EP1246940A2 - Procede de criblage pour substances chimiotherapeutiques - Google Patents

Procede de criblage pour substances chimiotherapeutiques

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Publication number
EP1246940A2
EP1246940A2 EP00991118A EP00991118A EP1246940A2 EP 1246940 A2 EP1246940 A2 EP 1246940A2 EP 00991118 A EP00991118 A EP 00991118A EP 00991118 A EP00991118 A EP 00991118A EP 1246940 A2 EP1246940 A2 EP 1246940A2
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EP
European Patent Office
Prior art keywords
cells
cd95l
promoter
test compound
chemotherapy
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00991118A
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German (de)
English (en)
Inventor
Peter Krammer
Sören EICHHORST
Min Li-Weber
Martina MÜLLER-SCHILLING
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Deutsches Krebsforschungszentrum DKFZ
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Deutsches Krebsforschungszentrum DKFZ
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the present invention relates to a method for examining whether a test compound is effective for chemotherapy, the method comprising the following steps: a) provision of a DNA sequence, preferably the AP-1 site shown in FIG. 5A, as part of a promoter the CD95L promoter, which is operably linked to the CD95L gene or a reporter gene, b) contacting the DNA sequence of a) with the test compound in a cellular assay, and c) determining the activation of the promoter, indicating activation that the test compound is effective for chemotherapy.
  • the conversion from normal cells to cancer cells takes place in several steps, which involve the mutation of cellular genes (proto-oncogenes and tumor suppressor genes) or the acquisition of viral oncogenes.
  • Cancer-relevant changes are the activation of proto-oncogenes by mutations, gene amplification, overexpression or chromosomal transpositions, and the inactivation of tumor suppressor genes by mutations, for example deletions.
  • the tumor suppressor genes turned out to be particularly interesting, since they obviously offer new possibilities in cancer therapy.
  • the present invention is therefore based on the technical problem of providing methods for identifying compounds which are effective for chemotherapy, for example in combating cancer.
  • Chemotherapeutic agents include compounds that can induce apoptosis in tumor cells. It was found that the CD95 (Apo-1 / Fas) / CD95L system is involved in the induction of apoptosis in lymphoid and non-lymphoid tissues, for example liver tissue and intestinal tissue, and plays a key role in the regulation of apoptosis. Upregulation of the ligand to CD95 (CD95L) is probably one of the most important mechanisms by which cancer drugs can induce apoptosis in tumors. In the course of the investigations which led to the present invention, it was found that the upregulation of CD95L is at least functionally relevant in liver cells and takes place at the level of the transcription regulation.
  • CD95L basal promoter 136 bp
  • An AP-1 element within the 5 'untranslated region (5'UTR) downstream of a "TATA box” is required to induce the promoter.
  • This increase in the transcription rate is transcription and translation dependent and is mediated by the binding of a Jun / Fos heterodimer to this AP-1 site.
  • chemotherapeutic agents induce the CD95 gene via a transcriptionally regulated p53-dependent mechanism.
  • chemotherapy drugs act on the SAPK / JNK pathway, which ultimately leads to the upregulation of CD95L.
  • the cell can either commit "suicide” or kill its neighboring cell using a mechanism known as "fratricide”. It was also shown that the effect of the chemotherapeutic agents on the CD95L promoter is delayed. A steep increase in mRNA levels and the promoter activity measured by activating the luciferase gene as a reprotergen occurs 20 to 25 hours after treatment with chemotherapeutic agents, and this increase continues afterwards.
  • chemotherapeutic agents eg 5-FU and etoposide
  • These observations can be used to improve chemotherapy for various tumors, for example, as already discussed above, can also be used to establish a screening system for isolating new chemotherapeutic agents, and can lead to an improved understanding of the side effects when administering chemotherapeutic agents to patients. It should also be possible to isolate or develop new chemotherapeutic agents that allow treatment of tumors that are already resistant to the previously known chemotherapeutic agents. It should also be possible to find chemotherapy drugs that have a higher specificity, resulting in fewer side effects on healthy cells.
