DT40 Cell Line Research Articles

Recruitment of two Ndc80 complexes via the CENP-T pathway is sufficient for kinetochore functions

To form functional kinetochores, CENP-C and CENP-T independently recruit the KMN (Knl1C, Mis12C, and Ndc80C) network onto the kinetochores. To clarify the functions of the KMN network on CENP-T, we evaluated its roles in chicken DT40 cell lines lacking the CENP-C-KMN network interaction. By analyzing mutants lacking both CENP-T-Mis12C and CENP-C-Mis12C interactions, we demonstrated that Knl1C and Mis12C (KM) play critical roles in the cohesion of sister chromatids or the recruitment of spindle checkpoint proteins onto kinetochores. Two copies of Ndc80C (N-N) exist on CENP-T via Mis12C or direct binding. Analyses of cells specifically lacking the Mis12C-Ndc80C interaction revealed that N-N is needed for proper kinetochore-microtubule interactions. However, using artificial engineering to directly bind the two copies of Ndc80C to CENP-T, we demonstrated that N-N functions without direct Mis12C binding to Ndc80C in native kinetochores. This study demonstrated the mechanisms by which complicated networks play roles in native kinetochores.

Characterisation of the spectrum and genetic dependence of collateral mutations induced by translesion DNA synthesis.

Translesion DNA synthesis (TLS) is a fundamental damage bypass pathway that utilises specialised polymerases with relaxed template specificity to achieve replication through damaged DNA. Misinsertions by low fidelity TLS polymerases may introduce additional mutations on undamaged DNA near the original lesion site, which we termed collateral mutations. In this study, we used whole genome sequencing datasets of chicken DT40 and several human cell lines to obtain evidence for collateral mutagenesis in higher eukaryotes. We found that cisplatin and UVC radiation frequently induce close mutation pairs within 25 base pairs that consist of an adduct-associated primary and a downstream collateral mutation, and genetically linked their formation to TLS activity involving PCNA ubiquitylation and polymerase κ. PCNA ubiquitylation was also indispensable for close mutation pairs observed amongst spontaneously arising base substitutions in cell lines with disrupted homologous recombination. Collateral mutation pairs were also found in melanoma genomes with evidence of UV exposure. We showed that collateral mutations frequently copy the upstream base, and extracted a base substitution signature that describes collateral mutagenesis in the presented dataset regardless of the primary mutagenic process. Using this mutation signature, we showed that collateral mutagenesis creates approximately 10-20% of non-paired substitutions as well, underscoring the importance of the process.

Metabolic adaptation to the chronic loss of Ca2+ signaling induced by KO of IP3 receptors or the mitochondrial Ca2+ uniporter

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP3Rs) and the mitochondrial Ca2+ uniporter (MCU) are key proteins that regulate intracellular Ca2+ concentration. Mitochondrial Ca2+ accumulation activates Ca2+-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP3R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca2+ signaling. Our results show that TKO cells (exhibiting total loss of Ca2+ signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca2+ signaling is dispensable for the bioenergetic needs of both IP3R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.

Monitoring the replication of structured DNA through heritable epigenetic change.

DNA can adopt non-B form structures that create significant blocks to DNA synthesis and seeking understanding of the mechanisms cells use to resolve such impediments continues to be a very active area of research. However, the ability to monitor the stalling of DNA synthesis at specific sites in the genome in living cells, of central importance to elucidating these mechanisms, poses a significant technical challenge. Replisome stalling is often transient with only a small fraction of events leading to detectable genetic changes, making traditional reporter assays insensitive to the stalling event per se. On the other hand, the imprint stalling leaves on the epigenome can be exploited as a form of biological 'tape recorder' that captures episodes of fork stalling as heritable changes in histone modifications and in transcription. Here we describe a detailed protocol for monitoring DNA structure-dependent epigenetic instability of the BU-1 locus in the avian cell line DT40, which has proved a sensitive tool for understanding the mechanisms by which structured DNA is replicated in a vertebrate system.

DUTPase inhibition confers susceptibility to a thymidylate synthase inhibitor in DNA-repair-defective human cancer cells.

