RESUMO
Diamond Blackfan anemia (DBA), a syndrome primarily characterized by anemia and physical abnormalities, is one among a group of related inherited bone marrow failure syndromes (IBMFS) which share overlapping clinical features. Heterozygous mutations or single-copy deletions have been identified in 12 ribosomal protein genes in approximately 60% of DBA cases, with the genetic etiology unexplained in most remaining patients. Unlike many IBMFS, for which functional screening assays complement clinical and genetic findings, suspected DBA in the absence of typical alterations of the known genes must frequently be diagnosed after exclusion of other IBMFS. We report here a novel deletion in a child that presented such a diagnostic challenge and prompted development of a novel functional assay that can assist in the diagnosis of a significant fraction of patients with DBA. The ribosomal proteins affected in DBA are required for pre-rRNA processing, a process which can be interrogated to monitor steps in the maturation of 40S and 60S ribosomal subunits. In contrast to prior methods used to assess pre-rRNA processing, the assay reported here, based on capillary electrophoresis measurement of the maturation of rRNA in pre-60S ribosomal subunits, would be readily amenable to use in diagnostic laboratories. In addition to utility as a diagnostic tool, we applied this technique to gene discovery in DBA, resulting in the identification of RPL31 as a novel DBA gene.
Assuntos
Precursores de RNA , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico , Proteínas Ribossômicas , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/metabolismo , Feminino , Humanos , Lactente , Células K562 , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismoRESUMO
Shwachman Diamond syndrome (SDS) is an inherited bone marrow failure syndrome typically characterized by neutropenia, exocrine pancreas dysfunction, metaphyseal chondrodysplasia, and predisposition to myelodysplastic syndrome and leukemia. SBDS, the gene affected in most cases of SDS, encodes a protein known to influence many cellular processes including ribosome biogenesis, mitotic spindle assembly, chemotaxis, and the regulation of reactive oxygen species production. The best characterized role for the SBDS protein is in the production of functional 60S ribosomal subunits. Given that a reduction in functional 60S subunits could impact on the translational output of cells depleted of SBDS we analyzed protein synthesis in yeast cells lacking SDO1, the ortholog of SBDS. Cells lacking SDO1 selectively increased the synthesis of POR1, the ortholog of mammalian VDAC1 a major anion channel of the mitochondrial outer membrane. Further studies revealed the cells lacking SDO1 were compromised in growth on non-fermentable carbon sources suggesting mitochondrial function was impaired. These observations prompted us to examine mitochondrial function in human cells where SBDS expression was reduced. Our studies indicate that reduced expression of SBDS decreases mitochondrial membrane potential and oxygen consumption and increases the production of reactive oxygen species. These studies indicate that mitochondrial function is also perturbed in cells expressing reduced amounts of SBDS and indicate that disruption of mitochondrial function may also contribute to SDS pathophysiology.
Assuntos
Doenças da Medula Óssea/metabolismo , Doenças da Medula Óssea/patologia , Insuficiência Pancreática Exócrina/metabolismo , Insuficiência Pancreática Exócrina/patologia , Lipomatose/metabolismo , Lipomatose/patologia , Mitocôndrias/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/metabolismo , Carbono/farmacologia , Linhagem Celular , Fermentação/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas/metabolismo , Proteômica , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Síndrome de Shwachman-DiamondRESUMO
Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 down-regulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNA interference that allows an inducible and graded down-regulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene.
Assuntos
Anemia de Diamond-Blackfan/genética , Modelos Animais de Doenças , Hemoglobinúria Paroxística/genética , Camundongos Transgênicos , Proteínas Ribossômicas/genética , Anemia Aplástica , Anemia de Diamond-Blackfan/patologia , Anemia de Diamond-Blackfan/fisiopatologia , Anemia Macrocítica/genética , Anemia Macrocítica/patologia , Anemia Macrocítica/fisiopatologia , Animais , Apoptose/fisiologia , Doenças da Medula Óssea , Transtornos da Insuficiência da Medula Óssea , Transplante de Medula Óssea , Divisão Celular/fisiologia , Células Cultivadas , Expressão Gênica/fisiologia , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/fisiologia , Hemoglobinúria Paroxística/patologia , Hemoglobinúria Paroxística/fisiopatologia , Leucopenia/genética , Leucopenia/patologia , Leucopenia/fisiopatologia , Camundongos , Fenótipo , Contagem de Plaquetas , RNA Interferente Pequeno/farmacologia , Proteínas Ribossômicas/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Shwachman-Diamond syndrome (SDS), associated with SBDS mutations, is characterized by pancreatic exocrine dysfunction and marrow failure. Sdo1, the yeast ortholog of SBDS, is implicated in maturation of the 60S ribosomal subunit, with delayed export of 60S-like particles from the nucleoplasm when depleted. Sdo1 is needed for release of the anti-subunit association factor Tif6 from 60S subunits, and Tif6 may not be recycled to the nucleus when Sdo1 is absent. METHODS: To clarify the role of SBDS in human ribosome function, TF-1 erythroleukemia and A549 lung carcinoma cells were transfected with vectors expressing RNAi against SBDS. RESULTS: Growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to controls. To analyze the effect of SBDS on 60S subunit maturation in A549 cells, subunit localization was assessed by transfection with a vector expressing a fusion between human RPL29 and GFP: we found a higher percentage of SBDS-depleted cells with nuclear localization of 60S subunits. Polysome analysis of TF-1 knockdown cells showed a decrease in free 60S and 80S subunits. We also analyzed the levels of eIF6 (human ortholog of Tif6) following near-complete knockdown of SBDS in TF-1 cells and found an approximately 20% increase in the amount of eIF6 associated with the 60S subunit. CONCLUSIONS: We conclude that knockdown of SBDS leads to growth inhibition and defects in ribosome maturation, suggesting a role for wild-type SBDS in nuclear export of pre-60S subunits. Furthermore, knockdown of SBDS may interfere with eIF6 recycling.
