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1.
Future Oncol ; 17(16): 1987-2003, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33682447

RESUMEN

Belantamab mafodotin (belamaf) is a BCMA-targeted antibody-drug conjugate recently approved as monotherapy for adults with relapsed/refractory multiple myeloma who have received ≥4 prior therapies. Belamaf binds to BCMA and eliminates myeloma cells by multimodal mechanisms of action. The cytotoxic and potential immunomodulatory properties of belamaf have led to novel combination studies with other anticancer therapies. Here, we describe the rationale and design of DREAMM-5, an ongoing Phase I/II platform study evaluating the safety and efficacy of belamaf combined with novel agents, including GSK3174998 (OX40 agonist), feladilimab (an ICOS; GSK3359609), nirogacestat (a gamma-secretase inhibitor; PF-03084014) and dostarlimab (a PD-1 blocker) versus belamaf monotherapy for patients with relapsed/refractory multiple myeloma. Clinical trial registration: NCT04126200 (ClinicalTrials.gov).


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígeno de Maduración de Linfocitos B/antagonistas & inhibidores , Mieloma Múltiple/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Receptores OX40/antagonistas & inhibidores , Proyectos de Investigación/normas , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados/administración & dosificación , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Resistencia a Antineoplásicos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Multicéntricos como Asunto , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/patología , Ensayos Clínicos Controlados Aleatorios como Asunto , Tetrahidronaftalenos/administración & dosificación , Valina/administración & dosificación , Valina/análogos & derivados , Adulto Joven
2.
Hum Mol Genet ; 23(19): 5243-50, 2014 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-24858910

RESUMEN

Retinoblastoma is a non-hereditary as well as an inherited pediatric tumor of the developing retina resulting from the inactivation of both copies of the RB1 tumor suppressor gene. Familial retinoblastoma is a highly penetrant genetic disease that usually develops by carrying germline mutations that inactivate one allele of the RB1 gene, leading to multiple retinoblastomas. However, large and complete germline RB1 deletions are associated with low or no tumor risk for reasons that remain unknown. In this study, we define a minimal genomic region associated with this low penetrance. This region encompasses few genes including MED4 a subunit of the mediator complex. We further show that retinoblastoma RB1 -/- cells cannot survive in the absence of MED4, both in vitro and in orthotopic xenograft models in vivo, therefore identifying MED4 as a survival gene in retinoblastoma. We propose that the contiguous loss of the adjacent retinoblastoma gene, MED4, explains the low penetrance in patients with large deletions that include both RB1 and MED4. Our findings also point to another synthetic lethal target in tumors with inactivated RB1 and highlight the importance of collateral damage in carcinogenesis.


Asunto(s)
Eliminación de Gen , Complejo Mediador/genética , Penetrancia , Proteína de Retinoblastoma/genética , Retinoblastoma/genética , Animales , Apoptosis/genética , Muerte Celular/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Hibridación Genómica Comparativa , Femenino , Expresión Génica , Técnicas de Inactivación de Genes , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Linaje , Interferencia de ARN , Retinoblastoma/mortalidad , Retinoblastoma/patología , Ensayo de Tumor de Célula Madre
3.
Clin Cancer Res ; 30(14): 3006-3022, 2024 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-38687588

