- Email: [email protected]
Fiber-substituted Conditionally Replicating Adenovirus Ad5F35 Induces Oncolysis of Human Bladder Cancer Cells in In Vitro Analysis
Basic and Translational Science Fiber-substituted Conditionally Replicating Adenovirus Ad5F35 Induces Oncolysis of Human Bladder Cancer Cells in In Vitro Analysis Akinobu Gotoh, Hisao Nagaya, Takeshi Kanno, Masatoshi Tagawa, and Tomoyuki Nishizaki OBJECTIVE
MATERIALS AND METHODS
RESULTS
CONCLUSION
To assess the oncolytic effect of fiber-substituted conditionally replicating adenovirus type 5 (Ad5) F35 vector on human bladder cancer cell lines such as 253J, 5637, KK-47, T24, TCCSUP, and UMUC-3 cells. Ad5F35 and Ad5 conditionally replicating adenovirus vectors containing the E1 gene controlled by the human midkine promoter (Ad5F35/MKp-E1 and Ad5/MKp-E1, respectively) were constructed. Reverse transcriptase-polymerase chain reaction and cell viability assay were performed in cells transfected with Ad5F35/MKp-E1 or Ad5/MKp-E1. Of the bladder cancer cells used, considerably lower expression of mRNA for Coxsackie and adenovirus receptor, an Ad5 receptor, was found with T24 and TCCSUP cells. However, the mRNA for CD46, an Ad35 receptor, was abundantly expressed in all the cell types. Ad5F35/ MKp-E1 induced oncolysis in a plaque formation unit-dependent manner for all the bladder cancer cells used, with greater efficacy than Ad5/MKp-E1 for T24, TCCSUP, and 253J cells. The results of the present study have shown that Ad5F35/MKp-E1 is more useful for the gene therapy of bladder cancer than Ad5/MKp-E1 is for some cell lines. UROLOGY 81: 920.e7e920.e11, 2013. 2013 Elsevier Inc.
A
n increasingly broad spectrum of molecules and pathways suitable for novel targeted therapies for bladder cancer has been identified from accumulating studies about the molecular mechanisms that drive urothelial transformation and the growth, invasion, and metastasis.1 Despite the huge clinical challenges, lesser improvement for the 5-year survival rate of bladder cancer has been obtained.2 Gene therapy using adenoviral vectors has been attempted in a variety of cancers.3 Less beneficial result than expected, however, has been obtained because of the limited gene transfer into solid tumor cells.4,5 In clinical tests, the safety and oncolytic effect of the first generation of adenovirus vectors have been confirmed, but the efficacy has been modest for the context of Financial Disclosure: The authors declare that they have no relevant financial interests. From the Laboratory of Cell and Gene Therapy, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan; Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, Nishinomiya, Japan; and Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chuo-ku, Chiba, Japan Reprint requests: Tomoyuki Nishizaki, M.D., Ph.D., Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501, Japan. E-mail: [email protected] Submitted: June 7, 2012, accepted (with revisions): December 12, 2012
920.e7
ª 2013 Elsevier Inc. All Rights Reserved
advanced tumors.3 Conditionally replicating adenovirus (CRAD) has been developed to reduce the side effects and obtain more beneficial efficacy.6,7 CRAD enables the gene therapy to induce tumor-specific cell death and amplify the oncolytic effect by replicating and spreading the surrounding cells.8-11 The CRAD vector used in the present study contains the E1 gene controlled by the midkine promoter. Midkine, a secreted heparin-binding growth factor, was identified as a product of the retinoic acid response gene, and it is expressed in a variety of cancer cells more highly than in normal cells.12 The CRAD vector has been shown to induce oncolysis of cancer without damaging normal liver.13,14 Adenovirus type 5 (Ad5) has been widely used for gene therapy.15 Ad5 attachment to target cells is mediated by the Coxsackie and adenovirus receptor (CAR). Low CAR expression in cancer cells is a large obstacle for gene therapies using Ad5.16 In contrast, adenovirus type 35 (Ad35) is infected into cells through the receptor CD46, which is abundantly expressed in cancer cells.17 We have earlier found that chimeric adenovirus Ad5F35 with substitution of the fiber knob on Ad5 by that on Ad35 exhibits a beneficial oncolytic effect on human renal carcinoma cells with low expression of CAR by 0090-4295/13/$36.00 http://dx.doi.org/10.1016/j.urology.2012.12.023
Table 1. Primers used for reverse transcriptase-polymerase chain reaction PCR Primer Midkine CAR CD46 GAPDH
Oligonucleotide Sequence 0
Sense: 5 -GCTTCCTCCTCCTCACCTC-30 Antisense: 50 -TCGCCTGCTGGCGCTCA-30 Sense: 50 -CAGAAGCTACATCGGCAGTAATCA-30 Antisense: 50 -CTCTGAGGAGTGCGTTCAAAGTC-30 Sense: 50 -GGTGTTGCTGCTGTACTCCTTCT-30 Antisense: 50 -CCAATGAGCTCCATAGCTTCAA-30 Sense: 50 -GAAGGTGAAGGTCGGAGTC-30 Antisense: 50 -GAAGATGGTGATGGGATTTC-30
CAR, Coxsackie and adenovirus receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PCR, polymerase chain reaction.
