The
Effects of Inhibiting P18INK4C Expression on the Invasion of Gastric
Adenocarcinoma Cell Line
WANG Chu, WANG
Jian-Hua, LI Feng, LU Jian*
(
Department of Biochemistry and Molecular Biology, Research Center for Human
Gene Therapy, Shanghai Second Medical University, Shanghai 200025, China )
Abstract Using
cDNA microarray with double dots of 4096 human genes, P18INK4C, a member of
CKI, was found down-regulated in a gastric adenocarcinoma metastatic cell line
(RF-48), compared with the corresponding primary cancer cell line (RF-1), which
implied that P18INK4C might be involved in cell invasion and metastatic
progression of human gastric adenocarcinoma. Antisense RNA expression plasmid
was applied to inhibit P18INK4C expression to study the effect of decreased
P18INK4C expression on cell migration, invasion and proliferation ability and
cell cycle of RF-1. Results showed that inhibition of P18INK4C expression could
obviously enhance cell invasion ability of RF-1, but had little effect on its
cell cycle and cell migration and proliferation ability. These results implied
that P18INK4C might play a pivotal role in regulating cell invasion, rather
than regulating cell cycle and proliferation in the progression of human
gastric adenocarcinoma as expected before.
Key words P18INK4C; human gastric
adenocarcinoma; invasion and metastasis
P18INK4C, a
protein of 18 kD, is a member of INK4 family of cyclin-dependent kinase
inhibitors (CKIs) which also include P15INK4A, P16INK4B and P19INK4D. These CKI
proteins could specifically bind not only monomeric CDK4 or CDK6 to prevent the
formation of CDK-cyclin complex, but also the CDK4/6-cyclin complex to form an
inactive ternary complex CKI-CDK-cyclin[1－8]. P18INK4C could also inhibit the activity of CDK-cyclin complex,
especially CDK6-cyclinD, by distorting the ATP binding site and misaligning
catalytic residues, which could block cell cycle progression[1,4,9].
Furthermore it distorted the cyclin-binding site of CDK as well, with the
cyclin remaining bound to CDK at a size-reduced interface[1].
P18INK4C gene
was localized on 1p32 which was frequently involved in chromosomal changes in
various tumors[10]. P18INK4C was considered as a potential candidate in tumor
therapy since its mutations, deletions and aberrant expressions existed in many
kinds of human cancers, such as breast cancer, oligodendrogliomas, Wilms tumor
and testicular cancer[11－15]. Recently, it was found that P18INK4C might be also correlated
with cancer invasion and metastasis. The research of Bartkova et al.[14] have
suggested that increased abundance of cyclinE and particularly down-regulation
or loss of P18INK4C might be one of the features of the progression from
pre-invasive carcinomas in situ (CIS) to invasive germ cell tumours of the human
testis.
Using cDNA
microarray with double dots of 4096 human genes, it was found that the
expression of P18INK4C gene was down-regulated in RF-48 which is the metastatic
cell line of a gastric adenocarcinoma patient, compared with RF-1 which is the
primary cancer cell line of the same person. Besides, we also found the
expression of cyclin E2 gene being up-regulated in RF-48 compared with
RF-1[16]. By using antisense RNA expression plasmid, we inhibited the P18INK4C
expression to study its effect on cell migration, invasion and proliferation
ability and cell cycle. Our findings indicated that down-regulated expression
of P18INK4C was associated with the enhanced invasion ability of RF-1 and this
effect was not the result of its function of cell cycle arrest. These implied
that P18INK4C might be involved in cell invasion and metastatic progression of
human gastric adenocarcinoma.
1 Materials
and Methods
1.1 Materials
RF-1, the
primary tumor cell line of a gastric adenocarcinoma patient, and RF-48 cell
lines, its relative metastatic counterpart were obtained from ATCC. E. coli DH5α and pcDNA3-GFP were from our lab.
RPMI 1640 and Opti-MEM�j were purchased from Gibco BRL, and FBS was from Hyclone; G418 was
bought from Roche; MTT was from Sigma; Trizol reagent for RNA extraction and
MLV reverse transcriptase were purchased from Gibco BRL; DNA extraction kit was
bought from Gentra; EX-Taq was from TaKaRa; HindIII, BamHI and T4 DNA ligase
were obtained from New England Biolabs; LipofectAMINETM 2000 was the product of
Invitrogen; transwell chambers (pore size: 8 μm) were bought from Costar; Matrigel�j basement membrane matrix (phenol-red free) was purchased from
Becton Dickinson; Northern blotting kit was from Boehringer Mannheim, Roche;
Western blotting kit and anti-mouse IgG-HRP were products from Wuhan Boshide
Inc; anti-P18 mouse monoclonal IgG antibody was bought from Santa Cruz
Biotechnology Inc.; RIPA kit was from Shanghai Shenergy Biocolor Inc.; Primers
were synthesized by Shanghai Sangon Inc.; DNA was sequenced by Shanghai
Genecore Inc.. All other chemicals were of analytical grade.
