CD8 follicular T cells localize throughout the follicle during germinal center reactions and maintain cytolytic and helper properties.

Follicular CXCR5+ PD-1+ CD8 T cells (CD8 Tfc) arise in multiple models of systemic autoimmunity yet their functional contribution to disease remains in debate. Here we define the follicular localization and functional interactions of CD8 Tfc with B cells during autoimmune disease. The absence of functional T regulatory cells in autoimmunity allows for CD8 Tfc development that then expands with lymphoproliferation. CD8 Tfc are identifiable within the lymph nodes and spleen during systemic autoimmunity, but not during tissue-restricted autoimmune disease. Autoimmune CD8 Tfc cells are polyfunctional, producing helper cytokines IL-21, IL-4, and IFNγ while maintaining cytolytic proteins CD107a, granzyme B, and TNF. During autoimmune disease, IL-2-KO CD8 T cells infiltrate the B cell follicle and germinal center, including the dark zone, and in vitro induce activation-induced cytidine deaminase in naïve B cells via IL-4 secretion. CD8 Tfc represent a unique CD8 T cell population with a diverse effector cytokine repertoire that can contribute to pathogenic autoimmune B cell response.

Activation-induced cytidine deaminase overexpression in double-hit lymphoma: potential target for novel anticancer therapy.

Activation-induced cytidine deaminase (AID) is one kind of the mutant enzymes, which target regulating the immunoglobulin (Ig) gene in Burkitt's lymphoma to initiate class switch recombination (CSR), resulting in c-Myc chromosomal translocation. However, it is not clear that whether AID induces c-Myc/IgH translocation in double-hit lymphoma (DHL) with c-Myc gene translocation. In this study, the AID in DHL tissues and classical diffuse large b-cell lymphoma (DLBCL) tissues were compared. The results suggested that AID is of important value in predicting DHL, stronger CSR of AID was observed in DHL patients, which exhibited AID overexpression and c-Myc gene translocation of DHL after CSR induction. It is concluded that AID directly induces CSR in DHL and may result in c-Myc gene translocation. Targeting AID may be a good treatment regimen for DHL.

Estrogen Reverses HDAC Inhibitor-Mediated Repression of Aicda and Class-Switching in Antibody and Autoantibody Responses by Downregulation of miR-26a.

Estrogen contributes to females' strong antibody response to microbial vaccines and proneness to autoimmunity, particularly antibody-mediated systemic autoimmunity, in females. We have hypothesized that this is due to estrogen-mediated potentiation of class switch DNA recombination (CSR) and somatic hypermutation (SHM). As we have shown, estrogen boosts AID expression, which is critical for both CSR and SHM, through upregulation of HoxC4, which together with NF-κB critically mediates Aicda (AID gene) promoter activation. We contend here that additional regulation of Aicda expression by estrogen occurs through epigenetic mechanisms. As we have shown, histone deacetylase inhibitors (HDIs) short-chain fatty acid (SCFA) butyrate and propionate as well as the pharmacologic HDI valproic acid upregulate miRNAs that silence AID expression, thereby modulating specific antibody responses in C57BL/6 mice and autoantibody responses in lupus-prone MRL/Faslpr/lpr mice. Here, using constitutive knockout Esr1-/- mice and B cells as well as conditional knockout Aicdacre/creEsr1flox/flox mice and B cells, we showed that the HDI-mediated downregulation of Aicda expression as well as the maturation of antibody and autoantibody responses is reversed by estrogen and enhanced by deletion of ERα or E2 inhibition. Estrogen's reversion of HDI-mediated inhibition of Aicda and CSR in antibody and autoantibody responses occurred through downregulation of B cell miR-26a, which, as we showed, targets Aicda mRNA 3'UTR. miR-26a was significantly upregulated by HDIs. Accordingly, enforced expression of miR-26a reduced Aicda expression and CSR, while miR-26a-sponges (competitive inhibitors of miR-26a) increased Aicda expression and CSR. Thus, our findings show that estrogen reverses the HDI-mediated downregulation of AID expression and CSR through selective modulation of miR-26a. They also provide mechanistic insights into the immunomodulatory activity of this hormone and a proof-of-principle for using combined ER inhibitor-HDI as a potential therapeutic approach.

