Pax5 Activates Oncogenic Transcription Factors of the Ets Family by Repressing the mir-15a/16-1 microRNA Cluster.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 346-346
Author(s):  
Elaine Y. Chung ◽  
Diana Cozma ◽  
Duonan Yu ◽  
Michael Dews ◽  
Erik A. Wentzel ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cell ◽  
Western Blotting ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Down Regulation ◽  
Protein Levels ◽  
Ets Family ◽  
Post Transcriptional Regulation

Abstract We have recently demonstrated that Pax5 promotes B-lymphomagenesis by upregulating key components of B-cell receptor signaling [Cozma et al, J Clin Inv, 117 (8), 2007]. Gene regulation by Pax5 often involves complex formation with other oncogenic transcription factors of the Ets family, namely Myb and Ets1. We determined that expression of these proteins themselves depends on the presence of Pax5, as seen in human diffuse large B-cell lymphomas with Pax5 knockdown and murine lymphomas with epigenetic silencing of Pax5 [Yu et al, Blood, 101:1950–1955, 2003; Johnson et al, Nat Immunol, 5:853–861, 2004]. Upon reconstitution with the Pax5 gene, Myb and Ets1 levels increase sharply. This occurs with little increase in steady-state mRNA levels, suggesting post-transcriptional regulation, possibly by microRNAs. To test this hypothesis, we compared miRNA profiles of Pax5-deficieint and sufficient cells and discovered that several miRNAs are indeed repressed by Pax5. Among them is the miR-15a/16-1 cluster whose predicted targets include both Myb and Ets1. Consistent with this prediction, forced expression of miR-15a/16 brings down Myb and Ets1 protein levels. This is accompanied by impaired Pax5 function and overall suppression of B-lymphomagenesis. Thus, Ets family members (along with previously identified bcl-2) are key targets of the miR-15a/16 locus, a tumor suppressor in chronic lymphocytic leukemia. Interplay between Pax5, Myb/Ets1, and miR-15a/16-1. (A) Upregulation of Myb and Ets 1 in tumors over-expressing Pax5ER fusion, as compared to control GFP-only neoplasms. (B) Down-regulation of Myb and Ets1 in Pax5 tumors engineered to over-express the miR-15a/16-1 cluster. All panels depict Western blotting. Interplay between Pax5, Myb/Ets1, and miR-15a/16-1. (A) Upregulation of Myb and Ets 1 in tumors over-expressing Pax5ER fusion, as compared to control GFP-only neoplasms. (B) Down-regulation of Myb and Ets1 in Pax5 tumors engineered to over-express the miR-15a/16-1 cluster. All panels depict Western blotting.

scholarly journals Targeted inhibition of eIF4A suppresses B-cell receptor-induced translation and expression of MYC and MCL1 in chronic lymphocytic leukemia cells

2021 ◽  
Author(s):  
Sarah Wilmore ◽  
Karly-Rai Rogers-Broadway ◽  
Joe Taylor ◽  
Elizabeth Lemm ◽  
Rachel Fell ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mrna Translation ◽  
Cell Receptor ◽  
Memory B Cells ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Mrna Stabilization

AbstractSignaling via the B-cell receptor (BCR) is a key driver and therapeutic target in chronic lymphocytic leukemia (CLL). BCR stimulation of CLL cells induces expression of eIF4A, an initiation factor important for translation of multiple oncoproteins, and reduces expression of PDCD4, a natural inhibitor of eIF4A, suggesting that eIF4A may be a critical nexus controlling protein expression downstream of the BCR in these cells. We, therefore, investigated the effect of eIF4A inhibitors (eIF4Ai) on BCR-induced responses. We demonstrated that eIF4Ai (silvestrol and rocaglamide A) reduced anti-IgM-induced global mRNA translation in CLL cells and also inhibited accumulation of MYC and MCL1, key drivers of proliferation and survival, respectively, without effects on upstream signaling responses (ERK1/2 and AKT phosphorylation). Analysis of normal naïve and non-switched memory B cells, likely counterparts of the two main subsets of CLL, demonstrated that basal RNA translation was higher in memory B cells, but was similarly increased and susceptible to eIF4Ai-mediated inhibition in both. We probed the fate of MYC mRNA in eIF4Ai-treated CLL cells and found that eIF4Ai caused a profound accumulation of MYC mRNA in anti-IgM treated cells. This was mediated by MYC mRNA stabilization and was not observed for MCL1 mRNA. Following drug wash-out, MYC mRNA levels declined but without substantial MYC protein accumulation, indicating that stabilized MYC mRNA remained blocked from translation. In conclusion, BCR-induced regulation of eIF4A may be a critical signal-dependent nexus for therapeutic attack in CLL and other B-cell malignancies, especially those dependent on MYC and/or MCL1.

