Human Non-functioning Pituitary Adenomas Research Articles

Cabergoline reduces cell viability in non functioning pituitary adenomas by inhibiting vascular endothelial growth factor secretion

Dopamine (DA) therapy of non-functioning pituitary adenomas (NFA) can result in tumor stabilization and shrinkage. However, the mechanism of action is still unknown. Previous evidence showed that DA can inhibit pituitary vascular endothelial growth factor expression (VEGF), that may be involved in pituitary tumor growth. The aim of our study was to clarify whether VEGF secretion modulation might mediate the effects of DA agonists on cell proliferation in human NFA. We assessed DA receptor subtype 2 (DR2) expression in 20 NFA primary cultures, where we also investigated the effects of a selective DR2 agonist, cabergoline (Cab), on VEGF secretion and on cell viability. All NFA samples expressed α-subunit and DR2 was expressed in 11 samples. In DR2 expressing tumors, Cab significantly reduced cell viability (-25%; P<0.05) and VEGF secretion (-20%; P<0.05). These effects were counteracted by treatment with the DA antagonist sulpiride. Cab antiproliferative effects were blocked by VEGF. Our data demonstrate that Cab, via DR2, inhibits cell viability also by reducing VEGF secretion in a selected group of NFA, supporting that DA agonists can be useful in the medical therapy of DR2 expressing NFA.

Overexpression of the Notch3 receptor in non-functioning pituitary tumours

Human non-functioning pituitary adenomas cause hypopituitarism or the compression of adjacent structures. At present, there is no available medical treatment for these tumours. The Notch3 pathway has an important role in the progression of non-functioning pituitary adenomas. We found, using reverse transcriptase–polymerase chain reaction, that Notch3 mRNA was significantly upregulated in clinically non-functioning pituitary adenomas, and, using Western blotting, that Notch3 protein was expressed at a higher level in non-functioning pituitary adenomas than in normal human pituitary tissue. In addition, using immunohistochemistry, we observed that Notch3 is highly expressed in the cytoplasm of adenoma cells compared to normal pituitary cells. These results suggest that the overexpression of Notch3 mRNA and protein by non-functioning pituitary adenomas may facilitate the growth of these tumours. Our results provide the first comprehensive analysis of Notch3 mRNA and protein overexpression in non-functioning pituitary tumours. This study provides new insights into the pathogenesis of human non-functioning pituitary adenomas and implicates the Notch3 pathway as a molecular therapeutic target for their treatment.

The Somatostatin Analogue Octreotide Confers Sensitivity to Rapamycin Treatment on Pituitary Tumor Cells

Rapamycin and its analogues have significant antiproliferative action against a variety of tumors. However, sensitivity to rapamycin is reduced by Akt activation that results from the ablative effects of rapamycin on a p70 S6K-induced negative feedback loop that blunts phosphoinositide 3-kinase (PI3K)-mediated support for Akt activity. Thus, sensitivity to rapamycin might be increased by imposing an upstream blockade to the PI3K/Akt pathway. Here, we investigated this model using the somatostatin analogue octreotide as a tool to decrease levels of activated Ser(473)-phosphorylated Akt (pAkt-Ser(473)) in pituitary tumor cells that express somatostatin receptors. Octreotide increased levels of phosphorylated insulin receptor substrate-1 that were suppressed by rapamycin, subsequently decreasing levels of pAkt-Ser(473) through effects on phosphotyrosine phosphatase SHP-1. Octreotide potentiated the antiproliferative effects of rapamycin in immortalized pituitary tumor cells or human nonfunctioning pituitary adenoma cells in primary cell culture, sensitizing tumor cells even to low rapamycin concentrations. Combined treatment of octreotide and rapamycin triggered G(1) cell cycle arrest, decreasing E2F transcriptional activity and cyclin E levels by increasing levels of p27/Kip1. These findings show that adjuvant treatment with a somatostatin analogue can sensitize pituitary tumor cells to the antiproliferative effects of rapamycin.

