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1.
Sci Rep ; 9(1): 4155, 2019 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-30858428

RESUMO

Missing in Metastasis (MIM), or Metastasis Suppressor 1 (MTSS1), is a highly conserved protein, which links the plasma membrane to the actin cytoskeleton. MIM has been implicated in various cancers, however, its modes of action remain largely enigmatic. Here, we performed an extensive in silico characterisation of MIM to gain better understanding of its function. We detected previously unappreciated functional motifs including adaptor protein (AP) complex interaction site and a C-helix, pointing to a role in endocytosis and regulation of actin dynamics, respectively. We also identified new functional regions, characterised with phosphorylation sites or distinct hydrophilic properties. Strong negative selection during evolution, yielding high conservation of MIM, has been combined with positive selection at key sites. Interestingly, our analysis of intra-molecular co-evolution revealed potential regulatory hotspots that coincided with reduced potentially pathogenic polymorphisms. We explored databases for the mutations and expression levels of MIM in cancer. Experimentally, we focused on chronic lymphocytic leukaemia (CLL), where MIM showed high overall expression, however, downregulation on poor prognosis samples. Finally, we propose strong conservation of MTSS1 also on the transcriptional level and predict novel transcriptional regulators. Our data highlight important targets for future studies on the role of MIM in different tissues and cancers.

2.
Blood ; 132(22): 2362-2374, 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30254128

RESUMO

ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in ARPC1B have been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in ARPC1B in 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper-immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α-directed migration. Gene transfer of ARPC1B in patients' T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8+ T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.

3.
Nat Commun ; 9(1): 1787, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29725010

RESUMO

In chronic lymphocytic leukemia (CLL), the non-hematopoietic stromal microenvironment plays a critical role in promoting tumor cell recruitment, activation, survival, and expansion. However, the nature of the stromal cells and molecular pathways involved remain largely unknown. Here, we demonstrate that leukemic B lymphocytes induce the activation of retinoid acid synthesis and signaling in the microenvironment. Inhibition of RA-signaling in stromal cells causes deregulation of genes associated with adhesion, tissue organization and chemokine secretion including the B-cell chemokine CXCL13. Notably, reducing retinoic acid precursors from the diet or inhibiting RA-signaling through retinoid-antagonist therapy prolong survival by preventing dissemination of leukemia cells into lymphoid tissues. Furthermore, mouse and human leukemia cells could be distinguished from normal B-cells by their increased expression of Rarγ2 and RXRα, respectively. These findings establish a role for retinoids in murine CLL pathogenesis, and provide new therapeutic strategies to target the microenvironment and to control disease progression.

4.
Blood ; 129(26): 3440-3451, 2017 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-28465341

RESUMO

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant CD5+ B lymphocytes in blood, bone marrow, and lymphoid organs. CD1d-restricted invariant natural killer T (iNKT) cells are innate-like T lymphocytes strongly implicated in tumor surveillance. We investigated the impact of iNKT cells in the natural history of the disease in the Eµ-Tcl1 (Tcl1) CLL mouse model and 68 CLL patients. We found that Tcl1-CLL cells express CD1d and that iNKT cells critically delay disease onset but become functionally impaired upon disease progression. In patients, disease progression correlates with high CD1d expression on CLL cells and impaired iNKT cells. Conversely, disease stability correlates with negative or low CD1d expression on CLL cells and normal iNKT cells, suggesting indirect leukemia control. iNKT cells indeed hinder CLL survival in vitro by restraining CD1d-expressing nurse-like cells, a relevant proleukemia macrophage population. Multivariable analysis identified iNKT cell frequency as an independent predictor of disease progression. Together, these results support the contribution of iNKT cells to CLL immune surveillance and highlight iNKT cell frequency as a prognostic marker for disease progression.


