The effects of estrogens on tumor growth and on prolactin and growth hormone mRNA expression in rat pituitary tissues.

Estrogens inhibit tumor growth and modify PRL and GH expression in the MtT/W15 transplantable rat pituitary tumor. The effects of estradiol (E2) and diethylstilbestrol (DES) on PRL and GH mRNA levels were investigated. Estrogens increased GH mRNA levels and decreased PRL mRNA levels as detected by in situ hybridization and Northern blot hybridization with oligonucleotide probes, while inhibiting tumor growth. Similar changes in immunoreactive GH and PRL were seen in the tumor cells. The pituitary glands of tumor-bearing rats treated with estrogen for 3 weeks were increased in weight with a concurrent increase in pituitary PRL mRNA when analyzed by dot blot hybridization. These results indicate that estrogens have an inhibitory effect on the growth of the MtT/W15 tumor and increase GH protein and mRNA levels, while causing PRL protein and mRNA levels to decrease. The pituitaries of tumor-bearing rats concomitantly undergo PRL cell hyperplasia with an increase in PRL mRNA. These results also demonstrate a paradoxical effect of estrogens on different pituitary tissues.     

Hybridization studies of cultured human pituitary prl and gh producing adenoma cells: Effects of thyrotropin-releasing hormone,somatostatin, and phorbol ester

The effects of the hypothalamic hormones, thyrotropin-releasing hormone (TRH), and somatostatin (SRIH), and of phorbol 12-myristate 13-acetate (PMA) on PRL and GH secretion and messenger RNA (mRNA) levels were analyzed in 10 GH and/or PRL producing adenomas after culturing the tumor cells in the presence of these secretagogues for 7 days. The expression of chromogranin A and B mRNAs was also examined. All four of the clinically diagnosed GH adenomas expressed or secreted both GH and PRL while four of six clinically diagnosed prolactinomas produced or secreted both PRL and GH. Prolactinomas had less than 10% of tumor cells expressing chromogranin A mRNA while more than 40% of the adenoma cells expressed chromogranin B mRNA. TRH stimulated PRL secretion and increased PRL mRNA levels while SRIH decreased GH secretion and mRNA expression in some cases. Unexpectedly, PMA stimulated PRL mRNA levels four- to sevenfold above control levels in two adenomas and generally stimulated chromogranin A and B mRNA expression but not GH mRNA, as determined by Northern hybridization and in situ hybridization analyses. These results indicate that cultured prolactinoma cells express significantly more chromogranin B mRNA than chromogranin A mRNA, and that PMA increases PRL mRNA expression in some prolactinomas, although the effect of PMA on various adenomas reflects the heterogeneity of these tumors with respect to protein kinase C stimulation.     

Coexpression of galanin and adrenocorticotropic hormone in human pituitary and pituitary adenomas.

Galanin is a neuropeptide that regulates the secretion of several pituitary hormones, including prolactin (PRL) and growth hormone (GH). Galaninlike immunoreactivity (Gal-IR) and galanin mRNA in the rat anterior pituitary is cell lineage specific, with predominant expression in lactotrophs and somatotrophs. The authors examined the cellular distribution of human Gal-IR in seven normal postmortem pituitaries and 62 pituitary tumors by immunoperoxidase staining. In contrast to the rat, Gal-IR in human anterior pituitaries was present in corticotrophs scattered throughout the gland, but not in lactotrophs, somatotrophs, thyrotrophs, or gonadotrophs. Distinct Gal-IR also was present in hyperplastic and neoplastic corticotrophs in 19 of 22 patients with Cushing's disease. In noncorticotroph cell tumors, unequivocal Gal-IR was present in 5 of 11 GH-secreting tumors associated with clinical acromegaly, 9 of 18 nonfunctioning pituitary adenomas, and 2 of 14 prolactinomas. Of these galanin-positive tumors, four of the five GH-secreting adenomas, six of the nine nonfunctioning adenomas, and both of the prolactinomas also contained adrenocorticotropic hormone immunoreactivity (ACTH-IR). Immunostaining and in situ hybridization on adjacent sections using an 35S-labeled probe complementary to human galanin mRNA demonstrated predominant galanin expression in normal corticotrophs. Immunoelectron microscopy confirmed the presence of Gal-IR in pituitary cells characteristic of corticotrophs in both normal and neoplastic pituitaries. Thus, as in the rat, galanin gene expression in the human pituitary is cell-type specific. Unlike the rat, however, human galanin gene expression is restricted to the corticotroph lineage. Studies of tumors confirmed the observed coexpression of galanin and adrenocorticotropic hormone. The divergent cell type specificity of galanin production in human and rat pituitaries reflects different patterns of gene activation in these two species. In addition, these results suggest that galanin in the human pituitary may participate locally in the regulation of the hypothalamic-pituitary-adrenal axis.     

