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Original Studies |
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-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 -subunit-producing pituitary tumor cell
lines, recombinant adenoviruses containing either the -subunit
promoter (AdGal; AdTK) 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 AdTK caused greater than 95%
cytotoxicity of GH3 and 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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
-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 -subunit
promoters to target genes to pituitary cell lines and transplanted
pituitary tumors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GH3 GH- and PRL-producing pituitary tumor cells and
HEK293 embryonic kidney cell lines were obtained from American Type
Culture Collection (Manassas, VA). The -subunit-secreting pituitary
tumor cell line, T3–1 (hereafter referred to 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. 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 -subunit promoter. The human GH promoter consists of
nucleotides -336 to +58 from the human GH 5'-flanking region, and the
human -subunit promoter consists of nucleotides -846 to +45 of the
human -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. 1
). 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. 1).
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-subunit
promoter (AdTK, AdGal). Individual clones of AdGHTK, AdGHGal,
AdTK, and AdGal 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, AdGal, 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 AdTK 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 AdTK-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. T3 cells (1 x
107) were also injected into male and female nude mice, but
no tumors developed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gal, and AdCMVGal
The efficiency of adenoviral vector-mediated gene transfer to
pituitary hormone producing GH3 and T3 cells was
assessed after infection with AdGHGal, AdGal, 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 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. 2B). The percentage of GH3
cells expressing ß-galactosidase increased with time and reached
95–100% by 4–5 days after infection (Fig. 2C). Similar results were
seen in T3 cells using the -subunit promoter-driven adenovirus,
AdGal. About 65–70% of T3 cells were stained blue at 48 h
(Fig. 2E), and the fraction of infected cells increased to 95–100% by
4–5 days after infection (Fig. 2F).
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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 T3 cells infected with
AdGal was 28.7% of the level of AdCMVGal (Fig. 3).
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Gal
vectors, they were each infected into the cell line producing the other
hormone. The AdGHGal vector exhibited 24.1% of the activity in T3
cells compared to GH3 cells. The AdGal vector was
expressed in GH3 cells at about 18.1% its
activity in T3 cells. In T47D breast cancer cells, AdGHGal
expression was undetectable, and AdGal showed 6.9% of the
ß-galactosidase activity seen in T3 cells (Fig. 3
).
Analysis of long term expression of the reporter gene demonstrated a
gradual increase in ß-gal activity in GH3 cells
and T3 cells infected with AdGHGal or AdGal, respectively, with
peak expression occurring 5–7 days after infection (Fig. 4). 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 AdGal.
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Western blot analyses were performed to examine the expression of
the HSV-TK protein 48 h after infection with either AdGHTK or
AdTK. As shown in Fig. 5, expression
of TK was detected in AdGHTK-infected GH3 (lane
2) and T3 cells (lane 6) and in AdTK-infected T3 (lane 4) and
GH3 cells (lane 5). The level of TK expression in
AdGHTK-infected T3 cells (lane 6) was weaker than that in
GH3 cells (lane 2), whereas AdTK expression
was similar in the two cell types (lanes 4 and 5). T47D cells infected
with AdGHTK (lane 8) or AdTK (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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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 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. 6A). 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. 6A). Similar results were
seen in T3 cells infected with the AdTK virus. There was 90%
cell death at a dose of 5 µg/mL of GCV and a MOI of 10 PFU/mL (Fig. 6C). At doses of 100–500 PFU/mL, the AdTK virus was toxic to T3
cells, even at 1 µg/mL GCV. Little or no cytopathic effect was seen
with the control AdGHGal and AdGal viruses, except at very high
amounts of the vectors (500 PFU/mL; Fig. 6, B and D).
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T3 cells
and the AdTK virus in GH3 cells to assess
whether TK expression from these pituitary hormone promoters is cell
type specific. As shown in Fig. 6, 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. 3).
In contrast, T47D breast cancer cells infected with AdGHTK or AdTK
did not show GCV sensitivity at low doses of adenoviral vectors (1–10
MOI; Fig. 6, G and H). These experiments indicate that AdGHTK or
AdTK 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. 7). In GH3 cells
infected with AdGHTK (MOI of 1), there was 70% growth inhibition after
8 days (Fig. 7A). 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 T3 cells when they were infected
with the AdTK virus (Fig. 7B). At 1 PFU/mL, AdTK caused 60%
growth inhibition, and there was greater than 95% inhibition of T3
cell growth at 5 and 10 PFU/mL. In contrast, there was no growth
inhibition of T3 cells infected with the AdGal virus.
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GH3 cells and 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 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. 8). 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. 9A),
whereas all eight tumors that were injected with AdGHTK, but were not
treated with GCV, increased in size (1299.9 ± 570.4
mm3; Fig. 9B). Tumors that were injected with the control
virus, AdGHGal, in the presence of GCV (1304.5 ± 432.2
mm3; Fig. 9C) or with vehicle in the presence of GCV
treatment (1627 ± 642.5 mm3; Fig. 9D) also increased
in size.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 -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 -subunit promoter have been
studied extensively. In transgenic mice, the mouse -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 -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 (-subunit, TSH, LH, and FSH) (3), we used -846 to +45 bp of
the human -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 T3
cells (25, 26), a finding consistent with our studies using adenoviral
vectors that carry the human -promoter.
An important question is whether the human GH and -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 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
-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 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 -subunit (33, 34), and
up to 59% stain positive for both GH and -subunit (35, 36, 37).
Similarly, about 30% of TSH-secreting tumors (producing the
-subunit in combination with the TSH ß-subunit) also produce GH
(38). Thus, there appears to be some overlap of -subunit and GH
expression in neoplastic pituitary cells.
Although the -subunit and GH promoters overlap in their
expression in different pituitary cell lines, there is evidence that
the GH and -subunit promoters are expressed preferentially in
GH3 and T3 cells, respectively. For example,
the -subunit promoter was expressed in GH3
cells at 18% of the level in T3 cells. Similarly, the activity of
the GH promoter in T3 cells was about 30% of that seen in
GH3 cells. In addition, AdGHGal and AdGal
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 AdTK. We
also did not detect AdGHGal or AdGal 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 AdTK (data not
shown).
Consistent with the ß-gal expression data, GH3
cells and T3 cells infected with AdGHTK or AdTK 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 AdTK 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.
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Acknowledgments |
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Footnotes |
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Received September 8, 1998.
Revised November 2, 1998.
Accepted November 11, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-subunit transgene in thyrotropes, gonadotropes, and
developing pituitary gland. Mol Endocrinol. 8:1420–1433.-subunit gene promoter. Endocrinology. 127:1352–1361.-gene in primary cultures of rat pituitary cells. Mol Endocrinol. 7:797–805. T3 cells
by increasing transcription and mRNA stability. Endocrinology. 134:2475–2481. subunit gene. J Biol Chem. 268:21984–21989.-subunit promoter by a
LIM-homeodomain transcription factor. Mol Cell Biol. 14:2985–2993.-subunit hypersecretion in patients with
pituitary tumors: clinically nonfunctioning and somatotroph adenomas. J Clin Endocrinol Metab. 70:859–864.-subunit by pituitary
tumors. J Clin Endocrinol Metab. 64:1187–1193. in GH-producing pituitary adenomas in acromegalic patients. Acta Neuropathol. 87:568–571.[Medline]
-subunit and prolactin
release by cultured pituitary adenoma cells from acromegalic patients:
correlation with GH release. Clin Endocrinol (Oxf). 30:601–611.[Medline]
subunit in human GH
secreting pituitary adenomas. Virchows Arch A Pathol Anat Histopathol. 411:323–330.[CrossRef][Medline]
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