anti-BrdU Antibody Research Articles

Abstract P6-08-05: Genome-wide identification of transcripts regulated by estrogen in MCF-7 cells using BrU-seq

Abstract Estrogen promotes estrogen receptor (ER)-positive breast cancer cell growth by modulating transcription through ligand activated ER. The spectrum of early ER target genes has been identified through gene expression microarray and more recently with RNA-seq analyses. These methods, although comprehensive, fall short in identifying many ER-regulated transcripts which are either not present in microarray; too abundant to determine estrogen regulated changes among steady-state pools of RNAs; or have very short half-lives and low abundance such as some non-coding RNAs, including RNAs generated by enhancers bound by ER (eRNAs). Global run-on sequencing (GRO-seq) has been used to identify ER target genes and eRNAs in breast cancer cells. It is technically cumbersome and is performed under non-physiological condition. We have applied BrU-seq technique to identify global transcripts regulated by estrogen in MCF-7 breast cancer cells. The newly synthesized RNAs in MCF-7 cells treated with or without estrogen for various time points, were labeled with 5'-bromo-uridine (BrU). The labeled RNAs were purified by immunoprecipitation using an anti-BrdU antibody and then subjected to RNA sequencing analysis, which provides greater sensitivity than regular RNA-seq analysis from steady-state total RNA samples. Robust estrogen-regulated transcripts can be detected within 30 min of treatment. Significantly more transcripts (both coding and non-coding RNAs) were identified as estrogen targets using BrU-seq analysis than RNA-seq analysis from total RNA pools. Many of these estrogen-regulated transcripts are located close to ER binding sites identified from ER ChIP-seq analysis, indicating a functional role for these ER binding sites in regulating RNA transcription. For estrogen targets identified through both analyses, the magnitude of expression changes (both up- and down-regulation) is usually greater from BrU-seq than from the regular RNA-seq analysis. The dynamics of RNA synthesis and degradation for some unstable RNAs (such as eRNAs) can be interrogated through our data set. Also, RNA pol II elongation rate can be deduced from some long ER target genes (>100 kb) such as GREB1. In addition to identifying estrogen targets, BrU-seq should be a very useful technique in assessing transcriptional changes triggered by various signal transduction pathways. Citation Format: Sun J, Capobianco E, Tsinoremas N, Lippman M. Genome-wide identification of transcripts regulated by estrogen in MCF-7 cells using BrU-seq [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-05.

Amyloid β Is Not the Major Factor Accounting for Impaired Adult Hippocampal Neurogenesis in Mice Overexpressing Amyloid Precursor Protein.

SummaryAdult hippocampal neurogenesis was impaired in several Alzheimer's disease models overexpressing mutant human amyloid precursor protein (hAPP). However, the effects of wild-type hAPP on adult neurogenesis and whether the impaired adult hippocampal neurogenesis was caused by amyloid β (Aβ) or APP remained unclear. Here, we found that neurogenesis was impaired in the dentate gyrus (DG) of adult mice overexpressing wild-type hAPP (hAPP-I5) compared with controls. However, the adult hippocampal neurogenesis was more severely impaired in hAPP-I5 than that in hAPP-J20 mice, which express similar levels of hAPP mRNA but much higher levels of Aβ. Furthermore, reducing Aβ levels did not affect the number of doublecortin-positive cells in the DG of hAPP-J20 mice. Our results suggested that hAPP was more likely an important factor inhibiting adult neurogenesis, and Aβ was not the major factor affecting neurogenesis in the adult hippocampus of hAPP mice.

