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Endocrinology Vol. 138, No. 11 4577-4583
Copyright © 1997 by The Endocrine Society

ARTICLES

Retrovirus-Mediated Herpes Simplex Virus Thymidine Kinase Gene Transduction Renders Human Thyroid Carcinoma Cell Lines Sensitive to Ganciclovir and Radiation in Vitro and in Vivo

Eijun Nishihara, Yuji Nagayama, Fumihiro Mawatari, Kunihiko Tanaka, Hiroyuki Namba, Masami Niwa and Shunichi Yamashita

Department of Nature Medicine, Atomic Bomb Disease Institute (E.J., H.N., S.Y.), Department of Pharmacology 1 (Y.N., K.T., M.N.), and First Department of Internal Medicine (H.M.), Nagasaki University School of Medicine, Nagasaki 852, Japan

Address all correspondence and requests for reprints to: Yuji Nagayama, M.D., Department of Pharmacology 1, Nagasaki University School of Medicine, Sakamoto 1–12-4, Nagasaki 852, Japan. E-mail: nagayama{at}net.nagasaki-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In an attempt to develop gene therapy for thyroid carcinomas, the present studies were undertaken to evaluate in vitro and in vivo therapeutic efficacy and toxicity of herpes simplex virus thymidine kinase (HSV-tk) gene and ganciclovir (GCV) treatment, a widely used prodrug/suicide gene therapy, in human thyroid carcinoma cell lines, FRO and WRO cells, using a means of retrovirus-mediated gene transduction. In vitro experiments demonstrated dose- and time-dependent cell killing by transduction of the HSV-tk gene followed by GCV treatment. The IC50 (the concentration required to elicit 50% growth inhibition) shifted from 250 to 0.5 mg/liter in FRO cells, and from 3,000 to 0.09 mg/liter in WRO cells with therapeutic indexes of 500 and 33,000, respectively. Treatment with 30 mg/liter GCV for 4 days led to complete cell death in HSV-tk tumor cells. Nontransduced cells mixed with transduced cells were also effectively killed by GCV (bystander effect). Low concentrations of GCV, which alone showed little cytotoxicity, enhanced radiation-induced cytotoxicity (radiosensitization). In vivo sc FRO-tk tumor models in nude mice also showed dose- and time-dependent tumor regression. The IC50 was less than 2 mg/kg, and treatment with 100 mg/kg GCV for 2 weeks completely eradicated all tumors. The bystander effect and radiosensitization were also obtained in vivo. These results suggest that the HSV-tk/GCV approach to human thyroid carcinoma cells appears to be very efficacious, with a wide therapeutic range, and exerts a bystander effect and radiosensitization both in vitro and in vivo. Thus, HSV-tk/GCV system, alone or in combination with radiotherapy, may be a promising suicide gene therapy for thyroid carcinomas.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID cancers are the most common endocrine malignancy, accounting for 1% of all cancers. In the case of well differentiated thyroid carcinomas, thyroidectomy followed by ablative radioiodine (131I) treatment and TSH suppression is generally recommended as the treatment of choice. Despite excellent good prognosis of this type of thyroid carcinomas in general, approximately 30% of patients have recurrences, and half of patients who developed recurrences eventually succumb to the disease (1, 2). In contrast, poorly differentiated or anaplastic thyroid carcinomas, a rare but highly lethal form of cancer with a median survival of less than 8 months, are refractory to the traditional treatments (1, 3). Furthermore, an increase in highly aggressive childhood thyroid cancers has recently been reported in the Republic of Belarus after the Chernobyl nuclear power plant accident, and the incidence of late onset of radiation-induced thyroid carcinomas is also suspected to be very high in the future (4, 5, 6, 7, 8). Therefore, development of novel therapeutic approaches for thyroid carcinomas are urgently warranted; gene therapy may be a good candidate.

Strategies for gene therapy include selective prodrug activation by suicide genes, inhibition of activated oncogenes by antisense or ribozyme, transfer of tumor suppressor genes, and cytokine gene transfer (cancer vaccination) (9). Gene therapy with prodrug/suicide gene combination involves the delivery of a suicide gene into cancer cells, making them sensitive to an appropriate prodrug. Herpes simplex virus thymidine kinase (HSV-tk) (10), which is the most widely used suicide gene (9), converts the antiviral prodrug of nucleotide analogs such as ganciclovir (GCV) and acyclovir to the monophosphorylated form, which is then converted to the toxic triphosphate form by endogenous cellar kinases that compete with normal nucleotides for DNA replication (10). Thus, the expression of HSV-tk gene in mammalian cells renders them sensitive to GCV, thereby killing them by interfering with DNA synthesis. Retrovirus- and adenovirus-mediated HSV-tk/GCV treatment has been used in various tumor model systems (9, 11).

