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Prediction of malignant behaviour of pheochromocytomas/sympathetic paragangliomas (PCCs/PGLs) is very difficult if not impossible on a histopathological basis. In a familial setting, it is well known that succinate dehydrogenase subunit B (SDHB)-associated PCC/PGL very often metastasise. Recently, absence of SDHB expression as measured through immunohistochemistry was shown to be an excellent indicator of the presence of an SDH germline mutation in PCC/PGL. SDHB loss is believed to lead to tumour formation by activation of hypoxia signals. To clarify the potential use of SDHB immunohistochemistry as a marker of malignancy in PCC/PGL and its association with classic hypoxia signalling we examined SDHB, hypoxia inducible factor-1α (Hif-1α) and its targets CA-9 and GLUT-1 expression on protein level using immunohistochemistry on a tissue micro array on a series of familial and sporadic tumours of 115 patients. Survival data was available for 66 patients. SDHB protein expression was lost in the tumour tissue of 12 of 99 patients. Of those 12 patients, 5 had an SDHB germline mutation, in 4 patients no germline mutation was detected and mutational status remained unknown in parts in 3 patients. Loss of SDHB expression was not associated with increased classic hypoxia signalling as detected by Hif-1α, CA-9 or GLUT-1 staining. Loss of SDHB expression was associated with an adverse outcome. The lack of correlation of SDHB loss with classic hypoxia signals argues against the current hypoxia hypothesis in malignant PCC/PGL. We suggest SDHB protein loss as a marker of adverse outcome both in sporadic and in familial PCC/PGL.

Adrenocortical cancer (ACC) is a rare and aggressive endocrine tumor. The patient presented with a large retroperitoneum tumor and lung metastases. Removal of the adrenocortical tumor with part of the transverse colon and tail of the pancreas, spleen and kidney was successfully performed following chemotherapy. Levels of serum neuron-specific enolase (NSE) were found to be markedly high before surgery and may be clinically useful markers for monitoring tumor status during management. Immunohistochemical studies showed that the cancer cells were positive for NSE and overexpression of p53. We identified a novel germ line variant of the 177 mutant (Pro to Arg; P177R) of p53 by genomic sequencing. The genetic and biochemical data presented in this case confirm the importance of screening for p53 status in ACC with inherited cancer syndrome.

Pheochromocytoma and paraganglioma are rare tumors of adrenals as well as the sympathetic and parasympathetic paraganglia. Clinical presentation of these tumors depends on localization, secretory profile and malignant potential. Four distinct syndromes--PGL1-4--are related to mutations in the succinate dehydrogenase gene--mitochondrial complex involved in electron transfer and Krebs cycle. Here we describe etiology, genetics, as well as clinical aspects of SDH-related tumors. We also describe recent discoveries in HIF-related pathway of tumorigenesis and mutations in new SDH-related genes that have improved our understanding of this disease., (Published by Elsevier Ltd.)

Background: Seladin-1 overexpression exerts a protective mechanism against apoptosis. Seladin-1 mRNA is variably expressed in normal human tissues. Adrenal glands show the highest levels of seladin-1 expression, which are significantly reduced in adrenal carcinomas (ACC). Since up to now seladin-1 mutations were not described, we investigated whether promoter methylation could account for the down-regulation of seladin-1 expression in ACC., Methods: A methylation sensitive site was identified in the seladin-1 gene. We treated DNA extracted from two ACC cell lines (H295R and SW13) with the demethylating agent 5-Aza-2-deoxycytidine (5-Aza). Furthermore, to evaluate the presence of an epigenetic regulation also 'in vivo', seladin-1 methylation and its mRNA expression were measured in 9 ACC and in 5 normal adrenal glands., Results: The treatment of cell lines with 5-Aza induced a significant increase of seladin-1 mRNA expression in H295R (fold increase, F.I. = 1.8; p = 0.02) and SW13 (F.I. = 2.9; p = 0.03). In ACC, methylation density of seladin-1 promoter was higher (2682 +/- 686) than in normal adrenal glands (362 +/- 97; p = 0.02). Seladin-1 mRNA expression in ACC (1452 +/- 196) was significantly lower than in normal adrenal glands (3614 +/- 949; p = 0.01)., Conclusion: On this basis, methylation could be involved in the altered pattern of seladin-1 gene expression in ACC.

Pheochromocytomas and paragangliomas are catecholamine-secreting tumors of neural crest origin caused by germline mutations in at least six distinct genes. This genetic heterogeneity has provided a rich source for both the discovery and functional characterization of new tumor-related genes. However, the genetic repertoire of these tumors is still not fully known, and current evidence points to the existence of additional pheochromocytoma susceptibility genes. Here, the unique contributions of three hereditary models of pheochromocytoma that can advance our knowledge of the disease pathogenesis are presented. The first model, loss of succinate dehydrogenase (SDH) function, illustrates how SDHB, C, or D mutations, components of the energy metabolism pathway, serve as a unique system to explore the pervasive metabolic shift of cancer cells towards glycolysis as a source of energy (also known as the Warburg effect) in contrast to the characteristic oxidative phosphorylation of normal cells. In the second model, mechanisms of tumorigenesis distinct from classical pheochromocytoma susceptibility genes are discussed in the context of a novel putative suppressor of neural crest-derived tumors, the KIF1B beta gene. Finally, NF1 loss is highlighted as a valuable study model to investigate the cell lineage selectivity of the Egln3-mediated developmental apoptotic defect of chromaffin precursor cells. Results from these studies may offer clues to understand the tissue specificity of hereditary pheochromocytoma syndromes. These distinct hereditary disease models illustrate how genetic-driven progress has the potential to narrow current gaps in our knowledge of pheochromocytoma and paraganglioma pathogenesis.

Background: Previous studies indicate that heparanase (HPA), an endoglycosidase involved in tumor angiogenesis and metastasis, is up-regulated in a variety of malignancies. However, the expression of HPA in neuroblastoma (NB), one of the most common extra cranial solid tumors in children, remains unknown. This study was undertaken to explore the expression and clinical significance of HPA in NB., Methods: Immunohistochemical staining was applied to detect the expression of HPA in 42 cases of NB. The relationships among HPA expression, international neuroblastoma staging system (INSS) stages, histopathological classification, and postoperative survival of the NB patients were analyzed., Results: The expression rate of HPA in NB was 61.9% (26/42), mainly in the cytoplasm of neuroblastoma cells. The expression rates of stage 1-2, stage 3-4 and stage 4S were 35.7%, 80.0% and 62.5%, respectively. The differences between stage 1-2 and stage 3-4 were significant (P<0.01). The expression of HPA was significantly higher in the NB cases that had one of the histopathological factors: age more than 1 year (P<0.01), poorer differentiation (P<0.01), and higher mitosis karyorrhexis index (P<0.01). The survival time of HPA-negative patients was significantly longer than that of HPA-positive patients (P<0.05)., Conclusion: Although these results indicate that heparanase might be correlated with development and progression of NB, a larger series of patients with a longer follow-up are probably needed to strengthen its role in assessment of NB prognosis.

Background: Germline mutations of the tumor suppressor genes SDHB, SDHC and SDHD play a major role in hereditary paraganglioma and pheochromocytoma. These three genes encode subunits of succinate dehydrogenase (SDH), the mitochondrial tricarboxylic acid cycle enzyme and complex II component of the electron transport chain. The majority of variants of the SDH genes are missense and nonsense mutations. To date few large deletions of the SDH genes have been described., Methods: We carried out gene deletion scanning using MLPA in 126 patients negative for point mutations in the SDH genes. We then proceeded to the molecular characterization of deletions, mapping breakpoints in each patient and used haplotype analysis to determine whether the deletions are due to a mutation hotspot or if a common haplotype indicated a single founder mutation., Results: A novel deletion of exon 3 of the SDHB gene was identified in nine apparently unrelated Dutch patients. An identical 7905 bp deletion, c.201-4429&#95;287-933del, was found in all patients, resulting in a frameshift and a predicted truncated protein, p.Cys68HisfsX21. Haplotype analysis demonstrated a common haplotype at the SDHB locus. Index patients presented with pheochromocytoma, extra-adrenal PGL and HN-PGL. A lack of family history was seen in seven of the nine cases., Conclusion: The identical exon 3 deletions and common haplotype in nine patients indicates that this mutation is the first Dutch SDHB founder mutation. The predominantly non-familial presentation of these patients strongly suggests reduced penetrance. In this small series HN-PGL occurs as frequently as pheochromocytoma and extra-adrenal PGL.

