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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 8 3531-3538
Copyright © 2003 by The Endocrine Society

Tumor-Specific Gene Therapy for Undifferentiated Thyroid Carcinoma Utilizing the Telomerase Reverse Transcriptase Promoter

Teiji Takeda, Hidehumi Inaba, Masanori Yamazaki, Satoru Kyo, Takahide Miyamoto, Satoru Suzuki, Takashi Ehara, Tomoko Kakizawa, Masahiro Hara, Leslie J. DeGroot and Kiyoshi Hashizume

Department of Aging Medicine and Geriatrics (T.T., H.I., T.M., S.S., T.K., M.H., K.H.), Shinshu University, Graduate School, Matsumoto, Nagano 390-8621, Japan; Thyroid Study Unit (M.Y., L.D.G.), Department of Medicine, University of Chicago, Chicago, Illinois 60637; Department of Obstetrics and Gynecology (S.K.), Kanazawa University, School of Medicine, Kanazawa, Ishikawa 920-0934, Japan; and Department of Pathology (T.E.), Shinshu University, School of Medicine, Matsumoto, Nagano 390-8621, Japan

Address all correspondence and requests for reprints to: Teiji Takeda, M.D., Ph.D., Department of Aging Medicine and Geriatrics, Shinshu University, Graduate School, 3-1-1 Asahi Matsumoto-city, Nagano-prefecture 390-8621, Japan. E-mail: teiji{at}hsp.md.shinshu-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A tumor-specific targeting system for cancer gene therapy was studied using the human telomerase reverse transcriptase (hTERT) promoter. Telomerase activity is increased in most tumors but not detected in most normal cells. We developed the recombinant adenovirus, carrying human herpes simplex virus thymidine kinase gene under the control of the hTERT promoter (AdhTERTtk) to obtain restricted expression of a suicide gene only in tumor cells. We found that transcriptional activity of hTERT was 2- to 9-fold higher in undifferentiated thyroid carcinoma cell lines than that of the Simian virus 40 promoter in transient transfection assay. Undifferentiated thyroid carcinoma cell lines were infected with AdhTERTtk, and sensitivity to ganciclovir (GCV) was analyzed. Cell viability was decreased in a GCV dose-dependent manner after treatment with AdhTERTtk/GCV. The cell-killing ability of AdhTERTtk in all thyroid or nonthyroid carcinoma cell lines tested was similar to AdCMVtk, which carries herpes simplex virus thymidine kinase gene driven by the cytomegalovirus promoter. However, normal cell lines were largely unaffected by AdhTERTtk/GCV, whereas these cells were also sensitive to GCV after infection with AdCMVtk. A xenograft model was established by transplanting human differentiated or undifferentiated thyroid carcinoma cells into Balb-C nude mice. The injections of AdhTERTtk into tumors and ip administration of GCV showed significant inhibition of tumor growth, similar to AdCMVtk/GCV treatment. Systemic administrations of adenovirus and GCV to normal rats demonstrated remarkable increase of serum liver transaminase levels and severe hepatic damages in pathological examinations in AdCMVtk-injected rats but not in the AdhTERTtk group. These results indicate that the AdhTERTtk/GCV system is a promising therapy for undifferentiated thyroid carcinoma, which is one of the most malignant tumors, without damage to normal tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID CARCINOMAS ARE the most common endocrine neoplasia. They are pathologically classified into papillary, follicular, anaplastic, medullary cancers, and malignant lymphoma. Papillary and follicular thyroid cancers are the two most frequent entities, usually referred to as differentiated thyroid carcinoma, with frequencies of 50–70% and 10–15%, respectively, of all thyroid carcinomas (1). Anaplastic thyroid carcinoma referred to undifferentiated thyroid carcinoma (UTC) is one of the most aggressive and lethal human cancers. Recent studies show that UTC constituted from 1.0% to 7.5% of thyroid cancers in the United States and Europe (2). The majority of UTC patients present with a rapidly growing thyroid mass. Clinicians may observe tumors double in volume over a period of several days to a week. Nearly half of the patients have symptoms of hoarseness and dyspnea, with some noting dysphagia or cervical pain. More than 50% of patients with UTC have distant metastasis at presentation, of which around 80% are in the lung, 15% in bone, and 13% in the brain (3, 4). This probably underestimates the predilection for aggressive distant spread because an autopsy study of 15 UTC patients revealed all to have pulmonary metastases, 80% with bone metastases, 60% to soft tissue of the neck, 27% to trachea, and 53% with other soft tissue metastases (5). With rare exceptions, nearly all patients with UTC die from their tumor within several months from the time of diagnosis.

