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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 2 786-794
Copyright © 1999 by The Endocrine Society

Original Studies

Targeted Expression of Toxic Genes Directed by Pituitary Hormone Promoters: A Potential Strategy for Adenovirus-Mediated Gene Therapy of Pituitary Tumors1

Eun Jig Lee, Leonard M. Anderson, Bayar Thimmapaya and J. Larry Jameson

Division of Endocrinology, Metabolism, and Molecular Medicine (E.J.L., J.L.J.) and the Department of Microbiology/Immunobiology (L.M.A., B.T.), Northwestern University Medical School, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: J. Larry Jameson, M.D., Ph.D., Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: ljameson{at}nwu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pituitary adenomas cause clinical manifestations because of mass effects and excess hormone production. This group of tumors represents a tractable target for gene therapy because they are rarely metastatic and because reductions in tumor size and function, in addition to those achieved after surgery, may be of clinical benefit. In this report we describe a strategy for targeting the expression of toxic genes to pituitary cells using adenoviral vectors. Pituitary hormone promoters (human GH or glycoprotein hormone {alpha}-subunit) were used to express either a marker gene [ß-galactosidase (ß-gal)] or a toxic gene [herpes simplex virus thymidine kinase (TK)]. In GH-producing GH3 cells and in {alpha}-subunit-producing pituitary tumor cell lines, recombinant adenoviruses containing either the {alpha}-subunit promoter (Ad{alpha}Gal; Ad{alpha}TK) or the GH promoter (AdGHGal; AdGHTK) were expressed at high levels. Using histological studies and assays for ß-gal activity, expression was shown to persist for at least 21 days, and it was relatively selective for pituitary cell lines. Cytotoxicity studies were performed using the TK-containing vectors and treatment with ganciclovir. Both AdGHTK and Ad{alpha}TK caused greater than 95% cytotoxicity of GH3 and {alpha}T3 cells, respectively, at a viral dose (multiplicity of infection, 5 plaque-forming units/cell) that induced minimal toxicity using control viruses. Little cellular toxicity was seen using a nonpituitary cell line (T47D breast tumor cells). The AdGHTK virus also caused marked reduction in the size of GH3 cell tumors that were propagated in nude mice. These studies suggest that adenoviral vectors carrying human pituitary gland specific promoters may be useful for developing gene therapy strategies for the treatment of pituitary adenomas.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PITUITARY adenomas are typically benign tumors that arise in the sella turcica of the brain. They comprise up to 15% of primary intracranial neoplasms (1). Pituitary adenomas create clinical manifestations because of the overproduction of hormones or because of mass effects of the enlarging tumor within the confined space of the sella turcica. There are five major types of pituitary tumors, reflecting the different cell types in the pituitary gland. These tumor types include ACTH-producing tumors that cause Cushing’s disease, prolactinomas that hypersecrete PRL resulting in reproductive abnormalities, GH-secreting tumors that cause the syndromes of acromegaly and gigantism, gonadotropin-secreting tumors that hypersecrete LH and/or FSH, and TSH-producing tumors that result in hyperthyroidism. Because of its anatomical location in the sella turcica, mass effects of pituitary adenomas frequently include visual field defects resulting from compression of the optic nerves, other cranial nerve deficits, hypopituitarism, and, occasionally, invasion into surrounding structures.

Current therapies for pituitary tumors include surgery, radiotherapy, and pharmacological approaches for a subset of tumor types (2). For microadenomas (<1 cm), transsphenoidal surgery is successful in the majority (70–90%) of cases in the hands of an experienced surgeon. However, larger macroadenomas are rarely cured by surgery alone (3, 4). Medical therapies are available for selected types of pituitary tumors. In the case of prolactinomas, dopamine agonists markedly reduce hormone secretion and reduce tumor size in most patients (5). However, dopamine agonists do not cure the tumors, and they recur if the medications are discontinued. Somatostatin analogs have also been used for the treatment of GH-secreting adenomas and for TSH-secreting adenomas. Finally, radiotherapy is used as an adjunctive treatment after surgery and in rare instances as primary treatment for pituitary tumors. Although radiotherapy is often effective, several years are usually required before it has full impact, and potential complications include hypopituitarism, secondary tumors, and occasional neural deficits (4).

Recently, gene therapy using adenoviral vectors to deliver the herpes simplex virus-thymidine kinase (HSV-TK) followed by ganciclovir (GCV) administration has been developed as a strategy for the treatment of various types of malignant tumors (6, 7). Partial efficacy has been shown in tumors carried in animal models (8, 9, 10, 11, 12, 13) as well as in trials of human malignancies. In general, these approaches have relied on viral promoters to achieve high level expression of the suicide gene (9, 10). One of the limitations of this type of adenoviral vector is that they can infect nontumorous cells as well as tumor cells, leading to potential toxic effects in normal tissues. However, this problem might be circumvented if toxic gene expression could be restricted to tumor cells using cell-specific promoters.