Thallium (Tl) is a highly toxic metal though yet its mechanisms are poorly understood. Previously, we demonstrated that rat pheochromocytoma (PC12) cells exposure to thallous (Tl(I)) or thallic (Tl(III)) cations leads to mitochondrial damage and reduced cell viability. In the present work we comparatively characterized the possible pathways involved in Tl(I)- and Tl(III)- (10-100 muM) mediated decrease in PC12 cells viability. We observed that these cations do not cause cell necrosis but significantly increased the number of cells with apoptotic features. Both cations lead to Bax oligomerization and caused apoptosis inducing factor (AIF), endonuclease G (Endo G), and cytochrome c release from mitochondria, but they did not activate caspase dependent DNAse (CAD). Tl(I)- and Tl(III)-dependent caspases 9 and 3 activation followed similar kinetics, with maximal effects at 18 h of incubation. In addition, Tl(I) promoted phosphatidylserine (PS) exposure. Tl(III) induced 2- and 18-fold increase in Fas content and caspase 8 activity, respectively. Together, experimental results show that Tl(I) and Tl(III) induce PC12 cells apoptosis, although differential pathways are involved. While Tl(I)-mediated cell apoptosis was mainly associated with mitochondrial damage, Tl(III) showed a mixed effect triggering both the intrinsic and extrinsic pathways of apoptosis. These findings contribute to a better understanding of the mechanisms underlying Tl-induced loss of cell viability in PC12 cells.

Objective: The aim of this study was to investigate the possible urinary markers of hormonal activity in patients with non-functioning adrenal incidentalomas. In order to evaluate the endocrine activity of aforementioned tumours, urinary steroid metabolite levels were analyzed in samples from patients and controls. Possible blocks in metabolic pathways of the examined hormones were determined by comparing selected urinary steroid metabolite sums and ratios in both groups of interest., Design: Urine samples were collected from 20 patients with non-functioning adrenal incidentalomas and from 25 controls matched in terms of age, sex and BMI. Excretion of 19 major urinary steroid metabolites was analyzed by gas chromatography. The results were subjected to statistical analysis., Results: In patients with adrenal incidentalomas sum of total urinary cortisol metabolites was significantly increased in respect to the control group. We also observed a shift towards tetrahydrocorticosterone, cortisol and etiocholanolone production in patients. No significant differences in production of other urinary steroid metabolites were noted in patients with adrenal incidentalomas in respect to control group., Conclusions: Our data suggests that not only urinary free cortisol but also its metabolite such as tetrahydrocortisol and other steroids including etiocholanolone and corticosterone tetrahydrometabolite might be urinary markers for the endocrine activity of adrenal incidentalomas. Enhanced levels of these urinary steroid metabolites indicate an impairment of 11beta-hydroxysteroid dehydrogenase activity and slightly increased activity of 5beta-reductase in patients with adrenal incidentalomas.

Myelolipomas of the adrenal gland are benign, nonfunctioning tumors. Patients with congenital adrenal hyperplasia sometimes develop large and bilateral myelolipomas. Although the precise pathogenesis of myelolipomas remains unclear, prolonged stimulation with high levels of adrenocorticotropic hormone (ACTH) or adrenal androgens are assumed to have a causative role. To clarify the role of ACTH and androgen in the pathogenesis of myelolipoma, we report a case of giant adrenal myelolipoma in a patient with poorly controlled congenital adrenal hyperplasia. A 43-year-old female was diagnosed with congenital adrenal hyperplasia at 6 years of age because of ambiguous genitalia. She had high plasma ACTH and 17-hydroxyprogesterone levels. Abdominal computed tomography showed a huge mass on the left adrenal gland, and an enlarged right adrenal mass. Genetic testing for CYP21A2 was performed and revealed that her genotype was IVS2-13A/C>G/I172N. Adrenalectomy for the left-side tumor was performed. Histological study revealed that the tumor consisted of fat cells and myeloid components, findings compatible with adrenal myelolipoma. Neither ACTH receptors nor androgen receptor was over-expressed in the tumor. Our finding that the tumor did not over-express ACTH or androgen receptor suggests a limited direct role for these hormones in the development of the myelolipoma.

Protein kinase R (PKR) is a serine/threonine-specific protein kinase implicated in the control of cell growth, differentiation, interferon-induced antiviral response, and induction of apoptosis. It is activated by various stress signals and growth factors. Activated PKR phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha), thereby inhibiting the initiation of translation. PKR also mediates the activation of several transcription factors (STAT1, p53, and NFkappaB) regulating both pro- and antiapoptotic mechanisms. In the present work, we studied the signaling pathways leading to PKR activation and apoptosis in PC12 rat pheochromocytoma cells, a model system of neuronal differentiation and cell death. We found that administration of various apoptosis inducing agents and conditions (serum starvation, anisomycin, LY294002, etoposide, and cisplatin) led to the proteolytic cleavage of PKR in PC12 cells. This cleavage was in strong correlation with the time kinetics of DNA fragmentation and morphological alterations characteristic of apoptosis. PKR was activated by the proteolytic cleavage: increased phosphorylation of eIF2alpha was found to run parallel with PKR cleavage. The activation of caspase-3 and caspase-9 was stimulated by all apoptosis inducing agents used in this study. The activation of caspase-3 preceded the cleavage of PKR after serum withdrawal, anisomycin and etoposide treatment, while coincided with it in cells treated with LY294002 or cisplatin. These observations suggest that early activation of caspase-3 is upstream of PKR proteolysis and that proteolytic activation of PKR may play a general role in the apoptosis of PC12 cells induced by various forms of cellular stress.

Cyclooxygenase-2 (COX-2) plays crucial roles in the development and invasion of tumors. Celecoxib, a selective COX-2 inhibitor, has been shown to be chemopreventive against cancer. However, to date, the mechanisms of these effects remain unclear. In this study, we investigate the effects of celecoxib on voltage-gated calcium channel (VGCC) currents in undifferentiated pheochromocytoma (PC12) cells using whole-cell patch clamp. Our results showed that celecoxib, instead of rofecoxib or NS-398, another selective COX-2 inhibitor, reversibly inhibited the current density of VGCC in a concentration-dependent manner, but had no apparent effects on the cells treated with nifedipine (1 microM), an L-type calcium channel blocker. Upon pre-incubation of PC12 cells with omega-conotoxia GVIA (1 microM), an N-type calcium channel blocker, omega-agatoxin IVA (1microM), a P/Q-type calcium channel blocker, or SNX-482 (1microM), a R-type calcium channel blocker, celecoxib (1microM) inhibited the currents by 36%, 28%, and 25%, respectively. Celecoxib up-shifted the current-voltage (I-V), and hyperpolarizedly shifted the inactivation curve, but did not markedly affect the activation curve. Intracellular application of H89, a protein kinase A inhibitor, failed to affect the celecoxib's VGCC currents inhibition. Taken together, our present results suggested that celecoxib inhibited L-type calcium channels in PC12 cells via a COX-2 independent pathway, which might be responsible for its clinical effects including anti-tumor.

