Expression of Activation-Induced Cytidine Deaminase and Transcripts of Immunoglobulin Variable Heavy Chains in Mature B-Cell Lines Lacking Immunoglobulin Protein Expression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2295-2295
Author(s):  
Yoshinobu Matsuo ◽  
Hans G. Drexler ◽  
Akira Harashima ◽  
Ayumi Okochi ◽  
Masanori Daibata ◽  
...  
Keyword(s):  
B Cells ◽  
Protein Expression ◽  
B Cell ◽  
Cell Lines ◽  
Cytidine Deaminase ◽  
Terminal Deoxynucleotidyl Transferase ◽  
B Cell Malignancies ◽  
Activation Induced Cytidine Deaminase ◽  
Variable Heavy ◽  
Rag 1

Abstract Expression of immunoglobulin (Ig) chains is a definitive marker of B-cell origin. Ig chains are generated during B-cell maturation by somatic rearrangement of Ig gene sequences. This rearrangement generally follows an orderly progression beginning with the heavy (H) chain genes and proceeding to the light (L) chain genes. The fact that Ig expression in B-cells generally reveals single L and H chains implies monoclonal cell expansion. Nevertheless, several exceptions have been reported, including double light chain positive cases. However, only little is known about mature B-cell malignancies lacking any Ig protein expression at all. In order to clarify the mechanisms of lacking any Ig protein expression in mature B-cell malignancies, we have analyzed the expression of activation-induced cytidine deaminase (AID), terminal deoxynucleotidyl transferase (TdT), recombination acting gene (RAG)-1 and RAG-2, transcription factors (TFs) and mRNA transcripts of Ig variable heavy (IgVH) chains using five both surface membrane (Sm) and cytoplasmic (Cy) Ig negative B-cell lines derived from acute lymphoblastic leukemia L3 type (ALL-L3), Burkitt’s lymphoma (BL) and pyothorax-associated lymphoma (PAL). BALM-9N and BALM-16 are ALL-L3, KATATA is BL, OPL-1 and OPL-3 are PAL-derived cell lines (EBV+). In cytogenetic analysis, t(8;14)(q24;q32) in BALM-9N and KATATA and t(8;22)(q24;q11) in BALM-16 were detected, respectively. Band 14q32 was not involved in the PAL cell lines. All cell lines showed rearrangement of IgH chain genes. Expression of RAG-1 and RAG-2 was variable, TdT was negative for all, AID was expressed in all cell lines except for PAL cell line OPL-3. The seven TFs BOB1/OBF1, E2A, IKAROS, OCT1, OCT2, PAX5 and PU1 were universally expressed at the mRNA level in all cell lines. Reverse transcriptase polymerase chain reaction elucidated the IgVH gene usage. BALM-9N = VH4; BALM-16 = VH2; KATATA = VH3; OPL-1 = VH3; OPL-3 = VH3. In summary, the present study demonstrates that Ig gene recombination, class switch recombination and somatic hypermutation machineries are not the cause for the lacking expression of both Sm/Cy Ig chains in mature B-cell malignancies. The lack of any Sm/Cy Ig expression in such mature B-cells may indicate rather a germinal center origin.

scholarly journals miR-181b negatively regulates activation-induced cytidine deaminase in B cells

2008 ◽  
Vol 205 (10) ◽  
pp. 2199-2206 ◽  
Author(s):  
Virginia G. de Yébenes ◽  
Laura Belver ◽  
David G. Pisano ◽  
Susana González ◽  
Aranzazu Villasante ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Molecular Mechanisms ◽  
Bystander Effect ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Fine Tuning ◽  
Functional Screening ◽  
Activation Induced Cytidine Deaminase ◽  
Activated B Cells

Activated B cells reshape their primary antibody repertoire after antigen encounter by two molecular mechanisms: somatic hypermutation (SHM) and class switch recombination (CSR). SHM and CSR are initiated by activation-induced cytidine deaminase (AID) through the deamination of cytosine residues on the immunoglobulin loci, which leads to the generation of DNA mutations or double-strand break intermediates. As a bystander effect, endogenous AID levels can also promote the generation of chromosome translocations, suggesting that the fine tuning of AID expression may be critical to restrict B cell lymphomagenesis. To determine whether microRNAs (miRNAs) play a role in the regulation of AID expression, we performed a functional screening of an miRNA library and identified miRNAs that regulate CSR. One such miRNA, miR-181b, impairs CSR when expressed in activated B cells, and results in the down-regulation of AID mRNA and protein levels. We found that the AID 3′ untranslated region contains multiple putative binding sequences for miR-181b and that these sequences can be directly targeted by miR-181b. Overall, our results provide evidence for a new regulatory mechanism that restricts AID activity and can therefore be relevant to prevent B cell malignant transformation.

