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Original Studies |
Molecular Medicine and Gene Therapy Unit, School of Medicine (S.W., T.D.S., R.A.D., F.B., M.J.P., A.T.L., T.C.M., P.R.L., M.G.C.), and School of Biological Sciences (I.D.M.), University of Manchester, Manchester M13 9PT, United Kingdom; and INIBIOLP-Histology "B", Faculty of Medicine (R.G.G.), National University of La Plata, La Plata, Argentina; and Department of Immunology, Hopital Pitie-Salpetriere (D.K.), Paris, France
Address correspondence and requests for reprints to: M. G. Castro, Molecular Medicine and Gene Therapy Unit, School of Medicine, University of Manchester, Room 1.302, Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdom. E-mail: mcastro{at}fs1.scg.man.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here, we tested the hypothesis that gene transfer using recombinant adenovirus vectors (RAds) expressing herpes simplex type 1-thymidine kinase (HSV1-TK) might provide a future alternative therapeutic approach for the treatment of pituitary prolactinomas (7). We have used sulpiride- and oestrogen-induced lactotroph hyperplasia within the rat AP gland as an in vivo prolactinoma animal model (8).
Suicide gene therapy uses the transfer of nontoxic proteins, which can cause cell death via the conversion of a nontoxic prodrug to a toxic product. A well characterized conditional cytotoxic approach uses the HSV1-TK, which converts the prodrug ganciclovir (GCV) to GCV monophosphate, which is in turn further phosphorylated by cellular kinases to GCV triphosphate, which is toxic to proliferating cells. One attractive feature of this system is the bystander effect (9, 10), whereby untransduced cells are also killed.
As an initial step toward determining the feasibility of using viral vectors to develop gene therapy for pituitary prolactinomas, we previously determined the infectivity of both normal and tumoral AP cells in culture using RAds expressing the marker gene ß-galactosidase (11). We determined that RAd infection and transgene expression was nontoxic for normal AP cells. We and others have demonstrated that RAds infect both quiescent and dividing tumor cells, but infection was more efficient in proliferating cells (11, 12).
Here, we show that RAd/HSV1-TK in the presence of the nucleoside analogue GCV is nontoxic for the normal AP cells in-vitro, but causes cell death in the pituitary tumor cell lines GH3, a PRL/GH-secreting cell line, and AtT20, a corticotrophic cell line. We also show that injection of RAd/HSV1-TK into pituitaries of rats bearing oestrogen-induced lactotroph hyperplasia, and subsequent treatment with GCV, decreases plasma PRL levels and reduces the mass of the pituitary gland.
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Materials and Methods |
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Male rats weighing 200–300 g were purchased from Charles River Laboratories, Inc., Margate, Kent, UK). All animals had free access to food and water, a 12-h light dark cycle, and constant housing temperature and humidity. Experiments were conducted according to the United Kingdom Animal (Scientific Procedures) Act of 1986.
Cell lines and culture conditions
AtT20 D16–16 cells (provided by Dr. F. Antoni, Medical Research Council Brain Metabolism Unit, Department of Pharmacology, University of Edinburgh, Edinburgh, UK) and GH3 cells (provided by Dr. S. Cockle, Department of Biochemistry and Physiology, University of Reading, Reading, UK) were grown in DMEM containing sodium pyruvate (1 mM), glutamine (2 mM), nonessential amino acids (0.2 mM), 10% horse serum, and 5% newborn calf serum at 37 C in a 5% CO2 atmosphere. Rat AP primary cell cultures were prepared as described previously (11).
Antibodies
The different cell types within the AP cultures were identified
by means of the following polyclonal antibodies: guinea pig antirat
ß-TSH (1:100), guinea pig antirat PRL (1:500), guinea pig antirat
-LH (1:100), guinea pig antihuman GH (1:500), rabbit antihuman
ß-FSH (1:100), and rabbit or sheep antihuman ACTH (1:500) (provided
by the NIDDK National Hormone and Pituitary Program, Bethesda, MD). The
antibody used to identify ß-galactosidase was rabbit polyclonal
anti-ß-galactosidase (1:500) (generated in the laboratory of R.G.G.);
rabbit anti-HSV1-TK antibody (1:1000) was kindly provided by M. Janicot
(Rhone-Poulenc-Rorer, France).
