Amyloid Deposits in Pituitaries and Pituitary Adenomas: Immunohistochemistry and In Situ Hybridization

The patterns of deposition and immunoreactivity of interstitial amyloid were studied in 11 pituitary glands obtained at autopsy and 9 surgically resected pituitary adenomas using Congo red staining and a panel of antisera directed against 5 major amyloid fibril proteins and all pituitary hormones. The deposition pattern of amyloid in pituitary glands differed from that in adenomas but all amyloid deposits showed an immunostaining with anti-amyloid λ-light chain. The remaining antisera were immunonegative.In situ hybridization using an oligodeoxyribonucleotide-probe complementary to the mRNA coding for the constant region of human λ-light chain yielded no hybridization signals in the pituitaries or pituitary adenomas, excluding local synthesis and secretion of immunoglobulins. Since no case studied suffered from generalized Aλ-amyloidosis and adsorption of immunoglobulins to the unknown amyloid fribril protein of the pituitary seems to be unlikely, crossreaction of the polyclonal antisera with an undefined antigen is probable. The similar immunostaining properties of amyloid deposits in “normal” pituitaries and pituitary adenomas suggest they both originate from the same precursor protein.     

Protein kinase C (PKC) activity and PKC messenger RNAs in human pituitary adenomas.

Protein kinase C (PKC) is involved in the differentiation and growth regulation of a variety of tissues including anterior pituitary gland cells. To determine the distribution of PKC in different types of adenomas, PKC activity was analyzed in human pituitary tumors and the effects of hypothalamic hormone stimulation on PKC activity were examined in cultured adenoma cells. Gonadotroph (LH/FSH) and null cell adenomas had significantly higher levels of particulate, soluble, and total PKC activity compared with growth hormone (GH) adenomas (P < 0.05). Chronic stimulation of null cell adenomas with gonadotropin hormone-releasing hormone or of one GH adenoma with GH-releasing hormone for 7 days did not significantly alter total PKC activity in pituitary cells cultured in serum-free medium. Localization of the calcium-dependent PKC isozymes (alpha, beta and gamma) by immunohistochemistry and in situ hybridization revealed predominantly PKC alpha in all adenomas and variable expression of PKC beta and gamma in some tumors. When the calcium-independent PKC isozymes (delta, epsilon, and zeta) were localized by in situ hybridization, normal and neoplastic pituitaries expressed abundant mRNA for PKC epsilon, whereas some tumors and one normal pituitary had a few cells positive for PKC zeta mRNA as evaluated by grain density and the number of cells labeled. These results indicate that there is a variable distribution of PKC mRNA isozymes in human pituitary adenomas and that normal pituitaries and pituitary adenoma cells express the mRNA for both the calcium-dependent and some of the calcium-independent PKC isozymes. Chronic treatment with the hypothalamic gonadotropin hormone-releasing hormone and GH-releasing hormone, which increased LH/FSH and GH secretion, respectively, did not increase PKC activity in cultured adenoma cells. The presence of calcium-dependent and calcium-independent PKC isozymes in normal and neoplastic pituitary cells indicates that PKC probably plays a major role in signal transduction in the human pituitary adenomas examined in this study.     

Neural cell adhesion molecule (NCAM) in normal and neoplastic human pituitary tissues: Analysis by immunohistochemistry and in situ hybridization

The expression of the neural cell adhesion molecule (NCAM) in 6 normal human pituitaries and 25 pituitary adenomas was investigated by immunohistochemistry and in situ hybridization. NCAM protein and mRNA were present in all normal and neoplastic human pituitary tissues. There were tumor type-specific differences in the distribution of NCAM in various pituitary adenomas. Growth hormone adenomas and prolactin-producing adenomas usually expressed lower levels of NCAM, compared to null cell and gonadotroph adenomas. Adreno-corticotropic hormone adenomas expressed the highest levels of NCAM mRNA. Six freshly dissociated pituitary adenomas were cultured in serum-free medium for 7 days to analyze the regulation of NCAM mRNA by in situ hybridization. The lower levels of NCAM expression in growth hormone and prolactin adenomas were not present in cells cultured for 7 days in serum-free medium on extracellular matrix. Phorbol 12-myristate 13-acetate (PMA) stimulated NCAM mRNA expression in 5 of 6 tumors. Gonadotropin-releasing hormone and growth hormone-releasing hormone increased NCAM expression in some adenomas. This study demonstrates that there is a variable expression of NCAM in pituitary adenomas and that hypotha-lamic hormones and PMA can regulate NCAM mRNA levels in neoplastic pituitary cells.     

