The herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) and the cytosine deaminase/5-fluorocytosine (CD/5-FC) systems have been widely applied in suicide gene therapy for cancer. Although suicide gene therapy has been successfully used
The cytosine deaminase/5-fluorocytosine (CD/5-FC) and the thymidine kinase/ganciclovir (TK/GCV) are the most common suicide gene therapy systems (
Recombinant adenoviral (Ad) vectors have been widely used as a gene delivery vehicle, since they can efficiently transfer genes into a wide spectrum of cell types at a high efficiency
In the present study, we investigated whether the suicide gene fusion
The human embryonic kidney epithelial 293 (HEK-293) and the breast cancer MCF-7 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). As previously described (
The recombinant Ad carrying the
Four million MCF-7 cells were inoculated in each well of 6-well plates. Cultures were maintained for 12 h, and cells were then infected with Ad-VEGFp-CD/TK at multiplicities of infection (MOI) of 20, 40, 60, 80, 100 and 200 pfu/cell, for 24 h. The number of GFP-positive cells was counted under an inverted fluorescence microscope (Leica, Mannheim, Germany).
The experimental group was infected for 24 h with the adenoviral vector at MOI 100. The control group was cultured in DMEM for 24 h. One day later, the cells in the two groups entered the logarithmic growth phase. The morphological changes of MCF-7 cells were examined under a phase contrast light microscope (Leica, Mannheim, Germany).
MCF-7 cells were incubated overnight in a 75-ml cell culture bottle. Then, the experimental group was incubated with the adenoviral vector Ad-VEGFp-CD/TK for 72 h at 37°C with 5% CO2. The control group was cultured in DMEM for 72 h. The cultured cells were harvested using trypsin and centrifuged for 10 min at 2,000 × g at room temperature. The pellets were next fixed overnight in 3% (v/v) glutaraldeyde at 4°C. The specimens were washed in phosphate-buffered saline (PBS) and post-fixed in 1% osmium tetraoxide for 20 min. Then, the specimens were dehydrated in a graded series of acetone dilutions. The area of interest in the resin block containing the embedded cells was selected using toluidine blue staining (Polysciences, Warrington, PA, USA), and later examined under a light microscope. Ultrathin sections of the selected area were performed using a Leica EM UC7 ultramicrotome (Leica). The stained samples were then observed using TEM (Philips, Eindhoven, The Netherlands). The nucleus-to-cytoplasm ratio was evaluated using the ratio of the volume size of the cell nucleus to the volume size of the cell cytoplasm.
MCF-7 cells were inoculated on 90-mm dishes and transduced with recombinant Ads for 24 h using the protocol of adenovirus vector infection. Cells were then lysed using lysis buffer (50 mM Tris/pH 8.0, 150 mM NaCl, 1% (w/v) Triton-X-100, 0.1% (w/v) SDS, 1% (w/v) sodium deoxycholate) and proteins were extracted from the MCF-7 cell lysate by centrifugation (Sigma, Deisenhofen, Germany) at 14,000 × g for 10 min. The extracted proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred onto a polyvinylidene fluoride membrane. Subsequently, the membrane was incubated with 30 g/l non-fat milk, and next, with sheep anti-CD (Biogenesis, Poole, UK) or goat anti-TK (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) antibodies overnight at 4°C. After a wash in Tris-buffered saline with Tween-20 (TBST), horseradish peroxidase-labeled rabbit anti-sheep or anti-goat IgG was added as the secondary antibody (Santa Cruz Biotechnology, Inc.), and incubated at room temperature for 1 h. The membrane was washed with TBST, and incubated with an enhanced chemiluminescence substrate (ECL; Merck KGaA, Darmstadt, Germany) for 1 min. Finally, the membrane was developed on an X-ray film (Fujifilm, Tokyo, Japan).
Transfected MCF-7 cells were inoculated on 24-pore plates at a density of 1×104/pore. The control group was untransfected MCF-7 cells. Three parallel pores were assayed for each group. MCF-7 cells were observed and counted each day for 7 consecutive days in order to establish the growth curve.
FCM analysis was used to assess the distribution of MCF-7 cells at the different cell cycle stages, as in (
The experimental data were processed with the SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Data were expressed as mean ± standard error of the mean (SEM). An independent samples t-test was used to compare the means between two groups. P<0.05 was considered to indicate statistically significant differences.
