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Experimental Therapeutics |
Department of Molecular Medicine, Osaka University Medical School, Osaka 565-0871 [T. Kij., T. O., K. N., T. Ku., T. F., H. G., I. T.]; Department of Internal Medicine, Osaka Prefectural General Hospital, Osaka 558-0056 [Y. T.]; and Osaka University, Osaka 565-0871 [T. Kis.], Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, a strategy called virus-directed enzyme/prodrug therapy has been used as an alternative treatment for localized brain tumors in clinical trials and has demonstrated promising results (7 , 8) . When so-called "suicide" genes, such as the Escherichia coli CD gene and the HSV-TK gene, are expressed in cancer cells, they convert the nontoxic prodrugs 5-fluorocytosine and GCV, respectively, to toxic compounds and induce apoptosis of cancer cells (9) . In applying virus-directed enzyme/prodrug therapy to refractory CEA-producing cancer, disseminated cancer cells must be controlled without affecting normal cells. Cell type-specific promoters have been shown to be effective tools for targeting tumor cells (10, 11, 12, 13, 14, 15, 16, 17) . They induce tumor cell-specific death by selective expression of the CD or the HSV-TK genes in tumor cells. However, few reports have demonstrated sufficient antitumor effects, even when tumor cells were introduced with specific promoter-suicide chimera genes in vivo by an Ad, the most efficient vector for introducing foreign genes into target cells (13 , 16) . It is conceivable that the activity of cell type-specific promoters, including the CEA promoter, is too low to induce sufficient drug sensitivity in vivo. Richards et al. (18) reported that multimerization of a part of the CEA promoter alone or ligated with its specific enhancer sequences resulted in copy number-related increases in promoter activity. We also analyzed by the CAT assay whether the activity of the CEA promoter was augmented by ligation of the enhancer sequence which extends from -6.1 to -4.0 kb upstream regions from the transcriptional start of the CEA gene or by tandem ligation of four copies of the 420-bp fragment of the CEA promoter sequence. These fragments, however, did not contribute to a significant increase in the CAT activity in CEA-producing LoVo cells.4
Cre recombinase (Cre) derived from bacteriophage P1 is a Mr 38,000 protein and mediates the site-specific excisional deletion of a DNA sequence that is flanked by a pair of loxP sites and is composed of 34 nucleotides (19)
. In 
-fetoprotein-producing hepatocellular carcinoma cells, the Cre/loxP system contributed to an 
100-fold increase in the magnitude of the LacZ gene expression compared with that obtained by infection with an Ad expressing the LacZ gene directly driven by the -fetoprotein promoter (20)
. To obtain enhanced expression of the HSV-TK gene exclusively in tumor cells and subsequent significant in vivo antitumor effects, we applied the Cre/loxP system to HSV-TK/GCV therapy for CEA-producing cancer. We constructed a Cre-producing Ad driven by the CEA promoter (Ad.CEA-Cre) and another Ad designed for inducible expression of the HSV-TK gene by Cre (Ad.lox-TK). Coinfection with these Ads induced selective GCV sensitivity at a higher level in vitro and augmented in vivo antitumor effects in human CEA-producing tumors, as compared with those obtained by infection with Ad.CEA-TK alone, an Ad that expresses the HSV-TK gene under the control of the CEA promoter.
Here, we demonstrate that the Cre/loxP system significantly enhances antitumor effects of the cell type-specific gene therapy against CEA-producing cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CEA Production by Tumor Cells.
Cells grown in culture medium were washed, suspended in PBS, and homogenized with a sonicator at 50 W for 2 min. The CEA content in the cell lysate was measured by solid-phase RIA. The lower limit of the detection of CEA was 0.5 ng/ml The total protein concentration was assayed by Lowry’s method (Special Reference Laboratory, Tokyo, Japan). Each value represents the former concentration divided by the latter one (ng/mg protein).
CAT Assay.