Detection of inhibin α and βA subunits (inhibin A, B, activin A,AB) in the human pituitary gland and in pituitary adenomas by immunohistochemistry and nonradioisotopicin situ hybridization

Inhibin and activin are gonadal hormones produced in human ovaries. They are known to act on anterior pituitary cells to regulate the synthesis and secretion of follicle-stimulating hormone (FSH). The purpose of the present study was to determine the localization of inhibin and activin subunits α and βA as endocrine markers in the human normal pituitary gland and pituitary adenomas, using immunohistochemistry andin situ hybridization (ISH) methods. Pituitary tissues from surgical and autopsy materials were fixed in 10% formalin and embedded in paraffin. Five normal pituitary glands and 79 pituitary adenomas were immunostained with the avidin-biotin peroxidase complex (ABC) method using polyclonal antibodies against inhibin and activin subunits α and βA. The other antibodies against anterior pituitary hormones used in this study were as follows: antigrowth hormone (anti-GH), antiprolactin (anti-PRL), antiadrenocorticotropic hormone (anti-ACTH), anti-FSHβ, antilutenizing hormone (anti-LH) β, antithyroid-stimulating hormone (anti-TSH) β, and antiglycoprotein α-subunit (anti-α-SU). We analyzed gene expressions of subunits α and βA by nonradioisotopic ISH in pituitary adenomas. In the normal human pituitary glands, inhibin and activin subunits α and βA immunoreactivities were found diffusely in the cytoplasm of anterior pituitary cells. The percentage of subunit α-immunopositive cells was 40% of the anterior pituitary cells. Subunit βA immunoreactivities were observed in about 15% of the anterior pituitary cells. By the double-staining method, subunit α immunoreactivity was detected in all types of anterior pituitary cells, and it was colocalized most frequently with GH and α-SU-positive cells. Subunit βA immunoreactivity was colocalized predominantly with PRL, FSH-β, LH-β, and α-SU. Among the 79 adenomas, 75 cases (94.9%) were positive for subunit α, and 50 cases (63.3%) were positive for subunit βA. Subunit βA was positive in tumor cells with the following incidences: GH adenomas, 3 of 14 (21.4%); PRL adenomas, 5 of 8 (62.5%); ACTH adenomas, 6 of 6 (100%); TSH adenomas, 7 of 7 (100%); nonfunctioning adenomas, 29 of 44 (65.9%), including gonadotropin-positive, 16 of 22 (80.0%). The ISH signals for subunits α and βA were strongly expressed in gonadotropin-positive adenomas among the nonfunctioning adenomas. The mRNA signals were low and infrequent in the GH-producing adenomas. Inhibin and activin subunit α localization did not demonstrate cell-type specificity in pituitary adenomas. In contrast, subunit βA demonstrated predominant positivity in the functioning pituitary adenomas (ACTH- and TSH-secreting) and nonfunctioning adenomas (including gonadotropin-positive adenomas). The present results suggest that the functional role of inhibin and activin in the differentiation of cells in normal human pituitary glands and adenomas is present in subunit βA.     