Effect of Everolimus on Cell Viability in Nonfunctioning Pituitary Adenomas

Pituitary adenomas can cause specific syndromes due to hormone excess and/or determine sellar mass symptoms. Pituitary cell growth can sometimes be influenced by medical therapy, such as for somatotroph adenomas treated with somatostatin analogs or prolactinomas treated with dopaminergic drugs. However, nonfunctioning pituitary adenomas (NFAs) are still orphans of medical therapy. Everolimus (RAD001), a derivative of rapamycin, is a well-known immunosuppressant drug, which has been recently shown to have antineoplastic activity in several human cancers. The objective of the study was to investigate the possible antiproliferative effects of RAD001 in human NFAs. We collected 40 NFAs that were dispersed in primary cultures, treated without or with 1 nm to 1 microm RAD001, 10 nm cabergoline, 10 nm SOM230 (a somatostatin receptor multiligand), and/or 50 nm IGF-I. Cell viability and apoptosis were evaluated after 48 h, and vascular endothelial growth factor (VEGF) secretion was assessed after an 8-h incubation. Somatostatin and dopamine subtype 2 receptor expression was investigated by quantitative PCR. In 28 cultures (70%), Everolimus significantly reduced cell viability (by approximately 40%; P < 0.05 vs. control), promoted apoptosis (+30%; P < 0.05 vs. control), inhibited p70S6K activity (-20%), and blocked IGF-I proliferative and antiapoptotic effects. In selected tissues cotreatment with SOM230, but not cabergoline, exerted an additive effect. Everolimus did not affect VEGF secretion but blocked the stimulatory effects of IGF-I on this parameter. Everolimus reduced NFA cell viability by inducing apoptosis, with a mechanism likely involving IGF-I signaling but not VEGF secretion, suggesting that it might represent a possible medical treatment of invasive/recurrent NFAs.

Folate receptor α regulates cell proliferation in mouse gonadotroph αT3-1 cells

We have previously found that the mRNA and protein levels of the folate receptor alpha (FRα) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FRα has not fully been determined. We investigated the effect of FRα over-expression in the mouse gonadotroph αT3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FRα were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNA staining. These observations indicate that over-expression of FRα promotes cell proliferation. These effects were abrogated in the same αT3-1 cells when transfected with a mutant FRα cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FRα over-expressing cells. In summary, our data suggests that FRα regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.

Efficacy of a dopamine-somatostatin chimeric molecule, BIM-23A760, in the control of cell growth from primary cultures of human non-functioning pituitary adenomas: a multi-center study

Dopamine D2 and somatostatin receptors (sstrs) were reported to affect non-functioning pituitary adenoma (NFPA) proliferation in vitro. However, the reported results differ according to the experimental conditions used. We established an experimental protocol allowing reproducible evaluation of NFPA cell proliferation in vitro, to test and compare the antiproliferative effects of dopamine and somatostatin analogs (alone or in combination) with the activity of the dopamine-somatostatin chimeric molecule BIM-23A760. The protocol was utilized by four independent laboratories, studying 38 fibroblast-deprived NFPA cell cultures. Cells were characterized for GH, POMC, sstr1-sstr5, total dopamine D2 receptor (D2R) (in all cases), and D2 receptor long and short isoforms (in 15 out of 38 cases) mRNA expression and for alpha-subunit, LH, and FSH release. D2R, sstr3, and sstr2 mRNAs were consistently observed, with the dominant expression of D2R (2.9+/-2.6 copy/copy beta-glucuronidase; mean+/-s.e.m.), when compared with sstr3 and sstr2 (0.6+/-1.0 and 0.3+/-0.6 respectively). BIM-23A760, a molecule with high affinity for D2R and sstr2, significantly inhibited [3H]thymidine incorporation in 23 out of 38 (60%) NFPA cultures (EC50=1.2 pM and Emax=-33.6+/-3.7%). BIM-23A760 effects were similar to those induced by the selective D2R agonist cabergoline that showed a statistically significant inhibition in 18 out of 27 tumors (compared with a significant inhibition obtained in 17 out of 27 tumors using BIM-23A760, in the same subgroup of adenomas analyzed), while octreotide was effective in 13 out of 27 cases. In conclusion, superimposable data generated in four independent laboratories using a standardized protocol demonstrate that, in vitro, chimeric dopamine/sstr agonists are effective in inhibiting cell proliferation in two-thirds of NFPAs.