1.2 Cell
culture
RF-1 (ATCC No:
CRL-1864), and RF-48 (ATCC No: CRL-1863) were suspending cell lines and
routinely cultured in a 37 ℃, 5% CO2 incubator with RPMI 1640 containing 10% FBS.
1.3 Construction
of antisense P18INK4C RNA expres-sion plasmid
Total RNA,
isolated from RF-1 using the Trizol reagent according to the manufacturer’s
instructions, was reversely transcribed and the product was used as the
template for PCR. The antisense P18INK4C gene (product size, 550 bp) was
amplified by the upstream primer (5′-AGTGGATCCCGTGAACAAGGGACCCT-AAAG-3′) and the downstream primer (5′-AGTAAG-CTTCGTTTATTGAAGATTTGTGGCTC-3′). BamHI and HindIII sites were underlined respectively. After
digested by the two restriction endonucleases, the partial antisense P18INK4C
gene and pcDNA3 were ligated by T4 ligase to form an antisense P18 RNA
expression plasmid, named as pcDNA3-antisense-p18.
1.4 Cell
transfection and screening
RF-1 was
transfected with pcDNA3-GFP and pcDNA3-antisense-p18 respectively using
Lipofect-AMINETM 2000 according to the manufacturer’s instructions.
Transfection rate was calculated according to the cells transfected with
pcDNA3-GFP by using FACS analysis. A cell suspension with a density of 106
cells/mL in RPMI 1640 growth medium with 10%FBS and without antibiotics was
added to a 6-well plate. 4 μg of pcDNA3-antisense-p18 or pcDNA3-GFP in 250 μL
of Reduced serum medium and 12 μL LipofectAMINETM 2000 in 250 μL Opti-MEM�jI medium were incubated for 5 min
and 20 min at room temperature respectively, then these tow solutions were
mixed, and added gently to each well containing RF-1 cells. Then the cells were
incubated for another 48 h before the cell activities to be detected.
Transfected cells were incubated for 2 d
and then screened with 500 mg/L G418 (active concentration). After 5 d, the
concentration of G418 was reduced to 250 mg/L and then maintained.
G418-resistant cell clones, named RF-1-anti-p18 and RF-1-GFP were isolated and
expanded respectively. Throughout the process of cell screening, the cells were
kept in RPMI 1640 containing 30% FBS.
1.5 Northern
blotting
All the process was referred to the
manufacturer’s instructions and the standard protocol of “Molecular Cloning: A
Laboratory Manual” (Cold Spring Harbor Laboratory Press, 1989). β-actin was
used as a control.
1.6 Western
blotting
Protein was
prepared by using RIPA kit and quantified by the method of BCA[17]. After
separated by 12% SDS-PAGE gels，30 μg of each protein
sample was transferred onto nitrocellulose membranes and probed with the mouse
monoclonal IgG antibody of P18INK4C and anti-mouse IgG-HRP. Bands were
visualized by DAB. The detailed procedure was referred to the standard protocol
of “Molecular Cloning: A Laboratory Manual” (Cold Spring Harbor Laboratory
Press, 1989) and the manufacturer’s instructions.
1.7 RT-PCR
Semi-quantitative
RT-PCR was adopted to detect the results of transfection and screening of
RF-1-anti-p18 and the change of P18INK4C expression at mRNA level. GAPDH was
used as an internal control. Total RNA, isolated from RF-1, RF-1-GFP and
RF-1-anti-p18 using the Trizol reagent, was reversely transcribed and the
products were used as the templates for PCR. neo (product size, 410 bp) was amplified
by the upstream primer (5′-GAAGGGACTGGCTGCTATTG-3′) and the downstream primer (5′-AGCCAACGCT-ATGTCCTGAT-3′). Endogenous P18INK4C gene in cells (product size, 452 bp) was
amplified by the upstream primer (5′-GGTGGAGTTCCTGGTGAAGC-3′) and the downstream primer (5′-CAACTTGGGTGTTG-AGAT-3′). GAPDH (product size, 233 bp) was amplified by the upstream primer
(5′-TGGGGAA-GGTGAAGGTCGG-3′) and the downstream primer (5′-CTGGAAGATGGTGATGGGA-3′).