The CAT assay was carried out as described previously (11)
. In brief, 5 µg of a plasmid with the SV40 promoter-CAT gene (pSV2CAT) or a plasmid with the CEA promoter-CAT gene (pCEACAT) was cotransfected with 10 µg of pSV-ß-galactosidase, a plasmid with the LacZ gene driven by the SV40 promoter as an internal control for transfection, into each cell line. 14C-labeled chloramphenicol and its acetyl form were quantitated in duplicate by liquid scintillation counting in Aquasol (DuPont-NEN, Boston, MA). The ratio of the count for the acetyl form to total count was calculated as the CAT activity. The CAT activity of pCEACAT was expressed as a percentage of that of pSV2CAT, a positive control plasmid.
Construction of Ads.
The recombinant replication-defective Ads were constructed by the cosmid-adenoviral DNA terminal protein complex method (21)
. The CEATK fragment prepared as the BglII-HindIII fragment from pCEATK (11)
was ligated into the SwaI site of pAxcw cassette cosmid and designated pAxosCEATK. pAxCAosTK and pAxCALNLosTK were constructed by subcloning the TK fragment from the BglII-PvuII site of pTK4 (11)
into the SwaI site of pAxCAwt and pAxCALNLw cassettes (20)
, respectively. The BglII-HindIII CEA promoter fragment from pCEACAT (11)
was also inserted into the SwaI site of pAxAwNCre cassette to produce pAxAosCEANCre (20)
. Cassette cosmids pAxcw, pAxCAwt, pAxCALNLw, and pAxAwNCre were all gifts from Dr. I. Saito (Tokyo University, Tokyo, Japan). Each expression cosmid and adenoviral DNA terminal protein complex were cotransfected into 293 cells by the calcium phosphate precipitation method to produce Ad through homologous recombination, and each Ad was isolated from a single plaque. As shown in Fig. 1A
, AxCAosTK (designated Ad.CAG-TK here), an Ad that contains the HSV-TK gene directly driven by the CAG promoter (consisting of the cytomegalovirus immediate early enhancer and a modified chicken ß-actin promoter; Ref. 22
), strongly expresses the HSV-TK gene in all types of infected cells, regardless of whether they produce CEA. The Ads AxosCEATK (Ad.CEA-TK) and AxAosCEANCre (Ad.CEA-Cre) are designed to express the HSV-TK and the Cre gene, respectively, exclusively in CEA-producing cells under the control of the CEA promoter. Ad.CEA-Cre possesses a SV40 poly(A) signal inserted upstream of the CEA promoter for prevention of nonspecific read-through transcription of the Cre gene and a NLS for keeping Cre recombinase inside the nucleus. AxCALNLosTK (Ad.lox-TK) is designed to express the HSV-TK gene under the control of the strong CAG promoter only after Cre excises the neomycin resistance gene in a loxP site-specific manner. AxCANLacZ (Ad.CAG-LacZ), with NLS-tagged ß-galactosidase (NLacZ) gene driven by the CAG promoter, and AxCALNLNZ (Ad.lox-LacZ), identical to Ad.lox-TK, except that the HSV-TK gene is replaced by the NLacZ gene, were also supplied by Dr. I. Saito (20
, 23) . Solutions of these Ads were prepared as described previously (24)
and stored at -80°C until use.
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In Vitro GCV Sensitivity of Tumor Cells Infected with Recombinant Ads.
One thousand A549 or 3 x 103 LoVo or VMRC-LCD cells were seeded in triplicate onto 96-well culture plates. Twenty-four h later, the culture medium was removed, and suspensions of Ad.CEA-TK alone, Ad.CAG-TK alone, or Ad.lox-TK in combination with Ad.CEA-Cre were distributed onto cell monolayers at a total moi of 30. After another 24 h of incubation, the medium was replaced with that containing various concentrations of GCV (F. Hoffmann-La Roche, Basel, Switzerland). After cultivation for an additional 6 days, MTT assay was carried out as described previously (11)
. The 50% growth inhibitory concentration (IC50) of GCV for each cell line was calculated using a curve-fitting parameter, and the results are represented as the means ± SD from three independent experiments. Student’s t test was used in the statistical analysis of IC50 in individual conditions. The difference was considered statistically significant at P < 0.05.