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.     

Effects of propylthiouracil on growth hormone and prolactin messenger ribonucleic acids in the rat pituitary

The effects of hypothyroidism induced by propylthiouracil (PTU) treatment on growth hormone (GH) and prolactin (PRL) messenger ribonucleic acid (mRNA) levels were analyzed in adult female rat adenohypophyses by in situ hybridization histochemistry and Northern hybridization analyses. Twenty-eight days of PTU treatment produced a significant decrease in GH mRNA levels and a smaller decrease in PRL mRNA determined by both in situ hybridization histochemistry and Northern hybridization analyses. A combined procedure of in situ hybridization histochemistry followed by immunochemistry on the same sections revealed mammosomatotropic cells expressing GH mRNA and PRL protein in the same pituitary cells from all treatment groups. Cells expressing GH mRNA and thyroid-stimulating hormone protein were not detected by this method. Immunochemical staining revealed a decrease in GH cells and an increase in thyroid-stimulating hormone cells in hypothyroid rats. Cells expressing both GH and thyroid-stimulating hormone protein were not detected by immunostaining. These results indicate that hypothyroidism produces significant decreases in GH mRNA and also decreases PRL mRNA and that mammosomatotropic cells can be detected in pituitaries from normal and hypothyroid rats.     

Detection of prolactin messenger RNA in rat anterior pituitary by in situ hybridization.

The effects of chronic diethylstilbestrol treatment on rat prolactin mRNA was analyzed by in situ hybridization histochemistry. Forty-day-old female rats were treated with 10 mg diethylstilbestrol in Silastic tubes for 3, 6, and 9 weeks. Estrogen treatment for 9 weeks increased pituitary wet weight (51.6 +/- 2.4 versus 7.9 +/- 0.31 mg for controls), serum prolactin (4155 +/- 571 versus 47.1 +/- 8.9 ng/ml for controls), and the percentage of immunoreactive prolactin cells (69% +/- 3% versus 34% +/- 2% for controls). In situ hybridization studies showed an increase in rat prolactin mRNA with increasing duration of estrogen treatment. After 9 weeks of estrogen treatment, there was a 2.3-fold increase in rat prolactin mRNA. 3H-cDNA was distributed diffusely throughout the anterior pituitary in both normal and hyperplastic pituitaries. There were no separate foci of adenomatous pituitary with increased labeling or with increased immunoreactive PRL cells. Although transplantable pituitary MtT/W15 tumors secreted very large amounts of PRL, compared with pituitaries from DES-treated rats, rat prolactin mRNA as evaluated by mean grain counts was considerably less in the MtT/W15 tumor than in DES-treated pituitary cells. These results show that in situ hybridization histochemistry can be used to detect changes in rat prolactin mRNA in tissue sections from the anterior pituitary with chronic estrogen treatment and that these pituitaries show a diffuse increase in immunoreactive prolactin cells and cellular prolactin mRNA, rather than distinct adenomatous areas within the glands.     

Pituitary Adenoma in Carney Complex: An Immunohistochemical,Ultrastructural, and Immunoelectron Microscopic Study

First described in 1985, Carney complex is a rare, heritable disorder featuring abnormal skin pigmentation, cardiac and cutaneous myxoma, melanotic schwannoma of psammomatous type, and endocrine abnormalities, including pituitary adenomas. Patients with the latter present with elevated growth hormone (GH) levels and acromegaly or gigantism. Prolactin (PRL) elevation may also be seen. The authors have investigated 2 resected pituitary adenomas from patients with Carney complex. One, a 19-year-old female acromegalic with elevated GH, IgF-1, and PRL levels, had a mammosomatotroph adenoma immunoreactive for GH and PRL. Ultrastructurally, GH and PRL were present in the same secretory granules. The second patient, a 27-year-old acromegalic, had a sparsely granulated GH cell adenoma that by immuno-electron microscopy revealed GH immunoreactivity only. The lack of morphologic similarity between the 2 adenomas indicates that pituitary tumors in patients with Carney complex may not exhibit the same phenotype.     

