Analysis of prolactin and growth hormone production in hyperplastic and neoplastic rat pituitary tissues by the hemolytic plaque assay

The reverse hemolytic plaque assay (RHPA) was used to detect hormone release from cultured normal, hyperplastic, and neoplastic rat pituitary cells. Hyperplastic pituitary cells were produced by s.c. diethylstilbestrol (DES) treatment (10 mg in Silastic tubes) for 3, 6, and 9 weeks. Neoplastic pituitary cells from rats with MtT/W15 transplantable tumors treated with DES for 3 weeks were also analyzed. Aliquots of the same cells were also analyzed by immunocytochemical staining. DES treatment resulted in an increase in prolactin (PRL)-producing cells in hyperplastic pituitaries compared to untreated pituitaries after 9 weeks of treatment by the RHPA [61.2 +/- 5.2 (SE) versus 32 +/- 3.0] and by immunocytochemical staining [70.9 +/- 2.4 versus 36 +/- 1.4]. The percentage of mammosomatotropic cells decreased from 11.3 +/- 3.8 to 4.2 +/- 2.6% in pituitary cells from these same groups of animals. After 3 weeks of DES treatment in rats with MtT/W15 tumor, there was an increase in growth hormone (GH)-producing cells and a decrease in PRL-producing cells when analyzed by the RHPA (control: percentage of GH, 36.3 +/- 6.2; percentage of PRL, 39.0 +/- 1.6 versus DES-treated tumors: percentage of GH of 68.2 +/- 1.9; and percentage of PRL, 3.2 +/- 1.8%). The percentage of mammosomatotropic cells declined from 12.4 +/- 2.3 to 0.77 +/- 2.4%. A combined procedure of RHPA followed by immunocytochemical staining on the same slides also revealed a decline in mammosomatotropic cells after chronic DES treatment in hyperplastic and neoplastic MtT/W15 tumor cells. These results show that DES has different effects on PRL and GH secretion and storage in hyperplastic pituitary and in the MtT/W15 pituitary tumor cells.     

Estrogen receptor levels and tumor growth in a series of pituitary clonal cell lines in rats.

Four kinds of in vitro clonal pituitary tumor cell lines named MtT/Se, MtT/SM, MtT/S and MtT/E, each of which shows different sensitivity to estrogen on proliferation, were inoculated into fat pad of ovariectomized rats and estrogen-loaded ovariectomized rats at 10(5) and 10(6) cells/site. They formed tumor with average latency ranging from 30 to 71 in ovariectomized rats and 13 to 63 days in estrogenized rats inoculated with 10(6) cells. MtT/Se was highly sensitive to estrogen for growth, and MtT/SM also grew well in estrogenized rats. With MtT/S and MtT/E, there was no significant shortening of average tumor latency in estrogenized rats. In vivo, the cytosolic estrogen receptor (ER) levels of MtT/Se, SM, S and E were measured to be 452 +/- 66, 370 +/- 115, 260 +/- 16 and 83 +/- 8 fmol/mg protein, respectively. In vitro, however, the lowest ER level was noted in MtT/Se. Histologically, all four tumors grown in rats were composed of homogeneous round cells, and MtT/Se contained particularly large nucleated cells. In MtT/E, the cells appeared to be changing into fibromatous cells. Three cell lines except MtT/E maintained the function of hormonal secretion in vivo as well as in vitro. Serum GH level was increased in rats with MtT/Se and MtT/S. Increased levels of both prolactin and growth hormone were measured in sera of rats with MtT/SM. Increases of hormones as well as tumor sizes were promoted by the estrogen.     