Pituitary tumors, because of characteristic expression of hormone genes, represent an attractive model for developing targeted expression of toxic genes. A variety of pituitary cell-type specific promoters have been identified. These include the promoters of the major pituitary hormones as well as receptors for hypothalamic releasing hormones and a number of transcription factors involved in the development of the pituitary gland. For example, transient gene expression studies (14) and transgenic experiments (15, 16) have documented restricted expression of the GH and glycoprotein hormone {alpha}-subunit promoters in pituitary cells (17, 18). In addition, the transcriptional activity of these promoters is relatively high, suggesting that they might be capable of delivering high levels of toxic genes to pituitary tumor cells. In this report, we describe experiments using adenoviruses containing the human GH and {alpha}-subunit promoters to target genes to pituitary cell lines and transplanted pituitary tumors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and culture conditions

GH3 GH- and PRL-producing pituitary tumor cells and HEK293 embryonic kidney cell lines were obtained from American Type Culture Collection (Manassas, VA). The {alpha}-subunit-secreting pituitary tumor cell line, {alpha}T3–1 (hereafter referred to {alpha}T3 cells), was provided by Dr. P. Mellon (University of California-San Diego). T47D breast cancer cells were provided by V. Craig Jordan (Northwestern University Medical School, Chicago, IL).

GH3 cells were grown in Optimem containing 5% FBS. HEK293 cells were maintained in DMEM supplemented with 10% FBS. {alpha}T3 cells were grown in DMEM-Ham’s F-12 supplemented with 10% FBS at 37 C with 5% CO2. T47D cells were cultured in RPMI with 10% FBS.

Generation of recombinant adenoviral vectors

The recombinant type 5 (Ad5 309/356) adenoviral vector has a backbone in which the E3 region has been deleted. This virus also carries a 2-bp deletion in the E4 6/7 open reading frame (19). Ad5 309/356 was constructed by ligating viral DNA fragments from 0–75.0 map units of dl 309 and 75.0–100 map units or dL 356. A cassette containing a pituitary hormone-specific promoter and ß-galactosidase (ß-gal) or the herpes simplex virus type 1 thymidine kinase (HSV-TK) gene was inserted in place of the E1 deletion. Two pituitary hormone-specific promoters were used: the human GH promoter and the human {alpha}-subunit promoter. The human GH promoter consists of nucleotides -336 to +58 from the human GH 5'-flanking region, and the human {alpha}-subunit promoter consists of nucleotides -846 to +45 of the human {alpha}-subunit 5'-flanking region. These sequences were derived by PCR from human genomic DNA, cloned into pGem3Zf(+) (Promega Corp., Madison, WI), and the sequences were verified by DNA sequencing. The HSV-TK sequences were derived from plasmid pPNT by PCR amplification using specific primers. The HSV-TK sequences were cloned into pGem7Zf(+) (Promega Corp.) and verified by DNA sequencing. The HSV-TK fragment was ligated to each promoter such that HSV-TK expression is under the control of pituitary hormone-specific promoters, and transcription is terminated by the simian virus 40 polyadenylation signal. Subsequently, the promoter-HSV-TK gene fragment was cloned into a pcDNA3-based vector downstream of adenoviral sequences between map units 0 and 1.2, which comprise the lefthand inverted terminal repeat and packaging signal of the adenovirus (Fig. 1Go). The Escherichia coli lacZ gene encoding ß-gal and the preceding nuclear localization signal were ligated to each of the promoter-containing plasmids by standard cloning techniques. Subsequently, the promoter-ß-gal fragments were cloned into a pcDNA3-based vector downstream of adenoviral sequences between map units 0 and 1.2 (Fig. 1Go).



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  		Figure 1. Generation of recombinant adenovirus vectors. The transfer plasmid containing the extreme lefthand adenoviral DNA sequences, promoters, and ß-gal or HSV-TK gene were digested with HindIII and XbaI to linearize DNA. The linearized fragments were ligated onto XbaI-digested Ad5 309/356 DNA representing 3.8–100 map units. The ligation products were then transfected into 293 cells, whereby the resulting recombinant adenoviral DNA was replicated and packaged into virions. The recombinant adenoviruses carried the HSV-TK or E. coli ß-gal gene fused to the human GH promoter (AdGHTK, AdGHGal) and the human {alpha}-subunit promoter (Ad{alpha}TK, Ad{alpha}Gal).

 
The adenoviral transfer plasmids were digested with HindIII and XbaI. The fragment containing the left end adenoviral sequences, the promoters, and the HSV-TK or ß-galactosidase genes were then ligated with XbaI-digested Ad5 309/356 DNA representing 3.8–100 map units. The Ad5 309/356 is a recombined virus deleted of the early region 3 (E3). The ligation products were then transfected into 293 cells to allow production of recombinant adenoviral DNA and packaging into virions. The recombinant adenoviruses contain the HSV-TK or E. coli ß-galactosidase genes fused to the human GH promoter (AdGHTK, AdGHGal) or the human {alpha}-subunit promoter (Ad{alpha}TK, Ad{alpha}Gal). Individual clones of AdGHTK, AdGHGal, Ad{alpha}TK, and Ad{alpha}Gal were purified and titrated using plaque assays. The expression cassette in the adenoviral vectors was confirmed by DNA sequencing of viral DNA using specific primers.

