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ClC-K/barttin channels are involved in the transepithelial transport of chloride in the kidney and inner ear. Their physiological role is crucial in humans because mutations in CLCNKB or BSND, encoding ClC-Kb and barttin, cause Bartter's syndrome types III and IV, respectively. In vitro experiments have shown that an amino acid change in a proline-tyrosine motif in the C-terminus of barttin stimulates ClC-K currents. The molecular mechanism of this enhancement and whether this potentiation has any in vivo relevance remains unknown. We performed electrophysiological and biochemical experiments in Xenopus oocytes and kidney cells co-expressing ClC-K and barttin constructs. We demonstrated that barttin possesses a YxxØ motif and, when mutated, increases ClC-K plasma membrane stability, resulting in larger currents. To address the impact of mutating this motif in kidney physiology, we generated a knock-in mouse. Comparing wild-type (WT) and knock-in mice under a standard diet, we could not observe any difference in ClC-K and barttin protein levels or localization, either in urinary or plasma parameters. However, under a high-sodium low-potassium diet, known to induce hyperplasia of distal convoluted tubules, knock-in mice exhibit reduced hyperplasia compared to WT mice. In summary, our in vitro and in vivo studies demonstrate that the previously identified PY motif is indeed an endocytic YxxØ motif in which mutations cause a gain of function of the channel. KEY POINTS: It is revealed by mutagenesis and functional experiments that a previously identified proline-tyrosine motif regulating ClC-K plasma membrane levels is indeed an endocytic YxxØ motif. Biochemical characterization of mutants in the YxxØ motif in Xenopus oocytes and human embryonic kidney cells indicates that mutants showed increased plasma membrane levels as a result of an increased stability, resulting in higher function of ClC-K channels. Mutation of this motif does not affect barttin protein expression and subcellular localization in vivo. Knock-in mice with a mutation in this motif, under conditions of a high-sodium low-potassium diet, exhibit less hyperplasia in the distal convoluted tubule than wild-type animals, indicating a gain of function of the channel in vivo.
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More than 20 years have passed since the identification of SLC3A1 and SLC7A9 as causative genes for cystinuria. However, cystinuria patients exhibit significant variability in the age of lithiasis onset, recurrence, and response to treatment, suggesting the presence of modulatory factors influencing cystinuria severity. In 2016, a second renal cystine transporter, AGT1, encoded by the SLC7A13 gene, was discovered. Although it was discarded as a causative gene for cystinuria, its possible effect as a modulatory gene remains unexplored. Thus, we analyzed its function in mouse models of cystinuria, screened the SLC7A13 gene in 34 patients with different lithiasic phenotypes, and functionally characterized the identified variants. Mice results showed that AGT1/rBAT may have a protective role against cystine lithiasis. In addition, among the four missense variants detected in patients, two exhibited a 25% impairment in AGT1/rBAT transport. However, no correlation between SLC7A13 genotypes and lithiasis phenotypes was observed in patients, probably because these variants were found in heterozygous states. In conclusion, our results, consistent with a previous study, suggest that AGT1/rBAT does not have a relevant effect on cystinuria patients, although an impact in patients carrying homozygous pathogenic variants cannot be discarded.
