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A low-cost template-free solution chemical route to highly porous nanocrystalline sponges of ZnO-EuO1.5 with 0-5 mol % Eu is presented. The process uses Zn- and Eu-acetate-nitrate and triethanolamine as precursors in methanol. After evaporation of the solvent and heating at 200 °C for 3 min, crystalline ZnO:Eu sponges with minor amounts of organic residues were obtained. Heating to 400 °C replaced the organics with carbonate, which in its turn was decomposed at temperatures below 600 °C, forming ZnO:Eu sponges. Samples heated to 200-1000 °C for 3 min were studied with XRD, SEM, TEM, TG, XPS, and IR spectroscopy. The ZnO:Eu crystallite sizes could be tuned from below 10 nm for sponges prepared at 200-500 °C, to over 100 nm range at 900 °C, without sintering of the overall microstructure. XRD showed the presence of hexagonal ZnO:Eu (or at 700-1000 °C, ZnO:Eu and cubic Eu2O3) as the only phases present. The ZnO:Eu had slightly larger unit cell dimensions than the literature value of ZnO for samples obtained at 200-600 °C, while the unit cells of samples obtained at higher temperatures were quite close to the value of undoped ZnO. XPS showed that Eu was mainly in its 3+ state and well-distributed within the sponges but segregated at the ZnO sponge surface upon heating at 700-1000 °C, in accordance with XRD studies showing Eu2O3 formation.

Small intestinal neuroendocrine tumors (SI-NETs) are small, slow growing neoplasms with loss of one copy of chromosome 18 as a common event. Frequently mutated genes on chromosome 18 or elsewhere have not been found so far. The aim of this study was to investigate a possible tumor suppressor role of the transmembrane receptor type tyrosine phosphatase PTPµ (PTPRM at 18p11) in SI-NETs. Immunohistochemistry, quantitative RT-PCR, colony formation assay and quantitative CpG methylation analysis by pyrosequencing were performed. Undetectable/very low levels of PTPRM or aberrant pattern of immunostaining, with both negative and positive areas, were detected in the majority of tumors (33/40), and a significantly reduced mRNA expression in metastases compared to primary tumors was observed. Both the DNA methylation inhibitor 5-aza-2′-deoxycytidine and the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) induced PTPRM expression in CNDT2.5 and KRJ-I SI-NET cells. CpG methylation of upstream regulatory regions, the promoter region and the exon 1/intron 1 boundary was detected by pyrosequencing analysis of the two cell lines and not in the analyzed SI-NETs. Overexpression of PTPRM in the SI-NET cell lines reduced cell growth and cell proliferation and induced apoptosis. The tyrosine phosphatase activity of PTPRM was not involved in cell growth inhibition. The results support a role for PTPRM as a dysregulated candidate tumor suppressor gene in SI-NETs and further analyses of the involved mechanisms are warranted.

Small intestinal neuroendocrine tumors (SI-NETs) are slow-growing tumors that seem genetically quite stable without highly recurrent mutations, but are epigenetically dysregulated. In contrast to the undetectable expression of the enhancer of zeste homolog 2 (EZH2) histone methyltransferase in the enterochromaffin cells of the small intestine, we found high and differential expression of EZH2 in primary SI-NETs and corresponding metastases. Silencing EZH2 in the SI-NET cell line CNDT2.5 reduced cell proliferation and induced apoptosis. Furthermore, EZH2 knockout inhibited tumor progression in a CNDT2.5 SI-NET xenograft mouse model, and treatment of SI-NET cell lines CNDT2.5 and GOT1 with the EZH2-specific inhibitor CPI-1205 decreased cell viability and promoted apoptosis. Moreover, CPI-1205 treatment reduced migration capacity of CNDT2.5 cells. The EZH2 inhibitor GSK126 also repressed proliferation of CNDT2.5 cells. Recently, metformin has received wide attention as a therapeutic option in diverse cancers. In CNDT2.5 and GOT1 cells, metformin suppressed EZH2 expression, and inhibited cell proliferation. Exposure of GOT1 three-dimensional cell spheroids to CPI-1205 or metformin arrested cell proliferation and decreased spheroid size. These novel findings support a possible role of EZH2 as a candidate oncogene in SI-NETs, and suggest that CPI-1205 and metformin should be further evaluated as therapeutic options for patients with SI-NETs.

An efficient, template-free solution-chemical route to nanostructured ZnO sponges is presented: A mixture of Zn(NO3)2·6H2O, Zn(OAc)2·2H2O, and triethanolamine in methanol was evaporated to a highly viscous liquid and rapidly heated to >200 °C for 1–3 min to achieve highly porous, nanocrystalline sponges of ZnO. The viscous precursor concentrate obtained on evaporation in air was characterized by TG, DSC, and IR spectroscopy, and the product ZnO sponges by XRD, SEM, TEM, and IR spectroscopy. The fast reaction forming ZnO started at 140 °C and finished within a few seconds. Scherrer analysis of the XRD peak broadening showed average crystallite sizes of 8 to 11 nm for ZnO prepared by annealing at 200–450 °C (3 min), while grain growth to 134 nm was observed from 500 to 900 °C (3 min). The ZnO powders obtained at 200–900 °C had cell dimensions of a = 3.25 A and b = 5.21 A, matching the ZnO literature data well. SEM and TEM analyses showed highly porous, bread-like 3D nanostructures built by ca. 30–70 nm thic...

Primary hyperparathyroidism (pHPT) is rarely caused by parathyroid carcinoma (PC, TET1andTET2play important roles in various cancers. Recently, we found that 5hmC was severely reduced in all of the analyzed PCs and with deranged expression of TET1 for the majority of PCs. Here, we have examined the expression of the TET2 protein in 15 5hmC-negative PCs from patients who had local invasion or metastases. Cell growth and cell migratory roles for TET2 as well as epigenetic deregulated expression were addressed. Immunohistochemistry revealed very low/undetectable expression of TET2 in all PCs and verified for two PCs that were available for western blotting analysis. Knockdown of TET2 in the parathyroid cell line sHPT-1 resulted in increased cell growth and increased cell migration. DNA sequencing ofTET2in PCs revealed two common variants and no obvious inactivating mutations. Quantitative bisulfite pyrosequencing analysis of theTET2promoter CpG island revealed higher CpG methylation level in the PCs compared to that in normal tissues and treatment of a PC primary cell culture with the DNA methylation inhibitor 5-aza-2′-deoxycytidine caused increased expression of the methylatedTET2gene. Hence, the data suggest that deregulated expression ofTET2by DNA hypermethylation may contribute to the aberrantly low level of 5hmC in PCs and further that TET2 plays a cell growth and cell migratory regulatory role and may constitute a parathyroid tumor suppressor gene.

Small intestinal neuroendocrine tumors (SI-NETs) are amine- and peptide-producing neoplasms. Most patients display metastases at the time of diagnosis; they have an unpredictable individual disease course and the tumors are often therapy resistant. Chromogranin A and 5-hydroxyindoleacetic acid are the biomarkers clinically used most often today, but there is a great need for novel diagnostic and prognostic biomarkers and new therapeutic targets. Sixty-nine biomarkers were screened in serum from 23 SI-NET patients and 23 healthy controls using the multiplex proximity ligation assay (PLA). A refined method, the proximity extension assay (PEA), was used to analyze 76 additional biomarkers. Statistical testing and multivariate classification were performed. Immunohistochemistry and ELISA were performed in an extended cohort. Using PLA, 19 biomarkers showed a significant difference in serum concentrations between patients and controls, and PEA revealed a difference in the concentrations of 17 proteins. Multivariate classification analysis revealed decoy receptor 3 (DcR3), trefoil factor 3 (TFF3), and midkine to be good biomarkers for the disease, which was confirmed by ELISA analysis. All 3 biomarkers were expressed in tumor tissue. DcR3 concentrations were elevated in patients with stage IV disease. High concentrations of DcR3 and TFF3 were correlated to poor survival. DcR3, TFF3, and midkine exhibited elevated serum concentrations in SI-NET patients compared to healthy controls, and DcR3 and TFF3 were associated with poor survival. DcR3 seems to be a marker for liver metastases, while TFF3 and midkine may be new diagnostic biomarkers for SI-NETs.