Total thyroidectomy may be attempted as soon as a diagnosis of UTC has been made. However, in most cases, infiltration of the soft tissues, or metastasis to lymph nodes or lungs, makes radical surgery impossible. Results of external radiotherapy and chemotherapy with several protocols are disappointing. The combination of radiotherapy and chemotherapy was tried as an initial treatment to control and reduce the primary tumor, giving the surgeon more chance to perform a radical thyroidectomy (1). The effect of these efforts are modest and limited. Thus, a novel approach of treatment for UTC is needed.

The suicide gene/prodrug system is one of the common strategies for cancer gene therapy. Several methods have been developed for delivering human herpes simplex virus thymidine kinase (HSVtk) genes to mammalian cells (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18). Recombinant adenoviruses, which have lost replicating activity, have advantages in gene delivering. They can be produced in high titers, and infection transduces genes with high efficacy in nearly every tissue without integration of the transduced genes into genomic DNA of host cells. Another advantage is that a favorable promoter can be chosen for the expression of transduced genes with this system. Several groups have reported adenovirus-mediated gene therapy models using HSVtk expression driven by strong nonspecific promoters (6, 7, 8, 9, 10, 11, 12, 13) or promoters with restricted expression (14, 15, 16, 17, 18).

As is well documented, tumor- or tissue-specific promoters are suitable for virus infection-induced gene therapy for cancers. However, promoters that can express transduced genes in a restricted manner in target cells may be too weak to kill the cells. To increase the cytotoxic sensitivity in thyroid cancers, many trials for improvement of methods have been studied. The Cre-loxP system has been introduced into adenovirus-mediated gene therapy (19) to enhance activity of thyroglobulin (TG) promoter-keeping tissue specificity (20). A specific enhancer sequence was fused to the TG promoter to enhance the promoter activity (21). A combination treatment with histone deacetylase inhibitor and sodium butyrate increased cell-killing effect of the HSVtk gene in vitro (21). We also demonstrated that a tandemly repeated TG core promoter enhanced cytotoxicity without losing tissue specificity for TG-producing thyroid cancers by HSVtk genes both in vitro and in vivo (22). These studies confirmed that infection with recombinant adenoviruses loading TGtk genes is available for gene therapy in TG-producing thyroid carcinomas. However, these strategies should not be effective for the treatment for UTC because UTC produces no or little TG.

Telomerase is a ribonucleoprotein enzyme that plays an important role for the replication of chromosomal ends or telomeres (23). Telomerase is highly active in approximately 90% of malignant tumors but is inactive in most normal cells (24, 25). The enzyme is composed of an essential RNA template (26), telomerase-associated protein (27, 28), and human telomerase reverse transcriptase (hTERT) (29, 30). Recently, the hTERT gene was cloned by several groups and found to have a key role in telomerase activity (31, 32). The hTERT expression is highly correlated with telomerase activity (29, 30) and extends the life span of human cells (33).

The promoter region of the hTERT gene has also recently been cloned and characterized (34, 35, 36). The promoter region is highly G/C rich and contains several binding sites for transcriptional factors, such as Sp1 and c-Myc, although it lacks TATA and CAAT boxes. Deletion analysis of the hTERT promoter revealed that a core promoter region, including approximately 200 bp upstream of the transcription start site, was most important for transcriptional activity in cancer cell lines but was not activated in normal cells. Tumor-specific gene therapy, using the hTERT promoter, which is highly activated in cancer cells but repressed in most normal cells, has been tried by several groups. The HSVtk (37), Bax (38, 39), Fas-associated death domain (FADD) (40), caspase-6 (41), caspase-8 (42), and nitro reductase (43) genes were expressed by the hTERT promoter.

