DOCSLIB.ORG

Modulation of (+)-Anti-BPDE Mediated P53 Accumulation by Inhibitors of Protein Kinase C and Poly(ADP-Ribose) Polymerase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Modulation of (+)-Anti-BPDE Mediated P53 Accumulation by Inhibitors of Protein Kinase C and Poly(ADP-Ribose) Polymerase Oncogene (1997) 14, 801 ± 809 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Modulation of (+)-anti-BPDE mediated p53 accumulation by inhibitors of protein kinase C and poly(ADP-ribose) polymerase Sundaresan Venkatachalam2,3, Mikhail Denissenko1,4 and Altaf A Wani1,2 1Department of Radiology and 2Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA The rapid accumulation of the p53 gene product is of the cell cycle occur after DNA damage, most likely considered to be an important component of the cellular for the ecient and optimal removal of DNA lesions response to a variety of genotoxins. In order to gain from the cellular genome before cell division (Grana insights on the biochemical pathways leading to p53 and Reddy, 1995; Canman et al., 1994; Guengerich, stabilization, the eect of (+) 7,8-dihydroxy-anti-9, 1988; Kastan et al., 1991). It is suggested that the 10-epoxy-7,8,9,10-tetrahydrobenzo(a)-pyrene [(+)-anti- programmed death of cells may be yet another mode BPDE] induced DNA damage on p53 protein levels necessary for the elimination of potential precursor was investigated in various repair-pro®cient and repair- cells escaping DNA repair (Canman et al., 1994; de®cient human cells. Brief exposure of normal human Homan and Liebermann, 1994). Recent reports have ®broblasts to 0.05 ± 1 mM (+)-anti-BPDE resulted in demonstrated that the p53 tumor suppressor protein elevated p53 protein levels as compared to the plays an integral role in such cellular response constitutive levels of control cells. The rapid induction pathways to DNA damage (Nelson and Kastan, response, detectable within a few hours, was sustained up 1994; Kastan et al., 1991, 1992). The rapid accumula- to a period of at least 24 h. Repair-pro®cient and repair- tion of transcriptionally active p53 tumor suppressor de®cient (XPA) human lymphoblastoid cells showed a protein upon genotoxic damage has been shown to be similar response. The poly(ADP-ribose) polymerase essential for G1-S cell cycle arrest. This occurs via the inhibitor, 3-aminobenzamide (3-AB), diminished the p53 induction of p21, a potent inhibitor of cyclin dependent induction response by concomitantly decreasing the kinases regulating the cell cycle (El-Deiry et al., 1994; extent of (+)-anti-BPDE induced DNA damage in cells Dulic et al., 1994; Nelson and Kastan, 1994; Kastan et pretreated with the inhibitor. However, the direct al., 1991, 1992). While p53 is shown to play a de®nite involvement of poly ADP-ribosylation was also apparent role in triggering apoptosis following DNA damage as 3-AB was able to attenuate (*50%) the p53 response (Kastan et al., 1992; Smith et al., 1994; El-Deiry et al., by post-damage inhibitor treatment of the cells. 1994; Price and Park, 1994; Lowe et al., 1993), cells Inhibition of cellular DNA replication by hydroxyurea de®cient in p53, have been shown to undergo apoptotic and AraC, in the presence or absence of DNA damage, cell death indicating the presence of other p53 also resulted in rapid p53 accumulation in repair- independent pathways (Nelson and Kastan, 1994; de®cient cells. On the contrary, inhibition of protein Venkatachalam et al., 1993). Besides this function, kinase C (PKC) by calphostin-C led to an abrogation of p53 can act as a transcriptional activator of repair (+)-anti-BPDE mediated p53 induction. Analysis of the related genes like gadd45 and very recent studies have downstream eects of carcinogen treatment showed that implicated a direct role of p53 in the excision repair the lymphoblastoid cells undergo DNA fragmentation process (Ford and Hanawalt, 1995). Thus, the indicative of apoptosis while ®broblasts exhibit cell cycle intracellular accumulation of p53 after DNA damage arrest at the G1-S boundary. mediates a wide range of interrelated processes that ensure the faithful propagation of parental genomic Keywords: DNA damage; p53 induction; apoptosis; cell sequences and the avoidance of mutations. In cycle arrest concordance with its pivotal role in guarding the genome, the p53 gene has been reported to be mutated in a majority of human cancers (Hollstein et al., 1991; Greenblatt et al., 1994). Introduction The increase in p53 levels following DNA damage of cells has been shown to result from an increase in half- Continuous exposure of cells to exogenous and life of the protein (Liu and Pelling, 1995; Abrahams et endogenous DNA damage is implicated in the al., 1995). While the biochemical and molecular pathogenesis of various cancers. A host of complex modulators responsible for the stabilization of cellular cellular DNA repair mechanisms have evolved to p53 are yet to be discerned, some studies suggest that counteract the deleterious eects of DNA damage in excision