Loss of 15-hydroxyprostaglandin dehydrogenase indicates a tumor suppressor role in pituitary adenomas

15-hydroxyprostaglandin dehydrogenase (15-PGDH) may function as a tumor suppressor that antagonizes the action of the cyclooxygenase-2 (COX-2) oncogene in several types of tumors. However, it is unknown if it has a role in the pituitary. Recently, our group found that 15-PGDH expression was low in prolactin (PRL) secreting adenomas (prolactinomas) and growth hormone (GH) secreting adenomas (GHomas) using fiber-optic BeadArray technology. In this study, we examined the relative expression of 15-PGDH and COX-2 mRNA in clinical specimens and examined the effects of 15-PGDH on GH3 rat pituitary tumor cell proliferation, apoptosis and hormone secretion. 15-PGDH expression was lower and COX-2 expression was higher in prolactinomas and GHomas compared with normal controls. Overexpressed 15-PGDH inhibited tumor cell proliferation and induced apoptosis. It had a significant suppressive effect on mRNA levels and on the secretion of PRL and GH in GH3 cells. The inhibition of cell proliferation was accompanied by the decreased expression of cox-2, matrix metalloproteinase-9 (MMP-9) and B cell leukemia/lymphoma-2 (Bcl-2). These data are suggestive of a previously unrecognized pathway in pituitary tumorigenesis, and this novel observation may shed light on therapeutic strategies for pituitary tumors.     

Stimulatory effects of retinoic acid on tumor growth and serum insulin-like growth factor-1 in rats bearing estrogen-responsive pituitary tumor MtT/Se

MtT/Se is one of 4 cell lines derived from an estrogen-dependent pituitary tumor, MtT/F84. The main difference between these tumor types is that MtT/F84 secretes both growth hormone (GH) and prolactin (PRL) whereas MtT/Se secretes only GH. MtT/Se grew slowly in ovariectomized (ovex) rats, but tumor growth was much faster in estrogen-treated ovex rats. Effects of dietary retinoic acid (RA) on tumor growth, serum GH and insulin-like growth factor-1 (IGF-1) levels were examined in ovex rats. Latency of tumor growth was shortened, and tumor take and weight were promoted by all-trans RA both in the presence and absence of exogenous estrogen. Serum GH and IGF-1 levels became increased in tumor-bearing rats whereas PRL levels remained unchanged. Serum IGF-1 levels exhibited a good correlation with tumor weights (r = 0.84). Our results suggest a close relationship between increase of tumor weight and stimulation of serum IGF-1 level by RA in tumor-bearing rats.     

Stimulatory Effects of Retinoic Acid on Tumor Growth and Serum Insulin-like Growth Factor-1 in Rats Bearing Estrogen-responsive Pituitary Tumor MtT/Se

MtT/Se is one of 4 cell lines derived from an estrogen-dependent pituitary tumor, MtT/F84. The main difference between these tumor types is that MtT/F84 secretes both growth hormone (GH) and prolactin (PRL) whereas MtT/Se secretes only GH. MtT/Se grew slowly in ovariectomized (ovex) rats, but tumor growth was much faster in estrogen-treated ovex rats. Effects of dietary retinoic acid (RA) on tumor growth, serum GH and insulin-like growth factor-1 (IGF-1) levels were examined in ovex rats. Latency of tumor growth was shortened, and tumor take and weight were promoted by all- trans RA both in the presence and absence of exogenous estrogen. Serum GH and IGF-1 levels became increased in tumor-hearing rats whereas PRL levels remained unchanged. Serum IGF-1 levels exhibited a good correlation with tumor weights ( r =0.84). Our results suggest a close relationship between increase of tumor weight and stimulation of serum IGF-1 level by RA in tumor-bearing rats.     

Rat prolactinoma cell growth regulation by epidermal growth factor receptor ligands

Epidermal growth factor (EGF) regulates pituitary development, hormone synthesis, and cell proliferation. Although ErbB receptor family members are expressed in pituitary tumors, the effects of EGF signaling on pituitary tumors are not known. Immunoprecipitation and Western blot confirmed EGF receptor (EGFR) and p185(c-neu) protein expression in GH3 lacto-somatotroph but not in adrenocorticotropic hormone-secreting AtT20 pituitary tumor cells. EGF (5 nmol/L) selectively enhanced baseline ( approximately 4-fold) and serum-induced (>6-fold) prolactin (PRL) mRNA levels, whereas gefitinib, an EGFR antagonist, suppressed serum-induced cell proliferation and Pttg1 expression, blocked PRL gene expression, and reversed EGF-mediated somatotroph-lactotroph phenotype switching. Downstream EGFR signaling by ERK, but not phosphoinositide-3-kinase or protein kinase C, mediated the gefitinib response. Tumors in athymic mice implanted s.c. with GH3 cells resulted in weight gain accompanied by increased serum PRL, growth hormone, and insulin growth factor 1. Gefitinib decreased tumor volumes and peripheral hormone levels by approximately 30% and restored normal mouse body weight patterns. Mice treated with gefitinib exhibited decreased tumor tissue ERK1/2 phosphorylation and down-regulated tumor PRL and Pttg1 mRNA abundance. These results show that EGFR inhibition controls tumor growth and PRL secretion in experimental lacto-somatotroph tumors. EGFR inhibitors could therefore be useful for the control of PRL secretion and tumor load in prolactinomas resistant to dopaminergic treatment, or for those prolactinomas undergoing rare malignant transformation.     