GH-, PRL-, POMC-, beta-TSH-, beta-LH-, beta-FSH-mRNA in gonadotroph adenomas of the pituitary by in situ hybridization in comparison with immunostaining and clinical data

In situ hybridization (ISH) enables the visualization of specific mRNA for pituitary hormones. Our collection consists of 40 surgically removed pituitary adenomas that were classified as follicle stimulating hormone/luteinizing hormone (FSH/LH) cell adenomas by structure and by immunostaining (IH) for all pituitary hormones. All forty adenomas were regarded as clinically inactive. The aim of our study was to examine nonfunctioning adenomas by ISH for demonostration of mRNAs for all pituitary hormones. The results were compared with proliferation markers, invasiveness and clinical data. ISH detected signals for all pituitary hormones at a range of 30% for prolactin (PRL) to 85% for proopiomelanocortin (POMC). mRNA for β-FSH was detected in 70% and β-LH mRNA in 43% of adenomas. Thirty-three percent of adenomas revealed negative mRNA detection for β-LH but positive hormone content. The majority of adenomas (75%) expressed more than two mRNAs simultaneously, mostly the combination of POMC mRNA together with β-FSH mRNA and one to four others. Comparison with clinical data showed no significant differences except for one adenoma with a high Ki-67 index (>2.1% positive nuclei). This adenoma showed very high signals for PRL and β-TSH mRNA.     

Pancreastatin secretion by pituitary adenomas and regulation of chromogranin B mRNA expression.

Pancreastatin, a carboxyl-terminal amidated peptide derived from chromogranin (Cg)A, inhibits secretion of insulin and parathyroid hormone. Our recent studies found significant amounts of immunoreactive pancreastatin in all pituitary adenomas except prolactin adenomas. To analyze the effects of pancreastatin on pituitary cell function, 17 cultured pituitary adenomas were examined for immunoreactive pancreastatin and pancreastatin secretion by the tumors. The effects of pancreastatin on pituitary hormone secretion and on pituitary hormone (follicle-stimulating hormone and prolactin), CgA, and CgB mRNA levels were also examined. Immunoreactive pancreastatin and CgA were present diffusely in gonadotroph and null cell adenomas, but only a few prolactin adenoma cells expressed pancreastatin or CgA. When cells were treated with hypothalamic peptides, gonadotroph adenomas were the only group that released increased amounts of pancreastatin in response to gonadotropin-releasing hormone (10(-7) mol/L). Pancreastatin (10(-7) mol/L) treatment did not stimulate pituitary hormone secretion significantly. In situ hybridization analyses showed that gonadotropin-releasing hormone and pancreastatin treatment led to significant increases in CgB and follicle-stimulating hormone mRNAs in gonadotroph adenomas, whereas CgA mRNA levels did not change significantly. These results show that there is a differential distribution of pancreastatin secretion in pituitary adenomas and that the hypothalamic hormone gonadotropin-releasing hormone and the CgA-derived peptide pancreastatin can regulate CgB mRNA in gonadotroph adenomas, suggesting an autocrine effect of pancreastatin on pituitary tumor function.     

Pituitary adenomas and granular cell tumors. Incidence, cell type, and location of tumor in 100 pituitary glands at autopsy.

Incidentally detected pituitary adenomas were investigated in 100 pituitary glands at autopsy to determine the number, cell type, and location of tumors, and the presence of coexisting granular cell tumors in the neurohypophysis. Pituitary glands were sagittally sectioned at 1.5-mm intervals in toto and embedded in 1 cassette to orient location of each tumor. Twenty-four pituitary glands harbored adenomas, most smaller than 3 mm and the largest 6 x 5 x 4 mm. Two pituitary glands contained double adenomas of immunocytochemically different cell types. Of the 26 adenomas, 10 had lactotrophs, 2 had mixed lactotrophs-somatotrophs, 1 had mixed lactotrophs-luteinizing hormone cells, and 12 were nonfunctioning. One adenoma with adenocorticotropic hormone cells was also detected. Thus 25 of 26 (96%) adenomas were either lactotrophic or nonfunctioning; this percentage is much higher than that of surgically resected tumors. Twenty-two tumors were contiguous with or adjacent to the capsule from which the adenomas originated. Nine granular cell tumors were noted in the neurohypophysis; 3 coexisted with pituitary adenomas. Fourteen additional cases revealed small granular cell nests. Thus the incidental finding of nonfunctioning pituitary adenomas is relatively common in adults (24% of cases in this study), and the coexistence of pituitary adenomas and granular cell tumors may suggest a possible histogenic connection between anterior and posterior pituitary tumorigenesis.     