improving the infection efficacy into cells.18 The present study examined the oncolytic effect of Ad5F35 on human bladder cancer cells. We found that Ad5F35 could be used as an efficient tool for gene therapy of bladder cancer.
MATERIAL AND METHODS Cells and Cell Culture The human bladder cancer cell lines used in the present study were 253J, 5637, KK-47, T24, TCCSUP, and UMUC-3 cells. KK-47 was generously provided by Dr. Seiji Naito (Department of Urology, Kyushu University, Fukuoka, Japan), and TCCSUP, T24, 253J, 5637, UMUC-3, and HEK293 human embryonic kidney cells were purchased from the American Type Culture Collection (Manassas, VA). The bladder cancer cells and HEK293 cells were grown in Roswell Park Memorial Institute-1640 (Life Technologies, Gaithersburg, MD) and Eagle minimum essential medium (Invitrogen, Carlsbad, CA), respectively, supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, penicillin (final concentration 100 U/mL), and streptomycin (final concentration 0.1 mg/mL), in a humidified atmosphere of 5% carbon dioxide and 95% air at 37 C.
Construction of CRAD Vector The CRAD vector containing the E1 gene under the control of the human midkine promoter was constructed by the following method. The cytomegalovirus promoter of the pShuttle2 vector (Clontech Laboratories, Mountain View, CA) was replaced with the 0.6-kb 50 -upstream regulatory region of the midkine gene and pS-MK/E1 vector was produced by inserting the E1 gene downstream of the midkine region. To construct pAd5F35, the Ad5 fiber region on the pAdeno-X vector (Clontech Laboratories) was substituted with the Ad35 fiber knob region on the RHSP vector (Avior Therapeutics, Seattle, WA). Ad5/MKp-E1 and Ad5F35/MKp-E1 were prepared by linking the pS-MK/E1 vector with pAdeno-X and pAd5F35, respectively. Adenovirus vectors were amplified in HEK293 and purified with Vivapure AdenoPACK (Sartorius AG, Goettingen, Germany). The number of virus particles/mL was estimated using the following formula: absorbance at 260 nm for purified adenovirus in the presence of 0.1% sodium dodecyl sulfate 1.1 1012.19 Determination of the infection unit (plaque formation unit [PFU]/mL) was accomplished using an Adeno-X titer kit. The PFU/virus particle ratio was 6.3 and 45.6 for Ad5 and Ad5F35, respectively. UROLOGY 81 (4), 2013
Reverse Transcriptase-Polymerase Chain Reaction Reverse transcriptase-polymerase chain reaction was performed using the primers listed in Table 1 and a previously described method.20 The expression levels for each mRNA were normalized by the levels for glyceraldehyde 3-phosphate dehydrogenase mRNA.
Oncolysis Assay Cells (4 104) were infected with Ad5/MKp-E1 or Ad5F35/ MKp-E1 at 0.01-100 virus particles/cell. At 5 days after infection, the cells were incubated with an Alamar Blue (Life Technologies), and the number of viable cells was counted at an absorbance of 560 nm with a high-throughput microplate spectrophotometer (SpectraMax Plus384, Molecular Devices, Sunnyvale, CA).
