Development of New Diffuse Large B Cell Lymphoma Mouse Models.

Diffuse large B cell lymphoma (DLBCL) is the most diagnosed, aggressive non-Hodgkin lymphoma, with ~40% of patients experiencing refractory or relapsed disease. Given the low response rates to current therapy, alternative treatment strategies are necessary to improve patient outcomes. Here, we sought to develop an easily accessible new xenograft mouse model that better recapitulates the human disease for preclinical studies. We generated two Luciferase (Luc)-EGFP-expressing human DLBCL cell lines representing the different DLBCL cell-of-origin subtypes. After intravenous injection of these cells into humanized NSG mice, we monitored the tumor growth and evaluated the organ-specific engraftment/progression period. Our results showed that human IL6-expressing NSG (NSG-IL6) mice were highly permissive for DLBCL cell growth. In NSG-IL6 mice, systemic engraftments of both U2932 activated B cell-like- and VAL germinal B cell-like-DLBCL (engraftment rate; 75% and 82%, respectively) were detected within 2nd-week post-injection. In the organ-specific ex vivo evaluation, both U2932-Luc and VAL-Luc cells were initially engrafted and expanded in the spleen, liver, and lung and subsequently in the skeleton, ovary, and brain. Consistent with the dual BCL2/MYC translocation association with poor patient outcomes, VAL cells showed heightened proliferation in human IL6-conditioned media and caused rapid tumor expansion and early death in the engrafted mice. We concluded that the U2932 and VAL cell-derived human IL6-expressing mouse models reproduced the clinical features of an aggressive DLBCL with a highly consistent pattern of tumor development. Based on these findings, NSG mice expressing human IL6 have the potential to serve as a new tool to develop DLBCL xenograft models to overcome the limitations of standard subcutaneous DLBCL xenografts.

Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma.

Expression of the serine/threonine kinase never in mitosis gene A (NIMA)-related kinase 2 (NEK2) is essential for entry into mitosis via its role in facilitating centrosome separation. Its overactivity can lead to tumorigenesis and drug resistance through the activation of several oncogenic pathways, including AKT. Although the cancer-enabling activities of NEK2 are documented in many malignancies, including correlations with poor survival in myeloma, breast, and non-small cell lung cancer, little is known about the role of NEK2 in lymphoma. Here, in tumors from patients with diffuse large B-cell lymphoma (DLBCL), the most common, aggressive non-Hodgkin lymphoma, we found a high abundance of NEK2 mRNA and protein associated with an inferior overall survival. Using our recently developed NEK2 inhibitor, NBI-961, we discovered that DLBCL cell lines and patient-derived cells exhibit a dependency on NEK2 for their viability. This compromised cell fitness was directly attributable to efficient NEK2 inhibition and proteasomal degradation by NBI-961. In a subset of particularly sensitive DLBCL cells, NBI-961 induced G2/mitosis arrest and apoptosis. In contrast, an existing indirect NEK2 inhibitor, INH154, did not prevent NEK2 autophosphorylation, induce NEK2 proteasomal degradation, or affect cell viability. Global proteomics and phospho-proteomics revealed that NEK2 orchestrates cell-cycle and apoptotic pathways through regulation of both known and new signaling molecules. We show the loss of NEK2-sensitized DLBCL to the chemotherapy agents, doxorubicin and vincristine, and effectively suppressed tumor growth in mice. These studies establish the oncogenic activity of NEK2 in DLBCL and set the foundation for development of anti-NEK2 therapeutic strategies in this frequently refractory and relapse-prone cancer.

Targeting a Novel G-Quadruplex in the CARD11 Oncogene Promoter with Naptho(2,1-b)furan-1-ethanol,2-nitro- Requires the Nitro Group.

The aggressive nature of the activated B cell such as (ABC) subtype of diffuse large B cell (DLBCL) is frequently associated with altered B cell Receptor (BCR) signaling through the activation of key components including the scaffolding protein, CARD11. Most inhibitors, such as ibrutinib, target downstream BCR kinases with often modest and temporary responses for DLBCL patients. Here, we pursue an alternative strategy to target the BCR pathway by leveraging a novel DNA secondary structure to repress transcription. We discovered that a highly guanine (G)-rich element within the CARD11 promoter forms a stable G-quadruplex (G4) using circular dichroism and polymerase stop biophysical techniques. We then identified a small molecule, naptho(2,1-b)furan-1-ethanol,2-nitro- (NSC373981), from a fluorescence-resonance energy transfer-based screen that stabilized CARD11 G4 and inhibited CARD11 transcription in DLBCL cells. In generating and testing analogs of NSC373981, we determined that the nitro group is likely essential for the downregulation of CARD11 and interaction with CARD11 G4, and the removal of the ethanol side chain enhanced this activity. Of note, the expression of BCL2 and MYC, two other key oncogenes in DLBCL pathology with known promoter G4 structures, were often concurrently repressed with NSC373981 and the highly potent R158 analog. Our findings highlight a novel approach to treat aggressive DLBCL by silencing CARD11 gene expression that warrants further investigation.