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.

Activation Induced Cytidine Deaminase (AID) Is Required for Germinal-Center Derived Lymphomagenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 223-223
Author(s):  
Laura Pasqualucci ◽  
Mara Compagno ◽  
Tongwei Mo ◽  
Paula Smith ◽  
Herbert C. Morse ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Somatic Hypermutation ◽  
Profile Analysis ◽  
Cytidine Deaminase ◽  
Receptor Gene ◽  
Activation Induced Cytidine Deaminase ◽  
Hodgkin's Lymphomas

Abstract Most B cell non-Hodgkin’s lymphomas (B-NHL) derive from germinal center (GC) B cells and their pathogenesis is associated with the accumulation of distinct genetic lesions, including chromosomal translocations and a more recently identified mechanism of genomic instability, termed aberrant somatic hypermutation. These alterations are thought to be due to mistakes occurring during two GC-associated immunoglobulin (Ig) genes remodeling processes: class switch recombination (CSR) and somatic hypermutation (SHM). However, this model has never been formally proven. To conclusively investigate the role of CSR and SHM in the pathogenesis of B-NHL, we examined whether lymphoma development in mice requires the function of activation induced cytidine deaminase (AID), a DNA editing enzyme expressed specifically in GC and activated B cells and essential for both processes. Three transgenic mouse models were generated by crossing lymphoma-prone mice (λMYC, λMYC/IμHABCL6 and IμHABCL6) with mice (AID−/−) that are unable to undergo both SHM and CSR. The λMYC mice develop a diffusely infiltrating monoclonal proliferation of pre-GC origin, with unmutated IgV genes and lack of BCL6 expression, and therefore presumably independent from AID-associated DNA remodeling events. Conversely, lymphomas in λMYC/IμHABCL6 and IμHABCL6 mice recapitulate GC/post GC-derived malignancies, in that the former display somatically mutated IgV genes and upregulation of post-GC markers (CD138) in most of the cases, while the latter develop a splenic lymphoproliferative syndrome that culminates, past 12 months of age, in clonal B cell lymphomas with DLBCL morphology and somatically mutated IgV genes (~70% of the animals) (Cattoretti et al., Cancer Cell 7:445–455, 2005). Mice were monitored for tumor incidence and survival, and a combination of histologic, immunophenotypic and gene expression profiling analysis was used for tumor characterization. As expected, no significant differences in event-free survival and lymphoma type were observed between AID-proficient and AID-deficient λMYC mice, in agreement with their pre-GC derivation. Conversely, a phenotypic shift of the tumor was observed in λMYC/IμHABCL6 mice when bred into an AID−/− background, with >80% of the cases (N=21/26) reverting to a pre-GC phenotype (loss of GC/post GC markers) undistinguishable from that of the λMYC and λMYC/AID−/− mice. Gene expression profile analysis on representative cases (N=10 λMYC/IμHABCL6 and 5 each for λMYC, λMYC/AIDKO, λMYC/IμHABCL6/AIDKO) confirmed significant phenotypic similarities between pre-GC derived λMYC lymphomas and the λMYC/IμHABCL6/AID −/− lymphomas, which co-segregated in a separate cluster from λMYC/IμHABCL6 tumors. Analogously, a significant reduction in DLBCL frequency was observed in the IμHABCL6/AIDKO cohort as compared to IμHABCL6 mice (N= 4/19, 21% vs 8/14, 57%; p=0.03). Taken together, these results indicate that GC-derived lymphomas cannot develop in the absence of AID, thereby providing direct support to the notion that AID-mediated mistakes in antigen receptor gene modification events (CSR and SHM) represent major contributors to B-NHL pathogenesis.

