Cell Cycle Profile Using the Cellometer Vision Image Cytometer
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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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. -
DNA Damage Activates a Spatially Distinct Late Cytoplasmic Cell-Cycle Checkpoint Network Controlled by MK2-Mediated RNA Stabilization
DNA Damage Activates a Spatially Distinct Late Cytoplasmic Cell-Cycle Checkpoint Network Controlled by MK2-Mediated RNA Stabilization The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Reinhardt, H. Christian, Pia Hasskamp, Ingolf Schmedding, Sandra Morandell, Marcel A.T.M. van Vugt, XiaoZhe Wang, Rune Linding, et al. “DNA Damage Activates a Spatially Distinct Late Cytoplasmic Cell-Cycle Checkpoint Network Controlled by MK2-Mediated RNA Stabilization.” Molecular Cell 40, no. 1 (October 2010): 34–49.© 2010 Elsevier Inc. As Published http://dx.doi.org/10.1016/j.molcel.2010.09.018 Publisher Elsevier B.V. Version Final published version Citable link http://hdl.handle.net/1721.1/85107 Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. Molecular Cell Article DNA Damage Activates a Spatially Distinct Late Cytoplasmic Cell-Cycle Checkpoint Network Controlled by MK2-Mediated RNA Stabilization H. Christian Reinhardt,1,6,7,8 Pia Hasskamp,1,10,11 Ingolf Schmedding,1,10,11 Sandra Morandell,1 Marcel A.T.M. van Vugt,5 XiaoZhe Wang,9 Rune Linding,4 Shao-En Ong,2 David Weaver,9 Steven A. Carr,2 and Michael B. Yaffe1,2,3,* 1David H. Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02132, USA 2Broad Institute of MIT and Harvard, Cambridge, MA 02132, USA 3Center for Cell Decision Processes, -
Dansylated Estramustine, a Fluorescent Probe for Studies Of
Proc. Natl. Acad. Sci. USA Vol. 82, pp. 8483-8487, December 1985 Cell Biology Dansylated estramustine, a fluorescent probe for studies of estramustine uptake and identification of intracellular targets (antimitotic drug/microtubules/microtubule-associated proteins) MARK E. STEARNS*t, DONALD P. JENKINS*, AND KENNETH D. TEWt *Department of Anatomy, Georgetown University Schools of Medicine and Dentistry, Washington, DC 20007; and tDepartments of Medicine and Pharmacology, Lombardi Cancer Center, Georgetown University, Washington, DC 20007 Communicated by Keith R. Porter, July 8, 1985 ABSTRACT Fluorescence-microscopic studies with of DnsEM was measured by their effect on the colony- dansylated estramustine (DnsEM) has permitted investigation forming ability of DU 145 human prostatic carcinoma cells. of the mechanism of estramustine (EM) uptake in live human The results presented here show that DnsEM is as cytotoxic prostatic tumor cells (DU 145). DnsEM appeared to enter cells to cells as EM and can be used as a direct probe for analysis rapidly at the peripheral cell margins. A progressive increase of EM's mechanism of action in live cells. in fluorescence was observed until the perinuclear material and cytoplasm were labeled brightly and the nucleoplasm was METHODS labeled faintly. Light microscopy showed that DnsEM is assimilated first in preexisting vesicles and then in numerous Dansylation of EM. The synthesis of DnsEM involved the newly created vesicles that accumulate in the cytoplasm and dansylation reaction as first introduced by Gray (9) and around the nucleus. Colony-forming assays showed EM and modified by Jenkins (10). Fifty milligrams of 5- DnsEM to be equally cytotoxic to cultured DU 145 cells. -
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. -
DNA Damage Response During Mitosis Induces Whole Chromosome Mis-Segregation Samuel F