In the present study, we studied tumor-specific gene therapy for UTC both in vitro and in vivo using the hTERT promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and cell cultures

FRTL5 cells, a normal rat thyroid cell line (44), WI-38 cells, established from normal human fibroblasts, and 293 cells, a human embryonal kidney cell line, were purchased from American Type Culture Collection (Manassas, VA). FTC-133 cells, a human follicular thyroid carcinoma cell line, were purchased from European Collection of Animal Cell Cultures (Wiltshire, UK). Continuous cell lines, 8505c and 8305c cells, derived from human UTCs, were obtained from Health Science Research Resources Bank (Osaka, Japan).

FRTL5 cells were grown in HamF-12K medium supplemented with 5% calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin (PCSM), and six hormones or growth factors (6H) (4 U/liter bovine TSH, 5 U/liter insulin, 5 U/liter transferrin, 2.5 nM hydrocortisone, 10 µg/liter somatostatin, and 10 µg/liter glycyl-L-histidyl-L-lysine acetate) as the cell bank recommended. The 293 cells were maintained in DMEM containing 10% fetal bovine serum (FBS), and PCSM. FTC-133 cells were grown in DMEM/F-12 supplemented with 10% FBS, PCSM, and 6H. Cultured 8505c and 8305c cells were grown in MEM, supplemented with 10% FBS, PCSM, and 6H. ARO cells, derived from a human UTC (45), were maintained in RPMI1640 medium containing 10% FBS, PCSM, and 6H. Rat fibroblasts were derived from skin of Fisher rats (Nippon SLC Co., Hamamatsu, Japan) and maintained in DMEM with 10% FBS, PCSM, and 6H.

Plasmid construction and preparation of recombinant adenovirus

The plasmid pBS-tk, which includes the HSVtk gene fused to a bovine GH gene polyadenylation signal (11), was a kind gift from Dr. S. L. C. Woo (Baylor College of Medicine, Houston, TX). The recombinant adenovirus, AdTKluc (46), carrying the luciferase gene driven by the thymidine kinase (TK) promoter, was kindly provided by Dr. S. Refetoff (University of Chicago, Chicago, IL). The plasmids, p{Delta}E1sp1B, pCA14, pBHG10, and pJM13, were purchased from Microbix Biosystems Inc. (Toronto, Canada).

To make the shuttle plasmid carrying the HSVtk gene driven by the hTERT promoter, the hTERT core promoter region (-181 to +77) was cut out from reporter plasmid, pGL3-181 (34), at Kpn I and Bgl II sites. This fragment was exchanged for the TG promoter of pBluS-TGtk (22), including the HSVtk gene under the control of the TG promoter, at Kpn I and BamH I sites (pBluS-hTERTtk). The Kpn I-Xba I fragment of the hTERTtk was inserted into pGEM7Z (Promega, Madison, WI) at the corresponding sites of the multiple cloning site. Then the BamH I-Xba I fragment, including the HSVtk gene driven by the hTERT core promoter, was subcloned into the multiple cloning site of p{Delta}E1sp1B (p{Delta}E1-hTERTtk). The hTERTtk gene was inserted reversely to the adenovirus sequence.

To generate replication-defective recombinant adenoviruses, p{Delta}E1-hTERTtk, was cotransfected with pBHG10, which includes the sequence of human adenovirus type 5 lacking E1 and E3 regions, into 293 cells by the calcium-phosphate precipitation method to produce AdhTERTtk. We created recombinant adenoviruses, carrying HSVtk genes under the control of the cytomegalovirus (CMV) promoter or the tandemly repeated TG promoters as previously reported (47). Each recombinant adenovirus was isolated from a single plaque. Integrity of the construct was checked by DNA digestion with proper restriction enzymes. Recombinant adenoviruses were expanded in 293 cells and purified by double cesium chloride gradient ultracentrifugations. After dialysis, the adenoviruses were stored in 10% glycerol at -80 C. Plaque-forming assays were repeated more than 3 times to determine the titer of each recombinant adenovirus.