APOBEC3B interaction with PRC2 modulates microenvironment to promote HCC progression.

OBJECTIVE: APOBEC3B (A3B), a cytidine deaminase acting as a contributor to the APOBEC mutation pattern in many kinds of tumours, is upregulated in patients with hepatocellular carcinoma (HCC). However, APOBEC mutation patterns are absent in HCC. The mechanism of how A3B affects HCC progression remains elusive. DESIGN: A3B -promoter luciferase reporter and other techniques were applied to elucidate mechanisms of A3B upregulation in HCC. A3B overexpression and knockdown cell models, immunocompetent and immune-deficient mouse HCC model were conducted to investigate the influence of A3B on HCC progression. RNA-seq, flow cytometry and other techniques were conducted to analyse how A3B modulated the cytokine to enhance the recruitment of myeloid--derived suppressor cells (MDSCs) and tumour--associated macrophages (TAMs). RESULTS: A3B upregulation through non-classical nuclear factor-κB (NF-κB)signalling promotes HCC growth in immunocompetent mice, associated with an increase of MDSCs, TAMs and programmed cell death1 (PD1) exprssed CD8+ T cells. A CCR2 antagonist suppressed TAMs and MDSCs infiltration and delayed tumour growth in A3B and A3BE68Q/E255Q- expressing mouse tumours. Mechanistically, A3B upregulation in HCC depresses global H3K27me3 abundance via interaction with polycomb repressor complex 2 (PRC2) and reduces an occupancy of H3K27me3 on promoters of the chemokine CCL2 to recruit massive TAMs and MDSCs. CONCLUSION: Our observations uncover a deaminase-independent role of the A3B in modulating the HCC microenvironment and demonstrate a proof for the concept of targeting A3B in HCC immunotherapy.

Pichia pastoris as a host for production and isolation of mutagenic AID/APOBEC enzymes involved in cancer and immunity.

AID/APOBEC3 enzymes are cytidine deaminases that mutate antibody and retroviral genes and also mediate extensive tumor genome mutagenesis. The study of purified AID/APOBEC3 proteins is challenged by difficulties with their expression and purification arising from genotoxicity in expression hosts, extensive non-specific protein-protein/DNA/RNA interactions and haphazard oligomerization. To date, expression hosts for purification of AID/APOBEC3 enzymes include bacteria, insect and mammalian cells. Here the establishment and optimization of a yeast expression/secretion system for AID/APOBEC3s are reported, followed by comparison with the same enzymes expressed in bacterial and mammalian hosts. AID and APOBEC3G were expressed successfully in Pichia pastoris, each either with an N-terminal GST tag, C-terminal V5-His tag or as untagged native form. It was verified that the yeast-expressed enzymes exhibit identical biochemical properties to those reported using bacterial and mammalian expression, indicating high fidelity of protein folding. It was demonstrated that the system can be adapted for secretion of the enzymes into the media which was used directly in various enzyme assays. The system is also amenable to elimination of bulky fusion tags, providing native untagged enzymes. Thus, P. pastoris is an advantageous expression factory for AID/APOBEC3 enzymes, considering the cost, time, efficiency and quality of the obtained enzymes. The first report is also provided here of a functionally active, untagged, secreted AID, which may become a useful research reagent. A comprehensive comparison is made of the effect of fusion tags and expression hosts on the biochemical actions of AID and APOBEC3G.

Ibrutinib therapy downregulates AID enzyme and proliferative fractions in chronic lymphocytic leukemia.

