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Polyamines, which are aliphatic compounds, exist in almost all living species and have important physiological functions
in the growth and differentiation of normal cells. In mammary cells, the intracellular polyamine biosynthetic pathway is mainly
regulated by the actions of 2 rate-limiting
enzymes[1]. The first of these, ornithine decarboxylase (ODC), is required for the
first step in polyamine synthesis, in which ornithine is decarboxylated to produce putrescine. The second rate-limiting
enzyme is AdoMetDC, which provides the aminopropyl donor dcSAM by decarboxylasting adenosyl-methionine. DcSAM
donates its propylamine moiety for the formation of spermidine and spermine by spermidine synthase and spermine synthase,
respectively. In addition, exogenous polyamine can also be imported into cells through an as yet unknown transport
mechanism[2].
The association between increased polyamine synthesis and cell proliferation and cancer has been known for
approxi-mately 40 years. High polyamine levels and elevated polya-mine synthetic enzyme activity have been detected in most kinds
of cancers[3_5]. In colorectal cancer, the activities of ODC and AdoMetDC and polyamine content are increased
3- to 4-fold over those found in the equivalent normal
tissue[6,7]. In addition, our earlier study demonstrated that ODC mRNA and protein
levels in colorectal cancer were higher than those in the equivalent normal tissue and, furthermore, they correlated with
Dukes stages[8,9]. Therefore, downregulation of ODC and AdoMetDC expression and depletion of polyamine content in
colorectal cancer with novel methods such as gene therapy has become a focus of research. Consequently, we constructed
a replication-deficient recombinant adenovirus to simultaneously downregulate both ODC and AdoMetDC and detected
whether it could inhibit colorectal cancer cell growth
in vitro.
Materials and methods
Cells and reagents HT-29 human colon cancer cells and HEK293 packaging cells (transformed human embryonic kidney
cells) were obtained from the Chinese Academy of Sciences (Shanghai, China). HT-29 cells were maintained in RPMI-1640
medium supplemented with 10% (v/v) heat-inactivated bovine serum and the antibiotics penicillin G potassium and
streptomycin sulfate in a humidified
CO2 incubator at 37 °C. HEK293 cells were grown in Dulbecco¡¯s modified Eagle¡¯s medium (Gibco
Grand Island, NY, USA ) containing 10% fetal bovine serum and penicillin and streptomycin. The shuttle vector pAdTrack
and Escherichia coli AdEasy-1 cells (E
coli BJ5183 cells transformed with the pAdEasy-1 vector) were provided by Dr Bert
Vogelstein (The John Hopkins Oncology Center, Baltimore, MD, USA) The polyamine standards (putrescine, spermidine,
spermine), internal standards (1,6-hexanediamine) and dansyl
chloride for high performance liquid chromatography (HPLC)
were purchased from Sigma (St Louis, MO, USA). Anti-ODC mouse monoclonal antibody and anti-AdoMetDC mouse
polyclonal antibody were prepared by our laboratory. Plasmid pTrack-ODCas containing a reverse 120 bp ODC gene was
constructed by Dr Yan ZHANG[10].
Amplification of AdoMetDC gene and construction of TA
clone Total RNA was extracted from colorectal cancer tissue
and then reverse transcribed to first strand cDNA from RNA templates by using a cDNA synthesis kit (TaKaRa, Japan).
Synthesized cDNAs were then used as the templates for polymerase chain reaction (PCR) for the amplification of the
AdoMetDC gene. The upstream PCR primer was
5¡¯-GGT CTA GAT TCG CTA GTC TCA CGG TGA T-3¡¯ and the downstream
primer was 5¡¯-GGC TCG AGT AAG CTT CCT GCT TGT CAG T-3¡¯ (enzyme recognition sites are underlined). The amplified
products were purified by using a Qiagen Gel Extraction kit (Qiagen, Germany) and then ligated into the TA cloning vector
(PMD-18T). The positive recombinant plasmid PMD-AdoMetDC was identified by dual digestion with endonucleases
XbaI and XhoI.
Construction of plasmid pTrack-ODC-AdoMetDCas
Both the recombinant TA vector PMD-AdoMetDC and the shuttle
vector pAdTrack-ODCas were digested with
XbaI and XhoI. The digested fragments were then separated with
electrophoresis and the desired bands of 250 bp and 9.3 kb were cut out from the gel and extracted by using a gel extraction kit. The two
purified DNA fragments were ligated with
T4 ligase at 16 °C overnight and then transformed into competent
DH5a cells. The positive clones (pTrack-ODC-AdoMetDCas) and their insert directions were confirmed by digestion and sequencing. The
sequencing primer was 5¡¯-TTC GCT AGT CTC ACG GTG AT-3¡¯.
