Combined radiation and gene therapy for brain tumors with ... - Nature

Cancer Gene Therapy (2002) 9, 840 – 845 D 2002 Nature Publishing Group All rights reserved 0929-1903 / 02 $25.00 www.nature.com / cgt

Combined radiation and gene therapy for brain tumors with adenovirus-mediated transfer of cytosine deaminase and uracil phosphoribosyltransferase genes Hirokazu Kambara,1 Takashi Tamiya,1 Yasuhiro Ono,1 Shinji Ohtsuka,1 Kinya Terada,1 Yoshiaki Adachi,1 Tomotsugu Ichikawa,1 Hirofumi Hamada,2 and Takashi Ohmoto1 1

Department of Neurological Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan; and 2Department of Molecular Medicine, Sapporo Medical University, Sapporo, Japan.

Radiation therapy is an established modality for the treatment of malignant gliomas. Several reports have shown the advantage of additional radiation in combination with gene therapy. In this study, we investigated the ability of radiation therapy to enhance 5 - fluorocytosine ( 5 - FC ) / cytosine deaminase ( CD ) plus uracil phosphoribosyltransferase ( UPRT ) gene therapy in malignant gliomas. In vitro study suggested evidence of a significant cytotoxic interaction between radiation therapy and 5 - FC / CD + UPRT gene therapy for glioma cells. In vivo experiments demonstrated that the combination of gene therapy and radiation possessed superior antitumor effect in comparison to single therapy. However, the adverse effects of radiation therapy in combination with the gene therapy were observed with respect to normal brain. This combination therapy may be feasible for the treatment of gliomas, although the radiation dose and area should be reduced in order to prevent side effects. Cancer Gene Therapy ( 2002 ) 9, 840 – 845 doi:10.1038/sj.cgt.7700506 Keywords: adenovirus vector; cytosine deaminase; gene therapy; glioma; radiation; uracil phosphoribosyltransferase

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ransduction of the Escherichia coli cytosine deaminase (CD ) gene into tumor cells followed by administration of 5- fluorocytosine (5 -FC ), deemed 5 -FC / CD gene therapy, was created as a suicide gene therapy for various cancers. The uracil phosphoribosyltransferase (UPRT ) gene, which is absent from mammalian cells, directly converts 5 -fluorouracil (5 -FU ) to 5- fluorouridine 50 -monophosphate.1,2 We previously demonstrated that coexpression of CD and UPRT genes generated a cooperative antitumor effect on experimental brain tumors.2 Radiation therapy has been commonly employed in glioma patients; moreover, it has enhanced the efficacy of chemotherapy, resulting in significant prolongation of patient survival. The present study investigated the ability of radiation therapy to enhance CD plus UPRT gene therapy for brain tumors. Additionally, we examined the untoward effects of this combined modality on normal brain.

Materials and methods Cell line and adenovirus vectors

Rat 9 L gliosarcoma cells were grown in Eagle’s minimum essential medium ( Gibco BRL, Grand Island, NY ) supplemented with 10% fetal bovine serum (Gibco BRL, Grand

Received June 11, 2002. Address correspondence and reprint requests to: Dr Takashi Tamiya, Department of Neurological Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. E - mail: [email protected] - u.ac.jp

Island, NY ) and antibiotics ( 40 U /mL penicillin G and 100 g /mL streptomycin ). Three recombinant replicationdeficient adenovirus vectors, AdCA - LacZ, AdCA -CD, and AdCA - UPRT, were constructed from a serotype 5 wild -type adenovirus ( Ad5 ) through insertion of a CAG promoter with E. coli LacZ gene, E. coli CD gene and E. coli UPRT gene, respectively, into the E1A region of the genome. The E1B and E3 regions were deleted. Viral titers were determined by end -point cytopathic effect assay on 293 cells and expressed as plaque -forming units ( pfu ) per milliliter. Chemoradiosensitivity

