Rat Pituitary Tumor Cell Line Research Articles

Epigenetic upregulation of galanin-like peptide mediates deoxynivalenol induced-growth inhibition in pituitary cells

Deoxynivalenol (DON) is an unavoidable contaminant in human food, animal feeds, and agricultural products. Growth retardation in children caused by extensive DON pollution has become a global problem that cannot be ignored. Previous studies have shown that DON causes stunting in children through intestinal dysfunction, insulin-like growth factor-1 (IGF-1) axis disorder and peptide YY (PYY). Galanin-like peptide (GALP) is an important growth regulator, but its role in DON-induced growth retardation is unclear. In this study, we report the important role of GALP during DON-induced growth inhibition in the rat pituitary tumour cell line GH3. DON was found to increase the expression of GALP through hypomethylationin the promoter region of the GALP gene and upregulate the expression of proinflammatory factors, while downregulate the expression of growth hormone (GH). Furthermore, GALP overexpression promoted proinflammatory cytokines, including TNF-α, IL-1β, IL-11 and IL-6, and further reduced cell viability and cell proliferation, while the inhibitory effect of GALP was the opposite. The expression of GALP and insulin like growth factor binding protein acid labile subunit (IGFALS) showed the opposite trend, which was the potential reason for the regulation of cell proliferation by GALP. In addition, GALP has anti-apoptotic effects, which could not eliminate the inflammatory damage of cells, thus aggravating cell growth inhibition. The present findings provide new mechanistic insights into the toxicity of DON-induced growth retardation and suggest a therapeutic potential of GALP in DON-related diseases.

DnBP-induced thyroid disrupting activities in GH3 cells via integrin αvβ3 and ERK1/2 activation

Di-n-butylphthalate (DnBP) exhibits alarming thyroid disrupting activities. However, the toxic mechanism of DnBP is not completely understood. In this study, we investigated the mechanism of DnBP in thyroid disruption. Rat pituitary tumor cell lines (GH3) were treated with DnBP in different scenarios, and cell viabilities, target gene transcriptions and protein levels were measured accordingly. The results showed that after treatment with DnBP (20 μmol/L), cell proliferation increased to 114.69% (p < 0.01) and c-fos gene was up-regulated by 1.57-fold (p < 0.01). Both nuclear thyroid hormone receptor β (TRβ) and membrane TR (integrin αv and integrin β3) genes were up-regulated by 1.31-, 1.08- and 2.39-fold (p < 0.01), respectively, the latter was inhibited by Arg-Gly-Asp (RGD) peptides; the macromolecular DnBP-BSA was unable to bind nuclear TRs, but still promoted cell proliferation to 104.18% and up-regulated c-fos by 2.99-fold (p < 0.01); after silencing TRβ gene, cell proliferation (106.64%, p < 0.05) and up-regulation of c-fos (1.23-fold, p < 0.01) were also observed. All of these findings indicated the existence of non-genomic pathway for DnBP-induced thyroid disruption. Finally, DnBP activated the downstream extracellular regulated protein kinases (ERK1/2) pathway, up-regulating Mapk1 (1.15-, p < 0.05), Mapk3 (1.26-fold, p < 0.01) and increasing protein levels of p-ERK (p < 0.01); notably, DnBP-induced ERK1/2 activation along with c-fos up-regulation were attenuated by PD98059 (ERK1/2 inhibitor). Taken together, it could be suggested that integrin αvβ3 and ERK1/2 pathway play significant roles in DnBP-induced thyroid disruption, and this novel mechanism warrants further investigation in living organisms.

The role of galectin-3 in the tumorigenesis and progression of pituitary tumors.