Deficiency in DNA repair proteins confers susceptibility to DNA damage, making cancer cells vulnerable to various cancer chemotherapies. 5‐Fluorouracil (5‐FU) is an anticancer nucleoside analog that both inhibits thymidylate synthase (TS) and causes DNA damage via the misincorporation of FdUTP and dUTP into DNA under the conditions of dTTP depletion. However, the role of the DNA damage response to its antitumor activity is still unclear. To determine which DNA repair pathway contributes to DNA damage caused by 5‐FU and uracil misincorporation, we examined cancer cells treated with 2ʹ‐deoxy‐5‐fluorouridine (FdUrd) in the presence of TAS‐114, a highly potent inhibitor of dUTPase that restricts aberrant base misincorporation. Addition of TAS‐114 increased FdUTP and dUTP levels in HeLa cells and facilitated 5‐FU and uracil misincorporation into DNA, but did not alter TS inhibition or 5‐FU incorporation into RNA. TAS‐114 showed synergistic potentiation of FdUrd cytotoxicity and caused aberrant base misincorporation, leading to DNA damage and induced cell death even after short‐term exposure to FdUrd. Base excision repair (BER) and homologous recombination (HR) were found to be involved in the DNA repair of 5‐FU and uracil misincorporation caused by dUTPase inhibition in genetically modified chicken DT40 cell lines and siRNA‐treated HeLa cells. These results suggested that BER and HR are major pathways that protect cells from the antitumor effects of massive incorporation of 5‐FU and uracil. Further, dUTPase inhibition has the potential to maximize the antitumor activity of fluoropyrimidines in cancers that are defective in BER or HR.

A DNA repair player, ring finger protein 43, relieves etoposide‐induced topoisomerase II poisoning

Ring finger protein 43 (RNF43) is an E3 ubiquitin ligase which is well-known for its role in negative regulation of the Wnt-signaling pathway. However, the function in DNA double-strand break repairs has not been investigated. In this study, we used a lymphoblast cell line, DT40, and mouse embryonic fibroblast as cellular models to study DNA double-strand break (DSB) repairs. For this purpose, we created RNF43 knockout, RNF43-/- DT40 cell line to investigate DSB repairs. We found that deletion of RNF43 does not interfere with cell proliferation. However, after exposure to various types of DNA-damaging agents, RNF43-/- cells become more sensitive to topoisomerase II inhibitors, etoposide, and ICRF193, than wild type cells. Our results also showed that depletion of RNF43 results in apoptosis upon etoposide-mediated DNA damage. The delay in resolution of γH2AX and 53BP1 foci formation after etoposide treatment, as well as epistasis analysis with DNAPKcs, suggested that RNF43 might participate in DNA repair of etoposide-induced DSB via non-homologous end joining. Disturbed γH2AX foci formation in MEFs following pulse etoposide treatment supported the notion that RNF43 also functions DNA repair in mammalian cells. These findings propose two possible functions of RNF43, either participating in NHEJ or removing the blockage of 5' topo II adducts from DSB ends.

Cullin 3 Is Crucial for Pro-B Cell Proliferation, Interacts with CD22, and Controls CD22 Internalization on B Cells.

B lymphocytes are important players of the adaptive immune system. However, not just activation of B cells but also regulation of B cell signaling is important to prevent hyperactivity and dysregulation of the immune response. Different mechanisms and proteins contribute to this balance. One of these is CD22, a member of the Siglec family. It is an inhibitory coreceptor of the BCR and inhibits B cell activation. Upon BCR stimulation, CD22-dependent inhibition of BCR signaling results in a decreased calcium mobilization. Although some CD22 binding partners have already been identified, the knowledge about the CD22 interactome is still incomplete. In this study, quantitative affinity purification-mass spectrometry enabled the delineation of the CD22 interactome in the B cell line DT40. These data will clarify molecular mechanisms and CD22 signaling events after BCR activation and revealed several new CD22-associated proteins. One new identified interaction partner is the E3 ubiquitin ligase cullin 3, which was revealed to regulate CD22 surface expression and clathrin-dependent CD22 internalization after BCR stimulation. Furthermore cullin 3 was identified to be important for B lymphocytes in general. B cell-specific cullin 3-deficient mice show reduced developing B cells in the bone marrow and a severe pro-B cell proliferation defect. Mature B cells in the periphery are also reduced and characterized by increased CD22 expression and additionally by preactivated and apoptotic phenotypes. The findings reveal novel functions of cullin 3 in B lymphocytes, namely regulating CD22 surface expression and internalization after B cell activation, as well as promoting proliferation of pro-B cells.

