Papers by Cristina Falcone
Nuclear factor (NF)-iB is a master regulator of pro-inflammatory genes and is upregulated in huma... more Nuclear factor (NF)-iB is a master regulator of pro-inflammatory genes and is upregulated in human immunodeficiency virus 1 (HIV-1) infection. Mechanisms underlying the NF-iB deregulation by HIV-1 are relevant for immune dysfunction in AIDS. We report that in single round HIV-1 infection, or single-pulse PMA stimulation, the HIV-1 Tat transactivator activated NF-iB by hijacking the inhibitor IiB-a and by preventing the repressor binding to the NF-iB complex. Moreover, Tat associated with the p65 subunit of NF-iB and increased the p65 DNA-binding affinity and transcriptional activity. The arginineand cysteine-rich domains of Tat were required for IiB-a and p65 association, respectively, and for sustaining the NF-iB activity. Among an array of NF-iB-responsive genes, Tat mostly activated the MIP-1a expression in a p65-dependent manner, and bound to the MIP-1a NF-iB enhancer thus promoting the recruitment of p65 with displacement of IiB-a; similar findings were obtained for the NF-iB-r...
The UN1 monoclonal antibody recognized the UN1 antigen as a heavily sialylated and O-glycosylated... more The UN1 monoclonal antibody recognized the UN1 antigen as a heavily sialylated and O-glycosylated protein with the apparent molecular weight of 100–120 kDa; this antigen was peculiarly expressed in fetal tissues and several cancer tissues, including leukemic T cells, breast, and colon carcinomas. However, the lack of primary structure information has limited further investigation on the role of the UN1 antigen in neoplastic transformation. In this study, we have identified the UN1 antigen as CD43, a transmembrane sialoglycoprotein involved in cell adhesion, differentiation, and apoptosis. Indeed, mass spectrometry detected two tryptic peptides of the membrane-purified UN1 antigen that matched the amino acidic sequence of the CD43 intracellular domain. Immunological cross-reactivity, migration pattern in monoand bidimensional electrophoresis, and CD43 gene-dependent expression proved the CD43 identity of the UN1 antigen. Moreover, the monosaccharide GalNAc-Olinked to the CD43 peptide...
Cell Death & Disease
Increasing evidence supports the involvement of IBTK in cell survival and tumor growth. Previousl... more Increasing evidence supports the involvement of IBTK in cell survival and tumor growth. Previously, we have shown that IBTK RNA interference affects the wide genome expression and RNA splicing in cell-type specific manner. Further, the expression of IBTK gene progressively increases from indolent to aggressive stage of chronic lymphocytic leukemia and decreases in disease remission after therapy. However, the role of IBTK in tumorigenesis has not been elucidated. Here, we report that loss of the murine Ibtk gene raises survival and delays tumor onset in Eμ-myc transgenic mice, a preclinical model of Myc-driven lymphoma. In particular, we found that the number of pre-cancerous B cells of bone marrow and spleen is reduced in Ibtk −/− Eμ-myc mice owing to impaired viability and increased apoptosis, as measured by Annexin V binding, Caspase 3/7 cleavage assays and cell cycle profile analysis. Instead, the proliferation rate of pre-cancerous B cells is unaffected by the loss of Ibtk. We observed a direct correlation between Ibtk and myc expression and demonstrated a Myc-dependent regulation of Ibtk expression in murine B cells, human hematopoietic and nonhematopoietic cell lines by analysis of ChIP-seq data. By tet-repressible Myc system, we confirmed a Mycdependent expression of IBTK in human B cells. Further, we showed that Ibtk loss affected the main apoptotic pathways dependent on Myc overexpression in pre-cancerous Eμ-myc mice, in particular, MCL-1 and p53. Of note, we found that loss of IBTK impaired cell cycle and increased apoptosis also in a human epithelial cell line, HeLa cells, in Myc-independent manner. Taken together, these results suggest that Ibtk sustains the oncogenic activity of Myc by inhibiting apoptosis of murine pre-cancerous B cells, as a cell-specific mechanism. Our findings could be relevant for the development of IBTK inhibitors sensitizing tumor cells to apoptosis.
