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1.
Stem Cells ; 35(6): 1614-1623, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28276606

RESUMEN

The imitation switch nuclear ATPase Smarca5 (Snf2h) is one of the most conserved chromatin remodeling factors. It exists in a variety of oligosubunit complexes that move DNA with respect to the histone octamer to generate regularly spaced nucleosomal arrays. Smarca5 interacts with different accessory proteins and represents a molecular motor for DNA replication, repair, and transcription. We deleted Smarca5 at the onset of definitive hematopoiesis (Vav1-iCre) and observed that animals die during late fetal development due to anemia. Hematopoietic stem and progenitor cells accumulated but their maturation toward erythroid and myeloid lineages was inhibited. Proerythroblasts were dysplastic while basophilic erythroblasts were blocked in G2/M and depleted. Smarca5 deficiency led to increased p53 levels, its activation at two residues, one associated with DNA damage (S15Ph °s ) second with CBP/p300 (K376Ac ), and finally activation of the p53 targets. We also deleted Smarca5 in committed erythroid cells (Epor-iCre) and observed that animals were anemic postnatally. Furthermore, 4-hydroxytamoxifen-mediated deletion of Smarca5 in the ex vivo cultures confirmed its requirement for erythroid cell proliferation. Thus, Smarca5 plays indispensable roles during early hematopoiesis and erythropoiesis. Stem Cells 2017;35:1614-1623.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Diferenciación Celular , Proteínas Cromosómicas no Histona/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Adenosina Trifosfatasas/deficiencia , Anemia/patología , Animales , Ciclo Celular , Proliferación Celular , Proteínas Cromosómicas no Histona/deficiencia , Daño del ADN/genética , Células Eritroides/citología , Eritropoyesis , Eliminación de Gen , Genotipo , Hematopoyesis , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína p53 Supresora de Tumor/metabolismo
2.
Int J Mol Sci ; 18(10)2017 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-29036928

RESUMEN

Primary cutaneous T-cell lymphomas (CTCL) affect the skin and tend to transform and spread. CTCL involves primarily the Mycosis fungoides (MF) and more aggressive Sezary syndrome (SS). Oncogenic microRNAs (miRs) are stable epigenetic inhibitors often deregulated in the tumour and detectable as biomarkers in non-cellular fractions of peripheral blood. The tumour-specific expression of miR-155, miR-203, and miR-205 was shown to correctly diagnose CTCL. We herein asked whether these microRNAs can be used as plasma biomarkers for clinical CTCL monitoring. Patients with CTCL (n = 10) and controls with non-malignant conditions (n = 11) repeatedly donated plasma samples every ca. five months. MicroRNAs were detected in the plasma samples by specifically-primed RT-PCR followed by multivariate analyses of the miR expression dynamics. We herein established the plasma miR-classifier for detecting CTCL based on the miR-155 upregulation and miR-203/miR-205 downregulation with 100% specificity and 94% sensitivity. The 3-miR-score in the consecutive samples coincided with the clinical outcome of MF and SS patients such as the therapy response or changes in the clinical stage or tumor size. Quantitation of the selected microRNAs in plasma is a specific and straightforward approach for evaluating CTCL outcome representing, thus, a valuable tool for CTCL diagnostics and therapy response monitoring.


Asunto(s)
Biomarcadores de Tumor , MicroARN Circulante , Linfoma Cutáneo de Células T/genética , MicroARNs/genética , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica , Humanos , Biopsia Líquida , Linfoma Cutáneo de Células T/sangre , Linfoma Cutáneo de Células T/diagnóstico , Linfoma Cutáneo de Células T/terapia , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Piel/patología , Resultado del Tratamiento
3.
Int J Cancer ; 139(10): 2252-60, 2016 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-27407063

