Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ia/genética , Mutación de Línea Germinal , Trastornos del Crecimiento/genética , Hipercalcemia/genética , Enfermedades Metabólicas/genética , Nefrocalcinosis/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Adolescente , Trastornos del Crecimiento/enzimología , Humanos , Hipercalcemia/enzimología , Masculino , Enfermedades Metabólicas/enzimología , Nefrocalcinosis/enzimología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimologíaRESUMEN
Loss of function mutations in the CYP24A1 gene, involved in vitamin D catabolism and in calcium homeostasis, are known to be the genetic drivers of both idiopathic infantile hypercalcemia (IIH) and adult renal stone disease. Recently, also defects in the SLC34A1 gene, encoding for the renal sodium-phosphate transporter NaPi-IIa, were associated with the disease. IIH typically affects infants and pediatric patients with a syndrome characterized by severe hypercalcemia, hypercalciuria, suppressed parathyroid hormone level and nephrolithiasis. In SLC34A1 mutated carriers, hypophosphatemia is also a typical biochemical tract. IIH may also persist undiagnosed into adulthood, causing an increased risk of nephrocalcinosis and renal complication. To note, a clinical heterogeneity characterizes IIH manifestation, principally due to the controversial gene-dose effect and, to the strong influence of environmental factors. The present review is aimed to provide an overview of the current molecular findings on the IIH disorder, giving a comprehensive description of the association between genotype and biochemical and clinical phenotype of the affected patients. We also underline that patients may benefit from genetic testing into a targeted diagnostic and therapeutic workflow.
Asunto(s)
Hipercalcemia/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Vitamina D3 24-Hidroxilasa/metabolismo , Genotipo , Humanos , Hipercalcemia/enzimología , Mutación , Fenotipo , Vitamina D3 24-Hidroxilasa/genéticaRESUMEN
G-protein subunit α-11 (Gα11 ) couples the calcium-sensing receptor (CaSR) to phospholipase C (PLC)-mediated intracellular calcium (Ca2+i ) and mitogen-activated protein kinase (MAPK) signaling, which in the parathyroid glands and kidneys regulates parathyroid hormone release and urinary calcium excretion, respectively. Heterozygous germline loss-of-function Gα11 mutations cause familial hypocalciuric hypercalcemia type 2 (FHH2), for which effective therapies are currently not available. Here, we report a novel heterozygous Gα11 germline mutation, Phe220Ser, which was associated with hypercalcemia in a family with FHH2. Homology modeling showed the wild-type (WT) Phe220 nonpolar residue to form part of a cluster of hydrophobic residues within a highly conserved cleft region of Gα11 , which binds to and activates PLC; and predicted that substitution of Phe220 with the mutant Ser220 polar hydrophilic residue would disrupt PLC-mediated signaling. In vitro studies involving transient transfection of WT and mutant Gα11 proteins into HEK293 cells, which express the CaSR, showed the mutant Ser220 Gα11 protein to impair CaSR-mediated Ca2+i and extracellular signal-regulated kinase 1/2 (ERK) MAPK signaling, consistent with diminished activation of PLC. Furthermore, engineered mutagenesis studies demonstrated that loss of hydrophobicity within the Gα11 cleft region also impaired signaling by PLC. The loss-of-function associated with the Ser220 Gα11 mutant was rectified by treatment of cells with cinacalcet, which is a CaSR-positive allosteric modulator. Furthermore, in vivo administration of cinacalcet to the proband harboring the Phe220Ser Gα11 mutation, normalized serum ionized calcium concentrations. Thus, our studies, which report a novel Gα11 germline mutation (Phe220Ser) in a family with FHH2, reveal the importance of the Gα11 hydrophobic cleft region for CaSR-mediated activation of PLC, and show that allosteric CaSR modulation can rectify the loss-of-function Phe220Ser mutation and ameliorate the hypercalcemia associated with FHH2. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.
