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Introduction
The polyamines, spermidine, spermine, and the diamine
precursor putrescine are positively-charged aliphatic amines
at physiological conditions, with a low-molecular weight and
a simple chemical structure. They interact with various
macromolecules, both electrostatically and covalently, and
as a consequence, have a variety of cellular effects. The
polyamines are typically met by the integrated contributions of
biosynthesis, catabolism, uptake, and export, each of which is sensitively
regulated by effector molecules that in turn are controlled by
intracellular polyamine pools[1]. Thus, ornithine
decarboxylase (ODC) and S-adenosylmethionine decarboxylase
(AdoMetDC) control biosynthesis, a polyamine transport
system modulates uptake, and spermidine/spermine
N1-acetyltransferase (SSAT) regulates polyamine catabolism and
export of the cell.
The polyamines are known to be critically involved in
cell growth and have been implicated in the process of cell
transformation[2,3]. The level of polyamines is high in cancer cells
and tissues, and rapid tumor growth has been
associated with remarkable elevation of polyamine
accumulation[4,5]. High polyamine levels have been detected in most
cancers[6_8]. In colorectal cancer, the polyamine content is increased 3-4-fold
more than those found in the equivalent normal
tissue[9,10].
The depletion of intracellular polyamine pools invariably
inhibits cell growth. This is usually accomplished by inhibiting
polyamine biosynthesis. Our previous research demonstrated
that the expression of ODC and AdoMetDC was increased in
colorectal cancer tissue[11,12], and the
downregulation of ODC and AdoMetDC could inhibit colorectal cancer
growth[13_15]. However, this might be more effectively achieved by the
activation of polyamine catabolism at the SSAT level, a
strategy first validated in MCF-7 breast carcinoma
cells[16]. Therefore, in the present study, we constructed a
recombinant adenovirus that could express human SSAT and
detected its inhibitory effect on colorectal cancer cell growth
in vitro.
Materials and methods
Cell culture and reagents Human colorectal
adenocarcinoma cell HT-29 and LoVo were preserved in our laboratory.
They were maintained in RPMI-1640 medium supplemented
with 10% (v/v) heat-inactivated bovine serum, 100 U/mL
penicillin, and 100 µg/mL streptomycin at 37 °C in a
humidified atmosphere of 5% CO2. HEK293 packaging cells
(transformed human embryonic kidney cells) were obtained
from the Chinese Academy of Science (Shanghai, China).
They were cultured in Dulbecco's modified Eagle's medium
(Gibco, Grand Island, NY, USA) containing 10% fetal bovine
serum, penicillin, and streptomycin. The pGL3-hTERT
plasmid was a gift from Dr Kou-juey WU (Department of
Pathology and Genetics & Development, Columbia University, New
York, USA). The shuttle vector pAdTrack and Escherichia
coli (E coli) AdEasy-1 cells (E
coli BJ5183 cells transformed with the pAdEasy-1 vector) were purchased from American
Type Culture Collection (Manassas, VA, USA). The MTS
(3-C4, 5-dimethylthiaol-2-yl)-5-(3-carboxy-methoxyphenyl)-
2-(-4-sulfophenyl)-2H-tetrazolium, inner salt)/phenazine
methosulfate (PMS) kit was purchased from Promega (Madison, WI, USA). The polyamines (spermidine and
spermine) were purchased from Sigma (St Louis, MO, USA).
The anti-SSAT mouse polyclonal antibody was purchased
from Abnova (Taipei, China). The β-actin antibody was
obtained from Santa Cruz Biotechnology (Santa Cruz, CA,
USA), and the ECL Western blotting detection system was
from Millipore (Bedford, MA, USA).
Amplification of SSAT gene and construction of TA
clone The total RNA was extracted from colorectal cancer tissue.
Complementary DNA was then synthesized using the cDNA
synthesis kit (MBI, Glen Burine, MD, USA). PCR was
performed to amplify the SSAT gene. The sequence of the SSAT
primers was as follows: forward primer, 5´-TAA CCA TGG
ATG GCT AAA TTC GTG ATC CGC-3´ and reverse primer,
5'-CGG TCT AGA TCA CTC CTC TGT TGC CAT TTT-3´.
