EP1034304A1

EP1034304A1 - Method for measuring the apoptosis

Info

Publication number: EP1034304A1
Application number: EP98959893A
Authority: EP
Inventors: Peter Steinlein; Johannes Hoffmann; Gabor Lamm; Gerhard Christofori
Original assignee: Boehringer Ingelheim International GmbH
Current assignee: Boehringer Ingelheim International GmbH
Priority date: 1997-11-28
Filing date: 1998-11-27
Publication date: 2000-09-13
Also published as: CA2311954A1; WO1999028499A1; JP2001525182A

Abstract

The invention relates to a method for quickly and easily measuring the apoptosis. Mammal cells are co-transfected with a plasmid coded for a fluorescent protein, and with a plasmid carrying an interested gene. The apoptosis is measured after incubation and mild fixation in which the DNA content of the cells is determined by means of DNA-binding colorants, and the portion of the transfected cells is determined by means of flow-through cytometry. The method can be used in order to identify substances which modulate the proapoptotic or antiapoptotic effect of genes or gene products.

Description

Procedure for measuring apoptosis

The invention relates to the field of biological test methods.

Apoptosis or programmed cell death (PCD) 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) • In order to investigate the effect of genes that play a role in apoptosis by means of transient transfection analyzes, however, most of these methods are either unsuitable or, in the case of the TUNEL method, too expensive and too complex.

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

A) transiently transfecting a population of mammalian cells

ai) with a plasmid containing a DNA sequence of interest, from which it is to be determined whether it or the polypeptide expressed thereby has a pro- or anti-apoptotic effect,

aii) or with a plasmid containing a DNA sequence of interest, from which it is to be determined whether or by which substances their pro- or anti-apoptotic effect or the effect of the polypeptide expressed thereby can be modulated,

b) and with a plasmid containing a DNA coding for a fluorescent marker protein,

B) that the cells are in a suitable nutrient medium, if appropriate in the presence of a test substance, incubated until the DNA sequence of interest or the expressed polypeptide has its potential effect on apoptosis,

C) 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,

D) that the proportion of apoptotic cells is determined by measuring the DNA content,

E) that the proportion of transfected cells is determined by measuring the cells with fluorescent marker protein,

F) and that by comparing the values obtained in steps D and E, the proportion of apoptical cells in the transfected subpopulation of the cells is determined.

The term “DNA sequence of interest” (hereinafter also referred to as “apoptosis test gene”) includes all DNA sequences which, as such or their translated products, directly or indirectly influence apoptosis. Examples of apoptosis-stimulating genes are p53, bax, E1A, examples of apoptosis-inhibiting genes are bcl-2, bcl-x, EIB 19K, the latter group also includes the so-called survival factors such as insulin-like growth factors (IGFs). Such apoptosis genes and their effects have been described in reviews (e.g. 4, 23, 24). 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.

In addition to the Green Fluorescent Protein (GFP), other fluorescent marker proteins can also be used. Examples include Blue Fluorescent Protein (BFP) (26) and Yellow Fluorescent Protein (YFP) (25). The properties listed above for GFP mutants are essential for the suitability of a marker protein for use in the method according to the invention.

Potential marker proteins as well as the type and amount of the plasmids coding therefor to be used in the assay and the most suitable transfection method can e.g. are tested as follows: 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). 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. Instead of propidium iodide, other DNA-binding dyes can also be used. Representatives of 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.

With regard to the transfection methods, 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.

Following the transfection, 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. With the previously available methods, 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.

In order to determine the suitable fixation conditions, the following procedure is expediently carried out: First, the fixation conditions that are optimal for measuring the fluorescence of the marker protein (strong fixation) and, on the other hand, the optimal fixation conditions for measuring the DNA content of the cells (weak fixation) are determined independently of one another . Starting from the Conditions with which the maximum measured values are obtained, 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.

Primary fixation with paraformaldehyde and subsequent treatment are preferred

(Secondary fixation / permeabilization) carried out with ethanol; this treatment has proven to be the most suitable in the course of the experiments carried out. The pre-fixation by means of 1 to 4% (w / v), in particular 2% parafor aldehyde takes place in an isotonic, buffered saline solution. Suitable are e.g. Standard solutions such as 100 mM NaCl, 3 mM MgCl2, 300 mM sucrose as well as commercially available physiologically compatible buffers.

