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Molecular Medicine and Gene Therapy Unit (T.D.S., S.W., J.S.-A., C.A.G., M.J.P., P.R.L., M.G.C.), School of Medicine, Stopford Building; School of Biological Sciences (I.M.); and Endocrine Sciences Research Group (J.R.E.D.), University of Manchester, Manchester M13 9PT, United Kingdom; and Laboratoire de Biologie et Therapeutique des Pathologies Immunitaires (D.K.), Universitè Pierre and Marie Curie, CNRS, Hôpital de la Pitiè Salpétrière, 75651 Paris, Cedex 13, France
Address all correspondence and requests for reprints to: Professor M. G. Castro, Molecular Medicine and Gene Therapy Unit, School of Medicine, Room 1.302, Stopford Building; University of Manchester, Manchester, Oxford Road, Manchester M13 9PT, United Kingdom. E-mail: mcastro{at}fsl.scg.man.ac.uk
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 5,000-bp human PRL promoter (hPrl) has been extensively studied and shown to allow pituitary-specific expression (4, 5). The specificity of the rat PRL promoter has been demonstrated in vivo using transgenic mice (6). The proximal region of the promoter (+33bp to -422 bp) is sufficient to provide lactotroph specific expression; the distal region (-1500 bp to -1800 bp) is required for high levels of expression. For this reason, we have decided to use the +14 bp to -4429 bp sequences containing the proximal element (-40 bp to -250 bp), the distal element (-1300 bp to -1700 bp), and a portion of the super distal region (-3500 bp to -5000 bp) of hPrl promoter for our studies.
Castro et al. (1997) (7) were the first to demonstrate the feasibility of using both adenoviruses and herpes simplex type-1 vectors to transfer genes into anterior pituitary cells in primary culture (8). We have also recently used herpes simplex type-1 thymidine kinase (HSV1-TK) encoded within a replication defective adenovirus vector under the control of a strong ubiquitous promoter (hCMV) to successfully inhibit pituitary lactotroph hyperplasia and reduce circulating PRL levels in an animal model (9).
In this paper, we demonstrate lactotrophic cell type- specific expression of the marker gene ß-galactosidase, and the conditional cytotoxic gene HSV1-TK, both driven by the hPrl promoter and encoded within recombinant adenovirus vectors. Cell type-specific expression was demonstrated in anterior pituitary tumor cell lines, anterior pituitary cells in primary culture, and the pituitary gland in vivo. Our results show, for the first time, transcriptional targeting of a marker transgene (ß-galactosidase), as well as the therapeutic transgene HSV1-TK, to a predetermined endocrine cell population within the AP gland in vivo.
When an adenovirus vector encoding HSV1-TK driven by the hPrl promoter was used in an in vivo model of estrogen/sulpiride induced lactotroph hyperplasia within the anterior pituitary (AP) gland in situ, the infection of the AP gland combined with the administration of ganciclovir, was not able to reduce neither the weight of the gland, the number of lactotrophic cells in vivo, nor the circulating PRL levels. This is in contrast to the use of an adenovirus expressing HSV1-TK under the transcriptional control of the human cytomegalovirus promoter (hCMV), employed in the same experimental paradigm. Such a vector, in combination with ganciclovir, was effective in reducing pituitary weight and also circulating PRL levels. Therefore, our results highlight that further engineering of these cell type-specific promoters will be needed to develop effective cell type-specific gene therapy strategies for the treatment of pituitary tumors using transcriptional targeted approaches.
Our results also have important implications for the design of gene therapy strategies for pituitary tumors. Our data demonstrate that both the choice of the in vivo animal model (e.g. anterior pituitary adenoma in situ vs. transplantable tumors), as well as the gene therapy strategy chosen (e.g. use of strong ubiquitous promoters vs. weaker but cell type-specific promoters), does influence the therapeutic outcome.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents and antibodies
Different cell types within the AP cultures were identified
using the following polyclonal antibodies: guinea-pig antirat ß-TSH
(1/100), guinea-pig antirat PRL (1/500), guinea-pig antirat ß-LH
(1/100), guinea-pig antihuman GH (1/500), sheep antihuman ß-FSH
(1/500), and sheep antihuman ACTH (1/500) (provided by NIDDK’s
National Hormone and Pituitary Program, Bethesda, MD). The antibody
used to identify ß-galactosidase was a rabbit polyclonal
anti-ß-galactosidase (1/750) (9); rabbit anti-HSV1-TK antibody
(1/1000) was kindly provided by M. Janicot, Rhone-Poulenc-Rorer, France
(10, 11).