STAT3 Silencing as a Novel Therapeutic Strategy for Activated B Cell-Type Diffuse Large B-Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 926-926
Author(s):  
Anna Scuto ◽  
Maciej Kujawski ◽  
Claudia Kowolik ◽  
Hua Yu ◽  
Stephen Forman ◽  
...  
Keyword(s):  
B Cell ◽  
Target Genes ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mrna Levels ◽  
Down Regulation ◽  
Protein Levels ◽  
Stat3 Signaling ◽  
Marrow Stroma ◽  
And Control

Abstract Abstract 926 Among the non-Hodgkin's lymphomas, the diffuse large B cell lymphoma (DLBCL) represents the most frequent (30%) of the aggressive lymphomas. Persistent STAT3 signaling contributes to malignant progression in many diverse human tumors. IL-6 and IL-10 are major activators of STAT3 signaling and are important in the pathophysiology of DLBCL. STAT3 has been found to be persistently active in activated B cells (ABC), which are non-germinal center-derived DLBCL cells. We studied the consequences of STAT3 inhibition on multiple biological functions in two representative human cell lines of this group, Ly3 and Ly10 cells. For this purpose, we established stably transduced STAT3 shRNA-expressing lentivirus Ly3 cells, control lentivirus Ly3 cells, STAT3 shRNA-expressing lentivirus Ly10 cells and control lentivirus Ly10 cells. The stable expression of STAT3 shRNA results in 40-50% reduction of total STAT3 protein levels in the STAT3 shRNA lentivirus Ly3 cells compared to the control lentivirus cells. STAT3 down-regulation induced inhibition of cell proliferation (approximately 40%). Ly3 cells respond to IL-10 more than to IL-6 in terms of proliferation; both cytokines induced less proliferation in the STAT3 shRNA lentivirus Ly3 cells compared to the control lentivirus Ly3 cells. Similar results were obtained in Ly10 cells, which respond more to IL-6 than to IL-10 in terms of proliferation. We analyzed by quantitative real-time PCR the mRNA levels of different STAT3 target genes and observed significant reduction in mRNA levels of Mcl-1, Bcl-xL and Survivin in STAT3 shRNA lentivirus Ly3 cells, as well as significant reduction of Cyclin D2 and up-regulation of STAT1 in shRNA lentivirus Ly10 cells. Comparison of these gene expression profiles with data obtained from other B-cell lymphoma cell lines revealed that silencing of STAT3 resulted in down-regulation of different STAT3 target genes in a cell-dependent manner. We also observed that both STAT3 and control lentivirus Ly3 cells have the same protein levels of c-Myc; nevertheless STAT3 silencing resulted in inhibition of IL-10-inducible upregulation of c-Myc. We next investigated the effect of STAT3 inhibition on adhesion to bone marrow stroma and chemotaxis. STAT3 shRNA lentivirus Ly3 cells adhered less to the stroma layer than control cells, and the longer they were cocultured with the stroma cells in the presence of serum-free media the more they lost the ability to adhere. Moreover, STAT3 shRNA lentivirus Ly3 cells had decreased capacity to migrate toward SDF-1 alpha, an important factor that mediates proliferation, survival, chemotaxis, migration and adhesion into bone marrow stroma. Radiation, in combination with chemotherapy, is one of the therapies used for DLBCL patients. We therefore investigated whether STAT3 down-regulation sensitized Ly3 cells to radiation. Radiation induced a higher accumulation of phospho-H2A.X (first sentinel event following DNA damage such as DSBs) and apoptosis in STAT3 shRNA lentivirus cells compared to control cells. Moreover, IL-6 and IL-10 protected the STAT3 shRNA lentivirus Ly3 cells less than the control cells from the induction of phospho-H2A.X following radiation. We further investigated the effect of STAT3 silencing in animal models of Ly3 lymphoma (Nude or NOD-SCID mice). Tumors in control lentivirus Ly3-bearing mice grew robustly, whereas tumors in STAT3 shRNA lentivirus Ly3-bearing mice regressed 5 days after injection. This tumor regression was associated with Caspase-3-dependent apoptosis, significant reduction of STAT3 target genes at the protein level such as Mcl-1, c-Myc and Survivin (approximately 40% to 60% inhibition), and reduction of cytokine production such as IL-10, IL-15, Leptin and Thrombopoietin. Taken together, these results suggest that inhibition of STAT3 is a potential promising approach in the therapy of ABC-type DLBCL. Disclosures: No relevant conflicts of interest to declare.