We estimated the transduction efficiencies of the adenoviral-mediated gene transfer in human breast cancer cells infected at different MOI of the Ad vector. As the MOI increased, the percentage of infected cells also increased (
MCF-7 cells were treated with the adenoviral vector at MOI 100 for 24 h. The infection toxicity was then evaluated by observations of the cell morphology (
As revealed by TEM, the ultrastructure of the transfected MCF-7 cells (
The expression of the CD and TK proteins was analyzed by western blot analysis. The results showed that the CD and TK proteins are not expressed in the control group, while they were strongly expressed in the experimental group (
MCF-7 cells were observed under a light microscope following an additional 72-h incubation with the adenoviral vector. The morphology of MCF-7 cells of the experimental group was similar to that of the control group. The growth of cells became relatively slow at one to two days after infection, while growth rates increased after two days. An increasing number of cells became cell growth arrested and eventually senescent from the fifth day onwards. Cell proliferation of the experimental group was reduced compared to the control group (
The proportion of cells at the different phases of the cell cycle is shown in
Adenoviral vectors are popular gene delivery vectors in clinical trials for gene therapy (
The VEGF protein has been shown to be upregulated in numerous types of cancer (
This study indicated that infection with the Ad-VEGFp-CD/TK vector exerts no prominent effect on cell proliferation in the human breast cancer cell line MCF-7 at a MOI of 100. Therefore, MOI 100 was selected as the working concentration. We found that infected MCF-7 cells have a lower growth rate than the uninfected cells. However, there was no significant difference (P>0.05) in cell proliferation between these two groups (
In summary, we achieved high-efficiency transduction of MCF-7 cells by a VEGF promoter-based adenoviral vector, and stable expression of the CD and TK proteins
This study was supported in part by the Shenzhen Council for Scientific and Technological Innovation grant JCYJ20130402151227177, and the Shenzhen Nanshan District Science and Technology Project grant 2012025.
Percentage of infected cells based on multiplicities of infection (MOI).
Morphology of MCF-7 cells prior to and following infection with the adenovirus, as observed under a phase contrast microscope (magnification, ×200). (A) Non-transfected cells and (B) experimental group of cells transfected with the adenoviral vector for 24 h.
Ultrastructural features of MCF-7 cells prior to and following infection with the adenovirus, as observed under an electron microscope (magnification, ×8,000). (A) Control group, untransfected cells and (B) experimental group, cells transfected with the adenovirus for 72 h.
Western blot analysis shows that cytosine deaminase (CD) and thymidine kinase (TK) are expressed in protein extracts prepared from MCF-7 cell lysates of infected MCF-7 cells, but not of control cells.
Growth curves of MCF-7 cells and of MCF-7 cells transfected with the adenoviral vector (MCF-7/CDTK).
Changes in the cell cycle of (A) untransfected and (B) transfected MCF-7 cells, as examined by flow cytometry.
Number of MCF-7 cells at different days of infection (mean ×105 ± SD, n=3).
Days of infection | |||||||
---|---|---|---|---|---|---|---|
| |||||||
Group | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
MCF-7 | 1.56±0.14 | 2.80±0.26 | 5.97±0.58 | 23.99±2.92 | 30.32±2.95 | 27.44±2.58 | 23.45±2.09 |
MCF-7/CDTK | 1.31±0.11 | 2.56±0.19 | 5.01±0.27 | 18.90±2.35 | 25.35±2.14 | 22.51±1.92 | 19.64±1.67 |
t | 2.463 | 1.265 | 2.574 | 2.349 | 2.363 | 2.659 | 2.469 |
P | 0.069 | 0.274 | 0.062 | 0.079 | 0.077 | 0.056 | 0.069 |
SD, standard deviation; MCF-7/CDTK, MCF-7 cells infected with the adenoviral vector; t, a parameter based on an analysis of t-test; P>0.05, between the MCF-7/CDTK group and the MCF-7 group.
Cell cycle changes in MCF-7 cells following infection with the adenoviral vector (mean% of cells ± SD, n=3).
Group | G0-G1 | G2-M | S |
---|---|---|---|
Control | 77.03±3.27 | 7.89±1.43 | 15.01±1.41 |
Transfected | 73.55±7.34 | 10.77±1.66 | 15.46±1.53 |
t | 0.749 | 2.279 | 0.375 |
P | 0.496 | 0.085 | 0.727 |
SD, standard deviation; control; untransfected cells; t, a parameter based on an analysis of t-test; P>0.05 between the transfected group and the control group.