GH3 Rat Pituitary Tumor Cells Research Articles

Alcohol metabolite acetic acid activates BK channels in a pH-dependent manner and decreases calcium oscillations and exocytosis of secretory granules in rat pituitary GH3 cells.

Acetaldehyde and acetic acid/acetate, the active metabolites of alcohol (ethanol, EtOH), generate actions of their own ranging from behavioral, physiological, to pathological/cancerogenic effects. EtOH and acetaldehyde have been studied to some depth, whereas the effects of acetic acid have been less well explored. In this study, we investigated the effect of acetic acid on big conductance calcium-activated potassium (BK) channels present in GH3 rat pituitary tumor cells in more detail. In whole cell voltage clamp recordings, extracellular application of acetic acid increased total outward currents in a dose-dependent manner. This effect was prevented after the application of the specific BK channel blocker paxilline. Acetic acid action was pH-dependent-in whole cell current and single BK channel recordings, open probability (Po) was significantly increased by extracellular pH reduction and decreased by neutral or base pH. Acetic acid hyperpolarized the membrane potential, whereas acidic physiological solution had a depolarizing effect. Moreover, acetic acid reduced calcium (Ca2+) oscillations and exocytosis of growth hormone contained secretory granules from GH3 cells. These effects were partially prevented by BK inhibitors-tetraethylammonium or paxillin. In conclusion, our experiments indicate that acetic acid activates BK channels in GH3 cells which eventually contribute to acetic acid-induced membrane hyperpolarization, cessation of Ca2+ oscillations, and decrease of growth hormone release.

Iodoacetic Acid Disrupting the Thyroid Endocrine System in Vitro and in Vivo.

Exposure to drinking water disinfection byproducts (DBPs) is potentially associated with adverse developmental effects. Iodoacetic acid (IAA), an unregulated DBP, has been shown to be cytotoxic, mutagenic, genotoxic, and tumorigenic. However, its endocrine-disrupting effects remain unknown. This study evaluated the IAA-induced disruption of the thyroid endocrine system using in vitro and in vivo assays. Rat pituitary tumor GH3 cells were treated with IAA in the presence and absence of triiodothyronine (T3). IAA exposure significantly reduced T3-activated GH3 cell proliferation, indicating the antagonistic activity of IAA in vitro. Sprague-Dawley rats were also subjected to IAA treatment through oral gavage for 28 consecutive days. IAA exposure significantly down-regulated the mRNA expression levels of the thyrotropin receptor (TSHR), the sodium/iodide symporter (NIS), and type I deiodinase and simultaneously reduced the protein expression levels of TSHR and NIS. IAA exposure decreased T3 levels but increased the weights of hypothalamus and the levels of thyrotropin releasing hormone and thyrotropin. In addition, IAA induced the formation of smaller and more depleted follicles or even vacuolization in the thyroid. These results suggested that IAA potentially disrupts the thyroid endocrine system both in vitro and in vivo.

Suppression of mTOR pathway and induction of autophagy-dependent cell death by cabergoline.

Cabergoline (CAB), the first-line drug for treatment of prolactinomas, is effective in suppressing prolactin hypersecretion, reducing tumor size, and restoring gonadal function. However, mechanisms for CAB-mediated tumor shrinkage are largely unknown. Here we report a novel cytotoxic mechanism for CAB. CAB induced formation of autophagosome in rat pituitary tumor MMQ and GH3 cells at the early stage through inhibiting mTOR pathway, resulting in higher conversion rates of LC3-I to LC3-II, GFP-LC3 aggregation, and increased autophagosome formation. Interestingly, CAB treatment augmented lysosome acidification and resulted in impaired proteolytic degradation within autolysosomes. This blocked the autophagic flux, leading to the accumulation of p62 aggregation and undigested autolysosomes. Knockdown of ATG7, ATG5, or Becn1, could significantly rescue the CAB-mediated cell death of MMQ cells (p < 0.05). CAB-induced autophagy and blockade of autophagy flux participated in antitumoral action in vivo. In conclusion, our study provides evidence that CAB concomitantly induces autophagy and inhibits the autophagic flux, leading to autophagy-dependent cell death. These findings elucidate novel mechanisms for CAB action.

