid and pcDNA3.1(þ)-myc-his vector plas-
mid were kind gifts from Prof. Masashi Idogawa, Sapporo Medical University
of Japan. Mouse monoclonal Ku80 (Ab-2) antibody was purchased from
Neomarkers (Thermo). c-H2AX (Ser139) (20E3) rabbit monoclonal antibody
was obtained from Cell Signaling Technology. Anti-p21
(Do-1), cdk2 (M2), cyclin E (C-19), cyclin A (M20), glyceraldehyde 3-phos-
phate dehydrogenase (H-12) and b-actin (C4) antibodies were purchased from
Santa Cruz Biotechnology (Santa Cruz).
Patients and specimens
One hundred pairs of human HCC and their corresponding adjacent liver
samples were obtained from 2008 to 2010 from patients who underwent liver
Abbreviations: DMEM, Dulbecco’s modiﬁed Eagle’s medium; DNA-PK,
DNA-dependent protein kinase; DSB, double-strand break; HBV, hepatitis B
virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; NHEJ, non-
homologous end joining; PARP-1, poly (adenosine diphosphate-ribose)
polymerase-1; PBS, phosphate-buffered saline; RNAi, RNA interference;
siRNA, small interfering RNA.
These authors contributed equally to this work.
ÓThe Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please email: email@example.com 538
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resection at the Hepatic Surgery Center of Tongji Hospital afﬁliated with
Huazhong University of Science and Technology. HCC was conﬁrmed patho-
logically, and all specimens were stored at 80°C until analysis. Informed
consent had been obtained from all patients. Clinicopathologic characteristics
for these patients, including age, sex, hepatitis history, alpha-fetoprotein, liver
cirrhosis, tumor number, tumor size, differentiation, preoperative Child-pugh
score, Union for International Cancer Control stage and vascular invasion were
shown in Table I. Tumor differentiation was evaluated by the same pathologist
according to the criteria proposed by Edmonson and Steiner (27). Liver
cirrhosis was graded into three stages according to the criteria described
previously by our group (28). HCC was staged according to the
tumor-node-metastasis staging system of the Union for International Cancer
Control. The medical ethics committee of Tongji Hospital approved this study.
Antigen retrieval of the tissue sections was performed in boiling citrate buffer
for 15 min. Peroxide blocking was conducted with 0.3% peroxide in absolute
methanol. After the slides had been incubated with primary antibodies (1:100
dilution) at 4°C overnight and washed twice with phosphate-buffered saline
(PBS), they were then incubated with secondary antibody (Dako, Denmark) at
37°C for 30 min. After washing, the color reaction was developed with dia-
minobenzidene work solution (Dako). Positive cells were identiﬁed as those
with nuclear staining. The percentage of positive cells was calculated by
dividing the number of positive cells by the total number of hepatocytes in
least 10 randomly chosen non-overlapping high-power (400) ﬁelds for each
case. Protein expression was graded on a scale from þto þþþ. The grade of
‘þ’ was given to cases whose percentage of positive cells was 25%, includ-
ing those with no positive cells. If the average percentage of positive cells was
25% but 50%, then the expression was graded as ‘þþ’, whereas the
percentage of positive cells 50% was graded as ‘þþþ’.
Western blot analysis
The cell cultures and tissues were lysed in RIPA lysis buffer (50 mM Tris–HCl
at pH 8.0, 1% NP40, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate,
0.02% sodium azide and 150 mM NaCl) containing Protease Inhibitor cocktail
(Roche, Switzerland) on ice. After the protein concentration was determined
by a bicinchoninic acid Kit (Pierce), 60 lg proteins were separated on pre-
casted 10% sodium dodecyl sulfate–polyacrylamide gels and then electrotrans-
ferred onto polyvinylidene diﬂuoride membranes (Millipore) in transfer buffer.
The blots were blocked in 5% non-fat milk and incubated overnight at 4°C with
primary antibodies (anti-Ku80 and p53 antibodies at 1:500 dilution; other
antibodies at 1:200). The blots were then incubated with horseradish peroxi-
dase-conjugated secondary antibody at 1:5000 dilution for 1 h at 37°C. The
signals were visualized using the enhanced chemiluminescence system (Pierce).
Protein expression was quantiﬁed by densitometry and normalized to b-actin
expression using Alpha View software.