Activation-induced cytidine deaminase localizes to G-quadruplex motifs at mutation hotspots in lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a molecularly heterogeneous group of malignancies with frequent genetic abnormalities. G-quadruplex (G4) DNA structures may facilitate this genomic instability through association with activation-induced cytidine deaminase (AID), an antibody diversification enzyme implicated in mutation of oncogenes in B-cell lymphomas. Chromatin immunoprecipitation sequencing analyses in this study revealed that AID hotspots in both activated B cells and lymphoma cells in vitro were highly enriched for G4 elements. A representative set of these targeted sequences was validated for characteristic, stable G4 structure formation including previously unknown G4s in lymphoma-associated genes, CBFA2T3, SPIB, BCL6, HLA-DRB5 and MEF2C, along with the established BCL2 and MYC structures. Frequent genome-wide G4 formation was also detected for the first time in DLBCL patient-derived tissues using BG4, a structure-specific G4 antibody. Tumors with greater staining were more likely to have concurrent BCL2 and MYC oncogene amplification and BCL2 mutations. Ninety-seven percent of the BCL2 mutations occurred within G4 sites that overlapped with AID binding. G4 localization at sites of mutation, and within aggressive DLBCL tumors harboring amplified BCL2 and MYC, supports a role for G4 structures in events that lead to a loss of genomic integrity, a critical step in B-cell lymphomagenesis.

An epigenetic breeding system in soybean for increased yield and stability.

Epigenetic variation has been associated with a wide range of adaptive phenotypes in plants, but there exist few direct means for exploiting this variation. RNAi suppression of the plant-specific gene, MutS HOMOLOG1 (MSH1), in multiple plant species produces a range of developmental changes accompanied by modulation of defence, phytohormone and abiotic stress response pathways along with methylome repatterning. This msh1-conditioned developmental reprogramming is retained independent of transgene segregation, giving rise to transgene-null 'memory' effects. An isogenic memory line crossed to wild type produces progeny families displaying increased variation in adaptive traits that respond to selection. This study investigates amenability of the MSH1 system for inducing agronomically valuable epigenetic variation in soybean. We developed MSH1 epi-populations by crossing with msh1-acquired soybean memory lines. Derived soybean epi-lines showed increase in variance for multiple yield-related traits including pods per plant, seed weight and maturity time in both glasshouse and field trials. Selected epi-F2:4 and epi-F2:5 lines showed an increase in seed yield over wild type. By epi-F2:6, we observed a return of MSH1-derived enhanced growth back to wild-type levels. Epi-populations also showed evidence of reduced epitype-by-environment (e × E) interaction, indicating higher yield stability. Transcript profiling of epi-lines identified putative signatures of enhanced growth behaviour across generations. Genes related to cell cycle, abscisic acid biosynthesis and auxin response, particularly SMALL AUXIN UP RNAs (SAURs), were differentially expressed in epi-F2:4 lines that showed increased yield when compared to epi-F2:6 . These data support the potential of MSH1-derived epigenetic variation in plant breeding for enhanced yield and yield stability.

MutS HOMOLOG1-derived epigenetic breeding potential in tomato.

Evidence is compelling in support of a naturally occurring epigenetic influence on phenotype expression in land plants, although discerning the epigenetic contribution is difficult. Agriculturally important attributes like heterosis, inbreeding depression, phenotypic plasticity, and environmental stress response are thought to have significant epigenetic components, but unequivocal demonstration of this is often infeasible. Here, we investigate gene silencing of a single nuclear gene, MutS HOMOLOG1 (MSH1), in the tomato (Solanum lycopersicum) 'Rutgers' to effect developmental reprogramming of the plant. The condition is heritable in subsequent generations independent of the MSH1-RNA interference transgene. Crossing these transgene-null, developmentally altered plants to the isogenic cv Rutgers wild type results in progeny lines that show enhanced, heritable growth vigor under both greenhouse and field conditions. This boosted vigor appears to be graft transmissible and is partially reversed by treatment with the methylation inhibitor 5-azacytidine, implying the influence of mobile, epigenetic factors and DNA methylation changes. These data provide compelling evidence for the feasibility of epigenetic breeding in a crop plant.

