JP2001525182A - Apoptosis assay - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for quickly and easily measuring apoptosis. Mammalian cells are co-transfected with a plasmid encoding the fluorescent protein and a plasmid carrying the gene. After culturing and gently fixing, apoptosis is determined by determining the DNA content of the cells using a DNA-binding stain and by determining the percentage of cells transfected by through-flow cytometry. Measure. This method can be used to identify substances that modulate the pro- or anti-apoptotic activity of a gene or gene product.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the field of biological testing. Apoptosis or programmed cell death (PCD) is a genetically controlled cell suicide mechanism to selectively remove unwanted cells (1-4). PCD is an essential process in many biological processes, including embryo and nerve development, regulation of the immune system, organ formation, tissue homeostasis, and prevention of diseases such as tumor growth and viral infections. is there. Apoptosis can be characterized by blistering of cell membranes, shrinking of cells, condensing nuclei, endonuclease cleavage of genomic DNA into fragments of internucleosomal length, and formation of apoptotic bodies.

Methods currently available for studying apoptosis include assessing morphological changes at the cellular level using light, electron or time-lapse microscopy in combination with fluorescent vital stains, membranes during apoptosis Based on the use of Annexin V that can be used to monitor the loss of phospholipid asymmetry (7),
Alternatively, they include assays for detection of DNA fragmentation by gel electrophoresis (8) or in situ labeling of DNA strand breaks ("nick end labeling") (TUNEL) (9). However, most of these methods are not suitable for studying the effects of genes that play a role in apoptosis by transient transfection analysis, or they are very expensive as in the case of the TUNEL method. And there is a lot of work. It was an object of the present invention to provide a new method for measuring apoptosis which overcomes the disadvantages of the known methods.

[0003] This problem was solved by an apoptosis assay characterized by the following features: A) a population of mammalian cells, ai) a DNA sequence or a polypeptide expressed thereby has a pro- or anti-apoptotic activity. A.2) a plasmid containing the DNA sequence to be tested for its presence; or aii) the pro- or anti-apoptotic activity of the DNA sequence or the activity of the polypeptide expressed thereby can be modulated, or by any substance B) a plasmid containing a DNA sequence which is to be examined for: a) a plasmid containing DNA encoding a fluorescent marker protein; and b) a cell containing the DNA sequence or expression in a suitable nutrient medium. Polypeptide has potential activity against apoptosis Until volatilizing, cultured; C) cells were collected and fixed, DN formed on the result during apoptosis
The fluorescent protein remains in the cells while the A fragments can diffuse out of the cells; D) the percentage of apoptotic cells is determined by measuring DNA content; E) the percentage of cells transfected Is determined by measuring cells having the fluorescent marker protein; and F) by comparing the values obtained in steps D and E, the percentage of apoptotic cells in the transfected subcellular population is determined. .

The expression of “DNA sequences” (hereinafter also referred to as “apoptosis test genes”) extends to all DNA sequences that directly or indirectly affect apoptosis, like or in their translation products. . An example of a gene that stimulates apoptosis is p5
3, bax, E1A and examples of genes that inhibit apoptosis are bcl-2, bcl-x, E1B
19K, the latter group further comprising so-called survival factors such as, for example, insulin-like growth factor (IGF). This type of apoptotic genes and their activities have been summarized in articles (eg, 4, 23, 24). The apoptosis test gene can be a known or unknown gene or a fragment thereof. Influencing apoptosis means both inhibiting and attenuating apoptosis, in addition to inducing and enhancing apoptosis.

[0005] The methods of the present invention show significant changes, such as those relating to transfected cells or markers used to measure apoptosis, and those relating to plasmids and transfection methods used to transfect cells. Have been. The method of the present invention has, as one of its essential components, a fluorescent marker protein used to indicate transient transfection of cells. A preferred marker protein is green fluorescent protein (GFP). GFP variants that are made for FACS analysis and are suitable for use within the scope of the present invention are known from the literature. One example of a suitable GFP variant is described in (10) ("Enhanced Green Fluorescent Protein", eGFP); however, within the scope of the present invention, it does not affect cell metabolism, Using conditions that remain localized at the intracellular level and emit a measurable fluorescent signal, and in particular the current method of fluorescence-activated throughflow cytometry (Fluorescence activated cell sorting (FACS)) Other variants can be used that satisfy the condition that they can be measured.