Neuroendocrine tumour of the small intestine (SI-NET), formerly known as midgut carcinoid tumour, is the most common small intestinal malignancy. The incidence is rising, with recent reports of 0.67 per 100 000 in the USA and 1.12 per 100 000 in Sweden. SI-NETs often present a challenge in terms of diagnosis and treatment, as patients often have widespread disease and are beyond cure by surgery. Somatostatin analogues provide the mainstay of medical treatment to control hormonal excess and increase the time to progression. Despite overall favourable prognosis (5-year overall survival of 65%), there is a need to find markers to identify both patients with worse outcome and new targets for therapy. Loss on chromosome 18 has been reported in 60-90% of SI-NETs, but mutated genes on this chromosome have failed detection. Recently, a putative tumour suppressor role has been suggested for TCEB3C occurring at 18q21 (encoding elongin A3), which may undergo epigenetic repression. CDKN1B has recently been revealed as the only recurrently mutated gene in SI-NETs but, with a frequency as low as 8%, its role as a driver in SI-NET development may be questioned. Integrated genomewide analysis including exome and whole-genome sequencing, gene expression, DNA methylation and copy number analysis has identified three novel molecular subtypes of SI-NET with differing clinical outcome. DNA methylation analysis has demonstrated that SI-NETs have significant epigenetic dysregulation in 70-80% of tumours. In this review, we focus on understanding of the genetic, epigenetic and molecular events that lead to development and progression of SI-NETs.

Primary hyperparathyroidism (pHPT) is a common endocrine disease characterized by excessive secretion of parathyroid hormone and an increased level of serum calcium. Overall, 80–85% of pHPT cases are due to a benign, single parathyroid adenoma (PA), and 15% to multiglandular disease (multiple adenomas/hyperplasia). Parathyroid carcinoma (PC) is rare, accounting for

A fast, low cost solution route to nickel dot coated NbC powder using Ni(NO3)26H2O, Ni(OAc)24H2O and triethanolamine (TEA) as precursors in methanol solvent was developed. After mixing the precursor solution with 50–300 ​nm sized NbC powder and evaporation of the solvent, heating to 150 ​°C, or higher, yielded NbC powder evenly covered with well-dispersed nickel metal dots, 6–25 ​nm in size, depending on the nickel loading and temperature. Coatings yielding 4 to 14 ​wt% nickel loading were studied at temperatures from 200 to 700 ​°C, using TG-DTA, XRD, SEM, XPS with Ar+-ion etching, TEM/DF, and STEM/HAADF/EDS.

European Neuroendocrine Tumor Society staging, together with the Ki67 grading system, has appeared as superior for classification of neuroendocrine tumors (NET). The management of small intestinal NET (SI-NET) has been overall controversial. Mesenteric metastases occur also with the smallest SI-NET, and the majority of patients risk to ultimately progress with liver metastases. 68Gallium (somatostatin receptor)/PET/CT has appeared as most sensitive for imaging, and fluorodeoxyglucose-PET is recommended to identify lesions with high proliferation. Our treatment policy for SI-NET is to initiate somatostatin analog treatment, and in order to prevent abdominal complications we recommend early intestinal resection for removal of primary tumors and clearance of lymph node metastases. Liver metastases are liberally treated by resection (or ablation), as this can efficiently palliate carcinoid syndrome-associated symptoms.

Until recently, the genetic landscape of small intestinal neuroendocrine tumors (SI-NETs) was limited to recurrent copy number alterations, most commonly a loss on chromosome 18. Intertumor heterogeneity with nonconcordant genotype in paired primary and metastatic lesions also is described, further contributing to the difficulty of unraveling the genetic enigma of SI-NETs. A recent study analyzing 55 SI-NET exomes nominated CDKN1B (p27) as a haploinsufficient tumor suppressor gene.This study aimed to determine the frequency of CDKN1B inactivation and to investigate genotype-phenotype correlations. It investigated 362 tumors from 200 patients. All samples were resequenced for mutations in CDKN1B using automated Sanger sequencing. The expression of p27 was investigated in 12 CDKN1B mutant and nine wild type tumors.Some 8.5 % (17/200) of patients had tumors with pathogenic mutations in CDKN1B including 13 insertion deletions, four nonsense variants, and one stop-loss variant. All variants with available nontumoral DNA were classified as somatic. Inter- and intratumor heterogeneity at the CDKN1B locus was detected respectively in six of ten and two of ten patients. Patients with CDKN1B mutated tumors had both heterogeneous disease presentation and diverse prognosis. Expression of the p27 protein did not correlate with CDKN1B mutation status, and no differences in the clinical characteristics between CDKN1B mutated and CDKN1B wild type tumor carriers were found.This study corroborates the finding of CDKN1B as a potential haplo-insufficient tumor suppressor gene characterized by inter- and intratumor heterogeneity in SI-NETs.

Background One-fifth of all patients with small-intestinal neuroendocrine tumors (SI-NETs) present with or develop peritoneal carcinomatosis (PC). Our aim was to determine the prognosis and genetic profiles of tumors in patients with PC compared with tumors in patients without PC. Methods We included SI-NET patients (cases with PC, n = 73, and controls without PC, n = 468) who underwent operation between 1985 and 2012. The Lyon prognostic index was used to correlate the amount of PC to survival. DNA samples from patients with ( n = 8) and without ( n = 7) PC were analyzed with a single-nucleotide polymorphism array (HumanOmni2.5 BeadChip, Illumina) to investigate genetic disparities between groups. Results Patients with PC had poorer survival (median 5.1 years) than controls (11.1 years). An advanced postoperative Lyon prognostic index was a negative prognostic marker for survival by multivariable analysis ( P = .042). Patients with and without PC clustered differently based on loss of heterozygosity and copy number variation data from single-nucleotide polymorphism array of the primary tumors ( P = .042). Conclusion SI-NET patients with PC have poor survival, which diminishes with increasing PC load after surgery. Clustering based on copy number variation and loss of heterozygosity data suggests different genotypes in primary tumors comparing patients with and without PC.

The genetic background in small intestinal neuroendocrine tumors is poorly understood, but several studies have revealed numerical imbalances. Loss of one copy of chromosome 18 is the most frequent genetic aberration in this tumor type, which indirectly suggests that a driver mutation may be present in the remaining allele. The aim of this study was to evaluate the mutation status on chromosome 18 in small intestinal neuroendocrine tumors. DNAs from 7 small intestinal neuroendocrine tumors were subjected to whole exome capture, followed by next generation sequencing and high resolution SNP array followed by copy number variation analysis. Exome capture sequencing generated an average coverage of 50.6-138.2. Only 19 genes were covered less than 8X. No tumor-specific somatic mutation was identified. Genomic profiling revealed loss of chromosome 18 in 5 out of 7 small intestinal neuroendocrine tumors and a number of other aberrancies. Loss of chromosome 18 is the most frequent genetic aberration in small intestinal neuroendocrine tumors, but no evidence for eventual mutations in the remaining allele. This suggests involvement of other mechanisms than point mutations in small intestinal neuroendocrine tumors tumorigenesis.

