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WO1998031787A1 - DEPISTAGE DE L'APOPTOSE $i(IN VIVO) - Google Patents

DEPISTAGE DE L'APOPTOSE $i(IN VIVO) Download PDF

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Publication number
WO1998031787A1
WO1998031787A1 PCT/US1998/001322 US9801322W WO9831787A1 WO 1998031787 A1 WO1998031787 A1 WO 1998031787A1 US 9801322 W US9801322 W US 9801322W WO 9831787 A1 WO9831787 A1 WO 9831787A1
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WO
WIPO (PCT)
Prior art keywords
embryo
cell death
test
compound
embryos
Prior art date
Application number
PCT/US1998/001322
Other languages
English (en)
Inventor
Lee L. Rubin
Christine De Leon Gatchalian
Original Assignee
Eisai Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eisai Co., Ltd. filed Critical Eisai Co., Ltd.
Priority to EP98903681A priority Critical patent/EP0964915A4/fr
Priority to JP53474098A priority patent/JP2001514738A/ja
Publication of WO1998031787A1 publication Critical patent/WO1998031787A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Definitions

  • This invention relates to apoptosis, which is associated with physiological or programmed cell death (PCD).
  • PCD programmed cell death
  • Apoptosis occurs in embryonic development, hormone deprivation of endocrine or other hormone- dependent or sensitive cells, cells responding to mild thermal or metabolic stress, and normal tissue turnover.
  • Compounds which affect PCD are potentially useful as therapeutics to treat a wide range of medical disorders, including cancer, AIDS, autoimmune disorders such as rheumatoid arthritis, and neurodegenerative diseases such as multiple sclerosis.
  • the first step is the transmission of information about the status of the cell from outside the cell to the cytoplasm, or from the cell membrane to the nucleus. This information may follow the appearance of an apoptosis-inducing factor such as the Fas ligand, or the disappearance of a survival-promoting factor, such as nerve growth factor (for some types of neurons).
  • the second step is gene transcription and translation into protein. This second step can be blocked by compounds such as actinomycin D or cycloheximide, compounds which block transcription or translation. In this manner, these agents block some types of apoptosis.
  • the effectors of cell death are activated; these effectors include (in many types of cells) cysteine proteases which cleave after aspartic acid residues; these agents are now termed caspases.
  • Caspase inhibitors can interrupt the programmed cell death chain of processes, by blocking this third step.
  • One aspect of the invention features a method of testing a compound for the ability to affect cell death.
  • This method includes: a) providing an osteichthes embryo which is translucent or transparent ( i.e., optically clear), b) contacting the compound with the clear embryo, and c) visually observing the pattern or extent of cell death in the embryo.
  • the visual observation can be accomplished by a variety of methods known to those in the art of cytology, including labeling cells which undergo programmed cell death or apoptosis in the living embryo for visualization microscopically.
  • one method of labeling is carried out by terminal deoxynucleotide transferase dUTP nick labeling.
  • Another aspect of the invention features a method which includes the steps of: a) providing an animal (e.g., a vertebrate, such as a fish, and preferably a zebrafish, Danio rerio) which, at an embryonic stage, has been contacted with an agent which increases apoptosis in cells of the animal, b) contacting the animal with the test compound, and c) determining whether the compound affects cell death in the animal.
  • the contacting step b) is carried out with the animal at an embryonic stage.
  • the determining step c) may include determining whether the compound affects cell death in a Rohon-Beard neuron.
  • the determining step c) may include determining whether the compound decreases or inhibits apoptosis (programmed cell death) in a Rohon-Beard neuron that has been contacted with an agent which increases apoptosis. A compound which decreases or inhibits apoptosis is said to have rescued a cell.
  • the determining step c) can also include using an antibody to label a cell, such as a Rohon-Beard cell, which undergoes cell death.
  • the invention also includes a test vertebrate embryo useful for screening compounds for the ability to affect cell death.
  • the test embryo is prepared by a) providing a vertebrate embryo (e.g., a zebrafish), and b) contacting the embryo with an agent (e.g., stauro-sporine) which increases apoptosis in cells of the animal.
  • This test embryo can be used in the methods disclosed herein for testing a compound for the ability to affect cell death.
  • test embryo of the invention can also be used in a variety of methods for obtaining information on cellular processes.
  • the cellular process which is investigated is selected from the group consisting of: a) neuronal cell function, b) neuronal connectivity, c) cell development, d) tissue development, and e) organ development.
  • This method includes a) providing at least two embryos, namely a test zebrafish embryo and a control zebrafish embryo, b) subjecting a test embryo to test conditions, and c) visually observing differences in cells of the test and control embryos.
  • the control embryo is treated such that the differences in visual observation result from application of the test conditions.
