Estrogen-induced Pituitary Tumors Research Articles

Decreased expression of carboxypeptidase E protein is correlated to estrogen-induction of rat pituitary tumors

Following the cleavage of peptide precursors by endopeptidases such as the proprotein convertases PC2 and PC3, carboxypeptidase E (CPE) functions to remove basic amino adds from the C-terminus of various pituitary hormones. We investigated the role of CPE in the differential sensitivity between rat strains to estrogen-induced pituitary tumors. Pituitary CPE protein levels were unchanged by diethylstilbestrol (DES) in tumor-resistant Sprague-Dawley (SD) rats. However, in tumor-susceptible Fischer 344 (F344) rats, DES decreased CPE protein levels such that by 7 and 8 weeks of treatment, CPE was barely detectable. One week withdrawal of DES caused an increase in CPE protein levels at 8 weeks. After 2 and 4 weeks of DES treatment, CPE protein levels in F344 rats decreased to 18 and 2.3% of control values, respectively, but no strain difference was observed in the protein levels of proprotein convertase 2 (PC2) or PC3. Additionally, Brown Norway (BN), F344, and F 1 hybrid (BN × F344) rats were treated with DES for 10 weeks. The level of pituitary CPE protein was not affected by DES in BN rats whereas F344 rats had only 8% of the level of CPE pituitary protein of BN rats. The pituitaries of F 1 rats, which had an intermediate weight response to DES, had an intermediate level of CPE protein (31% that of BN rats). Levels of CPE mRNA were not affected by DES in SD rats while in F344 rats DES tended to decrease levels of CPE mRNA after both 2 and 4 weeks of treatment, although the response was variable. It thus appears that pituitary CPE protein is differentially regulated by DES between tumor-resistant rats and F344 rats primarily at the post-transcriptional level. Furthermore, in F344 rats, levels of CPE protein are inversely correlated to increases in pituitary weight caused by DES treatment.

Triiodothyronine attenuates estradiol-induced increases in dopamine D-2 receptor number in rat anterior pituitary

Estrogens promote adenohypophyseal enlargement and tumor transformation, and thyroid hormones antagonize these effects. Hormone-induced pituitary enlargement may be mediated by alterations in pituitary dopaminergic function. The present study examined the effects of chronic (20 days) administration of estradiol benzoate (EB), triiodothyronine (T3), or EB and T3 (T3 + EB) on dopamine (D-2) receptors in rat anterior pituitary. D-2 receptor number increased after EB without altered receptor affinity. T3 alone did not affect D-2 receptor number in the anterior pituitary but significantly attenuated the effect of EB. T3 administration also inhibited EB-induced anterior pituitary hyperplasia. D-2 receptor upregulation by EB more likely could reflect a compensatory response to decreased receptor occupation. The present results suggest that D-2 receptors could play an important role in estrogen-induced adenohypophyseal tumor formation and hyperprolactinemia and that thyroid hormones may inhibit estrogen-induced pituitary tumor development via adenohypophyseal D-2 receptors.

Is estrogen-induced pituitary hyperplasia and hyperprolactinaemia mediated by angiotensin II?

Human and rat anterior pituitary glands (APG) contain local renin-angiotensin systems (RAS). In the rat APG renin, renin mRNA, angiotensin converting enzyme (ACE) and angiotensin II (A II) have been identified within gonadotrophs (Naruse et al. 1986; Deschepper et al. 1986; Thomas and Sernia 1990). Angiotensinogen (AGEN) and AGEN mRNA have also been found in the rat APG but their localization remains still unclear (Ganong and Deschepper 1989; Healy 1992). Main target sites for A II action in APG seem to be lactotrophs. Specific A II binding sites have been identified mainly on lactotrophs and A II has been shown to stimulate prolactin (PRL) secretion (Aguilera et al. 1982; Denef and Schramme 1985). Recently, we have found that A II stimulated the proliferation of lactotrophs isolated from the estrogen-induced pituitary tumors in the rat (Kunert-Radek and Pawlikowski 1992). Moreover, an ACE inhibitor, enalapril, was found to prevent the pituitary proliferative response to a single injection of estradiol in rats (Mucha et al. 1993). Estrogens are well known to stimulate the pituitary cell proliferation and tumorigenesis and the estrogen-induced APG hyperplasia involves mainly lactotrophs (Zondek 1936; Mietkiewski 1959; Lloyd et al. 1975). Estrogens have been also found to enhance the prolactin secretion (Nicoll and Meites 1962). On the other hand, estrogens were found to increase the AGEN plasma levels (Menard et al. 1970) and to enhance AGEN mRNA expression in the liver, brain (Kunapuli et al. 1987) and pituitary (Healy et al. 1992). Thus, a question arises whether A II is involved in the estrogen action on APG. The aim of the experiments presented below was to test the hypothesis in question.