The Human Pre-B Cell Receptor Signaling Cascade Is Regulated Via PI-3Kinase and MAPK Pathway

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1314-1314
Author(s):  
Kolandaswamy Anbazhagan ◽  
Vincent Fuentes ◽  
Eliane Bissac ◽  
Remy Nyga ◽  
Naomi Taylor ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
Nuclear Translocation ◽  
Accessory Pathway ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Signaling Cascade ◽  
Down Regulation ◽  
Bcr Signaling

Abstract Abstract 1314 Background: Pre-B cell receptor (pre-BCR) constitutes a major check point in the early steps of mouse and human B cell development. Several functions have been attributed to this receptor which include a delivery of proliferation and survival signals, increased sensitivity to interleukin-7 (IL-7) and down modulation of recombinase activating genes (RAG) and surrogate light chain (SLC) encoding genes. Pre-BCR is also involved in shaping the VH repertoire and preventing autoimmunity. Finally, there is increasing evidence that pre-BCR might be implicated in leukemogenesis. Most of the functions of pre-BCR have been predicted based on studies in knockout mice and leukemic cell lines. In a previous study we have shown that pre-BCR aggregation resulted in the activation of src and Syk kinases which in turn activated the PI-3K/Akt, Btk, PLCγ-2 and Ras/MAPK. In this study, we examined the pre-BCR signalling cascade using human normal primary pre-B cells with a particular focus on transcription factors activation and Rag modulation and their regulatory aspects. Methods: Pre-B cells were sorted from adult human bone marrow samples, treated or not with inhibitors of Syk (BAY61–3606), Akt (LY294002) and MEKK1 (UO126) prior to crosslink the pre-BCR by means of F(ab')2 anti-μHC. The effect of Pre-BCR signaling was examined by quantifying the transcript levels of Rag1, Rag2, E2A, EBF1, Pax5, FoxO1 and FoxO3, IRF4/8. Activation of transcription factors such as NF-κB p50, c-Fos, IRF4 and FoxO3A, was assessed by analyzing their nuclear translocation by immunofluorescence microscopy. Results: We show that NF-κB p50 is translocated into nucleus within 3h after pre-BCR stimulation. Crosslinking of pre-BCR also resulted in an enhancement of nuclear c-Fos translocation. BAY61-3606 (Syk inhibitor) treatment resulted in complete apoptosis (100 % cell death within 48h). Although treatment of normal pre-B cells with LY294002 or U0126 did not alter cell survival, nuclear translocation of pre-BCR-induced p50 NF-κB was prevented by former and enhanced by later. Conversely, c-Fos nuclear expression was inhibited by U0126 and slightly but consistently enhanced by LY294002 in association with a decrease in its cytoplasmic location. Pre-BCR stimulation also induced IRF4 translocation to the nucleus. Pre-BCR stimulation also resulted in the down regulation of Rag1 (− 48 %, P<0.01), Pax5 (− 40%, P<0.01) and E2A (− 35 %, P< 0.01) transcripts, whereas EBF1 and FoxO1 and 3 expression remained unchanged. In LY294002-treated cells, Rag1/Rag2 expression was up regulated (+130%, P< 0.01 and +251%, P< 0.01, respectively) following pre-BCR crosslinking, whereas in the presence of U0126 the pre-BCR induced Rag1/Rag2 down modulation remained unchanged. Conclusion: Our results indicate that the pre-BCR has the potential to promote pre-B cell proliferation, survival and differentiation by activating NF-kB, c-Fos and IRF4. It also has the ability to protect pre-B cells from genome instability by down-regulating Rag1/2, probably through down modulation of Pax5 and E2A. We bring evidence that PI-3 K/Akt pathway plays a crucial role in the regulation of the pre-BCR signaling cascade and that Akt-mediated NF-kB and c-Fos activation is antagonized by MAPK. Up-regulation of Rag transcripts upon Akt inhibition suggests either a feed-back negative loop or a dual effect of pre-BCR on Rag expression with an Akt-dependent Rag down regulation and an accessory pathway that enhances Rag expression. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Down-regulation of CXCR4 and CD62L in Chronic Lymphocytic Leukemia Cells Is Triggered by B-Cell Receptor Ligation and Associated with Progressive Disease