Streamlined human antibody generation and optimization by exploiting designed immunoglobulin loci in a B cell line

Monoclonal antibodies (mAbs) are widely utilized as therapeutic drugs for various diseases, such as cancer, autoimmune diseases, and infectious diseases. Using the avian-derived B cell line DT40, we previously developed an antibody display technology, namely, the ADLib system, which rapidly generates antigen-specific mAbs. Here, we report the development of a human version of the ADLib system and showcase the streamlined generation and optimization of functional human mAbs. Tailored libraries were first constructed by replacing endogenous immunoglobulin genes with designed human counterparts. From these libraries, clones producing full-length human IgGs against distinct antigens can be isolated, as exemplified by the selection of antagonistic mAbs. Taking advantage of avian biology, effective affinity maturation was achieved in a straightforward manner by seamless diversification of the parental clones into secondary libraries followed by single-cell sorting, quickly affording mAbs with improved affinities and functionalities. Collectively, we demonstrate that the human ADLib system could serve as an integrative platform with unique diversity for rapid de novo generation and optimization of therapeutic or diagnostic antibody leads. Furthermore, our results suggest that libraries can be constructed by introducing exogenous genes into DT40 cells, indicating that the ADLib system has the potential to be applied for the rapid and effective directed evolution and optimization of proteins in various fields beyond biomedicine.

Validation of the Reference Genes for the Gene Expression Studies in Chicken DT40 Cell Line.

The selection of a suitable reference gene assures a reliable gene expression analysis when using the qPCR method. Normalization of the reaction is based on the basic metabolism genes. These genes show a constant, unregulated expression in all cells and function throughout their lifetime. In the current study, seven reference gene candidates were screened using RT-qPCR, to determine the best-matched pair of reference genes in the chicken DT40 cell line. The DT40 was derived from bursal lymphoma cells that were subjected to RAV-1 bird retroviral infection. It is a simplified in vitro model that allows tracking the direct interaction of stimulants on the lymphoid population and profiling of the hepatocellular B cell transcriptome. The reference gene analysis was carried out using statistical tools integrating four independent methods—geNorm, Best Keeper, NormFinder, delta Ct and RefFinder. Based on the selected reference genes, the relative gene expression analysis was done using the ddCt method. Complete relative gene expression study on a panel of the target genes revealed that proper selection of reference genes depending on the tissue eliminate decreases in data quality. The SDHA and RPL4 genes constitute stable internal controls as reference genes when analyzing gene expression in the DT40 cell line.

Abstract NT-110: EFFICACY OF NOVEL TOPOISOMERASE 1 INHIBITORS AND THEIR SYNERGY WITH PARP INHIBITORS IN A MURINE MODEL FOR OVARIAN CANCER