At room temperature, exponentially growing cells in 96 -well plates were exposed to X -ray at a dose rate of 1.5 Gy /min employing an irradiation apparatus ( MBR - 1520A -TW; Hitachi Medico, Tokyo, Japan ). Radiation sensitivity of 9 L cells was assessed by measuring conversion of the tetrazolium salt, 3 -( 4,5- dimethylthiazol -2 -yl ) -2,5 -diphenyltetrazolium bromide (MTT ) ( Sigma, St. Louis, MO ), to formazan. Results were expressed as a growth ratio compared with the control. Additionally, the combined antitumor effect of 5- FC /CD +UPRT therapy and radiation was investigated in vitro. Cells were seeded in 96 -well plates at a density of 1000 cells/well. Seeded cells were subsequently infected with virus on day 0, exposed to 5 -FC on day 1, and irradiated on day 2. Six days later, sensitivity was assessed by MTT assay. Flow cytometric analysis of apoptosis

Cells were seeded in 6 -cm dish at a density of 5104 cells. Seeded cells were subsequently infected with virus at total

CD+UPRT gene therapy and radiation for gliomas H Kambara et al

841 Beginning on day 2, the rats received i.p. 5- FC at concentration of 500 mg /kg, or saline (control ) once per day for 2 weeks. On day 12, the rats were reanesthesized and subjected to whole brain irradiation to a dose of 15 Gy by using the irradiation apparatus at 1.5 Gy /min with customized shielding that allowed selective administration of radiation to the whole brain. Rats were observed and sacrificed near death. Rat brains were fixed, sliced into 50- m sections, and stained with hematoxylin – eosin ( HE ) and Kluver- Barrera (KB ) stainings for histological evaluation.

Results Chemoradiosensitivity

Figure 1 In vitro sensitivity of 9 L cells to radiation. Cells were seeded in 96 - well plates at a density of 1000 cells / well, followed by irradiation on day 2. Six days later, sensitivity was assessed by MTT assay. Radiation level of 10.2 Gy inhibits 50% of control in 9 L cells.

multiplicity of infection (MOI) of 50 on day 0, exposed to 5 -FC at a concentration of 10 M on day 1, and irradiated ( 10 Gy ) on day 2. Then we collected the cells on day 5. Apoptosis of treated tumor cells was detected using the APO - BRDU kit ( Pharmingen, San Diego, CA ), according to the manufacturer’s instructions. Briefly, the 30 -hydroxyl ends of the DNA in apoptotic cells were labeled with bromolated deoxyuridine triphosphate nucleotides ( Br-dUTP ) by terminal deoxynucleotidyl transferase, and the incorporated BrdU was stained with a fluorescein isothiocyanate ( FITC )– labeled anti -BrdU monoclonal antibody. After DNA was stained with propidium iodide, the cells were analyzed by FACScan.

In vitro experiments revealed the radiosensitivity of 9 L cells ( Fig 1). According to the linear curve of Figure 1, radiation doses of 10.2 Gy inhibited 50% of control in 9 L cells. Our previous study showed that 9 L cells were successfully transduced by adenovirus harboring LacZ, CD, or UPRT genes. AdCA -LacZ and AdCA - UPRT vectors were not cytotoxic at an MOI of less than 1000, whereas AdCA -CD displayed a relatively strong toxicity, the critical level of which occurred at an MOI of 100.1 Figure 2 illustrates the effects of combined modality treatments on 9 L cells. 9 L cells were infected with AdCA -CD and AdCA - UPRT following 5 -FC and radiation (5 Gy ) exposure. The cell survival of the CD / 5- FC + radiation group was significantly lower than that of the CD group; moreover, the cell survival of the CD + UPRT /5 -FC + radiation group was significantly lower than that of the CD +UPRT /5 -FC group.

In vivo antitumor activity

An approved rat brain tumor model was employed to evaluate the therapeutic efficacy of the combination of 5 -FC / CD + UPRT gene therapy and external irradiation. All experimental animals were housed and handled in accordance with the Okayama University Animal Research Committee guidelines. Male Wistar rats ( Charles River Laboratory, Osaka, Japan ), weighing 250– 300 g each, were used for animal experiments. In order to establish brain tumors, rats were anesthesized with intraperitoneal ( i.p. ) Nembutal ( 30 mg /kg ) and placed on a stereotactic apparatus ( Narishige, Tokyo, Japan ). As described previously,1 – 3 9 L cells (1105 cells / 5 L ) were slowly injected into the basal ganglia of the right cerebral hemisphere ( 4 mm lateral to the midline, 2 mm posterior to the coronal suture, 5 mm depth from the dura ) utilizing Hamilton syringes (Hamilton, Reno, NV ). Four days following tumor inoculation, all rats bearing brain tumors were reanesthesized and subsequently injected with AdCA -CD (5106 pfu / 5 L ) and AdCA UPRT ( 5106 pfu /5 L ) stereotactically at the tumor inoculation site based on the same coordinates.