Furthermore, in addition to its direct cytotoxic effect, there may be at least two advantages of HSV-tk/GCV therapy. The first is that not only HSV-tk-expressing cells but also nontransduced cells can be killed, a phenomenon called the bystander effect (12, 13, 14). The second is that phosphorylated GCV enhances the effect of radiation-induced cytotoxicity, a phenomenon termed radiosensitization (15, 16, 17).

The present study was designed to evaluate the efficacy and toxicity of retrovirus-mediated HSV-tk gene therapy for treatment of thyroid carcinomas. We first tested whether transduction of HSV-tk gene in conjunction with GCV led to cell killing in vitro and tumor growth suppression in vivo using two thyroid carcinoma cell lines, FRO and WRO cells. Furthermore, the bystander effect and the radiosensitization effect were examined both in vitro and in vivo to clarify the therapeutic advantage of HSV-tk/GCV treatment in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Human follicular and anaplastic thyroid carcinoma cell lines, WRO and FRO (18), were cultured with RPMI 1640 medium supplemented with 10% FBS and the appropriate antibiotics. The amphotropic and ecotropic retrovirus packaging line, PA317 and Psai-2, and mouse NIH-3T3 fibroblast cells were maintained in DMEM with 5% FBS.

Vector plasmid and retroviral transduction
The pLNCTK, which is a Moloney murine leukemia virus vector and contains the HSV-tk gene under the control of cytomegalovirus (CMV) promoter and neomycin phosphotransferase gene under control of simian virus 40 promoter, was provided by Dr. A. Ido (19). The pLNCTK was transfected into Psai-2 cells using Lipofectamine (Life Technologies, Grand Island, NY). Forty-eight hours later, the medium containing ecotropic recombinant retrovirus was infected into PA317 cells. The virus-infected PA317 cells were selected in medium with 800 mg/liter G418 (Geneticin, Wako, Osaka, Japan), and G418-resistant colonies were cloned with cloning cylinders. Supernatant from the producer cell line with a titer of ~5 x 107 colony-forming units/liter in NIH-3T3 cells was used to transduce FRO and WRO cells. The cells were incubated for 4 h and then selected with 800 mg/liter G418 for 2 weeks. The surviving cells were pooled and used for the subsequent experiments. FRO and WRO cells transduced with HSV-tk gene were designed for FRO-tk and WRO-tk, respectively.

Northern blot analysis
Total RNA was extracted by the guanidinium thiocyanate-phenol-chloroform method. Twenty micrograms of total RNA were subjected to Northern blot analysis as previously described (20). Membrane was sequentially hybridized with HSV-tk DNA and cyclophilin complementary DNA.

In vitro cytotoxic assays
The cells were seeded at density of 5 x 102 cells/well for FRO and 1 x 103 cells/well for WRO in 96-well microtiter plates. One day later (day 0), the cells were treated with various concentrations of GCV (Tanabe Seiyaku Co., Osaka, Japan) in 100 µl fresh medium. Medium was replaced with the same medium on day 2. The cell survival was quantitated with a commercially available cell counting kit (Wako) on day 4. Survival ratios were expressed as percentages relative to untreated control values.

To determine the in vitro bystander effect, the cell mixtures of transduced and parent cells at different ratios were seeded in 48-well culture plates at 3 x 104 cells/well. On the next day (day 0), when the cells were approximately 20–30% confluent, with most cells having visible contact with adjacent cells, GCV was added at concentrations that are not toxic to parental cells. Medium was replaced with the same medium every other day. On day 6, viable cells were counted using the trypan blue exclusion test.

For radiosensitization studies, the cells plated in 10-cm cell culture dishes at a low density were exposed to 0.1 mg/liter GCV for 48 h before and after a single dose of {gamma}-irradiation with an EXS-300 {gamma}-irradiator (200 kV; 15 mA; filter, 0.5 mm aluminum and 0.5 mm copper; 0.47 Gy/min; Toshiba, Tokyo, Japan). Two weeks later, clonogenic survival was determined by counting colonies larger than 5 mm. Data were normalized to control levels to account for drug toxicity.