Colocalization of prolactin and proliferating cell nuclear antigen in the anterior pituitary during estrogen-induced pituitary tumors.

Chronic estrogen treatment induces prolactin (PRL)-secreting pituitary tumors in laboratory animals. To determine earlier events of tumorigenesis, we studied cell proliferation in the pituitary following 7-30 days of estrogen administration in Fischer 344 rats. Immunohistochemical localization of proliferative cells by proliferating cell nuclear antigen (PCNA) staining and lactotropes by PRL staining revealed that estrogen treatment caused a time-dependent increase in the number of proliferative cells and lactotropes. Although the increase in lactotropic cell number paralleled the increase in PCNA-reactive cell number, only approximately 30% of lactotropes reacted simultaneously with the PCNA antibody. These results indicate that a subset population of lactotropes proliferates under the influence of estrogen during tumorigenesis.

Effect of angiogenesis inhibitor TNP-470 on vascular formation in pituitary tumors induced by estrogen in rats.

TNP-470 (TNP), an analog of fumagillin, inhibits cell proliferation and tumorigenesis in estrogen-induced pituitary tumors in rats. This study found that treatment with TNP or TNP and bromocriptine suppressed vascular formation in these pituitary tumors more than bromocriptine alone. TNP had a greater suppressive effect on vascular formation in pituitary tumors than in normal pituitary gland. Laminin and basic fibroblast growth factor (FGF) were detected around new blood vessels in normal pituitary gland and pituitary tumor. TNP did not completely suppress laminin or basic FGF expression. The effect of TNP on pituitary tumor may involve inhibition of vascular formation by mechanisms mediated by other factors in addition to basic FGF. Treatment with bromocriptine and TNP acts indirectly and directly on vascular formation and may provide a useful chemotherapeutic modality for pituitary tumors.

Restoration by bromocriptine of glucocorticoid receptors and glucocorticoid negative feedback on prolactin secretion in estrogen-induced pituitary tumors.

We previously reported a reduction of glucocorticoid receptors (GCR) in diethylstilbestrol-induced pituitary tumors (DES-T) in rats. Presently, we found that bromocriptine (BROM) treatment increased the levels of GCR in DES-T, demonstrated by steroid binding assays and immunocytochemistry using a monoclonal antibody against the type II GCR. We also found that the high content of nuclear estradiol receptors in the adenomata and the elevated levels of PRL in serum of DES-T were significantly reduced after BROM treatment. In parallel studies, PRL secretion was measured after administration of ether stress. In controls, serum PRL markedly increased after ether and this effect was blunted by prior dexamethasone (DEX) administration, due to the steroid negative feedback on PRL secretion. In animals with DES-T, ether stress had no effect on serum PRL, and the inhibition by DEX was lost unless they received BROM, which restored the negative feedback of DEX on serum PRL. Although increases of PRL titers in pituitary tumors may be due to estrogenic stimulation of lactotroph proliferation and function, coupled to absent dopaminergic inhibition on these cells, other mechanisms are possible. In this respect, inefficient steroid negative feedback on PRL synthesis due to down-regulation of GCR may contribute to hyperprolactinemia. This mechanism is supported from the restoration of GCR and steroid negative feedback on serum PRL by treatment of tumor-bearing rats with BROM.

