Pituitary Adenoma Cell Line Research Articles

Role of stromal cell-derived factor 1 (SDF1/CXCL12) in regulating anterior pituitary function

Chemokines are key factors involved in the regulation of immune response, through the activation and control of leukocyte traffic, lymphopoiesis and immune surveillance. However, a large number of chemokines and their receptors are expressed in central nervous system (CNS) cells, either constitutively or induced by inflammatory stimuli, playing a role in many neuropathological processes. Stromal cell-derived factor 1 (SDF1) is a chemokine whose extra-immunological localization and functions have been extensively studied. SDF1 and its receptor CXCR4 were identified in both neurons and glia of many brain areas, including the hypothalamus, as well as at the pituitary level. Importantly, SDF1 and CXCR4 expression is increased in brain tumors in which their activity induced tumor cell proliferation and brain parenchyma invasion. Despite their localization, to date very few reports addressed the role of CXCR4 and SDF1 in the modulation of the hypothalamus/pituitary axis and their possible involvement in the development of pituitary adenomas. In this review, we discuss previous literature data on the role of chemokines in normal and adenomatous pituitary cells, focusing on recent data from our group showing that CXCR4 activation controls proliferation and both prolactin and GH release in the pituitary adenoma cell line GH4C1 through a complex network of intracellular signals. Thus, the SDF1/CXCR4 system together with other chemokinergic ligand-receptor pairs, may represent a novel regulatory pathway for pituitary function and, possibly, be involved in pituitary adenoma development. These lines of evidence suggest that the inhibition of chemokine receptors may represent a novel pharmacological target for the treatment of pituitary adenomas.

Down-regulation of leucine-rich repeats and immunoglobulin-like domain proteins (LRIG1-3) in HP75 pituitary adenoma cell line

Three human leucine-rich repeats and immunoglobulin-like domains (LRIG) genes and proteins, named LRIG1-3, has been previously characterized and it was proposed that they may act as suppressors of tumor growth. The LRIG1 protein can inhibit the growth of tumors of glial cells and the down-regulation of the LRIG1 gene may be involved in the development and progression of the tumor. Real-time reverse transcription-polymerase chain reaction (RT-PCR) is a recently developed technique for quantitative assessment of specific RNA levels. In the current study, it was demonstrated that LRIG1-3 and EGFR mRNA was detected in human pituitary adenoma cell lines and a normal pituitary sample, with differences in the expression levels. Compared to the normal pituitary samples, the expression of LRIG1-3 in HP75 cell line was lower, but the expression of EGFR in HP75 cell line was higher. The results are consistent with LRIG1-3 being tumour suppressor genes, and LRIG genes decreasing the expression of EGFR. The ratio of EGFR/LRIG1 was increased at least 13-fold in HP75 cells compared with the normal pituitary cells, which was also the case for the ratio of EGFR/LRIG2 (14-fold increase in HP75) and EGFR/LRIG3 (11-fold increase in HP75). Further studies were needed to elucidate the explicit role of LRIG genes as negative regulators of oncogenesis in human pituitary adenoma.

Elevated Cell Invasion Is Induced by Hypoxia in a Human Pituitary Adenoma Cell Line

Pituitary adenoma tissues are hypovascular, and have a lower partial oxygen pressure compared with neighboring normal organs. In this study, we investigated whether hypoxia influences the cell invasiveness of the human pituitary adenoma cell line, HP-75. HP-75 cells were exposed to hypoxic (1-10% oxygen) or normoxic (21% oxygen) conditions for 24 hours. Gelatin and reverse zymogram assays were used to determine the enzyme activities of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP). Cell adhesion and Matrigel cell invasion were examined with a Boiden chamber. Finally, the mRNA gene expression profiles of cells exposed to hypoxia or normoxia were examined by cDNA microarray and confirmed with real-time RT-PCR and flow cytometry. The gelatin and reverse zymograms revealed that the activities of MMP and TIMP were not significantly altered by hypoxia. Matrigel cell invasion and cell adhesion to Matrigel or collagen type IV were increased by hypoxia (3.8- and 4.8-fold, respectively). The cDNA microarray analysis revealed that laminin β2 chain mRNA was specifically up-regulated under hypoxic conditions (4.96-fold). Finally, real-time RT-PCR and flow cytometry verified the elevated expression of laminin β-2 chain at the mRNA and protein levels under hypoxic conditions. RNA interference with siRNA targeting laminin β2 inhibited Matrigel invasion and adhesion to collagen type IV in a dose-dependent manner. Collectively, these results suggested that hypoxia (1% oxygen) enhanced the cell invasion properties of a pituitary adenoma cell line in association with elevated expression of laminin β2 and enhanced binding to collagen type IV.