Secondary antibodies used for either single or double immunolabeling were: swine antirabbit conjugated to fluorescein isothiocyanate (FITC) or donkey antirabbit R-phycoerythritin from DAKO Corp. Ltd. (High Wycombe, UK) and Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA), respectively; goat-antiguinea pig conjugated to FITC or Texas Red from Jackson ImmunoResearch Laboratories, Inc..
Recombinant adenoviruses
The RAdhCMV/HSV1-TK was designated RAd128 and has been described
in detail previously (13, 14). Production of high titer stocks,
purification with the fluorocarbon compound Arklone P (The Basic
Chemical Co. Ltd., High Wycombe, Bucks, UK), and titration of RAd128
(hCMV/HSV1-TK) were carried out as described previously (15). Viral DNA
was obtained as described by Revah et al. (16). To confirm
the presence of the transgene, viral DNA digestion with
HindIII and subsequent Southern blot hybridization were
performed using a BamHI HSV1-TK gene fragment, labeled by
random priming with digoxigenin-dUTP (Roche Molecular Biochemicals, Bell Lane, East Sussex, UK) (Fig. 1
). An adenovirus expressing the
Escherichia coli ß-galactosidase reporter gene under the
control of the hCMV promoter (RAd35) was used as a control virus. This
RAd has been described elsewhere (17, 18). After Arklone and caesium
chloride purification, the RAd stocks were assayed for the presence of
replication competent adenovirus by the supernatant rescue assay (19),
which can detect the presence of a single wild-type virus within
109 recombinant viruses. Viruses were also
assayed for the presence of endotoxin (lipopolysaccharide) by the
E-TOXATE assay (Sigma Chemical Co.), according to
the manufacturer’s instructions. The viruses used were designated
endotoxin-free, as defined by Cotten et al. (20). Viruses
were considered negative for lipopolysaccharide if levels were less
than 6 x 10-4 endotoxin units per dose of
adenovirus injected.
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Simultaneous detection of HSV1-TK protein and DNA content of GH3 and AtT20 cells after infection was performed by flow cytometry, as described previously (21). Fifty thousand cells were plated in 6-well plates and infected at increasing multiplicity of infection (MOI) (number of infectious virus particles/cell) (0, 1, 5, 20, 50, and 100) with RAd128. After 48 h of infection, the cells were exposed to 10 µM GCV and left for a further 72 h. Cells were harvested, fixed in 4% paraformaldehyde, permeabilized, and incubated in staining solution [PBS containing 200 mg/mL RNAse A, 10 mg/mL propidium iodide, 1% BSA ,and the polyclonal anti-HSV1-TK rabbit antiserum (1:1000)] overnight at room temperature. The cells were washed twice in PBS and incubated with a polyclonal swine antirabbit immunoglobulin antibody conjugated with FITC as secondary antibody (1:50), for 30 min at room temperature. Fluorescence intensity of 5000 cells was analyzed by flow cytometry using a FACScan (Becton Dickinson Co., Mountain View, CA). Propidium iodide intercalates into DNA after cell permeabilization, and apoptotic cells appear as a broad hypoploid DNA peak, well separated from the normal diploid distribution of DNA within nonapoptotic cells, during flow cytometry analysis (22).
Infection and detection of HSV1-TK within endocrine cells in primary AP cultures
Two weeks after plating, AP cells were infected with RAd128 at an MOI 30 for 48 h. The virus was then removed, and medium containing 0, 10, or 100 µM GCV (Cymevene; Roche Products Ltd., Welwyn Garden City, UK) was added. The primary cultures were then incubated for 3 additional days. Immunofluorescence detection of AP hormone cells and HSV1-TK was performed, as described previously (11); the cellular nuclei were stained using 4,6-diamidino-2-phenylindole (DAPI) (11, 23).
Induction of lactotroph hyperplasia and delivery of RAds into the AP gland
Rats were implanted with silastic pellets containing 15 mg 17ß-oestradiol and 50 mg sulpiride, prepared as described previously (8). The pellets were implanted sc in the lumbar region of each rat under anesthesia. Empty silastic pellets were implanted as controls.
For in vivo administration of RAds into the AP gland, rats were anesthetised with a mixture of Fluothane (Zeneca Pharmaceuticals Ltd, Cheshire, UK) and nitrous oxide (BOC Medical Gases) (2:1). Using a Hamilton syringe (Fisher Scientific, Leicester, UK) fitted with a 26-gauge needle, 1 x 109 pfu of either RAd35 or RAd128 (HSV1-TK) were injected in a volume of 10 µL into the pituitary gland using the transauricular route (24). Rats were given a 10 mL sc saline injection after 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 sacrificed using a lethal injection of pentobarbital and perfused 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), followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Pituitary, body, and testis weights were recorded. Before perfusion, trunk blood was collected.