Detection of apoptotic cells
The cells were plated at a density of 3 x 104 cells/well in six-well culture dishes and were incubated in the absence or presence of the IC50 of GCV. Four days later, nuclear morphological changes in cells were examined using the fluorescent DNA-binding dye, Hoechst 33258, as previously described (21). Cells were analyzed under fluorescence microscopy, and nuclei containing condensed chromatin were qualified as apoptotic cells. Three hundred cells were counted for each sample. Percent apoptotic cells were calculated as (total number of cells with apoptotic nuclei/total number of cells counted) x 100.

In vivo tumor studies
Five-week-old male nude mice (Charles-River Japan, Tokyo, Japan) were injected sc on both sides of flanks with 1 x 107 cells in 100 µl PBS. Two weeks later, ip injections of various concentrations of GCV were performed daily for 2 weeks. In some experiments, xenografted tumors were irradiated with an EXS-300 {gamma}-irradiator (200 kV; 15 mA; filter, 0.5 mm aluminum; 1.41 Gy/min). The perpendicular tumor diameters were measured using calipers, and tumor volumes (V) were calculated by the formula of rotational ellipsoid: V = A x B2/2, where A is the longer diameter, and B is the smaller diameter (22). The results were expressed as percentages relative to tumor size on day 0. None of the mice showed signs of wasting or other visible indications of toxicity.

All mice were maintained in Nagasaki University animal facility, and all animal studies were conducted in accordance with the principles and procedures outlined in the guide for the care and use of laboratory animals at Nagasaki University School of Medicine.

Statistical analysis was performed using unpaired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Virus infection of human thyroid carcinoma cell lines, FRO and WRO cells
Expression of the HSV-tk gene was confirmed with Northern blot analysis. As shown in Fig. 1Go, the 1.3-kb HSV-tk transcripts, which was initiated by CMV promoter and terminated by HSV-tk polyadenylation signal (19), as well as larger genomic viral RNA were comparably detected in both virus-transduced FRO and WRO cells, but not in the parental cells. Relative amounts of messenger RNA (mRNA) on the blot were estimated by rehybridization to cyclophilin complementary DNA.



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  		Figure 1. Northern blot analysis of HSV-tk transcripts in transduced and nontransduced FRO and WRO cells. Twenty micrograms of total RNA were analyzed for HSV-tk and cyclophilin mRNA expressions. Lane 1, Parental FRO cells; lane 2, parental WRO cells; lane 3, FRO-tk cells; lane 4, WRO-tk cells.

 
In vitro cytotoxicity
The cells expressing HSV-tk were first assayed for sensitivity to GCV by determining the IC50 (Fig. 2Go and Table 1Go). There was no significant difference in cell growth between the parental and transduced cells. In dose-response experiments (Fig. 2Go, A and B), the IC50 shifted from 250 mg/liter (mean of two independent experiments) in FRO cells to 0.5 mg/liter in FRO-tk cells, and from 3,000 mg/liter in WRO cells to 0.09 mg/liter in WRO-tk cells. Thus, wide therapeutic ranges were obtained in HSV-tk/GCV treatment for thyroid carcinoma cells; the therapeutic indexes calculated as the IC50 in parental cells/IC50 in transduced cells were approximately 500 in FRO cells and 33,000 in WRO cells (Table 1Go). The cell-killing effect of HSV-tk/GCV was also time dependent (Fig. 2Go, C and D). The parental FRO cells seem to be slightly more sensitive to GCV than the parental WRO cells, while FRO-tk cells are more resistant to HSV-tk/GCV treatment compared with WRO-tk cells, which somehow does not fit the fact that cell growth is more rapid in FRO cells than in WRO cells, because toxicity of GCV is at least in part dependent on cellular mitotic activity.



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  		Figure 2. Dose (A and B) and time (C and D) dependence of in vitro cytotoxicity of GCV in FRO and WRO cells. The cells were incubated with various doses of GCV for the indicated periods (4 days in A and B), followed by cell survival quantitation as described in Materials and Methods. Data are representative of at least two separate experiments; each point represents the mean ± SE (n = 6) and is expressed as a percentage relative to the value in untreated cells. A and C, FRO cells; B and D, WRO cells.