RESUMEN

PURPOSE: Bispecific antibodies (BsAb) directed against B-cell maturation antigen (teclistamab) or the orphan G protein-coupled receptor GPRC5D (talquetamab) induce deep and durable responses in heavily pretreated patients with multiple myeloma. However, mechanisms underlying primary and acquired resistance remain poorly understood. EXPERIMENTAL DESIGN: The anti-multiple myeloma activity of teclistamab and talquetamab was evaluated in bone marrow (BM) samples from patients with multiple myeloma. T-cell phenotype and function were assessed in BM/peripheral blood samples obtained from patients with multiple myeloma who were treated with these BsAb. RESULTS: In ex vivo killing assays with 41 BM samples from BsAb-naive patients with multiple myeloma, teclistamab- and talquetamab-mediated multiple myeloma lysis was strongly correlated (r = 0.73, P < 0.0001). Both BsAb exhibited poor activity in samples with high regulatory T-cell (Treg) numbers and a low T-cell/multiple myeloma cell ratio. Furthermore, comprehensive phenotyping of BM samples derived from patients treated with teclistamab or talquetamab revealed that high frequencies of PD-1+ CD4+ T cells, CTLA4+ CD4+ T cells, and CD38+ CD4+ T cells were associated with primary resistance. Although this lack of response was linked to a modest increase in the expression of inhibitory receptors, increasing T-cell/multiple myeloma cell ratios by adding extra T cells enhanced sensitivity to BsAb. Further, treatment with BsAb resulted in an increased proportion of T cells expressing exhaustion markers (PD-1, TIGIT, and TIM-3), which was accompanied by reduced T-cell proliferative potential and cytokine secretion, as well as impaired antitumor efficacy in ex vivo experiments. CONCLUSIONS: Primary resistance is characterized by a low T-cell/multiple myeloma cell ratio and Treg-driven immunosuppression, whereas reduced T-cell fitness due to continuous BsAb-mediated T-cell activation may contribute to the development of acquired resistance.


Asunto(s)
Anticuerpos Biespecíficos , Resistencia a Antineoplásicos , Mieloma Múltiple , Mieloma Múltiple/inmunología , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Humanos , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Resistencia a Antineoplásicos/inmunología , Antígeno de Maduración de Linfocitos B/inmunología , Linfocitos T Reguladores/inmunología , Femenino , Masculino , Linfocitos T/inmunología , Linfocitos T/metabolismo , Persona de Mediana Edad , Anciano , Receptores Acoplados a Proteínas G
4.
Mol Cancer Ther ; 20(10): 1941-1955, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34253590

RESUMEN

B-cell maturation antigen (BCMA) is an attractive therapeutic target highly expressed on differentiated plasma cells in multiple myeloma and other B-cell malignancies. GSK2857916 (belantamab mafodotin, BLENREP) is a BCMA-targeting antibody-drug conjugate approved for the treatment of relapsed/refractory multiple myeloma. We report that GSK2857916 induces immunogenic cell death in BCMA-expressing cancer cells and promotes dendritic cell activation in vitro and in vivo GSK2857916 treatment enhances intratumor immune cell infiltration and activation, delays tumor growth, and promotes durable complete regressions in immune-competent mice bearing EL4 lymphoma tumors expressing human BCMA (EL4-hBCMA). Responding mice are immune to rechallenge with EL4 parental and EL4-hBCMA cells, suggesting engagement of an adaptive immune response, immunologic memory, and tumor antigen spreading, which are abrogated upon depletion of endogenous CD8+ T cells. Combinations with OX40/OX86, an immune agonist antibody, significantly enhance antitumor activity and increase durable complete responses, providing a strong rationale for clinical evaluation of GSK2857916 combinations with immunotherapies targeting adaptive immune responses, including T-cell-directed checkpoint modulators.


Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antígeno de Maduración de Linfocitos B/antagonistas & inhibidores , Linfocitos T CD8-positivos/inmunología , Inmunoconjugados/farmacología , Muerte Celular Inmunogénica , Linfoma/tratamiento farmacológico , Mieloma Múltiple/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/química , Apoptosis , Antígeno de Maduración de Linfocitos B/inmunología , Proliferación Celular , Femenino , Humanos , Linfoma/inmunología , Linfoma/metabolismo , Linfoma/patología , Ratones , Ratones Endogámicos C57BL , Mieloma Múltiple/inmunología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Clin Cancer Res ; 27(19): 5195-5212, 2021 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-34321279

