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Cell lines originating from neuroendocrine tumors (NETs) represent useful experimental models to assess the control of synthesis and release of different hormones and hormone-like peptides, to evaluate the mechanisms of action of these agents in target tissues at the cellular and subcellular levels, and to study cell proliferation and tumor development, as well as the effect of different drugs on these complex processes. To date, the understanding of NET biology (with regard to their mechanisms of hormone secretion, cell proliferation and metastatic spread) has been hampered by the lack of appropriate animal models or cell lines for their study. In the present review, we aim to summarize the recent in vitro/in vivo data regarding cell lines derived from NETs which are most frequently employed in experimental neuroendocrinology.

Non-functioning pituitary adenomas (NFPAs) comprise a heterogeneous group, which are considered the most common pituitary tumor. As no clinically hormone hypersecretion is apparent, non-functioning pituitary adenomas are often diagnosed only when they are large enough to cause tumor mass effects, such as hypopituitarism, visual field defects or headaches. Efficient medical therapy for NFPAs is currently unavailable and surgical treatment of these tumors is not always satisfactory. Characterization of signaling regulatory events in the context of NFPAs may enable the development of new attractive novel strategies. Although data regarding gene expression profiling of signaling pathways in NFPAs have accumulated, studies aimed at fine-classification of NFPAs-specific signaling regulatory mechanisms and feedback loops are scarce.

Mitogen-activated protein kinase kinase (MEK) is a dual-specificity protein kinase that is located primarily in the cellular cytosol, both prior to and upon mitogenic stimulation. The existence of a nuclear export signal in the N-terminal domain of MEK [Fukuda, M., Gotoh, I., Gotoh, Y. & Nishida, E. (1996) J. Biol. Chem. 271, 20024-20028] suggests that there are circumstances under which MEK enters the nucleus and must be exported. Using mutants of MEK, we show that the deletion of the nuclear export signal sequence from constitutively active MEK caused constitutive localization of MEK in the nucleus of COS7 and HEK-293T cells. However, when the same region was deleted from a catalytically inactive MEK, cytoplasmic localization was observed in resting cells, which turned nuclear upon stimulation. Confocal microscopy of COS7 cells expressing the above mutants showed localization of the active MEK in the nuclear envelope and also in the cell periphery. The differences in cellular localization between the wild-type and mutant MEKs are not due to severe changes in specificity because the recombinant, constitutively active MEK that lacked its N-terminal region exhibited the same substrate specificity as the wild-type MEK, both in vitro and in intact cells. Taken together, our results indicate that upon mitogenic stimulation, MEK, like extracellular signal responsive kinase and p90RSK, is massively translocated to the nucleus. Rapid export from the nucleus, which is mediated by the nuclear export signal, is probably the cause for the cytoplasmic distribution observed with wild-type MEK.

Sequential activation of protein kinases within the mitogen-activated protein kinase (MAPK) cascades is a common mechanism of signal transduction in many cellular processes. Four such cascades have been elucidated thus far, and named according to their MAPK tier component as the ERK1/2, JNK, p38MAPK, and ERK5 cascades. These cascades cooperate in transmitting various extracellular signals, and thus control cellular processes such as proliferation, differentiation, development, stress response, and apoptosis. Here we describe the classic ERK1/2 cascade, and concentrate mainly on the properties of MEK1/2 and ERK1/2, including their mode of regulation and their role in various cellular processes and in oncogenesis. This cascade may serve as a prototype of the other MAPK cascades, and the study of this cascade is likely to contribute to the understanding of mitogenic and other processes in many cell lines and tissues.

Background Erythropoietin-producing hepatocellular (EPH) receptors are the largest known family of receptor tyrosine kinases characterized in humans. These proteins are involved in tissue organization, synaptic plasticity, vascular development and the progression of various diseases including cancer. The Erythropoietin-producing hepatocellular receptor tyrosine kinase member EphB6 is a pseudokinase which has not attracted an equivalent amount of interest as its enzymatically-active counterparts. The aim of this study was to assess the expression of EphB6 in pituitary tumors. Methods and Results Human normal pituitaries and pituitary tumors were examined for EphB6 mRNA expression using real-time PCR and for EphB6 protein by immunohistochemistry and Western blotting. EphB6 was highly expressed in non-functioning pituitary neuroendocrine tumors (NF-PitNETs) versus the normal pituitary and GH-secreting PitNETs. EphB6 mRNA expression was correlated with tumor size. Conclusions Our results suggest EphB6 aberrant expression in NF-PitNETs. Future studies are warranted to determine the role and significance of EphB6 in NF-PitNETs tumorigenesis.

Nitric oxide (NO), a highly reactive free radical, has been identified as a neurotransmitter in the central and peripheral nervous system. NO synthase (NOS) is the enzyme responsible for NO production from l-arginine and plays an important role in regulating the release of several hypothalamic peptides. In the pituitary, NO was found to increase growth hormone (GH) secretion in several in vitro and in vivo models. However, its role in human GH regulation is unknown. The aim of this study was to investigate the regulatory effects of NO on human GH and prolactin secretion using primary cell cultures of human fetal pituitaries and cultured hormone-secreting adenomas. Incubation of the human fetal pituitaries (21–24 wk gestation) in the presence of sodium nitroprusside (SNP; 1 mM), a NO donor, for 4 h resulted in a 50–75% increase in GH secretion, similar to the stimulatory effect evoked by growth hormone-releasing hormone (GHRH) (10 nM). However, fetal PRL secretion was not affected by SNP. GH release was also stimulated (40–70% increase) by SNP in 60% of the cultured GH-secreting adenomas studied. SNP-induced GH release was inhibited in both fetal and adenomatous cells by PT10, a NO scavenger. The addition of cGMP (0.1–1 mM), the second messenger of multiple NO actions, enhanced fetal and adenomatous GH secretion by 55–95%. Neuronal NOS (nNOS) was expressed in normal (fetal and adult) human pituitary tissues and in GH-secreting adenomas. Examination of its functional expression using l-arginine (1 µM) yielded a 35% increase in GH release from cultured GH-secreting adenoma. This response was blocked by a NOS inhibitor with high selectivity for the neuronal enzyme and by a guanylyl cyclase inhibitor. In conclusion, NO stimulates human GH in cultured fetal pituitaries and GH-secreting adenomas. Cyclic GMP is probably involved in this hormonal regulation.

PurposesTo test if the antibody array strategy could be utilized to simultaneously detect the secretion of multiple growth factors by human pituitary GH-adenomas and to measure octreotide-induced alterations.MethodsSpecimens of human pituitary adenomas were cultured and incubated with or without octreotide for 24 h. Conditional media were analyzed by human growth factor antibody array and VEGF concentrations were measured by ELISA. Media were also analyzed for GH concentrations. p21 expression levels were examined by Western blot of the specimens lysates.ResultsThe antibody arrays successfully identified growth factors secreted by GH-adenomas in vitro. Octreotide treatment induced both elevations and reductions in growth factors secretion. GH response to octreotide was measured, and in this small-sized study resistant and sensitive GH-adenomas presented with no unique secretome pattern of each of the groups. Octreotide-induced VEGF alterations analyzed by the antibody array and by ELISA were not fully matched.ConclusionsThis study suggests that the broad proteomic strategy of antibody arrays may be utilized to study the growth factors secretion pattern of GH-adenomas and its regulation by somatostatin analogs or other compounds.