WO1999028499A1 - Procede de mesure de l'apoptose - Google Patents

Procede de mesure de l'apoptose Download PDF

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Publication number
WO1999028499A1
WO1999028499A1 PCT/EP1998/007682 EP9807682W WO9928499A1 WO 1999028499 A1 WO1999028499 A1 WO 1999028499A1 EP 9807682 W EP9807682 W EP 9807682W WO 9928499 A1 WO9928499 A1 WO 9928499A1
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Prior art keywords
cells
apoptosis
dna
dna sequence
receptor
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PCT/EP1998/007682
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German (de)
English (en)
Inventor
Peter Steinlein
Johannes Hoffmann
Gabor Lamm
Gerhard Christofori
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Boehringer Ingelheim International Gmbh
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Publication date
Priority claimed from DE19752922A external-priority patent/DE19752922A1/de
Priority claimed from DE19805229A external-priority patent/DE19805229A1/de
Application filed by Boehringer Ingelheim International Gmbh filed Critical Boehringer Ingelheim International Gmbh
Priority to JP2000523374A priority Critical patent/JP2001525182A/ja
Priority to EP98959893A priority patent/EP1034304A1/fr
Priority to CA002311954A priority patent/CA2311954A1/fr
Publication of WO1999028499A1 publication Critical patent/WO1999028499A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • the invention relates to the field of biological test methods.
  • Apoptosis or programmed cell death is a genetically controlled cellular suicide mechanism for the selective elimination of unwanted cells (1-4).
  • PCD is an essential process in a variety of biological processes, including embryonic and neural development, immune system regulation, organogenesis, tissue homeostasis, and prevention of diseases such as tumor growth and viral infection.
  • Apoptosis is characterized by blistering of the plasma membrane, shrinking of the cells, condensation of the nucleus, endonucleolytic cleavage of genomic DNA into fragments of internucleosomal length and formation of apoptotic bodies.
  • the currently available methods for examining apoptosis are based on the assessment of morphological changes at the cellular level using light, electron or time-lapse microscopy in conjunction with fluorescent vital dyes, the use of Annexin V, by means of which the loss of membrane phospholipid asymmetry during the apoptosis can be monitored (7), or consist of assays for the detection of DNA fragmentation by means of gel electrophoresis (8) or by means of in situ labeling of DNA strand breaks (“nick-end labeling”) (TUNEL) (9) •
  • TUNEL in situ labeling of DNA strand breaks
  • the object of the present invention was to provide a new method for measuring apoptosis which eliminates the disadvantages of the known methods.
  • This object was achieved by a method for measuring apoptosis, which is characterized in that
  • the cells are harvested and fixed so that the fluorescent protein remains in the cells, while the DNA fragments formed during apoptosis can diffuse out of the cells,
  • DNA sequence of interest includes all DNA sequences which, as such or their translated products, directly or indirectly influence apoptosis.
  • apoptosis-stimulating genes are p53, bax, E1A
  • examples of apoptosis-inhibiting genes are bcl-2, bcl-x, EIB 19K
  • survival factors such as insulin-like growth factors (IGFs).
  • IGFs insulin-like growth factors
  • the apoptosis test gene can be known or unknown genes or fragments thereof. Influencing apoptosis means both induction and enhancement as well as blocking and weakening of apoptosis.
  • the method according to the invention allows great variability, e.g. with regard to the markers used for the determination of the transfected cells or for apoptosis, with regard to the plasmids and the transfection method used for the transfection of the cells.
  • the method according to the invention has, as one of its essential elements, a fluorescent marker protein which serves as evidence for the transient transfection of the cells.
  • the preferred fluorescent protein is the green fluorescent protein (GFP).
  • GFP mutants which are optimized with regard to FACS analysis and are suitable for use in the context of the present invention are known from the literature. An example of a suitable GFP mutant was described in (10) ("enhanced green fluorescent protein", eGFP); However, other mutants can be used within the scope of the present invention which fulfill the condition that they do not influence cell metabolism, that they remain localized intracellularly and that they deliver a measurable fluorescence signal, in particular that they are used by means of currently common fluorescence-activated flow cytometry methods Fluorescent Activated Cell Sorting (FACS)) are measurable.