Secondary antibodies used for either single or double immunolabeling were FITC or Texas Red-donkey antirabbit and FITC or Texas Red-goat anti-guinea-pig from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Generation of recombinant adenoviruses expressing transgenes under
the control of the human PRL promoter
The transgenes, HSV1-TK and ß-galactosidase, were excised from
the plasmids pMV60/HSV1-TK (10) and pMV12/lacZ (12) by
BamHI digestion and cloned into a plasmid derived from
pGEM9ZF (Promega Corp., Madison, WI), that contained the
neuronal specific enolase (NSE) promoter and the SV40 polyA generating
pGEM9ZF/NSE/HSV1-TK/polyA and pGEM9ZF/NSE/lacZ/polyA. The
HSV1-TK/polyA and lacZ/polyA cassettes were excised from
these plasmids by NspV/XhoI digestion and the
NspV site adapted to NcoI using a linker. The
adapted fragments were then cloned into the p5000 plasmid, containing
the 5kb hPRl, in the NcoI/XhoI sites (13). The
hPrl/HSV1-TK/polyA cassette was excised by
XbaI/XhoI digestion and cloned into the
XbaI/SalI site in p
E1sp1a (Microbix
Biosystems, Toronto, Ontario, Canada). The hPrl/lacZ/polyA cassette was
excised by MunI/XhoI digestion and cloned into
the EcoRI/XhoI site in pE1sp1a.
RAds were generated by cotransfection using the calcium phosphate coprecipitation method (14) with each shuttle vector and pBHG10 (Microbix Biosystems), and purified using double cesium chloride gradient as previously described (15). Viral DNA was obtained as described by Revah et al., 1996 (16). To confirm the presence of the transgenes, viral DNA digestion with HindIII and subsequent Southern blot hybridization was performed. The specific probes used were: the 2982 bp lacZ fragment excised using BamHI (+5033 to +8015) from p5000/hPrl/lacZ, and the 1131 bp HSV1-TK fragment excised using BamHI (+5033 to +6164) from p5000/hPrl/HSV1-TK. The probes were labeled by random priming with digoxigenin-dUTP as described by the manufacturers (Roche Molecular Biochemicals, Bell Lane, East Sussex, UK).
The RAd-hCMV/HSV1-TK (RAd128), and RAd-hCMV/ß-gal, (RAd35) have been described in detail previously (10, 11, 12). RAd-mCMV/ß-gal (RAd36) was constructed using the murine CMV promoter (-1336 to +36 bp) (17) driving the expression of the marker gene ß-galactosidase. RAd stocks were assayed and shown to be negative, for the presence of replication competent adenovirus or endotoxin (lipopolysaccharide) as previously described (18, 19).
Cell type-specific expression of ß-galactosidase in the pituitary
tumor cell lines, GH3 and AtT20s: X-gal histochemistry and enzymatic
activity from RAdPrl/lacZ, RAd35, and RAd36
GH3 and AtT20 cells were infected at multiplicity of infection
(MOI) (number of infectious virus particles/cell) 30 with RAd35, RAd36,
or RAd-Prl/lacZ. The cells were incubated for a further 2 days and
X-gal histochemistry was performed as described previously (7).
