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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-0633
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 11 6156-6161
Copyright © 2005 by The Endocrine Society

Expression of the Antiapoptotic Gene Seladin-1 and Octreotide-Induced Apoptosis in Growth Hormone-Secreting and Nonfunctioning Pituitary Adenomas

Paola Luciani, Stefania Gelmini, Emanuele Ferrante, Andrea Lania, Susanna Benvenuti, Silvana Baglioni, Giovanna Mantovani, Ilaria Cellai, Franco Ammannati, Anna Spada, Mario Serio and Alessandro Peri

Endocrine Unit (P.L., S.Be., S.Ba., I.C., M.S., A.P.) and Clinical Biochemistry Unit (S.G.), Department of Clinical Physiopathology, Center for Research, Transfer, and High Education on Chronic, Inflammatory, Degenerative, and Neoplastic Disorders (DENOthe), University of Florence, 50139 Florence, Italy; Neurosurgery Unit, Careggi Hospital (F.A.), 50139 Florence, Italy; and Institute of Endocrine Sciences, University of Milan, Ospedale Maggiore IRCCS (E.F., A.L., G.M., A.S.), 20122 Milan, Italy

Address all correspondence and requests for reprints to: Prof. Alessandro Peri, Endocrine Unit, Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy. E-mail: a.peri{at}dfc.unifi.it.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Seladin-1 (from selective Alzheimer’s disease indicator-1) is a recently discovered gene that has been found to be down-regulated in brain regions affected by Alzheimer’s disease. Seladin-1 effectively protects neurons against ß-amyloid-mediated toxicity and prevents apoptosis via inhibition of the activation of caspase-3, a key mediator of the apoptotic cascade. Although seladin-1 is expressed in the pituitary gland, no study addressed the expression or the function of this gene in pituitary adenomas.

Objective: The aim of the present study was to determine the expression level of the seladin-1 gene in pituitary tumors, i.e. GH-secreting and nonfunctioning pituitary adenomas (NFPA), and to determine whether differential expression might be associated with different somatostatin (sst)-induced apoptosis.

Results: We found by quantitative real-time RT-PCR that the expression level of seladin-1 was significantly higher in NFPA (n = 21) than in GH-secreting adenomas (n = 30; mean ± SE, 25.69 ± 6.39 vs. 8.02 ± 2.68 pg/µg total RNA; P = 0.006). Although the amount of activated caspase-3 did not differ between the two groups of tumors, in primary cell cultures, octreotide was able to increase apoptosis, evaluated by the level of cleaved cytokeratin 18 and the presence of apoptotic nuclei, in GH-secreting adenomas, but not in NFPA. This different response was not attributable to differences in the amount of transcript of sst receptors 2 and 5, which was similar in the two groups of tumors.

Conclusions: Our results suggest that differential seladin-1 expression in pituitary adenomas may be associated with a different apoptotic response to sst analogs.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
A FEW YEARS ago a novel gene was identified and found to be down-regulated in brain regions affected by Alzheimer’s disease (1). This gene was named seladin-1 (for selective Alzheimer’s disease indicator-1). Overexpression of seladin-1 conferred protection against ß-amyloid-mediated toxicity and from oxidative stress in neuroglioma H4 cells. In addition, seladin-1 effectively inhibited caspase-3 activity, a key mediator of apoptosis, and protected from apoptotic death (1). Accordingly, we have recently demonstrated in human fetal neuroblasts that estrogen and selective estrogen receptor modulators confer resistance to ß-amyloid-induced toxicity via increased seladin-1 expression and caspase-3 inhibition, thus indicating that this protein may act as a downstream effector of estrogen receptor-mediated neuroprotection (2). The seladin-1 gene has marked sequence homology to the Diminuto/Dwarf1 gene described in plants (i.e. Arabidopsis thaliana) and Caenorhabditis elegans (3). In plants, Diminuto/Dwarf1 is required for the synthesis of brassinosteroids, which are plant sterols essential for normal growth and development (4, 5). After its first identification, a subsequent study demonstrated that seladin-1 is the gene encoding 3ß-hydroxysterol {Delta}-24-reductase (DHCR24) (6), which converts desmosterol into cholesterol. Mutations of this gene have been found in desmosterolosis, a rare severe multiple congenital anomaly syndrome, including developmental and growth retardation (6).