Assuntos
Vigilância Imunológica , Leucemia Linfocítica Crônica de Células B/imunologia , Células T Matadoras Naturais/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos CD1d/sangue , Progressão da Doença , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Contagem de Linfócitos , Masculino , Camundongos , Pessoa de Meia-Idade , Prognóstico
5.
Oncotarget ; 8(7): 11219-11227, 2017 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-28061439

RESUMO

Chronic lymphocytic leukemia (CLL) remains incurable despite the introduction of new drugs. Therapies targeting receptors and pathways active specifically in malignant B cells might provide better treatment options. For instance, in B cell lymphoma, our group has previously shown that scavenger receptor type B-1 (SR-B1), the high-affinity receptor for cholesterol-rich high-density lipoproteins (HDL), is a therapeutic target. As evidence suggests that targeting cholesterol metabolism in CLL cells may have therapeutic benefit, we examined SR-B1 expression in primary CLL cells from patients. Unlike normal B cells that do not express SR-B1, CLL cells express the receptor. As a result, we evaluated cholesterol-poor synthetic HDL nanoparticles (HDL NP), known for targeting SR-B1, as a therapy for CLL. HDL NPs potently and selectively induce apoptotic cell death in primary CLL cells. HDL NPs had no effect on normal peripheral blood mononuclear cells from healthy individuals or patients with CLL. These data implicate SR-B1 as a target in CLL and HDL NPs as targeted monotherapy for CLL.


Assuntos
Apoptose/efeitos dos fármacos , Antígenos CD36/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Lipoproteínas HDL/metabolismo , Ligação Competitiva , Western Blotting , Antígenos CD36/antagonistas & inibidores , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipoproteínas HDL/síntese química , Lipoproteínas HDL/farmacologia , Masculino , Nanopartículas , Ligação Proteica
6.
J Immunol ; 197(6): 2522-31, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27534555

RESUMO

BCR signaling is a central pathogenetic pathway in chronic lymphocytic leukemia (CLL). Most CLL cells express BCRs of IgM and IgD isotypes, but the contribution of these isotypes to functional responses remains incompletely defined. We therefore investigated differences between IgM and IgD signaling in freshly isolated peripheral blood CLL cells and in CLL cells cultured with nurselike cells, a model that mimics the lymph node microenvironment. IgM signaling induced prolonged activation of ERK kinases and promoted CLL cell survival, CCL3 and CCL4 chemokine secretion, and downregulation of BCL6, the transcriptional repressor of CCL3 In contrast, IgD signaling induced activation of the cytoskeletal protein HS1, along with F-actin polymerization, which resulted in rapid receptor internalization and failure to support downstream responses, including CLL cell survival and chemokine secretion. IgM and IgD receptor downmodulation, HS1 and ERK activation, chemokine secretion, and BCL6 downregulation were also observed when CLL cells were cocultured with nurselike cells. The Bruton's tyrosine kinase inhibitor ibrutinib effectively inhibited both IgM and IgD isotype signaling. In conclusion, through a variety of functional readouts, we demonstrate very distinct outcomes of IgM and IgD isotype activation in CLL cells, providing novel insight into the regulation of BCR signaling in CLL.


Assuntos
Imunoglobulina D/metabolismo , Imunoglobulina M/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Linfócitos B/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Sobrevivência Celular/imunologia , Células Cultivadas , Microambiente Celular/imunologia , Quimiocina CCL3/imunologia , Quimiocina CCL3/metabolismo , Quimiocina CCL4/imunologia , Quimiocina CCL4/metabolismo , Regulação da Expressão Gênica , Humanos , Imunoglobulina D/imunologia , Imunoglobulina M/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Linfocítica Crônica de Células B/fisiopatologia , Linfonodos/citologia , Linfonodos/imunologia , Proteínas Tirosina Quinases/imunologia , Proteínas Proto-Oncogênicas c-bcl-6/genética , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia
7.
Oncotarget ; 7(27): 41725-41736, 2016 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-27203389