Structure-function correlations of human pituitary gonadotroph adenomas in vitro

Two pituitary gonadotroph adenomas were studied in vitro to characterize structure-function correlations. Both tumors were from men, aged 63 and 69 years, who had elevated blood levels of follicle-stimulating hormone (FSH) and normal blood luteinizing hormone (LH) and testosterone values. The surgically resected adenomas contained diffuse immunohistochemical positivity for beta-FSH, beta-LH, and alpha-subunit of glycoprotein hormones; by electron microscopy they were composed of well-differentiated gonadotrophs. In vitro, both tumors released FSH, LH, and alpha-subunit. Morphometric studies were performed on surgically resected and cultured adenoma cells. Compared with the surgical specimens, the cultured cells had decreased cytoplasmic volume densities of endoplasmic reticulum and Golgi apparatus and slightly increased cytoplasmic volume densities of secretory granules. Incubation with gonadotropin-releasing hormone (GnRH) for 2 and 24 hours increased FSH, LH, and alpha-subunit release by both tumors; morphometry after 2 consecutive days of exposure confirmed significant increases in cytoplasmic volume densities of endoplasmic reticulum and Golgi regions and marked decreases in that of secretory granules. There was no significant change in cell size, nuclear/cytoplasmic ratio, or secretory granule diameter. The two tumors differed in their response to gonadal steroids. Estradiol, testosterone, and progesterone stimulated release of FSH, LH, and alpha-subunit by one tumor and the morphologic changes paralleled the biochemical response; addition of testosterone suppressed the secretory and morphologic response to GnRH. The other tumor showed no significant response to estradiol or testosterone and addition of these steroids did not alter the response to GnRH. The results are consistent with the interpretation that GnRH stimulates not only release but also synthesis of gonadotropins by gonadotroph adenomas of men. The data also indicate variable sensitivity of these tumors to gonadal steroids with paradoxical stimulation alone and inhibition of response to GnRH. The structural changes correlate with the hormone release response in vitro.     

Pituitary Adenomas that Show a Faint GH-Immunoreactivity but Lack Fibrous Body: Pit-1 Adenoma with Endocrinologically Low Activity

Growth hormone (GH)-producing pituitary adenomas have been classified into densely and sparsely granulated adenomas. The latter are chromophobic with weak GH-positivity and characteristically possess fibrous body (FB), aggregation of cytokeratin filaments. We report eight cases of unusual chromophobic adenomas. GH-immunoreactivity was detected in most adenoma cells in five cases and scattered in three cases. However, it appeared much weaker than that seen in ordinary GH-producing adenomas because of spotty immunoreactivity. Although intracytoplasmic organelles were well-developed, secretory granules were small and sparse. FB was not identified in any cases. Thyroid-stimulating hormone was positive in four cases. Pit-1 protein was positive in all eight cases. A weak labeling with GH probe was detected in two of two cases examined by in situ hybridization. Acromegalic features were evident in four cases, while mild or absent in four cases. GH levels were below 5 μg/l in four cases and 5–10 μg/l in the remaining cases. Macroadenomas and invasive adenomas were seen in seven and six cases, respectively.     