2009 ◽  
Vol 69 (16) ◽  
pp. 6387-6395 ◽  
Author(s):  
Amalia Vlad ◽  
Pierre-Antoine Deglesne ◽  
Rémi Letestu ◽  
Stéphane Saint-Georges ◽  
Nathalie Chevallier ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Progressive Disease ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Down Regulation ◽  
Receptor Ligation

scholarly journals Constitutive Expression of the B7h Ligand for Inducible Costimulator on Naive B Cells Is Extinguished after Activation by Distinct B Cell Receptor and Interleukin 4 Receptor–mediated Pathways and Can Be Rescued by CD40 Signaling

2002 ◽  
Vol 196 (1) ◽  
pp. 97-108 ◽  
Author(s):  
Linda Liang ◽  
Evelyn M. Porter ◽  
William C. Sha
Keyword(s):  
B Cells ◽  
B Cell ◽  
Constitutive Expression ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Cytokine Signaling ◽  
Mrna Levels ◽  
Down Regulation ◽  
Inducible Costimulator ◽  
Activated B Cells

The recently described ligand–receptor pair, B7h–inducible costimulator (ICOS), is critical for germinal center formation and antibody responses. In contrast to the induced expression of the related costimulatory ligands B7.1 and B7.2, B7h is constitutively expressed on naive B cells and is surprisingly extinguished after antigen engagement and interleukin (IL)-4 cytokine signaling. Although signaling through both B cell receptor (BCR) and IL-4 receptor (R) converge on the extinction of B7h mRNA levels, BCR down-regulation occurs through Ca2+ mobilization, whereas IL-4R down-regulation occurs through a distinct Stat6-dependent pathway. During antigen-specific B cell activation, costimulation through CD40 signaling can reverse both BCR- and IL-4R–mediated B7h down-regulation. These data suggest that the CD40–CD40 ligand signaling pathway regulates B7h expression on activated B cells and may control whether antigen-activated B cells can express B7h and costimulate cognate antigen–activated T cells through ICOS.

SLP76 Is Ectopically Expressed in Chronic Lymphocytic Leukemia Cells and Involves in B-Cell Receptor Signaling

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1719-1719
Author(s):  
Yair Herishanu ◽  
Nili Dorozella ◽  
Mika Shapiro ◽  
Chava Perry ◽  
Ben-Zion Katz
Keyword(s):  
B Cell ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Tcr Signaling ◽  
Bcr Signaling