Transient transfection and reporter assay procedures

FRTL5 cells were plated in 24-well cell culture plates at densities of 4 x 104 cells/well 24 h before transfection. The reporter plasmids, pGL3-2xTGluc (22) and pGL3-181 (34) (500 ng/well) were transfected using the calcium-phosphate precipitation method. The same amount of pGL3-basic or pGL3-promoter plasmids (Promega) were also transfected as negative and positive controls, respectively. A plasmid-expressing ß-galactosidase (ß-gal) (Promega) (250 ng/well) was cotransfected for evaluating transfection efficiency. Cells were incubated with 0.5 ml medium containing transfection mixture for 12 h, and medium was changed. Incubation was continued for an additional 24 h. Then, medium was discarded and cells were washed twice with PBS. Cells in each well were harvested with 10 mM TrisCl (pH 7.4), 1 mM EDTA, and 150 mM NaCl and lysed with 50 µl of reporter assay reagent (Promega). Cell lysate (10 µl each) was used for luciferase and ß-gal assay. Luciferase activity was determined by luciferase assay system (Promega) using Lumat LB9501 (Berthold Japan KK, Tokyo, Japan). Assay for ß-gal activity was performed by the method described previously (48).

Cultured cells (WI-38, FTC-133, 8505c, ARO, and rat fibroblasts) were plated in 24-well culture plates at densities of 2 x 104 cells/well 24 h before transfection. The methods of cotransfection and reporter assays were the same as for FRTL5 cells. All data of luciferase activity were corrected for ß-gal activity to account for variation in transfection efficiency and expressed as a percentage of results using the pGL3-promoter. Each transfection was conducted in triplicate, and data represent the mean + SD from more than three individual experiments.

In vitro cytotoxic effect by adenovirus/GCV

Cultured cells were plated in 96-well plates at densities of 2 x 103 cells/well 12 h before infection with adenovirus. Cells were infected with 10 multiplicity of infection (MOI) of adenovirus for 2 h in minimal volume of medium supplemented with 2% FBS, PCSM, and 6H. Medium was changed to one containing 5% calf serum, PCSM, and 6H in the presence of various concentrations (0–100 µM) of ganciclovir (GCV) (F. Hoffmann-La Roche Ltd., Basel, Switzerland) and incubation was continued for 4 d. Then dimethylthiazoldiphenyltetra-zoliumbromide assay was performed by CellTiter 96TM nonradioactive cell proliferation assay (Promega), according to the protocol from the manufacturer, using Microplate reader model 550 (Bio-Rad, Hercules, CA). We determined that infection with 100 MOI of adenovirus for 12 h was suitable for FRTL5 cells because infectivity of adenovirus for FRTL5 cells was much lower than for other cell lines, as we reported previously (22). FRTL5 cells were plated in 96-well plates at higher densities (5 x 103 cells/well). The protocol after infection of FRTL5 cells with adenovirus was the same as for other cell lines.

Tumor growth inhibition by adenovirus/GCV in vivo

To establish a tumor-bearing animal model, cultured ARO or FTC-133 cells were harvested and injected sc into a Balb-C nu/nu mouse (Nippon SLC Co., Hamamatsu, Japan) using 5 x 106 cells in 50 µl PBS. Tumor volumes were measured with vernier calipers and calculated from the following formula: (length x width x height/2), which is derived from the formula for an ellipsoid (pd3/6). When the tumors reached an average size of 200 mm3, they were injected with 1 x 109 plaque-forming units (pfu) of AdTKluc, AdCMVtk, or AdhTERTtk at d 0 with a 1-ml insulin syringe. Intraperitoneal injections of GCV [80 mg/kg body weight (BW)] were started 24 h after the adenovirus injection at d 1. Administration of GCV was continued once a day until d 14. In other experiment, 100 or 200 mg/kg BW of GCV were given to the tumor-bearing nude mice to estimate the effect of doses of GCV for the suppression of tumor growth after infection with AdhTERTtk. Tumor volumes were measured as described above every 2 or 3 d. The data of tumor volumes were expressed as the percentage relative to the values of d 0 just before the injection of adenovirus in each group. Each point and bar shows the mean ± SE from more than five mice in each group.

Toxic effect by infection with adenoviruses

Fisher male rats were purchased from Nippon SLC Co. and maintained under standard conditions. Each adenovirus was injected into a jugular vein of a rat at the dose of 5 x 109 pfu in 200 µl PBS. GCV was given ip to rats infected or noninfected with AdCMVtk, AdhTERTtk, or Ad2xTGtk, 24 h after viral injection at the dose of 100 mg/kg BW per day. Administration of GCV was continued up to d 5. Three hundred microliters of blood was taken from the jugular vein of each rat 3 d after starting GCV treatment to measure serum glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels using a commercial kit (Sigma, St. Louis, MO). Rats were killed 24 h after the last GCV injection, and tissues of liver, kidney, spleen, testis, and thyroid were taken and fixed in 30% chloroform. The tissue specimens were embedded in paraffin, sectioned, and stained with hematoxylin-eosin for pathological examination.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transcriptional activation induced by the hTERT promoter

