RESUMO
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.
Assuntos
Epigênese Genética , Neoplasias Intestinais/genética , Intestino Delgado , Tumores Neuroendócrinos/genética , Variações do Número de Cópias de DNA , Perfilação da Expressão Gênica , Humanos , Mutação , RNA Mensageiro/genéticaRESUMO
OBJECTIVE/BACKGROUND: Aorto-bifemoral bypass remains the gold standard for treatment of aortoiliac occlusive disease (AIOD) in patients with advanced (TASC D) lesions, but has significant associated morbidity and mortality. Treatment with a unibody stent-graft positioned at the aortic bifurcation is a potential endovascular option for the treatment of AIOD. The current study examines the safety, efficacy, and early patency rates of the Endologix AFX unibody stent-graft for treatment of AIOD. METHODS: A multicenter retrospective review was conducted of patients treated exclusively for AIOD with the AFX device. Primary, assisted primary, and secondary patency rates were noted. Clinical improvement was assessed using Rutherford classification and ankle brachial index. Mean duration of follow-up was 22.2 ± 11.2 months. Ninety-one patients (56 males [62%]) were studied. RESULTS: Sixty-seven patients (74%) presented with lifestyle-limiting intermittent claudication and the remaining 24 (26%) had critical limb ischemia. Technical success was 100%. Complications included groin infection (n = 4 [4%]), groin hematoma (n = 4 [4%]), common iliac rupture (n = 4 [4%]), iliac dissection (n = 4 [4%]), and thromboembolic event (n = 3 [3%]; one femoral, one internal iliac artery, and one internal iliac with bilateral popliteal/tibial thromboemboli). Thirty-day mortality was 1% (1/91) resulting from a case of extensive pelvic thromboembolism. At 1 year, 73% of patients experienced improvement in Rutherford stage of -3 or greater compared with baseline. Nine patients (10%) required 16 secondary interventions. At all time points, primary patency rates were > 90%, assisted patency rates were > 98%, and secondary patency rates were 100%. CONCLUSION: This is the largest study to examine the use of the Endologix AFX unibody stent-graft for the treatment of AIOD. Use of the AFX stent-graft appears to be a safe and effective endovascular treatment for complex AIOD.
Assuntos
Doenças da Aorta/cirurgia , Prótese Vascular , Artéria Ilíaca/cirurgia , Stents , Enxerto Vascular/métodos , Idoso , Índice Tornozelo-Braço , Arteriopatias Oclusivas/cirurgia , Implante de Prótese Vascular/efeitos adversos , Implante de Prótese Vascular/métodos , Extremidades/irrigação sanguínea , Feminino , Humanos , Claudicação Intermitente/etiologia , Isquemia/etiologia , Masculino , Estudos Retrospectivos , Resultado do Tratamento , Enxerto Vascular/efeitos adversos , Enxerto Vascular/instrumentação , Grau de Desobstrução VascularRESUMO
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 <0.5-1% of pHPT cases. Secondary hyperparathyroidism (sHPT) is a complication of renal failure, with the development of parathyroid tumours and hypercalcaemia. Recurrent mutations in the MEN1 gene have been confirmed by the whole-exome sequencing in 35% of PAs, suggesting that non-protein-coding genes, regulatory elements or epigenetic derangements may also have roles in the majority of PAs. DNA translocations with cyclin D1 overexpression occur in PAs (8%). In PCs, mutations in CDC73/HRPT2 are common. Activation of the WNT/ß-catenin signalling pathway (accumulation of nonphosphorylated ß-catenin) by an aberrantly truncated LRP5 receptor has been seen for the majority of investigated PAs and sHPT tumours, and possibly by APC inactivation through promoter methylation in PCs. Promoter methylation of several other genes and repressive histone H3 lysine 27 trimethylation by EZH2 of the HIC1 gene may also contribute to parathyroid tumorigenesis. It is possible that a common pathway exists for parathyroid tumour development. CCND1 (cyclin D1) and EZH2 overexpression, accumulation of nonphosphorylated ß-catenin and repression of HIC1 have all been observed to occur in PAs, PCs and sHPT tumours. In addition, hypermethylation has been observed for the same genes in PAs and PCs (e.g. SFRP1, CDKN2A and WT1). Whether ß-catenin represents a 'hub' in parathyroid tumour development will be discussed.
