RESUMO
B-cell receptor (BCR) signaling and T-cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can use microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short noncoding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs, including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22) but also other candidates for a role in microenvironmental interactions. Notably, all 3 miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation and significantly shorter overall survival of CLL patients. We identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream nuclear factor-κB (NF-κB) signaling. In CLL, BCR represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40-NF-κB signaling. This regulatory loop is disrupted by BCR inhibitors (bruton tyrosine kinase [BTK] inhibitor ibrutinib or phosphatidylinositol 3-kinase [PI3K] inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29-TRAF4-CD40 signaling axis modulated by BCR activity.
Assuntos
Adenina/análogos & derivados , Antígenos CD40/metabolismo , Regulação Neoplásica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/patologia , MicroRNAs/genética , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-bcr/antagonistas & inibidores , Fator 4 Associado a Receptor de TNF/metabolismo , Adenina/farmacologia , Adulto , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Antígenos CD40/genética , Feminino , Seguimentos , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Taxa de Sobrevida , Fator 4 Associado a Receptor de TNF/genética , Células Tumorais CultivadasRESUMO
Mortar samples were prepared with sulphide-bearing aggregates and tested for the potential of aggregate oxidation followed by sulphate attack. Scanning Electron Microscopy and its associated Energy Dispersive X-Ray analysis were used to analyze the developed phases in the tested samples to confirm that the obtained expansion is attributable to sulphate attack. The Energy Dispersive X-Ray analysis helped identify sulphide phases in aggregates and evidence of sulphate attack in mortars. Ettringite was detected and confirmed in the mortars with sulphide-bearing aggregates suggesting that the test conditions are suitable for reproducing the damaging mechanism of sulphide oxidation.
Assuntos
Sulfatos , Sulfetos , Microscopia Eletrônica de Varredura , Sulfetos/análiseRESUMO
Samples were collected from a 20-year-old concrete suffering alkali-silica reaction for examination using scanning electron microscopy. The concrete was also exposed to cycles of freezing and thawing in service. The old concrete was processed and used as recycled concrete aggregate in new concrete tested under lab conditions. The study shows that backscattered electron imaging, energy dispersive x-ray analysis and x-ray mapping are helpful tools to identify the presence of alkali-silica reaction products. X-ray mapping was found helpful to confirm the presence of reaction products in cases where the products or the cracking are not very clear in the collected image. The study shows the presence of ettringite in the air voids of the concrete. The formed ettringite is believed to have no direct effect on the expansion due to the alkali-silica reaction as they form in an empty space without causing swelling pressure on the concrete. The effect of the ettringite formed in the air voids on the resistance to freezing/thawing was not confirmed in this study. Recycling the old affected concrete as aggregate in new concrete was found to cause significant expansion under lab conditions.
RESUMO
The Bruton tyrosine kinase (BTK) inhibitor ibrutinib has substantially improved therapeutic options for chronic lymphocytic leukemia (CLL). Although ibrutinib is not curative, it has a profound effect on CLL cells and may create new pharmacologically exploitable vulnerabilities. To identify such vulnerabilities, we developed a systematic approach that combines epigenome profiling (charting the gene-regulatory basis of cell state) with single-cell chemosensitivity profiling (quantifying cell-type-specific drug response) and bioinformatic data integration. By applying our method to a cohort of matched patient samples collected before and during ibrutinib therapy, we identified characteristic ibrutinib-induced changes that provide a starting point for the rational design of ibrutinib combination therapies. Specifically, we observed and validated preferential sensitivity to proteasome, PLK1, and mTOR inhibitors during ibrutinib treatment. More generally, our study establishes a broadly applicable method for investigating treatment-specific vulnerabilities by integrating the complementary perspectives of epigenetic cell states and phenotypic drug responses in primary patient samples.
Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Cromatina/fisiologia , Combinação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética/genética , Epigenômica/métodos , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Piperidinas , Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Análise de Célula Única/métodos , Serina-Treonina Quinases TOR/metabolismo , Quinase 1 Polo-LikeRESUMO
BACKGROUND: High UGT2B17 is associated with poor prognosis in untreated chronic lymphocytic leukaemia (CLL) patients and its expression is induced in non-responders to fludarabine-containing regimens. We examined whether UGT2B17, the predominant lymphoid glucuronosyltransferase, affects leukaemic drug response and is involved in the metabolic inactivation of anti-leukaemic agents. METHODS: Functional enzymatic assays and patients' plasma samples were analysed by mass-spectrometry to evaluate drug inactivation by UGT2B17. Cytotoxicity assays and RNA sequencing were used to assess drug response and transcriptome changes associated with high UGT2B17 levels. RESULTS: High UGT2B17 in B-cell models led to reduced sensitivity to fludarabine, ibrutinib and idelalisib. UGT2B17 expression in leukaemic cells involved a non-canonical promoter and was induced by short-term treatment with these anti-leukaemics. Glucuronides of both fludarabine and ibrutinib were detected in CLL patients on respective treatment, however UGT2B17 conjugated fludarabine but not ibrutinib. AMP-activated protein kinase emerges as a pathway associated with high UGT2B17 in fludarabine-treated patients and drug-treated cell models. The expression changes linked to UGT2B17 exposed nuclear factor kappa B as a key regulatory hub. CONCLUSIONS: Data imply that UGT2B17 represents a mechanism altering drug response in CLL through direct inactivation but would also involve additional mechanisms for drugs not inactivated by UGT2B17.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Biomarcadores Farmacológicos/metabolismo , Glucuronosiltransferase/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Antígenos de Histocompatibilidade Menor/genética , Adenina/efeitos adversos , Adenina/análogos & derivados , Adenina/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , NF-kappa B/genética , Piperidinas/efeitos adversos , Piperidinas/farmacologia , Purinas/efeitos adversos , Purinas/farmacologia , Quinazolinonas/efeitos adversos , Quinazolinonas/farmacologia , Vidarabina/efeitos adversos , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
Uridine diphospho glucuronosyltransferase 2B17 (UGT2B17) glucuronidates androgens and xenobiotics including certain drugs. The UGT2B17 gene shows a remarkable copy number variation (CNV), which predisposes for solid tumors and influences drug response. Here, we identify a yet undescribed UGT2B17 mRNA overexpression in poor-risk chronic lymphocytic leukemia (CLL). In total, 320 CLL patients and 449 healthy donors were analyzed. High (above median) UGT2B17 expression was associated with established CLL poor prognostic factors and resulted in shorter treatment-free and overall survival (hazard ratio ([death] 2.18; 95% CI 1.18-4.01; P = .013). The prognostic impact of mRNA expression was more significant than that of UGT2B17 CNV. UGT2B17 mRNA levels in primary CLL samples directly correlated with functional glucuronidation activity toward androgens and the anticancer drug vorinostat (R > 0.9, P < .001). After treatment with fludarabine containing regimens UGT2B17 was up-regulated particularly in poor responders (P = .030). We observed an exclusive involvement of the 2B17 isoform within the UGT protein family. Gene expression profiling of a stable UGT2B17 knockdown in the CLL cell line MEC-1 demonstrated a significant involvement in key cellular processes. These findings establish a relevant role of UGT2B17 in CLL with functional consequences and potential therapeutic implications.
