Contribution of Deregulated miRNAs to the Phenotypic Characteristic of Hodgkin Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 265-265
Author(s):  
Lu Ping Tan ◽  
Bart-Jan Kroesen ◽  
Enrico Tiacci ◽  
Gerben Duns ◽  
Erwin Seinen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Mirna Expression ◽  
Conflicts Of Interest ◽  
System Development ◽  
Cell Phenotype ◽  
Immune System Development

Abstract Abstract 265 In Hodgkin Lymphoma (HL), the Hodgkin Reed-Sternberg (HRS) cells are a minority of large mono- or multi-nucleated B cells characterized by a loss of B cell phenotype, constitutive NF-kB activation, a disturbed cell cycle and anti-apoptotic features. In this study we investigated the role of deregulated miRNA expression in the pathogenesis of HL. MiRNA in situ hybridization (ISH) in HL tissue was performed to determine expression of miRNAs previously reported to be highly abundant in HL cell lines, in HRS cells. Next we identified the miRNA-targetome of two HL cell lines by immunoprecipitation of RISC in untransfected and transfected cell lines. miRNA ISH confirmed expression of miR-17-5p, miR-24, miR-106a, miR-146a, miR-150, miR-155, miR-181b and miR-210 in HRS cells. Ago2-immunoprecipitation followed by microarray analysis of the co-immunoprecipitated mRNA revealed that the miRNA-targetome of HL comprises of about 2,500 genes. Inhibition of the anti-miR-17 seed family revealed that about 500 of these genes are regulated by miRNAs of the miR-17 seed family. Gene ontology (GO) analysis for the total miRNA-targetome of HL showed a significant enrichment of genes involved in the regulation of cell cycle, apoptosis, immune system development and NF-kB cascade. The miRNA-targetome of HL contained several genes known to be mutated in HRS cells, including A20, FAS, NFKB1A, NFKB1E, PERP and SOCS1. Also, using previously reported gene expression data, we defined a set of genes downregulated in HL cell lines (L428 and L1236) compared to germinal center B cells (GCB) and compared them to the miRNA-targetome of the same cell lines. This resulted in the identification of 149 genes in L428 and 183 genes in L1236 that were subjected to miRNA mediated repression. Unexpectedly, only a few of all the reported inactivated genes in HRS cells that might contribute to loss of B cell phenotype (MYBL1 and CXCR4) were found to be regulated by miRNAs in HL. In conclusion, we confirmed the expression of miRNAs in the HRS cells of HL tissue and identified miRNA repressed genes in HL. Our data indicated that aberrant miRNA expression contributes to the deregulation of apoptosis, cell cycle, and NF-kB pathways but not loss of B cell phenotype in HL. Disclosures: No relevant conflicts of interest to declare.

New Pathogenetic Insights Into Classical Hodgkin Lymphoma Revealed by Gene Expression Profiling of Microdissected Hodgkin/Reed-Sternberg Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 266-266 ◽  
Author(s):  
Enrico Tiacci ◽  
Verena Brune ◽  
Susan Eckerle ◽  
Wolfram Klapper ◽  
Ines Pfeil ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Expression Profiling ◽  
Expression Profiles ◽  
B Cell Lymphoma ◽  
Cell Phenotype

Abstract Abstract 266 Background. Previous gene expression profiling studies on cHL have been performed on whole tissue sections (mainly reflecting the prominent reactive background in which the few HRS cells are embedded), or on cHL cell lines. However, cultured HRS cells do not likely reflect primary HRS cells in all aspects, being derived from end-stage patients and from sites (e.g. pleural effusions or bone marrow) which are not typically involved by cHL and where HRS cells lost their dependence on the inflammatory microenvironment of the lymph node. Methods. ∼1000–2000 neoplastic cells were laser-microdissected from hematoxylin/eosin-stained frozen sections of lymph nodes taken at disease onset from patients with cHL (n=16) or with various B-cell lymphomas (n=35), including primary mediastinal B-cell lymphoma (PMBL) and nodular lymphocyte-predominant Hodgkin lymphoma (nLPHL). After two rounds of in vitro linear amplification, mRNA was hybridized to Affymetrix HG-U133 Plus 2.0 chips. Expression profiles were likewise generated from sorted cHL cell lines and several normal mature B-cell populations. Results. Primary and cultured HRS cells, although sharing hallmark cHL signatures such as high NF-kB transcriptional activity and lost B-cell identity, showed considerable transcriptional divergence in chemokine/chemokine receptor activity, extracellular matrix remodeling and cell adhesion (all enriched in primary HRS cells), as well as in proliferation (enriched in cultured HRS cells). Unsupervised and supervised analyses indicated that microdissected HRS cells of cHL represent a transcriptionally unique lymphoma entity, overall closer to nLPHL than to PMBL but with differential behavior of the cHL histological subtypes, being HRS cells of the lymphocyte-rich and mixed-cellularity subtypes close to nLPHL cells while HRS cells of NS and LD exhibited greater similarity to PMBL cells. HRS cells downregulated a large number of genes involved in cell cycle checkpoints and in the maintenance of genomic integrity and chromosomal stability, while upregulating gene and gene signatures involved in various oncogenic signaling pathways and in cell phenotype reprogramming. Comparisons with normal B cells highlighted the lack of consistent transcriptional similarity of HRS cells to bulk germinal center (GC) B cells or plasma cells and, interestingly, a more pronounced resemblance to CD30+ GC B cells and CD30+ extrafollicular B cells, two previously uncharacterized subsets that are transcriptionally distinct from the other mature B-cell types. Conclusions. Gene expression profiling of primary HRS cells provided several new insights into the biology and pathogenesis of cHL, its relatedness to other lymphomas and normal B cells, and its enigmatic phenotype. Disclosures: No relevant conflicts of interest to declare.