Enzymatic activity was tested by infecting GH3 and AtT20 cells at increasing MOIs (0, 3, 10, 30, 100, and 300) with RAd35, RAd36, or RAd-Prl/lacZ. The cells were incubated for 2 days and then harvested in lysis buffer [25 mM Tris-HCL (pH7.8), 6.7% (vol/vol) glycerol, 10 mM MgCl2, 0.01% (vol/vol) Triton X-100, 1 mM EDTA (pH8)]. The lysates were incubated with the O-nitrophenol-ß-D-galactopyranoside (ONPG) (4 mg/ml) substrate solution at 37 C for appropriate time intervals until color developed within the linear range of the standard curve. The reactions were stopped by the addition of 510 µl Na2CO3 and samples were read on a spectrophotometer (Amersham Pharmacia Biotech, St Albans, UK) at 420 nm. The enzyme activity was expressed as units of ß-galactosidase produced per cell. Each experimental condition was done in quadruplicate, and each experiment was repeated twice.
Cell type-specific expression of HSV1-TK and apoptosis in pituitary
tumor cell lines
GH3 and AtT20 cells were infected with RAdPrl/HSV1-TK at an MOI
30 for 48 h. The virus was then removed, and fresh medium was
added. The cells were then incubated for a further 2 days. HSV1-TK
transgene expression was assessed using immunofluorescence techniques
as described previously (7). Cellular nuclei were stained using
4,6-diamidino-2-phenylindole (DAPI).
Simultaneous detection of HSV1-TK protein and cellular DNA content within GH3 and AtT20 cells following infection with RAd-Prl/HSV-TK was performed by flow cytometry as described previously (9). Fifty thousand cells were plated in six-well plates and infected with RAdPrl/HSV1-TK at increasing MOI (0, 1, 5, 20, 50, and 100). Experiments were performed twice. After 48 h infection, the cells were fed with complete growth medium containing 10 µM ganciclovir (GCV; Roche Molecular Biochemicals, Welwyn Garden City, UK) and incubated for a further 72 h. Cells were harvested and then processed for DNA content and HSV1-TK immunoreactivity as described previously (9, 20, 21).
Infection and detection of HSV1-TK within endocrine cells in
primary AP cultures
AP cells were infected with RAd-Prl/HSV1-TK at an MOI 30, and
incubated for 48 h. The virus was removed, fresh complete growth
medium was added, and the primary cultures were incubated for 3
additional days. Colocalization of the HSV1-TK transgene within
specific cell types present within the primary AP culture was assessed
using immunofluorescence techniques as described previously (9); the
cellular nuclei were stained using DAPI. For the quantification of
endocrine and/or transduced cell types in vitro, 10 random
fields within each well were counted.
Animals
Male 8-week-old Buffalo rats were house bred at the University
of Manchester Biological Safety Unit. All animals had free access to
food and water, a 12-h light, 12-h dark cycle, and constant housing
temperature and humidity. Experiments were conducted according to the
United Kingdom Animal (Scientific Procedures) Act of 1986.
In vivo gene delivery to the anterior pituitary gland
Male 8-week-old Buffalo rats (house bred) were anesthetized with
halothane and placed in a sterotaxic frame. The skull was exposed,
bregma was identified, and a hole was drilled posterior to bregma until
revealing the superior sagittal sinus, and surrounding brain. A thin
surgical suture was passed underneath the vein, and the meninges
surrounding the vein were cut to allow the vein to be displaced
laterally without lesioning the vein wall or the surrounding cortex.
Intrapituitary injections were made using a 26-gauge Hamilton syringe
needle. The tip of the needle had been previously ground until the
opening of the needle was positioned at the base of the tip. Injections
were made at the following coordinates: antero-posterior from Bregma,
-0.57, -0.60, and -0.63 cm, and lateral on each side of the midline
at 0.05 cm. Thus, we made a total of six injections per pituitary
gland. Previous attempts to inject directly into the pituitary using
exclusively stereotaxic coordinates failed in reliably delivering
directly the RAds into the gland. Thus, we developed a modified
strategy as follows: the modified Hamilton needle was lowered at each
coordinate until touching the sphenoidal bone, and making contact with
the bottom of the rat equivalent of the sella turcica. This leaves the
opening of the needle within the pituitary gland, and adequate amounts
of recombinant vector were then injected. Under these conditions of
injection, the pituitary was transduced by recombinant adenoviruses in
100% of surgical attempts. At each of these six coordinates, 1 µl of
the recombinant vector [1 x108 (8) pfu] was
then delivered over 1 min per injection site. Animals were then given
10 ml of saline sc and allowed to recover. Forty-eight hours later,
animals were perfused transcardially with Tyrode solution (132
mM NaCl, 1.8 mM CaCl2,
0.32 mM
NaH2PO4, 5.56
mM glucose, 11.6 mM
NaHCO3, and 2.68 mM KCl),
pituitary glands were removed and placed in 4% paraformaldehyde
dissolved in 0.1 M PBS for 3 h. Tissue was then
paraffin embedded, sectioned using a microtome (5 µm) (Leica Corp.), and mounted onto 3-aminopropyltriethoxysilane (APES)
(Sigma)-coated glass slides.