We showed earlier that neurite outgrowth of rat pheochromocytoma PC12 cells was stimulated by royal jelly extract (PERJ) or its unique component, AMP N(1)-oxide, via adenosine A2a receptors. In this study, we found that stimulated neurite outgrowth occurred in medium supplemented with serum, but not in serum-free medium. The pentapeptide GRGDS, which includes the RGD sequence commonly shared by extracellular matrix (ECM) components, could attenuate the effect of serum, suggesting that integrin receptor signaling was essential for the neurite outgrowth induced by PERJ or AMP N(1)-oxide. PERJ or AMP N(1)-oxide also activated extracellular signal-regulated kinases 1 or 2 (ERK1/2); however, this activation was not associated with the neurite outgrowth. As it is known that Mn(2+) induces neurite outgrowth from PC12 cells and activates ERK1/2 through integrin signals and that activation of ERK1/2 is essential for Mn2+-induced neurite outgrowth, a difference in the mechanism between Mn(2+)-induced and PERJ- or AMP N(1)-oxide-induced neurite outgrowth is suggested. Furthermore, we demonstrated that PERJ contained no ECM component-like substances. These results demonstrate that AMP N(1)-oxide and its analogues were the only entities in PERJ with neurite outgrowth-inducing activity and that they required integrin signaling in addition to activation of A2a receptors to induce neurite outgrowth.

Pheochromocytomas in multiple endocrine neoplasia type 2 (MEN-2) express phenylethanolamine N-methyltransferase (PNMT), the enzyme that catalyzes conversion of norepinephrine to epinephrine, whereas those in von Hippel-Lindau (VHL) syndrome do not. Consequently, pheochromocytomas in MEN-2 produce epinephrine, whereas those in VHL syndrome produce mainly norepinephrine. This study examined whether transcription factors known to regulate expression of PNMT explain the different tumor phenotypes in these syndromes. Quantitative polymerase chain reaction (PCR) and Western blotting were used to assess levels of mRNA and protein for the glucocorticoid receptor, early growth response 1 (Egr-1), the Sp1 transcription factor (Sp1), and MYC-associated zinc finger protein (MAZ) in 6 MEN-2 and 13 VHL tumors. Results were cross-checked with data obtained using microarray gene expression profiling in a further set of 10 MEN-2 and 12 VHL tumors. Pheochromocytomas in MEN-2 and VHL syndrome did not differ in expression of the glucocorticoid receptor, Egr-1, Sp1, or MAZ as assessed by quantitative PCR and Western blotting. Microarray data also indicated no relevant differences in expression of the glucocorticoid receptor, Egr-1, MAZ, and the AP2 transcription factor. Thus, our results do not support a role for the above transcription factors in determining differences in expression of PNMT in pheochromocytomas from patients with VHL syndrome and MEN-2. Microarray analysis, however, did indicate differences in expression of genes involved in neural crest cell lineage and chromaffin cell development, consistent with differential survival of PNMT-expressing cells in the two syndromes.

The tumor suppressor PTEN is frequently inactivated in human cancers. A major downstream effector of PTEN is Akt, which is hyperactivated via PTEN inactivation. It is not known, however, whether diminished Akt activity is sufficient to inhibit tumorigenesis initiated by Pten deficiency. Here we showed that the deficiency of Akt1 is sufficient to dramatically inhibit tumor development in Pten+/- mice. Akt1 deficiency had a profound effect on endometrium and prostate neoplasia, two types of human cancer, in which PTEN is frequently mutated, and also affected thyroid and adrenal medulla tumors and intestinal polyps. Even haplodeficiency of Akt1 was sufficient to markedly attenuate the development of high-grade prostate intraepithelial neoplasia (PIN) and endometrial carcinoma. These results have significant implications for cancer therapy.

Background: Patients with advanced neuroblastoma have a poor prognosis. The antiapoptotic protein Akt has been implicated as a possible mediator of the resistance of human neuroblastoma cells to apoptosis; the proapoptotic protein Bid, is inhibited by activated Akt. Neuroblastoma has demonstrated responsiveness to immunotherapeutic approaches in preclinical studies, prompting investigation of new therapeutic strategies based on potentiation of the host immune response, including the use of systemic cytokines., Methods: We examined the antitumor efficacy and mechanisms of action of the central immunoregulatory cytokine interleukin-12 (IL-12) in mice bearing established orthotopic neuroblastoma tumors derived from murine TBJ and Neuro-2a cells. Cohorts of mice (10 mice/group) bearing established orthotopic neuroblastoma tumors were injected intraperitoneally with IL-12 or vehicle and monitored for survival. IL-12-induced apoptosis within the tumor microenvironment was investigated using ribonuclease protection assays, nuclear staining, and electron microscopy. Protein expression was determined via Western blot analysis and enzyme-linked immunosorbent assays. Confocal microscopy was used to examine the distribution of overexpressed Bid-enhanced green fluorescent protein fusion protein (Bid-EGFP) in TBJ cells. All statistical tests were two-sided., Results: IL-12 induced complete tumor regression and long-term survival of 8 (80%) of 10 mice bearing established neuroblastoma tumors compared with 1 (10%) of 10 control mice (P = .0055) and profound tumor cell apoptosis in vivo despite the fact that TBJ and Neuro-2a cells were resistant to receptor-mediated apoptosis in vitro. These cells expressed high levels of phosphorylated Akt, a key prosurvival molecule, and Akt inhibitors sensitized neuroblastoma cells to apoptosis mediated by IL-12-inducible cytokines including tumor necrosis factor-alpha and interferon-gamma in vitro. IL-12 increased the expression of proapoptotic genes and decreased Akt phosphorylation within established TBJ tumors in conjunction with activation and subcellular translocation of Bid., Conclusions: Our results suggest that IL-12 overcomes a potentially critical mechanism of tumor self-defense in vivo by inhibiting Akt activity and imply that IL-12 may possess unique therapeutic activity against tumors that express high levels of activated Akt.

The RET proto-oncogene encodes a receptor tyrosine kinase that is a main component of the signaling pathway activated by the glial cell line-derived neurotrophic factor family ligands. Gene targeting studies revealed that signaling through RET plays a crucial role in neuronal and renal organogenesis. It is well-known that germline mutations in RET lead to the human inherited diseases, multiple endocrine neoplasia type 2 (MEN 2) and Hirschsprung's disease, and that somatic rearrangements of RET cause papillary thyroid carcinoma. Due to marked advances in understanding of the molecular mechanisms of the development of MEN 2, a consensus on MEN 2 management associated with RET status is being reached and currently put into general use as a guideline. In this review, we summarize progress in the study of RET from bench to bedside, focusing on pathophysiology of neuroendocrine tumors.

Context: CYP11B2, the gene encoding aldosterone synthase, has several frequent polymorphisms. In particular, the Lys173Arg (K173R) polymorphism is in complete genetic linkage disequilibrium with the -344T/C polymorphism in the promoter of CYP11B2 that involves a binding site for the steroidogenic factor-1 transcription factor. These polymorphisms have been associated with cardiovascular parameters, including hypertension, but not directly with gene expression., Objective: The objective of this study was to correlate CYP11B2 genotype with gene expression in adrenal tissue., Design: We measured mRNA levels of CYP11B2 [presented as a ratio against glyceraldehyde-3-phosphate dehydrogenase (B2/G)] and CYP11B1 in relation to the K173R polymorphism., Subjects: We studied 28 subjects with aldosterone-producing adenomas (APA) and 18 subjects with normal adrenals., Main Outcome Measure: The main outcome measure was CYP11B2 expression levels., Results: Preoperative treatment with spironolactone or beta-blocker in five APA patients was associated with higher B2/G. The B2/G and B2/B1 ratios were much higher even in the remaining 23 APA patients than in subjects with normal adrenals. The B2/G and B2/B1 ratios in normal adrenals and APA were higher in the KK genotype than in the RR genotype. In patients with APA, urinary aldosterone excretion was higher in those with the KK genotype than in those with the KR genotype. Measurement of cDNA band intensities from normal and APA samples of the KR genotype revealed that the R173 allele was transcribed at levels 46.6 +/- 12.2% (mean +/- sd; n = 7) and 49.1 +/- 20.8% (n = 6), respectively, those of the K173 allele., Conclusions: A CYP11B2 haplotype including -344T and K173 is associated with higher gene expression than the -344C/R173 haplotype, supporting reported associations of -344T with higher aldosterone production and blood pressure.

