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and FasL induce apoptosis of normal and tumoral anterior pituitary cells
1 Centro de Investigaciones en Reproducción, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, Buenos Aires (C1121ABG), Argentina
2 Gene Therapeutics Research Institute, Cedars-Sinai Medical Center and Department of Medicine and Molecular and Medical Pharmacology, David Geffen School of Medicine, UCLA, 8700 Beverly Blvd, Davis Building, Suite 5090, Los Angeles, California 90048, USA
3 Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA
(Requests for offprints should be addressed to M G Castro; Email: Maria.Castro{at}cshs.org)
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Abstract |
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and FasL induce apoptosis of anterior pituitary cells. To further analyze the effect of these proapoptotic factors, we infected primary cultures from rat anterior pituitary, GH3 and AtT20 cells with first-generation adenoviral vectors encoding TNF-, FasL or, as a control, ß-galactosidase (ß-Gal), under the control of the human cytomegalovirus promoter. Successful expression of the encoded transgenes was determined by immunocytochemistry. Although we observed basal expression of TNF- and FasL in control cultures of anterior pituitary cells, fluorescence-activated cell sorting (FACS) cell cycle analysis showed that the overexpression of TNF- or FasL increases the percentage of hypodiploid lactotropes and somatotropes. Nuclear morphology and TUNEL staining revealed that the cells undergo an apoptotic death process. We detected strong immunoreactivity for TNFR1 and Fas in the somatolactotrope cell line GH3. TNF-, but not FasL, was expressed in control cultures of GH3 cells. The infection of GH3 cells with adenovirus encoding TNF- or FasL increased the percentages of hypodiploid and TUNEL-positive cells. TNF- or FasL immunoreactivity was not observed in the corticotrope cell line AtT20. However, adenovirus encoding TNF- or FasL efficiently transduced these cells and increased the percentages of hypodiploid and TUNEL-positive cells. The expression of ß-Gal was detected in all these cultures but did not affect cell viability. In conclusion, these results suggest that death signaling cascades triggered by TNF receptor 1 (TNFR1) and Fas are present in both normal and tumoral pituitary cells. Therefore, overexpression of proapoptotic factors could be a useful tool in the therapy of pituitary adenomas. |
Introduction |
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Death receptors, such as Fas (receptor for FasL), tumor necrosis factor receptor 1 (TNFR1; one of the receptors for TNF) and DR5 (receptor for TNF-related apoptosis-inducing ligand (TRAIL)) are type-I membrane proteins that have a conserved domain called the death domain. Upon binding to the specific ligand, death receptors form trimers (Ashkenazi & Dixit 1998, Evan & Littlewood 1998). The apopotic signal transduction cascade that is induced by death receptor activation has been well characterized. Ligation of Fas by FasL or TNFR1 by TNF-ß results in formation of a death-inducing signalling complex (DISC), which initiates a cascade of events that, through activation of caspases 8 and 10, leads to apoptosis (Scaffidi et al. 1999, Chen & Goeddel 2002). The initiator caspases can either directly activate executioner caspase 3 or cleave Bid, a proapoptotic member of the Bcl-2 family. The truncated Bid translocates to the mitochondria stimulating cytochrome c release (Gonzalvez et al. 2005). Cytochrome c, together with Apaf-1 activates caspase 9, which in turn activates caspase 3 (Cecconi 1999). Then, caspase 3 and other downstream caspases cleave subtrates involved in cell disassembling (Degterev et al. 2003).