Construction of pAdEasy-ODC-AdoMetDCas via homologous recombination in
bacteria The pTrack-ODC-AdoMetDCas plasmids were first digested with PmeI and then purified. Five micrograms of purified linearized plasmid was transformed into
highly competent cells AdEasy-1 prepared using
CaCl2 method for homologous recombination with pAdEasy-1. After
growth in L-agar plates containing 50 µg/mL kanamycin for 16_24 h, 10 to 20 of the smallest colonies were picked up and
grown in 2 mL L-Broth containing 50 µg/mL kanamycin overnight. The plasmid minipreps were prepared using a
conventional alkaline lysis method and were digested by
PacI or BamHI for identifying candidate clones that usually yield a large
fragment (approximately 30 kb), plus a small fragment of3.kb or 4.5 kb. The correct recombinant miniprep DNAs
(pAdEasy-ODC-AdoMetDCas) were next re-transformed into the
DH5a and purified using Qiagen¡¯s plasmid midi kit.
Viral production in 293 cells Recombinant adenoviral plasmids (pAdEasy-ODC-AdoMetDCas) were digested with
PacI, ethanol precipitated and resuspended in sterile
H2O. Fifteen micrograms of digested pAdEasy-ODC-AdoMetDCas was
transfected into 293 packaging cells by using standard Lipofectamine 2000 (Gibco) transfection according to the manufacturer¡¯s
instructions. Two handred and ninety three cells were harvested at 7_10 d post-transfection, then
repeatedly frozen at -80 °C and thawed in a
37 °C water bath for total 4 cycles. The samples were spun 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 2 genes ligated into the virus DNA
were also detected using PCR. The titer of purified adenovirus [as measured by green fluorescent protein (GFP) expression]
was 2×1010 pfu/mL[11].
Analysis of gene transduction efficiency in
vitro The efficiency of the adenovirus-mediated gene transfer was assessed
by using GFP. The HT-29 cells were infected with Ad-GFP at multiplicities of infection (MOI) of 5, 10, 20, 50, and 100 for 24 h.
The cells were washed with cold phosphate-buffered saline (PBS), and the GFP-positive cells were quantified using
fluorescence microscopy.
Measurement of ODC and AdoMetDC expression by Western blot
analysis After the HT-29 cells were treated with 50
MOI of PBS, Ad-GFP, and Ad-ODC-AdoMetDCas for 72 h, total cell lysates were prepared in extraction buffer containing 50
mmol/L Tris (pH 8.0), NP-40 (1%), aprotinin (1 µg/mL), sodium dodecylsulfate (0.1%), sodium azide (0.02%), NaCl (150
mmol/L), and phenylmethylsufonyl fluoride (100 µg/mL). Protein from these samples was qualified by using the bicinchoninic acid
assay and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA). After they were reacted with
appropriate antibodies in PBS containing 5% nonfat dry milk and 0.02% Tween-20, blots were incubated with horseradish
peroxidase-conjugated secondary antibodies and exposed to X-ray films (Kodak, Shantou, China) using a Western blotting luminol
reagent (Santa Cruz Biotechnology, USA).
Measurement of polyamine content Polyamine content was measured according to the method described
previous-ly[12]. A total of 1×106
HT-29 cells were treated with PBS, Ad-GFP and Ad-ODC-AdoMetDCas for 3 d and then harvested by
scraping. The cell pellets were permeabilized with 5% trichloroacetic acid for 1 h, and the polyamines in the supernatant were
separated by centrifugation. The precipitates left were measured for protein content. 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 separated and detected using reverse phase HPLC with a C18 column coupled to a scanning fluorescence
detector (excitation wavelength 336 nm, emission wavelength 520 nm). 1,6-Hexanediamine used as an internal standard was
dansylated and separated in the same way as the polyamines.
Measurement of cell growth Viable cell counts were used to observe the effect of adenovirus on cell proliferation. The
HT-29 cells were plated in 6-well tissue culture plates at a density of
5×104 cells/well. After 24 h, tumor cells were treated with
50 MOI Ad-GFP, Ad-ODC-AdoMetDCas or PBS as a mock control. Cells in each treatment group were plated in triplicate and
cultured for 6 d. Then, every 24 h cells were harvested by trypsinization and stained with 0.4% trypan blue (Gibco, USA) to
reveal the dead cells. Viable cells were then counted with hemocytometer.
Statistical analysis Data in this paper 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 by using the SPSS
(version 10.0) statistical software package.