Influence of the length and positioning of the antiestrogenic side chain of endoxifen and 4-hydroxytamoxifen on gene activation and growth of estrogen receptor positive cancer cells.

Tamoxifen has biologically active metabolites: 4-hydroxytamoxifen (4OHT) and endoxifen. The E-isomers are not stable in solution as Z-isomerization occurs. We have synthesized fixed ring (FR) analogues of 4OHT and endoxifen as well as FR E and Z isomers with methoxy and ethoxy side chains. Pharmacologic properties were documented in the MCF-7 cell line, and prolactin synthesis was assessed in GH3 rat pituitary tumor cells. The FR Z-isomers of 4OHT and endoxifen were equivalent to 4OHT and endoxifen. Other test compounds used possessed partial estrogenic activity. The E-isomers of FR 4OHT and endoxifen had no estrogenic activity at therapeutic serum concentrations. None of the newly synthesized compounds were able to down-regulate ER levels. Molecular modeling demonstrated that some compounds would each create a best fit with a novel agonist conformation of the ER. The results demonstrate modulation by the ER complex of cell replication or gene transcription in cancer.

β-Arrestin 1 and 2 and G Protein-Coupled Receptor Kinase 2 Expression in Pituitary Adenomas: Role in the Regulation of Response to Somatostatin Analogue Treatment in Patients With Acromegaly

Recent in vitro studies highlighted G protein-coupled receptor kinase (GRK)2 and β-arrestins as important players in driving somatostatin receptor (SSTR) desensitization and trafficking. Our aim was to characterize GRK2 and β-arrestins expression in different pituitary adenomas and to investigate their potential role in the response to somatostatin analog (SSA) treatment in GH-secreting adenomas (GHomas). We evaluated mRNA expression of multiple SSTRs, GRK2, β-arrestin 1, and β-arrestin 2 in 41 pituitary adenomas (31 GHomas, 6 nonfunctioning [NFPAs], and 4 prolactinomas [PRLomas]). Within the GHomas group, mRNA data were correlated with the in vivo response to an acute octreotide test and with the GH-lowering effect of SSA in cultured primary cells. β-Arrestin 1 expression was low in all 3 adenoma histotypes. However, its expression was significantly lower in GHomas and PRLomas, compared with NFPAs (P < .01). GRK2 expression was higher in PRLomas and NFPAs compared with GHomas (P < .05). In the GHoma group, GRK2 expression was inversely correlated to β-arrestin 1 (P < .05) and positively correlated to β-arrestin 2 (P < .0001). SSA treatment did not affect GRK2 and β-arrestin expression in GHomas or in cultured rat pituitary tumor GH3 cells. Noteworthy, β-arrestin 1 was significantly lower (P < .05) in tumors responsive to octreotide treatment in vitro, whereas GRK2 and SSTR subtype 2 were significantly higher (P < .05). Likewise, β-arrestin 1 levels were inversely correlated with the in vivo response to acute octreotide test (P = .001), whereas GRK2 and SSTR subtype 2 expression were positively correlated (P < .05). In conclusion, for the first time, we characterized GRK2, β-arrestin 1, and β-arrestin 2 expression in a representative number of pituitary adenomas. β-Arrestin 1 and GRK2 seem to have a role in modulating GH secretion during SSA treatment.