Assuntos
Neoplasias das Paratireoides/genética , Epigênese Genética , Humanos , Hiperparatireoidismo Primário/etiologia , Hiperparatireoidismo Primário/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , MicroRNAs/genética , Mutação , Neoplasias das Paratireoides/complicações , Neoplasias das Paratireoides/metabolismo , Via de Sinalização WntRESUMO
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.
Assuntos
Exoma , Neoplasias Intestinais/genética , Intestino Delgado/patologia , Tumores Neuroendócrinos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Intestinais/patologia , Tumores Neuroendócrinos/patologia , Análise de Sequência de DNARESUMO
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.
Assuntos
Neoplasias das Glândulas Suprarrenais/diagnóstico , Biomarcadores Tumorais/metabolismo , Feocromocitoma/diagnóstico , Estatmina/metabolismo , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/patologia , Medula Suprarrenal/metabolismo , Medula Suprarrenal/patologia , Adulto , Idoso , Biomarcadores Tumorais/genética , Western Blotting , Diagnóstico Diferencial , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Feocromocitoma/metabolismo , Feocromocitoma/patologia , Feocromocitoma/secundário , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatmina/genética , Adulto JovemRESUMO
Misregulation of the Wnt/beta-catenin signalling pathway is involved in the development and progression of many cancers. Recently, we presented evidence for aberrant accumulation of non-phosphorylated (stabilized) beta-catenin in benign parathyroid tumors from patients with primary hyperparathyroidism (pHPT) or HPT secondary to uremia (sHPT). Here we have used a human parathyroid hormone (PTH)-producing cell line (sHPT-1), established from a hyperplastic parathyroid gland removed at operation of a patient with sHPT, to further investigate the potential importance of beta-catenin in parathyroid tumorigenesis. Our studies demonstrate that efficient and specific knockdown of beta-catenin by small interfering RNA (siRNA) markedly decreased endogenous beta-catenin transcriptional activity as well as expression of the Wnt/beta-catenin target genes cyclin D1 and c-myc, known to be overexpressed in a substantial fraction of parathyroid tumors. Furthermore, siRNA to beta-catenin inhibited cellular growth and induced cell death. Growth and survival of the parathyroid tumor cells are thus dependent on maintained expression level of beta-catenin. The Wnt/beta-catenin signalling pathway, and beta-catenin in particular, presents a potential therapeutic target for HPT.
Assuntos
Neoplasias das Paratireoides/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Transdução de SinaisRESUMO
Vitamin D receptor (VDR) and 25-hydroxyvitamin D3 1-alpha-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-alpha-hydroxylase in thyroid follicular cells, with special emphasis on papillary thyroid carcinoma (PTC). VDR and 1-alpha-hydroxylase expression were increased in PTC compared with normal thyroid tissue and especially high in areas of lymphocyte infiltration. Expression of VDR and 1-alpha-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.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/biossíntese , Biomarcadores Tumorais/biossíntese , Carcinoma Papilar/metabolismo , Receptores de Calcitriol/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Adulto , Carcinoma Papilar/enzimologia , Carcinoma Papilar/patologia , Feminino , Humanos , Imuno-Histoquímica , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândula Tireoide/enzimologia , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/enzimologia , Neoplasias da Glândula Tireoide/patologiaRESUMO
The reaction of Eu metal with HOPr(i)/toluene solutions yielded the mixed Eu(2+)/Eu(3+) alkoxide [Eu(4)(OPr(i))(10)(HOPr(i))(3)] x 2HOPr(i) (1), in contrast to the other lanthanide metals, which exclusively yield trivalent lanthanide ions in the alkoxides formed. Metathesis between EuCl(3) and 3KOPr(i) and stoichiometric hydrolysis yielded the square-pyramidal Eu(5)O(OPr(i))(13) (2), and metathesis with EuCl(3) and 3KAl(OPr(i))(4) gave EuAl(3)(OPr(i))(12) (3). The structures of these compounds were determined by single-crystal X-ray diffraction. IR spectroscopic studies showed that the solid-state molecular structure of the three alkoxides remained close to intact in solution. Further characterizations were made with UV-vis spectroscopy, differential scanning calorimetry, and solubility studies. It was also found that 1 can be converted to 2 by oxidation with dioxygen, but 2 was not reduced by Eu metal to 1. The reactions of 2 and 1 with Al(4)(OPr(i))(12) in toluene/HOPr(i) solvent were studied by IR and UV-vis spectroscopy; 2 reacted completely to form 3 in 2 h at 75 degrees C, while 1 reacted to yield 3 and other unidentified Eu(2+) containing product(s) in the same time.
