anti-BrdU Antibody Research Articles

A method for in situ visualization of Protein-Nascent RNA interactions in single cell using Proximity Ligation Assay (IPNR-PLA) in mammalian cells

ABSTRACT Proximity ligation assay (PLA) is an immunofluorescence assay, which determines in situ interaction of two biomolecules present within 40 nm close proximity. Here, we describe a modification of PLA for visual detection of in situ protein interactions with nascent RNA in a single cell (IPNR-PLA). In IPNR-PLA, nascent RNA is labeled by incorporating 5-fluorouridine (FU), a uridine nucleotide analogue, followed by covalent cross-linking of the interacting partners in proximity to newly synthesized RNA. By using combination of anti-BrdU antibody, which specifically binds to FU, and primary antibody against a protein of interest, the IPNR reaction results in fluorescent puncta as a positive signal, only if the candidate proteins are in proximity to nascent RNA. We have validated this method by demonstrating known CDK9 and elongating RNA pol II interaction with nascent RNA. Finally, we used this method to test for the presence of DNA double strand breaks as well as Poly (ADP-ribose) polymerase 1 (PARP1), an RNA binding protein, in the vicinity of nascent RNA in cancer cells. The capability of performing parallel IF labeling and quantifiable multiparameter measurements within heterogeneous cell populations makes IPNR-PLA very attractive for use in biological studies. Overall, we have developed the IPNR-PLA method for analysis of protein association with nascent RNA with single-cell resolution, which is highly sensitive, quantitative, efficient, and requires little starting experimental material.

Investigation of the effects of mesenchymal stem cell administration on liver recovery in experimental hepatotoxicity model

Hepatotoxicity refers to liver dysfunction associated with certain medical drugs and chemicals. Studies have shown that mesenchymal stem cells have a positive effect on the improvement of liver diseases. The aim of this study was to investigate the potential protective effects of fetal kidney-induced mesenchymal stem cells on Doxorubicin-induced hepatotoxicity in rats. Sprague dawley rats were divided into three groups as control, sham, and treatment group. Intraperitoneal mesenchymal stem cells were treated with BrdU prior to transplantation so that they could be followed up after invivo transplantation. After completion of the experimental steps, the groups were monitored for 5 weeks. Then the rats were terminated and their livers were taken for histopathological and immunohistochemical evaluation. In immunohistochemical examinations performed with TNF-α, Caspase-3 and COX-2 primary antibodies, the most severe positivity was in the sham group, followed by the control and treatment groups. While the control and sham groups were found to be statistically similar in immunohistochemical staining with anti-BrdU antibody, the treatment group was found to be significantly different from the other groups (p<0.05). As a result, it has been revealed that mesenchymal stem cells administered intraperitoneally to rats with Doxorubicin-induced hepatotoxicity, prevent degeneration and necrosis in hepatocytes, and TNF-α, COX-2, and Caspase-3 levels were significantly decreased immunohistochemically, proving increased liver regeneration.

BrdU Incorporation Assay to Analyze the Entry into S Phase.

5-Bromo-2'-deoxyuridine (bromodeoxyuridine (BrdU)), a modified nucleotide and analog of thymidine, is commonly used for detecting proliferating cells. For detection, an anti-BrdU antibody (probe) with a fluorescent dye is applied to bind the BrdU label after DNA denaturation. In this protocol, we provide the BrdU labeling method for both in vitro and in vivo studies, along with immunocytochemistry (ICC)/immunofluorescence (IF) and immunohistochemistry (IHC) staining procedures, respectively. Multicolor staining is also presented as an option to detect the co-distribution of two or multiple antigens in the same sample, making it possible to visualize the location of different molecules at the same time.

Vitamin D Is Necessary for Murine Gastric Epithelial Homeostasis.