Curcumin suppresses HIF1A synthesis and VEGFA release in pituitary adenomas

Curcumin (diferuloylmethane), a polyphenolic compound derived from the spice plant Curcuma longa, displays multiple actions on solid tumours including anti-angiogenic effects. Here we have studied in rodent and human pituitary tumour cells the influence of curcumin on the production of hypoxia inducible factor 1α (HIF1A) and vascular endothelial growth factor A (VEGFA), two key components involved in tumour neovascularisation through angiogenesis. Curcumin dose-dependently inhibited basal VEGFA secretion in corticotroph AtT20 mouse and lactosomatotroph GH3 rat pituitary tumour cells as well as in all human pituitary adenoma cell cultures (n=32) studied. Under hypoxia-mimicking conditions (CoCl(2) treatment) in AtT20 and GH3 cells as well as in all human pituitary adenoma cell cultures (n=8) studied, curcumin strongly suppressed the induction of mRNA synthesis and protein production of HIF1A, the regulated subunit of the hypoxia-induced transcription factor HIF1. Curcumin also blocked hypoxia-induced mRNA synthesis and secretion of VEGFA in GH3 cells and in all human pituitary adenoma cell cultures investigated (n=18). Thus, curcumin may inhibit pituitary adenoma progression not only through previously demonstrated anti-proliferative and pro-apoptotic actions but also by its suppressive effects on pituitary tumour neovascularisation.

Differential effects of nerve growth factor on expression of dopamine 2 receptor subtypes in GH3 rat pituitary tumor cells

Nerve growth factor (NGF) can increase expression of dopamine 2 receptors (D2R) in GH3 rat pituitary tumor cells (GH3 cells). As D2R exists in long (D2L) and short (D2S) isoforms, effects of NGF on D2R subtypes have not been accurately evaluated. In this study, we compared mRNA levels of D2R subtypes in GH3 cells treated with or without NGF with real-time RT-PCR. In addition, we also evaluated the relationship between GH3 cell growth after bromocriptine treatment and mRNA levels of D2R subtypes. We found that D2R total, D2L, and D2S mRNA in GH3 cells were significantly increased after NGF treatment, compared with the vehicle group. Moreover, NGF increased the ratio of D2S to D2L. GH3 cell survival rate after bromocriptine treatment was negatively correlated with D2R total mRNA, and D2S may be more potent than D2L in inhibiting cell growth. Cell apoptosis rate was highly elevated in GH3 cells treated with NGF and bromocriptine, compared with the control group or the group treated with NGF or bromocriptine alone. Our data provide preliminary evidence that the effect of NGF was more prominent on expression of D2S than D2L, in addition, D2S might have a greater impact suppressing GH3 cells growth than D2L.

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.

Nitric oxide prevents phosphorylation of neuronal nitric oxide synthase at Serine1412 by inhibiting the Akt/PKB and CaM-K�II signaling pathways

Neuronal nitric oxide synthase (nNOS) is an important regulatory enzyme in the central nervous system catalyzing the production of NO, which regulates multiple biological processes in the central nervous system. However, the mechanisms by which nNOS activity is regulated are not completely understood. In the present study, the effects of protein kinases on the phosphorylation of nNOS in GH3 rat pituitary tumor cells were evaluated. We show that phosphorylation of nNOS at Ser1412 could be induced by the phosphatidylinositol 3-kinase/protein kinase B (Akt/PKB) agonist insulin, the calcium/calmodulin-dependent protein kinase II (CaM-K II) agonist A23187 or the cAMP-dependent protein kinase A (PKA) agonist IBMX, respectively. The phosphorylation levels of nNOS at Ser1412, induced by activation of Akt/PKB or CaM-K II, but not by PKA signaling, were reduced by pre-treatment with the NO donor diethylamine-NONOate. This inhibitory effect could be reversed by addition of a reducing reagent, dithiothreitol. Furthermore, the levels of phosphorylation of nNOS at Ser1412, induced by Akt/PKB or CaM-K II but not by PKA signaling, were enhanced by inhibition of nNOS activity with 7-nitroindazole. These findings suggest that the activation of nNOS can be catalyzed by at least three protein kinases, Akt/PKB, CaM-K II or PKA. NO generated from nNOS feedback prevents the activation of nNOS by inhibiting either Akt/PKB or CaM-K II but not PKA signaling.