[0006] Other fluorescent marker proteins than green fluorescent protein (GFP) can also be used. Examples are blue fluorescent protein (BFP) (26) and yellow fluorescent protein (YFP) (25). The above properties for the GFP variant are essential for the suitability of the marker protein used in the method of the invention. In addition to the above assays, the type and amount of marker proteins that may be used in the most appropriate transfection method and the plasmids encoding them can be tested, for example, as follows: The resulting plasmid is transiently transfected into mammalian cells, suitably in the same cells and under the same conditions used in the methods of the invention.
The suitability of the transfected marker protein is determined by a series of measurements where the transfection efficiency and the efficiency of the reproducible measurement are determined by FACS analysis.

[0007] The marker used for apoptosis is a DNA-binding stain, such as propidium iodide (PI), which results in a decrease in fluorescence in apoptotic subpopulations (14-17). This detection method is based on the principle that cellular genomic DNA is degraded by endonucleases during apoptosis. Small DNA fragments diffuse out of the cell; the DNA content is reduced to less than half the chromosome set ("sub-2N
") Is an indicator of apoptotic cells. Reduction in PI fluorescence in cells caused apoptosis, results in the appearance of the characteristic fluorescence peak at the portion of the G ₀ / G ₁ phase of the cell cycle ( "sub -2N- peak"). Other DNA-binding stains can be used instead of propidium iodide. Examples of suitable staining solutions of this type include, for example, DAPI (4 ', 6'-diamidino-2
-Phenylindole), acridine orange, ethidium bromide, etc. are commercially available. The most appropriate stain is determined by the stimulation of the cells for apoptosis and then by FACS or microscopic analysis whether the candidate stain can be used to reproducibly measure apoptosis. Can be.

[0008] One of the advantages of the method of the present invention is that the fluorescence and DNA content of the marker protein can be measured simultaneously, preferably by FACS analysis. Apparatus suitable for this is commercially available. The present invention can be applied to any culturable mammalian cells. Matching standard commercial FACS instruments to various cell types is a routine task for those skilled in the art. For transfection of cells with the marker gene on the one hand and the gene on the other hand, any vector which provides efficient and reproducible expression in mammalian cells is suitable. Some of the many available vectors, including those commercially available, have regulatory sequences that can achieve high expression rates in many mammalian cells. Examples are vectors containing the CMV- (cytomegalovirus), SV40- (cyamine virus), MSV (Moloney sarcoma virus) -promoter or other strong promoters not specific to the cell type.

The same or heterologous vectors can be used as carriers for marker genes and genes; to avoid competition between promoters during transcription, vectors with different promoters are used, depending on the cell type. Use can be advantageous.

With regard to the transfection method, the invention is not restricted in any way; in theory, all known methods for transient transfection of mammalian cells can be used. For example, the calcium phosphate method; commercially available cationic lipid methods such as, for example, Lipofectamine or Transfectam;
Based on receptor-mediated adenovirus-assisted endocytosis using polyethyleneimine and psoralen / UV-inactivated adenovirus described, for example, in U.S. Pat. Way. This transfection method uses a series of tests in which the transfection conditions, cell type, nutrient medium, etc. were changed, transfected with a fluorescent marker protein, and
Optimization can be achieved by measuring protein expression by CS analysis. Conditions optimized for this marker protein are used for co-transfection with the gene.

[0011] After transfection, the cells are cultured in an appropriate nutrient medium adapted for the particular cell type. These cells can optionally be stimulated for apoptosis, especially if the apoptosis test gene is examined for any inhibition of apoptosis. Suitable apoptosis stimulating agents are known from the literature and are commercially available; for example, staurosporine, daunomycin and etoposide. The suitability of the culture conditions and apoptosis stimulants is determined in preliminary tests.
With respect to culture, especially with respect to culture time, it is essential that apoptosis occurs to such an extent that a change can be measured by a measuring device such as FACS analysis. The fixation step performed after the culture is essential for performing the procedure of the present invention. An essential requirement for fixation is that small subgenomic DNA fragments (fragments of the nucleosome unit, that is, about 200 bp or more) generated during apoptosis can diffuse out of apoptotic cells, At the same time, the condition is such that the fluorescent marker protein stays inside the cell. In the methods available to date, the requirements on fixation, on the one hand for the measurement of the fluorescent marker protein and on the other hand, for the measurement of the DNA content of the cells, are completely opposite and therefore appear to be incompatible. Was impossible to combine. The present invention is the first to allow both measurements to be performed in the same population of cells using a suitable fixation step.

