The ret-Activating Ligand GDNF Is Differentiative and Not Mitogenic for Normal and Neoplastic Human Chromaffin Cells In Vitro

Activating mutations of the receptor tyrosine kinase, ret, are associated with multiple endocrine neoplasia type 2A (MEN 2A). However, the mechanisms leading to tumor development are unclear. Glial-derived neurotrophic factor (GDNF) activates wild-type ret via interaction with a second receptor, GFR a-l. We have utilized GDNF to stimulate normal and neoplastic chromaffin cells in order to ask whether ret activation is mitogenic. Cells from three normal adult adrenal medullas, one sporadic pheochromocytoma, and three MEN-2A pheochromocytomas were labeled with bromodeoxyuridine (BrdU) for 12 d in the presence or absence of GDNF or nerve growth factor (NGF), which is known to stimulate neurite outgrowth, but not proliferation in human chromaffin and pheochromocytoma cell cultures. Responses to GDNF and NGF were comparable, except for two MEN-2A pheochromocytomas that responded minimally to GDNF and robustly to NGF. These tumors responded to GDNF biochemically, as measured by phosphorylation of mitogen-activated protein kineses, despite their weak morphological responses. Our findings suggest that activation of ret may not be sufficient to produce chromaffin cell hyperplasia or neoplasia directly by stimulating cell proliferation. However the possibility that altered cell-cell or cell-substrate interactions might cause responses to become differ entiative rather than proliferative in vitro has not been ruled out. We also demonstrate, for the first time, that at least some human pheochromocytomas with an MEN-2A ret mutation respond to a normal ret ligand. This responsiveness could be mediated by a remaining normal ret allele or by other mechanisms.     

Different responses to mitogenic agents by Adult rat and human chromaffin cells in vitro

Adrenal medullary hyperplasia and pheochromocytomas occur frequently in laboratory rats, both in the courseof aging and in response to prolonged administration of a variety of drugs and other substances. In contrast, these lesions are rare in humans. Rat chromaffin cells proliferate throughout life, but the proliferative capacities of human chromaffin cells are unknown. To determine whether the difference in prevalence of adrenal lesions might be correlated with differences in cell proliferation, adrenal medullary cells from 3 patients undergoing radical nephrectomy were maintained in vitro for up to 2 weeks in control medium or in the presence of nerve growth factor (NGF) and/or tetradecanoyl phorbol acetate (TPA), an activator of protein kinase C. Both NGF and TPA are known mitogens for neonatal and adult rat chromaffin cells. At intervals, the cultures were pulsed for up to 36 hours with bromodeoxyuridine (BrdU) to label S-phase nuclei. They were then fixed and consecutively stained for BrdU and for tyrosine hydroxylase, to confirm that labeled cells were chromaffin cells. Cells from adult female F344 rats were similarly maintained. Human chromaffin cells labeled with BrdU were extremely rare (less than 0.1 %) under all culture conditions, and effects of NGF or TPA could not be demonstrated. Rat chromaffin cells showed little or no labeling with BrdU in control medium but, in contrast to their human counterparts studied, showed marked increases in the percentages of labeled cells in the presence of NGF (37% ± 3%), TPA (7% ± 1%), or both (31% ± 3%). The apparently lower responsiveness of human chromaffin cells to mitogenic signals, or responses to different types of signals, may contribute to the lower frequency of adrenal medullary hyperplasia and pheochromocytomas in humans compared to rats.     

Human pheochromocytomas show reduced p27Kip1 expression that is not associated with somatic gene mutations and rarely with deletions

Pheochromocytomas are neuroendocrine tumors arising in the neural crest-derived chromaffin cells of the adrenal gland or in extra-adrenal sympathetic ganglia (paragangliomas). In a rat model of multiple endocrine neoplasia (MEN), absence of functional p27Kip1 protein predisposes to pheochromocytoma and paraganglioma development. As no data is available regarding the involvement of p27Kip1 in human pheochromocytoma and/or paraganglioma, we set out to determine the expression pattern of p27Kip1 in those tumor types. A panel of 25 pheochromocytomas and 23 paragangliomas was collected. Two pheochromocytomas were from MEN2 patients. The paragangliomas included 15 tumors that developed at the carotid bifurcation, three in the jugulo–tympanic area, and five at other sites. Except for the MEN2 cases, all others were apparently sporadic. Immunohistochemistry for p27Kip1 and the proliferation marker Ki67 was performed. We found that p27Kip1 expression is reduced/lost in 56% of pheochromocytomas, but only in 18.1% of paragangliomas. Downregulation of p27Kip1 was not associated with increased proliferation. Cases showing reduced/lost p27Kip1 expression were screened for the presence of somatic mutations in CDKN1B (p27Kip1) and for allelic imbalance at the p27Kip1 locus. Three cases had allelic imbalance but none had mutations. In conclusion, pheochromocytomas display extreme reduction/loss of p27Kip1 expression at high frequency.     