Elevated Cell Invasion Is Induced by Hypoxia in a Human Pituitary Adenoma Cell Line

Pituitary adenoma tissues are hypovascular, and have a lower partial oxygen pressure compared with neighboring normal organs. In this study, we investigated whether hypoxia influences the cell invasiveness of the human pituitary adenoma cell line, HP-75. HP-75 cells were exposed to hypoxic (1-10% oxygen) or normoxic (21% oxygen) conditions for 24 hours. Gelatin and reverse zymogram assays were used to determine the enzyme activities of matrix metalloproteinases (MMP) and tissueinhibitor of metalloproteinases (TIMP). Cell adhesion and Matrigel cell invasion were examined with a Boiden chamber. Finally, the mRNA gene expression profiles of cells exposed to hypoxia or normoxia were examined by cDNA microarray and confirmed with real-time RT-PCR and flow cytometry. The gelatin and reverse zymograms revealed that the activities of MMP and TIMP were not significantly altered by hypoxia. Matrigel cell invasion and cell adhesion to Matrigel or collagen type IV were increased by hypoxia (3.8- and 4.8-fold, respectively). The cDNA microarray analysis revealed that laminin β2 chain mRNA was specifically up-regulated under hypoxic conditions(4.96-fold). Finally, real-time RT-PCR and flow cytometry verified the elevated expression of laminin β-2 chain at the mRNA and protein levels under hypoxic conditions. RNA interference with siRNA targeting laminin β2 inhibited Matrigel invasion and adhesion to collagen type IV in a dose-dependent manner. Collectively, these results suggested that hypoxia (1% oxygen) enhanced the cell invasion properties of a pituitary adenoma cell line in association with elevated expression of laminin β2 and enhanced binding to collagen type IV.

Matrix Metalloproteinase-9 Is Differentially Expressed in Nonfunctioning Invasive and Noninvasive Pituitary Adenomas and Increases Invasion in Human Pituitary Adenoma Cell Line

The complete resection of pituitary adenomas (PAs) is unlikely when there is an extensive local dural invasion and given that the molecular mechanisms remain primarily unknown. DNA microarray analysis was performed to identify differentially expressed genes between nonfunctioning invasive and noninvasive PAs. Gene clustering revealed a robust eightfold increase in matrix metalloproteinase (MMP)-9 expression in surgically resected human invasive PAs and in the (nonfunctioning) HP75 human pituitary tumor-derived cell line treated with phorbol-12-myristate-13-acetate; these results were confirmed by real-time polymerase chain reaction, gelatin zymography, reverse transcriptase-polymerase chain reaction, Western blot, immunohistochemistry, and Northern blot analyses. The activation of protein kinase C (PKC) increased both MMP-9 activity and expression, which were blocked by some PKC inhibitors (Gö6976, bisindolylmaleimide, and Rottlerin), PKC-alpha, and PKC-delta small interfering (si)RNAs but not by hispidin (PKC-beta inhibitor). In a transmembrane invasion assay, phorbol-12-myristate-13-acetate (100 nmol/L) increased the number of invaded HP75 cells, a process that was attenuated by PKC inhibitors, MMP-9 antibody, PKC-alpha siRNA, or PKC-delta siRNA. These results demonstrate that MMP-9 and PKC-alpha or PKC-delta may provide putative therapeutic targets for the control of PA dural invasion.