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.

Instead of ethanol, which has proven to be particularly suitable under the conditions chosen in the experiments carried out, 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. 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. In one embodiment, 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 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.

To determine such active synergisms, you can proceed as follows:

Starting from tumor cells, 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. Examples of 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). Examples of signal transmission molecules are Ras, Raf, phosphoinositol (3) kinase (= PI (3) kinase), MAP kinases, protein kinase type B and type C, phospholipase C, furthermore adapter molecules such as Shc, Grb-2 (33; 34; 35; 36; 37).

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. In the case of IGF-IR, 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.

The 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.

The 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. In particular, 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.

In order to find substances with the screening method that have the synergism with the inhibition of the survival factor function specifically for certain types of tumor, test cells derived from different types of tumor can be used in a parallel screening under otherwise identical experimental conditions.

As control cells for the specificity of the synergistic effect between the lack of survival factor function and chemotherapy, cells are used which naturally lack the survival factor function whose inhibition is to be determined in the assay. In principle, however, it is also possible to search for substances that increase the effect of apoptosis-inducing or enhancing molecules. Examples of this are members of the TNF receptor family (TNF receptors, Fas) and molecules of their signal transmission pathways (caspases). 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.

In the context of the present invention, 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 β-tumor cells is transmitted by the IGF receptor. For this purpose, a dominant-negative version of the human IGF-1 receptor (dnIGF-IR), which has an amino acid substitution in the ATP binding site (28), was transient in Wild-type ß tumor cells co-transfected with a plasmid carrying the green fluorescence protein (eGFP). The cells were then incubated with apoptotic stimuli and / or growth factors, harvested, fixed and stained with propidium iodide to determine the DNA content. 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 β-tumor cells with almost the same efficiency as the adenovirus EIA protein, one of the most potent apoptosis-inducing gene products at all. Thus, with the help of the assay method according to the invention, it could be shown that 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.

Furthermore, 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. For this, 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. For this application 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.

In order to validate the method according to the invention, 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. After the subsequent washing, 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.

In a further aspect, the invention relates to a kit for simple and routine implementation of the method.

Such a kit expediently contains the following components in several separate containers:

a) one or more components required for transfection; b) a plasmid containing the sequence coding for the fluorescent marker protein; c) an empty vector for the insertion of the DNA sequence of interest and for control measurements; d) the primary fixing solution, for example paraformaldehyde solution; e) the post-fixation / permeabilization solution, for example 70% ethanol; f) washing solution (s); g) a DNA binding dye.

The kit preferably contains polyethyleneimine and psoralen / UV-inactivated adenovirus as transfection components.

example 1

Established tumor cell lines (j3TC and / 3HC) derived from / 3-cell tumors (15) of transgenic mice in which the regulatory region of the insulin gene (Rip) expresses the large T antigen of simian virus were used for this example 40 (day) specifically induced in the pancreatic islet cells (16).

Approximately 80,000 cells were sown in a 6 cm well of a 6-well culture dish and in DMEM supplemented with 10% FCS (v / v), 2 mM glutamine, 100 international units penicillin and 100 μg / ml streptomycin to a confluence of 70% bred. The cells were treated with 1 μg of a plasmid coding for eGFP (“enhanced GFP”; pEGFP-Cl; Clontech) together with 1 μg 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 μl LipofectAMINE (GIBCO-BRL) as recommended by the manufacturer. After the transfection, 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. 32 hours after the transfection, 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-Nι_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.

After fixation, 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 μg / 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 μg / ml; Sigma, St. Louis, USA) stained. The other half of the sample was mixed with 50 μl 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) incubated for 1 h at 37 ° C, washed twice with 4 ml PBS, treated with RNase in PBS (50 μg / ml) for 30 min, washed twice with 4 ml HBS (from this step HBS was used instead of PBS used because DAPI in PBS tends to cause micro-precipitates), stained with DAPI in HBS (10 μg / ml; Sigma) for 20 min and analyzed on a Becton Dickinson FACS Vantage device. The FACS analysis of the Pl-stained cells was carried out with a Becton Dickinson FACScan device, which is equipped with a so-called "doublet discrimination module", with which cell aggregates are discriminated by calculating the pulse width and pulse width. The result of the tests is shown in Fig. 1. 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

424/44 narrowband filters collected for DAPI. Duplicates were excluded by means of pulsed processing. Cells expressing eGFP were selected and analyzed for Cy5 or PI fluorescence. The data were analyzed using the CELLQuest software (Becton Dickinson). Each bar represents the average of 3 transfections,

Standard deviations are indicated by error bars. Each measurement included 40,000 total events, selected by size and individual cells. The transfection efficiency was 20-30%. Figure 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).