Induction of lactotroph hyperplasia and delivery of RAds into the
AP gland
Male 8-week-old Buffalo rats were implanted with SILASTIC brand
pellets (Dow Corning Corp., Midland, MI) containing 15 mg
of 17ß-estradiol and 50 mg of sulpiride, prepared as previously
described (22). The pellets were implanted sc in the lumbar region of
each rat under anesthesia. Empty SILASTIC brand pellets were implanted
as controls. Three days later, the animals were anesthetized with
Fluothane and a total of 1 x 108 pfu in 6
µl of either RAd35, RAd128 or RAd-PRL/HSV1-TK was delivered to the
anterior pituitary gland as described above. Rats were given a 10ml sc
saline injection post surgery, and glucose was added to their drinking
water every 2 days to prevent dehydration. One day after surgery, all
animals received ip GCV injections at a dose of 25 mg/kg twice daily
for 7 days. The animals were killed using a lethal overdose of
pentobarbital and perfused with Tyrode’s solution. Pituitary, body,
and testis weights were recorded. Before perfusion, trunk blood was
collected. Pituitary glands were then placed in 4% paraformaldehyde
dissolved in 0.1 M PBS for 3 h. Tissue was then
paraffin embedded, sectioned using a microtome (5 µm), and mounted
onto APES-coated glass slides.
Immunohistochemical detection of transgene expression within the
anterior pituitary gland in vivo using fluorescence microscopy
Sections were deparaffinated using xylene for 5 min then
rehydrated through graded alcohols (100%, 95%, 85%, 70%, 50%
ethanol), 3 min each before being washed in saline (0.8% NaCl wt/vol)
for 5 min. The blocking solution was prepared using horse serum (10%
vol/vol or 1% vol/vol) diluted in PBS, containing 0.1% Triton X-100.
The sections were then incubated in (1): 10% blocking solution for
2 h at room temp (2); 1% blocking solution for 1 h at room
temp (3); primary antibody (diluted in 1% blocking solution) for
1 h at room temp (4); five washes in PBS containing 0.5% Triton
X-100 for 5 min (5); secondary antibody (diluted in 1% blocking
solution) for 1 h at room temp; and (6) five washes in PBS
containing 0.5% Triton X-100 for 5 min. The sections were stained with
DAPI for 15 min, washed twice in PBS for 5 min, once in
dH2O for 5 min, and mounted in Mowiol
(Calbiochem Nottingham, UK). Images were acquired
using Openlab software (Improvision, Coventry, UK) on an Olympus Corp. (Tokyo, Japan) Vanox microscope. For the quantification of
transduced or nontransduced pituitary cells, five fields within the
area of the anterior pituitary, which expressed transgenes encoded by
viral vectors, were counted.
Determination of hormone levels in peripheral blood
Rat plasma PRL, GH, and TSH-ß, concentrations were determined
using specific RIA kits provided by the National Hormone and Pituitary
Program, NIDDK, and Dr A. F. Parlow (Torrance, CA). Plasma ACTH
was measured using a specific immunoradiometric assay that has been
described previously (23).