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.

P21 Cip1 restrains pituitary tumor growth

As commonly encountered, pituitary adenomas are invariably benign. We therefore studied protective pituitary proliferative mechanisms. Pituitary tumor transforming gene (Pttg) deletion results in pituitary p21 induction and abrogates tumor development in Rb(+/-)Pttg(-/-) mice. p21 disruption restores attenuated Rb(+/-)Pttg(-/-) pituitary proliferation rates and enables high penetrance of pituitary, but not thyroid, tumor growth in triple mutant animals (88% of Rb(+/-) and 72% of Rb(+/-)Pttg(-/-)p21(-/-) vs. 30% of Rb(+/-)Pttg(-/-) mice developed pituitary tumors, P < 0.001). p21 deletion also accelerated S-phase entry and enhanced transformation rates in triple mutant MEFs. Intranuclear p21 accumulates in Pttg-null aneuploid GH-secreting cells, and GH(3) rat pituitary tumor cells overexpressing PTTG also exhibited increased levels of mRNA for both p21 (18-fold, P < 0.01) and ATM (9-fold, P < 0.01). PTTG is abundantly expressed in human pituitary tumors, and in 23 of 26 GH-producing pituitary adenomas with high PTTG levels, senescence was evidenced by increased p21 and SA-beta-galactosidase. Thus, either deletion or overexpression of Pttg promotes pituitary cell aneuploidy and p53/p21-dependent senescence, particularly in GH-secreting cells. Aneuploid pituitary cell p21 may constrain pituitary tumor growth, thus accounting for the very low incidence of pituitary carcinomas.

Involvement of bone morphogenetic protein-4 in GH regulation by octreotide and bromocriptine in rat pituitary GH3 cells

Here we investigated roles of the pituitary bone morphogenetic protein (BMP) system in modulating GH production regulated by a somatostatin analog, octreotide (OCT) and a dopamine agonist, bromocriptine (BRC) in rat pituitary somatolactotrope tumor GH3 cells. The GH3 cells were found to express BMP ligands, including BMP-4 and BMP-6; BMP type-1 and type-2 receptors (except the type-1 receptor, activin receptor-like kinase (ALK)-6); and Smad signaling molecules. Forskolin stimulated GH production in accordance with cAMP synthesis. BRC, but not OCT, suppressed forskolin-induced cAMP synthesis by GH3 cells. Individual treatment with OCT and BRC reduced forskolin-induced GH secretion. A low concentration (0.1 microM) of OCT in combination with BRC (1-100 microM) exhibited additive effects on reducing GH and cAMP production induced by forskolin. However, a high concentration (10 microM) of OCT in combination with BRC failed to suppress GH and cAMP production. BMP-4 specifically enhanced GH secretion and cAMP production induced by forskolin in GH3 cells. BRC, but not OCT, inhibited BMP-4-induced activation of Smad1,5,8 phosphorylation and Id-1 transcription and decreased ALK-3 expression. Of note, in the presence of a high concentration of OCT, the BRC effects suppressing BMP-4-Smad1,5,8 signaling were significantly impaired. In the presence of BMP-4, a high concentration of OCT also attenuated the BRC effects suppressing forskolin-induced GH and cAMP production. Collectively, a high concentration of OCT interferes with BRC effects by reducing cAMP production and suppressing BMP-4 signaling in GH3 cells. These findings may explain the mechanism of resistance of GH reduction to a combination therapy with OCT and BRC for GH-producing pituitary adenomas.