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination of the immunoglobulin genes. As a trade-off for its physiological function, AID also contributes to tumor development through its mutagenic activity. In chronic lymphocytic leukemia (CLL), AID is overexpressed in the proliferative fractions (PFs) of the malignant B lymphocytes, and its anomalous expression has been associated with a clinical poor outcome. Recent preclinical data suggested that ibrutinib and idelalisib, 2 clinically approved kinase inhibitors, increase AID expression and genomic instability in normal and neoplastic B cells. These results raise concerns about a potential mutagenic risk in patients receiving long-term therapy. To corroborate these findings in the clinical setting, we analyzed AID expression and PFs in a CLL cohort before and during ibrutinib treatment. We found that ibrutinib decreases the CLL PFs and, interestingly, also reduces AID expression, which correlates with dampened AKT and Janus Kinase 1 signaling. Moreover, although ibrutinib increases AID expression in a CLL cell line, it is unable to do so in primary CLL samples. Our results uncover a differential response to ibrutinib between cell lines and the CLL clone and imply that ibrutinib could differ from idelalisib in their potential to induce AID in treated patients. Possible reasons for the discrepancy between preclinical and clinical findings, and their effect on treatment safety, are discussed.

Polyomavirus T Antigen Induces APOBEC3B Expression Using an LXCXE-Dependent and TP53-Independent Mechanism.

APOBEC3B is a single-stranded DNA cytosine deaminase with beneficial innate antiviral functions. However, misregulated APOBEC3B can also be detrimental by inflicting APOBEC signature C-to-T and C-to-G mutations in genomic DNA of multiple cancer types. Polyomavirus and papillomavirus oncoproteins induce APOBEC3B overexpression, perhaps to their own benefit, but little is known about the cellular mechanisms hijacked by these viruses to do so. Here we investigate the molecular mechanism of APOBEC3B upregulation by the polyomavirus large T antigen. First, we demonstrate that the upregulated APOBEC3B enzyme is strongly nuclear and partially localized to virus replication centers. Second, truncated T antigen (truncT) is sufficient for APOBEC3B upregulation, and the RB-interacting motif (LXCXE), but not the p53-binding domain, is required. Third, genetic knockdown of RB1 alone or in combination with RBL1 and/or RBL2 is insufficient to suppress truncT-mediated induction of APOBEC3B Fourth, CDK4/6 inhibition by palbociclib is also insufficient to suppress truncT-mediated induction of APOBEC3B Last, global gene expression analyses in a wide range of human cancers show significant associations between expression of APOBEC3B and other genes known to be regulated by the RB/E2F axis. These experiments combine to implicate the RB/E2F axis in promoting APOBEC3B transcription, yet they also suggest that the polyomavirus RB-binding motif has at least one additional function in addition to RB inactivation for triggering APOBEC3B upregulation in virus-infected cells.IMPORTANCE The APOBEC3B DNA cytosine deaminase is overexpressed in many different cancers and correlates with elevated frequencies of C-to-T and C-to-G mutations in 5'-TC motifs, oncogene activation, acquired drug resistance, and poor clinical outcomes. The mechanisms responsible for APOBEC3B overexpression are not fully understood. Here, we show that the polyomavirus truncated T antigen (truncT) triggers APOBEC3B overexpression through its RB-interacting motif, LXCXE, which in turn likely modulates the binding of E2F family transcription factors to promote APOBEC3B expression. This work strengthens the mechanistic linkage between active cell cycling, APOBEC3B overexpression, and cancer mutagenesis. Although this mutational mechanism damages cellular genomes, viruses may leverage it to promote evolution, immune escape, and pathogenesis. The cellular portion of the mechanism may also be relevant to nonviral cancers, where genetic mechanisms often activate the RB/E2F axis and APOBEC3B mutagenesis contributes to tumor evolution.

APOBEC3B protein expression and mRNA analyses in patients with high-grade serous ovarian carcinoma.