In addition to expression in the brain, seladin-1 (or DHCR24) is expressed in various endocrine organs, such as adrenal cortex, testis, and ovary (1). Moreover, altered expression of this gene has been recently found in adrenal neoplasia (7, 8), consistent with a possible role of seladin-1 in cell behavior and survival. Although it has been demonstrated that the normal pituitary gland expresses seladin-1 (1), no study has addressed the expression and function of this protein in pituitary adenomas. Somatostatin (sst) analogs are usually effective in controlling GH secretion and determining tumor shrinkage in GH-secreting adenomas (9); however, a subset of these tumors shows unresponsiveness to medical treatment. This is the rule in nonfunctioning pituitary adenomas (NFPA), despite the fact that they usually express sst receptors (sstr), and the use of sst analogs has largely been discontinued (10). Admittedly, the reason for the unsatisfactory response to sst analogs in a relevant percentage of pituitary tumors has not yet been clearly elucidated.

The aim of this study was to investigate the expression of the seladin-1 gene, sstr (i.e. sstr2, -3, and -5), and the amount of activated caspase-3 in a large series of GH-secreting adenomas and NFPA. In addition, to determine whether the degree of sst-induced apoptosis in these tumors might be related to differential expression of seladin-1, the amount of cleaved cytokeratin 18 (CK18), a marker of caspase-3 activation (11, 12), and the presence of apoptotic nuclei were determined in primary cell cultures from GH-secreting adenomas and NFPA in both basal conditions and after exposure to the sst analog octreotide.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients and cell cultures

Thirty patients (18 men and 12 women) affected by acromegaly due to a GH-secreting pituitary adenoma and 21 patients (11 men and 10 women) affected by NFPA undergoing transsphenoidal surgery were included in the study after giving informed consent. For acromegalic patients, the age ranged from 18–63 yr (mean ± SE, 40.6 ± 4.6), the plasma GH level from 2.9–65.3 µg/liter (mean ± SE, 21 ± 4), and the plasma IGF-I level from 55.4–227.2 nmol/liter (mean ± SE, 129.6 ± 10.5). Three tumors were microadenomas, and 27 were macroadenomas. In patients with NFPA, the age range was 24–82 yr (mean ± SE, 52 ± 7), and all 21 tumors were macroadenomas. At the time of surgery, small adenoma fragments were fixed for light microscopy to check the adenomatous nature of the material, and the remaining tissues were quickly frozen and stored at –80 C.

In 10 tumors (five GH-secreting adenomas and five NFPA), a portion of the tissue was dispersed by 2-h collagenase (2 mg/ml) digestion at 37 C and then placed in the appropriate sterile medium for cell culture, as described previously (13). Local ethical approval was obtained for all studies.

Quantitative real-time RT-PCR for seladin-1 transcript

The measurement of seladin-1 transcript was performed by quantitative real-time RT-PCR, based on TaqMan technologies. Total RNA was obtained from tissues by acid guanidine thiocyanate-phenol-chloroform extraction. In addition, RNA from normal pituitary glands was obtained from BD Clontech (Mountain View, CA). Primers and probe were selected by the proprietary software Primer Express (Applied Biosystems, Foster City, CA). The sequence of seladin-1 primers and probe and the conditions of the real-time RT-PCR were described previously (8). A calibration curve was generated using serial dilutions of a single-stranded sense oligodeoxynucleotide spanning the sequence included between the primers, as described by Bustin (14). Because normalization to rRNA or to glyceraldehyde-3-phosphate-dehydrogenase as well as to other housekeeping genes has been clearly shown to be not accurate (14, 15), the results were expressed in terms of picograms of seladin-1 mRNA per microgram of total RNA. Each measurement was made in triplicate.