The anterior pituitary gland undergoes a process of cell renewal during the estrous cycle in the female rat (Hashi et al. 1995, Yin & Arita 2002). We have previously reported that TNFR1 and Fas activation induce apoptosis of anterior pituitary cells from female rats (Candolfi et al. 2002, 2004, 2005, Jaita et al. 2005b) suggesting that TNF- and FasL are involved in the maintenance of tissue homeostasis in the anterior pituitary gland and could have potential benefits for the treatment of pituitary diseases. Although major advances have been made in the therapy of pituitary tumors, only partial success has been possible and novel therapies are needed. Thus, we explored the effect of first-generation recombinant adenoviral vectors (RAd) encoding TNF- and FasL in normal and tumoral anterior pituitary cells. Our previous results have demonstrated the persistent high efficiency of transgene expression of adenoviral vectors encoding ß-galactosidase or herpes simplex virus (HSV) Type 1 thymidine kinase in the anterior pituitary gland (Castro et al. 1997, Southgate et al. 2000b, 2001, Smith-Arica et al. 2001). In the present work, we tested the ability of adenoviral vectors encoding TNF- (RAd-hCMV-TNF-) or FasL (RAd-hCMV-FasL) to infect and express these proapoptotic transgenes within anterior pituitary cells from two different rat strains and also in the pituitary cell lines GH3 and AtT20. We investigated the proapoptotic effect of the vectors expressing TNF- and FasL by fluorescence-activated cell sorting (FACS) analysis and the TUNEL method in both normal and tumoral pituitary cells. We found that adenoviral vectors encoding these proapoptotic genes are successfully expressed in pituitary cells, triggering the apoptotic cascade. Our results suggest that death receptors could possibly be considered as a target for the treatment of pituitary adenomas.
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Material and Methods |
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All culture media and supplements were obtained from Invitrogen, except fetal bovine serum (Biomeda, Foster City, CA, USA) and the materials indicated below. All TUNEL reagents were purchased from Roche. The primary antibodies against anterior pituitary hormones were purchased from Dr A Parlow at the National Hormone and Pituitary Program (Torrance, CA, USA). Secondary antibodies were obtained from DAKO (Glostrup, Denmark) and Chemicon International (Temecula, CA, USA). Avidin and biotin blocking solutions, fluorescein-avidin, diaminobenzidene (DAB), Vectastain ABC Elite kit and Vectashield were from Vector Laboratories Inc (Burlingame, CA, USA).
Adenoviral vectors
The RAd vectors used are based on adenovirus type 5 (Ad5), in which the left end of E1 and a portion of the E3 regions are deleted (E1–/E3–), and a cassette containing a recombinant exogenous gene and promoter is inserted in place of the E1 deletion. Three different vectors were used: RAd-hCMV-TNF- (Ehtesham et al. 2002), RAd-hCMV-FasL (Larregina et al. 1998, Morelli et al. 1999, Maleniak et al. 2001) and RAd-hCMV-ß-Gal (Castro et al. 1997). The RAds were grown and purified as previously described (Lowenstein et al. 1996, Southgate et al. 2000a).
Animals
Adult female Wistar and Sprague–Dawley rats were kept in controlled conditions of light (12 h light: 12 h darkness cycles) and temperature (20–25 °C). Standard laboratory chow and water were freely available and rats were kept in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Anterior pituitary glands were removed within minutes after rats were killed by breathing CO2, followed by physical induction of pneumothorax.
Cell culture
A pool of anterior pituitary cells from five rats was used for each primary cell culture. Anterior pituitary glands were washed several times with Dulbecco’s modified Eagle’s medium (DMEM) and cut into small fragments. Sliced fragments were dispersed enzymatically by successive incubations in DMEM supplemented with 3 mg/ml BSA, containing 2.5 mg/ml trypsin (Type I from bovine pancreas), 1 mg/ml DNase (deoxyribonuclease II, Type V from bovine spleen) and 1 mg/ml trypsin inhibitor (Type II-S from soybean), and finally dispersed by extrusion through a Pasteur pipette in Krebs buffer without Ca2+ and Mg2+. Dispersed cells were washed twice and resuspended in DMEM supplemented with 10 µl/ml MEM aminoacids, 2 mM glutamine, 5.6 µg/ml amphotericin B and 25 µg/ml gentamicin (DMEM-S or MEM-D-valine-S). Cell viability, as assessed by trypan blue exclusion, was over 90%. Cells were cultured in DMEM-S with 10% heat-inactivated fetal bovine serum for 1–2 days (37 °C, 5% CO2 in air) before RAd virus infection.
