J South Med Univ, 2013, 33(9): 1253-1259

doi 10.3969/j.issn.1673-4254.2013.09.01

1253

Original Article

Cisplatin induces cell cycle arrest and senescence via

upregulating P53 and P21 expression in HepG2 cells
QU Kai1#, LIN Ting1, 2#, WEI Jichao1, 3, MENG Fandi1, WANG Zhixin1, HUANG Zichao1, WAN Yong1, SONG
Sidong1, LIU Sinan1, CHANG Hulin1, DONG Yafeng4, LIU Chang1, 2*
Department of Hepatobiliary Surgery, 2Department of Surgical Intensive Care Units, First Affiliated Hospital, Xi'an Jiaotong University

College of Medicine, Xi'an 710061, China; 3Department of Neurosurgery, Shaanxi Provincial People's Hospital, Xi'an 710061, China;
Department of Obstetrics and Gynecology, Kansas University, Kansas City, Kansas 66160, USA

Abstract: Objective Cellular senescence as one of the important steps against tumor is observed in many cancer patients
receiving chemotherapy and is related to chemotherapeutic response. To investigate the effect of cisplatin on hepatocellular
carcinoma, we treated HepG2 cells exhibiting wild-type TP53 with gradient concentrations of cisplatin. Methods The
inhibitory effects of cisplatin on human hepatoma HepG2 cells were detected by MTT assay and colony formation test. The
changes in cell cycle were analyzed by flow cytometry, and cellular senescence was detected with senescence associated
-galactosidase (SA -gal) staining. The relative mRNA expression levels of TP53, P21 and P19 was estimated using
semi-quantitative real-time RT-PCR, and the protein expressions of P53 and P21 were detected using Western blotting. Results
Cisplatin induced irreversible proliferation inhibition and G1 phase arrest of HepG2 cells. Elevated levels of
senescence-associated -galactosidase was observed in HepG2 cells exposed to low doses of cisplatin. P19 expression
immediately increased following cisplatin exposure and reached the maximum level at 48 h, followed then by a rapid decrease
to the baseline level, whereas the expressions levels of TP53 and P21 mRNA increased continuously. Western blotting
confirmed P53 and P21 expression changes similar to their mRNA expressions during cisplatin-induced cellular senescence in
HepG2 cells. Conclusion Our results revealed a functional link between cisplatin and hepatocellular senescence. Cellular
senescence induced by cisplatin as a stabile senescent cellular model can be used for further research.
Key words: cisplatin; cellular senescence; hepatocellular carcinoma; P53; P21

Cellular senescence, a process leading to

irreversible arrest of cell division, was first described by
Hayflick in cultured human fibroblasts that lost the
ability to divide upon continuous subculture 1. Since
then, cellular senescence has been shown in various
mammalian tissues both in vitro and in vivo 2. The
growth arrest of cellular senescence can be triggered by
many different mechanisms, including recognition by
cellular sensors of DNA double-strand breaks leading to
the activation of cell cycle checkpoint responses and
recruitment of DNA repair foci. Different from normal
somatic cells, most tumors have extended or infinite life
spans. Many evidences suggested that cell senescence
was involved in neoplasms. Treatment-induced
senescence was shown to be one of the key determinants
of tumor response to therapy3.
Furthermore, tumor cells can also undergo
senescence and can be forced into this process by
various manipulations. Recent studies demonstrated that

chemotherapeutic drugs involved in cell cycle

progression, especially in mitosis and DNA replication,
could induce senescence-like morphological changes in
tumor cells4. Cisplatin, a widely used chemotherapeutic
drug, can bind to DNA to form intrastrand and
interstrand cross-links between purine bases and induce
DNA damage. Enhanced DNA damage repair was
thought to be relevant with cisplatin resistance in cancer
therapy5. While the major barriers limiting the use and
efficacy of cisplatin are its toxicity and resistance,
elucidation of the regulatory mechanisms that determine
different aspects of tumor senescence makes it possible
to design new therapeutic approaches to improving the
efficacy and to decreasing the side effects of cisplatin in
cancer therapy.
In this study, we utilized HepG2 cells to
investigate whether cisplatin was capable of inducing
senescence in hepatoma cell lines. We also studied the
biological characteristics of HepG2 cells with induced
senescence and the molecular mechanisms in this
process.