In Reed Sternberg Cells Suppression of the B-Cell Phenotype and Cell Proliferation Is Not Mediated by BLIMP-1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 24-24
Author(s):  
Catherine Burton ◽  
Sharon Barrans ◽  
Gareth Williams ◽  
Bradley Dickinson ◽  
Reuben Tooze ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
B Cell ◽  
Cell Cycle Arrest ◽  
Hodgkin Lymphoma ◽  
Tumour Cell ◽  
Plasma Cells ◽  
Cell Phenotype ◽  
Classical Hodgkin Lymphoma ◽  
Cycle Arrest

Abstract Expression of the transcriptional repressor BLIMP-1 is a pivotal event in the differentiation of post germinal centre B-cells to plasma cells. BLIMP-1 extinguishes the B-cell phenotype by down-regulation of PAX-5 with associated loss of BCL-6 and PU. and acting in concert with IRF-4 and XBP-1 induces the expression of the mature plasma cell phenotype. A key step in this process is the permanent exit of the differentiating plasma cell from the cell cycle which appears to be partly dependent on BLIMP-1 mediated repression of c-MYC. Reed Sternberg cells in Classical Hodgkin Lymphoma are known to be derived from post germinal centre B-cell which have undergone extinction of most elements of their B-cell phenotype but do not complete terminal differentiation to plasma cells. The primary aim of this study was to establish whether this loss of B-cell characteristics was mediated by BLIMP-1 and whether this was associated with exit from the cell cycle. 35 well characterised biopsies of nodular sclerosing Classical Hodgkin Lymphoma were studied using an extensive panel of B-cell and cell cycle markers. BLIMP-1 was identified using a BLIMP-1 alpha specific polyclonal antibody raised against the BLIMP-1 amino terminus which was validated by Western Blotting and immunocytochemical reactivity with normal plasma cells and squamous epithelia. All of the Reed Sternberg cells in all of the cases studied had the immunophenotype CD30+, IRF-4+, PU.1-. 15% cases showed PAX-5 a proportion of tumour cells and CD20 and or CD79 were found in 6% of cases. In 21% cases there was either nuclear or cytoplasmic expression of C/EBP alpha. All Reed Sternberg cells expressed Ki67 and around 50% of cells in all of the cases expressed the G2/M phase marker geminin with p53 and p21 uniformly expressed in the tumour cells. In contrast to the patterns seen in normal B-cell differentiation there was no evidence of BLIMP-1 or XBP-1 expression in any of the cases studied. To further clarify the reason for cell cycle arrest, interphase FISH using a panel of centromeric probes was performed on imprints preparations of lymph nodes replaced by Hodgkin lymphoma. This showed asymmetrical chromosome separation between the nuclear lobes of individual Reed Sternberg cells and heterogeneity of chromosome content within the mononuclear tumour cell fraction. These results indicate that extinction of the B-cell phenotype in Reed Sternberg cell is not dependent on the induction of BLIMP-1expression and that unlike B-cells undergoing differentiation to plasma cells most Reed Sternberg cells undergo cell cycle arrest mediated by normal p53 expression rather than permanently exiting from the cell cycle. Cell cycle arrest is likely to be related to asymmetrical chromosomal segregation within the tumour cell population. Previous experimental studies have shown that plasma cells differentiation is blocked in cells that are continuing to cycle. The results of this study raise the possibility that cell cycle arrest due to abberrant mitosis may be a key factor in determing the distinctive phenotype of Reed Sternberg cells through a mechansim that is independent of BLIMP-1 activation.