For the assessment of toxicity in the normal AP gland by the HSV1-TK therapy, 109 pfu of RAd128 (in 10 µL) was injected into the AP gland. Pituitaries were removed after perfusion postfixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 h and paraffin embedded. They were then sectioned (5 µm), mounted onto 3-aminopropyltriethoxysilane-coated slides deparaffinated with xylene for 10 min before rehydration through graded alcohols and stained for immunofluorescence using the technique, as described previously (11); the cellular nuclei were stained using DAPI (11, 23). Hematoxylin and eosin (H and E) staining was performed as described previously (14).
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 (National Institutes of Health, Bethesda, MD). Plasma ACTH was measured using a specific immunoradiometric assay that has been described previously (25).
Statistical analysis
The in vitro experimental results were analyzed using the Student’s t test. The in vivo results were analyzed using ANOVA, followed by the Student-Newman-Keuls multiple comparisons test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AP cells expressing HSV1-TK were identified using antibodies
against both individual pituitary hormones (i.e. PRL, GH,
ACTH, TSH, LHH) and HSV1-TK. We detected
HSV1-TK expression within all pituitary cell types (Fig. 2).
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). We also assessed the
cytotoxicity of RAd128/GCV within immunocytochemically identified
hormone-producing cells in the primary AP culture (Fig. 4). Quantitative analysis of AP cultures
after infection with RAd128 (MOI 30) in the presence of 100
µM GCV did not show apoptosis in any endocrine cell type,
either in the absence or presence of GCV (Table 1). These results indicate that the
treatment was not deleterious for normal pituitary cells in primary
culture.
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The expression of HSV1-TK was also assessed in both AtT20 and GH3
cells using immunofluorescence (Fig. 5).
Infection of these cells in the absence of GCV gave a strong
immunocytochemical staining; no signs of apoptotic nuclei were detected
at the concentration of virus used (MOI 30). HSV1-TK expression was
distributed both in the cytoplasm and nuclei of infected cells.
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shows a
gradual increase in HSV1-TK immunoreactivity, as detected by flow
cytometry in both cell lines. Levels of HSV1-TK immunoreactive protein
was maximal at MOI 100 in both GH3 and AtT20 cells, being substantially
higher in GH3 cells at all MOI tested (other than MOI 1) when compared
with AtT20 cells (P < 0.0001 for MOI 5, 20, 50, and
100).
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). Increasing the GCV concentration (0–300
µM) did not induce apoptosis in AtT20 cells, compared
with basal levels. In GH3 cells, however, GCV induced apoptosis at
concentration of 100 µM and higher (P <
0.0001). We, therefore, used 10 µM GCV for our
experiments to eliminate any potential apoptosis caused by GCV
alone.
Infection of GH3 cells with RAd128 in the presence of 10
µM GCV induced apoptosis, which was MOI dependent (Fig. 6C). Infection of GH3 with RAd128 in the absence of GCV induced
apoptosis at MOI 20 and above. When GH3 cells were infected with RAd35,
at the same MOI as RAd128, no apoptosis was observed, suggesting
apoptosis is due to expression of HSV1-TK. Fig. 6D shows the level of
apoptosis occurring in AtT20 cells in the presence and absence of 10
µM GCV with increasing MOI of RAd128. Apoptosis was MOI
dependent in the presence of GCV. In the absence of GCV, only basal
levels of apoptosis were observed indicating there was no toxicity from
the HSV1-TK transgene. RAd35 did not cause any cytotoxicity even at MOI
of 100 (data not shown).
In vivo gene therapy of oestrogen/sulpiride induced lactotroph hyperplasia using RAd128 in combination with GCV
Pituitary weights (Table 2) in
control animals averaged 10.6 ± 0.6 mg, whereas the O/S-implanted
animals treated with RAd35/GCV had pituitary weights of 23.9 ±
1.1 mg (P < 0.005). The group treated with RAd128/GCV
had pituitary weights of 17.5 ± 1.3 mg, a 26% reduction when
compared with the RAd35/GCV treated group (P <
0.05).