Transcriptional activity induced by the hTERT promoter was estimated by using the transient transfection system as related in Materials and Methods. When the luciferase activity, which was induced by the reporter plasmid containing the luciferase gene driven by the Simian virus 40 promoter (pGL3-promoter) (Promega), was defined as 100%, the activity induced by the hTERT promoter was increased to more than 200% in cancer cell lines (Fig. 1Go). Luciferase activity of 850% was observed in ARO cells, derived from a human UTC. Approximately 80% and 25% of the activity for the control was obtained in FRTL5 and a human fibroblast cell line (WI-38), respectively. When a tandemly repeated TG promoter was used (2xTGluc), the activity increased in FRTL5 cells and human follicular thyroid cancer cell lines but not in UTC cell lines. These results indicate that hTERT promoter works in a tumor-specific manner and has potential to strongly express a transgene in UTC cells, although the 2xTG promoter is not expected to work efficiently in UTCs.



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  		FIG. 1. Cell-specific transcriptional activity by the hTERT promoter. Indicated pGL3-hTERTluc, pGL3-2xTGluc, or pGL3-promoter plasmids were cotransfected with ß-gal plasmids into each cell line, and luciferase and ß-gal activity was determined, as described in Materials and Methods. The following cell lines were tested: WI-38 (human fibroblast), FRTL5 (rat normal thyroid), FTC-133 and WRO (human follicular thyroid carcinoma), 8505c, 8305c and ARO (human UTC), HepG2 (human hepatoma), COS1 (monkey kidney carcinoma), and dRLH-84 (rat hepatoma). After 24-h incubation, luciferase and ß-gal activity was determined. All data of luciferase activity were corrected for ß-gal activity to account for variation in transfection efficiency and expressed as percentages of the results using pGL3-promoter (shown as the control in the figure). Each transfection was conducted in triplicate, and data represent the mean + SD from more than three individual experiments.

 
Cytotoxic effect of GCV on cells infected with recombinant adenovirus

Recombinant expression vectors were constructed as shown in Fig. 2Go, and replication-defective recombinant adenoviruses were generated in 293 cells as related in Materials and Methods. The time course of cell viability in the presence of 100 µM GCV was estimated in FRTL5 cells infected with AdCMVtk. As a control, we used cells infected with AdTKluc. In a study of time course, GCV-induced cytotoxicity was significantly greater in the CMVtk group than that in the control during 2–6 d of incubation of the cells with GCV. A significant decrease in cell viability was observed from the second day of incubation in the CMVtk group (data not shown).



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  		FIG. 2. Schematic representation (not drawn to scale) of the structure of the plasmids and adenovirus vectors. The p{Delta}E1-hTERTtk carries the hTERT promoter fragment (from -181 to +71), which was fused to the HSVtk gene containing bovine GH gene polyadenylation signal (pA). The p{Delta}E1-CMVtk carries the same HSVtk gene fused to the CMV promoter. The p{Delta}E1-2xTGtk carries a tandemly repeated TG promoter, fused to the HSVtk gene. These plasmids were cotransfected into 293 cells with adenovirus plasmid, pBHG10 (E1 and E3 deleted) or pJM13 (E1 deleted), to generate replication-defective recombinant adenovirus. The terms in parentheses are the names of recombinant adenoviruses.

 
Cell-specific viability was examined in normal cell lines, FRTL5 (Fig. 3AGo), WI-38 (Fig. 3BGo), and rat fibroblasts (Fig. 3CGo), and thyroid cancer cell lines, ARO (Fig. 3DGo), 8505c (Fig. 3EGo), and FTC-133 (Fig. 3FGo). Cells were infected with 10 MOI (in case of FRTL5, 100 MOI were infected) of each adenovirus and incubated with 0–100 µM GCV for 4 d. Cell viability in the control group (TKluc) was not affected by GCV in any cell line. In contrast, the viability after AdCMVtk infection was decreased in all cells. Infection with AdhTERTtk showed decreased cell viability in thyroid cancer cell lines in a GCV dose-dependent manner, and the decrease was significant at 1.0 µM of GCV. In normal cell lines, however, decrease of cell viability by hTERTtk/GCV was not observed. The IC50s obtained by GCV treatment were 2 and 3 µM in ARO cells, 0.3 and 1 µM in 8505c cells, and 0.5 and 1.3 µM in FTC-133 cells after infection with AdCMVtk and AdhTERTtk, respectively. These results indicate that HSVtk/GCV system is useful for induction of tumor-specific cytotoxicity when AdhTERTtk is used as a vector and that the cytotoxic potency by AdhTERTtk is similar to that induced by AdCMVtk.