Specific Micro-RNA Expression Profile in Hodgkin Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 381-381
Author(s):  
Lu Ping Tan ◽  
Geert Harms ◽  
Tjasso Blokzijl ◽  
Rikst Nynke Schakel ◽  
Johan Gibcus ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Differentially Expressed ◽  
Cell Phenotype ◽  
Qrt Pcr

Abstract Introduction Classical Hodgkin lymphoma (cHL) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) differs not only in the form of histology and reactive background but also in the phenotypes of the tumor cells. Although tumor cells from both HL subtypes are originated from the germinal center (GC) B cell, gene expression studies show that lymphocytic and histiocytic (L&H) cells from NLPHL resembles normal B cells while Hodgkin/Reed-Sternberg cells (H/RS) from cHL demonstrate a loss of B cell phenotype and have significant overlap with primary mediastinal B cell lymphoma (PMBL). Recently, a new class of small RNAs, namely the micro-RNAs (miRNAs), has been identified. It is now known that at the post-transcriptional level, miRNAs negatively regulate gene expression in a sequence specific manner. Unique miRNAs expression patterns have been reported in various tissue types and also during a wide range of physiological states, such as cell proliferation, development, differentiation, apoptosis and hypoxia. As miRNAs play important roles in many cellular processes, it is proposed that there is a link between aberrant miRNA expression and loss of B cell phenotype in cHL. Methods In this study, miRNA profiles from cell lines of various B cell lymphoma subtypes were examined by qRT-PCR. Also, several B cell subsets were sorted from tonsil by FACS and the miRNA profiles studied by qRT-PCR. Some of the miRNAs are analyzed by in situ hybridization (ISH) in both HL tissue and tonsil samples. Results The miRNA profiling data indicated that cHL cell lines cluster together with PMBL while DEV, an NLPHL cell line, clusters together with CB. Upon validation of differentially expressed miRNAs on a cell line panel of 33 cell lines by monoplex qRT-PCR, 5/8 miRNAs identified as differentially expressed between cHL and GC B cells, were confirmed. Four out of six miRNAs differentially expressed between cHL and PMBL, were also confirmed as being differentially expressed in a larger cell panel. A high degree of overlap was observed between the most abundantly expressed miRNAs in the four HL cell lines. Expression of these miRNAs in HRS cells was verified by ISH in HL tissue samples. miRNA profiles of naive, GC and memory B cells display unique patterns. The overall miRNA expression levels were much lower than observed in the cell lines. Results of miRNA ISH in tonsil tissue demonstrated a specific staining pattern for each miRNA. These data indicate that miRNAs are particularly important for subsets of lymphocytes. Conclusion Several miRNAs that are expressed specifically in Hodgkin lymphoma have been identified. However, the effect of the aberrant expressions of these miRNAs in HL is yet to be elucidated, as the targets of these miRNAs remain unknown.

The TNF Family Members BAFF and APRIL Play an Important Role in Hodgkin Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 22-22 ◽  
Author(s):  
April Chiu ◽  
Xugang Qiao ◽  
Bing He ◽  
Elizabeth Hyjjek ◽  
Joong Lee ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Plasma Cells ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Dna Recombination ◽  
Classical Pathway ◽  
Bax Protein