RESUMEN

The development of novel agents has transformed the treatment paradigm for multiple myeloma, with minimal residual disease (MRD) negativity now achievable across the entire disease spectrum. Bone marrow-based technologies to assess MRD, including approaches using next-generation flow and next-generation sequencing, have provided real-time clinical tools for the sensitive detection and monitoring of MRD in patients with multiple myeloma. Complementary liquid biopsy-based assays are now quickly progressing with some, such as mass spectrometry methods, being very close to clinical use, while others utilizing nucleic acid-based technologies are still developing and will prove important to further our understanding of the biology of MRD. On the regulatory front, multiple retrospective individual patient and clinical trial level meta-analyses have already shown and will continue to assess the potential of MRD as a surrogate for patient outcome. Given all this progress, it is not surprising that a number of clinicians are now considering using MRD to inform real-world clinical care of patients across the spectrum from smoldering myeloma to relapsed refractory multiple myeloma, with each disease setting presenting key challenges and questions that will need to be addressed through clinical trials. The pace of advances in targeted and immune therapies in multiple myeloma is unprecedented, and novel MRD-driven biomarker strategies are essential to accelerate innovative clinical trials leading to regulatory approval of novel treatments and continued improvement in patient outcomes.


Asunto(s)
Mieloma Múltiple , Médula Ósea , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/tratamiento farmacológico , Neoplasia Residual/diagnóstico , Estudios Retrospectivos
6.
Nature ; 425(6960): 846-51, 2003 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-14520411

RESUMEN

Activation of the Hedgehog (Hh) signalling pathway by sporadic mutations or in familial conditions such as Gorlin's syndrome is associated with tumorigenesis in skin, the cerebellum and skeletal muscle. Here we show that a wide range of digestive tract tumours, including most of those originating in the oesophagus, stomach, biliary tract and pancreas, but not in the colon, display increased Hh pathway activity, which is suppressible by cyclopamine, a Hh pathway antagonist. Cyclopamine also suppresses cell growth in vitro and causes durable regression of xenograft tumours in vivo. Unlike in Gorlin's syndrome tumours, pathway activity and cell growth in these digestive tract tumours are driven by endogenous expression of Hh ligands, as indicated by the presence of Sonic hedgehog and Indian hedgehog transcripts, by the pathway- and growth-inhibitory activity of a Hh-neutralizing antibody, and by the dramatic growth-stimulatory activity of exogenously added Hh ligand. Our results identify a group of common lethal malignancies in which Hh pathway activity, essential for tumour growth, is activated not by mutation but by ligand expression.


Asunto(s)
Neoplasias Gastrointestinales/metabolismo , Neoplasias Gastrointestinales/patología , Regulación Neoplásica de la Expresión Génica , Transducción de Señal , Transactivadores/genética , Transactivadores/metabolismo , Animales , División Celular/efectos de los fármacos , Línea Celular Tumoral , Sistema Digestivo/citología , Sistema Digestivo/efectos de los fármacos , Sistema Digestivo/metabolismo , Sistema Digestivo/patología , Neoplasias Gastrointestinales/tratamiento farmacológico , Neoplasias Gastrointestinales/genética , Eliminación de Gen , Proteínas Hedgehog , Humanos , Ligandos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Desnudos , Mutación , Trasplante de Neoplasias , Receptores Patched , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Receptores de Superficie Celular , Transducción de Señal/efectos de los fármacos , Transactivadores/antagonistas & inhibidores , Trasplante Heterólogo , Alcaloides de Veratrum/farmacología , Alcaloides de Veratrum/uso terapéutico
7.
Sci Rep ; 10(1): 22155, 2020 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-33335114

RESUMEN

Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development. Here, we evaluate the effect of type I PRMT inhibition on arginine methylation in normal human peripheral blood mononuclear cells and utilize a broad proteomic approach to identify type I PRMT substrates. This work identified heterogenous nuclear ribonucleoprotein A1 (hnRNP-A1) as a pharmacodynamic biomarker of type I PRMT inhibition. Utilizing targeted mass spectrometry (MS), methods were developed to detect and quantitate changes in methylation of specific arginine residues on hnRNP-A1. This resulted in the development and validation of novel MS and immune assays useful for the assessment of GSK3368715 induced pharmacodynamic effects in blood and tumors that can be applied to GSK3368715 clinical trials.