Assuntos
Hematopoese/fisiologia , Proteínas/metabolismo , Ribossomos/fisiologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , Proteínas/genética , RNA Interferente Pequeno , TransfecçãoRESUMO
The RASSF1A tumor suppressor binds and activates proapoptotic MST kinases. The Salvador adaptor protein couples MST kinases to the LATS kinases to form the hippo pathway. Upon activation by RASSF1A, LATS1 phosphorylates the transcriptional regulator YAP, which binds to p73 and activates its proapoptotic effects. However, although serving as an adaptor for MST and LATS, Salvador can also bind RASSF1A. The functional role of the RASSF1A/Salvador interaction is unclear. Although Salvador is a novel tumor suppressor in Drosophila and mice, its role in human systems remains largely unknown. Here we show that Salvador promotes apoptosis in human cells and that Salvador inactivation deregulates the cell cycle and enhances the transformed phenotype. Moreover, we show that although the salvador gene is seldom mutated or epigenetically inactivated in human cancers, it is frequently down-regulated posttranscriptionally. Surprisingly, we also find that although RASSF1A requires the presence of Salvador for full apoptotic activity and to activate p73, this effect does not require a direct interaction of RASSF1A with MST kinases or the activation of the hippo pathway. Thus, we confirm a role for Salvador as a human tumor suppressor and RASSF1A effector and show that Salvador allows RASSF1A to modulate p73 independently of the hippo pathway.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster , Células HEK293 , Humanos , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação/fisiologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/genéticaRESUMO
In this issue of Blood, Devlin and colleagues use a new strategy to create a mouse model for the inherited bone marrow failure syndrome, DBA.The result, while recapitulating certain aspects of the disease and representing a positive step forward, also demonstrates that significant hurdles remain in faithfully creating a mammalian model for DBA.
RESUMO
BACKGROUND: Treatment options are limited for large, unresectable brain metastases. OBJECTIVE: To report a single institution series of staged stereotactic radiosurgery (SRS) that allows for tumor response between treatments in order to optimize the therapeutic ratio. METHODS: Patients were treated with staged SRS separated by 1 mo with a median dose at first SRS of 15 Gy (range 10-21 Gy) and a median dose at second SRS of 14 Gy (range 10-18 Gy). Overall survival was evaluated using the Kaplan-Meier method. Cumulative incidences were estimated for neurological death, radiation necrosis, local failure (marginal or central), and distant brain failure. Absolute cumulative dose-volume histogram was created for each treated lesion. Logistic regression and competing risks regression were performed for each discrete dose received by a certain volume. RESULTS: Thirty-three patients with 39 lesions were treated with staged radiosurgery. Overall survival at 6 and 12 mo was 65.0% and 60.0%, respectively. Cumulative incidence of local failure at 6 and 12 mo was 3.2% and 13.3%, respectively. Of the patients who received staged therapy, 4 of 33 experienced local failure. Radiation necrosis was seen in 4 of 39 lesions. Two of 33 patients experienced a Radiation Therapy Oncology Group toxicity grade > 2 (2 patients had grade 4 toxicities). Dosimetric analysis revealed that dose (Gy) received by volume of brain (ie, VDose(Gy)) was associated with radiation necrosis, including the range V44.5Gy to V87.8Gy. CONCLUSION: Staged radiosurgery is a safe and effective option for large, unresectable brain metastases. Prospective studies are required to validate the findings in this study.