Figure 2 In vitro sensitivity to 5 - FC following transduction with AdCA - CD or AdCA - CD and AdCA - UPRT and irradiation. Cells were seeded in 96 - well plates at a density of 1000 cells / well. Cells were subsequently infected with virus on day 0, exposed to 5 - FC on day 1, and irradiated on day 2. Six days later, sensitivity was assessed by MTT assay. At 12.5 M 5 - FC, percent of control differed significantly in the four treatment groups ( P < .01 ). Values were 65.71 ± 6.24%, 53.71 ± 3.8%, 43.27 ± 3.0%, and 33.43 ± 2.8% in the CD, CD + radiation, CD + UPRT, and CD + UPRT + radiation groups, respectively.

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842 bination with radiation, were investigated by flow cytometric analysis using the APO -BRDU kit. The results are shown in Figure 3. Treatment with CD gene therapy induced apoptosis in 21% cells ( Fig 3C ), combined therapy (CD gene therapy and radiation ) induced apoptosis in 60% ( Fig 3D ), CD plus UPRT gene therapy induced apoptosis in 41% (Fig 3E ), and combined therapy (CD plus UPRT gene therapy and radiation ) induced apoptosis in 73% (Fig 3F ). The results indicated that a positive cytotoxic interaction of CD plus UPRT gene therapy and radiation was mainly or partly caused by apoptotic mechanism and suggested that the combination of gene therapy and radiation could induce apoptosis synergistically. In vivo antitumor activity

Figure 3 Flow cytometric analysis of apoptotic cells after the treatment with CD plus UPRT gene therapy and radiation. After the treatment, 9 L cells were fixed, labeled with Br - dUTP, stained with FITC anti - BrDU antibody, and then analyzed by FACScan. The x - axis represents the propidium iodide – related fluorescence and the y - axis represents the Br - dUTP – related fluorescence. The dots above the x - axis line indicate the apoptotic cells. The percentage of apoptotic cells was also shown in each figure. A: CD + UPRT gene therapy ( not exposed to 5 - FC ) ( control ). B: CD + UPRT gene therapy ( not exposed to 5 - FC ) and radiation. C: CD gene therapy ( exposed to 5 - FC at concentration of 10 M ). D: CD gene therapy ( exposed to 5 - FC at concentration of 10 M ) and radiation. E: CD + UPRT gene therapy ( exposed to 5 - FC at concentration of 10 M ). F: CD + UPRT gene therapy ( exposed to 5 - FC at concentration of 10 M ) and radiation.

Concentrations of 5 -FC inhibiting 50% of control were 34, 23, 3.6, and 1.8 M in the CD, CD +radiation, CD +UPRT, and CD + UPRT +radiation groups, respectively. At 12.5 M 5 -FC, percent of control in the MTT assay differed significantly in the four treatment groups (P < .01). Values were 65.71 ± 6.24%, 53.71 ± 3.8%, 43.27 ± 3.0%, and 33.43 ± 2.8% in the CD, CD + radiation, CD + UPRT, and CD +UPRT + radiation groups, respectively (Fig 2). Synergistic cytotoxicity is commonly defined as cytotoxicity of greater magnitude with simultaneously delivered treatments relative to the summed cytotoxic effects of single treatments. Strict synergistic interaction between CD +UPRT infection plus 5 -FC and radiation treatments was not observed in this experimental model. However, a significant cytotoxic interaction of these treatments was indicated by the results. Induction of apoptosis with CD plus UPRT gene therapy and radiation

From the results of chemoradiosensitivity assays, we considered that radiation had an enhancing effect on induction of apoptosis by CD plus UPRT gene therapy. Treated 9 L cells with CD plus UPRT gene therapy only, or in com-

