AID is a poor prognostic marker of high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements.

Diffuse large B-cell lymphoma with MYC rearrangement is defined as double/triple-hit lymphoma (DHL/THL) or single-hit lymphoma (SHL) by the inclusion of the BCL2 and BCL6 rearrangements status. DHL/THL is called as "high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements" in the World Health Organization 2017 Classification of Tumors of Hematopoietic and Lymphoid Tissues. To find a prognostic biomarker of DHL/THL, we firstly examined 19 cases (molecular analysis series;10 cases of DHL/THL and 9 cases of SHL) with gene expression profile analysis. The gene expression profile analysis showed that the high expression of AICDA was associated with an adverse prognosis in DHL/THL, but not in SHL. Then, we evaluated immunohistochemical expression of AID, the protein product of AICDA, in 50 cases (molecular analysis series of 19 cases and additional immunohistochemistry series of 31 cases; 12 cases of DHL/THL and 19 cases of SHL) and confirmed that its expression was also associated with an adverse prognosis in DHL/THL. Therefore, AICDA and AID can be a predictor of an adverse clinical outcome in DHL/THL and immunohistochemistry of AID is useful to find DHL/THL-adverse prognosis group.

Human Herpes Simplex Virus-1 depletes APOBEC3A from nuclei.

APOBEC3 family of DNA-cytosine deaminases inactivate and mutate several human viruses. We constructed a human cell line that is inducible for EGFP-tagged APOBEC3A and found A3A predominantly in the nuclei. When these cells were infected with Herpes Simplex Virus-1, virus titer was unaffected by A3A expression despite nuclear virus replication. When A3A expression and virus infection were monitored, A3A was found predominantly to be nuclear in infected cells up to 3 h post-infection, but was predominantly cytoplasmic by 12 h. This effect did not require the whole virus, and could be reproduced using the UL39 gene of the virus which codes for a subunit of the viral ribonucleotide reductase. These results are similar to the reported exclusion of APOBEC3B by Epstein Barr virus ortholog of UL39, BORF2, but HSV1 UL39 gene product appears better at excluding A3A than A3B from nuclei.

Aberrant activation-induced cytidine deaminase expression in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia.

Activation-induced cytidine deaminase (AID) is expressed in germinal center B cells and plays a critical role in somatic hypermutation and class-switch recombination of immunoglobulin genes. Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) carries a poor prognosis and is specifically treated with tyrosine kinase inhibitors. Interestingly, AID has been shown to be aberrantly expressed and functional in Ph+ ALL and is thought to contribute to genetic instability. We hypothesized that AID might be detectable in routinely processed bone marrow biopsies by immunohistochemistry (IHC) and assist in identifying Ph+ ALL. We found that AID was expressed in 26 (70%) of 37 cases of Ph+ ALL but only 1 (2.9%) of 38 cases of Ph- ALL cases. There was a significant difference in AID expression between these 2 ALL groups (P < .001, Fisher exact test). The expression of AID was confirmed by RT-PCR (reverse-transcriptase polymerase chain reaction) and correlated with IHC scoring. AID protein is expressed in a large proportion of Ph+ ALL cases at levels detectable by IHC in clinical samples and might be useful to rapidly identify cases likely to have a BCR/ABL1 fusion.

Expression of Stathmin1 in gastric adenocarcinoma.

BACKGROUND: Over expression of Stathmin1 (STMN1), activation-induced cytidine deaminase (AID) and protein kinase C iota (PKCi) proteins participate in the regulation of carcinogenesis. In the present study, we investigated the expression of STMN1 in patients with gastric adenocarcinoma and also determined the correlation of STMN1 with AID and PKCi proteins. MATERIALS AND METHODS: This study was conducted in the Tokushima University Hospital between September 2009 and September 2010 on a total of 59 patients with gastric adenocarcinoma. Stathmin1, AID and PKCi protein expressions were evaluated by immuno-histochemistry in gastric adenocarcinoma. RESULTS: A strong expression of STMN1 was significantly associated with gender- and poorly differentiated gastric adenocarcinoma (p<0.05). A high mRNA level of STMN1 was found in the tumor tissue of gastric adenocarcinoma compared to non-tumor tissue (p<0.05). In addition, STMN1 expression was significantly correlated with AID and PKCi protein expressions in gastric adenocarcinoma (p<0.05). CONCLUSION: High mRNA level of the Stathmin1 gene was significantly expressed in gastric tumor tissue than non-tumor and strong expression of STMN1 protein is correlated with poorly-differentiated gastric adenocarcinoma.

Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia.

Activation-induced deaminase (AID) is a DNA-mutating enzyme that mediates class-switch recombination as well as somatic hypermutation of antibody genes in B cells. Due to off-target activity, AID is implicated in lymphoma development by introducing genome-wide DNA damage and initiating chromosomal translocations such as c-myc/IgH. Several alternative splice transcripts of AID have been reported in activated B cells as well as malignant B cells such as chronic lymphocytic leukemia (CLL). As most commercially available antibodies fail to recognize alternative splice variants, their abundance in vivo, and hence their biological significance, has not been determined. In this study, we assessed the protein levels of AID splice isoforms by introducing an AID splice reporter construct into cell lines and primary CLL cells from patients as well as from WT and TCL1(tg) C57BL/6 mice (where TCL1 is T-cell leukemia/lymphoma 1). The splice construct is 5'-fused to a GFP-tag, which is preserved in all splice isoforms and allows detection of translated protein. Summarizing, we show a thorough quantification of alternatively spliced AID transcripts and demonstrate that the corresponding protein abundances, especially those of splice variants AID-ivs3 and AID-ΔE4, are not stoichiometrically equivalent. Our data suggest that enhanced proteasomal degradation of low-abundance proteins might be causative for this discrepancy.

Duodenal follicular lymphoma lacks AID but expresses BACH2 and has memory B-cell characteristics.

We have reported previously that duodenal follicular lymphoma (FL) is distinct from nodal FL and showed more resemblance to mucosa-associated lymphoid tissue lymphoma, and that FL frequently involved the duodenal second portion. In the present study, we examined duodenal FLs and gastric/colonic FLs to clarify the clinicopathological and immunological differences between the tumor types. We analyzed 8 samples of gastric FL, 17 of duodenal ones, and 5 of colonic/rectal ones, and characterized them by immunohistochemistry, immunogenotyping, and histology. Gastric and colonic FLs presented in submucosal to subserosal areas, whereas duodenal ones presented in the mucosal to submucosal layers. Immunohistochemical analysis revealed that duodenal FLs exhibited the following phenotypes: CD10 (+), B-cell lymphoma 2 (BCL-2) (+), BCL-6 (+), activation-induced cytidine deaminase (AID) (-), BACH2 (+), CD27 (+), MUM-1 (-), Blimp-1 (-), and loose CD21 network (duodenal pattern). Gastric/colonic FLs exhibited the following phenotypes: CD10 (+), BCL-2 (+), BCL-6 (+), AID (+), BACH2 (+), CD27 (-), MUM-1 (-), Blimp-1 (-), and a dense CD21 network (nodal pattern). Expression of AID and CD27 in lymphoma cells and the CD21 network pattern were considerably different between duodenal FLs and gastric/colonic ones. Moreover, in situ hybridization revealed that, in the duodenal FLs, BACH2 was expressed at the periphery of the tumor follicle and tumor villi. The number of immunoglobulin heavy-chain variable domains VH4 and VH5 were higher in duodenal follicular lymphomoas than in gastric FLs. The lymphoma cells of duodenal FLs are different from those of gastric/colonic FLs, and duodenal FL is distinct even within the gastrointestinal tract. Somatic hypermutation in immunoglobulin genes and CD27 expression are hallmarks of memory B cells. We suggest that duodenal FL cells are in the memory B-cell stage, and require BACH2 instead of AID for ongoing mutation.

[Chronic inflammation and gastric cancer development].