Pituitary adenoma in Carney complex: an immunohistochemical,ultrastructural, and immunoelectron microscopic study

First described in 1985, Carney complex is a rare, heritable disorder featuring abnormal skin pigmentation, cardiac and cutaneous myxoma, melanotic schwannoma of psammomatous type, and endocrine abnormalities, including pituitary adenomas. Patients with the latter present with elevated growth hormone (GH) levels and acromegaly or gigantism. Prolactin (PRL) elevation may also be seen. The authors have investigated 2 resected pituitary adenomas from patients with Carney complex. One, a 19-year-old female acromegalic with elevated GH, IgF-1, and PRL levels, had a mammosomatotroph adenoma immunoreactive for GH and PRL. Ultrastructurally, GH and PRL were present in the same secretory granules. The second patient, a 27-year-old acromegalic, had a sparsely granulated GH cell adenoma that by immuno-electron microscopy revealed GH immunoreactivity only. The lack of morphologic similarity between the 2 adenomas indicates that pituitary tumors in patients with Carney complex may not exhibit the same phenotype.     

An Atypical Acidophil Cell Line Tumor Showing Focal Differentiation Toward Both Growth Hormone and Prolactin Cells

We report a case of giant pituitary adenoma in a child. Computerized tomography (CT) scan revealed a suprasellar extension tumor mass with hydrocephalus. There was no clinical evidence of acromegaly, gigantism, and other hormonal symptoms. Endocrinologic studies showed within normal value of serum growth hormone (GH: 4.2 ng/mL) and slightly increased levels of prolactin (PRL: 78 ng/mL) and other pituitary hormone values were within normal range. On suppression test by bromocryptin, both GH and PRL levels were reduced. Histopathological findings revealed that the tumor consisted of predominantly chromophobic and partly eosinophilic adenoma cells. Immunohistochemical staining detected GH and PRL in a small number of distinctly different adenoma cells, respectively. Nonradioactivein situ hybridization (ISH) also showed GH and PRL mRNA expression in identical immunopositive cells. Electron microscopy (EM) demonstrated adenoma cells with moderate or small numbers of two types of dense granules and without fibrous body which are characteristic of sparsely granulated GH-cell adenomas. The adenoma does not fit into any classification but may be an atypical acidophil cell line tumor showing focal differentiation toward both GH and PRL cells.     

Alpha-subunit immunoreactivity in plurihormonal pituitary adenomas of patients with acromegaly.

Twelve plurihormonal pituitary adenomas, removed surgically from 11 of 20 patients with acromegaly, were investigated. The mean age of the 11 patients was 45 yr. Seven patients with eight tumors were men. The tumors were immunostained for all known adenohypophysial hormones by the avidin-biotin-peroxidase complex (ABC) technique. All 12 adenomas were immunoreactive for growth hormone (GH) and alpha-subunit; prolactin (PRL) was detected in five tumors. Stains for the beta-subunits of glycoprotein hormones [thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH)] revealed immunopositivity for specific beta-subunits in ten adenomas. The close association between GH and alpha-subunit in pituitary tumors of acromegalic patients remains to be explained.     

HGH,PRL, and ACTH Gene Expression in Clinically Nonfunctioning Adenomas Detected with Nonisotopic In Situ Hybridization Method

In situ hybridization (ISH), which can manifest the specific gene expression of anterior pituitary hormones (mRNA), as well as immunohistochemistry (IHC), is needed to clarify the endocrine function of pituitary adenomas. With the aid of nonisotopic ISH, which has several advantages over isotopic ISH, we examined the expression of pituitary hormone mRNAs in 14 clinically nonfunctioning adenomas, which were considered to be a subtype of gonadotroph adenomas. Gene expression of growth hormone (GH; 4/14), prolactin (PRL; 5/14), adrenocorticotroph hormone (ACTH; 4/14), and gonadotropin were detected with our nonisotopic ISH studies. It is suggested from our ISH studies that some clinically nonfunctioning adenomas are composed of hormone (or subunit) producing cells and may be derived from plurihormonal primordial stem cells.     