Results
Identification of AdoMetDC gene and TA
clone The AdoMetDC gene was amplified by reverse transcription PCR and
then ligated into the TA clone vector PMD-18T. The PCR products were separated by agarose-gel electrophoresis, and an
approximately 205 bp band was obtained, which is consistent with the size of the AdoMetDC gene (gi: 5209326; Figure 1). A
fragment the same size as the AdoMetDC gene was also produced after the digestion of the constructed TA vector (Figure
1), which suggests that the PCR product was successfully inserted into PMD-18T.
Identification of recombinant shuttle vector pAdTrack-ODC-AdoMetDCas
To identify whether AdoMetDC had been inserted into the constructed shuttle vector, it was digested with
XbaI and XhoI, and 2 fragments (205 bp and 9.3 kb) were
found in 0.8% agarose gel electrophoresis (Figure 2). To identify whether ODC had been inserted, the vector was digested
with BglII and SalI, and 2 fragments (120 bp and 9.3 bp) were obtained (Figure 2). Furthermore, to identify whether AdoMetDC
plus ODC had been inserted, the vector was digested with
BglII and SalI, and 2 fragments (340 bp and 9.3 kb; Figure 2) were
produced. The restriction fragments generated in each case were consistent with the sizes of the inserted genes (ODC and
AdoMetDC) and the pAdTrack vector. These results suggest that the candidate shuttle vector contained both ODC and
AdoMetDC genes. The sequencing results further confirmed the insertion of the genes.
Identification of adenoviral backbone DNA
pAdEasy-ODC-AdoMetDCas The recombinant shuttle vector was
formed into E coli AdEasy-1 cells for homologous recombination with the pAdEasy-1 vector. Candidate clones were
digested with BamHI or PacI to verify proper recombination. The expected restriction fragments were generated in each case.
With BamHI, a 7 kb fragment was produced in addition to the 11.7 and 21.7 kb fragments generated from pAdEasy-1
sequences (Figure 3). With PacI digestion, two fragments (4.5 kb and 35 kb) were produced (Figure 3).
Production of adenovirus in 293 cells To produce the viruses, 15 µg of pAdEasy-ODC-AdoMetDCas was digested with
PacI to liberate the linear adenoviral genomes, and the resulting fragments were then transfected into 293 cells. GFP
transexpression was used to monitor the viral production process. As shown in Figure 4A, GFP expression was visible 24 h
after transfection, which indicated that the virus particles had been packaged. For amplification, the generated viral particles
were used to infect more 293 cells, and stronger GFP expression was found (Figure 4B).
Gene transduction efficiency in
vitro The Ad-GFP vector was used to estimate the gene transduction efficiency in
HT-29 cells. We found that 69±5.1% of HT-29 cells were GFP-positive with an MOI of 50, and there was no obvious toxicity.
Therefore, this MOI was adopted for further studies.
Inhibitory effect of Ad-ODC-AdoMetDCas on ODC and AdoMetDC gene expression in HT-29 cells
Western blot analysis was performed to detect the effect of replication-deficient Ad-ODC-AdoMetDCas infection on intracellular ODC and AdoMetDC
protein levels. Both ODC and AdoMetDC protein levels were significantly decreased in Ad-ODC-AdoMetDCas-treated
HT-29 cells when compared with Ad-GFP or PBS-treated cells (Figure 5).
Inhibitory effect of Ad-ODC-AdoMetDCas on polyamine content in HT-29 cells
After demonstrating that Ad-ODC-AdoMetDCas depressed ODC and AdoMetDC protein expression in HT-29 cells, we next evaluated whether the polyamine
concentration could be decreased accordingly by adenoviral gene transfer into tumor cells. Polyamines in
adenovirus-infected or -uninfected colorectal cancer cells were separated by ion-pairing reverse phase HPLC. Incubation with
Ad-ODC-AdoMetDCas resulted in depletion of all three polyamines to a very low level in HT-29 cells when compared with cells
incubated with Ad-GFP or PBS (Table 1).
Inhibitory effect of Ad-ODC-AdoMetDCas on HT-29 cell
growth Analysis of cell viability revealed significant inhibition
of cell proliferation in colorectal cancer cells treated with Ad-ODC-AdoMetDCas
(P<0.05) when compared with control cells
treated with Ad-GFP or PBS (Figure 6). This cell growth retardation remains for 7 d (data not shown).
Significant morphological changes were also observed 3 d after incubation with recombinant adenovirus. HT-29 cells
infected with Ad-ODC-AdoMetDCas had many vacuoles and particles in the cytoplasm and lost cell-to-cell contacts. In
addition, partial cells rounded up and detached from the plate, which indicated cell death (Figure 7A). In contrast, the
Ad-GFP-infected cells (Figure 7B) didn¡¯t have any obvious morphological changes relative to the uninfected cells (Figure 7C).