1.8 PCR
To verify the
insertion of antisense P18INK4C gene into the genome, PCR was performed using
genomic DNAs of RF-1, RF-1-GFP and RF-1-anti-p18 as the templates. The genomic
DNAs were prepared by using Gentra DNA extraction kit. The upstream primer was
5′-ACCCACTGCTTACTGGCTTATCG-3′, and the downstream primer was 5′-TTACAGACTTTGCTGG-AGTTTCA-3′. PCR product size was 314 bp.
1.9 MTT
assay of cell proliferation
MTT assay was
conducted to determine the cell proliferation. 103 cells in 2 mL of RPMI 1640
growth medium with 10%FBS were plated into a 96-well plate and incubated for 24
h. After that, 20 μL of 5 g/L MTT solution was added to each well and incubated
for another 4 h in a 37 °C incubator. 150 μL DMSO was added to dissolve the
crystal after the growth medium was removed. Then the absorbance of 200 μL
solution from each well was measured at 570 nm by a microplate reader. Each
sample was assayed in octuple for 7 d consecutively. Cell growth curves of
RF-1, RF-48, RF-1-GFP and RF-1-anti-p18 were obtained based on the
corresponding values of A570 respectively.
1.10 Cell
cycle analysis
The detailed
procedure was followed as previously reported[18]. 105 cells were collected by
centrifugation, washed, and suspended in ice-cold PBS. Cells were then fixed
with 70% ethanol for 10 min in room temperature, followed by staining with
propidium iodide (PI). Cell cycle was assessed by FACS.
1.11 Tumor
cell migration and invasion assay
Cell migration
assays were performed using 6.5 mm transwell chambers (pore size: 8 μm) as described
previously[19,20]. Briefly, conditioned NIH-3T3 medium was added to the bottom
well, and the filters were coated by the conditioned NIH-3T3 medium for 30 min
at 37 ℃. Cells were
resuspended in the appropriate buffer at a concentration of 106 cells/mL and
105 cells were added to the top well of transwell chambers. After 4 h
incubation, the cells that had not migrated were removed from the upper face of
the filters using cotton swabs, and the cells that had migrated to the lower
surface of the filters were fixed in methanol and stained by hematoxylin.
Migration was determined by the count of the cells that had migrated to the
lower surface of the filter with a microscope at ×400. Six visual fields were
counted for each essay. Assays were repeated for three times.
Matrigel invasive assays were performed
using 6.5 mm transwell chambers (pore size: 8 μm)[19,20]. Matrigel (Matrigel�j basement membrane matrix, phenol-red free, Becton Dickinson) was
diluted in serum-free medium, added to the top well of transwell chambers (19.6
μg/well), and dried under a sterile hood. The Matrigel was then reconstituted
with medium for 2 h at 37 °C before the addition of cells. Cells were
resuspended in serum-free medium containing 0.1% BSA. 2×105 cells were added to
each well. Conditioned NIH-3T3 medium was added to the bottom well of transwell
chambers. After 24 h, the cells that had not migrated were removed from the
upper face of the filters using cotton swabs, and the cells that had invaded to
the lower surface of the filters were fixed by methanol, and stained and
counted as described above, then the number of cells that invaded to the lower
side of the filter was measured as a parameter of the invasive ability of the
cells. Assays were repeated for three times.
1.12 Statistical
analysis
Data are
presented as x±s and significance was determined by the Student’s t-test.
2 Results
2.1 Analysis
of P18INK4C expression in RF-1 and RF-48
Using Northern
blotting and Western blotting, the expression of P18INK4C gene was found to be
down-regulated in RF-48, the metastatic cell line of a gastric adenocarcinoma
patient, compared with RF-1, the primary cancer cell line of the same person,
which confirmed the results of cDNA microarray[16] (Fig.1). These results
implied that the reduced expression of P18INK4C gene might be involved in the
progression of human gastric adenocarcinoma metastasis.
Fig.1 The
expression of P18INK4C in RF-1 and RF-48
(A) Northern blotting analysis of
P18INK4C and β-actin expressed in RF-1 and RF-48. 1, P18INK4C and β-actin
expressed in RF-1; 2, P18INK4C and β-actin expressed in RF-48. (B) Western
blotting analysis of P18INK4C expressed in RF-1 and RF-48. 1, P18INK4C
expressed in RF-48; 2, P18INK4C expressed in RF-1.