Galectin-3 (Gal-3), a β-galactoside-binding protein, has been implicated in cell proliferation, cell adhesion, and the progression and metastasis of various types of cancer. The present study investigated the involvement of Gal-3 in the tumorigenesis and progression of pituitary tumors using three rat pituitary tumor cell lines. Following transfection with Gal-3 expression and interference vectors, the impact of Gal-3 on proliferation, apoptosis and migration of pituitary tumor cells was been investigated. Meanwhile, BCL2 associated X, apoptosis regulator (Bax), caspase-3 and matrix metalloproteinase 7 (MMP7) protein expression levels were analyzed by western blotting. The results of the present study revealed that Gal-3 expression in GH3 and GH4C1 cells was higher than in RC-4B/C cells. Furthermore, Gal-3 was demonstrated to promote the proliferation and migration of GH3 and GH4C1 cells, and inhibit cell apoptosis. Caspase-3 and MMP7 protein expression was also increased by Gal-3, while Bax expression was decreased. These results suggested that Gal-3 serves an important function in the tumorigenesis and development of pituitary tumors, and it may be a useful target for the treatment of pituitary tumors in the future.

Pituitary Tumor Suppression by Combination of Cabergoline and Chloroquine.

The dopamine agonist cabergoline (CAB) has been used widely in the treatment of prolactinomas and other types of pituitary adenomas, but its clinical use is hampered by intolerance in some patients with prolactinoma and lack of effectiveness in other pituitary tumor types. Chloroquine (CQ) is an old drug widely used to treat malaria. Recent studies, including our own, have revealed that CAB and CQ are involved in induction of autophagy and activation of autophagic cell death. To test whether CAB and CQ can function cooperatively to suppress growth of pituitary adenomas as well as other cancers. In vitro studies using the rat pituitary tumor cell lines MMQ and GH3, human pituitary tumor cell primary cultures, and several human cancer cell lines showed that CQ enhanced suppression of cell proliferation by CAB. These results were confirmed in in vivo xenograft models in nude mice and estrogen-induced rat prolactinomas. To understand the mechanism of combined CAB and CQ action, we established a low-CAB-dose condition in which CAB was able to induce autophagy but failed to suppress cell growth. Addition of CQ to low-dose CAB blocked normal autophagic cycles and induced apoptosis, evidenced by the further accumulation of p62/caspase-8/LC3-II. The data suggest that combined use of CAB and CQ may increase clinical effectiveness in treatment of human pituitary adenomas, as well as other cancers, making it an attractive option in tumor and cancer therapies.

Negative Glucocorticoid Response-Like Element from the First Intron of the Chicken Growth Hormone Gene Represses Gene Expression in the Rat Pituitary Tumor Cell Line.

The effects of introns, especially the first intron, on the regulation of gene expression remains unclear. Therefore, the objective of the present study was to investigate the transcriptional regulatory function of intron 1 on the chicken growth hormone (cGH) gene in the rat pituitary tumor cell line (GH4-C1). Transient transfection using first-intron-inserted cGH complete coding sequences (CDSs) and non-intron-inserted cGH CDS plasmids, quantitative RT-PCR (qRT-PCR) and western blot assays were used to detect the expression of cGH. The reporter gene assay was also used to investigate the effect of a series of fragments in the first intron of cGH on gene expression in GH4-C1. All of the results revealed that a 200-bp fragment located in the +485/+684 region of intron 1 was essential for repressing the expression of cGH. Further informatics analysis showed that there was a cluster of 13 transcriptional factor binding sites (TFBSs) in the +485/+684 region of the cGH intron 1. Disruption of a glucocorticoid response-like element (the 19-nucleotide sequence 5′-AGGCTTGACAGTGACCTCC-3′) containing a T-box motif (TGACCT) located within this DNA fragment increased the expression of the reporter gene in GH4-C1. In addition, an electrophoretic mobility shift assay (EMSA) revealed a glucocorticoid receptor (GR) protein of rat binding to the glucocorticoid response-like element. Together, these results indicate that there is a negative glucocorticoid response-like element (nGRE) located in the +591/+609 region within the first intron of cGH, which is essential for the down-regulation of cGH expression.

Development of Automated Patch Clamp Assay for Evaluation of α7 Nicotinic Acetylcholine Receptor Agonists in Automated QPatch-16.