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). The testes weight was used as
a biological indicator of the effects of the oestrogen/sulpiride
implant. The oestrogen and sulpiride in the pellet caused a rise in
circulating PRL levels (Fig. 7). It has
been previously demonstrated that chronic oestrogen treatment directly
decreases the secretion of LH and FSH (28). LH and FSH are needed to
maintain normal testicular structure, and a lack of gonadotrophins in
turn causes the regression of spermatogenesis. It was unrealistic to
expect testis weight to change in the 9 days of the therapy because
implants were left in place throughout the experiment, with, therefore,
gonadtrophins remaining suppressed throughout the time course of the
therapy.
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).
Circulating GH levels in control rats were 81.7 ± 34.17 ng/mL,
which increased due to O/S implants, to 156.9 ± 27.38 ng/mL in
the RAd35/GCV-treated O/S-implanted group. Similar GH levels
(162.4 ± 57.35 ng/mL) were observed in the RAd128-treated
O/S-implanted group, indicating no toxic effect of the HSV1-TK/GCV
treatment on the GH-secreting cell population within the AP gland
in vivo (Fig. 7). No changes were observed in circulating
ACTH and TSH-ß levels in any of the experimental groups (Fig. 7).
We also assessed the effect of HSV1-TK/GCV therapy on cell viability in
AP cells in vivo within immunocytochemically identified
hormone-producing cells in the normal AP gland (Fig. 8). We failed to detect any nuclear
morphology indicative of apoptosis within HSV1-TK-positive endocrine
cells. These results indicate that the treatment was not deleterious
for normal endocrine pituitary cells in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first tested the cell killing efficiency of the conditional cytotoxic approach using HSV1-TK/GCV within AP tumor cell lines. In AtT20 cells, no apoptosis was observed after infection with RAd128 in the absence of GCV. However, in GH3 cells the levels of apoptosis increased with increasing MOI of RAd128, even in the absence of GCV. The levels of apoptosis did not increase in GH3 cells infected with RAd35 at the same MOI, suggesting this toxicity is not due to viral infection but due to the expression of the HSV1-TK protein. These results highlight endocrine tumor cell type-specific toxicity of HSV1-TK expression in the absence of GCV. Toxicity due to expression of HSV1-TK has also been observed in vivo in male transgenic mice expressing HSV1-TK, which were found to be sterile (29).
In the presence of GCV, both GH3 and AtT20 cells transduced with RAd128 underwent apoptosis. The bystander effect also played an important role in increasing the amount of apoptosis both in AtT20 and GH3 cells. At the lower MOI, the percentage of cells undergoing apoptosis was far greater than the percentage of cells expressing HSV1-TK. For example, at MOI 20, 31% GH3 and 7.5% AtT20 cells expressed the HSV1-TK transgene, whereas 80% apoptosis was observed in both cell lines. The bystander effect in the AtT20 cells is especially prominent in view of the lower percentage of cells expressing HSV1-TK. The in vitro bystander effect might be due to cellular transfer of phosphorylated GCV through gap junctions (9), or the uptake of apoptotic bodies by nontransduced cells (10). This implies that not all the tumor cells need to be expressing the transgene in order to have efficient cell killing.
In our studies, there was no indication of toxicity in vivo to the normal pituitary gland by the HSV1-TK/GCV therapy, as assessed by hormone levels not being altered after the treatment and through quantitative histological examination of hormone-producing cells. This is important because recent reports have demonstrated that HSV1-TK plus GCV can be cytotoxic to nondividing cells that display a high metabolic activity, such as thyroid secretory cells and hepatocytes (26, 27).
A gene therapy strategy for prolactinomas has been tested before in vitro by Freese et al. (30). Human lactotroph adenoma cell cultures were infected with an adenovirus expressing tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, observing a decrease in PRL secretion of 50%. Recently, an adenovirus expressing HSV1-TK under the control of the GH promoter has also been successfully used in a transplantable pituitary tumor model using GH3 cells (31). The advantages of using transcriptional-targeted approaches to limit transgene expression to a predetermined cell population have to be weighed against the levels of transgene expression, which can be achieved. It has been previously shown that cell type-specific promoters yield lower levels of transgene expression when compared with strong viral promoters (32). Therefore, the advantages of this approach will have to be assessed experimentally.