Asunto(s)
Cinacalcet/uso terapéutico , Subunidades alfa de la Proteína de Unión al GTP/genética , Mutación de Línea Germinal/genética , Hipercalcemia/congénito , Hipercalcemia/tratamiento farmacológico , Hipercalcemia/genética , Mutación con Pérdida de Función/genética , Adulto , Secuencia de Aminoácidos , Cinacalcet/farmacología , Femenino , Subunidades alfa de la Proteína de Unión al GTP/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Hipercalcemia/enzimología , Hipercalcemia/patología , Lactante , Recién Nacido , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Linaje , Fosforilación/efectos de los fármacos , Transducción de Señal , Fosfolipasas de Tipo C/metabolismoRESUMEN
OBJECTIVE: Small cell carcinoma of the ovary, hypercalcemic-type (SCCOHT) is a rare, extremely aggressive neoplasm that usually occurs in young women and is characterized by deleterious germline or somatic SMARCA4 mutations. We performed comprehensive genomic profiling (CGP) to potentially identify additional clinically and pathophysiologically relevant genomic alterations in SCCOHT. METHODS: CGP assessment of all classes of coding alterations in up to 406 genes commonly altered in cancer and intronic regions for up to 31 genes commonly rearranged in cancer was performed on 18 SCCOHT cases (16 exhibiting classic morphology and 2 cases exhibiting exclusive a large cell variant morphology). In addition, a retrospective database search for clinically advanced ovarian tumors with genomic profiles similar to SCCOHT yielded 3 additional cases originally diagnosed as non-SCCOHT. RESULTS: CGP revealed inactivating SMARCA4 alterations and low tumor mutational burden (TMB) (<6mutations/Mb) in 94% (15/16) of SCCOHT with classic morphology. In contrast, both (2/2) cases exhibiting only large cell variant morphology were hypermutated (TMB scores of 90 and 360mut/Mb) and were wildtype for SMARCA4. In our retrospective search, an index ovarian cancer patient harboring inactivating SMARCA4 alterations, initially diagnosed as endometrioid carcinoma, was re-classified as SCCOHT and responded to an SCCOHT chemotherapy regimen. CONCLUSION: The vast majority of SCCOHT demonstrate genomic SMARCA4 loss with only rare co-occurring alterations. Our data support a role for CGP in the diagnosis and management of SCCOHT and of other lesions with overlapping histological and clinical features, since identifying the former by genomic profile suggests benefit from an appropriate regimen and treatment decisions, as illustrated by an index patient.
Asunto(s)
Carcinoma de Células Pequeñas/genética , ADN Helicasas/genética , Hipercalcemia/genética , Proteínas Nucleares/genética , Neoplasias Ováricas/genética , Factores de Transcripción/genética , Adolescente , Adulto , Carcinoma de Células Pequeñas/sangre , Carcinoma de Células Pequeñas/enzimología , Carcinoma de Células Pequeñas/patología , Estudios de Cohortes , ADN Helicasas/metabolismo , Femenino , Silenciador del Gen , Mutación de Línea Germinal , Humanos , Hipercalcemia/enzimología , Hipercalcemia/patología , Persona de Mediana Edad , Proteínas Nucleares/metabolismo , Neoplasias Ováricas/sangre , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/patología , Factores de Transcripción/metabolismo , Transcriptoma , Adulto JovenRESUMEN
Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is a rare but aggressive and untreatable malignancy affecting young women. We and others recently discovered that SMARCA4, a gene encoding the ATPase of the SWI/SNF chromatin-remodelling complex, is the only gene recurrently mutated in the majority of SCCOHT. The low somatic complexity of SCCOHT genomes and the prominent role of the SWI/SNF chromatin-remodelling complex in transcriptional control of genes suggest that SCCOHT cells may rely on epigenetic rewiring for oncogenic transformation. Herein, we report that approximately 80% (19/24) of SCCOHT tumour samples have strong expression of the histone methyltransferase EZH2 by immunohistochemistry, with the rest expressing variable amounts of EZH2. Re-expression of SMARCA4 suppressed the expression of EZH2 in SCCOHT cells. In comparison to other ovarian cell lines, SCCOHT cells displayed hypersensitivity to EZH2 shRNAs and two selective EZH2 inhibitors, GSK126 and EPZ-6438. EZH2 inhibitors induced cell cycle arrest, apoptosis, and cell differentiation in SCCOHT cells, along with the induction of genes involved in cell cycle regulation, apoptosis, and neuron-like differentiation. EZH2 inhibitors suppressed tumour growth and improved the survival of mice bearing SCCOHT xenografts. Therefore, our data suggest that loss of SMARCA4 creates a dependency on the catalytic activity of EZH2 in SCCOHT cells and that pharmacological inhibition of EZH2 is a