Assuntos
Cistinúria , Litíase , Humanos , Animais , Camundongos , Cistinúria/genética , Cistinúria/patologia , Litíase/complicações , Cistina , Estudos Retrospectivos , Rim/patologiaRESUMO
Background Reabsorption of amino acids (AAs) across the renal proximal tubule is crucial for intracellular and whole organism AA homeostasis. Although the luminal transport step is well understood, with several diseases caused by dysregulation of this process, the basolateral transport step is not understood. In humans, only cationic aminoaciduria due to malfunction of the basolateral transporter y+LAT1/CD98hc (SLC7A7/SLC3A2), which mediates the export of cationic AAs, has been described. Thus, the physiologic roles of basolateral transporters of neutral AAs, such as the antiporter LAT2/CD98hc (SLC7A8/SLC3A2), a heterodimer that exports most neutral AAs, and the uniporter TAT1 (SLC16A10), which exports only aromatic AAs, remain unclear. Functional cooperation between TAT1 and LAT2/CD98hc has been suggested by in vitro studies but has not been evaluated in vivoMethods To study the functional relationship of TAT1 and LAT2/CD98hc in vivo, we generated a double-knockout mouse model lacking TAT1 and LAT2, the catalytic subunit of LAT2/CD98hc (dKO LAT2-TAT1 mice).Results Compared with mice lacking only TAT1 or LAT2, dKO LAT2-TAT1 mice lost larger amounts of aromatic and other neutral AAs in their urine due to a tubular reabsorption defect. Notably, dKO mice also displayed decreased tubular reabsorption of cationic AAs and increased expression of y+LAT1/CD98hc.Conclusions The LAT2/CD98hc and TAT1 transporters functionally cooperate in vivo, and y+LAT1/CD98hc may compensate for the loss of LAT2/CD98hc and TAT1, functioning as a neutral AA exporter at the expense of some urinary loss of cationic AAs. Cooperative and compensatory mechanisms of AA transporters may explain the lack of basolateral neutral aminoacidurias in humans.
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Sistema y+ de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Aminoácidos Neutros/metabolismo , Cadeias Leves da Proteína-1 Reguladora de Fusão/genética , Reabsorção Renal , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos Neutros/urina , Animais , Feminino , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Cadeias Leves da Proteína-1 Reguladora de Fusão/metabolismo , Túbulos Renais/fisiologia , Masculino , Camundongos KnockoutRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM genes. Previous work indicated that chloride currents mediated by the volume-regulated anion channel (VRAC) and ClC-2 channels were affected in astrocytes deficient in either Mlc1 or Glialcam. ClC-2 forms a ternary complex with GlialCAM and MLC1. LRRC8 proteins have been identified recently as the molecular components of VRAC, but the relationship between MLC and LRRC8 proteins is unknown. Here, we first demonstrate that LRRC8 and MLC1 are functionally linked, as MLC1 cannot potentiate VRAC currents when LRRC8A, the main subunit of VRAC, is knocked down. We determine that LRRC8A and MLC1 do not co-localize or interact and, in Xenopus oocytes, MLC1 does not potentiate LRRC8-mediated VRAC currents, indicating that VRAC modulation in astrocytes by MLC1 may be indirect. Investigating the mechanism of modulation, we find that a lack of MLC1 does not influence either mRNA or total and plasma membrane protein levels of LRRC8A; and neither does it affect LRRC8A subcellular localization. In agreement with recent results that indicated that overexpression of MLC1 decreases the phosphorylation of extracellular signal-regulated kinases (ERK), we find that astrocytes lacking MLC1 show an increase in ERK phosphorylation. In astrocytes with reduced or increased levels of MLC1 we observe changes in the phosphorylation state of the VRAC subunit LRRC8C. Our results thus reinforce previous suggestions that indicated that GlialCAM/MLC1 might modify signal transduction pathways that influence the activity of different proteins, such as VRAC.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Astrócitos/metabolismo , Cistos/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Astrócitos/química , Astrócitos/patologia , Proteínas de Ciclo Celular , Células Cultivadas , Cistos/patologia , Células HeLa , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Proteínas/análise , Proteínas/genética , Ratos , XenopusRESUMO
Colorectal cancer (CRC) is characterized by specific patterns of copy number alterations (CNAs), which helped with the identification of driver oncogenes and tumor suppressor genes (TSGs). More recently, the usage of single nucleotide polymorphism arrays provided information of copy number neutral loss of heterozygosity, thus suggesting the occurrence of somatic uniparental disomy (UPD) and uniparental polysomy (UPP) events. The aim of this study is to establish an integrative profiling of recurrent UPDs/UPPs and CNAs in sporadic CRC. Our results indicate that regions showing high frequencies of UPD/UPP mostly coincide with regions typically involved in genomic losses. Among them, chromosome arms 3p, 5q, 9q, 10q, 14q, 17p, 17q, 20p, 21q and 22q preferentially showed UPDs/UPPs over genomic losses suggesting that tumor cells must maintain the disomic state of certain genes to favor cellular fitness. A meta-analysis using over 300 samples from The Cancer Genome Atlas confirmed our findings. Several regions affected by recurrent UPDs/UPPs contain well-known TSGs, as well as novel candidates such as ARID1A, DLC1, TCF7L2 and DMBT1. In addition, VCAN, FLT4, SFRP1 and GAS7 were also frequently involved in regions of UPD/UPP and displayed high levels of methylation. Finally, sequencing and fluorescence in situ hybridization analysis of the gene APC underlined that a somatic UPD event might represent the second hit to achieve biallelic inactivation of this TSG in colorectal tumors. In summary, our data define a profile of somatic UPDs/UPPs in sporadic CRC and highlights the importance of these events as a mechanism to achieve the inactivation of TSGs.