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.     

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. An immunohistochemical study of hormone production and chromogranin localization.

Tumors from 42 surgically resected pituitaries and from 13 autopsy cases were studied immunohistochemically with polyclonal antisera to 7 anterior pituitary hormones and with a newly developed monoclonal antibody directed against human chromogranin for evaluation of the distribution of chromogranin in normal and neoplastic pituitaries. In addition, a prospective study was done for assessment of the prevalence, morphology, and endocrine cell types of pituitary tumors in 100 autopsy subjects. When these 55 pituitary adenomas were examined with monoclonal antibody (LK2H10) directed against human chromogranin, selective staining of normal adenohypophyseal cell types and pituitary tumors was observed. Most null-cell adenomas (12/14) were positive for chromogranin, whereas all prolactin (PRL)-producing adenomas (19/19) were negative. Growth hormone (GH) adenomas were focally positive (9/9). All oncocytomas (2/2), 1 thyrotropin (TSH) adenoma, and a follicle-stimulating hormone/luteinizing hormone adenoma were positive for chromogranin. One or more adenomas were present in 14% of the autopsy cases. The tumors occurred most frequently in patients in the fifth through the seventh decades of life. Immunohistochemical staining of 13 adenomas revealed 1 TSH, 1 ACTH, and 4 PRL-producing tumors, whereas 7 other tumors, which were null-cell or undifferentiated adenomas, failed to stain for any of the seven principle pituitary hormones. These results indicate that antibody LK2H10 to human chromogranin is useful in the immunohistochemical characterization of pituitary adenomas. Incidental pituitary microadenomas from autopsy-derived pituitaries most commonly produce PRL, or they belong to the null-cell or undifferentiated tumor group.     

Pituitary Hormone mRNA in Null Cell Adenomas and Oncocytomas by In Situ Hybridization Comparison with Immunohistochemical and Clinical Data

Null cell adenomas and oncocytomas are clinically inactive adenomas of the pituitary gland. They do not show any significant hormone content detectable by immunohistochemistry. This study aimed at demonstrating mRNAs for all main pituitary hormones in 32 null cell adenomas and 31 oncocytomas by non-isotopic in situ hybridization using digoxigenin-labeled oligonucleotide probes. The results were compared with immunohistochemical and clinical data. Immunohistochemistry (ABC method) was done with monoclonal antibodies against PRL, GH, FSH, LH, TSH, ACTH, alpha-subunit, and Ki-67 (mib-1). The signals for hormone production were detected in both adenoma types in a range from 42% for GH in oncocytomas to 78% for beta-FSH in null cell adenomas. However, these signals are apparently not effective on hormone production, as was shown by almost negative immunostaining. Owing to the simultaneous detection of at least two mRNAs in 78% of null cell adenomas and in 94% of oncocytomas, we assume that both tumor types originate from pluripotential precursor cells that are capable of producing various hormones. According to our data, it is unlikely that the signals influence the clinical behavior.     

Morphologically unclassified GH-producing adenoma showing galactorrhea without acromegaly

A 24-year-old woman with a large pituitary adenoma had amenorrhea and galactorrhea, but no physical stigmata of acromegaly despite slightly elevated serum growth hormone (GH) and normal serum prolactin (PRL) levels. Subtotal removal of the tumor cured galactorrhea and resulted in normalization of serum GH concentration. The question is raised whether amenorrhea and galactorrhea were related to excessive GH production in this patient. Absence of acromegaly might have been due to the short duration of the disease. The tumor was a chromophobic, periodic acid-Schiff-negative adenoma. Immunocytochemistry and in situ hybridization revealed focal GH immunoreactivity and diffuse, weak signal for GH messenger RNA. By electron microscopy, the tumor showed no features of GH or PRL-producing adenomas. Two different cell types could be distinguished: the majority were similar to null cells, whereas a small number of cells resembled somatotrophs and lactotrophs, possessing many secretory granules and exhibiting exocytosis. On the basis of its ultrastructure, this tumor can be classified as an atypical acidophil cell line adenoma in which adenomatous null cells transformed to the differentiated cells capable of producing GH.