RESUMEN

Richter syndrome represents the transformation of the chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, most frequently the diffuse large B-cell lymphoma (DLBCL). In this report we describe a patient with CLL, who developed a clonally-related pleomorphic highly-aggressive mantle cell lymphoma (MCL) after five cycles of a fludarabine-based second-line therapy for the first relapse of CLL. Molecular cytogenetic methods together with whole-exome sequencing revealed numerous gene alterations restricted to the MCL clone (apart from the canonical t(11;14)(q13;q32) translocation) including gain of one copy of ATM gene or emergence of TP53, CREBBP, NUP214, FUBP1 and SF3B1 gene mutations. Similarly, gene expression analysis revealed vast differences between the MCL and CLL transcriptome, including overexpression of cyclin D1, downregulation of cyclins D2 and D3, or downregulation of IL4R in the MCL clone. Backtracking analysis using quantitative PCR specifically detecting an MCL-restricted focal deletion of TP53 revealed that the pre-MCL clone appeared in the bone marrow and peripheral blood of the patient approximately 4 years before the clinical manifestation of MCL. Both molecular cytogenetic and sequencing data support the hypothesis of a slow development of the pre-MCL clone in parallel to CLL over several years, and thereby exclude the possibility that the transformation event occurred at the stage of the CLL relapse clone by mere t(11;14)(q13;q32) acquisition.


Asunto(s)
Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/patología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 14 , Femenino , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Leucemia Linfocítica Crónica de Células B/metabolismo , Pérdida de Heterocigocidad , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células del Manto/metabolismo , Persona de Mediana Edad , Translocación Genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
4.
Lab Invest ; 94(7): 806-17, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24862967

RESUMEN

Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma (NHL) associated with poor prognosis. Animal models of MCL are scarce. We established and characterized various in vivo models of metastatic human MCL by tail vein injection of either primary cells isolated from patients with MCL or established MCL cell lines (Jeko-1, Mino, Rec-1, Hbl-2, and Granta-519) into immunodeficient NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice. MCL infiltration was assessed with immunohistochemistry (tissues) and flow cytometry (peripheral blood). Engraftment of primary MCL cells was observed in 7 out of 12 patient samples. The pattern of engraftment of primary MCL cells varied from isolated involvement of the spleen to multiorgan infiltration. On the other hand, tumor engraftment was achieved in all five MCL cell lines used and lymphoma involvement of murine bone marrow, spleen, liver, and brain was observed. Overall survival of xenografted mice ranged from 22 ± 1 to 54 ± 3 days depending on the cell line used. Subsequently, we compared the gene expression profile (GEP) and phenotype of the engrafted MCL cells compared with the original in vitro growing cell lines (controls). We demonstrated that engrafted MCL cells displayed complex changes of GEP, protein expression, and sensitivity to cytotoxic agents when compared with controls. We further demonstrated that our MCL mouse models could be used to test the therapeutic activity of systemic chemotherapy, monoclonal antibodies, or angiogenesis inhibitors. The characterization of MCL murine models is likely to aid in improving our knowledge in the disease biology and to assist scientists in the preclinical and clinical development of novel agents in relapsed/refractory MCL patients.


Asunto(s)
Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Linfoma de Células del Manto/genética , Transcriptoma/genética , Anciano , Animales , Médula Ósea/metabolismo , Encéfalo/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Inmunohistoquímica , Inmunofenotipificación , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Estimación de Kaplan-Meier , Hígado/metabolismo , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Persona de Mediana Edad , Bazo/metabolismo , Trasplante Heterólogo , Células Tumorales Cultivadas
5.
Mol Cancer ; 13: 159, 2014 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-24972933