Animal Experiments.
On day 0, CEA-producing LoVo cells were inoculated s.c. into the flank of male BALB/c nu/nu mice at 5–7 weeks of age (Clea Japan, Tokyo, Japan). Mice were divided into eight groups: group I, Ad.CEA-Cre and Ad.lox-TK injection with GCV treatment; group II, Ad.CEA-Cre and Ad.lox-TK injection with 0.9% NaCl (saline) treatment; group III, Ad.CEA-TK injection with GCV treatment; group IV, Ad.CEA-TK injection with saline treatment; group V, PBS injection with GCV treatment; group VI, PBS injection with saline treatment; group VII, Ad.CEA-Cre and Ad.lox-LacZ injection with GCV treatment; and group VIII, Ad.CEA-Cre and Ad.lox-LacZ injection with saline treatment. Groups VII and VIII were excluded in the experiment shown in Fig. 4B
. When the average area of the inoculated tumors reached 20 mm2, Ads were injected i.t.. Each recombinant Ad was injected at a ratio of 1:1 in the coinfection groups (groups I, II, VII, and VIII). From the day after the first viral injection, mice received i.p. administration of 50 mg/kg body weight of GCV or saline twice a day for 2 weeks. The tumor size was measured every 3–4 days and expressed as the product of the perpendicular diameters of individual tumors. The treatment schedule including the number of inoculated cells and the dosage of Ads is specified in each experiment. The number of mice in each group is five in Fig. 4A
and seven in Fig. 4B
. Each animal experiment was repeated twice. Results are presented as the means ± SD. Student’s t test was used in the statistical analysis of the size of tumors in individual groups. The difference was considered statistically significant at P < 0.05.
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RESULTS |
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. LoVo and VMRC-LCD cells produced significant amounts of CEA protein (77.2 ± 12.7 and 204.1 ± 43.5 ng/mg protein, respectively), whereas secretion from A549 cells was below the detectable level. The activity of the CEA promoter was also analyzed using CAT assay (Table 1)
. The CAT activities of pCEACAT in LoVo and VMRC-LCD were 98.2 ± 0.7 and 61.7 ± 3.4% of that of pSV2CAT, respectively, whereas it was very low in A549 cells (4.2 ± 0.4%). A549 produced a significant amount of CEA in our previous experiments (11)
, but it became a non-CEA-producing cell line during a long-term cultivation. These results suggest that the CEA promoter shows selective activity in CEA-producing cancer cells, although the activity is not so strong.
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illustrates the mechanisms activating the HSV-TK gene specifically in CEA-producing cancer cells. When CEA-producing cells are infected simultaneously with Ad.CEA-Cre and Ad.lox-TK, the former Ad produces Cre recombinase under the control of the CEA promoter. Then the produced Cre excises the neomycin resistance gene between a pair of loxP sites out of the latter Ad and activates the HSV-TK gene driven by the strong CAG promoter. Therefore, administered GCV is phosphorylated and induces cell death exclusively in CEA-producing cells.
Determination of the Ideal Ratio of the Paired Ads to Achieve Maximum Expression of the LacZ Gene Using the Cre/loxP System.
To maximize the expression of the LacZ gene using the Cre/loxP system in vitro, we analyzed the ideal ratio of Ad.CEA-Cre to Ad.lox-LacZ. Cre released from Ad.CEA-Cre activates the LacZ gene driven by the CAG promoter. Human adenocarcinoma cell lines, LoVo and A549, were infected simultaneously with these coupled Ads at various ratios at a total moi of 30. The LacZ gene expression was quantified by ß-galactosidase assay (Fig. 2)
. In CEA-producing LoVo cells, the strongest ß-galactosidase activity (7.1 ± 0.3 µunits/cell) was obtained with Ads at a ratio of 1:1. No nonspecific ß-galactosidase activity was detected on infection with Ad.lox-LacZ alone. Moreover, in non-CEA-producing A549 cells, the ß-galactosidase activity remained at very low levels, regardless of the ratios of the paired Ads. The ß-galactosidase activity at each ratio increased in proportion to the total moi and reached the maximum level at a total moi of 30 (data not shown). Significant cell toxicity caused by infection with Ads was observed at a moi higher than 30 (data not shown). These results show that the Cre/loxP system allows specific expression of the LacZ gene in CEA-producing cells and confers the highest level of expression when these coupled Ads are used at a ratio of 1:1.