Cytotoxicity of Genetically Engineered Fusion Protein with CD154 Peptide Mimetic (OmpC-CD154p) on B-NHL Cell Lines.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4525-4525
Author(s):  
Bernardo Martinez-Miguel ◽  
Melisa A. Martinez-Paniagua ◽  
Sara Huerta-Yepez ◽  
Rogelio Hernandez-Pando ◽  
Cesar R. Gonzalez-Bonilla ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cells ◽  
Fusion Protein ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Genetically Engineered ◽  
B Cell Malignancies ◽  
Peptide Mimetic

Abstract The interaction between CD40, a member of the tumor necrosis factor super family, and its ligand CD154 is essential for the development of humoral and cellular immune responses. Selective inhibition or activation of this pathway forms the basis for the development of new therapeutics against immunologically-based diseases and malignancies. CD40 is expressed primarily on dendritic cells, macrophages and B cells. Engagement of CD40-CD154 induces activation and proliferation of B lymphocytes and triggers apoptosis of carcinoma and B lymphoma cells. Agonist CD40 antibodies mimic the signal of CD154-CD40 ligation on the surface of many tumors and mediate a direct cytotoxic effect in the absence of immune accessory molecules. CD40 expression is found on nearly all B cell malignancies. Engagement of CD40 in vivo inhibits B cell lymphoma xenografts in immune compromised mice. Several clinical trials have been reported targeting CD40 in cancer patients using recombinant CD154, mAbs and gene therapy, which were well tolerated and resulted in objective tumor responses. In addition to these therapies, CD54 mimetics have been considered with the objective to augment and potentiate the direct cytotoxic anti-tumor activity and for better accessibility to tumor sites. This approach was developed by us and we hypothesized that the genetic engineering of a fusion protein containing a CD154 peptide mimetic may be advantageous in that it may have a better affinity to CD40 on B cell malignancies and trigger cell death and the partner may be a carrier targeting other surface molecules expressed on the malignant cells. This hypothesis was tested by the development of a gene fusion of Salmonella typhi OmpC protein expressing the CD154 Trp140-Ser149 amino acid strand (Vega et al., Immunology2003; 110: 206–216). This OmpC-CD154p fusion protein binds CD40 and triggers the CD40 expressing B cells. In this study, we demonstrate that OmpC-CD154p treatment inhibits cell growth and proliferation of the B-NHL cell lines Raji and Ramos. In addition, significant apoptosis was achieved and the extent of apoptosis was a function of the concentration used and time of incubation. The anti-tumor effect was specific as treatment with OmpC alone had no effect. These findings establish the basis of the development of new fusion proteins with dual specificity (targeting the tumor cells directly or targeting the tumor cells and immune cells). The advantages of this approach over conventional CD40-targeted therapies as well as the mechanism of OmpC-CD154p-induced cell signaling and cell death will be presented.

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.

Expression of a Dysfunctional Activation Induced Cytidine Deaminase Is Correlated with Disease Progression in Splenic Marginal Zone Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2397-2397
Author(s):  
Gabriel Brisou ◽  
Laurent Jallades ◽  
Alexandra Traverse-Glehen ◽  
Francoise Berger ◽  
Aurélie Verney ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Marginal Zone ◽  
Cytidine Deaminase ◽  
Marginal Zone Lymphoma ◽  
Splenic Marginal Zone Lymphoma ◽  
Marginal Zone B Cells ◽  
Activation Induced Cytidine Deaminase ◽  
Splicing Variants