promising therapeutic strategy for treating this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Carcinoma de Células Pequeñas/enzimología , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/metabolismo , Hipercalcemia/enzimología , Neoplasias Ováricas/enzimología , Animales , Apoptosis/fisiología , Carcinoma Epitelial de Ovario , Puntos de Control del Ciclo Celular/fisiología , Línea Celular Tumoral , Transformación Celular Neoplásica , ADN Helicasas/deficiencia , Regulación hacia Abajo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Femenino , Histona Metiltransferasas , Humanos , Trasplante de Neoplasias , Neoplasias Glandulares y Epiteliales/enzimología , Proteínas Nucleares/deficiencia , Factores de Transcripción/deficiencia , Trasplante Heterólogo , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
In higher eukaryotes, cell proliferation is regulated by class I phosphatidylinositol 3-kinase (PI3K), which transduces stimuli received from neighboring receptors by local generation of PtdIns(3,4,5)P3 in cellular membranes. PI3K is a heterodimeric protein consisting of a regulatory and a catalytic subunit (p85 and p110 respectively). Heterologous expression of p110α in Saccharomyces cerevisiae leads to toxicity by conversion of essential PtdIns(4,5)P2 into futile PtdIns(3,4,5)P3, providing a humanized yeast model for functional studies on this pathway. Here, we report expression and functional characterization in yeast of all regulatory and catalytic human PI3K isoforms, and exploitation of the most suitable setting to functionally assay panels of tumor- and germ line-associated PI3K mutations, with indications to the limits of the system. The activity of p110α in yeast was not compromised by truncation of its N-terminal adaptor-binding domain (ABD) or inactivation of the Ras-binding domain (RBD). In contrast, a cluster of positively charged residues at the C2 domain was essential. Expression of a membrane-driven p65α oncogenic-truncated version of p85α, but not the full-length protein, led to enhanced activity of α, ß, and δ p110 isoforms. Mutations impairing the inhibitory regulation exerted by the p85α iSH2 domain on the C2 domain of p110α yielded the latter non-responsive to negative regulation, thus reproducing this oncogenic mechanism in yeast. However, p85α germ line mutations associated with short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome did not increase PI3K activity in this model, supporting the idea that SHORT syndrome-associated p85α mutations operate through mechanisms different from the canonical disruption of inhibitory p85-p110 interactions typical of cancer.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ia/genética , Mutación , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Trastornos del Crecimiento/enzimología , Trastornos del Crecimiento/genética , Trastornos del Crecimiento/patología , Humanos , Hipercalcemia/enzimología , Hipercalcemia/genética , Hipercalcemia/patología , Immunoblotting , Enfermedades Metabólicas/enzimología , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/patología , Modelos Biológicos , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Nefrocalcinosis/enzimología , Nefrocalcinosis/genética , Nefrocalcinosis/patología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoAsunto(s)
Hipercalcemia/etiología , Hipercalcemia/genética , Pancreatitis/etiología , Pancreatitis/genética , Complicaciones del Embarazo/enzimología , Complicaciones del Embarazo/genética , Vitamina D3 24-Hidroxilasa/genética , Enfermedad Aguda , Calcio/sangre , Femenino , Humanos , Hipercalcemia/enzimología , Mutación , Pancreatitis/enzimología , Embarazo , Recurrencia , Vitamina D/análogos & derivados , Vitamina D/sangre , Adulto JovenRESUMEN
Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare ovarian neoplasm that occurs in young women and has a poor prognosis. The histologic diagnosis of SCCOHT can be challenging due to its rarity and relatively nonspecific histologic features, which range from the classic, first-described small cell morphology to a pattern in which there are large cells with abundant eosinophilic cytoplasm. Many entities can be in the differential diagnosis and to date, immunohistochemical stains have shown no distinctive profile and have been of limited aid. SMARCA4 (also known as BRG1) mutations have recently been reported at high frequency in these tumors. SMARCA4 is an important component of the SWI/SNF complex that regulates gene expression through alteration of nucleosome conformation. Studies to date have suggested that immunohistochemical loss of expression of SMARCA4 is associated with the presence of a SMARCA4 mutation in most cases. In this study, the sensitivity and specificity of the immunohistochemical loss of SMARCA4 expression for the diagnosis of SCCOHT is examined in the context of the differential diagnosis with other primary or metastatic ovarian tumors. All but one of the SCCOHT showed loss of SMARCA4 expression (16/17; 94%), while of 279 other tumors tested, only two tumors (one clear cell carcinoma and one ovarian melanoma) showed loss of SMARCA4 expression. We conclude that SMARCA4 immunohistochemistry is highly sensitive and specific for a diagnosis of SCCOHT and is of clinical utility in the differential diagnosis of poorly differentiated ovarian tumors.