Localization of vascular endothelial growth factor (VEGF) receptors in normal and adenomatous pituitaries: detection of a non-endothelial function of VEGF in pituitary tumours

As for any solid tumour, pituitary adenoma expansion is dependent on neovascularization through angiogenesis. In this process, vascular endothelial growth factor (VEGF) and its receptors VEGFR-1, VEGFR-2 and neuropilin-1 (NRP-1) may play an outstanding role. The intention of this work was to study the expression/localization and possible function of VEGF receptors in pituitary adenomas. VEGF receptor mRNA and protein expression was studied by in situ hybridization, immunohistochemistry and RT-PCR in 6 normal human pituitaries, 39 human pituitary adenomas and 4 rodent pituitary adenoma cell lines. VEGFR-1 expressing somatotroph MtT-S cells were used as a model to study the role of VEGF on cell proliferation and to elucidate the underlying mechanism of action. In normal pituitaries, VEGFR-1 was detected in endocrine cells, whereas VEGFR-2 and NRP-1 were exclusively expressed in endothelial cells. In pituitary tumours, a heterogeneous VEGFR expression pattern was observed by IHC. VEGFR-1, VEGFR-2 and NRP-1 were detected in 24, 18 and 17 adenomas respectively. In the adenomas, VEGFR-1 was expressed in epithelial tumour cells and VEGFR-2/NRP-1 in vessel endothelial cells. Functional studies in VEGFR-1-positive MtT-S cells showed that the ligands of VEGFR-1 significantly stimulated cell proliferation. This effect was mediated through the phosphatidylinositol-3-kinase-signalling pathway and involves induction of cyclin D1 and Bcl-2. Based on our results, we speculate that the ligands of VEGF receptors, such as VEGF-A and placenta growth factor, not only play a role in angiogenesis in pituitary adenomas, but also affect the growth of pituitary tumour cells through VEGFR-1.

Enhancement of pituitary adenoma cell invasion and adhesion is mediated by discoidin domain receptor-1

The discoidin domain receptor-1 (DDR1) tyrosine kinases are a family of cell surface receptors that bind to several types of collagen and facilitate cell adhesion that is known association with several cancers. However, no previous study has examined the expression and function of DDR1 in pituitary adenoma. Tissue microarray analysis of DDR1 expression levels in 52 pituitary adenoma tissues revealed that DDR1 expression was significantly related to hormonal background (Kruskal-Wallis test; P < 0.0001). To further elucidate the function of DDR1 in pituitary adenoma, we developed DDR1 over- and under-expressing cell lines using DDR1 clone transfection and short interfering ribonucleic acids (siRNA)-based DDR1 gene silencing, respectively, in a human pituitary adenoma cell line (HP-75). Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting confirmed that expression of both DDR1 isoforms (DDR1a and DDR1b) was elevated by clone transfection and diminished by siRNA. Matrigel invasion assays revealed that cell invasion was increased in HP-75 cells over-expressing DDR1 and decreased in cells under-expressing DDR1. Consistent with this, zymography revealed that the activation levels of matrix metalloproteinase (MMP)-2 and -9 were increased and decreased in cells over- and under-expressing DDR1, respectively. Examination of in vitro cell adhesion to collagen types I, II, III, and IV with respect to MMP-2 and -9 expression revealed that DDR1 regulated cell adhesion to collagen type I, which was responsible for accelerating secretion of MMP-2 and -9 in DDR1 over-expressing cells. Taken together, these results strongly suggest that DDR1 mediates cell invasion-related signaling between collagen type I and MMP-2 and -9 in pituitary adenoma cells.

Anti-Apoptotic Action by Hypoxia Inducible Factor 1-Alpha in Human Pituitary Adenoma Cell Line, HP-75 in Hypoxic Condition

Hypoxia-inducible factor-1 (HIF-1) alpha is the major transcription factor involved in the adaptive response to hypoxia. The purpose of this study was to investigate whether HIF 1-alpha protects HP75 cells, pituitary adenoma cell line from hypoxia induced apoptosis. HP75 was transfected with siRNA targeting HIF 1-alpha mRNA sequences or scrambled RNA duplexes, followed by subjected to hypoxia (1% oxygen) for 24 h, compared with normoxia (21%). The efficacy of RNAi was assessed via real-time RT-PCR and immunohistochemistry. Apoptosis was determined by Tdt-mediated dUTP nick end-labeling (TUNEL) assay and agarose gel electrophoresis. Membrane cDNA microarray was examined to detect gene profiling among the cell in normoxia, hypoxia, or hypoxia following the RNAi. A significantly greater proportion of HP75 cells transfected with specific siRNA duplexes and subsequently exposed to hypoxia demonstrated apoptosis to a large extent when compared with non-transfected cells. Transfection with specific siRNA duplexes knocked down HIF 1-alpha mRNA and protein expression in hypoxia-exposed cells by approximately 80%, whereas transfection with scrambled siRNA duplexes had no noticeable effect on HIF 1-alpha expression. Microarray analysis indicated that HIF1-alpha down-regulated caspase-10. These findings strongly suggest that HIF 1-alpha exerts an antiapoptotic role in HP75 in hypoxia.