Abstract Introduction. B cell-activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL), a BAFF-related molecule, play a key role in the survival and proliferation of mature B cells. In addition, BAFF and APRIL cooperate with IL-4 to induce class switch DNA recombination (CSR) from IgM (or IgG) to IgG, IgA or IgE. This process requires activation-induced-cytidine deaminase (AID), a DNA-editing enzyme involved also in Ig somatic hypermutation and lymphomagenesis. BAFF and APRIL are usually produced by myeloid cells, including dendritic cells, macrophages and granulocytes, and engage three receptors preferentially expressed on B cells, including transmembrane activator and calcium modulator and cyclophylin ligand interactor (TACI), B cell maturation antigen (BCMA), and BAFF receptor (BAFF-R). Our previous studies show that BAFF and APRIL are EBV-inducible molecules implicated in B cell non-Hodgkin’s lymphoma (NHL). The scope of the present studies was to elucidate the expression and function of BAFF, APRIL, TACI, BCMA and BAFF-R in Hodgkin lymphoma (HL). Methods. Tissue sections from 5 primary EBV+ HL cases and 5 primary EBV− HL cases were analyzed for BAFF, APRIL, TACI, BCMA, and BAFF-R expression through immunohistochemistry. RS cells from 6 primary cases were microdissected and analyzed for the expression of AID and CSR byproducts by RT-PCR. The expression of BAFF, APRIL, TACI, BCMA, BAFF-R, AID, and CSR byproducts was also analyzed in 5 HL cell lines cultured in the presence or absence of recombinant BAFF, APRIL and cytokines as previously described1,2,3. Results. We found that the reactive infiltrate of primary HL tumors comprises non-malignant elements, such as macrophages, granulocytes and plasma cells, expressing BAFF and APRIL. Also a variable proportion of malignant CD30+ Reed-Sternberg (RS) cells from both EBV+ and EBV− HL cases express BAFF and APRIL. Unlike NHL B cells, which usually express BAFF-R, primary RS cells and RS cell lines lack BAFF-R, but express TACI and BCMA. In the presence of BAFF or APRIL, RS cell lines are rescued from spontaneous or induced apoptosis. This effect is associated with activation of NF-κB through a classical pathway. Increased RS cell survival is also associated with up-regulation of the pro-survival BCL-2 and BCL-XL proteins, and down-regulation of the pro-apoptotic BAX protein. Finally, in the presence of BAFF or APRIL and IL-4, RS cell lines up-regulate AID expression and increase their spontaneous CSR activity. Of note, AID expression extends to primary RS cells and is associated with ongoing CSR. Conclusions. Our studies indicate that BAFF and APRIL stimulate malignant RS cells through both autocrine and paracrine pathways. Engagement of TACI and BCMA receptors by BAFF and APRIL may enhance the expansion of RS cells by attenuating apoptosis through a mechanism involving NF-κB and BCL family proteins. By up-regulating AID, signals emanating from TACI and BCMA receptors might also introduce genomic instability. Finally, considering that TACI, BCMA and AID are B cell-specific molecules and that CSR is a process confined to B cells, our findings consolidate the notion that RS cells derive from a B cell precursor.

Comprehensive Analysis of Homeobox Gene Expression in Hodgkin Lymphoma Cell Lines Reveals Similarities to Prostate Carcinoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 966-966
Author(s):  
Stefan Nagel ◽  
Christof Burek ◽  
Hilmar Quentmeier ◽  
Corinna Meyer ◽  
Andreas Rosenwald ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Prostate Carcinoma ◽  
Homeobox Genes ◽  
Homeobox Gene ◽  
Cell Cycle Regulators ◽  
Rt Pcr

Abstract Homeobox genes code for transcription factors with essential regulatory impact on cellular processes during embryogenesis and in the adult. Increasingly, members of the circa 200 gene strong family are emerging as major oncogenic players, prompting our investigation into possible homeobox gene dysregulation in Hodgkin lymphoma (HL) in which no recurrent oncogene involvement has been known. Accordingly, we screened 6 well characterized HL cell lines (HDLM-2, KM-H2, L-1236, L-428, L-540, SUP-HD1) and 3 non-Hodgkin lymphoma (NHL) cell lines (RC-K8, RI-1, SC-1) for homeobox gene expression using Affymetrix U133-2.0 whole-genome oligonucleotide microarrays. Of 15 candidate genes thus shown to reveal HL-specific expression patterns, 5 homeobox genes were shortlisted as potentially key dysregulatory targets in HL after additional RT-PCR expression analysis relative to controls. While 3/5 homeobox genes were upregulated in HL (HOXB9, HOXC8, HLXB9), 2/5 were downregulated (BOB1, PAX5). Furthermore, cloning and sequencing RT-PCR products obtained with degenerate primers recognizing conserved homeobox motifs confirmed the predominant expression of HOXB9 in HL cells. However, fluorescence in situ hybridization (FISH) analysis of the HOXB locus (at 17q21) revealed no cytogenetic aberrations, indicating that its activation is conducted non-chromosomally in HL cells. Surprisingly, known target genes of HOXB9 and HOXC8 remained unperturbed, implying novel downstream effector pathways in HL cells. Antisense oligos directed against HOXB9 and forced expression experiments using cloned full length HOXB9 cDNA indicated its involvement in both proliferation and apoptosis. Cell cycle regulators BTG1, BTG2 and GEMININ have been described to interact with HOXB9 and may represent potential targets deserving investigation. We recently showed that HLXB9 promotes IL6 expression in HL cells in response to a constitutively active PI3K signalling pathway therein (Nagel et al., Leukemia19, 841–6, 2005). Our most recent data indicate that HLXB9 is also expressed in various NHL cell lines including anaplastic, diffuse and mediastinal large cell as well as follicular B-cell lymphomas while expression is notably absent from Burkitt, mantle cell and natural killer T-cell lymphomas reflecting their pathologic classification. Intriguingly, our data highlight unexpected similarities between HL and prostate cancer cells which together uniquely overexpress HOXB9, HOXC8 and HLXB9 (or its close homolog GBX2). Additional genes expressed in prostate carcinoma (HOXB13, PRAC1, PRAC2) were detected in two HL cell lines (KM-H2 and L-428) suggesting further parallels may be revealed. Detection of downregulated B-cell differentiation factors BOB1 and PAX5 in our panel of HL cell lines validated this approach. Both factors were previously implicated in oncogenesis of HL lacking IGH rearrangements and other key B-cell characteristics. In summary, we identified a unique homeobox gene expression pattern involving HOXB9, HOXB13, HOXC8 and HLXB9 in HL cell lines resembling that of prostate carcinoma cells. Overexpressed HOXB9 contributes to proliferation and protects against apoptosis in HL cells potentially via interacting with cell cycle regulators BTG1/2 and/or GEMININ.