 

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  		Table 1. Summary of in vitro efficacy of HSV-tk/GCV in FRO and WRO cells

 
We then evaluated whether nuclear fragmentations, a morphological characteristic of apoptosis, could be induced by GCV with Hoechst 33258 dye. As shown in Fig. 3Go, GCV treatment significantly increased the percentage of apoptotic cells in FRO-tk (8.4 ± 0.9% to 22.6 ± 1.7%; P < 0.01) and WRO-tk (2.5 ± 0.2% to 9.4 ± 2.4%; P < 0.01) cells, suggesting that the cytotoxicity induced by HSV-tk/GCV is in part mediated by an apoptotic mechanism. Representatives of intact FRO-tk cells and apoptotic cells are shown in Fig. 3Go, C and D.



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  		Figure 3. Apoptotic cell death induced by GCV in FRO-tk and WRO-tk cells. The cells were incubated in the presence or absence of the IC50 of GCV for 4 days, stained with Hoechst 33258, and visualized under fluorescence microscopy. Data presented in A and B are representative of at least two separate experiments; each point represents the mean ± SE of triplicate determinations. Three hundred cells were counted for each point. A, FRO-tk cells; B, WRO-tk cells; C, control FRO-tk cells; D, apoptotic FRO-tk cells with nuclear fragmentations. *, P < 0.01.

 
In vitro bystander effect
To investigate the in vitro bystander effect, HSV-tk-positive and -negative cells were mixed in different proportions and exposed to 50 mg/liter GCV for 6 days. A dose of 50 mg/liter GCV is not toxic to HSV-tk-negative cells (see Fig. 2Go). As shown in Fig. 4Go, the cell mixtures containing only 20% of the transduced cells showed more than 90% growth inhibition by GCV in FRO/FRO-tk cells, indicating that HSV-tk-expressing cells can exert an in vitro bystander effect. A less potent bystander effect was observed in WRO-tk cells; more than 90% growth inhibition was obtained in the cell mixture with 50% of transduced cells. Approximately equal amounts of connexin-43 mRNA expression, one of the commonly expressed connexins in the thyroids (23), was confirmed in FRO and WRO cells (data not shown).



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  		Figure 4. In vitro bystander effect in FRO and WRO cells. HSV-tk-positive cells were mixed with HSV-tk-negative cells in various proportions and incubated with 50 mg/liter GCV for 6 days. Viable cells were counted using the trypan blue exclusion test. Data are representative of at least two separate experiments; each point represents the mean ± SE (n = 6) and is expressed as percentage relative to the value in untreated cells. A, FRO cells; B, WRO cells.

 
In vitro radiosensitization
The extent of radiosensitization was assessed by comparing the percentage of survival cells after ionizing radiation with or without GCV administration in rapidly growing FRO-tk cells. To facilitate comparison, the levels of cytotoxicity were normalized to control levels to account for drug toxicity. The cells, cultured in the presence or absence of 0.1 mg/liter GCV, which alone produced only subtle cytotoxicity (see Fig. 2Go), were irradiated. As shown in Fig. 5Go, FRO-tk cells treated with 0.1 mg/liter GCV were radiosensitized at a single dose of 10 Gy; that is, the percent cell survival was significantly lower in the cells treated with irradiation and GCV than in those treated with irradiation alone (9.2 ± 1.8% vs. 1.0 ± 2.0%; P < 0.01).



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  		Figure 5. In vitro radiosensitization effect in FRO-tk cells. The cells were incubated in the presence or absence of 0.1 mg/liter GCV for 48 h before and after the administration of graded doses of ionizing radiation. Two weeks later, colonies larger than 5 mm were counted. Data are representative of at least two separate experiments and are normalized to control levels to account for drug toxicity; each point represents the mean ± SE (n = 4).

 
These in vitro data all together suggest that the HSV-tk/GCV regimen appears to be very efficacious with a wide therapeutic range for treatment of thyroid carcinoma cell lines. Therefore, we then progressed to in vivo experiments to elucidate whether these in vitro effects could be reproduced in vivo using sc tumor models in nude mice with FRO cells.