Abstract Abstract 2397 B cells can undergo at least two differentiation pathways, dependent of T cells or not, starting from follicular or marginal zone B cells respectively. The T-independent response, less understood than the germinal center reaction, is triggered by specific antigens and arises from marginal zone B cells. During this development, some B cells undergo somatic hypermutation (SHM) and class switch recombination (CSR), triggered by the same DNA editing enzyme called Activation Induced Cytidine Deaminase (AID). The splenic marginal zone lymphoma (SMZL) is a rare lymphoproliferative disorder characterized by a clonal expansion of B cells in the marginal zone of the spleen. These B-cells underwent SHM in roughly 60% of the cases but nearly none underwent CSR. These observations suggest that tumor clones originate from a particular activated B cell subset not transiting through the germinal center. In order to confirm this hypothesis, we focused our work on the status and impact of AID in this disease and worked on purified B cells extracted from spleen of well-characterized SMZL cases. We determined AID status by quantitative RT-PCR analysis on 27 SMZL samples and compared it with 5 controls. In the SMZL group the relative level of expression of AID is heterogeneous but two subgroups could be distinguished: one considered as expressing AID (14 cases out of the 27 analyzed), the remaining considered as not expressing AID. When we compared AID expression rate with occurrence of SHM and CSR, no clear correlation between AID expression and presence of SHM or CSR could be observed suggesting that AID, when expressed, is dysfunctional. To address this hypothesis, we first analyzed AID protein by immunohistochemistry and a good correlation between IHC signal and AID mRNA expression level has been observed. As AID gene was not mutated, we next focused our work on AID mRNA splicing variants as these variants exhibit different functions according to the domain of the protein they contain in a murine model. We found that SMZL B cells express various splicing variants of AID mRNA, some of those variants corresponding to the full length isoform (n = 6/17), and other variants corresponding to AID-ΔE4a (n = 2/17) or AID-ΔE4 (n = 7/17) isoforms known to be expressed in normal germinal center B cells as well as in Chronic Lymphocytic and Acute Lymphoblastic Leukemia. These findings indicate that although expressed at the mRNA and protein levels, AID may not be fully functional in SMZL cases. Finally we addressed the potential clinical significance of AID expression. We identified for that purpose a group of “progressive SMZL” patients that had received immuno-chemotherapy after splenectomy because of a significant risk of progression or transformation into aggressive large B cell lymphoma (n = 8/27) pre-empting outcome differences. We found a higher proportion of AID expressing patients in the defined “progressive SMZL” group (n = 7/8) as compared to the proportion found in the “indolent SMZL” group (n = 5/14, p = 0,03). Altogether, this data suggest that the B cell clone leading to SMZL originate from the marginal zone and support the hypothesis of a lymphoproliferative disorder affecting the T-independent response. AID expression in SMZL may reflect an advanced stage of the disease and could be correlated with the evolution of the lymphoma into a more clinically or pathologically aggressive form. Disclosures: No relevant conflicts of interest to declare.

Frequent Loss of the SLP-65 Adapter Protein in Childhood Acute B-Precursor Lymphoblastic Leukemia (ALL) with TEL/AML1 Rearrangement.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 741-741 ◽  
Author(s):  
Arndt Borkhardt ◽  
Christine Damm-Welk ◽  
Thomas Wossning ◽  
Bettina Storch ◽  
Uta Fuchs ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
Tumor Suppressor ◽  
Protein Expression ◽  
B Cell ◽  
Cell Lines ◽  
Sh2 Domain ◽  
B Cell Differentiation ◽  
Cell Leukemia ◽  
B Cell Leukemia

Abstract The adaptor protein SLP-65 plays an essential role during B cell differentiation. A crucial consequence of SLP-65 deficiency in mice is a high incidence of pre-B-cell leukemia, suggesting a tumor suppressor role for SLP-65 in pre-B-cells. While the link between SLP-65 deficiency and leukemia development is established in mice, experiments mainly using microarrays for gene expression profiling suggested normal expression of SLP-65 in human precursor B-cell ALL. This analysis however does not discriminate between normal and aberrant SLP-65 transcripts with the latter being unable to generate functional protein. To examine the correlation between SLP-65 deficiency and childhood precursor B-cell ALL, we determined SLP-65 expression in 119 precursor B-cell ALL samples by both RNA and protein methods. The expression of SLP-65 was compared to clinical and laboratory findings, cytogenetics as well as to the outcome data within this uniformly treated cohort of patients. Loss of slp-65 protein was significantly associated with the occurrence of the TEL/AML1 rearrangement (p=0.026) but not with any other clinical or cytogenetic feature. We found a profound disconnection between slp-65 mRNA and protein expression in 38 out of the 119 leukemic samples pointing to a posttranscriptional regulation of slp-65 (Table). To confirm that SLP-65 transcript expression does not automatically correlate with its protein expression, we analyzed a panel of human cell lines derived from precursor B-cell ALL patients. The cell lines HPB-NULL and BV-173 showed a deficiency in SLP-65 protein expression, although SLP-65 transcripts can easily be detected in both lines. Together, the data suggest that SLP-65 expression might be regulated at the posttranscriptional level and that the presence of SLP-65 transcripts does not necessarily lead to SLP-65 protein and function. In one particular patient, we found a truncated slp-65 transcript and the predicted slp-65 protein lacks its SH2 domain. We tested whether this SLP-65 protein lacking the SH2 domain is functional in pre-B cells. To this end, we transfected murine SLP-65 −/− pre-B cells with retroviral constructs for either wild-type (wt SLP-65) or truncated SLP-65 (SLP-65delSH2) and analysed pre-BCR downregulation, Ca2+ release and pre-B cell differentiation. The results showed that, in contrast to wt SLP-65, SLP-65delSH2 failed to induce any effects in the performed experiments. Together with previous findings showing that SLP-65-deficient mice develop pre-B cell leukemia, the data suggest that SLP-65 acts as a tumor suppressor that limits pre-B cell proliferation by inducing differentiation. Disconnection between slp-65 transcripts and protein expression total slp-65 protein+ (51 patients) slp-65 protein weak (19 patients) slp-65 protein- (49 patients) PCR+ 108 51(9 TEL/AML+, 42 TEL/AML-) 19 (9 TEL/AML+, 10 TEL/AML-) 38 (15 TEL/AML+, 23 TEL/AML-) PCR- 11 0 0 11 (T-ALL)