  • One embodiment of the present invention thus relates to a method for examining whether a test compound is effective for chemotherapy, the method comprising the following steps: a) providing a DNA sequence which the AP-1 site shown in FIG. 5A as part of a Promoter, preferably the CD95L promoter, which is operably linked to a reporter gene or the CDS95L gene; b) contacting the DNA sequence of a) with the test compound in a cellular assay; and c) determining activation of the promoter, activation indicating that the test compound is effective for chemotherapy.
  • AP-1 site includes the AP-1 site (a) with the sequence shown in Figure 5A and (b) with a sequence different from that shown in Figure 5A by one or more insertions, exchanges or Additions of nucleotide discriminating sequence, these changes do not lead to a loss of the AP-1 site in terms of activatability by the factors discussed here.
  • test compounds can be very different compounds, both naturally occurring as well as synthetic, organic and inorganic compounds, as well as polymers (e.g. oligopeptides, polypeptides, oligonucleotides and polynucleotides) as well as small molecules, antibodies, sugars, fatty acids, nucleotides and nucleotides. Analogs, analogs of naturally occurring structures (eg peptide "imitators", nucleic acid analogs etc.) and numerous other compounds.
  • polymers e.g. oligopeptides, polypeptides, oligonucleotides and polynucleotides
  • small molecules antibodies, sugars, fatty acids, nucleotides and nucleotides.
  • analogs of naturally occurring structures eg peptide "imitators", nucleic acid analogs etc.
  • the person skilled in the art can use customary cells, such as HepG2 or Hep3B, to produce the cellular assay and insert the DNA sequence of step a) into these by means of customary methods, such as transfection or electroporation.
  • customary methods such as transfection or electroporation.
  • Sambrook J. et al. supra referenced.
  • the connection can then e.g. be added via the medium. Care must be taken to ensure that the test compound comes into contact with the DNA sequence of step a) under such conditions that specific binding is made possible.
  • the values found in terms of the transcription rate are preferably compared with the values in a second test system that differs from the first only in the absence of the test compound.
  • suitable assay formats for the identification of compounds that affect the expression of CD95L are well known in the biotechnological and pharmaceutical industries and additional assays and variations of the assay provided above for illustration are obvious to those skilled in the art.
  • promoters such as the trail receptor promoter or the CD95 promoter are also suitable for the method according to the invention.
  • Changes in promoter activity can be measured by any suitable method. Changes in expression level can be examined using methods well known to those skilled in the art. This includes monitoring the mRNA concentration (eg using suitable probes or primers), immunoassays for the protein concentration (eg using the antibodies described below), RNAs e - Schu tzass ay s, Amplification assays or any other means suitable for detection known in the art. Further detection methods regarding the activation of the promoter depend on the specific properties of the respective reporter gene.
  • test compound is part of a substance library.
  • a large number of potentially useful molecules can be screened in a single test. For example, if a field of 1000 compounds is to be screened, in principle all 1000 compounds can be placed in a microtiter plate well and tested simultaneously. If a promoter activator is discovered, the pool of 1000 can then be divided into 10 pools of 100 and the process repeated until an individual activator is identified. In any event, the production and simultaneous screening of large banks of synthetic molecules can be carried out using well known combinatorial chemistry techniques, see for example van Breemen, Anal. Chem. 69. (1997), 2159-2164 and Lam, Anticancer Drug Des. 12th (1997), 145-167.
  • the method according to the invention can also be greatly accelerated as high throughput screening.
  • the assays regarding promoter activation described here can be modified accordingly for use in such a method. It it will be apparent to those skilled in the art that numerous methods are available for this purpose.
  • a large number of potentially useful promoter activity-modifying compounds can be screened in extracts from natural products as a starting material.
  • Such extracts can come from a large number of sources, for example the species fungi, actinomycetes, algae, insects, protozoa, plants and bacteria.