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Neoplasias Colorretais/genética , Genes Supressores de Tumor , Dissomia Uniparental/genética , Aberrações Cromossômicas , Neoplasias Colorretais/complicações , Neoplasias Colorretais/patologia , Humanos , Perda de Heterozigosidade , Dissomia Uniparental/patologiaRESUMO
The high recurrence rate of cystine lithiasis observed in cystinuria patients highlights the need for new therapeutic options to address this chronic disease. There is growing evidence of an antioxidant defect in cystinuria, which has led to test antioxidant molecules as new therapeutic approaches. In this study, the antioxidant l-Ergothioneine was evaluated, at two different doses, as a preventive and long-term treatment for cystinuria in the Slc7a9-/- mouse model. l-Ergothioneine treatments decreased the rate of stone formation by more than 60% and delayed its onset in those mice that still developed calculi. Although there were no differences in metabolic parameters or urinary cystine concentration between control and treated mice, cystine solubility was increased by 50% in the urines of treated mice. We also demonstrate that l-Ergothioneine needs to be internalized by its transporter OCTN1 (Slc22a4) to be effective, as when administrated to the double mutant Slc7a9-/-Slc22a4-/- mouse model, no effect on the lithiasis phenotype was observed. In kidneys, we detected a decrease in GSH levels and an impairment of maximal mitochondrial respiratory capacity in cystinuric mice that l-Ergothioneine treatment was able to restore. Thus, l-Ergothioneine administration prevented cystine lithiasis in the Slc7a9-/- mouse model by increasing urinary cystine solubility and recovered renal GSH metabolism and mitochondrial function. These results support the need for clinical trials to test l-Ergothioneine as a new treatment for cystinuria.
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Cistinúria , Ergotioneína , Litíase , Animais , Camundongos , Ergotioneína/farmacologia , Litíase/prevenção & controle , Cistinúria/tratamento farmacológico , Cistina , Antioxidantes/farmacologia , Camundongos Knockout , Masculino , Feminino , Camundongos Endogâmicos C57BL , Glutationa/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Mitocôndrias/efeitos dos fármacos , Estresse OxidativoRESUMO
Liver fibrosis is a major health problem with multiple associated complications, which, to date, has no effective treatment. Hepatic stellate cells are the main responsible cells for fibrosis formation; upon their activation, excess accumulation of extracellular matrix and collagen deposits occurs. The mitogen platelet-derived growth factor (PDGF) and its receptor ß (PDGFRß) play a major role in hepatic stellate cells activation and are, therefore, promising targets for antifibrotic therapies. Gold nanorods hold great potential for diseased liver treatments, since their passive hepatic accumulation enhances active targeting strategies, hence increasing therapeutic efficiency. In addition, gold nanorods have photothermal properties that, combined with specific cell delivery, can be exploited to induce localized near-infrared light-mediated thermal ablation. Here, we demonstrate that gold nanorods coated with anti-PDGFRß specifically target activated hepatic stellate cells in vivo. Additionally, gold nanorods-PDGFRß-mediated photothermal therapy decreases fibrosis, hepatic inflammation, and hepatocyte injury in the experimental model of CCl4-induced liver fibrosis in mice.