Abstract 2734: Click colorimetric EdU proliferation and TUNEL assay: Conversion of click chemistry from fluorescent to colorimetric for use with bright field microscopy

Abstract We present here the conversion of click chemistry fluorescence-based EdU proliferation assay and TUNEL apoptosis assay to use with bright field microscopy. Advantages of having a colorimetric signal are the retention of the click chemistry work flow while allowing for compatibility with standard H&E staining and protocols. Proliferation using copper-catalyzed labeling of the thymidine analog EdU (ethynyl deoxyuridine) was introduced in 2007. This click-based assay has advantages over the traditional antibody based BrdU assay. The small molecule detection is simple, rapid and gives a bright signal useful for multiplexing. Additionally, recent improvements to the chemistry allow for GFP and R-PE compatibly with the EdU and TUNEL assays. Several approaches for creating a colorimetric signal for EdU proliferation were attempted. The method presented here had the shortest protocol and uses a biotin-based click reaction followed by streptavidin -horseradish peroxidase (HRP) step. This two-step reaction creates an essentially covalent link between the EdU incorporated in the DNA and the enzyme reporter. Then standard HRP chromogens such as DAB were used to produce a signal marking the proliferating cells. Examples of proliferation using colorimetric EdU labeling in adult Zebrafish caudal fin, mouse cardiac, and rat intestine, mammary and uterine FFPE treated tissue are presented. Multiplexing with alkaline phosphatase for two color detection are also presented. Two color detection of EdU with BrdU or EdU with progesterone receptor (PR) are shown. Most HIER protocols are compatible with EdU detection. For EdU alone no HCl treatment is required. For EdU/BrdU double labeling the BrdU protocol for DNA revelation is used. Anti-BrdU mouse antibody clone MoBU-1 is used because of its lack of cross-reactivity with EdU. Additionally, we present the click chemistry-based apoptotic TUNEL assay colorimetric detection using the same approach. Clickable biotin adapters are used to couple HRP to the ethynyl tails created by TdT enzyme extension at 3′ end of double stranded breaks. We provide examples of this late stage indicator of apoptosis visualized with DAB substrate and stained with standard H&E and other histological stains. Citation Format: Scott T. Clarke, Carmen Finnessy, Fernada M. Bosada, Ben Armstrong, Michael O’Grady. Click colorimetric EdU proliferation and TUNEL assay: Conversion of click chemistry from fluorescent to colorimetric for use with bright field microscopy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2734.

The antibodies against 5-bromo-2'-deoxyuridine specifically recognize trifluridine incorporated into DNA.

Trifluridine (FTD) is a key component of the novel oral antitumor drug TAS-102 (also named TFTD), which consists of FTD and a thymidine phosphorylase inhibitor. FTD is supposed to exert its cytotoxicity via massive misincorporation into DNA, but the underlying mechanism of FTD incorporation into DNA and its correlation with cytotoxicity are not fully understood. The present study shows that several antibodies against 5-bromo-2′-deoxyuridine (BrdU) specifically cross-react with FTD, either anchored to bovine serum albumin or incorporated into DNA. These antibodies are useful for several biological applications, such as fluorescence-activated cell sorting, fluorescent immunostaining and immunogold detection for electron microscopy. These techniques confirmed that FTD is mainly incorporated in the nucleus during S phase in a concentration-dependent manner. In addition, FTD was also detected by immunohistochemical staining in paraffin-embedded HCT-116 xenograft tumors after intraperitoneal administration of FTD. Intriguingly, FTD was hardly detected in surrounding matrices, which consisted of fibroblasts with marginal expression of the nucleoside transporter genes SLC29A1 and SLC29A2. Thus, applications using anti-BrdU antibodies will provide powerful tools to unveil the underlying mechanism of FTD action and to predict or evaluate the efficacy and adverse effects of TAS-102 clinically.