  • Test conditions include mutagenesis-inducing conditions (e.g., radiation or chemical agents), apoptosis-inducing or increasing agents, apoptosis-inhibiting or decreasing compounds, labeling or staining of one or more types of cells, marker compounds which label or indicate the present of a metabolite, and ligands for a receptor.
  • mutagenesis-inducing conditions e.g., radiation or chemical agents
  • apoptosis-inducing or increasing agents e.g., apoptosis-inhibiting or decreasing compounds
  • labeling or staining of one or more types of cells e.g., marker compounds which label or indicate the present of a metabolite, and ligands for a receptor.
  • the test and control zebrafish embryos are pre-treated with an agent which affects programmed cell death.
  • the test conditions include a test compound (for example, either a cell death inhibitor, such as a caspase inhibitor, or a compound which increases cell death), and the observing step includes observing whether the test compound inhibits cell death in the test embryo, in other words, whether the test compounds rescues the test embryo.
  • the agent can be a protein kinase inhibitor, such as staurosporine.
  • the observing step can include observing neurons of the embryos for an interval of time sufficient to determine whether neurons in the test embryo which are saved from cell death develop or function normally.
  • the observing step can include observing or comparing Rohon-Beard neurons in the test and control embryos.
  • the invention also features a method for testing a compound for the ability to affect expression of a gene whose expression affects cell death, said method comprising the steps of: (a) providing an osteichthes test embryo which is translucent or transparent, wherein the gene is expressed in the embryo, wherein the gene either is not normally expressed in the osteichthes (embryo or adult), or is normally expressed in the osteichthes (embryo or adult) at a lower level than in the test embryo, (b) contacting the compound with the embryo, and (c) visually observing the pattern or extent of cell death in the embryo.
  • the osteichthes embryo can be a zebrafish embryo.
  • the gene can be a eukaryotic gene encoding a protein which inhibits cell death, such as a gene which encodes bcl-2, and is over-expressed in the embryo.
  • the invention in part is based on the observation that cells in the zebrafish embryo undergo apoptosis during normal development.
  • the dying cells can be identified by simply viewing the whole live embryo (e.g., using Nomarski optics, or by a vital stain such as acridine orange).
  • the dying cells can then be viewed and analyzed histologically, by staining the entire embryo using a method (the TUNEL method, explained in detail below), which detects DNA in the process of fragmenting during cell death.
  • the pattern of TUNEL-positive cells at about 24 hours of development is easily discernable and very reproducible.
  • the present studies have indicated that some of the dying cells are neurons, including Rohon-Beard sensory neurons, and thus the method provides an important tool for studying neuronal apoptosis.
  • Zebrafish are well suited for use in the method of the invention because of their rapid development, large brood size, external fertilization and, most importantly, the optical clarity of their embryos. Because the embryo is clear, apoptotic cells can be detected under the light microscope as highly refractive bodies, or stained using vital dyes such as acridine orange.
  • the amount of normal cell death in the developing fish embryo, as in other vertebrate embryos, is relatively small at any point in time.
  • the amount of cell death in these embryos can be greatly increased by briefly exposing the live embryos to a protein kinase inhibitor such as staurosporine.
  • a protein kinase inhibitor such as staurosporine.
  • Embryos can be treated with agents which inhibit apoptosis, e.g., caspase inhibitors, and the "saved" neurons can then be examined to determine whether they develop or function normally. Neural connectivity can also be observed using the system of the invention, as can cell, tissue, and embryonic development.
  • agents which inhibit apoptosis e.g., caspase inhibitors
  • caspase inhibitors e.g., caspase inhibitors
  • the invention can also be used (a) to test the effects of expression of a foreign gene whose expression ordinarily affects cell death, or (b) to screen for inhibitors of either a foreign gene whose expression ordinarily affects cell death or an endogenous gene that affects cell death.
  • the method involves the steps of: (a) providing an osteichthes (preferably zebrafish) test embryo which is translucent or transparent, and in which the cell death-affecting gene is expressed; the gene either is one which is not normally expressed in the species of which the embryo is a member, or is normally expressed at lower levels, and is over-expressed in the test embryo.
  • the test compound is contacted with the test embryo, and changes in the pattern or extent of cell death in the embryo brought about by the compound indicate its effect on cell death.
  • Compounds which inhibit cell death-blocking compounds are useful, e.g., as anti-tumor adjuvant therapeutics.
  • Any of the known genes which express proteins which inhibit or accelerate cell death can be used; one example is the bcl-2 gene, the overexpression of which can be expected to block apoptosis.
