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[Cancer Research 61, 6158-6162, August 15, 2001]
© 2001 American Association for Cancer Research

Experimental Therapeutics

Carcinoembryonic Antigen-specific Suicide Gene Therapy of Cytosine Deaminase/5-Fluorocytosine Enhanced by the Cre/loxP System in the Orthotopic Gastric Carcinoma Model1

Kentaro Ueda, Makoto Iwahashi2, Mikihito Nakamori, Masaki Nakamura, Ichiro Matsuura, Hiroki Yamaue and Hiroshi Tanimura

Second Department of Surgery, Wakayama Medical University, School of Medicine, Wakayama 641-8510, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor-specific gene delivery is crucial to achieving successful effects in suicide gene therapy. Carcinoembryonic antigen (CEA) promoter has been widely used for this purpose, but the expression level of tumor-specific promoters such as CEA promoter is generally low. In the previous study, we used the Cre/loxP system and showed that LacZ expression by the CEA promoter was remarkably enhanced and maintained its specificity using the Cre/loxP regulation system. In this study, the Cre/loxP system was first applied to augmentation of selective expression of the cytosine deaminase (CD) gene as a suicide gene therapy in CEA-producing cells. The double infection with AxCEANCre expressing Cre recombinase under the control of the CEA promoter and AxCALNLCD expressing the CD gene under the control of the CAG promoter by the Cre switching system rendered CEA-producing tumor cells 13-fold more sensitive to 5-fluorocytosine (5-FC) compared with the single infection with AxCEACD expressing CD gene driven by the CEA promoter. The therapeutic efficacy of the enhanced CD/5-FC suicide gene therapy was evaluated in orthotopic implantation models of human gastric carcinoma. Adenovirus vectors (1 x 109 plaque-forming units) were administered i.p. into mice three times, and then 5-FC was administered i.p. for the next 10 days. Tumor volume and weight in mice treated with AxCEANCre and AxCALNLCD/5-FC were significantly reduced as compared with those in mice treated not only with Mock (AxCALacZ) but also with AxCEACD/5-FC (P < 0.0001). This beneficial effect on tumor burden was also reflected in the overall survival. The survival periods of the mice treated with AxCEANCre and AxCALNLCD/5-FC were longer than those of mice treated with Mock or AxCEACD/5-FC (P < 0.01). These results suggested that application of the Cre/loxP system could provide a new approach for enhanced selective suicide gene therapy of CD/5-FC for the treatment of advanced gastric carcinoma.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gastric carcinoma is one of the leading causes of cancer-related death, and it remains endemic, especially in Asia, Africa, and parts of Europe (1 , 2) . Even extensive surgical operations combined with chemotherapy have not sufficiently improved the prognosis of patients with advanced gastric carcinoma (3 , 4) . Gene therapy could be one of the candidates for an innovative therapeutic approach for the treatment of advanced gastric carcinoma. Among the various strategies for cancer gene therapy, adenovirus-mediated enzyme/prodrug therapies, the so-called "suicide gene therapies," such as the combination of Escherichia coli CD3 gene transduction and 5-FC treatment (5 , 6) could be the most promising approaches for gastric cancer. Ad is a highly efficient gene expression system in vivo and is thus very useful for the delivery of a suicide gene (5 , 6) . Moreover, the gene product of CD transduced into cells converts nontoxic prodrug 5-FC to 5-FU, which has been widely prescribed for patients with gastric cancer and is reported to show obvious clinical benefits (7) . Tumor-specific gene delivery is crucial to achieve successful effects in suicide gene therapy because the expression of the targeted gene may cause toxicity in normal tissues with systemic administration of an Ad (8) . CEA promoter has been widely used for this purpose (8, 9, 10, 11, 12, 13) . However, the therapeutic efficacy of tumor-specific promoters such as the CEA promoter may be limited because the expression levels of these promoters are generally low (14 , 15) .

In the previous study, we reported that LacZ gene expression by the CEA promoter was remarkably enhanced and maintained its specificity using the Cre/loxP regulation system (16) both in vitro and in vivo (17) . Especially in vivo, we showed selective LacZ expression in gastric tumor tissue in orthotopic models by systemic administration of Ads (17) . In this study, we examined the therapeutic efficacy of the application of the Cre/loxP system for CD/5-FC suicide gene therapy in orthotopic models of CEA-producing gastric carcinoma.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Cell Culture.
Human gastric carcinoma cell lines MKN45 and MKN1 and human uterine cervical carcinoma HeLa cells were obtained from Riken Gene Bank (Ibaraki, Japan). Human colon carcinoma cell line LoVo was obtained from Osaka Institute for Feremetion (Osaka, Japan). Human embryonic kidney cell line 293 was purchased from the American Type Culture Collection (Manassas, VA). MKN45 and LoVo were CEA-producing cell lines, and MKN1 and HeLa were non-CEA-producing cell lines (17) . These cells were cultured as described previously (17) .