Abstract Camptothecin derivatives topotecan and irinotecan are currently the only FDA-approved topoisomerase I (Top1) inhibitors. Despite their wide usage they have many drawbacks: chemical instability, short half-life, dose-limiting bone marrow and gastrointestinal toxicity, and they are drug efflux substrates. The novel non-camptothecin Top1 inhibitors, indenoisoquinolines (LMP400, LMP776 and LMP744), have been developed to overcome these limitations. We explored whether treatment with LMPs could induce “synthetic lethality” in cells with impaired homologous recombination (HR) repair mechanism and whether it could be enhanced by combining them with PARP inhibitor, olaparib. Using the DT40 cell line, we assessed in vitro the role of HR in the cellular responses to the LMPs. We found that BRCA1-, BRCA2- and PALB2-deficient cells are 3 to 5 times more sensitive to the LMP compounds than wild type cells. Moreover, combination treatments showed a significant synergy between each of the three LMPs and olaparib. To confirm the differential response of Brca1 wild type or Brca1 deficient cells to LMP treatment we assessed compound potency in primary ovarian cancer cell lines derived from a genetically engineered mouse (GEM) model for serous epithelial ovarian cancer (SEOC). As in DT40 cells, we observed that murine Brca1 deficient cells were more sensitive to all 3 LMP compounds than Brca1 wild type cells. Our results suggest that Brca1 deficiency renders cells more sensitive to treatment with LMPs or combination of LMPs and olaparib, potentially directing selection of patients with impaired HR repair mechanism in the phase 2 clinical trials that are in preparation. To confirm this observation in vivo, we are performing efficacy studies using LMP compounds alone or in combination with olaparib in orthotopic allograft mouse models for ovarian cancer derived from the GEM model. In Brca1 deficient tumors, treatment with each of the 3 LMP compounds led to significant increase of survival compared to vehicle treated group. LMP400 had the greatest beneficial impact on survival and tumor regression. To confirm synergy between LMPs and olaparib we selected LMP400 to represent the LMPs and repeated the efficacy study using Brca1 deficient tumor model. We observed that suboptimal doses of LMP400 or olaparib used as single treatments resulted in some increase in survival, however, when combined they significantly increased survival due to tumor regression. Our data demonstrate that the LMPs synergize with olaparib and provide a rationale for personalized treatment and Phase 2 clinical trials with the indenoisoquinolines in combination with PARP inhibitors in HR-deficient ovarian cancers. Citation Format: Ludmila Szabova, Melanie Gordon, Laetitia Matzi, Yves Pommier, Zoe Weaver Ohler. EFFICACY OF NOVEL TOPOISOMERASE 1 INHIBITORS AND THEIR SYNERGY WITH PARP INHIBITORS IN A MURINE MODEL FOR OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-110.

The Indenoisoquinoline TOP1 Inhibitors Selectively Target Homologous Recombination-Deficient and Schlafen 11-Positive Cancer Cells and Synergize with Olaparib.

Irinotecan and topotecan are used to treat a variety of different cancers. However, they have limitations, including chemical instability and severe side effects. To overcome these limitations, we developed the clinical indenoisoquinolines: LMP400 (indotecan), LMP776 (indimitecan), and LMP744. The purpose of the study is to build the molecular rationale for phase II clinical trials. CellMinerCDB (http://discover.nci.nih.gov/cellminercdb) was used to mine the cancer cell lines genomic databases. The causality of Schlafen11 (SLFN11) was validated in isogenic cell lines. Because topoisomerase I (TOP1)-mediated replication DNA damage is repaired by homologous recombination (HR), we tested the "synthetic lethality" of HR-deficient (HRD) cells. Survival and cell-cycle alterations were performed after drug treatments in isogenic DT40, DLD1, and OVCAR cell lines with BRCA1, BRCA2, or PALB2 deficiencies and in organoids cultured from prostate cancer patient-derived xenografts with BRCA2 loss. We also used an ovarian orthotopic allograft model with BRCA1 loss to validate the efficacy of LMP400 and olaparib combination. CellMinerCDB reveals that SLFN11, which kills cells undergoing replicative stress, is a dominant drug determinant to the clinical indenoisoquinolines. In addition, BRCA1-, BRCA2-, and PALB2-deficient cells were hypersensitive to the indenoisoquinolines. All 3 clinical indenoisoquinolines were also synergistic with olaparib, especially in the HRD cells. The synergy between LMP400 and olaparib was confirmed in the orthotopic allograft model harboring BRCA1 loss. Our results provide a rationale for molecularly designed clinical trials with the indenoisoquinolines as single agents and in combination with PARP inhibitors in HRD cancers expressing SLFN11.