Plasma normetanephrine and metanephrine for detecting pheochromocytoma in von Hippel-Lindau disease and multiple endocrine neoplasia type 2.

BACKGROUND: The detection of pheochromocytomas in patients at risk for these tumors, such as patients with von Hippel-Lindau disease or multiple endocrine neoplasia type 2 (MEN-2), is hindered by the inadequate sensitivity of commonly available biochemical tests. In this study we evaluated measurements of plasma normetanephrine and metanephrine for detecting pheochromocytomas in patients with von Hippel-Lindau disease or MEN-2. METHODS: We studied 26 patients with von Hippel-Lindau disease and 9 patients with MEN-2 who had histologically verified pheochromocytomas and 50 patients with von Hippel-Lindau disease or MEN-2 who had no radiologic evidence of pheochromocytoma. Von Hippel-Lindau disease and MEN-2 were diagnosed on the basis of germ-line mutations of the appropriate genes. The plasma concentrations of normetanephrine and metanephrine were compared with the plasma concentrations of catecholamines (norepinephrine and epinephrine) and urinary excretion of catecholamines, metanephrines, and vanillylmandelic acid. RESULTS: The sensitivity of measurements of plasma normetanephrine and metanephrine for the detection of tumors was 97 percent, whereas the other biochemical tests had a sensitivity of only 47 to 74 percent. All patients with MEN-2 had high plasma concentrations of metanephrine, whereas the patients with von Hippel-Lindau disease had almost exclusively high plasma concentrations of only normetanephrine. One patient with von Hippel-Lindau disease had a normal plasma normetanephrine concentration; this patient had a very small adrenal tumor (<1 cm). The high sensitivity of measurements of plasma normetanephrine and metanephrine was accompanied by a high level of specificity (96 percent). CONCLUSIONS: Measurements of plasma normetanephrine and metanephrine are useful in screening for pheochromocytomas in patients with a familial predisposition to these tumors.     

Expression analysis of RET and the GDNF/GFRalpha-1 and NTN/GFRalpha-2 ligand complexes in pheochromocytomas and paragangliomas

Pheochromocytoma and its extra-adrenal counterpart paraganglioma are rare catecholamine producing tumors which usually occur sporadically but may also be a part of neuroendocrine tumor syndromes such as multiple endocrine neoplasia type 2A (MEN 2A). Activating mutations of the RET proto-oncogene which is the underlying cause of MEN 2A, is also seen in approximately 10% of sporadic pheochromocytomas. Glial cell line derived neurotrophic factor (GDNF) and neurturin (NTN) have been shown to function as independent ligands to RET, binding in a complex with the membrane-bound receptors GFRalpha-1 and GFRalpha-2 respectively. Here we have investigated the mRNA expression of RET and its ligand complexes, GDNF/GFRalpha-1 and NTN/GFRalpha-2, in a panel of pheochromocytomas and paragangliomas using mRNA in situ hybridization. RET expression was evident in normal adrenal medulla, and in 13/15 pheochromocytomas, including 5/5 MEN 2A associated tumors, but only in 1/10 paragangliomas. The frequent expression of RET in the pheochromocytomas suggest that this gene might be involved in the tumorigenesis. However, no expression of GDNF/GFRalpha-1 or NTN/GFRalpha-2 could be detected in any of the 25 tumors analyzed, suggesting that these ligand complexes are not important in the development of pheochromocytoma or paraganglioma.     