H. pylori-induced gastritis plays important roles in gastric cacrcinogenesis and cancer development is characterized by accumulation of various gene aberrations. Activation-induced cytidine deaminase (AID) is exclusively expressed in B lymphocytes and is essential for diversification of immunoglobulin genes. H. pylori-positive human gastritis mucosa ectopically express AID. H. pylori infection to gastric cells in vitro induced AID in association with induction of various gene mutations and deletions. H. pylori-induced AID is mediated by NFkappaB. Furthermore, AID gene introduction into gastric mucosal cells accelerated p53 gene mutations, and inhibition of AID using siRNA significantly reduced H. pylori-induced p53 gene mutations. Thus, H. pylori infection induces ectopic AID expression in the gastric mucosal cells via NFkappaB activation, resulting in various gene mutations and aberrations in the gastric mucosal cells leading to gastric cancer development.

E2A-positive gastric MALT lymphoma has weaker plasmacytoid infiltrates and stronger expression of the memory B-cell-associated miR-223: possible correlation with stage and treatment response.

Extranodal marginal-zone lymphoma of mucosa-associated lymphoid tissue of the stomach (gastric MALT lymphoma) is derived from memory B cells of the marginal zone. Normal memory B cells do not express markers of germinal-center B cells, such as E2A (immunoglobulin enhancer-binding factor E12/E47), B-cell chronic lymphocytic leukemia/lymphoma 6 (BCL6), or activation-induced cytidine deaminase (AID). E2A is a transcription factor that induces somatic hypermutations and blocks plasma cell differentiation. In 50 stage-I(E)/II(E1) gastric MALT lymphomas, we confirmed that all cases were BCL6(-)/AID(-), but a subset (50%, 25/50) was E2A(+). As E2A(-) and E2A(+) gastric MALT lymphomas had similar numbers of somatic hypermutations without intraclonal variations, which implied an origin from memory B cells, the expression of E2A was best regarded as a marker of aberrant follicular differentiation. Although the status of somatic hypermutation was not affected by E2A, E2A(+) gastric MALT lymphoma showed less plasmacytoid infiltrates and higher expressions of miRNA-223, a microRNA associated with memory B cells. Clinically, E2A(+) gastric MALT lymphomas were more likely to spread to perigastric lymph nodes and were less responsive to Helicobacter eradication therapy than were E2A(-) gastric MALT lymphomas. Taken together, aberrant E2A expression is a diagnostic feature of a subtype of gastric MALT lymphoma with weaker plasmacytoid infiltrates and stronger miR-223 expression. A prospective study would be necessary to verify the association between E2A expression and a poor response to Helicobacter eradication therapy.

The mechanisms regulating the subcellular localization of AID.

Activation induced deaminase (AID) is a unique enzyme that directly introduces mutations in the immunoglobulin genes to generate antibody diversity during the humoral immune response. Since this mutator enzyme poses a measurable risk of off-target mutation, which can be deleterious or transforming for a cell, several regulatory mechanisms exist to control its activity. At least three of these mechanisms affect AID subcellular localization. It was recently found that AID is actively imported into the nucleus, most likely through importin-α/ß recognizing a structural nuclear localization signal. However, AID is largely excluded from the nucleus in steady state thanks to two mechanisms. In addition to nuclear export through the exportin CRM1, a mechanism retaining AID in the cytoplasm exists. Cytoplasmic retention hinders the passive diffusion of AID into the nucleus playing an important role in the nuclear exclusion of AID. Subcellular localization of AID also determines its stability. The regulation of the nuclear fraction of AID by these many mechanisms has functional implications for antibody diversification.

Subepithelial dendritic B cells in orofacial granulomatosis.