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Células Pequeñas/enzimología , ADN Helicasas/análisis , Hipercalcemia/enzimología , Proteínas Nucleares/análisis , Neoplasias Ováricas/enzimología , Factores de Transcripción/análisis , Biopsia , Carcinoma de Células Pequeñas/patología , Diferenciación Celular , Diagnóstico Diferencial , Regulación hacia Abajo , Femenino , Humanos , Hipercalcemia/patología , Inmunohistoquímica , Neoplasias Ováricas/patología , Valor Predictivo de las Pruebas , Pronóstico , Estados UnidosAsunto(s)
Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Femenino , Humanos , Persona de Mediana Edad , /metabolismo , Disgerminoma/enzimología , Hipercalcemia/enzimología , Hipercalcemia/etiología , Neoplasias Ováricas/enzimología , Ovario/enzimología , /genética , /metabolismo , Disgerminoma/genética , Disgerminoma/patología , Macrófagos/enzimología , Macrófagos/patología , Glicoproteínas de Membrana/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Toll-LikeRESUMEN
CONTEXT: Mutations of the CYP24A1 gene encoding the 24-hydroxylase (24OHase) that inactivates metabolites of vitamin D can cause hypercalcemia in infants and adults; in vitro assays of 24OHase activity have been difficult. OBJECTIVE: We sought an alternative assay to characterize a CYP24A1 mutation in a young adult with bilateral nephrolithiasis and hypercalcemia associated with ingestion of excess vitamin D supplements and robust dairy intake for 5 years. METHODS: CYP24A1 exons were sequenced from leukocyte DNA. Wild-type and mutant CYP24A1 cDNAs were expressed in JEG-3 cells, and 24OHase activity was assayed by a two-hybrid system. RESULTS: The CYP24A1 missense mutation L409S was found on only one allele; no other mutation was found in exons or in at least 30 bp of each intron/exon junction. Based on assays of endogenous 24OHase activity and of activity from a transiently transfected CYP24A1 cDNA expression vector, JEG-3 cells were chosen over HepG2, Y1, MA10, and NCI-H295A cells for two-hybrid assays of 24OHase activity. The apparent Michaelis constant, Km(app), was 9.0 ± 2.0 nM for CYP24A1 and 8.6 ± 2.2 nM for its mutant; the apparent maximum velocity, Vmax(app), was 0.71 ± 0.055 d(-1) for the wild type and 0.22 ± 0.026 d(-1) for the mutant. As assessed by Vmax/Km, the L409S mutant has 32% of wild-type activity (P = .0012). CONCLUSIONS: The two-hybrid system in JEG-3 cells provides a simple, sensitive, quantitative assay of 24OHase activity. Heterozygous mutation of CYP24A1 may cause hypercalcemia in the setting of excessive vitamin D intake, but it is also possible that the patient had another, unidentified CYP24A1 mutation on the other allele.
Asunto(s)
Hipercalcemia/enzimología , Nefrolitiasis/enzimología , Técnicas del Sistema de Dos Híbridos , Vitamina D3 24-Hidroxilasa/metabolismo , Humanos , Hipercalcemia/genética , Masculino , Mutación , Nefrolitiasis/genética , Vitamina D3 24-Hidroxilasa/genética , Adulto JovenRESUMEN
The vitamin D signal transduction system involves a series of cytochrome P450-containing sterol hydroxylases to generate and degrade the active hormone, 1α,25-dihydroxyvitamin D3, which serves as a ligand for the vitamin D receptor-mediated transcriptional gene expression described in companion articles in this review series. This review updates our current knowledge of the specific anabolic cytochrome P450s involved in 25- and 1α-hydroxylation, as well as the catabolic cytochrome P450 involved in 24- and 23-hydroxylation steps, which are believed to initiate inactivation of the vitamin D molecule. We focus on the biochemical properties of these enzymes; key residues in their active sites derived from crystal structures and mutagenesis studies; the physiological roles of these enzymes as determined by animal knockout studies and human genetic diseases; and the regulation of these different cytochrome P450s by extracellular ions and peptide modulators. We highlight the importance of these cytochrome P450s in the pathogenesis of kidney disease, metabolic bone disease, and hyperproliferative diseases, such as psoriasis and cancer; as well as explore potential future developments in the field.