Background Primary hyperparathyroidism is characterized by enlarged parathyroid glands due to an adenoma (80–85 %) or multiglandular disease (~15 %) causing hypersecretion of parathyroid hormone (PTH) and generally hypercalcemia. Parathyroid cancer is rare (

Background Small intestinal neuroendocrine tumors (SI-NETs) originate from the enterochromaffin cells in the ileum and jejunum. The knowledge about genetic and epigenetic abnormalities is limited. Low mRNA expression levels of actin gamma smooth muscle 2 (ACTG2) have been demonstrated in metastases relative to primary SI-NETs. ACTG2 and microRNA-145 (miR-145) are aberrantly expressed in other cancers and ACTG2 can be induced by miR-145. The aim of this study was to investigate the role of ACTG2 in small intestinal neuroendocrine tumorigenesis. Methods Protein expression was analyzed in SI-NETs (n = 24) and in enterochromaffin cells by immunohistochemistry. The cell line CNDT2.5 was treated with the histone methyltransferase inhibitor 3-deazaneplanocin A (DZNep), the selective EZH2 inhibitor EPZ-6438, or 5-aza-2’-deoxycytidine, a DNA hypomethylating agent. Cells were transfected with ACTG2 expression plasmid or miR-145. Western blotting analysis, quantitative RT-PCR, colony formation- and viability assays were performed. miR-145 expression levels were measured in tumors. Results Eight primary tumors and two lymph node metastases displayed variable levels of positive staining. Fourteen SI-NETs and normal enterochromaffin cells stained negatively. Overexpression of ACTG2 significantly inhibited CNDT2.5 cell growth. Treatment with DZNep or transfection with miR-145 induced ACTG2 expression (>10-fold), but no effects were detected after treatment with EPZ-6438 or 5-aza-2’-deoxycytidine. DZNep also induced miR-145 expression. SI-NETs expressed relatively low levels of miR-145, with reduced expression in metastases compared to primary tumors. Conclusions ACTG2 is expressed in a fraction of SI-NETs, can inhibit cell growth in vitro, and is positively regulated by miR-145. Theoretical therapeutic strategies based on these results are discussed. Electronic supplementary material The online version of this article (doi:10.1186/s12902-016-0100-3) contains supplementary material, which is available to authorized users.

Primary hyperparathyroidism (pHPT) is a pathology associated with one or multiple hyperfunctioning parathyroid glands. The disease prevalence occurs in roughly 1-2% of the population primarily post-menopausal women. The molecular pathology of the disease is poorly understood. Elevated serum calcium levels in the setting of an inappropriately elevated parathyroid hormone level are indicative of the disease process. The ultimate treatment of the disease is to remove the hyperfunctioning gland.The aim of this thesis was to examine potential genetic and epigenetic aberrations that are potentially disease causing.The methylation signature of normal and pathological parathyroid tissue has yet to be investigated. DNA was bisulphite modified and analyzed using the Infinium HumanMethylation27 BeadChip. Distinct hierarchical clustering of genes with altered DNA methylation profiles in normal and pathologic parathyroid tissue was evident. DNA hypermethylation of CDKN2B, CDKN2A, WT1, SFRP1, SFRP2, and SFRP4 known to be important in the development of parathyroid tumors were associated with reduced gene expression in both benign and malignant parathyroid tumors.Familial primary hyperparathyroidism (FPHPT) may occur due to an underlying germ-line mutation in the MEN1, CASR, or HRPT2/CDC73 genes. Eighty-six young (≤45 years of age) patients with clinically non-syndromic PHPT underwent genetic analysis. Eight of 86 (9.3%) young patients with clinically non-familial PHPT displayed deleterious germ-line mutations in the susceptibility genes (4 MEN1, 3 CASR, and 1 HRPT2/ CDC73).Accumulation of non-phosphorylated active β -catenin has been reported to commonly occur in parathyroid adenomas from patients with primary hyperparathyroidism (pHPT). We assessed possible β-catenin stabilizing mutations in a large series of parathyroid adenomas. A total of one hundred and eighty sporadic parathyroid adenomas were examined for mutations in exon 3 of the CTNNB1gene. The mutation S33C (TCT >TGT) was detected by direct-DNA sequencing of PCR fragments in 1 out of 180 sporadic parathyroid adenomas (0.68 %).Eight matched tumor-constitutional DNA pairs from patients with sporadic parathyroid adenomas underwent whole-exome capture and high-throughput sequencing. Four of eight tumors displayed a frame shift deletion or nonsense mutations within the MEN1 gene, which was accompanied by loss of heterozygosity of the remaining wild-type allele. One tumor harbored a Y641N mutation of the histone methyltransferase EZH2 gene, previously linked to myeloid and lymphoid malignancy formation. Targeted sequencing in the additional 185 parathyroid adenomas revealed a high rate of MEN1 mutations (35%).

The genetic events leading the progression of midgut carcinoid tumors are largely unknown. The disease course varies from patient to patient, and there is a lack of reliable prognostic markers. In order to identify genes involved in tumor progression, gene expression profiling was performed on tumor specimens. Samples comprised 18 primary tumors, 17 lymph node (LN) metastases, and seven liver metastases from a total of 19 patients. Patients were grouped according to clinical data and histopathology into indolent or progressive course. RNA was subjected to a spotted oligo microarray and B-statistics were performed. Differentially expressed genes were verified using quantitative real-time PCR. Self-organizing maps demonstrated three clusters: 11 primary tumors separated in one cluster, five LN metastases in another cluster, whereas all seven liver metastases, seven primary, and 12 LN metastases formed a third cluster. There was no correlation between indolent and progressive behavior. The primary tumors with Ki67 >5%, with low frequency of the carcinoid syndrome, and a tendency toward shorter survival grouped together. Primary tumors differed in expression profile from their associated LN metastases; thus, there is evidence for genetic changes from primary tumors to metastases.ACTG2, GREM2, REG3A, TUSC2, RUNX1, TPH1, TGFBR2, andCDH6were differentially expressed between clusters and subgroups of tumors. The expression profile that assembles tumors as being genetically similar on the RNA expression level may not be concordant with the clinical disease course. This study reveals differences in gene expression profiles and novel genes that may be of importance in midgut carcinoid tumor progression.

Current studies suggest that short-term exposure of parathyroid glands to fibroblast growth factor 23 (FGF23) reduces parathyroid hormone secretion. However, patients with chronic kidney disease (CKD) develop secondary hyperparathyroidism despite high levels of serum FGF23, indicating a parathyroid FGF23 ‘resistance'. Here we analyzed the expression of the FGF23 receptors Klotho and FGF receptor 1 (FGFR1) in 88 hyperplastic parathyroid glands from 31 patients with CKD (including 21 renal allograft recipients), and their regulation in isolated bovine and human hyperplastic parathyroid cells. Glandular expression was variable, yet the Klotho and FGFR1 mRNA levels declined in parallel with the decreasing glomerular filtration rate, significantly decreasing over CKD stages. We found no association between the expression of Klotho, FGFR1, and the proliferation marker Ki67. In vitro treatment of bovine cells with FGF23 or calcium reduced the Klotho level, whereas active vitamin D 3 compounds increased its expression. Phosphate and parathyroid hormone had no effect. Treatment had less impact on Klotho in cultured human cells than in the bovine healthy cell model, whereas FGFR1 expression was induced in the hyperplastic cells. Thus parathyroid Klotho and FGFR1 decrease with declining renal function, possibly because of alterations in mineral metabolism related to the failing kidney. This could explain the observed parathyroid resistance to FGF23 in late CKD.