BACKGROUND: Orofacial granulomatosis (OFG) is a chronic, disfiguring, granulomatous inflammation of the lips and oral mucosa. The pathogenesis is unknown, but it has been linked previously to Crohn's disease (CD) and more recently to dietary sensitivity. The oral mucosa is an immunologically responsive site associated with the generation of protective mucosal and systemic immune responses to vaccination and also hyperresponsiveness to allergens in some individuals. Classically, immune responses in oral mucosa are considered to be mediated by mucosa-associated lymphoid tissues (MALT), secondary lymphoid follicles that are intimately associated with epithelia. METHODS: Immunohistochemistry was used to investigate the inflammatory infiltrate in OFG and control tissue samples. Polymerase chain reaction (PCR), cloning of PCR products, and sequencing were used to characterize the local immunoglobulin gene profile in OFG. RESULTS: We describe large, active, dendritic B cells in oral mucosa that were not associated with any organized lymphoid tissues in the local subepithelial microenvironment. They express activation induced cytidine deaminase, which is essential for immunoglobulin gene diversification by somatic hypermutation and class switch recombination. IgE is also expressed by these B cells. They do not align with any other previously described B-cell subset in secondary lymphoid tissues in terms of morphology, proliferative activity, or phenotype. CONCLUSIONS: These subepithelial dendritic B cells may contribute to the immune responsiveness of the oral mucosa, including IgE-mediated allergic responses. In patients with OFG, further understanding of the role these cells play in oral immunity may lead to novel therapeutic possibilities.

Speckled-like pattern in the germinal center (SLIP-GC), a nuclear GTPase expressed in activation-induced deaminase-expressing lymphomas and germinal center B cells.

We identified a novel GTPase, SLIP-GC, with expression limited to a few tissues, in particular germinal center B cells. It lacks homology to any known proteins, indicating that it may belong to a novel family of GTPases. SLIP-GC is expressed in germinal center B cells and in lymphomas derived from germinal center B cells such as large diffuse B cell lymphomas. In cell lines, SLIP-GC is expressed in lymphomas that express activation-induced deaminase (AID) and that likely undergo somatic hypermutation. SLIP-GC is a nuclear protein, and it localizes to replication factories. Reduction of SLIP-GC levels in the Burkitt lymphoma cell line Raji and in non-Hodgkin lymphoma cell lines resulted in an increase in DNA breaks and apoptosis that was AID-dependent, as simultaneous reduction of AID abrogated the deleterious effects of SLIP-GC reduction. These results strongly suggest that SLIP-GC is a replication-related protein in germinal center B cells whose reduction is toxic to cells through an AID-dependent mechanism.

Cholangiocarcinomas associated with long-term inflammation express the activation-induced cytidine deaminase and germinal center-associated nuclear protein involved in immunoglobulin V-region diversification.

Cholangiocarcinoma (CCA) represents a model of tumor development after long-term inflammation which causes DNA damage or impairs DNA repair mechanism. AID and GANP, both appearing in antigen-driven B cells, are involved in affinity maturation of the immunoglobulin V-region with increased somatic mutation. A normal cholangiocyte line showed the induction of AID transcripts after stimulation with TNF-alpha, whereas ganp transcripts appeared constitutively in this cell line. Next, we examined the expression of AID and GANP in clinical CCA specimens to obtain information whether their expression levels are associated with the malignant grade of CCA. AID expression was similarly detected in the clinical cases of both well-differentiated and poorly-differentiated CCAs. On the contrary, GANP expression was detected in CCA cells at a higher level in the nucleus of poorly-differentiated CCAs with shorter survivals than in that of well-differentiated CCAs. The high and low cases of nuclear GANP expression showed no change in the frequency of the TP53 mutations, however, further investigation by in vitro experiment demonstrated that the high GANP expression caused the increased number of gammaH2AX foci after DNA damage by ionizing-irradiation. These results suggest that GANP is involved in regulation of DNA repair mechanism and the abnormal over-expression of GANP together with AID might be associated with rigorous DNA damage, potentially causing the malignant development of CCAs during long-term inflammation.

The intersection of Epstein-Barr virus with the germinal center.

The current model of Epstein-Barr virus (EBV) infection and persistence in vivo proposes that EBV uses the germinal center (the GC model) to establish a quiescent latent infection in otherwise-normal memory B cells. However, the evidence linking EBV-infected cells and the GC is only indirect and limited. Therefore, a key portion of the model, that EBV-infected cells physically reside and participate in GCs, has yet to be verified. Furthermore, recent experiments suggested that upon infection of GC cells the viral growth latency transcription program is dominant and GC functionality and phenotype are ablated, i.e., EBV infection is not consistent with GC function. In this study we show that in vivo, EBV-infected B cells in the tonsils retain expression of functional and phenotypic markers of GC cells, including bcl-6 and AID. Furthermore, these cells are physically located in the GC and express a restricted form of latency, the default latency program. Thus, the EBV default latency transcription program, unlike the growth latency program, is consistent with the retention of GC functionality in vivo. This work verifies key components of the GC model of EBV persistence and suggests that EBV and the GC can interact to produce the latently infected memory cells found in the periphery. Furthermore, it identifies latently infected GC B cells as a potential pathogenic nexus for the development of the EBV-positive, GC-associated lymphomas Hodgkin's disease and Burkitt's lymphoma.

