Acute Hypoxia Induces Enkephalin Production and Release in an Adrenergic Cell Line Model of Neonatal Chromaffin Cell Responses to Hypoxic Stress.

OBJECTIVE: Prior to maturation of the human sympathetic nervous system, the neonatal adrenal medulla senses and responds to hypoxia. In addition to catecholamine release, the adrenal medulla synthesizes and stores opioid peptides, notably enkephalin (ENK). However, it is not known whether acute hypoxia evokes adrenal ENK production and release, as seen in the central nervous system (CNS). We hypothesize that acute hypoxia stimulates synthesis and release of ENK in chromaffin cells. STUDY DESIGN: Cultures of adrenergic mouse pheochromocytoma cells (MPC) 10/9/96CR were incubated in 10% oxygen (O2) at intervals of up to 60 minutes. ENK content and release were measured by Met-ENK enzyme-linked immunosorbent assay (ELISA). ENK messenger ribonucleic acid (mRNA) was analyzed by quantitative reverse-transcriptase polymerase chain reaction (PCR). RESULTS: Incubation of MPC 10/9 cells in 10% O2 evoked rapid release of epinephrine and of Met-ENK which increased approximately twofold in 15 minutes. Reduced [O2] also induced an overall increase (14%) in cellular ENK peptide content within 60 minutes. Acute hypoxia-stimulated release of Met-ENK was accompanied by increased mRNAENK expression in MPC 10/9s, a cell culture model of adrenergic chromaffin cells. CONCLUSION: We speculate that the ability of reduced [O2] to evoke ENK release from chromaffin cells may influence blood pressure regulation and heart contractility, thereby providing an adaptive survival advantage during neonatal asphyxia.

Tyrosine hydroxylase, chromogranin A, and steroidogenic acute regulator as markers for successful separation of human adrenal medulla.

Progress in high throughput "-omic" techniques now allows the simultaneous measurement of expression levels of thousands of genes and promises the improved understanding of the molecular biology of diseases such as cancer. Detection of the dysfunction of molecular pathways in diseases requires healthy control tissue. This is difficult to obtain from pheochromocytomas (PHEOs), rare chromaffin tumors derived from adrenal medulla. The two options for obtaining adrenal tissue are: (1) whole organ removal post-mortem or during radical nephrectomy; (2) removal during PHEO surgery. Access to high quality normal adrenal tissue is limited. Removal of whole adrenals during nephrectomy is rare, because of improved surgical techniques. For adrenals removed post-mortem, the lag time to proper organ perfusion causes uncontrolled tissue degradation. Adjacent normal adrenal tissue can almost never be obtained from resected PHEOs, because they often replace the entire medulla or are well-encapsulated. If a margin of normal adrenal is attached to a resected PHEO, it seldom contains any medulla. The clean separation of medulla and cortex is further complicated, because their border is convoluted, and because adult adrenal consists of approximately 90% cortex. Thus, the quality of separation has to be evaluated with specific medullary and cortical markers. We describe the successful dissection of highly pure, medullary tissue from adrenals snap-frozen upon resection during radical nephrectomy or after brain death. Separation quality has been verified by quantitative reverse transcription with polymerase chain reaction for the medullary enzymes, tyrosine hydroxylase, and chromogranin A, and for the cortical enzyme, steroidogenic acute regulator.

A xenograft and cell line model of SDH-deficient pheochromocytoma derived from Sdhb+/- rats.

Tumors caused by loss-of-function mutations in genes encoding TCA cycle enzymes have been recently discovered and are now of great interest. Mutations in succinate dehydrogenase (SDH) subunits cause pheochromocytoma/paraganglioma (PCPG) and syndromically associated tumors, which differ phenotypically and clinically from more common SDH-intact tumors of the same types. Consequences of SDH deficiency include rewired metabolism, pseudohypoxic signaling and altered redox balance. PCPG with SDHB mutations are particularly aggressive, and development of treatments has been hampered by lack of valid experimental models. Attempts to develop mouse models have been unsuccessful. Using a new strategy, we developed a xenograft and cell line model of SDH-deficient pheochromocytoma from rats with a heterozygous germline Sdhb mutation. The genome, transcriptome and metabolome of this model, called RS0, closely resemble those of SDHB-mutated human PCPGs, making it the most valid model now available. Strategies employed to develop RS0 may be broadly applicable to other SDH-deficient tumors.