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Esteroide Hidroxilasas/fisiología , Vitamina D/metabolismo , Secuencia de Aminoácidos , Animales , Predisposición Genética a la Enfermedad , Humanos , Hipercalcemia/enzimología , Hipercalcemia/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Insuficiencia Renal Crónica/enzimología , Insuficiencia Renal Crónica/genética , Esteroide Hidroxilasas/química , Deficiencia de Vitamina D/enzimología , Deficiencia de Vitamina D/genéticaRESUMEN
BACKGROUND: Loss-of-function mutations of vitamin D-24 hydroxylase have recently been recognized as a cause of hypercalcaemia and nephrocalcinosis/nephrolithiasis in infants and adults. True prevalence and natural history of this condition are still to be defined. METHODS: We describe two adult patients with homozygous mutations and six related heterozygous carriers. Mineral and hormonal data in these patients were compared with that in 27 patients with stage 2-3 chronic kidney disease and 39 healthy adult kidney donors. RESULTS: Probands had recurrent nephrolithiasis, chronic hypercalcaemia with depressed parathyroid hormone (PTH) and increased 1,25(OH)(2)D levels; carriers had nephrolithiasis (two of six), hypercalciuria (two of six) and high or normal-high 1,25(OH)(2)D (four of four). Corticosteroids did not reduce plasma and urine calcium levels, but ketoconazole did, indicating that 1,25(OH)(2)D production is not maximally depressed despite coexisting hypercalcaemia, high 1,25(OH)(2)D and depressed PTH, and that 1,25(OH)(2)D degradation through vitamin D-24 hydroxylase is a regulator of plasma 1,25(OH)(2)D levels. Both probands had vascular calcifications and high bone mineral content. One developed stage 3b renal failure: in this patient 1,25(OH)(2)D decreased within normal limits as glomerular filtration rate (GFR) fell and PTH rose to high-normal values, yet hypercalcaemia persisted and the ratio of 1,25(OH)(2)D to GFR remained higher than normal for any degree of GFR. CONCLUSIONS: This natural model indicates that vitamin D-24 hydroxylase is a key physiologic regulator of calcitriol and plasma calcium levels, and that balanced reduction of 1,25(OH)(2)D and GFR is instrumental for the maintenance of physiologic calcium levels and balance in chronic kidney diseases.
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Hipercalcemia/genética , Fallo Renal Crónico/genética , Vitamina D3 24-Hidroxilasa/genética , Estudios de Casos y Controles , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Hipercalcemia/complicaciones , Hipercalcemia/enzimología , Fallo Renal Crónico/enzimología , Masculino , Persona de Mediana Edad , Mutación , Hormona Paratiroidea/sangre , LinajeRESUMEN
The phosphatidylinositol 3 kinase (PI3K) pathway regulates fundamental cellular processes such as metabolism, proliferation, and survival. A central component in this pathway is the p85α regulatory subunit, encoded by PIK3R1. Using whole-exome sequencing, we identified a heterozygous PIK3R1 mutation (c.1945C>T [p.Arg649Trp]) in two unrelated families affected by partial lipodystrophy, low body mass index, short stature, progeroid face, and Rieger anomaly (SHORT syndrome). This mutation led to impaired interaction between p85α and IRS-1 and reduced AKT-mediated insulin signaling in fibroblasts from affected subjects and in reconstituted Pik3r1-knockout preadipocytes. Normal PI3K activity is critical for adipose differentiation and insulin signaling; the mutated PIK3R1 therefore provides a unique link among lipodystrophy, growth, and insulin signaling.