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In vivo antitumor effects of the combined treatments of gene therapy and radiation were analyzed using a 9 L brain tumor model. Survival analysis was conducted for 90 days following tumor inoculation. The median survival time ( MST ) of the control group ( saline i.p. injection exclusively, no radiation ) ( n= 10 ) and the saline with radiation group ( n =9 ) was 38 and 40 days ( Fig 4 ), respectively. All rats in these groups died within 62 days following tumor inoculation. Rats surviving for greater than 90 days included 3 of 10 animals in the 5 -FC ( 500 mg /kg ) without radiation group (n =10 ) and 7 of 10 animals in the 5 - FC ( 500 mg / kg) with radiation group (n = 10). The CD + UPRT /5- FC group and the CD + UPRT /5- FC with radiation group had significantly longer ( P< .01 ) survival than rats in other groups ( CD + UPRT /saline, CD +UPRT /saline with radiation ). In addition, the survival time of the CD +UPRT /5 -FC with radiation group was significantly longer (P=.0471) than the CD + UPRT/ 5- FC group (Fig 4).

Figure 4 Kaplan - Meier survival analysis of rats inoculated with intracerebral tumors. A total of 105 9 L cells were inoculated in rat brains. Four days later, the vectors AdCA - CD ( 510 6 pfu / 5 L ) + AdCA - UPRT ( 5106pfu / 5 L ) were injected followed by i.p. administration of 5 - FC ( 500 mg / kg ) or saline once daily for 14 days. The rats were reanesthesized and subjected to whole brain irradiation consisting of a total dose of 15 Gy. The rats were followed until 90 days following tumor inoculation. The CD + UPRT / 5 - FC group and the CD + UPRT / 5 - FC with radiation group had significantly longer ( P < .01 ) survival than rats in other groups ( CD + UPRT / saline, CD + UPRT / saline with radiation ). In addition, the survival time of the CD + UPRT / 5 - FC with radiation group was significantly longer ( P = .0471 ) than that of the CD + UPRT / 5 - FC group. ( ) CD + UPRT / saline ( n = 10 ). ( 4 ) CD + UPRT / saline with radiation ( n = 9 ). ( 5 ) CD + UPRT / 5 - FC ( n = 10 ). ( 6 ) CD + UPRT / 5 - FC with radiation ( n = 10 ).


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Figure 5 Rat brains treated with CD + UPRT / saline, CD + UPRT / 5 - FC, and CD + UPRT / 5 - FC with radiation were harvested on day 20. A: CD + UPRT / saline ( n = 4 ): huge 9 L tumors were detected in all rat brains. B: CD + UPRT / 5 - FC group ( n = 4 ): HE staining revealed small 9 L tumors in all rats, which were not lethal in size on day 20. C: CD + UPRT / 5 - FC with radiation group ( n = 4 ): no apparent 9 L tumors were detected in all rat brains.

For the histological evaluation of the combined therapy, 12 additional rats were divided into three groups: CD + UPRT without 5- FC, 5- FC /CD +UPRT with radiation, and 5 -FC / CD + UPRT without radiation. Treatment was identical to the aforementioned protocol. Animals were sacrificed on day 20. Presence of 9 L tumor and brain damage were examined in all sliced sections of resected brains. HE

staining showed huge 9 L tumors in all rats in the CD + UPRT without 5- FC group (Fig 5A ), and small 9 L tumors in all rats in the 5- FC /CD +UPRT without radiation group ( Fig 5B ). In the 5 -FC /CD +UPRT with radiation group, 9 L tumors were not apparent in all rats; however, focal brain damage was recognized, including necrotic change and surrounding demyelination, at the injection site of the basal

Figure 6 Brains from surviving rats treated with 5 - FC / CD + UPRT gene therapy plus radiation. A: HE staining demonstrated the absence of tumor cells at the injection sites. Several specimens displayed entire brain atrophy and enlargement of ventricles hydrocephalus caused by treatment with 5 - FC / CD + UPRT gene therapy and radiation. B: KB staining showed demyelination in the white matter injected by the tumor cells and adenovirus vectors. C: High magnification of KB staining clearly showed necrotic tissues surrounded by inflammatory cells ( macrophages, lymphocytes, and plasma cells ) and demyelination around the basal ganglia injected by the tumor cells and adenovirus vectors ( magnification, 200 ). D: KB staining showed no demyelination or necrotic tissue in the contralateral basal ganglia ( magnification, 200 ).

