RESUMEN
Richter's syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) into a high-grade B-cell malignancy. Molecular and functional studies have pointed out that CLL cells are close to the apoptotic threshold and dependent on BCL-2 for survival. However, it remains undefined how evasion from apoptosis evolves during disease transformation. Here, we employed functional and static approaches to compare the regulation of mitochondrial apoptosis in CLL and RS. BH3 profiling of 17 CLL and 9 RS samples demonstrated that RS cells had reduced apoptotic priming and lower BCL-2 dependence than CLL cells. While a subset of RS was dependent on alternative anti-apoptotic proteins and was sensitive to specific BH3 mimetics, other RS cases harbored no specific anti-apoptotic addiction. Transcriptomics of paired CLL/RS samples revealed downregulation of pro-apoptotic sensitizers during disease transformation. Albeit expressed, effector and activator members were less likely to colocalize with mitochondria in RS compared to CLL. Electron microscopy highlighted reduced cristae width in RS mitochondria, a condition further promoting apoptosis resistance. Collectively, our data suggest that RS cells evolve multiple mechanisms that lower the apoptotic priming and shift the anti-apoptotic dependencies away from BCL-2, making direct targeting of mitochondrial apoptosis more challenging after disease transformation.
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Apoptosis , Leucemia Linfocítica Crónica de Células B , Mitocondrias , Proteínas Proto-Oncogénicas c-bcl-2 , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Mitocondrias/metabolismo , Masculino , Femenino , Persona de Mediana EdadRESUMEN
Patients with chronic lymphocytic leukemia (CLL) relapsing on ibrutinib are often treated with the Bcl-2 inhibitor venetoclax. However, the transition from one agent to another poses some clinical challenges due to disease flares sometimes occurring right after ibrutinib interruption. Here, we describe three clinical vignettes highlighting two distinct patterns of ibrutinib-to-venetoclax transition. While patients following the favorable pattern transited to venetoclax without experiencing disease flare, the one patient who took the unfavorable path showed rapid disease rebound, with large-cell transformation occurring one week after ibrutinib interruption. A high burden of BTK and PLCG2 mutations was found only in patients with the favorable transition pattern, suggesting that removing BTK inhibition might be particularly harmful if CLL cells are progressing through mechanisms external to the BTK axis.
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Leucemia Linfocítica Crónica de Células B , Adenina/análogos & derivados , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Piperidinas/uso terapéutico , SulfonamidasRESUMEN
Mitochondria are critical organelles in the regulation of intrinsic apoptosis. As a general feature of blood cancers, different antiapoptotic members of the BCL-2 protein family localize at the outer mitochondrial membrane to sequester variable amounts of proapoptotic activators, and hence protect cancer cells from death induction. However, the impact of distinct anti-apoptotic members on apoptosis prevention, a concept termed anti-apoptotic dependence, differs remarkably across disease entities. Over the last two decades, several genetic and functional methodologies have been established to uncover the anti-apoptotic dependencies of the majority of blood cancers, inspiring the development of a new class of small molecules called BH3 mimetics. In this review, we highlight the rationale of targeting mitochondrial apoptosis in hematology, and provide a comprehensive map of the anti-apoptotic dependencies that are currently guiding novel therapeutic strategies. Cell-extrinsic and -intrinsic mechanisms conferring resistance to BH3 mimetics are also examined, with insights on potential strategies to overcome them. Finally, we discuss how the field of mitochondrial apoptosis might be complemented with other dimensions of precision medicine for more successful treatment of 'highly complex' hematologic malignancies.