Effects of selective somatostatin analogs and cortistatin on cell viability in cultured human non-functioning pituitary adenomas

Clinically "non-functioning" human pituitary adenomas (NFPA) constitute about 35% of pituitary adenomas. Somatostatin receptors (SSTR) expression in these adenomas has previously been described both in vitro and in vivo, without evidence for a correlation with tumor volume or the therapeutic efficacy of somatostatin analogs. This study was performed on 13 surgically removed pituitary macroadenomas, diagnosed before surgery as "non-functioning". In addition, 3 growth hormone (GH)-secreting adenomas served as controls. A specimen from each tumor was dispersed and digested to isolate and culture the tumor cells, and the in vitro effects of SSTR2 and SSTR5 selective analogs and Cortistatin (CST) (100nM) on cell viability were studied. The quantity of viable cells was estimated using the XTT method. RNA purification of tumor samples and subsequent RT-PCR studies for SSTR2 and SSTR5 expression were performed. Somatostatin analog with high affinity for SSTR2 reduced cell viability by 20-80% in 8 of 13 NFPAs studied, all expressing the SSTR2. The inhibitory effect on cell viability of SSTR5-selective analog was 15-80% in 10 of 13 NFPAs studied, all but three expressing the SSTR5. CST, however, effectively reduced cell viability in only 6 NFPAs. Cell viability was inhibited by all peptides studied in 2 out of 3 GH-secreting adenomas, expressing both receptors. The third adenoma responded to SSTR2 analog and expressed only SSTR2. These results suggest the involvement of SSTR2 and SSTR5 in the anti-proliferative effects of somatostatin; however, CST is less potent in reducing cell viability in these tumors.

Induction of GH, PRL, and TSHβ mRNA by transfection of Pit-1 in a human pituitary adenoma-derived cell line

The functional development of pituitary cells depends on the expression of a combination of transcription factors and co-factors. Pituitary-specific transcription factor-1 (Pit-1) is required for the expression of growth hormone (GH), prolactin (PRL), and the thyroid-stimulating hormone beta subunit (TSH beta) and acts synergistically with the estrogen receptor (ER) and GATA-binding protein 2 (GATA-2) to induce PRL and TSH beta expression, respectively. The glycoprotein hormone alpha subunit (alpha SU) is the first hormone to be expressed during pituitary development. In addition to being expressed in follicle-stimulating hormone, luteinizing hormone (LH), and TSH cells, alpha SU is reported to co-localize with GH in pituitary cells. These findings have led to the suggestion that the expression of Pit-1 in cells of the alpha SU-based gonadotropin cell lineage might also lead to the expression of GH. In this study, we transfected HP 75 cells (derived from a human non-functioning pituitary adenoma that expressed alpha SU and LH beta) with Pit-1 by using an adenovirus FLAG-Pit-1 construct. Most of the transfected cells expressed GH mRNA, with fewer cells expressing PRL and TSH beta mRNA. The HP 75 cells expressed the genes for ER and GATA-2, thus allowing their expression of GH, PRL, and TSH beta mRNA in response to Pit-1. These results support the hypothesis that GH can be induced in cells that possess an active alpha SU gene and shed light on the basic molecular mechanism that drives the development of GH, PRL, and TSH beta expression in the alpha SU-based gonadotroph lineage.