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844 ganglia ( Fig 5C ). Whole brain damage was not observed on day 20. Long -surviving rats of 5 -FC /CD + UPRT with radiation group exhibited no abnormal general findings in comparison to normal rats. Surviving rats were sacrificed and their brains were subjected to histological analysis. HE staining demonstrated the absence of tumor cells at the injection sites; moreover, necrotic tissues surrounded by inflammatory cells ( macrophages, lymphocytes, and plasma cells ) were revealed (Fig 6A ). KB staining clearly showed demyelination in the white matter around basal ganglia at the injection side ( Fig 6, B and C). On the contrary, no demyelination was observed in the white matter at the contralateral side ( Fig 6D ). Several samples displayed brain atrophy and enlargement of ventricles in not only the vector -injected side but also whole brains. Discussion

The primary limitations of systemic chemotherapy are a low local concentration of the therapeutic agent and systemic side effects. In order to overcome this problem, a prodrug/ enzyme system possesses the theoretical advantage of delivering high local concentrations of a therapeutic agent while minimizing systemic side effects, thus increasing the therapeutic window. The first trial employing the 5 -FC /CD system for malignancy was described in 1985.4 This gene therapy utilizing adenovirus vectors has been applied to several types of solid tumors, including colorectal,5,6 gastric,7 and hepatocellular cancers,8 as well as gliomas.1,9 Transduction of the UPRT gene has been reported to enhance chemosensitivity to 5 -FU or 5 -FC through the activation pathway to 5- FU.10 Simultaneous expression of CD and UPRT generated a cooperative effect, dramatically increasing the 5 -FC sensitivity of cells compared to the expression of CD alone.2,11 Furthermore, the association of UPRT with CD facilitated uptake of 5 -FC when the drug penetrates cells through passive diffusion as in mammalian cells by direct channeling of 5 -FU to 5 -FUMP.11 In a previous report, we also documented that UPRTtransduced 9 L cells were 16 times more sensitive to 5 -FU, whereas CD plus UPRT- transduced cells were 6000 times more sensitive to 5- FC than the parent 9 L cells.2 In 5 -FC / CD +UPRT gene therapy, only 1% transduction of 9 L cells with AdCA - CD and AdCA -UPRT affected cell growth; moreover, a maximal tumoricidal effect was seen when only 5 –10% of the tumor cells were transduced with the genes.2 This finding implied that 5- FC /CD +UPRT gene therapy affords a strong bystander effect, promising in vivo efficacy of gene therapy. In the brain tumor model, we obtained higher efficacy of 5 -FC /CD + UPRT gene therapy in comparison to the 5- FC /CD gene therapy.2 The roles for radiation therapy in the treatment of malignancy have involved utility as a single or multiple agents. However, radiation therapy is more commonly employed in an adjuvant or neo -adjuvant manner. To date, radiation therapy for gliomas is an established modality that permits patients’ survival advantage. Hence, the multimodality approach including radiation therapy is a common