The restriction sites were NcoI and
XbaI (enzyme recognition sites are underlined). The PCR products were purified using
a QIAquick gel extraction kit (Qiagen, Valencia, CA, Germany)
and linked to plasmid pMD-18T with a polyA linker. The
recombinant was transformed into E coli
DH5α and selected by selective culture medium containing ampicillin. The
positive recombinant plasmid was identified by dual digestion of
restrictive enzymes NcoI and XbaI.
Construction of plasmid pAdTrack-hTERT-SSAT
The pMD-18T-SSAT and pGL3-TERT plasmids were digested by
restrictive enzymes NcoI and XbaI. The digested fragments
were collected and purified using a gel extraction kit. The 2
fragments were ligated by T4 ligase at 16 °C overnight, and
then transformed into DH5α by CaCl2 method. Positive
colonies were selected by agar plates containing ampicillin and
confirmed by restriction enzyme mapping. The positive
recombinant plasmid pGL3-hTERT-SSAT was digested by
restrictive enzymes SalI and HindIII, and the small fragment
was ligated with pAdTrack, which was also digested by
SalI and HindIII. The positive clones (pAdTrack-hTERT-SSAT)
were confirmed by digestion.
Construction of pAdEasy-hTERT-SSAT plasmid
The pAdTrack-hTERT-SSAT plasmids were digested with
PmeI and then purified. In total, 5 µg purified linearized plasmid
was transformed into AdEasy-1 competent cells prepared by
CaCl2 method for the homologous recombinant with
pAdEasy-1. The reaction product was then plated onto
LB/kan (50 µg/mL) plates. After 24 h incubation at 37 °C,
approximately 24 of the smallest individual colonies were picked
and grown in 5 mL LB/kan for 16_18 h. The plasmids were
acquired using a conventional alkaline lysis method. The
candidate clones were identified by PacI digestion and yielded
a large (approximately 30 kb) and a small fragment (3.0
kb or 4.5 kb). BamHI was also used to identify the positive clones.
The correct recombinant miniprep DNA
(pAdEasy-hTERT-SSAT) were retransformed into the
DH5α and purified using Qiagen's plasmid midi kit.
Viral packaging and amplification in 293 cells
Recombinant adenoviral plasmids (pAdEasy-hTERT-SSAT) were
digested with PacI, ethanol precipitated, and resuspended
in sterile H2O. In total, 30 µg of digested
pAdEasy-hTERT-SSAT plasmids was transfected into 293 packaging cells using
Lipofectamine 2000 (Gibco, USA). The cells were cultured
for 7_10 d and harvested. The cells were then frozen at -80
°C and thawed in a 37 °C water bath; this procedure was
repeated 4 times. The samples were centrifuged at 12
000×g for 10 min, and the viral supernatant was stored at -80 °C.
For further amplification, more 293 cells needed to be
infected with these viral stocks. The recombinant virus
particles were purified by ultracentrifugation in cesium chloride
step gradients. The SSAT gene ligated into the virus DNA
was also detected by PCR. The titer of the purified
adenovirus was 2×108 pfu/mL.
Analysis of gene transduction efficiency in
vitro The efficiency of the adenovirus-mediated gene transfer was
assessed by MTS assay. The HT-29 and LoVo cells were seeded
at density of 5000 cells/well in 96-well plates and cultured
overnight. The cells were then infected with
Ad-hTERT-SSAT at multiplicities of infection (MOI) of 0, 10, 25, 50, 75,
and 100 for 24 h. After 72 h incubation, 20 µL MTS solution
(with PMS) was added to each well and maintained for 2 h at
37 °C. The optical density was measured at 490 nm.
