Molecular mechanisms of paclitaxel and NM-3 on human gastric ...

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RAPID COMMUNICATION
Molecular mechanisms of paclitaxel and NM-3 on human
gastric cancer in a severe combined immune defi ciency
mice orthotopic implantation model
Jin-Shui Zhu, Ming-Quan Song, Guo-Qiang Chen, Qin Li, Qun Sun, Qiang Zhang
Jin-Shui Zhu, Ming-Quan Song, Guo-Qiang Chen, Qin Li,
Molecular mechanisms of paclitaxel combined with NM-3
Qun Sun, Qiang Zhang, Department of Gastroenterology,
on the orthotopic implantation model with human gastric
Affi liated Sixth People's Hospital, Shanghai Jiaotong University,
cancer in severe combined immune defi ciency mice. World J
Shanghai 200233, China
Gastroenterol 2007; 13(30): 4131-4135
Guo-Qiang Chen, Chinese academy of science laboratory
animals' center, Shanghai Jiaotong University, Shanghai 200233,
http://www.wjgnet.com/1007-9327/13/4131.asp
China
Supported by Natural Science Foundation of Shanghai, No.
02ZB14072
Correspondence to: Jin-Shui Zhu Professor, Department of
Gastroenterology, Affiliated Sixth People's Hospital, Shanghai
INTRODUCTION
Jiaotong University, Shanghai 200233,
China. zhujs1803@hotmail.com
Gastric cancer is the common leading cause of cancer
Telephone: +86-21-64369181 Fax: +86-21-64837019
death worldwide. Its clinical behavior depends on its
Received: 2007-04-26 Accepted: 2007-05-12
capacity to establish metastases of the tumor, and
prognosis of advanced gastric cancers is poor. To date,
several molecules have been reported to play an important
role in gastroenterological tumorigenesis and metastasis[1-3],
Abstract
but the molecular mechanisms remain to be elucidated[1-3].
In previous studies using LMD, P27-based RNA
AIM: To explore the molecular mechanisms of action of
paclitaxel and NM-3 on human gastric cancer in severe
amplification, and cDNA microarray, we identified
combined immune defi ciency (SCID) mice.
some differentially expressed genes between primary
carcinoma cells and lymph node metastatic cells in two
METHODS: Human gastric cancer cells SGC-7901 were
patients. Moreover, we further identifi ed four differentially
implanted into SCID mice and mice were treated with
expressed genes in progression of gastric cancer in another
paclitaxel and NM-3. The effects of paclitaxel and NM-3
group of 15 patients by means of semiquantitative reverse
on apoptosis of human gastric cancer cells were analyzed
transcribed polymerase chain reaction (RT-PCR), and the
using fl ow cytometry, TUNEL assays, and DNA fragment
expression patterns of these four genes were similar to
analyses.
tumor suppressor genes or oncogenes.
It is now widely accepted that many malignant tumors
RESULTS: Apoptosis of SGC-7901 cells was successfully
contain subpopulations of heterogeneous cells. This
induced by paclitaxel, NM-3, and the combination
heterogeneity is exhibited in a wide range of genetic,
of paclitaxel and NM-3 24 h after injection as shown
biochemical and immunologic characteristics. It is likely
by the presence of apoptotic hypodiploid peaks on
that specific tumor cells or colonies within the larger
the flow cytometer before G1-S and a characteristic
heterogeneous tumor specimen are the forerunners of
apoptotic band pattern in the DNA electrophoresis. The
distant metastases[4]. Thus, many biologic differences might
apoptotic rate detected by TUNEL assay was found to be
exist between tumor cells in primary and metastatic lesions.
signifi cantly higher in the paclitaxel/NM-3 compared to
the control group (38.5% ± 5.14% vs 13.2% ± 1.75%,
Furthermore, the interaction of tumor cells with their
P < 0.01).
living environment may add more differences between
these two groups of cells[5]. As a result, tumor metastasis
related genes can be identified by comparing the gene
CONCLUSION: Paclitaxel in combination with NM-3 is
able to induce apoptosis of the human gastric cancer
expression profi les between them.
cells in SCID mice effectively and synergistically.