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Neoplasias Hematológicas , Medicina de Precisión , Apoptosis , Biología , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Humanos , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
The regulatory role of protein tyrosine kinases in ß1- and ß2-integrin activation and in the survival of chronic lymphocytic leukemia (CLL) cells is well established. In contrast, the involvement of protein tyrosine phosphatases in CLL biology was less investigated. We show that selective activation of the protein tyrosine phosphatase receptor type γ (PTPRG) strongly suppresses integrin activation and survival in leukemic B cells isolated from patients with CLL. Activation of PTPRG specifically inhibits CXCR4- as well as BCR-induced triggering of LFA-1 and VLA-4 integrins and mediated rapid adhesion. Triggering of LFA-1 affinity is also prevented by PTPRG activity. Analysis of signaling mechanisms shows that activation of PTPRG blocks chemokine-induced triggering of JAK2 and Bruton's tyrosine kinase protein tyrosine kinases and of the small GTP-binding protein RhoA. Furthermore, activated PTPRG triggers rapid and robust caspase-3/7-mediated apoptosis in CLL cells in a manner quantitatively comparable to the Bruton's tyrosine kinase inhibitor ibrutinib. However, in contrast to ibrutinib, PTPRG-triggered apoptosis is insensitive to prosurvival signals generated by CXCR4 and BCR signaling. Importantly, PTPRG activation does not trigger apoptosis in healthy B lymphocytes. The data show that activated PTPRG inhibits, at once, the signaling pathways controlling adhesion and survival of CLL cells, thus emerging as a negative regulator of CLL pathogenesis. These findings suggest that pharmacological potentiation of PTPRG tyrosine-phosphatase enzymatic activity could represent a novel approach to CLL treatment.
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Agammaglobulinemia Tirosina Quinasa/metabolismo , Adhesión Celular/fisiología , Supervivencia Celular/fisiología , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Adhesión Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Integrina alfa4beta1/metabolismo , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Classic Hodgkin lymphoma (cHL) is a unique lymphoid neoplasm characterized by extensive immune infiltrates surrounding rare malignant Hodgkin Reed-Sternberg (HRS) cells. Different subsets of T and NK cells have long been recognized in the cHL microenvironment, yet their distinct contribution to disease pathogenesis has remained enigmatic. Very recently, novel platforms for high dimensional analysis of immune cells, such as single-cell RNA sequencing and mass cytometry, have revealed unanticipated insights into the composition of T- and NK-cell compartments in cHL. Advances in imaging techniques have better defined specific T-helper subpopulations physically interacting with neoplastic cells. In addition, the identification of novel cytotoxic subsets with an exhausted phenotype, typically enriched in cHL milieu, is shedding light on previously unrecognized immune evasion mechanisms. This review examines the immunological features and the functional properties of T and NK subsets recently identified in the cHL microenvironment, highlighting their pathological interplay with HRS cells. We also discuss how this knowledge can be exploited to predict response to immunotherapy and to design novel strategies to improve PD-1 blockade efficacy.
RESUMEN
α-Bisabolol (BSB) is a plant-derived sesquiterpene alcohol able to trigger regulated cell death in transformed cells, while deprived of the general toxicity in several mouse models. Here, we investigated the involvement of lysosomal and mitochondrial compartments in the cytotoxic effects of BSB, with a specific focus on the BH3-only activator protein BID. We found that BSB particularly accumulated in cancer cell lines, displaying a higher amount of lipid rafts as compared to normal blood cells. By means of western blotting and microscopy techniques, we documented rapid BSB-induced BID translocation to lysosomes and mitochondria, both of them becoming dysfunctional. Lysosomal membranes were permeabilized, thus blocking the cytoprotective autophagic flux and provoking cathepsin B leakage into the cytosol. Multiple flow cytometry-based experiments demonstrated the loss of mitochondrial membrane potential due to pore formation across the lipid bilayer. These parallel events converged on neoplastic cell death, an outcome significantly prevented by BID knockdown. Therefore, BSB promoted BID redistribution to the cell death executioner organelles, which in turn activated anti-autophagic and proapoptotic mechanisms. This is an example of how xenohormesis can be exploited to modulate basic cellular programs in cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Lisosomas/metabolismo , Mitocondrias/metabolismo , Sesquiterpenos Monocíclicos/farmacología , Autofagia/efectos de los fármacos , Bencimidazoles/farmacología , Carbocianinas/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular , Gangliósido G(M1)/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Lisosomas/efectos de los fármacos , Microdominios de Membrana/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Mitocondrias/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacosRESUMEN
In classical Hodgkin lymphoma (cHL), the significance of the interplay between Hodgkin and Reed-Sternberg cells (HRS) and reactive T cells remains poorly defined. By immunohistochemistry on bioptic cHL specimens, we found that HRS and surrounding T lymphocytes stained positive for IL-17 in 40% of cases. IL-17 was detectable in a similar proportion of patients' sera and correlated with disease burden. Supernatants of KM-H2 and HDLM-2 cHL cell lines guided preferential chemotaxis of CCR6+ T lymphocytes. Coculture of cHL cell lines with PBMC promoted the enrichment of Th17 lymphocytes and Foxp3+/IL-17+ cells, whereas T regulatory cells slightly decreased. Soluble CD30 downmodulated membrane CD30 expression on T cells and contributed to their polarization shift by stimulating IL-17 production and reducing IFN-γ synthesis. Thus, HRS and a number of reactive CD4+ T cells, attracted by tumor-secreted chemokines, produce an IL-17 tumor-shaped inflammatory milieu in a cHL subset.