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Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Trastornos del Crecimiento/enzimología , Hipercalcemia/enzimología , Enfermedades Metabólicas/enzimología , Nefrocalcinosis/enzimología , Transducción de Señal , Adipocitos/metabolismo , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Índice de Masa Corporal , Diferenciación Celular , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Análisis Mutacional de ADN , Activación Enzimática , Exoma , Femenino , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Tamización de Portadores Genéticos , Predisposición Genética a la Enfermedad , Genética de Población/métodos , Trastornos del Crecimiento/patología , Humanos , Hipercalcemia/patología , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Masculino , Enfermedades Metabólicas/patología , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Nefrocalcinosis/patología , Linaje , Adulto Joven , Dominios Homologos srcRESUMEN
The small cell ovarian carcinoma of the hypercalcemic type (SCCOHT) represents an aggressive tumor with poor prognosis predominantly affecting young women and so far, no cell line or animal model is available to investigate this devastating disease. Biopsy material from a recurrent SCCOHT was subjected to an explant culture to obtain an adherent and continuously proliferating cell population. Morphological and functional characterization revealed a heterogeneous population (SCCOHT-1) of about 13 µm in diameter and approximately 36 h of doubling time. Flow cytometric analysis of surface markers demonstrated the expression of CD15, CD29, CD44 and CD90 paralleled by the presence of cytokeratins and vimentin. Cytogenetic analysis and high-resolution oligo-array comparative genomic hybridization (aCGH) demonstrated a stable karyotype including deletions of the PARK2, CSMD1, GRIN2B and ATF7IP genes. Following lentiviral transduction with a GFP vector, the labeled SCCOHT-derived cells were subjected to CCE to separate distinct subpopulations as evidenced by cell cycle analysis. Subcutaneous injection of these subpopulations into NOD/SCID mice exhibited hypercalcemia and a tumor development in 100% of the mice. Re-cultivation of the mouse tumors revealed an outgrowth of SCCOHT-derived phenotypes and all cell populations expressed high telomerase activity. Moreover, histopathological evaluation demonstrated close similarities between the mouse tumors and the original patient tumor. In conclusion, SCCOHT-1 cells provide a study platform to investigate this rare disease and to examine effective and sufficient therapeutic strategies for this rather unknown type of cancer.
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Carcinoma de Células Pequeñas/patología , Línea Celular Tumoral/ultraestructura , Hipercalcemia/patología , Neoplasias Ováricas/patología , Cariotipo Anormal , Adulto , Animales , Biomarcadores de Tumor/metabolismo , Calcio/sangre , Carcinoma de Células Pequeñas/sangre , Carcinoma de Células Pequeñas/enzimología , Línea Celular Tumoral/citología , Línea Celular Tumoral/metabolismo , Proliferación Celular , Forma de la Célula , Femenino , Inestabilidad Genómica , Proteínas Fluorescentes Verdes/biosíntesis , Humanos , Hipercalcemia/sangre , Hipercalcemia/enzimología , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Neoplasias Ováricas/sangre , Neoplasias Ováricas/enzimología , Proteínas Recombinantes/biosíntesis , Telomerasa/metabolismo , Carga TumoralRESUMEN
BACKGROUND: The most serious complication of subcutaneous fat necrosis (SCFN), a rare condition of the newborn characterized by indurated purple nodules, is hypercalcaemia. However, the mechanism for this hypercalcaemia remains unclear. OBJECTIVES: To determine whether the hypercalcaemia associated with SCFN involves expression of the vitamin D-activating enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase) in affected tissue. METHODS: Skin biopsies from two male patients with SCFN and hypercalcaemia were taken. The histological specimens were assessed using a polyclonal antibody against 1alpha-hydroxylase. RESULTS: Histology in both cases showed strong expression of 1alpha-hydroxylase protein (brown staining) within the inflammatory infiltrate associated with SCFN. This was consistent with similar experiments in other granulomatous conditions. CONCLUSIONS: Hypercalcaemia in SCFN appears to be due to abundant levels of 1alpha-hydroxylase in immune infiltrates associated with tissue lesions. This is consistent with previous observations of extrarenal 1alpha-hydroxylase in skin from other granulomatous conditions such as sarcoidosis and slack skin disease.