Assuntos
Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/cirurgia , Metástase Neoplásica/terapia , Radiocirurgia/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/mortalidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica/patologia , Radiocirurgia/mortalidade , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Introduction The roles of early whole brain radiotherapy (WBRT) and upfront stereotactic radiosurgery (SRS) alone in the treatment of melanoma patients with brain metastasis remain uncertain. We investigated the volumetric kinetics of brain metastasis development and associations with clinical outcomes for melanoma patients who received upfront SRS alone. Methods Volumetric brain metastasis velocity (vBMV) was defined as the volume of new intracranial disease at the time of distant brain failure (DBF) for the first DBF (DBF1) and second DBF (DBF2) averaged over the time since initial or most recent SRS. Non-volumetric brain metastasis velocity (BMV) was calculated for comparison. Results Median overall survival (OS) for all patients was 7.7 months. Increasing vBMVDBF1 was associated with worsened OS (hazard ratio (HR): 1.10, confidence interval (CI): 1.02 - 1.18, p = .01). Non-volumetric BMVDBF1 was not predictive of OS after DBF1 (HR: 1.00, CI: 0.97 - 1.02, p = .77). Cumulative incidence of DBF2 at three months after DBF1 was 50.0% for vBMVDBF1 > 4 cc/yr versus (vs) 15.1% for vBMVDBF1 ≤ 4 cc/yr, (Gray's p-value = .02). Cumulative incidence of salvage WBRT at three months after DBF1 was 50.0% for vBMVDBF1 > 4 cc/yr vs 2.3% for vBMVDBF1 ≤ 4 cc/yr (Gray's p-value < .001). Conclusion In melanoma patients with brain metastasis, volumetric BMV was predictive of survival, shorter time to second DBF, and the need for salvage WBRT. Non-volumetric BMV, however, did not predict for these outcomes, suggesting that vBMV is a stronger predictor in melanoma.
RESUMO
Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome caused by ribosomal protein haploinsufficiency. DBA exhibits marked phenotypic variability, commonly presenting with erythroid hypoplasia, less consistently with non-erythroid features. The p53 pathway, activated by abortive ribosome assembly, is hypothesized to contribute to the erythroid failure of DBA. We studied murine embryonic stem (ES) cell lines harboring a gene trap mutation in a ribosomal protein gene, either Rps19 or Rpl5. Both mutants exhibited ribosomal protein haploinsufficiency and polysome defects. Rps19 mutant ES cells showed significant increase in p53 protein expression, however, there was no similar increase in the Rpl5 mutant cells. Embryoid body formation was diminished in both mutants but nonspecifically rescued by knockdown of p53. When embryoid bodies were further differentiated to primitive erythroid colonies, both mutants exhibited a marked reduction in colony formation, which was again nonspecifically rescued by p53 inhibition. Cell cycle analyses were normal in Rps19 mutant ES cells, but there was a significant delay in the G2/M phase in the Rpl5 mutant cells, which was unaffected by p53 knockdown. Concordantly, Rpl5 mutant ES cells had a more pronounced growth defect in liquid culture compared to the Rps19 mutant cells. We conclude that the defects in our RPS19 and RPL5 haploinsufficient mouse ES cells are not adequately explained by p53 stabilization, as p53 knockdown appears to increase the growth and differentiation potential of both parental and mutant cells. Our studies demonstrate that gene trap mouse ES cells are useful tools to study the pathogenesis of DBA.
Assuntos
Anemia de Diamond-Blackfan/metabolismo , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Células-Tronco Embrionárias/fisiologia , Células Eritroides/citologia , Proteínas Ribossômicas/genética , Animais , Western Blotting , Ciclo Celular/fisiologia , Primers do DNA/genética , Haploinsuficiência , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Members of the tumor necrosis factor superfamily of receptors induce apoptosis by recruiting adaptor molecules through death domain interactions. The central adaptor molecule for these receptors is the death domain-containing protein Fas-associated death domain (FADD). FADD binds a death domain on a receptor or additional adaptor and recruits caspases to the activated receptor. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signals apoptosis through two receptors, DR4 and DR5. Although there is much interest in TRAIL, the mechanism by which FADD is recruited to the TRAIL receptors is not clear. Using a reverse two-hybrid system we previously identified mutations in the death effector domain of FADD that prevented binding to Fas/CD95. Here we show that these mutations also prevent binding to DR5. FADD-deficient Jurkat cells stably expressing these FADD mutations did not transduce TRAIL or Fas/CD95 signaling. Second site compensating mutations that restore binding to and signaling through Fas/CD95 and DR5 were also in the death effector domain. We conclude that in contrast to current models where the death domain of FADD functions independently of the death effector domain, the death effector domain of FADD comes into direct contact with both TRAIL and Fas/CD95 receptors.