Analysis of Gs{alpha} gene mutations

Genomic DNA was obtained from tissue homogenates by acidic guanidine thiocyanate-phenol-chloroform extraction, as described previously (16). Sequence analyses of the Gs{alpha} gene were directly performed by the dideoxynucleotide method and by automatic techniques (ABI PRISM 310, PerkinElmer, Norwalk, CT) on PCR products. The hot spots of the Gs{alpha} gene were amplified using intronic oligonucleotide primers, as described previously (16).

Quantitative real-time RT-PCR for sstr2, -3, and -5 transcripts

The primers and probes for sstr2 and -5 mRNA quantification were selected by the proprietary software Primer Express (Applied Biosystems) on the sequence NM_001050 (GenBank), and the corresponding sequences and the conditions for the real-time RT-PCR were described previously (17, 18). The amounts of sstr2 and sstr5 mRNA were measured by interpolation from a standard curve of threshold cycle values generated from known initial concentrations of RNA extracted from the neuroblastoma cell line CHP404, which overexpresses sstr2 mRNA, and from the colon carcinoma cell line HCT116, respectively. For the expression of sstr3, the standard curve was made by cloning in PCRII-TOPO vector (Invitrogen Life Technologies, Inc., Carlsbad, CA) an amplified insert obtained from the cDNA of Human Reference Total RNA (Stratagene, La Jolla, CA). The primers and the probe sequence for sstr3 were as follows: forward primer, 5'-GGATGGCATCAACCAGTTACA-3'; reverse primer, 5'-AGCGGGTGGGATGTACCA-3'; and probe, 5'-6-carboxyfluorescein-TATTCTGCCTGACTGTCATGAGCGTGGAC-tetramethylrhodamine-3'. Each measurement was run in triplicate, and the results were expressed in terms of number of copies per microgram of total RNA.

Determination of cleaved caspase-3

Total proteins were extracted from frozen pituitary tumor samples in those cases in which there was enough material left after RNA extraction (six GH-secreting adenomas and six NFPA) and were quantified using the bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, IL), as described previously (13). Twenty micrograms of protein was then resolved on a 10% SDS-PAGE gel and transferred onto nitrocellulose membranes (Transfer Blot, Bio-Rad Laboratories, Hercules, CA). A polyclonal antibody specific for cleaved caspase-3 (Asp175) was obtained from Cell Signaling Technology (Beverly, MA) and used according to the manufacturer’s indications. The membranes were finally treated with a chemiluminescent substrate and enhancer (LumiGLO, New England Biolabs, Beverly, MA) and exposed to x-ray films for 1–5 min.

The amount of cleaved caspase-3 in the above-mentioned pituitary adenomas was also determined by immunohistochemistry. Deparaffinized and rehydrated tissue sections were processed for antigen unmasking by boiling in 10 mM sodium citrate, pH 6.0, for 10 min, followed by 10 min at subboiling temperature. Subsequently, sections were exposed to the anticaspase-3 polyclonal antibody at a 1:200 dilution in Tris-buffered saline/0.1% Tween 20 for 24 h at 4 C. Then, incubation with a biotinylated secondary antibody (appropriately diluted in Tris-buffered saline/0.1% Tween 20, 1:50) for 1 h was performed, and the reaction product was visualized by avidin-biotin peroxidase-based detection protocol (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA). Finally, cells were counterstained with hematoxylin according to the manufacturer’s instructions. Neuroblastoma cells (SK-N-AS), treated with camptothecin (Sigma-Aldrich Corp., Milan, Italy), were used as the positive control.

Determination of cleaved CK18

Cleaved CK18 levels were measured using an M30-Apoptosense ELISA (Peviva-Alexis, Bromma, Sweden), an ELISA based on a monoclonal antibody (M30) that recognizes a neo-epitope on CK18, which is exposed after cleavage by caspases during apoptosis (19). Briefly, 105 cells obtained from pituitary tumors (five GH-secreting adenomas and five NFPA), enzymatically dispersed, were seeded in six-well plates and incubated in the presence or absence of 10 nM octreotide for 16 h at 37 C. Then, cell extracts were obtained according to the manufacturer’s instructions, and 25 µl cell extracts were used for the assay.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) analysis