Effect of agomelatine on adult hippocampus apoptosis and neurogenesis using the stress model of rats

Agomelatine (AG) is an agonist of melatonin receptors and an antagonist of the 5-HT2C-receptor subtype. The chronobiotic properties of AG are of significant interest due to the disorganization of internal rhythms, which might play a role in the pathophysiology of depression. The present study was designed to assess the effects of the antidepressant-like activity of AG, a new antidepressant drug, on adult neurogenesis and apoptosis using stress-exposed rat brains. Over the period of 1 week, the rats were exposed to light stress twice a day for 1h. After a period of 1 week, the rats were given AG treatment at a dose of either 10mg/kg or 40mg/kg for 15 days. The animals were then scarified, and the obtained tissue sections were stained with immuno-histochemical anti-BrdU, Caspase-3, and Bcl-2 antibodies. Serum brain-derived neurotrophic factor (BDNF) concentrations were measured biochemically using a BDNF Elisa kit. Biochemical BDNF analysis revealed a high concentration of BDNF in the serum of the stress-exposed group, but the concentrations of BDNF were much lower those of the AG-treated groups. Immuno-histochemical analysis revealed that AG treatment decreased the BrdU-positive and Bcl-2-positive cell densities and increased the Caspase-3-positive cell density in the hippocampus of stress-induced rats as compared to those of the stress group. The results of the study demonstrated that AG treatment ameliorated the hippocampal apoptotic cells and increased hippocampal neurogenesis. These results also strengthen the possible relationship between depression and adult neurogenesis, which must be studied further.

Deficiency of IκB Kinase β in Myeloid Cells Reduces Severity of Experimental Autoimmune Encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), peripherally developed myelin-reactive T lymphocytes stimulate myeloid cells (ie, microglia and infiltrated macrophages) to trigger an inflammatory reaction in the central nervous system, resulting in demyelination and neurodegeneration. IκB kinase β (IKKβ) is a kinase that modulates transcription of inflammatory genes. To investigate the pathogenic role of IKKβ in MS, we developed strains in which IKKβ was conditionally ablated in myeloid cells and established active or passive EAE in these animals. Deficiency of IKKβ in myeloid cells ameliorated EAE symptoms and suppressed neuroinflammation, as shown by decreased infiltration of T lymphocytes and macrophages and reduced inflammatory gene transcription in the spinal cord at the peak or end stage of EAE. Myeloid deficiency of IKKβ also reduced the transcription of Rorc or Il17 genes in T lymphocytes isolated from lymph nodes, spleen, and spinal cord of EAE mice. Moreover, cultured splenocytes isolated from myeloid IKKβ-deficient EAE mice released less IL-17, interferon-γ, and granulocyte-macrophage colony-stimulating factor after treatment with myelin peptide than splenocytes from IKKβ wild-type EAE mice. Thus, deficiency of myeloid IKKβ attenuates the severity of EAE by inhibiting both the neuroinflammatory activity and the activation of encephalitogenic T lymphocytes. These results suggest IKKβ may be a potential target for MS patients, especially when neuroinflammation is the primary problem.

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA. Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique.

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA. Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

Kinetics of Label Retaining Cells in the Developing Rat Kidneys.