Chemokine Stromal Cell-Derived Factor 1α Induces Proliferation and Growth Hormone Release in GH4C1 Rat Pituitary Adenoma Cell Line through Multiple Intracellular Signals

We used GH4C1 cells as a model to study the effects of the chemokine stromal cell-derived factor 1 (SDF1) in pituitary functions. In these cells, SDF1alpha induced proliferation and growth hormone secretion, suggesting a possible regulatory role for this chemokine at pituitary level. We evaluated the intracellular signaling involved in these effects: SDF1alpha increased cytosolic [Ca(2+)] and activated Pyk2, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) channels. To correlate these intracellular effectors with the proliferative and secretory effects, we inhibited their activity using BAPTA-AM (Ca(2+) chelator), 2'-amino-3'-methoxyflavone (PD98059; a mitogen-activated protein kinase kinase inhibitor), salicylate (Pyk2 inhibitor), and tetraethyl ammonium (K(+) channel blocker). All of these compounds reverted SDF1alpha-induced proliferation, suggesting the involvement of multiple intracellular pathways. Conversely, only BAPTA-AM reverted growth hormone secretion. To identify a possible cross-talk and a molecular ordering among these pathways, we tested these antagonists on SDF1alpha-dependent activation of ERK1/2, Pyk2, and BK(Ca) channels. From these experiments, we observed that the inhibition of [Ca(2+)](i) increase or BK(Ca) channel activity did not affect ERK1/2 activation by SDF1alpha; Pyk2 activation was purely Ca(2+)-dependent, not involving ERK1/2 or BK(Ca) channels; and BK(Ca) channel activity was antagonized by Pyk2 but not by ERK1/2 inhibitors. These data suggest that an SDF1alpha-dependent increase of [Ca(2+)](i) activates Pyk2, which in turn regulates BK(Ca) channel activity. Conversely, ERK1/2 activation is an independent phenomenon. In conclusion, we demonstrate that SDF1alpha causes both proliferation and growth hormone release from pituitary adenoma cells, suggesting that the activation of CXCR4 may represent a novel regulatory mechanism for growth hormone secretion and pituitary cell proliferation, which may contribute to pituitary adenoma development.

Secretoglobins in the human pituitary: high expression of lipophilin B and its down-regulation in pituitary adenomas

Secretoglobins are small secreted proteins, the expression of which has mostly been associated with secretory mucosal epithelia. Several secretoglobins have been implicated in the development of various human cancers. Allelic deletions of chromosome 11q13 correlates with the invasiveness of pituitary tumors. Intriguingly, several secretoglobin genes are located on 11q13; however, for most of these genes the expression in the pituitary and pituitary tumors have not been investigated. Antibodies specific for the secretoglobin lipophilin B (SCGB1D2, BU101) were developed and used in an immunohistochemical analysis of a human normal tissue microarray. Prominent lipophilin B immunoreactivity was found in the secretory cells of the anterior pituitary. Eight of nine analyzed pituitary adenomas showed a reduction in lipophilin B immunoreactivity compared to normal pituitary. However, there was no apparent association between lipophilin B immunoreactivity and hormone production or tumor invasiveness. Expression of eight different secretoglobin mRNAs were analyzed in normal pituitary and the pituitary adenoma cell line HP75 by highly specific quantitative real-time reverse transcription-PCR assays. Lipophilins B and C (SCGB2A1, mammaglobin B) were the most prominently expressed secretoglobin mRNAs in the pituitary. No secretoglobin mRNA was detected in the HP75 cells. The present report demonstrates, for the first time, lipophilin B expression in the pituitary and its apparent down-regulation in pituitary adenomas.