The α7 nicotinic acetylcholine receptor (α7 nAChR) is an important and challenging target for drug discovery in the area of neuropsychiatric disorders. The current screening for chemicals targeting α7 nAChRs is primarily achieved by the use of low-throughput assay two-electrode voltage clamp (TEVC) in nonmammalian Xenopus oocytes. Automated patch clamp system has emerged as an attractive approach compared to conventional electrophysiology. To develop a mammalian cell-based functional assay in an automated electrophysiology system, we in this study generated a stable expression of α7 nAChRs in GH3 cells that originated from a rat pituitary tumor cell line and utilized automated QPatch-16 to test a set of tool compounds and chemicals identified as α7 agonists by TEVC. For the improvement of evaluating weak or partial α7 nAChRs agonists, we achieved enhancement of the signal-to-noise ratio by the addition of a positive allosteric modulator PNU-120596, which only activates α7 current in the presence of agonist. This improved assay was further validated by using known α7 partial agonists, such as RG3487, EVP-6124, and A-P90. Using this validated assay, we were able to identify a novel agonist 140507C that partially activates α7 nAChRs. Taken together, our results validate the use of QPatch-16 for evaluation α7 partial agonists, demonstrating its utility as an effective tool for α7 ion channel drug discovery.

Butyrate increases intracellular calcium levels and enhances growth hormone release from rat anterior pituitary cells via the G-protein-coupled receptors GPR41 and 43.

Butyrate is a short-chain fatty acid (SCFA) closely related to the ketone body ß-hydroxybutyrate (BHB), which is considered to be the major energy substrate during prolonged exercise or starvation. During fasting, serum growth hormone (GH) rises concomitantly with the accumulation of BHB and butyrate. Interactions between GH, ketone bodies and SCFA during the metabolic adaptation to fasting have been poorly investigated to date. In this study, we examined the effect of butyrate, an endogenous agonist for the two G-protein-coupled receptors (GPCR), GPR41 and 43, on non-stimulated and GH-releasing hormone (GHRH)-stimulated hGH secretion. Furthermore, we investigated the potential role of GPR41 and 43 on the generation of butyrate-induced intracellular Ca2+ signal and its ultimate impact on hGH secretion. To study this, wt-hGH was transfected into a rat pituitary tumour cell line stably expressing the human GHRH receptor. Treatment with butyrate promoted hGH synthesis and improved basal and GHRH-induced hGH-secretion. By acting through GPR41 and 43, butyrate enhanced intracellular free cytosolic Ca2+. Gene-specific silencing of these receptors led to a partial inhibition of the butyrate-induced intracellular Ca2+ rise resulting in a decrease of hGH secretion. This study suggests that butyrate is a metabolic intermediary, which contributes to the secretion and, therefore, to the metabolic actions of GH during fasting.

IGF1 induces cell proliferation in human pituitary tumors – Functional blockade of IGF1 receptor as a novel therapeutic approach in non-functioning tumors

Insulin-like growth factor (IGF1) and its receptor display potent proliferative and antiapoptotic activities and are considered key players in malignancy. The objective of the study was to explore the role of IGF1 and its downstream pathways in the proliferation of non-functioning pituitary tumor cells and to develop a targeted therapeutic approach for the treatment of these tumors. Cultures of human non-functioning pituitary adenomas and the non-secreting immortalized rat pituitary tumor cell line MtT/E were incubated with IGF1, IGF1 receptor inhibitor or both, and cell viability, proliferation and signaling were examined. Our results show that IGF1 elevated cell proliferation and enhanced cell cycle progression as well as the expression of cyclins D1 and D3. IGF1 also induced the phosphorylation of ERK, Akt and p70S6K. On the other hand, the selective IGF1R inhibitor NVP-AEW541 abrogated IGF1-induced cell proliferation as well as IGF1 receptor phosphorylation and downstream signaling.