Statistical analysis
The in vitro and in vivo experimental
results were analyzed using either ANOVA, followed by the
Student’s-Neuman-Keuls multiple comparison test or the Student’s
t test where appropriate, using GraphPad Software, Inc. Instat Version 2 (GraphPad Software, Inc., San
Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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E1sp1a
containing the transgenes, i.e. HSV1-TK, or
ß-galactosidase under the control of +14 bp to -4429 bp or +14 bp to
-4152 bp of the hPrl promoter, respectively, and both followed by the
SV40 polyadenylation sequence. The expression construct was inserted
within the E1 region of the adenoviral genome. When DNA from these
recombinant RAds was digested with HindIII, and probed using
Southern blot hybridization, a band of the right size, indicating the
presence of the transgenes within the recombinant virus genome, was
observed, i.e. the 1406 bp HSV1-TK/polyA band, the 3257 bp
lacZ/polyA band, and the 5000 bp mCMV/lacZ,polyA band (Fig. 1
, A–C).
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). No expression of ß-galactosidase
was observed in the corticotrophic AtT20 cell line after infection with
RAd-Prl/lacZ. These results show that the PRL promoter can
restrict expression of the marker gene ß- galactosidase to
established lactotroph tumor cell lines in vitro.
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). Both
in GH3 cells and AtT20 cells, the levels of enzyme activity increased
with increasing MOIs used, of either RAd35 (hCMV/lacZ) or
RAd36 (mCMV/lacZ). After infection of GH3 cells with
RAd-Prl/lacZ, there was an increase in enzyme activity up to
MOI 300. After infection of AtT20 cells with RAd-Prl/lacZ, no enzyme
activity above basal levels was observed, even at the highest MOI used,
i.e. 300. In GH3 cells, at MOIs 100 and 300, there were 13-
and 24-fold more enzyme activity respectively (P <
0.005), in cells infected with RAd36 when compared with cells infected
with RAd35 (Fig. 3). Levels of transgene expressed under the control of
the mCMV promoter in GH3 cells were 46-fold (P <
0.005) and 70-fold (P < 0.005) stronger at MOIs of 100
and 300 MOIs (Fig. 3) compared with those expressed under the control
of the PRL promoter. In AtT20 cells at MOIs 100 and 300, the mCMV
promoter was 19-fold (P < 0.05) and 10-fold
(P < 0.005) stronger than the hCMV promoter (Fig. 3).
In GH3 cells at MOIs 100 and 300, the hCMV promoter was 3-fold
(P < 0.005) stronger than the PRL promoter at both
MOIs.
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). Infection of these
cells with RAd-Prl/HSV1-TK resulted in a strong immunocytochemical
staining in the lactotrophic GH3 cells; there was no increase, above
basal levels, of apoptotic nuclei at the concentration of virus used
(MOI 30) (Fig. 4). In AtT20 cells, we observed no HSV1-TK transgene
expression (Fig. 4).
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shows a gradual increase in HSV1-TK
immunoreactivity, as detected by flow cytometry in the GH3 cell line
only. The level of HSV1-TK immunoreactive protein was maximal at MOI
100 in the GH3 cells. HSV1-TK expression remained at basal levels in
AtT20 cells at all MOIs tested (Fig. 5A).
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). In the presence and absence of GCV (10 µM), even at
the highest MOI of RAd-Prl/HSV1-TK used, only basal levels of apoptosis
were observed in AtT20 cells indicating there was no HSV1-TK mediated
toxicity (Fig. 5C).
Cell type-specific expression of transgenes expressed under the
transcriptional control of the hPrl promoter in rat primary anterior
pituitary cell cultures and in the anterior pituitary gland in
vivo
When rat primary anterior pituitary cultures were infected with
RAd-Prl/HSV1-TK the expression of HSV1-TK was mainly restricted to
cells expressing PRL (Prl) (89% ± 7 of cells expressing HSV1-TK also
expressed PRL) (Fig. 6 and Table 1), and a subset of GH-producing cells
(11% ± 0.8 of total HSV1-TK immunoreactive cell population also
expressed GH) (Fig. 6 and Table 1). Transgene expression under the
control of the hPrl promoter was achieved in 60% ± 6.9 of the total
lactotrophic population in anterior pituitary cells in
vitro. Expression of the transgene HSV1-TK also observed in the
9% ± 0.9 of the total somatotrophic population possibly representing
the subpopulation of cells that co- express PRL and GH,
i.e. mammosomatotrophs. No expression of HSV1-TK was
observed in cells synthesizing FSH, LH, TSH, or ACTH. There was no
indication of apoptosis as assessed by the nuclear integrity after DAPI
staining (Fig. 6).