Simple SummaryThe gastric epithelium comprises multiple cell types that undergo continuous renewal through controlled proliferation, differentiation and death. Several vitamins, such as vitamin D (VD), are known to contribute to tissue homeostasis and numerous studies have shown the importance of VD in different body organ systems. However, VD’s normal function in the stomach is understudied. To better understand the role of VD in the murine stomach, we initially confirmed the expression of VD receptors (VDR) in the stomach. Mice were fed a VD-deficient diet for 3 months. The results showed that the proton pump of acid-secreting parietal cells was downregulated in vitamin D-deficient mice and contributed to an abnormal gastric physiology. Moreover, this diet increased the gastrin hormone gene expression and increased gastric epithelial cell proliferation. These findings suggest essential biological roles for VDR and VD in gastric epithelial homeostasis. Future research will be required to explore this phenomenon in humans.Unlike other organs, the importance of VD in a normal stomach is unknown. This study focuses on understanding the physiological role of vitamin D in gastric epithelial homeostasis. C57BL/6J mice were divided into three groups that were either fed a standard diet and kept in normal light/dark cycles (SDL), fed a standard diet but kept in the dark (SDD) or fed a vitamin D-deficient diet and kept in the dark (VDD). After 3 months, sera were collected to measure vitamin D levels by LC-MS/MS, gastric tissues were collected for immunohistochemical and gene expression analyses and gastric contents were collected to measure acid levels. The VDD group showed a significant decrease in the acid-secreting parietal cell-specific genes Atp4a and Atp4b when compared with the controls. This reduction was associated with an increased expression of an antral gastrin hormone. VDD gastric tissues also showed a high proliferation rate compared with SDL and SDD using an anti-BrdU antibody. This study indicates the requirement for normal vitamin D levels for proper parietal cell functions.

Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging.

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs for cancer treatment. These targets are poly(ADP-ribose) polymerases (PARPs), which initiate various forms of DNA repair. Inhibiting these enzymes using PARP inhibitors (PARPi) achieves synthetic lethality by conferring a therapeutic vulnerability in homologous recombination (HR)-deficient cells due to mutations in breast cancer type 1 (BRCA1), BRCA2, or partner and localizer of BRCA2 (PALB2). Cells treated with PARPi accumulate DNA double-strand breaks (DSBs). These breaks are processed by the DNA end resection machinery, leading to the formation of single-stranded (ss) DNA and subsequent DNA repair. In a BRCA1-deficient context, reinvigorating DNA resection through mutations in DNA resection inhibitors, such as 53BP1 and DYNLL1, causes PARPi resistance. Therefore, being able to monitor DNA resection in cellulo is critical for a clearer understanding of the DNA repair pathways and the development of new strategies to overcome PARPi resistance. Immunofluorescence (IF)-based techniques allow for monitoring of global DNA resection after DNA damage. This strategy requires long-pulse genomic DNA labeling with 5-bromo-2'-deoxyuridine (BrdU). Following DNA damage and DNA end resection, the resulting single-stranded DNA is specifically detected by an anti-BrdU antibody under native conditions. Moreover, DNA resection can also be studied using cell cycle markers to differentiate between various phases of the cell cycle. Cells in the S/G2 phase allow the study of end resection within HR, whereas G1 cells can be used to study non-homologous end joining (NHEJ). A detailed protocol for this IF method coupled to cell cycle discrimination is described in this paper.

EdU-Based Assay of Cell Proliferation and Stem Cell Quiescence in Skeletal Tissue Sections.