Supported by National Natural Science Foundation of China

MATERIALS AND METHODS

INTRODUCTION

Received: 2013-05-23

Accepted: 2013-06-23

(81201549, 81272644).
*Corresponding author: LIU Chang, Professor, PhD, Fax:
029-85323903; E-mail: liuchangdoctor@163.com.
#These authors contributed equally to this work.

Materials and Agents

RPMI-1640 medium and fetal calf serum were

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purchased from GIBCO (Canada). Cisplatin, MTT and

propidium iodide (PI) were purchased from Sigma
(USA). Annexin V-FITC apoptosis detection kit was
from BD Biosciences (USA). Senescence -galactosidase
(SA--Gal) staining kit was purchased from GENMED
(USA). The total RNA kit and primers of polymerase
chain reaction (PCR) were purchased from TaKaRa
(Japan).

formaldehyde/0.2% glutaraldehyde for 3-5 min at room

temperature. The cells were then washed twice with 1
PBS and incubated at 37 with fresh SA -gal staining
solution containing 0.4 g/L X-gal, 40 mmol/L citric acid/
sodium phosphate (pH 6.0), 5 mmol/L potassium
ferrocyanide, 5 mmol/L potassium ferrocyanide, 150
mmol/L NaCl and 2 mmol/L MgCl2. The staining was
achieved in 2-4 h.

Cell culture

Colony-forming assay

Human hepatoma cell line HepG2 was obtained

from Shanghai Institute of Biochemistry and Cell
Biology, Chinese Academy of Sciences (Shanghai,
China). HepG2 cells (5.0104 cells/ml) were cultured in
RPMI-1640 supplemented with 100 ml/L fetal bovine
serum containing 2.0 mmol/L glutamine and 20 mg
penicillin-streptomycin/ml in 50 ml/L CO2 at 37 , and
were allowed to adhere for 24 h.

Cellular proliferation was determined using a

colony-forming assay. Twenty-four hours after cisplatin
treatment, HepG2 cells were plated in 6-well tissue
culture plates at the density of 5103 cells/well. After 2
weeks of incubation, the cell colonies were stained with
crystal violet dissolved in methanol. Only the colonies
containing more than 50 cells were counted. The results
were reported as the mean number of colonies observed
in 5 randomly chosen microscope fields.

MTT assay for cell viability

HepG2 cells (5.0 104 cells/well) were seeded in
96-well plates and incubated with cisplatin at various
concentrations (0, 1.0, 2.0, 5.0, 10 and 20 g/ml) for 24,
48 and 72 h at 37 in the presence of 50 ml/L CO2.
Twenty milliliters of MTT solution (5 g/L) was then
added into each well and incubated for another 4 h.
Supernatants were removed and formazan crystals were
dissolved in 200 ml dimethylsulfoxide (DMSO). Finally,
the optical density was determined at 490 nm using a
POLARstar OPTIMA microplate reader (BMG
Labtechnologies, Ortenberg, Germany).
Senescence-associated -galactosidase (SA--Gal) assay
HepG2 cells (5.0 104 cells/well) were seeded in
6-well plates and incubated with the test substances for
different time lengths at 37 in 50 ml/L CO2. After
removing the medium, the cells were washed in 1
phosphate buffered saline (PBS) , and then fixed in 2%