Apoptosis was also evaluated by TUNEL analysis, using a commercially available detection kit (APO-DIRECT, Phoenix Flow Systems, Inc., San Diego, CA). Briefly, cells obtained from pituitary tumors (five GH-secreting adenomas and five NFPA), enzymatically dispersed, were seeded in coverslips (3 x 105 cells/coverslip) and incubated in the presence or absence of 10 nM octreotide for 16 h at 37 C. The cells were fixed in 1% paraformaldehyde in PBS (pH 7.4) and then treated with 70% iced ethanol for 30 min at –20 C. The cells were washed twice with the wash buffer and exposed for 60 min at 37 C to the DNA labeling solution provided with the kit. The cells were rinsed with the rinse buffer and observed using a transmission fluorescence microscope (307-148002, Leitz, Wetzlar, Germany) equipped with an E4 filter (Leica, Milan, Italy) by an oil immersion x100 magnification objective. Images were captured by a Canon digital camera using Remote Capture software (Canon, Tokyo, Japan) and edited by Photoshop version 5.0 (Adobe Systems, Inc., San Jose, CA).

Statistical analysis

Data were expressed as the mean ± SE. Statistical differences were analyzed using Student’s t test. Differences were considered statistically significant at the 0.05 level.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Seladin-1, sstr2, sstr3, and sstr5 expression in GH-secreting adenomas and NFPA

The amount of seladin-1 mRNA was assessed by real-time RT-PCR in a large series of GH-secreting adenomas (n = 30) and NFPA (n = 21). In the former group, significantly lower levels of seladin-1 transcript were observed (mean ± SE, 8.02 ± 2.68 vs. 25.69 ± 6.39 pg/µg total RNA; P = 0.006; Fig. 1Go). With regard to GH-secreting adenomas, no difference in the amount of seladin-1 mRNA was found between the eight tumors harboring a mutated (gsp+) and those with the wild-type (gsp–) Gs{alpha} gene (mean ± SE: gsp+, 7.07 ± 3.55; gsp–, 8.37 ± 3.46 pg/µg total RNA; P > 0.05). In addition, the level of seladin-1 mRNA was determined in total RNA extracted from normal pituitary gland samples and was found to be significantly lower than that in both groups of pituitary adenomas (mean ± SE, 0.21 ± 0.01 pg/µg total RNA; P < 0.01).



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  		FIG. 1. Amount of seladin-1 gene expression in NFPA and GH-secreting pituitary adenomas (GH-sec). *, P < 0.05.

 
In the same series of pituitary adenomas, sstr2, sstr3, and sstr5 transcripts were also measured by real-time RT-PCR. No significant difference was observed between GH-secreting adenomas and NFPA with regard to sstr2 and sstr5 (P > 0.05), whereas the level of expression of sstr3 was significantly lower in GH-secreting adenomas (P < 0.05), as shown in Fig. 2Go.



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  		FIG. 2. Amounts of sstr2, sstr3, and sstr5 expression in NFPA and GH-secreting adenomas. The scale on the left refers to sst2 and sstr5, whereas the scale on the right refers to sstr3. *, P < 0.05.

 
Cleaved caspase-3 in GH-secreting adenomas and NFPA

The amount of cleaved caspase-3 was determined in a subgroup of GH-secreting adenomas (n = 6) and NFPA (n = 6) as a marker of apoptotic activity. Western blot analysis revealed that the amount of activated caspase-3 was very low or not detectable in most cases, independent of the type of tumor (Fig. 3AGo). Accordingly, by immunohistochemical analysis, only a few isolated cells in either GH-secreting adenomas or NFPA were positive for cleaved caspase-3. A typical example is shown in Fig. 3BGo. Thus, under basal conditions the amount of apoptosis appeared minimal in both groups of tumors.