Apoptosis plays a crucial role in the proliferation and
turnover of cells in various malignant tumors, and it is
© 2007 WJG . All rights reserved.
enhanced by many anticancer drugs as cytotoxic drugs,
hormones, or some recombinant gene, medicine, etc. At
Key words: Gastric cancer; NM-3; Paclitaxel
present, there is no effective therapy for advanced gastric
cancer, as the other malignant tumor, gastric cancer is
Zhu JS, Song MQ, Chen GQ, Li Q, Sun Q, Zhang Q.
not only a disease with abnormal cell proliferation and
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4132 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol August 14, 2007 Volume 13 Number 30
differentiation, but also a disease with abnormal apoptosis.
normal saline, respectively. 24 h later mice were sacrifi ced
Thus, the enhanced induction of apoptosis in human
by cervical dislocation. Samples of the tumor were
gastric cancer cells will be needed to explore. Paclitaxel
immediately frozen in liquid nitrogen for later use. Part of
enhanced the expression of smad3 and smad4 in SCID
the fresh samples were immediately placed into Eppendorf
mice, smad3 and smad4 are kind of multifunction cyclin
tubes for fl ow cytometry analysis.
dependent kinase inhibitors. They play a negative role in
Ten mg of fresh tissue from every animal was minced
cell cycle regulation by inhibiting transition from G0/G1
with blades to millimeter sizes in tissue medium (RPMI
to S phase. Based on previous studies, we examined the
1640). The supernatant was separated and fi ltered through
apoptotic indices of human gastric cancer grafted into
a 50-mm nylon mesh. The fi ltered cells were collected by
SCID mice. We investigated its apoptotic effects on human
centrifugation and washed twice with PBS.
gastric cells, by which we explored its correlated anticancer
mechanisms and its synergistic effect combined with
Detection of cell apoptosis by TUNEL method
NM-3 in order to look for a novel therapy for advanced
Six cell suspensions (1 × 104 cells) of each group (NM-3,
gastric cancer.
paclitaxel, paclitaxel/NM-3, and saline) were placed
separately into 60-mm dishes containing cover glass slides
MATERIALS AND METHODS
(washed and high-pressure sterilized). Then the glass
slides were taken out, washed twice with PBS, and fixed
Materials
in methanol: freezing acetic acid (3:1) for 30 min. The
RPMI1640 media and TRI201 total RNA isolation kit
following procedures were carried out according to the kit
were purchased from Gibco BRL. The liposome, the
instruction. The average number of apoptotic cells was
trypsin, DMEM culture medium, Hepes and Csc1, 200
determined by counting 1000 cells on each glass slide and
bp DNA ladder, dNTP, Taq enzyme and the restriction
the apoptotic index (AI), i.e. the number of apoptotic cells
endonuclease were obtained from Sigma Co.
per 100 cancer cells, was calculated.
Drugs and reagents
Chemical staining of X-gal
Paclitaxel was obtained from the Chinese Academy of
1 × 106/L cells of four groups were fi xed by 0.5% glutaral
Science. NM-3 was provided by Doctor Robert, Huston
pentanediol for 15 min and washed thrice with PBS. X-gal
University, USA.
staining solution (20:1) was added and cells were incubated
at 37℃ for 4-24 h in a humidifi ed atmosphere containing
Animal model
50 mL/L CO2. Blue-stained cells, i.e. those with LacZ gene
Male severe combined immune deficiency (SCID) mice
expression, were counted under the microscope and the
were obtained from Shanghai Experimental Animal Center
percentage of the positive cells was calculated.
of Chinese Academy of Sciences. Animals used were
6-7 wk old and weighed 18-22 g. Human gastric cancer
Detection of the expression of the p27mt gene
SGC-7901 (Shanghai Tumor Institution No: 01842),
5 mg fresh tissue specimens of every mouse in four groups
a poorly differentiated adenocarcinoma cell line, was
were digested by 0.5 g/L trypsin. The cells were collected
originally derived from a primary tumor and maintained by
and washed twice with PBS. After cell lysis in 500 μL
passage in the subcutis of nude mice. Animal models were
SDS-PAGE cell lysis solution and boiling for 5 min,
made using orthotopic implantation of histological intact
supernatants were collected after centrifugation. Samples
tissue of human gastric carcinoma. Tumors were resected
were subjected to western blot analysis.
aseptically. Necrotic tissues were cut and the remaining
healthy tumor tissues were scissor minced into pieces
Detection of cells by fl ow cytometry
(about 5 mm × 7 mm in diameter) in Hank’s balanced salt
Fresh tissue specimens of the four groups were minced
solution. Each tumor piece was weighed and adjusted to
with blades to millimeter sizes in tissue medium (RPMI
50mg. Mice were anesthetized with 4.3% trichloraldehyde
1640). Then supernatant was separated and filtered
hydrate. An incision was made through the left upper
through a 50-mm nylon mesh. The filtered cells were
abdominal pararectal line. Then the peritoneal cavity was
collected by centrifugation, stained, washed twice with
carefully exposed and a part of the serosal membrane in
PBS, and cell suspension was adjusted to a density of
the middle of the greater curvature of the stomach was
106/L. 100 μL of cell suspension was mixed with 200 μL
mechanically injured using scissors. A tumor piece of 150
of DNA-PREPTM LPR, followed by detection using
mg was fixed on each injured site of the serosal surface.