Biological Significance of Lowered Tissue Oxygen Pressure in Pituitary Adenomas: A Review Article

Hypoxia, the disruption of oxygen homeostasis induced by low oxygen supply, is critical in the development and progression of a large number of tumors. Various solid tumors are basically in a hypoxic condition, when growth exceeds vascular supply. Under such conditions, cellular oxygen concentration redirects cellular biosynthetic pathways to promote adaptation and enable survival. Recently, a transcriptional factor called hypoxia-inducible factor (HIF)-1α has been shown to play a crucial role in the regulation of many genes involved in the hypoxia adaptive pathway, especially vascular endothelial growth factor (VEGF). Under hypoxic conditions, many tumor cells promote angiogenesis via HIF-1α. Meanwhile pituitary tumors are solid tumors in which the regional oxygen saturation is lower than that of normal pituitary gland and the vasculature is usually poor. Despite expression of HIF-1α was confined in pituitary adenomas, its function driving to angiogenesis, apoptotic induction, and cell invasion, even though these issues have been extended in the other malignant tumors in recent years, has not yet been discussed. We have investigated the expression of microvascular density, HIF-1α, and VEGF in primary human pituitary adenomas focusing on the co-localization, and subsequently in vitro study, elucidated gene profiling regulated by HIF-1α. Our previous studies indicated that HIF-1α immunoreactivity was confined to the nucleoplasm, but was present in both tumor cells and vascular endothelial cells. There was no difference in microvascular density by histotype. ACTH-producing adenomas showed the lowest level of HIF-1α, whereas PRL-producing adenomas and HIF-1α-positive microvessels showed the highest (p<0.001). There was no significant correlation in the expression levels of HIF-1α mRNA and VEGF mRNA in pituitary adenomas. Both of HIF-1α and VEGF proteins expressed in pituitary adenoma and they were, in part, co-localized. Transfection with specific siRNA duplexes knocked down HIF1-α mRNA and protein expression in hypoxia-exposed cells by approximately 80%. Microarray analysis indicated that HIF1-α down-regulated caspase-10, but up-regulated of laminin β2 (4.26 folds), SAP90 (3.34 folds), and BNIP3 (3.24 folds). Conclusively in these poorly vascularized tumors, HIF-1α may not mainly regulate the VEGF expression in pituitary adenoma. In vitro studies strongly suggest that HIF 1-α exerts an antiapoptotic role in HP-75 in hypoxia mediated by down-regulation of caspase-10 and that hypoxia can potentially enhance the cell invasion properties of a pituitary adenoma cell line through elevated expression of laminin β-2. The mechanism of tumor angiogenesis and cell invasion in pituitary adenomas may differ from that in the other cancer cells.

Bromocriptine-induced apoptosis in pituitary adenoma cells: relationship to p53 and bcl-2 expression.

In an attempt to understand the roles of the tumour suppressor gene p53 and the proto-oncogene bcl-2 in cell death and survival in pituitary adenomas, we investigated the relationship of their expression to the apoptotic response of two pituitary adenoma cell lines (GH3 and AtT-20) to bromocriptine. An MTT (3-4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasolium bromide) assay was performed after treatment with bromocriptine for various periods of time over a range of concentrations to determine the effect of this drug on cell growth. Bromocriptine inhibited growth of GH3 and AtT-20 cells in a dose dependent manner. DNA fragmentation was assessed in GH3 and AtT-20 cells exposed to 10 ug/ml bromocriptine- for 48 h and 72 h. The DNA of GH3 and AtT-20 cells showed nucleosomal fragmentation, indicative of apoptosis. When assayed 2 days after adding bromocriptine, approximately 60% of GH3 and 58% of AtT-20 cells treated with bromocriptine displayed typical apoptotic morphology, including condensed chromatin and fragmented nuclei. There was a time dependent increase in the proportion of all tumour cells undergoing apoptosis. Decreased expression of bcl-2 and accumulation of wild-type p53 were associated with bromocriptine induced apoptosis in pituitary adenoma cells. DNA analysis confirmed the results obtained by the protein study. Different expression of p53 and bcl-2 genes is consistent with the expression of these gene products. These findings show that bromocriptine activated wild-type p53 and suppressed bcl-2 favouring occurrence of apoptosis in pituitary adenoma cells. Copyright 1999 Harcourt Publishers Ltd.