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  		FIG. 3. A, Western blot analysis of cleaved caspase-3 in NFPA (lanes 1–6) and in GH-secreting adenomas (lanes 7–12). C+, Positive control (GH3 cells treated with 100 nM okadaic acid for 24 h). An immunoreactive signal is present in two NFPA (lanes 1 and 2) and in two GH-secreting adenomas (lanes 7 and 8). B, Immunohistochemical analysis of cleaved caspase-3 in NFPA and GH-secreting adenomas. Immunostained cells were rarely observed in both cases, as shown by the only positive cell in the left panel (NFPA) and by the small cluster of a few positive cells in the right panel (GH-secreting adenoma). Magnification, x40. Inset, Positive control; neuroblastoma (SK-N-AS) cells exposed to the effective inducer of apoptosis camptothecin (1 µM) show intense immunostaining.

 
Effect of octreotide on apoptosis

To determine whether a different degree of sst-induced apoptosis could be elicited in GH-secreting adenomas and NFPA, primary cell cultures were obtained from a subset of these tumors (five GH-secreting tumors and five NFPA) and were exposed to the sst analog octreotide. No significant difference in the expression of sstr2 and -5 was observed between the groups of cells. Similarly, under basal conditions, the amount of cleaved CK18 did not differ. Octreotide (10 nM; 16 h) induced a significant increase in cleaved CK18 levels, which is cleaved by caspases during apoptosis (11, 12), in GH-secreting cells (mean ± SE: after octreotide, 257.6 ± 31.1; basal, 169.5 ± 17 U/liter; P < 0.05), but not in NFPA cells (mean ± SE: after octreotide, 143.1 ± 5.7; basal, 127 ± 6.9, U/liter; P > 0.05; Fig. 4Go).



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  		FIG. 4. Amount of cleaved CK18 in basal conditions (bas) and after octreotide (oct) treatment of primary cell cultures from NFPA (A) and GH-secreting adenomas (B). *, P < 0.05.

 
Furthermore, because cells may also undergo apoptosis through a caspase-3-independent mechanism (20, 21), the degree of apoptosis in GH-secreting adenomas and NFPA was assessed by TUNEL analysis, which is able to reveal the total number of apoptotic cells independent of the pathway. Noticeably, the results were very similar to those obtained by determination of the amount of cleaved CK18. Representative images are shown in Fig. 5Go. These findings indicate that apoptosis cannot be effectively induced by octreotide in cells from NFPA.



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  		FIG. 5. Determination of apoptotic nuclei by TUNEL analysis in cells from NFPA (A and B) and GH-secreting adenomas (C and D) under basal conditions (A and C) and upon octreotide treatment (B and D). Green fluorescence (left) and bright images (right) from the same field are shown. A few sparse fluorescent nuclei were observed in NFPA cells under basal conditions (A) and after octreotide treatment (B). In cells from GH-secreting adenomas, a few apoptotic nuclei were observed under basal conditions (C), whereas nuclear fluorescence markedly increased after octreotide exposure, as shown in D, in which a cluster of fluorescent nuclei is visible. Magnification, x100.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
In the present study we focused on the expression of the recently described neuroprotective factor seladin-1 (1) in pituitary tumors. This protein, which is expressed in the normal pituitary gland (1), has antiapoptotic effects via inhibition of caspase-3 activation; therefore, we hypothesized that different expression levels of seladin-1 in GH-secreting adenomas and NFPA might account, at least in part, for the different apoptotic responses to sst analogs.

By assessing the amount of seladin-1 mRNA in a large series of GH-secreting tumors and NFPA using quantitative real-time RT-PCR based on TaqMan technologies, we found that the expression level of this gene was significantly higher in NFPA. In addition, the amount of expression of seladin-1 in the normal pituitary gland was lower than that in tumor tissues. This observation provides additional evidence for the presence in NFPA of signals resulting in a relatively more aggressive phenotype in comparison with functioning tumors (22). The high expression of the antiapoptotic factor seladin-1 in NFPA is consistent with previous studies showing reduced or absent expression of the antiproliferative gene ZAC in these tumors (23) and, conversely, an intense immunostaining for the proapoptotic Bax protein in functioning tumors (24). Because it has been demonstrated that seladin-1 expression is modulated via the cAMP/protein kinase A-dependent pathway (7, 8), the presence of a different amount of seladin-1 mRNA in GH-secreting adenomas harboring a wild-type or a mutated Gs{alpha} gene was investigated. In our series of GH-secreting tumors, no significant difference in the level of expression of seladin-1 between gsp+ (n = 8) and gsp– (n = 22) adenomas was found. This lack of difference is consistent with the idea that gsp mutations are associated with counteracting mechanisms, which are able to limit the cAMP-dependent signaling. In fact, both augmented cAMP degradation via increased phosphodiesterase expression and activity (16, 25) and reduced transcriptional activation through overexpression of the transcriptional repressor, inducible cAMP early repressor (26), have been reported in gsp+ tumors.