RESUMO

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of leukemic B cells in peripheral blood, bone marrow (BM) and lymphoid tissues, and by their recirculation between these compartments. We observed that circulating chromogranin A (CgA) and its N-terminal fragment (called vasostatin-1, CgA1-76), two neuroendocrine secretory polypeptides that enhance the endothelial barrier function, are present in variable amounts in the blood of CLL patients. Studies in animal models showed that daily administration of full-length human CgA1-439 (0.3 µg, i.v., or 1.5 µg/mouse, i.p.) can reduce the BM/blood ratio of leukemic cells in Eµ-TCL1 mice, a transgenic model, and decrease BM, lung and kidney infiltration in Rag2-/-γc-/- mice engrafted with human MEC1 CLL cells, a xenograft model. This treatment also reduced the loss of body weight and improved animal motility. In vitro, CgA enhanced the endothelial barrier integrity and the trans-endothelial migration of MEC1 cells, with a bimodal dose-response curve. Vasostatin-1, but not a larger fragment consisting of N-terminal and central regions of CgA (CgA1-373), inhibited CLL progression in the xenograft model, suggesting that the C-terminal region is crucial for CgA activity and that the N-terminal domain contains a site that is activated by proteolytic cleavage. These findings suggest that circulating full-length CgA and its fragments may contribute to regulate leukemic cell trafficking and reduce tissue infiltration in CLL.


Assuntos
Cromogranina A/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Idoso , Animais , Linhagem Celular Tumoral , Movimento Celular , Células Cultivadas , Cromogranina A/sangue , Cromogranina A/química , Progressão da Doença , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Inibidores da Bomba de Prótons/uso terapêutico
8.
Cell Rep ; 14(7): 1748-1760, 2016 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-26876171

RESUMO

The role of monocytes/macrophages in the development and progression of chronic lymphocytic leukemia (CLL) is poorly understood. Transcriptomic analyses show that monocytes/macrophages and leukemic cells cross talk during CLL progression. Macrophage depletion impairs CLL engraftment, drastically reduces leukemic growth, and favorably impacts mouse survival. Targeting of macrophages by either CSF1R signaling blockade or clodrolip-mediated cell killing has marked inhibitory effects on established leukemia also. Macrophage killing induces leukemic cell death mainly via the TNF pathway and reprograms the tumor microenvironment toward an antitumoral phenotype. CSF1R inhibition reduces leukemic cell load, especially in the bone marrow, and increases circulating CD20(+) leukemic cells. Accordingly, co-targeting TAMs and CD20-expressing leukemic cells provides a survival benefit in the mice. These results establish the important role of macrophages in CLL and suggest therapeutic strategies based on interfering with leukemia-macrophage interactions.


Assuntos
Apoptose/efeitos dos fármacos , Linfócitos B/imunologia , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Apoptose/imunologia , Linfócitos B/patologia , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ácido Clodrônico/farmacologia , Progressão da Doença , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/mortalidade , Lipossomos/farmacologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Cultura Primária de Células , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/imunologia , Transdução de Sinais , Análise de Sobrevida , Transplante Heterólogo , Microambiente Tumoral/efeitos dos fármacos
9.
Blood ; 127(16): 1987-97, 2016 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-26825709

RESUMO

Hypoxia-inducible transcription factors (HIFs) regulate a wide array of adaptive responses to hypoxia and are often activated in solid tumors and hematologic malignancies due to intratumoral hypoxia and emerging new layers of regulation. We found that in chronic lymphocytic leukemia (CLL), HIF-1α is a novel regulator of the interaction of CLL cells with protective leukemia microenvironments and, in turn, is regulated by this interaction in a positive feedback loop that promotes leukemia survival and propagation. Through unbiased microarray analysis, we found that in CLL cells, HIF-1α regulates the expression of important chemokine receptors and cell adhesion molecules that control the interaction of leukemic cells with bone marrow and spleen microenvironments. Inactivation of HIF-1α impairs chemotaxis and cell adhesion to stroma, reduces bone marrow and spleen colonization in xenograft and allograft CLL mouse models, and prolongs survival in mice. Of interest, we found that in CLL cells, HIF-1α is transcriptionally regulated after coculture with stromal cells. Furthermore, HIF-1α messenger RNA levels vary significantly within CLL patients and correlate with the expression of HIF-1α target genes, including CXCR4, thus further emphasizing the relevance of HIF-1α expression to CLL pathogenesis.