Expression and role of VEGF receptors in pituitary adenomas

Vascular endothelial growth factor (VEGF) is known to exert a highly specific mitogenic action on endothelial cells through two tyrosine kinase receptors: VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR) which are almost specifically expressed in endothelial cells. However, it has been shown that VEGF receptors may also be expressed by non-endothelial cells, especially by tumor cells. We have recently shown that pituitary adenoma cells produce variable amounts of VEGF and have now started to study the expression of VEGF receptors in normal human pituitary and pituitary adenomas by RT-PCR, in situ hybridisation and immunohistochemistry. Expression of VEGFR1 and VEGFR2 mRNA was found in normal pituitary and in the 49 adenomas (26 nonfunctioning, 9 somatotroph, 8 corticotroph and 6 lactotroph tumors) studied so far. The expression was variable and no correlation between VEGFR1 expression and tumor grade and vessels number was found. In normal pituitary, VEGFR1 immunoreactivity was observed to co-localize with ACTH-, FSH-, GH- and LH-secreting cells, but not in endothelial cells, suggesting that VEGF may affect function of endocrine cell types by paracrine mechanisms. In contrast, VEGFR2 immunoreactivity was detected only in the vascular formations, in both normal pituitary and pituitary adenomas. Immunohistochemical analysis for VEGFR2 in 33 pituitary adenomas (9 nonfunctioning, 7 somatotrophs, 6 corticotrophs and 11 lactotrophs) revealed immunoreactivity in the vascular formation of 18 tumors and we observed that somatotroph and corticotroph adenomas showed the highest number of cases with VEGFR2 immunoreactivity on vessels. However, we found no correlation between VEGFR2 expression and extent of vascularization. We have then observed that HP75, a human non-functioning pituitary adenoma cell line secreting gonadotropins, expresses both VEGFR-1 and –2 mRNA and human VEGF165 is able to stimulate cells proliferation in a dose-dependent manner. This finding could suggest again a possible paracrine/autocrine action of VEGF on endocrine and tumor cells.

Proteomics and transcriptomics analyses of secretagogin down-regulation in human non-functional pituitary adenomas.

In order to explore the presence of, and the potential role of, secretagogin in human pituitary adenomas, an analytical strategy that integrated comparative proteomics and comparative transcriptomics was used to detect the protein and the mRNA expression, respectively, of secretagogin in human non-functional pituitary adenomas compared to controls. Proteomics methods included two-dimensional gel electrophoresis, 2D gel image analysis, mass spectrometry [matrix-assisted laser desorption/ionization-time of flight-peptide mass fingerprinting (MALDI-TOF PMF) and liquid chromatography-electrospray ionization-quadrupole-ion trap tandem mass spectrometry (LC-ESI-Q-IT MS/MS)], and database analysis. Transcriptomics methods included the GeneChip microarray, image processing, and data analysis. The proteomics and transcriptomics data demonstrated that secretagogin was significantly down-regulated at the protein and mRNA levels, respectively, in the human non-functional (NF) pituitary adenomas (NF-, LH+, FSH+, and FSH+ + LH+). For the secretagogin protein, the expression level was NF- < FSH+ + LH+ < FSH+ < LH+ < Control, with a range of down-regulation of 2.2-6.9 fold in non-functional pituitary adenomas compared to controls, with a significant difference (p < 0.001). For secretagogin mRNA, the expression level was NF- < LH+ < FSH+ + LH+ < FSH+ < Control, with a range of down-regulation of 1.8-18.6 fold in non-functional pituitary adenomas compared to controls that was significant (p < 0.05). The secretagogin protein expression correlated significantly with its mRNA expression. Those results suggest that secretagogin might play a role in human non-functional pituitary adenomas. This novel finding may provide clues to clarify the basic molecular mechanisms of pituitary adenoma formation, and to identify new tumor-related markers.

Growth promoting effect of human plasma ultrafiltrate bioactive fraction (TBP) for human non-functioning pituitary adenoma cells in vitro.

We described before that the chromatographically purified "human plasma ultrafiltrate bioactive fraction" (humoral factor tentatively denoted as tumor basic protein--TBP) regulates in vitro release of ACTH from pituitary adenomas stimulating the hormone release from the tumors showing low hormonal activity in vitro and inhibiting ACTH production in vitro by highly hormonally active pituitary tumors. In this study we describe growth promoting effects (determined by 3H-TdR incorporation assay) of TBP (5 microg/l, i.e. 10% w/v plasma equivalent concentration) for 10 non-functioning pituitary tumors. The effects of TBP appeared to negatively correlate with the in vitro growth abilities of the tumors that were otherwise dependent on the duration of the clinical symptoms of the tumor presence. Hence, similar to its effects on hormonal activity of the pituitary tumors, TBP stimulated the growth of the tumors which did not express high spontaneous 3H-TdR intensity, but did not stimulate the cells with high capacity of spontaneous 3H-TdR incorporation. Moreover, all the tumors that were stimulated by TBP were nononcocytic adenomas while oncocytoma cells were not stimulated at all. Thus, TBP shows activity of humoral (plasma) factor involved in the growth regulation of pituitary adenomas that might be used to define the growth abilities of these tumors, especially in case of null cell adenomas and oncocytomas as were the tumors used in this study.