  • the embryo can be caused to express or over-express the cell death-affecting gene either via a transgenesis (the gene is inserted into embryo by standard microinjection techniques), or cells which have been transfected ex-vivo with the gene can be transplanted into the embryo.
  • the human sequence for bcl-2 was published in Cleary, M.L.
  • the invention offers ease of use compared to other vertebrate embryonic systems (such as rodent or avian), while the process of cell death is similar or identical.
  • compounds identified according to the invention which are effective anti-apoptotic agents are likely to be effective in mammalian systems as well.
  • a further advantage of the invention is that the embryos are treated while they are still alive and developing, and it is therefore possible to determine if cells that are prevented from dying develop normally, an important consideration in screening anti-cell death drug candidates.
  • the embryos used in the methods of the invention preferably are clear and large enough for easy microscopic visualization .
  • a number of osteichthes (bony fish) species are suitable, e.g., Medaka, Giant rerio.
  • the preferred species is Danio rerio, the zebrafish, which has large, clear, easily- visualized embryos, and which reproduces in large numbers.
  • the assay is carried out as follows. Ten zebrafish embryos, at 90% epiboly (about 9 hours), are placed in a tank containing standard fish H 2 0 (60 mg Instant Ocean/liter distilled water) and varying concentrations (0.001-1000 ⁇ M) of test compound. The embryos are incubated with the test compound overnight (until they reach about 22 hours of development), and examined in vitro and then prepared as follows for histologic examination.
  • the embryos are either acridine orange labelled or dechorionated and fixed in 4% paraformaldehyde made up in PBS. They can then be viewed with Nomarski optics or processed for TUNEL.
  • TUNEL terminal deoxynucleotide transferase
  • terminal deoxynucleotide transferase DNA polymerases target the multitude of new 3'OH ends generated by DNA fragmentation in both early stage and mo ⁇ hologically identifiable nuclei and apoptotic bodies.
  • TdT polymerases add digoxigenin-dUTP to the 3'OH ends of the PCD fragmented DNA, which can then be detected by anti-digoxigenin alkaline phosphase conjugate, and stained with substrate.
  • the TUNEL staining method is carried out as follows: Embryos are fixed and washed in PBT buffer. They are then treated with proteinase K, washed, and postfixed in paraformaldehyde. They are rinsed, fixed in methanol/acetic acid, rinsed again, and then subjected to the terminal transferase reaction. Embryos are incubated with terminal deoxytransferase (TdT) using reagents and conditions provided in the Apoptosis Detection Kit supplied by Oncor, Inc. Enzyme incubation is overnight at 37°C . The reaction is stopped and the embryos are rinsed in PBT.
  • TdT terminal deoxytransferase
  • Stop reaction by washing in working strength stop/wash buffer (prepare working strength stop/wash buffer by mixing 1 ml stop/wash buffer (S7100-4) with 17 ml distilled water) for 3 hours at 37°C. 2. Wash 3 x 5 minutes in PBT.
  • Diffuse PCD was observed in most regions during development, with concentrated regions of PCD which were localized spatially and temporally. The earliest PCD was detected in a few cells at 75% epipboly. Up to 12 somites, there is diffuse, seemingly random PCD, which then begins to concentrate toward the brain and tailbud. From 19 hours, a localized pattern of PCD was found in the lens and cornea of the eye, the otocyst, the cloacal opening, the olfactory placode, and portions of the nervous system, including the dorsoventral spinal cord. Diffuse apoptosis is believed to occur amidst tightly packed cells in order to allow their free movement during periods of gradual morphogenesis.
  • PCD may permit more radical mo ⁇ hogenesis.
  • localized cell death in the nervous system may clear the way for outgrowing axons; concentrated PCD in the olfactory placode coincides with the time that axons are exiting the placode and growing toward the telencephalon.
  • PCD Increase with Kinase Inhibitors and Decrease with Caspase Inhibitors
  • the protein kinase inhibitor staurosporine is used at a concentration of between 10 ⁇ M and 100 ⁇ M. Embryos at the 22 hour stage are incubated for 120 minutes, washed in PBS, and either labelled with acridine orange or fixed and viewed in Nomarski optics or processed for TUNEL.
  • EXAMPLE 1 An assay according to the invention was carried out with staurosporine-pretreated zebrafish embryos, using, as a test compound, a tripeptide inhibitor of ICE-like proteases, carboxybenzoyl Val-Ala-Asp fluoromethylketone (zVADfmk).
  • zVADfmk carboxybenzoyl Val-Ala-Asp fluoromethylketone
  • zFAfmk carboxybenzoyl Phe-Ala- fluoromethylketone
  • Embryos were fixed in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) for 4 hours, washed in PBS, incubated for 3 hours in PBS plus 3% goat serum and 0.1% Triton-X-100 (Sigma), and incubated overnight in a solution of monoclonal HNK-1 antibody (Sigma) diluted 1 : 1000. This was followed by washing in PBS plus 0.1% Triton (PBST) for at least one hour and incubating overnight in HRP-conjugated goat anti-mouse IgM diluted in PBST plus 1% goat serum.
  • PBS phosphate buffered saline
  • Double labeling cells with HNK- 1 antibody and TUNEL involved first HNK-1 antibody staining followed by TUNEL using the methods sequentially as already described.