BackgroundThe kidney is a specialized low-regenerative organ with several different types of cellular lineages. The BrdU label-retaining cell (LRCs) approach has been used as part of a strategy to identify tissue-specific stem cells in the kidney; however, because the complementary base pairing in double-stranded DNA blocks the access of the anti-BrdU antibody to BrdU subunits, the stem cell marker expression in BrdU-labeled cells are often difficult to detect. In this study, we introduced a new cell labeling and detection method in which BrdU was replaced with 5-ethynyl-2-deoxyuridine (EdU) and examined the time-dependent dynamic changes of EdU-labeled cells and potential stem/progenitor markers in the development of kidney.MethodsNewborn rats were intraperitoneally injected with EdU, and their kidneys were harvested respectively at different time points at 1 day, 3 days, 1 week, 2 weeks, and 6 weeks post-injection. The kidney tissues were processed for EdU and cellular markers by immunofluorescence staining.ResultsAt the early stage, LRCs labeled by EdU were 2176.0 ± 355.6 cells at day one in each renal tissue section, but dropped to 168 ± 48.4 cells by week 6. As time increased, the numbers of LRCs were differentially expressed in the renal cortex and papilla. At the postnatal day one, nearly twice as many cells in the cortex were EdU-labeled as compared to the papilla (28.6 ± 3.6% vs. 15.6 ± 3.4%, P<0.05), while there were more LRCs within the renal papilla since the postnatal week one, and at the postnatal week 6, one third as many cells in the cortex were EdU-labeled as compared to the papilla (2.5 ± 0.1% vs. 7.7 ± 2.7%, P<0.05). The long-term LRCs at 6-week time point were associated exclusively with the glomeruli in the cortex and the renal tubules in the papilla. At 6 weeks, the EdU-labeled LRCs combined with expression of CD34, RECA-1, Nestin, and Synaptopodin were discretely but widely distributed within the glomeruli; Stro-1 around the glomeruli; and α-smooth muscle actin (SMA) in arteries. Conversely, co-expression of CD34, RECA-1, and Nestin with the long term EdU-labeled LRCs was significantly lower in renal tubules (P<0.01), while Stro-1 and Synaptopodin were not detected.ConclusionOur data found that at 6-week time point, EdU-labeled LRCs existing in the glomeruli expressed undifferentiated podocyte and endothelial markers at high rates, while those in the renal tubules expressed Nestin and vascular markers at low rates. To understand the characterization and localization of these EdU-LRCs, further studies will be needed to test cell lineage tracing, clonogenicity and differentiation potency, and the contributions to the regeneration of the kidney in response to renal injury/repair.

Vnd and its target gene twine are required for cell cycle progression during embryonic nervous system development in Drosophila melanogaster

Twine is a homolog of CDC25 phosphatase regulating cell cycle progression. To elucidate a possible role of twine during embryonic neurogenesis, its expression levels and patterns were determined. twine was strongly expressed in the embryonic brain, and segmentally expressed along the ventral nerve cord during stage 12–14 in which neuroblasts and ganglionic mother cells are rapidly dividing. To examine whether twine is involved in the nervous system development, immunohistochemistry using neuron-specific antibodies was performed. The results showed the reduction of dorsal clusters in the peripheral nervous system and disorganization of neurons in the central nervous system. Moreover, consistent with our microarray data, real-time PCR results show that expression level of twine is proportional to that of vnd, suggesting that Vnd functions as an activator to regulate twine expression. This relationship is also confirmed by analyzing the expression patterns of twine in vnd loss-of-function and gain-of-function mutant background. Immunostaining using anti-phospho-histone H3 and anti-BrdU antibodies revealed that Twine regulates cell cycle progression in the developing embryonic nervous system. Down-regulation of vnd also caused a significant reduction of mitotic cells in embryonic neuroblasts, suggesting a role for vnd-twine in mitosis-associated nervous system development.

Effects of different forms of chitosan on intercellular junctions of mouse fibroblasts in vitro

Chitosan is a linear polysaccharide that has many biomedical applications. We compared the effects of chitosan, in both solution and membranous form, on intercellular adhesion of Swiss 3T3 mouse fibroblasts. Cells were grown as spheroidal cell cultures. Some control cell spheroids were cultured without chitosan and two experimental groups were cultured with chitosan. Chitosan in solution was used for one experimental group and chitosan in membranous form was used for the other. For each group, intercellular adhesion was investigated on days 5 and 10 of culture. Transmission electron microscopy revealed well-defined cellular projections that were more prominent in cells exposed to either membranous or solution forms of chitosan than to the chitosan-free control. Immunocytochemical staining of ICAM-1 and e-cadherin was used to determine the development of intercellular junctions. Compared to the weakly stained control, strong reactions were observed in both chitosan exposed groups at both 5 and 10 days. Cells were treated with 5-bromo-2-deoxyuridine (BrdU) and incubated with anti-BrdU primary antibody to assess proliferation. Both the solution and membranous forms of chitosan increased proliferation at both 5 and 10 days. Cellular viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The MTT assay indicated high cell viability; maximum viability was obtained with the solution form of chitosan at day 5. Chitosan exposure increased the number of intercellular junctions and showed a significant proliferative effect on 3T3 mouse fibroblasts.