  • FACS Fluorescent Activated Cell Sorting
  • GFP Green Fluorescent Protein
  • BFP Blue Fluorescent Protein
  • YFP Yellow Fluorescent Protein
  • the plasmids coding for the marker proteins are transiently transfected into mammalian cells, advantageously into the same cells and under the same conditions with which the method according to the invention is to be carried out.
  • the suitability of the transfected marker proteins is determined by series of measurements in which transfection efficiency and the efficiency of reproducible measurability are determined by FACS analysis.
  • a DNA-binding dye e.g. Propidium iodide (PI), which reduces fluorescence in the apoptotic subpopulation (14-17).
  • PI Propidium iodide
  • This detection method is based on the principle that the genomic DNA is degraded endonucleolytically in cells during apoptosis. The small DNA fragments diffuse out of the cell; the reduction in DNA content to less than double the set of chromosomes (“sub-2N”) is a hallmark of apoptotic cells.
  • the reduced fluorescence of PI in cells undergoing apoptosis results in the appearance of a characteristic fluorescence peak (“sub-2N peak”) in the region of the Go / Gi region of the cell cycle.
  • a characteristic fluorescence peak (“sub-2N peak”) in the region of the Go / Gi region of the cell cycle.
  • other DNA-binding dyes can also be used.
  • suitable dyes of this type are commercially available, for example DAPI (4 ', 6' -diamidino-2-phenylindole), acridine orange, ethidium bromide.
  • the most suitable dye can be determined by stimulating cells to apoptosis and then using FACS or microscopic analysis to determine whether apoptosis can be measured reproducibly with the dye candidate.
  • One of the advantages of the method according to the invention is that the fluorescence of the marker protein and the DNA content can be measured simultaneously, preferably by means of FACS analysis. Suitable devices are commercially available.
  • the invention is applicable to all mammalian cells that can be cultivated. It is routine for a person skilled in the art to adjust the commercially available FACS devices to different cell types.
  • All vectors which efficiently and reproducibly produce expression in mammalian cells are suitable for the transfection of the cells with marker gene on the one hand and gene of interest on the other hand.
  • Examples of the numerous available and also commercially available vectors contain regulatory sequences with the ability to achieve high expression rates in a large number of mammalian cells. Examples are vectors containing the CMV (cytomegalovirus), SV40 (simian virus), MSV (Moloney Sarcoma Virus) promoter or other strong, cell type-unspecific promoters.
  • Identical or different vectors can be used as carriers for the marker gene and the gene of interest; depending on the cell type, it may be advantageous to use vectors with different promoters in order to avoid competition between the promoters during transcription.
  • the invention is not subject to any restrictions; in principle all methods known for the transient transfection of mammalian cells can be used, e.g. Calcium phosphate, commercially available cationic lipids such as Lipofecta in or Transfectam, methods based on the receptor-mediated, adenovirus-assisted endocytosis, e.g. described in WO 93/07283, e.g. adenovirus inactivated with polyethyleneimm and psoralen / UV, as also described in (21).
  • the transfection method can be optimized by means of series experiments in which the transfection conditions, cell type, nutrient medium, etc. are varied by transfecting with a fluorescent marker protein and determining the expression of the protein by means of FACS analysis. The conditions optimized for the marker protein are used for the co-transfection with the gene of interest.
  • the cells are incubated in a suitable nutrient medium which is adapted to the respective cell type. If necessary, the cells are stimulated to apoptosis, especially if the apoptosis test gene is to be examined for an inhibition of apoptosis.
  • Suitable apoptosis stimuli are known from the literature and are commercially available, examples of which are staurosporine, daunomycin and etoposide.
  • the incubation conditions and the usefulness of a Apoptosis stimulants are determined in preliminary experiments. It is essential for the incubation, in particular for its duration, that apoptosis has taken place to an extent which enables a change to be detected by measurement, for example by means of FACS analysis.
  • the fixing step carried out after the incubation is essential for carrying out the method according to the invention.
  • An essential requirement for the fixation is that the conditions are selected so that the small subgenomic DNA fragments (internucleosomal fragments, ie those with a size of approximately 200 bp and a multiple thereof) that arise during apoptosis diffuse from the apoptotic cells can, but at the same time the fluorescent marker protein remains in the cell.
  • the combination of these measurements was not possible because the requirements for fixation with regard to measuring the fluorescent marker protein on the one hand and measuring the DNA content of the cells on the other were diametrically opposed and therefore seemed incompatible.