Construction of Ads.
Cosmid vector pAxCEANCre was constructed as described previously (17) . Briefly, the cis-acting sequences, which contain the CEA promoter (-299 to +69) region (18) , were cloned by PCR and ligated into the SwaI site of pAxAwNCre (16) to yield pAxCEANCre. pAxCACD and pAxCALNLCD were constructed by ligating the CD fragment into the SwaI site of the pAxCAwt and pAxCALNLw cassettes (16) , respectively, as described in our previous study (19) . Both the CEA promoter fragment and the CD gene fragment were subcloned into PCR2.1 (Invitrogen, Carlsbad, CA) to yield PCR2.1-CEACD. The Then the CEACD fragment was excised from PCR2.1 by EcoRI, blunt-ended, and ligated into the SwaI site of cassette cosmid pAxcw (16) to yield pAxCEACD.

The recombinant Ads, AxCEANCre, AxCACD, AxCALNLCD, and AxCEACD, were generated by the COS-TPC method (20) , and the viruses were propagated in 293 cells and concentrated and purified as described previously (Ref. 21 ; Fig. 1ACitation ).



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  		Fig. 1. A, structure of Ad. AxCEANCre, adenovirus expressing Cre recombinase under the control of CEA promoter. AxCALNLCD, adenovirus expressing the CD gene under the control of the CAG promoter by the Cre-mediated switching system. AxCEACD, adenovirus expressing the CD gene driven by the CEA promoter. AxCACD, adenovirus expressing the CD gene driven by the CAG promoter. B, strategy of the double infection. Cre recombinase expressed by the CEA promoter cuts off the stuffer gene (neo and SpA) from the AxCALNLCD genome and generates an activated CD gene expression driven by a strong CAG promoter.

 
5-FC Sensitivity of Tumor Cells Infected with the Recombinant Ads.
MKN45, LoVo, MKN1, or HeLa cells were seeded in triplicate in 96-well plates (Becton Dickinson, Franklin Lakes, NJ) at a density of 1 x 104 cells/well in 100 µl of medium. Twenty-four h later, the cells were infected with AxCEACD alone, AxCACD alone, or the combination of AxCEANCre and AxCALNLCD (3:2) at a moi of 50. After another 24 h of incubation, the medium was changed to fresh medium containing various concentrations of 5-FC (0, 10, 50, 100, 500, 1000, and 5000 µM; Sigma Chemical Co., St. Louis, MO). The cells were then cultured at 37°C in a humidified atmosphere of 5% CO2 for an additional 6 days, and the medium containing 5-FC was changed every other day in the meantime. Cell growth was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay that measures viable cell succinate dehydrogenase activity, as described previously (22) . The IC50 of 5-FC was calculated using a curve-fitting parameter, and the results are represented as the means ± SD from three independent experiments.

Experiment Design of in Vivo Gene Therapy for Orthotopic Gastric Carcinoma Models.
To investigate the efficacy of systemic Ad-mediated tumor-specific suicide gene therapy using the Cre/loxP system, orthotopic gastric carcinoma models were developed as described previously (17) . Briefly, s.c. MKN45 tumors in athymic female BALB/c nu/nu mice (CLEA, Tokyo, Japan) were removed and minced into pieces of about 2 mm in diameter. In 6–7-week-old athymic female BALB/c nu/nu mice, a part of the serosal membrane in the middle of the greater curvature of the glandular stomach was mechanically injured using scissors, and a tumor piece was fixed on each injured site of the serosal surface with a 6-0 Prolene (Ethicon, Somerville, NJ) transmural suture. These mice were randomly divided into the following five groups: (a) group A, mice treated with Mock (AxCALacZ) followed by PBS administration (n = 5); (b) group B, mice treated with Mock followed by 5-FC (500 mg/kg) administration (n = 5); (c) group C, mice treated with AxCEACD followed by 5-FC administration (n = 5); (d) group D, mice treated with AxCEANCre + AxCALNLCD (3:2) followed by 5-FC administration (n = 5); and (e) group E, mice treated with AxCACD followed by 5-FC administration (n = 5). The Ads (1 x 109 pfu) were injected i.p. once a day for 3 days at 5–7 days after tumor implantation. From the following day, 5-FC or PBS was administered i.p. once daily for the next 10 days. The mice were all sacrificed 4 weeks after tumor implantation, and the implanted gastric tumor was measured and weighed. Tumor volume was calculated as [length (mm) x width2 (mm)]/2.