Abstract In the last decade, the B-cell receptor (BCR) has emerged as a pivotal stimulus in CLL pathogenesis. The BCR responsiveness in CLL cells is heterogeneous among patients and correlates with disease aggressiveness. Here we show for the first time, that SLP76 a key scaffold protein in T-cell receptor (TCR) signaling, is ectopically expressed in CD19+ purified CLL cells (purity >95%) in the majority of patients, and is co-expressed with other components of the TCR pathway, including LCK and ZAP70. SLP76 mRNA levels correlated with its protein expression and SLP76 protein levels were higher in unmutated IGHV and ZAP70+ CLL cells. SLP76 was found to be functionally active in CLL cells, as it becomes phosphorylated in response to BCR engagement in a time dependent manner. Activation with anti-IgM antibody results in phosphorylation of SLP76 on the positive regulatory tyrosine residue Y128 residue with increased physical association with Btk, peaking after 15 minutes. The negative regulatory residue S376 became phosphorylated only after 45', concomitantly with downregulation of the tyrosine phosphorylation. SLP76 phosphorylation in response to BCR engagement did not correlate with total ZAP70 expression. Pre-incubation of CLL cells with the LCK kinase inhibitor LCKi and with the SYK inhibitor R406, inhibited SLP76 phosphorylation in response to BCR activation, while the BTK inhibitor-ibrutinib had no effect. These suggest that LCK and SYK, but not ZAP70, play a central role in the upstream signaling involved in SLP76 activation in CLL cells. Knockdown of SLP76 in CLL cells resulted in decreased induction of BTK and PLCγ2 phosphorylation after BCR activation with anti-IgM. Consistent with our findings that SLP76 in involved in BCR signaling in CLL cells, we found that high total SLP76 expression was associated with a shorter time to first progression or need for treatment. In conclusion, SLP76 is ectopically expressed in CLL cells and plays a role in BCR signaling in those cells. Disclosures No relevant conflicts of interest to declare.

BRCA1-Down-Regulation In Chronic Myeloid Leukemia (CML) Occurs Via The Deubiquitinase / Tumor Suppressor BRCA1-Associated Protein 1 (BAP1)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3813-3813
Author(s):  
Fatima Dkhissi ◽  
Djamel Aggoune ◽  
Julien Pontis ◽  
Marie Laure Bonnet ◽  
Marie Claude Meunier ◽  
...  
Keyword(s):  
Dna Repair ◽  
Tumor Suppressor ◽  
Western Blotting ◽  
Genetic Instability ◽  
Leukemic Cells ◽  
Mrna Levels ◽  
Down Regulation ◽  
Brca1 Protein ◽  
Protein Levels ◽  
Qrt Pcr

Abstract BCR-ABL has been shown to lead to a genetic instability in leukemic cells either directly by inducing oxidative stress or indirectly by compromising DNA repair mechanisms. BRCA1 is a major DNA repair gene as it promotes homologous recombination and plays thereby a critical role for preserving genomic integrity. We have previously reported that BCR-ABL down-regulates BRCA1 protein using a post-transcriptional mechanism (Deutsch et al, Blood 2003). The precise mechanism of this down regulation had not been established so far. BAP1 (BRCA1 associated protein-1) is a tumor suppressor gene that encodes a nuclear ubiquitin carboxy-terminal hydrolase, which interacts with BRCA1 protein and with many other cell cycle regulators. BAP1 is mutated in hereditary cancers and the overexpression of a deleted form of BAP1 has been shown to lead to a myelodysplastic syndrome in mice (Dey et al, Science 2012). In a gene profiling analysis of the human UT7 cells expressing BCR-ABL, we have discovered that the expression of BAP-1 is down-regulated as compared to parental UT7 cells. Using qRT-PCR and Western blotting analyses, we have confirmed the reduction of BAP1 transcript and protein levels in UT7 cells expressing BCR-ABL. This occurs in a tyrosine kinase dependent manner as exposure to Imatinib reverted BCR-ABL-associated BAP1 down-regulation. To determine the effects of BAP1 complementation in leukemic cells, we have transfected UT7-BCR-ABL cells with a BAP1 expression vector. The enforced expression of BAP1 in BCR-ABL expressing cells restored BRCA1 protein levels without affecting its mRNA level. As BAP1 is a deubiquitinase, we wondered whether there was an increased ubiquitination of BRCA1 in BCR-ABL expressing cells, due to a BAP1 deficiency. In the UT7-BCR-ABL model, we have performed immunoprecipitation of BRCA1 followed by Western blotting using anti-ubiquitin antibodies. These experiments revealed that BRCA1 was highly ubiquitinylated in BCR-ABL-expressing cells as compared to parental UT7 cells, explaining potentially its down-regulation in CML at the protein level via a proteasome-related mechanism. We next wished to validate these findings in primary human CML samples using qRT-PCR. In a cohort of newly diagnosed chronic phase CML patients before any therapy (n= 21) blood mRNA levels of BAP1 were significantly reduced ( p=0.0032, Mann Whitney Test ) as compared to normal controls (n= 8). Thus, our report reveals for the first time loss of BAP1 expression as a mechanism of the down-regulation of the DNA repair protein BRCA1 in CML. In addition to its contribution to genetic instability, BAP1 could be directly involved in the pathophysiology of CML due to its interactions with epigenetic factors such as polycomb proteins. The molecular mechanisms of BAP1 downregulation in BCR-ABL-expressing leukemic cells is under investigation. Disclosures: Guilhot: Novartis, BMS, Ariad, Pfizer: Honoraria. Turhan:BMS, Novartis: Honoraria, Research Funding.

Loss of PU.1 expression is associated with defective immunoglobulin transcription in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease

Blood ◽  
2002 ◽  
Vol 99 (8) ◽  
pp. 3060-3062 ◽  
Author(s):  
Franziska Jundt ◽  
Katharina Kley ◽  
Ioannis Anagnostopoulos ◽  
Kristina Schulze Pröbsting ◽  
Axel Greiner ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cell ◽  
Family Member ◽  
Hodgkin Disease ◽  
Immunoglobulin Genes ◽  
Down Regulation ◽  
Enhancer Activity ◽  
Ets Family

Abstract Immunoglobulin transcription is impaired in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin disease (cHD). We recently demonstrated that defective immunoglobulin promoter transcription correlates with the down-regulation of the B-cell transcription factors Oct2 and BOB.1/OBF.1. These results prompted us to investigate whether immunoglobulin enhancer activity is also impaired in HRS cells and whether as yet unidentified factors could be necessary for immunoglobulin enhancer activity in HRS cells of cHD. Here we analyzed 30 cases of cHD for expression of the Ets family member PU.1 that is known to collaborate with multiple transcription factors and to regulate expression of immunoglobulin genes. We show that PU.1 is not expressed in primary and cultured HRS cells. Reintroduction of PU.1 and Oct2 in cultured HRS cells restored the activity of cotransduced immunoglobulin enhancer constructs. Our study identifies PU.1 deficiency as a recurrent defect in HRS cells that might contribute to their impairment of immunoglobulin transcription.

Modulation of CD20 Expression in Rituximab-Sensitive (RSCL) and Rituximab Resistant Cell Lines (RRCL) Using IL-4 and Bryostatin-1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2619-2619 ◽  
Author(s):  
Ping-Chiao Tsai ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
Scott H. Olejniczak ◽  
Bangia Naveen ◽  
Myron S. Czuczman
Keyword(s):  
Transcription Factors ◽  
B Cell ◽  
Western Blotting ◽  
Flow Cytometric Analysis ◽  
Regulation Of Transcription ◽  
Bryostatin 1 ◽  
Down Regulation ◽  
Cd20 Antigen ◽  
Number Of Patients ◽  
Cd20 Expression