Reverse transcription-polymerase chain reaction (RTPCR) analysis

The total RNA was isolated from cultured HepG2
cells using the RNAfast200 Kit (Fastagen Biotech,
Shanghai, China). The cDNA was synthesized from the
total RNA (2 g) using random hexamer primers and
RevertAidTM First Strand cDNA Synthesis Kit
(Fermentas, USA) according to the manufacturer's
instructions. PCR was carried out on Applied
Biosystems Prism 7300 using the SYBR green dye
(TaKaRa). The mRNA expression was assayed in
triplicate and normalized to -actin mRNA expression.
The relative levels were calculated using the
comparative-Ct method (Ct method). The primer
pairs for different Prx isoforms and -actin (listed in
Tab.1) were synthesized by Sangon Biotech (Shanghai)
Co., Ltd. China. -actin was used as the internal control.
Real-time PCR was performed according to the protocols
described previously6.

Tab.1 Real-time PCR primers

Gene

Primer

Sequence (5'3')

5'-GCCCTCGTGCTGATGCTACT-3'

5'-CAGCGTGTCCAGGAAGCC-3'

P19
TP53
P21
-actin

5'- ATGAGCCGCCTGAGGTTGG-3'

5'-CAGCCTGGGCATCCTTGAGT-3'

5'-TGGCACCTCACCTGCTCTG-3'

5'-GTTTGGAGTGGTAGAAATCTGTCAT-3'

5'-ATCGTGCGTGTGACATTAAGGAG-3'

R
F: Forward; R: Reverse.

5'-AGGAAGGAAGGCTGGAAGAGTG-3'

Western blotting
HepG2 cells were plated in 6-well plates at 5 104
cells/well. After exposure to cisplatin for different time
lengths (0-120 h), the cells were lysed in ice-cold radio

Product size (bp)

Annealing temperature ()

254

57

572

58

373

57

540

58

immunoprecipitation assay (RIPA) lysis buffer (1%

NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 150
mmol/L NaCl, and 10 mmol/L Tris-HC1) containing a
protease inhibitor cocktail (Sigma Chemical Co., MO).
The total protein concentration was determined using a

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J South Med Univ, 2013, 33(9): 1253-1259

Bio-Rad protein assay reagent (Bio-Rad, Hercu1es, CA).

Equivalent amounts of protein (30 mg) were then
separated by 12% SDS-PAGE gels and transferred to
nitrocellulose membranes at 1 mA/cm2 for 1.5 h
(Bio-Rad, Hercules, CA, USA). After being blocked in
TRIS-buffered saline (TBS) containing 5% non-fat milk,
the membranes were incubated with the primary
antibodies at 4 for 12 h, and then with HRPconjugated anti-goat or anti-rabbit antibodies (Santa
Cruz Biotechnology, Santa Cruz, CA) at a 1 3000
dilution at room temperature for 1 h. Signals were
detected on X-ray film using the ECL detection system
(Pierce, Rockford, IL). Equal protein loading was
assessed by the expression of -actin.
Statistical analysis
All data were expressed as MeanSD and analyzed
using SPSS 11.0 software (SPSS Inc, Chicago, IL, USA).
Data analysis was performed using Student's t-test, and
P<0.05 was considered to indicate a statistically
significant difference. All the experiments were repeated
for 3 times.
RESULTS
Cisplatin induced irreversible inhibition of HepG2 cell
proliferation
The growth of HepG2 cells was evaluated using
MTT assay at 24, 48 and 72 h after treatment with
cisplatin at 0, 1.0, 2.0, 5.0, 10, or 20 g/ml. Polygonal
or fusiform HepG2 cells were observed in the control
group, but enlarged polygonal cells were found in
low-dose (1.0 and 2.0 g/ml) cisplatin treatment groups,
and small round cells were observed in high-dose (10
and 20 g/ml) treatment groups (Fig.1A). MTT assay
showed that cisplatin inhibited the growth of HepG2
cells in a dose-and time-dependent manner (Fig.1B).
HepG2 cells were incubated with cisplatin (1.0, 2.0 and
5.0 g/ml) for 48 h, and then re-cultured in drug-free
medium. MTT assay showed that growth arrest occurred
in 1.0, 2.0 and 5.0 g/ml cisplatin treatment groups
after drug withdrawal (Fig.1C). These ndings were con
rmed by clonogenic assays. Cisplatin also inhibited
anchorage-independent growth in HepG2 cells as shown
by a dose-dependent decrease in the number of colonies
on soft agar (Fig.1D and E).
Cisplatin arrested cell cycle in G1 phase in HepG2 cells
Flow cytometry was used to assess whether
cisplatin treatment caused alterations in cell cycle
progression of HepG2 cells. As shown in Fig.2A, the
cell cycle distributions varied in different cisplatin
treatment groups. The percentage of HepG2 cells in G1
phase in 2.0 and 5.0 g/ml cisplatin-treated cells was
increased relative to the control groups (P<0.05)
(Fig.2B). These results indicated that cisplatin
suppressed cell cycle progression in HepG2 cells.