  • the extracts showing activity can then be analyzed to isolate the active molecule. See, for example, Turner, J. Ethnopharmacol. 51 (1-3) (1996), 39-43 and Suh, Anticancer Res. 15 (1995) 233-239.
  • the DNA sequence containing the AP-1 site in step a) corresponds to the nucleic acid sequence shown in FIG. 5A from positions -36 to +100 or a fragment thereof (the fragment still the AP- 1-digit), most preferred from positions +84 to +91.
  • the reporter gene is a CAT, luciferase, LacZ or GFP gene.
  • a further embodiment of the present invention relates to a medicament which comprises a mixture of at least two compounds which have been identified according to the method according to the invention and / or which promote expression of the CD95L gene. Since it was shown in the examples below that the simultaneous administration of different compounds which are associated with the promotion of the expression of CD95L leads to synergistic effects, such a procedure is of particular therapeutic interest since, for example, the dosage of the individual substances is (greatly) reduced potential harmful side effects or at least can be reduced.
  • the medicament according to the invention is preferably in combination with a pharmaceutically acceptable carrier.
  • Suitable carriers include, for example, phosphate-buffered saline solutions, water, emulsions, for example oil / water emulsions, wetting agents, sterile solutions, etc.
  • the medicaments according to the invention can be administered orally or, preferably, parenterally.
  • Methods for parenteral administration include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
  • the appropriate dosage is determined by the attending physician and depends on various factors, for example the age, gender, weight of the patient, the stage of the disease, for example the tumor, the type of administration, etc.
  • the present invention relates to the use of the medicament according to the invention for the therapy of various diseases, in particular cancer and autoimmune diseases, such as diabetes, multiple sclerosis and rheumatoid arthritis.
  • HepG2 cells were grown to near confluence and then with 100 ⁇ g / ml 5-FU for the indicated periods either in the presence of an isotype-matched control antibody or in the presence of the anti-CD95L antibody NOK-1 (50 ⁇ g / ml ) incubated.
  • the same experiment was carried out with the addition of CD95-Fc (50 ⁇ g / ml).
  • Apoptosis was determined by PI exclusion and measurement of the forward / side scatter (FSC / SSC). The data shown represent mean values with standard deviations from three samples.
  • Various experiments were carried out with a similar result.
  • Figure 2 After stimulation with various chemotherapeutic agents, CD95L mRNA is induced and this induction is regulated at the transcription level
  • PCR analyzes of HepG2 and Hep3B cells were carried out. Total RNA was extracted and RT-PCR was performed as described in Example 1.
  • a + B HepG2 and Hep3B cells were incubated for 36 hours with the indicated concentrations of bleomycin or 5-FU.
  • C Hep3B cells were incubated with 100 ⁇ g / ml 5-FU for the periods indicated.
  • D + E 5- FU (100 ⁇ g / ml) was added to HepG2 cells for 48 hours and either the transcritical ion inhibitor actinomycin Cl (D) or the translation inhibitor cycloheximide (E) in the stated concentrations.
  • a 51 Cr release assay was used with Hep3B cells as effectors and SKW6.4 cells as target. Hep3B cells were grown in 96-well plates and treated with 5-FU. After 48 hours, the chemotherapeutic agents were removed from the culture medium and 51 Cr-labeled SKW6.4 cells were added either with a control antibody or with the anti-CD95L antibody NOK-1. After overnight incubation, the supernatants were measured in a gamma counter. The relative lysis was calculated as indicated in Example 1. E / T is the ratio between effector and target cells. Each series of concentrations was carried out in triplicate. The diagram represents an experiment from a series of independent experiments with the same result.
  • FIG. 4 The upregulation of the CD95L promoter by chemotherapeutic agents is potentiated by simultaneous stimulation with different chemotherapeutic compounds and is concentration-dependent
  • A Potentiation effect of different chemotherapeutic agents on the CD95L promoter.