Scientific Reports, 2015
Immune activation and chronic inflammation are hallmark features of HIV infection causing T-cell ... more Immune activation and chronic inflammation are hallmark features of HIV infection causing T-cell depletion and cellular immune dysfunction in AIDS. Here, we addressed the issue whether HIV-1 Tat could affect T cell development and acute inflammatory response by generating a transgenic mouse expressing Tat in lymphoid tissue. Tat-Tg mice showed thymus atrophy and the maturation block from DN4 to DP thymic subpopulations, resulting in CD4 + and CD8 + T cells depletion in peripheral blood. In Tat-positive thymus, we observed the increased p65/NF-κB activity and deregulated expression of cytokines/chemokines and microRNA-181a-1, which are involved in T-lymphopoiesis. Upon LPS intraperitoneal injection, Tat-Tg mice developed an abnormal acute inflammatory response, which was characterized by enhanced lethality and production of inflammatory cytokines. Based on these findings, Tat-Tg mouse could represent an animal model for testing adjunctive therapies of HIV-1-associated inflammation and immune deregulation. The Human Immunodeficiency Virus type-1 (HIV-1), the ethiological agent of the acquired immune deficiency syndrome (AIDS), preferentially infects CD4 + cells of the immune system such as T-lymphocytes, monocytes, macrophages and dendritic cells, causing chronic immune activation and inflammation 1,2. The HIV-1 Tat protein is essentially required for viral replication, and is a major pathogenic factor of AIDS-related immunological and neurological disorders 3. As transcriptional transactivator, Tat binds to the 5′-untranslated RNA leader region of HIV-1, interleukin-6 (IL-6) and TNF-α , and enhances their expression through interaction with transcription factors, such as TBP, NF-κ B and Sp1, and the histone acetyltransferases p300/CBP and P/CAF. Tat also recruits the cyclin T1/CDK9 complex to phosphorylate the carboxyl-terminus of the large subunit of RNA polymerase II, promoting elongation. Further, Tat affects cell signalling and metabolism by physical interaction with intracellular targets and membrane receptors, such as integrins, Flk1/KDR receptor and chemokine receptors 4,5 .
The Journal of biological chemistry, Jan 16, 2015
The human Inhibitor of Bruton's tyrosine kinase isoform α (IBtkα) is a BTB protein encoded by... more The human Inhibitor of Bruton's tyrosine kinase isoform α (IBtkα) is a BTB protein encoded by the IBTK gene, which maps to chromosomal locus 6q14.1, a mutational hot spot in lymphoproliferative disorders. Here, we demonstrate that IBtkα forms a CRL3(IBTK) complex promoting its self-ubiquitylation. We identified the tumor suppressor Pdcd4 as IBtkα interactor and ubiquitylation substrate of CRL3(IBTK) for proteasomal degradation. Serum-induced degradation of Pdcd4 required both IBtkα and Cul3, indicating that CRL3(IBTK) regulated the Pdcd4 stability in serum signalling. By promoting Pdcd4 degradation, IBtkα counteracted the suppressive effect of Pdcd4 on translation of reporter luciferase mRNAs with stem-loop structured or unstructured 5'UTR. IBtkα depletion by RNAi caused Pdcd4 accumulation, and decreased the translation of Bcl-xL mRNA, a well-known target of Pdcd4 repression. By characterizing CRL3(IBTK) as a novel ubiquitin ligase, this study provides new insights into regu...
Nucleic Acids Research, 2012
The plasmids pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65ΔC(1-318) and pRc/CMV-3xHA-p65ΔN(122-551) were gen... more The plasmids pRc/CMV-3xHA-p65, pRc/CMV-3xHA-p65ΔC(1-318) and pRc/CMV-3xHA-p65ΔN(122-551) were generated by PCR-amplification of p65 cDNA from pRc/CMVp65, followed by ligation to the HindIII/XbaI-digested pCMV4-3xHA (Addgene, Cambridge, MA, USA). The plasmids p3xFLAG-CMV-Tat T,N(23,24)A and p3xFLAG-CMV-Tat K(50,51)A were generated by sitedirected mutagenesis of p3xFLAG-CMV-Tat. The plasmids pGEX-2T-Tat T,N(23,24)A and pGEX-2T-Tat K(50,51)A were generated by PCR-amplification of Tat nucleotide sequence from p3xFLAG-CMV-Tat T,N(23,24)A and p3xFLAG-CMV-Tat K(50,51)A, followed by ligation to the BamHI/EcoRI-digested pGEX-2T
Nanoscale, 2010
1. Nanoparticles fabrication 2. Payload evaluation 3. Dissolution and release profiles 4. Multiva... more 1. Nanoparticles fabrication 2. Payload evaluation 3. Dissolution and release profiles 4. Multivalent loading 5. Targeting specifity on A20 Cells 6. Cell cycle analysis 7. In vitro cytotoxicity assay 8. In vivo cytotoxicity assay
Molecular Cancer Therapeutics, 2014
CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and different... more CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer-mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancerassociated mimotopes for immunotherapy. Mol Cancer Ther; 13(3); 1-11. Ó2013 AACR.