Estrogen Receptor Levels and Tumor Growth in a Series of Pituitary Clonal Cell Lines in Rats

Four kinds of in vitro clonal pituitary tumor cell lines named MtT/Se, MtT/SM, MtT/S and MtT/ E, each of which shows different sensitivity to estrogen on proliferation, were inoculated into fat pad of ovariectomized rats and estrogen-loaded ovariectomized rats at 10⁵ and 10⁶ cells/site. They formed tumor with average latency ranging from 30 to 71 in ovariectomized rats and 13 to 63 days in estrogenized rats inoculated with 106 cells. MtT/Se was highly sensitive to estrogen for growth, and MtT/SM also grew well in estrogenized rats. With MtT/S and MtT/E, there was no significant shortening of average tumor latency in estrogenized rats. In vivo , the cytosolic estrogen receptor (ER) levels of MtT/Se, SM, S and E were measured to be 452 ±66, 370 ±115, 260 ±16 and 83 ±8 fmol/ mg protein, respectively. In vitro , however, the lowest ER level was noted in MtT/Se. Histologically, all four tumors grown in rats were composed of homogeneous round cells, and MtT/Se contained particularly large nucleated cells. In MtT/E, the cells appeared to be changing into ftbromatous cells. Three cell lines except MtT/E maintained the function of hormonal secretion in vivo as well as in vitro . Serum GH level was increased in rats with MtT/Se and MtT/S. Increased levels of both prolactin and growth hormone were measured in sera of rats with MtT/SM. Increases of hormones as well as tumor sizes were promoted by the estrogen.     

Differential expression of galectin-3 in pituitary tumors

Galectin-3 (Gal-3), a beta-galactoside-binding protein, has been implicated in a variety of biological functions, including cell proliferation and differentiation, tumor cell adhesion, angiogenesis, apoptosis, tumor progression, and metastasis. We investigated the role of Gal-3 in the development and progression of pituitary tumors. Immunohistochemical and Western blot analysis of normal and neoplastic human pituitaries showed that only lactotroph (PRL) and corticotroph (ACTH) hormone-producing cells and tumors expressed Gal-3. Gal-3 was present in 24 of 38 (63.2%) PRL adenomas, 5 of 6 (83.3%) PRL carcinomas, 19 of 41 (46.3) ACTH adenomas, and 7 of 8 (87.5%) ACTH carcinomas, but not in 112 other pituitary adenomas and carcinomas. Pituitary folliculo-stellate cells, which have macrophage-type functions in the anterior pituitary, also expressed Gal-3. Hyperplastic and neoplastic pituitaries from p27(Kip1) (p27)-null mice, which produce mainly ACTH, showed increased Gal-3 expression levels compared with control mice. Treatment with transforming growth factor beta1, which regulates pituitary cell proliferation, reduced Gal-3 as well as p27 expression levels in cultured HP75 pituitary cells and Gal-3 in cultured pituitary cells from p27-null mice, suggesting that p27 is not necessary for the inhibitory effects of transforming growth factor beta1 on the cell cycle in the pituitary. The role of Gal-3 in pituitary cell function was examined by RNA interference experiments. Inhibition of Gal-3 gene expression by RNA interference decreased HP75 cell proliferation and increased apoptosis. These results indicate that Gal-3 has an important role in pituitary cell proliferation and tumor progression.     