To further examine whether the antiproliferative effect of Ad-ODC-AdoMetDCas can be antagonized by exogenous
polyamines, putrescine and spermidine were added to restore HT-29 cell proliferation. The growth arrest caused by
Ad-ODC-AdoMetDCas was not reversed by exogenous putrescine, though which was partially reversed by spermidine (Table 2).
Discussion
There are many biochemical alterations in colorectal cancer cells, but one of the most consistent changes is the elevation
of intracellular polyamine content[13]. Increases in ODC and polyamine content appear in both early and late stages during
the adenoma-carcinoma sequence[14]. Aberrations of tumor tissue metabolism render polyamine biosynthesis an attractive
target for anticancer drug
therapy[15,16]. Blockade of ODC and AdoMetDC enzyme activity by polyamine synthesis inhibitors
such as ODC suicide inhibitor alpha-difluoromethylornithine (DFMO) and AdoMetDC inhibitors
MGBG and SAM486A was found to be effective in arresting tumor cell growth in a preclinical
study[17]. However, due to their low efficiency, general
toxicity and side-effects, convincing antitumor effects were not found in subsequent clinical
studies[18_20]. Hence, the development of novel strategies for the blockade of polyamine synthesis such as gene therapy are necessary.
There are a number of viral and non-viral delivery routes and methods for gene transfer used in gene
therapy[21_24]. Among them, adenovirus-mediated gene transfer deserves particular attention as a means to deliver genes for cancer gene
therapy[25]. Adenoviruses are attractive vectors for cancer therapy because of their unparalleled capacity for gene transfer and
stability in vivo. Adenoviruses have the capacity to transfer genes to a broad spectrum of cell lines, and is not dependent on
active cell division. Additionally, high titers
(>1010 pfu/mL) can be generated, unlike retroviral vectors, which are produced
at relatively low titers (105 to
106 pfu/mL), thus making high MOI easy to achieve. Furthermore, adenovirus DNA is not
integrated into the host genome, and the risk of mutagenesis is low. Adenovirus cannot infect oocytes, therefore no female
germline transduction has been noted. To date, adenoviruses have been used in approximately 27% of the more than 600
gene therapy clinical protocols
reported[26]. Consequently, given this background, we constructed adenovirus vectors to
transfer antisense ODC and AdoMetDC genes to colorectal cancer.
In the present study we successfully constructed a replication-deficient recombinant adenovirus that can simultaneously
express antisense ODC and AdoMetDC genes. Western blot analysis demonstrated that Ad-ODC-AdoMetDCas infection
significantly reduced both ODC and AdoMetDC protein levels in HT-29 cells. Furthermore, with substantial decreases in
ODC and AdoMetDC expression, all 3 intracellular polyamines were depleted to a low level after Ad-ODC-AdoMetDCas
infection.
In addition, our data showed that Ad-ODC-AdoMetDCas infection had a significant effect in suppressing colorectal
cancer cell growth relative to vector infection. 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. Previous studies have shown that
DFMO induced growth arrest across a range of cancer cells, including
bladder[27], breast[28],
intestinal[29],
skin[30] and colon
cancer[31]. However, it is known that cell growth inhibition due to single blockade of ODC (eg by DFMO)
can be rescued by exogenous
putrescine[32]. The reason for this is that environmental putrescine that is
actively taken up by epithelial
cells[33] compensates for impaired putrescine synthesis, and then via AdoMetDC, is rapidly
converted to metabolically active spermidine and spermine. In contrast, cell growth arrest induced by Ad-ODC-AdoMetDCas
was not rescued by exogenous putre-scine. The reason may be the simultaneous inhibition of AdoMetDC by
Ad-ODC-AdoMetDCas, which would impair intracellular spermine biosynthesis and subsequently block DNA
synthesis[34]. Therefore, Ad-ODC-AdoMetDCas would be particularly useful in the treatment of colorectal cancers, which are reportedly "bathed" in
large amounts of luminal
putrescine[33].
In summary, our data provide evidence that adenovirus-mediated expression of both antisense ODC and AdoMetDC
depletes the polyamine pool and leads to significant suppression of colorectal cancer cell growth. Synergistic inhibition of
ODC and AdoMetDC by using a gene therapy approach might therefore represent a novel treatment option for colorectal
cancer.
Acknowledgement
We would like to thank Dr Bert VOGELSTEIN for providing the pAdEasy-1 adenovirus system.
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