RESUMO
Classical midgut carcinoids are rare intestinal neuroendocrine tumors that often present with metastases at diagnosis. In contrast to foregut carcinoids, midgut carcinoids are not related to the multiple endocrine neoplasia type 1 syndrome, and the mechanisms involved in their tumorigenesis are unknown. Eight classical midgut carcinoids were analyzed by genome-wide screening for loss of heterozygosity. Deletions on chromosome 18 were found in 88% of the tumors. DNA sequencing and immunohistochemical staining for Smad4/DPC4, which often is homozygously mutated in pancreatic and colon carcinomas, revealed no aberrations. In 1 tumor, a region telomeric to the Smad4/DPC4/DCC genes at 18q21 was deleted. Other chromosomes were affected in 3 lesions only. The high frequency of chromosome 18 deletions strongly indicates a genetic alteration of importance in classical midgut carcinoid tumorigenesis, apparently not involving the Smad4/DPC4 gene.
Assuntos
Tumor Carcinoide/genética , Deleção Cromossômica , Cromossomos Humanos Par 18 , Neoplasias Intestinais/genética , Adulto , Idoso , Alelos , Proteínas de Ligação a DNA/genética , Deleção de Genes , Homozigoto , Humanos , Imuno-Histoquímica , Perda de Heterozigosidade , Repetições de Microssatélites , Pessoa de Meia-Idade , Mutação , Neoplasias Pancreáticas/genética , Proteína Smad4 , Transativadores/genéticaRESUMO
The new tetranuclear alkoxide hexa-mu(2)-isopropoxy-1:2kappa(4)O;1:3kappa(4)O;1:4kappa(4)O-hexaisopropoxy-2kappa(2)O,3kappa(2)O,4kappa(2)O-trialuminium(III) neodymium(III), [Nd[Al(C(3)H(7)O)(4)](3)], has a metal-oxygen NdAl(3)O(12) core which consists of four metal atoms arranged in an approximately planar triangular geometry. The central Nd atom is six-coordinated by O atoms and the Al atoms are four-coordinated by O atoms.
RESUMO
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.
Assuntos
Alelos , Deleção de Genes , Perda de Heterozigosidade , Neoplasia Endócrina Múltipla Tipo 1/genética , Neoplasias Pancreáticas/genética , Adulto , Idoso , Tumor Carcinoide/genética , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 6 , Neoplasias Duodenais/genética , Marcadores Genéticos , Humanos , Pessoa de Meia-Idade , Neoplasias Pancreáticas/patologia , Neoplasias do Timo/genéticaRESUMO
OBJECTIVES: The cell cycle regulator cyclin D1 plays an important role in parathyroid tumourigenesis. The NciI polymorphism in exon 4 has recently been associated with early onset of hereditary nonpolyposis colorectal cancer and is a prognostic indicator of nonsmall cell lung cancer and squamous cell carcinomas. Furthermore, a limited study of 28 primary hyperparathyroidism (pHPT) patients displayed a tendency of NciI influence on HPT development. We hypothesized that the NciI polymorphism may relate to a risk of developing pHPT. DESIGN, SETTING AND SUBJECTS: We genotyped 182 patients with sporadic pHPT and matched controls for the cyclin D1 polymorphism. A total of 88 pHPT patients and controls were recruited via a health-screening. RESULTS: The frequency distribution of the NciI genotypes NN, Ni, and ii were in pHPT patients and controls 22, 44 and 34%, and 26, 49 and 25%, respectively. The calculated allele frequencies were A = 0.56; G = 0.44 in cases and A = 0.49; G = 0.51 in controls. The frequency distributions did not differ comparing cases and controls when subgrouped after age and menopausal status. The NciI genotypes were not significantly associated with age of the individuals, serum (s)-calcium, s-parathyroid hormone (PTH), bone mineral density (BMD) or parathyroid tumour weight in any of the groups of pHPT patients or controls. CONCLUSIONS: No significant differences in distribution of the genotypes could be detected between the groups, suggesting that the polymorphism has minor or no pathogenic importance in the development of pHPT. Our results suggest that determination of the NciI polymorphism in the cyclin D1 gene is not a clinically useful tool for prediction of pHPT.