Coulter Epics XL flow cytometer for 15 min. Cell cycle
The stomach was returned to the peritoneal cavity, and the
progression and cell apoptosis rate were analyzed.
abdominal wall and skin were closed.
After 12 d, when the tumor reached the size of 0.8-1.0
DNA fragment analysis
cm3, mice were randomly separated into four groups,
SGC-7901 cells, after being collected and washed twice
with ten mice per group. Body weight and tumor volume
with PBS, were lysed in 500 μL cell lysis solution [1%Np40,
of these orthotopic grafted mice in every group had no
20 mm/L EDTA, 50 mmol/L Tris-HCl (pH 7.5)] in the
obvious difference (P < 0.05). Via intraperitoneal injection
presence of 10 μL of protease K. Samples were heated
animals received paclitaxel (5 mg/kg), NM-3 (10 mg/kg),
in a 56℃ water bath for 1-2 h before extraction with
paclitaxel (5 mg/kg) combined with NM-3 (10 mg/kg), or
phenol/chloroform. After the DNA precipitate had
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Zhu JS et al . Paclitaxel and NM-3 effects on gastric cancer 4133
A
B
Table 1 Effects of paclitaxel/NM-3 on the cell cycle

300
300
progression (mean ± SD)

240
240
Group
G0/G1
S
G2/M
180
Counts
Control
24.13 ± 1.12
40.12 ± 1.09
32.17 ± 1.48
Counts
NM-3
34.42 ± 3.51
22.41 ± 2.13
18.23 ± 3.23
120 180

120
Paclitaxel
26.34 ± 0.35a
39.20 ± 0.39a
33.10 ± 0.89a
Paclitaxel/NM-3
87.42 ± 3.17b
9.12 ± 0.22b
15.12 ± 2.40b

60


60
a
0
0
P > 0.05 vs saline group; bP < 0.01 vs paclitaxel group).
0 200 400 600 800 1000
0 200 400 600 800 1000
Figure 1 Apoptotic curve line of multidrug-resistant SGC-7901 cells was induced
by NM-3 and NM-3 combined with paclitaxel, fi rst apoptotic peak (B) was elevated
Table 2 Effects of paclitaxel/NM-3 on the cell apoptosis (mean
compared with that of NM-3 group (A).
± SD)
Group
AI
A
B
Control
13.24 ± 7.75
NM-3
25.62 ± 6.46a
Paclitaxel
28.90 ± 5.38a
Paclitaxel/NM-3
38.51 ± 5.14b
aP < 0.05; bP < 0.01 vs control group.
and in the control group compared to the others, the
C
D
percentage of the S phase cells is higher, indicating
that transition time of cell cycle was shortened and cell
proliferation was active. However, the percentage of G0/
G1 phase cells increased and the cell cycle was arrested
in G0/G1 phase in the paclitaxel/NM-3 group, which
was significantly different between the control and the
paclitaxel group (Tables 1 and 2).
Figure 2 Detection of apoptotic cells by TUNEL method. A: The apoptotic cells
appeared yellow in the control group; B: The apoptotic cells appeared yellow in the
X-gal chemical staining:
NM-3 group; C: The apoptotic cells appeared yellow in the paclitaxel group; D: A
The adenovirus mediated gene transfer rate was evaluated
large number of apoptotic cells appeared in paclitaxel/NM-3 group.
by X-gal staining. The results showed that the infection
efficiency could reach 90%, indicating that recombinant
adenovirus could effectively transfer genes in vitro.
been washed once with 700 mL/L alcohol, 200 μL of
The expression of p27 protein was evaluated after
TE was added followed by an overnight incubation with
being injected into the cancer cells with paclitaxel
RNase (final concentration 50 μL/mL) at 37℃. The
in vitro: After the cells that could be used in experiment
final DNA was separated by agarose gel electrophoresis
were injected by paclitaxel/NM-3 for 24 h, these cells
(10 g/L) and visualized with the aid of an ultraviolet light
were collected and lysed with 1 × SDS PAGE cell lysis
lamp.
solution. After boiling at 100℃ for 5 min, the solution was
centrifuged. The supernatant was collected and the protein
Statistical analysis
was detected by TMB system western blot kit, KPL USA.
