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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 3 1407-1414
Copyright © 2002 by The Endocrine Society

Other Original Articles

Cell-Specific Viral Gene Therapy of a Hurthle Cell Tumor

Rusheng Zhang, Francis H. Straus and Leslie J. DeGroot

Thyroid Study Unit (R.Z., J.D.), Department of Medicine, Department of Pathology (F.H.S.), The University of Chicago, Chicago, Illinois 60637

Address all correspondence and requests for reprints to: Leslie J. DeGroot, M.D., Thyroid Study Unit, Mail Code 3090, The University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637.

Abstract

We evaluated the effectiveness of a replication-defective adenovirus-transducing thymidine kinase (TK) gene under the control of the rat Tg (rTg) promoter (AdrTgtk) in therapy of a human Hurthle cancer (XTC-1 cell) in vitro and in vivo. The ganciclovir (GCV) sensitivity of infected XTC-1 cells was assessed in vitro by H3-thymidine incorporation assay and Trypan-blue exclusion, and by an in vivo tumor development assay. Proliferation was strongly inhibited by adding GCV into the culture medium of infected cells, but not uninfected cells, proving cell infection and expression of TK in the XTC-1 cells. AdrTgtk, and also viruses that have the noncell-specific cytomegalovirus (CMV) promoter-directing expression of TK (AdCMVtk), or luciferase (AdCMVLuc), were used to transduce XTC-1 cells to evaluate killing effects. After infection with AdCMVtk or AdrTgtk, followed by GCV treatment, 70% of infected cells were killed in the presence of GCV, compared with less than 20% of cells infected by AdCMVLuc and treated with GCV.

In vivo toxicity was studied in BALB/c mice. When adenovirus is given iv, liver is the major organ infected. No significant changes of the serum transaminase levels and no histological abnormalities were found in animals treated with AdrTgtk/GCV given iv, compared with control animals. High levels of serum transaminases, lymphocyte infiltration, some Kupffer’s cell prominence, and extensive single-cell hepatocyte death were found in AdCMVtk/GCV-treated animals, indicating severe liver damage induced, as expected, by the noncell-specific CMV promoter.

XTL-1 cells (2 x 106) were injected sc into BALB/c-severe combined immunodeficient mice (BALB/c-SCID), and the mice developed tumors after 3 wk. After intratumoral injection of AdrTgtk and treatment with GCV, tumors stabilized in 15 of 17 mice within 3 wk, 9 tumors remained stabilized after 5 wk of treatment, and 2 disappeared during observation. In AdCMVLuc/GCV-treated control mice, almost all tumors grew continuously. The average tumor size in AdrTgtk-treated mice was significantly smaller than that of control animals after 2 wk of treatment.

Our data confirm the effectiveness and specificity of an adenovirus using rTg promoter to express TK, and support its future application to thyroid cancer gene therapy in humans.

MOST THYROID CANCERS are cured by surgery and 131I, but 10–18% of cases finally have distant metastases (1). Radioiodine and chemotherapy may be employed in these cases, but treatment may not be effective (2). Thus, a novel approach is necessary for treating metastatic differentiated thyroid carcinoma. Thyroid follicular cells and differentiated thyroid carcinoma cells have the unique capability to make Tg (3), because of the function of the tissue specific Tg promoter (4, 5, 6, 7). Transcription of the Tg gene is stimulated by TSH via cAMP (8, 9, 10). Several transcription factors are involved in the expression of Tg gene, including thyroid transcriptional factors -1 and -2 and Pax-8. These factors are DNA-binding proteins, which regulate thyroid-specific transcription and expression of the Tg gene (4).

Herpes simplex virus thymidine kinase and ganciclovir (TK/GCV) provide a well-characterized (11) and widely employed suicide gene/prodrug strategy for cancer gene therapy in both animal studies and clinical trails (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). The TK gene product efficiently phosphorylates nucleoside analogs, such as GCV, into GCV monophosphate, which is further phosphorylated to GCV diphosphate and GCV triphosphate by normal cell kinase. GCV triphosphate competes with deoxyguanosine triphosphate in DNA polymerization, causing arrest of DNA synthesis, DNA fragmentation, and thereby cell death (23).