Cell culture and transfection
The human HCC cell lines SMMC7721 with p53 wild-type background,
Hep3B with p53-null background and PLC/PRF/5 with p53-mutant back-
ground were obtained from the Center for Type Culture Collection of China
and maintained in Dulbecco’s modiﬁed Eagle’s medium (DMEM) (Gibco),
supplemented with 10% fetal bovine serum (Hyclone) and antibiotics (Invi-
trogen). SMMC7721, Hep3B and PLC/PRF/5 cells were cultured in 12-well
plates and transiently transfected with the Ku80 plasmid and empty vector
plasmid, respectively, by using Lipofectamine 2000 (Invitrogen) following
the manufacturer’s protocol. At the indicated time points, the cells were har-
vested for use in the western blot analysis and cell proliferation assays. For
stable transfections, 5 10
cells per well were seeded in a six-well plate for
24 h. Plasmid DNA was delivered into the cells using Lipofectamine 2000
following the manufacturer’s protocol. Brieﬂy, 4 lg of plasmid DNA was
mixed with 10 ll of Lipofectamine 2000 at room temperature for 30 min before
seeding in a well containing 1 ml of Opti-MEM medium (Invitrogen). After
culturing in medium containing 500 lg/ml of G418 (Calbiochem, Germany)
for 3 weeks, the individual clones were isolated. The positive cell clones that
stably expressed Ku80 protein were then determined using western blot. The
clones stably expressing Ku80 protein were maintained in medium containing
250 lg/ml of G418 for further experiments.
Cell proliferation assay
To assess serum-induced cell growth in culture, 10 000 cells were plated over-
night in triplicates in a 12-well plate, serum starved for 48 h and then stimu-
lated with the complete or selected growth media containing 10% fetal bovine
serum for additional 5 or 7 days. At each indicated time point, the cells were
trypsinized and counted. The number of cells, based on the average count of
the three wells, was compared among the different groups from a total of three
Soft agar colony formation assay
Soft agar colony formation assay was performed for the Ku80-expressing and
the vector-transfected clones as well as the parental SMMC7721 cells as de-
scribed previously (29). Brieﬂy, 1000 cells were equally divided into four wells
in a 24-well plate in medium containing 0.3% noble agar and grown for 14–21
days. The number of colonies was determined by direct counting using an
inverted microscope (Nikon, Japan).
Flow cytometry for cell cycle and apoptosis analysis
For cell cycle analysis, 1 10
Ku80-expressing or vector-transfected
SMMC7721 cells were serum starved for 48 h after washing with PBS for
three times. The cells were stimulated with DMEM containing 10% serum for
12, 24, 36 and 48 h, and the cells were harvested and washed with PBS. The
cells were then ﬁxed with 70% ethanol. Immediately prior to the analysis, the
cells were incubated with fresh propidium iodide containing RNase A for
30 min at 37°C. A total of 10
cells were analyzed from each sample on a
ﬂuorescence-activated cell sorting Calibur ﬂow cytometer (Becton Dickinson).
For apoptosis analysis, 1 10
cells per well were seeded in the six-well plates
and incubated with DMEM containing 10% serum. After 48 h, ﬂoating and
adherent cells were harvested and washed twice with pre-cold PBS. The cells
were stained for 15 min with Annexin V-ﬂuorescein isothiocyanate and propidium
iodide in 500 ll binding buffer and then analyzed by ﬂow cytometry within 1 h.
Table I. The correlation between Ku80 downregulation and
clinicopathologic parameters in the patients with HCC
Ku80 expression level P-value
Age (years) 0.339
50 53 30 23
.50 47 31 16
Male 81 52 29
Female 19 9 10
Liver cirrhosis 0.020
None/mild 28 12 16
Moderate/severe 72 49 23
39 17 22
61 44 17
Positive 6 5 1
Negative 94 56 38
Low 41 24 17
Moderate 40 27 13
High 19 10 9
Tumor multiplicity 0.509
Solitary 85 53 32
Multiple 15 8 7
AFP (ng/ml) 0.629
,25 31 20 11
25 69 41 28
Vascular invasion 0.211
Yes 3 3 2 3 1 0
No 67 38 29
Child–Pugh score 0.445
Stage (UICC) 0.797
I and II 42 25 17
III and IV 58 36 22
AFP, alpha-fetoprotein; UICC, Union for International Cancer Control.
Chi-square test or the Fisher exact test.
Statistically signiﬁcant (P,0.05).