APOBEC3 enzymes are part of the innate immune system and they are important in retroviral defense. The number of mutations in ovarian cancer increases with rising levels of APOBEC3B mRNA. We could confirm that APOBEC3B mRNA is upregulated in ovarian cancer cell lines and in ovarian cancer tissue. We evaluated APOBEC3B expression in histologically defined subtypes of ovarian cancer to identify its influence on overall survival (OS) and progression-free survival (PFS). Tissue microarrays from 219 patients with high-grade serous (HGSC), 61 with low-grade serous (LGSC), 62 with endometrioid (EC) and 55 with clear cell (CCC) ovarian carcinoma were stained using an antibody against APOBEC3B. Real-time quantitative PCR was performed to detect APOBEC3B mRNA levels in 274 cases of HGSC, in 11 cases of LGSC, in 47 cases of EC and in 29 cases of CCC. Tumor-infiltrating lymphocytes (TILs) have been evaluated in a previous project. APOBEC3B staining was cytoplasmic as well as nuclear and both were positively correlated (P<0.001). In HGSC a trend was detectable for positive cytoplasmic staining as favorable regarding OS (P=0.283) and PFS (P=0.137). High levels of APOBEC3B mRNA were associated with prolonged PFS in HGSC in univariate analyses (P=0.043) and multivariate analyses (HR 0.55; 95%CI 0.35-0.88; P=0.012). APOBEC3B cytoplasmic staining and APOBEC3B mRNA were positively correlated with TILs. APOBEC3B in HGSC is related to an active immune infiltrate. However, there is no evidence for APOBEC3B as a clinically relevant prognostic biomarker.

Activation-induced deaminase and its splice variants associate with trisomy 12 in chronic lymphocytic leukemia.

Activation-induced cytidine deaminase (AID) is a mutator enzyme essential for somatic hypermutation (SHM) and class switch recombination (CSR) during effective adaptive immune responses. Its aberrant expression and activity have been detected in lymphomas, leukemias, and solid tumors. In chronic lymphocytic leukemia (CLL) increased expression of alternatively spliced AID variants has been documented. We used real-time RT-PCR to quantify the expression of AID and its alternatively spliced transcripts (AIDΔE4a, AIDΔE4, AIDivs3, and AIDΔE3E4) in 149 CLL patients and correlated this expression to prognostic markers including recurrent chromosomal aberrations, the presence of complex karyotype, mutation status of the immunoglobulin heavy chain variable gene, and recurrent mutations. We report a previously unappreciated association between higher AID transcript levels and trisomy of chromosome 12. Functional analysis of AID splice variants revealed loss of their activity with respect to SHM, CSR, and induction of double-strand DNA breaks. In silico modeling provided insight into the molecular interactions and structural dynamics of wild-type AID and a shortened AID variant closely resembling AIDΔE4, confirming its loss-of-function phenotype.

Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages.

Cytidine deaminase (MtCDA), encoded by cdd gene (Rv3315c), is the only enzyme identified in nucleotide biosynthesis pathway of Mycobacterium tuberculosis that is able to recycle cytidine and deoxycytidine. An M. tuberculosis knockout strain for cdd gene was obtained by allelic replacement. Evaluation of mRNA expression validated cdd deletion and showed the absence of polar effect. MudPIT LC-MS/MS data indicated thymidine phosphorylase expression was decreased in knockout and complemented strains. The cdd disruption does not affect M. tuberculosis growth both in Mid- dlebrook 7H9 and in RAW 264.7 cells, which indicates that cdd is not important for macrophage invasion and virulence.

Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages

Cytidine deaminase (MtCDA), encoded by cdd gene (Rv3315c), is the only enzyme identified in nucleotide biosynthesis pathway of Mycobacterium tuberculosis that is able to recycle cytidine and deoxycytidine. An M. tuberculosis knockout strain for cdd gene was obtained by allelic replacement. Evaluation of mRNA expression validated cdd deletion and showed the absence of polar effect. MudPIT LC-MS/MS data indicated thymidine phosphorylase expression was decreased in knockout and complemented strains. The cdd disruption does not affect M. tuberculosis growth both in Mid- dlebrook 7H9 and in RAW 264.7 cells, which indicates that cdd is not important for macrophage invasion and virulence.

Establishment of induced pluripotent stem cells from normal B cells and inducing AID expression in their differentiation into hematopoietic progenitor cells.