Asunto(s)
Ligando CD30/inmunología , Linfocitos T CD4-Positivos/inmunología , Enfermedad de Hodgkin/inmunología , Interleucina-17/inmunología , Antígeno Ki-1/inmunología , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis , Ligando CD30/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Movimiento Celular , Proliferación Celular , Femenino , Estudios de Seguimiento , Enfermedad de Hodgkin/metabolismo , Enfermedad de Hodgkin/patología , Humanos , Interleucina-17/metabolismo , Antígeno Ki-1/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana Edad , Pronóstico , Células de Reed-Sternberg , Linfocitos T Reguladores/metabolismo , Adulto JovenRESUMEN
West Nile virus is a zoonotic agent causing life-threatening encephalitis in a proportion of infected patients. Older age, immunosuppression, and mutations in specific host genes (e.g., CCR5 delta-32 mutation) predispose to neuroinvasive infection. We report on two cases of severe West Nile encephalitis in recently-treated, different-aged, chronic lymphocytic leukemia patients. Both patients developed high-grade fever associated with severe neurological impairment. The younger one harboured germ-line CCR5 delta-32 mutation, which might have played a role in the pathogenesis of its neuroinvasive manifestations.
RESUMEN
Ibrutinib is increasingly adopted for treating lymphoid malignancies. While growing amounts of data pile up about Ibrutinib mechanism of action on neoplastic B cells, little is known about its impact on other immune cells. Here we investigated the effect of Ibrutinib on monocyte/macrophage functions. (1) Ibrutinib treatment of purified human monocytes affected both chemoattractant-triggered inside-out as well as integrin-mediated outside-in signaling events, thus provoking defective adhesion and spreading on purified integrin ligands, respectively. (2) In in vitro cell-culture experiments, Ibrutinib promoted a differentiation shift of monocytes to fibrocyte-like cells, characterized by the acquisition of a typical elongated cell morphology. Importantly, this clear-cut shape transition also occurred upon culturing monocytes with sera derived from Ibrutinib-treated patients, thus clearly suggesting that the drug concentrations achievable in vivo can generate the phenotypic shift. (3) Ibrutinib-induced fibrocyte-like cells showed adhesion deficiency, altered phagocytic properties, and, with respect to macrophages, they acquired the capability of generating larger amounts of reactive oxygen species, possibly displaying different metabolic activities. Taken together, our results indicate that Ibrutinib has profound effects on the monocyte/macrophage immunobiology. They may finally shed some light about the biological ground of several Ibrutinib-related toxicities.