Abstract 365: Indotecan (LMP400), indimitecan (LMP776) and LMP744, a new class of non-camptothecin topoisomerase I inhibitors selective for schlafen11-positive and BRCA-deficient cells that synergize with olaparib

Abstract To overcome the limitations of topoisomerase inhibitors, irinotecan and topotecan (chemical instability, drug efflux substrates, short half-life, dose-limiting bone marrow and gastrointestinal toxicity), we have developed the indenoisoquinolines (LMP400, LMP776 and LMP744). To rationally select patients for phase 2 clinical trials, we have taken two approaches. First, we mined the cancer cell lines genomic databases with CellMinerCDB and found that a dominant response determinant to the indenoisoquinolines is Schlafen 11 (SLFN11), a recently identified executor of cells undergoing replication stress. We validated this finding in isogenic cell lines. Second, because the major lesions generated by the trapping of TOP1 are replication-induced DNA double-strand breaks that are repaired by homologous recombination (HR), we determined whether the indenoisoquinolines exhibit a “synthetic lethality” in cells presenting BRCA1, BRCA2 or PALB2 deficiency. In addition, we tested wether this selectivity could be enhanced when combined with the PARP inhibitor, olaparib. Survival and cell cycle alterations were tested after treatment with the indenoisoquinolines as single agents in isogenic DT40, DLD1 and OVCAR cell lines, with BRCA1, BRCA2 or PALB2 deficiencies, and organoids cultured from patient-derived xenografts with BRCA2 loss, as well as in combination with olaparib. We found that BRCA1-, BRCA2- and PALB2-deficient cells are 3 to 5 times more sensitive to the indenoisoquinolines compared to isogenic cell lines. Moreover, combination treatments showed high synergy between all three indenoisoquinolines and olaparib. We also established the synergy between LMP400 (Indotecan) and olaparib in orthotopic allograft models derived from genetically engineered mouse models for serous epithelial ovarian cancer harboring BRCA1 loss. Better efficacy was observed with the combination over single agent treatments of LMP400 or olaparib. Our results provide a rationale for Phase 2 indenoisoquinoline clinical trials with the indenoisoquinolines in HR-deficient cancers as single agents and in combination with PARP inhibitors, and for measuring Schlafen 11 (SLFN11) as a clinical response determinant. Citation Format: Laetitia Marzi, Ludmila Szabova, Zoe Weaver Ohler, Shyam Sharan, Mike Beshiri, Junko Murai, Kathy Kelly, Yves Pommier. Indotecan (LMP400), indimitecan (LMP776) and LMP744, a new class of non-camptothecin topoisomerase I inhibitors selective for schlafen11-positive and BRCA-deficient cells that synergize with olaparib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 365.

A Novel DT40 Antibody Library for the Generation of Monoclonal Antibodies.

Early etiological diagnosis is very important for the control of sudden viral infections, and requires antibodies with both high sensitivity and high specificity. Traditional antibody preparation methods have limitations, such as a long and arduous cycle, complicated operation, and high expenses. A chicken lymphoma cell line, DT40, is known to produce IgM-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture. Here, the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro. Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase (AID) gene, AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected. In this study, we generated a novel AID-inducible DT40 cell line (DT40-H7), in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system, and an inducible AID gene, based on the Tet-Off expression system, was stably transfected. AID expression was controlled in DT40-H7 cells in a simple and efficient manner; gene conversion and point mutations were observed only when AID was expressed. Using the antibody library generated from this cell line, we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus. The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.

The Inducing Role and Molecular Basis of Bursal Hexapeptide (BHP) on Avian Immature B Cell.