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strategy for gliomas. Some reports have shown the advantage of additional radiation therapy to gene therapy, such as p53 gene12,13 and ganciclovir (GCV ) /herpes simplex virus thymidine kinase ( HSV-TK ) prodrug /enzyme therapies.14 – 18 Additionally, combined therapy with 5- FC /CD treatment plus radiation was reported to have a therapeutic advantage compared to 5- FC /CD therapy alone.5,16,19 – 21 Khil et al21 demonstrated that the CD gene, upon stable transfection into WiDr cells, was able to enhance radiation cell killing. Pederson et al20 reported the usefulness of 5 - FC /CD therapy combined with radiation therapy for cholangiocarcinoma cells in vitro and in vivo. Ohwada et al5 documented the utility of colon carcinoma xenografts. These reports indicated a positive cytotoxic interaction of 5- FC / CD therapy and radiation; however, to our knowledge, no studies exist regarding the combination of 5 -FC / CD + UPRT gene therapy and radiation. Our in vitro study suggested evidence of a significant positive cytotoxic interaction between radiation therapy and 5 -FC / CD + UPRT gene therapy for glioma cells. In addition, flow cytometric analysis of apoptosis indicated that the cytotoxic effect was synergistic. Our in vivo experiments showed that 5- FC / CD + UPRT plus radiation therapy provided a significant longer survival of rats than 5 -FC /CD + UPRT therapy alone. This finding indicated that radiation therapy demonstrated an additional survival effect on rats harboring brain tumors. We attempted to reduce the administration dose of 5- FC; however, doses of less than 500 mg /kg 5- FC did not have an effect on the survival of experimental rats (data not shown ). Previous investigations involving 5- FC /CD also employed 500 mg /kg (or more ) 5 -FC as the administration dose for animals. Neurotoxicity of prodrug/ enzyme systems is a critical problem in clinical trials. Goodman et al22 reported the toxicity of adenovirus -mediated GCV / HSV-TK gene therapy on infection in the brain of a baboon. Injection of high doses (1.5109 pfu ) of adenovirus caused animal death within a few days following GCV treatment. Coagulative necrosis, edema, and perivascular lymphocytosis were observed with intensive treatment. Smith et al23 also noted local toxicity of the same system in rat brains. In our previous study, necrosis, demyelination, and gliosis were recognized in 5 -FC / CD gene therapy for brain tumors.1 In order to relieve neurotoxicity in the current investigation, 5 - FC administration dosages were reduced, resulting in failure as stated above. Although the combination of 5- FC /CD gene therapy and radiation demonstrated superior antitumor effect relative to 5 -FC /CD gene therapy alone, neurotoxicity was present in brain tissues in our histological examination. The characteristic neurotoxicities observed in this investigation, brain atrophy and ventricular enlargement, were not focal damage at the tumor sites, but global and diffuse brain damage primarily attributable to radiation therapy. These neurotoxicities were not evident in early stage disease, but were clarified in late stage following treatment. Recently, stereotactic radiosurgery or radiotherapy can be applied for brain tumors to avoid the neurotoxicities of whole brain irradiation. According to previous reports concerning gene therapy in concert with radiation, radiation dosages consisting mainly of


15 –20 Gy were delivered in rat brain tumor models.12,17 Histological analysis of longitudinally surviving rats was not documented. Consequently, we were unable to compare brain alterations to the Kim and Broaddus data. Global brain damages observed in the current investigation were difficult to recognize symptomatically in rodents; however, these are important issues related to memory or cognitive functions in human patients. In order to prevent this type of damage, radiation field and dose should be considered in combination therapy. In summary, we reported that the combination of 5 -FC / CD +UPRT gene therapy and radiation displayed remarkable antitumor effect on gliomas. We should consider the adverse effects of radiation in combination with gene therapy for normal brain. Acknowledgments

We thank Hideki Wakimoto, Masako Arao, and Mayumi Konishi for their technical assistance and Judy Yamamoto for her English editorial assistance. This study was supported by Grants - in - Aid for Scientific Research from the Japan Ministry of Education, Science, Sports and Culture ( nos. 14370433 and 14571312 ). References 1. Ichikawa T, Tamiya T, Adachi Y, et al. In vivo efficacy and toxicity of 5 - fluorocytosine / cytosine deaminase gene therapy for malignant gliomas mediated by adenovirus. Cancer Gene Ther. 2000;7:74 – 82. 2. Adachi Y, Tamiya T, Ichikawa T, et al. Experimental gene therapy for brain tumors using adenovirus - mediated transfer of cytosine deaminase gene and uracil phosphoribosyltransferase gene with 5 - fluorocytosine. Hum Gene Ther. 2000;11:77 – 89. 3. Tamiya T, Ono Y, Wei MX, et al. Escherichia coli gpt gene sensitizes rat glioma cells to killing by 6 - thioxanthine or 6 thioguanine. Cancer Gene Ther. 1996;3:155 – 162. 4. Nishiyama T, Kawamura Y, Kawamoto K, et al. Antineoplastic effects in rats of 5 - fluorocytosine in combination with cytosine deaminase capsules. Cancer Res. 1985;45:1753 – 1761. 5. Ohwada A, Hirshowtiz EA, Cristal RG. Regional delivery of an adenovirus vector containing the Escherichia coli cytosine deaminase gene to provide local activation of 5 - fluorocytosine to suppress the growth of colon carcinoma metastatic to liver. Hum Gene Ther. 1996;7:1567 – 1576. 6. Hirshowitz EA, Ohwada A, Pascal WR, et al. In vivo adenovirus - mediated gene transfer of the Escherichia coli cytosine deaminase gene to human colon carcinoma - derived tumors induced chemosensitivity to 5 - fluorocytosine. Hum Gene Ther. 1995;6:1055 – 1063. 7. Lan K - H, Kanai F, Shiratori Y, et al. In vivo selective gene expression and therapy mediated by adenoviral vectors for human carcinoembryonic antigen - producing gastric carcinoma. Cancer Res. 1997;57:4279 – 4284.