Ku80 as tumor suppressor in HCC
at Johns Hopkins University, Eisenhower Library on September 12, 2016http://carcin.oxfordjournals.org/Downloaded from
c-H2AX immunoﬂuorescence and neutral comet assay
c-H2AX immunoﬂuorescence assay was performed as described previously
(30). Brieﬂy, after methanol ﬁxation, cells were permeabilized, then blocked in
3% bovine serum albumin/PBS and incubated with c-H2AX monoclonal an-
tibody at 1:500. Secondary antibodies labeled with Alexa 555 (Molecular
Probes) were added at 1:1000. Coverslips were mounted onto slides using
Hard Set Vectashield with 4#,6-diamidino-2-phenylindole (Vector Laborato-
ries). Cells were processed for comet tail formation using neutral comet assay
conditions according to the methods described previously (31). Comets were
analyzed using the measurement tool in OpenLab software.
Three 21-nucleotide small interfering RNA (siRNA) duplexes targeting differ-
ent coding regions of human p53, p21
and their scrambled sequence
siRNA (mock) were custom synthesized by Riobio Company (GuangZhou,
China). Three siRNA duplexes targeting p53 were named sip53-1, sip53-2
and sip53-3, and the duplexes targeting p21
were named sip21-1,
sip21-2 and sip21-3, respectively. For the RNAi knockdown, equal numbers
of cells were seeded in the plates containing medium without antibiotics for
24 h prior to the transfection. The siRNAs were introduced into the cells using
Lipofectamine 2000 in serum-free Opti-MEM, according to the manufacturer’s
instructions. The expression levels of p53 and p21
proteins were de-
termined by western blot. The two most efﬁcient siRNAs for knockdown were
chosen for further experiments. The transfected cells were grown in complete
medium at 37°C and 5% CO
and harvested at different time points for use in
the proliferation assay and cell cycle analysis described previously.
Growth in athymic immunocompromised mice
The Ku80-expressing and vector-transfected clone cells as well as parental
SMMC7721 cells were harvested and resuspended to 1 10
subcutaneously injected 1 10
cells in a total volume of 100 ll into the right
ﬂank of 4- to 6-week-old male athymic nude mice. Eight mice were injected
for each clone. Their tumors were monitored every 4 days by measuring the
tumor size using a caliper. The tumor volume was calculated by the formula,
/2, where Lrepresents the longest dimension and Wthe
shortest dimension of the tumor (32). Forty-four days after injection, the mice
were killed and their tumors removed and weighed. Tumor tissue fragments
were ﬁxed in 10% formalin. The expressions of Ku80, p53, p21, cyclin A,
cyclin E and cdk2 in xenograft tumor tissues were detected by immunohisto-
chemical staining as described previously.
All results represent the average from triplicate experiments, and all results are
expressed as the mean ± standard derivation. The associations between categorical
variables were assessed using the chi-square test or the Fisher’s exact test.
Analysis of variance was performed to determine the statistical signiﬁcance
among the groups. And a value of P,0.05 was considered statistically signiﬁcant.
Ku80 is frequently downregulated in HCC and the downregulation
was signiﬁcantly correlated with HBV infection and liver cirrhosis
Ku80 expressions in the 100 cases of paired HCC tissues and their
corresponding adjacent liver tissues were studied. Immunohistochemi-
cal staining indicated that Ku80 was expressed in the nucleus of
hepatocytes in liver tissues, and loss or decreased expression of Ku80
was frequently observed in HCC tissues (Figure 1A). There was a
signiﬁcant difference in Ku80 expression between the HCC tissues
and their corresponding adjacent liver tissues (Figure 1B; P,0.01).
The western blot further conﬁrmed that Ku80 protein expression in 61
HCC samples was lost or decreased compared with their adjacent liver
tissues (Figure 1C). The correlation between Ku80 expressions and
clinicopathologic parameters in the patients with HCC was shown in
Table I. The Ku80 downregulation was signiﬁcantly correlated with
elevated serum HBV-DNA load (P50.004) and the severity of liver
cirrhosis (P50.02). However, there was no signiﬁcant correlation
between Ku80 expressions and other clinicopathologic parameters,
such as age, sex, tumor size, tumor multiplicity, differentiation,
Fig. 1. Ku80 expression is frequently downregulated in HCC. (A) Representative immunostaining of Ku80 expression in human HCC tissues and corresponding
adjacent liver tissues. Arrows indicate positive nuclear staining for Ku80. (B) Semiquantitative analysis of Ku80 expressions in the 100 cases of paired HCC tissues
and their corresponding adjacent liver tissues. The P-value ,0.01 corresponds to the comparison of Ku80 expression between the HCC tissues and corresponding
adjacent liver tissues. (C) Western blot showing Ku80 expression in HCC tissues (T) and corresponding adjacent liver tissues (N) from HCC patients, and b-actin
was employed as an internal control.