Csplatin induced senescent phenotype in HepG2 cells

SA -gal, the most widely used biomarker for
senescent and aging cells, was used to detecte senescent
phenotype induced by exposure to cisplatin at different
concentrations (0, 1.0, 2.0, and 5.0 g/ml). SA -gal
staining positive cells usually show extended cell shape
with enlarged or multiple nuclei (Fig.3A). SA -gal
staining positivity was detected in the cytoplasm of these
cells (Fig.3B). We further exposed HepG2 cells to 1.0,
2.0 and 5.0 g/ml cisplatin and performed SA -gal
staining every 24 h. The percentage of cells with
positive SA--gal staining was the highest in 2.0 g/ml
cisplatin group (Fig.4A). In addition, cisplatin-induced
cellular senescence exhibited a time dependence, and
reached the highest percentage of (47.7 9.0)% at 96 h
(Fig.4B).
Expression of P19, TP53 and P21 genes in HepG2 cells
exposed to cisplatin
During treatment with 2.0 g/ml cisplatin for 120
h, HepG2 cells were collected every 24 h for analysis by
qRT-PCR. HepG2 cells exposed to cisplatin treatment
showed increased P19 mRNA levels with an increasing
level of senescence; P19 mRNA levels decreased
following arrival at the maximum level at 48 h (Fig.5A
and B). In contrast, TP53 and P21 mRNA levels both
increased continuously during cisplatin exposure and
reached the maximum levels at 120 h (Fig.5 C and D).
The data of Western blotting also showed that cisplatin
treatment resulted in increased p53 and p21 protein
expressions, which reached the maximum levels at 120
h (Fig.5 E and F).
DISCUSSION
Hepatocellular carcinoma (HCC) is the fifth most
common cancer worldwide and the third most common
cause of cancer mortality, responsible for about 50 0000
deaths each year. Most HCC cases occur in either
Eastern Asia (especially in China) or in sub-Saharan
Africa. Currently, the prognosis for HCC remains poor
as no effective therapy has been available 7. Cell
senescence is broadly defined as the physiological
program of terminal growth arrest, which is thought as
an important tumor suppressor mechanism. Recent
studies showed that transformation of liver carcinomas
involves events that inhibit the program of senescence,
and treatment-induced senescence was shown to be one
of the key determinants of tumor response to therapy in
vitro and in vivo 8. Clinical and preclinical studies
indicated that many manipulations, including
chemotherapeutic drugs and radiation, could induce
tumor senescence 9 10. The relative importance of
different pathways of cell death and senescence in tumor
cell response to anticancer drugs may vary depending on
the cell type, the nature of the drug and the conditions of
drug exposure. Several cellular senescent models
induced by H2O2 11, ionizing radiation 12 and genetic
manipulations 13 have been reported, but chemo-