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25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Necrosis Grasa/enzimología , Hipercalcemia/enzimología , Grasa Subcutánea/metabolismo , Vitamina D/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Necrosis Grasa/complicaciones , Necrosis Grasa/diagnóstico por imagen , Expresión Génica , Humanos , Hipercalcemia/diagnóstico por imagen , Hipercalcemia/etiología , Recién Nacido , Masculino , Ultrasonografía , Vitamina D/genéticaRESUMEN
Nephrogenic systemic fibrosis (NSF) is a disease of thickened, hard, hyperpigmented skin lesions with or without systemic fibrosis occurring in patients with renal insufficiency and associated with the administration of gadolinium-containing contrast. The pathogenesis of this disease is unclear, and there is no definitive treatment. We describe a 71-yr-old patient with stable chronic lymphocytic leukemia (CLL), end-stage renal disease (ESRD), and NSF who presented with hypercalcemia in 2006. Before onset of renal insufficiency in 2002, serum calcium, phosphorus, and PTH levels were normal. In 2004, the patient began hemodialysis, and he was diagnosed with NSF in 2005, shortly after undergoing an MRI with gadolinium contrast administration. Over the next 6 mo, albumin-corrected serum total calcium levels rose from 9.9 to 13.1 mg/dl (normal range, 8.5-10.5 mg/dl) with normal serum phosphorus levels. On admission in September 2006, 1,25-dihydroxyvitamin D [1,25(OH)(2)D] levels were elevated at 130.7 pg/ml (normal range, 25.1-66.1 pg/ml). Biopsy of an NSF lesion showed increased 25-hydroxyvitamin D(3)-1-alpha hydroxylase (CYP27B1) immunostaining compared with the biopsy from a normal control. This is the first reported association of NSF with hypercalcemia caused by elevated 1,25(OH)(2)D levels. This metabolic disturbance should be sought in future cases to determine a connection between NSF, 1,25(OH)(2)D metabolism, and CYP27B1 activation in the skin, which may shed light on the pathogenesis of this unusual local and systemic fibrosing disorder.
Asunto(s)
25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Hipercalcemia/complicaciones , Dermopatía Fibrosante Nefrogénica/complicaciones , Anciano , Difosfonatos/uso terapéutico , Humanos , Hipercalcemia/enzimología , Hipercalcemia/terapia , Leucemia Linfocítica Crónica de Células B/complicaciones , Leucemia Linfocítica Crónica de Células B/diagnóstico , Masculino , Dermopatía Fibrosante Nefrogénica/enzimología , Dermopatía Fibrosante Nefrogénica/terapia , Pamidronato , Diálisis RenalRESUMEN
We aim to test the hypothesis that hypercalcemia produces pulmonary edema (PE) and to elucidate the mechanism. Experimentations were carried out in conscious rats and isolated perfused rat lungs. We evaluated PE by lung weight changes, protein concentration in bronchoalveolar lavage, dye leakage, and microvascular permeability. Plasma nitrate/nitrite, methyl guanidine (MG), proinflammatory cytokines, procalcitonin levels, and histopathological examinations were evaluated. Immunochemical staining and reverse-transcriptase polymerase chain reaction (RT-PCR) were used to detect inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in the lungs. Hypercalcemia was produced in the conscious rat and isolated perfused lungs. Calcitonin and L-N(6) (1-iminoethyl)-lysine (L-Nil) were administered before hypercalcemia to observe their effects. Hypercalcemia caused severe PE in rats. Pathological and immunochemical examinations revealed hemorrhagic edema with iNOS activity in the alveolar macrophages and epithelial cells. RT-PCR showed an increase in iNOS mRNA expression. Hypercalcemia increased nitrate/nitrite, MG, proinflammatory cytokines and procalcitonin levels. Pretreatment with calcitonin or L-Nil prevented these changes. In conclusion, hypercalcemia caused PE in conscious rats and isolated perfused rat lungs. The increases in nitrate/nitrite, free radicals, proinflammatory cytokines, procalcitonin and iNOS activity suggest that hypercalcemia induces a sepsis-like syndrome. The effect of hypercalcemia on the lung may involve iNOS and NO.