Identifying and tracking proliferating and quiescent cells in situ is an important phenotyping component of skeletal tissues in development, physiology and disease. Among all the methods that exist, which include immunostaining for cell cycle-specific proteins, the gold standards use thymidine analogs. These compounds label proliferating cells by being incorporated into de novo-synthesized genomic DNA. 5-bromo-2'-deoxyuridine (BrdU) has traditionally been used for this purpose, but its detection is lengthy and requires harsh treatment of tissue sections to give access of anti-BrdU antibody to DNA. An alternative, more recently developed, uses 5-ethynyl-2'-deoxyuridine (EdU). This thymidine analog is detected by click chemistry, that is, covalent cross-linking of its ethynyl group with a fluorescent azide that is small enough to easily penetrate native tissues and reach DNA. In addition to being simple and quick, this EdU-based assay is compatible with other protocols, such as immunostaining, on the same tissue sections. We here describe an EdU-based protocol optimized to label and functionally assess actively proliferating cells as well as slowly dividing cells, including stem cells, in mouse skeletal tissues.

Detection of trifluridine in tumors of patients with metastatic colorectal cancer treated with trifluridine/tipiracil.

Trifluridine (FTD) is the active component of the nucleoside chemotherapeutic drug trifluridine/tipiracil (FTD/TPI), which is approved worldwide for the treatment of patients with metastatic gastrointestinal cancer. FTD exerts cytotoxic effects via its incorporation into DNA, but FTD has not been detected in the tumor specimens of patients. The purpose of this study was to detect FTD in tumors resected from metastatic colorectal cancer (mCRC) patients who were administered FTD/TPI. Another purpose was to investigate the turnover rate of FTD in tumors and bone marrow in a mouse model. Tumors and normal tissue specimens were obtained from mCRC patients who were administered FTD/TPI or placebo at Kyushu University Hospital. Tumors and bone marrow were resected from mice with peritoneal dissemination treated with FTD/TPI. To detect and quantitate FTD incorporated into DNA, immunohistochemical staining of paraffin-embedded specimens (IHC-p staining) and slot-blot analysis of DNA purified from these tissues were performed using an anti-BrdU antibody. IHC-p staining of proliferation and apoptosis markers was also performed. FTD was detected in metastatic tumors obtained from mCRC patients who were administered FTD/TPI, but who had discontinued the treatment several weeks before surgery. In a peritoneal dissemination mouse model, FTD was still detected in tumors 13days after the cessation of FTD/TPI treatment, but had disappeared from bone marrow within 6days. These results indicate that FTD persists longer in tumors than in bone marrow, which may cause a sustained antitumor effect with tolerable hematotoxicity.

Torin2 Exploits Replication and Checkpoint Vulnerabilities to Cause Death of PI3K-Activated Triple-Negative Breast Cancer Cells.

Frequent mutation of PI3K/AKT/mTOR signaling pathway genes in human cancers has stimulated large investments in targeted drugs but clinical successes are rare. As a result, many cancers with high PI3K pathway activity, such as triple-negative breast cancer (TNBC), are treated primarily with chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors, we find that one drug, Torin2, is unusually effective because it inhibits both mTOR and other PI3K-like kinases (PIKKs). In contrast to mTOR-selective inhibitors, Torin2 exploits dependencies on several kinases for S-phase progression and cell-cycle checkpoints, thereby causing accumulation of single-stranded DNA and death by replication catastrophe or mitotic failure. Thus, Torin2 and its chemical analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors.

The Membrane Scaffold CD82 Regulates Acute Myeloid Leukemia Dormancy and Bone Marrow Niche Interactions