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and 8 and
Table 2). The mCMV and the hCMV promoters
drove transgene expression of either HSV1-TK or ß-gal in all
endocrine cell populations encountered within the anterior pituitary
gland (Figs. 7 and 8 and Table 2). However, the hPrl promoter
restricted expression of HSV1-TK almost exclusively to Prl expressing
cells (approximately 50% of the lactotrophic population within the
transduced area expressed the transgene) (Fig. 8 and Table 2). A small
number of GH immunoreactive cells were also HSV1-TK immunoreactive
(0.5–1% of total GH immunoreactive cells). These cells are likely to
be mammosomatotrophs. There was no indication of apoptosis in the
normal pituitary glands infected with any of the recombinant adenovirus
vectors as assessed by the nuclear integrity after DAPI staining and
microscopic examination of all endocrine cell populations (results not
shown).
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), whereas in the E/S-implanted animals
treated with RAd35/GCV, the population rose to 23 ± 0.5% of the
total cell population within the area of the anterior pituitary, which
had been transduced by the vector (P < 0.005
vs. control). In the E/S-implanted animal group treated with
RAd128/GCV, we observed a decrease in the PRL producing cell population
to 15 ± 1% of the total cell population (P <
0.05 vs. E/S implanted, RAd35/GCV-treated group) (Table 2).
There was no change in the number of PRL positive cells in E/S
implanted animals treated with RAd-Prl/HSV1-TK. There were no
significant changes in GH, FSH, LH, ACTH, or TSH-ß cell populations
in any of the treated animal groups with respect to controls (Table 2). Pituitary weight in control animals averaged 11.3 ± 0.5 mg. whereas the E/S implanted animals treated with RAd35/GCV had a mean pituitary weight of 22.7 ± 1.3 mg (P < 0.0005 vs. controls). The E/S implanted group treated with RAd128/GCV had an average pituitary weight of 18.7 ± 0.5 mg, an 18% reduction when compared with the E/S implanted RAd35/GCV treated group (P < 0.05). The E/S implanted group treated with RAd-Prl/HSV1-TK/GCV had pituitary weight of 22.9 ± 0.3 mg (not significant vs. E/S implanted group treated with RAd35/GCV).
Plasma Prl in placebo-implanted control rats was 38 ± 4 ng/ml,
whereas in the E/S-implanted group treated with RAd35 and GCV,
circulating Prl levels increased to 660 ± 28.3ng/ml. In the E/S
implanted group treated with RAd128 and GCV, Prl levels were reduced to
330 ± 50.1ng/ml (P < 0.005 vs. E/S
implanted RAd35/GCV treated group), a reduction of 50% (Fig. 9). In the E/S-implanted group treated
with RAd-Prl/HSV1-TK and GCV, Prl levels were not significantly
reduced. No changes were observed in circulating ACTH, GH, and TSH-ß
levels in any of the experimental groups (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results indicate that the human PRL promoter encoded within recombinant adenovirus vectors can restrict transgene expression, e.g. HSV1-TK or ß-galactosidase, exclusively to lactotrophic cells both in vitro and in vivo. The restriction of expression of HSV1-TK to lactotrophic cells, using the PRL promoter, resulted in the cell type-specific induction of apoptosis in the lactotrophic tumor GH3 cell line, in the presence of GCV. In the corticotrophic AtT20 cell line, there was neither HSV1-TK expression, nor apoptosis in the presence of GCV, even at an MOI 100. Therefore, the human PRL promoter is able to target the expression of HSV1-TK to lactotroph tumor cells.