Subcellular Distribution of Cyclic Adenosine 3', 5'-Monophosphate-Binding Protein and Estrogen Receptors in Control Pituitaries and Estrogen-Induced Pituitary Tumors

Binding of cyclic adenosine 3',5'-monophosphate (cAMP) and steroid receptors was studied in cytoplasmic and nuclear fractions of pituitaries from castrated rats, in rats subjected to acute (60 min) or short-term (4 days) estradiol (E2) treatment, and in diethylstilbestrol-induced pituitary tumors (DES-T). E2 receptors were primarily in nuclear extracts in all animals that were given estrogens, whereas cytosolic receptors were low to absent. Contrarily, castrated rats showed high quantities of cytoplasmic receptor but little in nuclear sites. The progestin receptor was induced only in 4-day E2-treated rats and in DES-T. cAMP binding was stimulated in cytosol from 4-day E2-treated rats and in DES-T, but a significant reduction in binding was also noted in nuclear extracts from DES-T. Scatchard analysis for the cytosolic cAMP-binding activity demonstrated a two-component system, and the increased cAMP binding obtained in DES-T seemed to be caused by an increase in the low-affinity, high-capacity binder [regulatory type II (RII) subunit of protein kinase]. Suggestion of the preferential estrogenic induction of RII was also obtained by DEAE-cellulose chromatography, which provided separation of RI and RII subunits. The results suggest that sustained estrogenization leads to induction of cytosolic cAMP-binding protein and increased levels of nuclear E2 receptor. In DES-T, this effect resulted in an inverse subcellular distribution of both binding proteins, which may be related to abnormal growth of the pituitary, as has been postulated for hormone-dependent mammary tumors.

Localization of Immunoreactive Glandular Kallikrein in Lactotrophs of the Rat Anterior Pituitary

Glandular kallikrein (a trypsin-like serine protease) is an estrogen-induced and dopamine-repressed protein in the rat anterior pituitary which predominantly exists as a latent zymogen (prokallikrein). Its regulation, presence in estrogen-induced pituitary tumors in F344 rats, and expression in GH3 cells has suggested a localization in lactotrophs (prolactin-producing cells). This study examined the cellular origin of glandular kallikrein using immunocytochemical techniques. Anterior pituitaries from estrogen-treated rats were fixed and embedded in paraffin (for preparation of semi thick sections; 5 microns) or methacrylate (for preparation of thin sections; 1 micron). Glandular kallikrein immunostaining was readily detected in the perinuclear (Golgi) region of parenchymal cells of the anterior pituitary in both thin and semi thick sections. Two-color double immunoperoxidase staining of thin and semi thick sections indicated that glandular kallikrein was localized in cells containing prolactin (PRL) but not other pituitary hormones. Immunoperoxidase staining of consecutive serial thin sections with alternating antisera confirmed a localization of glandular kallikrein in lactotrophs. The results establish that glandular kallikrein is colocalized with PRL in lactotrophs of the rat anterior pituitary. This is consistent with the hypothesis that the function of anterior pituitary glandular kallikrein is linked to PRL in some fashion--possibly as a PRL-processing protease.

Regulation of GH and TSH release from hyperplastic and ectopic pituitaries: Effects of dopamine in vitro

This study was undertaken to examine the consequences of prolonged removal of the pituitary from hypothalamic control and of estrogen-induced pituitary tumors on the susceptibility of GH and TSH release to regulatory influences of dopamine (DA). Adult male Fischer 344 rats were treated with transplants of female anterior pituitaries under the renal capsule or with Silastic capsules containing diethylstilbestrol (DES). Capsules with DES remained in place until the animals were killed (DES-IN) or were removed 7 weeks prior to sacrificing the rats (DES-OUT). Both pituitary grafts and DES caused the expected elevation in plasma prolactin and suppression of plasma GH and TSH levels. Basal GH release in vitro was not affected by exposure to DES in vivo but was reduced by transplantation of the pituitary to an ectopic site. Treatment with DA in vitro suppressed GH release from the in situ pituitaries of control, DES treated and grafted rats but increased GH release from the ectopic pituitaries. Basal release of TSH in vitro was reduced in the pituitaries of DES-IN and DES-OUT animals but was not affected by the presence of pituitary transplants. No detectable TSH was released from the ectopic pituitaries in the absence of DA. DA decreased TSH release from the pituitaries of control, DES-OUT and DES-IN rats but not from the in situ pituitaries of grafted rats. In contrast, DA produced an increase in TSH release from ectopic pituitaries. These results demonstrate that somatotrophs and thyrotrophs removed from the hypothalamic influences on subjected to direct and indirect effects of DES exhibit abnormal responses to DA. We suspect that prolonged absence of normal pituitary control leads to the development of regulatory mechanism of pituitary hormone release which are different from those operating under physiological conditions.