The Pituitary in Gigantism

To compare the pituitary pathology of gigantism to that of acromegaly, 19 surgically resected lesions were studied from 10 males and 9 females, ages 13–49 (mean, 19 yr) with excessive height (≥95th percentile), onset of disease prior to puberty, elevated growth hormone (GH) levels despite glucose suppression, and a pathologically confirmed GH-producing pituitary mass. One patient had MEN-I. The lesions included 18 adenomas and 1 case of pure hyperplasia. The median, mean, and range of serum GH and prolactin (PRL) levels were 64, 235, 5–1000 ng/mL and 47, 146, 29–770 ng/mL, respectively. Of the 8 adenoma specimens accompanied by nontumoral pituitary (i.e., tissue wherein the presence of hyperplasia was assessable), 3 (37%) demonstrated both. Of the 18 tumors, 78% were macroadenomas and 22% were grossly invasive; their immunophenotypes included GH (5%), GH and PRL (19%), and GH-PRL and a glycoprotein hormone, usually TSH and/or α-subunit (76%). Of the 10 adenoma-containing lesions subject to electron microscopy (EM), 2 consisted of GH cells alone; 2 of mammosomatotroph (MS) cells alone; 1 of GH and MS cells; 1 of GH and PRL cells; 2 of GH, PRL, and MS cells; 1 of GH, PRL, and glycoprotein cells; and 1 was a subtype 3 adenoma. Ultrastructurally, GH cells and/or MS cells predominated in these lesions. Immuno-EM of one GH and PRL cell and of one GH-PRL-MS tumor showed GH and PRL to be present not only in single cells but within the same granules. Nine of 12 adenoma-associated lesions subject to combinedin situ hybridization (ISH) and immunostaining showed double labeling for PRL (or GH) mRNA and for GH (or PRL), respectively, features indicating MS differentiation. In the 4 lesions exhibiting hyperplasia, either alone (1) or in association with adenoma (3), EM showed MS cells in 3, and immuno-EM as well as combined immunohistochemistry and ISH showed double labeling for GH and PRL in both of the 2 cases studied. In summary, although in terms of their tinctorial characteristics and tumor size, the lesions of giants resemble those of acromegalics, those of the former are less often invasive and glycoprotein hormone containing, and more often contain ultrastructurally distinctive MS cells. The high frequency of adenoma with hyperplasia (37%) and the occurrence of hyperplasia alone (6%) is of particular notice since this finding is rare in patients with acromegaly. Hyperplasia is, however, seen in ectopic GH-releasing hormone production and the McCune-Albright syndrome. We conclude that the presence of MS is not rare in the pituitary lesions of patients with gigantism. Their presence may be a reflection of either hypothalamic dysfunction or of an intrinsic abnormality of pituitary cells.     

Pituitary oncocytoma. Indispensable role of electron microscopy in its identification

Two surgically resected pituitary adenomas that appeared to be chromophobe or poorly granulated acidophil adenomas by light microscopy were correctly identified as oncocytomas by electron microscopy. The tumor cells ahd markedly hyperplastic, moderately pleomorphic mitochondria in the cytoplasm of virtually all cells. Scattered secretory granules were displaced to the periphery of the cytoplasm but the secretion type of pituitary cell remained obscure. Histochemical and immunocytochemical methods imperfectly characterized these unusual tumors. Even though these neoplasms have been considered rare, they are now being recognized more often after ultrastructural study. Eventually their pathobiologic features may be better understood. One of our patients was 24 years old and is the youngest patient, to our knowledge, in whom a pituitary oncocytoma has been documented.     

Augmentation by thyroxine of human granulosa cell gonadotrophin-induced steroidogenesis

The objective of this study was to investigate the influenceof thyroid hormone on gonadotrophin-induced oestradiol and progesteronesecretion by human granulosa cells maintained in vitro. Granulosacells were obtained by aspiration of pre-ovulatory folliclesfrom women undergoing assisted reproductive technology. Ovulationinduction was performed with gonadotrophin-releasing hormoneagonist, human menopausal gonadotrophin and human chorionicgonadotrophin. Granulosa cells were maintained in vitro in adefined medium with added insulin. Between 48 and 72 h afterthe initiation of cell culture, oestradiol and progesteronesecretion into the medium was determined for granulosa cellsgrowing in serum-free medium with follicle-stimulating hormone(FSH)/luteinizing hormone (LH) and in serum-free medium withFSH/LH and thyroxine added in a concentration range of 10^–10–10^–7M. All concentrations of thyroxine used produced a statisticallysignificant increase in oestradiol (range 1.18–1.37 timesthe amount with FSH/LH alone) and progesterone (range 1.29–1.51times the amount with FSH/LH alone) secretion.     