repair coupled formation of transitory DNA humans (Sancar, 1995). In addition to the existence of strand breaks is an important initial factor in the DNA several detoxi®cation systems, delay at speci®c stages damage response pathway (Nelson and Kastan, 1994). Furthermore, DNA strand breaks that induce p53 protein levels also are known to activate various damage dependent cellular enzymes like poly(ADP- Correspondence: AA Wani ribose) polymerase and DNA dependent protein Present Addresses: 3Division of Molecular Virology, Baylor College kinases (Nelson and Kastan, 1994; Carson et al., of Medicine, Houston, TX 77030; 4Department of Biology, Beckman Research Institute, City of Hope, Duarte, CA 1986; Stierum et al., 1994; Anderson, 1993). Indirect Received 11 June 1996; revised 18 October 1996; accepted 18 October evidence from recent studies indicates that post- 1996 translational protein modi®cations, namely poly Modulation of DNA damage induced cellular p53 protein S Venkatachalam et al 802 ADP-ribosylation and phosphorylation may be in- indicated that the response was due to the interaction volved in the increased half-life of the p53 protein of the reactive epoxide metabolite with the genome (Whitacre et al., 1995; Khanna and Lavin, 1993). leading to DNA damage. The extent of p53 accumula- Benzo(a)pyrene is a major environmental pollutant tion increased proportionally with the dose of the present in automobile exhaust, cigarette smoke, carcinogen. However the p53 induction response was various foods and industrial wastes (Phillips, 1983). saturable at all the time points from treatment with The diol-epoxide metabolite, (+)-anti-BPDE reacts at doses higher than 0.5 mM (Figure 1, top and center). several nucleophilic sites in DNA and the covalent N2- Time course experiments revealed that the tumor dG-anti-BPDE adduct comprises more than 80% of suppressor protein begins to accumulate as early as the total adduct population (Osborne et al., 1981). 2.5 h and remains at elevated levels for at least 24 h The DNA base alterations arising from exposure to after exposure (Figure 1, center). Control cells treated (+)-anti-BPDE have been implicated in mutagenesis, with vehicle alone did not indicate any change in the carcinogenesis and cellular transformation (Harris, intracellular levels of the p53 protein. Cell lysates from 1991; Conney, 1982). In the present report we have transformed HeLa cells, containing high constitutive used (+)-anti-BPDE as a model DNA damaging levels of p53, served as a positive control for speci®c agent to study the modulation of the p53 induction antibody based detection by Western blotting. The p53 response by inhibitors of poly(ADP-ribose) polymer- protein speci®c binding of antibodies was further ase and PKC. The results indicate that the down established by using extracts of p53 null HL-60 cells regulation of PKC activity leads to an abrogation of which demonstrated absence of any immuno-reactive p53 protein elevation following exposure to (+)-anti- protein bands at 53 Kd (data not shown). BPDE. Inhibition of poly(ADP-ribose) polymerase activity by 3-aminobenzamide results in an attenua- tion of p53 accumulation in various cultured human cells exposed to carcinogenic (+)-anti-BPDE via, (a) a le I grinogen dose dependent indution of @CAEntiEfhi concomitant decrease in the absolute adduct levels hxe dduts in humn ells upon pre-incubation of the cells with 3-AB and (b) a T wA eddutsGIH nuleotides @m direct decrease in the protein accumulation upon post- hose QFUTCHFRS damage incubation of the cells with the PARP HFHS VFUTCIFPV inhibitor. The study also demonstrates that the HFI HFS RWFSCHFQT IHUCIHFWP formation of DNA stand breaks might not be the IFH only necessary factor in the p53 accumulation xorml lympholstoid ells were treted with the indited doses response triggered by (+)-anti-BPDE. Lastly the data of the rinogen for QH minD lysed nd proessed for hxe are presented to show that the downstream eects of isoltionF he representtive ddut levels of smples orresponding DNA damage i.e. cell cycle arrest and apoptosis occur to the experiment shown in ®gure I @ottomAD were quntitted y in a cell type dependent fashion. the nonEompetitive immunoEslot lot ssy s desried in mterils nd methodsF he levels represent the menCsFdF of replite mesurements Results Time 0 2.5 10 Dose dependence and time course of p53 accumulation [hr] BPDE after (+)-anti-BPDE treatment m 0.00 0.05 0.50 M 0.10 0.50 1.00 0.00 0.01 0.05 [ ] HeLa 0.00 0.01 0.05 0.10 0.50 1.00 DNA damage serves as an important trigger to activate p53 the cellular responses such as cell cycle arrest, DNA repair and apoptosis (Nelson and Kastan, 1994; BPDE Kastan et al., 1991). To establish a dose-response m relationship between (+)-anti-BPDE induced DNA [ M] 0.0 0.05 0.5 Time damage and p53 induction, excision repair-pro®cient 0 2.5 5 10 [hr] 24 0 2.5 5 0 24 human ®broblast and lymphoblastoid cells were treated 10 24 with increasing concentrations of the carcinogen (0.05 p53 to 1.0 mM).