In vivo cytotoxicity
FRO or FRO-tk cells (1 x 107) were inoculated sc into both flanks of each mouse. Two weeks later (day 0 in Fig. 6Go), when small tumor nodules were present, mice were treated with various doses of GCV (0–100 mg/kg BW) for 2 weeks. There was no significant difference in the tumor sizes between FRO and FRO-tk cells on day 0. As shown in Fig. 6Go, daily administration of 100 mg/kg GCV did not inhibit the growth of FRO cells. In contrast, GCV treatment resulted in dose- and time-dependent tumor regression in HSV-tk-transduced cells. Tumor volumes in mice treated with 0, 2, 10, and 100 mg/kg GCV for 2 weeks were 1148 ± 287%, 315 ± 21%, 50 ± 22%, and 0%, respectively, compared with that on day 0; the IC50 was less than 2 mg/kg, and all tumors were completely eradicated in mice treated with 100 mg/kg GCV, in which tumors did not recur in the subsequent 2 weeks (data not shown).



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  		Figure 6. Dose and time dependence of the in vivo cytotoxic effect of GCV in FRO and FRO-tk cells inoculated in nude mice. Two weeks after inoculations, various doses of GCV were administered. The tumor sizes were measured three times a week for 2 weeks. Data are representative of two separate experiments; each point represents the mean ± SE (n = 4–6) and is expressed as a percentage relative to tumor size on day 0. The actual tumor sizes were 213.5 ± 43.7 and 220.9 ± 27.4 mm3 in FRO and FRO-tk cells, respectively, on day 0 and 2536.2 ± 634.0, 695.8 ± 46.4, 117.8 ± 48.6, and 0 mm3 in FRO-tk cells treated with 0, 2, 10, and 100 mg/kg·day GCV, respectively, on day 14.

 
In vivo bystander effect
Mice inoculated with the cell mixtures of FRO and FRO-tk cells at different ratios were used to evaluate the in vivo bystander effect. As shown in Fig. 7Go, more than 70% and 90% of tumor growth inhibitions were observed in tumors containing only 20% and 50% of FRO-tk cells, respectively, demonstrating an in vivo bystander effect.



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  		Figure 7. In vivo bystander effect in FRO cells. Tumors composed of a mixture of transduced and nontransduced FRO cells in various proportions were treated with 100 mg/kg·day GCV for 2 weeks. Data are representative of two separate experiments; each point represents the mean ± SE (n = 4 to 6) and is expressed as a percentage relative to volumes of tumors composed of only the parental FRO cells.

 
In vivo radiosensitization
Finally, mice with tumors composed of FRO-tk cells were treated with either GCV (5 mg/kg for 4 days), ionizing radiation (40 Gy), or both to evaluate the cooperative therapeutic effect of radiation and HSV-tk/GCV. GCV was given from days 0–3, and a single dose of 40 Gy was administered on day 2 (Fig. 8Go). GCV treatment alone did not affect tumor size, and 40 Gy ionizing radiation alone elicited approximately 85% tumor growth inhibition compared with that in untreated tumors. The combination of these two regimens produced significantly profound tumor regression compared with the effect of radiation alone (78 ± 27% vs. 178 ± 7% tumor volumes compared with day 0; P < 0.05). This cooperative effect of tumor regression is probably due to the radiosensitization effect observed in vitro.



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  		Figure 8. In vivo radiosensitization in FRO-tk cells. Tumors of FRO-tk cells were treated with 25 mg/kg GCV for 4 days, a single dose of 40 Gy, or both. Tumor sizes were measured three times a week for 2 weeks. Data are representative of two separate experiments; each point represents the mean ± SE (n = 4–6) and is expressed as percentage relative to the tumor size on day 0. *, P < 0.05.

 
Therefore, all of the in vitro effects of HSV-tk/GCV therapy on thyroid carcinoma cells (direct cytotoxic effect, bystander effect, and radiosensitizing effect) were confirmed in in vivo experiments.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present studies we evaluated the in vitro and in vivo therapeutic efficacy of the HSV-tk/GCV system, a widely used prodrug/suicide gene therapy, for treatment of thyroid carcinoma cells using a means of retrovirus-mediated gene transduction. In vitro dose-dependent experiments showed that transduction of the HSV-tk gene into thyroid carcinoma cell lines, FRO and WRO cells, rendered them 3–5 orders of magnitude more sensitive to GCV. Furthermore, complete in vitro cell death and in vivo tumor eradication were observed with 30 mg/liter and 100 mg/kg GCV, respectively. These concentrations can be achievable clinically (24). Our data also indicate that apoptosis contributes at least partly to GCV induction of cell death, which is advantageous to the bystander effect (see below).