Hypoxia Inhibits Expression of Prolactin and Secretion of Cathepsin-D by the GH4C1 Pituitary Adenoma Cell Line

Diminished oxygen concentration within growing tumors may stimulate neovascularization by inducing both up-regulation of angiogenic factors and down-regulation of antiangiogenic agents. A potentially important molecule in the growth of pituitary adenomas is prolactin (PRL), which can be cleaved by cathepsin-D to yield a 16-kDa form (16K-PRL) with potent antiangiogenic effects. We examined the expression of PRL in cultured GH4C1 pituitary adenoma cells after exposure to hypoxia (0.1% oxygen) for periods of 12 to 36 hours. In contrast to increased expression of the angiogenic factor vascular endothelial growth factor in hypoxic cells, PRL mRNA and levels of intracellular and secreted PRL were significantly reduced under hypoxia. The reduction was not attributable to a general suppression of either transcription or protein synthesis. Although 16K-PRL was not evident in conditioned medium at physiologic pH, lowering the pH to mimic the acidic tumor microenvironment resulted in generation of 16K-PRL, which was sharply reduced in medium drawn from hypoxic cells. Production of 16K-PRL was blocked by the cathepsin-D inhibitor pepstatin-A, and the reduced 16K-PRL formation in hypoxic-conditioned medium correlated with a decrease in secretion of cathepsin-D and its precursor, procathepsin-D. Thus, hypoxia acts upon GH4C1 cells to increase vascular endothelial growth factor expression, decrease PRL synthesis, and suppress conversion of PRL to 16K-PRL via inhibition of cathepsin-D proteolysis. These mechanisms may act in concert to stimulate angiogenesis in prolactinomas.

Expression and role of VEGF receptors in pituitary adenomas

Vascular endothelial growth factor (VEGF) is known to exert a highly specific mitogenic action on endothelial cells through two tyrosine kinase receptors: VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR) which are almost specifically expressed in endothelial cells. However, it has been shown that VEGF receptors may also be expressed by non-endothelial cells, especially by tumor cells. We have recently shown that pituitary adenoma cells produce variable amounts of VEGF and have now started to study the expression of VEGF receptors in normal human pituitary and pituitary adenomas by RT-PCR, in situ hybridisation and immunohistochemistry. Expression of VEGFR1 and VEGFR2 mRNA was found in normal pituitary and in the 49 adenomas (26 nonfunctioning, 9 somatotroph, 8 corticotroph and 6 lactotroph tumors) studied so far. The expression was variable and no correlation between VEGFR1 expression and tumor grade and vessels number was found. In normal pituitary, VEGFR1 immunoreactivity was observed to co-localize with ACTH-, FSH-, GH- and LH-secreting cells, but not in endothelial cells, suggesting that VEGF may affect function of endocrine cell types by paracrine mechanisms. In contrast, VEGFR2 immunoreactivity was detected only in the vascular formations, in both normal pituitary and pituitary adenomas. Immunohistochemical analysis for VEGFR2 in 33 pituitary adenomas (9 nonfunctioning, 7 somatotrophs, 6 corticotrophs and 11 lactotrophs) revealed immunoreactivity in the vascular formation of 18 tumors and we observed that somatotroph and corticotroph adenomas showed the highest number of cases with VEGFR2 immunoreactivity on vessels. However, we found no correlation between VEGFR2 expression and extent of vascularization. We have then observed that HP75, a human non-functioning pituitary adenoma cell line secreting gonadotropins, expresses both VEGFR-1 and –2 mRNA and human VEGF165 is able to stimulate cells proliferation in a dose-dependent manner. This finding could suggest again a possible paracrine/autocrine action of VEGF on endocrine and tumor cells.

Positive feedback of protein kinase C proteolytic activation during apoptosis.