Targeting pheochromocytoma/paraganglioma with polyamine inhibitors.

BACKGROUND: Pheochromocytomas (PCCs) and paragangliomas (PGLs) are neuroendocrine tumors that are mostly benign. Metastatic disease does occur in about 10% of cases of PCC and up to 25% of PGL, and for these patients no effective therapies are available. Patients with mutations in the succinate dehydrogenase subunit B (SDHB) gene tend to have metastatic disease. We hypothesized that a down-regulation in the active succinate dehydrogenase B subunit should result in notable changes in cellular metabolic profile and could present a vulnerability point for successful pharmacological targeting. METHODS: Metabolomic analysis was performed on human hPheo1 cells and shRNA SDHB knockdown hPheo1 (hPheo1 SDHB KD) cells. Additional analysis of 115 human fresh frozen samples was conducted. In vitro studies using N1,N11-diethylnorspermine (DENSPM) and N1,N12- diethylspermine (DESPM) treatments were carried out. DENSPM efficacy was assessed in human cell line derived mouse xenografts. RESULTS: Components of the polyamine pathway were elevated in hPheo1 SDHB KD cells compared to wild-type cells. A similar observation was noted in SDHx PCC/PGLs tissues compared to their non-mutated counterparts. Specifically, spermidine, and spermine were significantly elevated in SDHx-mutated PCC/PGLs, with a similar trend in hPheo1 SDHB KD cells. Polyamine pathway inhibitors DENSPM and DESPM effectively inhibited growth of hPheo1 cells in vitro as well in mouse xenografts. CONCLUSIONS: This study demonstrates overactive polyamine pathway in PCC/PGL with SDHB mutations. Treatment with polyamine pathway inhibitors significantly inhibited hPheo1 cell growth and led to growth suppression in xenograft mice treated with DENSPM. These studies strongly implicate the polyamine pathway in PCC/PGL pathophysiology and provide new foundation for exploring the role for polyamine analogue inhibitors in treating metastatic PCC/PGL. PRéCIS: Cell line metabolomics on hPheo1 cells and PCC/PGL tumor tissue indicate that the polyamine pathway is activated. Polyamine inhibitors in vitro and in vivo demonstrate that polyamine inhibitors are promising for malignant PCC/PGL treatment. However, further research is warranted.

A Previously Unrecognized Monocytic Component of Pheochromocytoma and Paraganglioma.

We describe a consistently present, previously unrecognized, population of monocytes in pheochromocytomas and paragangliomas. Although sustentacular cells are generally recognized as a common component of these tumors, differential immunohistochemical staining for CD163 and S100 shows that monocytes can in fact be more numerous. These cells frequently resemble sustentacular cells topographically and cytologically, possibly explaining why they have not been previously noticed. They contribute to the tumor proteome and may have implications for tumor biology. No correlations were identifiable between the presence of these cells and any clinical characteristics of the tumors in the present study. A possible association with genotype is suggested by immunoblot showing high expression of CD163 protein in tumors with succinate dehydrogenase mutations.

Metastasis-associated gene expression profile of liver and subcutaneous lesions derived from mouse pheochromocytoma cells.

The development of metastatic cancer is associated with overexpression or downregulation of specific genes and cell regulatory pathways. Some of these genes and pathways may be involved in invasion and dissemination of tumor cells, while others may promote seeding, survival or growth of cells at specific distant sites. In this investigation, gene expression profiles of nonmetastasizing tumors generated by injecting mouse pheochromocytoma cells (MPCs) subcutaneously were compared to those of liver tumors generated by injecting the cells intravenously. Both were compared to the cultured parental cell line. Tumors in the liver have a route of spread, anatomical distribution, and growth environment similar to naturally metastasizing pheochromocytomas, while intravenous injection of cells bypasses the initial steps of metastasis occurring spontaneously from a primary tumor. Eight genes were upregulated in liver tumors, 15 in subcutaneous tumors and seven in both compared to the cultured cells. Using quantitative real-time PCR, expression of five genes (Metap2, Reck, S100a4, Timp2, and Timp3) was verified as significantly lower in liver tumors than in subcutaneous tumors. Downregulation of these genes has been previously been associated with malignancy of pheochromocytomas. These findings indicate that different microenvironments can differentially affect the expression of metastasis-related genes in pheochromocytomas, and that overexpression or underexpression of these genes need not be present when the tumor cells are initially disseminated. The hepatic localization of tumors formed by intravenously injected MPC cells and the tumors' gene expression profile resembling that of naturally occurring pheochromocytoma metastases support the use of this model to study pheochromocytoma metastasis.