Arabidopsis MSH1 mutation alters the epigenome and produces heritable changes in plant growth.

Plant phenotypes respond to environmental change, an adaptive capacity that is at least partly transgenerational. However, epigenetic components of this interplay are difficult to measure. Depletion of the nuclear-encoded protein MSH1 causes dramatic and heritable changes in plant development, and here we show that crossing these altered plants with isogenic wild type produces epi-lines with heritable, enhanced growth vigour. Pericentromeric DNA hypermethylation occurs in a subset of msh1 mutants, indicative of heightened transposon repression, while enhanced growth epi-lines show large chromosomal segments of differential CG methylation, reflecting genome-wide reprogramming. When seedlings are treated with 5-azacytidine, root growth of epi-lines is restored to wild-type levels, implicating hypermethylation in enhanced growth. Grafts of wild-type floral stems to mutant rosettes produce progeny with enhanced growth and altered CG methylation strikingly similar to epi-lines, indicating a mobile signal when MSH1 is downregulated, and confirming the programmed nature of methylome and phenotype changes.

Effect of Si-Miao-Yong-An on the stability of atherosclerotic plaque in a diet-induced rabbit model.

ETHNOPHARMACOLOGICAL RELEVANCE: Si-Miao-Yong-An (Trade name: Mai-Luo-Ning), a Chinese herbal formulation comprising Flos Lonicerae Japonicae, Radix Scrophulariae Ningpoensis, Radix Angelicae Sinensis and Radix Glycyrrhizae Uralensis, has been used in treating ischemic cardiovascular and cerebrovascular diseases for many years. Clinical and experimental studies have shown that Si-Miao-Yong-An can inhibit the inflammatory response and antagonize the blood clotting process. AIM OF THE STUDY: To investigate the effect of Si-Miao-Yong-An on atherosclerotic plaque stability in rabbit model. MATERIALS AND METHODS: Seventy male rabbits were divided into four groups. Rabbits in the normal group were fed with normal diet, while rabbits in model group and drug treatment groups were fed with high cholesterol diet, underwent BSA-induced immunologic injury and balloon-induced mechanical injury. After atherosclerotic rabbits were treated with simvastatin or Si-Miao-Yong-An for 16 weeks, blood and aorta in four groups were collected for analysis. RESULTS: Si-Miao-Yong-An reduced the level of triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) in blood after treatment for 16 weeks. Compared with model group, Si-Miao-Yong-An decreased the content of many inflammatory cytokines in blood and plaque. Morphological analysis of abdominal aorta showed that Si-Miao-Yong-An increased fibrous cap thickness and smooth muscle cells, reduced lipid core area and macrophages, and contributed to inhibit matrix degradation and inflammatory response. CONCLUSION: In this study, we provided evidence for that Si-Miao-Yong-An could promote the stability of atherosclerotic plaque in the rabbit model, indicating that this medicine was a reasonable drug treating cardiovascular diseases in clinical.

The chloroplast triggers developmental reprogramming when mutS HOMOLOG1 is suppressed in plants.

Multicellular eukaryotes demonstrate nongenetic, heritable phenotypic versatility in their adaptation to environmental changes. This inclusive inheritance is composed of interacting epigenetic, maternal, and environmental factors. Yet-unidentified maternal effects can have a pronounced influence on plant phenotypic adaptation to changing environmental conditions. To explore the control of phenotypy in higher plants, we examined the effect of a single plant nuclear gene on the expression and transmission of phenotypic variability in Arabidopsis (Arabidopsis thaliana). MutS HOMOLOG1 (MSH1) is a plant-specific nuclear gene product that functions in both mitochondria and plastids to maintain genome stability. RNA interference suppression of the gene elicits strikingly similar programmed changes in plant growth pattern in six different plant species, changes subsequently heritable independent of the RNA interference transgene. The altered phenotypes reflect multiple pathways that are known to participate in adaptation, including altered phytohormone effects for dwarfed growth and reduced internode elongation, enhanced branching, reduced stomatal density, altered leaf morphology, delayed flowering, and extended juvenility, with conversion to perennial growth pattern in short days. Some of these effects are partially reversed with the application of gibberellic acid. Genetic hemicomplementation experiments show that this phenotypic plasticity derives from changes in chloroplast state. Our results suggest that suppression of MSH1, which occurs under several forms of abiotic stress, triggers a plastidial response process that involves nongenetic inheritance.