RESULTS Expression of Midkine, CAR, and CD46 Midkine mRNA was expressed in all the bladder cancer cells, with the lowest expression for UMUC-3 cells, and the expression levels tended to be lower than for the HEK293 cells (Fig. 1A,B). Of the bladder cancer cells used, considerably lower expression of the CAR mRNA was found with T24 and TCCSUP cells (Fig. 1A,C). However, the CD46 mRNA was abundantly expressed in all the cell types, with the expression levels mostly greater than for HEK293 cells (Fig. 1A,D). Oncolytic Effects of Ad5/MKp-E1 and Ad5F35/MKpE1 For the HEK293 cells, Ad5F35/MKp-E1 reduced cell viability in a PFU-dependent manner to an extent similar to that for Ad5/MKp-E1 (Fig. 2A). For the bladder cancer cell lines, Ad5F35/MKp-E1 reduced cell viability in a PFU-dependent manner to an extent apparently greater than Ad5/MKp-E1 for T24, TCCSUP, and 253J cells (Fig. 2B,E,F). For 5637, KK-47, and UMUC-3 cells, Ad5F35/MKp-E1 exhibited an oncolytic effect, with similar or slightly lesser efficacy compared with that for Ad5/MKp-E1 (Fig. 2C,D,G).
COMMENT Midkine is abundantly expressed in a variety of cancer cells originating from the esophagus, stomach, colon, 920.e8
Figure 1. Expression of mRNA for midkine, Coxsackie and adenovirus receptor (CAR), and CD46. (A) Reverse transcriptasepolymerase chain reaction products shown. Signal intensities for each mRNA sample normalized by intensities for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. Each column represents mean standard error of mean normalized expression of mRNA for (B) midkine, (C) CAR, and (D) CD46 (n ¼ 4 independent experiments).
liver, breast, and pancreas. Also, the expression in nonmalignant cells is quite limited, with moderate expression in the kidney and weak expression in the lung, colon, and thyroid gland.21-25 The midkine promoter, accordingly, could be used for cancer gene therapy. A similar or lower expression of midkine mRNA was found with all bladder cancer cells compared with the expression for HEK293 cells. We constructed the Ad5F35 CRAD vector encoding the E1 gene under the control of the 0.6-kb midkine promoter (Ad5F35/MKp-E1). Ad5 and Ad35 infect into cells by targeting CAR and CD46, respectively. Studies have shown that expression of CAR disappears in aggressive bladder cancer26 and decreases as an early event in bladder carcinogenesis.27 In contrast, CD46 expression increases in bladder cancer cells.28 In the present study, the bladder cancer cells, except for the T24 and TCCSUP cells, expressed CAR mRNA, with the quantity similar to, or greater than, that in the HEK293 cells. The bladder cancer cells, except for the UMUC-3 cells, however, expressed CD46 mRNA more highly than did the HEK293 cells. This indicates that Ad35 tends to more easily infect human bladder cancer cells than does Ad5. Receptors that mediate attachment of the adenovirus fiber knobs to the host cell have been shown.29 Species A 920.e9
and C-F adenoviruses such as Ad5 and Ad12 interact with the receptor CAR. The Ad5 knob and the CAR D1-2 domains form a complex. Species B adenoviruses such as Ad11 and Ad35 attach to CD46. The Ad35 knob and the CD46 short consensus repeat 1-4 domains form a complex. Species D adenoviruses such as Ad37 attach to GD1a, which is linked to an unidentified glycoprotein. Species B adenoviruses such as Ad3, Ad7, and Ad14 attach exclusively to DSG-2. In the present study, Ad5F35/MKp-E1 reduced cell viability in a PFU-dependent manner for all the bladder cancer cells, with the potential greater than Ad5/MKp-E1 did for T24, TCCSUP, and 253J cells. The profound effect was obtained with T24 and TCCSUP cells, with abundant expression of CD46 mRNA and particularly lower expression of CAR mRNA. In contrast, the oncolytic effect of Ad5F35/MKp-E1 on the 253J and UMUC-3 cells was much weaker than that for the other bladder cancer cells, although these cells abundantly expressed both CAR and CD46 mRNA. Taken together, these results suggest that the oncolytic potential of Ad5F35/MKp-E1 for human bladder cancer cells might depend on the expression levels, not only of CD46, but also some Ad5F35/MKp-E1-targeted apoptosis-related molecules. To address this question, we are currently performing additional experiments. UROLOGY 81 (4), 2013
Figure 2. Oncolytic effects of adenovirus (Ad)5/MKp-E1 and Ad5F35/MKp-E1. Cell viability for (A) HEK293, (B) 253J, (C) 5637, (D) KK-47, (E) T24, (F) TCCSUP, and (G) UMUC-3 cells assayed 5 days after infection using Alamar Blue. Each point represents mean standard error of mean percentage of basal levels (viabilities of cells uninfected; n ¼ 6 independent experiments).