Somatostatin and its analog lanreotide inhibit the proliferation of dispersed human non-functioning pituitary adenoma cells in vitro.

Somatostatin is a powerful inhibitor of hormone secretion and cell proliferation. Treatment with somatostatin analogs in humans causes a reduction in size and secretory activity of some endocrine tumors, including somatotropic pituitary adenomas. Less studied are the effects of somatostatin agonists on non-functioning pituitary adenomas (NFPAs). In this study we characterized the effects of somatostatin and its analog lanreotide on the proliferation of NFPAs in vitro and the intracellular mechanisms involved. Twenty-three NFPA post-surgical specimens were analyzed for somatostatin receptor (SSTR) expression and 12 of them were cultured in vitro to study somatostatin's effects on cell proliferation, assessed by means of [(3)H]thymidine uptake, and the intracellular signaling. One or more SSTR subtypes were expressed in 90% of the adenomas tested. Somatostatin and lanreotide treatment inhibited phorbol myristate acetate (PMA)-induced cell proliferation. Vanadate pretreatment reversed somatostatin and lanreotide inhibition of PMA-induced DNA synthesis suggesting an involvement of tyrosine phosphatase in this effect. In the only adenoma tested, somatostatin directly induced a tyrosine phosphatase activity. Somatostatin and lanreotide caused also a significant inhibition of voltage-sensitive calcium channel activity induced by 40mmol/l K(+) depolarization in microfluorimetric analysis. These data show that somatostatin and lanreotide inhibit human NFPA cell proliferation in vitro, and suggest that activation of tyrosine phosphatases and inhibition of the activity of voltage-dependent calcium channels may represent intracellular signals mediating this effect.

Spontaneous and agonist-induced calcium oscillations in single human nonfunctioning adenoma cells

The effects of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) on cytosolic free calcium concentration ([Ca(2+)](i)) were investigated in 20 human nonfunctioning pituitary adenomas. We divided these tumors into three classes according to their response pattern to hypothalamic peptides. In type I adenomas (8 out of 20 adenomas), GnRH and GAP mobilized intracellular calcium ions stored in a thapsigargin (TG)-sensitive store. For the same concentration of agonist, two distinct patterns of GnRH-GAP-induced Ca(2+) mobilization were observed (1) sinusoidal oscillations, and (2) monophasic transient. The latter is followed by a protein kinase C (PKC)-dependent increase in calcium influx through L-type channels. In type II adenomas (7 out of 20 adenomas), GnRH and GAP only stimulate calcium influx through dihydropyridine-sensitive Ca(2+) channels by a PKC-dependent mechanism. TG (1 μM) did not affect [Ca(2+)](i) in these cells, suggesting that they do not possess TG-sensitive Ca(2+) pools. All the effects of GnRH and GAP were blocked by an inhibitor of phospholipase C (PLC), suggesting that they were owing to the activation of the phosphoinositide turnover. Type I and type II adenoma cells showed spontaneous Ca(2+) oscillations that were blocked by dihydropyridines and inhibition of PKC activity. GnRH and GAP had no effect on the [Ca(2+)](i) of type III adenoma cells that were also characterized by a low resting [Ca(2+)](i) and by the absence of spontaneous Ca(2+) fluctuations. K(+)-induced depolarization provoked a reduced Ca(2+) influx, whereas TG had no effect on the [Ca(2+)](i) of type III adenoma cells. The variety of [Ca(2+)](i) response patterns makes these cells a good cell model for studying calcium homeostasis in pituitary cells.