In contrast with protein kinase Calpha (PKCalpha) and PKCepsilon, which are better known for promoting cell survival, PKCdelta is known for its pro-apoptotic function, which is exerted mainly through a caspase-3-dependent proteolytic activation pathway. In the present study, we used the rat GH3B6 pituitary adenoma cell line to show that PKCalpha and PKCepsilon are activated and relocalized together with PKCdelta when apoptosis is induced by a genotoxic stress. Proteolytic activation is a crucial step used by the three isoforms since: (1) the catalytic domains of the PKCalpha, PKCepsilon or PKCdelta isoforms (CDalpha, CDepsilon and CDdelta respectively) accumulated, and this accumulation was dependent on the activity of both calpain and caspase; and (2) transient expression of CDalpha, CDepsilon or CDdelta sufficed to induce apoptosis. However, following this initial step of proteolytic activation, the pathways diverge; cytochrome c release and caspase-3 activation are induced by CDepsilon and CDdelta, but not by CDalpha. Another interesting finding of the present study is the proteolysis of PKCdelta induced by CDepsilon expression that revealed the existence of a cross-talk between PKC isoforms during apoptosis. Hence the PKC family may participate in the apoptotic process of pituitary adenoma cells at two levels: downstream of caspase and calpain, and via retro-activation of caspase-3, resulting in the amplification of its own proteolytic activation.

Tamoxifen-induced growth arrest and apoptosis in pituitary tumor cells in vitro via a protein kinase C-independent pathway

Protein kinase C (PKC), a kinase family involved in cell signal transduction, is overexpressed in most pituitary adenoma cells. We studied the effect of tamoxifen, an estrogen receptor antagonist and also a protein kinase inhibitor, on pituitary tumor cell proliferation and the induction of apoptosis; and we compared its effects with those of another PKC inhibitor, staurosporine. Tamoxifen induced growth arrest and apoptosis in a mouse pituitary adenoma cell line, AtT20, and in low-passage human primary pituitary tumor cell cultures. Staurosporine also inhibited pituitary tumor cell growth. PKC activity in AtT20 cells was inhibited by staurosporine and by prolonged treatment with phorbol myristate acetate, which down-regulates PKC activity, but not by tamoxifen, at the dosages used to induce apoptosis. Our findings suggest that tamoxifen induces apoptosis in AtT20 cells independent of a classical PKC isozyme pathway.

Light-Induced Photoactivation of Hypericin Inhibits Cellular Growth in Pituitary Adenoma Cell Line AtT20/D16v-F2 (Hypericin Inhibits Cellular Growth of AtT20/D16v-F2)

Cultivation with 4-8 mumol/l hypericin (specific proteinkinase C inhibitor) activated by light induced high inhibition of the rate of HL-60 cell growth. When hypericin treated cells were not exposed to light growth inhibition was insignificant. Cultivation with light activated hypericin in concentration 16 mumol/l slightly inhibited growth rate of AtT20/D16v-F2 cells. AtT20/D16v-F2 cells did not proliferate in presence of light activated 32 mumol/l hypericin. Evidence of apoptosis was found in HL-60 cells treated with 1-8 mumol/l light activated hypericin, with maximum of apoptotic cells detected after 8 mumol/l light activated hypericin. Light activated hypericin induces both apoptosis and necrosis in dose and time dependent manners in HL-60 cells, but causes only necrosis in AtT20/D16v-F2 cells.

Involvement of p38 mitogen-activated protein kinase activation in bromocriptine-induced apoptosis in rat pituitary GH3 cells.

Bromocriptine, a dopamine D(2) receptor agonist, is a therapeutic agent for patients with prolactinoma and hyperprolactinemia. In this study we demonstrated that bromocriptine induced activation of p38 mitogen-activated protein (MAP) kinase, with concomitant induction of apoptosis in rat pituitary adenoma cell line GH3 cells. Treatment of GH3 cells for 48 h with bromocriptine increased the p38 MAP kinase activity up to 3- to 5-fold and simultaneously increased the number of apoptotic cells. Inclusion in the medium of SB212090 or SB203580, specific p38 MAP kinase inhibitors, completely abolished the bromocriptine-induced activation of p38 MAP kinase and significantly reduced the number of apoptotic cells. The bromocriptine-induced p38 MAP kinase activation was not prevented by S(-)-eticropride hydrochloride, a specific D(2) receptor antagonist. Treatment with either epidermal growth factor (EGF) or thyrotropin-releasing hormone (TRH), which stimulates p44/42 MAP kinase, rescued cells from the bromocriptine-induced apoptosis, with concomitant inhibition of the bromocriptine-induced p38 MAP kinase activation. These results suggest that bromocriptine induces apoptosis in association with p38 MAP kinase activation, and that the p44/42 MAP kinase signaling through EGF and TRH receptors has an opposing effect on p38 MAP kinase activation as well as on apoptosis induced with bromocriptine in GH3 cells.