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Hipertermia , Cirrose Hepática , Animais , Células Estreladas do Fígado/patologia , Fígado/patologia , Cirrose Hepática/patologia , Cirrose Hepática/terapia , Camundongos , Receptor beta de Fator de Crescimento Derivado de PlaquetasRESUMO
BACKGROUND: Aneuploidy, centrosome abnormalities and gene amplification are hallmarks of chromosome instability (CIN) in cancer. Yet there are no studies of the in vivo behavior of these phenomena within the same bladder tumor. METHODS: Twenty-one paraffin-embedded bladder tumors were analyzed by conventional comparative genome hybridization and fluorescence in situ hybridization (FISH) with a cyclin D1 gene (CCND1)/centromere 11 dual-color probe. Immunofluorescent staining of alpha, beta and gamma tubulin was also performed. RESULTS: Based on the CIN index, defined as the percentage of cells not displaying the modal number for chromosome 11, tumors were classified as CIN-negative and CIN-positive. Fourteen out of 21 tumors were considered CIN-positive. All T1G3 tumors were included in the CIN-positive group whereas the majority of Ta samples were classified as CIN-negative tumors. Centrosome clustering was observed in six out of 12 CIN-positive tumors analyzed. CCND1 amplification in homogeneously staining regions was present in six out of 14 CIN-positive tumors; three of them also showed amplification of this gene in double minutes. CONCLUSIONS: Complex in vivo behavior of CCND1 amplicon in bladder tumor cells has been demonstrated by accurate FISH analysis on paraffin-embedded tumors. Positive correlation between high heterogeneity, centrosome abnormalities and CCND1 amplification was found in T1G3 bladder carcinomas. This is the first study to provide insights into the coexistence of CCND1 amplification in homogeneously staining regions and double minutes in primary bladder tumors. It is noteworthy that those patients whose tumors showed double minutes had a significantly shorter overall survival rate (p < 0.001).
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Biomarcadores Tumorais/genética , Centrossomo/patologia , Instabilidade Cromossômica , Cromossomos Humanos Par 11 , Ciclina D1/genética , Amplificação de Genes , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Adulto , Idoso , Centrossomo/química , Hibridização Genômica Comparativa , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Estimativa de Kaplan-Meier , Masculino , Micronúcleos com Defeito Cromossômico , Pessoa de Meia-Idade , Mitose , Estadiamento de Neoplasias , Inclusão em Parafina , Prognóstico , Fatores de Tempo , Tubulina (Proteína)/análise , Neoplasias da Bexiga Urinária/química , Neoplasias da Bexiga Urinária/mortalidadeRESUMO
STUDY TYPE: Prognosis (case series) Level of Evidence 4. OBJECTIVE: To estimate the relative risk of developing a second primary neoplasm, in particular lung cancer, after having non-muscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS: Patients were included in the study if they had developed NMIBC between 1995 and 2003. All clinical data were extracted from the medical records of our institution's database. The interval between neoplasms, smoking habits, histological subtypes and survival were also analysed. Patient follow-up was >or=5 years. RESULTS: We found 231 patients with NMIBC, 39 of which had a second primary neoplasm: 10 lung cancer, one pancreas, one gastric, one pharynx, one liver, one parathyroid, one oesophageal, five basal cell carcinoma, three larynx, two colon, three rectal and 10 prostate. In patients with lung cancer, NMIBC was the first primary tumour. Overall, the median (range) interval between occurrence of NMIBC and lung cancer was 54.2 (8-168) months. For the relationship between the observed and expected cases of lung cancer, after normalizing our frequencies to the sex ratio and age of our group of patients, the risk of lung cancer was 10.27-fold higher in patients with NMIBC as compared with the general population of Catalonia (95% confidence interval 4.92-18.88). CONCLUSION: We consider that an annual examination for the detection and prevention of lung cancer must be included in clinical guides for patients with NMIBC. This proposal is reinforced by the finding that death in our group of patients with both tumours was always derived from lung cancer and not from bladder cancer.