Pheochromocytomas of the adrenal medulla may be life-threatening catecholamine-producing tumors which are malignant in about 10% of cases. Differential diagnosis between malignant and benign tumors is dependent on the development of metastasis or extensive local invasion. A number of genetic aberrations have been described in pheochromocytomas, but no marker associated to malignancy has been reported. We applied an expression microarray containing 7770 cDNA clones and analysed the expression profiles in eleven tumors compared to normal adrenal medulla. Stathmin (STMN1, Op18) was most conspiciously overexpressed among the differentially expressed genes. RT-PCR analysis further confirmed mRNA overexpression, 6 to 8-fold for benign and malignant tumors, and 16-fold for metastases. Stathmin protein overexpression was observed by immunohistochemistry, and distinct differential protein expression between benign and malignant/metastasis specimens was confirmed by Western blot analysis. The results introduce stathmin as a possible diagnostic marker for malignant pheochromocytomas, and further evaluations are warranted.

Primary hyperparathyroidism (HPT) is often caused by a benign parathyroid tumor, adenoma; less commonly by multiglandular parathyroid disease/hyperplasia; and rarely by parathyroid carcinoma. Patients with multiple tumors require wider exploration to avoid recurrence and have increased risk for hereditary disease. Secondary HPT is a common complication of renal failure. Improved knowledge of the molecular background of parathyroid tumor development may help select patients for appropriate surgical treatment and can eventually provide new means of treatment. The present contribution summarizes more recent knowledge of parathyroid molecular genetics.A literature search and review was made to evaluate the level of evidence concerning molecular biology and genetics of primary, secondary, and familial HPT according to criteria proposed by Sackett, with recommendation grading by Heinrich et al.Most parathyroid adenomas and hyperplastic glands are monoclonal lesions. Cyclin D1 gene (CCND1) translocation and oncogene action occur in 8% of adenomas; cyclin D1 overexpression is seen in 20% to 40% of parathyroid adenomas and in 31% of secondary hyperplastic glands. Somatic loss of one MEN1 allele is seen in 25% to 40% of sporadic parathyroid adenomas, half of which have inactivating mutation of the remaining allele. Inactivating somatic HRPT2 mutations are common in parathyroid carcinoma, often causing decreased expression of the protein parafibromin involved in cyclin D1 regulation. Aberrant regulation of Wnt/beta-catenin signaling may be important for parathyroid tumor development.Molecular genetic studies of parathyroid tumors are well designed basic experimental studies providing strong level III evidence, with data frequently confirmed by subsequent studies.

Context: The type I membrane protein Klotho was recently shown to mediate PTH secretion in parathyroid cells in response to low extracellular calcium. In contrast, Klotho inhibits PTH secretion indirectly through the action of fibroblast growth factor-23. Abnormal Klotho expression in parathyroid disorders remains to be elucidated. Objective: The aim of the study was to determine: 1) Klotho expression in parathyroid adenomas from patients with primary hyperparathyroidism (pHPT) compared to normal tissue; and 2) its relation to the serum calcium and PTH levels. Design: Surgically removed parathyroid glands (n = 40) and four normal parathyroid tissue specimens were analyzed for Klotho mRNA and protein levels by quantitative real-time PCR and immunohistochemistry. In vitro effects of calcium on Klotho mRNA expression were studied in bovine parathyroid cells. Results: Klotho mRNA levels were significantly decreased (n = 23) or undetectable (n = 17) in parathyroid adenomas compared to normal tissues (P < 0.001). Reduced Klotho protein expression was confirmed by immunohistochemistry. Klotho mRNA levels were inversely correlated to serum calcium (r = −0.97; P < 0.0001), and calcium dose-dependently decreased Klotho mRNA expression in normal parathyroid cells in vitro (P < 0.01). Serum calcium was the only significant marker of Klotho expression in multivariate analysis with calcium, phosphate, PTH, and adenoma weight as independent variables. Conclusions: Parathyroid Klotho expression is decreased or undetectable in pHPT. We provide evidence that 1) serum calcium is strongly associated with parathyroid Klotho expression in pHPT; and 2) abnormal PTH secretion in hypercalcemic pHPT subjects is mediated by Klotho-independent mechanisms.

Fibroblast growth factor-23 (FGF23) is a novel, bone-produced hormone that regulates renal phosphate (Pi) reabsorption and calcitriol metabolism. Disorders of mineral and bone metabolism, such as autosomal dominant hypophosphatemic rickets (ADHR) and hyperostosis-hyperphosphatemia syndrome (HHS), witness the importance of well-balanced serum levels of FGF23. Patients with chronic kidney disease (CKD) are highly morbid due to Pi retention/hyperphosphatemia and calcitriol deficiency, which lead to elevated serum levels of parathyroid hormone (PTH) and secondary hyperparathyroidism (sHPT). As a response to hyperphosphatemia, CKD patients have also remarkably high serum FGF23 levels, which are associated with cardiovascular risk factors and increased mortality in CKD. The overall aim of this dissertation was to discern a possible role of FGF23 in parathyroid biology. Our in vitro experiments on isolated bovine parathyroid cells demonstrate that FGF23 directly and dose-dependently suppresses the PTH production and secretion, while increasing the expression of the 25-hydroxyvitamin D3-activating enzyme 1α-hydroxylase. We investigated possible expressional changes in the FGF23 receptor co-factor Klotho in hyperparathyroid disorders and found that Klotho expression is decreased or absent and inversely correlated to serum calcium (Ca) in adenomas of primary HPT (pHPT). In the hyperplastic parathyroid glands of sHPT, Klotho expression declines in parallel with the kidney function and correlates with the glomerular filtration rate. Moreover, Klotho expression is suppressed by Ca and FGF23, increased by calcitriol, but unaffected by Pi and PTH in vitro. Finally, we identified a novel missense mutation in the gene encoding GALNT3, which is normally involved in the post-translational glycosylation of FGF23, as the cause of aberrant FGF23 processing in a patient with HHS. In summary, we provide evidence for a novel bone/parathyroid axis in which FGF23 functions as a direct, negative regulator of the PTH production. High extracellular Ca is a major determinant of the Klotho expression in pHPT, whereas the Klotho levels in sHPT may be attributed to a combination of the high FGF23 and Ca, and low calcitriol levels associated with CKD. Hence, the decreased Klotho expression in sHPT could explain the concomitantly high FGF23 and PTH levels, as well as the failure of FGF23 to prevent or mitigate the development of sHPT in CKD.

Background and objective: 1,25-dihydroxyvitamin D3 [1α,25(OH)2D3, calcitriol], and its less calcaemic synthetic analogues have therapeutic potential in several diseases, including hyperparathyroidism (HPT). We have suggested that non-1α-hydroxylated (nonactive) vitamin D analogues may present an alternative in tumour cells expressing 25-hydroxyvitamin D3 1α-hydroxylase (1α-hydroxylase). The aim of this study was to investigate biological effects of a non-1α-hydroxylated vitamin D analogue in normal and tumour parathyroid cells. Patients and methods: Effects of vitamin D analogues and ketoconazole on parathyroid hormone (PTH) secretion (radioimmunoassay) and PTH mRNA expression (reverse transcription-polymerase chain reaction) were studied in primary bovine parathyroid cells. Proliferation of tumour cells isolated from HPT patients was determined by thymidine incorporation Results: EB1285, non-1α-hydroxylated precursor of the vitamin D analogue EB1089, suppressed PTH secretion and PTH mRNA level as well as increased expression of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) in bovine parathyroid cells. EB1285 also inhibited cell proliferation of parathyroid tumour cells from primary (pHPT) and secondary HPT (sHPT) patients. Combined treatment with the cytochrome P450-dependent enzyme inhibitor ketoconazole and EB1285 or with active vitamin D compounds potentiated the suppressive effect on PTH secretion from bovine parathyroid cells. Ketaconazole alone displayed PTH suppression and increased 24-hydroxylase expression. Conclusion: The results support the idea that a non-1α-hydroxylated vitamin D analogue may elicit vitamin D receptor (VDR) effects in 1α-hydroxylase expressing parathyroid tumour cells. Further studies are warranted to elucidate whether precursor vitamin D analogues as well as inhibitors of 24-hydroxylase present therapeutic alternatives in patients suffering from HPT.