  • Hep3B cells were transfected with the -36 / + 100 construct and the luciferase activity was determined after 48 hours of treatment with 5 ⁇ M etoposide (eto), 100 ⁇ g / ml 5-FU or a combination of both agents. Each bar represents the calculated value from treated / untreated cells.
  • the transfection efficiency in A and B was monitored by cotransfection of a Renilla luciferase construct that was under the control of a basal promoter.
  • Figure 5 A region encompassing the nucleotides +20 to +100 within the 5'UTR of the CD95L gene is for the
  • A Overview of the 5'UTR of the CD95L gene. Bars represent the -36 / + 100 and -36 / + 100 constructs. The boxed sequence is the AP-1 site near the first ATG codon. The arrow shows the translation start point.
  • B Hep3B cells were transfected with the constructs described in A and the luciferase activity was measured after 48 hours of treatment with 5-FU (100 ⁇ g / ml). A representative experiment of five independent experiments (triple samples) is shown. Pnull.luc is a construct used as a negative control without a promoter. The transfection efficiency was monitored by cotransfection with a Renilla luciferase construct.
  • FIG. 6 The -36 / + 100 and -36 / + 19 constructs behave differently with respect to the kinetics of activation and inducibility by cotransfection with c-iun and c-fos.
  • A Cotrans fect ion experiments e in Hep3B cells with expression vectors for c-jun and c-fos. The cotransfected cells were then either treated with 100 ⁇ g / ml 5-FU for 48 hours or left untreated. The transfection efficiency was determined either by cotransfection of an expression vector for chlora phenicoltransferase (CAT) or Renilla luciferase (both under the control of a basal promoter) normalized.
  • CAT chlora phenicoltransferase
  • Renilla luciferase both under the control of a basal promoter
  • the luciferase activity was measured using the "dual luciferase” assay (Promega) according to the manufacturer's instructions, the CAT activity was determined by means of a commercially available CAT ELISA. Average values (with standard deviation) from various independent experiments are shown.
  • B “Dual luciferase” assay with Hep3B cells which had been cotransfected with the CD95L promoter construct described and a Renilla luciferase expression vector (as a control for the transfection efficiency). The cells were harvested at the times indicated. In addition, the protein content of the transfected cells was determined. The values are mean values
  • FIG. 7 Nuclear extracts from liver cell lines treated with chemotherapeutic agents shift the mobility of an oligonucleotide which contains the AP-1 sequence in the CD95L promoter
  • EMSA and "subershift" analyzes of the +73 / +99 region of the human CD95L promoter sequence were carried out.
  • Nuclear extracts from HepG2 and Huh7 cells were prepared as described in Example 1. The cells were either treated with 100 ⁇ g / ml 5-FU for 48 hours or left untreated.
  • EMSA analyzes were carried out as described recently.
  • Antibodies against c-Jun or c-Fos were added for the "supershift” analyzes.
  • the isotype-matched anti-C / EBP antibody was used as a control. 0: negative control without nuclear extracts; -: untreated cells; +: treated cells. Antibodies were added as indicated.
  • the AP-1 site in the basal promoter was mutated as indicated.
  • Hep3B cells were treated with the -36 / + 100 construct (CD95L prom wt) or the APX4 construct (mutated -36 / + 100 CD95L. luc; CD95L prom mut) transfected.
  • the transfection efficiency was monitored by cotransfection with Renilla luciferase. After transfection, the cells were treated with 5-FU (100 ⁇ g / ml) for 48 hours. The luciferase activity was measured and the multiplication of the induction was calculated. A representative experiment (out of a total of 5 experiments) is shown.
  • A Influence of DN c-jun.
  • Hep3B cells were labeled with the -36 / + 100-, -36 / + 19- or pnull. Luc construct transfected. The cells were cotransfected with either a control plasmid (c) or an expression construct for dominant-negative c-jun (+). As a control, c-jun was additionally cotransfected in an experiment. After the transfection was completed, the cells were divided. One half was treated with 100 ⁇ g / ml 5-FU, the other half remained untreated. The bars show the multiplication of the induction, which was calculated as follows: RLU (treated cells) / RLU (untreated cells).