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Carcinoma de Células de Transição , Detecção Precoce de Câncer , Neoplasias Pulmonares/diagnóstico , Segunda Neoplasia Primária/diagnóstico , Neoplasias da Bexiga Urinária , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células de Transição/mortalidade , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Segunda Neoplasia Primária/mortalidade , Fatores de Risco , Neoplasias da Bexiga Urinária/mortalidadeRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare genetic disorder belonging to the group of vacuolating leukodystrophies. It is characterized by megalencephaly, loss of motor functions, epilepsy, and mild mental decline. In brain biopsies of MLC patients, vacuoles were observed in myelin and in astrocytes surrounding blood vessels. There is no therapy for MLC patients, only supportive treatment. We show here a preclinical gene therapy approach for MLC using the Mlc1 knock-out mouse. An adeno-associated virus coding for human MLC1 under the control of the glial fibrillary acidic protein promoter was injected in the cerebellar subarachnoid space of Mlc1 knock-out and wild-type animals at 2 months of age, before the onset of the disease, as a preventive approach. We also tested a therapeutic strategy by injecting the animals at 5 months, once the histopathological abnormalities are starting, or at 15 months, when they have progressed to a more severe pathology. MLC1 expression in the cerebellum restored the adhesion molecule GlialCAM and the chloride channel ClC-2 localization in Bergmann glia, which both are mislocalized in Mlc1 knock-out model. More importantly, myelin vacuolation was extremely reduced in treated mice at all ages and correlated with the amount of expressed MLC1 in Bergmann glia, indicating not only the preventive potential of this strategy but also its therapeutic capacity. In summary, here we provide the first therapeutic approach for patients affected with MLC. This work may have also implications to treat other diseases affecting motor function such as ataxias.
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Astrócitos/patologia , Cerebelo/patologia , Cistos/patologia , Cistos/terapia , Terapia Genética/métodos , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/terapia , Fatores Etários , Animais , Astrócitos/ultraestrutura , Cerebelo/ultraestrutura , Cistos/genética , Células HEK293 , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Humanos , Camundongos , Camundongos KnockoutRESUMO
Cataract, the loss of ocular lens transparency, accounts for â¼50% of worldwide blindness and has been associated with water and solute transport dysfunction across lens cellular barriers. We show that neutral amino acid antiporter LAT2 (Slc7a8) and uniporter TAT1 (Slc16a10) are expressed on mouse ciliary epithelium and LAT2 also in lens epithelium. Correspondingly, deletion of LAT2 induced a dramatic decrease in lens essential amino acid levels that was modulated by TAT1 defect. Interestingly, the absence of LAT2 led to increased incidence of cataract in mice, in particular in older females, and a synergistic effect was observed with simultaneous lack of TAT1. Screening SLC7A8 in patients diagnosed with congenital or age-related cataract yielded one homozygous single nucleotide deletion segregating in a family with congenital cataract. Expressed in HeLa cells, this LAT2 mutation did not support amino acid uptake. Heterozygous LAT2 variants were also found in patients with cataract some of which showed a reduced transport function when expressed in HeLa cells. Whether heterozygous LAT2 variants may contribute to the pathology of cataract needs to be further investigated. Overall, our results suggest that defects of amino acid transporter LAT2 are implicated in cataract formation, a situation that may be aggravated by TAT1 defects.
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This study describes the health services available in Catalonia, Spain as part of the situation analysis of the healthcare map, setting a starting point for the process of adapting services to the needs of the population. It also includes an analysis of the geographic accessibility to healthcare centres in the public health system, through the use of a geographic information system (GIS), with geo-referencing variables and calculations of travel times and distances. The principal results show, on one hand, the adaptation of the Catalan healthcare network to the distribution of the population, with a high level of geographic proximity of the services to the population, and a high degree of capillarity, principally in primary healthcare; and on the other hand, the importance that GIS tools and procedures may acquire in healthcare planning is highlighted.