Resistance to the Novel Translation Inhibitor Silvestrol Is Mediated by Elevated Mcl-1 Expression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1737-1737
Author(s):  
David M. Lucas ◽  
Ellen J. Sass ◽  
Ryan B. Edwards ◽  
Li Pan ◽  
Gerard Lozanski ◽  
...  
Keyword(s):  
Drug Resistance ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Multi Drug Resistance ◽  
Parental Line

Abstract Abstract 1737 Poster Board I-763 We previously reported the efficacy and B-cell selectivity of the natural product silvestrol in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), using both primary cells and B-cell lines. We also showed that silvestrol inhibits translation, resulting in rapid depletion of the short half-life protein Mcl-1 followed by mitochondrial damage and apoptosis. Cencic et al. reported that silvestrol directly blocks translation initiation by aberrantly promoting interaction of eIF4A with capped mRNA (PLoS One 2009; 4(4):e5223). However, the loss of Mcl-1 in breast and prostate cancer cell lines is delayed relative to what we observe in B-leukemias (48 hr vs. 4-6 hr in CLL and ALL cells). Additionally, silvestrol does not reduce Mcl-1 expression in normal T-cells to the same extent that it does in B-cells, potentially explaining in part the relative resistance of T-cells to this agent. We therefore investigated cell-type differences, as well as the importance of Mcl-1, in silvestrol-mediated cytotoxicity. We incubated the ALL cell line 697 with gradually increasing concentrations of silvestrol to generate a cell line (697-R) with resistance to 30 nM silvestrol (IC50 of parental 697 < 5 nM). No differences between 697-R and the parental line were detected upon detailed immunophenotyping. However, cytogenetic analysis revealed a balanced 7q;9p translocation in 697-R not present in the parental 697 cell line that may be related to the emergence of a resistant clone. We also detected no difference in expression of multi-drug resistance proteins MDR-1 and MRP, which can contribute to resistance to complex amphipathic molecules such as silvestrol. In contrast, we found that baseline Mcl-1 protein expression is strikingly increased in 697-R cells relative to the parental line, although these cells still show similar percent-wise reduction in Mcl-1 upon re-exposure to 80 nM silvestrol. To investigate whether this resistance to silvestrol is reversible, 697-R cells were maintained without silvestrol for 6 weeks (∼18 passages). During this time, viability remained near 99%. Cells were then treated with 30 nM silvestrol. Viability was 94% at 48 hr post-treatment and returned to 99% within a week, while parental 697 cells with the same treatment were completely dead. Baseline Mcl-1 levels remained elevated in 697-R even with prolonged silvestrol-free incubation. These results indicate that the resistance phenotype is not rapidly reversible, as is seen with transient upregulation of multi-drug resistance or stress-response proteins. Additionally, silvestrol moderately induces the transcription of several pro-apoptotic Bcl-2 family members and results in elevated levels of these proteins despite its translation inhibitory activity. Interestingly, no such activity is detected in silvestrol-treated normal T-cells. Together, these results support the hypothesis that in B-cells, silvestrol induces cell death by altering the balance of pro- and anti-apoptotic factors, and that increased Mcl-1 protein can force the balance back toward survival. This work further underscores the importance of Mcl-1 in silvestrol-mediated cytotoxicity. We are now investigating the mechanism of Mcl-1 upregulation in 697-R cells to identify a factor or pathway that can be targeted therapeutically to circumvent resistance. Silvestrol is currently undergoing preclinical pharmacology and toxicology investigation by the U.S. National Cancer Institute Drug Development Group at the Stage IIA level to facilitate its progression to Phase I clinical testing. Disclosures No relevant conflicts of interest to declare.