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. The lowest IC50 was obtained in both LoVo and VMRC-LCD cells when coinfection with the paired Ads was performed at a ratio of 1:1. These results were compatible with those obtained by infection with Ad.CEA-Cre and Ad.lox-LacZ (Fig. 2)
. The IC50s of each cell line (0.17 ± 0.04 and 1.40 ± 0.50 µM, respectively) were 8.4- and 3.3-fold lower than that obtained by infection with Ad.CEA-TK alone (1.43 ± 0.21 and 4.63 ± 0.71 µM, respectively; P < 0.01) and 2488- and 316-fold lower than the values for nontransduced cell lines (423.4 ± 40.4 and 443.3 ± 55.1 µM, respectively). The IC50s of these CEA-producing cells conferred by the Cre/loxP system were comparable with those obtained by infection with Ad.CAG-TK, which rendered all of these cell lines highly sensitive to GCV, regardless of CEA production. In LoVo and VMRC-LCD cells, the lowest IC50s were only 2.1- and 1.5-fold higher than the value for those infected with Ad.CAG-TK (0.17 ± 0.04 and 0.08 ± 0.02 µM, respectively). Infection with Ad.lox-TK alone did not significantly change GCV sensitivity in CEA-producing cells. Moreover, coinfection with Ad.CEA-Cre and Ad.lox-TK did not induce significant change in sensitivity of non-CEA-producing A549 cells. These results show that coinfection with Ad.CEA-Cre and Ad.lox-TK remarkably augments selective GCV sensitivity in CEA-producing cancer cells compared with infection by Ad.CEA-TK alone and that the magnitude of enhancement in GCV sensitivity correlates with the activity of the CEA promoter rather than the amount of CEA protein produced (Table 1)
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. When a low dose of Ads (2x108 pfu per injection) was injected (Fig. 4A)
, significant regression of tumor growth was observed only in the mice treated with Ad.CEA-Cre and Ad.lox-TK followed by GCV (group I; group I versus groups III-VIII, P < 0.005). In three of five mice (60%) in this group, tumors were completely eradicated, and two other mice showed transient regression of tumor during GCV treatment. Nevertheless, the other treatment including Ad.CEA-TK/GCV (group III) did not have any significant effects on tumor growth (group III versus group VII, P > 0.05). To further augment antitumor effects, we administered an increased dose of Ads to the mice (5 x 108 pfu per injection; Fig. 4B
). All mice treated with Ad.CEA-Cre and Ad.lox-TK followed by GCV injection (group I) showed immediate regression of inoculated tumors. Six of seven mice (86%) became free of palpable tumor by the end of the treatment schedule, and the tumor in the rest of the mice in this group remained a small nodule. On the other hand, Ad.CEA-TK/GCV (group III) as well as Ad.CEA-Cre and Ad.lox-TK/saline treatment (group II) slightly retarded the growth of tumors but did not eradicate them (group III versus group IV, P < 0.05, from day 24 to day 44, and group II versus group VI, P < 0.01, from day 24 to day 44, respectively). Thus, antitumor effects observed in the Ad.CEA-Cre and Ad.lox-TK/GCV treatment group (group I) were more remarkable compared with those of the Ad.CEA-TK/GCV treatment group (group III; group I versus group III, P < 0.0001). We did not observe any apparent side effects including body weight loss or death in these treatment cycles. These results suggest that the Cre/loxP system remarkably enhances antitumor effects on CEA-producing tumors in this cancer gene therapy model. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The activity of the CEA promoter in CEA-producing LoVo cells was comparable with that of the SV40 promoter determined by CAT assay (Table 1)
. Ad.CEA-TK rendered LoVo cells 296-fold more sensitive to GCV in vitro in comparison with uninfected cells (Fig. 3)
. However, repeated i.t. injection of Ad.CEA-TK followed by administration of GCV at most retarded the growth of the inoculated tumors (Fig. 4)
