Abstract
Background: The transduction efficacy of adenovirus serotype 5 (Ad5) vector in high-grade human bladder cancer cells is generally extremely low due to the non-expression of coxsackie and adenoviral receptor (CAR). We investigated whether fiber-modified adenovirus vector containing an RGD motif in the HI loop of the adenovirus fiber knob could increase the transduction efficiency of Ad5 into human bladder cancer cells in vitro. Materials and Methods: We examined the expressions of CAR, and of αv, β3 and β5 integrin, and the transduction efficacy of fiber-modified adenovirus vector in four human bladder cancer cell lines (TCC-SUP, 253J, T24 and KK47). Results: The expression of CAR was lower and those of αv and β3 integrin were higher in four human cancer cell lines compared with the control cell line, KK47. The transduction efficacy of fiber-modified adenovirus vector increased by 20- to 470-fold compared with Ad5. Conclusion: Fiber-modified adenovirus vector may be useful in order to establish new effective gene therapy strategies for the treatment of high-grade human bladder cancer.
Adenoviruses are useful vectors for cancer gene delivery because of the high gene transfer efficacy, high titer production, and safety (1, 2). Recombinant adenovirus 5 (Ad5) has been widely used in gene transfer experiments and clinical gene therapy. Entry of Ad5 into the host cell is initiated by the knob domain of the fiber protein binding to the cell receptor, coxsackie and adenoviral receptor (CAR) (3). This is followed by a secondary interaction, where an Arg-Gly-Asp (RGD) motif in the penton base interacts with an αv-containing integrin, particularly αvβ3 and/or αvβ5 integrin. Binding to αv integrin results in endocytosis of the virus particle via clathrin-coated pits (4). However, the loss or decrease of CAR expression has been observed in various types of cancer, including bladder cancer (5-7). In an effort to target bladder cancer one needs to either increase the expression of CAR or to increase the transduction efficacy of Ad5 vector using CAR-independent tropism. Mizuguchi and Hayakawa have developed vectors with improved tropism by altering the fiber protein (8).
Fiber-modified adenovirus vector containing an RGD motif in the HI loop of the fiber knob (Ad5RGD vector) is capable of CAR-independent tropism in target cell expressing αvβ3 or αvβ5 integrin on the membrane (9). We showed that the Ad5RGD vector is capable of inducing much higher transduction efficacy for human renal cell carcinoma cells than the Ad5 vector (10)
In the present study, we examined the transduction efficacy of the Ad5 and Ad5RGD vectors in several human bladder cancer cell lines.
Materials and Methods
Cell lines and cell culture. Established cell lines derived from human bladder carcinoma cell lines, namely, TCC-SUP, 253J and T24, were obtained from the American Type Culture Collection (Manassas, VA, USA); the KK47 human bladder cancer cell line was generously provided by Dr. Seiji Naito (Department of Urology, Kyushu University, Fukuoka, Japan). In the present study, we maintained TCC-SUP, 253J, T24 and KK47 cells in Roswell Park Memorial Institute-1640 medium (Life Technologies, Inc., Gaithersburg, MD, USA), containing 10% fetal bovine serum and antibiotics (50 μg/ml streptomycin sulfate and 50 IU/ml of penicillin). All cell lines were maintained at 37°C in a humidified incubator with an atmosphere of 5% CO2 and 97% relative humidity, and were subcultured on reaching 80% confluence using trypsin-EDTA. The cells were transferred two or three times a week into fresh growth medium.
Adenovirus vector preparation. We examined the transduction efficacy of Ad5-LacZ, constructed as previously described (11), and of Ad5RGD-LacZ, containing an RGD peptide in the HI loop of the fiber knob (9, 10) (Figure 1). The viruses were purified by double cesium chloride gradient ultracentrifugation using standard methods. Serial dilutions of the viruses were used to infect HEK 293 cell (RIKEN Bioresource Center, Tsukuba, Japan) for a plaque assay. Titers of adenovirus vectors were assessed using the 50% tissue culture infectious dose method and were expressed as plaque-forming units (pfu)/ml (Ad5-LacZ, 3.6×1011 pfu/ml; Ad5RGD-LacZ, 1.1×1011 pfu/ml).