Analysis of mammosomatotropic cells in normal and neoplastic human pituitaries

The reverse hemolytic plaque assay (RHPA) was used to analyze hormone secretion in normal and neoplastic human pituitary cells. Immunocytochemical (ICC) staining for PRL and GH with the peroxidase method and ultrastructural ICC with colloidal gold labeling were used along with the RHPA to analyze for mammosomatotropic (MS) cells in these dissociated and cultured pituitary cells. MS cells were identified in both normal and neoplastic pituitary tissues. Quantitation of plaque areas, showed that PRL cells from normal pituitaries had significantly larger plaque areas than PRL cells from PRL-producing adenomas or from mixed PRL-GH producing adenomas.     

Hormonal secretion by adult pituitary cells in culture

Cells isolated from 24 autopsied adult human pituitary glands by enzymatic dissociation and Percoll density gradient methods were maintained in culture for up to 10 months. Using rabbit antisera specific for individual pituitary hormones and immunoperoxidase staining, we found a large number of cultured pituitary cells to react positively to growth hormone, prolactin, adrenocorticotropin, follicle-stimulating hormone, luteinizing hormone, and thyrotropin immunoreactions. Electron microscopic examination of cultured pituitary cells demonstrated the occurrence of secretory granules in cytoplasm characteristic of in vivo human pituitary cells. The hormone secretion in culture by these pituitary cells was confirmed by the detection of prolactin, thyrotropin, and follicle-stimulating hormone in the culture medium by radioimmunoassay.     

A Modular Tissue Engineering Construct Containing Smooth Muscle Cells and Endothelial Cells

Human umbilical vein endothelial cells (HUVEC) were seeded on sub-mm sized collagen cylinders containing embedded umbilical vein smooth muscle cells (UVSMC). These cylindrical “modules” are intended to be used as a vascularized construct, in which HUVEC lined channels are created by the random packing of the modules in situ or within a larger container. Embedding UVSMC cultured in medium containing 10% FBS had an adverse effect on subsequently seeded HUVEC junction morphology; HUVEC/UVSMC co-culturing was done in HUVEC medium (2% FBS with the addition of 0.03 mg/mL endothelial cell growth supplement) as compared to HUVEC seeded on collagen-only modules. In contrast, embedding UVSMC cultured in serum-free medium prior to embedding improved EC junction morphology. Such serum-free culturing, also prevented the UVSMC induced contraction of the collagen modules. On the other hand, embedding serum-free cultured UVSMC promoted HUVEC proliferation and NO secretion compared to those modules embedded with 10% serum cultured UVSMC. These results suggest, not surprisingly, that embedded UVSMC phenotype plays an important role in the seeded HUVEC phenotype, and that the response can be modulated by the UVSMC culture medium serum concentration. These studies were undertaken with a view to using the UVSMC to modulate the thrombogenicity of the HUVEC. Exploration of this outcome awaits further studies directed to understanding the mechanism of the cellular interactions.     

Detection of gonadotropin-releasing hormone receptor in normal human pituitary cells and pituitary adenomas using immunohistochemistry