The measurement of ß-galactosidase enzyme activity levels enabled us to quantitate and compare the levels of transgene expression directed by either the PRL promoter, or the constitutive mCMV or hCMV promoters. The mCMV promoter has been reported to be stronger in a variety of human and murine immortalized cell lines in vitro (25) or in the brain in vivo (26). Of the three promoters tested, i.e. hCMV, mCMV, and hPrl, the mCMV elicited the highest levels of transgene expression in both pituitary tumor cell lines. In GH3 cells, the mCMV promoter elicited approximately 50-fold higher expression when compared with the hPrl promoter.
The strength of the promoter used would determine the amount of virus needed to achieve a therapeutic effect. By using a stronger promoter, less virus would be needed to achieve adequate levels of transgene expression (25, 26). This would therefore reduce toxic side effects due to high doses of viral vector that have been previously shown to cause acute cytotoxicity (27) as well as chronic inflammation in the brain (10).
Our work has shown that sequences +14 bp to -4152 bp or -4429 bp of the human PRL promoter can restrict transgene expression from an adenoviral vector to lactotrophic cells in primary anterior pituitary cultures and importantly within the anterior pituitary gland in vivo. However, transgene expression was also detected in a subpopulation of GH expressing cells both in primary culture and in vivo. These cells are probably mammosomatotrophs, which are thought to be the transitional intermediates of lactotrophs and somatotrophs (28), and synthesize both GH and PRL.
Lee et al. (1999), (3) have recently shown that the GH and
the 
-glycoprotein subunit promoters could restrict the expression of
transgenes to somatotroph and null-cell tumor cell lines, respectively.
They also showed that the size of sc tumors, derived from implanted GH3
cells in nude mice, could be reduced by the delivery of an adenovirus
expressing HSV1-TK, under the control of the GH promoter, when combined
with the administration of GCV (3).
We decided to explore the in vivo delivery of HSV1-TK driven by the human PRL promoter encoded within recombinant adenovirus vectors to the anterior pituitary in a rat model of estrogen/sulpiride induced pituitary hyperplasia. This enabled us to demonstrate for the first time that, although the human PRL promoter is able to restrict transgene expression mainly to lactotrophic cells within the anterior pituitary gland in vivo, the level of expression of the therapeutic transgene (HSV1-TK) was not sufficient to achieve a measurable therapeutic outcome.
In contrast, the ubiquitous hCMV promoter driving expression of HSV1-TK, which we used as a positive control for this experiment, was able to elicit a beneficial therapeutic outcome. This confirms our previous results showing that delivery of HSV1-TK driven by the hCMV promoter to rats bearing estrogen-induced lactotroph hyperplasia, into the AP gland via the transauricular route followed by subsequent treatment with GCV decreased plasma PRL levels and reduced the mass of the pituitary gland (9). Neither RAd-Prl/HSV1-TK nor RAd-hCMV/HSV1-TK (RAd128) caused deleterious effects on circulating levels of other anterior pituitary hormones, suggesting that the treatment was nontoxic to the normal endocrine anterior pituitary cells in situ (9), although hormone secretion in response to secretagogues was not tested. Even if the hCMV promoter is not specific to the hyperplasic lactotrophic cells, its toxic effects ought to be restricted to those cells, which are actively dividing. In the estrogen/sulpiride model used, only lactotrophic cells are actively dividing. Although the hCMV promoter has no cell-type specificity, in our model only the lactotrophic cells are eliminated, due to the molecular mechanism of cell killing of HSV1-TK in combination with GCV, which only affects actively dividing cells.
The lessons to be learned from the in vivo experiment described in this paper are very important, not only in terms of the implications for the development of gene therapy strategies for pituitary disease, but also for gene therapy in general. Although cell type-specific promoters are efficient in restricting transgene expression to predetermined cell types, in this case such a promoter was not strong enough to produce a beneficial therapeutic outcome. Furthermore, a nonspecific promoter used in conjunction with a cell killing mechanism that selectively kills actively dividing cells, provided the restricted elimination of hyperplasic lactotroph pituitary cells, without adversely affecting other pituitary cell types.