Assuntos
Ciclina D1/genética , Hiperparatireoidismo/genética , Polimorfismo Genético , Idoso , Alelos , Análise de Variância , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
Methylation of CpG residues in mammalian genomes is a mechanism of vital importance for many cellular functions, which all relate to gene expression. In this study we describe the identification of a CpG island in the 5'-region of the gene encoding human megalin/LRP-2, a receptor capable of binding multiple ligands, which is involved in the regulation of calcium metabolism. Southern blot analysis and genomic bisulfite sequencing revealed that the CpG island is methylated in a non-expressing cell line, largely unmethylated in an expressing cell line and unmethylated in human parathyroid tissue. In addition, we show that artificial methylation of LRP-2 promoter reporter plasmids leads to strong transcriptional repression, in vitro as well as in transfected cells. No evidence for aberrant LRP-2 gene methylation in parathyroid adenomas, in which the LRP-2 protein is generally down-regulated, was found.
Assuntos
Adenoma/genética , Ilhas de CpG , Metilação de DNA , Glicoproteínas de Membrana/genética , Neoplasias das Paratireoides/genética , Sequência de Bases , Primers do DNA , Complexo Antigênico da Nefrite de Heymann , Humanos , Regiões Promotoras Genéticas , Transcrição Gênica/genéticaRESUMO
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.
Assuntos
Adenoma/genética , Hiperparatireoidismo Secundário/genética , Hiperparatireoidismo/genética , Glândulas Paratireoides/metabolismo , Neoplasias das Paratireoides/genética , Receptores de Calcitriol/genética , Adenoma/patologia , Adenoma/cirurgia , Idoso , Cálcio/sangue , Humanos , Hiperparatireoidismo/metabolismo , Hiperparatireoidismo Secundário/metabolismo , Pessoa de Meia-Idade , Hormônio Paratireóideo/sangue , Neoplasias das Paratireoides/patologia , Neoplasias das Paratireoides/cirurgia , RNA Mensageiro/genéticaRESUMO
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.
Assuntos
Distúrbios do Metabolismo do Cálcio/genética , Proteínas de Ligação ao Cálcio/genética , Cromossomos Humanos Par 3 , Hipercalcemia/genética , Mutação Puntual , Adulto , Idoso , Substituição de Aminoácidos , Cálcio/urina , Linhagem Celular , Mapeamento Cromossômico , DNA/sangue , Éxons , Feminino , Marcadores Genéticos , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Mutagênese Sítio-Dirigida , Linhagem , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Megalin/low density lipoprotein receptor-related protein 2 (LRP-2) is an endocytic receptor expressed in highly specialized cell types such as parathyroid cells and epithelia of the kidney. Previous experiments identified a nonconsensus TATA element, with the sequence TAGAAAA, as crucial for accurate and efficient transcription from the LRP-2 promoter. Here we show that, in addition to the TAGA element, promoter sequences downstream of the transcription start site contribute significantly to transcription both in vitro and in transfected cells. Deletion and point mutational analyses reveal that the promoter region located between positions +5 and +11 (sequence TTTTGGC) is of particular importance. Complementation experiments in nuclear extracts lacking transcription factor IID (TFIID) activity show that TATA-binding protein-associated factors of TFIID are essential for the function of LRP-2 downstream promoter sequences. Interestingly, DNase I footprinting studies show that the downstream region between positions +5 and +11 does not significantly affect overall TFIID affinity to the promoter but that it profoundly affects the topology of the TFIID x promoter complex not only downstream of the transcription start site, but in particular in the TATA box region. Our observations suggest a model for a novel downstream sequence function, in which TATA-binding protein-associated factor-promoter interactions downstream of the transcription start site modulate TFIID-DNA interactions in the TATA box region.