To determine the appropriate fixation conditions, it is preferred to follow the following procedure: first, on the one hand the best fixation conditions for measuring the fluorescence of the marker protein (strong fixation), and on the other hand the cells The optimal fixation conditions (potentially weak fixation) for measuring the DNA content of each are determined individually. Reagents (fixed reagents, salts, buffers) and their concentrations are started in such a way that the maximum measured value is obtained and the efficiency is minimized when the two measurement operations are performed simultaneously. The fixed condition is changed with respect to the fixed time. Preferably, the primary fixation is carried out with paraformaldehyde and the subsequent treatment with ethanol (secondary fixation / permeabilisation); this treatment has proven to be optimal in the tests performed. 1-4% (weight /
Volume), especially primary fixation with 2% paraformaldehyde, is performed in isotonic buffered saline. In addition to standard solutions such as 100 mM NaCl, 3 mM MgCl ₂ , 300 mM sucrose, commercially available physiologically acceptable standard buffers are suitable. Instead of paraformaldehyde, it is also possible to use other reagents, such as those commonly used in immunohistochemistry. Examples of conventional fixing reagents can be found in the relevant text (27), which includes formaldehyde or chloroform / acetone.

Making the cell membrane slightly permeable, instead of ethanol, which has proven to be particularly suitable under selected conditions in tests carried out on a secondary fixation following the primary fixation with paraformaldehyde, It is theoretically possible to use other reagents, for example surfactants. Transient expression of genes that modulate apoptosis, such as members of the Bcl family or survival factor signaling components, followed by quantitative analysis of apoptosis using the methods of the invention, allows compounds to be specific for the function of apoptosis-modulating genes. It allows the testing of compounds to see if they are able to influence them globally. The method of the present invention can be automated, for example, by appropriate modifications of equipment such as sample preparation and FACS analysis, which makes the method, for example, high throughput screening (Hig
h Throughput Screening), which is suitable for large-scale measurements.

This form of the method is used to identify pharmaceutically active substances that can modulate apoptosis as a function of the expression of a particular gene (apoptosis test gene). A gene whose effect on apoptosis is presumed to be modulated by the test substance is transiently transfected into the test cells, and the test cells are cultured with the test substance from many available substances. The modulating effect of the test substance on the activity of the test gene is directly measured using a measuring device. This type of method can be used for the following screening applications: a) the search for survival factors and their signaling inhibitors in tumor cells, as well as inhibitors of anti-apoptotic gene products; b) in tumor cells The search for specific survival factors and chemicals that inhibit their signaling in synergy with chemotherapy; c) the search for chemotherapeutics that act synergistically with the inhibition of survival factors. In one embodiment, the method of the present invention comprises a survival factor (IGF-I, IGF-I
I, activation of corresponding receptors by FGF (fibroblast growth factor), receptor ligands such as PDGF (platelet-derived growth factor), and subsequent apoptosis of these factors as mediated by a signal cascade Used in screening methods to determine the effect on This assay can be used in a screen to modulate the activity of a known or perhaps unidentified natural survival factor for a course of tumor therapy that enhances apoptosis of tumor cells. The aim of such a method is to identify substances which act synergistically with the inhibition of tumor-specific survival factors, in particular with respect to apoptosis.

To detect such a synergistic effect, the following methods are used: Starting from the tumor cells, the DNA encoding the dominant negative version of the receptor for the survival factor or such a DNA A test cell in which survival factor function is inhibited is prepared by introducing into a cell and expressing a DNA encoding a dominant negative signaling molecule of the receptor. Examples of receptors include the IGF-1 receptor (29), F
GF receptor (30), PDGF receptor (31), EGF-growth factor receptor (32; EGF receptor, Her-2 /
new / ErbB-2, ErbB-3, ErbB-4). Examples of signaling molecules are Ras, Raf, phosphoinositol (3) kinase (= PI (3) -kinase), MAP kinase, protein kinases B and C, phospholipase C, and also Shc, Grb-2. (33; 34; 35; 36; 37). Suitable receptor variants are characterized in that the receptor actually binds the ligand, but the functional domain of the receptor has been modified such that this binding no longer results in activation of the signal cascade. In the case of IGF-1R, this modification includes a complete deletion of the receptor kinase domain or a mutation in the ATP binding site (28). Signaling molecules are modified by inactivation of domains necessary for signal transduction, such as the catalytic domain of an enzyme or the protein binding site of an adapter molecule, or by mutation.

