Fernandez-Luna, J.L. (J.L.)
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- Lack of Bcr-Abl point mutations in chronic myeloid leukemia patients in chronic phase before imatinib treatment is not predictive of response(Ferrata Storti Foundation, 2003) Montiel-Duarte, C. (Cristina); Andreu, E.J. (Enrique José); Fernandez-Luna, J.L. (J.L.); Larrayoz, M.J. (María J.); Prosper-Cardoso, F. (Felipe); Calasanz-Abinzano, M.J. (Maria Jose); Odero, M.D. (Maria Dolores); Aguirre-Ena, X. (Xabier); Fontalba, A. (A.)
- NALP1 is a transcriptional target for cAMP-response-element-binding protein (CREB) in myeloid leukaemia cells(Biochemical Society, 2004) Andreu, E.J. (Enrique José); Fernandez-Luna, J.L. (J.L.); Richard, C. (Carlos); Sanz, C. (C.); Prosper-Cardoso, F. (Felipe); Calasanz-Abinzano, M.J. (Maria Jose)NALP1 (also called DEFCAP, NAC, CARD7) has been shown to play a central role in the activation of inflammatory caspases and processing of pro-IL1β (pro-interleukin-1β). Previous studies showed that NALP1 is highly expressed in peripheral blood mononuclear cells. In the present study, we report that expression of NALP1 is absent from CD34+ haematopoietic blast cells, and its levels are upregulated upon differentiation of CD34+ cells into granulocytes and to a lesser extent into monocytes. In peripheral blood cells, the highest levels of NALP1 were observed in CD3+ (T-lymphocytes), CD15+ (granulocytes) and CD14+ (monocytes) cell populations. Notably, the expression of NALP1 was significantly increased in the bone marrow blast cell population of some patients with acute leukaemia, but not among tissue samples from thyroid and renal cancer. A search for consensus sites within the NALP1 promoter revealed a sequence for CREB (cAMP-response-element-binding protein) that was required for transcriptional activity. Moreover, treatment of TF1 myeloid leukaemia cells with protein kinase C and protein kinase A activators induced CREB phosphorylation and upregulated the mRNA and protein levels of NALP1. Conversely, ectopic expression of a dominant negative form of CREB in TF1 cells blocked the transcriptional activity of the NALP1 promoter and significantly reduced the expression of NALP1. Thus NALP1 is transcriptionally regulated by CREB in myeloid cells, a mechanism that may contribute to modulate the response of these cells to pro-inflammatory stimuli.
- Induction of Nod2 in Myelomonocytic and Intestinal Epithelial Cells via Nuclear Factor-kB Activation(JBC Papers in Press, 2002) Pipaon, C. (Carlos); Inoharas, N. (N.); Fernandez-Luna, J.L. (J.L.); Nuñez, G. (Gabriel); Gutierrez, O. (Olga); Prosper-Cardoso, F. (Felipe); Fontalba, A. (A.); Ogura, Y. (Yasunori)Nod2, a member of the Apaf1/Nod protein family, confers responsiveness to bacterial products and activates NF-kB, a ranscription factor that plays a central role in innate immunity. Recently, genetic variation in Nod2 has been associated with susceptibility to Crohn’s disease. Here, we report that expression of Nod2 is induced upon differentiation of CD34+ hematopoietic progenitor cells into granulocyte or monocyte/macrophages. In peripheral blood cells, the highest levels of Nod2 were observed in CD14+ (monocytes), CD15+ (granulocytes), and CD40+/CD86+ (dendritic cells) cell populations. Notably, stimulation of myeloblastic and epithelial cells with bacterial lipopolysaccharide or TNF resulted in up-regulation of Nod2. A search for consensus sites within the Nod2 promoter revealed a NF-kB binding element that was required for transcriptional activity in response to TNF . Moreover, ectopic expression of p65 induced transactivation, whereas that of dominant-negative I B blocked the transcriptional activity of the Nod2 promoter. Upon stimulation with TNF or lipopolysaccharide, both p50 and p65 subunits of NF-kB were bound to the Nod2 promoter. Thus, Nod2 expression is enhanced by proinflammatory cytokines and bacterial components via NF-kB, a mechanism that may contribute to the amplification of the innate immune response and susceptibility to inflammatory disease.
- Involvement of miRNAs in the differentiation of human glioblastoma multiforme stem-like cells(Public Library of Science, 2013) Grande, L. (Lara); Huse, J.T. (Jason T.); Alonso-Roldán, M.M. (Marta María); Nogueira, L. (Lorena); Martinez-Climent, J.A. (José Ángel); Tejada-Solis, S. (Sonia); Aznar, M.A. (María Ángela); Aldaz-Arrieta, B. (Beatriz); Diez-Valle, R. (Ricardo); Fernandez-Luna, J.L. (J.L.); Raquel; Sagardoy, A. (Ainara); Guruceaga, E. (Elizabeth)Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process.