Load more

Recommended publications
  • DNA Damage Checkpoint Dynamics Drive Cell Cycle Phase Transitions
    bioRxiv preprint doi: https://doi.org/10.1101/137307; this version posted August 4, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. DNA damage checkpoint dynamics drive cell cycle phase transitions Hui Xiao Chao1,2, Cere E. Poovey1, Ashley A. Privette1, Gavin D. Grant3,4, Hui Yan Chao1, Jeanette G. Cook3,4, and Jeremy E. Purvis1,2,4,† 1Department of Genetics 2Curriculum for Bioinformatics and Computational Biology 3Department of Biochemistry and Biophysics 4Lineberger Comprehensive Cancer Center University of North Carolina, Chapel Hill 120 Mason Farm Road Chapel Hill, NC 27599-7264 †Corresponding Author: Jeremy Purvis Genetic Medicine Building 5061, CB#7264 120 Mason Farm Road Chapel Hill, NC 27599-7264 [email protected] ABSTRACT DNA damage checkpoints are cellular mechanisms that protect the integrity of the genome during cell cycle progression. In response to genotoxic stress, these checkpoints halt cell cycle progression until the damage is repaired, allowing cells enough time to recover from damage before resuming normal proliferation. Here, we investigate the temporal dynamics of DNA damage checkpoints in individual proliferating cells by observing cell cycle phase transitions following acute DNA damage. We find that in gap phases (G1 and G2), DNA damage triggers an abrupt halt to cell cycle progression in which the duration of arrest correlates with the severity of damage. However, cells that have already progressed beyond a proposed “commitment point” within a given cell cycle phase readily transition to the next phase, revealing a relaxation of checkpoint stringency during later stages of certain cell cycle phases.
    [Show full text]
  • Epithelial Cell Death Analysis of Cell Cycle by Flow Cytometry White Paper
    Epithelial Cell Death Analysis of Cell Cycle by Flow Cytometry White Paper Authors: Savithri Balasubramanian, John Tigges, Vasilis Toxavidis, Heidi Mariani. Affiliation: Beth Israel Deaconess Medical Center Harvard Stem Cell Institute a Beckman Coulter Life Sciences: Epithelial Cell Death Analysis of Cell Cycle by Flow Cytometry PRINCIPAL OF TECHNIQUE Background: Cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication (replication). In cells without a nucleus (prokaryotic), cell cycle occurs via a process termed binary fission. In cells with a nucleus (eukaryotes), cell cycle can be divided in two brief periods: interphase—during which the cell grows, accumulating nutrients needed for mitosis and duplicating its DNA—and the mitosis (M) phase, during which the cell splits itself into two distinct cells, often called «daughter cells». Cell-division cycle is a vital process by which a single-celled fertilized egg develops into a mature organism, as well as the process by which hair, skin, blood cells, and some internal organs are renewed. Cell cycle consists of four distinct phases: G1 phase, S phase (synthesis), G2 phase (collectively known as interphase) and M phase (mitosis). M phase is itself composed of two tightly coupled processes: mitosis, in which the cell’s chromosomes are divided between the two daughter cells, and cytokinesis, in which the cell’s cytoplasm divides in half forming distinct cells. Activation of each phase is dependent on the proper progression and completion of the previous one. Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called G0 phase.