Primary hyperparathyroidism (pHPT) resulting from parathyroid tumors is a common endocrine disorder with incompletely understood etiology, affecting about 1% of the adult population, with an even higher prevalence for elderly individuals. In renal failure, secondary hyperparathyroidism (sHPT) occurs with multiple tumor development as a result of calcium and vitamin D regulatory disturbance.Aberrant Wnt/beta-catenin signaling with accumulation of beta-catenin in the cytoplasm/nucleus is involved in the development of a variety of neoplasms. The aim of this study was to evaluate whether the Wnt/beta-catenin signaling pathway is activated in parathyroid adenomas of pHPT and in hyperplastic glands from uremic patients with sHPT.Immunohistochemistry, Western blotting, real-time quantitative RT-PCR, and DNA sequencing were performed.beta-Catenin was accumulated in all analyzed parathyroid tumors (n = 47) from patients with pHPT and from patients with HPT secondary to uremia. The accumulation included nonphosphorylated, stabilized (transcriptionally active) beta-catenin. The overexpression was not related to increased beta-catenin mRNA levels. A protein-stabilizing mutation in exon 3 of beta-catenin (S37A) was detected in three of 20 pHPT tumors (15%). No mutation was detected in secondary hyperplastic glands (n = 20), and no evidence for truncated adenomatosis polyposis coli proteins was found in adenomas and secondary hyperplastic glands. Mutations in other Wnt signaling components leading to beta-catenin accumulation, other than in beta-catenin itself, are therefore anticipated. The beta-catenin target gene c-myc was overexpressed in a substantial fraction of the parathyroid tumors.Our results strongly suggest that modifications in the Wnt/beta-catenin signaling pathway may be involved in the development of hyperparathyroidism.

Vitamin D receptor (VDR) and 25-hydroxyvitamin D3 1-α-hydroxylase expression have recently been shown to be upregulated in several tumors and thought to represent an important endogenous response to tumor progression. Little is known about the expression of these proteins in thyroid carcinoma, although previous reports have documented evidence of the biological effect of vitamin D in thyroid cells. Using paraffin-embedded and frozen sections of papillary thyroid carcinoma, we utilized real-time quantitative RT-PCR and immunohistochemistry to characterize the expression of VDR and 1-α-hydroxylase in thyroid follicular cells, with special emphasis on papillary thyroid carcinoma (PTC). VDR and 1-α-hydroxylase expression were increased in PTC compared with normal thyroid tissue and especially high in areas of lymphocyte infiltration. Expression of VDR and 1-α-hydroxylase in PTC may be compatible with an overall favorable prognosis for this tumor type and may constitute important prerequisites for using vitamin D and/or vitamin D analogs in the treatment of PTC.

The cytochrome P450 mitochondrial enzyme 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase) of renal tubule cells hydroxylates the major circulating form of vitamin D (25(OH)D3) to the active systemic hormone 1,25(OH)2D3. Local production of 1,25(OH)2D3 appears to occur also at other sites where 1alpha-hydroxylase is expressed for autocrine/paracrine regulation. To reduce risks of hypercalcemia during treatment with vitamin D, we have previously suggested use of non-1alpha-hydroxylated vitamin D analogues to target tissues where 1alpha-hydroxylase is expressed, including the parathyroid glands in secondary hyperparathyroidism. The present study was undertaken to examine expression of 1alpha-hydroxylase in breast cancer and to investigate whether a non-1alpha-hydroxylated vitamin D analogue displayed biological function. In addition, expression of the 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) and the vitamin D receptor (VDR) was investigated.The expression of 1alpha-hydroxylase, 24-hydroxylase and VDR was investigated in breast cancer specimens (n = 19) and normal breast tissues (n = 10) by immunohistochemistry and/or RT-PCR. Consecutive cryosections of 6 mum essentially free of immune cells were used in the analyses. The effect of vitamin D analogues on transcriptional activation was analyzed in transiently transfected MCF-7 breast cancer cells.1alpha-hydroxylase protein was demonstrated in 79% and 100% of breast cancer specimens and normal breast, respectively. The overall relative mRNA levels of 1alpha-hydroxylase and 24-hydroxylase in normal breast compared to breast tumors were: 1alpha-hydroxylase, 1 +/- 0.07 versus 0.7 +/- 0.05, respectively (p0.001); 24-hydroxylase, 1 +/- 0.08 verus 2.1 +/- 0.2, respectively (p0.001). The VDR was expressed in 95% of the tumors as expected, with mRNA levels of 1 +/- 0.09 and 1.4 +/- 0.12 (p0.05) in breast cancer and normal breast, respectively. The ketoconazole-sensitive transcription activation potential of the non-1alpha-hydroxylated vitamin D analogue prodrug of EB1089 (EB1285) was demonstrated in MCF-7 cells, which express 1alpha-hydroxylase. The activity of EB1285 was about 20% of 1,25(OH)2D3.These results demonstrate nearly normal expression levels of 1alpha-hydroxylase, 24-hydroxylase and VDR in the majority of investigated breast cancer specimens. A non-1alpha-hydroxylated vitamin D analogue displayed activity in breast cancer cells. Such analogues may present future therapeutic options for proliferative disorders where 1alpha-hydroxylase is expressed.

Carbon-silica composite films have been fabricated using sol-gel synthesis of silica-carbon precursor composites, followed by carbonization in an inert atmosphere. Four categories of samples were studied. These were the tetraethyl orthosilicate-only (TEOS-only), methyl trimethoxysilane (MTES), acetic acid anhydride (Ac2O) and soot (SOOT) samples. The carbon-silica films, spin-coated on aluminium and steel substrates, have been investigated for selective solar absorber functionality. Optical measurements were performed on these samples to determine solar absorptance and thermal emittance of each. The morphology of the films was studied by electron microscopy. Electron energy-loss spectroscopy (EELS) mapping was used to determine the carbon distribution. An optimum performance of 0.88 for solar absorptance and 0.41 for thermal emittance has been achieved. The sol-gel technique produced films with very flat surfaces and uniform thicknesses in the 1 μm range. The fine structure showed homogeneous mixing of the carbon and silica in the TEOS-only samples while the separate additions of MTES and Ac2O resulted in segregation of composition of the silica and carbon at nano-scale. The addition of 20 wt% MTES or 15 wt% Ac2O to the TEOS-only sols also helped to reduce the cracks in the TEOS-only samples. The addition of soot in place of the carbon precursor did not yield a net advantage.