Oncocytic adrenal cortical tumor with cytoplasmic inclusions and hyaline globules.

Adrenal cortical tumors, particularly oncocytic tumors, have been reported to contain a variety of intracytoplasmic and intramitochondrial inclusions. Oncocytic cortical tumors can also morphologically mimic pheochromocytomas. We report an unusual, partially oncocytic cortical neoplasm with nesting architecture, intranuclear inclusions, and hyaline globules reminiscent of pheochromocytoma, together with numerous, small, brightly eosinophilic, periodic acid-Schiff-positive cytoplasmic inclusions and typical cytoplasmic lipid droplets. Ultrastructural study revealed oncocytes containing numerous mitochondria with intramitochondrial crystals and lipid droplets. Immunohistochemistry and immunoblots were utilized to further characterize the tumor. Immunohistochemistry demonstrated immunoreactivity of both the eosinophilic inclusions and the hyaline globules for adipose differentiation-related protein (ADRP), which is one of a group of proteins associated with storage of neutral lipids in many cell types. Immunoblots confirmed the presence of ADRP and demonstrated an imbalance between ADRP and perilipin, another neutral lipid-associated protein, in tumor tissue compared to normal adrenal cortex. The findings suggest that mitochondrial dysfunction in oncocytic cortical tumors may lead to abnormal processing of proteins related to the lipid-storing functions of the adrenal cortex, resulting in unusual cytoplasmic inclusions and extracellular globules resembling the globules in pheochromocytomas. The finding of ADRP as a constituent of inclusions in adrenal cortical tumors has not been previously reported.

A unique model for SDH-deficient GIST: an endocrine-related cancer.

We describe a unique patient-derived xenograft (PDX) and cell culture model of succinate dehydrogenase-deficient gastrointestinal stromal tumor (SDH-deficient GIST), a rare mesenchymal tumor that can occur in association with paragangliomas in hereditary and non-hereditary syndromes. This model is potentially important for what it might reveal specifically pertinent to this rare tumor type and, more broadly, to other types of SDH-deficient tumors. The primary tumor and xenografts show a very high proliferative fraction, and distinctive morphology characterized by tiny cells with marked autophagic activity. It is likely that these characteristics resulted from the combination of the germline SDHB mutation and a somatic KRAS G12D mutation. The most broadly relevant findings to date concern oxygen and oxidative stress. In paragangliomas harboring SDHx mutations, both hypoxic signaling and oxidative stress are putative drivers of tumor growth. However, there are no models for SDH-deficient paragangliomas. This related model is the first from a SDHB-mutated human tumor that can be experimentally manipulated to study mechanisms of oxygen effects and novel treatment strategies. Our data suggest that tumor growth and survival require a balance between protective effects of hypoxic signaling vs deleterious effects of oxidative stress. While reduced oxygen concentration promotes tumor cell survival, a further survival benefit is achieved with antioxidants. This suggests potential use of drugs that increase oxidative stress as novel therapies. In addition, autophagy, which has not been reported as a major finding in any type of SDH-deficient tumor, is a potential target of agents that might trigger autophagic cell death.

Pathology of Human Pheochromocytoma and Paraganglioma Xenografts in NSG Mice.