    [Show full text]
  • Cell Cycle Profile Using the Cellometer Vision Image Cytometer
    Measuring Drug Effect on Cell Cycle Profile Using the Cellometer Vision Image Cytometer Nexcelom Bioscience LLC. | 360 Merrimack Street, Building 9 | Lawrence, MA 01843 T: 978.327.5340 | F: 978.327.5341 | E: [email protected] | www.nexcelom.com 1001276 Rev. A Measuring Drug Effect on Cell Cycle Profile Using the Cellometer Vision Image Cytometer Introduction Cell cycle analysis is a commonly used assay in both clinical diagnosis and biomedical research. This analysis distinguishes cells in different phases of cell cycle and is often used to determine cellular response to drugs and biological stimulations [1, 2]. Because this assay is based on measuring the DNA content in a cell population, it can also be used to analyze DNA fragmentation during apoptosis, requiring multicolor fluorescent staining of biomarkers and DNA [3]. Recently, a small desktop imaging cytometry system (Cellometer Vision) has been developed by Nexcelom Bioscience LLC for automated cell concentration and viability measurement using bright-field (BR) and fluorescent (FL) imaging methods [4]. The system can perform rapid cell enumeration using disposable counting slides. The software utilizes a novel counting algorithm for accurate and consistent measurement of cell concentration and viability on a variety of cell types [5]. By developing fluorescent-based cell cycle assays, the Cellometer imaging cytometry can provide a quick, simple, and inexpensive alternative for biomedical research, which may be beneficial for smaller research laboratories and clinics. In this work, we demonstrate new applications of the Cellometer Vision for fluorescence- based cell population analysis as an alternative for flow cytometry. Cell cycle analysis was performed by inducing specific arrest in G0/G1, S, and G2/M phase of Jurkat cell population with aphidicolin, etoposide, and nocodazole, respectively [6-8].
    [Show full text]
  • Introduction to DNA Cell Cycle Analysis
    INTRODUCTION TO CELL CYCLE ANALYSIS Written by Dr. Peter Rabinovitch, M.D., Ph.D. Published by Phoenix Flow Systems, Inc. 6790 Top Gun St. #1 San Diego, CA. USA 92121 858 453-5095 fax 858 453-2117 The Biological Cell Cycle .........................2 DNA Analysis and the Flow Cytometric Cell Cycle ....................3 Diploid and Aneuploid DNA Contents......5 Cell Cycle Analysis of DNA Content Histograms.........................6 Fitting of Background “Debris” and Effects of Nucleus Sectioning..................9 Fitting and Correction for the Effects of Cellular or Nuclear Aggregation.........16 Quantitation of Background, Aggregates And Debris.....................................................31 Analysis of Apoptosis.............................31 Beyond Single Parameter Analysis: DNA vs. Immunofluorescence................32 Basics of DNA Cell Cycle Analysis www.phoenixflow.com Page 1 This chapter is organized into progressively more advanced sections. Feel free to skip ahead to the level appropriate for your background. THE BIOLOGICAL CELL CYCLE Reproduction of cells requires cell division, with production of two daughter cells. The most obvious cellular structure that requires duplication and division into daughter cells is the cell nucleus - the repository of the cell's genetic material, DNA. With few exceptions each cell in an organism contains the same amount of DNA and the same complement of chromosomes. Thus, cells must duplicate their allotment of DNA prior to division so that each daughter will receive the same DNA content as the parent. The cycle of increase in components (growth) and division, followed by growth and division of these daughter cells, etc., is called the cell cycle. The two most obvious features of the cell cycle are the synthesis and duplication of nuclear DNA before division, and the process of cellular division itself - mitosis.