To further investigate the therapeutic effect against gastric carcinoma in this model, we observed the survival time of mice treated with the protocols described above (five mice in each group). If possible, each animal was examined to evaluate the cause of death.

Evaluation of Adverse Effects by Ads and 5-FC Treatment.
To evaluate the adverse effects by Ad-mediated CD/5-FC suicide gene therapy, serum samples of mice from each group described above were collected for the purpose of measuring serum AST and ALT levels when the mice were sacrificed at 4 weeks after tumor implantation. Serum AST and ALT levels of normal mice (n = 5) were also measured as controls. In addition, the livers from mice treated with AxCEANCre and AxCALNLCD/5-FC or AxCACD/5-FC were removed, fixed, and stained with H&E for pathological examination.

Statistical Analysis.
Quantitative results were expressed as the mean ± SD of the mean. Statistical analysis was performed by using ANOVA and Fisher’s test. The survival rates were estimated using the Kaplan-Meier method, and the differences were analyzed by using the log-rank test to compare the resulting curves of the treatment groups. P < 0.05 was considered statistically significant. StatView 5.0 software (Abacus Concepts, Inc., Berkeley, CA) was used for all statistical analyses.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Strategy of Enhancing Specific CD Gene Expression in CEA-producing Cells by the Cre/loxP System.
To enhance CD gene expression while maintaining cell specificity, we used the double infection method (14 , 16 , 17) . The regulator vector AxCEANCre expresses a Cre recombinase tagged with a nuclear localization signal (NCre) under the control of the CEA promoter. Cells were infected with this vector along with target vector AxCALNLCD, which bears an on/off switching unit for activation by NCre. The switching unit in this target vector contains a stuffer sequence with polyadenylation signal between the CAG promoter (23) and the CD gene blocking the expression of CD. The stuffer is flanked by a pair of loxP sites, allowing its excision by Cre followed by high-level expression of CD under the control of the CAG promoter, which shows very strong activity for gene expression (23) . Cre recombinase is expressed only in CEA-producing cells by the CEA promoter; therefore, the enhanced expression of CD was expected to occur in a tissue-specific manner (Fig. 1B)Citation .

Selective Enhanced 5-FC Sensitivity by Double Infection with AxCEANCre and AxCALNLCD in Vitro.
To analyze the selective enhanced 5-FC sensitivity of the CEA promoter using the Cre/loxP system, CEA-producing cell lines (MKN45 and LoVo) and non-CEA-producing cell lines (MKN1 and HeLa) were infected with AxCEANCre and AxCALNLCD (3:2), AxCEACD, or AxCACD at a moi of 50 and cultured in medium containing various concentrations of 5-FC for 6 days. In the preliminary study, the optimal ratio of AxCEANCre:AxCALNLCD was examined in MKN45 and LoVo cells, and the results showed that it was 3:2 in each cell (data not shown).

Double infection with AxCEANCre and AxCALNLCD rendered MKN45 and LoVo more sensitive to 5-FC compared with single infection with AxCEACD (Fig. 2, A and B)Citation . The IC50 of 5-FC for each cell line obtained by this double infection was 224.7 ± 48.7 and 312.1 ± 32.0 µM, respectively. On the other hand, the IC50 of 5-FC for each cell line obtained by single infection with AxCEACD was 2981 ± 466 and 3863 ± 381 µM, respectively. Thus, these results demonstrated a 12–13-fold higher efficacy of AxCEANCre and AxCALNLCD as compared with AxCEACD in CEA-producing cell lines (P < 0.0001). The IC50 values of 5-FC for MKN45 and LoVo obtained by AxCACD infection were 29.5 ± 6.0 and 59.0 ± 16.0 µM, respectively, and were lower than those obtained by double infection (Table 1)Citation . On the other hand, in non-CEA-producing cell lines (MKN1 and HeLa), strong cytotoxic effects were recognized only by the infection with AxCACD, and double infection with AxCEANCre and AxCALNLCD did not induce cytotoxicity, demonstrating that the specificity of the CEA promoter using the Cre/loxP system was strictly maintained in vitro (Table 1Citation ; Fig. 2, C and DCitation ). Moreover, even in the absence of 5-FC, about 30% of tumor cells died of AxCACD infection at a moi of 50 (Fig. 2)Citation .