Biological Functions of Protein Disulfide Isomerase as a Target of Phenolic Endocrine-disrupting Chemicals

Endocrine disrupting chemicals (EDCs) are widespread in the environment and suspected of interfering with endocrine homeostasis. Bisphenol A (BPA), 2,2-bis(4-hydroxyphenyl)propane, an EDC, has been widely used throughout the world as an industrial chemical. BPA is a developmental, neural, and reproductive toxicant that mimics estrogen and can interfere with growth and body function. In a recent study, a BPA-binding protein was isolated from P2 fractions of rat brain, and identified as protein disulfide isomerase (PDI). PDI has been studied extensively as a key enzyme involved in the formation of the correct pattern of disulfide bonds in proteins. PDI is also known to be a membrane-associated 3,3',5-triiodo-L-thyronine (T3)-binding protein. BPA inhibits the binding of T3 to PDI and isomerase activity of PDI. PDI has four consecutive domains, a, b, b', and a', each with a thioredoxin fold. The domains a and a' have a catalytically active Cys-Gly-His-Cys motif, while b and b' have substrate-binding sites. To address the chemical structural requirements of BPA for the inhibitory effect of PDI, several BPA analogs were tested, suggesting that the phenolic structure is important for BPA to affect PDI functions. To investigate the biological effects of BPA on PDI functions, GH3 cells were used. GH3 is a rat pituitary tumor cell line and releases growth hormone (GH) via the T3 receptor. Over-expression of PDI suppresses the T3-induced release of GH and when the cells were treated with BPA together with T3, the amount of GH released was much increased. Thus, PDI plays an important role in the hypothalamic-pituitary-thyroid axis. In this review, the possible involvement and biological significance especially in the central nervous system, of PDI as a target of phenolic environmental chemicals, are discussed.

Proliferative and anti-proliferative effects of dietary levels of phytoestrogens in rat pituitary GH3/B6/F10cells - the involvement of rapidly activated kinases and caspases

BackgroundPhytoestogens are a group of lipophillic plant compounds that can have estrogenic effects in animals; both tumorigenic and anti-tumorigenic effects have been reported. Prolactin-secreting adenomas are the most prevalent form of pituitary tumors in humans and have been linked to estrogen exposures. We examined the proliferative effects of phytoestrogens on a rat pituitary tumor cell line, GH3/B6/F10, originally subcloned from GH3 cells based on its ability to express high levels of the membrane estrogen receptor-α.MethodsWe measured the proliferative effects of these phytoestrogens using crystal violet staining, the activation of several mitogen-activated protein kinases (MAPKs) and their downstream targets via a quantitative plate immunoassay, and caspase enzymatic activities.ResultsFour phytoestrogens (coumestrol, daidzein, genistein, and trans-resveratrol) were studied over wide concentration ranges. Except trans-resveratrol, all phytoestrogens increased GH3/B6/F10 cell proliferation at some concentration relevant to dietary levels. All four phytoestrogens attenuated the proliferative effects of estradiol when administered simultaneously. All phytoestrogens elicited MAPK and downstream target activations, but with time course patterns that often differed from that of estradiol and each other. Using selective antagonists, we determined that MAPKs play a role in the ability of these phytoestrogens to elicit these responses. In addition, except for trans-resveratrol, a serum removal-induced extrinsic apoptotic pathway was blocked by these phytoestrogens.ConclusionPhytoestrogens can block physiological estrogen-induced tumor cell growth in vitro and can also stimulate growth at high dietary concentrations in the absence of endogenous estrogens; these actions are correlated with slightly different signaling response patterns. Consumption of these compounds should be considered in strategies to control endocrine tumor cell growth, such as in the pituitary.

Membrane estrogen receptor-α-mediated nongenomic actions of phytoestrogens in GH&lt;sub&gt;3&lt;/sub&gt;/B&lt;sub&gt;6&lt;/sub&gt;/F&lt;sub&gt;10&lt;/sub&gt; pituitary tumor cells