As a positive control for expression and detection of ß-galactosidase activity, parallel studies were carried out using AdCMVGal, an adenovirus vector similar in design, but with the cytomegalovirus (CMV) promoter, nuclear localization signal, and ß-galactosidase as the reporter gene.

Evaluation of efficiency of recombinant adenoviral infection and recombinant gene expression

The transduction efficiency of adenoviral vectors in pituitary tumor cell lines was tested using AdGHGal, Ad{alpha}Gal, and AdCMVGal. Cells were plated in 12-well culture plates at a density of 2 x 105 cells/well. The next day, infections of cell lines were carried out by the addition of viral solutions to cell monolayers and incubation at 37 C for 1 h with brief agitation every 15 min. After the addition of culture medium, infected cells were returned to the 37 C incubator, and medium was changed 24 h later. For studies using a range of multiplicity of infection (MOI) doses, triplicate wells were infected with each virus at plaque-forming units (PFU) of 1, 5, 10, or 25 for 48 h. For long term expression studies, cells were infected using a MOI of 5 for 1, 2, 3, 5, 7, 10, 14, or 21 days. Cells were fixed with 1.0% glutaraldehyde for 10 min, washed with phosphate-buffered saline (PBS; pH 7.4), and then incubated with X-gal substrate solution (10.0 mmol/L potassium ferrocyanide, 10.0 mmol/L potassium ferricyanide, 1 mmol/L MgCl2, 20% Nonidet P-40, and 0.1% X-Gal in PBS) at 37 C for 2 h to evaluate ß-gal expression at the histological level.

Triplicate wells of infected cells were also used to measure ß-gal activity using O-nitrophenyl ß-D-galactopyranoside (ONPG) as a substrate (Sigma Chemical Co., St. Louis, MO). Culture medium was aspirated, cell lysis solution was added, and lysates were mixed with the ONPG substrate solution and incubated in 37 C for 2 h. The reaction was stopped with 100 µL 1 mol/L Na2CO3. Absorption was measured at 405 nm, and ß-gal activity was calculated using a standard curve.

Western blot analysis

Cells were plated in six-well culture dishes at a density of 1.5 x 106 cells/well. The next day, cells were infected with AdGHTK or Ad{alpha}TK at a MOI of 10 PFU/cell. Forty-eight hours after infection, cell cultures were washed twice with PBS, then scraped into 0.3 mL sample buffer containing 20 mmol/L Tris-HCl (pH 7.5), 2 mmol/L dithiothreitol, 0.5 mol/L KCl, 1 mmol/L phenylmethylsulfonylfluoride, and 20% glycerol. After three cycles of freezing in a dry ice-ethanol bath and thawing in a 37 C water bath, samples were centrifuged at 12,000 x g for 20 min at 4 C, and the supernatants were stored at -70 C until assayed. Solubilized samples were separated by SDS-PAGE using 4.5% stacking and 10% separating gels cast in a minigel apparatus (Hoeffer, San Francisco, CA). After electrophoresis, the gels were rinsed with transfer buffer containing 25 mmol/L Tris base, 192 mmol/L glycine, and 20% methanol. Electroblotting of the proteins to nitrocellulose paper was performed at 30 mA overnight at 4 C. Residual gels were stained with Coomassie blue (0.012% Coomassie blue, 40% methanol, and 7% acetic acid) to evaluate the transfer of proteins.

After electroblotting, nonspecific protein binding was blocked using 3% milk in PBS for 2 h. Antibody against HSV-TK (rabbit polyclonal specific for HSV-TK; provided by Dr. S. M. Albelda, University of Pennsylvania Medical Center, Philadelphia, PA) was diluted 1:800 in 3% milk in PBS and then incubated overnight at 4 C followed by three 10-min washes in 0.1% Tween-20 in PBS. Anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Promega Corp.; 1:5000) was added for 1 h at room temperature. After three washes in 0.1% Tween-20 in PBS, chemiluminescent substrate solution (LumiGlo, Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added and incubated for 1 min. Samples were blot-dried and exposed to film for 3 min.

GCV sensitivity of AdGHTK- and Ad{alpha}TK-infected cells

The sensitivity of adenovirus-infected cells to GCV was measured with a nonradioactive cell proliferation assay according to the manufacturer’s protocol (CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay, Promega Corp.). The day after plating 4 x 103 cells in triplicate wells of 96-well plates, adenoviral vectors were infected at different MOIs (0, 1, 5, 10, 100, and 500 PFU/cell). Sixteen hours after infection, increasing concentrations (0, 1, 5, 10, and 50 µg/mL) of GCV were added, and fresh medium containing GCV was added every 2 days. Cell viability was assayed on day 4 after adding GCV. The percent survival of cells is presented as the fraction of the absorbance found in the GCV-treated cells divided by that in the cells without GCV treatment (mean ± SD). For long term studies of infected tumor cells, MOIs of 0, 1, 5, and 10 were used, and 5 µg GCV were added to each well. Cell viability was assayed on days 0, 2, 4, 6, and 8 after adding GCV.