  • Relative luciferase units were normalized by Renilla luciferase activity using the "dual luciferase" system. Three independent experiments were carried out, one of which is shown.
  • B Influence of the JNK / SAPK cascade. Co-transfection experiments with a control vector (pUCSV) or dominant-negative mutants for the stress-activated protein kinases JNKK-1, MEKK-1, MKK3 and MKK6 were carried out. The induction multiplication values were calculated as in A. One of three independent experiments is shown.
  • FIG. 10 C-jun is upregulated in Hep3B cells and human primary hepatocytes after treatment with chemotherapeutic agents
  • a + B Hep3B cells were seeded on LabTek TM culture supports and grown for two days. The cells were then either left untreated (A) or 40 hours with 5-FU (100 ⁇ g / ml) treated (B), fixed and stained as described.
  • C + D Human primary hepatocytes were isolated as described in Example 1 and sown on culture carriers. The cells were then either left untreated (C) or treated with 5-FU (50 ⁇ g / ml) for 40 hours (D), fixed and stained with an antibody specific for c-Jun.
  • HepG2 which is derived from human liver blastoma and expresses wild-type p53 at low concentration
  • Huh7 which is derived from human hepatocellular carcinoma and expresses a mutated form of p53 in which a point mutation of codon 220 leads to a shorter half-life of p53
  • Hep3B which is derived from a human hepatocellular carcinoma and is deficient for p53
  • SKW6.4 a human T cell leukemia cell line.
  • HepG2, Hu7 and HepG3 cells were grown in DMEM (Gibco BRL, Eggenstein, Germany) containing 10% heat inactivated fetal calf serum (FCS) (Gibco BRL), 10 mM HEPES (Gibco BRL), 5 mM L-glutamine (Gibco BRL) and 100 ⁇ g / ml gentamycin (Gibco BRL) had been supplemented.
  • FCS heat inactivated fetal calf serum
  • HEPES Gibco BRL
  • 5 mM L-glutamine Gabco BRL
  • 100 ⁇ g / ml gentamycin Gabco BRL
  • SKW6.4 cells were kept in RPMI medium (Gibco BRL) containing 10% FCS (Gibco BRL), 10 mM HEPES (Gibco BRL), 2 mM L-glutamine (Gibco BRL) and 100 ⁇ g / ml gentamycin (Gibco BRL) included.
  • the cells were washed twice with WME and in maintenance medium to a density of 1 , 0- l, 5xl0 5 living cells / cm 2 sown on collagen-coated culture plates (collagen type I, Serva Biochemicals, Germany). Viability was determined using the trypan blue exclusion method Maintenance medium WME was used, which had been supplemented with the following compounds: 5 mM L-glutamine (Flow Laboratory, Germany), 0.6% glucose, 0.02M HEPES, 50 ⁇ g gentamycin (Sigma), 100 ⁇ g / ml penicillin (Flow Laboratory), 100 ⁇ g / ml streptomycin (Flow Laboratory), 37 ⁇ M inosine (Serva), 17.4% DMSO (Merck, Darmstadt, Germany) and 0.14 U / ml insulin (Serva) the maintenance medium was supplemented with 10% FCS (Gibco BRL). The cells were at 37 ° C. and 5% C0 2 incubated.
  • FCS Gibco BRL
  • CD95L promoter Plasmids The serial deletion constructs of the CD95L promoter were in the pTATA. Luc vector (available from T. Wirth, Institute for Medical Radiation and Cell Research, Wuerzburg, Germany) or cloned into the pGL2 base vector (Promega, Madison, Wisconsin, USA). The deletion constructs from 2269 / + 100 to -36 / + 100 were prepared as recently described (Li-Weber et al., European Journal of Immunology 28 (1998), 2373). The -36 / + 19 vector was constructed as follows: The -36 / 100-CD95L promoter fragment was cut with Psp5II and the smaller fragment obtained again in the pTATA. Luc vector cloned.