Asunto(s)
Antineoplásicos/farmacocinética , Biomarcadores , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteínas Represoras/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/farmacocinética , Antineoplásicos Inmunológicos/farmacología , Arginina/metabolismo , Células Cultivadas , Cromatografía Liquida , Monitoreo de Drogas , Activación Enzimática , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ribonucleoproteína Nuclear Heterogénea A1/sangre , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Espectrometría de Masas , Metilación , Ratones , Terapia Molecular Dirigida , Neoplasias/sangre , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Represoras/genética , Especificidad por Sustrato
8.
Sci Rep ; 8(1): 9711, 2018 06 26.
Artículo en Inglés | MEDLINE | ID: mdl-29946150

RESUMEN

Evasion of the potent tumour suppressor activity of p53 is one of the hurdles that must be overcome for cancer cells to escape normal regulation of cellular proliferation and survival. In addition to frequent loss of function mutations, p53 wild-type activity can also be suppressed post-translationally through several mechanisms, including the activity of PRMT5. Here we describe broad anti-proliferative activity of potent, selective, reversible inhibitors of protein arginine methyltransferase 5 (PRMT5) including GSK3326595 in human cancer cell lines representing both hematologic and solid malignancies. Interestingly, PRMT5 inhibition activates the p53 pathway via the induction of alternative splicing of MDM4. The MDM4 isoform switch and subsequent p53 activation are critical determinants of the response to PRMT5 inhibition suggesting that the integrity of the p53-MDM4 regulatory axis defines a subset of patients that could benefit from treatment with GSK3326595.


Asunto(s)
Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Empalme del ARN/genética , Proteína p53 Supresora de Tumor/metabolismo , Empalme Alternativo/genética , Antineoplásicos , Arginina/análogos & derivados , Arginina/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Nucleares snRNP/metabolismo
9.
Novartis Found Symp ; 264: 51-58; discussion 58-62, 227-30, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-15773747

RESUMEN

Loss of emerin, a nuclear membrane protein, causes Emery-Dreifuss muscular dystrophy (EDMD), characterized by muscle weakening, contractures of major tendons and potentially lethal cardiac conduction system defects. Emerin has a LEM-domain and therefore binds barrier-to-autointegration factor (BAF), a conserved chromatin protein essential for cell division. BAF recruits emerin to chromatin and regulates higher-order chromatin structure during nuclear assembly. Emerin also binds filaments formed by A-type lamins, mutations in which also cause EDMD. Other partners for emerin include nesprin-1alpha and transcriptional regulators such as germ cell-less (GCL). The binding affinities of these partners range from 4nM (nesprin-1alpha) to 200 nM (BAF), and are physiologically significant. Biochemical studies therefore provide a valid means to predict the properties of emerin-lamin complexes in vivo. Emerin and lamin A together form stable complexes with either BAF or GCL in vitro. BAF, however, competes with GCL for binding to emerin in vitro. These and additional partners, notably actin and nuclear myosin II, suggest disease-relevant roles for emerin in gene regulation and the mechanical interity of the nucleus.