The Bursa of Fabricius is an acknowledged central humoral immune organ unique to birds, which provides an ideal research model on the immature B cell development. In this article, our motivation is to study the role on sIgM and establish the molecular basis and functional processes of Bursal Hexapeptide (BHP) in avian immature B cells DT40 cell lines. In this article, we detected the expressions of sIgM mRNA with qPCR in DT40 cells with BHP treatment, and investigated the gene expression profiles of BHP-treated DT40 cells, employing microarray analyses. Also, to validate the differentially expressed genes, we performed KEGG pathway and Gene Ontology analysis in the BHP-treated DT40 cells. Finally, we comparatively analyzed the similar regulated genes and their involved immune functional processes between DT40 cell and mouse immature B cell line WEHI231 cell with BHP treatment. Following the proposed framework, we proved that the BHP enhanced the mRNA expression levels of IgM in DT40 cells, and induced 460 upregulated genes and 460 downregulated genes in BHP-treated DT40 cells. The pathway analysis showed that the differentially regulated genes in DT40 cell line with BHP treatment were involved in 12 enrichment pathways, in which Toll-like receptor signaling pathway was the vital pathways, and cytokine-cytokine receptor interaction and Jak-STAT signaling pathway were another two important pathways in BHP-treated DT40 cells. Moreover, BHP induced the immune related biological processes in BHP-treated DT40 cells, including T cell related, cytokine related, lymphocyte related, and innate immune response GO terms. Finally, the comparatively analysis showed that there were two downregulated genes GATA3 and IFNG to be found co-existed among the differentially expressed genes in BHP-treated DT40 cell and WEHI231 cells, which shared some same immune related functional processes in both cell lines. After the applying the framework, we proved the inducing roles and the gene expression profiles of BHP on avian immature B cells, and verified some molecular basis from the KEGG and GO analysis. These results provided the insight for mechanism on immature B cell differentiation, and offer the essential direction for the vaccine improvement.

Brca1 is involved in tolerance to adefovir dipivoxil‑induced DNA damage.

Nucleos(t)ide analogues (NAs) are currently the most important anti‑viral treatment option for patients with chronic hepatitis B (CHB). Adefovir dipivoxil (ADV), a diester pro‑drug of adefovir, has been widely used for the clinical therapy of hepatitis B virus infection. It has been previously reported that adefovir induced chromosomal aberrations (CAs) in the in vitro human peripheral blood lymphocyte assay, while the genotoxic mechanism remains elusive. To evaluate the possible mechanisms, the genotoxic effects of ADV on the TK6 and DT40 cell lines, as well as DNA repair‑deficient variants of DT40 cells, were assessed in the present study. A karyotype assay revealed ADV‑induced CAs, particularly chromosomal breaks, in wild‑type DT40 and TK6 cells. A γ‑H2AX foci formation assay confirmed the presence of DNA damage following treatment with ADV. Furthermore, Brca1‑/‑ DT40 cells exhibited an increased sensitivity to ADV, while the knockdown of various other DNA damage‑associated genes did not markedly affect the sensitivity. These comprehensive genetic studies identified the genotoxic capacity of ADV and suggested that Brca1 may be involved in the tolerance of ADV‑induced DNA damage. These results may contribute to the development of novel drugs against CHB with higher therapeutic efficacy and less genotoxicity.

The immunomodulatory peptide bursopentin (BP5) enhances proliferation and induces sIgM expression in DT40 cells.

In the recent past, many studies have been focused on extracts of BF and multiple biologically active factors and their effects on humoral immune system in chickens and birds. However, the mechanism of those immunomodulatory peptides on the B lineage cells proliferation and antibody production in chicken is fairly unknown. DT40 cell line, an avian leucosis virus-induced chicken pre-B cell line, expresses immunoglobulin M (IgM) isotype B cell reporter in the plasma membrane. There are many evidences suggesting that DT40 cells are best characterized as a bursal stem cell line. Because of the unique characteristics of DT40 cell line, it has been widely used to observe biological processes of pre-B lymphocyte cell within living cells. The chicken B cell line DT40 was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium and cytotoxicity was studied. Also, effect of BP5 on cell proliferation and cell cycle distribution of DT40 cells was studied. Also, the effect of BP5 on sIgM mRNA expression was studied by using real-time PCR. To investigat the effects of Bursopentin (Cys-Lys-Arg-Val-Tyr, BP5) on a chicken promyelocyte cell line DT40, assays of cell proliferation, cell cycle distribution, detection of surface immunoglobulin G (sIgM) mRNA expression and gene microarray analysis were performed. The results showed that BP5 displayed concentration-dependent effects on the proliferation, cell cycle, and sIgM mRNA expression in DT40 cells. And the analysis of expression profiles identified a signature set of 3022 genes (1254 up regulated genes, 1762 down regulated genes), which clearly discriminated the BP5-treated DT40 cells from control with high certainty (P≤0.02). The results of microarray analysis were confirmed by quantitative reverse transcription-polymerase chain reaction for 12 of the differentially expressed genes. Theses findings showed the immuno-activity effect of BP5 on B lymphocyte and indicated that BP5 treatment regulated eight signaling pathways, in which Toll-like signaling pathway was the most significant enrichment pathway.