In vitro real-time quantitative reverse transcription-PCR assay. Total cellular RNA was isolated from all cell lines using a TaKaRa RNA extraction KIT (Takara Bio Inc., Shiga, Japan), and was reverse transcribed using a reverse transcription kit (TaKaRa RNA PCR Kit Ver. 3.0), following the manufacturer's protocol. The resulting cDNA was amplified with CAR, αv integrin, β3 integrin, β5 integrin, and glyceraldehyde -3-phosphate dehydrogenase (GAPDH) sequence-specific primers (40 cycles: 95°C for 15 s, 60°C for 1 min) using TaqMan chemistry in the StepOnePlus Real-Time PCR System v2.0 (Applied Biosystems Japan Ltd., Tokyo, Japan). Table I shows the sequences of the TaqMan probes and primers for CAR, αv integrin, β3 integrin, β5 integrin, and GAPDH. All primers and probes were purchased from Biosearch Technologies Japan (Tokyo, Japan).
Transduction efficacy of adenovirus vectors. In order to determine the transduction efficacy in each cell line, 2.5×104 cells were prepared in a 24-well plate and infected with Ad5-LacZ or Ad5RGD-LacZ. After 48 h, the transduction efficacy was assessed by β-galactosidase (β-gal) staining and expressed as blue titer units (btu)/ml.
Statistical analysis. Statistical significance was determined by using analysis of variance (ANOVA) and Bonferroni correction, with p<0.01 considered to be statistically significant.
Results
Relative quantification of mRNA expression of CAR. The mean relative quantification of CAR mRNA expression detected in the cell lines used in this study, is shown in Figure 2. In order to normalize for differences in the amount of total RNA, GAPDH was used as an endogenous RNA control. The relative quantification was calculated by dividing by the value of RNA obtained for T24 cells. The levels of CAR mRNA expression were considerably higher in KK47 cells compared with the other cell lines.
Relative quantification of mRNA expressions of αv, β3 and β5 integrins. The mean relative quantifications of αv integrin, β3 integrin and β5 integrin mRNA expression detected in the cell lines used in this study are shown in Figure 3. The relative quantification was calculated by dividing by the value obtained for T24 cells. αv and β5 integrin mRNA were found to be uniformly expressed among the four cell lines. β3 Integrin mRNA expression in 253J cells was found to be approximately four-fold higher than that of T24 and TCC-SUP cells. On the other hand KK47 was found to have minimal expression of β3 integrin mRNA.
Transduction efficacy of adenovirus vectors. In order to assess the transduction efficacy in all cell lines, cells were infected with both Ad5-LacZ and Ad5RGD-LacZ. The transduction efficacy for each cell line was significantly increased by 470-, 20-, and 23-fold in T24, TCC-SUP, and 253J cells, respectively, using the RGD-bearing adenovirus, compared with the Ad5-LacZ (p<0.01) (Figure 4).
Discussion
Ad5 vectors are one of the most studied vectors for gene therapy, as safety data for Ad5 have been excellent. However, the main disadvantage of the current therapies is that low transduction efficacy of Ad5 vectors limits the efficacy of treatment. Thus for successful cancer gene therapy, transduction efficacy of Ad5 vectors needs to be improved. In the present study, we attempted to increase the transduction efficacy of adenovirus vectors in bladder cancer cells.
Our present results revealed that the transduction efficiency in each of the bladder cancer cell lines tested, paralleled the relative quantification of CAR mRNA expression. To increase transduction efficacy of adenovirus vectors, we tested the Ad5RGD vector. Indeed, our data revealed that the fiber-modified Ad5RGD vector achieved significantly higher transduction levels in all human bladder cancer cells as compared to the Ad5 vector itself. Oncolytic adenoviruses are being considered as a new therapeutic option for treatment of refractory disseminated cancer, including bladder cancer. Previously we demonstrated an antitumor effect in KK47 cells both in vitro and in vivo using an oncolytic Ad5 vector containing the E1a gene, controlled by the tumor-specific midkine promoter (12). However, the antitumor effect was considerably lower in T24 cells compared with KK47 cells in vitro. One reason for such a result for T24 cells is the low transduction efficiency of Ad5 vector into these cells because of their low CAR expression. Therefore, our results also suggest that if we can construct an oncolytic Ad5 virus containing the E1a gene controlled by midkine promoter, which has an inserted RGD motif, it may be possible to achieve a higher antitumor effect in bladder cancer cells with low CAR expression, including T24 cells, compared with gene therapy using a conventional Ad5 vector.
In this study, we demonstrated a dramatic increase in transduction efficacy in bladder cancer cells using an adenovirus vector containing the RGD motif on the HI loop of the fiber knob. Therefore, it may be preferable to use the fiber-modified adenovirus vector described in this study to target bladder cancer cells, and by applying our findings, it may be possible to establish new effective gene therapy strategies for the treatment of bladder cancer.
Acknowledgements
This study was supported by Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science. The Authors wish to thank Satoko Kodama for her help in the completion of the manuscript.
- Received March 30, 2012.
- Revision received May 14, 2012.
- Accepted May 15, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved