WO1998059069A2 - Identification d'agents cancerogenes grace a un mammifere avec inhibition de la poly(adp-ribose)-polymerase - Google Patents

Identification d'agents cancerogenes grace a un mammifere avec inhibition de la poly(adp-ribose)-polymerase Download PDF

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Publication number
WO1998059069A2
WO1998059069A2 PCT/DE1998/001797 DE9801797W WO9859069A2 WO 1998059069 A2 WO1998059069 A2 WO 1998059069A2 DE 9801797 W DE9801797 W DE 9801797W WO 9859069 A2 WO9859069 A2 WO 9859069A2
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WIPO (PCT)
Prior art keywords
polymerase
adp
ribose
poly
mammal
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PCT/DE1998/001797
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German (de)
English (en)
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WO1998059069A3 (fr
Inventor
Jan-Heiner KÜPPER
Alexander BÜRKLE
Leon Van Gool
Harald Zur Hausen
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Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
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Priority to JP50359699A priority Critical patent/JP2002507885A/ja
Priority to EP98941257A priority patent/EP1015573A2/fr
Publication of WO1998059069A2 publication Critical patent/WO1998059069A2/fr
Publication of WO1998059069A3 publication Critical patent/WO1998059069A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases

Definitions

  • the present invention relates to a method for identifying carcinogenic agents.
  • the very widespread Arnes test also called the Salmonella typhimurium test, is based on the mutagenicity of substances in bacteria (Clonfero et al., Med. Lav. 81, pp. 3-10 (1 990)).
  • Another well-known in vitro test is the SOS chromotest (Quillardet et al., Mutat. Res. 297, pp. 235-279 (1 993)), which is based on the induction of the bacterial SOS system by genotoxic agents.
  • the sensitivity of both tests is comparable, but they have the fundamental disadvantage that the genotoxic effects of substances in bacteria and higher organisms can be different and the results are therefore not transferable to the mammalian organism.
  • the object of the present invention is therefore to provide a method with which carcinogenic agents can be reliably identified.
  • the method according to the invention is carried out using a mammal, preferably a rodent, particularly preferably using a mouse, in which there is a fault in the DNA repair.
  • the DNA repair disorder is based on the trans-dominant inhibition of poly (ADP-ribose) polymerase (PARP), an enzyme involved in DNA repair processes.
  • PARP poly (ADP-ribose) polymerase
  • the inhibition of PARP is preferably based on the expression of a dominant negative mutant of poly (ADP-ribose) polymerase, preferably the transgenic expression of such a mutant in a mammal, thereby producing a transgenic animal which is also the subject of the present invention.
  • the PARP has a DNA binding domain (abbreviated DBD), which enables binding to DNA strand breaks and leads to an enzyme activity of the PARP, which enables repair of the strand breaks.
  • DBD DNA binding domain
  • the dominant negative PARP mutant has a deletion, so that only the DNA binding domain of the PARP is expressed. This inhibits PARP enzyme function and thus DNA repair.
  • the expression of this PARP mutant has no influence on cell division and cell vitality in the absence of genotoxic stress. However, if genotoxic (chemical or physical) agents are applied, PARP inhibition leads to a considerable increase in the sensitivity of the cells to these treatments. The presence of the PARP mutant then leads to an increased genetic instability after carcinogen treatment, which manifests itself in increased recombination and increased gene amplification.
  • the disruption of the PARP function leads to an increased mutagenicity of genotoxic agents.
  • the disruption of cellular PARP function leads to an increased rate of various genetic changes (mutations, recombinations, gene amplification) after treatment with a carcinogen. These different genetic changes, depending on the nature of the carcinogenic agent and the type of its application, allow different ways of tumor development (eg oncogene amplification, tumor suppressor gene mutation or combinations thereof).
  • the mammal used in the method according to the invention advantageously has a skin-specific expression of the dominant-negative PARP mutant, although of course any other organ-specific expression is also possible.
  • the skin is the preferred organ for carcinogenesis studies because of its easy accessibility and controllability.
  • Any promoter known to the person skilled in the art that allows tissue-specific expression, preferably in the skin, can be used to control the transgene; the cytokeratin 14 promoter is preferably used, which allows expression in the cell-dividing basal layer, from which skin tumors preferably arise (Vassar et al., Proc. Natl. Acad. Sei USA 86, pp. 1,563-1,567 (1,989 )).
  • a fragment is preferably used to produce the transgenic mammal, which has the following structure (see FIG. 1):
  • the transgenic mammal is made according to the method described by Hogan et al. ("Manipulating the mouse embryo: A laboratory manual", Cold Spring Habor Laboratory, New York (1986)). The microinjection of a corresponding DNA fragment into fertilized mouse oocytes and subsequent implantation into hypophysical females is particularly suitable for this. It offspring arise which contain the transgene and are passed on to their offspring (DBD line).
  • the method according to the invention is carried out in the form of an in vivo assay. 10-1 5-week-old transgenic animals from the DBD line are selected and acclimatized accordingly for the test. 1 0- 1 5 animals (female or male) are required for each treatment. Since the skin is an important target organ of the carcinogenesis studies, the potentially carcinogenic chemical agents are applied topically twice a week in 50-200 ⁇ ⁇ solvents, e.g. water, physiological saline, acetone or ethanol). For the investigation of potentially carcinogenic physical agents, corresponding applications are also carried out twice a week. These treatments can last up to 20 weeks. To enable tumor growth, an additional time of up to 40-80 weeks after the last application is estimated.
  • solvents e.g. water, physiological saline, acetone or ethanol
  • DMBA 2-dimethylbenzanthracene
  • the corresponding solvent which was also used to dissolve the test substance, can be applied as a negative control.
  • the animals are weighed regularly throughout the test period and the application site is examined. Papillomas and other skin tumors are examined macroscopically once a week and measured if necessary. When tumors have reached a critical size (depends on the species, location of the tumor and the national animal welfare conditions), the animals are killed and tumor tissue is removed for histological and molecular biological characterization. Primary cultures of the tumors can also be created.
  • a sensitivity gain can be achieved with the method according to the invention using the transgenic mammal which has a disturbance in DNA repair by trans-dominant inhibition of PARP activity, whereby the problem of overdosing and producing false positive results is reduced.
  • the method of the present invention circumvents the problem of the pathway towards a predetermined tumorigenesis.
  • the PARP inhibition leads to a fundamental disruption of the DNA repair with the consequence of an increase in the genetic instability (mutation, recombination, gene amplification rate) after carcinogen treatment, which then promotes tumor development in various ways.
  • Gene DBD coding sequence of the DNA binding domain of human poly (ADP-ribose) polymerase (EC 2.4.2.30)
  • p-A polyadenylation signal of human
  • the plasmid pKDinoDBD (see FIG. 1) was cut with the restriction enzyme Not I. After separation of the restriction fragments on a 1% agarose gel, a 3.6 kB fragment containing the expression cassette of pKDinoDBD was isolated and by means of a commercially available kit (for example "Gene Clean” R; Dianova, Hamburg) according to the manufacturer's instructions prepared. This fragment was adjusted to a concentration of 2 ng / ⁇ l in 10 mM Tris-HCl (pH 7.6), 0.25 mM EDTA. F1 females from the cross between the mouse strains C57BL / 6 x DBA2 were subjected to superovulation by hormone administration.
  • PCR cycles were carried out, each with 200 ng of genomic DNA, denaturing in each case 300 seconds at 95 ° C., annealing for 60 seconds at 60 ° C. and polymerizing at 72 ° C. for 20 seconds.
  • a positive female (ear tag # 354) was identified. Protein material was obtained from the tail biopsy of this animal and examined for expression of the transgene by means of Western blot. Both the monoclonal anti-DBD antibody CM 10 (Lamarre et al., Biochim. Biophys. Acta 950, S. 147-160 (1 988)) and the anti-FII rabbit serum directed against the DBD (Küpper et al., J. Biol. Chem. 265, p. 1 8721 -1 8724 (1 990)). With both antibodies, the 45 kDa DNA binding domain (DBD) was detectable in the Western blot, so that the evidence for the expression of the transgene was provided. The Founder-DBD-Mouse # 354 was mated with DBA2 males and the offspring were analyzed. The transgene is passed on to the offspring so that the line DBD # 354 is stable.
  • CM 10 Lamarre et al., Biochim. Biophys. Act
  • mice DBD line # 354 described in Example 1 1 2 week old animals of the mouse DBD line # 354 described in Example 1 are acclimated to the test site over three weeks.
  • Female animals are kept in groups of 5 animals / cage, male animals are kept individually under specific pathogen-free (SPF) conditions. The animals are fed according to standard (# D10010 feed from Research Diets, New Brunswick, New Jersey, USA and water ad libitum). 10-1 5 animals (male or female) are required for each treatment.
  • the putative carcinogenic chemicals to be tested are taken up in several dilution stages in physiological saline or acetone and 100 ⁇ l of each dilution is applied topically twice a week.
  • DMBA 2-dimethylbenzanthracene
  • Acetone is applied as a negative control.
  • the treatment is carried out for 1 5 weeks.
  • the animals are weighed weekly and examined at the application site. Visible tumor growth can be expected in the group of the positive control 1 2 weeks after the end of the treatments. Experience has shown that the tumors grow rapidly (within a further 1 2 weeks). After reaching a critical tumor size, the animals are killed by cervical dislocation and the tumor is removed. As expected, there was no tumor growth in the group of animals treated with solvent even after 60 weeks. Alternatively, a so-called initiation doctoral protocol can be used.
  • a very low dose of an initiating (mostly DNA-damaging) carcinogen is administered, followed by repeated applications of a non-carcinogenic tumor promoter (Becker et al., Cancer Res. 56, pp. 3244-3249, 1 996).
  • a non-carcinogenic tumor promoter Becker et al., Cancer Res. 56, pp. 3244-3249, 1 996
  • methyl nitrosourea (20 ⁇ mol in 100 ⁇ l acetone; applied topically once
  • TPA tumor promoter tetradecanoyl-phorbol acetate
  • negative controls are animals that have only been given acetone instead of the methyl nitrosourea, but then followed by the usual TPA treatment.
  • the chemicals to be tested for carcinogenicity are also applied instead of the methyl nitrosourea, again followed by the usual TPA treatment.
  • visible tumor growth in the mouse of the DBD line # 354 described in Example 1 is to be expected after 9 weeks at the latest.