Functional and morphological characteristics of transplantable rat pituitary tumors established in nude mice

Two strains of transplantable rat pituitary tumors, MtT SA5 and MtT SA6, have been established in female nude mice from a single original pituitary tumor which had spontaneously occurred in a female Wistar rat at 759 days of age. MtT SA5 tumor produces prolactin (PRL), growth hormone, and adrenocorticotropic hormone, and MtT SA6 tumor secretes PRL and growth hormone. Additionally, both tumors induce severe nephropathy and promote pathogenicity of murine hepatitis virus, resulting in hepatic necrosis. Electron micrographs of MtT SA5 and MtT SA6 tumors revealed three and two types of cells, respectively, in reference to secretory granules. The tumors seem to consist of mixed population, each cell secreting each hormone. Since marked adrenal enlargement and relatively low serum corticosterone levels were found in MtT SA5-bearing rats, it is suggested that MtT SA5 tumor secretes adrenocorticotropic hormone and/or its related peptides which induce adrenal hyperplasia with little or no stimulation of corticoid production. In nude mice bearing MtT SA5 tumor, concentrations of growth hormone in serum and tumor tissue were exceedingly higher than those of PRL, while they were in the same magnitude in MtT SA6-bearing nude mice. We also found that PRL levels in serum and tumor tissue of MtT SA5-bearing nude mice were much higher in males than females, although those of MtT SA5-bearing rats were not significantly different in both sexes.     

The in vitro and in vivo effects of human growth hormone administration on tumor growth of rats bearing a transplantable rat pituitary tumor (7315b)

The direct effects of human GH and IGF-I on PRL secretion and cell proliferation were studied on PRL secreting rat pituitary tumor 7315b cells in vitro, as well as the effects in vivo of human GH administration on body weight, IGF-I levels and tumor size in rats bearing this transplantable tumor. In the in vitro studies IGF-I levels above 5 nM stimulated PRL release in a dose-dependent manner while GH, in concentrations of 0.23–45 nM, did not affect PRL release. Cell proliferation was stimulated by IGF-I in a dose-dependent manner from 0.5 nM onwards, while GH did not have an effect. The in vivo studies showed that 1 mg GH/rat/day prevented tumor-induced cachexia and normalized the suppressed IGF-I levels without stimulating tumor growth. It is concluded that tumor-induced cachexia can be prevented by exogenous GH administration without an increase in tumor mass, even if a tumor model is used whose cultured tumor cells respond to exposure to IGF-I with a mitotic response.     

CUL4A is overexpressed in human pituitary adenomas and regulates pituitary tumor cell proliferation

Cullin 4A (CUL4A) encodes a core subunit of an E3 ubiquitin ligase that targets proteins for ubiquitin-mediated degradation, and aberrant expression of the CUL4A is found in many tumor types. However, its roles and clinicopathologic significance in pituitary adenomas are not clear. The aim of this study was to investigate the possible role of CUL4A in pituitary tumorigenesis. Immunohistochemistry was used to examine CUL4A expression in human normal pituitaries and pituitary tumors with respect to various clinicopathologic factors in pituitary adenomas. Cell proliferation was assessed by MTT and colony formation, and migration and invasion were analyzed by Transwell and Matrigel assays after CUL4A overexpression or knockdown in pituitary tumor cells. Overexpression of CUL4A was frequently observed in pituitary adenomas compared with normal adenohypophysial tissue and significantly associated with tumor progressiveness and invasion. CUL4A overexpression in GH3 adenoma cells increased colony numbers, cell viability and cell invasion and silencing CUL4A in AtT20 adenoma cells decreased cell proliferation, migration and invasion. Mechanistically, CUL4A could modulate the expression of p53, p21, and p27 in pituitary tumor cells. In addition, high levels of CUL4A expression also significantly inversely correlated with the p53 protein level in human pituitary adenomas. Our results indicate that CUL4A enhances pituitary cell proliferation, migration and invasion and may thus contribute to pituitary tumor development and progression.     