2.2 Analysis
of the effects of cell transfection and screening
Using the total
RNA of RF-1, RF-1-GFP and RF-1-anti-p18 as templates, RT-PCR was adopted to
amplify the neo gene from these three kind cells respectively. It was found
that neo could only be amplified from RF-1-GFP and RF-1-anti-p18 (Fig.2), which
indicated the process of cell transfection and screening was successful.
Fig.2 RT-PCR
analysis of transfection and screening of RF-1-GFP and RF-1-anti-p18
1, 100 bp DNA ladder marker; 2, neo
expressed in RF-1; 3, neo expressed in RF-1-GFP; 4, neo expressed in
RF-1-anti-p18. GAPDH was used as an internal control.
To verify the
insertion of antisense P18INK4C gene into the genome, PCR was performed using
genomic DNAs of RF-1, RF-1-GFP and RF-1-anti-p18 as the templates. The upstream
primer was designed complementary to the cytomegalo virus (CMV) promoter
sequence in the pcDNA3, whereas the downstream primer was complementary to the
antisense P18INK4C sequence. The desired PCR product (314 bp) was obtained from
RF-1-anti-p18 (Fig.3), which indicated the integration of antisense P18INK4C
gene into the genome of RF-1.
Fig.3 PCR
detection of a 314 bp fragment from the genomic DNAs of RF-1, RF-1-GFP and
RF-1-anti-p18
1, 100 bp DNA ladder marker;
2, PCR result of RF-1; 3, PCR result of RF-1-GFP; 4, PCR result of
RF-1-anti-p18.
2.3 Expression
of P18INK4C in RF-1-anti-p18
Through
analyzing the expression of P18INK4C in RF-1, RF-1-GFP and RF-1-anti-p18, the
effect of antisense P18INK4C could be evaluated. The expression of endogenous P18INK4C,
amplified by RT-PCR, was obviously inhibited in RF-1-anti-p18, compared with
RF-1 and RF-1-GFP (Fig.4). The result of Western blotting also showed that the
protein level in RF-1-anti-p18 was lower than that in the other two (Fig.4).
All of these results indicated that the expression of P18INK4C was inhibited by
antisense P18INK4C.
Fig.4 The
expression of P18INK4C in RF-1, RF-1-GFP and RF-1-anti-p18
(A) RT-PCR analysis of P18INK4C
expressed in RF-1, RF-1-GFP and RF-1-anti-p18. 1, 100 bp DNA ladder marker; 2,
P18INK4C expressed in RF-1; 3, P18INK4C expressed in RF-1-GFP; 4, P18INK4C
expressed in RF-1-anti-p18. (B) Western blotting analysis of P18 expressed in
RF-1, RF-1-GFP and RF-1-anti-p18. 1, P18INK4C expressed in RF-1; 2, P18INK4C
expressed in RF-1-GFP; 3, P18INK4C expressed in RF-1-anti-p18.
2.4 Cell
proliferation and cell cycle analysis
The
proliferation ability of RF-1, RF-48, RF-1-GFP and RF-1-anti-p18 was evaluated
by MTT assay. According to the A570 values of 7 d, cell growth curves were made
(Fig.5). There was no detectable difference in the proliferation ability among
them, which, at the same time, suggested that the proliferation of
RF-1-anti-p18 cells was not affected by the change in the expression of
P18INK4C.
Fig.5 Cell
growth curves of RF-1, RF-1-GFP, RF-1-anti-p18 and RF-48 in 7 d
The cell growth curve was
measured by MTT assay. Values were expressed as x±s， n= 8.
Using FACS, cell
cycle of RF-1、RF-48、RF-1-GFP and RF-1-anti-p18 were analyzed.
No significant difference of cell cycle distribution was found among these
different cells (Fig.6).
Fig.6 Cell cycle
analysis of RF-1, RF-1-GFP, RF-1-anti-p18 and RF-48 by FACS
1,
RF-1; 2, RF-1-GFP; 3, RF-1-anti-p18; 4, RF-48.
2.5 Cell
migration and invasion abilities in vitro
According to the
results of cell migration assay in vitro, it was found that RF-1,
RF-1-GFP and RF-1-anti-p18 could all migrate to the lower surface of the
transwell filter under the same condition. As to the migrated cells stained by
hematoxylin under a microscope at ×400, there were (91±3) cells of RF-1, (95±3)
cells of RF-1-GFP and (94±3) cells of RF-1-anti-p18 (Fig.7), and no significant
difference was found among the groups, which implied that cell migration
abilities of these different cells were similar.