Assuntos
Glucuronosiltransferase/genética , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/genética , Idoso , Antineoplásicos/uso terapêutico , Sequência de Bases , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Dosagem de Genes , Técnicas de Silenciamento de Genes , Glucuronosiltransferase/metabolismo , Humanos , Estimativa de Kaplan-Meier , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , RNA Interferente Pequeno/genética , Fatores de Risco , Transcriptoma , Regulação para Cima/efeitos dos fármacos , Vidarabina/análogos & derivados , Vidarabina/uso terapêuticoRESUMO
Chronic lymphocytic leukaemia (CLL) cells express constitutively activated NOTCH2 in a protein kinase C (PKC)- dependent manner. The transcriptional activity of NOTCH2 correlates not only with the expression of its target gene FCER2 (CD23) but is also functionally linked with CLL cell viability. In the majority of CLL cases, DNA-bound NOTCH2 complexes are less sensitive to the γ-secretase inhibitor (GSI) DAPT. Therefore, we searched for compounds that interfere with NOTCH2 signalling at the transcription factor level. Using electrophoretic mobility shift assays (EMSA), we identified the Aspergillum-derived secondary metabolite gliotoxin as a potent NOTCH2 transactivation inhibitor. Gliotoxin completely blocked the formation of DNA-bound NOTCH2 complexes in CLL cells independent of their sensitivity to DAPT. The inhibition of NOTCH2 signalling by gliotoxin was associated with down regulation of CD23 (FCER) expression and induction of apoptosis. Short time exposure of CLL cells indicated that the early apoptotic effect of gliotoxin is independent of proteasome regulated nuclear factor κB activity, and is associated with up regulation of NOTCH3 and NR4A1 expression. Gliotoxin could overcome the supportive effect of primary bone marrow stromal cells in an ex vivo CLL microenvironment model. In conclusion, we identified gliotoxin as a potent NOTCH2 inhibitor with a promising therapeutic potential in CLL.
Assuntos
Apoptose/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Gliotoxina/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Receptor Notch2/antagonistas & inibidores , Ativação Transcricional/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/patologia , Técnicas de Cocultura , Análise Mutacional de DNA , Depressão Química , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Concentração Inibidora 50 , Leucemia Linfocítica Crônica de Células B/sangue , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/biossíntese , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptor Notch3 , Receptores Notch/biossíntese , Receptores Notch/genética , Células Estromais/fisiologia , Fatores de Transcrição/fisiologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/patologiaRESUMO
Evidence suggests that tumor microenvironment is critically involved in supporting survival of chronic lymphocytic leukemia (CLL) cells. However, the molecular mechanisms of this effect and the clinical significance are not fully understood. We applied a microenvironment model to explore the interaction between CLL cells and stromal cells and to elucidate the role of phosphatidylinositol 3 kinase (PI3-K)/Akt/phosphatase and tensin homolog detected on chromosome 10 (PTEN) cascade in this process and its in vivo relevance. Primary human stromal cells from bone marrow, lymph nodes, and spleen significantly inhibited spontaneous apoptosis of CLL cells. Pan-PI3-K inhibitors (LY294002, wortmannin, PI-103), isotype-specific inhibitors of p110α, p110ß, p110γ, and small interfering RNA against PI3-K and Akt1 counteracted the antiapoptotic effect of the stromal cells. Induction of apoptosis was associated with a decrease in phosphatidylinositol-3,4,5-triphosphate, PI3-K-p85, and dephosphorylation of phosphatidylinositol-dependent kinase-1 (PDK-1), Akt1, and PTEN. Freshly isolated peripheral blood mononuclear cells from patients with CLL (n = 44) showed significantly higher levels of phosphorylated Akt1, PDK-1, PTEN, and CK2 than healthy persons (n = 8). CK2 inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole, apigenin, and 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazol) decreased phosphorylation of PTEN and Akt, induced apoptosis in CLL cells, and enhanced the response to fludarabine. In conclusion, bone marrow microenvironment modulates the PI3-K/Akt/PTEN cascade and prevents apoptosis of CLL cells. Combined inhibition of PI3-K/Akt and recovery of PTEN activity may represent a novel therapeutic concept for CLL.