A major impediment to the development of effective treatments for metastatic or unresectable pheochromocytomas and paragangliomas has been the absence of valid models for pre-clinical testing. Attempts to establish cell lines or xenografts from human pheochromocytomas and paragangliomas have previously been unsuccessful. NOD-scid gamma (NSG) mice are a recently developed strain lacking functional B-cells, T-cells, and NK cells. We report here that xenografts of primary human paragangliomas will take in NSG mice while maintaining their architectural and immunophenotypic characteristics as expressed in the patients. In contrast to grafts of cell lines and of most common types of primary tumors, the growth rate of grafted paragangliomas is very slow, accurately representing the growth rate of most pheochromocytomas and paragangliomas even in metastases in humans. Although the model is therefore technically challenging, primary patient-derived xenografts of paragangliomas in NSG mice provide a potentially valuable new tool that could prove especially valuable for testing treatments aimed at eradicating the small tumor deposits that are often numerous in patients with metastatic paraganglioma.

Case Report of a Prolactinoma in a Patient With a Novel MAX Mutation and Bilateral Pheochromocytomas.

Pheochromocytomas are neuroendocrine tumors that can arise sporadically or be inherited as a familial disease, and they may occur in isolation or as part of a multitumor syndrome. Familial disease typically presents in younger patients with a higher risk of multifocality. Recently, the tumor suppressor MYC-associated factor X (MAX) gene has been implicated as a cause of familial isolated pheochromocytoma and paraganglioma. We describe a patient with a pituitary prolactinoma and bilateral pheochromocytomas who tested positive for a germline MAX mutation. Interestingly, the patient also had mild primary hyperparathyroidism that resolved upon resection of the pheochromocytomas despite the absence of parathyroid hormone staining in the tumors. To our knowledge, this case is the first report of prolactinoma in a patient with a MAX mutation, which suggests the possibility of germline MAX mutations also contributing to the development of prolactinomas.

Gene expression profiling of rat pheochromocytoma.

Sporadic and syndrome-associated human pheochromocytomas exhibit a spectrum of common and distinctive phenotypic markers. Animal models may contribute to understanding of common denominators leading to development and progression of pheochromocytoma, and to mechanisms that underlie distinctive phenotypes. Rat pheochromocytomas are common, in contrast to their human counterparts, and their frequency is increased by a variety of genotoxic or nongenotoxic agents. Toxicological studies of rats are therefore a potentially rich source of information on pheochromocytoma biology. To compare the molecular profiles of rat and human pheochromocytomas and to identify pathways potentially involved in pathogenesis of rat pheochromocytomas, we conducted a gene expression profiling study comparing 31 pheochromocytomas obtained from the National Toxicology Program to normal adult rat adrenal medulla. The microarray chips were generated from 31,769-oligomer set representing over 27,200 unique Mouse Ensembl genes. The analysis showed over 1,900 genes that were up- or downregulated in the tumors. More than half of the former are involved in protein synthesis and signal transduction, including oncogenes of the RAS family and several heat shock proteins and chaperones. Downregulated genes included receptors and tumor-suppressor genes, including NF2 and Dmbt1. Specific genes related to neuroendocrine function were either upregulated or downregulated in subsets of tumors. Cross-comparison with a human pheochromocytoma database showed greater than 60% correlation. Results of this study reveal both generic and specific parallels between rat and human pheochromocytomas.

Nicotine stimulates expression of the PNMT gene through a novel promoter sequence.

Transcription of the gene encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28) accelerates in response to hormonal and neural stimuli. Cholinergic stimulation through neuronal nicotinic receptors constitutes the primary means for neural regulation of PNMT expression in the adrenal medulla (AM). Therefore, the regulatory sequence conveying responsiveness of the PNMT gene to nicotinic stimuli has been characterized in the 5' upstream region of the rat PNMT promoter. Functional analyses using nested deletion and substitution mutations of the PNMT promoter map the nicotine responsive region to a sequence spanning -633 to -595 bp, designated the PNMT nicotine-responsive element (NicRE). Sequences at the 5' (-633 to -620) and 3' (-599 to -595) ends of this region are essential to convey nicotine responsiveness to PNMT promoter constructs expressed in primary bovine chromaffin cells and in selected lines derived from mouse pheochromocytomas and human neuroblastomas. Profiles of nuclear proteins associating with PNMT promoter sequences also change following nicotine treatment of these cells. Electrophoretic mobility shift and DNase I footprinting analyses distinguish multiple sites of DNA-protein interactions within the NicRE region. Because the PNMT promoter does not contain a cAMP responsive element (the site through which nicotine stimulation is mediated for other catecholamine-synthesizing and AM genes), the NicRE of the PNMT gene must therefore be distinct. Thus, nicotinic cholinergic stimuli appear to regulate expression of the epinephrine-synthesizing gene PNMT through a previously uncharacterized regulatory element.