Ethosuximide Induces Hippocampal Neurogenesis and Reverses Cognitive Deficits in an Amyloid-β Toxin-induced Alzheimer Rat Model via the Phosphatidylinositol 3-Kinase (PI3K)/Akt/Wnt/β-Catenin Pathway

Neurogenesis involves generation of new neurons through finely tuned multistep processes, such as neural stem cell (NSC) proliferation, migration, differentiation, and integration into existing neuronal circuitry in the dentate gyrus of the hippocampus and subventricular zone. Adult hippocampal neurogenesis is involved in cognitive functions and altered in various neurodegenerative disorders, including Alzheimer disease (AD). Ethosuximide (ETH), an anticonvulsant drug is used for the treatment of epileptic seizures. However, the effects of ETH on adult hippocampal neurogenesis and the underlying cellular and molecular mechanism(s) are yet unexplored. Herein, we studied the effects of ETH on rat multipotent NSC proliferation and neuronal differentiation and adult hippocampal neurogenesis in an amyloid β (Aβ) toxin-induced rat model of AD-like phenotypes. ETH potently induced NSC proliferation and neuronal differentiation in the hippocampus-derived NSC in vitro. ETH enhanced NSC proliferation and neuronal differentiation and reduced Aβ toxin-mediated toxicity and neurodegeneration, leading to behavioral recovery in the rat AD model. ETH inhibited Aβ-mediated suppression of neurogenic and Akt/Wnt/β-catenin pathway gene expression in the hippocampus. ETH activated the PI3K·Akt and Wnt·β-catenin transduction pathways that are known to be involved in the regulation of neurogenesis. Inhibition of the PI3K·Akt and Wnt·β-catenin pathways effectively blocked the mitogenic and neurogenic effects of ETH. In silico molecular target prediction docking studies suggest that ETH interacts with Akt, Dkk-1, and GSK-3β. Our findings suggest that ETH stimulates NSC proliferation and differentiation in vitro and adult hippocampal neurogenesis via the PI3K·Akt and Wnt·β-catenin signaling.

Inhibitory Effect of Berberine on Zeste Homolog 2 (Ezh2) Enhancement in Human Esophageal Cell Lines

Purpose: To investigate the inhibitory effect of berberine treatment on enhancement of zeste of homolog 2 (Ezh2) expressions in KYSE450 human esophageal cancer cells.Methods: Transwell motility chambers were used to analyze cell migration and invasion. Bio-Rad protein assay was used for the determination of protein concentration. Chemiluminescence with ECL system was employed for the detection of protein bands as per the manufacturer’s protocol. Staining was carried out with Alexa-Fluor 647 mouse anti-BrdU antibody. Flow cytometry was performed after adding DAPI. Annexin-V/DAPI staining and flow cytometry were used for the quantification of apoptotic cell death. Total RNA was isolated from KYSE450 cells using an RNA isolation kit.Results: Berberine-induced inhibition of Ezh2 expression led to inhibition of cell proliferation by G1 phase cell cycle arrest and induced anti-invasive properties of KYSE450 cells in Boyden chamber assays. There was 92 % reduction in invasive tendency of KYSE450 cells following treatment with berberine. Histone methylation inhibitor, 3-deazaneoplanocin A (DZNep), also led to a similar effect on cell proliferation of KYSE450 cells. Berberine treatment also resulted in strong transcriptional reduction of the AXL receptor kinase. The results of qRT-PCR and FACS analyses showed significant inhibition of AXL mRNA and protein expression in KYSE450 carcinoma cells after treatment with berberine.Conclusion: Berberine may be an effective therapeutic agent in the treatment of esophageal carcinoma.Keywords: Berberine, Histone methylation inhibitor, Anti-invasive, Cell proliferation, Human Esophageal cancer

Time-Dependent Changes of Cell Proliferation After Laser Photocoagulation in Mouse Chorioretinal Tissue.