Assuntos
Comunicação Celular/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Leucemia Linfocítica Crônica de Células B/patologia , Microambiente Tumoral/genética , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Adesão Celular/genética , Quimiotaxia de Leucócito/genética , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Baço/metabolismo , Baço/patologia , Células Estromais/metabolismo , Células Estromais/patologia
11.
PLoS One ; 10(6): e0130195, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26110819

RESUMO

Immortalized cell lines representative of chronic lymphocytic leukemia (CLL) can assist in understanding disease pathogenesis and testing new therapeutic agents. At present, very few representative cell lines are available. We here describe the characterization of a new cell line (PCL12) that grew spontaneously from the peripheral blood (PB) of a CLL patient with progressive disease and EBV infection. The CLL cell origin of PCL12 was confirmed after the alignment of its IGH sequence against the "original" clonotypic sequence. The IGH gene rearrangement was truly unmutated and no CLL-related cytogenetic or genetic lesions were detected. PCL12 cells express CD19, CD20, CD5, CD23, low levels of IgM and IgD and the poor-outcome-associated prognostic markers CD38, ZAP70 and TCL1. In accordance with its aggressive phenotype the cell line is inactive in terms of LYN and HS1 phosphorylation. BcR signalling pathway is constitutively active and anergic in terms of p-ERK and Calcium flux response to α-IgM stimulation. PCL12 cells strongly migrate in vitro in response to SDF-1 and form clusters. Finally, they grow rapidly and localize in all lymphoid organs when xenotrasplanted in Rag2-/-γc-/- mice. PCL12 represents a suitable preclinical model for testing pharmacological agents.


Assuntos
Antígenos CD5/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Animais , Linhagem Celular Tumoral , Rearranjo Gênico , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Camundongos , Transplante de Neoplasias , Fenótipo , Proteína-Tirosina Quinase ZAP-70/metabolismo
12.
J Vis Exp ; (92): e51942, 2014 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-25350848

RESUMO

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease's biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as "spots" on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.


Assuntos
Proteínas Sanguíneas/metabolismo , Eletroforese em Gel Bidimensional/métodos , Leucemia Linfocítica Crônica de Células B/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Linfócitos B/patologia , Proteínas Sanguíneas/análise , Movimento Celular/fisiologia , Citoesqueleto/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Dados de Sequência Molecular , Fosforilação , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
13.
Semin Cancer Biol ; 24: 43-8, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23831274

RESUMO

The investigation on the mechanisms that govern the development and progression of cancer is constantly swaying between "seed" and "soil". Chronic lymphocytic leukemia (CLL) makes no exception. Its natural history, including response to treatment and drug resistance, is determined both by causal and influential genes and by the relationships that leukemic cells entertain with their supportive microenvironments. Therefore dissecting the role of microenvironment may provide new strategies of diagnosis and treatment. CLL, though phenotypically homogeneous, is clinically heterogeneous and despite major therapeutic advances remains incurable. Conceivably the host of new non-genotoxic drugs that operate at the forefront between tumor cells and their milieu will modify the present therapeutic perspective by re-shaping the tumor cell/microenvironment cross talk.