Kallikrein gene expression in estrogen-induced pituitary tumors

Anterior pituitary kallkrein-like enzyme activity, immunoreactivity and mRNA levels have previously been shown to be regulated by estrogen, in parallel with prolactin. In this study, we have examined the relationship between kallikrein and prolactin mRNA levels in estrogen-induced pituitary tumors. Treatment of Fischer 344 rats with diethylstilbestrol implants for 3, 5 and 7 weeks produced a dramatic increase in kallikrein mRNA levels and a modest increase in prolactin mRNA levels. These changes were partially reversed by bromocriptine treatment, and completely reversed by bromocriptine plus estrogen withdrawal. Using a panel of oligonucleotide probes specific for various members of the rat kallikrein gene family, we have shown that the kallikrein-like gene expressed appears to be true kallikrein.

Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA.

The administration of high levels of estrogen is a well established method for producing prolactin-secreting pituitary tumors in rodents but the mechanism of tumor induction is not clear. In this paper we describe a cDNA clone (pEIC) which has been isolated from an estrogen-induced pituitary tumor cDNA library. The mRNA transcript corresponding to the pEIC clone is 0.9 kilobase in length and is not detectable in normal pituitaries but is expressed as early as 3 h after estrogen stimulation. Nucleotide sequence analysis of two 700-base pair recombinant clones shows that they encode a 124-amino acid protein which is 70% identical to the porcine galanin precursor. The sequence of 29 amino acid residues coded for by the pEIC cDNA clone is 88% identical with porcine galanin with only three amino acid substitutions near the C terminus. This extensive homology suggests that the pEIC cDNA clone codes for rat galanin or a protein belonging to the galanin gene family. These results provide the first evidence of a physiological regulator (estrogen) of the expression of the galanin gene. They also imply that galanin is secreted by prolactin-secreting tumors. Because intracerebroventricular injection of galanin can stimulate prolactin secretion and galanin inhibits hypothalamic dopamine release, it is conceivable that galanin may play a role in the induction of prolactin-secreting tumors.

Binding of steroids in nuclear extracts and cytosol of rat pituitary and estrogen-induced pituitary tumors

We have determined binding sites for estrogen, progestin, androgen and glucocorticoid in anterior pituitaries from Sprague-Dawley rats, a strain with low estrogen sensitivity, and in diethyl-stilbestrol-induced pituitary tumors in Fischer 344 rats, a strain with high estrogen sensitivity. Binding sites differ in their quantity and subcellular distribution. Cytosolic sites for [ 3H]estradiol in normal pituitaries from untreated rats were high prevailing over sites for other hormones, but they were depleted in the tumors due to their retention in nuclei under the influence of estrogen. Unoccupied nuclear sites for estrogen in normal glands also prevailed over sites for other steroids, and were similar to those in tumors. Second, the progestin site labeled with [ 3H]R 5020 was concentrated 5.7-fold in cytosol and 8.5-fold in nuclei of the tumors over the values found in glands from normal males estrogenized for 3 days. Third, glucocorticoid receptors labeled with [ 3H]dexamethasone were predominantly cytosolic in normal glands, but very low in cytosol and more evident in nuclear extracts from the tumors, the reverse of the profile found in normal pituitaries. Last, limited and comparable amounts of androgen receptors were measured in the subcellular fractions of both tissues. It is suggested that the subcellular distribution of some steroid receptors may be controlled in part by the cell population of the tissue and its degree of genetic activity.