The bystander effect was readily observed in both in vitro and in vivo experiments. Thus, 20–50% transduced cells led to more than 90% cell death. As phosphorylated GCV cannot pass freely through the plasma membrane, the bystander effect appears to be mediated by transport of phosphorylated GCV through gap junctions (12, 13) and/or phagocytosis of apoptotic vesicles (14). The gap junction is constituted by a family of proteins, called connexins (23), and is the route for intercellular exchange of small hydrophilic molecules. Thus, expression of connexin-43 mRNA and induction of apoptotic cells after GCV administration probably contribute to this phenomenon (12, 13, 14, 25). This phenomenon is clinically very important, as genetic modification of an entire tumor is at present impossible. Further, HSV-tk/GCV-induced tumor immunity in immune-competent cells (26, 27, 28), which cannot be expected in the nu/nu mice used in the present studies, may augment the in vivo bystander effect in clinical settings.

Our data also demonstrated that GCV enhances the sensitivity of HSV-tk-transduced cells to ionizing radiation. Such a prodrug-induced radiosensitization has recently been shown in glioma cells expressing the HSV-tk gene or the Escherichia coli cytosine deaminase gene (15, 16, 17). Kim et al. (15) speculated that possible mechanisms for enhancement of radiation-induced cytotoxicity by GCV and 5-fluorocytosine may be to modify the DNA structure so as to make it more sensitive to radiation, to inhibit the repair of DNA lesions produced by irradiation, or both. From a clinical point of view, internal radioiodine treatment with 131I as well as external radiation have long been widely used in patients with differentiated thyroid carcinomas whose intrinsic ability to concentrate iodine is preserved. Such tumors may benefit from the combination of HSV-tk gene therapy and radioiodine treatment. In this regard, it is of particular interest is to use a radiation-inducible promoter, such as egr-1, thymidine kinase, and tissue-type plasminogen activator (29), to express a radiosensitizer, such as HSV-tk, in a tumor-specific manner.

As it has been reported that killing of HSV-tk-modified tumors by GCV may not be always complete (27, 30, 31) and that the bystander effect and radiosensitization cannot be seen in all types of cancer (12, 17), our data suggest that the HSV-tk/GCV system seems to be an efficacious therapy for thyroid carcinoma cells. Thus, HSV-tk/GCV treatment may be a promising approach for thyroid carcinomas. It should be noted here, however, that as selection of an optimal prodrug/suicide gene system may vary in each cell type, it will be necessary to compare the relative efficacies of several prodrug/suicide gene therapy approaches (32, 33), including Escherichia coli cytosine deaminase/5-fluorocytosine, human deoxycytisine kinase/1-ß-D-arabinofuranosylcytosine, etc., in treating thyroid carcinomas in the future.

While the present studies were in progress, Zeiger et al. (34) reported their preliminary results demonstrating GCV-mediated killing of a rat normal thyroid cell line FRTL5, which stably expressed HSV-tk gene, in a tissue-specific manner using the thyroid-specific thyroglobulin promoter, which enables tissue-specific, but not tumor-specific, ablation of the thyroid glands (35). Their strategy may be applicable for systemic treatment of distant metastatic thyroid carcinomas. In contrast, our studies indicate the potential of direct injection of recombinant virus encoding a suicide gene, which is derived from a viral promoter, as local treatment for thyroid carcinomas; because the thyroid glands are situated in anterior neck, regional neck lymph node metastases are often seen in patients with papillary type of thyroid carcinomas, and more importantly, death is caused by local invasion in most patients with anaplastic carcinomas (1) that do not usually express any differentiated markers of the thyroid cells. Virus-derived promoters, for example the CMV promoter used here, are indeed stronger than mammalian promoters.

In summary, the present studies demonstrate that the HSV-tk/GCV combination efficiently kills thyroid carcinoma cells not only by a direct cytotoxic effect but also by a bystander effect and enhances radiation-induced cell death both in vitro and in vivo. The very wide therapeutic range observed suggests that the HSV-tk/GCV regimen may be a good candidate for a novel approach in the treatment of thyroid carcinomas.


   Acknowledgments
 
We thank Dr. Akio Ido for kindly providing the plasmid pLNCTK, and Tanabe Seiyaku Co. (Osaka, Japan) for GCV.