These mutants provided for use in the screening method can be used to transfect the mutant cells into test cells by current methods in a series of tests.
And determination of the degree of apoptosis of test cells using the method of the present invention, to determine their presence in test cells (eg, a fibroblast cell line created in a factor-dependent manner by transfection with an appropriate wild-type receptor) in a preliminary test. Apoptosis Optimized in terms of inducible or augmenting activity. Certain functional domains of this variant may optionally have optimal inhibition of wild-type receptor and subsequent signaling,
As well as further modifying by current molecular biology methods until the maximum level of apoptosis of the resulting test cells is achieved. In screening, test cells are incubated with known chemotherapeutic drugs or substances from the pool being studied for potential chemotherapeutic activity, and their effect on apoptosis is determined by the method of the invention. In particular, this type of screening procedure is designed to find some synergistic activity between inhibition or lack of survival factor function in tumor cells and a known chemotherapeutic agent or potential chemotherapeutic substance.

In order to use this screening method to find a substance that exerts a synergistic effect with inhibition of survival factor function specific to a specific tumor type, cells derived from various tumor types are used. It can be used in a parallel screening test under the same experimental conditions. Cells used as control cells for the specificity of synergistic activity between lack of survival factor function and chemotherapy are cells that are naturally deficient in a particular survival factor function, the inhibition of which is examined in the assay. In theory, however, it is also possible to look for substances which increase the activity of the apoptosis-inducing or -increasing molecule. Examples are members of the TNF receptor family (TNF receptors, Fas) and the molecular caspases of their signaling pathways. The test principle is exactly the same as the apoptosis-inhibiting survival factor, except that the wild-type or constitutively active form of these apoptotic molecules is expressed in tumor cells. Therefore, the focus of this screening is mainly on the search for synergism that leads to increased apoptosis of tumor cells. This provides a prerequisite for therapies such that the dose of chemotherapeutic agent can be significantly reduced without affecting the outcome of the treatment, while at the same time inhibiting the survival function of the tumor cells. Decreasing the chemotherapeutic dose is a significant benefit to the patient, as a result of which the toxic side effects of chemotherapy are greatly reduced.

Within the scope of the present invention, the survival factor IGF-, which results in the survival of β-tumor cells
The method of the present invention is used to determine whether the signal mediated by II was transmitted by the IGF receptor. For this purpose, the dominant negative form of the human IGF-1 receptor (dnIGF-1R), which has an amino acid substitution at the ATP-binding site (dnIGF-1R), converts green fluorescent protein (eGFP) into wild-type β-tumor cells. Transiently co-transfected with the retained plasmid. These cells are then
Cultured with apoptosis stimulants and / or growth factors, collected, fixed and stained with propidium iodide, the DNA content is measured. Using FACS analysis, single transfected cells expressing eGFP were detected, and apoptotic cells were identified with a DNA content of less than 2N in this population. It has been found that transfection with a plasmid encoding dnIGF-1R results in a dramatic increase in apoptosis in both untreated wild-type β-tumor cells and such cells treated with daunomycin or etoposide. ing. dnIGF-1R has been shown to enhance apoptosis in β-tumor cells with almost the same efficiency as the adenovirus-EIA protein, where one of the most potent apoptosis-inducing gene products is present. Thus, by using the assays of the present invention, tumor cells are more sensitive to apoptosis stimulators when IGF-1R signaling is disrupted, and as in IGF-II-deficient tumor cells. Could show greater sensitivity to chemotherapeutic drugs.

In addition, the methods of the present invention can be used to determine whether a known gene modulates apoptosis in various cell types, and to what extent. Another use of the method of the invention is the expression cloning of genes that modulate apoptosis. At this time, the complete cDNA expression library was transiently transfected into cells. The method of the present invention is capable of measuring the effect of gene expression within 24-48 hours. Thus, it is possible to analyze and isolate cells while they are alive. To this end, the method of the invention is modified using FACS sorting to isolate single cells that deviate from the background of apoptosis measured. Plasmids transfected into these cells are isolated, amplified and selected for further transfection steps. Thus, a plasmid containing the apoptosis-modulating gene is isolated. The corresponding gene is then characterized by sequencing and other tests for expression and function.