Assuntos
Carcinogênese/genética , Leucemia Linfocítica Crônica de Células B/genética , Microambiente Tumoral , Animais , Citoesqueleto/genética , Modelos Animais de Doenças , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Transdução de Sinais
14.
Blood ; 121(19): 3879-88, S1-8, 2013 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-23460614

RESUMO

B-cell receptor (BCR) triggering and responsiveness have a crucial role in the survival and expansion of chronic lymphocytic leukemia (CLL) clones. Analysis of in vitro response of CLL cells to BCR triggering allowed the definition of 2 main subsets of patients and lack of signaling capacity was associated with constitutive activation of extracellular-regulated kinases 1/2 (ERK1/2) and nuclear factor of activated T cells c1 (NF-ATc1), consistent with the idea that at least one group of CLL patients derives from the abnormal expansion of anergic B cells. In the present work, we further investigated the anergic subset of CLL (defined as the one with constitutive ERK1/2 phosphorylation) and found that it is characterized by low levels of surface immunoglobulin M and impairment of calcium mobilization after BCR engagement in vitro. Chronic BCR triggering promoted CLL cell survival selectively in phosphorylated ERK1/2 samples and the use of mitogen-activated protein kinase and NF-AT signaling inhibitors specifically induced apoptosis in this group of patients. Apoptosis induction was preceded by an initial phase of anergy reversal consisting in the loss of ERK phosphorylation and NF-AT nuclear translocation and by the restoration of BCR responsiveness, reinforcing the idea that the anergic program favors the survival of leukemic lymphocytes.


Assuntos
Linfócitos B , Anergia Clonal/imunologia , Leucemia Linfocítica Crônica de Células B/terapia , Terapia de Alvo Molecular/métodos , Animais , Antineoplásicos/uso terapêutico , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/fisiologia , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Cadeias gama de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/imunologia , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células Tumorais Cultivadas
15.
Blood ; 121(12): 2264-73, 2013 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-23325840

RESUMO

HS1 (hematopoietic cell-specific Lyn substrate-1) is a cytoskeletal interactor in the B-cell receptor (BCR) signaling pathway whose phosphorylation correlates with prognosis in Chronic Lymphocytic Leukemia (CLL). The differentially phosphorylated sites and the kinases that regulate HS1 activity in CLL remain poorly understood. We demonstrate that HS1 activity is differentially regulated by LYN kinase that, in a subset of patients, phosphorylates HS1 on Tyrosine (Y)397, resulting in its activation. This correlates with increased cytoskeletal functionality in terms of migration, adhesion and F-actin polymerization. In these patients, LYN is also activated on Y396 residue and its inhibition with the tyrosine kinase inhibitor Dasatinib abrogates HS1-Y397 phosphorylation. This results in the reduction of HS1 activation along with that of cytoskeletal effector VAV1 and the downstream kinase ERK also in the presence of BCR and CXC chemokine receptor CXCR4 stimulation. Interestingly, targeting the LYN/HS1 axis in vitro leads to the concomitant reduction of cytoskeletal activity, BCR signaling and cell survival in the subset of patients with activated LYN/HS1. In a transplantable mouse model based on the EµTCL1 transgenic mouse, LYN/HS1 signaling inhibition interferes with CLL progression and lymphoid organ infiltration. Thus LYN/HS1 axis marks distinct signaling profiles and cytoskeletal-related features that may represent valuable targets for cytoskeleton-targeted therapeutic intervention in CLL.


Assuntos
Antineoplásicos/uso terapêutico , Proteínas Sanguíneas/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Quinases da Família src/antagonistas & inibidores , Animais , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dasatinibe , Ativação Enzimática/efeitos dos fármacos , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/genética , Leucemia Linfocítica Crônica de Células B/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/administração & dosagem , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Tiazóis/administração & dosagem , Tiazóis/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/genética , Quinases da Família src/metabolismo
16.
Blood ; 118(25): 6618-25, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21876118