Treatment of tumor-bearing mice with adenoviral vectors in vivo

All studies involving the use of nude mice were approved by the Northwestern University Medical School animal care and use committee. GH3 cells (1 x 107) were injected into the flank area of adult (7-week-old) athymic male nude mice (Harlan Sprague-Dawley, Inc., Indianapolis, IN). Three weeks after injection of GH3 cells, tumors of about 1.0 cm in diameter developed, and the mice were divided into four groups: 1) AdGHTK injection and GCV treatment (n = 8), 2) AdGHTK injection without GCV treatment (n = 8), 3) AdGHGal injection and GCV treatment (n = 8), and 4) injection of dialysis buffer used for adenovirus preparation with GCV treatment (n = 8). Adenoviral vectors (0.5 x 109 PFUs) in a total volume of 100 µL dialysis buffer were injected into a growing tumor from three directions on 2 successive days. The following day, GCV was administered ip at 100 mg/kg once daily for 10 days. The size of the tumor was measured every 2 days with calipers in three dimensions. Tumor size is presented as cubic millimeters. {alpha}T3 cells (1 x 107) were also injected into male and female nude mice, but no tumors developed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gene transfer efficiency of AdGHGal, Ad{alpha}Gal, and AdCMVGal

The efficiency of adenoviral vector-mediated gene transfer to pituitary hormone producing GH3 and {alpha}T3 cells was assessed after infection with AdGHGal, Ad{alpha}Gal, or AdCMVGal adenoviruses. Expression of ß-galactosidase was detected by X-gal staining, and the enzymatic activity of ß-galactosidase was quantitated in cell lysates. Consistent with the strong activity of the CMV promoter, 95–100% of GH3 cells and {alpha}T3 cells were stained blue at 48 h after infection with AdCMVGal (MOI of 5–10 PFU/cell; data not shown). The intensity of X-gal staining was even stronger using a greater viral titer. Using the GH promoter-driven adenovirus, AdGHGal (MOI of 10 PFU/cell), 50–60% of GH3 cells were stained at 48 h after infection (Fig. 2BGo). The percentage of GH3 cells expressing ß-galactosidase increased with time and reached 95–100% by 4–5 days after infection (Fig. 2CGo). Similar results were seen in {alpha}T3 cells using the {alpha}-subunit promoter-driven adenovirus, Ad{alpha}Gal. About 65–70% of {alpha}T3 cells were stained blue at 48 h (Fig. 2EGo), and the fraction of infected cells increased to 95–100% by 4–5 days after infection (Fig. 2FGo).



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  		Figure 2. The expression of ß-gal in GH3 cells and {alpha}T3 cells infected with AdGHGal or Ad{alpha}Gal (MOI of 10). Cells were fixed with 1.0% glutaraldehyde for 10 min, washed with PBS (pH 7.4), and then incubated with X-gal substrate solution at 37 C for 2 h. A, GH cells without virus; B, GH3 cells at 2 days after infection of AdGHGal; C, GH3 cells at 5 days after infection of AdGHGal; D, {alpha}T3 cells without virus; E, {alpha}T3 cells at 2 days after infection of Ad{alpha}Gal; F, {alpha}T3 cells at 5 days after infection of Ad{alpha}Gal.

 
The intensity of X-gal staining was variable even though 100% of cells were stained blue. ß-Gal activity in cell lysates was therefore measured to obtain a more quantitative index of the level of expression. Results with AdGHGal and the Ad{alpha}Gal vectors are expressed relative to the ubiquitously expressed AdCMVGal (MOI of 10 PFU/mL) to allow comparisons between different cell types. GH3 cells infected with AdGHGal at an MOI of 10 PFU/cell had 16.2% of the activity of AdCMVGal. The activity of {alpha}T3 cells infected with Ad{alpha}Gal was 28.7% of the level of AdCMVGal (Fig. 3Go).



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  		Figure 3. Cell-specific expression of adenoviral vectors. GH3 cells, {alpha}T3 cells, and T47D cells were infected with AdCMVGal, AdGHGal, and Ad{alpha}Gal at a MOI of 10. The ß-gal activity was assayed at 48 h after infection. The activity of cells infected with AdGHGal and Ad{alpha}Gal was calculated as a percentage of the activity of AdCMVGal-infected cells. Results are expressed as the mean ± SD.

 
In an effort to assess the cell specificity of the AdGHGal and Ad{alpha}Gal vectors, they were each infected into the cell line producing the other hormone. The AdGHGal vector exhibited 24.1% of the activity in {alpha}T3 cells compared to GH3 cells. The Ad{alpha}Gal vector was expressed in GH3 cells at about 18.1% its activity in {alpha}T3 cells. In T47D breast cancer cells, AdGHGal expression was undetectable, and Ad{alpha}Gal showed 6.9% of the ß-galactosidase activity seen in {alpha}T3 cells (Fig. 3Go).