Ad5 vector containing the RGD motif in the adenovirus fiber knob (Ad5RGD) induces oncolysis of renal cancer cells by targeting the RGD receptor integrin.30 UROLOGY 81 (4), 2013
The transduction efficacy of Ad5RGD vector for renal cancer cells is much lower than that of Ad5F35/MKpE1.30 Ad5F35/MKp-E1, accordingly, could exhibit a more 920.e10
beneficial oncolytic effect on bladder cancer cells than the Ad5/MKp-E1 or Ad5RGD vector.
CONCLUSION The results of the present in vitro study have shown that the Ad5F35 vector could be potentially useful for the development of gene therapy for bladder cancer. References 1. Black PC. Molecular signaling and the role of targeted therapies in bladder cancer. Minerva Urol Nefrol. 2012;64:7-17. 2. Winer E, Gralow J, Diller L, et al. Clinical cancer advances 2008: major research advances in cancer treatment, prevention, and screening—a report from the American Society of Clinical Oncology. J Clin Oncol. 2009;27:812-826. 3. Guse K, Cerullo V, Hemminki A. Oncolytic vaccinia virus for the treatment of cancer. Expert Opin Biol Ther. 2011;11:595-608. 4. Bauerschmitz GJ, Barker SD, Hemminki A. Adenoviral gene therapy for cancer: from vectors to targeted and replication competent agents (review). Int J Oncol. 2002;21:1161-1174. 5. Kanerva A, Hemminki A. Modified adenoviruses for cancer gene therapy. Int J Cancer. 2004;110:475-480. 6. Ganly I, Kirn D, Eckhardt G, et al. A phase I study of Onyx-015, an E1B attenuated adenovirus, administered intratumorally to patients with recurrent head and neck cancer. Clin Cancer Res. 2000;6: 798-806. 7. Kirn D. Clinical research results with dl1520 (Onyx-015), a replication-selective adenovirus for the treatment of cancer: what have we learned? Gene Ther. 2001;8:89-98. 8. Biederer C, Ries S, Brandts CH, et al. Replication-selective viruses for cancer therapy. J Mol Med (Berl). 2002;80:163-175. 9. Alemany R, Balague C, Curiel DT. Replicative adenoviruses for cancer therapy. Nat Biotechnol. 2000;18:723-727. 10. Heise C, Kirn DH. Replication-selective adenoviruses as oncolytic agents. J Clin Invest. 2000;105:847-851. 11. Ring CJ. Cytolytic viruses as potential anti-cancer agents. J Gen Virol. 2002;83:491-502. 12. Adachi Y, Reynolds PN, Yamamoto M, et al. Midkine promoterbased adenoviral vector gene delivery for pediatric solid tumors. Cancer Res. 2000;60:4305-4310. 13. Rodriguez R, Schuur ER, Lim HY, et al. Prostate attenuated replication competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-specific antigen-positive prostate cancer cells. Cancer Res. 1997;57:2559-2563. 14. Terao S, Shirakawa T, Kubo S, et al. Midkine promoter-based conditionally replicative adenovirus for targeting midkine-expressing human bladder cancer model. Urology. 2007;70:1009-1013.