BackgroundEstradiol (E2) mediates various intracellular signaling cascades from the plasma membrane via several estrogen receptors (ERs). The pituitary is an estrogen-responsive tissue, and we have previously reported that E2 can activate mitogen-activated protein kinases (MAPKs) such as ERK1/2 and JNK1/2/3 in the membrane ERα (mERα)-enriched GH3/B6/F10 rat pituitary tumor cell line. Phytoestrogens are compounds found in plants and foods such as soybeans, alfalfa sprouts, and red grapes. They are structurally similar to E2 and share a similar mechanism of action through their binding to ERs. Phytoestrogens bind to nuclear ERs with a much lower affinity and therefore are less potent in mediating genomic responses. However, little is known about their ability to act via mERs to mediate nongenomic effects.MethodsTo investigate the activation of different nongenomic pathways, and determine the involvement of mERα, we measured prolactin (PRL) release by radio-immunoassay, MAPK activations (ERK1/2 and JNK1/2/3) via a quantitative plate immunoassay, and intracellular [Ca2+] by Fura-2 fluorescence imaging in cells treated with E2 or four different phytoestrogens (coumestrol, daidzein, genistein, and trans-resveratrol).ResultsCoumesterol and daidzein increased PRL release similar to E2 in GH3/B6/F10 cells, while genistein and trans-resveratrol had no effect. All of these compounds except genistein activated ERK1/2 signaling at 1–10 picomolar concentrations; JNK 1/2/3 was activated by all compounds at a 100 nanomolar concentration. All compounds also caused rapid Ca2+ uptake, though in unique dose-dependent Ca2+ response patterns for several aspects of this response. A subclone of GH3 cells expressing low levels of mERα (GH3/B6/D9) did not respond to any phytoestrogen treatments for any of these responses, suggesting that these nongenomic effects were mediated via mERα.ConclusionPhytoestrogens were much more potent in mediating these nongenomic responses (activation of MAPKs, PRL release, and increased intracellular [Ca2+]) via mERα than was previously reported for genomic responses. The unique non-monotonic dose responses and variant signaling patterns caused by E2 and all tested phytoestrogens suggest that complex and multiple signaling pathways or binding partners could be involved. By activating these different nongenomic signaling pathways, phytoestrogens could have significant physiological consequences for pituitary cell functions.

Effects of DTPA on zinc flux in leukemia cell lines

Previous studies from our laboratory with Zn‐65 have shown that H4IIE (rat hepatoma) and GH3 (rat pituitary tumor) cell lines reduce zinc efflux in the presence of the extracellular metal chelator DTPA (diethylenetriaminepentaacetic acid). Primary rat hepatocytes and anterior pituitary cells give out more zinc under the same conditions. This may reflect the greater zinc requirements of the rapidly dividing transformed cells. Here, we examined the effects of DTPA on radioactive Zn‐65 flux in C58(NT) (rat thymic lymphoma) and HL‐60 (human promyelocytic leukemia) cell lines. When these cells were co‐incubated with DTPA (50 µM) and Zn‐65, uptake of label was reduced. But when cells were pre‐labeled with Zn‐65 and then incubated with DTPA, the chelator increased efflux of Zn‐65. Despite being transformed, these cell lines behaved similarly to primary cells. Since the leukemia cells grow in suspension unlike the monolayer cell lines previously examined, we determined whether non‐adherence contributed to the difference in response to zinc deprivation. Experiments were repeated with H4IIE cells kept in suspension by growth in roller bottles. However, even under these conditions, DTPA enhanced retention of cellular zinc in the hepatoma cells. Thus, leukemia cells differ from other transformed cell lines in their homeostatic response to zinc deprivation and this is not due to their inability to form a monolayer.

Functional expression of CXCR4 in somatotrophs: CXCL12 activates GH gene, GH production and secretion, and cellular proliferation