Replication-defective recombinant adenovirus has several advantages as a gene-delivering system, as described previously (24). Others, and our group, using different adenoviral vectors, have successfully treated tumors in various animal models (24, 25, 26, 27, 28, 29, 30, 31). However, suicide genes using adenoviral vectors can induce severe adverse effects, because the vectors can be expressed in liver and other organs, especially when TK expression is controlled by a strong ubiquitously expressed promoter (31, 32, 33). Even if the adenovirus is given intratumorally, liver is the main target of virus leaked from the injection site (31). This problem can limit the clinical application of the TK/GCV system in patients. To control the expression of foreign genes and reduce possible side effects, tissue-specific promoter and enhancer systems are employed to obtain restricted expression (34, 35, 36, 37, 38, 39, 40, 41, 42).

Considering the unique property of the thyroid cancer cells, we constructed an adenovirus vector expressing TK under the control of rat Tg (rTg) promoter (43). During in vitro studies, this vector showed expression specifically in a thyroid cell line, and minimally in cells from other tissues. During in vitro killing studies, the vector specifically killed the infected Tg-producing thyroid cells in vitro but not cells from other tissues.

In the present study, we demonstrate that the adenovirus-transducing TK gene under the control of the rTg promoter (AdrTgtk) causes in vitro killing of a human Hurthle cell line, has minimal toxicity in vivo in animals, and has a potent antitumor effect on tumors developed in severe combined immunodeficient BALB/c-SCID mice.

Materials and Methods

Animals

Four- to 6-wk-old BALB/c or BALB/c-SCID mice were obtained from The Jackson Laboratory and maintained at the Carlson Biocontainment Suite, under standard conditions, according to the Guidelines of the Animal Research Center.

Cell lines and cell cultures

XTC-1 cells, a human Hurthle cell line, were kindly provided by Dr. Orlo H. Clark at the University of California, San Francisco-Mt. Zion Medical center. XTC-1 cells are maintained in DMEM/Ham’s F12 medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% FBS, TSH (10 mU/ml), insulin (10 µg/ml), 100 U/ml penicillin, and 100 µg/ml streptomycin. FRTL-5 cells, a differentiated normal rat thyroid cell line (44), were maintained in F12 Coon’s modified medium supplemented with 5% FBS and a 6-hormone mixture (45) consisting of TSH (10 mU/ml), insulin (10 µg/ml), hydrocortisone (10-8 M), transferrin (5 µg/ml), glycyl-histidyl-L-lysine acetate (10 ng/ml), somatostatin (10 µg/ml), 100 U/ml penicillin, and 100 µg/ml streptomycin. Rat and human medullary thyroid carcinoma cell lines (rMTC and hMTC) were purchased from American Type Culture Collection (Rockville, MD). Rat MTC cells were maintained in DMEM medium supplemented with 10% horse serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (complete DMEM medium). Human MTC cells were maintained in complete RPMI-1640 medium (Life Technologies, Inc.) containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. 293 cells were purchased from Microbix Biosystems Inc. (Ontario, Canada) and were maintained in complete MEM medium (containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin). Cos-1 cells (a SV40-transformed African green monkey kidney cell line), Hep G2 cells (a human hepatoblastoma cell line), Hela cells (a human epitheloid carcinoma cell line), and GH3 cells (a rat pituitary tumor cell line) were maintained in complete DMEM medium. CA77 cells, a rat C cell line, were maintained in a 1:1 mixture of DMEM and F12 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. T98G cells, a human glioblastoma cell line, were maintained in MEM supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 1 mM nonessential amino acids.

Construction of recombination-defective adenoviral vectors

Replication-defective adenoviral vectors containing the luciferase (Luc) or TK gene, under the transcriptional control of the human cytomegalocyte viral immediate early promoter/enhancer systems (CMV), or rTg promoter (AdCMVLuc, AdCMVtk, AdrTgtk), were constructed as reported (43, 48). Rescued virus was plaque-purified and amplified in 293 cells. The viral preparations were purified by two CsCl density centrifugations, dialyzed, and stored in 10% glycerol at -80 C. The titers of the viral stocks were determined by plaque assay using 293 cells (46, 47).

Infection of cells with adenoviral vectors in vitro

Cells were harvested during exponential growth of the cell culture. The cells were washed with serum-free medium, counted, and centrifuged (1000 rpm for 5 min). Viability was examined by trypan-blue dye exclusion and always showed more than 95% living cells. Adenoviral vectors were added to the cell pellets at various multiplicities of infection (moi) and incubated for 1 h at 37 C in a minimum volume (usually 500 µl of 2% FBS) to permit efficient infection (standard infection condition). Cells were then washed three times with serum-free medium to remove free viral particles. Washed cells were resuspended in fresh complete medium and incubated at 37 C.