  • the present invention makes it possible for the first time to carry out both measurements in the same cell population with the aid of a suitable fixing step.
  • the fixation conditions that are optimal for measuring the fluorescence of the marker protein strong fixation
  • the optimal fixation conditions for measuring the DNA content of the cells weak fixation
  • the fixing conditions with regard to the reagents fixing reagent, salts, buffers
  • their concentration and the fixing time are changed in such a way that the efficiency is impaired as little as possible when both measurement processes are carried out simultaneously.
  • paraformaldehyde instead of paraformaldehyde, other reagents, such as are customary, e.g. used in immunohistochemistry. Examples of common fixatives that can be found in the relevant manuals (27) are formaldehyde or chloroform / acetone.
  • secondary fixation following primary fixation with paraformaldehyde can in principle be used with other reagents which enable weak permeabilization of the cell membrane, such as detergents.
  • Other reagents which enable weak permeabilization of the cell membrane such as detergents.
  • the transient expression of genes that modulate apoptosis, for example Bcl family members or components of survival factor signal transduction, and the subsequent quantitative analysis of apoptosis by means of the method according to the invention make it possible to test chemical compounds for their ability to be specific to influence the function of the apoptosis-modulating genes.
  • the method according to the invention can be adapted by appropriate equipment, e.g. sample preparation and FACS analysis, which they use for large-scale measurements, e.g. in high throughput screening methods.
  • the method in this form is used to identify pharmaceutically active substances that can modulate apoptosis depending on the expression of certain genes (apoptosis test genes).
  • the gene whose effect on apoptosis is to be modulated by the test substance is transiently transfected into test cells and the test cells are incubated with a test substance from a supply of substances.
  • the modulating effect of a test substance on the activity of the test gene is recorded directly by measurement.
  • Such methods can be used for the following screening applications: a) search for inhibitors of survival factors and their signal transduction, as well as inhibitors of anti-apoptotic gene products in tumor cells; b) Search for chemicals that inhibit certain survival factors and their signal transduction in tumor cells synergistically with chemotherapy; c) Search for chemotherapy drugs that work synergistically with the inhibition of survival factors.
  • the method according to the invention is used in a screening process in order to investigate the effect of tumor cell survival factors (receptor ligands such as IGF-I, IGF-II, FGFs (Fibroblast Growth Factors), PDGFs (Platelet Derived Growth Factors) on apoptosis, as mediated by the activation of the corresponding receptors by these factors and the subsequent signal cascade
  • tumor cell survival factors receptor ligands such as IGF-I, IGF-II, FGFs (Fibroblast Growth Factors), PDGFs (Platelet Derived Growth Factors)
  • the assay method can be used in a screening to determine the effect of natural, known or possibly still to be identified survival factors with regard to tumor therapy in the course of which apoptosis
  • the aim of such a method is above all to identify substances which act synergistically with regard to apoptosis with the inhibition of tumor-specific survival factors.
  • test cells are produced in which the survival factor function is inhibited by introducing and expressing DNAs coding for dominant-negative versions of receptors of the survival factors or for dominant-negative signal transmission molecules of such receptors in the cells.
  • receptors are the IGF-1 receptor (29), FGF receptors (30), PDGF receptors (31), receptors of the EGF growth factors (32; EGF receptor, Her-2 / new / ErbB-2, ErbB-3, ErbB-4).
  • Suitable mutant receptors are characterized in that the functional domains of the receptor are modified in such a way that the receptor binds the ligand, but this binding no longer results in the activation of the signal cascade.
  • the modification is the complete absence of the receptor kinase domain or a mutation in the ATP binding site (28).
  • Signaling molecules can be changed by changing the domain necessary for the transmission of the signal, e.g. the catalytic domain of an enzyme or the protein binding site of an adapter molecule is inactivated by mutation.
  • mutants intended for use in a screen are optimized in preliminary tests for their apoptosis-inducing or enhancing effect in test cells (e.g. fibroblast cell lines, which were made factor-dependent by transfection with the respective wild-type receptor) according to standard methods, in that the mutants in the test cells are transfected and the extent of the apoptosis of the test cells is measured using the method according to the invention. If necessary, the respective functional domains of the mutants are further expanded using common molecular biological methods changed until an optimal inhibition of the wild type receptor and its subsequent signal transmission and thus a maximum degree of apoptosis of the test cells is reached.