This study investigated the time course of cell proliferation after laser photocoagulation and analyzed the cell types of proliferating cells. C57BL/6J mice received unilateral laser photocoagulation. Intraperitoneal bromodeoxyuridine (BrdU) injection was performed, and mice were divided into two groups according to the injection paradigm: group 1 with continuous injection and group 2 with periodic injection. Each group was again divided into four subgroups according to injection period: 0 to 3 days (n = 11), 0 to 7 days (n = 14), 0 to 14 days (n = 6), and 0 to 28 days (n = 6) after laser photocoagulation for group 1; and 0 to 3 days (n = 11), 4 to 7 days (n = 6), 8 to 14 days (n = 6), and 15 to 28 days (n = 6) after laser photocoagulation for group 2. The eyes were examined with immunohistochemistry using anti-BrdU antibody and other various antibodies for identification of proliferating cells. Manual cell counting and flow cytometry were performed for quantification. In group 1, the number of BrdU+ cells showed marked increase during the first 3 days of laser lesioning, reaching its maximum after 7 days (P < 0.05). Group 2 also demonstrated peak proliferation during the first 3 days, but a significantly reduced number of BrdU+ cells were detected during 4 to 7 days, 8 to 14 days, and 15 to 28 days of laser treatment (P < 0.05). BrdU+ cells colocalized with CD11b, F4/80, iba1, RPE65, CD31, and glial fibrillary acidic protein (GFAP) labeling, and CD11b+, F4/80+, and iba1+ cells constituted the main fraction of BrdU+ cells. Laser photocoagulation induced cell proliferation mostly during the first 3 days, and many proliferating cells were identified as inflammatory cells, RPE cells, endothelial cells, and Müller cells.

Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining.

Determination of incorporation of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle. However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S-phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by 5-ethynyl-2'-deoxyuridine (EdU) which is incorporated into DNA. The nucleotide-exposed ethynyl residue was then derivatized by a copper-catalyzed cycloaddition reaction ("click chemistry" coupling) using a BrdU azide probe. The resulting DNA-bound bromouracil moieties were then detected by commercial anti-BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is more sensitive than traditional BrdU and allows multicolor and multiplex analysis in flow cytometry (FCM) and image-based cytometry.

Immunofluorescence-based methods to monitor DNA end resection.

Double-strand breaks (DSBs) are the most deleterious among all types of DNA damage that can occur in the cell. These breaks arise from both endogenous (e.g., DNA replication stress) and exogenous insults (e.g., ionizing radiation). DSBs are principally repaired by one of two major pathways: nonhomologous end joining (NHEJ) or homologous recombination (HR). NHEJ is an error-prone process that can occur in all phases of the cell cycle, while HR is limited to the S and G2 phases of the cell cycle when a sister chromatid is available as a template for error-free repair. The first step in HR is "DNA end resection," a process during which the broken DNA end is converted into a long stretch of 3'-ended single-stranded DNA (ssDNA). In recent years, DNA end resection has been identified as a pivotal step that controls "repair pathway choice," i.e., the appropriate choice between NHEJ and HR for DSB repair. Therefore, methods to quantitatively or semiquantitatively assess DNA end resection have gained importance in laboratories working on DNA repair. In this chapter, we describe two simple immunofluorescence-based techniques to monitor DNA end resection in mammalian cells. The first technique involves immuno-detection of replication protein A (RPA), an ssDNA-binding protein that binds to resected DNA. The second technique involves labeling of genomic DNA with 5-bromo-2'-deoxyuridine (BrdU) that can be detected by anti-BrdU antibody only after the DNA becomes single stranded due to resection. These methods are not complicated, do not involve sophisticated instrumentation or reporter constructs, and can be applied to most mammalian cell lines and, therefore, should be of broad utility as simple ways of monitoring DNA end resection in vivo.