The Role of DNA Repair in the Pathogenesis of Activation Induced Cytidine Deaminase Dependent B Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 397-397
Author(s):  
Xiwen Gu ◽  
Carmen J. Booth ◽  
David G. Schatz ◽  
Matthew P. Strout
Keyword(s):  
Dna Repair ◽  
B Cells ◽  
B Cell ◽  
Median Survival ◽  
Cell Lymphoma ◽  
Cytidine Deaminase ◽  
B Cell Lymphoma ◽  
Enzyme Activation ◽  
Activation Induced Cytidine Deaminase

Abstract Abstract 397 Upon antigenic stimulation of B cells, germinal centers (GCs) are formed where somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes serve to diversify the immune response. SHM and CSR are initiated by the enzyme activation induced cytidine deaminase (AID) through the conversion of C/G base pairs to U-G mismatches. These mismatches are processed by UNG-dependent base excision repair (BER) and MSH2-dependent mismatch repair (MMR) pathways to yield mutations (for SHM) and DNA strand lesions (for CSR). Despite this essential role in immune diversification, the intrinsic activity of AID as a DNA mutator poses a threat to genomic integrity. Indeed, aberrant targeting of AID activity is associated with translocations and point mutations of proto-oncogenes associated with B cell malignancies. A specific dependence on AID in the pathogenesis of lymphomas of GC B cell origin is exemplified in Iμ-Bcl6 knock-in mice. These mice develop a diffuse large B cell lymphoma (DLBL) that resembles the human disease but are protected from development of this lymphoma when crossed onto an Aid-deficient background. To investigate the role of Aid-associated DNA repair in the pathogenesis of this disease, we crossed Iμ-Bcl6 mice onto a background deficient in BER (Ung−/−) and MMR (Msh2−/−). Young healthy Iμ-Bcl6 and Iμ-Bcl6 Ung−/−Msh2−/− mice displayed a normal number and distribution of B cells and normal architecture of lymphoid organs. Five of 28 Iμ-Bcl6 mice (17.9%) became sick starting at ∼12 months of age. Historically, median survival in these mice has not been reached and ∼80% survive to 15 months. In contrast, 21 of 28 Iμ-Bcl6 Ung−/−Msh2−/−mice (75%) developed disease with an onset of ∼3 months and had a median survival of 6.2 months (p<0.0001). All 5 of the Iμ-Bcl6 mice and the majority of Iμ-Bcl6 Ung−/−Msh2−/−mice developed B cell lymphoma with splenic involvement and variable nodal involvement. Five of the Iμ-Bcl6 Ung−/−Msh2−/−mice developed other cancers (3 T cell lymphomas, 1 pre-B cell lymphoma and 1 colon adenocarcinoma). Tumors from both genotypes expressed a mature B cell phenotype (B220+ IgM+ Igκ+ CD138-) and morphology revealed loss of normal lymphoid architecture with infiltration by lymphoid blasts. Additional staining demonstrated expression of at least one GC marker (Fas, GL7 and/or PNA). Similar to Iμ-Bcl6 mice, while many of the Iμ-Bcl6 Ung−/−Msh2−/−tumors had clonal mutated Ig heavy chain gene variable regions, two of the tumors were identified as oligoclonal, suggesting a preceding lymphoproliferative stage. In the absence of Ung and Msh2, Aid-generated U-G mismatches are not recognized and are simply replicated, causing only C/G to T/A transition mutations and no strand lesions. Thus, as expected, all Ig mutations in Iμ-Bcl6 Ung−/−Msh2−/−mice were C/G to T/A transitions. Lymphomas from Iμ-Bcl6 mice have been found to harbor numerous chromosome translocations and aneuploidies. Although additional analyses are underway, spectral karyotyping of 3 Iμ-Bcl6 Ung−/−Msh2−/−tumors revealed 2 with normal cytogenetics and 1 with a 40–41,XX,t(2;17),+15,+19. Surprisingly, sequence analysis of several known Aid target genes (cMyc, Pim1, RhoH, Pax5, Cd79a, Fas, H2ax and OcaB) in tumors from 3 Iμ-Bcl6 Ung−/−Msh2−/− mice did not identify any clonal mutations. However, non-clonal C/T to T/A transition mutations in cMyc were present at a frequency of 1.2 × 10−4, suggestive of ongoing Aid activity. The presence of Aid activity but absence of off-target Aid-mediated clonal SHM suggests that either other genes are targeted by Aid or that Aid has a secondary role in lymphomagenesis such as epigenetic reprogramming, as has been shown in iPS cells. Nonetheless, the incidence of Aid-dependent lymphomagenesis in the absence of Aid-associated DNA repair is significantly increased and the latency is greatly shortened. Altogether, this data suggests that Aid-associated BER and MMR pathways afford a protective effect against the development of Aid-dependent GC B cell lymphomas such as DLBL. To investigate the role of the individual Aid-associated DNA repair pathways, we have also generated Iμ-Bcl6 Ung−/− and Iμ-Bcl6 Msh2−/− single knockout mice. These studies are ongoing but preliminary results suggest that while the effect of Ung and Msh2 deficiency on lymphomagenesis may be synergistic, Msh2 might play a more critical role in preventing Aid-mediated genomic instability. Disclosures: No relevant conflicts of interest to declare.