Dysregulation of Pax5 Activity Contributes to the Extinction of the B-Cell Phenotype in Reed-Sternberg Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 517-517 ◽  
Author(s):  
Graham P Collins ◽  
Jennifer C Paterson ◽  
Gillian E May ◽  
Rajeev Gupta ◽  
Teresa Marafioti ◽  
...  
Keyword(s):  
B Cells ◽  
Protein Expression ◽  
B Cell ◽  
Binding Site ◽  
Cell Lines ◽  
Target Genes ◽  
Epigenetic Modification ◽  
Methylation Status ◽  
Cell Phenotype ◽  
Transfected Cells

Abstract Hodgkin/Reed-Sternberg cells (HRS cells) are thought to be derived from post-germinal centre B-cells and yet have down-regulated the B-cell phenotype. The B-cell transcription factor Pax5 is important in the maintenance of B-cell identity and we demonstrate that it is down-regulated in HRS cells lines and in HRS cells of the majority of primary classical Hodgkin Lymphoma (cHL) cases. Specifically, 3/30 cases were negative for Pax5, 16/30 were weakly positive, 10/30 cases were moderately positive and 1/30 showed Pax5 staining of equivalent intensity to infiltrating, polyclonal B-cells. In order to functionally test the relevance of a reduced Pax5 expression level, the cHL cell lines L428 and L1236 were stably transfected with Pax5 using a lentiviral transfection system. Transfection of L1236 resulted in up-regulation of CD79a protein expression. However, CD79a was not upregulated in L428 and expression of the Pax5 target genes Cd19 and Blnk was unaffected by Pax5 transfection in both cell lines. Chromatin immunoprecipitation demonstrated that Pax5 failed to bind the high affinity binding site within the Cd19 promoter in the cHL lines despite high levels of Pax5 expression, appropriately localised to the nucleus. Pax5 could, however, bind synthetic oligonucleotide corresponding to this site (as shown by electrophoretic mobility shift assays) raising the possibility that epigenetic modification in vivo may be responsible for the failure to bind DNA. Bisulphite genome sequencing confirmed that in cHL cell lines, the region surrounding the Pax5 binding site in the Cd19 promoter was extensively methylated. Moreover, histone modification analysis also demonstrated an absence of markers of accessible, active chromatin (di- and trimethylated H3K4) and an enrichment of a marker indicating closed, repressive chromatin (trimethylated H3K27). Within the Cd79a promoter, previous studies have implicated the methylation status of a single cytosine residue within the binding site for a Pax5-Ets1 complex to be an important determinant of activation of the Cd79a gene. Interestingly, this residue was shown to be largely methylated in L428 cells but largely unmethyated in L1236 cells, providing a likely mechanism for the differential activation of this gene by transfected Pax5 protein. To investigate whether the observed epigenetic changes were responsible for preventing Pax5 binding and activity at the Cd19 and Cd79a promoters, Pax5 transfected cHL cell lines were cultured in the presence of the demethylating agent 5-aza-2-deoxycytidine. Up-regulation of Cd19 and Cd79a expression was significantly greater in Pax5 transfected cells than in control transfected cells. To conclude: our data suggests that dysregulation of Pax5 activity (at the levels of protein expression and epigenetic modification of the Pax5 binding sites) is important in mediating the extinction of the B-cell programme in HRS cells.

Repression of Mir-106b Family Members Does Not Alter Cell Cycle Progression in Hodgkin Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4722-4722
Author(s):  
Johan H Gibcus ◽  
Lu Ping Tan ◽  
Rikst Nynke Schakel ◽  
Geert Harms ◽  
Peter Moeller ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Cycle Arrest ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Target Genes ◽  
Family Members ◽  
Luciferase Reporter ◽  
P21 Protein ◽  
Cycle Arrest

Abstract MicroRNAs (miRNAs) are 19–25 nucleotide long RNA molecules derived from precursor genes that inhibit the expression of target genes by binding to their 3′ UTR region. Expression of miRNAs is often tissue specific and miRNA profiling has shown specific miRNA expression patterns in both B-cell development and lymphomagenesis. Hodgkin lymphoma is derived from pre-apoptotic germinal center B-cells, although a general loss of B cell phenotype is noted. Using quantitative RT-PCR and miRNA microarray, we determined the miRNA profile of HL and compared this with the profile of a panel of B-cell non-Hodgkin lymphomas (NHL). The two methods showed a very good correlation for the expression levels of the individual miRNAs. Using a large panel of cell lines, we confirmed differential expression between HL and other B-cell lymphoma derived cell lines for 27 miRNAs. The HL specific miRNAs included miR-155, miR-21 and miR-106b seed family members miR-17-5p, miR-20a, miR-93, miR-106a and miR- 106b. Next, we performed target gene validation of predicted target genes for miR-17-5p, which is highly expressed in HL. Using luciferase reporter assays with stabilized anti-sense miR17-5p oligonucleotides, we showed that GPR137B, RAB12 and RBJ are likely miR-17-5p target genes in two different HL cell lines. Previous publications indicated that miR-106b seed family members negatively regulate the cyclin-dependent kinase inhibitor 1A (p21/CIP1) resulting in cell cycle arrest at G1. Consistent with these findings, we show that the miR-106b family members are highly expressed in L428, whereas p21 is not. However, inhibition of the miR-106b seed family members in L428 does not result in elevated p21 protein expression. Furthermore, there is no cell cycle arrest, growth reduction or increase in cell death and apoptosis after inhibition of the miR-106b seed family members. Thus, we conclude that blocking of the miR-106b seed family members does not necessarily lead to indiction of p21 protein. This suggests an additional regulatory layer of p21 expression in L428 cells.