Endocrine and Antineoplastic Actions of Growth Hormone-Releasing Hormone Antagonists

Potent antagonists of growth hormone-releasing hormone (GHRH) have been developed for the treatment of disorders caused by excessive GHRH or growth hormone (GH) production and for therapy of cancers. GHRH antagonists suppressed the release of GH and insulin-like growth factor (IGF)-I in transgenic mice overexpressing human (h) GHRH gene, an animal model of human acromegaly. It was also shown in GH3 rat pituitary tumor cells overexpressing the human pituitary GHRH receptor (pGHRH-R) that GHRH antagonists can inhibit c-AMP production and GH secretion through the human receptor. These observations indicate that GHRH antagonists could be used clinically in disorders characterized by excessive GHRH/GH secretion. Many recent studies demonstrate that GHRH antagonists can inhibit tumor growth by several mechanisms. By indirect action through pGHRH-Rs these antagonists suppress circulating GH/IGF-I level, which results in the inhibition of cancers that depend on GH and/or IGF-I as growth factors. However, GHRH antagonists are also effective inhibitors of tumor IGF-II production, which is a potent mitogen but independent of GH. GHRH antagonists can inhibit tumor cell proliferation by direct action on tumor cell receptors, suppressing the IGF-II and other growth factor production of tumor cells. In addition, various human tumors and tumor cell lines secrete GHRH peptide and respond to GHRH with proliferation. This finding suggests that GHRH functions as an autocrine growth factor and that GHRH antagonists can block its effects on tumor growth. Recently, we demonstrated the expression of hGHRH-R and its splice variants in various human cancers. Antiproliferative action of GHRH antagonists on these cancers indicates that the direct inhibitory effects of GHRH antagonists are mediated by tumoral GHRH receptors.

Methylmercury inhibition of type II 5′-deiodinase activity resulting in a decrease in growth hormone production in GH3 cells

We have recently reported that methylmercury (MeHg) inhibits type II iodothyronine 5′-deiodinase (D2) activity in mouse neuroblastoma NB41A3 cells. In the present study, we determined the biological significance of D2 inhibition by MeHg. GH3 rat pituitary tumor cells were treated with MeHg and D2 activity and production of a thyroid hormone-responsive gene product, growth hormone (GH) were analyzed. MeHg inhibited D2 activity and decreased thyroxine (T4)-induced, but not 3,3′,5-triiodothyronine (T3)-induced GH secretion in GH3 cells. Our results suggest that MeHg inhibition of D2 activity might be involved in the inhibition of GH production in GH3 cells. Thus, D2 inhibition could be a novel mechanism involved in MeHg toxicity.

The regulatory mechanism by which interleukin-6 stimulates GH-gene expression in rat GH3 cells

The present study was performed to elucidate the effect of interleukin (IL)-6 on the human GH (hGH)-gene expression in GH3 rat pituitary tumor cells using stable transfection of the hGH promoter fused to a luciferase reporter gene. Our results showed that IL-6 (10(2)-10(4) U/ml) stimulated GH secretion and synthesis, and promoted the luciferase expression in stably transfected GH3 cells with the maximal action of 1.99 times above the control by 10(4) U/ml IL-6. Among the inhibitors of signaling transduction pathways, mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor PD98059 (40 microM) and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 microM) completely blocked the stimulatory effect of IL-6. Western blot analysis demonstrated that IL-6 indeed increased the activation of phosphorylated MEK and p38 MAPK in GH3 cells. Neither overexpression of Pit-1 nor inhibiting Pit-1 expression affected IL-6 induction of hGH-promoter activity. To identify the DNA sequence that mediated the effect of IL-6, six deletion constructs of hGH promoter were created. The stimulatory effect of IL-6 was abolished following deletion of the -196 to -132 bp fragment. In conclusion, our data show that IL-6 promotes GH secretion and synthesis by rat pituitary GH3 cells. The stimulatory effect of IL-6 on hGH-gene promoter appears to require the activation of MEK and p38 MAPK, and a fragment of promoter sequence that spans the -196 to -132 bp of the gene, but may be unrelated to Pit-1 protein.