To test the method according to the invention, first in Example 1, on the one hand with the GFP plasmid and on the other hand with the plasmid containing the pro-apoptotic or anti-apoptotic gene sequence or with the control plasmid Transfected established tumor cell lines were used. After transfection, the cells were treated with an apoptosis stimulator after a certain period of time (control cells were not treated). Thereafter, detached cells were collected and combined with trypsinized adherent cells, washed and fixed. After washing, these cells are treated with the method of the invention and fluorescent Cy5-dCTP.
(5-Amino-propargyl-2'-deoxycytidine-5'-conjugated to Cy5 fluorescence stain)
(Triphosphoric acid) so as to allow comparison with the conventional TUNEL method. It has been found that the method of the invention reliably detects the pro- or anti-apoptotic effect of the predicted gene product and that the addition of an apoptosis stimulant enhances the observed effect. Under selected conditions, the sensitivity of the method of the invention is slightly lower than that of the TUNEL method, but normalized as a ratio of the maximum apoptotic value in a particular assay achieved by the pro-apoptotic gene. Apoptosis values are virtually the same. Compared with the TUNEL method, the method of the present invention has advantages in terms of speed, convenience and economy.

The versatility and reliability of the method of the invention was confirmed by the use of a non-transformed rat fibroblast cell line that responded less to apoptosis stimulators than established tumor cell lines. The method of the invention makes it possible to quickly, efficiently and reproducibly establish the potential role of the gene product in apoptosis. In another aspect, the present invention relates to a kit for performing a convenient method as a routine operation. A kit of this type will contain the following components in a number of individual containers to suit the purpose: a) one or more components required for transfection; b) encoding the fluorescent marker protein C) an empty vector for inserting specific DNA sequences and for control measurements; d) a primary fixative, such as a paraformaldehyde solution; e) a secondary fixative / permeate solution. F) wash solution; g) DNA-binding stain. A preferred kit contains polyethyleneimine and psoralen / UV-inactivated adenovirus as components of the transfection.

Example 1 In this example, the regulatory region of the insulin gene (Rip) specifically induces the expression of the large T-antigen (Tag) of siamin virus 40 in islet β cells (16).
), Established tumor cell lines (βTC and βHC) derived from β-cell tumors of transgenic mice (15).

Approximately 80,000 cells are seeded in 6 cm wells of a 6-well culture dish and 10% (vol / vol) FCS, 2 mM glutamine, 100 International Units (IU) penicillin and 100 μg / ml streptomycin are added. Cultured in supplemented DMEM to reach 70% confluency. These cells contain 1 μg of a plasmid encoding eGFP (“enhanced GFP”; pEGFP-C1; Clontech), 1 μg of a control plasmid (pMEX; (22)), and the pro-apoptotic adenovirus gene E1A. LipofectAMIN with the pCMV plasmid or the pCMV plasmid (17,18) containing the anti-apoptotic adenovirus gene E1B-19K
Transfection was performed using 10 μl E (GIBCO-BRL) according to the manufacturer's instructions. After transfection, the cells were left in complete medium for 16 hours, after which the cells were either treated or untreated with an apoptosis stimulator (staurosporine 800 ng / ml, Sigma) for another 16 hours (19, 20). 32 hours after transfection, detached cells are combined with trypsinized adherent cells and mixed with 4 ml of PBS.
Washed twice and fixed at room temperature for 30 minutes (2% paraformaldehyde, 100 mM NaCl, 300
ml sucrose, 3 mM MgCl ₂ , 1 mM EGTA (ethylene glycol-bis- (2-aminoethyl) -tetraacetic acid), 10 mM PIPES (piperazine-N ₁ N ¹ -bis [z-ethanesulfonic acid])
, PH 6.8). They were then washed twice with 4 ml of PBS and fixed with ice-cold 70% EtOH for 14 hours.