GH3 and AtT20 cells (Castro et al. 1997) were cultured in MEM containing 10 µl/ml non-essential aminoacids, 10 µl/ml L-glutamine, 10 µl/ml penicillin/streptomycin and 10% fetal calf serum 24 h prior to RAd virus infection.
Anterior pituitary cells, as well as GH3 and AtT20 cells were seeded onto coverslides in 24-well tissue culture plates (10 x 104 cells/0.5 ml per well) for the TUNEL method or immunocytochemistry, and onto 24-well tissue culture plates (25 x 104 cells/0.5 ml per well) for flow cytometry (FACS). Then, cells were infected for 48 h with different doses of viral vectors expressed as infectious units/cell (IU/cell) of adenoviral vectors expressing human TNF- (RAd-hCMV-TNF), murine FasL (RAd-hCMV-FasL) or the Escherichia coli enzyme, ß-galactosidase (RAd-hCMV-ß-Gal) under the control of the human cytomegalovirus (hCMV) promoter (Southgate et al. 2000b, Windeatt et al. 2000). After this period, cells were fixed for immunocytochemistry and TUNEL or flow cytometric analysis.
Immunocytochemistry of TNFR1, TNF-, Fas, FasL and ß-Gal
We identified the presence of TNFR1, TNF-, Fas, FasL and ß-Gal in anterior pituitary cells by immunocytochemistry using rabbit anti-TNFR1 antibody (1:50; Stressgen, CA, USA), mouse anti-Fas antibody (1:50; Stressgen), rabbit anti-TNF- (1:50; Endogen, IL, USA), rabbit anti-FasL antibody (1:25; Santa Cruz Biotech, CA, USA), and rabbit anti-ß-Gal (1:1000, developed in our laboratory (Southgate et al. 2000b)) respectively. Briefly, after the culture period, cells were fixed with 4% formaldehyde in PBS for 30 min followed by an incubation with 10% normal donkey serum in PBS with 0.2% Triton X-100. After incubation with 0.3% H2O2 to inactivate endogenous peroxidase, or with avidin and biotin blocking solutions, cells were incubated overnight with primary antibodies in PBS containing 1% donkey serum. To detect ß-Gal, cells were incubated with the primary antibody for 1 h. After rinsing, slides were incubated for 1 h with the corresponding biotinylated donkey anti-mouse or anti-rabbit IgG at a 1:200 dilution in the same buffer. Biotinylated antibodies were labeled with Vectastain ABC Elite kit, followed by staining with DAB and glucose oxidase (Sigma), and dehydration through graded ethanol solutions or with 2 µg/ml fluorescein-conjugated avidin in 10 mM HEPES buffer, pH 7.5. Finally, slides were mounted with Vectashield, a mounting medium for fluorescence, containing DAPI (4', 6-diamino-2-phenylindole dihydrochloride; Sigma) for DNA staining; slides were visualized in a fluorescence microscope (Axiophot, Carl Zeiss, Jena, Germany) or a confocal microscope (Leica, Germany) or mounted with DPX-mounting medium (Fluka, Switzerland) for light microscopy. Control slides were incubated with the corresponding IgG subtype instead of primary antibody.
Microscopic determination of DNA fragmentation by the TUNEL method
The TUNEL method was performed as previously described (Candolfi et al. 2002). Briefly, after the culture period, cells were fixed with 4% formaldehyde in PBS for 30 min and permeabilized by microwave irradiation. DNA strand breaks were labeled with digoxigenin-dUTP using terminal deoxynucleotidyl transferase (0.18 U/µl) according to the manufacturer’s protocol. The incorporation of nucleotides into the 3'-OH end of damaged DNA was detected with an anti-digoxigenin-fluorescein antibody.