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  		Fig. 2. In vitro 5-FC sensitivity of tumor cells infected with AxCEANCre and AxCALNLCD. A, MKN45; B, LoVo; C, MKN1; D, HeLa. Tumor cells were infected with AxCEANCre (moi, 30) + AxCALNLCD (moi, 20; {blacksquare}), AxCEACD (moi, 50; {triangleup}), and AxCACD (moi, 50; {circ}). The next day, the medium was changed to fresh medium containing various concentrations of 5-FC (0–5000 µM). The cells were then cultured for another 6 days, and the medium containing 5-FC was changed every other day. Cell growth was then assessed by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Each point represents the mean ± SD (bars) of three independent assays.

 

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  		Table 1 5-FU sensitivity of parental cells and 5-FC sensitivity of adenovirus-infected cells

 
Therapeutic Efficacy of Double Administration of AxCEANCre and AxCALNLCD followed by 5-FC Treatment in Orthotopic Gastric Carcinoma Model.
To evaluate the in vivo therapeutic efficacy of double administration of AxCEANCre and AxCALNLCD followed by 5-FC treatment on CEA-producing gastric carcinoma, we used orthotopic gastric carcinoma models. The orthotopic models with gastric carcinoma were developed and randomly grouped for treatment with Mock (AxCALacZ)/PBS, Mock/5-FC, AxCEACD/5-FC, AxCEANCre + AxCALNLCD (3:2)/5-FC, or AxCACD/5-FC. The Ads (1 x 109 pfu) were i.p. administered to the mice three times at 5–7 days after tumor implantation. From the following day, 5-FC or PBS was administered i.p. for the next 10 days. All of the mice were sacrificed 4 weeks after tumor implantation, and the volume and the weight of the implanted gastric tumors in each animal were examined. At this time, there were no metastases in any mice. As shown in Table 2Citation , tumor volume and weight in mice treated with AxCEACD/5-FC (120.8 ± 33.6 mm3 and 141.0 ± 22.7 mg) were reduced as compared to with those in mice treated with Mock/PBS (195.2 ± 27.5 mm3 and 200.7 ± 23.8 mg) or Mock/5-FC (207.6 ± 31.2 mm3 and 196.2 ± 23.8 mg; P < 0.001). The tumor volume and weight in mice treated with AxCEANCre and AxCALNLCD (10.9 ± 1.7 mm3 and 49.6 ± 5.7 mg) were significantly reduced as compared with those in the three groups mentioned above (P < 0.0001). The tumor volume and weight in mice treated with AxCACD/5-FC (15.8 ± 6.6 mm3 and 92.0 ± 45.1 mg) were similar to those in the AxCEANCre and AxCALNLCD/5-FC group, but two of five mice treated with AxCACD/5-FC died before the end of 4 weeks after tumor implantation.


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  		Table 2 In vivo antitumor effect of enhanced CEA-specific CD/5-FC suicide gene therapy in orthotopic gastric carcinoma models

 
The beneficial effects of AxCEANCre and AxCALNLCD/5-FC on tumor burden were also reflected in the overall survival (Fig. 3)Citation . The median survival time of the mice treated with AxCEANCre and AxCALNLCD/5-FC was longer (150 ± 22 days) than that of the mice treated with Mock/PBS (73 ± 10 days), Mock/5-FC (86 ± 14 days), or AxCEACD/5-FC (81 ± 8 days). Survival was significantly better for mice treated with AxCEANCre and AxCALNLCD/5-FC as compared with the other three groups (log-rank P < 0.01). The mice treated with AxCACD/5-FC showed a median survival time of just 104 ± 57 days; additionally, two of these mice died of systemic toxicity as early as 12–24 days after tumor implantation, when there was little growth of the transplanted tumors. The remaining mice died directly of tumors or metastases such as peritoneal dissemination, as indicated by the results of necropsies; however, there were no liver metastases and no lymph node metastases in these mice.