The interaction of chemokine (C-X-C motif) ligand 12 (CXCL12) and its receptor CXCR4 may play an important role in the regulation of anterior pituitary function. In this study, we investigated the expression of CXCL12 and CXCR4 and their role in normal rat pituitary and GH-producing GH3 tumor cell line. RT-PCR analysis and immunohistochemistry revealed that CXCR4 was expressed in normal rat anterior pituitary and GH3 tumor cells. Double immunofluorescent staining showed the complete colocalization of CXCR4 with GH in rat pituitary, indicating that CXCR4 is specifically expressed in rat somatotrophs. Using rat primary pituitary cell cultures and GH releasing hormone receptor expressing stable GH3 cells (GH3-GHRHR), we evaluated the function of CXCL12 compared with GHRH. CXCL12 stimulated GH gene activation in both primary rat anterior pituitary cells and GH3-GHRHR cells. CXCL12 also stimulated GH secretion from primary rat pituitary cells in a dose-dependant manner. BrdU incorporation was increased in response to CXCL12 addition in GH3 cell culture, indicating CXCL12-induced cell proliferation. CXCL12-dependent phosphorylation of ERK1/2 was also confirmed by western blot analysis, supporting the evidence that MAPK is an intracellular mediator of CXCL12/CXCR4 interaction in GH3 cell proliferation. In conclusion, these results indicate that CXCL12/CXCR4 interaction plays an important role in GH production, secretion, and the proliferation of somatotrophs.

RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1α during Hypoxia

SUMO conjugation to proteins is involved in the regulation of diverse cellular functions. We have identified a protein, RWD-containing sumoylation enhancer (RSUME), that enhances overall SUMO-1, -2, and -3 conjugation by interacting with the SUMO conjugase Ubc9. RSUME increases noncovalent binding of SUMO-1 to Ubc9 and enhances Ubc9 thioester formation and SUMO polymerization. RSUME enhances the sumoylation of IkB in vitro and in cultured cells, leading to an inhibition of NF-kB transcriptional activity. RSUME is induced by hypoxia and enhances the sumoylation of HIF-1alpha, promoting its stabilization and transcriptional activity during hypoxia. Disruption of the RWD domain structure of RSUME demonstrates that this domain is critical for RSUME action. Together, these findings point to a central role of RSUME in the regulation of sumoylation and, hence, several critical regulatory pathways in mammalian cells.

Differential regulation of thyroid hormone receptor-mediated function by endocrine disruptors

It is well known that endocrine disruptors (EDs) act as anti-estrogenic agents and affect the function of reproductive organ. EDs are also thought to affect thyroid hormone (TH) system which is important for biological functions such as growth, development and metabolism. However, it is still not clear how EDs are able to regulate TH receptor (TR)-mediated functions. In this study, therefore, the modulatory effects of representative EDs such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyl (Aroclor 1254) and bisphenol A (BPA) were examined using TR-expressing GH3 cells (a rat pituitary gland epithelial tumor cell line) activated by triiodothyronine (T3). EDs tested significantly blocked T3 binding to TR in a dose-dependent manner. Biochemical characterization by Scatchard and Lineweaver-Burk plot analyses indicated that TCDD and aroclor 1254 bound to TH receptors in a competitive inhibitory manner, whereas BPA bound to TH receptors in a non-competitive pattern. The different inhibitory mode of action by EDs was also found in regulating TR-mediated production of prolactin (PRL). Aroclor 1254 exposure for 48 h enhanced T3-mediated PRL production, but BPA down-regulated. These results suggest that the EDs (TCDD, Aroclor 1254 and BPA) could differentially bind to TR and distinctly regulate the action of TR function, even though EDs are structurally similar.

T-Screen as a tool to identify thyroid hormone receptor active compounds

The T-Screen represents an in vitro bioassay based on thyroid hormone dependent cell proliferation of a rat pituitary tumour cell line (GH3) in serum-free medium. It can be used to study interference of compounds with thyroid hormone at the cellular level, thus bridging the gap between limitations of assays using either isolated molecules (enzymes, transport proteins) or complex in vivo experiments with all the complex feedback mechanisms present. Compounds are tested both in the absence and presence of thyroid hormone (EC 50 concentration of T 3) to test for both agonistic and antagonistic potency. Thyroid hormones (3,3′-5-triiodothyronine: T 3 and 3,3′,5,5′-tetraiodothyroxine: T 4) and compounds resembling the structure of thyroid hormones (3,3′-5-triiodothyroacetic acid: Triac; 3,3′,5,5′-tetraiodothyroacetic acid: Tetrac) induced cell growth, with the rank order Triac > T 3 > Tetrac > T 4 (relative potency = 1.35 > 1 > 0.29 > 0.07), which is identical to published affinities of these compounds for nuclear thyroid hormone receptors. Exposure to 5,5′-diphenylhydantoin (DPH) in the presence of 0.25 nM T 3 resulted in up to 60% decreased cell growth at 200 μM DPH. No effect of DPH on basal metabolic activity of GH3 cells was observed at this concentration. Fentinchloride (IC 50 = 21 nM) decreased cell growth induced by 0.25 nM T 3, whereas parallel exposure to these concentrations in the absence of T 3 did not alter basal metabolic activities of GH3 cells. Apolar sediment extracts from the Dommel (34%) and Terneuzen (14%) decreased cell growth in the presence of 0.25 nM T 3, whereas the extract from Hoogeveen increased cell growth (26%) and the extract from North Sea Channel had no effect. The T-Screen proved to be a fast and functional assay for assessing thyroid hormone receptor active potencies of pure chemicals or environmental mixtures.