Asunto(s)
Endotelina-1 , Mieloma Múltiple , Humanos , Pirimidinas , Receptor de Endotelina A , SulfonamidasRESUMEN
Bruton's tyrosine kinase (BTK) regulates the B-cell receptor (BCR) signaling pathway, which, in turn, plays a critical role in B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis. The BTK-specific inhibitor Ibrutinib blocks BCR signaling and is now approved as effective B-CLL therapy. Chemokines, such as the homeostatic chemokine CXCL12, play a central role in B-CLL pathogenesis and progression, by regulating CLL cell interaction with the stromal microenvironment, leading to cells survival and proliferation. In this study, we investigated, in normal versus CLL B-lymphocytes, the role of BTK in signal transduction activated by the CXCL12-CXCR4 signaling axis and its involvement in rapid integrin activation. We show that BTK is rapidly activated by CXCL12 in healthy as well as CLL B-lymphocytes, with a kinetic of tyr-phosphorylation coherent with rapid adhesion triggering. BTK inhibition prevents CXCL12-induced triggering of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. Furthermore, BTK inhibition blocks the activation of the small GTP-binding protein RhoA, controlling integrin affinity. Very importantly, we show that BTK tyr-phosphorylation and activation by CXCL12 depends on upstream activation of JAK2 tyrosine kinase. A comparative analysis of 36 B-CLL patients demonstrates that JAK2-dependent BTK regulatory role on integrin activation by CXCL12 is fully conserved in CLL cells. Finally, we show that the JAK2-BTK axis also regulates signaling to integrin activation by BCR. Thus, BTK and JAK protein tyrosine kinases (PTKs) manifest a hierarchical activity both in chemokine- as well as BCR-mediated integrin activation and dependent adhesion, potentially suggesting the possibility of combined therapeutic approaches to B-CLL treatment.
RESUMEN
CXCL12/CXCR4 axis relies on both heterotrimeric Gi protein and ß-arrestin coupling to trigger downstream responses. G protein activation allows for calcium flux, chemotaxis and early extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation, whereas ß-arrestin recruitment leads to late signaling, receptor desensitization and internalization. Together they may regulate the balance between transactivation and transinhibition of epithelial growth factor receptor 1 (HER1). Since we have previously noted significant differences between CXCL12 and its structural variant [N33A]CXCL12 in CXCR4 signaling, we sought to better characterize them by performing cAMP inhibition and ß-arrestin recruitment assays, as well as functional tests that separately investigate G protein and ß-arrestin-induced responses. [N33A]CXCL12 showed reduced potency both in Gαi coupling and ß-arrestin recruitment as compared to the wild type chemokine, acting as an unbiased ligand. While these findings translated into reduced potency within Gαi-dependent functions, ß-arrestin-dependent modules were affected in a more peculiar way. Unlike CXCL12, the mutant analogue did not restore HB-EGF-stimulated HER1 from CXCR4-induced transinhibition, and did not trigger the late wave of ERK1/2 phosphorylation. Instead, CXCR4 internalization was not impaired upon [N33A]CXCL12 stimulation. These differences highlight the novel opportunity to dissect CXCL12 signaling within the ß-arrestin layer, in which the mutant chemokine clearly favors the internalization module over the other pathways. Such functional selectivity has an impact on HER1 activation status and may play a relevant part in the crosstalk between tyrosine kinase and seven transmembrane receptors.
RESUMEN
The sesquiterpene α-bisabolol (α-BSB) is a cytotoxic agent against acute leukemia and chronic myeloid leukemia cells. Here the profile of α-BSB citotoxicity was evaluated ex vivo in primary mononuclear blood cells isolated from 45 untreated B-chronic lymphocytic leukemia (B-CLL) patients. We studied the effects of α-BSB by flow cytometric and western blotting techniques with the following findings: (1) α-BSB was an effective proapoptotic agent against B-CLL cells (IC50 42 ± 15 µM). It was also active, but to a lesser extent, on normal residual B cells and monocytes (IC50 68 ± 34 and 74 ± 28 µM, respectively; p < 0.01), while T-cells, though not achieving IC50, were nevertheless decreased. (2) Lipid raft content positively correlated with α-BSB cell sensitivity, while neither the phenotype of B-CLL cells nor the disease clinical stage did affect the sensitivity to α-BSB. (3) Flow cytometry analysis evidenced the induction of pores in mitochondrial and lysosomal membrane after 3- to 5-hour exposure of B-CLL cells to α-BSB, leading to apoptosis; in contrast, western blotting analysis showed inhibition of the autophagic flux. Therefore, according to cellular selectivity, α-BSB is a cytotoxic agent preferentially active against leukemic cells, while its lower activity on normal B cells, monocytes and T cells may account for an additive anti-inflammatory effect targeting the leukemia-associated pro-inflammatory microenvironment. Consistent with the observed effects on intracellular processes, α-BSB should be regarded as a dual agent, both activating mitochondrial-based apoptosis and inhibiting autophagy by disrupting lysosomes.