We tested a gene therapy strategy in vivo using HSV1-TK, followed by GCV treatment in an oestrogen/sulpiride-induced lactotroph hyperplasia model. This is one of the best models available at present, together with the spontaneous prolactinomas of aged rats, for testing the effects of gene therapy in a pituitary prolactinoma in situ. The side effects on other nontarget cell populations (i.e. the somatotrophs, corticotrophs, and so forth) can, therefore, be determined by analyzing circulating hormone levels after treatment. The models using sc transplantable tumor cells [e.g. MMQ (33) and GH3 cells (31)] give less meaningful information for the effectiveness of gene therapy strategies for pituitary adenomas because they are a homogeneous highly proliferating cell population implanted at a heterotopic site.
After HSV1-TK/GCV treatment, pituitary weight decreased by 26% (P < 0.05) in the RAd128/GCV-treated group compared with the RAd35/GCV treated group. The pituitary weight probably did not return to normal because the oestrogen/sulpiride pellets were present during the duration of the treatment, so stimulation of the lactotroph cell population was maintained. The continued presence of the O/S pellet also explains the lack of change in testes weights between the O/S implanted group treated with RAd128/GCV and the RAd35/GCV group. Oestrogen, as well as causing prolactin to rise, directly decreases LH and FSH secretion, which are needed to maintain normal testicular structure (28). A lack of gonadotrophins in turn causes the regression of spermatogenesis in our model and determines testis weight loss. The oestrogen/sulpiride implants were left in place throughout the experiment. Thus, gonadtrophins remained suppressed, even if in the RAd128 + ganciclovir group PRL levels were restored to almost normal values. Even if the reduction in spermatogenesis would solely be due to increased PRL levels, which it is not, spermatogenesis in the rat takes up to 52 days. It is, thus, unrealistic to expect testis weight to change in 9 days even if PRL had a spermatogenic target. Importantly, the role of PRL in affecting rat spermatogenesis is marginal (34). Therefore, the lack of change in the testes of RAd128-treated rats is entirely predictable.
Circulating PRL levels were significantly reduced (60%; P < 0.01) in the RAd128/GCV-treated group when compared with the oestrogen/sulpiride group treated with RAd35/GCV, another strong indication that the treatment was successful. There were no effects on GH, ACTH, and TSH hormone levels, which indicates that the HSV1-TK/GCV treatment is not toxic to other hormone-producing cell populations within the AP gland. The reason why lactotroph cells are selectively affected is that this is the only population of cells within the pituitary gland that is actively proliferating in response to the oestrogen-sulpiride treatment. This is not the case for other endocrine cells within the pituitary gland, such as corticotrophic or thyrotrophic cells in vivo, which are normally quiescent and are not stimulated to divide through the oestrogen/sulpiride treatment used by us. This correlates with our measurements of circulating hormone levels, which demonstrate that only PRL levels are reduced, whereas all other hormone levels tested remained uninfected. This has important implications because any gene therapy for pituitary adenomas has to avoid toxic side effects to other normal cell populations within the gland. The results reported here show that suicide gene therapy using the HSV1-TK transgene could be developed as a useful treatment to complement current therapies for prolactinomas or other pituitary tumors that do not respond to current available treatments. To this end, it will be important to test these therapies in animal models of malignant prolactinomas and assess both their safety and efficiency.
Gene therapy is currently being used to treat very serious diseases, for which current therapies fail. Thus, initially, gene therapy would be applied to pituitary tumors that cannot be treated by other means. In the future, as gene therapy techniques become more efficient and less toxic, gene therapy will be applied to patients suffering from nonterminal conditions. Under these circumstances, and given that for many patients with otherwise nonlife-threatening pituitary adenomas, treatment is inefficient when evaluated over long periods, it is likely that gene therapy techniques could provide therapeutic advantages. Although the cost of gene therapy will be initially high, it is likely that, as with other new technologies, prices will fall with time. The number of patients is also likely to be higher than the number of patients suffering from some rare disorders being targeted by gene therapy. Furthermore, the long-term inflammatory and immunogenic potential of gene therapy in the pituitary gland will have to be evaluated in the context of recent data from our laboratory on the development of gene therapy strategies for the treatment of brain glioblastoma (14).
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this study and should be
considered first authors.
3 Present address: INIBIOLP-Histology "B", Faculty of Medicine,
National University of La Plata, La Plata, Argentina.
4 Present address: E1504, Biomedical Science Tower, 200 Lothrop
Street, Pittsburgh, Pennsylvania 15213-2582.
Received August 18, 1999.
Revised November 23, 1999.
Accepted December 3, 1999.
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