Double-strand break repair processes drive evolution of the mitochondrial genome in Arabidopsis.

BACKGROUND: The mitochondrial genome of higher plants is unusually dynamic, with recombination and nonhomologous end-joining (NHEJ) activities producing variability in size and organization. Plant mitochondrial DNA also generally displays much lower nucleotide substitution rates than mammalian or yeast systems. Arabidopsis displays these features and expedites characterization of the mitochondrial recombination surveillance gene MSH1 (MutS 1 homolog), lending itself to detailed study of de novo mitochondrial genome activity. In the present study, we investigated the underlying basis for unusual plant features as they contribute to rapid mitochondrial genome evolution. RESULTS: We obtained evidence of double-strand break (DSB) repair, including NHEJ, sequence deletions and mitochondrial asymmetric recombination activity in Arabidopsis wild-type and msh1 mutants on the basis of data generated by Illumina deep sequencing and confirmed by DNA gel blot analysis. On a larger scale, with mitochondrial comparisons across 72 Arabidopsis ecotypes, similar evidence of DSB repair activity differentiated ecotypes. Forty-seven repeat pairs were active in DNA exchange in the msh1 mutant. Recombination sites showed asymmetrical DNA exchange within lengths of 50- to 556-bp sharing sequence identity as low as 85%. De novo asymmetrical recombination involved heteroduplex formation, gene conversion and mismatch repair activities. Substoichiometric shifting by asymmetrical exchange created the appearance of rapid sequence gain and loss in association with particular repeat classes. CONCLUSIONS: Extensive mitochondrial genomic variation within a single plant species derives largely from DSB activity and its repair. Observed gene conversion and mismatch repair activity contribute to the low nucleotide substitution rates seen in these genomes. On a phenotypic level, these patterns of rearrangement likely contribute to the reproductive versatility of higher plants.

MutS HOMOLOG1 is a nucleoid protein that alters mitochondrial and plastid properties and plant response to high light.

Mitochondrial-plastid interdependence within the plant cell is presumed to be essential, but measurable demonstration of this intimate interaction is difficult. At the level of cellular metabolism, several biosynthetic pathways involve both mitochondrial- and plastid-localized steps. However, at an environmental response level, it is not clear how the two organelles intersect in programmed cellular responses. Here, we provide evidence, using genetic perturbation of the MutS Homolog1 (MSH1) nuclear gene in five plant species, that MSH1 functions within the mitochondrion and plastid to influence organellar genome behavior and plant growth patterns. The mitochondrial form of the protein participates in DNA recombination surveillance, with disruption of the gene resulting in enhanced mitochondrial genome recombination at numerous repeated sequences. The plastid-localized form of the protein interacts with the plastid genome and influences genome stability and plastid development, with its disruption leading to variegation of the plant. These developmental changes include altered patterns of nuclear gene expression. Consistency of plastid and mitochondrial response across both monocot and dicot species indicate that the dual-functioning nature of MSH1 is well conserved. Variegated tissues show changes in redox status together with enhanced plant survival and reproduction under photooxidative light conditions, evidence that the plastid changes triggered in this study comprise an adaptive response to naturally occurring light stress.

[Effect of bushen kangshuai tablet on NF-kappaB signal pathway and inflammation factors in rabbits with experimental atherosclerosis].