GCV sensitivity assay

Three methods were employed for assaying GCV sensitivity. AdCMVtk-infected, AdrTgtk-infected, or wild-type cells were incubated in 6-well plates using 1 x 105 cells. After 24 h of incubation, GCV was added in various concentrations. Five days later, cells were harvested, and the living cells were counted by trypan-blue exclusion.

A 3H-thymidine incorporation assay was also used to evaluate GCV sensitivity. AdCMVtk-infected, AdrTgtk-infected, or noninfected XTC-1 cells were incubated in sextuplet in 96-well plates (5 x 103/180 µl/well) for 24 h, and then GCV was added at various concentrations. Forty-eight hours after incubation, 0.5 µCi 3H-thymidine (20 µl) was added to each well, and samples were incubated for another 24 h. Cells were harvested after detaching them from the plates by three freeze-thaw cycles. Incorporation of 3H-thymidine was determined in a scintillation counter. The amount of incorporation of 3H-thymidine reflects the extent of cell proliferation, and this is inhibited by TK/GCV treatment.

The expression of AdrTgtk in infected cells was also evaluated by an in vitro infection/in vivo killing study. XTC-1 cells were infected in vitro, at 10 moi for 1 h, by AdrTgtk, or AdCMVtk that served as a positive control, or AdCMVLuc or noninfected cells that served as the negative controls. Infected cells were then injected sc into the flank of BALB/c-SCID mice at 2 x 106 cells/mouse. Two days later, after the injection, GCV was administered ip at 80 mg/kg body weight per day for 5 consecutive days. The development of tumors was checked once a week for 8 wk.

In vivo toxicity assay

Different adenoviral vectors were injected into 4- to 6-wk-old BALB/c mice, iv or sc, at a dose of 1–5 x 109 plaque-forming units (pfu) in 100 µl 0.9% sodium chloride solution. GCV was administered 48 h after the viral injection, at a dose of 80 mg/kg body weight per day for 5 consecutive days. Treated animals were inspected every day for their behavior and killed 24 h after the last GCV treatment. Serum was saved from each animal to test liver function, and tissues were harvested for pathological examination.

A commercial kit was used for quantitative colorimetric determination of glutamic-oxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) (Sigma, St. Louis, MO). The procedure from the company was followed.

At the time of necropsy, tissues were harvested and put into Zamboni’s fixative solution (Newcomer Supply, Middleton, WI). The specimens were then embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

Development of XTC-1 tumors in BALB/c-SCID mice

XTC-1 cells were harvested in the exponential growth phase and washed three times in serum-free medium. Then 2 x 106 cells were injected sc into the right flank of each mouse. Tumors usually developed in 3 wk.

In vivo antitumor efficiency study

Adenoviral vectors were directly injected into the tumors using 2 x 109 pfu in 100 µl of 0.9% sodium chloride. Forty-eight hours after the viral treatment, GCV was administrated ip at 80 mg/kg body weight per day for 7 consecutive days. Tumor size was measured every 4 d. The sc tumor volumes were determined from the formula: v = a2b/2, where a is the shortest diameter and b is the longest diameter of the tumor.

Statistical calculations

The t test was used to analyze the data; P < 0.05 was considered significant.

Results

Sensitivity of infected XTC-1 cells

To determine the effect of the TK/GCV system on XTC-1 cells, XTC-1 cells were transduced with AdCMVtk or AdrTgtk at various moi (Fig. 1Go). As calculated from the raw data, GCV alone, at 1 µM, inhibited cell proliferation by 8.5%; whereas at same GCV concentration proliferation of cells infected at 10 moi was inhibited by 23.4%. At a GCV concentration of 100 µM and infected at 10 moi, cell proliferation was inhibited by 67% (27.9% more than in the uninfected control). When infected at 20 moi, proliferation was significantly inhibited by 33.9% at a GCV concentration of 1 µM, or by 99% at a GCV concentration of 100 µM, 28.7% more or 59.9% more than in the uninfected control cells, respectively. These results demonstrate the high sensitivity of the TK/GCV system in infected XTC-1 cells.