Asunto(s)
Actinas/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas de la Membrana/fisiología , Distrofias Musculares/metabolismo , Membrana Nuclear/metabolismo , Timopoyetinas/fisiología , Animales , Humanos , Proteínas Nucleares
10.
Mol Oncol ; 9(3): 657-74, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25497280

RESUMEN

BACKGROUND: Breast cancer is a heterogeneous disease with different molecular subtypes that have varying responses to therapy. An ongoing challenge in breast cancer research is to distinguish high-risk patients from good prognosis patients. This is particularly difficult in the low-grade, ER-positive luminal A tumors, where robust diagnostic tools to aid clinical treatment decisions are lacking. Recent data implicating chromatin regulators in cancer initiation and progression offers a promising avenue to develop new tools to help guide clinical decisions. METHODS: Here we exploit a published transcriptome dataset and an independent validation cohort to correlate the mRNA expression of selected chromatin regulators with respect to the four intrinsic breast cancer molecular subtypes. We then perform univariate and multivariate analyses to compare the prognostic value of a panel of chromatin regulators to Ki67, a currently utilized proliferation marker. RESULTS: Unsupervised hierarchical clustering revealed a gene cluster containing several histone chaperones and histone variants highly-expressed in the proliferative subtypes (basal-like, HER2-positive, luminal B) but not in the luminal A subtype. Several chromatin regulators, including the histone chaperones CAF-1 (subunits p150 and p60), ASF1b, and HJURP, and the centromeric histone variant CENP-A, associated with local and metastatic relapse and poor patient outcome. Importantly, we find that HJURP can discriminate favorable and unfavorable outcome within the luminal A subtype, outperforming the currently utilized proliferation marker Ki67, as an independent prognostic marker for luminal A patients. CONCLUSIONS: The integration of chromatin regulators as clinical biomarkers, in particular the histone chaperone HJURP, will help guide patient substratification and treatment options for low-risk luminal A breast carcinoma patients.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , Autoantígenos/metabolismo , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteína A Centromérica , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Análisis por Conglomerados , Estudios de Cohortes , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Componente 2 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Análisis Multivariante , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Modelos de Riesgos Proporcionales , ARN Mensajero/genética , ARN Mensajero/metabolismo , Resultado del Tratamiento
11.
Cell Rep ; 8(1): 190-203, 2014 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-25001279

RESUMEN

Centromeres, epigenetically defined by the presence of the histone H3 variant CenH3, are essential for ensuring proper chromosome segregation. In mammals, centromeric CenH3(CENP-A) deposition requires its dedicated chaperone HJURP and occurs during telophase/early G1. We find that the cell-cycle-dependent recruitment of HJURP to centromeres depends on its timely phosphorylation controlled via cyclin-dependent kinases. A nonphosphorylatable HJURP mutant localizes prematurely to centromeres in S and G2 phase. This unregulated targeting causes a premature loading of CenH3(CENP-A) at centromeres, and cell-cycle delays ensue. Once recruited to centromeres, HJURP functions to promote CenH3(CENP-A) deposition by a mechanism involving a unique DNA-binding domain. With our findings, we propose a model wherein (1) the phosphorylation state of HJURP controls its centromeric recruitment in a cell-cycle-dependent manner, and (2) HJURP binding to DNA is a mechanistic determinant in CenH3(CENP-A) loading.


Asunto(s)
Autoantígenos/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Ciclo Celular , Línea Celular Tumoral , Proteína A Centromérica , Proteínas de Unión al ADN/química , Humanos , Datos de Secuencia Molecular , Fosforilación , Unión Proteica
12.
Curr Opin Cell Biol ; 23(3): 266-76, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21470840

RESUMEN

Classical heterochromatin chromosomal landmarks, such as centromeres and telomeres, are characterized by specific chromatin signatures. Among these, the incorporation of histone variants has recently emerged as an important feature. Using the centromere as a paradigm, we consider the role of histone variant dynamics in locus-specific chromatin organization. We describe the distinct location and dynamics of CenH3, H3.3, and H2AZ at the centromere during the cell cycle. This leads us to present the current view concerning modes of incorporation at this chromosomal landmark. Finally, we highlight the importance of histone variants in the crosstalk between centric and pericentric domains for maintaining centromere identity.