Analysis of long term expression of the reporter gene demonstrated a gradual increase in ß-gal activity in GH3 cells and {alpha}T3 cells infected with AdGHGal or Ad{alpha}Gal, respectively, with peak expression occurring 5–7 days after infection (Fig. 4Go). Thereafter, the activity decreased, but it was still maintained until 21 days after infection. For cells infected with AdCMVGal, ß-gal activity increased sharply and peaked earlier, but also decreased faster than in cells infected with AdGHGal or Ad{alpha}Gal.



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  		Figure 4. Long term course of lacZ gene expression of GH3 and {alpha}T3 infected with adenoviral vectors (5 PFU/cell of AdCMVGal and 10 PFU/cell of AdGHGal and Ad{alpha}Gal). Triplicate wells were used to measure lacZ gene expression by assessing ß-galactosidase activity using ONPG as substrate. Infected cell lysates were added to the ONPG substrate solution and incubated at 37 C for 2 h. The reaction was stopped with 100 µL 1 mol/L Na2CO3. Absorption was measured at 405 nm, and the activities of individual wells were calculated by a standard ß-gal activity curve. Results are expressed as the mean ± SD.

 
Expression of TK

Western blot analyses were performed to examine the expression of the HSV-TK protein 48 h after infection with either AdGHTK or Ad{alpha}TK. As shown in Fig. 5Go, expression of TK was detected in AdGHTK-infected GH3 (lane 2) and {alpha}T3 cells (lane 6) and in Ad{alpha}TK-infected {alpha}T3 (lane 4) and GH3 cells (lane 5). The level of TK expression in AdGHTK-infected {alpha}T3 cells (lane 6) was weaker than that in GH3 cells (lane 2), whereas Ad{alpha}TK expression was similar in the two cell types (lanes 4 and 5). T47D cells infected with AdGHTK (lane 8) or Ad{alpha}TK (lane 9) did not express the TK protein. These results demonstrate that the viral vectors express TK protein and confirm that expression occurs in a pituitary cell-preferential manner.



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  		Figure 5. Western blot analysis of HSV-TK protein. Cell extracts were analyzed for expression of the HSV-TK protein with a rabbit polyclonal antibody 48 h after infection with 10 PFU/cell of AdGHTK or Ad{alpha}TK. The anti-HSV-TK antibody recognized a band at 46 kDa. Each sample was prepared using equal numbers of cells, and equal loading was confirmed by Coomassie blue staining (not shown). Lanes 1, 2, and 5, GH3 cells; lanes 3, 4, and 6, {alpha}T3 cells; lanes 7–9, T47D.

 
Ganciclovir sensitivity of GH3 cells and {alpha}T3 cells infected with adenoviral vectors

Adenoviral vectors expressing the TK gene were used to investigate the sensitivity of tumor cell lines to expression of the toxic gene. To activate TK-mediated cytotoxicity, cells were exposed for 4 days to a range of GCV doses, and cell viability was determined using a cell proliferation assay. Without viral infection, high doses of GCV (50 µg/mL) induced partial cytopathic effects (~25% cell death) using either GH3 or {alpha}T3 cells (data not shown). In addition, cytopathic effects were seen when adenoviral vectors were infected at high MOIs (>100 PFU/cell). For these reasons, experiments were performed using varying amounts of adenoviral vectors and a range of GCV doses. As controls, the cytotoxicity of the TK-expressing vectors was compared to that of the ß-Gal-expressing vectors.

Using the AdGHTK virus, cytotoxicity increased in proportion to the amount of virus and in response to increasing amounts of GCV (Fig. 6AGo). For example, at a MOI of 10 PFU/mL, cytotoxicity of GH3 cells infected with AdGHTK occurred with GCV concentrations as low as 1 µg/mL, and complete cell death occurred using 10 µg/mL GCV (Fig. 6AGo). Similar results were seen in {alpha}T3 cells infected with the Ad{alpha}TK virus. There was 90% cell death at a dose of 5 µg/mL of GCV and a MOI of 10 PFU/mL (Fig. 6CGo). At doses of 100–500 PFU/mL, the Ad{alpha}TK virus was toxic to {alpha}T3 cells, even at 1 µg/mL GCV. Little or no cytopathic effect was seen with the control AdGHGal and Ad{alpha}Gal viruses, except at very high amounts of the vectors (500 PFU/mL; Fig. 6Go, B and D).



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  		Figure 6. GCV sensitivity of cells infected with adenoviral vectors. After infection of cell lines at different MOI of adenoviral vectors, cells were treated with varying doses of GCV for 4 days, and the cell viability was determined by a cell proliferation assay. The percent survival of cells was presented as a percentage of the absorbance found in the GCV-treated cells divided by that in the cells without GCV treatment (mean ± SD). A, GH3 cells infected with AdGHTK; B, GH3 cells infected with AdGHGal; C, {alpha}T3 cells infected with Ad{alpha}TK; D, {alpha}T3 cells infected with Ad{alpha}Gal; E, GH3 cells infected with Ad{alpha}TK; F, {alpha}T3 cells infected with AdGHTK; G, T47D cells infected with AdGHTK; H, T47D cells infected with Ad{alpha}TK.