Abstract While the use of rituximab in combination with chemotherapy resulted in an improved survival among various subtypes of B-cell lymphomas, a significant number of patients fail to respond or relapse as a consequence of intrinsic or acquired resistance. A change in CD20 antigen density expression is a potential mechanism to explain rituximab resistance. Moreover, several groups of investigators are focused in understanding the mechanisms that regulate CD20 expression and to develop therapeutic strategies to up-regulate CD20 expression (i.e. IL-4, GM-CSF or Bryostatin-1). Up-regulation of CD20 in DB and Ramos cells by Bryostatin-1 was found to be PKC and Erk dependent. In an attempt to characterize the mechanisms responsible for rituximab resistance we developed several RRCL derived from rituximab-sensitive RL and Raji cells. We have demonstrated a significant down-regulation of CD20mRNA and CD20 surface antigen in RRCL when compared to RSCL. In our present work we evaluated the mechanisms involved in the mRNA down-regulation of CD20 and the potential of IL-4 and Bryostatin-1 in modulating CD20 antigen expression among a panel of RRCL. To this end, RSCL and RRCL were treated with either 5ng/ml of IL-4 or 3 different doses of Bryostatin-1(1, 3 or 5ng/ml). Nuclear and cytosolic extract were also obtained from RSCL RL and RRCL RL-4RH after 24, 48 and 74 hrs exposure to IL-4 (5ng/ml) or control. Differences in the expression of key regulatory transcription factors for B-cell lymphocyte development (PU.1, Oct-2, Pax5, E2A and EBF) were studied by Western Blotting. Previously, we found a significant down-regulation of CD20 antigen in the RRCL. An up-regulation of cytosolic and surface CD20 was detected in RRCL exposed to either IL-4 or Bryostatin by western blotting and flow cytometric analysis. Besides, a higher expression of Pax5, PU.1, EBF was found in nuclear fractions of RRCL when compared to RSCL. In vitro exposure of RRCL to IL-4 decreases the expression of PU.1, Oct-2, Pax5 and EBF in nuclear extracts from RSCL or RRCL when compared with controls treated cells. Our data suggest that rituximab resistance is associated with the up-regulation of transcription factors PU.1, Oct-2, Pax5 and EBF and concomitant suppression of CD20 antigen. Future study of how these agents induce CD20 expression in RRCL will provide a potential means to reversing resistance towards rituximab treatment.

scholarly journals Restoration of Noradrenergic Function in Parkinson’s Disease Model Mice

ASN NEURO ◽  
2021 ◽  
Vol 13 ◽  
pp. 175909142110097
Author(s):  
Kui Cui ◽  
Fan Yang ◽  
Turan Tufan ◽  
Muhammad U. Raza ◽  
Yanqiang Zhan ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Transcription Factors ◽  
Disease Model ◽  
Neuronal Loss ◽  
Mrna Levels ◽  
Gene Therapies ◽  
Protein Levels ◽  
Dopaminergic Systems ◽  
And Function

Dysfunction of the central noradrenergic and dopaminergic systems is the primary neurobiological characteristic of Parkinson’s disease (PD). Importantly, neuronal loss in the locus coeruleus (LC) that occurs in early stages of PD may accelerate progressive loss of dopaminergic neurons. Therefore, restoring the activity and function of the deficient noradrenergic system may be an important therapeutic strategy for early PD. In the present study, the lentiviral constructions of transcription factors Phox2a/2b, Hand2 and Gata3, either alone or in combination, were microinjected into the LC region of the PD model VMAT2 Lo mice at 12 and 18 month age. Biochemical analysis showed that microinjection of lentiviral expression cassettes into the LC significantly increased mRNA levels of Phox2a, and Phox2b, which were accompanied by parallel increases of mRNA and proteins of dopamine β-hydroxylase (DBH) and tyrosine hydroxylase (TH) in the LC. Furthermore, there was considerable enhancement of DBH protein levels in the frontal cortex and hippocampus, as well as enhanced TH protein levels in the striatum and substantia nigra. Moreover, these manipulations profoundly increased norepinephrine and dopamine concentrations in the striatum, which was followed by a remarkable improvement of the spatial memory and locomotor behavior. These results reveal that over-expression of these transcription factors in the LC improves noradrenergic and dopaminergic activities and functions in this rodent model of PD. It provides the necessary groundwork for the development of gene therapies of PD, and expands our understanding of the link between the LC-norepinephrine and dopamine systems during the progression of PD.

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