Western blot analysis of SSAT protein After the HT-29
and LoVo cells were treated with phosphate-buffered saline
(PBS), Ad-green fluorescent protein (GFP), and
Ad-hTERT-SSAT by 50 and 25 MOI in 1640 medium containing 5% fetal
bovine serum for 72 h. They were then collected with a cell
scraper and washed with ice-cold PBS 3 times. The total cell
lysates were prepared in extraction buffer containing 0.05
mol/L Tris (pH 8.0), 0.15 mol/L NaCl, 0.02% sodium azide,
0.1% SDS, 100 mg/mL phenylmethylsulfonyl fluoride, 1 mg/mL
aprotinin, and 1% NP-40. Sample protein concentrations
were qualified by the bicinchoninic acid protein assay. The
proteins were subjected to 5% stacking and 12% resolving
SDS-PAGE gels and transferred to polyvinylidene difluoride
membranes (Millipore, USA). After incubation with the
appropriate antibodies in PBS containing 5% non-fat dry milk
and 0.02% Tween-20, the membranes were incubated with a
secondary antibody conjugated with horseradish peroxidase.
The membranes were then reacted with luminol substrate for
5 min and exposed to X-ray films (AGFA, Mortsel, Belgium).
The results were analyzed using SmartView analysis
software (Smarview Enterprise Imaging Solutions, Lake Forest,
CA, USA).
Measurement of polyamine content Polyamine content
was measured by HPLC analysis. HT-29 and LoVo cells were
infected with Ad-GFP and Ad-hTERT-SSAT by 50 and 25
MOI for 3 d and then harvested by scraping. Intracellular
polyamines were extracted from cell pellets with 10%
trichloroacetic acid and then the polyamines were mixed with a
2-fold volume of dansyl chloride and dansylated in the
presence of sodium carbonate for 20 min at 70 °C. Dansylated
polyamines were quantified using reverse-phase HPLC.
Effect on cell growth To observe the effect of the
adenovirus on cell growth in vitro, the MTS assay was used to
draw cell growth curves. The cells were inoculated at a
density of 4000 cells/well in 96-well plates and cultured overnight.
Owing to different infective efficiency, HT-29 and LoVo cells
were then treated with PBS, Ad-GFP, and Ad-hTERT-SSAT
by 50 and 25 MOI. All experiments were performed 6 times.
After 24, 48, 72, 96, and 120 h, 20 µL MTS solution (with
PMS) was added to each well and maintained for 2 h at 37 °C.
The optical density was measured at 490 nm. Cell viability
was assessed by absorbance at 490nm.
Colony-forming assay The HT-29 and LoVo cells were
infected with Ad-GFP or Ad-hTERT-SSAT by 50 and 25 MOI.
After 24 h, the cells were trypsinized, counted, and seeded
for the colony-forming assay on 6-well culture plates
(1×103 cells/well) in triplicate, respectively. After incubation for 14
d, the colonies were stained with Giemsa. Colonies containing
more than 50 cells were scored and the plating efficiency
was calculated.
Exogenous polyamine supplementation
experiment HT-29 and LoVo cells were inoculated at a density of
1×104 cells/well in 6-well plates and cultured overnight. The cells were
then infected with Ad-GFP and Ad-hTERT-SSAT by 50 and
25 MOI. At the same time, the medium of the infected cells
Ad-hTERT-SSAT was supplemented with spermidine (50
µmol/L) or spermine (25 µmol/L), respectively. The medium
was changed daily to keep the concentration of the
exogenous polyamine concentration stable. After 72 h, the cells
were then harvested by trypsinization and stained with 0.4%
trypan blue (Gibco, USA) to reveal the dead cells. Viable
cells were then counted with a hemocytometer.
Statistical analysis Data are presented as the mean±SD
from 3 separate experiments. Student's t-test was used to
compare the data and P<0.05 was taken as the level of
significance. All results were analyzed using the SPSS
statistical software package (Chicago, IL, USA).
Results
Identification of adenoviral backbone DNA
pAdEasy-hTERT-SSAT The SSAT gene was amplified by RT-PCR.
The PCR products were separated by electrophoresis and
an approximately 516 bp band was obtained, which is
consistent with the size of the SSAT gene (gi: 4506788, Figure
1A). A fragment approximately 500 bp was also obtained
after the constructed TA vector was digested (Figure 1A). It
demonstrated that the PCR product was successfully inserted
into pMD-18T.