  • test cells e.g. fibroblast cell lines, which were made factor-dependent by transfection with the respective wild-type receptor
  • test cells are incubated in the screen with known chemotherapeutic agents or with substances from a pool which are to be investigated with regard to their potential chemotherapeutic effect, and the effect on apoptosis is examined using the method according to the invention.
  • the aim of such a screen is to determine a synergistic effect between the inhibition or the lack of the survival factor function in tumor cells and known chemotherapeutic agents or potentially chemotherapeutically active substances.
  • test cells derived from different types of tumor can be used in a parallel screening under otherwise identical experimental conditions.
  • TNF receptors TNF receptors, Fas
  • caspases TNF receptors, Fas
  • the test principle is identical to that for the apoptosis-inhibiting survival factors, with the difference that wild-type or constitutively active versions of these apoptosis molecules are expressed in the tumor cells.
  • the main focus of application in screening procedures is the search for synergisms that lead to an increase in tumor cell apoptosis. This creates the prerequisite for therapeutic approaches in which the dose of chemotherapeutic agents can be significantly reduced without inhibiting the success of the therapy while simultaneously inhibiting the tumor cell survival function. Lowering the chemotherapeutic dose brings decisive advantages for the patient because it can greatly reduce the toxic side effects of chemotherapy.
  • the method according to the invention was used to investigate whether the signal mediated by the survival factor IGF-II and which causes the survival of ⁇ -tumor cells is transmitted by the IGF receptor.
  • IGF-II a dominant-negative version of the human IGF-1 receptor
  • eGFP green fluorescence protein
  • Facs analysis was used to identify transfected individual cells expressing eGFP and to identify the apoptotic cells in this population by their DNA content of less than 2N. It was shown that transfection with plasmids which code for the dnIGF-IR results in a dramatic increase in apoptosis both in untreated and in wild-type ß tumor cells treated with daunomycin or etoposide. It was found that dnIGF-IR increases apoptosis in ⁇ -tumor cells with almost the same efficiency as the adenovirus EIA protein, one of the most potent apoptosis-inducing gene products at all.
  • tumor cells are more sensitive to apoptotic stimuli when the IGF-IR signal transmission is interrupted and that, like IGF-II-deficient tumor cells, they have an increased sensitivity to chemotherapeutic agents.
  • known genes can be examined with the method according to the invention to determine whether and to what extent they modulate apoptosis in different cell types.
  • Another application of the method according to the invention is the expression cloning of genes that modulate apoptosis.
  • a complete cDNA Expression library transiently transfected into cells.
  • the method according to the invention is able to measure the influence of gene expression within 24 to 48 hours. It is therefore possible to analyze cells and isolate them while they are still alive.
  • the method is modified by isolating single cells that deviate from an apoptosis background to be determined by FACS sorting.
  • the plasmids transfected into these cells are isolated, amplified and selected in further rounds of transfection. Plasmids containing an apoptosis-modulating gene are isolated in this way.
  • the corresponding genes are then characterized by sequencing and further expression and function studies.
  • Example 1 established tumor cells were first used in Example 1, which were transfected on the one hand with a GFP plasmid, and on the other hand with a plasmid containing a pro-apoptotic or an anti-apoptotic gene sequence, or a control plasmid. Following the transfection, the cells were treated with an apoptotic stimulus after a break (control cells remained untreated). The detached cells were then collected and combined with the trypsinized adherent cells, washed and fixed.
  • the cells were divided in order to compare the method according to the invention with the conventional TUNEL method, which uses fluorescent Cy5-dCTP (5-amino-propargyl-2'-deoxycytidine 5'-triphosphate coupled to Cy5 fluorescent dye) enable. It was shown that the method according to the invention reliably proves the expected pro- or anti-apoptotic effect of the gene products and that the addition of the apoptotic stimulus increases the observed effect. Although the sensitivity of the method according to the invention was somewhat less than that of the TUNEL method under the selected conditions, the normalized apoptosis value, expressed as the ratio of the maximum apotosum values of the respective assay achieved with the pro-apoptotic gene, was almost identical. Compared to the TUNEL method, the method according to the invention has the advantage of speed, simplicity and cheapness.
  • the broad applicability and reliability of the method according to the invention was confirmed by using an untransformed rat fibroblast cell line which reacts less to apoptotic stimuli than the established tumor cell lines.