- Nuclear factor κ B is activated in myelodysplastic bone marrow cells(Ferrata Storti Foundation, 2002) Fernandez-Luna, J.L. (J.L.); Richard, C. (Carlos); Sanz, C. (C.); Prosper-Cardoso, F. (Felipe)
- Characterization of 8p21.3 chromosomal deletions in B-cell lymphoma: TRAIL-R1 and TRAIL-R2 as candidate dosage-dependent tumor suppressor genes(American Society of Hematology, 2005) Benito-Boilos, A. (Alberto); DeLeeuw, R. (R.); Dyer, M.J.S. (Martin J. S.); Rosenwald, A. (Andreas); Pinkel, D. (Daniel); Karran, E.L. (E. L.); Martinez-Climent, J.A. (José Ángel); Blesa, D. (David); Gesk, S. (Stefan); Fernandez-Luna, J.L. (J.L.); Balasas, T. (T.); Siebert, R. (Reiner); Mestre-Escorihuela, C. (Cinta); Esteller, M. (Manel); Staudt, L.M. (Louis M.); Rubio-Moscardo, F. (Fanny); Climent, J. (Javier); Schilhabel, M. (Markus); Martinez, J.I. (José I.)Deletions of chromosome 8p are a recurrent event in B-cell non-Hodgkin lymphoma (B-NHL), suggesting the presence of a tumor suppressor gene. We have characterized these deletions using comparative genomic hybridization to microarrays, fluorescence in situ hybridization (FISH) mapping, DNA sequencing, and functional studies. A minimal deleted region (MDR) of 600 kb was defined in chromosome 8p21.3, with one mantle cell lymphoma cell line (Z138) exhibiting monoallelic deletion of 650 kb. The MDR extended from bacterial artificial chromosome (BAC) clones RP11-382J24 and RP11-109B10 and included the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor gene loci. Sequence analysis of the individual expressed genes within the MDR and DNA sequencing of the entire MDR in Z138 did not reveal any mutation. Gene expression analysis and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) showed down-regulation of TRAIL-R1 and TRAIL-R2 receptor genes as a consistent event in B-NHL with 8p21.3 loss. Epigenetic inactivation was excluded via promoter methylation analysis. In vitro studies showed that TRAIL-induced apoptosis was dependent on TRAIL-R1 and/or -R2 dosage in most tumors. Resistance to apoptosis of cell lines with 8p21.3 deletion was reversed by restoration of TRAIL-R1 or TRAIL-R2 expression by gene transfection. Our data suggest that TRAIL-R1 and TRAIL-R2 act as dosage-dependent tumor suppressor genes whose monoallelic deletion can impair TRAIL-induced apoptosis in B-cell lymphoma.
- Identification of c-Kit gene mutations in patients with polycythemia vera(Elsevier, 2006) Fernandez-Luna, J.L. (J.L.); Real, P.J. (Pedro J.); Richard, C. (Carlos); Prosper-Cardoso, F. (Felipe); Aguirre-Ena, X. (Xabier); Fontalba, A. (A.)Imatinib mesylate has recently been reported to have clinical activity in the treatment of polycythemia vera (PV), suggesting the involvement of one of the kinases targeted by this inhibitor, including c-Kit and PDGFR. Activating c-Kit mutations have been identified in patients with mastocytosis and other myeloid disorders such as acute myeloid leukemia. Thus, we wanted to analyze the presence of mutations of c-Kit in polycythemia vera patients. We found that 7 out of 20 patients carried missense mutations in the c-Kit gene whereas no sequence variation was detected in 15 healthy controls.
- Imatinib inhibits proliferation of Ewing tumor cells mediated by the stem cell factor/KIT receptor pathway, and sensitizes cells to vincristine and doxorubicin-induced apoptosis(American Association for Cancer Research, 2004) Alava, E. (Enrique) de; Martin-Algarra, S. (Salvador); Gonzalez, I. (Iranzu); Sierrasesumaga, L. (Luis); Andreu, E.J. (Enrique José); Fernandez-Luna, J.L. (J.L.); Inoges, S. (Susana); Gaboli, M. (M.); Panizo, A. (Ángel); Prosper-Cardoso, F. (Felipe); Pardo, J. (Javier); Fontalba, A. (A.)Purpose and Experimental Design: The stem cell factor/ KIT receptor loop may represent a novel target for molecular- based therapies of Ewing tumor. We analyzed the in vitro impact of KIT blockade by imatinib in Ewing tumor cell lines. Results: KIT expression was detected in 4 of 4 Ewing tumor cell lines and in 49 of 110 patient samples (44.5%) by immunohistochemistry and/or Western blot analysis. KIT expression was stronger in Ewing tumors showing EWSFLI1 nontype 1 fusions. Despite absence of c-kit mutations, constitutive and ligand-inducible phosphorylation of KIT was found in all tumor cell lines, indicating an active receptor. Treatment with KIT tyrosine kinase inhibitor imatinib (0.5–20 M) induced down-regulation of KIT phosphorylation and dose response inhibition of cell proliferation (IC50, 12–15 M). However, imatinib administered alone at doses close to IC50 for growth inhibition (10 M) did not induce a significant increase in apoptosis. We then analyzed if blockade of KIT loop through imatinib (10 M) was able to increase the antitumor in vitro effect of doxorubicin (DXR)and vincristine (VCR), drugs usually used in Ewing tumor treatment. Addition of imatinib decreased in 15–20 and 15–36% of the proliferative rate of Ewing tumor cells exposed to DXR and VCR, respectively, and increased in 15 and 30% of the apoptotic rate of Ewing tumor cells exposed to the same drugs. Conclusions: Inhibition of Ewing tumor cell proliferation by imatinib is mediated through blockade of KIT receptor signaling. Inhibition of KIT increases sensitivity of these cells to DXR and VCR. This study supports a potential role for imatinib in the treatment of Ewing tumor.