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  		Fig. 3. Therapeutic effect of double administration of AxCEANCre and AxCALNLCD followed by 5-FC treatment on survival periods in orthotopic gastric carcinoma models. The nude mice, which were orthotopically implanted with human gastric carcinoma, were randomly divided into the following five groups according to treatment schedules: {square}, Mock (AxCALacZ) followed by PBS (n = 5); {diamondsuit}, Mock followed by 5-FC (n = 5); {triangleup}, AxCEACD followed by 5-FC (n = 5); {blacksquare}, AxCEANCre + AxCALNLCD (3:2) followed by 5-FC (n = 5); and {circ}, AxCACD followed by 5-FC (n = 5). The Ads (1 x 109 pfu) were administered i.p. into the mice three times a day at 5–7 days after tumor implantation. From the following day, the mice received i.p. administration of 5-FC (500 mg/kg) or PBS for the next 10 days.

 
Toxicity of Ad-mediated Suicide Gene Therapy.
The serum levels of AST and ALT, which were collected at 4 weeks after tumor implantation, were remarkably high in mice treated with AxCACD/5-FC. On the other hand, the serum levels of AST and ALT in mice treated with AxCEANCre and AxCALNLCD/5-FC were similar to those in normal mice. There was a significant difference in serum AST and ALT between the AxCACD/5-FC group and the other groups (P < 0.0001 and P < 0.05; Table 3Citation ).


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  		Table 3 The data of hepatic toxicity of mice with various treatment protocols

 
Macroscopic examination showed that the livers of mice treated with AxCACD/5-FC were remarkably swollen, although the livers of the mice treated with AxCEANCre and AxCALNLCD/5-FC seemed to be normal. Microscopically, swelling of hepatic cells and slight necrosis of hepatic cells with lymphocyte infiltration were observed (data not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recently, it has been reported that the application of the Cre/loxP system remarkably enhances the selective expression of therapeutic gene using tissue-specific promoters such as the CEA promoter and the thyroglobulin promoter (15 , 24) . In those studies, the HSV-TK gene is transduced by Ads followed by GCV treatment as suicide gene therapy, and the Cre/loxP system increases the sensitivity of tumor cells to GCV up to 5–10-fold (24) or 8.5-fold (15) compared with the specific promoter alone. In this study, the Cre/loxP system was first applied to augmentation of selective expression of the CD gene in CEA-producing tumor cells. Double infection with AxCEANCre and AxCALNLCD rendered CEA-producing cells such as MKN45 and LoVo 13-fold more sensitive to 5-FC compared with single infection with AxCEACD. These results suggested that the enhancement of the Cre/loxP system in CD/5-FC suicide gene therapy seemed to be superior to that in HSV-TK/GCV therapy, at least in gastrointestinal tumor cells.

Many studies concerning Ad-mediated suicide gene therapy using selective expression have been reported, and in most of these studies, s.c. tumor models were used, with recombinant adenoviruses being directly injected into the tumors (11 , 13 , 15 , 24) . The strategy of tissue-specific gene expression plays a very important role in suicide gene therapy in terms of avoiding adverse effects, but the benefits of tissue-specific gene targeting cannot be properly evaluated by local administration of Ads. In cases where Ads can be injected directly into the tumor, selective gene expression is not required at all. However, patients with advanced gastric carcinoma usually have metastatic tumors in the abdominal cavity (1 , 2) , and direct injection of the therapeutic agents into the tumors is generally impossible. Therefore, the systemic administration of Ads should be selected according to the circumstances, and moreover, animal models that closely resemble human patients are also required for evaluation of the therapeutic efficacy. We thus developed orthotopic implantation models of human gastric carcinoma and treated them with the systemic administration of Ads described in this study. The orthotopic models that had been developed for analyzing biological characterization of carcinoma cells are correlated with the clinical behavior of cancer (17 , 25 , 26) ; therefore, we thought that these models would allow us to accurately determine the efficacy of the in vivo gene therapy.