Adrenergic Differentiation and Ret Expression in Rat Pheochromocytomas

Pheochromocytomas are catecholamine-producing tumors of the adult adrenal medulla. They are rare in humans and most other species but common in laboratory rats. However, the relevance of rat pheochromocytomas as a model for their human counterparts is uncertain. Previous studies of spontaneous and drug-induced rat pheochromocytomas and the PC12 pheochromocytoma cell line suggested a distinctive noradrenergic phenotype, possibly reflecting origin from a progenitor not present in the adult human adrenal. In this study, we studied 31 pheochromocytomas derived from test and control male and female rats in toxicologic studies for expression of the epinephrine-synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT) and the receptor tyrosine kinase Ret. PNMT, which defines adrenergic chromaffin cells, is frequently expressed in human pheochromocytomas, often in tumors that also overexpress RET. We also tested for the expression of the cell cycle checkpoint protein p27^Kip1, which recently was reported absent in pheochromocytomas from a strain of rats with a hereditary mixed multiple endocrine neoplasia (MEN)-like syndrome. Using immunoblots, we demonstrated PNMT expression in almost 50% of the 31 tumors, although often at lower levels than in normal rat adrenal medulla. The majority of tumors overexpressed Ret. There was no apparent correlation between PNMT and Ret. However, in this study, PNMT expression was strongly associated with tumors arising in female rats, while overexpression of Ret did not show a sex predilection. Robust expression of p27^Kip1 was seen in all tumors from the toxicologic studies and also in a small sample of pheochromocytomas from Long–Evans rats, which were reported to have a mixed MEN-like syndrome in the 1980s. The present results show that rat pheochromocytomas have greater phenotypic diversity than previously believed and greater similarity to their human counterparts with respect to these two important markers. Loss of p27^Kip1 does not appear to account for the high frequency of pheochromocytomas in commonly utilized rat strains.     

Tissue Localization of Nerve Growth Factor Receptors: trk A and Low-Affinity Nerve Growth Factor Receptor in Neuroblastoma,Pheochromocytoma, and Retinoblastoma

The immunohistochemical localization of nerve growth factor receptor (NGFR)—high-affinity NGFR (trk A) and low-affinity NGFR (LNGFR)—was investigated in 23 neuroblastoma group tumors, 18 pheochromocytomas, 2 mixed neuroendocrine-neural tumors, and 16 retinoblastomas.trk A was expressed in the tumor cells of all neuroblastomas, pheochromocytomas, and retinoblastomas. Immunoreactive intensity was especially strong in the larger ganglionic tumor cells of ganglioneuroblastoma and ganglioneuroma. Messenger RNA (mRNA) oftrk A was also strongly expressed in the ganglionic cells of ganglioneuroblastomas and chromaffin cells of pheochromocytomas byin situ hybridization method. LNGFR was negative in the tumor cells of neuroblastoma; however, it showed strong immunoreactivity in ganglionic tumor cells and Schwann cells of ganglioneuroblastoma/ganglioneuroma, and sustentacular cells of pheochromocytoma. Although normal retina expressed bothtrk A and LNGFR, tumor cells of retinoblastoma were positive for onlytrk A but negative for LNGFR. Such differences in the expression oftrk A and LNGFR may reflect neuron-glial interactions in the survival and maturation of the sympathetic nerves, retina, and tumors in these tissues.     

Discordant effects of rapamycin on proliferation and p70S6 kinase phosphorylation in normal and neoplastic rat chromaffin cells

Normal adult rat chromaffin cells show a robust proliferative response in vitro to nerve growth factor (NGF) and other mitogens. In contrast, PC12 rat pheochromocytoma cells proliferate in the absence of exogenous mitogens and undergo neuronal differentiation in response to NGF. We demonstrate in this work that the antiproliferative drug rapamycin suppresses normal chromaffin cell proliferation. This effect is blocked by FK 506, indicating that it occurs via interaction of rapamycin with its intracellular binding protein, FKBP. Rapamycin must be added within 2 days of mitogen stimulation in order to be fully effective. PC12 cells are refractory to the antiproliferative effect of rapamycin, although rapamycin does exert its expected inhibitory effect in PC12 cells on both basal and NGF-stimulated activation of one of its biochemical targets, the 70-kDa S6 protein kinase (p70S6K). The discordant findings suggest that a proliferative signal normally requiring activation of p70S6K either is unnecessary in PC12 cells or is provided by a downstream or cross-communicating pathway. They also suggest that p70S6K does not participate in the morphological responses of PC12 cells to NGF. Determining the basis for rapamycin resistance in PC12 cells might help to identify signaling abnormalities involved in the pathogenesis of pheochromocytoma.     