  • DN-MKK3, DN-MKK6 and pUCSV constructs have already been described (Angel et al., Nature 332 (1988), 166).
  • Dominant negative c-jun cells ( ⁇ aa 1-192) and the empty control vector pCMV were provided by D. Bohmann (Leppa et al., EMBO Journal 17 (1998), 4404).
  • Mutations in the +90 API site on the -36 / + 100 construct (APX-4) were introduced using the "QuikChange" mutagenesis kit (Stratagene Corp., La Jolla, California, USA).
  • the following primers (MWG Biotech GmbH, Ebersberg, Germany) were used for the mutagenesis reaction: APX-4 / Sense: 5 '-CCG TTT GCT GGG GCT GGC CTA ATT AAC CAG CTG CCT CTA GAG G-3'; APX-4 / Antisense: CCT CTA GAG GCA GCT GGT TAA TTA GGC CAG CCC CAG CAA ACG G-3 '.
  • the underlined nucleotides represent mutated sites compared to the wild type sequence of the CD95L promoter. The mutations were checked and confirmed by automated sequencing (TOPLAB GmbH, Kunststoff, Germany).
  • Neutralizing anti-CD95L antibodies (NOK-1) and isotype-matched control antibodies were obtained from Pharmingen (Hamburg, Germany). Antibodies directed against c-jun, c-fos and CEBP for "supershift" analyzes were from Santa Cruz Biotechnology Inc. (Heidelberg, Germany). The cells were treated with mAb IgG3-anti-APO-1 at a concentration of 100 ng / ml as described recently (Berndt et al., PNAS USA 95 (1998), 12556).
  • Cell cultures were treated with bleomycin (cell pharm GmbH, Hanover, Germany) in a dose range from 1 ⁇ g / ml to 1 mg / ml, 5-FU (ribosepharm GmbH, Kunststoff, Germany) at one Concentration from 10 ⁇ g / ml to 150 ⁇ g / ml, etoposide (Bristol-Myers Sqibb GmbH, Kunststoff, Germany) in a dose range of 1 to 5 ⁇ M or cisplatin (ribosepharm GmbH, Munich, Germany) in a dose range of 0.5 ⁇ g / ml to 2.0 ⁇ g / ml treated. The cells were incubated with the different chemotherapeutic agents for 2 to 64 hours.
  • the clinically relevant concentrations in human cancer therapy are as follows: bleomycin: 1.5 ⁇ g / ml to 3.0 ⁇ g / ml; 5-FU: -; Etoposide: -; Cisplatin: 0.4 ⁇ g / ml to 1.6 ⁇ g / ml.
  • the cells were trypsinized with 1% trypsin-EDTA for 5 min, washed twice with PBS and stained with 2.5 ⁇ g / ml propidium iodide (PI; Sigma). The dye uptake was measured with a "FACScan” cytometer (Becton Dickinson GmbH, Heidelberg, Germany) using the "CellQuest” software. Changes in the forward / side scatter (FSC / SSC) of the cell population were evaluated at the same time. To quantify DNA fragmentation, the supernatants were centrifuged at 200 x g, the cells trypsinized and washed as indicated above.
  • Hep3B cells effector cells
  • SKW6.4 cells target cells
  • Na 2 51 Cr0 4 100 ⁇ Ci; NEN, Neu-Isenburg, Germany
  • Primers were purchased from MWG Biotech GmbH, Ebersberg, Germany. They had the following sequences: CD95L / Sense: 5'ATG TTT CAG CTC TTC CAC CTA CAG A-3 '; CD95L / Antisense: 5 '-CCA GAG AGA GCT CAG ATA CGT TGA C-3', whereby a PCR product with a length of 500 bp was obtained.