Assuntos
Glicoproteínas de Membrana/genética , Regiões Promotoras Genéticas , Fatores de Transcrição TFII/metabolismo , Transcrição Gênica , Sequência de Bases , DNA , Pegada de DNA , Complexo Antigênico da Nefrite de Heymann , Humanos , Conformação Molecular , Fator de Transcrição TFIID , Fatores de Transcrição TFII/químicaRESUMO
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.
Assuntos
Histona Desacetilases/metabolismo , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-jun/farmacologia , Proteínas Proto-Oncogênicas , Ativação Transcricional/efeitos dos fármacos , Regulação para Baixo , Biblioteca Gênica , Inibidores de Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/farmacologia , Luciferases , Neoplasia Endócrina Múltipla Tipo 1/genética , Mutação , Proteínas de Neoplasias/farmacologia , Ligação Proteica , TransfecçãoRESUMO
Pancreatic endocrine tumors occur sporadically and as part of the multiple endocrine neoplasia type 1 (MEN 1) and von Hippel-Lindau (VHL) syndromes. The MEN1 locus on 11q13 and a candidate tumor suppressor locus on 3p are known to be hemi- or homozygously mutated in a subset of these tumors. Chromosome arm 18q harbors the SMAD4/DPC4 tumor suppressor gene that is frequently deleted and inactivated in tumors of the exocrine pancreas. We have analyzed 22 nonfamilial and 16 MEN 1-associated pancreatic endocrine tumors for loss of heterozygosity (LOH) at 3p, 11q13, and 18q. LOH at 3p was revealed in 45% and 36% of tumors from 31 patients with nonfamilial and MEN 1-associated disease, respectively. The corresponding proportions for 11q13 were 55% and 91%, and for 18q 27% and 25%, respectively. A striking relation between LOH at 11q13 and 3p and a malignant phenotype was found for the nonfamilial tumors. None of the six benign tumors (all of them insulinomas) had allelic loss at 3p or 11q13, whereas 92% (P < 0.01) of the malignant tumors (including malignant insulinomas) had such deletions. Besides the 11q13 abnormality, more than half of the MEN 1-associated tumors had additional genetic lesions affecting 3p or 18q. LOH analysis of several tumors from two MEN 1 patients suggested different clonal origin of the lesions. Sequencing of the SMAD4/DPC4 gene did not identify mutations in coding regions or at exon/intron boundaries in tumors with LOH at 18q. The data indicate involvement of tumor suppressor genes on 3p and 18q, in addition to the MEN1 gene at 11q13, in the tumorigenesis of both nonfamilial and MEN 1-associated pancreatic endocrine tumors.
Assuntos
Aberrações Cromossômicas/genética , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 18 , Cromossomos Humanos Par 3 , Neoplasia Endócrina Múltipla Tipo 1/genética , Neoplasias Pancreáticas/genética , Biomarcadores Tumorais/genética , Transtornos Cromossômicos , Marcadores Genéticos/genética , Humanos , Perda de Heterozigosidade/genéticaRESUMO
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.
Assuntos
Códon de Iniciação/genética , Hiperparatireoidismo/genética , Glândulas Paratireoides/metabolismo , Polimorfismo Genético , RNA Mensageiro/biossíntese , Receptores de Calcitriol/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Cálcio/sangue , Cálcio/metabolismo , Citratos , Desoxirribonucleases de Sítio Específico do Tipo II/biossíntese , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Feminino , Genótipo , Humanos , Hiperparatireoidismo/fisiopatologia , Masculino , Pessoa de Meia-Idade , Glândulas Paratireoides/fisiopatologia , Hormônio Paratireóideo/biossíntese , Hormônio Paratireóideo/genética , Receptores de Calcitriol/biossínteseRESUMO
Vitamin D and retinoids are important regulators of differentiation and proliferation in a number of tissues, and have been implicated as chemopreventive agents in several different tumors. While vitamin D is known to be important for regulation of parathyroid function and proliferation, it has recently been established that parathyroid cells also are targets for retinoids. Hyperparathyroidism (HPT) is a common disorder, and evaluation of the disease has indicated high prevalence of subclinical disease, as well as clear benefits of offering treatment for the disease. This review summarizes the data so far gathered concerning parathyroid cells, vitamin D and retinoids, with clear implication on prospects of possible medical treatment of hyperparathyroidism.