Characterization of Pheochromocytomas in a Mouse Strain with a Targeted Disruptive Mutation of the Neurofibromatosis Gene Nf1

Patients with neurofibromatosis type 1 (NF1) show an increased frequency of pheochromocytomas. TheNF1 gene encodes a GTPase-activating protein that controls the activity ofras proteins in intracellular signalling. A mouse strain with a knockout mutation of Nf1, the murine counterpart ofNF1, has recently been constructed. This mutation, designated Nf1ⁿ³¹, has been shown to be associated with the frequent development of pheochromocytomas in heterozygous animals. Pheochromocytomas are extremely rare in wild-type mice. We have characterized the tumors to assess their relevance as a model for human pheochromocytomas. The frequency of pheochromocytomas was determined in inbred compared to outbred mice carrying the Nf1ⁿ³¹ mutation. Paraffin sections of pheochromocytomas from seven mice were stained immunohistochemically for the catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) to infer their profiles of catecholamine synthesis, and for chromogranin A (CGA) to infer their content of secretory granules. Cultured cells from a representative tumor were studied in vitro to assess proliferation and neuronal differentiation. Pheochromocytomas arose in approx 15% of Nf1ⁿ³¹ mice with a mixed genetic background, but were absent in inbred mice. Approximately one-fourth of the tumors were bilateral. The tumors exhibited variable morphology. All included cells that appeared well differentiated and resembled normal chromaffin cells in that they expressed TH, PNMT, and CGA. Focal neuronal differentiation was also observed. In cell culture, the tumor cells ceased to proliferate and the majority underwent terminal differentiation into TH-positive cells with neuronal morphology. The phenotype of pheochromocytomas in mice with the Nf1ⁿ³¹ mutation resembles that of human pheochromocytomas, particularly with respect to their ability to produce epinephrine, as inferred from positive staining for PNMT. The tumors also resemble both normal and neoplastic human adrenal medulla with respect to their extensive differentiation into neuron-like cells in vitro. This change in phenotype may be related toras activation. These neoplasms may be valuable both as models for the pathobiology of adrenal medullary neoplasia, and as a source of epinephrine-producing pheochromocytoma cells lines, for which adequate models currently do not exist.     

Investigation of the genes for RET and its ligand complex,GDNF/GFRα-1, in small cell lung carcinoma

RET is a receptor tyrosine kinase expressed in neuroendocrine cells and in tumors of these cell types. RET activation may be mediated by a ligand complex comprising glial cell line-derived neurotrophic factor (GDNF) and GDNF family receptor alpha-1 (GFRα-1). Activating RET mutations are found in the inherited cancer syndrome multiple endocrine neoplasia type 2 and in a subset of the related sporadic tumors, medullary thyroid carcinoma and pheochromocytoma, both being derived from neuroendocrine tissues. In one small study, mutations were identified in another tumor with neuroendocrine features, small cell lung carcinoma (SCLC). To determine whether RET mutations contribute to the pathogenesis of SCLC, we examined a panel of 54 SCLC cell lines. No mutations were identified in RET exons 10, 11, and 13–16, regions previously implicated in SCLC or other neuroendocrine tumors. We further examined the expression pattern of RET and the genes encoding the components of its ligand complex GDNF and GFRα-1 , in 21 SCLC lines by using RT-PCR. Although we found no consistent pattern of expression for these three genes, RET was expressed in 57% of SCLC lines. Thus, although RET mutations appear unlikely to be an important step in the tumorigenesis of SCLC, the frequent expression of this gene suggests that RET may have a mitogenic role in a subset of SCLC cell lines. Genes Chromosomes Cancer 21:326–332, 1998. © 1998 Wiley-Liss, Inc.     