Received May 16, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Braverman LE, Utiger RD 1996 Werner and Ingbar’s The Thyroid. A Fundamental and Clinical Text, ed 7. Lippincott Raven, Philadelphia
  2. Hardy KJ, Walker BR, Lindsay RS, Kennedy RL, Seckl JR, Padfield PL 1995 Thyroid cancer management. Clin Endocrinol (Oxf) 42:651–655[Medline]
  3. Sweeney PJ, Haraf DJ, Recant W, Kaplan EL, Vokes EE 1996 Anaplastic carcinoma of the thyroid. Ann Oncol 7:739–744[Free Full Text]
  4. Ito M, Yamashita S, Ashizawa K, Namba H, Hoshi M, Shibata Y, Sekine I, Nagataki S, Shigematsu I 1995 Childhood thyroid diseases around Chernobyl evaluated by fine needle aspiration cytology. Thyroid 5:365–368[Medline]
  5. Ito M, Yamashita S, Ashizawa K, Hara T, Namba H, Hoshi M, Shibata Y, Sekine I, Kotova L, Panasyuk G, Demidchik E, Nagataki S 1996 Histopathological characteristics of childhood thyroid cancer in Gomel, Belarus. Int J Cancer 65:29–33[CrossRef][Medline]
  6. Karaoglon A, Desmet G, Kelly GN, Menzel HG 1996 The radiological consequences of the Chernobyl accident. European Commission (EUR16544EN), pp 1–1192
  7. Souchkevitch G, Tsyb AF 1996 Scientific Report of International Programme on the Health Effects of the Chernobyl Accident. WHO, Geneva, pp 1–443
  8. Williams ED, Becker D, Dimidchik EP, Nagataki S, Pinchera A, Toronko ND 1996 Effects of the thyroid in populations exposed to radiation as a result of the Chernobyl accident. Background paper session 2. International Conference One Decade after Chernobyl. EC/IAEA/WHO, Vienna, 1–25
  9. Freeman SM, Whartenby KA, Freeman JL, Abboud CN, Marrogi AJ 1996 In situ use of suicide genes for cancer therapy. Semin Oncol 23:31–45[Medline]
  10. Moolten FL 1986 Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer Res 46:5276–5281[Abstract/Free Full Text]
  11. Weichselbaum RR, Kufe D 1997 Gene therapy of cancer. Lancet 349:10–12
  12. Mesnil M, Piccoli C, Tiraby G, Willecke K, Yamasaki H 1996 Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins. Proc Natl Acad Sci USA 93:1831–1835[Abstract/Free Full Text]
  13. Elshami AA, Saavedra A, Zhang H, Kucharczuk JC, Spray DC, Fishman GI, Amin KM, Kaiser LR, Albelda SM 1996 Gap junctions play a role in the "bystander effect" of the herpes simplex virus thymidine kinase/ganciclovir system in vitro. Gene Ther 3:85–92[Medline]
  14. Freeman SM, Abboud CN, Whartenby KA, Packman CH, Koeplin DS, Moolten FL, Abraham GN 1993 The bystander effect: tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res 53:5274–5283[Abstract/Free Full Text]
  15. Kim JH, Kim SH, Brown SL, Freytag SO 1994 Selective enhancement by an antiviral agent of the radiation-induced cell killing of human glioma cells transduced with HSV-tk gene. Cancer Res 54:6053–6056[Abstract/Free Full Text]
  16. Kim JH, Kim SH, Kolozsvary A, Brown SL, Kim OB, Freytag SO 1995 Selective enhancement of radiation response of herpes simplex virus thymidine kinase transduced 9L gliosarcoma cells in vitro and in vivo by antiviral agents. Int J Radiat Oncol Biol Physiol 33:861–868[CrossRef][Medline]
  17. Khil MS, Kim JH, Mullen CA, Kim SH, Freytag SO 1996 Radiosensitization by 5-fluorocytosine of human colorectal carcinoma cells in culture transduced with cytosine deaminase gene. Clin Cancer Res 2:53–57[Abstract]
  18. Namba H, Hara T, Tukazaki T, Migita K, Ishikawa N, Ito K, Nagataki S, Yamashita S 1995 Radiation-induced G1 arrest is selectively mediated by the p53-WAF1/Cip1 pathway in human thyroid cells. Cancer Res 55:2075–2080[Abstract/Free Full Text]
  19. Ido A, Nakata K, Kato Y, Nakao K, Murata K, Fujita M, Ishii N, Tamaoki T, Shiku H, Nagataki S 1996 Gene therapy for hepatoma cells using a retrovirus vector carrying herpes simplex thymidine kinase under the control of human alpha-fetoprotein gene promoter. Cancer Res 55:3105–3109[Abstract/Free Full Text]