Data were analyzed by t test and a P value < 0.05 was
Followed by X-gal chemical staining, there was high
considered statistically significant. All statistical analyses
expression of a 27 kDa protein in the paclitaxel/NM-3
were performed using SPSS 11.5 for windows.
group while only slight traces were seen (endogenous
expression) in the paclitaxel and in the control group.
RESULTS
Thus, the human mutant p27 recombinant adenovirus
constructed in the present study expresses the p27 gene
Detection of cell apoptosis by TUNEL method
properly in SGC-7901 cells and the protein product could
The nucleus of apoptotic cells was dark stained, dark
be expressed at a high level in cells.
brown and the cytoplasm was concentrated and the cell
shrank. The AI of samples of the paclitaxel/NM-3 and the
PCR identifi cation and effect of paclitaxel/NM-3 on p27mt
control group was 82.6% (± 3.12%) and 5.0% (± 0.35)%),
target gene
respectively. This difference was statistically signifi cant (P
The pathological change of SGC-7901 cells and their
< 0.05) showing that paclitaxel/NM-3 could obviously
culture fl uid were collected and centrifuged. Five milliliters
induce apoptosis of gastric cancer cells (Figures 1 and 2).
of the supernatant was mixed with 1 mg of protease K,
Cell cycle progression is shown in Table1. Whereas the
2 mL of 1% SDS, 10 mmoL/L EDTA and 20 mmol/L
number of cells in G0/G1 phase is lower in the paclitaxel
Tris-HCl to be digested for 2 h. After being precipitated by
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4134 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol August 14, 2007 Volume 13 Number 30
dehydrated alcohol, the DNA was collected. PCR reaction
prohibited the viability of the astrocyte when these cells
was carried out after adding the forward and reverse
were transfected with exogenous gene p27 carried by
primers. Finally, a 275-bp target fragment was amplified,
adenovirus. Zhang D[13] reported that the upregulation
which showed that paclitaxel, paclitaxel/NM-3 had already
of the expression of the p27 by retinoic acid signifi cantly
played an inhibitory role on the p27mt target gene.
inhibited the growth of the oophoroma cell. Koh TY[14]
found high expression of p27 and raised the expression
Detection of cells by fl ow cytometry
level of cyclinD1 and cyclinE in the cell lines SUN-1066,
After the animals had been treated with NM-3, paclitaxel,
SUN-1041, SUN-1076 derived from cephalocervical
paclitaxel/NM-3, or saline for 24 h, the detection of
squamous cell carcinoma by transfecting these cells with
apoptotic cells was carried out by flow cytometry and
p27kip1 carried by reconstructed adenovirus, the cancer
repeated six times. The mean value of hypodiploid cells
cells’ proliferation were significantly prohibited and
was: 8.23%, 4.67%, 41.0%, and 1.96%, respectively.
the cell cycle analysis showed that the cancer cells were
Statistical analysis revealed a signifi cant difference between
mainly stopped at the G1-S stage in their study. All this
the paclitaxel/NM-3 group and the paclitaxel group and
showed that p27 was a very important gene related to the
the NM-3 group, (paclitaxel/NM-3 vs paclitaxel group,
development of carcinoma and had signifi cant impact on
paclitaxel/NM-3 group, NM-3 group and paclitaxel group
the onset, development and prognosis of the tumor.
vs normal saline group, P < 0.01; paclitaxel group vs NM-3
Nowadays, functional reconstruction of anti-oncogene
group, P > 0.05).
has been a reasonable strategy of gene therapy for tumor.
Sasaki T[15] found that p27mt had stronger suppressive
Detection of DNA fragment
effects than p27wt on apoptosis and cell proliferation
The result of DNA electrophoresis showed intact genomic
when they applied adenovirus-induced p27mt and p27mt
DNA in samples obtained from the paclitaxel and the
to transfect the cholangiocarcinoma cell lines TFK-1
normal control group while there was an obvious 180-200
and HuCCT-1. Park KH[16,17] got the same results when
bp diploid ‘trapezia’ pattern in samples derived from
they used adenovirus mediated p27mt and p27wt to
NM-3 or the paclitaxel/NM-3 injected group, which was in
transfect lung cancer cell lines NCI H460, NCI H1264,
concordance with the characteristic changes of apoptosis.