After fixation, cells were washed twice with 4 ml of PBS and split. Half of the sample was treated with RNase A (Sigma, St. Louis, USA) (50 μg / ml) using PBS as a solvent for 30 min.
Washed twice with 1 l of PBS and stained with propidium iodide in PBS (PI; 50 μg / ml; Sigma, St. Louis, USA) 30 minutes before FACS analysis. Half of the remaining sample was added to 50 μl of TdT reaction mixture (terminal deoxynucleotidyl transferase; Boehringer Mannheim; 200 mM potassium cacodylate, 25 mM Tris
-HCl, pH6.6,0.25mg / ml bovine serum albumin, 1mM CoCl _2; 0.25nmol FluoroLink
Incubate at 37 ° C. for 1 hour with Cy5AP3-dCTP (Amersham, 12.5 units TdT), wash twice with 4 ml of PBS, and wash with RNase (50 μg / ml) in PBS.
Treat for 30 minutes, wash twice with 4 ml of HBS (DAPI has a tendency to cause microprecipitation in PBS, so use HBS instead of PBS after this step).
Stained with API (10 μg / ml; Sigma) for 20 minutes, and Vectom Dickinson F
Analysis was performed using an ACS Vantage instrument. FACS analysis of the PI-stained cells was performed by Vectom Dickinson, equipped with a so-called "doublet discrimination module", in which cell aggregates were identified by calculating pulse width and pulse width. This was performed using a FACScan apparatus. The test results are shown in FIG. FIG. 1A shows the number of apoptotic βHC13T tumor cells (% apoptosis) in all eGFP-positive cell groups. Black bars indicate measurement of sub-2N DNA content.
(GFP / PI); open bars indicate incorporation of fluorescent Cy5AP3-dCTP during TdT reaction (GFP / TUN
EL). The addition of staurosporine is noted. Excitation wavelength 488 for eGFP and PI
nm, the excitation wavelength for Cy5 was 647 nm, and the UV wavelength 51-364 nm was used for DAPI. Emission fluorescence was measured using a 530/20 nm narrow band filter for eGFP, a 610 nm blocking filter for PI, and 67 for Cy5.
The 5/20 nm narrow band filter was used, and the DAPI was collected using a 424/44 narrow band filter. Doublets were excluded by pulse processing. eGFP
-Expressing cells were selected and analyzed for Cy5- or PI-fluorescence. Data is CELLQu
Analysis was performed using est software (Vectom Dickinson). Each bar represents the average of three transfections, and the standard deviation is represented by an error bar. Each measurement consists of 40,000 total events selected according to size and single cell. Transfection efficiency was 20-30%.

FIG. 1B shows the normalized percentage of apoptosis for various constructs.
Apoptosis index was normalized using the following formula: (% apoptosis in X / eG
% Apoptosis in FP-C1 / E1A) × 100. The apoptosis index was normalized for each of the detection methods used and for each subsequent transfection treatment (+/- staurosporine).

Example 2 In this example, a non-transformed rat fibroblast cell line called Rat1A was used. These cells were isolated from LipofectAMINE as described in Example 1 or from a polyethylenimine (PEI 2000) -DNA-adenovirus complex (WO 93/93).
/ 07283) was used for transient transfection. Furthermore, with regard to the treatment of the cells and the determination of apoptosis, on the one hand the method of the invention was used and on the other hand the TUNEL method was used, the procedure being exactly the same as that shown in Example 1. A comparison of the different transfection and apoptosis assays is shown in the table. Each value represents the average of three transfections; standard deviation is shown in (sd).
The efficiency of these transfection methods was 25-30%.

Example 3 In this example, wild-type β-tumor cells (15) were used. A dominant-negative IGF-1 receptor construct was used that was under the control of the CMV promoter and had the ATP binding site codon 1003 mutated from lysine to alanine, which was described in (28). Have been. As shown in the previous example, wild-type β-tumor cells were seeded in 6-well culture dishes in triplicate to a density of 80,000 cells. 24 hours later
These cells were transformed with pEGFP-C1 and a control plasmid (FIG. 2: pMEX / ctr), or pEGFP-C1.
C1 and adenovirus-E1A (FIG. 2: E1A), adenovirus-E1B-19K (FIG. 2: E1B)
-19K) or an expression plasmid encoding either dominant-negative IGF-1R. 36 hours after transfection, daunomycin (FIG. 2: shaded bars) or etoposide (FIG. 2: open bars) was added to the medium at a concentration of 1 μM or 10 μM. Transfected but untreated cells are indicated by black bars in FIG. Twelve hours after treatment with daunomycin or etoposide, cells were harvested, fixed, and treated with propidium iodide as described in the previous examples. In addition, apoptotic cells were determined using the method described above.
For each measurement, 40,000 events were collected; transfection efficiency was 25-30%. Standard deviation is indicated by an error bar.