Pituitary Adenylate Cyclase-Activating Polypeptide Inhibits Transforming Growth Factor-β1-Induced Apoptosis in a Human Pituitary Adenoma Cell Line

Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from hypothalamic tissues based on its ability to stimulate cAMP production in cultured anterior pituitary cells. Recent studies have suggested a functional role for PACAP in the apoptosis of brain cells. However, the role of PACAP in regulating apoptosis in human pituitary adenomas has not previously been examined. Analysis of the cultured human pituitary adenoma cell line HP75, which expresses all three major PACAP receptors, showed that both PACAP-38 and PACAP-27 inhibited TGF-beta1-induced apoptosis. Treatment with the PACAP receptor antagonists PACAP 6-38 (PACAP type I receptor antagonist) and (p-chloro-D-Phe(6), Leu(17))-VIP (PACAP type II receptor antagonist) blocked the effects of PACAP-38 on the inhibition of transforming growth factor-beta1 (TGF-beta1)-induced apoptosis, confirming the specificity of the role of PACAP. Treatment with forskolin but not phorbol 12-myristate 13-acetate (PMA) also inhibited TGF-beta1-induced apoptosis. TGF-beta1 treatment was associated with an increase in mitogen-activated protein kinase (MAP kinase) when analyzed by Western blotting, but PACAP inhibition of TGF-beta1-induced apoptosis was not associated with activation of MAP kinase. Immunocytochemical analysis of the cell cycle cyclin-dependent kinase inhibitor p27 showed that treatment with TGF-beta1, forskolin, PMA, and PACAP increased p27 expression in cultured HP75 cells. These results indicate that PACAP is a highly specific inhibitor of TGF-beta1-induced apoptosis in the HP75 human pituitary adenoma cell line and that PACAP, TGF-beta1, forskolin, and PMA all stimulate expression of the TGF-beta-regulated cell cycle protein p27 in the HP75 human pituitary adenoma cell line. The HP75 cell line can be used as a model to study the regulation of apoptosis in human pituitary cells.

Bromocriptine-induced apoptosis in pituitary adenoma cells: relationship to p53 and bcl-2 expression

In an attempt to understand the roles of the tumour suppressor gene p53 and the proto-oncogene bcl-2 in cell death and survival in pituitary adenomas, we investigated the relationship of their expression to the apoptotic response of two pituitary adenoma cell lines (GH3 and AtT-20) to bromocriptine. An MTT (3–4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasolium bromide) assay was performed after treatment with bromocriptine for various periods of time over a range of concentrations to determine the effect of this drug on cell growth. Bromocriptine inhibited growth of GH3 and ArT20 cells in a dose dependent manner. DNA fragmentation was assessed in GH3 and AtT-20 cells exposed to 10 ug/ml bromocriptinefor 48h and 72 h. The DNA of GH3 and ART-20 cells showed nucleosomal fragmentation, indicative of apoptosis. When assayed 2 days after adding bromocriptine, approximately 60% of GH3 and 58% of AtT-20 cells treated with bromocriptine displayed typical apoptotic morphology, including condensed chromatin and fragmented nuclei. There was a time dependent increase in the proportion of all tumour cells undergoing apoptosis. Decreased expression of bcl-2 and accumulation of wild-type p53 were associated with bromocriptine induced apoptosis in pituitary adenoma cells. DNA analysis confirmed the results obtained by the protein study. Different expression of p53 and bcl-2 genes is consistent with the expression of these gene products. These findings show that bromocriptine activated wild-type p53 and suppressed bcl-2 favouring occurrence of apoptosis in pituitary adenoma cells.