Flow cytometric analysis (FACS)
Cultured cells were harvested with trypsin-EDTA, fixed with 75% ice-cold ethanol and permeabilized with 0.1% Triton-X100. Afterwards, cells were incubated in the presence of propidium iodide (50 µg/ml) and RNAse (10 µg/ml) in PBS with 0.1% sodium azide. In the case of normal anterior pituitary cells, prior to incubation with propidium iodide, cells were incubated for 1 h with anti-rat prolactin (NHPP-IC, 1:1000) or anti-rat growth hormone (NHPP-IC, 1:2000) guinea pig primary antibodies in PBS containing 1% donkey serum and 0.1% sodium azide, followed by an incubation for 1 h with fluorescein-conjugated donkey anti-guinea pig secondary antibody. The percentage of hypodiploid cells (Sub-G1) was determined using a FACScan (Becton Dickinson, Franklin Lakes, NJ, USA).
Statistical analysis
Percentages of hypodiploid (Sub-G1) cells, as analyzed by FACS, were expressed as means ± S.E. and evaluated by ANOVA followed by Dunnet’s multiple comparison test. The number of apoptotic cells, as identified by the TUNEL method, was analyzed in duplicate slides from at least two independent experiments. Results were expressed as the percentage of apoptotic cells ±95% confidence limits (CL) of the total number of cells counted in each specific condition. Confidence intervals for proportions were analyzed by the 
2 test. P<0.05 was used as the cut-off point for significance. All experiments were performed at least twice.
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Results |
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and FasL transgene expression in normal anterior pituitary cells
Our previous work showed that both TNF- and FasL induce apoptosis of anterior pituitary cells (Candolfi et al. 2002, 2004, 2005, Jaita et al. 2005b). Now, we overexpressed TNF- and FasL in primary cultures of anterior pituitary cells by infecting them with first-generation adenoviral vectors encoding TNF- (RAd-hCMV-TNF-), FasL (RAd-hCMV-FasL) or, as a control, ß-Gal (RAd-hCMV-ß-Gal), under the control of the human cytomegalovirus promoter (hCMV). This promoter was previously shown to successfully drive expression of ß-Gal in anterior pituitary cells and pituitary cell lines as well (Castro et al. 1997). All the encoded transgenes were efficiently transduced in infected anterior pituitary cells as detected by immunocytochemistry (Fig. 1
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or FasL induces apoptosis of anterior pituitary cells, we determined the percentage of hypodiploid cells in cultures of anterior pituitary cells from female Wistar rats after infection with increasing doses of RAd-hCMV-TNF-, RAd-hCMV-FasL or RAd-hCMV-ß-Gal. FACS cell cycle analysis showed that the infection of anterior pituitary cells with RAd-hCMV-TNF- and RAd-hCMV-FasL increases the percentage of hypo-diploid cells in a dose-dependent manner (Fig. 2A). In contrast, the expression of ß-Gal had no cytotoxic effects in these cultures, even at the highest dose tested (Fig. 2A). Microscopic analysis of the nuclear morphology of RAd-hCMV-TNF-- or RAd-hCMV-FasL-infected cultures of anterior pituitary cells revealed that cells exhibited characteristic features of an apoptotic death process, such as TUNEL staining in condensed and/or fragmented nuclei (data not shown). FACS cell cycle analysis of specific subpopulations of anterior pituitary cells infected with increasing concentrations of RAd-hCMV-TNF-, RAd-hCMV-FasL or RAd-hCMV-ß-Gal showed that adenoviral-driven expression of TNF- or FasL exerts a dose-dependent propapoptotic effect in lactotropes (Fig. 2B). The infection with 50 infectious units/cell (IU/cell) of RAd-hCMV-FasL induced apoptosis of somatotropes, whereas a higher dose (100 IU/cell) of RAd-hCMV-TNF- was required to induce an apoptotic effect in this cell subpopulation (Fig. 2C).
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and RAd-hCMV-FasL was also observed in anterior pituitary cells from female Sprague–Dawley rats infected with 50 IU/cell of RAd-hCMV-TNF- and RAd-hCMV-FasL. TNF- and FasL overexpression increased the percentage of hypodiploid (Fig. 3A) and TUNEL-positive (Fig. 3B) anterior pituitary cells, whereas the expression of ß-Gal did not exert cytotoxic effects (Fig. 3A and B). Figure 3C shows that only anterior pituitary cells successfully infected with RAd-hCMV-TNF- exhibit apoptotic nuclear morphology.