Fig.7 Comparison
of cell migration ability of RF-1, RF-1-GFP and RF-1-anti-p18.
Cell number in each visual
field was counted under a phase contrast microscope at ×400. Values are
expressed as x±s, n=3.
Under the same
condition we analyzed the cell invasion ability of the RF-1, RF-1-GFP and
RF-1-anti-p18 in vitro. There were (15±2) cells of RF-1, (16±1) cells of
RF-1-GFP and (34±3) cells of RF-1- anti-p18 each visual field under a phase
contrast microscope at ×400(Fig.8). We could find significant difference among
the cell numbers. The result suggested that RF-1-anti-p18 could easily migrate
though Matrigel, compared with the other two.
Fig.8 Comparison
of cell invasion abilities of RF-1, RF-1-GFP and RF-1-anti-p18
Cell number in each visual field was
counted under a phase contrast microscope at ×400. Values were expressed as
x±s, *P<0.01 vs. RF-1-anti-p18, n=3.
3 Discussion
Invasion and
metastasis of tumor cells were the main causes of cancer patients’
death[21,22]. The mechanisms of acquiring the capabilities of invasion and
metastasis were different from the primary mechanisms of inducing malignant
transformation. In various experimental systems, the expression of a
considerable amount of genes affected metastatic ability, and several model
systems had been established to study mechanisms that induced tumor cells to
metastasize[23－29]. However,
despite recent progress in the knowledge about metastatic pathways, the
mechanisms hadn’t been fully understood yet.
In order to
identify the possible metastasis-associated genes of human gastric
adenocarcinoma, cDNA microarray with double dots of 4096 human genes was
performed and the expression of P18INK4C was found to be down-regulated in
RF-48, the metastatic cell line of a gastric adenocarcinoma patient, compared
with RF-1, the primary cancer cell line[16]. The result was also substantiated
by Northern blotting and Western blotting. Bartkova et al.[14] demonstrated
that P18INK4C was considerably reduced or absent at the protein level in
invasive germ cell tumors (GCTs), which was different from its abundant
expression in the CIS cells, and suggested that down-modulation or loss of
P18INK4C might contribute to progression from pre-invasive lesions to overt
germinal tumors. All of these gave us a hint that the down-regulation of
P18INK4C expression might be associated with the progression of invasion and
metastasis of gastric adenocarcinoma.
By using
antisense RNA technology, we successfully inhibited the expression of P18INK4C
gene in RF-1 with pcDNA-antisense-p18 and studied its effects on cell migration
and invasion ability as well as cell cycle and proliferation. The results of
cell migration and invasion assay indicated that although the migration
abilities of RF-1, RF-1-GFP and RF-1-anti-p18 were similar, RF-1-anti-p18
apparently migrate though Matrigel and filter more easily than the other two
cells. But these results could not assure us that the invasion ability of
RF-1-anti-p18 was actually enhanced because the mechanism of cell invasion
assay was that the invasion ability was determined by the total number of the
cells having migrated through Matrigel and 8 μm pores to the lower surface of
the filter. As a negative regulator of cell cycle, reduced expression of
P18INK4C gene may increase the number of the cells having migrated through the
transwell filter by increasing the total cell number of RF-1-anti-p18 in a
certain period without affecting cell invasion ability. Recent studies
indicated that although P18INK4C, as a member of INK4 family, could block cell
cycle progression at G1/S phrase, it did not play a pivotal role in controlling
cell cycle and proliferation due to the compensatory roles by the Cip/Kip family
of CKI, and loss of P18INK4C did not appear to confer any proliferative
advantage to some kind of cell lines[15, 30]. Our results of MTT assay and cell
cycle analysis also showed that there were no significant difference in cell
cycle and proliferation among RF-1, RF-1-GFP and RF-1-anti-p18. Therefore,
through all the researches above, it can be concluded that the inhibition of
P18INK4C expression could enhance the invasion ability of RF-1 without
affecting its migration ability, which implied that P18INK4C might play an
important role in regulating cell invasion ability in the progression of human
gastric adenocarcinoma metastasis. Besides, the primary function of P18INK4C
might not be to regulate cell cycle and proliferation during the development of
gastric adenocarcinoma from CIS to invasive cancer.
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Received: March 3, 2003Acccepted: April 4,
2003
*Corresponding author: Tel,
86-21-63846590-776441; e-mail, [email protected]