Asunto(s)
Centrómero/metabolismo , Cromosomas/metabolismo , Histonas/metabolismo , Animales , Ciclo Celular , Centrómero/química , Cromosomas/química , Heterocromatina , Histonas/química , Histonas/genética , Humanos
13.
Nucleus ; 2(6): 580-90, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22127260

RESUMEN

Defects in the nuclear envelope or nuclear 'lamina' networks cause disease and can perturb histone posttranslational (epigenetic) regulation. Barrier-to-Autointegration Factor (BAF) is an essential but enigmatic lamina component that binds lamins, LEM-domain proteins, DNA and histone H3 directly. We report that BAF copurified with nuclease-digested mononucleosomes and associated with modified histones in vivo. BAF overexpression significantly reduced global histone H3 acetylation by 18%. In cells that stably overexpressed BAF 3-fold, silencing mark H3-K27-Me1/3 and active marks H4-K16-Ac and H4-Ac5 decreased significantly. Significant increases were also seen for silencing mark H3-K9-Me3, active marks H3-K4-Me2, H3-K9/K14-Ac and H4-K5-Ac and a mark (H3-K79-Me2) associated with both active and silent chromatin. Other increases (H3-S10-P, H3-S28-P and silencing mark H3-K9-Me2) did not reach statistical significance. BAF overexpression also significantly influenced cell cycle distribution. Moreover, BAF associated in vivo with SET/I2PP2A (protein phosphatase 2A inhibitor; blocks H3 dephosphorylation) and G9a (H3-K9 methyltransferase), but showed no detectable association with HDAC1 or HATs. These findings reveal BAF as a novel epigenetic regulator and are discussed in relation to BAF deficiency phenotypes, which include a hereditary progeria syndrome and loss of pluripotency in embryonic stem cells.


Asunto(s)
Ciclo Celular , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Histonas/metabolismo , Lámina Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Progeria/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , Animales , Proteínas de Unión al ADN/genética , Células HeLa , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histonas/genética , Humanos , Laminas/genética , Laminas/metabolismo , Lámina Nuclear/genética , Proteínas Nucleares/genética , Progeria/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Xenopus laevis
14.
Nat Genet ; 43(3): 220-7, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21317888

RESUMEN

HP1 enrichment at pericentric heterochromatin is considered important for centromere function. Although HP1 binding to H3K9me3 can explain its accumulation at pericentric heterochromatin, how it is initially targeted there remains unclear. Here, in mouse cells, we reveal the presence of long nuclear noncoding transcripts corresponding to major satellite repeats at the periphery of pericentric heterochromatin. Furthermore, we find that major transcripts in the forward orientation specifically associate with SUMO-modified HP1 proteins. We identified this modification as SUMO-1 and mapped it in the hinge domain of HP1α. Notably, the hinge domain and its SUMOylation proved critical to promote the initial targeting of HP1α to pericentric domains using de novo localization assays, whereas they are dispensable for maintenance of HP1 domains. We propose that SUMO-HP1, through a specific association with major forward transcript, is guided at the pericentric heterochromatin domain to seed further HP1 localization.


Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Heterocromatina/metabolismo , Sumoilación , Animales , Centrómero/metabolismo , Homólogo de la Proteína Chromobox 5 , Ratones , Estructura Terciaria de Proteína , ARN , Secuencias Repetitivas de Ácidos Nucleicos
15.
PLoS One ; 4(9): e7050, 2009 Sep 16.
Artículo en Inglés | MEDLINE | ID: mdl-19759913