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and FasL transgene expression in murine anterior pituitary cell lines
To determine whether TNF- and FasL exert an apoptotic effect in tumoral anterior pituitary cells, we infected the murine anterior pituitary cell lines GH3 and AtT20 with adenoviral vectors encoding these ligands. Since the expression of death receptors has not been previously reported in the somatolactotrope cell line GH3, we first determined the presence of both TNFR1 and Fas in these cells. We detected strong immunoreactivity for TNFR1 (Fig. 4A, left panel) and Fas (Fig. 4A, right panel) in this cell line. The expression of receptors for TNF- and FasL in the murine corticotrope cell line AtT20 was previously reported (Kobayashi et al. 1997). TNF- but not FasL immunoreactivity was detected in control cultures of GH3 cells. However, after infection with RAd-hCMV-FasL, these cells showed a strong expression of FasL (Fig. 4B). Although we did not detect TNF- or FasL immunoreactivity in control AtT20 cells, RAd-hCMV-TNF- and RAd-hCMV-FasL efficiently transduced these cells (Fig. 4C).
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and FasL increased the percentage of hypodiploid GH3 cells in a dose-dependent manner, whereas ß-Gal expression had no cytotoxic effect even at the highest dose tested (Fig. 5A). Infection of GH3 cells with 50 IU/cell of RAd-hCMV-TNF- or RAd-hCMV-FasL also increased the percentage of TUNEL-positive cells (Fig. 5A). Overexpression of both vectors in AtT20 cells increased the percentage of hypodiploid cells whereas infection with RAd-hCMV-ß-Gal did not induce apoptosis (Fig. 5B). In addition, RAd-hCMV-TNF- or RAd-hCMV-FasL strongly enhanced the percentage of AtT20 TUNEL-positive cells (Fig. 5B).
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Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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and FasL induce apoptosis of both normal and tumoral anterior pituitary cells. Considering that the adenoviral vector encoding ß-Gal did not exert a cytotoxic effect in any of the cell types studied in our experimental conditions, the present results indicate that the proapoptotic effect of the virus encoding TNF- or FasL is specifically due to the expression of these proapoptotic factors.
Previous reports indicate that anterior pituitary cells produce TNF- under basal conditions (Gatti & Bartfai 1993, Goujon et al. 1996). Although we found cells expressing TNF- in control anterior pituitary cultures, hCMV promoter was effective in driving the expression of TNF- within anterior pituitary cells, increasing TNF- immunoreactivity. Two different types of receptors for TNF- have been described: TNFR1 (55 kDa) and TNFR-2 (75 kDa). Although both receptors are coexpressed in the majority of cells, each one can be predominant in specific cell types (Ksontini et al. 1998). Only TNFR1 presents an intracellular death domain that triggers an apoptotic cascade (Aggarwal & Natarajan 1996). Both TNFR1 and TNFR-2 are expressed in the mouse anterior pituitary gland (Lewis et al. 1991, Wolvers et al. 1993) but, in the pituitary of the rat only one binding site was detected for TNF- (Watanobe & Yoneda 2003), with an affinity compatible with that of TNFR1 (Lewis et al. 1991, Wolvers et al. 1993). The cell type/s of the anterior pituitary that expresses this death receptor has not been identified yet. However, our previous results have shown that TNF- induces apoptosis of lactotropes and somatotropes from female rats (Candolfi et al. 2002, 2005) suggesting that at least these cell subpopulations express TNFR1. In fact, our present data show that overexpression of TNF- in primary anterior pituitary cell cultures induced apoptosis of prolactin- and growth hormone-bearing cells. We previously detected the expression of FasL and Fas in several anterior pituitary cell types from female rats, mainly in lactotropes and somatotropes (Jaita et al. 2005b). In addition, we reported that Fas activation induces apoptosis of lactotropes (Jaita et al. 2005b) and somatotropes as well (Jaita et al. 2005a). Our present results show that viral vectors encoding FasL exert very potent apoptotic effects in lactotropes and somatotropes. Although we cannot rule out that TNF- or FasL could also induce apoptosis in other anterior pituitary subpopulations, our previous studies showed that TNF- does not exert apoptotic effects in gonadotropes and corticotropes (Candolfi et al. 2002).