[0028]

[Table 1]

──────────────────────────────────────────────────の Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), CA, JP, MX, US (72) Inventor Ram Gabo, Germany Day 80796 München Hohenzollenstraße 120 (72) Inventor Christophorie Gerhard Austria Ar 1230 Wien au Gustig Greimleweg 17F Terms (reference) 2G045 AA40 BB20 BB22 BB24 CB02 CB17 CB30 DA12 DA13 DA36 FA11 FA16 FA37 FB12 FB20 HA09 HA20 4B024 AA01 AA11 CA01 DA02 EA04 GA11 HA11 4B063 QA01 QA05 QA19 QQ08 QQ42 QR33 QR59 QR66

Claims (18)

[Claims] 1. A method for measuring apoptosis, characterized by: A) a population of mammalian cells is examined for ai) whether a DNA sequence or a polypeptide expressed thereby has pro- or anti-apoptotic activity. A plasmid containing a DNA sequence; or aii) a DNA sequence to be investigated for which pro- or anti-apoptotic activity of the DNA sequence or the activity of the polypeptide expressed thereby can be modulated or by which substance. B) the cells are transiently transfected with a plasmid containing DNA encoding the fluorescent marker protein; and Cultivated to exert potential activity; C) Cells are harvested and fixed Is, DN formed on the result during apoptosis
The fluorescent protein remains in the cells while the A fragments can diffuse out of the cells; D) the percentage of apoptotic cells is determined by measuring DNA content; E) the percentage of cells transfected Is determined by measuring cells having the fluorescent marker protein; and F) by comparing the values obtained in steps D and E, the percentage of apoptotic cells in the transfected subcellular population is determined. . 2. The method according to claim 1, wherein said transfection of said cells is carried out with polyethyleneimine and an inactivated adenovirus.
The described method. 3. The method according to claim 1, wherein the fluorescent polypeptide defined in Ab) is a green fluorescent protein. 4. The method according to claim 1, wherein said DNA content is measured by a DNA-binding stain. 5. The method according to claim 1, wherein the staining solution is propidium iodide.
The method of claim 4. 6. The method according to claim 1, wherein the culturing in step B) is performed in the presence of a test substance. 7. The method according to claim 1, wherein the culturing in step B) is carried out in the presence of a substance that stimulates apoptosis. 8. The primary fixation of step C) is performed with paraformaldehyde,
The second fixation / permeabilization of the cells is performed with ethanol.
The method according to any one of claims 7 to 10. 9. The method according to claim 1, wherein the measurements defined in steps D and E are performed in one step using fluorescence-activated through-flow cytometry analysis. . 10. Kit for carrying out the method according to claim 1, characterized in that several individual containers contain the following components: a) one or more components necessary for transfection B) a plasmid containing a sequence encoding a fluorescent marker protein; c) an empty vector for inserting specific DNA sequences and for control measurements; d) a primary fixation solution; e) a secondary fixation / permeation solution; f) Wash solution; g) DNA-binding stain. 11. Polyethyleneimine and psoralen / UV as component a)
11. The kit of claim 10, comprising an inactivated adenovirus. 12. The kit according to claim 10, comprising as component b) a plasmid encoding a green fluorescent protein. 13. The kit of claim 10, comprising approximately 2% paraformaldehyde solution as component d) and about 70% ethanol as component e). 14. Use of the method according to claim 1 for identifying a substance that modulates the pro- or anti-apoptotic activity of a gene or gene product. 15. The use according to claim 14, for identifying a therapeutically effective substance exhibiting a synergistic activity with inhibition or deficiency of a survival factor function derived from a tumor cell,
The cell is a tumor cell and the DNA sequence of aii) is a dominant-negative form of a receptor for a survival factor specific for the tumor cell or a dominant-negative form of a signaling molecule of such a receptor. And wherein the cells are cultured in the presence of the test substance. 16. Use according to claim 15, characterized in that the DNA sequence of aii) is the dominant-negative form of the IGF-1 receptor. 17. The use according to claim 15, wherein the DNA sequence of aii) is a dominant-negative form of the FGF receptor. 18. The use according to claim 15, wherein the DNA sequence of aii) is a dominant-negative form of the PDGF receptor.