An efficient, low-cost solution-chemical route to nanophase cobalt as dense films and sponges has been developed. Co(NO3)2·6H2O and Co(OAc)2·4H2O were mixed with triethanolamine in methanol, and the solution was evaporated in air to a viscous liquid. Heating at 10 C min-1 to 470 and 500 C yielded highly porous sponges consisting of 3-6 and 9-11 nm sized face centered cubic (fcc) cobalt crystallites, respectively. Spin-coating on alumina and aluminum substrates with a 1 M Co-precursor solution and subsequent heating at 10 C min-1 to 500 C yielded dense films of

In the absence of metastases or overgrowth to adjacent organs, the lack of reliable markers for malignancy is a well-recognized problem for clinicians managing patients with endocrine tumors. Apart from inactivation of the multiple endocrine neoplasia type 1 (MEN1) gene, the molecular mechanisms involved in tumorigenesis of the endocrine organs and MEN1-associated nonendocrine lesions are vastly unknown. To try to learn more about down-stream effects on MEN1 gene inactivation, we used the BON1 cells, showing low levels of endogenous menin, and transfected them with a MEN1 gene construct. On restoring the menin expression, we recorded inhibition of cell growth. We also performed macroarray and present data on differentially expressed genes in the transfected cells, after corroboration by Northern blots and quantitative PCR. JunD was up-regulated in menin-expressing clones, whereas delta-like protein 1/preadipocyte factor-1 (involved in differentiation and growth of the pancreatic endocrine cells), proliferating cell nuclear antigen, and QM/Jif-1 (a negative regulator of c-Jun) became down-regulated. These findings might contribute to the understanding of the tissue-specific features of MEN1. We also show that homozygous inactivation of the MEN1 gene statistically correlates to higher expression of delta-like protein 1/preadipocyte factor-1, proliferating cell nuclear antigen, and QM/Jif-1, as well as lower MEN1 expression, in a limited sample of malignant endocrine pancreatic tumors.

Hyperparathyroidism (HPT) refers to states of excessive production of parathyroid hormone (PTH). The eukaryotic cell cycle is driven forward by cyclins and their cyclin-dependent kinase (CDK) partners. Cyclin-dependent kinase inhibitors (CKIs), which generally inhibit cell cycle progression, modulate the activity of cyclin-CDK complexes.In order to quantify the expression of the CKI genes p18, p21, and p27 semiquantitative RT-PCR with mRNA specific-primers was performed on four normal parathyroid biopsies, 31 parathyroid adenomas of primary HPT and 13 hyperplastic glands from uraemic patients with secondary HPT.Parathyroid adenomas and secondary hyperplastic glands were obtained from 31 and 13 randomly selected patients undergoing parathyroidectomy in the clinical routine, respectively. Four normal parathyroid gland biopsies were obtained at surgery for pHPT or from normocalcemic patients undergoing thyroidectomy for goitre.The relative p27 expression (p27/GAPDH) was significantly reduced in parathyroid adenomas compared to normal parathyroid gland biopsies. Furthermore, 42% and 53% of the parathyroid adenomas displayed undetectable p18 and p21 expression levels, respectively. All 13 adenomas that lacked p18 expression showed undetectable p21 expression. The p18 expression was significantly lower in tumours of uraemic sHPT as compared to normal parathyroids and an undetectable expression level was observed for p21 and p27 in 61% and 53%, respectively.Parathyroid adenomas and secondary hyperplastic glands exhibit aberrant reduced expression of the CKIs p18, p21, and p27. This suggests that deranged collaboration of different CKIs may contribute to the development of both primary and secondary HPT.

Several vitamin D analogues, with reduced hypercalcemic and hyperphosphatemic toxicity at therapeutic dosages, are in clinical use for prevention and treatment of secondary hyperparathyroidism (HPT) in chronic renal failure. We have performed a first in vitro evaluation of five vitamin D analogues displaying less calcemic activity in normal rats, considerably more antiproliferative ability and higher transcription activation potential than 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), with the future prospects in mind to identify even more effective and less calcemic vitamin D analogues for treatment of HPT. The vitamin D analogues EB1089 and GS1590 have polyunsaturated side-chains, whereas HEP187, MC1598 and CB1393 display altered stereochemistry at carbon 20. In primary cultures of bovine parathyroid cells EB1089, GS1590, CB1393 and MC1598 as well as the comparative controls 1,25(OH)(2)D(3), 22-oxacalcitriol (OCT, maxacalcitol), 19-nor-1,25(OH)(2)D(2) (paricalcitol) and 1alpha(OH)D(2) (doxercalciferol) significantly suppressed PTH secretion or reduced PTH mRNA level at 10(-8), 10(-10), and 10(-11)M for all compounds except for MC1598 at the lowest concentration. The analogue HEP187 displayed no PTH suppressive activity. We conclude that the vitamin D analogues EB1089, GS1590 and CB1393 may be suitable for treatment of hyperparathyroidism secondary to uremia and that further evaluation in vivo should be considered.

Summary objective Glial cells missing (Gcm) was first identified as a binary switch between neuronal and glial determination in Drosophila. Two homologues of DrosophilaGcm have been identified in mice and humans, namely Gcm1 and Gcm2. Mouse Gcm2 is restricted to parathyroid tissues and Gcm2 was recently identified as a master regulatory gene of parathyroid gland development as Gcm2 knockout mice lack parathyroid glands. design/patients To identify Gcm2 transcripts in human non-neural tissues and to examine whether Gcm2 is involved in parathyroid gland tumorigenesis we analysed Gcm2 transcript levels in several non-neural tissues by reverse transcriptase polymerase chain reaction (RT-PCR) and performed real-time quantitative RT-PCR analysis on five normal glands, 15 parathyroid adenomas of primary hyperparathyroidism (HPT) and nine hyperplastic glands of secondary HPT. results We found high Gcm2 mRNA expression in human parathyroid glands in comparison with other non-neural tissues and underexpression in parathyroid adenomas but not in lesions of HPT secondary to uraemia. conclusion Because adenomas demonstrate lower Gcm2 expression than normal glands we suggest that reduced expression of Gcm2 contributes to parathyroid gland tumorigenesis. We speculate that a proper expression level of the Gcm2 transcription factor could be important for maintaining a fully differentiated state of the parathyroid cell.

Active vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], plays a pivotal role in calcium homeostasis and bone metabolism. Circulating levels of 1,25(OH)(2)D(3) are thought to be dependent mainly on the activity of the renal cytochrome P450 enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase), which is potently induced by PTH. However, 1alpha-hydroxylase activity or expression has also been reported at several extrarenal sites, at which local synthesis of 1,25(OH)(2)D(3) appears to fulfill autocrine or paracrine functions. This includes tissues such as placenta and brain that also express LRP-2/megalin, an endocytic receptor for multiple ligands, which is involved in the renal uptake of the substrate for 1alpha -hydroxylase, 25-hydroxyvitamin D(3). We have previously demonstrated LRP-2/megalin in parathyroid cells, and here we present results from RT-PCR and immunohistochemical analyses showing coincident expression of 1alpha-hydroxylase in normal and pathological parathyroid tissue. With real-time quantitative RT-PCR analysis, the expression of 1alpha-hydroxylase mRNA was higher in the majority of parathyroid adenomas and secondary hyperplastic glands but lower in parathyroid carcinomas, compared with normal parathyroid tissue. The findings imply that in addition to feedback control by circulating 1,25(OH)(2)D(3) levels, parathyroid cells may also be influenced by local 1alpha -hydroxylase activity with possible growth regulatory and differentiating effects.