Germline NF1, c-RET, SDH, and VHL mutations cause familial pheochromocytoma. Pheochromocytomas derive from sympathetic neuronal precursor cells. Many of these cells undergo c-Jun-dependent apoptosis during normal development as NGF becomes limiting. NF1 encodes a GAP for the NGF receptor TrkA, and NF1 mutations promote survival after NGF withdrawal. We found that pheochromocytoma-associated c-RET and VHL mutations lead to increased JunB, which blunts neuronal apoptosis after NGF withdrawal. We also found that the prolyl hydroxylase EglN3 acts downstream of c-Jun and is specifically required among the three EglN family members for apoptosis in this setting. Moreover, EglN3 proapoptotic activity requires SDH activity because EglN3 is feedback inhibited by succinate. These studies suggest that failure of developmental apoptosis plays a role in pheochromocytoma pathogenesis.

Mutations in VHL, RET, NF1, SDHB, SDHC, and SDHD can give rise to pheochromocytoma/paraganglioma. These different genetic lesions may all act by decreasing the activity of a 2-oxoglutarate-dependent oxygenase, SM-20/EglN3/PHD3, resulting in reduced apoptosis of neural crest cells during development.

In a previous study, we show that stimulation of chemotaxis in rat pheochromocytoma PC12 cells by nerve growth factor (NGF) and epidermal growth factor (EGF) requires activation of the RAS-ERK signaling pathway. In this study, we compared the threshold levels of ERK activation required for EGF and NGF-stimulated chemotaxis in PC12 cells. The threshold ERK activity required for NGF to stimulate chemotaxis was approximately 30% lower than that for EGF. PD98059 treatment inhibited EGF stimulation of growth and chemotaxis; however, stimulation of chemotaxis required an EGF concentration approximately 10 times higher than for stimulation of PC12 cell growth. Thus, ERK-dependent cellular functions can be differentially elicited by the concentration of EGF. Also, treatment of PC12 cells with the PI3-K inhibitor LY294002 reduced ERK activation by NGF; thus, higher NGF concentrations were required to initiate chemotaxis and to achieve the same maximal chemotactic response seen in untreated PC12 cells. Therefore, the threshold NGF concentration to stimulate chemotaxis could be adjusted by the crosstalk between the ERK and PI3-K pathways, and the contributions of PI3-K and ERK to signal chemotaxis varied with the concentrations of NGF used. In comparison, LY294002 treatment had no effect on ERK activation by EGF, but the chemotactic response was reduced at all the concentrations of EGF tested indicating that NGF and EGF differed in the utilization of ERK and PI3-K to signal chemotaxis in PC12 cells.

Background: Inactivation of the human type Ialpha regulatory subunit (RIalpha) of cyclic AMP dependent protein kinase (PKA) (PRKAR1A) leads to altered kinase activity, primary pigmented nodular adrenocortical disease (PPNAD), and sporadic adrenal and other tumours., Methods and Results: A transgenic mouse carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline responsive promoter (the Tg(Prkar1a*x2as)1Stra, Tg(tTAhCMV)3Uh or tTA/X2AS line) developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia and other features reminiscent of PPNAD, including late onset weight gain, visceral adiposity, and non-dexamethasone suppressible hypercorticosteronaemia, with histiocytic, epithelial hyperplasias, lymphomas, and other mesenchymal tumours. These lesions were associated with allelic losses of the mouse chromosome 11 Prkar1a locus, an increase in total type II PKA activity, and higher RIIbeta protein levels; the latter biochemical and protein changes were also documented in Carney complex tumours associated with PRKAR1A inactivating mutations and chromosome 17 PRKAR1A locus changes., Conclusion: We conclude that the tTA/X2AS mouse line with a downregulated Prkar1a gene replicates several of the findings in Carney complex patients and their affected tissues, supporting the role of RIalpha as a candidate tumour suppressor gene.

Flavonoids and related structures (e.g., flavones, isoflavones, flavanones, catechins) exert various biological effects, including anticarcinogenic, antioxidant and (anti-)estrogenic effects, and modulation of sex hormone homeostasis. A key enzyme in the synthesis of estrogens from androgens is aromatase (cytochrome P450 19; CYP19). We investigated the effects of various natural and synthetic flavonoids on the catalytic activity and promoter-specific expression of aromatase in H295R human adrenocortical carcinoma cells. Natural flavones were consistently more potent inhibitors than flavanones. IC(50) values for 7-hydroxyflavone, chrysin, and apigenin were 4, 7, and 20 microM, respectively; for the flavanones 7-hydroxyflavanone and naringenin the IC(50) values were 65 and 85 microM, respectively. The steroidal aromatase inhibitor (positive control) 4-hydroxyandrostenedione had an IC(50) of 20 nM. The inhibition by apigenin and naringenin coincided with some degree of cytotoxicity at 100 microM. The natural flavonoid derivative rotenone (IC(50) 0.3 microM) was the most potent aromatase inhibitor tested. Several synthetic flavonoid and structurally related quinolin-4-one analogs inhibited aromatase activity. The most potent inhibitor was 4'-tert-butyl-quinolin-4-one (IC(50) 2 microM), followed by two 2-pyridinyl-substituted alpha-naphthoflavones (IC(50)s 5 and >30 microM). The two 2-pyridinyl-substituted gamma-naphthoflavones consistently produced biphasic concentration-response curves, causing about 1.5-fold aromatase induction at concentrations below 1 microM and inhibition above that level (IC(50)s 7 and >30 microM). The natural flavone quercetin and isoflavone genistein induced aromatase activity 4- and 2.5-fold induction, respectively, at 10 microM. This coincided with increased intracellular cAMP concentrations and increased levels of the cAMP-dependent pII and to a lesser extent 1.3 promoter-specific aromatase transcripts. These results shed light on the structure-activity relationships for aromatase inhibition as well as mechanisms of induction in human H295R cells.

Neuroblastoma is the single most common and deadly tumor of childhood and is often associated with therapy resistance. Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to prostaglandins. COX-2 is up-regulated in several adult epithelial cancers and is linked to proliferation and resistance to apoptosis. We detected COX-2 expression in neuroblastoma primary tumors and cell lines but not in normal adrenal medullas from children. Treatment of neuroblastoma cells with nonsteroidal anti-inflammatory drugs, inhibitors of COX, induced caspase-dependent apoptosis via the intrinsic mitochondrial pathway. Treatment of established neuroblastoma xenografts in nude rats with the dual COX-1/COX-2 inhibitor diclofenac or the COX-2-specific inhibitor celecoxib significantly inhibited tumor growth in vivo (P < 0.001). In vitro, arachidonic acid and diclofenac synergistically induced neuroblastoma cell death. This effect was further pronounced when lipooxygenases were simultaneously inhibited. Proton magnetic resonance spectroscopy ((1)H MRS) of neuroblastoma cells treated with COX inhibitors demonstrated accumulation of polyunsaturated fatty acids and depletion of choline compounds. Thus, (1)H MRS, which can be performed with clinical magnetic resonance scanners, is likely to provide pharmacodynamic markers of neuroblastoma response to COX inhibition. Taken together, these data suggest the use of nonsteroidal anti-inflammatory drugs as a novel adjuvant therapy for children with neuroblastoma.