Suppression of testosterone and estradiol-17beta-induced dysplasia in the dorsolateral prostate of Noble rats by bromocriptine

We, and others, have previously described the histological changes that occur in the prostate gland of intact Noble (NBL) rats following prolonged hormonal treatment. Dysplasia, a pre-neoplastic lesion, develops specifically in the dorsolateral prostates (DLPs) of NBL rats treated for 16 weeks with a combined regimen of testosterone (T) and estradiol-17beta (E2) (T + E2-treated rats). Concurrent with DLP dysplasia induction, the dual hormone regimen also elicits hyperprolactinemia, in addition to an elevation of nuclear type II estrogen binding sites (type II EBS), no alteration in estrogen receptors (ER), and marked epithelial cell proliferation in the dysplastic foci. The aim of this study was to investigate whether the dual hormone action is mediated via E2-induced hyperprolactinemia. Bromocriptine (Br), at a dose of 4 mg/kg body wt per day, was used to suppress pituitary prolactin (PRL) release. Serum PRL levels were lowered from values of 341 +/- 50 ng/ml in T + E2-treated rats to 32 +/- 10 ng/ml in Br co-treated animals. The latter values were comparable to those in untreated control rats. In addition, Br co-treatment effectively inhibited the evolution of dysplasia (six out of eight rats) and the often associated inflammation (five out of eight rats) in most animals. In contrast, Br co-treatment did not suppress the T + E2- induced type II EBS elevation nor alter ER levels in the DLPs of these rats, when compared with T + E2-treated rats. These data extend the many previous studies that have detailed marked influences of PRL on rat prostatic functions. However, the current study is the first to implicate PRL in prostatic dysplasia induction in vivo.      

人垂体腺瘤细胞原代培养的纯化

背景与目的：较纯的人垂体腺瘤细胞对垂体腺瘤的生物学特性、病因、发病机制等的研究非常重要，本文探讨原代培养垂体腺瘤的细胞纯化方法。方法：采用三种原代培养方法对垂体腺瘤细胞进行培养，方法Ⅰ为目前常用的垂体瘤细胞培养方法，方法Ⅱ结合使用右旋颉氨酸（D-valine）替代左旋颉氨酸（L-valine）的DMEM D-valine培养液，方法Ⅲ采用反复贴壁法结合DMEM D-valine培养液培养。观察其细胞形态变化和生长特征。免疫组织化学染色法检测培养细胞生长激素（growth hormone，GH）、泌乳素（prolactin，PRL）的表达。结果：3种方法均能成功培养出垂体腺瘤细胞，呈圆形或椭圆形，其纯度随培养时间的延长逐渐下降，培养第20天其平均纯度分别为40％、46％、96％。方法Ⅲ明显高于方法Ⅰ和方法Ⅱ，差异具有显著性意义（P〈0．01）：腺瘤细胞分别呈GH、PRL阳性表达，成纤维细胞表达Ⅰ型胶原。结论：反复贴壁法结合DMEM D-valine培养液培养法可得到纯度达95％以上的人垂体腺瘤细胞，纯化后的细胞可分别呈GH和PRL阳性表达，为进一步研究人垂体腺瘤的发病机理、药物治疗和颅外移植等方面奠定了实验基础。     

Overexpression of vascular endothelial growth factor164 and its co-receptor neuropilin-1 in estrogen-induced rat pituitary tumors and GH3 rat pituitary tumor cells

We have shown previously that the VEGF system plays a crucial role in regulation of tumor angiogenesis during the development of estrogen-induced prolactin-secreting pituitary tumors in Fisher 344 rats. Studies also suggested that both endothelial and non-endothelial cells expressed VEGF. However, several questions concerning the VEGF signals in regulation of estrogen-induced angiogenesis in rat pituitary remained unanswered. VEGF exists in a number of isoforms in human and rodent tissue (i.e., VEGF206h/205r, VEGF189h/188r, VEGF165h/164r, VEGF145h/144r and VEGF121) that differ in their molecular masses and biological activities. The VEGF isoforms bind with two tyrosine-kinase receptors, KDR/flk-1 and flt-1. In addition, VEGF165 binds with a newly identified co-receptor, neuropilin-1, which is expressed in human endothelial cells and several types of non-endothelial cells including tumor cells. The present study was undertaken to elucidate which isoforms of VEGF are predominantly expressed in normal Fisher 344 rat pituitaries, estrogen-induced prolactin secreting rat pituitary tumors and in prolactin secreting rat pituitary tumor cell line (GH3 cell line). To identify the isoform, RT-PCR with primer pairs derived from exon 1 and exon 8 of the VEGF gene, cloning, sequencing and Western blot analysis were performed. The status of neuropilin-1 in the rat pituitaries (normal and transformed) and GH3 pituitary tumor cell line has also been investigated using RT-PCR and Western blot analysis. These studies demonstrate that normal rat pituitaries, estrogen-induced rat pituitary tumors and GH3 pituitary tumor cells expressed VEGF164 and co-receptor, neuropilin-1. The VEGF164 was the predominant form in all of these cells. The VEGF164 and neuropilin-1 mRNA and protein levels were significantly higher in the estrogen-induced pituitary tumors and GH3 tumor cell line, as compared to normal pituitary. The data suggest that both VEGF164 and neuropilin-1 may actively participate in modulation of tumor angiogenesis and the development of pituitary tumors in Fisher 344 rats.     