S.Wei et al.
at Johns Hopkins University, Eisenhower Library on September 12, 2016http://carcin.oxfordjournals.org/Downloaded from
alpha-fetoprotein levels, tumor vascular invasion, Child-pugh score and
HCC stage (P.0.05).
Ku80 overexpression suppresses cell growth and colony formation in
To study the effects of Ku80 expression on cell growth, the
pcDNA3.1(þ)-myc-his-Ku80 plasmid and the vector plasmid were tran-
siently transfected into three HCC cell lines Hep3B, PLC/PRF/5 and
SMMC7721, respectively. Cell proliferation of the Ku80-transfected
cells, the vector-transfected cells and the parental cells was studied.
Ku80-transfected SMMC7721 cells (a Ku80-deﬁcient HCC cell line)
grew at signiﬁcantly slower rates than the vector-transfected control cells
(P,0.01). However, there was no signiﬁcant difference in proliferation
between the Ku80-tranfected cells and the vector-tranfected cells for
both Hep3B and PLC/PRF/5 cell lines (P.0.05; Supplementary data
are available at Carcinogenesis Online). Subsequently, SMMC7721 was
chosen for further stable transfection study. Ku80-expressing and
vector-transfected stable SMMC7721 cell clones were then generated.
The western blot analysis conﬁrmed that Ku80 clones 18, 26 and 33
expressed high protein levels of Ku80, whereas the clones with the vector
and the parental SMMC7721 cells lacked Ku80 expression (Figure 2A).
The growth curves of the Ku80-expressing cells, the vector-transfected
cells and the parental SMMC7721 cells were determined using the cell
proliferation assay. As shown in Figure 2B, on days 5 and 7, Ku80-18
and Ku80-26 cells grew at signiﬁcantly slower rates than the control
cells (P,0.01). The number of cells in the Ku80-expressing clones
decreased by 62.0–71.4% on day 7 compared with those in the vector
clone and SMMC7721 cells (Figure 2B). Furthermore, the soft
agar assay suggested that the colony number of the Ku80-expressing
clones was signiﬁcantly lower by 45.5–54.5% than those of the vector-
transfected clone and the parental cells (Figure 2C). The statistics graph
showed that there was a signiﬁcant difference among the different cell
types (Figure 2D; P,0.01).
Ku80 overexpression causes S-phase arrest in SMMC7721 cells
To explore the mechanism underlying the cell growth suppression
caused by Ku80 overexpression, the cell cycle distributions of the
Ku80-expressing and the vector-transfected SMMC7721 cells were
analyzed. After synchronization through serum starvation for 48 h, the
Ku80-18 clones and vector clones were stimulated with DMEM con-
taining 10% serum for 0, 12, 24, 36 and 48 h. Flow cytometry analysis
demonstrated signiﬁcantly increased numbers of cells from the Ku80-
18 clone in S phase at all time points (Figure 3A and B) compared
with those in the vector clone (Figure 3A and C). Under serum-starved
conditions, the average percentages of vector-transfected clone cells
and Ku80-expressing clone cells in S phase were 26.73 ± 1.79 and
48.60 ± 3.89%, respectively. After serum stimulation for 12, 24, 36
and 48 h, the percentages of vector-transfected clone cells in S phase
were 34.06 ± 2.23, 39.60 ± 5.18, 34.70 ± 4.26 and 32.72 ± 3.50%,
respectively, whereas the corresponding percentages of Ku80-
expressing cells were 69.14 ± 5.88, 73.24 ± 6.02, 81.08 ± 7.89 and
76.87 ± 8.06%, respectively. There was a signiﬁcant difference in cell
percentages in S phase between the vector-transfected and the Ku80-
expressing cells (Figure 3D; P,0.01). The expressions of cell cycle
regulators associated with S-phase regulation were further investi-
gated. The expression levels of cdk2 and cyclin A were signiﬁcantly
decreased in the Ku80-expressing cells (P,0.05), whereas the ex-
pression levels of p53 and p21
signiﬁcantly increased (P,
0.05). The expression level of cyclin E remained unchanged (P.
0.05) (Figure 3E).