Microrna-155 Controls The RB/E2F Axis In Normal and Malignant B Cells Via The Non-Canonical TGFβ1-SMAD5 Signaling Module

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 241-241 ◽  
Author(s):  
Daifeng Jiang ◽  
Ricardo Aguiar
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
Developmental Regulation ◽  
G1 Arrest ◽  
Malignant Cells ◽  
Germinal Center B Cells ◽  
In Cells

Abstract MicroRNA-155 (miR-155) plays pleiotropic roles in the biology of normal and malignant B cells. MiR-155 knockout (KO) mice have fewer germinal center B cells, while overexpression of this miRNA is associated with aggressive DLBCL. Although several miR-155 targets have been identified, a mechanism that unifies the features of loss and gain of miR-155 function in normal and malignant cells remains to be described. In B cells, TGFβ signals are suppressive indicating that deregulation of this pathway may interfere with the developmental regulation of lymphocytes and contribute to the pathogenesis of B cell malignancies. Earlier, we described the direct targeting of the transcription factor SMAD5 by miR-155, and uncovered the presence of non-canonical signaling model in B cell lymphomas whereby TGFβ1, a cytokine that typically activates SMAD2/3, phosphorylated SMAD5. Herein, we used the miR-155 KO mice and genetically modified DLBCL cell lines to investigate which downstream effectors of TGFβ signals are disrupted by the miR-155/SMAD5 interaction, thus shedding light on the phenotypes associates with miR-155 loss and gain of function. We confirmed the phosphorylation of SMAD5 by TGFβ1 in DLBCL cell lines, and demonstrated for the first time that this non-canonical signal is also present in untransformed normal mature B cells. We stably expressed miR-155 in the TGFβ1-responsive DLBCL cell lines Ly7, Ly18 and DHL5, and readily detected suppression of SMAD5, but not of other SMADs. TGFβ1 cytostatic activities include up-regulation of p15 and p21, which are primarily found in the context of SMAD2/3 activation. However, we found that stable expression of miR-155, and downregulation of SMAD5, significantly limited TGFβ1-dependent induction of both p15 and p21 in DLBCL. TGFβ1-mediated upregulation of p15 and p21 limits the activity of cyclin/CDK complexes, enriches for hypophosphorylated (active) RB, and promotes cell cycle arrest. We measured the effects of miR-155 in this process, and found that the accumulation of hypophosphorylated RB following TGFβ1 exposure was blunted in miR-155 expressing cells, resulting in an impaired G0/G1 arrest. The impact of miR-155 on TGFβ1 activity was also detectable by directly measuring the phosphorylation levels of RB’s Ser780 residue. Active pRB blocks cell cycle progression at least in part by binding to and inhibiting the E2F family of transcriptional regulators. Thus, we performed co-immunoprecipitation experiments and quantified the levels of RB-bound E2F1. In these assays, following TGFβ1 exposure we found a markedly decreased pRB-E2F1 complex formation in miR-155 expressing cells when compared to their controls. In agreement with these data, DLBCL cell lines expressing miR-155 displayed higher levels of free E2F1. Together, these data suggested the existence of a miR-155-SMAD5-p15/p21 axis that regulates TGFβ1 effects towards RB and E2F in DLBCL. To confirm the specific role of each component in this circuit, we used an RNAi strategy to transiently or stably knockdown (KD) SMAD5, p15 or p21 in our DLBCL models. In control RNAi cells, exposure to TGFβ1 led to decrease in RB phosphorylation, whereas these effects were abrogated upon KD of each of these genes, resulting in accumulation of hyperphosphorylated RB, a phenocopy of miR-155 expression. To define if the interplay between miR-155/SMAD5 and RB was also present in non-malignant cells, we purified mature B lymphocytes from miR-155 WT and KO mice. Examination of four pairs of mice, showed a higher expression of SMAD5 in cells from miR-155 KO than WT mice. In addition, TGFβ1-mediated suppression of phospho-RB was consistently more pronounced in miR-155 KO than in WT B cells, which resulted in a significantly higher G0/G1 arrest in cells lacking this miRNA. Of note, in absence of TGFβ1 there was no significant difference in cell cycle profile of mature B cells from miR-155 WT and KO mice. We concluded that an unrestrained TGFβ activity, secondary to SMAD5 upregulation, may help explain the deficient germinal center B cells formation found in miR-155 KO mice. Together, our findings demonstrate that miR-155 overexpression is a novel model for deregulation of the lymphomagenic RB/E2F axis, and define an unsuspected role for the non-canonical TGFβ1 activation of SMAD5 in the developmental regulation of mature B cells. Disclosures: No relevant conflicts of interest to declare.