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15. Hamilton MM, Byrnes GA, Gall JG, et al. Alternate serotype adenovector provides long-term therapeutic gene expression in the eye. Mol Vis. 2008;14:2535-2546. 16. Okegawa T, Sayne JR, Nutahara K, et al. A histone deacetylase inhibitor enhances adenoviral infection of renal cancer cells. J Urol. 2007;177:1148-1156. 17. Gaggar A, Shayakhmetov DM, Lieber A. CD46 is a cellular receptor for group B adenoviruses. Nat Med. 2003;9:1408-1412. 18. Acharya B, Terao S, Suzuki T, et al. Improving gene transfer in human renal carcinoma cells: utilization of adenovirus vectors containing chimeric type 5 and type 35 fiber proteins. Exp Ther Med. 2010;1:537-540. 19. Li Q, Kawamura K, Yamanaka M, et al. Upregulated p53 expression activates apoptotic pathways in wild-type p53-bearing mesothelioma and enhances cytotoxicity of cisplatin and pemetrexed. Cancer Gene Ther. 2012;19:218-228. 20. Honda M, Kanno T, Fujita Y, et al. Mesothelioma cell proliferation through autocrine activation of PDGF-bb receptor. Cell Physiol Biochem. 2012;29:667-674. 21. Tsutsui J, Kadomatsu K, Matsubara S, et al. A new family of heparin-binding growth/differentiation factors: increased midkine expression in Wilms tumor and other human carcinomas. Cancer Res. 1993;53:1281-1285. 22. Aridome K, Tsutsui J, Takao S, et al. Increased midkine gene expression in human gastrointestinal cancers. Jpn J Cancer Res. 1995;86:655-661. 23. Garver RI Jr, Radford DM, Donis-Keller H, et al. Midkine and pleiotrophin expression in normal and malignant breast tissue. Cancer. 1994;74:1584-1590. 24. Koide N, Hada H, Shinji T, et al. Expression of the midkine gene in human hepatocellular carcinomas. Hepatogastroenterol. 1999;46: 3189-3196. 25. Muramatsu T. Midkine (MK), the product of a retinoic acid responsive gene, and pleiotrophin constitute a new protein family regulating growth and differentiation. Int J Dev Biol. 1993;37: 183-188. 26. Matsumoto K, Shariat SF, Ayala GE, et al. Loss of coxsackie and adenovirus receptor expression is associated with features of aggressive bladder cancer. Urology. 2005;66:441-446. 27. Buscarini M, Quek ML, Gilliam-Hegarich S, et al. Adenoviral receptor expression of normal bladder and transitional cell carcinoma of the bladder. Urol Int. 2007;78:160-166. 28. Varela JC, Atkinson C, Woolson R, et al. Upregulated expression of complement inhibitory proteins on bladder cancer cells and antiMUC1 antibody immune selection. Int J Cancer. 2008;123: 1357-1363. 29. Cupelli K, Stehle T. Viral attachment strategies: the many faces of adenoviruses. Curr Opin Virol. 2011;1:84-91. 30. Terao S, Acharya B, Suzuki T, et al. Improved gene transfer into renal carcinoma cells using adenovirus vector containing RGD motif. Anticancer Res. 2009;29:2997-3001.
UROLOGY 81 (4), 2013
MATERIALS AND METHODS
RESULTS
CONCLUSION
To assess the oncolytic effect of fiber-substituted conditionally replicating adenovirus type 5 (Ad5) F35 vector on human bladder cancer cell lines such as 253J, 5637, KK-47, T24, TCCSUP, and UMUC-3 cells. Ad5F35 and Ad5 conditionally replicating adenovirus vectors containing the E1 gene controlled by the human midkine promoter (Ad5F35/MKp-E1 and Ad5/MKp-E1, respectively) were constructed. Reverse transcriptase-polymerase chain reaction and cell viability assay were performed in cells transfected with Ad5F35/MKp-E1 or Ad5/MKp-E1. Of the bladder cancer cells used, considerably lower expression of mRNA for Coxsackie and adenovirus receptor, an Ad5 receptor, was found with T24 and TCCSUP cells. However, the mRNA for CD46, an Ad35 receptor, was abundantly expressed in all the cell types. Ad5F35/ MKp-E1 induced oncolysis in a plaque formation unit-dependent manner for all the bladder cancer cells used, with greater efficacy than Ad5/MKp-E1 for T24, TCCSUP, and 253J cells. The results of the present study have shown that Ad5F35/MKp-E1 is more useful for the gene therapy of bladder cancer than Ad5/MKp-E1 is for some cell lines. UROLOGY 81: 920.e7e920.e11, 2013. 2013 Elsevier Inc.