  20. Nagayama Y, Tanaka K, Namba H, Hara T, Yamashita S, Taniyama K, Miwa M 1996 Involvement of G-protein coupled receptor kinase 5 in homologous desensitization of the thyrotropin receptor. J Biol Chem 271:10143–1014[Abstract/Free Full Text]
  21. Ting YT, Namba H, Hara T, Takamura N, Nagayama Y, Fukata S, Ishikawa N, Kuma K, Ito K, Yamashita S 1997 p53 induced by ionizing radiation mediates DNA end-jointing activity, but not apoptosis of thyroid cells. Oncogene 14:1511–1519[CrossRef][Medline]
  22. Janik P, Briand P, Hartmann NR 1975 The effect of estrone-progesterone treatment on cell proliferation kinetics of hormone-dependent GR mouse mammary tumors. Cancer Res 35:3698–3704[Abstract/Free Full Text]
  23. Meda P, Pepper MS, Traub O, Willecke K, Gros D, Beyer E, Nicholson B, Paul D, Orci L 1993 Differential expression of gap junction connexins in endocrine and exocrine glands. Endocrinology 133:2371–2378[Abstract]
  24. Hayden FG, Douglas RG 1990 Antiviral agents. In: Mandell GL, Dopuglas RG, Bennett JE (eds) Principles and Practice of Infectious Diseases, ed 3, Churchill Livingstone, New York, pp 371–393
  25. Colombo BM, Benedetti S, Ottolenghi S, Mora M, Pollo B, Poli G, Finocchiaro G 1995 The "bystander effect:" association of U87 cell death with ganciclovir-mediated apoptosis of nearby cells and lack of effect in athymic mice. Hum Gene Ther 6:763–772[Medline]
  26. Barba D, Hardin J, Sadelain M, Gage FH 1994 Development of anti-tumor immunity following thymidine kinase-mediated killing of experimental brain tumors. Proc Natl Acad Sci USA 91:4348–4352[Abstract/Free Full Text]
  27. Vile RG, Nelson JA, Castleden S, Chong H, Hart IR 1994 Systemic gene therapy of murine malanoma using tissue specific expression of the HSVtk gene involves an immune component. Cancer Res 54:6228–6234[Abstract/Free Full Text]
  28. Hall SJ, Mutchnik SE, Chen S-H, Woo SLC, Thompson TC 1997 Adenovirus-mediated herpes simplex virus thymidine kinase gene and ganciclovir therapy leads to systemic activity against spontaneous and induced metastasis in an orthotopic mouse model of prostate cancer. Int J Cancer 70:183–187[CrossRef][Medline]
  29. Buchsbaum DJ, Raben D, Stackhouse MA, Khazaeli MB, Rogers BE, Rosenfeld ME, Liu T, Curiel DT 1996 Approaches to enhance cancer radiotherapy employing gene transfer methods. Gene Ther 3:1042–1068[Medline]
  30. Moolten FL, Wells JM, Heyman JR, Evans RM 1990 Lymphoma regression induced by ganciclovir in mice bearing a herpes thymidine kinase transgene. Hum Gene Ther 1:125–134[Medline]
  31. Zhang L, Wikenheiser KA, Whitsett JA 1997 Limitation of retrovirus-mediated HSV-tk gene transfer to pulmonary adenocarcinoma cells in vitro and in vivo. Hum Gene Ther 8:563–574[Medline]
  32. Beck C, Cayeux S, Lupton SD, Dorken B, Blandenstein T 1995 The thymidine kinase/ganciclovir-mediated "suicide" effect is variable in different tumor cells. Hum Gene Ther 6:1525–1530[Medline]
  33. Hoganson DK, Batra RK, Olsen JC, Boucher RC 1996 Comparison of the effects of three different toxin genes and their levels of expression on cell growth and bystander effect in lung adenocarcinoma. Cancer Res 56:1315–1323[Abstract/Free Full Text]
  34. Zeiger MA, Takiyama Y, Bishop J, Ellison AR, Saji M, Levine MA 1996 Adenoviral infection of thyroid cells: a rationale for gene therapy for metastatic thyroid carcinoma. Surgery 120:921–925[CrossRef][Medline]
  35. Wallace H, Ledent C, Vassart G, Bishop JO, Al-Shawi R 1991 Specific ablation of thyroid follicle cells in adult transgenic mice. Endocrinology 129:3217–3226[Abstract]



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