To identify whether the SSAT gene had been inserted
into the plasmid pGL3-hTERT, the constructed plasmid
pGL3-hTERT-SSAT was digested with NcoI and
XbaI, and 2 fragments (516 bp and 4.5 kb) were found in 1% agarose gel
electrophoresis (Figure 1B). The pGL3-hTERT-SSAT
construct was also digested by SalI and
HindIII, and 2 fragments (1.5 kb and 3.5 kb) were observed (Figure 1B). The
pAdTrack-hTERT-SSAT plasmids were digested by
SalI and HindIII to identify whether the fragment (-hTERT-SSAT-) was
inserted, and 2 fragments (1.5 kb and 8.3 kb) were found
(Figure 1C). These results suggested that the shuttle
plasmid pAdTrack-hTERT-SSAT was successfully constructed.
The recombinant shuttle vector was transformed into
E coli AdEasy-1 cells for homologous recombination with the
pAdEasy-1 vector. Candidate clones were digested with
PacI or BamHI to identify proper recombination. With
PacI digestion, 2 fragments (4.5 kb and 35 kb) were produced
(Figure 1D). With BamHI, a 7 kb fragment was produced in
addition to the 11.7 and 21.7 kb fragments generated from
the pAdEasy-1 sequences (Figure 1D).
Production of the adenovirus in 293
cells In order to package the viruses, 30
µg of digested pAdEasy-hTERT-SSAT plasmids was transfected into 293 packaging cells.
The viral production process was monitored by GFP expression. GFP expression could be observed 24 h after
transfection (Figure 2A), which indicated that the virus
particles had been packaged. The viruses were identified by
PCR with the supernatant fluid using the SSAT primers, and
an approximately 516 bp band was obtained (Figure 3). To
acquire more viruses, the generated viral particles were used
to infect more 293 cells, and a stronger GFP expression was
observed (Figure 2B).
Gene transduction efficiency in
vitro The results (Figure 4A, 4B) showed that there was dose-dependent growth
inhibition in both HT-29 and LoVo cell lines. It reflected the
transduction efficiency of the adenovirus in different cell
lines of colorectal cancer cells. The LoVo cell line was more
sensitive to infection of Ad-hTERT-SSAT. Owing to the
differential sensitivities of various cell lines, we chose 50 and
25 MOI of the adenovirus to infect HT-29 and LoVo cells.
Under these conditions, Ad-hTERT-SSAT was more
suppressive of growth than the control Ad-GFP virus, while Ad-GFP
had no obviously toxic effects on the cells.
Effect of Ad-hTERT-SSAT on SSAT gene expression in
HT-29 cells To detect the effect of replication-deficient
Ad-hTERT-SSAT infection on intracellular SSAT protein levels,
Western blot analysis was performed and the results showed
that SSAT protein levels were significantly increased in
Ad-hTERT-SSAT-treated HT-29 cells when compared with
Ad-GFP or PBS-treated cells (Figure 5).
Inhibitory effect of Ad-hTERT-SSAT on polyamine
content in colorectal cancer cells After proving that
Ad-hTERT-SSAT could increase SSAT protein expression in colorectal
cancer cells, we next evaluated whether the polyamine
concentration decreased accordingly. Polyamines in
adenovirus-infected or -uninfected colorectal cancer cells were separated
by ion-pairing, reverse-phase HPLC. As shown in Table
1, Ad-hTERT-SSAT decreased the content of spermidine and
spermine in HT-29 and LoVo cells, which was correlated with
the acceleration of polyamine catabolism.
Inhibitory effect of Ad-hTERT-SSAT on colorectal
cancer cells We examined the inhibitory effects of
Ad-hTERT-SSAT on the growth of HT-29 and LoVo cells
in vitro using the MTS assay and drew the cell growth curves (Figure 6A,
6B). The expression of SSAT affected the growth of both
HT-29 and LoVo cells. Ad-hTERT-SSAT inhibited their
proliferation when compared with the control groups (treated
with Ad-GFP or PBS).