MDM2 is a novel E3 ligase for HIV-1 Vif.

The human immunodeficiency virus type 1 (HIV-1) Vif plays a crucial role in the viral life cycle by antagonizing a host restriction factor APOBEC3G (A3G). Vif interacts with A3G and induces its polyubiquitination and subsequent degradation via the formation of active ubiquitin ligase (E3) complex with Cullin5-ElonginB/C. Although Vif itself is also ubiquitinated and degraded rapidly in infected cells, precise roles and mechanisms of Vif ubiquitination are largely unknown. Here we report that MDM2, known as an E3 ligase for p53, is a novel E3 ligase for Vif and induces polyubiquitination and degradation of Vif. We also show the mechanisms by which MDM2 only targets Vif, but not A3G that binds to Vif. MDM2 reduces cellular Vif levels and reversely increases A3G levels, because the interaction between MDM2 and Vif precludes A3G from binding to Vif. Furthermore, we demonstrate that MDM2 negatively regulates HIV-1 replication in non-permissive target cells through Vif degradation. These data suggest that MDM2 is a regulator of HIV-1 replication and might be a novel therapeutic target for anti-HIV-1 drug.

Activation-induced cytidine deaminase links bile duct inflammation to human cholangiocarcinoma.

UNLABELLED: Chronic inflammation plays a critical role in oncogenesis in various human organs. Epidemiological studies have demonstrated that patients with primary sclerosing cholangitis have a predisposition to develop cholangiocarcinoma (CC). However, the molecular mechanisms that account for the development of bile duct carcinomas are not well defined. We recently provided evidence that activation-induced cytidine deaminase (AID), a member of the DNA/RNA editing enzyme family, is implicated in human tumorigenesis via its mutagenic activity. We found here that ectopic AID production is induced in response to tumor necrosis factor-alpha (TNF-alpha) stimulation via the IkappaB kinase-dependent nuclear factor-kappaB (NF-kappaB) activation pathway in human cholangiocarcinoma-derived cells. Aberrant expression of AID in biliary cells resulted in the generation of somatic mutations in tumor-related genes, including p53, c-myc, and the promoter region of the INK4A/p16 sequences. In human tissue specimens, real-time reverse transcription polymerase chain reaction (RT-PCR) analyses revealed that AID was increased significantly in 28 of 30 CC tissues (93%), whereas only trace amounts of AID were detected in the normal liver. Immunohistochemistry showed that all of the CC tissue samples examined showed overproduction of endogenous AID protein in cancer cells. Moreover, immunostaining for AID was detectable in 16 of 20 bile epithelia in the tissues underlying primary sclerosing cholangitis. CONCLUSION: The proinflammatory cytokine-induced aberrant production of AID might link bile duct inflammation to an enhanced genetic susceptibility to mutagenesis, leading to cholangiocarcinogenesis.

Frequent occurrence of B-cell lymphomas in angioimmunoblastic T-cell lymphoma and proliferation of Epstein-Barr virus-infected cells in early cases.

Secondary lymphomas occurring in the setting of angioimmunoblastic T-cell lymphoma (AILT) are considered to be rare. Their occurrence has been attributed to Epstein-Barr virus (EBV)-associated lymphoproliferations. A previous study detected a dysregulated hypermutation process in B-cells of AILT. The present study aimed at estimating the frequency of B-cell lymphomas in AILT. By studying the expression of EBV and activation-induced cytidine deaminase (AID) as an indicator of hypermutating cells, we assessed whether B-cell lymphoproliferations in AILT were strictly associated with EBV and whether hypermutation might contribute to lymphomagenesis. Among 161 cases of AILT, diagnosed between 1996 and 2005 at the lymph node registry, Frankfurt, Germany, 19 cases were detected that also had B-cell non-Hodgkin lymphoma (NHL) and two cases had classical Hodgkin lymphoma (HL). EBV was detected in tumour cells of 7/18 NHL and both HL, suggesting that factors other than EBV contribute to lymphomagenesis. AID was expressed in AILT in large cells disseminated in the tissue, implying that the process of somatic hypermutation is ongoing in AILT, although the GC architecture is disrupted. This might be relevant in the development of secondary lymphomas.