Assuntos
Apoptose , Células da Medula Óssea/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células da Medula Óssea/patologia , Caseína Quinase II/metabolismo , Células Cultivadas , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
Marrow stromal cells (MSCs) provide important survival and drug resistance signals to chronic lymphocytic leukemia (CLL) cells, but current models to analyze CLL-MSC interactions are heterogeneous. Therefore, we tested different human and murine MSC lines and primary human MSCs for their ability to protect CLL cells from spontaneous and drug-induced apoptosis. Our results show that both human and murine MSCs are equally effective in protecting CLL cells from fludarabine-induced apoptosis. This protective effect was sustained over a wide range of CLL-MSC ratios (5:1 to 100:1), and the levels of protection were reproducible in 4 different laboratories. Human and murine MSCs also protected CLL cells from dexamethasone- and cyclophosphamide-induced apoptosis. This protection required cell-cell contact and was virtually absent when CLL cells were separated from the MSCs by micropore filters. Furthermore, MSCs maintained Mcl-1 and protected CLL cells from spontaneous and fludarabine-induced Mcl-1 and PARP cleavage. Collectively, these studies define common denominators for CLL cocultures with MSCs. They also provide a reliable, validated tool for future investigations into the mechanism of MSC-CLL cross talk and for drug testing in a more relevant fashion than the commonly used suspension cultures.
Assuntos
Células da Medula Óssea/metabolismo , Comunicação Celular/fisiologia , Resistencia a Medicamentos Antineoplásicos , Leucemia Linfocítica Crônica de Células B/metabolismo , Células Estromais/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Western Blotting , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Citometria de Fluxo , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Células Estromais/citologia , Células Estromais/efeitos dos fármacosRESUMO
The clinical benefit of the addition of granulocyte colony-stimulating factor (G-CSF) to standard immunochemotherapy of chronic lymphocytic leukemia (CLL) with fludarabine, cyclophosphamide, and rituximab (FCR) is still unclear. In this retrospective study we analyzed the outcome of 32 consecutive patients with CLL during treatment with FCR. Sixteen patients received G-CSF for treatment of CTC grade 3 or 4 neutropenia or febrile neutropenia at some point during therapy and 16 did not. Both groups were well balanced for clinical and biological risk factors. Overall response rates were not significantly different (94% vs. 75%; p=0.144). Interestingly, a significantly better progression-free survival (100% vs. 35.4% at 24 months; p<0.001) and even overall survival (100% vs. 77.8% at 24 months; p=0.022) was observed in patients receiving G-CSF. While the underlying cause remains to be elucidated, these data strongly suggest an association of the addition of G-CSF to FCR therapy with final patient outcome.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Fármacos Hematológicos/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/fisiopatologia , Idoso , Anticorpos Monoclonais Murinos/administração & dosagem , Anticorpos Monoclonais Murinos/efeitos adversos , Anticorpos Monoclonais Murinos/uso terapêutico , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Estudos de Coortes , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Ciclofosfamida/uso terapêutico , Monitoramento de Medicamentos , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Masculino , Pessoa de Meia-Idade , Neutropenia/induzido quimicamente , Projetos Piloto , Proteínas Recombinantes , Estudos Retrospectivos , Rituximab , Análise de Sobrevida , Vidarabina/administração & dosagem , Vidarabina/efeitos adversos , Vidarabina/análogos & derivados , Vidarabina/uso terapêuticoRESUMO
One characteristic of chronic lymphocytic leukaemia (CLL) lymphocytes is high expression of CD23, which has previously been identified as a downstream target for NOTCH2 signalling. The mechanisms regulating NOTCH2-dependent CD23 expression, however, are largely unknown. This study showed that peripheral CLL cells overexpressed transcriptionally active NOTCH2 (N2(IC)), irrespective of their prognostic marker profile. When placed in culture, NOTCH2 activity was spontaneously decreased in 25 out of 31 CLL cases (81%) within 24 h. DNA-bound N2(IC) complexes could be maintained by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or by gamma-interferon (IFN-gamma), two CLL characteristic inducers of CD23 expression. Inhibition of PKC-delta by RNA interference or by rottlerin antagonised PMA-induced NOTCH2 activation and also suppressed NOTCH2 activity in CLL cases with constitutively activated NOTCH2 signalling. In 23 out of 29 CLL cases tested (79%), DNA-bound N2(IC) complexes were found to be resistant to the gamma-secretase inhibitor (GSI) DAPT, suggesting that GSIs will be only effective in a subset of CLL cases. These data suggest that deregulation of NOTCH2 signalling is critically involved in maintaining the malignant phenotype of CLL lymphocytes and point to a link between PKC-delta and NOTCH2 signalling in the leukemic cells.