Our results contrast with those published previously showing tumor regression in a GH transplantable tumor model in nude mice (3). We believe the different results depend on the particular model used. The transplantable model employs a very rapidly growing tumor in which HSV1-TK would elicit a very strong effect. We believe that our model represents more closely the situation that would be encountered in a human pituitary tumor in which the proliferative index is low. Because the efficiency of the HSV1-TK plus GCV system depends on both the efficiency of virus transduction and the rate of cell proliferation, we predict higher levels of HSV1-TK expression to be required to efficiently reduce lactotrophic cells’ proliferation and PRL hypersecretion in our in situ tumor model. This explains, why HSV1-TK expressed under the control of the hCMV promoter, but not, under control of the hPrl promoter, showed a significant therapeutic benefit. Again our results indicate the importance of properly designed preclinical studies before these therapeutic approaches are taken into the clinic.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this work and should be
considered first authors.
3 Training Fellow supported by Action Research (UK).
4 Funded by a BBSRC Studentship.
5 Fellow of The Lister Institute of Preventive Medicine.
Received December 28, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M Candolfi, G Jaita, D Pisera, L Ferrari, C Barcia, C Liu, J Yu, G Liu, M G Castro, and A Seilicovich Adenoviral vectors encoding tumor necrosis factor-{alpha} and FasL induce apoptosis of normal and tumoral anterior pituitary cells. J. Endocrinol., June 1, 2006; 189(3): 681 - 690. [Abstract] [Full Text] [PDF]
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 W. Xiong, S. Goverdhana, S. A. Sciascia, M. Candolfi, J. M. Zirger, C. Barcia, J. F. Curtin, G. D. King, G. Jaita, C. Liu, et al. Regulatable Gutless Adenovirus Vectors Sustain Inducible Transgene Expression in the Brain in the Presence of an Immune Response against Adenoviruses J. Virol., January 1, 2006; 80(1): 27 - 37. [Abstract] [Full Text] [PDF]
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 G. Jaita, M. Candolfi, V. Zaldivar, S. Zarate, L. Ferrari, D. Pisera, M. G. Castro, and A. Seilicovich Estrogens Up-Regulate the Fas/FasL Apoptotic Pathway in Lactotropes Endocrinology, November 1, 2005; 146(11): 4737 - 4744. [Abstract] [Full Text] [PDF]
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E. J. Lee and J L. Jameson Gene therapy of pituitary diseases J. Endocrinol., June 1, 2005; 185(3): 353 - 362. [Abstract] [Full Text] [PDF]
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C. Roche, A. J Zamora, D. Taieb, E. Lavaque, R. Rasolonjanahary, H. Dufour, C. Bagnis, A. Enjalbert, and A. Barlier Lentiviral vectors efficiently transduce human gonadotroph and somatotroph adenomas in vitro. Targeted expression of transgene by pituitary hormone promoters J. Endocrinol., October 1, 2004; 183(1): 217 - 233. [Abstract] [Full Text] [PDF]
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L. Barzon, M. Boscaro, and G. Palu Endocrine Aspects of Cancer Gene Therapy Endocr. Rev., February 1, 2004; 25(1): 1 - 44. [Abstract] [Full Text] [PDF]
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M. S. Sarac, S. Windeatt, M. G. Castro, and I. Lindberg Intrapituitary Adenoviral Administration of 7B2 Can Extend Life Span and Reverse Endocrinological Deficiencies in 7B2 Null Mice Endocrinology, June 1, 2002; 143(6): 2314 - 2323. [Abstract] [Full Text] [PDF]
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E. J. Lee, W. R. Duan, M. Jakacka, B. D. Gehm, and J. L. Jameson Dominant Negative ER Induces Apoptosis in GH4 Pituitary Lactotrope Cells and Inhibits Tumor Growth in Nude Mice Endocrinology, September 1, 2001; 142(9): 3756 - 3763. [Abstract] [Full Text] [PDF]
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 E. J. Lee, F. Martinson, T. Kotlar, B. Thimmapaya, and J. L. Jameson Adenovirus-Mediated Targeted Expression of Toxic Genes to Adrenocorticotropin-Producing Pituitary Tumors Using the Proopiomelanocortin Promoter J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3400 - 3409. [Abstract] [Full Text] [PDF]
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