High prevalence of herpes simplex virus DNA in temporal arteritis biopsy specimens.

Giant cell arteritis (GCA) affecting the cranial arteries is a disease of unknown cause that causes blindness, stroke, and other morbidity. Its sudden onset and segmental distribution are suggestive of diseases that involve viral reactivation, and cranial arteries are known to be innervated by ganglia that harbor herpes simplex virus (HSV). We used a high-sensitivity polymerase chain reaction assay to test for HSV DNA in specimens from 39 consecutive temporal artery biopsies performed for suspected GCA. HSV DNA was detected in 21 (88%) of 24 histologically positive and 8 (53%) of 15 histologically negative specimens (P = .027; Fisher exact test). Analysis of 10 renal artery samples from age-matched control subjects using the same assay showed no detectable HSV DNA. We conclude that detectable HSV DNA is correlated with histologically confirmed GCA in this patient population.

Microarray-based comparative genomic hybridization of pheochromocytoma cell lines from neurofibromatosis knockout mice reveals genetic alterations similar to those in human pheochromocytomas.

Somatic genetic aberrations have been identified in both sporadic pheochromocytomas and those associated with familial tumor syndromes; however, individual variations between human tumors and the absence of in vitro human pheochromocytoma models hinder efforts to understand the roles of those aberrations in tumorigenesis. Pheochromocytomas occur frequently in neurofibromatosis knockout mice and we have recently developed cell lines from those tumors. The availability of multiple tumors from genetically identical animals provides a powerful tool for understanding the pathobiology of pheochromocytomas. For the present investigation, we performed a genomic scanning analysis of four mouse pheochromocytoma cell lines by standard cytogenetics and microarray-based comparative genomic hybridization in order to identify genetic common denominators. All of the lines showed losses of most or all of chromosome 9; three lines lost most or all of chromosome 4. Mouse chromosome 4 is homologous to human chromosome 1p, which is the most frequent deletion in human pheochromocytomas. Mouse chromosome 9 shows large areas of homology to human 3p, 3q, and 11q, which are also frequently deleted. These comparisons suggest that genetic mechanisms in the genesis of pheochromocytomas may be similar across species. Additional changes that may be specific to this model included complete or partial gains of chromosome 12 as seen in 3 of the 4 lines analyzed by array CGH.

Hypoxia activates multiple transcriptional pathways in mouse pheochromocytoma cells.

Mouse pheochromocytoma cells (MPCs) provide an excellent model system for investigating the effects of hypoxia on catecholamine enzyme genes and on transcription factors mediating stress responses. RT-PCR detects rapid, transient increases in PNMT mRNA in hypoxic MPC 712 cells. Additionally, elevation of mRNAs encoding transcription factors hypoxia inducible factor 1 (HIF-1) alpha subunit and Egr-1 are evident within 60 min incubation in anoxia. Therefore, hypoxia elicits rapid transcriptional responses in numerous genes expressed by chromaffin cells.

Plasticity of pheochromocytoma cell lines from neurofibromatosis knockout mice.

Adrenergic mouse pheochromocytoma (MPC) cells from heterozygous neurofibromatosis knockout mice show little or no expression of the NGF receptor trk A and do not undergo neuronal differentiation in response to NGF. However, they express high levels of receptor tyrosine kinase, Ret, and GDNF family receptor alpha(1) (GFRalpha(1)) in vivo and in vitro and respond to glial cell line-derived neurotrophic factor (GDNF). In addition, they form short processes in response to PACAP or cyclic AMP. Morphological effects of GDNF, PACAP, or cyclic AMP are similar to those of NGF, PACAP, or cyclic AMP on PC12 cells, and all three agents cause downregulation of PNMT mRNA. The MAP kinase kinase inhibitor U0126 inhibits both baseline proliferation and stimulated process outgrowth, consistent with a model in which sustained low-level ERK activation drives proliferation, and more intense activation drives neuronal differentiation. The sensitivity of MPC cells to U0126 both may reflect mechanisms that cause pheochromocytomas in neurofibromatosis and aid in their clarification.