In our experience, it was very hard to demonstrate the survival benefit of gene therapy in orthotopic models, even if tumor reductions were observed (27) . The systemic administration of Ads to the orthotopic implantation models seems to have difficulty in attaining a high transduction efficiency of targeting gene into the tumor tissue. In fact, the transduction rate of LacZ gene to the implanted gastric tumor tissues was just 15% in orthotopic models receiving i.p. administration of Ads expressing LacZ gene using the Cre/loxP system in our previous study (17) . Besides, the study of biodistribution of Ads has demonstrated that the transduced gene expression in the stomach is only around 10% of that in the liver after i.p. administration to mice (28) . Nevertheless, in this study, tumor volume and weight in mice treated with AxCEANCre and AxCALNLCD followed by 5-FC administration were significantly reduced as compared with those in mice treated not only with Mock but also with AxCEACD, and it should be noticed that this beneficial effect on tumor burden was obviously reflected in the prolongation of survival, although AxCEACD, which also showed a significant effect on the suppression of tumor growth, could not provide any survival benefit. The following reasons may account for the efficient enhancement of the Cre/loxP system in CD/5-FC gene therapy in in vivo treatment models: (a) the bystander effect (29) of CD/5-FC therapy is much stronger than that of HSV-TK/GCV therapy (30) because the neighbor cell killing effect of CD/5-FC therapy is mediated by rapid diffusion of 5-FU through cell membranes, and direct cell to cell contact is not required (31 , 32) ; and (b) 5-FU has been generally used for the treatment of patients with gastrointestinal cancers and has shown clinical efficiency (7 , 33) . In additional, the sensitivity of tumor cells to 5-FU was generally high (22) . The benefits described above were also confirmed by the results of in vitro experiments in this study, which showed that the Cre/loxP system enhanced the therapeutic efficacy much more remarkably in CD/5-FC suicide gene therapy than in HSV-TK/GCV therapy (15 , 24) .

Although tumor growth inhibition in the AxCACD/5-FC group was similar to that in the AxCEANCre and AxCALNLCD/5-FC group, two of five mice in the AxCACD/5-FC group had died by the end of 4 weeks after tumor implantation. These deaths in the early period were thought to be due to severe hepatic toxicity, which was strongly suggested by the elevated level of AST and ALT in the serum and by histological findings on the liver. Lan et al. (8) also reported that severe hepatic toxicity was observed when an Ad-expressing CD gene under the control of a strong CAG promoter, which has almost the same construct as that used in this study, was i.p. administered. On the other hand, no remarkable adverse effects were observed in the treatment with AxCEANCre and AxCALNLCD followed by 5-FC administration, suggesting that the specificity of the CEA promoter was strictly maintained and that CEA promoter activity was even enhanced. In the in vitro experiment, AxCACD infection at a moi of 50 brought about 30% tumor inhibition, even without 5-FC treatment. This result suggested that only expression of the CD gene in the nonspecific manner could cause the toxicity, which was much more severe when followed by 5-FC treatment. It was emphasized that the strategy of selective strong gene expression is very important for CD/5-FC gene therapy.

The evident efficacy of the application of the Cre/loxP system for the enhancement of selective CD/5-FC suicide gene therapy was demonstrated in this study, but there was a problem in that no complete tumor regressions were observed. Regression of primary tumors and metastatic tumors was found for less than 3 months, and the tumors increased thereafter in this study. The disadvantage of the Cre/loxP enhancing system is that two different vectors need to be transduced into the same cells at the same time, which obviously decreases the expression efficiency. Additional extensive studies are required for clinical applications such as developing gutted Ads (34) that allow two different expression cassettes of both regulator unit and target unit to be inserted in the same vector, leading to reduction of immunogenicity and improvement of transduction efficacy in vivo.

In conclusion, the application of the Cre/loxP system strongly enhanced the selective suicide gene therapy of CD/5-FC in CEA-producing tumor cells and achieved survival improvement in orthotopic implantation models of human gastric carcinoma, implying future clinical applications for the treatment of highly advanced gastric carcinoma.


   ACKNOWLEDGMENTS
 
We thank Dr. I Saito (Tokyo University, Tokyo, Japan) for cosmid vectors and Dr. J Miyazaki (Osaka University, Osaka, Japan) for the CAG promoter.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grant-in-Aid 11671180 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Back

2 To whom requests for reprints should be addressed, at Second Department of Surgery, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8510, Japan. Phone: 81-73-441-0613; Fax: 81-73-446-6566; E-mail: makoto{at}wakayama-med.ac.jp Back

3 The abbreviations used are: CD, cytosine deaminase; 5-FC, 5-fluorocytosine; Ad, adenovirus vector; 5-FU, 5-fluorouracil; CEA, carcinoembryonic antigen; moi, multiplicity of infection; pfu, plaque-forming unit(s); AST, aspartate aminotransferase; ALT, alanine aminotransferase; HSV-TK, herpes simplex virus thymidine kinase; GCV, ganciclovir. Back

Received 8/25/00. Accepted 6/ 7/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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