Summary objective Parathyroid adenomas frequently harbour deletions of genomic DNA at chromosome regions 1p, 6q and 11q. In this study we related clinical characteristics in 56 patients with primary hyperparathyroidism (pHPT) to loss of heterozygosity (LOH) in these chromosome regions. design LOH analysis was performed on 56 sporadic parathyroid tumours using a total of 18 microsatellite markers for chromosome regions 1p, 6q and 11q. LOH was identified, for either radioactive or fluorescent labelled markers, as total absence or reduction of ≥ 50% of the signal intensity of an allele in the tumour DNA vs. constitutional DNA. patients Twenty-one of the patients were recruited by a population-based screening for pHPT and the remaining pHPT patients were gathered from routine clinical practice. results In total, 27%, 23% and 23% of the tumours showed LOH at 1p, 6q and 11q, respectively. LOH at both 1p and 11q was more common in the screening-detected pHPT patients compared to those recruited from clinical practice (38%vs. 20%; P= 0·02 and 43%vs. 11%; P= 0·001, respectively), while allelic loss at 6q was more prevalent in the latter group (11%vs. 31%; P= 0·001). No apparent relationships between LOH at 1p, 6q, and 11q and clinical characteristics, such as glandular weight, serum levels of PTH or calcium, were demonstrated. Moreover, additional LOH analysis of chromosome 1p suggested a putative parathyroid tumour suppressor gene(s) in the region between markers DS214 and D1S503, spanning approximately 6 cM. conclusion A high frequency of LOH at 1p and 11q in tumours of screening-detected pHPT patients is intriguing, and may suggest that inactivation of known (the MEN1 gene) and putative tumour suppressor genes at these chromosomal regions is associated with a more benign disease.

Multiple endocrine neoplasia type 1 (MEN1) is an inherited syndrome with multiple tumors of the endocrine pancreas, the parathyroid, the pituitary, and other tissues. The MEN1 gene at 11q13 is homozygously mutated in the majority of MEN1 tumors. Here we present a genome-wide loss of heterozygosity (LOH) screening of 23 pancreatic lesions, one duodenal tumor, and one thymic carcinoid from 13 MEN1 patients. Multiple allelic deletions were found. Fractional allelic loss varied from 6-75%, mean 31%. All pancreatic tumors displayed LOH on chromosome 11, whereas the frequency of losses for chromosomes 3, 6, 8, 10, 18, and 21 was over 30%. Different lesions from individual patients had discrepant patterns of LOH. Intratumoral heterogeneity was revealed, with chromosome 6 and 11 deletions in most tumor cells, whereas other chromosomal loci were deleted in portions of the analyzed tumor. Chromosome 6 deletions were mainly found in lesions from patients with malignant features. Fractional allelic loss did not correlate to malignancy or to tumor size. Our findings indicate that MEN1 pancreatic tumors fail to maintain DNA integrity and demonstrate signs of chromosomal instability.

Familial hyperparathyroidism (HPT), characterized by hypercalcemia and hypercalciuria, and familial benign hypocalciuric hypercalcemia (FHH) are the most common causes of hereditary hypercalcemia. The calcium-sensing receptor (CaR) regulates PTH secretion and renal calcium excretion. Heterozygous inactivating mutations of the gene cause FHH, whereas CaR gene mutations have not been demonstrated in HPT. In a kindred with 20 affected individuals, the hypercalcemic disorder segregated with inappropriately higher serum PTH and magnesium levels and urinary calcium levels than in unaffected members. Subtotal parathyroidectomy revealed parathyroid gland hyperplasia/adenoma and corrected the biochemical signs of the disorder in seven of nine individuals. Linkage analysis mapped the condition to markers flanking the CaR gene on chromosome 3q. Sequence analysis revealed a mutation changing phenylalanine to leucine at codon 881 of the CaR gene, representing the first identified point mutation located within the cytoplasmic tail of the CaR. A construct of the mutant receptor (F881L) was expressed in human embryonic kidney cells (HEK 293), and demonstrated a right-shifted dose-response relationship between the extracellular and intracellular calcium concentrations. The hypercalcemic disorder of the present family is caused by an inactivating point mutation in the cytoplasmic tail of the CaR and displays clinical characteristics atypical of FHH and primary HPT.

Vitamin D, via its receptor (VDR), inhibits the hormone secretion and proliferation of parathyroid cells. Vitamin D deficiency and reduced parathyroid VDR expression has been associated with development of hyperparathyroidism (HPT) secondary to uremia. VDR polymorphisms may influence VDR messenger RNA (mRNA) levels and have been coupled to an increased risk of parathyroid adenoma of primary HPT. VDR mRNA relative to glyceraldehyde-3-phosphate dehydrogenase mRNA levels were determined by RNase protection assay in 42 single parathyroid adenomas of patients with primary HPT, 23 hyperplastic glands of eight patients with uremic HPT, and 15 normal human parathyroid glands. The adenomas and hyperplasias demonstrated similar VDR mRNA levels, which were reduced (42 ± 2.8% and 44 ± 4.0%) compared with the normal glands (P < 0.0001). Comparison of parathyroid adenoma with a normal-sized parathyroid gland of the same individual (n = 3 pairs) showed a 20–58% reduction in the tumor. Nodularly enlarged glands represent a more advanced form of secondary HPT and showed greater reduction in the VDR mRNA levels than the diffusely enlarged glands (P < 0.005). The reduced VDR expression is likely to impair the 1,25(OH)2D3-mediated control of parathyroid functions, and to be of importance for the pathogenesis of not only uremic but also primary HPT. Circulating factors like calcium, PTH, and 1,25(OH)2D3 seem to be less likely candidates mediating the decreased VDR gene expression in HPT.

Routes to nano-phase nickel in the forms of highly porous sponges and dense films have been developed. Nickel acetate and nitrate were mixed with triethanolamine and methanol, and evaporated to a concentrated liquid. Heat-treatment at 10°C/min to 170°C under N2 atmosphere yielded a highly porous sponge consisting of

Recently the multiple endocrine neoplasia type 1 (MEN-1) tumor suppressor gene was cloned. MEN-1 encodes a nuclear protein, called menin, of hitherto unknown function. In order to investigate the biological function of menin we employed the yeast two-hybrid system to identify menin-interacting proteins. Here we report that menin functions as a transcriptional repressor through interaction with the transcription factor JunD. The interaction is mediated via the N-terminal transcription activation domain of JunD, and the C-terminal part of menin. In transient co-transfection experiments, expression of menin leads to specific repression of JunD transcriptional activity, which is dependent on the integrity of the menin C-terminal region. C-Terminal truncations of the protein not only abolish repression, but increase JunD transcriptional activity, implying the existence of a functional domain separate from the JunD-binding region. Menin-mediated repression is relieved by the histone deacetylase inhibitor trichostatin A, indicating that deacetylation of histones is an essential component of this repression mechanism, as has recently been demonstrated for the retinoblastoma protein. Missense, in-frame deletions, frameshift and nonsense mutations lead to inactivation of menin or possibly to truncated proteins. This would result in loss of repression of menin/JunD target genes, as well as non-target genes through indirect mechanisms, deregulation of cellular growth control and endocrine tumorigenesis.

Vitamin D regulates parathyroid cell proliferation and secretion of PTH. Increased prevalence of the polymorphic vitamin D receptor (VDR) alleles b, a, and T has been reported in sporadic primary hyperparathyroidism (PHPT), suggesting that these genetic variants may predispose to the disease. Recently, another polymorphism in the VDR gene was related to bone mineral density, and this VDR-FokI polymorphism causes different lengths of the VDR, implying possible functional consequences. The VDR-FokI polymorphism was studied in 182 postmenopausal women with sporadic PHPT and in matched controls. No significant differences in distribution of the VDR-FokI genotypes could be detected between the groups, although there was a tendency toward overrepresentation of the F allele in the PHPT patients (P = 0.05). There were no significant associations with age, serum calcium, serum PTH, bone mineral density, or parathyroid tumor weight. The VDR genotypes were unrelated to VDR and PTH messenger ribonucleic acid levels in the parathyroid adenomas of 42 PHPT patients. In 23 PHPT patients, the Ca2+-PTH set-points were determined in vivo and were unrelated to the VDR alleles. We suggest that the VDR-FokI polymorphism has at most a minor pathogenic importance in the development of PHPT.