3D genomic architecture reveals that neocentromeres associate with heterochromatin regions.

The centromere is an important genomic locus for chromosomal segregation. Although the centromere is specified by sequence-independent epigenetic mechanisms in most organisms, it is usually composed of highly repetitive sequences, which associate with heterochromatin. We have previously generated various chicken DT40 cell lines containing differently positioned neocentromeres, which do not contain repetitive sequences and do not associate with heterochromatin. In this study, we performed systematic 4C analysis using three cell lines containing differently positioned neocentromeres to identify neocentromere-associated regions at the 3D level. This analysis reveals that these neocentromeres commonly associate with specific heterochromatin-rich regions, which were distantly located from neocentromeres. In addition, we demonstrate that centromeric chromatin adopts a compact structure, and centromere clustering also occurs in vertebrate interphase nuclei. Interestingly, the occurrence of centromere-heterochromatin associations depend on CENP-H, but not CENP-C. Our analyses provide an insight into understanding the 3D architecture of the genome, including the centromeres.

Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2S short isoform.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.

Surface IgM λ light chain isinvolved in thebinding and infection of infectious bursal disease virus (IBDV) to DT40 cells.

Infectious bursal disease virus (IBDV) is an important immunosuppressive virus in chickens. Surface immunoglobulin M (sIgM)-bearing B lymphocytes act as the major targets of IBDV in the bursa of Fabricius, and sIgM may function as one of the membrane binding sites responsible for IBDV infection. Recently, using the virus overlay protein binding assay, the chicken λ light chain of sIgM was identified to specifically interact with IBDV in a virulence-independent manner in vitro. To further investigate sIgM λ light chain-mediated IBDV binding and infection in pre-B cells, the cell line DT40, which is susceptible to both pathogenic and attenuated IBDV, was used. Based on the RNA interference strategy, the DT40 cell line whose λ light chain of sIgM was stably knocked down, herein termed DT40LKD, was generated by the genomic integration of a specific small hairpin RNA and a green fluorescence protein co-expression construct. Flow cytometry analysisindicated that the binding of IBDV to DT40LKD cells was significantly reduced due to the loss of sIgM λ light chain. In particular, reduced viral replication was observed in IBDV-incubated DT40LKD cells, and no viral release into cell culture medium was detected by the IBDV rapid diagnostic strips. In addition, the rescue of sIgM λ light chain expression restored viral binding and replication in DT40LKD cells. These results show that sIgM λ light chain appears to be beneficial for IBDV attachment and infection, suggesting that sIgM acts as a binding site involved in IBDV infection.

ESCO1/2's roles in chromosome structure and interphase chromatin organization.

ESCO1/2 acetyltransferases mediating SMC3 acetylation and sister chromatid cohesion (SCC) are differentially required for genome integrity and development. Here we established chicken DT40 cell lines with mutations in ESCO1/2, SMC3 acetylation, and the cohesin remover WAPL. Both ESCO1 and ESCO2 promoted SCC, while ESCO2 was additionally and specifically required for proliferation and centromere integrity. ESCO1 overexpression fully suppressed the slow proliferation and centromeric separation phenotypes of esco2 cells but only partly suppressed its chromosome arm SCC defects. Concomitant inactivation of ESCO1 and ESCO2 caused lethality owing to compromised mitotic chromosome segregation. Neither wapl nor acetyl-mimicking smc3-QQ mutations rescued esco1 esco2 lethality. Notably, esco1 esco2 wapl conditional mutants showed very severe proliferation defects associated with catastrophic mitoses and also abnormal interphase chromatin organization patterns. The results indicate that cohesion establishment by vertebrate ESCO1/2 is linked to interphase chromatin architecture formation, a newly identified function of cohesin acetyltransferases that is both fundamentally and medically relevant.