First continuous human pheochromocytoma cell line: KNA. Biological, cytogenetic and molecular characterization of KNA cells

Pheochromocytomas are rare tumours, with an incidence of 1–2 per million which arise from chromaffin cells of the adrenal medulla. They occur sporadically or as part of dominantly inherited cancer syndromes like multiple endocrine neoplasia 2 (MEN2A and 2B) and others. Continuous cell lines, not available so far, are essential tools for studies in these tumours. A continuous cell line (KNA) was established from a sporadic pheochromocytoma of the right adrenal gland of a 73-year-old woman. The KNA cells grow as suspensions of spheroids and show the morphological and immunocytochemical characteristics of neuronal chromaffin cells, such as neuroendocrine granules, and positive reactions to chromogranin- and related peptide-, neuron specific enolase and vasoactive intestinal peptide antibodies. Neurite-like processes are formed after addition of nerve growth factor. Chromosomal analyses revealed a diploid (46,XX, n=50) to hypodiploid (43–45,XX, n=15) karyotype. In hypodiploid metaphases most frequently #19, #17, #21 and #22 were missing. Chromosome arms 1p and 4q showed apparently consistent interstitial deletions: 6q, 8q, 13q and 22q showed clonal interstitial deletions. The cell line shows a heterozygous sequence variant TGC (cysteine) to TGG (tryptophan) in codon 611 in exon 10 of the RET proto-oncogene. So far, PC-12, a rat adrenal pheochromocytoma, has been the only continuous pheochromocytoma cell line available. KNA represents the first report on a human continuous pheochromocytoma cell line, the first report of structural chromosome aberrations in pheochromocytomas and the first report of a RET mutation TGC to TGG in exon 10 of the RET proto-oncogene in a sporadic pheochromocytoma. © 1998 Chapman and Hall     

Genetics of endocrine tumours

Major advances have been made in the understanding of the genetic mechanisms underlying endocrine tumorigenesis, through the study of several syndromes of genetic predisposition and the identification of the genes involved. The syndrome of type 1 multiple endocrine neoplasia (MEN-1) is one of the best known; this autosomal dominant hereditary syndrome predisposes to the development of endocrine tumors of the pituitary, the parathyroids, the foregut and the adrenals. The responsible gene, known as MEN-1, encodes an original protein, menin, involved in several major cellular functions, such as the control of cell proliferation and differentiation. Type 2 multiple endocrine neoplasia (MEN-2) is an autosomal dominant hereditary syndrome associated with the development of medullary carcinomas of the thyroid, pheochromocytomas and hyperparathyroidism; the corresponding gene, RET, encodes a transmembrane receptor with tyrosine kinase activity. Endocrine tumors are also associated with non Hippel-Lindau disease and with phacomatoses, such as type 1 neurofibromatosis and tuberous sclerosis. Finally, isolated familial syndromes of endocrine tumors have been described: isolated familial hyperparathyroidism type II (HRPT2), associated with alterations in a gene coding for an original protein, parafibromin, or isolated familial syndromes of pheochromocytomas and paragangliomas (PRG) associated with mutations in the genes SDHB, SDHC or SDHD, which encode succinate-dehydrogenase subunits. The understanding of the genetic mechanisms underlying these syndromes of predisposition is essential for the diagnosis and management of these patients and their family; it also gives insight on the molecular mechanisms of endocrine tumorigenesis.     

A comparative immunohistochemical study of spontaneous and chemically induced pheochromocytomas in B6C3F1 mice

Spontaneously occurring and chemically induced pheochromocytomas are rare in mice. That the mouse pheochromocytoma is a more appropriate animal model than that of the rat for study of human medullary adrenal tumors has been suggested. The expression of phenylethanolamine-N-methyltransferase (PNMT), the enzyme responsible for production of epinephrine from norepinephrine, is common to both mouse and human pheochromocytomas. This investigation assessed the expression of the immunohistochemical markers PNMT, tyrosine hydroxylase (TH), and chromogranin A (CGA) in spontaneously occurring and chemically induced pheochromocytomas in the B6C3F1 mouse. Spontaneous tumors were derived from control animals from 10 different studies and the pheochromocytomas from treated groups from 4 different studies. All tumors were positive for maximal TH expression. A highly significant difference in PNMT expression (p<0.01) occurred between spontaneously occurring pheochromocytomas classified as benign or “malignant” by the criteria of toxicologic pathology. Chemically induced tumors showed intermediate PNMT staining. A marked reduction in CGA expression occurred in pheochromocytomas induced by technical grade pentachlorophenol, compared to the other three chemicals and the spontaneously occurring tumors. These findings suggest that immunohistochemistry is a reliable tool in investigating the functional capabilities of pheochromocytomas in mice. PNMT expression is a tightly regulated component of the chromaffin cell phenotype and appears to be readily lost in mouse pheochromocytomas, particularly those with aggressive characteristics.     