OBJECTIVE: To observe the intervening effect and acting mechanism of Bushen Kangshuai Tablet (BKT) on rabbits' atherosclerosis (AS). METHODS: Thirty-six white Japanese rabbits were randomly divided into four groups: 6 in the normal control group, and each 10 in the model group, the BKT group and the simvastatin group. The AS model was established by high fatty diet feeding from the 1st to the 10th week, combined with immune injury at the 2nd week and femoral arterial balloon tearing at the 4th week. The medication of BKT and simvastatin was given during the whole 10-week course. Levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), interleukin-1 (IL-1), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-alpha) were measured at the beginning and the 3rd, 6th and 10th weekend of the experiment. The nuclear factor-kappa B (NF-kappaB) expression in the aortic wall, ratio of plaque area to intima area (PA/IA), intima thickness (IT), aortic intima/media thickness ratio (IT/MT) and intima hyperplasia index (IHI) were measured at the terminal of the experiment. The correlation analysis was conducted between serum lipids, inflammation factors and IHI. RESULTS: Compared with the normal control group, all indices of the blood lipids and Inflammation factors measured at various time points, and the PA/IA, IT/MT ratios as well as IHI in the model group were higher (P<0.01, P<0.05). Compared with the model group, the levels of IL-1, MCP-1, TNF-alpha, NF-kappaB as well as the ratios of PA/IA, IT/MT and IHI were lower in the two treated groups at all time points after treatment (P<0.01 or P<0.05). Pearson correlation analysis showed positive correlation between TC with IL-1, and TNF-alpha with IHI in the model group (P< 0.05); also between TC with IHI, IL-1 with IHI, and TNF-alpha with IHI and IL-1 in the BKT group (P<0.05), while no correlation between blood lipids with inflammation factors was observed. CONCLUSION: BKT could suppress the inflammation reaction in rabbits to prevent AS formation, the action is not directly correlated with the blood lipid level.

Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila.

We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH2-terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.

Chromator, a novel and essential chromodomain protein interacts directly with the putative spindle matrix protein skeletor.

We have used a yeast two-hybrid interaction assay to identify Chromator, a novel chromodomain containing protein that interacts directly with the putative spindle matrix protein Skeletor. Immunocytochemistry demonstrated that Chromator and Skeletor show extensive co-localization throughout the cell cycle. During interphase Chromator is localized on chromosomes to interband chromatin regions in a pattern that overlaps that of Skeletor. However, during mitosis both Chromator and Skeletor detach from the chromosomes and align together in a spindle-like structure. Deletion construct analysis in S2 cells showed that the COOH-terminal half of Chromator without the chromodomain was sufficient for both nuclear as well as spindle localization. Analysis of P-element mutations in the Chromator locus shows that Chromator is an essential protein. Furthermore, RNAi depletion of Chromator in S2 cells leads to abnormal microtubule spindle morphology and to chromosome segregation defects. These findings suggest that Chromator is a nuclear protein that plays a role in proper spindle dynamics during mitosis.

Leech filamin and Tractin: markers for muscle development and nerve formation.

The Lan3-14 and Laz10-1 monoclonal antibodies recognize a 400 kDa antigen that is specifically expressed by all muscle cells in leech. We show that the antigen recognized by both antibodies is a member of the filamin family of actin binding proteins. Leech filamin has two calponin homology domains and 35 filamin/ABP-repeat domains. In addition, we used the Laz10-1 antibody to characterize the development of the segmentally iterated dorsoventral flattener muscles. We demonstrate that the dorsoventral flattener muscle develops as three discrete bundles of myofibers and that CNS axons pioneering the DP nerve extend only along the middle bundle. Interestingly, the middle dorsoventral muscle anlage is associated with only non-neuronal expression of the L1-family cell adhesion molecule Tractin. This expression is transient and occurs at the precise developmental stages when DP nerve formation takes place. Based on these findings we propose that the middle dorsoventral muscle anlagen provides a substrate for early axonal outgrowth and nerve formation and that this function may be associated with differential expression of distinct cell adhesion molecules.

Proteolytic cleavage of the ectodomain of the L1 CAM family member Tractin.

Tractin is a member of the L1 family of cell adhesion molecules in leech. Immunoblot analysis suggests that Tractin is constitutively cleaved in vivo at a proteolytic site with the sequence RKRRSR. This sequence conforms to the consensus sequence for cleavage by members of the furin family of convertases, and this proteolytic site is shared by a majority of other L1 family members. We provide evidence with furin-specific inhibitor experiments, by site-specific mutagenesis of Tractin constructs expressed in S2 cells, as well as by Tractin expression in furin-deficient LoVo cells that a furin convertase is the likely protease mediating this processing. Cross-immunoprecipitations with Tractin domain-specific antibodies suggest that the resulting NH(2)- and COOH-terminal cleavage fragments interact with each other and that this interaction provides a means for the NH(2)-terminal fragment to be tethered to the membrane. Furthermore, in S2 cell aggregation assays we show that the NH(2)-terminal fragment is necessary for homophilic adhesion and that cells expressing only the transmembrane COOH-terminal fragment are non-adhesive. However, tethering of exogeneously provided Tractin NH(2)-terminal fragment to S2 cells expressing only the COOH-terminal fragment can functionally restore the adhesive properties of Tractin.