    [Show full text]
  • Analysis of Apoptosis by Propidium Iodide Staining and Flow Cytometry
    PROTOCOL Analysis of apoptosis by propidium iodide staining and flow cytometry Carlo Riccardi & Ildo Nicoletti Department of Clinical and Experimental Medicine, School of Medicine, University of Perugia, and Foundation IBiT, 06122 Perugia, Italy. Correspondence should be addressed to C.R. ([email protected]) Published online 9 November 2006; doi:10.1038/nprot.2006.238 Since its introduction, the propidium iodide (PI) flow cytometric assay has been widely used for the evaluation of apoptosis in s different experimental models. It is based on the principle that apoptotic cells, among other typical features, are characterized by DNA fragmentation and, consequently, loss of nuclear DNA content. Use of a fluorochrome, such as PI, that is capable of binding and labeling DNA makes it possible to obtain a rapid (the protocol can be completed in about 2 h) and precise evaluation of cellular DNA content by flow cytometric analysis, and subsequent identification of hypodiploid cells. The original protocol enhanced the capacity for a rapid, quantitative measure of cell apoptosis. For this reason, since its publication, the PI assay has been widely used, as natureprotocol / demonstrated by the large number of citations of the original paper and/or the continuous use of the method in many laboratories. m o c . e r u INTRODUCTION t a n Apoptosis is a common form of cell death in eukaryotes, playing a (sub-G ) peak, which can be easily discriminated from the narrow . 1 w fundamental role during embryogenesis, in the homeostatic control peak of cells with normal (diploid) DNA content in the red w w / of tissue integrity, tumor regression and immune response devel- fluorescence channels.
    [Show full text]
  • DNA Ploidy Cell Cycle Analysis AHS – M2136
    Corporate Medical Policy DNA Ploidy Cell Cycle Analysis AHS – M2136 File Name: dna _ploidy_cell_cycle_analysis Origination: 1/2019 Last CAP Review: 3/2021 Next CAP Review: 3/2022 Last Review: 7/2021 Description of Procedure or Service S-phase fraction (SPF) is an assessment of how many cells a re a ctively synthesizing DNA (UIHC, 2016). It is used as a measure of cell proliferation, particularly for cancer (Pinto, André, & Soares, 1999). ***Note: This Medical Policy is complex and technical. For questions concerning the technical language and/or specific clinical indications for its use, please consult your physician. Policy DNA ploidy cell cycle analysis is not covered. BCBSNC will not reimburse for non-covered services or procedures. Benefits Application This medical policy relates only to the services or supplies described herein. Please refer to the Member's Benefit Booklet for availability of benefits. Member's benefits may vary according to benefit design; therefore member benefit language should be reviewed before applying the terms of this medical policy. When DNA Ploidy Cell Cycle Analysis is covered Not Applicable. When DNA Ploidy Cell Cycle Analysis is not covered Reimbursement is not allowed for the measurement of flow cytometry-derived DNA content (DNA index) or cell prolifera tive a ctivity (S-phase fraction or % S-phase) for prognostic or therapeutic purposes in the routine clinical management of cancers. Policy Guidelines Cancer is the uncontrolled growth and spread of abnormal cells and is increasingly shown to be initiated, propagated, and maintained by somatic genetic events (Johnson et al., 2014). In 2020, an expected 1,806,590 Americans will be diagnosed with new cancer cases and 606,520 Americans will die from the disease (Siegel, Miller, & Jema l, 2020).
    [Show full text]
  • Image-Based Analysis of Cell Cycle Using PI
    Application Note Image-Based Analysis of Cell Cycle Cell Cycle Using PI Cell-Based Assays Introduction: Cell Cycle prior to staining. All of the cells are then stained. Cells preparing for division will contain increasing The process of DNA replication and cell division is amounts of DNA and display proportionally known as the cell cycle. The cell cycle is typically increased fluorescence. Differences in fluorescence divided into five phases: G0, the resting phase, G1, intensity are used to determine the percentage of the normal growth phase, S, the DNA replication cells in each phase of the cell cycle. phase, G2, involving growth and preparation for mitosis, and M, mitosis. 1Collins, K., et al. (1997). Cell Cycle and Cancer. Proc. Natl. Acad. Sci. V.94, pp2776-2778. 2000 2n M Experimental Procedure 1800 G2 1600 G1 Jurkat cells were used to analyze cell cycle kinetics G0 following treatment with the cell-cycle-arresting 1400 S drugs etoposide and nocodazole. Jurkat cells were 1200 incubated with media only (control), etoposide (0.12, 0.6, and 3 µM), or nocodazole (0.004, 0.02, 0.1 1000 µg/mL) for 24 hours. Control and drug-treated cells 800 were fixed then stained with propidium iodide. 6 600 For each sample, 20 µl of cell sample (at ~4 x 10 4n cells / mL) was loaded into a Cellometer Imaging 400 Chamber, inserted into the Vision CBA Analysis 200 System, and imaged in both bright field and fluorescence. The fluorescence intensity for each cell 0 was measured. 0 2000 4000 6000 8000 10000 Results There are at least two types of cell cycle control mechanisms.