RESUMEN

BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma associated with poor prognosis. Implementation of high-dose cytarabine (araC) into induction therapy became standard-of-care for all newly diagnosed younger MCL patients. However, many patients relapse even after araC-based regimen. Molecular mechanisms responsible for araC resistance in MCL are unknown and optimal treatment strategy for relapsed/refractory MCL patients remains elusive. METHODS: Five araC-resistant (R) clones were derived by long-term culture of five MCL cell lines (CTRL) with increasing doses of araC up to 50 microM. Illumina BeadChip and 2-DE proteomic analysis were used to identify gene and protein expression changes associated with araC resistance in MCL. In vitro cytotoxicity assays and experimental therapy of MCL xenografts in immunodeficient mice were used to analyze their relative responsiveness to a set of clinically used anti-MCL drugs. Primary MCL samples were obtained from patients at diagnosis and after failure of araC-based therapies. RESULTS: Marked downregulation of deoxycytidine-kinase (DCK) mRNA and protein expression was identified as the single most important molecular event associated with araC-resistance in all tested MCL cell lines and in 50% primary MCL samples. All R clones were highly (20-1000x) cross-resistant to all tested nucleoside analogs including gemcitabine, fludarabine and cladribine. In vitro sensitivity of R clones to other classes of clinically used anti-MCL agents including genotoxic drugs (cisplatin, doxorubicin, bendamustine) and targeted agents (bortezomib, temsirolimus, rituximab) remained unaffected, or was even increased (ibrutinib). Experimental therapy of immunodeficient mice confirmed the anticipated loss of anti-tumor activity (as determined by overall survival) of the nucleoside analogs gemcitabine and fludarabine in mice transplanted with R clone compared to mice transplanted with CTRL cells, while the anti-tumor activity of cisplatin, temsirolimus, bortezomib, bendamustine, cyclophosphamide and rituximab remained comparable between the two cohorts. CONCLUSIONS: Acquired resistance of MCL cells to araC is associated with downregulation of DCK, enzyme of the nucleotide salvage pathway responsible for the first phosphorylation (=activation) of most nucleoside analogs used in anti-cancer therapy. The data suggest that nucleoside analogs should not be used in the therapy of MCL patients, who relapse after failure of araC-based therapies.


Asunto(s)
Cladribina/farmacología , Citarabina/farmacología , Desoxicitidina Quinasa/metabolismo , Desoxicitidina/análogos & derivados , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Linfoma de Células del Manto/enzimología , Vidarabina/análogos & derivados , Animales , Anticuerpos Monoclonales de Origen Murino/farmacología , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Western Blotting , Línea Celular Tumoral , Células Clonales , Desoxicitidina/farmacología , Electroforesis en Gel Bidimensional , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/genética , Espectrometría de Masas , Ratones , Proteómica , Rituximab , Vidarabina/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Gemcitabina
6.
Blood ; 117(14): 3816-25, 2011 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-21296997

RESUMEN

Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In this study, we present evidence that MYB (v-myb myeloblastosis viral oncogene homolog) is overexpressed in a subset of B-CLL patients. MYB physically associates with the promoter of miR-155 host gene (MIR155HG, also known as BIC, B-cell integration cluster) and stimulates its transcription. This coincides with the hypermethylated histone H3K4 residue and spread hyperacetylation of H3K9 at MIR155HG promoter. Our data provide evidence of oncogenic activities of MYB in B-CLL that include its stimulatory role in MIR155HG transcription.


Asunto(s)
Leucemia Linfocítica Crónica de Células B/genética , MicroARNs/genética , Proteínas Oncogénicas v-myb/fisiología , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Células HeLa , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Análisis por Micromatrices , Proteínas Oncogénicas v-myb/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Transcripción Genética/fisiología , Transfección , Células Tumorales Cultivadas
7.
Comput Biol Med ; 154: 106539, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36689856