To investigate whether apoptosis is also involved in the Ku80-induced
cell growth inhibition, ﬂow cytometry was performed for apoptosis
analysis. Our data indicated that the apoptotic rates in SMMC7721,
the vector-transfected cells, Ku80-18 and Ku80-26 clone cells were
1.81 ± 0.15, 1.83 ± 0.25, 9.44 ± 1.52 and 9.26 ± 1.72%, respectively
(Figure 3F). There was a signiﬁcant difference in cell apoptotic rate
between Ku80-18 or Ku80-26 clone cells and SMMC7721 or the vec-
tor-transfected cells (P,0.01; Figure 3G). These data indicate that
Fig. 2. Overexpression of Ku80 in SMMC7721 cells signiﬁcantly inhibits cell growth in vitro and colony formation in soft agar. (A) Western blot showing Ku80
expression in the Ku80-transfected SMMC7721 clones, Ku80-18, Ku80-26 and Ku80-33, and no Ku80 expression in the vector-transfected clone or parental
SMMC7721 cells. b-actin was included as a loading control for each sample. (B) Direct cell counting indicated signiﬁcant suppression of proliferation of the
Ku80-expressing clones Ku80-18 and Ku80-26 on days 5 and 7 of culture compared with that of the vector-transfected clone and parental SMMC7721 cells (P,
0.01). The mean and standard deviation are shown for each cell line. (C) Photomicrographs of representative ﬁelds showing that the Ku80-expressing SMMC7721
cell clones (Ku80-18 and Ku80-26) formed fewer and smaller colonies in soft agar than those of the vector-transfected clone and parental SMMC7721 cells. (D)
Quantiﬁcation of colony number. The asterisk denotes a statistically signiﬁcant decrease in colony number (P,0.01). The results shown represent the average
value and standard deviation of triplicate experiments.
Ku80 as tumor suppressor in HCC
apoptosis is also involved in the Ku80-induced cell growth inhibition
but the apoptotic rates in the Ku80-expressing cells are very low
Ku80 overexpression decreases DNA damage and increases the
genomic stability of SMMC7721 cells
c-H2AX assay indicated that the numbers of c-H2AX foci in the
nuclei of Ku80-18 and Ku80-26 cells were signiﬁcantly decreased
compared with SMMC7721 cells or the vector-transfected cells
(Figure 4A and B; P,0.05). Neutral comet assay also indicated that
Ku80-18 and Ku80-26 clones exerted a signiﬁcantly lower number of
comet tails indicative of DNA damage in comparison with the control
SMMC7721 cells or the vector-transfected cells (Figure 4A and C;
P,0.05). Our data indicated that Ku80 overexpression was able to
decrease DNA damage and increase the genomic stability of
The Ku80-induced growth suppression and cell cycle arrest depend on
the p53 pathway
Ku80 overexpression induced the upregulation of p53 and p21
in the SMMC7721 cells, suggesting that the Ku80-induced growth
suppression and S-phase arrest might be associated with the p53
pathway. To verify this assumption, we employed the speciﬁc RNAi
technique to suppress the expression of p53 and its downstream
. After three siRNA duplexes speciﬁc for p53
were transfected into the Ku80-18 clones, the protein
expression of p53 or p21
signiﬁcantly decreased, but no
change in p53 or p21
expression was observed in the scram-
bled siRNA (mock)-transfected Ku80-18 cells. The sip53-2, sip53-3,
sip21-1 and sip21-3 siRNA duplexes exhibited higher suppressive efﬁ-
ciency and were subsequently chosen for further study (data not shown).
The Ku80-18 clone transfected with different concentrations (30, 50
and 100 nmol/l) of sip53-2, sip53-3, sip21-1 or sip21-3 had decreased
p53 or p21
expression, respectively, in a dose-dependent
manner at 72 h after transfection (data not shown). Further study
indicated that the sip53-2- and sip53-3-induced p53 suppression started
24 h after transfection and lasted for 168 h (Figure 5A), whereas the
sip21-1- and sip21-3-induced p21
inhibition started 24 h after
transfection and lasted for 120 h (Figure 5B). The cell cycle analysis
indicated that S-phase arrest was overcome in the sip53-2-, sip53-3-,
sip21-1- or sip21-3-transfected Ku80-18 cells because the percentages
of these cells in S phase decreased to the same levels as those of the
vector control cells after 72 h of culture. In addition, there was no
signiﬁcant difference between the percentages of mock and sip53-2-,
sip53-3-, sip21-1- or sip21-3-transfected vector control cells in S phase
at all time points after transfection (Figure 5C and D; P.0.05). Cell
proliferation assay demonstrated that the proliferation of the Ku80-18
cells was signiﬁcantly increased after transfection with sip53-2 or
sip53-3 compared with the mock-transfected Ku80-18 cells or the
Fig. 3. Ku80 overexpression results in a consistent increase in the percentages of cells in S phase. (A) The Ku80-expressing clone (Ku80-18) and the vector-
transfected clones (vector) were synchronized by serum starvation for 48 h and then stimulated with DMEM containing 10% serum for 0, 12, 24, 36 and 48 h. The
distribution of cells in G
, S and G
/M phases is represented graphically for Ku80-18 (B) and vector cells (C) after serum starvation and at each time point after
serum stimulation. (D) At each time point after serum stimulation, signiﬁcantly more Ku80-18 cells were in S phase than vector control cells (P,0.01), even after
serum starvation (P,0.05). The result represents the average value and standard deviation from triplicate experiments. (E) Western blot showing the expression
levels of Ku80, p53, p21, cyclin A, cyclin E and cdk2 in Ku80-expressing cells and control cells, whereas b-actin was included as a loading control for each
sample. (F) Flow cytometry study indicating the apoptotic rates in Ku80-expressing cells and control cells. (G) There was a signiﬁcant difference in cell apoptotic
rate between Ku80-18 or Ku80-26 clone cells and SMMC7721 or the vector-transfected cells (P,0.01).