    [Show full text]
  • Multiplex Cell Fate Tracking by Flow Cytometry
    Protocol Multiplex Cell Fate Tracking by Flow Cytometry Marta Rodríguez-Martínez 1,*, Stephanie A. Hills 2, John F. X. Diffley 2 and Jesper Q. Svejstrup 1,* 1 Mechanisms of Transcription Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK 2 Chromosome Replication Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK * Correspondence: [email protected] (M.R.-M.); [email protected] (J.Q.S.) Received: 18 June 2020; Accepted: 15 July 2020; Published: 17 July 2020 Abstract: Measuring differences in cell cycle progression is often essential to understand cell behavior under different conditions, treatments and environmental changes. Cell synchronization is widely used for this purpose, but unfortunately, there are many cases where synchronization is not an option. Many cell lines, patient samples or primary cells cannot be synchronized, and most synchronization methods involve exposing the cells to stress, which makes the method incompatible with the study of stress responses such as DNA damage. The use of dual-pulse labelling using EdU and BrdU can potentially overcome these problems, but the need for individual sample processing may introduce a great variability in the results and their interpretation. Here, we describe a method to analyze cell proliferation and cell cycle progression by double staining with thymidine analogues in combination with fluorescent cell barcoding, which allows one to multiplex the study and reduces the variability due to individual sample staining, reducing also the cost of the experiment. Keywords: BrdU; EdU; fluorescent cell barcoding 1. Introduction Assessing cell proliferation and cell cycle distribution is often at the basis of cell behavior studies.
    [Show full text]
  • Blue Intensity Matters for Cell Cycle Profiling in Fluorescence DAPI
    Laboratory Investigation (2017) 97, 615–625 © 2017 USCAP, Inc All rights reserved 0023-6837/17 Blue intensity matters for cell cycle profiling in fluorescence DAPI-stained images Anabela Ferro1,2, Tânia Mestre3, Patrícia Carneiro1,2, Ivan Sahumbaiev3, Raquel Seruca1,2,4 and João M Sanches3 In the past decades, there has been an amazing progress in the understanding of the molecular mechanisms of the cell cycle. This has been possible largely due to a better conceptualization of the cycle itself, but also as a consequence of technological advances. Herein, we propose a new fluorescence image-based framework targeted at the identification and segmentation of stained nuclei with the purpose to determine DNA content in distinct cell cycle stages. The method is based on discriminative features, such as total intensity and area, retrieved from in situ stained nuclei by fluorescence microscopy, allowing the determination of the cell cycle phase of both single and sub-population of cells. The analysis framework was built on a modified k-means clustering strategy and refined with a Gaussian mixture model classifier, which enabled the definition of highly accurate classification clusters corresponding to G1, S and G2 phases. Using the information retrieved from area and fluorescence total intensity, the modified k-means (k = 3) cluster imaging framework classified 64.7% of the imaged nuclei, as being at G1 phase, 12.0% at G2 phase and 23.2% at S phase. Performance of the imaging framework was ascertained with normal murine mammary gland cells constitutively expressing the Fucci2 technology, exhibiting an overall sensitivity of 94.0%. Further, the results indicate that the imaging framework has a robust capacity to both identify a given DAPI-stained nucleus to its correct cell cycle phase, as well as to determine, with very high probability, true negatives.
    [Show full text]
  • Downregulation of Cell Cycle and Checkpoint Genes by Class I HDAC Inhibitors Limits Synergism with G2/M Checkpoint Inhibitor MK-1775 in Bladder Cancer Cells
    G C A T T A C G G C A T genes Article Downregulation of Cell Cycle and Checkpoint Genes by Class I HDAC Inhibitors Limits Synergism with G2/M Checkpoint Inhibitor MK-1775 in Bladder Cancer Cells Michèle J. Hoffmann * , Sarah Meneceur †, Katrin Hommel †, Wolfgang A. Schulz and Günter Niegisch Department of Urology, Medical Faculty, Heinrich-Heine-University Duesseldorf, Moorenstr. 5, 40225 Duesseldorf, Germany; [email protected] (S.M.); [email protected] (K.H.); [email protected] (W.A.S.); [email protected] (G.N.) * Correspondence: [email protected]; Tel.: +49-21-1811-5847 † Contributed equally. Abstract: Since genes encoding epigenetic regulators are often mutated or deregulated in urothelial carcinoma (UC), they represent promising therapeutic targets. Specifically, inhibition of Class-I histone deacetylase (HDAC) isoenzymes induces cell death in UC cell lines (UCC) and, in contrast to other cancer types, cell cycle arrest in G2/M. Here, we investigated whether mutations in cell cycle genes contribute to G2/M rather than G1 arrest, identified the precise point of arrest and clarified the function of individual HDAC Class-I isoenzymes. Database analyses of UC tissues and cell lines revealed mutations in G1/S, but not G2/M checkpoint regulators. Using class I-specific HDAC inhibitors (HDACi) with different isoenzyme specificity (Romidepsin, Entinostat, RGFP966), cell cycle arrest was shown to occur at the G2/M transition and to depend on inhibition of HDAC1/2 Citation: Hoffmann, M.J.; rather than HDAC3. Since HDAC1/2 inhibition caused cell-type-specific downregulation of genes Meneceur, S.; Hommel, K.; Schulz, encoding G2/M regulators, the WEE1 inhibitor MK-1775 could not overcome G2/M checkpoint W.A.; Niegisch, G.