RESUMEN

Model-based reconstruction employing the time separation technique (TST) was found to improve dynamic perfusion imaging of the liver using C-arm cone-beam computed tomography (CBCT). To apply TST using prior knowledge extracted from CT perfusion data, the liver should be accurately segmented from the CT scans. Reconstructions of primary and model-based CBCT data need to be segmented for proper visualisation and interpretation of perfusion maps. This research proposes Turbolift learning, which trains a modified version of the multi-scale Attention UNet on different liver segmentation tasks serially, following the order of the trainings CT, CBCT, CBCT TST - making the previous trainings act as pre-training stages for the subsequent ones - addressing the problem of limited number of datasets for training. For the final task of liver segmentation from CBCT TST, the proposed method achieved an overall Dice scores of 0.874±0.031 and 0.905±0.007 in 6-fold and 4-fold cross-validation experiments, respectively - securing statistically significant improvements over the model, which was trained only for that task. Experiments revealed that Turbolift not only improves the overall performance of the model but also makes it robust against artefacts originating from the embolisation materials and truncation artefacts. Additionally, in-depth analyses confirmed the order of the segmentation tasks. This paper shows the potential of segmenting the liver from CT, CBCT, and CBCT TST, learning from the available limited training data, which can possibly be used in the future for the visualisation and evaluation of the perfusion maps for the treatment evaluation of liver diseases.


Asunto(s)
Tomografía Computarizada de Haz Cónico , Tomografía Computarizada por Rayos X , Tomografía Computarizada de Haz Cónico/métodos , Artefactos , Hígado/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador/métodos
8.
Med Phys ; 49(6): 3624-3637, 2022 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-35396720

RESUMEN

BACKGROUND: CT perfusion imaging (CTP) is used in the diagnostic workup of acute ischemic stroke (AIS). CTP may be performed within the angio suite using flat detector CT (FDCT) to help reduce patient management time. PURPOSE: In order to significantly improve FDCT perfusion (FDCTP) imaging, data-processing algorithms need to be able to compensate for the higher levels of noise, slow rotation speed, and a lower frame rate of current FDCT devices. METHODS: We performed a realistic simulation of FDCTP acquisition based on CTP data from seven subjects. We used the time separation technique (TST) as a model-based approach for FDCTP data processing. We propose a novel dimension reduction in which we approximate the time attenuation curves by a linear combination of trigonometric functions. Our goal was to show that the TST can be used even without prior assumptions on the shape of the attenuation profiles. RESULTS: We first demonstrated that a trigonometric basis is suitable for dimension reduction of perfusion data. Using simulated FDCTP data, we have shown that a trigonometric basis in the TST provided better results than the classical straightforward processing even with additional noise. Average correlation coefficients of perfusion maps were improved for cerebral blood flow (CBF), cerebral blood volume, mean transit time (MTT) maps. In a moderate noise scenario, the average Pearson's coefficient for the CBF map was improved using the TST from 0.76 to 0.81. For the MTT map, it was improved from 0.37 to 0.45. Furthermore, we achieved a total processing time from the reconstruction of FDCTP data to the generation of perfusion maps of under 5 min. CONCLUSIONS: In our study cohort, perfusion maps created from FDCTP data using the TST with a trigonometric basis showed equivalent perfusion deficits to classic CT perfusion maps. It follows, that this novel FDCTP technique has potential to provide fast and accurate FDCTP imaging for AIS patients.


Asunto(s)
Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Humanos , Circulación Cerebrovascular/fisiología , Imagen de Perfusión/métodos , Accidente Cerebrovascular/diagnóstico por imagen , Tomografía Computarizada por Rayos X/métodos
10.
Cells ; 11(2)2022 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-35053339

RESUMEN

The mechanisms by which myelodysplastic syndrome (MDS) cells resist the effects of hypomethylating agents (HMA) are currently the subject of intensive research. A better understanding of mechanisms by which the MDS cell becomes to tolerate HMA and progresses to acute myeloid leukemia (AML) requires the development of new cellular models. From MDS/AML cell lines we developed a model of 5-azacytidine (AZA) resistance whose stability was validated by a transplantation approach into immunocompromised mice. When investigating mRNA expression and DNA variants of the AZA resistant phenotype we observed deregulation of several cancer-related pathways including the phosphatidylinosito-3 kinase signaling. We have further shown that these pathways can be modulated by specific inhibitors that, while blocking the proliferation of AZA resistant cells, are unable to increase their sensitivity to AZA. Our data reveal a set of molecular mechanisms that can be targeted to expand therapeutic options during progression on AZA therapy.