S.Wei et al.
vector-tranfected control cells on days 5 and 7 of culture (Figure 5E;
P,0.01). It also indicated that the cell proliferation of the sip21-1- and
sip21-3-transfected Ku80-18 cells were increased compared with the
mock-transfected Ku80-18 cells or vector-transfected control cells on
days 4 and 5 of culture (Figure 5F; P,0.01). There was no signiﬁcant
difference in cell numbers between the mock- and sip53-2- or
sip53-3-transfected vector-transfected control cells as well as between
the mock- and sip21-1- or sip21-3-transfected vector-transfected
control cells at all time points after transfection (Figure 5E and F;
Ku80 overexpression suppresses xenografts tumor growth in nude mice
Thirty-two 4- to 6-week-old male athymic nu/nu mice were divided
into four groups of eight mice each. The mice received 1 10
cells of Ku80-expressing clones (Ku80-18 and Ku80-26), the
vector-transfected clone and the parental SMMC7721 cells,
respectively, by subcutaneous injection into the right ﬂank. Tumor
volumes were measured with calipers every 4 days after injection.
As shown in Figure 6A, when the tumors were removed from the
killed mice on day 44 after injection, the mean volumes of the tumors
derived from the Ku80-18 (1.66 ± 0.39 cm
) and Ku80-26 (1.50 ± 0.31
) clones were signiﬁcantly smaller than those derived from the
vector-transfected (3.19 ± 0.39 cm
) or the parental SMMC7721 (3.04
± 0.51 cm
) cells (P,0.01). In addition, compared with the control
groups, Ku80-expressing subcutaneous tumors (Ku80-18 and Ku80-
26) grew at signiﬁcantly slower rates and were smaller at all time
points examined since day 24 after injection (Figure 6B; P,0.01).
The weights of the tumors derived from the Ku80-18 and Ku80-26
cells were signiﬁcantly lower than those from the vector-transfected
clone and the parental cells when the mice were killed (Figure 6C;
P,0.01). Immunohistochemical staining study indicated that Ku80,
p53 and p21 expressions were increased in the tumors derived from
the Ku80-transfected cells compared with those in the tumors from
the vector-transfected cells (P,0.01), whereas cyclin A and cdk2
expressions were decreased (P,0.05) and cyclin E expression in
tissue remained unchanged (P.0.05) (Figure 6D). The alteration
pattern of these protein expressions in xenograft tumor tissues was
consistent with that of those protein expressions identiﬁed in vitro in
the Ku80-expressing cells and the vector-transfected cells.
Genome instability is the hallmark of all forms of cancers because the
mammalian genome is at constant risk from genotoxic factors and
accumulates various mutations until malignant transformation occurs
(29). Chromosomal instability is one of the salient features in HCC
development and has been observed in 90% of HCC patients (33).
HCC is closely correlated with cirrhosis following infection with
HBV or HCV, and liver cirrhosis is associated with genotoxic DNA
damage and mutations of known DNA repair genes (34). Under
chronic genotoxic stress, failure of liver cells to initiate DNA repair
and growth control may contribute to liver carcinogenesis. Hepatitis B
virus X protein, an important oncogenic molecule of HBV, has been
shown to signiﬁcantly inhibit the ability of cells to repair damaged
DNA by downregulating a series of DNA repair molecules, such as
Xeroderma pigmentosum group B, Xeroderma pigmentosum group D
and human DNA glycosylase alpha (hMYHalpha) (35,36). HCV
infection has also been implicated in impairing DNA damage repair
by repair enzymes, including Ku70 and GADD45b(37,38). These
studies suggested that infection with HBV or HCV may cause
the downregulation of these DNA damage repair enzymes, and the
disruption of the DNA damage repair process might be involved in
the mechanism for HBV- or HCV-associated HCC carcinogenesis.