A
n increasingly broad spectrum of molecules and pathways suitable for novel targeted therapies for bladder cancer has been identified from accumulating studies about the molecular mechanisms that drive urothelial transformation and the growth, invasion, and metastasis.1 Despite the huge clinical challenges, lesser improvement for the 5-year survival rate of bladder cancer has been obtained.2 Gene therapy using adenoviral vectors has been attempted in a variety of cancers.3 Less beneficial result than expected, however, has been obtained because of the limited gene transfer into solid tumor cells.4,5 In clinical tests, the safety and oncolytic effect of the first generation of adenovirus vectors have been confirmed, but the efficacy has been modest for the context of Financial Disclosure: The authors declare that they have no relevant financial interests. From the Laboratory of Cell and Gene Therapy, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan; Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, Nishinomiya, Japan; and Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chuo-ku, Chiba, Japan Reprint requests: Tomoyuki Nishizaki, M.D., Ph.D., Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501, Japan. E-mail: [email protected] Submitted: June 7, 2012, accepted (with revisions): December 12, 2012
920.e7
ª 2013 Elsevier Inc. All Rights Reserved
advanced tumors.3 Conditionally replicating adenovirus (CRAD) has been developed to reduce the side effects and obtain more beneficial efficacy.6,7 CRAD enables the gene therapy to induce tumor-specific cell death and amplify the oncolytic effect by replicating and spreading the surrounding cells.8-11 The CRAD vector used in the present study contains the E1 gene controlled by the midkine promoter. Midkine, a secreted heparin-binding growth factor, was identified as a product of the retinoic acid response gene, and it is expressed in a variety of cancer cells more highly than in normal cells.12 The CRAD vector has been shown to induce oncolysis of cancer without damaging normal liver.13,14 Adenovirus type 5 (Ad5) has been widely used for gene therapy.15 Ad5 attachment to target cells is mediated by the Coxsackie and adenovirus receptor (CAR). Low CAR expression in cancer cells is a large obstacle for gene therapies using Ad5.16 In contrast, adenovirus type 35 (Ad35) is infected into cells through the receptor CD46, which is abundantly expressed in cancer cells.17 We have earlier found that chimeric adenovirus Ad5F35 with substitution of the fiber knob on Ad5 by that on Ad35 exhibits a beneficial oncolytic effect on human renal carcinoma cells with low expression of CAR by 0090-4295/13/$36.00 http://dx.doi.org/10.1016/j.urology.2012.12.023
Table 1. Primers used for reverse transcriptase-polymerase chain reaction PCR Primer Midkine CAR CD46 GAPDH
Oligonucleotide Sequence 0
Sense: 5 -GCTTCCTCCTCCTCACCTC-30 Antisense: 50 -TCGCCTGCTGGCGCTCA-30 Sense: 50 -CAGAAGCTACATCGGCAGTAATCA-30 Antisense: 50 -CTCTGAGGAGTGCGTTCAAAGTC-30 Sense: 50 -GGTGTTGCTGCTGTACTCCTTCT-30 Antisense: 50 -CCAATGAGCTCCATAGCTTCAA-30 Sense: 50 -GAAGGTGAAGGTCGGAGTC-30 Antisense: 50 -GAAGATGGTGATGGGATTTC-30
CAR, Coxsackie and adenovirus receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PCR, polymerase chain reaction.
improving the infection efficacy into cells.18 The present study examined the oncolytic effect of Ad5F35 on human bladder cancer cells. We found that Ad5F35 could be used as an efficient tool for gene therapy of bladder cancer.