Mechanism of Action of Pituitary Adenylate Cyclase‐Activating Polypeptide (PACAP) in Human Nonfunctioning Pituitary Tumors

Several evidence suggest that pituitary adenylate cyclase activating polypeptides (PACAP-38 and -27) could function as hypophysiotropic factors. Both peptides interact with either the type I receptor, which preferentially binds the two PACAPs and has a much lower affinity for vasoactive intestinal polypeptide (VIP) or the type II receptor, which binds the two PACAPs and VIP with a nearly equal affinity. In addition to the stimulation of adenylyl cyclase (AC) activity, in different cell types PACAP causes an increase of cytosolic calcium levels ([Ca2+]i), consequent to phospholipase-C activation. In the present study, we investigated the effect of PACAP on cAMP formation and [Ca2+]i levels in 16 human nonfunctioning pituitary adenomas (NFPA). PACAP-38 increased cAMP formation in all tumors; the peptide stimulated either AC activity in membrane preparations from 26 +/- 10 to 214 +/- 179 pmol/mg prot/min (P < 0.01) or cAMP efflux from 12 +/- 5.4 to 73.2 +/- 32 pmol/well (P < 0.01) in cultured cells. The effect, detectable at concentrations higher than 1-10 pM, was maximal at 0.1-10 nM. While PACAP-38 and PACAP-27 were nearly equally effective and potent, 100-fold higher concentrations of VIP were required to obtain similar AC activation. GHRH and CRH were ineffective in any NFPA. The PACAP effect was not antagonized by a VIP antagonist, while PACAP fragment 6-27 amide partially reduced the stimulatory effects of both PACAP-27 and VIP in 2 out of 3 tumors tested. PACAP-38 caused a [Ca2+]i rise in cells obtained from 7 NFPA (from 110 +/- 34 to 151 +/- 40 nM [Ca2+]i, P < 0.05) while in the remaining 7 the peptide was ineffective at any concentrations tested (from 1 nM to 10 microM). In the responsive tumors, PACAP-38 effect was not consequence of phospholipase-C activation since removal of extracellular Ca2+ as well as blockade of L-type Ca2+ channels by dihydropyridine antagonists abolished [Ca2+]i increase triggered by the peptide. These data indicate that PACAP is by far the most potent activator of cAMP formation in NFPA and suggest a possible modulatory action of this peptide on cell growth.

Abnormal transduction of dopamine signal in human nonfunctioning pituitary adenomas.

It is well established that dopamine (DA) plays an important role in inhibiting anterior pituitary function. DA receptors present in the pituitary show the pharmacological and biochemical characteristics of the D2 receptor; in fact, they are coupled to the inhibition of both adenylyl cyclase (AC) activity and the reduction of cytosolic free Ca2+ levels ([Ca2+]i) suggesting the involvement of different G-proteins. While the DA receptors present in human PRL-omas display these characteristics, no information is available on the coupling mechanism(s) of DA receptors expressed in nonfunctioning pituitary adenomas (NF-PA). In the present study, the effect of DA on AC activity and [Ca2+]i was investigated in 8 NFPAs surgically removed by the transphenoidal route. DA, at concentrations between 0.01 and 10 mumol/l, had no effect on cAMP formation in any tumor (from 27.6 +/- 11.9 to 27.9 +/- 11.0 pmol/mg prot/min; NS). By contrast, DA was effective in reducing [Ca2+]i levels either in resting conditions or after TRH stimulation in 5 out of 8 tumors, suggesting that NFPA express DA receptors with a defective transduction mechanism. As in these tumors SRIH caused the expected inhibition of both AC activity (from 31.4 +/- 9.3 to 24.4 +/- 11.0 pmol/mg prot/min; p < 0.005) and [Ca2+]i levels, it is likely that the lack of DA action on AC activity may be due to functional/structural properties of DA receptors expressed in NFPA, instead of a defect at the level of Gi proteins. In conclusion, these data indicate that DA receptors expressed in NFPA show a defective transduction mechanism, leading to a partial inhibitory response.

Immunodetection of chorionic gonadotropin and its subunits in human nonfunctioning pituitary adenomas.