 
We also evaluated the effects of the AdGHTK virus in {alpha}T3 cells and the Ad{alpha}TK virus in GH3 cells to assess whether TK expression from these pituitary hormone promoters is cell type specific. As shown in Fig. 6Go, E and F, the GCV sensitivity induced by these viruses was similar to that seen in cell lines that actively express the hormone promoters. These findings are consistent with the expression of ß-Gal by these promoters in both cell lines (Fig. 3Go). In contrast, T47D breast cancer cells infected with AdGHTK or Ad{alpha}TK did not show GCV sensitivity at low doses of adenoviral vectors (1–10 MOI; Fig. 6Go, G and H). These experiments indicate that AdGHTK or Ad{alpha}TK viruses confer dose-dependent GCV sensitivity to pituitary, but not breast cancer, cell lines. However, because the promoters are active in both pituitary cell lines, their effects are not specific for the types of hormones produced by these cell lines.

The long term effects of the adenoviral vectors on cell growth were assessed at a dose of GCV (5 µg/mL) that is not toxic in the absence of viral infection. Cells were infected with varying doses of virus (MOIs of 0, 1, 5, and 10), and cell viability was determined during an 8-day period of treatment with GCV (Fig. 7Go). In GH3 cells infected with AdGHTK (MOI of 1), there was 70% growth inhibition after 8 days (Fig. 7AGo). At higher doses of AdGHTK (5 and 10 PFU/mL), there was greater than 95% tumor cell growth inhibition. The growth rate of GH3 cells was not affected by the AdGHGal virus, indicating that growth inhibition is caused by the expression of the TK gene. Similar results were seen in {alpha}T3 cells when they were infected with the Ad{alpha}TK virus (Fig. 7BGo). At 1 PFU/mL, Ad{alpha}TK caused 60% growth inhibition, and there was greater than 95% inhibition of {alpha}T3 cell growth at 5 and 10 PFU/mL. In contrast, there was no growth inhibition of {alpha}T3 cells infected with the Ad{alpha}Gal virus.



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  		Figure 7. Long term effect of GCV on growth of GH3 cells and {alpha}T3 cells infected with adenoviral vectors. MOIs of 0, 1, 5, and 10 of viruses were achieved, and 5 µg GCV were added to each well. The cell viability was assayed on days 0, 2, 4, 6, and 8 after adding GCV. Results are expressed as the mean ± SD. A, GH3 cells infected with AdGHTK and AdGHGal; B, {alpha}T3 cells infected with Ad{alpha}TK and Ad{alpha}Gal.

 
Effect of the AdGHTK virus on GH3 cell tumors in vivo

GH3 cells and {alpha}T3 cells were injected sc into nude mice in an effort to develop an in vivo model for assessing the effects of the recombinant adenoviruses. Tumors (601.7 ± 217.1 mm3) developed with the GH3 cells, but not with {alpha}T3 cells (data not shown). Therefore, subsequent experiments were performed using tumors developed from GH3 cells.

Injection of the GH3 cell tumors with AdGHTK virus, and treatment with GCV caused a dramatic regression in the tumors (Fig. 8Go). To quantitate the effect of the virus, several different tumors were injected with AdGHTK or with the nontoxic control, AdGHGal. All eight GH3 cell tumors that received AdGHTK demonstrated regression after GCV treatment (Fig. 9AGo), whereas all eight tumors that were injected with AdGHTK, but were not treated with GCV, increased in size (1299.9 ± 570.4 mm3; Fig. 9BGo). Tumors that were injected with the control virus, AdGHGal, in the presence of GCV (1304.5 ± 432.2 mm3; Fig. 9CGo) or with vehicle in the presence of GCV treatment (1627 ± 642.5 mm3; Fig. 9DGo) also increased in size.



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  		Figure 8. Injection of adenoviral vectors into GH3 cell tumors in nude mice. Tumors were injected 1.0 x 109 PFUs of AdGHTK or AdGHGal adenoviral vectors followed by treatment with GCV (100 mg/kg, ip, once daily for 10 days). Excised tumors are shown in the panels at the right.

 


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  		Figure 9. Effect of injection of adenoviral vectors into preestablished GH3 cell tumors of nude mice. The growing tumors received an intratumoral injection of 1.0 x 109 PFUs of the indicated adenoviral vectors, and GCV was administered ip at 100 mg/kg once daily for 10 days. Tumor size (presented as cubic millimeters) was measured before and 10 days after GCV treatment. A, AdGHTK and GCV treatment; B, AdGHTK without GCV treatment; C, AdGHGal and GCV treatment; D, dialysis buffer used for adenovirus preparation and GCV treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Gene therapy is an appealing strategy for the treatment of pituitary tumors for several reasons. Pituitary adenomas are biologically benign, relatively localized, and rarely metastasize. Therefore, direct administration of adenoviruses has a greater chance of reaching most of the tumor cells. In contrast to more aggressive forms of cancer, incomplete destruction of pituitary tumor cells is unlikely to be associated with rapid recurrence or metastases. Although an ultimate goal is to completely eliminate residual pituitary tumor cells, a partial reduction of tumor size would be of clinical benefit by virtue of reducing tumor mass effects on surrounding structures and should also lower hormone levels.