Acute Myeloid Leukemia (AML) patients respond favorably to induction chemotherapy treatment, however, due to a high rate of minimal residual disease, a significant majority of patients fatally relapse. The bone marrow niche can provide a protective environment for leukemic stem cells (LSCs) and is often the primary site for minimal residual disease after chemotherapy. Therefore, in order to improve long-term patient outcomes, it is critical to expand our focus to the development of therapies that are specifically directed towards the AML cell population that is residing within the bone marrow. Previous studies from our lab have established the membrane-scaffold protein, CD82, as a positive regulator of AML adhesion and signaling within the bone marrow. In this study, we test the hypothesis that specific bone marrow niche interactions and signaling mediated by CD82 overexpression promote AML cell dormancy and contribute to minimal residual disease. To assess the effects of CD82 expression on AML cell dormancy, we first measured leukemia engraftment using luciferase engineered AML cells and preclinical mouse models. Luciferase engineered cells differentially expressing CD82 were intravenously injected into immunocompromised NOD-scid IL2rgnull (NSG) mice, and bioluminescence intensity (BLI) was longitudinally assessed as a measure of tumor burden using the IVIS® Spectrum in vivo imaging system. At 4 weeks of tumor growth, animals were sacrificed and AML cells were isolated from the blood and bone marrow and quantified by flow cytometry (hCD45+,GFP+). Our results suggest that CD82 overexpressing cells have a reduced tumor engraftment potential when compared to CD82 knockdown and control AML cells. To confirm that the reduced engraftment observed was not due to disrupted bone marrow trafficking, we completed homing experiments where bone marrow was harvested 16 hours post intravenous injection. Homing studies demonstrated that CD82 overexpressing cells display comparable bone marrow homing to control cells and increased bone marrow homing when compared to the CD82 knockdown cells. Interestingly, CD82 knockdown cells demonstrate a disruption in homing. Next, we went on to investigate how CD82 expression contributes to AML cell cycle activation in the bone marrow. Following engraftment, mice were injected with 100mg/kg of BrdU as a measure of proliferation and additional BrdU was also supplemented in the drinking water untill the time of harvest. Three days post injection, the animals were sacrificed and AML cells were isolated from the bone marrow and identified by flow cytometry (hCD45+,GFP). BrdU incorporation was measured with anti-BrdU specific antibodies. We find that over 60% of CD82 knockdown and control AML cells displayed increased BrdU incorporation, whereas only 30% of CD82 overexpressing cells were BrdU positive. These results suggest that CD82 overexpression may promote a more quiescent phenotype in AML cells localized within the bone marrow. Recognizing that specific bone marrow regions can play a critical role in providing cues for AML cell survival and growth, we investigated how CD82 expression impacts AML cell localization within the bone marrow. Femoral bones from NSG mice with established leukemia were fixed, cryoembedded and cryosectioned. The thick femur sections were immunostained and analyzed using confocal microscopy to assess AML cell (CD45+,GFP+) localization within the bone marrow with respect to endothelial and osteoblastic niches. Our preliminary analysis suggests that while CD82 knockdown cells infiltrate the entire bone marrow, CD82 overexpressing AML cells distribute to the marrow periphery near osteoblastic regions. Future studies will be focused on elucidating the specific interacting partners of AML cells that are promoted by CD82 and the impact of the N-linked glycosylation pattern of CD82. Collectively, our findings demonstrate a role of CD82 in AML cell dormancy, potentially by fostering specific interactions within bone marrow niche, which may contribute to increased minimal residual disease and reoccurrence. Disclosures No relevant conflicts of interest to declare.

Aberrant Function of the C-Terminal Tail of HIST1H1E Accelerates Cellular Senescence and Causes Premature Aging

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

Cell Cycle Analysis Using In Vivo Staining of DNA-Synthesizing Cells.

The thymidine analogues BrdU (5-bromo-2´-deoxyuridine) and EdU (5-ethynyl-2´-deoxyuridine) are routinely used for determination of the cells synthesizing DNA in the S-phase of the cell cycle. Availability of the anti-BrdU antibody clone MoBu-1 detecting only BrdU allowed to develop a method for the sequential DNA labelling by these two thymidine analogues for determining the cell cycle kinetic parameters.In the current step-by-step protocol, we present` two approaches optimized for in vivo study of the cell cycle and the limitations that such approaches imply: (1) determination of the cell flow rate into the G2-phase by dual EdU/BrdU DNA-labelling method and (2) determination of the outflow of DNA-labelled cells arising from the mitosis.

Effects of Fragrance-Loaded Mesoporous Silica Nanocolumns on Central Nervous System.