In an effort to clarify the role of telomerase in the pathogenesis of pheochromocytomas and neuroblastomas, and to test whether its component could serve as a marker of malignancy, we measured telomerase reverse transcriptase (TERT) mRNA in 31 human pheochromocytoma tissue samples (5 malignant, 23 benign and 3 suspected malignant) and 16 neuroblastoma tissues (9 unfavorable and 7 favorable). All cases were classified by both the clinical course and histopathological examination. Malignancy was defined as the presence of metastasis and/or extensive local invasion. TERT mRNA was determined by nested PCR and a real-time PCR system (LightCycler). By nested PCR methods, 5 of the 5 malignant pheochromocytoma samples were positive (sensitivity = 100%), and 21 of 23 benign pheochromocytoma samples were negative (specificity = 91%) in pheochromocytomas. Four out of five malignant tumors were positive for either hTERT expression or Ki-67/MIB-1 immunoreactivity. In the neuroblastoma tissues, 9 of the 9 unfavorable samples were positive (sensitivity = 100%), and only 2 of 7 favorable samples were negative (specificity = 29%). We also determined the expression of the hTERT mRNA by real-time PCR to quantitate the mRNA. The mean values of hTERT mRNA by real time PCR in benign and malignant pheochromocytomas were 2 and 26 arbitrary units (AU), respectively. The difference was not significant by the U-test. The mean values of hTERT mRNA in favorable and unfavorable neuroblastoma were 203 and 497 AU, respectively. This difference was also not significant (U-test). N-Myc mRNA expression correlated with the expression of hTERT mRNA in the neuroblastoma samples (r = 0.534, p = 0.0317). Thus, hTERT mRNA might be a potential marker for estimating the malignancy of pheochromocytomas and neuroblastomas.

Germ-line mutations in the genes encoding succinate dehydrogenase complex subunits B (SDHB) and D (SDHD) have been reported in familial paragangliomas and apparently sporadic phaeochromocytomas (ASP), but the genotype-phenotype relationships of these mutations are unknown. Eighty-four patients (all but 2 followed up for 8.8 +/- 5.7 years) with ASP (57 with adrenal tumors, 27 with extra-adrenal, multiple, malignant, or recurrent tumors) were screened for the major susceptibility genes for phaeochromocytoma (RET, VHL, SDHD, and SDHB). Thirty-three tumors were available for molecular analysis, enzyme assays, and immunohistochemistry. No (0%) RET and 2 (2.4%) VHL mutations were detected. Only two coding single nucleotide polymorphisms in the SDHD gene (G12S and H50R) were found in 6 patients (7%). Conversely, six deleterious mutations in the SDHB gene were identified in 8 patients (9.5%). Ectopic site and recurrence or malignancy were strongly associated with SDHB mutations (7 of 8, 87%, versus 20 of 76, 26%; P = 0.001). Somatic DNA analysis indicated a loss of heterozygosity at chromosome 1p36 (SDHB locus) in 16 of 33 cases (48%). A loss of heterozygosity at the SDHB locus was found in all tumors with SDHB mutation, and assays of respiratory chain enzymes showed a complete loss of complex II catalytic activity. The vascular architecture of tumors with SDHB mutations displayed features typical of malignancy. These data strongly suggest that SDHB gene is a tumor suppressor gene and that the identification of germ-line mutations in SDHB gene in patients with ASPs should be considered as a high-risk factor for malignancy or recurrence.

Pheochromocytomas and paragangliomas are tumors of the autonomic nervous system; pheochromocytomas are tumors of the adrenal medulla, and paragangliomas are extra-adrenal tumors arising from either the sympathetic nervous system or parasympathetic ganglia. It has previously been estimated that approximately 10%-15% of pheochromocytomas are due to hereditary causes. However, our increased understanding of the three hereditary syndromes (neurofibromatosis 1, multiple endocrine neoplasia type 2, and von Hippel-Lindau syndrome) in which pheochromocytoma is found and the recent discovery that mutations in genes in the succinate dehydrogenase family (SDHB and SDHD) predispose to pheochromocytoma have necessitated a re-evaluation of the genetic basis of pheochromocytoma. These studies indicate that the frequency of germline mutations associated with isolated pheochromocytoma is higher than previously estimated, with both hospital-based series and a large population-based series indicating that the frequency of germline mutations in RET, VHL, SDHB, and SDHD taken together approximates 20%. In all patients with pheochromocytoma, including those with known hereditary syndrome or a positive family history, the frequency of germline mutations in these four genes together approaches 30%. Given the frequency of germline mutations, consideration should be given to genetic counseling for all patients with pheochromocytoma and is particularly important for individuals with a positive family history, multifocal disease, or a diagnosis before age 50. Identification of patients with hereditary pheochromocytoma is important because it can guide medical management in mutation-positive patients and their families. This review provides an overview of the known genetic syndromes that are commonly associated with pheochromocytoma, examines recent data on the association of germline mutations in the succinate dehydrogenase gene family with pheochromocytoma, and suggests guidelines for the genetic evaluation of pheochromocytoma patients.

Pheochromocytoma, a catecholamine-producing tumor of the chromaffin tissue, may present with various features. Herein, we report case of 66-year-old woman with pheochromocytoma accompanying hyperamylasemia and acute abdomen. She was admitted to another hospital due to myocardial infarction 5 months ago. At that time, pheochromocytoma was suggested on the basis of hormonal studies, but she refused surgical resection. When she came to our hospital, serum amylase level was 703 U/L. Subsequent studies revealed pancreatic type isoenzyme, and elevated lipase level. After normalization of serum amylase level, she undertook laparoscopic adrenalectomy. On pathologic examination, pheochromocytoma was confirmed. There are several cases of pheochromocytoma with hyperamylasemia. In general, the source of hyperamylasemia was thought to be pulmonary endothelial cells under ischemic damage caused by potent vasoconstrictive action of circulating catecholamines. In our case, analysis of isoenzymes and serum lipase level suggest that hyperamylasemia can originate from the pancreas. Thus, pancreatitis also should be considered when serum amylase level is elevated in pheochromocytoma.

Background: Serotonin is the principal endocrine product of carcinoid tumors, but simultaneously increased production of catecholamines has been described in these tumors. As it is not clear whether these tumors contain specific enzymes for catecholamine synthesis, we aimed to detect catecholamine-synthesizing enzymes [tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT)] in midgut carcinoid tumors and pheochromocytoma and to correlate enzyme expression to serotonin production as well as catecholamines and metabolites excreted in urine., Methods: Paraffin-embedded tumor specimens from 21 midgut carcinoid patients and 20 pheochromocytoma patients (10 sporadic and 10 MEN type IIa-related tumors) were stained for TH, DBH, and PNMT, using a three-step biotin-avidin-peroxidase method., Results: TH was demonstrated in 9 (43%) of 21 carcinoids and in all (100%) of 20 pheochromocytomas, DBH in 8 (38%) carcinoids and in 15 (75%) pheochromocytomas, and PNMT in 7 (33%) carcinoids and in 13 (65%) pheochromocytomas. Increased urinary excretion of catecholamines and metabolites was observed in 10 (48%) carcinoid patients and in all pheochromocytoma patients. No clinically relevant association between enzyme expression and urinary excretion of catecholamines and metabolites was found., Conclusions: Catecholamine-synthesizing enzymes are present in many carcinoid tumors. This finding possibly indicates the existence of a catecholamine-synthesizing pathway in carcinoids similar to that found in pheochromocytoma.

Phaeochromocytomas arising in adrenal or extra-adrenal sites and paragangliomas of the head and neck, in particular of the carotid bodies, occur sporadically and also in a familial setting. In addition to mutations in RET and VHL in familial disease, germline mutations in SDHD and SDHB genes that encode subunits of mitochondrial complex II have also been associated with the development of familial phaeochromocytomas. To further investigate the role of SDHD and SDHB in the development of these tumours we determined the occurrence of germline SDHD and SDHB mutations in four patients with a family history of phaeochromocytoma with associated head and neck paraganglioma, one patient with a family history of phaeochromocytoma only and two patients with apparently sporadic extra-adrenal phaeochromocytoma, one of whom had early onset disease. Secondly, we investigated whether somatic SDHB mutations correlated with loss of heterozygosity at 1p36 in a subgroup of 11 sporadic and three MEN 2-associated RET-mutation-positive phaeochromocytomas. Novel SDHB mutations were identified in the probands from four families and two apparently sporadic cases (six of seven probands studied), including two missense mutations, a single nonsense and frameshift mutation, as well as two splice site mutations, one of which was shown to have partial penetrance resulting in 'leaky' splicing. Further, five intronic polymorphisms in SDHB were found. No SDHD mutations were identified. In addition, no somatic SDHB mutations were found in the remaining allele of the 11 sporadic adrenal phaeochromocytomas with allelic loss at 1p36 or the three MEN 2-associated RET-mutation-positive phaeochromocytomas. Therefore, we conclude that SDHB has a major role in the pathogenesis of familial phaeochromocytomas, but the possible role of SDHB in sporadic tumours showing allelic loss at 1p36 has yet to be ascertained.