Supplementary information for: DNA damage response during mitosis induces whole chromosome mis-segregation Samuel F. Bakhoum1,2,5,6,*, Lilian Kabeche1,2,6, John P. Murnane3, Bassem I. Zaki4, Duane A. Compton1,2,* SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure S1. A, Example of an anaphase spindle with lagging chromosomes and the number of lagging chromosomes per anaphase spindle as a function of IR dose. B, Example of a multipolar anaphase spindle and the percentage of anaphase spindles with acentric chromatin as a function of IR dose. C, Example of an anaphase spindle with acentric chromatin fragments and the number of multipolar mitoses as a function of IR dose. Circles represent mean ± s.e.m, n = 150 cells, 3 experiments. Representative mitotic spindles are stained for Hec1 to denote kinetochores (green), DNA (blue), and microtubules (red). Scale bar 5-μm. Supplementary Figure S2. IR does not dramatically alter spindle geometry of irradiated mitotic cells. Examples of monopolar (A) and multipolar (B) Spindles of U251 cells stained for kinetochores (green), DNA (blue), and microtubules (red). Scale bar, 5-μm. Graphs represent the percentage of monopolar (A) and multipolar (B) spindles as a function of IR dose. Circles represent mean ± s.e.m, n = 150 cells, 3 experiments. Supplementary Figure S3. IR does not significantly affect sister chromatid cohesion. A, Experimental schema of assessing sister chromatid cohesion after IR exposure. Noc, Nocodazole. Bar graphs show percentage of mitotic spreads containing chromosomes with intact sister- chromatid cohesion or uncohesed chromosomes as a function of IR dose. n>300 mitotic spreads, p>0.14, χ2-test for all cell lines B, Experimental schema of assessing sister chromatid cohesion after Doxorubicin exposure. -
Asymmetric Mitosis: Unequal Segregation of Proteins Destined for Degradation
Asymmetric mitosis: Unequal segregation of proteins destined for degradation Luis C. Fuentealba*, Edward Eivers*, Douglas Geissert, Vincent Taelman, and E. M. De Robertis† Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662 Communicated by Joseph G. Gall, Carnegie Institution of Washington, Baltimore, MD, March 27, 2008 (received for review February 21, 2008) Mitotic cell division ensures that two daughter somatic cells inherit identical genetic material. Previous work has shown that signaling by the Smad1 transcription factor is terminated by polyubiquiti- nylation and proteasomal degradation after essential phosphory- lations by MAPK and glycogen synthase kinase 3 (GSK3). Here, we show that, unexpectedly, proteins specifically targeted for protea- somal degradation are inherited preferentially by one mitotic daughter during somatic cell division. Experiments with dividing human embryonic stem cells and other mammalian cultured cell lines demonstrated that in many supposedly equal mitoses the segregation of proteins destined for degradation (Smad1 phos- phorylated by MAPK and GSK3, phospho--catenin, and total polyubiquitinylated proteins) was asymmetric. Transport of pSmad1 targeted for degradation to the centrosome required functional microtubules. In vivo, an antibody specific for Mad phosphorylated by MAPK showed that this antigen was associated preferentially with one of the two centrosomes in Drosophila embryos at cellular blastoderm stage. We propose that this re- markable cellular property may be explained by the asymmetric inheritance of peripheral centrosomal proteins when centrioles separate and migrate to opposite poles of the cell, so that one mitotic daughter remains pristine. We conclude that many mitotic divisions are unequal, unlike what was previously thought. -
108654V1.Full.Pdf
bioRxiv preprint doi: https://doi.org/10.1101/108654; this version posted February 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Evaluation of anticancer and anti-mitotic properties of quinazoline and quinazolino-benzothiadiazine derivatives Thoukhir B. Shaik,a,b M. Shaheer Malik,c Zaki S. Seddigid, Sunitha R Routhu,a Ahmed Kamala* a Department of Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology, Hyderabad-500007, India b Department of Biotechnology, Acharya Nagarjuna university, Nagarjuna nagar, Guntur, A.P, India c Science and Technology Unit, Umm Al-Qura University, 21955 Makkah, Saudi Arabia d Department of Environmental Health, College of Public Health and Health informatics, Umm Al-Qura University. *Corresponding author - Phone: +91 40 27193157; Fax: +91-40-27193189. E-mail: [email protected]. Summary: The present study describes the exploration of small molecules based on heterocyclic scaffolds for tubulin target based development of anticancer agents. Abstract Cancer is one of the major health and social-economic problems despite considerable progress in its early diagnosis and treatment. Owing to the emergence and increase of multi drug resistance to various conventional drugs, and the continuing importance on health-care expenditure, many researchers have focused to develop novel and effective anticancer compounds. In the present study, a series of in-house synthesized quinazoline and quinazolino-benzothiadiazine derivatives were investigated for their anticancer efficacy against a panel of five cancer (DU145, MCF7, HepG2, SKOV3 and MDA-MB-231) and one normal (MRC5) cell lines. -