MATERIAL AND METHODS Cells and Cell Culture The human bladder cancer cell lines used in the present study were 253J, 5637, KK-47, T24, TCCSUP, and UMUC-3 cells. KK-47 was generously provided by Dr. Seiji Naito (Department of Urology, Kyushu University, Fukuoka, Japan), and TCCSUP, T24, 253J, 5637, UMUC-3, and HEK293 human embryonic kidney cells were purchased from the American Type Culture Collection (Manassas, VA). The bladder cancer cells and HEK293 cells were grown in Roswell Park Memorial Institute-1640 (Life Technologies, Gaithersburg, MD) and Eagle minimum essential medium (Invitrogen, Carlsbad, CA), respectively, supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, penicillin (final concentration 100 U/mL), and streptomycin (final concentration 0.1 mg/mL), in a humidified atmosphere of 5% carbon dioxide and 95% air at 37 C.
Construction of CRAD Vector The CRAD vector containing the E1 gene under the control of the human midkine promoter was constructed by the following method. The cytomegalovirus promoter of the pShuttle2 vector (Clontech Laboratories, Mountain View, CA) was replaced with the 0.6-kb 50 -upstream regulatory region of the midkine gene and pS-MK/E1 vector was produced by inserting the E1 gene downstream of the midkine region. To construct pAd5F35, the Ad5 fiber region on the pAdeno-X vector (Clontech Laboratories) was substituted with the Ad35 fiber knob region on the RHSP vector (Avior Therapeutics, Seattle, WA). Ad5/MKp-E1 and Ad5F35/MKp-E1 were prepared by linking the pS-MK/E1 vector with pAdeno-X and pAd5F35, respectively. Adenovirus vectors were amplified in HEK293 and purified with Vivapure AdenoPACK (Sartorius AG, Goettingen, Germany). The number of virus particles/mL was estimated using the following formula: absorbance at 260 nm for purified adenovirus in the presence of 0.1% sodium dodecyl sulfate 1.1 1012.19 Determination of the infection unit (plaque formation unit [PFU]/mL) was accomplished using an Adeno-X titer kit. The PFU/virus particle ratio was 6.3 and 45.6 for Ad5 and Ad5F35, respectively. UROLOGY 81 (4), 2013
Reverse Transcriptase-Polymerase Chain Reaction Reverse transcriptase-polymerase chain reaction was performed using the primers listed in Table 1 and a previously described method.20 The expression levels for each mRNA were normalized by the levels for glyceraldehyde 3-phosphate dehydrogenase mRNA.
Oncolysis Assay Cells (4 104) were infected with Ad5/MKp-E1 or Ad5F35/ MKp-E1 at 0.01-100 virus particles/cell. At 5 days after infection, the cells were incubated with an Alamar Blue (Life Technologies), and the number of viable cells was counted at an absorbance of 560 nm with a high-throughput microplate spectrophotometer (SpectraMax Plus384, Molecular Devices, Sunnyvale, CA).
RESULTS Expression of Midkine, CAR, and CD46 Midkine mRNA was expressed in all the bladder cancer cells, with the lowest expression for UMUC-3 cells, and the expression levels tended to be lower than for the HEK293 cells (Fig. 1A,B). Of the bladder cancer cells used, considerably lower expression of the CAR mRNA was found with T24 and TCCSUP cells (Fig. 1A,C). However, the CD46 mRNA was abundantly expressed in all the cell types, with the expression levels mostly greater than for HEK293 cells (Fig. 1A,D). Oncolytic Effects of Ad5/MKp-E1 and Ad5F35/MKpE1 For the HEK293 cells, Ad5F35/MKp-E1 reduced cell viability in a PFU-dependent manner to an extent similar to that for Ad5/MKp-E1 (Fig. 2A). For the bladder cancer cell lines, Ad5F35/MKp-E1 reduced cell viability in a PFU-dependent manner to an extent apparently greater than Ad5/MKp-E1 for T24, TCCSUP, and 253J cells (Fig. 2B,E,F). For 5637, KK-47, and UMUC-3 cells, Ad5F35/MKp-E1 exhibited an oncolytic effect, with similar or slightly lesser efficacy compared with that for Ad5/MKp-E1 (Fig. 2C,D,G).