Fragrances have rapid effect on our central nervous system, such as making the spirit of relaxation, relieving tensions and refreshing ourselves. However, the release of fragrances is not stable. Here, we added photo-driven mesoporous silica nanocolumns loaded with sandela 803 (MS-C@S803) into wallpaper to obtain fragrant wallpaper (MS-C-W@S803). We then analysed the effects of MS-C-W@S803 and pure sandela 803 treated wallpaper (W@S803) on the CNS of mice and explored the internal mechanism of these effects. Besides, we evaluated the short-term (7 days) and long-term (30 days) effects of the fragrance treated wallpaper. In behaviouristics level, we detected the anxiolytic effects via elevated plus maze and open field test. In tissue level, we analysed the neural activity in hippocampus, hypothalamus and olfactory bulb regions via measuring the electrophysiological signal. In cell level, we tested the nerve regeneration in hippocampus, substantia nigra and corpus striatum via immunofluorescence staining with the anti-BrdU antibody. In molecule level, we measured the expression of dopamine, acetylcholine, γ-aminobutyric acid (GABA) and N-methyl-D-aspartic acid (NMDA) via liquid chromatography-mass spectrometry. Finally, we find that MS-C-W@S803 had anxiolytic effects on the CNS of mice, and the effects were more significant as time progresses.

Planar cell polarity gene Fuz triggers apoptosis in neurodegenerative disease models.

Planar cell polarity (PCP) describes a cell-cell communication process through which individual cells coordinate and align within the plane of a tissue. In this study, we show that overexpression of Fuz, a PCP gene, triggers neuronal apoptosis via the dishevelled/Rac1 GTPase/MEKK1/JNK/caspase signalling axis. Consistent with this finding, endogenous Fuz expression is upregulated in models of polyglutamine (polyQ) diseases and in fibroblasts from spinocerebellar ataxia type 3 (SCA3) patients. The disruption of this upregulation mitigates polyQ-induced neurodegeneration in Drosophila We show that the transcriptional regulator Yin Yang 1 (YY1) associates with the Fuz promoter. Overexpression of YY1 promotes the hypermethylation of Fuz promoter, causing transcriptional repression of Fuz Remarkably, YY1 protein is recruited to ATXN3-Q84 aggregates, which reduces the level of functional, soluble YY1, resulting in Fuz transcriptional derepression and induction of neuronal apoptosis. Furthermore, Fuz transcript level is elevated in amyloid beta-peptide, Tau and α-synuclein models, implicating its potential involvement in other neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. Taken together, this study unveils a generic Fuz-mediated apoptotic cell death pathway in neurodegenerative disorders.

Abstract 2886: ChIP-seq analysis to explore DNA replication profile in trifluridine-treated human colorectal cancer cells in vitro