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Acessibilidade aos Serviços de Saúde/organização & administração , Administração de Serviços de Saúde , Regionalização da Saúde/métodos , Sistemas de Informação Geográfica , EspanhaRESUMO
Age-related hearing loss (ARHL) is the most common sensory deficit in the elderly. The disease has a multifactorial etiology with both environmental and genetic factors involved being largely unknown. SLC7A8/SLC3A2 heterodimer is a neutral amino acid exchanger. Here, we demonstrated that SLC7A8 is expressed in the mouse inner ear and that its ablation resulted in ARHL, due to the damage of different cochlear structures. These findings make SLC7A8 transporter a strong candidate for ARHL in humans. Thus, a screening of a cohort of ARHL patients and controls was carried out revealing several variants in SLC7A8, whose role was further investigated by in vitro functional studies. Significant decreases in SLC7A8 transport activity was detected for patient's variants (p.Val302Ile, p.Arg418His, p.Thr402Met and p.Val460Glu) further supporting a causative role for SLC7A8 in ARHL. Moreover, our preliminary data suggest that a relevant proportion of ARHL cases could be explained by SLC7A8 mutations.
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Mutação , Presbiacusia/genética , Presbiacusia/patologia , Sistema y+ de Transporte de Aminoácidos/deficiência , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Cadeias Leves da Proteína-1 Reguladora de Fusão/deficiência , Cadeias Leves da Proteína-1 Reguladora de Fusão/genética , Deleção de Genes , Testes Genéticos , Humanos , CamundongosRESUMO
Mantle-cell lymphoma (MCL) is genetically characterized by 11q13 chromosomal translocations involving the CCND1 gene. We have characterized five MCL cell lines, JVM-2, GRANTA-519, REC-1, JEKO-1, and NCEB-1, combining metaphase and array comparative genomic hybridization, multicolor-FISH, and molecular analysis. Our results revealed common gained regions at 2p14, 9q31.2-qter, 11q13.1-q21, 13q14-q21.2, 13q34-qter and 18q21.1-q22.1, and losses at 1p21.2-p31.1, 2p11.2, 8p21.2-pter, 9p21.3-pter, 11q23.3-qter, 17p11.2-pter, and 17q21.2-q22.2. All cell lines except JVM-2, displayed moderate or high numerical chromosome instability. In addition, an ongoing level of chromosome rearrangements was observed in REC-1. Surprisingly, NCEB-1 carried several stable mouse chromosomes and showed expression of both human and murine bcl-2 protein. Our findings indicate that these cell lines represent three patterns of chromosome evolution in MCL and may be useful to understand the pathogenesis of this neoplasm.
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Aberrações Cromossômicas , Linfoma de Célula do Manto/genética , Animais , Linhagem Celular Tumoral , Análise Citogenética/métodos , Genoma Humano , Humanos , Hibridização in Situ Fluorescente/métodos , Cariotipagem , Metáfase , Camundongos , Hibridização de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodosRESUMO
Most tumors show abnormal karyotypes involving either chromosome rearrangements and/or aneuploidies. The aim of our study is to measure the rate of both structural and numerical chromosome instability in two colorectal cancer cell lines: HCT116, and SW480 and its single subclones. To determine structural instability, we measured the nonclonal chromosome alterations of the last cell division by means of multicolor-fluorescence in situ hybridization (FISH). To quantify numerical instability, we used centromere-specific DNA probes to simultaneously detect chromosome loss and nondisjunctional events in binucleated cells obtained by cytokinesis-block micronucleus assay (CBMN). After clonal episodes, the structural chromosome instability rate increased significantly, confirming the large contribution of structural rearrangements to the heterogeneity of cancer cells. On the other hand, the aneuploidy rate was high and conserved in both the parental SW480 cell line and its subclones. The ability to differentiate chromosome loss and nondisjunction by the CBMN assay allowed us to conclude that no significant differences were detected among these events. Analysis of nucleoplasmic bridges, micronuclei, and nuclear blebs also demonstrated the differences among the structural instability rates of the parental cell line and its subclones. Overall, our results demonstrate the prevalence of structural over numerical chromosome instability in the subclones when comparing them with their parental cell line, confirming the contribution of ongoing chromosomal reorganizations in the generation of tumor cell heterogeneity.