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  • Biochemistry (AREA)
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Abstract

Un animal est utilisé pour tester des composés ayant la capacité d'influer sur la mort cellulaire. Ce procédé consiste à: a) produire un embryon d'animal, b) mettre l'embryon en contact avec un inhibiteur de protéine kinase de façon à accroître l'apoptose dans les cellules de l'animal. L'invention porte également sur les procédés d'utilisation de l'animal de test.
PCT/US1998/001322 1997-01-22 1998-01-22 DEPISTAGE DE L'APOPTOSE $i(IN VIVO) WO1998031787A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP98903681A EP0964915A4 (fr) 1997-01-22 1998-01-22 DEPISTAGE DE L'APOPTOSE $i(IN VIVO)
JP53474098A JP2001514738A (ja) 1997-01-22 1998-01-22 生体内アポプトシススクリーニング

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9701245.4 1997-01-22
GBGB9701245.4A GB9701245D0 (en) 1997-01-22 1997-01-22 Method of screening compounds

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WO1998031787A1 true WO1998031787A1 (fr) 1998-07-23

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042606A1 (fr) * 1998-02-23 1999-08-26 Phylonix Pharmaceuticals, Inc. Procedes de criblage destines a determiner l'activite d'agents au moyen de poissons teleosteens
EP1160570A1 (fr) * 2000-05-27 2001-12-05 ARTEMIS Pharmaceuticals GmbH Methode pour identifier des agents et des genes qui influencent la function cardiovasculaire
WO2001092874A1 (fr) * 2000-05-27 2001-12-06 Artemis Pharmaceuticals Gmbh Methode d'identification d'agents et de genes qui influencent la fonction cardio-vasculaire
WO2001040503A3 (fr) * 1999-12-06 2002-07-18 Axxima Pharmaceuticals Ag Methode d"identification et de quantification des inhibiteurs de kinase
US6761876B2 (en) 1998-12-01 2004-07-13 Phylonix Pharmaceuticals, Inc. Methods for introducing helterologous cells into fish
EP1379868A4 (fr) * 2001-04-04 2007-10-03 Zygogen Llc Modeles de poisson zebre transgenique destines a des maladies neurodegeneratives
US7482507B2 (en) 1998-02-23 2009-01-27 Phylonix Pharmaceuticals, Inc. Methods of screening agents for activity using teleosts
US7767880B2 (en) 2006-09-01 2010-08-03 Phylonix Pharmaceuticals, Inc. Methods of screening agents for activity using teleosts
WO2017011653A1 (fr) * 2015-07-15 2017-01-19 The University Of Florida Research Foundation, Inc. Procédés de décellularisation d'un tissu