  • the method according to the invention enables the potential role of a gene product in apoptosis to be determined quickly, effectively and reproducibly.
  • the invention relates to a kit for simple and routine implementation of the method.
  • kit expediently contains the following components in several separate containers:
  • the kit preferably contains polyethyleneimine and psoralen / UV-inactivated adenovirus as transfection components.
  • the cells were treated with 1 ⁇ g of a plasmid coding for eGFP (“enhanced GFP”; pEGFP-Cl; Clontech) together with 1 ⁇ g of a control plasmid (pMEX; (22)), a pCMV plasmid, containing the pro-apoptotic Adenovirus gene EIA or a pCMV plasmid containing the anti-apoptotic adenovirus gene E1B-19K (17, 18) transfected using 10 ⁇ l LipofectAMINE (GIBCO-BRL) as recommended by the manufacturer.
  • a plasmid coding for eGFP (“enhanced GFP”; pEGFP-Cl; Clontech) together with 1 ⁇ g of a control plasmid (pMEX; (22)
  • pCMV plasmid containing the pro-apoptotic Adenovirus gene EIA
  • E1B-19K 17, 18
  • the cells were left to rest in complete medium for 16 h, after which the cells were either left untreated or treated with an apoptotic stimulus (800 ng / ml staurosporin; Sigma) (19, 20) for a further 16 h.
  • an apoptotic stimulus 800 ng / ml staurosporin; Sigma
  • the detached cells were combined with trypsinized, adherent cells, washed twice with 4 ml of PBS and fixed at room temperature for 30 min (2% paraformaldehyde, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA ( Ethylene glycol bis (2-aminoethyl) tetraacetic acid), 10 mM PIPES (piperazine-N ⁇ _N -bis [z-ethane sulfonic acid]) pH 6.8). The mixture was then washed twice with 4 ml of PBS and fixed in ice-cold 70% EtOH for 14 h.
  • 2% paraformaldehyde 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA ( Ethylene glycol bis (2-aminoethyl) tetraacetic acid), 10 mM PIPES (piperazine-N
  • the cells were washed twice with 4 ml PBS and divided. Half of the sample was treated with RNase A (Sigma, St. Louis, USA) (50 ⁇ g / ml) in PBS for 30 min, washed twice with 4 ml PBS and 30 min before FACS analysis with propidium iodide in PBS (PI ; 50 ⁇ g / ml; Sigma, St. Louis, USA) stained.
  • RNase A Sigma, St. Louis, USA
  • TdT reaction mixture terminal deoxynucleotidyl transferase; Boehringer Mannheim; 200 mM potassium cocodylate, 25 mM Tris-HCl pH 6.6, 0.25 mg / ml bovine serum albumin, 1 mM C0CI2; 0.25 nmol FluoroLink Cy5AP3-dCTP [Amersham] , 12.5 units TdT
  • TdT reaction mixture terminal deoxynucleotidyl transferase; Boehringer Mannheim; 200 mM potassium cocodylate, 25 mM Tris-HCl pH 6.6, 0.25 mg / ml bovine serum albumin, 1 mM C0CI2; 0.25 nmol FluoroLink Cy5AP3-dCTP [Amersham] , 12.5 units TdT
  • Fig. 1A shows the number of apoptotic / 3HC 13T tumor cells (% apoptosis) in the entire eGFP-positive cell population.
  • the black bars show the determination of the sub-2N DNA content (GFP / PI); the white bars indicate the incorporation of fluorescent Cy5AP3-dCTP during the TdT reaction (GFP / TUNEL). The addition of staurosporine is indicated.
  • An excitation wavelength of 488 nm was used for eGFP and PI, an excitation wavelength of 647 nm for Cy5 and UV with a wavelength range of 51-364 nm for DAPI.
  • the emission fluorescence was measured using a 530/20 nm narrow band filter for eGFP, a 610 nm blocking filter for PI, a 675/20 nm narrow band filter for Cy5 and one
  • FIG. 1B shows the normalized percentage of apoptosis for the different constructs.
  • Apoptosis index was normalized using the following function: (% apoptosis in X /% apoptosis in eGFP-Cl / ElA) x 100. The apoptotic index was normalized for each of the detection methods used and for each post-transfection treatment (+/- staurosporin).