Effect of mesenchymal stem cells transplantation on glycaemic profile & their localization in streptozotocin induced diabetic Wistar rats.

Background & objectives:Bone marrow is a rich source of adult stem cells that can differentiate into various cell types. Administration of mesenchymal stem cells (MSCs) in irradiated diabetic rat model has transiently shown to decrease blood glucose level. This study examines the effect of high dose and multiple injections of MSCs on glycemic profile, their localization and regeneration of islet in diabetic Wistar rat.Methods:The study was carried out in male Wistar rats categorized into three groups (n=6, in each group): Group 1 as control, group 2 streptozotocin (STZ) (50 mg/kg) induced diabetic group and group 3 experimental group; 5-bromo-2-deoxyuridine (BrdU) labelled allogenic MSCs were injected in the non-irradiated diabetic rat of the experimental group through tail vein. The blood glucose profile was subsequently monitored at regular intervals. Rats were sacrificed on day 45 and pancreas was examined for localization of BrdU labelled stem cells by immunofluorescence and islet-neogenesis by immunohistochemistry.Results:There was a significant reduction in blood glucose level after administration of MSCs in the experimental group (P<0.001). The presence of BrdU labelled MSCs in islet suggested their localization in the pancreas. Co-expression of anti-BrdU and anti-insulin antibody indicated trans-differentiation/fusion into insulin producing cells evidenced by significant increase in total number of islet (P=0.004) and insulin positive cells (P<0.0001) in experimental group.Interpretation & conclusions:Our results showed that the MSCs administration in non-irradiated diabetic Wistar rat reduced hyperglycaemia and was accompanied by increased islet-neogengesis, possibly through trans- differentiation/fusion.

Abstract 1022: Development and preclinical testing of AMG 780, a fully human antibody targeting angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2)

Abstract Targeting the angiopoietins with trebananib (a peptibody that inhibits the interaction between the endothelial cell-selective Tie2 receptor and its ligands Ang1 and Ang2) in combination with paclitaxel was associated with clinical benefit in a phase 3 ovarian cancer clinical trial (TRINOVA 1). Herein, we describe the generation of AMG 780, a fully human IgG2 antibody targeting Ang1 and Ang2. We compared AMG 780 to trebananib in affinity/neutralization ELISA assays and in vivo studies assessing inhibition of tumor growth and endothelial cell proliferation. AMG 780 is a derivative of an Ang2-binding antibody (Ab X). Ab X was generated by panning a human phage display Fab library (Dyax Corporation) against recombinant Ang2. The Ab X Fab was affinity matured by individually substituting every possible amino acid at every position in the light and heavy chain complementarity determining regions (CDRs). Resulting phage clones were interrogated for improved Ang1- and Ang2-binding activity. Clones with improved potency were converted to full antibodies. The most improved heavy chain clones were paired with the most improved light chain clones. The resulting IgGs were tested for neutralization of the interaction between the angiopoietins and their receptor, Tie2, as measured by homogenous time-resolved fluorescence (HTRF). Three resulting fully human IgG2 antibodies (AMG 780, Ab Y, and Ab Z) and the parental clone (Ab X) with potent Ang2 inhibitory activities (IC50 [nM], 0.05-0.10) and a range of Ang1 inhibitory activities (IC50 [nM], 0.31-547.00) were selected for xenograft studies. Mice were implanted with 5x106 Colo205 (human colon carcinoma) cells. When tumors were approximately 200 mm3, 300 µg of each of the four antibodies intraperitoneally or 14 µg of trebananib subcutaneously were administered twice weekly. For viable tumor fraction analyses, treated tumors at the end of the xenograft study were harvested and processed for paraffin histology. To examine endothelial cell proliferation, treated tumors were enzyme digested and stained with anti-CD31, anti-CD45, and anti-BrdU antibodies. The percentage of BrdU-positive tumor-associated endothelial cells (CD31high/CD45neg) was determined by flow cytometry. Relative to an isotope control antibody, all four IgG2 antibodies and trebananib inhibited tumor growth (P &amp;lt; 0.0001), viable tumor fraction (P &amp;lt; 0.0001), and tumor endothelial cell proliferation (P &amp;lt; 0.002). Tumor xenografts in those animals receiving AMG 780 exhibited the most pronounced inhibition in tumor growth (66%), viable tumor fraction (81%), and endothelial cell proliferation (84%). The inhibitory activities of AMG 780 (IC50 [nM]: Ang1, 4.5; Ang2, 0.06) were similar to those of trebananib (IC50 [nM]: Ang1, 3.5; Ang2, 0.03). Based on these findings, AMG 780 was selected for clinical development and currently is being evaluated in a first-in-human phase 1 trial. Citation Format: James V. Bready, Kyung Lee, Rick Jacobsen, Kevin Graham, Juan Estrada, Stephen A. Kaufman, Dongyin Yu, Angela Coxon, Jon Oliner. Development and preclinical testing of AMG 780, a fully human antibody targeting angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1022. doi:10.1158/1538-7445.AM2014-1022