Assuntos
Leucemia Linfocítica Crônica de Células B/imunologia , Proteínas de Neoplasias/metabolismo , Proteína Quinase C-delta/metabolismo , Receptor Notch2/metabolismo , Receptores de IgE/metabolismo , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Humanos , Interferon gama/imunologia , Leucemia Linfocítica Crônica de Células B/genética , Prognóstico , Proteína Quinase C-delta/antagonistas & inibidores , Interferência de RNA , Receptor Notch2/genética , Transdução de Sinais , Acetato de Tetradecanoilforbol/imunologia , Células Tumorais CultivadasRESUMO
NOTCH signaling represents a promising therapeutic target in chronic lymphocytic leukemia (CLL). We compared the anti-neoplastic effects of the nuclear NOTCH2 inhibitor gliotoxin and the pan-NOTCH γ-secretase inhibitor RO4929097 in primary CLL cells with special emphasis on the individual roles of the different NOTCH receptors. Gliotoxin rapidly induced apoptosis in all CLL cases tested, whereas RO4929097 exerted a variable and delayed effect on CLL cell viability. Gliotoxin-induced apoptosis was associated with inhibition of the NOTCH2/FCER2 (CD23) axis together with concomitant upregulation of the NOTCH3/NR4A1 axis. In contrast, RO4929097 downregulated the NOTCH3/NR4A1 axis and counteracted the spontaneous and gliotoxin-induced apoptosis. On the cell surface, NOTCH3 and CD23 expression were mutually exclusive, suggesting that downregulation of NOTCH2 signaling is a prerequisite for NOTCH3 expression in CLL cells. ATAC-seq confirmed that gliotoxin targeted the canonical NOTCH signaling, as indicated by the loss of chromatin accessibility at the potential NOTCH/CSL site containing the gene regulatory elements. This was accompanied by a gain in accessibility at the NR4A1, NFκB, and ATF3 motifs close to the genes involved in B-cell activation, differentiation, and apoptosis. In summary, these data show that gliotoxin recovers a non-canonical tumor-suppressing NOTCH3 activity, indicating that nuclear NOTCH2 inhibitors might be beneficial compared to pan-NOTCH inhibitors in the treatment of CLL.
Assuntos
Gliotoxina/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Receptor Notch2/antagonistas & inibidores , Receptor Notch3/agonistas , Adulto , Idoso , Idoso de 80 Anos ou mais , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzazepinas/administração & dosagem , Benzazepinas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Feminino , Gliotoxina/administração & dosagem , Humanos , Lectinas Tipo C/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/agonistas , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Receptor Notch3/genética , Receptor Notch3/metabolismo , Receptores de IgE/antagonistas & inibidores , Elementos Reguladores de Transcrição , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Rabbit mesenchymal stem cells (rMSCs) are promising agents for the preservation of genetic biodiversity in domestic rabbit breeds. However, rMSCs must meet certain requirements to be used for cryopreservation in animal gene banks. Currently, there are numerous discrepancies in the published data regarding the rMSC phenotype, which may complicate efforts to evaluate their purity and suitability for reuse after cryopreservation in gene and tissue banks. We propose a combined approach (flow cytometry, PCR, differentiation and ultrastructure studies) for the characterization and recovery of rMSCs after cryopreservation. Flow cytometric analyses of rMSCs confirmed the expression of CD29, CD44, vimentin, desmin and α-SMA. RT-PCR revealed the expression of other markers at the mRNA level (SSEA-4, CD73, CD90, CD105, CD146 and CD166). rMSCs showed efficient multilineage differentiation into adipo-, chondro- and osteogenic lineages, SOX2 expression (pluripotency) and typical MSC morphology and ultrastructure. The confirmed rMSCs were subsequently used for cryopreservation. Efficient recovery of rMSCs after cryogenic freezing was demonstrated by high cell viability, normal ultrastructure of reseeded rMSCs, high expression of CD29 and CD44 and lineage differentiation capacity. The proposed combined approach could be used for characterization, cryopreservation and recovery of rMSCs as genetic resources for native rabbit breeds.