Example 2

In this example, 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%.

Example 3

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). As described in the previous examples, wild-type β-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. 2: EIA), adenovirus, E1B-19K (Fig. 2: E1B-19K) or the dominant-negative IGF-IR. 36 h after the transfection, daunomycin (FIG. 2: hatched bars) or etoposide (FIG. 2: white bars) were added to the culture medium in a concentration of 1 μM or 10 μM. 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.

table

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Claims

claims

1) Method for measuring apoptosis, characterized in that

A) transiently transfected a population of mammalian cells

b) and with a plasmid containing a DNA coding for a fluorescent marker protein,

B) the cells are incubated in a suitable nutrient medium until the DNA sequence of interest or the expressed polypeptide has had its potential effect on apoptosis,

C) 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, D) determining the proportion of apoptotic cells by measuring the DNA content,

E) the proportion of the transfected cells is determined by measuring the cells with fluorescent marker protein,

F) and by comparing the values obtained in steps D and E the proportion of apoptical cells in the transfected subpopulation of the cells is determined.

2. The method according to claim 1, characterized in that one carries out the transfection of the cells with polyethyleneimine and inactivated adenovirus.

3. The method according to claim 1, characterized in that the fluorescent polypeptide defined in A b) is the green fluorescent protein.

4. The method according to claim 1, characterized in that the DNA content is measured with a DNA-binding dye.

5. The method according to claim 4, characterized in that the dye is propidium iodide.

6. The method according to any one of claims 1 to 5, characterized in that one carries out the incubation according to step B) in the presence of a test substance.

Method according to one of claims 1 to 6, characterized in that the incubation according to step B) is carried out in the presence of a substance which stimulates apoptosis.

Method according to one of claims 1 to 7, characterized in that the primary fixation in step C is carried out with paraformaldehyde and the secondary fixation / permeabilization of the cells with ethanol.

Method according to one of claims 1 to 8, characterized in that the measurements defined in steps D and E are carried out in one step by means of fluorescence-activated flow cytometry analysis.

10. Kit to perform the procedure after

Claim 1, characterized in that it contains the following components in several separate containers:

a) one or more components required for transfection;

b) a plasmid containing the sequence coding for the fluorescent marker protein;

c) an empty vector for the insertion of the DNA sequence of interest and for control measurements;

d) the primary fixative;

e) the secondary fixation / permeabilization solution; f) washing solution (s);

g) a DNA binding dye.

11. Kit according to claim 10, containing as component a) polyethyleneimine and psoralen / UV-inactivated adenovirus.

12. Kit according to claim 10, containing as component b) a plasmid coding for green fluorescent protein.

13. Kit according to claim 10, containing as components d) an approximately 2%

Paraformaldehyde solution and as component e) about 70% ethanol.

14. Use of the method according to claim 1 for identifying substances which modulate the pro- or anti-apoptotic effect of genes or gene products.

15. Use according to claim 14 for identifying therapeutically active substances which have a synergistic effect with the inhibition or lack of the survival factor function of tumor cells, characterized in that the cells are tumor cells, that the DNA sequence according to aii) is a dominant negative version of a receptor for a tumor cell survival factor or one

Signal transmission molecule of such a receptor and that the cells are incubated in the presence of the test substance.

16. Use according to claim 15, characterized in that the DNA sequence according to aii) is a dominant-negative version of the IGF-1 receptor.

17. Use according to claim 15, characterized in that the DNA sequence according to aii) is a dominant-negative version of an FGF receptor.

18. Use according to claim 15, characterized in that the DNA sequence according to aii) is a dominant-negative version of a PDGF receptor.