The aim of the present study was to investigate the therapeutic potential of a double suicide gene, thymidine kinase (TK) combined with cytosine deaminase (CD), mediated by generation of 5-polyamidoamine dendrimers (G5-PAMAM-D) on human Tenon's capsule fibroblasts (HTFs) as an anti-scarring agent. The pAcGFP1-Hyg-TK-CD plasmid was transfected into HTFs, and reverse-transcription polymerase chain reaction (RT-PCR) was used to detect TK-CD expression. MTT cell proliferation assay was used to evaluate the cytotoxic effects of ganciclovir (GCV) and 5-flurocytosine (5-FC) on HTFs. The optimal concentration of GCV and 5-FC in TK-CD transfected HTFs (HTF-TK-CD) was selected by accessing the lowest and highest cytotoxicity caused, respectively. The morphological changes of transfected HTFs following treatment with GCV and 5-FC were observed by light and transmission electron microscopy. Results demonstrated that the double suicide gene TK-CD mediated by the G5-PAMAM-D delivery system was successfully expressed in HTFs as determined by RT-PCR. A concentration of 3 µg/ml GCV and 200 µg/ml 5-FC was identified as optimal for these prodrugs. The growth rate and number of HTF-TK-CD cells decreased following treatment with GCV and 5-FC as revealed by light microscopy. Additionally, the prodrugs GCV and 5-FC not only demonstrated toxicity on transfected HTFs but also exerted a ‘bystander effect’. The present study illustrated that the double suicide gene TK-CD delivery mediated by G5-PAMAM-D was effective in reducing HTF proliferation and inducing cell apoptosis. Furthermore, TK-CD delivery mediated by G5-PAMAM-D may be used as an anti-scarring agent and provide a therapeutic potential for patients requiring glaucoma filtration surgery.
Glaucoma filtration surgery has been established as an effective therapeutic method for intraocular pressure control (
It has been reported that transfer of the herpes virus thymidine kinase (TK) gene into tumor cells could lead to cell death following administration of a prodrug (
Viral vectors have been mostly used for suicide gene therapy; however, certain limitations remain. For example, retroviruses only transduce a dividing cell, lentiviruses may induce oncogenesis and herpes simplex virus (HSV) may cause inflammation (
HTFs were obtained from Tenon's capsule tissues of 5 patients (age range, 36–61 years; mean age, 47.4±12.3 years; 4 males and 1 female, sample collection between October 2015 and January 2016) undergoing surgery to treat glaucoma at Tianjin Eye Hospital (Tianjin, China). Tissues were prepared for culture in 35 mm Petri tissue-culture dishes that contained Dulbecco's modified Eagle's medium (DMEM; Tianjin Haoyang Biotechnology Co., Ltd., Tianjin, China), supplemented with 10% fetal bovine serum (Tianjin Haoyang Biotechnology Co., Ltd.) Cells were incubated at 37°C in a humidified environment of 95% air and 5% CO2. Following tissue culture, third to sixth generation cells were prepared for experiments. The present study was approved by the Institutional Review Board of Tianjin Eye Hospital (Tianjin, China) and the Human Research Ethics Committee of Tianjin Eye Hospital (Tianjin, China). Patient informed consent was obtained prior to initiation of the study.
The restriction enzymes
The instruments that were used in the present study were as follows: GeneAmp9600PCR (PerkinElmer, Inc., Waltham, MA, USA), OLYMPUS Ix53 optical microscope (Olympus Corporation, Tokyo, Japan), an OLYMPUS IX71 inverted fluorescence microscope and camera system (Olympus Corporation), BD FACS Calibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) and Philips EM400ST transmission electron microscope (Philips Healthcare, Amsterdam, The Netherlands).
G5-PAMAM-D, plasmid pAcGFP1-Hyg-CD and
The TK gene fragment was amplified from the plasmid pMD18-TK by PCR with Probest DNA polymerase (New England Biolabs, Inc.). The thermocycling conditions for the reaction were as follows: 94°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 52°C for 30 sec, 72°C for 90 sec; followed by 72°C for 10 min. The following primers were used: TK-S1, forward primer, 5′-GCT
The TK gene fragment was further amplified by PCR with Probest DNA polymerase. The thermocycling conditions for the reaction were as described above. The following primers were used: TK-S2, forward primer, 5′-CCG
A total of 2.0 µg/ml pAcGFP1-Hyg-TK-CD plasmid and PAMAM-D were mixed (mass ratio, 1:2) and incubated for 30 min at 25°C. Logarithmic growth phase HTFs were trypsinized and plated in 24-well plates (5×104/well). After 24 h when the cell confluency reached 70–75%, cells were divided into experimental (G5-PAMAM-D/TK-CD), positive control (Lipofectamine 2000 transfection and Lipo/TK-CD) and negative control (pAcGFP1-Hyg-TK-CD plasmid alone) groups, and each assay was repeated in triplicate. Lipofectamine® 2000 was purchased from Invitrogen (Thermo Fisher Scientific, Inc.).