Characterization of lincRNA BALIR-6 in MLL rearranged B-lymphoblastic leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3730-3730
Author(s):  
Norma Iris Rodriguez-Malave ◽  
Weihong Yan ◽  
Giuseppe Basso ◽  
Martina Pigazzi ◽  
Dinesh S. Rao
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Microarray Data ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Regulate Gene Expression ◽  
In Cis ◽  
Regulate Gene

Abstract A new class of non-coding RNA, known as long intergenic non-coding RNAs (lincRNAs), has only recently been described. These lincRNAs have been found to play a role in various molecular processes within the cell including gene regulation, acting as sinks for microRNAs, and regulating splicing, implicating them in development and oncogenic processes. B lymphoblastic leukemia (B acute lymphoblastic leukemia; B-ALL), a malignancy of precursor B-cells, harbors mutations and translocations that result in a dysregulated gene expression. Interestingly, dysregulated expression of lincRNAs has been found in various cancers, but has not yet been described in B-ALL. Recently, we completed a gene expression profiling study in human B-ALL samples, which showed differential lincRNA expression in samples with particular cytogenetic abnormalities. This led us to hypothesize that lincRNAs may be related to disease pathogenesis. Here, we describe a promising lincRNA from our microarray data designated B-ALL associated long intergenic RNA 6 (BALIR-6). Expression of BALIR-6 is highest in patient samples carrying the MLL rearrangement (n=16; when compared to patients with TEL-AML1-translocated, n=39; E2A-PBX1-translocated, n=8; BCR-ABL-translocated, n=3; and cytogenetically normal cases, n=56; 1-way ANOVA p<0.0001) and showed significant variance in the expression level based on the immunophenotype (1-way ANOVA p=0.0004). BALIR-6 is located on chromosome 3p24.3 in humans, and exists in a syntenic gene block in with neighboring genes SATB1 and TBC1D5, and is conserved in mammals. Rapid Amplification of cDNA Ends (RACE) uncovered multiple transcript isoforms; from these, three were cloned out and sequenced, corresponding to the genomic locus as predicted. In B-ALL cell lines, BALIR-6 expression was highest in RS411 cells, which carry the MLL rearrangement, when compared to other B-ALL cell lines. This suggests that the cell lines may show a similar expression pattern to human B-ALL samples. To study the functional role of BALIR-6 we utilized siRNA in a mmu-miR-155 expression cassette to knockdown the transcript. In RS411 cells we observed a reduction in proliferation by MTS assay. Additionally, we observed an increase Sub-G0 cells and a decrease in G2-M phase cells by propidium iodide staining, suggesting an increase in apoptosis. Conversely, overexpression of BALIR-6 in a mouse pre-B cell line (70Z/3), leads to an increase in proliferation. Interestingly, during normal B cell development, BALIR-6 is dynamically expressed, with high expression in pre-B cells and subsequent downregulation, suggesting that a normal role during development is being hijacked in patients with B-ALL. Mechanistically, a few recent studies have described that lincRNAs can regulate gene expression in cis. To explore whether BALIR 6 regulates surrounding genes in cis, we analyzed microarray data of MLL rearranged B-ALL samples, finding that expression of BALIR-6 correlates with expression of surrounding genes SATB1 and TBC1D5. Interestingly for SATB1, this correlation is also seen in human B cell developmental stages. Altering BALIR-6 expression by siRNA mediated knockdown or overexpression causes an effect on the expression of surrounding genes SATB1 and TBC1D5. Previous findings have shown that dysregulated SATB1 has been seen in a variety of malignancies, suggesting a mechanism for how BALIR-6 may produce the changes in cell growth and apoptosis described above. Altogether, these results identify a novel and interesting RNA transcript with the potential to regulate gene expression and pathogenesis in B-ALL with MLL rearrangement, suggesting novel diagnostic, prognostic, and therapeutic implications. Disclosures: No relevant conflicts of interest to declare.

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