51例垂体腺瘤体外培养和免疫组化的研究

对51例垂体腺瘤手术标本进行体外培养和免疫组化研究。结果显示垂体腺瘤细胞体外培养有分泌功能.成活率达88％。血清激素水平高,免疫组化呈阳性的腺瘤细胞分泌激素一般也多。部分无功能腺瘤细胞也具有分泌功能,与免疫组化阳性程度不成正比。除催乳素腺瘤外,大多数腺瘤可同时分泌两种激素。实验结果提示细胞培养结合免疫组化、血清激素水平和临床症状,可以对垂体腺瘤作出形态与功能统一的、较完善的分类,有助于临床诊断和治疗。     

Evidence for a novel pituitary factor that potentiates the mitogenic effect of estrogen in human breast cancer cells

Estrogen, prolactin, and other tissue-derived factors are implicated in the etiology and pathophysiology of human breast cancer (HBC). In a previous study, we demonstrated that a factor(s) secreted by rat pituitary tumor cells (GH3) synergizes with estrogen to induce growth of HBC cells (T-47D) transplanted into athymic nude mice. The present studies were carried out to characterize further this pituitary growth factor. Pituitary tumor cell lines (GH3, GH1, 235-1, and AtT-20) and normal rat pituitaries were transplanted s.c. into estrogen-treated (estradiol valerate injection, 500 micrograms/14 days) athymic nude mice which also received T-47D cells. The influence of the presence of these normal and tumorous pituitary cells on growth (size and weight) of T-47D tumors was monitored for 49 to 56 days. The results indicate that factor(s) from normal rat pituitary glands as well as from the GH1 and GH3 but not 235-1 and AtT-20 pituitary tumor cells were able to potentiate the growth of T-47D tumors in estrogenized mice. To ascertain whether or not prolactin and/or growth hormone are responsible for the growth-promoting activity, purified human and ovine growth hormone and ovine prolactin were administered to estrogenized athymic nude mice either by daily s.c. injection (100 micrograms/day) or by constant infusion using Alzat osmotic minipumps (1.25 and 5.0 micrograms/h) for 29 to 56 days. None of these treatments stimulated the growth of the T-47D tumors, suggesting that prolactin, growth hormone, and their intermediates may not be directly involved. We further determined whether the factor from pituitary tumor cells was present in serum-free conditioned medium and could stimulate the growth of HBC cells in vitro. Conditioned medium from GH3 and GH1 but not from 235-1 and AtT-20 pituitary cells significantly stimulated growth of T-47D cells in the presence of estradiol (10(-10) M) after 12 days of culture in a serum-free medium (Dulbecco's modified Eagle's medium containing bovine serum albumin, 0.5 mg/ml). Optimal serum-free growth of T-47D cells (2-fold above control) was observed in the presence of estradiol (10(-10) M) and conditioned medium (30% v/v) from 48-h cultures of GH3 cells. The bovine serum albumin concentration of the serum-free medium (Dulbecco's modified Eagle's medium) was also important: optimal T-47D cell proliferation was observed with BSA between 0.5 and 2.0 mg/ml. Conditioned medium preparations from serum-pretreated flasks (without cells) from GH3 cell monolayers for zero time and from actinomycin D plus cycloheximide-inhibited GH3 cells were inactive.(ABSTRACT TRUNCATED AT 400 WORDS)     