After 48 h of transfection, transfection efficiency was assessed by observation of green fluorescent protein expression. Total cell RNA was extracted from transfected HTFs 48 h after transfection with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and then one-step reverse transcription (RT)-PCR was performed using a Transcriptor One-step RT-PCR kit (Roche Diagnostics, Basel, Switzerland), according to the manufacturer's protocol. The thermocycling conditions for the RT reaction were as follows: 42°C for 2 h, 95°C for 5 min, preserved at 20°C. PCR reaction conditions were as follows: 95°C for 5 min, followed by 32 cycles of 94°C for 50 sec, 52°C for 50 sec, 72°C for 1 min, then 72°C for 8 min. The following primers were used: Forward, 5-GGGTCTAGAATGGCTTCGTACCCC-3 and reverse, 5-TCTGTTAACTCAGTTAGCCTCCCCCATCTCCCG-3. The TK-CD PCR product was analyzed by 1% agarose gel electrophoresis and an Image Master Total Lab gel imaging analytical system version 2.0 (TotalLab Ltd., Newcastle upon Tyne, UK) was used to analyze nucleic acid bands.
MTT colorimetric assay was used to evaluate the l
To evaluate the bystander effect of the TK-CD gene, untransfected cells were mixed in an increased proportion of HTF-TK-CD cells at 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%. The cells were then plated into 96-well plates at lx104 cells/well in four replicate groups.
To each well, 3 µg/ml GCV and/or 200 µg/ml 5-FC were added in DMEM. After 72 h at 37°C, the cell inhibition ratio was determined using the MTT colorimetric assay, where the cell inhibition rate (%) was calculated according to the following formula: (1-experimental group OD value/blank control group OD value) x100.
A total of 1×105 HTFs cells or HTF-TK-CD cells were divided into groups A, B, C and D and plated into 6-well culture plates. GCV and/or 5-FC in DMEM media was added as follows: Group A, GCV 3 µg/ml; group B, 5-FC 200 µg/ml; group C, GCV 3 µg/ml and 5-FC 200 µg/ml; and group D, DMEM without prodrug. Each condition was prepared in duplicate. Furthermore, the morphological changes of the cells were observed using an inverted microscope after 24 h (37°C) of prodrug treatment.
For transmission electron microscopy, after 72 h (37°C) of cell culture, cells were washed with PBS three times, trypsinized and collected. Cells were then centrifuged at 251.5 × g (room temperature) for 2 min, washed with 4°C precooled PBS twice, fixed in 4°C precooled 3% glutaraldehyde for 4 h and rinsed with PBS for 10 min three times. Subsequently, cells were fixed with 1% osmium tetroxide for 2 h at room temperature, rinsed with PBS for 10 min three times, dehydrated with 30–100% alcohol and embedded (at 37°C) with SPI-PON 812 resin. Following this, the samples were cut into slices (100 nm thick) with a LKBV-type ultramicrotome, double-stained with 3% uranyl acetate for 3 min and 2% lead citrate for 20 min at room temperature and observed under a transmission electron microscope (magnification, ×3,400 or ×5,700).
Statistical analysis was performed using SPSS version 19.0 (IBM Corp., Armonk, NY, USA). Data were presented as the mean ± standard deviation. The effects of different concentrations of GCV and 5-FC on the two groups were analyzed using linear regression analysis. Differences between the combined and individual treatment groups in cell survival were compared using two-way analysis of variance (ANOVA) for each concentration. The Student-Newman-Keuls (SNK) method was used for pair-wise comparisons and comparisons among various groups were performed using Dunnett's test. P<0.05 was considered to indicate a statistically significant difference.
To verify the constructed plasmid (pAcGFP1-Hyg-TK-CD), the plasmid was digested with
The MTT assay in HTF cells indicated that the cell survival rate significantly decreased as the concentration of GCV and/or 5-FC added increased (all P<0.05;
Therefore, 200 µg/ml of 5-FC was selected for subsequent experiments as this concentration could prevent 5-FC-mediated cytotoxicity and effectively exert an inhibitory effect on the suicide gene. Similarly, 3 µg/ml of GCV was also selected based on the same criteria. In total, 200 µg/ml 5-FC and/or 3 µg/ml GCV represented an optimal concentration and were used in the present study.