Flow cytometric determination of 5-bromo-2'-deoxyuridine pharmacokinetics in blood serum after intraperitoneal administration to rats and mice.

5-Bromo-2'-deoxyuridine (BrdU) is a marker that is widely used to label S-phase cells in neurobiological research in most common doses 50 or 100 mg/kg per single intraperitoneal (i.p.) injection. However, the important data regarding its pharmacokinetics in rodents are still missing. The aim of our study was to investigate the BrdU level in serum after a single i.p. injection to adult rats (doses: 50 or 100 mg/kg) and adult mice (50 mg/kg). The animals were killed at selected time-points after the BrdU injection, and proliferating tumour cells (cell lines HCT-116 and HL-60) were co-cultivated with isolated blood sera. BrdU incorporated in the DNA of the S-phase tumour cells was stained with an anti-BrdU antibody and analysed using flow cytometry. In rats, the efficacies of BrdU labelling of S-phase cells in both in vitro and in vivo conditions were compared in the 50 and 100 mg/kg groups. According to our results, BrdU was in saturated concentration to label almost all S-phase cells for 60 min in both doses and was detectable in blood serum until 120 min after the single i.p. injection. However, the 100 mg/kg dose of BrdU did not provide a prolonged staining period to offset the potentially higher toxicity in comparison with the 50 mg/kg dose. In mice, due to their faster metabolism, the concentration of BrdU in blood serum was sufficient to label the whole population of S-phase cells for only 15 min after the i.p. injection, then dropped rapidly.

Homeobox Transcription Factor VentX Regulates Differentiation and Maturation of Human Dendritic Cells

Dendritic cells (DCs) are specialized antigen presentation cells that play critical roles in the initiation and regulation of immune responses. The molecular determinants of DC differentiation and maturation are target of extensive investigation. VentX is a human homeobox transcriptional factor that regulates proliferation and differentiation of hematopoietic cells. In the current study, we report that ablation of VentX expression in monocytes significantly impaired their differentiation into DCs. Conversely, overexpression of VentX in monocytic THP1 cells accelerated their differentiation toward DCs. We showed that VentX regulates DC differentiation, in part, through modulating IL6 expression. Clinically, we found that VentX expression was elevated in intestinal lamina propria DCs (LPDCs) of inflamed mucosa from inflammatory bowel disease patients. Knockdown experiments suggested that VentX is essential for the maturation of LPDCs. In addition, corticosteroid treatment markedly decreased VentX expression in LPDCs and enforced expression of VentX counteracted the effects of corticosteroid on DCs maturation. Our data suggest that VentX is a critical transcriptional regulator of DC differentiation and maturation, and a potential target of immune regulation and therapy.