Studies on estrogen induced pituitary tumor in the rat with special reference to the relationship of the tuberoinfundibular dopamine neuron system.

Pituitary tumors were experimentally induced in female Wistar rats by repeated injections of estradiol dipropionate. The hypothalamus and pituitary tumors were studied simultaneously by fluorescence histochemistry and immunohistochemistry. The pituitary gland became larger with a concomitant increase of serum prolactin in proportion to the dose of estrogen. Estrogen-induced pituitary tumor exhibited a proliferating prolactin cells by the peroxidase immunohistochemical method. Ultramicroscopical findings showed that these tumor cells were in an extremely hyperfunctional state. The dopamine neuronal perikarya in the hypothalamic arcuate nucleus and their terminals in the external layer of the median eminence were examined by fluorescence histochemistry in the rats bearing estrogen induced pituitary tumor and it was concluded that in our experimental conditions, estrogen effected directly on pituitary rather than on the hypothalamus and consequently dopamine synthesis in the arcuate neurons and its release into portal capillaries were accelerated simultaneously in order to inhibit prolactin secretion from tumor cells.

Decreased tuberoinfundibular and pituitary dopamine and dihydroxyphenylacetic acid concentrations in rats with estrogen-induced pituitary tumors

The majority of pituitary adenomas are prolactin (PRL)-secreting, but it is still uncertain whether their pathogenesis results from a central nervous system (CNS) disturbance or autonomous lactotroph growth and function. We have measured dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) concentrations in rats bearing estradiol-induced PRL-secreting pituitary tumors. Female rats, injected at 3-week intervals with 2 mg estradiol valerate (EV), had increased plasma prolactin concentrations, up to 3 micrograms/ml, at 31 weeks. Inversely, there was a reduction of DA and DOPAC in the median eminence and arcuate nucleus as well as a decreased DOPAC/DA ratio in the arcuate nucleus. DA-containing nuclei in the other parts of the brain were not affected. Anterior pituitary weight increased while its DA concentration decreased during estradiol treatment. In the neurohypophysis, DA concentrations were unchanged while DOPAC and the ratio DOPAC/DA decreased following the estrogen treatment. Our data suggest that rats with primary estrogen-induced PRL-secreting tumors have a defect in the CNS-DA neurotransmission.

Effects of a luteinizing hormone-releasing hormone analog on the growth of estrogen-induced prolactin-secreting rat pituitary tumors and its influence on their dopamine receptors.

Effects of a luteinizing hormone-releasing hormone analog on the growth of estrogen-induced prolactin-secreting rat pituitary tumors and its influence on their dopamine receptors.

Melanocyte-stimulating hormone and prolactin secretion in cell culture of estrogen-induced pituitary tumors in the hamster

Pituitary cells from hamsters bearing diethylstilbestrol induced renal adenocarcinomas were cultured in vitro. Dispersed cells in plastic dishes were viable for up to two weeks in Dulbecco's modified Eagle's medium supplemented with 17.5% of 6:1 horse serum to fetal calf serum. The secretion of α-melanocyte stimulating hormone and prolactin into the medium were measured by radioimmunoassay. The concentrations of both were elevated by day 3 in the medium from the hyperplastic pituitaries obtained from the estrogen treated, tumor bearing hamsters. Neither DES (10 8M) nor tamoxifen (10 −7M) influenced the secretion of either hormone and neither altered either cell number or DNA synthetic activity as measured by thymidine incorporation. The secretion of hormones and the growth of the pituitary cells were, however, decreased by charcoal treatment of the serum. The results suggest that the elevation of serum α-MSH and prolactin observed in DES implanted hamsters is due to pituitary secretion of the hormones but that DES probably does not act directly on the pituitary to control the secretion.

Defective central nervous system dopaminergic function in rats with estrogen-induced pituitary tumors, as assessed by plasma prolactin concentrations.