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  		Figure 1. GCV sensitivity of AdrTgtk-infected XTC-1 cells. XTC-1 cells were infected, at different moi, for 1 h, in 500 µl infection solution. Then infected cells were incubated in sextuplet in 96-well plates (5 x 103/180 µl/well) for 24 h, and GCV was added at concentrations of 0–100 µM. Forty-eight hours after incubation, 0.5 µCi 3H-thymidine (20 µl) was added to each well, and samples were incubated for another 24 h. Cells were harvested after detaching them from the plates by three freeze-thaw cycles. Incorporation of 3H-thymidine was determined in a scintillation counter. The amount of incorporation of 3H-thymidine reflects the extent of cell proliferation, and this is inhibited by TK/GCV treatment. GCV alone inhibits part of the cell proliferation, especially at a high concentration. Inhibition of the cell proliferation is related directly to viral infection at each level of GCV treatment.

 
The GCV sensitivity of infected XTC-1 cells was also examined in vivo. AdrTgtk or AdCMVtk cells infected in vitro at 10 moi were injected sc into BALB/c-SCID mice using 2 x 106 cells per mouse. Forty-eight hours after the injection, GCV was administered in the amount of 80 mg/kg body weight per day for 5 consecutive days. Animals with AdrTgtk- or AdCMVtk-infected cells treated by administration of PBS, and AdCMVLuc-infected cells treated with GCV, served as controls. No animals developed tumors in the AdrTgtk- or AdCMVtk-infected GCV-treated group, and all animals in the control groups developed tumors (Fig. 2Go).



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  		Figure 2. In vitro infection/in vivo killing effect of TK/GCV system. XTC-1 cells were infected in vitro, at 10 moi, for 1 h, with different vectors. After washing, the infected cells and uninfected control cells were injected sc into BALB/c-SCID mice at 2 x 106 cells per mouse. GCV was administrated 2 d after the injection for 5 consecutive days at 80 mg/kg body weight per day. Noninfected (N) XTC-1 cells served as the control cells. Injected mice were observed for 8 wk for the development of tumor. No tumor developed in vivo after in vitro infection with AdrTgtk or AdCMVtk.

 
Cell-specific killing effect by AdrTgtk

To determine the specificity of expression of AdrTgtk, XTC-1 and other cells were transduced with AdrTgtk or AdCMVtk at various moi levels. After 6 d of incubation in GCV-containing medium, more than 65% of AdrTgtk-infected and 85% of AdCMVtk-infected XTC-1 cells were killed (Fig. 3Go). In all other cell lines, AdrTgtk had no greater effect than the control vector AdCMVLuc (Fig. 4Go). When different cell lines were infected with AdCMVtk, all cell lines were killed after incubation in GCV-containing medium (Fig. 4Go). This result shows the cell-specific killing effect by AdrTgtk and the nonspecific killing effect by AdCMVtk.



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  		Figure 3. Killing effect of AdrTgtk/GCV system in vitro. XTC-1 cells were infected with different vectors, at 100 moi, for 1 h, in 1 ml solution. Thirty-six hours after the infection, GCV was added, at the final concentration of 100 µM, and cells were incubated for 6 d. Living cells were determined by trypan-blue exclusion. Cell treatment is shown below each column. *, P < 0.05.

 


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  		Figure 4. Specific killing effect of AdrTgtk/GCV system in vitro. Different cell lines were infected with different vectors, at 100 moi, for 1 h, in 1 ml solution. Thirty-six hours after the infection, GCV was added, at the final concentration of 100 µM, and cells were incubated for 6 d. Living cells were determined by trypan-blue exclusion. The cell lines studied are indicated on the abscissa.

 
In vivo cytotoxicity

To evaluate the in vivo toxicity of AdrTgtk virus, AdrTgtk and AdCMVtk were administrated by either tail vein or sc at 1–5 x 109 pfu in 100 µl of 0.9% sodium chloride solution. Five animals were used for each treatment. Two days after the treatment, all animals received GCV at 80 mg/kg body weight per day for 5 consecutive days. The animals were inspected every day. One day after the last administration of GCV, animals were killed. Serum was saved for the liver enzyme assays, and tissue was used for pathological studies. No animal showed overt adverse effect upon treatment with AdrTgtk or in the control groups. In contrast, one animal in the AdCMVtk-treated group died, and others showed reduced activity and/or an unhealthy coat. Their livers appeared atrophic and yellow. There was no significant change of the serum transaminase levels in AdrTgtk-treated animals, compared with in control animals, whereas animals treated with AdCMVtk, iv, showed very high levels of transaminases, indicating liver injury after AdCMVtk/GCV treatment (Fig. 5Go).