We have reported that excessive steroid hormone production in adrenal cortical tumors results from the disordered expression and activity of specific steroidogenic enzymes. Since no genetic mutations in these steroidogenic enzymes have as yet been identified, disordered expression at the transcription level may be crucial for excessive hormone production in adrenocortical tumors. Nuclear receptors SF-1 and COUP-TF/DAX-1 have been shown to activate and repress, respectively, the transcription of CYP17 gene in a mutually exclusive manner in Y-1 cells. Interestingly, the expression of COUP-TF and DAX-1 is significantly decreased in the cortisol-producing adenomas, in which CYP17 is overexpressed. Conversely, DAX-1 is highly expressed in deoxycorticosterone-producing adenomas, where CYP17 expression is almost absent. These expression profiles indicate the possibility that COUP-TF and DAX-1 play important roles in the transcriptional repression of CYP17 in adrenal tumors. To clarify the mechanisms of COUP-TF-mediated repression, we therefore screened for COUP-TF-interacting proteins using a yeast two-hybrid system from a cortisol-producing adenoma cDNA library. We then cloned a novel COUP-TF-interacting protein-1 (CIP-1), which interacts with COUP-TFI, COUP-TFII, and SF-1. Functionally, CIP-1 can act as a transcriptional co-repressor for COUP-TF repression activity. CIP-1 expression profiles parallel those of COUP-TFI in steroidogenic tissues, strongly suggesting that, together, COUP-TFI and CIP-1 play an important role in steroidogenesis., (Copyright 2003 S. Karger AG, Basel)

Phaeochromocytoma is a tumour of the adrenal medulla, which, although rare, is a major cause of correctable hypertension with a prevalence of 0.1-0.5% in the hypertensive population. Clinical symptoms include attacks of paroxysmal headache, sweating, palpitations, stress and a sense of imminent death. Often associated with the above is an increase in blood pressure. Despite the fact that the underlying genetic mechanisms of phaeochromocytoma have been well investigated, they are still incompletely understood. In approximately 80% of cases the tumour occurs sporadically, but it may occur in association with type 2 multiple endocrine neoplasia, type 1 neurofibromatosis or von Hippel-Lindau disease. Molecular evidence suggests that other genes such as SDHD or SDHB may control its development; the possibility of other putative phaeochromocytoma genes is currently being investigated., (Copyright 2003 S. Karger AG, Basel)

Adrenal gland as a major source of enkephalins on the periphery can be affected by a rare adrenal gland tumor, adrenal pheochromocytoma. It has been demonstrated that this tumor might be associated with altered concentration of enkephalin-like peptides. The effect of these peptides can be either prolonged or abbreviated by two neutrophil membrane bound enzymes; aminopeptidase N (APN) and neutral endopeptidase (NEP). We assumed that altered enkephalin level in pheochromocytoma patients (but not in patients with non-functional adenomas or tumors of different origin) might result in differently regulated APN and/or NEP activity. We measured APN and NEP activity on surface of neutrophils, level of lipid peroxidation (LPO) in plasma and enkephalin concentration in plasma in patients with pheochromocytomas, non-functional adenomas, malignant renal tumors and healthy controls. Catheholamines and vanyllmandelic acid (VMA) were measured in 24-h urine of pheochromocytoma patients. NEP and APN activity on neutrophils from all pheochromocytoma patients was significantly increased as compared with healthy controls, non-functional adenomas and malignant renal tumors. In all pheochromocytoma patients NEP activity was reduced almost to the control level after surgery. At the same time APN activity was in some patients up- and in others down-regulated. In comparison, elevated levels of cateholamines and VMA were found after multiple determinations in 6 out of 10 pheochromocytoma patients. Although preliminary, this study has shown specifically and consistently up-regulated NEP activity on neutrophils from pheochromocytoma patients, and uniformly decreased NEP activity in these patients after adrenalectomy.

Introduction: Extracellular matrix (ECM) degradation is an essential step that allows tumor cells to penetrate a tissue barrier and become metastatic. Heparanase-1 (HPR) is an endoglycosidase that specifically degrades heparan sulfate proteoglycans, a chief component of the ECM. Previous studies have demonstrated HPR expression in various malignancies and that there is differential HPR expression between benign and malignant tumors. Currently, there is no technique that can reliably predict the malignant behavior of some pheochromocytomas. This study tests whether HPR is differentially expressed in malignant and benign pheochromocytomas., Methods: Paraffin-embedded specimens from 29 pheochromocytomas were evaluated. The tissues were collected from surgical specimens over a 10-year period from 26 patients (8 males, 18 females) with a mean age of 47 years (range 19-78 years, median 47 years). One female patient underwent 3 separate operations for malignant pheochromocytoma and thus provided 3 specimens. Another female patient had both the primary tumor and a liver metastasis processed, and therefore provided 2 specimens. Patient charts and pathology reports were reviewed to classify the pheochromocytomas as either benign or malignant. Based on clinical behavior and/or pathological evidence of metastasis or invasion into surrounding tissues, 10 specimens were malignant and 19 had benign behavior. As a control, normal adrenal tissue from 3 nephrectomy specimens was included in the study, as was tissue from 1 adrenocortical adenoma. All 33 specimens were tested for HPR gene expression by in situ hybridization (ISH) with an antisense RNA probe and immunohistochemistry (IHC) with an anti-HPR antibody. Statistical analysis was done using the chi(2) test of proportions to determine if HPR expression correlated with malignancy using ISH, IHC, or both tests together., Results: Using ISH, the percentage of HPR expression in the malignant pheochromocytomas was 50% while HPR expression in the benign tumors was 21% (P = 0.11). Using IHC, the percentage of HPR expression in the malignant pheochromocytomas was 80% while HPR expression in the benign tumors was 32% (P = 0.01). Considering both tests cumulatively, all 10 malignant pheochromocytomas stained positive for HPR by ISH and IHC, while only 37% of the benign tumors were positive for HPR expression (P = 0.001). The one adrenal adenoma and the 3 normal adrenal glands processed stained negative for HPR expression by both ISH and IHC., Conclusions: HPR expression is higher in malignant pheochromocytomas than in benign pheochromocytomas or normal tissue. HPR may contribute to the invasive characteristics of malignant pheochromocytomas and might be used as a marker to distinguish malignant from benign pheochromocytomas. HPR expression might also be used as a prognostic tool in guiding long-term patient follow-up.

Three genes encoding for mitochondrial complex II proteins are linked to hereditary paraganglioma. We have recently shown that an inactivation of the SDHD gene is associated with a complete loss of mitochondrial complex II activity and a stimulation of the angiogenic pathway (Gimenez-Roqueplo, A. P., J. Favier, P. Rustin, J. J. Mourad, P. F. Plouin, P. Corvol, A. Rötig, and X. Jeunemaitre, 2001, Am J Hum Genet 69:1186-1197). Here, we relate the case of a malignant sporadic pheochromocytoma induced by a germline missense mutation of the SDHB gene. Within the tumor, a loss of heterozygosity at chromosome 1pter led to a null SDHB allele and to a complete loss of complex II enzymatic activity. In situ hybridization and immunohistochemistry experiments showed a high expression of hypoxic-angiogenic responsive genes, similar to that previously observed in inherited-SDHD tumors. This observation highlights the role of the complex II mitochondrial genes in the oxygen-sensing pathway and in the regulation of angiogenesis of neural crest-derived tumors.