RESUMO

Monoclonal B-cell lymphocytosis (MBL) is classified as chronic lymphocytic leukemia (CLL)-like, atypical CLL, and CD5(-) MBL. The number of B cells per microliter divides CLL-like MBL into MBL associated with lymphocytosis (usually detected in a clinical setting) and low-count MBL detected in the general population (usually identified during population screening). After a median follow-up of 34 months we reevaluated 76 low-count MBLs with 5-color flow cytometry: 90% of CLL-like MBL but only 44.4% atypical CLL and 66.7% CD5(-) MBL persisted over time. Population-screening CLL-like MBL had no relevant cell count change, and none developed an overt leukemia. In 50% of the cases FISH showed CLL-related chromosomal abnormalities, including monoallelic or biallelic 13q deletions (43.8%), trisomy 12 (1 case), and 17p deletions (2 cases). The analysis of the T-cell receptor ß (TRBV) chains repertoire showed the presence of monoclonal T-cell clones, especially among CD4(high)CD8(low), CD8(high)CD4(low) T cells. TRBV2 and TRBV8 were the most frequently expressed genes. This study indicates that (1) the risk of progression into CLL for low-count population-screening CLL-like MBL is exceedingly rare and definitely lower than that of clinical MBL and (2) chromosomal abnormalities occur early in the natural history and are possibly associated with the appearance of the typical phenotype.


Assuntos
Linfócitos B/metabolismo , Aberrações Cromossômicas , Leucemia Linfocítica Crônica de Células B/genética , Linfocitose/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/patologia , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 17/genética , Progressão da Doença , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Hibridização in Situ Fluorescente , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/metabolismo , Contagem de Linfócitos , Linfocitose/diagnóstico , Linfocitose/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/metabolismo , Linfócitos T/patologia , Recombinação V(D)J/genética
17.
Semin Cancer Biol ; 20(6): 384-90, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20816789

RESUMO

CLL-like monoclonal B-cell lymphocytosis (MBL) shares a unique immunophenotype with chronic lymphocytic leukemia (CLL), and represents the vast majority of clonal B-cell expansions found in the peripheral blood of otherwise healthy subjects. Along with the improvement of laboratory techniques and the widespread availability of multiparameter flow cytometry, the finding of tiny aberrant B-cell populations became more frequent, prompting the need for clinical and biological definition of the nature of this condition and its relationship with leukemia development. MBL seems to be a melting-pot containing several entities, identical in terms of phenotype but with extremely different risks of leukemia development (from low to none) that seem to correlate with the number of B lymphocytes. CLL-like MBL observed in the clinical setting ("Clinical MBL"), usually being characterized by lymphocytosis, demonstrated a sizeable, even if low (1.1-1.4% per year), risk of leukemic progression, but represents a minority of all MBL cases. The vast majority of CLL-like MBL are detected in general population screenings and do not likely have a risk of CLL that is substantially higher than that of unaffected individuals. Interestingly, MBL frequency increases with age, being virtually undetectable under 40 years of age but being present in 50-75% of the people older than 90 years. It has been proposed that MBL could be interpreted as an epiphenomenon of a chronic and persistent antigenic stimulation. The (rare) possibility to evolve into a frank leukemia might then depend on biological and molecular factors insofar unknown that may modify the modality of cell reaction as well as the potential to acquire further genetic abnormalities. Therefore, the real challenge of the next years in the MBL research field is not to increase the sensitivity of detection, neither to implement screening protocols to be applied to the general population, rather to unravel the biologic features that, at individual level, will identify those (few) cases that are at risk of developing a progressive disease.


Assuntos
Linfócitos B/imunologia , Linfocitose/imunologia , Distribuição por Idade , Animais , Linfócitos B/patologia , Senescência Celular , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Linfocitose/patologia
18.
Leuk Lymphoma ; 51(8): 1371-4, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20687794

RESUMO

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in primary and secondary lymphoid tissues of CD5+ B cells that have the same B cell receptor (BCR) rearrangement. Genetic alterations and different stimuli originating from the microenvironment cooperate in the selection and expansion of the malignant clone. Molecular and functional analyses suggest that stimulation through the BCR affects the destiny of leukemic cells in terms of life or death. Microenvironmental signals are crucial for this process, inducing proliferation and leading to the survival and accumulation of leukemic cells within lymphoid organs. Nevertheless, a number of major biological issues still remain to be solved, including the relationships between cell proliferation and cell accumulation within lymphoid organs as well as the mechanisms that regulate CLL cell migration and recirculation between peripheral blood and lymphoid tissues. We focused on the role played by the cytoskeleton, given its relevance in controlling cellular shape, mobility, and homing. We hypothesize that hematopoietic cell-specific Lyn substrate 1 (HS1), a putative prognostic marker in CLL that interacts with distinct cytoskeleton adapters in leukemic B-lymphocytes, could regulate the CLL cell cytoskeleton.