Inherited endocrinopathies: an update

Inherited endocrinopathies, including multiple endocrine neoplasia type 1 (MEN-1), multiple endocrine neoplasia type 2 syndromes (MEN-2A, MEN-2B, familial medullary thyroid carcinoma), and inherited syndromes with pheochromocytoma (von Hippel–Lindau disease, neurofibromatosis type 1, others), comprise a heterogeneous group of cancer susceptibility syndromes that affect one or more components of the endocrine system. During the past several years, novel findings regarding genotype–phenotype correlation have highlighted the importance of establishing a genetic diagnosis in the treatment of these diseases. Here, we present a case-based review of recent advances in the genetics, diagnosis and management of inherited endocrinopathies.     

NGF和GDNF对大鼠嗅鞘细胞体外增殖和分化的影响

本研究旨在比较神经生长因子(NGF)和胶质细胞源性神经营养因子(GDNF)对大鼠嗅鞘细胞(OECs)体外增殖和分化的影响。在体外分离培养大鼠OECs的培养液中不加神经营养因子(对照组)或加入不同的神经营养因子(NGF组和GDNF组),倒置显微镜下进行观察,并通过S-100免疫荧光组织化学染色检测各组大鼠OECs体外增殖和分化情况。结果显示:NGF组和GDNF组OECs增殖和分化明显好于对照组(P<0.01),NGF组又明显好于GDNF组(P<0.01)。这些结果提示NGF能促进大鼠OECs体外增殖和分化。     

Chromaffin cell mitogenesis by neurturin and glial cell line-derived neurotrophic factor.

Neurturin and glial cell line-derived neurotrophic factor are novel mitogens for normal adult rat chromaffin cells in vitro. These neurotrophic factors differ from the previously described adult chromaffin cell mitogens, nerve growth factor and basic fibroblast growth factor, in that their effects are potentiated by depolarization and activation of protein kinase C. Neurturin and glial cell line-derived neurotrophic factor signal via the receptor tyrosine kinase, ret, but may also act independently of ret. Both depolarization and phorbol esters act synergistically with neurturin to up-regulate ret protein expression in chromaffin cell cultures, suggesting a mechanism for potentiation of mitogenesis. However, a direct role for ret in mitogenesis has not been established. Stimulation by neurturin causes increased phosphorylation of extracellular signal-regulated kinases 1 and 2 in cultured chromaffin cells, and mitogenesis is prevented by inhibitors of their phosphorylation. Inhibitors of phosphatidylinositol 3-kinase also prevent mitogenesis.The present findings suggest the hypothesis that neurotrophic factors and neurally derived signals might cooperatively regulate chromaffin cell proliferation in vivo in the rat. In addition, trans-synaptic stimulation might provide a route by which epigenetic factors could influence the development of adrenal medullary hyperplasia in humans with hereditary multiple endocrine neoplasia syndromes 2A and 2B by affecting expression and/or activation of ret.     

Molecular and cellular biology of pheochromocytomas and extra-adrenal paragangliomas

Efforts to understand the pathobiology of pheochromocytomas and extra-adrenal paragangliomas have been spurred by genetic and gene expression profiling studies showing genotype-phenotype correlations in familial pheochromocytoma/paraganglioma syndromes and in some sporadic tumors. The current challenge is to relate catalogs of genetic and phenotypic markers to cell biology. Hypothetical bases for genotype-phenotype correlations include: cell of origin, pathway dependence, and functional and anatomic context. A further consideration is phenotype plasticity. Cross talk between signaling pathways provides a general framework for understanding how mutations of apparently unrelated genes might lead to the same type of tumor, and also suggests that targeted therapies might be directed either at a specific mutated gene or a downstream signal transducer. However, precise mechanisms of tumorigenesis remain unknown. An intriguing hypothesis proposes that mutations of RET, NF1, VHL, or SDH predispose to hereditary pheochromocytoma/ paraganglioma by causing defective apoptotic culling of cells that would normally be destroyed during embryogenesis. An argument favoring survival rather than mitogenesis as a common denominator is that the same mutations are rare in sporadic pheochromocytomas/paragangliomas, suggesting they only need to act during a limited developmental window. An implication is that tumor precursors could be identified and eradicated in individuals who carry the mutations.     