  • the primers span all three introns of CD95L, which makes it easier to distinguish between cDNA and genomic DNA.
  • each reverse-transcribed mRNA was checked by means of a ⁇ -actin PCR, the following PCR primers being used: actin / sense: 5 '-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3'; and ⁇ -actin / antisense: 5 '-CTA GAA TTT GCG GTG GAC GAT GGA GGG-3', whereby a PCR product with a length of 600 bp is obtained.
  • PCR products were analyzed on 1.5-2% TBE (Tris-Borate-EDTA) agarose gels.
  • the cells were lysed in 200 ⁇ l passive lysis buffer (Promega). After a 20 minute incubation at room temperature, the cells were scraped off the plates. The lysates were subjected to two freeze / thaw cycles and then cell debris was separated by centrifugation. The supernatants were measured in a w Duolumat "device (from Berthold, Wildbach, Germany) using the" dual luciferase "assay system from Promega. A renal luciferase expression vector pRenilla, controlled by a basal promoter, was used to normalize the transfection efficiencies.
  • Renilla luciferase was measured in the "dual luciferase" assay system and the CAT expression was determined by means of a commercially available ELISA (Röche Diagnostics). In addition, the amount of protein was measured in the Biorad protein assay (Munich, Germany) and used to normalize the protein content of the transfected cells.
  • the AP-1 site at +90 double-stranded 01 igonucleotide ide containing the CD95L promoter was determined using T4 polynucleotide kinase (MBI Fermentas, St. Leon-Roth, Germany) with [ ⁇ - 32 P] ATP (5000 Ci / mmol; Amersham GmbH, Braunschweig, Germany).
  • the single-stranded oligonucleotides had the following sequences: Sense: 5 '-GGG CTG GCC TGA CTC ACC
  • the binding reactions were carried out at 4 ° C. for 30 min using 5 ⁇ g of nuclear protein in a buffer which
  • Cultivated Hep3B cells or freshly isolated human primary hepatocytes were plated on Lab-Tek TM chamber supports (Renner GmbH, Darmstadt, Germany). After culturing for at least two days, the cells were treated with chemotherapeutic agents. Subsequently, a
  • Example 2 Chemotherapeutic agents induce apoptosis in liver cell cancer cell lines via the CD95 / CD95L system
  • HepG2 cell cultures were treated at 70% confluency with the 5-FU either in the absence or presence of one of the CD95L blocking compounds and apoptosis was assessed by PI exclusion and forward / side scatter analysis
  • Example 3 CD95L is up-regulated after administration of chemotherapeutics
  • CD95L mRNA is up-regulated after treatment with bleomycin, a chemotherapeutic from the group of antibiotics. All concentrations used were in the clinically relevant range.
  • the upregulation was observed in both HepG2 (wt p53) and Hep3B (p53 - / -) cells, and is therefore independent of p53, since the Hep3B cell line lacks this protein.
  • 5-FU and etoposide can upregulate CD95L in both HepG2 and Hep3B cells depending on time and dose (FIGS. 2B and 2C).
  • the upregulation of CD95L is delayed and occurs after 20 to 25 hours in relation to the occurrence of apoptosis in liver cell cancer cell lines.
  • blocking experiments were carried out either with the transcription inhibitor Actinomycin Cl or with the tr a n s 1 a t i o n s i n h i b i t o r cycloheximide.
  • Example 5 CD95L is up-regulated in liver cells after stimulation with chemotherapeutic agents by activating the newly identified AP-1 element.
  • a computational search for B in nigest 11 s for transcription factors revealed a consensus sequence for the dimeric factor AP-1. The exact location of this sequence is shown in Figure 5A (boxed). To confirm that this site is in fact an AP-1 functional site transfected together with the -36 / + 100 and -36 / + 19 luciferase reporter constructs c-jun and c-fos constructs. As shown in Figure 6A, cotransfection with c-jun and c-fos resulted in significant stimulation of the -36 / + 100 construct containing the AP-1 site but not the -36 / + 19 construct, the this position at +90 is missing.