Asunto(s)
Hipercalcemia/enzimología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico/metabolismo , Edema Pulmonar/enzimología , Animales , Calcitonina/farmacología , Permeabilidad Capilar/efectos de los fármacos , Citocinas/sangre , Hipercalcemia/complicaciones , Hipercalcemia/patología , Lisina/análogos & derivados , Lisina/farmacología , Masculino , Metilguanidina/sangre , Nitratos/sangre , Óxido Nítrico Sintasa de Tipo III , Nitritos/sangre , Edema Pulmonar/etiología , Edema Pulmonar/patología , Ratas , Ratas Sprague-Dawley , Síndrome de Respuesta Inflamatoria Sistémica/enzimología , Síndrome de Respuesta Inflamatoria Sistémica/etiología , Síndrome de Respuesta Inflamatoria Sistémica/patologíaRESUMEN
Humoral hypercalcemia of malignancy (HHM) is a common paraneoplastic disorder usually associated with increased synthesis of parathyroid hormone-related peptide (PTHrP). Unlike non-cancer forms of hypercalcemia, HHM does not routinely involve increased circulating levels of the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Dysgerminomas are a notable exception to this rule, previous reports having described hypercalcemia with elevated serum 1,25(OH)2D3. To investigate the etiology of this form of HHM we have characterized expression and activity of the enzyme that catalyzes synthesis of 1,25(OH)2D3, 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase), in a collection of 12 dysgerminomas. RT-PCR analyses indicated that mRNA for 1alpha-hydroxylase was increased 222-fold in dysgerminomas compared to non-tumor ovarian tissue. Parallel enzyme assays in tissue homogenates showed that dysgerminomas produced fivefold higher levels of 1,25(OH)2D3 compared to normal ovarian tissue. Immunolocalization studies indicated that 1alpha-hydroxylase was expressed by both tumor cells and by macrophages within the inflammatory cell infiltrate associated with dysgerminomas. The immunological nature of the increased 1,25(OH)2D3 production observed in dysgerminomas was further emphasized by correlation between expression of 1alpha-hydroxylase and the endotoxin recognition factors CD14 and toll-like receptor 4 (TLR4). These data suggest that inflammatory mechanisms associated with dysgerminomas are the underlying cause of the increased expression and activity of 1alpha-hydroxylase associated with these tumors. We further postulate that this autocrine/paracrine action of 1alpha-hydroxylase may lead to increased circulating levels of 1,25(OH)2D3 and a form of HHM which is distinct from that seen with PTHrP-secreting tumors.
Asunto(s)
25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Disgerminoma/enzimología , Hipercalcemia/enzimología , Hipercalcemia/etiología , Neoplasias Ováricas/enzimología , Ovario/enzimología , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Disgerminoma/genética , Disgerminoma/patología , Femenino , Humanos , Receptores de Lipopolisacáridos/metabolismo , Macrófagos/enzimología , Macrófagos/patología , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
Alkaline phosphatase (AP) is the classic marker of bone formation, especially in cancer patients, but the interpretation of its measurement is complicated by the existence of various circulating isoenzymes, especially of liver origin. The introduction of a mass measurement of the bone isoenzyme of AP (BAP) by an immunoradiometric assay has markedly improved the sensitivity and the specificity of the determination. We measured BAP and other markers of bone turnover in 46 patients with tumour-induced hypercalcaemia (TIH), which is an interesting model for evaluating markers of bone formation because of the uncoupling between bone formation and bone resorption found by histomorphometric techniques. The extent of bone metastatic involvement was evaluated by planimetry on bone scintigraphy. Mean (+/- S.D.) BAP concentrations were slightly higher in patients with TIH than in healthy subjects, 15.5 +/- 8.5 versus 12.4 +/- 3.5 micrograms/L (P < 0.05). However, the scatter of the data in TIH patients was quite marked. Increased values (10/46 patients, 22%) occurred only in patients with bone metastases. Total AP, gamma GT and BGP levels, as well as markers of bone resorption, were not significantly different between patients with or without bone metastases. BAP levels were significantly correlated with AP (rs = 0.63; P < 0.01) but not with BGP levels nor with markers of bone resorption. BAP levels were also correlated with the extent of bone uptake at scintigraphy (rs = 0.54; P < 0.01), but this was not the case for total AP or BGP. In the 36 patients re-evaluated when normocalcemic after pamidronate therapy, BAP levels increased from 16.3 +/- 9.2 to 22.2 +/- 21.3 micrograms/L (P < 0.05) but there were no significant changes in AP or BGP concentrations. In summary, our data confirm the existence of an uncoupling in bone turnover in TIH and indicate that cancer hypercalcaemia is another pathological condition characterised by a discordance between BAP and BGP concentrations. BAP levels appear to be a better reflection of bone metastatic involvement than total AP or BGP and their short-term increase after pamidronate therapy could reflect the recently described effects of bisphosphonates on osteoblasts.