  • RatlA an untransformed rat fibroblast cell line called RatlA was used.
  • the cells were transiently transfected using either, as described in Example 1, LipofectAMINE or polyethyleneimine (PEI 2000) DNA adenovirus complexes (WO 93/07283). Otherwise, the procedure for treating the cells and determining apoptosis using the method according to the invention on the one hand and the TUNEL method on the other hand was carried out in exactly the same way as described in Example 1.
  • the comparison of the different transfection methods and apoptosis measurement methods is shown in the table. Each value represents the average of 3 transfections; the standard deviation is given (see below). The efficiency in the transfection methods was 25-30%.
  • Wild-type ß tumor cells (15) were used in this example.
  • the dominant-negative IGF-1 receptor construct used which is under the control of the CMV promoter and in which codon 1003 is mutated from lysine to alanine in the ATP binding site, was described by (28).
  • wild-type ⁇ -tumor cells were seeded in triplicates in 6-well culture plates at a density of 80,000 cells. 24 hours later, the cells were co-transfected with pEGFP-Cl and a control plasmid (FIG. 2: pMEX / ctr) or with pEGFP-Cl and an expression plasmid, coding for either adenovirus EIA (FIG.
  • adenovirus E1B-19K (Fig. 2: E1B-19K) or the dominant-negative IGF-IR.
  • daunomycin FIG. 2: hatched bars
  • etoposide FIG. 2: white bars
  • Transfected but untreated cells are shown in Fig. 2 by black bars. 12 hours after treatment with daunomycin or etoposide, the cells were harvested, fixed and treated with propidium iodide, as described in the previous examples.
  • the determination of the apoptotic cells was also carried out according to the methods described above. 40,000 events were collected for each measurement; the transfection efficiency was 25-30%. The standard deviation is indicated by error bars.

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Abstract

L'invention concerne un procédé permettant de mesurer rapidement et simplement l'apoptose. Des cellules de mammifère sont cotransfectées avec un plasmide qui code pour une protéine fluorescente et également avec un plasmide qui porte un gène d'intérêt. Après incubation et fixation douce, l'apoptose est mesurée par détermination de la teneur en ADN des cellules, au moyen d'un colorant fixant l'ADN, et de la proportion de cellules transfectées, par cytométrie en flux. Ce procédé peut être mis en oeuvre pour l'identification de substances qui modulent l'effet pro-apoptotique ou anti-apoptotique de gènes ou de produits géniques.
PCT/EP1998/007682 1997-11-28 1998-11-27 Procede de mesure de l'apoptose WO1999028499A1 (fr)

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Application Number Priority Date Filing Date Title
JP2000523374A JP2001525182A (ja) 1997-11-28 1998-11-27 アポトーシス測定法
EP98959893A EP1034304A1 (fr) 1997-11-28 1998-11-27 Procede de mesure de l'apoptose
CA002311954A CA2311954A1 (fr) 1997-11-28 1998-11-27 Procede de mesure de l'apoptose

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE19752922A DE19752922A1 (de) 1997-11-28 1997-11-28 Verfahren zur Messung der Apoptose
DE19752922.4 1998-02-10
DE19805229.4 1998-02-10
DE19805229A DE19805229A1 (de) 1998-02-10 1998-02-10 Verfahren zur Messung der Apoptose

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WO2003067255A1 (fr) * 2002-02-04 2003-08-14 Hemolytics Ag Procede de criblage a debit eleve de composes ayant une activite non-, pro-, ou anti-apoptotique ou proliferative ou necrotique
CN110208515A (zh) * 2019-05-31 2019-09-06 山东省农业科学院农产品研究所 鸡骨香挥发油抗肿瘤活性及其相关机制的测试方法及应用

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003008648A1 (fr) * 2001-07-16 2003-01-30 Deltagen Proteomics, Inc. Procedes pour identifier des agents qui provoquent un phenotype cellulaire, et compositions associees
WO2003067255A1 (fr) * 2002-02-04 2003-08-14 Hemolytics Ag Procede de criblage a debit eleve de composes ayant une activite non-, pro-, ou anti-apoptotique ou proliferative ou necrotique
CN110208515A (zh) * 2019-05-31 2019-09-06 山东省农业科学院农产品研究所 鸡骨香挥发油抗肿瘤活性及其相关机制的测试方法及应用

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