RESUMEN

Nuclear lamin filaments and associated proteins form a nucleoskeletal ("lamina") network required for transcription, replication, chromatin organization and epigenetic regulation in metazoans. Lamina defects cause human disease ("laminopathies") and are linked to aging. Barrier-to-autointegration factor (BAF) is a mobile and essential component of the nuclear lamina that binds directly to histones, lamins and LEM-domain proteins, including the inner nuclear membrane protein emerin, and has roles in chromatin structure, mitosis and gene regulation. To understand BAF's mechanisms of action, BAF associated proteins were affinity-purified from HeLa cell nuclear lysates using BAF-conjugated beads, and identified by tandem mass spectrometry or independently identified and quantified using the iTRAQ method. We recovered A- and B-type lamins and core histones, all known to bind BAF directly, plus four human transcription factors (Requiem, NonO, p15, LEDGF), disease-linked proteins (e.g., Huntingtin, Treacle) and several proteins and enzymes that regulate chromatin. Association with endogenous BAF was independently validated by co-immunoprecipitation from HeLa cells for seven candidates including Requiem, poly(ADP-ribose) polymerase 1 (PARP1), retinoblastoma binding protein 4 (RBBP4), damage-specific DNA binding protein 1 (DDB1) and DDB2. Interestingly, endogenous BAF and emerin each associated with DDB2 and CUL4A in a UV- and time-dependent manner, suggesting BAF and emerin have dynamic roles in genome integrity and might help couple DNA damage responses to the nuclear lamina network. We conclude this proteome is a rich source of candidate partners for BAF and potentially also A- and B-type lamins, which may reveal how chromatin regulation and genome integrity are linked to nuclear structure.


Asunto(s)
Cromatina/química , Proteínas de la Membrana/metabolismo , Lámina Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteoma/química , Secuencia de Aminoácidos , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HeLa , Histonas/química , Humanos , Datos de Secuencia Molecular , Poli(ADP-Ribosa) Polimerasas/química , Estructura Terciaria de Proteína , Proteína 4 de Unión a Retinoblastoma/metabolismo , Homología de Secuencia de Aminoácido
16.
J Biol Chem ; 280(51): 42252-62, 2005 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-16203725

RESUMEN

Barrier to autointegration factor (BAF) is an essential conserved double-stranded DNA-binding protein in metazoans. BAF binds directly to LEM domain nuclear proteins (e.g. LAP2, Emerin, and MAN1), lamin A, homeodomain transcription factors, and human immunodeficiency virus type 1-encoded proteins. BAF influences higher order chromatin structure and is required to assemble nuclei. BAF also facilitates retroviral preintegration complex insertion into target DNA in vitro, through unknown mechanisms. We report that BAF binds directly and selectively to linker histone H1.1 (among three subtypes tested) and core histone H3 with affinities of approximately 700 nm and approximately 100-200 nm, respectively, in vitro and in vivo. Mutations at the bottom and top surfaces of the BAF dimer disrupted or enhanced, respectively, this binding and affected H1 and H3 similarly. Biochemical studies showed that C-terminal residues 108-215 of histone H1.1 and the N-terminal tail plus helix alphaN in the core of histone H3.1 were each necessary and sufficient to bind BAF. Based on its interactions with histones and DNA, we propose BAF might bind nucleosomes in vivo.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Proteínas de Unión al ADN/genética , Inmunoprecipitación , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Unión Proteica , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transfección
17.
Blood ; 105(3): 1003-9, 2005 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-15454491

RESUMEN

DNA damage activates the monoubiquitination of the Fanconi anemia (FA) protein, FANCD2, resulting in the assembly of FANCD2 nuclear foci. In the current study, we characterize structural features of FANCD2 required for this intranuclear translocation. We have previously identified 2 normal mRNA splice variants of FANCD2, one containing exon 44 sequence at the 3' end (FANCD2-44) and one containing exon 43 sequence (FANCD2-43). The 2 predicted FANCD2 proteins differ in their carboxy terminal 24 amino acids. In stably transfected FANCD2(-/-) fibroblasts, FANCD2-44 and FANCD2-43 proteins were monoubiquitinated on K561. Only FANCD2-44 corrected the mitomycin C (MMC) sensitivity of the transfected cells. We find that monoubiquitinated FANCD2-44 was translocated from the soluble nuclear compartment into chromatin. A mutant form of FANCD2-44 (FANCD2-K561R) was not monoubiquitinated and failed to bind chromatin. A truncated FANCD2 protein (Exon44-T), lacking the carboxy terminal 24 amino acids encoded by exon 44 but retaining K561, and another mutant FANCD2 protein, with a single amino acid substitution at a conserved residue within the C-terminal 24 amino acids (D1428A), were monoubiquitinated. Both mutants were targeted to chromatin but failed to correct MMC sensitivity. Taken together, our results indicate that monoubiquitination of FANCD2 regulates chromatin binding and that D1428 within the carboxy terminal acidic sequence encoded by exon 44 is independently required for functional complementation of FA-D2 cells. We hypothesize that the carboxy terminus of FANCD2-44 plays a critical role in sensing or repairing DNA damage.