Anti-proliferative effects and cell death mediated by two isoforms of dopamine D2 receptors in pituitary tumor cells

Stable rat pituitary tumor cell lines expressing two isoforms of the dopamine D2 receptor, D2L (long) and D2S (short) (the GH3D2L and GH3D2S cell lines, respectively), were established, and the signaling pathway underlying the anti-proliferative and cell death effects of dopaminergic agonists was examined in these cells. After either dopamine or quinpirole treatment, the cell viability decreased significantly only in GH3D2L cells and GH3D2S cells, but not in GH3 cells where D2 receptors are absent. Treatment with haloperidol, a specific D2 receptor antagonist, rescued the dopamine-mediated decreased cell viability in both the GH3D2L and GH3D2S cells. Treatment of these cells with dopamine decreased the DNA synthesis rate, as demonstrated by the incorporation of 5-bromo-2′-deoxyuridine (BrdU). Dopamine-induced cell death was observed in the GH3D2L and GH3D2S cells, and was accompanied by DNA laddering and caspase-3 activation, which were blunted by haloperidol, indicating that dopamine-induced cell death in these cells is mediated by the dopamine D2 receptors. D2 receptor-mediated cell death in these cells correlated with the sustained and enhanced activation of p38 mitogen-activated protein kinase (MAPK) and the extracellular-signal regulated kinase (ERK)1/2 pathways. Treatment with SB203580, which is a specific p38 MAPK inhibitor and PD98059, which is an inhibitor of MEK1/ERK signaling, selectively abrogates dopamine-induced cell death. It was further shown that p38 MAPK and ERK activation was inhibited by the antioxidant, N-acetylcysteine (NAC), and that a treatment with haloperidol completely blocked the p38 and ERK activation induced by dopamine. These results suggest that dopamine induces an anti-proliferative effect and cell death via the dopamine D2 receptors, by means of the p38 MAPK and ERK pathways involving oxidative stress, in the pituitary tumor cells.

Change in expression of basic fibroblast growth factor mRNA in a pituitary tumor clonal cell line.

To study pituitary tumor formation, we used a rat pituitary tumor cell line, MtT/E, which was derived from an estrogen-induced rat prolactinoma. MtT/E cells are known not to produce any pituitary hormone; however, they do produce the Pit-1 protein, which is known to be a common transcription factor in thyrotropes, somatotropes, and mammotropes. Although MtT/E is a clonal cell line, it exhibits two distinct phenotypes, fibroblastic (F-) and epithelial (E-) cells. We obtained subclonal cell lines from MtT/E cells with characters similar to those of F- and E-cells and called them MtT/E-G1 and MtT/E-B3, respectively. To examine tumor formation by these cells, we implanted them into female Fischer rats. One month later, typical pituitary tumors had appeared in MtT/E-B3-implanted rats; however, tumor formation by MtT/E-G1 was delayed. Interestingly, the tumors formed by MtT/E-B3 cells were intensely vascularized. To examine changes in tumor cell morphology, we performed primary culture and found that spindle-shaped cells appeared. These spindle-shaped cells were immunopositive for the Pit-1 protein, which suggests that they originated from MtT/E-B3 cells. Interestingly, reverse transcriptase polymerase chain reaction showed that both tumors and the cells obtained in primary culture expressed basic fibroblast growth factor (bFGF). By contrast, the original MtT/E-B3 cells did not express bFGF. These results suggested that MtT/E-B3 cells show a change in phenotype during tumor formation; that is, epithelial-type cells change into bFGFexpressing fibroblastic cells. These phenomena, especially the appearance of bFGFexpressing cells in tumor tissue, may explain the extensive angiogenesis in the tumors formed by MtT/E-B3 cells.