Asunto(s)
Azacitidina/farmacología , Resistencia a Antineoplásicos , Modelos Biológicos , Animales , ADN de Neoplasias/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Ratones , Ratones SCID , Anotación de Secuencia Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Transcriptoma/genética
11.
Oncotarget ; 8(67): 111966-111978, 2017 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-29340104

RESUMEN

Azacitidine (AZA) for higher risk MDS patients is a standard therapy with limited durability. To monitor mutation dynamics during AZA therapy we utilized massive parallel sequencing of 54 genes previously associated with MDS/AML pathogenesis. Serial sampling before and during AZA therapy of 38 patients (reaching median overall survival 24 months (Mo) with 60% clinical responses) identified 116 somatic pathogenic variants with allele frequency (VAF) exceeding 5%. High accuracy of data was achieved via duplicate libraries from myeloid cells and T-cell controls. We observed that nearly half of the variants were stable while other variants were highly dynamic. Patients with marked decrease of allelic burden upon AZA therapy achieved clinical responses. In contrast, early-progressing patients on AZA displayed minimal changes of the mutation pattern. We modeled the VAF dynamics on AZA and utilized a joint model for the overall survival and response duration. While the presence of certain variants associated with clinical outcomes, such as the mutations of CDKN2A were adverse predictors while KDM6A mutations yield lower risk of dying, the data also indicate that allelic burden volatility represents additional important prognostic variable. In addition, preceding 5q- syndrome represents strong positive predictor of longer overall survival and response duration in high risk MDS patients treated with AZA. In conclusion, variants dynamics detected via serial sampling represents another parameter to consider when evaluating AZA efficacy and predicting outcome.

12.
J Cancer Res Clin Oncol ; 141(5): 887-99, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25367136

RESUMEN

PURPOSE: Here, we studied whether amplicon next-generation deep sequencing (NGS) could improve the detection of emerging BCR-ABL1 kinase domain mutations in chronic phase chronic myeloid leukemia (CML) patients under tyrosine kinase inhibitor (TKI) treatment and discussed the clinical relevance of such sensitive mutational detection. METHODS: For NGS data evaluation including extraction of biologically relevant low-level variants from background error noise, we established and applied a robust and versatile bioinformatics approach. RESULTS: Results from a retrospective longitudinal analysis of 135 samples of 15 CML patients showed that NGS could have revealed emerging resistant mutants 2-11 months earlier than conventional sequencing. Interestingly, in cases who later failed first-line imatinib treatment, NGS revealed that TKI-resistant mutations were already detectable at the time of major or deeper molecular response. Identification of emerging mutations by NGS was mirrored by BCR-ABL1 transcript level expressed either fluctuations around 0.1 %(IS) or by slight transcript level increase. NGS also allowed tracing mutations that emerged during second-line TKI therapy back to the time of switchover. Compound mutants could be detected in three cases, but were not found to outcompete single mutants. CONCLUSIONS: This work points out, that next-generation deep sequencing, coupled with a robust bioinformatics approach for mutation calling, may be just in place to ensure reliable detection of emerging BCR-ABL1 mutations, allowing early therapy switch and selection of the most appropriate therapy. Further, prospective assessment of how to best integrate NGS in the molecular monitoring and clinical decision algorithms is warranted.


Asunto(s)
Antineoplásicos/uso terapéutico , Benzamidas/uso terapéutico , Proteínas de Fusión bcr-abl/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Mutación/efectos de los fármacos , Piperazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/uso terapéutico , Adulto , Anciano , Biología Computacional , Femenino , Humanos , Mesilato de Imatinib , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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