Assuntos
Bancos de Espécimes Biológicos/normas , Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Coelhos/genética , Animais , Antígenos CD/metabolismo , Células da Medula Óssea/metabolismo , Criopreservação , Células-Tronco Mesenquimais/metabolismo , Fenótipo , RNA Mensageiro/genética , Coelhos/classificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Few data are available concerning the prevalence of autoimmune disease or chronic infections in chronic lymphocytic leukemia patients at diagnosis as well as their clinical outcome. We studied the frequency of such chronic conditions in relation to prognostic markers. A history of autoimmune disease or chronic infection was found in 21% of 186 chronic lymphocytic leukemia patients (12% in autoimmune diseases, 9% in chronic infections). Patients with a history of chronic stimulation were more likely to have unmutated IgV(H) genes (p<0.002), unfavorable or intermediate risk cytogenetics (11q, 17p deletions, trisomy 12) (p<0.001), and higher CD38 expression (p=0.004). Autoimmune conditions (n=22) were characterized by female predominance (55.0%) with a high frequency of unmutated IgV(H) (53,8%). Median time to first treatment was 83 months for the chronic stimulation group compared to 128 months for the non-chronic stimulation group (n.s.). Patients suffering from chronic conditions at chronic lymphocytic leukemia diagnosis are likely to have poor prognostic markers, particularly unmutated IgV(H) genes.
Assuntos
Autoimunidade , Infecções/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Doença Crônica , Genes de Imunoglobulinas , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Infecções/etiologia , Leucemia Linfocítica Crônica de Células B/complicações , Estudos RetrospectivosRESUMO
Rabbits have many hereditary diseases common to humans and are therefore a valuable model for regenerative disease and hematopoietic stem cell (HSC) therapies. Currently, there is no substantial data on the isolation and/or enrichment of rabbit HSCs. This study was initiated to evaluate the efficiency of the commercially available anti-CD34 and anti-CD133 antibodies for the detection and potential enrichment of rabbit HSCs from peripheral blood. PBMCs from rabbit and human blood were labelled with different clones of anti-human CD34 monoclonal antibodies (AC136, 581, and 8G12) and rabbit polyclonal CD34 antibody (pCD34) and anti-human CD133 monoclonal antibodies (AC133 and 293C3). Flow cytometry showed a higher percentage of rabbit CD34+ cells labelled by AC136 in comparison to the clone 581 and pCD34 (P < 0.01). A higher percentage of rabbit CD133+ cells were also detected by 293C3 compared to the AC133 clone (P < 0.01). Therefore, AC136 clone was used for the indirect immunomagnetic enrichment of rabbit CD34+ cells using magnetic-activated cell sorting (MACS). The enrichment of the rabbit CD34+ cells after sorting was low in comparison to human samples (2.4% vs. 39.6%). PCR analyses confirmed the efficient enrichment of human CD34+ cells and the low expression of CD34 mRNA in rabbit positive fraction. In conclusion, the tested antibodies might be suitable for detection, but not for sorting the rabbit CD34+ HSCs and new specific anti-rabbit CD34 antibodies are needed for efficient enrichment of rabbit HSCs. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1278-1289, 2018.