In the colony-forming assay, plating efficiencies in
HT-29, Ad-GFP-infected HT-29, and Ad-hTERT-SSAT-infected
HT-29 were 65.5%±8.0%, 60.4%±5.6%, and 8.5%±3.2%,
respectively; Plating efficiencies in LoVo, Ad-GFP-infected
LoVo, and Ad-hTERT-SSAT-infected LoVo were 62.5%±9.6%,
58.7%±8.6%, and 30.5%±5.4%, respectively (Figure 7). The
plating efficiency in Ad-hTERT-SSAT-infected cells was
significantly decreased compared with their 2 controls
(P<0.05).
To further examine whether the antiproliferative effect of
Ad-hTERT-SSAT can be antagonized by exogenous
polyamines, spermidine and spermine were added to restore HT-29 and
LoVo cell proliferation. The growth arrest caused by
Ad-hTERT-SSAT was partially reversed by spermidine and
spermine (Table 1).
Discussion
There are many biochemical alterations in colorectal
cancer cells, but one of the most consistent changes is the
elevation of the intracellular polyamine
content[17]. The depletion of intracellular polyamine pools invariably inhibits cell growth.
Although this is usually accomplished by inhibiting polyamine
biosynthesis, this might be more effectively achieved by the
activation of polyamine catabolism at the SSAT level, a
strategy first validated in MCF-7 breast carcinoma
cells[16]. Therefore, the polyamine catabolism pathway may become
another potent and attractive target for cancer therapy.
SSAT is the principle catabolic enzyme responsible for
the regulation of the intercellular polyamine content in
mammalian cells. SSAT transfers an acetyl group from
acetyl-CoA to the N1 positions of spermidine and spermine. The
corresponding N1-acetyl derivatives are either excreted
outwards or undergo further metabolism by polyamine oxidase,
ultimately yielding putrescine, which is a pathway opposite to
that of polyamine synthesis. SSAT is thought to play a key
role in maintaining a properly balanced ratio of polyamine in
cells and in preventing the over accumulation of higher
polyamines that may become
cytotoxic[17,19]. Whereas SSAT activity is normally very low in cells and tissues, the enzyme is
strongly induced by elevations in intracellular polyamines and
in response to terminally-alkylated polyamine analogs that
are currently undergoing clinical trials as cancer
chemotherapeutic agents[20,21]. A number of independent lines of
evidence suggest that non-steroidal anti-inflammatory drugs
(NSAIDs) can prevent colorectal cancer. Martin
et al found that aspirin was able to induce SSAT expression and decrease
intracellular polyamine levels in HT-29 colon adenocarcinoma
cells[22]. They also demonstrated that the induction of polyamine
catabolism leading to the reduction in intracellular polyamines
could be one of the general mechanisms for the chemopreventive
actions of different NSAIDs in colon
cancer[23].
There are a number of viral and non-viral delivery routes
and methods for gene transfer used in gene
therapy[24_27]. Due to its unparalleled capacity for gene transfer, stability,
high titers, and low risk of
mutagenesis[28], adenovirus vectors were chosen in our study to transfer the SSAT gene into
colorectal cancer.
In the present study, we successfully constructed a
replication-deficient recombinant adenovirus that can
simultaneously express the SSAT gene. Western blot analysis
demonstrated that Ad-hTERT-SSAT significantly increased SSAT
protein levels in HT-29 and LoVo cells. Furthermore, with a
substantial increase in SSAT expression, spermidine and
spermine decreased to low levels after Ad-hTERT-SSAT
infection. In addition, our data showed that Ad-hTERT-SSAT
had a significant inhibitory effect on colorectal cancer cell
growth. Polyamines are known to play a key role in
maintaining a high cell proliferation rate. A reduction in
polyamines may contribute to the suppression of cancer growth. The
cell growth inhibition due to Ad-hTERT-SSAT infection can
be partially reversed by exogenous polyamines.
In summary, the experimental data presented here
demonstrate that the adenovirus-mediated expression of SSAT
induces polyamine catabolism and leads to the significant
suppression of colorectal cancer cell growth. Although many
details must be elucidated in its antitumor effect, adenoviral
vector-mediated SSAT provides a novel treatment option
for colorectal cancer.
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