B cell derived induced pluripotent stem cells (BiPSCs) were recently established from peripheral blood B cells by the simultaneous transfection of Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) and C/EBPα using a Sendai virus vector. Here, using a different method, we established BiPSCs with immunoglobulin heavy chain (IgH) gene rearrangement from normal B cells purified from lymph nodes. The critical points of our method are pre-stimulation of B cells with IL-21 and CD40-ligand (CD40L), followed by consecutive transfection of highly concentrated Yamanaka factors using a retroviral vector. Following each transfection the cells were centrifuged onto a retronectin coated plate and the activated by IL-4, IL-2, and CD40L. Furthermore, we established BiPSCs (BiPSC-A) in which activation-induced cytidine deaminase (AID) could be induced using the doxycycline-controlled. Both the parental BiPSC and BiPSC-A showed the capability of differentiating into hematopoietic progenitor cells (HPCs) based on confirmation of CD34 expression and colony-formation from CD34-positive cells. The findings that BiPSC-A can differentiate into HPCs suggest that there is a possibility that induction of AID expression would result in chromosomal translocations in the process of differentiation from BiPSCs, and therefore that these BiPSCs could be useful in elucidating the tumor origin of abnormal B cells in myelomagenesis.

B-Myb Induces APOBEC3B Expression Leading to Somatic Mutation in Multiple Cancers.

The key signature of cancer genomes is the accumulation of DNA mutations, the most abundant of which is the cytosine-to-thymine (C-to-T) transition that results from cytosine deamination. Analysis of The Cancer Genome Atlas (TCGA) database has demonstrated that this transition is caused mainly by upregulation of the cytosine deaminase APOBEC3B (A3B), but the mechanism has not been completely characterized. We found that B-Myb (encoded by MYBL2) binds the A3B promoter, causing transactivation, and this is responsible for the C-to-T transitions and DNA hypermutation in breast cancer cells. Analysis of TCGA database yielded similar results, supporting that MYBL2 and A3B are upregulated and putatively promote C-to-T transitions in multiple cancer types. Moreover, blockade of EGF receptor with afatinib attenuated B-Myb-A3B signaling, suggesting a clinically relevant means of suppressing mutagenesis. Our results suggest that B-Myb-A3B contributes to DNA damage and could be targeted by inhibiting EGF receptor.

Cytidine deaminase Apobec3a induction in fallopian epithelium after exposure to follicular fluid.

OBJECTIVE: Ovarian carcinomas that originate from fallopian epithelial cells are suggested to arise due to repeated exposure to ovulatory follicular fluid (FF). Mechanistic explanation(s) for how this occurs are unknown. Here, we sought to understand if FF exposure to fallopian epithelial cells could induce DNA damage and expression of a known family of DNA mutators, apolipoprotein B mRNA editing enzyme, catalytic polypeptide (APOBEC) cytidine deaminases. METHODS: Follicular fluid and matched patient plasma samples were obtained from donors. Fallopian epithelial cells (FT33-TAg, FT189, FT190, and FT194) were cultured with FF or plasma for 24h, and cell proliferation and DNA damage were assessed. Effects of FF on Apobec gene expression were determined by qRT-PCR and western blot analyses. Fallopian epithelial cells were transfected with an APOBEC3A expression vector and DNA damage was assessed. RESULTS: Follicular fluid exposure increased epithelial cell proliferation as measured by three independent methods, and DNA damage accumulation as assessed using three independent measures. This effect was specific to FF, as matched patient plasma did not have the same effects. Increased expression of Apobec3a was observed in fallopian epithelial cells following exposure to 5 of 8 patient FF samples, and transient overexpression of APOBEC3A was sufficient to induce double strand DNA breaks. CONCLUSIONS: Follicular fluid can induce cell proliferation and DNA damage accumulation in cultured fallopian epithelial cells. Increased expression of APOBEC3A, a known DNA mutator, may explain the high incidence of DNA damage after FF exposure. The role of Apobec3a in ovulation-induced inflammation warrants further investigation.

Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage.

Foreign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA agonists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers interferon α and ß production through the RNA polymerase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A. By catalyzing C→U editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromosomal DNA damage in the form of CG→TA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer.

Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor.