This is the first report demonstrating that the somatolactotrophic tumor GH3 cell line expresses both TNFR1 and Fas. In the corticotrophic tumor AtT20 cell line, expression of both death receptors has already been reported (Kobayashi et al. 1997, Huang et al. 2005). In GH3 and AtT20 cells, overexpression of either TNF- or FasL strongly enhanced cell death by apoptosis, suggesting that death receptor-apoptotic pathways are conserved in both tumoral cell lines. Since GH3 and AtT20 cells express TNFR1 and Fas, TNF- or FasL gene transfer into transformed pituitary cells may become a feasible therapeutic approach for the treatment of pituitary tumors.
Successful transgene delivery to the anterior pituitary gland has been achieved using first-generation recombinant adenoviral vectors (Castro et al. 1997, Windeatt et al. 2000, Lee et al. 2001a,b, Southgate et al. 2001, Williams et al. 2001). Although the safety of intrapituitary administration of adenoviral vectors remains controversial (Davis et al. 2001, Southgate et al. 2001, Carri et al. 2005), several transgenes have been shown to be promising gene therapy targets for pituitary adenomas: such as HSV Type 1 thymidine kinase (HSV1-TK) (Lee et al. 1999, Windeatt et al. 2000), tyrosine hydroxylase (Williams et al. 2001) and the diphtheria toxin gene (Lee & Jameson 2002) – all of which have been shown to reduce pituitary growth and hyperprolactinemia in murine pituitary tumor models (Lee & Jameson 2005). Considering that the TNFR1/ TNF- and Fas/FasL systems can act in an auto/paracrine manner, one advantage of TNF- or FasL gene transfer is that if not all the tumor cells are successfully transduced, the transduced cells, even if few in number, could induce apoptosis in the neighboring cells. Although this could result in toxic effects on normal surrounding tissue, the low diffusibility of replication-defective adenoviral vectors throughout the pituitary gland (Davis et al. 2001, Southgate et al. 2001, Carri et al. 2005) ensures the expression of the transgene only inside the tumor mass. We previously described the low toxicity of RAd-hCMV-FasL in the brain (Ambar et al. 1999), which was neither associated with local morphologic changes (such as apoptosis of brain parenchyma cells) nor with systemic alterations; suggesting that the overexpression of death receptor ligands in the pituitary would constitute a safe approach for the treatment of pituitary adenomas. Nevertheless, adenoviral vectors can be engineered to have even lower toxicity. The incorporation of pituitary hormone promoters (Castro et al. 1997, Lee et al. 2000, 2001b, Davis et al. 2001, Southgate et al. 2001) into viral vectors has shown to drive transgene expression selectively in specific endocrine pituitary cell populations in vivo (Southgate et al. 2001, Roche et al. 2004). On the other hand, inducible promoter systems driving transgene expression have allowed for tight regulation of transgene expression in the pituitary gland (Smith-Arica et al. 2001, Williams et al. 2001).
In conclusion, considering that apoptosis plays a central role in regulation of pituitary tissue homeostasis, the imbalance between cell death and proliferation in favor of cell survival could result in tumor formation. One of the commonly employed strategies in experimental gene therapy for cancer is to target death receptors specifically to trigger apoptosis in tumor cells (Bianco et al. 2003, Nakanishi et al. 2003, Rubinchik et al. 2003, Sanlioglu et al. 2004). Our results indicate that forced overexpression of TNF- or FasL induces apoptosis of normal and tumoral pituitary cells and suggest that TNF- and FasL gene transfer may be suitable to promote apoptosis of TNFR1 or Fas positive pituitary tumoral cells.
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Funding |
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References |
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Received in final form 10 February 2006
Accepted 8 March 2006
Made available online as an Accepted Preprint 24 March 2006
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