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Control

1 g/ml

2 g/ml

5 g/ml

10 g/ml

20 g/ml

B
1 g/ml Cis

1.2

Control

1.5

1 g/ml Cis

5 g/ml Cis
10 g/ml Cis

0.8

20 g/ml Cis

0.6
0.4

*
0.2

The values of OD at 490 nm

The values of OD at 490 nm

2 g/ml Cis
1.0

0.0

5 g/ml Cis

**
0.9

*
0.6

0.3
0

2 g/ml Cis
1.2

24

48
Time (h)

Control

24

72
1 g/ml Cis

48
Time (h)

72

500

2 g/ml Cis

5 g/ml Cis

Colony number

400

300

200

**

**

2.0
Cisplatin (g/ml)

5.0

100

0
1.0
Control

Fig.1 Effect of cisplatin on the growth of HepG2 cells. A: Observation of cell morphology after cisplatin treatment at the doses of 0,
1.0, 2.0, 5.0, 10, and 20 g/ml; B: Inhibition of cell growth shown by MTT assay; C: Irreversible growth arrest of HepG2 cells cultured
in drug-free medium following exposure to different doses of cisplatin; D, E: Inhibitory effect of cisplatin on colony formation of
HepG2 cells. The results are given as MeanSD from three experiments. *P<0.05, **P<0.01 vs control group; Cis: Cisplatin.

therapeutic senescent model for HCC is rare14.

Induction of cell apoptosis is generally the most
prominent at the highest drug doses, whereas low doses
of chemotherapeutic drugs have a more pronounced
cytostatic effect of senescence with transient growth

arrest3. In this study, we treated HepG2 cells with a

gradient concentration (0, 1.0, 2.0, 5.0, 10 and 20 g/
ml) of cisplatin. We found significant differences in
growth inhibition between low dose (1.0, 2.0, and 5.0 g/
ml) groups and high dose (10 and 20 g/ml) groups.

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J South Med Univ, 2013, 33(9): 1253-1259

A
1 g/ml Cis

600

Control

Number

Number

400

200

400

200

0
0

50

100
FL2-A

150

200

50

100
FL2-A

150

200

5 g/ml Cis

2 g/ml Cis
600

Number

Number

400
400

200
200

0
0

80

50

100
FL2-A

150

* *

200

Control

Cell percentage (%)

1 g/ml Cis
2 g/ml Cis

60

5 g/ml Cis
40

*
20

**

50

150

200

Fig.2 Effect of cisplatin on cell cycle

of HepG2 cells. A: Distribution of cell
cycle in HepG2 cells after cisplatin
treatment for 48 h; B: Accumulated
percentages of HepG2 cells in G1
phase treated with 2.0 and 5.0 g/ml
cisplatin. The results are given as
Mean SD from three experiments.
*P<0.05, **P<0.01 vs control group.

0
G0/G1

100
FL2-A

G2/M

1257

After removal of the drug,

HepG2 cells underwent
prolonged growth arrest and
G1
phase
arrest.
Subsequently, we detected
the cellular senescence in
these treated HepG2 cells.
The results showed that
HepG2 cells treated with
2.0
g/ml
cisplatin
presented with a dominant
senescent phenotype, and
compared with the other
groups, approximately 50%
of the senescent cells could
be harvested. These results
were
consistent
with
previous studies that found
cell senescence was more
common than apoptosis in
tumor cell lines exposed to
chemotherapeutic
drugs
with different effects 3.
Senescence
was
more
prominent at less cytotoxic
drug doses (such as 1.0-5.0
g/ml cisplatin), which
could be readily induced
and maintained for a long
period of time in patients
plasma
by
continuous
Therefore,
infusion 15.

Nuclei

Cytoplasm

Fig.3 SA -gal staining of

cisplatin-treated HepG2 cells. A:
SA -gal staining positive cells,
which exhibit extended shape
and
enlarged
cell
nuclei
compared with negative cells; B:
SA -gal staining was positive in
the cytoplasm of HepG2 cells.