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  		FIG. 3. GCV dose-dependent cytotoxicity after infection with recombinant adenovirus. The cell lines used were described in Fig. 1Go. FRTL5 (A) or other indicated cells (B–F) were infected with 100 or 10 MOI of adenoviruses, respectively, and treated with GCV at indicated concentrations in 96 well-plates. Four days after infection, cell viability was measured as described in Materials and Methods. All data of cell viability were expressed as a percentage relative to the untreated controls in the absence of GCV. Each point and bar shows the mean ± SD from more than three independent experiments. Open square, TKluc; closed circle, CMVtk; open triangle, hTERTtk; closed triangle, 2xTGtk.

 
When Ad2xTGtk was used to infect FRTL5 and FTC-133 cells, cytotoxicity was induced after treatment with GCV, and the potency was similar to that with AdCMVtk. However, Ad2xTGtk/GCV did not induce cytotoxicity for ARO and 8505c cells. This result suggests that Ad2xTGtk cannot be useful for gene therapy for UTCs. Infection with AdhTERTtk to cancer cell lines originated from other tissues was also performed, and cells were incubated with GCV for 4 d. dRLH-84 (rat hepatoma), COS 1(monkey kidney carcinoma), HepG2 (human hepatoma), and Panc-1 (human pancreatic carcinoma) were infected with adenoviruses and incubated with GCV. A similar result was obtained to that with ARO and 8505c cells (data not shown), indicating that AdhTERTtk also has the potential to be an effective vector for gene therapy for these carcinomas.

Effect of viral infections on tumor growth in vivo

Differentiated (FTC-133) or undifferentiated (ARO) thyroid carcinoma cells were transplanted to nude mice, and the changes in the tumor volume were estimated in vivo during and after treatment with adenovirus/GCV. As shown in Fig. 4AGo, in ARO-transplanted nude mice, tumor volumes increased in a time-dependent manner even during injection of GCV in the control (TKluc) group. In contrast, tumor growth was significantly inhibited by infection with AdhTERTtk after the beginning of GCV. The inhibition of tumor growth in this group continued after the period of GCV treatment, and the growth curve of tumors was similar to that of the CMVtk group. The similar results were observed in mice, transplanted with FTC-133 cells (Fig. 4BGo). These results indicated that we confirmed in vivo the data of cytotoxicity assays in vitro. We further studied inhibition of tumor growth in vivo by AdhTERTtk with various doses of GCV. AdTKluc or AdhTERTtk (1 x 109 pfu) was injected into tumors of ARO-transplanted Balb-C nude mice and 100 or 200 mg/kg BW of GCV was given ip to the mice once a day for 14 d. As shown in Fig. 5Go, almost the same results were observed as in Fig. 4AGo. There were no significant differences in tumor growth curves among the mice infected with AdhTERTtk and treated with 80, 100, and 200 mg/kg BW of GCV (Figs. 4AGo and 5Go). Some mice, even infected with AdTKluc, were dead during administration of 200 mg/kg BW of GCV. These results suggested that 80 or 100 mg/kg BW of GCV administration leads nearly maximal effects for tumor growth inhibition in the condition of 1 x 109 pfu of AdhTERTtk injection.



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  		FIG. 4. Tumor growth inhibition by adenovirus/GCV in vivo. Human undifferentiated thyroid carcinoma, ARO (A), or differentiated thyroid carcinoma, FTC-133 (B), cells were transplanted into Balb-C nu/nu mice. Adenoviruses (1 x 109 pfu) were injected into tumors at d 0 and 80 mg/kg BW of GCV was given ip once a day from d 1 to d 14. Tumor volumes were expressed as a percentage relative to the values of d 0 in each group and plotted. Each point and bar shows the mean ± SE from more than five mice in each group. Statistical significance was evaluated by ANOVA. Open square, TKluc; closed circle, CMVtk; open triangle, hTERTtk. *, P < 0.05, compared with the TKluc group.