Gonadotropin-releasing hormone (GnRH), which is a well-known regulator of gonadotroph function, has recently been considered to be a paracrine factor involved in the control of somatotroph, lactotroph, and corticotroph cells. GnRH action is initiated by binding to a specific cell surface receptor, the gonadotropin-releasing hormone receptor (GnRHR), which is expressed by follicle-stimulating hormone/luteinizing hormone (FSH/LH) cells. Using in situ hybridization techniques, GnRHR messenger ribonucleic acid (mRNA) has recently been detected in normal human anterior pituitary gland and in various pituitary adenomas, including FSH/LH-cell, growth hormone (GH)-cell, adrenocorticotropic hormone (ACTH)-cell, and null-cell adenomas. However, immunohistochemical studies indicating the specific cell distribution of GnRHR in normal pituitary cells have never been reported. The aim of the present investigation was to evaluate the immunohistochemical expression of GnRHR in different types of normal pituitary cells and related tumors. Using double-label immunohistochemical techniques on formalin-fixed and paraffin-embedded tissues and specific antibodies directed against pituitary hormones and GnRHR, we found GnRHR immunoreactivity not only in FSH/LH cells, but also in GH- and thyroid-stimulating hormone (TSH) cells. GnRHR was detected in FSH/LH-cell, GH-cell, mixed GH- and prolactin (PRL)-cell, and α-subunit (α-SU)/null-cell adenomas. The findings of this study suggest that the interaction between GnRH and GnRHR may play a role in paracrine/autocrine regulation of different types of normal pituitary cells and pituitary adenomas. Received: 24 January 2000 / Accepted: 12 April 2000     

A human pituitary adenoma cell line proliferates and maintains some differentiated functions following expression of SV40 large T-antigen

Human pituitary cells proliferate very slowly in vitro. Only a few cell lines have been established, and these have been used mainly for short-term studies. To obtain immortalized cell lines of human pituitary adenomas for in vitro studies, we infected adenoma cells with a replication-defective recombinant human adenovirus, which contains an SV40 early large T-antigen. One of the cell lines (HP75), which has been studied in culture during 60 passages, has been extensively characterized. It expressed the large T-antigen protein and its mRNA, as well as the genes for FSH-β, LH-β and α-subunit (α-SU) of gonadotropin hormone. The HP75 cell line also expressed the genes of various members of the chromogranin (Cg)/secretogranin (Sg) family, including CgA as well as the prohormone convertases PC1/3 and PC2. CgA was processed to pancreastatin in vitro, which was secreted into the culture medium. Treatment with phorbol 12-myristate 13-acetate (PMA), TGF-β1, and forskolin increased CgA expression in the cells and stimulated pancreastatin secretion into the medium while inhibiting cell growth. The HP75 cell line also expressed TGF-β mRNA isoforms (β1, β2, β3) and the mRNAs for the receptors for TGF-β (RI, RII, and RIII). The cells responded to TGF-β1 in vitro by increasing CgA protein expression and pancreastatin secretion. TGF-β-RII protein and mRNA expression were both increased by PMA. Ultrastructural studies showed that the HP75 cells had very few dense-core secretory granules and a poorly developed Golgi complex. After treatment with TGF-β1 and PMA, there was an increase in the development of rough endoplasmic reticulum and the Golgi complex. This is the first report of the development of an immortalized human pituitary cell line that retains some differentiated functions. HP75 can be used to study TGF-β and CgA functions in pituitary cells. Replication-defective recombinant human adenovirus with an SV40 large T-antigen insert can be used to generate other immortalized human pituitary cell lines for in vitro studies.     

Folliculostellate cells in pituitary adenomas: Studies of hormonal profile and tumor vascularity

The hormonal immunoreactivity and vascularity of pituitary adenomas containing folliculostellate (FS) cells have been compared with those of tumors in which such cells were not identified. FS cells were present in variable numbers in 36 of 92 tumors. Adenomas immunoreactive for growth hormone (GH), adrenocorticotropic hormone (ACTH), or prolactin (PRL) contained FS cells in 40–50% of cases. Those immunoreactive for glycoprotein hormones and alphasubunit contained FS cells in 67–85% of cases, a statistically significant correlation. When alpha-subunit was also present in GH-, GH/PRL-, and ACTH-immunoreactive tumors, a higher proportion contained FS cells (57–91%). These data suggest a correlation between the presence of FS cells and glycoprotein immunoreactivity in pituitary adenomas. Vascular channels identified by the binding of the lectinUlex europaeus were quantified in the two types of tumors. Those containing FS cells were not more vascular than those without FS cells, which suggests that FS cells do not play a significant role in the regulation of intratumoraf vascularization in human pituitary adenomas.     