Role of specific protein kinase C isozymes in mediating epidermal growth factor, thyrotropin-releasing hormone, and phorbol ester regulation of the rat prolactin promoter in GH4/GH4C1 pituitary cells.

Epidermal growth factor (EGF) and TRH both produce enhanced prolactin (PRL) gene transcription and PRL secretion in GH4 rat pituitary tumor cell lines. These agents also activate protein kinase C (PKC) in these cells. Previous studies have implicated the PKCepsilon isozyme in mediating TRH-induced PRL secretion. However, indirect studies using phorbol ester down-regulation to investigate the role of PKC in EGF- and TRH-induced PRL gene transcription have been inconclusive. In the present study, we examined the role of multiple PKC isozymes on EGF- and TRH-induced activation of the PRL promoter by utilizing general and selective PKC inhibitors and by expression of genes for wild-type and kinase-negative forms of the PKC isozymes. Multiple nonselective PKC inhibitors, including staurosporine, bisindolylmaleimide I, and Calphostin C, inhibited both EGF and TRH induced rat PRL promoter activity. TRH effects were more sensitive to Calphostin C, a competitive inhibitor of diacylglycerol, whereas Go 6976, a selective inhibitor of Ca(2+)-dependent PKCs, produced a modest inhibition of EGF but no inhibition of TRH effects. Rottlerin, a specific inhibitor of the novel nPKCdelta isozyme, significantly blocked both EGF and TRH effects. Overexpression of genes encoding PKCs alpha, betaI, betaII, delta, gamma, and lambda failed to enhance either EGF or TRH responses, whereas overexpression of nPKCeta enhanced the EGF response. Neither stable nor transient overexpression of nPKCepsilon produced enhancement of EGF- or TRH-induced PRL promoter activity, suggesting that different processes regulate PRL transcription and hormone secretion. Expression of a kinase inactive nPKCdelta construct produced modest inhibition of EGF-mediated rPRL promoter activity. Taken together, these data provide evidence for a role of multiple PKC isozymes in mediating both EGF and TRH stimulated PRL gene transcription. Both EGF and TRH responses appear to require the novel isozyme, nPKCdelta, whereas nPKCeta may also be able to transmit the EGF response. Inhibitor data suggest that the EGF response may also involve Ca(2+)-dependent isozymes, whereas the TRH response appears to be more dependent on diacylglycerol.

Iron lactate induction of pancreatic and endometrial proliferative lesions and a lack of increased tumors in a 104-week carcinogenicity study in F344 rats

Iron lactate has been used as a food additive for iron supplementation. The present study was conducted to determine whether it might have carcinogenic potential. A total of 150 male and 150 female Fischer 344 rats were divided into three groups and fed basal diet containing 0, 1 or 2% of iron lactate for 104 weeks. No iron lactate-induced tumors were observed in any groups, although the incidences of pancreatic acinar cell and endometrial gland hyperplasias were increased in males and females, respectively, in the 2% group. Thus our in vivo animal data indicate that iron lactate lacks carcinogenicity in male and female F344 rats. However, estrogenic effects might be concluded based on the data for endometrial lesions. In a second experiment, an estrogen responsive rat pituitary tumor cell line, MtT/Se, and a human breast cancer cell line, MCF-7, were therefore employed to examine the estrogenic potential of iron lactate with regard to receptor binding affinity and ERE-reporter gene activation. Results in both cases were negative. Further investigations are needed to elucidate the mechanisms of induction of pancreatic and endometrial proliferative lesions by iron lactate.