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AMSTERDAM A., ET AL.: "REQUIREMENTS FOR GREEN FLUORESCENT PROTEIN DETECTION IN TRANSGENIC ZEBRAFISH EMBRYOS.", GENE., ELSEVIER, AMSTERDAM., NL, vol. 173., 1 January 1996 (1996-01-01), NL, pages 99 - 103., XP002912389, ISSN: 0378-1119, DOI: 10.1016/0378-1119(95)00719-9 *
MUNDLE S. D., ET AL.: "TWO IN SITU LABELING TECHNIQUES REVEAL DIFFERENT PATTERNS OF DNA FRAGMENTATION DURING SPONTANEOUS APOPTOSIS IN VIVO AND INDUCED APOPTOSIS IN VITRO.", ANTICANCER RESEARCH - INTERNATIONAL JOURNAL OF CANCER RESEARCH AND TREATMENT, INTERNATIONAL INSTITUTE OF ANTICANCER RESEARCH, GR, vol. 15., 1 January 1995 (1995-01-01), GR, pages 1895 - 1904., XP002912390, ISSN: 0250-7005 *
See also references of EP0964915A4 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7687682B2 (en) 1998-02-23 2010-03-30 Phylonix Pharmaceuticals, Inc. Methods of screening agents for activity using teleosts
US7408095B2 (en) 1998-02-23 2008-08-05 Phylonix Pharmaceuticals, Inc. Methods for introducing heterologous cells into fish to determine if the heterologous cells contain cancer cells or a pathogen
US8993834B2 (en) 1998-02-23 2015-03-31 Phylonix Pharmaceuticals, Inc. Methods of screening agents for activity using teleosts
US7951989B2 (en) 1998-02-23 2011-05-31 Phylonix Pharmaceuticals, Inc. Methods of screening agents for activity using teleosts
WO1999042606A1 (fr) * 1998-02-23 1999-08-26 Phylonix Pharmaceuticals, Inc. Procedes de criblage destines a determiner l'activite d'agents au moyen de poissons teleosteens
EP1878799A3 (fr) * 1998-02-23 2008-01-23 Phylonix Pharmaceuticals Inc. Procédés de criblage d'agents pour l'activité utilisant des téléosts
EP1548123A1 (fr) * 1998-02-23 2005-06-29 Phylonix Pharmaceuticals Inc. Procédés de criblage destinés a determiner l'activité d'agents au moyen de poissions téléosteens
US7482507B2 (en) 1998-02-23 2009-01-27 Phylonix Pharmaceuticals, Inc. Methods of screening agents for activity using teleosts
US6299858B1 (en) 1998-02-23 2001-10-09 Phylonix Pharmaceuticals, Inc. Methods of screening agents for activity using teleosts
US7435870B2 (en) 1998-02-23 2008-10-14 Phylonix Pharmaceuticals, Inc. Methods of screening agents for activity using teleosts
US6761876B2 (en) 1998-12-01 2004-07-13 Phylonix Pharmaceuticals, Inc. Methods for introducing helterologous cells into fish
WO2001040503A3 (fr) * 1999-12-06 2002-07-18 Axxima Pharmaceuticals Ag Methode d"identification et de quantification des inhibiteurs de kinase
WO2001092874A1 (fr) * 2000-05-27 2001-12-06 Artemis Pharmaceuticals Gmbh Methode d'identification d'agents et de genes qui influencent la fonction cardio-vasculaire
EP1160570A1 (fr) * 2000-05-27 2001-12-05 ARTEMIS Pharmaceuticals GmbH Methode pour identifier des agents et des genes qui influencent la function cardiovasculaire
EP1379868A4 (fr) * 2001-04-04 2007-10-03 Zygogen Llc Modeles de poisson zebre transgenique destines a des maladies neurodegeneratives
US7767880B2 (en) 2006-09-01 2010-08-03 Phylonix Pharmaceuticals, Inc. Methods of screening agents for activity using teleosts
WO2017011653A1 (fr) * 2015-07-15 2017-01-19 The University Of Florida Research Foundation, Inc. Procédés de décellularisation d'un tissu
US10898609B2 (en) 2015-07-15 2021-01-26 University Of Florida Research Foundation, Inc. Tissue decellularization methods

Also Published As

Publication number Publication date
GB9701245D0 (en) 1997-03-12
JP2001514738A (ja) 2001-09-11
EP0964915A1 (fr) 1999-12-22
EP0964915A4 (fr) 2003-05-07

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