NCI H358 and NCI H157, and a spongioblast line. In
our study, mutant p27 was used to transfect gastric cancer
cells and the high expression of this gene was proven
DISCUSSION
by p27 polyclonal antibody. This result supported that
Since paclitaxel was extracted from Taxul which has special
adenovirus with reconstructed p27mt could transfect
anti-tumor effect, paclitaxel had cell toxic side-effects as
the target gene into a gastric cancer cell which derived
Taxol either. We found that paclitaxel could inhibit the
from human gastric adenocarcinoma tissue, developed
activation of CyclinE-CDK2 complex and the activation
into tumor in SCID mice and expressed endogenous
of CyclinD-CDK4 and CyclinA-C DK2 complex as well.
p27. By flow cytometry, rate of apoptosis up to 41.0% in
In addition, it could also down-regulate the expression of
paclitaxel/NM-3 group was proven which had signifi cant
CyclinB, because the arrest of G
difference compared to the control group, DNA analysis
0/G1 was mainly caused by
p27 whose accumulation could be induced by exogenous
showed 180-200bp DNA ladder. By TUNEL technique,
signal. Gastric tumorigenesis was closely correlated with
a value of apoptotic index up to 82.6% was detected
translocation, deletion and mutation of p27 gene and its
which showed significant difference compared to the
expression or activity changes[5,6]. At present, gastric cancer
control group. The results showed that the gene p27 was
cells treated by paclitaxel/NM-3 in SCID mice were not
an important gene related to the occurance of the large
reported. If the expression level of p27 mt in gastric
gastric carcinoma and the down-regulation of p27 may
cancer cell was down-regulated or inhibited by p27, thus
be the main cause of cell differentiation dysfunction and
the DNA damaged cell could not transit form form G1
apoptosis dysfunction. Upregulating the expression of
phase to S phase directly, that would induced the apoptosis
p27 by mutant p27, which promotes the apoptosis of the
of human gastric cancer cells in SCID mice.
tumor cell, could serve as a new scheme in the treatment
Paclitaxel was first found in 1998, the researchers
of advanced gastric carcinoma. Circle analysis showed
revealed it effected on cell contact inhibition with
that the cleavage of tumor cells was stopped at G1 stage
TGF-β[4]. The degradation of the p27 protein is mainly
via suppressing the activity of the cyclin/CDK kinase by
caused by phosphorylation of the threonine residue at
p27mt. Winteringham LH[18] thought that the accumulation
position 187 which is mediated by ubiquitin[7-9]. Kudo
of p27 played an important role in apoptotic mechanisms
and his colleagues[10] found that if the 187th threonine of
of the gastric cell cycle arrest at the initiation of cell
p27 which was mediated by p27 protein, could inhibit
differentiation. Whether other apoptotic factors might
cell growth obviously, these inhibitory effects were more
affect this process should be explored further.
obvious on mutant p27 (T187A) than on wild-type
Recently, although the apoptosis of the human gastric
p27. The target phosphorylation site of CDK would be
cancer cell was successfully induced by the application of
protected from phosphorylaton. A replication-deficient
NM-3 and other gene therapy, but the apoptosis of human
recombinant adenovirus was constructed which carried
gastric cancer cells induced by paclitaxel/NM-3 had not
p27mt to study apoptosis of the gastric cancer cell line,
been reported in the SCID mice model[19,20], it was very
by which it expected to find a more effective p27 gene
useful experimental support of tumor-suppressed function
to treat gastric cancer. Koguchi K[12] reported that they
for human gastric cancer treated by paclitaxel/NM-3. The
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Zhu JS et al . Paclitaxel and NM-3 effects on gastric cancer 4135
effi cacy of this method and the mechanism of apoptosis
T. Transfection of p27(Kip1) threonine residue 187 mutant
strongly indicate that paclitaxel/NM-3 is synergistically
type gene, which is not influenced by ubiquitin-mediated
effective against human gastric carcinomas.
degradation, induces cell cycle arrest in oral squamous cell
carcinoma cells. Oncology 2002; 63: 398-404
11 Hurteau JA, Brutkiewicz SA, Wang Q, Allison BM, Goebl MG,
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