Stimulatory Effect of Interleukin-1β on Growth Hormone Gene Expression and Growth Hormone Release from Rat GH3 Cells

Our previous studies demonstrated that interferon gamma increases the human (h) growth hormone (GH) gene promoter activity in rat pituitary GH3 cells, and its regulatory mechanism may be different from the classical GH-releasing hormone-induced regulatory mechanism. Interleukin-1β (IL-1β) is thought to induce the release of GH by pituitary cells, but whether or not and by which mechanisms IL-1β regulates GH synthesis remains unclear. The purpose of our study was thus to investigate the effect of IL-1β on the hGH gene expression in GH3 rat pituitary tumor cells using stable transfection of the hGH promoter fused to a luciferase reporter gene. Our results showed that IL-1β (10–10⁴ U/ml) increased GH secretion and synthesis and that 10² to 10⁴ U/ml IL-1β promoted the luciferase expression in stable GH3 cells, with a maximal action of 1.61 times over that of controls. Among inhibitors of intracellular signaling transduction pathways, mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor PD98059 (40 µM) and p38 MAPK inhibitor SB203580 (5 µM)blocked completely the stimulatory effect of IL-1β, and the phosphoinositide 3-kinase inhibitor LY294002 (10 µM) blocked partially the induction of IL-1β. Western blot analysis demonstrated that IL-1β increased the activation of phosphorylated MEK and p38 MAPK in GH3 cells. Neither overexpression of Pit-1 nor inhibiting Pit-1 expression affected IL-1β induction of hGH promoter activity. To identify the DNA sequence that mediated the effect of IL-1β, six deletion constructs of hGH promoter were created. The stimulatory effect of IL-1β was abolished following deletion of the –196- to –132-bp fragment. In conclusion, our data show that IL-1β promotes GH secretion and synthesis by rat pituitary GH3 cells. The stimulatory effect of IL-1β on the hGH gene promoter appears to require the activation of MEK, p38 MAPK, and phosphoinositide 3-kinase and a fragment of promoter sequence that spans the –196- to –132-bp fragment of the gene, but is unrelated to the Pit-1 protein.

The Role of Poly (Adenosine 5′-Diphosphate-Ribose) Polymerase in the Response of Pituitary Tumor Cells to Reactive Oxygen Species

As an enzyme implicated in the stress response, we investigated poly(ADP-ribose) polymerase (PARP) in the response of GH3 rat pituitary tumor cells to oxidants. These cells are unusual in that they undergo rapid cell death (90 min) with low doses of the prooxidant, H2O2 (50-200 microm), whereas at higher doses (1 mm), death occurs some hours later (4-5 h). Measurement of PARP activity shows that low doses of H2O2 (50-200 microm) fail to increase the activity of PARP, whereas at 0.5 and 1 mm, the enzyme becomes activated. In parallel with the activation of PARP, cellular ATP concentrations fall at high H2O2 doses and the PARP inhibitors, 3-aminobenzamide and nicotinamide (NIC) partially prevent this fall. Using NIC to inhibit PARP activity, we show that treatment of cells with NIC before the addition of H2O2 (0.5-1 mm), results in rapid cell death (90 min). In contrast, prior exposure to H2O2 (0.5-1 mm) for 1 h, before withdrawal and exposure to 1 mm NIC, allows cell survival for many hours. These data suggest that PARP is involved in blocking rapid death of GH3 cells in response to oxidants. In contrast to other cell types tested here, in which inhibitor studies show that PARP is activated at low H2O2 doses and this decreases the extent of apoptosis, GH3 cells are unable to sufficiently activate PARP to prevent rapid cell death.

Diethylenetriaminepentaacetic acid enhances thyroid hormone action by a transcriptional mechanism.