8. Kanai F, Lan K - H, Shiratori Y, et al. In vivo gene therapy for alfa - fetoprotein – producing hepatocellular carcinoma by adenovirus - mediated transfer of cytosine deaminase gene. Cancer Res. 1997;57:461 – 465. 9. Dong Y, Wen P, Manome Y, et al. In vivo replication - deficient adenovirus vector – mediated transduction of the cytosine deaminase gene sensitizes glioma cells to 5 - fluorocytosine. Hum Gene Ther. 1996;7:713 – 720. 10. Kanai F, Kawakami T, Hamada H, et al. Adenovirus - mediated transduction of Escherichia coli uracil phosphoribosyltransferase gene sensitizes cancer cells to low concentration of 5 - fluorouracil. Cancer Res. 1998;58:1946 – 1951. 11. Tiraby M, Cazaux C, Baron M, et al. Concomitant expression of E. coli cytosine deaminase and uracil phosphoribosyltransferase improves the cytotoxicity of 5 - fluorocytosine. FEMS Microbiol Lett. 1998;167:41 – 49. 12. Broaddus WC, Liu Y, Steele LL, et al. Enhanced radiosensitivity of malignant glioma cells after adenoviral p53 transduction. J Neurosurg. 1999;91:997 – 1004. 13. Badie B, Goh CS, Klaver J, et al. Combined radiation and p53 gene therapy of malignant glioma cells. Cancer Gene Ther. 1999;6:155 – 162. 14. Valerie K, Brust D, Farnworth J, et al. Improved radiosensitization of rat glioma cells with adenovirus - expressed mutant herpes simplex virus - thymidine kinase in combination with acyclovir. Cancer Gene Ther. 2000;7:879 – 884. 15. Marples B, Scott SD, Hendry JH, et al. Development of synthetic promoters for radiation - mediated gene therapy. Gene Ther. 2000;7:511 – 517. 16. Rogulski KR, Kim JH, Kim SH, et al. Glioma cells transduced with an E. coli CD / HSV- 1 TK fusion gene exhibit enhanced metabolic suicide and radiosensitivity. Hum Gene Ther. 1997;8:73 – 85. 17. Kim SH, Kim JH, Kolozsvary A, et al. Preferential radiosensitization of 9 L glioma cells transduced with HSV- tk gene by acyclovir. J Neuro - Oncol. 1997;33:189 – 194. 18. Kim JH, Kim SH, Kolozsvary A, et al. Selective enhancement of radiation response of herpes simplex virus thymidine kinase transduced 9 L gliosarcoma cells in vitro and in vivo by antiviral agents. Int J Radiat Oncol Biol Phys. 1995;33: 861 – 868. 19. Freytag SO, Rogulski KR, Paielli DL, et al. A novel three pronged approach to kill cancer cells selectively: concomitant viral, double suicide gene, and radiotherapy. Hum Gene Ther. 1998;9:1323 – 1333. 20. Pederson LC, Bushsbaum DJ, Vickers SM, et al. Molecular chemotherapy combined with radiation therapy enhances killing of cholangiocarcinoma cells in vitro and in vivo. Cancer Res. 1997;57:4325 – 4332. 21. Khil MS, Kim JH, Mullen CA, et al. Radiosensitization by 5 - fluorocytosine of human colorectal carcinoma in culture transduced with cytosine deaminase gene. Clin Cancer Res. 1996;2:53 – 57. 22. Goodman JC, Trask TW, Chen S - H. Adenoviral - mediated thymidine kinase gene transfer into the primate brain followed by systemic ganciclovir: pathologic, radiologic, and molecular studies. Hum Gene Ther. 1996;7:1241 – 1250. 23. Smith JG, Raper SE, Wheeldon EB. Intracranial administration of adenovirus - expressing HSV- TK in combination with ganciclovir produces a dose - dependent, self - limiting inflammatory response. Hum Gene Ther. 1997;8:943 – 9564.

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