Arsenic trioxide induces p53-dependent apoptotic signals in myeloma cells with SiRNA-silenced p53: MAP kinase pathway is preferentially activated in cells expressing inactivated p53.

Mutations in p53 are the most common genetic abnormality in cancers. Arsenic trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia (APL) and is being tested in phase II studies in various types of cancers. We have shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging primarily the intrinsic apoptotic pathway in cells expressing w.t. p53 and the extrinsic apoptotic pathway in cells expressing mutant p53. To further establish the differential apoptotic signals of ATO in relation to p53 functional status we studied the activation of the intrinsic and the extrinsic apoptotic pathways in IM9 myeloma cells expressing w.t. p53 following silencing of p53 and p21 with the corresponding SiRNAs-GFP constructs. In untransfected cells or in cells transfected with GFP-empty vector construct we observed weak apoptosis concomitant with mild depolarization of mitochondrial membrane, depletion of reduced glutathione and release of cytochrome c. Following silencing of p53 or p21 we observed extensive apoptosis concomitant with extensive depolarization of mitochondrial membrane and depletion of reduced glutathione. We also observed in these cells activation of the extrinsic apoptotic pathway through upregulation of APO2/TRAIL and APO2/TRAIL-R2, activation of caspase 8, degradation of FLIP-L and release of apoptosis inducing factors from mitochondria, instead of cytochrome c. In addition, we observed marked activation of the MAP kinase pathway and dephosphorylation of Akt in p53 or p21 silenced cells. Hence, silencing of p53 or p21 in IM9 myeloma cells results in diversion of apoptosis to the extrinsic pathway and sensitization of myeloma cells to ATO.

Monocyte fructose 1,6-bisphosphatase and cytidine deaminase enzyme activities: potential pharmacodynamic measures of calcitriol effects in cancer patients.

PURPOSE: To determine, in peripheral blood monocytes (PBM), whether the enzymatic activities of fructose 1,6-bisphosphatase (FBPase), cytidine deaminase (CDDase) and 24-hydroxylase (CYP24), enzymes regulated by calcitriol are useful pharmacodynamic (PD) measures of calcitriol effects in cancer patients. METHODS: Cancer patients enrolled in a phase I clinical trial of calcitriol and carboplatin were studied. Baseline and calcitriol-induced changes in FBPase, CDDase and CYP24 activities were measured in PBM collected before, 6, 24, and 48 h after administration of calcitriol, prior to carboplatin, in doses ranging from 4 to 11 mug daily for 3 consecutive days (QDx3). Normal FBPase, CYP24 and CDDase activities were measured in PBM from untreated healthy volunteers. RESULTS: Baseline activities in PBM from cancer patients and healthy volunteers were (median and range): 1.0 (0.0-43.5) and 4.4 (3.1- 8.2) nmol/min/mg protein for FBPase (P = 0.002); 2.5 (0.9-9.3) and 0.8 (0.4-2.0) fmol/h/10(6) cells for CYP24 (P = 0.016), and 5.6 (2.5-22.3) and 6.6 (1.1-47.4) nmol/min/mg protein for CDDase (P > 0.05), respectively. All calcitriol doses achieved peak serum calcitriol levels > x3 the physiological levels, increased cancer patient PBM FBPase activity to normal levels and decreased CDDase activity to undetectable levels within 48 h, with no significant change in CYP24 activity. These enzyme activity changes were not associated with hypercalcemia. CONCLUSIONS: Calcitriol treatment-induced increase in FBPase and decrease in CDDase activities in cancer patient PBM are potential early and sensitive non-hypercalcemia PD measures of calcitriol effects.