Assuntos
Antígeno AC133/imunologia , Anticorpos/imunologia , Antígenos CD34/imunologia , Células-Tronco Hematopoéticas/imunologia , Animais , Citometria de Fluxo , CoelhosRESUMO
In 2008 the Ludwig Boltzmann Cluster Oncology (LBC ONC) was established on the basis of two previous Ludwig Boltzmann Institutes working in the field of hematology and cancer research. The general aim of the LBC ONC is to improve treatment of hematopoietic neoplasms by eradicating cancer-initiating and disease-propagating cells, also known as leukemic stem cells (LSC) in the context of leukemia. In a first phase, the LBC ONC characterized the phenotype and molecular aberration profiles of LSC in various malignancies. The LSC phenotypes were established in acute and chronic myeloid leukemia, in acute lymphoblastic leukemia and in chronic lymphocytic leukemia. In addition, the concept of preleukemic (premalignant) neoplastic stem cells (pre-L-NSC) was coined by the LBC ONC and was tested in myelodysplastic syndromes and myeloproliferative neoplasms. Phenotypic characterization of LSC provided a solid basis for their purification and for the characterization of specific target expression profiles. In a second phase, molecular markers and targets were validated. This second phase is ongoing and should result in the development of new diagnostics parameters and novel, more effective, LSC-eradicating, treatment strategies; however, many issues still remain to be solved, such as sub-clonal evolution, LSC niche interactions, immunologic control of LSC, and LSC resistance. In the forthcoming years, the LBC ONC will concentrate on developing LSC-eradicating strategies, with special focus on LSC resistance, precision medicine and translation of LSC-eradicating concepts into clinical application.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Humanos , Células-Tronco NeoplásicasRESUMO
The mechanisms that lead to reticulin fibrosis of bone marrow (BM) in hairy cell leukemia (HCL) are not fully understood. We therefore investigated the involvement of TGF-beta1, a potent fibrogenic cytokine, in this process. Immunoassays revealed that TGF-beta1 is present at higher concentrations in BM, serum, and plasma of HCL patients in comparison with healthy donors (P < 0.001). RT-PCR and immunofluorescence studies showed that TGF-beta1 is overexpressed at the mRNA and protein levels in peripheral blood, spleen, and BM mononuclear cells and that hairy cells (HCs) are the main source of TGF-beta1. Active TGF-beta1 correlated significantly with grades of BM fibrosis, infiltration with HCs, and serum procollagen type III aminoterminal propeptide (PIIINP). Ex vivo studies demonstrated that TGF-beta1 significantly enhances the production and deposition of reticulin and collagen fibers by BM fibroblasts. In addition, BM plasma of HCL patients increased the synthesis of type I and type III procollagens, the main components of reticulin fibers, at the mRNA and protein levels. This fibrogenic activity of BM plasma was abolished by neutralizing anti-TGF-beta1 antibodies. These results show, for the first time to our knowledge, that TGF-beta1 is highly expressed in HCs and is directly involved in the pathogenesis of BM reticulin fibrosis in HCL.
Assuntos
Medula Óssea/metabolismo , Leucemia de Células Pilosas/metabolismo , Mielofibrose Primária/patologia , Reticulina/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea , Células Cultivadas , Colágeno/metabolismo , Colágeno Tipo III/química , Ensaio de Imunoadsorção Enzimática , Feminino , Fibroblastos/metabolismo , Fibrose , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imunoensaio , Leucócitos Mononucleares/metabolismo , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Peptídeos/química , RNA Mensageiro/metabolismo , Radioimunoensaio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1RESUMO
Deregulation of NOTCH2 signaling is implicated in a wide variety of human neoplasias. The current concept of targeting NOTCH is based on using gamma secretase inhibitors (GSI) to regulate the release of the active NOTCH intracellular domain. However, the clinical outcome of GSI remains unsatisfactory. Therefore we analyzed human solid tumor derived cell lines for their nuclear NOTCH activity and evaluated the therapeutic potential of the NOTCH2 transactivation inhibitor gliotoxin in comparison to the representative GSI DAPT. Electrophoretic mobility shift assays (EMSA) were used as a surrogate method for the detection of NOTCH/CSL transcription factor complexes. The effect of gliotoxin on cell viability and its clinical relevance was evaluated in vitro and in a melanoma xenograft mouse model. Cell lines derived from melanoma (518A2), hepatocellular carcinoma (SNU398, HCC-3, Hep3B), and pancreas carcinoma (PANC1) express high amounts of nuclear NOTCH2. Gliotoxin efficiently induced apoptosis in these cell lines whereas the GSI DAPT was ineffective. The specificity of gliotoxin was demonstrated in the well differentiated nuclear NOTCH negative cell line Huh7, which was resistant to gliotoxin treatment in vitro. In xenotransplanted 518A2 melanomas, a single day dosing schedule of gliotoxin was well tolerated without any study limiting side effects. Gliotoxin significantly reduced the tumor volume in early (83 mm3 vs. 115 mm3, p = 0.008) as well as in late stage (218 mm3 vs. 576 mm3, p = 0.005) tumor models. In conclusion, NOTCH2 appears to be a key target of gliotoxin in human neoplasias and gliotoxin deserves further evaluation as a potential therapeutic agent in cancer management.