Zinc is thought to be required as a structural component of the thyroid hormone (triiodothyronine, T3) receptor (TR). However, we have previously demonstrated that use of diethylenetriaminepentaacetic acid (DTPA) to restrict zinc availability to cultured cells actually potentiates rather than inhibits thyroid hormone action. In this article, the mechanisms underlying these effects of DTPA have been investigated. Treatment of GH3 rat pituitary tumor cells with DTPA in the presence of T3 resulted in twofold greater concentrations of growth hormone (GH) mRNA. Addition of actinomycin D to inhibit transcription showed that GH mRNA was actually less stable in the presence of DTPA, eliminating mRNA stabilization as a possible mechanism underlying this effect. Cycloheximide was able to block the induction by DTPA, showing a requirement for protein synthesis. Transient transfection of a GH promoter/luciferase reporter construct into GH3 cells revealed an inhibitory effect of DTPA on luciferase activity. However, when cells were stably transfected with the same construct, a T3-dependent stimulation of luciferase activity by DTPA was observed, mimicking the effects seen with the endogenous mRNA. Thus, the GH promoter does mediate the effects of DTPA, but stable integration into chromosomal material is required.

2-Methoxyestradiol Exhibits a Biphasic Effect on VEGF-A in Tumor Cells and Upregulation Is Mediated Through ER-α: A Possible Signaling Pathway Associated with the Impact of 2-ME2 on Proliferative Cells

2-Methoxyestradiol (2-ME2) was reported to elicit both stimulation and inhibition of tumor angiogenesis and growth depending on the dosage used. However, the mechanism(s) of the biphasic action of 2-ME2 has been elusive. Here we describe a regulatory role of vascular endothelial growth factor-A (VEGF-A) in the biphasic effects on estrogen receptor (ER)+ GH3 rat pituitary tumor cells and MCF-7 human breast tumor cells depending on the dosage of 2-ME2 used. We observed that acute exposure to 2-ME2, irrespective of dosage, did not alter cellular proliferation, but enhanced the VEGF-A mRNA level. As the treatment duration increased, biphasic effect was elicited. A concentration of 1 μM 2-ME2 increased both cell proliferation and VEGF-A levels in these cells, whereas higher doses exhibited reversed impact. A low dose of 2-ME2 also increased the VEGF-A mRNA expression in ER-α-transfected human mammary epithelial cells (HMECs). The effect was reversed in ER- cells. The enhanced expression of VEGF-A mRNA could be blocked by the pure estrogen antagonist, ICI 182,780, reveal that the upregulation of VEGF-A expression by 2-ME2 is mediated through ER-α. Furthermore, the biphasic effect of 2-ME2 on cell proliferation can be modulated by administrating VEGF-A antibodies or VEGF-A proteins. Studies also demonstrate that the VEGF-A protein, induced by 2-ME2, is functionally active and upregulates the proliferation of adjacent endothelial cells.

17α-Estradiol-induced VEGF-A expression in rat pituitary tumor cells is mediated through ER independent but PI3K-Akt dependent signaling pathway

17α-E 2, a weak estrogen exhibited both agonistic and antagonistic effects, and caused a time- and dose-dependent induction of VEGF-A mRNA expression in GH3 rat pituitary tumor cells. This effect was unaffected by the presence of the pure estrogen receptor antagonist ICI 182,780 but was specifically blocked by a protein synthesis inhibitor puromycin. Inhibition of phosphatidylinositol-3 kinase (PI3K) activity by wortmannin decreased the effect of 17α-E 2 on VEGF-A mRNA expression. This inhibitor also blocked the increase in phosphorylation of Akt induced by exposure to 17α-E 2. In contrast, exposure to the MAP kinase inhibitor, U0126, had no impact on 17α-E 2-induced VEGF-A mRNA expression. Taken together, these studies indicate that like potent estrogens 17α-E 2 up-regulates VEGF-A mRNA expression in estrogen responsive GH3 rat pituitary tumor cells, but this induction is not mediated through a classical estrogen receptor pathway. PI3K-Akt signaling pathway is required for the induction of VEGF-A mRNA in GH3 cells by 17α-E 2.