Pancreatic endocrine tumors (PETs) arise sporadically or are associated with multiple endocrine neoplasia type 1 (MENi) syndrome or von Hippel-Lindau syndrome. About 90% of patients with familial MENi display detectable MEN1 gene (menin) mutations. The cyclin-dependent kinase inhibitor p27 (CDKN1 B) is a downstream target of menin and has been recently shown to be responsible for the multiple endocrine neoplasia-like syndrome in rats, where affected animals develop multiple tumors and hyperplasia in endocrine tissues, including the pancreatic islets of Langerhans. A germline nonsense truncation mutation of p27 has been recently described in a suspected MENi family without MENi mutation, raising the possibility that p27 mutation could be responsible for MENi phenotype. Somatic MENi mutations occur at low frequency in sporadic PETs; here, we subjected p27 to mutational analysis in 27 sporadic PETs. As an additional menin target, analysis of the p18(CDKN2C) gene was included. In the p27 gene, one common polymorphism (V1 09G) and one novel polymorphism (g/a) in the noncoding part of exon 2 were identified. Three known polymorphisms were found in the p18 gene. These data suggest that p27 and p18 are unlikely to present classic tumor-suppressor genes in sporadic PETs.

Biochemical signs and severity of symptoms of primary hyperparathyroidism (pHPT) differ among patients, and little is known of any coupling of clinical characteristics of nonfamilial pHPT to genetic abnormalities in the parathyroid tumors. Mutations in the recently identified MEN1 gene at chromosome 11q13 have been found in parathyroid tumors of nonfamilial pHPT. Using microsatellite analysis for loss of heterozygosity (LOH) at 11q13 and DNA sequencing of coding exons, the MEN1 gene was studied in 49 parathyroid lesions of patients with divergent symptoms, operative findings, histopathological diagnosis, and biochemical signs of nonfamilial pHPT. Allelic loss at 11q13 was detected in 13 tumors, and 6 of them demonstrated previously unrecognized somatic missense and frameshift deletion mutations of the MEN1 gene. Many of the detected mutations would most likely result in a nonfunctional menin protein, consistent with a tumor suppressor mechanism. Clinical and biochemical characteristics of HPT were apparently unrelated to the presence or absence of LOH and the MEN1 gene mutations. However, the demonstration of LOH at 11q13 and MEN1 gene mutations in small parathyroid adenomas of patients with slight hypercalcemia and normal serum PTH levels suggest that altered MEN1 gene function may also be important for the development of mild sporadic pHPT.

Vitamin D receptor (VDR) and parathyroid hormone mRNA levels correspond to polymorphic VDR alleles in human parathyroid tumors

Primary hyperparathyroidism (pHPT) resulting from parathyroid tumors is a common endocrine disorder with incompletely understood etiology. In renal failure, secondary hyperparathyroidism (sHPT) occurs with multiple tumor development as a result of calcium and vitamin D regulatory disturbance. The aim of this study was to investigate a potential role of the histone 3 lysine 27 methyltransferase EZH2 in parathyroid tumorigenesis. Parathyroid tumors from patients with pHPT included adenomas and carcinomas. Hyperplastic parathyroid glands from patients with HPT secondary to uremia and normal parathyroid tissue specimens were included in this study. Quantitative RT-PCR, western blotting, bisulfite pyrosequencing, colony formation assay, and RNA interference were used.EZH2was overexpressed in a subset of the benign and in all malignant parathyroid tumors as determined by quantitative RT-PCR and western blotting analyses. Overexpression was explained byEZH2gene amplification in a large fraction of tumors.EZH2depletion by RNA interference inhibited sHPT-1 parathyroid cell line proliferation as determined by tritium–thymidine incorporation and colony formation assays.EZH2depletion also interfered with the Wnt/β-catenin signaling pathway by increased expression of growth-suppressiveAXIN2, a negative regulator of β-catenin stability. Indeed,EZH2contributed to the total level of aberrantly accumulated transcriptionally active (nonphosphoylated) β-catenin in the parathyroid tumor cells. To our knowledgeEZH2gene amplification presents the first genetic aberration common to parathyroid adenomas, secondary hyperplastic parathyroid glands, and parathyroid carcinomas. This supports the possibility of a common pathway in parathyroid tumor development.

Endocrine tumors arise from endocrine glands. Most endocrine tumors are benign but malignant variants exist. Several endocrine neoplasms display loss of parts of chromosome 11 or 18, produce hormones and responds poorly to conventional chemotherapeutics. The multiple endocrine neoplasia syndromes are mainly confined to endocrine tumors. This opens the question if there exists a single or several endocrine tumor genes.The aim of the study was to describe genetic derangements in endocrine tumors.Paper I: Investigation of mutational status of SDHAF2 in parathyroid tumors. SDHAF2 is located in the proximity of 11q13, a region that frequently displays loss in parathyroid tumors. We established that mutations in SDHAF2 are infrequent in parathyroid tumors.Paper II: Study of SDHAF2 gene expression in a cohort of benign pheochromocytomas (PCC) (n=40) and malignant PCC (n=10). We discovered a subset of benign PCC (28/40) and all malignant PCC (10/10) with significantly lower SDHAF2 expression. Benign PCC with low SDHAF2 expression and malignant tumors consistently expressing low levels of SDHAF2 were methylated in the promoter region. SDHAF2 expression was restored in vitro after treatment with 5- aza-2-deoxycytidine.Paper III: HumanMethylation27 array (Illumina) covering 27578 CpG sites spanning over 14495 genes were analyzed in a discovery cohort of 10 primary small neuroendocrine tumors (SI-NETs) with matched metastases. 2697 genes showed different methylation pattern between the primary tumor and its metastasis. We identified several hypermethylated genes in key regions. Unsupervised clustering of the tumors identified three distinct clusters, one with a highly malignant behavior.Paper IV: Loss of chromosome 18 is the most frequent genetic aberration in SI-NETs. DNA from SI-NETs were subjected to whole exome capture sequencing and high resolution SNP array. Genomic profiling revealed loss of chromosome 18 in 5 out of 7 SI-NETs. No tumor-specific somatic mutation on chromosome 18 was identified which suggests involvement of other mechanisms than point mutations in SI-NET tumorigenesis.Paper V: The cost for diagnostic genetic screening of common susceptibility genes in PCC is expensive and labor intensive. Three PCC from three patients with no known family history were chosen for exome capture sequencing. We identified three variants in known candidate genes. We suggest that exome-capture sequencing is a quick and cost-effective tool.

Adrenal aldosterone-producing adenomas (APAs) constitutively produce the salt-retaining hormone aldosterone and are a common cause of severe hypertension. Recurrent mutations in the potassium channel KCNJ5 that result in cell depolarization and Ca2+ influx cause ~40% of these tumors1. We found five somatic mutations (four altering glycine 403, one altering isoleucine 770) in CACNA1D, encoding a voltage-gated calcium channel, among 43 non-KCNJ5-mutant APAs. These mutations lie in S6 segments that line the channel pore. Both result in channel activation at less depolarized potentials, and glycine 403 mutations also impair channel inactivation. These effects are inferred to cause increased Ca2+ influx, the sufficient stimulus for aldosterone production and cell proliferation in adrenal glomerulosa2. Remarkably, we identified de novo mutations at the identical positions in two children with a previously undescribed syndrome featuring primary aldosteronism and neuromuscular abnormalities. These findings implicate gain of function Ca2+ channel mutations in aldosterone-producing adenomas and primary aldosteronism.