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  		Figure 5. Serum transaminase levels of virus-treated animals. AdCMVLuc, AdCMVtk, or AdrTgtk were administered iv or sc at doses of 2 x 109 pfu in 100 µl of 0.9% sodium chloride solution. GCV was given ip, 48 h after the viral injection, at the dose of 80 mg/kg body weight per day for 5 consecutive days. Serum was harvested 24 h after the last GCV treatment. The serum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic-pyruvic transaminase (SGPT) levels were determined using a commercial kit. The groups include control (N) without virus, with or without GCV, and the three viruses given sc or iv, always with GCV.

 
Pathological changes of liver in virus-treated animals

Liver, lung, spleen, kidney, and thyroid were examined in the control and differently treated animals. The liver of animals treated with AdCMVtk showed lymphocyte triaditis and marked interstitial inflammation; hepatocyte ballooning degeneration, with large vesicular nuclei; and prominent chromatin clumping (Fig. 6AGo). Animals treated with AdrTgtk showed mild triaditis and minimum interstitial inflammation. No hepatocyte damage is evident (Fig. 6BGo), compared with control animals (Fig. 6Go, C and D). There was no clear pathologic change in lungs, spleen, kidney, or thyroid in any of the animals (data not shown).



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  		Figure 6. Pathologic changes of liver of adenoviral vector-treated animals. AdCMVtk, AdrTgtk, or AdCMVLuc was administered iv, at 2 x 109, in 100 µl of 0.9% sodium chloride solution. GCV was given ip, 48 h after the viral administration, at the dose of 80 mg/kg body weight per day for 5 consecutive days. Twenty-four hours after the last GCV treatment, animals were killed, and various tissues were obtained for pathological examination. A, Liver lobule from AdCMVtk-treated animal. Triaditis and marked interstitial inflammation was observed; extensive single-cell hepatocyte death and large vesicular reactive hepatocyte nuclei were observed (magnification, x175). B, Liver lobule from AdrTgtk-treated animal; portal triad on left with central vein upper left and lower middle field. Mild triaditis is evident, with little interstitial inflammation; hepatocytes look good (magnification, x175). C, Liver lobule from AdCMVLuc-treated animal (portal triad on right and central vein on left); usual mouse liver cord, no evidence of inflammation or parenchyma change was observed (magnification, x175). D, Liver lobule from nontreated control animal. As in C, no abnormality was found in the control normal liver.

 
Antitumor efficacy in vivo

The antitumor effect of AdrTgtk was evaluated by direct injection of vector into XTC-1 tumors in the BALB/c-SCID model. An efficient antitumor effect was obtained in treated animals (Fig. 7Go). A significant difference in tumor size between AdrTgtk- and AdCMVLuc-treated groups was found, 2 wk after treatment of tumors.



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  		Figure 7. Antitumor effect of AdrTgtk/GCV system in vivo. XTC-1 tumors were developed in SCID-BALB/c mice. Then, tumors were treated by intratumor injection of different vectors at 2 x 109 pfu. Two days after the viral treatment, GCV was administrated at 80 mg/kg body weight per day for 7 consecutive days. The tumor volume was measured every 4 d. Averages and SDs of tumor volumes are shown. The average volumes in AdCMVtk- or AdrTgtk-treated animals are significantly smaller, 12 d after the treatment, than that of AdCMVLuc-treated control animals (P < 0.05).

 
Discussion

A concern when employing adenoviral gene therapy is that normal tissue cells may be damaged by virus leaked from treated tumors when using strong nontissue-specific promoters, such as CMV or SV40 promoters. Liver is the main target organ for adenovirus when administered iv or leaked from tumor after direct injection (31, 32, 33, 49, 50). Ninety percent of adenovirus homes to liver (32, 33, 49, 50). Therefore, strategies to reduce liver toxicity are essential when applying this system to human cancer patients.

Several groups have reported cell-specific effect of HSVtk/GCV systems for cancer gene therapy, using tissue-specific promoters in adenoviral vectors (35, 36, 37). Restricted expression can be difficult to achieve, even using tissue-specific promoters. Imler et al. (51) failed to demonstrate cell-specific expression strictly paralleling that of endogenous human cystic fibrosis transmembrane conductance regulator (CFTR) with the CFTR promoter. Ring et al. (52) had a similar experience. They transduced TK expression in tumor cells with recombinant adenovirus, retrovirus, and plasmid vector under the control of the ERBB2 promoter. All of the vectors were capable of sensitizing ERBB2-positive cells to the action of GCV. In contrast to the retroviral and plasmid vectors, transduction with the adenoviral vector also resulted in the sensitization of ERBB2-negative cells to the GCV. The E1A enhancer in the adenovirus may be responsible for expression of TK in ERBB2-negative cells (52).