Ret protein expression in adrenal medullary hyperplasia and pheochromocytoma.

Ret is a developmentally regulated tyrosine kinase involved in formation and maintenance of the nervous system. Ret mutations predisposing to pheochromocytomas and medullary thyroid carcinomas occur in multiple endocrine neoplasia (MEN) syndromes 2A and 2B. Biochemical studies have demonstrated overexpression of Ret mRNA and protein in pheochromocytomas compared to normal adrenal medulla. However, the cellular distribution of Ret in the normal human adrenal and in hyperplastic lesions that antecede pheochromocytomas are unclear. The present investigation was undertaken to resolve the histological distribution of Ret in the normal human adrenal, in pheochromocytomas evolving from adrenal medullary hyperplasia in MEN2A and in sporadic pheochromocytomas. Ret expression was studied by immunohistochemistry using both a polyclonal and a monoclonal antibody, with confirmation by immunoblotting of representative cases. Only occasional cells stained for Ret in the normal adrenal, consistent with the distribution in adult adrenals of other species. Heterogeneous, progressively increased Ret expression was observed during the evolution of pheochromocytomas. In both normal and neoplastic adrenal, the most intense immunoreactivity was observed in cells with neuron-like features. Our finding that Ret is not expressed at high levels in the early stages of disease suggests that elucidation of mechanisms that regulate Ret expression is required for understanding the pathobiology of MEN2A. The association of high-level Ret expression with neuronal morphology suggests that the variable overexpression of Ret in pheochromocytomas might in part be an epiphenomenon, reflecting the known phenotypic plasticity of these tumors.

Cytocidal activities of topoisomerase 1 inhibitors and 5-azacytidine against pheochromocytoma/paraganglioma cells in primary human tumor cultures and mouse cell lines.

There is currently no effective treatment for metastatic pheochromocytomas and paragangliomas. A deficiency in current chemotherapy regimens is that the metastases usually grow very slowly. Drugs that target dividing tumor cells have therefore had limited success. To improve treatment, new strategies and valid experimental models are required for pre-clinical testing. However, development of models has itself been hampered by the absence of human pheochromocytoma/paraganglioma cell lines for cultures or xenografts. Topoisomerase 1 (TOP1) inhibitors are drugs that interfere with mechanisms that maintain DNA integrity during transcription in both quiescent and dividing cells. We used primary cultures of representative human tumors to establish the cytotoxicity of camptothecin, a prototypical TOP1 inhibitor, against non-dividing pheochromocytoma/paraganglioma cells, and then employed a mouse pheochromocytoma model (MPC) to show that efficacy of low concentrations of camptothecin and other TOP1 inhibitors is increased by intermittent coadministration of sub-toxic concentrations of 5-azacytidine, a DNA methylation inhibitor that modulates transcription. We then tested the same drugs against a clonal MPC derivative that expresses CMV reporter-driven luciferase and GFP, intended for in vivo drug testing. Unexpectedly, luciferase expression, bioluminescence and GFP expression were paradoxically increased by both camptothecin and SN38, the active metabolite of irinotecan, thereby masking cell death. Expression of chromogranin A, a marker for neuroendocrine secretory granules, was not increased, indicating that the drug effects on levels of luciferase and GFP are specific to the GFP-luciferase construct rather than generalized cellular responses. Our findings provide proof of principle for use of TOP1 inhibitors against pheochromocytoma/paraganglioma and suggest novel strategies for enhancing efficacy and reducing toxicity by optimizing the combination and timing of their use in conjunction with other drugs. The paradoxical effects of TOP1 inhibitors on luciferase and GFP dictate a need for caution in the use of CMV promoter-regulated constructs for cancer-related imaging studies.