The cytidine deaminase APOBEC3B (A3B) underlies the genetic heterogeneity of several human cancers, including cervical cancer, which is caused by human papillomavirus (HPV) infection. We previously identified a region within the A3B promoter that is activated by the viral protein HPV16 E6 in human keratinocytes. Here, we discovered three sites recognized by the TEAD family of transcription factors within this region of the A3B promoter. Reporter assays in HEK293 cells showed that exogenously expressed TEAD4 induced A3B promoter activation through binding to these sites. Normal immortalized human keratinocytes expressing E6 (NIKS-E6) displayed increased levels of TEAD1/4 protein compared to parental NIKS. A series of E6 mutants revealed that E6-mediated degradation of p53 was important for increasing TEAD4 levels. Knockdown of TEADs in NIKS-E6 significantly reduced A3B mRNA levels, whereas ectopic expression of TEAD4 in NIKS increased A3B mRNA levels. Finally, chromatin immunoprecipitation assays demonstrated increased levels of TEAD4 binding to the A3B promoter in NIKS-E6 compared to NIKS. Collectively, these results indicate that E6 induces upregulation of A3B through increased levels of TEADs, highlighting the importance of the TEAD-A3B axis in carcinogenesis.IMPORTANCE The expression of APOBEC3B (A3B), a cellular DNA cytidine deaminase, is upregulated in various human cancers and leaves characteristic, signature mutations in cancer genomes, suggesting that it plays a prominent role in carcinogenesis. Viral oncoproteins encoded by human papillomavirus (HPV) and polyomavirus have been reported to induce A3B expression, implying the involvement of A3B upregulation in virus-associated carcinogenesis. However, the molecular mechanisms causing A3B upregulation remain unclear. Here, we demonstrate that exogenous expression of the cellular transcription factor TEAD activates the A3B promoter. Further, the HPV oncoprotein E6 increases the levels of endogenous TEAD1/4 protein, thereby leading to A3B upregulation. Since increased levels of TEAD4 are frequently observed in many cancers, an understanding of the direct link between TEAD and A3B upregulation is of broad oncological interest.

Plastic restriction of HIV-1 replication in human macrophages derived from M1/M2 polarized monocytes.

M1/M2 cytokine-dependent polarization of primary human MDMs has been shown to contain CCR5-dependent (R5) HIV-1 replication. In this study, a similar effect was achieved when monocytes were first polarized toward M1 or M2 and were infected 7 d after their differentiation into MDMs, regardless of whether the cytokines were removed 18 h after cell stimulation or were left in culture. Unlike polarized MDMs, no significant down-regulation of CD4 from the cell surface was observed in MDMs derived from M1/M2-polarized monocytes. A second stimulation of MDMs differentiated from M1/M2 monocytes with the opposite polarizing cytokines converted the virus replication profile according to the new stimuli. The expression of M1 and M2 markers (i.e., APOBEC3A and DC-SIGN, respectively) was induced by MDM stimulation with the opposite cytokines, although it also persisted in cells according to their first stimulatory condition. Thus, stimulation of monocytes with M1- and M2-inducing cytokines leads to a restriction of HIV-1 replication when these cells are infected several days later as differentiated MDMs. These observations imply that activation of circulating monocytes significantly influences their capacity to either support or restrict HIV-1 replication, once extravasated, and eventually to become infected as tissue macrophages.

Activation-Induced Cytidine Deaminase Links Ovulation-Induced Inflammation and Serous Carcinogenesis.

In recent years, the notion that ovarian carcinoma results from ovulation-induced inflammation of the fallopian tube epithelial cells (FTECs) has gained evidence. However, the mechanistic pathway for this process has not been revealed yet. In the current study, we propose the mutator protein activation-induced cytidine deaminase (AID) as a link between ovulation-induced inflammation in FTECs and genotoxic damage leading to ovarian carcinogenesis. We show that AID, previously shown to be functional only in B lymphocytes, is expressed in FTECs under physiological conditions, and is induced in vitro upon ovulatory-like stimulation and in vivo in carcinoma-associated FTECs. We also report that AID activity results in epigenetic, genetic and genomic damage in FTECs. Overall, our data provides new insights into the etiology of ovarian carcinogenesis and may set the ground for innovative approaches aimed at prevention and early detection.