RESUMEN
New effective treatments are needed to improve outcomes for multiple myeloma (MM) patients. Receptors with restricted expression on plasma cells (PCs) represent attractive new therapeutic targets. The endothelin-1 (EDN1) axis, consisting of EDN1 acting through EDN-receptor A (EDNRA) and B (EDNRB), was previously shown to be overexpressed in several tumours, including MM. However, there is incomplete understanding of how EDN1 axis regulates MM growth and response to therapy. Besides EDNRA, the majority of MM cell lines and primary malignant PCs express high levels of EDNRB and release EDN1. Similarly, bone-marrow microenvironment cells also secrete EDN1. Investigating the extent of epigenetic dysregulation of EDNRB gene in MM, we found that hypermethylation of EDNRB promoter and subsequent down-regulation of EDNRB gene was observed in PCs or B lymphocytes from healthy donors compared to EDNRB-expressing malignant PCs. Pharmacological blockade with the dual EDN1 receptor antagonist bosentan decreased cell viability and MAPK activation of U266 and RPMI-8226 cells. Interestingly, the combination of bosentan and the proteasome inhibitor bortezomib, currently approved for MM treatment, resulted in synergistic cytotoxic effects. Overall, our data has uncovered EDN1-mediated autocrine and paracrine mechanisms that regulate malignant PCs growth and drug response, and support EDN1 receptors as new therapeutic targets in MM.
Asunto(s)
Antagonistas de los Receptores de la Endotelina A/farmacología , Mieloma Múltiple/sangre , Receptor de Endotelina A/sangre , Adulto , Anciano , Anciano de 80 o más Años , Comunicación Autocrina/fisiología , Bortezomib/farmacología , Bosentán , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Metilación de ADN , ADN de Neoplasias/genética , Sinergismo Farmacológico , Endotelina-1/sangre , Endotelina-1/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida/métodos , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Células Plasmáticas/metabolismo , Regiones Promotoras Genéticas , Receptor de Endotelina A/genética , Sulfonamidas/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/patologíaRESUMEN
A new immunological investigation was carried out to study the association between non-Hodgkin lymphoma and Simian virus 40 (SV40). To this end, a new indirect ELISA was employed with two mimotopes from SV40 large T antigen (Tag), the viral oncoprotein, to analyse for specific reactions to antibodies in sera from non-Hodgkin lymphoma patients and controls, represented by healthy subjects (HS) and breast carcinoma (BC) patients. This study allowed us to assay a new sera collection from non-Hodgkin lymphoma patients (NHL, n = 254). To verify the association between NHL and SV40 Tag, two totally independent cohorts were analysed: NHL1 n = 150 and NHL2 n = 104. The epidemiological survey included sera from HS1, n = 150; HS2, n = 104 and BC, n = 78. This new indirect ELISA revealed that antibodies against SV40 Tag mimotopes are detectable in NHL1 and NHL2 sera with a prevalence of 37 and 36%, respectively. The prevalence of SV40-antibodies detected in both NHL1 and NHL2 cohorts differs statistically from controls, at 19% for HS1 (p < 0.01), HS2 (p < 0.05) and BC patients (p < 0.05). This study, carried out with an immunological assay with specific Tag oncoprotein mimotopes of Simian virus 40, reports the presence of IgG antibodies against the large Tumour antigen in non-Hodgkin lymphomas for the first time. Our immunological data with two independent NHL cohorts show a statistically significant association between Simian virus 40 Tag and non-Hodgkin lymphoma. These results suggest that SV40-positive non-Hodgkin lymphomas could be treated differently from those tested SV40-negative.