  • oligonucleotides which comprised the consensus AP-1 site (CCTGACTC) in the CD95L promoter. These oligonucleotides could be extracted from 5-FU-treated HepG2 cells and 5-FU-treated Huh7- Cells (another liver cell carcinoma cell line) were shifted with regard to their mobility, with only a slight mobility shift being observed with extracts from untreated cells (FIG. 7).
  • this AP-1 element was mutated by site-directed mutagenesis.
  • the consensus wild-type sequence CTGACTCA was mutated to CTAATTAA at three different positions.
  • These mutations destroy the artefact of an ⁇ P-1-uciferase reporter construct.
  • the introduction of these mutations into the - 36 / + 100 reporter construct almost completely nullified the inducibility of the construct after treatment with 100 ⁇ g / ml 5-FU for 48 hours (FIG. 8). This is further evidence of the importance of the AP-1 site in chemotherapy-induced apoptosis.
  • Example 6 Involvement of the stress-activated protein kinase pathway (SAPK / JNK) in the chemotherapy-induced upregulation of CD95L.
  • SAPK / JNK stress-activated protein kinase pathway
  • the SAPK / JNK cascade in the target cells is involved in many factors that trigger cell stress.
  • Chemotherapeutic agents are strong stress factors both in vivo and in vitro. It was therefore investigated whether this system of successively activated kinases is involved in the reaction to chemotherapeutic agents.
  • the final goal of the SAPK cascade is the phosphorylation of c-Jun.
  • AP-1 is an acute phase protein and is normally activated within a few minutes after a given stimulus, it was examined whether an increased activation of AP-1 can be observed in a time frame that that in the above experimental System found upregulation of CD95L fits.
  • Hep3B cells were immunostained 40 hours after treatment with 5-FU with an antibody against c-Jun. After immunofluorescence staining, the intensity of the staining and the spatial distribution of positive signals were examined. The specificity of the staining was confirmed by blocking experiments using antigenic peptides. Untreated Hep3B cells showed slight positive signals (Figure 10A). After treatment with 5-FU for 40 hours, the cells AP-1 accumulated strongly in the nucleus, which can be seen as point-like positive signals in FIG.

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Abstract

Procédé qui permet de déterminer si un composé à l'essai est efficace pour une chimiothérapie. Ledit procédé consiste (a) à disposer d'une séquence d'ADN qui comprend le site AP-1 représenté à la figure 5A en tant que partie d'un promoteur, de préférence du promoteur CD95L, lié de manière fonctionnelle avec un gène reporter ou avec le gène CD95L, (b) à mettre en contact la séquence d'ADN de l'étape (a) avec le composé à l'essai dans un milieu cellulaire et (c) à déterminer l'activation du promoteur, la présence d'une activation indiquant que le composé à l'essai est efficace pour une chimiothérapie.
EP00991118A 1999-12-23 2000-12-22 Procede de criblage pour substances chimiotherapeutiques Withdrawn EP1246940A2 (fr)

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DE19962828A DE19962828A1 (de) 1999-12-23 1999-12-23 Screeningsverfahren für Chemotherapeutika
DE19962828 1999-12-23
PCT/DE2000/004643 WO2001048238A2 (fr) 1999-12-23 2000-12-22 Procede de criblage pour substances chimiotherapeutiques

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GB0205794D0 (en) 2002-03-12 2002-04-24 Montelius Lars G Mems devices on a nanometer scale
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Title
HOLTZ-HEPPELMANN ET AL.: "Transcriptional regulation of the human FasL promoter-enhancer region", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 8, 1998, pages 4416 - 4423 *
HOLTZ-HEPPELMANN ET AL: "Transcriptional regulation of the human FasL promoter-enhancer region", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 8, 1998, pages 4416 - 4423 *
See also references of WO0148238A3 *

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