APOBEC3B is an enzymatic source of molecular alterations in esophageal squamous cell carcinoma.

APOBEC3B belongs to the cytidine deaminase apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3) family of enzymes and induces C to T transitions of target DNA by cytidine deamination. Recently, several mutations in various cancers have been linked to APOBEC3B, suggesting a crucial role for this protein in carcinogenesis and cancer development. However, the significance of APOBEC3B in esophageal squamous cell carcinoma (ESCC) remains uncertain. In addition, the APOBEC3B immunoreactivity in cancer tissues is uncertain. Recently, we have demonstrated that PIK3CA mutation and the methylation level of long interspersed nucleotide element 1 (LINE-1) (a surrogate marker of global DNA methylation level) are prognostic markers and have crucial role on malignancy in ESCC patients. This study aims to clarify the impact of APOBEC3B on the clinical, pathological, and molecular features of ESCC. We evaluated APOBEC3B expression in ESCC and investigated the relationships among the immunoreactivity of APOBEC3B, clinical and pathological features, and the molecular features of ESCC (PIK3CA mutation, p53 expression, and LINE-1 methylation level). The immunoreactivity and mRNA level of APOBEC3B were significantly higher in cancer tissues than in noncancerous esophageal mucosae (P = 0.050). APOBEC3B expression was significantly correlated with PIK3CA mutation (P = 0.013), particularly with C to T transitions of PIK3CA (P = 0.041). Moreover, a high expression of APOBEC3B was significantly associated with LINE-1 hypomethylation (P = 0.027). Given the crucial roles of PIK3CA mutation and LINE-1 methylation levels, our findings might provide new insights into the biological mechanisms of ESCC tumorigenesis and progression.

Chemoprotection of murine hematopoietic cells by combined gene transfer of cytidine deaminase (CDD) and multidrug resistance 1 gene (MDR1).

BACKGROUND: Hematologic toxicity represents a major side effect of cytotoxic chemotherapy frequently preventing adequately dosed chemotherapy application and impeding therapeutic success. Transgenic (over)expression of chemotherapy resistance (CTX-R) genes in hematopoietic stem- and progenitor cells represents a potential strategy to overcome this problem. To apply this concept in the context of acute myeloid leukemia and myelodysplasia, we have investigated the overexpression of the multidrug resistance 1 (MDR1) and the cytidine deaminase (CDD) gene conferring resistance to anthracyclines and cytarabine (Ara-C), the two most important drugs in the treatment of these diseases. METHODS: State-of-the-art, third generation, self-inactivating (SIN) lentiviral vectors were utilized to overexpress a human CDD-cDNA and a codon-optimized human MDR1-cDNA corrected for cryptic splice sites from a spleen focus forming virus derived internal promoter. Studies were performed in myeloid 32D cells as well as primary lineage marker negative (lin(-)) murine bone marrow cells and flow cytometric analysis of suspension cultures and clonogenic analysis of vector transduced cells following cytotoxic drug challenge were utilized as read outs. RESULTS: Efficient chemoprotection of CDD and MDR1 transduced hematopoietic 32D as well as primary lin(-) cells was proven in the context of Ara-C and anthracycline application. Both, CTX-R transduced 32D as well as primary hematopoietic cells displayed marked resistance at concentrations 5-20 times the LD50 of non-transduced control cells. Moreover, simultaneous CDD/MDR1 gene transfer resulted in similar protection levels even when combined Ara-C anthracycline treatment was applied. Furthermore, significant enrichment of transduced cells was observed upon cytotoxic drug administration. CONCLUSIONS: Our data demonstrate efficient chemoprotection as well as enrichment of transduced cells in hematopoietic cell lines as well as primary murine hematopoietic progenitor cells following Ara-C and/or anthracycline application, arguing for the efficacy as well as feasibility of our approach and warranting further evaluation of this concept.