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Instabilidade Cromossômica , Citocinese , Hibridização in Situ Fluorescente , Testes para Micronúcleos , Neoplasias/genética , Aneuploidia , Linhagem Celular Tumoral , Centrômero/genética , Neoplasias Colorretais/genética , Sondas de DNA , Rearranjo Gênico , Células HCT116 , Humanos , Não Disjunção GenéticaRESUMO
In this study, we summarized cytogenetic and comparative genomic hybridization (CGH) results, mutation analysis of the MET gene, and immunohistochemistry results of tumors from three patients in the same family who were affected by hereditary papillary renal carcinoma (HPRC). All three patients showed germline mutations in the tyrosine kinase domain of the MET proto-oncogene, and developed bilateral and multiple papillary renal tumors. DNA mutation analysis showed an increased dosage of the mutant allele in six tumors from two patients but not in two tumors from the third patient. In addition to the recurrent chromosomal alterations found in papillary renal carcinomas, cytogenetic analyses revealed the presence of an identical chromosomal translocation, t(2;15)(q13;p11), in two different tumors from the same patient. Moreover, the same pattern of autosomal trisomies (+7, +12, +13, +17) was detected by CGH analysis in tumors from different siblings. Taking into account that the presence of an identical structural chromosomal aberration in two tumors and the gain of chromosome 13 are unusual chromosomal changes in this type of tumor, we can conclude that our results confirm those of other authors and suggest that the genetic predisposition to HPRC might predispose the acquisition of genomic alterations in specific chromosomes or chromosomal regions.
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Carcinoma Papilar/genética , Carcinoma de Células Renais/genética , Aberrações Cromossômicas , Neoplasias Renais/genética , Carcinoma Papilar/patologia , Carcinoma de Células Renais/patologia , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 7 , Humanos , Neoplasias Renais/patologia , Masculino , Mutação , Hibridização de Ácido Nucleico/métodos , Linhagem , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-met/genéticaRESUMO
Cancer cells progress through the accumulation of genetic alterations. Familial adenomatous polyposis (FAP) tumors provide an excellent model to unravel the molecular steps underlying malignant transformation. Global genomic damage was assessed in 56 adenomas and 3 carcinomas from six FAP patients and compared with that of sporadic adenomas and carcinomas. Evolutive trees were traced after application of maximum likelihood clustering and split decomposition methods to the analysis of comprehensive genetic profiles generated by diverse molecular approaches: arbitrarily primed PCR, comparative genomic hybridization, and flow cytometry. Overall, genomic damage as assessed by arbitrarily primed PCR was lower in familial adenomas than in sporadic adenomas and carcinomas. Comparative genomic hybridization data also show a low number of alterations in the majority of FAP adenomas. Tumors of the same patient were likely to share specific genetic alterations and may be grouped into one or two clusters. Putative common pathways were also identified, which included tumors of up to three different patients. According to our data, FAP tumors accumulate specific genetic alterations and in a preferred order that is characteristic of each individual. Moreover, the particular genetic background and environmental conditions of a FAP patient restrain the molecular evolution portrait of synchronous tumors.