In this report, we describe initial attempts to target a toxic gene to rodent pituitary tumor cell types. We have shown that 0.4 kb of the human GH promoter and 0.9 kb of the human {alpha}-subunit promoter are sufficient to express the ß-gal and TK genes. Parallel studies of these two genes offer several advantages. The ß-gal gene allows determination of infection efficiency using histological analyses and allows quantitation of promoter activity based on the level of expressed enzyme activity. On the other hand, the TK gene confers cytotoxicity in the presence of GCV, more closely mimicking the ultimate goal of cell killing in pituitary tumors.

The choice of promoter fragments for these studies was based in part on known functional sequences involved in expression and cell specificity, but also on practical limitations involving the amount of DNA that can be inserted into the modified adenoviral genome. In the case of the GH promoter, the cell type-specific expression of the 5'-flanking region has been confirmed in several lines of transgenic mice (16). The rat GH promoter, between -181 and +2 bp, was sufficient to be expressed specifically in the pituitary of transgenic mice (15). Expression was seen in some lactotrophs as well as somatotropes. In other transgenic models, GH promoter activity was greater when sequences were extended to -310 bp (20). Transient expression studies using 500 bp of the human GH promoter demonstrated efficient expression in GH3 cells (14). Deletion of the human GH gene to -285 bp preserved cell-specific expression, but further deletions caused loss of specific expression (21). For this reason, the human GH promoter used in this study (-336 to +58) contains this region. However, it is unclear at present whether this fragment confers maximal expression and cell specificity.

The expression and regulation of the {alpha}-subunit promoter have been studied extensively. In transgenic mice, the mouse {alpha}-promoter is strongly expressed in gonadotrope and thyrotrope cells using promoter sequences between 0.48–4.6 kb (22). Experiments using the diphtheria toxin gene linked to 313 bp of the bovine {alpha}-promoter caused ablation of gonadotope cells, but not thyrotrope cells, suggesting that distinct regulatory elements may be involved in thyrotrope- and gonadotrope-specific expression (18). In thyrotrope tumor cells, additional upstream sequences (-507 bp) are required for thyrotrope-specific expression (23). Because it is of interest to target each of the various types of glycoprotein hormone-producing tumors ({alpha}-subunit, TSH, LH, and FSH) (3), we used -846 to +45 bp of the human {alpha}-promoter sequence, which should be sufficient for expression in gonadotrope and thyrotrope cells (24). This promoter sequence has been shown previously to be expressed strongly in {alpha}T3 cells (25, 26), a finding consistent with our studies using adenoviral vectors that carry the human {alpha}-promoter.

An important question is whether the human GH and {alpha}-subunit promoters target gene expression in a cell type-specific manner. Our studies provide mixed evidence in this regard. Using ß-gal as an indicator, it is clear that each of these promoters is expressed in both GH3 cells and {alpha}T3 cells. Thus, promoter activity is not completely restricted to cell lines that produce the respective hormones. This finding is not unexpected for several reasons. First, these cell lines are not fully differentiated, a feature of most cells that proliferate rapidly in culture. Second, the {alpha}-promoter is known to be expressed in GH3 cells (27, 28, 29). To our knowledge, there are no previous studies of GH promoter activity in {alpha}T3 cells. However, these cells are thought to represent an early pluripotential progenitor of the pituitary cell lineage (30). Recently, several transcription factors, such as LH-2 (31) and Ptx1 (32), have been shown to be expressed in multiple pituitary cell types and to regulate the expression of several different pituitary hormone promoters. These and other pituitary transcription factors might account for some degree of leaky expression of the hormone promoters.

It is noteworthy that a substantial fraction (~37%) of human GH-secreting pituitary tumors cosecrete the {alpha}-subunit (33, 34), and up to 59% stain positive for both GH and {alpha}-subunit (35, 36, 37). Similarly, about 30% of TSH-secreting tumors (producing the {alpha}-subunit in combination with the TSH ß-subunit) also produce GH (38). Thus, there appears to be some overlap of {alpha}-subunit and GH expression in neoplastic pituitary cells.

Although the {alpha}-subunit and GH promoters overlap in their expression in different pituitary cell lines, there is evidence that the GH and {alpha}-subunit promoters are expressed preferentially in GH3 and {alpha}T3 cells, respectively. For example, the {alpha}-subunit promoter was expressed in GH3 cells at 18% of the level in {alpha}T3 cells. Similarly, the activity of the GH promoter in {alpha}T3 cells was about 30% of that seen in GH3 cells. In addition, AdGHGal and Ad{alpha}Gal expression was much lower in the nonpituitary T47D breast cancer cells. Consistent with the ß-gal results, there was no GCV-induced cytotoxicity when T47D cells were infected with AdGHTK or Ad{alpha}TK. We also did not detect AdGHGal or Ad{alpha}Gal expression in the livers of rats injected in the tail vein with 1 x 1010 PFU of virus (data not shown). By comparison, a similar amount of AdCMVGal was strongly expressed in liver, lung, and other organs. Further studies of the cell specificity of these promoters are required, because leaky expression of targeted genes could lead to undesired toxicity to normal tissues.