 


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  		FIG. 5. Tumor growth inhibition by AdhTERTtk combined with administration of higher doses of GCV in vivo. AdhTERTtk (1 x 109 pfu) was injected into ARO tumors, established in Balb-C nu/nu mice, at d 0 and 100 or 200 mg/kg BW of GCV was given ip once a day from d 1 to d 14. Tumor volumes were expressed as a percentage relative to the values of d 0 in each group and plotted. Each point and bar shows the mean ± SE from more than five mice in each group. Statistical significance was evaluated by ANOVA. *; P < 0.05, compared with the TKluc group; NS, not significant. GCV100 and GCV200 mean that 100 and 200 mg/kg BW of GCV was given to mice, respectively.

 
Toxic effect of HSVtk/GCV system

Toxic effects of AdhTERTtk/GCV or AdCMVtk/GCV were analyzed in rats. As markers for liver damage, serum GOT and GPT levels were measured during HSVtk/GCV treatment. Enzyme levels drastically increased in rats treated with AdCMVtk/GCV. On the other hand, no significant elevation of transaminase levels was observed in animals given Ad2xTGtk or AdhTERTtk followed by GCV (Fig. 6AGo). These results indicate that infection with Ad2xTGtk and AdhTERTtk do not induce damage of hepatocytes, although AdCMVtk does. Pathological examination revealed hepatic necrosis associated with cell ballooning and lymphocyte infiltration in rats treated with AdCMVtk/GCV (Fig. 6BGo, top). In these animals, damage was also observed in kidneys and spleens (data not shown). In contrast, no abnormal pathological findings were observed in livers after treatment with AdhTERTtk/GCV (Fig. 6BGo, bottom).



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  		FIG. 6. Rat in vivo liver toxicity assays after an iv injection with adenovirus and ip administrations with GCV. Each adenovirus was injected into a jugular vein of a Fisher rat and GCV was ip given once a day for 5 d. Serum levels of GOT (A, top) and GPT (A, bottom) were measured 3 d after starting GCV treatment. Statistical significance was evaluated by t test. *; P < 0.01, compared with mock group. B, Pathological examination of rat livers after treatment with adenovirus/GCV as related above. Liver samples were collected 1 d after the last GCV ip administration, and specimens were stained with HE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Specificity and efficacy are most important for cancer gene therapy using a suicide gene/prodrug system. As is well documented, specific promoters, which can induce restricted expression of transduced genes only in target cells, are suitable for adenovirus-mediated gene therapy for cancers. However, specific promoters may be too weak to be used for killing target cells. To increase the cytotoxic sensitivity, many improvements of methods have been studied (19, 20, 21). We have previously reported that a tandemly repeated core TG promoter enhanced efficacy of HSVtk/GCV for TG-producing thyroid cancers without losing tissue specificity (22). We also constructed a tandemly repeated (double or triple) hTERT core promoter fused to the luciferase gene in the pGL3-Basic reporter plasmid. We found that transcriptional activity by the tandemly repeated hTERT promoters stayed at the same levels as using a single hTERT promoter in transient transfection studies (data not shown). Thus, it is suggested that mechanism of induction of transcription, between TG and hTERT promoters, is different.

The mechanisms by which the hTERT core promoter works so differentially in tumors and normal cells are still unclear. It has been reported that c-Myc (34, 35, 36, 49, 50, 51), Sp1 (52), and estrogen (53, 54) activated hTERT promoter, but p53 (55), WT-1 (56), and Mad1 (57) were the factors for down-regulation of the promoter activity. In our data of cytotoxicity assay in vitro, normal rat thyroid FRTL5 cells and rat fibroblasts were partially killed at high concentrations of GCV (Fig. 3Go, A and C). However, an in vivo toxicity study demonstrated that the hTERT promoter has dramatic specificity, compared with the CMV promoter (Fig. 6Go, A and B). No pathological abnormalities were observed in spleen, kidney, lung, testis, and thyroid tissues in the same studies (data not shown). These results indicate that the adenovirus-mediated hTERTtk/GCV system is not harmful for normal tissues in vivo. The telomerase activity has been detected in some human normal cells, such as germ cells (58), lymphocytes (59), and hematopoietic progenitor cells (60), suggesting that the hTERTtk/GCV system may affect these cells. However, telomerase expression is a transient response to proliferative stimuli in these cells, and efficiency of transduction by adenovirus in these cells is limited in vivo (61). Thus, our results and reports from other groups suggest that side effects by hTERTtk/GCV system should be minimal in clinical trials. Moreover, we found that the hTERT core promoter is as potent for expression of a protein of interest as is the CMV promoter, both in vitro and in vivo.