Human nonfunctioning pituitary adenomas (NFPA) may produce CG in addition to the classical glycoprotein hormones (LH, FSH, and TSH). The aim of the present study was to localize LH beta, FSH beta, TSH beta, alpha-subunit (alpha SU), CG, and its beta-subunit (beta SU) in NFPA using a highly specific immunohistochemical technique. Nine NFPA, obtained at surgery, were processed for both electron microscopy and immunohistochemistry. Three tumors resulted oncocytomas, and six were null cell. Using an immunofluorescence technique, all tumors were positive for at least one glycoprotein; in particular, seven adenomas were markedly positive for CG beta, whereas only two were positive for the intact CG. No association among LH beta, FSH beta, and CG beta positivity could be demonstrated in the different adenomas. In the seven tumors positive for both CG beta and alpha SU, double fluorescence labeling demonstrated that six cases localized CG beta and alpha SU in different cells, but only one tumor showed the two subunits colocalized in the same cells. These data confirm that pituitary tumors synthesize both alpha SU and beta SU of glycoprotein hormones; in particular, the present study indicates that the majority of NFPA is able to synthesize CG, particularly its beta SU. Moreover, the localization of CG beta and alpha SU in different tumoral cells might account for the preferential expression of beta SU and alpha SU instead of the intact hormonal molecules in NFPA.

Immunodetection of chorionic gonadotropin and its subunits in human nonfunctioning pituitary adenomas

Immunodetection of chorionic gonadotropin and its subunits in human nonfunctioning pituitary adenomas

In vitro detection of glycoprotein production and secretion by human nonfunctioning pituitary adenomas.

This study, carried out on 9 nonfunctioning pituitary adenomas, was undertaken in order to evaluate the ability of these tumors to synthesize and release gonadotropins and/or free alpha-subunit (alpha-SU) of glycoproteins. The morphological study included electron microscopy and immunofluorescence analysis while hormone release was evaluated by the reverse hemolytic plaque assay (RHPA) and measurements in culture media. By electron microscopy in all tumors (6 null cell adenomas and 3 oncocytomas), it was possible to identify rough endoplasmic reticulum, Golgi apparatus and secretory granules. By immunofluorescence, 5 of 6 tumors were immunoreactive for one or more gonadotropin subunits; in particular, 5 adenomas were positive for alpha-SU and LH-beta, and 3 for FSH-beta. By the RHPA, about 1% of cells obtained from one single tumor formed plaques for LH-beta and alpha-SU while the remaining tumors were negative. Similarly, the study of media concentrations of LH, FSH and alpha-SU in 2 h culture revealed very low amounts of released hormones. In these experimental conditions no modification was observed after the addition of stimulatory agents such as TRH, GnRH and VIP. The present study clearly indicates that although the large majority of nonfunctioning tumors are positive for gonadotropins their secretory capacity is very low in both basal and stimulated conditions.

Secretion of polypeptide growth factors by human nonfunctioning pituitary adenoma cells in culture.

The growth-promoting activities of tumor-conditioned media (TU-CM) obtained from 23 cultured human nonfunctioning pituitary adenomas were studied in vitro. TU-CM obtained from adenoma cell cultures increased both cell counts (range: 108-179%; control = growth in serum-free medium = 100%) and 3H-thymidine incorporation (112-139%) of rat pituitary cell cultures, indicating that TU-CM contains growth-stimulating substances. TU-CM was also able to stimulate the growth of normal fibroblasts (3H-thymidine incorporation: 164-178%; cell counts: 145-157%) and endothelial cells (3H-thymidine incorporation: 131-149%; cell counts: 181-217%), suggesting the presence of - possibly angiogenic-growth factors that act on these cell types. However, the growth of hormone-producing cells was also stimulated, since TU-CM increased 3H-thymidine incorporation into rat pituitary cells in the presence of D-Val-MEM, a medium specifically inhibiting growth of fibroblasts. Addition of neutralizing antibodies against transforming growth factor alpha (TGF-alpha), epidermal growth factor (EGF), insulin-like growth factor I (IGF-I) and basic fibroblast growth factor (bFGF), either alone or in different combinations, reduced the growth-promoting activity of TU-CM on rat pituitary cells (range: 96-71%; control = growth effect of TU-CM without antibodies = 100%), strongly indicating the presence of these growth factors in TU-CM. All 4 antibodies together completely inhibited the growth-stimulatory activity of TU-CM, strongly suggesting that these growth factors play the major role among growth-stimulating substances in TU-CM. This is the first study giving evidence that TGF-alpha, EGF, IGF-I and bFGF are secreted by nonfunctioning adenoma cells indicating that the growth factors could be involved in growth regulation of pituitary adenomas by paracrine or autocrine mechanisms.