Blood, 2010
B-cell lymphoma is a clonal expansion of neoplastic cells that may result in fatal outcomes. Here... more B-cell lymphoma is a clonal expansion of neoplastic cells that may result in fatal outcomes. Here, we report the in vivo targeting and growth inhibition of aggressive A20 murine B-cell lymphoma by idiotype-specific peptide pA20-36. pA20-36 was selected from random peptide libraries and bound specifically to the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, as shown by histology and positron emission tomographic analysis. BCR cross-linking of A20 cells with pA20-36 resulted in massive apoptosis of targeted tumor cells and in an increased survival of the diseased animals without any detectable evidence of toxicity. The pA20-36 treatment reverted the immune suppression of the tumor microenvironment as shown by reduced expression of vascular endothelial growth factor, interleukin-10, and transforming growth factor-β cytokines together with a lower number of CD11b+Gr-1+ inhibitor myeloid-derived suppressor cells and Foxp3+CD4+ Treg cells. Furthermore, pA20-36 tr...
Blood, 2011
The inhibitor of Bruton tyrosine kinase γ (IBtkγ) is a negative regulator of the Bruton tyrosine ... more The inhibitor of Bruton tyrosine kinase γ (IBtkγ) is a negative regulator of the Bruton tyrosine kinase (Btk), which plays a major role in B-cell differentiation; however, the mechanisms of IBtkγ-mediated regulation of Btk are unknown. Here we report that B-cell receptor (BCR) triggering caused serine-phosphorylation of IBtkγ at protein kinase C consensus sites and dissociation from Btk. By liquid chromatography and mass-mass spectrometry and functional analysis, we identified IBtkγ-S87 and -S90 as the critical amino acid residues that regulate the IBtkγ binding affinity to Btk. Consistently, the mutants IBtkγ carrying S87A and S90A mutations bound constitutively to Btk and down-regulated Ca2+ fluxes and NF-κB activation on BCR triggering. Accordingly, spleen B cells from Ibtkγ−/− mice showed an increased activation of Btk, as evaluated by Y551-phosphorylation and sustained Ca2+ mobilization on BCR engagement. These findings identify a novel pathway of Btk regulation via protein kin...
Molecular & Cellular …, 2011
The UN1 monoclonal antibody recognized the UN1 antigen as a heavily sialylated and O-glycosylated... more The UN1 monoclonal antibody recognized the UN1 antigen as a heavily sialylated and O-glycosylated protein with the apparent molecular weight of 100120 kDa; this antigen was peculiarly expressed in fetal tissues and several cancer tissues, including leukemic T ...
International Journal of Molecular Sciences, 2012
The Bridging Sheet domain of HIV-1 gp120 is highly conserved among the HIV-1 strains and allows H... more The Bridging Sheet domain of HIV-1 gp120 is highly conserved among the HIV-1 strains and allows HIV-1 binding to host cells via the HIV-1 coreceptors. Further, the bridging sheet domain is a major target to neutralize HIV-1 infection. We rationally designed four linear peptide epitopes that mimic the three-dimensional structure of bridging sheet by using molecular modeling. Chemically synthesized peptides BS3 and BS4 showed a fair degree of antigenicity when tested in ELISA with IgG purified from HIV + broadly neutralizing sera while the production of synthetic peptides BS1 and BS2 failed due to their high degree of hydrophobicity. To overcome this limitation, we linked all four BS peptides to the COOH-terminus of GST protein to test both their antigenicity and immunogenicity. Only the BS1 peptide showed good antigenicity; however, no envelope specific antibodies were elicited upon mice immunization. Therefore we performed further analyses by linking BS1 peptide to the NH2-terminus of the E2 scaffold from the Geobacillus Stearothermophylus PDH complex. The E2-BS1 fusion peptide showed good antigenic results, however only one immunized rabbit elicited good antibody titers towards both the monomeric and oligomeric viral envelope glycoprotein (Env). In addition, moderate neutralizing antibodies response was elicited against two HIV-1 clade B and one clade C primary isolates. These preliminary data validate the peptide mimotope approach as a promising tool to obtain an effective HIV-1 vaccine.
PLoS ONE, 2013
Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and s... more Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and spatial control of gene expression. Initiation of protein synthesis requires at least 13 translation initiation factors to assemble the 80S ribosomal initiation complex. Loss of translation control may result in cell malignant transformation. Here, we asked whether translational initiation factors could be regulated by NF-kB transcription factor, a major regulator of genes involved in cell proliferation, survival, and inflammatory response. We show that the p65 subunit of NF-kB activates the transcription of eIF4H gene, which is the regulatory subunit of eIF4A, the most relevant RNA helicase in translation initiation. The p65dependent transcriptional activation of eIF4H increased the eIF4H protein content augmenting the rate of global protein synthesis. In this context, our results provide novel insights into protein synthesis regulation in response to NF-kB activation signalling, suggesting a transcription-translation coupled mechanism of control.
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Papers by Cristina Falcone