Wallace et al. (53) established transgenic mice in which TK was expressed mainly in the thyroid and testis under the control of bovine Tg promoter. After administering GCV, thyroid follicular cells were selectively ablated, suggesting the usefulness of the Tg promoter in gene therapy of thyroid follicle tumors.

Zeiger et al. (54) also demonstrated the feasibility of using adenovirus as a vector to treat differentiated thyroid carcinomas using the Tgtk/GCV system. In those studies, FRTL-5 cells, which were stably transfected with Tgtk genes, were killed in culture after the administration of 5 µg/ml GCV. Braiden et al. (55) reported that TK gene under the control of Tg promoter could induce cell-specific cytotoxicity. They first made cell lines expressing TK gene by infection with a retroviral vector harboring the TK gene under the control of Tg promoter. These Tgtk-expressing cells were cultured and used for study of in vitro cytotoxicity and transplantation in nude mice.

We have explored the feasibility of using an adenovirus vector with the Tg promoter controlling the expression of TK gene for cell-specific expression in a human thyroid tumor cell line. Previously, we demonstrated that, in adenovirus vectors, the Tg promoter can induce cell-specific expression of TK, Luc, and LacZ. Luc expression was found in Tg-producing FRTL-5 cells when cells were infected with AdrTgLuc reporter vector, but no expression was found in other cell lines tested. In a GCV-sensitivity study, FRTL-5 cells were killed by AdrTgtk/GCV treatment, whereas other cultured cell lines did not show any specific effect from the AdrTgtk/GCV treatment (43).

In this study, we developed a human Hurthle tumor model in BALB/c-SCID mice. In our in vitro studies, we demonstrated that, in the adenoviral vectors, the cell-specific promoter (rTg promoter) could induce TK expression only in the XTC-1 cells. Specific expression of TK induced killing only in the thyroid cell lines after the administration of GCV. When in vitro infected cells were injected into the BALB/c-SCID mice, no tumor developed in the AdrTgtk-infected group after treatment with GCV, indicating uniform infection of XTC-1 human Hurthle cancer cells, effective expression of TK in vivo, and the efficacy of the TK/GCV system. During toxicity studies, AdrTgtk did not induce significant side effects in BALB/c mice after iv or sc administration and GCV treatment, whereas AdCMVtk given in identical dosage caused severe liver damage. In the in vivo antitumor study, AdrTgtk demonstrated an antitumor effect similar to AdCMVtk. All tumors treated in vivo stabilized after treatment, and some of them were completely destroyed.

Our data confirm the utility of controlled gene expression using a cell-specific promoter in cancer gene therapy, and efficacy in an in vivo model of human thyroid cancer. We believe such vectors should be useful in thyroid cancer gene therapy in patients currently untreatable by conventional methods.

Acknowledgments

We are grateful to Dr. Bernard Roizman for the use of his laboratory; to the Marjorie B. Kovler Viral Oncology Laboratories, at The University of Chicago, for producing and preparing adenoviral vectors; to Dr. Cyprian Gardine for his careful reading of the manuscript and important suggestions; to Miss Myrna Zimberg for her excellent secretarial assistance; and to all the fellows in our laboratory for their advice and useful discussion during this project.

Footnotes

This work was supported by a Center of Excellence Award from Knoll Pharmaceutical Co. (Mount Olive, NJ), by the Elsa U. Pardee Foundation, and by the David Wiener Research Fund.

Abbreviations: AdCMVLuc, Adenovirus-transducing Luc gene under the control of CMV promoter; AdCMVtk, adenovirus-transducing TK gene under the control of CMV promoter; AdrTgtk, adenovirus-transducing TK gene under the control of rTg promoter; CFTR, cystic fibrosis transmembrane conductance regulator; CMV, cytomegalovirus; GCV, ganciclovir; Luc, luciferase; moi, multiplicities of infection; pfu, plaque-forming units; rTg, rat Tg; SCID mice, severe combined immunodeficient mice; SGOT, serum glutamic-oxaloacetic transaminase; SGPT, serum glutamic-pyruvic transaminase; TK, thymidine kinase.

Received June 19, 2001.

Accepted November 15, 2001.

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