Several groups have recently reported usefulness of gene expression under the control of the hTERT promoter for cancer gene therapy. The caspase-8 (42), caspase-6 (41), and FADD (40) genes were fused to hTERT promoter, and the plasmid was directly injected into tumors of prostatic carcinoma, glioma, and glioma cells, respectively. The transduced genes caused significant inhibition of tumor growth by inducing apoptosis. However, they did not have the efficient gene transduction because they needed a large amount of the genes given directly into tumors. Recombinant adenoviruses were generated including HSVtk (37) and Bax (38, 39) genes fused to the hTERT promoter. These authors showed tumor-specific cytotoxicity for osteosarcoma, lung carcinoma, and fibrosarcoma, respectively, in vitro and in vivo. These reports confirm that the hTERT promoter is useful for gene therapy of a broad spectrum of cancers.

Based on these reports, we targeted UTC, which is one of the most aggressive and malignant human tumors. It has been reported that transduction of the wild-type p53 gene, as widely tried in other tumors, prevented growth of the cell lines derived from UTCs (62, 63, 64). It is also reported that expression of p53 increased sensitivity to chemotherapeutic agents, especially doxorubicin. However, it is unclear whether the effect is due to an additive effect of p53 and doxorubicin or enhanced effect of doxorubicin with p53. Double infection with adenoviruses carrying the thyroid transcription factor-1 gene and the TG promoter-controlled HSVtk gene revealed cell-specific cytotoxicity for UTC cell lines in vitro (65). However, high titers of adenoviruses (totally 200–600 MOI) were needed for killing cells. Ten MOI of adenovirus was enough for killing UTC cell lines in our studies in vitro. The differences of results may be due to the difference of infectivity of adenovirus to UTC cell lines tested and the more powerful effect of the hTERT promoter than the TG promoter. More recently the ONYX-015 adenovirus was tested for treatment of UTC cell lines (66). UTC cells were inhibited by the ONYX-015 adenovirus, and it showed a synergistic effect with chemotherapeutic agents for UTC cell lines. In our data, AdhTERTtk is also effective for both differentiated and undifferentiated thyroid carcinomas in vitro and in vivo. However, further studies are needed to increase cell-killing ability, keeping tumor specificity to increase the number of tumor-free mice after treatment with AdhTERTtk/GCV. Increasing the dose of GCV may not be effective from the results of Fig. 5Go. It might be important to increase expression of TK, increase bystander effect, increase infectivity of adenovirus, and efficacious administration of GCV, such as continuous administration of GCV because of its short half-life.

As a novel approach for UTCs, we have described here the adenovirus-meditated hTERTtk/GCV system. The system showed powerful and selective effect to kill UTCs without damage to normal tissues. Thus, this system has the potential to be a beneficial therapy for UTCs.


   Acknowledgments
 
We are grateful to Dr. S. L. C. Woo for providing with pBS-tk plasmid and Dr. S. Refetoff for the recombinant adenovirus AdTKluc. We also thank F. Hoffmann-La Roche Ltd., Switzerland, for supplying ganciclovir.


   Footnotes
 
Abbreviations: ß-gal, ß-Galactosidase; BW, body weight; CMV, cytomegalovirus; FBS, fetal bovine serum; GCV, ganciclovir; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; 6H, six hormones or growth factors; HSVtk, human herpes simplex virus thymidine kinase; hTERT, human telomerase reverse transcriptase; MOI, multiplicity of infection; PCSM, penicillin and streptomycin; pfu, plaque-forming units; TG, thyroglobulin; TK, thymidine kinase; UTC, undifferentiated thyroid carcinoma.

Received November 25, 2002.

Accepted May 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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