The cytotoxic effects of the TK gene can be activated by treatment with the synthetic nucleoside analog GCV. TK converts GCV into triphosphate-GCV, which results in the termination of DNA synthesis (39, 40). In principle, treatment with GCV provides an additional dimension of therapeutic opportunity by allowing the activity of the toxic gene to be modulated by the dose of the nucleoside analog. An important advantage of the TK gene is that the cytotoxicity induced by triphosphate-GCV only affects cells that are dividing. Therefore, one might expect minimal toxicity for normal pituitary cells, which have a low mitotic index. Consistent with this idea, we did not observe GCV-induced cytotoxicity in primary rat pituitary cell cultures infected with AdGHTK and Ad{alpha}TK (data not shown).

Consistent with the ß-gal expression data, GH3 cells and {alpha}T3 cells infected with AdGHTK or Ad{alpha}TK at a MOI of 5–10 PFU/cell showed cytotoxicity at 5–10 µg/mL GCV. The therapeutic dosage of GCV for human CMV isolates tested in vitro in several cell lines ranges from 0.2–3 µg/mL (39). Higher concentrations of GCV (>10 µg/mL) inhibit the growth of some mammalian cells in vitro. In our study, 50 µg/mL GCV induced some cell death in the absence of viral infection. These findings suggest that the amounts of TK expression produced by the AdGHTK or Ad{alpha}TK are sufficient to sensitize pituitary cells to therapeutic doses of GCV.

One of the benefits of using TK as a toxic gene is its ability to generate an in vivo "bystander" effect. In a dose-effect study, it has been shown that complete regression of tumors was observed when only 10% of the tumor cells expressed TK (41). This effect reflects the fact that the triphosphate nucleoside analogs can be transferred from one cell to another through gap junctions. In our in vivo studies, AdGHTK caused significant regression of GH3 cell tumors 10 days after GCV treatment. All other groups, including AdGHTK injection in the absence of GCV, demonstrated continued growth of the tumors. In other studies, 2 x 109 PFUs of adenoviral vectors were cytopathic to hepatocellular carcinomas when injected into tumors (83 mm3) in vivo (8). In our study, effective tumor regression was achieved without an apparent cytotoxic effect of the adenoviral vectors alone, even though the initial tumor volume (601.7 ± 217.1 mm3) was much greater, and the dose of the viral vectors was lower. These findings may reflect the impact of the bystander effect to induce regression of the AdGHTK-injected GH3 cell tumors or the efficacy of the GH promoter. Further studies to optimize the doses of virus and GCV will be of interest.

In cell culture experiments, high doses of the adenoviral vectors were cytopathic, even without expression of the TK gene. At a high multiplicity of infection, the E1 region becomes unnecessary for viral replication (42). This may reflect the high level of expression of viral gene products, some of which are cytotoxic or trigger host cellular immune responses (43, 44). Although this effect is not necessarily deleterious in the context of tumor cell destruction, it could lead to inadvertent toxicity in normal cells. Recently, the E4 region of adenovirus has been shown to be necessary for viral replication (45). The E4 region contains several open reading frames that encode a variety of regulatory proteins that shut off host protein synthesis and induce normal viral DNA replication (46, 47). In the future, deletion of the E1 and E4 regions should be advantageous compared to the current E1-deleted vector.

Although many hurdles remain before gene therapy might be applied to human pituitary tumors, it represents a potentially valuable adjunct to existing therapeutic options. Because of the advantages of local administration of adenoviral vectors, gene therapy might be used initially in conjunction with neurosurgery. Recently, there have been a number of important advances in specific medical therapy of pituitary tumors (2). Dopamine agonists represent an effective modality for the treatment of prolactinomas, and somatostatin analogs provide a useful means for suppressing GH and TSH production by pituitary tumors. Efforts to develop specific antagonists for the GH receptor also represent a potentially promising treatment for GH hypersecretion (48). Thus, there are several emerging strategies for designing specific therapies for different types of pituitary tumors, and ultimately, it may be possible to use independent approaches for more effective management of pituitary tumors.

In conclusion, we have demonstrated that adenoviral vectors containing pituitary hormone promoters efficiently transfer marker genes as well as a toxic gene to pituitary tumor cells in vitro and in vivo. These or similar adenoviral vectors have potential for gene therapy of human pituitary adenomas.


   Acknowledgments
 
We thank Richard Yu and Youngkyu Park for helpful discussions and advice. We are grateful to Tom Kotlar, Leah Sabacan, and Kristina Stanfield for DNA sequencing and assistance with animal experiments.


   Footnotes
 
1 This work was supported in part by an institutional grant from the Northwestern Memorial Foundation and by a Center of Excellence grant from Knoll Pharmaceutical Co.. Back

Received September 8, 1998.

Revised November 2, 1998.

Accepted November 11, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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