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Polipose Adenomatosa do Colo/genética , Instabilidade Cromossômica , Adulto , Idoso , Evolução Molecular , Feminino , Citometria de Fluxo , Genes APC , Humanos , Cariotipagem , Masculino , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico , Filogenia , Reação em Cadeia da Polimerase/métodos , Polimorfismo GenéticoRESUMO
Cystinuria is an aminoaciduria caused by mutations in the genes that encode the two subunits of the amino acid transport system b0,+, responsible for the renal reabsorption of cystine and dibasic amino acids. The clinical symptoms of cystinuria relate to nephrolithiasis, due to the precipitation of cystine in urine. Mutations in SLC3A1, which codes for the heavy subunit rBAT, cause cystinuria type A, whereas mutations in SLC7A9, which encodes the light subunit b0,+AT, cause cystinuria type B. By crossing Slc3a1-/- with Slc7a9-/- mice we generated a type AB cystinuria mouse model to test digenic inheritance of cystinuria. The 9 genotypes obtained have been analyzed at early (2- and 5-months) and late stage (8-months) of the disease. Monitoring the lithiasic phenotype by X-ray, urine amino acid content analysis and protein expression studies have shown that double heterozygous mice (Slc7a9+/-Slc3a1+/-) present lower expression of system b0,+ and higher hyperexcretion of cystine than single heterozygotes (Slc7a9+/-Slc3a1+/+ and Slc7a9+/+Slc3a1+/-) and give rise to lithiasis in 4% of the mice, demonstrating that cystinuria has a digenic inheritance in this mouse model. Moreover in this study it has been demonstrated a genotype/phenotype correlation in type AB cystinuria mouse model providing new insights for further molecular and genetic studies of cystinuria patients.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Cistinúria/genética , Padrões de Herança , Mutação , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animais , Cistinúria/complicações , Cistinúria/metabolismo , Modelos Animais de Doenças , Rim/metabolismo , Rim/patologia , Litíase/etiologia , Litíase/patologia , Masculino , Camundongos , Camundongos Knockout , FenótipoRESUMO
The aim of the study was to test the multicolor fluorescence in situ hybridization technique (M-FISH) in seven multiple myeloma (MM) and eight monoclonal gammopathy (MGUS) patients. None of the eight MGUS patients had chromosomal abnormalities by conventional cytogenetics. In two of these patients structural abnormalities of chromosomes 2, 11 and 19 were found by M-FISH. However, these findings were not confirmed by conventional in situ hybridization. M-FISH only showed numerical chromosomal abnormalities in one out of the three MM cases with a normal karyotype. In the two MM cases with complex karyotype, M-FISH demonstrated the origin of the marker chromosomes. M-FISH is a useful technique to identify the origin of the marker chromosomes in MM. In contrast, MM or MGUS patients with normal karyotypes by conventional cytogenetics did not show structural abnormalities by M-FISH.
Assuntos
Coloração Cromossômica , Cromossomos Humanos/ultraestrutura , Mieloma Múltiplo/genética , Paraproteinemias/genética , Artefatos , Aberrações Cromossômicas , Estudos de Viabilidade , Feminino , Humanos , Cariotipagem , Masculino , Mieloma Múltiplo/patologia , Paraproteinemias/patologia , Sensibilidade e EspecificidadeRESUMO
The cytogenetic and molecular cytogenetic characterization of the human cell line A-431 derived from a vulvar epidermoid carcinoma is presented. A combination of karyotyping, fluorescence in situ hybridization (FISH) with chromosome- and/or region-specific probes, M-FISH, RxFISH, and comparative genomic hybridization (CGH) analysis was used. Six marker chromosomes with rearrangements involving insertions of single or double nucleolar organizing regions (NORs) and/or homogeneously staining regions containing active and overexpressed NORs and regions of centromeric heterochromatin were found: der(6), der(7), der(17), der(21), dic(13;14), and dic(14;18). The chromosomal origin of 14 other marker chromosomes was elucidated. Amplification of the C-MYC oncogene at 8q24 was revealed in two marker chromosomes: dup(8)(q24) and der(15)t(8;15)(q22;p11). Confirming previous reports, amplification of the cyclin D1 gene within an abnormal chromosome 11, that is, der(11)t(7;11)(p15;q21), was also detected. Loss of the TP53 tumor suppressor gene was evidenced over two der(17). Good concordance was found among karyotyping, FISH analysis, and CGH. Although reasons for NOR amplification or ectopic location in the epidermal carcinoma A-431 cell line are not clear yet, our data suggest that these phenomena play a supporting role with regard to other amplified genes. Thus, the A-431 cell line would be an appropriate model to study the different mechanisms involved in human tumorigenesis.