scholarly journals FTY720 demonstrates promising preclinical activity for chronic lymphocytic leukemia and lymphoblastic leukemia/lymphoma

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 275-284 ◽  
Author(s):  
Qing Liu ◽  
Xiaobin Zhao ◽  
Frank Frissora ◽  
Yihui Ma ◽  
Ramasamy Santhanam ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Raji Cell ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
B Cell Malignancies ◽  
Pp2a Activation

FTY720 is an immunosuppressant developed to prevent organ transplant rejection. Recent studies indicate an additional role for FTY720 in inducing cell apoptosis. We demonstrate here that FTY720 mediates toxic effects in cell lines representing different B-cell malignancies and primary B cells from patients with chronic lymphocytic leukemia (CLL). In contrast to previous reports in T-cell lines, FTY720-induced toxicity in the Raji cell line and primary CLL B cells is independent of activation of caspases or poly(ADP-ribose) polymerase processing. Further, pancaspase inhibitor Z-VAD-fmk failed to rescue these cells from apoptosis mediated by FTY720. FTY720 induced down-regulation of Mcl-1 but not Bcl-2 in CLL B cells. Overexpression of Bcl-2 failed to protect transformed B cells from FTY720-induced apoptosis, suggesting a Bcl-2–independent mechanism. Interestingly, FTY720 induced protein phosphatase 2a (PP2a) activation and downstream dephosphorylation of ERK1/2, whereas okadaic acid at concentrations that inhibited the FTY720-induced PP2a activation also resulted in inhibition of FTY720-mediated apoptosis and restoration of baseline ERK1/2 phosphorylation in primary CLL cells, indicating a role for PP2a activation in FTY720-induced cytotoxicity. Further, FTY720 treatment resulted in significant prolonged survival in a xenograft severe combined immunodeficiency (SCID) mouse model of disseminated B-cell lymphoma/leukemia. These results provide the first evidence for the potential use of FTY720 as a therapeutic agent in a variety of B-cell malignancies, including CLL.

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