. On the other hand, i.t. injection of Ad.CAG-TK, which has the strong CAG promoter, with GCV treatment completely eradicated s.c. LoVo tumors in all animals.4 It is conceivable that expression of the HSV-TK gene at a higher level is necessary for complete rejection of tumors. To augment selective expression of the HSV-TK gene in CEA-producing cells, we applied the Cre/loxP system. Coinfection with Ad.CEA-Cre and Ad.lox-TK significantly enhanced the sensitivity of CEA-producing LoVo and VMRC-LCD cells to GCV. The IC50s of these cells were only 2.1- and 1.5-times as high as those of Ad.CAG-TK-transfected LoVo and VMRC-LCD cells, respectively. Moreover, infection with Ad.lox-TK alone did not induce nonspecific GCV sensitivity in CEA-producing cells, nor did coinfection with these Ads significantly change GCV sensitivity in Non-CEA-producing cells (Fig. 3)
. GCV treatment following i.t. injection of Ad.CEA-Cre combined with Ad.lox-TK induced immediate regression of established CEA-producing LoVo tumors and resulted in complete tumor rejection in six of seven animals (Fig. 4B)
. This treatment significantly suppressed the growth of the other CEA-producing VMRC-LCD tumors as well in all mice but did not show any antitumor effects on non-CEA-producing A549 tumors (data not shown). The following reasons might account for the enhanced antitumor effects. (a) A limited amount of induced Cre recombinase effectively processes a large number of molecules on the target Ad genome (23
, 26)
. (b) The activity of the CAG promoter is far stronger than that of the CEA promoter. In fact, Ad.CAG-TK made LoVo cells 18-fold more sensitive to GCV compared with Ad.CEA-TK (Fig. 3)
. (c) Cre is efficiently activated inside the nucleus by NLS with an effective initiator codon of Ad.CEA-Cre (23)
.
Marginal retardation of tumor growth was observed in mice injected i.t. with a higher dose of Ad.CEA-Cre combined with Ad.lox-TK then administered saline (Fig. 4B)
. When LoVo cells were coinfected with Ad.CEA-Cre and Ad.lox-TK or infected with Ad.CAG-TK alone in vitro at a total moi of 30, the viability of the infected cells decreased to 70 or 50% of that of uninfected cells, respectively (data not shown). Other reports also showed that i.t. injection of Ads containing the CAG promoter significantly retarded the growth of s.c. tumors (16)
. We speculate that infection by a higher dose of Ads with the CAG promoter interferes with the transcription of genes necessary for cell survival and induces apoptosis (12
, 16)
.
Death related to severe hepatic toxicity is reported when animals receive administration of Ad expressing the HSV-TK gene driven by the cytomegalovirus promoter followed by GCV treatment (27 , 28) . In another report, reverse transcriptase-PCR analysis detected expression of transduced genes predominantly in the liver, the kidneys, and the lung, and histological examination revealed zonal necrosis in the periportal area of the liver when an Ad expressing the CD gene under the control of the CAG promoter was i.p. administered then 5-fluorocytosine given (29) . We did not observe any side effects including elevation of transaminase or creatinine in the serum of animals treated with i.p. injection of Ad.CEA-Cre and Ad.lox-TK followed by GCV administration (data not shown). Thus, application of the Cre/loxP system is expected to not only enhance antitumor effects at a reduced dosage of Ads but also avoid damage to normal tissues. Another advantage in application of this system is that it might be no longer needed to specify the enhancer or silencer sequences in the genome for each cell type-specific promoter. Our ultimate goal is to control metastasized tumor cells without affecting normal cells. We are now planning to evaluate the efficacy and side effects of this system in the peritonitis carcinomatosa model in mice, mimicking pleuritis for lung cancer and peritonitis for gastrointestinal cancers in future clinical applications to human beings.