Assuntos
Proteínas Sanguíneas/metabolismo , Citoesqueleto/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Movimento Celular , Forma Celular , Humanos , Receptores de Antígenos de Linfócitos B , Transdução de Sinais
19.
Blood ; 116(18): 3537-46, 2010 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-20530793

RESUMO

The function of the intracellular protein hematopoietic cell-specific Lyn substrate-1 (HS1) in B lymphocytes is poorly defined. To investigate its role in migration, trafficking, and homing of leukemic B lymphocytes we have used B cells from HS1(-/-) mice, the HS1-silenced human chronic lymphocytic leukemia (CLL) MEC1 cell line and primary leukemic B cells from patients with CLL. We have used both in vitro and in vivo models and found that the lack of expression of HS1 causes several important functional effects. In vitro, we observed an impaired cytoskeletal remodeling that resulted in diminished cell migration, abnormal cell adhesion, and increased homotypic aggregation. In vivo, immunodeficient Rag2(-/-)γ(c)(-/-) mice injected with HS1-silenced CLL B cells showed a decreased organ infiltration with the notable exception of the bone marrow (BM). The leukemic-prone Eµ-TCL1 transgenic mice crossed with HS1-deficient mice were compared with Eµ-TCL1 mice and showed an earlier disease onset and a reduced survival. These findings show that HS1 is a central regulator of cytoskeleton remodeling that controls lymphocyte trafficking and homing and significantly influences the tissue invasion and infiltration in CLL.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos B/patologia , Proteínas Sanguíneas/metabolismo , Movimento Celular , Proteínas de Ligação a DNA/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linfócitos B/citologia , Proteínas Sanguíneas/genética , Medula Óssea/patologia , Adesão Celular , Linhagem Celular Tumoral , Citoesqueleto/patologia , Citoesqueleto/ultraestrutura , Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Inativação Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Tumorais Cultivadas
20.
Blood ; 115(19): 3949-59, 2010 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-20203269

RESUMO

We investigated functional relationships between microRNA 221/222 (miR-221/222) cluster and p27, a key regulator of cell cycle, in chronic lymphocytic leukemia (CLL). The enforced expression of miR-221/222 in the CLL cell line MEC1 induced a significant down-regulation of p27 protein and conferred a proliferative advantage to the transduced cells that exhibited faster progression into the S phase of the cell cycle. Accordingly, expression of miR-221/miR-222 and p27 was found to be inversely related in leukemic cells obtained from peripheral blood (PB) of 38 patients with CLL. Interestingly, when miR-221/222 and p27 protein were evaluated in different anatomic compartments (lymph nodes or bone marrow) of the same patients, increased expression of the 2 miRNAs became apparent compared with PB. This finding was paralleled by a low expression of p27. In addition, when CLL cells were induced in vitro to enter cell cycle (eg, with cytosine phosphate guanine oligodeoxynucleotide), a significant increase of miR-221/222 expression and a marked down-regulation of p27 protein were evident. These data indicate that the miR-221/222 cluster modulates the expression of p27 protein in CLL cells and lead to suggest that miR-221/222 and p27 may represent a regulatory loop that helps maintaining CLL cells in a resting condition.


Assuntos
Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/genética , Leucemia Linfocítica Crônica de Células B/genética , MicroRNAs/genética , Western Blotting , Ciclo Celular , Imunofluorescência , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
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