The bystander effect of the TK-CD suicide gene was further evaluated. As indicated in
The effect of 5-FC and GCV on HTF cell morphology was accessed by light microscopy. The morphology of HTF cells following treatment with 3 µg/ml GCV and/or 200 µg/ml 5-FC was comparable to non-treated HTF cells (
The ultrastructure of the HTF cells was further evaluated using a transmission electron microscope. As indicated in
Suicide gene therapy is a gene-directed enzyme prodrug therapy that is based on the introduction of a viral or bacterial gene that encodes a metabolic enzyme into target cells, which allows the conversion of a non-toxic compound into a lethal drug, causing the death of target cells (
It has been reported that suicide gene therapy may inhibit HTF proliferation when delivered using a viral vector, more evidently for single suicide gene transfection (
Double suicide gene therapy represents the genetic integration of two types of suicide genes that express a fusion gene product with a double gene encoding enzyme activity in the target cell (
In the present study, by observing HTF cells transfected with TK-CD and exposed to the prodrug GCV and 5-FC, deterioration of cell growth and shrinkage were observed, as well as a reduction in the number of cells. For the GCV and 5-FC combination compared with prodrug alone, cell growth was diminished and there were fewer cells left following prodrug treatment. HTF-TK-CD cell nuclear chromatin margination, cytoplasmic condensation and cell deformation were observed by transmission electron microscopy. Additionally, typical apoptotic bodies were visible while the cell membrane was intact, indicating that the cells were in an ongoing apoptotic process. Altogether, these observations indicated that the TK-CD double suicide gene system could initiate apoptosis in HTF cells.
While G5-PAMAM-D-mediated gene transfection efficiency requires further improvement, the notable characteristic of the suicide gene/prodrug system is the existence of a ‘bystander effect’, whereby the transduction of a small fraction of target cells with the suicide gene may result in widespread target cell death, including non-transduced cells, causing exacerbation of the lethal effect. A preliminary study of the mechanism of the ‘bystander effect’ was reported previously (
In summary, the present study demonstrated that the G5-PAMAM-D nanoparticle-mediated double suicide gene TK and CD system inhibited HTF cell proliferation
The present study was supported by Tianjin Municipal Science and Technology Commission (grant no. 13JCYBJC21500).
TK and CD gene expression in HTF cells. (A) The plasmid map showing the expression vector, pAcGFP1-Hyg, carrying the TK and CD genes. (B) The 1,617 bp fragment of the TK-CD gene was confirmed by restriction enzyme digestion of the plasmid with
Effect of 5-FC and GCV on cell survival rate (%) of HTF and HTF-TK-CD cells was evaluated by adjusting the concentration of prodrugs in the MTT assay. 5-FC, 5-flurocytosine; GCV, ganciclovir; HTFs, human Tenon's capsule fibroblasts; TK, thymidine kinase; CD, cytosine deaminase.
Effect of 5-FC and GCV on the inhibition of cell proliferation in HTF cells mixed with an increased number of HTF-TK-CD cells. Proliferation was expressed as a percentage of the inhibition for HTFs transfected with the TK-CD gene (control group, set as 100%) *P<0.05 vs. control group, by Dunnett's test (n=4). 5-FC, 5-flurocytosine; GCV, ganciclovir; HTFs, human Tenon's capsule fibroblasts; TK, thymidine kinase; CD, cytosine deaminase.
Effect of 5-FC and GCV on HTF cell morphology was accessed by light microscopy. (A) Normal HTF cells demonstrated typical array polarity (magnification, ×100). (B) HTF cells treated with GCV, 5-FC or GCV/5-FC (magnification, ×100) (C) HTF-TK-CD cells treated with GCV, 5-FC or GCV/5-FC (magnification, ×100). 5-FC, 5-flurocytosine; GCV, ganciclovir; HTFs, human Tenon's capsule fibroblasts; TK, thymidine kinase; CD, cytosine deaminase.
Effect of 5-FC and GCV on HTF cells was accessed by transmission electron microscopy. (A and B) Ultrastructure of normal HTFs. (C and D) Larger regular chromatin ball typical for apoptosis was observed in HTFs-TK-CD treated with the prodrug for 72 h (apoptotic body, black arrows). Scale bar, 2 µm. Cells were double-stained with 3% uranyl acetate and lead citrate. Magnification: A, ×3,400; B, ×5,700; C, ×3,400; D, ×3,400. 5-FC, 5-flurocytosine; GCV, ganciclovir; HTFs, human Tenon's capsule fibroblasts; TK, thymidine kinase; CD, cytosine deaminase.