Progranulin (pgrn; PC-cell-derived growth factor, epithelin precursor, or acrogranin) has been identified recently as an autocrine regulator of tumorigenesis in several cancer cells including SW-13 adrenal carcinomas and some breast cancers, but how pgrn promotes tumor progression is not well understood. SW-13 cells do not form tumors in nude mice but become highly tumorigenic when their pgrn expression is elevated, and this provides a useful model in which to investigate the role of pgrn in tumorigenesis. Here we show that, in SW-13 cells, the level of pgrn expression is a major determinant of the intrinsic activity of the mitogen-activated protein kinase, phosphatidylinositol 3'-kinase, and focal adhesion kinase signaling pathways. Pgrn stimulates the invasion of SW-13 cells across Matrigel-coated filters, increases the expression of matrix metalloproteinase 13 and 17, protects against anoikis, and overcomes the inhibition of cell growth imposed on SW-13 cells by interstitial type-I collagen. Inhibition of the mitogen-activated protein kinase and phosphatidylinositol 3'-kinase signaling pathways impairs each of the pgrn-dependent biological responses tested, but to different extents. The ability of pgrn to stimulate cell division, invasion, and survival demonstrates that pgrn regulates multiple steps in carcinomal progression, and suggests that the pgrn system may be a possible future therapeutic target.

Familial paraganglioma is a dominantly inherited disorder characterised by the development of highly vascular tumours in the head and neck. Recently, a relationship between hereditary tumours derived from the autonomic nervous system and germline mutations in the gene encoding succinate dehydrogenase complex subunit D (SDHD) is increasingly a subject of study. Familial paraganglioma syndrome is embryologically related to phaeochromocytoma, another neuroendocrine tumour that shows great aetiological and genetic heterogeneity. Some hereditary phaeochromocytomas may be associated with germline mutations in VHL, RET and NF1 genes in genetic disorders such as von Hippel-Lindau disease (VHL), multiple endocrine neoplasia type 2 (MEN 2) and neurofibromatosis type 1 (NF 1), respectively. However, there are many cases that cannot be explained by mutations in these genes. In this report, we describe two previously unreported mutations in two patients from 25 unrelated kindreds with phaeochromocytoma and/or paraganglioma disorders and with or without familial antecedents: a mutation featuring the change of tryptophan to a termination codon in exon 2, and a 4-bp deletion in exon 4 that results in a truncated protein. We also describe one missense substitution of uncertain significance. The patients had previously tested negative for germline mutations in VHL and RET genes and had not been previously selected. The involvement of SDHD mutations in familial phaeochromocytoma and/or paraganglioma predisposition is of considerable interest since other studies have shown these alterations to be associated with highly expressed angiogenic factors.

Several types of endocrine tumors show frequent somatic deletions of the distal part of chromosome arm 11q, where the tumor-suppressor gene SDHD (succinate-ubiquinone oxidoreductase subunit D), constitutionally mutated in paragangliomas of the head and neck, is located. In this study, we screened 18 midgut carcinoids, 7 Merkel cell carcinomas, 46 adrenal pheochromocytomas (37 sporadic and 9 familial), and 7 abdominal paragangliomas for loss of heterozygosity (LOH) and/or mutations at the SDHD gene locus. LOH was detected in 5 out of 8 (62%) informative midgut carcinoids, in 9 out of 30 (30%) sporadic pheochromocytomas, in none of the familial pheochromocytomas (0%), and in 1 out of 6 (17%) abdominal paragangliomas. No sequence variants were detected in the pheochromocytomas or paragangliomas. However, two constitutional putative missense mutations, H50R and G12S, were detected in two midgut carcinoids, which were both associated with LOH of the other allele. The same sequence variants were also detected in two Merkel cell carcinomas. In addition, the S68S polymorphism was found to coexist with the G12S sequence variant in both cases. In conclusion, we show that alterations of the SDHD gene seem to be involved in the tumorigenesis of both midgut carcinoids and Merkel cell carcinomas., (Copyright 2002 Wiley-Liss, Inc.)

Background: 11beta-Hydroxysteroid dehydrogenase types 1 and 2 (11betaHSD1 and 11betaHSD2) are two isoenzymes that convert inactive glucocorticoids (e.g., cortisone) to their active forms (e.g., cortisol) and vice versa. Abundant evidence indicates that 11betaHSD2 is expressed as mRNA and protein in both adrenal cortex and adrenal tumors. In contrast, 11betaHSD1 has been investigated to a much lesser degree. We therefore studied and compared the expression and activity of the two isoenzymes in the human adrenal cortex (HAC) and cortisol-secreting adenomas (CSAs)., Methods: Six HAC and six CSA specimens were studied. 11betaHSD1 and 11betaHSD2 gene expression was studied by conventional and semiquantitative reverse transcription-polymerase chain reaction. 11betaHSD1 and 11betaHSD2 activity was assayed by measuring the capacity of both microsomal fraction and tissue fragments to convert [3H]cortisone to [3H]cortisol and vice versa. Steroid hormones were separated and purified by high-performance liquid chromatography, and cortisol concentration was measured by radioimmunoassay., Results: Semiquantitative reverse transcription-polymerase chain reaction and enzymatic assay demonstrated higher 11betaHSD1 expression and activity and lower 11betaHSD2 expression and activity in CSAs than in HACs. CSA slices secreted larger amounts of cortisol than did HAC specimens, and the cholesterol side-chain-cleaving enzyme inhibitor aminoglutethimide, by blocking the early step of steroid synthesis, reduced cortisol secretion by approximately 70%. Aminoglutethimide decreased [3H]cortisol production from [3H]cortisone and increased [3H]cortisone production from [3H]cortisol in both tissues, thereby annulling differences in 11betaHSD1 and 11betaHSD2 activity between HACs and CSAs., Conclusion: Collectively, our findings indicate that 1) both 11betaHSD isoenzymes are expressed as mRNA and proteins in the HAC and CSA, with 11betaHSD1 upregulated and 1betaHSD2 downregulated in CSAs; and 2) 11betaHSD1 and 11betaHSD2 activity is positively and negatively correlated with the intracellular concentration of steroid hormones. Hence, 11betaHSD isoenzymes could act as amplifiers of the secretagogue effect of agonists and could contribute to the elevated hormonal secretion of CSAs.

Objective: As a result of the widespread use and the enhanced quality of high-resolution radiological techniques [computed tomography (CT), magnetic resonance imaging (MRI)] a high frequency (4-10%) of adrenal incidentalomas has been detected in the general population. It is still debated whether undiagnosed 21-hydroxylase (21-OH) deficiency, accounting for more than 90% of congenital adrenal hyperplasia (CAH) cases, predisposes for adrenal tumours. We therefore performed an analysis of the prevalence of 21-OH germline mutations in patients with non-functional adrenal incidentalomas., Subjects and Methods: Fifty Austrian patients with non-functional adrenal adenomas detected by CT for unrelated reasons were screened by PCR-based sequencing for the most common point mutations and by Southern blot analysis for large gene deletion/conversion events of the 21-OH gene., Results: Heterozygosity for large gene conversions was shown in 5 (10%), for Q318 point mutations in 2 (4%) and for the Intron2splice mutation in 1 (2%) of the 50 patients with adrenal adenomas. One (2%) patient (70 years of age), identified to have a chimeric CYP21AB gene with a junction site before Intron 2 on one and a large (30 kb) deletion on the other allele, was diagnosed to be affected by CAH., Conclusion: 21-OH mutation screening indicates a higher frequency of classic CAH carriers (16%) and of manifest CAH (2%) due to 21-OH-deficiency among patients with adrenal adenomas than in the general population (1-2% carrier frequency for classic CAH).