    [Show full text]
  • Flow Cytometry (Ploidy Determination, Cell Cycle Analysis, DNA Content Per Nucleus)
    Medicago truncatula handbook version November 2006 Flow cytometry (ploidy determination, cell cycle analysis, DNA content per nucleus) Sergio J. Ochatt INRA, C.R. Dijon, Unité de Génétique et Ecophysiologie des Légumineuses, BP 86510, 21065 Dijonn Cedex France – [email protected] Table of contents 1. General considerations and theory of flow cytometry 2. Optics, measurements, fluorescent dyes, signal processing 3. Data collection and analysis 4. Uses of flow cytometry for the characterization of Medicago truncatula tissues and plants 5. Other uses and future of flow cytometry in Medicago truncatula 6. References 1. General considerations and theory of flow cytometry During recent years, flow cytometry has established itself as a useful, quick novel method to determine efficiently, reproducibly and at a reduced cost per sample the relative nuclear DNA content and ploidy level of a large number of species (plants and animals), that has also been used to sort cells with different traits from a mixed cell population (e.g. as when separating heterokaryons from parental protoplasts in protoplast fusion experiments for somatic hybrid production) and, more recently, for the analysis of the composition in various chemical components of different tissues (such as apoptotic markers bound to cell compartments). This chapter will focus on the protocols and uses of flow cytometry applied to Medicago truncatula. Basically, a flow cytometer is a fluorescence microscope which analyses moving particles in a suspension. These are excited by a source of light (U.V. or laser) and in turn emit an epi- fluorescence which is filtered through a series of dichroic mirrors (Figure 1). Then, the in- built programme of the equipment converts these signals into a graph plotting the intensity of the epi-fluorescence emitted against the count of cells emitting it at a time given.
    [Show full text]
  • Apoptosis, Cell Cycle, and Cell Proliferation
    Tools for Assessing Cell Events Apoptosis, Cell Cycle, and Cell Proliferation Life, Death, and Cell Proliferation The balance of cell proliferation and apoptosis is important for both development and normal tissue homeostasis. Cell proliferation is an increase in the number of cells as a result of growth and division. Cell proliferation is regulated by the cell cycle, which is divided into a series of phases. Apoptosis, or programmed cell death, results in controlled self-destruction. Several methods have been developed to assess apoptosis, cell cycle, and cell proliferation. BD Biosciences offers a complete portfolio of reagents and tools to allow exploration of the cellular features of these processes. Over the years, multicolor flow cytometry has become essential in the study of apoptosis, cell cycle, and cell proliferation. Success of the technology results from its ability to monitor these processes along with other cellular events, such as protein phosphorylation or cytokine secretion, within heterogeneous cell populations. BD Biosciences continues to innovate in this area with new products such as the BD Horizon™ cell proliferation dyes (VPD450 and CFSE), BD Horizon™ fixable viability stains, and popular reagents such as antibodies to cleaved PARP and caspase-3 available in new formats and for different types of applications. In addition to flow cytometry products, BD Biosciences carries a broad portfolio of reagents for determination and detection of apoptotic and proliferative events by immunohistochemistry, cell imaging, and Western blot. As part of our commitment to maximize scientific results, BD Biosciences provides a variety of tools to assist customers in their experimental setup and analysis. These include a decision tree to guide in the selection of the most suitable methods for a specific study.
    [Show full text]