RESUMO
Inhibitor of proteasome, bortezomib (BOR), although highly active in vitro, showed unexpectedly low efficacy in vivo in patients with B-CLL when used alone. We studied the in vitro cytotoxic effects of BOR in combination with anti-CD20 (rituximab, RIT) or anti-CD52 (campath, CAM) monoclonal antibodies on B-CLL cells. Both BOR+RIT and BOR+CAM combinations exerted additive cytotoxicity, triggering caspase-dependent apoptosis. The treatment significantly modified expression of several apoptosis-regulating proteins, including upregulation of Bax or downregulation of Bcl-2 and Mcl-1 by BOR+RIT, as well as downregulation of Bcl-2 and XIAP by BOR+CAM. These data suggest the feasibility of concomitant use of those agents for the treatment of B-CLL patients.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Pirazinas/farmacologia , Idoso , Idoso de 80 Anos ou mais , Alemtuzumab , Anticorpos Monoclonais Humanizados , Anticorpos Monoclonais Murinos , Proteínas Reguladoras de Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Bortezomib , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , RituximabRESUMO
The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous with some patients requiring early therapy whereas others will not be treated for years. The evaluation of an individual CLL patient's prognosis remains a problematic issue. The presence or absence of somatic mutations in the IgVH genes is currently the gold-standard prognostic factor, but this technique is labor intensive and costly. Genomic studies uncovered that 70 kDa zeta-associated protein (ZAP-70) expression was associated with unmutated IgVH genes and ZAP-70 protein expression was proposed as a surrogate for somatic mutational status. Among the available techniques for ZAP-70 detection, flow cytometry is most preferable as it allows the simultaneous quantification of ZAP-70 protein expression levels in CLL cells and residual normal lymphocyte subsets. However, several factors introduce variability in the results reported from different laboratories; these factors include the anti-ZAP-70 antibody clone and conjugate, the staining procedure, the gating strategy, and the method of reporting the results. The need for standardization of the approach led to the organization of an international working group focused on harmonizing all aspects of the technique. During this workshop, a technical consensus was reached on the methods for cell permeabilization and immunophenotyping procedures. An assay was then designed that allowed comparison of two clones of anti-ZAP-70 antibody and the identification of the expression of this molecule in B, T, and NK cells identified in a four multicolor analysis. This procedure was applied to three stabilized blood samples, provided by the UK NEQAS group to all participating members of this study, in order to minimize variability caused by sample storage and shipment. Analysis was performed in 20 laboratories providing interpretable data from 14 centers. Various gating strategies were used and the ZAP-70 levels were expressed as percentage positive (POS) relative to isotype control or normal B-cells or normal T-cells; in addition the levels were reported as a ratio of expression in CLL cells relative to T-cells. The reported level of ZAP-70 expression varied greatly depending on the antibody and the method used to express the results. The CLL/T-cell ZAP-70 expression ratio showed a much lower interlaboratory variation than other reporting strategies and is recommended for multicenter studies. Stabilization results in decreased expression of CD19 making gating more difficult and therefore stabilized samples are not optimal for multicentric analysis of ZAP-70 expression. We assessed the variation of ZAP-70 expression levels in fresh cells according to storage time, which demonstrated that ZAP-70 is labile but sufficiently stable to allow comparison using fresh samples distributed between labs in Europe. These studies have demonstrated progress toward a consensus reporting procedure, and further work is underway to harmonize the preparation and analysis procedures.