DNA damage repair molecule Ku80 is a key molecule in repairing
DSBs through NHEJ and maintaining genome stability, and previous
studies revealed that the deletion of both DNA damage repair
molecules, Ku80 and PARP-1, promoted HCC development in
a transgenic mouse model (25). Decreased Ku80 has been reported
to be involved in transforming growth factor-a/c-myc-associated
hepatocarcinogenesis in a mouse model (39). Our study found that
Ku80 expression was frequently downregulated in human HCC tis-
sues compared with adjacent liver tissues, and Ku80 downregulation
was signiﬁcantly correlated with elevated HBV-DNA load and
the severity of liver cirrhosis. Furthermore, the overexpression of
Fig. 4. Ku80 overexpression decreases DNA damage and increases the genomic stability of SMMC7721 cells. (A)c-H2AX foci localization and comet tail
formation. (B) Quantiﬁcation of c-H2AX-positive cells (P,0.05). (C) Quantitation of neutral comet assay (P,0.05).
Ku80 as tumor suppressor in HCC
Ku80 in human HCC cell line SMMC7721 cells suppressed the
in vitro cell proliferation and xenograft tumor growth in nude mice
through inducing cell cycle S-phase arrest. In addition, Ku80
overexpression was able to decrease DNA damage and increase
the genomic stability of the SMMC7721 cells. These data concluded
that Ku80 functioned as a tumor suppressor gene in human
HCC, and the downregulation of Ku80 played an important role in
Ku80 is one of the subunits of DNA-dependent protein kinase (DNA-
PK). Previous studies showed that DNA-PK acts upstream of p53 in
response to DNA damage, allowing p53 to bind to speciﬁc DNA
sequences and preventing MDM2 from inhibiting the p53-dependent
transactivation (40). DNA-PK downregulation by RNAi reduced
the accumulation of p53 by affecting the stability of p53 through Akt/
Protein Kinase B and Glycogen Synthase Kinase-3 phosphorylation (41).
A previous protein–protein interaction analysis suggested that the p53
DNA-binding domain may be an interacting partner of Ku80 in the
nucleus (42). Our study indicated that the overexpression of Ku80 was
associated with upregulation of p53 and p21
cells (with wild-type p53 background). In contrast, the study of
Holcomb (43) indicated that Ku80 deletion led to p53 activation in the
mice and reduced multiple intestinal adenoma and
verse role in a particular type of tissue or tumor. The underlying
regulating mechanism between Ku80 and p53 still needs to be
further deﬁned in the future.
Fig. 5. Ku80-induced growth suppression and cell cycle arrest depends on the p53 pathway. The western blot showed that p53 suppression induced by sip53-2 and
sip53-3 in the Ku80-18 clone started at 24 h after transfection and lasted for 168 h (A), whereas the p21
inhibition induced by sip21-1 and sip21-3 also
started at 24 h after transfection and lasted for 120 h. (B) The cells transfected with scrambled siRNA served as controls (mock). The cell cycle analysis indicated
that the percentages of vector cells transfected with sip53-2 and sip53-3 (C) or sip21-1 and sip21-3 (D) in S phase were comparable with that of the cells mock
transfected at all time points. In contrast, the percentages of Ku80-18 cells in S phase signiﬁcantly decreased past 48 h after transfection with sip53-2 and sip53-3
or sip21-1 and sip21-3 (P,0.01). (Eand F) The cell proliferation assay further showed that the knockdown of p53 or p21
by sip53-2 and sip53-3 or
sip21-1 and sip21-3 overcame the growth suppression induced by Ku80 in the Ku80-18 clone cells after days 5 or 4 of culture (P,0.01).
S.Wei et al.
In addition, when p53 or p21
expression was inhibited by
using RNAi, the S-phase arrest and cell growth suppression induced
by the overexpression of Ku80 were overcome. On the contrary, Ku80
overexpression was unable to suppress cell proliferation in p53-null
(Hep3B cell line) or p53-mutant (PLC/PRF/5 cell line) cells. These
observations strongly suggested that the S-phase arrest and cell
growth suppression induced by Ku80 overexpression in SMMC7721
cells were dependent on the p53 pathway. These results are supported
by a previous study that suggested that DNA-PK was involved in the
downregulation of cell proliferation and cell cycle progression
-to-S transition) through E2F-1-responsible genes (44). DNA-
dependent protein kinase catalytic subunit, a subunit of DNA-PK,
has also been demonstrated to function as negative feedback to pre-
vent excessive growth and tumor formation through the negative reg-
ulation of Akt upon ﬁbroblast growth factor-2 treatment (45).