Abstract Background: Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which was approved for the treatment of patients with metastatic colorectal cancer refractory to standard chemotherapies. FTD, an antineoplastic thymidine analogue, is efficiently incorporated into the genomic DNA of tumor cells. However, the molecular mechanisms underlying its cytotoxic effects remain unclear. Therefore, the DNA replication profile in FTD-treated cells was comprehensively analyzed to explore the mode of FTD incorporation into the genomic DNA. Method: We generated the DNA replication profile in HCT-116 human colorectal cancer cells as follows: asynchronous cultures of HCT-116 were exposed to 5 μM 5-bromo-2′-deoxyuridine (BrdU), a non-cytotoxic control drug or FTD, for 4 h, harvested, and subjected to DNA immunoprecipitation using an anti-BrdU antibody. An input and immunoprecipitated DNA from each experiment were sequenced, and the sequence reads generated using Illumina sequencing were aligned uniquely to the human genome (hg19), allowing up to two mismatches using the BWA algorithm with default settings. The density of aligned reads along the genome was calculated and stored in a bigWig file for visualization in genome browsers. Peak finding and gene ontology analysis were performed using MACS and Genomic Regions Enrichment of Annotations Tool (GREAT). The 60-bp sequences surrounding the summits of the top 1000 peaks were analyzed for conserved motifs using the online program MEME. Results: In an antibody validation step, we observed significant FTD peaks over input DNA. We identified 6043 and 5080 high confident FTD and BrdU peaks in HCT-116 cells, respectively. Interestingly, 2911 of 6043 FTD peaks were uncommon to BrdU peaks, while 3232 peaks were common in FTD and BrdU. The FTD and BrdU peaks were not randomly distributed throughout the genome. Most FTD and BrdU peaks were ±5 kb or further away from transcription start sites. We identified the genes associated with distant peaks of FTD using GREAT. FTD peaks occurred near genes involved in extracellular matrix structural composition such as ACAN, BGN, CHAD, and COL12A1, while BrdU peaks were near genes involved in cysteine-type endopeptidase inhibitor activity such as ARRB1, BIRC7, LCN1, and CST1. Dinucleotide repeats such as TGTGTG were observed as conserved motifs in FTD-peak sequences. Conclusion: Global FTD incorporation patterns can delineate active replication loci, namely FTD preferentially incorporating loci, in human cancer cells. The DNA replication profile in FTD-treated cells differed from that in BrdU-treated cells. Thus, differences in the incorporation patterns of FTD and BrdU into the genomic DNA revealed the mechanisms of antitumor activities of FTD. Citation Format: Takashi Kobunai, Kazuaki Matsuoka, Teiji Takechi. ChIP-seq analysis to explore DNA replication profile in trifluridine-treated human colorectal cancer cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2886.

DNA in Squid Synaptosomes.

The synthesis of brain metabolic DNA (BMD) is modulated by learning and circadian oscillations and is not involved in cell division or DNA repair. Data from rats have highlighted its prevalent association with the mitochondrial fraction and its lack of identity with mtDNA. These features suggested that BMD could be localized in synaptosomes that are the major contaminants of brain mitochondrial fractions. The hypothesis has been examined by immunochemical analyses of the large synaptosomes of squid optic lobes that are readily prepared and identified. Optic lobe slices were incubated with 5-bromo-2-deoxyuridine (BrdU) and the isolated synaptosomal fraction was exposed to the green fluorescent anti-BrdU antibody. This procedure revealed that newly synthesized BrdU-labeled BMD is present in a significant percent of the large synaptosomes derived from the nerve terminals of retinal photoreceptor neurons and in synaptosomal bodies of smaller size. Synaptosomal BMD synthesis was strongly inhibited by actinomycin D. In addition, treatment of the synaptosomal fraction with Hoechst 33258, a blue fluorescent dye specific for dsDNA, indicated that native DNA was present in all synaptosomes. The possible role of synaptic BMD is briefly discussed.

UTX Affects Neural Stem Cell Proliferation and Differentiation through PTEN Signaling.

SummaryNeural stem cell (NSC) proliferation and differentiation in the developing brain is a complex process precisely regulated by intrinsic and extrinsic signals. Although epigenetic modification has been reportedly involved in the regulation of the cerebral cortex, whether UTX, an H3K27me3 demethylase, regulates the development of cerebral cortex during the embryonic period is unclear. In this study, we demonstrate that Utx deficiency by knockdown and conditional knockout increases NSC proliferation and decreases terminal mitosis and neuronal differentiation. Furthermore, we find that impairment of cortical development caused by lack of Utx is less significant in males than in females. In addition, UTX demethylates H3K27me3 at the Pten promoter and promotes Pten expression. P-AKT and P-mTOR levels are increased in the Utx conditional knockout cortices. Finally, Utx or Pten overexpression can rescue the impairment of brain development caused by Utx loss. These findings may provide important clues toward deciphering brain diseases.

Identifying Quiescent Stem Cells in Hair Follicles.