Asunto(s)
Anticuerpos Antivirales/inmunología , Antígenos Virales de Tumores/inmunología , Linfoma no Hodgkin/inmunología , Proteínas Oncogénicas/metabolismo , Virus 40 de los Simios/inmunología , Adulto , Animales , Femenino , Humanos , Linfoma no Hodgkin/patología , Ratones , Ratones Transgénicos , Persona de Mediana Edad , PrevalenciaRESUMEN
Tumor dormancy is a poorly understood stage in cancer progression characterized by mitotic cycle arrest in G0/G1 phase and low metabolism. The cells survive in a quiescent state and wait for appropriate environmental conditions to begin proliferation again giving rise to metastasis. Despite their key role in cancer development and metastasis, the knowledge about their biology and origin is still very limited due to the poorness of established in vitro models that faithfully recapitulated tumor dormancy. Using at least three cycles of 1% O2 hypoxia and reoxygenation, we establish and characterize the hypoxia-resistant human breast cancer cell line chMDA-MB-231 that can stably survive under 1% O2 condition by entering into dormant state characterized by arrest in G0/G1 phase and low metabolism. This dormant state is reversible since once replaced in normoxia the cells recover the proliferation rate in 2 weeks. We show that chronic hypoxia induces autophagy that may be the survival mechanism of chMDA-MB-231 cells. Furthermore, the data in this work demonstrate that cycling hypoxic/reoxygenation stress selects MDA-MB-231 population that presents the cancer stem-like phenotype characterized by CD24- /CD44+ /ESA+ expression and spheroid forming capacity. We believe that our study presents a promising approach to select dormant breast cancer cells with stem-like phenotype using the hypoxia/reoxygenation regimen that may represent an area with profound implications for therapeutic developments in oncology. J. Cell. Biochem. 118: 3237-3248, 2017. © 2017 Wiley Periodicals, Inc.
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Neoplasias de la Mama/metabolismo , Fase G1 , Células Madre Neoplásicas/metabolismo , Fase de Descanso del Ciclo Celular , Neoplasias de la Mama/patología , Hipoxia de la Célula , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Metástasis de la Neoplasia , Células Madre Neoplásicas/patologíaRESUMEN
BACKGROUND: Differentiation, proliferation, and chemotaxis responses stem from biological programs that recognize checkpoints at the level of membrane receptor internalization and shedding. Therefore, receptor trafficking represents a crucial regulator of cell functions. METHODS: Here, we present a survey of analyses of receptor internalization vs. shedding based on simple flow cytometry-based techniques. A relevant basic observation is that a fluorochrome-bearing antibody bound to a specific receptor that is translocated from the membrane to the cytoplasm continues to emit light, i.e., the cell remains equally positive for that marker even if the receptor is strongly downregulated or no longer detectable on the membrane. In contrast, fluorescence is lost following receptor shedding. RESULTS: The combined uses of standardized hyperosmolar sucrose, acidic treatment and flow cytometry staining at different times allows for fully informative studies of the internalization or shedding pattern of a given receptor. The procedure can be simplified into a straightforward, simple-to-use, and flexible flow cytometry method based on two sequential steps with in-between receptor stimulation. This method obviates the need for time-consuming fluorescence techniques and even confocal microscopy. CONCLUSION: We validate this procedure via comparisons of three receptors, i.e., CXCR4, CD30, and CD25, with membrane trafficking patterns that are involved in biological functions that are relevant to immunity and cancer. © 2016 International Clinical Cytometry Society.