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. -
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. -
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). -
Inhibition of P34cdc2kinase Activation, P34cdc2tyrosine Dephosphorylation, and Mitotic Progression in Chinese Hamster Ovary Cells Exposed to Etoposide1
[CANCER RESEARCH 52. 1817-1822. April 1. 1992] Inhibition of p34cdc2Kinase Activation, p34cdc2Tyrosine Dephosphorylation, and Mitotic Progression in Chinese Hamster Ovary Cells Exposed to Etoposide1 Richard B. Lock2 The J. Graham Brown Cancer Center, Departments of Medicine and Biochemistry, University of Louisville, Louisville, Kentucky 40292 ABSTRACT which is regulated by a complex series of phosphorylation/ II.M"''- kinase, an enzyme essential for mitosis in mammalian cells, dephosphorylation reactions (17, 18) and by its association with cyclin B (19). The high degree of conservation between human may play a role in etoposide-induced G2 phase arrest of Chinese hamster and Schizosaccharomyces pombe p34tdc2 proteins [63% amino ovary cells. In this study, etoposide is shown to cause inhibition of a specific p3-4"''-'kinase activation pathway, that of tyrosine dephosphoryl- acid homology (20)] and its identification as a component of ation. Exposure of asynchronous!}' dividing cells to etoposide caused a both maturation promoting factor and the growth-associated simultaneous rapid decline of both mitotic index and p.V4"''-'kinase histone HI kinase (21-24) suggest a central role for p34cdc2in activity, suggesting that the kinase was not activated and that the arrest mitotic progression. However, more direct evidence for its role point was in late »...phase. Using synchronized cells, p34cdc2kinase in mitosis of mammalian cells was obtained by the inhibition exhibited maximal activity at the Gz/M transition. Activation of the of cell division following microinjection of affinity-purified kinase and the onset of mitosis were accompanied by increased electro- p34«ic2antibodiesinto serum-stimulated rat fibroblasts (25) and phoretic mobility and tyrosine dephosphorylation of the p34"'' ' protein. -
(12) Patent Application Publication (10) Pub. No.: US 2016/0244766A1 Bettencourt Et Al
US 20160244766A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0244766A1 Bettencourt et al. (43) Pub. Date: Aug. 25, 2016 (54) COMPOSITIONS AND METHODS FOR Related U.S. Application Data INHIBITING EXPRESSION OF THE ALAS1 (60) Provisional application No. 61/983,720, filed on Apr. GENE 24, 2014, provisional application No. 61/887,288, (71) Applicants: ALNYLAM PHARMACEUTICALS, filed on Oct. 4, 2013. INC., Cambridge, MA (US); ICAHN SCHOOL OF MEDCINEAT MOUNTSINAI, New York, NY (US) Publication Classification (72) Inventors: Brian Bettencourt, Groton, MA (US); (51) Int. Cl. Kevin Fitzgerald, Brookline, MA (US); CI2N IS/II3 (2006.01) William Querbes, Cambridge, MA CI2O I/68 (2006.01) (US); Robert J. Desnick, New York, NY A619/00 (2006.01) (US); Makiko Yasuda, New York, NY (52) U.S. Cl. (US) CPC .......... CI2N 15/I 137 (2013.01); A61 K9/0019 (2013.01); C12O 1/6876 (2013.01); C12N (73) Assignees: ALNYLAM PHARMACEUTICALS, 23 10/14 (2013.01); C12N 23.10/321 (2013.01); INC., Cambridge, MA (US); ICAHN CI2N 23.10/315 (2013.01); C12N 23.10/351 SCHOOL OF MEDCINEAT (2013.01) MOUNTSINAI, New York, NY (US) (21) Appl. No.: 15/027,176 (57) ABSTRACT (22) PCT Filed: Oct. 3, 2014 The invention relates to double-stranded ribonucleic acid (86). PCT No.: PCT/US2O14/05916O (dsRNA) compositions targeting the ALAS1 gene, and meth S371 (c)(1), ods of using Such dsRNA compositions to alter (e.g., inhibit) (2) Date: Apr. 4, 2016 expression of ALAS1. Patent Application Publication Aug. 25, 2016 Sheet 1 of 61 US 2016/024476.6 A1 Mitochondria