Hair follicle stem cells (HFSCs) are noted for their relative quiescence and therefore can be distinguished from other cells by their differential history of cell division. Replicating cells can be labeled by pulsing the animals repeatedly with 5-bromo-2'-deoxyuridine (BrdU) or tritiated thymidine ([3H]TdR), thymidine analogs that get incorporated into DNA during DNA synthesis. Because dividing cells dilute the label after each cell division, frequently dividing cells will lose the label over time while slow cycling cells will retain the label and thus are termed label retaining cells (LRCs). [3H]TdR can be visualized by autoradiography and BrdU can be detected by immunofluorescence with anti-BrdU antibodies. Alternatively, a well-established tet-regulatable transgenic mouse model can be used to express histone H2B-GFP in epithelial proliferative cells and their dilution and retention of the GFP signal can be followed. In this chapter, we detail the steps to perform BrdU pulse-chase and H2B-GFP pulse-chase experiments to identify quiescent cells in the hair follicle.

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.

Abstract 2490: Rad51 suppresses innate immune response by blocking MRE11-mediated degradation of newly replicated genome

Abstract Purpose of the study: Eukaryotic cells accrue DNA damage as a result of endogenous metabolic activities such as DNA replication, recombination errors or environmental exposures such as ionizing radiation, ultra-violet light and chemical mutagens. Unrepaired DNA damage leads to tumorigenesis. Rad51 is a multifunctional protein that plays a central role in DNA replication and homologous recombination repair. It is known that defects in Rad51 function can cause cancer. The goal of this study is to identify a novel role for Rad51 outside of its known functions in DSB repair and replication fork processing. Methods: Since Rad51 knockout is lethal to cells, we generated an inducible system in which we can down-regulate Rad51 expression in HT1080 cells after Doxycycline treatment. To determine the effect of Rad51-knockdown in global gene expression pattern, we carried out unbiased microarray gene expression analysis and after induction of DNA damage and replication stress by radiation. ssDNA and dsDNA in the cytosolic fractions were quantified using Quant-iT OliGreen and PicoGreen Assay Kits. For cytoplasmic BrdU detection, exponentially grown cells were labeled with BrdU for 18-20 h and then immunostained with anti-BrdU antibody. Additionally, we measured the expression and post-translational modification of proteins involved in innate immune signaling by western blotting. We also employed DNA fiber assay to determine the role of Rad51 in replication fork processing. Results: We found that defects in Rad51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. Mechanistically, the unprotected newly replicated genome in the absence of Rad51 is degraded by the exonuclease activity of Mre11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data revealed that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, Rad51 is also implicated in the suppression of innate immunity. Conclusion: Rad51 plays a novel role in immunity outside its known functions in DSB repair and replication fork processing. We discovered that the lack of Rad51 leads to the upregulation of innate immune response pathway genes upon DNA damage and replication induced by irradiation. We found that in the absence of Rad51 the newly replicated genome is degraded by the exonuclease activity of Mre11. We also showed that these degraded nascent DNA fragments are exported to the cytoplasm, triggering innate immune response signaling. Our study reveals a previously unidentified role for Rad51 in triggering an innate immune response, and places Rad51 at the hub of new interconnections between DNA replication, DNA repair, and immunity. Funding: This work was supported by NIH R01AG053341 grants. Citation Format: Kalayarasan Srinivasan, Souparno Bhattacharya, Salim Abdisalaam, Shibani Mukherjee, Asaithamby Aroumougame. Rad51 suppresses innate immune response by blocking MRE11-mediated degradation of newly replicated genome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2490. doi:10.1158/1538-7445.AM2017-2490

Identification of CD146 as a novel molecular actor involved in systemic sclerosis

We highlight for the first time that CD146/sCD146 is involved in fibrotic process during SSc. sCD146 could thus constitute a new biomarker to assess disease activity, and potentially a new target for therapeutic applications.