J South Med Univ, 2013, 33(9): 1253-1259

80

**

60

*
40

20

1.0
Control

2.0
Cisplatin (g/ml)

SA -gal-positive cell percentage (%)

SA -gal-positive cell percentage (%)

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80

60

**

**

40

*
20

5.0

1.0
Control

2.0
Cisplatin (g/ml)

5.0

Fig.4 Percentage of cisplatin-treated HepG2 cells positive for SA -gal staining. A: Positive percentage of HepG2 cells
treated with 1.0, 2.0 and 5.0 g/ml cisplatin; B: Positive percentage of HepG2 cells treated with 2.0 g/ml cisplatin for 24
to 120 h. The results are given as MeanSD from three experiments. *P<0.05, **P<0.01 vs control group.

Control

24

Cisplatin treatment (h)

48
72
96
120

P19

TP53

P21

1.5

P19 relative expression

1.0

0.5

0.0
-actin

2.5

P21 relative expression

TP53 relative expression

Control

2.0
1.5
1.0
0.5
0.0

24

Cisplatin treatment (h)

48
72
96

120

5.0
4.0
3.0
2.0
1.0
0.0

Control

24

E
Control

24

Cisplatin treatment (h)

48
72
96
Cisplatin treatment (h)
48
72
96

Control

120

24

F
Control

120

P53

P21

-actin

-actin

24

Cisplatin treatment (h)

48
72
96
Cisplatin treatment (h)
48
72
96

120

120

Fig.5 Expression of P19, P53 and P21 in HepG2 cells exposed to 2.0 g/ml cisplatin. A: Expressions of P19, TP53 and P21
mRNA detected by RT-PCR; B: Results of real-time PCR showing increased P19 mRNA expression to the maximum level
at 48 h, followed by a drastic decline to the baseline level; C, D: TP53 and P21 mRNA expressions which increased from 24
h and reached the maximum level at 120 h; E, F: Western blotting for detecting P53 and P21 expressions in HepG2 cells
during cisplatin exposure (0-120 h). The results are given as MeanSD from three experiments. M: 50 bp marker.

senescence may be a significant determinant of tumor

response to continuous infusion protocols.
Recent studies have established that P53 functions

as the master regulator of senescence in various tumor

types and is capable of promoting tumor regression by
triggering senescence16, 17. DNA damage induced by cli-

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J South Med Univ, 2013, 33(9): 1253-1259

nically relevant concentrations of cisplatin could cause

irreversible growth arrest involving P53 activation 18.
P21, as a p53-regulated cyclin-dependent kinase
inhibitor, is also involved in cellular damage by drugs
that induce transient growth arrest19, and its expression
increases significantly along with upregulated P53
levels following 2.0 g/ml cisplatin exposure. P19, as a
well known positive regulator of p53 stability 20,
increased immediately in response to 2.0 g/ml
cisplatin up to the maximum level before the change of
TP53 mRNA, followed by a drastic decrease to the
baseline level. These data suggest that p53 and p21 play
a central role in the onset of senescence, whereas P19
may be involved in triggering senescence.
In conclusion, senescence of HCC cells is more
pronounced following exposure to cisplatin at moderate
concentrations and involves the status of P53, P19, and
P21. Establishment of cisplatin-induced cellular
senescence may therefore help to understand the
mechanisms of chemotherapy for HCC for better
manipulation of this process.
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P
P53
53
P21
21
HepG
HepG22

1 12 13 1 1
1
1
1
1
1
4
12
1 2 ICU 7100613
7100614 66160

MTT HepG2

-
RT-PCR TP53
P21 P19 mRNA
Western blotting
P53 P21 HepG2 2.0 g/ml
HepG2-

P19
48 h

P53P21

P53
P21
2013-05-23
8120154981272644
E-mail: joanne8601@163.com
E-mail: youlan@stu.xjtu.edu.cn
029-85323903E-mail: liuchangdoctor@163.com