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Citometría de Flujo/métodos , Subunidad alfa del Receptor de Interleucina-2/inmunología , Antígeno Ki-1/inmunología , Linfocitos/inmunología , Receptores CXCR4/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Quimiocina CXCL12/genética , Quimiocina CXCL12/inmunología , Endocitosis/efectos de los fármacos , Espacio Extracelular/metabolismo , Fluoresceína-5-Isotiocianato/química , Colorantes Fluorescentes/química , Expresión Génica , Humanos , Subunidad alfa del Receptor de Interleucina-2/genética , Antígeno Ki-1/genética , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Ligandos , Linfocitos/efectos de los fármacos , Linfocitos/patología , Presión Osmótica , Ficoeritrina/química , Cultivo Primario de Células , Unión Proteica , Transporte de Proteínas/efectos de los fármacos , Receptores CXCR4/genética , Sacarosa/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The sesquiterpene α-bisabolol (α-BSB) has been shown to be an effective cytotoxic agent for a variety of human cancer cells in culture and animal models. However, much of its intracellular action remains elusive. We evaluated the cytotoxic action of α-BSB against CML-T1, Jurkat and HeLa cell lines, as preclinical models for myeloid, lymphoid and epithelial neoplasias. The approach included single cell analysis (flow cytometry, immunocytology) combined with cytotoxicity and proliferation assays to characterize organelle damage, autophagy, cytostatic effect, and apoptosis. The study focuses on the relevant steps in the cytotoxic cascade triggered by α-BSB: (1) the lipid rafts through which α-BSB enters the cells, (2) the opening of pores in the mitochondria and lysosomes, (3) the activation of both caspase-dependent and caspase-independent cell death pathways, (4) the induction of autophagy and (5) apoptosis. The effectiveness of α-BSB as an agent against tumor cells is grounded on its capability to act on different layers of cell regulation to elicit different concurrent death signals, thereby neutralizing a variety of aberrant survival mechanisms leading to treatment resistance in neoplastic cell.
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Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Lisosomas/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Sesquiterpenos/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células HeLa , Humanos , Células Jurkat , Lisosomas/metabolismo , Mitocondrias/metabolismo , Sesquiterpenos MonocíclicosRESUMEN
Chemokines participate to B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis by promoting cell adhesion and survival in bone marrow stromal niches and mediating cell dissemination to secondary lymphoid organs. In this study we investigated the role of JAK protein tyrosine kinases (PTK) in adhesion triggering by the CXC chemokine CXCL12 in normal versus CLL B-lymphocytes. We demonstrate that CXCL12 activates JAK2 in normal as well as CLL B-lymphocytes, with kinetics consistent with rapid adhesion triggering. By using complementary methodologies of signal transduction interference, we found that JAK2 mediates CXCL12-triggered activation of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. We also show that JAK2 mediates the activation of the small GTP-binding protein RhoA, in turn controlling LFA-1 affinity triggering by CXCL12. Importantly, comparative analysis of 41 B-CLL patients did not evidence JAK2 functional variability between subjects, thus suggesting that JAK2, differently from other signaling events involved in adhesion regulation in B-CLL, is a signaling molecule downstream to CXCR4 characterized by a conserved regulatory role. Our results reveal JAK2 as critical component of chemokine signaling in CLL B-lymphocytes and indicate JAK inhibition as a potentially useful new pharmacological approach to B-CLL treatment.
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Quimiocina CXCL12/metabolismo , Integrinas/metabolismo , Janus Quinasa 2/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos B/metabolismo , Western Blotting , Adhesión Celular/fisiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Persona de Mediana Edad , ARN Interferente Pequeño , Transducción de Señal/fisiología , TransfecciónRESUMEN
The CD30/CD30L signalling system has been implicated in the pathogenesis of several autoimmune and inflammatory conditions. In rheumatoid arthritis (RA), soluble CD30 (sCD30) levels reflect the recruitment of CD30(+) T cells into the inflamed joints and correlate with a positive response to immunosuppressive therapy. The aim of our report was to clarify the role of CD30/CD30L signalling system in the pathogenesis of RA. Our analysis of the CD30L(+) T cell subsets in peripheral blood (PB) and synovial fluid (SF) of RA patients and of the related cytokine profiles suggests the involvement of CD30/CD30L signalling in polarization of T cells towards a Th17 phenotype with proinflammatory features. Moreover, in RA SF nearly 50% of Treg cells express CD30, probably as an attempt to downmodulate the ongoing inflammation. We also show here that the engagement of CD30L on neutrophils stimulated with CD30/Fc chimera may play a crucial role in RA inflammation since activated neutrophils release IL-8, thus potentially amplifying the local inflammatory damage. In conclusion, the results obtained suggest that the complex CD30/CD30L signalling pathway is implicated in the pathogenesis and progression of RA synovitis through a concerted action on several immune effector cells.