Recognition of gonadotroph adenomas in women

BACKGROUND. Pituitary adenomas that arise from the gonadotroph cells are being recognized with increasing frequency in men, but they are still rarely recognized in women. This rarity could be the result of an actual difference in occurrence or of greater difficulty in recognition. The tumors are usually recognized in men more than 50 years old, but elevated serum gonadotropin levels in women of that age could be produced by normal gonadotroph cells. METHODS. Because the stimulation of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and the beta subunit of LH (LH beta) by thyrotropin-releasing hormone (TRH) is a characteristic of gonadotroph adenomas in men, we administered TRH to 16 women with apparently nonsecreting pituitary macroadenomas and measured serum FSH, LH, LH beta, and the glycoprotein hormone alpha subunit every 15 minutes for 90 minutes before and 90 minutes after. The results were compared with the responses in 16 healthy women matched for age and in 10 women with macroadenomas secreting prolactin, growth hormone, or corticotropin. The tumors from 12 of the women with nonsecreting adenomas were cultured, and the secretion of FSH, LH, and LH beta in culture was determined. RESULTS. Eleven of the 16 women with apparently nonsecreting adenomas had significant increases in serum LH beta in response to TRH, 3 had FSH responses, and 4 had LH responses. None of the 16 healthy women and none of the 10 women with secreting macroadenomas had LH beta, FSH, or LH responses to TRH. Ten of the 12 adenomas that were cultured secreted readily detectable amounts of FSH, LH, and LH beta, and their secretion in vitro correlated with the patients' responses to TRH in vivo. CONCLUSIONS. Most apparently nonsecreting pituitary macroadenomas in women arise from gonadotroph cells. The majority of these can be recognized, even in postmenopausal women, by the serum LH beta responses to TRH, and some can be recognized by the responses of serum FSH and LH.     

Downregulation of E-cadherin and its undercoat proteins in pituitary growth hormone cell adenomas with prominent fibrous bodies

The cadherin-catenin complex regulates cellular adhesion and motility, and genetic alterations in these molecules play a critical role in multistage tumorigenesis. In this study, the expression of three major type I classic cadherins—E-, N-, and P-cadherin—and their undercoat proteins—α-, β-, and γ-catenin, and pp120–was investigated in 127 pituitary adenomas and 10 normal adenohypophyseal glands using an immunohistochemical technique with highly specific monoclonal antibodies. In normal pituitary glands, E-cadherin, catenins, and pp 120 were strongly expressed on almost all hormone-producing cell-cell boundaries, N-cadherin was weakly immunoreactive on a few cell-cell boundaries, and P-cadherin was negative. In pituitary adenomas, a correlation was not identified among expression of E-cadherin, catenins, or pp120 with patient age, sex, hormone level, tumor size, and/or invasiveness, respectively. Expression of E-cadherin, catenins, and pp 120 was significantly reduced in 24 growth hormone (GH) cell adenomas with prominent fibrous bodies compared with the other subtypes of pituitary adenomas and normal pituitary glands (p<0.0001, respectively). Methylation-specific polymerase chain reaction analysis revealed that the E-cadherin gene promoter region was methylated in 6 of 16 (37.5%) GH cell adenomas with prominent fibrous bodies examined, 2 of which displayed total methylation, but not in 10 GH cell adenomas without fibrous bodies. No mutation of exon 3 of the β-catenin gene was found in 16 GH cell adenomas with prominent fibrous bodies or in 10 other subtypes of pituitary adenomas that showed unremarkable intracellular presence of β-catenin protein. In conclusion, the decreased expression of the E-cadherin-catenin complex and methylation of the E-cadherin gene promoter region only in GH cell adenomas with prominent fibrous bodies may be an event associated with the formation of fibrous bodies.