Autopsy case of prostate cancer with multiple endocrine neoplasia 2A

We describe an autopsy case of a 65-year-old man with prostate cancer accompanied by multiple endocrine neoplasia 2A (MEN 2A), including malignant pheochromocytomas, thyroid medullary carcinomas and parathyroid hyperplasia. Metastatic lesions from the prostate primary were identified using immunohistochemistry for prostate specific antigen within both primary and metastatic pheochromocytomas in the liver. To investigate the affinity of prostate cancer for pheochromocytoma cells, immunohistochemistry was carried out using a number of antibodies and both tumors were positive for N-cadherin. Interestingly, pheochromocytomas, thyroid medullary carcinomas and prostate cancer were all positive for the anti-RET antibody. The immunohistochemical results suggest that the cell affinity may, in part, result from cell-cell adhesion via N-cadherin. Although prostate cancer is rarely associated with MEN, RET activation may have participated in the tumorigenesis of this case.     

Catecholamine release after physical exercise. A new provocative test for early diagnosis of pheochromocytoma in multiple endocrine neoplasia type 2

A simple and practical provocative test is needed for early asymptomatic pheochromocytoma, which is a major risk for patients with multiple endocrine neoplasia type 2 (MEN-2). We measured plasma catecholamines before and after submaximal exercise in 26 MEN-2 gene carriers, eight of whom with asymptomatic pheochromocytoma, nine with medullary thyroid carcinoma and 10 after uni- or bilateral adrenalectomy. Seventeen clinically healthy individuals and 11 patients with neurovegetative lability and symptoms mimicking pheochromocytoma served as controls. Plasma adrenaline, noradrenaline and dopamine increased after exercise except for adrenaline after bilateral adrenalectomy. The post-exercise levels of adrenaline and the adrenaline/dopamine ratio were significantly higher in the pheochromocytoma patients compared to the healthy controls and the patients with neurovegetative lability, while the patients with medullary thyroid carcinoma represented an intermediate group with a high probability of developing adrenal tumors. The present method is a physiological test with a high sensitivity and specificity. It is practical and well suited for repeated examinations and seems to be of value for the detection of early pheochromocytoma in MEN-2 patients. Furthermore, the test could be used in the differential diagnosis between pheochromocytoma and neurovegetative lability.     

Up-regulation of ret by reserpine in the adult rat adrenal medulla

The receptor tyrosine kinase, ret, is activated by glial cell line-derived neurotrophic factor, neurturin and related ligands that bind to glycosylphosphatidylinositol-tailed receptors GFRalpha1-4. Ret expression is developmentally regulated and detectable only at very low levels in adult adrenal medulla. However, mutations of ret that cause constitutive activation or alter signal transduction give rise to adrenal medullary hyperplasia and pheochromocytomas in humans with hereditary multiple endocrine neoplasia (MEN) syndromes 2A and 2B and in animal models. These discordant observations pose the conundrum of how a molecule barely detectable in the adult adrenal can contribute to development of adrenal medullary pathology that typically occurs in adults. We recently reported that depolarization and phorbol esters that activate protein kinase C act synergistically with neurturin to up-regulate ret protein and mRNA expression in adult rat chromaffin cell cultures. Those findings suggested that ret expression in vivo is not static and might be regulated in part by neurally derived signals. We show here that the anti-hypertensive agent reserpine, which is known to cause a reflex increase in trans-synaptic stimulation of chromaffin cells, increases expression of ret mRNA and protein in adult rat adrenal medullary tissue in vivo. Elevated ret protein levels are detectable both by immunoblots and immunohistochemistry, which shows immunoreactive ret in chromaffin cells and neurons after reserpine administration. The finding that ret expression is subject to up-regulation by environmental signals in vivo suggests that epigenetic factors might influence the development of adrenal medullary disease by affecting the expression of ret. It is known that long-term administration of reserpine leads to the development of adrenal medullary hyperplasia and pheochromocytomas in rats. Our findings suggest potential utility of the rat model for studying the roles of ret in the adrenal medulla and the mechanisms of its involvement in MEN 2 and other pheochromocytoma syndromes.