Moreover, the p53 tumor suppressor was reported to inhibit cellular
proliferation by inducing cell cycle arrest and apoptosis in response to
cellular stresses (46). Our study has indicated that apoptosis is also
involved in the Ku80-induced tumor growth inhibition. Considering
the low apoptotic rates in the Ku80-expressing cells, cell apoptosis
would only play a minor role in the Ku80-induced cell growth inhibi-
tion. Cell cycle arrest plays a major role in the Ku80-induced cell
growth suppression in SMMC7721 cells.
Our data showed that Ku80 downregulation was signiﬁcantly cor-
related with elevated serum HBV-DNA load and the severity of liver
cirrhosis. HBV is the most common cause of cirrhosis in most of the
countries (47). It was believed that cirrhosis is a consequence of the
immune-mediated liver damage induced by chronic HBV infection
(48). About 10–30% of patients with chronic hepatitis B developed
liver cirrhosis in the end-stage of disease and at least 90% of HCCs are
associated with the liver cirrhosis (47,49). However, the molecular
mechanism for the Ku80 downregulation in human HCC is still not
Fig. 6. Ku80 overexpression inhibits SMMC7721 cell-derived tumor growth in vivo.(A) The subcutaneous tumors derived from the Ku80-18 and Ku80-26 clones
were smaller in size than those from the vector-transfected clones and parental SMMC7721 cells. (B) The tumor growth curve showed that the tumors derived from
the Ku80-18 and Ku80-26 cells grew signiﬁcantly slower than those from the control cells at all time points past 24 days after injection (P,0.01). The mean and
SD for tumor volumes were determined for each group. (C) When the mice were killed at 44 days after injection, the tumor weights of the Ku80-18 and Ku80-26
groups were lower than those of the control groups (P,0.01). (D) The expression status of Ku80, p53, p21, cyclin A, cyclin E and cdk2 in the xenograft tumor
tissues. Arrows indicate positive nuclear staining for these molecules.
Ku80 as tumor suppressor in HCC
clear. A recent study found that two genetic variants in Ku80,
rs16855458 and rs9288516, were identiﬁed to be signiﬁcantly asso-
ciated with HCC risk (50). Furthermore, the effects of rs16855458 and
rs9288516 were more signiﬁcant in HBsAg-infected subjects than
non-infected subjects (50). It is more reasonable to believe that
Ku80 gene mutation is a main cause resulting in Ku80 downregulation
in HCC. Previous studies suggested that the HhaI site (GCGC) in the
Ku80 promoter may be involved in regulating Ku80 gene expression
through a methylation–demethylation mechanism by HhaI methylase
(51), and Ku80 expression could be activated in primary cultured
retinal neurocytes after in vitro treatment with 5-azacytidine, whereas
the methylation of 179 bp in the Ku80 promoter induced Ku80
silencing in retinal neurocytes (52). Hence, the DNA methylation
status of the Ku80 gene in HBV-associated HCC deserves further
investigation. Furthermore, whether HBV infection is directly in-
volved in the regulation of Ku80 expression in the pathogenesis of
HCC remains to be answered in the future study.
In summary, our study found that Ku80 was downregulated in
human HCC and the Ku80 downregulation was signiﬁcantly
correlated with elevated HBV-DNA load and liver cirrhosis. We have
suggested an underlying mechanism in which Ku80 functions as a
tumor suppressor in HCC by inducing S-phase arrest through a p53-
dependent pathway. An increased understanding of the molecular
mechanism of Ku80 downregulation in HCC and further search for
possible strategies to induce Ku80 expression may provide a new
therapeutic target for HCC in the future.
Supplementary data can be found at http://carcin.oxfordjournals.org/.
This work was supported by fundings from the National Natural Sci-
ence Foundation of China (No. 81172293; No. 30972901) and the New
Century Excellent Talent Foundation of China Ministry of Education
(No. NCET-04-0701) awarded to Prof. Z.-Y.H. and by grants from the
state key project on infection disease of China (Grant No.
2012ZX10002016-004 and 2012ZX10002010-001-004), the Chinese
Ministry of Public Health for Key Clinical Projects (No. 
493–51) and Hubei Province for the Clinical Medicine Research Centre
of Hepatic Surgery (2007) awarded to Prof. X.-P.C.
Conﬂict of Interest Statement: None declared.
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Received July 13, 2011; revised December 19, 2011;
accepted December 27, 2011
Ku80 as tumor suppressor in HCC