Recently, a combination of HSV-TK/GCV gene therapy with cytokines, including interleukin 2 (30 , 31) , interleukin 4 (32) , and granulocyte-macrophage colony-stimulating factor (31) , has been shown to enhance antitumor effects. Modified Ad.lox-TK in which the neomycin resistance gene is replaced by these cytokine genes would enhance antitumor effects. When these Ads are used simultaneously with Ad.CEA-Cre, not only is the HSV-TK gene expressed specifically in CEA-producing tumor cells, but the cytokines released nonspecifically from surrounding normal cells also induce antitumor immunity.
In conclusion, the Cre/loxP system augments possible clinical application of gene therapy specific for CEA-producing cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture in Japan. 
2 To whom requests for reprints should be addressed, at Department of Molecular Medicine, Osaka University Medical School, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan.
3 The abbreviations used are: CEA, carcinoembryonic antigen; CD, cytosine deaminase; HSV-TK, herpes simplex virus thymidine kinase; GCV, ganciclovir; Ad, adenoviral vector; CAT, chloramphenicol acetyltransferase; TK, thymidine kinase; NLS, nuclear localization signal; moi, multiplicity of infection; pfu, plaque-forming unit(s); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; i.t., intratumoral(ly).
4 T. Kijima, unpublished observations.
Received 2/18/99. Accepted 8/ 6/99.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-fetoprotein-producing hepatocellular carcinoma by adenovirus-mediated transfer of cytosine deaminase gene. Cancer Res., 57: 461-465, 1997.This article has been cited by other articles:
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, adenoviral DNA terminal protein complexes; 
, deletions of adenoviral early regions. CAG, CAG promoter; CEA, CEA promoter; NCre, NLS-tagged Cre gene; SpA, SV40 early poly(A) signal; GpA, rabbit ß-globin poly(A) signal; *, loxP site. B, the Cre/loxP system mediates high level expression of the HSV-TK gene specifically in CEA-producing cancer cells. Cre recombinase expressed by Ad.CEA-Cre excises the neo gene with SpA at the loxP sites out of Ad.lox-TK and activates the HSV-TK gene driven by the strong CAG promoter.
, group I, Ad.CEA-Cre and Ad.lox-TK injection with GCV treatment; 
, group II, Ad.CEA-Cre and Ad.lox-TK injection with saline treatment; •, group III, Ad.CEA-TK injection with GCV treatment; 
, group VI, PBS injection with saline treatment; 
, group VII, Ad.CEA-Cre and Ad.lox-LacZ injection with GCV treatment; 
, group VIII, Ad.CEA-Cre and Ad.lox-LacZ injection with saline treatment. Each Ad was injected at a ratio of 1:1 in the coinfection groups (groups I, II, VII, and VIII). Data points, tumor area, shown as the mean of the products of the perpendicular diameters of each tumor; bars, SD. The difference was considered statistically significant at P < 0.05. N.S., not significant. The numbers of inoculated LoVo cells were 2 x 106 (A) and 3 x 106 (B). Ad was i.t. injected at 2 x 108 pfu on days 8, 15, and 18 (A) and 5 x 108 pfu on days 3, 8, and 13 (B). GCV was i.p. administered at 50 mg/kg twice a day on days 9–22 (A) and on days 4–17 (B). The numbers of mice in each group were n = 5 (A) and n = 7 (B). Groups VII and VIII were excluded in B. Ad.CEA-Cre and Ad.lox-TK/GCV treatment (group I) completely eradicated tumors in three of five (A) and six of seven (B) mice, but Ad.CEA-TK/GCV treatment (group III) at most retarded the growth of inoculated tumors (B).
), Ad.CEA-TK alone (
), or Ad.lox-TK in combination with Ad.CEA-Cre at various ratios (