COMMENT Midkine is abundantly expressed in a variety of cancer cells originating from the esophagus, stomach, colon, 920.e8
Figure 1. Expression of mRNA for midkine, Coxsackie and adenovirus receptor (CAR), and CD46. (A) Reverse transcriptasepolymerase chain reaction products shown. Signal intensities for each mRNA sample normalized by intensities for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. Each column represents mean standard error of mean normalized expression of mRNA for (B) midkine, (C) CAR, and (D) CD46 (n ¼ 4 independent experiments).
liver, breast, and pancreas. Also, the expression in nonmalignant cells is quite limited, with moderate expression in the kidney and weak expression in the lung, colon, and thyroid gland.21-25 The midkine promoter, accordingly, could be used for cancer gene therapy. A similar or lower expression of midkine mRNA was found with all bladder cancer cells compared with the expression for HEK293 cells. We constructed the Ad5F35 CRAD vector encoding the E1 gene under the control of the 0.6-kb midkine promoter (Ad5F35/MKp-E1). Ad5 and Ad35 infect into cells by targeting CAR and CD46, respectively. Studies have shown that expression of CAR disappears in aggressive bladder cancer26 and decreases as an early event in bladder carcinogenesis.27 In contrast, CD46 expression increases in bladder cancer cells.28 In the present study, the bladder cancer cells, except for the T24 and TCCSUP cells, expressed CAR mRNA, with the quantity similar to, or greater than, that in the HEK293 cells. The bladder cancer cells, except for the UMUC-3 cells, however, expressed CD46 mRNA more highly than did the HEK293 cells. This indicates that Ad35 tends to more easily infect human bladder cancer cells than does Ad5. Receptors that mediate attachment of the adenovirus fiber knobs to the host cell have been shown.29 Species A 920.e9
and C-F adenoviruses such as Ad5 and Ad12 interact with the receptor CAR. The Ad5 knob and the CAR D1-2 domains form a complex. Species B adenoviruses such as Ad11 and Ad35 attach to CD46. The Ad35 knob and the CD46 short consensus repeat 1-4 domains form a complex. Species D adenoviruses such as Ad37 attach to GD1a, which is linked to an unidentified glycoprotein. Species B adenoviruses such as Ad3, Ad7, and Ad14 attach exclusively to DSG-2. In the present study, Ad5F35/MKp-E1 reduced cell viability in a PFU-dependent manner for all the bladder cancer cells, with the potential greater than Ad5/MKp-E1 did for T24, TCCSUP, and 253J cells. The profound effect was obtained with T24 and TCCSUP cells, with abundant expression of CD46 mRNA and particularly lower expression of CAR mRNA. In contrast, the oncolytic effect of Ad5F35/MKp-E1 on the 253J and UMUC-3 cells was much weaker than that for the other bladder cancer cells, although these cells abundantly expressed both CAR and CD46 mRNA. Taken together, these results suggest that the oncolytic potential of Ad5F35/MKp-E1 for human bladder cancer cells might depend on the expression levels, not only of CD46, but also some Ad5F35/MKp-E1-targeted apoptosis-related molecules. To address this question, we are currently performing additional experiments. UROLOGY 81 (4), 2013
Figure 2. Oncolytic effects of adenovirus (Ad)5/MKp-E1 and Ad5F35/MKp-E1. Cell viability for (A) HEK293, (B) 253J, (C) 5637, (D) KK-47, (E) T24, (F) TCCSUP, and (G) UMUC-3 cells assayed 5 days after infection using Alamar Blue. Each point represents mean standard error of mean percentage of basal levels (viabilities of cells uninfected; n ¼ 6 independent experiments).
Ad5 vector containing the RGD motif in the adenovirus fiber knob (Ad5RGD) induces oncolysis of renal cancer cells by targeting the RGD receptor integrin.30 UROLOGY 81 (4), 2013
The transduction efficacy of Ad5RGD vector for renal cancer cells is much lower than that of Ad5F35/MKpE1.30 Ad5F35/MKp-E1, accordingly, could exhibit a more 920.e10
beneficial oncolytic effect on bladder cancer cells than the Ad5/MKp-E1 or Ad5RGD vector.
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