Asunto(s)
Cromatina/genética , Anemia de Fanconi/genética , Proteínas Nucleares/genética , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Daño del ADN/genética , Reparación del ADN/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi , Variación Genética , Células HeLa , Humanos , Microscopía Fluorescente , Mitomicina/farmacología , Unión Proteica , Empalme del ARN , ARN Mensajero/genética , Eliminación de Secuencia , Ubiquitina/metabolismo
18.
Blood ; 100(13): 4649-54, 2002 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-12393398

RESUMEN

Fanconi anemia (FA) is an autosomal recessive chromosomal instability syndrome characterized by congenital abnormalities, progressive bone marrow failure, and cancer predisposition. Although patients with FA are candidates for bone marrow transplantation or gene therapy, their phenotypic heterogeneity can delay or obscure diagnosis. The current diagnostic test for FA consists of cytogenetic quantitation of chromosomal breakage in response to diepoxybutane (DEB) or mitomycin C (MMC). Recent studies have elucidated a biochemical pathway for Fanconi anemia that culminates in the monoubiquitination of the FANCD2 protein. In the current study, we develop a new rapid diagnostic and subtyping FA assay amenable for screening broad populations at risk of FA. Primary lymphocytes were assayed for FANCD2 monoubiquitination by immunoblot. The absence of the monoubiquitinated FANCD2 isoform correlated with the diagnosis of FA by DEB testing in 11 known patients with FA, 37 patients referred for possible FA, and 29 healthy control subjects. Monoubiquitination of FANCD2 was normal in other bone marrow failure syndromes and chromosomal breakage syndromes. A combination of retroviral gene transfer and FANCD2 immunoblotting provides a rapid subtyping assay for patients newly diagnosed with FA. These new FA screening assays would allow efficient testing of broad populations at risk.


Asunto(s)
Western Blotting , Anemia de Fanconi/genética , Pruebas Genéticas , Linfocitos/química , Proteínas Nucleares/sangre , Procesamiento Proteico-Postraduccional , Enfermedades de la Médula Ósea/sangre , Línea Celular Transformada , Rotura Cromosómica , Cromosomas Humanos/efectos de los fármacos , ADN Complementario/genética , Compuestos Epoxi/farmacología , Anemia de Fanconi/sangre , Anemia de Fanconi/clasificación , Anemia de Fanconi/diagnóstico , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi , Heterogeneidad Genética , Vectores Genéticos/genética , Humanos , Microscopía Fluorescente , Mitomicina/farmacología , Proteínas Nucleares/metabolismo , Retroviridae/genética , Transfección , Ubiquitina/metabolismo
19.
Acta pediátr. Méx ; 6(4): 173-6, oct.-dic. 1985. tab
Artículo en Español | LILACS | ID: lil-27815

RESUMEN

Se presenta la experiencia con 17 niños en quienes se implantó una prótesis testicular por diversas causas y la repercusión psicológica de esta intervención que tienen los pacientes, sobre todo los mayores de 12 años y los padres


Asunto(s)
Lactante , Humanos , Trastornos de la Personalidad/etiología , Prótesis e Implantes , Testículo/anomalías
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