The Rit and Rin proteins comprise a distinct and evolutionarily conserved subfamily of Ras-related small GTPases. Although we have defined a role for Rit-mediated signal transduction in the regulation of cell proliferation and transformation, the function of Rin remains largely unknown. Because we demonstrate that Rin is developmentally regulated and expressed in adult neurons, we examined its role in neuronal signaling. In this study, we show that stimulation of PC6 cells with either epidermal growth factor or nerve growth factor (NGF) results in rapid activation of Rin. This activation correlates with the onset of Ras activation, and dominant-negative Ras completely inhibits Rin activation induced by NGF. Further examination of Ras-mediated Rin activation suggests that this process is dependent upon neuronally expressed regulatory factors. Expression of mutationally activated H-Ras fails to activate Rin in non-neuronal cells, but results in potent stimulation of Rin-GTP levels in a variety of neuronal cell lines. Furthermore, although constitutively activated Rin does not induce neurite outgrowth on its own, both NGF-induced and oncogenic Ras-induced neurite outgrowth were inhibited by the expression of dominant-negative Rin. Together, these studies indicate that Rin activation is a direct downstream effect of growth factor-dependent signaling in neuronal cells and suggest that Rin may function to transduce signals within the mature nervous system.

Background: Overexpression of manganese superoxide dismutase (MnSOD) cDNA via plasmid transfection leads to growth inhibition in vitro and in vivo in various human cancers. Polyphenolic compounds such as catechin isolated from tea bush Camellia sinensis has been shown to have anticancer effect in vitro. This study evaluated the effect of catechin on the MnSOD activity and mRNA level of pheochromocytoma cells (PC-12)., Methods: PC-12 cells were incubated with different concentrations of catechin at short-term (2 days) and long-term (7 days) in Dulbecco modified Eagle medium. The activity of superoxide dismutase (SOD) was measured and its mRNA was assayed by Northern blotting., Results: After incubation for 2 days, catechin slightly but significantly increased the activity of copper/zinc superoxide dismutase (CuZnSOD). However, it did not show any significant effect at 7 days. The MnSOD activity showed significant changes in both short-term and long-term. The amount of mRNA also showed similar changes., Conclusions: Catechin is a natural antioxidant which has been shown to have antitumor effect in basic and epidemiological studies. The present data suggest that catechin can increase MnSOD gene expression in PC-12, which might have beneficial effect in tumor prevention.

The neurotransmitter norepinephrine is metabolized by monoamine oxidase into an aldehyde intermediate that is further metabolized to the stable glycol derivative, 3,4-dihydroxyphenylglycol (DHPG). In this study, the possible role of aldose reductase in reducing this aldehyde intermediate in human sympathetic neurons has been examined. DHPG is formed when norepinephrine is incubated with aldose reductase in the presence of monoamine oxidase. DHPG metabolism is inhibited by the monoamine oxidase inhibitor, pargyline which prevents the deamination of norepinephrine, and by the aldose reductase inhibitor AL 1576, which inhibits DHPG formation without affecting the deamination of norepinephrine. Although similar formation of DHPG was observed with human liver aldehyde reductase, the production of DHPG was more effective with aldose reductase than aldehyde reductase. Two peaks of reductase activity corresponding to aldose reductase and aldehyde reductase were observed when sympathetic ganglia were chromatofocused. Molecular modeling studies indicate that the energy-minimized structure of 3,4-dihydroxymandelaldehyde bound to aldose reductase is similar to that of glyceraldehyde where the 2'-hydroxyl group forms hydrogen bonds with Trp111 and NADPH. These results suggest that aldose reductase may be important in metabolizing the potentially toxic aldehyde intermediate formed from norepinephrine in human sympathetic ganglia.

Objective: The prevalence of steroid secretion abnormalities was studied by evaluating the 17-hydroxyprogesterone (17-OHP) and 11-deoxycortisol (S) responses to adrenocorticotropic hormone (ACTH) stimulation in 48 patients with 'nonfunctioning' incidentalomas and in 10 patients with 'subclinical' Cushing's syndrome., Methods: In all patients the cortisol, 17-OHP, and S levels were measured after ACTH test. Eight patients were reinvestigated after surgery., Results: In patients with nonfunctioning lesions, the ACTH test induced 17-OHP and S peaks higher than in normals (p < 0.005). In 10 cases an augmented rise of 17-OHP and S was observed. In patients with subclinical Cushing's syndrome, the 17-OHP peak after ACTH was greater than in patients with nonfunctioning lesions and in normals (p < 0.005); the S peak was also higher than in controls (p < 0.005). In 7 of 8 operated patients, the exaggerated 17-OHP peak was normalized., Conclusions: A combined impairment of different enzyme activities is frequently present in adrenal incidentalomas; the alteration of enzymatic pathways can also coexist with the presence of partial cortisol autonomy., (Copyright 2002 S. Karger AG, Basel)

Some species of marine sponge have been shown to produce metabolites with endocrine-altering and cell growth regulatory properties. Since cell division and differentiation are controlled, in part, by the mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK/ERK) cascade, we tested extracts (1.0mg/ml) from six shallow water marine species obtained in the Florida Keys for effects on MAPK/ERK(l,2) (sub-variant of EC 2.7.1.37) activity in incubations with SW-13 human adrenal carcinoma cells in culture. In these short-term incubations, extracts from two species, the purple bleeding sponge (Iotrochota birotulata) and the West Indian bath sponge (Spongia barbara), significantly inhibited MAPK/ERK(1,2) activity (to 51 and 44% of control levels, respectively) without altering cell survival. Western blots for phosphorylated and total ERK showed that ERK(2) predominated over ERK(1) by a factor of about 4:1 and that the phosphorylated forms of these isozymes were strongly suppressed by active extracts from both sponges. Another species, the green sponge (Haliclona veridis), whose extract has been shown previously to activate guanylate cyclase and to inhibit adenylate cyclase in a variety of mammalian tissues, was found not to affect MAPK/ERK(1,2) in human adrenal carcinoma cultures but did lyse and kill most of these cultured cells. Extracts from the sheepswool sponge (Hippospongia lachne) and the bleeding sponge (Oligoceras hemorrhages) did not significantly affect either MAPK/ERK(1,2) activity or the survival of attached cells. An extract from the fire sponge (Tedania ignis) did not alter MAPK/ERK(1,2) activity but did modestly decrease cell viability. These studies document for the first time species-specifc effects of marine sponge extracts on the MAPK/ERK(1,2) cascade and on the growth and survival of human adrenal carcinoma cells in culture.

Pheochromocytomas are rare tumors of the adrenal medulla or the paraganglion system. There are no histological or chemical markers available that define the malignant behavior of these tumors; so far only the discovery of metastases reveals malignancy. Cyclooxygenase (Cox) is the key enzyme in conversion of arachidonic acid to PGs, and two isoforms, Cox-1 and Cox-2, have been identified. Cox-2 has been associated with carcinogenesis, and it is overexpressed in many human malignancies. We have now investigated the expression of Cox-2 in normal adrenal gland, in 92 primary pheochromocytomas and in six metastases using immunohistochemistry and Northern blot and Western blot analyses. Cox-2 protein was expressed in the adrenal cortex, whereas the medulla was negative as detected by immunohistochemistry. Interestingly, all malignant pheochromocytomas (n = 8), regardless of the primary location of the tumor, showed moderate or strong Cox-2 immunoreactivity, whereas 75% of the benign adrenal tumors (n = 36) showed no or only weak immunopositivity. The staining was negative or weak in 79% of the adrenal tumors that showed histologically suspicious features (n = 24), but had not metastasized. Most of the pheochromocytoma samples studied also expressed low levels of Cox-2 mRNA. Our data show that normal adrenal medulla does not express Cox-2 immunohistochemically. However, strong Cox-2 protein expression was found in malignant pheochromocytomas, whereas most benign tumors expressed Cox-2 only weakly. To our knowledge, this is the first report on Cox-2 expression in pheochromocytomas and enhanced expression in malignant pheochromocytomas. These findings suggest that negative or weak Cox-2 expression in pheochromocytomas favors benign diagnosis.