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Abstract

Selon ce procédé d'identification d'agents cancérogènes, on administre les agents potentiellement cancérogènes à un mammifère qui présente une perturbation de la réparation de l'ADN par inhibition de la poly(ADP-ribose)-polymérase.
PCT/DE1998/001797 1997-06-24 1998-06-24 Identification d'agents cancerogenes grace a un mammifere avec inhibition de la poly(adp-ribose)-polymerase WO1998059069A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP50359699A JP2002507885A (ja) 1997-06-24 1998-06-24 ポリ（ａｄｐリボース）ポリメラーゼの阻害での哺乳類を使用する発癌性因子の同定方法
EP98941257A EP1015573A2 (fr) 1997-06-24 1998-06-24 Procede pour identifier des agents cancerogenes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19726824.2 1997-06-24
DE1997126824 DE19726824C1 (de) 1997-06-24 1997-06-24 Verfahren zur Identifizierung kanzerogener Agenzien

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WO1998059069A2 true WO1998059069A2 (fr) 1998-12-30
WO1998059069A3 WO1998059069A3 (fr) 1999-04-29

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995024379A1 (fr) * 1994-03-09 1995-09-14 Newcastle University Ventures Limited Analogues de benzamides utiles en tant qu'inhibiteurs de l'enzyme parp (adp-ribosyltransferase, adprt) de reparation de l'adn
DE4433130A1 (de) * 1994-09-16 1996-03-21 Deutsches Krebsforsch Identifizierung von DNA-schädigenden Substanzen durch Poly(ADP-Ribose)-Polymerase überexprimierende Zellinien
WO1996018737A2 (fr) * 1994-12-16 1996-06-20 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Vecteurs et virus utilises pour la therapie genique
EP0757102A1 (fr) * 1995-08-04 1997-02-05 Plant Genetic Systems N.V. Transformation génétique utilisant un inhibiteur de PARP

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995024379A1 (fr) * 1994-03-09 1995-09-14 Newcastle University Ventures Limited Analogues de benzamides utiles en tant qu'inhibiteurs de l'enzyme parp (adp-ribosyltransferase, adprt) de reparation de l'adn
DE4433130A1 (de) * 1994-09-16 1996-03-21 Deutsches Krebsforsch Identifizierung von DNA-schädigenden Substanzen durch Poly(ADP-Ribose)-Polymerase überexprimierende Zellinien
WO1996018737A2 (fr) * 1994-12-16 1996-06-20 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Vecteurs et virus utilises pour la therapie genique
EP0757102A1 (fr) * 1995-08-04 1997-02-05 Plant Genetic Systems N.V. Transformation génétique utilisant un inhibiteur de PARP

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
K]PPER, J.H. ET AL.: "Trans-dominant inhibition of poly(ADP-ribosyl)ation potentiates carcinogen-induced gene amplification in SV40-transformed Chinese Hamter cells " CANCER RESEARCH, Bd. 56, Nr. 12, 15. Juni 1996, Seiten 2715-2717, XP002090238 MD US *
K]PPER, J.H. ET AL.: "Trans-dominant inhibition of poly(ADP-Ribosyl)ation sensitizes cells against gamma-irradiation and N-methyl-N'-Nitro-N-nitroguanidine but does not limit DNA replication of a polyomavirus replicon" MOLECULAR AND CELLULAR BIOLOGY, Bd. 15, Nr. 6, Seiten 3154-3163, XP002090237 WASHINGTON US *
MOLINETE, M. ET AL.: "Overproduction of the poly(ADP-ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells" EMBO JOURNAL, Bd. 12, Nr. 5, 1993, Seiten 2109-2117, XP002090239 EYNSHAM, OXFORD GB *

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JP2002507885A (ja) 2002-03-12
EP1015573A2 (fr) 2000-07-05
DE19726824C1 (de) 1999-03-04
WO1998059069A3 (fr) 1999-04-29

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