Abnormal expression of 11 beta-hydroxysteroid dehydrogenase type 2 in human pituitary adenomas: a prereceptor determinant of pituitary cell proliferation

The physiological effects of glucocorticoids (GCs) are, at least in part, mediated by inhibition of cell proliferation. Two isozymes of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) interconvert cortisol (F) and inactive cortisone (E), and are thus able to modulate GC action at an autocrine level. Previously, we have demonstrated absent expression of 11 beta-HSD2 in normal pituitaries; however, in a small number of pituitary tumors analysed, 11 beta-HSD2 was readily demonstrable. Here we have used real-time RT-PCR to quantify expression of mRNA for 11 beta-HSD1 and 2 in 105 human pituitary tumors and have performed enzyme expression and activity studies in primary pituitary cultures. Overall, pituitary tumors expressed lower levels of 11 beta-HSDl mRNA compared with normals (0.2-fold, P<0.05). In contrast, expression of 11 beta-HSD2 mRNA was 9.8-fold greater in tumors than in normals (P<0.001). Enzyme assays showed significant 11 beta-HSD2 activity (71.9+/-22.3 pmol/h/mg protein (mean+/-s.d.)) but no detectable 11 beta-HSDl activity. Proliferation assays showed that addition of glycyrrhetinic acid (an 11 beta-HSD2 inhibitor) resulted in a 30.3+/-7.7% inhibition of cell proliferation. In summary, we describe a switch in expression from 11 beta-HSDl to 11 beta-HSD2 in neoplastic pituitary tissue. We propose that abnormal expression of 11 beta-HSD2 acts as a proproliferative prereceptor determinant of pituitary cell growth, and may provide a novel target for future tumor therapy.     

Prolactin release-inhibitory effects of progesterone, megestrol acetate, and mifepristone (RU 38486) by cultured rat pituitary tumor cells

The prolactin (PRL) release-inhibitory effects of progesterone, dexamethasone, megestrol acetate, and mifepristone (RU 38486) were studied in cultured pituitary tumor cells prepared from the 7315a and 7315b tumor. Both tumors contain similar numbers of estrogen and progesterone receptors, while only the 7315a tumor also has glucocorticoid receptors. PRL release by the 7315a tumor was stimulated by low concentrations of dexamethasone (10(-10)-10(-9) M) and was inhibited in a dose-dependent manner by higher concentrations (-86% by 10(-7) M). In contrast only 10(-6) and 10(-5) M dexamethasone inhibited PRL release by the 7315b cells by 14 and 24%, respectively. Progesterone caused a dose-dependent inhibition of PRL release, which was similar in the 7315a and b tumor cells. Progesterone (10(-9) M) inhibited PRL release by 62% and this inhibition was completely prevented by 100 nM estradiol, which was stimulatory by itself (+48%). Mifepristone inhibited PRL release by both tumors in a dose-dependent manner, but more powerfully in the 7315a tumor; 10(-6) M concentrations of the compound inhibited PRL release by 52% in the 7315a and by 26% in the 7315b tumor cells. Megestrol acetate inhibited PRL release in both tumors in a dose-dependent manner, but more powerfully in the 7315b tumor; a 10(-8) M concentration of the compound inhibited PRL release by 54% in the 7315b tumor and by 14% in the 7315a tumor. In the 7315a tumor 10(-9) M megestrol acetate even stimulated PRL release, suggesting a dexamethasone-like glucocorticoid effect of the drug on this tumor. Thereafter the interaction of mifepristone and megestrol acetate on PRL release was investigated. In the 7315a tumor cells different combinations of both drugs neutralized each other's inhibitory effects on PRL release, while both drugs had additional inhibitory effects on PRL release by 7315b tumor cells. Changes in PRL release by the cultured pituitary tumor cells were in all instances closely correlated with changes in the PRL content, the protein content, and the DNA content of the tumor cells. This suggests that the inhibitory effect of the compounds studied on PRL release is paralleled by an inhibitory effect on the number of pituitary tumor cells. These studies show the importance of the presence of glucocorticoid receptors in the effectiveness and mechanism of action of the antitumor effects of megestrol acetate and mifepristone.(ABSTRACT TRUNCATED AT 400 WORDS)