We next measured the amount of cleaved, i.e. activated, caspase-3 in pituitary tumors to determine whether there was an inverse relationship between seladin-1 expression and caspase-3 activation. However, under basal conditions, caspase-3 appeared minimally activated in both GH-secreting adenomas and NFPA; therefore, it was not possible to establish any relationship with the amount of seladin-1 expression. It might be hypothesized that the low degree of apoptosis in the former group of tumors may be due at least in part to the observed lower expression of sstr3, which is able to promote apoptosis (27), despite the presence of a low level of expression of seladin-1. In our model, the different capabilities of GH-secreting adenomas and NFPA to undergo apoptosis was well documented in primary cell cultures exposed to the sst analog octreotide. In fact, octreotide markedly increased apoptosis in cells from GH-secreting tumors, but not in NFPA cells, as assessed by the amount of cleaved CK18 and by TUNEL analysis. It is worth noting that this different response was not attributable to differences in the amount of sstr2 and sstr5 transcripts, which were similar in the two groups of tumors, thus confirming that the poor response to sst analogs in NFPA (10) does not appear to be related to absent or reduced expression of these sstr subtypes (28, 29, 30). The octreotide-induced caspase activation observed in these experiments is in keeping with recent data concerning sstr signaling. In fact, although initially only sstr3 was thought to trigger phosphotyrosine phosphatase-dependent apoptosis accompanied by activation of p53 and of the proapoptotic protein Bax (27), subsequent studies have shown that sstr2, to which octreotide binds with high affinity, is also able to induce apoptotic cell death in leukemia and pancreatic adenocarcinoma cells by activating the caspase cascade (31, 32). Therefore, our hypothesis is that the different levels of seladin-1 expression may account at least in part for the different apoptotic responses to sst analogs observed in GH-secreting adenomas and NFPA. This hypothesis appears to be substantiated by our very recent finding that upon silencing seladin-1 expression in the neuroblastoma cell line SK-N-AS (which expresses seladin-1 and sstr2 and -5), apoptosis significantly increased. The number of apoptotic cells further increased after octreotide treatment (Cellai, I., P. Luciani, S. Benvenuti, S. Baglioni, M. Serio, and A. Peri, unpublished observations).

In conclusion, the present study showed for the first time that the seladin-1 gene is highly expressed in NFPA compared with GH-secreting adenomas and suggested that this protein, by efficiently inhibiting caspase-3 activation, may interfere with the apoptotic response to sstr agonists in NFPA. In view of these results, it is tempting to speculate that seladin-1 might be targeted to design pharmacological strategies to enforce caspase-3 activation. This hypothesis is in keeping with other innovative drug-based strategies for pituitary adenomas, such as the recent proposal to use peroxisome proliferator-activated receptor-{gamma} ligands to suppress hormone secretion and induce apoptosis (33, 34). The latter effect might be particularly advisable in NFPA, in which the poor response to conventional pharmacological interventions leaves an open window for novel strategies.


   Footnotes
 
This work was supported in part by a grant from Ente Cassa di Risparmio di Firenze and a grant from Ministero dell’Istruzione, dell’Università e della Ricerca.

First Published Online August 9, 2005

Abbreviations: CK18, Cytokeratin 18; NFPA, nonfunctioning pituitary adenoma; sst, somatostatin; sstr, sst receptor; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

Received March 23, 2005.

Accepted August 2, 2005.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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