Mature cycling female rats were each injected at 3-week intervals with 2 mg estradiol valerate (EV) in sesame oil. Controls received an equivalent volume of sesame oil. The administration of EV induced, at first, hyperplasia and then, 10 weeks after the first EV injection, adenomatous changes in the pituitary gland. Concurrently, there was a striking rise in plasma PRL concentrations, which reached values of about 2 μg/ml at 25 weeks. Acute administration of nomifensine (NOM), an indirectly acting dopamine (DA) agonist, to EV-injected rats suppressed baseline PRL levels at 1 week but failed to significantly decrease PRL levels at 4, 7, 10, and 25 weeks. Unlike NOM, bromocriptine, a direct DA agonist, strikingly reduced baseline PRL levels in EV-injected rats 1,10, and 25 weeks after the first EV injection. The administration of domperidone (DOM), a potent blocker of DA receptors, inconsistently increased plasma PRL at 1 week and was ineffective at the following time intervals. Since both NOM and DOM are d...

Pituitary hormones and prolactin-releasing activity in rats with primary estrogen-induced pituitary tumors.

Pituitary tumors were induced in Wistar-strain female rats by repeated injections of depot estradiol preparations. Fifty-five, 130, and 200 days after the last injection, eight or nine experimental animals and the same number of untreated control animals were sacrificed. Pituitary weight, histology, and content of DNA, PRL, GH, TSH, and LH, hypothalamic content of PRL-releasing activity (PRL-RA) and immunoreactive TRH, and serum levels of PRL, GH, TSH, LH, T3, T4, and estradiol were determined. The pituitaries in the estrogen-treated rats increased markedly in weight and DNA content, and formed tumors. There was a striking increase in serum levels of PRL and a less marked increase in PRL content of the pituitary. Hypothalamic PRL-RA was higher than in the controls 55 and 200 days after the last estrogen injection at a time when serum estradiol levels were similar to those of controls. On the other hand, TSH, LH, and, to a slight degree, GH were suppressed. Short term administration of estrogen resulted in...

Biochemical and Ultrastructural Studies on Estrogen-Induced Pituitary Tumors in F344 Rats<xref ref-type="fn" rid="FN2">2</xref>

The hormone function, the metabolism of nucleic acids, and the ultrastructure of estrogen-induced pituitary tumors and of normal glands were examined in male F344 rats. The tumors had a high capacity for prolactin (PRL) synthesis, and the plasma levels of PRL were elevated 65-fold to 100-fold in the tumor-bearing animals. Uridine uptake and phosphorylation to nucleotides, as well as uridine incorporation into total RNA, were similar in tumors and normal glands, whereas [3H]thymidine incorporation into DNA was double in the former group as compared to the latter. After a [3H]uridine pulse, labeled RNA turnover was different in tumors and normal glands. Electron microscopy of the tumors revealed hypertrophy, degranulation, and hyperplasia of cells producing PRL with proliferation of their ergastoplasm in whorls. Other pituitary cell types were reduced in number. It is suggested that the whorl configuration caused the high rate of protein and PRL synthesis as well as the changes in RNA metabolism displayed by the tumors.

Changes of growth hormone-containing cells during tumorigenesis and subpassages of estrogen-induced pituitary tumors in rats.

The changes of growth hormone-containing cells during the tumorigenesis and subpassages of the estrogen-induced pituitary tumors were studied with peroxidaselabeled antibody method.In a primary pituitary tumor induced with estrogen in a female W/Fu rat, growth hormone-containing cells were rather small and compressed away by the tumorous growths to localize mainly to the peripheral areas, corresponding with the unchanged acidophils in the routinely stained sections.The cells positively stained with the peroxidase-labeled antibody were scattered in a transplantable mammotropic pituitary tumor (MtT/F4). Since this tumor is well known to produce growth hormone in addition to prolactin and adrenocorticotropic hormone, a few non-functional growth hormone-containing cells which remained in the primary pituitary tumor were considered to have transformed to secrete growth hormone during the further subpassages.Further, the changes and variations of the hormonal activities of the estrogeninduced pituitary tumors were discussed from the view point of the different cellular populations.